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

Abstract

The present invention relates to novel synergistic compositions comprising known “clean-label” antimicrobial substances as well as processes for stabilizing food substances against typical food borne spoilage microorganisms and/or pathogens. It is a further object of the invention to provide novel combinations of “clean label” antimicrobials which may be employed in such a process.

Claims

1. An antimicrobial composition comprising rosemary essential oil, cultured dextrose and buffered vinegar, wherein the rosemary essential oil is present at a concentration of 0.5% or less, the cultured dextrose is present at a concentration of 0.112% or less, and the buffered vinegar is present at a concentration of 0.044% or less, and wherein the composition exhibits synergistic antimicrobial activity.

2. The antimicrobial composition of claim 1, wherein the cultured dextrose constituent is a fermentation product of sugar sources such as corn, cane sugar, or dairy based sources including skim milk.

3. The antimicrobial composition of claim 1, wherein the buffered vinegar constituent is a fermentation product of corn and cane sugar, but may also be applied to general vinegar compounds having acetic acid as a main component.

4. The antimicrobial composition of claim 1, wherein the buffered vinegar constituent is selected from general vinegar compounds having acetic acid as a main component.

5. A stabilized food, beverage, cosmetic and/or nutritional supplement comprising the antimicrobial composition of claim 1.

6. A method for stabilizing foods, beverages, cosmetics and/or nutritional supplements comprising incorporating an effective amount of the antimicrobial composition of claim 1, wherein the composition exhibits synergistic antimicrobial activity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows antimicrobial testing of rosemary essential oil (RO) and cultured dextrose (CD).

(2) FIG. 2 shows antimicrobial testing of rosemary essential oil (RO) and buffered vinegar (BV).

(3) FIG. 3 shows antimicrobial testing of cultured dextrose (CD) and buffered vinegar (BV).

(4) FIG. 4 shows antimicrobial testing of rosemary essential oil (RO), cultured dextrose (CD) and buffered vinegar (BV).

(5) FIG. 5 shows antimicrobial testing of A) rosemary essential oil (RO), cultured dextrose (CD), and the combination of RO and CD; B) rosemary essential oil (RO), buffered vinegar (BV), and the combination of RO and BV; and C) cultured dextrose (CD), buffered vinegar (BV), and the combination of CD and BV.

(6) FIG. 6 shows antimicrobial testing of A) buffered vinegar (BV) and pimento leaf oil (PO); B) cultured dextrose (CD) and clove oil (CO); and C) cultured dextrose (CD) and thyme oil (TO).

(7) FIG. 7 shows the synergistic antimicrobial activity of encapsulated rosemary essential oil, cultured dextrose, and buffered vinegar against Salmonella cocktail in Fresh Ground Turkey.

(8) FIG. 8 shows the Synergistic antimicrobial activity of encapsulated rosemary essential oil, cultured dextrose, and buffered vinegar against coliforms in Fresh Ground Turkey.

(9) FIG. 9 shows the Synergistic antimicrobial activity of dried rosemary extract, cultured dextrose, and buffered vinegar against total aerobic bacteria in Fresh Ground Turkey.

(10) FIG. 10 shows the synergistic antimicrobial activity of dried rosemary extract, cultured dextrose, and buffered vinegar against Listeria in chicken salad.

DETAILED DESCRIPTION OF THE INVENTION

(11) Microbial Strains and Culture Conditions

(12) Salmonella enterica serovar typhimurium (ATCC® 14028™) was obtained from ATCC (Manassas, Va.). Alternatively, lactic acid bacteria including Propionibacterium spp., Lactococcus lactis, Lactobacillus reuteri, may be used in the assay as representative microbial sources. Brain heart infusion broth (BD, Sparks, Md.) was used as a growth medium and the pH was adjusted to pH 5.2 with HCl. Chloramphenicol (Sigma, St. Louis, Mo.) was used as a positive control. All cultures were incubated at 37° C. with agitation at 220 rpm for 24 hours. Growth was determined by measuring the optical density at 600 nm (OD.sub.600) with 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.

(13) Cultured Dextrose is prepared by fermentation of corn sugar or cane sugar or alternative sugar sources.

(14) Buffered Vinegar is prepared by the fermentation of corn and cane sugar, but may also be applied to general vinegar compounds having acetic acid as main component.

(15) Antimicrobial Synergy Determination

(16) Percentage inhibition was calculated by the following equation:

(17) $\mathrm{Percentage}\mathrm{inhibition}\left(\%\right)=\frac{\mathrm{Delta}\mathrm{growth}\left(\mathrm{growth}\mathrm{control}\right)-\mathrm{Delta}\mathrm{growth}\left(\mathrm{treatment}\right)}{\mathrm{Delta}\mathrm{growth}\left(\mathrm{growth}\mathrm{control}\right)}\times 100$

(18) All experiments were performed in triplicate.

(19) Test for Two-Way Synergy

(20) Rosemary essential oil (RO), buffered vinegar (BV), and cultured dextrose (CD) was tested for synergistic antimicrobial activity against S. typhimurium. Prior to synergy testing, minimum inhibitory concentration (MIC) of each compound was determined. The concentrations showing sub-inhibitory or no inhibitory effect were used for synergy test.

(21) Two-way synergy was determined by the following equations:
Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose mix]>Percentage inhibition of [a% rosemary essential oil]+Percentage inhibition of [b% cultured dextrose]
Percentage inhibition of [a% rosemary essential oil and c% buffered vinegar mix]>Percentage inhibition of [a% rosemary essential oil]+Percentage inhibition of [c% buffered vinegar]
Percentage inhibition of [b% cultured dextrose and c% buffered vinegar mix]>Percentage inhibition of [b% cultured dextrose]+Percentage inhibition of [c% buffered vinegar]
Test for Three-Way Synergy

(22) The concentration of rosemary essential oil, buffered 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 not showing 100% inhibition against S. typhimurium.

(23) 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% buffered vinegar mix]>Percentage inhibition of [a% rosemary essential oil]+Percentage inhibition of [b% cultured dextrose]+Percentage inhibition of [c% buffered vinegar]
Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose and c% buffered vinegar mix]>Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose mix]+Percentage inhibition of [c% buffered vinegar]
Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose and c% buffered vinegar mix]>Percentage inhibition of [a% rosemary essential oil and c% buffered vinegar mix]+Percentage inhibition of [b% cultured dextrose]
Percentage inhibition of [a% rosemary essential oil and b% cultured dextrose and c% buffered vinegar mix]>Percentage inhibition of [b% cultured dextrose and c% buffered vinegar mix]+Percentage inhibition of [a% rosemary essential oil]
Antimicrobial Testing in Food Models
Microbial Strains and Culture Conditions

(24) 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 obtained from ATCC (Manassas, Va.). Strains were initially grown in Brain heart infusion broth (BD, Sparks, Md.) at 37° C. with agitation at 220 rpm for 24 hours before inoculating in food matrices. All 4 strains of Salmonella were individually grown and then combined as a cocktail prior to inoculation.

(25) Listeria monocytogenes ATCC® 19115™, Listeria monocytogenes ATCC® 19115™, Listeria monocytogenes ATCC® 19115™ were obtained from ATCC (Manassas, Va.). Strains were initially grown in Brain heart infusion broth (BD, Sparks, Md.) at 37° C. with agitation at 220 rpm for 24 hours before inoculating in food matrices. All 3 strains of Listeria monocytogenes were individually grown and then combined as a cocktail prior to inoculation.

(26) Ground Turkey Model Preparation (for Salmonella and Coliforms Challenge Test)

(27) Fresh ground turkey (80% lean and 20% fat) was purchased from a local store and transferred immediately to the laboratory. Ground turkey was coarsely ground (16 mm) and then finely ground (5 mm). Antimicrobial compounds were directly added to the ground turkey in a sterile stomacher bag and homogenized twice for 1 min at 230 rpm using stomacher (Seward Stomacher 400 Circulator).

(28) Chicken Salad Model Preparation (for Listeria Challenge Test)

(29) Fresh chicken breast, celery, mayonnaise, table salt, black pepper were purchased from a local store and transferred to the laboratory. Chicken breast was boiled in hot water until the internal temperature reached 74° C. and cut into 2 cm cubes. Celery was washed under cool water and dried and cut into 1 cm pieces. Chicken salad model was made by mixing 1640 g of cooked chicken cube, 105 g of celery, 180 g of mayonnaise, 2 g of black pepper, and 10 g of table salt. Antimicrobial compounds were directly added to chicken salad in a sterile stomacher bag and homogenized twice for 1 min at 230 rpm using stomacher (Seward Stomacher 400 Circulator).

(30) Microbial Sampling

(31) Food samples (25 grams) were transferred to a sterile stomacher bag and filled up with 0.1% peptone water to 250 grams. The mixtures were homogenized for 1 min at 230 rpm and serial dilution was performed to appropriate level and the diluents were plated on selective agar medium followed by 24 hours incubation at 37° C. For enumerating Salmonella, sample diluents were plated on XLD (Xylose Lysine Deoxycholate) agar medium and the red colonies with black center were counted. Yellow colonies appearing on XLD agar are gram-negative and lysine decarboxylase-negative bacteria and were considered as coliforms. Total plate count was determined by plating the samples on Total Plate Count (TPC) agar and incubating at 30° C. for 24 hours. Listeria monocytogenes count was determined by plating samples on Listeria selective agar (LSA) and incubating at 37° C. for 24 to 48 hours.

EXAMPLES

(32) The following examples illustrate the invention without limiting its scope.

Example 1—Two-Way Synergy Between Rosemary Essential Oil and Cultured Dextrose

(33) Salmonella enterica serovar typhimurium (ATCC® 14028™) was cultured at 37° C. with agitation at 220 rpm for 24 hours with brain heart infusion broth as a growth medium and the pH was adjusted to pH 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. Delta growth was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. Test antimicrobials and their percent inhibition were provided as noted below and in FIG. 1:

(34) TABLE-US-00001 Concentration Inhibition Test Substance (%) (%) 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—Two-Way Synergy Between Rosemary Essential Oil and Buffered Vinegar

(35) Salmonella enterica serovar typhimurium (ATCC® 14028™) was cultured at 37° C. with agitation at 220 rpm for 24 hours with brain heart infusion broth as a growth medium and the pH was adjusted to pH 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. Delta growth was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. Test antimicrobials and their percent inhibition were provided as noted below and in FIG. 2:

(36) TABLE-US-00002 Concentration Inhibition Test Substance (%) (%) Chloramphenicol 0.02 99 Rosemary Essential Oil 0.50 0 Buffered Vinegar 0.0625 1 Rosemary Essential Oil + 0.5 55 Buffered Vinegar 0.0625

Example 3—Two-Way Synergy Between Cultured Dextrose and Buffered Vinegar

(37) Salmonella enterica serovar typhimurium (ATCC® 14028™) was cultured at 37° C. with agitation at 220 rpm for 24 hours with brain heart infusion broth as a growth medium and the pH was adjusted to pH 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. Delta growth was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. Test antimicrobials and their percent inhibition were provided as noted below and in FIG. 3:

(38) TABLE-US-00003 Concentration Inhibition Test Substance (%) (%) Chloramphenicol 0.02 98 Cultured Dextrose 0.16 3 Buffered Vinegar 0.0625 0 Cultured Dextrose + 0.16 100 Buffered Vinegar 0.0625

Example 4—Three-Way Synergy Among Rosemary Essential Oil, Cultured Dextrose, and Buffered Vinegar

(39) In an effort to evaluate the potential for three-way synergy among the respective antimicrobial test substances, the concentration of such substances was reduced and three-way synergy was compared with two-way synergy among the test antimicrobials utilizing the reduced concentration. Salmonella enterica serovar typhimurium (ATCC® 14028™) was cultured at 37° C. with agitation at 220 rpm for 24 hours with brain heart infusion broth as a growth medium and the pH was adjusted to pH 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. Delta growth was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. Test antimicrobials and their percent inhibition were provided as noted below and in FIGS. 4 and 5:

(40) TABLE-US-00004 Concentration Inhibition Test Substance (%) (%) Chloramphenicol 0.02 99 Rosemary Essential Oil 0.50 0 Cultured Dextrose 0.112 0 Buffered Vinegar 0.044 0 Rosemary Essential Oil + 0.5 100 Cultured Dextrose + 0.112 Buffered Vinegar 0.044 Rosemary Essential Oil + 0.5 22 Cultured Dextrose 0.112 Rosemary Essential Oil + 0.5 24 Buffered Vinegar 0.044 Cultured Dextrose + 0.112 64 Buffered Vinegar 0.044

Example 5—Two-Way Synergy Among Various Essential Oils in Combination with Cultured Dextrose, and Buffered Vinegar

(41) In an effort to evaluate the potential for synergy among the respective antimicrobial test substances and various other essential oils selected from clove, thyme and pimento leaf, sub-inhibitory concentrations of the additional essential oil extracts were evaluated singly and in combination with cultured dextrose and buffered vinegar. As the essential oils under evaluation demonstrated antagonistic or non-synergistic effects over the inhibition observed with cultured dextrose and buffered vinegar, the concentration of the cultured dextrose and buffered vinegar was increased over the sub-inhibitory concentrations utilized in the three-way synergy experiments with rosemary essential oil. Salmonella enterica serovar typhimurium (ATCC® 14028™) was cultured at 37° C. with agitation at 220 rpm for 24 hours with brain heart infusion broth as a growth medium and the pH was adjusted to pH 5.2 with HCl. Chloramphenicol was used as a positive control. Growth was determined by measuring the optical density at 600 nm. Delta growth was calculated by subtracting the growth measured at 0 hours from the growth measured at 24 hours. Test antimicrobials and their percent inhibition were provided as noted below and in FIG. 6:

(42) TABLE-US-00005 Concentration Inhibition Test Substance (%) (%) Chloramphenicol 0.02 100 Cultured Dextrose 0.2 90 Buffered 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 Pimento Leaf Oil 0.05 14 Pimento Leaf Oil + 0.05 32 Buffered Vinegar 0.065

Example 6—Synergistic Antimicrobial Activity of Encapsulated Rosemary Essential Oil, Cultured Dextrose, and Buffered Vinegar Against Salmonella Cocktail and Coliforms in Fresh Ground Turkey

(43) 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 heart infusion broth (BD, Sparks, Md.) at 37° C. with agitation 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 antimicrobial added (control), 2) 0.3% cultured dextrose and buffered vinegar blend mixed at 50:50 ratio (0.3% CDV), 3) 5% encapsulated rosemary essential oil with 20% loading yield (5% RO), and 4) 0.3% CDV+5% RO. After the addition of antimicrobial treatments, Salmonella cocktail was inoculated to the ground turkey with a target initial population around 4 log CFU/g. Ground turkey samples were then vacuum packaged and stored at 10° C. for 14 days period and checked for Salmonella population by plating the samples on XLD agar media and counting the red colonies with black centers. Yellow colonies appearing on XLD agar are gram-negative and lysine decarboxylase-negative bacteria and were considered as coliforms. All experiments were performed in duplicate, and the average value is reported.

(44) The ground turkey treated with 0.3% CDV+5% RO showed the best result by inhibiting the growth of Salmonella very effectively compared to the other treatment groups. The results are shown in FIG. 7. A bactericidal effect (decrease in Salmonella counts) was observed when comparing day 14 (3.1 log CFU/g) to day 0 (3.8 log CFU/g). Especially in day 4, the rosemary oil alone (5% RO) did not show any inhibitory effect against Salmonella, but when rosemary oil was combined with cultured dextrose and buffered vinegar, a clear synergistic antimicrobial effect was observed.

(45) The combination of cultured dextrose, buffered vinegar, and encapsulated rosemary essential oil also performed the best and showed synergistic antimicrobial effect against coliforms. The results are shown in FIG. 8.

Example 7—Synergistic Antimicrobial Activity of Dried Rosemary Extract, Cultured Dextrose, and Buffered Vinegar Against Spoilage Microorganisms in Fresh Ground Turkey

(46) 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 antimicrobial added (control), 2) 1% cultured dextrose and buffered vinegar blend mixed at 50:50 ratio (CDV), 3) 1% blend of 2.2% dried rosemary extract, 85% cultured dextrose, 13.85% Maltodextrin and 0.7% Silica (NCD), and 4) 1% blend of 2.2% dried rosemary extract, 50% Cultured Dextrose, 35% buffered vinegar, 13.85% Maltodextrin, and 0.7% Silica (1% NCDV). Ground turkey samples were then vacuum packaged and stored at 4° C. for a 17 day period and checked for total plate count by plating the samples on Total Plate Count (TPC) agar. All the experiments were performed in duplicate, and the average value is reported.

(47) 1% NCD was the most effective antimicrobial agent for inhibiting total aerobic bacteria. Although the average value of the 1% NCD treatment showed slight increase of total plate count at day 5 compared to day 0, the difference were not statistically significant. The ground turkey treated with a combination of NCD was more effective in inhibiting the growth of total aerobic bacteria compared to the CDV without the rosemary extract (FIG. 9) showing 1.6 log differences at day 17. On the other hand, NCD treatment did not perform well compared to the other two treatments.

(48) When dried rosemary extract was used alone at the same concentration (2.2%), there were no antimicrobial activity observed (data not shown) proving that the combined effect was synergistic.

(49) To identify which microorganisms were inhibited by the additional synergistic effect with the addition of dried rosemary extract in the synergistic combination, the colonies that grow on the TPC agar from 1% CDV treated ground turkey sample but not from the 1% NCDV treated sample were isolated and identified by 16s rRNA sequencing. All the colonies isolated were identified as Pseudomonas spp.

Example 8—Synergistic Antimicrobial Activity of Dried Rosemary Extract, Cultured Dextrose, and Buffered Vinegar Against Listeria monocytogenes in Chicken Salad

(50) Listeria monocytogenes ATCC® 19115™, Listeria monocytogenes ATCC® 19115™, Listeria monocytogenes ATCC® 19115™ were obtained from ATCC (Manassas, Va.). Strains were initially grown in Brain heart infusion broth (BD, Sparks, Md.) at 37° C. with agitation at 220 rpm for 24 hours before inoculating in food matrices. All 3 strains of Listeria monocytogenes were individually grown and then combined as a cocktail. Chicken salad was prepared and mixed with each antimicrobial treatment: 1) control treatment with no antimicrobial added (control), 2) 1% cultured dextrose and buffered vinegar blend mixed at 50:50 ratio (CDV), 3) 1% blend of 2.2% dried rosemary extract, 85% cultured dextrose, 13.85% Maltodextrin and 0.7% Silica (NCD), and 4) 1% blend of 2.2% dried rosemary extract, 50% Cultured Dextrose, 35% buffered vinegar, 13.85% Maltodextrin, and 0.7% Silica (1% NCDV). After the addition of antimicrobial treatments, Listeria cocktail was inoculated to the chicken salad with a target initial population around 4 log CFU/g. Chicken salad samples were then stored at 4° C. for 28 days period and checked for Listeria monocytogenes population by plating the samples on LSA agar media and counting the black colonies. All the experiments were performed in duplicate, and the average value is reported.

(51) The combination of dried rosemary extract, cultured dextrose, and buffered vinegar (NCDV) treatment performed better than the combination of culture dextrose and buffered vinegar (CDV) or the combination of dried rosemary extract and cultured dextrose (NCD). At day 21, 1% NCD had a higher Listeria monocytogenes count compared to the other two treatments and at day 28, 1% CDV and 1% NCD treatments had higher Listeria monocytogenes counts compared to 1% NCDV. When dried rosemary extract was used alone at the same concentration (2.2%), there were no antimicrobial activity observed (data not shown) proving that the combined effect was synergistic.

(52) The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

(53) All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.