U.S. patent application number 17/400396 was filed with the patent office on 2022-03-10 for bioflavonoid impregnated materials.
The applicant listed for this patent is CITROX BIOSCIENCES LIMITED. Invention is credited to Howard Thomas.
Application Number | 20220073487 17/400396 |
Document ID | / |
Family ID | 49117880 |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220073487 |
Kind Code |
A1 |
Thomas; Howard |
March 10, 2022 |
BIOFLAVONOID IMPREGNATED MATERIALS
Abstract
Cellulosic fibrous materials are described which are impregnated
with a bioflavonoid composition, the bioflavonoid content of the
composition comprising at least naringin and neohesperidin. The use
of such impregnated materials is also described, for example as
paper or bamboo towels and cardboard, as well as the process for
impregnating the materials.
Inventors: |
Thomas; Howard; (Cambridge,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CITROX BIOSCIENCES LIMITED |
Cambridgeshire |
|
GB |
|
|
Family ID: |
49117880 |
Appl. No.: |
17/400396 |
Filed: |
August 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14422759 |
Feb 20, 2015 |
|
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PCT/GB2013/052218 |
Aug 22, 2013 |
|
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17400396 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08J 7/065 20130101;
C07D 311/32 20130101; Y10T 428/31971 20150401; Y10T 428/265
20150115; C08J 7/123 20130101; C09D 5/14 20130101; D06M 15/03
20130101; D06M 13/10 20130101; Y10T 428/3188 20150401; D06M 13/165
20130101; D06M 10/025 20130101; Y10T 428/3179 20150401; A41D
13/1236 20130101; A01N 43/16 20130101; B05D 1/02 20130101; D06M
16/00 20130101; Y10T 428/24355 20150115; B65D 81/28 20130101; B65D
65/42 20130101; A41B 11/00 20130101; D06M 13/12 20130101; A23B 4/10
20130101; B05D 1/18 20130101; A01N 25/10 20130101 |
International
Class: |
C07D 311/32 20060101
C07D311/32; C08J 7/06 20060101 C08J007/06; D06M 15/03 20060101
D06M015/03; D06M 13/12 20060101 D06M013/12; D06M 16/00 20060101
D06M016/00; D06M 13/10 20060101 D06M013/10; D06M 10/02 20060101
D06M010/02; A01N 25/10 20060101 A01N025/10; A01N 43/16 20060101
A01N043/16; A23B 4/10 20060101 A23B004/10; B05D 1/02 20060101
B05D001/02; B05D 1/18 20060101 B05D001/18; B65D 65/42 20060101
B65D065/42; B65D 81/28 20060101 B65D081/28; C09D 5/14 20060101
C09D005/14; A41B 11/00 20060101 A41B011/00; A41D 13/12 20060101
A41D013/12; D06M 13/165 20060101 D06M013/165 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 24, 2012 |
GB |
1215171.8 |
Oct 19, 2012 |
GB |
1218829.8 |
Claims
1-17. (canceled)
18. A dry respiratory mask for use in reducing viral transmission
by air, which comprises cellulosic fibres impregnated with a
mixture of bioflavonoids comprising at least 70% naringin and
neohesperidin.
19. The dry respiratory mask of claim 18, wherein the naringin and
neohesperidin together form at least 75% of the bioflavonoid
content.
20. The dry respiratory mask of claim 18, wherein the naringin and
neohesperidin together form between 75% and 80% of the bioflavonoid
content.
21. The dry respiratory mask of claim 18, wherein the mixture of
bioflavonoids further comprises one or more compounds selected from
the group of neoeriocitrin, isonaringin, hesperidin, neodiosmin,
naringenin, poncirin and rhiofolin.
22. The dry respiratory mask of claim 18, wherein the mixture of
bioflavonoids comprises neoeriocitrin, isonaringin, hesperidin,
neodiosmin, naringenin, poncirin and rhiofolin.
23. The dry respiratory mask of claim 18, wherein the mixture of
bioflavonoids is uniform throughout the mask.
24. A method for preparing a dry respiratory mask suitable for
reduction of viral transmission by air, which comprises preparing a
respiratory mask from cellulosic fibres and impregnating said mask
with a mixture of flavonoids which comprises at least 70% of
naringin and neohesperidine, and drying said mask.
25. The method as claimed in claim 24, wherein said impregnating
comprises immersing or spraying said mask of cellulosic fibres.
26. The method as claimed in claim 24, wherein drying comprises
mechanically drying.
27. The method as claimed in claim 24, wherein the mixture of
bioflavonoids further comprises one or more compounds selected from
the group of neoeriocitrin, isonaringin, hesperidin, neodiosmin,
naringenin, poncirin and rhiofolin.
28. The method as claimed in claim 24, wherein the mixture of
bioflavonoids is uniform throughout the mask.
29. A method of reducing aerial viral transmission which comprises
use of a dry respiratory mask, as claimed in claim 18.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/422,759, filed Feb. 20, 2015, which is a 35
U.S.C. .sctn. 371 filing of International Patent Application No.
PCT/GB2013/052218, filed Aug. 22, 2013, which claims priority to
Great Britain Patent Application Nos. 1218829.8, filed Oct. 19,
2012, and 1215171.8, filed Aug. 24, 2012, the entire disclosures of
which are hereby incorporated herein by reference.
FIELD
[0002] The present invention relates to bioflavonoid impregnated
cellulosic fibrous materials, processes for impregnating the
materials and their uses. In particular, the invention relates to
bioflavonoid impregnated cellulosic fibrous materials such as
paper, paper towels, bamboo fibre and cardboard and articles formed
from such materials.
BACKGROUND
[0003] Cellulosic fibrous materials such as paper are used in a
wide variety of applications, ranging from domestic use to
commercial use in, for example, hospitals, schools, kitchens and
laboratories in the form of, for example, paper towels or face
masks or even garments such as bamboo fibre socks.
[0004] Some materials would benefit from having antimicrobial
properties. These include for example, cardboard, paper, cleaning
wipes, paper towels or face masks or even garments.
[0005] GB2468836 discloses compositions comprising bioflavonoid
compounds and their antibacterial, antifungal and antiviral
activity but no suggestion was made that they could be used in
impregnating fibres and materials.
SUMMARY OF THE INVENTION
[0006] The present invention relates to cellulosic materials
impregnated with a bioflavonoid composition.
[0007] According to a first aspect of the invention there is
provided a material impregnated with a bioflavonoid composition,
the bioflavonoid content of the composition comprising at least
naringin and neohesperidin.
[0008] Especially preferred is when the major part of the
bioflavonoid content of the composition comprises naringin and
neohesperidin. Preferably, naringin and neohesperidin together form
at least 50% wt/wt, more preferably at least 70% wt/wt, for example
at least 75% wt/wt, for example 75%-80% wt/wt of the bioflavonoid
content of the composition (excluding other biomass).
[0009] The bioflavonoid content of the composition may further
comprise one or more compounds of Formula (I):
##STR00001##
wherein R.sup.1 is a hydroxyl or methoxyl and R.sup.2 is hydrogen,
hydroxyl or methoxyl and X is hydrogen or a saccharide.
[0010] A preferred option is when R.sup.2 is hydrogen and R.sup.1
is in the 3- or 4-position. Another option is when R.sup.1 is
3-hydroxy and R.sup.2 is 4-methoxyl. Preferably, X is H. More
preferably, X is a saccharide.
[0011] In preferred embodiments, X is a disaccharide. Suitable
disaccharides include combinations of two monosaccharides,
preferably pyranoses, linked by a glycosidic bond, for example
rhamnose and glucose, for example L-rhamnose and D-glucose.
[0012] Suitable disaccharides can have the structure:
##STR00002##
wherein one of R.sup.3 and R.sup.4 is H and the other OH or both
are H or both are OH. Preferably R.sup.3 is H and R.sup.4 is OH so
that the disaccharide is rutinose.
[0013] Favoured aglycones of bioflavonoids for use in this
invention are the disaccharides
6-O-(alpha-L-rhamnopyranosyl)-beta-D-glucopyranose, also known as
rutinose, and
2-O-(alpha-L-rhamnopyra-nosyl)-beta-D-glucopyra-rose.
[0014] Suitable compounds of Formula (I) include neoeriocitrin,
isonaringin, hesperidin, neodiosmin, naringenin, poncirin and
rhiofolin, in addition to naringin and neohesperidin. One of these
compounds may be present in addition to naringin and neohesperidin,
although a mixture of two or more of these compounds is
particularly preferred.
[0015] Such mixtures can be obtained by extraction from bitter
oranges and the end product is called Citrus aurantium amara
extract. Particularly preferred are the mixtures of bioflavonoid
obtained from the extract of crushed whole immature bitter oranges.
The mixtures can also be derived from the starting material
comprised of the pith of immature, bitter (blood/red) oranges such
as Seville oranges that are classed as `inedible` and from which
the pips, flesh and oily skin have been substantially removed or
remain undeveloped.
[0016] Suitable mixtures can include 2, 3, 4, 5, 6, 7, 8, 9 or more
compounds of Formula (I). A mixture comprising 2, 3, 4, 5, 6, 7, 8,
or 9 of the above named bioflavonoids is preferable, for example
containing 3, or containing 4, or containing 5, or containing 6, or
containing 7, or containing 8, or containing 9 of said
bioflavonoids.
[0017] It is presently believed that mixtures of such bioflavonoids
have advantages over the use of a single bioflavonoid. It is
particularly advantageous that extract of bitter oranges is
employed without the need for isolating individual bioflavonoids.
In an extract from bitter oranges biomass may be associated with up
to 40-60% wt/wt, preferably about 55% wt/wt based on the weight of
the bioflavonoid content of the composition. The biomass comprises
pectins and other sugar derived materials. If it is desired to
avoid biomass, other solubilising agents such as dextrines, for
example cyclodextrin, may be employed if desired.
[0018] A particular advantage of many compositions described herein
is that they may employ compounds of natural origin. Thus, for
example, it is preferred to employ compounds of Formula (I) from
bitter oranges. However synthetically or semi-synthetically
obtained compounds may be employed if desired instead of the ones
directly extracted from natural sources although this tends to be
less favourable in view of cost.
[0019] The compositions may further comprise oleuropein. Preferably
this is obtained from extraction from the leaf of the olive, for
example Olea europaea. Such extracts typically contain 5% to 80%
wt/wt, more preferably 10% to 70%, for example 20% wt/wt of
oleuropein.
[0020] The wt/wt ratio of bioflavonoids to oleuropein can be 5:1 to
1:4, preferably 2:1 to 1:2, more preferably 1:2 to 1:1 and even
more preferably 3:2. In addition to the bioflavonoid content of the
composition, the composition may further comprise one or more fruit
acids, for example citric acid, malic acid, and ascorbic acid. One
or more of the acids are preferably neutralized with a suitable
base, such as a quaternary ammonium base, for example a choline
base, such as choline carbonate, bicarbonate or, preferably,
hydroxide. More preferably, citric, malic and ascorbic acids are
all used in the preparation of the composition, and especially
preferred is when these are fully neutralized to provide citrate,
malate and/or ascorbate salts. Especially preferred is choline
ascorbate.
[0021] It has been found that the composition described herein is
particularly effective in the presence of one or more organic
acids. In one embodiment, the composition further comprises one or
more organic acids.
[0022] A surprisingly effective organic acid is salicylic acid or
its pharmaceutically acceptable salt optionally together with a
further organic acid or pharmaceutically acceptable salt.
[0023] The salicylic acid may be obtained from willow bark extract.
Alternatively, methods for synthesising salicylic acid are known to
those skilled in the art.
[0024] Sometimes it is preferred that the salicylic acid is in the
form of the acid rather than its salt.
[0025] Similarly, a further organic acid if present is similarly in
the form of the acid rather than its salt. Suitable further organic
acids include acids of up to 8 carbon atoms which are monobasic
(i.e. one CO.sub.2H group), di-basic or tri-basic acid which
optionally contain 1, 2 or 3 hydroxyl groups. Such further organic
acid may be one or more of citric acid, malic acid, latic acid,
tartaric acid, fumaric acid and the like.
[0026] Such compositions can provide an approximately neutral or
acid pH, when used, for example from 3 to 8, more aptly 3.5 to 7,
for example 4 to 5.
[0027] At present it is preferred to employ salicylic acid and
citric acid in the compositions.
[0028] Such compositions may include a solubilising agent, for
example, salicylic acid such as a dextrin such as cyclodextrin.
[0029] The compositions described herein have an extremely
favourable safety and environmental profile. As well as showing
extremely effective antimicrobial activity, the compositions are
also non-toxic, non-corrosive, renewable and completely
biodegradable. The compositions disclosed in WO 2012/017186 (herein
incorporated by reference) are the preferred compositions of the
present invention.
[0030] The cellulosic fibrous materials of the invention may be
composed of paper or cardboard or bamboo fibres. Paper is defined
as a material produced from a cellulose pulp which may be derived
from wood, rags or grasses. The paper may be in the form of a paper
towel, towelette, cloth, wipe or pad. Paper towels have a variety
of applications, for example, paper towels are used to dry a
person's hands after washing, also known as hand towels. Paper
towels or wipes are also used for cleaning purposes to wipe down
surfaces in a hospital, laboratory or a kitchen, for example, and
can also be known as kitchen roll, kitchen paper or kitchen wipes.
Pads are cellulosic fibre sponges and have application in personal
hygiene and in medical kits. Wipes are produced as air-laid paper
where the fibres are carried and formed to the structure of paper
by air.
[0031] The paper may be treated with softeners, lotions or added
perfume to create a desirable "feel" or texture.
[0032] Bamboo materials may be formed of bamboo fibre which is a
cellulose fibre extracted or fabricated from natural bamboo. Bamboo
is a sustainable crop and, as a natural product derived entirely
from plant cellulose, bamboo fibre is biodegradable by
microorganisms in soil and also by sunlight. Preferably, the bamboo
materials of the present invention are formed of 100% bamboo fibres
although mixtures with other cellulose fibres are also
contemplated.
[0033] The bamboo may also be in the form of a paper towel,
towelette, wipe or pad which may have the same applications as
paper towels. The bamboo fibres may also be used as a clothing
fabric, optionally in combination with other known fibres, to make
garments, such as socks and hospital gowns. For example, socks made
from bamboo fibres impregnated with the bioflavonoid compositions
described herein can help reduce foot odour. The bioflavonoid
impregnated bamboo fibres are activated when they come into contact
with moisture from the foot. For hospital gowns, the bioflavonoid
composition is activated when the gowns come into contact with, for
example, blood or urine.
[0034] Fabrics made from bamboo fibres which are impregnated with
the bioflavonoid compositions described herein are very useful in
hospital or care home environments. For example, the bamboo fabric
can be used for bedding sheets, surgical drapes, curtains and the
like where it is desirable to use a material with antimicrobial
properties.
[0035] Paper fibre fabrics can be used instead of the bamboo fibre
fabrics described herein; however, the bamboo fibre fabrics are
preferred as these fabrics are more durable than paper fibre
fabrics.
[0036] Paper towels, bamboo towels and the like, may be heated, for
example by using a microwave, in order to provide a hot towel.
These hot towels may be disposable and/or re-heatable and can be
used in restaurants, hotels and on planes.
[0037] The bioflavonoid impregnated paper and/or bamboo fibres of
the invention can also be provided in the form of a face mask, such
as a respiratory mask or surgical mask, to provide the user with
enhanced protection against inhaling bacteria and viruses or to
prevent or reduce the spread of bacteria and viruses. The face
masks may be reusable or disposable. Methods of manufacturing face
masks are well known in the art.
[0038] Bioflavonoid impregnated bamboo and/or paper fibres can be
used in the form of single or multi-ply food pads. Such food pads
are often found in the bottom of food packaging and can also be
referred to as napkins or blankets. The use of these food pads is
particularly desirable in food packaging containing food with a
short shelf life, for example meat or fruit. The food product, for
example, the meat or fruit generally sit on top of the food pad
within the packaging. The bioflavonoid impregnated food pad
provides a dramatic reduction in the number of bacteria such as
Salmonella, E. coli and Campylobacter which cause foods such as
meat and fresh fruit to decay, reducing their shelf life. The
bioflavonoid impregnated food pads are particularly suitable in the
packaging of meats, including poultry (e.g. chicken or turkey),
lamb, beef and pork; fish, including salmon and prawns; and fruits
including soft fruits such as blackberries, raspberries,
loganberries, strawberries and the like.
[0039] Cardboard is heavy duty paper and may include a single thick
sheet of paper or more complex configurations such as multiple
corrugated and uncorrugated layers which tend to by more durable
than regular paper. The cardboard of the present invention will
generally be of a depth of less than about 1 cm. The impregnated
cardboard can be used in packaging, for example food packaging.
[0040] The cellulosic fibrous materials of the present invention
are provided in a dry form and are activated when they are wetted,
i.e. when the material comes into contact with moisture, such as a
liquid. The liquid may be, for example, water, body fluids, for
example sweat, blood or urine, fruit juice, cooking juices and the
like. The materials can be wetted before being applied to a surface
to be cleaned, for example, by applying water to the material
before using on a surface. Alternatively, the materials are
activated during use, for example, when drying hands moisture is
transferred onto the material or when using the material to wipe
down a wet surface.
[0041] The materials are provided in a substantially dry form and
are preferably dried by heating to constant mass.
[0042] Preferably, the amount of bioflavonoid coating impregnated
in the material is uniform throughout the material.
[0043] The bioflavonoid compositions described herein are
biodegradable and can be impregnated into biodegradable materials
such as biodegradable paper, bamboo fibres and the like to provide
environmentally friendly products.
[0044] The bioflavonoid compositions described herein show activity
against a wide range of organisms including gram positive bacteria,
gram negative bacteria, fungi, virus, protazoans and insect
parasites. The compositions may be employed against difficult
bacteria such as methicillin resistant Staphylococcus aureus
(MRSA), Clostridium difficile (C. diff), Helicobacter pylori (H.
pylori), and vancomycin resistant enterobacteria. The compositions
may also be used against norovirus and other pathogens whereby
transmission is by contact on air. In particular, the compositions
described herein show activity against E. coli, S. aureus,
Salmonella, B. subtilis and P. aeruginosa.
[0045] According to a second aspect of the invention, there is
provided a process for impregnating the materials described herein
with the bioflavonoid compositions described herein. Impregnation
is the partial or total saturation of a material, although total
saturation is preferred. In particular the material is a thin
material. A thin material is defined as having a depth of less than
about 1 cm. Impregnation may be after manufacture of the thin
material or it may occur during manufacture of the thin material,
for example, impregnation of the cellulose fibres before being
formed into the material.
[0046] If impregnating pre-formed cellulosic fibrous material, the
process involves immersing the material, in the bioflavonoid
composition to totally or partially saturate the material with the
composition. The material may then be rolled, squeezed or wrung to
remove any excess of the composition. The material is then dried,
either by air drying naturally, oven drying or by mechanical
drying. The equipment used to mechanically dry materials will be
known to those skilled in the art as will alternative drying
methods. The process results in a dry material which can then be
packaged as desired and later activated by wetting.
[0047] Alternatively, the cellulosic fibres used to produce the
material may first be immersed in the bioflavonoid composition to
totally or partially saturate the fibres with the composition which
are then dried either before or after being formed into materials
such as paper or cardboard by methods known in the art.
[0048] Alternatively, the materials may be impregnated by spraying
the bioflavonoid composition onto the materials so that the
composition impregnates the outer surface region of the material to
achieve at least partial impregnation. Spraying may also be used to
impregnate the fibres during manufacture or extraction, before
being formed into the materials of the invention.
[0049] A particular method for impregnating paper towels is
disclosed in Example 3. Fibrous bamboo products may also be
impregnated in the same way as disclosed in Example 3.
[0050] Preferably, the processes described above provide uniform
bioflavonoid impregnation throughout the cellulosic fibrous
material. A concentration of between 0.005 and 0.75%, preferably
between 0.005 and 0.5%, more preferably between 0.025% and 0.5%,
even more preferably between 0.025 and 0.1% of the bioflavonoid
composition is used. The compositions described herein are water
soluble and water can be used to dilute the bioflavonoid
composition to the desired concentration.
[0051] According to a third aspect of the invention, there is
provided a method of reducing the bacterial load on a surface. The
method of reducing the bacterial load on a surface is provided by
two mechanisms. Firstly, the kill is achieved by the action of the
bioflavonoid compositions and then secondly the contaminants are
mechanically removed by the material itself via the action of
placing on and wiping the surface, i.e. mechanical wiping.
[0052] The surface may be any bioactive surface and could be either
a human or non-human surface. For example, a human surface may
include the skin on the hands, feet or face. A non-human surface
may include any surface of sanitary importance which may carry a
contaminant, for example, the surfaces found in schools, bathrooms,
kitchens, factories, for example food factories, laboratories,
hospitals and the like.
[0053] For food contact the Environmental Protection Agency (EPA)
requires the active to effect a 5 log reduction of the challenge
organism in 30 seconds. Preferably, the materials of the present
invention effect at least a 5 log reduction of the bacteria load on
a surface in 30 seconds.
[0054] According to a fourth aspect of the invention, there is
provided a packaged product wherein the product is formed of a dry
cellulosic fibrous material impregnated with a bioflavonoid
composition. The product and the bioflavonoid composition are as
described in the first aspect of the invention.
[0055] The cellulosic product may be individually packaged.
Alternatively, the product may be packaged as part of a multi-pack.
Known packaging methods and materials may be used to package the
products of the present invention, for example conventional filmic
agents or cardboard boxes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] In order that the invention may be more fully understood it
will now be described, by way of example only, and with reference
to the following Figure(s), in which:
[0057] FIG. 1 is a graph showing the results of the effects of
different dilutions of the Citrox BC active dried onto Bounty.RTM.
brand paper towels on S. aureus activity.
[0058] FIG. 2 is a graph showing the results of the effects of
different dilutions of the Citrox BC active dried onto Bounty.RTM.
brand paper towels on E. coli activity.
DETAILED DESCRIPTION
[0059] The bioflavonoid content may comprise 40-50%, for example
about 45% wt/wt of the bioflavonoid composition. A suitable source
of a bioflavonoid composition is herein referred to as "HPLC 45" or
"Citrox BC" of which about 45% (of the total composition of HPLC
45/Citrox BC) comprises bioflavonoids. The bioflavonoids are in
admixture with biomass residues of extraction from bitter oranges,
such as pectins, sugars and minor organic acids, which make up the
remaining 55%. HPLC 45 is available from Exquim (a company of Grupo
Ferrer) as Citrus Bioflavonoid Complex 45% HPLC.
TABLE-US-00001 TABLE 1 The mixture of bioflavonoids in HPLC 45 %
bioflavonoid in mixture Bioflavonoid with biomass Neoeriocitrin 1.1
Isonaringin 1.2 Naringin 23.4 Hesperidin 1.4 Neohesperidin 12.5
Neodiosmin 1.4 Naringenin 1.5 Poncirin 2.0 Other (Rhiofolin)
0.5
EXAMPLES
[0060] Staphylococcus aureus was chosen as a representative gram
positive organism. This organism is found on mammalian skin and is,
therefore, shed into the surrounding environment. E. coli was
chosen as the representative of the gram negative enteric bacteria.
This organism is found in the digestive tract of birds, mammals and
reptiles. Its presence in the environment signals fecal
contamination. Pseudomonas aeruginosa was chosen to represent the
non-enteric gram-negative bacteria. This genera of bacteria is
present in water with related species representing major plant
pathogens and human opportunistic pathogens. Bacillus subtilis was
chosen as the representative gram positive spore-formers. This
bacterium is found in soil and water but is also ubiquitous in the
environment. This species forms endospores as a survival mechanism.
Bacterial endospores are the most resistant form of life on Earth
and, therefore, represent an ongoing concern for sanitation,
disinfection and sterilisation processes. Endospores represent the
"ultimate" challenge for any antimicrobial agent.
Example 1: Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC)
[0061] Procedure
[0062] A pure culture of a single microorganism is grown in an
appropriate broth. The culture is standardized using standard
microbiological techniques to have a concentration of very near 1
million cells per millilitre. The more standard the microbial
culture, the more reproducible the test results. The antimicrobial
agent is diluted a number of times, 1:1, using sterile diluents.
After the antimicrobial agent has been diluted, a volume of the
standardised inoculums equal to the volume of the diluted
antimicrobial agent is added to each dilution vessel, bringing the
microbial concentration to approximately 500,000 cells per
millilitre. The inoculated, serially diluted antimicrobial agent is
incubated at an appropriate temperature for the test organism for a
pre-set period, usually 18 hours. After incubation, the series of
dilution vessels is observed for microbial growth, usually
indicated by turbidity and/or a pellet of microorganisms in the
bottom of the vessel. The last tube in the dilution series that
does not demonstrate growth corresponds with the minimum inhibitory
concentration (MIC) of the antimicrobial agent.
[0063] In order to differentiate between a microbiostatic agent
(bacteria are not killed just inhibited) and a microbiocidal agent
(bacteria are killed) an MBC test is performed. When a
microbiostatic agent is removed or neutralized, previously
inhibited bacteria begin to grow again. Each well showing no
growth/turbidity in the MIC test is sub-cultured on media that
contains no biocide. Any microbial growth resulting from this test
indicates that, at that concentration, the active is
microbiostatic. If the subculture results in no bacterial regrowth,
then, at that concentration, the active is microbiosidal. The range
of concentration of Citrox BC active tested was 0.075-0.75%.
Discussion of Results
[0064] The MIC test is an established "screen" for the biostatic
(and possibly also biocidal) activity of liquid antimicrobials. It
is often used to find the appropriate concentrations of an
antimicrobial active to use for further efficacy testing.
Performing both the MIC and MBC test will enable one to
differentiate between a biocidal or biostatic mode of action.
Depending on the concentration of active used and the contact time
an active will often demonstrate both biostatic and biocidal modes
of action.
[0065] The range of Citrox BC active tested was 0.075%-0.75%. For
P. aeruginosa, no MIC value was obtained as all concentrations of
the Citrox BC active tested showed no turbidity (Table 2).
[0066] MCB testing showed that all concentrations were also
bactericidal for B. subtilis, there was also no MIC value obtained
demonstrating that inhibition of growth took place at all
concentrations tested. The MBC value obtained for B. subtilis was
0.315% Citrox BC active. This means that concentrations ranging
from 0.075% to 0.315% are bacteristatic and all concentrations of
the Citrox BC active greater than or equal to 0.315% are
bactericidal.
[0067] These results indicate that gram negatives like P.
aeruginosa are more easily killed by the Citrox BC active than the
gram positive B. subtilis.
TABLE-US-00002 TABLE 2 MIC/MBC Testing % Citrox MIC(G/NG) MBC
(CFU/mL) BC P.a B.s P.a B.s 0 G G 0 0 0.075 NG G 0 4.2 .times.
10.sup.2 0.095 NG G 0 3.1 .times. 10.sup.2 0.115 NG G 0 3.3 .times.
10.sup.2 0.135 NG G 0 3.5 .times. 10.sup.2 0.155 NG G 0 3.6 .times.
10.sup.2 0.175 NG G 0 2.4 .times. 10.sup.2 0.195 NG G 0 1.5 .times.
10.sup.2 0.215 NG G 0 1.3 .times. 10.sup.2 0.235 NG G 0 1.6 .times.
10.sup.2 0.255 NG G 0 40 0.275 NG G 0 40 0.295 NG G 0 1 0.315 NG NG
0 0 0.335 NG NG 0 0 0.355 NG NG 0 0 0.375-0.750 NG NG 0 0 G =
Growth, NG = No Growth P.a. = Psuedomonas aeruginosa B.s. =
Bacillus subtilis
Example 2: Time Kill Test
[0068] Procedure
[0069] All timed kill tests were performed using a standard viable
count procedure. Reference NB X34689.
[0070] The following neutralising solution was used in all kill
tests.
[0071] Tween 80--3%
[0072] Saponin--3%
[0073] Histidine--0.1%
[0074] Cysteine--0.1%
[0075] Rationale
[0076] A timed kill test assesses the amount of time it takes to
kill a defined population of microorganisms. A wide variety of
microorganisms are killed by the Citrox BC active. An important
first step in characterising this active for use in an
antimicrobial towel is to verify the kill claims. Claims for
efficacy are based on the number of bacterial killed within a
defined time frame. The most rigorous claims are those made for
food contact where the active must affect a 5 log reduction of the
challenge organism in 30 seconds.
Discussion of Results
Example 2(a): Timed Kill Test: 10 Minute Contact Time
[0077] Bacterial kill kinetics are affected by bacterial numbers,
the concentration of active used and the contact time. In order to
determine the most effective range of the Citrox BC active, S.
aureus was used in a 10 minute kill test to assess the efficacy of
various concentrations of the Citrox BC active. A >6.56 log
reduction was observed for all concentrations (0.45-0.65%) of the
Citrox BC active tested (Table 3).
[0078] When B. subtilis was used as a challenge organism, 0.7%
Citrox BC was required to effect a >5 log reduction in 10
minutes (Table 4). Based on previous tests, 0.5% active is the most
effective for general use.
TABLE-US-00003 TABLE 3 Time Kill Test: S. aureus, 10 min. % Citrox
Log10 Log BC CFU/mL CFU/mL Reduction 0 7.4 .times. 10.sup.6 6.86 0
0.45 <2 0.3 6.56 0.5 <2 0.3 6.56 0.55 <2 0.3 6.56 0.6
<2 0.3 6.56 0.65 <2 0.3 6.56 0.65 + 6.6 .times. 10.sup.6 6.81
0.05 neutralizer
TABLE-US-00004 TABLE 4 Time Kill Test: B. subtilis, 10 min. %
Citrox Log10 Log BC CFU/mL CFU/mL Reduction 0 1.1 .times. 10.sup.6
6.04 NA 0.5 2.9 .times. 10.sup.4 4.4 1.64 0.7 <2 0.3 5.74
Example 2(b): Timed Kill Test: 30 Second Contact Time
[0079] Timed kill studies using E. coli, P. aeruginosa and S.
aureus were performed using 0.5% Citrox BC active with a contact
time of 30 seconds. Log reductions of >6.4 were seen for all
organisms (Table 5). This confirms that this active would meet the
criteria for use in food contact situations.
TABLE-US-00005 TABLE 5 Time Kill Test: 30 seconds E. coli P.
aeruginosa S. aureus % Citrox BC 0 0.5 0 0.5 0 0.5 CFU/mL 5.1
.times. <2 6.4 .times. <2 7.4 .times. <2 10.sup.6 10.sup.7
10.sup.6 Log10 CFU/mL 6.7 <0.3 7.8 <0.3 6.8 <0.3 Log
Reduction NA 6.5 NA 7.3 NA 6.5
Example 2(c): Timed Kill Test: Sporicidal Activity
[0080] As stated above, the ultimate test for any antimicrobial
active is the ability to kill spores. Any chemical or process that
kills a bacterial spore is, by definition, a sterilant. In order to
assess if the Citrox BC active was sporicidal, a kill test was
performed on an actual spore suspension. Citrox BC, over a range
0.5% to 1.5%, was tested over a 1 hour time period. There were some
limitations to this test. The spore suspension (B. subtilis, ATCC
6633, 6.4.times.10.sup.4 CFU/pellet, Microbiologics) in the test
was only at .about.2.times.10.sup.4 CFU/ml, limiting the log
reduction calculation. The lyophilized pellets were found to
contain charcoal, a substance known to neutralise the bioflavonoid
component of the Citrox BC active. With those limitations,
approximately a 2 log reduction in spores was demonstrated. This
indicates that the Citrox BC active has definite activity against
spores. Spore suspensions at a higher titer without a charcoal
additive should be used to investigate this activity further.
Example 3: Surface Testing Using Paper Towel Impregnated with
Citrox BC Active
[0081] 1) Procedure: Adding Citrox BC to Paper Towel
[0082] Bounty.RTM. ("Bounty" is a registered trademark of Procter
& Gamble) brand paper towels were used to make the dry
antimicrobial towels. Bounty.RTM. paper towels are a conventional,
commercially available paper towel product. Citrox BC active
concentrate was diluted to desired concentrations. One paper towel
was immersed completely into the diluted active and then wrung out
by hand. The towel was dried overnight.
[0083] 2) Procedure: Weight of Citrox BC Active Dried onto
Bounty.RTM. Brand Paper Towel
[0084] Bounty.RTM. brand paper towels were dried to a constant
weight in a 54.degree. C. oven. Various dilutions of the Citrox BC
active were dried onto Bounty.RTM. brand paper towels as described
above. The towels were dried at room temperature overnight. The
treated towels were then dried to a constant weight at 54.degree.
C. The weight difference between the untreated and treated towels
is presumed to be the weight of the Citrox BC active.
[0085] 3) Procedure: For Testing Affect of Administration of
Impregnated Paper Towels to a Surface
[0086] Using the lab bench top as a representative hard, non-porous
surface, a grid was marked off using tape. Cotton-tipped swabs
saturated with a broth culture of the challenge organism were used
to inoculate the surface and air dried. Paper towels treated with
dilutions of the Citrox BC active were wetted and then used to
clean the inoculated bench top. The bench top was visibly wet for 3
minutes (contact time) and then allowed to completely air dry.
RODAC (Replicate Organism Detection and Counting) plates were used
to sample the cleaned surface for surviving bacteria. The plates
were incubated overnight at a temperature appropriate to the
challenge organism. Colonies were counted and the number used to
calculate CFU/cm.sup.2. Results were calculated by averaging the
counts from five 3''.times.3'' "grid squares".
[0087] 4) Procedure: RODAC Sampling
[0088] A RODAC plate is used to touch the surface to be sampled
after which the plate is incubated at an appropriate temperature.
There are nutrients in the media that promote the growth of a
variety of microbes. Lecithin and Polysorbate 80 are incorporated
in the agar and function as disinfectant/sanitizer neutralisers.
The type and number of microorganisms is detected by the appearance
of colonies on the surface of the agar medium. Collection of
samples from the same area before and after cleaning and treatment
with a disinfectant permits the evaluation of sanitary
procedures.
[0089] Results
[0090] Paper towels wetted with water and containing no Citrox BC
active were assessed for the ability to remove bacteria from a
contaminated hard surface. The results for this control (i.e.
unimpregnated paper towels) are shown by the bar labelled "0" in
FIGS. 1 and 2. FIGS. 1 and 2 show the results for both S. aureus
and E. coli. Paper towels containing dilutions of the Citrox BC
active greater than 1:200 were able to reduce the levels of S.
aureus from >50 CFU/cm.sup.2 to <1 CFU/cm.sup.2. The paper
towels containing dilutions of the Citrox BC active greater than
1:200 were able to reduce the levels of E. coli from >7
CFU/cm.sup.2 to <1 CFU/cm.sup.2.
[0091] These results show that a dry antimicrobial towel are
activated by wetting.
Discussion of Results
[0092] Different dilutions of the Citrox BC active were dried onto
Bounty.RTM. brand paper towels. These treated towels were used to
decontaminate a lab bench heavily inoculated with bacteria. The
ability of the treated towels to affect a decrease of contaminants
on the lab bench was evaluated using RODAC plates.
[0093] A method was developed to assess ability of a paper towel
impregnated with the Citrox BC active to reduce bacterial numbers
on a contaminated hard surface. RODAC plates are recommended for
the detection and enumeration of microorganisms present on surfaces
of sanitary importance. RODAC plates are specially constructed so
that an agar medium can be overfilled producing a dome-shaped
surface that can be pressed on a surface for sampling its microbial
content. RODAC plates are used in a variety of programs to
establish and monitor cleaning techniques and schedules.
[0094] When using a paper towel plus an antimicrobial active, one
must keep in mind that removal of bacteria from a contaminated
surface occurs by two mechanisms: first is the kill achieved by the
action of the antimicrobial active and second is mechanical removal
of the contaminants by the paper towel itself.
[0095] Lab scale antibacterial towels were used to calculate the
weight of the Citrox BC active dried onto the towels. The weight of
active present on the towel (Table 6) can be used as a starting
point for cost analysis.
TABLE-US-00006 TABLE 6 Weight of Citrox BC active dried onto Bounty
.RTM. paper towel Ave. wt. Citrox Pre- Post- of BC BC treatment
treatment active Dilution Dry Weight Dry Weight Difference
(g/towel) Std Dev 1:150 4.32547 4.31821 -0.00726 0.00388 0.006875
4.31226 4.32089 0.00863 4.31476 4.32400 0.00924 4.32424 4.32607
0.00183 4.32625 4.33321 0.00696 1:100 4.30877 4.35320 0.04443
0.05244 0.010257 4.30530 4.36125 0.05595 4.32380 4.37021 0.04641
4.30800 4.35446 0.04646 4.30435 4.37329 0.06895 1:50 4.31739
4.49804 0.18065 0.20090 0.046566 4.38309 4.51604 0.13295 4.31517
4.54191 0.22674 4.31539 4.52472 0.20933 4.32305 4.57787 0.25482
* * * * *