CA1340850C - Nisin compositions for use as enhanced, broad range bactericides - Google Patents

Abstract

Bacteriocin compositions comprising lanthionine containing bacteriocins and non-bactericidal agents.
When the bacteriocin compositions are combined with a suitable carrier with each component present in sufficient quantities such that the composition is effective against Gram negative bacteria in addition to Gram positive bacteria, they become enhanced, rapid acting, broad range bactericides suitable for a variety of applications.

Description

1340~~~1 Nisin is a polypeptide with antimicrobial properties wh:Lch is produced in nature by various strains of the bacterium Streptococcus lactis. It is a known food preservative which inhibits the outgrowth of spores of certain species of Gram positive Bacilli.
Although sometimes mistakenly and imprecisely referred to as an antibiotic, nisin is more correctly classified as a bacaeriocin, i.e. a proteinaceous substance produced by bacteria and which has anti-bacterial activity only towards species closely related to the species of its origin. Nisin is a naturally-occurring preservative found in low concen-tration in milk and cheese, and is believed to be completely no:n-toxic and non-allergenic to humans.
Nisin hays recently been recognized as safe by the FDA as a direct food ingredient in pasteurized cheese spread, pasteurized processed cheese spread, and pasteurized o.r pasteurized processed cheese spread with fruits, ~regetables, or meats. Furthermore, since it is a polypcsptide, any nisin residues remaining in foods are quickly digested.
A summar5r of nisin's properties appears in Hurst, A., Advances in Applied Microbiolo y 27:85-123 (1981). This publication describes what is generally known about nisin. Nisin, produced by Streptococcus lactis, is available commercially as an impure preparation, rfisapl.in'", from Aplin & Barrett Ltd., Dorset, England and can be obtained by isolating naturally-occurring nisin from cultures of Streptococcus lacti;a and then concentrating the nisin 2 1340~~U
according to known methods. There are also reported methods for producing nisin using altered strains of Streptococcus. See Gonzalez et al., U.S. Pat. No.
4,716,115, :issued December 29, 1987. It should also be possible to produce nisin by recombinant DNA
technology.
Nisin has been applied effectively as a preserva-tive in dairy products, such as processed cheese, cream and milk. '.the use of nisin in processed cheese products hay; been the subject of recent patents. See U.S. Pat. Nos. 4,..'i84,199 and 4,597,972. The use of nisin to inhibit the growth of certain Gram positive bacteria has, been well documented. However, its complete success and acceptance as a food preservative has heretofore been hampered by the belief that nisin was ineffective against Gram negative and many Gram positive bacteria. Gram negative bacteria are almost always present in conjunction with Gram positive bacteria and are a. major source of food spoilage and contamination. See Taylor, U.S. Pat. No. 5,584,199, issued April 22, 1986 and Taylor, U.S. Pat. No.
4,597,972, issued July 1, 1986; Tsai and Sardine, "Conjugal Tr,~nsfer of Nisin Plasmid Genes from Streptococus Lactis 7962 to Leuconostoc Dextranicum 181, Applied and Environmental Microbiolo y, Feb.
1987, p. 352; "A Natural Preservative," Food Engineering :Cnt'1" May 1987, pp. 37-38; "Focus on Nisin," Food Manufacture, March 1987, p. 63.
Summary of the Invention It has now been found that contrary to prior teaching, compositions comprising nisin, in combina-tion with various non-bactericidal agents have enhanced, broad range bactericidal activity against Gram negatives bacteria as well as enhanced activity against a broader range of Gram positive bacteria than nisin alone. The s,nhanced bactericidal activity against Gram positive bacteria occurs in a pH range -3- 1340~~U
broader than previously taught. The invention provides bacteriocin compositions of nisin or other, lanthionine containing bacteriocins, in combination with various non-bactericidal agents for example chelating agents or surfactants. A preferred aspect of the invention further provides the compositions dissolved or suspended in a suitable carrier to yield enhanced broad rangE~ bactericides.
Detailed Description of the Invention Specifically, it has been found that a solution of about 0.1 ug/ml to 300 ug/ml of nisin in the presence of about 0~.1 mM to 20 mM of a chelating agent, for example EDTA, virtually eliminates the growth of Gram negative bacteria such as Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Bacterioides yin ivalis, Actinobacillus actinomycetes-comitans, and Klebsiella pneumonia_e and is more active towards Gram ~?ositive bacteria such as Staphylococcus aureus, Strepi~ococcus mutans, Listeria monocytogenes Streptococcus a alactiae and Coryneform bacteria than nisin alone. Although the enhancement of nisin activity by chelator was concentration dependent, contrary to e~cpectations, concentrations of EDTA in excess of 20mr4 were inhibitory to the bactericidal activity of n:Lsin. However, in the presence of a proteinaceous carrier, and polyvalent polymers such as serum albumin, collagen, gelatin, casein and keratin, the inhibition of nisin by concentrations of EDTA
above 20mM was: significantly reduced, thereby extending the usefu:L range of EDTA enhancement of nisin.
It has also bean found that a solution of about 0.1 ug/ml to 300 l~g,im1 nisin and about 0.1 mM to 20 mM
of a chelating~ ageni~ will further enhance the effec-tiveness of nisin acLainst Gram negative and Gram positive bacteria in the presence of about 0.01% to 1.0% of surfactant. Additionally, it has been found Y..,~...

13408~~

that, in the presence of surfactant alone, nisin has enhanced activity against Gram positive bacteria.
In the present invention, suitable chelating agents include, but are not limited to, EDTA, CaEDTA, CaNa2EDTA, <~nd other alkyldiamine tetraacetates, EGTA
and citrate., Surfactants, valuable as cleansing agents, suil~able :for combination with nisin, with or without EDT~~, inc:Lude, but are not limited to, the nonionic surfactants Tweens, Tritons, and glycerides, ionic surfacaants such as fatty acids, quaternary compounds, anionic: surfactants such as sodium dodecyl sulphate and amphoteric surfactants such as cocamidopro~~yl bet:aine and emulsifiers.
Since Gram positive and Gram negative bacteria are almost always found together in foods, the effectiveness of t:he nisin compositions towards Gram negative bacteria such as Salmonella ty himurium, Escherichia Coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacterioides gingivalis, Actinobacillus actinomycetescomit:ans, and other Gram negative pathogens and Gram positive bacteria will be of great use. The bactericides are particularly suited for the control and prevention of contamination of raw ingre-dients, processed foods and beverages by bacterial pathogens and other microbial spoilage organisms.
Potential fo~~d related uses include treatment of meats, especially poultry, eggs, cheese and fish and treatment of food packaging and handling equipment.
Further uses include as food preservative, such as in processed cheese, .cream, milk, dairy products and in cleaning pou:Ltry, fish, meats, vegetables, and dairy and food processing equipment. The use of the nisin compositions should not be limited to food related uses and the nisin compositions should be useful in any situation in which there is a need or desire to eliminate Gram negative and Gram positive bacteria.
The com~~ositions can be dissolved in a suitable carrier for example: an aqueous solvent or buffer or _5- f 34(~8~~1 suspended i:n any suitable liquid, colloidal or poly-meric matri:K to create bactericides. The compositions or bactericides can be incorporated into ointments or coatings for medicinal uses such as the treatment of infections, wound dressings or surgical implants and as a broad ;spectrum disinfectant for skin or oral rinses, dis:Lnfect~~nt scrubs, wipes or lotions. The bactericide:a can be used for cleaning medical instru-ments, in pre-operative surgical scrubs and the like.
The bactericides are particularly useful in circum-stances where environmental disinfection is desired but where chemical germicidals are precluded because of the risk; of corrosive or otherwise toxic residues.
Unlike the acaivity of most broad spectrum germi-cidals which. is compromised by the presence of complex organic matter, the compositions of the present inven-tion are effective as bactericides in the presence of organic matter, such as milk or serum.
Nisin was known to optimally inhibit the growth of a few closely related Gram positive bacteria, particularly certain Gram positive spore forming bacilli at pH 5Ø The bactericidal activity of nisin in solution with a chelating agent was surprisingly rapid and greatly enhanced towards a broad range of Gram positive bacteria at pH values greater than pH

5.0, and, moreover, was activated towards Gram nega-tive bacteri~~ at both acidic and basic pH, preferably in the range pH 5.0 to 8Ø This unexpectedly rapid and broad-ranged bactericidal activity of chelator-activated nisin makes it suitable for use as, among other things,, a disinfectant.
Nisin bE:longs to the class of peptide bacterio-cins containing lanthionine. Also included among that class are subtilin,, epidermin, cinnamycin, duramycin, ancovenin and Pep Vii. These bacteriocin peptides are each producef. by dpfferent microorganisms. However, subtilin obtained from certain cultures of Bacillus subtilis, and epidermin obtained from certain cultures of Staphylococcus :epidermidis, have been found to have molecular structures very similar to that of nisin (see Hurst, pp. 85~-86, and Schnell et al., Nature, 333:276-278). It .is therefore believed that because of the molecular similarities, other lanthionine containing peptide bacteriocins will be equally as effective as nisin in combination with chelating agents and non-ionic surfactants in eliminating Gram negative and Gram ~~ositive bacterial contaminations.
The effectiveness of the nisin, and by extension other lanthionine containing peptide bacteriocin, compositions as bactericides against Gram negative bacteria is ~;urpri:~ing, since the prior art generally teaches away from i~his activity of nisin. The enhanced activity of nisin against Gram positive bacteria in the prE~sence of EDTA at a pH greater than 5.0 is unexpected since it was previously believed that nisin activit~r is optimal at pH 5Ø Further-more, the discovery of such effectiveness of the nisin and lanthionine containing peptide bacteriocin compo-sitions as bactericides fulfills a long-felt need in the science of food preservation, which has suffered from the absence of: an acceptable, natural, non-toxic agent effective against a broad range of bacteria.
In order to demonstrate the superior and unexpected rapid acaivity of the composition con-taining nisin, EDT~1, and/or various surfactants against both Gram negative and Gram positive bacteria, a num-ber of experiments were conducted with the bacteri-cides. These experiments are meant as illustration and are not intended to limit this invention. It is to be expected that other, lanthionine containing peptide bacteriocins would be effective substitutes for nisin and that chelating agents other than EDTA
will be effective substitutes for EDTA.
All tests in t:he following examples were per-formed at 3?oc:. The efficacy of the enhanced broad range bactericides was determined by assaying -'- 13~~8~~
bactericidal activity as measured by the percent bacterial survival after treatment with the bacteri-cide. Generally, after incubation of a 107 cell per ml suspension of target species with the novel bactericide for spE;cified lengths of time, bacteria were collected by <:entrifugation for 2 minutes. The bacterial pellet was washed free of the bactericide with a rescue buffE:r, termed herein Phage buffer (50mM
Tris-HC1 buffer pH 7.8, 1mM MgS04, 4mM CaCl2, 0.1 M
Nacl, and 0.1% gelatin), resuspended and serially diluted into Phage buffer, and 100 1 of the suspended bacteria were. spread on nutrient agar plates. Surviv-ing bacteria were determined by scoring colony forming units (CFU) after incubation for 24-48 hours at 37~C.
An effective bactericide according to this invention is one which allow:a less than 0.1% of the initial viable count of thE: bacteria to survive.
Example 1 Activity of Nisin and a Chelating Agent Against Gram tdegative Bacteria (S. typhimurium) As shown in Table 1, two tests were conducted in 20mM Tris, pH 8.0 at 37~C to show the effect of the bactericide containing nisin and the chelating agent EDTA alone. Test ~'1, a control, was conducted without EDTA and shows the effect of nisin alone toward the Gram negative bacts:rium S. typhimurium. The increased concentrations of nisin do exhibit some activity, but even the activity of the higher concentrations in the absence of ED'rA, 1.6% survival at 100 ~g/ml nisin, is wholly inadequate for a food preservative. The level of bactericidal activity obtained from nisin and EDTA
is significant.

_g_ mT nr ~ i Initial Viable Test Bacteria EDTA Nisin (ug/ml) Count _ ~M~ 0 10 30 50 100 300 Percentage S. typhimurium Survival at 3 hours 1 3.0 x 106 0 100 51.3 - 7.0 1.6 -2 5. 7 x 106 20 2 . 5 - 10 3 - O10 4 < 10 4 Test #2 (Table 1), conducted using nisin plus 20mM EDTA, demonstrates the surprising activity of the nisin composition in eliminating the target Gram negative bacteria.
Table 1 shows that in test #2 at a concentration of 20mM EDTA and 30 a g/ml of nisin, the bactericide has a marked bactericidal activity towards S. typhimurium, while at nisin concentrations of 100 i.~g/ml and grE:ater, the nisin and EDTA bactericide virtually el~~minates the bacteria (percentage survival less than 10 4 which indicates no surviving bacteria in the assay). Thus, the combination of EDTA and nisin demonstrates a synergistic activity of greater than 1000 times that of nisin alone.
Example 2 ~~ctivi~ty of Nisin, a Chelating ~~gent .and a Surfactant Against Gram ~legati.ve Bacteria (S. typhimurium) Four tee>ts (Table 2) were conducted to determine the effect on S. typhimurium of the bactericide containing nisin a;nd both EDTA and the surfactant Triton* X-100 in 20~~nM Tris, pH 8 . 0 at 37 ~. The control (Test #1) is identical to the control of Example 1 (Table 1).
* Trade-mark _g_ Initial Viable Triton Test Bacteria. EDTA X-100 Nisin (ug/ml) Count . (mM) (%) 0 10 30 50 100 300 Percentage S. typhimurium Survival at 3 hours 1 3.0x106 0 0 100 51.3 - 7.0 1.6 2 3.0x106 0 1.0 37.4 93.0 - 64.0 47.0 3 5.7x106 20 0.1 0.03 - <10 3 - -4 5.7x106 20 1.0 <10 4 - <10 4 - <-10 4 <10 4 Test #2 (Table: 2) was conducted using nisin and 1.0% Triton X-100, but without EDTA. The presence of the detergent alone inhibits the activity of the nisin towards the Gram negative bacteria and nisin was ineffective. However, in tests #3 and #4 (Table 2), which represent they invention, the presence of 20mM
EDTA in combination with Triton X-100 is a bactericide which markedly increases the bactericidal activity of nisin towards Sty~phimurium. Indeed the combination of Triton X-100 with EDTA but without nisin was effective, although. to a lesser degree than in the presence of nisin. While in both tests #3 and #4 (Table 2) the nisin. combinations were very effective, the concentration of 1.0% Triton X-100 (test #4, Table 2) was most effective.
The presence of the non-ionic surfactant, Triton X-100, in combination with EDTA, enhances the activity of nisin toward Gram negative bacteria even more than the bactericide containing nisin and EDTA alone (Example 1).

~13408~0 f w Example 3 Activity of Nisin, a Chelating Agent and ~~ Surfactant Against Gram Negative Bacteria (S. typhimurium) Table 3 shows the enhanced activity toward S. typhimurium of the bactericide containing nisin, 20mM of the c~helating agent EDTA and the non-ionic surfactant T~aeen* 20 in 20 mM Tris, pH 8 . 0 at 37° C. As with Triton*:~-100 (Example 2) the combination of nisin and EDTA with (1~) of Tween*20 is most effective.

Initial Viable Test Bacteria EDTA Tweer~20 Nisin (ug/ml) # Count _ (mM) (o) 0 10 30 ' 50 100 300 Percentage S. typhimurium Survival at 3 hours 1 3.0x106 0 0 100 51.3 - 7.0 1.6 -2 5.7x106 20 0 2. 5 - <10 3 - < 10 4 < 10 4 3 4. 3x106 20 1. 0 <10 2 - <10 4 - < 10 4 < 10 4 Example 4 Act:Lvity of Nisin, a Chelating Agent and a Surfactant Against Gram Negative Bacteria (Escherichia coli) Ths~ effect of the bactericide containing nisin and ED'7~A towards the Gram negative bacteria E. coli was demonstrated, as shown in Table 4.
* Trade-mark -11- 13408~U

Initial Viable Triton Test Bacteria EDT.A X-100 Nisin (ug/ml) # Count _ ~~ (%) 0 30 100 300 Percentage E. coli Survival at 2 hours 1 1.0 x 107 ~D 0 100 27 25 8.5 2 1.0 x 1(17 20 0 14.5 0.86 0.01 0.001 3 1.0 x 107 0 1.0 100 - 30 -4 1.0 x 107 20 1.0 1.2 0.8 0.05 < 10 The test:a, with and without EDTA, were performed in 20mM '.iris buffer solution, pH 8.0 at 37°C, with an initial viable count of 1 x 107 E. coli cells/ml. The effects of the bactericide were mea-sured as a function of percentage bacteria survival after 2 hours..
In test #1, (control, Table 4) without EDTA, nisin exhibited lii~tle meaningful activity toward the elimination o~f E. c:oli. In test #2 (Table 4), how-ever, where 20mM EI)TA was present, the bactericidal composition exhibited substantial activity towards the E. coli bacteria. The activity increased in effec-tiveness as the concentration of nisin was increased.
The combination of nisin with EDTA as a bactericide demonstrates a 100(1 fold synergistic increase in effectiveness towards E. coli. In tests #3 and #4 (Table 4), it can be seen that Triton X-100 has no significant bactericidal activity towards E. coli. In fact, Triton X-100 appears to inhibit nisin activity towards Gram :negative bacteria as was found with S. typhimurium (Table 2). However, the overall enhancement o:f nisin by EDTA substantially reverses the inhibitor~~ effects of Triton X-100 as seen in Tables 2 and ~I.

It thus appears that the bactericide con-taining nisin and a chelating agent, such as EDTA, is an effective food ;preservative towards various types of Gram negative bacteria even in the presence of surfactants:
Example 5 Activity of Nisin and a Chelating Agent: Against Gram Negative Bacteria (Klebsiella neumoniae) The effect of the bactericide containing nisin and EDTA alone towards the Gram negative bacteria K.
pneumoniae was demonstrated, as shown in Table 5.

Initial ViablE~ Triton Nisin a g/ml Bacteria EDTA X-100 Test Count - ~~ (%) 0 30 100 300 Survival at 2 hours 2 107 21) 0 22 0.5 1.1 0.085 The two tests,, one with and one without EDTA
(control), were performed in 20 mM Tris buffer, pH 8.0 at 37°C with an initial viable count of 107 cells/ml of K. pneumoniae. The effect was measured as a function of percentage bacterial survival after 2 hours.
In test #1, (control, Table 5) without EDTA, nisin exhibited litale meaningful bactericidal activity toward K. pneumoniae. In test #2 (Table 5), however, where 20mNt EDTA was present, the bactericide exhibited substantial activity towards _K. pneumoniae.
The activity increased in effectiveness as the concen-tration of ni;sin was increased.

~ ~~a8~0 Example 6 Nisin Activity Against Gram Negative Bacteria (Salmonella typhimurium) is Dependent on Chelator Concentration The date in Table 6 demonstrate that the enhanced activation ojE nisin towards Gram negative bacteria (S. typhimur:Lum is dependent on the concentration of EDTA in eithE~r 50 mM sodium acetate, pH 5.0, or 20 mM
Tris, pH 8.0 at 37°C.
, TABLE 6 Initial Viable Test Bacaerial Nisin EDTA(mM) # ~H Count - ug/ml 0 0.2 2.0 10 50 100 % Survival at 2 hours 1 5.0 .!x106 0 100 - 38.7 15.2 3.5 -2 5.0 :fx106 100 0.6 10 4 10 4 0.004 0.02 -3 8.0 °ix106 0 100 - 8.7 14 11.4 45 4 8.0 _°ix106 100 4 10 4 10 4 10 4 0.6 30 In test~c #1 and #3, (controls, Table 6) using EDTA concentration: up to 100 mM without nisin exhibited litale meaningful activity towards _S.
typhimurium a.t either pH 5.0 (#1) or pH 8.0 (#3). In tests #2 and #4 (T<~ble 6), however, where 100 g/ml nisin was preaent in combination with EDTA, the bactericides exhib:Lted substantial activity towards S. typhimurium. The activity of the bactericides was similar at both acp~dic pH (5.0) and basic pH (8.0), despite the fact that the activity of nisin alone towards Gram positive bacteria is optimal at pH 5Ø
The enhancement of nisin by EDTA was concentra-tion dependent, being optimal in the range 0.2 mM to 10 mM at pH values 5.0 and 8Ø Surprisingly, at concentrations greater than 10 mM EDTA, the enhance-ment of nisin by EL>TA becomes reduced; the reduction -14- 13~~~~~
of activation is significantly greater at pH 8.0 than at pH 5Ø
Example 7 Nisin anc~ a Chelating Agent Against Gram Negat9_ve Bacteria (S. ty himurium) The enhancement of the activity of nisin by EDTA
towards Gram negative bacteria in the presence of biological t:Lssue 'was demonstrated with S. typhimurium on chicken muscle, and is shown in Table 7.

Test Nisin EDTA(mM) # pH ug/ml_0 0.1 0.3 1 3 10 20 30 lOC

Survivals at 2 hours 1 5.0 0 11.8 - - - - 6.4 - - -2 5.0 300 0.1 0.2 0.05 0.01 0.003 0.016 0.03 0.02 0.0 3 8.0 0 100 - - - - 5.2 - - -4 8.0 300 7.5 0.1 0.02 0.02 0.09 0.47 0.5 - 2.2 5 8.0 300b 0.02 0.09 0.0002 < 10-4 0.0004 0.003 - 0.03 0.0 a Unadhered cells b Contains 1°s Bovine serum albumin (BSA) Incubations were performed in either 50mM sodium acetate, pH 5.0, on 20 mM Tris, pH 8.0 at 37°C.
Cubes of chic3~:en muscle were cleansed with sodium hypochlorite and povidone iodine prior to use. To inoculate the tissue, the cubes of chicken muscle were dipped into a 108 cells/ml suspension of S. typhimurium in 20 mM Tris HC1, pH 8Ø Excess moisture was removed from dipped cubes by tapping.
The chicken samples were placed into sufficient buffer containing th~s nisin composition to cover the tissue and incubated for 2 hours at 37°C after which the tissue was removed to sufficient Phage buffer to cover 13~0~~~

the tissue. The bacteria remaining in the test solution wer~a collected by centrifugation, washed with Phage buffer, and combined with bacteria washed from the tissue b:Y Phage buffer. The combined samples (termed "unadhered" cells) were serially diluted and 100 ~ aliquots were plated for determination of surviving bacteria.
In test, #1 and #3 (Table 7), in the absence of nisin at either pH 5 or pH 8, EDTA alone has no significant effect on the survival of S. typhimurium.
In tests #2 ~~nd #4 (Table 7), however, where 300u g/ml nisin was present, the bactericides exhibited substan-tial activit~t towards S. typhimurium on chicken muscle at both pH 5..0 and pH 8Ø
The enh<~ncement of nisin by EDTA was concentra-tion dependent, the optimal concentration being in the range 0.3mM i.o lOmM EDTA at both pH values 5.0 and 8Ø At con<:entrations greater than lOmM EDTA at pH
8.0, the act»vation of nisin by EDTA is reduced.
However, as 9:s shown in test #5 (Table 7), in the presence of J_.0 % :bovine serum albumin at pH 8.0, the efficacy of nisin 'towards S. typhimurium on chicken muscle is expressed throughout the range of EDTA
concentrations up to 100mM.
Thus, bactericides containing nisin and low concentrations of chelating agent, such as EDTA in the range O.lmM t:o 20m1~I, can be extremely effective for the elimination or prevention of contamination of food by Gram negative b~~cteria.
Example 8 Titration of Nisin Activity Against Gram Negative Bacteria (S. ty himurium) At the optimal concentration of chelating agent, the efficacy of ths= bactericide in Tris buffer towards Gram negative bacteria was demonstrated to be substan-tial, as is shown ~ln Table 8.

~~~~~~o Initial Viable Test Bacteri<~1 EDTA BSA Nisin ug/ml # Count - ~~ % 0 0.1 0.3 1.0 3.0 10 30 100 % Survival at 2 hours 1 6x106 0 0 100 - - - - 51.3 - 1.6 2 6x106 1.0 1.0 63 0.7 0.08 0.01 0.05 0.01 <10 4 -In test #2 (Table 8), it can be seen that as little as O.:i a g/ml of nisin, with 1.0 mM EDTA in 20mM
Tris at pH 8..0 in 'the presence of 1% bovine serum albumin (HSA), significantly reduced the survival of S. typhimurium. The bactericide is as active towards Gram negativE~ bacteria as nisin alone is towards Gram positive Stre toco~~ci.
Example 9 Titration of Nisin Activity Against Gram a~egati.ve Bacteria (S. typhimurium) At the optima:L concentration of chelating agent, the efficacy of a bactericide towards Gram negative bacteria in t:he presence of biological tissue was demonstrated with S. typhimurium on chicken muscle, and is shown in Tahle 9.

Initial Viable Test Bacterial 1~DTA BSA Nisin ~g/ml # ~H Count. _mM (%) 0 10 100 200 300 % Survival at 2 hours 1 8.0 3x107 0 0 100 - - - -2 8.0 3x107 ".L.O 1.0 27 0.26 0.008 0.007 0.006 Cubes of chic)';en muscle were cleansed with sodium hypochlorite and povidone iodine prior to use. To inoculate the tissue, the cubes of chicken muscle x_ -17- I340~~~1 were dipped :into a 108 cells/ml suspension of S.
typhimurium :in 20 mM Tris HC1, pH 8Ø Excess mois-ture was removed from dipped cubes by tapping. The tissue was placed into sufficient buffer containing the nisin compositions to cover the tissue, and incubated for 2 hours at 37°C after which the tissue was removed i~o sufficient Phage buffer to cover the tissue. The bacteria remaining in the test solution were collecte=d by centrifugation, washed with Phage buffer, and c:ombined with bacteria washed from the tissue by Phage buffer. The combined samples (termed "unadhered" c:ells) were serially diluted and 100 aliquots were: plated for determination of surviving bacteria.
Example 10 Nisin EDTA and Methyl Paraben ~~ctivi~ty Against Gram Negative Bacteria (S. ty himurium) A bactericide containing nisin and EDTA, when combined with a known food preservative, methyl paraben, was demonstrated to be exceptionally effective towards Gram negative bacteria, as shown in Table 10.
mn nr ~ ~ n Initial.
Viable Test Bacterial N:isin EDTAb % Methyl Paraben # Count - a c~ml- mM 0 0.1 1.0 ~ Survivals at 2 hours 1 3x106 0 10 11.8 1.0 10 4 2 3x106 :f 00 10 0 . 03 < 10 3 X10 50 mM Na acetate buffer, pH 5.0 c Unadhered cells _18_ 13~08~0 Cubes oi= chicken muscle were cleaned with sodium hypochlorite and povidone iodine prior to use. To inoculate the. tissue, the cubes of chicken muscle were dipped into a 108 cells/ml suspension of S.
typhimurium in 50 ~mM sodium acetate buffer, pH 5Ø
Excess moisture wars removed from dipped cubes by tapping. The tissue was placed into sufficient buffer containing nisin compositions to cover the tissue, and incubated for 2 hours at 37°C after which the tissue was removed t:o sufficient Phage buffer to cover the tissue. The bacteria remaining in the test solution were collectead by centrifugation, washed with Phage buffer, and combined with bacteria washed from the tissue by Phage buFfer. The combined samples (termed "unadhered" cells) were serially diluted and 100 aliquots werE: plated for determination of surviving bacteria.
In test #1 (Table 10), methyl paraben in the presence of 7.0 mM IEDTA was shown to be effective towards St~~phimurium only at a concentration of 1.0~. In te~;t #2 (Table 10), however, in the presence of 300 a g/ml nisin, the effectiveness of methyl paraben and nisin itowards S. typhimurium was substan-tially improved.
The compositions containing nisin and EDTA
significantly improve the utility of the food preser-vative methyl paraben. Furthermore, the bactericides may lead to substantial reductions in the concentra-tions, or eliminate the need for these commonly recognized, though less desirable, food preservatives such as methyl paraben.

_1g-l~~o~~u Example 11 Ni:~in and Chelating Agent Activity ~~gainst Gram Positive Bacteria (Staphylococcus aureus) The act»vation of nisin by a chelating agent is pH-dependent.. The data in Table 11 confirm that at pH 5.0, nisin is somewhat more bactericidal towards S. aureus than is nisin at pH 8Ø At pH 5.0, EDTA
does not enhance nisin activity towards S. aureus and at concentrations of EDTA greater than 10 mM, EDTA is inhibitory to the :bactericidal activity of nisin.
However, the bactericidal activity of nisin activated by EDTA at pH 8.0 is significantly greater than the bactericidal activity of nisin alone, or in combina-tion with ED~CA at ;pH 5Ø
Influence of pH on the Effects of EDTA on Nisin Bactericidal Activity towards Staphvloccus aureus Nisin EDTA mM
p H a g/ml 0 0.1 0.3 1.0 3.0 10 30 100 Survival 2 hra 8.0 0 100 - 100 81 100 100 100 -8.0 3.0 7.4 0.173 0.01 0.2 0.4 3 56 -5.0 0 100 - - - 100 - - -5.0 3.0 0.6 1.0 1.3 1.4 1.8 - 34 80 a Initial viable count: 8.0 x 106 cfu/ml Incubations were performed in 50 mM sodium acetate buffer, pH 5.0 or 20 mM Tris-HC1 buffer, pH 8.0 at 37° C.
The bactericidal activity of nisin alone is reported (see Hurst:) to be greatest at pH 5.0 or ~.. ._ _ -20- 1340$0 lower, and data presented in Table 11 support this.
On the basis of this information it was believed that the bactericidal activation of nisin by EDTA towards S. aureus would likewise be greatest at lower pH.
However, as can bE~ seen in Table 11 and contrary to expectation:c (see Table 6), EDTA was not observed to enhance nisin activity towards Gram positive bacteria at pH 5Ø HowevEar, inhibition of nisin activity by high concentrations of EDTA was still observed at pH
5Ø Thus, the acaivation of nisin by a chelating agent occur, only within a range of chelator concen-trations and., with respect to Gram positive bacteria, is dependent. upon pH with the preferred pH range greater than pH 5Ø
Example 12 Nisin and Chelating Agent Activity Against Gram Positive Bacteria The effects of EDTA on the bactericidal activity of nisin at pH 8.0 are not limited to S. aureus, an important human pathogen, but are also observed with Streptococcus mutans, responsible for dental plaque (Table 12A), Listeria monocytogenes, a foodborne pathogen (Table 12B), and with a mixed population of axillary Cor,neform bacteria, contributors to body odor (Table :12C) .

-21- 1340~~ p Table 12A
The Effects of EDTA on the Bactericidal Activity of Nisin towards Streptococcus mutans Nisin EDTA mM
pH ug/ml 0 0.01 0.1 0.3 1.0 3.0 10 30 100 Survival after 2 bra 8.0 0 1.00 -- - - - - - - _ 8.0 0.1 4.3 1..8 0.04 0.02 0.06 1 25 100 100 a Initial viable count: 6.0 x 106 cfu/ml Incubations were performed in 20 mM Tris-HC1, pH 8.0 at 37 °C.
Table 12B
The Effecta of EDTA on the Bactericidal Activity~towards Listeria monocytogenes Nisin EDTA mM
pH a g/ml 0 0.1 0.3 1.0 3.0 10 30 100 Survival after 2hra 8.0 0 100 - - g4 - - - -8.0 3.0 0.71 0.04 0.04 0.02 0.1 0.64 10 14 a Initial viable count: 6.0 x 106 cfu/ml Incubations were performed in 20 mM Tris-HC1, pH 8.0 at 37Q C.

Table 12C
The F:ffect;s of EDTA on Nisin Bactericidal Activity towards Coryneform bacteria Nisin ~ EDTA mM
pH a g/ml 0 0.1 0.3 1.0 3.0 10 Survival 2hra 8.0 0 100 - 4.6 3.6 8 36 8.0 3 ~ 0.22 0.03 0.0009 0.1 -- 0.16 a Initial viable count: 1.0 x 106 cfu/ml Incubation: were performed in 20 mM Tris-HC1, pH 8.0 at 37° C.
Example 13 Rapid Bactericidal Activity of Nisi:n Activated by Chelator The bact:erici~de comprising nisin with EDTA is rapidly bactE~ricidal as is illustrated by the data presented in Table 13A. Suspensions of the Gram positive bacterium S. mutans at 107 cells/ml were incubated in 20 mM Tris buffer, pH 7.3 at 37°C with a range of concentrations of nisin activated by 1 mM
EDTA. The suspensions were incubated for various times ranging from 0.5 to 60 minutes with the bacteri-cides. The bactericidal efficacy of the bactericides was estimated by determining the percent survival of bacteria. Enhanced by EDTA, as little as 10 ug/ml of the nisin in this formulation is able to reduce the bacterial load by 6 logs within 1 minute.
Rapid bactericidal activity is a prerequisite for effective disinfection. Thus, the compositions are predicted to be effective bactericides particularly as demonstrated here, as a component of a mouthwash, -23- ~~~o~Jo rinse, toothpaste, or other similar dentrifice active against plaque forming S. mutans.
The activity of nisin enhanced by EDTA against Gram negative: bactcaria after 2-3 hours was shown in Examples 1-7. Rapid bactericidal activity of nisin enhanced by E;DTA i:a also seen towards Gram negative bacteria and this :~s illustrated by the data in Table 13B.
Kineti~~s of Bactericidal Activity towards Streptococcus mutans of Nisin Enhanced by EDTA
Incubation Nisin a g/ml with 1.0 mM EDTA
Time 0 1 3 10 30 100 (Minutes) ~ Survivals 0.5 - _ _ - - <10 4 1 - - - < 10 4 < 10 4 < 10 4 3 100 0.5 0.002 < 10 4 < 10 4 -- 0.03 < 10 4 < 10 4 -30 - - < 10 4 - - _ 60 100 0.003 - - -a Control viable cell count: 1.0 x 10~ cfu/m1 Incubations were performed in 20 mM Tris-HC1, pH 7.3 at 3 7 oC~.

-24- 1340~3J~

Rapid Bactericidal Activity towards Escherichia coli of Nisin Enhanced by EDTA
Nisin ug/ml mM EDTA 0 0.3 1.0 3 10 30 100 survival at 1 mina 1.0 100 100 56 0.37 0.013 0.015 0.008 a Initial viable count: 1.0 x 107 cfu/m1 Incubations. were performed in 20 mM Tris, pH 7.0 at 37° C.
Example 14 Effect of Divalent Cations on EDTA
E~nhanceament of Nisin Activity Divalent. cations bind to EDTA and other chelating agents and would bsa expected to neutralize the activa-tion of nisin. by EI)TA. However, as can be seen by the data in Table 14, t;he bactericidal activity of nisin against S. mutans Ls enhanced by 1 mM EDTA even in the presence of 1 mM Ca2+ ion; only above 3 mM was Ca2+
ion inhibitory to EDTA-activated nisin. This is particularly important in mouthwash applications where calcium ion concentrations are relevant.

...._ - 2 5 -1340~~0 i Rapid Bactericidal Activity towards Streptococcus mutans of Nisin Activated by EDTA in ths~ presence of Divalent Cation CaCl3 mM
Nisin I 0 0.1 0.3 1.0 3 10 % survival at 1 min a 3 2.9 3E 0.0042 0.0042 0.052 18 30E 0.0019 0.0003 0.0004 0.06 6.8 4 <

100 < 1a~ 10 <10 0. 0001 1. 5 E 1 mM Na2EDTA
a Initial viable count 1.0 X 102 cfu/ml.
Incubations pert°ormed in 10% Fetal Calf Serum at 37° C.
Example 15 tJisin and Surfactant Activity A ainst: Gram Positive Bacteria The bactericidal activity of nisin can also be significantly enhanced when combined with a surfactant alone. This is be~;t illustrated at a limiting nisin concentration (0.2 ~g/m1) as shown in Table 15A. At concentrations up t:o 0.1%, the food grade surfactant monolaurin has little significant bactericidal activity towards Streptococcus agalactiae in the complex medium milk:. Nisin, at concentrations up to 0.2 ~g/ml, likewise: does not exhibit significant bactericidal ,activity in milk. However, the combina-tion of the t~~ao agents, 0.1% monolaurin and nisin a -26- 134(~~~ f~
0.2 ug/m1, is extremely potent towards S. agalactiae.
This bactericide i:> over 100 times more active than what would be expecaed for the additive effect and 10,000 times more active than either of the components individually. Thu:a, when the application of nisin is limited by its available activity, a bactericide comprising nisin w~_th a surfactant can be expected to be more useful.
An example of where the application of nisin is limited by its ava~:lable activity is illustrated by the data in Table 7~5B. Although nisin, and particu-. larly the bactericide comprising nisin and EDTA, is bactericidal towards L. monocytogenes, the data in Table 15B demonstrate that in a complex medium like milk the available nisin activity towards this organism is restricaed. However, the bactericide comprised of nisin with the glyceride, monooleate, is effective in milk towards this foodborne pathogen even though monooleate by itself had no bactericidal activity towards this organism.

13408~0 Table 15A
Nisi.n Bactericidal Activity towards Streptococcus agalactiae in milk at 37°C
(Act:ivati~on of nisin by monolaurin) Nisin Monolaurin ( 1~g/ml ) ( % ) 0 0.01 0.1 % survival at 2ha 0 100 100 4.5 0.02 100 100 0.2 0.2 2.2 0.05 0.0008 a Initial visable counts 6.0 X 107 cfu/ 1.
Incubations were' in milk at 37 °C.
Table 15B
Nis:Ln Bactericidal Activity towards Listeria monocytogenes in milk at 37°C
(Act:ivation of nisin by monooleate) Nisin % Monooleate g/ml 0 0.1 1.0 % Survival 2 hra 100 0.56 10 3 10 a Initial viable count 5.0 x 107 cfu/m1 Incubations werE: in milk at 37°~ C.

Claims (26)

1. A composition comprising a lanthionine containing bacteriocin and a chelating agent.

2. A composition comprising a lanthionine containing bacteriocin and a surfactant.

3. A composition comprising a lanthionine containing bacteriocin, a chelating agent and a surfactant.

4. The composition as defined in claim 1, 2 or 3 wherein the lanthionine containing bacteriocin is selected from the group consisting of nisin, subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5.

5. The composition as defined in claim 1 wherein the chelating agent is selected from the group consisting of alkyldiamine tetra-acetates, CaEDTA, Na2CaEDTA, EGTA and citrate.

6. The composition as defined in claim 3 wherein the chelating agent is selected from the group consisting of alkyldiamine tetra-acetates, CaEDTA, Na2CaEDTA, EGTA and citrate.

7. The composition as defined in claim 5 or 6 wherein the alkyldiamine tetra-acetate is EDTA and the bacteriocin is nisin.

8. The composition as defined in claim 2 or 3 wherein the surfactant is selected from the group consisting of glycerides, fatty acids, emulsifiers, quaternary compounds, amphoteric surfactants and nonionic surfactants.

9. The composition as defined in claim 1 also containing a food preservative.

10. An enhanced broad range bactericide comprising a carrier, a lanthionine containing bacteriocin and a chelating agent.

11. An enhanced broad range bactericide comprising a carrier and a lanthionine containing bacteriocin and a surfactant.

12. An enhanced broad range bactericide comprising a carrier, a lanthionine containing bacteriocin, a chelating agent and a surfactant.

13. The enhanced broad range bactericide as in claim 10 wherein the lanthionine containing bacteriocin selected from the group consisting of nisin, subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5 and the chelating agent selected from the group consisting of alkyldiamine tetra-acetates, EGTA and citrate are present in quantities such that the bactericide has enhanced effectiveness against at least one of the bacteria from the group consisting of Staphylococcus aureus, Streptococcus mutans, Listeria monocytogenes, Streptococcus agalactiae, Coryneform bacteria, Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacteroides gingivalis and Actinobacillus actinomycetescomitans.

14. The enhanced bread range bactericide as in claim 11 wherein the lanthionine containing bacteriocin selected from the group consisting of nisin, subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5 and the chelating agent selected from the group consisting of alkyldiamine tetraacetates, EGTA and citrate are present in quantities such that the bactericide has enhanced effectiveness against at least one of the bacteria from the group consisting of Staphylococcus aureus, Streptococcus mutans, Listeria monocytogenes, Streptococcus agalactiae, Coryneform bacteria, Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacteroides gingivalis and Actinobacillus actinomycetescomitans.

15. The enhanced broad range bactericide as in claim 12 wherein the lanthionine containing bacteriocin selected from the group consisting of nisin, subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5 and the chelating agent selected from the group consisting of alkyldiamine tetraacetates, EGTA and citrate are present in quantities such that the bactericide has enhanced effectiveness against at least one of the bacteria from the group consisting of Staphylococcus aureus, Streptococcus mutans, Listeria monocytogenes, Streptococcus agalactiae, Coryneform bacteria, Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacteroides gingivalis and Actinobacillus actinomycetescomitans.

16. The enhanced broad range bactericide of claims 13, 14 or 15 wherein the alkyldiamine tetra-acetate is EDTA.

17. The enhanced broad range bactericide as in claim 11 wherein the surfactant is selected from the group consisting of glycerides, fatty acids, quaternary compounds, emulsifiers, amphoteric surfactants and nonionic surfactants and is present in an amount sufficient such that the bactericide has enhanced effectiveness against at least one of the bacteria from the group consisting of Gram negative and Gram positive bacteria.

18. The enhanced broad range bactericide as in claim 12 wherein the surfactant is selected from the group consisting of glycerides, fatty acids, quaternary compounds, emulsifiers, amphoteric surfactants and nonionic surfactants and is present in an amount sufficient such that the bactericide has enhanced effectiveness against at least one of the bacteria from the group consisting of Gram negative and Gram positive bacteria.

19. The enhanced broad range bactericide of claims 13, 14 or 15 wherein the concentration of nisin is between about 0.1 µg/ml and 300.0 µg/ml and the concentration of chelating agent is between about: 0.1 mM and 20 mM.

20. The enhanced broad range bactericide as in claims 17 or 18 wherein the concentration of surfactant is between about 0.01% and 1.0%.

21. A composition comprising nisin and a chelating agent.

22. A composition comprising nisin and a surfactant.

23. A composition comprising nisin, a chelating agent, and a surfactant.

24. A bactericide comprising nisin and a chelating agent.

25. A bactericide comprising nisin and a surfactant.

26. A bactericide comprising nisin, a chelating agent, and a surfactant.