MICROORGANISM STRAIN PEDIOCOCCUS ACIDILACTICI TAK 589 COCCOBEST AS AN ANTIMICROBIAL AND ANTIOXIDANT PROBIOTIC

Abstract

The microorganism strain Pediococcus acidilactici TAK 589 CoccobEst is a new antimicrobial and antioxidant probiotic, CoccobEst inhibits the growth of the enteropathogens L. monocytogenes, E. coli, S. enteritidis, S. typhimurium, S. sonnei, S. aureus, E. faecalis and C. sakazakii, but does not inhibit lactobacilli of natural intestinal microbiota. The strain's antimicrobial properties are enhanced by periplasmic peptidoglycan hydrolases with proteolytic activity. CoccobEst is used as a zootechnieai feed additive to prevent bacterial infections in the gastrointestinal tract of farm animals and pets and to prevent and reduce diarrhea. CoccobEst has highly active antioxidant defense system, increasing the redox activity of glutathione and reducing oxidative stress in cells. Therefore, CoccobEst is used as a food supplement or functional food for humans to prevent bacterial infections of the gastrointestinal tract and to prevent and reduce oxidative stress. In addition, CoccobEst is used as a starter culture in fermentation of vegetables.

Claims

1. (canceled)

2. (canceled)

3. A composition comprising lyophilized Pediococcus acidilactici TAK 589 Coccobest DSM 32372.

4. The composition of claim 3, wherein the composition is a probiotic food and beverage additive.

5. The composition of claim 3, wherein the composition is a zootechnical feed additive for preventing bacterial infections of the gastrointestinal tract and preventing and/or reducing diarrhea in farm animals and pets.

6. The composition of claim 3, wherein the compositions is a component of functional food or beverage additive to prevent bacterial infections of the gastrointestinal tract and prevent and/or reduce oxidative stress in humans.

7. (canceled)

8. The composition of claim 3, wherein the composition is a technological starter culture for fermentation of vegetables.

9. The composition of claim 3, wherein the composition is an aqueous solution.

10. The composition of claim 3, wherein the composition comprises at least one of milk replacer, milk, starter culture, or concentrated feed.

11. A method to prevent bacterial infections of the gastrointestinal tract and to prevent and/or reduce diarrhea in an animal, said method comprising administering the composition of claim 3 to the animal.

12. A method to prevent bacterial infections of the gastrointestinal tract and prevent and/or reduce oxidative stress in a human, the method comprising administering the composition of claim 3 to the human.

13. The method of claim 11, wherein the bacterial infection is caused by Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, Salmonella enterica serovar Typhimurium, Shigella sonnei, Staphylococcus aureus, Enterococcus faecalis and Cronobacter sakazakii.

14. The method of claim 12, wherein the bacterial infection is caused by Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, Salmonella enterica serovar Typhimurium, Shigella sonnei, Staphylococcus aureus, Enterococcus faecalis and Cronobacter sakazakii.

15. A method to inhibit a enteropathogens without suppression of lactobacilli, said method comprising administering the composition according to claim 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG. 1. The survival of Pediococcus acidilactici TAK 589 Coccobest in log 10 system in diluted buttermilk when incubated at high temperatures for 10 to 20 minutes.

[0070] FIG. 2. Inhibition in anaerobic environment. The antimicrobial effect of the Pediococcus acidilactici TAK 589 Coccobest on pathogens compared to the control strains of Pediococcus spp. isolated from the intestine. On the modified MRS agar using the streak line method in anaerobic (5/90/5 CO.sub.2/O.sub.2/N.sub.2) environment after 24 hours of incubation (growth suppression of the target micro-organisms in mm).

[0071] FIG. 3. Inhibition in microaerobic environment. The antimicrobial effect of the Pediococcus acidilactici TAK 589 Coccobest on pathogens compared to the control strains of Pediococcus spp. isolated from the intestine. On the modified MRS agar using the streak line method in microaerobic (10% CO.sub.2) environment after 24 hours of incubation (growth suppression of the target micro-organisms in mm).

DESCRIPTION OF EMBODIMENTS

Example 1. Antagonistic Effect on Pathogenic Micro-Organisms

[0072] Objective: Testing the antagonistic properties of Pediococcus acidilactici TAK 589 Coccobest against the most common pathogenic micro-organisms.

[0073] Method: In order to assess the antagonistic properties of Pediococcus acidilactici TAK 589 Coccobest against pathogens. the streak line method was used; the growth inhibition zone of pathogens caused by the TAK 589 Coccobest and Pediococcus spp. control strains was measured in millimeters (Hütt et al. 2006. Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol. 100: 1324-1332).

[0074] The antagonistic activity of Pediococcus spp. strains was assessed in regard to eight pathogen strains in different environments: Listeria monocytogenes ATCC 51774; Escherichia coli ATCC 25922; Salmonella enteritidis ATCC 13076: Salmonella enterica serovar Typhimurium; Shigella sonnei ATCC 25931; Staphylococcus aureus ATCC 25923; and Enterococcus faecalis ATCC 29212. First. Pediococcus acidilactici TAK 589 Coccobest was cultivated in an IG 150 incubator (Jouan, France) for 24 hours in MRS (Oxoid. UK) in microaerobic (10% CO.sub.2) environment at 37° C. The pathogenic strains were grown for 24 hours on agar supplemented with blood in aerobic environment at 37° C. In order to assess the antimicrobial properties, 20 μL of five different Pediococcus strains, cultivated for 24 hours, were cultured onto modified MRS agar medium (does not contain triammonium sulphate C.sub.6H.sub.17O.sub.7N.sub.3 and sodium acetate C.sub.2H.sub.9O.sub.5Na) along the middle line and cultivated in microaerobic and anaerobic (5/90/5 CO.sub.2/O.sub.2/N.sub.2) environments simultaneously at 37° C. The pathogen strains that were going to be tested were seeded in two perpendicular lines in opposite directions and incubated for 24 hours in microaerobic and anaerobic environments at 37° C. The growth-free zone was measured to determine the inhibition of pathogens. The control strains of Pediococcus spp. isolated from the intestines and belonging in the BioCC microbial culture collection were used as a basis for comparison.

[0075] Results: Pediococcus acidilactici TAK 589 Coccobest had the strongest inhibitory properties against the growth of pathogens in microaerobic and anaerobic environments; the effect of other strains was intermediate or low (FIG. 2, FIG. 3).

Example 2. The Effect of Administration to Calves (Experiment 1)

[0076] Objective: Studying the effect of Pediococcus acidilactici TAK 589 Coccobest on the intestinal microbiota of calves and on the incidence of diarrhoea.

[0077] Method: The experiment included 8 Estonian male calves of Holstein breed. New-born calves were divided into test and control groups (both groups: n=4). The calves in the test group received Pediococcus acidilactici TAK 589 Coccobest as an aqueous solution prepared with lyophilized powder in a daily dose of 1×10.sup.10 CPU from the first day until the calves were five days old. Stool samples were collected from the calves on days 2 and 5 of their life, and additionally, on day 8 after birth in order to assess the aftereffects. Stool samples were collected from the rectum with gloved hands into sterile cups which were stored at −20° C. and later at −80° C. The total quantity of lactobacilli and the quantity of E. coli and coliform micro-organisms were measured in the samples. In addition, the incidence of diarrhoea and general health indicators were assessed according to health scoring criteria (University of Wisconsin-Madison, School of Veterinary Medicine, Calf Health Scoring Chart and Calf Health Scoring Criteria).

[0078] Results: The control group had seven samples out of 16 positive for diarrhoea (44%) and the test group three samples out of 16 (19%). Administering Pediococcus acidilactici TAK 589 Coccobest reduced the quantity of E. coli in the stool samples of the test group by 1.6 logarithms by day 5 and 2.5 logarithms by day 8 after birth. The quantity of coliform micro-organisms reduced in the test group stool samples by 0.3 logarithms by day 5 and 0.8 logarithms by day 8. At the same time, the quantity of lactobacilli did not decrease in the stool samples of the test group.

Example 3. The Effect of Administration to Calves (Experiment 2)

[0079] Objective: Studying the effect of Pediococcus acidilactici TAK 589 Coccobest on the intestinal enterobacterial microbiota of calves and the incidence of diarrhoea.

[0080] Method: The experiment included 32 Estonian female calves of Holstein breed. New-born calves were divided into test and control groups (both groups: n=16) and were given colostrum for the first three days of their lives. After the day 3 and until day 16, the calves were given a mixture of milk from a local farm and complete milk replacer feed (MRF), prepared with MRF powder (20% fat, 22% protein, 47.6% lactose, and 16.4% casein) with a ratio of 140 g to 890 mL of warm water (40° C.). Starting from day 17. calves were given only MRF to drink. Pediococcus acidilactici TAK 589 Coccobest (1.2-3.2×10.sup.7 CFU/mL) was added to the MRF of the calves in the test group starting from day 3.

[0081] Between days 4 and 10, the calves received 6 liters of MRF per day, and after that, 8 liters a day until being weaned (approximately on day 70). During the experiment, a veterinarian recorded the consistency of the calf stool daily according to stool assessment criteria (University of Wisconsin-Madison, School of Veterinary Medicine, Calf Health Scoring Chart and Calf Health Scoring Criteria). There were no differences in the birth weight and Ig levels between the test and control group calves (Table 11). Calf stool samples were collected from the rectum on days 2, 7, 18 and 30 after birth with a gloved hand into sterile cups which were stored at −20° C. and later at −80° C. The general quantity of E. coli, coliform micro-organisms and enterobacteria in the samples was determined according to ISO standards.

TABLE-US-00012 TABLE 11 The health indicators of test and control group calves after birth Test group Control group (n = 16) (n = 18) p-value Time of birth 4.03. - 11.05.2015 6.03. - 10.05.2015 — Birth weight, kg 42.9 ± 3.37 42.6 ± 3.54 0.843 Ig level, g/L 34.8 ± 5.47 35.4 ± 10.2 0.838

[0082] Results: Between the days 15-65 of their lives, only one test group calf out of 16 had diarrhoea (6.3%) as opposed to seven control group calves out of 16 (43.8%). Moreover, two control group calves died. Administering Pediococcus acidilactici TAK 589 Coccobest decreased the quantity of E. coli in the stool samples of the test group by one logarithm by day 30. The quantity of coliform bacteria and enterobacteria reduced in the stool samples of the test group by 1.1 and 1.3 logarithms respectively by day 30. At the same time, the quantity of E. coli in the stool samples of the control group increased by 0.5 logarithms by day 30. The quantity of coliform bacteria and enterobacteria in the stool samples of the control group increased by 0.4 logarithms by day 30.

Example 4. Fermentation of Vegetables with the TAK 589 Coccobest Strain (Experiment 1)

[0083] Objective: Testing the suitability of Pediococcus acidilactici TAK 589 Coccobest for the fermentation of vegetables, e.g., white cabbage, or Brassica oleracea var. capitata f. alba.

[0084] Method: Cabbage was grated with a food processor into thin strips, table salt (1.7%) was added to it and mixed in carefully. Afterwards, Pediococcus acidilactici TAK 589 Coccobest. cultivated for 24 hours and having the final strain density of 10.sup.7 CFU/g, was mixed with the cabbage. Cabbages were fermented for 9 days at 18° C. Spontaneously fermented cabbage was used as a control. The pH level of the cabbage juice was measured on days 2, 5, and 9.

[0085] Results: Adding Pediococcus acidilactici TAK 589 Coccobest helped accelerate the fermentation of cabbage. The base pH level of the grated cabbage was 5.60. Compared to spontaneous fermentation, the pH level of the grated cabbage decreased faster (Table 12). The fast decrease in the pH level helped suppress the undesirable microflora of the cabbage, incl. lactic bacteria that create slime (e. g. Leuconostoc mesenteroides).

TABLE-US-00013 TABLE 12 The pH decrease of grated cabbage during the fermentation period pH on testing day Grated cabbage 1. 2. 5. 9. Control 5.60 5.15 3.82 3.80 TAK 589 5.60 4.65 3.67 3.71

[0086] During both tests, the fermented cabbage had no defects. tasted sour, and had a crunchy texture. Pediococcus acidilactici TAK 589 Coccobest is suitable for use in fermentation as a technological starter culture in order to direct the fermentation of cabbage.

Example 5. Fermentation of Vegetables with the TAK 589 Coccobest Strain (Experiment 2)

[0087] Objective: Testing the suitability of Pediococcus acidilactici TAK 589 Coccobest for the fermentation of vegetables, e. g. cucumbers, or Cucumis sativus L, grown outdoors.

[0088] Method: A solution consisting of 1 tablespoon of table salt and 1 tablespoon of sugar per 1 liter of water was prepared for the fermentation of Estonian cucumbers grown outdoors. A culture of Pediococcus acidilactici TAK 589 Coccobest, cultivated for 24 hours, was added to the solution with the final density of 10.sup.7 CFU/g. Cucumbers (4-5 cucumbers, 400 mL liquid) were fermented at 30° C. for 24 hours. At the end of fermentation, the pH level was measured and the cucumbers cooled down to 4° C.

[0089] Results: At the end of the fermentation, the pH level of spontaneously fermented cucumbers was 4.34. The pH level of cucumbers fermented with Pediococcus acidilactici TAK 589 Coccobest was 3.85. Both the control cucumbers and the cucumbers fermented with Pediococcus acidilactici TAK 589 Coccobest had no defects. They tasted sour. Cucumbers fermented with Pediococcus acidilactici TAK 589 Coccobest did not have the bitter aftertaste of the spontaneously fermented cucumbers. Therefore. Pediococcus acidilactici TAK 589 Coccobest is suitable for use as a technological starter culture to direct the fermentation when fermenting cucumbers.