Bifidobacterium longum strain biocc1719 DSM 34239 and its use as food supplement, beverage additive and for functional food

Abstract

A probiotic bacterium BIOCC1719 DSM 34239 is used in food supplements, beverage additives and for production of functional food. The use of the bacterium allows to reduce oxidative stress, prevent bacterial infections caused by Listeria monocytogenes, Pseudomonas aeruginosa, Cronobacter sakazakii, Staphylococcus epidermidis and Bacillus cereus, and increase the relative abundunce of genera Lactobacillus, Biofidobacterium, Faecalibacterium, Roseburia and Ruminococcus in gastrointestinal tract. Since the strain produces bioactive compounds with health promoting properties, e.g. fatty acids, essential amino acids, non-proteinogenic amino sulfonic acid, group B vitamins and acylcarnitines, it is possible to use the compositions comprising said strain to enrich the diet. The bacteria is also used as a postbiotic ingredient in food supplements, beverage additives and for production of functional food. For preparation of postbiotic, the viable cells of strain were inactivated. The functional food products, beverages and food supplements can contain the bacterium in a live form, in an inanimate form or both.

Description

[0001] BIFIDOBACTERIUM LONGUM STRAIN BIOCC1719 DSM 34239 AND ITS USE AS FOOD SUPPLEMENT, BEVERAGE ADDITIVE AND FOR FUNCTIONAL FOOD

[0002] TECHNICAL FIELD

[0003] The present invention belongs to the field of biotechnology, food industry and medicine. The probiotic bacterium of the invention relates to food supplements, beverage additives and functional food for prevention of bacterial infections and reduction of oxidative stress, and for use as a postbiotic for enrichment of diet.

[0004] BACKGROUND ART

[0005] Western lifestyle and refined high-fat, high-sugar diet has led to altering the composition of the host microbiota, which in turn has led to deficiency of certain micronutrients and adverse effect also on systemic human health. The use and movement of micronutrients in the intestine is bidirectional, as balanced gut microbes use dietary micronutrients for their growth and in turn supply the host with various metabolites of microbial origin, which are essential for human health (Hadadi, N., Berweiler, V., Wang, H., & Trajkovski, M. (2021). Intestinal microbiota as a route for micronutrient bioavailability. Current Opinion in Endocrine and Metabolic Research, 20, 100285. doi:10.1016 / j.coemr.202L 100285). Many various microbial metabolites have been well-characterised mostly in preclinical studies (in vitro, ex vivo, animal model) regarding their health-promoting properties (A. Sarkar, S. Mandal (2016). Bifidobacteria - Insight into clinical outcomes and mechanisms of its probiotic action. Microbiological Research 192 159-171; Plaza- Diaz J., Ruiz-Ojeda F. J., Gil-Campos M., Gil A. Mechanisms of Action of Probiotics. Adv Nutr 2019;10:S49-S66.; Latif A, Shehzad A, Niazi S, Zahid A, Ashraf W, Iqbal MW, Rehman A, Riaz T, Aadil RM, Khan IM, Ozogul F, Rocha JM, Esatbeyoglu T and Korma SA (2023) Probiotics: mechanism of action, health benefits and their application in food industries. Front. Microbiol. 14:1216674. doi: 10.3389 / fmicb.2023.1216674).

[0006] Bifidobacteria are commensals of the gastrointestinal tract (GIT) of various a living being. They quickly colonize the infant intestine during the first weeks of human life, reaching the highest proportion in the colon during the first 12 months after birth. Bifidobacterium longum is one of the earliest and one of the most successful colonizers of the gastrointestinal tract of infants (Schell, M. A.; Karmirantzou, M.; Snel, B.; Vilanova, D.; Berger, B.; Pessi, G.; Zwahlen, M. -C.; Desiere, F.; Bork, P.; Delley, M.; Pridmore, R. D.; Arigoni, F. (2002). "The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract". Proceedings of the National Academy of Sciences. 99 (22): 14422-14427. doi: 10.1073 / pnas.212527599; Arboleya S, Bottacini F, O’ Connell -Mother way M et al (2018) Gene-trait matching across the Bifidobacterium longum pan-genome reveals considerable diversity in carbohydrate catabolism among human infant strains. BMC Genomics 19:33. https: / / doi.org / 10.1186 / S12864-017-4388- 9).

[0007] The abundance of bifidobacteria within the human gut decreases with age. Although bifidobacteria represent only 3-6% of the adult gastrointestinal microbiota, they confer several positive health benefits to the human host, contributing gut barrier functions, gut, and immune homeostasis, protecting against pathogens by competitive exclusion, during carbohydrate fermentation produce organic acids which decrease the luminal pH and serve as co-substrates for butyrate-producing colon bacteria through cross-feeding interactions etc. (Riviere, A., Selak, M., Lantin, D., Leroy, F., & De Vuyst, L. (2016). Bifidobacteria and Butyrate-Producing Colon Bacteria: Importance and Strategies for Their Stimulation in the Human Gut. Frontiers in Microbiology, 7. doi: 10.3389 / fmicb.2016.00979; Nogal, A., Louca, P., Zhang, X., Wells, P.M., Steves, C.J., Spector, T.D., Falchi, M., Valdes, A.V., and Menni, C. (2021). Circulating Levels of the Short-Chain Fatty Acid Acetate Mediate the Effect of the Gut Microbiome on Visceral Fat. Frontiers in Microbiology, 12. doi.org / 10.3389 / fmicb.2021.711359).

[0008] Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill, C., Guarner, F., Reid, G., Gibson, G. R., Merenstein, D. J., Pot, B., et al. (2014). Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat. Rev. Gastroenterol. Hepatol. 11, 506-514).

[0009] Due to their positive health benefits to the human host, strains of different bifidobacteria species, incl. B. longum have most widely used in a live form as probiotics for modulation of gut microbiome and alleviating various conditions like constipation, lactose intolerance, immunomodulation, fighting oral infections, treatment of diarrhoeas of different ethology, enteritis, etc. (Wong C.B., Odamaki T., Xiao J. (2019) Beneficial effects of Bifidobacterium longum subsp. longum BB536 on human health: Modulation of gut microbiome as the principal action. Journal of Functional Foods 54 506-519; Chen J, Chen X and Ho CL (2021) Recent Development of Probiotic Bifidobacteria for Treating Human Diseases. Front. Bioeng. Biotechnol. 9:770248. doi: 10.3389 / fbioe.2021.770248).

[0010] Gut microbiota diversity, especially the diversity of lactic acid bacteria (such as species of bifidobacteria and lactobacilli, also used as probiotics) are essential for producing and promoting the production of different bioactive molecules e.g., modulating the bile acid pool, crucial for lipid metabolism, immune system and for also modulating the shape of gut microbiota composition. However, not all mechanisms nor health benefits for a host are related to viable bacteria. Non- viable bacteria, their cell components, and low-molecular-weight bioactive compounds (e.g., amino acid derivatives, short chatty fatty acids etc.), produced during fermentation also have an impact on human health.

[0011] According to the International Scientific Association for Probiotics and Prebiotics (ISAPP), a preparation of inanimate microorganisms and / or their components conferring a health benefit on the (human or animal) host is defined by the term "postbiotic“ (Salminen, S., Collado, M.C., Endo, A., Hill, C., Lebeer, S., Quigley, E.M.M., Sanders, M.E., Shamir, R., Swann, J.R., Szajewska, H., et al. (2021). The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol., 18, 649-667). According to ISAPP, ‘inanimate’ means, that live microorganisms were present but have now been killed (inactivated).

[0012] As on some occasions, applying an inanimate microbial culture and / or its bioactive metabolites into functional food product / supplement have technological (e.g., longer product shelf life, no viability loss during the manufacture and shelf life) or consumer-related (e.g., lesser subjective side-effects like bloating and flatulence, more favourable absorption of bioactive molecules etc) advantages over products containing live microbes, research and development of postbiotic products is of rising interest Aguilar-Toala J.E., Garcia- Varelab R., Garciac H.S., Mata-Harod V., Gonzalez-Cordova A.F., Vallejo-Cordoba B., Hernandez-Mendoza A. (2018) Postbiotics: An evolving term within the functional foods field Trends in Food Science & Technology 75 105- 114).

[0013] Postbiotic formulation must contain inactivated microbial cells or cell components, with or without its metabolites / functional bioactive compounds, generated in a food matrix during fermentation prior to inactivation of the microbe.

[0014] The spectrum of metabolites produced by the microbe into the specific growth medium / food matrix depends on the strain- specific properties of the microbe and the growth substrate characteristics. The formation or de novo synthesis specific bioactive compounds can be influenced by supplementing the medium with specific precursors or combining the activities of different microbes.

[0015] Postbiotics can be differentiated by their composition (e.g., butyrate, propionate, vitamins / cofactors, organic acids etc.) or by their physiological functions (immunomodulation, antiinflammatory, hypocholesterolemic, anti-hypertensive, antioxidative effects etc) (Aguilar-Toala J.E., Garcia- Varelab R., Garciac H.S., Mata-Harod V., Gonzalez-Cordova A.F., Vallejo-Cordoba B., Hernandez-Mendoza A. (2018) Postbiotics: An evolving term within the functional foods field Trends in Food Science & Technology 75 105-114). The positive effect on the host can be local and / or systemic. The postbiotic composition or composition containing bioactive molecules can be developed for targeting one or more health aspect. For example, a in the patent application WO2023111270A1 of Biopolis S.L. is disclosed a postbiotic pharmaceutical or nutritional composition (in liquid form or in solid form) comprising non-viable strains Bifidobacterium longum CECT 7347 and Lactobacillus rhamnosus CECT 8361 for the use in the treatment and / or prevention of anxiety disorders. The Hyproca Nutrition Co Ltd and Jinlac Biotech Co Ltd patent application CN115161348A describes a composition comprising fermentate by strains of Bifidobacterium longum subsp. infantis BLL02 Lactobacillus salivarius AP-32, L. plantarum LPL28, and L. acidophilus TYCA06 of milk- or soy-based substrates. The composition is claimed to have several health effects: inhibiting the growth of Escherichia coli, Salmonella, Staphylococcus aureus, and / or Helicobacter pylori, lowering blood sugar, regulating immunity, relieving diarrhoea, and lowering uric acid. The cells of bacteria used for fermentation are removed by centrifugation and discarded, resulting cell-free supernatants (fermentate), which are spray-dried and mixed with other components. The composition does not meet the postbiotic criteria, as it contains bioactive molecules without inactivated microbial cells or cell components.

[0016] Also, probiotic and postbiotic combinations could be developed. For example, Nestle SA patent applications W02023021141A1 describes a developed dried fermentate i.e., supernatant or postbiotic for oral application in the form of a supplement or a nutritional composition (capsule or a tablet), prepared using different strains of Bifidobacterium lactis. The product can be used as a bifidogenic factor (i.e., for enhancing the growth of bifidobacteria in the gastrointestinal tract of the consumer) or combined with one or more probiotics or prebiotics (dietary fibre). The composition is meant for subjects at risk of a gastrointestinal disease (like antibiotic-associated diarrhoea, Helicobacter pylori infection, an inflammatory bowel disease, irritable bowel syndrome, lactose intolerance, chemotherapy-induced diarrhoea, necrotizing enterocolitis etc.) and / or the subject has a low abundance of bifidobacteria in their gastrointestinal tract and / or faeces. The patent application CN114259058A of Hangzhou Yi Xun Technology Co., Ltd. describes a composition with the function of slow carbon and fat reduction, comprising viable probiotic bacteria, heat-inactivated bifidobateria Bifidobacterium longum, Bifidobacterium lactis, Lactobacillus plantarum S58 prebiotics, polydextrose, trehalose, and dietary fibre .

[0017] Intraspecific genomic diversity gives the species B. longum a competitive advantage and persistence in the host gut microbiome and at the same time, ensures a large stock of strainspecific characteristics within this species, which in turn gives the opportunity to discover novel strains with previously undescribed pleiotropic health effects. DISCLOSURE OF THE INVENTION

[0018] The invention relates to the isolated probiotic microorganism Bifidobacterium longum strain BIOCC1719 DSM 34239, its lyophilized (freeze-dried), spray-dried and inanimate forms, and use of aforesaid strain and compositions comprising said strain for use as a food supplement, beverage additive and for production of functional food.

[0019] The probiotic strain microorganism Bifidobacterium longum BIOCC1719 DSM 34239 and the compositions comprising freeze-dried or spray-dried aforementioned microorganism is used in order to improve mammalian's microbiota composition by increasing relative abundance of lactic-, acetic- and / or butyric acid producing genera Lactobacillus, Bifidobacterium, Faecalibacterium, Ruminococcus and Roseburia in mammalian gastrointestinal tract, thus increasing butyric acid bioavailability for colon epithelial cells, enhancing the health status of colonocytes and integrity of the intestinal barrier.

[0020] Based on investigations into antimicrobial properties, Bifidobacterium longum BIOCC1719 DSM 34239 and the compositions comprising said strain inhibit the growth and action of enteropathogens, thus preventing bacterial infections of mammalian gastrointestinal tract. Said enteropathogens are selected from the group comprising Listeria monocytogenes, Pseudomonas aeruginosa, Cronobacter sakazakii, Staphylococcus epidermidis and Bacillus cereus.

[0021] Another object of the invention is the the microorganism Bifidobacterium longum strain BIOCC1719 DSM 34239 for use for preventing and / or reducing oxidative stress and low-grade inflammation in humans. The microorganism Bifidobacterium longum BIOCC1719 DSM 34239 possesses antioxidative activity and increases in serum the concentration of a sulphur-containing cysteine, a precursor to the antioxidant glutathione, and increases the concentration of antiinflammatory polyamine spermidine in blood.

[0022] The next object of the invention is the microorganism Bifidobacterium longum strain BIOCC1719 DSM 34239 as well as composition comprising said strain for use as a postbiotic ingredient (i.e., in inanimate non-viable / inactivated form) for use as a food supplement, beverage additive and for production of functional food. Another object of invention is a composition comprising freeze-dried or spray-dried probiotic microorganism Bifidobacterium longum strain BIOCC1719 DSM 34239 for use in food supplement, beverage additive or for production of functional food for enrichment of diet with bioactive compounds produced by the said strain: fatty acid, short chain fatty acids, medium chain fatty acids, dicarboxylic acid, organic acid, essential amino acids, non-proteinogenic amino sulfonic acid, group B vitamins and short and long chain acylcamitines. Said fatty acid is docosahexaenoic acid, said short chain fatty acids are lactic acid, succinic acid, acetic acid and butyric acid), said medium chain fatty acids are caproic acid and caprylic acid, said dicarboxylic acid is succinic acid, said organic acid lactic acid, said essential amino acids are methionine, threonine, histidine, lysine, valine, leucine, phenylalanine, and tryptophan, said non-proteinogenic amino sulfonic acid is taurine, said group B vitamins are B l, B2, B6, B7, B 12, said short chain acylcarnitines are C3-0H (Hydroxypropionylcarnitine), C3:l (propenoylcarnitine), C4:l (butenylcamitine), C5-M-DC (methylglutarylcarnitine), C5-DC (C6-0H) (glutarylcarnitine, (hydroxyhexanoylcamitine)), said long chain acylcarnitines are C14 (tetradecanoylcarnitine), C14:2 (tetradecadienoylcarnitine), C16-0H (Hydroxyhexadecanoylcamitine), C16:l (hexadecenoylcarnitine), C18 (octadecanoylcamitine) .

[0023] DESCRIPTION OF THE STRAIN

[0024] The microorganism strain Bifidobacterium longum BIOCC1719 DSM 34239 was isolated from a stool sample of a 2-month-old full-term breastfed child. The strain was identified by MALDI- TOFF MS (Bruker MicroFlex Biotyper, USA) as Bifidobacterium longum ssp. longum. Identification was confirmed by pulsed-field gel electrophoresis (PFGE) analysis in comparison with type strain.

[0025] Bifidobacterium longum BIOCC1719 was deposited in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under number DSM 34239 on April 12, 2022.

[0026] PHYSIOLOGICAL-BIOCHEMICAL CHARACTERISTICS

[0027] Bifidobacterium longum BIOCC1719 DSM 34239 is a Gram-positive, catalase and oxidase negative, pleomorphic rod-shaped bacterium. After 48 hours of cultivation on TPY (tryptic soy) agar (Biolife, Italy) anaerobically (5 / 5 / 90 CO2 / H2 / N2), the colonies of Bifidobacterium longum BIOCC1719 DSM 34239 are 1.5-2 mm in diameter, creamy-white, convex, shiny, with a regular border. The optimum growth temperature of the strain is 37 °C. The optimum pH is 6.5.

[0028] According to the API (Analytical Profile Index) 50 CHE System (bioMerieux, France) system, Bifidobacterium longum BIOCC1719 DSM 34239 utilises L-arabinose, D-xylose, D-galactose, D-glycose, D-fructose, D-maltose, D-lactose, D-melibiose, D-saccharose, D-melezitose, D- raffinose, to a lesser extent D-mannose, D-turanose. Based on API ZYM test kit, the strain has esterase, esterase lipase, leucine arylamidase, acid phosphatase peptidase, Naptolas-BI- phosphatse, alfa-galactosidase, beta-galactosidase, alfa-glucosidase, cystine arylamidase, N- acetyl-beta-glucosaminidase, alfa-mannosidase activities. Anitibotic resistance profile

[0029] Antibiotic susceptibility of Bifidobacterium longum BIOCC1719 DSM 34239 was analysed according to standard ISO 10932 s by the microtiter plate method (Sensititre™ EULAC BI 1, EULAC BI 2, Thermo Ficher Scientific, USA). The minimum inhibitory concentrations (MIC) were compared with the epidemiological breakpoints recommended by the European Food Safety Authority (EFSA). A microorganism strain is defined as susceptible when it is inhibited at a concentration of a specific antimicrobial equal or lower than the established cut-off value (S < x mg / L). A microorganism strain is defined as resistant when it is not inhibited at a concentration of a specific antimicrobial higher than the established cut-off value (R > x mg / L). The strain Bifidobacterium longum BIOCC1719 DSM 34239 showed no resistance to the tested antibiotics (Table 1).

[0030] Table 1. Cut-off values (mg / L) of antibiotics for Bifidobacterium longum BIOCC1719 DSM

[0031] 34239 a Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal 2018; 16(3):5206, 24 pp. https: / / doi. org / 10.2903 / j.efsa.2018.5206 [retrieved on 24.November 2023].

[0032] FUNCTIONAL PROPERTIES OF STRAIN

[0033] Antimicrobial properties

[0034] The antimicrobial properties of metabolites, produced by the strain Bifidobacterium longum BIOCC1719 DSM 34239 were evaluated by the well-diffusion method on plate count agar (Biolife, Italy) using the cell-free supernatant of 24 hours -old culture of anaerobically (5 / 5 / 90 CO2 / H2 / N2) cultivated strain in TPY broth (Condalab, Spain). Pathogen growth inhibition zone (mm) around the well containing lOOpL of Bifidobacterium longum BIOCC1719 DSM 34239 supernatant was measured.

[0035] Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis and Staph aureus, Bacillus cereus, Cronobacter sakazakii, Salmonella enterica subsp. enterica serovar Enteriditis, Listeria monocytogenes and L. innocua were used as target pathogens.

[0036] The supernatant of Bifidobacterium longum BIOCC1719 DSM 34239 expressed high antagonistic activity (inhibition zone > 6 mm) against P. aeruginosa, Staph, epidermidis, Cronobacter sakazakii and B. cereus, moderate activity (inhibition zone 5.0-5.9 mm) against Listeria monocytogenes and low activity (< 4.9 mm) against E. coli, S. enterica subsp. enterica serovar Enteriditis, L. innocua and Staph, aureus.

[0037] Tolerability of Bifidobacterium longum BIOCC1719 DSM 34239 to simulated gastric juice, bile and pancreatin

[0038] Bifidobacterium longum BIOCC1719 DSM 34239 was grown in MRS broth for 24h at 37 °C anaerobically, harvested cells were washed twice with lx PBS solution, diluted in Ringer's solution, and added to MRS broth, the pH of which was adjusted pH 6.4, pH 3.0 and pH 2.0 with HC1. The suspensions were incubated at 37 °C for two hours. At pH 6.5, Bifidobacterium longum BIOCC1719 DSM 34239 survived without any loss of viability, at both lower pH values the strain suffered average 3-log viability loss. In MRS broth with 0.5% pepsin (pH 3.0), 2-log loss in viability was registered during two hours of testing. Presence of 0.3% or 0.5 or 2.0% bile in MRS broth (pH 6.5) and MRS broth (pH 3) with 0.5% pancreatin had no negligible effect on the survival of Bifidobacterium longum BIOCC1719 DSM 34239.

[0039] Total antioxidative capacity of Bifidobacterium longum BIOCC1719 DSM 34239

[0040] The antioxidative capacity of Bifidobacterium longum BIOCC1719 DSM 34239 lyophilized and frozen cell culture was tested. The lyophilized and frozen cell culture of the strain Bifidobacterium longum BIOCC1719 DSM 34239 were cultured in TPY broth (Biolife, Italy) for 24 hours at 37 °C under anaerobic conditions. The bacterial suspension was centrifuged at 4 °C for 10 minutes at 5000 x g, the supernatant was separated and filter- sterilized using a 0.2 pm filter. Antioxidant capacity in hydrophilic environment was measured using ABTS+( (2,2'- Azinobis-(3-Ethylbenzthiazolin-6-Sulfonic Acid)) radical scavenging assay. The ABTS+ was dissolved in water at a concentration of 7 mmol / L. ABTS radical cation was generated by mixing the ABTS stock solution with (final concentration) of 2.45 mmol / L of potassium persulfate. Subsequently, the ABTS+ solution was diluted with ultrapure water to achieve an absorbance of 0.7 ± 0.02 at 734 nm before analysis. Each measurement was performed at three times. The results were expressed as equivalents of Trolox or TEAC (Trolox Equivalent Antioxidant Capacity).

[0041] The anti-lipid peroxidation activity was measured by using the thiobarbituric acid (TBA) method. The absorbance was determined at 534 nm using a Synergy HTX multimode reader (BioTek Instruments, USA). The inhibition rate was calculated by using the following equation: [1- (A sample) / (A 534control)] x 100. Isotonic saline was used in the control samples. A higher numerical value (%) of TAA indicates higher antioxidative activity as described by Re et al. in 1999 (Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine 26 (9), 1231-1237).

[0042] Table 2. Antioxidative capacity of Bifidobacterium longum BIOCC1719 DSM 34239

[0043] The strain Bifidobacterium longum strain BIOCC1719 DSM 34239 possesses antioxidative capacity in hydrophilic and lipophilic environments (Table 2). The freeze-drying process did not affect the antioxidative capacity of the strain.

[0044] Glutathione concentrations in Bifidobacterium longum BIOCC1719 DSM 34239

[0045] Cells of Bifidobacterium longum strain BIOCC1719 DSM 34239, cultivated in TPY broth anaerobically for 24 h (cell count 8xl06CFU / mL) and 48 h (cell count 9.3xl07CFU / mL) were harvested by centrifugation and supernatant removed as described in Songisepp et al. (Songisepp E., Stsepetova J., Ratsep M., Kuus L., Piir A., Kilk K., Mikelsaar M. 2022 Polyfunctional metabolic properties of the human strain Lactiplantibacillus plantarum Inducia (DSM 21379): Experimental and clinical approaches J Functional Foods. 92: 105064). Measurement was carried out using cell lysate treated with 5% 5-sulfo-salicylic acid (Sigma, USA) to eliminate protein from the sample. Glutathione was measured, using a colorimetric detection kit (Invitrogen™ EIAGSHC, Invitrogen, USA). The concentration of oxidized glutathione (GSSG) was read from the standard curve. Reduced glutathione (GSH) concentrations were determined by subtracting GSSG concentration from values obtained from nontreated samples and standards. The amount of GSH (pg / mL) was calculated as the difference between total glutathione (TGSH) and GSSG (TGSH- GSSG = GSH). The glutathione red / ox ratio was expressed as GSSG / GSH. Table 3 Glutathione concentrations (pM / mL) in cell lysate of 24 h and 48 h old culture of

[0046] Bifidobacterium longum BIOCC1719 DSM 34239

[0047] TGSH - total glutathione (oxidized plus reduced form), GSH - reduced glutathione, GSSG - oxidized glutathione, GSSG / GSH - glutathione redox potential.

[0048] Bifidobacterium longum strain BIOCC1719 DSM 34239 can recycle / regenerate glutathione, the main intracellular antioxidant. Reduced glutathione is an important cellular scavenger of hydroxyl radicals and helps to recycle and preserve all other antioxidants.

[0049] Production of organic acids, ethanol, and B-group vitamins by Bifidobacterium longum BIOCC1719 DSM 34239 during whey fermentation

[0050] A suspension from the 24 h old Bifidobacterium longum BIOCC1719 DSM 34239 culture was inoculated in inoculation dose of 3xl05CFU / mL into pasteurized (for 5 min at 72.0 °C) sweet whey and demineralised sweet whey, with or without 0.5g / L betaine hydrochloride (Sigma- Aldrich, Germany), L-threonine 0.2 g / L (Sigma-Aldrich, Germany) or 0.5 g / L betaine hydrochloride and L-threonine 0.2 g / L combination and incubated at 37 °C for 24 h. The experiment was carried in two repetitions. Acetic acid, butyric acid, lactic acid, propanoic acid, isovaleric acid, valeric acid and hexanoic acid) and ethanol were determined by gas chromatograph Agilent 6890A, capillary column CP-Wax 52 CB (30 m x 0.25 mm, 0.25 pm), flame ionization detector at 280 °C. The oven temperature program 175 °C 1 min, increased by 20 °C / min to 190 °C, and held for 5 min. Results were expressed as mg / g sample.

[0051] The analysis of B-group vitamins was done using Shimadzu HPLC system with ACE 3 C18-AR column (Catalogue No: ACE-119-1546). Vitamin B6 was identified and detected using Fluorescence detector. Forms of vitamin B6 were detected and identified at excitation wavelength: 290 nm and emission wavelength of 395 nm. Pyridoxine hydrochloride (98%) (P9755-25G, Sigma- Aldrich), pyridoxal- 5 -phosphate monohydrate (99%) (228170050, Acros Organics), pyridoxal hydrochloride (99%) from (352710050, Acros Organics) and pyridoxamine dihydrochloride (98%) (J62679 Alfa Aesar) were used as etalons. After 24h fermentation with Bifidobacterium longum BIOCC1719 DSM 34239, in demineralised sweet whey similar drop from initial pH 6.8 to pH 4.7 occurred regardless the presence or absence of betaine or betaine and L-threonine combination. The average increase of viable counts of Bifidobacterium longum BIOCC1719 DSM 34239 was 3-logs. The pH of all versions of sweet whey remained after 24h fermentation higher, being an average pH 5.8. A 2-log increase in viable counts of the Bifidobacterium strain was detected.

[0052] In all versions of fermented sweet whey, ethanol concentrations remained below detection limits. During fermentation, acetic acid concentration raised to 0.198 mg / mL in sweet whey without additives. In the presence of betaine, L-threonine, betaine, and L-threonine combination, acetic acid concentration remained lower: from 0.126 mg / mL to 0.160 mg / mL. The same phenomenon was detected in the case of butyric acid, concentration of which in sweet whey without additives after fermentation was 0.011 mg / mL. In the presence of betaine, L-threonine, betaine, and L- threonine combination, the butyric acid was detected, concentration of which remained between 0.005 mg / mL and 0.008 mg / mL.

[0053] In fermented demineralised whey and in fermented demineralised whey with L-threonine, concentrations of ethanol and acetic acid were relatively high, values for ethanol: 0.106 mg / mL and 0.079 mg / mL, respectively. Respective values for acetic acid were 0.968 mg / mL and 0.940 mg / mL.

[0054] In comparison of control i.e., unfermented demineralised whey, fermentation with Bifidobacterium longum BIOCC1719 DSM 34239 doubled the concentration of niacinamide (B3’) (in control: 0.0002 mg / mL vs 0.0004 mg / mL in fermented version).

[0055] Supplementation with L-threonine induced the increased concentration of the major form of vitamin B6 pyridoxine to 0.000074 mg / mL in demineralised sweet whey fermented with Bifidobacterium longum BIOCC1719 DSM 34239 in comparison with unfermented L-threonine supplemented control, where the vitamin remained undetected. Supplementation with betaine and L-threonine combination induced the increase cyano-cobalamin (vitamin B 12) concentration to 0.000036 mg / mL in demineralised sweet whey fermented with Bifidobacterium longum BIOCC1719 DSM 34239 in comparison with unfermented control, treated with betaine and L- threonine combination, where the vitamin remained undetected.

[0056] Fermentation by means of Bifidobacterium longum BIOCC1719 DSM 34239 sweet whey supplemented solely with L-threonine but also with betaine and L-threonine combination enhanced the increase of another structural analogues of vitamin B6, pyridoxal. Pyridoxal concentration increased with L-threonine (0.00068 mg / mL) and with betaine and L-threonine combination (0.00061 mg / mL) supplemented versions, compared with unfermented and accordingly treated whey (respective pyridoxal values of which were O.OOO38 mg / mL and O.OOO33 mg / mL).

[0057] Example 1. Effect of administration of Bifidobacterium longum BIOCC1719 DSM 34239 on gastrointestinal microbiota and blood serum parameters in a mouse model

[0058] The aim of the study was to prove on mice model the biosafety and the probiotic properties of Bifidobacterium longum BIOCC1719 DSM 34239 upon administration in live form in certain daily doses to mammals. The experiment started with 16 mice in test group, and 16 mice on control group. Bifidobacterium longum BIOCC1719 DSM 34239 was administered with drinking water in daily dose of 1010CFU per mouse per day. Control animals received plain water. Half of the mice of both groups were sacrificed on day 15 of the experiment and the remaining 8 on day 30. Mice were weighed and their food consumption was assessed once a week, faecal and blood samples were collected at start and at the end of the trial (i.e., on day 15 or on day 30).

[0059] Changes for behaviour, physical activity, and general health and body weight of mice as well as food and water consumption were observed daily.

[0060] For histological analysis, tissue sections of the liver, spleen, kidneys, and lungs were fixed in 10% of formaldehyde and embedded in paraffin. The microtome-cut tissue samples were stained with hematoxylin and eosine. In the ileum and colon, the count of lymphatic nodes was evaluated.

[0061] DNA from faecal samples was extracted according to the QIAamp DNA Stool Kit protocol (QIAGEN (2010) QIAamp® DNA Stool Handbook https: / / www.qiagen.com / us / resources / download.aspx2id-c8fe97e7 -78cc-4275-bbac-

[0062] V2c9b7c^^^^ [retrieved on 19 December 2022].

[0063] DNA 16S amplicon sequencing was performed by Novogene ( http s : / / www . n ovogene . com / eu -

[0064] 44L6rqIJG0cIDlg-G3hLKiEY4gDg8z.xS NEz()tjTJZzT5bhxjTlCBoC14UQAvD BwE) [retrieved on 08 May 2023]. The V3-V4 region was sequenced with primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) pairs with end reads on the llumina NovaSeq 6000 platform. Sequencing data were processed in the Qiime2 program (version 2021.4).

[0065] Short chain fatty acids (SCFA) from faecal samples were extracted according to a modified procedure of Zhao et al. (Zhao G., Nyman M. and Jonsson A. (2006). Rapid determination of short-chain fatty acids in colonic contents and faeces of humans and rats by acidified waterextraction and direct-injection gas chromatography. Biomed Chromatogr. 20: 674 - 682). SCFA were determined by gas chromatograph Agilent 6890A, capillary column CP-Wax 52 CB (30 m x 0.25 mm, 0.25 pm), flame ionization detector at 280 °C. The oven temperature program 175 °C 1 min, increased by 20°C / min to 190°C, and held for 5 min. Acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, valeric acid, hexanoic acid solutions were used for identification and calibration. Results were expressed as mg / 1 g sample.

[0066] SCFA from blood serum were measured on Xevo TQ-XS mass spectrometry (Waters, MA, USA) coupled to ACQUITY ultra-performance liquid chromatography (Waters, MA, USA). SCFA was derivatized with 2-nitrophenylhydrazine by treating 50 pL sample with 50 pL internal standards (2H4acetic acid and 2H1 lhexanoic acid), 100 pL 200mmol / L 2-nitrophenylhydrazine and 20 pL 120 mmol / L l-(3-dimethylaminopropyl)-3-ethylcarbodiimide. After 1 hour incubation the mixture was centrifuged 10 min at 21,000 xg. SCFA were separated on an ACQUITY Premier BEH Cl 8 Column with VanGuard FIT, 1.7 pm, and 2.1 x 100 mm column using water and acetonitrile with 0.1% formic acid as eluents.

[0067] Targeted metabolic profiling of blood serum of mice was carried out by mass-spectrometry using MxP® Quant 500 kit (biocrates life sciences ag, Austria, https: / / biocrates.com / ) . Biocrates MxP®Quant 500 allows the determination of up to 630 metabolites from 26 biochemical classes from one sample, which enables the analysis of different mechanisms of action and metabolic capacity of bacteria.

[0068] Biosafety of Bifidobacterium longum BIQCC1719 DSM 34239

[0069] The mice stayed in good condition during the trial, no changes in behaviour, characteristics of the fur and digestion were detected. No translocation of bacteria to blood or organs was detected.

[0070] Effect of Bifidobacterium longum BIQCC1719 DSM 34239 on faecal microbiota groups

[0071] The number of anaerobic genera of bifidobacteria and Ruminococcus increased after administration of Bifidobacterium longum BIOCC1719 DSM 34239 for 15 and 30 days in daily dose of 1010CFU with drinking water in the test group (Table 4 and 5). In contrast to the control group, already the short intervention increased the relative abundance of lactic acid producing genus Lactobacillus in the test group (Table 4). Similar effects were also observed after 30 days of administration of Bifidobacterium longum BIOCC1719 DSM 34239 (Table 5). Table 4. Effect of 15-day administration of Bifidobacterium longum BIOCC1719 DSM 34239 on relative abundance of faecal microbiota genera

[0072] *Test group received Bifidobacterium longum BIOCC1719 DSM 34239 with drinking water in daily dose of IO10CFU per mouse per day

[0073] Additionally, administration of Bifidobacterium longum BIOCC1719 DSM 34239 resulted also in an increase in the abundance of butyrate-producing bacteria of the genera Roseburia, Faecalibacterium and Ruminococcaceae, which preferentially colonize the mucus layer, increasing the butyrate bioavailability for colon epithelial cells (Table 4 and 5). At the same time, a decrease in proinflammatory genera Desulfovibrio and Bacteroides was recorded (Table 4 and 5).

[0074] Table 5. Effect of 30-day administration of Bifidobacterium longum BIOCC1719 DSM 34239 on relative abundance of faecal microbiota genera *Test group received Bifidobacterium longum BIOCC1719 DSM 34239 with drinking water in daily dose of IO10CFU per mouse per day Effect of Bifidobacterium longum BIQCC1719 DSM 34239 on serum metabolites and short chain fatty acid content in faces

[0075] In the test group higher faecal acetate level, esp. after 30-day administration of Bifidobacterium longum BIOCC1719 DSM 34239 was recorded in comparison to control group (Table 6 and 7). Higher faecal butyrate levels were recorded in the test group in comparison to control group after 30 days of Bifidobacterium longum BIOCC1719 DSM 34239 consumption (Table 7).

[0076] Table 6. Short chain fatty acid (SCFA) content in faeces (pmol / g) and in blood serum (pmol / L) of mice after 15 days of administration of Bifidobacterium longum BIOCC1719 DSM 34239 *Test group received Bifidobacterium longum BIOCC1719 DSM 34239 with drinking water in daily dose of IO10CFU per mouse per day

[0077] Serum butyric acid levels in test group after 15 days of Bifidobacterium longum BIOCC1719 DSM 34239 consumption were lower, and significantly lower (p = 0.016) in comparison with control group at the end of 30-day feeding, indirectly indicating the better health status of colonocytes and integrity of the intestinal barrier (Table 6 and 7).

[0078] Table 7. Short chain fatty acid (SCFA) content in faeces (pmol / g) and in blood serum (pmol / E) of mice after 30 days of administration of Bifidobacterium longum BIOCC1719 DSM 34239 *Test group received Bifidobacterium longum BIOCC1719 DSM 34239 with drinking water (daily dose IO10CFU / mouse / day)

[0079] An increase in one of primary bile acids, cholic (CA) and one of the secondary bile acids, deoxy cholic (DC A) acid (produced from the CA) was observed in serum of mice after 15 and 30 days of administration of Bifidobacterium longum BIOCC1719 DSM 34239 (Table 8). The increase of DC A is associated with microbial bile salt hydrolase activity of intestinal bacteria, which in turn is one of the main mechanisms of hypocholesterolemic effect due to the more efficient removal of bile acids from the body.

[0080] In comparison with control group, in the test group fed with Bifidobacterium longum BIOCC1719 DSM 34239, lower content of one of the atherosclerosis risk markers, p-cresol-SO4 was detected at both timepoints of the feeding experiment i.e., on day 15 and on day 30) (Table 8). Higher concentration of amino acids cysteine, tyrosine and aspartic acid was observed in serum of mice after 15 and 30 days, arginine after 15-day and tryptophan after 30 days administration of Bifidobacterium longum BIOCC1719 DSM 34239 in comparison with the control group (Table 8). An increase anti-inflammatory polyamine spermidine in serum of mice was registered after 15- and 30-days administration of Bifidobacterium longum BIOCC1719 DSM 34239 (Table 8).

[0081] Table 8 The concentration of metabolites (pmol / L, median (quartile)) in serum of mice on day

[0082] 15 and on day 30 of administration of Bifidobacterium longum BIOCC1719 DSM 34239

[0083] *Test group received Bifidobacterium longum BIOCC1719 DSM 34239 with drinking water in daily dose of IO10CFU per mouse per day.

[0084] When administered in live form, strain Bifidobacterium longum BIOCC1719 DSM 34239 possesses probiotic properties, positively influencing blood parameters and the gastrointestinal microbiota by increasing the relative abundance of bacteria that produce health supporting bioactive compounds.

[0085] Example 2. Bioactive compounds produced by Bifidobacterium longum BIOCC1719 DSM 34239 in different growth media

[0086] A suspension from the 24 h old Bifidobacterium longum BIOCC1719 DSM 34239 culture was inoculated in final inoculation dose 3xl05CFU / mL into TPY broth (a laboratory reference growth medium for bifidobacteria), cow milk (fat content 2.5%) and reconstituted demineralised sweet whey (10% of demineralised whey powder dissolved in water, sterilised by autoclaving at 115 °C, 0.5 atm for 10 min) and incubated microaerobically at 37 °C for 24 h). Targeted metabolic profiling of fermented reconstituted demineralised sweet whey was carried out by mass-spectrometry using MxP® Quant 500 kit (biocrates life sciences ag, Austria, https : / / biocrates .com / ) . B -group vitamins were analysed as described by Yang and Rainville (2021) (Yang J. and Rainville P.D. (2021) Enhancing the LC-MS / MS Analysis of B-group Vitamins with MaxPeak High Performance Surfaces Technology. Waters application notes. https: / / www.waters.com / content / dam / waters / en / a p-notes / 2021 / 720007264 / 7200Q7264-en.pdf. [retrieved on 08 November 20231). Vitamins were separated on a ACQUITY Premier BEH C18 Column with VanGuard FIT, 1.7 pm, 2.1 x 100 mm column using 20mmol / L ammonium formate buffer in water and methanol with 0.1% formic acid.

[0087] SCFA and medium-chain fatty acids (MCFA) were measured on Xevo TQ-XS mass spectrometry (Waters, USA) coupled to ACQUITY ultra-performance liquid chromatography (Waters, USA). SCFA and MCFA were derivatized with 2-nitrophenylhydrazine by treating 50 pF sample with 50 pF internal standards (2H4acetic acid and 2Hl lhexanoic acid); 100 pF 200 mmol / U 2-nitrophenylhydrazine and 20 pF 120 mmol / U l-(3-dimethylaminopropyl)-3- ethylcarbodiimide. After 1 hour incubation the mixture was centrifuged 10 min at 21.000 xg and MCFA were separated on a ACQUITY Premier BEH C18 Column with VanGuard FIT 1.7 pm; 2.1 x 100 mm column using water and acetonitrile with 0.1% formic acid as eluents.

[0088] Sterile TPY broth, unfermented milk and whey served as controls.

[0089] The baseline concentrations of B vitamins in unfermented sterile TPY, CM and RDSW were considered 100%. To calculate effect of Bifidobacterium longum BIOCC1719 DSM 34239, the change in the concentration achieved after 24 h- fermentation the was calculated from the baseline.

[0090] The 24 h cultivation of Bifidobacterium longum BIOCC1719 DSM 34239 in TPY broth, increased the concentrations of thiamine pyrophosphate (vitamin B 1 co-enzyme) for 0.028 mg / E, i.e., 313.37% and vitamin B12 for 148.651 mg / L, i.e., 10.03% in comparison with sterile control (Table 9).

[0091] In the milk the highest increase (at least 20% compared to control i.e., sterile milk) was detected in vitamins B l, B2, B7, and forms of vitamin B6 pyridoxine and pyridoxamine concentrations, indicating that fermented with Bifidobacterium longum BIOCC1719 DSM 34239 milk products could be a good source for dietary B vitamins. In fermented with Bifidobacterium longum BIOCC1719 DSM 34239 reconstituted whey an 744% increase in cyanocobalamin concentration compared to control. Also, an increase in concentration of riboflavin (B2) 40.057 mg / L (42.09 %) and pyridoxal (B6 vitamin) 1.107 mg / L (25.28 %) was detected in comparison with control. Whey fermented with strain Bifidobacterium longum BIOCC1719 could be used as an additive in various food products in to increase the concentration of B vitamins like cyanocobalamin (B12), riboflavin (B2) and pyridoxal (B6). Table 9. Concentrations (mg / L) of B -group vitamins, or co-enzyme in TYP broth, cow milk and reconstituted demineralised sweet whey after 24 h cultivation of Bifidobacterium longum BIOCC1719 DSM 34239 and in uncultivated controls. Fermentation with Bifidobacterium longum BIOCC1719 DSM 34239 increased in TPY broth and milk the amount of essential amino acids leucine, and phenylalanine and tryptophan in TPY broth (Table 10). Reconstituted demineralised sweet whey fermentation with Bifidobacterium longum BIOCC1719 DSM 34239 increased histidine, lysine, and threonine concentrations. Cultivating Bifidobacterium longum BIOCC1719 DSM 34239 increased methionine in TPY broth, milk, and reconstituted demineralised sweet whey (Table 10). Tabel 10. Concentration of essential amino acids in TPY broth, milk and reconstituted demineralised sweet whey and after 24 h of fermentation (absolute concentrations, |amol / L).

[0092] * Sterile TPY broth; **Unfermented milk; *** Unfermented reconstituted sweet whey; His -

[0093] Histidine; Leu - Leucine; Lys - Lysine; Met - Methionine; Thr - Threonine; Phe - Phenylalanine; Trp - Tryptophan; Vai - Valine.

[0094] Cultivating Bifidobacterium longum BIOCC1719 DSM 34239 in TPY broth for 24 h increased the amount of acetic acid (a SCFA with antimicrobial and anti-inflammatory properties) for 442% from the initial 17.57 pmol / mL) and non-proteinogenic amino sulfonic acid taurine for 45% % from the initial 1.425 pmol / mL in control).

[0095] Also, Bifidobacterium longum BIOCC1719 DSM 34239 increased amounts of DHA (docosahexaenoic acid, 22:6co3, a very long omega-3 polyunsaturated fatty acid (PUFA), with cardiovascular health supporting properties) in comparison with respective controls in all tested media, with the respective increase in TPY was 30%, to 0.773 pmol / mL from 0.5945 pmol / mL in control, lower in milk (21% from 1.47pmol / mL in control to 1.77 pmol / mL) and in reconstituted demineralised sweet whey 17%, from 0.96pmol / mL in control to 1.12 pmol / mL).

[0096] In TPY broth, Bifidobacterium longum BIOCC1719 DSM 34239 utilised bile acids for growth. In milk, an increase in cholic acid (CA) for 52% from 0.123 pmol / mL to 0.187 pmol / mL and deoxycholic acid (DCA) for 141% from 0.123 pmol / mL in control to 0.053 pmol / mL was detected. DCA increased (122%) also in reconstituted demineralised sweet whey from 0.0185 pmol / mL in control to 0.041 pmol / mL. The DCA is the end-product of an enzymatic process, carried out by the salt hydrolase (bsh) activity. Results imply the bile salt hydrolase activity of the strain BBIOCC1719. The clinical efficacy for humans of probiotic bacteria possessing bsh activity i.e., deconjugation of bile acids lies in lowering cholesterol levels by decreasing cholesterol absorption by enterocytes and enhancing its faecal excretion (Begley, M; Hill, C; Gahan, Cormac G. M. (2006). "Bile Salt Hydrolase Activity in Probiotics". Applied and Environmental Microbiology. 72 (3): 1729-1738; Bourgin, M.; Kriaa, A.; Mkaouar, H.; Mariaule, V.; Jablaoui, A.; Maguin, E.; Rhimi, M. (2021) Bile Salt Hydrolases: At the Crossroads of Microbiota and Human Health. Microorganisms 9, 1122).

[0097] Fermentation with Bifidobacterium longum BIOCC1719 DSM 34239 increased amount of medium chain fatty acids, caproic acid (34% from inital 0.004 pmol / mL) in TPY and, also a slight increase was registered in milk. Caprylic acid increased in milk for 42% from inital concentration of 0.031 pmol / mL in sterile milk. Both are digestion improving, freely absorbable nutrients, a quick energy source for all tissue cells. Caprylic acid has also antibacterial, antiviral, antifungal, anti-inflammatory properties linked to prevention of genitourinary tract infections (incl. UTI, bladder, Candida) and oral infections like gingivitis.

[0098] The highest increase (1408%) was detected in lactic acid in TYP broth from initial 366718 pmol / mL to 53631 pmol / mL, 383% in milk from 348.5 pmol / mL to 1335 pmol / mL and 129% in reconstituted demineralised sweet whey from 818.5 pmol / mL to 1055 pmol / mL. Succinic acid was also produced by BL1719 into all growth media during growth, increasing the initial amount 503.5 pmol / mL for 282% to 1419.9 pmol / mL in TYP, in milk the increase was 38% from 126.5 pmol / mL to 48 pmol / mL and 64% in reconstituted demineralised sweet whey from 30.5 pmol / mL to 19.58 pmol / mL.

[0099] Acylcarnitines are fatty acid metabolites, essential for many cellular energy metabolism pathways. Culturing BLBIOCC1719 in the three tested media increase in the concentrations of 15 short chain acylcamitines (SCAC; C2-C5) was detected. The threshold determined for TPY broth was exceeded for C5-DC (C6-OH) (Glutarylcarnitine (Hydroxyhexanoylcarnitine), where the increase was from 0.109 pmol / L to 0.153 pmol / L, increase 41%).

[0100] In fermented milk, concentrations of nine SCAC increased: C4:l (butenylcarnitine, from control 0.004 pmol / L to 0.014 pmol / L, increase 300%), C5-M-DC (methylglutarylcarnitine, from 0.001 pmol / L to 0.007 pmol / L, increase 600%) and increase 28% for C5-OH (C3-DC-M) (hydroxyvalerylcarnitine (methylmalonylcarnitine), from control 0.124 pmol / L to 0.158 pmol / L).

[0101] In reconstituted demineralised sweet whey, remarkably high increase (819%) was detected after fermentation with Bifidobacterium longum BIOCC1719 DSM 34239 in C3-OH (3-Hydroxy- propionylcamitine, from control 0.019 pmol / L to 0.170 pmol / L in fermented reconstituted demineralised sweet whey. Additionally, concentrations of C5:1-DC (glutaconylcarnitine) increased from 0.034 pmol / L in control to 0.067 pmol / L, increase 100%, C3:l (Propenoylcarnitine, from 0.044 pmol / L in control to 0.074 pmol / L, increase 70%) and C5-M- DC (glutaconylcamitine, from 0.054 pmol / L in control to 0.074 pmol / L, increase 37%).

[0102] In long chain acylcarnitine (LCAC, C14-C18) group, Bifidobacterium longum BIOCC1719 DSM 34239 increased in the three tested media the concentrations of 9 LCAC. For TPY broth the threshold was exceeded for C14:2 (tetradecadienoylcarnitine, increase 33.0%, the increase was from 0.021 pmol / L to 0.028 pmol / L) and for C18 (octadecanoylcarnitine, increase 64%, from 0.011 pmol / L to 0.018 pmol / L).

[0103] In fermented milk, concentrations with Bifidobacterium longum BIOCC1719 DSM 34239, two LCAC increased: C14 (tetradecanoylcarnitine, increase 50%, from 0.032 pmol / L to 0.048 pmol / L) and for C16-OH (hydroxyhexadecanoylcamitine, increase 88%, from 0.008 pmol / L to 0.015 pmol / L). In reconstituted demineralised sweet whey, increase was detected after fermentation in C14:2 (tetradecadienoylcarnitine, increase 38%, from 0.023 pmol / L to 0.031 pmol / L) and in C16:l (hexadecenoylcarnitine, increase 33% from 0.009 pmol / L to 0.012 pmol / L).

[0104] Both groups of acylcarnities, SCAC (C2-C5) and LCAC (C14-C18) have been shown to possess positive effect on human metabolic health (M. Dambrova, Makrecka-Kuka M., Kuka J., Vilskersts R., Nordberg D., Attwood M. M., Smesny S., Sen Z.D., Guo A. C., Oler E., Tian S., Zheng J., Wishart D. S., Liepinsh E., Schibth H.B. (2022). Acylcarnitines: Nomenclature, Biomarkers, Therapeutic Potential, Drug Targets, and Clinical Trials. Pharmacological Reviews 74: 506-551).

[0105] During the growth in TPY broth, milk and reconstituted sweet whey, Bifidobacterium longum BIOCC1719 DSM 34239 produces large rage of bioactive compounds with health promoting properties.

[0106] Example 3. Semi-industrial scale preparation of fermented whey using Bifidobacterium longum BIOCC1719 DSM 34239.

[0107] A suspension from the 24 h old Bifidobacterium longum BIOCC1719 DSM 34239 culture was inoculated in final inoculation dose 3xl05CFU / mL into pasteurized (for 5 min at 72.0 °C) sweet whey and pasteurized demineralised sweet whey and incubated at 37 °C for 24 h. After 24h fermentation with Bifidobacterium longum BIOCC1719 DSM 34239, the initial pH of sweet whey decreased from 6.22 ± 0.2 to 5.69 ± 0.13 and pH of demineralised sweet whey decreased from 6.22 ± 0.2 to 4.49 ± 0.30. In both sweet whey and demineralised sweet whey, the viable counts of the Bifidobacterium longum BIOCC1719 DSM 34239 increase for 3-log. Example 4. Survival of the strain Bifidobacterium longum BIOCC1719 DSM 34239 during semi-industrial scale spray drying of fermented whey and fermented demineralised whey. Preparation of postbiotic Bifidobacterium longum BIOCC1719 DSM 34239

[0108] After 24 h the fermentation of sweet whey and demineralised sweet whey comprising the strain Bifidobacterium longum BIOCC1719 DSM 34239 carried out as describe in Example 3, the fermentation was stopped by cooling the whey to +4 °C till spray drying. The fermented whey was warmed to room temperature (approx. +20 °C) and spray dried in semi-industrial scale. Spray drying parameters were as follows: target inlet temperature 180 °C; target outlet temperature 83 °C; material pumping speed 30 %; aspiration (drying air flow rate) 60%; spray air pressure 5.0 bar / 600 L / h and spray nozzle was 0.5mm.

[0109] The yield of whey powder from the spray drying of 1 Liter of fermented whey is approx. 98 g.

[0110] From fermented sweet whey, the resulting powder has a slightly salty-sweet-sour pleasant taste and slightly granular texture. The powder is easily soluble.

[0111] The resulting powder from demineralised fermented sweet whey has a slightly sour-sweet, pleasant taste and a slightly finer texture compared to fermented sweet whey powder. The powder is easily soluble.

[0112] The strain Bifidobacterium longum BIOCC1719 DSM 34239 survived the spray-drying process: in both sweet whey and demineralised sweet whey powders the viable counts of the strain were IxlO5CFU / g.

[0113] For preparation of postbiotic, the viable cells of Bifidobacterium longum BIOCC1719 DSM 34239 were inactivated with pasteurization before spray-dying.

[0114] In order to meet the postbiotic criteria, i.e., the inanimate state of Bifidobacterium longum BIOCC1719 DSM 34239 in the whey powder, the fermented whey is pasteurized in the range of 80-85 °C (preferably 82 °C + 2) for 30-35 minutes (preferably 30 minutes) and cooled down to temperature 20-30 °C.

[0115] Example 5. Functional food products based on the value-added powdered whey, fermented by the strain Bifidobacterium longum BIOCC1719 DSM 34239.

[0116] The functional food product (e.g. value-added powdered whey) can contain the strain Bifidobacterium longum BIOCC1719 DSM 34239 in a live form, in an inanimate form or both. 1. Dry base of functional instant porridge Recipe

[0117] Technology of dry base preparation for functional porridge.

[0118] Dry components are weighed and mixed thoroughly to ensure even distribution of components and packaged in hermetically sealed serving-sized (60.6 g) cups.

[0119] Preparation of porridge ready for consumption:

[0120] Add 200 ml boiling water to the dry basis of the porridge, leave steeping for at least 3 min. Before consumption mix again. Season with desired toppings (berries; nuts; honey; etc).

[0121] 2. Dry base of functional chicory-carob drink

[0122] The drink is suitable as a healthy coffee substitute for adults and as a cocoa drink / hot chocolate substitute for children or for people, who do not like coffee / can’t drink coffee due to health concerns.

[0123] Recipe

[0124] Technology of dry base preparation for functional porridge

[0125] Dry components are weighed and mixed thoroughly to ensure even distribution of components and packaged in hermetically sealed 200 ml bottle.

[0126] Preparation of chicory-carob drink for consumption

[0127] Pour 150 ml of cold or hot milk or water on the base of the chicory-carob drink (if desired, milk and water can be replaced it with herbal drink) and shake the bottle intensively for 10 seconds to ensure proper dissolution of components.

Claims

CLAIMS1. An isolated probiotic microorganism strain Bifidobacterium longum BIOCC1719 DSM 34239.

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

3. The inanimate microorganism of claim 1.

4. The microorganism of any one of claims 1, 2 or 3 for use as a food supplement, beverage additive or for production of functional food.

5. The microorganism of any one of claims 1 or 2 for use as a food supplement, beverage additive or for production of functional food in order to improve composition of microbiota of mammalian by increasing the relative abundance of genera Lactobacillus, Biofidobacterium, Faecalibacterium, Roseburia and Ruminococcus in mammalian gastrointestinal tract.

6. The microorganism of any one of claims 1 or 2 for use as a food supplement, beverage additive or for production of functional food in order to prevent bacterial infections of mammalian gastrointestinal tract.

7. The microorganism according to claim 6, wherein the microorganisms that cause bacterial infections are Listeria monocytogenes, Pseudomonas aeruginosa, Cronobacter sakazakii, Staphylococcus epidermidis and Bacillus cereus.

8. The microorganism of any one of claims 1 or 2 in order to prevent and / or reduce oxidative stress in humans.

9. The microorganism of claim 3 for use as a postbiotic ingredient in food supplement, beverage additive or for production of functional food.

10. A composition comprising the microorganism strain of any one of claim 1 or 2.

11. The composition comprising the microorganism strain of claim 3.

12. The composition of claim 11 for use as a postbiotic ingredient in food supplement, beverage additive or for production of functional food.

13. The composition of claim 10 for use in food supplement, beverage additive or for production of functional food for enrichment of human diet with bioactive compounds produced by the microorganism of claim 1 or 2.

14. The composition of claim 13 where said bioactive compounds belong to fatty acids, short chain fatty acids, medium chain fatty acids, dicarboxylic acids, organic acids, essential amino acids, non-proteinogenic amino sulfonic acid, group B vitamins and acylcarnitines.

15. The composition of claim 14 where the said fatty acid is docosahexaenoic acid, said short chain fatty acids are acetic acid and butyric acid, said medium chain fatty acids are caproic acid and caprylic acid, said dicarboxylic acid is succinic acid, said organic acid is lactic acid, said essential amino acids are methionine, threonine, histidine, lysine, valine, leucine, phenylalanine, and tryptophan, said non-proteinogenic amino acid is taurine, said group B vitamins are Bl, B2, B6, B7, B12, said acylcamitines are short chain acylcamitines (C2-C5) and long chain acylcamitines (C14-C18).

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