RU2092180C1 - Antibacterial composition - Google Patents

Abstract

FIELD: medicine, food industry. SUBSTANCE: composition has the following components, wt.-%: nisine, 0.00001-0.3; and EDTK, 0.003-0.7 and can contain additionally surface-active substance (Triton X-100, Tween-20 or monolaurin) taken at amount 0.01-1.0 wt.-%. The obtained composition exhibits the broad effect spectrum. EFFECT: enhanced effectiveness. 2 cl, 19 tbl

Description

 The invention is a partial continuation of application N 209861, filed June 22, 1988. Nisin is a polypeptide with antimicrobial properties, which is produced in nature by different strains of Streptococcus lactis bacteria.

 It is a well-known food preservative that slows down the growth of spores of certain types of gram-positive bacteria.

 Although in some sources it is mistakenly and inaccurately called an antibiotic, nisin is more appropriate for bacteriocins, i.e. a protein substance produced by bacteria that has antibacterial activity only against those species that are close to their species by origin.

 Nisin is a naturally occurring preservative that is found in small concentrations in milk and cheese and is considered completely non-toxic and does not cause allergic reactions in humans. The Food and Drug Administration has recognized it as safe for direct use as a food ingredient in cheese pastes, processed cheese pastes, in cheeses and pastes with fruits, vegetables or meat. In addition, since it is a polypeptide, any nisin residues remaining in foods are quickly digested.

 A full description of the properties of nisin is given in [1]. This publication describes everything that is currently generally known about nisin.

 The nisin produced by Streptococcus lactis is commercially available as an unclean preparation, nizaplin TH, from Aplin Barrett Ltd. Dorset, England, and it can be obtained by isolating nisin from cultures of Streptococcus lactis, followed by concentration by known methods. Such methods for producing nisin have been published by using altered strains of Streptococcus (see US Pat. No. 4,716,115 to Jouzaler et al, December 29, 1987) Nisin can also be obtained using recombinant DNA technology.

 Nisin has been used effectively as a preservative in dairy products such as cheeses, cream and milk. The use of nisin in cheese making and cheese products is the subject of recent patents (see US patents NN 4584199 and 4597972). The use of nisin to inhibit the growth of certain gram-positive bacteria is well documented. However, its complete success and acceptability as a food preservative is hindered by the idea that nisin is ineffective against gram-negative and many gram-positive bacteria. Gram-negative bacteria are almost always present along with gram-positive and are the main source of food spoilage (see Taylor, US Pat. No. 5,584,199, issued April 22, 1986, and Taylor, US Pat. No. 4,597,972, issued July 1, 1986. Tsai and Saudine , "Conjugae Trausfer of Nisin Plasmid Genes from Streptococcus lactis 7962 to Leuconostoc Dextranieum 181, Applied and Environmentae Microbiology, Feb. 1987, p. 352;" A Naturae Preservative ", Food Engineering Int'l, May, 1987, pp. 37 37 ; "Focus on Nisin" Food Manufacture, March, 1987, p. 63).

 It has now been found that, contrary to previous estimates, compositions comprising nisin, in combination with various antibacterial drugs, have a wide range of bactericidal activity against gram-negative bacteria, as well as increased activity against a wider range of gram-positive bacteria than nisin in pure form.

 Increased bacterial activity against gram-positive bacteria exists in a wider range of pH values than previously indicated. The invention provides compositions based on nisin or other lanthionin-containing bactericides in combination with various non-bactericidal preparations, for example, chelating agents or surfactants.

 The invention also provides these compositions in dilute or suspended form in a suitable vehicle, resulting in wider spectrum bactericides.

 In particular, it was found that a solution with a nisin content of 0.1300 μg / ml in the presence of 0.1-20 mM, which promotes the formation of a chelating agent, such as EDTA, virtually eliminates the growth of gram-negative bacteria such as Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Bacterioides gingivalis, Actinobacillus actinomycetescomitans u Klebsiella pneumonial and is more active against gram-positive bacteria such as Staphylococcus aureus, Streptococcus mutans, Listriamonocytogenes Streptococinum agalact.

 Although the enhancement of activity with the aid of a chelating compound-forming substance is concentration dependent, in contrast to the expected EDTA concentration above 20 mM, the bactericidal activity of nisin was lowered. However, in the presence of a protein carrier and polyvalent polymers such as serum albumin, collagen, gelatin, casein and keratin, the decrease in the activity of nisin by the presence of EDTA at a concentration of more than 20 mM decreases, thus providing a useful range of concentrations of EDTA in which the presence of EDTA enhances the activity of nisin.

 It was also found that a solution comprising 0.1 300 μg / ml nisin and 0.1 20 mM providing a chelate compound of the substance further increases the effectiveness of nisin against gram-positive and gram-negative bacteria in the presence of 0.01 1.0% Surfactant.

 In addition, it was found that in the presence of surfactant only, nisin has an increased activity against gram-positive bacteria.

In the invention, suitable chelating agents are EDTA, Ca EDTA, Ca a ₂ EDTA and other alkyldiamines tetraacetates, EGTA and citrates. Surfactants valuable as brighteners and suitable for combination with nisin, with or without EDTA include non-ionic surfactants Tweens, Tritons and glycerides, ionic surfactants such as fatty acids, quaternary compounds, anionic surfactants such as sodium dodecyl sulfate, and amphoteric surfactants such like cocamidopropyl betaine, and emulsifiers.

 Since gram-positive and gram-negative bacteria are almost always present together in food products, the effectiveness of nisin-based compositions against gram-negative bacteria such as Salmonella typhimurium, Escherichia coli, Klebsiella pneumonial, Pseuclomonas aeruginosa, Bacterioides gingivalis, Actinobactesus actinomy andomy other gram-negative pathogenic and gram-positive bacteria are very valuable.

 These bactericides, in particular, are intended to prevent spoilage of raw ingredients, prepared foods and drinks by pathogenic bacteria and other microorganisms. In relation to food products, processing of meat, especially poultry, eggs, cheeses and fish, and processing of food packaging and equipment used are used.

 In addition, food preservatives are used, such as in ready-made cheeses, cream, milk, dairy products and in cleaning poultry, fish, meat, vegetables, dairy products and food processing equipment. The use of nisin-based compositions should not be limited to applications in the food industry; they could be used in any area where there is a need to destroy gram-negative and gram-positive bacteria.

 These compositions may be dissolved in a suitable vehicle, aqueous solvent or buffer, or suspended in any suitable liquid, colloidal or polymeric medium to produce a bactericide.

 These compositions or bactericides can be included in ointments or dressings for medical use such as treating infections, dressings for wounds or surgical implants and as a number of skin antiseptics and gargles, disinfecting sterile products, cleaning agents and cosmetics.

 These bactericides can be used to disinfect medical instruments, preoperative floor wipes, etc. These bactericides are especially useful in such circumstances when it is necessary to disinfect the medium, but where chemical preparations cannot be used because of the danger of corrosive or other toxic effects.

 Unlike most broad-spectrum germicides, which are regulated by the presence of a complex of organic compounds, the compositions of the invention are effective as bactericides in the presence of an organic substance such as milk or whey.

 It is known that nisin optimally inhibits the growth of several related gram-positioning bacteria, in particular some gram-positive spore-forming bacteria at pH 5.0. The bactericidal effect of nisin in solution with a chelating agent was surprisingly fast and greatly enhanced against a number of gram-positive bacteria at pH greater than 5.0 and moreover, it was activated against gram-negative bacteria at both basic and acidic pH, preferably in range 5.0 to 8.0.

 This unexpectedly fast and wide range of bactericidal activity of the nisin activated by the chelating agent makes it suitable for use, among other areas, as a disinfectant.

 Nisin belongs to the class of peptide bactericides containing lanthionine. Subtiline, epidermis, cinnamycin, duramycin, ankovenin and Pep 5 also belong to this class. Each of these peptide bactericides is produced by different microorganisms. However, it was found that subtiline obtained by culturing Bacillus subtilis and epidermis obtained by culturing certain cultures of Staphy lococcus epidepmidis have a molecular structure very close to the molecular structure of nisin (see Hurst; pp. 85 86 and Schnell et al, Nature, 333 , 276 278).

 Therefore, it is believed that as a result of molecular identity, other lanthionins containing peptide bactericides will also be effective, like nisin, in combination with chelating agents and non-ionic surfactants in the fight against lesions caused by gram-positive and gram-negative bacteria.

 The effectiveness of nisin and other lanthionine-containing peptide bactericides and compositions with them against gram-negative bacteria is surprising, since the prior art indicates the absence of such activity in nisin. The enhanced activity of nisin against gram-positive bacteria in the presence of ethylenediaminetetraacetic acid (EDTA) at pH greater than 5.0 is unexpected since it was previously believed that nisin activity was optimal at pH 5.0.

 In addition, the discovery of the effectiveness of nisin and lanthion-containing peptide bacteriocidal compositions such as bactericides satisfies the long-awaited need for food preservation, which suffered from a lack of an acceptable, natural, non-toxic agent effective against a wide range of bacteria.

 To demonstrate the excellent and unexpected rapid activity of a composition containing nisin, EDTA and / or various surfactants against gram-positive and gram-negative bacteria, a series of experiments were conducted with these bactericides. These experiments are illustrative and are not intended to limit the invention. It can be expected that other lanti-containing peptide bacteriocins will be effective nisin substitutes and that chelating agents other than EDTA will be effective substitutes for EDTA.

All tests in the following examples were carried out at 37 ^o C. The effectiveness of the increased wide range of bactericides was determined by measuring the bactericidal activity as a percentage of the surviving bacteria after treatment of the bactericides. Usually, after growing 10 ⁷ cells per ml of a suspension of the target species with a new bactericide during this time interval, the bacteria were collected by centrifugation for 2 minutes.

The in vitro bacterial pellet was washed to the bactericide removal state with a rescue buffer, hereinafter referred to as Phage buffer (50 mM Tris-HCl pH 7, 8, 1 mM MgSO ₄ buffer; 4 mM CaCl ₂ ; 0.1 M NaCl and 0.1% gelatin) , resuspended and diluted with releases in Phage buffer, and then 100 ml of suspended bacteria were sprayed onto plates with nutrient agar medium.

Bacterial survival was determined by counting colony forming units (CFU) after growing for 24–28 hours at 37 ^° C. An effective bactericide in accordance with the invention is one that allows less than 0.1% of the initial number of viable bacteria to survive.

Example 1. The activity of nisin and a chelating agent against gram-negative bacteria (S. typhimurium)
As shown in the table. 1, two tests were carried out in 20 mM Tris, pH 8.0 at 37 ^° C to exert exposure to a bactericide containing nisin and one chelating agent EDTA. Test 1, control, was performed without EDTA and shows the effect of one nisin on gram-negative bacteria S. typhimurium.

 Increased concentrations of nisin do show some activity, but even this activity of increased concentrations in the absence of EDTA gives only 1.6% survival at 100 mg / ml nisin, which is not consistent with the goals of the food preservative. The level of bactericidal activity obtained from nisin and EDTA is significant.

 Test 2 (Table 1) was performed using nisin plus 20 mM EDTA, and it shows the excellent activity of the nisin-based composition in killing target gram-negative bacteria.

Tab. 1 shows that in test 2, at a concentration of 20 mM EDTA and 30 mg / ml nisin, the bactericide exhibits significant bactericidal activity against S. typhimurium, while at concentrations of nisin 100 mg / ml and higher, the bactericide based on nisin and EDTA actually kills bacteria (survival rate less than 10 ^-4 , which indicates the absence of surviving bacteria in the sample). Thus, the combination of EDTA and nisin shows a synergistic activity that is 1000 times higher than the activity of nisin alone.

Example 2. The activity of nisin, a chelating agent and a surfactant against gram-negative bacteria (S. typhimurium)
Four tests (Table 2) were conducted to determine the effect on S. typhimurium of a bactericide containing nisin and EDTA and surfactant Triton X-100 in 20 mM Tris, pH 8.0 at 37 ^o C. The control test (test 1) is identical to the control the test of example 1 (table. 1).

 The test (Table 2) was performed using nisin and 1.0% Triton X-100, but without EDTA. The presence of a single detergent suppresses the activity of nisin against gram-negative bacteria, and nisin was ineffective. However, in tests 3 and 4 (Table 2), which represent the invention, the presence of 20 mM EDTA in combination with Triton X-100 is a bactericide that significantly increases the bactericidal activity of nisin against S. typhimurium.

 In addition, the combination of Triton X-100 with EDTA, but without nisin, was effective, although to a lesser extent than in the presence of nisin. Although in both tests 3 and 4 (Table 2) nisin combinations were very effective, a concentration of 1.0% Triton X-100 (Test 4, Table 2) was most effective.

 The presence of a non-ionic surfactant, Triton X-100 in combination with EDTA enhances the activity of nisin against gram-negative bacteria even more than the bactericide containing only nisin and EDTA (example 1).

Example 3. The activity of nisin, a chelating agent and a surfactant against gram-negative bacteria (S. typhimurium)
Tab. 3 shows an increased activity against S. typhimurium bactericide containing nisin, 20 mM EDTA chelating agent and Tween 20 nonionic surfactant in 20 mM Tris, pH 8.0 at 37 ^o C. As in the case of Triton X-100 (example 2), the combination of nisin and EDTA with (1%) Tween 20 is more effective.

Example 4. The activity of nisin, a chelating agent and a surfactant against gram-negative bacteria (Escherichia coli)
The effect of a bactericide containing nisin and EDTA on gram-negative bacteria E. coli is shown in table. 4.

Tests with and without EDTA were performed in 20 mM Tris buffer solution, pH 8.0 at 37 ^o C with an initial number of viable E. coli bacteria of 1 • 10 ⁷ cells / ml. The effects of bactericides were measured as a function of the percentage of bacterial survival after 2 hours.

 In test 1 (control, Table 4), without EDTA, nisin exhibits low non-significant activity against E. coli. In test 2 (Table 4), however, with 20 mM EDTA, the bactericidal composition exhibits significant activity against E. coli bacteria.

 Activity increases in effectiveness as the concentration of nisin increases. The combination of nisin with EDTA as a bactericide shows a 1000-fold synergistic increase in efficacy against E. coli. In tests 3 and 4 (Table 4), it can be seen that Triton X-100 does not have significant bactericidal activity against E. coli.

 In fact, Triton X-100 appears to suppress the activity of nisin against gram-negative bacteria, as was established with C. typhimurium (Table 2). However, the overall increase in nisin using EDTA significantly changes the inhibitory effects of Triton X-100, as can be seen in the table. 2 and 4.

 Thus, it is obvious that a bactericide containing nisin and a chelating agent such as EDTA is an effective food preservative against various gram-negative bacteria even in the presence of surfactants.

Example 5. The activity of nisin and a chelating agent against gram-negative bacteria (Klebsiella pneumonial)
The effect of a bactericide containing nisin and EDTA on gram-negative K. pneumonial bacteria is shown in Table. 5.

Two tests, one with EDTA and the other without (control), were carried out in 20 mM Tris buffer, pH 8.0 at 37 ^° C with an initial number of viable bacteria of 10 ⁷ cells / ml K. pneumonial. The effect was measured as a function of the percentage of surviving bacteria after 2 hours.

 In test 1 (control, Table 5) without EDTA, nisin exhibits little insignificant bactericidal activity against K. pneumonial. In test 2 (Table 5), in the presence of 20 mM EDTA, the bactericide exhibits significant activity against K. pneumonial. Activity increases in effectiveness with increasing concentration of nisin.

Example 6. The activity of nisin against gram-negative bacteria (Salmonella typhimurium) depends on the concentration of the chelator
The data in the table. 6 show that the increased activity of nisin against gram-negative bacteria (S. typhimurium) depends on the concentration of EDTA in 50 mm sodium acetate, pH 5.0 or 20 mm Tris, pH 8.0 at 37 ^o C.

 In tests 1 and 3 (control, table. 6), using concentrations of EDTA without nisin, a small insignificant activity against S. typhimurium is shown at pH 5.0 (1) or 8.0 (3). In tests 2 and 4 (Table 6), where 10 mg / ml nisin was presented in combination with EDTA, bactericides showed significant activity against S. typhimurium. The activity of bactericides is similar to acidic pH (5.0) and basic pH (8.0), contrary to the fact that the activity of nisin alone against gram-positive bacteria is optimal at pH 5.0.

 The amplification of nisin by EDTA depends on the concentration, being optimal in the range of 0.2 to 10 mM at pH 5.0 and 8.0. Surprisingly, at concentrations greater than 10 mM EDTA, the amplification of nisin with EDTA decreases; this decrease in activity is significantly greater at pH 8.0 than at pH 5.0.

Example 7. Nisin and a chelating agent against gram-negative bacteria (S. typhimurium)
Enhanced nisin activity with EDTA against gram-negative bacteria in the presence of biological tissue was shown using S. typhimurium on chicken muscle and is shown in table. 7.

a) unattached cells;
b) contains 1% bovine serum albumin (BSA).

Cultivation was carried out in 50 mm sodium acetate, pH 5.0 or 20 mm Tris, pH 8.0 at 37 ^o C.

Before use, the cubes of the chicken muscle were disinfected with sodium hypochlorite and povidone iodine. To infect tissue, chicken cubes were immersed in a suspension of 10 ⁸ cells / ml S. typhimurium in 20 mM Tris HCl, pH 8.0.

Excess moisture is removed from submerged cubes by suction. Chicken tissue samples were placed in a sufficient buffer containing a nisin-based composition to cover the tissue, and grown for 2 hours at 37 ^° C, after which this tissue was placed in a Phage buffer sufficient to cover the tissue. The bacteria remaining in the test solution were collected by centrifugation, washed with Phage buffer and combined with bacteria washed from the tissue using Phage buffer. The pooled samples (the term "non-adherent cells") were diluted with outlets and 100 ml aliquots were placed in plates to determine bacterial survival.

 In tests 1 and 3 (Table 7), in the absence of nisin at pH 5.0 and 8.0, one EDTA did not significantly affect the survival of S. typhimurium. In tests 2 and 4 (Table 7), where 300 mg / ml nisin was present, bactericides showed significant activity against S. typhimurium on chicken muscle at pH 5.0 and 8.0.

 The amplification of nisin by EDTA depends on the concentration, and the optimal concentration is in the range of 0.3 mm 10 mm EDTA at pH 5.0 and 8.0. At concentrations greater than 10 mM EDTA at pH 8.0, activation of nisin EDTA decreases. However, as shown in test 5 (Table 7), in the presence of 1.0% BSA at pH 8.0, the effectiveness of nisin against S. typhimurium in chicken muscle is expressed in the concentration range of EDTA up to 10 mM.

 Thus, bactericides containing nisin and low concentrations of a chelating agent, such as EDTA in the range of 0.1 to 20 mM, can be extremely effective in eliminating or preventing food contamination by gram-negative bacteria.

Example 8. Titration of the activity of nisin against gram-negative bacteria (S. typhimurium)
The optimal concentration of the chelating agent, the effectiveness of the bactericide in Tris buffer against gram-negative bacteria were significant, as shown in the table. eight.

 In test 2 (Table 8), it can be seen that 0.3 μg / ml nisin with 1.0 mM EDTA in 20 mM Tris at pH 8.0 in the presence of 1% bovine serum albumin (BSA) significantly reduces the survival of S. typhimurium . Said bactericide is also active against gram-negative bacteria, like pure nisin against gram-positive Streptococci.

Example 9. Titration of the activity of nisin against gram-negative bacteria (S. typhimurium)
At the optimal concentration of the chelating agent, the effectiveness of the bactericide against gram-negative bacteria in the presence of biological tissue was demonstrated on S. typhimurius on chicken muscle and is presented in table. 9.

Cubes of chicken muscle were washed with sodium hypochlorite and providone iodine before use. To inoculate tissue, cubes of chicken muscle were immersed in a suspension of 10 ⁸ cells / ml S. typhimurium in 20 mM Tris HCl, pH 8.0. Excess moisture was drained. The tissue was placed in a sufficient buffer containing nisin composition to cover the tissue, and incubated for 2 hours at 37 ^° C, after which the tissue was transferred to the Phage buffer so that the solution covered the tissue.

 The bacteria remaining in the subsequent solution was collected by centrifugation, washed with Phage buffer and combined with bacteria washed with tissue with Phage buffer. The pooled samples (called "unattached" cells) were repeatedly diluted, and 100 μl aliquots were placed on the plate to determine the number of surviving bacteria.

Example 10. The activity of nisin, EDTA and methylparaben against gram-negative bacteria (S. typhimurium)
It was shown that a bactericide containing nisin, EDTA in combination with the well-known food preservative methylparaben, is extremely effective against gram-negative bacteria, as shown in table. 10.

b) 50 mM in acetate buffer, pH 5.0;
c) unattached cells
Cubes of chicken muscle were washed with sodium hypochlorite and providone iodine before use. To inoculate tissue, cubes of chicken muscle were immersed in a suspension of 10 ⁸ cells / ml S. typhimurium in 50 mM sodium acetate buffer at pH 5.0.

Excess moisture was lowered. The tissue was placed in a sufficient amount of buffer containing nisin compositions so that the solution covered the tissue and incubated for 2 hours at 37 ^° C, after which the tissue was transferred to a Phage buffer that covered the tissue. The bacteria remaining in the test solution were collected by centrifugation, washed with Phage buffer and combined with bacteria washed from the tissue with Phage buffer. The pooled samples (called "non-attached" cells) were diluted many times, and 100 μl aliquots were placed on plates to determine the number of surviving bacteria.

 Test 1 (Table 10) showed that in the presence of 10 mM EDTA, methylparaben is effective against S. typhimurium only at a concentration of 1.0%. In Test 2 (Table 10), however, it is seen that in the presence of 300 μg / ml nisin the effectiveness of methylparaban and nisin against S. typhimurium has increased significantly.

 Compositions containing nisin and EDTA significantly improve the use of the food preservative methylparaben. In addition, bactericides can significantly reduce concentrations or eliminate the need for well-known, albeit little described, food preservatives such as methyl paraben.

Example 11. The activity of nisin and a chelating agent against gram-positive bacteria (Staphylococcus aureus)
The activation of nisin by a chelating agent depends on the pH of the medium. The data given in table. 11 confirm that at pH 5.0 nisin is more a bactericidal agent against S. acreus than at pH 8.0. At pH 5.0, EDTA does not increase nisin activity against S. acreus, and at concentrations of EDTA greater than 10 mM EDTA reduces the bactericidal activity of nisin. However, the bactericidal activity of nisin activated with EDTA at pH 8.0 is significantly greater than the bactericidal activity of pure nisin or nisin in combination with EDTA at pH 5.0.

From the data table. 11 shows that
a) the initial number of live 8.0 • 10 ⁶ cells / ml

Incubation was carried out in 50 mm sodium acetate buffer, pH 5.0 or 20 mm Tris-HCl buffer, pH 8.0, at 37 ^o C.

 It was reported that the bactericidal activity of pure nisin (see. Hurst) is maximum at pH 5.0 or lower, and the data table. 11 confirm this. Based on this information, we believe that activation of the bactericidal activity of nisin using EDTA against S. acreus will also be greater at lower pH.

 However, from the table. 11, it can be seen that, in contrast to expectations (see Table 6), EDTA does not increase nisin activity against gram-positive bacteria at pH 5.0. However, a decrease in the activity of nisin at high concentrations of EDTA was also observed at pH 5.0. Thus, the activation of nisin by a chelating agent exists only in the range of concentrations of the chelating agent, in relation to gram-positive bacteria, it depends on pH with a preferred pH range of more than 5.0.

Example 12. The activity of nisin and chelating agent against gram-positive bacteria
The effect of EDTA on the bactericidal activity of nisin at pH 8.0 is not limited to S. aureus, an important pathogenic species for humans, but is also observed in the case of Streptococcus mutans responsible for plaque (Table 12A), a foodborne pathogenic species (Table 12B) and in the case of a mixed population of axillary bacteria Coryneform responsible for body odor (Table 12C).

From the data of table 12A shows that
a) the initial number of live 6.0 • 10 ⁶ cells / ml
Incubation was carried out in 20 mm Tris-HCl, pH 8.0 at 37 ^o C.

From the data table. 12V follows that
a) the initial number of live 1.0x10 ⁶ cells / ml
Incubation was carried out in 20 mm Tris-HCI, pH 8.0 at 37 ^o C.

Example 13. Acceleration of the bactericidal activity of nisin by activation with a chelating agent
The bactericide, including nisin with EDTA, acts much faster, as shown in table 13A. Suspensions of gram-positive bacteria S. mutans at a concentration of 10 ⁷ cells / ml were incubated in 20 mm Tris buffer, pH 7.3 at a temperature of 37 ^o C at several concentrations of nisin activated with 1 mm EDTA. Suspensions were incubated for various times from 0.5 to 60 minutes with a bactericide.

 The bactericidal efficacy of bactericides was measured by determining the percentage of surviving bacteria. Activated EDTA Nisin at such a low concentration as 10 μg / ml in this composition can reduce the number of bacteria by 6 orders of magnitude in 1 min.

 Fast bactericidal action is necessary for effective disinfection. So, it is predicted that these compositions will be effective bactericides, in particular, as shown in this application, as a component for rinsing the mouth, rinses, toothpaste or other similar toothpastes, powders, active against plaque formed by C. mutans.

 EDTA activated nisin activity against gram-negative bacteria after 2 3 hours is shown in Examples 1-7. The fast bactericidal effect of nisin activated by EDTA against gram-negative bacteria is also observed, and this is shown by the data in table. 13B.

As follows from the data table. 13A,
a) control number of viable cells 1.0 x 10 ⁷ cells / ml
Incubation was carried out in 20 mm Tris-HCl, pH 7.3 at 37 ^o C.

As follows from the data table. 13B,
a) initial number of viable cells 1.0 x 10 ⁷ cells / ml
Incubation was carried out in 20 mm Tris-HCl, pH 7.3 at 37 ^o C.

Example 14. The effect of divalent cations on the ability of EDTA to activate nisin activity
Divalent cations bind EDTA and other chelating agents, and one would expect that they neutralize the ability of EDTA to activate nisin. However, as can be seen from the data given in table. 14, the bactericidal activity of nisin against S. mutans is activated by 1 mM EDTA even in the presence of 1 mM Ca ²⁺ ion, and only at concentrations above 3 mM Ca ^{2+ ions} inhibit the ability of EDTA to activate nisin. This is especially important for rinsing the oral cavity, where the concentration of calcium ions is significant.

From the data table. 14 shows that
E) 1mM Na ₂ EDTA
a) initially viable amount of 1.0 x 10 ² cells / ml
Incubation was carried out in 10% fetal serum at 37 ^o C.

Example 15. The activity of a composition based on nisin with a surfactant against gram-positive bacteria
The bactericidal activity of nisin can also be significantly increased by the introduction of a single surfactant. This is best illustrated with limited concentrations of nisin (0.2 μg / ml), as shown in Table 15A. At concentrations up to 0.1%, food grade surfactants monolaurin has low bactericidal activity against Streptococcus agalactial in a complex dairy medium.

 Nisin at a concentration of up to 0.2 μg / ml also does not show significant activity in milk. However, a composition in which both of these agents are combined, 0.1% monolaurin and 0.2 μg / ml nisin, is very active against S. agalactial. This bactericide is more than 100 times more active than can be expected for an additional effect, and 10,000 times more active than each of the components separately. Thus, when the use of nisin is limited by its activity, it can be expected that a bactericide including nisin with surfactants will be more useful.

 An example when the use of nisin is limited by its available activity is illustrated by the data given in table. 15B. Although nisin, and in particular a bactericide including nisin and EDTA, is a bactericide against L. monocytogenes, the data in Table 15B show that in a complex medium such as milk, the nisin activity against this microorganism is limited.

 However, a bactericide, including nisin along with glyceride, a monooleate, is effective in milk against this pathogenic foodborne microorganism, even if the monooleate itself does not have bactericidal activity against this microorganism.

From the data of table.15A shows that
a) initially viable 6.0 x 10 ⁷ cells / ml.

Claims (1)

 1. Antimicrobial composition containing nisin, characterized in that, in order to expand the spectrum of exposure of the composition to bacteria and thus increase bactericidal activity, it additionally contains ethylenediaminetetraacetic acid (EDTA) in the following ratio of components, wt. Nisin 0.00001 0.3
EDTA 0.003 0.7
2. The composition according to claim 1, characterized in that it further comprises 0.01 to 1.0 wt. surfactant triton X-100, tween 20 or monolaurin.

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