Novel Weissella sibaria bacterial strain, compositions containing the same, pharmaceutical compositions for use, nutritional supplements, probiotic preparations, bacterial starter cultures for bread making, sourdough, bread, methods for bread making, methods for microbial production of dextran, bacterial preparations, and uses of this strain.

Abstract

The object of the present invention is a novel Weissella sibaria bacterial strain deposited as KKP 2094p, compositions containing the same, pharmaceutical compositions for pharmaceutical use, nutritional supplements, probiotic preparations, bacterial starter cultures for making bread, sourdough, bread, methods for making bread, methods for microbial production of dextran, bacterial preparations, and uses of this strain in particular for the prevention and / or treatment, preferably for the treatment, of intestinal cancer, preferably colorectal cancer.

Description

Technical Field

[0001] The object of the present invention is a novel strain of Weissella cibaria deposited on February 6, 2023 (06.02.2023) as KKP 2094p in the IAFB collection (KKP collection), i.e., the IAFB-SRI Collection of Industrial Microorganisms of prof. Waclaw Dabrowski (IBPRS-PIB Instytut Biotechnologii Przemyslu Rolno-Spozywczego im. prof. Waclawa Dabrowskiego-Panstwowy Instytut Badawczy w Warszawie, Poland, IAFB-Institute of Agricultural and Food Biotechnology of prof. Waclaw Dabrowski-State Research Institute in Warsaw, Poland, IAFB-SRI) (IAFB, Culture Collection of Industrial Microorganisms, prof. Waclaw Dabrowski, IAFB-SRI). The object of the present invention is also a composition containing Weissella cibaria KKP 2094p; a pharmaceutical composition for use; a dietary supplement; a probiotic preparation; a bacterial starter culture for making bread (manufacturing bakery products); sourdough; bread (bakery); a method for manufacturing bread (bakery); a method for the microbial production of dextran; a bacterial preparation; and in particular, the use of this strain for the prevention and / or treatment, preferably treatment, of intestinal cancer, preferably colorectal cancer.

[0002] The biomass of the Weissella sibaria KKP 2094p strain is used for the biotechnological production of dextran, for sourdough production, and for probiotic applications. This strain was selected for its optimal technical effect in the biotechnological production of dextran, its favorable technical properties in the production of sourdough and sourdough bread, its favorable health-promoting properties for probiotic applications, and its ability to survive under conditions that mimic the gastrointestinal tract.

[0003] The object of the present invention is also the use of the Weissella sibaria KKP 2094p bacterial strain for the production of dextran, for the construction of starter cultures for the manufacture of sourdough and sourdough bread, and as a probiotic strain having desirable properties in the skin and gastrointestinal environment. The Weissella sibaria KKP 2094p strain exhibits high dextran production efficiency, antibacterial and anticancer activity, high resistance to bile salts and low pH, and can be used for the biotechnological production of dextran as a strain with desirable technical properties for the manufacture of sourdough and sourdough bread, as well as as a probiotic strain. [Background technology]

[0004] Bacteria of the genus Weissella were first discovered in fermented sausage in 1993. This genus belongs to the Lactobacillaceae family, which is a well-known group of lactic acid bacteria (LAB) (Collins et al., 1993). Weissella species are Gram-positive and catalase-negative, and their cocci or rod-shaped cells do not form endospores. Weissella strains and species have been isolated from various environments for a long time and have a variety of uses. Fermented food products, such as yogurt, sourdough, and kimchi, are sources from which Weissella bacteria can be isolated (Mun and Chang, 2020; Valerio et al., 2020). Weissella strains play an important role in the formation of flavor and texture in food during fermentation.

[0005] The majority of scientific literature on species of the genus Weissella, particularly Weissella confusa and Weissella sibaria, focuses on the production and characterization of extracellular polysaccharides (EPS) (Hu and Gaenzle, 2018).

[0006] Weissera Confusa and Weissera Sivaria can produce large amounts of dextran. These polysaccharides increase the elasticity of bread, and their use in gluten-free baking appears promising (Wolter et al., 2014). In cosmetic products, they are used as humectants and rheological modifiers (Yildiz and Karatas, 2018). Dextran has a soothing effect and improves skin hydration. It is an emulsion stabilizer. It maintains moisture on the skin surface and prevents cosmetics from drying out.

[0007] EPS produced by LABs has been popular for many years due to their potential functional and technical characteristics (Lynch et al., 2018). The genera Weissella and Leuconostoc have been shown to produce fairly large amounts of EPS in LABs (Kavitake et al., 2016). In particular, Weissella is involved in the production of a considerable amount of dextran (Kim et al., 2008). Weissella hellenica, Weissella confuza, and Weissella sibaria are EPS-producing strains of the Weissella genus, which produce many EPS with different structures and chemical compositions. Weissella bacteria use the extracellular polysaccharide synthesis pathway to produce glucans, dextran, levan, galactans, and fructans, which are homo- or hetero-polysaccharides (Zhou et al., 2018). EPS production is influenced by many sugars, including monosaccharides such as glucose, mannose, galactose, fructose, and oligosaccharides: lactose; saccharose; and raffinose. The unique technical characteristics of LAB EPS allow them to occupy an important position in the food industry as effective emulsifiers, thickeners, stabilizers, or texture improvers (Han et al., 2016; Zhang et al., 2018). Buksa et al. recently demonstrated that 1.5% dextran reduces the formation of resistant starch in starch paste during storage. These studies provide new information on the inhibition of resistant starch formation by the use of EPS, which is efficiently produced in sourdough and can thereby improve the properties of sourdough bread (Buksa et al., 2021).

[0008] EPS also possesses remarkable biological properties resulting from its prebiotic, antioxidant, antiviral, anticancer, and immunomodulatory activity (Badel et al., 2011; Saadat et al., 2019; Xu et al., 2017). To be classified as a prebiotic, non-digestible substances, particularly EPS, must stimulate the growth of probiotic bacteria more than the bacteria present in the gut. Dextran produced by Weissella sivariata was shown to stimulate the growth of Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium animalis, Bifidobacterium bifidum, and Bifidobacterium infantis more than Escherichia coli and Enterobacter aerogenes (Baruah et al., 2016). This activity confirmed the classification of dextran as a prebiotic compound.

[0009] Literature data for various strains of lactic acid bacteria indicate that the amount of EPS produced is usually higher than 1 g / l (Torino et al., 2015; Vasanthakumari et al., 2015), and in some cases, it reaches approximately 10 g / l (Zeidan et al., 2017). The highest dextran production efficiency was recorded at 24.8 g / l for 27 strains of Weissella sibaria isolated from kimchi (Kavitake et al., 2020). A recent analysis of EPS produced by four selected strains of Weissella confusa / sibaria showed a difference in EPS production efficiency ranging from 3.2 g / l to 47.1 g / l, making it possible to determine the type of EPS produced as dextran (Buksa et al., 2021).

[0010] The availability of novel LAB strains with pro-technological properties is of paramount importance to industry. Therefore, researchers are focusing on exploring wild-type strains of natural origin to design novel, industrially interesting starters, supplements, or bioprotective cultures, and probiotics. Recent advances in microbiome research have increased interest in and paved the way for studying the mechanisms of action of probiotics (Tao et al., 2017). In addition to previously identified Lactobacillus bacteria, there are many other non-pathogenic LAB strains with well-known GRAS (Generally Recognized as Safe) status provided by the FDA in the United States or QPS (Qualified Presumption of Safety) status in Europe that can be considered when selecting novel probiotic strains. There is growing evidence that selected strains of the Weissella genus, which play a vital role in many industrial and food fermentation processes, possess probiotic activity. Weissella bacteria are known to inhabit the intestines of vertebrates, including humans (Lee et al., 2012). To date, these bacteria have been found to affect intestinal permeability (Prado et al., 2020), reduce depression (Sandes et al., 2020), regenerate intestinal epithelial cells (Prado et al., 2020), affect metabolism (Elshaghabee et al., 2020), and kill harmful bacteria (Dey and Kang, 2020). Furthermore, it is known that some strains of the genus Weissella have beneficial effects on human health, particularly in maintaining a healthy oral cavity (Kang et al., 2019), treating atopic dermatitis and some cancers (Teixeira et al., 2021), and possessing antitoxic, anticancer, and immunomodulatory activity (Yu et al., 2019a; 2019b; Le et al., 2020). However, these LAB strains were not previously included in the list of species with QPS status issued by the EFSA (European Food Safety Authority). The use of these bacteria as probiotics requires safety analysis (EFSA, 2018).This is the reason for the limited use of this group of LABs in the food industry or as commercially available starter cultures as probiotic strains.

[0011] For microorganisms to be used as probiotics, they must be non-pathogenic and non-toxic, and in particular, they must not possess antibiotic resistance genes on their mobile genetic material. In addition, these microorganisms must possess functional characteristics that have beneficial effects on host health. The ability to survive and thrive in gastrointestinal conditions, especially under high concentrations of bile salts and low pH, as well as the ability to adhere to mucus, extracellular matrix, or intestinal epithelial cells, are the most important characteristics of probiotics that support their activity (Lee et al., 2015). Weissella sivaria can produce several substances with antibacterial activity. A probiotic strain for oral use (Weissela sivaria CMU) has been demonstrated to exhibit antibacterial activity against pathogenic bacteria such as Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, which are involved in the production of organic acids, H2O2, oleic acid, and proteins, such as N-acetylmuraminase (Lim et al., 2018). In other studies, the same strains (Weissella sibaria CMU) and Weissella sibaria CMS1 strain exhibited antibacterial activity and inhibited biofilm formation by pathogenic bacteria that cause upper respiratory tract infections, such as strains of Streptococcus pyogenes, Staphylococcus aureus, Streptococcus pneumoniae, and Moraxella catarrhalis (Yeu et al., 2021). A study by Patrone et al. (2021) showed that the culture supernatants of Weissella sibaria SP7 and SP19 strains inhibited the growth of Escherichia coli ATCC 25,922 and Salmonella enterica UC3605, and that this was associated with low pH.Weissella sivaria strains may not only exhibit antagonistic effects against pathogenic bacteria but also possess an increased ability to effectively reduce the level of fungal infections (Ndagano et al., 2011; Lan et al., 2012).

[0012] Currently, the increasing problem of antibiotic resistance among many bacterial pathogens necessitates novel strategies for detecting and developing antibiotics or other antimicrobial agents as alternatives to conventional antibiotics. Staphylococcus aureus, resistant to many antibiotics, is one of the most common causes of severe hospital-acquired infections, with the gastrointestinal tract being a significant source of transmission (Boyce et al., 2007; Onanuga and Temedie, 2011). At the same time, Staphylococcus is recognized as a pathogen causing antibiotic-associated diarrhea in humans (Boyce and Havill, 2005). Human colonization by conditional bacterial pathogens such as Staphylococcus aureus is a major risk factor for invasive infection. In recent years, there has been an increase in the number of infections caused by both Staphylococcus aureus and Staphylococcus epidermidis, as well as other coagulase-negative Staphylococcus. These bacteria can cause inflammation of the joints, tendons, endocardium, bones, lungs, urinary system, prosthetic joints, heart valves, catheters, food poisoning, and skin infections, such as impetigo, folliculitis, boils, and staphylococcal exfoliative arthritis (SSSS syndrome - staphylococcal scalded skin syndrome), but they can also be secondary skin lesions, such as ulcers (Sokolowska-Wojdylo, 2021). In this context, it is highly desirable to develop preparations based on natural ingredients that maintain skin hygiene by protecting the natural symbiotic microbiota and oral probiotics from colonization by pathogenic bacteria, without disrupting their composition, thereby limiting the development of Staphylococcus in the human gastrointestinal tract and thus minimizing the risk of transmitting multiple antibiotic-resistant pathogens from one of their main reservoirs in the environment.

[0013] From the publication of Polish Patent No. 238153, a starter culture containing strains of Lactobacillus plantarum B / 00117, Lactobacillus plantarum B / 00118, and Lactobacillus brevis is known and described in Set 11, and is used to produce sourdough from whole wheat and rye flour and to bake bread therein. Similarly, a second known starter culture for acidifying rye flour is Lesaffre's bacterial-yeast culture LV2 containing Saccharomyces chevalieri and Lactobacillus brevis (data from Lesaffre Bio-Corporation SA, Lodz). [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] Polish Patent No. 238153 [Non-patent literature]

[0015] [Non-Patent Document 1] Collins et al. (1993). Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for the Leuconostoc paramesenteroides group of species. J Appl Bacteriol, pp. 75:595-603 [Non-Patent Document 2] Mun and Chang(2020).Characterization of Weissella koreensis SK isolated from kimchi fermented at low temperature(around 0 degrees C) based on complete genome sequence and corresponding phenotype.Microorganisms, 8, 1147 [Non-Patent Document 3] Valerio et al. (2020).Effect of Amaranth and Quinoa Flours on exopolysaccharide production and protein profile of liquid sourdough fermented by Weissella cibaria and Lactobacillus plantarum.Front Microbiol 11, page 967 [Non-Patent Document 4] Hu and Gaenzle(2018)Effect of temperature on production of oligosaccharides and dextran by Weissella cibaria 10 M.Int J Food Microbiol 280:27~34 [Non-Patent Document 5] Wolter et al. (2014) Evaluation of exopolysaccharide producing Weissella cibaria MG1 strain for the production of sourdough from various flours.Food Microbiol 37:44~50 [Non-Patent Document 6] Yildiz and Karatas(2018).Microbial exopolysaccharides: Resources and bioactive properties.Process Biochemistry, 72:41~6 [Non-Patent Document 7] Lynch et al. (2018). Lactic acid bacteria exopolysaccharides in foods and beverages: isolation, properties, characterization, and health benefits, Annu Rev Food Sci Technol, 9:155~76 [Non-Patent Document 8] Kavitake, et al. (2016).Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food.International journal of biological macromolecules, 86, pp. 681-689 [Non-Patent Document 9] Kim et al. (2008).Characterization of exopolysaccharide(EPS)produced by Weissella hellenica SKkimchi3 isolated from kimchi.J Microbiol, 46(5):535-41 [Non-Patent Document 10] Zhou et al. (2018) Exopolysaccharides of lactic acid bacteria: structure, bioactivity and associations: a review, Carbohydr Polym, 207: 317-32 [Non-Patent Document 11] Han et al. (2016). Improvement of the texture of yogurt by use of exopolysaccharide producing lactic acid bacteria, BioMed Res Int, page 7945675 [Non-Patent Document 12] Zhang et al. (2018). Characterization of a yogurt-quality improving exopolysaccharide from Streptococcus thermophilus AR333. Food Hydrocoll, 81: 220 - 228 pages

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[0016] In consideration of the state of the art described, the object of the present invention is to overcome the disadvantages shown and to provide a selected novel strain of Lactobacillus weissella that has high-efficiency EPS production, desirable technical properties for sourdough and bread production, and at the same time additional desirable health-promoting properties for probiotic and anti-cancer applications, the ability to survive under conditions mimicking the gastrointestinal tract, and proven safety for use (acceptable level of antibiotic resistance, no antibiotic resistance genes, no toxicity genes). [Means for solving the problem]

[0017] This invention relates to a novel Weissella sibaria bacterial strain deposited with the IAF Collection (KKP Collection) (IAFB Collection of Industrial Microorganisms of prof. Waclaw Dabrowski, State Research Institute in Warsaw, Poland) under accession number KKP 2094p.

[0018] The present invention also relates to a composition containing a novel Weissella sibaria bacterial strain deposited with the IAF Collection (KKP Collection) (IAFB Collection of Industrial Microorganisms of prof. Waclaw Dabrowski, State Research Institute in Warsaw, Poland) under accession number KKP 2094p.

[0019] The composition is preferably a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, and the composition is preferably in the form of a liquid, solid, powder, tablet, or capsule intended for oral administration.

[0020] The present invention further relates to pharmaceutical compositions for pharmaceutical use, comprising the Weissella sibaria bacterial strain and / or the composition of the present invention, as well as a pharmaceutically acceptable carrier.

[0021] Preferably, the pharmaceutical composition for use as a pharmaceutical is for use in the prevention and / or treatment of intestinal cancer, preferably colorectal cancer, and most preferably intestinal / colorectal adenocarcinoma.

[0022] The present invention also relates to a nutritional supplement comprising the Weissella sibaria bacterial strain of the present invention and / or the composition of the present invention, the nutritional supplement preferably in the form of a liquid, solid, powder, tablet, or capsule intended for oral administration.

[0023] The present invention also relates to a probiotic preparation comprising the Weissella sibaria bacterial strain of the present invention and / or a composition of the present invention, wherein the probiotic preparation preferably further comprises a prebiotic substance preferably selected from oligosaccharides, polysaccharides, fructooligosaccharides, lactulose, inulin, indigestible starch, cellulose, hemicellulose and pectin, and the preparation preferably further comprises a postbiotic, wherein the postbiotic is preferably selected from short-chain fatty acids, enzymes, lipopolysaccharides, teicoic acid, vitamins, butyric acid, acetate, propionate, muramyl dipeptide, indole, teicoic acid and lactosepin.

[0024] The probiotic preparation is preferably in the form of a liquid, solid, powder, tablet, or capsule intended for oral administration.

[0025] The present invention also relates to a bacterial starter culture for bread making, comprising the Weissella sibaria strain of the present invention.

[0026] The present invention also relates to a sourdough for making bread, which contains the bacterial starter culture of the present invention.

[0027] The present invention also relates to a method for making bread, comprising the step of adding the bacterial starter culture and / or sourdough of the present invention.

[0028] The present invention also relates to bread containing the bacterial starter culture of the present invention and / or the sourdough of the present invention, or bread produced using them or mixtures thereof.

[0029] The present invention also relates to a method for microbial production of dextran, comprising the step of growing the Weissella sibaria bacterial strain and / or composition of the present invention in a dextrose-free (glucose-free) liquid medium supplemented with an organic nitrogen source containing saccharose, amino acids and short peptides, B vitamins, and inorganic salts.

[0030] In a preferred method, the culture is carried out in MRS medium (DeMan-Rogosa-Sharpe broth) or another medium for lactic acid bacteria, preferably supplemented with 5-10±2% by weight of saccharose.

[0031] The present invention also relates to bacterial preparations comprising the Weissella sibaria strain of the present invention and / or compositions of the present invention for use as an active ingredient in probiotics, therapeutic preparations, functional foods, and dietary supplements, and for use as an active ingredient in drugs intended for the prevention and / or treatment of intestinal cancer, preferably colon cancer.

[0032] The present invention also relates to the use of the Weissella sibaria bacterial strain and / or compositions of the present invention as functional additives in functional foods and functional additives in food and beverages, as well as as active ingredients in probiotic preparations, bacterial preparations, nutritional supplements, and pharmaceutical compositions.

[0033] The present invention also relates to the use of the Weissella sibaria bacterial strain and / or the composition of the present invention in the food industry as an emulsifier, thickener, stabilizer, or texture improver for food products.

[0034] The present invention also relates to the use of the Weissella sibaria bacterial strain and / or the composition of the present invention for the microbial production of extracellular polysaccharides (EPS), preferably dextran.

[0035] The present invention also relates to the use of the Weissella sibaria bacterial strain and / or the composition and / or the bacterial starter culture of the present invention for the production of sourdough for bread.

[0036] The present invention also relates to the use of the Weissella sibaria bacterial strain and / or the composition of the present invention and / or the sourdough of the present invention for making bread (bakery products).

[0037] The Weissella sibaria IBB3394 strain, deposited as KKP 2094p, which is the object of this invention, possesses favorable technical and functional properties and is advantageous over currently known and used strains. Similar to the previously described Weissella confusa / sibaria EPS_3-IBB3326 strain (Buksa et al., 2021), which has high EPS production efficiency, this strain was isolated from bakery sourdough and, based on similar results obtained by the inventors, has the ability to produce EPS (dextran) at a level of 50 g / l (unpublished data). The amount of EPS produced by strains IBB3326 and IBB3394 is higher than that described for other lactic acid bacteria strains, with the exception of Weissella confusa VP30 strain (Jin et al., 2019), which produces dextran at a level of 60 g / l. However, it should be emphasized that the yield of live EPS for strain VP30 was not comparable due to the considerably high content of impurities. The above-described strains Weissella sibaria 27 and Weissella confuza VP30, which have high dextran production efficiency, were isolated from kimchi and children's excrement, respectively, and were unable to adapt to the sourdough environment. Here, the favorable effect of EPS on the quality of sourdough bread has been demonstrated previously. In addition to its ability to produce dextran, the Weissella sibaria IBB3394 (KKP 2094p) strain is also characterized by other demonstrated functional properties (probiotic properties): antibacterial and anticancer activity, exceptionally high resistance to bile salts and low pH compared to other Weissella confuza / sibaria strains isolated from various sources tested by the inventors, and the presence of genes for the vitamin B1 recovery pathway as well as all genes for the vitamin B2 and K2 production pathways. The probiotic Weissella sivariata strains described in the literature do not exhibit such high dextran production efficiency, antimicrobial and anticancer activity, and useful physicochemical properties as the selected strain of Weissella sivariata IBB3394 (KKP 2094p) of the present invention.Furthermore, it should be emphasized that in the case of Weissella sibaria IBB3394 (KKP 2094p) of the present invention, probiotic genome analysis showed antibiotic resistance at a level consistent with the values ​​recommended by EFSA, while confirming the absence of antibiotic resistance genes and toxicity genes. In the tested cell lines: human intestinal epithelium (colorectal adenocarcinoma) Caco-2 (ATCC HTB-37) and human fetal kidney epithelial cells HEK293 (ATTC CRL-1573), the lack of cytotoxicity of extracellular polysaccharides produced by the Weissella sibaria IBB3394 (KKP 2094p) strain was also demonstrated, demonstrating the anticancer properties of Weissella sibaria IBB3394 (KKP 2094p) particularly against intestinal cancer, especially colon cancer.

[0038] The essence of the present invention is a strain of Lactobacillus weissella sibaria IBB3394 (KKP 2094p) characterized by the presence of all genes for selected vitamin production or recovery pathways, high extracellular polysaccharide production efficiency, antibacterial and anticancer activity, excellent acidification properties, and very good survival under conditions that mimic the digestive tract, all of which are proven safe.

[0039] The Weissella sibaria strain (as designated by us, IBB3394, as used herein) was deposited on February 6, 2023, as a deposit for patent purposes, under accession number KKP 2094p, in the IAFB collection (KKP collection) (Culture Collection of Industrial Microorganisms, prof. Waclaw Dabrowski IAFB-SRI), specifically in the IAFB-SRI Collection of Industrial Microorganisms of prof. Waclaw Dabrowski (IBPRS-PIB Instytut Biotechnologii Przemyslu Rolno-Spozywczego im.prof.Waclawa Dabrowskiego-Panstwowy Instytut Badawczy w Warszawie, Polska, IAFB-Institute of Agricultural and Food Biotechnology of prof. Waclaw Dabrowski-State Research Institute in Warsaw, Poland, IAFB-SRI).

[0040] The present invention's Weissella sibaria KKP 2094p, a novel, unknown, and previously undescribed strain of lactic acid bacteria, exhibits desirable technical properties for the production of sourdough and sourdough bread, as well as probiotic and anticancer properties, characterized by exceptionally high dextran production efficiency, possessing antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermides, as well as against undesirable spore-forming bacteria in bread (antagonist activity against Bacillus cereus and Bacillus brevis, and specific activity against Lysynibacillus fusiformis), inhibiting the proliferation of colon cancer cells, being highly resistant to bile salts and low pH, possessing all genes for the vitamin B2 and K2 production pathways as well as the vitamin B1 recovery pathway, and also exhibiting an acceptable level of antibiotic resistance. Probiotic genome analysis of this strain did not detect any antibiotic resistance genes or toxicity genes, confirming its safety.

[0041] The Weissela sibaria KKP 2094p strain was isolated from bakery sourdough.

[0042] The present invention also relates to the use of strains for the bioengineered production of dextran.

[0043] Preferably, the culture medium for Weissella sibaria KKP 2094p must contain saccharose, an organic source of nitrogen containing amino acids and short peptides, vitamin B, and necessary inorganic salts.

[0044] For example, a suitable culture medium for culturing bacteria of the genus Weissella that enable EPS production is commercially available MRS medium (DeMan-Rogosa-Sharpe broth) supplemented with saccharose.

[0045] In a preferred embodiment of the dextran production method, the culture of Weissela sivaria KKP 2094p is carried out in dextrose-free (glucose-free) MRS medium supplemented with saccharose, preferably at a concentration of 10% by weight, to enable maximum efficiency of dextran production.

[0046] The present invention relates to a method for obtaining dextran from the culture of the Weissella sibaria KKP 2094p strain of the present invention.

[0047] Preferably, the resulting culture was diluted with chilled sterile water (1:1, v / v), and the post-culture liquid was separated from the cells by centrifugation (15°C, 4°C, 15 min), after which EPS was extracted from the resulting supernatant. EPS precipitation was carried out in two steps, with 4 volumes of chilled 75% ethanol added at each step. After the first step, the sample was centrifuged, the precipitate was suspended in water, and the second portion of ethanol was added.

[0048] The present invention also relates to the use of the Weissella sibaria KKP 2094p strain due to the favorable technical properties demonstrated by this strain (dextran production, antagonist activity against undesirable spore-forming bacteria in bread) for the production of sourdough and sourdough bread.

[0049] Antagonist activity was tested against undesirable spore-forming bacteria (Bacillus cereus, Bacillus brevis, and Lysinibacillus fusiformis) in the bread.

[0050] The present invention also relates to the use of strains due to their probiotic properties.

[0051] In a preferred embodiment, the Weissella sibaria KKP 2094p strain exhibited antagonist activity against Staphylococcus aureus and Staphylococcus epidermides strains, but did not inhibit the growth of strains corresponding to the lactic acid bacterium Lactococcus lactis (L. lactis).

[0052] The antagonist activity of the overnight culture, cell suspension in PBS, and post-culture supernatant of the Weissella sibaria KKP 2094p strain was demonstrated against Staphylococcus aureus IBB4005 (i.e., the multidrug-resistant Staphylococcus aureus strain NCTC9789[PS80], which exhibits resistance to AMP Pen Asa Cad Mer). The most favorable activity was observed for the overnight culture and cell suspension in PBS.

[0053] The Weissella sibaria KKP 2094p strain has been shown to possess desirable characteristics for a probiotic bacterium: tolerance to low pH, enabling the bacteria to pass into further sections of the gastrointestinal tract; and tolerance to bile salts, ensuring bacterial survival in the presence of pancreatic juice.

[0054] Dextran produced by the Weissella sibaria KKP 2094p strain has been shown to be neither cytotoxic to human Caco-2 cell lines nor non-cancerous renal epithelial cells HEK293, nor to exhibit antagonist activity against tested strains of Staphylococcus aureus and Lactococcus lactis bacteria.

[0055] The Weissella sibaria KKP 2094p strain inhibited the proliferation of colon cancer cells, and its demonstrated anticancer activity was shown to be stronger than that of known lactic acid bacteria strains with confirmed antiproliferative activity (Lactococcus lactis deposited in PCM as B / 00311 and B / 00312) (Polish Patent No. 241568, Salanski et al., 2022).

[0056] The Weissella sibaria KKP 2094p strain was sequenced, and probiotic genome analysis confirmed the absence of antibiotic resistance and toxicity genes, thus confirming its potential for probiotic and medical use.

[0057] Bioinformatics analysis of the Weissela sibaria KKP 2094p strain was performed using recommended programs and databases, in accordance with EFSA recommendations.

[0058] In this application, the term "biomass" should be understood as the biomass of cells of the bacterial Weissella sibaria KKP 2094p strain obtained as a result of cell proliferation in a liquid or solid medium having a composition suitable for the requirements of lactic acid fermentation bacterial culture.

[0059] In this application, the term “potentially probiotic” should be understood, in accordance with available literature data, as a characteristic demonstrated in in vitro studies, with possible beneficial effects on host health, particularly: EPS production, activity against Staphylococcus aureus and Staphylococcus epidermides bacteria, reduction of environmental pH, tolerance to low pH and the presence of bile salts.

[0060] In this application, the term "antimicrobial activity" should be understood as an antagonist effect against Staphylococcus aureus and Staphylococcus epidermides bacteria, as well as against undesirable spore-forming bacteria in bread (Bacillus cereus, Bacillus brevis, and Lysinibacillus fusiformis).

[0061] In this application, the term “confirmed safety” should be understood as the acceptable level of resistance to the nine antibiotics recommended by EFSA: ampicillin (AM), chloramphenicol (CL), clindamycin (CM), kanamycin (KM), gentamicin (GM / CN), streptomycin (SM), tetracycline (TC), vancomycin (VA), and erythromycin (EM), as well as the absence of antibiotic resistance genes and toxicity genes in progenomic analysis performed in accordance with EFSA recommendations using the recommended programs and databases.

[0062] In this application, the term "cytotoxicity" is understood to mean cytotoxicity exceeding 10% and resulting cell survival below 90%, according to the values ​​proposed by EL-Adawi et al. (2012) for EPS produced by lactic acid bacteria.

[0063] In this application, the term “overnight culture” should be understood as a culture of a bacterial strain established from a single colony in a suitable medium, incubated for several hours.

[0064] In this application, the term "supernatant" should be understood as the liquid remaining after culture, separated from bacterial cells.

[0065] In this application, the term “cell extract” should be understood as the liquid obtained after disruption of bacterial cells using glass beads, separated from an overnight culture by centrifugation and suspended in PBS buffer.

[0066] In this application, the term "cell suspension in PBS" should be understood as bacterial cells obtained from an overnight culture by centrifugation, washed, and resuspended in PBS buffer. [Modes for carrying out the invention]

[0067] The Weissella sibaria IBB3394 strain, deposited as KKP 2094p in this invention, was isolated as shown in Example 1 by searching for Weissella strains capable of producing extracellular polysaccharides (EPS) from various environments (plants, fermented plants, fermented dairy products). The Weissella sibaria KKP 2094p strain was identified as Weissella confuza / sibaria by sequencing of its 16S rRNA gene. The species affiliation of the Weissella sibaria KKP 2094p strain was then determined to be Weissella sibaria by genome sequencing and mass spectrometry using a MALDI TOF instrument. The Weissella sibaria KKP 2094p strain was selected from five isolated strains of the Weissella genus and classified into the group of strains with the highest antagonist activity against Staphylococcus aureus, as performed as described in Example 2 (Figure 1), while simultaneously confirming the lack of such activity against representative lactic acid bacteria (Figure 2). Based on the results of the analysis, which showed exceptionally high tolerance to low pH and the presence of bile salts compared to other tested strains under the conditions used in Example 3 to check the survival of the strain under conditions mimicking the gastrointestinal tract (Figure 3), it was compared to strains of this species previously isolated from natural bakery sourdough. Antagonist activity of Weissella sibaria KKP 2094p strain against other Staphylococcus aureus strains and Staphylococcus epidermides strains was confirmed (Example 4). This example also demonstrated the Weissella sibaria KKP 2094p strain's ability to acidify the environment. The potential use of the Weissella sibaria KKP 2094p strain as a probiotic strain with activity against Staphylococcus aureus has been demonstrated and confirmed in Example 5 (Figure 4) for both the strain's overnight culture and cell suspension in PBS. Although at lower levels, this type of activity was observed in the supernatant, indicating that the inhibitory effect against Staphylococcus aureus is not simply caused by lowering the pH of the environment. Furthermore, the absence of antagonist activity in the cell extract case suggests an important, yet-to-be-known, role in keeping the cells of this strain alive.In Example 6, the Weissella sivaria KKP 2094p strain demonstrated an exceptionally strong inhibitory effect on the proliferation of colorectal cancer cells (Figure 5), suggesting the potential for use in the treatment of gastrointestinal cancer.

[0068] The sourizing ability of the Weissella sibaria KKP 2094p strain of the present invention, isolated from bakery sourdough (adapted to live in such an environment) as demonstrated in Example 7 (Bacillus cereus, Bacillus brevis, and Lysinibacillus fusiformis), as well as its antagonist activity against undesirable spore-forming bacteria in sourdough and bread, as observed in Example 4, confirms the potential use of the Weissella sibaria KKP 2094p strain due to its favorable technical properties for the production of sourdough and sourdough bread. The use of this strain for this purpose is also favorable due to its ability to produce EPS, which plays an important role in the formation of the rheological and structural properties of the bread.

[0069] A selected strain of Weissella sibaria KKP 2094p capable of producing EPS was used in Example 8 to produce EPS, yielding the highest demonstrated biotechnological yield of dextran production in a medium containing 10% saccharose.

[0070] In Example 9, newly obtained EPS and EPS stored under refrigerated conditions for 6 months were checked for antagonist activity against Staphylococcus aureus and lactic acid bacteria strains (Figure 6). The EPS concentration range used, from 0 to 20 mg / ml, did not significantly inhibit the growth of either of the indicator strains, thus demonstrating that such EPS did not possess significant antagonist activity against the tested bacterial strains.

[0071] Extracellular polysaccharides used as food and feed additives must be safe for humans and animals. The cytotoxicity of extracellular polysaccharides produced by lactic acid bacteria should not exceed 10%, which should be understood as the percentage of viable cells not falling below 90% (EL-Adawi et al., 2012). The cytotoxicity of extracellular polysaccharides derived from the Weissella sibaria (KKP 2094p) strain was tested against two human cell lines from the American Type Culture Collection: Caco-2 colorectal adenocarcinoma (ATCC HTB-37) and non-neogeneic HEK293 renal epithelial cells (ATTC CRL-1573), as described in Example 10. Treatment of the cell lines with extracellular polysaccharide concentrations of 2.5–30% did not result in statistically significant inhibition of growth, thus confirming the lack of cytotoxicity of the tested EPS (Figures 7–10).

[0072] Safety analyses were also performed on this strain. In Example 11, antibiotic susceptibility testing confirmed the level of resistance at an acceptable level, in accordance with EFSA guidelines. In cases of Weissella where no specified limit is given, the results should be compared, as recommended, with those for the most phylogenetically related bacteria (heterofermentative lactic acid bacteria and Leuconostoc species). In addition, the safety of the strain (no antibiotic resistance genes and no toxic factors) was confirmed by probiotic genome analysis described in Example 12.

[0073] In Example 13, the Weissela sibaria KKP 2094p strain was successfully used to produce sourdough bread, which scored well compared to bread obtained without this strain.

[0074] Examples of embodiments of the present invention are shown in the drawings. [Brief explanation of the drawing]

[0075] [Figure 1]Figure 1 shows the results of the antagonist activity test of Weissella strains against Staphylococcus aureus IBB4005. From left to right, counting from top to bottom, plate (a) is 3714(1), 3715(2), 3716(3), 3717(4), 3277(5), 3278(6), 3279(7), 3385(8), 3386(9), 3387(10), 3388(11), 3389(12), 3393(13), 3394(14) (i.e., Weissella sibaria IBB3394(KKP) Plate (b) contains 2094p) and the K-control strain (Lactococcus lactis IBB1339, which produces nisin). Plate (b), on the other hand, contains 3287(15), 3325(16), 3326(17), 3327(18), 3382(19), 3383(20), 3384(21), 3280(22), 3281(23), 3282(24), 3284(25), 3285(26), 3286(27) and the K-control strain (Lactococcus lactis IBB1339, which produces nisin). [Figure 2] Figure 2 shows the results of the antagonist activity test of Weissella strains against Lactococcus lactis IL1403. From left to right, counting from top to bottom, plate (a) is 3714(1), 3715(2), 3716(3), 3717(4), 3277(5), 3278(6), 3279(7), 3385(8), 3386(9), 3387(10), 3388(11), 3389(12), 3393(13), 3394(14) (i.e., Weissella sibaria IBB3394(KKP) Plate (b) contains 2094p) and the K-control strain (Lactococcus lactis IBB1339, which produces nisin). Plate (b), on the other hand, contains 3287(15), 3325(16), 3326(17), 3327(18), 3382(19), 3383(20), 3384(21), 3280(22), 3281(23), 3282(24), 3284(25), 3285(26), 3286(27) and the K-control strain (Lactococcus lactis IBB1339, which produces nisin). [Figure 3]Figure 3 shows the response of Weissella strains to stress associated with the presence of bile salts and low pH. [Figure 4] Figure 4 shows the effect of Weissella sibaria IBB3394 (KKP 2094p) (live cells suspended in PBS buffer) on the survival of Staphylococcus aureus IBB4005. The number of Staphylococcus bacteria, expressed as a percentage, is presented against the number in the control in MRS (for culture and supernatant) or PBS (for cells and extract). Calculations were made for measurements taken after the start of incubation (T0), 1 hour (T1h), and 3 hours (T3h). [Figure 5] Figure 5 shows the inhibitory effect of the Weissella species IBB3394 (KKP 2094p) strain on the proliferation of Caco-2 colorectal adenocarcinoma cells compared to the effects of two other lactic acid bacteria strains, including the IBB109 strain, which has been previously confirmed to have antiproliferative activity. The proliferation of Caco-2 cells incubated with the bacterial strain was measured against a sterile Caco-2 cell culture (100%) using a BrdU colorimetric assay. Error bars represent the standard error. [Figure 6] Figure 6 shows the results of a test on the effect of EPS produced by Weissella species IBB3394 (KKP 2094p) on the growth of Staphylococcus aureus IBB4005(a) and Lactococcus lactis IL1403(b). Fresh EPS and EPS samples stored under refrigerated conditions for 6 months (20, 10, 5, 2.5, 0 mg / ml) were on the line, while positive controls with nisin of bacteriocin at various concentrations (100, 50, 20, 10, 5, 2.5 mg / ml) were in the lower part of the plate, from left to right. [Figure 7] Figure 7 shows the results of a study on the cell survival of Caco-2 colorectal adenocarcinoma cell lines after incubation with various concentrations of extracellular polysaccharides isolated from cultures of Weissella species IBB3394 (KKP 2094p), for cell lines that were not co-incubated with extracellular polysaccharides. Error bars represent the standard error. [Figure 8]Figure 8 shows the results of viability tests of the HEK293 kidney epithelial cell line after incubation with various concentrations of extracellular polysaccharides isolated from cultures of Weissella species IBB3394 (KKP 2094p), compared to cell lines that were not co-incubated with extracellular polysaccharides. Error bars represent the standard error. [Figure 9] Figure 9 shows the results of cytotoxicity tests of extracellular polysaccharides isolated from cultures of Weissella species IBB3394 (KKP 2094p) against Caco-2 colorectal adenocarcinoma cells, for cell lines that were not co-incubated with extracellular polysaccharides. Error bars represent the standard error. [Figure 10] Figure 10 shows the results of cytotoxicity tests of extracellular polysaccharides isolated from cultures of Weissella species IBB3394 (KKP 2094p) against the HEK293 kidney epithelial cell line for cell lines that were not co-incubated with extracellular polysaccharides. Error bars represent the standard error.

[0076] The publications cited herein and the references provided therein are incorporated herein by reference in their entirety. The following examples illustrate the present invention without limiting them.

[0077] Description of the Embodiment The following embodiments are provided merely to illustrate the present invention and to clarify its various aspects, and are not intended to limit it, nor should they be considered equivalent to the entirety of its scope as defined in the appended claims. [Examples]

[0078] Example 1 Isolation and identification of EPS-producing strains A search for bacterial strains capable of producing EPS was conducted in dextrose-free solid MRS selective medium supplemented with 10% saccharose. Sourdough, fermented cabbage and cucumber juice, and liquids obtained after thoroughly rinsing plant fragments in PBS buffer (pH 7.4) or suspending fermented dairy products were seeded using the streaking plate method. The plates were incubated under aerobic conditions at 30°C for a minimum of 48 hours. Single colonies of mucoid bacteria were subcultured in solid MRS medium to obtain pure cultures of the isolated bacteria, which were deposited in the IBB PAN Collection (COLIBB, Poland).

[0079] Species identification was determined by sequencing of the 16S rRNA gene. Among the EPS-producing bacteria isolated from saccharose-containing media, 12 strains belonging to the genus Leuconostoc and 5 strains belonging to Weissella confusa / Sibaria were detected. In a further stage of the search, after determining the remarkable functional characteristics of Weissella sibaria strain IBB3394, its species affiliation was confirmed as Weissella sibaria by genome sequencing and mass spectrometry using a MALDI TOF instrument. When this strain was deposited with the IAF (KKP) (IAFB Collection of Industrial Microorganisms of prof. Waclaw Dabrowski), it was deposited as KKP 2094p.

[0080] Example 2 Screening of Weissella species for antagonist activity against pathogenic Staphylococcus aureus strains. The antagonist activity of five strains of the genus Weissella, isolated as described in Example 1 against the pathogenic Staphylococcus aureus strain IBB4005 (Figure 1) and the representative lactic acid bacterium Lactococcus lactis strain IL1403 (Figure 2), as well as 22 strains previously isolated from natural bakery sourdough (Table 1), was analyzed by dropping them onto a plate together with the indicator strain against nisin-producing Lactococcus lactis strain IBB1339 as a positive control.

[0081] [Table 1]

[0082] Weissella species, Lactococcus lactis IBB1339 (control strain) and IL1403 (indicator strain), derived from the IBB PAN strain collection (COLIBB, Poland), were streaked onto plates containing MRS, GM17, and BHI medium, respectively, and incubated at 30°C for 48 hours. Staphylococcus aureus IBB4005 (indicator strain) was also seeded in BHI medium (BSL2 laboratory). Single colonies from each plate were inoculated into liquid medium and incubated overnight. Working bacterial cultures were grown as follows: for the genus Weissella, in MRS broth at 30°C; for Lactococcus lactis IBB1339, in GM17 liquid medium at 30°C; and for the indicator strains Staphylococcus aureus IBB4005 and Lactococcus lactis IL1403, in BHI broth under aerobic conditions, at 37°C with shaking and at 30°C without shaking. 100 μl of overnight culture (indicator strains: Staphylococcus aureus IBB4005, Lactococcus lactis IL1403) was inoculated into 5 ml of soft BHI agar heated to 55°C, immediately poured onto a large BHI solid medium plate, and spread evenly to cover the surface. After the agar had cured, 5 μl of overnight cultures of the tested Weissella species and the positive control (nisin-producing Lactococcus lactis strain IBB1339) were applied to the designated positions on the plate. The droplets were absorbed into the plate and incubated overnight at 37°C and 30°C for Staphylococcus aureus IBB4005 and Lactococcus lactis IL1403, respectively. The experiment was performed in three biological replicates. Antagonism against the indicator strain was evaluated by observing the growth inhibition zone.

[0083] The size and permeability of such zones varied depending on the strain. Overall, four distinct effects were observed: i) a clear inhibitory zone, ii) an inhibitory zone with visible growth of the test strain in the center, iii) a blurred / indistinct zone with a slight increase in the index and visible growth of the test strain in the center, and iv) no inhibitory zone with growth around the test strain. The first two effects were classified as strong antagonist activity and marked (+), the third effect was bacteriostatic (+ / -), and the last effect was characterized as no antagonist activity (-). Results obtained from three replicates (Table 2) showed that some Weissella strains could inhibit the growth of Staphylococcus aureus, some were bacteriostatic, and others did not inhibit the growth of Staphylococcus aureus. At the same time, the test strains did not inhibit the growth of the lactic acid bacterium Lactococcus lactis IL1403 strain (Table 2).

[0084] [Table 2]

[0085] Example 3 Survival study of Weissella species under low pH and in the presence of bile salts One of the key defining characteristics of probiotic bacteria is their ability to survive in the gastrointestinal tract. The resistance of five Weissella strains isolated as described in Example 1 and twelve strains previously isolated from wild sourdough to bile salts (bile salt stress) and low pH (acid stress) was tested as follows: Fresh, grown colonies from each bacterial isolate on IST-MRS medium [90% ISO-Sensitest broth (Oxoid) and 10% MRS (Merck)] were suspended in PBS, pH=7.4 (PBS404, BioShop) and OD600. nmThe cells were diluted to 0.5 in 1 ml of PBS, pH=7.4 (control); PBS, pH=3 (acid stress); and PBS, pH=7.4 (bile salt stress) containing 3 g / l of two bile salts: sodium cholate and sodium deoxycholate (B8756, Sigma-Aldrich). The samples were incubated at 37°C for 1 hour. After incubation, the tubes were centrifuged (3 minutes, 4°C, 10,000 rpm), the supernatant was discarded, and the cell pellet was suspended in 0.5 ml of PBS buffer, pH 7.4. 10 -1 ~10 -6 Dilutions within the specified range were prepared for each sample. 20 μl of each dilution was spotted onto an IST-MRS solid substrate plate. The plate was incubated under aerobic conditions at 30°C for 48 hours. Screening experiments to characterize the strains under conditions mimicking the gastrointestinal tract were performed in three biological replicates, and the average for each condition was calculated. Susceptibility to stressors was expressed as an order of magnitude reduction in the number of bacterial colony-forming units (CFU) / ml compared to the control condition (pH PBS = 7.4). The results were presented in a chart (Figure 3), and based on these, the IBB3394 isolate (later deposited as KKP 2094p) was selected as the most resistant and unique strain to bile salts and low pH.

[0086] Example 4 Testing of selected Weissella strains for antagonist activity against Staphylococcus aureus and Staphylococcus epidermides strains. Six strains of Lactobacillus 'Weissella' (Confusa / Sibaria) selected from the Collection of Bacterial Strains of the IBB PAN (COLIBB) were tested for the following:

[0087] • To investigate the potential effects of pH reduction on inhibiting the growth of Staphylococcus aureus and Staphylococcus epidermides strains, • Antagonist activity against live cells of three pathogenic strains of Staphylococcus aureus, including multidrug-resistant strains. Antagonist activity against living cells of two strains of Staphylococcus epidermides, which represent bacterial species that cause opportunistic infections (but do not cause infection in healthy individuals).

[0088] When tested Weissella strains spotted on solid BHI medium containing bromocresol purple (an acidity indicator), the medium underwent a color change from violet to purple to yellow, indicating acidification of the medium around the droplet zone. The pH value of the overnight culture ranged from 4.23 to 4.89.

[0089] Antagonist activity testing was performed by dropping 5 μl of liquid overnight culture of Weissella strains onto large plates (BHI with bromocresol purple) containing indicator strains of Staphylococcus aureus IBB4002, IBB4005, and IBB4009, as well as Staphylococcus epidermides 6PII6 and 5L03, inoculated in loan form. The plates were incubated at 37°C for 24 hours. Growth inhibition zones were observed around the tested Weissella strains for all indicator strains of Staphylococcus aureus (Table 3) and Staphylococcus epidermides (Table 4).

[0090] [Table 3]

[0091] [Table 4]

[0092] Example 5 Antagonist activity of Weissella sibaria IBB3394 (KKP 2094p) strain against live Staphylococcus aureus strains in various preparations (culture, supernatant, cell extract, live cells suspended in PBS buffer). The experiment was conducted with the following various variants: • Overnight culture of Weissela sibaria IBB3394 (KKP 2094p) strain - 900 μl • Supernatant - 900 μl of Weissella sibaria IBB3394 (KKP 2094p) liquid overnight culture - centrifuged at 13000 RPM for 3 minutes, then the supernatant was filtered through a 0.45 μm filter. The extract-pellet from Weissella sibaria IBB3394 cells (KKP 2094p), precipitated by centrifugation (3 minutes, 13,000 RPM), was suspended in 900 μl of PBS buffer. The cells were then disrupted using glass beads (3 × 1 minute, with cooling interruptions on ice after 1 minute each), and the mixture was centrifuged at 4°C at 14,000 RPM for 10 minutes. The liquid on the beads was collected and added to PBS (approximately 100 μl) to a final volume of 900 μl. • Cell suspension in PBS - 900 μl of liquid overnight culture of Weissella sibaria IBB3394 (KKP 2094p) was centrifuged at 13000 RPM for 3 minutes, the cell pellet was then suspended in 900 μl of sterile PBS, centrifuged again at 13000 RPM for 3 minutes, and the resulting precipitate was suspended in 900 μl of sterile PBS.

[0093] Dilution (100,000x) of overnight liquid culture of Staphylococcus aureus indicator strain in sterile PBSIBB4005.

[0094] Diluted Staphylococcus overnight cultures with individual variants of Weissella sibaria IBB3394 strain (10 -5 Incubate 100 μl of diluted PBS or MRS medium (as a control) and 900 μl of MRS medium at approximately 30°C for 3 hours. Immediately after the start of incubation (T0), 1 hour (T0). 1h ) and 3 hours (T 3h) Then, in two replicates, 100 μl of each variant was seeded onto an LB plate. The plates were incubated at 42°C (to eliminate LAB growth) for 24 or 48 hours (optionally with cultures and cells overnight). The experiment was performed in two technical replicates and at least two biological replicates. The results are presented graphically (Figure 4), where the number of Staphylococcus bacteria, expressed as a percentage, is relative to the number in the control (in MRS or PBS).

[0095] A clear antagonist effect was observed in the overnight culture and in cells suspended in PBS buffer. Furthermore, the supernatant showed anti-staphylococcus activity, but such an effect was not observed in the cell extract.

[0096] Example 6 Antiproliferative activity of IBB3394 (KKP 2094p) strain against Caco-2 colorectal adenocarcinoma cells The inhibition of colon cancer cell proliferation was tested by culturing the Weissella strain IBB3394 in MRS medium and following the method described by Salanski et al. (2022). The BrdU assay results showed that strain IBB3394 strongly inhibited the proliferation activity of Caco-2 tumor cells (Figure 5).

[0097] Example 7 Testing of the IBB3394 (KKP 2094p) strain for antagonist activity against undesirable spore-forming bacteria (Bacillus cereus, Bacillus brevis, and Lysinibacillus fusiformis) in bread. The antagonist activity of strain IBB3394 (KKP 2094p) was tested against spore-forming bacteria that spoil bread, following the method described for strains isolated from rye sourdough (Litwinek et al., 2022). For this purpose, spore-forming indicator bacteria (Bacillus cereus, Bacillus brevis, and Lysinibacillus fusiformis) were inoculated into Luria-Bertani (LB) liquid medium (Difco Laboratories, Franklin Lakes, New Jersey, USA) and incubated overnight. The overnight culture was then inoculated into liquefied and chilled solid LB medium, which was immediately poured onto plates. After the medium solidified, 5 μl of the test strain, grown in liquid MRS medium, was seeded onto selected locations on the plates. Droplets were allowed to absorb onto the plates, and the plates were incubated overnight at 30°C. Antagonism against the strain was visualized by observing growth inhibition zones. The presence of an inhibitory zone was represented as "++", "+", or "+ / -" depending on its size, while the absence of antagonist activity was represented as "-". The results obtained were compared with those obtained for *Bacillus weissella* strains isolated from rye flour sourdough (Litwinek et al., 2022). From the analysis, it was found that strain IBB3394 (KKP 2094p) contained a unique component against *Lysinibacillus fusiformis* strains and exhibited antagonist activity rated "+" against all indicator strains. This activity was higher than that of strains isolated from rye sourdough, where slightly weak inhibition of grown *Bacillus brevis* was observed in all test strains, and antagonist activity against *Bacillus cereus* was observed in 4 out of 7 strains, while no such activity was observed against *Lysinibacillus fusiformis*.

[0098] Example 8 Characterization of extracellular polysaccharides (EPS) produced by IBB3394 (KKP 2094p) The yield of extracellular polysaccharide (EPS) production by IBB3394 was determined for strains grown in dextrose-free MRS medium containing 5% or 10% saccharose. EPS production, followed by extraction, isolation, and characterization, was performed according to the method described by Buksa et al. (2021). The results of the production yield analysis and EPS characterization are presented in Table 5. The yield of raw EPS produced by the IBB3394 strain was higher in the medium containing 10% saccharose, at approximately 50 g / l. As a result of the isolation of raw EPS in the SEC column, a fraction with a high molar mass was detected and identified as dextran.

[0099] [Table 5]

[0100] Example 9 Analysis of the effect of EPS produced by the Weissella species IBB3394 (KKP 2094p) on Staphylococcus aureus IBB4005 and Lactococcus lactis IL1403. The antagonist activity of EPS produced by strain IBB3394 was evaluated using a method similar to that described for determining the antagonist activity of Weissella strains in Example 2. In this example, fresh EPS at specified concentrations (20, 10, 5, 2.5, 0 mg / ml) and EPS stored under refrigerated conditions for 6 months were spotted onto plates with the indicator strain against positive controls—nisin at various concentrations (100, 50, 20, 10, 5, 2.5 mg / ml). The results obtained using different concentrations of nisin showed that EPS from Weissella IBB3394 did not inhibit the growth of Staphylococcus aureus at most of the concentrations tested, except for a concentration of 20 mg / ml which showed very little inhibition (Figure 6a). Furthermore, it was observed that there were no EPS concentrations that inhibited the growth of Lactococcus lactis IL1403 (Figure 6b).

[0101] Example 10 Cytotoxicity study of extracellular polysaccharides (EPS) isolated from cultures of Weissella species IBB3394 (KKP 2094p) The study was carried out according to the method described by Vosough et al., 2021, with modifications. Caco-2 adenocarcinoma cell line (ATCC HTB-37) and HEK293 fetal kidney epithelial cell line (ATCC CRL-1573) were cultured under the optimal conditions recommended by ATCC, i.e., 37°C, 5% CO2, and 95% humidity. The culture medium used (hereinafter referred to as cell line medium) was MEM (Minimum Essential Medium; Gibco) enriched with 10% fetal bovine serum, amino acid solution (NEAA - non-essential amino acid solution) (1×), 1 mM sodium pyruvate, penicillin (100 U / ml), and streptomycin (100 μg / ml). The cell count of the cell line was determined in a Thoma chamber. The cells were diluted in the cell line medium mentioned above and 10 5 Cell density was obtained, and 100 μl of cell suspension per well was applied to a 96-well plate and incubated for 24 hours under optimal conditions. 100 μl of extracellular polysaccharide suspensions at concentrations of 30, 20, 10, 5, and 2.5 mg / ml in the cell line medium were seeded onto 96-well plates containing Caco-2 or HEK293 cells 24 hours after culture, and the medium was collected beforehand. The plates were incubated at 37°C, 5% CO2, and 95% humidity for 24 hours, and the cell viability of the cell line was then evaluated using a colorimetric method with the commercially available Cell Growth Kit I, MTT (Roche). Absorbance measurements were performed at wavelengths of 570 and 680 nm. The experiment was performed in 10 technical replicates and 3 biological replicates. Controls consisting of Caco-2 and HEK293 cell cultures were treated with extracellular polysaccharide-free medium. Viability was expressed using the following formula:

[0102]

number

[0103] Due to the high viscosity of the extracellular polysaccharide suspension, a concentration of 30 mg / ml was selected as the highest concentration usable in this experiment. Statistical analysis was performed using the Shapiro-Wilk test and multivariate analysis of variance (ANOVA).

[0104] The results are shown in the figures (Figures 7-10). Treatment of cell lines with extracellular polysaccharide concentrations of 2.5-30% did not result in statistically significant inhibition of growth, thus confirming the lack of cytotoxicity of the tested EPS.

[0105] Example 11 Antibiotic sensitivity test The safety of selected strains of the genus Weissella, species IBB3394, was assessed by testing for antibiotic susceptibility using E test strips (bioMerieux), as recommended by the manufacturer. Test strains were seeded on IST-MRS solid medium and incubated under aerobic conditions at 30°C. Using the resulting colonies, bacterial suspensions were prepared in PBS using an OD600nm=0.25-McFarland scale 1. These suspensions were then spread across the plate surface using swabs in different directions to ensure uniform distribution of the strains. Antibiotic strips were placed on the plate surface, and the plates were incubated under aerobic conditions at 30°C for 48 hours. Resistance to nine antibiotics was determined (following EFSA recommendations): ampicillin (AM), vancomycin (VA), gentamicin (GM), kanamycin (KM), streptomycin (SM), erythromycin (EM), clindamycin (CM), tetracycline (TC), and chloramphenicol (CL).

[0106] The results are shown in Table 6. The minimum inhibitory concentrations (MICs) for all test antibiotics were shown to be below the limits recommended by the EFSA.

[0107] [Table 6]

[0108] Example 12 Genome analysis of the IBB3394 (KKP 2094p) strain DNA was isolated from the Weissella strain IBB3394 and then subjected to whole-genome sequencing using DNA sequencing and synthesis facilities at IBB PAN using the Illumina MiSeq platform (Illumina, San Diego, USA) and a GridION sequencer (Oxford Nanopore Technologies, Oxford, Great Britain).

[0109] Various servers, software, and databases were used for bioinformatics analysis. The RAST service was used to determine coded sequences (CDS) and non-coding RNAs (Aziz et al., 2008). Overall genome sequencing analysis of strain IBB3394 showed that the strain had a 2,488,843 bp circular DNA chromosome and a 44.8% GC content, but the total number of coded sequences (CDS) was 2,313. Genome sequencing analysis of the strain indicated that it belongs to the Weissella sibaria species. The KEGG database (Kanehisa et al., 2022) and the automated annotation service BlastKOALA were used for gene function analysis (Kanehisa et al., 2016). The strain was found to encode 172 different metabolic pathways. Thirty complete pathway modules were found, among others, including complete vitamin biosynthesis pathways, such as the riboflavin (vitamin B2), menaquinone (vitamin K2), and thiamine (vitamin B1) salvage pathways. Safety analysis was performed by searching for putative toxic factors using VirulenceFinder. The evaluation was conducted by comparing the whole genome sequence of IBB3394 with known toxic genes from the genera Enterococcus, Escherichia coli, Staphylococcus aureus, and Listeria. The results indicated the absence of toxic genes in the IBB3394 genome. Consequently, IBB3394 was found to lack toxic or pathogenic genes associated with well-known pathogenic Enterococcus, Escherichia coli, Staphylococcus aureus, and Listeria. The results were also confirmed by whole-genome sequencing analysis using PathogenFinder, another bacterial pathogenicity prediction web server. This tool did not classify the IBB3394 strain as a pathogen. Genomic analysis using various bioinformatics tools recommended by EFSA, such as ResFinder, CARD, and RGI, as well as KEGG, did not reveal the presence of antibiotic resistance genes (ARGs) in the IBB3394 strain. Sequencing analysis confirmed the safety of using the strain as a probiotic, as well as for industrial, food, and pharmaceutical applications.

[0110] Example 13 Manufacturing of sourdough bread containing Weissela sibaria IBB3394 (KKP 2094p) Bread made with added baker's yeast was prepared using whole-grain rye flour.

[0111] To produce sourdough, the starter culture described in Polish Patent No. 238153 was used, consisting of a starter (set 11) containing Lactobacillus plantarum B / 00117, Lactobacillus plantarum B / 00118, and Lactobacillus brevis strains in a 1:1:1 ratio, a commercially available starter culture LV2 [containing Saccharomyces chevalieri and Lactobacillus brevis, starter culture (bacteria and yeast) by Lesaffre for acidifying rye flour, data obtained from Lesaffre Bio-Corporation SA, Lodz] as a control, and the same strain as set 11 supplemented with Weissella sibaria IBB3394 (KKP 2094p) strain in a 1:1:1 ratio as a test sample.

[0112] The sourdough was prepared as described in Example 3 of Polish Patent No. 238153.

[0113] The bread (bakery product) was prepared according to the following recipe (for a loaf of bread weighing 0.5 kg). Preparation of raw materials and their quantities for approximately 1 kg of dough: Rye flour type 2000 0.5kg • Sourdough made from rye flour (type 2000) 0.17kg • Bread yeast 0.006 kg • Salt 0.012 kg • Water (temperature 38℃) approximately 0.31kg Proper dough manufacturing and preparation • Kneading the dough (shaping the dough) - Time - Slow rotation 7 minutes • Kneading the dough (shaping the dough) - Time - Fast rotation 3 minutes • The appropriate dough temperature is up to 35℃. Proper dough division, formation, and shaping • Weight of individual piece: 0.5kg • Time required to divide and shape the dough into small pieces: 45 minutes • Proper dough fermentation time for small pieces: up to 90 minutes Firing process • Firing temperature: 200℃ • Baking time: 60 minutes

[0114] The bread was evaluated using the method described in Polish Patent No. 238153. The resulting bread was sensorily evaluated and analyzed according to PN-A-74108:1996 [Standard] using a point method with a panel of proven sensory sensitivities. After baking, the moisture content and texture profile of the crumb of the baked-loaf bread were analyzed. The microbiological stability of the produced bread was also determined. The resulting bread loaves were stored for 7 days to determine the changes that occur during bread staling. Individual results obtained for two types of bread, consisting of test bread produced using Set 11 supplemented with the Weissella sibaria IBB3394 (KKP 2094p) strain, and controls produced using Set 11 and a commercially available culture (LV2), are presented in Tables 7-11.

[0115] Compared to the control, bread supplemented with sourdough made from Weissella sibaria IBB3394 (KKP 2094p) showed increased crumb elasticity while maintaining improvements in crust crispness and the resulting taste and aroma. Bread supplemented with Weissella sibaria IBB3394 (KKP 2094p) was characterized by similar loaf volume and higher crumb moisture content, and significantly lower crumb firmness and chewiness compared to the control bread. The sourdough bread supplemented with Weissella sibaria IBB3394 (KKP 2094p) exhibited the same microbiological stability (3 days) as the control bread. The resulting bread received a higher sensory evaluation rating than the control bread.

[0116] [Table 7]

[0117] [Table 8]

[0118] [Table 9]

[0119] [Table 10]

[0120] [Table 11]

[0121] Bread prepared using the Set 11+IBB3394 culture was characterized by the lowest hardness of all tested loaves, regardless of the storage day, indicating a more delicate bread crumb. Furthermore, the hardness in the case of bread made with Set 11+IBB3394 increased by 16% compared to day 1, and in the case of the commercially available LV2 culture, increased by 28%, indicating a much slower deterioration process, which is influenced by extracellular polysaccharides produced during sourdough fermentation.

[0122] Literature:

[0123] [Table 12] TIFF2026509356000014.tif253167TIFF2026509356000015.tif93169

Claims

1. A new strain of Weissella cibaria has been deposited with the IAFB (IAFB Collection of Industrial Microorganisms of prof. Waclaw Dabrowski, Warsaw, Poland) under accession number KKP 2094p.

2. A composition containing a novel Weissella sibaria bacterial strain deposited with the IAFB under accession number KKP 2094p.

3. The composition according to claim 2, characterized in that it further comprises a pharmaceutically acceptable carrier, preferably the composition is in the form of a liquid, solid, powder, tablet, or capsule intended for oral administration.

4. A pharmaceutical composition comprising a Weissella sibaria bacterial strain as defined in claim 1 and / or a composition as defined in claim 3, and a pharmaceutically acceptable carrier, for use as a pharmaceutical.

5. A pharmaceutical composition for use according to claim 4, characterized in that it is for use as a pharmaceutical for the prevention and / or treatment of intestinal cancer, preferably colorectal cancer, and most preferably colorectal adenocarcinoma.

6. A dietary supplement comprising the Weissella sibaria bacterial strain defined in claim 1 and / or the composition defined in claim 3, preferably in the form of a liquid, solid, powder, tablet, or capsule intended for oral administration.

7. A probiotic preparation comprising the Weissella sibaria bacterial strain defined in claim 1 and / or the composition defined in claim 3, wherein the probiotic preparation preferably further comprises a prebiotic substance preferably selected from oligosaccharides, polysaccharides, fructooligosaccharides, lactulose, inulin, indigestible starch, cellulose, hemicellulose and pectin, and the probiotic preparation preferably also comprises a postbiotic, preferably the postbiotic being selected from short-chain fatty acids, enzymes, lipopolysaccharides, teicoic acid, vitamins, butyric acid, acetate, propionate, muramyl dipeptide, indole, teicoic acid and lactosepin.

8. The probiotic preparation according to claim 7, characterized in that it is in the form of a liquid, solid, powder, tablet, or capsule intended for oral administration.

9. A bacterial starter culture for making bread, characterized in that the culture contains the Weissella sibaria strain as defined in claim 1.

10. A sourdough for making bread, characterized by comprising a bacterial starter culture as defined in claim 9.

11. A method for producing bread, comprising the step of adding a bacterial starter culture as defined in claim 9 and / or sourdough as defined in claim 10.

12. Bread, characterized by comprising a bacterial starter culture as defined in claim 9 and / or a sourdough starter as defined in claim 10.

13. A method for microbial production of dextran, comprising the step of culturing a bacterial strain of Weissella sibaria as defined in claim 1 and / or a composition as defined in claim 2 in a dextrose-free (glucose-free) liquid medium supplemented with an organic nitrogen source containing saccharose, amino acids and short peptides, vitamin B, and inorganic salts.

14. The method according to claim 13, characterized in that the culture is carried out in MRS medium (DeMan-Rogosa-Sharp broth), a culture medium for lactic acid bacteria, preferably with 5 to 10 ± 2 weight percent of saccharose added.

15. A bacterial preparation comprising the Weissella sibaria strain defined in claim 1 and / or the composition defined in claim 2, for use as an active ingredient in probiotics, therapeutic preparations, functional foods, and dietary supplements, and for use as an active ingredient in drugs intended for the prevention and / or treatment of intestinal cancer, preferably colon cancer.

16. The use of the Weissella sibaria bacterial strain defined in claim 1 and / or the composition defined in claim 2 as a functional additive in functional foods, a functional additive in food and beverages, a probiotic preparation, a bacterial preparation, a nutritional supplement, and an active ingredient in a pharmaceutical composition.

17. Use in the food industry of the Weissella sibaria bacterial strain defined in claim 1 and / or the composition defined in claim 2 as an emulsifier, thickener, stabilizer, or texture improver for food products.

18. Use of the Weissella sibaria bacterial strain defined in claim 1 and / or the composition defined in claim 2 for microbial production of extracellular polysaccharides (EPS), preferably dextran.

19. Use of the Weissella sibaria bacterial strain defined in claim 1 and / or the composition defined in claim 2 and / or the bacterial starter culture defined in claim 9 for the production of sourdough for bread.

20. Use of the Weissella sibaria bacterial strain defined in claim 1 and / or the composition defined in claim 2 and / or the sourdough defined in claim 10 for making bread.

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