Antimicrobial compositions

Abstract

A composition includes hesperdin and/or a Lamiaceae extract wherein a majority of the volatile components have been removed from the Lamiaceae extract. A method for applying the composition to a food such as meat, fish or poultry, including processed and fresh or unprocessed meat, poultry and fish is also provided.

Claims

1. A composition comprising pure hesperidin and a deodorized Lamiaceae extract, wherein a majority of volatile oil components having been removed from the Lamiaceae extract, the Lamiaceae extract containing a Lamiaceae phenolic diterpene selected from carnosic acid and carnosol and mixtures thereof, and the composition comprises from 24% to 48% by weight Hesperidin and from 1.28% to 2.56% by weight Lamiaceae extract.

2. The composition according to claim 1, wherein the Lamiaceae extract is selected from rosemary extract, oregano extract, thyme extract, sage extract, mint extract, Salvia extract, Rosmarinus extract, Lepechinia extract, Oreganum extract, Thymus extract, Hyssopus extract and mixtures thereof.

3. The composition according to claim 1, further comprises a carrier selected from arabic gum, dextrose, salt, mono & diglycerides of fatty acids, MPG, Polysorbate 80, dextrose, vegetable oil, glucose syrup, glycerin, decaglycerol monooleate, fatty acid esters, benzyl alcohol, ethyl alcohol, propylene, glycol, polysorbates, sorbitans, sorbitan trioleate, capric/caprylic triglycerides, and combinations thereof.

4. The composition according to claim 1, further comprises a carrier of maltodextrin having a structure distinct from a starch from which the maltodextrin originates.

5. The composition according to claim 3, wherein the composition is in the form of a dry powder.

6. The composition according to claim 3, further comprises one or more flavorings and adjuvants.

7. The composition according to claim 3, wherein the composition is in liquid form.

8. A food product comprising a food and the composition of claim 1.

9. The food product of claim 8, wherein the food is selected from the group consisting of meat, poultry and fish.

10. The food product of claim 9, wherein the meat, poultry and fish are fresh meat, poultry and fish.

11. The food product according to claim 8, wherein the Lamiaceae extract containing Lamiaceae phenolic diterpene in an amount of 12 ppm or more of Lamiaceae phenolic diterpene selected from carnosic acid, carnosol, and mixtures thereof, and 56 ppm or more of hesperidin.

12. The food product according to claim 11, containing between 12 and 170 ppm of Lamiaceae phenolic diterpene and between 56 and 5381 ppm of hesperidin.

13. The food product according to claim 8, containing between 12 and 170 ppm of Lamiaceae phenolic diterpene and between 56 and 5381 ppm of hesperidin.

14. A packaged food product comprising a food according to claim 8, wherein the food is packaged in an atmosphere comprising 20% or more oxygen.

15. A packaged food product comprising a food according to claim 8, wherein the food is packaged in a standard atmospheric environment.

16. A method for processing a food comprising: applying to or incorporating into a food, a composition comprising hesperidin and a deodorized Lamiaceae extract, wherein a majority of the volatile oil components have been removed from the Lamiaceae extract, the Lamiaceae extract containing a Lamiaceae phenolic diterpene selected from carnosic acid and carnosol and mixtures thereof, and the composition comprises from 24% to 48% by weight Hesperidin and from 1.28% to 2.56% by weight Lamiaceae extract.

17. The method of claim 16, further comprising packaging the food in an atmosphere that contains 20% or more oxygen.

18. The method of claim 16, wherein the food is selected from the group consisting of fresh meat, fish or poultry.

19. The method according to claim 16, wherein applying to or incorporating into a food product comprises applying the composition, in the form a dry powder, to the food.

20. The method according to claim 16, wherein applying to or incorporating into a food product comprises applying a single composition comprising both the Lamiaceae extract and hesperidin to or incorporate into the food.

21. A food product comprising food in combination with deodorized Lamiaceae extract essentially free of native essential oil which contains carnosic acid, carnosol, and hesperidin, wherein the food product comprises a composition comprising from 24% to 48% by weight Hesperidin and from 1.28% to 2.56% by weight Lamiaceae extract, and wherein hesperidin is present in the food in an amount between 56 and 5381 ppm.

22. The food product according to claim 21, containing between 12 and 170 ppm of Lamiaceae phenolic diterpene and between 56 and 5381 ppm (%) hesperidin.

23. The food product according to claim 21, wherein the Lamiaceae extract and hesperidin is in the form of a composition which additionally contains a carrier selected from the group consisting of arabic gum, dextrose, salt, mono & diglycerides of fatty acids, MPG, Polysorbate 80, dextrose, vegetable oil, glucose syrup, glycerin, decaglycerol monooleate, fatty acid esters, benzyl alcohol, ethyl alcohol, propylene, glycol, polysorbates, sorbitans, sorbitan trioleate, capric/caprylic triglycerides, dextrose and combinations thereof.

24. The food product according to claim 23, wherein the composition is in dry form.

25. The food product according to claim 23, wherein the composition is in liquid form.

26. A method for preparing a food product, comprising: applying to or incorporating into a food, a combination of deodorized Lamiaceae extract essentially free of native essential oil and is an antimicrobial which contains carnosic acid, carnosol, and hesperidin, wherein the combination of deodorized Lamiaceae extract essentially free of native essential oil and hesperidin comprises from 24% to 48% by weight Hesperidin and from 1.28% to 2.56% by weight Lamiaceae extract, and wherein hesperidin is present in the food in an amount between 56 and 5381 ppm.

27. The method claim 26, wherein the bacterial growth inhibited, decreases and/or limited is listeria.

28. The method claim 27, wherein the bacterial growth inhibited is listeria.

29. The composition of claim 1, wherein the pure hesperidin is 80-99% hesperidin.

30. The method of claim 26, wherein the food has enhanced color preservation and/or antimicrobial properties as compared with preservation using Lamiaceae extract or Hesperidin individually.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is adapted from Basic Principles of Pharmacology, (Tulane University), in which the top portion is Summation: Compounds A and B Produce Equal Effects, And Their Affects Are Additive When Combined and the bottom portion is Synergism: The Combination of Half the Dose of Compound A and Compound B Produces a Response Greater Than A or B Alone.

(2) FIG. 2 is a graph showing Listeria monocytogenes growth in meat in accordance with the present invention.

(3) FIG. 3 is a graph showing antilisterial synergy effects of combinations of hesperidin and rosemary extract in accordance with the present invention.

(4) FIG. 4 is a graph showing antimicrobial surface response of different combinations of rosemary extract and hesperidin inhibiting and decreasing Listeria monocytogenes growth, in accordance with the present invention.

(5) FIG. 5 is a bar chart showing red color values of the meat in accordance with the present invention with regard to combinations of rosemary extract and hesperidin in accordance with the present invention.

(6) FIG. 6 is a bar chart showing red color values of meat in accordance with the present invention with regard to various combinations of rosemary and punica extract in accordance with the present invention.

(7) FIG. 7 is a chart showing inhibition of Listeria monocytogenes growth by plant extracts in minced beef.

(8) FIG. 8 is a graph showing inhibition of Listeria monocytogenes growth by plant extracts in minced beef at 8 C. at day 9.

(9) FIG. 9 is a chart showing inhibition of Listeria monocytogenes growth by plant extracts in minced beef at 8 C. at day 6.

(10) FIG. 10 is a chart showing inhibition of Listeria monocytogenes e by plant extracts in minced beef at 8 C. at day 9 in accordance with the present invention.

(11) FIG. 11 is a graph showing in accordance with the present invention, antimicrobial surface response of different combinations of rosemary and Punica extract inhibiting and decreasing Listeria monocytogenes growth.

DETAILED DESCRIPTION OF THE INVENTION

(12) Compositions in accordance with this disclosure include Lamiaceae extract and hesperidin and methods for using compositions for extending the shelf life of food including meat, fish and poultry (both fresh/unprocessed and processed) without impacting the taste.

(13) The present invention in another form, includes compositions comprising Lamiaceae extract and Punica extract and methods for using these compositions for extending the shelf life of food including meat, fish and poultry (both fresh/unprocessed and processed) without impacting the taste.

(14) The present methods and compositions are based on a discovery that rosemary extracts rich in phenolic diterpenes, alone, or in combination with hesperidin or with Punica extracts rich in ellagic acid and in punicalagins, preserve the red color of the meat for commercially significant period. The present inventors discovered that treating meat with pure hesperidin, that is a flavonoid, extracted from citrus peels and then purified, prevents the growth of Listeria monocytogenes in meat. Lamiaceae extracts comprising phenolic diterpenes in combination with hesperidin or with Punica extract comprising ellagic acid and punicalagins, synergistically provided novel solutions for suppressing the growth of microorganisms for a commercially desirable period and for preserving the red color of the meat without impacting the meat taste. Compositions of this invention have been found to inhibit the growth of Gram positive microorganisms. Compositions of this invention, have been found to inhibit the growth of Listeria. Compositions of this invention, have been found to inhibit the growth of Listeria monocytogenes.

(15) Combinations comprising: plant extracts standardized in phenolic diterpenes carnosic acid and carnosol, and hesperidin, or plant extracts standardized in phenolic diterpenes carnosic acid and carnosol, and plant extracts standardized in ellagic acid and punicalagins, that would have been used to synergistically prevent Listeria monocytogenes growth in food including meat, fish and poultry (both fresh/unprocessed and processed), without impacting the food taste and that synergistically improve the preservation of the food color (e.g. meat color), could not be retrieved from the prior art.

(16) None of the prior art on the antimicrobial use of the combination of rosemary or other Lamiaceae extracts comprising phenolic diterpenes with hesperidin or with Punica extracts comprising punicalagins and ellagic acid, either anticipates or renders obvious the present methods and compositions. The prior art focuses on the use of herb essential oils or on the use of organic acids, such as citric acid. The rosemary extracts used in the present disclosure are processed in a manner that makes them essentially free of the native essential oil and rich in phenolic diterpenes. The prior art neither anticipates nor renders obvious the synergistic combination of Lamiaceae extracts rich in phenolic diterpenes and Punica extracts rich in punicalagins and ellagic acid.

(17) The prior art neither anticipates nor renders obvious the synergistic combination of Lamiaceae extracts rich in phenolic diterpenes and hesperidin. The prior art neither anticipates nor renders obvious the surprisingly beneficial antimicrobial effect of the combination of Lamiaceae extracts comprising phenolic diterpenes with Punica extracts comprising punicalagins and ellagic acid, or with hesperidin, on Gram positive organisms: Listeria monocytogenes. The prior art neither anticipates nor renders obvious the surprisingly beneficial color preservation effect of the combination of Lamiaceae extracts comprising phenolic diterpenes with Punica extract comprising punicalagins and ellagic acid, or with hesperidin.

(18) The prior art neither anticipates nor renders obvious the absence of the impact on food taste of the combination of Lamiaceae extracts comprising phenolic diterpenes with Punica extract comprising punicalagins and ellagic acid, or with hesperidin.

(19) Other flavonoids that have the same effect including but are not limited to: narigin, isocurametin, neohesperidin, hesperidin, poncirin, nebiletin, and tangeretin.

(20) Definitions

(21) The following are a list of definitions used throughout this disclosure:

(22) Effective amount is the amount necessary in order to achieve a specific effect, in accordance with what one of ordinary skill in the art would be readily able to determine through routine experimentation. For example, with regard to the present disclosure, an effective amount of a composition comprising hesperidin and a Lamiaceae extract to be applied to a food product or foodstuff, e.g. meat, fish and poultry (both fresh/unprocessed and processed), to extend the longevity of the food, e.g. red color to the fresh meat, fish and poultry, is an amount which is determined to provide the red color longevity based on known parameters which include, but are not limited to the concentration of hesperidin and a Lamiaceae extract, the volume and/or surface area of the meat, fish and poultry, and the atmospheric environment conditions of the meat, fish and poultry. Similarly, the effective amounts of rosemary/Punica to extend the longevity of red color to the meat, fish and poultry are determined in a similar way.

(23) Food, food product and foodstuff mean products that people or animals eat. The food, food product and foodstuff include, but are not limited to fresh and/or unprocessed meat, fish and poultry and processed meat, fish and poultry.

(24) Fresh meat, fish, and poultry means meat fish and poultry, entire carcasses, cut portions thereof, and ground portions thereof. Fresh meat, fish, and poultry includes both unprocessed meat, fish and poultry as well as meat, fish, and poultry that includes additives such as polyphosphates, salt, water, flavors, broths, added proteins, sugar, starches and the like which are incorporated into the meat, fish or poultry. It is important to distinguish fresh meat, fish or poultry which may contain these ingredients, from processed meat, fish and poultry which includes cured meat, fish and poultry, which may contain the same ingredients, but also contain one or more of the following: erythorbates, erythorbic acid, ascorbates, ascorbic acid, nitrites, nitrates or cultures. Fresh meat, fish and poultry are to be distinguished from, and as opposed to, and does not include cured meat, fish or poultry, known as processed meat, fish and poultry.

(25) Hesperidin means a compound extracted from nature or synthesized.

(26) Lamiaceae extract means extract from a plant of the Lamiaceae family, preferably rosemary, sage, oregano, thyme, mints, and the following genera: Salvia, Rosmarinus, Lepechinia, Oreganum, Thymus, Hyssopus and mixtures thereof. The most preferred is rosemary.

(27) Meat, fish and poultry means both a) processed meat, fish and poultry and b) unprocessed meat, fish and poultry.

(28) Phenolic diterpenes means carnosic acid, carnosol, methylcarnosate, and other phenolic diterpene derivatives (rosmanol, isorosmanol, 11,12-di-O-methylisorosmanol, 12-O-methylcarnosic acid, rosmanol-9-ethyl ether, circimaritin, Methylated monooxidized product of carnosic acid, genkwanin, epirosmanol, epiisorosmanol, carnosic acid derivative, epirosmanol ethyl ether, cryptotanshinone) and mixtures thereof.

(29) Processed such as processed foodstuff and processed meat, fish and poultry are products resulting from the processing of food, such as meat, fish or poultry or from the further processing of such processed products, so that the cut surface shows that the product no longer has the characteristics of fresh meat, fish or poultry. Processing means any action that substantially alters the initial product, including heating, smoking, curing, maturing, drying, marinating, extraction, extrusion or a combination of those processes. Processes include non-heat treated and heat-treated processes.

(30) Punica extract means extract from a plant of the Punica genus, preferably Punica granatum and Punica protopunica, and mixtures thereof. The most preferred is Punica granatum.

(31) Pure hesperidin extract means a hesperidin extract that has a concentration of at least 80% hesperidin.

(32) Unprocessed (such as meat, fish and poultry) means not having undergone any treatment resulting in a substantial change in the original state of the foodstuffs (e.g. meat, fish and poultry). However, the foodstuffs may have been for example divided, parted, severed, boned, minced, skinned, pared, peeled, ground, cut, cleaned, trimmed, deep-frozen, frozen, chilled, milled or husked, packed or unpacked. Unprocessed foodstuff, including meat, fish and poultry include untreated raw meat, fish and poultry, as well as fresh meat, fish and poultry that has been comminuted or minced, that has had foodstuffs seasons or additives added to it or that has undergone processing insufficient to modify the internal muscle fiber of the meat, fish or poultry and thus eliminate the characteristics of fresh meat, fish or poultry.

(33) In the development of the present method and composition, it was discovered that hesperidin has an antilisterial effect in meat when prepared within certain ranges of concentrations.

(34) In the development of the present method and composition, it was discovered that rosemary extract comprising phenolic diterpenes combined with hesperidin or with Punica extract has a superior effect on suppressing the growth of Listeria monocytogenes in meat than when extracts are applied alone.

(35) In the development of the present method and composition, it was discovered that certain mixtures of extracts of the rosemary combined with hesperidin or with Punica extract comprising punicalagins and ellagic acid, provide a synergistic antilisterial effect when prepared within certain ranges of concentration ratios.

(36) In the development of the present method and composition, it was discovered that rosemary extract comprising phenolic diterpenes combined with hesperidin or with Punica extract has a superior effect on preserving the red color in meat than when extracts are applied alone.

(37) In the development of the present method and composition, it was discovered that certain mixtures of extracts of the rosemary combined with hesperidin or with Punica extract comprising punicalagins and ellagic acid, provide a synergistic red color preservation effect in meat when prepared within certain ranges of concentration ratios.

(38) Mixtures of Extracts Rich in Phenolic Diterpenes and Hesperidin or Punica Extract

(39) Phenolic diterpenes such as carnosic acid or carnosol occur specifically in Lamiaceae. To date, carnosic acid has been identified in only a few species, all exclusive of the Lamiaceae. To the best of the inventors' knowledge, only seven out of seventy (70) genera of the Mentheae tribe contain carnosic acid: Salvia (Brieskorn and Dumling, 1969), Rosmarinus (Luis and Johnson, 2005), Lepechinia (Bruno et al., 1991), Oreganum (Hossain et al., 2010) and Thymus (Achour et al., 2012). It may be present in Hyssopus where one of its possible derivatives, rosmanol-9-ethyl ether (7), was identified (Djarmati et al., 1991). Carnosic acid also occurs as a minor compound in one genus of the Ocimeae tribe, Ocimum (Jayasinghe et al., 2003). Brieskorn, C. H., Dumling, H. J., 1969. Carnosolsaure, der wichtige antioxydativ wirksame Inhaltsstoff des Rosmarin-und Salbeiblattes. Zeitschrift fur Lebensmittel-Untersuchung and Forschung 141, 10-16; Luis, J. C., Johnson, C. B., 2005; Bruno, Maurizio, et al. Abietane diterpenoids from Lepechinia meyeni and Lepechinia hastata. Phytochemistry 30.7 (1991): 2339-2343; Hossain, Mohammad B., et al. Characterization of phenolic composition in Lamiaceae spices by LC-ESI-MS/MS. Journal of agricultural and food chemistry 58.19 (2010): 10576-10581; Achour, S., Khelifi, E., Attia, Y., Ferjani, E., Noureddine Hellah A., 2012. Concentration of Antioxidant Polyphenols from Thymus capitatus extracts by Membrane Process Technology. Journal of food science 77, C703-C709; Djarmati, Z., Jankov, R. M., Schwirtlich, E., Djulinac, B., Djordejevic, A., 1991. High antioxidant activity of extracts obtained from sage by supercritical CO.sub.2 extracton. Journal of the American Oil Chemists Society 68, 731-734; Jayasinghe, C., Gotoh, N., Aoki, T., Wada, S., 2003. Phenolic composition and antioxidant activity of sweet basil (Ocimum basilicum L.). Journal of agricultural and food chemistry 51, 4442-4449. Seasonal variations of rosmarinic and carnosic acids in rosemary extracts. Analysis of their in vitro antiradical activity. Spanish Journal of Agricultural Research 3, 106-112.

(40) Here these phenolic diterpenes were extracted from rosemary with the aim of extracting and concentrating essentially phenolic diterpenes: 44-85%. Thus obtained extract was then deodorized in order to get rid of essential oils and volatile compounds that impact the food taste.

(41) Rosemary Extract

(42) Rosemary (Rosmarinus officinalis) leaves can be extracted with various solvents and yield extracts that are rich in different compounds. For instance, aqueous extracts are rather abundant in rosmarinic acid whereas extractions using organic solvents rather yield in extracts rich in phenolic diterpenes such as carnosic acid and carnosol. The detailed procedure to prepare the composition of Rosemary extract was described in the U.S. Pat. No. 5,859,293 and WO 96/34534, both herein incorporated by reference.

(43) The rosemary leaf was extracted with acetone at room temperature. After the extraction was completed, the acetone extract was filtered to separate the solution from rosemary leaf and concentrated under reduced pressure to make concentrated native extract. At this time, the concentrated extract can be dried directly in a vacuum oven under mild heat to make a powdered extract, which is a composition comprising about 15%-30% carnosic acid and 1%-3% carnosol. Alternatively, to the concentrated native extract, aqueous sodium carbonate (NaHCO.sub.3) was added to dissolve carnosic acid and other organic acids, while base insoluble substances were precipitated out.

(44) The solution was filtered to separate from solid, and the filtrate was further concentrated under reduced pressure. Once finishing concentration is achieved, phosphoric acid (H.sub.3PO.sub.4) was added and the acid insoluble substances (including carnosic acid, carnosol, and carnosic derivatives) were precipitated from the concentrated solution. Charcoal active is used during the process to decolorize the rosemary extract in solution before filtration. Through filtering, the precipitated solid was subsequently separated from liquid and rinsed with water to remove impurities.

(45) Last, the solid was dried in a vacuum oven and then milled into powder to make a composition containing about 40-65% carnosic acid, 2-10% carnosol, and 2-10% 12-O-methylcarnosic acid. Here used extract contained >48% carnosic acid+carnosol. A last step was done to deodorize the rosemary extract. It corresponded to a subsequent extraction of the previous solid with a mix of acetone/hexane. The purpose of this step was the elimination of fatty molecules and of volatile compounds. The filtrate was concentrated under reduced pressure and was directly formulated on liquid carrier.

(46) Within the present specification and claims, this extract standardized in phenolic diterpenes carnosic acid and carnosol, will be referred to either as rosemary, or rosemary extract or rosemary (powder) or rosemary (liquid).

(47) Hesperidin Extraction

(48) Dried immature fruits (citrus aurantium L.) were exposed to a vapor in order to remove pectins prior to the extraction with water. Subsequently, sodium hydroxide and calcium hydroxide were added in the solution to stabilize the pH value. Following the filtration step, an acidification of the filtrate was induced using HCl. Upon this step the hesperidin precipitates, the liquid solution is removed and the precipitate is dried. The final product (pure hesperidin) contains 90% to 99% hesperidin, preferably more than 95% of hesperidin as measured by HPLC.

(49) The obtained extracts contain essentially hesperidin (>80%) and are considered to be pure. Throughout this disclosure, this extract standardized in hesperidin at >95%, will be referred to as hesperidin or hesperidin (powder) or hesperidin (liquid).

(50) Punica Extraction

(51) Pomegranate skin bitter (Punica granatum L.) was extracted with ethanol/water. The extract was filtered, then concentrated. The extract was mixed with a carrier, in this example with maltodextrin prior to drying. Different drying technologies can be applied. This extract was standardized in following polyphenols: punicalagins (>7.5% by HPLC) and ellagic acid (1.5-2.5%) as determined by HPLC.

(52) Throughout this disclosure, this extract standardized in punicalagins (>7.5% by HPLC) and ellagic acid (1.5-2.5%), will be referred to either as Punica, or Punica extract or Punica (powder) or Punica (liquid).

(53) Preparation of Products and Mixtures of Rosemary Extract/Hesperidin and Rosemary Extract/Punica Extract

(54) Plant extracts and their combinations were dried into powders. Maltodextrin was used in order to insure the suitable drying process of combinations of extracts. Maltodextrins are commonly used excipients or carriers for drying processes.

(55) Maltodextrins are defined as starch hydrolysis products with dextrose equivalent less than 20. Dextrose equivalent (DE value) is a measure of the reducing power of starch derived oligosaccharides expressed as percentage of D-glucose on dry matter of hydrolysate and is inverse value of average degree of polymerisation (DP) of anhydro glucose units. As products of starch hydrolysis, maltodextrins contain linear amylose and branched amylopectin degradation products, therefore they are considered as D-glucose polymers joined by a-(1,4) and a-(1,6) linkages.

(56) Although maltodextrins are derived from a natural compound (starch), their structure is different from the initial structure of the natural molecule they derive from (starch). This difference is induced by the hydrolysis process. Thus, maltodextrin structure does not occur in nature.

(57) Other possible excipients or carriers include maltodextrin, arabic gum, dextrose, salt, mono & diglycerides of fatty acids, MPG, Polysorbate 80, vegetable oil, mono & diglycerides of fatty acids, glucose syrup, glycerin, water and alcohol.

(58) Compositions Were Added to the Raw Minced Beef Meat at 15% fat.

(59) In the course of the work leading to the present method and composition, mixtures of rosemary extract and of hesperidin or of Punica extract, in a number of varying concentration ratios were tested for antilisterial effectiveness using the classical microbiological methods. Bacterial enumeration in all here studied samples was performed on the Aloa medium according to the standardized method (NF EN ISO 11-290). The growth of Listeria monocytogenes was evaluated in meat without any antilisterial agent and without any plant extract (control). The data of listerial growth in a control meat are represented in FIG. 2. It will be noted that after 6 days of growth Listeria grew slightly, only by 0.29 log CFU/mL. After 9 days of growth, Listeria grew by 2.42 log CFU/mL. Experiments on meat were conducted in modified atmosphere packagings (MAP) that contained more than 20% O.sub.2, more precisely 70% O.sub.2 and 30% CO.sub.2.

(60) Following the meat manufacture, a batch of meat was sampled straight after the mincing process and transported in refrigerated conditions to the laboratory. In the laboratory, the meat was sampled into 2 kg samples and conditioned in vacuum at 20 C., 24 h prior to experimentation, the 2 kg meat samples were transferred at 2-4 C. and kept at this temperature for 24 h3 h until the core temperature attained 1 C.

(61) At this stage the 2 kg meat samples were inoculated with Listeria monocytogenes in a laboratory of a biosafety of level 3 so that the contamination by other microorganisms was avoided. Any further supplementation to the meat was conducted in such a laboratory. Following the homogenization of the inoculum at 4 C., the inoculated 2 kg meat samples were supplemented with plant extracts and homogenized. Plant extracts were in powder form and were added as such to the meat. To keep them as dry powders, plant extracts were supplemented with maltodextrin prior to drying process.

(62) Plant extracts could be added as lipophilic or hydrophilic liquids, or combinations thereof, to the meat. To do so, plant lipophilic or hydrophilic extract need to be solubilized or liquid, undried extracts could be used directly without undergoing the drying step.

(63) Immediately after the supplementation of plant extracts and homogenization, two pieces of 100 g of thus formed minced meat were placed together in trays. Control meat pieces, without extract treatment, followed the same procedure.

(64) Trays were then conditioned under modified atmosphere of 20% or more of oxygen, preferably 70% O.sub.2 and 30% CO.sub.2 at 4 or at 8 C. Packaged meat was stored in the dark for a stated amount of time.

(65) A series of experiments involving rosemary and hesperidin extracts, rosemary and Punica extracts, typical antilisterial compounds (Sodium lactate or Sodium acetate) and untreated control were conducted. Mixtures or alone extracts of rosemary and of hesperidin were added at 1.18% to the meat. Mixtures or alone extracts of rosemary and of Punica were added at 0.48% to the meat. Typical antilisterial compounds, Sodium lactate and Sodium acetate, were added at classic concentrations 25 g/kg and 3 g/kg, respectively, in separate experiments.

(66) Combinations of extracts were prepared and added to the meat according to the following proportions and doses prior to testing:

(67) TABLE-US-00001 Contol LM 0.5R R 0.5H H 0.5R + 0.5H 0.5R + H R + 0.5H R + H Composition of Rosemary 0.00 1.28 2.56 0.00 0.00 1.28 1.28 2.56 2.56 extracts (%) extract Carnosic acid 0.00 0.56 1.13 0.00 0.00 0.56 0.56 1.13 1.13 Carnosic acid + 0.00 0.62 1.24 0.00 0.00 0.62 0.62 1.24 1.24 carnosol Hesperidin 0.00 0.00 0.00 24.00 49.00 24.00 48.00 24.00 48.00 extract Hesperidin 0.00 0.00 0.00 22.80 46.55 22.80 45.60 22.80 45.60 Composition in Rosemary 0 151 302 0 0 151 151 302 302 minced beef extract (ppm) Carnosic acid 0 66 133 0 0 66 66 133 133 Carnosic acid + 0 73 146 0 0 73 73 146 146 carnosol Hesperidin 0 0 0 2832 5782 2832 5664 2832 5664 extract Hesperidin 0 0 0 2690 5493 2690 5381 2690 5381 R: rosemary extract; H: hesperidin; 0.5R: half concentration of rosemary extract; 0.5H: half concentration of hesperidin; R: rosemary extract; P: Punica extract; 0.5R: half concentration of rosemary extract; 0.5P: half concentration of Punica extract

(68) TABLE-US-00002 Control LM 0.5R R 0.5P P 0.5R + 0.5P 0.5R + P R + 0.5P R + P Composition of Rosemary extract 0.00 3.33 6.65 0.00 0.00 3.33 3.33 6.65 6.65 extracts (%) Carnosic acid 0.00 1.47 2.93 0.00 0.00 1.47 1.47 2.93 2.93 Carnosic acid + 0.00 1.61 3.22 0.00 0.00 1.61 1.61 3.22 3.22 carnosol Pomegranate extract 0.00 0.00 0.00 13.50 7.00 13.50 27.00 13.50 27.00 Ellagic acid 0.00 0.00 0.00 0.27 0.54 0.27 0.54 0.27 0.54 Punicalagins 0.00 0.00 0.00 1.22 2.43 1.22 2.43 1.22 2.43 Composition in Rosemary extract 0 160 319 0 0 160 160 319 319 minced beef Carnosic acid 0 70 140 0 0 70 70 140 140 (ppm) Carnosic acid + 0 77 154 0 0 77 77 154 154 carnosol Pomegranate extract 0 0 0 648 1296 648 1296 648 1296 Ellagic acid 0 0 0 13 26 13 26 13 26 Punicalagins 0 0 0 58 117 58 117 58 117

(69) Immediately after the supplementation and the homogenization, two pieces of 100 g minced meat in shape of hamburgers were placed in trays. The trays were then conditioned in a modified atmosphere containing 70% O.sub.2 and 30% CO.sub.2 and stored at 8 C. until analysis of Listerial growth and of organoleptic features, including the red color. Such analyses were conducted on the 0.sup.th, 6.sup.th and 9.sup.th day of storage.

(70) The growth of Listeria monocytogenes was evaluated in meat in refrigerated conditions for each extract or compound and for their combinations. The growth of Listeria monocytogenes was measured at the beginning of the experiment, at rd of the commercial shelf life (6 days) and at the time point corresponding to the commercial duration of the shelf life (9 days). Logarithmic values of Listerial growth (log CFU/mL) were calculated for each experiment and treatment. Differences of logarithmic values of Listerial growth (log CFU/mL) between the meat treated with plant extracts and the untreated control were calculated to yield a final result. The more negative value was obtained, the higher was the antilisterial effect of the extract or of the combination of extracts. In meat science microbiology, for a given time, values are considered to be significant between two series when a difference of 0.5 Log 10 CFU.Math.g.sup.1 is observed (Chaillou et al., 2014); (Guide pour la validation de mthodes d'essais microbiologiques et l'valuation de leur incertitude de mesure dans les domaines de la microbiologie alimentaire et de l'environnement), Schweizerische Eidgenossenschaft, Confdration suisse, Dpartement fdral de l'conomie, de la formation et de la recherche DEFR, Document No. 328, April 2013, Rev. 03). In microbiology, it will be noted that a treatment has a significant antibacterial effect if its effect exceeds 0.5 log CFU/mL as compared to the untreated control.

(71) During the listerial growth, color of meat was monitored and images were taken straight after the addition of the extract (on the day 0) and on the 6.sup.th day of growth). Images were taken under standardized light conditions of exposure and using a system called PackShot Creator. Indeed, this professional equipment consists of an optimized light box containing four fluorescent tubes diffusing homogeneous light, resulting in images always taken under the same conditions, with minimal reflection.

(72) Each picture representing the sample at a different time scale was loaded in the open source image analysis program ImageJ. The software is commonly used in the food industry to measure different food parameters such as color or density (Reineke et al. The Influence of Sugars on Pressure Induced Starch Gelatinization, Procedia Food Science, 1, 2011, 2040-3046; Kelkar et al. Developing novel 3D measurement techniques and prediction method for food density determination, Procedia Food Science, 1, 2011, 483-491). In order to obtain representative values of the red color, the color unit red (R) out of the three color units red (R) green (G) and blue (B) of the RGB model was used, and the color was measured of each pixel of a line that was drawn across the sample. The inbuilt RGB profile plot plugging was used to determine the different color values of each pixel along this line, notably the values of the red color. The results are presented as variation of the different color value as a function of the pixel number along this line. The results were statistically analyzed for significant differences using ANOVA test at p<0.05. Thus, per sample, more than 1000 pixels were analyzed.

(73) In order to evaluate the effect of plant extracts on the color of the meat, red color of the treated meat was compared to an untreated control. The effect was calculated by [red color in meat with extract][red color in control meat (without extract)]. Negative effect means that the addition of extracts does not preserve the red color of the meat. Positive effect means that the addition of extracts improves the red color of the meat as compared to the control.

(74) Mixtures of Rosemary and Hesperidin

(75) Growth of Listeria monocytogenes in Raw Meat

(76) Results of such testing at 6.sup.th and 9.sup.th day are presented in FIG. 7 and FIG. 8. Data are means of 2 to 6 replicates. Data represent log differences in L. monocytogenes growth in treated meat as compared to inoculated controls (non-treated meat). Data were statistically analyzed for significance at p<0.05 using ANOVA. Different letters indicate significant differences at p<0.05.

(77) Results of such testing at 6.sup.th day using rosemary extract and/or hesperidin are set forth in the following Table 1.

(78) TABLE-US-00003 TABLE 1 Rosemary extract (%) Hesperidin (%) Expected effect Measured effect R 0 0.08 0 H 0.09 R H 0.08 to 0.17 0.07* *Unexpected effect

(79) Rosemary extract and/or hesperidin: full concentration effects on Listeria monocytogenes growth after 6 days of growth in meat: [(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]

(80) It will be noted that at such short duration (six (6) days of growth in cold conditions), the difference in listerial growth in meat treated with plant extracts as compared with untreated meat, expressed in log, did not attain 0.5 log, which means that in such short time of growth, antilisterial effects could not be appreciated. It will be noted that at such short duration (six (6) days of growth in cold conditions), Listeria monocytogenes grew in control meat only by 0.29 log CFU/mL (FIG. 2).

(81) It will be noted that when combined, the measured effect of the combination of rosemary extract and of hesperidin does not correspond to a synergistic effect at the above concentrations after 6 days of growth as the combinatory effect is unexpectedly antagonistic.

(82) When concentrations were halved, the following expected effects calculated from the table above and measured effects were obtained and shown in Table 2.

(83) TABLE-US-00004 TABLE 2 Rosemary extract (%) Hesperidin (%) Expected effect Measured effect 0.5R 0 0.04 0.04* 0 0.5H 0.045 0.37* 0.5R 0.5H 0.045 to 0.085 0.11* R 0.5H 0.045 to 0.125 0.07 0.5R H 0.04 to 0.13 0* *Unexpected effect

(84) Rosemary extract and hesperidin: half concentrations and combinations of half and full concentrations effects on Listeria monocytogenes growth after 6 days of growth in meat:[(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]

(85) It will be noted that at such short duration (6 days of growth in cold conditions), the difference in listerial growth in meat treated with plant extracts as compared with untreated meat, expressed in log, did not attain 0.5 log, which means that in such short time of growth, antilisterial effects could not be appreciated. It will be noted that at such short duration (6 days of growth in cold conditions), Listeria monocytogenes grew in control meat only by 0.29 log CFU/mL (FIG. 2).

(86) It will be noted though, that, in the above Table 2, hesperidin applied at a half dose alone has surprisingly a greater antilisterial effect than at the full dose. Unexpected effect is signified by a star. On the other hand, effects of half dose of rosemary and of combination of half dose rosemary and full dose hesperidin were antagonistic from what was expected. Finally, effects of combination of half dose rosemary and half dose hesperidin and of combination of full dose rosemary and half dose hesperidin remained within additional range, as expected.

(87) Results of such testing at 9.sup.th day using rosemary extract and/or hesperidin are set forth in the following Table 3.

(88) TABLE-US-00005 TABLE 3 Rosemary extract (%) Hesperidin (%) Expected effect Measured effect R 0 1.12 0 H 0.64 R H 1.76 0.85 *Unexpected effect

(89) Rosemary extract and hesperidin: full concentration effects on Listeria monocytogenes after 9 days of growth in meat:[(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]

(90) It will be noted that after nine (9) days of growth in cold conditions, the difference in listerial growth expressed in log CFU/mL exceeded 0.5 log CFU/mL, which means that antilisterial effects of all extracts and their concentrations and combinations presented in the above table could be appreciated within the commercially desirable period.

(91) TABLE-US-00006 TABLE 4 Rosemary extract (%) Hesperidin (%) Expected effect Measured effect 0.5R 0 0.56 1.61* 0 0.5H 0.32 0.71* 0.5R 0.5H 2.35 1.64 R 0.5H 1.83 1.68 0.5R H 2.28 0.82 *Unexpected effect

(92) Rosemary extract and hesperidin: half concentration and combinations of half and full concentrations effects on Listeria monocytogenes growth after 9 Days of Growth in meat:[(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]

(93) After nine (9) days of growth in meat, extracts alone or their combinations at all tested concentrations inhibited the growth of Listeria monocytogenes by more than 0.5 log which means that they had an antilisterial effect in meat.

(94) Unexpectedly in view of the prior art, hesperidin had an antilisterial effect at all tested concentrations. Further, unexpectedly, rosemary extract or hesperidin alone had a greater antilisterial effect when used at half concentrations as compared to full concentrations. Still further, unexpectedly, rosemary extract combined with hesperidin at half concentrations had a greater antilisterial effect than each extract alone at full concentration. This is synergy (FIG. 3).

(95) Different concentrations and their response surfaces were analyzed using surface response methodology factorial experimental design that was designed at three levels. These results are shown in FIG. 4. They indicate the following concentration ranges that provide antilisterial response in meat which is determined as: [(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]<0.5 as provided in Table 5.

(96) TABLE-US-00007 TABLE 5 Extract Concentration (%) Hesperidin 0.5-48.0 Rosemary extract 0.2-3.0 Concentration ranges that provide antilisterial response in meat (%)

(97) It will be noted that to insure antilisterial effect, any of the above extract concentrations (Table 5) can be added in combination or alone to the meat. The total percentage of the added extract, alone or in combination, to the meat did not exceed 1.18%.

(98) During the listerial growth, color of meat was monitored and images were taken straight after addition of the extract (on the day 0) and on the 6.sup.th day of growth).

(99) Each picture representing the sample at a different time scale was loaded in the open source image analysis program ImageJ. The software is commonly used in the food industry to measure different food parameters such as color or density (Reineke et al. 2011; Kelkar et al. 2011. In order to obtain representative values of the three color units red (R), green (G) and blue (B) of the RGB models, a line was drawn across the sample. The inbuilt RGB profile plot plugin was used to determine the different color values of each pixel along this line. The results are presented as variation of the different color value as a function of the pixel number along this line. The results were statistically analyzed for significant differences using ANOVA test at p<0.05. Per sample, more than 1000 pixels were analyzed.

(100) Red Color of the Raw Meat

(101) The color of the meat was appreciated by a panel of sensorial analysis. This panel distinguished the meat color between bright red, red, brown and green hues. All meat samples were bright red on the day 0 of experiments.

(102) On the 6.sup.th day, the overall panel appreciation described the color of different meat samples subjected to different meat treatments as following:

(103) TABLE-US-00008 Meat color at Day 6 Control brown Sodium brown acetate Sodium brown lactate 0.5R brown R red 0.5H green H brown 0.5R + 0.5H red 0.5R + H brown R + 0.5H red R + H brown

(104) During the listerial growth, color of meat supplemented or not with plant extracts was monitored and images were taken straight after addition of the extract (on the day 0) and on the 6.sup.th day of growth).

(105) Results of such monitoring at 6.sup.th day using rosemary extract and hesperidin alone or in combination are set forth in the following Table 6:

(106) TABLE-US-00009 TABLE 6 Rosemary extract (%) Hesperidin (%) Expected effect Measured effect R 0 11.05 0 H 10.69 R H 21.75 15.39

(107) Rosemary extract and hesperidin of full concentration effects on red meat color after 6 days of growth in meat. The effect was calculated using: [red color of meat with extract][red color of control meat (without extract)]

(108) Contrary to the reports from the prior art, unexpectedly, Rosemary extract better preserved the red color of the meat as compared to the control. Hesperidin had slightly lower but similar effect.

(109) The combination effect remains within the additional range and therefore was not found to be synergistic at these concentrations.

(110) The combination of extracts as compared to the control significantly improves the preservation of the red color more than each extract alone.

(111) TABLE-US-00010 TABLE 7 Rosemary extract (%) Hesperidin (%) Expected effect Measured effect 0.5R 0 5.53 14.143* 0 0.5H 5.35 1.63* 0.5R 0.5H 12.51 17.16* R 0.5H 9.42 15.03* 0.5R H 16.22 13.33 *Unexpected effect

(112) Rosemary Extract and Hesperidin at Full and Half Concentrations and Combinations of Half and Full Concentrations Effects on Red Meat Color after 6 Days of Growth in Meat. Each Effect Was Calculated Using: [Red Color of Meat with Extract][Red Color of Control Meat (Without Extract)]

(113) FIG. 5 shows that when the concentration in hesperidin added to meat is halved, it significantly decreased the preservation of the red color of meat as compared to the control.

(114) Adding rosemary significantly improved the preservation of the meat color as compared to the untreated control meat. Unexpectedly, halving rosemary concentration provoked a greater effect in red color preservation of meat than the full rosemary concentration. In addition, unexpectedly, halving hesperidin concentration did not yield in a preservation effect of the red color as expected but at this concentration, hesperidin deteriorated the preservation of the red color as compared to the control. Further, unexpectedly, the effect on the preservation of the red color of the meat of the combination of full concentration of rosemary and of half concentration of hesperidin, exceeded the expected additional effects of rosemary at full concentration or of hesperidin at halved concentration alone. This is synergy.

(115) Still further, unexpectedly, the effect on the preservation of the red color of the meat of the combination of half a concentration of rosemary and of half a concentration of hesperidin, exceeded the expected additional effects of half a concentration of rosemary or of hesperidin at halved concentration alone. This is synergy.

(116) As to the rosemary extract at halved concentration and hesperidin at full concentration, their combination effect remained within the additional range and therefore was not found to be synergistic.

(117) FIG. 5 shows that all combinations between rosemary and hesperidin at any here presented concentration significantly improved the preservation of the red color of the meat as compared to the control and as compared to typical antilisterial compounds such as sodium acetate and sodium lactate.

(118) Mixtures of Rosemary and Punica

(119) Growth of Listeria monocytogenes in Raw Meat

(120) Results of such testing at 6.sup.th and 9.sup.th day are presented in FIG. 9 and FIG. 10. Data are means of 2 to 6 replicates. Data represent log differences in L. monocytogenes growth in treated meat as compared to inoculated controls (non-treated meat). Data were statistically analyzed for significance at p<0.05 using ANOVA. Different letters indicate significant differences at p<0.05.

(121) TABLE-US-00011 TABLE 8 Rosemary extract (%) Punica extract (%) Expected effect Measured effect R 0 0.26 0 P 0.05 R P 0.31 0.12 *Unexpected effect

(122) Rosemary extract and/or Punica extract: full concentration effects on Listeria monocytogenes growth after 6 days of growth in meat: [(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]

(123) It will be noted that at such short duration (6 days of growth in cold conditions), the difference in listerial growth in meat treated with plant extracts as compared with untreated meat, expressed in log, did not attain 0.5 log, which means that in such short time of growth, antilisterial effects could not be appreciated.

(124) It will be noted that when combined, the measured effect of the combination of rosemary extract and of Punica extract did not correspond to a synergistic effect.

(125) When concentrations were halved, the following expected effects calculated from the table above and measured effects were obtained:

(126) TABLE-US-00012 TABLE 9 Rosemary extract (%) Punica extract (%) Expected effect Measured effect 0.5R 0 0.13 0.28 0 0.5P 0.025 0.27 0.5R 0.5P 0.55 0.11* R 0.5P 0.53 0.03* 0.5R P 0.33 0.26* *Unexpected effect

(127) Rosemary extract and/or Punica extract: half concentrations and combinations of half and full concentrations effects on Listeria monocytogenes growth after 6 days of growth in meat: [(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]

(128) It will be noted that at such short duration (6 days of growth in cold conditions), the difference in listerial growth expressed in log did not attain 0.5 log, which means that in such short time of growth, antilisterial effects could not be appreciated.

(129) Unexpected Effect is Signified by a Star

(130) Unexpectedly, the antilisterial effect of the combination of half a concentration of rosemary and of half concentration of Punica, exceeded the expected additional effects of rosemary at halved concentration or of Punica at halved concentration, alone. This is synergy.

(131) Further, unexpectedly, the antilisterial effect of the combination of full concentration of rosemary and of half concentration of Punica, exceeded the expected additional effects of rosemary at full concentration or of Punica at halved concentration, alone. This is synergy.

(132) Still further, unexpectedly, the antilisterial effect of the combination of half a concentration of rosemary and of full concentration of Punica, exceeded the expected additional effects of rosemary at halved concentration or of Punica at full concentration, alone. This is synergy.

(133) Results of such testing at 9.sup.th day using rosemary extract and/or Punica extract are set forth in Table 10.

(134) TABLE-US-00013 TABLE 10 Punica Rosemary extract (%) extract (%) Expected effect Measured effect R 0 0.39 0 P 0.53 R P 0.92 0.63 *Unexpected effect

(135) Rosemary extract and Punica extract: full concentration effects on Listeria monocytogenes growth after 9 days of growth in meat: [(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]

(136) As mentioned above, in microbiology, it will be noted that a treatment has an antibacterial effect if its effect exceeds 0.5 log CFU/mL as compared to the untreated control. It will be noted that after 9 days of growth in cold conditions, compared to the control, the difference in listerial growth expressed in log CFU/mL exceeded 0.5 log CFU/mL when the meat was treated with full concentrations of Punica or of the combination of full concentration of rosemary and of full concentration of Punica. Rosemary alone at the full concentration did not significantly inhibit the listerial growth as compared to the untreated control meat. However, combining rosemary at full concentration with Punica at full concentration had a greater antilisterial effect than when extracts were used alone and enabled to exceed the threshold of 0.5 log CFU/mL that is required for a significant effect in antilisterial growth.

(137) TABLE-US-00014 TABLE 11 Rosemary extract (%) Punica extract (%) Expected effect Measured effect 0.5R 0 0.195 0.35* 0 0.5P 0.265 0.84* 0.5R 0.5P 1.19 1.47* R 0.5P 1.23 1.45* 0.5R P 0.88 0.69 *Unexpected effect

(138) Rosemary extract and Punica extract: half concentrations and combinations of half and full concentrations effects on Listeria monocytogenes growth after 9 days of growth in meat: [(log(CFU/mL) in meat treated with plant extracts)(log(CFU/mL) control meat (without treatment))]

(139) After 9 days of growth in meat, all but one extracts alone or their combinations at almost all tested concentrations inhibited the growth of Listeria monocytogenes by more than 0.5 log CFU/mL as compared to the control which means that they had an antilisterial effect in meat. Only rosemary extract alone when tested at half a concentration did not attain the difference of 0.5 log CFU/mL as compared to the control.

(140) Unexpectedly, Punica extract alone had a greater antilisterial effect when used at half concentration as compared to a full concentration. Further, unexpectedly, when used at a half concentration, rosemary extract had a greater antilisterial effect than expected. Still further, unexpectedly, the antilisterial effect of the combination of half concentration of rosemary and of half concentration of Punica extract, exceeded their expected additional effects of rosemary at halved concentration or of Punica extract at halved concentration alone. This is synergy.

(141) In addition, unexpectedly, the antilisterial effect of the combination of full concentration of rosemary and of half concentration of Punica extract, exceeded the expected additional effects of rosemary at full concentration or of hesperidin at halved concentration, alone. This is synergy.

(142) Further, unexpectedly, rosemary extract combined with Punica extract at half concentrations had a greater antilisterial effect than each extract alone at full concentration. This is synergy (See e.g., FIG. 1).

(143) Different concentrations and their response surfaces were analyzed using surface response methodology factorial experimental design that was designed at three levels. These results are shown in FIG. 11. They indicate the following concentration ranges that provide antilisterial response in meat which is determined as [log(CFU/mL) in meat treated with plant extracts][log(CFU/mL) control meat (without plant extract)]1 as shown in Table 12.

(144) TABLE-US-00015 TABLE 12 Extract Extract (%) Punica extract 5.0-24.0 Rosemary extract 0.5-8.0 *Extract %

(145) It will be noted that to insure antilisterial effect, any of the above extract concentrations (Table 12) can be added in combination or alone to the meat. The total percentage of the added extract, alone or in combination, to the meat did not exceed 0.18%.

(146) Red Color of the Raw Meat

(147) The color of the meat was appreciated by a panel of sensorial analysis. This panel distinguished the meat color between bright red, red, brown and green hues. All meat samples were bright red on the day 0 of experiments.

(148) On the 6.sup.th day, the overall panel appreciation described the color of different meat samples subjected to different meat treatments as following:

(149) TABLE-US-00016 Meat color at Day 6 Control brown Sodium acetate brown Sodium lactate brown 0.5R brown R brown 0.5P red P red 0.5R + 0.5P red 0.5R + P brown R + 0.5P red R + P brown

(150) During the listerial growth, color of meat supplemented or not with plant extracts was monitored and images were taken straight after addition of the extract (on the day 0) and on the 6.sup.th day of growth). Red pixels were quantified as explained above in the Methods section.

(151) Results of such monitoring at 6.sup.th day using rosemary extract and Punica extract alone or in combination are set forth in the following:

(152) TABLE-US-00017 TABLE 13 Rosemary Punica extract extract (%) (%) Expected effect Measured effect R 0 3.72 0 P 5.60 R P 9.32 1.27* *Unexpected effect

(153) Rosemary extract and Punica extract: full concentration effects on the preservation of the red color of meat after 6 days of growth in meat. The effect was calculated by: [red color of meat with extract][red color of control meat (without extract)]

(154) At the above concentrations (Table 13), when added alone, rosemary or Punica extract deteriorated the preservation of the red color of the meat as compared to the control. It was therefore expected that when combined, these extracts would even further deteriorate the preservation of the red color of the meat. Unexpectedly, when combined, rosemary and Punica extracts improved the preservation of the red color of the meat as compared to the control.

(155) Unexpectedly, the effect on the preservation of the red color of the meat of the combination of full concentration of rosemary and of full concentration of Punica, exceeded the expected additional effects of rosemary at full concentration or of full concentration of Punica alone. This is synergy.

(156) TABLE-US-00018 TABLE 14 Rosemary Punica extract extract (%) (%) Expected effect Measured effect 0.5R 0 1.86 1.07* 0 0.5P 2.80 0.34* 0.5R 0.5P 0.73 4.45* R 0.5P 4.04 2.71* 0.5R P 4.53 6.37* *Unexpected effect

(157) Rosemary extract and Punica extract: full and half concentrations and combinations of half and full concentrations effects on the preservation of the red color of the meat after 6 days of Listerial growth in meat. Each effect was calculated by: [red color of meat with extract][red color of control meat (without extract)]

(158) As full concentrations of rosemary and of Punica extracts deteriorated the preservation of the red color of the meat, it was expected that halved concentrations would have also deteriorated the preservation of the red color of the meat. Unexpectedly, halving the added rosemary concentration significantly improved the preservation of the red color of the meat as compared to the untreated control.

(159) Adding rosemary significantly improved the preservation of the meat color as compared to the untreated control meat. Unexpectedly, halving the concentration of the added Punica extract did not deteriorate as much as expected the preservation of the red color of the meat.

(160) Further, unexpectedly, the improvement of the preservation of the red color of the combination of half concentration of rosemary and of half concentration of Punica extract, exceeded their expected additional effects of rosemary at halved concentration or of Punica extract at halved concentration alone. This is synergy.

(161) In addition, unexpectedly, the improvement of the preservation of the red color of the combination of full concentration of rosemary and of half concentration of Punica extract, exceeded the expected additional effects of rosemary at full concentration or of hesperidin at halved concentration, alone. This is synergy.

(162) Still further, unexpectedly, the improvement of the preservation of the red color of the combination of full concentration of Punica extract and of half concentration of rosemary extract, exceeded the expected additional effects of Punica extract at full concentration or of rosemary extract at halved concentration, alone. This is synergy.

(163) Unexpectedly, the combination of full concentration of Punica extract and of half concentration of rosemary extract improved the preservation of the color of the meat whereas it was expected that the preservation of the color be deteriorated upon application of such combination.

(164) FIG. 6 shows that all combinations between rosemary and Punica extract at any here presented concentration significantly improved the preservation of the red color of the meat as compared to the control and as compared to classic antilisterial agents such as Sodium acetate and Sodium lactate.

(165) One of ordinary skill in the art will recognize that additional embodiments are also possible without departing from the teachings of the presently-disclosed subject matter. This detailed description, and particularly the specific details of the exemplary embodiments disclosed herein, is given primarily for clarity of understanding, and no unnecessary limitations are to be understood therefrom, for modifications will become apparent to those skilled in the art upon reading this disclosure and can be made without departing from the spirit and scope of the presently-disclosed subject matter.

REFERENCES

(166) Numerous references have been cited throughout this disclosure. All references cited in this disclosure including the three listed both are incorporated by reference. Kai Reineke, Henning Weich, Dietrich Knorr, The Influence of Sugars on Pressure Induced Starch Gelatinization, Procedia Food Science, Vol. 1, (2011), pages 2040-2046. Shivangi Kelkar, Scott Stella, Carol Boushey, Martin Okos, Developing novel 3D measurement techniques and prediction method for food density determination, Procedia Food Science, Vol. 1, (2011), pages 483-491. S. Chaillou, S. Christieans, M. Rivollier, I. Lucquin, M. G. Champomier-Vergs, M. Zagorec; Quantification and efficiency of Lactobacillus sakei strain mixtures used as protective cultures in ground beef; Meat Science 97, (3) (2014), pages 332-338.