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

1-38. (canceled)

39. An antimicrobial composition comprising an effective amount of a phenolic diterpene and of hesperidin.

40. An antibacterial composition comprising an effective amount of a phenolic diterpene and of hesperidin.

41. The composition according to claim 40 wherein the composition is effective against Gram positive bacteria selected from the group consisting of Bacillus cereus, Staphylococcus aureus, Streptococcus mutans, Listeria monocytogenes, Clostridium perfringens, Enterococcus hirae and Mycobacterium bovis.

42. The composition according to claim 40 wherein the composition is effective against Gram negative bacteria selected from the group consisting of Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium and Enterobacter cloacae.

43. An anti-fungal/anti-yeast composition comprising an effective amount of a phenolic diterpene and of hesperidin.

44. The composition according to claim 43 wherein the composition is effective against yeast selected from the group consisting of Saccharomyces cerevisiae and Candida albicans.

45. The antimicrobial composition according to claim 39 comprising pure hesperidin and a Lamiaceae extract, wherein a majority of volatile oil components from the lamiaceae extract having been removed.

46. The antibacterial composition according to claim 40 comprising pure hesperidin and a Lamiaceae extract, wherein a majority of volatile oil components from the lamiaceae extract having been removed.

47. The antimicrobial composition according to claim 45, wherein the pure hesperidin contains from 80% to 99% hesperidin.

48. The antibacterial composition according to claim 46, wherein the pure hesperidin contains from 80% to 99% hesperidin.

49. The anti-fungal/anti-yeast composition according to claim 43 comprising pure hesperidin and a Lamiaceae extract, wherein a majority of volatile oil components from the lamiaceae extract having been removed.

50. The anti-fungal/anti-yeast composition according to claim 49, wherein the pure hesperidin contains from 80% to 99% hesperidin.

51. A food product comprising a food and the composition of claim 39.

52. A food product comprising a food and the composition of claim 40.

53. The food product according to claim 51, wherein the food is selected from the group consisting of fresh meat, fish and poultry.

54. The food product according to claim 52, wherein the food is selected from the group consisting of fresh meat, fish and poultry.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] 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.

[0083] FIG. 2 is a graph showing Listeria monocytogenes growth in meat in accordance with the present invention.

[0084] FIG. 3 is a graph showing antilisterial synergy effects of combinations of hesperidin and rosemary extract in accordance with the present invention.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] FIG. 7 is a chart showing inhibition of Listeria monocytogenes growth by plant extracts in minced beef.

[0089] FIG. 8 is a graph showing inhibition of Listeria monocytogenes growth by plant extracts in minced beef at 8 C. at day 9.

[0090] FIG. 9 is a chart showing inhibition of Listeria monocytogenes growth by plant extracts in minced beef at 8 C. at day 6.

[0091] 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.

[0092] 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.

[0093] FIG. 12 is a graph showing Listeria monocytogenes in poultry sausages.

[0094] FIG. 13 is a graph showing inhibition of Listeria growth in poultry sausages by an extract combination of R/P in accordance with the present invention.

[0095] FIG. 14 is a graph showing inhibition Listeria growth in poultry sausage by extract combination of R/H.

[0096] FIG. 15 is a graph showing Listeria monocytogenes in pork sausage (control).

[0097] FIG. 16 is a graph showing inhibition of Listeria growth in pork sausages by an extract combination of R/P.

[0098] FIG. 17 is a graph showing inhibition of Listeria growth in pork saugsage by an extract combination of R/H.

[0099] FIG. 18 is a graph showing growth of Listeria monocytogenes in smoked salmon (control).

[0100] FIG. 19 is a graph showing inhibition of Listeria monocytogenes in smoked salmon on the 30.sup.th day of growth.

[0101] FIG. 20 is a graph showing inhibition of Listeria monocytogenes in smoked salmon on the 30.sup.th day of grown.

DETAILED DESCRIPTION OF THE INVENTION

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] Other flavonoids that have the same effect including but are not limited to: narigin, isocurametin, neohesperidin, hesperidin, poncirin, nebiletin, and tangeretin.

Definitions

[0110] The following are a list of definitions used throughout this disclosure:

[0111] 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.

[0112] 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.

[0113] 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.

[0114] Hesperidin means a compound extracted from nature or synthesized.

[0115] 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.

[0116] Meat, fish and poultry means both a) processed meat, fish and poultry and b) unprocessed meat, fish and poultry.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] Pure hesperidin extract means a hesperidin extract that has a concentration of at least 80% hesperidin.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

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

[0127] 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.

[0128] 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.

[0129] Rosemary Extract

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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).

[0135] Hesperidin Extraction

[0136] 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.

[0137] 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).

[0138] Punica Extraction

[0139] 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.

[0140] 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).

[0141] Preparation of Products and Mixtures of Rosemary Extract/Hesperidin and Rosemary Extract/Punica Extract

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] Compositions Were Added to the Raw Minced Beef Meat at 15% fat.

[0147] 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.

[0148] 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., 24h 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] Combinations of extracts were prepared and added to the meat according to the following proportions and doses prior to testing:

TABLE-US-00001 Control 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 Control LM 0.5R R 0.5P P 0.5R + 0.5P 0.5R + P R + 0.5P R + P Composition Rosemary extract 0.00 3.33 6.65 0.00 0.00 3.33 3.33 6.65 6.65 of 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 27.00 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 Rosemary extract 0 160 319 0 0 160 160 319 319 in 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 R: rosemary extract; H: hesperidin extract; 0.5R: half concentration of rosemary extract; 0.5H: half concentration of hesperidin extract R: rosemary extract; P: Punica extract; 0.5R: half concentration of rosemary extract; 0.5P: half concentration of Punica extract

[0155] 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.

[0156] 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 fderal 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.

[0157] 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.

[0158] 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.

[0159] 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.

Mixtures of Rosemary and Hesperidin

[0160] Growth of Listeria monocytogenes in Raw Meat

[0161] 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.

[0162] Results of such testing at 6.sup.th day using rosemary extract and/or hesperidin are set forth in the following Table 1.

TABLE-US-00002 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 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))]

[0163] 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).

[0164] 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.

[0165] When concentrations were halved, the following expected effects calculated from the table above and measured effects were obtained and shown in Table 2.

TABLE-US-00003 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 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))]

[0166] 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).

[0167] 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.

[0168] Results of such testing at 9.sup.th day using rosemary extract and/or hesperidin are set forth in the following Table 3.

TABLE-US-00004 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 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))]

[0169] 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.

TABLE-US-00005 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 0.88 1.64* R 0.5H 1.83 1.68 0.5R H 2.28 0.82 *Unexpected effect 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))]

[0170] 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.

[0171] Unexpectedly in view of the prior art and in view of data in vitro, 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).

[0172] 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.

TABLE-US-00006 TABLE 5 Extract Extract proportion in combination (%) Hesperidin 0.5-48.0 Rosemary extract 0.2-3.0 Extract proportions in combination that provide antilisterial response in meat (%)

[0173] 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%.

[0174] 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).

[0175] 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.

[0176] Red Color of the Raw Meat

[0177] 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.

[0178] On the 6.sup.th day, the overall panel appreciation described the color of different meat samples subjected to different meat treatments as following:

TABLE-US-00007 Meat color at Day 6 Control brown Sodium acetate brown Sodium lactate brown 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

[0179] 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).

[0180] 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:

TABLE-US-00008 TABLE 6 Rosemary extract (%) Hesperidin (%) Expected effect Measured effect R 0 11.05 0 H 10.69 R H 21.75 15.39

[0181] 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)]

[0182] 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.

[0183] The combination effect remains within the additional range and therefore was not found to be synergistic at these concentrations.

[0184] The combination of extracts as compared to the control significantly improves the preservation of the red color more than each extract alone.

TABLE-US-00009 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 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)]

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

Mixtures of Rosemary and Punica

[0190] Growth of Listeria monocytogenes in Raw Meat

[0191] 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.

TABLE-US-00010 TABLE 8 Rosemary Punica extract (%) extract (%) Expected effect Measured effect R 0 0.26 0 P 0.05 R P 0.31 0.12 *Unexpected effect 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))]

[0192] 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.

[0193] 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.

[0194] When concentrations were halved, the following expected effects calculated from the table above and measured effects were obtained:

TABLE-US-00011 TABLE 9 Rosemary Punica extract (%) 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 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))]

[0195] 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.

[0196] Unexpected Effect is Signified by a Star

[0197] 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.

[0198] 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.

[0199] 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.

[0200] Results of such testing at 9.sup.th day using rosemary extract and/or Punica extract are set forth in Table 10.

TABLE-US-00012 TABLE 10 Rosemary Punica extract (%) extract (%) Expected effect Measured effect R 0 0.39 0 P 0.53 R P 0.92 0.63 *Unexpected effect 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))]

[0201] 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.

TABLE-US-00013 TABLE 11 Rosemary Punica extract (%) 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 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))]

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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).

[0206] 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.

TABLE-US-00014 TABLE 12 Extract Extract (%) Punica extract 5.0-24.0 Rosemary extract 0.5-8.0 * Extract %

[0207] 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%.

[0208] Red Color of the Raw Meat

[0209] 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.

[0210] On the 6.sup.th day, the overall panel appreciation described the color of different meat samples subjected to different meat treatments as following:

TABLE-US-00015 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

[0211] 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.

[0212] 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:

TABLE-US-00016 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 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)]

[0213] 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.

[0214] 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.

TABLE-US-00017 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

[0215] 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)]

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] In vitro experiments:

[0225] Antimicrobial activities of Rosemary, Punica and hesperidin extracts alone and in combination Plant extracts of rosemary, hesperidin, Punica and their combinations rosemary/hesperidin, rosemary/Punica were prepared in 10% at DMSO.sub.50% final according to the following protocol in which combinations of extracts were prepared, completed up to 100% with maltodextrin and added to the test solutions according to the following proportions and doses prior to testing.

TABLE-US-00018 TABLE 15 R/H Composition of Rosemary extract 3.05 extracts (%) Carnosic acid 1.34 Carnosic acid + carnosol 1.48 Hesperidin extract 56.7 Hesperidin 53.87 R: rosemary extract; H: hesperidin extract

TABLE-US-00019 TABLE 16 R P R/P Composition of Rosemary extract 5.33 0 5.33 extracts (%) Carnosic acid 2.35 0 2.35 Carnosic acid + carnosol 2.58 0 2.58 Pomegranate extract 0 21.60 21.60 Ellagic acid 0 0.43 0.43 Punicalagins 0 1.94 1.94 R: Rosemary extract; P: Punica extract

[0226] Preparation of the Working/Test Solution

[0227] 200 mg of the extract (combination) were mixed with 1 ml 100% DMSO, vortexed, sonicated for 10 min at power 100%, 45 kHz, normal mode, well vortexed, sonicated again and diluted 1:2 in sterile water at a final concentration of 100 mg/ml DMSO.sub.50% final.

[0228] These preparations at 100 mg/ml were prepared in sterile 5 ml Eppendorf tube. A sample was taken prior to preparing Minimum Bactericidal Concentration (MBC) plates. MBC, Minimal Fungicidal Concentration (MFC) and Minimal Inhibitory Concentration (MIC).

[0229] Principle

[0230] The minimum bactericidal concentration (MBC) is the lowest sample concentration required to kill at least 99.99% of the inoculum (4 log 10). The minimum bactericidal concentration (MBC) is the lowest concentration of an antibacterial agent required to kill a particular bacterium. It can be determined from broth dilution minimum inhibitory concentration (MIC) tests by subculturing to agar plates that do not contain the test agent. The MBC is identified by determining the lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by 99.9%. The MBC is complementary to the MIC; whereas the MIC test demonstrates the lowest level of antimicrobial agent that inhibits growth, the MBC demonstrates the lowest level of antimicrobial agent that results in microbial death. This means that even if a particular MIC shows inhibition, plating the bacteria onto agar might still result in organism proliferation because the antimicrobial did not cause death. Antibacterial agents are usually regarded as bactericidal if the MBC is no more than four times the MIC. This test was based on the count of microorganisms in wells displaying little or no growth visually and then plated.

[0231] Samples prepared in 10% DMSO.sub.50% .sub.final were tested at concentrations of 2.5, 0.5 and 0.1%, against one equivalent DMSO control (respectively 12.5, 2.5 and 0.5%).

[0232] In the case of yeast, fungicide minimum concentrations (CMF) were performed as the WCD. In the case of the strain A. brasiliensis, a fungus, the mere presence of growth was interpreted as absence of fungicidal activity. The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after adapted period of incubation. Minimum inhibitory concentrations are typically used to determine the potency of new antimicrobial agents, such as plant extracts or their combinations. The minimal inhibitory concentration or MIC is the lowest concentration that is sufficient to inhibit microbial growth by here tested extracts. This test is based on visual observation of the wells that contain microbial strain that is studied with tested extracts or without (control). Time length and conditions of microbial growth were conducted according to classical methods well known by one ordinary skilled in the art.

[0233] The tests were performed on the following bacterial strains E. coli, Pseudomonas aeruginosa, Salmonella enterica ser typhimurium, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Streptococcus mutans, Clostridium perfringens, Enterococcus hirae, Enterobacter cloacae, Moraxella bovis and two yeasts Saccharomyces cerevisiae and Candida albicans and mold Aspergillus brasiliensis. The evaluation of bactericidal and fungicidal activity was conducted according to the internal procedure in rich nutrient media , with a calibrated inoculum of 10.sup.5-10.sup.6 CFU/ml for bacteria and yeasts and 10.sup.5 spores/ml for A. brasiliensis.

[0234] Prior to antimicrobial evaluation of extracts, the sterility of extracts was verified by plating. The absence of any microbial growth was compulsory. Microbial cultures were conducted classically as summarized in Table 17.

TABLE-US-00020 TABLE 17 Growth conditions and collections of microorganisms Organism Collection Culture conditions Bacillus cereus ATCC 11.778 TS-30 C.-24 h Staphylococcus CIP 4.83 TS-37 C.-18 h aureus Listeria ATCC 19.115 TS-37 C.-24 h monocytogenes Salmonella CIP 103.799 TS-37 C.-18 h typhimurium Escherichia coli CIP 53.126 TS-37 C.-18 h Pseudomonas CIP 82.118 TS-37 C.-18 h aeruginosa Streptococcus ATCC 35.668 COS 5% sang/Broth Streptos- mutans 37 C.-24 h Enterococcus ATCC 8043 Broth TS-37 C.-24 h hirae Enterobacter CIP 103.475 Broth TS-30 C.-24 h cloacae Moraxella bovis CIP 70.40 T TS/Broth Col 10% horse serum-30 C.-24 h Clostridium ATCC 13.124 RCM/37 C./Anaerobiosis/48 h perfringens Candida albicans UMIP 48.72 Sabouraud/30 C./48 h Saccharomyces UMIP 1181.79 Sabouraud/30 C./48 h cerevisiae Aspergillus niger IP 1431.83 Sabouraud/30 C./48 h

[0235] Results

TABLE-US-00021 TABLE 18 MBC and MFC concentrations (0.1; 0.5 or 2.5%) of Rosemary (R), Punica (P), Rosemary/Punica (R/P) and Rosemary/Hesperidin (R/H) as effective against the here presented microorganisms. R/H R/P S. aureus 0.5 0.1 S. mutans 2.5 0.5 S. typhimirium 2.5 0.1 P. aeruginosa 2.5 0.1 E. coli 2.5 0.5 L. monocytogenes 0.5 B. cereus 2.5 0.1 C. perfringens 0.5 0.1 E. hirae 2.5 E. cloacae 0.5 M. bovis 0.1 0.1 R/H combination of rosemary and hesperidin extracts according to Table 15; R: rosemary extract, P: Punica extract, R/P: combination of rosemary and Punica extracts according to Table 16. Empty cells: minimal concentrations (if any) would be higher than the highest concentration here measured (2.5%).

TABLE-US-00022 TABLE 19 Unexpected/Synergistic MBC and MFC effects of R and P (% in test solution) Expected Measured MBC/MFC MBC/MFC MBC/MFC MBC/MFC of R (%) of P (%) of R/P (%) of R/P (%) S. typhimirium 0.5 0.5 0.25-0.5 0.1* P. aeruginosa 0.5 0.5 0.25-0.5 0.1* C. perfringens 0.03 >0.016 >0.016 0.0078* *Unexpected result Unexpectedly, when applied in combination, the minimal concentration of the combination R/P to decrease microbial growth by 4 log, S. typhimirium and P. aeruginosa is surprisingly low; it is 2.5-5 times lower than expected. Surprisingly, when the two extracts are combined, their activity is synergistically increased by 2.5-5 times.

[0236] Unexpectedly, the anti-salmonella effect of the combination of rosemary and Punica extracts, exceeded their expected additional effects when extracts were applied alone. Indeed, it would have been expected that to achieve the same anti-salmonella effect and decrease microbial growth by 4 log, MBC would have been at best halved when the extracts are combined as compared to MBC when extracts were applied alone. Surprisingly, here, MBC were decreased by 2.5-5 times. This is synergy.

[0237] Further unexpectedly, the anti-pseudomonas effect of the combination of rosemary and Punica extracts, exceeded their expected additional effects when extracts were applied alone. Indeed, it would have been expected that to achieve the same anti-pseudomonas effect and decrease microbial growth by 4 log, MBC would have been at best halved when the extracts are combined as compared to MBC when extracts were applied alone. Surprisingly, here, MBC were decreased by 2.5-5 times. This is synergy.

[0238] Still further unexpectedly, the anti-clostridium effect of the combination of rosemary and Punica extracts, exceeded their expected additional effects when extracts were applied alone. Indeed, it would have been expected that to achieve the same anti-clostridium effect and decrease microbial growth by 4 log, MIB would have been at best halved when the extracts are combined as compared to MIB when extracts were applied alone. This is synergy. Note, Punica extract had a MIC effect of 0.016%. This implies that its MBC must be higher than MIC, meaning higher than 0.016%. Either way, it is further unexpected that the MBC of the combination of rosemary and Punica extracts is more than twice lower than the MIC of an extract alone.

TABLE-US-00023 TABLE 20 Unexpected/Synergistic MIC effects of R and P (% in test solution) Expected Measured MIC MIC MIC MIC of R (%) of P (%) of R/P (%) of R/P (%) S. mutans 0.25 >1 0.25 0.0625* S. aureus 0.125 0.5 0.125-0.325 0.0625* C. albicans 1 >1 1 0.25* S. cerevisiae 0.25 >1 0.25 0.0625*

[0239] Unexpectedly, when applied in combination, the minimal concentration of the combination R/P to inhibit microbial growth of bacteria or of yeast, S. mutans, S. aureus, C. albicans and S. cerevisiae is surprisingly low; it is 2-4 times lower than expected. Surprisingly, when the two extracts are combined, their activity is synergistically increased by 2.5-5 times than expected.

[0240] Further unexpectedly, the anti-streptococcus effect of the combination of rosemary and Punica extracts, exceeded their expected additional effects when extracts were applied alone. Indeed, it would have been expected that to achieve the same anti-streptococcus effect and inhibit microbial growth, MIC would have been at best at the lowest MIC as compared to MIC when extracts were applied alone. Surprisingly, here, MIC were decreased by 4 times than expected. This is synergy.

[0241] Still further unexpectedly, the anti-staphylococcus effect of the combination of rosemary and Punica extracts, exceeded their expected additional effects when extracts were applied alone. Indeed, it would have been expected that to achieve the same anti-staphylococcus effect and inhibit microbial growth, MIC would have been at best halved when the extracts are combined as compared to MIC when extracts were applied alone. Surprisingly, here, MIC were decreased by 2-4.5 times than expected. This is synergy.

[0242] Further unexpectedly, the anti-candida effect of the combination of rosemary and Punica extracts, exceeded their expected additional effects when extracts were applied alone. Indeed, it would have been expected that to achieve the same anti-candida effect and inhibit microbial growth, MIC would have been at best at the lowest MIC as compared to MIC when extracts were applied alone. Surprisingly, here, MIC were decreased by 4 times than expected. This is synergy.

[0243] And, still further unexpectedly, the anti-saccharomyces effect of the combination of rosemary and Punica extracts, exceeded their expected additional effects when extracts were applied alone. Indeed, it would have been expected that to achieve the same anti-saccharomyces effect and inhibit microbial growth, MIC would have been at best at the lowest MIC as compared to MIC when extracts were applied alone. Surprisingly, here, MIC were decreased by 4 times than expected. This is synergy.

[0244] It is noted that P. aeruginosa and S. typhimirium are Gram negative bacteria. C. perfringens, S. mutans, S. aureus and L. monocytogenes are Gram positive bacteria. C. albicans and S. cerevisiae are yeast.

[0245] Rosemary in combination with Punica extract clearly exhibits synergistic antimicrobial effects against the growth of Gram positive (including L. monocytogenes (in meat), C. perfringens, S. mutans, S. aureus), Gram negative (including S. typhimirium, P. aeruginosa) bacteria and yeast (including C. albicans, Saccharomyces cerevisiae).

TABLE-US-00024 TABLE 21 Unexpected/Synergistic effects of the combination of extracts R/P on different microorganisms are here summarized Nature of Gram Antimicrobial Microorganism microorganism (+ or ) effect L. monocytogenes Bacteria G+ Unexpected, Synergy S. typhimirium Bacteria G Unexpected, Synergy P. aeruginosa Bacteria G Unexpected, Synergy C. perfringens Bacteria G+ Unexpected, Synergy S. mutans Bacteria G+ Unexpected, Synergy S. aureus Bacteria G+ Unexpected, Synergy C. albicans Yeast Unexpected, Synergy S. cerevisiae Yeast Unexpected, Synergy

[0246] Note that hesperidin in combination with rosemary had no bactericidal effect in vitro on Listeria monocytogenes even at highest here tested concentrations: 2.5%.

[0247] Yet, surprisingly, such extract combination significantly and efficiently inhibited the growth of Listeria monocytogenes in meat products in synergistic manner.

[0248] Similar observation could be drawn when comparing hesperidin effects in vitro, as reported in prior art against Listeria monocytogenes growth and in meat (this study). Indeed, although hesperidin in vitro did not have antilisterial effects, it inhibited listerial growth in beef meat.

[0249] Use of Rosemary/Punica and Rosemary/Hesperidin Extracts in Meat/Poultry/Fish Matrices

[0250] Processing Methods, Extract Incorporation, Listerial Contamination and Growth Inhibition

[0251] Fresh pork sausages and poultry were produced according to standard recipes used by industry. As far as fish and seafood are concerned, here exampled by smoked salmon, its processing was also done according to the industrial procedures.

[0252] Meat/Poultry/Fish Batches

[0253] Whenever possible, to overcome the variability of raw materials in terms of pH and endogenous flora (rate and nature of the constituent flora) for each meat/category, the processing was carried out on 3 batches of raw materials originating from different meat/poultry/fish suppliers/(salmon) farms. Plant extracts or their combinations were incorporated at the beginning of each recipe, together with basic processing ingredients. The plant extracts or their combinations were incorporated in food matrices at 1-3 different concentrations, as explained in Tables 23, 24, 26, 27, 29, and 30 below.

[0254] As far as every product category is concerned, controls, i.e. products not comprising plant extracts, were tested.

[0255] Nature and Origin of Strains of Listeria monocytogenes/Bacterial Preparation Conditions

[0256] Two strains of Listeria monocytogenes in mixture (50/50) were studied according to the Standard Operating Procedure NF V01-009 and concerning any meat/poultry/fish product. As far as tests in meat products are concerned: the reference strain CIP 7838 (serovar 4b) and a so-called field strain isolated from pork (ADIV collection) serotype 1/2a, the one that predominates in more than 50% on the fresh pork were used.

[0257] As far as poultry meat is concerned, the reference strain 7838 was coupled to a strain of Listeria monocytogenes isolated from poultry carcass (ADIV collection; serotype 1/2b). As far as tests in salmon are concerned: the reference strain CIP 7838 was mixed with a strain isolated from salmon that ADIV was donated by a partner of its network. As far as any here studied food matrix is concerned, the two strains were prepared and inoculated according to the guidelines of Standard Operating Procedures NF V01-009 (Version 2014).

[0258] Frozen strains, preserved as cryobilles (80 C.), were revived and cultivated individually. Each strain was revived by transplanting 0.1 mlin 10 ml of BHI culture medium (for 24 h at 30 C.). Two successive subcultures have resulted in a pre-culture of each strain. Subsequently, each strain was again cultured for 24 hours at 30 C. The latter culture was used in matrix product contamination (at end of the exponential phase or early stationary phase). After two successive centrifugations, the pellets were suspended in 10 ml of buffered peptone water (BPW), counts were performed on BHI medium and the bacterial solution was kept at 0 C. for 24 hours prior to inoculation. After reading of the bacterial growth, the concentrations were then adjusted so as to inoculate the food matrix product with the mixture of the two strains (50/50) at 2-3 log cfu/g.

[0259] Inoculation Method

[0260] As specified in the NF V01-009, the products inoculation mode should match the reality of industrial contamination. Thus, sausages (pork or poultry) were infected by inoculation in the mass of the meat mix to simulate contamination from meat As the contamination of smoked salmon occurs during the handling of raw materials or when slicing/packaging, here contamination of the surface of salmon slices was applied.

[0261] The inoculation method on the surface of the products has been developed by ADIV and provided an accurate weight of the inoculum, calculated in a way to obtain the desired concentration at the surface of the products. Irrespectively of the method of inoculation (ground or surface), in order to maintain the adequate water activity of food products, the aqueous volume to be added to the food matrix was calculated so as not to exceed a weight/volume ratio of 1/100 (NF V01-009).

[0262] Meat/Poultry/Fish Product Elaboration and Extract Incorporation

[0263] Pork Sausages

[0264] Pork sausages were manufactured according to a conventional process, in compliance with the code of practice, from lean pork (86%) and pork fat (14%). The initial meat mix intended for sausage manufacturing was obtained by grinding/mincing fat and lean parts of pork meat at low-temperature trough a grid (6 mm grid). Then, here tested plant extracts were added whenever applicable, according to Table 24 and Table 25. After homogenization, the mix was then stuffed into natural casings (sheep menus, 24/26 diameter).

[0265] Contamination/Packaging

[0266] For a given batch, a meat mix of 42 kg was prepared and then divided into different sets of 5 kg each for the production of test series. Five (5) kg of meat mix were added to a mix prepared that did not contain any antimicrobial agent nor additive (Mix Fraiche 230 South at 23 g/kg) and 50 g water/kg. After homogenization, thus obtained mixture was divided into parts. A portion without artificial listerial contamination, was placed in trays (6 sausages per tray) that were packaged in MAP conditions (70% O.sub.2/30% CO.sub.2) with a conservation time scenario of conservation time at 4 C. and of conservation time at 8 C. The other part was artificially contaminated by Listeria monocytogenes at an average rate of 3 log cfu/g (inoculation in the ground meat mass) before being packaged and conserved as described above. The contaminated meat samples that did not comprise plant extracts were termed control.

[0267] Analytical Monitoring During the Conservation

[0268] Microbiological analyses were performed on D0 and JDLC (D14) and they concerned the enumeration of Listeria monocytogenes, the total mesophilic acidifying flora (FAM) and the lactic flora. At each point of analysis, a single repeat batch was performed by analysis of a tray (n=3 for the 3 batches).

[0269] Manufacturing of Poultry Sausages

[0270] Poultry sausages were produced according to the same experimental conditions as described for pork sausages. The only difference remains in the nature of the raw materials used and the nature of compounds that may have been added. Indeed, in this case, the lean was made using upper parts of chicken thighs 85% and fat represented by the skin.

[0271] The ground meat mix did not include any classical antimicrobial but contained a mix from which any ingredients having antimicrobial or antioxidative activity were removed (Mix Chipo Flight 310 to 31 g/kg). Thus prepared mixture either comprised plant extracts, or not (controls) and followed the same experimental conditions and listerial/bacterial analyses as described for pork.

[0272] Preparation and Inoculation of Smoked Salmon

[0273] The manufacture of smoked salmon was conducted according to classical procedures well known from and available to a person skilled in the art. As for meat products, three different batches of salmon from three different origins were made during the same week. The products were packaged sliced on trays under vacuum (about 200 g per tray) on the industrial site prior to listerial/bacterial inoculation and analysis.

[0274] Tested seafood, i.e. smoked salmon, were incorporated extract combinations of Rosemary and hesperidin extracts, and of Rosemary and Punica extracts according to proportions and concentrations described in Tables 29 and 30. The salmon was weighed and then surface-inoculated by spraying, with the mixture of the two strains (50/50) at an average rate of 30.5 log/g. After inoculation, the plates were again vacuum packed and put into storage at 8 C. for 30 days. Listerial growth was monitored upon inoculation (day 0) and on the 30.sup.th day of growth. Non-inoculated controls were kept at cold storage for all meet/poultry/fish matrices tested here for comparison purposes.

[0275] pH, total mesophilic acidifying flora, lactic flora were measured throughout all experiments in beef, pork, poultry meat or fish. The presence of here tested botanical extracts in these food matrices had no significant impact on pH, total mesophilic acidifying flora, lactic flora.

EXAMPLE

Antimicrobial Effects of R/P and R/H in Poultry Sausages

[0276] Classical method, available to persons skilled in the art and based on the protocol described for minced beef and above, was applied to processed poultry sausages.

[0277] As described above, briefly, three different batches of fresh poultry meet processed into sausages, comprising R/P and R/H extract combinations at different concentrations were inoculated with Listeria monocytogenes. Controls did not comprise plant extracts. Fresh poultry meat samples were kept in cool conditions and Listerial growth was measured on the 14.sup.th day.

TABLE-US-00025 TABLE 22 Initial Listerial load per batch of poultry sausages Batch L. monocytogenes (cfu/g) Batch 1 1.95E+03 Batch 2 2.93E+03 Batch 3 2.75E+03

[0278] Extracts were prepared as follows: Half quantities of R (0.5 R) and half quantities of H (0.5H) or half quantities of P (0.5P) extracts as defined above, were mixed together according to the Table 21 and, the mix was completed up to 100% by maltodextrin. These powdered mixes were added to the poultry fresh meat upon processing into sausages in proportions as described in Tables 23 and 24. Tables 23 and 24 also indicate final contents in extracts and extract compounds in % and in ppm, in extract and in the meat matrix.

[0279] Note that 0.5% of 0.5R+0.5H would correspond to the quantities in extract compounds termed 0.5R+0.5H that were tested in minced beef. Further note that 0.3% of 0.5R+0.5P would correspond to the quantities in extract compounds termed 0.5R+0.5P that were tested in minced beef. All obtained data were analyzed and expressed (i.e. delta log etc) according to explanations communicated regarding minced beef.

TABLE-US-00026 TABLE 23 Control 0.5R + 0.5H Composition Rosemary extract 0.00 3.05 of extracts Carnosic acid 0.00 1.34 (%) Carnosic acid + carnosol 0.00 1.48 Hesperidin extract 0.00 56.70 Hesperidin 0.00 53.87 0.6% Composition Rosemary extract 0 183 in poultry Carnosic acid 0 80 (ppm) Carnosic acid + carnosol 0 89 Hesperidin extract 0 3402 Hesperidin 0 3232

TABLE-US-00027 TABLE 24 Control LM 0.5R + 0.5P Composition Rosemary extract 0.00 5.33 of extracts Carnosic acid 0.00 2.35 (%) Carnosic acid + carnosol 0.00 2.58 Pomegranate extract 0.00 21.60 Ellagic acid 0.00 0.43 Punicalagins 0.00 1.94 0.2% 0.4% Composition Rosemary extract 0 107 213 in poultry Carnosic acid 0 47 94 (ppm) Carnosic acid + carnosol 0 52 103 Pomegranate extract 0 432 864 Ellagic acid 0 9 17 Punicalagins 0 39 78

[0280] Results:

[0281] L. monocytogenes has clearly grown in control meat (FIG. 12.). When combinations of extracts were added to the meat, they efficiently inhibited Listerial growth (FIG. 13 and FIG. 14). Those data confirm the data found in minced beef which clearly demonstrated synergistic antilisterial effect of rosemary and hesperidin, and of rosemary and Punica extracts.

EXAMPLE

Antimicrobial Effects of R/P and R/H in Pork Sausages

[0282] Classical method, available to people of ordinary skill in the art and based on the protocol described for minced beef and above, was applied to processed pork sausages.

[0283] Briefly, three different batches of fresh pork meet processed into sausages, comprising R/P and R/H extract combinations at different concentrations were inoculated with Listeria monocytogenes. Controls did not comprise plant extracts. Fresh pork meat samples were kept in cool conditions (8 C.) and Listerial growth was measured on the 14.sup.th day.

TABLE-US-00028 TABLE 25 Initial Listerial load per batch Batch L. monocytogenes (cfu/g) Batch 1 1.55E+03 Batch 2 2.96E+03 Batch 3 2.36E+03

[0284] Extracts were prepared as follows: Half quantities of R (0.5 R) and half quantities of H (0.5H) or half quantities of P (0.5P) extracts as defined above, were mixed together according to the Table 24 and, the mix was completed up to 100% by maltodextrin. These powdered mixes were added to the pork fresh meat upon processing into sausages in proportions as described in Tables 26 and 27. Tables 26 and 27 also indicate final contents in extracts and extract compounds in % and in ppm, in extract and in the meat matrix.

[0285] Note that 0.5% of 0.5R+0.5H would correspond to the quantities in extract compounds termed 0.5R+0.5H that were tested in minced beef. Also note that 0.3% of 0.5R+0.5P would correspond to the quantities in extract compounds termed 0.5R+0.5P that were tested in minced beef. All obtained data were analyzed and expressed (i.e. delta log etc) according to explanations given above regarding minced beef.

TABLE-US-00029 TABLE 26 Control 0.5R + 0.5H Composition Rosemary extract 0.00 3.05 of extracts Carnosic acid 0.00 1.34 (%) Carnosic acid + carnosol 0.00 1.48 Hesperidin extract 0.00 56.70 Hesperidin 0.00 53.87 Control 0.4% 0.5% 0.6% Composition Rosemary extract 0 122 153 183 in pork Carnosic acid 0 54 67 80 (ppm) Carnosic acid + carnosol 0 59 74 89 Hesperidin extract 0 2268 2835 3402 Hesperidin 0 2155 2694 3232 Control 0.5R + 0.5H Composition Rosemary extract 0.00 3.05 of extracts Carnosic acid 0.00 1.34 (%) Carnosic acid + carnosol 0.00 1.48 Hesperidin extract 0.00 56.70 Hesperidin 0.00 53.87 0.4% 0.5% 0.6% Composition Rosemary extract 0 122 153 183 in pork Carnosic acid 0 54 67 80 (ppm) Carnosic acid + carnosol 0 59 74 89 Hesperidin extract 0 2268 2835 3402 Hesperidin 0 2155 2694 3232

TABLE-US-00030 TABLE 27 Control LM 0.5R + 0.5P Composition Rosemary 0.00 5.33 of extracts extract (%) Carnosic acid 0.00 2.35 Carnosic 0.00 2.58 acid + carnosol Pomegranate 0.00 21.60 extract Ellagic acid 0.00 0.43 Punicalagins 0.00 1.94 0.2% 0.3% 0.4% Composition Rosemary 0 107 160 213 in pork extract (ppm) Carnosic acid 0 47 71 94 Carnosic 0 52 77 103 acid + carnosol Pomegranate 0 432 648 864 extract Ellagic acid 0 9 13 17 Punicalagins 0 39 58 78

[0286] Results:

[0287] L. monocytogenes has clearly grown in control meat (FIG. 15). When combinations of extracts were added to the meat, they efficiently inhibited Listerial growth at all here tested concentrations (FIG. 16 and FIG. 17). Those data confirm the data found in minced beef which clearly demonstrated a synergistic antilisterial effect of rosemary and hesperidin, and of rosemary and Punica extracts.

[0288] Classical method, available to persons skilled in the art and based on the protocol described for minced beef and above, was applied to smoked salmons.

[0289] As described above, briefly, three different batches of smoked salmon, comprising R/P and R/H extract combinations at different concentrations were inoculated with Listeria monocytogenes. Controls did not comprise plant extracts. Smoked salmon samples were kept in cool conditions and Listerial growth was measured on the 14.sup.th day.

[0290] Initial Listerial load per batch of poultry sausages was as presented in Table 28:

TABLE-US-00031 TABLE 28 Initial Listerial load in smoked salmon Batch L. monocytogenes (cfu/g) Batch 1 1.46E+03 Batch 2 4.93E+03 Batch 3 5.64E+03

TABLE-US-00032 TABLE 29 Control 0.5R + 0.5H Composition Rosemary extract 0.00 3.05 of extracts Carnosic acid 0.00 1.34 (%) Carnosic acid + carnosol 0.00 1.48 Hesperidin extract 0.00 56.70 Hesperidin 0.00 53.87 Control 0.4% 0.5% Composition Rosemary extract 0 122 153 in smoked Carnosic acid 0 54 67 salmon Carnosic acid + carnosol 0 59 74 (ppm) Hesperidin extract 0 2268 2835 Hesperidin 0 2155 2694

TABLE-US-00033 TABLE 30 Control LM 0.5R + 0.5P Composition Rosemary extract 0.00 5.33 of extracts Carnosic acid 0.00 2.35 (%) Carnosic acid + carnosol 0.00 2.58 Pomegranate extract 0.00 21.60 Ellagic acid 0.00 0.43 Punicalagins 0.00 1.94 0.3% Composition Rosemary extract 0 160 in smoked Carnosic acid 0 71 salmon Carnosic acid + carnosol 0 77 (ppm) Pomegranate extract 0 648 Ellagic acid 0 13 Punicalagins 0 58

[0291] Note that 0.3% of 0.5R+0.5P corresponds to the same proportions and dosage of extract combination added in minced beef and termed 0.5R+0.5P.

[0292] Results:

[0293] L. monocytogenes has clearly grown in control smoked salmon (FIG. 18). When combinations of extracts were added to the meat, they efficiently inhibited Listerial growth (FIG. 19 and FIG. 20). Those data confirm the data found in minced beef which clearly demonstrated a synergistic antilisterial effect of rosemary and hesperidin, and of rosemary and Punica extracts.

REFERENCES

[0294] Numerous references have been cited throughout this disclosure. All references cited in this disclosure including the three additional references listed below are incorporated by reference.

[0295] Kai Reineke, Henning Weich, Dietrich Knorr, The Influence of Sugars on Pressure Induced Starch Gelatinization, Procedia Food Science, Vol. 1, (2011), pages 2040-2046.

[0296] 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.

[0297] S. Chaillou, S.Christieans, M. Rivollier, I. Lucquin, M. C. 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.