Yarrowia lipolytica PT3309 from healthy human intestinal tract and application thereof

Abstract

The application discloses a strain of intestinal fungus Yarrowia lipolytica (Yarrowia lipolytica) PT3309 from healthy people and an application thereof, and belongs to the field of functional microorganisms. The strain of the Yarrowia lipolytica PT3309 has a strain preservation number of GDMCC 65342 and a preservation date of October 24, 2024. The Yarrowia lipolytica PT3309 can tolerate a human gastrointestinal environment and can be efficiently adhered to intestinal epithelial cells Caco2. The Yarrowia lipolytica PT3309 and biomass thereof can relieve weight loss, hematochezia and diarrhea of an inflammatory bowel disease model mouse, improve colon pathological changes, reduce expression of inflammatory factors such as IL-1β, IL-6 and TNF-α, and restore a colon epithelial barrier. The application provides the Yarrowia lipolytica PT3309, which can be used for preparing functional bacterial agents, foods and drugs for relieving inflammatory bowel diseases, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a strain of intestinal fungus Yarrowia lipolytica PT3309 derived from healthy human individuals and its application in the prevention and treatment of inflammatory bowel disease, belonging to the field of functional microbial technology. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory bowel disease characterized by recurrent diarrhea, bloody and mucous stools, abdominal pain, and tenesmus, severely impacting patients' quality of life. IBD is classified into ulcerative colitis (UC) and Crohn's disease (CD). Due to its recurrent and persistent nature, it is often referred to as "green cancer." Currently, emerging industrialized countries like my country are experiencing an accelerated rise in IBD incidence, with a projected rapid increase in the next decade. With an aging population and low birth rates, my country's public health resources will face a severe challenge from the IBD epidemic. Current conventional treatments for IBD include aminosalicylic acid preparations, immunosuppressants such as glucocorticoids, and biologics such as infliximab. However, these treatments often only provide temporary relief from IBD symptoms and are frequently accompanied by adverse reactions such as drug resistance, hormone dependence, and immunosuppression, leading to decreased patient adherence and a vicious cycle of relapse-remission-relapse. Therefore, developing long-acting and safe IBD treatments to improve the quality of life and health of my country's large IBD population is a long-term and urgent challenge for medical researchers.

[0003] The gut microbiota plays a crucial role in the development and progression of IBD, and changes in the gut microbiota are key clinical events in the pathological process of IBD. Changes in the abundance and diversity of the gut microbiota directly drive the intestinal immune response and persistently induce chronic inflammation. Simultaneously, remodeling the gut microbiota using probiotics has made fecal microbiome transplantation (FMT) an important strategy for the prevention and treatment of IBD. Current research on the influence of gut microbiota on disease progression often focuses on gut bacteria; however, gut fungi, as an important gut microbiota community, play a key role in the development and progression of the disease, but are often overlooked due to limitations in technology and insufficient understanding. Yeast lipophila PT3309 is a functional gut fungus obtained through systematic analysis of the gut fungal community in healthy individuals using large-scale culture omics technology; its biological functions, particularly its effects on IBD, have not been reported. Therefore, this invention comprehensively utilizes microbiological and pharmacological experimental techniques to discover probiotic resources from human gut fungi that can be used for the prevention and treatment of IBD. Summary of the Invention

[0004] To address the above problems, the purpose of this invention is to provide a functional intestinal fungus for the prevention and treatment of inflammatory bowel disease.

[0005] In a first aspect, the present invention provides a strain of Yersinia lipophila ( Yarrowia lipolytica PT3309, deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC 65342) on October 24, 2024, is a strain isolated from fecal samples of healthy individuals. It exhibits resistance to gastric acid, intestinal fluid, and high intestinal adhesion.

[0006] Secondly, the Yeast PT3309 is used to prepare products for the prevention or treatment of inflammatory bowel disease (IBD), regulating the balance of intestinal flora, possessing antioxidant activity, and repairing the intestinal barrier.

[0007] Furthermore, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0008] Furthermore, the therapeutic effects of the product include at least one of the following aspects:

[0009] 1. Relieves symptoms of weight loss, rectal bleeding, and diarrhea;

[0010] 2. Improves colonic mucosal damage and pathological changes;

[0011] 3. Reduces the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the colon;

[0012] 4. Upregulates the expression of tight junction proteins Claudin-1 and ZO-1, repairing the intestinal epithelial barrier function.

[0013] Thirdly, the product may take the form of:

[0014] 1. Probiotic preparations: composed of live bacteria of Yersinia lipophila PT3309, freeze-dried bacterial powder, heat-inactivated bacterial biomass, or bacterial metabolites;

[0015] 2. Drugs or drug compositions: consisting of live bacteria, lyophilized bacterial powder, heat-inactivated bacterial biomass or strain metabolites of Yersinia lipolytica PT3309, and pharmaceutically acceptable carriers (such as milk powder, lactose, cyclodextrin, maltose, glucose, glycerol, monosodium glutamate, vitamin C, mannose, galactose, mannitol or methylcellulose), in dosage forms including oral preparations, suppositories or injections;

[0016] 3. Fermented foods: produced by fermentation with Yersinia lipophila PT3309, including solid foods, liquid foods, semi-solid foods, dairy products, fruit and vegetable foods, and freeze-dried foods.

[0017] 4. A functional food comprising live bacteria of Yersinia lipolytica PT3309, freeze-dried bacterial powder, heat-inactivated bacterial biomass, or bacterial metabolites, wherein the functional food is prepared by fermentation or by directly adding bacterial cells, and is selected from fermented dairy products, fermented vegetable and fruit juices, biscuits, beverages, or nutritional supplements.

[0018] Fourthly, the Yersinia lipophila PT3309 achieves prevention and treatment of inflammatory bowel disease through the following mechanism:

[0019] 1. It regulates the balance of intestinal flora, specifically increasing the abundance of beneficial bacteria such as Lactobacillus and inhibiting the proliferation of harmful bacteria such as Enterobacteriaceae;

[0020] 2. Antioxidant effect: Reduces intestinal oxidative stress levels;

[0021] 3. Immune regulation: Inhibits the release of pro-inflammatory factors and restores immune homeostasis.

[0022] 4. Repairing intestinal epithelial barrier function: Upregulating the expression of tight junction proteins such as Claudin-1 and ZO-1, restoring intestinal epithelial barrier function.

[0023] Technical Efficacy: Animal experiments have shown that *Yersinia lipolyticis* PT3309 can significantly alleviate IBD symptoms in mice, improve colonic shortening and histopathological damage, reduce inflammatory factor levels, and achieve long-term protection by repairing the intestinal epithelial barrier function. This strain has application potential in functional foods, probiotic preparations, and drug development.

[0024] This invention isolates and identifies a strain of intestinal fungus, *Yarrowia lipolytica* PT3309, from fecal samples of healthy individuals. This fungus exhibits resistance to gastric acid, intestinal fluid, and high intestinal adhesion. *Yarrowia lipolytica* PT3309 significantly alleviates symptoms of inflammatory bowel disease in mice, reduces colonic pathological changes, inhibits the expression of colonic IL-1β, IL-6, and TNF-α, upregulates the expression of tight junction proteins such as Claudin-1 and ZO-1, and restores intestinal epithelial barrier function. It shows broad application prospects in the prevention and treatment of inflammatory bowel disease. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Scanning electron microscope image of Yersinia lipophila PT3309;

[0027] Figure 2 The growth curve of Yersinia lipophila PT3309;

[0028] Figure 3 The graph shows the therapeutic effects of live Yersinia lipophila PT3309 bacteria and heat-inactivated bacterial biomass in Example 3 on alleviating the inflammatory bowel disease phenotype in wild-type mice compared to the model group. In the graph, A represents the change in mouse body weight; B represents the mouse diarrhea score; and C represents the mouse bloody stool condition.

[0029] Figure 4 The effect of Yersinia lipophila PT3309 on colonic permeability in wild-type inflammatory bowel disease mice in Example 3; where A is the fluorescence image of FITC-dextran in plasma, and B is the fluorescence quantification image of FITC-dextran in plasma;

[0030] Figure 5 The effect of Yersinia lipophila PT3309 on colonic lesions in mice with inflammatory bowel disease in Example 3; where A is an endoscopy image and B is an HE-stained section of colonic tissue;

[0031] Figure 6 The effect of Yersinia lipophila PT3309 on colon length in mice with inflammatory bowel disease in Example 3; where A is a colon photograph and B is the colon length data statistics;

[0032] Figure 7 The expression of inflammatory factors IL-1β, IL-6, and TNF-α in the colon tissue of mice after colonization with Yersinia lipophila PT3309 in Example 3;

[0033] Figure 8 The figures for Example 4 show the body weight, diarrhea score, and bloody stool of mice treated with antibiotic-induced gut microbiota clearance (ABX) using Yersinia lipophila PT3309. A represents the change in mouse body weight; B represents the bloody stool; and C represents the mouse diarrhea score.

[0034] Figure 9 The effect of Yersinia lipophila PT3309 on colonic permeability in ABX inflammatory bowel disease mice in Example 4; where A is the fluorescence image of FITC-dextran in plasma, and B is the fluorescence quantification image of FITC-dextran in plasma;

[0035] Figure 10 The results of the effect of Yersinia lipophila PT3309 on colonic lesions in ABX inflammatory bowel disease mice in Example 4 are shown; where A is a colonoscopy image and B is an HE-stained section of colonic tissue.

[0036] Figure 11The results show the colon length of ABX mice after colonization with Yersinia lipophila PT3309 in Example 4. In this figure, A is a colon photograph, B is the colon length data statistics, and C is the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the colon tissue. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1: Isolation and Identification of Strains

[0040] Fresh human fecal samples were serially diluted with sterile water and spread onto MTB medium (penicillin 100 U / mL, streptomycin 100 μg / mL) and incubated at 32°C for 5-10 days. Once distinct colonies were observed, colonies of different morphologies were picked and placed on fresh MTB plates, and incubated at 32°C for another 3 days. After monoclonal colonies were observed, monoclonal strains were further cultured to obtain purified monoclonal enteric fungi. The strain of this invention is a white, round colony with a smooth surface (sample number PT3309). The bacterial culture was placed in a 50% glycerol aqueous solution and stored at -80°C. Simultaneously, nucleic acids were extracted from the isolated strain using a DNA extraction kit. RT-PCR was performed using 18S rDNA primers, and 18S rRNA gene sequencing analysis was conducted on the isolated strain. Based on BLAST comparison with the National Center for Biotechnology Information (NCBI) nucleic acid database, the species was identified as Yarrowia lipolytica (99.92% homology). Figure 1 The image shows a scanning electron microscope (SEM) image of *Yersinia lipophila* PT3309. The *Yersinia lipophila* PT3309 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection (accession number: GDMCC 65342, address: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province).

[0041] Nucleotide sequence of 18S rRNA (IS2625-1)

[0042]

[0043] Example 2: Characterization of Yeast PT3309

[0044] 1. Growth curve determination

[0045] A 1% inoculum was taken from a PT3309 bacterial suspension with a concentration of 1×10⁸ CFU / mL and inoculated into MTB medium. The medium was then incubated in a shaker at 32°C and 100 rpm. The absorbance of the fermentation broth at OD600 was measured every 2 hours using a microplate reader. The results showed ( Figure 2 The PT3309 strain enters the logarithmic growth phase after 10 hours and reaches the plateau phase after 30 hours.

[0046] 2. Tolerance test

[0047] Table 1. Survival rates of Yersinia lipophila PT3309 and CICC1675 in simulated gastric and intestinal fluids (*, P<0.05)

[0048]

[0049] Overnight cultured *Yersinia lipolytica* PT3309 and *Yersinia lipolytica* CICC1675 (reference strain) were washed three times with PBS buffer, centrifuged, and resuspended in PBS. The resuspended bacterial suspension was divided into two aliquots: one was plated and counted as N0; the other aliquot (500 μL) was added to 4.5 mL of artificial gastric fluid and artificial intestinal fluid, respectively, and incubated at 32°C and 100 rpm for 1 h and 3 h, respectively, and then plated again to count the bacteria (N1). Viability (%) = N1 / N0 × 100%. Artificial gastric fluid preparation: 0.2 g NaCl and 0.35 g pepsin were dissolved in 100 mL triple-distilled water, pH adjusted to 3, and filtered for sterilization. Artificial intestinal fluid preparation: 0.2 g NaCl and 0.1 g trypsin were dissolved in 100 mL triple-distilled water, pH adjusted to 8 (solution a). 1.2 g ox bile salts were dissolved in 100 mL triple-distilled water, pH adjusted to 8 (solution b). Solution A and solution B were mixed at a 1:2 ratio and filtered for sterilization. CICC1675, purchased from the China Industrial Microbial Culture Collection Center (https: / / www.china-cicc.org / cicc / detail2 / ?sid=1824), was used as a control. The results showed that PT3309 exhibited strong tolerance to both artificial gastric and intestinal fluids, suggesting it can withstand the digestive environment of the gastrointestinal tract and function better. Compared to the CICC1675 standard strain, PT3309 survived better in artificial gastric fluid, while their tolerance to intestinal fluids was essentially the same (Table 1).

[0050] 3. Adhesion test of colonic epithelioid cells

[0051] Human colon cancer cells (Caco2) were seeded into 6-well plates. The complete culture medium consisted of 20% FBS, 1% Sodium Pyruvate, 1% MEM NEAA, 1% GlutaMAX, and 77% MEM. The medium was changed every 2 days until the cells reached a polarized state. Overnight cultured PT3309 and CICC1675 cells were washed twice with PBS, resuspended in MEM medium, and divided into two portions. One portion was counted as N0, and the other portion was incubated with 500 μL of Caco2 cells per well. The cells were incubated at 37°C for 2 hours, then washed three times with PBS. 1 mL of trypsin was added to each well, and the cells were digested at 37°C for 3 minutes. After digestion, the cells were mixed by pipetting and plated, and the cells were counted as N1. Adhesion rate (%) = N1 / N0 × 100%. The results showed that PT3309 had stronger adhesion to colonic epithelial-like cells compared to the standard strain, which facilitated longer colonization in the intestine and prolonged the therapeutic effect.

[0052] Table 2. Adhesion rates of Yersinia lipolyticis PT3309 and CICC1675 to Caco 2 cells (*, P<0.05)

[0053]

[0054] Example 3: The alleviating effect of Yersinia lipophila PT3309 on inflammatory bowel disease in wild-type mice.

[0055] Male C57BL / 6J mice aged 6-8 weeks were randomly divided into four groups (blank group, model group, model group + PT3309 colonization group, and model group + PT3309 biomass group) after 1 week of acclimatization, with 6 mice in each group.

[0056] From day 1 to 14, PT3309 bacterial culture was cultured for 2 days, then resuspended in physiological saline at a dose of 5 × 10⁹ CFU / kg and administered to mice by gavage. The PT3309 biomass group was prepared with the same dose of bacterial culture, centrifuged, washed twice with physiological saline, dried in a 60℃ oven to constant weight, dissolved in physiological saline, and administered to mice by gavage. The normal control group and the inflammatory bowel disease model group were each administered the same volume of sterile physiological saline by gavage. From day 15 to 23, the blank group received normal drinking water, while the other three groups received drinking water containing 3% sodium dextran sulfate (DSS) to establish a colitis model. After modeling began, mouse weight, diarrhea, and bloody stools were recorded daily. The results are as follows: Figure 3 As shown, A represents mouse weight change; B represents mouse diarrhea score; and C represents rectal bleeding or bloody stool in mice. Figure 3 It can be seen that, compared with the model group mice, the inflammatory bowel disease status of PT3309-colonized mice was significantly improved. Similarly, mice administered PT3309 biomass by gavage also showed improvements in weight loss, diarrhea, and bloody stools compared with the model group.

[0057] On day 23, mice in each group were administered FITC-dextran fluorescent reagent (200 mg / kg) by gavage. Blood was collected 4 hours later to detect the fluorescence intensity in the plasma and to assess intestinal permeability. The results are as follows: Figure 4 A and 4B. Compared with the model group mice, the fluorescence intensity of FITC-dextran in the blood of PT3309-colonized mice was significantly reduced, indicating that PT3309 colonization improved DSS-induced intestinal damage in mice. All mice in each group underwent colonoscopy, and the results are as follows: Figure 5 As shown in Figure A, compared with the model group mice, the colonic wall of PT3309-colonized mice was smoother, with fewer bleeding points and improved ulcer lesions. Colonic tissue was taken for HE staining, and the results are as follows. Figure 5 As shown in Figure B, compared with the model group mice, the PT3309-colonized mice showed significantly reduced histopathological damage to the colon, improved submucosal edema, and decreased inflammatory cell infiltration. After euthanizing the mice in each group, the colon and cecum were harvested, photographed on graph paper, and the colon length was calculated. One of the phenotypic characteristics of ulcerative colitis is intestinal shortening. Figure 6 As shown in A and 6B, the colon length of PT3309-colonized mice was significantly restored. The colon tissue was immediately stored at -80℃ after photographing. 30 mg of the frozen colon tissue was taken, 300 μL of protein lysis buffer was added, and the tissue was ground and lysed on ice for 1 h. Colon tissue protein was extracted, and protein concentration was determined using a BCA protein quantification kit. Western blot analysis was used to detect the expression of tight junction proteins Occludin and Claudin-1. The results showed that PT3309 colonization improved the decrease in tight junction protein expression in the colon of DSS-induced mice, restoring the intestinal barrier. 20 mg of the frozen colon tissue was taken, and total RNA was extracted. RT-PCR was used to detect the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α. Results ( Figure 7 The results showed that the levels of inflammatory factors in PT3309-colonized mice were significantly lower than those in the model group. Similarly, gavage administration of heat-inactivated PT3309 biomass significantly improved colitis-related symptoms in mice, indicating that PT3309 biomass can effectively alleviate DSS-induced colitis in mice. However, compared to PT3309, live bacteria colonization has a sustained effect in the intestine, and the treatment effect in the PT3309 colonization group was more significant.

[0058] Example 4: The alleviating effect of Yersinia lipophila PT3309 on inflammatory bowel disease in ABX mice.

[0059] Male C57BL / 6J mice aged 6-8 weeks were randomly divided into two groups (model group and model group + PT3309 colonization) after 1 week of acclimatization, with 6 mice in each group.

[0060] On days 1-3, ampicillin (1g / L), vancomycin (0.5g / L), neomycin (1g / L), metronidazole (1g / L), fluconazole (0.5g / L), and amphotericin B (0.1g / L) were added to the drinking water of both groups of mice to clear the intestinal flora, and these mice were then known as ABX mice.

[0061] From day 4 to day 18, PT3309 bacterial suspension (5×10⁹ CFU / kg) was administered to mice by gavage (model + PT3309 group); the model group was administered the same volume of sterile saline by gavage.

[0062] From days 19 to 25, both groups of mice were given drinking water containing 3% DSS to establish a colitis model. Mouse weight, diarrhea score, and fecal blood loss were recorded during the modeling process. Results are as follows: Figure 8 As shown, A represents the change in mouse body weight; B represents the condition of bloody stools in mice; and C represents the diarrhea score in mice. Figure 8 It can be seen that, compared with the model group mice, mice colonized with Yersinia lipophila PT3309 showed significant relief of inflammatory bowel disease symptoms.

[0063] On day 25, mice in each group were given 200 mg / kg of FITC-dextran fluorescent reagent. Blood was collected 4 hours later to detect the fluorescence intensity in the plasma, which reflects the intestinal permeability of the mice. The results are as follows. Figure 9 Compared with the model group mice, PT3309 colonization significantly improved DSS-induced intestinal injury. All mice underwent colonoscopy. Results are as follows: Figure 10 As shown in Figure A, compared with the model group mice, mice colonized with *Yersinia lipolyticis* PT3309 showed reduced colonic bleeding points and improved ulcer lesions. Colonic tissue was harvested, fixed in paraformaldehyde fixative for 24 hours, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (HE). The inflammation of the colonic tissue was observed under a microscope. HE-stained sections are shown below. Figure 10 As shown in Figure B, compared with the model group, the PT3309 group mice had better colonic villi integrity and no obvious inflammatory infiltration. Pathological sections indicated that *Yersinia lipolytica* PT3309 had a significant alleviating effect on colonic lesions in ABX inflammatory bowel disease mice. After sacrificing both groups of mice, the colon and cecum were harvested, photographed on graph paper, and the colon length was counted. The results showed that the colon length of the *Yersinia lipolytica* PT3309-colonized mice was significantly longer than that of the model group mice. Figure 11(A is a colon photograph, B is a colon length chart), indicating that *Yersinia lipolyticis* PT3309 has an ameliorative effect on colon length in ABX mice with inflammatory bowel disease. The expression of tight junction proteins Occludin and Claudin-1 was detected in frozen colon tissue; the results showed that PT3309 colonization improved the decrease in tight junction protein expression in the colon of ABX mice induced by DSS, restoring the intestinal barrier. The expression of inflammatory factors such as IL-1β, IL-6, and TNF-α was detected in frozen colon tissue, and the results are as follows. Figure 11 As shown in Figure C, the levels of inflammatory factors in ABX mice colonized with Yersinia lipolyticis PT3309 were significantly lower than those in the model group. These results indicate that the therapeutic effect of PT3309 on inflammatory bowel disease can act independently, without depending on the gut microbiota environment.

Claims

1. A strain of Yarrowia lipolytica PT3309, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC 65342, on October 24, 2024.

2. The use of Yersinia lipolyticis PT3309 (accession number GDMCC 65342) in the preparation of drugs for the prevention and / or treatment of inflammatory bowel disease, characterized in that... The drug comprises live bacteria of Yersinia lipophila PT3309 and / or heat-inactivated bacterial biomass.

3. The application according to claim 2, characterized in that, The inflammatory bowel disease mentioned refers to ulcerative colitis or Crohn's disease.

4. The application according to claim 2 or 3, characterized in that, The drug is used to improve colonic pathological damage, reduce the expression of colonic inflammatory factors, and / or restore intestinal barrier function.

5. A probiotic preparation, characterized in that, The probiotic preparation contains live bacteria of Yersinia lipophila PT3309 as described in claim 1, and the probiotic preparation is not used for the prevention or treatment of disease.

6. The probiotic preparation according to claim 5, characterized in that, The live bacteria of *Yersinia lipophila* PT3309 are in the form of bacterial suspension or freeze-dried live bacterial powder; the live count of *Yersinia lipophila* PT3309 in the probiotic preparation is ≥1×10⁻⁶. 6 CFU / g.

7. A pharmaceutical composition for the prevention and / or treatment of inflammatory bowel disease, characterized in that, The product comprises live bacteria and / or heat-inactivated bacterial cell biomass of Yersinia lipophila PT3309 as described in claim 1, and a pharmaceutically acceptable carrier.

8. A functional food, characterized in that, The functional food contains live bacteria and / or heat-inactivated bacterial biomass of Yeast PT3309 as described in claim 1, and is a fermented dairy product or fermented vegetable and fruit juice, and is not used for the prevention or treatment of diseases.

Images