CA2055984A1 - Synergistic effect of lantibiotics in combination with selected agents against gram negative bacteria - Google Patents
Synergistic effect of lantibiotics in combination with selected agents against gram negative bacteriaInfo
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- CA2055984A1 CA2055984A1 CA002055984A CA2055984A CA2055984A1 CA 2055984 A1 CA2055984 A1 CA 2055984A1 CA 002055984 A CA002055984 A CA 002055984A CA 2055984 A CA2055984 A CA 2055984A CA 2055984 A1 CA2055984 A1 CA 2055984A1
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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
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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
~g's~
IN COMBINATION WITH SELECTED AGENTS
AGAINST GRAM NEGATIV~_BACTERIA
Background of khe Invention Nisin, a member of the group of antimicrobials known as lanthionine bacteriocins, is an antimicrobial polypeptide produced by certain strains of Lactococcus lactis (formerly Streptococcus lactis). It is manufactured through the pure-culture fermentation of these bacteria with subsequent purification and dryingO M. Doyle in Food Technol. 1988 42: (4~
169-171, describes the inhibitory effects of nisin on the growth and survival of L. monocytoqenes.
The term "lantibiotics" was coined by Schnell et al. (1988 Nature 333: 276-273) 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, duramycin and ancovenin. These ribosomally synthe-sized peptide bacteriocins contain from 19 to 34 amino acids and are produced by various microbes including Staphlococcus species, Bac~11us species and Streptomyces species.
MS ~1644 -2- 2~`~9~
The combination of nisin with other material~ to enhance its antimicrobial activity has been reported.
Thus, International patent application WO 89J12399 discloses the combination of ni~in with chelating agents and~or surfactants for use against Gram positive and gram negative ~acteria.
Published European patent application 0,384,319 discloses the synergistic effect of nisin with chelating agents against L. monocytoqenes. This reference discloses the use of certain amino acids and monocarboxylic acids in combination with nisin.
While numerous references describe synergistic combinations of nisin in combination with other ingredients as being effective against Gram positive bacteria, the activity of nisin, e ther alone or in combination with other materials against Gram negative bacteria is not widely reported.
Summary of the In~ention The present invention involves a method for inhibiting the growth of Gram negative bacteria in an environment where their growth is undesirable which method comprise~ introducing to said envixonment a synergistically effective combination of a lanthionine barteriocin and a synergist therefore wherein said synergist is selected from the group consisting 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 me*al salt thereof, phenolic antioxidant antimicrobials, ~enzoic MS #1644 ~3~
or sorbic acid and a suitable alkali metal or alkaline earth metal salts thereof.
Description of the Invention The present invention relates to the observat,ion tha~ a lanthionine bacteriocin in combination with certain additives has provided a composition which inhibits the growth of undesirable Gram negative bacteria to a greater degree than either material by itself and that the combined inhibitory effect of these substances is greater ~han the additive effect observed when using the lantibiotic or other compo-sition individually.
Various classes of compositions which act synergistically with the lantibiotics have been id~ntified. Among these are amino acids and in particular glycine.
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-carboxylic acids containing from 1 to 8 carbon atoms.These acids include lactic, propionic, acetic, malic, tartaric, succinic, maleic, oxalic and fumaric acid.
Suita~la salts, such as the alkali and alkaline earth metal salts, e.g. sodium, potassium and calcium can be employed. Sorbic acid and suitable salts thereof also exhibit synergistic activity against Gram negative bacteria when used in combination with a lantibiotic such as nisin.
The mechanism of action of lantibiotics such as nisin has not been resolved, but many investigators MS #1644 ~59~
have linked ~he lethality of ~hese peptides to irreversible changes in the cell membranes of sus-ceptible organisms. The resiskance of some organisms to nisin is thought to be due to the lack o~ access of the peptide to its site of action, either because of physical exclusion or the lack of appropriate receptor molecules. The lantibiotic synergists of the present invention appear to alleviate this inhibition of the access to the taxget microorganism although the mechanism of action i5 not understood.
The present invention is predicated on the discovery that a lantibiotic and certain ~ynergists therefore, in combination, inhibit the growth of undesirable Gram negative bacteria to a grea~r extent than either material by itself and that the combined inhibitory effect of these substances is greater than the additive effect obser~ed when using the lanti-biotic or synergist individually.
The lantibiotics are generally regarded as being ineffective against Gram negative bacteria in the absence of a potentiator. In the previously mentioned WO 89/12399, chelating agents and surfactants are described as being suitable potentiating agents for the control of Gram negative species. It has now been discovered that amino acids and certain mono-and di- carboxylic organic acids as well as sorbic acid and salts thereof act as potentiators which render lantibiotics such as nisin effective against Gram negative bacteria. In a typical example, when using a lantibiotic such as nisin and a synergist therefore, the total amount of nisin will typically range from about 0.1 IU/gm (or IU/ml in liquid MS #1644 systems) of the substrate material being treated up to as much as 2,000 IU/gm or IU/ml in a liquid system. In most applications, a nisin concen~ration of from 50 to 500 IU/gm will be sufficient due to the boost it will receive from the synergist. As used herein, the term IU is intended to refer to Interna-tional Unit as determined by the assay of Tramer and Fowler in J. Sci~ Food and Agric., 15(8) 522 (1964).
The amount of synergist to be employed will depend on the particular composition involved. In yeneral the amount of synergist in terms of weight percent of the substrate being treated will be within the ranges set out in Table A. Lanthionine bacteriocins other than nisin can be expected to be efficacious at these concen~rations although some routine experimentation may be necessary to determine the concentration of a particular lantibiotic acid synergist therefore for optimal synergistic effect.
TABLE A
20 Class of Range of Preferred Synerqist Suqqested Use Ranqe Amino Acids 0.001% - 5~ 0.03% - 0.1%
Aliphatic mono- 0.001~ - 25% 0.01% - 1 and di- carboxylic 25 organic acids Sorbic Acid ~ Salts 0.001% 2% 0.02% - 0.3%
MS #1644 9~ ~
Surface 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 lanthionineisynergist combination can be incorporated into films or gels which are applied to the substrate's surface or the combination can be incorporated directly into the environment keing 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 lanti-biotic and synergist can approach their solubility limits to provide a finished product which, after application and diffusion, contains residual levels of lantibiotic/synergist which are within the desired ranges. When treating the material's surface, some routine experimentation may be needed to ascertain the most effective concentra1ion.
Environments capable of being treated with the compositions of the present :invention include, but are not limited to, meats and meat products, such as chicken, turkey, ham, beef, salami, sausages and smoked meat products; mayonnaise; dairy products such as cheese, milk and yogurt; oils, fish and fish products; soft drinks including natural juices;
animal Eeeds and other high protein products. In addition to use in Eoods, the compositions of the present invention have non food uses such as inclusion in mouthwashes, denture cleaners, ointments, creams and shampoos.
MS #1644 _7_ ~5~
Amony the problematical Gram negative bacteria, Salmonalla typhimurium is a particular pest because -of its ubiquitous sontamination of fresh poultry.
Other undesirable Gram negative bacteria whose growth can be inhibited by the synergistic combinations of the present invention include other species of Salmonella such as enteriditis and species from the -genera Shiqella and Camylobacter as well as various strains of Escherichia coli.
The presen~ invention is further illustrated by the following examples in which International Units of nisin are expresses as U nisin. Nisin for these studies was prepared from Nisaplin~ (Aplin & Barrett) by diafiltration and ultrafiltration resulting in a preparation which is essentially salt free, EXAMPLE I
The synergistic bacteriocidal activity of nisin and glycine against Salmonel:La tvPhimurium ATCC 14028 was evaluated at pH 3.5, 4.5 and 5.5 in 10 mM citrate buffer (C~B). Nisin was tested at 500 and 1000 IU/ml while glycine concentrations 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 pH evaluated. Treatments were prepared in 0.9 ml quantities in 1 ml microtubes.
The treatment time was initiated with the addition o 0.1 ml of S. ty~himurium in its logrithmic growth phase at approximately 1 x 10' CFU/ml. The treatment time was 60 minutes at 37C. Treatments were then serially diluted in phosphate buffered saline (0.01 MS #1644 -8~ 5~ ~
M, pH 7.0) using microtiter plates. Enumeration of surviving bacteria was done with tryptic soy agar spread plates after 48 hours of incubation at 37C.
The results of this experiment are set out in Tables 1 and 2 where synergy is identi~isd with a "~" when the bacteriocidal effects of the combination of test compound and nisin was observed to be greater than the additive log1O reductions of the individual components of the combination followed by the amount of the increase in bacteriocidal activity in terms of "% over". The following calculation was used where T
= test compound alone, N = nisin alone and T + N =
combination.
loglO reduction T ~ N> (logl~ reduction T +
loglO reduction N) = synergy For purposes of the above calculation, any negative log reductions were assumed to be 0. Any estimations where greater than (~) or less than (<) are reported, exceeded the detection limits of the system and where not used for calculation of synergy, except in obvious cases, e.g. where the minimum effect which could have occurred was synergistic.
The results of this experiment are summarized in Table 1 where international units of nisin are e~pressed as U nisin.
MS #1644 Ac~i~ity ~ nisin ln ~u~b~na~ion ~ith gly~ine (GLY) ag~insS S_ 5YDr~ iY~ in 10 ~ ~i~xa~ buS~2r ~ ~elect~d p~'s Tr~at~ent LoglO CFU/~l L~gl, syn2rgy/% ovar U~l nisin/ reduction llM GI.Y
pa 3.S
0/0 ~.30 __ 0~6.4 5.34 -0.04 0 Otl6 ~.48 -O.la 0/4a 6.00 -0.70 0/100 6.34 -1.04 0/254 4.50 0.80 lOOOfO 3.95 1.35 1000/6.4 3.80 1.50 +/11.1 1000~16 4.81 0.49 1000/40 ~.54 0.76 1000/100 4.04 1.26 ' 000/2~0 1.78 3.52 +/63.7 p~ 4.5 / 7.45 __ 0~6.4 7. ~3 0.02 0/16 7.48 -o . o~
0/40 7.49 -0.04 0/100 >7.48 ~-0.03 0/250 7.40 0.05 500/0 5.73 1.72 500/6.4 6.28 1.17 500/16 5.99 1.46 500/40 6.00 1.45 500/100 6.34 1.11 5~0~250 5. ~8 1.47 p~ 5.5 0/0 7.65 __ 0/6.4 >7 ~ 48 <0.17 0~16 ~7.4a c0.17 0/40 7.53 0.12 - 0/100 7.36 0.29 0~250 7.38 0.27 500~0 3.08 4.57 500/6.4 3. ~0 4.35 500/16 3.23 4.42 5~0/~0 2.91 4.74 ~/1.07 500/100 ~ Z .80 4.85 500/250 2.34 5.31 +/9.7 MS #1644 -1o- 2~5~
High concentrations of glycine were bacteriocidal to S. t~phimurium at all three pH levels. Detectable synergy with nisin occurred at pH 3.5 and 5.5.
EXAMPLE II
.
The inhib1tory effect of nisin and organic acids against S. typhimurium was determined by the procedure described in Example I.
The results of these experiments are set out in Table 2 (lactic acid) and ~able 3 (propionic acid).
MS #1644 2~ 9~ ~
Ac~i~y o~ nisi~ a~ioYI 17ith 12cl:ic acid ~IA) agz~ 3t ~-. S~=D~ in 10 ~ cit:ra~ b~2er zlt ~elected pll'~
~gl, CPlJ/ml I.og,O Syn~rgy/% over Il/~ in/ redllctior p~ 3.S
0/0 7.08 __ 0~6.4 ~.2~ -0.15 0 0/16 6.81 0.27 0/40 6.15 0.93 0/100 ~2.00 >5.08 0/250 ~2.00 ~.08 1000/0 6.63 0~45 1000/6.4 3.53 3.55 ~/689 1000/'6 6.36 0.72 1000~0 ~.53 1.55 +/12.3 1000/'~0 ~2.00 >5.08 1000~250 ~2.00 >5.0a p~ 4.5 / 7.20 --0/6.4 7.45 -0.25 0/16 7.26 -0.06 0/40 ~.43 -0.23 0/100 7.4a -0.28 0/250 7.28 -0.08 500/0 6.45 0.75 500/6.4 6.56 0.64 500/16 6.60 0.60 500/40 >7.00 <0.2 500/100 ~7.00 <0.2 500/250 >7.00 <0.2 p~ 5.5 0/0 7.34 __ 0/~ . 4 ~.35 -0.02 0/16 7 .4a -0.14 0~40 7.49 -0.15 0/100 7.36 -0.02 0/2S0 7.45 -0.11 4~ 50~0 4.68 2.66 50/6.4 3.g7 3.37 ~/26.7 S0/16 3.79 3.55 +/33~4 50/40 4~34 3.00 ~/12.8 50/100 ~7,00 ~0,3~
50/250 >7.00 ~0.34 MS #1644 -12- ~ ~S5~3~
~ Ct~Yity 0~ n~in i~ ~o~bination ~th propio~ic acid (PAl ag~nst S~ iY~d~ in 10 ~ cit~ate bufr~r a~ ~elected p~'s Tr~atment Lag,~ CF~/~l LoqID Syneryy/% ovor ~/~1 ni~in/ r~duction mH P~
~
p~ 3.5 0/0 6.95 __ Of6.4 6.79 0.16 0/16 6.~6 0.09 0/40 6.99 -0.04 0/lOo 6.20 0.75 0/2S0 ~2.00 >4.95 1000/0 6.40 0.55 1000/6.4 6.29 0.56 1000~16 6.13 0.82 +/28.1 1000/40 5.89 1.06 +/9.27 1000/100 4.g8 1.97 -/79.
1000/250 ~1.00 ~5.95 ~0 p~ 4.5 0/0 ~.26 --0/6.4 7.22 0.04 0/16 7.29 -0.03 0~40 7.28 -0.02 2X 0/100 7.41 -0.15 0/250 7.31 0.05 500/0 6.50 0.76 500/6.4 6.g3 0.73 500/16 6.59 0.67 500/40 >6.00 ~1.26 500~100 >6.00 <1.26 500/250 >6.00 <1.26 p~ 5.5 0/0 7.~2 __ 0/6.4 7.43 -0.01 o/16 7.46 -0.04 0/40 ?.53 -0.11 0/100 7.5g -0.17 0/Z50 7.46 -0.04 4a 500/0 4.55 2087 500/6.4 5~11 2.31 500/16 6.34 1.08 500/40 >6.00 <1.42 00~100 >6.00 <1.42 S00/Z50 >6.0~ <1.~2 MS #1644 The higher concentratLons of lactic and propionic acids alone were bacteriocidal to the test organism a~ pH 3.5. Synergy with lactic acid occurred at all pH levels evaluated at concentrations between 6.4 and 40 mM while propionic acid/nisin synergy occurred only at pH 3.5.
In general monocarboxylic acids of 3-8 carbon atoms, i.e. propionic, butyric, pentanoic, hexanoic, heptanoate and octanoate can bs us~d in conjunction with lantibiotic in order to enhance its effectiveness in controlling Gram negative bacteria.
EXAMPLE I I I
The activity of nisin and potassium sorbate against S~ typhimurium was determined as described in Example I. The results of this experiment are summarized in Table 4. The results of this experiment are set out in Table 4.
MS #1644 ~5~
A~tivity ~ ni~in in ~GmbiD~tia~ ~i~h po~a~iu~
~xb~ K~qORB~TE) ~ga~ns~ S_ $Y5 dh~l UI} in 10 ~ ~itr~t~ burrer at s~l~ted p~'s Tr~tEe~t L~g~O C~l L~gl. Syn~rqy~% ov~r ~/~1 nisin/ reduction ~+gO~13Aq`E
P~ 3.~
0/0 6.91 __ 0 0~0.00256 6.96 -0,05 0/0.0064 6.g2 -0.01 0/0.016 7.00 -o.og 0/0.04 6.47 0.44 0~0.1 ~2.00 >4.91 100/0 6.~8 0.23 100/O.OOZ56 6.65 0~26 +/13 100/0.0064 6.61 0.30 +/30.4 100/0.016 6.69 0.22 LOO/0.04 6.12 0.79 +/243 100/0.1 3.45 3.46 P~ ~.5 0/0 7.01 __ 0/0.002~6 7.05 _0,04 o/0.00~4 7.1g -0.14 0/0.016 7.21 -0.20 0/0.04 7.21 -0.20 0/0.1 7.23 -0.22 50~0 6.~1 0.20 SO/0.00256 6.75 0.26 +/30.0 50~0.0064 >7.00 <1.00 50/0.016 6.81 0.20 50/0.04 >7.00 <1.00 50/0.1 >7.00 ~1.00 p~ 5.5 0/0 7.S8 --O/O.nO256 7.38 0~20 0~0.0064 6.86 0.72 0/0.016 7.17 0.41 0~0.04 7.26 0.32 0/0.1 7.26 0.32 50/0 >7.00 ~0.5~
50/0.00256 >7.00 ~0.58 50/0.0064 >7.00 <0.58 50/0.016 >7.00 <0.58 50/0.04 >7.00 ~0.58 50/0.1 >7.00 <0.58 MS ~1644 -15- ~'5~59~
Potassium sorbate by itself was bacteriocidal at pH 5.5. Synergism with nisin was observed at various concentrations at pH 3.5 and 4.5. Data at pH 5.5 was inconclusive.
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 order to determine the optimal pH range for use of a lantibiotic/synergist system in a particular envi-ronment being treated.
MS #1644
IN COMBINATION WITH SELECTED AGENTS
AGAINST GRAM NEGATIV~_BACTERIA
Background of khe Invention Nisin, a member of the group of antimicrobials known as lanthionine bacteriocins, is an antimicrobial polypeptide produced by certain strains of Lactococcus lactis (formerly Streptococcus lactis). It is manufactured through the pure-culture fermentation of these bacteria with subsequent purification and dryingO M. Doyle in Food Technol. 1988 42: (4~
169-171, describes the inhibitory effects of nisin on the growth and survival of L. monocytoqenes.
The term "lantibiotics" was coined by Schnell et al. (1988 Nature 333: 276-273) 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, duramycin and ancovenin. These ribosomally synthe-sized peptide bacteriocins contain from 19 to 34 amino acids and are produced by various microbes including Staphlococcus species, Bac~11us species and Streptomyces species.
MS ~1644 -2- 2~`~9~
The combination of nisin with other material~ to enhance its antimicrobial activity has been reported.
Thus, International patent application WO 89J12399 discloses the combination of ni~in with chelating agents and~or surfactants for use against Gram positive and gram negative ~acteria.
Published European patent application 0,384,319 discloses the synergistic effect of nisin with chelating agents against L. monocytoqenes. This reference discloses the use of certain amino acids and monocarboxylic acids in combination with nisin.
While numerous references describe synergistic combinations of nisin in combination with other ingredients as being effective against Gram positive bacteria, the activity of nisin, e ther alone or in combination with other materials against Gram negative bacteria is not widely reported.
Summary of the In~ention The present invention involves a method for inhibiting the growth of Gram negative bacteria in an environment where their growth is undesirable which method comprise~ introducing to said envixonment a synergistically effective combination of a lanthionine barteriocin and a synergist therefore wherein said synergist is selected from the group consisting 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 me*al salt thereof, phenolic antioxidant antimicrobials, ~enzoic MS #1644 ~3~
or sorbic acid and a suitable alkali metal or alkaline earth metal salts thereof.
Description of the Invention The present invention relates to the observat,ion tha~ a lanthionine bacteriocin in combination with certain additives has provided a composition which inhibits the growth of undesirable Gram negative bacteria to a greater degree than either material by itself and that the combined inhibitory effect of these substances is greater ~han the additive effect observed when using the lantibiotic or other compo-sition individually.
Various classes of compositions which act synergistically with the lantibiotics have been id~ntified. Among these are amino acids and in particular glycine.
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-carboxylic acids containing from 1 to 8 carbon atoms.These acids include lactic, propionic, acetic, malic, tartaric, succinic, maleic, oxalic and fumaric acid.
Suita~la salts, such as the alkali and alkaline earth metal salts, e.g. sodium, potassium and calcium can be employed. Sorbic acid and suitable salts thereof also exhibit synergistic activity against Gram negative bacteria when used in combination with a lantibiotic such as nisin.
The mechanism of action of lantibiotics such as nisin has not been resolved, but many investigators MS #1644 ~59~
have linked ~he lethality of ~hese peptides to irreversible changes in the cell membranes of sus-ceptible organisms. The resiskance of some organisms to nisin is thought to be due to the lack o~ access of the peptide to its site of action, either because of physical exclusion or the lack of appropriate receptor molecules. The lantibiotic synergists of the present invention appear to alleviate this inhibition of the access to the taxget microorganism although the mechanism of action i5 not understood.
The present invention is predicated on the discovery that a lantibiotic and certain ~ynergists therefore, in combination, inhibit the growth of undesirable Gram negative bacteria to a grea~r extent than either material by itself and that the combined inhibitory effect of these substances is greater than the additive effect obser~ed when using the lanti-biotic or synergist individually.
The lantibiotics are generally regarded as being ineffective against Gram negative bacteria in the absence of a potentiator. In the previously mentioned WO 89/12399, chelating agents and surfactants are described as being suitable potentiating agents for the control of Gram negative species. It has now been discovered that amino acids and certain mono-and di- carboxylic organic acids as well as sorbic acid and salts thereof act as potentiators which render lantibiotics such as nisin effective against Gram negative bacteria. In a typical example, when using a lantibiotic such as nisin and a synergist therefore, the total amount of nisin will typically range from about 0.1 IU/gm (or IU/ml in liquid MS #1644 systems) of the substrate material being treated up to as much as 2,000 IU/gm or IU/ml in a liquid system. In most applications, a nisin concen~ration of from 50 to 500 IU/gm will be sufficient due to the boost it will receive from the synergist. As used herein, the term IU is intended to refer to Interna-tional Unit as determined by the assay of Tramer and Fowler in J. Sci~ Food and Agric., 15(8) 522 (1964).
The amount of synergist to be employed will depend on the particular composition involved. In yeneral the amount of synergist in terms of weight percent of the substrate being treated will be within the ranges set out in Table A. Lanthionine bacteriocins other than nisin can be expected to be efficacious at these concen~rations although some routine experimentation may be necessary to determine the concentration of a particular lantibiotic acid synergist therefore for optimal synergistic effect.
TABLE A
20 Class of Range of Preferred Synerqist Suqqested Use Ranqe Amino Acids 0.001% - 5~ 0.03% - 0.1%
Aliphatic mono- 0.001~ - 25% 0.01% - 1 and di- carboxylic 25 organic acids Sorbic Acid ~ Salts 0.001% 2% 0.02% - 0.3%
MS #1644 9~ ~
Surface 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 lanthionineisynergist combination can be incorporated into films or gels which are applied to the substrate's surface or the combination can be incorporated directly into the environment keing 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 lanti-biotic and synergist can approach their solubility limits to provide a finished product which, after application and diffusion, contains residual levels of lantibiotic/synergist which are within the desired ranges. When treating the material's surface, some routine experimentation may be needed to ascertain the most effective concentra1ion.
Environments capable of being treated with the compositions of the present :invention include, but are not limited to, meats and meat products, such as chicken, turkey, ham, beef, salami, sausages and smoked meat products; mayonnaise; dairy products such as cheese, milk and yogurt; oils, fish and fish products; soft drinks including natural juices;
animal Eeeds and other high protein products. In addition to use in Eoods, the compositions of the present invention have non food uses such as inclusion in mouthwashes, denture cleaners, ointments, creams and shampoos.
MS #1644 _7_ ~5~
Amony the problematical Gram negative bacteria, Salmonalla typhimurium is a particular pest because -of its ubiquitous sontamination of fresh poultry.
Other undesirable Gram negative bacteria whose growth can be inhibited by the synergistic combinations of the present invention include other species of Salmonella such as enteriditis and species from the -genera Shiqella and Camylobacter as well as various strains of Escherichia coli.
The presen~ invention is further illustrated by the following examples in which International Units of nisin are expresses as U nisin. Nisin for these studies was prepared from Nisaplin~ (Aplin & Barrett) by diafiltration and ultrafiltration resulting in a preparation which is essentially salt free, EXAMPLE I
The synergistic bacteriocidal activity of nisin and glycine against Salmonel:La tvPhimurium ATCC 14028 was evaluated at pH 3.5, 4.5 and 5.5 in 10 mM citrate buffer (C~B). Nisin was tested at 500 and 1000 IU/ml while glycine concentrations 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 pH evaluated. Treatments were prepared in 0.9 ml quantities in 1 ml microtubes.
The treatment time was initiated with the addition o 0.1 ml of S. ty~himurium in its logrithmic growth phase at approximately 1 x 10' CFU/ml. The treatment time was 60 minutes at 37C. Treatments were then serially diluted in phosphate buffered saline (0.01 MS #1644 -8~ 5~ ~
M, pH 7.0) using microtiter plates. Enumeration of surviving bacteria was done with tryptic soy agar spread plates after 48 hours of incubation at 37C.
The results of this experiment are set out in Tables 1 and 2 where synergy is identi~isd with a "~" when the bacteriocidal effects of the combination of test compound and nisin was observed to be greater than the additive log1O reductions of the individual components of the combination followed by the amount of the increase in bacteriocidal activity in terms of "% over". The following calculation was used where T
= test compound alone, N = nisin alone and T + N =
combination.
loglO reduction T ~ N> (logl~ reduction T +
loglO reduction N) = synergy For purposes of the above calculation, any negative log reductions were assumed to be 0. Any estimations where greater than (~) or less than (<) are reported, exceeded the detection limits of the system and where not used for calculation of synergy, except in obvious cases, e.g. where the minimum effect which could have occurred was synergistic.
The results of this experiment are summarized in Table 1 where international units of nisin are e~pressed as U nisin.
MS #1644 Ac~i~ity ~ nisin ln ~u~b~na~ion ~ith gly~ine (GLY) ag~insS S_ 5YDr~ iY~ in 10 ~ ~i~xa~ buS~2r ~ ~elect~d p~'s Tr~at~ent LoglO CFU/~l L~gl, syn2rgy/% ovar U~l nisin/ reduction llM GI.Y
pa 3.S
0/0 ~.30 __ 0~6.4 5.34 -0.04 0 Otl6 ~.48 -O.la 0/4a 6.00 -0.70 0/100 6.34 -1.04 0/254 4.50 0.80 lOOOfO 3.95 1.35 1000/6.4 3.80 1.50 +/11.1 1000~16 4.81 0.49 1000/40 ~.54 0.76 1000/100 4.04 1.26 ' 000/2~0 1.78 3.52 +/63.7 p~ 4.5 / 7.45 __ 0~6.4 7. ~3 0.02 0/16 7.48 -o . o~
0/40 7.49 -0.04 0/100 >7.48 ~-0.03 0/250 7.40 0.05 500/0 5.73 1.72 500/6.4 6.28 1.17 500/16 5.99 1.46 500/40 6.00 1.45 500/100 6.34 1.11 5~0~250 5. ~8 1.47 p~ 5.5 0/0 7.65 __ 0/6.4 >7 ~ 48 <0.17 0~16 ~7.4a c0.17 0/40 7.53 0.12 - 0/100 7.36 0.29 0~250 7.38 0.27 500~0 3.08 4.57 500/6.4 3. ~0 4.35 500/16 3.23 4.42 5~0/~0 2.91 4.74 ~/1.07 500/100 ~ Z .80 4.85 500/250 2.34 5.31 +/9.7 MS #1644 -1o- 2~5~
High concentrations of glycine were bacteriocidal to S. t~phimurium at all three pH levels. Detectable synergy with nisin occurred at pH 3.5 and 5.5.
EXAMPLE II
.
The inhib1tory effect of nisin and organic acids against S. typhimurium was determined by the procedure described in Example I.
The results of these experiments are set out in Table 2 (lactic acid) and ~able 3 (propionic acid).
MS #1644 2~ 9~ ~
Ac~i~y o~ nisi~ a~ioYI 17ith 12cl:ic acid ~IA) agz~ 3t ~-. S~=D~ in 10 ~ cit:ra~ b~2er zlt ~elected pll'~
~gl, CPlJ/ml I.og,O Syn~rgy/% over Il/~ in/ redllctior p~ 3.S
0/0 7.08 __ 0~6.4 ~.2~ -0.15 0 0/16 6.81 0.27 0/40 6.15 0.93 0/100 ~2.00 >5.08 0/250 ~2.00 ~.08 1000/0 6.63 0~45 1000/6.4 3.53 3.55 ~/689 1000/'6 6.36 0.72 1000~0 ~.53 1.55 +/12.3 1000/'~0 ~2.00 >5.08 1000~250 ~2.00 >5.0a p~ 4.5 / 7.20 --0/6.4 7.45 -0.25 0/16 7.26 -0.06 0/40 ~.43 -0.23 0/100 7.4a -0.28 0/250 7.28 -0.08 500/0 6.45 0.75 500/6.4 6.56 0.64 500/16 6.60 0.60 500/40 >7.00 <0.2 500/100 ~7.00 <0.2 500/250 >7.00 <0.2 p~ 5.5 0/0 7.34 __ 0/~ . 4 ~.35 -0.02 0/16 7 .4a -0.14 0~40 7.49 -0.15 0/100 7.36 -0.02 0/2S0 7.45 -0.11 4~ 50~0 4.68 2.66 50/6.4 3.g7 3.37 ~/26.7 S0/16 3.79 3.55 +/33~4 50/40 4~34 3.00 ~/12.8 50/100 ~7,00 ~0,3~
50/250 >7.00 ~0.34 MS #1644 -12- ~ ~S5~3~
~ Ct~Yity 0~ n~in i~ ~o~bination ~th propio~ic acid (PAl ag~nst S~ iY~d~ in 10 ~ cit~ate bufr~r a~ ~elected p~'s Tr~atment Lag,~ CF~/~l LoqID Syneryy/% ovor ~/~1 ni~in/ r~duction mH P~
~
p~ 3.5 0/0 6.95 __ Of6.4 6.79 0.16 0/16 6.~6 0.09 0/40 6.99 -0.04 0/lOo 6.20 0.75 0/2S0 ~2.00 >4.95 1000/0 6.40 0.55 1000/6.4 6.29 0.56 1000~16 6.13 0.82 +/28.1 1000/40 5.89 1.06 +/9.27 1000/100 4.g8 1.97 -/79.
1000/250 ~1.00 ~5.95 ~0 p~ 4.5 0/0 ~.26 --0/6.4 7.22 0.04 0/16 7.29 -0.03 0~40 7.28 -0.02 2X 0/100 7.41 -0.15 0/250 7.31 0.05 500/0 6.50 0.76 500/6.4 6.g3 0.73 500/16 6.59 0.67 500/40 >6.00 ~1.26 500~100 >6.00 <1.26 500/250 >6.00 <1.26 p~ 5.5 0/0 7.~2 __ 0/6.4 7.43 -0.01 o/16 7.46 -0.04 0/40 ?.53 -0.11 0/100 7.5g -0.17 0/Z50 7.46 -0.04 4a 500/0 4.55 2087 500/6.4 5~11 2.31 500/16 6.34 1.08 500/40 >6.00 <1.42 00~100 >6.00 <1.42 S00/Z50 >6.0~ <1.~2 MS #1644 The higher concentratLons of lactic and propionic acids alone were bacteriocidal to the test organism a~ pH 3.5. Synergy with lactic acid occurred at all pH levels evaluated at concentrations between 6.4 and 40 mM while propionic acid/nisin synergy occurred only at pH 3.5.
In general monocarboxylic acids of 3-8 carbon atoms, i.e. propionic, butyric, pentanoic, hexanoic, heptanoate and octanoate can bs us~d in conjunction with lantibiotic in order to enhance its effectiveness in controlling Gram negative bacteria.
EXAMPLE I I I
The activity of nisin and potassium sorbate against S~ typhimurium was determined as described in Example I. The results of this experiment are summarized in Table 4. The results of this experiment are set out in Table 4.
MS #1644 ~5~
A~tivity ~ ni~in in ~GmbiD~tia~ ~i~h po~a~iu~
~xb~ K~qORB~TE) ~ga~ns~ S_ $Y5 dh~l UI} in 10 ~ ~itr~t~ burrer at s~l~ted p~'s Tr~tEe~t L~g~O C~l L~gl. Syn~rqy~% ov~r ~/~1 nisin/ reduction ~+gO~13Aq`E
P~ 3.~
0/0 6.91 __ 0 0~0.00256 6.96 -0,05 0/0.0064 6.g2 -0.01 0/0.016 7.00 -o.og 0/0.04 6.47 0.44 0~0.1 ~2.00 >4.91 100/0 6.~8 0.23 100/O.OOZ56 6.65 0~26 +/13 100/0.0064 6.61 0.30 +/30.4 100/0.016 6.69 0.22 LOO/0.04 6.12 0.79 +/243 100/0.1 3.45 3.46 P~ ~.5 0/0 7.01 __ 0/0.002~6 7.05 _0,04 o/0.00~4 7.1g -0.14 0/0.016 7.21 -0.20 0/0.04 7.21 -0.20 0/0.1 7.23 -0.22 50~0 6.~1 0.20 SO/0.00256 6.75 0.26 +/30.0 50~0.0064 >7.00 <1.00 50/0.016 6.81 0.20 50/0.04 >7.00 <1.00 50/0.1 >7.00 ~1.00 p~ 5.5 0/0 7.S8 --O/O.nO256 7.38 0~20 0~0.0064 6.86 0.72 0/0.016 7.17 0.41 0~0.04 7.26 0.32 0/0.1 7.26 0.32 50/0 >7.00 ~0.5~
50/0.00256 >7.00 ~0.58 50/0.0064 >7.00 <0.58 50/0.016 >7.00 <0.58 50/0.04 >7.00 ~0.58 50/0.1 >7.00 <0.58 MS ~1644 -15- ~'5~59~
Potassium sorbate by itself was bacteriocidal at pH 5.5. Synergism with nisin was observed at various concentrations at pH 3.5 and 4.5. Data at pH 5.5 was inconclusive.
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 order to determine the optimal pH range for use of a lantibiotic/synergist system in a particular envi-ronment being treated.
MS #1644
Claims (18)
1. A method for inhibiting the growth of Gram negative bacteria in an environment where their growth is undesirable which method comprises intro-ducing to said environment a synergistically effec-tive combination of a lanthionine bacteriocin and a synergist therefore wherein said synergist is selected from the group consisting 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, and sorbic acid or a suitable alkali metal or alkaline earth metal salt thereof.
2. The method of Claim 1 wherein the amino acid is glycine, the aliphatic organic acid is lactic or propanoic acid and the sorbic acid salt is sodium sorbate.
3. The method of Claim 1 wherein the lanthionine bacteriocin is nisin, subtilin, pep 5, epidermin, gallidermin, cinnamycin, Ro09-0198, duramycin or ancovenin.
4. The method of Claim 1 wherein the lanthionine bacteriocin is nisin.
5. The method of Claim 1 wherein the Gram negative bacteria is from the species Salmonella typhimurium.
MS #1644
MS #1644
6. The method of Claim 1 wherein the lanthionine bacteriocin is introducad 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, and c) 0.001 to 2% for sorbic acid and salts thereof.
a) 0.001 - 5% for amino acids, b) 0.001 - 25% for aliphatic mono- and di-carboxylic organic acids, and c) 0.001 to 2% for sorbic acid and salts thereof.
7. 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) 0.02 to 0.3% for sorbic acid and salts thereof.
a) 0.03 - 0.1% for amino acids, b) 0.01 - 1% for aliphatic mono- and di-carboxylic acids, c) 0.02 to 0.3% for sorbic acid and salts thereof.
8. A method of inhibiting the growth of Salmonella typhimurium in an environment where its growth is undesirable which method comprises introducing to said environment a synergistically effective combi-nation of nisin and a synergist therefore selected MS #1644 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 of alkaline earth metal salt thereof and sorbic acid or a suitable alkali metal or alkaline earth metal salt thereof.
9. The method of Claim 8 wherein the syneryist is glycine.
10. The method of Claim 9 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 glycine is introduced at a level of from 0.001 to 5 weight percent.
11. The method of Claim 10 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.
12. The method of Claim 8 wherein the aliphatic, mono- or di- carboxylic acid is lactic or propionic acid.
13. The method of Claim 12 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 car-boxylic acid is introduced in an amount of from 0.001 to 25 weight percent.
14. The method of Claim 13 wherein the nisin is introduced in an amount of from 50 to 500 IU/gm and MS #1644 the carboxylic acid is introduced at a level of from 0.01 to 1 weight percent.
15. The method of Claim 8 wherein the sorbic acid salt is potassium sorbate.
16. The method of Claim 15 wherein the nisin is introduced in an amount of from 0.1 to 2,000 IU/gm of the environment being treated and the potassium sorbate is introduced in an amount of from 0.001% to 2% 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 potassium sorbate is introduced in an amount of from 0.02% to 0.3% weight percent.
18. A solid or liquid product suitable for ingestion by humans or other animals which comprises an ingestible substrate containing a synergistically effective Gram negative bacteria inhibiting combina-tion 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 and sorbic acid or a suitable alkali metal or alkaline earth metal salt thereof.
MS #1644
MS #1644
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63239790A | 1990-12-21 | 1990-12-21 | |
| US632,397 | 1990-12-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2055984A1 true CA2055984A1 (en) | 1992-06-22 |
Family
ID=24535369
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002055984A Abandoned CA2055984A1 (en) | 1990-12-21 | 1991-11-21 | Synergistic effect of lantibiotics in combination with selected agents against gram negative bacteria |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPH04295431A (en) |
| CA (1) | CA2055984A1 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994005251A1 (en) * | 1992-09-10 | 1994-03-17 | Sara Lee/De N.V. | Mouth-care products with anti-bacterial activity |
| 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 |
| 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 |
| EP1238591A1 (en) * | 2001-03-05 | 2002-09-11 | Nutrinova Nutrition Specialties & Food Ingredients GmbH | Preparation which contains sorbic acid and bacteriocins as a feed additive in animal raising |
| WO2004110425A1 (en) * | 2003-06-12 | 2004-12-23 | Nutrinova Nutrition Specialties & Food Ingredients Gmbh | Use of sorbic aid for prevention of bacterial infections and as performance enhancer for animals |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1629724A1 (en) | 2004-08-27 | 2006-03-01 | PURAC Biochem BV | The use of glycine and/or a glycine derivative as antibacterial agent in foods and/or drinks |
-
1991
- 1991-11-21 CA CA002055984A patent/CA2055984A1/en not_active Abandoned
- 1991-12-19 JP JP3353962A patent/JPH04295431A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994005251A1 (en) * | 1992-09-10 | 1994-03-17 | Sara Lee/De N.V. | Mouth-care products with anti-bacterial activity |
| 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 |
| 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 |
| EP1238591A1 (en) * | 2001-03-05 | 2002-09-11 | Nutrinova Nutrition Specialties & Food Ingredients GmbH | Preparation which contains sorbic acid and bacteriocins as a feed additive in animal raising |
| DE10110431A1 (en) * | 2001-03-05 | 2002-09-19 | Nutrinova Gmbh | Bacteriocin-containing sorbic acid preparation as a feed additive in livestock rearing |
| US6780447B2 (en) | 2001-03-05 | 2004-08-24 | Nutrinova Nutrition Specialties & Food Ingredients Gmbh | Bacteriocin-containing sorbic acid product as addition to feedstuffs in agricultural livestock rearing |
| WO2004110425A1 (en) * | 2003-06-12 | 2004-12-23 | Nutrinova Nutrition Specialties & Food Ingredients Gmbh | Use of sorbic aid for prevention of bacterial infections and as performance enhancer for animals |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04295431A (en) | 1992-10-20 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 19940521 |