Composition comprising a bacteriocin and an extract from a plant of the Labiatae family

Abstract

The present invention provides a composition comprising (a) an antimicrobial material; and (b) an extract obtained from or obtainable from a plant of the Labiatae family, wherein (a) and (b) are different; wherein the composition contains phenolic diterpenes in an amount of greater than 1.0 wt. %, based on the composition, and wherein when the antimicrobial material consists of nisin, the composition comprises carvacrol in an amount of less than 0.075 wt. % based on the composition and carvone in an amount of less than 15 wt. % based on the composition.

Claims

1. A method for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein: the method comprises contacting the material with a protectant composition comprising nisin and an extract obtained from or obtainable from rosemary; and the protectant composition comprises phenolic diterpenes in an amount of greater than 1.0 wt. % based on the protectant composition.

2. A method according to claim 1, wherein the protectant composition comprises carvacrol in an amount of less than 0.04 wt. %, based on the protectant composition.

3. A method according to claim 1, wherein the protectant composition comprises carvone in an amount of less than 0.075 wt. %, based on the protectant composition.

4. A method according to claim 1, wherein the protectant composition comprises thymol in an amount of less than 0.1 wt. %, based on the protectant composition.

5. A method according to claim 1, wherein the protectant composition comprises phenolic diterpenes in an amount of greater than 2.0 wt. %, based on the protectant composition.

6. A method according to claim 1, wherein: the extract comprises a phenolic triterpene; and the total amount of phenolic triterpene, based on the protectant composition, is greater than 3.5 wt. %.

7. A method according to claim 6, wherein the phenolic triterpene is selected from betulinic acid, oleanolic acid, and ursolic acid.

8. A method according to claim 1, wherein: the extract comprises rosmarinic acid; and the amount of rosmarinic acid, based on the protectant composition, is greater than 3.5 wt. %.

9. A method according to claim 1, wherein the nisin and the extract are present in the protectant composition in an amount to provide a microbicidal or microbiostatic synergistic effect.

10. A method according to claim 9, wherein the nisin and the extract are present in the protectant composition in an amount to provide a microbicidal synergistic effect.

11. A method according to claim 9, wherein the microbicidal or microbiostatic effect is a bactericidal or bacteriostatic effect.

12. A method according to claim 11, wherein the bactericidal or bacteriostatic effect is with respect to Gram-positive bacteria.

13. A method according to claim 1, wherein the material is a foodstuff.

14. A method according to claim 13, wherein the foodstuff is selected from raw meat, cooked meat, raw poultry products, cooked poultry products, raw seafood products, cooked seafood products, ready to eat meals, pasta sauces, pasteurised soups, mayonnaise, salad dressings, oil-in-water emulsions, margarines, low fat spreads, water-in-oil emulsions, dairy products, cheese spreads, processed cheese, dairy desserts, flavoured milks, cream, fermented milk products, cheese, butter, condensed milk products, ice cream mixes, soya products, pasteurised liquid egg, bakery products, confectionery products, fruit products, and foods with fat-based or water-containing fillings.

15. A method according to claim 1, wherein the protectant composition further comprises an emulsifier selected from polysorbates, monoglycerides, diglycerides, acetic acid esters of mono-diglycerides, tartaric acid esters of mono-diglycerides and citric acid esters of mono-diglycerides.

16. A method according to claim 1, wherein the protectant composition further comprises a chelator.

17. A method according to claim 16, wherein the chelator is selected from EDTA, citric acid, monophosphates, diphosphates, triphosphates and polyphosphates.

18. A method according to claim 1, wherein the protectant composition further comprises a lytic enzyme.

19. A method for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a foodstuff, wherein: the method comprises forming a protected foodstuff by a process comprising contacting the foodstuff with a protectant composition comprising nisin and an extract obtained from or obtainable from rosemary, the protected foodstuff comprises a phenolic diterpene having a concentration of at least about 0.00084% w/w, and the protected foodstuff comprises nisin at a concentration of at least about 25 IU/g.

20. A method of claim 19, wherein: the protected foodstuff comprises a phenolic diterpene having a concentration of at least about 0.0012% w/w, and the protected foodstuff comprises nisin at a concentration of at least about 50 IU/g.

Description

(1) The present invention will now be described in further detail by way of example only with reference to the accompanying figures in which:

(2) FIG. 1 is a graph showing synergistic enhancement of nisin cidal activity against Listeria monocytogenes in chicken soup at 25? C. by a selectively extracted rosemary extract

(3) FIG. 2 is a graph showing synergistic enhancement of nisin control of Listeria monocytogenes growth in chilled chicken soup by a selectively extracted rosemary extract (GRE09)

(4) FIG. 3 is a graph showing synergistic enhancement by a selectively extracted rosemary extract of nisin control of B. cereus spore outgrowth in chilled chicken soup. Minimal detection limit was 100 cfu/g. For the length of the testing period, the samples containing the combination of nisin and rosemary had Bacillus counts at or below 100 cfu/g.

(5) FIG. 4 is a graph showing combined effect of nisin, selectively extracted rosemary extracts and rosemary extract components against L. monocytogenes in chicken soup at 20? C. (Minimum detection limit 100 cfu/g)

(6) FIG. 5 is a graph showing synergistic enhancement of nisin activity by selectively extracted extracts of rosemary or rosmarinic acid against Listeria monocytogenes in a chicken soup at ambient temperature.

(7) FIG. 6 is a graph showing a demonstration of synergy between nisin and phenolic diterpene-containing rosemary extract. Inhibition of L. monocytogenes at 8? C.

(8) FIG. 7 is a graph showing a demonstration of synergy between nisin and phenolic diterpene-containing rosemary extract. Inhibition of B. cereus at 15? C.

(9) FIG. 8 is a graph showing enhanced nisin growth inhibitory activity by a phenolic diterpene-containing rosemary extract. Control of L. monocytogenes in carbonara sauce at 8? C.

(10) FIG. 9 is a graph showing enhanced nisin growth inhibitory activity by a phenolic diterpene-containing rosemary extract. Control of B. cereus spores in carbonara sauce at 15? C.

(11) FIG. 10 is a graph showing enhanced cidal effect of a nisin and phenolic diterpene-containing rosemary extract against L. monocytogenes in chicken soup at 20? C. a) pH 4.5

(12) FIG. 11 is a graph showing enhanced cidal effect of a nisin and phenolic diterpene-containing rosemary extract against L. monocytogenes in chicken soup at 20? C. b) pH 6.7

(13) The present invention will now be described in further detail in the following examples.

EXAMPLES

Experimental Evidence of Benefit

(14) In vitro studies described herein have shown synergy between nisin and extracts of Rosmarinus officinalis containing >3.5% phenolic diterpenes, increasing the efficacy of nisin significantly. This enhanced activity was also observed in food models, increasing nisin kill and growth control of Gram-positive bacteria. The experimental studies also demonstrated that the phenolic diterpenes camosic acid and camosol were implicated in this synergy. The results also indicated that rosmarinic acid may also enhance nisin activity, although this synergistic effect was not as strong as that observed with the phenolic diterpenes.

(15) I) In Vitro Demonstration of Nisin and Deodorised Rosemary Extract Synergy

(16) Materials:

(17) GUARDIAN? Rosemary Extract 09 (Danisco) (GRE09). This is a water dispersible deodorised rosemary extract containing 4% phenolic diterpenes and <1% essential oils, extracted from rosemary leaves, combined with the carriers polyoxyethylene sorbitan monooleate (Tween 80) and propylene glycol. A commercial extract of nisin at potency of 1?10.sup.6 IU/g: NISAPLIN? Natural Antimicrobial (Danisco).

(18) Test Strains:

(19) Bacillus cereus 204, B. cereus Campden, B. cereus NCTC2599, B. subtilis Campden, Listeria monocytogenes 272, L. monocytogenes NCTC12426, L. monocytogenes S23, Lactobacillus sake 272, Escherichia coli S15, E. coli CRA109, Salmonella Typhimurium S29, Pseudomonas fluorescens 3756.

(20) Method of Microbial Growth Curve Analysis.

(21) A 100,000 ppm GRE09 solution was prepared in water and filter sterilised (0.2 ?m). Further dilutions were prepared in sterile deionised water at 1,250-20,000 ppm. Brain Heart Infusion broth (Oxoid) was prepared and GRE09 stock solutions were added to give the following test solutions of GRE09; 125, 250, 500, 750, 1000, 1250, 1500, 2000 ppm. A 10,000 IU/ml nisin solution was prepared, filter sterilised and a range of stock solutions then prepared. A range of nisin concentrations was then prepared in Brain Heart Infusion broth. A fully automated microbial growth analyser was used to determine microbial growth curves (Microbiology Reader Bioscreen C analyser linked to a PC with installed software BioLink v 5.30; Labsystem Oy, Finland). Tests were prepared in honeycomb 2 (HC 2) microtitre/cuvette plates with a capacity of 100 wells per plate. The wells were loaded with 270 ?l of the prepared media and inoculated at a level of 10.sup.3 CFU (colony forming units)/ml with 30 ?l of microbial suspension. Incubation time and temperature was as appropriate for the test organism. This test allowed suitable test levels for the compounds to be determined. The rosemary extract and nisin were then tested in combination, using the same procedure. Nisin solutions were prepared at 50-1000 IU/ml in broth as above. GRE09 solutions were prepared at 250, 500 and 1000 ppm as above. Combinations of all these test levels were prepared and tested in the Bioscreen as before.

(22) Results:

(23) The minimum inhibitory concentration of nisin alone, rosemary extract GRE09 alone and the two in combination in the Bioscreen after 48 h at 30? C. is shown in Table 1. The minimal inhibition was taken as the lowest concentration that caused total inhibition of the bacteria after 48 h at 30? C. Synergy was observed between nisin and the rosemary extract GRE09 against all Gram-positive bacteria but no significant effect was observed against Gram-negative bacteria. This can be determined from the table by comparing the column of data showing MIC levels of nisin alone, GRE09 alone and the two combined. The latter column gave levels much lower than the other two for Gram-positive bacteria (Bacillus, Listeria) but not for Gram-negative bacteria (E. coli, Salmonella).

(24) TABLE-US-00001 TABLE 1 Synergy tests of nisin and the rosemary extract GRE09 MIC in broth after 48 h at 30? C. (total Other test levels of the inhibition) combination causing total Nisin GRE09 MIC of nisin (IU/ml) + inhibition Test organism (IU/ml) (ppm) GRE09 (ppm) Nisin (IU/ml) + GRE09 (ppm) B. cereus 204 500 >1000 50 + 250 50 + 500 50 + 1000 100 + 250 100 + 500 100 + 1000 200 + 250 200 + 500 200 + 1000 B. cereus 500 >1000 50 + 250 50 + 500 NCTC2599 50 + 1000 100 + 250 100 + 500 100 + 1000 200 + 250 200 + 500 200 + 1000 B. subtilis 100 >1000 50 + 250 50 + 500 Campden 50 + 1000 100 + 250 100 + 600 100 + 1000 200 + 250 200 + 500 200 + 1000 L. monocytogenes >500 >1000 50 + 250 50 + 500 S23 50 + 1000 100 + 250 100 + 500 100 + 1000 200 + 250 200 + 500 200 + 1000 L. monocytogenes >500 >1000 50 + 250 50 + 500 272 50 + 1000 100 + 250 100 + 500 100 + 1000 200 + 250 200 + 500 200 + 1000 L. monocytogenes >500 >1000 50 + 250 50 + 500 12426 50 + 1000 100 + 250 100 + 500 100 + 1000 200 + 250 200 + 500 200 + 1000 E. coli S15 >500 >1000 >1000 + >1000 E. coli CRA109 >500 >1000 >1000 + >1000 S. Typhimurium >500 >1000 >1000 + >1000 S29 Ps. fluorescens >500 >1000 >1000 + >1000 3756
II) Demonstration of Nisin and Rosemary Extract GRE09 Synergy in Food
A) Synergy Against Listeria monocytogenes
Test Compounds:

(25) GRE09 at 0.1%, 0.5%, NISAPLIN? (Danisco).

(26) Test Strains:

(27) a cocktail was prepared of L. monocytogenes strains NCTC12426, NCTC5105, NCC FSM60 and CRA3930. The Listeria strains were grown at 30? C. on Brain heart infusion agar overnight then inoculated into broth at 30? C. overnight. A volume of each broth was mixed together to give a cocktail of strains with a cell concentration of approximately 10.sup.9 CFU/ml.

(28) Media:

(29) A chilled pasteurised chicken soup was used as a food model because it was a good mix of different food components including vegetables, dairy products and poultry meat. It was comprised of a chicken stock with the addition of chicken, cream, vegetables, flour and seasonings. The pH was 6.12. After addition of nisin and rosemary extract GRE09, the soup was pasteurised at a core temperature of 80? C. for 2 minutes. The Listeria cocktail was diluted to 10.sup.4 CFU/ml and inoculated into soup tests to give a final cell count of approximately 10.sup.2 CFU/g (growth inhibitory tests) and 10.sup.7 CFU/ml (cidal tests). The latter test was incubated at 25? C. for 2 h and then tested by viable count enumeration to estimate the extent of cidal activity. The growth test was incubated at 8? C. with regular sampling to estimate bacteriostatic activity.

(30) Results.

(31) The rosemary extract GRE09 alone at 0.5% showed no listericidal activity. Nisin at 250 IU/g caused a 1 log drop in Listeria numbers after 2 h, but only a slight delay in growth after 24 h (FIG. 1). In comparison the combination of the two test products at these levels caused a 2-3 log drop in Listeria numbers after 2 h. After 24 h the cells still had not recovered to their initial inoculum level. This was a particularly harsh test for any preservative system, since the test medium was a rich food model, the incubation temperature was at ambient and the bacterial numbers high. Therefore any enhanced nisin activity was a good indication of synergy.

(32) Incubation for the bacteriostatic test was for 43 days: results of this are shown in FIG. 2 and Table 2. The nisin/rosemary synergy was again clearly demonstrated in the food model against the Listeria cocktail. For example, Listeria growth reached 10.sup.6 CFU/ml after 13 days in the presence of 100 IU/ml nisin; after 10 days in the presence of 0.1% GRE09 but only after a much longer period, 34 days, in the presence of the combination of these two ingredients. Similarly, Listeria growth reached 10.sup.6 CFU/ml after 13 days in the presence of 100 IU/ml nisin; after 20 days in the presence of 0.5% GRE09. The combination of the two components resulted in no growth being observed by then end of the test period.

(33) TABLE-US-00002 TABLE 2 Summary of growth inhibition of Listeria in chilled chicken soup (Trial lasted 43 days) Days until Test conditions growth reached 10.sup.6 CFU/ml Control 6 Nisin at 100 IU/ml 13 Nisin at 250 IU/ml 27 Rosemary extract GRE09 at 0.1% 10 Rosemary extract GRE09 at 0.5% 20 Nisin (100 IU/ml) + GRE09 at 0.1% 34 NISAPLIN (100 IU/ml) + GRE09 at 0.5% >43 NISAPLIN (250 IU/ml) + GRE09 at 0.1% >43 NISAPLIN (250 IU/ml) + GRE09 at 0.5% >43 During the test period (a) NISAPLIN (100 IU/ml) + GRE09 at 0.5%, (b) NISAPLIN (250 IU/ml) + GRE09 at 0.1%, and (c) NISAPLIN (250 IU/ml) + GRE09 at 0.5% did not give any total aerobic viable counts above 100 cfu/g.
B) Synergy Against Bacillus cereus
Test Strains:

(34) a cocktail of Bacillus spores was prepared as an inoculum, using Bacillus cereus strain 204, Bacillus cereus strain 199, B. cereus strain Campden, and B. cereus strain ABC 4/9.

(35) Additions of the test compounds were made to chicken soup, prepared as above. The soup was pasteurised at 70? C. for 2 minutes, cooled and inoculated with approximately 10.sup.3 CFU/g of a cocktail of Bacillus cereus spores. Incubation was for 56 days. Results are shown in FIG. 3 and summarised in Table 3. Bacteriostatic synergy between the nisin and rosemary extract GRE09 was evident. For example, spoilage (i.e. 10.sup.6 CFU/ml) resulted after 13 days in the presence of 25 IU/ml nisin, and after 10 days in the presence of 300 ppm GRE09. In the presence of both these ingredients, no spoilage had occurred by the end of the trial (56 days).

(36) TABLE-US-00003 TABLE 3 Summary of results of chilled chicken soup trial inoculated with Bacillus cereus spores (Trial lasted 70 days). Days until growth Test conditions reached 10.sup.6 CFU/ml Control 6 Nisin at 25 IU/ml 13 Rosemary extract GRE09 at 300 ppm 10 Rosemary extract GRE09 at 600 ppm 13 Nisin (25 IU/ml) + GRE09 at 300 ppm >70 NISAPLIN (25 IU/ml) + GRE09 at 600 ppm >70
C) Synergy Against Clostridium sporogenes
Test Strains:

(37) a cocktail of Clostridium spores was prepared as an inoculum, using Clostridium sporogenes strain Campden, Clostridium sporogenes 1.221, and Clostridium sporogenes NCIMB1793.

(38) Additions of the test compounds were made to chicken soup, prepared as above. The soup was pasteurised at 70? C. for 2 minutes and transferred to sterile test tubes. These were inoculated with a cocktail of heat-shocked Clostridium sporogenes spores, at a level of 2.2?10.sup.2 CFU/g, then anaerobic conditions were created by plugging the tubes with agar. The samples were incubated at 37? C. and checked daily for gas production (observed by blowing of the gas plug and by the distinctive clostridial odour). Results for a 27 day incubation period, demonstrating synergy, are shown in Table 4. For example, synergy was clearly seen by the combined effect of 50 IU/ml nisin and 300 ppm GRE09, which prevented growth for 27 days (the length of the trial), whereas the individual ingredients both prevented clostridial growth for 2 days (the same as the control).

(39) TABLE-US-00004 TABLE 4 Summary of results of chicken soup trial inoculated with Clostridium sporogenes spores incubated at 37? C. (Trial lasted 27 days). Days until growth Test conditions observed (gas production) Control 2 Nisin at 25 IU/ml 2 Nisin at 50 IU/ml 2 Nisin at 100 IU/ml 7 Rosemary extract GRE 09 at 300 ppm 2 Rosemary extract GRE 09 at 600 ppm 2 Nisin (25 IU/ml) + GRE 09 at 300 ppm 3 Nisin (50 IU/ml) + GRE 09 at 300 ppm >27 Nisin (100 IU/ml) + GRE 09 at 300 ppm >27 NISAPLIN (25 IU/ml) + GRE 09 at 600 ppm 10 NISAPLIN (100 IU/ml) + GRE 09 at 600 ppm >27
III) Demonstration of In Vitro Synergy with Different Deodorised, Selectively Extracted Rosemary Extracts and Rosmarinic Acid

(40) Growth curves of Listeria monocytogenes and B. cereus strains in laboratory media were analysed as described above using the Bioscreen C analyser. Minimal inhibitory concentrations (MIC) were determined for the test compounds used singly or in combination after 24 h at 30? C. Results are shown in Table 5. The test compounds comprised nisin (as Nisaplin?; Danisco), GRE09 (Danisco), pure rosmarinic acid (RA; Sigma) and a range of deodorised rosemary extracts. These had been prepared by selected extraction with either organic solvents or CO.sub.2 to obtain extracts containing 28% phenolic diterpenes (28RE; Danisco) and a rosemary extract containing 6% rosmarinic acid (6RA; Danisco). Enhanced nisin activity was evident with a combination of nisin combined with pure rosmarinic acid (RA; this may have partly been due to low pH levels), a combination of nisin with a rosemary extract containing 6% rosmarinic acid (6RA) and a combination of nisin with a deodorised rosemary extract containing 28% phenolic diterpenes and <1% essential oils (28RE). The known nisin synergy with Tween 80 was also observed. The other carrier propylene glycol did not enhance nisin activity. The synergies can be observed, as before, by comparing the MIC levels for nisin alone, the other test compound, and the two together (see Table 5).

(41) TABLE-US-00005 TABLE 5 MIC after growth at 30? C. in laboratory medium MIC in Bioscreen after 24 h at 30? C. Individual Test organism components Combination with nisin L. monocytogenes Nisin at strain S23 1000 IU/ml 0.1% GRE09 0.05% GRE09 + 50 IU/ml nisin 1% RA 0.25% RA + 250 IU/ml nisin 0.5% RA + 100 IU/ml nisin 0.75% RA + 50 IU/ml nisin 1% 6RA <0.1% 6RA + 250 IU/ml nisin 0.5% 6RA + 50 IU/ml nisin L. monocytogenes 500 IU/ml nisin strain 272 0.25% GRE09 <0.05% GRE09 + 50 IU/ml nisin 1% of RE28 <0.05% RE28 + 50 IU/ml nisin >2% Tween 80 0.5% Tween 80 + 250 IU/ml nisin L. monocytogenes 250 IU/ml nisin strain 0.25% GRE09 <0.05% GR 09 + 50 IU/ml nisin NCTC12426 1% of RE28 <0.05% RE28 + 50 IU/ml nisin >2% Tween 80 0.5% Tween 80 + 100 IU/ml nisin B. cereus 500 IU/ml nisin Campden spores 0.1% GRE09 0.05% GRE09 + 50 IU/ml 1% RA 0.5% RA + 250 IU/ml nisin 0.75% RA + 100 IU/ml nisin 0.75% RA + 50 IU/ml nisin 1% 6RA 0.25% 6RA + 100 IU/ml nisin 0.5% 6RA + 50 IU/ml nisin
IV) Demonstration of Synergy for Nisin Activity with Different Deodorised Rosemary Extract Components in Food
Test Strains:

(42) Listeria monocytogenes strains 272, CRA3930 and NCTC12426

(43) The chicken soup model was used as before. The following samples were tested: GRE09, deodorised rosemary extracts containing 28% or 70% phenolic diterpenes (RE28 and RE70; Danisco), a water soluble rosemary extract containing 6% rosmarinic acid (6RA; Danisco) and pure rosmarinic acid (RA; Sigma). Additions to the soup were made as appropriate. The soup was pasteurised (70? C./2 minutes), the pH recorded and the soup was then inoculated with a cocktail of Listeria cells prepared as described before. The tests were incubated at 20? C. and viable count enumeration performed after 0, 2, 4 and 24 h at 20? C. Initial Listeria levels were 1.3?10.sup.5 CFU/ml. The test was repeated at two nisin levels and over different time periods. The pH of the soup without addition was pH 6.06-6.20. Addition of rosmarinic acid at 0.1% resulted in a slight pH drop to pH 5.75. Addition of 6% RA resulted in a soup pH of pH 5.75-5.78. Addition of 0.5% RE28 resulted in a soup pH of pH 5.98. Addition of 0.5% RE70 resulted in a soup pH of pH 6.10. Addition of 0.5% GRE09 resulted in a soup pH of pH 6.02-6.09.

(44) Results, shown in FIGS. 4 and 5, indicate that all the deodorised extracts tested and rosmarinic acid contributed to synergy with nisin in achieving kill of Listeria cells. This could not be attributed to the drop in pH caused by some of the additions. The additional synergy with Tween 80 was observed in GRE09. The results indicate that the antioxidant compounds carnosol and camosic acid, present at 28 and 70% in two of the extracts tested, synergistically enhanced the cidal and growth inhibitory activity of nisin against Listeria monocytogenes. A nisin synergy with rosmarinic acid was evident but not as strong.

(45) V) Demonstration of Synergistic Enhancement of Nisin's Growth Inhibitory Activity in Different Food Systems Using a Blend of Nisin with a Phenolic Diterpene-Containing Rosemary Extract

(46) A) Pasteurised Chicken Soup Tests

(47) Method:

(48) Different additions of nisin (as Nisaplin?, Danisco), a Rosemary extract containing 28% phenolic diterpenes (RE28), and a blend of nisin with the Rosemary extract at levels of 50 IU/mg and 4.2% phenolic diterpenes were added to commercial chicken soup that contained no other preservatives. After addition of the compounds the soup (pH 5.8) was pasteurised at a core temperature of 70? C. for 2 minutes. The soup was cooled to ambient temperature and either inoculated with a cocktail of stationary phase cells of Listeria monocytogenes strains or spores of Bacillus cereus. The strain cocktails comprised: L. monocytogenes strains NCIMB12426, strain 358, strain 272, strain CRA3930. The B. cereus cocktail comprised strains 204, 199, ABC4/9 and 3.046. Initial inoculum levels were approximately 10.sup.2-10.sup.3 CFU/g. Bacillus tests were incubated at 15? C., Listeria tests were incubated at 8? C. Microbiological analysis was conducted at regular intervals (Milk Plate count Agar, Oxford Listeria Selective agar).

(49) Results:

(50) The results, shown as the time taken for bacterial numbers to reach 10.sup.6 CFU/g, are summarised in Table 5. The full data are shown in FIGS. 6 and 7. The results show that the Rosemary extract alone had no activity against Bacillus, and only slight activity against Listeria. The Rosemary extract significantly enhanced the growth inhibitory activity of nisin.

(51) TABLE-US-00006 TABLE 5 Summary of results demonstrating nisin/phenolic diterpene synergy against Listeria and Bacillus in a pasteurised chicken soup Days until Phenolic growth reached 10.sup.6 CFU/g Nisin diterpene L. monocytogenes B. cereus Test content content at 8? C. at 15? C. Control 0 3 2 RE28 at 75 ppm 0 IU/g 21 ppm 5 2 NISAPLIN at 100 IU/g 0 ppm 6 3 100 mg/kg NISAPLIN at 250 IU/g 0 ppm 16 6 250 mg/kg Nisin/Rosemary 100 IU/g 8.4 pm 15 >26 blend A Nisin/Rosemary 250 IU/g 21 ppm 52 >26 blend B
B) Pasteurised Meat Pasta Sauce Tests
Method:

(52) The sauce was prepared from lean minced beef (50%), tomatoes and juice (48.9%), starch (0.5%), salt (0.4%) and sucrose (0.2%). The beef was fried for 5 minutes until brown, then the dry ingredients mixed in followed by the tomatoes with juice. The sauce was simmered for 10 minutes and allowed to cool before blending to a smooth consistency. Final pH was 5.13. Additions were made of nisin, rosemary extract and blends. The sauce was pasteurised to a core temperature of 80? C. for 2 minutes. A cocktail of Listeria monocytogenes strains (as above) were inoculated after pasteurisation and the tests incubated at 8? C.

(53) Results:

(54) The results, shown as the time taken for bacterial numbers to reach 10.sup.6 CFU/g, are summarised in Table 6. These show that the rosemary extract alone had no activity against Bacillus, and only slight activity against Listeria. The rosemary extract significantly enhanced the growth inhibitory activity of nisin.

(55) TABLE-US-00007 TABLE 6 Summary of results demonstrating nisin/phenolic diterpene synergy against Listeria in a pasteurised meat sauce at 8? C. Phenolic Test Nisin diterpene Days until 10.sup.6 CFU/g Control 0 0 4 RE28 at 60 ppm 0 IU/g 16.8 ppm 5 NISAPLIN at 100 IU/g 0 ppm 11 100 mg/kg Nisin/Rosemary 100 IU/g 8.4 pm >76 blend A
C) Carbonara Pasta Sauce Tests
Method:

(56) A commercial chilled pasteurised sauce was used, containing cream, smoked bacon, cheese, mascarpone, butter, starch, onion, garlic puree. Protein 7 g, carbohydrate 6 g, fat 17 g. Additions of test compounds were made prior to the pasteurisation (core temperature of 70? C. for 10 minutes). Inoculations were made once the sauce had cooled. Samples were analysed regularly for microbial numbers.

(57) Results:

(58) These are shown in FIGS. 8 and 9. As before, the phenolic diterpene-containing extract (8.4 ppm) synergistically enhanced the nisin growth inhibitory activity against Listeria cells and Bacillus spores. The rosemary extract alone showed no activity.

(59) VI) Demonstration of Synergistic Enhancement of Nisin's Cidal Activity in a Food System Using a Blend of Nisin with Phenolic Diterpene-Containing Rosemary Extract

(60) Method:

(61) The diluted chicken soup (pH 6.2) was prepared as above, and split into 2 batches with one batch being adjusted to pH 4.5 with HCl. Appropriate additions of nisin, rosemary extract and blends were made, then the soup was pasteurised. A cocktail of Listeria strains was inoculated to give an initial inoculum of 10.sup.5 CFU/g. Viable cells were enumerated by microbiological analysis at 0 and 2 h.

(62) The test blends contained 1) 100 IU/g nisin+30 ppm rosemary extract (i.e. 8.4 phenolic diterpenes), and 2) 150 IU/g nisin+45 ppm rosemary extract (i.e. 12.6 phenolic diterpenes).

(63) Results:

(64) The results demonstrated that the presence of the phenolic diterpene containing rosemary extract synergistically enhanced the cidal activity of nisin (FIGS. 10 and 11), particularly at more acidic conditions (FIG. 10). The rosemary extract alone had no significant cidal effect.

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(67) All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology, food science or related fields are intended to be within the scope of the following claims