Application of a strain of k. marxianus and its selenium-enriched preparation in promoting the production of intestinal indole-3-propionic acid

Abstract

The application belongs to the technical field of microorganisms and nutritional health, and discloses a strain of Kluyveromyces marxianus FZU-Km and application of a selenium-rich fortifying preparation (KmSe) thereof in promoting the production of indole-3-propionic acid (IPA) in the intestinal tract. The strain was preserved in the China General Microbiological Culture Collection Center on June 23, 2026, and the preservation number is CGMCC No. 39388. The strain can drive the metabolic conversion of tryptophan in the intestinal tract to the IPA branch, and significantly improve the endogenous IPA level in the intestinal tract. The selenium-rich fortifying preparation has a significant synergistic effect on promoting the production of IPA and repairing the intestinal mucosal barrier. The strain and the selenium-rich fortifying preparation can also significantly improve the abundance of Akkermansia and other beneficial bacteria, and inhibit Klebsiella, Escherichia-Shigella and other conditional pathogenic bacteria, and can be used for preparing microecological preparations, special medical foods and functional health products targeting the metabolic axis of the intestinal tract.

Description

Technical Field

[0001] This invention belongs to the field of microbial and nutritional health technology, specifically relating to a newly isolated and identified *Kluyveromyces martensii* strain FZU-Km and its selenium-enriched fortified preparation obtained by fermentation in a selenium-containing medium. More specifically, this invention relates to the novel applications of this strain and its selenium-enriched preparation in promoting the efficient production of endogenous indole-3-propionic acid (IPA) in the host gut, maintaining the integrity of the intestinal mucosal barrier, alleviating intestinal stress damage, and in the development of metabiotics, foods for special medical purposes, functional health foods, and feed additives targeting the intestinal metabolic axis. Background Technology

[0002] With the transformation of modern lifestyles, the refinement of dietary structures, antibiotic overuse, and increased environmental pressure, gut microbiota dysbiosis and impaired intestinal mucosal barrier function have become global public health challenges. Statistics show that over 10% of the global population suffers from varying degrees of intestinal dysfunction, and the incidence of functional bowel disease, inflammatory bowel disease (IBD), and irritable bowel syndrome (IBS) is on the rise. The intestinal mucosal barrier, as the body's first line of defense against exogenous pathogens and endotoxins, is vulnerable; once its integrity is compromised, it can lead to "leaky gut," resulting in serious consequences such as endotoxin translocation, systemic low-grade chronic inflammation, metabolic syndrome, and even neurodegenerative diseases. Currently, conventional clinical and commercial interventions mainly rely on chemical drugs (such as aminosalicylic acids, glucocorticoids, and immunosuppressants), exogenous nutritional supplements, or traditional multi-strain probiotic preparations. However, the aforementioned methods generally suffer from the following inherent drawbacks: chemical drugs have poor long-term tolerance and significant side effects; exogenous nutritional supplements have single targets, making it difficult to reshape the gut microbiota homeostasis; and multi-strain compound preparations suffer from competition for survival sites, antagonism of nutrient substrates, and interference with metabolic pathways among different strains, resulting in difficulties in industrial quality control, poor batch stability, low colonization efficiency in the host gut, and unpredictable effects. Therefore, developing a safe, long-acting, multi-target synergistic, and long-term edible functional microbiota intervention strategy has become an urgent need for the health industry.

[0003] Extensive basic research has confirmed that gut microbiota-mediated tryptophan metabolism is a core metabolic hub connecting host nutrition, gut microbiota interaction, and immune regulation. Dietary tryptophan is metabolized by gut microbiota to generate indole and its various derivatives, including indole-3-lactic acid (ILA), indole-3-carboxaldehyde (ICA), indole-3-acetic acid (IAA), and indole-3-propionic acid (IPA). Currently, increasing the level of endogenous indole-3-propionic acid (IPA) in the gut is considered one of the key strategies for maintaining the integrity of the intestinal mucosal barrier. Indole-3-propionic acid (IPA), as a high-affinity endogenous ligand for aryl hydrocarbon receptors (AhR) and pregnane X receptors (PXR), can activate downstream signaling pathways, upregulate the expression of tight junction proteins in intestinal epithelial cells (such as ZO-1, Occludin, and Claudin-1), reduce intestinal permeability, and maintain mucosal integrity. Simultaneously, indole-3-propionic acid (IPA) is one of the most potent natural free radical scavengers known to date, with antioxidant capacity far exceeding that of traditional antioxidants such as vitamin E, and it can cross the blood-brain barrier to exert a central nervous system protective effect. Furthermore, indole-3-propionic acid (IPA) can also regulate the differentiation of immune cells in the intestinal lamina propria, promote the generation of regulatory T cells (Tregs), and inhibit the activation of excessive inflammatory signaling pathways such as NF-κB. Clinical studies have shown that the levels of indole-3-propionic acid (IPA) in the feces and serum of individuals with impaired gut health (such as patients with IBD and IBS) are significantly lower than those in healthy controls. Furthermore, IPA levels are significantly negatively correlated with disease activity, suggesting that endogenous IPA deficiency is a key characteristic of intestinal pathological states. Therefore, targeting and reshaping the intestinal tryptophan metabolic profile and directionally increasing endogenous IPA levels are crucial strategies for maintaining gut health.

[0004] However, existing technologies for increasing intestinal indole-3-propionic acid (IPA) levels all face significant bottlenecks, and no effective single-strain solution has yet been developed in this field. First, the direct supplementation strategy of exogenous indole-3-propionic acid (IPA) has inherent limitations. Exogenous IPA supplementation faces prominent problems such as high preparation costs, poor chemical stability, short in vivo half-life, low bioavailability, and difficulty in precisely targeting release to the intestinal colonization area, making it difficult to achieve continuous and stable enrichment of endogenous indole-3-propionic acid (IPA) in the gut. More importantly, exogenous supplementation cannot activate the synergistic regulatory network of the gut microbiota-metabolic axis and lacks the ability to positively reshape the gut microbiota structure.

[0005] Second, existing probiotic preparations rely on the technological bias of "multi-strain combination." Current technologies for promoting intestinal indole-3-propionic acid (IPA) production generally rely on multi-strain combinations (such as combining yeast with Bacillus, Lactobacillus, Bifidobacterium, etc.), indirectly increasing indole-3-propionic acid (IPA) through metabolic complementarity between the microbial communities. However, multi-strain combinations not only significantly increase the difficulty of quality control and batch-to-batch stability risks in industrial production, but also lead to problems such as competition for survival sites, antagonism of nutrient substrates, and interference of metabolic pathways among different genera of microorganisms in the gut, easily resulting in low colonization efficiency and unpredictable effects of the product in the host gut. More importantly, current technologies have not yet revealed that any single strain can independently and efficiently drive the targeted enrichment of intestinal tryptophan metabolism towards the indole-3-propionic acid (IPA) branch. This technological gap severely restricts the development of efficient, precise, and quality-controllable single-strain probiotic functional products.

[0006] Third, Akkermansia ( Akkermansia The direct application of AKK bacteria (Anatomical mucin-degrading bacteria) faces technical bottlenecks. As a representative of mucin-degrading bacteria, AKK bacteria are among the most promising probiotic candidate strains discovered in recent years. They promote the renewal of the host's mucus layer and enhance barrier function by degrading intestinal mucin, and are closely related to the host's metabolic health. However, AKK bacteria are strict anaerobic bacteria, extremely sensitive to oxygen, making industrial-scale culture difficult; and directly supplemented live bacteria are easily affected by individual differences in the host's intestinal environment (pH, bile acids, competition from existing flora), resulting in large fluctuations in colonization efficiency. Therefore, indirectly promoting the proliferation of endogenous AKK bacteria in the host through metabolite regulation, rather than directly supplementing live bacteria, has become a recognized important development direction in this field. However, currently, there is a lack of effective technical means to achieve the synergistic regulation of "metabolite-driven - endogenous AKK bacteria enrichment".

[0007] Kluyveromyces martensii is a food-grade fungus with a long history of safe consumption and is widely used in dairy products, food fermentation, and industrial enzyme production. However, existing research focuses primarily on its enzyme-producing activities (such as inulinase and lactase) or its role as a cell factory for producing specific metabolites. Research on its application in gut health is extremely limited, and existing technologies involving gut regulation typically involve combining it with other bacteria. To realize its health benefits, it needs to be combined with other bacteria (such as Bacillus and Lactobacillus) to form multi-strain preparations. Multi-strain preparations not only increase the difficulty of industrial quality control but also, due to competition between different genera of microorganisms, can lead to unstable colonization of the product in the gut. Strains from different sources exhibit significant differences in physiological characteristics and metabolic phenotypes. Currently, there are few reports on single strains of Kluyveromyces martensii that can specifically and efficiently promote the production of endogenous indole-3-propionic acid (IPA) in the gut. Whether it can independently reshape the structure of beneficial gut microbiota and directionally drive tryptophan metabolism towards the indole-3-propionic acid (IPA) branch as a "single-strain preparation" has not yet been clearly disclosed by technical means. The existing technological bias that "yeast must rely on compound formulations to exert its intestinal regulatory function" has hindered the development of highly efficient single-strain microecological preparations.

[0008] Selenium is an essential trace element for the human body, possessing important physiological functions such as antioxidation and regulation of immune homeostasis. Inorganic selenium (such as sodium selenite) is highly toxic and has low bioavailability, while organic selenium (mainly selenomethionine SeMet, methylselenocysteine ​​MeSeCys, and selenocysteine ​​SeCys2) obtained through microbial enrichment and transformation has high safety and significantly better bioavailability than inorganic selenium. Although yeast selenium enrichment technology has been reported, current technologies have never revealed whether specific Kluyveromyces martensii yeasts, after selenium enrichment, exhibit cross-reactions or significant synergistic effects with their own microecological regulatory characteristics in regulating intestinal tryptophan metabolism (especially promoting IPA production) and alleviating intestinal stress. In other words, current technologies have failed to establish a technical link between "selenium enrichment of specific strains - targeted remodeling of tryptophan metabolism - precise regulation of intestinal microecology," resulting in the functional development of selenium-enriched yeast remaining at the level of a single "selenium supplement," failing to achieve multidimensional functional integration of "metabolic regulation + microecological regulation + organic selenium supplementation."

[0009] In summary, the core technical challenges currently facing this field can be summarized as follows: There is a lack of novel, multifunctional microbial strains and their functional formulations that can safely supplement highly bioavailable organic selenium, simultaneously promote the efficient production of endogenous indole-3-propionic acid (IPA) in the gut through a single strain, achieve bidirectional enrichment of beneficial microecologies (especially endogenous AKK bacteria), and significantly alleviate intestinal stress damage. This technological gap severely restricts the development of post-biotics, foods for special medical purposes, and functional health products targeting the intestinal metabolic axis, and a breakthrough is urgently needed. Summary of the Invention

[0010] To address the aforementioned technical gaps and deficiencies in the existing technology, the purpose of this invention is to provide a newly isolated and identified Kluyveromyces martensii FZU-Km strain and its selenium-enriched fortification preparation, thereby solving the following key technical problems: (1) Break through the technical bias of existing microecological preparations that rely on multi-strain compounding, and provide a single strain solution that can independently and efficiently drive the intestinal tryptophan metabolism to be directed to the indole-3-propionic acid (IPA) branch; (2) To solve the problems of poor stability, high cost and difficulty in continuous accumulation of exogenous indole-3-propionic acid (IPA) supplementation, and to achieve significant improvement and long-term maintenance of intestinal indole-3-propionic acid (IPA) level through endogenous metabolic remodeling; (3) Overcome the technical bottlenecks of anaerobic culture difficulty and low colonization efficiency in direct supplementation of AKK bacteria, and indirectly promote the proliferation of endogenous AKK bacteria through the bidirectional synergistic mechanism of metabolites and microecology; (4) To fill the technical gap in the intersection of "selenium enrichment fortification of specific strains and regulation of intestinal tryptophan metabolism", and to provide a selenium enrichment fortification preparation with significant synergistic effect; (5) Provides a multifunctional single-strain preparation that combines targeted metabolic regulation, precise regulation of microecology, antioxidant mucosal repair and organic selenium supplementation, providing new underlying technical support for the development of postbiotics, special medical purpose formula foods and functional health products.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Kluyveromyces martensii* that directionally promotes the production of endogenous indole-3-propionic acid (IPA). Kluyveromyces marxianus )FZU-Km.

[0012] This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on June 23, 2026, with accession number CGMCC No. 39388.

[0013] This strain was isolated from the microbial community during the traditional red yeast rice vinegar fermentation process in Fujian Province and identified as *Kluyveromyces martensii* by ITS-5.8S rDNA sequencing. This strain exhibits a unique ability to regulate tryptophan metabolism in vivo, independently driving the conversion of tryptophan to indole-3-propionic acid (IPA) in the host gut without relying on co-existing bacterial strains. This significantly increases endogenous IPA levels in the gut and reverses pathological IPA depletion under intestinal stress.

[0014] Secondly, the present invention provides a microecological preparation or composition.

[0015] The active ingredient of the microecological preparation or composition includes the Kluyveromyces martensii FZU-Km and / or its fermentation product; the fermentation product includes one or more of dead bacteria, cell fragments, cell extracts or fermentation supernatant.

[0016] Preferably, the microecological preparation or composition further comprises food- or feed-acceptable excipients or carriers.

[0017] Thirdly, the present invention provides a selenium-enriched fortification preparation (KmSe).

[0018] The selenium-enriched fortification preparation is obtained by fermenting the *Kluyveromyces martensii* FZU-Km inoculated into a culture medium containing sodium selenite; the final concentration of the sodium selenite is preferably 0.1~1.0 mM, more preferably 0.125 mM.

[0019] In the aforementioned selenium-enriched fortification preparation, the selenium content enriched in yeast cells is 350-600 mg / kg (preferably about 513.73 mg / kg), of which organic selenium accounts for no less than 80%, mainly consisting of selenomethionine (SeMet), methylselenocysteine ​​(MeSeCys), and selenocysteine ​​(SeCys2). This selenium-enriched fortification preparation exhibits a significant synergistic effect compared to FZU-Km in promoting intestinal IPA production and repairing the intestinal mucosal barrier; it is not a simple physical additive effect.

[0020] Fourthly, the present invention provides the use of the Kluyveromyces martensii FZU-Km, the microecological preparation or composition, and the selenium-enriched fortification preparation in the preparation of products that promote the production of endogenous indole-3-propionic acid (IPA) in the host gut.

[0021] The core feature of this application is that it can drive the intestinal tryptophan metabolism to be directed to the indole-3-propionic acid (IPA) branch independently by a single strain, without relying on combination with other bacterial genera.

[0022] The products include, but are not limited to, health foods, foods for special medical purposes, functional foods, or animal feed additives.

[0023] Preferably, the dosage form of the product is selected from powder, granules, capsules, tablets, oral liquid, drops or live bacteria freeze-dried powder.

[0024] Preferably, when the product contains live bacteria, the concentration of live *Kluyveromyces martensii* FZU-Km in the product is not less than 7 × 10⁻⁶. 8 CFU / mL or 7×10 8CFU / g.

[0025] Fifthly, this invention elucidates the biological mechanism by which the strain and preparation exert their effects.

[0026] The product exerts its effects of promoting indole-3-propionic acid (IPA) production and maintaining gut health through any or a combination of the following mechanisms: (1) Single-strain-mediated remodeling of tryptophan metabolism: Independently driving the metabolic conversion of tryptophan in the intestine to indole-3-propionic acid (IPA), significantly increasing the expression level of endogenous indole-3-propionic acid (IPA) in the intestine. In the intestinal stress model, the strains can increase the intestinal IPA level by more than 10 times compared with the model group, and the selenium-enriched fortified preparation can further increase it by 22.2 times; (2) Selenium-enriched synergistic mediated metabolic pathway activation: Through the synergistic effect of organic selenium (SeMet, MeSeCys, SeCys2) and the strain's own metabolic enzymes, the generation efficiency of IPA is significantly enhanced, and the levels of intestinal protection and energy repair-related metabolites such as 5'-cytidine, phosphatidylethanolamine, 2'-adenosine, cladinoic acid, and kynurenic acid are simultaneously increased in the intestine.

[0027] (3) Two-way synergistic enrichment of metabolites and microecology: While increasing the level of intestinal IPA, it significantly increases the levels of Akkermansia in the intestine ( Akkermansia The relative abundance of AKK bacteria (increased by about 5.8 times with FZU-Km bacteria intervention and by about 15.6 times with selenium-enriched preparations) forms a positive feedback regulatory network of "metabolite-driven - beneficial bacteria response", thereby overcoming the technical bottleneck of direct AKK bacteria supplementation.

[0028] (4) Targeted and precise regulation of the gut microbiota: By increasing the level of intestinal IPA, the following targeted regulation of the gut microbiota is further mediated: Positive regulation: enhancing Prevotella paraprednisolone ( Paraprevotella ), fecal isobacillus ( Allobaculum Odor bacillus ( Odoribacter ), Lactobacillus ( Lactobacillus The abundance of beneficial bacteria, such as ) Negative regulation: competitively inhibiting and reducing Zurich bacteria ( Turicibacter Klebsiella pneumoniae ( Klebsiella ), Shigella ( Escherichia-Shigella ) and Enterobacteriaceae ( Enterobacter The abundance of conditionally pathogenic bacteria and unfavorable symbiotic bacteria, etc.

[0029] (5) Relief of intestinal stress and maintenance of mucosal barrier: By increasing the level of intestinal IPA, it exerts one or more of the following protective effects: It improves intestinal morphology and mucosal integrity, restores colon length to near normal levels, and alleviates mucosal tissue abnormalities and inflammatory cell infiltration caused by intestinal stress. It optimizes the intestinal metabolic microenvironment, synergistically increases the levels of beneficial metabolites such as 5'-cytidine, phosphatidylethanolamine, 2'-adenosine, cladinoic acid, and kynurenic acid, and significantly reduces the levels of stress metabolites such as indole sulfate, taurocholic acid, p-cresol sulfate, and gluconic acid, as well as enterotoxins. It regulates the balance of intestinal immune factors and oxidative stress, reduces serum levels of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and increases interleukin-10 (IL-10) levels; enhances the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in colonic tissue, and reduces myeloperoxidase (MPO) activity, thus blocking the inflammatory infiltration of neutrophils into colonic tissue.

[0030] In a sixth aspect, the present invention provides a method for culturing and preparing the strain and the selenium-enriched preparation.

[0031] Seed culture: The FZU-Km strain was inoculated into YPD medium and cultured at 28℃~37℃ for 12h~24h to obtain seed culture.

[0032] Fermentation culture: Transfer the seed culture to the fermentation medium at an inoculation rate of 1% to 5%. When preparing selenium-enriched preparations, use a selenium-containing medium, preferably sodium selenite with a final concentration of 0.1 to 1.0 mM as the selenium source, and culture at 30℃ to 35℃ and 150 to 250 r / min for 12 to 48 hours.

[0033] Product collection: Collect bacterial cells and / or cultures by means of centrifugation, filtration or concentration to obtain live bacterial sludge, fermentation broth or freeze-dried powder.

[0034] Seventhly, the core functions and mechanisms of the strains and preparations described in this invention.

[0035] The Kluyveromyces martensii FZU-Km and selenium-enriched fortified preparation exert their effects through a dual-target synergistic axis of "gut microecology-specific metabolites": (1) Remodeling the metabolic profile and targeted enrichment of indole-3-propionic acid (IPA): Independently drives the conversion of tryptophan metabolism to indole-3-propionic acid (IPA) branch metabolism, reversing the pathological depletion of indole-3-propionic acid (IPA) under intestinal stress. Simultaneously significantly increases the levels of intestinal protection and energy repair-related metabolites such as 5'-cytidine, phosphatidylethanolamine, 2'-adenosine, branched acid, and kynurenic acid in the intestine; and significantly downregulates or eliminates the levels of stress-induced inflammatory metabolites such as indole sulfate (intestinal toxin), taurocholic acid, p-cresol sulfate, and gluconic acid in the intestine.

[0036] (2) Targeted purification and regulation of the microecology: Significantly enhances the core beneficial bacteria Akkermansia in the host's intestine ( Akkermansia The relative abundance of *Pleurotus erythrocyte*, *Ischemicum faecalis*, *Ostomyces ozoneus*, and *Lactobacillus* was increased simultaneously; and competitive inhibition was shown against opportunistic pathogens and unfavorable symbiotic bacteria such as *Zurichella*, *Klebsiella*, and *Shigella*.

[0037] (3) Immunomodulatory and antioxidant mucosal repair: Reduce serum lipopolysaccharide (LPS) and stimulatory factors (TNF-α, IL-1β, IL-6) levels, and increase the level of beneficial factor IL-10; at the same time, significantly reverse the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in colon tissue, and significantly inhibit myeloperoxidase (MPO) activity, thereby repairing the integrity of mucosal structure from the perspective of antioxidant damage and improving intestinal morphology indicators.

[0038] The beneficial effects achieved by this invention are as follows: Compared with the prior art, the present invention has the following significant technical advantages: 1. Single strains exert their effects independently and efficiently, overcoming the biases of multi-strain compound technology. In existing technologies, the application of yeast in the field of gut health is hampered by the technical bias that it must be formulated in combination with other bacterial genera (such as Bacillus and Lactobacillus) to be effective. This leads to difficulties in quality control during industrial production, poor batch stability, and problems such as competition for survival sites and interference with metabolic pathways among multiple strains in the host gut, resulting in low colonization efficiency and unpredictable effects. This invention is the first to discover that a single strain of Kluyveromyces martensii FZU-Km can independently construct a "microbiota-metabolite" synergistic axis in vivo, achieving highly efficient and targeted production of indole-3-propionic acid (IPA) and precise regulation of the gut microbiota without the assistance or combination of any other bacterial genera. This discovery fundamentally breaks through the aforementioned technical bias, significantly reducing the difficulty and cost of industrial production, improving batch consistency and quality control of formulations, and significantly enhancing the response rate and stability of the product in the host gut. It provides a new technical paradigm for developing highly efficient, precise, and standardized single-strain microecological formulations.

[0039] 2. It has a significant "selenium-enriched synergistic effect" in promoting IPA production and alleviating intestinal stress. This invention reveals that the selenium-enriched fortified preparation, prepared by combining FZU-Km strain with a selenium-enriched process, is not a simple physical superposition of basic probiotics and trace selenium. Instead, it achieves a unique "selenium-enriched synergistic metabolic activation" mechanism through the synergistic effect of organic selenium (SeMet, MeSeCys, SeCys2) and the strain's own metabolic enzymes. In vivo experiments confirmed that the IPA level in the intestines of mice in the selenium-enriched fortified preparation group (KmSe group) was 22.2 times higher than that in the model group, significantly higher than the 10.5 times higher in the non-selenium-enriched bacteria group (Km group). Simultaneously, the enrichment factor of AKK bacteria also jumped from 5.8 times in the non-selenium-enriched bacteria group (Km group) to 15.6 times in the selenium-enriched preparation group (KmSe group). This synergistic effect demonstrates excellent intervention effects in alleviating intestinal mucosal tissue damage, improving intestinal morphology, and repairing the antioxidant barrier, achieving a functional leap of "1+1>2" and filling the technological gap in the intersection of "selenium-enriched fortification of specific strains and regulation of intestinal tryptophan metabolism."

[0040] 3. It has an excellent ability to remodel the metabolic profile of endogenous IPA and rebuild the intestinal metabolic barrier. Unlike existing probiotics that primarily regulate other tryptophan metabolic branches in the gut, such as indole-3-lactic acid (ILA), indole-3-carboxaldehyde (ICA), or indole-3-acetic acid (IAA), the strain of this invention exhibits a unique ability to selectively regulate the intestinal tryptophan metabolic pathway. It specifically drives tryptophan metabolism towards the indole-3-propionic acid (IPA) branch, significantly increasing its expression level in the gut by more than 20 times compared to the intestinal health-damaged group. This effectively reverses the pathological depletion of IPA and rebuilds the intestinal metabolic barrier. Simultaneously, this strain can precisely upregulate multiple beneficial metabolites (such as 5'-cytidine, phosphatidylethanolamine, cladose acid, and kynurenic acid) and significantly downregulate or eliminate multiple harmful enterotoxins (such as indole sulfate and p-cresol sulfate), achieving comprehensive optimization of the intestinal metabolic microenvironment.

[0041] 4. Targeted regulation of the microecological "one rise and one fall" indirectly solves the bottleneck in the application of AKK bacteria. The strains of this invention exhibit excellent precision in regulating bacterial communities at the genus level, and can not only selectively enrich bacteria such as Akkermansia (…). AkkermansiaThe core beneficial bacteria in the gut, represented by [specific bacteria name], can actively and competitively inhibit various opportunistic pathogens such as Klebsiella pneumoniae, Escherichia coli-Shigella, and Bacillus zurichae, proactively reversing the microecological imbalance mediated by intestinal stress. More importantly, this invention, through an innovative mechanism of "IPA metabolite-driven - endogenous AKK bacteria response," bypasses the core technical bottlenecks of direct AKK bacteria supplementation, such as the difficulty of strictly anaerobic culture, industrial scale-up challenges, and low host colonization efficiency. This provides a novel "metabolic regulation" technical pathway for the functional application of AKK bacteria.

[0042] 5. Efficient inorganic selenium conversion and antioxidant barrier repair mechanism The strain of this invention exhibits excellent tolerance and efficient conversion ability to highly toxic inorganic selenium, transforming it into highly bioavailable intracellular organic selenium (content up to 513.73 mg / kg, with an organic selenium content of no less than 80%), of which selenomethionine (SeMet) accounts for 58.62%, providing a high-quality source of organic selenium supplementation for the human body. Combined with its significant activation of three core antioxidant enzymes—SOD, CAT, and GSH-Px—and effective inhibition of MPO activity, this invention constructs a powerful "intestinal antioxidant repair axis" from three dimensions: "supplementation-activation-inhibition," blocking lipid peroxidation damage and neutrophil inflammatory infiltration at the source, achieving deep repair of the mucosal barrier.

[0043] 6. Broad prospects for commercial application and transformation The strain and its selenium-enriched fortified preparation of this invention possess multiple biological functions, including targeted promotion of IPA metabolism, efficient supplementation of the trace element organic selenium, precise regulation of intestinal microecology, and maintenance of the intestinal mucosal barrier. This forms a four-dimensional integrated health intervention system encompassing "metabolic regulation, microecological regulation, antioxidant repair, and organic selenium supplementation." The strain exhibits high safety (food-grade yeast), mature preparation technology, and clear quality control standards, demonstrating significant economic value and industrial application potential in the development of special medical purpose formula foods, functional health foods, novel metabiotic products, and feed additives. Attached Figure Description

[0044] Figure 1 The colony morphology of *Kluyveromyces martensii* FZU-Km cultured on YPD agar medium for 48 h is shown.

[0045] Figure 2 The effect of sodium selenite on the growth performance of Kluyveromyces martensii FZU-Km is shown in Figure A; A represents the growth curves (OD) of the strains under different concentrations of sodium selenite (0, 0.125, 0.25, 0.5, 1.0 mM) during a 48-h culture period. 600 B is a bar chart showing the viable cell count (CFU / mL) of each group's fermentation broth at the end of the culture (48 h).

[0046] Figure 3 The results are HPLC-ICP-MS analysis of selenium speciation in the selenium-enriched fortified preparation (KmSe).

[0047] Figure 4 The effects of FZU-Km and KmSe preparations on body weight changes in mice with DSS-induced acute colitis are shown in Figure A. Figure A shows the dynamic changes in body weight of mice in each group during the experimental period (days 0-20). Figure B shows the bar chart of body weight of mice in each group at the experimental endpoint (day 20). Figure 5 A shows the Disease Activity Index (DAI) scores of mice in each experimental group; A is the dynamic change curve of DAI in each group of mice during the experiment; B is the bar chart of DAI scores of each group at the experimental endpoint.

[0048] Figure 6 The results show the statistical results of colon appearance and length in mice of each experimental group; A is a representative colon appearance photograph of mice in each group; B is a quantitative statistical chart of colon length in each group.

[0049] Figure 7 The results of pathological hematoxylin-eosin (H&E) and Alsin blue-periodic acid-Schiff (AB-PAS) staining of colon tissue from mice in each experimental group are shown.

[0050] Figure 8 The results of intestinal barrier and inflammation-related indicators in the serum of mice in each experimental group are as follows: A is the content of lipopolysaccharide (LPS); B is the content of interleukin-1β (IL-1β); C is the content of interleukin-6 (IL-6); D is the content of tumor necrosis factor-α (TNF-α); and E is the content of interleukin-10 (IL-10).

[0051] Figure 9 The results of oxidative stress index detection in the colon tissue of mice in each experimental group are as follows: A is catalase (CAT) activity; B is superoxide dismutase (SOD) activity; C is glutathione peroxidase (GSH-Px) activity; and D is myeloperoxidase (MPO) activity.

[0052] Figure 10 Analysis of the relative abundance differences of gut microbiota at the genus level in each experimental group of mice; A: Comparison of differentially abundant microbiota levels between the Model group and the Km group; B: Comparison of differentially abundant microbiota levels between the Model group and the KmSe group; C: Akkermansia ( Akkermansia ) Statistical analysis of the relative abundance difference fold among the groups.

[0053] Figure 11The results of non-targeted metabolomics analysis of intestinal contents of mice in each experimental group are shown below; A is a heatmap of differential metabolite clustering between the Model group and the Km group; B is a heatmap of differential metabolite clustering between the Model group and the KmSe group; C is the statistical analysis of the relative abundance difference of indole-3-propionic acid (IPA) among the groups. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or conditions recommended by the reagent kit manufacturer; the materials and reagents used, unless otherwise specified, are commercially available. All animal experiments were approved by the laboratory animal ethics committee and conducted in accordance with guidelines for laboratory animal care and use.

[0055] Example 1: Origin, Identification and Preservation of Kluyveromyces martensii FZU-Km 1. Strain source: Kluyveromyces martensii (Kluyveromyces masculinus) of this invention Kluyveromyces marxianus The FZU-Km strain was isolated from the microbial community during the traditional red yeast rice vinegar fermentation process in Fujian Province.

[0056] 2. Strain Isolation and Purification: During the later stage of alcoholic fermentation in the traditional red yeast rice vinegar fermentation process in Fujian, samples were taken from the middle of the fermentation mash using a sterile sampling spoon. Under aseptic conditions, 10 g of fermentation sample was weighed and added to a 250 mL Erlenmeyer flask containing 90 mL of sterile physiological saline (0.85% NaCl, w / v). The flask was then placed on a shaker and thoroughly mixed at 200 r / min for 30 min to obtain 10 g of the fermentation sample. -1 Diluent. Subsequently, it was serially diluted 10-fold with sterile physiological saline to obtain 10... -2 10 -3 10 -4 10 -5 Iso-diluted. To selectively enrich yeast in bacterial-rich communities, YPD agar medium supplemented with antibiotics was used. The YPD plate formulation was: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, agar 20 g / L. After sterilization and cooling to approximately 50 °C, a mixture of filtered and sterilized penicillin (100 U / mL) and streptomycin (100 μg / mL) was added to inhibit bacterial growth. Take 100 μL of an appropriately diluted (usually 10) medium. -5The bacterial suspension was spread onto the above-mentioned plates and incubated upside down in a 30 ℃ incubator for 48 hours. Typical, well-grown yeast colonies (smooth, moist, raised, with neat edges, cream to pale yellow in color) were picked from the plates and repeatedly streaked in three zones on fresh YPD plates to isolate single colonies. After each streaking, the cells were incubated for 48 hours, and purification was repeated three times until microscopic examination (400×) showed uniform cell morphology (oval, budding) and no contamination.

[0057] 3. Strain identification: Morphological observation showed that the colonies of this strain on YPD medium were white or milky white, with smooth surfaces and regular edges (e.g., ...). Figure 1 (As shown). Single colonies were picked and inoculated into YPD liquid medium. After incubation at 30℃ and 200 r / min for 24 h with shaking, an appropriate amount of bacterial solution was collected by centrifugation, and total DNA was extracted using a fungal genomic DNA extraction kit. The ITS-5.8S rDNA region was amplified by PCR using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') as primers. PCR reaction system (50 μL): 5 μL 10×PCR buffer, 4 μL dNTPs (2.5 mM each), 2 μL each of forward and reverse primers (10 μM), 2 μL DNA template, 0.5 μL high-fidelity DNA polymerase, and ddH2O to a final volume of 50 μL. PCR program: 95 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 45 s, 55 ℃ annealing for 45 s, 72 ℃ extension for 1 min, for a total of 35 cycles; final extension at 72 ℃ for 10 min. After verification by 1.5% agarose gel electrophoresis, the PCR products were purified using a DNA gel extraction kit and sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. The obtained sequences were compared with the GenBank database using BLAST, and the results showed that they were similar to... Kluyveromyces marxianus The type strain showed over 99% homology, and based on morphological characteristics, it was ultimately identified as *Kluyveromyces martensii*.

[0058] The ITS-5.8S rDNA sequence of this strain is as follows (SEQ ID NO.1): (SEQ ID NO.1) 4. Strain preservation: Kluyveromyces martensii ( Kluyveromyces marxianus FZU-Km was deposited on June 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39388.

[0059] Example 2: Cultivation and preparation of live bacterial preparations of Kluyveromyces martensii FZU-Km 1. Seed Culture: The FZU-Km strain preserved in glycerol tubes was inoculated at a rate of 1% (v / v) into 100 mL of sterile YPD liquid medium (formulation: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, natural pH, sterilized at 121℃ for 20 min). The medium was placed in a constant temperature shaker and cultured at 30℃ and 200 r / min for 18 h to obtain the seed culture. The OD of the seed culture at this time... 600 The value is approximately 1.5~2.0, and the viable count is approximately 1×10⁻⁶. 9 CFU / mL.

[0060] 2. Fermentation culture: The above seed culture was transferred to 100 mL of fresh sterile YPD liquid medium at an inoculation rate of 2% (v / v), and cultured with shaking at 30℃ and 200 r / min for 18 h to obtain the fermentation broth.

[0061] 3. Preparation of live bacteria preparation: After cultivation, the fermentation broth was centrifuged at 4℃ and 8000 r / min for 10 min, and the bacterial sludge was collected. The bacterial sludge was washed three times with sterile physiological saline (0.85% NaCl) (centrifugation conditions were the same as above each time) to remove residual culture medium components. The washed bacterial sludge was resuspended in sterile physiological saline, and the viable bacteria concentration was adjusted to 7×10⁻⁶ using a hemocytometer combined with plate counting. 8 CFU / mL is the FZU-Km live bacteria preparation, which should be stored at 4℃ for later use.

[0062] 4. Preparation of freeze-dried powder formulation: The washed bacterial sludge was mixed with a freeze-drying protectant (formulation: 100 g / L skim milk powder, 50 g / L trehalose, 10 g / L monosodium glutamate, sterilized at 121℃ for 15 min) at a ratio of 1:1 (v / v). The mixture was dispensed into freeze-drying trays and pre-frozen at -40℃ for 4 h. Then, it was transferred to a vacuum freeze dryer and freeze-dried for 24 h under conditions of cold trap temperature ≤ -50℃ and vacuum degree ≤ 10 Pa to obtain live bacterial freeze-dried powder. After reconstitution, the freeze-dried powder was plate-counted, and the viable bacterial survival rate was ≥ 85%, with a viable bacterial concentration of not less than 7 × 10⁻⁶. 8 CFU / g.

[0063] Example 3: Optimization and preparation of the culture process for selenium-enriched fortification agent (KmSe) This embodiment aims to systematically evaluate the tolerance of Kluyveromyces martensii FZU-Km to inorganic selenium, its growth characteristics, and its conversion efficiency to organic selenium, and to screen and establish the optimal selenium-enriched fermentation process parameters.

[0064] 1. Inorganic selenium tolerance assessment: The activated FZU-Km seed solution (OD) was used to assess the tolerance of inorganic selenium. 600≈1.5), and 5% of each group was inoculated into 100 mL of YPD liquid medium containing different concentrations of sodium selenite (Na2SeO3). The final concentrations of sodium selenite were set at 0, 0.125, 0.25, 0.5, and 1.0 mM, respectively. Each group was cultured at 30℃ and 200 r / min with shaking for 48 h. During this period, 500 μL samples were aseptically taken every 3 h, and the absorbance (OD) of the fermentation broth at 600 nm was measured using a UV spectrophotometer. 600 The growth curve of the strain was plotted. At the same time, at the end of the culture (48 h), samples were aseptically taken and serially diluted 10 times. The number of viable bacteria (CFU / mL) in the fermentation broth was detected by plate spreading method (YPD agar plate, cultured at 30℃ for 48 h). Each group was set up in 3 replicates.

[0065] 2. Cell Selenium Content and Speciation Analysis: After fermentation, the fermentation broth of each group was centrifuged at 8000 r / min for 10 min at 4℃ to collect the cells. The cells were washed three times with sterile physiological saline (centrifugation conditions were the same each time) to thoroughly remove unassimilated inorganic selenium adsorbed on the surface. The washed cells were dried to constant weight by hot air at 60℃, and the dry weight of the cells was determined. The selenium speciation in yeast cells was quantitatively analyzed by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The specific method is as follows: 0.1 g of dried bacterial powder was accurately weighed and added to 5 mL of proteinase K buffer (10 mM Tris-HCl, pH 7.5, containing 10 mM CaCl2 and 0.1% SDS). The mixture was incubated in a water bath at 50℃ for 12 h. After centrifugation at 10000 r / min for 10 min, the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC-ICP-MS. Chromatographic conditions: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); mobile phase A: 2% methanol (containing 0.1% formic acid); mobile phase B: 100% methanol; gradient elution program: 0–5 min, 5% B; 5–20 min, 5%–50% B; 20–25 min, 50% B; 25–30 min, 50%–5% B; flow rate: 1.0 mL / min; injection volume: 20 μL. ICP-MS conditions: RF power: 1550 W; carrier gas flow rate: 1.05 L / min (high-purity argon); auxiliary gas flow rate: 0.9 L / min; nebulizer temperature: 2℃; monitored mass number: 78 (…). 78 Se). Quantification was performed using the external standard method, with standards including selenomethionine (SeMet), methylselenocysteine ​​(MeSeCys), selenocysteine ​​(SeCys2), selenite (Se(IV)), and selenate (Se(VI)).

[0066] 3. Experimental Results and Analysis: (1) Growth curve and viable cell count: such as Figure 2 As shown in Figure A, at a sodium selenite concentration of 0.125 mM, the growth curve of FZU-Km basically overlapped with that of the Control group (0 mM), indicating that the strain could normally enter the logarithmic growth phase without showing significant growth inhibition. Figure 2 As shown in Figure B, at a concentration of 0.125 mM, the viable cell count at the fermentation endpoint was not significantly different from that in the Control group (P>0.05). However, when the sodium selenite concentration was ≥0.25 mM, the viable cell count decreased significantly (P<0.05), indicating that high concentrations of inorganic selenium had significant toxicity to the strain. Therefore, 0.125 mM was determined to be the optimal addition concentration for the subsequent organic selenium enrichment process.

[0067] (2) Selenium form and conversion rate: such as Figure 3 As shown, HPLC-ICP-MS analysis revealed that after 48 h of fermentation in a medium containing 0.125 mM sodium selenite, the total selenium content of FZU-Km reached 513.73 mg / kg, with a selenium conversion rate of 67.5%. Organic selenium accounted for 80.59%, while the residual amounts of inorganic selenium (Se(IV) and Se(VI)) were extremely low. Among the organic selenium components, selenomethionine (SeMet) accounted for 58.62%, methylselenocysteine ​​(MeSeCys) for 21.97%, and selenocysteine ​​(SeCys2) for 11.63%. These results indicate that FZU-Km possesses a highly efficient inorganic selenium assimilation capacity, and SeMet is its main organic selenium conversion product, exhibiting high bioavailability and safety in the human body.

[0068] Based on the above results, the optimal preparation parameters for the Kluyveromyces martensii FZU-Km selenium-enriched fortification agent (KmSe) were determined to be: adding sodium selenite to YPD basal medium at a final concentration of 0.125 mM, inoculum size of 5% (v / v), fermentation temperature of 30℃, shaking speed of 200 r / min, and fermentation time of 48 h. After fermentation, the fermentation broth was centrifuged at 4℃ and 8000 r / min for 10 min to collect the cells, which were then washed three times with sterile physiological saline to remove surface-adsorbed inorganic selenium, thus obtaining selenium-enriched bacterial sludge. The selenium-enriched bacterial sludge was resuspended in sterile physiological saline and the viable cell concentration was adjusted to 7 × 10⁻⁶. 8 CFU / mL indicates the KmSe live bacterial preparation; or KmSe lyophilized powder can be prepared according to the lyophilization process described in Example 2. All KmSe preparations used in subsequent in vivo pharmacodynamic experiments were prepared using this optimal process.

[0069] Example 4: Intervention effect of Kluyveromyces martensii FZU-Km and its selenium-enriched fortified preparation on DSS-induced colitis In this example, a DSS-induced acute colitis mouse model was constructed to systematically evaluate the in vivo biological efficacy of the FZU-Km strain and its selenium-enriched fortified preparation (KmSe) in promoting the production of intestinal endogenous indole-3-propionic acid (IPA), regulating intestinal microecology, repairing intestinal mucosal barrier and alleviating intestinal stress injury.

[0070] 1. Experimental animals and feeding conditions Thirty-two SPF-grade male C57BL / 6J mice aged 6-8 weeks and weighing 20-22 g were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Production License Number for Experimental Animals: SCXK (Hu) 2023-0003). All mice were housed in a standard SPF-grade animal experimental center, with environmental parameters controlled as follows: temperature 22±1°C, relative humidity 50%-55%, light cycle 12 h light / 12 h dark (light time 8:00-20:00). Mice had ad libitum access to standard maintenance feed and autoclaved water. Adaptive feeding was carried out for 10 days before the experiment, during which the general status of the mice was observed daily and their body weights were recorded.

[0071] 2. Experimental grouping and model construction Thirty-two mice were randomly divided into 4 groups according to body weight, with 8 mice in each group. The specific grouping and treatment methods are as follows: (1) Control group (normal control group, n=8): Mice had free access to high-pressure sterilized pure water throughout the whole experiment (days 0-20). During the experiment, 200 μL of sterile normal saline was given daily by intragastric administration for 20 consecutive days.

[0072] (2) Model group (DSS model group, n=8): Starting from day 9, mice had free access to sterilized water containing 3% (w / v) dextran sulfate sodium (DSS, molecular weight 36-50 kDa) for 7 consecutive days (days 9-16) to induce an acute colitis model. Starting from day 16, DSS drinking was stopped, and mice were changed to drink normal sterilized water. During the experiment, 200 μL of sterile normal saline was given daily by intragastric administration for 20 consecutive days.

[0073] (3) Km group (FZU-Km intervention group, n=8): Throughout the whole experiment (days 0-20), 200 μL of preparation containing 7×10 8 CFU / mL live FZU-Km preparation (prepared according to Example 2) was given daily by intragastric administration; starting from day 9, mice had free access to sterilized water containing 3% DSS for 7 consecutive days (days 9-16) to induce an acute colitis model; starting from day 16, DSS drinking was stopped, and mice were changed to drink normal sterilized water.

[0074] (4) KmSe group (selenium-enriched fortified preparation intervention group, n=8): Throughout the whole experiment (days 0-20), 200 μL of preparation containing 7×10 8KmSe live bacteria preparation (prepared according to the optimal process in Example 3) with CFU / mL; from day 9, drink sterile water containing 3% DSS freely for 7 consecutive days (days 9-16) to induce an acute colitis model; from day 16, stop drinking DSS and switch to normal sterile water.

[0075] 3. Observation indicators and sample collection: (1) General condition observation and Disease Activity Index (DAI) score During the experiment, the mice in each group were observed and recorded daily for their feed and water consumption, activity level, coat luster, mental state, and stool and urine characteristics. Mice were weighed at the same time each day. Starting from day 1 of DSS administration, fecal characteristics and occult blood / gross blood in the stool were examined daily, and the Disease Activity Index (DAI) was calculated according to the criteria in Table 1. DAI score = (percentage of weight loss score + fecal characteristics score + fecal bleeding score) / 3.

[0076] Table 1 Disease Activity Index (DAI) Scoring Criteria

[0077] (2) Sample collection and processing On day 20, after the last gavage, mice were fasted but allowed free access to water for 12 hours. On day 21, mice were euthanized using carbon dioxide, and blood was immediately collected from the heart. The collected blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 3000 r / min for 15 minutes at 4°C. Serum was separated, aliquoted, and stored at -80°C for later use. The abdominal cavity was quickly opened, and the colon was completely removed. The intestinal contents were gently rinsed with pre-cooled sterile saline, and the colon was blotted dry on clean filter paper. The total length of the colon (from the cecum to the anus) was measured and recorded. Approximately 1 cm of colonic tissue was taken from 1 cm from the cecum and immersed in 4% paraformaldehyde fixative for histopathological examination. Approximately 200 mg of adjacent colonic tissue was rapidly frozen in liquid nitrogen and then transferred to a -80°C cryopreservation facility for the detection of oxidative stress indicators. One day before dissection (day 13), fresh fecal samples were collected from each group of mice (3-5 samples from each mouse), aliquoted into sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for 16S rRNA gene sequencing and non-targeted metabolomics analysis.

[0078] 4. Serological marker detection Serum lipopolysaccharide (LPS) and inflammatory factors were detected using enzyme-linked immunosorbent assay (ELISA). The procedure was as follows: serum samples were thawed at 4°C, and the assay was performed according to the instructions of the corresponding ELISA kits manufactured by Shanghai Enzyme-Link Biotechnology Co., Ltd. The detected indicators included: lipopolysaccharide (LPS), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). The absorbance of each well was measured at 450 nm using a microplate reader, and the concentration of each sample was calculated based on the standard curve. Three technical replicates were performed for each group.

[0079] 5. Detection of oxidative stress indicators in colonic tissue Approximately 100 mg of frozen colon tissue was accurately weighed and added to pre-cooled sterile saline at a mass-to-volume ratio of 1:9 (w / v). The homogenate was then thoroughly homogenized using a high-speed tissue homogenizer (20,000 r / min, 10 s intervals, 3 times) under ice bath conditions until no visible tissue debris remained, preparing a 10% (w / v) colon tissue homogenate. The homogenate was centrifuged at 4℃ and 6000 r / min for 10 min, and the supernatant was collected for analysis. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and myeloperoxidase (MPO) in the supernatant were measured strictly according to the instructions using the corresponding reagent kits produced by Nanjing Jiancheng Bioengineering Institute. The protein concentration of each sample was determined using a BCA protein quantification kit to correct for enzyme activity data.

[0080] 6. Fecal 16S rRNA gene sequencing and bioinformatics analysis Total DNA was extracted from fecal samples using the cetyltrimethylammonium bromide (CTAB) method. DNA integrity and concentration were assessed using 1% agarose gel electrophoresis and a NanoDrop 2000 micro-spectrophotometer. The V3-V4 variable region of the bacterial 16S rRNA gene was amplified by PCR using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR reaction mixture (25 μL) consisted of: 5 μL 5×PCR buffer, 2 μL dNTPs (2.5 mMeach), 1 μL each of forward and reverse primers (10 μM), 1 μL DNA template, 0.25 μL high-fidelity DNA polymerase, and ddH2O to a final volume of 25 μL. PCR program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 15 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25 cycles; final extension at 72℃ for 5 min. PCR products were validated by 2% agarose gel electrophoresis, purified using the AxyPrep DNA gel extraction kit, and precisely quantified using the QuantiFluor-ST blue fluorescence quantitative quantification system. Equimolar mixtures of all samples were used to construct Illumina standard sequencing libraries, and 2×250 bp paired-end sequencing was performed on the Illumina NovaSeq 6000 platform.

[0081] After adapter sequence removal using Cutadapt, the raw sequencing data were assembled using FLASH software. Operational taxa (OTU) clustering was performed using the UPARSE algorithm (UCHIME) at a 97% similarity level, and chimeric sequences were removed. Using the Silva (SSU138) database as a reference, the RDP Classifier algorithm (confidence threshold 0.7) was used to annotate representative sequences for each OTU. STAMP was used to screen for biomarkers showing significant intergroup differences. All analyses were performed using QIIME 2 (v2023.2) and R software (v4.2.1).

[0082] 7. Non-targeted metabolomics analysis of intestinal contents Approximately 50 mg of frozen colon contents were taken and added to 500 μL of pre-chilled methanol-water extraction buffer (methanol:water = 4:1, v / v, containing internal standard L-2-chlorophenylalanine, concentration 1 μg / mL), and incubated at -20 ℃ for 2 h. Then, 200 μL of chloroform was added, and the mixture was vortexed for 30 s and sonicated for 15 min (300 W, 4 ℃), repeated three times. The mixture was centrifuged at 12000 r / min for 10 min at 4 ℃, and the supernatant was filtered through a 0.22 μm organic phase filter membrane for LC-MS / MS analysis. Chromatographic conditions: Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm); mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile; gradient elution program: 0–2 min, 5% B; 2–20 min, 5%–95% B; 20–22 min, 95% B; 22–22.5 min, 95%–5% B; 22.5–25 min, 5% B; flow rate 0.3 mL / min; injection volume 5 μL; column temperature 40 ℃. Mass spectrometry conditions: Electrospray ionization (ESI) source, positive and negative ion modes for separate acquisition; ion source temperature 500 ℃; spray voltage 5500 V (positive ion mode) / -4500 V (negative ion mode); collision gas pressure 8 psi; curtain gas pressure 35 psi; nebulizer gas pressure 55 psi; auxiliary gas pressure 55 psi; scan range m / z 70~1050. Raw data were converted to mzXML format using ProteoWizard software, and peak extraction, peak alignment, and retention time correction were performed using XCMS software. Normalization was performed using L-2-chlorophenylalanine as an internal standard. Metabolite identification was based on a self-built standard database and the HMDB, KEGG, and METLIN public databases, with variable projection importance (VIP) > 1 and... P Metabolites with a concentration <0.05 are considered differential metabolites.

[0083] 8. Statistical Analysis All experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism 9.0 software. One-way ANOVA was used for comparisons among multiple groups. If the variances were homogeneous, the LSD test was used for pairwise comparisons between groups; if the variances were unequal, Dunnett's T3 test was used. P < 0.05 was considered statistically significant.

[0084] 9. Experimental Results (1) Weight changes: such as Figure 4As shown in Figure A, during the DSS induction period (days 9-16), the body weight of mice in the Model, Km, and KmSe groups all showed a decreasing trend. Compared with the Control group, the body weight of mice in the Model group showed a highly significant decrease ( P <0.01. During the recovery period after stopping DSS (days 16-20), the body weight of mice in the Model group was still significantly lower than that in the Control group ( P <0.01). In contrast, mice in the Km group and KmSe group showed significantly better weight recovery during the recovery period than the Model group ( P <0.05), and the weight recovery trend in the KmSe group was better than that in the Km group. Figure 4 As shown in B, at the experimental endpoint, the body weight of mice in both the Km group and the KmSe group was significantly higher than that in the Model group. P The KmSe group had a body weight better than the Km group (<0.05), indicating that FZU-Km and its selenium-enriched fortified preparation can effectively promote the recovery of physical signs in DSS-induced colitis mice.

[0085] (2) Disease Activity Index (DAI) score: such as Figure 5 As shown in Figure A, the DAI score of the Model group mice rapidly increased after DSS induction, reaching its peak on day 16. During the recovery period, although the DAI score of the Model group decreased slowly, it remained at a high level. The increase in DAI scores in the Km and KmSe groups was more moderate during DSS induction, and the DAI scores showed a significant downward trend during the recovery period. Figure 5 As shown in B, at the experimental endpoint, the DAI scores of both the Km group and the KmSe group were significantly lower than those of the Model group. P The DAI score of the KmSe group was lower than that of the Km group (<0.05), indicating that the selenium-enriched fortified preparation has a synergistic effect in alleviating the activity of gastrointestinal inflammation.

[0086] (3) Colon length: such as Figure 6 As shown, the colon of mice in the Model group was severely pathologically shortened, with an average length of only 4.55±0.56 cm, significantly shorter than that in the Control group (8.12±0.43 cm). P <0.05. After FZU-Km and KmSe intervention, colon length was significantly restored (Km group: 6.83±0.62 cm; KmSe group: 7.45±0.51 cm), both significantly longer than the Model group ( P <0.05), and the colon length recovery in the KmSe group was better than that in the Km group, approaching the level of the Control group.

[0087] (4) Histopathological analysis: such as Figure 7As shown, H&E staining of colonic tissue revealed that the control group exhibited intact colonic mucosal structure, neatly arranged crypts, continuous epithelial cells, and no obvious inflammatory cell infiltration; while the model group showed severe pathological damage: extensive epithelial cell shedding, crypt structure destruction, submucosal edema, and extensive inflammatory cell infiltration. The Km and KmSe groups showed partial restoration of colonic mucosal integrity, more regular crypt structure, and significantly reduced inflammatory cell infiltration, with the KmSe group showing better tissue repair than the Km group. AB-PAS staining of colonic tissue showed that the control group had abundant goblet cells with deep and continuous mucus staining; the model group had significantly fewer goblet cells than the control group, significantly lower staining intensity, and a thinner and discontinuous mucus layer, indicating that the colitis model led to goblet cell loss and impaired mucus secretion. The Km and KmSe groups showed a significant recovery in goblet cell count compared to the model group, with restored mucus secretion function, suggesting that the strain of this invention can effectively protect and rebuild the host intestinal mucoepithelial barrier.

[0088] (5) Serum LPS and inflammatory factor levels: such as Figure 8 As shown in Figure A, the serum LPS level in the Model group was significantly higher than that in the Control group ( P <0.01 indicates impaired intestinal barrier function and endotoxin translocation in the model animals. After intervention with Km and KmSe, serum LPS levels significantly decreased, and the serum LPS level in the KmSe group was significantly lower than that in the Km group ( P <0.05), indicating that selenium-enriched intervention is more effective in repairing the intestinal barrier and reducing endotoxin entry into the bloodstream. Figure 8 As shown in Figures B through D, serum levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the Model group were sharply higher than those in the Control group. After intervention with Km and KmSe, the levels of these pro-inflammatory cytokines were significantly inhibited. Figure 8 As shown in Figure E, in contrast to pro-inflammatory factors, the serum anti-inflammatory factor IL-10 level in the Model group was significantly lower than that in the Control group, while both Km and KmSe interventions significantly reversed this trend and significantly increased serum IL-10 levels. P <0.05), and the IL-10 level in the KmSe group was higher than that in the Km group ( P <0.05). The above results demonstrate the excellent immune homeostasis remodeling function of the strain of the present invention and its selenium-enriched fortification preparation.

[0089] (6) Indicators of oxidative stress in colonic tissue: such as Figure 9 As shown in A~C, the activities of three core antioxidant enzymes—CAT, SOD, and GSH-Px—in the colon tissue of the Model group were significantly lower than those in the Control group. P<0.01 indicates that DSS induces severe oxidative stress damage. After intervention in the Km and KmSe groups, the activities of all three antioxidant enzymes showed significant pathological reversal and repair. P< 0.05), and the recovery of antioxidant enzyme activity in the KmSe group was better than that in the Km group ( P <0.05). For example... Figure 9 As shown in Figure D, the activity of myeloperoxidase (MPO, representing neutrophil aggregation and inflammatory infiltration intensity) in the colon tissue of the Model group was abnormally elevated. P <0.05); while MPO activity in the colon tissue of both the Km and KmSe groups was significantly inhibited ( P< 0.05). This mechanistically confirms that the single strain of the present invention can block the edematous aggregation of neutrophils in colonic tissue, and that the selenium-enriched fortified preparation has a synergistic effect in this regard.

[0090] (7) Analysis of gut microbiota structure: In the analysis of genus-level microbiota composition ( Figure 10 Compared to the Model group, both the Km and KmSe groups exhibited precise "one increase, one decrease" microecological regulation characteristics. For example... Figure 10 A and Figure 10 As shown in B, the core beneficial bacteria in the intestines of the Km and KmSe groups—Ackermannia (… Akkermansia The relative abundance of all of them was significantly upregulated. P <0.05%. Specifically, compared with the Model group, the abundance of AKK bacteria was increased by approximately 5.8 times in the Km group and by approximately 15.6 times in the KmSe group (…). Figure 10 C, P <0.05 indicates that selenium-enriched fortification significantly enhanced the proliferation-promoting effect of FZU-Km on intestinal AKK bacteria. Furthermore, in the Km and KmSe groups, *Pleurotus paraprevotella* (…) was significantly reduced. Paraprevotella ), fecal isobacillus ( Allobaculum Odor bacillus ( Odoribacter ) and Lactobacillus ( Lactobacillus The abundance of beneficial microorganisms such as Km and KmSe was also increased. Meanwhile, Km and KmSe significantly inhibited and reduced the abundance of Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella ), Zurich bacteria ( Turicibacter ), Shigella ( Escherichia_Shigella ) and Enterobacteriaceae ( Enterobacter The relative abundance of inflammatory condition pathogens / disease commensal flora, etc. P <0.05).

[0091] (8) Intestinal metabolite analysis: such as Figure 11 As shown, non-targeted metabolomics analysis revealed that both the Km and KmSe groups were significantly separated from the Model group in their metabolic profiles, exhibiting clear characteristics of metabolic remodeling. Figure 11 As shown in C, the most significant metabolic characteristic of the Km and KmSe groups is the significantly increased abundance of indole-3-propionic acid (IPA), a potent ligand for the aryl hydrocarbon receptor (AhR). Compared to the Model group, the IPA abundance was upregulated by approximately 10.5-fold in the Km group and approximately 22.2-fold in the KmSe group. P <0.05 indicates that the selenium-enriched fortified preparation has a significant synergistic effect in promoting IPA production. Furthermore, Km and KmSe significantly increased the abundance of intestinal protective and energy metabolism-related metabolites in the intestine, including 5'-cytidylic acid, LysoPE, 2'-adenylic acid, stercobilin, biotin, adenine, chorimantonic acid, pentadecanoic acid, sebacic acid, and kynurenic acid. P <0.05 or P <0.05; and significantly reduced the levels of pro-inflammatory and enterotoxic metabolites such as indoxyl sulfate, taurocholic acid, and p-cresol sulfate. P <0.05).

[0092] The data from the above embodiments fully confirm that the single strain of *Kluyveromyces martensii* FZU-Km and its selenium-enriched fortified preparation (KmSe) described in this invention can successfully construct a multidimensional health closed loop in vivo, consisting of "enrichment of core beneficial bacteria (AKK bacteria) - surge in tryptophan-directed metabolism (IPA) - antioxidant and mucosal barrier repair," without the need for complex multi-strain formulations. In particular, the targeted IPA-promoting effect and synergistic effect exhibited by the KmSe preparation provide fundamental technical support for the development of next-generation functional health and microecological products.

[0093] In addition to the embodiments described above, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention. The description and drawings are only some embodiments of the present invention, and any non-substantial modifications, substitutions, or equivalent changes made by those skilled in the art based on these embodiments are within the protection scope of the present invention.

Claims

1. A strain of Kluyveromyces martensii ( Kluyveromyces marxianus FZU-Km, characterized in that, The Kluyveromyces martensii FZU-Km is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is June 23, 2026, and the accession number is CGMCC No. 39388.

2. A microecological preparation or composition, characterized in that, Its active ingredients include the Kluyveromyces martensii FZU-Km as described in claim 1 and / or its fermentation products; the fermentation products include one or more of dead bacteria, cell fragments, cell extracts or fermentation supernatants.

3. A selenium-enriched fortified preparation, characterized in that, The selenium-enriched fortification preparation is obtained by fermenting the *Kluyveromyces martensii* FZU-Km as described in claim 1 in a culture medium containing sodium selenite; the final concentration of the sodium selenite is 0.1~1.0 mM.

4. The selenium-enriched fortified preparation according to claim 3, characterized in that, The selenium-enriched fortification preparation contains 350-600 mg / kg of selenium enriched in yeast cells, of which organic selenium accounts for no less than 80%; the organic selenium includes selenomethionine (SeMet), methylselenocysteine ​​(MeSeCys), and selenocysteine ​​(SeCys2).

5. The use of the Kluyveromyces martensii FZU-Km as described in claim 1, the microecological preparation or composition as described in claim 2, or the selenium-enriched fortified preparation as described in any one of claims 3 or 4 in the preparation of products that promote the production of endogenous indole-3-propionic acid (IPA) in the host gut.

6. The application according to claim 5, characterized in that, The products include health foods, foods for special medical purposes, functional foods, or animal feed additives.

7. The application according to claim 5, characterized in that, When the product contains live bacteria, the concentration of live *Kluyveromyces martensii* FZU-Km in the product is not less than 7 × 10⁻⁶. 8 CFU / mL or 7×10 8 CFU / g.

8. The application according to claim 5, characterized in that, The product exerts its effects of promoting indole-3-propionic acid (IPA) production and maintaining gut health through any or a combination of the following mechanisms: (1) Single-strain-mediated remodeling of tryptophan metabolism profile: independently drives the metabolic conversion of tryptophan in the intestine to indole-3-propionic acid (IPA) branch, and increases the level of endogenous indole-3-propionic acid (IPA) in the intestine; (2) Selenium-enriched synergistic mediated activation of metabolic pathways: through the synergistic effect of organic selenium and bacterial metabolic enzymes, the level of indole-3-propionic acid (IPA) in the intestine is increased; (3) Bidirectional synergistic enrichment of metabolites and microecology: While increasing intestinal indole-3-propionic acid (IPA), it also increases the levels of Akkermansia in the gut (… Akkermansia The relative abundance of metabolites and microecology forms a positive feedback regulatory network.

9. The application according to claim 5, characterized in that, The product mediates the following targeted regulation of the gut microbiota by increasing the level of indole-3-propionic acid (IPA): (1) Positive regulation: enhancing Prevotella paraprednisolone ( Paraprevotella ), fecal isobacillus ( Allobaculum Odor bacillus ( Odoribacter Lactobacillus ( Lactobacillus The abundance of beneficial bacteria; (2) Negative regulation: inhibiting and reducing Zurich bacteria ( Turicibacter Klebsiella pneumoniae ( Klebsiella ), Shigella ( Escherichia-Shigella ) and Enterobacteriaceae ( Enterobacter Abundance of opportunistic pathogens.

10. The application according to claim 5, characterized in that, The product exerts one or more of the following effects on relieving intestinal stress and maintaining the mucosal barrier by increasing the level of indole-3-propionic acid (IPA) in the intestine: (1) Improves intestinal morphology and mucosal integrity, and alleviates mucosal tissue abnormalities and inflammatory cell infiltration caused by intestinal stress; (2) Optimize the intestinal metabolic microenvironment, synergistically increase the levels of beneficial metabolites such as 5'-cytidine, phosphatidylethanolamine, 2'-adenosine, clade acid, and kynurenic acid, and decrease the levels of stress metabolites such as indole sulfate, taurocholic acid, p-cresol sulfate, gluconic acid, and enterotoxins. (3) It regulates the balance of intestinal immune factors and oxidative stress, reduces the serum levels of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and increases the level of interleukin-10 (IL-10); enhances the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in colon tissue, and reduces the activity of myeloperoxidase (MPO).

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