Synergistic antimicrobial effects between rosemary extract, cultured dextrose, and buffered vinegar

Abstract

The present invention relates to an antimicrobial composition characterized in that it comprises rosemary essential oil, cultured dextrose, and pH-regulating vinegars, wherein the rosemary essential oil is present at a concentration of 1% or less, the cultured dextrose is present at a concentration of 1% or less, and the pH-regulating vinegar is present at a concentration of 0.1% or less, and wherein the composition exhibits synergistic antimicrobial activity.

Description

Synergistic antimicrobial effects between rosemary extract, cultured dextrose, and buffered vinegar FIELD OF INVENTION The present invention relates to antimicrobial processes and novel antimicrobial compositions that can be used in such processes. In the search for a new and powerful clean-label antimicrobial solution, three different classes of antimicrobials were tested, including plant extracts (such as plant essential oils), cultured dextrose, and pH-controlled vinegar, for synergistic antimicrobial effects. Remarkable synergies were observed when rosemary extract or rosemary essential oil, cultured dextrose, and pH-controlled vinegar were combined. Combinations of two and / or three of these antimicrobials showed a synergistic antimicrobial effect against Salmonella entericia subsp. enterica serovar Typhimurium. BACKGROUND OF THE INVENTION Chemical preservatives, in combination with various processing aids, have traditionally been applied to food systems to prevent food spoilage microorganisms or foodborne pathogens. However, increasing consumer demand for natural, additive-free food products has led the food industry to seek effective, clean-label antimicrobial solutions that maintain food safety and stability. Cultured dextrose is a commercially available food additive produced by the fermentation of sugar sources such as corn, cane sugar, or dairy-based sources, including skim milk. Cultured dextrose is composed of various fermentation metabolites, the main active components of which are antimicrobial peptides and organic acids. The microorganisms involved in the fermentation are primarily probiotic bacteria, such as propionic acid bacteria (including Propionibacterium freudenreichii) and lactic acid bacteria (including Lactococcus lactis). The main application of cultured dextrose is to inhibit the growth of bacteria, yeast, and mold in food systems. Cultured dextrose could be an effective preservative in various applications, including dairy, baked goods, and culinary products.However, the product often imparts salty notes to the product when used in effective doses. Growing evidence that plant materials have potential for antimicrobial activity has led researchers to study different extracts to inhibit bacteria, yeasts, and molds in various applications. Plant secondary metabolites are natural compounds known for their role in plant defense mechanisms and possess multiple biological activities, including antimicrobial effects. These include alkaloids, flavonoids, tannins, terpenes, quinones, and resins. One of the main limitations to using plant extracts as antimicrobial agents in the food and feed industry is that they often require a high effective dose and, therefore, negatively affect the organoleptic properties of the applied matrices. Essential oils are a mixture of volatile compounds extracted from plant biomass and have been identified as natural antimicrobial agents, with their activity understood to arise primarily from phenolic components. However, the use of essential oils as food preservatives is limited due to their strong flavor profile, which negatively affects the organoleptic properties of the food in which they are used. pH-regulated vinegar is another clean-label alternative to chemical preservatives in today's food industry, especially for meat and poultry. pH-regulated vinegar is known to inhibit the growth of foodborne pathogens as well as common spoilage organisms. The impact of vinegar's flavor can be reduced by buffering it with sodium- or potassium-based alkalis. However, the concentration of pH-regulated vinegar required to exhibit antimicrobial activity often imparts vinegary or acidic notes to the final products and negatively impacts their organoleptic properties. The present invention identifies synergistic combinations that could effectively suppress the growth of spoilage microorganisms or pathogens at lower concentrations and, therefore, reduce the impact of the flavor associated with the constituent antimicrobials. These synergistic combinations are applicable in meats and poultry, sauces and dressings, salads, hummus, seafood, cosmetics, and / or nutritional supplements. OBJECT OF THE INVENTION It is an object of the present invention to provide novel synergistic compositions comprising known clean-label antimicrobial substances, as well as processes for stabilizing food substances against typical foodborne spoilage microorganisms and / or pathogens. The synergistic composition and process presented are designed to replace current synthetic antimicrobials and processes using synthetic antimicrobials, such as sodium diacetate, potassium lactate, and sodium benzoate. It is a further object of the invention to provide novel combinations of clean-label antimicrobials that can be employed in such a process. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to antimicrobial compositions comprising at least two antimicrobial constituents selected from the group consisting of plant extracts (including essential plant oils), cultured dextrose, and pH-regulated vinegars, wherein the composition exhibits synergistic antimicrobial activity. Another aspect of the invention relates to an antimicrobial composition of this type in which the plant extract component is a rosemary extract. Another aspect of the invention relates to such an antimicrobial composition wherein the rosemary extract is a flavorless rosemary extract low in essential oil (e.g., in the range of 0.06-0.63% w / w). Another aspect of the invention relates to an antimicrobial composition of this type in which the component of the plant extract is a plant essential oil. Another aspect of the invention relates to an antimicrobial composition of this type in which the essential vegetable oil component is rosemary essential oil. Another aspect of the invention relates to such an antimicrobial composition wherein the cultured dextrose constituent is a fermentation product of sugar sources such as corn, cane sugar, or dairy-based sources, including skimmed milk. Another aspect of the invention relates to such an antimicrobial composition where the pH-regulated vinegar component is a fermentation product of corn and cane sugar, but can also be applied to general vinegar compounds having acetic acid as the main component. In one embodiment, the pH-regulated vinegar component is selected from general vinegar compounds that have acetic acid as the main component. A modality antimicrobial composition comprises rosemary extract and cultured dextrose. A modality antimicrobial composition comprises rosemary extract and pH-regulated vinegar. A modality antimicrobial composition comprises rosemary essential oil and cultured dextrose. A modality antimicrobial composition comprises rosemary essential oil and pH-regulated vinegar. An antimicrobial composition of the modality comprises cultured dextrose and pH-regulated vinegar. A modality antimicrobial composition comprises rosemary extract, cultured dextrose, and pH-regulated vinegar. A modality antimicrobial composition comprises rosemary essential oil, cultured dextrose, and pH-regulated vinegar. Another aspect of the invention relates to a stabilized food, beverage, cosmetic and / or nutritional supplement comprising the antimicrobial composition. Another aspect of the invention relates to a method for stabilizing food, beverages, cosmetics and / or nutritional supplements comprising incorporating an effective amount of the antimicrobial composition, wherein the composition exhibits synergistic antimicrobial activity. In one embodiment of the method, the antimicrobial composition comprises two antimicrobial constituents. In one modality of the method, the antimicrobial composition comprises three antimicrobial constituents. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows antimicrobial tests of rosemary essential oil (RO) and cultured dextrose (CD), and the combination of RO and CD. S. Typhimurium growth was measured by the change in optical density (600 nm). The percentage on the bar indicates growth inhibition. Figure 2 shows antimicrobial tests of rosemary essential oil (RO), pH-regulated vinegar (BV), and the combination of RO and BV. S. Typhimurium growth was measured by the change in optical density (600 nm). The percentage on the bar indicates growth inhibition. Figure 3 shows the antimicrobial test of cultured dextrose (CD), pH-regulated vinegar (BV), and the combination of CD and BV. S. Typhimurium growth was measured by the change in optical density (600 nm). The percentage on the bar indicates growth inhibition. Figure 4 shows antimicrobial tests of rosemary essential oil (RO), cultured dextrose (CD), pH-regulated vinegar (BV), and the combination of the three compounds. S. Typhimurium growth was measured by the change in optical density (600 nm). The percentage on the bar indicates growth inhibition. Figure 5 shows the antimicrobial assay of A) rosemary essential oil (RO), cultured dextrose (CD), and the combination of RO and CD; B) rosemary essential oil (RO), pH-controlled vinegar (BV), and the combination of RO and BV; and C) cultured dextrose (CD), pH-controlled vinegar (BV), and the combination of CD and BV. S. Typhimurium growth was measured by the change in optical density (600 nm). The percentage on the bar indicates growth inhibition. / orccn / zznz / q / uili Figure 6 shows the antimicrobial assay of A) pH-controlled vinegar (BV), pepper leaf oil (PO), and the combination of BV and PO; B) cultured dextrose (CD), clove oil (CO), and the combination of CD and CO; and C) cultured dextrose (CD), thyme oil (TO), and the combination of CD and TO. S. Typhimurium growth was measured by the change in optical density (600 nm). The percentage on the bar indicates growth inhibition. Figure 7 shows the synergistic antimicrobial activity of encapsulated rosemary essential oil, cultured dextrose, and pH-regulated vinegar against Salmonella cocktail in fresh ground turkey. Figure 8 shows the synergistic antimicrobial activity of encapsulated rosemary essential oil, cultured dextrose, and pH-regulated vinegar against coliforms in fresh ground turkey. Figure 9 shows the synergistic antimicrobial activity of dried rosemary extract, cultured dextrose, and pH-regulated vinegar against total aerobic bacteria in fresh ground turkey. Figure 10 shows the synergistic antimicrobial activity of dried rosemary extract, cultured dextrose, and pH-regulated vinegar against Listeria in chicken salad. DETAILED DESCRIPTION OF THE INVENTION Microbial strains and culture conditions Salmonella enterica serovar Typhimurium (ATCC® 14028™) was obtained from ATCC (Manassas, VA). Alternatively, lactic acid bacteria, including Propionibacterium spp., Lactococcus lactis, and Lactobacillus reuteri, may be used in the assay as representative microbial sources. Brain and heart infusion broth (BD, Sparks, MD) was used as the culture medium, and the pH was adjusted to 5.2 with HCl. Chloramphenicol (Sigma, St. Louis, MO) was used as a positive control. All cultures were incubated at 37°C with shaking at 220 rpm for 24 hours. Growth was determined by measuring the optical density at 600 nm (OD600) using a BIOTEK PowerWave HT 340 spectrophotometer (BIOTEK, Winooski, VT). Delta growth was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. / occcn / zznz / q / uιλι Cultured dextrose is prepared by fermenting corn sugar or cane sugar or alternative sugar sources. pH-regulated vinegar is prepared by fermenting corn and cane sugar, but it can also be applied to general vinegar compounds that have acetic acid as the main component. Determination of antimicrobial synergy The percentage of inhibition was calculated using the following equation: Percentage of inhibition (%) Delta growth (growth control) — Delta growth (treatment) Delta growth (growth control) x 100 All experiments were performed in triplicate. Bidirectional synergy test Rosemary essential oil (RO), pH-regulated vinegar (BV), and cultured dextrose (CD) were analyzed to determine their synergistic antimicrobial activity against *5. typhimurium*. Prior to the synergy test, the minimum inhibitory concentration (MIC) of each compound was determined. Concentrations showing sub-inhibitory or no inhibitory effect were used for the synergy test. The bidirectional synergy was determined using the following equations: - Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose mixture] > Percentage inhibition of [a% rosemary essential oil] + Percentage inhibition of [b% cultured dextrose] - Percentage of inhibition of [a% rosemary essential oil and c% pH-regulated vinegar mixture] > Percentage of inhibition of [a% rosemary essential oil] + Percentage of inhibition of [c% pH-regulated vinegar] - Percentage of inhibition of [b% cultured dextrose and c% pH-regulated vinegar mixture] > Percentage of inhibition of [b% cultured dextrose] + Percentage of inhibition of [c% pH-regulated vinegar] / occcn / zznz / q / uli Three-way synergy test The concentration of rosemary essential oil, pH-regulated vinegar, and cultured dextrose used for the three-way synergy test was designed to have slight synergistic antimicrobial effects when two agents were combined, but without showing 100% inhibition against S. typhimurium. Three-way synergy was determined when all of the following conditions were met: - Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose and c% pH-regulated vinegar mixture] > Percentage inhibition of [a% rosemary essential oil] + Percentage inhibition of [b% cultured dextrose] + Percentage inhibition of [c% pH-regulated vinegar] - Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose and c% pH-regulated vinegar mixture] > Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose mixture] + Percentage inhibition of [c% pH-regulated vinegar] - Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose and c% pH-regulated vinegar mixture] > Percentage inhibition of [a% rosemary essential oil and c% pH-regulated vinegar mixture] + Percentage inhibition of [b% cultured dextrose] - Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose and c% pH-regulated vinegar mixture] > Percentage inhibition of [b% cultured dextrose and c% pH-regulated vinegar mixture] + Percentage inhibition of [a% rosemary essential oil] Antimicrobial testing in food models Microbial strains and culture conditions Salmonella enterica subsp. enterica serovar Typhimurium (ATCC® 14028™), Salmonella enterica subsp. enterica serovar Typhimurium (ATCC® 700720™), Salmonella enterica subsp. enterica serotype Enteritidis (ATCC® 4931™), and Salmonella enterica subsp. enterica serovar Newport (ATCC® 6962™) were obtained from ATCC (Manassas, VA). The strains were initially cultured in brain and heart infusion broth (BD, Sparks, MD) at 37°C with shaking at 220 rpm for 24 hours prior to inoculation into food matrices. The four Salmonella strains were cultured individually and then combined as a cocktail prior to inoculation. Listeria monocytogenes ATCC® 19115™, Listeria monocytogenes ATCC® 19115™, and Listeria monocytogenes ATCC® 19115™ were obtained from ATCC (Manassas, VA). The strains were initially cultured in brain and heart infusion broth (BD, Sparks, MD) at 37°C with shaking at 220 rpm for 24 hours prior to inoculation into food matrices. The three Listeria monocytogenes strains were cultured individually and then combined as a cocktail prior to inoculation. Preparation of ground turkey model (for Salmonella and coliform exposure testing) Fresh ground turkey (80% lean and 20% fat) was purchased from a local store and immediately transferred to the laboratory. The ground turkey was coarsely ground (16 mm) and then finely ground (5 mm). Antimicrobial compounds were added directly to the ground turkey in a sterile peristaltic homogenizer and homogenized twice for 1 min at 230 rpm using a peristaltic homogenizer (Seward Stomacher 400 Circulator). Preparation of the chicken salad model (for the Listeria challenge test) Fresh chicken breast, celery, mayonnaise, table salt, and black pepper were purchased from a local store and transported to the laboratory. The chicken breast was boiled in hot water until the internal temperature reached 74 °C and then cut into 2 cm cubes. The celery was washed in cold water, dried, and cut into 1 cm pieces. The chicken salad sample was prepared by mixing 1640 g of cooked chicken cubes, 105 g of celery, 180 g of mayonnaise, 2 g of black pepper, and 10 g of table salt. The antimicrobial compounds were added directly to the chicken salad in a sterile peristaltic homogenizer and homogenized twice for 1 min at 230 rpm using a peristaltic homogenizer (Seward Stomacher 400 Circulator). / occcn / zznz / q / uιλι Microbial sampling Food samples (25 grams) were transferred to a sterile peristaltic homogenizer and filled with 0.1% peptone water to a final volume of 250 grams. The mixtures were homogenized for 1 minute at 230 rpm, and serial dilutions were performed to the appropriate level. The diluents were plated onto selective agar medium followed by 24 hours of incubation at 37 °C. To enumerate Salmonella, the sample diluents were plated onto XLD (xylose lysine deoxycholate) agar, and red colonies with black centers were counted. Yellow colonies appearing on XLD agar were Gram-negative, lysine decarboxylase-negative bacteria and were considered coliforms. The total plate count was determined by plating the samples onto Total Plate Count (TPC) agar and incubating at 30 °C for 24 hours. The Lisieria monocytogenes count was determined by plating the samples on Listeria selective agar (LSA) and incubating at 37 °C for 24 to 48 hours. EXAMPLES The following examples illustrate the invention without limiting its scope. EXAMPLE 1 - Bidirectional synergy between rosemary essential oil and cultured dextrose Salmonella enterica serovar Typhimurium (ATCC® 14028™) was cultured at 37°C with shaking at 220 rpm for 24 hours using brain heart infusion broth as the culture medium, and the pH was adjusted to 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. The growth delta was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. The test antimicrobials and their percentage inhibition are provided below and in Figure 1. / occcn / zznz / q / uιλι Test Substance Concentration (%) Inhibition (%) Chloramphenicol 0.02 97 Rosemary Essential Oil 0.50 9 Cultured Dextrose 0.16 0 Rosemary Essential Oil + 0.5 98 Cultured Dextrose 0.16 EXAMPLE 2 - Bidirectional synergy between rosemary essential oil and pH-regulated vinegar Salmonella enterica serovar Typhimurium (ATCC® 14028™) was cultured at 37°C with shaking at 220 rpm for 24 hours using brain heart infusion broth as the growth medium, and the pH was adjusted to 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. The growth delta was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. The test antimicrobials and their percentage inhibition are provided below and in Figure 2: / orccn / zznz / q / uili Test Substance Concentration (%) Inhibition (%) Chloramphenicol 0.02 99 Rosemary Essential Oil 0.50 0 pH-Regulated Vinegar 0.0625 1 Rosemary Essential Oil + 0.5 55 pH-Regulated Vinegar 0.0625 EXAMPLE 3 - Bidirectional synergy between cultured dextrose and pH-regulated vinegar Salmonella enterica serovar Typhimurium (ATCC® 14028™) was cultured at 37°C with shaking at 220 rpm for 24 hours using brain heart infusion broth as the culture medium, and the pH was adjusted to 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. The growth delta was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. The test antimicrobials and their percentage inhibition are provided below and in Figure 3: Test Substance Concentration (%) Inhibition (%) Chloramphenicol 0.02 98 Cultured Dextrose 0.16 3 pH-Controlled Vinegar 0.0625 0 Cultured Dextrose + 0.16 100 pH-Controlled Vinegar 0.0625 EXAMPLE 4: Three-way synergy between rosemary essential oil, cultured dextrose, and pH-regulated vinegar In an effort to evaluate the potential for three-way synergy among the respective antimicrobial test substances, the concentration of these substances was reduced, and the three-way synergy was compared to the two-way synergy among the test antimicrobials using the reduced concentration. Salmonella enterica serovar Typhimurium (ATCC® 14028™) was cultured at 37°C with shaking at 220 rpm for 24 hours using brain heart infusion broth as the culture medium, and the pH was adjusted to 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. The growth delta was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. The test antimicrobials and their percentage inhibition are provided below and in Figures 4 and 5: Test Substance Concentration (%) Inhibition (%) Chloramphenicol 0.02 99 Rosemary Essential Oil 0.50 0 Cultured Dextrose 0.112 0 pH-Controlled Vinegar 0.044 0 Rosemary Essential Oil + 0.5 100 Cultured Dextrose + 0.112 pH-Controlled Vinegar 0.044 Rosemary Essential Oil + 0.5 22 Cultured Dextrose 0.112 Rosemary Essential Oil + 0.5 24 pH-Controlled Vinegar 0.044 Cultured Dextrose + 0.112 64 pH-Controlled Vinegar 0.044 EXAMPLE 5: Bidirectional synergy between several essential oils in combination with cultured dextrose and pH-regulated vinegar In an effort to evaluate the potential synergy between the respective antimicrobial test substances and several other selected essential oils from clove, thyme, and pepper leaf, sub-inhibitory concentrations of the additional essential oil extracts were evaluated individually and in combination with cultured dextrose and pH-adjusted vinegar. Since the essential oils under evaluation demonstrated antagonistic or non-synergistic effects on the inhibition observed with cultured dextrose and pH-adjusted vinegar, the concentrations of cultured dextrose and pH-adjusted vinegar were increased above the sub-inhibitory concentrations used in the three-way synergy experiments with rosemary essential oil. Salmonella enterica serovar Typhimurium (ATCC® 14028™) was cultured at 37°C with shaking at 220 rpm for 24 hours using brain heart infusion broth as the culture medium, and the pH was adjusted to 5.2 with HCl.Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. The delta growth was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. The test antimicrobials and their percentage inhibition are provided as indicated below and in Figure 6. Test Substance Concentration (%) Inhibition (%) Chloramphenicol 0.02 100 Cultured Dextrose 0.2 90 pH-Controlled Vinegar 0.065 26 Clove Essential Oil 0.025 53 Clove Essential Oil + 0.025 92 Cultured Dextrose 0.2 Thyme Essential Oil 0.03 47 Thyme Essential Oil + 0.03 91 Cultured Dextrose 0.2 Pepper Leaf Oil 0.05 14 Pepper Leaf Oil + 0.05 32 pH-Controlled Vinegar 0.065 EXAMPLE 6: Synergistic antimicrobial activity of encapsulated rosemary essential oil, cultured dextrose, and pH-regulated vinegar against the Salmonella and coliform cocktail in fresh ground turkey Salmonella enterica subsp. enterica serovar Typhimurium (ATCC® 14028™), Salmonella enterica subsp. enterica serovar Typhimurium (ATCC® 700720™), Salmonella enterica subsp. enterica serovar Enteritidis (ATCC® 4931™), Salmonella enterica subsp. enterica serovar Newport (ATCC® 6962™) were individually cultured in brain and heart infusion broth (BD, Sparks, MD) at 37 °C with shaking at 220 rpm for 24 hours and then combined as a cocktail. Fresh ground turkey was prepared and mixed with each antimicrobial treatment: 1) control treatment with no added antimicrobial (control), 2) a mixture of 0.3% cultured dextrose and pH-adjusted vinegar mixed in a 50:50 ratio (0.3% CDV), 3) 5% rosemary essential oil encapsulated with a 20% loading yield (5% RO), and 4) 0.3% CDV + 5% RO. After the addition of antimicrobial treatments, the Salmonella cocktail was inoculated into the ground turkey with a target initial population of approximately 4 log CFU / g.The ground turkey samples were then vacuum-packed and stored at 10°C for 14 days. The Salmonella population was verified by plating the samples on XLD agar and counting the red colonies with black centers. Yellow colonies appearing on XLD agar were Gram-negative, lysine decarboxylase-negative bacteria and were considered coliforms. All experiments were performed in duplicate, and the average value is reported. Ground turkey treated with 0.3% CDV + 5% RO showed the best results, inhibiting Salmonella growth very effectively compared to the other treatment groups. The results are shown in Figure 7. A bactericidal effect (decreased Salmonella counts) was observed when comparing day 14 (3.1 log CFU / g) with day 0 (3.8 log CFU / g). Specifically on day 4, rosemary oil alone (5% RO) showed no inhibitory effect against Salmonella, but when rosemary oil was combined with cultured dextrose and pH-adjusted vinegar, a clear synergistic antimicrobial effect was observed. The combination of cultured dextrose, pH-regulated vinegar, and encapsulated rosemary essential oil also performed better and showed a synergistic antimicrobial effect against coliforms. The results are shown in Figure 8. EXAMPLE 7: Synergistic antimicrobial activity of dried rosemary extract, cultured dextrose, and pH-regulated vinegar against spoilage microorganisms in fresh ground turkey Fresh ground turkey (80% lean and 20% fat) was coarsely ground (16 mm) and then finely ground (5 mm) and mixed with each antimicrobial treatment: 1) control treatment with no added antimicrobial (control), 2) 1% cultured dextrose and pH-regulated vinegar mixture mixed in a 50:50 ratio (CDV), 3) 1% mixture of 2.2% dried rosemary extract, 85% cultured dextrose, 13.85% maltodextrin and 0.7% silica (NCD), and 4) mixture of 1% dried rosemary extract, 2.2% cultured dextrose, 35% pH-regulated vinegar, 13.85% maltodextrin and 0.7% silica (1% NCDV). Then, the ground turkey samples were vacuum-packed and stored at 4 °C for 17 days, and the total plate count was verified by plating the samples onto Total Plate Count (TPC) agar. All experiments were performed in duplicate, and the average value is reported. NCD at 1% was the most effective antimicrobial agent for inhibiting total aerobic bacteria. Although the mean value of the NCD 1% treatment showed a slight increase in total plaque count on day 5 compared to day 0, the difference was not statistically significant. Ground turkey treated with a combination of NCD was more effective at inhibiting the growth of total aerobic bacteria compared to ground turkey without rosemary extract (Figure 9), showing a 1.6 log difference on day 17. On the other hand, the NCD treatment did not perform well compared to the other two treatments. When dried rosemary extract was used alone at the same concentration (2.2%), no antimicrobial activity was observed (data not shown), demonstrating that the combined effect was synergistic. To identify which microorganisms were inhibited by the additional synergistic effect of adding dried rosemary extract to the synergistic combination, colonies growing on TPC agar from the ground turkey sample treated with 1% CDV but not from the ground turkey sample treated with 1% NCDV were isolated and identified by 16S rRNA sequencing. All isolated colonies were identified as Pseudomonas spp. / occcn / zznz / q / uιλι EXAMPLE 8 - Synergistic antimicrobial activity of dried rosemary extract, cultured dextrose, and pH-regulated vinegar against Listeria monocytogenes in chicken salad Listeria monocytogenes ATCC® 19115™, Listeria monocytogenes ATCC® 19115™, and Listeria monocytogenes ATCC® 19115™ were obtained from ATCC (Manassas, VA). The strains were initially cultured in brain and heart infusion broth (BD, Sparks, MD) at 37°C with shaking at 220 rpm for 24 hours prior to inoculation into food matrices. The three Listeria monocytogenes strains were cultured individually and then combined as a cocktail. Chicken salad was prepared and mixed with each antimicrobial treatment: 1) control treatment with no added antimicrobial (control), 2) mixture of 1% cultured dextrose and pH-regulated vinegar mixed in a 50:50 ratio (CDV), 3) 1% mixture of 2.2% dried rosemary extract, 85% cultured dextrose, 13.85% maltodextrin and 0.7% silica (NCD), and 4) 1% mixture of 2.2% dried rosemary extract, 50% cultured dextrose, 35% pH-regulated vinegar, 13.85% maltodextrin and 0.7% silica (1% NCDV).After the addition of antimicrobial treatments, a Listeria cocktail was inoculated into chicken salad with a target initial population of approximately 4 log CFU / g. The chicken salad samples were then stored at 4 °C for 28 days, and the Listeria monocytogenes population was assessed by plating the samples on LSA agar and counting the black colonies. All experiments were performed in duplicate, and the average value is reported. The combination of treatment with dried rosemary extract, cultured dextrose, and pH-regulated vinegar (NCDV) performed better than the combination of cultured dextrose and pH-regulated vinegar (CDV) or the combination of dried rosemary extract and cultured dextrose (NCD). On day 21, the 1% NCD treatment had a higher Listeria monocytogenes count compared to the other two treatments, and on day 28, the 1% CDV and 1% NCD treatments had higher Listeria monocytogenes counts compared to the 1% NCDV treatment. When dried rosemary extract was used alone at the same concentration (2.2%), no antimicrobial activity was observed (data not shown), demonstrating that the combined effect was synergistic. The scope of the present invention is not limited by the specific embodiments described herein. In fact, several modifications of the invention, in addition to those described herein, will be obvious to those skilled in the art from the foregoing description. It is intended that such modifications fall within the scope of the appended claims. All patents, applications, publications, test methods, literature, and other materials cited in this document are incorporated herein by reference.

Claims

1. An antimicrobial composition characterized in that it comprises at least two antimicrobial constituents selected from the group consisting of plant extracts, cultured dextrose and pH-regulated vinegars, wherein the composition exhibits synergistic antimicrobial activity.

2. The antimicrobial composition according to claim 1, further characterized in that the plant extract component is a rosemary extract.

3. The antimicrobial composition according to claim 1, further characterized in that the component of the plant extract is a plant essential oil.

4. The antimicrobial composition according to claim 3, further characterized in that the vegetable essential oil is rosemary essential oil.

5. The antimicrobial composition according to any of claims 1 to 4, further characterized in that the cultured dextrose constituent is a fermentation product of sugar sources such as corn, cane sugar, or dairy-based sources, including skimmed milk.

6. The antimicrobial composition according to any of claims 1 to 5, further characterized in that the pH-regulated vinegar component is a fermentation product of corn and cane sugar, but can also be applied to general vinegar compounds having acetic acid as the main component.

7. The antimicrobial composition according to any of claims 1 to 5, further characterized in that the pH-regulated vinegar component is selected from general vinegar compounds having acetic acid as the main component.

8. The antimicrobial composition according to claim 1, further characterized in that it comprises rosemary extract and cultured dextrose. / occcn / zznz / q / uili 9. The antimicrobial composition according to claim 1, further characterized in that it comprises rosemary essential oil and pH-regulated vinegar.

10. The antimicrobial composition according to claim 1, further characterized in that it comprises rosemary essential oil and cultured dextrose.

11. The antimicrobial composition according to claim 1, further characterized in that it comprises rosemary essential oil and pH-regulated vinegar.

12. The antimicrobial composition according to claim 1, further characterized in that it comprises cultured dextrose and pH-regulated vinegar.

13. The antimicrobial composition according to claim 1, further characterized in that it comprises rosemary extract, cultured dextrose and pH-regulated vinegar.

14. The antimicrobial composition according to claim 1, further characterized in that it comprises rosemary essential oil, cultured dextrose, and pH-regulated vinegar.

15. A stabilized food, beverage, cosmetic and / or nutritional supplement characterized in that it comprises an antimicrobial composition in accordance with any of claims 1 to 14.

16. A method for stabilizing food, beverages, cosmetics and / or nutritional supplements, characterized in that it comprises incorporating an effective amount of the antimicrobial composition according to any of claims 1 to 14, wherein the composition exhibits synergistic antimicrobial activity.

17. The method according to claim 16, further characterized in that the antimicrobial composition comprises two antimicrobial constituents.

18. The method according to claim 16, further characterized in that the antimicrobial composition comprises three antimicrobial constituents.