Meyerozyma guilliermondii, preparation method of inactivated vaccine thereof and application in prevention and treatment of alcoholic liver disease

Abstract

The application belongs to the field of microorganism eukaryotes, and the strain preservation date is August 18, 2025, and the preservation number is CGMCC NO.35650. The vaccine is prepared by isolating and inactivating the Meyerozyma guiliardmi yeast ML001, the vaccine can significantly reduce the alcohol-induced ALT and AST levels in mice, reduce liver lipid accumulation, down-regulate fatty acid synthesis genes Fasn and Acly and up-regulate fatty acid oxidation genes Ppara and Cpt1a , thereby effectively inhibiting lipid synthesis and activating fatty acid oxidation, reducing alcoholic liver injury, and having no obvious toxicity to main organs such as heart, spleen, lung and kidney, and showing good safety and clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of microbial eukaryotes, specifically relating to a strain of Saccharomyces cerevisiae ML001, a method for preparing its inactivated vaccine, and its application in the prevention and treatment of alcoholic liver disease. Background Technology

[0002] Alcohol-associated liver disease (ALD) is a liver disease caused by long-term heavy drinking. Approximately 7.5 million people worldwide have alcohol abuse problems, and long-term excessive drinking poses a significant threat to health. ALD includes various pathological phenotypes: it begins with alcoholic fatty liver (hepatic steatosis), gradually progressing to alcoholic hepatitis (liver inflammation and damage), and may eventually lead to liver fibrosis, cirrhosis, and even hepatocellular carcinoma.

[0003] Despite a deepening understanding of ALD, current treatment options remain very limited. Abstinence from alcohol is currently the most effective measure; early-stage, mild cases can recover with nutritional support in addition to abstinence. Patients with alcoholic hepatitis (ASH) require abstinence in addition to anti-inflammatory and hepatoprotective medications. Unfortunately, no drugs 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. Although glycyrrhizic acid preparations, pentoxifylline, and S-adenosylmethionine have been used to treat ALD, their clinical application is limited due to numerous side effects and poor efficacy.

[0004] In recent years, microbial therapy has become a research hotspot, and regulating gut microbiota is considered a new direction for ALD treatment, with related clinical trials underway. There are three main methods for regulating gut microbiota: antibiotic therapy, probiotic / prebiotic supplementation, and fecal microbiota transplantation (FMT). However, clinical research on these methods is still limited, and ALD treatment targeting gut microbiota regulation is still under exploration. Furthermore, there are currently no studies targeting fungi for the prevention and treatment of ALD. Although existing research has found a link between fungi and alcoholic liver disease, in-depth research into the potential applications of fungi in the prevention and treatment of alcoholic liver disease is of great significance for overcoming the limitations of ALD treatment methods and developing new prevention and treatment strategies. Summary of the Invention

[0005] Technical problem solved: This invention addresses the lack of strategies for the prevention and treatment of alcoholic liver disease by providing a strain of Saccharomyces cerevisiae, a method for preparing its inactivated vaccine, and its application in the prevention and treatment of alcoholic liver disease.

[0006] Technical solution: A strain of Meyerozyma guilliermondii ML001, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 18, 2025, with accession number CGMCC NO. 35650, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] An inactivated vaccine for the prevention or treatment of alcoholic liver disease, wherein the active ingredient of the vaccine is a bacterial cell obtained by inactivating the aforementioned *Saccharomyces cerevisiae* ML001.

[0008] The aforementioned vaccine is a freeze-dried powder formulation.

[0009] A method for preparing the above-mentioned inactivated vaccine includes the following steps: (1) Seed culture: the above-mentioned yeast ML001 is inoculated into a liquid culture medium and cultured by shaking at 25-40 ℃ to obtain seed liquid; (2) Cell collection and washing: the cells in the seed liquid obtained in step (1) are collected by centrifugation and washed with buffer solution; (3) Inactivation treatment: the washed cells are resuspended and inactivated by heating at 65-80 ℃; (4) Freeze-dried preparation: the inactivated cells are collected by centrifugation, resuspended and pre-frozen and vacuum freeze-dried to obtain freeze-dried vaccine powder.

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

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

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

[0013] The process after step (4) also includes: (5) vaccine reconstitution: the lyophilized vaccine powder is reconstituted using an adjuvant to prepare a vaccine suspension.

[0014] The above-mentioned inactivated vaccine is used in the preparation of drugs for the prevention or treatment of alcoholic liver disease.

[0015] Beneficial effects: This invention prepares a vaccine by isolating and inactivating Saccharomyces giardiformis ML001. This vaccine can significantly reduce alcohol-induced ALT and AST levels in mice, reduce lipid accumulation in the liver, 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 showing no obvious toxicity to major organs such as the heart, spleen, lungs, and kidneys, demonstrating good safety and clinical translation potential. Attached Figure Description

[0016] Figure 1 Macroscopic colony morphology of the preserved strain of yeast ML001 of this invention on an upright plate of PDA medium and its microscopic morphological characteristics under an optical microscope.

[0017] Figure 2 The effects of the inactivated Saccharomyces cerevisiae ML001 vaccine of this invention on plasma liver function indicators in mice with alcoholic liver disease are shown in the following figures: the left figure is a bar chart of ALT level and the right figure is a bar chart of AST level.

[0018] Figure 3 The following are the histopathological examination results of the major organs of mice after treatment with the inactivated Saccharomyces cerevisiae ML001 vaccine of this 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 of the real-time quantitative PCR results of the expression levels of lipid metabolism-related genes mRNA in mouse liver tissue after treatment with the inactivated Saccharomyces cerevisiae ML001 vaccine of this invention. Detailed Implementation

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

[0021] Example 1: Isolation and Identification of Saccharomyces cerevisiae ML001

[0022] 1.1 Strains Isolation

[0023] Under aseptic conditions, euthanized mice were disinfected with 75% (vol / vol) ethanol, and their livers were completely removed using sterile surgical instruments and immediately placed in pre-chilled sterile culture dishes. The livers were cut into small pieces and transferred to 2 mL EP tubes containing 1 mm diameter sterile magnetic beads. 1 mL of sterile PBS was added, and the tubes were tightly capped. The EP tubes were fixed in a tissue homogenizer, set to a frequency of 60 Hz, and run for 1 min with a 30 s interval, repeating 2-3 times to obtain a homogenized liver slurry. All homogenates were spread onto PDA agar plates and incubated at 30 ℃ for 7 days. Colony growth was observed and recorded.

[0024] 1.2 Strain Identification

[0025] The isolated *Saccharomyces giardi* ML001 was inoculated into PDA medium and cultured at 30 °C for 2 days. The colonies were round and smooth (see...). Figure 1Single colonies were picked and transferred into 1.5 mL EP tubes containing 20 µL TE buffer and a small amount of sterile magnetic beads. The tubes were boiled at 100 °C for 5 min, immediately frozen at -20 °C for 5 min, and this process was repeated once. The tubes were then centrifuged at 12,000 rpm for 5 min. The supernatant was used as a PCR template, and PCR amplification was performed using primers ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. The PCR program was as follows: 94 °C pre-denaturation 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 products were sequenced by a third-party sequencing institution. The obtained ITS sequence was identified as *Meyerozyma guilliermondii* by BLAST comparison with the NCBI Nucleotide database.

[0026] Example 2: Preparation of Saccharomyces cerevisiae ML001 vaccine

[0027] 2.1 Seed Culture

[0028] Under aseptic conditions, a single colony of *Saccharomyces cerevisiae* ML001 was picked and inoculated into 5 mL of YPS liquid medium. The culture was carried out at 30 ℃ and 180 rpm with shaking for 48 h, yielding a viable cell concentration of approximately 1.2 × 10⁻⁶. 8 Seed culture of CFU / mL.

[0029] 2.2 Collection and washing of bacterial cells

[0030] Take 5 mL of the above seed culture, centrifuge at 4000 rpm for 10 min to collect the bacterial cells; discard the supernatant, resuspend in sterile PBS and wash twice, centrifuge again to obtain pure bacterial cells.

[0031] 2.3 Inactivation treatment

[0032] The washed bacterial cells were resuspended in 1 mL of sterile water, dispensed into 1.5 mL EP tubes at a rate of 200 μL / tube, and heated in a 70 ℃ metal bath for 3 h to inactivate the bacteria, followed by natural cooling to room temperature.

[0033] 2.4 Lyophilized Formulations

[0034] The inactivated bacterial cells were collected by centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in 100 μL of sterile water. After pre-freezing at -80℃ for 30 min, the cells were transferred to a vacuum freeze dryer and dried for 4 h to obtain the vaccine freeze-dried powder.

[0035] 2.5 Vaccine reconstitution

[0036] Dissolve the above lyophilized powder in 1 mL of vaccine adjuvant and vortex thoroughly to obtain the Saccharomyces cerevisiae ML001 vaccine suspension for later use.

[0037] Example 3: Verification of the immunoprotective effect of Saccharomyces cerevisiae ML001 vaccine on mice with alcoholic liver disease.

[0038] 3.1 Laboratory Animals and Grouping

[0039] Twelve SPF-grade female mice (20 ± 2 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a strain of C57BL / 6N. They were randomly divided into two groups of six mice each: A) vaccine immunization group; B) adjuvant control group.

[0040] The disease model used was a mouse model of alcoholic liver disease induced by chronic alcohol feeding followed by a single acute alcohol gavage, also known as the NIAAA model. The vaccine group received an intramuscular injection of 100 μL of the inactivated *Saccharomyces cerevisiae* ML001 vaccine of this invention on days 0, 7, and 14 of the experiment, while the adjuvant group received an equal volume of adjuvant. Model construction began on day 14. For the first 5 days, the mice were fed an adaptive liquid diet. On day 19, the diet was changed to a Lieber-DeCarli liquid diet containing 5% (vol / vol) ethanol, which was continued for 10 days. On the morning of day 11, the mice were weighed, and then gavage was administered a 31.5% (vol / vol) ethanol solution at a volume of (mouse weight (g) × 20) μL. Nine hours later, the mice were euthanized, and various indicators were measured.

[0041] 3.2 Liver function index measurement

[0042] At the end of the experiment, mice in each group were euthanized, and inferior vena cava blood was rapidly collected. The plasma was separated by centrifugation at 3000 × g for 10 min, and the activities of ALT and AST in the plasma were measured. Results are as follows: Figure 2 As shown, compared with the adjuvant control group, the levels of ALT and AST in the vaccine immunization group were significantly reduced (P < 0.05), suggesting that the vaccine of the present invention can effectively inhibit alcohol-induced liver injury.

[0043] 3.3 Histopathological examination

[0044] Liver, heart, spleen, lungs, and both kidneys were collected, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned (5 μm thick) for hematoxylin-eosin (H&E) staining. Results are as follows: Figure 3As 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 no significant differences were found in the histological morphology of the heart, spleen, lungs and kidneys. This indicates that the vaccine of the present invention can significantly protect against alcohol-induced fatty liver and has no toxic effects on major organs, and has good safety.

[0045] 3.4 Real-time quantitative PCR (qPCR)

[0046] 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 by SYBR Green qPCR. 18S rRNA was used as an internal control, and the primer sequences are shown in Sequence Listing 1.

[0047] Table 1 List of qPCR primers

[0048]

[0049] The results are as follows Figure 4 As shown, compared with the adjuvant control group, the expression of fatty acid synthesis genes (Fasn, Acly) was significantly downregulated in the vaccine-immunized group, indicating that the lipid synthesis pathway was inhibited; the expression of fatty acid oxidation genes (Ppara, Cpt1a) was significantly upregulated in the vaccine-immunized group, indicating that the fatty acid oxidation pathway was activated. Therefore, the vaccine of this invention exerts a protective effect against alcohol-induced fatty liver by inhibiting lipid synthesis and promoting fatty acid oxidation.

[0050] 3.5 Statistical Methods

[0051] Data are expressed as Mean ± SEM. Two-tailed unpaired Student's t-test was performed using GraphPad Prism 9.0; P < 0.05 was considered statistically significant.

Claims

1. A strain of *Saccharomyces cerevisiae* ( Meyerozyma guilliermondii ML001, characterized in that, The strain has been preserved in the China General Microbiological Culture Collection Center on August 18, 2025, and the preservation number is 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 body obtained by inactivating the Meyerozyma guilliermondii ML001 in claim 1.

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

4. A method for the preparation of an inactivated vaccine according to claim 2 or 3, characterized in that, The method comprises the following steps: (1) Seed culture: inoculate the Meyerozyma guilliermondii ML001 in claim 1 into a liquid culture medium, and cultivate at 25-40 ℃ with oscillation to obtain a seed liquid; (2) Bacteria body collection and washing: centrifugally collect the bacteria body in the seed liquid obtained in step (1), and wash with a buffer solution; (3) Inactivation treatment: resuspend the washed bacteria body, and heat-inactivate at 65-80 ℃; (4) Freeze-dried preparation: centrifugally collect the inactivated bacteria body, resuspend, and then pre-freeze and vacuum freeze-dry to obtain a vaccine freeze-dried powder.

5. The method of claim 4, wherein, The liquid culture medium in step (1) is YPS culture medium, the oscillation culture speed is 180 rpm, the culture time is 48 hours, and the culture temperature is 30 ℃.

6. The method of claim 4, wherein, The buffer solution in step (2) is a PBS buffer solution.

7. The method of claim 4, wherein, The temperature for heat-inactivation in step (3) is 70 ℃, and the inactivation time is 3 hours.

8. The method of claim 4, wherein, After step (4), the method further comprises: (5) Vaccine reconstitution: reconstitute the vaccine freeze-dried powder with an adjuvant to prepare a vaccine suspension.

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

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