WO1993004588A1

WO1993004588A1 - Composition and method of attenuation of ionic strength inhibition of lantibiotics

Info

Publication number: WO1993004588A1
Application number: PCT/US1992/007462
Authority: WO
Inventors: Mark W. Collison; Terry F. Farver; Richard E. Willits
Original assignee: Haarmann & Reimer Corporation
Priority date: 1991-09-05
Filing date: 1992-09-03
Publication date: 1993-03-18
Also published as: AU2591592A

Abstract

An improved antimicrobial composition comprising a lanthionine containing bacteriocin and an attenuator capable of reducing ionic strength inhibition and chosen from the group consisting of polyols, saccharides and polyethylene glycol and a method of decreasing inhibition of antimicrobial activity found in high ionic strength food systems. Particularly effective attenuators include glucose and sorbitol. Such attenuators are particularly effective for use with nisin. Temperature elevation of the treatment also enhanced antimicrobial activity.

Description

COMPOSITION AND METHOD OF ATTENUATION OF

IONIC STRENGTH INHIBITION OF LANTIBIOTICS

FIELD OF THE INVENTION Nisin, one of the lanthionine containing bacteriocins, has reduced antimicrobial efficacy in many food systems due to high salt content. This invention provides an improved antimicrobial composition and an improved method of use. The composition includes a lanthionine containing bacteriocin and an attenuator, capable of reducing ionic strength inhibition, chosen from the group consisting of polyols, saccharides and polyethylglycol.

BACKGROUND J.F. Back et al., Bioche ., 18:23, 5191-5196, 1979 studied the stabilization of proteins against heat denaturation in the presence of sugars and polyols. One of the proteins studied was lysozyme.

Polyols (polyhydroxy alcohols) and sugars such as sorbitol, glucose and sucrose were studied and it was found that glucose and sorbitol were about equally effective and that sucrose was slightly less so (page 5192, bottom column 2 and page 5193 top column 1) . Neither Nisin nor any inhibition due to a high ionic strength environment were mentioned. The stabilizatio described is apparently due to changes in the structur of water which lead to increased hydrophobic interac- tions in the hydrophobic core of the protein, resultin a higher temperature required to unfold and denature t protein. Lanthionine containing bacteriocins such as nisin are too small to have a folding structure which i stabilized by hydrophobic interactions. Therefore, the knowledge that polyols and sugars may stabilize protein structures would not lead one of skill in the art to th use of such compounds to decrease the antimicrobial inhibition by high ionic strength.

R. G. Bell & K. M. DeLacy, J. App. Bacteriology, 5 , 127-132, 1985, studied the effect of nisin-sodium chloride interactions on the outgrowth of Bacillus licheniformis spores. They concluded that salt appears to antagonize the sporicidal action of nisin by inter¬ fering with nisin adsorption onto the spore. However, they did not suggest any method or composition which would alleviate the effect of salt.

Attempts have been made to increase the antimi¬ crobial effectiveness of bacteriocins such as nisin wit additives to antimicrobial composition containing nisin. wo 89/12399, published 28 December 1989, discloses that when a lanthionine containing bacteriocin is combined with a chelating agent or a surfactant such a composi¬ tion is effective against Gram negative bacteria in addition to Gram positive bacteria, and that such bacteriocins become enhanced, rapid acting, broad range bactericides suitable for a variety of applications. Chelating agents named include EDTA, CaEDTA, CaNa-EDTA and other alkyldiamine tetracetates, EGTA and citrate. EP 0 384 319, published 19 August 90, suggests an antimicrobial composition comprising a Streptococcus- derived or Pediococcus-derived bacteriocin or a syn¬ thetic equivalent antibacterial agent in combination with a chelating agent. Chelating agents are defined therein as synonymous with sequestering agents and as including carboxylic acids, polycarboxylic acids, amin acids and phosphates. A long list of specific compoun is given which includes sorbitol Work was done with frankfurters. However, there is no experimental data given for a combination of any antibacterial and sor¬ bitol. There is no mention in either of the above patent applications of inhibition of nisin or any lanthionine containing bacteriocin by high ionic strength or of any way to overcome such inhibition.

DESCRIPTION OF THE FIGURES

The data obtained on the attenuation of ionic strength inhibition of nisin by polyethylene glycol (PEG) from the experiment disclosed in Example 1 is shown in a graph at Figure 1. Increasing ionic streng conditions, as provided by increasing sodium chloride from 200 to 800 millimolar (mM) , is graphed versus the log of cells per milliliter (ml) with a control (no nisin or PEG) shown by circles; 10 IU/ml nisin shown by squares; and 10 IU/ml nisin plus 10% weight/volume (w/v PEG 8000 shown by triangles.

The data obtained on the attenuation of ionic strength inhibition of nisin by sorbitol from the experiment disclosed in Example 2 is shown in a graph a Figure 2. Increasing amounts of sorbitol in percent weight/volume (w/v) are graphed versus the log of cells per milliliter (ml) with a buffer control (no nisin or sorbitol) shown by circles; 10 IU/ml nisin shown by squares; and 10 IU/ml nisin plus 800 mM sodium chloride shown by triangles. The data obtained on the attenuation of ionic strength inhibition of a nisin plus lysozyme combinatio by sorbitol from the experiment disclosed in Example 3 is shown in a graph at Figure 3. Increasing amounts of sorbitol in percent weight/volume (w/v) are graphed versus the log of cells per milliliter (ml) with a buffer control (no nisin/lysozyme or sorbitol) shown by circles; 10 IU/ml nisin plus 1 mg/ml lysozyme shown by squares; and 10 IU/ml nisin plus 1 mg/ml lysozyme plus 800 mM sodium chloride shown by triangles. The data obtained on the attenuation of ionic strength inhibition of subtilin (a lanthionine contain¬ ing bacteriocin produced by Bacillus subtilis) by sorbitol from the experiment disclosed in Example 4 is shown in a graph at Figure 4. Increasing amounts of sorbitol in percent weight/volume (w/v) are graphed versus the log of cells per milliliter (ml) with a buffer control (no subtilin or sorbitol) shown by circles; 100 IU/ml subtilin shown by squares; and 100 IU/ml subtilin plus 800 mM sodium chloride shown by triangles.

The data obtained on the effect of package incuba¬ tion temperature on efficacy of a nisin/ lysozyme/- glucose treatments on frankfurters from the experiment disclosed in Example 9 is shown in a graph at Figure 5. The effect of increasing temperature is graphed versus the log reduction of cells per frank when treated with a composition containing 20,000 IU/ml nisin plus 24 mg/ml lysozyme and 25% w/v glucose. SUMMARY OF THE INVENTION

An antimicrobial composition comprising a lanthi^¬ onine containing bacteriocin and an effective amount o an attenuator capable of decreasing the ionic strength inhibition of a lanthionine containing bacteriocin whi attenuator is chosen from the group consisting consist ing of polyols, saccharides and polyethylene glycol. The composition may be mixed with a food directly or applied to the surface. It has also been found that elevating the temperature of the treatment composition increases the efficacy of nisin, with and without lysozyme, even without an attenuator. Elevated temper ture also further enhances the efficacy of a compositi including an attenuator. A preferred method of improving the antimicrobial efficacy of lanthionine containing bacteriocins, in¬ cludes the steps of:

a. adding between about 15 to 30 percent weight/ volume of an attenuator capable of decreasin ionic strength inhibition of a lanthionine containing bacteriocin which attenuator is chosen from the group consisting of glucose and sorbitol to an antimicrobial composition containing nisin forming an antimicrobial mixture;

b. heating the mixture so formed to about 60 degrees;

c. applying the heated mixture to a food system; and d. rapidly cooling the food to a suitable stor temperature.

DETAILED DESCRIPTION OF THE INVENTION

Lanthionine containing bacteriocins such as nisin have found limited use as food preservatives to date. One possible cause of such limitation is that the high salt content of many foods, for example hot dogs, chee and many other prepared foods, inhibits the activity o these antimicrobials so that their use is either inef- fective or commercially unfeasible.

Testing was done with nisin (with and without lysozyme) in 10 mM sodium phosphate buffer at pH 6.00 and at 4 degrees Celsius against Listeria monocytoqene Scott A, since Listeria is an organism which has been particularly identified as a problem with such foods a this strain has been implicated in several instances o food poisoning.

It has been found that if ionic strength (as sodi chloride) is greater than 100 mM, the antimicrobial activity of nisin is impaired. Significant inhibition of activity is found at 200 mM sodium chloride. This effect is expected to be similar for other lanthionine containing bacteriocins such as for example subtilin, PEP 5, epidermin and gallidermin. An "attenuator" as defined herein is a compound or mixture of compounds which is capable of reducing ionic strength inhibition of lanthionine containing bacterio¬ cins. With the addition of an effective amount of an attenuator, the inhibition of antimicrobial activity wa shown to be decreased. Attenuators may be chosen from the group consisting of polyols, saccharides and polyethylene glycol. Polyols are defined herein to include those compounds having at least a four carbon backbone and two or more hydroxyl groups. Saccharides are defined herein to include those sugars containing four or more carbon atoms or polymers of such sugars containing four or more carbon atoms. Polyethylene glycol is defined herein as a compound of that class with a minimum molecular weight of 106. Preferred polyethylene glycols have a molecular weight in the range of between about 6000 and 10,000; most preferred are polyethylene glycols having a molecular weight abou 8000. Examples of such attenuators include, but are no limited to, sorbitol, mannitol, sorbitol, xylitol, maltitol, maltose, maltotriose, erythrose, threose, ribose, xylose, arabinose, glucose, lactose, fructose and sucrose, and polyethylene glycol. Preferred polyol are sorbitol and mannitol. Preferred saccharides are glucose, fructose, sucrose and lactose. Particularly preferred attenuators are sorbitol and glucose. Although the concentration of attenuator required to decrease the inhibitory effect of high ionic strengt will vary to some degree depending on the food system o interest and the concentration of the lanthionine containing bacteriocin used, the disclosure herein and the examples will provide one of skill in the art with sufficient guidance to determine a suitable combination and concentration. A concentration of from about 0.5% to about 50% weight/volume (w/v) of sorbitol or glucose provides an improvement in the antimicrobial activity of lanthionine containing bacteriocins in a high ionic strength environment. A concentration of about 25% w/v is preferred against Listeria monocytogenes in a ionic strength environment greater than or equal to about 200 mM as provided by sodium chloride. For other saccha¬ rides and polyols of lesser solubility, concentrations near the solubility limit of the compound may be desir able, with preferred concentrations in the range of 15 to 25%. For polyethylene glycols, concentrations in t range of 0.5% to 50% provide an improvement in the antimicrobial activity of lanthionine containing bac¬ teriocins in a high ionic strength environment. A concentration of 10% w/v is preferred for polyethylene glycol of 8000 dalton molecular weight against Listeri monocytoqenes in an ionic strength environment greater than or equal to 200 mM (sodium chloride) .

It was found that the addition of such attenuators to nisin, with or without lysozyme, does not affect the antimicrobial activity unless there is additionally a high ionic strength environment. In fact, when the hig salt content is not present, the addition of mannitol, lactose or fructose to nisin alone in buffer, inhibits antimicrobial activity. it has also been found that the antimicrobial activity of a composition including a lanthionine containing bacteriocin can be enhanced if the composi¬ tion is applied at an elevated treatment temperature. This effect was also seen in a high ionic strength environment. For a combination of nisin and glucose or sorbitol, an elevated temperature of between 40 and 65 degrees Celsius provides better antimicrobial efficacy, preferably a temperature of about 60 degrees Celsius. The same temperature ranges provide increased efficacy of a nisin/lysozyme combination with and without an attenuator. A preferred method of improving the anti¬ microbial efficacy of lanthionine containing bacterio¬ cins, comprises the steps of: a. adding between about 15 to 30 percent weight volume of an attenuator capable of decreasin ionic strength inhibition of a lanthionine containing bacteriocin which attenuator is chosen from the group consisting of glucose and sorbitol to an antimicrobial composition containing a lanthionine containing bacterio cin, forming an antimicrobial mixture;

b. heating the mixture so formed to about 60 degrees;

c. applying the heated mixture to a food system and

d. rapidly cooling the food to a suitable stora temperature.

The antimicrobial composition of the invention ma be incorporated directly into a food or food component by mixing or may be applied to the surface. Treatment may be accomplished by suspending and storing a food stuff to be treated in a solution of the composition. Alternatively, the food stuff may be dipped in the solution or the solution may be sprayed onto the surfa of the food stuff. The composition may also be incorp rated into films or gels which are applied to the surface of the food stuff. In film coating applicatio such as dips, films, gels or casings, the initial concentration of the components of the composition can approach their solubility limits to provide a finished product which after application and diffusion contains residual levels of the components which are within the proscribed limits. When treating the food stuff sur^¬ face, some routine experimentation may be needed to ascertain the most effective contractions of the compo nents. in addition to treatment of food stuffs, other fo preparation and food packaging equipment and materials may be treated. A method of improving the antimicrobi efficacy of lanthionine containing bacteriocins, com¬ prises the steps of:

a. adding an effective amount of an attenuator capable of decreasing ionic strength inhibi¬ tion of a lanthionine containing bacteriocin which compound is chosen from the group consisting of polyols, saccharides and poly- ethylene glycol to an antimicrobial composi¬ tion containing a lanthionine containing bacteriocin; and

b. applying mixture of lanthionine containing bacteriocin and attenuator to a food stuff.

EXAMPLES

Example 1: Attenuation of Nisin Inhibition with PEG

8000

A Listeria monocytoqenes inoculum was prepared as 7 10 colony forming units (cfu) per ml in 10 mM citrate buffer, pH 5.5 that was previously diluted from a 6 hour, 3% subculture of an overnight, 35 C culture. The culture broth was brain heart infusion. Treatments wer prepared such that when 100 ml inoculum was added into 900 ml of treatment solutions, the resulting mixtures zero time contained 10⁶ CFU/ml in 10 mM citrate buffer pH 5.5. Nisin was constant at 10 IU/ml with or withou polyethylene glycol (PEG 8000) at 10% w/v. Sodium chloride concentration ranged from zero to 800 mM in 2 mM increments. At zero time, the treatments were plac into a 37°C incubator for 60 minutes. At 60 minutes, the cell number was determined by plating onto brain heart infusion agar. Figure 1 shows the inhibition of nisin's antimicrobial activity against Listeria as the concentration of sodium chloride increases with nisin constant at 10 IU/ml. The addition of 10% w/v Peg 800 to nisin attenuates the ionic strength inhibition.

Example 2: Attenuation Of Nisin Inhibition With Sorbitol

The same procedure was followed as Example 1. Increasing sorbitol concentration from zero to 18% w/v did not influence the cell suspension (Listeria monocy- toqenes) as shown by control in Figure 2. 10 IU/ml nisin decreased the cell suspension count to below the detection limit. The addition of 800 mM sodium chlorid without sorbitol completely inhibited nisin activity against Listeria. As sorbitol increased to greater tha 6% w/v, attenuation of ionic strength inhibition is evident and continues with increasing sorbitol concen¬ tration. Example 3: Attenuation of Nisin Plus Lysozyme

Inhibition By Sorbitol

All conditions and procedures were the same as Example 2 except the treatment solution contained 10 IU/ml nisin plus 1 mg/ml lysozyme. Nisin/lysozyme treatment decreased the cell suspension count below the detection limit, as shown in Figure 3. The addition of 800 mM sodium chloride without sorbitol inhibited most but not all of the antimicrobial activity against Listeria. As sorbitol was increased to greater than 2% w/v, attenuation of ionic strength inhibition is eviden and continues with increasing sorbitol concentration.

Example 4: Attenuation Of Subtilin Inhibition With

Sorbitol

The experiment outlined in Example 2 was repeated except that nisin was replaced with 100 IU/ml subtilin. Figure 4 reveals that the effect of sorbitol on ionic strength inhibition of subtilin is similar to that of nisin.

Example 5: Attenuation Of Nisin Inhibition With

Glucose, Sorbitol Or Maltodextrins

The same procedure was followed as Example 1. Nisin was constant at 10 IU/ml. Sodium chloride, when added, was 800 mM. The attenuation of ionic strength inhibition of nisin against Listeria monocytoqenes by glucose, sorbitol or a maltodextrin, Maltrin® M365,

(corn syrup solids 36 D.E., available from Grain Pro^¬ cessing Corporation, Muscatine, Iowa) were compared a level of 15% w/v. The results in Log(cfu/ml) for e treatment in Table 1.

Table 1

Recovered Listeria monocytogenes after 60 min. at 37°

Treatment Loq(cfu/m

Control (cells only) 6.24 Nisin (without NaCl) 1.00

Nisin/NaCl 5.98

Nisin/NaCl: Glucose 4.68

Nisin/NaCl: Sorbitol 4.78

Nisin/NaCl: M365 5.17

Compared on a weight basis, glucose and sorbitol were more effective than maltodextrins (M365), but all attenuated the ionic strength inhibition.

Example 6: Attenuation Of Ionic Strength Inhibition O Nisin And Nisin Plus Lysozyme By Polyols And Saccharid

A cold-adapted, stationary phase inoculum of o

Listeria monocytoqenes Scott A was prepared as 10 cfu/ml in 10 mM sodium phosphate buffer, pH 6.0 and stored on ice. At zero time, 0.2 ml of the dilute Listeria culture was added to 1.8 ml treatment solutio previously equilibrated to 4°C in a water bath. Treat ments were incubated 60 minutes at 4°C and analyzed by 10-tube most probable number (MPN) procedure using microtest plates for dilutions. [For reference to calculation of cell numbers by the MPN procedure, see J.C. de Man, European Journal of Applied Microbiology 67-78 (1975). Log reduction in microbial numbers were calculated by subtracting the base ten logarithm of th MPN for the treated sample from the logarithm of the M for an inoculum control. This calculation method was used consistently on the data herein unless otherwise noted. ]

The effect of polyols and saccharides on antimi¬ crobial efficacy at low ionic strength, was tested on treatment solutions containing 10 mM sodium phosphate buffer, pH 6.0, nisin at 1.5 IU/ml with and without lysozyme at 0.25 mg/ml. The addition the saccharides o polyols at 15% w/v to the nisin or nisin plus lysozyme treatments did not enhance the antimicrobial activity, as shown in Table 2. None of the additives had an appreciable effect on the nisin plus lysozyme samples. Glucose, sorbitol, and sucrose added to nisin without lysozyme at low ionic strength did not appear to influ¬ ence the antimicrobial activity, but mannitol, lactose, and fructose all appeared to have an inhibitory effect on nisin alone but not on nisin plus lysozyme mixtures.

Table 2

Effect of Saccharides and Polyols at Low Ionic Strength

Additive Additive

(15% w/v) +

Lysozyme

(0.25 Log Reduction with Log Reduction with mg/ml) Nisin (1.5 IU/ml) Nisin (1.5 IU/ml)

The effect of polyols and saccharides on antimi^¬ crobial efficacy at high ionic strength was tested on treatment solutions containing 10 mM sodium phosphate buffer, pH 6.0, 200 mM sodium chloride, nisin at 20 IU/ml with and without lysozyme at 0.5 mg/ml. Additio of 200 mM sodium chloride to produce high ionic streng significantly inhibited both nisin and nisin plus lysozyme treatments, as shown in Table 2. Nisin at 20 IU/ml with or without 0.5 mg/ml lysozyme produced a lo reduction in cell number of about 0.2 in the presence 200 mM sodium chloride, while much lower levels of nisi produced much greater log reductions at low ionic strength (Table 1). The addition the saccharides or polyols at 15% w/v to the nisin or nisin plus lysozyme treatments significantly attenuated the ionic strength inhibition of nisin with or without lysozyme. All compounds tested significantly improved the antimicrob¬ ial effect of nisin or nisin plus lysozyme in the high ionic strength environment.

In summary, all the carbohydrates and polyols tested (glucose, lactose, fructose, sucrose, sorbitol. mannitol) attenuated ionic strength inhibition of nisi with and without lysozyme against Listeria. At low ionic strength, these compounds did not improve the antimicrobial activity of nisin with or without lyso- zyme, and some of the compounds tested (mannitol, lactose, fructose) appeared to inhibit the antimicrobia activity of nisin alone.

Example 7: Effect Of Glucose On Antimicrobial Effectiveness Of Nisin Plus Lysozyme On Frankfurters

A cold-adapted, stationary phase inoculum of

7 Listeria monocytoqenes Scott A was prepared as 10 cfu/ml in 50 mM sodium chloride and stored on ice.

Frankfurters were dipped in duplicate into the inoculum solution for 15 seconds with gentle agitation and drained 5 seconds. Franks were briefly dipped into treatment solutions at 0°C, removed and vacuum packed i plastic bags. Packaged samples were incubated at 4°C for 30 minutes. Two franks were removed from each treatment bag, 310 ml phosphate buffered saline (PBS) was added and franks were stomached 2 minutes in a

Stomacher® Lab Blender (Tekmar® Company, Cincinnati,

OH) . Samples of the stomached frank were diluted in PBS and analyzed by a 10-tube most probable number (MPN) procedure. Cells in dilutions were detected using a 2-stage enrichment/detection procedure: 48 hour growth at 30°C in UVM medium followed by 48 hour growth at 35°C in Fraser's medium, with positive tubes being indicated by blackening of the Fraser's medium. Where necessary, positive tubes were confirmed for Listeria monocytoqenes by streaking a 10 μl loop of culture from a positive Fraser's tube onto Modified Oxford Medium, and incuba ing the plates for 24 or 48 hours at 35°C. Positive plates were identified by the presence of typical, relatively large circular translucent colonies on a blackened background. This procedure is a modificati of the USDA method for Listeria detection.

The effect of adding 0, 15% or 25% glucose to treatments was examined in frankfurters treated with 20,000 IU/ml nisin and 10 mg/ml lysozyme in 10 mM phosphate buffer, pH 6.0. As shown in Table 4, additi of 15% or 25% glucose enhanced the antimicrobial effec tiveness of the nisin/lysozyme treatment on frank¬ furters.

Table 4 Influence of Glucose on Antimicrobial Effectiveness of Nisin/Lysozyme Treatments on Frankfurters

This study indicates the effects of glucose added at 15% or 25% to nisin plus lysozyme treatments are on the order of a few tenths of a log. This data indicat 25% glucose was of greater benefit than 15% glucose in enhancing treatment efficacy. Example 8: Effects Of Increased Treatment Temperatur And Comparison Of Sorbitol With Glucose On Antimicrobi Efficacy Of Nisin Plus Lysozyme On Frankfurters

The effect of increased treatment temperature was tested using procedures similar to Example 7. Frank¬ furters were dip inoculated at 0°C as before, dip treated with treatments either at 0°C or 60°C, vacuum packed and incubated 30 minutes at 4°C. 60°C treatmen were held at 60°C for exactly 10 minutes prior to dip treatment. Analysis of surviving cell numbers was as i Example 7. The results are shown in Table 5.

Table 5

Effects Of Elevated Treatment Temperature On Antimicrobial Efficacy Of Nisin-Lysozyme On Frankfurter

All treatment solutions contain 10 mM phosphate, p 6.0. The composition codes used in the table below are

20N/10L/G: 20,000 IU/ml nisin, 10 mg/ml lysozyme, 25% w/v glucose.

35N/10L/G: 35,000 IU/ml nisin, 10 mg/ml lysozyme, 25% w/v glucose.

20N/10L/S: 20,000 IU/ml nisin, 10 mg/ml lysozyme, 25% w/v sorbitol.

35N/10L/S: 35,000 IU/ml nisin, 10 mg/ml lysozyme, 25% w/v sorbitol. 0°C Log MPN Std. dev. Log Reduction

Treatments Cells/Frank

INOCULUM 6.64 0.07

20N/10L/G 4.32 0.18 2.33

35N/10L/G 3.84 0.24 2.80

20N/10L/S 3.86 0.22 2.78

35N/10L/S 3.51 0.26 3.13

60°C Log MPN Std. dev. Log Reduction Treatments Cells/Frank

INOCULUM 6.51 0.09

20N/10L/G 3.76 0.26 2.76

35N/10L/G 3.52 0.22 3.00

20N/10L/S 3.49 0.25 3.03

35N/10L/S 3.39 0.26 3.12

Statistical Analysis: Overall Effects

Comparison 90% Confidence 95% Confidence Interval Interval

Sorbitol-Glucose 0.17-0.42 0.15-0.45 60°C - 0°C 0.09-0.35 0.07-0.37 35,000-20,000 IU/ml Nisin 0.16-0.42 0.13-0.44

A statistical analysis of the data found the overall effects of increased treatment temperature, substitution of sorbitol for glucose, and increased nisin concentration were significant at the 95% confi¬ dence level.

In conclusion, these experiments indicate elevated treatment temperature can give an appreciable increase in efficacy of nisin antimicrobial activity.

The magnitude of the difference between 20,000 and 35,000 IU/ml nisin in Table 4 is similar in magnitude to the effect of addition of 25% glucose to the treatments, as shown by the data in Table 3. Sorbitol also added increased efficacy over glucose. Example 9: Effect Of Elevated Product Incubation

Temperature On Antimicrobial Effectiveness On

Nisin/Lysozyme/Glucose Of Franks

Frankfurters were inoculated, treated and analyzed as described in Example 7. After inoculation and treatment at 0°C, franks were incubated at temperatures between 0°C and 12°C. Increased incubation temperature of the treated frankfurters resulted in increased antimicrobial efficacy of the nisin/lysozyme/glucose treatment, as shown in Figure 5.

Claims

WHAT IS CLAIMED IS:

1. An antimicrobial composition comprising a lanthionine containing bacteriocin and an effective amount of an attenuator capable of decreasing the ion strength inhibition of a lanthionine containing bac- teriocin which attenuator is chosen from the group consisting consisting of polyols, saccharides and polyethylene glycol.

2. The composition of claim 1 in which the atte ator is a polyol chosen from the group consisting of sorbitol and mannitol.

3. The composition of claim 1 in which the atte ator is a saccharide chosen from the group consisting glucose, fructose, sucrose and lactose.

4. The composition of claim 1 in which the atte ator is polyethylene glycol.

5. The composition of claim 1 in which the atte ator is chosen from the group consisting of sorbitol glucose.

6. The composition of claim 1 in which the atte ator is present in a concentration from about 0.5 percent weight per volume to about the solubility lim of the attenuator.

7. The composition of claim 1 in which the lant onine containing bacteriocin is nisin.

8. The composition of claim 7 which additionally includes lysozyme.

9. An antimicrobial composition comprising a synergistic combination of nisin and lysozyme and an attenuator in an effective amount to decrease ionic strength inhibition of nisin and lysozyme which attenua tor is chosen from the group consisting of sorbitol and glucose.

10. The composition of claim 9 in which the concentration of the attenuator is from about 0.5% percent weight/volume to about 50% weight volume.

11. The composition of claim 10 in which the concentration of the attenuator is about 25 percent weight volume.

12. A method of improving the antimicrobial efficacy of lanthionine containing bacteriocins, com- prising the steps of:

13. The method of claim 12 wherein the mixture is applied by incorporation into the food stuff or by surface application.

14. The method of claim 12 in which the attenuator is a polyol chosen from the group consisting of sorbitol and mannitol.

15. The method of claim 12 in which the attenuator is a saccharide chosen from the group consisting of glucose, fructose, sucrose and lactose.

16. The method of claim 12 in which the attenuator is polyethylene glycol.

17. The method of claim 12 in which the attenuator is chosen from the group consisting of sorbitol and glucose.

18. The method of claim 12 in which the mixture of lanthionine containing bacteriocin and attenuator is applied at a temperature above the temperature of storage of the food.

19. The method of claim 12 in which the mixture of of lanthionine containing bacteriocin and attenuator is heated to about 60 degrees C prior to application to the food. 1 20. A method of improving the antimicrobial efficacy of lanthionine containing bacteriocins, com- 3 prising the steps of:

a. adding between about 15 to 30 percent weight/- 5 volume of an attenuator capable of decreasing ionic strength inhibition of a lanthionine

I containing bacteriocin which attenuator is chosen from the group consisting of glucose

9 and sorbitol to an antimicrobial composition containing nisin, forming an antimicrobial I mixture;

b. heating the mixture so formed to about 60 3 degrees;

c. applying the heated mixture to a food system; 5 and

d. rapidly cooling the food to a suitable storage 7 temperature.