JPH04295431A - Synergistic effect of lunch biotics used in combination with selected medicine on gram-negative bacterium - Google Patents

Abstract

PURPOSE: To provide a method for synthetically effectively controlling the growth of gram-negative bacteria such as Salmonella typhimurium with lantinonine bacteriocin including nisin and other synergists. CONSTITUTION: There is disclosed a method for inhibiting the growth of undesired gram-negative bacteria with lanthionine bacteriocin in combination with a substance selected from amino acids such as glycine, 1-8C aliphatic mono-or-di- carboxylic acids such as lactic acid and propionic acid, sorbic acid and their salts as a synergist for the lanthionine bacteriocin.

Description

[Detailed description of the invention]

0001

[Prior art] Lanthionine bacteriocin (lan
Nisin, a member of the class of antibacterial agents known as thionin bacteriocin,
is) [formerly Streptococcus lactis (St
It is an antibacterial polypeptide produced from a strain of Streptococcus lactis (reptococcus lactis). It is produced by fermentation with pure cultures of these bacteria followed by purification and drying. FoodTec
hnol. 1988 42: (4) 169-171, M. Doyle is L. describes the inhibitory effects of nisin on the growth and survival of P. monocytogenes.

[0002] “Lantibiotics”
The term ``otics'' was coined by Schnell et al. [1988 Nature Nature) 333:2
76-278]. This class of bacteriocins includes subtilin, pep5, epidermin,
gallidermin, cinnamycin, Ro09-0198,
Contains duramycin and ancovenin. These ribosomally synthesized polypeptide bacteriocins contain 19 to 34 amino acids and are synthesized by a variety of microorganisms, including Staphylococcus sp., Bacillus sp., and Streptomyces sp. produced.

Combinations of nisin with other substances have been reported to enhance its antibacterial activity. Thus, international patent application WO 89/12399 describes the combination of nisin with chelating agents and/or surfactants for use against Gram-positive and Gram-negative bacteria.

Published European Patent Application No. 0,384,3
No. 19 is L. discloses the synergistic effect of nisin and chelating agents against S. monocytogenes. This document discloses the use of certain amino acids and monocarboxylic acids in combination with nisin.

[0005]

[Problems to be Solved by the Invention] Numerous documents describe synergistic combinations of nisin and other ingredients as being effective against Gram-positive bacteria; There are few reports regarding the activity of nisin against Gram-negative bacteria when used in combination with nisin.

[0006]

SUMMARY OF THE INVENTION The present invention includes a method for inhibiting the growth of Gram-negative bacteria in an environment where such growth is undesirable; Aliphatic mono- and diorganic carboxylic acids containing 8 carbon atoms or their suitable alkali metal or alkaline earth metal salts, phenolic antioxidant antibacterial agents, benzoic acid or sorbic acid and their suitable alkali metal or alkali comprising introducing a synergistically effective combination of lanthionine bacteriocin synergists selected from the group consisting of earth metal salts.

The present invention provides compositions in which a lanthionine bacteriocin in combination with certain additives inhibits the growth of undesirable Gram-negative bacteria to a greater extent than the bacteriocin itself, and the combination of these substances. This is related to the observation that the inhibitory effect is greater than the additive effect observed when using lantibiotics or other compositions individually.

Various classes of compositions that act synergistically with lantibiotics have been identified. Among these, mention may be made inter alia of amino acids and especially glycine.

Another class of agents that have been found to produce synergistic antimicrobial effects in combination with lantibiotics are aliphatic mono- and dicarboxylic acids containing from 1 to 8 carbon atoms. These acids are lactic acid, propionic acid,
Contains acetic acid, malic acid, tartaric acid, succinic acid, maleic acid, oxalic acid and fumaric acid. Suitable salts can be used, such as alkali and alkaline earth metal salts, such as sodium, potassium and calcium salts. Sorbic acid and its appropriate salts also exhibit synergistic activity against Gram-negative bacteria when used in combination with lantibiotics such as nisin.

Although the mechanism of action of lantibiotics such as nisin is not understood, many researchers have linked the lethality of these peptides to irreversible changes in the cell membranes of susceptible organisms. . The resistance of some microorganisms to nisin is believed to be due to lack of proximity of the peptide to the site of action, either through physical exclusion or lack of a suitable acceptor molecule. The lantibiotic synergistic effect of the present invention appears to be to alleviate proximate inhibition on the target microorganism, although its mechanism of action is not yet understood. The present invention demonstrates that the use of lantibiotics and certain synergists in combination inhibits the growth of unwanted Gram-negative bacteria to a greater extent than the lantibiotics themselves, and that the combined inhibitory effect of these substances It is based on the discovery that tics or synergists are greater than the additive effects seen when used individually.

[0011] Lantibiotics are generally considered to be ineffective against Gram-negative bacteria in the absence of synergists. In the aforementioned WO 89/12399, chelating agents and surfactants are described as suitable synergists for controlling Gram-negative bacterial species. It has been newly discovered that amino acids and certain mono- and diorganic carboxylic acids and sorbic acid and their salts act as synergists that make lantibiotics such as nisin effective against Gram-negative bacteria. It was done. In a typical example, when using a lantibiotic such as nisin and synergists therefor, the total amount of nisin is typically about 0.1 IU per gram of substrate material being treated. (or 0.1 IU per ml in liquid systems) to 2,000 IU or 1 m in liquid systems
It ranges from around 2,000 IU per liter. For most uses, a concentration of nisin in the range 50 to 500 IU/g is sufficient, depending on the enhancing effect received from the synergist. The term IU used in the text is from J.
Sci. Food and Agriculture. , 15 (8
) 522 (1964)
r) and international units as measured by Fowler's method of analysis. The amount of synergist used will depend on the particular composition involved. The amount of synergist, generally expressed as a weight percent of the substrate being treated, is shown in Table A.
within the range listed. Although some routine experimentation may be required to determine the concentration of a particular lantibiotic-acid synergist for optimal synergy, lanthionine bacteriocins other than nisin are
It can be considered effective at the indicated concentrations.

0012

[Table 1] Table A Class of synergist Suggested range of use Preferred range Amino acid
0.001%-5%
0.03%-0.1% aliphatic mono-
0.001%-25% 0.01%-1%
and di-carboxylic acid sorbic acid & salt 0.001%-2
% 0.02%-0.3% Surface treatment of the target substrate can be carried out by suspending the material to be treated in a lanthionine/synergist solution. Alternatively,
The material can also be dipped into the solution or sprayed onto the surface of the material. The lanthionine/synergist combination can be incorporated into a film or gel that is applied to the surface of a substrate, or in the environment in which it is processed, such as by adding the combination to milk used in cheese production. Can be directly mixed with. For use in film coatings such as dips, films, gels or casings, the lantibiotics/synergist is added to the final product so that after coating and spreading the final product contains a residual concentration of lantibiotic/synergist within the desired range. The initial concentrations of the biotic and synergist can be close to the solubility limit. Treating the surface of the material may require some routine experimentation to ascertain the most effective concentration.

Environments that can be treated with the compositions of the invention include meat and meat products such as chicken, turkey, ham, beef, salami, sausages and smoked meat products; mayonnaise; cheese, milk and yogurt. dairy products; oils, fish and seafood products; soft drinks, including natural juices; animal feed and other high protein products. In addition to their use in foods, the compositions of the present invention have non-food uses such as mouth rinses, denture cleaners, ointments, creams and shampoos.

Among the problematic Gram-negative bacteria, Salmonella typhimurium (Salmonella ty-p.
himurium) is particularly harmful due to its universal contamination, especially in new poultry. Other undesirable Gram-negative bacteria, the growth of which can be inhibited by the synergistic combinations of the present invention, include S. enteriditis.
) and other species of Salmonella such as Shigella
era) genus and Camylobacter (Camylobacter)
er) species and genera from the genus Echelchia coli (Esh
Erichia coli).

The invention is further illustrated by the following examples, in which the international unit of nisin is expressed as U nisin. Nisin in these studies was produced by diafiltration (diafiltration), which yields an essentially salt-free preparation.
filtration) and ultrafiltration.
Aplin & Barrett].

[0016]

[Example 1] Salmonella Typhimurium ATCC14
The synergistic bactericidal activity of nisin and glycine against 028 was determined in 10 mM citrate buffer (CAB) at pH
It was evaluated as 3.5, 4.5 and 5.5. Nisin has glycine concentrations of 6.4, 16, 40, 100 and 250 m
M was tested at 500 and 1000 IU/ml. Control tests were conducted in CAB. All stock solutions, culture dilutions and treatments were prepared in CAB at the evaluated pH. The treatment solution is 0.9m in a 1ml micro test tube.
It was prepared in an amount of l. Processing time is approximately 1×107CFU/
0.1 ml of S. ml in its growth phase. It was started with the addition of Typhimurium. The treatment time was 60 minutes at 37°C. Next, the processing solution was poured into a microtiter (
Using a phosphate buffered saline (microtiter) plate
0.01M, pH 7.0). The viable bacteria were counted after 48 hours at 37°C on trypsin-digested soybean agar spread plates.
(ead plates). The results of this experiment are displayed in Tables 1 and 2, where it was observed that the bactericidal efficacy of the combination of test compound and nisin was greater than the additive log10 reduction of the individual components of the combination. Sometimes a "+" is used to identify the amount of increase in bactericidal activity as "% over." The following formula was used where T=test compound alone, N=nisin alone and T+N=combination.

log10 reduction T + N > (log10 reduction T + log10 reduction N) = synergy For the purposes of the calculations above, any negative logarithmic reduction is 0
It was assumed that Estimates reported to be greater than (>) or less than (<) are above the detection limits of the system, and estimates other than in obvious cases, e.g. in the case of the smallest possible effect, are It was synergistic.

The results of this experiment are summarized in Table 1, in which the unit of nisin is expressed as U nisin.

[0019]

Table 2 Table 1 S in 10mM citrate buffer at specified pH
．． Active treatment method of nisin in combination with glycine (Gly) against Typhimurium
LOG10 LOG10 decrease
Synergy/Excess %U/ml Nisin/C
FU/mlmM Gly

p
H3.5 0/0 5.30
--0/6.4
5.34 -0.040
/16 5.48
-0.180/40
6.00 -0.700/
100 6.34
-1.040/250
4.50 0.801000
/0 3.95
1.351000/6.4 3.8
0 1.50
+/11.11000/16 4.81
0.491000/40
4.54 0.7
61000/100 4.04
1.261000/250 1
．． 78 3.52
+/63.7 pH4.5 0/0 7.45
--0/6.4
7.43 0.02
0/16 7.48
-0.030/40
7.49 -0.040
/100 >7.48
<-0.030/250
7.40 0.05500/0
5.73
1.72500/6.4 6.
28 1.17
500/16 5.99
1.46500/40
6.00 1.4550
0/100 6.34
1.11500/250 5.
98 1.47 pH5
．． 5 0/0 7.65
--0/6.4
>7.48 <0.170/
16 >7.48
<0.170/40
7.53 0.120/1
00 7.36
0.290/250
7.38 0.27500/
0 3.08
4.57500/6.4 3
．． 30 4.35
500/16 3.23
4.42500/40
2.91 4.74
+/1.07500/100
2.80 4.8550
0/250 2.34
5.31 +/9.7 high concentrations of glycine caused S.D. at all three pH values. It was bactericidal against Typhimurium. Detectable synergy with nisin occurred at pH 3.5 and 5.5.

[0020]

[Example 2] S. The inhibitory effect of nisin and organic acids on Typhimurium was determined by the procedure described in Example 1.

The results of these experiments are displayed in Table 2 (lactic acid) and Table 3 (propionic acid).

[0022]

Table 3 Table 2 S in 10mM citrate buffer at specified pH
．． Active treatment method of nisin in combination with lactic acid (LA) against Typhimurium
LOG10 LOG10 decrease
Synergy/excess %U/ml nisin/CFU/
mlmM LA

pH3
．． 5 0/0 7.08
--0/6.4
7.23 -0.150
/16 6.81
0.270/40
6.15 0.93
0/100 <2.00
>5.080/250
<2.00>5.081000/
0 6.63
0.451000/6.4 3.53
3.55 +
/6891000/16 6.36
0.721000/40
5.53 1.55
+/12.31000/100
<2.00 >5.08
1000/250 <2.00
>5.08 pH4.5 0/0 7.20
--0/6.4
7.45 -0.250
/16 7.26
-0.060/40
7.43 -0.230/
100 7.48
-0.080/250
7.28 -0.08
500/0 6
．． 45 0.75500/6.
4 6.56
0.64 500/16
6.60 0.605
00/40 >7.00
<0.2500/100 >7.00
<0.2500/250
>7.00 <0.2 pH
5.5 0/0 7.34
--0/6.4
7.36 -0.020
/16 7.48
-0.140/40
7.49 -0.150/
100 7.36
-0.020/250
7.45 -0.11500/0
4.68
2.66500/6.4 3.
97 3.37
+/26.7500/16 3.
79 3.55
+/33.4500/40 4.
34 3.00
+/12.8500/100 >7.00
<0.34500/250
>7.00 <0.34

0
023]

Table 4 Table 3 S in 10mM citrate buffer at specified pH
．． Active treatment method of nisin in combination with propionic acid (PA) against Typhimurium
LOG10 LOG10 decrease Synergy/excess %U/ml nisin/
CFU/mlmM PA


pH3.5 0/0 6.95
--0/6.4
6.79 0.16
0/16 6.86
0.090/40
6.99 -0.04
0/100 6.20
0.750/250
<2.00 >4.95100
0/0 6.40
0.551000/6.4 6.
29 0.661000/16
6.13 0
．． 82 +/28.11000/40
5.89 1.
06 +/9.271000/100
4.98 1.97
+/79.11000/250
<1.00 >5.95
pH4.5 0/0 7.26
--0/6.4
7.22 0.04
0/16 7.29
-0.030/40
7.28 -0.020
/100 7.41
-0.150/250
7.31 -0.05
500/0
6.50 0.76500/6
．． 4 6.53
0.73 500/16
6.59 0.67
500/40 >6.00
<1.26500/100>6.
00 <1.26500/250
>6.00 <1.26
pH5.5 0/0 7.42
--0/6.4
7.43 -0.010
/16 7.46
-0.040/40
7.53 -0.110/
100 7.59
-0.170/250
7.46 -0.04500/0
4.55
2.87500/6.4 5.
11 2.31500/16
6.34
1.08 500/40
>6.00 <1.42500/
100 >6.00 <
1.42500/250 >6.00
<1.42 High concentrations of lactic acid and propionic acid alone were bactericidal against the test microorganisms at pH 3.5. Synergy with lactic acid occurred at all pH values at concentrations between 6.4 and 40 mM, whereas propionic acid/nisin synergy was only seen at pH 3.5.

Monocarboxylic acids, generally of 3-8 carbon atoms,
Thus, propionic acid, butyric acid, pentanoic acid, hexanoic acid, heptanoate and octanoate can be used with lantibiotics to increase their effectiveness in controlling Gram-negative bacteria.

[0025]

[Example 3] S. The activity of nisin and potassium sorbate against Typhimurium was determined as described in Example 1. The results of this experiment are summarized in Table 4, and the results obtained in this experiment are displayed in Table 4.

[0026]

Table 5 Table 4 S in 10mM citrate buffer at specified pH
．． Potassium sorbate (K+S) against Typhimurium
Nisin activation treatment method combined with ORBATE)
LOG10
LOG10 decrease Synergy/excess %U/m
Nisin/CFU/mlK+SORBATE


pH3.5 0/0 6.91
--0/0.00256
6.96 -0.050/0
．． 0064 6.92
-0.010/0.016
7.00 -0.090/0.04
6.47
0.440/0.1 <
2.00 >4.91100/0
6.68
0.23100/0.00256 6.65
0.26 +/1
3100/0.0064 6.65
0.30 +/30.41
00/0.016 6.69
0.22 100/0.04
6.12 0
．． 79 +/243100/0.1
3.45 3
．． 46 pH4.5 0/0 7.01
--0/0.00256
7.05 -0.040/0
．． 0064 7.15
-0.140/0.016
7.21 -0.200/0.04
7.21
-0.200/0.1
7.23 -0.2250/0
6.81
0.2050/0.00256 6.7
5 0.26 +
/30.050/0.0064 >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 pH5.
5 0/0 7.58
--0/0.00256
7.38 0.200/
0.0064 6.86
0.720/0.016
7.17 0.410/0.
04 7.26
0.320/0.1
7.26 0.3250
/0 >7.00
<0.5850/0.00256 >7
．． 00 <0.5850/0.006
4 >7.00 <0.58
50/0.016 >7.00
<0.5850/0.04
>7.00 <0.5850/0.1
>7.00
<0.58 Potassium sorbate itself is bactericidal at pH 5.5. Synergy with nisin was observed at various concentrations at pH 3.5 and 4.5. Data for pH 5.5 were uncertain.

The pH range in which the synergism is particularly effective depends on the environment in question. Therefore, even if a particular synergist exhibits activity in a particular pH range, it may
Sometimes a range is optimal. For this reason, lantibiotics/
A certain amount of routine experimentation will be required to determine the optimal pH range for use in the synergist system.

The main features and aspects of the invention are as follows.

1. A method for inhibiting the growth of Gram-negative bacteria in an environment in which the growth of Gram-negative bacteria is undesirable, the method comprising: containing a lanthionine bacteriocin and amino acids, aliphatic mono- and diorganic carbon atoms containing from 1 to 8 carbon atoms; Synergy of the lanthionine bacteriocin with a synergist selected from the group consisting of acids or their suitable alkali metal or alkaline earth metal salts, and sorbic acid and its suitable alkali metal or alkaline earth metal salts. 1. A method comprising introducing a combination that is clinically effective.

2. 2. The method according to 1 above, wherein the amino acid is glycine, the aliphatic organic acid is lactic acid or propionic acid, and the sorbate is sodium sorbate.

3. The method according to 1 above, wherein the lanthionine bacteriocin is nisin, subtilin, pep5, epidermin, gallidermin, cinnamycin, Ro09-0198, duramycin, and ancovenin.

4. The method according to 1 above, wherein the lanthionine bacteriocin is nisin.

5. The method according to 1 above, wherein the Gram-negative bacterium is a bacterium derived from Salmonella typhimurium species.

6. Lanthionine 0.1 to 2,000 I per gram of the environment in which the bacteriocin is processed
The amount of synergist introduced in the amount of U and expressed in weight % of the environment is: a) for amino acids 0.001-5% b) for aliphatic mono- and diorganic carboxylic acids 0.001- 25
%, and c) In the case of sorbic acid and its salts: The method according to 1 above, wherein sorbic acid and its salts are introduced at 0.001 to 2%.

7. Lanthionine bacteriocin 50
introduced at a concentration of from 500 IU/g to which the synergist is expressed in weight % of the environment to be protected: a
) for amino acids 0.03-0.1%b) for aliphatic mono- and diorganic carboxylic acids 0.01-1%,
and c) In the case of sorbic acid and its salts 0.02 to 0
．． The method according to claim 1, wherein the method is introduced at a concentration of 3%.

8. A method for inhibiting the growth of Salmonella typhimurium in an environment in which the growth of Salmonella is undesirable, the method comprising: comprising a lanthionine bacteriocin and an amino acid, an aliphatic mono- and diorganic carboxylic acid containing 1 to 8 carbon atoms, or a synergistically effective agent of the lanthionine bacteriocin selected from the group consisting of sorbic acid and a suitable alkali metal or alkaline earth metal salt of A method comprising introducing a combination.

9. 9. The method according to 8 above, wherein the synergist is glycine.

10. Nisin is introduced in an amount of 0.1 to 2,000 IU per gram of the environment being treated;
and glycine is introduced at a concentration of 0.001 to 5% by weight.

11. Nisin 50 to 500 IU/
g, and the amino acids are introduced in an amount of 0.03 to 0.g.
11. The method according to claim 10, wherein the method is introduced at a concentration of 1% by weight.

12. 8. The method according to 8 above, wherein the aliphatic mono- and dicarboxylic acid is lactic acid or propionic acid.

13. Nisin is introduced in an amount of 0.1 to 2,000 IU per gram of the environment being treated;
and the carboxylic acid is introduced in a concentration of 0.001 to 25% by weight.

14. Nisin 50 to 500 IU/
g, and the carboxylic acid is introduced in an amount of 0.01 to 1
14. The method according to claim 13, wherein the method is introduced at a concentration of % by weight.

15. 9. The method according to 8 above, wherein the sorbate is potassium sorbate.

16. Nisin is introduced in an amount of 0.1 to 2,000 IU per gram of the environment being treated;
and potassium sorbate are introduced in a concentration of 0.001 to 2% by weight.

17. Nisin 50 to 500 IU/
g, and potassium sorbate was introduced in an amount of 0.02 g.
17. The method according to claim 16, wherein the method is introduced at a concentration of 0.3% to 0.3% by weight.

18. lanthionine bacteriocin,
The class consisting of amino acids, aliphatic mono- and diorganic carboxylic acids containing 1 to 8 carbon atoms or their suitable alkali metal or alkaline earth metal salts, and sorbic acid and its suitable alkali metal or alkaline earth metal salts. suitable for ingestion by humans and other animals, comprising a synergistically effective Gram-negative bacteria inhibiting combination with a lanthionine bacteriocin synergist selected from solid or liquid products.

Claims (3)

[Claims] 1. A method for inhibiting the growth of Gram-negative bacteria in an environment where the growth of Gram-negative bacteria is undesirable, the method comprising: a lanthionine bacteriocin; an amino acid; an aliphatic monomer containing 1 to 8 carbon atoms; - and a diorganic carboxylic acid or a suitable alkali metal or alkaline earth metal salt thereof, and sorbic acid and a suitable alkali metal or alkaline earth metal salt thereof; A method comprising introducing a synergistically effective combination with an agent. 2. A method of inhibiting the growth of Salmonella typhimurium in an environment in which the growth of Salmonella enterica is undesirable, the method comprising: a lanthionine bacteriocin; an amino acid; an aliphatic mono-containing from 1 to 8 carbon atoms; a synergist for the lanthionine bacteriocin selected from the group consisting of diorganic carboxylic acids or their suitable alkali metal or alkaline earth metal salts; and sorbic acid and its suitable alkali metal or alkaline earth metal salts. A method comprising introducing a synergistically effective combination of. 3. Lanthionine bacteriocin and amino acids, aliphatic mono- and diorganic carboxylic acids containing 1 to 8 carbon atoms or their suitable alkali metal or alkaline earth metal salts, and sorbic acid and its suitable alkalis. an ingestible carrier comprising a synergistically effective Gram-negative bacteria inhibiting combination with a lanthionine bacteriocin synergist selected from the group consisting of metal or alkaline earth metal salts. , solid or liquid products suitable for consumption by humans and other animals.