U.S. patent application number 14/795139 was filed with the patent office on 2015-11-26 for liquid nisin compositions.
The applicant listed for this patent is DSM IP ASSETS B.V.. Invention is credited to Ben Rudolf DE HAAN, Johannes Martinus Jacobus VISSER.
Application Number | 20150335030 14/795139 |
Document ID | / |
Family ID | 41168228 |
Filed Date | 2015-11-26 |
United States Patent
Application |
20150335030 |
Kind Code |
A1 |
VISSER; Johannes Martinus Jacobus ;
et al. |
November 26, 2015 |
LIQUID NISIN COMPOSITIONS
Abstract
The present invention relates to liquid nisin compositions
having a high anti-microbial activity. The invention further
relates to a method for preparing the liquid nisin compositions as
well as their use as a preservative in food products.
Inventors: |
VISSER; Johannes Martinus
Jacobus; (Echt, NL) ; DE HAAN; Ben Rudolf;
(Echt, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DSM IP ASSETS B.V. |
Heerlen |
|
NL |
|
|
Family ID: |
41168228 |
Appl. No.: |
14/795139 |
Filed: |
July 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13055981 |
Jan 26, 2011 |
9107443 |
|
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PCT/EP2009/060413 |
Aug 12, 2009 |
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14795139 |
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Current U.S.
Class: |
514/2.4 |
Current CPC
Class: |
A01N 63/10 20200101;
A23K 30/00 20160501; A23L 3/3526 20130101; A23L 3/34635 20130101;
A23V 2002/00 20130101; A23L 2/44 20130101; A23B 5/16 20130101 |
International
Class: |
A01N 63/02 20060101
A01N063/02; A23K 3/00 20060101 A23K003/00; A23L 3/3526 20060101
A23L003/3526 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 2008 |
EP |
08162201.1 |
Claims
1. An aqueous suspension of nisin having a pH of 2 to 12 and
comprising 0.2 to 5% (w/w) xanthan gum.
2. The suspension according to claim 1, comprising 0.01 to 5% (w/w)
nisin.
3. The suspension according to claim 1, further comprising at least
one compound selected from the group consisting of an additional
antimicrobial compound, a surfactant, a pH adjusting agent, an
anti-foaming agent and a cryoprotectant.
4. A method of preparing the suspension according to claim 1, the
method comprising the steps of: adding nisin and xanthan gum,
either separately or as a powder composition, to an aqueous
solution, mixing to obtain a suspension comprising 0.2 to 5% (w/w)
xanthan gum, and if necessary, adjusting the pH of the suspension
to 2 to 12.
5. A powder composition for use in the method according to claim 4
comprising nisin and xanthan gum.
6. A treatment liquid for treatment of a food, feed or agricultural
product comprising the aqueous suspension according to claim 1.
7. A preservative for a food, feed or agricultural product
comprising the suspension according to claim 1.
8. A method for preserving a food, feed or agricultural product,
wherein the suspension according to claim 1 is applied to the food,
feed or agricultural product.
9. The method of claim 8, wherein the suspension is applied to the
food, feed or agricultural product by spraying, dipping, immersion
or brushing.
10. A method for producing a solid nisin composition comprising the
step of subjecting the suspension according to claim 1 to a drying
step, lyophilisation step, crystallisation step or precipitation
step.
11. A food, feed or agricultural product comprising the suspension
according to claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/055,981, filed Jan. 26, 2011, which is a 371 of
PCT/EP2009/060413, filed Aug. 12, 2009, which claims priority to EP
08162201.1, filed Aug. 12, 2008, the contents of which are
incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the invention
[0003] The present invention relates to liquid nisin compositions,
to methods for preparing the compositions, to their use as a
preservative and to methods for preserving food wherein the
compositions are used.
[0004] 2. Description of Related Art
[0005] The need for improved food preservation methods is great. It
has been estimated that a large part of the world'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.
[0006] 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 poisonings is still increasing in most of
the countries. Food preservation techniques, e.g. heat processing,
freezing, ultrasound, irradiation, and high pressure treatment,
significantly reduce microbial load but of particular concern is
the evidence that processed foods are contaminated with
micro-organisms following processing and prior to packaging. Of
rising concern in the food industry are microbial problems related
to various foods such as dairy and meat products, fresh and chilled
foods and seafood.
[0007] Especially food products in the pH range of 4.5 to 7.0 are
known to be susceptible to microbial spoilage by micro-organisms,
including pathogens and spore forming bacteria. At lower pH levels,
yeasts, moulds and acid-tolerant bacteria are most relevant.
Mostly, processed foods are not consumed directly after processing,
thereby permitting bacteria surviving the production process or
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.
[0008] Furthermore, the recent trend for minimally processed foods
with the intrinsic nutritional and sensory qualities of raw and
fresh foods has raised the safety risk. Milder preservation
treatments, such as high hydrostatic pressure and
pulsed-electric-field techniques have proven to be successful, but
often rely on effective hurdles, i.e. cold chain and addition of
natural antimicrobials.
[0009] There has been extensive research conducted in the field of
food safety to develop effective anti-microbial product designs,
which result in a combination of compositions, processing and
shelf-life conditions.
[0010] Nisin is a peptide-like antibacterial substance produced by
Lactococcus lactis subsp. lactis. It comprises 34 amino acids and
is active against mainly 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 survives
pasteurisation temperatures with minimal loss of activity.
[0011] Usually, nisin is obtained by fermentation of a species of
Lactococcus lactis and is further formulated as a dry powder that
can be used as a preservative as such or after having first being
solved into a suitable solvent. Delvoplus.RTM. and Nisaplin.RTM.
are brand names for a nisin powder containing 1 million IU per
gram. They are distributed by DSM and Danisco, respectively. These
powdered nisin products have several drawbacks: dust is generated
upon handling, and dosing and mixing small amounts of powders into
products is difficult. Therefore, liquid nisin compositions which
do not have the drawbacks described above are commercially
preferred.
[0012] Liquid nisin compositions as such are known in the art.
Although liquid nisin compositions have been reported to have
activity against gram-positive bacteria (see Mota-Meira et al.
(2000), Montville et al. (1999), U.S. Pat. No. 5,584,199 and U.S.
Pat. No. 4,597,972) and even gram-negative bacteria (see EP 0 453
860, U.S. Pat. No. 5,260,271 and U.S. Pat. No. 5,559,096), there is
still a need for liquid nisin compositions having an improved
antimicrobial activity, particularly against gram-positive bacteria
found in the food industry.
SUMMARY
[0013] Surprisingly, nisin compositions having a very high activity
against gram-positive bacteria have now been found. Due to their
high antimicrobial activity only low amounts of the compositions
are needed for effective action against bacteria e.g. gram-positive
bacteria. The compositions have good microbiological stability
which in combination with their good physical and chemical
stability makes the compositions suitable for prolonged storage and
ergo gives them a long shelf life. In addition, the compositions of
the invention can have a low turbidity, which makes them suitable
for use in food applications, wherein addition of low turbidity
additives is of importance. In the light of the above
characteristics, the compositions of the invention can
advantageously be employed as food preservatives.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0014] According to a first aspect the invention provides a method
for preparing a liquid nisin composition, preferably an aqueous
liquid nisin composition. The method comprises the steps of: a)
preparing a first liquid nisin containing composition having a pH
of about 1.5 to about 12, preferably about 3 to about 10,
preferably about 3.5 to about 9.5, more preferably about 4 to about
9, yet more preferably about 4.5 to about 8.5, even more preferably
about 5 to about 8, most preferably about 5.5 to about 7.5, and in
particular about 5.5 to below 7, b) isolating solid compounds from
the prepared first liquid nisin containing composition, c)
contacting the isolated solid compounds with a solution having a pH
of about 0 to about 5, preferably about 0.5 to about 4.5, more
preferably about 1 to about 4, even more preferably about 1.5 to
about 3.5, most preferably about 1.5 to about 3 and in particular
about 2 to about 3 to prepare a second liquid nisin composition,
and d) removing solid compounds from the second liquid nisin
composition. Step d is optional, but in a preferred embodiment it
is performed in the method of the invention.
[0015] In a further embodiment the method of the invention
comprises the step of: e) adjusting the pH of the second liquid
nisin composition to a desired pH-value such as a pH between 2 and
6, e.g. a pH between 2 and 3 or a pH between 5 and 6.
[0016] Optionally, at least one of the additional functional
compounds mentioned below can be added before, during or after at
least one of the steps of the method of the invention. For
instance, a cryoprotectant, e.g. glycerol, can be added during step
c, such that the second liquid nisin composition comprises 35% to
60% w/w cryoprotectant. In another example, a compound that
decreases or diminishes foam formation and/or an additional
antimicrobial compound, e.g. an organic acid or a salt thereof, can
be added before step b. In a preferred embodiment however at least
one additional functional compound is added after step d and prior
to step e, or during or after step e. In an embodiment step a
comprises mixing nisin with an aqueous solution to prepare a first
liquid nisin containing composition having a final inorganic salt
(e.g. NaCl) concentration of 1.5 M or below, preferably 0.05 M to
1.5 M and more preferably 0.1 M to 1.5 M. The first liquid nisin
containing composition has a pH of about 1.5 to about 12,
preferably about 3 to about 10, preferably about 3.5 to about 9.5,
more preferably about 4 to about 9, even more preferably about 4.5
to about 8.5, yet even more preferably about 5 to about 8 and most
preferably about 5.5 to about 7.5 and in particular about 5.5 to
below 7. Any source of nisin can be suspended and/or dissolved in
the aqueous solution. In a preferred embodiment the nisin is a
powder, preferably a dry powder. For example, commercially
available nisin powder compositions such as Delvoplus.RTM. and
Nisaplin.RTM. can be used. The source may comprise nisin A, nisin Z
or a combination thereof. The aqueous solution may be a buffer
solution, e.g. a phosphate buffer such as
NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4. Other suitable buffers can of
course also be used. These include, but are not limited to, acetate
buffers, lactate buffers, citrate buffers, glycine/HCl buffers and
any combination thereof.
[0017] Solid compounds can be separated/isolated from the first
liquid nisin containing composition by well-known isolation
techniques. In a preferred embodiment step b is performed by means
of centrifugation, filtration or any combination thereof.
[0018] Subsequently, a second liquid nisin composition can de
prepared by e.g. contacting, e.g. dissolving or mixing or
suspending, the isolated solid compounds with/in a solution,
preferably an aqueous solution, having a pH of about 0 to about 5,
preferably about 0.5 to about 4.5, more preferably about 1 to about
4, even more preferably about 1.5 to about 3.5, most preferably
about 1.5 to about 3 and in particular about 2 to about 3. In an
embodiment an additional functional compound mentioned below is
added during this step.
[0019] Next, the second liquid nisin composition can be purified by
removing e.g. the remaining debris and/or non-nisin proteins or
parts thereof. This purification step can be performed by
well-known isolation techniques. In a preferred embodiment step d
is performed by means of centrifugation, filtration or any
combination thereof.
[0020] The above-described method results in a liquid nisin
composition having a much higher activity against micro-organisms,
particularly gram-positive bacteria, than liquid nisin compositions
described in the prior art. In other words, the method of the
present invention results in liquid nisin compositions having a
much lower minimum inhibitory concentration (MIC) against
micro-organisms, particularly gram-positive bacteria, than liquid
nisin compositions described in the prior art.
[0021] Therefore, a nisin composition obtainable by a method
according to the invention is another part of the present
invention. The nisin composition may be solid, but preferably it is
a liquid composition.
[0022] In an embodiment the nisin compositions of the invention
have a MIC of 1.0 .mu.g/ml or less against at least one
gram-positive bacterium. MIC refers to the minimum concentration of
a compound or composition necessary to inhibit growth of the
organism tested. Preferably, the MIC is an average of at least
three independent repetitions. Compositions of the present
invention having a MIC of 1.0 .mu.g/ml or less when tested for
growth inhibition of at least one gram-positive bacterium in the
assay described herein. In an embodiment the compositions of the
invention have a MIC of 0.5 .mu.g/ml or less, preferably a MIC of
0.1 .mu.g/ml or less, more preferably a MIC of 0.05 .mu.g/ml or
less, even more preferably a MIC of 0.01 .mu.g/ml or less, yet even
more preferably a MIC of 0.005 .mu.g/ml or less, particularly a MIC
of 0.001 .mu.g/ml or less, more particularly a MIC of 0.0005
.mu.g/ml or less against at least one gram-positive bacterium and
most particularly a MIC of 0.0001 .mu.g/ml or less against at least
one gram-positive bacterium. Gram-positive bacteria include, but
are not limited to, Micrococcus sp., Listeria sp., Bacillus sp.,
Staphylococcus sp., Clostridium sp., Streptococcus sp.,
Lactobacillus sp. and Lactococcus sp. In an embodiment the
gram-positive bacterium is selected from the group consisting of
Bacillus, Lactococcus, Staphylococcus, Listeria and Micrococcus.
Suitable species within the genera Bacillus, Lactococcus,
Staphylococcus, Listeria and Micrococcus include, but are not
limited to, B. subtilis, L. lactis, S. aureus, L. innocua and M.
luteus, respectively. Within the species given suitable strains
include, but are not limited to, Bacillus subtilis ATCC 31578,
Lactococcus lactis ATCC 19257, Staphylocoocus aureus ATCC 27661,
Listeria innocua LMD 92.20 and Micrococcus luteus B212,
respectively. In a preferred embodiment the compositions of the
present invention have a MIC of 0.5 .mu.g/ml or less, preferably a
MIC of 0.1 .mu.g/ml or less, more preferably a MIC of 0.05 .mu.g/ml
or less, even more preferably a MIC of 0.01 .mu.g/ml or less, yet
even more preferably a MIC of 0.005 .mu.g/ml or less, particularly
a MIC of 0.001 .mu.g/ml or less and more particularly a MIC of
0.0005 .mu.g/ml or less against at least one strain of M. luteus,
preferably M. luteus B212.
[0023] Nisin activity can be measured using the following bio-assay
well-known to the skilled person (see Pongtharangkul and Demirci,
2004), including pre-treating the nisin composition at low pH.
Briefly, M. luteus B212 containing agar plates (Iso-sensitest agar)
are prepared using a freshly grown culture. After drying, a vacuum
pump is used to create small holes in the agar. Samples and
dilutions thereof (10 .mu.l) are transferred into the holes and
allowed to diffuse into the agar for 18 hours at 5.degree. C.
Subsequently, the agar plates are incubated for 24 hours at
30.degree. C. and the inhibition zones around the sample containing
holes are measured. Parallel to the samples, controls with known
amounts of nisin (0-1600 IU/ml) are included. Their inhibition
zones are used to prepare a calibration curve required to determine
the nisin levels of the samples. All steps are carried out
aseptically. The IU for nisin has already been defined as follows.
The World Health Organization Committee on Biological
Standardization, Twenty second report. World Health Organization
Technical Report Series, No. 444 in 1970, 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 nisin powder
products containing 1 million IU per gram, are distributed by DSM
and Danisco, respectively. By means of the above assay the nisin
concentration in samples can be determined.
[0024] The MIC of nisin compositions can be measured by means of
the following MIC assay. Nisin activity is measured using the
standard microdilution broth assay, well-known to the skilled
person. Briefly, a Micrococcus luteus B212 containing Iso-sensitest
broth is prepared using a freshly grown culture. The number of
cells per ml is determined using a counting chamber. Preferably, a
cell count of 10.sup.3 is used. 100 .mu.l of inoculum is added to
each well of a 96-well microtiter plate. 100 .mu.l of a nisin
composition is added to the first well (A1) and mixed properly by
pipetting up and down three times. A serial dilution is made by
transferring 100 .mu.l of the first well to the next well (A2) and
diluted properly. This is repeated until each component is serially
diluted in 36 wells. Next, plates are incubated at 30.degree. C.
for 7 days and read each day for bacterial growth. MIC
concentrations are the lowest concentration completely inhibiting
growth.
[0025] In an embodiment the compositions of the invention have a pH
of about 0 to about 5, preferably about 0.5 to about 4.5, more
preferably about 1 to about 4, even more preferably about 1.5 to
about 3.5, most preferably about 1.5 to about 3 and in particular
about 2 to about 3. At such pH conditions, the microbiological
stability of the compositions of the invention is good and the MIC
of the compositions is low and stable during storage.
[0026] In a further embodiment the compositions according to the
invention comprise 0.01 to 5%, preferably 0.05 to 2.5%, more
preferably 0.1 to 1.0%, most preferably 0.15 to 0.5% and in
particular 0.2 to 0.3% (w/w) nisin.
[0027] The nisin compositions of the invention may comprise a low
amount of salts such as inorganic salts e.g. NaCl. It is to be
understood that the additional functional compounds mentioned below
(e.g. antimicrobial compounds such as organic acids or their salts)
are not meant to be included within the definition of "salt". In an
embodiment the compositions of the invention comprise a salt, e.g.
inorganic salt, to nisin ratio of 100:1 to 1:100, preferably 50:1
to 1:100, more preferably 25:1 to 1:100 and in particular 10:1 to
1:100. In an embodiment the nisin compositions of the invention are
essentially free of salts, preferably inorganic salts such as e.g.
NaCl. The inorganic salt may be any suitable, food grade inorganic
salt. Examples of inorganic salts are NaCl, Na.sub.2SO.sub.4,
(Ca).sub.3(PO.sub.4).sub.2, KNO.sub.B, KCl and MgCO.sub.3. The
concentration of these salts in the compositions is 100 mg/ml or
less, preferably 50 mg/ml or less, more preferably 25 mg/ml or less
and in particular 15 mg/ml or less. The salt concentration may be
measured by separate cationic analysis, by atomic absorption
anionic analysis, by HPLC or preferably by determination of the ash
content by ignition (550.+-.25.degree. C.). Nisin compositions
having a low concentration of inorganic salts are very attractive,
since they will not interfere with the food matrix to give
undesired reactions and alterations of taste and/or structure.
[0028] The nisin compositions of the invention may comprise low
amounts of components other than nisin and salt. These components
may be proteins or parts thereof. It is to be understood that the
additional functional compounds mentioned below (e.g. antimicrobial
compounds, anti-foaming agents, surfactants, etc.) are not meant to
be included within the definition of "components other than nisin
and salt". In an embodiment the compositions of the invention
comprise a non-nisin component to nisin ratio of 100:1 to 1:100,
preferably 10:1 to 1:100 and more preferably 2:1 to 1:100. In an
embodiment the nisin compositions of the invention are essentially
free of these components. The components may originate from the
biomass produced during the nisin fermentation process using
Lactococcus lactis. The nisin concentration may first be measured
by the assay described above. Subsequently, total protein
concentration may be estimated using classical assays known to the
skilled person. The non-nisin protein concentration may be
estimated by subtracting the nisin concentration from the total
protein concentration.
[0029] In yet another embodiment the compositions of the invention
are clear liquid compositions. Clear liquid nisin compositions can
be used on and/or in any type of product. In view of their clarity,
they can advantageously be used in products wherein clarity is of
importance such as jelly-based products e.g. jelly dessert, fruit
juices, beverages and surface applications on food products. Clear
liquid compositions as used herein are liquid compositions having a
turbidity of 0 to 100 FUN, preferably 0 to 50 FUN, more preferably
0 to 25 FUN and particularly 0 to 10 FUN. Turbid liquid
compositions are liquid compositions having a turbidity of above
100 FUN. The turbidity in FUN (Formazine Nephelometric Unit) can be
determined with a light scattering method and can be measured using
a Nephla turbidity photometer with measuring method DIN EN
27027/ISO 7027. Clear as well as turbid liquid nisin compositions
can be prepared by means of the method according to the invention.
A clear liquid composition is prepared, if a liquid nisin
containing composition having a pH of about 5 or higher, preferably
a pH of about 5 to about 9, is prepared in step a of the method of
the invention. A turbid liquid composition is prepared, if a liquid
nisin containing composition having a pH of below about 5,
preferably a pH of about 1.5 to below about 5, or a pH of above
about 9, preferably a pH of above about 9 to about 12 is prepared
in step a of the method of the invention. Both the clear and the
turbid liquid nisin compositions have the above-described high
activity against micro-organisms, in particular gram-positive
bacteria.
[0030] A method wherein the final inorganic salt (e.g. NaCl)
concentration of the first liquid nisin containing composition
(i.e. the liquid nisin composition prepared in step a of the method
according to the invention, see above) is above 1.5 M has several
disadvantages compared to a method wherein the final inorganic salt
concentration of the first liquid nisin containing composition is
1.5 M or below. Firstly, the first liquid nisin composition having
a final inorganic salt concentration of above 1.5 M shows a
decreased separation performance in centrifugation (i.e. has lower
sedimentation rate) in comparison to liquid nisin compositions with
a final inorganic salt concentration of 1.5 M or below. Secondly,
the resulting final liquid nisin composition that is prepared by
performing the method according to the present invention (i.e.
steps a to c and optionally steps d and e, see above) wherein the
first liquid nisin containing composition has a final inorganic
salt concentration of above 1.5 M has several disadvantages:
[0031] It is turbid;
[0032] It has a lower purity than final liquid nisin compositions
that have been made by means of a method according to the present
invention wherein the first liquid nisin containing composition
contains a final inorganic salt concentration of 1.5 M or
below;
[0033] It has a lower antimicrobial activity than final liquid
nisin compositions that have been made by means of a method
according to the present invention wherein the first liquid nisin
containing composition contains a final inorganic salt
concentration of 1.5 M or below; and
[0034] It has a higher risk to precipitate than final liquid nisin
compositions that have been made by means of a method according to
the present invention wherein the first liquid nisin containing
composition contains a final inorganic salt concentration of 1.5 M
or below
[0035] The liquid nisin compositions of the invention have at least
one of the advantages listed below compared to liquid nisin
preparations known in the prior art:
[0036] the compositions of the invention have a better
antimicrobial efficacy compared to liquid nisin compositions of the
prior art, and/or
[0037] the compositions of the invention are essentially free of
salts such as e.g. inorganic salts e.g. NaCl and essentially free
of other non-nisin components. As a result thereof, in food
applications, the use of the compositions of the invention do not
interfere with the food matrix to give undesired reactions and
alterations of the taste and/or structure are avoided, and/or
[0038] the compositions of the invention can be clear, i.e. have a
low turbidity (between 0 and 100 FNU). Such compositions do not
interfere with the colour and/or clarity of the products to which
they are applied.
[0039] According to another embodiment, the compositions of the
invention further comprise at least one additional functional
compound including, but not limited to, an additional antimicrobial
compound such as an acid e.g. sorbic acid, propionic acid, benzoic
acid, acetic acid, lactic acid, citric acid, cinnamic acid, or a
salt of any of these acids, a glucose oxidase, natamycin, lysozyme,
poly-L-lysine, nystatin, lucensomycin, amphotericin B, filipin,
pediocin; a surfactant e.g. SDS, Tween, fatty acids; a pH adjusting
agent such as HCl or NaOH or a buffering agent e.g. a phosphate
salt or acetate salt; a cryoprotectant such as glycerol or
propanediol; a thickening agent e.g. xanthan gum, guar gum, Arabic
gum, tragacanth gum, gellan gum, locust bean gum, carrageenan gum,
rhamxan gum, alginate, starch, carboxymethyl cellulose,
carboxyethyl cellulose, hydroxypropylmethyl cellulose,
hydroxypropyl cellulose, methyl cellulose, polyvinyl alcohol,
polyethylene glycol, polypropylene glycol. Moreover, the
compositions of the invention may comprise agents that decrease or
diminish foam formation. The additional compounds may be added to
the compositions of the invention in solid or liquid form and may
be mixed well in advance or directly prior to use. Using at least
one additional antimicrobial compound/preservative in the nisin
compositions of the invention is expected to further stabilize it
microbiologically and therefore may be beneficial for its
shelf-life.
[0040] The activity of nisin present in an aqueous liquid
composition can be substantially increased by removing impurities.
Moreover, the solubilisation rate of nisin in aqueous compositions
is increased by the removal of impurities such as e.g. inorganic
salts. Nisin may be partly bound to the impurities resulting in
nisin which is non-available for its preservative activity. In
other words, nisin has a limited bioavailability in the presence of
impurities. As used herein, the term "bioavailability" refers to
the availability, amount (e.g., concentration), or activity of
nisin in a liquid, semi-solid or solid formulation. Impurities such
as non-nisin proteins or other non-nisin components, cell wall
debris and salts may have a negative effect on the solubilisation
rate of nisin. Approximately, less than 50% of the nisin present in
such liquid formulation is found to be available as a preservative
in case these impurities are present. The impurities have been
found to be present in commercially available nisin products.
Commercially available nisin contains in general 5-25% on non-nisin
protein and cell debris. These impurities originate from the
production process of the nisin. In the recovery, purification or
reformulation following the fermentation salts are often used which
still are present in the final nisin formulation.
[0041] In a further aspect the invention relates to an aqueous
suspension of nisin comprising a thickening agent. Of course, two
or more different thickening agents can also be used. The
suspensions of the invention comprise 0.01 to 5%, preferably 0.05
to 2.5%, more preferably 0.1 to 1%, most preferably 0.15 to 0.5%
and in particular 0.2 to 0.3% (w/w) nisin. The suspensions of the
invention comprise 0.01 to 5%, preferably 0.05 to 5%, more
preferably 0.1 to 5%, most preferably 0.2 to 5% and in particular
0.5 to 5% (w/w) thickening agent. The thickening agent is selected
from the group consisting of xanthan gum, guar gum, Arabic gum,
tragacanth gum, gellan gum, locust bean gum, carrageenan gum,
rhamxan gum, alginate, starch, carboxymethyl cellulose,
carboxyethyl cellulose, hydroxypropylmethyl cellulose,
hydroxypropyl cellulose, methyl cellulose, polyvinyl alcohol,
polyethylene glycol and polypropylene glycol. In a preferred
embodiment the thickening agent is a gum such as xanthan gum. The
pH of the suspension according to the invention is about 2 to about
12, preferably about 2 to about 11, more preferably about 2 to
about 10, even more preferably about 2 to about 9, yet even more
preferably about 2 to about 8, most preferably about 2 to about 7
and in particular about 2 to about 6. The suspension of the
invention is stable. "Stable suspension" as used herein means a
physically stable suspension, i.e. a suspension that shows 50% or
less, preferably 40% or less, more preferably 30% or less, even
more preferably 20% or less, most preferably 10% or less and in
particular 0% sedimentation after storage at room temperature for 9
days at pH 5. The physical stability of the suspensions can be
measured by methods known in the art such as the sedimentation
assay as shown herein (see Example 9).
[0042] In an embodiment the suspension according to the invention
further comprises at least one additional functional compound
selected from the group consisting of an additional antimicrobial
compound, a surfactant, a pH adjusting agent, and a cryoprotectant.
Examples of suitable additional antimicrobial compounds are acids
such as sorbic acid, propionic acid, benzoic acid, acetic acid,
lactic acid, citric acid, cinnamic acid, or salts of any of these
acids, a glucose oxidase, natamycin, lysozyme, poly-L-lysine,
nystatin, lucensomycin, amphotericin B, filipin, pediocin. Examples
of suitable surfactants are SDS, Tween, fatty acids, to name just a
few. Examples of suitable pH adjusting agents are among others HCI
or NaOH or buffering agents such as phosphate salts and acetate
salts. Examples of suitable cryoprotectants are glycerol and
propanediol. Moreover, the suspensions of the invention may
comprise agents that decrease or diminish foam formation. The
additional compounds may be added to the suspensions of the
invention in solid or liquid form and may be mixed well in advance
or directly prior to use.
[0043] In a further embodiment the invention relates to a method of
preparing a suspension according to the invention, the method
comprising the steps of: a) adding nisin and a thickening agent,
either separately or as a powder composition, to an aqueous
solution (e.g. water), and b) mixing to obtain a suspension. If
necessary, the pH of the suspension can be adjusted to a pH of
about 2 to about 12, preferably about 2 to about 11, more
preferably about 2 to about 10, even more preferably about 2 to
about 9, yet even more preferably about 2 to about 8, most
preferably about 2 to about 7 and in particular about 2 to about
6.
[0044] The nisin and thickening agent can be added separately to
the aqueous solution. They can be in powder form or in liquid form.
Alternatively, nisin and the thickening agent can be present in one
powder composition and this powder composition can be added to the
aqueous solution. So, in a further embodiment the invention relates
to a powder composition comprising nisin and a thickening agent.
Nisin and/or the thickening agent can be added together with an
additional functional compound described above to the aqueous
solution and then mixed to obtain a suspension. Alternatively, the
additional functional compounds can be added after the suspension
comprising nisin and thickening agent has been obtained. In a
further embodiment nisin is first added to the aqueous solution,
followed by an additional functional compound and thereafter the
thickening agent is added and the solution is mixed to obtain a
suspension. In yet a further embodiment a thickening agent is first
added to the aqueous solution, followed by an additional functional
compound and thereafter nisin is added and the solution is mixed to
obtain a suspension. In again a further embodiment the additional
functional compound is first added to the aqueous solution,
followed by addition of a thickening agent and/or nisin.
[0045] Another aspect of the invention is concerned with the use of
an aqueous suspension according to the invention for preparation of
a treatment liquid for treatment of a food, feed or agricultural
product. The treatment liquid can be prepared by mixing an aqueous
solution with the suspension according to the invention. Treatment
of the food, feed or agricultural product can be done by spraying,
dipping, immersion, brushing to name just a few.
[0046] According to a further aspect, the invention provides the
use of a composition or suspension according to the invention as a
preservative in and/or on food, feed or agricultural products.
Hereafter, the term "suspension" also includes a treatment liquid
prepared from a suspension according to the invention. The
compositions and suspensions of the invention do not have drawbacks
associated with powder formulations: they are more easy-to-use
(ease of dosing) and there is no dust formation when using them.
Additionally, foam formation and dissolving problems that occur
when solubilising nisin powder into a solvent are prevented.
Effective levels of nisin to preserve food products range from 1 to
1500 IU/g or 0.025 to 37.5 ppm of nisin. The compositions and
suspensions according to the invention can be used alone, but also
in combination with other antimicrobial compositions, e.g.
compositions comprising organic acids or salts thereof, lysozyme.
The antimicrobial compositions can be applied to food, feed or
agricultural products before, during or after application of the
compositions or suspensions according to the invention.
[0047] In a further aspect the invention pertains to a container
comprising 1 to 1000 litre of a composition or suspension according
to the invention. The container can be a bottle, bag or tank, to
name just a few.
[0048] According to a further aspect, the invention provides a
method for preserving food, feed or agricultural products, wherein
the nisin compositions or suspensions of the invention are being
used, e.g. applied in and/or on the respective products. The nisin
compositions and suspensions can be applied by spraying, dipping,
immersion, brushing, to name just a few methods. In case the
substrate/product is a liquid or semi-liquid, they may be directly
added. The compositions or suspensions may even leave a coating,
e.g. an antimicrobial coating, on the substrate they are applied
to/on. Optionally, in a further step, the product may also be
pasteurised/sterilized. This step may of course also be performed
before application of the nisin compositions or suspensions of the
invention. All types of food products may be treated with the
compositions or suspensions of the invention. The food products may
be dairy food products; food products containing or derived from
eggs, meats, especially poultry e.g. freshly slaughtered poultry,
vegetables, crustacean and fish; rice products such as boiled rice
products; bakery food products; beverages; chilled food products;
clear food products such as jelly-based food products such as jelly
desserts; juices; spreads; jam; canned fruit and other canned
products; food products wherein the compositions or suspensions of
the invention are applied to on the surface. Dairy food products
include, but are not limited to, processed cheese, milk, clotted
cream, dairy desserts, ice cream mixes, dressing and yoghurts. The
compositions and suspensions according to the invention can also be
used in the treatment of food packaging and handling equipment and
can be included in/on packaging materials used for packaging of
food, feed or agricultural products. The compositions and
suspensions of the invention may also be used as a disinfectant for
cleaning surfaces and cooking utensils in food processing plants
and any area in which food is prepared or served such as hospitals,
nursing homes, restaurants, especially fast food restaurants,
delicatessens and the like. The compositions and suspensions
according to the invention are capable of inhibiting bacterial
growth in products for an extended period of time, for example at
least about 1 day, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 400,
500, 600, 700, 800, 900 days and preferably at least about 1000
days. The compositions and suspensions according to the invention
can be used to prevent bacterial growth, e.g. the growth of
Gram-positive bacteria such as Staphylococcus, Streptococcus,
Listeria, and Colyneform bacteria. It can even be used to prevent
growth of Gram-negative bacteria such as Gram negative bacteria
such as Salmonella, Shigella, Escherichia Coli, Klebsiella,
Pseudomonas, Bacterioides, and Actinobacillus bacteria.
[0049] Ergo, a food, feed, or agricultural product comprising a
nisin composition or suspension according to the invention is
another part of the invention.
[0050] In yet another aspect, the invention pertains to a method
for producing a solid, e.g. powder, nisin composition comprising
the step of subjecting the liquid nisin composition according to
the invention to e.g. a drying step, lyophilisation step,
crystallisation step (followed if necessary by filtration or
centrifugation) or a precipitation step (followed if necessary by
filtration or centrifugation), to name just a few. The steps may be
performed immediately after step c, d or e of the method for
preparing the nisin compositions of the invention as described
above. They may also be done after the liquid nisin compositions of
the invention have been stored for a period of time. The resulting
solid/powder nisin compositions can be mixed with powder
compositions comprising other suitable compounds such as e.g. the
additional functional compounds described above.
[0051] The invention is further illustrated by the following
examples, which should not be construed as limiting the scope of
the invention.
EXAMPLES
Example 1
Preparation of Liquid Nisin Compositions
[0052] The following liquid nisin compositions were prepared:
Composition A: Ten grams of nisin powder Nisaplin.RTM. (Danisco,
Denmark) containing 2.5% w/w nisin and at least 50% w/w NaCl was
dissolved in an aqueous HCl solution (pH 2.0-3.0; total volume 100
ml).
[0053] Composition B: Ten grams of nisin powder Nisaplin.RTM.
(Danisco, Denmark) containing 2.5% w/w nisin and at least 50% w/w
NaCl was dissolved in an aqueous HCl solution (pH 5.5-6.5; total
volume 100 ml).
[0054] Compositions C and D: Ten grams of nisin powder
Nisaplin.RTM. (Danisco, Denmark) containing 2.5% w/w nisin and at
least 50% w/w NaCl was dissolved in a buffered aqueous solution of
0.2 M sodiumdihydrogenphosphate and disodiumhydrogenphosphate (pH
7.0; total volume 100 ml; in addition composition having a pH of 6
and compositions having a pH of 6.5 were made). The mixture was
subsequently mixed for about 15 minutes. The mixture was
centrifuged at 4,500.times.g for 15 minutes at 10.degree. C. and a
pellet containing nisin was obtained. Subsequently, the pellet was
dissolved in an aqueous citric acid solution (pH 2.0 to 3.0; total
volume 100 ml). The mixture was stirred for 15 minutes. The
obtained solution containing nisin was centrifuged at 4,500.times.g
for 15 minutes at 10.degree. C. to remove remaining solid
components. The obtained liquid compositions were either kept at a
pH of 2.0 to 3.0 (Composition C) or the pH was adjusted to a pH
between 5.5 and 6.5 by addition of NaOH (Composition D).
[0055] Compositions E and F: The preparation of compositions E and
F was identical to the preparation of compositions C and D with the
proviso that the pellet was dissolved in an aqueous HCl solution
having a pH of 2.0 to 3.0.
[0056] Compositions G and H: Ten grams of nisin powder
Nisaplin.RTM. (Danisco, Denmark) containing 2.5% w/w nisin and at
least 50% w/w NaCl was dissolved in an aqueous HCl solution (pH
2.0-3.0; total volume 100 ml). The obtained mixture was dialysed
for 24 hours in an aqueous HCl solution of pH 2.0 to 3.0. Next, the
dialysed mixture was centrifuged at 4,500.times.g for 15 minutes at
10.degree. C. The obtained liquid compositions were either kept at
a pH of 2.0 to 3.0 (Composition G) or the pH was adjusted to a pH
between 5.5 and 6.5 by addition of NaOH (Composition H).The
obtained compositions were used in the following experiments.
Example 2
MIC Assay
[0057] For the MIC assay freshly cultured Micrococcus luteus cells
(B212) and Pseudomonas aeruginosa cells (ATCC 9027) were obtained
from an overnight culture grown in Iso Sensitest Broth (Oxoid) and
Plate Count Broth (Difco), respectively, at 30.degree. C. A stock
suspension of 4.3.times.10.sup.5 and 2.5.times.10.sup.4 colony
forming units CFU/ml, respectively, was prepared in physiological
saline. 30 .mu.l of the respective stock solution was added to 30
ml of Iso Sensitest Broth (suspension A) and Plate Count Broth
(suspension B), respectively. Then, 100 .mu.l of suspension A was
transferred to each well of a first 96-wells microtiter plate and
100 .mu.l of suspension B was transferred to each well of a second
96-wells microtiter plate. Nisin compositions were prepared
according to Example 1. 100 .mu.l of a nisin composition was used
in a standard micro dilution broth assay to determine the Minimal
Inhibition Concentration (MIC) of each nisin composition. The
results, presented in Table 1, show that the nisin compositions C,
D, E and F show the highest activity (i.e. lowest MIC) against both
micro-organisms (a gram-positive micro-organism, i.e. M. luteus,
and a gram-negative micro-organism, i.e. P. aeruginosa) at pH 2.5
and pH 6.0. The MIC of compositions C and E is between about 40--to
about 100-fold lower than the MIC of compositions A and G
(compositions all having a pH of 2.5), while the MIC of
compositions D and F is significantly lower than the MIC of
compositions B and H (compositions all having a pH of 6.0). The MIC
of compositions C and D prepared with 0.1 M phosphate buffer at pH
7 was comparable to the MIC of compositions C and D prepared with
0.2 M phosphate buffer at pH 6 or pH 6.5.
[0058] In a separate experiment the MIC concentrations of
compositions A and C were compared for freshly cultured Bacillus
subtilis (ATCC 31578), Staphylococcus aureus (ATCC 27661),
Lactococcus lactis (ATCC 19257) and Listeria innocua (LMD92.20).
The experiment was done identically to the experiment described
above, with the proviso that the stock suspension prepared
contained 1.0.times.10.sup.6, 1.3.times.10.sup.6,
7.7.times.10.sup.4, and 2.8.times.10.sup.5 CFU/ml of the respective
micro-organism, respectively. The results show that the MIC of
composition C for the gram-positive micro-organisms tested is
between about 5- to about 300-fold lower than the MIC of
composition A (data not shown). The MIC of composition C prepared
with 0.1 M phosphate buffer at pH 7 was comparable to the MIC of
composition C prepared with 0.2 M phosphate buffer at pH 6 or pH
6.5.
Example 3
Use of Liquid Nisin Compositions in a Baking Application
[0059] Two cakes were prepared. For each cake 1000 grams of
Moscovisch Powder Damco was mixed with 800 grams of liquid eggs and
100 grams of water. One cake was prepared by adding 120 mg nisin
powder Nisaplin.RTM. (Danisco, Denemark) to the liquid eggs. A
second cake was prepared by adding the 1.2 gram of composition C to
the liquid eggs. The same amounts of nisin were used in both cakes
(150 mg nisin/l egg). The mixture was mixed during 10 minutes in a
Hobart mixer in the third gear, and baked in a 170.degree. C. oven
for 25 minutes. Both cakes were baked the same way.
[0060] The baked cake comprising composition C showed a fine,
woolly crumb structure, while the cake comprising nisin powder
showed a hard irregular crumb structure. This clearly shows that
baked products wherein the compositions of the invention are used
have a better structure than baked products wherein nisin powder is
used. The structure of a bakery product is improved by adding a
nisin composition according to the invention when compared to
adding nisin powder. The example also shows that the compositions
according to the invention can be added to the product before
baking.
Example 4
Use of Liquid Nisin Compositions in a Beverage Application
[0061] In this experiment composition A and C were tested for their
ability to decrease the viable count of different contaminating
micro-organisms in a beverage application. Compositions A and C
were prepared according to Example 1, using nisin powder from
Silver Elephant, China. The beverage used was a malt drink, Pony
from Bavaria, Colombia. For the experiment freshly cultured
Listeria monocytogenes cells (LMD 92.20), Leuconostoc oenos cells
(ML-34) and Leuconostoc mesenteroides cells isolated from a
contaminated product were obtained from an overnight culture grown
at 30.degree. C. in Plate Count Broth (Difco). Stock suspensions of
3.8.times.10.sup.5, 5.7.times.10.sup.6 and 7.2.times.10.sup.5
CFU/ml, respectively, were prepared in physiological saline. 25
.mu.l of the respective stock solutions was added to 25 ml of
beverage spiked with composition A or C. The nisin concentration
tested was 0.5 ppm for Listeria monocytogenes cells, 4 ppm for
Leuconostoc oenos cells and 2 ppm for Leuconostoc mesenteroides
cells. A control comprising no nisin was included for each
micro-organism. The samples were incubated at room temperature and
the total count of micro-organisms (in CFU/ml) was measured at
different time intervals using well known methods.
[0062] The results are shown in Table 2. They clearly demonstrate
that for each of the three different micro-organisms tested
composition C reduces the viable cell count to below the detection
limit of 1 CFU/ml in less than one day, while composition A needs
two or three days to accomplish this. So, composition C is at least
24-48 hours faster than composition A.
Example 5
Use of Liquid Nisin Compositions in a Food Application Model
[0063] In this experiment compositions A and C were tested for
their ability to decrease the viable count of Listeria
monocytogenes in a food application model. After decrease of the
viable cell count to below the detection limit of 10 CFU/ml the
nisin compositions were also tested for their ability to maintain
this low level and prevent outgrowth of surviving cells.
Compositions A and C were prepared according to Example 1, using
nisin from Silver Elephant, China. The application model was
prepared using Plate Count Broth (Difco). The pH of the medium was
set at pH 7.0 with HCl and autoclaved for 15 minutes at 121.degree.
C. For the experiment freshly cultured Listeria monocytogenes cells
(LMD 92.20) were obtained from an overnight culture grown in Plate
Count Broth (Difco) at 30.degree. C. A stock suspension of
3.4.times.10.sup.7 CFU/ml was prepared in physiological saline. 250
.mu.l of the stock solution was added to 25 ml of model medium
spiked with composition A or C. The nisin concentration tested was
2.5 .mu.g/ml for the experiment at 10.degree. C. and 6.25 and 12.5
.mu.g/ml for the experiment at room temperature. A control
comprising no nisin was included for each temperature tested. The
samples were incubated at 10.degree. C. and at room temperature and
the total count of micro-organisms (in CFU/ml) was measured at
different time intervals using well known methods.
[0064] The results clearly demonstrate that composition C inhibits
the outgrowth of Listeria monocytogenes in a food application model
at 10.degree. C. for more than 25 days, while composition A only
inhibits the outgrowth for four days (see Table 3). The results
further show that composition C inhibits the outgrowth of Listeria
monocytogenes in a food application model at room temperature for
more than 25 days, while composition A only inhibits the outgrowth
for two or three days (see Table 4).
Example 6
Preparation of Liquid Nisin Compositions on Pilot Scale Using
Centrifugation
[0065] 100 kg of nisin powder (Silver Elephant, China) containing
2.5% w/w nisin and at least 50% w/w NaCl was dissolved in water.
The pH was set to 7.0 with NaOH. The mixture was subsequently mixed
for about one hour. An anti-foaming agent (Clerol FBA 3107) was
added at 1.5 g/kg mixture. The mixture was subjected continuous
centrifugation at 10.degree. C. at 12,000.times.g with a feed rate
of 200 l/h. The concentrate containing nisin solids was recovered.
Subsequently, the concentrate was dissolved in an aqueous HCl
solution (pH 2.0 to 3.0; mass of complete mix: 1000 kg). The
mixture was stirred for at least one hour. The obtained solution
containing nisin was again subjected to continuous centrifugation
at 10.degree. C. at 12,000.times.g with a feed rate of 200 l/h
followed by depth filtration (with a filter having a pore size of 3
micron) and sterile filtration to remove remaining solid
components. The obtained liquid composition was kept at a pH of 2.0
to 3.0. The turbidity of the final product was 21 FNU, i.e. a clear
liquid composition was obtained. The obtained liquid nisin
composition had a good antimicrobial activity, i.e. a MIC
comparable to the MIC of composition C (see Example 2).
[0066] The above process was also performed without performing the
second continuous centrifugation step. The final product had the
same properties as the final product described above.
[0067] In addition, the process was performed without the second
continuous centrifugation step and without the depth filtration
step. The resulting product also had the same properties as the
final product described above.
Example 7
Preparation of Liquid Nisin Compositions on Pilot Scale Using
Filtration
[0068] 100 kg of nisin powder (Silver Elephant, China) containing
2.5% w/w nisin and at least 50% w/w NaCl was dissolved in a
buffered aqueous solution of 0.2 M sodiumdihydrogenphosphate and
disodiumhydrogenphosphate (pH 7.0; mass of complete mix: 1000 kg).
The mixture was subsequently mixed for about one hour. The mixture
was filtered by dead-end filtration at 10.degree. C. using Dicalite
BF filter aid. The filter cake containing nisin solids was
recovered. Subsequently, the filter cake was dissolved in an
aqueous citric acid solution (pH 2.0 to 3.0; mass of complete mix:
1000 kg). The mixture was stirred for at least one hour. The
obtained solution containing nisin was subjected to dead-end
filtration, depth filtration and sterile filtration at 10.degree.
C. to remove remaining solid components. The obtained liquid
composition was kept at a pH of 2.0 to 3.0. The turbidity of the
final product was 25 FNU, i.e. a clear liquid composition was
obtained. The obtained liquid nisin composition had a good
antimicrobial activity, i.e. a MIC comparable to the MIC of
composition C (see Example 2).
Example 8
Preparation of Liquid Nisin Compositions Using Filtration at Low
pH
[0069] 100 g of nisin powder (Silver Elephant, China) containing
2.5% w/w nisin and at least 50% w/w NaCl was dissolved in water.
The pH was set to 4.0 with NaOH. The mixture was subsequently mixed
for about one hour. The mixture was filtered by dead-end filtration
at 10.degree. C. using Dicalite BF filter aid. The filter cake
containing nisin solids was recovered. Subsequently, the filter
cake was dissolved in an aqueous HCl solution (pH 2.0 to 3.0). The
mixture was stirred for at least one hour. The obtained solution
containing nisin was filtered using dead-end filtration, depth
filtration and sterile filtration at 10.degree. C. to remove
remaining solid components. The obtained liquid composition was
kept at a pH of 2.0 to 3.0. The turbidity of the final product was
123 FNU, i.e. a turbid liquid composition was obtained. The
obtained liquid nisin composition had a good antimicrobial
activity, i.e. a MIC comparable to the MIC of composition C (see
Example 2).
Example 9
Preparation of Stable Nisin Powder Suspension
[0070] Nisin powder (Silver Elephant, China) containing 2.5% w/w
nisin and at least 50% w/w NaCl was suspended in water. Various
thickening agents at various amounts were added. The pH was set at
either pH 2 or pH 5 with HCl and NaOH solutions. The physical
stability of the suspensions was analysed after storage for 9 days
at room temperature by analyses of the height of the sedimentation
front in a 50 ml tube containing 47.5 ml of the suspension. The
results are depicted in the Table 5. The nisin concentration in all
suspensions was 0.25% w/w. Sedimentation is expressed as the
percentage clear liquid that was observed (i.e. the liquid that did
not contain particles). 0% indicates that no sedimentation has
occurred and that the suspension therefore has a good physically
stability. The results show that at pH 2 and pH 5 the suspensions
are physically stable when xanthan gum is used at a concentration
higher than 0.05% (w/w). The results further show that at pH 2 and
pH 5 the suspensions are physically stable when CMC or alginate are
used at a concentration of 1% (w/w) or higher, while for HPMC a
concentration of 3% (w/w) or higher leads to physical stable nisin
suspensions.
TABLE-US-00001 TABLE 1 MIC values of nisin compositions in .mu.g/ml
against M. luteus and P. aeruginosa. Name M. luteus P. aeruginosa
Composition A 0.015-0.0077 >64800 Composition B 0.5-0.23
>61200 Composition C 0.00012-0.00006 1900-1000 Composition D
0.012-0.006 6400-3200 Composition E 0.0004-0.0002 >50000
Composition F 0.05-0.025 >52000 Composition G 0.03-0.015
>32000 Composition H 0.16-0.08 >41000
TABLE-US-00002 TABLE 2 Days until respective micro-organism is
reduced till below the detection limit of 1 CFU/ml in a beverage
application with different nisin compositions. Listeria Leuconostoc
Leuconostoc monocytogenes oenos mesenteroides Control >4 >4
>4 Composition A 3 3 2 Composition C <1 <1 <1
TABLE-US-00003 TABLE 3 Amount of days that outgrowth of Listeria
monocytogenes is inhibited below the detection limit of 10 CFU/ml
in a food application model at 10.degree. C. with different nisin
compositions. Amount of days that outgrowth is inhibited at
10.degree. C. Control <1 Composition A 4 Composition C
>25
TABLE-US-00004 TABLE 4 Amount of days that outgrowth of Listeria
monocytogenes is inhibited below the detection limit of 10 CFU/ml
in a food application model at room temperature with different
nisin compositions. Amount of days that outgrowth is inhibited at
room temperature Control <1 Composition A (250 .mu.g/ml) 2
Composition A (500 .mu.g/ml) 3 Composition C (250 .mu.g/ml) >25
Composition C (500 .mu.g/ml) >25
TABLE-US-00005 TABLE 5 Physical stability of nisin powder
suspension with different thickening agents. Concentration
Percentage Percentage thickening agent sedimentation sedimentation
Thickening agent (% w/w) pH 2 (%) pH 5 (%) No thickening agent 0 79
77 Xanthan gum 0.05 79 79 0.1 40 40 0.2 0 0 0.4 0 0 1 0 0 3 0 0 5 0
0 CMC 0.05 92 93 0.2 89 87 0.4 87 79 1 0 0 3 0 0 5 0 0 HPMC 0.05 81
94 0.2 83 87 0.4 83 87 1 73 66 3 40 11 5 5 0 Alginate 0.05 72 94
0.2 77 89 0.4 63 58 1 42 0 3 0 0 5 0 0
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Nisin A depletes intracellular ATP and acts in bactericidal manner
against Mycobacterium smegmatis. Letters in Appl. Microbial.
28:189-193.
[0072] Mota-Meira M, LaPointe G, Lacroix C and Lavoie MC (2000),
MICs of Mutacin B-NY266, Nisin A, Vancomycin, and Oxacillin against
bacterial pathogens. Antimicrobial Agents and Chemotherapy
44:24-29.
[0073] Pongtharangkul T and Demirci A (2004). Evaluation of agar
diffusion bioassay for nisin quantification, Appl. Microbial.
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