Marine-sourced S.coagulans strain W-1 as well as preparation and application of marine-sourced S.coagulans strain W-1

Abstract

The invention relates to the technical field of microorganisms, in particular to a marine-derived Weizmannia coagulans strain W-1 as well as a preparation and application thereof, the Weizmannia coagulans strain W-1 has acid resistance and can keep activity in gastric juice with the pH value of 2.5, and the preparation of the Weizmannia coagulans strain W-1 synergistically improves intestinal health through triple mechanisms of bacteriostasis, barrier repair and brain intestinal axis regulation and control, so that the intestinal health is improved. And the viable bacterium rate is 65.2% after being stored at normal temperature for 3 months, and excellent stability is shown.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a strain of Weizmannella coagulans ( Weizmannia coagulans ) strain W-1 and its preparation and application. Background Art

[0002] Irritable bowel syndrome (IBS) is a functional, chronic gastrointestinal disorder with a complex and incompletely elucidated pathophysiology. Currently, effective clinical treatments are lacking. In recent years, with the advancement of intestinal microbiome research, a wealth of evidence has demonstrated a significant association between structural and functional disturbances in the gut microbiome and the pathogenesis of IBS. Imbalances in the gut microbiome can contribute to the development and progression of IBS by influencing host immune function, neural regulation, and metabolic activity. Consequently, probiotics and their metabolites have been considered promising intervention strategies for IBS due to their multiple mechanisms of action, including regulating intestinal microbiome balance, improving intestinal mucosal barrier function, and modulating the brain-gut axis. However, current research has yet to identify the optimal therapeutic approach for specific probiotic strains, their metabolites, or combinations thereof, and their mechanisms of action require further systematic investigation.

[0003] Existing studies have shown that probiotics can improve the clinical symptoms of irritable bowel syndrome through multiple mechanisms, including regulating the balance of intestinal flora composition, enhancing intestinal motility, reducing visceral hypersensitivity, regulating host immune responses, and affecting microbiome-brain-gut axis signaling. These synergistic mechanisms can effectively alleviate common clinical symptoms of IBS patients, such as abdominal distension, abdominal pain, and gastrointestinal dysfunction. Among them, Weizmannella coagulans ( Weizmannia coagulans ) is a Gram-positive, spore-forming, rod-shaped probiotic that combines the biological characteristics of lactic acid bacteria and Bacillus. Compared to Bifidobacterium and Lactobacillus, this strain can form a highly stress-resistant spore structure under adverse environmental conditions, giving it significant stability advantages during industrial production, storage, and gastrointestinal delivery. Its spore morphology is resistant to erosion by gastric acid and bile salts, ensuring effective colonization in the intestine. It also exerts beneficial effects such as regulating the intestinal microecology, enhancing digestive function, and modulating the immune system by lowering intestinal pH and inhibiting the proliferation of opportunistic pathogens.

[0004] Currently, research on the application of Weizmannella coagulans is primarily focused on aquaculture, plant protection, and animal husbandry, with strain sources largely limited to terrestrial environments. For example, patent CN115487214A discloses a strain of Weizmannella coagulans BC99 isolated from the intestines of infants, which can alleviate intestinal inflammation in IBS mice by increasing the abundance of beneficial bacteria. Patent CN114196600B describes Lactobacillus plantarum AR495 (isolated from rice wine lees) as improving IBS symptoms by inhibiting mast cell activation and the PAR2-TRPV1 signaling pathway. Patent CN116083324A reports on Bifidobacterium animalis subspecies Lactobacillus BA79 (isolated from human breast milk), and patent CN116004486A discloses Bacteroides fragilis BFS17 (isolated from the intestines of healthy humans), both of which show potential to regulate intestinal flora disorders and alleviate IBS-related symptoms. It is worth noting that the existing probiotic resources with IBS relief function are still mainly concentrated in terrestrial ecosystems, and the development of marine-derived probiotics is obviously insufficient.

[0005] The marine environment has special physical and chemical conditions that are significantly different from terrestrial ecosystems, including extreme factors such as high salinity, high pressure and low temperature. This unique selection pressure has prompted marine microorganisms to evolve highly diverse physiological characteristics. Among them, marine-derived lactic acid bacteria groups may exhibit probiotic functional characteristics that are significantly different from terrestrial strains due to their long-term adaptation to special habitats. Based on this, there is an urgent need to systematically develop microbial resources in marine ecosystems, specifically covering a variety of habitat samples such as seawater environments, the surface and intestines of marine organisms, and seabed sediments. Through targeted screening technology, marine-derived probiotic strains with significant intervention effects on irritable bowel syndrome were obtained. This move can not only enrich the existing probiotic germplasm resource library, but also provide a material basis for the development of new IBS treatment plans based on marine microorganisms, and has important theoretical research and clinical application value. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a marine-derived Weizmannella coagulans ( Weizmannia coagulans ) W-1, isolated from dolphin stomach, deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCCNO: 31804, and the deposit date is September 2, 2024.

[0007] The Weizmannella coagulans W-1 is acid-resistant and can maintain activity in gastric juice at pH 2.5.

[0008] The second aspect of the present invention provides use of the Weizmannella coagulans W-1 described in the first aspect in preparing a preparation for improving intestinal health.

[0009] In some preferred embodiments, the preparation is used for: (1) Inhibiting intestinal pathogens, wherein the pathogens include at least one of Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, Shigella dysenteriae and Streptococcus agalactiae.

[0010] (2) Upregulating the expression of tight junction proteins and aquaporins in intestinal epithelial barrier function regulatory proteins; optionally, the tight junction proteins in the preparation include the transmembrane protein claudin-1 and the cytoplasmic scaffold protein ZO-1, and the aquaporins include the transmembrane protein AQP3. By synergistically upregulating the expression of tight junction proteins and aquaporins, on the one hand, the connection between epithelial cells is enhanced to reduce intestinal permeability, and on the other hand, intestinal water transport is regulated to maintain water-salt balance, thereby jointly improving intestinal barrier function and alleviating the symptoms of irritable bowel syndrome.

[0011] (3) Reducing the level of serum brain-gut axis signaling molecules; optionally, the brain-gut axis signaling molecules include corticosterone, 5-hydroxytryptamine, and substance P. By reducing the level of brain-gut axis-related signaling molecules, intestinal nerve sensitivity is reduced, thereby improving the visceral pain and diarrhea symptoms caused by irritable bowel syndrome.

[0012] The third aspect of the present invention provides a microbial preparation comprising the Weizmannella coagulans W-1 described in the first aspect as an active ingredient and a pharmaceutically acceptable carrier, wherein the carrier comprises trehalose and skim milk.

[0013] In some preferred embodiments, the carrier further comprises glycerol.

[0014] In some preferred embodiments, the formulation is in the form of a lyophilized powder.

[0015] In some preferred embodiments, the lyophilized powder is used to prepare medicines, foods or health products.

[0016] In some preferred embodiments, the dosage form of the drug is capsule, tablet or powder.

[0017] In some preferred embodiments, the food comprises a fermented dairy product or a solid beverage.

[0018] In some preferred embodiments, the fermented dairy product is yogurt.

[0019] In some preferred embodiments, the health product comprises capsules or powders.

[0020] In some preferred embodiments, the Weizmannella coagulans W-1 in the preparation is a living bacterium or spore.

[0021] In some preferred embodiments, the number of viable cells of Weizmannella coagulans W-1 in the preparation is 1×108 ~1×10 11 CFU / g.

[0022] In some preferred embodiments, the mass ratio of trehalose to skim milk is 1:1-3:1.

[0023] In some preferred embodiments, the mass ratio of trehalose to skim milk is 1:1.

[0024] The fourth aspect of the present invention provides a method for preparing the microbial preparation of the third aspect, comprising culturing Weizmannella coagulans W-1 to the logarithmic growth phase, inducing spore formation, and mixing the bacteria with a carrier.

[0025] In some preferred embodiments, the method comprises the following steps: ① Bacterial culture: Weizmannella coagulans W-1 was inoculated into MRS liquid medium with a pH of 6.0-7.5 and cultured at 37°C under aerobic conditions until OD 600 =0.6-1.0, obtain the bacterial solution in the logarithmic growth phase; ② Spore induction: Transfer the bacterial solution obtained in step ① to an induction medium containing 0.1-0.1 mM MnSO4 and continue to culture at 37°C under aerobic conditions for 48-72 hours to form spores; ③ Bacteria-carrier mixing: The spore-forming bacteria obtained in step ② are collected by centrifugation and mixed with the pharmaceutically acceptable carrier described in the third aspect to prepare a microbial preparation.

[0026] Beneficial effects: (1) The Weizmannella coagulans W-1 provided by the present invention is isolated from the acidic environment of the dolphin stomach and can maintain a high survival rate in simulated gastric fluid at pH 2.5, which is significantly better than conventional terrestrial Weizmannella coagulans strains. This characteristic enables it to effectively tolerate the acidic environment of the human stomach and ensures that a sufficient number of live bacteria reach the intestine to exert their effects.

[0027] (2) The Weizmannella coagulans W-1 provided by the present invention synergistically upregulates the expression of tight junction protein claudin-1 / ZO-1 and water channel protein AQP3, thereby enhancing the connection between epithelial cells and regulating water transport, effectively reducing intestinal permeability and exerting a significant effect on improving intestinal health.

[0028] (3) The Weizmannella coagulans W-1 provided by the present invention can significantly reduce the levels of stress marker corticosterone, intestinal neurotransmitter 5-HT and pain conduction medium substance P in serum, and relieve abdominal pain and diarrhea symptoms in patients with irritable bowel syndrome.

[0029] (4) The microbial preparation provided by the present invention exhibits excellent stability (viable bacteria rate >65.2% after 3 months of storage at room temperature), which is far superior to conventional probiotics that require cold chain transportation. The carrier system used further ensures the survival rate of spores by forming a protective matrix, giving the preparation a significant advantage in industrial production and clinical application.

[0030] (5) The microbial preparation provided by the present invention can significantly improve the quality of yogurt. Its highly active strains can effectively maintain the probiotic properties of yogurt. At the same time, by optimizing the carrier formula, the original flavor and texture of yogurt are maintained, solving the problems of live bacteria attenuation and taste separation caused by post-addition of probiotic yogurt, and providing an innovative solution for the development of functional dairy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the antibacterial result diagram of Weizmannella coagulans W-1; Figure 2 Figure 2 is the weight change of mice; Figure 3 This is a graph showing changes in water content in mouse feces; Figure 4 is a comparison chart of serum corticosterone levels; Figure 5 is a comparison chart of serum serotonin levels; Figure 6 This is a comparison chart of serum substance P levels; Figure 7 This is a histopathological section of mouse colon tissue; Figure 8 This is a comparison chart of the expression of tight junction protein claudin-1; Figure 9 This is a comparison chart of tight junction protein ZO-1 expression; Figure 10 This is a comparison chart of the expression of water channel protein AQP3. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0033] Explanation of terms: (1) Weizmannella coagulans ( Weizmannia coagulans ): Gram-positive Bacillus, belonging to the Firmicutes, has both the metabolic characteristics of lactic acid bacteria and the stress resistance of Bacillus. Its spore structure can withstand extreme pH, high temperature and digestive fluid erosion, and is widely used in probiotic preparations.

[0034] (2) Irritable bowel syndrome (IBS): a functional bowel disease with pathological characteristics including visceral hypersensitivity, brain-gut axis disorder and intestinal barrier damage. It is clinically divided into diarrhea type (IBS-D), constipation type (IBS-C) and mixed type (IBS-M).

[0035] (3) Intestinal pathogens: Pathogenic microorganisms that colonize the intestine and destroy the microecological balance. They are divided into invasive, toxin-producing and adherent-invasive types according to their pathogenic mechanisms. Their virulence factors include adhesins, endotoxins and type III secretion system.

[0036] (4) Escherichia coli ( Escherichia coli Enterobacteriaceae are classified according to pathogenicity into commensal, pathogenic, and conditionally pathogenic (e.g., UPEC). Serotyping is based on O (lipopolysaccharide), H (flagellar), and K (capsular) antigens. This example uses pathogenic or conditionally pathogenic Escherichia coli.

[0037] (5) Salmonella Enteritidis ( Salmonella Enteritidis ): A member of the Salmonella serogroup D, it is mainly transmitted through poultry eggs. Its pathogenicity depends on the type III secretion system encoded by the SPI-1 / SPI-2 virulence island. The clinical manifestations are self-limited gastroenteritis, and bacteremia may occur in severe cases.

[0038] (6) Listeria monocytogenes ( Listeria monocytogenes ): Gram-positive short rod, the only foodborne pathogen that can grow at 4°C. Its internalin (InlA / B) mediates cell invasion, and its ActA protein induces actin polymerization to form a "comet tail" movement. It is highly pathogenic to pregnant women, newborns and immunocompromised people.

[0039] (7) Shigella dysenteriae ( Shigella dysenteriae Shigella serogroup A: Gram-negative bacilli, which secrete invasion plasmid antigen (Ipa protein) through the type III secretion system to destroy colon epithelial cells and produce Shiga toxin (Stx) to cause bloody diarrhea. It is the main pathogen of bacterial dysentery.

[0040] (8) Streptococcus agalactiae ( Streptococcus agalactiae ): Group B Streptococcus (GBS), a Gram-positive coccus containing capsular polysaccharide antigens, is an important conditional pathogen of neonatal sepsis and meningitis. Its virulence factors include CAMP factor, β-hemolysin and adhesion proteins.

[0041] (9) Intestinal epithelial barrier function regulatory proteins: a complex composed of tight junction proteins, adherens junction proteins and cytoskeletal proteins, which maintains the selective permeability of the intestinal mucosa by regulating the expression of transmembrane proteins and paracellular pathways.

[0042] (10) Claudin-1: A four-transmembrane protein with a molecular weight of 23 kDa. It is the main structural protein of tight junctions and regulates the permeability of intestinal epithelial cells by forming intercellular ion-selective channels. Its downregulation is associated with intestinal leakiness in inflammatory bowel disease (IBD).

[0043] (11) ZO-1: A cytoplasmic scaffold protein with a molecular weight of 220 kDa, it connects claudins to the actin cytoskeleton through the PDZ domain, maintains the stability of tight junctions, and participates in the regulation of the TGF-β / Smad signaling pathway.

[0044] (12) AQP3: A transmembrane protein with a molecular weight of 28 kDa, belonging to the water-glycerol channel protein subfamily, expressed in the colon basement membrane, and participates in intestinal water reabsorption and cell volume regulation by bidirectionally transporting water molecules and glycerol. Its abnormal function can lead to diarrhea or dehydration.

[0045] (13) Brain-gut axis: A bidirectional neuro-endocrine-immune regulatory network composed of the central nervous system (CNS), autonomic nervous system (ANS), enteric nervous system (ENS) and intestinal microorganisms. It realizes the functional interaction between the brain and the intestine through the vagus nerve, HPA axis and neurotransmitters. Its disorder is closely related to diseases such as irritable bowel syndrome and depression.

[0046] (14) Brain-gut axis signaling molecules: including neuropeptides, neurotransmitters, hormones and microbial metabolites, which mediate brain-gut communication through paracrine / endocrine pathways.

[0047] (15) Corticosterone: The main glucocorticoid in rodents, secreted by the zona fasciculata of the adrenal cortex, inhibits the CRH / ACTH negative feedback loop by activating GR receptors, and regulates intestinal permeability and immune balance in stress response.

[0048] (16) 5-HT: 90% is synthesized by enterochromaffin cells (EC cells), which activate ENS neurons through 5-HT3 / 4 receptors, regulating intestinal motility and secretory reflexes; central 5-HT affects mood through SERT reuptake outside the blood-brain barrier and is the target of SSRI antidepressants.

[0049] (17) Substance P (SP): A neuropeptide of the tachykinin family, encoded by the TAC1 gene, which activates mast cells to release histamine and TNF-α through the NK1 receptor, mediating neurogenic inflammation and visceral pain sensitization. It is abnormally highly expressed in irritable bowel syndrome and fibromyalgia.

[0050] (18) Fermented dairy products: Dairy products (yogurt, kefir, etc.) fermented by lactic acid bacteria (such as Lactobacillus bulgaricus and Streptococcus thermophilus). They contain probiotics, active peptides (such as κ-casomorphin) and lactic acid, and have the effects of regulating bacterial flora, enhancing calcium absorption and reducing lactose intolerance.

[0051] (19) Viable bacterial count (CFU / g): refers to the number of viable bacteria that can form visible colonies per gram of sample. It is the core indicator for evaluating the potency of probiotic preparations and must be determined using the standard plate count method.

[0052] (20) Carrier: refers to the matrix material used to protect the activity of bacteria in probiotic preparations. It must meet the following requirements: pharmaceutical inertness, protective function and processing suitability. Common carriers also include starch, mannitol, etc.

[0053] (21) Trehalose: a non-reducing disaccharide (α-D-pyranose glucopyranosyl-α-D-pyranose glucoside), which effectively maintains the integrity of cell membranes and the spatial conformation of proteins during the freeze-drying process by forming a glassy matrix and a "water replacement" mechanism, thereby preventing ice crystal damage and oxidative stress.

[0054] (22) Skimmed milk: A dairy product that has milk fat removed by centrifugation. It contains casein (80%) and whey protein (20%). Its calcium phosphate micelle structure and lactose can effectively prevent freezing damage, and synergistically with trehalose can increase the spore survival rate to more than 90%.

[0055] (23) Logarithmic growth phase: The second stage of the bacterial growth curve, at this time μ = μmax, the bacteria grow at a rate of 2 n Exponential proliferation, OD 600 Value 0.4-0.8 (about 10 8 CFU / mL) is the best harvest period, at which the bacteria have vigorous metabolism but have not exhausted nutrients.

[0056] (24) Induction medium: A medium that triggers spore formation through specific ingredients. In this example, the key parameters include: MnCl2 concentration, carbon-nitrogen ratio, and redox potential, which can induce more than 90% of vegetative cells to transform into spores.

[0057] (25) MRS medium (de Man, Rogosa and Sharpe Medium): Chinese name “de Man, Rogosa and Sharpe medium”, a selective medium specifically used for the culture of lactic acid bacteria, containing peptone, beef extract, yeast extract, glucose and Tween 80. Its low pH value (6.2-6.5) can inhibit the growth of miscellaneous bacteria. It is a standard medium for the isolation and proliferation of Weizmannia coagulans.

[0058] (26) Inhibition zone: In the agar diffusion method, the antibacterial substances secreted by probiotics inhibit the growth of pathogens and form a transparent area. The diameter of the area quantitatively reflects the strength of the antibacterial activity.

[0059] (27) Inflammatory factors: Cell signaling molecules secreted by immune cells, including pro-inflammatory factors (such as IL-1β, IL-6, TNF-α) and anti-inflammatory factors (such as IL-10). The imbalance of these factors can lead to low-grade inflammation of the intestinal mucosa. Probiotics can improve the intestinal inflammatory state by downregulating pro-inflammatory factors and upregulating anti-inflammatory factors.

[0060] (28) IL-1β (interleukin 1β): A pro-inflammatory factor produced by macrophages that enhances the inflammatory response by activating the NF-κB pathway. Its expression is elevated in the colon tissue of patients with irritable bowel syndrome.

[0061] (29) IL-6 (interleukin 6): A multifunctional cytokine that acts as a pro-inflammatory mediator to induce acute phase protein synthesis and as an anti-inflammatory regulator to inhibit TNF-α production. Its serum concentration is positively correlated with the severity of irritable bowel syndrome symptoms.

[0062] (30) IL-10 (interleukin 10): a key anti-inflammatory cytokine secreted by regulatory T cells that maintains immune tolerance by inhibiting the function of Th1 cells and antigen-presenting cells.

[0063] (31) TNF-α (tumor necrosis factor α): A key pro-inflammatory cytokine secreted by macrophages, it promotes inflammatory responses by activating the NF-κB signaling pathway, leading to epithelial cell apoptosis and barrier function damage in intestinal inflammation.

[0064] (32) Protein expression: refers to the synthesis level of a specific protein in cells or tissues, which can be quantitatively detected by methods such as Western blot or immunohistochemistry, reflecting the post-transcriptional regulation and functional status of genes.

[0065] (33) Live bacteria survival rate: refers to the ratio of the number of live bacteria before and after treatment. It is used to evaluate the tolerance of probiotics to adverse conditions such as gastric acid and bile salts and is a core indicator of formulation stability.

[0066] (34) Pepsin: A digestive enzyme secreted by gastric chief cells. It is most active at pH 2.5 and can degrade proteins. It is the first digestive barrier that probiotics need to overcome after oral administration.

[0067] (35) Bile salts: amphiphilic molecules synthesized by the liver and stored in the gallbladder. They promote lipid digestion through emulsification and have antibacterial activity, affecting the intestinal survival of probiotics.

[0068] (36) Scanning electron microscopy (SEM): A microscopic analysis technique that uses a focused electron beam to scan the sample surface and obtain a high-resolution three-dimensional morphological image of the sample surface by detecting secondary electron and backscattered electron signals. Its resolution can reach the nanometer level and is widely used in the characterization of microstructures in fields such as materials science and biology.

[0069] (37) Rifaximin: A non-absorbable antibiotic, a rifamycin derivative, which exerts a broad-spectrum antibacterial effect by specifically inhibiting bacterial RNA polymerase. It is mainly used to treat traveler's diarrhea, hepatic encephalopathy and irritable bowel syndrome. Its local intestinal action characteristics can reduce systemic side effects.

[0070] (38) LB medium (Luria-Bertani Medium): Chinese name "Luria-Bertani medium", a universal bacterial liquid / solid culture medium, the main components of which are peptone, yeast extract and sodium chloride, pH 7.0-7.4, widely used for the culture and amplification of Gram-negative bacteria such as Escherichia coli.

[0071] (39) XLD medium (Xylose Lysine Deoxycholate Agar): Chinese name "Xylose Lysine Deoxycholate Agar Medium", a selective identification medium containing xylose, lysine and sodium deoxycholate. It uses phenol red as an indicator to distinguish Salmonella from Shigella and is used for the isolation and identification of intestinal pathogens.

[0072] (40) PALCAM medium (Polymyxin-Acriflavine-LiCl-Ceftazidime-Aesculin-Mannitol Agar): Chinese name: "Polymyxin-Acriflavine-Lithium Chloride-Ceftazidime-Aesculin-Mannitol Agar Medium", a selective culture medium for Listeria, containing polymyxin B, ceftazidime and aesculin, and is identified by the black hydrolysis zone and gray-green colonies unique to Listeria.

[0073] (41) MacConkey Agar: A culture medium for the identification of intestinal bacteria containing bile salts and neutral red. It can distinguish lactose-fermenting bacteria from non-fermenting bacteria and inhibit the growth of Gram-positive bacteria.

[0074] (42) Columbia Blood Agar: A nutrient-rich, non-selective medium containing 5-10% defibrinated sheep blood that supports the growth of fastidious bacteria and allows for preliminary identification by hemolysis type (α / β / γ).

[0075] (43) TSA medium (Tryptic Soy Agar): Chinese name: "Tryptic Soy Agar Medium", a general solid culture medium composed of tryptone, soy peptone, sodium chloride and agar, pH 7.3±0.2. Its high nutritional properties support the growth of most non-fastidious bacteria and is commonly used for microbial isolation and culture, purity testing and strain preservation.

[0076] (44) TSB medium (Tryptic Soy Broth): Chinese name: “Tryptic Soy Broth”, a general-purpose liquid culture medium, the main components of which are tryptone, soy peptone, sodium chloride, and dipotassium hydrogen phosphate, with a pH of 7.3±0.2. Its high nutritional properties support the growth of a variety of fastidious and non-fastidious bacteria, including Salmonella enteritidis, and is commonly used for bacterial amplification, drug sensitivity testing, and biofilm research.

[0077] (45) BHI liquid medium (Brain Heart Infusion Broth): Chinese name: “Brain Heart Infusion Broth”, a high-nutrient liquid medium containing bovine brain extract and bovine heart extract, supplemented with peptone, glucose, and sodium chloride, pH 7.4±0.2. It is suitable for culturing pathogens with demanding nutritional requirements and can also be used for the enrichment of microorganisms in blood specimens.

[0078] (46) Manganese chloride: A divalent manganese ion compound that acts as a spore formation inducer in microbial culture and promotes morphological differentiation by activating spore-specific sigma factors.

[0079] (47) Biosafety Cabinet: A type of laboratory equipment that uses a high-efficiency filtration system and directional airflow design to protect personnel, samples, and the environment when handling pathogenic microorganisms. It is divided into three levels, of which Level II is the most commonly used. It is suitable for handling most pathogens and can prevent microbial leakage and cross contamination. It is an essential safety protection equipment for microbiology laboratories.

[0080] (48) Bovine serum albumin solution: An aqueous solution prepared from albumin extracted from bovine serum. In this study, 5% bovine serum albumin solution was used as an immunohistochemical blocking solution. Its nonspecific protein adsorption properties blocked residual sites on the surface of tissue sections, effectively reducing the nonspecific binding background of the antibody while maintaining the specific binding ability of the primary antibody to the target protein.

[0081] Sources: (1) Culture medium: MRS culture medium was purchased from Beijing Solebeau Technology Co., Ltd.; LB culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; XLD culture medium was purchased from Guangdong Huankai Microbiology Technology Co., Ltd.; PALCAM culture medium was purchased from Hangzhou Microbiology Reagent Co., Ltd.; MacConkey agar plates were purchased from Shanghai Yihua Clinical Medicine Technology Co., Ltd.; Columbia blood agar plates were purchased from Guangdong Huankai Microbiology Technology Co., Ltd.

[0082] (2) Chemical reagents: Manganese chloride was purchased from Sinopharm Chemical Reagent Co., Ltd.; trehalose was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; skim milk was purchased from Beijing Aoboxing Biotechnology Co., Ltd.; pepsin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; bile salts were purchased from Xilong Science Co., Ltd.; rifaximin was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; 4% paraformaldehyde was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; DAB colorimetric solution was purchased from Wuhan Bosted Bioengineering Co., Ltd.; hematoxylin was purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.; 3% hydrogen peroxide solution was purchased from Xilong Science Co., Ltd.; physiological saline was purchased from Sichuan Kelun Pharmaceutical Co., Ltd.; sterile PBS buffer (pH 7.4) was purchased from Beyotime Biotechnology Co., Ltd.; 5% bovine serum albumin solution was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0083] (3) Instruments and equipment: Scanning electron microscope (SEM) was purchased from JEOL Ltd.; ELISA tester was purchased from Bio-Rad, Inc.; microscope was purchased from Nikon Instruments (China) Co., Ltd.; electronic balance was purchased from Mettler-Toledo (China) Co., Ltd.; centrifuge was purchased from Hunan Hercy Instrument Equipment Co., Ltd.; biosafety angle rotor was purchased from Hunan Hercy Instrument Equipment Co., Ltd.; constant temperature incubator was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.; biosafety cabinet was purchased from Suzhou Antai Air Technology Co., Ltd.; microplate reader was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0084] (4) Yogurt: Mengniu plain yogurt available on the market.

[0085] (5) Mice: This study used 40 healthy adult male C57BL / 6 mice, aged 8-10 weeks, with an initial body weight of 20±2 g. All mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.

[0086] Bacterial solution preparation: (1) Preparation of Weizmannella coagulans W-1 culture medium: Weizmannella coagulans W-1 in the cryopreserved tube was streaked onto the surface of MRS solid culture medium under sterile conditions and cultured in a 37°C constant temperature incubator for 48 hours to obtain single colonies. After the colonies were formed, single colonies with typical morphology were selected and transferred to liquid MRS culture medium at a 1% to 5% inoculum volume. The culture was shaken at 37°C and 150 rpm for 24 hours to prepare W-1 culture medium.

[0087] (2) Preparation of coagulated Weizmannella W-1 bacterial suspension: The W-1 bacterial suspension was subcultured twice under the same conditions, each time for 24 hours. After the culture was completed, the bacterial suspension was centrifuged at 4°C and 3000 r / min for 10 minutes, the bacterial precipitate was collected, and washed twice with sterile PBS buffer (pH 7.4) to remove the residual culture medium. Finally, the bacterial suspension was resuspended in PBS and the concentration was adjusted to about 1×10 9 CFU / mL, and obtain a uniform and stable W-1 bacterial suspension.

[0088] (3) Preparation of Weizmannella coagulans BC99 bacterial suspension: The frozen BC99 strain was streaked onto MRS solid medium under sterile conditions and placed in a 37°C constant temperature incubator for anaerobically cultured for 48 hours. A single colony was picked and inoculated into MRS liquid medium and cultured at 37°C and 150 rpm for 24 hours until the logarithmic growth phase. The bacterial suspension was centrifuged at 3000×g for 10 minutes to collect the cells, washed twice with sterile PBS buffer (pH 7.4), and finally resuspended to 1×10 9 CFU / mL is reserved.

[0089] (4) Preparation of 5% Escherichia coli culture: Take the Escherichia coli strain and streak it on LB solid plate in a Class II biosafety cabinet and culture it at 37℃ for 24 hours. Pick a single colony in the biosafety cabinet and inoculate it into LB liquid culture medium aseptically. Seal the culture bottle and move it out of the biosafety cabinet. Culture it at 37℃ and 200 rpm for 12 hours until the logarithmic growth phase. Return the culture liquid to the biosafety cabinet for subsequent processing: centrifuge at 5000×g for 5 minutes, wash twice with sterile PBS buffer (pH 7.4), and finally adjust the concentration to 1×10 8 CFU / mL. Aliquot the bacterial solution into sealed cryopreservation tubes, disinfect the outer surface, and transfer to -80℃ for storage or use immediately in antibacterial experiments.

[0090] (5) Preparation of 5% Enteritidis Salmonella liquid: Operate in a Class II biosafety cabinet, inoculate the standard strain of Enteritidis Salmonella into XLD medium, and culture at 37°C for 24 hours. Pick typical black colonies in the safety cabinet, transfer to sealed TSB liquid culture medium, immediately seal the bottle mouth and move out of the safety cabinet, and culture at 37°C and 180 rpm for 16 hours. The bacterial liquid treatment is strictly carried out in the safety cabinet: centrifuge at 4000×g for 8 minutes using a biosafety angle rotor, wash twice with pre-cooled saline containing 0.85% NaCl, and finally adjust the concentration to 5×10 7 CFU / mL. Aliquots of bacterial solution must be labeled with a biohazard symbol, and waste must be sterilized by autoclaving at 121°C.

[0091] (6) Preparation of 5% Listeria monocytogenes culture liquid: The whole process was completed in a Class II biosafety cabinet. The Listeria monocytogenes strain was cultured on PALCAM selective medium at 30°C for 48 hours. Typical gray-green colonies were selected in the safety cabinet and inoculated into a BHI liquid culture medium bottle with a filter membrane. After sealing, the culture was shaken at 30°C and 150 rpm for 18 hours until the stable period. The bacterial liquid was strictly processed in the safety cabinet: centrifuged at 6000×g for 10 minutes using a biosafety angle rotor, and resuspended to 1×10 7 CFU / mL.

[0092] (7) Preparation of 5% Shigella dysenteriae bacterial solution: All operations must be performed in a Class II biosafety cabinet. Use the Shigella dysenteriae strain, streak on a MacConkey agar plate, and culture at 37°C for 24 hours. Pick colorless and transparent colonies in the safety cabinet, inoculate them into GN broth, and use sealed culture bottles to culture at 37°C for 18 hours. The entire bacterial solution processing process is completed in the safety cabinet: centrifugation at 4000×g for 10 minutes, washing twice with 0.9% sterile saline, avoiding vortexing when resuspending, and adjusting the final concentration to 1×10 8 CFU / mL. All items must be autoclaved after the experiment, and spills should be handled according to BSL-2 emergency procedures.

[0093] (8) Preparation of 5% Streptococcus agalactiae liquid: The operation was carried out in a Class II biosafety cabinet. The Streptococcus agalactiae strain was streaked on a Columbia blood agar plate and cultured in a 5% CO2 incubator at 37°C for 24 hours. β-hemolytic colonies were picked in the safety cabinet and inoculated into a THB liquid culture medium bottle with a breathable membrane. After sealing the bottle, the culture was incubated at 37°C and 5% CO2 for 12 hours. The bacterial liquid was processed strictly in the safety cabinet: centrifuged at 3000×g for 8 minutes using a biosafety angle rotor, washed with PBS at pH 7.2, and adjusted to 5×10 7 CFU / mL. Pay special attention to the possibility that clinical isolates may carry drug-resistant genes. All waste should be sterilized at 121°C for 30 minutes.

[0094] Reagent preparation: (1) Preparation of simulated gastric fluid: Use 0.3% pepsin + 0.2% NaCl, and adjust the pH to 2.5 with HCl. After sterilization by filtration through a 0.22 μm filter membrane, store at 4°C until use. All operations are performed under sterile conditions.

[0095] (2) Preparation of simulated intestinal fluid: 0.3% bile salt is dissolved in PBS. 0.1% trypsin can be optionally added to enhance the simulation effect. The solution is sterilized at 121°C for 15 minutes before use to ensure a sterile operating environment.

[0096] (3) Preparation of induction medium: Add 0.1-1 mM MnSO4 to MRS basal medium, and supplement 1 mM CaCl2 if necessary to increase the spore formation rate. The medium is sterilized at 121°C for 15 min and then divided into packages. After incubation at 37°C for 48-72 h, spore formation is observed. Strictly maintain sterile conditions throughout the process.

[0097] Example 1: Antibacterial experiment Take a Weizmannella coagulans W-1 culture medium and place it in a sterilized LB solid medium in a clean bench. Allow it to cool to approximately 50°C. Then, inoculate it with 5% culture medium of Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, Shigella dysenteriae, and Streptococcus agalactiae. Shake well and pour onto a plate. After the LB solid medium cools, punch a well and add 100 μL of the Weizmannella coagulans W-1 culture medium. Incubate at 37°C for 24 hours, and observe and measure the size of the inhibition zone.

[0098] The experimental results are shown in Table 1: Table 1 Record of inhibition zone size of different pathogenic bacteria

[0099] The experimental results are shown in Table 1 and Figure 1 As shown, Weizmannella coagulans W-1 exhibited significant antibacterial activity against all five enteric pathogens tested. The largest inhibition zone diameter was observed against Listeria monocytogenes, followed by Streptococcus agalactiae, Escherichia coli, and Salmonella Enteritidis. The inhibitory effect against Shigella dysenteriae was relatively weak. The differences in antibacterial activity among the strains were statistically significant (p < 0.05).

[0100] Conclusion: The experiments confirmed that Weizmannella coagulans W-1 can effectively inhibit the growth of several common intestinal pathogens, with the most significant inhibitory effect on Listeria monocytogenes. This broad-spectrum antibacterial activity suggests that Weizmannella coagulans W-1 has the potential to improve intestinal health by regulating the balance of intestinal flora.

[0101] Example 2: Construction of an irritable bowel syndrome animal model Restraint stress was used to induce an irritable bowel syndrome mouse model. The specific procedures were as follows: Mice were randomly divided into four groups (n=10): (a) normal group, (b) IBS model group, (c) positive control group (treated with rifaximin), and (d) probiotic group (treated with Weizmannella coagulans W-1). All mice were housed in a standard environment with a constant temperature and a 12-hour light / dark cycle, with free access to food and water. After 7 days of adaptive feeding, mice in the normal group, except for the normal group, were subjected to 2 hours of restraint stress daily at a fixed time for 3 consecutive weeks to establish an IBS model. One week after model establishment, the positive control group began daily oral administration of rifaximin solution, while the probiotic group began daily oral administration of 0.2 mL of Weizmannella coagulans W-1 solution. Body weight and fecal characteristics of the mice were regularly monitored during the experiment.

[0102] During the experiment, all the following operations were performed in an experimental environment with strictly controlled temperature (25±1°C) and humidity (50±5%). Weight changes and fecal characteristics of each group of mice were systematically monitored: The mice's weight was measured using an electronic balance at a fixed time each week, ensuring that the mice were fasting for 4 hours before measurement. Each weighing was performed individually in a clean weighing box. The data was recorded after the reading stabilized. The measurement was repeated three times for each mouse and the average was taken. Fecal sample collection and testing were performed simultaneously: fresh feces were collected immediately after weighing, and the wet weight index (W1) was recorded using a precision analytical balance. The collected feces were placed in an oven and dried at 60°C for dehydration, and the dry weight index (W2) was recorded. Finally, the moisture content of the mouse feces was calculated.

[0103] Fecal moisture content% = (W1−W2) / W1× 100% The experimental results are as follows Figure 2 and Figure 3 As shown, while all groups of mice showed an increasing weight trend, the weight gain in the model group, positive control group, and probiotic group subjected to restraint stress was significantly lower than that in the normal control group. Compared with the normal control group, the stressed group mice exhibited typical symptoms of irritable bowel syndrome, such as increased fecal water content and abnormal fecal morphology. After treatment with Weizmannella coagulans W-1, the fecal water content of the probiotic group mice was significantly reduced, and diarrhea symptoms were significantly improved.

[0104] Conclusion: This study successfully established an IBS mouse model through 2 weeks of restraint stress. This model exhibited clinical symptoms similar to those of human IBS patients, including restricted weight gain and altered stool characteristics. This study also confirmed the ameliorative effect of Weizmannella coagulans W-1 on the diarrheal symptoms of IBS.

[0105] Example 3: Comparison of brain-gut axis signaling molecule levels After orbital bleeding, whole blood samples were incubated at 4°C for 30 minutes and then centrifuged at 3000 rpm for 10 minutes to separate serum. Serum concentrations of corticosterone, serotonin, and substance P were measured using an enzyme-linked immunosorbent assay (ELISA) at 450 nm using a microplate reader. Three replicates were performed for each sample, and the data were averaged.

[0106] The experimental results are as follows Figure 4-6 As shown, decreased serum corticosterone levels indicate that Weizmannella coagulans W-1 can effectively alleviate stress responses and enhance the body's anti-stress capacity. Decreased 5-HT and SP levels confirm that Weizmannella coagulans W-1 can effectively inhibit the overproduction of sensitizing factors such as 5-HT and SP, improving intestinal nerve sensitivity. Furthermore, all indicators in the Weizmannella coagulans W-1 group approached those of the normal control group.

[0107] Conclusion: This experiment confirmed that Weizmannella coagulans W-1 can regulate the brain-gut axis function through multiple targets and has important potential to regulate intestinal function through neuro-endocrine pathways.

[0108] Example 4: Weizmannella coagulans W-1 intervention improves colon tissue damage in a mouse model of irritable bowel syndrome Immediately after killing mice, colon tissue was harvested 2 cm from the anus and fixed with 4% paraformaldehyde for 24 hours. Dehydrated with graded ethanol, the sections were embedded in paraffin, and sections were prepared. Following hematoxylin staining, the sections were observed under a light microscope for morphological changes in the colon, with a particular focus on assessing mucosal integrity, epithelial cell arrangement, and inflammatory cell infiltration.

[0109] The experimental results are as follows Figure 7 As shown, the colonic tissue of mice in the normal group showed intact mucosal structure, with neatly arranged crypts and no inflammatory cell infiltration. In contrast, the model group displayed typical pathological damage characteristics, including mucosal epithelial damage (indicated by red arrows in the figure), disordered crypt structure, and significant inflammatory cell infiltration. Notably, after intervention with Weizmannella coagulans W-1, the colonic histopathological changes in the treated mice were significantly improved, with the mucosal structure basically restored to normal and the epithelial cells tightly arranged.

[0110] Conclusion: This experiment confirmed that Weizmannella coagulans W-1 has the function of maintaining tight junctions between epithelial cells, inhibiting inflammatory cell infiltration and promoting the recovery of mucosal barrier function.

[0111] Example 5: Effects of expression of tight junction proteins and aquaporins This study systematically evaluated the effects of probiotic intervention on the expression of key intestinal mucosal barrier proteins using immunohistochemistry. Colonic tissue from each group of mice was fixed with 4% paraformaldehyde, and tissue sections were prepared using standard paraffin-embedding procedures. To accurately detect target protein expression, sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity, and nonspecific binding sites were blocked with 5% bovine serum albumin solution. The sections were then incubated with primary antibodies against claudin-1, ZO-1, and AQP3, respectively, overnight at 4°C. Following secondary antibody incubation, target protein expression was visualized using the DAB colorimetric system, and cell nuclei were counterstained with hematoxylin. Quantitative analysis was performed using ImageJ software.

[0112] The experimental results are as follows Figure 8-10 As shown, compared with the model group, the expression levels of tight junction proteins claudin-1 and ZO-1 in the W-1 group were upregulated, indicating that probiotics can effectively repair damaged intestinal epithelial tight junction structure. Increased expression of the aquaporin AQP3 confirms its ability to improve intestinal water transport function. The expression levels of each indicator in the W-1 group were close to those of the normal control group.

[0113] Conclusion: This experiment confirmed that Weizmannella coagulans W-1 enhances the junction between epithelial cells and reduces intestinal permeability by upregulating the expression of tight junction proteins; at the same time, it regulates the expression of aquaporins to improve the balance of intestinal water and salt metabolism, thereby improving the diarrhea symptoms of irritable bowel syndrome.

[0114] Example 6: Comparison of inflammatory cytokines in colon tissue Mouse colon tissue was homogenized in pre-chilled saline and centrifuged at 3500 rpm for 10 minutes at 4°C. The supernatant was collected. The levels of inflammatory factors (IL-1β, IL-6, IL-10, and TNF-α) in the supernatant were assayed using enzyme-linked immunosorbent assay (ELISA). A standard curve and replicate controls were established to ensure assay accuracy.

[0115] Table 2 Contents of inflammatory factors in colon tissue homogenates of mice in each group (unit: pg / mL)

[0116] Note: In the same column, a represents a significant difference compared with the normal group, and b represents a significant difference compared with the model group. (P<0.05) The experimental results are shown in Table 2. The levels of proinflammatory factors IL-1β, IL-6 and TNF-α in the model group were significantly higher than those in the normal group. After intervention with Weizmannella coagulans W-1, the levels of proinflammatory factors in the W-1 group were reduced by 8.9%-18.4% compared with the model group (p<0.05). At the same time, the content of anti-inflammatory factor IL-10 increased by 1.08 times (p<0.01). All indicators were close to the levels of the normal group.

[0117] Conclusion: This study confirmed that Weizmannella coagulans W-1 can inhibit the release of pro-inflammatory factors and promote the secretion of anti-inflammatory factors, thereby effectively alleviating irritable bowel syndrome.

[0118] Example 7: Comparative experiment on gastrointestinal fluid tolerance of marine and terrestrial Weizmannella coagulans To verify the gastrointestinal adaptability of marine-derived Weizmannella coagulans W-1, we selected marine-derived Weizmannella coagulans W-1 as the experimental group and terrestrial-derived Weizmannella coagulans BC99 (CGMCC NO: 21801, isolated from the intestines of infants) as the control group. Both strains were cultured to the logarithmic growth phase. After washing with PBS, the cells were incubated in simulated human gastric fluid (pH 2.5) containing 0.3% pepsin at 37°C with shaking for 2 hours. The cells were then transferred to simulated human intestinal fluid (containing 0.3% bile salts, pH 6.8) for another 4 hours. Viable cell counts were determined periodically.

[0119] The experimental results are shown in Table 3: Table 3 Comparison of the survival rates of marine and terrestrial Weizmannella coagulans at different treatment times

[0120] The experimental results are shown in Table 3. The marine-derived Weizmannella coagulans W-1 has a significant advantage in gastrointestinal fluid tolerance, which can ensure that a sufficient number of bacteria can survive and function in the human body.

[0121] Example 8: Preparation and stability test of microbial preparation The microbial preparations were prepared by the following method: ① Bacterial culture: Weizmannella coagulans W-1 was inoculated into MRS liquid medium at pH 6.0 and cultured at 37°C under aerobic conditions until OD 600 =0.8, obtain bacterial solution in logarithmic growth phase; ② Spore induction: Transfer the bacterial solution obtained in step ① to an induction medium containing 0.1 mM MnSO4 and continue to culture at 37°C under aerobic conditions for 48 hours to form spores; ③ Bacteria-carrier mixing: The spore-forming bacteria obtained in step ② are collected by centrifugation and mixed with the pharmaceutically acceptable carrier described in the third aspect to prepare a microbial preparation, which is in the form of a lyophilized powder.

[0122] Take 100 mg of lyophilized powder and add 900 μL of sterile saline (containing 0.1% Tween 80), vortex for 3 minutes to completely resuspend. Then perform 10-fold serial dilution, select 10 -6 , 10 -7 , 10 -8Three appropriate dilutions were prepared, and 100 μL of each dilution was evenly spread on a pre-dried TSA plate. Three parallels were set for each dilution, and a sterile saline blank control was also set up. The plates were placed in a 37°C constant temperature incubator, incubated upright for 1 hour, and then inverted for 48 hours before counting. The number of colonies at each dilution was recorded using a colony counter and calculated according to the formula: Viable bacteria count (CFU / g) = (average colony count × dilution factor × 10) / sample volume (g) The experimental results are shown in Table 4: Table 4. Viable bacteria counts of Weizmannella coagulans W-1 lyophilized powder at different dilutions

[0123] The prepared lyophilized powder was divided into sterile vials and subjected to long-term stability test at a constant temperature of 25°C. Samples were taken for testing at 0, 30, 60, 90, 180, 270, and 365 days of storage, and three independently packaged samples were randomly selected as parallel samples at each time point. During the test, the samples were graded diluted with phosphate buffer containing 0.1% (w / v) Tween 80, with a dilution factor of 10. -6 100 μL was evenly spread on a tryptone soy agar plate and cultured in a 37°C constant temperature incubator for 48 hours before colony counting. The number of viable bacteria was calculated according to the above formula. The average of three parallel experiments was taken as the average colony count.

[0124] The experimental results are shown in Table 5: Table 5 Evaluation of the effect of storage time on microbial activity

[0125] The experimental results are shown in Table 4-5. The three dilutions (10 -6 ~10 -8 ) have good consistency in the counting results, and the viable bacteria count results are within 10 8 -10 11 The viable bacterial count declined slowly in the first 90 days (survival rate 65.2%), but accelerated significantly after 180 days, reaching a survival rate of 7.8% after 365 days, possibly due to the gradual depletion of the protective agent or oxidative damage.

[0126] The same method as in this example was used, except that strain W-1 was replaced with terrestrial BC99. Experimental results showed that the relative initial survival rate of terrestrial BC99 was: 0 day: 100%; 30 days: 93.5%; 60 days: 79.8%; 90 days: 63.1%; 180 days: 37.4%; 270 days: 18.8%; and 365 days: 7.1%.

[0127] Conclusion: The experimental results confirm that the microbial preparation obtained by the preparation method of the present invention has excellent stability, far superior to conventional probiotics that require cold chain transportation, and fully meets the requirements of industrial production and clinical application.

[0128] Example 9: Optimization of freeze-drying protective agent and stability test of microbial preparation Based on the pre-prepared coagulant Weizmannella W-1 bacterial solution, the effects of five different formulations of freeze-dried protective agents on bacterial survival were systematically investigated. Five groups of protective agent systems were set up in the experiment: the first group was a composite protective agent (10% trehalose + 10% skim milk), the second group was a single protective agent of 15% trehalose, the third group was a single protective agent of 15% skim milk, the fourth group used 10% glycerol as a positive control, and the fifth group did not add any protective agent as a negative control. All protective agents were prepared with sterile saline and mixed with the W-1 bacterial solution in the logarithmic growth phase to keep the final bacterial concentration at 1×10 9 CFU / g. Prefreeze at -80°C for 2 hours, then freeze-dry at -50°C with a vacuum of ≤0.1 mbar for 24 hours. Then, perform room temperature storage experiments, sampling and testing at 0, 7, 15, 30, 60, and 90 days. The survival rate of Weizmannella coagulans W-1 was calculated using the following formula: Survival rate (%) = [(CFU / g after freeze-drying) / (CFU / g before freeze-drying)] × 100% The experimental results are shown in Table 6: Table 6 Experimental comparison of the effects of different freeze-dried protective agents on the survival rate of Weizmannella coagulans W-1

[0129] The experimental results, shown in Table 6, showed that the composite protective agent group demonstrated the best protective performance. After 90 days of freeze-drying, the bacterial survival rate reached 82.4%, significantly higher than that of the other groups (p<0.05). Specifically, the survival rates of the 15% skim milk group were 60.5%, the 15% trehalose group was 30.1%, and the 10% glycerol control group was only 12.7%. The group without protective agent had virtually no survival.

[0130] Conclusion: In the composite protective agent composed of 10% trehalose and 10% skim milk, trehalose protects the cell structure by forming a stable glassy matrix, while skim milk provides a protein protective layer. The synergistic effect of the two significantly improves the anti-freeze-drying damage ability of the bacteria, providing reliable technical parameters for the industrial production of freeze-dried preparations of Weizmannella coagulans W-1.

[0131] Example 10: Application of microbial preparations There were two groups: group I (commercial yogurt control) and group II (commercial yogurt + 1×10 9The samples of Group II were added with bacterial powder after fermentation and then dispensed into 100 mL sterilized cups and refrigerated at 4°C for 24 h. The samples were blindly scored by a 10-person professional taste evaluation panel (0-5 points, including taste, smell, texture and acceptance). A pH meter was used to measure acidity changes, and MRS medium (anaerobic culture at 37°C for 48 h) was used to detect the number of viable bacteria. All tests were performed on 0 and 21 days of storage, and the experiments were repeated three times.

[0132] The sensory evaluation results are shown in Table 7. The acidity and pH value results are shown in Table 7: Table 7 Comparison of sensory evaluation, acidity and pH index of yogurt in group I and group II

[0133] The experimental results are shown in Table 7. The sensory score of group II added with Weizmannella coagulans W-1 freeze-dried powder was significantly better than that of the control group after storage for 14 days, and the acidity change was not significantly different from that of the control group.

[0134] Conclusion: The addition of Weizmannella coagulans W-1 freeze-dried powder can improve the sensory quality of yogurt, maintain a good viable cell count, and does not affect the physicochemical properties of the product. It is recommended to be added as a functional ingredient after fermentation. The shelf life can reach more than 21 days when stored at 4℃.

[0135] The marine-derived Weizmannella coagulans W-1 provided in the above examples exhibits excellent gastrointestinal tolerance. This strain enhances intestinal barrier function by broadly inhibiting pathogens and upregulating tight junction proteins (claudin-1 / ZO-1) and aquaporin (AQP3). It also modulates brain-gut axis signaling molecules (reducing corticosterone, 5-HT, and substance P levels) to alleviate visceral hypersensitivity and significantly reduces the expression of pro-inflammatory cytokines (IL-1β / IL-6 / TNF-α). This microbial preparation exhibits excellent stability and can be used in pharmaceuticals, foods, and health supplements. Through a triple mechanism of "bacteriostasis, barrier repair, and neural regulation," it synergistically improves irritable bowel syndrome, providing a novel marine microbial resource for the treatment of intestinal diseases.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A marine-derived strain of Weizmannella coagulans ( Weizmannia coagulans ) W-1, characterized in that, It was isolated from dolphin stomach and deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCCNO:31804.

2. Use of the Weizmannella coagulans W-1 according to claim 1 in the preparation of a preparation for improving intestinal health.

3. The use according to claim 2, characterized in that The preparation is used for: Inhibiting intestinal pathogens, wherein the pathogens include at least one of Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, Shigella dysenteriae and Streptococcus agalactiae; and / or Upregulating the expression of tight junction proteins and aquaporins in intestinal epithelial barrier function regulatory proteins; optionally, the tight junction proteins in the preparation include claudin-1 and ZO-1, and the aquaporins include AQP3; and / or Reducing the level of serum brain-gut axis signaling molecules; optionally, the brain-gut axis signaling molecules comprise corticosterone, serotonin, and substance P.

4. A microbial preparation, characterized in that The invention comprises the Weizmannella coagulans W-1 according to claim 1 as an active ingredient and a pharmaceutically acceptable carrier, wherein the carrier comprises trehalose and skim milk, and optionally further comprises glycerol.

5. The microbial preparation according to claim 4, characterized in that The preparation is in the form of a freeze-dried powder; optionally, the freeze-dried powder is used to prepare medicines, foods or health products.

6. The microbial preparation according to claim 5, characterized in that The dosage form of the medicine is capsule, tablet or powder; and / or, the food comprises a fermented dairy product or a solid beverage, and the fermented dairy product is preferably yogurt; and / or, the health product comprises capsules or powder.

7. The microbial preparation according to claim 4, characterized in that The Weizmannella coagulans W-1 in the preparation is a viable bacterium or spore; and / or the number of viable bacteria of Weizmannella coagulans W-1 in the preparation is 1×10 8 ~1×10 11 CFU / g.

8. The microbial preparation according to claim 4, characterized in that The mass ratio of trehalose to skim milk is 1:1-3:1, preferably 1:

1.

9. A method for preparing the microbial preparation according to claim 4, characterized in that: The method comprises culturing Weizmannella coagulans W-1 to a logarithmic growth phase, inducing spore formation, and mixing the bacteria with a carrier.

10. The method according to claim 9, characterized in that The following steps are involved: ① Bacterial culture: Weizmannella coagulans W-1 was inoculated into MRS liquid medium with a pH of 6.0-7.5 and cultured at 37°C under aerobic conditions until OD 600 =0.6-1.0, obtain the bacterial solution in the logarithmic growth phase; ② Spore induction: Transfer the bacterial solution obtained in step ① to an induction medium containing 0.1-1 mM MnSO4 and continue to culture at 37°C under aerobic conditions for 48-72 hours to form spores; ③ Bacteria-carrier mixing: The spore-forming bacteria obtained in step ② are collected by centrifugation and mixed with the pharmaceutically acceptable carrier according to claim 4 to prepare a microbial preparation.

Images