U.S. patent application number 14/124429 was filed with the patent office on 2014-06-26 for long chain glycolipids useful to avoid perishing or microbial contamination of materials.
This patent application is currently assigned to IMD NATURAL SOLUTIONS GMBH. The applicant listed for this patent is Jens Bitzer, Barbel Kopcke, Jana Moldenhauer, Kathrin Reinhardt, Marc Stadler. Invention is credited to Jens Bitzer, Barbel Kopcke, Jana Moldenhauer, Kathrin Reinhardt, Marc Stadler.
Application Number | 20140178444 14/124429 |
Document ID | / |
Family ID | 46210197 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140178444 |
Kind Code |
A1 |
Stadler; Marc ; et
al. |
June 26, 2014 |
LONG CHAIN GLYCOLIPIDS USEFUL TO AVOID PERISHING OR MICROBIAL
CONTAMINATION OF MATERIALS
Abstract
The invention relates to the use of, and methods of use
employing, certain glycolipid compounds as defined in detail below
and having preservative or antimicrobial properties, novel
compounds of the glycolipid class, and related invention
embodiments. The compounds have the formula I ##STR00001## wherein
m is 3 to 5, n is 2 to 5, o is 0 or 1 and p is 3 to 17, with the
proviso that the sum m+n+o+p is not less than 14; and R is a
carbohydrate moiety bound via one of its carbon atoms to the
binding oxygen, and/or a physiologically, especially
pharmaceutically or nutraceutically or cosmetically, acceptable
salt thereof, or an ester thereof, as such or in the form of a
composition, where the compound may be present in open chain form
and/or in the form of a lactone (FIG. 1).
Inventors: |
Stadler; Marc;
(Niederkirchen, DE) ; Bitzer; Jens; (Dortmund,
DE) ; Kopcke; Barbel; (Dortmund, DE) ;
Reinhardt; Kathrin; (Dortmund, DE) ; Moldenhauer;
Jana; (Dortmund, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Stadler; Marc
Bitzer; Jens
Kopcke; Barbel
Reinhardt; Kathrin
Moldenhauer; Jana |
Niederkirchen
Dortmund
Dortmund
Dortmund
Dortmund |
|
DE
DE
DE
DE
DE |
|
|
Assignee: |
IMD NATURAL SOLUTIONS GMBH
Dortmund
DE
|
Family ID: |
46210197 |
Appl. No.: |
14/124429 |
Filed: |
June 6, 2012 |
PCT Filed: |
June 6, 2012 |
PCT NO: |
PCT/EP2012/002399 |
371 Date: |
February 12, 2014 |
Current U.S.
Class: |
424/401 ;
424/400; 424/55; 424/59; 424/65; 426/532; 510/119; 514/25; 514/777;
536/18.2 |
Current CPC
Class: |
A23B 5/14 20130101; A23L
2/44 20130101; A61P 31/10 20180101; C07H 15/04 20130101; A61K 8/33
20130101; A61Q 5/02 20130101; A23B 9/26 20130101; A23B 7/154
20130101; A61K 8/365 20130101; A01N 63/30 20200101; A61Q 17/005
20130101; A01N 43/16 20130101; A61K 8/37 20130101; A23B 4/20
20130101; C12P 19/44 20130101; A61P 31/04 20180101; A61K 8/9728
20170801 |
Class at
Publication: |
424/401 ; 514/25;
536/18.2; 424/400; 424/65; 514/777; 424/55; 424/59; 426/532;
510/119 |
International
Class: |
A01N 43/16 20060101
A01N043/16; A61Q 5/02 20060101 A61Q005/02; A23L 2/44 20060101
A23L002/44; C07H 15/04 20060101 C07H015/04; A61K 8/33 20060101
A61K008/33 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 2011 |
EP |
11004776.8 |
Claims
1. Method of preservating a material or imparting antimicrobial
properties to said material, said method comprising adding to said
material an effective amount therefor of a compound of the formula
I, or a mixture of two or more such compounds of the formula I,
##STR00055## wherein m is 3 to 5, n is 2 to 5, o is 0 or 1 and p is
3 to 17, with the proviso that the sum m+n+o+p is not less than 14;
and R is a carbohydrate moiety bound via one of its carbon atoms to
the binding oxygen, and/or a physiologically, especially
pharmaceutically or nutraceutically or cosmetically, acceptable
salt thereof, or an ester thereof, where the compound may be
present in open chain form and/or in the form of a lactone, where
said material is selected from the group consisting of a cosmetic,
a food, a beverage, a pharmaceutical, a medical device, and an
active packaging material.
2. Method according to claim 1, where the material to which the
compound is added is a cosmetic, a food or a beverage.
3. Method according to claim 1, where in the compound of the
formula I m is 3 to 5, n is 2 to 5, o is 0 or 1, p is 5 to 15 and R
is a moiety of the subformula ##STR00056## wherein the rings A, B
and C are monosaccharide moieties each independently from the
others with 5 or 6 ring members, wherein one or more of the
hydroxyl groups may be acylated.
4. Method according to claim 1, wherein the compound or mixture of
compounds of the formula I comprises at least one compound selected
from the group of compounds with the following formulae:
##STR00057## ##STR00058## ##STR00059## ##STR00060## or a
physiologically acceptable salt thereof.
5. Method according to claim 1, where at least one additional
preservative is added.
6. Method according to claim 1, where the compound or compounds of
the formula I, a physiologically acceptable salt thereof, and/or an
ester thereof, is added in the form of an extract from a natural
source or obtained from such an extract.
7. Method according to claim 6, where the source of the extract is
Dacryopinax spathularia strain FU50088, Ditiola radicata strain
MUCL 53180, Ditiola nuda strain CBS 173.60 or Femsjonia luteo-alba
(=Ditiola pezizaeformis) strain MUCL 53500.
8. A compound or a mixture of compounds of the formula I:
##STR00061## wherein m is 3 to 5, n is 2 to 5, o is 0 or 1 and p is
3 to 17, with the proviso that the sum m+n+o+p is not less than 14;
and R is a carbohydrate moiety bound via one of its carbon atoms to
the binding oxygen, and/or a physiologically, especially
pharmaceutically or nutraceutically or cosmetically, acceptable
salt thereof, or an ester thereof, where the moiety R carries at
least one hydroxyl group esterified with an acid with 3 or more
carbon atoms, where the compound may be present in open chain form
and/or in the form of a lactone, or a physiologically acceptable
salt, and/or an ester thereof.
9. A compound of the formula I according to claim 8, which is
selected from the group consisting of compounds represented in the
following table: ##STR00062## ##STR00063## and physiologically
acceptable salts and esters thereof.
10. A preservative or antimicrobial composition, comprising as
active agent a compound or a mixture of compounds of the formula I,
a physiologically acceptable salt thereof, and/or an ester thereof,
as shown or defined in claim 8, alone or with another additive,
optionally a carrier material.
11. The composition according to claim 10, which is a precursor of
a beverage.
12. An extract comprising one or more compounds of the formula I, a
physiologically acceptable salt thereof, and/or an ester thereof,
as shown or defined in claim 8.
13. A method of enhancing microbial stability of a material,
comprising adding one or more compounds of the formula I, a
physiologically acceptable salt thereof, and/or an ester thereof,
as shown or defined in claim 8 to a material, selected from the
group consisting of a cosmetic, a food, a beverage, a
pharmaceutical, a medical device, and an active packaging
material.
14. A material comprising, as or within a coating and/or as
admixture, an additive in the form of a compound or a mixture of
compounds of the formula I, a physiologically acceptable salt
thereof and/or an ester thereof, as defined in claim 8, which
material is a cosmetic, a food, a beverage, a pharmaceutical, a
medical device, or an active packaging material.
15. The material according to claim 14, where the beverage is
selected from the group consisting of water, flavoured water,
fortified water, a flavoured beverage, carbonated water, a juice,
cola, lemon-lime, ginger ale, root beer beverages which are
carbonated in the manner of soft drinks, a syrup, a diet beverages,
a carbonated soft drink, a fruit juice, other fruit containing
beverages which provide the flavor of fruit juices and contain
greater than 0% fruit juice but less than 100% fruit juice, fruit
flavored beverages, vegetable juices, vegetable containing
beverages, which provide the flavor of any of the aforementioned
vegetable juices and contain greater than 0% vegetable juice but
less than 100% vegetable juice, isotonic beverages, non-isotonic
beverages, soft drinks containing a fruit juice, coffee, tea, tea
beverages prepared from tea concentrate, extracts, or powders,
drinkable dairy products, hot chocolate, chocolate powders/mixes,
drinkable soy products, non-diary milks, alcoholic beverages, fruit
smoothies, horchata, sport drinks, energy drinks, health drinks,
wellness drinks, shakes, protein drinks, drinkable soy yogurts, low
acid beverages, acidified beverages, nectars, tonics, frozen
carbonated beverages, frozen uncarbonated beverages, liquid meal
replacements, infant formulations, and combinations or mixtures
thereof; or the material is a cosmetic selected from a cream,
emulsion, lotion, gel or oil for the skin; a face masks, a tinted
base, a make-up powder, an after-bath powder, a hygienic powder, a
toilet soap, a deodorant soap, a perfumes, a toilet water, an eau
de Cologne, a bath or shower preparation; a depilatory; a
deodorant, an anti-perspirant, a hair care product; a setting
product; a cleansing product; a conditioning product; a
hairdressing product; a shaving products; a product for making up
and removing make-up from the face and the eyes, a product intended
for application to the lips, a products for care of the teeth
and/or the mouth; a product for nail care and/or make-up, a product
for external intimate hygiene, a sunbathing product, a product for
tanning without sun, a skin-whitening product, an anti-wrinkle
product, a tampon, a sanitary towel, a diaper and a
handkerchief.
16. A material obtained by the method according to claim 1.
Description
SUMMARY OF THE INVENTION
[0001] The invention relates to the use of, and methods of use
employing, certain glycolipid compounds as defined in detail below
and having preservative or antimicrobial properties, novel
compounds of the glycolipid class, and related invention
embodiments.
[0002] These and related invention embodiments are described below
and in the claims which are incorporated into the specification by
reference.
BACKGROUND OF THE INVENTION
[0003] Bacteria and other microbial organisms cause food and
beverage products, cosmetic and home care products as well as other
products to go bad, thereby reducing the shelf life or useful life
of such products or goods. Thus, numerous efforts have been made to
reduce the deleterious effects of microbial contaminants in food
and beverage products, cosmetics, dressing material and other
materials, e.g. medical devices such as implants.
[0004] Other food preservatives such as salt, sugar and vinegar
have been used for generations and while relatively safe to use,
their preservative effect is limited in both duration of effect and
the types of food and beverages for which they can be used. In
addition, at higher levels, preservatives such as salt and vinegar
can affect the taste of the product.
[0005] Commonly used preservatives for cosmetics include
antimicrobial agents such as quaternary ammonium compounds,
alcohols, chlorinated phenols, parabens and paraben salts,
imidazolidinyl urea, phenoxyethanol, p-hydroxybenzoate, small
carboxylic acids like benzoic acid, sorbic acid, salicylic acid,
formic acid, proponic acid or corresponding salts.
Formaldehyde-releasers and isothiazolinones may also be used.
[0006] However, these materials often may not be tolerated or, e.g.
in the case of formaldehyde, may even be toxic and even
carcinogenic, or they may cause allergies or food intolerance.
[0007] Another preservative e.g. used in food and especially
beverages is sulfuric acid, while in meat products, e.g. sausages,
preserved meat and meat, stabilizers which decrease water activity
such as potassium and/or sodium nitrites and nitrates are often
added. Also smoke is often used for preserving meat products, with
the undesirable side effect of formation of polycyclic aromatic
hydrocarbons which have carcinogenic properties.
[0008] Food and beverages have varying degrees of sensitivity to
microbiological spoilage depending on intrinsic factors of the food
or beverage such as pH, nutrient content (e.g., juice, vitamin, or
micronutrient content), carbonation level, Brix (an indicator of
sugar content), water quality (e.g., alkalinity and/or hardness),
and preservatives.
[0009] Spoilage events occur when microorganisms are able to
overcome the product's intrinsic protection factors and grow. The
microorganisms' ability to overcome these hurdles can be influenced
by, among other things, initial contamination level, temperature,
water content, e.g. water activity, and package integrity. Of
special importance are also recurrent contaminations of cosmetics,
e.g. by hand contact during normal use.
[0010] A number of organisms are responsible for spoiling a variety
of beverages materials, including cold-filled beverages. Yeasts
such as Saccharomyces, Zygosaccharomyces, Candida, and Dekkera spp.
are most common. Also, acidophilic bacteria such as Lactobacillus,
Leuconostoc, Gluconobacter, and Zymomonas spp., and molds like
Penicillium, Aspergillus and Mucor spp. can spoil various water
containing materials.
[0011] Other materials and also other types of beverages are
susceptible to spoilage by microorganisms. Spores of acidophilic,
thermophilic bacteria, such as Alicyclobacillus spp., and heat
resistant mold spores of Byssochlamys, its anamorphic (asexual)
stages Paecilomyces, and Neosartorya spp. can survive
pasteurization and may spoil non-carbonated, hot-filled products
such as sport drinks and teas. Also, packaged waters are
susceptible to contamination by molds.
[0012] In cosmetic, personal care and home care products spoilage
occurs by a variety of micro-organisms, ranging from Gram-positive
bacteria (e.g. Staphylococcus spp.), Gram-negative bacteria (e.g.
Escherichia coli, Pseudomonas spp.) to yeasts (e.g. Candida
albicans) and common molds (e.g. Aspergillus niger). Microbial
growth in or on these products depends on several instrinsic
factors such as water activity of the formulation (minimum water
activity requirements for growth or proliferation range from 0.99
for Acinetobacter species down to 0.61 for some fungal species),
formulation composition, pH value (e.g., optimum pH for the growth
of most yeasts and molds is between 4.0 and 6.0), and processing
conditions such as temperature. While high temperatures, e.g.
80.degree. C. for 20 minutes, may reduce microbial contaminations
during processing, it is important to prevent inactivation or
degradation of the preservatives in the formulation. Furthermore,
product packaging, solubility of the preservative and its
antimicrobial susceptibility profile will influence preserving
efficacy and, consequently, the shelf life of the products.
[0013] Protection against microbiological spoilage of sensitive
products can be achieved using chemical preservatives and/or
processing techniques such as hot filling, tunnel pasteurization,
ultra-high temperature (UHT), or pasteurization followed by aseptic
packaging, and/or pasteurization followed by chilling the beverage.
Generally, beverages with a pH<4.6 can be chemically preserved,
heat processed, and filled into packages such that the product is
not re-contaminated. For example, process techniques such as
cold-filling, followed by chemical preservatives or pasteurization
with cold filling, may be used to preserve a cold-filled beverage.
In a similar manner, this same beverage may be processed using
non-preserved techniques such as hot filling, tunnel
pasteurization, pasteurization followed by aseptic filling, or
requiring the beverage to be chilled, i.e., under refrigeration
following the pasteurization step. Beverages having a pH 2-4.6 must
be processed such that spores are destroyed using ultra-high
temperatures followed by aseptic filling into packages or by using
a retort.
[0014] Current preservation systems for acidic, shelf-stable,
carbonated and non-carbonated food or beverages, e.g. soft drinks,
generally rely on weak acid preservatives (e.g., benzoic and/or
sorbic acid). Benzoic and sorbic acids (and salts thereof)
effectively inhibit yeasts, bacteria, and molds with some
exceptions. Weak acids in beverages exist in equilibrium between
their dissociated and undissociated forms, which is dependent upon
the dissociation constant of the acid (pKa) and the beverage's pH.
The pKa for benzoic acid is 4.19 and the pKa of sorbic acid is
4.76. A beverage pH below the pKa of the involved acid pushes the
equilibrium towards the undissociated form. The undissociated form
is more efficacious against microorganisms; therefore, weak acid
preservatives are most effective in the low pH range.
[0015] The preservation properties of weak acids may be enhanced by
the addition of preservative enhancers, such as chelating
compounds, to the material to be preserved, e.g. a food, beverage
or cosmetic preparation. For example, common chelating compounds
added include calcium disodium ethylenediaminetetraacetic acid
(EDTA) or one or more of the polyphosphates such as sodium
hexametaphosphate (SHMP).
[0016] In high nutrient, non-carbonated products, such as those
beverages containing juice, vitamins, and/or minerals, the weak
acids are more likely to exert inhibition if used in conjunction
with preservative enhancers. Also weak acid preservation systems,
however, have limitations:
[0017] Genetic adaptation and subsequent resistance by
microorganisms is one of the biggest concerns (see Piper. et al.,
Microbiol. (2001) 147: 2635-2642). Certain yeasts such as Z.
bailii, Z. bisporus, Candida krusei, and S. cerevisiae have
specific genes that enable them to resist the weak acid
preservatives and grow. This happens despite the presence of
preservatives and regardless of the co-presence of EDTA or SHMP.
Some bacteria such as Gluconobacter spp. are also thought to be
preservative resistant. The levels of weak acids necessary to
overcome this resistance have been shown to be far beyond
regulatory limits on use levels. Most often, spoilage of preserved
teas, juice-containing beverages, and carbonated beverages is due
to preservative resistant micro-organisms.
[0018] Medium chain saturated fatty acids and their esters with
oligohydroxylated compounds have been found to possess inhibitory
effects against several bacteria and fungi. The minimum inhibitory
concentration values reach a maximum with a chain length of about 8
to 12 carbon atoms (Varvaresou, Int. J. Cosmetic Sci (2009) 31:
163-75).
[0019] In addition, the other process techniques for low acid
beverages (i.e., pH 4.6) have limitations. Such low acid beverages
should be thermally-treated sufficiently to destroy spores of
Clostridium botulinum and Bacillus species (B. cereus, B. subtilis
and others). Examples of such processes include UHT and retort.
Even after such processing, the beverage products should be handled
in a way to prevent post-processing contamination. Research,
however, suggests that there may still be various strains of
microorganisms that can survive those different processing
techniques. To that end, those processing techniques may not
eliminate the potential for spoilage.
[0020] Other chemical preservatives can likewise cause adverse side
effects when consumed. Thus, many existing preservatives must be
regulated and have legally imposed upper limits on usage. In
addition, many preservatives, such as sodium benzoate,
proprionates, aromatic benzenes, organic acids, propylene glycol
and glycerol, for example, when used at levels sufficient for
antimicrobial effects, impart an unpleasant taste on the beverage
or food, masking or altering to some degree the taste expected by
the consumer. Weak acids can impart throat or mouth burn when used
at high levels. Although there are certain shelf-stable beverages
where this attribute may be acceptable, this sensory perception is
often considered negative. Similarly, polyphosphates used in weak
acid preservation systems can have some limitations. For example,
polyphosphates can impart off-flavors to a beverage.
[0021] Certain emollient solvents exhibit synergistic action when
combined with essential oils or ingredients against microorganisms
as noted in WO 03/034994, which is incorporated by reference in its
entirety. The emollient solvents used as preservatives in cosmetics
do not usually produce skin reactions, and in addition, render the
skin smooth and silky.
[0022] In a series of publications Nishida et al. reported on
compounds of an unidentified strain of "Basidiomycetes sp." to
produce glycolipids which are assumed to exhibit inhibitory
activity against Gram-positive bacteria (Nishida, Tetrahedron Lett
1988, 29(41): 5287-90; Chem Pharm Bull 1990, 38(9): 2381-9; J
Antibiot 1991, 44(5): 541-5; Chem Pharm Bull 1991, 39(11): 3044-7;
Proc "Symposium on the chemistry of natural organic compounds"
1987, 29: 729-36; ibid 1990, 32: 253-9) and against infection by
polio and herpes virus (J Chrom 1994, 664(2): 195-202; J Mass
Spectrom Soc Jpn 1995, 43(1): 27-36; ibid 37-44). Much effort was
done in the structure elucidation and the results were presented in
detail in the above cited publications; nevertheless the authors
did not present any data of the suggested antimicrobial activity.
Later on, one of the compounds isolated by Nishida et al. (Glykenin
IVA) was reported as antifungal agent from a Dacrymyces sp.
(Wunder, A., Diss. 1995, Univ. Kaiserslautern, Germany). Mierau (Z
Naturforsch 2003; 58c: 541-6) made reference to this work. In
neither of the cited publications, details on the biological data
and the identity of the producer organisms were reported.
[0023] JP 2006-176438 A and J. Antibiot. 2007, 60, 633-639 disclose
F-19848 A, a glyocolipid obtained from the fermentation broth of
the fungus strain Dacrymyces sp. SANK 20204, as inhibitor of
hyaluronic acid binding receptor CD44 and as being useful for
treating or preventing degenerative arthritis or a disease caused
by the degenerative arthritis.
[0024] Biosurfactants are produced extracellularly or as a part of
the cellular membrane by various organisms such as bacteria and
fungi. Their structures usually contain a hydrophobic non-polar
moiety that consists of unsaturated, saturated, and/or oxidized
lipids or fatty acids, and a hydrophilic component, which may be
composed of amino acids, carbohydrates, phosphates or cyclic
peptides. They are generally classified into glycolipids,
lipopeptides, phospholipids, fatty acids and polymeric compounds
according to their chemical structures. Biosurfactants are produced
by a wide range of microorganisms and therefore differ in their
chemical structure. Some biosurfactants have antimicrobial activity
against bacteria, yeasts, molds or viruses. Moreover, they can
prevent microbial colonization of surfaces such as those of
implanted medical devices through their ability to disrurpt
biofilms on these surfaces.
[0025] Sophorolipids, rhamnolipids and mannosyl-erythritol lipids
are the most widely used glycolipid biosurfactants in
cosmetics.
[0026] Rhamnolipids are known for their efficiency in remove of
nosocomial microbes in biofilms. A biofilm is characterized by a
strong adherence activity of the engaged microorganisms. Known
biosurfactants with anti-adhesive or biofilm disrupting activity
are produced by Lactobacillus acidophilus, L. fermentum,
Lactococcus lactis, Streptococcus thermophilus, Bacillus subtilis,
B. licheniformis, Brevibacterium aureum, Pseudomonas aeruginosa,
and P. putida.
[0027] The genus Lactobacillus produces the lipopeptide surlactin,
the genus Bacillus produces lipopeptides belonging to the
fengycin-like and surfactin-like families of secondary metabolites.
Another lipopeptide has been isolated from Brevibacterium aureum.
Streptococcus thermophilus is a producer of yet unidentified
glycolipids, which contain sizeable amounts of nitrogen (Rodrigues,
Colloids & Surfaces B: Biointerfaces (2006) 53: 105-112).
Further known producers of biosurfactants exhibiting activity
against microorganisms involved in biofilms are Pseudomonas
aeruginosa which produces rhamnolipids, and P. putida, which
produces lipopeptides. The nitrogen containing biosurfactants
produced by Streptococcus mitis were not yet identified.
Lactococcus lactis produces a low molecular weight [467 Da]
biosurfactant consisting of methyl-2-O-methyl-beta-d-xylopyranoside
with octadecanoic acid. (Saravanakumari & Mani, Bioresour
Technol (2010) 101:8851-8854).
[0028] A good overview of different classes of known biosurfactants
is given by Rahman et al. (Biotechnology (2008): 360-70) presenting
great advantages of theses compounds against synthetic compounds
such as a lower toxicity, higher biodegradability, a better
environmental compatibility and their ability to tolerate extreme
temperature, pH and salinity. Nevertheless the reported
difficulties in the production teach the limitation of industrial
usage as being relative low yields, high fermentation costs and
difficult isolation procedures within industrial processes.
Acceptable economic production of biosurfactants in large scale is
suggested by the use of industrial wastes as process media.
[0029] As commonly understood in the art, the definitions of the
terms "preserve", "preservative," and "preservation" do not provide
a standard time period for how long the matter to be preserved is
kept from spoilage, decomposition, or discoloration. The time
period for "preservation" can vary greatly, depending on the
subject matter. Without a stated time period, it can be difficult
or impossible to infer the time period required for a composition
to act as a "preservative".
[0030] In summary, many preservatives and preservation methods have
undesirable side effects, such as toxicity, allergenicity,
carcinogenicity, occasionally formation of resistance, and/or often
are not accepted by the consumers in a time where natural
preservation is preferred over preservation with synthetic or other
products having a negative health image.
[0031] Accordingly, a great need exists for effective, relatively
inexpensive, non-toxic, naturally derived preservative compositions
that avoid disadvantages as mentioned and are capable of reducing
microbial contamination and concomitant spoilage in a wide range of
perishable food, beverages, cosmetics, and other consumer goods,
but without appreciably altering the taste, color, odor, or
function of the product.
GENERAL DESCRIPTION OF THE INVENTION
[0032] Surprisingly, new compounds were found and isolated from
strains of Dacryopinax spathularia and other fungal strains
belonging to the Dacrymycetaceae family, and for them as well as
for compounds of the same class it has been found that they have
novel and useful properties. The compounds are described in more
detail below.
[0033] The class of compounds of the formula I, both insofar as
they are not novel as well as they are novel, is not known in the
art for any spoilage/perishing preventing activity.
[0034] Very surprisingly therefore, it has been found that these
compounds exhibit a strong inhibition activity against
microorganisms which are responsible for spoiling or deterioration
of orally consumable products (such as food products and beverages)
or cosmetic compositions. For example, an extract of Dacryopinax
according to the present invention can show a broader activity
spectrum than the corresponding single components of the produced
glycolipid complex (glycolipid mixture) that have been isolated to
purity. Such multi-component mixtures exhibit a remarkable long
term activity against numerous important spoilage microbes,
including e.g. Zygosaccharomyces and Bacillus species.
[0035] Further surprisingly, the microorganisms used in the present
invention allow producing large amounts of the compounds of formula
I in a cost effective production process.
[0036] They are, for example, able to reduce growth of microbial
contaminants such as bacteria, yeasts, molds and other
microorganisms and their spores, especially those which are
temperature resistant e.g. thermophilic or heat resistant, or
acidophilic e.g. microorganisms which tolerate a lower pH value
that cause spoilage of food, beverages, cosmetics and other
materials.
[0037] The glycolipids do not exhibit a distinctive taste or
unpleasant mouth feeling and therefore this application relates to
the use of glycolipids in materials which get in contact with the
oral cavity of a human. This application also relates to
synergistic combinations of antimicrobial ingredients that can be
used in orally consumable compositions, such as food and beverages,
without imparting off-flavors.
[0038] Yet surprisingly it was found that the use of a simple
medium only using dextrose or glucose as carbon source and a small
amount of yeast extract can be superior against typical complex
media known from the literature. However, also the use of other
media or growth substrates is included. Furthermore it was found
that this culture medium supported particularly high production
rates at relatively low biomass production, which facilitated
downstream processing of the crude glycolipid products, allowing
their easy recovery from the culture fluid by precipitation.
[0039] The glycolipids of the present invention can be shown to
demonstrate a broad antimicrobial spectrum and can be incorporated
as additives into various materials as a preservative or an agent
with preserving activity, especially as cosmetic additive and/or
food additive and/or beverage additive based on this antimicrobial
activity.
[0040] Although the mechanism of action for the glycolipids is
unknown, these compounds can, without that this is intended to mean
a comprehensive and concluding definition of their properties, be
considered as biosurfactants and, additionally, might influence the
cell membranes of microorganisms. Other known biosurfactants are
rhamnolipids, sophorolipids, lipopeptides like chlamydocin,
surfactin, lichenysin G, etc. as cited by Mukherjee (in
"Biosurfactins", R Sen ed., Springer, 2010, chapter 4, "Microbial
Surfactants and Their Potential Applications"). The cited
biosurfactants differ from the compounds of formula I of the
present invention. Although Mukherjee listed compounds named as
glycolipids, the structures of these compounds differ significantly
from those of the present invention:
[0041] The compounds useful according to the invention are in
general characterized
by a long chain fatty acid with at least 20 carbon atoms; by a
carbohydrate moiety which is not attached to the acid group: by an
alpha hydroxyl group; and by at least one further hydroxyl group,
in the "center" of the fatty acid carbon chain, clearly separated
from both the alpha position and the glycosyl substituent.
[0042] Especially compounds of the formula I represented below
which are esterified in their carbohydrate moiety by other acids
than acetic acid, in particular esterified by isovaleric acid, are
not known in the art and thus novel.
DETAILED DESCRIPTION OF THE INVENTION
[0043] In a first embodiment, the invention relates to the use of a
compound of the formula I, or a mixture of two or more such
compounds of the formula I,
##STR00002##
wherein m is 3 to 5, n is 2 to 5, o is 0 or 1 and p is 3 to 17,
with the proviso that the sum m+n+o+p is not less than 14; and R is
a carbohydrate moiety bound via one of its carbon atoms to the
binding oxygen, and/or a physiologically, especially
pharmaceutically or nutraceutically or cosmetically, acceptable
salt thereof, or an ester thereof, as such or in the form of a
composition, where the compound may be present in open chain form
and/or in the form of a lactone, as agent with preservative or
antimicrobial properties, comprising adding the agent to a
material, where said material is preferably selected from the group
consisting of a cosmetic, a food, a beverage, a pharmaceutical, a
medical device, a home care, and an active packaging material.
[0044] Preferred is the use, where in the compound of the formula
I, m is 3 to 5, n is 2 to 5, o is 0 or 1, p is 5 to 15 and R is a
moiety of the subformula
##STR00003##
wherein the rings A, B and C are monosaccharide moieties each
independently from the others with 5 or 6 ring members, wherein one
or more of the hydroxyl groups may be acylated.
[0045] In other terms: A compound for use according to the
invention is especially a linear carboxylic acid with at least 20
carbon atoms, preferably 22 to 28, 24 to 26, in particular 26,
substituted at position 2, that means the alpha position, with a
hydroxyl group; substituted with a second hydroxyl group at the
omega-5, omega-6 or omega-7 position which is itself substituted by
a carbohydrate, e.g. as defined below, and one additional (a third)
hydroxyl group between the omega and the alpha substituents which
is separated from the second one by two to five methylene groups,
where optionally this third hydroxyl group has a vicinal hydroxyl
group, in the direction of the acidic end.
[0046] In another embodiment of the invention, the compound or
compounds of the formula I, a physiologically acceptable salt
thereof, and/or an ester thereof, is added in the form of an
extract from a natural source or obtained from such an extract.
Preferably, the source of the extract is a fungus belonging to
family Dacrymycetaceae, a species of the genera Dacryopinax,
Ditiola, Guepiniopsis and/or Femsjonia, more especially Dacryopinax
spathularia, Dacrymyces sp., Dacrymyces stillatus, Dacrymyces
chrysocomus, Guepiniopsis buccina and/or Femsjonia luteo-alba
(=Ditiola pezizaeformis). Especially preferred are Dacryopinax
spathularia strain MUCL 53181, Dacryopinax spathularia strain MUCL
53182, Ditiola radicata strain MUCL 53180, Ditiola nuda strain MUCL
53179, Dacrymyces chrysocomus strain CBS280.84 and Femsjonia
luteo-alba (=Ditiola pezizaeformis) strain MUCL 53500.
[0047] In our investigations we found Dacryopinax spathularia
strain MUCL 53181 to be the best strain for identified so far for
producing the compounds of formula I and mixtures of two or more
compounds of formula I, in particular those described in detail
hereinafter, particularly the compounds of formula I exhibiting the
strongest antimicrobial activity against yeasts and molds. Thus, in
a further aspect the present invention also relates to Dacryopinax
spathularia strain MUCL 53181 as such.
[0048] Another embodiment relates to the use as described above or
below of one or more compounds of the formula I, where the material
to which such compound(s) are applied is subjected to a heat
treatment before, during or after addition of the compound(s) of
the formula I, a physiologically acceptable salt thereof and/or an
ester thereof.
[0049] In another embodiment, the invention relates to a novel
compound of the formula I, or a mixture of two or more compounds of
the formula I including a novel compound of the formula I, where
the compound or compounds may be present in open chain form and/or
in the form of a lactone, and/or a pharmaceutically or
nutraceutically or cosmetically acceptable salt thereof, as
such.
[0050] Another embodiment of the invention relates to a compound or
a mixture of compounds of the formula I shown above or as defined
above or below, where the moiety R carries at least one hydroxyl
group esterified with an acid with 3 or more carbon atoms, a
physiologically acceptable salt, and/or an ester thereof,
especially wherein the acid is a C.sub.3-C.sub.10-alkanoic acid,
especially isovaleric acid; a physiologically acceptable salt,
and/or an ester thereof, more especially a compound selected from
the group of compounds represented by the following formulae:
##STR00004## ##STR00005##
and/or a physiologically acceptable salt, and/or an ester of the
acid group thereof.
[0051] In yet another embodiment, the present invention relates to
a preservative or antimicrobial composition, comprising as active
agent a compound or a mixture of compounds of the formula I, a
physiologically acceptable salt thereof, and/or an ester thereof,
as shown or defined in any one of claims 1, 3, 4 and 8 to 15, alone
or with another additive, such as a carrier material, where the
preservative composition is especially for use in a cosmetic, a
food, a beverage, a pharmaceutical, a medical device, or an active
packaging material, especially in the form of a powder or a liquid,
e.g. a composition which is a coating or film. The composition, in
a more specific embodiment of the invention, may be a precursor of
a beverage, especially a concentrate, a syrup or a powder.
[0052] In another embodiment, the composition defined in the
preceding paragraph is an antimicrobial composition for enhancing
the stability against microorganisms, especially where at least one
microorganism is selected from the group consisting of mold, yeast
and bacteria.
[0053] In another embodiment, the composition according to any one
of the two preceding paragraphs is a preservative or antimicrobial
composition for a pharmaceutical, a medical device, a food
container, a beverage container, or especially a food, a beverage,
a cosmetic, or a home care product.
[0054] In another embodiment, the composition according to any one
of the two preceding paragraphs is a biofilm inhibiting agent and
used as such by administering, or in methods comprising
administering, one or more compounds of the formula I, or a
composition comprising it, to surfaces or materials coming into
contact with surfaces. This way biofilms on various materials
including medical devices, teeth, containers, home care products,
pipes or mains or other liquid conducting or containing devices and
the like can be avoided.
[0055] In yet another embodiment of the invention, the composition
according to any one of the preceding four paragraphs comprises an
additional preservative.
[0056] The invention, in yet another embodiment, also relates to an
extract comprising one or more compounds of the formula I, a
physiologically acceptable salt thereof, and/or an ester thereof,
as shown or defined above or below.
[0057] A further invention embodiment relates to a method of
enhancing microbial stability of a material, comprising adding to
said material one or more compounds of the formula I, a
physiologically acceptable salt thereof, and/or an ester thereof,
as shown or defined above or below, preferably a material selected
from the group consisting of a cosmetic, a food, a beverage, a
pharmaceutical, a home care, a medical device, and an active
packaging material, especially a beverage, or a food, or a
cosmetic.
[0058] Another embodiment of the invention relates to a material
comprising, as or within a coating and/or as admixture, an additive
in the form of a compound or a mixture of compounds of the formula
I, a physiologically acceptable salt thereof and/or an ester
thereof, as defined above or below. This material must be other
than the fungus from which the compound or compounds of the formula
I are extracted. In another embodiment of the invention, the
material is a cosmetic, a food, a beverage, a pharmaceutical, a
home care, a medical device, or an active packaging material,
especially a beverage, a beverage precursor, especially a
concentrate, syrup or powder, a food or a cosmetic. In another
embodiment, such material comprises an additional preservative.
[0059] Another invention embodiment relates to a material according
to the preceding paragraph, which is obtained after heat
treatment.
[0060] The invention also relates to a method of extracting and/or
isolating one or more compounds of the formula I, especially as
described below and/or in the Examples.
[0061] The invention also relates to the embodiments in the claims
and especially the dependent claims which are herewith incorporated
by reference into the description.
[0062] The compounds of the present invention or useful according
to the present invention of the formula I, according to present
knowledge, are produced only by fungi of the family
Dacrymycetaceae, particularly by fungi of the genera Dacryopinax
(e.g. D. spathularia), Dacrymyces, Ditiola (e.g. Ditiola radicata
or Ditiola nuda), Guepiniopsis and Femsjonia (e.g. F. luteo-alba).
All these fungi have in common that they belong to family
Dacrymycetaceae. All species of Dacrymycetaceae hitherto known are
wood-inhabiting saprotrophs, which may either cause brown rot or
white rot (Seifert, Mycologia 75 (1983): 1011-1018). Even though
they can be isolated easily from spores of basidiocarps growing in
the field and readily grow in culture, relatively few strains of
Dacrymycetes are deposited in public collections.
[0063] The fungal genus Dacryopinax G. W. Martin was erected by
Martin (Lloydia 11 (1948): 111-122) and currently comprises 23
accepted taxa, including 22 species and one variety (fide Mycobank;
http://www.mycobank.org). According to the latter database, the
genus is presently classified in the Basidiomycota, class
Dacrymycetes, order Dacrymycetales, family Dacrymycetaceae. Members
of the Dacrymycetes (previously often referred to as order
Dacrymycetales before it was elevated to class rank), are
characterized by their unique basidial morphology with two
equidiametrous epibasidia, thus shaping the basidium like a tuning
fork. In addition, they have dolipores with continuous
parenthesomes.
[0064] These common morphological and ultrastructural features were
supported very well in various molecular phylogenetic studies, as
reflected by Hibbett (Mycologia 98, 917-925, 2006) and references
cited therein. Most species of the Dacrymycetes still belong to
order Dacrymycetales, family Dacrymycetaceae. The latter family
comprises eight genera, which have been traditionally separated on
the basis of macroscopical (primarily relating to the basidiocarp
habit) and microscopical (e.g., the wall thickness of marginal
hyphae in the sterile parts of basidiocarps) morphological
characters of the fruiting bodies (i.e., basidiocarps). However,
this classification is not unequivocal and therefore gave rise to
various alternative taxonomic concepts over the past decades. For
instance, McNabb (N. Z. J. Bot 3, 59-72, 1965), gave the first
comprehensive treatment of Dacryopinax and circumscribed its
basidiocarp habit as follows; "Fructifications extremely variable
in shape, stipitate with a spathulate, petaloid, flabellate,
cupulate, obliquely cupulate, inversely cupulate, foliose, or
occasionally lobed and somewhat morchelloid pileus" This
circumscription, which is still valid until today, suggests that
Dacryopinax is a complex genus. In addition, recent molecular
phylogentic studies by Shirouzu et al (Mycoscience 48:388-394, 2007
and Persoonia 23, 16-34, 2009) suggested that convergent
evolutionary developments in the Dacrymycetales and Dacrymycetaceae
might have given rise to development of similar morphological
features, hence the basidiocarps that are characteristic of
Dacryopinax and other genera of Dacrymycetaceae might have evolved
independently more than once.
[0065] The currently accepted type species of Dacryopinax is
Dacryopinax elegans. However, by far the most cited species in the
literature is Dacryopinax spathularia (Schwein Fr.) G. W. Martin.
This species was first described from South Carolina, USA, and had
been treated under different names (i.e., Merulius spathularius,
Guepinia spathularia) before Martin proposed the genus Dacryopinax.
Under Guepinia spathularia (which is an invalid, later synonym of a
plant genus name and therefore had to be abandoned), this fungus
was already reported to occur in various tropical and subtropical
regions of the world, including Northern Australia, New Zealand,
Asia and America by Saccardo, P. A., Sylloge Fungorum 6, p 808
(1888) and has since then been reported from numerous other
countries of the world. Dacryopinax spathularia possesses an
unusual geographical distribution. According to McNabb (N.Z.J. Bot
3 (1965): 59-72) it is widely distributed throughout both
hemispheres, but has never been found in Europe, though it occurs
in North Africa and eastern Russia. The species is characterised by
having variable, albeit typically spathulate basidiocarps of up to
2.5 mm height, uniseptate spores, and thick-walled, cylindrical
abhymenial hairs.
[0066] Dacryopinax spathularia is one of the species in the family
and order which is capable of producing comparably large
basidiocarps. It was reported to be used as "edible mushroom" by
the indigenous population of Cameroon (Van Dyck et al., Ambio 32
(2003): 19-23). The entire fungal family Dacrymycetaceae does not
contain any poisonous species, even though the basidiocarps of most
species are rather inconspicuous and/or have a tough, rubbery
consistence that prevents their culinary use. Interestingly, the
cultures of certain Dacrymyces species, which can be regarded as
closely related to Dacryopinax, have been patented for their
utility in production of carotene pigments (U.S. Pat. No.
2,974,044). Carotenoids are also apparently the only secondary
metabolites that were so far reported from a species of
Dacryopinax, and their production in the cultures of D. spathularia
have been studied in detail by Vail & Lilly (Mycologia 60
(1968): 902-907).
[0067] The fungal strains that produce the compounds of the present
invention were characterised by morphological methodology, using
phase contrast microscopy of cultures grown on solid YMG medium,
and by molecular phylogenetic methods. Since the LSU or 28S/5.8S
nuc-rDNA had recently been reported to be informative for the
phylogenetic assessments of the Dacrymycetes by Shirouzu et al
(Persoonia 23, 16-34, 2009), and the authors of the latter paper
published numerous reliable reference sequence data, this region of
the DNA was chosen for comparison upon characterisation of the
strains of Dacrymycetes that are the subject of the present
invention.
[0068] DNA for PCR was isolated from YMG cultures. The 28S/5.8S
nuc-rDNA regions were then amplified using primers LR7 and 5.8SR
(Vilgalys Lab, Duke University, Durham, USA,
http://www.biology.duke.edu/fungi/mycolab/primers.htm), using the
PCR Taq PCR Core Kit (Qiagen, Hilden), and applying a standard
thermal profile with an annealing temperature of 53.degree. C.
Amplification products were purified using SigmaSpin Post-Reaction
Clean-Up columns (Sigma-Aldrich), using the protocol supplied by
the manufacturer. Nucleotide sequences were obtained by cycle
sequencing using a DNA Cycle Sequencing Kit (Jena Bioscience, Jena,
Germany) and 5' IRD700-labelled primer LROR (Vilgalys Lab).
Labelled primers were custom synthesized by Eurofins MWG Operon,
Ebersberg, Germany). The cycle sequencing products were then
analysed using a LI-COR 4200 (Li-Cor Bioscience, Lincoln, NB)
genetic analyser. In the following the characteristics of five
strains that were identified of producers of the glycolipids that
are subject of the current invention are briefly summarised.
[0069] Strain FU50088 was isolated from the sporocarp of an
unidentified basidiomycete growing on wood in French Guiana by
Sergej Buchet in 2002, provided to Bayer Healthcare AG, and
selected for fermentation in order to provide extracts that are
suitable for natural products screening. On YMG agar at 23.degree.
C., the culture attained about 10 mm diameter after 10 days of
incubation. The mycelium at first appeared velvety and white, but
soon attained a strong yellowish color. The occasional presence of
clamp connections revealed that the fungus belongs to the
Basidiomycota. After 5 days of incubation, conidiogenous cells
appeared in abundance on the vegetative hyphae, showing
polyblastic, sympodial conidiogenesis, producing subglobose hyaline
conidia, averaging 5-6.times.2.5-3 .mu.m in size. These
characteristics were found to be largely in agreement with the data
reported by Shirouzu et al (Persoonia 23, 16-34, 2009). The LSU
nucrDNA sequence of this strain FU50088 is included here as
sequence <SEQ ID NO: 1>.
[0070] The strain was studied in comparison with an authentic
strain of Dacryopinax spathularia, CBS 197.63, originating from
Africa, which was obtained from the Centrallbureau voor
Schimmelcultures, Utrecht, The Netherlands. Its morphological
characteristics, as well as its secondary metabolite production
were largely in accordance with that of strain FU50088.
Furthermore, a high degree of homology was observed between the
5.8S/ITS nrDNA and 28S nrDNA sequences of the two aforementioned
strains and reference DNA sequence data that had been published on
the Internet by specialists in the taxonomy and phylogeny of
Basidiomycota, under the name of Dacryopinax spathularia or
synonyms thereof. Therefore, strain FU50088 was identified to
belong to the species Dacryopinax spathularia by morphological,
molecular phylogenetic and chemotaxonomic methodology and is
referred herein as this species.
[0071] The reference strain Dacryopinax spathularia CBS 197.63,
collected from Bangui, Central African Republic, isolated by J.
Boidin and deposited with CBS in April 1963, resembled Dacryopinax
spathularia strain FU50088 in its growth and morphological
characteristics. However, its pigmentation was not as intense, and
even in aged cultures, the mycelia only turned pale yellow. The
conidia were subglobose to ovoid, measuring 3-6.5 (-8).times.2.5-4
.mu.m. The LSU nucrDNA sequence of this reference strain CBS 197.63
is included here as sequence <SEQ ID NO: 2>.
[0072] Three other strains that were not assigned to the genus
Dacryopinax but are members of family Dacrymycetaceae as well were
obtained from public culture collections, studied and found to
produce the compounds of the invention. Their history and
characteristics are given below:
[0073] Ditiola nuda strain CBS 173.60 was isolated in Shirokane,
Tokyo, Japan, from a petiole of the plant Shiia sieboldii according
to the information provided in the CBS catalogue and deposited with
CBS by K. Tubaki in 1960. The strain showed similar growth
characteristics to Dacryopinax spathularia FU50088 and like those
of the latter strain; its conidia measured 6-5.times.2.5-3 .mu.m.
The LSU nucrDNA sequence of Ditiola nuda strain CBS 173.60 is
included here as sequence <SEQ ID NO: 3>.
[0074] Ditiola radicata strain CBS 126.84 was isolated from
sporocarps growing on gymnosperm wood collected in August 1982 in
Canada, Alberta, Banff National Park, C Level Cirque Trail, by
Keith A. Seifert and deposited with CBS in 1984. The strain showed
similar growth and morphological characteristics to Dacryopinax
spathularia FU50088, but had smaller conidia (4-5.times.1.5-2
.mu.m). The LSU nucrDNA sequence of Ditiola radicata strain CBS
126.84 is included here as sequence <SEQ ID NO: 4>.
[0075] Ditiola pezizaeformis strain ATCC13299 was originally
deposited with ATCC as Femsjonia luteo-alba. The strain had been
used in an U.S. Pat. No. 2,974,044 and claimed to be a producer of
carotenoids. However, according to the current taxonomy, Femsjonia
luteo-alba is a synonym of the valid, internationally accepted
name, Ditiola pezizaeformis, as reported by Reid (A monograph of
the British Dacrymycetales. Transactions of the British Mycological
Society 62 (1974): 433-494) and in accordance with current entries
in Mycobank and other taxonomic databases and monographs of
Basidiomycetes. The strain ATCC13299 also showed similar growth and
morphological characteristics to strain FU50088, and its conidia
were elongate-ellipsoid to subglobose, 5-6.5.times.1.5-2 .mu.m. The
LSU nucrDNA sequence of Ditiola pezizaeformis strain ATCC13299 is
included here as sequence <SEQ ID NO: 5>.
[0076] Dacryopinax spathularia strain FU50088 has been deposited
under the Budapest Treaty at BCCM/MUCL, Mycotheque de l'Universite
catholique de Louvain, Place Croix du Sud 3, B-1348
Louvain-la-Neuve, Belgium, under the designation number MUCL 53181
on Oct. 11, 2010.
[0077] Other strains which produce the compounds of the invention
have also been deposited under the Budapest Treaty at BCCM/MUCL:
Dacryopinax spathularia, CBS 197.63 under the designation number
MUCL 53182 on Oct. 11, 2010, Ditiola radicata, CBS 126.84 under the
designation number MUCL 53180 on Oct. 11, 2010, Ditiola nuda, CBS
173.60 under the designation number MUCL 53179 on Oct. 11, 2010,
and Femsjonia luteo-alba Fr.1849, ATCC13299 under the designation
number MUCL 53500 at May 19, 2011.
[0078] Surprisingly the compounds of the invention e.g. compounds
of the formula I exhibit a strong, long term inhibitory activity
against organisms involved in spoilage of pharmaceutical
nutraceutical, nutritional, cosmeceutical, and/or cosmetic
preparations or compositions. Said compounds are especially useful
against acidophilic spoilage yeasts, which are involved in spoiling
or deterioration of beverages. Even more surprisingly these
compounds are able to inhibit the growth of thermophilic molds,
which are difficult to control with standard sterilizing and/or
pasteurizing processes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] FIG. 1: Atom numbering of the compounds of the invention for
signal assignments of analytical processes.
[0080] FIG. 2: Typical HPLC-MS of an extract of FU50088 (MUCL
53181) produced following the example 1B Fermentation c) 200 l
fermentation; C Preparation of extracts c) Preparation of a
sedimentation product. Adjustment of signals according the "Adapted
method" of Example 3. The numbers in brackets "[.]" represent the
corresponding compound in Table 1.
[0081] FIG. 3: Typical HPLC-ELSD chromatogram of an extract of
Ditiola pezizaeformis strain ATCC13299 (MUCL 53500), peak
annotation according to Table 16 below.
[0082] FIG. 4: Typical HPLC-MS of an extract [X8] obtained from
Dacryopinax spathularia strain FU50088 (MUCL 53181) following
example 8D), annotation of signals according the "Improved method"
of Example 8C), for details see Table 24A below.
APPLICATIONS & DEFINITIONS
[0083] The general expressions, within the present disclosure,
preferably have the following meaning, where in each embodiment
one, more than one or all more general expressions may,
independently of each other, be replaced with the more specific
definitions, thus forming special, e.g. preferred, embodiments of
the invention, respectively:
[0084] Preferably, the compounds of the formula I are natural
compounds, that is, compounds that are present in and can be
isolated or extracted from natural sources (especially those
mentioned in detail above and below) without chemical synthesis
steps (though they may also be prepared or modified by chemical
synthesis, e.g. acylated or the like), or be modified by certain
downstream processing procedures (e.g. permethylated under
influence of acidic methanol) and are thus present as extracts or
purified components of extracts, and not derivatives only
obtainable by chemical synthesis.
[0085] They can also be present and used as part of an extract
which is obtainable by extracting a fungus or a part from an
appropriate fungus of the genus Dacryopinax.
[0086] "Substantially" means preferably that the corresponding
impurities are present only in trace amounts, e.g. in less than 5%
by weight, less than 4% by weight, less than 3% by weight, less
than 2% by weight, less than 1% by weight, less than 0.5% by weight
or less than 0.2% by weight, in relation to the complete weight of
the corresponding dry extract or compound of the formula I or
mixture of compounds of the formula I.
[0087] In the context of the present invention, the terms
"essentially consists of" or "essentially consisting of" mean that
the total weight share is 90 wt. % or more, preferably 95 wt. % or
more, more preferably 98 wt. % or more, most preferably 99 wt. % or
more, in each case based on the total amount used. For example, a
"mixture essentially consisting of" means that the total amount of
the constituents as defined in the respective case is 90 wt. % or
more, preferably 95 wt. % or more, more preferably 98 wt. % or
more, most preferably 99 wt. % or more, in each case based on the
total weight of the mixture
[0088] The term "Glycolipid" can be replaced with "glycosylated
fatty acid" as well, where it is used with regard to the compounds
of the formula I.
[0089] "A compound of the formula I" or "compound(s) of the formula
I" can refer to one or more compounds of the formula I, that is one
compound or a mixture of compounds of the formula I, or to the USE
of a compound of the formula I, where reference to compound(s) of
the formula I always includes the compound(s) as such or in the
form of a salt (especially a physiologically, that is, e.g.,
pharmaceutically, nutraceutically or cosmetically) acceptable salt,
a solvate and/or a tautomer thereof, or in the lactone form. In all
cases this means that either only one compound (in substantially
pure form or as a direct extract or a further enriched extract) or
a mixture of two or more compounds of the formula I (which mixture
is preferred) can be present, e.g. in an extract or pharmaceutical,
nutraceutical or cosmetical formulation according to the invention,
or that it or they can be of use according to the invention.
[0090] The compounds of the formula I may also be esterified at
their free carboxyl group shown in formula I on the right hand side
with alcohols, e.g. alcohols with 1 to 10 carbon atoms, such as
alkanols, e.g. C.sub.1-C.sub.7alkanols, such as methanol or
ethanol, phenyl-C.sub.1-C.sub.4alkanols, such as benzyl alcohol, or
the like. Preferred are the compounds not esterified at the
carboxyl group in formula I on the right hand side.
[0091] Preferably, the total weight share of the compound or all
compounds of the formula I in an extract or mixture of compounds of
the formula I or a purified compound of the formula I that is of
use according to the invention in the final extract, mixture or
compound (direct or further enriched) is in the range from 0.01% to
100% by weight, more preferably from 1% to 100% or to 99% by
weight, in another embodiment from 5% to 100% or to 99% by weight,
or from 20% to 100% or to 95% by weight, or e.g. from 50% to 100%
or to 90% by weight.
[0092] Wherever used in the invention "%" e.g. percent is defined
by the weight portion of the part of interest of the total weight,
e.g. % is meant as % by weight; except where otherwise explicitly
defined.
[0093] For the purpose of the invention the term "carbohydrate" is
used in conformity with the IUPAC recommendations (Pure and Applied
Chemistry, 1995, 67, 1307). The term "carbohydrate having 3 to 30
(preferably 6 to 18) carbon atoms bound via one of its oxygen
atoms" especially refers to mono, oligo- or polysaccharidyl
moieties bound via one of their oxygen atoms. The carbohydrates
forming the basis for such moieties include, but are not limited
to, monosaccharides, disaccharides, further oligosaccharides, or
polysaccharides.
[0094] Monosaccharide for example includes, but is not limited to,
aldotrioses such as glyceraldehyde, ketotrioses such as
dihydroxyacetone, aldotetroses such as erythrose and threose,
ketotetroses such as erythrulose, aldopentoses such as arabinose,
lyxose, ribose and xylose, and desoxypentoses such as deoxyribose;
ketopentoses such as ribulose and xylulose; hexoses, especially
aldohexoses such as allose, altrose, galactose, glucose, gulose,
idose, mannose and talose, or ketohexoses such as fructose,
psicose, sorbose and tagatose, or desoxyhexoses such as rhamnose,
cymarose, fucose, 2-desoxyglucose or 2-deoxygalactose; heptoses
such as mannoheptulose, sedoheptulose; octoses such as octolose,
2-keto-3-deoxy-manno-octonate; nonoses such as sialoseallose.
[0095] Disaccharides for example include, but are not limited to,
trehalose, sucrose, kojibiose, sophorose, nigerose, laminaribiose,
maltose, cellobiose, isomaltose, gentiobiose, lactose, melibiose,
neohsperidose, rutinose, primeverose, sambubiose, xylobiose,
lathyrose and mannobiose.
[0096] Oligosaccharides for example include, but are not limited
to, raffinose, nystose, panose, cellotriose, maltotriose,
maltotetraose, xylobiose, galactotetraose, isopanose, cyclodextrin
(alpha-CD) or cyclomaltohexaose, beta-cyclodextrin (beta-CD) or
cyclomaltoheptaose and gamma-cyclodextrin (gamma-CD) or
cyclomaltooctaose. Polysaccharides for example include, but are not
limited to, xylan, mannan, galactan, glucan, arabinan, pustulan,
gellan, guaran, xanthan, and hyaluronan. Some examples include, but
not limited to, starch, glycogen, cellulose, inulin, chitin,
amylose and amylopectin.
[0097] In the case of di-, tri- and oligo-saccharides the bonds
between the carbohydrate subunits may include various possible
types, e.g. preferably in the form of glycosidic connections of the
1.fwdarw.2, 1.fwdarw.3, 1.fwdarw.4 and 1.fwdarw.6 types, in
particular the glycosidic connections are of the 1.fwdarw.2
type.
[0098] Especially preferred are trisaccharide carbohydrate
moieties, especially of the formula
##STR00006##
wherein the rings A, B and C are monosaccharide moieties each
independently from the others with 5 or 6 ring members, wherein one
or more of the hydroxyl groups may be acylated or etherified.
[0099] Preferably the carbohydrate moieties without substitutents
resulting from acylation or etherification have 15 to 18 carbon
atoms and they are especially selected from the
hexapyranosyl-pentapyranosyl-pentapyranosid type such as
beta-D-glucopyranosyl-(1.fwdarw.2)-beta-D-xylopyranosyl-(1.fwdarw.2)-beta-
-D-xylopyranosid or the hexapyranosyl-pentapyranosyl-hexapyranosid
type such as
beta-D-glucopyranosyl-(1.fwdarw.2)-beta-D-xylopyranosyl-(1.fwdarw-
.2)-beta-D-glucopyranosid.
[0100] The carbohydrates may carry one, more or all hydroxyl groups
in modified form, e.g. as etherified hydroxyl or especially
esterified hydroxyl as defined below, respectively, for example in
a form acylated by a C.sub.2-C.sub.10-alkanoic acid, e.g.
acetylated, e.g. mono- or di or tri- or tetra-acetylated, form. A
particularly preferred modified form is represented by those
compounds of the formula I which have one or more hydroxyl groups
in the carbohydrate moiety that is or are acylated by an isovaleryl
(3-methyl-butanoyl) moiety--these compounds are novel and thus also
as such form an invention embodiment.
[0101] Individual compounds of formula I with an acyl substituent
with more than 2 carbon atoms, such as (and preferably) an
isovaleryl substituent (and preferably one single isovaleryl
substituent), in the carbohydrate moiety R typically exhibit a
stronger antimicrobial activity, particularly against yeasts and
molds, especially against yeasts and molds of relevance regarding
food, beverage and/or cosmetic spoilage, and/or a broader activity
spectrum than the corresponding compounds with an acetyl
substituent in the carbohydrate moiety R.
[0102] Further esters may be acetates; propionates; butyrates;
isobyturates; valerates such as n-pentanoate) or 2-methyl butyrate,
or the unsaturated derivatives such as but not limited to
2-methyl-2-butenoate (e.g. angeloate or tiglate),
3-methyl-2-butenoate or 3-methyl-3-butenoate (senecioate), or
hydroxylated derivatives such as 2-methyl-3-hydroxy butyrate or
2-hydroxymethyl butyrate; or hexenoates such as n-hexanoate
(caproate), isohexanoates such as but not limited to
2-methylvalerate, 3-methylvalerate, 4-methylvalerate, 2,3-dimethyl
butyrate, or the unsaturated derivatives e.g. 2-ethyl-2-butyrate,
2-methyl-2-pentenoate, 4-methyl-2-pentenoate; or aminoacyl, e.g.
alanyl, cysteinyl, aspartyl, glutamyl, phenylalanyl, glycyl,
histidyl, isoleucyl, lysyl, leucyl, methionyl, asparaginyl,
pyrrolysinyl, prolyl, glutaminyl, arginyl, seryl, threonyl,
selenocysteyl, valyl, tryptophanyl or tyrosinyl. The acylated forms
may preferably be natural products, but they can also be products
of chemical or enzymatic acylation, e.g. using active forms of the
acids and, where required to avoid reaction of other functional
groups, introduction and, especially to obtain the final product,
removal of protecting groups ("Pg").
[0103] The protection of such functional groups by such protecting
groups ("Pg"), the protecting groups themselves, and their removal
reactions are described for example in standard reference works,
such as J. F. W. McOmie, "Protective Groups in Organic Chemistry",
Plenum Press, London and New York 1973, in T. W. Greene and P. G.
M. Wuts, "Protective Groups in Organic Synthesis", Third edition,
Wiley, New York 1999, in "The Peptides"; Volume 3 (editors: E.
Gross and J. Meienhofer), Academic Press, London and New York 1981,
in "Methoden der organischen Chemie" (Methods of organic
chemistry), Houben Weyl, 4th edition, Volume 15/I, Georg Thieme
Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit,
"Aminosauren, Peptide, Proteine" (Amino acids, peptides, proteins),
Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in
Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and
Derivate" (Chemistry of carbohydrates: monosaccharides and
derivatives), Georg Thieme Verlag, Stuttgart 1974. Especially
preferred protecting groups are hydroxyl protecting groups, such as
tert-butyldimethylsilyl, methyl, methoxymethyl, or trityl.
[0104] The chemical acylation can take place with the corresponding
acid as such or preferably in the form of a reactive derivative.
Reactive (or active) derivatives used as such include the
halogenides, e.g. chlorides, or nitrophenyl esters, e.g. the
2,4-dinitrophenyl esters, or acid anhydrides (symmetric or e.g.
with acetic acid) of the carboxy groups of the acids to be
reacted.
[0105] For in situ formation, customary coupling agents may be
applied. Such reagents are known to the person skilled in the art
and can be deduced conveniently from many sources, e.g. Aldrich
ChemFiles--Peptide Synthesis (Aldrich Chemical Co., Inc.,
Sigma-Aldrich Corporation, Milwaukee, Wis., USA) Vol. 7 No. 2, 2007
(see
http://www.sigmaaldrich.com/etc/medialib/docs/Aldrich/Brochure/al_chemfil-
e_v7_n2.Par.0001.File.tmp/al_chemfile_v7_n2.pdf). Among the
possible coupling agents for amide and ester bond synthesis the
following may be mentioned:
[0106] Triazoles, uronium or hexafluorophosphonium derivatives,
e.g. 1-hydroxy-benzotriazole (HOBt), Carbodiimides, e.g.
dicyclohexylcarbodiimide, active ester forming agents, e.g.
2-mercaptobenzothiazole (2-MBT), azide forming agents, e.g.
diphenyl phosphoryl azide, acid anhydrides, such as propane
phosphonic acid anhydride, acid halogenation agents, e.g.
1-chloro-N,N,2-trimethyl-1-propenylamine, or the like, or mixtures
of two or more such agents.
[0107] While the compounds of the formula I are preferably obtained
by extraction in the form of extracts from natural sources or in
further enriched or purified from such extracts (see below), they
can also be obtained by chemical synthesis methods.
[0108] For example, the compounds may be synthesized chemically
e.g. by a convergent strategies. The glycoside part and the
unbranched long-chain .alpha.-hydroxy carboxylic acid part of the
molecules are build up separately, with the hydroxyl groups and the
carboxylic acid moieties being protected by suitable protecting
groups. Afterwards, both building blocks are connected via building
a glycosidic linkage using methods described in the scientific
literature. Finally, removal of the protecting groups will lead to
the desired compounds.
[0109] The following Reaction schemes provides an Example of a
possible synthesis pathway:
##STR00007## ##STR00008## ##STR00009## ##STR00010##
[0110] Synthetic methods for glycosylation reactions--including
protection, activation and deprotection strategies--are described,
e.g., in the following literature: [0111] J. McMurry; Organic
Chemistry, 5th ed.; Brooks/Cole; 2000, 1031; D E. Levy, P. Fugedi;
The organic chemistry of sugars; Taylor & Francis, 2006,
181-197; S. Bufali, P. Seeberger, Org. React. 2006, 68, 303; G.-J.
Boons, K. J. Hale, Organic synthesis with carbohydrates. Blackwell
Publishing, 2000; R. R. Schmidt, J. Michel, Angew. Chem. Int. Ed.
Engl. 1980, 19, 731-732; X. M. Zhu, R. R. Schmidt, Angew. Chem.
Int. Ed. 2009, 48, 1900-1934; R. R. Kale et al, Angew. Chem. Int.
Ed. 2008, 47, 1265-1268; N. Miguel, S. Vignando, G. Russo, L. Lay,
Synlett 2004, 2, 341-343; W. Koenigs, E. Knorr, Chem. Ber. 1901,
34, 957-981; Fraser-Reid, B.; Tatsuta, K.; Thiem, J., Hrsg.,
Glycoscience--Chemistry and Chemical Biology, Springer: Berlin,
(2001); Lindhorst, T. K., Essentials of Carbohydrate Chemistry and
Biochemistry, Wiley-VCH: Weinheim, (2000); Bochov, A. F.; Zaikov,
G. E., Chemistry of the O-Glycosidic Bond: Formation and Cleavage,
Pergamon Press: Oxford, (1979); Fraser-Reid, B.; Wu, Z.; Udodong,
U. E.; Ottosson, H., J. Org. Chem., (1990) 55, 6068-6070; Lemieux,
R. U.; Morgan, A. R., Can. J. Chem., (1965) 43, 2190-2198; Sinay,
P., Pure Appl. Chem., (1991) 63, 519-528 Toshima, K.; Tatsuta, K.,
Chem. Rev., (1993) 93, 1503-1531; Evans, W. L.; Reynolds, D. D.;
Talley, E. A., Adv. Carbohydr. Chem., (1951) 6, 27-81
[0112] Synthetic methods for dihydroxylation reactions are
described, e.g., in the following literature: [0113] R. Bruckner:
Reaktionsmechanismen, 2. Aufl, Spektrum Verlag, Heidelberg/Berlin
2003, 750-758; M. H. Junttila, O. E. O. Hormi, J. Org. Chem., 2004,
69, 4816-4820; M. H. Junttila, O. O. E. Hormi, J. Org. Chem., 2009,
74, 3038-3047; B. M. Choudary, N. S. Chodari, K. Jyothi, M. L.
Kantam, J. Am. Chem. Soc., 2002, 124, 5341-5349; G. M. Mehltretter,
S. Bhor, M. Klawonn, C. Dobler, U. Sundermeier, M. Eckert, H.-C.
Militzer, M. Beller, Synthesis, 2003, 295-301; L. C. Branco, C. A.
M. Afonso, J. Org. Chem., 2004, 69, 4381-4389; Krauch, H.; Kunz,
H., Reaktionen der Organischen Chemie, 6. Aufl.; Huthig: eidelberg,
(1997); S. 434-436; Hudlicky, T.; Fan, R.; Luna, H.; Olivo, H.;
Price, J., Pure Appl. Chem., (1992) 64, 1109-1113; Jacobsen, E. N.,
Acc. Chem. Res., (2000) 33, 421-431; Kolb, H. C.; VanNieuwenhze, M.
S.; Sharpless, K. B., Chem. Rev., (1994) 94, 2483-2547.
[0114] Synthetic methods for reactions connecting two building
blocks via double bond generation are described, e.g., in the
following literature: [0115] Ivin, K. J.; Mol, J. C., Olefin
Metathesis and Metathesis Polymerization, Academic Press: New York,
(1997); Grubbs, R. H., Handbook of Metathesis, Wiley-VCH: Weinheim,
(2003); Bd. 1-3; Furstner, A.; Langemann, K., Synthesis, (1997),
792-803; Furstner, A., Angew. Chem., (2000) 112, 3140-3172;
Blakemore, P. R., J. Chem. Soc., Perkin Trans. 1, (2002),
2563-2585; Staden, L. F., van; Gravestock, D.; Ager, D. J., Chem.
Soc. Rev., (2002) 31, 195-200; Wittig, G., Angew. Chem., (1980) 92,
671-675; Schlosser, M.; Christmann, K., Synthesis, (1969), 38-39;
Maryanoff, B. E.; Reitz, A. B., Chem. Rev., (1989) 89, 863-927;
Murphy, P. J.; Brennan, J., Chem. Soc. Rev., (1988) 17, 1-30;
Boutagy, J.; Thomas, R., Chem. Rev., (1974) 74, 87-99; Clayden, J.;
Warren, S., Angew. Chem., (1996) 108, 261-291; Bruckner, R.,
Reaktionsmechanismen, 2. Aufl.; Spektrum: Heidelberg, (2003); Ager,
D. J., Synthesis, (1984), 384-398; Mukaiyama, T.; Asami, M., Top.
Curr. Chem., (1985) 127, 133-167.
[0116] Further, the present glycolipid compounds of the formula I
comprise all stereoisomers, such as those which may exist due to
asymmetric carbons on the various substituents, including
enantiomeric forms and diastereomeric forms. Individual
stereoisomers of the glycolipid derivatives of the present
invention may, for example, be substantially free of other isomers,
or may be admixed, for example, as racemates or with all other,
more than one other, or two to less than all other selected
stereoisomers, e.g. diastereomers.
[0117] Especially the vicinal dihydroxy group in the fatty acid
part is in one embodiment of the invention to be understood as syn
and/or anti configurated.
[0118] To the extent that compounds the formula I and salts thereof
may exist in their tautomeric form, all such tautomeric forms are
contemplated herein as part of the present invention
embodiments.
[0119] As the final carboxyl group of the carboxylic acid chain may
also form a lactone with one of the hydroxyl groups present on the
rest of a molecule of the formula I, compounds of the formula I may
also be present in the lactone form, either purely or in admixture
with the open chain form.
[0120] The salts of compound(s) of the formula I are especially
physiologically acceptable salts, that is, salts that have no
disturbing toxical, allergenic and/or mutagenic properties on human
or animal cells. Such salts can be selected from those known in the
art, e.g. using calcium, sodium, magnesium, or ammonium as
counterions of the carboxylic group or the salts mentioned
below.
[0121] Where salt-forming groups (e.g. acidic groups, such as
carboxylic acid groups, or basic groups, such as amino or imino
groups) are present within them, the glycolipid compounds of the
formula I may be in the free form or in the form of salts. The term
"salt(s)", as employed herein, denotes acidic and/or basic salts
formed with inorganic and/or organic acids and bases. In addition,
when a compound of the formula I contains both a basic moiety and
an acidic moiety, "inner salts" may be formed and are included
within the term "salt(s)" as used herein. Pharmaceutically or
nutraceutically or cosmetically acceptable (i.e., non-toxic,
physiologically acceptable) salts are preferred, although other
salts are also useful, e.g., in isolation or purification steps
which may be employed during preparation. Salts of compounds of the
formula I may be formed, for example, by reacting a compound of the
formula I with an amount of acid or base, such as an equivalent
amount, in a medium such as one in which the salt precipitates or
in an aqueous medium followed by lyophilisation or followed by the
addition of a water miscible organic solvent. Also ion exchangers
can be used to form salts from free forms or free forms from salts
of a compound of the formula I. "Free form" refers to "form without
salt-forming counterions", e.g. in non-salt form.
[0122] Where two active groups with different charge are present,
also internal or zwitterionic salts can be formed.
[0123] Where the compounds of the formula I (or glycolipids of the
formula I or the like) are mentioned in the present disclosure,
this also comprises the corresponding (especially physiologically
acceptable) salts thereof, also where not explicitly stated, as
well as the esters, as well as the lactones, or mixtures of two or
more of these forms.
[0124] The compounds of the formula I which contain an acidic
moiety (e.g. carboxyl (--COON) groups) may form salts with a
variety of organic and inorganic bases. Exemplary basic salts
include ammonium salts, non-toxic metal salts derived from metals
of groups Ia, Ib, IIa and IIb of the Periodic Table of Elements,
e.g. alkali metal salts such as sodium, lithium, or potassium
salts, alkaline earth metal salts such as calcium or magnesium
salts, or salts with other metals, such as zinc, salts with organic
bases (for example, organic amines) such as unsubstituted or
hydroxy-substituted mono-, di- or tri-alkylamines, especially
mono-, di- or tri-lower alkylamines, or quaternary ammonium
compounds, for example with alkyl amines, e.g. t-butyl amine,
N-methyl-N-ethylamine, diethylamine, triethylamine, mono-, bis- or
tris-(2-hydroxy-lower alkyl)amines, such as mono-, bis- or
tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine or
tris(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-lower
alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)-amine or
tri-(2-hydroxyethyl)-amine, or N-methyl-D-glucamine; or cyclic
amines such as piperidine, N-lower alkyl piperidine e.g.
N-methyl-piperidine, piperazine, or quaternary ammonium salts
formed via common processes out of the above define amines, such as
tetrabutylammonium salts, or with benzathines, dicyclohexylamines,
N-methyl-D-glucamines, N-methyl-D-glucamides, purines, caffeine,
theobromine, hydrabamine, choline, betaine, or salts with amino
acids such as arginine, lysine, histidine and the like. Also salts
with salt-forming pharmaceutical and/or nutraceutical carrier
materials are possible and encompassed by the invention.
Isolation
[0125] In order to obtain the compounds of the formula I, as such
or comprised in an extract, preferably the desired substances of
the formula I are isolated from natural sources, either with
subsequent chemical modification (e.g. acylation) or preferably
without such chemical modification.
[0126] The purpose of the extraction and especially isolation step
is to retain the desired substances. Desired substances in the
present context are any substances that directly or indirectly
contribute to the preservative properties of the composition, with
the proviso that one or more compounds of the formula I are also
included. The isolation can be performed by isolating or separating
the one or more compounds of the formula I according to chemical
and/or physical properties. Examples of chemical properties include
affinity for one or more compounds and chemical stability. Examples
of physical properties include mass or size, charge, solubility,
polarity, distribution, absorption to surfaces, melting point, and
the like.
[0127] Natural compounds of the formula I, or extracts comprising
one or more thereof, for USE in or according to the present
invention are isolated from one or more cultures, especially liquid
cultures, of mushrooms of the genera listed above or below, e.g.
with the genetic characteristics provided in detail below.
[0128] By the term "extract", either a direct extract (in liquid or
preferably dried form), e.g. obtained as described below, or
preferably a further enriched extract (obtainable e.g. by one or
more further purification steps after extraction, e.g.
chromatography, for example as described below) containing one or
more, preferably two or more compounds of the formula I is
meant.
[0129] The compound(s) of the formula I in the form of an extract
and extracts according to the invention can be obtained especially
preferably by extraction of liquid cultures, especially liquid or
solid mycelial cultures, of mushrooms of the genus Dacryopinax,
e.g. mushrooms or parts thereof of the species Dacryopinax,
mushrooms of the genus Ditiola, e.g. mushrooms or parts thereof,
and/or mushrooms of the genus Femsjonia luteo-alba, e.g. mushrooms
or parts thereof, especially the species and more especially the
deposited strains as defined above.
[0130] Extracts according to the invention or useful according to
the invention may be manufactured according to any suitable
process, preferably comprising extraction of one or more compounds
of the formula I. The term "extract" wherever used also includes
precipitates, e.g. manufactured as described below.
[0131] For example, the extraction of one or more compounds and/or
mixture of compounds of the formula I from a cultured mushroom
(especially from submerged mycelial culture) or mushroom part of
the genera mentioned above by means of a lipophilic (preferably
non-aqueous) solvent.
[0132] Extraction thus may take place with a non polar or weakly
polar (meaning less polar than water) solvent or solvent mixture,
meaning that the preferred obtainable or obtained extracts
according to the invention are lipophilic extracts.
[0133] Preferably, the polarity is defined by an E.sub.T(30) value
of 56 kcal/mol or lower (at 25.degree. C. and 1 bar), e.g. of 52
kcal/mol or lower (water has an E.sub.T(30) of 63.1). The
E.sub.T(30) method is based on a method published by Reichart et
al. and makes use of the stabilisation of the ground state of the
betaine dye 2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)phenolate,
CAS No 10081-39-7, in apolar solvents leading to a higher energy
for the transition from the ground state (HOMO) to the first
excited state (LUMO) of the molecule (see K. Dimroth, J Lieb Ann d
Chemie (1963) 661(1): 1-37, DOI 10.1002/jlac.19636610102).
[0134] Examples of appropriate solvents are organic solvents (two
or more of which can also be mixed), e.g. a ketone or an ester,
such as acetone and/or ethyl acetate, an ether, e.g. a cyclic ether
such as dioxane, and/or (also in a specific embodiment) an alcohol
e.g. ethanol, and/or a liquid or superfluid gas, especially
superfluid CO.sub.2.
[0135] Alternatively, the extract may be obtained by bringing a
culture supernatant to a slightly alkaline pH, e.g. by adding an
alkalimetal hydroxide, such as sodium or potassium hydroxide,
separating of any solid material, e.g. the mycelia or other solid
components, e.g. by microfiltration, acidifying the filtrate by
addition of an acid, e.g. an organic or inorganic acid, such as a
hydrohalogenide, such as hydrochloride, to an appropriate pH, e.g.
lower than the pKa (which may, for compounds of the formula I, be
assumed to lie in the range of about 4.0 to 5.0, e.g. 4.2 to 4.5),
removing the supernatant from an obtained precipitate (which
comprises the compounds of the formula I) and optionally washing
the precipitate and/or extracting the compounds of the formula I at
an appropriate pH into a less polar solvent, e.g. one as mentioned
above. This process (also leading to what is called an "extract" in
the present disclosure) is especially preferred as it leads to high
yield and helps to avoid the use of solvents, thus being both
economically and ecologically advantageous.
[0136] The addition according to the use or method of the invention
preferably takes place by mixing the resulting extract or isolated
compound(s) of the formula I into such a material or by
impregnating or coating it with the compound(s) of the formula as
such or in an appropriate (e.g. liquid) composition.
[0137] For preservative or antimicrobial compositions, further
processing steps may precede and/or follow, such as drying (e.g.
freeze-drying, spray-drying, fluid bed or spouted bed or
evaporation), granulation, agglomeration, concentrating (e.g. to
syrups, formed via concentration and/or with the aid of
thickeners), pasteurizing, sterilizing, freezing, dissolving,
dispersing, filtering, centrifuging, confectioning, and the
like.
[0138] The compounds of the formula I have surprisingly been found
to show especially preservative or antimicrobial purposes (the term
"antimicrobial" especially referring to treated materials which are
not treated to avoid perishing of themselves but are to be used in
a form not contaminated by microbes, e.g. implants or the like,
while the term "preservative activity" also includes antimicrobial
activity, but also other stabilizing activity, e.g. by
emulsification or acidification due to addition of the compound(s)
of the formula Ito perishable goods).
[0139] The preservative and antimicrobial properties can
conveniently be shown by methods known in the art, e.g. as
described below and in the Examples.
[0140] In such tests, antimicrobial and preservative activity can
be shown.
[0141] The term "enhance the stability against microorganisms"
refers to inhibiting the growth or killing microorganisms, thus
providing a material equipped according to the invention with one
or more compounds of the formula I with protection against
microbial damage, films or degradation.
[0142] Among the materials to which one or more compounds of the
formula I can be added, the following may be mentioned: A material
selected from the group consisting of a cosmetic, a home care
product, a food, a beverage, a pharmaceutical, a medical device,
and an active packaging material. Also semi-finished products or
precursors are included, e.g. especially in the case of beverages
or foods, ready-to-use powders or concentrates.
[0143] Food & Beverages
[0144] The term "food" sometimes also named "foodstuff" means
articles used for food or (drink) for man or other animals, chewing
gum, and articles used for components of any such article. The term
"food" especially refers to materials, usually of plant, animal or
other organism origin, that comprise body nutrients, such as
carbohydrates, fats, proteins, vitamins and/or minerals, and is
ingested and assimilated by the human or animal organism to produce
energy, stimulate growth and maintain life. Usually, food has a
rather solid form, but may also be near liquid, e.g. in the case of
yoghurt or the like. "Food" includes a raw, cooked, or processed
edible substance, ice, or ingredient used or intended for use or
for sale in whole or in part for human or animal consumption, or
chewing gum.
[0145] "Beverage" means a liquid product for drinking, usually
including water, which may be consumed to quench thirst, to provide
nutrition, for pleasure or relish purposes and/or for other
functional purposes (e.g. to administer medicines or other
functional materials).
[0146] Among the liquids for human or animal consumption those can
be mentioned which are labelled as juice, drink (including soft
drink, such as lemonade), non-alcoholic or alcoholic beverage,
and/or cocktail.
[0147] "Juice" means the aqueous liquid expressed or extracted from
one or more fruits or vegetables, purees of the edible portions of
one or more fruits or vegetables, or any concentrates of such
liquid or puree.
[0148] The use as an agent with preservative properties of a
compound or compounds of the formula I includes also the use in
precursor products of beverages, e.g. concentrates, syrups and/or
powders will reconstitute to a beverage in the sense of the
invention by the addition of water.
[0149] The term "infant formula" means a food which purports to be
or is represented for special dietary use solely as a food for
infants by reason of its simulation of human milk or its
suitability as a complete or partial substitute for human milk.
[0150] Beverages can be alcoholic and/or non alcoholic, carbonated
and/or non carbonated
[0151] Beverages include non-dairy milks, and the like.
[0152] Beverages may include water, flavoured water, fortified
waters, flavoured beverages, carbonated water, e.g. flavoured
seltzer or soda waters, juices, cola, lemon-lime, ginger ale, and
root beer beverages which are carbonated in the manner of soft
drinks, as well as beverages that provide health or wellness
benefits from the presence of metabolically active substances, such
as vitamins, amino acids, proteins, carbohydrates, lipids, or
polymers thereof, where such products may also be formulated to
contain milk, coffee, or tea (e.g. green tea) or other botanical
solids, syrup, diet beverages, carbonated soft drinks, fruit
juices, e.g. orange juice, grapefruit juice, apple juice, red grape
juice, white grape juice, pear juice, concord grape juice,
pineapple juice, pomegranate juice, cranberry juice, passion fruit
juice, lime juice, lemon juice, mango juice, guava juice, banana
juice, red and black currant juice, cashew apple juice, cantaloupe
melon juice, apricot juice, blackberry juice, lingonberry juice,
dewberry juice, gooseberry juice, crabapple juice, prune juice,
plum juice, kiwi juice, strawberry juice, blueberry juice, red
raspberry juice, black raspberry juice, cherry juice, watermelon
juice, peach juice, nectarine juice, loganberry juice, honeydew
melon juice, papaya juice, boysenberry juice, youngberry juice,
rhubarb juice, guanabana juice, acai juice, goji juice, fig juice,
elderberry juice, date juice, carambola juice, acerola juice,
quince juice, bilberry juice, tangerine juice, fruit containing
beverages, e.g. fruit drinks which provide the flavor of any of the
e.g. aforementioned fruit juices and contain greater than 0% fruit
juice but less than 100% fruit juice, fruit flavored beverages,
vegetable juices, e.g. tomato juice, beet juice, carrot juice,
celery juice, vegetable containing beverages, which provide the
flavor of any of the aforementioned vegetable juices and contain
greater than 0% vegetable juice but less than 100% vegetable juice,
isotonic beverages, non-isotonic beverages, soft drinks containing
a fruit juice, coffee, tea, tea beverages prepared from tea
concentrate. extracts, or powders, drinkable dairy products, e.g.
drinkable yogurts (drink yoghurt), kefir or buttermilk, hot
chocolate, chocolate powders/mixes, drinkable soy products,
non-diary milks, e.g. coconut milk, alcoholic beverages, e.g. malt
beverages, wine, beer, distilled liquors, spirits, sparkling wine,
champagne or liqueurs, fruit smoothies, horchata (vegetable and/or
rice components made into a beverage), sport drinks, energy drinks,
health drinks, shakes, protein drinks (e.g. dairy, soy, rice or
other), drinkable soy yogurts, low acid beverages as defined in US
21 C.F.R. Part 113. Acidified beverages as defined in US 21 C.F.R.
Part 114, nectars, tonics, frozen carbonated beverages, frozen
uncarbonated beverages, liquid meal replacements, infant
formulations, and combinations or mixtures thereof.
[0153] It is also possible to formulate such beverages to contain
one or more nutraceuticals. Herein, a nutraceutical is a substance
that has been shown to possess, minimally, either a general or
specific health benefit or sense of wellness as documented in
professional journals or texts. Nutraceuticals, however, do not
necessarily act to either cure or prevent specific types of medical
conditions.
[0154] Apart from one or more compounds of the formula I, the foods
or beverages may comprise further customary additives for food
and/or beverages.
[0155] For the purpose of the invention "additives" in the sense of
"sweeteners" are substances used to impart a sweet taste to foods
(this term in the present paragraph also including beverages) or in
table-top sweeteners; "antioxidants" are substances that hinder the
oxidation of components, e.g. avoiding that the material becomes
rancid; "colors" are substances which add or restore color in a
food, and include natural constituents of foods and natural sources
which are normally not consumed as foods as such and not normally
used as characteristic ingredients of food. Preparations obtained
from foods and other edible natural source materials obtained by
physical and/or chemical extraction resulting in a selective
extraction of the pigments relative to the nutritive or aromatic
constituents are colors within the meaning of this Regulation;
"preservatives" are substances which prolong the shelf-life of
foods by protecting them against deterioration caused by
micro-organisms and/or which protect against growth of pathogenic
micro-organisms; "antioxidants" are substances which prolong the
shelf-life of foods by protecting them against deterioration caused
by oxidation, such as fat rancidity and color changes; "carriers"
are substances used to dissolve, dilute, disperse or otherwise
physically modify a food additive or a flavouring, food enzyme,
nutrient and/or other substance added for nutritional or
physiological purposes to a food without altering its function (and
without exerting any technological effect themselves) in order to
facilitate its handling, application or use; "acids" are substances
which increase the acidity of a foodstuff and/or impart a sour
taste to it; "acidity regulators" are substances which alter or
control the acidity or alkalinity of a foodstuff; "anti-caking
agents" are substances which reduce the tendency of individual
particles of a foodstuff to adhere to one another; "anti-foaming
agents" are substances which prevent or reduce foaming; "bulking
agents" are substances which contribute to the volume of a
foodstuff without contributing significantly to its available
energy value; "emulsifiers" are substances which make it possible
to form or maintain a homogenous mixture of two or more immiscible
phases such as oil and water in a foodstuff; "emulsifying salts"
are substances which convert proteins contained in cheese into a
dispersed form and thereby bring about homogenous distribution of
fat and other components; "firming agents" are substances which
make or keep tissues of fruit or vegetables firm or crisp, or
interact with gelling agents to produce or strengthen a gel;
"flavor enhancers" are substances which enhance the existing taste
and/or odor of a foodstuff; "foaming agents" are substances which
make it possible to form a homogenous dispersion of a gaseous phase
in a liquid or solid foodstuff; "gelling agents" are substances
which give a foodstuff texture through formation of a gel; "glazing
agents" (including lubricants) are substances which, when applied
to the external surface of a foodstuff, impart a shiny appearance
or provide a protective coating; "humectants" are substances which
prevent foods from drying out by counteracting the effect of an
atmosphere having a low degree of humidity, or promote the
dissolution of a powder in an aqueous medium; "modified starches"
are substances obtained by one or more chemical treatments of
edible starches, which may have undergone a physical or enzymatic
treatment, and may be acid or alkali thinned or bleached;
"packaging gases" are gases other than air, introduced into a
container before, during or after the placing of a foodstuff in
that container; "propellants" are gases other than air which expel
a foodstuff from a container; "raising agents" are substances or
combinations of substances which liberate gas and thereby increase
the volume of a dough or a batter; "sequestrants" are substances
which form chemical complexes with metallic ions; "stabilizers" are
substances which make it possible to maintain the physico-chemical
state of a foodstuff; stabilizers include substances which enable
the maintenance of a homogenous dispersion of two or more
immiscible substances in a foodstuff, substances which stabilize,
retain or intensify an existing color of a foodstuff and substances
which increase the binding capacity of the food, including the
formation of cross-links between proteins enabling the binding of
food pieces into re-constituted food; "thickeners" are substances
which increase the viscosity of a foodstuff; "flour treatment
agents" are substances, other than emulsifiers, which are added to
flour or dough to improve its baking quality.
[0156] Among known preservatives for food and beverages (also named
additional (chemical) preservatives herein), the following may be
mentioned, without excluding others: benzoic acid, benzoic acid
sodium salt, benzoic acid potassium salt, benzoic acid calcium
salt, propionic acid, salicylic acid, sorbic acid, sorbic acid
sodium salt, sorbic acid potassium salt, sorbic acid calcium salt,
ethyl para-hydroxybenzoate, sodium ethyl para-hydroxybenzoate,
propyl para-hydroxybenzoate, sodium propyl para-hydroxybenzoate,
methyl para-hydroxybenzoate, sodium methyl para-hydroxybenzoate,
sulphur dioxide, sodium sulphite, sodium hydrogen sulphite, sodium
metabisulphite, potassium metabisulphite, calcium sulphite, calcium
hydrogen sulphite, biphenyl or diphenyl, orthophenyl phenol, sodium
orthophenyl phenol, thiabendazole, nisin, natamycin or pimaracin,
formic acid, sodium formate, calcium formate, hexamethylene
tetramine or hexamine, formaldehyde, dimethyl dicarbonate, sodium
nitrite, potassium nitrite, sodium nitrate, potassium nitrate,
acetic acid, sodium acetates, e.g. sodium hydrogen acetate,
potassium acetate, calcium acetate, ammonium acetate, lactic acid,
propionic acid, sodium propinate, potassium propionate, calcium
propionate, oric acid, sodium tetraborate (borax), invertase,
lysozyme.
[0157] Preferably, however, no such preservatives are added in the
embodiments of the present invention.
[0158] The consumables of the present invention, e.g., beverages,
may have a pH ranging from 1.5 to 10, e.g. from about 1.5 to about
4.6 It is known in the art that the pH of a beverage may be a
factor in maintaining a shelf-stable beverage, as the growth of
some microorganisms may be hindered under acidic conditions. This,
however, is not the case for acidophilic microorganisms such as
Lactobacillus, Saccharomyces and Candida which thrive in such an
acidic environment. Utilizing the present invention allows the
composition to maintain microbial stability even in view of these
acidophilic microorganisms.
[0159] For an acidic beverage (pH<4.6), the acidity of the
beverage can be adjusted to and maintained within the recited range
by known and conventional methods in the art. For example, the pH
can be adjusted using one or more acidulant(s), also named acidity
regulator(s), e.g. as defined below.
[0160] In addition, the use of acidity regulators may assist in
microbial inhibition at the same time as maintaining the pH of the
beverage. Compositions of the present invention, however, may
inherently have a desirable pH without the use of any acidity
regulator or other components to modify the pH. Thus, the
incorporation of at least one acidity regulator is optional in
compositions of the present invention.
[0161] Moreover, the amounts of the acidity regulator(s), which may
be present in the composition according to the present disclosure,
are those conventionally used in beverage compositions. For
example, at least one acidulant may be present in an amount ranging
from about 0.01% to about 1% by weight relative to the
composition.
[0162] An aspect of the invention is directed to preserving a broad
range of beverage products that possess a pH of less than 7.5, in
particular less than about 4.6, such as 2.5 to 4.6 against spoilage
by yeast, mold and a range of acid tolerant bacteria. Preservation
of product can be accomplished merely through the addition of the
chemical agents described herein, but it is also possible to
supplement the action of the chemicals with purely physical forms
of preservation such as alteration of product temperature, various
wavelengths of irradiation, pressure or combinations thereof. In
certain exemplary embodiments, the pH of the beverage product
comprising the preservative system is e.g., about 4.6 or less,
about 2.5 to about 4.4, about 2.6 to about 4.5.
[0163] The acidity regulator(s) may be in an undissociated form or
in their respective salt form such as potassium, sodium, or
hydrochloride salts, or be a mixture, thus forming a kind of buffer
for an intended pH. Among acidity regulators (pH regulators),
organic and inorganic acids to be used in adjusting the pH of a
composition of the present invention such as a beverage may be
mentioned, e.g. acetic acid, sodium acetates, e.g. sodium hydrogen
acetate, potassium acetate, calcium acetate, ascorbic acid, sodium
ascorbate, potassium ascorbate, carbon dioxide, sodium carbonates
incl. sodium hydrogen carbonate (bicarbonate of soda) and sodium
sesquicarbonate, potassium carbonates, e.g. potassium hydrogen
carbonate, ammonium carbonates, e.g. ammonium hydrogen carbonate,
magnesium carbonates, e.g. magnesium hydroxide carbonate (syn.
magnesium hydrogen carbonate), malic acid, fumaric acid, sodium
fumarate, potassium fumarate, calcium fumarate, calcium citrates
incl. mono, di or tri calcium salts, triammonium citrate,
ammoniumferrocitrate, sodium malates, e.g. sodium hydrogen malate,
potassium malate, calcium malates, e.g. calcium hydrogen malate,
adipic acid, sodium adipate, potassium adipate, succinic acid,
1,4-heptonolactone, potassium chloride, calcium chloride, ammonium
chloride or ammonia solution, magnesium chloride, stannous
chloride, sodium sulphates, e.g. sodium hydrogen sulphate,
potassium sulphates, e.g. potassium hydrogen sulphate, calcium
sulphate, ammonium sulphate, magnesium sulphate or Epsom salts,
copper sulphate, aluminium sulphate, aluminium sodium sulphate,
aluminium potassium sulphate, aluminium ammonium sulphate, sodium
hydroxide, potassium hydroxide, calcium hydroxide, ammonium
hydroxide, magnesium hydroxide, calcium oxide, magnesium oxide,
sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide,
dicalcium diphosphate, tartaric acid, sodium tartaric acid,
potassium tartaric acid, gluconic acid, glucono-delta-lactone, or
mixtures of two or more thereof, may be mentioned. Note that the
compounds of the formula I, due to their acid/base properties, can
also be used to regulate the pH of a composition comprising
them.
[0164] Among the emulsifiers, the following may be mentioned:
lecithins; metatartaric acid, calcium tartrate; alginic acid and
the sodium, potassium, ammonium and calcium salts, propane-1,2-diol
alginate; agar; Carrageenan; processed eucheuma seaweed; locust
bean gum; carob gum; guar gum; tragacanth; acacia gum; gum arabic;
xanthan gum; Karaya gum; Tara gum; Gellan gum; glycerol; Konjac,
konjac gum, konjac glucomannane; soybean emicellulose; Cassia gum;
polyoxyethylene (8) stearate; polyoxyethylene sorbitan monolaurate,
polysorbate 20; polyoxyethylene sorbitan monooleate, polysorbate
80; polyoxyethylene sorbitan monopalmitate, polysorbate 40;
polyoxyethylene sorbitan monostearate, polysorbate 60;
polyoxyethylene sorbitan tristearate, polysorbate 65; pectins and
amidated pectin; ammonium phosphatides; sucrose acetate
isobutyrate; glycerol esters of wood rosins; diphosphates and
salts, disodium, trisodium diphosphate, tetrasodium diphosphate,
dipotassium diphosphate, tetrapotassium diphosphate, dicalcium
diphosphate, calcium dihydrogen diphosphate; triphosphates and
salts, pentasodium, pentapotassium; polyphosphates and salts,
sodium, potassium, sodium calcium, calcium, sodium aluminium,
aluminium; beta-cyclodextrine; cellulose, podered or
microcrystalline and derivatives, methyl-, ethyl-, hydroxypropyl-,
hydroxypropyl methyl-, ethyl methyl-, carboxy methyl-, crosslinked
sodium carboxy methyl-, enzymatically hydrolysed carboxy methyl-;
sodium, potassium and calcium salts of fatty acids; magnesium salts
of fatty acids; mono- and diglycerides of fatty acids; acetic acid
esters of mono- and diglycerides of fatty acids; lactic acid esters
of mono- and diglycerides of fatty acids; citric acid esters of
mono- and diglycerides of fatty acids; tartaric acid esters of
mono- and diglycerides of fatty acids; mono- and diacetyltartaric
acid esters of mono- and diglycerides of fatty acids; mixed acetic
and tartaric acid esters of mono- and diglycerides of fatty acids;
sucrose esters of fatty acids; sucroglycerides; polyglycerol esters
of fatty acids; polyglycerol polyricinoleate; propane-1,2-diol
esters of fatty acids; thermally oxidised soya bean oil interacted
with mono- and diglycerides of fatty acids; sodium
stearoyl-2-lactylate; calcium stearoyl-2-lactylate; stearyl
tartrate; sorbitan monostearate; sorbitan tristearate; sorbitan
monolaurate; sorbitan monooleate; sorbitan monopalmitate;
invertase; silicon dioxide (Silica); magnesium stearate, calcium
stearate; oxidized starch; acetylated distarch phosphate; starch
sodium octenyl succinate; acetylated oxidised starch, or mixtures
of two or more thereof.
[0165] As anti-oxidants, among others the following may be
mentioned:
ascorbic acid and salts, sodium, calcium; fatty acid esters of
ascorbic acid; tocopherols, alpha-tocopherol, gamma-tocopherol,
delta-tocopherol; propyl gallate; octyl gallate; dodecyl gallate;
erythorbic acid, sodium erythorbate; tertiary-butyl hydroquinone
(TBHQ); butylated hydroxyanisole (BHA); butylated hydroxytoluene
(BHT); extracts of rosemary; 4-hexylresorcinol, or mixtures of two
or more thereof.
[0166] Such additives may be present in relative amounts,
considering the complete composition of the food or beverage
product concerned, in amounts summing up to from 0.01% up to 90% by
weight, e.g. from 0.05% to 50% by weight, e.g. from 0.1% to 5% by
weight or from 0.2% to 20% by weight.
[0167] Cosmetics
[0168] The compounds of the formula I, in view of their
preservative properties, are also useful in supporting or providing
preservation of cosmetics.
[0169] The term "cosmetic" is here intended to mean
(1) an article, e.g. a mixture or substance or product, intended to
be placed in contact with the various external parts of the human
body or animal body (epidermis, hair system, nails, lips and
external genital organs), e.g. which can be rubbed, poured,
sprinkled, or sprayed on, or otherwise applied to the human body or
any part thereof, including the oral cavity and the teeth, skin and
hair, with a view exclusively or mainly to cleaning them, perfuming
them, changing their appearance and/or correcting body odors,
and/or protecting them or keeping them in good condition.; and (2)
articles intended for use as a component of any such articles;
explicitly including soap.
[0170] The term cosmetics as used here also includes "personal care
products" and "personal hygiene products", such as menstrual care
products, handkerchief tissues and the like.
[0171] For example, cosmetic products include but are not limited
to:
Creams, emulsions, lotions, gels and oils for the skin (hands,
face, feet, etc.); face masks (with the exception of peeling
products); tinted bases (liquids, pastes, powders); make-up
powders, after-bath powders, hygienic powders, etc.; toilet soaps,
deodorant soaps, etc.; perfumes, toilet waters and eau de Cologne;
bath and shower preparations (salts, foams, oils, gels, etc.);
depilatories; deodorants and anti-perspirants; hair care products,
(hair tints and bleaches; products for waving, straightening and
fixing); setting products; cleansing products (lotions, powders,
shampoos, hand washing products, (hand) disinfecting products);
conditioning products (lotions, creams, oils); hairdressing
products (lotions, lacquers, brilliantines); shaving products
(creams, foams, lotions, etc.); products for making up and removing
make-up from the face and the eyes; products intended for
application to the lips; products for care of the teeth and the
mouth (e.g. gargles, mouthwash or toothpastes); products for nail
care and make-up; products for external intimate hygiene;
sunbathing products; products for tanning without sun;
skin-whitening products; anti-wrinkle products, tampons, sanitary
towels, wet wipes, diapers or handkerchiefs.
[0172] Depending on the field of application, certain above
mentioned cosmetic products may also be used in the medical filed,
in particular certain washing products are suitable as disinfecting
products, such as hand disinfecting products or instrument
disinfecting products.
[0173] In all cases, the antimicrobial properties of the compounds
of the formula I may provide, as additional benefit in the sense of
a bonus effect, their antimicrobial efficiency to the cosmetic
properties of the formulations, although the purely cosmetic use of
the corresponding cosmetics is preferably predominant.
[0174] The cosmetics may comprise, in relationship to their
intended use, various active and inactive ingredients, named
"cosmetic additives" in the following.
[0175] Among the cosmetic additives, the following, without
limiting the scope of possible additives, may be mentioned:
"Abrasives" are substances which remove materials from various body
surfaces or aids mechanical tooth cleaning or improves gloss,
"absorbents" are substances which take up water- and/or oil-soluble
dissolved or finely dispersed substances, "anti-cakings" are
substances which allow free flow of solid particles and thus avoids
agglomeration of powdered cosmetics into lumps or hard masses,
"anti-corrosives" are substances which prevent corrosion of the
packaging, "anti-dandruffs" are substances which help to control
dandruff, "anti-foaming" are substances which suppress foam during
manufacturing or reduce the tendency of finished products to
generate foam, "anti-microbials" are substances which help to
control the growth of micro-organisms on the skin, "anti-oxidants"
are substances which inhibit reactions promoted by oxygen, thus
avoiding oxidation and rancidity, "anti-perspirants" are substances
which reduce perspiration, "anti-plaques" are substances which help
to protect against plaque, "anti-seborrhoeics" are substances which
help to control sebum production, "anti-statics" are substances
which reduce static electricity by neutralising electrical charge
on a surface, "astringents" are substances which contract the skin,
"bindings" are substances which provide cohesion in cosmetics,
"bleachings" are substances which lightens the shade of hair or
skin, "bufferings" are substances which stabilize the pH of
cosmetics, "bulkings" are substances which reduce bulk density of
cosmetics, "chelatings" are substances which react and form
complexes with metal ions which could affect the stability and/or
appearance of cosmetics, "cleansings" are substances which help to
keep the body surface clean, "cosmetic colorants" are substances
which color cosmetics and/or imparts color to the skin and/or its
appendages (e.g. hair or nails) (e.g. dyes or pigments, e.g.
lactoflavin, caramel capsanthin, capsorubin, beetroot red,
anthocyanins, bromothymol blue, bromocresol green, acid red,
aluminium, magnesium, calcium and zinc stearates); "denaturants"
are substances which render cosmetics unpalatable, mostly added to
cosmetics containing ethyl alcohol; "deodorants" are substances
which reduce or mask unpleasant body odors, "depilatories" are
substances which remove unwanted body hair; "detanglings" are
substances which reduce or eliminate hair intertwining due to hair
surface alteration or damage and, thus, helps combing; "emollients"
are substances which soften and smooth the skin; "emulsifiers" are
substances which promote the formation of intimate mixtures of
non-miscible liquids by altering the interfacial tension; "emulsion
stabilizers" are substances which help the process of
emulsification and improves emulsion stability and shelf-life;
"film formings" are substances which produce, upon application, a
continuous film on skin, hair or nails; "foamings" trap numerous
small bubbles of air or other gas within a small volume of liquid
by modifying the surface tension of the liquid; "foam boosters" are
substances which improve the quality of the foam produced by a
system by increasing one or more of the following properties:
volume, texture and/or stability; "gel formers" are substances
which give the consistency of a gel (a semi-solid preparation with
some elasticity) to a liquid preparation; "hair conditioners" are
substances which leave the hair easy to comb, supple, soft and
shiny and/or imparts volume, lightness, gloss, etc; "hair dyes" are
substances which color hair; "hair fixers" are substances which
permit physical control of hairstyle; "hair waving or
straighteners" are substances which modify the chemical structure
of the hair, allowing it to be set in the style required;
"humectants" are substances which hold and retain moisture;
"hydrotropers" are substances which enhance the solubility of
substance which is only slightly soluble in water; "keratolytics"
are substances which help to eliminate the dead cells of the
stratum corneum; "masking agents" are substances which reduce or
inhibit the basic odor or taste of the product; "moisturing"
compounds increase the water content of the skin and helps keep it
soft and smooth; "nail conditioners" are substances which improve
the cosmetic characteristics of the nail; "opacifiers" are
substances which reduce transparency or translucency of cosmetics;
"oral care" provides cosmetic effects to the oral cavity, e.g.
cleansing, deodorising, protecting; "oxidizers" are substances
which change the chemical nature of another substance by adding
oxygen or removing hydrogen; "pearlescents" are substances which
impart a nacreous appearance to cosmetics; "plasticizers" are
substances which soften and make supple another substance that
otherwise could not be easily deformed, spread or worked out;
"preservatives" (additional preservatives) are substances which
inhibit primarily the development of micro-organisms in cosmetics;
"propellants" are substances which generate pressure in an aerosol
pack, expelling contents when the valve is opened, some liquefied
propellants can act as solvents; "reducers" are substances which
change the chemical nature of another substance by adding hydrogen
or removing oxygen; "refatters" are substances which replenish the
lipids of the hair or of the top layers of the skin; "refreshers"
are substances which impart a pleasant freshness to the skin; "skin
conditioners" are substances which maintain the skin in good
condition; "skin protectors" are substances which help to avoid
harmful effects to the skin from external factors; "smoothers" are
substances which seek to achieve an even skin surface by decreasing
roughness or irregularities; "solvents" are substances which
dissolve other substances; "soothers" are substances which helps
lightening discomfort of the skin or of the scalp; "stabilizers"
are substances which improve ingredients or formulation stability
and shelf-life; "surfactants" are substances which lower the
surface tension of cosmetics as well as aids the even distribution
of the product when used; "tanning" is a process which darkens the
skin with or without exposure to UV; "tonics" are substances which
produce a feeling of well-being on skin and hair; "UV absorbers"
are substances which protect the cosmetic product from the effects
of UV light; "UV filters" are substances which filter certain UV
rays in order to protect the skin or the hair from harmful effects
of these rays; "viscosity controllers" are substances which
increase or decrease the viscosity of cosmetics.
[0176] In one embodiment, the formulations can be or comprise or
contain cosmetic additives such as those conventionally used in
cosmetic preparations, e.g. sunscreens, preservatives,
bactericides, fungicides, virucides, cooling substances, insect
repellents (e.g. DEET, IR 3225, Dragorepel), plant extracts,
antiinflammatory substances, wound healing accelerators (e.g.
chitin or chitosan and its derivatives), film-forming substances
(e.g. polyvinylpyrrolidones or chitosan or its derivatives),
customary antioxidants, vitamins (e.g. vitamin C and derivatives,
tocopherols and derivatives, vitamin A and derivatives),
2-hydroxycarboxylic acids (e.g. citric acid, malic acid, L-, D- or
DL-lactic acid), skin colorants (e.g. walnut extracts or
dihydroxyacetone), active ingredients for promoting hair growth
(e.g. minoxidil, diphencyprone, hormones, caffeine, finasteride,
phytosterols such as beta-sitosterol, biotin, or extracts of
Cimicifuga racemosa, Eugenia caryophyllata or Hibiscus
rosa-sinensis, barley, hops, or rice or wheat hydrolysates), skin
care products (e.g. cholesterol, ceramides, pseudoceramides),
softening, moisturizing and/or moisture-retaining substances (e.g.
glycerol or urea), fats, oils, saturated fatty acids,
monounsaturated or polyunsaturated fatty acids, alpha-hydroxy
acids, polyhydroxy fatty acids or their derivatives (e.g. linoleic
acid, alpha-linolenic acid, gamma-linolenic acid or arachidonic
acid and their respective natural or synthetic esters), waxes or
other conventional constituents of a cosmetic or dermatological
formulation, such as alcohols, polyols, polymers, foam stabilizers,
electrolytes, organic solvents, silicone derivatives or chelating
agents (e.g. ethylenediaminetetraacetic acid and derivatives),
antidandruff substances (e.g. climbazole, ketoconazole,
piroctonoleamine, zinc pyrithione), hair care products, perfumes,
antifoams, dyestuffs, pigments with a coloring action, thickeners
(advantageously silicon dioxide, aluminium silicates such as
bentonites, polysaccharides ortheir derivatives, e.g. hyaluronic
acid, guar kernel flour, xanthan gum, hydroxypropyl methyl
cellulose or allulose derivatives, particularly advantageously
polyacrylates such as carbopols, or polyurethanes), surface-active
substances, emulsifiers, plant parts and plant extracts (e.g.
arnica, aloe, beard lichen, ivy, stinging nettle, ginseng, henna,
camomile, marigold, rosemary, sage, horsetail or thyme), animal
extracts, e.g. royal jelly or propolis, proteins, protein
hydrolysates, yeast extracts, hop and wheat extracts, peptides or
thymus extracts.
[0177] Among the "preservatives" (additional preservatives) for
cosmetic preparations, the following may be mentioned: benzoic
acid, formic acid and their sodium salt; propionic acid, salicylic
acid, sorbic acid, other weak acids, e.g. free fatty acids, esters
and derivatives thereof, undec-10-enoic acid and their salts;
formaldehyde incl. para formaldehyde; biphenyl-2-ol and its salts;
zinc pyrithione; inorganic sulphites and hydrogen-sulphites;
chlorobutanol; 4-hydroxybenzoic acid and its salts and esters;
3-acetyl-6-methylpyran-2,4 (3H)-dione (dehydracetic acid) and its
salts; 3,3'-dibromo-4,4'-hexamethylenedioxydibenzamidine
(dibromohexamidine) and its salts (including isethionate);
thiomersal; phenylmercuric salts (including borate); hexetidine;
5-bromo-5-nitro-1,3-dioxane; bronopol; 2,4-dichlorobenzyl alcohol;
triclocarban; 4-chloro-m-cresol; triclosan; 4-chloro-3,5-xylenol;
3,3'-bis(1-hydroxymethyl-2,5-dioxoimidazolidin-4-yl)-1,1'-methylenediurea
(imidazolidinyl urea); poly (1-hexamethylenebiguanide
hydrochloride; 2-phenoxyethanol; hexamethylenetetramine
(methenamine); methenamine 3-chloroallylochloride;
1-(4-chlorophenoxy)-1-(imidazol-1-yl)-3,3-dimethylbutan-2-one;
1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione; benzyl
alcohol; 1-hydroxy-4-methyl-6(2,4,4-trimethylpentyl) 2-pyridone and
its monoethanolamine salt;
6,6-dibromo-4,4-dichloro-2,2'-methylenediphenol (bromochlorophen);
4-isopropyl-m-cresol; mixture of
5-chloro-2-methyl-isothiazol-3(2H)-one and
2-methylisothiazol-3(2H)-one with magnesium chloride and magnesium
nitrate; 2-benzyl-4-chlorophenol (clorophene); 2-chloroacetamide;
chlorhexidine and its digluconate, diacetate and dihydrochloride;
1-phenoxypropan-2-ol; alkyl (C12-C22) trimethyl ammonium, bromide;
4,4-dimethyl-1,3-oxizalidine;
N-(hydroxymethyl)-N-(dihydroxymethyl-1,3-dioxo-2,5-imidazolidinyl-4)-N'-(-
hydroxymethyl) urea and chloride;
1,6-Di-(4-amidinophenoxy)-n-hexane (Hexamidine) and its salts
(including isethionate and p-hydroxybenzoate); glutaraldehyde
(pentane-1,5-dial); 5-ethyl-3,7-dioxa-1-azabicyclo[3.3.0]octane;
3-(p-chlorophenoxy)-propane-1,2 diol (chlorphenesin); sodium
hydroxymethylamino acetate (sodium hydroxymethylglycinate); silver
chloride deposited on titanium dioxide; benzethonium chloride;
benzalkonium chloride, bromide and saccharinate; benzylhemiformal;
iodopropynyl butylcarbamate (IPBC) 3-iodo-2-propynylbutylcarbamate;
methylisothiazolinone, sodium hexametaphosphate,
ethylenediaminetetraacetic acid, peptides such as polylysine,
lauric arginate, cultured dextrose, neem oil, eugenol, p-cymene,
thymol, carvacrol, linalool, hydroxycinnamic acid, cinnamic acid,
cinnamic aldehyde, tea tree oil, fingerroot extract, acai powder,
4-hydroxybenzyl isothiocyanate and/or white mustard seed essential
oil, ferulic acid, or mixtures of two or more thereof.
[0178] Other preserving agents such 1,3-diols which are named in WO
2011/023582 and/or benzaldehydes such as those disclosed in WO
2009/000097, or extracts from Scutellaria baicalensis such as those
disclosed in KR 20030012821 are also understood as preservatives.
Preferred are Scutellaria baicalensis extracts comprising
dimethoxytetrahydroxyflavone and/or baicaleine
(5,6,7-trihydroxyflavone), e.g. as obtainable by extraction with a
solvent selected from propylene glycol, glycerine, 1,3-butylene
glycol, water, ethanol, and mixtures thereof.
[0179] The cosmetics according to the invention preferably comprise
only natural preservatives, or no preservatives are added in view
of the preservative properties of the compound(s) of the formula
I.
[0180] The invention also comprises cosmetics, especially their
use, comprising beyond one or more of the compounds of the formula
I also "other natural antimicrobially active agents", e.g.
proteins, corresponding peptides alone or in combination, natural
essential oils or derivatives thereof, such as oil from anis,
lemon, orange, grapefruit, rosemary, thyme, lavender, tee tree,
citron, wheat, lemon grass, cedar, cinnamon, eucalyptus,
peppermint, basil, fennel, menthol, Ocmea origanum, Hydastis
carradensis, Krameria lappacea, Podophyllum spp., Curcuma longa, or
mixtures of two or more such oils.
[0181] In certain embodiments of the invention, essential oils are
used in combination with emollient solvents and AHAs. Essential
oils ("EOs"), as defined herein, are volatile oils obtained from
plant or animal sources, or their synthetic equivalents, and are
composed of complex mixtures of several constituents as
monoterpenes and sesquiterpene hydrocarbons, monoterpene and
sesquiterpene; alcohols, esters, ethers, aldehydes, ketones, oxides
and the like. Examples of EOs include but are not limited to:
bergamot oil, clary sage oil, sage oil, almond oil, ylang-ylang
oil, neroli oil, sandalwood oil, frankincense oil, ginger oil,
peppermint oil, lavender oil, jasmine absolute, geranium oil
bourbon, spearmint oil, elove oil, patchouli oil, rosemary oil,
rosewood oil, sandalwood oil, tea tree oil, vanilla oil, lemongrass
oil, cedarwood oil, balsam oils, tangerine oil, Hinoki oil, Hiba
oil, ginko oil, eucalyptus oil, lemon oil, orange oil, thyme oil,
savory oil, oregano oil, and sweet orange oil. Botanicals, such as
camphor and cinnamon may also be used. Individual constituents
("ICs") of essential oils may be natural or entirely or partially
synthetic, and include, but are not limited to, l-citronellol,
alpha-amylcinnamaldehyde, lyral, geraniol, famesol,
hydroxycitronellal, isoeugenol, eugenol, eucalyptol, linalool,
citral, thymol, limonene and menthol. Additionally,
sesquiterpenoids such as nerolidol, farnesol, bisabolol and
apritone may also be used in the present invention. Mixtures of one
or more EO, one or more IC, and one or more EO as well as one or
more IC, are encompassed by the present invention.
[0182] Possible UV filters include but are not limited to:
phenylen-1,4-bis-(2-benzimidazyl)-3,3'-5,5'-tetrasulfonic acids;
2-phenylbenzimidazol-5-sulfonic acid and corresponding salts;
1,4-di(2-oxo-10-sulfo-3-bornylidenmethyl)-benzene and corresponding
salts; 4-(2-oxo-3-bornylidenmethyl)benzenesulfonic acid and its
salts; 2-methyl-5-(2-oxo-3-bornylidenmethyl)sulfonic acid and its
salts;
2,2'-methylen-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)--
phenol);
2-(2H-benzotriazol-2-yl)-4-methyl-6-[2-methyl-3-[1,3,3,3-tetramet-
hyl-1-[(trimethylsilyl)oxy]disiloxanyl]propyl]-phenol;
3-(4-methylbenzyliden)campher; 3-benzylidencampher;
4-(tert-butyl)-4'-methoxydibenzoylmethane;
2-(4'-diethylamino-2'-hydroxybenzoyl)-benzoic acid methyl ester;
terephthalidendicamphersulfonic acid; 4-(dimethylamino)-benzoic
acid (2-ethylhexyl)ester; 4-(dimethylamino)benzoic acid amylester;
4-ethoxybenzalmalonic acid (2-ethylhexyl)ester;
2-hydroxy-4-methoxybenzophenone,
2-hydroxy-4-methoxy-4'-methylbenzophenone;
2,2'-dihydroxy-4-methoxybenzophenone;
2-ethylhexyl-2-hydroxybenzoate; 3-(4-(2,2-bis
ethoxycarbonylvinyl)-phenoxy)propenyl)-methoxysiloxan/dimethylsiloxan-cop-
olymer; dioctylbutylamidotriazone (INCI:
Diethylhexyl-Butamidotriazone);
2,4-bis-[5-1(dimethylpropyl)benzoxazol-2-yl-(4-phenyl)-imino]-6-(2-ethylh-
exyl)-imino-1,3,5-triazine (CAS RN 288254-16-0);
4,4',4''-(1,3,5-triazin-2,4,6-triyltriimino)-tris-benzoic acid
tris(2-ethylhexylester) (also:
2,4,6-tris-[anilino-(p-carbo-2'-ethyl-t-hexyloxy)]-1,3,5-triazine
(INCI: Ethylhexyl Triazone);
2,4-bis-{[4-(2-ethyl-hexyloxy)-2-hydroxy]-phenyl}-6-(4-methoxphenyl)-1,3,-
5-triazine (INCI: Bis-Ethylhexyloxyphenol Methoxyphenyl Triazin);
2,4,6-tris-(biphenyl)-1,3,5-triazine;
2,4-bis-(4'-di-neopentylamino-benzalmalonat)-6-(4''-butylaminobenzoat)-s--
triazine,
4-dicyanomethylen-2,6-dimethyl-1,4-dihydropyridin-N-(ethyloxysul-
fate ester salt), titan dioxides, zink oxides, merocyanine,
piperazine derivatives as mentioned in WO 2011/042088 without being
limited to these.
[0183] Possible solvents include but are not limited to: alcohols
such as methanol, ethanol, butanol, pentanol (amyl alcohol),
ethylene glycol, propylene glycol, glycerol, butyl acetate,
dimethlsulfoxide, acetone, methyl ethyl ketone, hydrocarbons such
as hexane, pentane, oils such as zea mays oil, or the like.
[0184] The cosmetics may be solid, e.g. of a waxy appearance or the
like, or liquid or in the form of pastes or creams, e.g. as
emulsions, solutions or suspensions, e.g. oil in water or water in
oil (O/W or W/O) mixtures. They can thus form e.g. a solution, an
emulsion of the water-in-oil (W/O) type or oil-in-water (O/W) type,
or a multiple emulsion, for example of the water-in-oil-in-water
(W/O/W) type, a gel, a hydrodispersion, a solid stick or else an
aerosol.
[0185] The possible surfactants include but are not limited to
customary ones, e.g. anionic, non-ionic, amphoteric tensides, such
as soaps or sodium dodecylsulfate, or the substances disclosed in
WO 2011/023582.
[0186] It is to be mentioned here that in view of their molecular
structures the compounds of the formula I can also contribute
surfactant properties to a composition according to the invention,
so that this use is a preferred embodiment of the invention.
[0187] The composition comprising one or more compounds of the
formula I according to the invention can be applied to the skin or
lips or other body surfaces, e.g. hair, nails or teeth, according
to the use for which it is intended. It can thus be used in a
method for the cosmetic treatment of said body surfaces, e.g. the
skin, comprising the application of the composition according to
the invention to said body surface, e.g. the skin, for example for
the purpose of toning it up, of regenerating it or of smoothing out
its e.g. wrinkles in skin and/or for combating ageing, e.g. of the
skin, or the damaging effects of UV radiation and/or for
strengthening skin tissues, teeth, hair and/or nails against
attacks from the surroundings.
[0188] In an alternative form, the composition according to the
invention can be used for the manufacture of a dermatological
preparation.
[0189] Home Care Products
[0190] As possible home care products to be equipped with one or
more of the compounds of the formula I, among others, laundry
detergents, dishwashing detergents, fabric softeners, hard surface
cleaner or bleach compositions; surface, laundry and/or dish
cleaners, laundry soaps, air fresheners and odor eliminators,
insect repellents, laundry detergents, fabric softeners, bleaching
agents, organic cleaners, degreasers, stain removers, window and
glass cleaners, bathroom and toilet bowl cleaners, floor cleaners,
carpet cleaners, pet odor removers, cat litter deodorizers, car
refresheners, furniture polishes, waterless hand cleaners,
disinfectants, spray deodorizers, food processing plant cleaners,
coloring matters or other like home care applications may be
mentioned.
[0191] The home care products have customary compositions, For
example, in addition to surfactants, conventional solvents, dyes,
preservatives, emulsifying agents, perfumes, antibacterial agents,
thickeners, conditioners, antistatic agents, silicone surfactants,
and other like ingredients that are typically present in
conventional home care formulations may be comprised. Mixtures
and/or combinations of the aforementioned additional formulating
agents may also be employed in the present invention. The amounts
of the additional formulating agents that may be employed in the
present invention are within ranges that are well known to those
skilled in the art and further formulating is performed using
processes that are also well known in the art.
[0192] Pharmaceuticals
[0193] Pharmaceuticals comprise one or more pharmaceutically active
agents and a pharmaceutically acceptable carrier material.
[0194] Examples of such pharmaceuticals (pharmaceutical
compositions) are e.g. solid (tablet, capsule, powder, medical
chewing gum, lozenge, suppository) or liquid formulations (e.g.
injection solution, infusion solution, syrup, drinkable solution),
a spray, or a pasty material, e.g. a gel or a cream.
[0195] Among the possible active ingredients, all drugs known in
the art may be added, e.g. (without that this enumeration is
intended to be limiting) bronchodilators, antipyretics, analgetics,
antiphlogistics, antiarrhythmics, blood-pressure reducing agents,
vasodilators, anticholinergics, antiarteriosclerotics, enzymes,
antibodies, secretolytics, ulcer preparations, antiproliferative
agents, vasoconstrictors, expectorants, antitussiva, mucolytics, or
secretomotorics; in particular, free of antiallergics (including
those referred to hereinbefore), such as .alpha.-sympathicometics
(in particular Phenylephrin, Ephedrin, Tetryzolin, Naphazolin,
Oxymetozolin, Xylometazolin or Tramazolin), antihistamines,
non-steroidal or steroidal anti-inflammatory active substances (in
particular Triamcinolone acetonide, glucocorticoids, such as
Prednisolone, Triamcinolonacetonide, Clomethasone, Dexamethasone,
or Fluticasone), .beta..sub.2 sympathomimetics; mast cell
stabilizers, aromatase inhibitors; antiestrogens; topoisomerase I
inhibitors; topoisomerase II inhibitors; microtubule active
compounds; alkylating compounds; histone deacetylase inhibitors;
compounds which induce cell differentiation processes;
cyclooxygenase inhibitors; MMP inhibittors; mTOR inhibitors;
antineoplastic antimetabolites; platin compounds; compounds
targeting/decreasing a protein or lipid kinase activity and further
anti-angiogenic compounds; compounds which target, decrease or
inhibit the activity of a protein or lipid phosphatase; gonadorelin
agonists; anti-androgens; methionine aminopeptidase inhibitors;
bisphosphonates; biological response modifiers; antiproliferative
antibodies; heparanase inhibitors; inhibitors of Ras oncogenic
isoforms; telomerase inhibitors; proteasome inhibitors; compounds
used in the treatment of hematologic malignancies; compounds which
target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors
such as 17-AAG (17-allylamino-geldanamycin, NSC330507), 17-DMAG
(17-dimethylaminoethylamino-17-demethoxy-gelda-namycin, NSC707545),
IPI-504, CNF1010, CNF2024, CNF1010 from Conforma Therapeutics;
temozolomide (TEMODAL.RTM.); kinesin spindle protein inhibitors,
such as SB715992 or SB743921 from GlaxoSmithKline, or
pentamidine/chlorpromazine from CombinatoRx; MEK inhibitors such as
ARRY142886 from Array PioPharma, AZD6244 from AstraZeneca, PD181461
from Pfizer, leucovorin, EDG binders, antileukemia compounds,
ribonucleotide reductase inhibitors, S-adenosylmethionine
decarboxylase inhibitors, antiproliferative anti-bodies or other
chemotherapeutic compounds; tricyclics, e.g. benzodiazepines
including mitochondrial benzodiazepine-ligands MAO inhibitors,
SSRI's, SNRI's, NK receptor antagonists, CRF-receptor antagonists,
5HT7 receptor-antagonists, mGlu receptor
agonists/antagonist/modulators, GABA-A or GABA-A/B receptor
agonist/antagonists or modulators, vasopressin receptor
antagonists, herbal medicine such as St. John's Wort, 5-HT1A
receptor agonists, vasopressin receptor-antagonists,
acetylcholine-esterase inhibitors, such as rivastigmine or
donepezil, mixed acetylcholine/butyrylcholine esterase-inhibitors.
nicotinic-alpha7-receptor agonists, typical or atypical
antipsychotics, such as clozapine or haloperidol,
nicotinic-alpha7-receptor agonists, antimanic agents (e.g. lithium,
Carbamazepine, Valproate) or any atypical or typical antipsychotic;
or the like; pharmaceutically acceptable salts thereof, if
salt-forming groups are present; or combinations of two or more of
the aforementioned active substances or their pharmaceutically
acceptable salts.
[0196] Pharmaceutical compositions comprising one or more active
ingredients and one or more compounds of the formula I in
association with at least one pharmaceutical acceptable carrier or
diluent may be manufactured in conventional manner by mixing with a
pharmaceutically acceptable carrier or diluent.
[0197] The invention relates also to pharmaceutical compositions
comprising an antimicrobially effective amount, especially an
amount effective in the treatment of one of the above-mentioned
disorders, of one or more compounds of the formula I, a
pharmaceutically acceptable salt thereof, and/or an ester thereof,
together with one or more pharmaceutically acceptable carriers that
are suitable for topical, enteral, for example oral or rectal, or
parenteral administration and that may be inorganic or organic,
solid or liquid. There can be used for oral administration
especially tablets or gelatin capsules that comprise the active
ingredient together with diluents, for example lactose, dextrose,
mannitol, and/or glycerol, and/or lubricants and/or polyethylene
glycol. Tablets may also comprise binders, for example magnesium
aluminum silicate, starches, such as corn, wheat or rice starch,
gelatin, methylcellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidone, and, if desired, disintegrators, for example
starches, agar, alginic acid or a salt thereof, such as sodium
alginate, and/or effervescent mixtures, or adsorbents, dyes,
flavorings and sweeteners. It is also possible to use the
pharmacologically active compounds of the present invention in the
form of parenterally administrable compositions or in the form of
infusion solutions. The pharmaceutical compositions may be
sterilized and/or may comprise excipients, for example
preservatives, stabilizers, wetting compounds and/or emulsifiers,
solubilizers, salts for regulating the osmotic pressure and/or
buffers. The present pharmaceutical compositions, which may, if
desired, comprise other pharmacologically active substances are
prepared in a manner known per se, for example by means of
conventional mixing, granulating, confectioning, dissolving or
lyophilizing processes, and comprise approximately from 1% to 99%
by weight, especially from approximately 1% to approximately 60%,
active ingredient(s) and 0.001 to 10, 0.01 to 8, 0.02 to 6 or 0.03
to 5 weight percent of the compound(s) of the formula I, a
pharmaceutically acceptable salt thereof and/or an ester thereof.
Also the use of their preservative or antimicrobial properties in
said pharmaceutical compositions by the addition of one or more
compounds of the formula I is included.
[0198] Additives, both in the case of foods and of cosmetics, as
well as in the case of pharmaceuticals (the term pharmaceuticals
also including nutraceuticals), may exhibit more than one property
as selected from the above lists or other not cited properties,
e.g. preservatives may also act as acidity regulators and vice
versa, or e.g. antioxidants may act as preservatives as well as
acidity regulators, or other thinkable multi functional uses.
[0199] Medical Devices
[0200] Medical devices are especially devices intended for use in
the diagnosis of disease or other conditions, or in the cure,
mitigation, treatment, or prevention of disease, in man or other
animals, and mean e.g. any instrument, apparatus, appliance,
material or other article, whether used alone or in combination,
including the software necessary for its proper application
intended by the manufacturer to be used for human beings for the
purpose of: [0201] diagnosis, prevention, monitoring, treatment or
alleviation of disease, [0202] diagnosis, monitoring, treatment,
alleviation of or compensation for an injury or handicap, [0203]
investigation, replacement or modification of the anatomy or of a
physiological process, [0204] control of conception, and which does
not achieve its principal intended action in or on the human body
by pharmacological, immunological or metabolic means, but which may
be assisted in its function by such means.
[0205] Among the medical devices, among others, e.g. implants,
prosthesis, plasters (=adhesive tapes), (wound) dressing materials,
bandages, cotton wool, gauze bandages, surgical instruments, tooth
brushes, syringes, syringe needles, medication containers, infusion
bottles, infusion tubes, valves or multiports used in infusion,
infusion needles, infusion assemblies, surgical instruments,
catheters, artificial or natural tissues or membranes, tooth
brushes, and the like may be mentioned.
[0206] Possible implants include but are not limited to:
a) Surgical meshes or other 2-dimensionally extended or extendable
materials (such as membranes), such as polypropylene meshes (e.g.
BARD MESH.RTM. from Bard Inc., SURGIPRO.RTM. from US Surgical,
Inc., TRELEX.RTM. from Boston Scientific, PROLENE.RTM. or
MERSILENE.RTM. from Ethicon, Inc.), polyester meshes (e.g.
MERSILENE.RTM. from Ethicon), expanded polytetrafluoroethylen
meshes (e.g. SOFT TISSUE PATCH.RTM. from W.L. Gore &
Associates, Inc), polyamide materials or the like, e.g. for the
repair of hernias; or such meshes or materials 2-dimensionally
extended or extendable materials, such as polyglactin
(e.g.VICRYL.RTM. from Ethicon, Inc.), polyglykolate (e.g.
DEXON.RTM. from US Surgical, Inc.), polydioxanone (PDS),
polyglyconate (e.g. MAXON.RTM., Davis & Geck, Gosport, UK) or
collagene materials, e.g. COOK SURGISIS.RTM. from Cook Biomedical,
Inc.; other possible membrane or mesh materials include
FLUORO-TEX.RTM. Pericardial and Peritoneum Surgical Membrane or
FLUORO-TEX Dura Substitute (each from C.R. Bard) orr PRECLUDE.RTM.
Pericardial Membrane PRECLUDE.RTM. Peritoneal Membrane and
PRECLUDE.RTM. Dura Substitute Membrane each from W.L. Gore &
Associates, silicone elastomers, such as SILASTIC Rx.RTM. Medical
Grade Sheeting from Dow Corning or mikroporous Polypropylen from
Celgard, Inc., for example where the implant is used to seal
compartmenting tissues or structures in the body, such as
peritoneum, pleura, diaphragma, lung, pericard or the like; b)
electrode coatings, e.g. for electrodes of pacemakers or neural or
muscular stimulation or the like, e.g. made from tungsten, silicon,
platinum-iridium or stainless steel or combinations thereof; c)
degradable or non-degradable bone or cartilage implants; d)
orthopaedic implants, such as hard tissue, bone or joint replacing
implants, for example for hip or knee or other joint repair, e.g.
implants made from stainless steel, cobalt-chromium alloys,
titanium or titanium alloys, pure titanium, tantalum, plastics
materials such as polyethylene, polypropylene, polylactate, carbon
fibre, ceramics or compounds of two or more such materials; e)
screws, nails, threads, plates or other hard fixation materials for
hard tissues, e.g. from the materials mentioned under d); f) oral,
such as dental implants, e.g. from the materials mentioned under
d); g) Bone fillers, such as bone cement composites, hydroxyl
apatite composites or polycaprolactone (Blurr plug); h) implants
coming into contact with blood, such as vascular grafts, e.g. from
biocompatible plastics materials, such as extended
polytetrafluoroethylene or poly[ethylene terephthalate], stents
(e.g. from metals or metal alloys, such as (e.g. 316L) stainless
steel, cobalt-chromium-nickel-molybdenum-iron alloy, Tantalum,
shape memory alloys, e.g. nitinol, or (e.g. shape memory) polymer
materials, such as polyethylene or polyurethane), heart or venous
valves (e.g. from polymer or metal or natural materials or
combinations thereof, e.g. pyrolytic carbon, titanium coated with
pyrolytic carbon, and the sewing ring cuff is e.g. teflon,
polyester or dacron), stent/valve combinations, or continuous
accesses e.g. to veins, or to the peritoneum e.g. for peritoneal
dialysis or the like; i) implants for delivery of signals or
chemical substances, e.g. drugs, coming into contact with tissue
and/or body fluids, e.g. pumps for delivery of drugs or pacemakers;
j) organs or tissues for transplantation (e.g. to decrease the
expression of antigens evoking transplant rejection), especially
autografts, allografts, heterografts or xenografts; k) skin
substitutes or wound coating materials, such as natural (e.g.
keratinocytes in combination with human fibroblasts in bovine type
I collagen or other ECM proteins and cytokines, such as
Apligraf.RTM. (Organogenisis Inc.), or from synthetics, or
combinations with natural materials e.g. synthetic polysiloxanes
with bovine type I collagen and chondroitin-6-sulphate (e.g.
Integra.RTM. (Johnson & Johnson Medica Care Life)) or
preferably from synthetics alone or combined with human dermal
tissue (e.g. Tanscyte.RTM. (Advanced Tissue Sciences Inc.)),
allografts, collagen (e.g. in reconstituted form) or the like; l)
suturing materials (especially for internal sutures not accessible
from the outside), e.g. from absorbable or non-absorbable
synthetics or natural materials (e.g. cat gut).
[0207] Especially preferred are artificial implants made from
metals, metal alloys, synthetic materials (=polymers) (either
degradable or non-degradable), carbon fibres, boranes, ceramics,
glass or bone replacement materials, especially of the types a) to
i) or k) to l) mentioned above, including composites of two or more
such materials.
[0208] The implants may be for permanent (e.g. in the case of joint
replacement) or transitory (e.g. in the case of fixing devices or
skin replacements) insertion or other administration.
[0209] Especially here the biofilm inhibiting usefulness of the
compound(s) of formula I and of compositions comprising them is of
advantage.
[0210] Active Packaging Materials
[0211] Among the active packaging materials, e.g. food or beverage
or pharmaceutical or surgical packaging material having a spoilage
preventing/preservative effect e.g. against colonialisation by
bacterial or other microorganismic films or against spoiling of
materials coming into contact with other perishable products can be
mentioned, e.g. cans, wraps, foils, bottles, mugs, cartons, tubs,
bags, cartridges, tubes, sachets, ampoules, sacks, or the like.
[0212] Both with regard to medical devices and to active packaging
materials, as well as with regard to personal care products used
having a predetermined shape, the application of compound(s) of the
formula I is especially by coating e.g. on surfaces coming into
contact with perishable products or a human or an animal, or bulk
integration (e.g. by mixing of starting materials and/or
impregnation of final products) into the material.
[0213] The compound(s) of the formula I to medical devices and
active packaging materials can especially be applied (alone or in
combination with appropriate carrier materials) on surfaces coming
into contact with perishable products or a human or an animal, e.g.
in the form of a coating, or applied by bulk integration into the
material.
[0214] The materials equipped (which form an embodiment of the
invention) or to be equipped with one or more compounds of the
formula I may comprise the compound(s) of the formula I, a
physiologically acceptable salt thereof and/or an ester thereof,
either in admixture to the bulk of the material, or (in the case of
products with a stable surface) by covalent and/or non-covalent
attachment to (parts or the whole of) said surface.
[0215] For covalent attachment, the surface must either expose or
be chemically modified to expose functional groups which would
allow for covalent bonding of the compound of the formula I either
directly or via a spacer molecule.
[0216] In the case of the covalently bound (at least bivalent)
linker molecules, these may allow covalent or non-covalent binding
of the compounds of the formula I.
[0217] The linker in the covalent attachment method can be any
linker.
[0218] Covalent binding of the compounds of the formula I with or
without linkers can take place directly by reacting their
precursors with the surfaces without activation or to activated
surfaces on the implants or other products. Examples are
1. epoxy- or activated ester-functionalized surfaces, where
reaction with OH-- or amino groups in the linker precursors is
possible. 2. Where the linker precursors are organic compounds
which are furnished terminally with a thiol group, they can be
bound e.g. via gold-plated surfaces or maleinimide-layered
surfaces. 3.) Linker precursors which, during the process of
manufacture, are furnished terminally with a carboxyl or phosphate
group, can be activated to active esters or the like, e.g. with
EDC, so that an OH--, SH-- or amino-reactive on the surface can be
bound. 4.) Precursors for homo- or preferably hetero-bifunctional
cross-linker which can be bound to reactive groups at the surface,
such as carboxyl, epoxy, OH, SH, aldehyde or amino groups; or other
known methods. 5.) Other directly functionalized surfaces, e.g.
especially polymers with plasma-coated aldehydes.
[0219] Other possible activations for both covalent and
non-covalent binding include but are not limited to glow-discharged
surfaces, electrostically charged surfaces, and/or roughened
surfaces. Also coating with materials, e.g. gels, varnishes, paints
or the like, comprising the compound(s) of the formula I, is a
method for providing their surfaces with these compounds.
[0220] In the (preferred) case of the non-covalent attachment, the
material can be any substrate. This substrate could be a synthetic
polymer (i.e. polyacrylate, polylactide-co-glycolide, polyethylene,
or polypropylene), carbon fibre, glass, boranes, metal (i.e.
titanium or stainless steel), natural polymer (i.e. collagen or
alginate), or any other surface that is capable of supporting a
coating, e.g. in solid or fibre form, respectively. Composites of
two or more such materials are also included. The non-covalent can,
for example, be via adsorption, integration into a coating matrix
or the like.
[0221] The compounds of the formula I in the embodiments of the
invention, in a further embodiment, can be used also where the
materials (products) with which they are associated (e.g. by mixing
in) require a heat treatment, e.g. to achieve pasteurization
sterilization or the like.
[0222] Thus, in one embodiment, the one or more compounds of the
formula I is/are heat stable. In one example the compound(s) of the
formula I fully or partially retain(s) structure and activity e.g.
regarding its preservative properties after heating. Heating of the
antimicrobial composition can be performed at 60-130.degree. C.,
such as in the range of 60-65.degree. C., 65-70.degree. C.,
70-75.degree. C., 75-80.degree. C., 80-85.degree. C., 85-90.degree.
C., 90-95.degree. C., 95-100.degree. C., 100-105.degree. C.,
105-110.degree. C., 110-115.degree. C., 115-120.degree. C.,
120-125.degree. C., 125-130.degree. C.
[0223] In one embodiment heating is performed at about
65-75.degree. C., more preferred at about 70.degree. C. In one
embodiment heating is performed at about 90-110.degree. C., more
preferred at about 100.degree. C.
[0224] In yet another embodiment heating is performed at about
120-125.degree. C., more preferred at about 121.degree. C.
[0225] According to the present invention, heating can be performed
for shorter or longer periods of time, such as from a minute to
several hours. Heating can for example be performed for a few
minutes such as in the range of about 1-5 minutes, 5-10 minutes,
10-15 minutes, 15-20 minutes, 20-25 minutes, 25-30 minutes, 30
minutes-to 1 hour.
[0226] For the purpose of the invention other known preserving
agents or known preservatives may be added to the pharmaceutical
incl. nutraceutical and cosmeceutical, nutritive or cosmetic
product as well as to the composition.
[0227] Preferred combinations of the compounds of the invention for
use in food and beverages are with weak organic acids, especially
preferred are combinations with sorbic acid and/or benzoic acid and
their appropriate salts, or with natural preservatives.
[0228] Among possible preferred combinations of the compounds of
the invention for cosmetic use are combinations with
C.sub.1-C.sub.4 alkyl para-hydroxybenzoate or its salts, e.g.
methylparaben, ethylparaben, propylparaben, isopropylparaben,
butylparaben, isobutylparaben and their appropriate salts,
benzylparaben, benzoic acid or its salts, e.g. sodium benzoate,
N-(3-chloroallyl)hexaminium chloride, alcohols or polyols, such as
ethanol, propylene glycol, benzyl alcohol or 2-phenoxyethanol,
benzalkonium chloride, chloroacetamide, thimerosal, benzalkonium
chloride, cetylpyridinium chloride, N-(3-chloroallyl)hexaminium
chloride, formaldehyde donors, such as imidazolidinyl urea,
diazolidinyl urea, or DMDM hydantoin, isothiazolinones, such as
KATHON.RTM. CG, available commercially from Rohm & Haas,
Philadelphia, Pa., which contains a chloro-substituted
isothiazolinone (methylchloroisothiazolinone), other chlorinated
aromatic compounds, such as chlorphenesin, phenoxyethanol, vicinal
diols, such as a 1,2-alkane diol or a glyceryl monoether, such as
glyceryl laurate, decyl glycoside isothiazolinone compounds, such
as methylisothiazolinone, e.g. 2-methyl-3(2H)isothiazolinone,
propionic acid and its salts, undec-10-enoic acid and salts,
Scutellaria baicalensis extracts (such as e.g. available from
BMB-FS, or the like), or mixtures of two or more such
preservatives.
[0229] In all materials, the compound(s) of the formula I can also
be used as emulsifiers, in addition to use of their preservative
properties.
[0230] The compound or compounds of the formula I are preferably
comprised, taking the weight or the material to which it is added
and the compound(s) of formula I as 100 weight %, in a relative
weight share of 0.00001 to 10 weight percent.
[0231] In the foods or beverages according to the invention, the
compound or compounds of the formula I are preferably
added/comprised in a concentration e.g. in the range from 50 to
20000 ppm, e.g. from 100 to 1000 ppm, for example from 10 to 120
ppm, such as from 30 to 60 ppm, or e.g. from 0.1-150 ppm, where ppm
refers to weight parts per million.
[0232] "Minimal inhibitory concentration" (MIC) is a term for which
no standard time period is routinely defined or understood. In the
medical fields, MIC is frequently employed to designate the
concentration of a substance which prohibits the growth of a single
type of microorganism in over-night incubation as compared to a
positive control without the substance. However, the rest of the
scientific community has adopted the term MIC to mean any of a
number of conditions of period of incubation and degree of
inhibition.
[0233] Even within the medical field, it is recognized that an MIC
value developed over a period of 24 hours incubation may not be the
same value developed after 48 hours or longer. In other words, a
substance may exhibit an observable MIC during the first 24 hours
of an experiment, but exhibit no measurable MIC relative to the
positive control after 48 hours.
[0234] The following table gives some of the compounds of the
formula I that are of interest in the various embodiments of the
present invention.
TABLE-US-00001 TABLE 1 Name Cpd. (if known: CAS Num number): ber
2,17,18- trihydroxy- hexacosanoic acid 22-O- [6-isovaleroyl-
hexapyranosyl- (1.fwdarw.2)-5-acetyl- pentapyranosyl-
(1.fwdarw.2)-penta- pytanosid]- [1] ##STR00011## [2] ##STR00012##
Glykenin-IIC 134528-36-2 [3] ##STR00013## [4] ##STR00014## [5]
##STR00015## Glykenin IIIB or Glykenin IIIC 134528-37-3,
134479-71-3 [6] ##STR00016## Glykenin IVC or IVB 134479-72-4,
134528-38-4 [7] ##STR00017## [8] ##STR00018## [9] ##STR00019## [11]
##STR00020## [12] ##STR00021## [13] ##STR00022## [14] ##STR00023##
Glykenin DGC or Glykenin DGB 112965-51-2, 112848-53-0 [16]
##STR00024## [17] ##STR00025## Antibiotic F 19848A 895129-04-1 [10]
##STR00026## [18] ##STR00027## [19] ##STR00028## [20] ##STR00029##
[21] ##STR00030##
[0235] The preserving properties of the compounds of the invention
e.g. compounds of the formula I can be evaluated according methods
cited in the art such as WO 2010/062548. For example, they can be
determined for beverages using the method described in Example 3 in
WO 2010/062548 which is incorporated herein by reference. For
example, a single preparation of base beverage is employed to
prepare each of five tests and consists of 4% apple juice, 68 g/l
sucrose, 52 g/l glucose, 2 g/l fructose in batch water which is
formulated to 90 ppm hardness with clalcium chloride and magnesium
chloride. A pH of 3.4 is achieved through combinations of malic
acid and sodium malate for all preparations regardless of the
presence or absence of compounds of the formula I. The total
combined quantity of sodium malate and malic acid is near constant,
but the ratio of malic acid and malate may vary slightly given the
presence of compound of the formula I. It is relevant that the
beverage employed for testing does not naturally contain any
substance with measurable antimicrobial activity such as in
essential oils. Where required, compound of the formula I is
supplemented from separately prepared stock solutions. Dimethyl
dicarbonate is delivered by means of a hypodermic needle (Hamilton
syringe) through septum that seals the test vessel against loss of
moisture. Dimethyl dicarbonate stock solution consists of 1 ml
dimethyl dicarbonate (1.25 g) in 49 ml of 100% ethanol (25 mg/ml).
Hence, a microliter of stock contains 25 microgram of dimethyl
dicarbonate. Each of the five tests employs the same bio-indicator
organisms; Growth (+) versus no growth (-) is established by visual
inspection or spectrophotometrically (see e.g. Examples below). The
organism names and their strain numbers, as well as incubation
times and details on deviating assay conditions, if any were used,
are mentioned in the Examples.
[0236] Among the preferred embodiments of the invention, the
following are to be mentioned:
A) The first embodiment of the invention is that defined in claim
1, or a method comprising the use mentioned therein. B) Especially
preferred is said use or method, where the material to which the
agent is added is a cosmetic, a food or a beverage. C) Another
invention embodiment related to the use or method according to
paragraph A) or B), where in the compound of the formula I m is 3
to 5, n is 2 to 5, o is 0 or 1, p is 5 to 15 and R is a moiety of
the subformula
##STR00031##
wherein the rings A, B and C are monosaccharide moieties each
independently from the others with 5 or 6 ring members, wherein one
or more of the hydroxyl groups may be acylated, preferably by a
C.sub.2-C.sub.10alkanoic, more preferably a
C.sub.3-C.sub.10alkanoic acid. D) Another invention embodiment
relates to the use or method according to paragraph A) above,
wherein the compound or mixture of compounds of the formula I
comprises, preferably, at least one compound selected from the
group of compounds mentioned in table 1, or a physiologically
acceptable salt thereof. E) Another invention embodiment relates to
the use or method according to any one of paragraphs A) to D)
above, wherein the compound or compounds of the formula I, or a
physiologically acceptable salt, or a physiologically acceptable
ester thereof, is added to enhance the stability against
microorganisms. F) Another invention embodiment relates to the use
or method according to paragraph E) above, where the microorganism
is at least one microorganism selected from the group consisting of
mold, yeast and bacteria of a beverage or a food or a cosmetic. G)
Another invention embodiment relates to the use or method according
to any one of paragraphs A) to F) above, where at least one
additional preservative is added. H) Another invention embodiment
relates to the use or method according to any one of paragraphs A)
to G) above, where the compound or compounds of the formula I, a
physiologically acceptable salt thereof, and/or an ester thereof,
is added in the form of an extract (this term including a
precipitate) from a natural source or obtained from such an
extract. I) Another invention embodiment relates to the use or
method according to paragraph H), where the source of the extract
is a Dacryopinax, a Ditiola and/or a Femsjonia fungus. J) Another
invention embodiment relates to the use or method according to
paragraph H), where the source of the extract is Dacryopinax
spathularia, Dacrymyces sp., Ditiola radicata, Ditiola nuda and/or
Femsjonia luteo-alba (=Ditiola pezizaeformis). K) Another invention
embodiment relates to the use or method according to paragraph J)
above, where the source of the extract is Dacryopinax spathularia
strain FU50088, Ditiola radicata strain MUCL 53180, Ditiola nuda
strain CBS 173.60 or Femsjonia luteo-alba (=Ditiola pezizaeformis)
strain MUCL 53500. L) Another invention embodiment relates to the
use or method according to any one of paragraphs A) to K), where
the material is subjected to a heat treatment before, during or
after addition of the compound(s) of the formula I, a
physiologically acceptable salt thereof and/or an ester thereof, as
defined in any one of paragraphs A), C), D) or H) to K), especially
heating the material to a temperature from 60 to 130.degree. C. M)
Another invention embodiment relates to a compound or a mixture of
compounds of the formula I shown in paragraph A) or as defined in
any one of paragraphs C), D) or H) to L), where the moiety R
carries at least one hydroxyl group esterified with an acid with 3
or more carbon atoms, a physiologically acceptable salt, and/or an
ester thereof. N) Another invention embodiment relates to the
compound or compound mixture of paragraph M), wherein the acid is a
C.sub.3-C.sub.10-alkanoic acid, especially isovaleric acid; a
physiologically acceptable salt, and/or an ester thereof. O)
Another invention embodiment relates to a compound of the formula I
shown in claim 1, selected from the group of compounds represented
in Table 1 with the following compound numbers: [1], [12], [13],
[14], [17] and [18], and in a broader aspect from compound [4], a
physiologically acceptable salt, and/or an ester thereof. P) A
further embodiment of the invention relates to a preservative or
antimicrobial composition, comprising as active agent a compound or
a mixture of compounds of the formula I, a physiologically
acceptable salt thereof, and/or an ester thereof, as shown or
defined in any one of paragraphs A), C), D) and H) to O), alone or
with another additive, such as a carrier material, where the
preservative composition is especially for use in a cosmetic, a
food, a beverage, a pharmaceutical, a medical device, or an active
packaging material. Q) Another invention embodiment relates to the
composition according to paragraph P) which is a powder. R) Another
invention embodiment relates to the composition according to
paragraph P) which is a liquid. S) Yet another invention embodiment
relates to the composition according to paragraph P) which is a
coating or film. T) Another invention embodiment relates to the
composition according to any one of paragraphs P) to S), wherein
the preservative or antimicrobial composition is for enhancing the
stability against microorganisms. U) Another invention embodiment
relates to the composition according to paragraph T), wherein the
microorganisms are at least one microorganism selected from the
group consisting of mold, yeast and bacteria. V) Another invention
embodiment relates to the composition according to any one of
paragraphs P) to U), being a preservative or antimicrobial
composition for a pharmaceutical, a medical device, a food
container, a beverage container, or especially a food, a beverage
or a cosmetic or a home care product. W) Another invention
embodiment relates to the composition according to any one of
paragraphs P) to V), which comprises an additional preservative. Y)
Another invention embodiment relates to the composition according
to any one of paragraphs P) to W), which is a precursor of a
beverage, especially a concentrate, a syrup or a powder. Z) Another
invention embodiment relates to an extract comprising one or more
compounds of the formula I, a physiologically acceptable salt
thereof, and/or an ester thereof, as shown or defined in any one of
paragraphs A), C), D) or H) to O). AA) Another invention embodiment
relates to a method of enhancing microbial stability of a material,
comprising adding one or more compounds of the formula I, a
physiologically acceptable salt thereof, and/or an ester thereof,
as shown or defined in any one of paragraphs A), C), D) or H) to O)
to a material, preferably a material selected from the group
consisting of a cosmetic, a food, a beverage, a pharmaceutical, a
medical device, and an active packaging material. BA) Another
invention embodiment relates to the method of paragraph AA),
wherein the material is a beverage or a food. CA) Another invention
embodiment relates to the method of paragraph AA), wherein the
material is a cosmetic. DA) Another invention embodiment relates to
a material comprising, as or within a coating and/or as admixture,
an additive in the form of a compound or a mixture of compounds of
the formula I, a physiologically acceptable salt thereof and/or an
ester thereof, as defined in any one of paragraphs A), C), D) or H)
to O). EA) Another invention embodiment relates to the material of
paragraph DA), which is a cosmetic, a food, a beverage, a
pharmaceutical, a medical device, or an active packaging material.
FA) Another invention embodiment relates to the material according
to paragraph DA) which is a beverage. GA) Another invention
embodiment relates to the material in the form of a compound or a
mixture of compounds of the formula I, a physiologically acceptable
salt thereof and/or an ester thereof, according to paragraph FA),
where the beverage is selected from the group consisting of water,
flavoured water, fortified water, a flavoured beverage, carbonated
water, a juice, cola, lemon-lime, ginger ale, root beer beverages
which are carbonated in the manner of soft drinks, a syrup, a diet
beverages, a carbonated soft drink, a fruit juice, other fruit
containing beverages which provide the flavor of fruit juices and
contain greater than 0% fruit juice but less than 100% fruit juice,
fruit flavored beverages, vegetable juices, vegetable containing
beverages, which provide the flavor of any of the aforementioned
vegetable juices and contain greater than 0% vegetable juice but
less than 100% vegetable juice, isotonic beverages, non-isotonic
beverages, soft drinks containing a fruit juice, coffee, tea, tea
beverages prepared from tea concentrate, extracts, or powders,
drinkable dairy products, hot chocolate, chocolate powders/mixes,
drinkable soy products, non-diary milks, alcoholic beverages, fruit
smoothies, horchata, sport drinks, energy drinks, health drinks,
wellness drinks, shakes, protein drinks, drinkable soy yogurts, low
acid beverages, acidified beverages, nectars, tonics, frozen
carbonated beverages, frozen uncarbonated beverages, liquid meal
replacements, infant formulations, and combinations or mixtures
thereof. HA) Another invention embodiment relates to the material
according to paragraph DA) which is a beverage precursor,
especially a concentrate, syrup or powder. IA) Another invention
embodiment relates to the material according to paragraph DA) which
is a food. JA) Another invention embodiment relates to the material
according to paragraph DA) which is a cosmetic. KA) Another
invention embodiment relates to the material in the form of a
cosmetic according to paragraph JA) which is cream, emulsion,
lotion, gel or oil for the skin; a face masks, a tinted base, a
make-up powder, an after-bath powder, a hygienic powder, a toilet
soap, a deodorant soap, a perfumes, a toilet water, an eau de
Cologne, a bath or shower preparation; a depilatory; a deodorant,
an anti-perspirant, a hair care product; a setting product; a
cleansing product; a conditioning product; a hairdressing product;
a shaving products; a product for making up and removing make-up
from the face and the eyes, a product intended for application to
the lips, a products for care of the teeth and/or the mouth; a
product for nail care and/or make-up, a product for external
intimate hygiene, a sunbathing product, a product for tanning
without sun, a skin-whitening product, an anti-wrinkle product, a
tampon, a sanitary towel, a diaper or a handkerchief. LA) Another
invention embodiment relates to the cosmetic material according to
any one of paragraphs JA) and LA), which comprises one or more
additives selected from the group consisting of abrasives,
absorbents, anti-cakings, anti-corrosives, anti-dandruffs,
anti-foamings, anti-microbials, anti-oxidants, anti-perspirants,
anti-plaques, anti-seborrhoeics, anti-statics, astringents,
bindings, bleachings, bufferings, bulkings, chelatings, cleansings,
cosmetic colorants, denaturants, deodorants, depilatories,
detanglings, emollients, emulsifiers, emulsion stabilizers, film
formings, foamings, foam boosters, gel formers, hair conditioners,
hair dyes, hair fixers, hair waving or straighteners, humectants,
hydrotropers, keratolytics, masking agents, moisturing, nail
conditioners, opacifiers, oral care, oxidizers, pearlescents,
plasticizers, preservatives, propellants, skin protectors,
smoothers, solvents, soothers, stabilizers, surfactants, tanning,
tonics, UV absorbers, UV filters, and viscosity controllers. MA)
Another invention embodiment relates to the cosmetic material
according to any one of paragraphs JA) and LA), where the
formulations can be or comprise or contain cosmetic additives
selected from sunscreens, preservatives, bactericides, fungicides,
virucides, cooling substances, insect repellents, plant extracts,
antiinflammatory substances, wound healing accelerators,
film-forming substances, customary antioxidants, vitamins,
2-hydroxycarboxylic acids, skin colorants, active ingredients for
promoting hair growth, skin care products, softening, moisturizing
and/or moisture-retaining substances, fats, oils, saturated fatty
acids, monounsaturated or polyunsaturated fatty acids, alfa-hydroxy
acids, polyhydroxy fatty acids or their derivatives, waxes,
alcohols, polyols, polymers, foam stabilizers, electrolytes,
organic solvents, silicone derivatives or chelating agents,
antidandruff substances, hair care products, perfumes, antifoams,
dyestuffs, pigments with a coloring action, thickeners,
surface-active substances, emulsifiers, plant parts and plant
extracts, animal extracts, proteins, protein hydrolysates, yeast
extracts, hop and wheat extracts, peptides and thymus extracts. NA)
Another invention embodiment relates to the material according to
any one of paragraphs DA) to MA) comprising an additional
preservative. PA) Another invention embodiment relates to the
material according to any one of paragraphs DA) to NA) which is
obtained after heat treatment, especially at 60 to 130.degree. C.
QA) Another invention embodiment relates to a compound of the
formula I or a mixture of such compounds, according to any one of
paragraphs A), B) and H) to O) or a composition comprising them,
especially according to any one of paragraphs P) to S) mentioned
above, as a biofilm inhibiting agent and its corresponding use,
e.g. by administering, or in methods comprising administering, one
or more compounds of the formula I, or a composition comprising
them, to surfaces or materials coming into contact with
surfaces.
[0237] In a preferred embodiment, the invention relates to (the use
of) one or more compounds of the above formula I, one or more
physiologically acceptable salts of a compound of the above formula
I, or a mixture thereof,
as agent with preservative properties against (i) Gram-positive
bacteria and/or (ii) fungi, wherein the [0238] (i) Gram-positive
bacteria are selected from the group consisting of the genera
Bacillus, Brevibacterium, Lactobacillus, Micrococcus,
Staphylococcus, Streptococcus, Clostridium, Chlamydia,
Enterococcus, Listeria, Corynebacterium, Leuconostoc, Pediococcus,
Propionibacterium, and the fungi preferably are selected from the
group consisting of [0239] (ii-a) fungi from the families
Trichocomaceae, Arthrodermataceae and Mucoraceae, more preferably
molds (moulds) of the genera Aspergillus, Botryotinia,
Byssochlamys, Magnaporthe, Paecilomyces, Neosartorya, Mucor,
Penicillium, Rhizopus, Talaromyces, and Trichophyton, [0240] (ii-b)
yeasts from the order Saccharomycetales, preferably yeasts from the
families Saccharomycetaceae or Pichiaceae, more preferably from the
group consisting of the genera Brettanomyces, Candida, Dekkera,
Pichia, Saccharomyces, and Zygosaccharomyces, preferably comprising
adding the agent to a material, where said material is selected
from the group consisting of a cosmetic product, a food product, a
beverage, a pharmaceutical product, a medical device, a medical
hygiene product, a home care product, and an active packaging
material.
[0241] In a more preferred embodiment, the invention relates to
(the use of) one or more compounds of the above formula I, one or
more physiologically acceptable salts of a compound of the above
formula I, or a mixture thereof,
wherein m is 3 to 5, n is 3, o is 0 or 1 and p is 11 to 14,
preferably m is 3 to 5, n is 3, o is 0 or 1 and p is 12 or 13, and
R is a trisaccharide carbohydrate moiety of the subformula
##STR00032##
bound via a carbon atom to the binding oxygen (via the bond
indicated by the dotted line), wherein ring A is a xylopyranoside
moiety, ring B is a xylopyranosyl moiety, and ring C is
glucopyranosyl moiety, and wherein one or more of the hydroxyl
groups of said rings are esterified with a
C.sub.2-C.sub.10-alkanoic acid, preferably a
C.sub.3-C.sub.10-alkanoic acid, more preferably a
C.sub.3-C.sub.6-alkanoic acid, as agent with preservative
properties against (i) Gram-positive bacteria and/or (ii) fungi, as
indicated above, preferably comprising adding the agent to a
material, where said material is preferably selected from the group
consisting of a cosmetic product, a food product, a beverage, a
pharmaceutical product, a medical device, a medical hygiene
product, a home care product, and an active packaging material.
[0242] As already mentioned, and especially due to their excellent
and generally superior antimicrobial properties (in particular
regarding yeasts and molds), preferred compounds or mixtures of
compounds of the formula I shown above are defined by a
trisaccharide carbohydrate moiety R carrying at least one hydroxyl
group esterified with an acid with 3 or more carbon atoms,
particularly wherein the acid is a C.sub.3-C.sub.10-alkanoic acid,
especially wherein the acid is a C.sub.3-C.sub.6-alkanoic acid,
and/or a physiologically acceptable salt thereof.
[0243] In a particularly preferred embodiment, the invention
relates to (the use of) one or more compounds of the above formula
I, one or more physiologically acceptable salts of a compound of
the above formula I, or a mixture thereof (preferably as defined in
one of the preferred or particularly preferred embodiments herein)
as agent with preservative properties against [0244] bacteria
selected from the group consisting of Bacillus subtilis, Bacillus
cereus, Brevibacterium epidermidis, Brevibacterium linens,
Chlamydia trachomatis, Clostridium perfringens, Clostridium
botulinum, Clostridium sporogenes, Corynebacterium xerosis,
Corynebacterium variabile, Corynebacterium minutissimum,
Enterococcus faecalis, Lactobacillus plantarum, Listeria
monocytogenes, Listeria welshimeri, Micrococcus luteus,
Propionibacterium acnes, Staphylococcus aureus, Staphylococcus
epidermidis, Streptococcus mutans, and Streptococcus pneumoniae,
and/or [0245] filamentous fungi selected from the group consisting
of Aspergillus brasiliensis, Aspergillus flavus, Aspergillus
fumigatus, Aspergillus niger, Botrytis cinerea, Byssochlamys fulva,
Magnaporthe grisea (Magnaporthe oryzae, Pyricularia oryzae), Mucor
plumbeus, Rhizopus arrhizus, Rhizopus nigricans, Rhizopus
stolonifer, and Talaromyces luteus, and/or [0246] yeast selected
from the group consisting of Brettanomyces bruxellensis,
Brettanomyces naardenensis, Candida albicans, Candida glabrata,
Candida lusitaniae, Candida tropicalis, Dekkera bruxellensis,
Dekkera naardenensis, Saccharomyces cerevisiae, Zygosaccharomyces
bailiff, Zygosaccharomyces bisporus, Zygosaccharomyces florentinus,
and Zygosaccharomyces rouxii.
[0247] Within the scope of the present text, the terms "for oral
consumption", "orally consumable" or "food product" and the like in
particular refer to materials which are intended to be swallowed by
a human being in an unchanged (i.e. by direct oral consumption,
"ready-to-eat", "ready-to-drink") or processed state and then to be
digested.
[0248] The term "ready-to-use" product refers to a product, the
composition of which, in terms of the substances which determine
the flavour, is (essentially) complete. The term "ready-to-use"
product includes carbonated and non-carbonated liquids and viscous
or semisolid products. Examples of "ready-to-use" products include
deep-frozen products which, prior to consumption, are be defrosted
and heated before consumption. The ready-to-use products may also
be "ready-to-eat" or "ready-to-drink", like e.g. carbonated
beverages, flavoured milk, (water) ice, yoghurts, and the like, or
may have to be diluted with water before oral consumption, which is
for example the case for beverage syrups.
[0249] Yeasts are able to grow in orally consumable compositions,
such as foods and beverages, with a low pH values (generally pH 5.0
or lower), and in the presence of sugars, organic acids or other
easily metabolized carbon sources. During their growth, yeasts
metabolize some food components and produce metabolic products.
This causes the physical, chemical, and sensory properties of an
orally consumable composition to change, and the composition is
spoiled. The growth of yeast within orally consumable compositions
is often seen on their surface, as in cheeses or meats, or by the
fermentation of sugars in beverages, such as juices, and
semi-liquid products, such as syrups and jams.
[0250] Of particular relevance in the context of orally consumable
compositions are Aspergillus niger, Brettanomyces bruxellensis,
Brettanomyces naardenensis, Dekkera bruxellensis, Dekkera
naardenensis, Saccharomyces cerevisiae, Zygosaccharomyces bailii,
Zygosaccharomyces bisporus, Zygosaccharomyces florentinus, and
Zygosaccharomyces rouxii.
[0251] The yeasts of the Zygosaccharomyces genus have had a long
history as spoilage yeasts within the food industry. This is due
mainly to the fact that these species can grow in the presence of
high sucrose, ethanol, acetic acid, sorbic acid, benzoic acid, and
sulphur dioxide concentrations (which are some of the commonly used
preservatives in orally consumable compositions).
[0252] Clostridium botulinum is a Gram-positive bacterium that
produces several toxins, inter alia neurotoxins that cause the
flaccid muscular paralysis seen in botulism. Botulism poisoning can
occur due to improperly preserved food or canned food that was not
processed using correct preservation times and/or pressure. Mainly
slightly acidic or neutral food is at risk which was stored under
anaerobic conditions (generally pH>4.6) and storage temperatures
above 10.degree. C. The latter is generally given for canned foods,
such as meat and fish preserves, mayonnaise, but also slightly
acidic fruit or vegetables.
[0253] Bacillus cereus may be harmful to humans and cause foodborne
illness (severe nausea, vomiting and diarrhea), particularly in
foods like meat, milk, spices, seasonings, fruits, vegetables,
cereal and cereal products, rice products and (ready-to-eat) rice
dishes. Bacillus cereus is also known to cause chronic skin
infections and keratitis.
[0254] Bacillus subtilis is known to cause disease in severely
immunocompromised patients, and it may cause food poisoning.
Bacillus subtilis spores can survive the extreme heat during
cooking. Bacillus subtilis strains are responsible for causing
ropiness in spoiled bread dough.
[0255] Micrococcus luteus is found in soil, dust, water and air,
and is part of the normal flora of the mammalian skin and mucosae.
It is further a food spoiling bacterium and often found on spoiled
meat. In immunocompromised patients, Micrococcus luteus may cause
infections.
[0256] Propionibacterium acnes is largely commensal and part of the
healthy adult human skin flora. It lives primarily on fatty acids
in sebum secreted by sebaceous glands in the follicles and is
linked to the skin condition acne. Propionibacterium acnes can also
cause chronic blepharitis and endophthalmitis, the latter
particularly following intraocular surgery.
[0257] Dental plaque is a biofilm formed by colonizing bacteria
trying to attach themselves to a smooth tooth surface. A
microorganism significantly contributing dental plaque and tooth
decay is Streptococcus mutans.
[0258] The rice blast fungus Magnaporthe grisea (syn.: Pyricularia
olyzae; conidial stage/anamorph: Pyricularia grisea) attacks
leaves, grains, and other parts of rice plants.
[0259] Athlete's foot (tinea pedis) is a communicable disease
caused by parasitic molds in the genus Trichophyton, predominantly
Trichophyton rubrum and/or Trichophyton mentagrophytes. These can
also cause skin infections on other areas of the body, most often
under toenails (onychomycosis) or on the groin (tinea cruris).
[0260] Mucor species are often involved in the composting of plants
and plant residues and are found on foods such as milk, butter,
cheese and tomatoes. Mucor plumbeus has a worldwide distribution in
soil. As spoilage germ, Mucor plumbeus is mainly found on fermented
foods (such as bread, beer, wine, cheese, yoghurt, kefir, salami),
and on grain.
[0261] Mucormycosis (sometimes also referred to as Zygomycosis) is
the term used to describe fungal infections caused by fungi in the
order Mucorales, inter alia by species in the Mucor genus. These
rare yet serious and potentially life-threatening fungal infections
usually affect the face, oropharyngeal (nose/mouth) cavity,
gastrointestinal tract or the skin. Individuals with immune
disorders (immunocompromised) are more prone to this type of fungal
infection.
[0262] Rhizopus is a genus of fungi found on plants and on various
other organic substrates, including mature fruits and vegetables,
jellies, syrups, bread, peanuts and tobacco. Some Rhizopus species
are opportunistic agents of human zygomycosis (fungal infection)
and can be fatal. Rhizopus infections are also an associated
complication of diabetic ketoacidosis.
[0263] Rhizopus arrhizus is the most common cause of mucormycosis
in humans and occasionally infects other animals.
[0264] Rhizopus nigricans is a fungus commonly known as bread mold
and is the most common species of Rhizopus. It is found on old
food. The spores, dispersed in hot dry weather, contain allergenic
proteins, which can produce respiratory and nasal symptoms. Food
handling workers are particularly at risk if they are mold
allergic.
[0265] Rhizopus stolonifer (black bread mold) is a widely
distributed mold and is most commonly found growing on bread and
soft fruits such as bananas and grapes, and causes damage to the
surface where it lives. It is capable of causing opportunistic
infections of humans.
[0266] Staphylococcus aureus is the most common species to cause
staphylococcal infections. Staphylococcus aureus can cause a range
of illnesses, from minor skin infections to life-threatening
diseases such as pneumonia, meningitis, osteomyelitis,
endocarditis, toxic shock syndrome, bacteremia, and sepsis.
Staphylococcus aureus strains are also responsible for food
poisoning through the production of an enterotoxin, particularly in
meat, meat products (e.g. luncheon meats, cold meats, sausages),
milk, milk products, such as cheese).
[0267] Clostridium perfringens is widely present in nature and can
be found as a normal component of decaying vegetation, but also in
the intestinal tract of humans. Clostridium perfringens bacteria
often cause of foodborne illness, particularly in poorly prepared
meat and poultry. Often, meat is well prepared, but too far in
advance of consumption. Since Clostridium perfringens forms spores
that can withstand cooking temperatures, upon standing or storage
germination ensues and infective bacterial colonies develop.
Clostridium perfringens causes a wide range of symptoms: it is a
very common cause of food poisoning and the most common bacterial
agent for gas gangrene, which is necrosis, putrefaction of tissues,
and gas production.
[0268] Fungi of the genus Aspergillus may cause infections causing
a variety of diseases called aspergillosis (common forms are
allergic bronchopulmonary aspergillosis, pulmonary aspergilloma and
invasive aspergillosis).
[0269] Aspergillus flavus is a common mold in the environment, and
can cause storage problems in stored grains. It can also be a human
pathogen, associated with aspergillosis and other infections.
[0270] Aspergillus fumigatus is one of the most common Aspergillus
species to cause disease in individuals with an immunodeficiency.
In immunocompromised individuals, such patients receiving
immunosuppressive therapy for autoimmune or neoplastic disease,
organ transplant recipient, and people with AIDS or leukemia, the
fungus is more likely to become pathogenic and cause aspergillosis.
Aspergillus fumigatus primarily causes invasive infection in the
lung (e.g. chronic pulmonary infections) and represents a major
cause of morbidity and mortality in these individuals.
[0271] Aspergillus niger causes black mold on certain fruits and
vegetables such as grapes, onions, and peanuts, and is a common
contaminant of food. For example, Aspergillus niger causes a common
postharvest disease of onions. Aspergillus niger is less likely to
cause human disease than some other Aspergillus species, but, if
large amounts of spores are inhaled, a serious lung disease
(aspergillosis can occur). Aspergillus niger is one of the most
common causes of otomycosis (fungal ear infections).
[0272] Chlamydia infection is one of the most common sexually
transmitted infections in humans, and caused by the bacterium
Chlamydia trachomatis. Chlamydia is a major infectious cause of
human genital and eye disease. Chlamydia conjunctivitis or trachoma
is a common cause of blindness worldwide. Both sexes can display
urethritis, proctitis, trachoma, and infertility. If untreated,
chlamydial infections can cause serious health problems. Chlamydia
trachomatis is also an important neonatal pathogen, where it can
lead to infections of the eye (trachoma) and pulmonary
complications.
[0273] Enterococcus faecalis inhabits the gastrointestinal tracts
of humans and other mammals. It may cause endocarditis and
bacteremia, urinary tract infections, meningitis, and other
infections in humans (e.g. in root canal-treated teeth). It can
even cause life-threatening infections in humans, especially in
hospital environment.
[0274] Listeria may be been found in uncooked meats, uncooked
vegetables, fruit, pasteurized or unpasteurized milk, foods made
from milk, and processed foods. Pasteurization and sufficient
cooking kill Listeria; however, contamination may occur after
cooking and before packaging. For example, processing plants
producing ready-to-eat foods, such as hot dogs, deli meats, fish
products, cheeses, milk, and deli salads, follow extensive
sanitation policies and procedures to prevent Listeria
contamination. The major human pathogen in the Listeria genus is
Listeria monocytogenes. It is usually the causative agent of
listeriosis, a serious bacterial infection caused by eating food
contaminated with Listeria monocytogenes.
[0275] Yeasts of the Candida genus are a group of opportunistic
pathogens that causes oral and vaginal infections in humans, known
as candidiasis. The pathogenic yeasts of candidiasis are Candida
albicans, Candida tropicalis, Candida stellatoidea, Candida
glabrata, Candida krusei, Candida parapsilosis, Candida
guilliermondii, Candida viswanathii, and Candida lusitaniae, of
these Candida albicans being the most important and most
relevant.
[0276] Candida glabrata is the second most common Candida pathogen
after Candida albicans, also causing infections of the urogenital
tract, and of the bloodstream (candidemia). Candida glabrata has
been shown to be a highly opportunistic pathogen and is especially
prevalent in immunocompromised individuals and elderly. Candida
glabrata can also adhere to biotic and abiotic surfaces, thereby
forming microbial "biofilms" on e.g. urinary catheters or
indwelling intravenous catheters. It may also cause problems with
dental devices, such as dentures.
[0277] Additionally, in particular Staphylococcus aureus, Candida
albicans, Aspergillus brasiliensis and Aspergillus niger are likely
microbiological contaminants of cosmetic formulations.
[0278] Species of certain bacteria such as Staphylococcus
epidermidis, Corynebacterium xerosis, Corynebacterium minutissimum
and Brevibacterium epidermidis are largely responsible for the
formation of underarm and/or foot odor, or body odor in general.
Brevibacterium linens inter alia causes foot odor.
[0279] In another aspect the present invention relates to one or
more compounds of formula I and/or the physiologically acceptable
salts thereof, particularly one or more compounds selected from the
group consisting of [1], [2], [3], [4], [5], [6], [7], [8], [9],
[10], [11], [12], [13], [14], [18], [19], [20], [21] and the
physiologically acceptable salts thereof, for use in the
prophylactic and/or therapeutic treatment of a disorder, disease or
condition selected from the group consisting of
mycoses (fungal infections), preferably Aspergillus, Candida or
Mucor associated mycoses, particularly Candida associated
mycoses.
[0280] Particularly relevant and preferably treated are mycoses
selected from the group consisting of candidiasis [in particular
oral (thrush) Candida infections, infections of the urogenital
(e.g. vaginal) tract by Candida bacteria (in particular by Candida
albicans and/or Candida glabrata), diaper candidiasis (Candida
associated diaper dermatitis, diaper rash)], invasive candidiasis
(particularly candidemia (infections of the bloodstream)),
aspergillosis and mucormycosis.
[0281] The compounds of formula I and/or the physiologically
acceptable salts thereof, particularly those selected from the
group consisting of [1], [2], [3], [4], [5], [6], [7], [8], [9],
[10], [11], [12], [13], [14], [18], [19], [20], [21] and the
physiologically acceptable salts thereof, are particularly
beneficial for use in the prophylactic treatment of a disorder,
disease or condition mentioned above.
[0282] As used herein, the term "effective amount" or "effective
dose" refers to the (preferably oral) administration of an
effective dose of one or more compounds of formula I and/or the
physiologically acceptable salts thereof that produces the effects
for which it is administered.
[0283] As used herein, the term "therapeutical" or
"therapeutically" refers to the (in particular oral) administration
of a therapeutically effective dose of one or more compounds of
formula I and/or the physiologically acceptable salts thereof
(preferably in form of a mixture, a composition or a material as
defined in the context of the present invention) that produces the
effects for which it is administered, i.e. that will elicit the
biological or medical response (in vitro or in vivo, preferably in
vivo in a mammal, particularly in vivo in human being) that is
being sought, in particular the amelioration and/or alleviation of
the symptoms of the disorder, disease or condition being treated up
to and including complete cure.
[0284] As used herein, the term "prophylactic" or
"prophylactically" refers to the (in particular oral)
administration of a prophylactically effective dose of one or more
compounds of formula I (preferably in form of a mixture, a
composition or a material as defined in the context of the present
invention) that produces the effects for which it is administered,
i.e. that will elicit the biological or medical response (in vitro
or in vivo, preferably in vivo in a mammal, particularly in vivo in
a human being) that is being sought, in particular the prevention
of the onset of a disorder, disease or condition in individuals at
risk for such disorder, disease or condition as mentioned
herein.
[0285] The present invention also relates to a method of reducing
the activity and/or number of pathogenic Gram-positive bacteria
and/or pathogenic fungi in an immunocompromised individual,
comprising the following step:
administering (preferably orally or topically) to a mammal,
particularly an immunocompromised mammal, particularly an
immunosuppressed human being, an effective total amount of [0286]
one or more compounds of formula I as defined herein, and/or one or
more physiologically acceptable salts thereof, particularly one or
more compounds selected from the group consisting of [1], [2], [3],
[4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [18],
[19], [20], [21] and the physiologically acceptable salts thereof,
or [0287] an extract or material according to the present
invention, preferably in a preferred or particularly preferred
embodiment according to the present invention.
[0288] Preferably, the pathogenic Gram-positive bacteria are
selected from the group consisting of the genera Bacillus,
Clostridium, Listeria, Micrococcus and Staphylococcus, more
preferably selected from the group consisting of Bacillus cereus,
Clostridium sporogenes, Clostridium perfringens, Listeria
monocytogenes, Micrococcus luteus, Staphylococcus aureus,
Staphylococcus epidermidis.
[0289] Preferably, the pathogenic fungi are selected from the group
consisting of the genera Aspergillus and Candida, more preferably
selected from the group consisting of Aspergillus flavus,
Aspergillus fumigatus, Candida albicans, Candida glabrata, Candida
lusitaniae, and Candida tropicalis.
[0290] The present invention also relates to a method for the
prophylactic and/or therapeutic treatment of a disease, disorder or
condition, comprising the following step:
administering (preferably orally or topically) to a mammal,
particularly an immunocompromised mammal, particularly an
immunosuppressed human being, an effective total amount of [0291]
one or more compounds of formula I as defined herein, and/or one or
more physiologically acceptable salts thereof, particularly one or
more compounds selected from the group consisting of [1], [2], [3],
[4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [18],
[19], [20], [21], and the physiologically acceptable salts thereof,
or [0292] an extract or material according to the present
invention, preferably in a preferred or particularly preferred
embodiment according to the present invention.
[0293] The compounds of formula I (for use) according to the
present invention and/or the physiologically acceptable salts
thereof (preferably the preferred or particularly preferred
compounds of formula I defined above) show a comparatively weak
activity against Gram-negative bacteria, such as Escherichia coli,
Pseudomonas aeruginosa, Pseudomonas putida or Salmonella
typhimurium. In our own investigations regarding such Gram-negative
bacteria generally MIC values >100 ppm were observed, typically
in the range of 200-500 ppm.
[0294] Thus, in a preferred embodiment, a material according to the
present invention, preferably a cosmetic or a pharmaceutical,
comprises (i) one or more compounds of formula I (for use)
according to the present invention and/or the physiologically
acceptable salts thereof, and (ii) one or more agents exhibiting an
antimicrobial activity against Gram-negative bacteria, preferably
selected from the group consisting of Triclosan.RTM.
(2,4,4'-trichloro-2'-hydroxydiphenyl ether), chlorhexidine,
chlorhexidine salts (preferably chlorhexidine diacetate,
chlorhexidine dichloride, chlorhexidine digluconate), octenidine,
octenidine dihydrochloride, 2-bromo-2-nitropropane-1,3-diol,
polyaminopropyl biguanide, imidazolidinyl urea, diazolidinyl urea,
chlorphenesin, DMDM hydantoin, sodium hydroxymethylglycinate,
phenoxyethanol, isothiazolinones (preferably methylisothiazolinone,
methylchloroisothiazolinone), benzalkonium chloride
(alkyldimethylbenzylammonium chloride, preferably
N-octyl-N-benzyl-N,N-dimethylammonium chloride,
N-decyl-N-benzyl-N,N-dimethylammonium chloride,
N-dodecyl-N-benzyl-N,N-dimethylammonium chloride,
N-tridecyl-N-benzyl-N,N-dimethylammonium chloride,
N-tetradecyl-N-benzyl-N,N-dimethylammonium chloride,
N-hexadecyl-N-benzyl-N,N-dimethylammonium chloride,
N-octadecyl-N-benzyl-N,N-dimethylammonium chloride), and
lantibiotics (preferably those disclosed in U.S. Pat. No. 7,960,505
B2).
[0295] In some cases, a material according to the present
invention, preferably a cosmetic or a pharmaceutical, comprises (i)
one or more compounds of formula I (for use) according to the
present invention and/or the physiologically acceptable salts
thereof, and (ii) one or more parabens (para-hydroxybenzoic acid
esters) and/or the salts thereof, preferably one, two or more
parabens selected from the group consisting of methylparaben,
ethylparaben, propylparaben, isopropylparaben, butylparaben,
isobutylparaben, benzylparaben and the physiologically acceptable
salts (preferably the sodium salts) thereof.
[0296] Compounds of the formula I, mixtures thereof, and the
physiologically acceptable salts thereof are preferred in the
context of the present invention, wherein R is a moiety of
formula
##STR00033##
wherein R.sup.z denotes H or C.sub.2-C.sub.6-alkanoyl, preferably
H, acetyl or C.sub.5-alkanoyl, more preferably H, acetyl or
isovaleryl, most preferably isovaleryl, R.sup.y denotes H or
C.sub.2-C.sub.6-alkanoyl, preferably H or acetyl, and R.sup.x,
independent of each other, each denote H or
C.sub.2-C.sub.6-alkanoyl, preferably H or acetyl, with the proviso
that at least one of R.sup.x, R.sup.y and R.sup.z is not
hydrogen.
[0297] Compounds of the formula I, mixtures thereof, and the
physiologically acceptable salts thereof are more preferred in the
context of the present invention, wherein R is a moiety of
formula
##STR00034##
wherein R.sup.z denotes H, acetyl or isovaleryl, most preferably
isovaleryl, R.sup.y denotes H or C.sub.2-C.sub.6-alkanoyl,
preferably H or acetyl, and R.sup.x, independent of each other,
each denote H or C.sub.2-C.sub.6-alkanoyl, preferably H or acetyl,
with the proviso that at least one of R.sup.y and R.sup.z is not
hydrogen, preferably R.sup.z is not hydrogen.
[0298] Individual compounds of formula I with an acyl substituent
with more than 2 carbon atoms, such as an isovaleryl substituent,
in the trisaccharide carbohydrate moiety R exhibit a stronger
antimicrobial activity, particularly against yeasts and molds,
especially against yeasts and molds of relevance regarding food,
beverage and/or cosmetic spoilage, and/or a broader activity
spectrum than the corresponding compounds with an acetyl
substituent in the trisaccharide carbohydrate moiety R.
[0299] It was also found in own investigations that mixtures
comprising (i) two, three, four, five, six or more compounds of the
above preferred formula I, (ii) two, three, four, five, six or more
physiologically acceptable salts of a compound of the above
preferred formula I, or a mixture thereof, or (iii) one, two, three
or more compounds of the above preferred formula I and one, two,
three or more physiologically acceptable salts of a compound of the
above preferred formula I typically showed a broader activity
spectrum and/or stronger long term inhibitory activity,
particularly against yeasts and molds, especially against yeasts
and molds of relevance regarding food, beverage and/or cosmetic
spoilage, in comparison to individual compounds of (preferred)
formula I or a physiologically acceptable salt thereof.
[0300] Said stronger long term inhibitory activity--which typically
is bactericidal and/or fungicidal, and not merely bacteriostatic
and/or fungistatic) and/or broader long term inhibitory activity
spectrum of mixtures according to the present invention against
organisms involved in spoilage of preparations or compositions with
a high water content (generally 50 wt. % or more, based on the
total weight of the preparation or composition) is especially
useful against acidophilic spoilage yeasts, which are for example
involved in spoiling of beverages. These mixtures according to the
present invention are particularly able to inhibit the growth of
thermophilic molds, which are difficult to control with standard
sterilizing and/or pasteurizing processes.
[0301] Especially preferred in the context of the present invention
in view of the excellent properties is a mixture of three or more
compounds of formula I or the physiologically acceptable salts
thereof, said mixture comprising
(a) one or more compounds of formula I or a physiologically
acceptable salt thereof, wherein one group of R.sup.x, R.sup.y, and
R.sup.z is not hydrogen, (b) one or more compounds of formula I or
a physiologically acceptable salt thereof wherein two groups of
R.sup.x, R.sup.y, and R.sup.z are not hydrogen, and (c) one or more
compounds of formula I or a physiologically acceptable salt thereof
wherein three groups of R.sup.x, R.sup.y, and R.sup.z are not
hydrogen, wherein preferably [0302] the total amount of the
compounds of group (a) is 2 wt. % or more, preferably 3 wt. % or
more, more preferably 5 wt. % or more, in each case based on the
total weight of the mixture, [0303] and/or [0304] said mixture
comprises one, two, three or all compounds [5], [8], [12], and
[14], and/or [0305] the total amount of the compounds of group (b)
is 20 wt. % or more, preferably 25 wt. % or more, more preferably
30 wt. % or more, even more preferably 35 wt. % or more, most
preferably 40 wt. % or more, in each case based on the total weight
of the mixture, [0306] and/or [0307] said mixture comprises one,
two, three, four, five or all compounds [1], [6], [7], [10], [13],
and [18], more preferably comprises [1] and/or [7], particularly
more preferably [1] and [7], [0308] and/or [0309] said mixture
comprises compound [1], preferably in a total amount of 1 wt. % or
more, more preferably 2 wt. % or more, even more preferably 4 wt. %
or more, particularly preferably 5 wt. % or more, [0310] and/or
[0311] said mixture comprises compound [7], preferably in a total
amount of 5 wt. % or more, more preferably 8 wt. % or more, even
more preferably 10 wt. % or more, particularly preferably 12 wt. %
or more, and/or [0312] the total amount of the compounds of group
(c) is 1 wt. % or more, preferably 1.5 wt. % or more, more
preferably 2 wt. % or more, in each case based on the total weight
of the mixture, [0313] and/or [0314] said mixture comprises [3]
and/or [9], preferably compound [9].
[0315] In a preferred mixture according to the present invention,
preferably the average degree of acylation is as follows:
1.1 to 2.2 acetyl groups per molecule, preferably 1.3 to 1.9 acetyl
groups per molecule, and/or 0.1 to 1.0 isovaleryl groups per
molecule, preferably 0.15 to 0.6 isovaleryl groups per molecule,
wherein the average degree of acylation preferably is determined
via .sup.1H-NMR quantification using an average molecular weight
for the glycolipids of 985 g/mol. As internal standard preferably
1,3,5-trichlorbenzene may be used. The .sup.1H-NMR signal used for
said quantification was that of the hydrogen atom bound to the
carbon atom in position C-2 (i.e. (CH)OH, the carbon atom bearing
the alpha-hydroxy group relative to the carboxylic acid group at
C-1).
[0316] A mixture according to the present invention, an extract
according to the present invention, a material according to the
present invention, and/or a composition according to the present
invention preferably comprises less than 25 wt. % of Glykenin IVA
((2S,16R,17S,21R)-2,16,17-trihydroxy-21-[[2-O-[2-O-(6-O-acetyl-.beta.-D-g-
lucopyranosyl)-.beta.-D-xylopyranosyl]-4-O-acetyl-.beta.-D-xylopyranosyl]o-
xy]hexacosanoic acid), more preferably less than 20 wt. % of
Glykenin IVA, particularly preferably less than 15 wt. % of
Glykenin IVA, in each case based on the total amount of compounds
of formula I and the physiologically acceptable salts thereof.
[0317] Preferably, a mixture comprises compounds [1] and [7], and
the physiologically acceptable salts thereof, wherein more
preferably the total amount of
compound [1] and the physiologically acceptable salts thereof is 1
wt. % or more, more preferably 2 wt. % or more, more preferably 3
wt. % or more, and/or compound [7] and the physiologically
acceptable salts thereof is 1 wt. % or more, more preferably 2 wt.
% or more, more preferably 3 wt. % or more, and/or the compounds
[1] and [7], and the physiologically acceptable salts thereof is 5
wt. % or more, more preferably 8 wt. % or more, even more
preferably 12 wt. % or more, the weight percentages in each case
relating to the total weight of the mixture,
[0318] More preferably, a mixture according to the present
invention comprises
a total amount of 1-20 wt. %, preferably 2-15 wt. %, of compound
[1] and the physiologically acceptable salts thereof, a total
amount of 0-10 wt. %, preferably 0.5-5 wt. %, of compound [5] and
the physiologically acceptable salts thereof, a total amount of
0-10 wt. %, preferably 0.5-5 wt. %, of compound [6] and the
physiologically acceptable salts thereof, a total amount of 2-75
wt. %, preferably 5-50 wt. %, of compound [7] and the
physiologically acceptable salts thereof, a total amount of 0-12
wt. %, preferably 1-8 wt. %, of compound [8] and the
physiologically acceptable salts thereof, a total amount of 0-12
wt. %, preferably 1-8 wt. %, of compound [9] and the
physiologically acceptable salts thereof, a total amount of 0-12
wt. %, preferably 1-10 wt. %, of compound [10] and the
physiologically acceptable salts thereof, a total amount of 0-10
wt. %, preferably 0.5-6 wt. %, of compound [12] and the
physiologically acceptable salts thereof, a total amount of 0-8 wt.
%, preferably 0.25-4 wt. %, of compound [13] and the
physiologically acceptable salts thereof, a total amount of 0-8 wt.
%, preferably 0.25-5 wt. %, of compound [18] and the
physiologically acceptable salts thereof, and additionally
preferably one, several or all of the following further parameters
apply: [0319] said mixture comprises a total amount of compounds of
formula I and the physiologically acceptable salts thereof of 75
wt. % or more, preferably 80 wt. % or more, more preferably 85 wt.
% or more, [0320] said mixture comprises less than 8 wt. %,
preferably less than 6 wt. %, more preferably less than 4 wt. %, of
compounds of formula I without any acyl substituents in the
trisaccharide carbohydrate moiety R (particularly less than 2.0 wt.
% of compound [16]), [0321] said mixture comprises Glykenin IVA in
an amount of 25 wt. % or less, preferably of 20 wt. % or less, more
preferably of 15 wt. % or less, [0322] said mixture comprises
proteins in a total amount of 2 wt. % or less, preferably of 1.25
wt. % or less, more preferably of 1.0 wt. % or less, and/or [0323]
said mixture comprises water in a total amount of 4 wt. % or less,
preferably of 3 wt. % or less, more preferably of 2 wt. % or less,
the percentages in each case relating to the total weight of the
mixture.
[0324] Particularly preferably, a mixture according to the present
invention comprises
a total amount of 3-15 wt. %, of compound [1] and the
physiologically acceptable salts thereof, a total amount of 0.5-5
wt. % of compound [5] and the physiologically acceptable salts
thereof, a total amount of 0.5-5 wt. % of compound [6] and the
physiologically acceptable salts thereof, a total amount of 10-35
wt. % of compound [7] and the physiologically acceptable salts
thereof, a total amount of 1-8 wt. % of compound [8] and the
physiologically acceptable salts thereof, a total amount of 1-8 wt.
% of compound [9] and the physiologically acceptable salts thereof,
a total amount of 1-10 wt. % of compound [10] and the
physiologically acceptable salts thereof, a total amount of 0.5-6
wt. % compound [12] and the physiologically acceptable salts
thereof, a total amount of 0.25-4 wt. % of compound [13] and the
physiologically acceptable salts thereof, and a total amount of
0.25-5 wt. % of compound [18] and the physiologically acceptable
salts thereof, and additionally preferably one, two, three, four or
all of the following further parameters apply: [0325] said mixture
comprises a total amount of compounds of formula I and the
physiologically acceptable salts thereof of 85 wt. % or more, more
preferably of 90 wt. % or more, [0326] said mixture comprises less
than 5 wt. %, preferably less than 3 wt. %, of compounds of formula
I without any acyl substituents in the trisaccharide carbohydrate
moiety R (particularly less than 1.0 wt. % of compound [16]),
[0327] said mixture comprises Glykenin IVA in an amount of 20 wt. %
or less, preferably of 15 wt. % or less, [0328] said mixture
comprises proteins in a total amount of 1.25 wt. % or less,
preferably of 0.95 wt. % or less. and/or [0329] said mixture
comprises water in a total amount of 3 wt. % or less, preferably of
2 wt. % or less, the percentages in each case relating to the total
weight of the mixture.
[0330] Such a particularly preferred mixture according to the
present invention had the following average degree of
acylation:
1.4 to 1.8 acetyl groups per molecule, and 0.2 to 0.5 isovaleryl
groups per molecule.
[0331] In view of their broad activity spectrum and particularly
high efficacy (which was found to be superior to otherwise
identical compounds of formula I not carrying a hydroxyl group
esterified with isovaleric acid), particularly against yeasts and
molds, especially against yeasts and molds of relevance regarding
food, beverage and/or cosmetic spoilage, the compounds of formula
I, mixtures thereof, and the physiologically acceptable salts
thereof are especially preferred in the context of the present
invention wherein
m is 3, 4 or 5, n is 3, o is 0 or 1 and p is 11 to 14 (preferably p
is 12 or 13), and R is a moiety of formula
##STR00035##
wherein R.sup.y denotes H or C.sub.2-C.sub.6-alkanoyl, preferably H
or acetyl, and R.sup.x, independent of each other, each denote H or
C.sub.2-C.sub.6-alkanoyl, preferably H or acetyl.
[0332] Particularly preferred are compounds of formula I of the
following formulae
##STR00036##
more preferably of the following formulae
##STR00037##
wherein in each of said formulae R.sup.y denotes H or
C.sub.2-C.sub.6-alkanoyl, preferably H or acetyl, and each R.sup.x,
independently of the other R.sup.x, denotes H or
C.sub.2-C.sub.6-alkanoyl, preferably H or acetyl.
[0333] Particularly preferred are the following compounds of
formulae [1a], [12a], [13a], [18a], [19a], [20a], and [21a],
mixtures thereof, and the physiologically acceptable salts
thereof
##STR00038## ##STR00039## ##STR00040##
[0334] Particularly preferred are the following compounds of
formulae [1b], [12b], [13b] and [18b], mixtures thereof, and the
physiologically acceptable salts thereof
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047##
[0335] Particularly preferred are the following compounds of
formulae [1c], [12c], [13c] and [18c], mixtures thereof, and the
physiologically acceptable salts thereof
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
##STR00053## ##STR00054##
[0336] In a preferred embodiment, the present invention relates to
a composition comprising or consisting of three, four, five, six,
seven, eight, nine, ten, eleven, twelve or more compounds of
formula I,
wherein the total amount of said compounds of formula I is greater
than 75 wt. %, preferably greater than 80 wt. %, more preferably
greater than 85 wt. %, particularly preferably greater than 90 wt.
%, in each case based on the total weight the composition, and
preferably [0337] a total amount of 90 wt. % or less, more
preferably of 75 wt. % or less, even more preferably of 50 wt. % or
less, particularly preferably of 25 wt. % or less, especially
preferably of 10 wt. % or less, and most preferably of 5 wt. % or
less of liquid (at 25.degree. C. and 1013 mbar) diluents, in
particular water, in each case based on the total weight of the
composition, and/or [0338] a total amount of 5 wt. % or less, more
preferably of 2 wt. % or less, particularly preferably of 1.25 wt.
% or less, and most preferably of 0.8 wt. % or less of proteins, in
each case based on the total weight of the composition, and/or
[0339] a total amount of 5 wt. % or less, more preferably of 3 wt.
% or less, particularly preferably of 2 wt. % or less of sugar
alcohols and mono- or disaccharides, in each case based on the
total weight of the composition, and/or [0340] a total amount of 5
wt. % or less, more preferably of 4 wt. % or less, even more
preferably of 3 wt. % or less, particularly preferably of 2 wt. %
or less, most preferably of 1 wt. % or less of cells and cell
material with a size in at least one dimension of greater than 3
micrometer (.mu.m), preferably with a size in at least one
dimension of greater than 2 .mu.m, more preferably with a size in
at least one dimension of greater than 1 .mu.m, of fungi of the
Dacrymycetaceae family, in each case based on the total weight of
the composition.
[0341] In a particularly preferred embodiment, the present
invention relates to a composition comprising or consisting of one
or more, preferably two, three, four, five, six, seven, eight,
nine, ten, eleven, twelve or more physiologically acceptable salts
of one or more, preferably two, three, four, five, six, seven,
eight, nine, ten, eleven, twelve or more compounds of formula
I,
wherein the total amount of said physiologically acceptable salts
of the compounds of formula I is greater than 70 wt. %, preferably
greater than 80 wt. %, more preferably greater than 90 wt. %,
particularly preferably greater than 95 wt. %, in each case based
on the total amount of compounds of formula I and the
physiologically acceptable salts thereof, and preferably [0342] a
total amount of 90 wt. % or less, more preferably of 75 wt. % or
less, even more preferably of 50 wt. % or less, particularly
preferably of 25 wt. % or less, especially preferably of 10 wt. %
or less, and most preferably of 5 wt. % or less of liquid (at
25.degree. C. and 1013 mbar) diluents, in particular water, in each
case based on the total weight of the composition, and/or [0343] a
total amount of 5 wt. % or less, more preferably of 3 wt. % or
less, particularly preferably of 2 wt. % or less, and most
preferably of 1 wt. % or less of proteins, in each case based on
the total weight of the composition, and/or [0344] a total amount
of 20 wt. % or less, more preferably of 15 wt. % or less,
particularly preferably of 10 wt. % or less, most preferably of 5
wt. % or less of sugar alcohols and mono- or disaccharides, in each
case based on the total weight of the composition, and/or [0345] a
total amount of 5 wt. % or less, more preferably of 4 wt. % or
less, even more preferably of 3 wt. % or less, particularly
preferably of 2 wt. % or less, most preferably of 1 wt. % or less
of cells and cell material with a size in at least one dimension of
greater than 10 micrometer (.mu.m), preferably with a size in at
least one dimension of greater than 5 .mu.m, more preferably with a
size in at least one dimension of greater than 3 .mu.m, most
preferably with a size in at least one dimension of greater than 2
.mu.m, of fungi of the Dacrymycetaceae family, in each case based
on the total weight of the composition.
[0346] In a especially preferred embodiment, the present invention
relates to a composition comprising or consisting of three, four,
five, six, seven, eight, nine, ten, eleven, twelve or more
physiologically acceptable salts of one or more, preferably two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve or
more compounds of formula I,
wherein the total amount of said physiologically acceptable salts
of the compounds of formula I is greater than 85 wt. %, more
preferably greater than 90 wt. %, particularly preferably greater
than 95 wt. %, in each case based on the total amount of compounds
of formula I and the physiologically acceptable salts thereof, and
additionally [0347] a total amount of 25 wt. % or less, preferably
of 10 wt. % or less, and particularly preferably of 5 wt. % or less
of water, in each case based on the total weight of the
composition, [0348] a total amount of 2 wt. % or less, preferably
of 1 wt. % or less of proteins, in each case based on the total
weight of the composition, [0349] a total amount of 10 wt. % or
less, most preferably of 5 wt. % or less of sugar alcohols and
mono- or disaccharides, in each case based on the total weight of
the composition, and [0350] a total amount of 2 wt. % or less, most
preferably of 1 wt. % or less of cells and cell material with a
size in at least one dimension greater than 2 .mu.m, more
preferably with a size in at least one dimension of greater than 1
.mu.m, most preferably with a size in at least one dimension of
greater than 0.7 .mu.m of fungi of the Dacrymycetaceae family, in
each case based on the total weight of the composition.
[0351] Preferably, the determination of the total amount of
nitrogen and the total protein content is performed according to
the Kjeldahl method, preferably according to ISO 5549: 1978.
[0352] It was found in our own investigations that said preferred
or particularly preferred compositions according to the present
invention comprising one or more physiologically acceptable salts
of one or more compounds of formula I show improved, in particular
longer, storage stability in comparison to the corresponding
compounds of formula I in free acid form. It was also found in our
own investigations that such compositions have a superior, i.e.
higher, solubility in aqueous-alcoholic solvents or water in
comparison to the corresponding compounds of formula I in free acid
form. For example, while it is not possible to obtain a stable 1
wt. % solution of a mixture of compounds of formula I in free acid
form in water, it is easily possible to produce a 10 wt. % solution
of a mixture of compounds of formula I in their salt form. Thus,
the one or more physiologically acceptable salts of one or more
compounds of formula I have excellent formulation properties, in
particular regarding aqueous food, beverage and cosmetic materials,
in particular with a water content of 50 wt. % or more, which are
superior in comparison to the corresponding compounds of formula I
in free acid form. Additionally, said compositions and solutions
showed very good skin compatibility.
[0353] Particularly preferably, the trisaccharide carbohydrate
moiety R in the compounds of formula I--without including any
substituents resulting from acylation of hydroxyl groups of said
trisaccharide carbohydrate moiety R--is a
beta-D-glucopyranosyl-(1.fwdarw.2)-beta-D-xylopyranosyl-(1.fwdarw.2)-beta-
-D-xylopyranoside moiety. Further, of said trisaccharide
carbohydrate moiety R preferably one, two, three or four hydroxyl
groups are esterified by a C.sub.2-C.sub.10-alkanoic acid, i.e.
said trisaccharide carbohydrate moiety R being in mono- or di- or
tri- or tetra-acylated form (i.e. esterified with an
C.sub.2-C.sub.10-alkanoic acid). It was found in our own
investigations that compounds of formula I without any acyl
substituents in the trisaccharide carbohydrate moiety R (such as
compound [16]) showed significantly inferior antimicrobial
activities, in particular regarding Gram-positive bacteria, but
also to a noticeable extent regarding yeasts and molds.
[0354] Therefore, a mixture according to the present invention, an
extract according to the present invention, a material according to
the present invention, and/or a composition according to the
present invention preferably comprises less than 15 wt. % of
compounds of formula I without any acyl substituents in the
trisaccharide carbohydrate moiety R (particularly compound [16]),
more preferably less than 10 wt. %, particularly preferably less
than 5 wt. %, in each case based on the total amount of compounds
of formula I and the physiologically acceptable salts thereof.
[0355] In a particularly preferred embodiment relates to the
compounds of the formula I carrying one, two, three or four
acylated hydroxyl groups in the trisaccharide carbohydrate moiety
R, said acyl moiety preferably being a C.sub.3-C.sub.6-alkanoic
acid, more preferably an isovaleryl (3-methyl-butanoyl) moiety.
These compounds were found to exhibit superior antimicrobial
activity regarding certain Gram-positive bacteria and regarding
fungi, yeasts and molds.
[0356] Preferably, such a mixture comprises, essentially consists
of or consists of alkali and/or alkaline earth salts of two or more
compounds of formula I, more preferably of sodium and/or potassium
and/or calcium and/or magnesium salts thereof, in particular the
sodium and/or potassium and/or calcium and/or magnesium salts of
one, two, three, four, five, six, seven, eight, nine or more of the
compounds selected from the group consisting of compounds [1], [4],
[5], [6], [7], [8], [9], [10], [12], [13], [14], and [18], wherein
the total amount of water preferably is less than 5 wt. %, more
preferably less than 3 wt. %, and particularly preferably less than
1 wt. %, in each case based on the total weight of the mixture.
[0357] Preferably such a mixture comprises the sodium and/or
potassium and/or calcium and/or magnesium salts of one, two, three,
four, five, six, seven, eight or more of the compounds selected
from the group consisting of [1], [4], [5], [6], [7], [8], [9],
[10], [12], [13], [14], and [18], wherein the total amount of salts
of the compounds of formula I is greater than 70 wt. %, preferably
greater than 75 wt. %, more preferably greater than 80 wt. %,
particularly preferably greater than 85 wt. %, in each case based
on the total weight of the mixture.
[0358] Particularly preferably such a mixture comprises the sodium
and/or potassium and/or calcium and/or magnesium salts of one, two,
three, four or all of the compounds selected from the group
consisting of compounds [1], [7], [12], [13], and [18], wherein the
total amount of said salts of the compounds [1], [7], [12], [13],
and [18] is greater than 10 wt. %, preferably greater than 15 wt.
%, more preferably greater than 20 wt. %, in each case based on the
total weight of the mixture.
[0359] Preferably, such a mixture according to the present
invention is either in solid (at 25.degree. C. and 1013 mbar) form,
preferably in powder form with a total residual water content of 5
wt. % or less, preferably of 3 wt. % or less, more preferably of 1
wt. % or less, or is in the form of an aqueous or aqueous-alcoholic
solution, wherein the total amount compounds of formula I and the
physiologically acceptable salts thereof is in the range of 1 to 40
wt. %, more preferably in the range of 2 to 33 wt. %, even more
preferably in the range of 3 to 25 wt. %, and most preferably in
the range of 5 to 20 wt. %, in each case based on the total weight
of the mixture.
[0360] Further purification of an extract according to the present
invention, a mixture according to the present invention, and in
particular of a composition comprising or consisting of one or more
physiologically acceptable salts of one or more compounds of
formula I (as defined above) may be further purified according to
methods and materials described in U.S. Pat. No. 6,051,212, WO
96/38057 and/or JP 2006-176438 A, preferably centrifugation and/or
filtration (including ultrafiltration and/or microfiltration),
preferably using one or more sorbent (absorbent or adsorbent)
materials selected from the group consisting of activated carbons,
charcoal, ion exchange resins (preferably a weakly basic or weakly
acidic ion exchange resin, macroporous ion exchange resins in turn
being preferred), silica, alumina, kieselgur (diatomaceous earth,
e.g. celite), glass particles, glass wool, glass fibers, zeolites
(such as zeolite A, zeolite X, zeolite Y), silicates and
aluminosilicates (preferably clays and clay minerals like
bentonite, kaolinite, montmorillonite, smectite, illite,
chlorite).
[0361] In a further aspect the present invention relates to a
method of producing an extract comprising a compound of formula I
or a mixture of two or more compounds of formula I (for use)
according to the present invention, in particular a mixture
comprising one or more compounds selected from the group consisting
of compounds [1], [2], [3], [4], [5], [6], [7], [8], [9], [10],
[11], [12], [13], [14], [18], [19], [20], and [21], comprising the
following steps: [0362] preferably providing a fungus of the family
Dacrymycetaceae, preferably a fungus of the genera Dacryopinax,
Dacrymyces, Ditiola, Femsjonia or Guepiniopsis, more preferably a
fungus of the species Dacryopinax spathularia, [0363] carrying out
a fermentation process such that one, two or more compounds of
formula I are produced (preferably by said fungus of the family
Dacrymycetaceae), [0364] setting the pH value of the fermentation
broth to a value below 4, preferably to a pH value in the range of
from 1 to 3.5, more preferably to a pH value in the range of from
1.5 to 3, [0365] keeping the resulting reaction mixture at a pH
value below 4, preferably at a pH value in the range of from 1 to
3.5, more preferably to a pH value in the range of from 1.5 to 3,
thereby partially or essentially precipitating one, two or more
compounds of formula I, and [0366] washing the resulting
precipitate comprising, essentially consisting of or consisting of
one, two or more compounds of formula I, preferably with an aqueous
diluent, more preferably with water, particularly preferably with
demineralized water, [0367] optionally suspending the precipitate
comprising, essentially consisting of or consisting of one, two or
more compounds of formula I in an aqueous diluent, more preferably
in water, particularly preferably in demineralized water, [0368]
optionally removing water from the resulting product, preferably
freeze-drying the resulting product, thereby preferably yielding an
extract comprising, essentially consisting of or consisting of one,
two or more compounds of formula I in solid (and preferably
essentially water-free) form.
[0369] In a further aspect the present invention relates to a
method of producing one or more physiologically acceptable salts of
one or more compounds of formula I according to the present
invention, in particular one or more physiologically acceptable
salts of one, two or more compounds selected from the group
consisting of compounds [1], [2], [3], [4], [5], [6], [7], [8],
[9], [10], [11], [12], [13], [14], [18], [19], [20], and [21],
comprising the following steps: [0370] providing a fungus of the
family Dacrymycetaceae, preferably a fungus of the genera
Dacryopinax, Dacrymyces, Ditiola, Femsjonia or Guepiniopsis, more
preferably a fungus of the species Dacryopinax spathularia, [0371]
carrying out a fermentation process such that a compound of formula
I or a mixture of two or more compounds of formula I is produced by
said fungus, [0372] setting the pH value of the fermentation broth
to a value below 4, preferably to a pH value in the range of from 1
to 3.5, more preferably to a pH value in the range of from 1.5 to
3, [0373] keeping the resulting reaction mixture at a pH value
below 4, preferably at a pH value in the range of from 1 to 3.5,
more preferably to a pH value in the range of from 1.5 to 3,
thereby partially or essentially precipitating a compound of
formula I or a mixture of two or more compounds of formula I,
[0374] preferably washing the resulting precipitate (pellet)
preferably with an aqueous diluent, more preferably with water,
particularly preferably with demineralized water, [0375] suspending
the resulting precipitate (pellet) in an aqueous diluent, more
preferably in water, particularly preferably in demineralized
water, [0376] setting the pH value of the suspension to a value in
the range of 4.5 to 7.5, preferably in the range of 5 to 7,
particularly preferably in the range of 5.5 to 6.5, by adding an
inorganic base (solution or suspension), preferably sodium
hydroxide, potassium hydroxide, calcium hydroxide, and/or magnesium
hydroxide, and [0377] removing water from the resulting product,
preferably drying, particularly freeze-drying the resulting
product, thereby obtaining one or more physiologically acceptable
salts of one or more compounds of formula I according to the
present invention as a solid, preferably a powder, and preferably
in essentially water-free form.
[0378] Preferably, the fermentation is carried out in the absence
of an effective amount of visible light (i.e. light with a
wavelength in the range 380 to 750 nm), more preferably in the
absence of an effective amount of visible light and ultraviolet
light, most preferably in the absence of an effective amount of
light. Due to such measure the production of carotenoids, in
particular beta-carotene, is minimized or avoided (in contrast to
U.S. Pat. No. 2,974,044).
[0379] Preferably, the fermentation is carried out with one or more
fungi selected from the group consisting of
Dacryopinax aurantiaca, Dacryopinax crenata, Dacryopinax dennisii,
Dacryopinax elegans, Dacryopinax felloi, Dacryopinax fissus,
Dacryopinax foliacea, Dacryopinax formosus, Dacryopinax imazekiana,
Dacryopinax indacocheae, Dacryopinax lowyi, Dacryopinax macrospora,
Dacryopinax martinii, Dacryopinax maxidorii, Dacryopinax
parmastoensis, Dacryopinax petaliformis, Dacryopinax spathularia,
Dacryopinax sphenocarpa, Dacryopinax taibaishanensis, Dacryopinax
xizangensis, Dacryopinax yungensis, Dacrymyces ancyleus, Dacrymyces
aureosporus, Dacrymyces australis, Dacrymyces capitatus, Dacrymyces
chrysocomus, Dacrymyces chrysospermus, Dacrymyces cupularis,
Dacrymyces dictyosporus, Dacrymyces enatus, Dacrymyces
flabelliformis, Dacrymyces intermedius, Dacrymyces Dacrymyces
macnabbii, Dacrymyces minor, Dacrymyces novae-zelandiae, Dacrymyces
ovisporus, Dacrymyces paraphysatus, Dacrymyces pinacearum,
Dacrymyces punctiformis, Dacrymyces stillatus, Dacrymyces
subarcticus, Dacrymyces tortus, Dacrymyces variisporus, Ditiola
abieticola, Ditiola brasiliensis, Ditiola coccinea, Ditiola nuda,
Ditiola oblique, Ditiola orientalis, Ditiola pezizaeformis, Ditiola
radicata, Guepiniopsis alpina, Guepiniopsis buccina, Guepiniopsis
estonica, Guepiniopsis oresbia, Guepiniopsis ovispora, Guepiniopsis
pedunculata, and Guepiniopsis suecica.
[0380] In a preferred embodiment, a material according to the
present invention comprising an above defined (preferred or
particularly preferred) total amount of water are selected from the
group consisting of an O/W-emulsion, a hydrodispersion, a
suspension, a solution, or a hydrogel.
[0381] Preferably, a material according to the present invention
comprises a high proportion of water, preferably water in a total
amount of 50 wt. % or more, more preferably of 60 wt. % or more,
even more preferably 65 wt. % or more, particularly preferably 70
wt. % or more, and most preferably 75 wt. % or more, in each case
based on the total weight of the material.
[0382] Preferably, the total amount of water is in the range of 70
to 99.5 wt. %, more preferably in the range of 75 to 99 wt. %, and
most preferably in the range of 80 to 98 wt. %, in each case based
on the total weight of the material.
[0383] The total amount of water of an orally consumable material
according to the present invention, in particular of a
ready-to-drink composition according to the present invention, is
60 wt. % or more, preferably 70 wt. % or more, more preferably 75
wt. % or more, even more preferably 80 wt. % or more, in each case
based on the total weight of the orally consumable material.
[0384] Preferably, the total amount of water, preferably of
non-deionized water, particularly of drinking water or mineral
water, of an orally consumable material according to the present
invention, in particular of a ready-to-drink composition according
to the present invention, is in the range of from 82 wt. % to 98
wt. %, more preferably in the range of from 83 wt. % to 96 wt. %,
even more preferably in the range of from 84 wt. % to 95 wt. %, and
most preferably in the range of from 85 wt. % to 94 wt. %, in each
case based on the total weight of the orally consumable
material.
[0385] In a preferred embodiment, a material according to the
present invention comprising an above defined (preferred or
particularly preferred) total amount of water has a pH-value at
25.degree. C. of 6.8 or less, preferably in the range of 1.5 to
6.5, more preferably in the range of 2.0 to 6.0, even more
preferably in the range of 2.1 to 5.8, particularly preferably in
the range of 2.2 to 5.0, and most preferably in the range of 2.3 to
4.5.
[0386] The pH-value of an orally consumable material according to
the present invention, in particular of a ready-to-drink
composition according to the present invention, when measured at
25.degree. C. (and preferably at 1013 mbar) preferably is in the
range of from 1.5 to 6.5, preferably in the range of from 1.8 to
6.0, more preferably in the range of from 2.0 to 5.5, even more
preferably in the range of from 2.0 to 5.0, particularly preferably
in the range of from 2.1 to 4.4, especially preferably in the range
of from 2.2 to 4.2, and most preferably in the range of from 2.3 to
3.9.
[0387] It should be emphasized that the one or more compounds of
the above formula I, one or more physiologically acceptable salts
of a compound of the above formula I, or a mixture thereof are
stable (preferably at temperatures of 40.degree. C. or lower) for a
long period of time (generally more than 16 weeks at 25.degree. C.)
in such aqueous materials at the acidic pH-values indicated
hereinbefore, i.e. the compounds of the above formula I are not
decomposed or degraded to an appreciable or significant extent.
[0388] In a preferred embodiment, a material according to the
present invention (preferably comprising an above defined preferred
or particularly preferred total amount of water and having a
pH-value in a preferred or particularly preferred range indicated
above) suitable for oral consumption, preferably a food or a
beverage, comprises [0389] one or more organic food acids and/or
the physiologically acceptable salt thereof, preferably selected
from the group consisting of acetic acid, adipic acid, caffeotannic
acid, citric acid, iso-citric acid, maleic acid, fumaric acid,
galacturonic acid, glucuronic acid, glyceric acid, glycolic acid,
lactic acid, malic acid, oxalic acid, pyruvic acid, quinic acid,
succinic acid, tannic acid, tartaric acid, and the physiologically
acceptable salts thereof, preferably the sodium and/or potassium
and/or calcium and/or magnesium salts thereof thereof, and/or
[0390] one or more edible inorganic acids and/or the
physiologically acceptable salt thereof, preferably selected from
the group consisting of phosphoric acid, pyrophosphoric acid,
polyphosphoric acids, and bisphosphonic acids (in particular those
explicitly mentioned in paragraph [0050] of US 2010/0151104 A1),
and the physiologically acceptable salts thereof, and/or [0391] one
or more high potency sweeteners, preferably selected from the group
consisting of magap, sodium cyclamate, acesulfame K, neohesperidin
dihydrochalcone, saccharin sodium salt, aspartame, superaspartame,
neotame, alitame, sucralose, stevioside, rebaudiosides (preferably
rebaudioside A), lugduname, carrelame, sucrononate, sucrooctate,
monatin, phyllodulcin, hernandulcin, dihydrochalcone glycosides,
glycyrrhizin, glycyrrhetinic acid and its sweet tasting
physiologically acceptable salts, preferably glycyrrhetinic acid
ammonium salt, mogrosides, liquorice extracts (Glycyrrhizza glabra
ssp.), Lippia dulcis extracts, Momordica ssp. extracts (in
particular Momordica grosvenori [Luo Han Guo]), Hydrangea dulcis
extracts and Stevia ssp. (e.g. Stevia rebaudiana) extracts, [0392]
preferably one or more high potency sweeteners in a total amount
isosweet to or sweeter than a 1.0 wt. % solution of sucrose in
water, more preferably in a total amount isosweet to or sweeter
than a 2.0 wt. % solution of sucrose in water, even more preferably
in a total amount isosweet to or sweeter than a 3.0 wt. % solution
of sucrose in water, particularly preferably in a total amount
isosweet to or sweeter than a 4.0 wt. % solution of sucrose in
water, and/or [0393] one or more sweet tasting mono- or
disaccharides, preferably selected from the group consisting of
sucrose, lactose, maltose, glucose, and fructose, the total amount
of said sweet tasting mono- or disaccharides being in the range of
1.5 to 19 wt. %, preferably in the range of 2.5 to 16 wt. %, more
preferably in the range of 3.5 to 14 wt. %, particularly preferably
in the range of 4.5 to 13 wt. %, and most preferably in the range
of 5.5 to 12 wt. %, in each case based on the total weight of the
material, and preferably one, two, three, four, five or more
flavouring agents, preferably having a molecular weight in the
range of 120 to 300 g/mol, more preferably in the range of 130 to
280 g/mol.
[0394] The one or more compounds of formula I and/or the
physiologically acceptable salts thereof, especially as defined in
one of the preferred or particularly preferred embodiments, and the
mixtures as defined in one of the preferred or particularly
preferred embodiments, allow the prevention of spoilage of
materials with such a high proportion of water (and preferably a
pH-value at 25.degree. C. in the range of 2.2 to 4.6) by
microorganisms, within a sealed container for a period of at least
12 weeks, preferably at least 16 weeks at 25.degree. C. Thus, a
reduction or substitution of conventional preservatives (that may
pose health and/or environmental concerns) is possible.
[0395] The one or more compounds of formula I and/or the
physiologically acceptable salts thereof, especially as defined in
one of the preferred or particularly preferred embodiments, and the
mixtures as defined in one of the preferred or particularly
preferred embodiments, allow to be used together in other known
beverage preserving agents in an additive or synergistic manner to
reduce the amount of preservative required and so improve the
inventive beverage's sensory impact over beverages having
conventional preservatives. Such other known beverage preserving
agents are preferably selected from the group consisting of
ethyl-N-alpha-lauroyl-L-arginate (LAE) and its hydrochloride,
dimethyl dicarbonate, trans-cinnamic acid, EDTA (ethylene diamine
tetraacetic acid) and its physiologically acceptable salts,
preferably the sodium and/or calcium salts thereof, EDDS (ethylene
diamine-N,N'-disuccinic acid) and its physiologically acceptable
salts, preferably the sodium and/or calcium salts thereof,
polyphosphoric acid and its physiologically acceptable salts
(preferably comprising or consisting of sodium hexametaphosphate),
bisphosphonic acids and bis-phosphonates (in particular those
explicitly mentioned in paragraph [0050] of US 2010/0151104 A1),
and the mixtures thereof.
[0396] The total amount of ethyl-N-alpha-lauroyl-L-arginate and its
hydrochloride preferably is in the range of 1 to 25 ppm, more
preferably 2 to 12 ppm, based on the total weight of the
beverage.
[0397] The total amount of dimethyl dicarbonate preferably is in
the range of 20 to 500 ppm, more preferably 50 to 250 ppm, based on
the total weight of the beverage.
[0398] The total amount of trans-cinnamic acid preferably is in the
range of 1 to 40 ppm, more preferably 2 to 30 ppm, based on the
total weight of the beverage.
[0399] The total amount of EDTA (ethylene diamine tetraacetic acid)
and its physiologically acceptable salts is in the range of 0.5 to
50 ppm, more preferably 1 to 30 ppm, based on the total weight of
the beverage.
[0400] The total amount of EDDS (ethylene diamine-N,N'-disuccinic
acid) and its physiologically acceptable salts is in the range of 1
to 500 ppm, more preferably 20 to 450 ppm, based on the total
weight of the beverage.
[0401] The total amount of polyphosphoric acid and its
physiologically acceptable salts is in the range of 10 to 1500 ppm,
based on the total weight of the beverage.
[0402] As already mentioned above, the compounds of formula I,
preferably the compounds [1], [2], [3], [4], [5], [6], [7], [8],
[9], [10], [11], [12], [13], [14], [18], [19], [20], [21], and the
physiologically acceptable salts thereof do not exhibit a
distinctive taste or unpleasant mouth feeling, in particular no
off-flavours, in particular in the total amounts used in the
ready-to-use product.
[0403] The total amount of the compounds of formula I, preferably
of the compounds [1], [2], [3], [4], [5], [6], [7], [8], [9], [10],
[11], [12], [13], [14], [18], [19], [20], [21] and the
physiologically acceptable salts thereof, more preferably of the
compounds [1], [4], [5], [6], [7], [8], [9], [10], [12], [13],
[14], [18], and the physiologically acceptable salts thereof,
preferably is in the range of 0.1 to 1000 ppm, more preferably of
0.5 to 500 ppm, particularly preferably of 1 to 250 ppm, and most
preferably of 2 to 150 ppm, in each case based on the total weight
of the orally consumable material according to the present
invention, particularly a food or beverage according to the present
invention.
[0404] Therefore, the compounds of formula I and the
physiologically acceptable salts thereof are preferably used in
orally consumable materials, particularly foods or beverages,
according to the invention, in combination with one or more
flavouring agents, preferably having a molecular weight in the
range of 120 to 300 g/mol, more preferably in the range of 130 to
280 g/mol.
[0405] Preferably, one, two, three or more of said flavouring
agents are fresh, sweet, fruity, spicy and/or herbal flavouring
agents, preferably selected from the group consisting of menthol
(preferably L-menthol, D-menthol, racemic menthol, isomenthol,
neoisomenthol, neomenthol), isomenthone, menthone, peppermint oil,
L-carvone, D-carvone, spearmint oil, cineol, eucalyptus oil,
cinnamaldehyde (preferably trans-cinnamaldehyde), cinnamic alcohol,
cinnamon bark oil, cinnamon leaf oil, methyl cinnamate,
benzaldehyde, furfural, furfuryl alcohol, methyl salicylate,
wintergreen oil, thyme oil, thymol, carvacrol, clove oil, camphene,
p-cymene, alpha-terpinene, borneol, eugenol, anise oil, star anise
oil, anethole (preferably trans-anethole), anisole, cis-3-hexenol,
cis-3-hexenyl acetate, D-limonene, L-limonene, linalool, citral,
geraniol, geranyl acetate, nerol, citronellol, citronellal,
alpha-phellandrene, beta-phellandrene, alpha-pinene, beta-pinene,
vanilla extract, vanillin, ethylvanillin,
2-hydroxy-4-methoxybenzaldehyde,
2,5-dimethyl-4-hydroxy-3(2H)-furanone,
2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone,
2-ethyl-5-methyl-4-hydroxy-3(2H)-furanone,
5-ethyl-2-methyl-4-hydroxy-3(2H)-furanone,
3-hydroxy-4,5-dimethyl-2(5H)-furanone, maltol, ethylmaltol,
coumarin, butyrolactone, gamma-undecalactone, gamma-nonalactone,
4-methyl-delta-lactone, massoia lactone, sotolon,
delta-decalactone, tuberolactone, methyl sorbate,
2-hydroxy-3-methyl-2-cyclopentenones, n-butyl acetate, isoamyl
acetate, ethyl butyrate, n-butyl butyrate, isoamyl butyrate, ethyl
3-methyl-butyrate, ethyl n-hexanoate, allyl n-hexanoate, n-butyl
n-hexanoate, ethyl n-octanoate, ethyl-3-methyl-3-phenylglycidate,
ethyl-2-trans-4-cis-decadienoate, 4-(p-hydroxyphenyl)-2-butanone,
1,1-dimethoxy-2,2,5-trimethyl-4-hexane, 2,6-dimethyl-5-hepten-1-al,
and phenylacetaldehyde.
[0406] Preferably, one, two, three or more of said flavouring
agents are sweet, fruity and/or spicy flavouring agents, preferably
selected from the group consisting of trans-cinnamaldehyde,
cinnamic alcohol, methyl cinnamate, benzaldehyde, furfural,
furfuryl alcohol, camphene, p-cymene, alpha-terpinene, borneol,
eugenol, trans-anethole, anisole, cis-3-hexenol, cis-3-hexenyl
acetate, D-limonene, L-limonene, linalool, citral, geraniol,
geranyl acetate, nerol, citronellol, citronellal,
alpha-phellandrene, beta-phellandrene, alpha-pinene, beta-pinene,
vanilla extract, vanillin, ethylvanillin,
2-hydroxy-4-methoxybenzaldehyde,
2,5-dimethyl-4-hydroxy-3(2H)-furanone,
2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone,
2-ethyl-5-methyl-4-hydroxy-3(2H)-furanone,
5-ethyl-2-methyl-4-hydroxy-3(2H)-furanone,
3-hydroxy-4,5-dimethyl-2(5H)-furanone, maltol, ethylmaltol,
coumarin, gamma-undecalactone, gamma-nonalactone,
4-methyl-delta-lactone, massoia lactone, sotolon,
delta-decalactone, tuberolactone, methyl sorbate, n-butyl acetate,
isoamyl acetate, ethyl butyrate, n-butyl butyrate, isoamyl
butyrate, ethyl 3-methyl-butyrate, ethyl n-hexanoate, allyl
n-hexanoate, n-butyl n-hexanoate, ethyl n-octanoate,
ethyl-3-methyl-3-phenylglycidate, ethyl-2-trans-4-cis-decadienoate,
4-(p-hydroxyphenyl)-2-butanone, 2,6-dimethyl-5-hepten-1-al, and
phenylacetaldehyde.
[0407] The present invention also relates to foods, such as meat,
meat products, fish and seafood products, with an increased shelf
life stability and an increased resistance against the growth of
Gram-positive bacteria. The preparation process for manufacturing
foodstuffs using the compounds of formula I and/or the
physiologically acceptable salts thereof according to the invention
comprises, for example, combining an uncooked meat, meat products,
fish or seafood product with one or more phosphates, one or more
lactates, lactic acid, and preferably further and/or a flavouring
agent, followed by further processing such as packing or cooking.
In this specific aspect, the present invention makes use of
phosphates which are functional in protein activation of meat, meat
products, fish or seafood and have also properties to absorb
lactates and flavouring agents.
[0408] In this context the present invention relates to a foodstuff
treatment composition, particularly for the treatment of meat, meat
products, fish and seafood products, said composition comprising
(i) one or more compounds of formula I and/or the physiologically
acceptable salts thereof, (ii) lactic acid and/or lactates, and
(iii) one or more phosphates.
[0409] In a preferred embodiment of the invention said foodstuff
treatment composition comprises
(i) one, two, three or more of compounds [1], [2], [3], [4], [5],
[6], [7], [8], [9], [10], [11], [12], [13], [14], [18], [19], [20],
[21] and/or the physiologically acceptable salts thereof,
preferably in a total amount of 0.0001 to 1 wt. %, more preferably
in a total amount of 0.0005 to 0.5 wt. %, even more preferably in a
total amount of 0.001 to 0.1 wt. %, particularly preferably in a
total amount of 25 to 500 ppm, (ii) sodium lactate and/or potassium
lactate, preferably in a total amount of 0.5 to 25 wt. %, more
preferably in a total amount of 1 to 20 wt. %, even more preferably
in a total amount of 1.5 to 15 wt. %, (iii) one or more sodium
and/or potassium phosphates selected from the group consisting of
sodium and/or potassium orthophosphates, pyrophosphates
(diphosphates), metaphosphates, and polyphosphates
(hexametaphosphates), preferably in a total amount of 1 to 45 wt.
%, more preferably in a total amount of 2 to 30 wt. %, even more
preferably in a total amount of 3 to 25 wt. %, (iv) preferably
water, more preferably water in a total amount of 50 wt. % or more,
even more preferably in a total amount of 60 wt. % or more, and
optionally (v) one or more further constituents selected from the
group consisting of sodium chloride, sodium nitrite, potassium
nitrite, sodium nitrate, potassium nitrate, calcium lactate, sodium
diacetate, acetic acid, sodium acetate, sodium diacetate, potassium
acetate, potassium diacetate citric acid, and sodium citrate,
wherein the amounts in each case are based on the total weight of
the foodstuff treatment composition.
[0410] In another preferred embodiment of the invention the one or
more sodium and/or potassium phosphate salts of constituent (iii)
are selected from trisodium phosphate (Na.sub.3PO.sub.4),
tetrasodium pyrophosphate (Na.sub.4P.sub.2O.sub.7), sodium
tripolyphosphate (Na.sub.5P.sub.3O.sub.10), tripotassium phosphate
(K.sub.3PO.sub.4), tetrapotassium pyrophosphate
(K.sub.4P.sub.2O.sub.7), potassium tripolyphosphate
(K.sub.5P.sub.3O.sub.10), and sodium hexametaphosphate
(NaPO.sub.3).sub.6.
[0411] These foodstuff treatment compositions are particularly
useful for the treatment of meat, meat products, fish and seafood
products, to increase the resistance of the foodstuff against the
growth of bacteria, in particular of the genera Listeria
(particularly Listeria monocytogenes), Lactobacillus, Clostridia,
Micrococcus (particularly Micrococcus luteus), and/or Bacillus
(particularly Bacillus cereus), whereby the foodstuff treatment
composition is applied to the food in an amount to achieve a total
amount of 0.25 to 6 wt %, preferably of 0.5 to 4 wt. %, of
constituents (ii) lactic acid and lactates and (iii) phosphates in
the final treated foodstuff.
[0412] In a preferred embodiment, a material according to the
present invention (preferably comprising an above defined preferred
or particularly preferred total amount of water and having a
pH-value in a preferred or particularly preferred range indicated
above) is a cosmetic product suitable for topical application onto
the mucous membrane (mucosa) and/or the epidermis of a mammal,
preferably in the form of an O/W-lotion, a milk, a (hydro)gel, a
body care and/or hair care product (such as preferably a shower gel
and/or a shampoo, a hair conditioning product, or a deodorant),
comprises [0413] one or more surfactants not corresponding to
formula I as defined in the context of the present invention,
preferably one or more surfactants selected from the group
consisting of anionic tensides, cationic tensides, non-ionic
tensides, amphoteric (zwitterionic) tensides, and biosurfactants,
and/or [0414] one or more mono-, di- or triols having 2 to 14
carbon atoms, preferably one or more di- or triols having 3 to 12
carbon atoms, wherein preferably the total amount of mono-, di- and
triols is 1 wt. % or more, more preferably in the range of 1.1 to
30 wt. %, and/or [0415] one or more fragrance substances,
preferably a mixture of three, five, eigh for more fragrance
substances, more preferably a perfume, preferably fragrance
substances in a total amount of 0.1 to 3 wt. %, more preferably in
a total amount of 0.15 to 2 wt. %, even more preferably in a total
amount of 0.2 to 1 wt. %, wherein the percentages in each case are
based on the total weight of the cosmetic product.
[0416] Preferably one, several or all mono-, di- or triols having 2
to 14 carbon atoms are selected from the group consisting of
ethanol, 1-propanol, 2-propanol, ethylene glycol, 1,2-propylene
glycol, glycerol (glycerin), 1,3-propandiol,
2-methyl-1,3-propandiol, trimethylolpropane, 1,2-butandiol,
1,3-butandiol, 1,4-butandiol, 1,2,3-butantriol, 1,2,4-butantriol,
1-pentanol, 2-pentanol, 3-pentanol, 1,2-pentandiol, 1,3-pentandiol,
1,5-pentandiol, 1-hexanol, 2-hexanol, 3-hexanol, 1,2-hexandiol,
1,3-hexandiol, dipropylene glycol, 1-octanol, 2-octanol, 3-octanol,
1,2-octandiol (caprylyl glycol), 1,3-octandiol,
2-methyl-5-cyclohexylpentanol, 2-methyl-4-phenyl-2-butanol,
4-methyl-4-phenyl-2-pentanol (dimethyl phenyl 2-butanol),
1-decanol, 2-decanol, 1,2-decandiol,
3-(2-ethylhexyloxy)propane-1,2-diol (ethylhexylglycerin,
octoxyglycerin), 1-dodecanol, 2-dodecanol, 1,2-dodecandiol,
1,12-dodecandiol, 1-tetradecanol, 2-tetradecanol,
1,2-tetradecandiol and 1,14-tetradecandiol, wherein preferably the
total amount of mono-, di- and triols having 2 to 14 carbon atoms
is 0.5 wt. % or more, more preferably 1.0 wt. % or more, even more
preferably 1.25 wt. % or more, and preferably is in the range of
1.25 to 25 wt. %, particularly in the range of 1.5 to 20 wt. %, in
each case based on the total weight of the cosmetic product.
[0417] Preferably one, several or all di- or triols having 3 to 12
carbon atoms are selected from the group consisting of
1,2-propylene glycol, glycerol (glycerin), 1,3-propandiol,
2-methyl-1,3-propandiol, trimethylolpropane
(2-(hydroxymethyl)-2-ethylpropane-1,3-diol), 1,2-butandiol,
1,4-butandiol, 1-pentanol, 2-pentanol, 1,2-pentandiol,
1,5-pentandiol, 1-hexanol, 2-hexanol, 1,2-hexandiol, dipropylene
glycol, 1-octanol, 2-octanol, 1,2-octandiol,
2-methyl-4-phenyl-2-butanol, 4-methyl-4-phenyl-2-pentanol,
1-decanol, 1,2-decandiol, 1-dodecanol, 1,2-dodecandiol,
1,12-dodecandiol, and 3-(2-ethylhexyloxy)propane-1,2-diol
(ethylhexylglycerin), wherein preferably the total amount of di-
and triols having 3 to 12 carbon atoms is 0.5 wt. % or more, more
preferably 1.0 wt. % or more, even more preferably is in the range
of 1.25 to 15 wt. %, particularly in the range of 1.5 to 10 wt. %,
in each case based on the total weight of the cosmetic product.
[0418] The anionic tensides, cationic tensides non-ionic tensides,
amphoteric (zwitterionic) tensides, and biosurfactants are
preferably selected from [0419] anionic tensides based on permanent
anions (sulfate, sulfonate, phosphate) or pH-dependent anions
(carboxylate), preferably sulfates [alkyl sulfates, such as
ammonium lauryl sulfate, sodium lauryl sulfate (SDS, sodium dodecyl
sulfate), alkyl ether sulfates, such as sodium laureth sulfate,
also known as sodium lauryl ether sulfate (SLES), sodium myreth
sulfate], sulfonates [docusates, such as dioctyl sodium
sulfosuccinate, sulfonate fluorosurfactants
(perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate), alkyl
benzene sulfonates], phosphates [alkyl aryl ether phosphate, alkyl
ether phosphate], carboxylates [alkyl carboxylates, fatty acid
salts (soaps), such as sodium stearate, sodium lauroyl sarcosinate,
carboxylate fluorosurfactants, such as perfluorononanoate,
perfluorooctanoate (PFOA or PFO)], [0420] cationic tensides based
on pH-dependent primary, secondary, or tertiary amines (such as
octenidine dihydrochloride, quaternary ammonium cations, preferably
alkyltrimethylammonium salts (such as cetyl trimethylammonium
bromide (hexadecyl trimethyl ammonium bromide), cetyl
trimethylammonium chloride, cetylpyridinium chloride, benzalkonium
chloride, benzethonium chloride, dimethyldioctadecylammonium
chloride and dioctadecyldimethylammonium bromide), [0421]
zwitterionic (amphoteric) tensides based on primary, secondary, or
tertiary amines or quaternary ammonium cation with sulfonates (such
as (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)
(CHAPS), sultaines (such as cocamidopropyl hydroxysultaine),
carboxylates (e.g. from amino acids, imino acids), betaines (such
as cocamidopropyl betaine), and phosphates (such as lecithins),
[0422] nonionic tensides, preferably fatty alcohols (such as cetyl
alcohol, stearyl alcohol, cetostearyl alcohol (essentially
consisting of cetyl and stearyl alcohols), oleyl alcohol),
polyoxyethylene glycol alkyl ethers
CH.sub.3--(CH.sub.2).sub.10-16--(O--C.sub.2H.sub.4).sub.1-25--OH
(such as octaethylene glycol monododecyl ether, pentaethylene
glycol monododecyl ether), polyoxypropylene glycol alkyl ethers
CH.sub.3--(CH.sub.2).sub.10-16--(O--C.sub.3H.sub.6).sub.1-25--OH,
glucoside alkyl ethers
CH.sub.3--(CH.sub.2).sub.10-16--(O-glucoside).sub.1-3-OH (such as
decyl glucoside, lauryl glucoside, octyl glucoside),
polyoxyethylene glycol octylphenol ethers
C.sub.8H.sub.17--(C.sub.6H.sub.4)--(O--C.sub.2H.sub.4).sub.1-25--OH
(such as Triton X-100), polyoxyethylene glycol alkylphenol ethers
C.sub.9H.sub.19--(C.sub.6H.sub.4)--(O--C.sub.2H.sub.4).sub.1-25--OH
(such as nonoxynol-9), glycerol alkyl esters (such as glyceryl
laurate), polyoxyethylene glycol sorbitan alkyl esters (such as
polysorbates, preferably polysorbate 20, polysorbate 40,
polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85
and/or polysorbate 120; these are all commercially available, e.g.
under the brand names Canarcel.RTM. or Tween.RTM.), sorbitan alkyl
esters, cocamide MEA, cocamide DEA, dodecyldimethylamine oxide,
block copolymers of polyethylene glycol and polypropylene glycol
(such as poloxamers (commercially available, e.g. under the brand
name Pluronic.RTM.)), and polyethoxylated tallow amine (POEA),
[0423] biosurfactants, preferably sophoroplipids, rhamnolipids,
mannosyl-erythritol lipids, and lipopeptides (preferred
lipopeptides are chlamydocin, surfactin, lichenysin G, and
fengycin-like lipopeptides).
[0424] Preferably, one, two, three, four, five or more fragrance
substances with one or more notes selected from the group fresh,
floral (flowery), aldehydic, watery, fruity, sweet, woody, musky,
green and herbal, more preferably with one or more notes selected
from the group floral (preferably rose and/or lily-of-the-valley
(muguet)), aldehydic, vanilla, citrus, sandalwood, and musk.
[0425] The epidermis refers to the outermost layers of cells in the
skin of a mammal, in particular certain parts of the body of a
human being, namely hand, arm, foot, head and axillary region
(particularly the armpit). The mucous membrane (mucosa) lines
cavities that are exposed to the external environment and internal
organs, particularly at the nostrils, the mouth (oral cavity), the
lips, the eyelids, and the genital area.
[0426] A cosmetic deodorant composition according to the present
invention preferably comprises [0427] an antimicrobial effective
amount of one or more compounds of formula I and/or the
physiologically acceptable salts thereof, preferably 2 ppm or more,
more preferably 5 ppm or more, even more preferably 10 ppm or more,
[0428] water, preferably in a total amount of 10 wt. % or more,
more preferably in a total amount of 50 wt. % or more, particularly
preferably in a total amount of 60 wt. % or more, [0429] one or
more alcohols selected from the group consisting of ethanol,
1,2-propylene glycol, glycerol (glycerin), 2-methyl-1,3-propandiol,
1,2-butandiol, 1,3-butandiol 1,4-butandiol, 1,2,4-butantriol,
1-pentanol, 1,2-pentandiol, 1,5-pentandiol, 1,2-hexandiol,
1-octanol, 1,2-octandiol, and 3-(2-ethylhexyloxy)propane-1,2-diol
(ethylhexylglycerin, octoxyglycerin), preferably in a total amount
of 1.5 wt. % or more, more preferably in a total amount of 2.5 wt.
% or more, wherein the amounts indicated in each case relate to the
total weight of the cosmetic deodorant composition.
[0430] Such a cosmetic deodorant composition according to the
present invention preferably additionally comprises one or more
constituents selected from the group consisting of antiperspirants,
fragrance substances, and further surfactants.
[0431] Antiperspirants inhibit the secretion of sweat. As
antiperspirants astringent metal salts are generally used, in
particular inorganic and organic metal salts of the elements
aluminum, zinc, magnesium, tin and zirconium as well as mixtures
thereof are used. Frequently, aluminum and zirconium salts and
their mixtures are also used in complex form, with propylene
glycol, polyethylene glycol or glycerin being used as complexing
agents. One ore more antiperspirants are preferably selected from
the group consisting of aluminium chlorohydrate; aluminium
sesquichlorohydrate, aluminium chlorohydrex propylene glycol,
aluminium dichlorohydrex propylene glycol, aluminium
sesquichlorohydrex propylene glycol, aluminium chlorohydrex
polyethylene glycol, aluminium dichlorohydrex polyethylene glycol,
aluminium sesquichlorohydrex polyethylene glycol, aluminium
chloride, aluminium zirconium chlorohydrate, aluminium zirconium
trichlorohydrate, aluminium zirconium tetrachlorohydrate, aluminium
zirconium pentachlorohydrate, aluminium zirconium
octachlorohydrate, aluminium zirconium trichlorohydrex-glycerin,
aluminium zirconium tetrachlorohydrex-glycerin, aluminium zirconium
pentachlorohydrex-glycerin, aluminium zirconium
octachlorohydrex-glycerin, basic aluminium chloride, zirconium
hydroxychloride, zirconium chloride.
[0432] Preferably one or more of the further surfactants are
selected from the group consisting of anionic tensides, cationic
tensides, non-ionic tensides, or amphoteric tensides explicitly
mentioned above.
[0433] Examples of preferred materials according to the present
invention, particularly orally consumable materials, are [0434]
fruit or vegetable containing products (preferably products
containing juice, extract, puree, mash, pulp, concentrate, dried
parts of lemon, lime, grapefruit, orange, sweet orange, bitter
orange, bergamot, mandarin, apple, pear, prickly pear, peach,
apricot, fig, pineapple, prune, mango, melon, plum, kiwi, lychee,
banana, cherry, sweet cherry, strawberry, raspberry, red currant,
black currant, blackberry, blueberry, marionberry, passion fruit,
grapes (white grape, red grape, green grape, purple grape),
pomegranate, acerola, tomato, carrot, parsnip, pumpkin, lettuce,
cabbage, fermeted cabbage, bean, pea, potato, bell pepper, red
chilli, green chilli, onion, celery, cucumber, leek, broccoli,
cauliflower, radish, aubergine, zucchini), [0435] soy based
products (preferably soy milk, soy drinks, soy yoghurts), [0436]
non-alcoholic beverages and syrups (preferably lemonades, beverage
concentrates (syrups), non-carbonated soft drinks, and carbonated
soft drinks), [0437] alcoholic beverages, [0438] products
containing 50 wt. % or more of water and one or more other extracts
from herbs and/or spices (preferably selected from the group
consisting of vanilla, cinnamon, anise, fennel, clove, cardamom,
tamarind, nutmeg, allspice, black pepper, licorice, ginger, rose
hip, green tea, red tea, rooibos tea, mate tea, honeybush tea,
pu-erh tea, oolong tea, black tea, coffee bean, cocoa bean,
peppermint, spearmint, and wintergreen), [0439] non-frozen
fermented or non-fermented dairy or dairy-based products
(preferably milk, quark, cream cheese, cheese, custards, puddings,
mousses, milk based drinks, drink yoghurts, and yoghurts), [0440]
frozen products (preferably ice-cream, frozen yoghurt, sorbet, ice
milk, frozen custard, water-ices, granitas, sherbets. and frozen
fruit purees), [0441] doughs and batters (preferably pancake
batter, wafle dough, cake doughs, bread dough, bun dough, pasta
doughs), [0442] O/W-emulsions (spreads, sauces, and (salad)
dressings).
[0443] In a preferred embodiment, the one or more compounds of
formula I and/or the physiologically acceptable salts thereof (for
use) according to the present invention, the extracts according to
the present invention are combined with one or more ingredients
selected from the group consisting of lactic acid, lactose,
sucrose, calcium salts (preferably calcium phosphate, calcium
gluconate, calcium lactate, and calcium chloride), calcium oxide,
magnesium salts, magnesium oxide, iron salts (preferably ferrous
fumarate, ferrous succinate, iron sucrate-malate, iron
fructate-malate, iron sucrate-citrate, iron fructatecitrate, iron
sucrate-ascorbate, iron fructate-ascorbate, and mixtures thereof),
vitamin A (particularly retinol (vitamin A1)), vitamin B6, vitamin
B12, vitamin C, vitamin D, vitamin E, thiamine, niacin, biotin,
riboflavin, pantothenic acid, phytic acid, daidzein, genistein,
proteins (preferably casein, caseinates (preferably sodium
caseinate), milk protein, milk protein hydrolyzate, milk protein
isolate, whey protein, whey protein hydrolyzate, whey protein
isolate, soy protein, soy protein hydrolyzate, soybean protein
isolate), milk powder, soy powder, polyunsaturated fatty acids
[preferably omega-3-, omega-6- and/or omega-9-fatty acids,
preferably selected from the group consisting of docosahexaenoic
acid (DHA, all-cis-docosa-4,7,10,13,16,19-hexaenoic acid),
eicosatetraenoic acid (ETA, all-cis-8,11,14,17-eicosatetraenoic
acid), eicosatetraenoic acid (ETA,
all-cis-8,11,14,17-eicosatetraenoic acid), stearidonic acid (SDA,
all-cis-6,9,12,15-octadecatetraenoic acid), docosapentaenoic acid
(DPA; clupanodonic acid, all-cis-7,10,13,16,19-docosapentaenoic
acid), linoleic acid, .alpha.-linolenic acid
(all-cis-9,12,15-octadecatrienoic acid), and .gamma.-linolenic
acid], soy oil, butterfat, (refined) fish oil, algal oil, squid
oil, flaxseed oil, grape seed oil, and triglycerides derived from
the fatty acids myristic acid, palmitic acid and/or oleic acid,
thereby forming preferred materials according to the present
invention, particularly materials suitable for oral
consumption.
[0444] Preferably, a material according to the present invention
(preferably comprising an above defined preferred or particularly
preferred total amount of water and having a pH-value in a
preferred or particularly preferred range indicated above) suitable
for oral consumption comprises a total amount of glutamic acid and
sodium glutamate of less than 0.2 wt. %, preferably of less than
0.15 wt. %, more preferably of less than 0.1 wt. %, particularly
preferably of less than 0.05 wt. %, and most preferably is free of
glutamic acid and sodium glutamate.
[0445] Examples of stabilizers and/or thickeners which may be part
of a (preferably orally consumable) material according to the
present invention are preferably selected from the group consisting
of carbohydrate polymers (polysaccharides, preferably starches,
polydextrose (E-number E1200), physically modified starches,
chemically modified starches (preferably oxidized starch (E-number
E1404), monostarch phosphate (E-number E1410), distarch phosphate
(E-number E1412), phosphated distarch phosphate (E-number E1413),
acetylated distarch phosphate (E-number E1414), acetylated starch
(starch acetate esterified with acetic anhydride; E-number E1420),
acetylated distarch adipate (E-number E1422), hydroxy propyl starch
(E-number E1440), hydroxy propyl distarch phosphate (E-number
E1442) starch sodium octenyl succinate (E-number E1450), and
acetylated oxidized starch (E-number E1451)), cyclodextrins,
celluloses, modified celluloses (preferably methylcellulose,
ethylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxyethyl methylcellulose,
hydroxypropyl methylcellulose), gum Arabic (gum acacia), gum
ghatti, gum tragacanth, gum karaya, carrageenan, guar gum, carob
gum (carob flour, locust bean gum, E-number E410), alginates,
pectins, inulin and xanthan gum.
[0446] In a further preferred embodiment the total amount of
compounds of formula I and the physiologically acceptable salts
thereof in an orally consumable material according to the present
invention, in particular of a ready-to-drink composition according
to the present invention, is in the range of from 1 to 200 ppm,
more preferably in the range of from 2 to 150 ppm, even more
preferably in the range of from 5 to 100 ppm, in each case based on
the total weight of the orally consumable material.
[0447] Preferably, the total amount of glucose, fructose and
sucrose of an orally consumable material according to the present
invention, in particular of a ready-to-drink composition according
to the present invention, is in the range of from 1.5 to 15.0 wt.
%, more preferably in the range of from 2.0 to 12.0 wt. %, even
more preferably in the range of from 2.5 to 10.5 wt. %,
particularly preferably in the range of from 3.0 to 9.5 wt. %, in
each case based on the total weight of the orally consumable
material.
[0448] Glucose, fructose and sucrose are commercially readily
available from various sources and in various forms, and may be
obtained from suitable plant sources, for example from sugar beet
(Beta vulgaris ssp., sugar fractions, sugar syrup, molasses), from
sugar cane (Saccharum officinarum ssp., e.g. molasses, sugar
syrups), from sugar maple (Acer ssp.), from agave (agave thick
juice), sorghum, certain palm trees, invert sugar syrup, high
fructose corn syrup (HFCS, also called glucose-fructose syrup, e.g.
made from wheat or corn starch), or fruit concentrates (e.g. from
apples or pears, apple syrup, pear syrup).
[0449] In a preferred embodiment, an orally consumable material
according to the present invention, in particular a ready-to-drink
composition according to the present invention, preferably
comprises one, two, three or more fruity flavouring agents having a
molecular weight in the range of 135 to 190 g/mol, more preferably
a molecular weight in the range of 135 to 180 g/mol, said fruity
flavouring agents preferably imparting a flavour note selected from
the group consisting of lemon, lime, grapefruit, orange, sweet
orange, bitter orange, bergamot, mandarin, apple, pear, prickly
pear, peach, apricot, pineapple, prune, mango, melon, plum, kiwi,
lychee, banana, cherry, sweet cherry, strawberry, raspberry, red
currant, black currant, blackberry, blueberry, passion fruit,
grape, pomegranate, acerola, coconut, vanilla and mixtures
thereof.
[0450] Preferably, an orally consumable material according to the
present invention, in particular of a ready-to-drink composition
according to the present invention, comprises one or more organic
food acids (i.e. organic acids suitable for oral consumption),
preferably selected from the group consisting of acetic acid,
adipic acid, caffeotannic acid, citric acid, iso-citric acid,
maleic acid, fumaric acid, galacturonic acid, glucuronic acid,
glyceric acid, glycolic acid, lactic acid, malic acid, oxalic acid,
pyruvic acid, quinic acid, succinic acid, tannic acid, tartaric
acid, and the physiologically acceptable salts thereof, preferably
the sodium and/or potassium and/or calcium and/or magnesium salts
thereof.
[0451] Preferred physiologically acceptable salts of phosphoric
acid are for example sodium acetate, monosodium phosphate, disodium
phosphate, monopotassium phosphate, dipotassium phosphate, sodium
hexametaphosphate, and sodium bis-phosphonates.
[0452] Preferably, an orally consumable material according to the
present invention, in particular a ready-to-drink composition
according to the present invention, comprises one or more acids
selected from the group consisting of citric acid, tartaric acid,
lactic acid, malic acid, maleic acid, fumaric acid, phosphoric
acid, pyrophosphoric acids, polyphosphoric acids, bisphosphonic
acids and the physiologically acceptable salts thereof.
[0453] A more preferred orally consumable material according to the
present invention, in particular a ready-to-drink composition
according to the present invention, comprises [0454] sucrose,
and/or [0455] a mixture of glucose and fructose, wherein the amount
of fructose is in the range of from 30 to 95 wt. %, preferably 40
to 92 wt. %, based on the total amount of glucose and fructose in
the orally consumable material.
[0456] Another preferred orally consumable material according to
the present invention, in particular a ready-to-drink composition
according to the present invention, comprises one or more further
constituents suitable for consumption selected from: [0457] one or
more emulsifiers, and/or [0458] one or more antioxidants and
optionally one or more substances for intensifying the
antioxidative effect of said antioxidants, and/or [0459] one or
more preservatives, and/or [0460] one or more vitamins and the
physiologically acceptable salts or esters thereof, and/or [0461]
one or more coloring agents, and/or [0462] one or more weighting
agents, and/or [0463] one or more sugar alcohols, and/or [0464] one
or more high potency sweeteners, preferably one or more naturally
occurring high potency sweeteners, and/or [0465] one or more
stabilizers and/or thickeners.
[0466] Preferably, an orally consumable material according to the
present invention, in particular a ready-to-drink composition
according to the present invention, comprises one or more further
constituents suitable for oral consumption, particularly [0467] one
or more emulsifiers, preferably selected from the group consisting
of lecithins (preferably naturally occurring lecithins,
particularly lecithin from egg or soy), phospholipids (preferably
phosphatidylcholines), monoacylglycerols, and diacylglycerols,
and/or [0468] one or more antioxidants and optionally one or more
substances for intensifying the antioxidative effect of said
antioxidants, and/or [0469] one or more preservatives (preferably
selected from the group consisting of benzoic acid, sodium
benzoate, potassium benzoate, sorbic acid, sodium sorbate, sodium
sorbate, butylated hydroxyanisole (BHA), and butylated
hydroxytoluene (BHT)), preferably in a total amount of from 0.05 to
0.5 wt. %, more preferably of from 0.1 to 0.3 wt. %, based on the
total weight of the composition, and/or [0470] one or more vitamins
and the physiologically acceptable salts or esters thereof,
preferably selected from the group consisting of vitamin A, vitamin
A palmitate, vitamin B1, vitamin B2 (riboflavin), vitamin B3
(niacin), vitamin B6, vitamin B9 (folic acid) vitamin B12, vitamin
C (ascorbic acid), monosodium ascorbate, monopotassium ascorbate,
calcium diascorbate, magnesium diascorbate, ascorbyl palmitate,
ascorbyl stearate, vitamin D, and vitamin E, vitamin E acetate,
vitamin E palmitate, vitamin H (biotin), vitamin K, and/or [0471]
one or more coloring agents, preferably selected form the group
consisting of carotenes (E-number E160a, preferably beta-carotene),
paprika extract (E-number E160c), red beet juice powder (comprising
betanine, beetroot red, E-number E162), annatto (E-number E160b),
anthocyanins (E-number E163), chlorophylls (E-number E140),
turmeric (E-number E100, comprising curcumin), tartrazine (FD&C
Yellow No. 5, E-number E102), amaranth (E-number E123), titanium
dioxide (E-number E171), iron oxides and iron hydroxides (E-number
E172), erythrosine (E-number E127), caramel color (E-number E150,
preferably E150d), FD&C yellow No. 6 (E-number E110), allura
red (FD&C red No. 40, E-number E129), FD&C green No. 3
(fast green, E-number E143), FD&C blue No. 1 (brilliant blue,
E-number E133) and FD&C blue No. 2 (indigotine, E-number E132),
and/or [0472] one or more bitter tasting substances selected from
the group consisting of quinine, neohesperidin, hesperidin,
naringin, quercitrin, phloridzin, phloretin-2-O'-xyloglucoside,
caffeic acid, chlorogenic acid, neochlorogenic acid,
cryptochlorogenic acid, limonoids (preferably limonin or nomilin
from citrus fruits), lupolones from hops, humulones from hops,
gallic and ellagic acid esters of carbohydrates (preferably
pentagalloylglucose), catechins and epicatechins (preferably
selected from the group consisting of galloylated catechins,
galloylated epicatechins, gallocatechins or epigallocatechins,
galloylated gallocatechins or galloylated epigallocatechins),
theaflavins (in particular theaflavin, isotheaflavin,
neotheaflavin), galloylated theaflavins, and procyanidines
(=proanthocyanidines) (in particular Procyanidin B1, Procyanidin
B2, Procyanidin A2, Procyanidin B5, and Procyanidin C1), and/or
[0473] one or more stabilizers and/or thickeners, preferably
selected from the group consisting of sodium octenyl succinate,
carboxymethyl cellulose, maltodextrin, gum Arabic, guar gum, carob
gum, alginates, pectin, and xanthan gum.
[0474] Examples of stabilizers and/or thickeners which may be part
of an orally consumable material according to the present
invention, in particular of a ready-to-drink composition according
to the invention, are preferably selected from the group consisting
of carbohydrate polymers (polysaccharides), cyclodextrins,
starches, degraded starches (starch hydrolysates), chemically or
physically modified starches (preferably starch sodium octenyl
succinate, E1450), modified celluloses (preferably carboxymethyl
cellulose), gum Arabic (gum acacia), gum ghatti, gum tragacanth,
gum karaya, carrageenan, guar gum, carob gum (carob flour),
alginates, pectin, inulin and xanthan gum.
[0475] If an orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, comprises one or more thickeners, then the
total amount of thickeners preferably is in the range of from
0.0025 to 1 wt. %, more preferably in the range of from 0.01 to
0.4. %, even more preferably in the range of from 0.015 to 0.2. %,
in each case based on the total weight of the composition.
[0476] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, preferably comprises 200 ppm or more
hydrogen carbonate (HCO.sub.3.sup.-), more preferably 250 ppm or
more hydrogen carbonate, even more preferably 300 ppm or more
hydrogen carbonate, and particularly preferably 400 ppm or more
hydrogen carbonate, in each case based on the total weight of the
orally consumable material.
[0477] If an orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, is carbonated, the total amount of carbon
dioxide (CO.sub.2) preferably is in the range of from 0.02 to 5.0
wt. %, more preferably in the range of from 0.05 to 3 wt. %, even
more preferably in the range of from 0.1 to 2.5 wt. %, particularly
preferably in the range of from 0.2 to 2.0 wt. %, most preferably
in the range of from 0.25 to 1.5 wt. %, in each case based on the
total weight of the orally consumable material.
[0478] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, may additionally comprise lactose and/or
maltose, and/or one or more sugar alcohols such as dulicitol,
fucitol, maltitol, erythritol, isomaltitol (E 953), lactitol (E
966), maltitol, mannitol (E421), sorbitol (E420), xylitol (E967),
and mixtures thereof.
[0479] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, may additionally comprise one or more high
potency sweeteners are preferably selected from the group
consisting of sodium cyclamate, acesulfame K, neohesperidin
dihydrochalcone, saccharin, saccharin sodium salt, aspartame,
superaspartame, neotame, alitame, sucralose, magap, lugduname,
carrelame, sucrononate, sucrooctate, miraculin, curculin, monellin,
mabinlin, thaumatin, curculin, brazzein, pentadin, or extracts or
fractions thereof obtained from natural sources containing said
amino acids and/or proteins, neohesperidin dihydrochalcone,
steviolgylcoside, stevioside, steviolbioside, rebaudiosides
(preferably rebaudioside A, rebaudioside B, rebaudioside C,
rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G,
rebaudioside H, dulcoside, rubusoside), suavioside A, suavioside B,
suavioside G, suavioside H, suavioside I, suavioside J, baiyunoside
1, baiyunoside 2, phlomisoside 1, phlomisoside 2, phlomisoside 3,
phlomisoside 4, abrusoside A, abrusoside B, abrusoside C,
abrusoside D, cyclocaryoside A and cyclocaryoside I, oslandin,
polypodoside A, strogin 1, strogin 2, strogin 4, selligueanin A,
dihydroquercetin-3-acetate, perillartine, telosmoside A.sub.15,
periandrin I-V, pterocaryoside, cyclocaryoside, mukurozioside,
bryoside, bryonoside, bryonodulcoside, carnosifloside,
scandenoside, gypenoside, trilobtain, phloridzin, dihydroflavanol,
hematoxylin, cyanin, chlorogenic acid, albiziasaponin, telosmoside,
gaudichaudioside, mogroside, hernandulcine, monatin, glycyrrhetin
acid, glycyrrhizin, phyllodulcin, or the physiologically acceptable
salts thereof, preferably the respective potassium, sodium, calcium
or ammonium salts thereof, liquorice extracts (Glycyrrhizza glabra
ssp.), Lippia dulcis extracts, Momordica ssp. extracts or
individual substances (in particular Momordica grosvenori [Luo Han
Guo] and the mogrosides obtained therefrom), Hydrangea dulcis or
Stevia ssp. (e.g. Stevia rebaudiana) extracts or individual
substances.
[0480] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, preferably comprises one or more high
potency sweeteners, preferably selected from the group indicated
above, more preferably selected from the group consisting of
aspartame, neotame, superaspartame, advantame, saccharin,
sucralose, cyclamate, acesulfam, tagatose, monellin, stevioside,
rebaudioside A, rebaudioside C, rebaudioside D, rubusosid,
phyllodulcin, hernandulcin, thaumatin, brazzein, miraculin,
glycyrrhizin, glycyrrhetinic acid, the physiologically acceptable
salts (preferably the sodium, potassium or calcium salts) of the
these compounds.
[0481] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, preferably comprises a total amount of less
than 4.5 wt. % fats and fatty oils (i.e. triglycerides), more
preferably less than 3.5 wt. % fats and fatty oils, even more
preferably less than 2.0 wt. % fats and fatty oils, particularly
preferably less than 1.0 wt. % fats and fatty oils, and most
preferably less than 0.5 wt. % fats and fatty oils, in each case
based on the total weight of the orally consumable material.
[0482] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, preferably comprises a total amount of less
than 5.0 wt. % proteins, more preferably less than 4.0 wt. %
proteins, even more preferably less than 3.0 wt. % proteins,
particularly preferably less than 1.0 wt. % proteins, and most
preferably less than 0.5 wt. % proteins, in each case based on the
total weight of the orally consumable material.
[0483] Preferred orally consumable materials according to the
present invention are alcoholic or non-alcoholic beverages
(preferably coffee-containing beverages, tea-containing beverages,
cocoa-containing beverages, wine-containing drinks, beer-containing
drinks, fruit-containing soft drinks, isotonic drinks, soft drinks,
energy drinks, nectars, fruit and vegetable juices, instant
beverage powders after dilution in water, beverage concentrates,
beverage syrups, fountain syrups, smoothies), dairy products
(preferably flavoured milk, yoghurts, yoghurt drinks, kefir,
buttermilk drinks, milk shakes, milk mix beverages), ice products
(water ice, ice cream), fruit preparations (preferably sorbets,
fruit sauces, fruit fillings, fruit ice creams), vegetable products
(preferably soy milk products, ketchup, sauces), emulsions
(preferably mayonnaise, remoulade, dressings, bakery flavour
emulsions), jams, jellies, bakery fillings, pickle brine, frozen
juice compositions, sour confections, fruit pie fillings, desserts,
marinades, and soups.
[0484] A (preferably acidic) beverage of this invention may be
prepared, for example, from a corresponding (preferably acidic)
syrup composition based on a dilution or a throw of the (preferably
acidic) syrup. Those skilled in the art recognize that a common
throw for a soft drink, e.g. a cola-flavoured carbonated soft
drink, is 1+5 so that a preparer uses one part of cola syrup and
five parts water to prepare the (preferably acidic) beverage from
the (preferably acidic) syrup. The amount of (preferably acidic)
syrup employed to prepare the (preferably acidic) beverage of this
invention will of course vary depending on the concentration of the
syrup and the desired end product. Such amount can be readily
determined by those of ordinary skill in the art.
[0485] In a preferred embodiment, an orally consumable material
according to the present invention, in particular a ready-to-drink
composition according to the present invention, is clear. The term
"clear" in the context of the present invention refers to a
composition of matter having a turbidity of less than 25 FNU
(Formazin Nephelometric Units) as measured according to DIN EN ISO
7027-Water quality-Determination of turbidity (ISO 7027:1999).
[0486] Preferably, an orally consumable material according to the
present invention, in particular a ready-to-drink composition
according to the present invention, has a turbidity of less than 12
FNU, more preferably of less than 6 FNU, preferably measured with a
Hach Turbidimeter 2100N IS.
[0487] Preferred orally consumable materials according to the
present invention are in particular clear or turbid (carbonated or
non-carbonated) beverages, preferably selected from the group
consisting of lemonade, carbonated soft drinks, tea, ice-tea,
beer-lemonade mixes, cola, beer-cola mixes, whey drink lemonade,
tea, beer-lemonade mixtures, cola drinks, beer-cola mixes, and whey
drinks, and the concentrates for producing said beverages.
[0488] An orally consumable material according to the present
invention (as defined above) at 20.degree. C. and 1013 mbar
preferably is pourable, and more preferably liquid.
[0489] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the invention, preferably has dynamic viscosity value of smaller
than 1250 mPa s (mPa s=milli Pascal seconds; equal to
cP=centiPoise), preferably of smaller than 600 mPa s, more
preferably of smaller than 250 mPa s, particularly preferably of
smaller than 100 mPa s, especially preferably of smaller than 50
mPa s, and most preferably of smaller than 25 mPa s, in each case
measured at 20.degree. C. and at a shear rate of D=10 s.sup.-1,
e.g. as determined with a Brookfield.RTM. viscometer according to
DIN 53018.
[0490] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the invention, preferably has dynamic viscosity value in the range
of from 1 to 1000 mPa s (mPa s=milli Pascal seconds; equal to
cP=centiPoise), preferably in the range of 2 to 500 mPa s, more
preferably in the range of 2 to 125 mPa s, particularly preferably
in the range of 3 to 50 mPa s, especially preferably in the range
of 3 to 25 mPa s, in each case measured at 20.degree. C. according
to DIN 53018.
[0491] The flavour of an orally consumable material according to
the present invention, in particular of a ready-to-drink
composition according to the present invention, preferably is
selected from the group consisting of berries, citrus fruits,
pomaceous fruit, spices, herbs, mints, teas, coffees, milk and/or
milk products, and more particularly preferably selected from the
group consisting of cola, lemon, lime, lemon-lime, grapefruit,
orange, sweet orange, bitter orange, bergamot, mandarin, apple,
pear, prickly pear, peach, apricot, pineapple, prune, mango, melon,
plum, kiwi, lychee, banana, cherry, sweet cherry, strawberry,
raspberry, red currant, black currant, blackberry, blueberry,
passion fruit, grape, pomegranate, acerola, vanilla, cinnamon,
anise, fennel, clove, cardamom, tamarind, nutmeg, allspice, black
pepper, honey, licorice, ginger ale, ginger, root beer, rose hip,
green tea, red tea, rooibos tea, mate tea, honeybush tea, pu-erh
tea, oolong tea, black tea, kombucha, milk, coffee, espresso,
cocoa, chocolate, hazelnut, walnut, almond, peppermint, spearmint,
wintergreen and mixtures thereof.
[0492] In a preferred embodiment, an orally consumable material
according to the present invention, in particular a ready-to-drink
composition according to the present invention, comprises one or
more amino carboxylic acids and/or one or more amino sulfonic
acids, preferably gamma-amino butyric acid and/or taurine
(2-aminoethanesulfonic acid).
[0493] An orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, may preferably comprise one or more fruit
derived ingredients, in particular fruity flavours, fruit juices,
fruit purees and fruit juice concentrates.
[0494] Fruit juices or fruit juice concentrates that can be used
are preferably derived from citrus fruits such as orange, lemon,
grapefruit and tangerine, and other fruits such as apple, pear,
grape, apricot and pineapple. Furthermore, fruit juices and fruit
juice concentrates may be derived from soft fruits like blackberry,
gooseberry, currant, blueberry, elderberry, strawberry and
raspberry.
[0495] Preferably, an orally consumable material according to the
present invention, in particular a ready-to-drink composition
according to the present invention, is an emulsion. Densities of
the disperse phase, which are preferred for an adequate
stabilization and avoidance of ringing, preferably lie in the range
of from 0.92 to 1.06 g/ml, more preferably in the range of from
0.94 to 1.03 g/ml. "Ringing" is the formation of a ring around the
neck of a (beverage) container which is sought to be avoided.
[0496] If an orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, comprises one or more weighting agents,
these are preferably selected from the group consisting of sucrose
acetate isobutyrate (SAIB, E 444), estergum (E 445), dammar gum,
and brominated vegetable oils in an amount not exceeding the
respective legally authorized concentrations.
[0497] In a preferred embodiment, an orally consumable material
according to the present invention, in particular a ready-to-drink
composition according to the present invention, is a cloudy
(turbid) emulsion, preferably comprising one or more clouding
agents, such as titanium dioxide, palm oil, or terpene oils like
limonene.
[0498] If an orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, is an emulsion, e.g. prepared as described
in U.S. Pat. No. 5,616,358, EP 2 025 250 or EP 1 151 677.
[0499] If an orally consumable material according to the present
invention, in particular a ready-to-drink composition according to
the present invention, is a cloudy (turbid) emulsion, the D90
particle (droplet) size of the disperse phase (as measured by laser
diffraction) is in the range of from 0.15 to 1.0 .mu.m (microns),
in some preferred embodiments in the range of from 0.35 to 0.5
.mu.m, in some other preferred embodiments in the range of from 0.6
to 0.75 .mu.m.
[0500] In a preferred embodiment, a beverage according to the
present invention comprises a terpene oil, more preferably a
terpene citrus oil. Preferred terpene oils in the context of the
present invention comprise or consist of orange, lemon and/or
grapefruit oils and fractions thereof, preferably limonene
(especially D-limonene) and/or orange oil terpenes.
[0501] A material according to the present invention, preferably a
food or cosmetic product according to the present invention, may
additionally comprise one or more physiological cooling agents,
preferably selected from the group consisting of menthone
derivatives (preferably L-menthone glycerol ketal),
p-menthane-3,8-diol, cubebol, isopulegol and its esters (preferably
L-(-)-isopulegol, L-(-)-isopulegol acetate), menthyl ethers
(preferably (L-menthoxy)-1,2-propanediol,
(L-menthoxy)-2-methyl-1,2-propanediol, L-menthyl-methyl ether),
menthyl esters (preferably menthyl formate, menthyl acetate,
menthyl isobutyrate, menthyl lactate, L-menthyl-L-lactate,
L-menthyl-D-lactate, L-menthyl-(2-methoxy)acetate,
L-menthyl-(2-methoxyethoxy)acetate, L-menthyl pyroglutamate),
menthyl carbonates (preferably L-menthyl propylene glycol
carbonate, L-menthyl ethylene glycol carbonate, L-menthyl glycerol
carbonate or mixtures thereof), semi-esters of menthols with a
dicarboxylic acid or derivatives thereof (preferably menthyl
oxamate, menthyl-N-methyloxamate, menthyl-N-ethyloxamate,
mono-L-menthyl succinate, mono-L-menthyl glutarate, mono-L-menthyl
malonate, O-L-menthyl succinic acid ester-N,N-(dimethyl)amide,
O-L-menthyl succinic acid ester amide),
2,3-dimethyl-2-(2-propyl)-butanoic acid derivatives (preferably
2,3-dimethyl-2-(2-propyl)-butanoic acid-N-methyl amide [WS-23]),
menthane carboxylic acid amides (preferably L-menthane carboxylic
acid-N-ethyl amide [WS-3],
N.sup..alpha.-(L-menthanecarbonyl)glycine ethyl ester [WS-5],
menthane carboxylic acid-N-(4-methoxphenyl)-amide [WS-12],
L-menthane carboxylic acid-N-tert-butyl amide [WS-14], L-menthane
carboxylic acid-N-(4-cyanophenyl)amide, N-(4-cyanomethylphenyl)
p-menthanecarboxamide), L-menthane carboxylic
acid-N-(alkoxyalkyl)amides, L-menthane carboxylic
acid-N-(alkylthioalkyl)amides, and pyrrolidone derivatives of
cycloalkyldione derivatives (preferably
3-methyl-2(1-pyrrolidinyl)-2-cyclopenten-1-one).
[0502] In one aspect of the present invention, preferred cosmetic
products of the invention are oral hygiene products (oral care
products). Preferred oral hygiene products are creams, gels,
pastes, foams, emulsions, suspensions, aerosols, sprays or chewing
gums. Such formulations serve to clean and care for the tooth
substance and oral cavity and to freshen the breath. More preferred
oral hygiene products are toothpastes, tooth gels, 2-in-1 tooth
gels, mouthwashes, mouth rinses, gargles and mouth or breath
sprays.
[0503] A cosmetic, in particular an oral hygiene oral product
according to the invention can contain further auxiliary substances
such as are conventionally used in such preparations, for example
further preservatives, abrasives, antibacterial agents,
anti-inflammatory agents, irritation-preventing agents,
irritation-inhibiting agents, further antimicrobial agents,
antioxidants, astringents, antiseptic agents, antistatics, binders,
buffers, support materials, chelating agents, cell stimulants,
cleansing agents, conditioning agents, further surface-active
substances, deodorising agents, softeners, emulsifiers, enzymes,
essential oils, film formers, foaming agents, foam stabilisers,
substances to prevent foaming, gelling agents, moisturizing
substances, moisture-retaining substances, bleaching agents,
optical brighteners, dirt-repelling agents, lubricants, opacifiers,
brighteners, polymers, powders, proteins, silicones, skin-calming
agents, skin-cleansing agents, skin care agents, skin-healing
agents, skin-cooling agents, skin-warming agents, stabilisers,
thickeners, vitamins, oils, waxes, fats, phospholipids, saturated
fatty acids, mono- or polyunsaturated fatty acids, alpha-hydroxy
acids, polyhydroxy fatty acids, dyes, colour-protecting agents,
pigments, aroma substances, perfumes, and other conventional
constituents, such as alcohols, polyols, electrolytes, organic
solvents, sweeteners, sugar substitutes, silicas, calcium
carbonate, calcium hydrogen phosphate, aluminium oxide, fluorides,
zinc, tin, potassium, sodium and strontium salts, pyrophosphates,
hydrogen peroxide, and hydroxyapatites.
[0504] Examples of aroma substances which can preferably be part of
an oral hygiene product according to the invention are: anethole,
menthol, menthone, isomenthone, menthyl acetate, menthyl
propionate, menthofuran, mintlactone, eucalyptol (1,8-cineol),
limonene, eugenol, eugenol acetate, isoeugenol methyl ether,
thymol, pinene, sabinene hydrate, 3-octanol, carvone,
gamma-octalactone, gamma-nonalactone, germacrene-D, viridiflorol,
1,3E,5Z-undecatriene, isopulegol, piperitone, 2-butanone, ethyl
formate, 3-octyl acetate, isoamyl isovalerianate, hexanol, hexanal,
cis-3-hexenol, linalool, alpha-terpineol, cis- and trans-carvyl
acetate, p-cymol, damascenone, damascone, rose oxide, fenchol,
acetaldehyde diethylacetal, 1-ethoxyethyl acetate, cis-4-heptenal,
isobutyraldehyde, isovaleraldehyde, cis-jasmone, methyl
dihydrojasmonate, anisaldehyde, methyl salicylate,
2'-hydroxpropiophenone, menthyl methyl ether, myrtenyl acetate,
2-phenylethyl alcohol, 2-phenylethyl isobutyrate, 2-phenylethyl
isovalerate, cinnamaldehyde, geraniol, nerol. In the case of chiral
compounds the aroma substances can be used as a single enantiomer
or a mixture of enantiomers, e.g. in the form of a racemate.
[0505] Aroma substances which preferably are part of an oral
hygiene product according to the invention are preferably selected
from the group consisting of aniseed oil, basil oil, bitter almond
oil, camphor oil, citronella oil, citrus oils, eucalyptus
citriodora oil, eucalyptus oil, camomile oil, spearmint oil,
limette oil, mandarin oil, clove oil, orange oil, peppermint oil,
sage oil, thyme oil, wintergreen oil, cinnamon oil, cinnamon bark
oil, l-menthol, menthone, isomenthone, 1,8-cineol (eucalyptol),
carvone, alpha-terpineol, methyl salicylate,
2'-hydroxpropiophenone, and menthyl methyl ether.
[0506] Another group of preferred cosmetic products of the
invention are sanitary articles, preferably selected from the group
consisting of wet wipes, sanitary towels, diapers, tampons,
handkerchiefs and refreshing tissues containing one or more
compounds of formula I (to be used) according to the invention
and/or one or more physiologically acceptable salts thereof. Said
sanitary articles according to the invention preferably contain a
material according to the present invention, in particular in one
of the preferred variants described herein.
[0507] In a non-woven system, preferably at least one layer
comprises an absorbent non-woven fabric or a porous polymer which
is impregnated with a solution or suspension comprising one or more
compounds of formula I (to be used) according to the invention
and/or one or more physiologically acceptable salts thereof, and
preferably comprises one or more additional other active substances
(such as skin-soothing and/or skin-moisturizing agents).
[0508] The following Examples illustrate the invention without
limiting its scope.
EXAMPLES
TABLE-US-00002 [0509] Abbreviation Definition ppm = .mu.g/mL Part
per million identical with micro gram per millilitre ACN
Acetonitrile CFU Colony forming units ELSD Evaporative Light
Scattering Detection HPGPC High Performance Gel Permeation
Chromatography HPLC High Performance Liquid Chromatography HR-ESIMS
High Resolution Electrospray Ionisation Mass Spectrometry LCT
Liquid Chromatography Time of Flight MS Mass Spectrometry TFA
Trifluoroacetic acid UV/Vis Ultraviolet/visible light
Example 1
Cultivation
A) Media:
[0510] 1) SDB (Sabouraud Dextrose Broth, Ref. 238230, Difco.TM.
Lawrence, Kans., USA, containing 0.5% Peptic Digest of Animal
Tissue, 0.5% Pancreatic Digest of Casein, 2.0% Dextrose, pH 5.6.
[0511] 2) YMG (Yeast-Malt-Glucose) medium: D-glucose 0.4% (Merck,
Darmstadt, Germany, Ref. K25252846 831), malt extract 1% (Carl
Roth, Karlsruhe, Germany, Ref. X976.2), yeast extract 0.4% (Merck,
Darmstadt, Germany, Ref. 1.11926.1000), pH 6.3. [0512] 3) PDB
(Potato Dextrose Broth): 2.0% D-glucose, 0.4% mashed potatoes
(Pfanni, Hamburg, Germany). [0513] 4) Cornmeal medium (CM): 2.0%
corn meal (Neuform, Zarrentin, Germany), 1.0% D-glucose. [0514] 5)
GM1 (Glucose-Yeast medium 1): 2.0% D-glucose, 0.5% yeast extract.
[0515] 6) GM2 (Glucose-Yeast medium 2): 2.0% D-glucose, 0.1% yeast
extract. [0516] 7) Malt medium 1: 2.0% malt extract, 0.5% yeast
extract. [0517] 8) Malt medium 2: 2.0% malt extract without yeast
extract. [0518] 9) Apple juice medium: 10% apple juice (common
commercial product: clear juice, EAN 20009717, Fruchtstern.RTM.,
trademark by Netto Marken-Discount AG & Co. KG,
Maxhutte-Haidhof, Germany), 4.83% glucose, 5.94% fructose, 0.23%
sucrose, 0.1% (v/v) hardness solution (prepared from 4.4 g
CaCl.sub.2*2H.sub.2O+3.04 g MgCl.sub.2*6H.sub.2O dissolved in 100
ml water), 0.2% (v/v) 1M sodium malate, 0.2% (v/v) 1M maleic acid;
adjusted to pH 3.2-3.4 with 1M maleic acid. [0519] 10) Trypticase
Soy Yeast Extract medium: 3% Trypticase soy broth (Difco, Lawrence
USA, Ref. 211825), 0.3% Yeast extract (Merck, Darmstadt Germany,
Ref. 111926).
B) Fermentation (Exemplarily Presented Using the Strain
FU50088)
a) Seed Cultures (Shake Flask Cultures)
[0520] One ml of a cryo vial containing a mycelial suspension of
FU50088 in 10% glycerol was retrieved from liquid nitrogen and,
after thawing, used to inoculate 200 ml Erlenmeyer flasks
containing 50 ml of sterile YMG medium and propagated on a rotary
shaker at 240 rpm and 23.degree. C. for 72 h. Thereafter, each two
ml of the primary seed culture were transferred into batches of two
500 ml Erlenmeyer flasks containing 200 ml of the same medium and
propagated on a rotary shaker at 140 rpm and 23.degree. C. for 120
h. These flasks served as secondary seed cultures.
b) Fermentation in 30 l Scale
[0521] A 40 l Biostat LP42 fermentor (Bioengeneering, Wald,
Switzerland) containing 301 of medium was sterilized in situ (1 h
at 121.degree. C. and 1.1 bar) and inoculated with 400 ml of the
secondary seed culture. The production culture was grown under
stirring (240 rpm) and aeration (0.2 vvm (volumes of air per minute
per volume of batch)) at 30.degree. C.
[0522] Furthermore at each point of incubation time listed above,
HPLC analysis of crude extracts prepared from 20 ml samples taken
under sterile conditions and extracted with equal amounts of ethyl
acetate served as a means of detection and estimation of
glycolipids. For this purpose, the ethyl acetate extracts are dried
over anhydrous sodium sulfate, evaporated to dryness, re-dissolved
in 2-propanol and analyzed using the HPLC systems described below
in HPLC-MS methods "fermentation control".
TABLE-US-00003 TABLE A Dependency of yield on nutrient composition
Production Extractable Diameter of inhibition Culture maximum [h
Mycelial dry material zone (mm)** medium fermentation] pH value
weigth [g/l] [g/l] B. subtilis S. cerevisiae SDB 192 4.6 6.2 0.26
18 10 YMG 216 6.0 4.8 0.13 12 0 PDB 312 4.7 1.7 0.76 18 9 CM 216
4.6 * 0.65 17 14 GM1 312 4.7 4.3 0.69 20 13 GM2 192 4.1 3.3 1.26 20
13 MM1 192 5.2 12.8 0.24 18 11 MM2 192 4.4 8.0 1.07 19 15 *Mycelial
weight was not determined (medium contaning solid constitutents)
**Inhibition zones caused after 24 h by 50 .mu.g of ethyl acetate
extract dissolved in methanol on a paper disk (6 mm diameter)
[0523] For optimisation of culture media, Dacryopinax spathularia
was propagated in a series of 500 ml shake flask batches containing
each 200 ml of culture media as described above (Example 1B) under
"a) Shake flask cultures (including "Seed cultures). During
fermentation, samples were taken, and pH, mycelial dry weight,
amount of extractable material and biological activities of crude
ethyl acetate extracts of the culture broth against Bacillus
subtilis and Zygosaccharomyces bailii in the agar diffusion assay
were determined. The results (Table A) show that, even though the
glycolipids are produced in a variety of different culture media,
GM2 medium showed the highest specific biological activity with
respect to the mycelial dry weight and the fermentation time. As it
could even be observed during fermentation by microscopic control
that the glycolipids sticked to the mycelial hyphae during the
production phase, it was deemed favorable to use GM2 medium for
large scale fermentation, in order to direct the majority of the
products to be located in the culture broth, especially in view of
the precipitation experiments that were finally found the most
effective and ecologically friendly way of obtaining the desired
compounds. In addition, the maximum of production was obtained
earlier than in most other fermentation media.
c) Fermentation in 200 l Scale
[0524] The fermentation was performed in a 3001 fermentor
(Bioengineering Type P, equipped with four Ekato Intermig.RTM.
impellers) containing 200 l of GM2 medium, sterilized under steam
for 45 min at 121.degree. C., inoculated with the above described
20 l seed culture. To prevent foaming, 0.03 ml/l of Clarol FBA
3003K (Cognis, Monheim, Germany) as anti-foam agent were added; no
additional supply of antifoam was necessary during fermentation.
The fermentation was performed at ca. 33.degree. C., under
agitation (75 rpm) and aeration (0.2 vvm). The fermentation was
stopped after 300 h when the free glucose had almost been consumed
and the oxygen partial pressure had dropped to 20%.
C) Preparation of Extracts
a) Preparation of Mycelial Extract:
[0525] The cultures from 10 shake flasks were harvested. The
culture fluid was separated by filtration from the mycelia. The wet
mycelia were extracted two times with equal volumes of acetone for
each 30 min in an ultrasonic bath. This acetone was evaporated in
vacuo at 40.degree. C. and the remaining aqueous phase was diluted
to 700 ml with water. This phase was extracted three times with
equal volumes of ethyl acetate (EtOAc). The combined organic phases
were dried over anhydrous Na.sub.2SO.sub.4 and evaporated in vacuo
to yield 329 mg of a crude extract.
b) Preparation of the Culture Fluid Extract:
[0526] 1000 g Amberlite.RTM. XAD16 (Sigma-Aldrich, St. Louis, Mo.
63103, CAS 90003-69-4, Batch-No. 099K0079) were added to 301
culture filtrate (from a 30 l fermenter, GM2 medium) and incubated
over night under stirring (60 rpm). The resin was harvested by
filtration and the dry resin was incubated with two liters of
methanol and incubated for 30 min in an ultrasonic bath.
Thereafter, the methanol eluate was removed by filtration. This
methanol elution process was repeated twice. The methanol eluates
(ca. 6 l) were combined evaporated in vacuo and the resulting oily
residue redissolved in 500 ml distilled water. The pH was adjusted
with 2 M HCl to pH 2.9, and the resulting suspension was extracted
three times each with equal amounts of ethyl acetate. The combined
organic phases were dried over sodium sulfate, evaporated in vacuo
(40.degree. C.) to yield 25 g of an amorphous, light brown crude
extract.
c) Preparation of a Sedimentation (Precipitation) (Downstream
Processing) Product
[0527] The culture broth resulting from a 2001 fermentation was
alkalized from an initial pH value of 4.5 to pH 8 with 1 N sodium
hydroxide solution to allow the glycolipids that partially stick to
the cells under acidic conditions to become largely released from
the mycelia. After 1 h the mycelia were separated using a Westfalia
KA1-06-525 separator, and in addition the culture broth was
filtrated through a Pall (Dreieich Germany) 0.1 .mu.m polysulfone
membrane filter casettes, diameter of fibers 1.4 mm, total area 24
m.sup.2) microfiltration system to completely retain the mycelia.
The product was sedimented (precipitated) by acidifying the
filtrate with 2 N hydrochloric acid to pH 3 and incubating for 16 h
under cooling to 11.degree. C. The fluid was removed by decantation
and subsequent centrifugation (4500 rpm, 15 min, Typ Jouan SA LR
5.22 (Jouan, Paris France), resulting in a whitish-grey gel. This
crude product was washed immediately with water (pH 7), centrifuged
again at (4500 rpm for 15 min) and freeze-dried. This process
yielded 380 g dry glycolipid which was further characterized by
HPLC-MS (see e.g. HPLC chromatogram in FIG. 2).
D) Isolation of Compounds
a) Flash Chromatography
[0528] 10 g of the raw product were dissolved in 5 ml of methanol
and bound onto 10 g of Chromabond XTR (Kieselguhr for liquid-liquid
extraction Macherey-Nagel, Article No. 730 595.500, Duren, Germany)
and the solvent was evaporated in vacuo. This dried material was
applied onto a Biotage Isolera (Uppsala, Sweden) MPLC system, using
a Chromabond.RTM. flash (120 Nucleodur 100-20 C18ec; 130.times.40
mm) (trademark by Macherey-Nagel) as stationary phase.
[0529] The column was equilibrated with ACN/water (1:5) and then
eluted using the following conditions at a flow of 20 ml/min: 3:30
min, ACN/water (1:5) isocratic; 9 min, ACN/water (1:1) gradient;
19:00 min, ACN/water (1:1) isocratic; 29:00 min ACN/water (2:1)
gradient; 49:00 min ACN/water (2:1) isocratic; 59:00 min pure ACN
gradient.
[0530] Small aliquots of the fractions were taken, evaporated and
dissolved in 2-propanol to concentrations of 5 mg/ml and analyzed
by HPLC-MS using the "Adapted Method" as described in Example 3.
The fractions were combined according to HPLC-MS results and
concentrated in vacuo.
[0531] Fractions containing the compounds of the formula I elute
according the following table.
TABLE-US-00004 TABLE 2 Flash chromatographic elution profile of
compounds of the formula I Elution time Containing compound Yield
[min] number [mg] 17-26 [16] 686 27-29 [14] 791 32-33 [12] 505
34-35 [13] 542 36-38 [1] 1256 41-48 [18] 570
b) Purification by HPLC
[0532] All these separation steps were performed with [0533] CA:
Waters SunFire C18 preparative HPLC column (7 .mu.m, length 250
mm*diameter 19 mm) [0534] CB: Kromasil C18 (7 .mu.m, length 250
mm*diameter 40 mm)+pre column (Kromasil C18, 7 .mu.m, length 50
mm*diameter 20 mm) [0535] CC: Kromasil C8 (7 .mu.m, length 250
mm*diameter 40 mm) [0536] CD: lnertsil ODS-3 C18 (5 .mu.m, length
250 mm*diameter 40 mm) as stationary phase. The eluent was built up
with ACN and water using a flow rate of 10 ml/min. The maximal
capacity of this column is about 600 mg. Therefore it was necessary
to perform several identical purifications in series in order to
purify larger amounts.
[0537] The separations were monitored by a diode array detector at
200 and 210 nm. Five ml fractions were taken using an automatic
fraction collector and finally combined according to UV absorption
(200 and 210 nm), concentrated in vacuo and subjected to HPLC-MS,
to assess their purity.
[0538] For those skilled in the art it is obvious to adapt the
elution methodology to the retention necessities of each compound,
e.g. less polar compounds will elute later, consequently it is
possible to start with a higher content of ACN in the eluent.
Sometimes it is better to use another slope of the gradient or it
is necessary to use an isocratic system.
TABLE-US-00005 TABLE 3 HPLC purification elution profiles of
compounds of the formula I Purified Retention Column Gradient
compound time [min] code flow Purity (NMR) [16] I CB 10 min: ACN
50% 85% (NMR) 6.5-8.5 30 min: ACN 50% .fwdarw. 80% 45 min: ACN 80%
50 min: ACN 80% .fwdarw. 85% 70 min: ACN 85% 90 min: ACN 85%
.fwdarw. 100% 20 ml/min [14] I CC 15 min: MeOH 20% .fwdarw. 50%,
49.5-54 70 min: MeOH 50% .fwdarw. 100%, 100 min: MeOH 100%, 20
ml/min II CD 15 min: ACN 20% .fwdarw. 45%, 75% (NMR) 26.5-28.5 30
min: ACN 45% 35 min: ACN 45% .fwdarw. 50%, 50 min: ACN 50% 55 min:
ACN 50% .fwdarw. 55%, 70 min: ACN 55% 90 min: ACN 55% .fwdarw.
100%, 100 min: ACN 100% 20 ml/min [12] I CB 10 min: ACN 50% 95%
(NMR) 19.5-21 30 min: ACN 50% .fwdarw. 80% 45 min: ACN 80% 50 min:
ACN 80% .fwdarw. 85% 70 min: ACN 85% 90 min: ACN 85% .fwdarw. 100%
20 ml/min [13] I CC 15 min: MeOH 20% .fwdarw. 50%, 55-56 70 min:
MeOH 50% .fwdarw. 100%, 100 min: MeOH 100%, 20 ml/min II CD 15 min:
AC N 20% .fwdarw. 50%, 85% (NMR) 35-36 30 min: ACN 50% 35 min: ACN
50% .fwdarw. 55%, 50 min: ACN 55% 55 min: ACN 55% .fwdarw. 60%, 70
min: ACN 60% 90 min: ACN 60% .fwdarw. 100%, 100 min: ACN 100% 20
ml/min [1] I CB 10 min: ACN 50% 95% (NMR) 35-36 min. 30 min: ACN
50% .fwdarw. 80% 45 min: ACN 80% 50 min: ACN 80% .fwdarw. 85% 70
min: ACN 85% 90 min: ACN 85% .fwdarw. 100% 20 ml/min [18] I CA 15
min: ACN 20% .fwdarw. 50%, 42 min 35 min: ACN 50%, 40 min: ACN 50%
.fwdarw. 100%, 50 min: ACN 100%, 20 ml/min II CA 40 min: ACN 40%
.fwdarw. 100%, 93% (NMR) 17 min 45 min: ACN 100%, 20 ml/min
[0539] Exemplarily the purification of compound [1] is described
herein in detail: The elution profile was running from: 10 min
isocratic ACN 50%; 30 min gradient ACN 50%.fwdarw.80%; 45 min
isocratic ACN 80%; 50 min gradient ACN 80%.fwdarw.85%; 70 min
isocratic ACN 85%; 90 min gradient ACN 85%.fwdarw.100% with a flow
of 20 ml/min using column CB (Kromasil C18). Within a retention
time of 35-36 min compound [1] eluted from the above described
system. The purity was determined as 95% by .sup.1H-NMR.
E) Definition of Extracts
[0540] Extracts containing compounds of the formula I are defined
with their preparation procedures, e.g. the use of the above
explained processes.
TABLE-US-00006 TABLE 4 Preparation Extract Fermentation of extracts
id Strain process B process C Full process code [X1] MUCL53181
example 1, B b) example 1, C c) MUCL53181 example 1, B b), C c)
[X2] MUCL53181 example 1, B c) example 1, C c) MUCL53181 example 1,
B c), C c) [X3] MUCL53181 example 1, B a) example 1, C a) MUCL53181
example 1, B a), C a) [X4] MUCL53182 example 1, B a), example 1, C
b) MUCL53182 example 1, B a), C b) [X5] MUCL53179 example 1, B a),
example 1, C a) MUCL53179 example 1, B a), C a) [X6] MUCL53500
example 1, B b), example 1, C c) MUCL53500 example 1, B b), C
c)
[0541] Exemplarily the codation of [X1] is described herein in
detail: The use of the strain FU50088 of the species Dacryopinax
spathularia in a fermentation procedure as described above in the
example 1 "Cultivation" with a process as described in this example
under section B "fermentation" using a 30 l fermentor as described
in this section under subsection b) "30 l fermentations" followed
by a preparation procedure as described in the same example 1 with
a process as described under section C "Preparation of extracts"
using a precipitation process as described in this section under
subsection c) "Preparation of a sedimentation product" is coded as
"an extract of FU50088 according example 1 using a production
process B b) and an extraction process C c)" or in short terms
"extract FU50088 example 1, B b), C c)".
Example 2
Structural Characterisation
Compound No. [1]
[0542] The molecular structure was elucidated by thorough
interpretation of high resolution mass spectrometric data and 1D
and 2D NMR spectra. The structural characterisation follows the
general methodology which is known to the person skilled in the art
and described in more detail in the scientific literature
(examples: Nishida et al., J. Antibiot. 1991, 44, 541; Nishida et
al, Chem. Pharm. Bull. 1991, 39, 3044).
[0543] The numbering of the atoms is shown in FIG. 1.
[0544] Chemical Formula: C.sub.49H.sub.88O.sub.21
[0545] Exact Mass: 1012.5818 Da
[0546] Molecular Weight: 1013.2104 Da
[0547] HR-ESIMS: found m/z 1013.5874; calculated m/z 1013.5891 for
[M+H].sup.+
[0548] NMR spectra were obtained in CD.sub.3OD at 293 K on a Bruker
DRX spectrometer operating at 500 MHz proton frequency. The
residual solvent peak was used as internal reference
(.delta..sub.H=3.30; .delta..sub.C=49.0). The assigned NMR data are
summarized in Tables 5-14.
TABLE-US-00007 TABLE 5 NMR data for Compound No. [1] aglycon moiety
carbohydrate moiety atom .delta..sub.C .delta..sub.H, mult. atom
.delta..sub.C .delta..sub.H, mult. 1 178.1 -- 1' .sup. 102.5 4.38,
d (4.7) 2 71.4 4.09, dd (7.6, 4.4) 2' .sup. 84.5 3.25, m 3 35.4
1.63, 1.74, m 3' .sup. 77.1 3.52, m 4 26.2 1.42, m 4' .sup. 70.6
3.50, m 5 30.7 1.33, m 5' .sup. 66.6 3.16, 3.83, m 6-14 30.8 1.30,
m 1''.sup. 104.2 4.61, d (7.1) 15 26.9 1.33, 1.53, m 2''.sup. 83.1
3.51, m 16 33.5 1.33, 1.60, m 3''.sup. 74.4 3.80, m 17 76.0 3.36, m
4''.sup. 72.9 4.69, m 18 76.0 3.36, m 5''.sup. 63.5 3.23, 3.96, m
19 33.5 1.33, 1.60, m 1''' 105.4 4.67, d (7.6) 20 22.6 1.36, 1.62,
m 2''' 75.6 3.25, m 21 35.9 1.54, m 3''' 11.1 3.37, m 22 80.1 3.62,
m 4''' 71.4 3.30, m 23 35.4 1.43, 1.45, m 5''' 76.0 3.53, m 24 28.5
1.33, 1.38, m 6''' 64.4 4.21, 4.49, m 25 24.2 0.92, 1.32, m
4''--O--COCH.sub.3 172.5 -- 26 14.6 0.92, t (6.8)
4''--O--COCH.sub.3 20.8 2.06, s
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 174.8 --
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 44.2 2.29, d (7.1)
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 26.9 2.10, m
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 23.0 0.98, d (6.8)
TABLE-US-00008 TABLE 6 NMR data for Compound No. [13] aglycon
moiety carbohydrate moiety atom .delta..sub.C .delta..sub.H, mult.
atom .delta..sub.C .delta..sub.H, mult. 1 178.3 -- 1' .sup. 102.3
4.40, d (6.8) 2 71.5 4.08, m 2' .sup. 84.0 3.27, m 3 35.4 1.62,
1.74, m 3' .sup. 77.2 3.51, m 4 26.1 1.42, m 4' .sup. 70.7 3.50, m
5 30.6 1.33, m 5' .sup. 66.5 3.17, 3.86, m 6-14 30.2 1.29, m
1''.sup. 104.1 4.60, d (6.8) 15 26.9 1.33/1.52, m 2''.sup. 83.4
3.44, t (7.9) 16 33.4 1.32/1.60, m 3''.sup. 77.2 3.55, m 17 76.0
3.36, m 4''.sup. 70.7 3.50, m 18 76.0 3.36, m 5''.sup. 66.5 3.17,
3.86, m 19 33.4 1.32/1.60, m 1''' 105.4 4.71, d (7.6) 20 22.4
1.36/1.62, m 2''' 75.8 3.36, m 21 35.8 1.55, m 3''' 75.5 3.57, m 22
80.1 3.62, m 4''' 72.0 4.83, m 23 35.2 1.45, m 5''' 73.6 3.72, m 24
28.3 1.33, m 6''' 63.4 4.19, m 25 24.0 1.31, m 4'''--O--COCH.sub.3
171.8 -- 26 14.5 0.91, t (6.5) 4'''--O--COCH.sub.3 20.9 2.06, s
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 174.9 --
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 44.0 2.26, d (7.1)
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 26.7 2.08, m
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 22.9 0.96, d (7.0)
TABLE-US-00009 TABLE 7 NMR data for Compound No. [16] aglycon
moiety carbohydrate moiety atom .delta..sub.C .delta..sub.H, mult.
atom .delta..sub.C .delta..sub.H, mult. 1 178.3 -- 1' .sup. 102.3
4.43, d (6.5) 2 71.0 4.09, dd (7.1, 4.1) 2' .sup. 83.6 3.31, m 3
35.8 1.62, 1.74, m 3' .sup. 76.3 3.57, m 4 26.1 1.42, m 4' .sup.
70.7 3.51, m 5 30.5 1.33, m 5' .sup. 66.1 3.18, 3.86, m 6-14 30.2
1.29, m 1''.sup. 104.2 4.61, m 15 27.0 1.33/1.52, m 2''.sup. 84.0
3.44, m 16 33.4 1.32/1.60, m 3''.sup. 77.3 3.53, m 17 75.7 3.36, m
4''.sup. 70.8 3.51, m 18 75.7 3.36, m 5''.sup. 66.8 3.18, 3.86, m
19 33.5 1.32/1.60, m 1''' 105.7 4.62, d (7.2) 20 22.6 1.36/1.62, m
2''' 75.9 3.25, m 21 35.8 1.55, m 3''' 77.3 3.35, m 22 80.1 3.62, m
4''' 71.1 3.31, m 23 35.4 1.45, m 5''' 78.8 3.31, m 24 28.4 1.33, m
6''' 62.4 3.71, 3.90, m 25 24.1 1.31, m 26 14.5 0.91, t (6.5)
TABLE-US-00010 TABLE 8 NMR data for Compound No. [17] aglycon
moiety carbohydrate moiety atom .delta..sub.C .delta..sub.H, mult.
atom .delta..sub.C .delta..sub.H, mult. 1 176.4 -- 1' .sup. 102.3
4.44, d (6.8) 2 71.7 4.13, dd (7.6,4.6) 2' .sup. 83.6 3.31, m 3
35.3 1.64, 1.74, m 3' .sup. 76.4 3.57, m 4 25.8 1.42, m 4' .sup.
70.6 3.51, m 5 30.6 1.33, m 5' .sup. 66.2 3.19, 3.86, m 6-14 30.2
1.29, m 1''.sup. 104.2 4.61, d (6.5) 15 26.9 1.33/1.52, m 2''.sup.
84.0 3.43, t (7.8) 16 33.4 1.33/1.60, m 3''.sup. 77.3 3.53, m 17
76.0 3.35, m 4''.sup. 70.9 3.51, m 18 76.0 3.35, m 5''.sup. 66.7
3.17, 3.86, m 19 33.4 1.33, m 1''' 105.9 4.62, d (7.4) 20 22.4
1.35, m 2''' 75.9 3.25, m 21 35.8 1.53, m 3''' 77.7 3.36, m 22 80.2
3.63, m 4''' 70.9 3.31, m 23 33.4 1.46, m 5''' 78.8 3.31, m 24 28.5
1.34, m 6''' 62.5 3.71, 3.89, m 25 23.8 1.33, m 26 14.5 0.91, t
(7.0) OMe 52.4 3.71, s
TABLE-US-00011 TABLE 9 NMR data for Compound No. [12] aglycon
moiety carbohydrate moiety atom .delta..sub.C .delta..sub.H, mult.
atom .delta..sub.C .delta..sub.H, mult. 1 178.0 -- 1' .sup. 102.5
4.39, d (6.8) 2 71.5 4.09, dd (7.4, 4.4) 2' .sup. 84.4 3.26, m 3
35.5 1.62, 1.73, m 3' .sup. 77.4 3.52, m 4 26.1 1.42, m 4' .sup.
71.0 3.51, m 5 30.6 1.32, m 5' .sup. 66.5 3.18, 3.86, m 6-14 30.8
1.30, m 1''.sup. 104.3 4.55, d (6.8) 15 26.8 1.33, 1.52, m 2''.sup.
83.4 3.43, m 16 33.6 1.34, m 3''.sup. 76.9 3.55, m 17 76.0 3.36, m
4''.sup. 71.0 3.51, m 18 76.0 3.36, m 5''.sup. 66.5 3.18, 3.86, m
19 33.5 1.34, 1.49, m 1''' 105.4 4.65, d (6.8) 20 22.6 1.36, 1.61,
m 2''' 75.7 3.28, m 21 35.8 1.53, m 3''' 77.7 3.38, m 22 80.1 3.61,
m 4''' 71.5 3.30, m 23 35.4 1.47, 1.56, m 5''' 75.7 3.52, m 24 28.4
1.33, m 6''' 64.4 4.21, 4.49, m 25 24.1 1.32, m
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 174.9 -- 26 14.5 0.91, t
(6.8) 6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 44.2 2.28, d (7.1)
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 26.8 2.10, m
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 22.9 0.97, d (6.6)
TABLE-US-00012 TABLE 10 NMR data for Compound No. [14] aglycon
moiety carbohydrate moiety atom .delta..sub.C .delta..sub.H, mult.
atom .delta..sub.C .delta..sub.H, mult. 1 178.0 -- 1' .sup. 102.3
4.42, d (6.3) 2 71.5 4.09, dd (7.4, 4.6) 2' .sup. 83.2 3.30, m 3
35.8 1.62, 1.74, m 3' .sup. 76.8 3.57, m 4 26.1 1.42, m 4' .sup.
70.7 3.50, m 5 30.5 1.33, m 5' .sup. 66.3 3.17, 3.85, m 6-14 30.2
1.29, m 1''.sup. 104.1 4.69, d (7.1) 15 26.9 1.34, 1.55, m 2''.sup.
83.5 3.44, t (7.9) 16 33.4 1.33, m 3''.sup. 74.2 3.55, m 17 76.0
3.36, m 4''.sup. 72.9 3.50, m 18 76.0 3.36, m 5''.sup. 63.4 3.17,
3.86, m 19 33.4 1.33, 1.63, m 1''' 105.8 4.71, d (7.6) 20 22.5
1.38, 1.63, m 2''' 75.66 3.36, m 21 35.8 1.53, m 3''' 77.2 3.57, m
22 80.2 3.63, m 4''' 71.1 4.83, m 23 35.2 1.40, m 5''' 78.7 3.72, m
24 28.4 1.32, m 6''' 62.5 4.19, m 25 23.8 1.32, m
4'''--O--COCH.sub.3 172.2 -- 26 14.5 0.91, t (6.5)
4'''--O--COCH.sub.3 20.8 2.06, s
TABLE-US-00013 TABLE 11 NMR data for Compound No. [10] aglycon
moiety carbohydrate moiety atom .delta..sub.C*.sup.) .delta..sub.H,
mult. atom .delta..sub.C*.sup.) .delta..sub.H, mult. 1 179.1 -- 1'
.sup. 102.0 4.39, d (6.8) 2 72.2 3.97, dd (7.4, 4.4) 2' .sup. 84.0
3.25, m 3 35.4 1.60, 1.73, m 3' .sup. 76.9 3.50, m 4 26.1 1.42, m
4' .sup. 70.4 3.49, m 5-13 30.3 1.28, m 5' .sup. 66.3 3.17, 3.84, m
14 26.3 1.31, 1.42, m 1''.sup. 103.9 4.63, d (6.8) 15 38.2 1.35,
1.42, m 2''.sup. 83.2 3.52, m 16 72.1 3.50, m 3''.sup. 74.1 3.79, m
17 38.2 1.35, 1.42, m 4''.sup. 72.6 4.68, m 18 22.0 1.35, 1.51, m
5''.sup. 63.2 3.23, 3.97, m 19 35.7 1.51, m 1''' 105.2 4.66, d
(6.8) 20 79.8 3.61, m 2''' 75.4 3.27, m 21 34.6 1.44, 1.54, m 3'''
77.4 3.36, m 22 30.3 1.28, m 4''' 71.0 3.33, m 23 30.3 1.28, m 5'''
75.6 3.52, m 24 32.8 1.28, m 6''' 64.1 4.22, dd (11.7, 5.2), 4.46,
d (11.7 25 23.4 1.33, m 4''--O--COCH.sub.3 171.8 -- 26 13.9 0.91, t
(6.8) 4''--O--COCH.sub.3 20.5 2.06, s 6'''--O--COCH.sub.3 172.6 --
6'''--O--COCH.sub.3 20.7 2.10, s *.sup.)carbon chemical shifts
obtained from HSQC/HMBC experiments.
TABLE-US-00014 TABLE 12 NMR data for Compound No. [18] aglycon
moiety carbohydrate moiety atom .delta..sub.C*.sup.) .delta..sub.H,
mult. atom .delta..sub.C*.sup.) .delta..sub.H, mult. 1 178.1 -- 1'
.sup. 102.5 4.38, d (6.8) 2 71.5 4.07, m 2' .sup. 84.4 3.25, m 3
35.5 1.63, 1.74, m 3' .sup. 77.1 3.52, m 4 26.1 1.43, m 4' .sup.
70.6 3.51, m 5-13 30.6 1.30, m 5' .sup. 66.5 3.11, 3.84, m 14 26.0
1.35, 1.42, m 1''.sup. 104.3 4.62, d (6.5) 15 38.4 1.34, m 2''.sup.
83.3 3.52, m 16 72.4 3.50, m 3''.sup. 74.4 3.80, m 17 38.4 1.34, m
4''.sup. 72.9 4.69, m 18 22.3 1.35, 1.51, m 5''.sup. 63.5 3.23,
3.97, m 19 35.8 1.51, m 1''' 105.4 4.66, d (7.6) 20 80.2 3.61, m
2''' 75.6 3.26, m 21 34.9 1.44, 1.54, m 3''' 111 3.36, m 22 30.6
1.30, m 4''' 71.5 3.31, m 23 30.6 1.30, m 5''' 75.9 3.53, m 24 33.4
1.27, m 6''' 64.4 4.21, dd (11.7, 5.5), 4.56, d (11.7) 25 23.8
1.32, m 4''--O--COCH.sub.3 172.2 -- 26 14.5 0.91, t (6.8)
4''--O--COCH.sub.3 20.8 2.06, s 6'''--O--COCH.sub.3 174.8 --
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 44.2 2.29, d (7.1)
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 26.8 2.10, m
6'''--O--COCH.sub.2CH(CH.sub.3).sub.2 22.9 0.98, d (6.5)
TABLE-US-00015 TABLE 13 NMR data for Compound No. [7] aglycon
moiety carbohydrate moiety atom .delta..sub.C .delta..sub.H, mult.
atom .delta..sub.C .delta..sub.H, mult. 1 178.1 -- 1' .sup. 102.5
4.47, d (6.8) 2 71.5 4.09, m 2' .sup. 85.3 3.33, m 3 35.4 1.64,
1.75, m 3' .sup. 74.2 3.77, t (9.1) 4 26.1 1.43, m 4' .sup. 73.0
4.71, m 5 30.7 1.33, m 5' .sup. 63.5 3.25, 3.95, m 6-14 30.7 1.30,
m 1''.sup. 104.8 4.64, d (7.4) 15 26.9 1.33, 1.53, m 2''.sup. 84.7
3.40, m 16 33.4 1.34, 1.59, m 3''.sup. 77.6 3.53, m 17 76.0 3.37, m
4''.sup. 70.9 3.51, m 18 76.0 3.37, m 5''.sup. 66.9 3.17, 3.84, m
19 33.4 1.34, 1.59, m 1''' 106.1 4.64, d (7.6) 20 22.6 1.36, 1.61,
m 2''' 76.0 3.27, m 21 35.8 1.54, m 3''' 77.6 3.37, m 22 80.1 3.63,
m 4''' 71.1 3.33, m 23 34.8 1.47, m 5''' 75.9 3.55, m 24 28.4 1.34,
m 6''' 64.6 4.18, dd (12.0, 5.7), 4.41, d (11.4) 25 23.8 1.33, m
4'--O--COCH.sub.3 172.2 -- 26 14.5 0.92, t (6.8) 4'--O--COCH.sub.3
20.9 2.04, s 6'''--O--COCH.sub.3 172.7 -- 6'''--O--COCH.sub.3 21.0
2.09, s
TABLE-US-00016 TABLE 14 NMR data for Compound No. [6] aglycon
moiety carbohydrate moiety atom .delta..sub.C .delta..sub.H, mult.
atom .delta..sub.C .delta..sub.H, mult. 1 178.2 -- 1' .sup. 102.4
4.41, d (6.8) 2 70.7 4.12, m 2' .sup. 84.2 3.30, m 3 34.2 1.67,
1.78, m 3' .sup. 77.1 3.55, t (9.1) 4 24.6 1.46, m 4' .sup. 70.9
3.51, m 5 30.8 1.33, m 5' .sup. 66.4 3.21, 3.87, m 6-14 30.8 1.30,
m 1''.sup. 104.2 4.62, d (7.1) 15 27.0 1.33, 1.53, m 2''.sup. 83.6
3.40, m 16 34.5 1.34, 1.59, m 3''.sup. 77.3 3.53, m 17 75.7 3.37, m
4''.sup. 70.6 3.51, m 18 75.7 3.37, m 5''.sup. 66.7 3.17, 3.84, m
19 34.0 1.59, m 1''' 105.5 4.73, d (7.6) 20 22.5 1.48, 1.55, m 2'''
75.7 3.37, m 21 35.7 1.56, m 3''' 75.2 3.59, m 22 80.2 3.63, m 4'''
71.9 4.87, m 23 34.5 1.52, m 5''' 73.5 3.73, m 24 28.4 1.36, m 6'''
64.6 4.15, dd (11.0, 5.6), 4.22, d (11.7) 25 24.1 1.35, m
4'''--O--COCH.sub.3 172.2 -- 26 14.5 0.93, t (6.8)
4'''--O--COCH.sub.3 20.9 2.09, s 6'''--O--COCH.sub.3 172.7 --
6'''--O--COCH.sub.3 20.9 2.09, s
Example 3
HPLC-UV-MS-ELSD Analysis
A General Methods
a) "Standard Method"
[0549] LC-MS/UVELSD analyses were performed using an Agilent HP1100
(Agilent, Waldbronn, Germany) liquid chromatograph coupled with a
LCT mass spectrometer (Waters Corporation, Milford, Mass., USA) in
the positive and negative electrospray ionization (ESI) mode and a
Sedex 75 Evaporative Light Scattering Detector (Sedere, Alfortville
Cedex, France). A Waters symmetry column (Waters Symmetry.RTM.
(Trademark by Waters) C18, 3.5 .mu.m, 2.1 mm.times.150 mm, Waters
GmbH, Eschborn, Germany) was used as stationary phase with a flow
rate of 0.4 ml/min at 40.degree. C. Mobile phase A: 0.1% formic
acid in water, mobile phase B: 0.1% formic acid in acetonitrile;
gradient: 0-1 min. 98% A, from 1-21 min. to 100% B, from 21-27 min
100% B. The UVN is spectra were recorded between 200-500 nm, the
LC-MS (Liquid Chromatography-Mass Spectrometry coupling) spectra
were recorded in the range of molecular weights between 160 and
1.600 Da.
b) "Adapted Method"
[0550] LC-MS/UVELSD analyses were performed using an Agilent HP1100
(Agilent, Waldbronn, Germany) liquid chromatograph coupled with a
LCT mass spectrometer (Waters Corporation, Milford, Mass., USA) in
the positive and negative electrospray ionization (ESI) mode and a
Sedex 75 Evaporative Light Scattering Detector (Sedere, Alfortville
Cedex, France). A Waters symmetry column (Waters Symmetry.RTM.
(Trademark by Waters) C18, 3.5 .mu.m, 2.1 mm.times.150 mm, Waters
GmbH, Eschborn, Germany) was used as stationary phase with a flow
rate of 0.4 ml/min at 40.degree. C. Mobile phase A: 0.1% formic
acid in water, mobile phase B: 0.1% formic acid in acetonitrile;
gradient: 0 min: 55% A, from 0-14 min. to 100% B, from 14-16 min
100% B. The UV/Vis spectra were recorded between 200-500 nm, the
LC-MS (Liquid Chromatography-Mass Spectrometry coupling) spectra
were recorded in the range of molecular weights between 160 and
1.600 Da.
c) "Fermentation Control"
[0551] HPLC system: Agilent 1100 analytical HPLC system including
pumps and autosampler, DAD (200-500 nm) and ELSD detectors; column
oven at 40.degree. C.; column: Waters Symmetry.RTM. C18 3.5 .mu.m
(2.1.times.150 mm); solvents: deionised water (A) and acetonitrile
(B) with 0.1% formic acid each. The flow was adjusted to 0.4 ml/min
by using a temperature of 40.degree. C. The gradient applied was
optimized for separation and resolution of the glycolipid pattern:
0 to 14 min: from 45% to 100% (B); 14 to 16 min: 100% (B); 16 to
16.1 min: from 100% to 45% (B); 16.1 to 20 min: 45% (B). For HPLC
analysis, samples were dissolved in 2-propanol.
B HPLC-MS of Pure Compounds
TABLE-US-00017 [0552] TABLE 15 Retention times and MS signals for
compounds of the formula I, HPLC prepared according to the methods
specified in Section A of this Example.. HPLC HPLC Compound
"Standard "Adapted No. method" method" m/z [16] 14.09 2.94 [M +
H].sup.+ 887; [M acid].sup. 459 [14] 14.93 4.73 [M + H].sup.+ 929;
[M acid].sup. 459 [6] 15.15 4.99 [M + H].sup.+ 971; [M acid].sup.
459 [17] 15.39 5.55 [M + H].sup.+ 901; [M acid].sup. 473 [7] 15.44
5.68 [M + H].sup.+ 971; [M acid].sup. 459 [12] 15.79 6.07 [M +
H].sup.+ 971; [M acid].sup. 459 [3] 16.32 6.84 [M + H].sup.+ 1013;
[M acid].sup. 459 [13] 16.52 7.06 [M + H].sup.+ 1013; [M acid].sup.
459 [1] 17.00 7.48 [M + H].sup.+ 1013; [M acid].sup. 459 [4] 17.50
8.38 [M + H].sup.+ 1054; [M acid].sup. 459 [10] 17.67 8.50 [M +
H].sup.+ 955; [M acid].sup. 443 [18] 19.36 10.43 [M + H].sup.+ 997;
[M acid].sup. 443
C Characterization of Extracts
[0553] a) Extract of Ditiola pezizaeformis Strain ATCC13299
TABLE-US-00018 TABLE 16 HPLC-MS analysis of an extract obtained
from Ditiola pezizaeformis strain ATCC13299 (HPLC-ELSD chromatogram
presented in FIG. 3) HPLC Compound "Adapted Peak No. method" m/z 1
3.55 [M + H].sup.+ 943; [M acid].sup. 431 2 4.80 [M + H].sup.+ 985;
[M acid].sup. 431 3 [7] 5.63 [M + H].sup.+ 971; [M acid].sup. 459 4
5.83 [M + H].sup.+ 985; [M acid].sup. 431 5 [12] 6.08 [M + H].sup.+
971; [M acid].sup. 459 6 6.67 [M + H].sup.+ 927; [M acid].sup. 415
7 [1] 7.49 [M + H].sup.+ 1013; [M acid].sup. 459 8 7.90 [M +
H].sup.+ 969; [M acid].sup. 415 9 8.64 [M + H].sup.+ 969; [M
acid].sup. 415 10 8.86 [M + H].sup.+ 969; [M acid].sup. 415 11 9.58
[M + H].sup.+ 983; [M acid].sup. 429 12 [18] 10.51 [M + H].sup.+
997; [M acid].sup. 443
[0554] All shown signals were unequivocally assigned to be
glycolipids by HPLC-MS/UV.
b) Extracts of Dacryopinax spathularia Strain MUCL53181 Extract
[X1]: Glycolipid mixture prepared according to Example 1 from 30 l
fermentation Extract [X2]: Glycolipid mixture prepared according to
Example 1 from 200 l fermentation
TABLE-US-00019 TABLE 17 HPLC-MS analysis (using the "Adapted
method") of extracts obtained from Dacryopinax spathularia strain
MUCL 53181 (HPLC-ELSD chromatogram for [X2] is presented in FIG. 2)
Ret. Nominal time MW Identified compound [min] [Da] class [X1] [X2]
3.8 942 Glycolipid derivative * 0.4 4.3 928 Glycolipid derivative *
0.3 4.6 928 [14] * 0.4 4.9 984 Glycolipid derivative 0.3 5.1 970
[6] 0.9 1.3 5.6 970 [7] 72.5 25.1 6.1 970 [12] 8.3 12.3 6.5 998
Glycolipid derivative * 0.7 6.6 1012 Glycolipid derivative 0.9 *
6.8 1012 [3] * 10.8 7.0 998 Glycolipid derivative * * 7.1 1012 [13]
2.6 10.7 7.5 1012 [1] 7.8 29.8 7.8 1012 Glycolipid derivative 1.3
4.3 8.4 1054 [4] * 8.5 954 [10] 5.2 2.2 8.8 954 + 1054 Glycolipid
derivatives * 0.3 8.9 954 Glycolipid derivative * * 9.8 982
Glycolipid derivative * 0.4 10.0 996 Glycolipid derivative * 0.5
10.4 996 [18] 0.4 0.6 11.3 1038 Glycolipid derivative * * Peak area
below detection limit of ELSD, but compound was detected by ESI MS
signals.
Example 4
Biological Activities
A. Determination of Minimal Inhibition Concentration
a) Non-Pathogenic Microorganisms
[0555] Various nonpathogenic bacteria, yeasts, and filamentous
fungi were obtained from public culture collections and maintained
as recommended in the catalogues and protocols of the respective
institutions. Saccharomyces cerevisiae strain HT10 and Mucor
plumbeus were originally taken from the culture collection of
InterMed Discovery GmbH but deposited with MUCL as reference
strains for antimicrobial susceptibility testing. These strains
were maintained under liquid nitrogen and, prior to the screening,
on YMG agar.
[0556] Prior to the screening the yeast and bacterial strains were
grown over night on SDB (medium 1), except for Bacillus subtilis,
which was grown on YMG agar (medium 2) for 1 week to prepare spore
suspensions. Likewise, the filamentous fungi were pre-incubated on
YMG agar for 2-3 weeks to create inoculum for spore suspensions.
The spores were then rinsed from the surface of the flasks using
0.9% saline, checked for viability by microscopic control and by
plating on agar plates, and diluted to the desired concentration of
spores. For all experiments, freshly prepared spore suspensions
were used. The initial concentrations for the bioassays were
adjusted to 1.times.10.sup.5 CFU (i.e. cells or spores,
respectively) per ml. Standards (preservative agents benzoic acid
and sorbic acid; antibiotics: penicillin G, streptomycin sulphate,
amphopthericin B) served as positive controls.
[0557] To adjust the initial titer prior to the bioassays, the CFU
per ml was determined under the microscope using a counting chamber
type "Brand Neubauer improved"; BRAND GmbH & Co KG, Wertheim,
Germany). This microscopic control also served as means for
assessment of the viability of the cells.
[0558] The actual assays were carried out in Greiner type Bio-one
suspension culture plates (96 Well flat-bottom sterile, No
655185).
[0559] The compounds or extracts to be tested from stock material,
including standards, were dissolved in appropriate volumes of DMSO
prior to the test and diluted into the microtiter plates, using a
final concentration of 1.5%. Aside from regular incubation time for
determination of MIC (18-24 h), the stability of the inhibitory
effects was also studied at prolonged incubation times. For this
purpose, each microtiter plate vial was filled with 200 .mu.l of
the cell suspensions and the test plates were incubated in an
incubator (Heraeus HERA cell) at 28.degree. C. an absolute humidity
of 95%, to prevent evaporation of the solvent. Under such
conditions, no notable evaporation of the microtiter plates was
observed for up to several weeks. MICs were generally determined in
a traditional manner, by checking the MTP optically and determining
the dilution of each individual compound where no visible growth
had occurred. However, the OD.sub.630 was also determined using a
plate reader, in those cases where it appeared difficult to observe
the MIC with the naked eye. In some instances, the OD.sub.630 of
the plates could be monitored and determined using the plate reader
for up to four weeks. However, in general, the long term
experiments were run for at least 168 hours. For determination of
OD.sub.630, the microtiter plates were scanned using a SPECTROstar
Omega (BMG LABTECH, Offenburg, Germany) plate reader, except for
the filamentous fungi, where a PHERAstar plus (BMG LABTECH,
Offenburg, Germany) plate reader was used in "Wellscan" mode
(orbital averaging at 4 mm), since this instrument provided more
reliable data if mycelial colonies had arisen from the initial
spore suspensions. MIC in the optical readout was determined using
the following formula:
Inhibition [ % ] = 100 - ( ( x _ OD 630 [ sample ] ) .times. 100 (
x _ OD 630 [ control ] ) ) ##EQU00001##
[0560] The MIC values reported relate to the concentration causing
at least 80% inhibition as compared to the positive control.
Results:
TABLE-US-00020 [0561] TABLE 18 MIC values of standards in different
media: sorbic acid [SA] and benzoic acid [BA] [SA] [SA] [BA] [BA]
day SDB applejuice SDB applejuice Bacteria B. subtilis 2 250 250
(ATCC6633) 7 250 250 16 250 250 28 500 250 L. plantarum 2 >1000
31.3 >1000 250 (DSM12028) 7 >1000 500 >1000 >1000 16
>1000 >1000 28 >1000 >1000 L welshimeri 2 500 500 7 500
500 16 250 250 28 500 250 Filamentous fungi A. niger 2 250 500
(ATCC16404) 7 1000 >1000 16 1000 >1000 28 1000 >1000 M.
plumbeus 2 250 1000 (MUCL49355) 7 500 1000 16 1000 >1000 28 1000
>1000 Yeast D. bruxellensis 2 500 250 500 125 (DSM70726) 7 500
500 1000 125 16 >1000 500 >1000 250 28 >1000 >1000 D.
naardenensis 2 250 500 (DSM70743) 7 500 500 16 500 500 28 500 500
B. fulva 2 62.5 62.5 (DSM62097) 7 500 500 16 500 500 28 500 500 Z.
bailii 2 500 >1000 (DSM70492) 7 1000 >1000 16 >1000
>1000 28 >1000 >1000
TABLE-US-00021 TABLE 19 SDB medium: pure compounds day [1] [12]
[13] [6] [16] [18] [10] [14] Bacteria B. subtilis (ATCC6633) 2 3.1
3.1 3.1 1.6 3.1 3.1 <0.8 7 3.1 3.1 3.1 1.6 3.1 3.1 <0.8 16
6.3 6.3 3.1 6.3 3.1 3.1 <0.8 28 6.3 6.3 3.1 6.3 6.3 3.1 <0.8
Filamentous fungi A. niger (ATCC16404) 2 6.3 6.3 6.3 12.5 3.1 3.1
50 7 12.5 12.5 12.5 25 >100 6.3 50 16 12.5 12.5 62.5 25 >100
6.3 28 12.5 >100 25 12.5 50 >100 6.3 M. plumbeus (MUCL49355)
25 50 12.5 25 >100 25 12.5 50 50 100 25 25 >100 >100 50
>100 Yeast D. bruxellensis (DSM70726) 2 12.5 12.5 6.3 25 >100
3.1 3.1 7 25 25 12.5 25 >100 12.5 6.3 16 25 25 12.5 50 >100
100 6.3 28 25 25 25 50 >100 100 6.3 D. naardenensis 2 (DSM70743)
12.5 100 12.5 25 50 3.1 3.1 25 7 25 12.5 25 >100 50 6.3 100 1 25
50 12.5 25 >100 >100 6.3 28 25 >100 12.5 25 >100
>100 6.3 B. fulva (DSM62097) 2 3.1 6.3 3.1 3.1 6.3 1.6 1.6 7 6.3
12.5 3.1 6.3 6.3 1.6 1.6 16 6.3 12.5 3.1 6.3 6.3 1.6 1.6 28 31.3
12.5 15.6 6.3 31.3 7.8 1.6 Z. bailii (DSM70492) 2 25 >100 12.5
25 >100 >100 6.3 7 25 >100 12.5 25 >100 >100 12.5 16
12.5 >100 12.5 25 >100 >100 12.5 28 25 >100 12.5 25
>100 >100 12.5
TABLE-US-00022 TABLE 20 SDB medium: extracts day [X1] [X2] [X3]
[X4] [X5] [X6] Bacteria B. subtilis 2 <3.9 <3.9 <3.9
>500 7.8 <3.9 (ATCC6633) 7 <3.9 <3.9 <3.9 >500
7.8 <3.9 16 <3.9 <3.9 <3.9 <3.9 15.6 <3.9 28
<3.9 <3.9 <3.9 <3.9 31.3 7.8 Filamentous fungi A. niger
2 <3.9 <3.9 <3.9 <3.9 125 <3.9 (ATCC16404) 7 7.8 7.8
7.8 15.6 500 7.8 16 7.8 7.8 7.8 7.8 >500 7.8 28 7.8 7.8 7.8 15.6
>500 7.8 M. plumbeus 2 7.8 7.8 7.8 15.6 62.5 7.8 (MUCL49355) 7
15.6 15.6 15.6 31.3 125 15.6 16 15.6 31.3 31.3 31.3 >500 31.3 28
62.5 31.3 62.5 125 >500 250 Yeast D. naardenensis 2 7.8 <3.9
7.8 7.8 125 7.8 (DSM70743) 7 7.8 7.8 15.6 15.6 500 15.6 16 7.8 7.8
15.6 15.6 >500 31.3 28 7.8 15.6 15.6 15.6 >500 15.6 B. fulva
2 <3.9 <3.9 <3.9 <3.9 15.6 <3.9 (DSM62097) 7 <3.9
<3.9 <3.9 <3.9 62.5 <3.9 16 <3.9 <3.9 <3.9
<3.9 62.5 <3.9 28 <3.9 <3.9 <3.9 <3.9 62.5
<3.9
TABLE-US-00023 TABLE 21 SDB vs apple juice medium: pure compounds
and extracts day [1] [13] [6] [10] [X1] [X2] [X3] [X4] [X5] [X6]
Bacteria L. plantarum 2 (DSM12028) 12.5 6.3 25 3.1 >100 <3.9
7.8 <3.9 250 >500 SDB 7 25 12.5 25 6.3 >100 <3.9 15.6
500 7.8 16 25 12.5 50 6.3 15.5 <3.9 31.3 <3.9 >500 7.8 28
25 6.3 25 6.3 15.6 31.3 31.3 <3.9 >500 7.8 L. plantarum 2
(DSM12028) 6.3 <0.8 6.3 <0.8 <3.9 <3.9 <3.9 <3.9
62.5 <3.9 apple juice 7 >100 <0.8 50 <0.8 62.5 >500
<3.9 >500 259 125 Yeast Z. bailii (DSM70492) 2 25 12.5 25 6.3
7.8 7.8 7.8 15.6 125 >500 SDB 7 25 12.5 25 12.5 7.8 7.8 7.8 15.6
125 >500 16 12.5 12.5 25 12.5 7.8 7.8 7.8 15.6 250 15.6 28 25
12.5 25 12.5 15.6 7.8 7.8 15.6 250 15.6 Z. bailii (DSM70492) 2 6.3
3.1 3.1 3.1 <3.9 <3.9 >500 <3.9 31.3 <3.9 apple
juice 7 6.3 3.1 6.3 <3.9 <3.9 >500 <3.9 31.3 <3.9 16
6.3 3.1 3.1 3.1 <3.9 <3.9 <3.9 <3.9 31.3 <3.9 28 6.3
3.1 3.1 3.1 <3.9 <3.9 <3.9 <3.9 31.3 <3.9 D.
bruxellensis 2 (DSM70726) 12.5 6.3 25 3.1 7.8 <3.9 7.8 7.8 250
7.8 SDB 7 25 12.5 25 6.3 15.6 7.8 15.6 31.3 >500 15.6 16 25 12.5
50 6.3 15.6 7.8 15.6 15.6 >500 31.3 28 25 12.5 25 6.3 15.6 15.6
15.6 31.3 >500 31.3 D. bruxellensis 2 (DSM70726) 6.3 3.1 3.1 3.1
<3.9 <3.9 <3.9 <3.9 15.6 <3.9 apple juice 7 25 6.3
3.1 3.1 <3.9 <3.9 <3.9 <3.9 31.3 <3.9 16 6.3 6.3 3.1
3.1 <3.9 <3.9 <3.9 <3.9 31.3 <3.9 28 6.3 3.1 3.1 3.1
<3.9 <3.9 <3.9 <3.9 31.3 <3.9 Note: Apple juice
medium per se shows already limited growth which is based mainly on
the low concentration of nitrogen compounds available and needed
for growth.
[0562] It is evident and remarkable that independently of the
extraction method (e.g. X1-X3) comparable results are achieved.
Bacteria
[0563] Bacillus subtilis (ATCC6633) Clostridium perfringens
(ATCC13124) Corynebacterium variabile (DSM20132) Corynebacterium
variabile (ATCC15753) Escherichia coli (ATCC9637) Lactobacillus
plantarum (DSM12028) Pseudomonas putida (ATCC17484) Staphylococcus
aureus (ATCC 6538P)
Filamentous Fungi (<<Molds>>)
[0564] Aspergillus fumigatus (ATCC1028) Aspergillus niger
(ATCC16404) Byssochlamys fulva (DSM62097) Mucor plumbeus
(MUCL49355)
Yeasts
[0565] Dekkera bruxellensis (DSM70726) Dekkera naardenensis
(DSM70743) Saccharomyces cerevisiae (HT10) Zygosaccharomyces bailii
(DSM70492) Zygosaccharomyces bailii (ATCC60484) Zygosaccharomyces
bisporus (DSM70415) Zygosaccharomyces florentinus (DSM70506)
Zygosaccharomyces rouxii (NCYC381)
b) Pathogenic Microorganisms
[0566] Several samples of extracts and pure compounds were tested
against Staphylococcus aureus (ATCC 6538P), Clostridium perfringens
(ATCC 13124) or Aspergillus fumigatus (ATCC 1028). The test was
performed by Ricerca Biosciences, LLC (Taiwan): S. aureus (cat no
604000) with Mueller-Hinton Broth medium using 1% DMSO as vehicle
for an incubation time of 20 hours at 36.degree. C. (di Modugno,
Antimicrob Agents Chemother (1994) 38: 2362-8); C. perfringens (cat
no 620700) with Reinforced Clostridial Medium using 1% DMSO as
vehicle for an incubation time of 2 days at 36.degree. C. (di
Modugno, ibid); A. fumigatus (cat no 640010) with Potato Dextrose
Broth medium using 1% DMSO as vehicle for an incubation time of 3
days at 28.degree. C. (Turner, Antimicrob Agents Chemother (1989)
33(2): 215-22). MICs were detected via turbidity measurements in
all cases.
[0567] The average mol weight was set to 1000 g/mol, the presented
data in the following table 22 are given in mg/ml (ppm).
TABLE-US-00024 TABLE 22 MICs [ppm] of extracts and pure compounds
against human pathogenic microorganisms. S. aureus C. perfringens
A. fumigatus D. spathularia MUCL 60 20 53181 extract D. spathularia
MUCL 60 60 53182 extract [1] 200 60 20 [12] 200 60 20
Example 5
Sensory Evaluation
[0568] The extract [X2] was dissolved in water to final
concentrations of 10 ppm and 100 ppm. These two test samples were
presented together with a pure water sample (as negative control)
to two test persons. The three samples were blinded prior to the
taste evaluation.
[0569] None of the test persons were able to discriminate between
pure water and the sample containing 10 ppm of the test compound.
The sample containing 100 ppm of the test compound was described
with a diffuse taste comparable with water which was stored for a
longer time in an open PET bottle. No bitter, spicy or otherwise
unpleasant taste was observed.
[0570] In a second trial one test person tried the pure dry powder
of the above test compound. Even after this application no further
adverse or unpleasant taste was named.
Example 6
Optical Rotation Values
[0571] Optical rotation values were determined in methanolic
solutions using a Schmidt-Haensch, Unipol L 1000 polarimeter
equipped with a silica glass micro cuvette (100 mm length; 1 ml
sample volume).
TABLE-US-00025 TABLE 23 Specific optical rotation values in
methanol Compound/ extract alpha.sup.D.sub.20 c [g/100 mL] [1]
-20.0 0.34 [13] -22.3 0.36 [16] +23.5 0.22 [17] n.d. [12] -19.1
0.93 [14] n.d. [10] -18.8 0.52 [18] -23.0 0.95 [7] n.d. [6] -29.4
0.64 [X2] -25.5 0.68
Example 6
Apple Juice Based Beverage
[0572] (cf. WO 2010/148045)
[0573] A 2% fruit juice based non-carbonated beverage of pH 3.4 and
about 12 Brix is formed by combining the following ingredients.
TABLE-US-00026 Ingredient % Composition Water ca. 84 (added to
final 100%) Apple juice concentrate 0.372 of concentrate to provide
single strength concentration of about 2% Sucrose 6.8 Glucose 5.2
Fructose 0.2 Compound extract [X2] 0.000001-0.5% Malic acid 0.134
Sodium malate 0.013 CaCl2--2H2O 0.011 MgCl2--6 H2O 0.003 EDTA
0.003
[0574] A pH of 3.4 is achieved through combinations of malic acid
and sodium malate. The total combined quantity of sodium malate and
malic acid is near constant, but the ratio of malic acid and malate
varied slightly depending on the content of compound extract
[X2].
[0575] The above mixture is inoculated with test organisms and
incubated for weeks at ambient temperature.
Example 7
Semifinished Products
[0576] A) The following mixtures can be used as concentrates for
preservative efforts in different foods or beverages.
TABLE-US-00027 Compound Concentration in % Variation A B C Water
ca. 70 ca. 84 ca. 50 Sucrose, acetate 2 5 isobutyrate Xanthan gum
0.16 Gum arab 6 4 Beeswax 1 TWEEN 20 5 Ethanol 10 Glycerol 5 Citric
acid 5 Orange oil 5 Compound [X2] 0.1 0.1 0.1
[0577] Compound extract [X2] is used as concentrated solution
(concentrate) in DMSO which will be diluted to a final DMSO content
of (concentrate) in the semi finished products. The compound
extract [X2] solution is premixed with the appropriate alcohol, in
variation C together with the orange oil, in variation B together
with the beeswax, whereas the thickeners are mixed with an
appropriate volume of water. The two mixtures are vigorously
stirred, combined by continuing the stirring and filled up with
water to the final volume (100%).
B) The following mixture is to be used as concentrate for an apple
juice beverage (9 liter)
TABLE-US-00028 Ingredients Amount Sucrose 10% Clear juice (apple)
10% Flavor emulsion (variant C 0.2% of the above table) Citric acid
0.15%.sup. Water ca. 7.2 I
[0578] The semifinished products are tested against microorganisms
e.g. mold, yeasts and/or bacteria.
Example 8
Production and Isolation of a Mixture of Compounds of Formula I and
a Mixture of Sodium Salts of Compounds of Formula I
[0579] A) Fermentation Using the Strain FU50088 (Dacryopinax
spathularia Strain MUCL 53181)
a) Seed Cultures (Shake Flask Cultures)
[0580] One ml of a cryo vial containing a mycelial suspension of
strain MUCL 53181 in 10% glycerol was retrieved from liquid
nitrogen and, after thawing, used to inoculate 200 ml Erlenmeyer
flasks containing 50 ml of sterile YMG medium and propagated on a
rotary shaker at 240 rpm and 23.degree. C. for 72 h. Thereafter,
each two ml of the primary seed culture were transferred into
batches of two 500 ml Erlenmeyer flasks containing 200 ml of the
same medium and propagated on a rotary shaker at 140 rpm and
28.degree. C. for 90 h. These flasks served as secondary seed
cultures.
b) Fermentation in 301 Scale
[0581] A 40 l Biostat LP42 fermentor (Bioengineering, Wald,
Switzerland) containing 301 of GM2 medium was sterilized in situ (1
h at 121.degree. C. and 1.1 bar) and inoculated with 1500 ml of the
secondary seed culture. The production culture was grown under
stirring (240 rpm) and aeration (0.2 vvm (volumes of air per minute
per volume of batch) at 30.degree. C. for 200 h.
B) Downstream Processing and Isolation
B-1) Isolation of a Mixture of Compounds of Formula I
[0582] The culture broth resulting from the 301 fermentation of
Example 8A) b) was alkalized from an initial pH value of 4.5 to a
pH value of 8 with 5 N sodium hydroxide solution to allow the
glycolipids that partially stick to the cells under acidic
conditions to become largely released from the mycelia. After 1 h
the mycelia were separated from the culture broth by
centrifugation, and then the culture broth was filtrated
additionally through a Pall T1000 depth filter (Dreieich, Germany)
to remove cell clusters and filamentous material. The product was
sedimented (precipitated) by acidifying the filtrate with 6 N
hydrochloric acid to pH 2.2 and subsequent storing for 20 h at
4.degree. C. The fluid was removed by decantation, subsequent
centrifugation (4500 rpm, 15 min, Type Jouan SA LR 5.22 (Jouan,
Paris, France) and then discarded, resulting in a whitish-grey gel.
This crude product was washed immediately with 11 of slightly basic
demineralized water (set to a pH-value of 8 with sodium hydroxide)
and centrifuged at (4500 rpm for 15 min). The supernatant was
removed and the remaining pellet suspended in 0.5 l demineralized
water. After decantation the residue was freeze-dried to yield a
slightly beige-grey powder (residual water content: 1.36%
(according to the Karl Fischer method), the total protein content
was below 1% (<1%) (Kjeldahl method according to ISO 5549:
1978). This process yielded a total of 87 g of glycolipids which
subsequently were characterized by HPLC-MS (see [X7] in Table
24).
B-2) Isolation of a Mixture of Sodium Salts of Compounds of Formula
I
[0583] The culture broth resulting from the 301 fermentation of
Example 8A) b) was alkalized from an initial pH value of 4.5 to a
pH value of 8 with 5 N sodium hydroxide solution to allow the
glycolipids that partially stick to the cells under acidic
conditions to become largely released from the mycelia. After 1 h
the mycelia were separated from the culture broth in a separator,
and then the culture broth was pumped through a combined filter
assembly: first though a depth filter with a pore size of 0.65
.mu.m and then through a membrane filter with a pore size of 0.45
.mu.m to remove not only filamentous material and cell clusters but
also cells. The product was sedimented (precipitated) by acidifying
the filtrate with 6 N hydrochloric acid to pH 2 and then stored for
16 h at 4.degree. C. The fluid was removed by decantation,
subsequent centrifugation (4500 rpm, 15 min, Type Jouan SA LR 5.22
(Jouan, Paris, France) and then discarded, resulting in a
whitish-grey gel. This crude product was washed immediately with 1
l demineralized water and centrifuged at (4500 rpm for 15 min). The
supernatant was removed, the remainder suspended in 0.5 l
demineralized water and the pH adjusted to a pH value of about 6
with 5 N sodium hydroxide solution. Finally, the resulting solution
was freeze-dried to yield a total of 93 g of sodium salts of
glycolipids in dry form as a slightly beige powder (residual water
content: 1.2% (according to the Karl Fischer method), the total
protein content was below 1% (<1%) (Kjeldahl method according to
ISO 5549: 1978).
C) HPLC-MS Analysis--"Improved Method"
[0584] HPLC-MS analyses were performed using a Dionex Ultimate.RTM.
3000 RSLC (Thermo Fisher GmbH, Idstein; Germany) liquid
chromatograph coupled with a amaZon SL ion trap mass spectrometer
(Bruker Daltonik GmbH, Bremen, Germany) in the negative
electrospray ionization (ESI) mode and a Sedex 85 ELSD (Sedere,
Alfortville Cedex, France). A Nucleoshell RP18 column (2.7 .mu.m, 2
mm.times.150 mm, Macherey-Nagel GmbH & Co. KG, Duren, Germany)
was used as stationary phase with a flow rate of 0.4 ml/min at
40.degree. C. Mobile phase A: 0.1% formic acid in water, mobile
phase B: 0.1% formic acid in acetonitrile; gradient: 0 min: 70% A,
from 0-40 min. to 40% A, from 40-42 min to 0% A, from 42-48 min. 0%
A, from 48-49 min to 70% A, from 49-55 min 70% A. The LC-MS (Liquid
Chromatography-Mass Spectrometry coupling) spectra were recorded in
the range of molecular weights between 700 and 1.200 Da.
TABLE-US-00029 TABLE 24 HPLC-MS analysis (using the "Improved
method") of the extract [X7] obtained from Dacryopinax spathularia
strain MUCL 53181 according to Example 8 B-1) Ret. Molecular time
weight [X7]* [min] [Da] Identified compound (type) in wt. % 13.49
886 [16] 0.1 14.20 886 Glycolipid without acyl groups 0.1 14.46 942
Glycolipid 1.6 15.46 928 Glycolipid 0.4 15.79 928 Glycolipid 1.2
16.27 928 Glycolipid 0.2 16.55 928 Glycolipid 1.2 16.98 928; 956
Glycolipids 1.1 17.38 928 [14] 0.8 17.49 970 Glycolipid 0.2 17.78
970 Glycolipid 0.9 18.26 970 Glycolipid 1.6 18.58 970 Glycolipid
0.5 19.00 970 [6] 2.3 19.73 970 Glycolipid 16.9 19.98 970 [7] 22.6
20.66 970; 984 Glycolipids 1.1 20.90 970 Glycolipid 3.4 21.44 970
Glycolipid 2.3 21.69 970 [12] 2.8 22.02 984 Glycolipid 0.3 22.57
970; 998 Glycolipids 1.3 22.86 926; 998 Glycolipids 1.2 23.09 1012
Glycolipid 1.0 23.91 1012 [5] 1.1 24.02 1012 [9] 2.4 24.19 1012
Glycolipid 0.4 24.73 1012 [13] 1.0 25.00 1012 Glycolipid 4.0 25.96
1012 [8] 1.9 26.22 1012 [1] 6.7 26.34 1012 Glycolipid 3.0 27.18
954; 1012 Glycolipids 0.7 27.33 1012 Glycolipid 0.4 27.89 968
Glycolipid 0.4 29.08 954 [10] 5.4 29.51 954; 968; 1054 Glycolipids
0.9 29.81 1054 [4] 1.6 30.52 954 Glycolipid 0.3 30.93 1054
Glycolipid 0.3 33.65 982 Glycolipid 0.5 34.46 996 Glycolipid 1.1
36.04 996 [18] 1.5 43.86 No Glycolipid 1.1 *Some minor peaks of
[X7] are not listed in Table 24.
D) Fermentation in 400 Ml Scale
[0585] A 1 l Erlenmeyer flask containing 400 ml of sterilized
medium (2.0% D-glucose, 0.5% malt extract) was inoculated with 2 ml
of the secondary seed culture from Example 8A a). The production
culture was grown on a rotary shaker at (200 rpm) at 32.degree. C.
for 360 h.
[0586] Work-up after 360 h of fermentation yielded a total of 5.6
g/l of glycolipids of formula I which subsequently were
characterized by HPLC-MS (see [X8] in Table 24A).
TABLE-US-00030 TABLE 24A HPLC-MS analysis (using the "Improved
method" of Example 8 C)) of the extract [X8] obtained from
Dacryopinax spathularia strain MUCL 53181 according to Example 8 D)
(the HPLC-ESI chromatogram for [X8] is presented in FIG. 4) Ret.
Molecular time weight [X8]* [min] [Da] Identified compound (type)
in wt. % 13.08 886 Glycolipid without acyl groups 0.4 13.49 886
[16] 0.5 14.20 886 Glycolipid without acyl groups 0.4 14.46 942
Glycolipid 0.6 15.46 928 Glycolipid 0.5 15.79 928 Glycolipid 1.0
16.27 928 Glycolipid 0.2 16.55 928 Glycolipid 1.6 16.98 928; 956
Glycolipids 1.0 17.38 928 [14] 0.1 17.49 970 Glycolipid 1.4 17.78
970 Glycolipid 1.9 18.26 970 Glycolipid 0.3 18.58 970 Glycolipid
1.5 19.00 970 [6] 1.9 19.73 970 Glycolipid 12.7 19.98 970 [7] 12.4
20.66 970; 984 Glycolipids 1.4 20.90 970 Glycolipid 5.5 20.99 870
Glycolipid without acyl groups 1.5 21.44 970 Glycolipid 2.1 21.69
970 [12] 3.0 22.02 984 Glycolipid 0.4 22.57 970; 998 Glycolipids
2.9 23.09 1012 Glycolipid 0.9 23.91 1012 [5] 1.7 24.02 1012 [9] 2.8
24.19 1012 Glycolipid 0.8 24.73 1012 [13] 1.2 25.00 1012 Glycolipid
4.6 25.96 1012 [8] 2.1 26.22 1012 [1] 8.8 26.34 1012 Glycolipid 3.6
27.18 954; 1012 Glycolipids 1.3 27.33 1012 Glycolipid 4.4 27.89 968
Glycolipid 0.5 29.08 954 [10] 4.7 29.51 954; 968; 1054 Glycolipids
1.1 29.81 1054 [4] 0.2 30.52 954 Glycolipid 0.6 30.93 1054
Glycolipid 0.6 33.65 982 Glycolipid 0.6 34.46 996 Glycolipid 0.7
36.04 996 [18] 2.2 43.86 No Glycolipid 1.0 *Some minor peaks of
[X8] are not listed in Table 24A.
E) Fermentation in 400 ml Scale
[0587] A 1 l Erlenmeyer flask containing 400 ml of sterilized
medium (4.0% D-glucose, 0.2% yeast extract) was inoculated with 6
ml of the secondary seed culture from Example 8A a). The production
culture was grown on a rotary shaker at (200 rpm) at 32.degree. C.
for 240 h.
[0588] Work-up after 240 h of fermentation yielded a total of 6.2
g/l of glycolipids of formula I.
Example 9
Biological Activities
[0589] In analogy to the methods described in Example 4 above,
further biological activity MIC data were determined for several
pure (purity>94 wt. %) compounds and for the mixture of
compounds of formula I (extract [X8] as described in detail in
Example 8D).
[0590] The following Tables 25-27 represent the corresponding MIC
values of several pure compounds after 48 h against various
microorganisms.
[0591] In summary, Tables 25-27 demonstrate that individual
compounds of formula I without any acyl substituents in the
trisaccharide carbohydrate moiety R (such as the case for compound
[16]) typically exhibit a significantly weaker antimicrobial
activity, particularly against yeasts and molds, than the
corresponding compounds being mono- or diacylated in the saccharide
moiety.
[0592] Said Tables also demonstrate that individual compounds of
formula I with an acyl substituent with more than 2 carbon atoms,
such as an isovaleryl substituent, in the trisaccharide
carbohydrate moiety R (such as the case for compounds [12], [13]
and [1]) typically exhibit a stronger antimicrobial activity,
particularly against yeasts and molds, and/or a broader activity
spectrum than the corresponding compounds with one acetyl
substituent in the trisaccharide carbohydrate moiety R (such as
compound [14]).
TABLE-US-00031 TABLE 25 MIC values [ppm] after 48 h of pure
compounds against Gram-positive bacteria Bacteria [16] [14] [12]
[13] [1] Bacillus subtilis (ATCC6633) 3.1 1.6 1.6 3.1 3.1
Propionibacterium acnes 100 (ATCC6919) Clostridium perfringens 60
60 (ATCC13124) Corynebacterium variabile 1.6 1.6 <0.8 1.6 1.6
(DSM20132) Lactobacillus plantarum 50 25 25 25 25 (DSM12028)
TABLE-US-00032 TABLE 26 MIC values [ppm] after 48 h of pure
compounds against filamentous fungi (molds) Filamentous fungi [16]
[14] [12] [13] [1] Aspergillus fumigatus 20 20 (ATCC1028)
Aspergillus niger (ATCC16404) 25 50 6.3 12.5 3.1 Byssochlamys fulva
(DSM62097) 6.3 6.3 3.1 3.1 Mucor plumbeus (MUCL49355) >100 50
6.3 12.5 6.3 Talaromyces luteus (CBS348.51) 50 50 25 25 12.5
Pyricularia oryzae (DSM62938) 25 25 25 6 6 Dekkera bruxellensis
>100 50 6.3 6.3 6.3 (DSM70726) Dekkera naardenensis 50 25 6.3
12.5 6.3 (DSM70743)
TABLE-US-00033 TABLE 27 MIC values [ppm] after 48 h of pure
compounds against yeasts Yeasts [16] [14] [12] [13] [1]
Saccharomyces cerevisiae (HT10) >100 50 6.3 12.5 6.3
Zygosaccharomyces bailii 25 25 6.3 6.3 6.3 (DSM70492)
Zygosaccharomyces bisporus >100 50 12.5 25 12.5 (DSM70415)
Zygosaccharomyces florentinus >100 50 6.3 12.5 6.3 (DSM70506)
Zygosaccharomyces rouxii 100 25 6.3 12.5 6.3 (NCYC381) Candida
albicans (ATCC10231) 30
[0593] The following Table 28 represents the MIC values [ppm] of
mixture [X8] and pure compound [12] after 48 h against various
microorganisms.
TABLE-US-00034 TABLE 28 MIC values [ppm] of mixture [X8] and pure
compound [12] after 48 h [X8] [12] Bacteria Bacillus cereus
(ATCC11778) 12.5 12.5 Bacillus subtilis (ATCC6633) 1.6
Propionibacterium acnes (ATCC6919) 60 Clostridium perfringens
(ATCC13124) 60 Clostridium sporogenes (ATCC3584) 50 50 Enterococcus
faecalis (ATCC19433) 50 50 Listeria welshimeri (DSM15452) 25 25
Listeria monocytogenes (ATCC19111) 50 50 Corynebacterium variabile
(DSM20132) <0.8 Lactobacillus plantarum (DSM12028) 25
Staphylococcus aureus (ATCC6538) 25 Filamentous fungi Aspergillus
fumigatus (ATCC1028) 20 Aspergillus niger (ATCC16404) 6.3
Byssochlamys fulva (DSM62097) 3.1 Mucor plumbeus (MUCL49355) 6.3
Talaromyces luteus (CBS348.51) <3.9 Pyricularia oryzae
(DSM62938) 6 Dekkera bruxellensis (DSM70726) 6.3 Dekkera
naardenensis (DSM70743) 12.5 Yeasts Saccharomyces cerevisiae (HT10)
12.5 Zygosaccharomyces bailii (DSM70492) 6.3 Zygosaccharomyces
bisporus (DSM70415) 12.5 Zygosaccharomyces florentinus (DSM70506)
6.3 Zygosaccharomyces rouxii (NCYC381) 6.3 Candida albicans
(ATCC10231) 12.5 25 Candida glabrata (ATCC36583) 20
[0594] At a concentration of 25 ppm [X8], the observed inhibition
of Bacillus cereus was 90%. At a concentration of 12.5 ppm [X8],
the observed inhibition of Candida albicans was 90-100%. At a
concentration of 50 ppm [X8], the observed inhibition of
Clostridium sporogenes was 80-90%. At a concentration of 100 ppm
[X8], the observed inhibition of Staphylococcus aureus was 90%.
[0595] The following Table 29 shows the results of several bacteria
count tests performed with mixture [X8] for various microorganisms
in comparison with an untreated control. Tests were performed at
37.degree. C. at a pH of 7.4 in full medium. The respective
bacteria count (Ba.C) is indicated in colony forming units/ml
(CFU/ml).
TABLE-US-00035 TABLE 29 Bacteria count tests performed with mixture
[X8] Sample with Untreated Initial [X8] Ba.cndot.C control
Ba.cndot.C Ba.C after after after after [X8] 0 h 2 h 6 h 2 h 6 h
Listeria 100 5 .times. 10.sup.5 1 .times. 10.sup.4 1 .times.
10.sup.4 1.1 .times. 10.sup.6 5 .times. 10.sup.7 monocytogenes ppm
(ATCC19111) Staphylococcus 100 1 .times. 10.sup.5 1 .times.
10.sup.4 5 .times. 10.sup.3 5 .times. 10.sup.5 1 .times. 10.sup.8
aureus ppm (ATCC6538) Enterococcus 200 5 .times. 10.sup.4 1 .times.
10.sup.4 1 .times. 10.sup.4 5 .times. 10.sup.5 1 .times. 10.sup.8
faecalis ppm (ATCC19433) Bacillus cereus 50 1 .times. 10.sup.5 1
.times. 10.sup.2 <10 2 .times. 10.sup.6 7 .times. 10.sup.8
(ATCC11778) ppm Micrococcus 500 1 .times. 10.sup.8 2 .times.
10.sup.2 2 .times. 10.sup.1 >1 .times. 10.sup.8 >1 .times.
10.sup.9 luteus ppm
Example 10
Sugar-Free Beverage Compositions
TABLE-US-00036 [0596] A B C D Ingredient in wt. % in wt. % in wt. %
in wt. % Potassium sorbate -- -- 0.03 -- Sodium benzoate 0.03 --
0.015 -- Phosphoric acid 0.048 0.06 0.025 0.16 Citric acid 0.016
0.012 0.025 0.02 Caffeine 0.013 0.009 -- -- Sucralose -- -- 0.008
0.0052 Aspartame 0.030 0.018 -- -- Acesulfame K 0.0095 0.0055 0.019
-- Na cyclamate -- 0.050 0.045 0.009 Na Saccharin -- 0.006 0.01 --
Erythritol 0.20 -- -- 0.45 Dimethylpolysiloxane -- -- 0.00075
0.0008 Lemon-orange flavor -- -- 0.25 0.15 Cola flavor 0.35 0.42 --
0.20 Caramel color (E150d) 0.009 0.009 -- 0.008 Extract [X7] of
0.0003 -- -- 0.0001 Example 8 B-1) Extract [X8] of -- 0.0005 0.0004
-- Example 8 D) Pure compound [1] -- -- -- 0.0003 Drinking water Ad
100 Ad 100 Ad 100 Ad 100 The beverage compositions A and C each
were carbonated with 3.8 volumes of carbon dioxide after filling
into bottles. The beverage compositions B and D each were
carbonated with with 3.0 volumes of carbon dioxide after filling
into bottles.
[0597] The beverage compositions A and C each were carbonated with
3.8 volumes of carbon dioxide after filling into bottles. The
beverage compositions B and D each were carbonated with 3.0 volumes
of carbon dioxide after filling into bottles.
Example 11
Tea Beverage Compositions
TABLE-US-00037 [0598] A B C D Ingredient in wt. % in wt. % in wt. %
in wt. % Sucrose 3.30 -- -- -- Trisodium citrate 0.50 0.50 0.25 --
Citric acid 0.40 0.60 0.50 0.80 Malic acid -- 0.20 0.30 0.50
Ascorbic acid 0.20 0.10 0.15 0.20 Water soluble green 2.50 2.25 --
-- tea powder Water soluble Ceylon -- -- 2.75 2.40 black tea powder
Sucralose -- 0.08 0.05 -- Acesulfame K 0.0035 0.0035 0.0045 0.0055
Aspartame -- -- -- 0.025 Rebaudioside A 0.01 -- 0.005 -- Peach
flavor -- -- 0.40 -- Lemon flavor -- -- -- 0.70 Jasmine tea flavor
-- 0.45 -- -- Colorant 0.007 0.009 0.009 0.008 Extract [X7] of
0.0003 0.0002 Example 8 B-1) Extract [X8] of -- 0.0005 0.0004
0.0003 Example 8 D) Drinking water Ad 100 Ad 100 Ad 100 Ad 100
Example 12
Beverage Compositions
TABLE-US-00038 [0599] A B C D Ingredient in wt. % in wt. % in wt. %
in wt. % Sucrose 7.70 10.40 6.00 4.00 Erythritol 0.22 -- -- 0.40
Tagatose 0.44 -- -- 0.30 Trehalose -- -- 3.00 -- Fructose -- --
1.00 1.00 Citric acid, anhydrous 0.30 0.235 0.21 0.21 Sodium
benzoate -- -- 0.04 0.05 Potassium sorbate -- -- 0.02 0.03 Ascorbic
acid 0.10 0.15 0.10 0.125 Lemon oil, cold pressed 0.08 -- -- --
Orange oil, cold pressed 0.02 0.09 0.09 0.09 FD&C Yellow 5
0.003 0.007 0.007 0.007 FD&C blue No. 1 0.006 -- 0.002 0.002
Rebaudioside A -- 0.008 -- 0.02 Titanium dioxide -- 0.08 -- --
Extract [X7] of 0.0003 -- -- -- Example 8 B-1) Extract [X8] of --
0.0005 0.0001 0.0003 Example 8 D) Pure compound [1] -- -- 0.0002 --
Tap water Ad 100 Ad 100 Ad 100 Ad 100 The beverage compositions A
and C each were carbonated with 4 volumes of carbon dioxide after
filling into bottles. The beverage composition B was carbonated
with 2 volumes of carbon dioxide after filling into bottles.
[0600] The beverage compositions A and C each were carbonated with
4 volumes of carbon dioxide after filling into bottles. The
beverage composition B was carbonated with 2 volumes of carbon
dioxide after filling into bottles.
Example 13
Yogurt Drink Compositions
[0601] Skimmed milk and whole milk were mixed in proportions to
give milk with 1.1% fat, then 5 wt. % of sucrose were added
thereto, and heated to 82.degree. C. for 30 minutes. After cooling
to 42.degree. C., 0.7% of a commercially available starter culture
of Bifidobacterium bifidum and 0.5% of a starter culture of
Streptococcus thermophilus culture were added, and the mixture
cultured at 39.degree. C. until the pH-value of the mixture reached
4.4. The resulting firm yoghurt curd was then broken by stirring,
and split in two portions (portion A and portion B).
[0602] Preparation of LiqYog A: To stirred portion A were added 0.4
wt. % (based on the mass of firm yoghurt) of high methoxyl citrus
pectin as 5 wt. % solution in water and the mixture cooled with
stirring to 5.degree. C. This product was then passed through a
sterilized homogenizer at 40 bar to give a liquid yoghurt having a
dynamic viscosity of 380 mPas at 10.degree. C. Thereto were first
added with stirring 45 ppm of the extract [X7] of Example 8B-1),
and then 9.0 wt. % of a pasteurized peach pulp, in both cases based
on the total weight of the liquid yoghurt. The resulting mixture
was homogenized giving LiqYog A which was transferred into a glass
container, and stored at 7.degree. C. for 10 days.
[0603] Preparation of LiqYog B: To stirred portion B were added 0.6
wt. % (based on the mass of firm yoghurt) of high methoxyl apple
pectin as 5 wt. % solution in water and the mixture cooled with
stirring to 5.degree. C. This product was then passed through a
sterilized homogenizer at 15 bar to give a liquid yoghurt having a
dynamic viscosity of 600 mPas at 10.degree. C. Thereto were first
added with stirring 95 ppm of the extract [X8] of Example 8D), and
then 7.5 wt. % of a pasteurized strawberry-blueberry puree, in both
cases based on the total weight of the liquid yoghurt. The
resulting mixture was homogenized giving LiqYog B which was stored
at 8.degree. C. before further processing.
Example 14
Mouthwashes
TABLE-US-00039 [0604] A B C Ingredient in wt. % in wt. % in wt. %
Ethanol (96% in water) 8.00 5.00 -- Glycerin 8.00 10.00 12.00
1,2-Propylene Glycol -- -- 2.00 Sodium Fluoride 0.05 0.13 0.10
Poloxamer 407 (Pluronic F-127 .RTM., 1.40 -- 0.60 BASF) PEG-40
hydrogenated castor oil and -- 1.00 0.50 propylene glycol
(Cremophor .RTM. CO 40, BASF) Sodium Phosphate buffer (pH 7.0) 1.10
-- 1.00 Blue colorant (1% in water) 0.10 0.05 -- Red colorant (1%
in water) -- 0.05 0.08 Sorbic acid 0.025 -- -- Benzoic acid 0.025
-- -- 4-Hydroxybenzoic acid methylester -- -- 0.06 Na Salt Sodium
Saccharinate 0.10 -- 0.12 Sorbitol (70% in water) -- 3.00 --
Eucalyptus oil-menthol-wintergreen 0.15 0.10 -- flavour
Peppermint-spearmint flavour -- 0.10 0.20 (menthol content: 58%)
Cetylpyridinium Chloride 0.05 -- 0.07 Hydrogen Peroxide (30% in
water) -- -- 3.00 Extract [X7] of 0.0005 -- -- Example 8 B-1)
Extract [X8] of -- 0.001 0.0003 Example 8 D) Pure compound [1] --
-- 0.0001 Deionized water Ad 100 Ad 100 Ad 100
Example 15
Perfume Oils for Use in Cosmetic Compositions
TABLE-US-00040 [0605] Perfume oil P1 Perfume oil P2 Ingredient
parts by weight parts by weight Acetophenone, 10% in DPG 10.00 --
n-Undecanal 15.00 5.00 Allylamylglycolate, 20.00 -- 10% in DPG
Amylsalicylate 25.00 15.00 Benzyl acetate 60.00 40.00 Citronellol
80.00 50.00 D-Limonen 60.00 10.00 Dihydromyrcenol 60.00 15.00
Eucalyptus oil 10.00 -- Geraniol 40.00 60.00 Nerol 20.00 20.00
Geranium oil 15.00 15.00 Hexenol cis-3, 10% in DPG 5.00 15.00
Hexenyl salicylate cis-3 20.00 -- Indole, 10% in DPG 10.00 --
Alpha-Ionone 15.00 15.00 Vanillin 5.00 -- Lilial
(2-methyl-3-(4-tert- 60.00 -- butyl-phenyl)propanal) Linalool 40.00
60.00 Methylphenyl acetate 10.00 -- Phenylethyl alcohol 255.00
30.00 Styrene acetate 20.00 20.00 Terpineol 30.00 --
Tetrahydrolinalool 50.00 50.00 Cinnamon alcohol 10.00 --
Ambrettolide -- 25.00 p-tert-Butyl cyclohexyl 20.00 80.00 acetate
Exaltolide -- 50.00 Galaxolide, 50% in IPM 30.00 120.00
Hexadecanolide -- 10.00 Iso E Super 20.00 75.00 Musk indanone --
70.00 Coumarine -- 20.00 Patchouli oil -- 70.00 Vetiveryl acetate
-- 50.00 Methyl dihydrojasmonate 20.00 90.00 DPG = Dipropylene
glycol; IPM = Isopropyl myristate
Example 16
Roll-on Deodorant Emulsions
TABLE-US-00041 [0606] A B C Ingredient in wt. % in wt. % in wt. %
PEG-40 Stearate 5.00 4.00 5.50 Ethylhexylglycerin 1.00 1.20 --
(Octoxyglycerin) Cetyl Alcohol 2.00 1.00 1.70 Stearyl Alcohol --
1.00 0.50 Mineral oil 2.00 2.00 2.00 Aluminum Hydrochlorate powder
-- 8.00 15.00 Polysorbate 80 0.80 1.00 1.20 Glycerin 2.50 1.50 1.50
Mg-Aluminium Silicate 0.80 0.80 0.80 Talc 1.50 -- -- 1,2-Pentandiol
-- -- 0.60 1,2-Octandiol -- 0.75 0.20 2-Benzyl heptanol -- 0.10 --
2-Methyl-4-phenyl-2-butanol -- 0.10 0.30 4-Methoxybenzyl alcohol
0.10 -- -- 4-Methyl-4-phenyl-2-pentanol 0.05 -- 0.10 Extract [X7]
of 0.001 -- 0.0004 Example 8 B-1) Extract [X8] of -- 0.0005 0.0004
Example 8 D) Perfume oil P1 according 0.70 -- 0.65 to Example 14
Perfume oil P2 according 0.10 0.60 -- to Example 14 Water Ad 100 Ad
100 Ad 100
Example 17
Deodorant Microemulsions
TABLE-US-00042 [0607] A B C Ingredient in wt. % in wt. % in wt. %
Glycerin Isostearate 1.80 2.00 1.80 Octoxyglycerin -- 0.80 0.90
Ceteareth-15 5.25 5.50 5.00 Isotridecyl Isononanoate 3.30 3.50 3.80
Cyclomethicon 6.80 6.40 6.00 L-Menthyl-L-Lactate -- 0.20 0.10
Octyldodecanol -- 0.40 -- Aluminium Chlorohydrate -- 5.00 9.00
Triclosan .RTM. -- -- 0.25 Extract [X7] of 0.0012 -- 0.0004 Example
8 B-1) Extract [X8] of -- 0.0005 0.0006 Example 8 D) Perfume oil P1
according 0.65 0.10 0.45 to Example 14 Perfume oil P2 according --
0.40 0.25 to Example 14 Water Ad 100 Ad 100 Ad 100
Example 18
Shampoo Compositions
TABLE-US-00043 [0608] A B C Ingredient in wt. % in wt. % in wt. %
C.sub.10-30 Alkyl acrylate crosspolymer 0.60 0.60 0.60 Sodium
Hydroxide, 15% in water 0.10 0.12 0.10 Disodium EDTA 0.10 0.10 0.10
1,2-Decandiol 1.00 0.50 1.00 1,2-Dodecandiol -- 0.70 --
1-Tetradecanol -- -- 0.50 Glycol Distearate, Laureth-4, 3.00 3.00
3.00 Cocamidopropyl Betaine Sodium Laureth Sulfate (SLES), 12.00
14.00 10.00 53% in water Sodium Cocamphoacetate 5.00 5.00 7.00
Ammonium Cocoyl Isethionate 10.00 8.00 9.00 Acetamide MEA 1.00 1.50
0.50 Palmitamide MEA 0.50 -- 0.50 Phenoxyethanol -- 0.70 0.30
Extract [X7] of 0.0015 -- 0.0003 Example 8 B-1) Extract [X8] of --
0.0010 0.0006 Example 8 D) 2-Methyl-4-phenyl-2-butanol 0.08 -- --
Perfume oil P1 according to 0.20 0.40 0.25 Example 14 Perfume oil
P2 according to -- -- 0.15 Example 14 Water Ad 100 Ad 100 Ad
100
Example 19
Clear Shampoo Compositions with UV-Protection
TABLE-US-00044 [0609] A B C Ingredient in wt. % in wt. % in wt. %
Polyquaternium-7 0.50 0.50 0.65 Disodium Phenyl Dibenzimidazole
0.50 -- -- Tetrasulfonate Butyl Methoxydibenzoylmethane
(Avobenzone) 0.25 -- 0.80 Phenylbenzimidazole Sulfonic Acid, Sodium
0.80 1.00 1.20 Salt Amino Methyl Propanol 0.50 0.60 0.40 Sodium
Laureth Sulfate (SLES), 28% in water 30.00 25.00 20.00
Cocoamidopropyl Betaine 5.00 6.00 7.50 Propylene Glycol, PEG-55
Propylene Glycol 0.80 0.80 0.80 Dioleate Panthenol 1.00 0.40 --
Allantoin -- 0.25 0.50 Methylparaben, Ethylparaben, -- 0.30 0.55
Butylparaben, Propylparaben, Isopropylparaben, Isobutylparaben,
Benzylparaben Phenoxyethanol -- 0.25 -- 1,2-Propylene Glycol 1.00
-- 0.30 Glycerin -- 1.00 0.60 Sodium Chloride 0.70 0.60 0.70
Extract [X7] of 0.0025 -- 0.0004 Example 8 B-1) Extract [X8] of --
0.0010 0.0005 Example 8 D) Perfume oil P1 according to 0.40 0.35
0.25 Example 14 Perfume oil P2 according to -- 0.10 0.15 Example 14
Water Ad 100 Ad 100 Ad 100
Example 20
Wetting Compositions for Wet Wipes
TABLE-US-00045 [0610] A B C D Ingredient in wt. % in wt. % in wt. %
in wt. % Ceterayl isononanoate, Ceterareth-20, Stearyl 3.00 -- --
2.50 alcohol, Glyceryl stearate, Glycerin, Ceterareth-21, Cetyl
palmitate Mineral oil 3.00 -- 3.00 -- Paraffinum liquidum -- --
4.00 -- Ethylhexyl ethylhexanoate -- -- 5.00 -- Cetearyl
ethylhexanoate -- -- 3.50 -- PEG-40 Hydrogenated castor oil,
Trideceth-9, -- 2.00 -- -- Propylene glycol, water Citric acid,
anhydrous -- 0.01 -- -- Vitamin E Acetate (Tocopheryl Acetate) 0.10
-- -- 0.08 Phenoxyethanol 0.40 -- -- 0.10 Imidazolinyl urea -- 0.10
-- -- Diazolidinyl urea -- -- 0.15 0.05 Benzethonium chloride --
0.05 -- 0.05 (-)-Alpha-Bisabolol, natural -- -- 0.09 0.10 Chamomile
extract -- 0.35 -- -- Allantoin 0.10 -- 0.08 -- Acrylates/C10-30
Acrylates Copolymer -- -- 0.20 -- Glycerin 0.50 -- -- --
1,2-Pentandiol 2.00 -- -- 0.50 1,2-Hexandiol 1.00 -- -- 1.50
1,2-Propylene glycol -- 8.00 -- 2.00 Extract [X7] of 0.0012 --
0.0004 0.0001 Example 8 B-1) Extract [X8] of -- 0.0010 0.0004
0.0003 Example 8 D) Lavender oil -- -- -- 0.05 Perfume oil P1
according to -- 0.10 0.25 -- Example 14 Perfume oil P2 according to
-- 0.01 -- -- Example 14 Water Ad 100 Ad 100 Ad 100 Ad 100 The
emulsions/lotions A, C and D were each applied separately to a
nonwoven fabric to produce wet wipes (premoistened wipes). Solution
B was applied to woven fabric sheets.
Example 21
Wetting Compositions for Wet Wipes
TABLE-US-00046 [0611] A B C D Ingredient in wt. % in wt. % in wt. %
in wt. % Sodium chloride 1.25 2.10 2.60 1.50 Zinc chloride 0.50
0.50 -- 0.25 Glycerin 1.00 1.30 0.50 1.50 1,2-Butylene glycol --
0.40 0.80 -- DMDM Hydantoin -- 0.10 -- 0.10 Phenoxyethanol -- --
0.40 -- Imidazolinyl urea 0.10 -- -- 0.05 Cyclodextrins -- 0.15 --
0.20 Acyl Glutamate 1.00 -- 0.90 1.10 (surfactant) Dimethiconol and
TEA 0.70 0.50 0.60 0.80 Dodecylbenzene sulfonate PEG-75 Lanolin
0.25 0.75 0.50 0.60 Polysorbate 20 0.22 0.40 0.30 0.50 Malic acid
0.05 0.07 0.10 0.10 Extract [X7] of 0.0008 -- 0.0002 0.0003 Example
8 B-1) Extract [X8] of -- 0.0006 0.0002 0.0004 Example 8 D)
Chamomile extract -- -- -- 0.10 Rose and lavender -- -- -- 0.05
perfume oil Perfume oil P1 0.08 0.15 -- -- according to Example 14
Water Ad 100 Ad 100 Ad 100 Ad 100 The compositions A-D were each
applied separately to a nonwoven fabric to produce wet wipes.
Example 22
Cosmetic O/W--Lotions
TABLE-US-00047 [0612] A B C D Ingredient in wt. % in wt. % in wt. %
in wt. % Paraffin oil 5.00 5.00 5.00 5.00 Isopropyl palmitate 5.00
5.00 5.00 5.00 Cetyl alcohol 2.00 -- 1.00 2.00 Stearyl alcohol --
2.00 1.00 -- Beeswax 2.00 2.00 1.00 2.00 Ceteareth-20 2.00 2.00
2.00 2.00 PEG-20-glyceryl stearate 1.50 1.50 1.50 1.50 Glycerine
3.00 3.00 4.00 2.00 1,2-Butylene glycol/ -- 0.50 -- 1.50
1,2-Propylene glycol (1:1) Phenoxyethanol -- 0.25 -- 0.15 Extract
[X7] of 0.001 -- 0.0004 0.0007 Example 8 B-1) Extract [X8] of --
0.0005 0.0006 -- Example 8 D) Perfume oil P1 according 0.75 0.10
0.45 -- to Example 14 Perfume oil P2 according -- 0.40 0.25 0.70 to
Example 14 Water Ad 100 Ad 100 Ad 100 Ad 100
Example 23
Cosmetic O/W Sunprotection Compositions
TABLE-US-00048 [0613] A B C Ingredient in wt. % in wt. % in wt. %
Glyceryl Oleate Citrate, Caprylic/Capric Triglyceride 2.00 -- 2.00
Potassium Cetyl Phosphate -- 1.50 -- Cetearyl alcohol -- 1.00 --
C12-15 - Alkyl Benzoate -- 2.50 -- Homosalate 5.00 2.00 5.00 Butyl
Methoxydibenzoylmethane (Avobenzone) 3.00 3.00 3.00 Ethylhexyl
Salicylate 3.00 4.00 3.00 Octocrylene -- 5.50 -- Isoamyl
p-Methoxycinnamate -- 3.00 -- Diisopropyl Adipate 6.00 -- 6.00
Ethylhexyl Isononanoate -- 2.00 -- Diethylhexyl 2,6 Naphthalate
1.50 -- 2.00 Disodium EDTA 0.10 0.10 0.10 Vitamin E Acetate
(Tocopheryl Acetate) 0.50 0.50 0.50 Micronized rutile (TiO.sub.2),
Alumina (9.5%) and -- 3.00 0.80 Simethicone (2%) Dimethicone 1.00
2.00 1.30 (-)-Alpha-Bisabolol, natural 0.10 0.10 0.15 Allantoin
0.10 -- 0.15 Acrylates/C10-30 Acrylates Copolymer 0.25 -- 0.25
Carbomer -- 0.20 -- Xanthan Gum -- 0.20 -- Sodium Cetearyl Sulfate
-- 0.60 -- Phenylbenzimidazole Sulfonic Acid, Sodium Salt, 35% 2.50
3.00 2.00 solution in water, neutralized with triethanolamine
Disodium Phenyl Dibenzimidazole Tetrasulfonate, 30% -- 2.00 --
solution in water, neutralized with triethanolamine
Tris-Hydroxyaminomethane 0.45 -- 0.50 Glycerin 4.00 1.00 3.00
1,2-Butylene glycol and 1,3-Butylene glycol (1:1) 3.00 -- 2.00
Extract [X7] of 0.0016 -- 0.0004 Example 8 B-1) Extract [X8] of --
0.0004 0.0004 Example 8 D) Perfume oil P1 according to Example 14
0.25 0.20 0.15 Perfume oil P2 according to Example 14 -- 0.10 0.25
Water Ad 100 Ad 100 Ad 100 Compositions A and C are sunsprays,
composition B is a sunscreen softcream.
[0614] Compositions A and C are sunsprays, composition B is a
sunscreen softcream.
Example 24
Hand Sanitizing Composition
[0615] A clear liquid composition having a pH-value of 5.6 was
prepared consisting of 2-propanol (45 wt. %), 1-propanol (30 wt.
%), lactic acid (0.3 wt. %), 1-tetradecanol, medium chain
triglycerides, glycerol, sodium lactate, extract [X7] of Example
8B-1) (25 ppm), and water.
Example 25
Hand Sanitizing Composition
[0616] A clear liquid composition having a pH-value of 5.3 was
prepared consisting of 1-propanol (40 wt. %), 2-propanol (28 wt.
%), citric acid (0.2 wt. %), lactic acid (0.15 wt. %), 1-dodecanol,
medium chain triglycerides, glycerol, 1,2-propylene glycol, extract
[X8] of Example 8D) (45 ppm), and water.
Example 26
Hand Sanitizing Composition
[0617] A composition was prepared consisting of ethanol (55 wt. %),
1-propanol (10 wt. %), 1,2-propylene glycol (6 wt. %), 1,3-butylene
glycol, lactic acid, extract [X8] of Example 8D) (15 ppm), and
water.
Example 27
Medical Sanitizing/Disinfecting Compositions
TABLE-US-00049 [0618] A B C D Ingredient in wt. % in wt. % in wt. %
in wt. % Benzalkonium chloride 2.00 2.50 0.10 0.15
Cocopropylenediamine guanidinium acetate 14.00 14.00 0.25 --
Phenoxypropanol 35.00 30.00 0.50 --
Tetrakis-(2-hydroxypropyl)-N,N,N',N'- 5.00 -- 0.08 --
ethylenediamine Disodium EDTA -- 1.00 0.10 0.05
Laurylpropylenediamine -- 4.50 -- 0.10 Maleic acid 0.45 0.15 0.02
0.01 Citric acid -- 0.25 -- 0.01 Tridecyl ethoxylate-12EO 15.00 --
0.15 0.05 Tridecyl ethoxylate-5EO -- 10.00 0.10 0.20 Ethanol 8.00
10.00 0.25 0.20 1-Propanol 1.00 -- -- 0.10 Extract [X7] of 0.10 --
0.0004 0.0007 Example 8 B-1) Extract [X8] of -- 0.08 0.0006 --
Example 8 D) Perfume oil P1 according to 1.55 0.90 0.05 -- Example
14 Perfume oil P2 according to -- 0.40 -- -- Example 14 Water Ad
100 Ad 100 Ad 100 Ad 100 Compositions A and B are concentrates
which are diluted with water in a ratio (concentrate:water) in the
range of 1:80 to 1:20 to give a ready-to-use solution. Compositions
C and D are ready-to-use solutions.
Example 28
Ready-to-Use Sanitizing/Disinfecting Compositions
TABLE-US-00050 [0619] A B C D Ingredient in wt. % in wt. % in wt. %
in wt. % Benzalkonium chloride 0.20 0.05 -- -- Ethylhexylglycerin
(Octoxyglycerin) 0.25 0.20 0.25 -- Octenidine dihydrochloride --
0.07 -- 0.10 Laureth-35 0.02 -- 0.01 -- Sodium gluconate -- 0.40 --
0.30 Glycerol 2.10 2.50 1.50 3.00 Mecetroniumetilsulfate -- -- 0.15
-- Phenoxypropanol -- -- -- 1.00 Cocoamidopropyl betaine -- 0.30
0.25 -- 2-Propanol 0.50 -- -- 0.45 Extract [X7] of 0.002 -- 0.0006
0.0007 Example 8 B-1) Extract [X8] of -- 0.001 0.0003 -- Example 8
D) Lemon perfume oil -- 0.10 0.05 -- Water Ad 100 Ad 100 Ad 100 Ad
100
Sequence CWU 1
1
51256DNADacryopinax spathularia FU50088, MUCL 53181 1cccctagtaa
ctgcgagtga agcgggaaaa gctcaaattt aaaatccctt cggggagttg 60taatctagag
acgtgttttc ggtcgttgcc tcggacaagt cccttggaac agggcgtcat
120agagggtgag aatcccgtac ttgccgagct cccaatgact atgtgataca
cgttcgaaga 180gtcgagttgt ttgggaatcg agctcaaaat gggtgtgaaa
ctccatctaa agctaaatat 240tggcgagaga ccgata 2562256DNADacryopinax
spathularia CBS197.83, MUCL 53182 2cccctagtaa ctgcgagtga agcgggaaaa
gctcaaattt aaaatccctt ccgggagttg 60taatctagag acgtgttttc ggtcgttgcc
tcggacaagt cccttggaat agggcgtcat 120agagggtgag aatcccgtac
ttgccgagct cccaatgact atgtgataca cgttcgaaga 180gtcgagttgt
ttgggaatgc agctcaaaat gggtggtaaa ctccatctaa agctaaatat
240tggcgagaga ccgata 2563254DNADitiola nuda CBS173.60, MUCL 53179
3cccctagtaa ctgcgagtga agcgggaaaa gctcaaattt gaaatccttc gggagttgta
60atctagagaa gtgttttcgg tcgttgcctc ggacaagtcc cttggaacag ggcgtcatag
120agggtgagaa tcccgtcctt gccgagctac caacgtctat gtgatgcgct
ctcgaagagt 180cgagttgttt gggaatgcag ctcaaaatgg gtggtaaact
ccatctaaag ctaaatattg 240gcgagagacc gata 2544254DNADitiola radicata
CBS126.84, MUCL 53180 4cccctagtaa ctgcgagtga agcgggaaaa gctcaaattt
agaacccttc gggaattgta 60atctagagaa gtgttttcgg tcgttgcctc ggacaagtcc
cttggaacag ggcgtcatag 120agggtgagaa tcccgtcctt gccgagctac
caacgtctat gtgatgcgct ctcgaagagt 180cgagttgttt gggaatgcag
ctcaaaatgg gtggtaaact ccatctaaag ctaaatattg 240gcgagagacc gata
2545272DNADitiola pezizaeformis ATCC13299,
MUCLmisc_feature(52)..(53)n is a, c, g, or t 5gcggaggaaa agaaactaac
aaggattccc ctagtaactg cgagtgaagc gnngaaaagc 60tcaaatttaa aagccttcgg
gcgttgtaat ctatagaagt gttttcggtc gttgcctcgg 120ataagtctct
tggaacagag tgtaaagagg gtgagaatcc cgttcttgcc gagctaccaa
180cgtctatgcg atacatttca aagagtcgag ttgtttggga atgcagctca
aaatcgggtg 240gtaaactcca tctaaagcta aatattcggc cg 272
* * * * *
References