Microbial strain Pediococcus acidilactici tak 589 coccobest as an antimicrobial and antioxidant probiotic

Abstract

The microbial strain Pediococcus acidilactici TAK 589 CoccobEst is a novel antibacterial 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 S. sakazakii, but not the lactic acid bacteria of the natural intestinal microbiota. The antibacterial properties of the strain are enhanced by a periplasmic peptidoglycan hydrolase protein. CoccobEst is used as a zootechnical feed additive to prevent bacterial infection in the gastrointestinal tract of livestock and pets, and prevent and reduce diarrhea. CoccobEst has high activity of the antioxidant defense system, enhances the redox activity of glutathione, and reduces intracellular oxidative stress. Therefore, CoccobEst can be used as a food supplement or functional food for humans to prevent bacterial infection in the gastrointestinal tract and prevent and reduce oxidative stress. In addition, CoccobEst can be used as a starter culture for vegetable fermentation.

Description

[Technical Field]

[0001] Technical Field The present invention relates to the field of biotechnology. The probiotic lactic acid bacteria of the present invention are used to enhance the natural immunity of the host organism and for the prevention and alleviation of various diseases. [Background technology]

[0002] The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) define probiotics as live microorganisms that, when administered in sufficient amounts as dietary supplements or incorporated into functional foods, have a favorable effect on the microbiological balance of the gastrointestinal tract and are therefore beneficial to the health of the host. Functional foods or compositions contain additional ingredients, including probiotics, or biologically active preparations that have health benefits or reduce the risk of disease.

[0003] The gut microbiota, which varies significantly between species and specimens, and its interactions with the host, are altered by genetic and environmental factors and disease. Favorably influencing the balance of the gut microbiota can enhance immune, psychological, and metabolic resistance and therapeutic response. Administration of probiotics promotes the colonization resistance of the gut microbiota and enhances resistance to stress and intestinal infections (Sassone-Corsi et al. 2015. How Beneficial Microbes Cooperate with Immunity to Provide Colonization Resistance to Pathogens. J. Immunol. 194:4081-4087 (Non-Patent Document 1)).

[0004] Many probiotics are lactic acid bacteria or lactofermentative bacteria, including the four-lobed homofermentative Pediococcus genus, which can adapt to a wide range of pH levels, temperatures, and osmolality, allowing them to colonize the intestinal tract.

[0005] When selecting probiotics suitable for human or animal administration, the principles of safety, functionality and technical properties are followed (FAO / WHO 2001. Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria; EFSA Panel on Additives and Products or Substances used in Animal Feed. 2011. Technical guidance: Tolerance and efficacy studies in target animals. EFSA J. 9:2175).

[0006] Regarding safety, it is essential to exclude bacterial strains associated with antibiotic resistance, pathogenicity, and infectious diseases. The functionality of the isolated strains, i.e., their ability to colonize the intestine and their biological activity, is more important than their origin (Saarela et al. 2000. Probiotic bacteria: Safety, functional and technological properties. J. Biotechnol. 84:197-215). Technological properties include preserving the biological activity and sensory properties of the probiotic strains during the production and storage process, including ensuring their survival after passage through the intestinal tract.

[0007] Problems caused by administering antibiotics to animals Animals born in the wild acquire their intestinal microbiota from their mothers. However, modern animal husbandry methods may not provide sufficient contact between mothers and their young, which may prevent the young animals' intestinal microbiota from developing rapidly enough. This does not provide sufficient protection for newborn animals against pathogens, and the stress of changes in physical and emotional environment, such as being separated from their mothers or being weaned, makes young animals particularly susceptible to infectious diseases (Corcionivoschi et al. 2010. The Effect of Probiotics on Animal Health., for a review, see J Anim Sci Biotechnol. 43:1).

[0008] Antibiotics have been used in livestock farming for decades to prevent infections and promote growth. As a result, natural selection and genetic mutation have significantly promoted the selection of antibiotic-resistant pathogens, leading to the horizontal transfer of antibiotic resistance genes not only among farm animals but also from agricultural land to groundwater (FAO. 2016; YSBajagai et al. FAO Animal Production and Health Paper No. 179, Probiotics in Animal Nutrition - Production, Impact and Regulation (Non-Patent Document 5); Rome; Chee-Sanford et al. 2001; Chee-Sanford et al. 2001, Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities, Appl Environ Micro-biol. 67:1494-1502 (Non-Patent Document 6)).

[0009] The spread of antibiotic-resistant pathogens among livestock poses a threat to human health as they can travel up the food chain and cause serious outbreaks. The outbreaks in Denmark and the United States in the 1990s were directly linked to the spread of multidrug-resistant Salmonella enterica serovar Typhimurium DT104 in pork and quinolone-resistant Campylobacter jejuni in poultry (Molbak et al. 1999; An outbreak of multidrug-resistant, quinolone-resistant S. enterica Typhimurium DT104. N Engl J Med. 341:1420-1425; Smith et al. 1999). Quinolone-resistant Campylobacter jejuni infections in Minnesota, 1992-1998. N Engl J Med. 340:1525-1532 (Non-Patent Document 8).

[0010] Probiotic administration in veterinary medicine Regulation (EC) No 1831 / 2003 of the European Parliament and of the Council has prohibited the supplementation of animal feed with antibiotics for the prevention of diseases (including gastrointestinal infections) and for growth promotion since 2006. This regulation classifies probiotics, as intestinal flora stabilizers, as zootechnical nutritional additives for various animal species.

[0011] Probiotic additives not only reduce coliform bacteria and associated diarrhea in calves, but also increase calf intake, promote growth, and reduce morbidity (Wallace and Newbold, 1995. Bacteriology in Animal Feed and Animal Feeding, VCH Verlagsgesellschaft mbH). Furthermore, probiotics promote resistance to intestinal colonization (Jatkauskas and Vrotniakien, 2010. Effects of probiotic dietary supplementation on diarrhea patterns, fecal microbiota, and performance of early weaned calves, Vet Med Praha, 55:494-503).

[0012] Probiotics are more effective in the early stages of calf development (7-10 days), when symptoms of disease appear more frequently and complications can become more severe (Kawakami et al. 2010. Feeding of Lactic Acid Bacteria and Yeast on Growth and Diarrhea of ​​Holstein Calves, J Anim Vet Adv. 9:1112-1114). Therefore, it is necessary to suppress the spread of pathogens in the animal's intestines as early as possible after birth.

[0013] Administration of pediococci as a probiotic to livestock Pediococcus acidilactici CNCM MA 18 / 5M, which is administered to piglets and chickens to increase their weight, is classified as an intestinal flora stabilizer in the European Union (Commission Implementing Regulation (EU) No. 413 / 2013 of 6 May 2013 (Non-Patent Document 12)).

[0014] Administration of this strain to piglets within 42 days of birth has a positive effect on weight gain, protects the small intestinal mucosa, and increases colonization and stress resistance at weaning (Di Giancamillo et al. 2008. Effects of orally administered probiotic Pediococcus acidilactici on the small and large intestine of weaning piglets. A qualitative and quantitative micro-anatomical study. Histol. Histopathol. 23:651-664 (Non-Patent Document 13)).

[0015] When administered to chicken, Pediococcus acidilactici CNCM MA 18 / 5M had a positive effect on egg weight, shell thickness, feed efficiency, yolk fatty acid composition, and cholesterol levels, and reduced the number of defective eggs (Mikulski et al. 2012. Effects of dietary probiotic Pediococcus acidolactici supplementation on performance, nutrient digestibility, egg traits, egg yolk cholesterol, and fatty acid profile in laying hens. Poultry Science, 91:2691-2700).

[0016] Some probiotic strains of Pediococcus acidilactici have been shown to be capable of regulating the synthesis of protein signaling molecules, thereby suppressing inflammatory processes in the body.

[0017] Pediococcus acidilactici LDTM 5201, isolated from a Korean national dish, inhibits the transcription of IL-8 cytokine, which induces an inflammatory response at the site of infection. Furthermore, when administered through fermented dairy products or as a food additive, this strain amplifies the transcription of IL-10 cytokine, a mediator of anti-inflammatory responses in the body (KR 20180117761, Seoul National University R&DB Foundation, 2018 (Patent Document 4)). Administration of Pediococcus acidilactici NRRL B-50517 has a similar effect of suppressing the development of inflammatory responses by increasing the transcription of IL-10 cytokines and M2 macrophages in the body and decreasing the transcription of IL-6 and IL-23 cytokines (U.S. Patent Application Publication No. 2019 / 16266397, Imagilin Technology LLC, 2019).

[0018] Pediococcus acidilactici has been used in several compositions of probiotic bacterial species containing additional digestive-influencing ingredients such as inulin, adsorbents, and inhibitors of intestinal motility to reduce and prevent the occurrence of diarrhea in calves (EP20110711323, Technische Universitat Munchen, 2011).

[0019] On the use of pediococci in the fermentation of plant matter. Fermentation is one of the simplest methods for preserving vegetables. Adding table salt to chopped vegetables promotes the separation of juices, which serve as a nutrient medium for the developing microorganisms. Fermentation requires anaerobic conditions to prevent the development of undesirable microorganisms. Any errors in the vegetable fermentation technique, as well as the influence of microorganisms present in the vegetables' natural microflora, can lead to softening or sliminess in the final product, or changes in product color (Pudnir and Jain, 2010. Changes in microflora of sauerkraut during fermentation and storage. World J Dairy Food Sci 5:221-225). Several factors limit the use of starter cultures in vegetable fermentation: indigenous flora of the vegetables, 1-2% salt concentration, and temperature fluctuations. Furthermore, the starter culture must not adversely affect the sensory characteristics of the final product.

[0020] The dominant lactic acid bacteria species in vegetable fermentations are Leuconostoc mesenteroides, Lactobacillus brevis, Pediococcus pentosaceus, and Lactobacillus plantarum (Biotechnology Applications in Traditional Fermented Foods. 1992. Report of an ad hoc panel of the board on science and technology for international development. National Academy Press, Washington, DC (Non-Patent Document 16); Plengvidhya et al. 2007. DNA Fingerprinting of Lactic Acid Bacteria in Sauerkraut Fermentations. Appl. Environ. Microbiol. 73:7697-7702 (Non-Patent Document 17)).

[0021] In this regard, consuming a fermented functional food prepared from soybeans, turmeric, and the fruit of Hovenia spp., containing Pediococcus acidilactici IOB 701 and other probiotic strains, is known to alleviate the aftereffects of alcohol consumption, support liver activity in alcohol decomposition, and reduce the risk of liver damage (CN 201910930726) (CN 201910930726, Tianjin Chuangyuan Biotechnology Co., Ltd., 2019 (Patent Document 3)).

[0022] The above shows that Pediococcus acidilactici strains with various properties have already been studied and used, but different strains of the same species do not have identical properties due to intraspecific differences caused by genetic mutations or unique properties of individual strains.

[0023] Pediococcus isolated from various environments—silage, fecal samples, milk, fermented vegetables, and juice—have widely varying strain-specific susceptibilities to antibiotics (Singla et al. 2018. Antibiotic susceptibility profile of Pediococcus spp. From diverse sources. 3 Biotech. 8:489). For example, when several strains of Pediococcus acidilactici were tested, widespread resistance to common antibiotics was found (Daniels et al. 2007. Susceptibility of Pediococcus spp. to antimicrobial agents. J. Appl. Microbiol. 102:384-389). Therefore, closely related probiotic bacterial strains can differ depending on their genotype, phenotype, and functionality (Barros et al. 2001. Phenotypic and Genotypic Characterization of Pediococcus Strains Isolated from Human Clinical Sources. J. Clin. Microbiol. 39:1241-1246).

[0024] The main prerequisite for the expression of specific strain-specific properties is the presence of the genes responsible for them within the cells of the microorganism (Marteau, 2011. Evidence of Probiotic Strain Specificity Makes Extrapolation of Results Impossible from a Strain to Another, Even from the Same Species, Annals of Gastroenterology & Hepatology (Non-Patent Document 21)). [Prior art documents] [Patent documents]

[0025] [Patent Document 1] U.S. Patent Application Publication No. 2019 / 16266397 (US 201916266397) [Patent Document 2] European Patent Application Publication No. 20110711323 (EP20110711323) [Patent Document 3] Chinese Patent Application Publication No. 201910930726 (CN 201910930726) [Patent Document 4] Korean Patent Application Publication No. 20180117761 (KR 20180117761) [Non-patent literature]

[0026] [Non-Patent Document 1] Sassone-Corsi et al.2015. How Beneficial Microbes Cooperate with Immunity to Provide Colonization Resistance to Pathogens. J.Immunol.194:4081-4087 [Non-patent document 2] Health and Nutritional Properties of Probiotics in Food including Powdered Milk with Live Active Acid Bacteria; EFSA Panel on Additives and Products or Substances used in Animal Feed. 2011. Technical guidance: Tolerance and efficacy studies in target animals. EFSA J. 9:2175 [Non-patent document 3] Probiotic bacteria:Safety, functional and technological properties). J.Biotechnol.84:197-215 [Non-patent document 4] The Effect of Probiotics on Animal Health.) As a review, J Anim Sci Biotechnol. 43:1 [Non-patent document 5] FAO.2016.YSBajagai et al. Probiotics in Animal nutrition - Production, impact and regulation by FAO Animal Production and Health Paper No.179 [Non-patent document 6] Rome; Chee-Sanford et al. 2001. Chee-Sanford et al. 2001. Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities. Appl Environ Micro-biol. 67:1494-1502 [Non-Patent Document 7] This has been directly linked to the spread of Salmonella enterica serovar Typhimurium DT104 and quinolone-resistant Campylobacter jejuni in poultry (Molbak et al. 1999; An outbreak of multidrug-resistant, quinolone-resistant S. enterica serovar Typhimurium DT104. N Engl J Med. 341:1420-1425 [Non-patent document 8] Smith et al. 1999. (Quinolone-resistant Campylobacter jejuni infections in Minnesota), 1992-1998 Investigation Team. N Engl J Med. 340:1525-1532 [Non-Patent Document 9] Wallace and Newbold.1995.Bacteriology in Animal Feed and Animal Feeding.VCH Verlagsgesellschaft mbH [Non-Patent Document 10] Effects of probiotic dietary supplementation on diarrhoea patterms, fecal microbiota and performance of early weaned calves), Vet Med Praha.55:494-503 [Non-Patent Document 11] Kawakami et al. 2010. Feeding of Lactic Acid Bacteria and Yeast on Growth and Diarrhea of ​​Holstein Calves. J Anim Vet Adv. 9:1112-1114 [Non-Patent Document 12] Pediococcus acidilactici) CNCM MA 18 / 5M is a European Commission regulation implementing Regulation (EU) No. 413 / 2013 of 6 May 2013 [Non-Patent Document 13] Effects of orally administered probiotic Pediococcus acidilactici on the small and large intestine of weaning piglets. Aqualitative and quantitative micro-anatomical study). Histol. Histopathol.23:651-664 [Non-Patent Document 14] Mikulski et al. 2012. Effects of dietary probiotic Pediococcus acidilactici supplementation on performance, nutrient digestibility, egg traits, egg yolk cholesterol, and fatty acid profile in laying hens. Poultry Science, 91:2691-2700 [Non-Patent Document 15] Changes in microflora of sauerkraut during fermentation and storage). World J Dairy Food Sci 5:221-225 [Non-Patent Document 16] Biotechnology Applications to Traditional Fermented Foods. 1992. Report of an ad hoc panel of the board on science and technology for international development. National Academy Press, Washington, DC [Non-Patent Document 17] Plengvidhya et al. 2007. DNA Fingerprinting of Lactic Acid Bacteria in Sauerkraut Fermentations. Appl. Environ. Microbiol. 73:7697-7702 [Non-Patent Document 18] Antibiotic susceptibility profile of Pediococcus spp. From diverse sources). 3 Biotech.8:489 [Non-Patent Document 19] Daniels et al. 2007. Susceptibility of Pediococcus spp. to antimicrobial agents. J Appl Microbiol. 102: 384-389 [Non-Patent Document 20] Barros et al. 2001. Phenotypic and Genotypic Characterization of Pediococcus Strains Isolated from Human Clinical Sources.J. Clin.Microbiol.39:1241-1246 [Non-Patent Document 21] Marteau. 2011. Evidence of Probiotic Strain Specificity Makes Extrapolation of Results Impossible from a Strain to Another, Even from the Same Species. Annals of Gastroenterology & Hepatology [Non-Patent Document 22] Quality control and preprocessing of metagenomic datasets).Bioinformatics,27:863-864 [Non-Patent Document 23] Brettin et al. 2015. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. 5:8365 [Non-Patent Document 24] Tatusov. 2000. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28: 33-36 [Non-Patent Document 25] Sharma et al. 2016. Prediction of peptidoglycan hydrolases - a new class of antibacterial proteins. BMC Genomics. 17:1 [Non-Patent Document 26] Garcia-Cano et al. 2011. Detection, cellular localization and antibacterial activity of two lytic enzymes of Pediococcus acidilactici ATCC 8042. J Appl Microbiol. 111: 607-615 [Non-Patent Document 27] Marchler-Bauer et al.2016.CDD / SPARCLE: Function Classification of proteins via subfamily domain architectures.Nucleic Acids Res. 45:D200-D203 [Non-patent document 28] It is considered a standard of high safety compared to P. acidilactici strains (Daniels et al. 2007. Susceptibility of Pediococcus spp. to antimicrobial agents. J Appl Microbiol. 102: 384-389 [Non-Patent Document 29] EFSA Journal.2012.10(6):2740 [Non-Patent Document 30] Partial Characterization of Bacteriocin Produced by Halotolerant Pediococcus acidilactici Strain QC38 Isolated from Traditional Cotija Cheese.). Pol J Microbiol.65: 279-285 [Non-Patent Document 31] Hutt et al. 2006. Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol. 100:1324-32 [Non-Patent Document 32] Smirnova and Oktyabrsky. 2005. Glutathione in Bacteria. Biokhimiya 70:1199-211 [Non-Patent Document 33] Hutt et al. 2006. Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol. 100:1324-1332 [Non-Patent Document 34] University of Wisconsin-Madison,School of Veterinary Medicine, Calf Health Scoring Chart and Calf Health Scoring Criteria Summary of the Invention [Problem to be solved by the invention]

[0027] For this reason, there is a need for strains of Pediococcus acidilactici that have desirable properties and are suitable for use in the fermentation of human food and animal feed, as well as plant matter. [Means for solving the problem]

[0028] The present invention relates to the isolated bacterial strain Pediococcus acidilactici TAK 589 Coccobest, its lyophilized form, compositions containing the strain, and its use as a probiotic additive in foods or beverages, as a veterinary additive for livestock and pets, and as an additive in functional foods or beverages for humans. The purpose of the present invention is to propose a novel probiotic bacterial strain with antibacterial and antioxidant activity suitable for correcting and stabilizing the gastrointestinal microflora, thereby preventing the onset of bacterial infections, and preventing and / or reducing oxidative stress in the body. Additionally, Pediococcus acidilactici TAK 589 Coccobest is suitable for use as a technical starter culture in the fermentation of plant matter. [Brief explanation of the drawings]

[0029] [Figure 1] Survival in log10 system of Pediococcus acidilactici TAK 589 Coccobest in diluted buttermilk after 10-20 min incubation at elevated temperatures. [Figure 2] Inhibition under anaerobic conditions. The antimicrobial effect of Pediococcus acidilactici TAK 589 Coccobest against pathogens was compared with control strains of Pediococcus spp. isolated from the intestine. Growth inhibition of target microorganisms in mm was measured on modified MRS agar using the streak-line method in an anaerobic (5 / 90 / 5 CO₂ / O₂ / N₂) environment after 24 hours of incubation. [Figure 3]Inhibitory effect in a microaerobic environment. The antibacterial effect of Pediococcus acidilactici TAK 589 Coccobest against pathogens was compared with control strains of Pediococcus spp. isolated from the intestine. Growth inhibition of target microorganisms in mm was measured on modified MRS agar using the streak-line method in a microaerobic environment (10% CO2) after 24 hours of incubation. DETAILED DESCRIPTION OF THE INVENTION

[0030] DISCLOSURE OF THE INVENTION Pediococcus acidilactici TAK 589 Coccobest, in the form of a freeze-dried culture of the microorganism, is suitable for administration as a food additive to prepare functional foods for humans and / or livestock and pets (including fish and birds). Other ingredients in compositions containing Pediococcus acidilactici TAK 589 Coccobest can include complete milk replacers, milk, starter cultures, concentrates, premixes, and other ingredients.

[0031] The strains of the present invention can be used to prevent bacterial infections in the gastrointestinal tract of humans, livestock, and pets, and thus to prevent and / or alleviate diarrhea caused by microorganisms including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Salmonella enterica serovar Typhimurium, Shigella sonnei, Enterococcus faecalis, and Cronobacter sakazakii.

[0032] The antibacterial and antioxidant properties of Pediococcus acidilactici TAK 589 Coccobest allow it to be safely used in several areas of biotechnology, including the fermentation of plant matter or in probiotic dietary supplements for humans.

[0033] Pediococcus acidilactici TAK 589 Coccobest has been deposited with the Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure under DSM number 32372 (2016.09.23).

[0034] Morphological properties Pediococcus acidilactici TAK 589 Coccobest strain was isolated from diluted feces of calves (10 -2 ~10 -7 Pediococcus acidilactici TAK 589 was isolated from fecal samples of healthy calves during a gastrointestinal microbiota study by culturing it in saline (0.9% NaCl) and plating it onto MRS agar (OXOID, UK) in an IG 150 incubator (Jouan, France) under a microaerophilic atmosphere (10 / 5 / 85 CO₂ / O₂ / N₂) at 37°C for 48 hours. Cultured microbial colonies were described and counted to determine the total microbial abundance. Pediococcus acidilactici TAK 589 is a Gram-positive coccobacillus with cells that form pairs or tetrads.

[0035] Physiological and biochemical properties Pediococcus acidilactici TAK 589 Coccobest is cultivated in MRS broth by incubation in a microaerobic environment for 24 to 48 hours, resulting in a uniformly turbid growth. After 48 hours of incubation on MRS agar at 37°C under a microaerobic environment (10 / 5 / 85 CO₂ / O₂ / N₂), the microbial colonies are 1.5 to 2.5 mm in diameter, have regular edges, and are grayish-white convex. The optimal growth temperature for this strain is 37°C to 45°C, but it can also grow at 7°C and 15°C. The optimal pH level for the culture medium is 6.5.

[0036] The TAK 589 Coccobest strain was identified as Pediococcus acidilactici based on its biochemical activity using API CHL 50 (bioMérieux, France) medium and a Maldi Biotyper mass spectrometer (Bruker Daltonik, Germany) to determine the carbohydrate fermentation profile of the strain's fermented products: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, D-trehalose, gentiobiose, tagatose, and sodium gluconate.

[0037] Genomic and molecular characterization DNA fragments from samples obtained by whole-genome sequencing of Pediococcus acidilactici TAK 589 Coccobest (Omega Bioservices, USA) were sorted according to Phred quality score (<20) and length (<40 bp) using PRINSEQ web software (Schmieder and Edwards, 2011. Quality control and preprocessing of metagenomic datasets. Bioinformatics, 27:863-864). Genomes were fully assembled and annotated from samples that passed quality control using the PATRIC software based on the RAST server (Brettin et al. 2015. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. 5:8365).

[0038] Based on these results, the nucleotide structure of the genome of Pediococcus acidilactici TAK 589 Coccobest was determined, and 1,947 protein-coding DNA sequences were identified (Table 1).

[0039] [Table 1]

[0040] The 1,947 protein-coding sequences identified during the genome annotation were aligned to orthologous genes in a database based on the COG server (Tatusov. 2000. COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28:33-36 (Non-Patent Document 24)); 1,677 sequences were classified into 19 major categories according to function (Table 2).

[0041] [Table 2]

[0042] A periplasmic peptidoglycan hydrolase (PGH) gene sequence was identified in the genome of Pediococcus acidilactici TAK 589 Coccobest, and the enzyme site has proteolytic activity and inhibits the growth of a wide range of microorganisms in vitro (Sharma et al. 2016). Prediction of peptidoglycan hydrolases - a new class of antibacterial proteins. BMC Genomics. 17:1 (Non-Patent Document 25); Garcia-Cano et al. 2011. Detection, cellular localization, and antibacterial activity of two lytic enzymes of Pediococcus acidilactici ATCC 8042. J. Appl Microbiol. 111: 607-615 (Non-Patent Document 26)).

[0043] The identified PGH sequence (GenBank AKD44141.1) was aligned with the NCBI Conserved Domains Database, confirming the two active sites identified in previous studies. The first belongs to a family of proteins that recognize the peptidoglycan layer and are capable of extracellular hydrolysis of peptidoglycan by cleaving the amide tubule bonds in the cell membrane. The second active site belongs to the glucosaminidase superfamily, which has a similar effect (Marchler-Bauer et al. 2016. CDD / SPARCLE: Function Classification of proteins via subfamily domain architectures. Nucleic Acids Res. 45:D200-D203 (Non-Patent Document 27)).

[0044] Thus, Pediococcus acidilactici TAK 589 Coccobest contains at least two lytic enzymes with active sites that enhance the strain's antibacterial activity. The inhibitory effect on microbial growth attributed to the PGH enzymes described above has been demonstrated in vitro, and the antibacterial properties of the present invention have been demonstrated in vivo. In both cases, delivery of antibacterial effects against several widespread pathogens, including Listeria monocytogenes, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli, has been demonstrated.

[0045] Antibiotic susceptibility The antimicrobial susceptibility of Pediococcus acidilactici TAK 589 Coccobest to antibiotics was tested using the VetMIC™ panel (SVE, Sweden) according to ISO 10932 standards. The minimum inhibitory concentrations were determined according to the epidemiological cutoff values ​​recommended by the European Food Safety Authority (EFSA) and compared with other pediococcids of animal origin in the BioCC microbial culture collection (Table 3). A microbial strain is considered susceptible if it is inhibited at a concentration equal to or lower than the cutoff value (S ≤ × mg / L) of a specific antimicrobial compound. A microbial strain is considered resistant if it is inhibited at a concentration higher than the cutoff value (R > × mg / L).

[0046] The Pediococcus acidilactici TAK 589 Coccobest strain was not resistant to the antibiotics used in this study, and is therefore considered a benchmark for increased safety compared to several other P. acidilactici strains that have been tested and found to be resistant to common antibiotics (Daniels et al. 2007. Susceptibility of Pediococcus spp. to antimicrobial agents. J Appl Microbiol. 102: 384-389).

[0047] [Table 3]

[0048] antibacterial The antimicrobial efficacy of various P. acidilactici strains against pathogens can vary widely. For example, strain QC38 has been shown to exhibit low inhibitory activity against E. coli and S. enterica serovar Typhimurium and intermediate inhibitory activity against S. aureus (Morales-Estrada et al. 2016). Partial Characterization of Bacteriocin Produced by Halotolerant Pediococcus acidilactici Strain QC38 Isolated from Traditional Cotija Cheese. Pol J Microbiol. 65: 279-285 (Non-Patent Document 30) At the same time, the inhibitory effect of the strain of the present invention on these pathogens is strong.

[0049] Based on in vivo studies, Pediococcus acidilactici TAK 589 Coccobest inhibits pathogenic microorganisms, including Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, Salmonella enterica serovar Typhimurium, Shigella sonnei, Staphylococcus aureus, Enterococcus faecalis, and Cronobacter sakazakii (Example 1).

[0050] The streak line method was used to evaluate the antibacterial activity of Pediococcus acidilactici TAK 589 Coccobest against pathogenic bacteria (Hutt et al. 2006. Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol. 100:1324-32 (Non-Patent Document 31)). To measure the antagonistic activity against the target microorganism, the width of the inhibition zone was measured in millimeters, and the arithmetic mean and standard deviation were calculated based on the results of the samples used, and the antagonistic activity of the strain was evaluated based on these values.

[0051] Pediococcus acidilactici TAK 589 Coccobest strain has strong antagonistic properties against selected pathogens after 24-48 hours of incubation, but this strain does not inhibit lactic acid bacteria belonging to the natural intestinal flora (Tables 4 and 5).

[0052] [Table 4]

[0053] [Table 5]

[0054] Antioxidant properties Oxidative stress in the body is caused by factors that promote the chain reaction of free radicals, i.e., pro-oxidants. The continuous and unregulated production and action of pro-oxidants causes oxidative damage to lipids, proteins, nucleic acids, and carbohydrates, and is one of the causes of many diseases (myocardial infarction, stroke, arteriosclerosis, and cancer). Among pro-oxidants, reactive oxygen species are included, and the accumulation of these reactive oxygen species is one of the main reasons for cell damage.

[0055] Antioxidants are enzymes or substances that can inhibit or prevent the formation of prooxidants and chain reactions starting at very low concentrations. Both specific antioxidant enzymes and antioxidants (e.g., glutathione) affect the body. In addition, antioxidants are absorbed from food. In polluted and stressful environments, antioxidant protection requires additional support. Among other things, the function of intestinal probiotics releases antioxidants into the bloodstream.

[0056] Gene sequences for antioxidant enzymes and transcription factors were identified in the genome of Pediococcus acidilactici TAK 589 Coccobest, including NADH peroxidase (EC 1.11.1.1), hydrogen peroxide stress response regulator (PerP), thioredoxin (Trx), thioredoxin reductase (EC 1.8.1.9), glutathione reductase (EC 1.8.1.7), thiol peroxidase (EC 1.11.1.15), methionine sulfoxide reductase A and B (EC 1.8.4.11 and EC 1.8.4.12), coenzyme A disulfide reductase (EC 1.8.1.1), and manganese catalase (EC 1.11.1.6).

[0057] To evaluate the effects of the identified antioxidant enzymes, they were tested in vitro and compared with other pediococci from animal origin included in the BioCC microbial culture collection (Table 6).

[0058] Pediococcus acidilactici TAK 589 Coccobest strain was cultured in MRS broth for 24 hours and centrifuged at 10,000×G at 4°C for 5 minutes. The supernatant was then removed and the resulting product was suspended in 1 ml of MQ water. The density of the suspension was 10 at an OD600 of 1.1. 9 To obtain the lysate, lysis buffer (Qiagen, Netherlands) containing SDS (CAS# 151-21-3) was added to the suspension, and the bacterial cells were mechanically disrupted at room temperature for 15 minutes.

[0059] Total antioxidant activity (TAA) was determined using a commercially available kit (Cayman Chemicals, USA). The method is based on the oxidation of ABTS (CAS# 28752-68-3) by metmyoglobin and the quantification of inhibitory antioxidants in the samples. The absorbance of samples and standards was measured at a wavelength of 750 nm using a Synergy HTX spectrophotometer (BioTek, USA).

[0060] Oxidized (GSSG) and reduced (GSH) glutathione were determined using a colorimeter kit (Invitrogen, USA). To remove proteins in the lysates, samples were treated with 5% benzoic acid (CAS# 97-05-2). To quantify oxidized glutathione, free glutathione and other thiols were blocked with 2-vinylpyridine (CAS# 100-69-6). The absorbance of samples and standards was measured at 405 nm using a Synergy HTX spectrophotometer.

[0061] Hydrogen peroxide was measured using a colorimeter kit (Thermo Scientific, USA). The method is based on the oxidation of Mohr's salt (CAS# 24389-93-3) by hydrogen peroxide, which is enhanced by sorbitol (CAS# 50-70-4). The oxidized Mohr's salt binds to xylenol (CAS# 1611-35-4) in the sample. The absorbance of the samples and standards was measured at 595 nm using a Synergy HTX spectrophotometer.

[0062] The results showed that hydrogen peroxide content was low in samples containing Pediococcus acidilactici TAK 589 Coccobest strain in both living cell-containing and cell-free environments (Table 6). H2O2 is a precursor for the formation of reactive radicals in the body, diffusing through cellular structures and becoming a powerful oxidant in acidic environments.

[0063] Reduced glutathione levels measured in a cell-free environment are highest in Pediococcus acidilactici TAK 589 Coccobest strain, and it is one of the most important traps for reactive oxygen species. The redox activity of glutathione, i.e., its ability to suppress autooxidation and maintain its reduced form, makes it the most important redox buffer within the cell. Changes in glutathione form directly affect the antioxidant activity of the cell (Smirnova and Oktyabrsky. 2005. Glutathione in Bacteria. Biokhimiya 70:1199-211 (Non-Patent Document 32)). The GSSG / GSH ratio, an indicator of intracellular oxidative stress, was lowest in the Pediococcus acidilactici TAK 589 Coccobest strain compared to other pediococcids of animal origin in the BioCC microbial culture collection.

[0064] In addition, the measured total antioxidant activity was highest in the cell lysate of Pediococcus acidilactici TAK 589 Coccobest (Table 6).

[0065] [Table 6]

[0066] Maintaining biological activity When using the powder for the preparation of a complete milk replacer feed (MRF) for calves, this powder is diluted with water at 40-50°C, which is why the ability of the probiotic strain to maintain its biological activity at high temperatures is essential.

[0067] To demonstrate this, freeze-dried cultures of Pediococcus acidilactici TAK 589 Coccobest were stirred into diluted buttermilk and heated to 85°C for 20 minutes. After heating, the cultures were immediately plated on MRS agar and incubated at 37°C for 48 hours, after which the microbial wells were counted. During the course of the experiment, we found that the number of Pediococcus acidilactici TAK 589 Coccobest remained unchanged when heated to 60°C for 10–20 minutes, but decreased by one logarithm at 70°C (Figure 1).

[0068] Maintaining biological activity over long periods at various temperatures, in addition to those used in food processing, is important for the preservation and storage of probiotic lactic acid bacteria strains. For a continuous 36-month storage challenge test, a culture of Pediococcus acidilactici TAK 589 Coccobest was used as a freeze-dried powder (Table 7).

[0069] [Table 7]

[0070] The biological activity of Pediococcus acidilactici TAK 589 Coccobest strain was also tested in the acidic environment of the stomach and in contact with bile acids. This strain was cultured in MRS broth at 37°C for 24 hours in a microaerobic environment (10 / 5 / 85 CO₂ / O₂ / N₂). To measure its resistance to bile acids and gastric acid, three more MRS broths were prepared. 3 g / L bile extract (Sigma, USA) was added to the first, and 1 M HCl solution was added to the second until the environmental pH level reached 3. Finally, 3 g / L pepsin (EC 3.4.23.1; Sigma, USA) was added to the third in 1 M HCl solution, bringing the environmental pH level to 2. A pre-cultured bacterial culture was added to the modified MRS broth and then incubated at 37°C in a microaerobic environment.

[0071] The viability of Pediococcus acidilactici TAK 589 Coccobest strain was demonstrated by inoculation every hour (Table 8).

[0072] [Table 8]

[0073] Ability to colonize the intestine The ability of Pediococcus acidilactici TAK 589 Coccobest to pass through the digestive tract was demonstrated as part of a study conducted in Estonia using 32 Holstein heifer calves (Example 3). Newborn calves were divided into test and control groups and fed 6 liters of a complete milk replacer (MRF) mixture prepared with milk and powder from a local dairy farm from day 4 to day 16 of age. The MRF fed to calves in the test group was supplemented with the probiotic strain Pediococcus acidilactici TAK 589 Coccobest. Fecal samples were collected from the rectum of the calves by gloved hands at the end of the first and second weeks into sterile cups and stored at -20°C, followed by storage at -80°C.

[0074] The amount of Pediococcus acidilactici TAK 589 Coccobest was measured in fecal samples collected from calves by qPCR using strain-specific primers and gene probes. DNA was extracted from fecal samples according to the protocol of the QIAamp DNA Stool Mini Kit (Qiagen, Germany). DNA concentration was checked using a Synergy HTX spectrophotometer (BioTek, USA). To determine the strain of Pediococcus acidilactici TAK 589 Coccobest, an aliquot of DNA sample from each calf was prepared at a concentration of 10 ng / μL in a final volume of 35 μL. For DNA amplification, strain-specific primers and TaqMan diagnostic gene probes were designed against a region of the gene sequence essential for bacterial activity, the transporter protein BioY (WP_002832434.1), belonging to the biotin biosynthetic chain, strain-specific for Pediococcus acidilactici TAK 589 Coccobest (Table 9).

[0075] [Table 9]

[0076] A 2 μL aliquot of DNA (~20 ng) used in the qPCR reaction was dried. The DNA was pipetted into a microplate and dried overnight under sterile conditions. Next, 20 μL of the previously prepared reaction mixture was added to each sample. Two blanks were added to the plate to control for assay purity and possible false positives. The reaction mixture per sample contained the following components:

[0077] 4 μL PrimeTime® Mini qPCR Assay (Integrated DNA Technologies, USA)

[0078] 4 μL 5× HOT FIREPol® Probe qPCR Mix (Solis BioDyne, Estonia)

[0079] 12 μL MQ deionized H2O

[0080] A QuantStudio thermocycler (Thermo Fisher Scientific, USA) was used for the qPCR reactions. QuantStudio Design and Analysis Software (Thermo Fisher Scientific, USA) was used to read the PCR plates and analyze the results. The qPCR reaction conditions were as follows:

[0081] [Table 10]

[0082] Pediococcus acidilactici TAK 589 Coccobest in fecal samples collected from calves was tested with the aid of fluorescence produced by TaqMan strain-specific gene probes. During PCR, primers and gene probes bind in a strain-specific manner by joining or mismatching at specific sequences. The gene probe is conjugated to a fluorophore at its 5' end and a quencher at its 3' end. The quencher is active only when in proximity to the fluorophore. When a new DNA strand is synthesized, the fluorophore at the 5' end of the gene probe is released. With each new cycle, the fluorophore is released in proportion to the number of amplicons. This results in increasingly intense fluorescence, allowing for the quantification of strain-specific amplicons in the reaction mixture. A cycle threshold is recorded when a fluorescence threshold is exceeded. The specificity of the diagnostic gene probe for Pediococcus acidilactici TAK 589 Coccobest strain was evaluated using control samples and reaction mixtures containing genomic DNA.

[0083] The presence of Pediococcus acidilactici (TAK 589 Coccobest) strain was tested in 9 of 10 fecal samples from calves in the test group at week 1 and week 2 of age, and in 7 of 11 fecal samples (Table 10). The presence of this strain in fecal samples from calves in the control group was tested in 1 of 13 fecal samples at week 1 and 1 of 7 fecal samples at week 2. Therefore, it can be said that Pediococcus acidilactici (TAK 589 Coccobest) can successfully colonize the intestines of calves.

[0084] [Table 11] [Example]

[0085] Description of the embodiment Example 1. Antagonism regarding pathogenic microorganisms Objective: To test the antagonistic properties of Pediococcus acidilactici TAK 589 Coccobest against the most common pathogenic microorganisms.

[0086] Methods: To evaluate the antagonistic properties of Pediococcus acidilactici TAK 589 Coccobest against pathogenic bacteria, the streak-line method was used; the zone of growth inhibition of the pathogen caused by TAK 589 Coccobest and a Pediococcus spp. control strain was measured in millimeters (Hutt et al. 2006. Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol. 100:1324-1332 (Non-Patent Document 33)).

[0087] The antagonistic activity of Pediococcus spp. strains was evaluated against eight pathogen strains in different environments.

[0088] Listeria monocytogenes ATCC 51774; Escherichia coli ((E. coli) 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

[0089] Pediococcus acidilactici TAK 589 Coccobest was cultured for 24 hours at 37°C in a microaerophilic (10% CO2) MRS (Oxoid, UK) in an IG 150 incubator (Jouan, France). The pathogens were grown on blood-supplemented agar plates at 37°C for 24 hours under aerobic conditions. To evaluate the antibacterial properties, 20 μL of each of the five Pediococcus strains cultured for 24 hours was placed along the centerline on a modified MRS agar plate (triammonium sulfate C6H). 17 The cultures were grown on a medium containing 100% CO₂ / O₂ / N₂ (without O7N3 and sodium acetate C2H9ON5Na) and simultaneously incubated at 37°C in microaerophilic and anaerobic (5 / 90 / 5 CO₂ / O₂ / N₂) environments. The pathogen strains to be tested were inoculated in two perpendicular lines, one above the other, and incubated at 37°C in microaerophilic and the other in anaerobic environments for 24 hours. The growth-free zone was measured to determine the inhibition of the pathogens. As a standard of comparison, a control strain of Pediococcus spp. isolated from the intestine and belonging to the BioCC Microbial Culture Collection was used.

[0090] result Pediococcus acidilactici TAK 589 Coccobest showed the strongest inhibitory properties against the growth of pathogenic bacteria in microaerobic and anaerobic environments, while the effects of the other strains were intermediate or low (Figures 2 and 3).

[0091] Example 2. Effects of administration to calves (Experiment 1) the purpose: To investigate the effect of Pediococcus acidilactici TAK 589 Coccobest on the intestinal flora and the occurrence of diarrhea in calves.

[0092] Methods: The experiment included eight Estonian Holstein male calves. Newborn calves were divided into a test group and a control group (n = 4 in each group). Calves in the test group were administered 1 × 10 HCl as an aqueous solution prepared from freeze-dried powder from day 1 until the calves were 5 days old. 10 Calves were fed Pediococcus acidilactici TAK 589 Coccobest at a daily dose of 100 CFU. Feces were collected from calves on days 2 and 5 of age, and on day 8 of age for evaluation of sequelae. Fecal samples were collected rectally with gloved hands into sterile cups and stored at -20°C, followed by storage at -80°C. The total amount of lactic acid bacteria, Escherichia coli (E. coli), and coliform microorganisms in the samples were measured. In addition, the incidence of diarrhea and general health indicators were evaluated according to the Calf Health Scoring Chart and Calf Health Scoring Criteria (University of Wisconsin-Madison, School of Veterinary Medicine, Non-Patent Document 34).

[0093] Results: Seven of 16 samples from the control group were positive for diarrhea (44%), while three of 16 samples from the test group were positive for diarrhea (19%). Pediococcus acidilactici TAK 589 Administration of Coccobest reduced the amount of Escherichia coli (E. coli) in fecal samples from the test group by 1.6 logs by day 5 and by 2.5 logs by day 8 of age. Additionally, the amount of coliform organisms in the test group's fecal samples was reduced by 0.3 logs by day 5 and 0.8 logs by day 8. At the same time, the amount of lactobacillus was not reduced in the test group's fecal samples.

[0094] Example 3. Effects of administration to calves (Experiment 2) the purpose: Objective To investigate the effect of Pediococcus acidilactici TAK 589 Coccobest on the intestinal microflora and incidence of diarrhea in calves.

[0095] method: The study involved 32 Estonian Holstein female calves. Newborn calves were divided into test and control groups (n = 16 in each group) and received colostrum for the first 3 days of life. From day 3 to day 16, calves were fed a mixed diet of milk and complete milk replacer (MRF) from a local farm (prepared by mixing 140 g of MRF powder (20% fat, 22% protein, 47.6% lactose, 16.4% casein) with 890 mL of warm water (40°C). From day 17, calves were fed only MRF. From day 3, Pediococcus acidilactici TAK 589 Coccobest (1.2–3.2 × 10 7 CFU / mL) was added.

[0096] Calves were fed 6 liters of MRF per day from days 4 to 10, and then 8 liters per day until weaning (approximately day 70). During the experiment, a veterinarian recorded the calves' stool consistency daily according to a stool evaluation standard (University of Wisconsin-Madison, School of Veterinary Medicine, Calf Health Scoring Chart and Calf Health Scoring Criteria (Non-Patent Document 34)). There were no differences in birth weight and Ig levels between calves in the test and control groups (Table 11). Calf stool samples were collected rectally by gloved hand into sterile cups on days 2, 7, 18, and 30 of age and stored at -20°C, followed by storage at -80°C. The general amounts of Escherichia coli (E. coli), coliform organisms and Enterobacteriaceae in the samples were determined according to ISO standards.

[0097] [Table 12]

[0098] Results: Between days 15 and 65 of life, only one of 16 calves (6.3%) in the test group developed diarrhea, compared with seven of 16 calves (43.8%) in the control group. Additionally, two calves died in the control group. Pediococcus acidilactici (TAK 589) Administration of Coccobest reduced E. coli counts in fecal samples from the test group by 1 log by day 30. Coliform bacteria and Enterobacteriaceae counts were reduced by 1.1 log and 1.3 log, respectively, in fecal samples from the test group by day 30. Concurrently, E. coli counts increased by 0.5 log in fecal samples from the control group by day 30. E. coli and Enterobacteriaceae counts increased by 0.4 log in fecal samples from the control group by day 30.

[0099] Example 4. Vegetable fermentation with TAK 589 Coccobest strain (Experiment 1). the purpose: To test the compatibility of Pediococcus acidilactici TAK 589 Coccobest with vegetables, such as fermented Chinese cabbage, or Brassica oleracea var. capitata f. alba.

[0100] method: The cabbage was grated into thin pieces in a food processor, and then table salt (1.7%) was added and mixed carefully. After that, the cabbage was cultured for 24 hours and then simmered for 10 minutes. 7 Pediococcus acidilactici TAK 589 Coccobest with a final strain density of CFU / g was mixed with cabbage. The cabbage was fermented at 18°C ​​for 9 days. Naturally fermented cabbage was used as a control. The pH level of the cabbage juice was measured on days 2, 5, and 9.

[0101] Results: Addition of Pediococcus acidilactici TAK 589 Coccobest accelerated the fermentation of cabbage. The base pH level of the grated cabbage was 5.60. Compared to natural fermentation, the pH level of the grated cabbage decreased more quickly (Table 12). The faster decrease in pH level helps suppress undesirable microflora in the cabbage, including slime-producing lactic acid bacteria (e.g., Leuconostoc mesenteroides).

[0102] [Table 13]

[0103] In all tests, the fermented cabbage was defect-free, had a sour taste and a chewy texture. Pediococcus acidilactici TAK 589 Coccobest is suitable for use in fermentations as a technical starter culture for carrying out cabbage fermentations.

[0104] Example 5. Fermentation of vegetables with TAK 589 Coccobest strain (Experiment 2). the purpose: To test the suitability of Pediococcus acidilactici TAK 589 Coccobest for the fermentation of outdoor grown vegetables such as cucumber, i.e. Cucumis sativus L.

[0105] method: A solution of 1 tablespoon of table salt and 1 tablespoon of sugar per liter of water was prepared for the fermentation of outdoor-grown Estonian cucumbers. A 24-hour culture of Pediococcus acidilactici TAK 589 Coccobest was added to 10 7 The solution had a final density of CFU / g. Cucumbers (4-5 cucumbers, 400 mL of solution) were fermented at 30°C for 24 hours. At the end of the fermentation, the pH level was measured and the cucumbers were cooled to 4°C.

[0106] Results: At the end of fermentation, the pH level of the naturally fermented cucumbers was 4.34. The pH level of the cucumbers fermented with Pediococcus acidilactici TAK 589 Coccobest was 3.85. Neither the control cucumbers nor the cucumbers fermented with Pediococcus acidilactici TAK 589 Coccobest had any defects. They had a sour taste. The cucumbers fermented with Pediococcus acidilactici TAK 589 Coccobest did not have the bitter aftertaste of the naturally fermented cucumbers. Pediococcus acidilactici TAK 589 Coccobest is therefore suitable for use as a technical starter culture for carrying out fermentation when fermenting cucumbers.

Claims

1. Isolated probiotic microorganism Pediococcus acidilactici TAK 589 Coccobest DSM 32372.

2. The microorganism of claim 1 in freeze-dried form.

3. A composition comprising the microorganism of claim 1 or 2.

4. 3. Use of a microorganism according to claim 1 or 2 as a probiotic food and drink additive.

5. 10. Use of a microorganism according to claim 1 or 2 as a zootechnical feed additive to prevent bacterial infections of the digestive tract and to prevent and / or alleviate diarrhea in livestock and pets.

6. 3. Use of the microorganism according to any one of claims 1 or 2 as a functional food or drink additive for preventing bacterial infections of the gastrointestinal tract and for preventing and / or reducing oxidative stress in humans.

7. The microorganisms causing bacterial infections include Listeria monocytogenes, Escherichia coli, Salmonella Enteritidis, Salmonella Enterica serovar Typhimurium, Shigella sonnei, Staphylococcus aureus, Enterococcus faecalis, and Cronobacter sakazakii.

7. Use of a microorganism according to claim 5 or 6, wherein the microorganism is a microorganism according to claim 1 or 2.

8. 3. Use of a microorganism according to claim 1 or 2 as a technical starter culture for the fermentation of vegetables.

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