S-nitrosothiol compounds and their combinations with nitrosamine blocking agents for safe food preservation

Abstract

The invention relates to a process for treating food, in particular for curing meat, comprising adding to the food a compound of Formula III, which is a derivative of S-nitroso-cysteine. Use of the compound of Formula III as food preservative and compositions for curing meat are also provided by the invention.

Claims

1. A process for curing food while suppressing nitrosamine formation, the process comprising the step of adding to the food S-nitroso-N-acetylcysteine or a physiologically acceptable salt or ester thereof, whereby the food is cured while suppressing nitrosamine formation.

2. A process according to claim 1, further comprising adding an additional inhibitor of nitrosamine formation to the food.

3. A process according to claim 2, wherein the additional inhibitor of nitrosamine formation is ascorbic acid or a physiologically acceptable salt or ester thereof.

4. A process according to claim 1, wherein the food is meat and wherein the S-nitroso-N-acetylcysteine or a physiologically acceptable salt or ester thereof is added to the meat to produce a cured meat product.

5. A process according to claim 4, further comprising adding nitrite to the meat, such that the nitrite is present in a concentration in the meat of below 150 ppm.

6. A process according to claim 5, wherein nitrite is added such that the nitrite is present in a concentration in the meat of below 120 ppm.

7. A process according to claim 1, devoid of nitrite addition.

8. A process according to claim 1, further comprising a step of providing low-oxygen environment by creating vacuum or purging with inert gas to replace oxygen with inert gas during preparation, packaging or storage of the food product.

9. The process according to claim 1, achieving at least one of reduction of microorganisms, improvement of product color and contribution to flavor.

10. The process according to claim 1, wherein the food is meat and the process achieves suppressing nitrosamine formation.

11. The process according to claim 1, wherein the food is meat and the process achieves reduction of microorganisms and improving the meat color.

12. A composition for curing meat while suppressing nitrosamine formation, comprising S-nitroso-N-acetylcysteine or a physiologically acceptable salt or ester thereof, and at least one meat additive.

13. A composition according to claim 12, wherein the meat additive is selected from the group consisting of sodium chloride, potassium chloride, sugar, antioxidants, inhibitors of nitrosamine formation comprising one or more of ascorbic acid and physiologically acceptable salts or esters thereof, flavor additives and a mixture thereof.

14. A composition according to claim 13, which is a curing salt blend consisting of from 0.5 to 20% by weight S-nitroso-N-acetylcysteine or a physiologically acceptable salt or ester thereof, and 80 to 99.5% by weight sodium chloride.

15. A composition according to claim 13, comprising a compound of S-nitroso-N-acetylcysteine or a physiologically acceptable salt or ester thereof, sodium chloride and/or one or more sugars, and optionally one or more additional inhibitors of nitrosamine formation.

16. A composition according to claim 13, which is a brine solution comprising water, the compound of S-nitroso-N-acetylcysteine or a physiologically acceptable salt or ester thereof, sodium chloride, sugars and optionally one or more additional inhibitors of nitrosamine formation.

17. A cured meat product with suppressed nitrosamine formation, comprising at least 10 ppm of S-nitroso-N-acetylcysteine or a physiologically acceptable salt or ester thereof, and optionally nitrite, wherein the concentration of nitrite, if present, is below 150 ppm.

18. Meat product according to claim 17, wherein the nitrite is present in a concentration of below 120 ppm.

19. Meat product according to claim 18, which is nitrite free, said product comprising at least 100 ppm of added S-nitroso-N-acetylcysteine or a physiologically acceptable salt of ester thereof.

20. Meat product according to claim 17, which further comprises at least 20 ppm ascorbic acid or physiologically acceptable salt or ester thereof.

21. The meat product according to claim 17, wherein the product is selected from smoked poultry, including smoked poultry breasts and smoked poultry rolls; corned beef; ham; bacon; and meat-based sausages.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a bar diagram illustrating N-nitroso-methylaniline production at 37° C. (30 min reaction) by a reaction of N-methylaniline with different agents described in Example 1 hereinbelow. Arrows indicate amount below detection limit.

(2) FIG. 2 is a bar diagram illustrating N-nitroso-methylaniline production at 37° C. (30 min reaction) by a reaction of N-methyl aniline with different agents described in Example 2 hereinbelow.

(3) FIG. 3 shows the kinetics of N-nitroso-methylaniline formation for a reaction at pH 6.2 of N-methylaniline with S-nitroso-cysteine (2.5 mM) (upper curve) and a reaction of N-methylaniline with S-nitroso-N-acetyl-cysteine (2.5 mM) (lower curve).

(4) FIG. 4 is a bar diagram illustrating N-nitroso-methylaniline production at 4° C. (24 h reaction) by a reaction of N-methylaniline with different agents described in Example 3 hereinbelow.

(5) FIGS. 5A-5F show the results of a study where N-nitroso-methylaniline was formed by a reaction of N-methylaniline with different agents in a meet model system under various conditions.

(6) FIGS. 6A-6C illustrate the effect of the additives on the color of the meat.

(7) FIG. 7 is a bar diagram showing aerobic colony count after three months of naturally occurring bacteria, in smoked beef shoulder pastrami prepared with nitrite or S-nitroso-N-acetylcysteine (plates were incubated at 30° C. for 24 hours then colonies were counted and CFU calculated).

(8) FIG. 8 is a bar diagram showing aerobic colony count after 0-4 weeks of naturally occurring bacteria, in ground beef shoulder treated with nitrite, S-nitroso-cysteine (Cys-SNO) or S-nitroso-N-acetylcysteine (NAC-SNO) (plates were incubated at 30° C. for 24 hours then colonies were counted and CFU calculated).

(9) FIG. 9 is a bar diagram showing bacterial growth after 24 h at 37° C. determined by measuring the OD at 595 nm of C. perfringens suspension treated with 2.5 mM NAC-SNO or DDW as a control.

EXAMPLES

(10) Materials

(11) Trisodium citrate, hydrochloric acid (HCl) 37%, N-methylaniline, L-ascorbic acid (AA), sodium nitrite, L-cysteine and N-acetyl-L-cysteine were purchased from Sigma-Aldrich (USA). Acetonitrile, water and sodium chloride were purchased from J.T. Baker (USA). Citric acid was obtained from Bio-Lab Ltd. (Israel). Citrate buffer was prepared by mixing appropriate amounts of 0.1 M citric acid and 0.1 M trisodium citrate, pH was adjusted using HCl. All solvents were HPLC grade. Fresh ground beef was purchased at a local supermarket.

(12) Methods

(13) The HPLC separation was performed on a Merck/Hitachi HPLC system consisting of a LaChrom L 7100 pump and a LaChrom DAD L 7450 detector. The system was operated with a data processor working with a LaChrom interface D 7000 and the EZChrom Elite 3.3.1 software. The mixture was separated on a Kinetex EVO C18 column (250×4.6 mm, 5 μm) using a gradient elution system, solvent A was 5% acetonitrile in water (v/v) and B was acetonitrile. Gradient conditions were initial=0% B, 25 min=70% B and 30 min=100% B. The flow rate was constant at 1.0 mL min.sup.−1. The injection loop volume was 20 μL, absorbance was monitored at 272 nm.

Preparation 1

Preparation of S-nitrosothiols

(14) S-nitrosothiols were synthesized by reacting equimolar amounts of sodium nitrite and the corresponding thiol, that is, either L-cysteine or N-acetyl-L-cysteine, in HCl (1 M). The reaction took place under nitrogen at room temperature for 5 minutes by which time a characteristic red color developed.

(15) The so-formed S-nitrosothiols: S-nitroso-cysteine and S-nitroso-acetyl-cysteine, were quantified by measuring UV absorbance at 334 nm. Extinction coefficients were estimated from the UV spectra of non-purified S-nitrosothiols on the basis of the amount of thiol used in the reaction and assuming that the reaction went to completion. S-nitroso-cysteine and S-nitroso-acetyl-cysteine were not isolated from the reaction mixtures and were used in the form of the solutions in the experiments reported below.

Example 1

Formation of N-nitroso-N-methylaniline from the Reaction of N-methylaniline with Sodium Nitrite (Comparative), S-nitroso-cysteine (Comparative) and S-nitroso-N-acetylcysteine (of the Invention) at 37° C.

(16) Reaction mixtures were prepared by mixing in citrate buffer, at pH=3.0 simulating stomach conditions and at pH=6.2 simulating meat conditions, N-methylaniline (0.25 mM) and NaCl (0.25 mM) together with an equimolar amount of the tested nitrosilating agent, used either alone or in combination with ascorbic acid, as set out below:

(17) TABLE-US-00001 1. Sodium nitrite (0.25 mM); 2. Sodium nitrite (0.25 mM) + ascorbic acid (1 mM); 3. S-nitroso-cysteine (0.25 mM); 4. S-nitroso-cysteine (0.25 mM) + ascorbic acid (1 mM); 5. S-nitroso-cysteine (0.25 mM) + ascorbic acid (2 mM); 6. S-nitroso-N-acetylcysteine (0.25 mM) 7. S-nitroso-N-acetylcysteine (0.25 mM) + ascorbic acid (1 mM) 8. S-nitroso-N-acetylcysteine (0.25 mM) + ascorbic acid (2 mM)

(18) The reaction vessel was placed in a water bath at 37° C. for 30 minutes. The reaction mixture was subsequently transferred to HPLC for analysis of the reaction product, N-nitroso-methylaniline. Results of the HPLC analysis, for the reaction at pH 3.0 and for the reaction at pH 6.2, are shown in the form of bar diagrams in FIG. 1 (with arrows indicating results below detection limit).

(19) The HPLC analysis indicates that S-nitroso-N-acetylcysteine, either alone or in combination with ascorbic acid, is able to suppress the formation of nitrosamine (that is, N-nitroso-methylaniline) much more effectively than sodium nitrite or S-nitrosocysteine.

Example 2

Formation of N-nitroso-N-methylaniline from the Reaction of N-methylaniline with Sodium Nitrite (Comparative), S-nitroso-cysteine (Comparative) and S-nitroso-N-acetylcysteine (of the Invention) at 37° C.

(20) The experiment of Example 1 was repeated with a tenfold increase of concentration of the reactants. That is, reaction mixtures were prepared by mixing in citrate buffer, at pH=3.0 and at pH=6.2, N-methylaniline (2.5 mM) and NaCl (2.5 mM) together with an equimolar amount of the tested nitrosilating agent, used either alone or in combination with ascorbic acid, as set out below:

(21) TABLE-US-00002 1. Sodium nitrite (2.5 mM); 2. Sodium nitrite (2.5 mM) + ascorbic acid (2.5 mM); 3. S-nitroso-cysteine (2.5 mM); 4. S-nitroso-cysteine (2.5 mM) + ascorbic acid (2.5 mM); 5. S-nitroso-N-acetylcysteine (2.5 mM) 6. S-nitroso-N-acetylcysteine (2.5 mM) + ascorbic acid (2.5 mM).

(22) Results of the HPLC analysis, for the reaction at pH 3.0 and for the reaction at pH 6.2, are presented in the form of bar diagrams in FIG. 2. S-nitroso-N-acetylcysteine emerges victorious also from this study, that is, from the experiments conducted in the high concentration regime.

(23) FIG. 3 shows the kinetics of N-nitroso-methylaniline formation from the reaction of S-nitroso-cysteine (2.5 mM) and N-methyl aniline (2.5 mM) at pH 6.2 (the upper curve) and from the reaction of S-nitroso-N-acetylcysteine (2.5 mM) and N-methylaniline (2.5 mM) at pH 6.2 (the lower curve), as monitored by measuring UV absorbance at 270 nm over.

(24) It is seen from the absorbance versus time plots that S-nitroso-cysteine reacts rapidly with N-methylaniline to produce progressively increasing amounts of nitrosamine. In contrast, nitrosamine formation is significantly lesser for the reaction of S-nitroso-N-acetylcysteine and N-methyl aniline and remains unchanged after a brief period time.

Example 3

Formation of N-nitroso-N-methylaniline from the Reaction of N-methylaniline with Sodium Nitrite (Comparative), S-nitroso-cysteine (Comparative) and S-nitroso-N-acetylcysteine (of the Invention) at 4° C.

(25) The experiment of Example 2 was repeated, but this time the reaction took place at 4° C. for 24 h and pH 6.2, to simulate the conditions that are used for long-term storage of meat. That is, reaction mixtures were prepared by mixing in citrate buffer, at pH=6.2, N-methylaniline (2.5 mM) and NaCl (2.5 mM) together with an equimolar amount of the tested nitrosilating agent, used either alone or in combination with ascorbic acid, as set out below:

(26) TABLE-US-00003 1. Sodium nitrite (2.5 mM); 2. Sodium nitrite (2.5 mM) + ascorbic acid (2.5 mM); 3. S-nitrosocysteine (2.5 mM); 4. S-nitrosocysteine (2.5 mM) + ascorbic acid (2.5 mM); 5. S-nitroso-N-acetylcysteine (2.5 mM) 6. S-nitroso-N-acetylcysteine (2.5 mM) + ascorbic acid (2.5 mM).

(27) The reaction vessel was kept at 4° C. for twenty four hours. Results of the HPLC analysis of N-nitroso-methylaniline levels are presented in the form of a bar diagram in FIG. 4. It is apparent that under storage conditions, the combination of S-nitroso-N-acetylcysteine and vitamin C performs surprisingly well, seeing that it is able to reduce the formation of N-nitroso-methylaniline very effectively.

(28) (In bar diagrams appended, the position of the bars, from left to right, corresponds to the order of experiments reported).

Example 4

Formation of N-nitroso-N-methylaniline from the Reaction of N-methylaniline with Sodium Nitrite (Comparative), S-nitroso-cysteine (Comparative) and S-nitroso-N-acetylcysteine (of the Invention) in a Meat Model System

(29) Meat contains substances that may affect N-nitrosamines formation (e.g., ascorbic acid, free L-Cys and myoglobin). The purpose of the study reported herein was to measure N-nitrosamines formation from the secondary amine N-methylaniline added to ground beef together with the tested nitrosilating agents.

(30) A set of experiments labeled A, B, C, D, E and F was conducted as described specifically below. In general, N-Methylaniline (0.25 mM) and NaCl (0.25 mM) were mixed into 1 gr of ground beef. Then the tested additive: sodium nitrite, S-nitroso-cysteine or S-nitroso-N-acetylcysteine (2.5 mM) was added. The concentrations were calculated as the final concentrations in the meat per Kg, using Kg=L. In one experiment (F) the experimental conditions were extended to mimic realistic meat processing (increased amount of sodium chloride and adding ascorbic acid).

(31) The meat was processed according to an acceptable industrial meat preparation practice (GMP—Good Manufacture Practice), i.e., the meat was heated to 72° C. (in a water bath for two minutes to reach that temperature in the center of the product), then transferred to 4° C. for cooling (30 minutes) or storage (24 to 216 hours). Some of the samples were packed in vacuum bags using MULTIVAC C100, in order to assess oxygen effect on N-nitrosamine formation in meat (see Experiments D and E), whereas the rest of the samples were placed into 4 mL closed glass vials.

(32) The meat was eventually homogenized in citrate buffer. 3 ml of citrate buffer (pH 6.2) were added to the meat and the mixture was homogenized using Kinematica Polytron PT 3000. In one experiment (C), intended to assess formation of N-nitrosamines upon digestion in an acidic stomach environment, citrate buffer at pH 3 was used to homogenize the meat. The homogenate at pH 3.0 was then incubated in a water bath at 37° C. for 30 min to simulate stomach digestion conditions.

(33) Acetonitrile (2 ml) was added to the homogenate and vortexed for N-nitrosamine extraction. The mixture was then centrifuged for 10 min at 4696× g using Thermo Scientific Heraeus Megafuge 16R. The supernatant was filtered using a 13 mm, 0.45 μm, PTFE syringe filter for HPLC analysis.

(34) More specifically, six different types of experiments were conducted according to the procedure outlined above with certain variations, and the results are shown in FIGS. 5A-5F, respectively: A) The meat was heated at 72° C., cooled down to 4° C. (30 minutes) and then immediately homogenized in citrate buffer pH=6.2. B) The meat was heated at 72° C., cooled down to 4° C., stored at 4° C. for twenty four hours and then homogenized in citrate buffer pH=6.2. C) The meat was heated at 72° C., cooled down to 4° C., stored at 4° C. for twenty four hours, homogenized in citrate buffer pH=3.0 and incubated at 37° C. for thirty minutes. D) After packaging in vacuum bag (vacuum to 100 mbar), the meat was heated to 72° C., stored at 4° C. for different time periods, and then homogenized in citrate buffer pH=6.2. E) After packaging in vacuum bag (vacuum to 8 mbar), the meat was heated to 72° C., cooled to 4° C. and then immediately homogenized in citrate buffer pH=6.2 or after storage for different time periods. F) The meat was also treated with 2% (w/w) NaCl and 3.27 mM ascorbic acid (AA, 576 ppm). The meat product was heated in a water bath at 75° C. for 5 min (temperature measured at the center of the product was 72° C. for 3 min) and stored at 4° C. for 216 hr. After storing the meat was homogenized without or with incubation in citrate buffer pH 3.0 for 30 min at 37° C. (stomach medium).

(35) The results of these experiments are presented in the graphs of FIGS. 5A-5F, respectively; in the graphs Cys-SNO stands for S-nitroso-cysteine and NAC-SNO stands for S-nitroso-N-acetylcysteine.

(36) Experiments A-C: The bar diagrams indicate that immediately after heating and cooling (FIG. 5A), N-nitrosamines were formed mostly by S-nitroso-cysteine, ˜28 μM, whereas both sodium nitrite and S-nitroso-N-acetylcysteine were able to keep N-nitrosamines formation levels below 10 μM. After a 24 h storage period at 4° C. (FIG. 5B) formation by nitrite and S-nitroso-N-acetylcysteine increased to 15 μM and 13 μM, respectively. N-nitrosamines formation by S-nitroso-cysteine was significantly higher than nitrite and S-nitroso-N-acetylcysteine. After consumption meat is digested in the acidic stomach pH. The results of the experiment mimicking these conditions are shown in FIG. 5C. N-nitrosamines formation by nitrite had increased considerably, by eightfold to 115 μM. Formation by S-nitroso-cysteine increased to 47 μM. However, formation by S-nitroso-N-acetylcysteine continued to remain rather low at 15 μM NA. The results indicate the ability of S-nitroso-N-acetylcysteine to suppress formation of N-nitrosamines in an acidic environment better than nitrite.

(37) Experiments D-E: The effect of oxygen was assessed by packing the treated meat in vacuum bags prior to heating. The vacuumed meat was then heated to 72° C. and stored at 4° C., and N-nitrosamine formation was measured. At a vacuum of 100 mbar (FIG. 5D), after 24 hr storage all three treatments formed ˜19 μM. However, during storage, N-nitrosamines level increased to ˜50 μM after 192 h storage period in meat samples that were treated with sodium nitrite and S-nitroso-cysteine, whereas N-nitrosamines formation did not change in the S-nitroso-N-acetylcysteine-added sample, and remained ˜20 μM. At stronger vacuum conditions of 8 mbar (FIG. 5E) N-nitrosamine formation by nitrite was twofold higher than the other agents after 216 hr. N-nitrosamine formation by nitrite and S-nitroso-cysteine was reduced by ˜40% in anaerobic conditions compared to aerobic conditions whereas, N-nitrosamine formation by S-nitroso-N-acetylcysteine remained low and the same.

(38) Experiment F: The results of the experiment mimicking meat processing conditions that include ascorbic acid and sodium chloride addition to the meat (FIG. 5F) show that the transfer of the meat to simulated stomach medium at pH 3.0 for only 30 min generated high concentration of N-nitrosamine by nitrite in the presence of ascorbic acid: N-nitrosamines levels increased by more than fourfold, from ˜20 μM to 90 μM in the simulated stomach medium. The concentration of N-nitrosamines that were generated in the meat product by S-nitroso-cysteine and S-nitroso-N-acetylcysteine almost did not change after the transfer to simulated stomach medium, at pH 3.0.

(39) In summary, from the perspective of restraining N-nitrosamines formation, S-nitroso-N-acetylcysteine clearly emerges as the best additive from the study conducted in a meat model system.

Example 5

The Effect of Sodium Nitrite (Comparative), S-nitroso-cysteine (Comparative) and S-nitroso-N-acetylcysteine (of the Invention) on the Color of the Meat

(40) Nitrite is known to be responsible for the characteristic pink color associated with cured meat products and flavor stabilization. The color development and color stability of ground beef treated S-nitroso-cysteine and S-nitroso-N-acetylcysteine after heating to 72° C. was compared to the color of ground beef treated with nitrite. A portable colorimeter (X-rite) was used to measure Hunter color lightness, redness and yellowness (L*, a*, b*) values of ground beef samples treated with the different additives and heated in a microwave oven (Dow, S-Korea, at 800 W) to 72° C. in the center. The instrument was standardized using white and black standard plates.

(41) The results are given in the form of bar diagram in FIGS. 6A and 6B. There was a significant difference in total color difference (ΔE*) (FIG. 6A) and in redness (Δa*) (FIG. 6B) between the meat treated with S-nitrosocysteine or S-nitroso-N-acetylcysteine, and meat treated with sodium nitrite. The meat treated with either S-nitroso-cysteine or S-nitroso-N-acetylcysteine was twofold redder and better color looking than the meat treated with sodium nitrite (there was no difference in yellowness (Δb*) between the treatments and hence these results are not shown). In the RSNO treatment (either S-nitroso-cysteine or S-nitroso-N-acetylcysteine) under aerobic conditions the color remained stable over a period of 24 h, whereas the color in nitrite-treated meat decreased over time (see FIG. 6A). The differences are readily visible also from the photo appended as FIG. 6C.

Example 6

Antimicrobial Activity of Sodium Nitrite (Comparative) and S-nitroso-N-acetylcysteine (of the Invention) in Smoked Beef Shoulder

(42) Smoked beef shoulder manufacturing process: brine ingredients [water, modified starch (E-1442), maltodextrin, salt, flavor enhancers (monosodium glutamate, yeast extract), preservatives (potassium lactate and potassium acetate and either sodium nitrite or S-nitroso-N-acetylcysteine), phosphate, carrageenan, seasoning, antioxidants (oregano extract, ascorbic acid)] were mixed to form the brine solution. S-nitroso-N-acetylcysteine was added to the brine in the form of freshly prepared aqueous solution (amount equivalent to 150 ppm sodium nitrite and 2% NaCl in the final product).

(43) Next, the brine was added at 50% (w/w) to previously cleaned from fat and cut into large cubes beef shoulder. Then the meat was tumbled under vacuum. In order to emulsify the meat, about 23% of the meat was transferred to an industrial cutter. Afterwards the emulsion was mixed with the rest of the tumbled meat in a mixer for several minutes. The meat was then stuffed into fibrous casing, which was transferred to steam cooking till the center of the product reached a temperature of 72° C. The product was then rapidly chilled to 4° C., sliced and packed in a low oxygen modified atmosphere. The pastrami was stored at 4° C. for three months.

(44) Aerobic colony count of naturally occurring bacteria in smoked beef shoulder pastrami: 5 gr of the pastrami manufactured in a meat factory as described above were homogenized in a sterile grinder with 45 ml of sterile saline, then further diluted with saline. The diluents were plated on BHI agar plates and incubated at 30° C. for 24 hr then colonies were counted and CFU calculated.

(45) The results shown in the form of bar diagram in FIG. 7 indicate that the antimicrobial activity of nitrite and S-nitroso-N-acetylcysteine is comparable, seeing that after three months, upon expiration of the product's shelf life, the treated pastrami developed similar concentration of bacteria: nitrite treated pastrami developed 8*10.sup.3 CFU/ml and S-nitroso-N-acetylcysteine-treated pastrami 7*10.sup.3 CFU/ml.

Example 7

Antimicrobial Activity of Sodium Nitrite (Comparative), S-nitroso-cysteine (Comparative) and S-nitroso-N-acetylcysteine (of the Invention) in Ground Beef

(46) 10 gr of ground beef shoulder was mixed with 1 ml (25 mM) aqueous solution of the tested preservative (Sodium nitrite, S-nitroso-cysteine and S-nitroso-N-acetylcysteine) or DDW for control and 1 ml of NaCl solution (10% w/v). The final concentrations were 2.5 mM preservative (where Kg meat=L) and 1% (w/w) NaCl. The meat was divided to 1 gr portions and placed in open vacuum bags, then heated in a water bath at 75° C. for 15 min and sealed by MULTIVAC C100 at a vacuum of 8 mbar. The bags were transferred to 4° C. for cooling and storage. After time periods of 1 hr, 1 week and 4 weeks elapsed, the bags were opened in a hood and 1 gr of meat was homogenized in a sterile grinder with 9 ml of sterile saline, then the homogenate was diluted further with saline and the diluents were plated on BHI agar plates. The plates were incubated at 30° C. for 24 hours then colonies were counted and CFU calculated.

(47) The results are shown in the form of a bar diagram in FIG. 8. It is seen that after 1 hour of cooling at 4° C., all treatments had a similar concentration of bacteria ˜10.sup.2-˜10.sup.3. After 1 week of storage at 4° C. nitrite-treated meat bacteria count had a minor increase to 5.8*10.sup.3 cfu/ml while S-nitroso-cysteine and S-nitroso-N-acetylcysteine remained ˜2.0*10.sup.2, all three treatments achieving significantly lower concentration of bacteria compared to control (3.3*10.sup.6). After 4 weeks the nitrite treated meat had a bacteria concentration of ˜10.sup.8 whereas Cys-SNO and NAC-SNO treated meat had a lower bacteria concentration.

Example 8

The Effect of NAC-SNO on Clostridium perfringens

(48) C. perfringens cells were grown overnight at 37° C. in fluid thioglycollate medium (FTM). Then the bacterial suspension was diluted in FTM to reach optical density (OD) of 0.1 A at 595 nm. The suspension was divided to tubes (7 ml per tube), then 70 μl of NAC-SNO was added to the tubes to a final concentration of 2.5 mM. DDW was added as a control. The tubes were gently mixed and incubated for 24 h at 37° C. Bacterial growth was determined by measuring the OD at 595 nm. The results are shown in the form of a bar diagram in FIG. 9.