U.S. patent application number 14/035319 was filed with the patent office on 2014-01-23 for antimicrobial composition.
This patent application is currently assigned to DSM IP ASSETS B.V.. The applicant listed for this patent is DSM IP ASSETS B.V.. Invention is credited to Ben Rudolf DE HAAN, Nicole Liliane DUTREUX, Jacobus STARK.
Application Number | 20140023762 14/035319 |
Document ID | / |
Family ID | 35448207 |
Filed Date | 2014-01-23 |
United States Patent
Application |
20140023762 |
Kind Code |
A1 |
DUTREUX; Nicole Liliane ; et
al. |
January 23, 2014 |
Antimicrobial Composition
Abstract
The present invention describes a process to preserve a
water-containing product such as a foodstuff, which comprises
adding natamycin and nisin to the product, whereby at least 90%
(w/v) of natamycin is dissolved and/or less than 10% (w/v) of
natamycin is present in crystal form.
Inventors: |
DUTREUX; Nicole Liliane;
(DELFT, NL) ; DE HAAN; Ben Rudolf; (VOORBURG,
NL) ; STARK; Jacobus; (ROTTERDAM, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DSM IP ASSETS B.V. |
Heerlen |
|
NL |
|
|
Assignee: |
DSM IP ASSETS B.V.
Heerlen
NL
|
Family ID: |
35448207 |
Appl. No.: |
14/035319 |
Filed: |
September 24, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11663347 |
Mar 21, 2007 |
8574645 |
|
|
PCT/EP2005/054663 |
Sep 19, 2005 |
|
|
|
14035319 |
|
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Current U.S.
Class: |
426/310 ;
426/532; 514/2.3 |
Current CPC
Class: |
A23B 4/22 20130101; A23L
3/3463 20130101; A01N 43/90 20130101; A23L 3/34635 20130101 |
Class at
Publication: |
426/310 ;
426/532; 514/2.3 |
International
Class: |
A23L 3/3463 20060101
A23L003/3463; A01N 43/90 20060101 A01N043/90 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 23, 2004 |
EP |
04104633.5 |
Mar 8, 2005 |
EP |
05101792.9 |
Apr 26, 2005 |
EP |
05103375.1 |
Claims
1. A process to preserve a water-containing product such as a food
product, which comprises applying in the product an anti-microbial
composition including natamycin and nisin, whereby at least 90%
(w/v) of natamycin is dissolved and/or less than 10% (w/v) of
natamycin is present in solid form.
2. The process according to claim 1, whereby natamycin is present
in the product in an amount of 1 to 20 ppm natamycin based on the
water content of the product.
3. The process according to claim 1, whereby an amount of 0.25 to
20 ppm of nisin is present in the product.
4. A process to preserve a product such as a food product, which
comprises applying on the product an anti-microbial composition
including natamycin and nisin, and said composition comprises
between 0.01 and 10 ppm natamycin.
5. The process according to claim 4, whereby nisin is present in an
amount ranged between 0.0001 and 1 ppm.
6. The process according to claim 1, whereby nisin and natamycin
are added at the same time.
7. The process according to claim 1, whereby natamycin and nisin
are applied at the surface of the product.
8. The process according to claim 7, whereby the anti-microbial
composition is applied by spraying or dipping.
9. A water-containing product such as a food product comprising an
anti-microbial composition including natamycin and nisin, whereby
at least 90% (w/v) of natamycin is dissolved and/or less than 10%
(w/v) of natamycin is present in solid form.
10. The product according to claim 9, whereby natamycin is present
in an amount of 1 to 20 ppm based on the water content of the
product.
11. The product according to claim 10, whereby said product
comprises 0.25 to 20 ppm of nisin.
12. A product such as a food product comprising an anti-microbial
composition including natamycin and nisin, whereby natamycin and
nisin are present at the surface of the product.
13. The product according to claim 12, whereby natamycin is present
in an amount ranged between 0.01 and 10 ppm.
14. The product of claim 12, whereby nisin is present in an amount
ranged between 0.0001 and 1 ppm.
15. Use of natamycin to obtain a product, such as a food product
comprising an anti-microbial composition including natamycin and
nisin, whereby at least 90% (w/v) of natamycin is dissolved and/or
less than 10% (w/v) of natamycin is present in solid form.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/663,347, filed 21 Mar. 2007, which is the US national phase
of international application PCT/EP2005/054663 filed 19 Sep. 2005,
which claims benefit of EP 04104633.5, filed 23 Sep. 2004; European
Application 05101792.9, filed 8 Mar. 2005; and EP 05103375.1, filed
26 Apr. 2005, the entire content, all of which are hereby
incorporated by reference in their entities.
FIELD OF THE INVENTION
[0002] The present invention discloses a method to prevent or delay
microbial growth in or on foodstuffs, agricultural products,
pharmaceutical products and cosmetics. More specifically the method
comprises using as an ingredient or applying to a food surface an
anti-microbial composition including natamycin as anti-fungal agent
and nisin as anti-bacterial agent.
BACKGROUND OF THE INVENTION
[0003] The need for improved food preservation methods is great. It
has been estimated that about one quarter of the word's food supply
is lost as a result of microbial spoilage and food-borne microbial
infections represent a constant and serious threat to human
health.
[0004] Several bacterial species that may contaminate and grow in
foodstuffs and crops are pathogenic or produce toxins and cause a
range of food-poisoning diseases. Despite substantial improvement
in the technology and hygiene, food products may be exposed to
spoilage and pathogenic bacteria in the food-handling environment
and the number of food poisoning is still increasing in most of the
countries.
[0005] Fungal spoilage can lead to serious economic losses. Several
food products e.g. agricultural products, dairy and meat products,
fruits and vegetables and derived products, bakery products and
cosmetics are very susceptible to fungal growth. Examples of dairy
products are cheese, cottage cheese, ricotta and yogurt. Dried
cured sausages are an example of meat products. Examples of
agricultural products are crops such as cereals, nuts, fruits,
vegetables and flower bulbs. Spoilage by fungi does not only affect
the quality of the product, but also represents a health risk. It
is well known that some fungal species, which grow on e.g. dairy
products and sausages, can produce mycotoxins. Some mycotoxins are
extremely dangerous as they can cause lethal diseases. Therefore
the outgrowth of unwanted fungi in and on food products should
always be prevented.
[0006] Food preservation techniques, e.g. heat processing,
freezing, ultrasound, irradiation, and modified atmosphere
packaging, significantly reduce microbial load but of particular
concern is the evidence that processed foods are being contaminated
with microorganisms following processing and prior to packaging. Of
rinsing concern in the food industry is microbial spoilage of
various foods such as dairy and meat products, dressings, spreads,
margarines and seafood. Especially food products in the 2.0 to 7.0
pH range are known to be susceptible to microbial spoilage by
yeast, mould, acid tolerant bacteria and/or mesophilic or
themophitic spore forming and non-spore forming bacteria.
[0007] Mostly, processed foods are not eaten directly after
processing thereby permitting bacteria, yeast or mould introduced
by post-contamination to grow. Since food consumption may occur
without reheating the processed foods to sufficient temperatures
for sufficient time, there is a risk of food poisoning or food
spoilage. Furthermore, the recent trend for minimally processed
foods with the intrinsic nutritional and sensory qualities of raw
and fresh foods has raised a new safety risk. Milder preservation
treatments, such as high hydrostatic pressure and pulsed electric
fields have been proved to be successful but rely on effective
hurdles i.e. cold chain and addition of natural
anti-microbials.
[0008] There has been extensive research conducted in the field of
food safety to develop effective anti-microbial compositions, which
function as anti-fungal and anti-bacterial compositions.
[0009] In U.S. Pat. No. 5,895,680 by Cirigliano et al., the
preservative system including natamycin and nisin is stated. The
instability of aqueous natamycin solution is compensated by
increased natamycin concentrations. In U.S. Pat. No. 5,895,680 it
is taught that natamycin has to be present in amount of at least
1.5 times the maximum solubility based on the water content present
in the foodstuff. The reason behind this is that it is believed
that soluble natamycin is not stable in aqueous surroundings and
that therefore always solid (for example crystal or amorphous)
natamycin has to be present for the anti fungal activity.
DESCRIPTION OF THE INVENTION
[0010] The object of this invention is to provide a novel natural
microbiocidal and/or microbiostatic composition and a method for
preserving foodstuffs, agricultural goods, cosmetics and
pharmaceutical products by employing a novel combination of
microbiocidal and microbiostatic components in or on the
foodstuffs. The above subject may be achieved by incorporating a
combination of polyene antifungal agent and a bacteriocin into
foodstuffs and/or by treating the surface of foodstuffs with the
novel composition of polyene and bacteriocin.
[0011] The polyene antifungal agent may be, for example, natamycin,
lucensomycin, nystatin or amphotericin. Examples of bacteriocins
are nisin, pediocin, reuterin and sakacin. According to one aspect
of this invention the polyene antifungal agent is natamycin. A
prefered embodiment of the invention is the combination of
natamycin with nisin or pediocin.
[0012] In a first aspect of the invention, there is provided a
process to preserve products, such as food products, which
comprises applying in or on said product an anti-microbial
composition including natamycin and nisin.
[0013] According to a first preferred embodiment, the invention
relates to a process to preserve a water-containing product such as
a food product, which comprises applying in the product an
anti-microbial composition including natamycin and nisin, whereby
at least 90% (w/v) of natamycin is dissolved and/or less than 10%
(w/v) of natamycin is present in solid form. Preferably, at least
95% (w/v) of natamycin is dissolved and/or less than 5% (w/v) of
natamycin is present in solid form. More preferably, at least 97%
(w/v) of natamycin is dissolved and/or less than 3% (w/v) of
natamycin is present in solid form. Most preferably, almost all
natamycin is dissolved and almost no natamycin is present in solid
form.
[0014] The total amount of natamycin present is either dissolved in
water or present in a solid form. Solid form of natamycin means
`natamycin not dissolved in water`. The solid form of natamycin may
preferably comprise natamycin particles. Natamycin particles are
natamycin crystals, which, for example, may have the following
forms: needle-formed crystals, disc-formed crystals or the like.
The natamycin particles usually have diameters ranging from 0.5-20
micrometer. The diameter of the natamycin particle is the largest
distance from one part of the particle to the other end of the
particle. Needle-formed natamycin particles with diameters of more
than 40 micrometer have been observed. Diameters may be determined
using a microscope. Preferably, natamycin particles have an average
particle diameter of at least 2 micrometer, more preferably the
natamycin particles have an average particle diameter of at least 5
micrometer and most preferably the natamycin particles have an
average particle diameter of at least 10 micrometer. The presence
of solid form of natamycin may be determined by microscopy.
[0015] Throughout the application, natamycin concentration is
measured using the International Dairy Federeation (IDF) Standard
140A:1992. Nisin concentration is measured using the well-known
agar diffusion test.
[0016] Nisin is a peptide-like antibacterial substance produced by
microorganisms such as Lactococcus lactis subsp. lactis. It is
active against gram-positive bacteria. Nisin is non-toxic and is
free of side effects. Nisin is a Generally Recognized as Safe
substance and is widely used in a variety of foods. Examples of
such products are processed cheese, milk, clotted cream, dairy
desserts, ice cream mixes, liquid egg, hot-baked flour products,
dressings and beer. Nisin is heat-stable and can stand
sterilization temperatures with minimal loss of activity. The World
Health Organization Committee on Biological Standardization has
established an international reference preparation of nisin, and
the international unit (IU hereinafter) is defined as 0.001 mg of
this preparation. Delvoplus.RTM. and Nisaplin.RTM., brand names for
a nisin concentrate containing 1 million IU per gram, are
distributed by DSM and Danisco, respectively. Effective levels of
nisin to preserve food products range from 10 to 800 IU/g or 0.25
to 20 ppm of nisin.
[0017] Natamycin has been used for more than 30 years to prevent
outgrowth of fungi on cheeses and sausages. Natamycin is on the
market under the brand name of Delvocid.RTM. or Natamax.RTM., a
powder composition containing 50% (w/w) of natamycin and 50% (w/w)
of lactose. Natamycin has a MIC (Minimal Inhibition Concentration)
of less than 10 ppm for most food born fungi while its solubility
in water is from 30 to 50 ppm. Natamycin can easily be applied to
prevent spoilage by fungi in food products. However, the low
solubility of natamycin has limited its use to the surface
treatment of cheese and fluids e.g juices, lemonades, wine and
yoghurt. Surprisingly we found natamycin, when introduced into
foodstuffs at concentrations whereby almost no crystal form or
solid form of natamycin is present for example in concentrations
ranging from 1-20 ppm, based on the water content of the product
wherein it is used, to be extremely effective against yeast and
mould, completely inhibiting or significantly delaying their
outgrowth. Natamycin can be added as solid, suspension on any of
other form to a water-containing product in an amount such that all
natamycin will dissolve. Surprisingly we have found that natamycin
dissolved in water-containing product for example food products and
preferably in dairy products, is stable while in literature it is
mentions that dissolved natamycin in water is unstable.
[0018] According to a preferred embodiment, the process of the
invention is applied to a food product, which is a water-containing
product, wherein natamycin and nisin are present in the product and
wherein natamycin is present in an amount of about 1 to 20 ppm
natamycin based on the water content of the product, preferably
about 1 to 15 ppm. Preferably, an amount of about 0.25 to 20 ppm of
nisin is present in the product, more preferably about 1 to 15 ppm
of nisin.
[0019] According to a second preferred embodiment, the process of
the invention is a process for preserving a product such as a food
product, which comprises applying on the product an anti-microbial
composition including natamycin and nisin, whereby natamycin is
present in an amount ranged between 0.01 and 10 ppm. More
preferably, nisin is present in an amount ranged between 0.0001 and
1 ppm.
[0020] Applying the anti-microbial composition on the product
means, applying it on the surface of the product. The surface of a
product, such as a food product is defined as being the outside
layer of the product, which may be in contact with oxygen. For
example if the food product is cheese, the cheese surface is the
outside layer of cheese or parts of cheese, even in the sliced,
shredded or grated form. The term cheese surface includes the
outside of the whole cheese, disregarding whether a ring has been
formed or not.
[0021] Surprisingly, we found that natamycin and nisin, when
applied at the surface of product such as a food product in
concentrations ranging from 0.01 to 10 ppm for natamycin and 0.0001
and 1 ppm for nisin were found both extremely effective against
yeast and mould for natamycin and Gram positive bacteria for nisin,
completely inhibiting or significantly delaying their
outgrowth.
[0022] Nisin and natamycin may be added separately or at the same
time to the product. This addition may be performed simultaneously
or subsequently. According to a preferred embodiment, nisin and
natamycin are added at the same time to the product.
[0023] An anti-microbial composition comprising natamycin and nisin
is preferably added to the product after the reduction of size has
occurred at the end of the processing. More preferably, the
anti-microbial composition is added at the end of the processing
before packaging.
[0024] The anti-microbial compositions according to the invention,
which may be used for the treatment of food product such as cheese
or sausages may be, for example, liquids for treatment by immersion
or dipping and/or by spraying, or coating emulsions like that of
the polyvinyl acetate type or of the oil in water (o/w) or water in
oil (w/o) type.
[0025] Anti-microbial compositions of the invention for the
treatment of agricultural products such as flower bulbs, (kernel)
grain and vegetables may be, for example, aqueous systems which may
be used by methods known per se, e.g. dipping or spraying.
[0026] Examples of pharmaceutical compositions for topical
application in which a solution of the anti-microbial composition
of the invention may be incorporated are lotions, creams and
ointments. The amount of fungicide, e.g. natamycin, in a liquid
composition for immersion treatment may be from 0.001% to 2% w/w.
Preferably, the amount is from 0.01% to 1% w/w. The amount of
nisin, in a liquid composition for immersion treatment may be from
0.0001 to 1% w/w. Preferably the amount of nisin is from 0.001 to
1% w/w, more preferably from 0.01 to 0.05% w/w. In principle, the
immersion liquid may be of any kind. When an aqueous system is
used, the addition of a surfactant may be of advantage, in
particular for treating objects with a hydrophobic surface.
[0027] In a coating emulsion according to the invention, the amount
of fungicide, e.g. natamycin, may be from 0.001% to 2% w/w,
preferably from 0.01% to 1% w/w and more preferably from 0.01% to
0.5% w/w. The amount of nisin, in a coating emulsion may be from
0.0001 to 1% w/w. Preferably the amount of nisin is from 0.001 to
1% w/w, more preferably from 0.01 to 0.05% w/w. The coating
emulsion may be of the o/w or w/o type. Particularly preferred are
emulsions prepared from coating emulsions commonly employed In the
food industry. For example, for the treatment of hard cheeses an
aqueous polymer emulsion of the polyvinyl acetate type may be
used.
[0028] A composition comprising natamycin and nisin can be added to
the product at any moment or step of the processing. Natamycin and
nisin can be added in combination or separately, in the end product
before packaging, during processing or in any ingredients used to
prepare the products. The advantage of the present invention is
that it allows the production of microbiologically stable and safe
foodstuffs products with low concentration of anti-microbials. The
innovative composition can be used to preserve microbiological
safety and/or stability in all kind of food products such as dairy
products, ice cream mixes, hot-baked flour products, spreads,
margarines, sauce, dressings or any other foodstuffs distributed at
ambient or chilled temperatures. Preferred food products have pH
ranging from pH 2 to pH 7.0. Preferred food products are dairy
products and more especially cottage cheese, ricotta, cream cheese,
sour cream and dairy desserts. The addition of natamycin and nisin
to food products is furthermore expected to reduce or completely
eliminate moulds, yeasts and bacterial outgrowth on the food
products in the time frame comprised between the end of the
processing of the product, size reduction included and commercial
sale. This time frame varies with the type of food, the
distribution and sale conditions. Preferably, the product to be
treated is such that its surface will be in contact with oxygen at
the end of its processing and/or later on if a reduction of size
occurs. Preferably the product is a food product such as dairy
products, ice cream mixes, hot-baked flour products, spreads,
margarines, sauce, dressings or any other food product distributed
at ambient or chilled temperatures.
[0029] According to another preferred embodiment, the
anti-microbial composition of the invention comprising natamycin
and nisin has a pH ranging from pH 2 to pH 7.0, more preferably
from 3 to 5. According to another preferred embodiment, the
anti-microbial composition of the invention further comprises water
and/or salt and/or any component selected from the group consisting
of a solvent, a surfactant, a carrier, a food acid, a thickener
such as xanthan, any other food grade anti-microbial compound.
Preferred solvents, surfactants and carrier are already described
in WO 95/08918. Preferred thickeners were described in U.S. Pat.
No. 5,962,510 and U.S. Pat. No. 5,552,151. Preferred food acids
such as an organic acidic anti-fungal agent and/or any additional
acid are described in EP 608 944 B1. The content of the patents and
patent applications cited in this paragraph is fully incorporated
by reference in this context.
[0030] According to a second aspect of the invention, there is
provided a product, such as a food product comprising an
anti-microbial composition including natamycin and nisin.
[0031] According to a first preferred embodiment, the product is a
water-containing product such as a food product, which comprises an
anti-microbial composition including natamycin and nisin, whereby
at least 90% (w/v) of natamycin is dissolved and/or less than 10%
(w/v) of natamycin is present in solid form. Preferably, at least
95% (w/v) of natamycin is dissolved and/or less than 5% (w/v) of
natamycin is present in solid form. More preferably, at least 97%
(w/v)of natamycin is dissolved and/or less than 3% (w/v) of
natamycin is present in solid form. Most preferably, almost all
natamycin is dissolved and almost no natamycin is present in solid
form.
[0032] Preferably, natamycin is present in the product in an amount
of 1 to 20 ppm based on the water content of the product, more
preferably natamycin is present in an amount ranged between 0.01
and 10 ppm. According to another preferred embodiment, the product
comprises 0.25 to 20 ppm of nisin, more preferably nisin is present
in an amount ranged between 0.0001 and 1 ppm.
[0033] According to a second preferred embodiment, natamycin and
nisin are present at the surface of the product. Preferably,
natamycin is present in an amount ranged between about 0.01 and 10
ppm. Preferably natamycin is present in an amount ranged between
about 0.05 and 7 ppm, even more preferably about 0.1 and 5 ppm and
most preferably about 0.5 ppm and 4 ppm. According to another
preferred embodiment, nisin is present in an amount ranged between
about 0.0001 and 1 ppm, more preferably about 0.0005 and 0.75 ppm,
even more preferably about 0.001 and 0.5 ppm, even more preferably
about 0.005 and 0.5 ppm and most preferably about 0.007 and 0.4
ppm.
[0034] According to a third aspect of the invention, there is
provided the use of natamycin to obtain a product, such as a food
product comprising an anti-microbial composition including
natamycin and nisin.
[0035] According to a first preferred embodiment of the invention,
the invention relates to the use of natamycin to obtain a
water-containing product, such as a food product comprising an
anti-microbial composition including natamycin and nisin, whereby
at least 90% (w/v) of natamycin is dissolved and/or less than 10%
(w/v) of natamycin is present in solid form. The product has been
defined earlier in the description.
[0036] According to a second preferred embodiment, natamycin and
nisin are applied at the surface of the product.
[0037] Before, during or after the addition of natamycin and nisin
to the product, other ingredients such as colorants, texturals etc.
can be added as well to the product.
[0038] The invention will further now be illustrated by examples,
which should not be construed as limiting the scope of the
invention.
EXAMPLES
Example 1
Natamycin Stability in Cottage Cheese
[0039] This example describes the stability of low natamycin
concentration in cottage cheese containing nisin versus a lower
stability in water with similar pH.
[0040] Cottage cheese was prepared following the standard procedure
for cottage cheese making. The dry curd was obtained by addition of
a starter culture to pasteurized skim milk. After acidification
till pH 4.4 the curd was pasteurized and rinced with water. The dry
curd was mixed with the dressing at the ratio 66% curd and 44%
dressing. Before mixing the curd and the dressing, the
preservatives Delvocid.RTM. and Delvoplus.RTM. were added to the
dressing. The preservative concentrations in the end products were
2.5 and 5 ppm of natamycin (5 and 10 ppm of Delvocid.RTM.) and 1.25
and 2.5 ppm of nisin (50 and 100 ppm of Delvoplus.RTM.). The
moisture content of cottage cheese (dry curd mixed with the
dressing) was 82%.
[0041] The procedure described in the International IDF
(International Dairy Federation) Standard 140A:1992 for cheese was
used to determine the concentration of natamycin in the cottage
cheese samples. In total 12 different samples were analysed in
triplicate. The remaining percentage of natamycin was calculated
using the average of the triplicate. The results presented in Table
1 show that natamycin was stable in cottage cheese. After 6 weeks
of storage at refrigerated temperature, the average stability of
natamycin in cottage cheese was of 92%.
TABLE-US-00001 TABLE 1 Stability of natamycin in cottage cheese
stored at 4-7.degree. C. 0 week 3 weeks 6 weeks Remaining 100 96 86
percentage 100 94 89 of natamycin 100 88 97 100 103 92 100 96 88
100 102 91 100 98 93 100 99 90 100 99 93 100 100 97 100 97 87 100
96 98 Average 100 98 92
Example 2
Antifungal Activity of Low Natamycin Concentration in Cottage
Cheese Containing Nisin
[0042] This example describes the antifungal activity of low
natamycin concentration (2.5 and 5 ppm) in cottage cheese, in
presence of nisin. Cottage cheese was prepared as described above.
After adding the dressing to the dry curd, the cottage cheese was
inoculated with yeasts and fungal cells, Kluyveromyces marxianus
CBS 1557 and Penicillium roqueforti CBS 304.97, respectively.
Following the artificial contamination, natamycin and/or nisin was
added to the cottage cheese at two different concentrations, 2.5
and 5 ppm for natamycin and 1.25 and 2.50 ppm for nisin. The
antifungal activity of natamycin against the yeast and the mould
cells was evaluated after 3 and 6 weeks of incubation at 4.degree.
C. through the enumeration of the viable cells in the inoculated
samples, in presence or not of preservatives. The number of viable
yeast of fungal cells was also determined in a negative sample, non
inoculated cottage cheese without preservatives and in a positive
control, sample artificially contaminated with yeast and fungal
cells without preservative. The results are presented in the tables
2 and 3. It is clear that natamycin present at 2.5 or 5 ppm was
effective in preventing the growth of K. marxianus and P.
roqueforti for up to 6 weeks at 4-7.degree. C.
TABLE-US-00002 TABLE 2 Antifungal activity against Kluyveromyces
marxianus CBS 1557 of natamycin in presence or absence of nisin,
after 0, 3 and 6 weeks of incubation at 4-7.degree. C.
Concentration Log CFU/g of (ppm) K. marxianus Natamycin Nisin 0
week 3 weeks 6 weeks 0.00 0.00 1.80 3.50 4.50 2.50 0.00 1.70 1.70
<1.00 2.50 1.25 2.00 0.80 <1.00 2.50 2.50 1.90 0.80 <1.00
5.00 0.00 1.30 <1.00 <1.00 5.00 1.25 1.90 1.10 <1.00 5.00
2.50 1.70 <1.00 <1.00
TABLE-US-00003 TABLE 3 Antifungal activity against Penicillium
roqueforti CBS 304.97 of natamycin in presence or absence of nisin,
after 0, 3 and 6 weeks of incubation at 4-7.degree. C.
Concentration Log spores/g of (ppm) P. roqueforti Natamycin Nisin 0
week 3 weeks 6 weeks 0.00 0.00 1.80 2.70 5.40 2.50 0.00 1.70 1.70
<1.00 2.50 1.25 1.60 <1.00 <1.00 2.50 2.50 1.60 <1.00
<1.00 5.00 0.00 1.60 <1.00 <1.00 5.00 1.25 1.60 <1.00
<1.00 5.00 2.50 1.70 <1.00 <1.00
Example 3
Antibacterial Activity of Nisin in Cottage Cheese Containing Low
Natamycin Concentration
[0043] This example describes the antibacterial activity of nisin
(1.25 and 2.5 ppm) in cottage cheese, in presence of low natamycin
concentration. Cottage cheese was prepared as described above.
After adding the dressing to the dry curd, the cottage cheese was
inoculated with yeasts, fungal cells and cells of Listeria innocua
LM35. Following the artificial contamination, the cottage cheese
was added with two nisin concentrations 1.25 and 2.5 ppm and/or in
combination with natamycin at the concentration 2.5 or 5 ppm. The
antibacterial activity of nisin against L. innocua was evaluated
after 3 and 6 weeks of incubation at 4.degree. C. through the
enumeration of the viable cells in the inoculated samples, in
presence or not of preservatives. The number of viable bacterial
cells was also determined in a negative sample, non inoculated
cottage cheese without preservatives and in a positive control,
sample artificially contaminated with bacteria.
[0044] The results presented in the table 4 indicate that nisin
inhibited the growth of L. innocua in presence or not of
natamycin.
TABLE-US-00004 TABLE 4 Antifungal activity against Listeria innocua
LM 35 of natamycin in presence or absence of nisin, after 0, 3 and
6 weeks of incubation at 4-7.degree. C. Concentration Log CFU/g of
(ppm) L. innocua Natamycin Nisin 0 week 3 weeks 6 weeks 0.00 0.00
2.00 3.10 6.30 2.50 0.00 1.90 2.50 4.50 2.50 1.25 2.00 <1.00
<2.00 2.50 2.50 1.90 <1.00 <1.00 5.00 0.00 1.90 1.90 4.70
5.00 1.25 1.60 <1.00 <1.00 5.00 2.50 2.10 <1.00
<1.00
Material and Methods Used in Coming Examples
[0045] Non-contaminated brine was obtained from the Food Innovation
Centre of DSM Food Specialties, P.O. Box 1, 2600 MA, Delft, The
Netherlands.
[0046] Freshly made 2 kg brined wheels of Gouda cheeses were
obtained from the Food Innovation Centre of DSM Food Specialties,
P.O. Box 1, 2600 MA, Delft, The Netherlands. The contamination of
the cheeses was tested by evaluating the presence of yeast cells of
the type Debaromyces hansenii ATCC 10623) and by the presence of
Lactobacillus salt-resistant strains, which are both normally found
in brine baths. In this example, Lactobacillus sakei ATCC 15521 was
used.
[0047] The source of natamycin used in the anti-microbial
composition is Delvocid.RTM., containing 50% active (w/w)
natamycin, DSM Food Specialties, P.O. Box 1, 2600 MA, Delft, The
Netherlands.
[0048] The source of nisin used in the anti-microbial composition
is Delvoplus.RTM., containing 2.5% active (w/w) nisin, DSM Food
Specialties, P.O. Box 1, 2600 MA, Delft, The Netherlands.
[0049] Sodium chloride (NaCl) was purchased by Gaches Chimie
France, 31750 Escalquens, France.
Example 4
Preparation of Several Anti-Microbial Compositions
[0050] This example describes a method to prepare several dipping
anti-microbial compositions suitable for use as a food coating to
prevent microbial growth on cheese. These anti-microbial
compositions were prepared by adding 1 gram of Delvocid.RTM. and/or
0.5 gram of Delvoplus.RTM. with 100 gram of NaCl to 800 ml of
tapwater. 0.2% xanthan was further added. The pH of the composition
was adjusted to 4.5 by adding citric acid. The different
ingredients were homogenized used an electric top stirrer (type RW
20 DZM, from Janke & Kunkel equiped with a Ruston stirrer).
Finally the weight of the mixture was adjusted to one kilo by
addition of tap water. The final compositions were stored at
18.degree. C. in the dark.
[0051] Three anti-microbial compositions have been prepared: [0052]
one containing 500 ppm natamycin [0053] one containing 12.5 ppm
nisin [0054] one containing 500 ppm natamycin and 12.5 ppm nisin:
anti-microbial composition of the invention
Example 5
Method for Testing the Efficacy of the Anti-Microbial
Compositions
[0055] This example describes the method used to test the efficacy
of the compositions, described in example 4, to protect cheeses
contaminated from the brine. A freshly brined wheel of Gouda cheese
was dipped in a brine solution containing 10.sup.6 CFU per ml of
the salt resistant yeast Debaromyces hansenii ATCC 10623 and/or
10.sup.4 CFU per ml of salt-resistant Lactobacillus sakei ATCC
15521 for one minute and accordingly flushed with tap water and
accordingly dipped in the compositions as described in example 4.
Five samples of cheese were used for each anti-microbial
composition tested. The cheese contained: [0056] 2.5 ppm natamycin
or [0057] 0.06 ppm nisin or [0058] 2.5 ppm natamycin and 0.06 ppm
nisin (composition of the invention) or [0059] no anti-microbial
compound in the composition (control)
[0060] The cheeses were accordingly vacuum packed in standard
polyethylene (PE) bags with a Kramer & Grebe compack machine
and stored at 18.degree. C. in the dark until the moment of
analysis.
[0061] The efficacy of the different compositions was expressed as
the difference between the amount of yeasts and lactobacilli found
on the surface of the different tested cheeses and the determined
surface contamination for the control cheese. The control cheese is
a contaminated cheese coated with a composition, which does not
contain any anti-microbial compound.
Example 6
Determination of the Contamination Levels on the Cheese Surface
After Removal of the Protection Foil
[0062] For each five tested cheese, 20 g of cheese were sampled
from the total cheese surface and suspended with 180 ml of citrate
buffer at 45.degree. C. Following homogeneization, 1 ml of cheese
suspension was further diluted in physiological salt and plated out
on specific agar, OGY (oxytetracycline yeast extract)+4% NaCL for
the yeast and TGV (tryptone glucose meat extract)+4% NaCl, for the
lactobacilli. The plates were incubated for 3 days at 30.degree. C.
for the yeast and 37.degree. C. for the lactobacilli.
Example 7
Anti-Microbial Efficacy of the Different Compositions
[0063] Anti-microbial compositions were prepared as described in
example 4 and tested according to example 5. The cheeses were
stored for 3 months between 6 and 10.degree. C. and the
contamination levels for each cheese were determined following the
method of the example 6. The results are listed in the table
below.
TABLE-US-00005 TABLE 5 Average contamination levels (expressed in
CFU/cm2) Treatment with Delvocid and Average No anti- No anti-
Delvoplus (2.5 ppm contamination microbial microbial Treatment with
Treatment with natamycin level in treatment treatment Delvocid (2.5
ppm Delvoplus (0.06 ppm and cfu's/cm.sup.2 t = 0 t = 3 months
natamycin) nisin) 0.06 ppm nisin) Yeast 10.sup.4 10.sup.6 0
10.sup.6 0 Lactobacilli 10.sup.2 10.sup.3 10.sup.4 0 0
* * * * *