Strain of Yarrowia guilliermondii, preparation method of inactivated vaccine of Yarrowia guilliermondii and application in prevention and treatment of alcoholic liver diseases

Abstract

The invention discloses Yarrowia guilliermondii, a preparation method of an inactivated vaccine of the Yarrowia guilliermondii and application of the Yarrowia guilliermondii in prevention and treatment of alcoholic liver diseases, and belongs to the field of microorganism eukaryotes, the preservation date of the Yarrowia guilliermondii is August 18, 2025, and the preservation number is CGMCC NO. According to the present invention, the vaccine is prepared by separating and inactivating the Yarrowia guilliermondii ML001, and the vaccine can significantly reduce the alcohol-induced ALT and AST levels in mice, reduce the liver lipid accumulation, reduce the fatty acid synthesis genes Fasn and Acly, and increase the fatty acid oxidation genes Ppara and Cpt1a so as to effectively inhibit the lipid synthesis and activate the fatty acid oxidation, moreover, the traditional Chinese medicine composition has no obvious toxicity to main organs such as heart, spleen, lung, kidney and the like, and shows good safety and clinical transformation potential.

Description

Technical Field

[0001] The present invention belongs to the field of microbial eukaryotes, and particularly relates to a strain of Mayer yeast ML001, a preparation method of an inactivated vaccine thereof, and application of the inactivated vaccine in preventing and treating alcoholic liver disease. Background Art

[0002] Alcohol-associated liver disease (ALD) is a liver disease caused by long-term, heavy drinking. Approximately 7.5 million people worldwide suffer from alcohol abuse, and long-term, excessive drinking poses significant health risks. ALD encompasses multiple pathological phenotypes, beginning with alcoholic fatty liver (fatty degeneration of the liver) and progressing to alcoholic hepatitis (liver inflammation and damage), ultimately leading to liver fibrosis, cirrhosis, and even hepatocellular carcinoma.

[0003] Despite a growing understanding of ALD, current treatment options remain very limited. Alcohol abstinence is currently the most effective measure. Early-stage mild patients can recover with nutritional support and abstinence. However, patients with alcoholic hepatitis (ASH) require the addition of anti-inflammatory and hepatoprotective medications to abstinence. Unfortunately, no medications are currently approved specifically for the treatment of ALD. While glucocorticoids have anti-inflammatory effects, they increase the risk of infection and gastrointestinal bleeding, limiting their clinical applicability. While glycyrrhizic acid preparations, pentoxifylline, and S-adenosylmethionine have been used to treat ALD, their clinical application is limited by their numerous side effects and poor efficacy.

[0004] In recent years, microbial therapy has become a research hotspot, and regulating intestinal microorganisms is regarded as a new direction for the treatment of ALD, and related clinical trials are underway. There are three main methods to regulate intestinal microorganisms: antibiotic treatment, probiotic / prebiotic supplementation, and fecal microbiota transplantation. However, clinical studies on these methods are still relatively few, and ALD treatment targeting the regulation of intestinal microorganisms is still under exploration. In addition, there are currently no studies targeting fungi for the prevention and treatment of ALD. Although studies have found a link between fungi and alcoholic liver disease, in-depth research on the potential application of fungi in the prevention and treatment of alcoholic liver disease is of great significance for breaking through the limitations of ALD treatment methods and developing new prevention and treatment strategies. Summary of the Invention

[0005] Technical Problem Solved: The present invention addresses the problem of lack of strategies for preventing and treating alcoholic liver disease, and provides a strain of Meyer yeast, a method for preparing an inactivated vaccine thereof, and its application in preventing and treating alcoholic liver disease.

[0006] Technical solution: A strain of Meyer yeast ( Meyerozyma guilliermondii) ML001, the strain has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, the deposit date is August 18, 2025, the deposit number is CGMCC NO. 35650, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] An inactivated vaccine for preventing or treating alcoholic liver disease, wherein the active ingredient of the vaccine is the bacteria obtained by inactivating the Mayerella guilleriae ML001.

[0008] The above-mentioned vaccine is in the form of freeze-dried powder.

[0009] A method for preparing the above-mentioned inactivated vaccine comprises the following steps: (1) seed culture: inoculating the Mayer yeast ML001 described in claim 1 into a liquid culture medium, and culturing with shaking at 25-40°C to obtain a seed solution; (2) bacterial cell collection and washing: centrifuging and collecting the bacterial cells in the seed solution obtained in step (1) and washing them with a buffer solution; (3) inactivation treatment: resuspending the washed bacterial cells and heating them at 65-80°C for inactivation; (4) freeze-dried preparation: centrifuging and collecting the inactivated bacterial cells, resuspending them, pre-freezing them, and vacuum freeze-drying them to obtain a vaccine freeze-dried powder.

[0010] The liquid culture medium in step (1) is YPS culture medium, the shaking culture speed is 180 rpm, the culture time is 48 hours, and the culture temperature is 30°C.

[0011] The buffer solution in step (2) is PBS buffer solution.

[0012] The temperature for heat inactivation in step (3) is 70°C and the inactivation time is 3 hours.

[0013] After step (4), the method further includes: (5) vaccine reconstitution: reconstitution of the vaccine lyophilized powder with an adjuvant to prepare a vaccine suspension.

[0014] Use of the inactivated vaccine in the preparation of a medicament for preventing or treating alcoholic liver disease.

[0015] Beneficial effects: The vaccine prepared by isolating and inactivating the yeast ML001 can significantly reduce the levels of ALT and AST induced by alcohol in mice, reduce liver lipid accumulation, and downregulate fatty acid synthesis genes. Fasn and Acly and upregulate fatty acid oxidation genes Ppara and Cpt1a , thereby effectively inhibiting lipid synthesis and activating fatty acid oxidation, alleviating alcoholic liver damage, and having no obvious toxicity to major organs such as the heart, spleen, lungs, and kidneys, showing good safety and clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention provides the macroscopic colony morphology of the preserved strain Meyer yeast ML001 on an upright plate of PDA culture medium and its microscopic morphological characteristics under an optical microscope.

[0017] Figure 2 This figure shows the effects of the inactivated vaccine of Mycobacterium guilliermonensis ML001 of the present invention on the plasma liver function indicators of mice with alcoholic liver disease model: the left figure is a statistical histogram of ALT levels, and the right figure is a statistical histogram of AST levels.

[0018] Figure 3 These are the histopathological examination results of the main organs of mice after treatment with the inactivated vaccine of Mycobacterium guilliermondii ML001 of the present invention: A is a liver tissue section, B is a spleen tissue section, C is a heart tissue section, D is a lung tissue section, and E is a kidney tissue section.

[0019] Figure 4 This is a comparison chart of the real-time fluorescence quantitative PCR statistical results of the mRNA expression levels of lipid metabolism-related genes in the liver tissue of mice after treatment with the inactivated vaccine of Meyer yeast ML001 of the present invention. DETAILED DESCRIPTION

[0020] The following examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0021] Example 1 Isolation and Identification of Meyer yeast ML001 1.1 Strain Isolation Under sterile conditions, euthanized mice were disinfected with 75% (vol / vol) ethanol. The livers were then completely removed using sterile surgical instruments and immediately placed in ice-cold sterile culture dishes. The livers were cut into small pieces and transferred to 2 mL EP tubes containing sterile 1 mm diameter magnetic beads. 1 mL of sterile PBS was added, and the caps were tightened. The EP tubes were secured to a tissue grinder set to 60 Hz, with a single 1-minute cycle and a 30-second pause, repeated 2-3 times to obtain a uniform liver homogenate. The resulting homogenate was then spread onto PDA culture plates and incubated in a 30°C incubator for 7 days. Colony growth was observed and recorded.

[0022] 1.2 Strain identification The isolated Mycobacterium guillierensis ML001 was inoculated into PDA medium and cultured at 30°C for 2 days. The colonies were round and smooth (see Figure 1A single colony was picked and transferred into a 1.5 mL EP tube containing 20 µL TE buffer and a small amount of sterile magnetic beads. The tube was boiled at 100°C for 5 min and immediately frozen at -20°C for 5 min. This was repeated once and then centrifuged at 12,000 rpm for 5 min. The supernatant was used as a PCR template and PCR amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') as shown in SEQ ID NO.1 and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') as shown in SEQ ID NO.2. The PCR program was as follows: initial denaturation at 94°C for 10 min; 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. The amplified product was sequenced by a third-party sequencing organization. The resulting ITS sequence was compared against the NCBI Nucleotide database by BLAST and identified as Meyer yeast ( Meyerozyma guilliermondii ).

[0023] Example 2 Preparation of Mayer yeast ML001 vaccine 2.1 Seed culture Under sterile conditions, a single colony of Mycobacterium guillierensis ML001 was picked and inoculated into 5 mL of YPS liquid medium. The culture was shaken at 30 °C and 180 rpm for 48 h to obtain a viable bacterial concentration of approximately 1.2 × 10 8 CFU / mL of seed solution.

[0024] 2.2 Bacteria collection and washing Take 5 mL of the above seed solution and centrifuge at 4000 rpm for 10 min to collect the bacteria; after discarding the supernatant, resuspend and wash twice with sterile PBS, and centrifuge again to obtain pure bacteria.

[0025] 2.3 Inactivation treatment The washed bacteria were resuspended in 1 mL of sterile water and dispensed into 1.5 mL EP tubes at 200 μL / tube. The tubes were heated inactivated in a 70 °C metal bath for 3 h and then cooled naturally to room temperature.

[0026] 2.4 Lyophilized preparations The inactivated bacteria were collected by centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in 100 μL sterile water; after pre-freezing at -80°C for 30 min, the cells were transferred to a vacuum freeze dryer and dried for 4 h to obtain the vaccine freeze-dried powder.

[0027] 2.5 Vaccine reconstitution Take 1 mL of vaccine adjuvant to dissolve the above lyophilized powder and vortex thoroughly to mix to obtain the Mayer yeast ML001 vaccine suspension for later use.

[0028] Example 3 Verification of the immune protection effect of the ML001 vaccine of Meyeriella guilliermondii on mice with alcoholic liver disease 3.1 Experimental animals and groups Twelve SPF female mice (20 ± 2 g) from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., strain C57BL / 6N, were randomly divided into two groups, with 6 mice in each group: A) vaccine immunization group; B) adjuvant control group.

[0029] The disease model employed was a mouse model of alcoholic liver disease (ALD) characterized by chronic alcohol feeding followed by a single acute alcohol gavage, also known as the NIAAA model. The vaccine group received intramuscular injections of 100 μL of the inactivated Mayer yeast ML001 vaccine of the present invention on days 0, 7, and 14 of the experiment, while the adjuvant group received an equal volume of adjuvant. Model establishment began on day 14. For the first 5 days, the mice were fed an adaptive liquid diet. On day 19, they were switched to a Lieber-DeCarli liquid diet containing 5% (vol / vol) ethanol, which was continued for 10 days. On the morning of day 11, mice were weighed and then gavaged with (mouse weight (g) × 20) μL of 31.5% (vol / vol) ethanol solution. Nine hours later, the mice were euthanized, and various indicators were measured.

[0030] 3.2 Determination of liver function indicators At the end of the experiment, mice in each group were euthanized, and blood from the inferior vena cava was quickly collected and centrifuged at 3000 × g for 10 min to separate the plasma. The activities of ALT and AST in the plasma were determined. Figure 2 As shown: Compared with the adjuvant control group, the ALT and AST levels in the vaccine immunization group were significantly reduced ( P < 0.05), indicating that the vaccine of the present invention can effectively inhibit alcohol-induced liver damage.

[0031] 3.3 Histopathological examination The liver, heart, spleen, lungs, and bilateral kidneys were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned (5 μm thick) for hematoxylin-eosin (H&E) staining. Figure 3 As shown: Compared with the adjuvant control group, the number of lipid droplets in the liver of mice in the vaccine immunization group was significantly reduced, and there was no significant difference in the histological morphology of the heart, spleen, lung, and kidney, indicating that the vaccine of the present invention can significantly protect against alcohol-induced fatty liver, has no toxic effects on major organs, and has good safety.

[0032] 3.4 Real-time quantitative PCR (qPCR) Approximately 30 mg of liver tissue was collected, and total RNA was extracted using the TRIzol method. After reverse transcription, the expression of lipid metabolism-related genes was detected using SYBR Green qPCR. 18S rRNA was used as an internal reference. qPCR primer sequences are shown in Table 1.

[0033] Table 1 qPCR primer list

[0034] The results are as follows Figure 4 As shown: Compared with the adjuvant control group, the fatty acid synthesis gene ( Fasn、Acly ) expression was significantly downregulated, suggesting that the lipid synthesis pathway was inhibited; the fatty acid oxidation gene ( Ppara 、 Cpt1a ) expression was significantly upregulated, indicating activation of the fatty acid oxidation pathway. Therefore, the vaccine of the present invention exerts its protective effect against alcohol-induced fatty liver by inhibiting lipid synthesis and promoting fatty acid oxidation.

[0035] 3.5 Statistical methods The data are expressed as Mean ± SEM, and two-tailed unpaired Student's t test was performed using GraphPad Prism 9.0; P A difference of < 0.05 was considered statistically significant.

Claims

1. A strain of Meyer yeast ( Meyerozyma guilliermondii )ML001, characterized in that, The strain has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit date being August 18, 2025, and the deposit number being CGMCC NO. 35650.

2. An inactivated vaccine for preventing or treating alcoholic liver disease, characterized in that: The active ingredient of the vaccine is the bacteria obtained by inactivating the Mayer yeast ML001 according to claim 1.

3. The inactivated vaccine according to claim 2, characterized in that The vaccine is in the form of a freeze-dried powder.

4. A method for preparing the inactivated vaccine according to claim 2 or 3, characterized in that: The following steps are involved: (1) Seed culture: inoculate the Mayer yeast strain ML001 described in claim 1 into a liquid culture medium and culture it with shaking at 25-40°C to obtain a seed solution; (2) Bacterial cell collection and washing: collect the bacterial cells in the seed solution obtained in step (1) by centrifugation and wash them with buffer; (3) Inactivation treatment: resuspend the washed bacterial cells and heat inactivate them at 65-80°C; (4) Freeze-dried preparation: collect the inactivated bacterial cells by centrifugation, resuspend them, pre-freeze them and vacuum freeze-dry them to obtain vaccine freeze-dried powder.

5. The method according to claim 4, characterized in that The liquid culture medium in step (1) is YPS culture medium, the shaking culture speed is 180 rpm, the culture time is 48 hours, and the culture temperature is 30°C.

6. The method according to claim 4, characterized in that The buffer solution in step (2) is PBS buffer solution.

7. The method according to claim 4, characterized in that The temperature for heat inactivation in step (3) is 70°C and the inactivation time is 3 hours.

8. The method according to claim 4, characterized in that After step (4), the method further includes: (5) vaccine reconstitution: reconstitution of the vaccine lyophilized powder with an adjuvant to prepare a vaccine suspension.

9. Use of the inactivated vaccine according to claim 2 or 3 in the preparation of a medicament for preventing or treating alcoholic liver disease.

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