CA2058455A1 - Synergistic effect of lantibiotics in combination with selected agents against gram positive bacteria - Google Patents
Synergistic effect of lantibiotics in combination with selected agents against gram positive bacteriaInfo
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- CA2058455A1 CA2058455A1 CA002058455A CA2058455A CA2058455A1 CA 2058455 A1 CA2058455 A1 CA 2058455A1 CA 002058455 A CA002058455 A CA 002058455A CA 2058455 A CA2058455 A CA 2058455A CA 2058455 A1 CA2058455 A1 CA 2058455A1
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- Prior art keywords
- nisin
- amount
- acid
- environment
- weight percent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
Abstract
ABSTRACT OF THE DISCLOSURE
Disclosed is a method for inhibiting the growth of undesirable gram positive bacteria which involves the use of a lanthionine bacteriocin together with a synergist therefore as an antimicrobial treatment.
MS #1643
Disclosed is a method for inhibiting the growth of undesirable gram positive bacteria which involves the use of a lanthionine bacteriocin together with a synergist therefore as an antimicrobial treatment.
MS #1643
Description
20~8~5 SYNERGISTIC EFFECT OF LANTIBIOTICS
IN COMBINATION WITH SELECTED AGENTS
AGAINST GRAM POSITI~E BACTERIA
Back~round of the Invention s Nisin, a member of the group of antimicrobials known as lanthionine bacteriocins, is an antimicrobial polypeptide produced by certain strains of Lactococcus lactis (formerly Stre~tococcus lactis). It is manu-factured through the pure-culture fermentation of these bacteria with subsequent purification and drying. M. Doyle in Food Technol. 1988 42: (4) 169-171, describes the inhlbitory effects of nisin on the growth and survival of L. monocvto~enes.
The term "lantibiotics~' was coined by Schnell et al. (1988 Nature 333: 276-278) to describe a group of bacteriocins including nisin which contain the amino acid lanthionine and other "non-protein" amino acids.
This class of bacteriocins includes subtilin, pep 5, epidermin, gallidermin, cinnamycin, RoO9-0198, dura-mycin and ancovenin. These ribosomally synthesized peptide bacteriocins contain from l9 to 34 amino acids and are produced by various microbes including Sta~hlococcus species, Bacillus species and Stre~to-mvces species.
The combination of nisin with other materials to enhance its antimicrobial activity has been reported.
MS ~1643 ' -2- 20~8~
Thus, International patent applicatior, iO 89/12399 discloses the combination of nisin wit~, chelating agents and/or surfactants for use against Gram posi-tive and gram negative bacteria.
Campden Yoods Drink Research Assoc ation, Tech-nical Memorandum No. 520 (March 1989) rspor~s increased efficacy of nisin in combination with cltric acid and acetic and antagonism of nisin in the presence of citric acid and lactic acid against Bacillus spore outgrowth.
- Mikolajcik et al. in J. Dairy Sci. ~8:1522-1524 disclose the efficacy of nisin as a seoricidal agent against Bacillus spores in the presence of L-alanine, an amino acid which is recognized as a germination inducer of Bacillus spores. This pro~erty was used to render the spores more susceptible to nisin.
Shehata reports the use of nisin, sorbate and sodium benzoate as antimicrobials in Ain Shams Univ.
Fac. Agric. Res. ~ull. 0:1554.
Published European patent application 0,384,319 discloses the synergistic effect of nisin with chelatinq agents against L. monocvtoqenes. This reference discloses the use of certain amino ac s and mono-carboxylic acids in combination with r.lsin.
Summary of the Invention The present invention involves a method for inhibiting the growth of gram positive bacteria in an environment where their growth is undesirable which method comprises introducing to said environment a synergistically effective combination of a lanthionine MS #1643 _3- 2~8~
bacteriocin and a synergist therefore, said synergist being selected from the group consisting of amino acids, aliphatic mono- and di- carboxyiic organic acids containing from 1 to 8 carbon atoms or a suitable salt thereof, phenolic antioxidant antimicrobials, benzoic acid or a suitable salt thereof and carbohy-drate or modified carbohydrate food gums.
Description of the Invention The present invention relates to the observation that a lanthionine bacteriocin in combination with certain additives has provided a composition which inhibits the growth of undesirable gram positive bacteria to a greater degree than either ~aterial by itself and that the combined inhibitory effect of these substances is greater than the additive effect observed when using the lantlobiotic or other compo-sition individually.
Various classes of compositions which act syner-gistically with the lantibiotics have been identified.
Among these are amino acids and in particular an amino acid selected from the group of glycine, aspartic acid, leucine and lysine. The synergistic effect of these amino acids with lantibio~ics does not rely on the induction of spore outgrowth, as the prior art implies, since this synergy has been demonstrated with a microorganism, L. monocYtoqenes, which does not form spores.
Another class of agents which has been found to produce a synergistic antimicrobial effect in combi-nation with a lantibiotic are aliphatic mono- and di-MS #1643 _4_ 2~ 5~
carboxylic acids containing from 3 to 8 carbon atoms.These acids include lactic, propionic, ascorbic, isoascorbic, acetic, malic, tartaric, succinic, maleic, oxalic and fumaric acid. Suitable salts, such as the alkali and alkaline earth metal salts, e.g.
sodium, potassium and calcium can be employed.
Phenolic antioxidant antimicrobials that demon-strate synergistic antimicrobial activity in combi-nation with lantibiotics include tertiary butyl hydroxyanisole (BHA), 1 to 7 carbon aliphatic esters of parahydroxy benzoic acid, particularly the methyl ester known as methylparabens, tertiary butyl hydro-quinone (TBHQ) and butylated hydroxytoluene (BHT). In addition, benzoic acid and suitable salts thereof, particularly the sodium salt, exhibit the desired synergistic effect.
Another class of synergists include the carbo-hydrate or modified carbohydrate food gums. This class of composition is characterized in that they are 2~ water soluble carbohydrate polymers and derivatives of carbohydrate polymers which often contain positively or negatively charged side groups. Included within this class of materials are for example, alginates, guar gum, gum arabic, xanthan gum, chitosan and derivated carbohydrate polymers such as propylene glycol alginate, methyl cellulose and carboxymethyl-cellulose.
The mechanism of action of lantibiotics such as nisin has not been resolved, but many investigators have linked the lethality of these peptides to irre-versible changes in the cell membranes of susceptible organisms. The resistance of some organisms to nisin MS #1643 -5- 2~84~
is thought to be due to the lack of access of the peptide to its site of action, either because of physical exclusion or the lack of appropriate receptor molecules. ~he lantibiotic synergists of the present invention appear to alleviate this inhibition of the access to the target microorganism although the mechanism of action is not understood. The present invention is predicated on the discovery that a lantibiotic and certain synergists therefore, in combination, inhibit the growth of undesirable gram positive bacteria to a greater extent than either material by itself and that the combined inhibitory effect of these substances is greater than the additive effect observed when using the lantibiotic or synergist individually.
When using nisin, or other lantibiotic, as an anti-microbial agent a loading level of from 200 to 300 International Units (IU) per gram of the substrate being treated is usually recommended to achieve maximal effect. The discovery that lantibiotics (such as nisin) and the supplemental component act in a synergistic way to inhibit the growth of gram positive bacteria facilitates the use of lantibiotic bacteriocins with greater efficacy and economy than if they were used by themselves. In a typical example, to reduce in number or eliminate the undesirable gram positive bacteria using a lantibiotic such as nisin and the synergist, the total amount of nisin will preferably range from about 0.1 IU/gm (or IU/ml in liquid systems) of the substrate material being treated up to a~ much as 2,000 IU/gm or IU/ml in a liquid system. In most applications, a nisin concentration of from 50 to 500 MS #1643 -6- 2~5845~
IU/gm will be sufficient due to the boost it will receive from the synergist. As used ~.erein, the term IU is intended to refer to internationai unit as determined by the method of Tramer and Fowler in J.
Sci. Food & Agric., 15 (8) 522 (1964). The amount of synergist to be employed will depend cn the particular composition involved. In general the amount of synergist in terms of weight percent of the substrate being treated will be within the ranges set out in Table A.
TABLE A
Class of Range of Preferred Synerqist Suqqested Use Ranqe Amino Acids 0.001% - 5% 0.03% - 0.1%
15 Aliphatic mono- 0.001% - 25% 0.01% - 1%
& di- carboxylic organic acids Phenolic 0.0001% - 2% 0.005% - 0.02%
Antioxidants 20 Benzoic acid, 0.0001% - 2% 0.03% - 0.1%
salts and esters Carbohydrate 0.001% - 50% 0.02% - 2.5 food gums MS #1643 .
:
2 0 ~
Lanthionine bacteriocins other than nisin and the synergists therefore can be expected t~ be efficacious at these concentrations although some reutine experi-mentation may be necessary to determine the concen-tration of a particular lantibiotic for optimalsynergistic effect.
5urface treatment of a target substrate can be accomplished by suspending the material to be treated in a lanthionine/synergist solution. Alternatively, the material may be dipped in the solution or it can be sprayed onto the surface of the material. The lanthionine/synergist combination can re incorporated into films or gels which are applied to the substrates' surface or the combination can be incorporated directly ~s into the environment being treated such as by adding it to milk to be used in making cheese. In film coating applications such as dips, films, gels or casings, the initial concentrations of the lantibiotic and synergist can approach their solubility limits to provide a finished product which, after application and diffusion, contains residual levels of lanti-biotic/synergist which are within the desired ranges.
When treating the material's surface, some routine experimentation may be needed to ascer~ain the most effective concentration.
Environments capable of being treated with the compositions of the present invention include, but ar~
not limited to, substrates such as meats and meat products, such as chicken, turkey, ham, beef, salami, sausages and smoked meat prducts; mayonnaise; dairy products such as cheese, milk and yogurt; oils, fish and fish products; soft drinks including natural MS #1643 -8- 2058~5~
juices; animal feeds and other high protein products.
In addition to use in foods, the compositions of the present invention have non-food uses such as inclusion in mouthwashes, denture cleaners, ointments, creams and shampoos.
Among the problematical gram positive bacteria, L. monoc~toqenes is a particular pest because of its ability to grow at refrigerator temperatures and its pathologic nature which can result in serious conse-quences when ingested. Thus, L. monocYto~enes hasbeen implicated in several fatalities in recent years, especially following outbreaks of listerosis associ-ated with milk, cole slaw and soft ripened cheese.
Other undesirable gram positive bacteria whose growth can be inhibited by the synergistic combinations of the present invention include for example species of Clo_tridia, e.g. C. botulinum and C. perfrinqens;
Bacilli, e.g. B. cereus and B. subtilis; Lactococcus lactis, subsp. cremoris and organisms of the genera Stre~tococcus, Sta~hvlococcus, Pediococcus, Listeria and Micrococcus.
The present invention is further illustrated by the following examples in which International Units of nisin are expressed as U nisin. Nisin for these studies was prepared from Nlsaplin~ (Aplin h Barrett) which contains about 1 x 10~ U/gm nisin, 7S% by weight NaCl and 22.5% by weight milk solids by diafiltration and ultrafiltration resulting in a preparation which is essentially salt free.
MS #1643 205845~
_9_ EXAMPLE I
The bacteriocidal activity of nisin and glycine against L. monocvtoqenes Scott A was evaluated at pH
IN COMBINATION WITH SELECTED AGENTS
AGAINST GRAM POSITI~E BACTERIA
Back~round of the Invention s Nisin, a member of the group of antimicrobials known as lanthionine bacteriocins, is an antimicrobial polypeptide produced by certain strains of Lactococcus lactis (formerly Stre~tococcus lactis). It is manu-factured through the pure-culture fermentation of these bacteria with subsequent purification and drying. M. Doyle in Food Technol. 1988 42: (4) 169-171, describes the inhlbitory effects of nisin on the growth and survival of L. monocvto~enes.
The term "lantibiotics~' was coined by Schnell et al. (1988 Nature 333: 276-278) to describe a group of bacteriocins including nisin which contain the amino acid lanthionine and other "non-protein" amino acids.
This class of bacteriocins includes subtilin, pep 5, epidermin, gallidermin, cinnamycin, RoO9-0198, dura-mycin and ancovenin. These ribosomally synthesized peptide bacteriocins contain from l9 to 34 amino acids and are produced by various microbes including Sta~hlococcus species, Bacillus species and Stre~to-mvces species.
The combination of nisin with other materials to enhance its antimicrobial activity has been reported.
MS ~1643 ' -2- 20~8~
Thus, International patent applicatior, iO 89/12399 discloses the combination of nisin wit~, chelating agents and/or surfactants for use against Gram posi-tive and gram negative bacteria.
Campden Yoods Drink Research Assoc ation, Tech-nical Memorandum No. 520 (March 1989) rspor~s increased efficacy of nisin in combination with cltric acid and acetic and antagonism of nisin in the presence of citric acid and lactic acid against Bacillus spore outgrowth.
- Mikolajcik et al. in J. Dairy Sci. ~8:1522-1524 disclose the efficacy of nisin as a seoricidal agent against Bacillus spores in the presence of L-alanine, an amino acid which is recognized as a germination inducer of Bacillus spores. This pro~erty was used to render the spores more susceptible to nisin.
Shehata reports the use of nisin, sorbate and sodium benzoate as antimicrobials in Ain Shams Univ.
Fac. Agric. Res. ~ull. 0:1554.
Published European patent application 0,384,319 discloses the synergistic effect of nisin with chelatinq agents against L. monocvtoqenes. This reference discloses the use of certain amino ac s and mono-carboxylic acids in combination with r.lsin.
Summary of the Invention The present invention involves a method for inhibiting the growth of gram positive bacteria in an environment where their growth is undesirable which method comprises introducing to said environment a synergistically effective combination of a lanthionine MS #1643 _3- 2~8~
bacteriocin and a synergist therefore, said synergist being selected from the group consisting of amino acids, aliphatic mono- and di- carboxyiic organic acids containing from 1 to 8 carbon atoms or a suitable salt thereof, phenolic antioxidant antimicrobials, benzoic acid or a suitable salt thereof and carbohy-drate or modified carbohydrate food gums.
Description of the Invention The present invention relates to the observation that a lanthionine bacteriocin in combination with certain additives has provided a composition which inhibits the growth of undesirable gram positive bacteria to a greater degree than either ~aterial by itself and that the combined inhibitory effect of these substances is greater than the additive effect observed when using the lantlobiotic or other compo-sition individually.
Various classes of compositions which act syner-gistically with the lantibiotics have been identified.
Among these are amino acids and in particular an amino acid selected from the group of glycine, aspartic acid, leucine and lysine. The synergistic effect of these amino acids with lantibio~ics does not rely on the induction of spore outgrowth, as the prior art implies, since this synergy has been demonstrated with a microorganism, L. monocYtoqenes, which does not form spores.
Another class of agents which has been found to produce a synergistic antimicrobial effect in combi-nation with a lantibiotic are aliphatic mono- and di-MS #1643 _4_ 2~ 5~
carboxylic acids containing from 3 to 8 carbon atoms.These acids include lactic, propionic, ascorbic, isoascorbic, acetic, malic, tartaric, succinic, maleic, oxalic and fumaric acid. Suitable salts, such as the alkali and alkaline earth metal salts, e.g.
sodium, potassium and calcium can be employed.
Phenolic antioxidant antimicrobials that demon-strate synergistic antimicrobial activity in combi-nation with lantibiotics include tertiary butyl hydroxyanisole (BHA), 1 to 7 carbon aliphatic esters of parahydroxy benzoic acid, particularly the methyl ester known as methylparabens, tertiary butyl hydro-quinone (TBHQ) and butylated hydroxytoluene (BHT). In addition, benzoic acid and suitable salts thereof, particularly the sodium salt, exhibit the desired synergistic effect.
Another class of synergists include the carbo-hydrate or modified carbohydrate food gums. This class of composition is characterized in that they are 2~ water soluble carbohydrate polymers and derivatives of carbohydrate polymers which often contain positively or negatively charged side groups. Included within this class of materials are for example, alginates, guar gum, gum arabic, xanthan gum, chitosan and derivated carbohydrate polymers such as propylene glycol alginate, methyl cellulose and carboxymethyl-cellulose.
The mechanism of action of lantibiotics such as nisin has not been resolved, but many investigators have linked the lethality of these peptides to irre-versible changes in the cell membranes of susceptible organisms. The resistance of some organisms to nisin MS #1643 -5- 2~84~
is thought to be due to the lack of access of the peptide to its site of action, either because of physical exclusion or the lack of appropriate receptor molecules. ~he lantibiotic synergists of the present invention appear to alleviate this inhibition of the access to the target microorganism although the mechanism of action is not understood. The present invention is predicated on the discovery that a lantibiotic and certain synergists therefore, in combination, inhibit the growth of undesirable gram positive bacteria to a greater extent than either material by itself and that the combined inhibitory effect of these substances is greater than the additive effect observed when using the lantibiotic or synergist individually.
When using nisin, or other lantibiotic, as an anti-microbial agent a loading level of from 200 to 300 International Units (IU) per gram of the substrate being treated is usually recommended to achieve maximal effect. The discovery that lantibiotics (such as nisin) and the supplemental component act in a synergistic way to inhibit the growth of gram positive bacteria facilitates the use of lantibiotic bacteriocins with greater efficacy and economy than if they were used by themselves. In a typical example, to reduce in number or eliminate the undesirable gram positive bacteria using a lantibiotic such as nisin and the synergist, the total amount of nisin will preferably range from about 0.1 IU/gm (or IU/ml in liquid systems) of the substrate material being treated up to a~ much as 2,000 IU/gm or IU/ml in a liquid system. In most applications, a nisin concentration of from 50 to 500 MS #1643 -6- 2~5845~
IU/gm will be sufficient due to the boost it will receive from the synergist. As used ~.erein, the term IU is intended to refer to internationai unit as determined by the method of Tramer and Fowler in J.
Sci. Food & Agric., 15 (8) 522 (1964). The amount of synergist to be employed will depend cn the particular composition involved. In general the amount of synergist in terms of weight percent of the substrate being treated will be within the ranges set out in Table A.
TABLE A
Class of Range of Preferred Synerqist Suqqested Use Ranqe Amino Acids 0.001% - 5% 0.03% - 0.1%
15 Aliphatic mono- 0.001% - 25% 0.01% - 1%
& di- carboxylic organic acids Phenolic 0.0001% - 2% 0.005% - 0.02%
Antioxidants 20 Benzoic acid, 0.0001% - 2% 0.03% - 0.1%
salts and esters Carbohydrate 0.001% - 50% 0.02% - 2.5 food gums MS #1643 .
:
2 0 ~
Lanthionine bacteriocins other than nisin and the synergists therefore can be expected t~ be efficacious at these concentrations although some reutine experi-mentation may be necessary to determine the concen-tration of a particular lantibiotic for optimalsynergistic effect.
5urface treatment of a target substrate can be accomplished by suspending the material to be treated in a lanthionine/synergist solution. Alternatively, the material may be dipped in the solution or it can be sprayed onto the surface of the material. The lanthionine/synergist combination can re incorporated into films or gels which are applied to the substrates' surface or the combination can be incorporated directly ~s into the environment being treated such as by adding it to milk to be used in making cheese. In film coating applications such as dips, films, gels or casings, the initial concentrations of the lantibiotic and synergist can approach their solubility limits to provide a finished product which, after application and diffusion, contains residual levels of lanti-biotic/synergist which are within the desired ranges.
When treating the material's surface, some routine experimentation may be needed to ascer~ain the most effective concentration.
Environments capable of being treated with the compositions of the present invention include, but ar~
not limited to, substrates such as meats and meat products, such as chicken, turkey, ham, beef, salami, sausages and smoked meat prducts; mayonnaise; dairy products such as cheese, milk and yogurt; oils, fish and fish products; soft drinks including natural MS #1643 -8- 2058~5~
juices; animal feeds and other high protein products.
In addition to use in foods, the compositions of the present invention have non-food uses such as inclusion in mouthwashes, denture cleaners, ointments, creams and shampoos.
Among the problematical gram positive bacteria, L. monoc~toqenes is a particular pest because of its ability to grow at refrigerator temperatures and its pathologic nature which can result in serious conse-quences when ingested. Thus, L. monocYto~enes hasbeen implicated in several fatalities in recent years, especially following outbreaks of listerosis associ-ated with milk, cole slaw and soft ripened cheese.
Other undesirable gram positive bacteria whose growth can be inhibited by the synergistic combinations of the present invention include for example species of Clo_tridia, e.g. C. botulinum and C. perfrinqens;
Bacilli, e.g. B. cereus and B. subtilis; Lactococcus lactis, subsp. cremoris and organisms of the genera Stre~tococcus, Sta~hvlococcus, Pediococcus, Listeria and Micrococcus.
The present invention is further illustrated by the following examples in which International Units of nisin are expressed as U nisin. Nisin for these studies was prepared from Nlsaplin~ (Aplin h Barrett) which contains about 1 x 10~ U/gm nisin, 7S% by weight NaCl and 22.5% by weight milk solids by diafiltration and ultrafiltration resulting in a preparation which is essentially salt free.
MS #1643 205845~
_9_ EXAMPLE I
The bacteriocidal activity of nisin and glycine against L. monocvtoqenes Scott A was evaluated at pH
3.5, 4.5 and 5.5 in 10 mM citrate buffer (CAB). Nisin was tested at 0.2 or 2 IU/ml while glycine concentra-tions were 6.4, 16, 40, 100 and 250 mM. Controls were run in CAB. All stock solutions, culture dilutions and treatments were prepared in CAB at the p~ evaluated.
Treatments were prepared in 0.9 ml quantities in l ml microtubes with treatment ~eing initiated by the addition of 0.1 ml of L. monocvtoqenes Scott A at approximately lx 10' CFU/ml. After treatment for 60 minutes at 37C the test samples were serially diluted in phosphate buffered saline (O.OlM, pH 7.0) using microtiter plates. Enumeration of surviving bacteria was carried out with tryptic soy agar spread plates which were incubated for 48 hours at 37C after inoculation. Synergy was noted when the bacteriocidal effects of the combination of a test compound and nisin was greater than that of the additive loglO
reductions of the individual compounds. The following calculation was used where:
T = test compound N = nisin T+N = combination loglO reduction T+N~(loglO reduction T+loglO
reduction N) = synergy Results are set out in tables 1 (aspartic acid) and 2 where synergistic combinations are identifled with a MS #1643 -lO- 20584~5 "+" to designate the observation that ~he bacteriocidal effects of the combination were greater than those of the additive log reductions alone.
For purposes of the above calcuiation, any negative log reductions were assumed te be 0. Any estimations where greater than (>) or less than (<) are reported, exceeded the detection limits of the system and were not used for calculation of synergy, except in obvious cases, e.g. where the minimum effect which could have occurred was synergistic.
MS #1643 -11- 2058~
Activity of Nisin in Combination with Aspartic Acid (ASP) Against L. mono~vtoqenes ~ Log" C~ Log" Syr~ o ~r ~ol nis~n~ r~oc2~0n ~11 a~lP
p~ 3.5 0/0 6.26 - -0/0.0096 6.11 0.15 0 0~0.024 6.20 0.06 0/0.06 6.30 -0.04 0/0.15 6. zo 0.06 0/0.375 6.20 0.06 0.2~0 6.15 0.1:
0.2/0.0096 6.04 0.2 0.2/0.024 6.15 0.1:
0.2/0.06 6.1a 0.08 0.2/0. lS 5.97 0.29 +/70.6 0.2/0.375 5.26 1.00 f/488 p~ ~.5 0/0 6.04 0/0.0096 6.15 -0.1 -0/0.024 6.23 -O . lg 0/0.06 6.34 -0.30 0/0.15 6.38 -0.34 0/0.37S 6.04 o . Oo 0.2/0 5.97 0.07 0.2~0.0096 6.04 0.00 0.2~0.024 6.00 0.04 0.2~0.06 6.11 -0.07 0.2/O.lS 6.08 -0.0 0.2~0.375 6.04 o .00 p~ 5.S
o/o 6.26 ~
0/0.0096 6.38 -0.12 0~0.024 6.32 -0.06 0/0.06 6.32 -0.06 0~0. lS 6. ~0 -0.14 0~0.3~5 6.41 -0. lS
0.2~0 6.26 o.oO
0.2~0.0096 6.18 0.08 0.2/0.024 6.20 0.06 0.2/0.06 6.28 -0.02 0.2/O.lS 6.34 -0.08 0.2/0.37S 6.26 0 - 00 MS #1643 -12- 2~84~
Activity of Nisin in Combination With Glycine Against L. monocvtoqenes Tr~t~ nt Lcg,. CF~l Loq,. Syn~rgy/% ov~r ~ 1 ni~n/ rsduction .Y
pH 3.5 / 4.30 __ 0/6.4 4~34 ~0~04 ~/16 4.62 -0.32 0/40 4.99 -0.69 0/100 4.75 ~0-45 0/250 4.74 -0.44 0.2/o 2.48 1.82 0.2/6.4 2.62 l.6a 0.2/16 2.96 1.34 0.2/40 4.80 -0.50 0.2~100 4.83 -0.53 0.2/250 4.79 _0.49 P~ ~5 0/0 5.34 __ 0/6.4 5.18 0.16 0/~6 S.23 0.11 0/~0 5.1a 0.16 0/100 S.ll 0.23 0/250 3.62 1.72 0.2/0 4.38 0.96 0~2/6.4 4.28 1.06 0.2/16 4.11 1.23 +/15.0 0.2/40 4.00 1.34 +/19.6 0.2/100 3.89 1.45 +/21.q 0.2~250 2.88 2.46 p~ 5.5 0/0 5.72 __ 0/6.4 5.48 0.24 0/16 5.46 0.26 o/~o 5.46 0.26 0/100 5.49 0.23 0/250 5.41 0.31 4~ 0.2/o 5.38 0.34 0.2~6.4 S.28 0.44 0.2~16 5.41 0.31 0.2/~0 5.00 0.72 +/20 0.2/100 5.~5 0.57 0.2/250 4.92 0.80 +/23 MS #1643 2058~
Tables 1 and 2 represent examples of the type of data used for synergy determination. In each case, under several conditions, the decrease in bacterial population (logLO reduction) when nisin and the synergist were combined significantly exceeded the sum of the effects of nisin alone and the synergist alone.
With synergists such as glycine, which are ionic in nature, very high concentrations sometimes appear inhibitory. This is due to ionic strength inhibition ~0 of nisin and occurs with all ionic compounds at high concentration.
EXAMPLE II
Nisin~Orqanic Acids The nisin-organic acid synergy was initially demonstrated in a diffusion plate assay. The assay was prepared by the method described by Fowler et al in Technical Series of the Society for Applied Bacte-riology No. 8 (1975) which uses Micrococcus luteus as an indicator organism. Nisin or nisin plus the synergist were used in the wells of the plate and the resulting zone diameter was measured. Antimicrobial activity is related to the diameter of the zone of growth inhibition around wells both in cases where the nisin/organic acid mixture was used and where the control (containing nisin only) was added to the well.
The zone of growth inhibition diameters were measured for both the control and the wells containing nisin/-organic acid of which 2 replicates were tested. From MS #1643 20~84~5 these measurements, the effective nisln units/ml were calculated as follows:
The zone diameter with nisin plus the organic acid synergist was calculated and compared to a nisin standard curve. The amount of nisin alone which would have produced a zone of equivalent size was determined, and this value was reported as "Effec~ive Nisin Units/ml". Since none of the test synergists produced a zone of measurable size alone, any increase in zone size represents a synergistic effect. ~he degree of synergy is related to the "Effective ~isin Units".
The results of this experiment are summarized in Table 3A.
Zone Effective SamPle Dlameter Nisin Units/ml 10 U~ml nisin Control/Sample 10 U/ml 10 U/ml nisin + 1% Citric Acid 1.63/2.05 18.1 U/ml 20 + 1~ Lactic Acid 1.63/1.88 14.2 U/ml + 1% Ascorbic Acid 1.63/1/89 14.4 U/ml + 1% Isoascorbic Acid 1.63/1.79 12.5 U/ml The nisin-organic acid synergy was also determined using Listeria monoCYtOqeneS as the target organism.
The assay medium was Brain ~eart Infusion medium containing 1% ~ifco Agar. The medium was adjusted to pH 6.0 and melted whereupon 1% Tween 20 was added.
This is part of the diffusion plate medium and is MS #1643 -15- 2~58~5~
present in each of the reported experiments. L.
monocytoqenes was added at about 2X10S cells/ml (4 ml of 107 cells/ml to each 200 ml agar). ~his inoculated medium was poured into a 243 x 243 x 18 mm culture plate and allowed to cool, whereupon 7 mm circular holes were cut into the agar and removed to provide circular wells. The solutions were adjusted to pH 6.0 with NaOH or HCl prior to use and added to the wells where they were allowed to incubate overnight. A
variety of organic acids (in the form of their sodium salts) were tested in conjunction with nisin at 10,000 U/ml. The results of this experimen~ are set out in Table 3B. Referring to Table 3B, 2% lactic acid provided a 50% increase in efficacy of a 10,000 IU/ml nisin sample by producing a zone equivalent to what 15,000 units/ml nisin would produce while 2% propionic acid caused a 21-fold increase in effective activity of 10,000 units/ml nisin (resulting in a zone size comparable to 212,000 IU/ml nisin alone).
MS #1643 -16- 20~
Effective SamPle Zone DiameterNisin Units/ml 10,000 U/ml Nisin Control/Sample 10,000 10,000 U/ml Nisin +2~ Citric Acid 2.01/2.20 26,300 +2% Lactic Acid 2.01/2.11 15,000 +2% Propionic Acid 2.17/2.78 212,000 +2~ Acetic Acid 2.17/2.40 27,300 +2% Malic Acid 2.17/2.35 20,800 +2% Tartaric Acid 2.17/2.38 24,500 +2% Succinic Acid 2.17t2.36 22,000 +2% Maleic Acid 2.09/2.20 16,400 . +2% Oxalic Acid 2.09/2.21 17,400 +2% Fumaric Acld 2.09/2.30 29,500 From Table 3 it can be determined that all of the organic acids were, to some degree, synergistic with nisin. Lactic acid, maleic acid and oxalic acid provided less than a doubling of apparent activity of 10,000 units/ml nisin, while all the remaining acids except propionic produced an apparent activity increase of between 2 and 3 fold. Propionic acid is unusually effective since it wa~ observed to produce a greater than 21-fold increase in activity. This trend would be expected to continue with the use of longer chain mono- carboxylic acids. Accordinly, mono- carboxylic acids containing from 3 to about 8 carbon atoms, e.g.
MS #1643 2~58~
propionic, butyric, pentanoic, hexanoic, heptanoic and octanoic acids are preferred organic acids for use as synergists of lantibiotics such as nisin.
EXAMPLE III
Selected food gums were combined with nisin to determine the synergistic activity of the resulting combinations against L. monocYtoqenes. Food gums which exhibited synergy were commercial preparations of guar gum, algin and propylene glycol alginate.
10 Nisin was evaluated at 1,000 IU/ml and 100 IU~ml both by itself and in combination with the food gums.
The food gums were added to 25 mM citrate buffer, pH 6.0, at 0.125% (w/v) and autoclaved prior to use.
Nisin stock solutions were prepared in the citrate buffer and filter sterilized. Treatment solutions were prepared in the citrate buffer to contain 0.1% of the food gum in combination with l,000 or 100 IUtml nisin. Controls were prepared to contain only nisin, only the food gum or only buffer. L. monocvtoaenes was added to each test sample at approximately 5 x 10 CFU/ml. The treatment times were 30 minutes with 1,000 IU/ml nisin and 15 minutes with lO0 IU/ml nisin.
Surviving populations were enumerated on ~rain Heart Infusion agar spread plates in triplicate and incubated for 24 hours at 35C. Synergy wa~ noted when the log reductions of the nisin/food gum was greater than the sum of the log reductions of the food gum and nisin controls.
The data generated during these experiments are presented in Table 4.
MS #1643 -18- 205845~ `
Trial 1 (1,000 U/ml nisin, 30 minutes) Log1OLog~O Reduction SamPle CFU/mlfrom control 5 Control 6.53 --Nisin control 6.41 0.12 Propylene glycol alginate (PGA) 6.58 -O.OS
PGA + nisin 4.97 1.56 10 Guar gum 6.48 0.05 Guar gum + nisin 4.75 1.78 Algin 6.11 0.42 Algin + nisin 5.11 1.42 Trial 2 (1,000 U/ml nlsin, 15 minutes) LoglOLoglO Reduction SamPle CFU/mlfrom control Control 6.28 ---Nisin control 6.10 0.18 Propylene glycol alginate (PGA) 6.36 -0.08 PGA + nisin 5.53 0.75 Guar gum 6.30 -0.02 Guar gum + nisin 5.30 0.98 Algin 6.26 0.02 Algin + nisin 5.43 0.85 MS #1643 205845~
From the data of Table 4 it can be determined that in most cases the food gums tested showed little or no antimicrobial effect by themselves. However, when the food gums, all of which are characterized as S being water soluble carbohydrate polymers or deriva-tlves thereof were combined with nisin, the effect of the combination of nisin and the food ~um was signif-icantly greater than the sum of the effect of the nisin and food gum alone.
EXAMPTE IV
The synergism of sodium benzoate with nisin was determined using an experimental procedure essentially as described in Example I.
The results of this experiment are summarized in Table 5. Synergism with nisin was observed at pH 3.5 and 4.5 while the effect was less than additive at pH
5.5.
MS #1643 -20- 20~8~
hctivity Or ni~in in cc nin tion vit~ ~od us c~n~o t-( Na~H~ZCAT~t agninst L ~SrC~Dn=Y~CI in 10 ~
citr~t- ku~S~r at l-Ct8d pH'-sr uO nt Loq CYC~l Log~. Synorgy/% over 13~1 nlsin/ roduc210n % II~I~D~ZOAS~
p8 3.S
o~o s.sa -_ 0 0/0.00256 S .91 0.07 o/0.0064 5.96 - Z9 0/0.016 S .91 0 - 07 0/0.04 1.70 4.28 0/0.1 ~2.00 ~3.98 0.2~0 5.~6 0.5Z
0.2/0.00256 5.38 0.60 +~1.69 0.2/0.0064 s . ~2 0.66 0.2/0.016 5.~6 0.52 0.2 /0. 04 5 . 1 0 0 . 88 0.2/o . l 2.06 3.92 p~ ~..5 0/0 5.92 - _ 0/0.00256 s . gs -0.03 o/0.0064 6.02 -0.10 0/0.0~6 5.90 0.02 0~0.04 5.38 0.54 0~0.1 5.20 0.?2 0.2~0 ~.84 0.08 0.2~0.002~6 ~ .89 0.03 0.2~0.0064 ~ .71 0.21 0.2~0.016 5.80 0. ~2 +/20 0.2~0.04 5.18 0.74 +/19.4 0.2~0.1 0.70 5.22 +/552 p~ 5.S
O/O 6.94 --0/0.00256 6.63 0.31 o/0.0064 6.33 0.61 0/0.016 6.34 0.60 0~0.0~ 6.24 o.70 0/0.1 6.24 0.70 0.2/0 6.1~ 0.77 0.2~0.00256 6.15 0.79 0.2~0.006~ 6.06 0. ~t8 0.2~0.016 6.07 0. ~t7 0.2~0.0~ 6.07 o.~7 0.2~0.1 6.32 0.~2 MS #1643 -21- 2~
From Table 5, it can be determined that under appropriate conditions there is observed synergistic activity between nisin and benzoate under various pH
conditions. Failure to observe a synergistic response ; at pH 5.5 was probably due to the low concentration of undissociated benzoic acid since the undissociated form is the active antimicrobial form.
In a similar manner, the methyl ester of para-hydroxy benzoic acid (methyl parabens) was tested for its antimicrobial effect against L. monocYtoqenes in combination with nisin. This experiment was carried out in accordance with the prodecure outlined for Example I.
The results of this experiment are set out in Table 6.
MS #1643 2 0 ~
ActlYlty o~ ni~in in co DmntiOn Vit~ n t~ylpnr~conn ~)I~PAR~B~q) ~ ln t L~ 9~LCY=~9~=9~ i~ 10 U~
c~t~t - buS~r ~t ~ ce ~ p~ ~
Sren~ Dllt Log CF~Dl Lo9Syn r9y/% ov~r 1l/~ ni~n/ r~duct~on % I~-PAEIA~I
p~ 3.5 0/0 6.06 --0 0/0.00256 5.69 0.37 0/0.0064 6.11 -0.06 0/0.016 5.42 0.64 0~0.04 5.35 0. ~1 0/0.1 3.94 2.12 0.2~0 5.12 0.93 0.2/0.002S6 5.19 0.86 0.2~0.0064 5.22 0.8~
0.2/0.016 5.01 1.05 0.2~0.04 4.94 1.12 0.2/0.1 3.04 3.01 p~ ~.5 0/0 6.06 --0/0.00256 6. - 6 -0.10 0/0.0064 6.14 -0. ~3 o/~ . 016 6.20 -0.14 0/0.04 6.14 -0. oa 0/0.1 6.03 0.03 0.2/0 ~.9~ 0.11 0.2/0.002S6 5.92 0. ~4~/27.3 o . 2/0.0064 6. ~30 0.06 0.2/0.016 6.18 -0.12 0.2~0.04 6.2a -0.22 0.2tO .1 5.84 0. Z21 /5~ .1 p~ 5.5 oto 6.37 --0/0.00256 6.27 0.10 0/0.006~ 6.37 0.00 0/0.016 6.6 S -0.28 0/0.04 6.28 0.09 0/0.1 6.50 -0.13 0.2/0 6.16 0.21 0.2/0.002S6 6.2-3 0.09 0.2/0.006~ 6.27 0.10 0. ~/0.016 6.22 0.15 0.2/0.04 6.0S 0.~2 I~6.7 0.2/0.1 5~2~ 38 MS #1643 2 ~ 5 5 Synergy was observed at each pH level in this experiment. In an earlier experiment slnergy was observed at pH 4.5 and 5.5 but not at '.5. The failure to observe synergy at pH 3.5 was probably due to normal experimental variation since the magnitude of synergy at pH 3.5 was relatively small.
The pH range where a particular synergist is effective will depend upon the environment being considered. Thus, while a specific synergist may show activity at a particular pH range in a buffer system, a different pH range may be optimal in certain food systems. For this reason, a certain amount of routine experimentation may be necessary in oraer to determine the optimal pH range for use of a lant~biotic/synergist system in a particular environment being treated.
MS #1643
Treatments were prepared in 0.9 ml quantities in l ml microtubes with treatment ~eing initiated by the addition of 0.1 ml of L. monocvtoqenes Scott A at approximately lx 10' CFU/ml. After treatment for 60 minutes at 37C the test samples were serially diluted in phosphate buffered saline (O.OlM, pH 7.0) using microtiter plates. Enumeration of surviving bacteria was carried out with tryptic soy agar spread plates which were incubated for 48 hours at 37C after inoculation. Synergy was noted when the bacteriocidal effects of the combination of a test compound and nisin was greater than that of the additive loglO
reductions of the individual compounds. The following calculation was used where:
T = test compound N = nisin T+N = combination loglO reduction T+N~(loglO reduction T+loglO
reduction N) = synergy Results are set out in tables 1 (aspartic acid) and 2 where synergistic combinations are identifled with a MS #1643 -lO- 20584~5 "+" to designate the observation that ~he bacteriocidal effects of the combination were greater than those of the additive log reductions alone.
For purposes of the above calcuiation, any negative log reductions were assumed te be 0. Any estimations where greater than (>) or less than (<) are reported, exceeded the detection limits of the system and were not used for calculation of synergy, except in obvious cases, e.g. where the minimum effect which could have occurred was synergistic.
MS #1643 -11- 2058~
Activity of Nisin in Combination with Aspartic Acid (ASP) Against L. mono~vtoqenes ~ Log" C~ Log" Syr~ o ~r ~ol nis~n~ r~oc2~0n ~11 a~lP
p~ 3.5 0/0 6.26 - -0/0.0096 6.11 0.15 0 0~0.024 6.20 0.06 0/0.06 6.30 -0.04 0/0.15 6. zo 0.06 0/0.375 6.20 0.06 0.2~0 6.15 0.1:
0.2/0.0096 6.04 0.2 0.2/0.024 6.15 0.1:
0.2/0.06 6.1a 0.08 0.2/0. lS 5.97 0.29 +/70.6 0.2/0.375 5.26 1.00 f/488 p~ ~.5 0/0 6.04 0/0.0096 6.15 -0.1 -0/0.024 6.23 -O . lg 0/0.06 6.34 -0.30 0/0.15 6.38 -0.34 0/0.37S 6.04 o . Oo 0.2/0 5.97 0.07 0.2~0.0096 6.04 0.00 0.2~0.024 6.00 0.04 0.2~0.06 6.11 -0.07 0.2/O.lS 6.08 -0.0 0.2~0.375 6.04 o .00 p~ 5.S
o/o 6.26 ~
0/0.0096 6.38 -0.12 0~0.024 6.32 -0.06 0/0.06 6.32 -0.06 0~0. lS 6. ~0 -0.14 0~0.3~5 6.41 -0. lS
0.2~0 6.26 o.oO
0.2~0.0096 6.18 0.08 0.2/0.024 6.20 0.06 0.2/0.06 6.28 -0.02 0.2/O.lS 6.34 -0.08 0.2/0.37S 6.26 0 - 00 MS #1643 -12- 2~84~
Activity of Nisin in Combination With Glycine Against L. monocvtoqenes Tr~t~ nt Lcg,. CF~l Loq,. Syn~rgy/% ov~r ~ 1 ni~n/ rsduction .Y
pH 3.5 / 4.30 __ 0/6.4 4~34 ~0~04 ~/16 4.62 -0.32 0/40 4.99 -0.69 0/100 4.75 ~0-45 0/250 4.74 -0.44 0.2/o 2.48 1.82 0.2/6.4 2.62 l.6a 0.2/16 2.96 1.34 0.2/40 4.80 -0.50 0.2~100 4.83 -0.53 0.2/250 4.79 _0.49 P~ ~5 0/0 5.34 __ 0/6.4 5.18 0.16 0/~6 S.23 0.11 0/~0 5.1a 0.16 0/100 S.ll 0.23 0/250 3.62 1.72 0.2/0 4.38 0.96 0~2/6.4 4.28 1.06 0.2/16 4.11 1.23 +/15.0 0.2/40 4.00 1.34 +/19.6 0.2/100 3.89 1.45 +/21.q 0.2~250 2.88 2.46 p~ 5.5 0/0 5.72 __ 0/6.4 5.48 0.24 0/16 5.46 0.26 o/~o 5.46 0.26 0/100 5.49 0.23 0/250 5.41 0.31 4~ 0.2/o 5.38 0.34 0.2~6.4 S.28 0.44 0.2~16 5.41 0.31 0.2/~0 5.00 0.72 +/20 0.2/100 5.~5 0.57 0.2/250 4.92 0.80 +/23 MS #1643 2058~
Tables 1 and 2 represent examples of the type of data used for synergy determination. In each case, under several conditions, the decrease in bacterial population (logLO reduction) when nisin and the synergist were combined significantly exceeded the sum of the effects of nisin alone and the synergist alone.
With synergists such as glycine, which are ionic in nature, very high concentrations sometimes appear inhibitory. This is due to ionic strength inhibition ~0 of nisin and occurs with all ionic compounds at high concentration.
EXAMPLE II
Nisin~Orqanic Acids The nisin-organic acid synergy was initially demonstrated in a diffusion plate assay. The assay was prepared by the method described by Fowler et al in Technical Series of the Society for Applied Bacte-riology No. 8 (1975) which uses Micrococcus luteus as an indicator organism. Nisin or nisin plus the synergist were used in the wells of the plate and the resulting zone diameter was measured. Antimicrobial activity is related to the diameter of the zone of growth inhibition around wells both in cases where the nisin/organic acid mixture was used and where the control (containing nisin only) was added to the well.
The zone of growth inhibition diameters were measured for both the control and the wells containing nisin/-organic acid of which 2 replicates were tested. From MS #1643 20~84~5 these measurements, the effective nisln units/ml were calculated as follows:
The zone diameter with nisin plus the organic acid synergist was calculated and compared to a nisin standard curve. The amount of nisin alone which would have produced a zone of equivalent size was determined, and this value was reported as "Effec~ive Nisin Units/ml". Since none of the test synergists produced a zone of measurable size alone, any increase in zone size represents a synergistic effect. ~he degree of synergy is related to the "Effective ~isin Units".
The results of this experiment are summarized in Table 3A.
Zone Effective SamPle Dlameter Nisin Units/ml 10 U~ml nisin Control/Sample 10 U/ml 10 U/ml nisin + 1% Citric Acid 1.63/2.05 18.1 U/ml 20 + 1~ Lactic Acid 1.63/1.88 14.2 U/ml + 1% Ascorbic Acid 1.63/1/89 14.4 U/ml + 1% Isoascorbic Acid 1.63/1.79 12.5 U/ml The nisin-organic acid synergy was also determined using Listeria monoCYtOqeneS as the target organism.
The assay medium was Brain ~eart Infusion medium containing 1% ~ifco Agar. The medium was adjusted to pH 6.0 and melted whereupon 1% Tween 20 was added.
This is part of the diffusion plate medium and is MS #1643 -15- 2~58~5~
present in each of the reported experiments. L.
monocytoqenes was added at about 2X10S cells/ml (4 ml of 107 cells/ml to each 200 ml agar). ~his inoculated medium was poured into a 243 x 243 x 18 mm culture plate and allowed to cool, whereupon 7 mm circular holes were cut into the agar and removed to provide circular wells. The solutions were adjusted to pH 6.0 with NaOH or HCl prior to use and added to the wells where they were allowed to incubate overnight. A
variety of organic acids (in the form of their sodium salts) were tested in conjunction with nisin at 10,000 U/ml. The results of this experimen~ are set out in Table 3B. Referring to Table 3B, 2% lactic acid provided a 50% increase in efficacy of a 10,000 IU/ml nisin sample by producing a zone equivalent to what 15,000 units/ml nisin would produce while 2% propionic acid caused a 21-fold increase in effective activity of 10,000 units/ml nisin (resulting in a zone size comparable to 212,000 IU/ml nisin alone).
MS #1643 -16- 20~
Effective SamPle Zone DiameterNisin Units/ml 10,000 U/ml Nisin Control/Sample 10,000 10,000 U/ml Nisin +2~ Citric Acid 2.01/2.20 26,300 +2% Lactic Acid 2.01/2.11 15,000 +2% Propionic Acid 2.17/2.78 212,000 +2~ Acetic Acid 2.17/2.40 27,300 +2% Malic Acid 2.17/2.35 20,800 +2% Tartaric Acid 2.17/2.38 24,500 +2% Succinic Acid 2.17t2.36 22,000 +2% Maleic Acid 2.09/2.20 16,400 . +2% Oxalic Acid 2.09/2.21 17,400 +2% Fumaric Acld 2.09/2.30 29,500 From Table 3 it can be determined that all of the organic acids were, to some degree, synergistic with nisin. Lactic acid, maleic acid and oxalic acid provided less than a doubling of apparent activity of 10,000 units/ml nisin, while all the remaining acids except propionic produced an apparent activity increase of between 2 and 3 fold. Propionic acid is unusually effective since it wa~ observed to produce a greater than 21-fold increase in activity. This trend would be expected to continue with the use of longer chain mono- carboxylic acids. Accordinly, mono- carboxylic acids containing from 3 to about 8 carbon atoms, e.g.
MS #1643 2~58~
propionic, butyric, pentanoic, hexanoic, heptanoic and octanoic acids are preferred organic acids for use as synergists of lantibiotics such as nisin.
EXAMPLE III
Selected food gums were combined with nisin to determine the synergistic activity of the resulting combinations against L. monocYtoqenes. Food gums which exhibited synergy were commercial preparations of guar gum, algin and propylene glycol alginate.
10 Nisin was evaluated at 1,000 IU/ml and 100 IU~ml both by itself and in combination with the food gums.
The food gums were added to 25 mM citrate buffer, pH 6.0, at 0.125% (w/v) and autoclaved prior to use.
Nisin stock solutions were prepared in the citrate buffer and filter sterilized. Treatment solutions were prepared in the citrate buffer to contain 0.1% of the food gum in combination with l,000 or 100 IUtml nisin. Controls were prepared to contain only nisin, only the food gum or only buffer. L. monocvtoaenes was added to each test sample at approximately 5 x 10 CFU/ml. The treatment times were 30 minutes with 1,000 IU/ml nisin and 15 minutes with lO0 IU/ml nisin.
Surviving populations were enumerated on ~rain Heart Infusion agar spread plates in triplicate and incubated for 24 hours at 35C. Synergy wa~ noted when the log reductions of the nisin/food gum was greater than the sum of the log reductions of the food gum and nisin controls.
The data generated during these experiments are presented in Table 4.
MS #1643 -18- 205845~ `
Trial 1 (1,000 U/ml nisin, 30 minutes) Log1OLog~O Reduction SamPle CFU/mlfrom control 5 Control 6.53 --Nisin control 6.41 0.12 Propylene glycol alginate (PGA) 6.58 -O.OS
PGA + nisin 4.97 1.56 10 Guar gum 6.48 0.05 Guar gum + nisin 4.75 1.78 Algin 6.11 0.42 Algin + nisin 5.11 1.42 Trial 2 (1,000 U/ml nlsin, 15 minutes) LoglOLoglO Reduction SamPle CFU/mlfrom control Control 6.28 ---Nisin control 6.10 0.18 Propylene glycol alginate (PGA) 6.36 -0.08 PGA + nisin 5.53 0.75 Guar gum 6.30 -0.02 Guar gum + nisin 5.30 0.98 Algin 6.26 0.02 Algin + nisin 5.43 0.85 MS #1643 205845~
From the data of Table 4 it can be determined that in most cases the food gums tested showed little or no antimicrobial effect by themselves. However, when the food gums, all of which are characterized as S being water soluble carbohydrate polymers or deriva-tlves thereof were combined with nisin, the effect of the combination of nisin and the food ~um was signif-icantly greater than the sum of the effect of the nisin and food gum alone.
EXAMPTE IV
The synergism of sodium benzoate with nisin was determined using an experimental procedure essentially as described in Example I.
The results of this experiment are summarized in Table 5. Synergism with nisin was observed at pH 3.5 and 4.5 while the effect was less than additive at pH
5.5.
MS #1643 -20- 20~8~
hctivity Or ni~in in cc nin tion vit~ ~od us c~n~o t-( Na~H~ZCAT~t agninst L ~SrC~Dn=Y~CI in 10 ~
citr~t- ku~S~r at l-Ct8d pH'-sr uO nt Loq CYC~l Log~. Synorgy/% over 13~1 nlsin/ roduc210n % II~I~D~ZOAS~
p8 3.S
o~o s.sa -_ 0 0/0.00256 S .91 0.07 o/0.0064 5.96 - Z9 0/0.016 S .91 0 - 07 0/0.04 1.70 4.28 0/0.1 ~2.00 ~3.98 0.2~0 5.~6 0.5Z
0.2/0.00256 5.38 0.60 +~1.69 0.2/0.0064 s . ~2 0.66 0.2/0.016 5.~6 0.52 0.2 /0. 04 5 . 1 0 0 . 88 0.2/o . l 2.06 3.92 p~ ~..5 0/0 5.92 - _ 0/0.00256 s . gs -0.03 o/0.0064 6.02 -0.10 0/0.0~6 5.90 0.02 0~0.04 5.38 0.54 0~0.1 5.20 0.?2 0.2~0 ~.84 0.08 0.2~0.002~6 ~ .89 0.03 0.2~0.0064 ~ .71 0.21 0.2~0.016 5.80 0. ~2 +/20 0.2~0.04 5.18 0.74 +/19.4 0.2~0.1 0.70 5.22 +/552 p~ 5.S
O/O 6.94 --0/0.00256 6.63 0.31 o/0.0064 6.33 0.61 0/0.016 6.34 0.60 0~0.0~ 6.24 o.70 0/0.1 6.24 0.70 0.2/0 6.1~ 0.77 0.2~0.00256 6.15 0.79 0.2~0.006~ 6.06 0. ~t8 0.2~0.016 6.07 0. ~t7 0.2~0.0~ 6.07 o.~7 0.2~0.1 6.32 0.~2 MS #1643 -21- 2~
From Table 5, it can be determined that under appropriate conditions there is observed synergistic activity between nisin and benzoate under various pH
conditions. Failure to observe a synergistic response ; at pH 5.5 was probably due to the low concentration of undissociated benzoic acid since the undissociated form is the active antimicrobial form.
In a similar manner, the methyl ester of para-hydroxy benzoic acid (methyl parabens) was tested for its antimicrobial effect against L. monocYtoqenes in combination with nisin. This experiment was carried out in accordance with the prodecure outlined for Example I.
The results of this experiment are set out in Table 6.
MS #1643 2 0 ~
ActlYlty o~ ni~in in co DmntiOn Vit~ n t~ylpnr~conn ~)I~PAR~B~q) ~ ln t L~ 9~LCY=~9~=9~ i~ 10 U~
c~t~t - buS~r ~t ~ ce ~ p~ ~
Sren~ Dllt Log CF~Dl Lo9Syn r9y/% ov~r 1l/~ ni~n/ r~duct~on % I~-PAEIA~I
p~ 3.5 0/0 6.06 --0 0/0.00256 5.69 0.37 0/0.0064 6.11 -0.06 0/0.016 5.42 0.64 0~0.04 5.35 0. ~1 0/0.1 3.94 2.12 0.2~0 5.12 0.93 0.2/0.002S6 5.19 0.86 0.2~0.0064 5.22 0.8~
0.2/0.016 5.01 1.05 0.2~0.04 4.94 1.12 0.2/0.1 3.04 3.01 p~ ~.5 0/0 6.06 --0/0.00256 6. - 6 -0.10 0/0.0064 6.14 -0. ~3 o/~ . 016 6.20 -0.14 0/0.04 6.14 -0. oa 0/0.1 6.03 0.03 0.2/0 ~.9~ 0.11 0.2/0.002S6 5.92 0. ~4~/27.3 o . 2/0.0064 6. ~30 0.06 0.2/0.016 6.18 -0.12 0.2~0.04 6.2a -0.22 0.2tO .1 5.84 0. Z21 /5~ .1 p~ 5.5 oto 6.37 --0/0.00256 6.27 0.10 0/0.006~ 6.37 0.00 0/0.016 6.6 S -0.28 0/0.04 6.28 0.09 0/0.1 6.50 -0.13 0.2/0 6.16 0.21 0.2/0.002S6 6.2-3 0.09 0.2/0.006~ 6.27 0.10 0. ~/0.016 6.22 0.15 0.2/0.04 6.0S 0.~2 I~6.7 0.2/0.1 5~2~ 38 MS #1643 2 ~ 5 5 Synergy was observed at each pH level in this experiment. In an earlier experiment slnergy was observed at pH 4.5 and 5.5 but not at '.5. The failure to observe synergy at pH 3.5 was probably due to normal experimental variation since the magnitude of synergy at pH 3.5 was relatively small.
The pH range where a particular synergist is effective will depend upon the environment being considered. Thus, while a specific synergist may show activity at a particular pH range in a buffer system, a different pH range may be optimal in certain food systems. For this reason, a certain amount of routine experimentation may be necessary in oraer to determine the optimal pH range for use of a lant~biotic/synergist system in a particular environment being treated.
MS #1643
Claims (29)
1. A method for inhibiting the growth of gram positive bacteria in an environment where their growth is undesirable which method comprises introducing to said environment a synergistically effective combina-tion of a lanthionine bacteriocin and a synergist therefore said synergist being selected from the group of amino acids, aliphatic mono- and di- carboxylic organic acids containing from 1 to 8 carbon atoms or a suitable alkali metal or alkaline earth metal salt thereof, phenolic antioxidant antimicrobials, benzoic acid or a suitable alkali metal or alkaline earth metal salt thereof and carbohydrate or modified carbohydrate food gums.
2. The method of Claim 1 wherein the amino acid is glycine, leucine, lysine or aspartic acid, the organic acid is lactic, propionic, ascorbic, isoascorbic, acetic, malic, tartaric, succinic, maleic, oxalic or fumaric acid, the phenolic antioxidant antimicrobial is tertiary butyl hydroxyanisole, a 1 to 7 carbon atom aliphatic ester of para hydroxy benzoic acid, tertiary butyl hydroquinone or butylated hydroxytoluene, the salt of benzoic acid is sodium benzoate, and the food gum is an alginate, guar gum, gum arabic, xanthan gum, chitosan, methyl cellulose, propylene glycol alginate and carboxymethylcellulose.
3. The method of Claim 1 wherein the lanthionine is nisin, subtilin, pep 5, epidermin, gallidermin, cinnamycin, Ro09-0198, duramycin and ancovenin.
MS #1643
MS #1643
4. The method of Claim 2 wherein the amino acid is lysine or aspartic acid.
5. The method of Claim 2 wherein the phenolic antioxidant antimicrobial is the methyl ester of para hydroxy benzoic acid.
6. The method of Claim 2 wherein the food gum is propylene glycol alginate, guar gum or sodium alginate.
7. The method of Claim 1 wherein the organic acid is propionic acid.
8. The method of Claim 1 wherein the bacteria is from the species Listeria monocytogenes.
9. The method of Claim 1 wherein the lanthionine bacteriocin is introduced in an amount of from 0.1 to 2,000 IU/gm of the environment being treated and the amount of the synergist in terms of weight percent of the environment is:
a) 0.001 - 5% for amino acids, b) 0.001 - 25% for aliphatic mono- and di-carboxylic organic acids, c) 0.0001 - 2% for phenolic antioxidant anti-microbials, d) 0.001 - 2% for benzoic acid, salts and esters thereof and e) 0.001 - 50% for the food gums.
MS #1643
a) 0.001 - 5% for amino acids, b) 0.001 - 25% for aliphatic mono- and di-carboxylic organic acids, c) 0.0001 - 2% for phenolic antioxidant anti-microbials, d) 0.001 - 2% for benzoic acid, salts and esters thereof and e) 0.001 - 50% for the food gums.
MS #1643
10. The method of Claim 9 wherein the lanthionine bacteriocin is introduced at a level of from 50 to 500 IU/gm and the synergist therefore in terms of weight percent of the environment to be protected is:
a) 0.03 - 0.1% for amino acids, b) 0.01 - 1% for aliphatic mono- and di-carboxylic acids, c) 0.005 - 0.02% for phenolic antioxidant antimicrobials, d) 0.03 - 0.1% for benzoic acid, salts and esters thereof, and e) 0.02 - 2.5% for the food gums.
a) 0.03 - 0.1% for amino acids, b) 0.01 - 1% for aliphatic mono- and di-carboxylic acids, c) 0.005 - 0.02% for phenolic antioxidant antimicrobials, d) 0.03 - 0.1% for benzoic acid, salts and esters thereof, and e) 0.02 - 2.5% for the food gums.
11. A method of inhibiting the growth of Listeria monocytogenes in an environment where its growth is undesirable which method comprises introducing to said environment a synergistically effective combination of nisin and a synergist therefor selected from the group of amino acids, aliphatic mono- and di- carboxylic organic acids containing from 1 to 8 carbon atoms or a suitable alkali metal or alkaline earth metal salt thereof, phenolic anitoxidant antimicrobials, benzoic acid or a suitable alkali metal or alkaline earth metal salt thereof and carbohydrate or modified carbohydrate food gums.
12. The method of Claim 11 wherein the amino acid is glycine or aspartic acid.
13. The method of Claim 11 wherein the nisin is introduced in an amount of from 0.1 to 2,000 IU per MS #1643 gram of the environment being treated and the amino acid is introduced at a level of from 0.001 to 5 weight percent.
14. The method of Claim 13 wherein the nisin is introduced in an amount of from 50 to 500 IU/gm and the amino acid is at a level of from 0.03 to 0.1 weight percent.
15. The method of Claim 10 wherein the aliphatic, mono- or di- carboxylic acid is lactic, propionic, acetic, malic, tartaric, succinic, maleic, oxalic or fumaric acid.
16. The method of Claim 15 wherein the nisin is introduced in an amount of from 0.1 to 2,000 IU per gram of the environment being treated and the mono- or di- carboxylic acid is introduced in an amount of from 0.001 to 25 weight percent.
17. The method of Claim 16 wherein the nisin is introduced in an amount of from 50 to 500 IU/gm and the mono- or di- carboxylic acid is introduced at a level of from 0.01 to 1 weight percent.
18. The method of Claim 10 wherein the alkali metal salt of benzoic acid is sodium benzoate.
19. The method of Claim 18 wherein the nisin is introduced in an amount of from 0.1 to 2,000 IU per gram of the environment being treated and the sodium MS #1643 benzoate is introduced in an amount of from 0.001 to 2 weight percent.
20. The method of Claim 19 wherein the nisin is introducedin an amount of from 50 to 500 IU/gm and the sodium benzoate is introduced in an amount of 0.03 to 0.1 weight percent.
21. The method of Claim 10 wherein the phenolic antioxidant antimicrobial is a 1 to 7 carbon atom ester of para hydroxy benzoic acid, tertiary butyl hydroquinone or butylated hydroxy toluene.
22. The method of Claim 21 wherein the nisin is introduced in an amount of from 0.001 to 2,000 IU per gram of the environment being treated and the phenolic antioxidant antimicrobial is introduced in an amount of from 0.001 to 2 weight percent.
23. The method of Claim 22 wherein the nisin is introduced in an amount of from 50 to 500 IU per gram and the phenolic antioxidant antimicrobial is intro-duced in an amount of from 0.005 to 0.02 weight percent.
24. The method of Claim 22 wherein the phenolic antioxidant antimicrobial is the methyl ester of para hydroxy benzoic acid.
25. The method of Claim 20 wherein the food gum is an alginate, guar gum, gum arabic, xanthan gum, chitosan, methyl cellulose, algin or carboyxmethylcellulose.
MS #1643
MS #1643
26. The method of Claim 25 wherein the nisin is introduced in an amount of from 0.1 to 2,000 IU per gram of the environment being treated and the food gum is introduced in an amount of from 0.001 to 50 weight percent.
27. The method of Claim 25 wherein the nisin is introduced in an amount of from 0.1 to 2,000 IU per gram of the environment being treated and the food gum is introduced in an amount of from 0.02 to 2.5 weight percent.
28. The method of Claim 27 wherein the food gum is propylene glycol alginate, guar gum or algin.
29. A solid or liquid product suitable for ingestion by humans or other animals which comprises an ingest-ible substrate containing a synergistically effective, gram positive bacteria inhibiting combination of a lanthionine bacteriocin and a synergist therefore which is selected from the group of amino acids, aliphatic mono- and di- carboxylic organic acids containing from 1 to 8 carbon atoms or a suitable alkali metal or alkaline earth metal salt thereof, benzoic acid or a suitable alkali metal or alkaline earth metal salt thereof and carbohydrate or modified carbohydrate food gums.
MS #1643
MS #1643
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63338090A | 1990-12-21 | 1990-12-21 | |
| US633,380 | 1990-12-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2058455A1 true CA2058455A1 (en) | 1992-06-22 |
Family
ID=24539404
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002058455A Abandoned CA2058455A1 (en) | 1990-12-21 | 1991-11-21 | Synergistic effect of lantibiotics in combination with selected agents against gram positive bacteria |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPH04334307A (en) |
| CA (1) | CA2058455A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994012150A1 (en) * | 1992-11-24 | 1994-06-09 | Smithkline Beecham Plc | Mouthcare compositions |
| US5368845A (en) * | 1993-01-07 | 1994-11-29 | Colgate Palmolive Company | Oral composition |
| WO1995001154A1 (en) * | 1993-07-01 | 1995-01-12 | Unilever N.V. | Oral care compositions |
| WO1997010801A2 (en) * | 1995-09-20 | 1997-03-27 | Ambi Inc. | Nisin compositions to prevent the promotion of tooth decay by suppressing formation of acid from foods by oral bacteria |
| DE10028998A1 (en) * | 2000-02-17 | 2001-08-23 | Bode Chemie Gmbh & Co Kg | Cleaning and disinfection systems for medical instruments |
| EP1621085A1 (en) | 2004-07-30 | 2006-02-01 | Kraft Foods Holdings, Inc. | Methods for preserving food products |
| US11008277B2 (en) * | 2016-06-13 | 2021-05-18 | Syneurx International (Taiwan) Corp. | Co-crystals of sodium benzoate and uses thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002330740A (en) * | 2001-05-08 | 2002-11-19 | Yamaya Communications:Kk | Method for spore bearing bacterium sterilization |
| JP2002330715A (en) * | 2001-05-08 | 2002-11-19 | Yamaya Communications:Kk | Method for producing koji |
| WO2006090903A1 (en) * | 2005-02-24 | 2006-08-31 | Sumitomo Chemical Company, Limited | Composition for controlling harmful organism |
| JP5374260B2 (en) * | 2009-07-10 | 2013-12-25 | 静岡商工会議所 | Agricultural materials |
-
1991
- 1991-11-21 CA CA002058455A patent/CA2058455A1/en not_active Abandoned
- 1991-12-13 JP JP3351248A patent/JPH04334307A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994012150A1 (en) * | 1992-11-24 | 1994-06-09 | Smithkline Beecham Plc | Mouthcare compositions |
| US5368845A (en) * | 1993-01-07 | 1994-11-29 | Colgate Palmolive Company | Oral composition |
| WO1995001154A1 (en) * | 1993-07-01 | 1995-01-12 | Unilever N.V. | Oral care compositions |
| WO1997010801A2 (en) * | 1995-09-20 | 1997-03-27 | Ambi Inc. | Nisin compositions to prevent the promotion of tooth decay by suppressing formation of acid from foods by oral bacteria |
| WO1997010801A3 (en) * | 1995-09-20 | 1997-04-24 | Applied Microbiology Inc | Nisin compositions to prevent the promotion of tooth decay by suppressing formation of acid from foods by oral bacteria |
| DE10028998A1 (en) * | 2000-02-17 | 2001-08-23 | Bode Chemie Gmbh & Co Kg | Cleaning and disinfection systems for medical instruments |
| EP1621085A1 (en) | 2004-07-30 | 2006-02-01 | Kraft Foods Holdings, Inc. | Methods for preserving food products |
| US11008277B2 (en) * | 2016-06-13 | 2021-05-18 | Syneurx International (Taiwan) Corp. | Co-crystals of sodium benzoate and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04334307A (en) | 1992-11-20 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 19940521 |