PL163983B1 - Germicide - Google Patents

Abstract

1. A bactericidal agent comprising a bacteriocin and excipients, characterized in that it comprises a bacteriocin comprising lanthionine in a concentration of between 0.1 gg/ml and 300 gg/ml and a chelating agent in a concentration of between 0.1 mM and 20 mM.

Description

The subject of the invention is an extended-spectrum bactericidal agent containing a lanthionine-containing bactericide.

Nisin, one of the baclocins, is a polypeptide with antimicrobial properties that is naturally produced by various strains of the bacterial Stokptacoccus lactis. It is used as a food preservative that inhibits the growth of spores of certain strains of Gram-positive Bacilli.

Although sometimes mistakenly and inaccurately called an antibiotic, nisin is correctly classified as a bacteriocin, a protein substance produced by bacteria that has antibacterial activity only against strains closely related to the strains from which it originates. Nisin is a naturally occurring, natural protective substance found in low concentrations in milk and cheese, and is believed to be completely harmless and non-allergenic in humans.

Nisin has been determined by FDA testing to be a safe substance that can be used in food products such as pasteurized cheese spreads, pasteurized processed cheese spreads, and pasteurized processed cheese spreads with added fruit, vegetables, or meat. Furthermore, because nisin is a polypeptide, any remaining nisin in food products is rapidly digested.

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A summary of the properties of nisin appeared in A. Hurst, Advances in Applied Microbiology 27:85-123 (1981). This publication covers everything known about nisin in general. Nisin produced by Streptococcus lactis is commercially available as an impure preparation, Nisaplin, manufactured by Aplin and Barrett Ltd., Dorset, UK, and can be obtained by isolating naturally occurring nisin from a culture of Streptococcus lactis and then concentrating the nisin by known methods. Numerous methods for producing nisin using modified strains of Streptococcus are also known (see U.S. Patent No. 4,767,667). Nisin can also be produced using recombinant DNA technology.

Nisin has been used effectively as a preservative in dairy products such as processed cheese, cream, and milk. The use of nisin in products such as processed cheese has been the subject of U.S. patents. Nos. 4,584 (11) and 4,517,172. The use of nisin to inhibit the growth of certain Gram-positive bacteria has been well documented. However, complete success and full acceptance as a food preservative has, so far, been hindered by the belief that nisin is ineffective against Gram-negative and many Gram-positive bacteria. Gram-negative bacteria are almost always present in association with Gram-positive bacteria and are a primary source of food spoilage and contamination (see U.S. Patent Nos. 5,584 (11) and 4,517,172, Tsau and Sandine, Conjugal Transfer of Nisin Plasmid Genes from Streptococcus Lactis 7162 to Leuconostoc Dextranicum i8i, Applied and Environmental Microbiology, February (187, p. 352, A Natural Preservative, Food Engineering Int'l, May (184, pp. 37-38; Focus on Nisin, Food Manufacture, March (187, p. 63.

Contrary to previous knowledge, nisin-containing agents combined with various nonbactericidal agents were found to have better and broader bactericidal activity against Gram-negative bacteria, as well as better activity against a wider range of Gram-positive bacteria than nisin alone. Greater bactericidal activity against Gram-positive bacteria occurred over a wider pH range than previously reported.

The subject of the invention is a bactericidal agent containing a bacteriocin containing lanthionine in a concentration between 0.( pg/ml and 300 pg/ml and a chelating agent in a concentration between 0.( mM and 20 mM. The agent according to the invention may further contain a surfactant in a concentration between 0.0(% and (.0%). According to the invention, such agents dissolved or suspended in a suitable carrier provide a bactericidal substance with a better and broader effect.

It was found that a solution of about 0.5 pg/ml to 300 pg/ml of bacteriocin in the presence of about 0.5 mM to 20 mM of a chelating agent, for example, ethylenediaminetetraacetic acid (EDTA), actually eliminates the growth of Gram-negative bacteria such as Salmonella typhimurium, Escherichia coli, Pseudomones aeruginosa, E. coli, Pseudomones aeruginosa, E. bacterilides gingivalis, Actinobacillus actinomycetescomitans, and Klebsiella pneumoniae, and is more active against Gram-positive bacteria such as Staphylococcus aureus, Streptococcus mutans, Listeria miltogenes, Streptococcus agalactiae, and Ceryneform bacteria than nisin itself. Although the increase in nisin activity induced by the chelator is concentration dependent, contrary to the As expected, EDTA concentrations exceeding 20 mM inhibited the bactericidal activity of nisin. However, in the presence of a proteinaceous carrier and multivalent polymers such as serum albumin, collagen, gelatin, casein, and keratin, nisin inhibition induced by EDTA concentrations above 20 mM was significantly reduced, thereby expanding the range of utility of the nisin-containing agent improved by the addition of EDTA.

It was found that a solution of 0.( pg/ml to 300 pg/ml nisin and 0.( mM to 20 mM chelating agent will have further improved nisin efficacy against Gram-negative and Gram-positive bacteria in the presence of 0.0(% to (.0% surfactant). It was further found that in the presence of surfactant alone, nisin has enhanced activity against Gram-positive bacteria.

Suitable chelating agents for use in the compositions of the invention are: EDTA, CaEDTA, CaNa2EOTA and other alkylamine tetraacetates, EGTA and citrate, surfactants suitable as components of the combination with nisin and with or without EDTA are: nonionic surfactants such as Tweens, Tritonyl and glycerides, ionic surfactants such as fatty acids, quaternary compounds, anionic surfactants such as sodium dodecylsulfonate and amphoteric surfactants such as cylcamylpropyloobetaine and emulsifiers.

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Since Gram-negative and Gram-positive bacteria are almost always present in food, the effectiveness of a nisin-containing agent against Gram-negative bacteria such as Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacterioides gingivalis, Actinobacillus actinomycetescomintans, and other Gram-negative and Gram-positive pathogens would be highly useful. Antibacterial agents are particularly useful for reducing and preventing contamination of raw ingredients, processed foods, and beverages by bacterial pathogens and other spoilage microorganisms. Potential food applications include meat processing, particularly poultry, eggs, cheese, and fish, and use in packaging and handling equipment used in food handling. Further uses of the composition according to the invention include protecting food such as processed cheeses, creams, milk and dairy products, washing poultry, fish, meat and vegetables, and dairy equipment. The use of nisin-containing compositions should not be limited to food applications, as they can be useful in any situation where the elimination of gram-negative and gram-positive bacteria is necessary or desired.

The agent may be soluble in a suitable carrier, for example, an aqueous or buffer solution, or suspended in any suitable liquid, colloidal, or polymeric vehicle to provide a bactericidal preparation. Such an agent or bactericidal preparation may be used for disinfecting surgical instruments, wiping, or washing. Bactericidal preparations may be used for cleaning surgical instruments, surgical suite equipment, and the like. Bactericidal preparations are particularly useful in circumstances where environmental disinfection is required, but the use of chemical disinfectants is prohibited due to the risk of corrosion or the toxic effects of their residues.

Unlike broad-spectrum disinfectants, whose activity is limited by the presence of complex organic matter, the preparations of the agent according to the invention are bactericidal also in the presence of organic matter, such as milk or blood serum.

Nisin has been known to optimize the growth inhibition of a number of related Gram-positive bacteria, especially against certain Gram-positive spore-forming bacteria at pH 5.0. The activity of nisin in solution with a chelating agent is unexpectedly accelerated and improved against many Gram-positive bacteria at pH values greater than 5.0, and is further enhanced against Gram-negative bacteria at both acidic and alkaline pH values in the pH range 5.0 to 8.0. This unexpectedly accelerated and enhanced bactericidal activity of chelator-activated nisin makes it suitable for use, among other things, as a disinfectant.

Nisin belongs to the class of lanthionine-containing peptide bacteriocins. This class also includes subtilin, epidermin, cinnamycin, duramycin, ancovenin, and Pep 5. All of these bacteriocin peptides are produced by different microorganisms. However, subtilin obtained from certain cultures of Bacillus subtilis and epidermin obtained from certain cultures of Staphylococcus epidermidis have been found to have a very similar molecular structure to that of nisin (see Hurst, pp. 85-36 and Schnell et al., Nature, 333:276-278). Therefore, it is believed that, because of their similar molecular structure, other lanthionine-containing protein bacteriocins will be as effective as nisin in combination with chelating agents and possibly nonionic surfactants in eliminating gram-negative and gram-positive bacterial contaminants.

The effectiveness of nisin, and by extension, other lanthionine-containing bacteriostatic proteins, as bactericidal agents against Gram-negative bacteria is surprising, as such activity was not apparent from the prior art. The increase in nisin activity against Gram-positive bacteria in the presence of EDTA at pH higher than 5.0 is unsurprising, as nisin activity was previously believed to be optimal at pH 5.0. Furthermore, the discovery of such activity of nisin and lanthionine-containing bacteriostatic proteins satisfies a long-felt need in the food safety field, stemming from the lack of an effective, natural, and harmless agent active against a wide range of bacteria.

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To demonstrate the excellent and unexpectedly rapid activity of a preparation containing nisin, as well as other bacteriocins and EDTA or other chelators and possibly various surfactants against both gram-negative and gram-positive bacteria, numerous experiments were conducted with these bactericidal preparations. These experiments are illustrative and demonstrate that nisin substitutes, i.e., protein bacteriocins containing lanthionine, can be expected to be effective, and furthermore, that EDTA substitutes as a chelating agent will also be effective.

All tests carried out in the following examples were performed at 37°C. The effectiveness of improving the range of bactericidal activity was determined by assessing the bactericidal activity measured by the percentage of bacteria surviving the bactericidal treatment. Typically, after incubating a suspension of 108 cells/ml of the test strains with the new bactericidal agent for a specified time, the bacteria were separated by centrifugation for 2 minutes. The bacterial plates were washed free from the test bactericidal agent preparation with a purifying buffer, referred to here as Phage (50 mM Tris-HCl, pH 7.8, 1 mM MgSO4, 1 mM CaCl4, 0.1 M NaCl, and 0.1% gelatin), resuspended, and serially diluted with Phage buffer, after which 100 ml aliquots of the bacterial suspension were plated onto nutrient agar plates. The degree of bacterial survival was determined by counting colony-forming units (cfu) after incubation for 24-48 hours at 37°C. The effectiveness of the agent according to the invention allows the survival of less than 0.1% of the initial number of viable bacteria.

Example i. Activity of nisin and chelating agent against Gram-negative bacteria (S. typhimurium).

As shown in Table 1, two studies were conducted at 20 mM Tris, pH 8-0, 37°C, to evaluate the effectiveness of a bactericidal agent containing nisin and EDTA alone as a chelating agent. Control study 1 was conducted without EDTA and demonstrated the activity of nisin alone against the Gram-negative bacterium S. typhimurium. Elevated concentrations of nisin show some activity, but even at higher concentrations, and in the absence of EDTA, the 1.6% survival rate at 100 pg/ml nisin is completely insufficient for food preservation. The level of bactericidal activity obtained with nisin and EDTA is significant.

Table 1

Study Number Initial count of viable bacteria EDTA (mM) Nisin (pg/ml) 0 10 30 50 100 300 1 2 3.0 x K)6 5.7 x U)6 0 20 Pr< 100 2.5 icent S. tyf 51.3 Jhimurium, 10' ³ <that's what I've been through) 7.0 'ly after 3 hours; 1.6 < 10-4 tinach <10-4

Study No. 2 in Table 1, conducted with nisin and 20 mM EDTA, showed unexpected activity of the nisin-containing preparation in eliminating the targeted Gram-negative bacteria.

Table 1 shows that in study No. 2, at a concentration of 20 mM EDTA and 30 pg/ml nisin, the bactericide has significant activity against S. typhimurium, whereas at nisin concentrations of 1000 pg/ml and higher, the bactericide containing nisin and EDTA actually eliminated bacteria (the percentage of surviving bacteria was less than 10, indicating no viable bacteria in the sample). This indicates that the combination of EDTA and nisin exhibited synergistic activity greater than 1000-fold compared to the activity of nisin alone.

Example ii. Activity of nisin, a chelating and surfactant agent, against Gram-negative bacteria (S. typhimurium).

Four studies (Table 2) were conducted to determine the effect on S. typhimurium of a bactericide containing nisin and both EDTA and the surfactant Triton Χ-100 in 20 mM Tris, pH 8.0, and 37°C. Control study 1 was identical to control study 1 in Table 1.

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Table 2

Study Number Initial count of viable bacteria EDTA (mM) Triton Χ-100 (%) Nisin (pg/ml) 0 10 30 50 100 300 Percentage of S. typhimurium that survived after 3 hours 1 3.0 x 10 ⁶ 0 0 100 51.3 - 7.0 1.6 - 2 3.0 x 10 ^ć 0 1.0 37.4 93.0 - 64.0 47.0 - 3 5.7 x 10 ⁶ 20 0.1 0.03 - lo' ⁵ - - - 4 5.7 x 10 ⁶ 20 1.0 10-4 - 10-4 - 10-4 10-4

Study 2 in Table 2 was conducted using nisin and 1.0% Triton X-100 but without EDTA. The presence of detergent alone inhibits nisin activity against Gram-negative bacteria, and nisin is ineffective. However, in studies 3 and 4 (Table 2) demonstrating the composition of the invention, the presence of 20 mM EDTA in combination with Triton X-100 provides a bactericidal agent that significantly increases the bactericidal activity of nisin against S. typhimurium. In fact, the combination of Triton X-100 with EDTA but without nisin was effective, but to a lesser extent than in the presence of nisin. Although nisin concentrations were very effective in both studies 3 and 4 (Table 2), the most effective concentration was 1.0% Triton X-100 (study 4, Table 2).

Example III. Activity of nisin, a chelating agent and surfactant, against Gram-negative bacteria (S. typhimurium).

Table 3 shows the improved activity against S. typhimurium of a bactericide containing nisin, 20 mM chelating agent, EDTA and the non-ionic surfactant Tween 20 in 10 mM Tris at pH 8.0 and 37*C. As in the case of Triton Χ-100 (Example II), the combination of nisin and EDTA with (1%) Tween was the most effective.

Table 3

Study Number Initial count of viable bacteria EDTA (mM) Tween 20% Nisin (pg/ml) 0 10 30 50 100 300 1 3.0 x 106 0 0 Slingshots 100 nt S. typhi 51.3 murium, which re survived 7.0 after 3 hours 1.7 oh 2 5.7 x 10 ⁶ 20 0 2.5 < 10-3 < ^10-4 <10-4 3 4.3 x Π)6 20 1.1 <10-2 - < 10-4 - < 10-4 <10-4

Example IV. Activity of nisin, a chelating agent and surfactant, against Gram-negative bacteria (Escherichia coli).

The effectiveness of the bactericidal agent containing nisin and EDTA against Gram-negative E. coli bacteria is presented in Table 4.

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Table A

Study Number Initial count of viable bacteria Triton EDTA (mM) X-100 Nisin (pg/ml) 0 30 100 300 1 1.0 x 10? 0 0 Percent 100 E. coli, which 2? survived 2 25 hours 8.5 2 1.0 x 10? 20 0 14.5 0.86 0.01 0.001 3 1.0 x 10? 0 1.0 100 - 30 - 4 1.0 x 10? 20 1.0 1.2 0.8 0.05 < 10-4

Studies with and without EDTA were performed in 20 mM Tris buffer, pH 8.0, at 37°C with an initial viable count of 1 x 10⁵ E. coli/ml. Bactericidal activity was measured as a function of the percentage of bacteria surviving for 2 hours.

In study No. 1 (control, Table 4) without EDTA, nisin showed little activity in eliminating E. coli. However, in study No. 2 (Table 4), when 20 mM EDTA was present, the bactericidal composition showed significant activity against E. coli bacteria. The effectiveness increases with increasing nisin concentration. The combination of nisin with EDTA as a bactericidal agent shows a 1000-fold synergistic increase in effectiveness against E. coli. In studies No. 3 and No. 4 (Table 4), it can be seen that

-T.r_i.ton Χ-100 has no significant bactericidal activity against JE. coli. In fact, Tnton

Χ-100 appears to inhibit nisin activity against Gram-negative bacteria, as observed for S. typhimuΓlun (Table 2). However, the combined enhancement of nisin activity by EDTA practically reverses the effect of Tnton Χ-100 inhibition, as seen in Tables 2 and 4.

It follows that a bactericide containing nisin and a chelating agent such as EDTA is an effective food preservative against various types of Gram-negative bacteria, even in the presence of surfactants.

Example V. Activity of nisin and chelating agent against Gram-negative bacteria (Klebsiella pneumoniae).

The activity of the bactericidal agent containing nisin and EDTA alone against the Gram-negative bacteria K. pneumoniae is presented in Table 5.

Table 5

Study Number Initial count of viable bacteria m Triton Χ-100 (%) Nisin (pg/ml) 0 30 100 300 1 10? 0 0 P 100 survival after 2 hours 50 38 2 10? 20 0 22 0.5 1.1 0.085

Two studies, one with and one without EDTA (control), were performed in 20 mM Tris buffer, pH 8.0, at 37°C, with an initial viable K. pneumoniae count of 107/ml. The result was determined as a function of the percentage of bacteria surviving for more than 2 hours.

In study 1 (control, Table 5) without EDTA, nisin showed little bactericidal activity against K. pneumoniae. However, in study 2 (Table 5), in the presence of 20 mM EDTA, the bactericidal agent showed significant activity against K. pneumoniae. The activity increased as the nisin concentration increased.

Example VI. The activity of nisin against Gram-negative bacteria (Salmonella typhimurium) depends on the chelator concentration.

The data in Table 6 show that the improvement in nisin activity against Gram-negative bacteria (S. typhimurium) depends on the EDTA concentration in either 50 mM sodium acetate, pH 5.0, or 20 mM Tris, pH 8.0, at 37°C.

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Table 6

Study Number PH Initial count of viable bacteria Nisin pg/ml EDTA (mM) Oh 0.2 2.0 10 50 100 1 2 3 4 5.0 5.0 8.0 0.0 3 x 10 ⁶ 3 x 10 ^æ 5 x 10 ⁶ 5 x 10 ⁶ 0 100 0 100 100 0.6 100 4 % p 10-4 10'4 survival after 38.7 104 8.7 10'4 2 hours 15.2 0.004 14 10-4 h 3.5 0.02 11.4 0.6 45 30

In studies 1 and 3 (controls, Table 6), EDTA concentrations up to 100 mM without nisin resulted in low activity against S. typhimurium at both pH 5.0 (no. 1) and pH 8.0 (no. 3). However, in studies 2 and 4 (Table 6), when 100 pg/ml nisin was present in combination with EDTA, the bactericidal agents showed significant activity against S. typhimurium. Bactericidal activity was similar at both pH levels, acidic pH (5.0) and alkaline pH (8.0), even though the activity of nisin alone against Gram-positive bacteria is optimal at pH 5.0.

The enhancement of nisin efficacy by EDTA is concentration-dependent, being optimal in the range of 0.2 mM to 10 mM at pH values of 5.0 and 8.0. Surprisingly, at concentrations higher than 10 mM EDTA, the enhancement of nisin efficacy by EDTA is reduced. This decrease in activity is much greater at pH 8.0 than at pH 5.0.

Example VII. Nisin and chelating agent against Gram-negative bacteria (S. typhimurium).

The enhancement of nisin activity by EDTA against Gram-negative bacteria in the presence of biological tissue was demonstrated using S. typhimurium on chicken muscle. Data are summarized in Table 7.

Table 7

Study Number PH Nisin pg/ml EDTA (mM) 0 0.1 0.3 1 3 10 20 30 100 1 5.0 0 11.8 % over life after 2 hours 6.4 1 ^a 2 5.0 300 0.1 0.2 0.05 0.01 0.003 0.016 0.03 0.02 0.07 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 3.0 300 ^b 0.02 0.09 0.0002 10-4 0.0004 0.003 - 0.03 0.09

a Non-compact cells contain 1% bovine serum albumin (BSA)

Incubations were carried out either in 50 mM sodium acetate, pH 5.0, or in 20 mM Tris, pH 8.0, at 37°C.

Chicken muscle pieces were cleaned with sodium hypochlorite and iodine in polyvinylpyrrolidone before use. To inoculate the tissue, chicken muscle pieces were immersed in a suspension of cells/ml of S. typhimurium in 20 mM Tris HCl, pH 8.0. Excess moisture was removed from the immersed pieces by removing the liquid. Chicken samples were placed in an appropriate buffer containing the appropriate amount of nisin, and the tissue was covered and incubated for 2 hours at 37°C. After this time, the tissue was transferred to the appropriate amount of Phage buffer to cover it. Bacteria remaining in the solution used for the study were separated by centrifugation, washed with Phage buffer, and combined with bacteria washed from the tissue with Phage buffer. The combined samples

163,983 (referred to as aierwarSe cells) were serially diluted with 100 .1 οοι^.β nanoaaoeo .a nSydel to determine the number of surviving bacteria.

In tests no. 1 and no. 3 (Table 7), in the absence of azithromycin, at pH 10-5 and 10-8, amoo

EDTA showed no significant effect on the survival of S. nypdamuΓίum. . aa^iiu or . 1 or 4 (Table

7) however, in which there was 300 pg alryay, the bactericidal agents showed significant activity against S. typhi.πluΓluy on chicken muscle at both pH 5.0 and pH 0.0.

The improvement in antifungal efficacy by EDTA was concentration-dependent. Ozzy Ryan and ρsnariike's concentration ranged from 0.3 mM to 10 mM EDTA, both at pH 5.0 and pH 0.0. At concentrations higher than 10 mM EDTA at pH 0.0, EDTA activation of antifungal activity was limited. However, as shown in study no. 5 (Table 7), in the presence of 1.0% bovine serum albumin at pH 0.0, the antifungal efficacy against S. typhlyurluy on chicken muscle was accelerated over the EDTA concentration range up to 100 mM.

Thus, antibacterial agents containing air and a low concentration of a chelating agent such as EDTA in the range of 0.1 mM to 20 mM can be extremely effective in eliminating or preventing food contamination caused by Gram-negative bacteria.

Example VIII. Grading of the activity of the drug against Gram-negative bacteria (S. tnphlyurluy).

At the optimal concentration of the chelating agent, the bactericidal effectiveness in Tris buffer against Gram-negative bacteria appears to be significant, as shown in Table 0.

Table 0

Study Number Initial count of viable bacteria EDTA (mM) BSA (%) jg/ml 0 0.1 0.3 1.0 3.0 10 30 100 1 6 x 10 ⁶ 0 0 100 % fr life pc 2 hours iach 51.3 1-6 2 6 x 10 ⁶ 1.0 1.0 62 0.7 0.00 0.01 0.05 0.01 CIO' ⁴ -

In study no. 2 (Table 0), it can be concluded that just 0.3 pg/ml nirin with 1.0 mM EDTA in 20 mM Tois, at pH 0.0, in the presence of 1% bovine serum albumin (BSA) significantly reduces the survival time of S. Snphlmurluy. The bactericidal agent is as active against Gram-negative bacteria as aisin alone is against Gram-positive Stoeptococci bacteria.

Example IX. Assessment of the activity of aisin against Goam-ujkmnyy bacteria (S. typhimurium).

Beyond the optimal concentration of the stimulant, the effectiveness of the bactericidal agent against Gram-negative bacteria in the presence of biological tissue was demonstrated for S. typtiimurium on chicken muscle, as shown in Table 9.

Table 9

Study Number pH Initial number of viable cells EDTA (y+) BSA (%) Nlryar pg/ml 0 10 100 200 300 1 0.0 3 x 107 0 0 100 % over life after 2 hours 2 0.0 3 x 10 ⁷ 1.0 1.0 27 0.26 0.000 0.007 0.006

Chicken muscle pieces were cleaned with sodium hypochlorite and iodine in polyvinylpyrrolidate before use. To purify the tissue, chicken muscle pieces were immersed in a suspension

ABOUT

100 cells/ml S. Syphlyuoluy in 20 mM Tois HCl, pH 0.0. Excess moisture was removed from the immersed pieces by removing the liquid. The tissue was placed in an appropriate buffer containing the appropriate

163,983 sufficient nisin was added to cover the tissue and incubated for 2 hours at 37°C, after which the tissue was removed and coated with the appropriate Phage buffer. Bacteria remaining in the test solution were separated by centrifugation, washed with Phage buffer, and combined with bacteria washed from the tissue with Phage buffer. The combined samples (referred to as non-compact cells) were serially diluted, and 100 µl/aliquot were plated to determine the number of surviving bacteria.

Example X. Activity against Gram-negative bacteria (S. typhimurium) of nisin, EDTA and methylparaben.

A microbicide containing nisin and EDTA, which combines with the known food preservative methylparaben, has been shown to be effective against Gram-negative bacteria only, as shown in Table 10.

Table 10

Study Number Initial count of viable bacteria Nisin (g/ml) EDTA ^b (mM) % Methylparaben 0 0.1 1.0 1 3 x ^()6 0 10 % pr 11.8 life after 2 hours 1.0 zinach ^c 1O-4 2 3 x 1θ6 300 10 0.03 < 10-3 < 10-4

b50 mM sodium acetate buffer, pH 5.0 cells not compacted

Chicken muscle pieces were cleaned with sodium hypochlorite and iodine in polyvinylpyrrolidone before use. To inoculate the tissue, chicken muscle pieces were immersed in a suspension

O cells/ml of S. typhimurium in 50 mM sodium acetate buffer, pH 5.0. Excess moisture was removed from the immersed pieces by draining the liquid. The tissue was placed in an appropriate buffer containing sufficient nisin to cover the tissue and incubated for 2 hours at 37°C, after which the tissue was removed and covered with the appropriate Phage buffer. Bacteria remaining in the test solution were separated by centrifugation, washed with Phage buffer, and combined with bacteria washed from the tissue with Phage buffer. Pooled samples (referred to as non-compact cells) were serially diluted, and 100-µl aliquots were plated to determine the number of surviving bacteria.

In study 1 (Table 10), methylparaben in the presence of 10 mM EDTA was effective against S. typhimurium only at a concentration of 1.0%. In study 2 (Table 10), however, in the presence of 300 pg/ml nisin, the efficacy of methylparaben and nisin against S. typhimurium was significantly increased.

Nisin- and EDTA-containing formulations significantly improve the usability of the food preservative methylparaben. Furthermore, these substances can significantly reduce the concentrations or eliminate the need for known, but less desirable, food preservatives such as methylparaben.

Example XI. Activity against Gram-positive bacteria (Staphylococcus aureus) of nisin and a chelating agent.

The activation of nisin by the chelating agent is pH dependent. The data in Table 11 confirm that at pH 5.0, nisin is somewhat more bactericidal against S. aureus than at pH 8.0. At pH 5.0, EDTA does not enhance nisin activity against S. aureus, and at EDTA concentrations higher than 10 mM, EDTA is an inhibitor of nisin's bactericidal activity. However, the bactericidal activity of nisin enhanced by EDTA at pH 8.0 is significantly higher than that of nisin alone or in combination with EDTA at pH 5.0.

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Table 11

The effect of pH on the action of EDTA on the bactericidal activity of nisin against Staphyllcoccus aureus

Lowlands EDTA mm PH ug/ml Oh 0,( 0.3 (,0 3.0 (0 30 100 % Survival after 2 hours 8.0 0 00 - (00 8( H)0 HU HU - 8.0 3.0 7.4 0.03 0.0( 0.2 0.4 3 56 - 5.0 0 (00 - - - (00 - - - 5.0 3.0 0.6 (,0 (,3 (,4 (,8 - 34 80

aNumber of initially viable bacteria: 8.0 x ^6 cfu/ml

Incubations were carried out in 50 mM sodium acetate buffer, pH 5.0 or 20 mM Tris-HCl buffer, pH 8.0 at 37*C.

The bactericidal activity of nisin itself, according to literature reports (see Hurst), is greatest at pH 5.0 or lower, and the data in Table 1 confirm this. On this basis, it was thought that the activation of nisin bactericidal activity by EDTA against S. aureus would be greatest at lower pH. However, as can be seen from Table K, contrary to these expectations (see Table 6), EDTA was not observed to improve nisin activity against Gram-positive bacteria at pH 5.0. However, inhibition of nisin activity by high concentrations of EDTA was still observed at pH 5.0. This indicates that activation of nisin by a chelating agent occurs only within a certain range of chelating agent concentrations, and with respect to Gram-positive bacteria, it is pH-dependent, with a preferred pH range higher than pH 5.0.

Example XII. Activity of nisin and chelating agent 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 bacterial pathogen, but are also observed on Streptococcus mutans^, responsible for dental caries (Table (2A)), Listeria monocytogenes, a foodborne pathogen (Table (28)), and on mixed populations of axillary Coryneform bacteria, responsible for body odor (Table (2C)).

Table (2A

Effects of EDTA on the bactericidal activity of nisin against Streptococcus Putans

Lowlands EDTA mm pH ug/ml 0 0.0( 0,( 0.3 (,0 3.0 30 (00 X experiences after 2 hours 8.0 0 00 - - - - - - - - 8.0 0,( 4.3 (,8 0.04 0.02 0.06 25 H)0 (00

aThe number of initially viable bacteria is 6.0 · i06 cfu/ml

Incubation was carried out with 20 mM Tris-HCl, pH 8.0 at 37'C.

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Table 128

Effects of EDTA on the bactericidal activity of nisin against Listeria monocySogenes

Lowlands EDTA rtM pH pg/ml 0 0.1 0.3 1.0 3.0 10 30 100 8.0 0 100 % pr life after 84 2 hours ³ 8.0 3.0 0.71 0.04 0.04 0.02 0.1 0.64 10 14

and 6

Number of initially viable bacteria: 6.0 10 cfu/ml

Incubations were carried out in 20 mM Tris-HCl, pH 8.0 at 37*C.

Table 12C

Effects of EDTA on the bactericidal activity of nisin against Coryneform bacteria

Lowlands EDTA mm pH pg/ml 0 0.1 0.3 1.0 3.0 10 h survival after 2 hours ^a 8.0 0 100 - 4.6 3.6 8 36 0.0 3 0.22 0.03 0.0009 0.1 - 0.16

a) 6

Number of initially viable bacteria: 10 10 cfu/n^l.

Incubations were carried out in 20 mM Tris-HCl, pH 8.0, at 37°C.

Example XIII. Rate of bactericidal activity of nisin activated by chelaSor.

The bactericidal preparation containing nisin with EDTA is a rapidly acting bactericidal agent, as illustrated by the data in Table 13A. Suspensions of Gram-positive S. mutans bacteria at a concentration of 1q7 cells/ml were incubated in 20 mM Tris buffer, pH 7.3, at 37°C, with the indicated concentrations of nisin activated with 1 mM EDTA. The suspensions were incubated for various times ranging from 0.5 to 60 minutes with the bactericidal preparation. The bactericidal efficacy of the bactericidal preparation was assessed by determining the percentage of surviving bacteria. EDTA-activated nisin as little as 10 pg/ml in this preparation can reduce bacterial counts six-fold within 1 minute.

Rapid bactericidal activity is a prerequisite for effective disinfection. Therefore, the preparations presented herein are intended as effective bactericidal substances for mouthwashes, rinses, toothpastes, or other similar tooth cleaning agents active against cariogenic S. mutans.

The effective activity of EDTA-activated nisin against Gram-negative bacteria after 2-3 hours is shown in Examples 1-7. The rapid activity of EDTA-enhanced nisin against Gram-negative bacteria is also evident and is illustrated by the data in Table 138.

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Table 13A

Kinetics of bactericidal activity of EDTA-activated nisin against Streptococcus mutans

Incubation time (minutes) Nisin pg/ml with 1.0 mM EDTA ° ¹ ’ .. 10 ³⁰ 100 0.5 % over life ^and < 10‘4 1 - - - < 10-4 <10-4 <110-4 3 100 0.5 0.002 <110-4 <10-4 - 15 - 0.03 <10-4 <104 - - 30 - - CIO' ⁴ - - - 60 100 0.003 - - - -

Number of control, live cells: 1.0 · ¹⁰⁷ cfu/ml, incubation was carried out in 20 mM Tris-HCl, pH 7.3 at 37°C.

Table 136

Rapid bactericidal activity against Escherichia coli of EDTA-activated nisin

mM Nisin pg/ml 0 0.3 1.0 3 10 30 100 Survival S after 1 minutea 1.0 100 100 56 0.37 0.013 0.015 0.008

1.0 10 ⁷ cfu/ml s, pH 7.0 at temperature

Initial number of viable bacteria: incubation was carried out in 20 mM Tri *C.

Example XiV. The effect of divalent cations on E0TA improving nisin activity.

Divalent cations bind to EDTA and other chelating agents, and one might expect EDTA-activated nisin to be neutralized. However, as can be seen from the data in Table 14, the bactericidal activity of nisin against S. mutans is enhanced by 1 mM EDTA even in the presence of 1 mM Ca ⁺⁷ , and only concentrations above 3 mM Ca ⁺⁷ inhibited EDTA-activated nisin. This is particularly important for oral applications, which involve high concentrations of calcium ions.

Table 14

Rapid bactericidal activity against Streptococcus mutans of EDTA-activated nisin in the presence of a divalent cation

Lowlands CaCl ₂ mM 0 0.1 0.3 1.0 3 10 % survival after 1 minute a 0 100 3 2.9 3E 0.0042 0.0042 0.052 18 30 ^e 0.0019 0.0003 0.0004 0.06 6.8 100 ^E 10-4 10-4 10-4 0.0001 1.5

163 983 ^Ε 1 mM Na ₂ EDTA a 2

Initial number of viable bacteria: 1.0 10 cfu/ml

Incubations were carried out in 10% calf fetal fluid at 3°C.

Example Χν. Activity of nisin and surfactant against Gram-positive bacteria.

The bactericidal activity of nisin can also be significantly improved by the addition of surfactant alone. This is best illustrated by the limited nisin concentration (0.2 µg/ml) shown in Table 15A. At concentrations up to 0.1% of the permitted food surfactant, monolaurate, has negligible bactericidal activity against Streptococcus agalactiae in a complex milk environment. Nisin, at concentrations up to 0.2 µg/ml, similarly exhibits no significant bactericidal activity in milk. However, the combination of these two agents, 0.1H monolaurate and nisin at a concentration of 0.2 µg/ml, is remarkably effective against S. agalactiae. This bactericidal preparation is more than 100 times more active than would be expected if the effect were additive and 10,000 times more active than either of the individual components. Therefore, if the use of nisin is limited by the activity that can be achieved, a bactericide containing nisin with a surfactant would be expected to be more effective.

A case in which the use of nisin is limited by its achievable activity is illustrated by the data in Table 158. Although nisin, and particularly a bactericide containing nisin and EDTA, is bactericidal against L. monocytogenes, the data in Table 15B show that in a complex environment similar to milk, the achievable activity of nisin against this microorganism is limited. However, a bactericide composed of nisin with the glyceride monooleate is effective in milk against the foodborne pathogen, even though monooleate itself has no bactericidal activity against this microorganism.

Table 15A

Bactericidal activity of nisin against Streptococcus agalactiae in milk at 3?*C (activation of nisin with monolaurate)

Lowlands % Monolaurate 0 | 0.01 0.1 % survival at 2 hours and 0 100 100 4.5 0.02 100 100 0.2 0.2 2.2 0.05 0.0008

Initial number of live bacteria: 6.0 · 10? cfu/ml Incubations were carried out in milk at 3?°C.

Table 158

Bactericidal activity of nisin against Listeria monocytogenes in milk at 3?’C (activation of nisin with monooleate)

Nisin (pg/ml) % Monooleate 0 0.1 1.0 % 2nd hour experience and 0 100 6? 63 100 0.56 10-3 10-4

and ?

Initial count of viable bacteria: 5.0 x 10' cfu/ml

Incubations were carried out in milk at 31*C.

163 983

Example XVI. The following Tables 16A - 16G illustrate the biocidal activities of various formulations of the agent according to the invention.

Table 16A

Subtilin and chelating agent activity against Gram-negative bacteria Salmonella typ Kimurium

Substrate Subtilin (ug/ml) E0TA iM Buffer pH 8, ppm 0 5 % survival 2 hours ³ 100 ^and 5 0.008 0.0004 0.004 0.04 11.3 Buffer pH 5.0° 5 0.06 0.04 0.0003 0.0003 0.08 6

a Initial number of viable bacteria: 3 x 106 cfu/ml ^b 20 mM Tris-HCl, pH 8.0 c 50 mM Na acetate, pH 5.0

Table 16B

Activity of nisin and chelating agents against Escherichia coli

Nisin (ug/ml) 0 Chelating agent 1 mM b EDTA DTPA COTA EGTA 100 100a 0.61 1-minute survival rate 4.2 | 4.3 10.9

and 7

Initial viable count: 1 x 10' cfu/ml b 10-mM Tris-HCl, pH 7.3

Table 16C

Activity of nisin and CaNajEOTA against Streptococcus mutans

Substrate Nisin (ug/ml) Cal^EDTA mM 0 1 1-minute survival rate 10% FCSb 3.0 3.0 0.052

Initial viable count: 3 x 106 cfu/ml b 10% calf fetal fluid

163 983

Table 16D

Activity of nisin and citrate against Streptococcus mutans

Substrate Ni^na (pg/ml) Citrate mM 0 0.1 1 3 10 100 Buffer about 2-hour survival rate pH 7.3 ^b 0.3 3.65 1.25 0.1 0.42 0.2 0.35

Initial number of viable bacteria: 3 x 1(]6 cfu/ml b 10 mM Tois-HCl, pH 7.3

Table 16E

Activity of methylene blue and citrate against Escheoichia coli

Substrate Lowlands % citrate (pg/ml) Oh 0.1 0.3 1 3 Buffer about 0 100 % survival 100 1 minute 56 73 100 PH 3.5 100 100 0.0025 0.0006 0.56 14.6

Initial count of viable bacteria: 5 x ¹⁰⁷ cfu/ml

Table 16F

Activity of ndrin and nonionic surfactants against Escheoichia coli

Substrate Ni^na (pg/ml) + 1 mM EDTA Tweeny 0 Tw2O Tw4O Tw0O Buffer about 0 100a % Survival 100 a 1 minute 100 100 pH 7.0b 3 0.25 0.001 0.007 0.007

a Initial number of viable bacteria: 1 x 1O ⁷ cfu/ml b 20 mM Tris-HCl, pH 7.0

Table 16G

Activity of nlznan and αιafaScoycrac^a surfactant against 3SrepSococcus mutans

Substrate Nisin (pg/ml) + 1 ml EDTA % kokaradopoopniabkSaina 0 0.1 % survival 1 yinuSya 10% FCS ^b 0 100 11 3 30—.— ..and 0.07

³ Initial viable count: 1 x ¹⁰⁷ cfu/ml 10% calf fetal fluid.

Publishing Department of the Polish Patent Office. Circulation: 90 copies. Price: PLN 10,000.

Claims (7)

Patent claims 1. A bactericidal agent containing bacteriocin and excipients, characterized in that it comprises a bacteriocin containing lanthionine in a concentration between 0.1 µ / ml and 300 pg / ml and a chelating agent in a concentration between 0.1 mM and 20 mM. 2. a bolt according to claim The process of claim 1, further comprising a surfactant concentration between 0.01% and 1.0%. 3. an agent according to claim 1 The method of claim 1 or 2, characterized in that the lanthionine-containing bacteriocin is a substance selected from the group consisting of nisin, subtiline, epideomine, cinnamicin, ankowenin and Pep 5. i. an agent according to claim 1 The method of claim 1, wherein the chelating agent is a substance selected from the group consisting of alkyldiamine tetraacetates, such as ethylenediaminetetraacetic acid (EOTA), CaEDTA, NajCa EDTA or citrate. 5. middle ełgg slr. 4, Znameenny that eea ^ak o eerracctan alkylddiamin contains ethylkiodiayinoSetrrocSawy acid (EDTA) and as a bacterium it contains lowland. 6. blower according to claim 2. characteristic t y m. that the surfactant is a substance selected from the group consisting of ToiSann, Tweeny, Glycerides, fatty acids, emulsifiers, quaternary compounds, AmfaSeoncrae and anionic surfactants. 7. bolt according to claim A compound as claimed in claim 1 or 2, characterized in that it contains lanSlonin-containing bαkteriacin selected from the group consisting of mine, subtinine, epidine, cinnamycin, duramycin, αcavaline and Pep 5, and a chelating agent selected from the group consisting of tetrαactαin alkllodlaylate and citrate in such amounts that the germicide is more effective against at least one bacterium selected from the group consisting of Stap ^ 'coccus aureus, Streptococcu s muisnn, Listeri a monocytogenme, Streptococcu s agataca, Cornyeform bacteria, Salmonella typhimurium, Es ^ erich ^ coli , Klebsien a ρηθυΐϋ ^,., Pseudornonas aeruginosa, Bacterioides gingiualis and A ^^ both ^ l ^ s actlaamycetescomitans. 8. the center of the eel. ast ^ z. ,, known t m m, e. a ^ o. ildlaodrine xraaosαan contains (EDTA) Ethylene DαminoSetoacetic Acid. * * *