Wickerhamomyces anomalus Hta-3 and application thereof

Abstract

The invention relates to the technical field of microbial fermentation, and particularly provides Wickerhamomyces anomalus Hta-3 and application thereof, the Wickerhamomyces anomalus Hta-3 is preserved in China General Microbiological Culture Collection Center on June 6, 2025, and the preservation number is CGMCC No.34786; the content of flavone in the fermented hippophae rhamnoides fruit juice can be remarkably increased by 28.79%, and the fermented hippophae rhamnoides fruit juice has a certain repairing effect on acute alcoholic injury, reduces the content of proinflammatory factors IL-6 and TNF-alpha, relieves inflammatory response of the liver, improves the SOD activity of the liver and the protein expression quantity of ALDH2 and Bcl-2, enhances the oxidative stress ability of the liver, reduces liver injury caused by excessive drinking, and has a good application prospect. The acute alcoholic injury of the mouse is relieved; in addition, through fermentation, the taste of the Tibetan hippophae rhamnoides fruit juice can be improved, the shelf life of the hippophae rhamnoides fruit juice is prolonged, transformation of traditional fermented products is facilitated, and the economic benefits of the products are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to an abnormal Wickham yeast Hta-3 and an application thereof. Background Art

[0002] Tibetan sea buckthorn (Hippophae thibetana) belongs to the genus Hippophae of the family Elaeagnaceae. It is drought-resistant and salt-alkali-resistant and is often planted in Northwest and North my country for soil and water conservation and windbreak and sand fixation, and has important ecological significance. As a plant with both medicinal and edible properties, it contains a variety of bioactive substances and is known as a "treasure trove of bioactive substances." Its main component, sea buckthorn flavonoids, has the effects of protecting the cardiovascular system, improving the blood system, enhancing immunity, anti-oxidation, anti-cancer, anti-allergy, and antibacterial, and is widely used in industries such as food, cosmetics, and nutritional preparations. However, Tibetan sea buckthorn fruit has a high water content of approximately 70%, making it difficult to store at room temperature and susceptible to mechanical damage and microbial infection during picking and transportation. In addition, sea buckthorn fruit has a sour and astringent taste and does not taste good when eaten directly, making it unpopular among the public. Therefore, deep processing of sea buckthorn fruit to improve the taste of sea buckthorn products is a technical problem that needs to be urgently addressed by those skilled in the art.

[0003] In recent years, microbial fermentation has become a popular food processing method. The addition of microorganisms to food production aids preservation, improves sensory properties, alters nutritional and bioactive properties, and helps maintain and restore the human intestinal microbiome. Studies have found that the bioactive components of most edible and medicinal plants are complex, and some natural products cannot be directly absorbed by humans and animals. Furthermore, the content of most bioactive components in these plants is low, even below 1%, and some substances are toxic to humans and animals. However, the use of microbial fermentation technology can enhance the efficacy of edible and medicinal plants, reduce toxicity, produce new chemical components, increase the utilization rate of active ingredients, and enhance their pharmacological activity. Therefore, the use of microbial fermentation in food holds great promise for development.

[0004] In order to solve the above technical problems, the inventors screened a Wickerhamomyces anomalus Hta-3 in the research process, which is used for fermenting Tibetan Hippophae rhamnoides, and the fermentation conditions are optimized, the flavone content in the fermented Tibetan Hippophae rhamnoides fruit juice can be significantly increased by 28.79%, and has a certain repair effect on acute alcohol damage, reduces the contents of pro-inflammatory factors IL-6 and TNF-α, reduces the inflammatory reaction of the liver, increases the SOD activity, ALDH2 and Bcl-2 protein expression of the liver, enhances the oxidative stress capacity of the liver, reduces the liver damage caused by excessive drinking, and relieves acute alcohol damage in mice; in addition, the fermentation can improve the taste of Tibetan Hippophae rhamnoides juice, prolong the shelf life of Hippophae rhamnoides juice, and is more helpful for the transformation of traditional fermentation products and the improvement of economic benefits of products. SUMMARY

[0005] The primary object of the present application is to provide a Wickerhamomyces anomalus Hta-3, which is deposited with the China General Microbiological Culture Collection Center on June 6, 2025, and the deposit number is CGMCC No.34786.

[0006] The second object of the present application is to provide a microbial agent, which comprises the Wickerhamomyces anomalus Hta-3.

[0007] The third object of the present application is to provide the application of the Wickerhamomyces anomalus Hta-3 or the microbial agent in fermenting Hippophae rhamnoides.

[0008] The fourth object of the present application is to provide a Hippophae rhamnoides product, which is obtained by fermenting Hippophae rhamnoides with the Wickerhamomyces anomalus Hta-3.

[0009] The fifth object of the present application is to provide the application of the Hippophae rhamnoides product in preparing an alcoholism-relieving product.

[0010] The sixth object of the present application is to provide the application of the Hippophae rhamnoides product in preparing a liver damage-treating drug.

[0011] The seventh object of the present application is to provide the application of the Hippophae rhamnoides product in preparing an antioxidant product.

[0012] The eighth object of the present application is to provide the application of the Hippophae rhamnoides product in preparing a liver-protecting product.

[0013] The ninth object of the present application is to provide the application of the Hippophae rhamnoides product in preparing an anti-inflammatory product.

[0014] The application provides an abnormal Wickerhamomyces anomalus Hta-3, which is preserved in the China General Microbiological Culture Collection Center on June 6, 2025, and has a preservation number of CGMCC No.34786; and specifically discloses the application in fermenting a Tibetan Hippophae rhamnoides product. The flavone content in the Tibetan Hippophae rhamnoides juice after fermentation of the abnormal Wickerhamomyces anomalus Hta-3 can be increased by 28.79%, and the flavone content has a certain repairing effect on acute alcohol damage, reduces the contents of pro-inflammatory factors IL-6 and TNF-alpha, reduces the inflammatory reaction of the liver, increases the SOD activity, ALDH2 and Bcl-2 protein expression of the liver, enhances the oxidative stress capacity of the liver, reduces the liver damage caused by excessive drinking, and relieves acute alcohol damage of mice. In addition, the fermentation can improve the taste of the Tibetan Hippophae rhamnoides juice, prolong the shelf life of the Tibetan Hippophae rhamnoides juice, is more helpful for the transformation of traditional fermentation products, and improves the economic benefits of products. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Effect of different natural fermentation times on flavone content in Hippophae rhamnoides fruits

[0016] Figure 2 Morphological characteristics of the strain Hta-3

[0017] Figure 3 Phylogenetic tree of the strain Hta-3

[0018] Figure 4 Change of flavone content in Hippophae rhamnoides under different fermentation conditions

[0019] Figure 5 Response surface and contour graph of the influence of different two-factor interactions on flavone content in Hippophae rhamnoides

[0020] Figure 6 Effect of different treatments on liver index of miceNote: compared with the blank group * P<0.05; compared with the blank group * P<0.01; compared with the blank group ** P<0.001; compared with the model group # P<0.05; compared with the model group ## P<0.01; compared with the model group ### P<0.001;

[0021] Figure 7 Effect of different treatments on inflammatory factors in liver tissues of miceNote: compared with the blank group * P<0.05; compared with the blank group * P<0.01; compared with the blank group ** P<0.001; compared with the model group #P<0.05; compared with the model group ## P<0.01; compared with the model group ### P < 0.001;

[0022] Figure 8 Effects of different treatments on oxidative stress in mouse liver tissue Note: Compared with the blank group * P<0.05; compared with the blank group* * P<0.01; compared with the blank group* ** P<0.001; compared with the model group # P<0.05; compared with the model group ## P<0.01; compared with the model group ### P < 0.001;

[0023] Figure 9 Effects of different treatments on alcohol metabolism in mouse liver tissue Note: Compared with the blank group * P<0.05; compared with the blank group* * P<0.01; compared with the blank group* ** P<0.001; compared with the model group # P<0.05; compared with the model group ## P<0.01; compared with the model group ### P < 0.001;

[0024] Figure 10 Effects of different treatments on apoptosis of mouse liver cells Note: Compared with the blank group * P<0.05; compared with the blank group* * P<0.01; compared with the blank group* ** P<0.001; compared with the model group # P<0.05; compared with the model group ## P<0.01; compared with the model group ### P < 0.001; DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to specific embodiments.

[0026] In the following examples, the PDA solid culture medium (g / L) was formulated as follows: 200 g fresh potato, 20 g glucose, 20 g agar, diluted to 1 L with distilled water, set to natural pH, and sterilized at 121° C. for 20 min.

[0027] The Tibetan seabuckthorn fruits in the following examples were picked from Tianzhu County, Wuwei City, Gansu Province.

[0028] Unless otherwise specified, other experimental materials and instruments can be purchased through commercial channels.

[0029] Example 1. Screening, purification and identification of strains

[0030] Natural seabuckthorn fermentation: Wash Tibetan seabuckthorn berries 2-3 times with saline to remove dust and bacteria from the surface. After washing, place in a well-ventilated area to air dry. Take 9g of dried seabuckthorn berries and add sugar in a ratio of 3:1 (weight of Tibetan seabuckthorn berries to weight of sugar). Place in a sterile fermentation bottle and mix thoroughly. Add 3-5 drops of purified water and seal the bottle for fermentation. Open the vent valve every 5 days for ventilation. Determine the flavonoids, total phenolics, and vitamin C contents on the 5th, 10th, 15th, 20th, 25th, and 30th day of fermentation.

[0031] Yeast isolation and purification: Take 2g of Tibetan sea buckthorn fruit with the best fermentation time and grind it in a sterile mortar, dilute it with 10 -6 times, spread 100 μL of the diluted fermentation liquid on PDA medium and culture in a 28°C incubator for 2-4 days; pick single colonies of different morphologies and streak inoculate them on PDA medium, repeat the separation and purification three times, pick single colonies for microscopic examination, and store the purified strains at 4°C for later use.

[0032] Yeast screening: Use the isolated and purified single strain to ferment the pasteurized sea buckthorn juice at a fermentation temperature of 28°C. After 5 days of fermentation, the sea buckthorn flavonoid content is measured, and the strain with the best sea buckthorn flavonoid enhancement effect is selected as the fermentation bacteria for the subsequent fermentation of Tibetan sea buckthorn juice.

[0033] Morphological identification of yeast: A preliminary judgment is made based on the size, color, surface smoothness, neatness of edges, consistency of color between the front and back sides, and edge and center parts of the yeast colony in the "Handbook of Characteristics and Identification of Yeast", and then its morphological structure is observed under a microscope.

[0034] Molecular biological identification and phylogenetic tree construction of yeast: Yeast DNA was extracted according to the rapid extraction kit procedure for fungal genomic DNA. PCR reaction system (50 μL), template DNA 2 μL, 26S rDNA primers:

[0035] NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3',

[0036] NL4: 5'-GGTCCGTGTTTTCAAGACGG--3'

[0037] 1 μL of each was added to 25 μL of Taq enzyme premix, and then double-distilled water was added to 50 μL. PCR reaction conditions included 35 cycles of pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 5 minutes and a 4°C incubation. After PCR amplification, 2 μL of the amplified product was examined on a 1% agarose gel for the presence of the specific target band. The PCR product was then sent to Shanghai Sangon Biotechnology Service Co., Ltd. for sequencing.

[0038] The sequenced gene sequences were entered into NCBI for BLAST homology comparison, and the phylogenetic tree was constructed using the Neighbor-Joining method in MEGA7 software for further species identification (the phylogenetic tree was tested using the bootstrap method, and the bootstrap data set was 1000).

[0039] Depend on Figure 1 It is known that the flavonoid content of sea buckthorn fruit is highest at 10 days of natural fermentation, so the strain was isolated at 10 days. Screening of the isolated strains showed that inoculation with strain Hta-3 significantly increased the flavonoid content in sea buckthorn juice. The colonies of this fungus are milky white and round, with a large diameter, a slightly raised center, irregular edges, and radially raised and wrinkled surfaces. The cells are sausage-shaped, with Candida-type hyphae and are multi-lateral budding ( Figure 2 ). A phylogenetic tree of strains was constructed based on 26S rDNA gene sequences. Figure 3 As shown, strain Hta-3 and abnormal Wickerham yeast clustered in a branch and had the closest relationship. Therefore, the strain was identified as abnormal Wickerham yeast, with the Latin name Wickerhamomyces anomalus, and named as abnormal Wickerham yeast (Wickerhamomyces anomalus) Hta-3. It was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on June 6, 2025, with the deposit number CGMCC NO.34786. The deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Tel: 010-64807596; Fax: 010-64807596.

[0040] In the following examples, Wickerhamomyces anomalus Hta-3 is abbreviated as Wickerhamomyces anomalus Hta-3.

[0041] Example 2: Preparation of fermented Tibetan sea buckthorn juice

[0042] The preparation method of Tibetan sea buckthorn juice is as follows:

[0043] (1) Weigh 100 g of ripe, plump, and mechanically undamaged Tibetan sea buckthorn fruit, rinse repeatedly with physiological saline to remove dust and bacteria from the peel, and dry in a ventilated place for later use;

[0044] (2) The washed and dried Tibetan sea buckthorn fruit was squeezed with a juicer, filtered twice with 100-mesh medical degreased gauze, and the residue was discarded. Three times the volume of purified water and 20% w / v white sugar were added to the filtrate and mixed to obtain Tibetan sea buckthorn juice;

[0045] (3) Sterilize the Tibetan sea buckthorn juice obtained in (2) at 63°C for 30 min, cool to room temperature, and then refrigerate at 4°C for later use;

[0046] (4) picking the abnormal Wickham yeast Hta-3 stored on the slant, streaking on PDA solid medium for activation, and then culturing in liquid medium at 28°C and 180 rpm for 48 h to obtain a suspension of abnormal Wickham yeast Hta-3;

[0047] (5) The bacterial suspension obtained in (4) was inoculated with 1% of the volume of the Tibetan sea buckthorn juice, and cultured at 28° C. and 180 rpm for 5 days to obtain fermented Tibetan sea buckthorn juice.

[0048] Example 3: Optimization of fermentation time

[0049] 15 mL of the Tibetan sea buckthorn juice in Example 2 was taken into a sterile fermentation bottle, and the bacterial suspension was inoculated into the sea buckthorn juice at a rate of 1% of the inoculum. 20% of white sugar was added and cultured at 30°C and 180 rpm for 3, 4, 5, 6, and 7 days, respectively. Three replicates were set for each treatment, and the flavonoid content in the Tibetan sea buckthorn juice after fermentation was determined.

[0050] On the 3rd, 4th, 5th, 6th, and 7th day of fermentation, 5 mL of fermented sea buckthorn juice was collected, diluted to 25 mL with 40% ethanol, and ultrasonicated at 4000 Hz for 20 minutes. After ultrasonication, the fermented juice was centrifuged at 5000 r / min for 20 minutes. 2 mL of the supernatant was collected and placed in a test tube. 1 mL of 5% NaNO2 solution was added, mixed well, and allowed to stand in the dark for 6 minutes. 1 mL of 10% Al(NO3)3 solution was added, mixed well, and allowed to stand in the dark for 6 minutes. 10 mL of 10% NaOH solution was added, diluted to 10 mL with 30% ethanol, and allowed to stand in the dark for 15 minutes. The absorbance was measured at 510 nm. The flavonoid content was calculated using the formula.

[0051] Flavonoid content (mg / L) = (A5 10-0.0163) / 0.0162×D f

[0052] Among them, A5 10 represents the absorbance value of the sample at 510nm, D f is the dilution multiple.

[0053] Depend on Figure 4 It can be seen from a that among the five fermentation time periods, the flavonoid content in the fermented Tibetan sea buckthorn juice reached the highest when the fermentation time was 5 days, so 5 days was selected as the fermentation time for subsequent fermentation condition optimization.

[0054] Example 4: Optimization of fermentation temperature

[0055] 15 mL of the Tibetan sea buckthorn juice in Example 2 was taken into a sterile fermentation bottle, and the bacterial liquid was inoculated into the Tibetan sea buckthorn juice at a 1% inoculum amount, and 20% white sugar was added. The temperatures were set at 26°C, 28°C, 30°C, 32°C, and 34°C, respectively, and the shaker was cultured at a speed of 180 r / min for 5 days. Three replicates were set for each treatment, and the flavonoid content in the Tibetan sea buckthorn juice after fermentation was determined.

[0056] After 5 days, 5 mL of fermented sea buckthorn juice was taken, diluted to 25 mL with 40% ethanol, and sonicated at 4000 Hz for 20 minutes. After sonication, the fermented juice was centrifuged at 5000 r / min for 20 minutes. 2 mL of the supernatant was placed in a test tube, 1 mL of 5% NaNO2 solution was added, the mixture was mixed and allowed to stand in the dark for 6 minutes, 1 mL of 10% Al(NO3)3 solution was added, the mixture was mixed and allowed to stand in the dark for 6 minutes, 10 mL of 10% NaOH solution was added, the mixture was diluted to 10 mL with 30% ethanol, the mixture was allowed to stand in the dark for 15 minutes, and the absorbance was measured at 510 nm. The flavonoid content was calculated using the formula.

[0057] Flavonoid content (mg / L) = (A5 10-0.0163) / 0.0162×D f

[0058] Among them, A5 10 represents the absorbance value of the sample at 510nm, D f is the dilution multiple.

[0059] Depend on Figure 4 b It can be seen that among the five fermentation temperatures, when the temperature is 28°C, the flavonoids content in the fermented Tibetan sea buckthorn juice reaches the highest, so 28°C is selected as the fermentation temperature for subsequent fermentation condition optimization.

[0060] Example 5: Optimization of sugar content

[0061] 15 mL of the Tibetan sea buckthorn juice in Example 2 was taken into a sterile fermentation bottle, and the bacterial suspension was inoculated into the sea buckthorn juice at a rate of 1% of the inoculum. 5%, 10%, 15%, 20%, and 25% of white sugar were added and mixed, and the mixture was cultured at 30°C and 180 r / min for 5 days. Three replicates were set for each treatment, and the flavonoid content in the Tibetan sea buckthorn juice after fermentation was determined.

[0062] After 5 days, take 5 mL of fermented sea buckthorn juice, add 40% ethanol to 25 mL, and sonicate at 4000 Hz for 20 minutes. After sonication, centrifuge the fermented juice at 5000 r / min for 20 minutes. Take 2 mL of the supernatant and place it in a test tube. Add 1 mL of 5% NaNO2 solution, mix well, and let it stand in the dark for 6 minutes. Add 1 mL of 10% Al(NO3)3 solution, mix well, and let it stand in the dark for 6 minutes. Add 10 mL of 10% NaOH solution, and dilute to 10 mL with 30% ethanol. Let it stand in the dark for 15 minutes. Measure the absorbance at 510 nm. Substitute this into the formula to calculate the flavonoid content.

[0063] Flavonoid content (mg / L) = (A510-0.0163) / 0.0162×D f

[0064] Among them, A5 10 represents the absorbance value of the sample at 510nm, D f is the dilution multiple.

[0065] Depend on Figure 4 c It can be seen that among these five sugar contents, when the sugar content is 15%, the flavonoid content in the fermented Tibetan sea buckthorn juice reaches the highest. Therefore, 15% white sugar was added to the sea buckthorn juice to optimize the subsequent fermentation conditions.

[0066] Example 6: Optimization of inoculation amount

[0067] 15 mL of the Tibetan sea buckthorn juice in Example 2 was taken into a sterile fermentation bottle, and the abnormal Wickham yeast Hta-3 culture liquid was added according to the inoculation ratio of 0.5%, 1%, 1.5%, 2% and 2.5%, respectively, and 20% white sugar was added. The fermentation temperature was set to 30°C and the shaking incubator was 180 rpm for 5 days. Three replicates were set for each treatment, and the flavonoid content in the Tibetan sea buckthorn juice after fermentation was determined.

[0068] After 5 days, 5 mL of fermented sea buckthorn juice was taken, diluted to 25 mL with 40% ethanol, and sonicated at 4000 Hz for 20 minutes. After sonication, the fermented juice was centrifuged at 5000 r / min for 20 minutes. 2 mL of the supernatant was placed in a test tube, 1 mL of 5% NaNO2 solution was added, the mixture was mixed and allowed to stand in the dark for 6 minutes, 1 mL of 10% Al(NO3)3 solution was added, the mixture was mixed and allowed to stand in the dark for 6 minutes, 10 mL of 10% NaOH solution was added, the mixture was diluted to 10 mL with 30% ethanol, the mixture was allowed to stand in the dark for 15 minutes, and the absorbance was measured at 510 nm. The flavonoid content was calculated using the formula.

[0069] Flavonoid content (mg / L) = (A5 10-0.0163) / 0.0162×D f

[0070] Among them, A5 10 represents the absorbance value of the sample at 510nm, D f is the dilution multiple.

[0071] Depend on Figure 4 It can be seen from the data that among the five inoculation doses, when the inoculation dose was 1.5%, the flavonoid content in the fermented Tibetan sea buckthorn juice reached the highest. Therefore, 1.5% was selected as the optimal inoculation dose for subsequent fermentation condition optimization.

[0072] Example 7: Response surface methodology optimization

[0073] On the basis of the single-factor experiment, with the seabuckthorn flavonoids content (Y) as the response value and the fermentation time (A), fermentation temperature (B), initial sugar content (C) and inoculation amount (D) that have significant effects on the seabuckthorn flavonoids content as the four factors, 29 response surface model (RSM) experiments with four factors and three levels were designed using DesignExpert8.0.6.1 software. The factors and levels of the Box-Behnken experiment are shown in Table 1. The experimental results and analysis are shown in Table 2.

[0074] Table 1 Central composite experimental factorial design

[0075]

[0076] The flavonoid content (Y) of seabuckthorn juice fermented with abnormal Wickham yeast Hta-3 was used as the response value. According to the response surface design, the quadratic polynomial regression equation of the relationship between the flavonoid content (Y) of seabuckthorn and fermentation time (A), fermentation temperature (B), sugar content (C) and inoculation amount (D) was obtained as Y=3959.49-308.92A-25.07B-146.33C+26.42D+347.53AB-128.04AC-36.58AD+166.65BC-69.1BD-136.17CD-609.61A2 -252.93B 2 -361.66C 2 -362.68D 2 .

[0077] Table 2 Box-Behnken experimental design quadratic model variance analysis

[0078]

[0079] The reliability and variance analysis of the regression model are shown in Table 2. It can be seen that the model F = 14.68, P < 0.0001, indicating that the regression equation is extremely significant. The non-significant term indicates that the model has good simulation. The adjusted multiple correlation coefficient R 2 Adj = 0.8725, indicating that 87.25% of the change in the content of sea buckthorn flavonoids (Y) is due to fermentation time, fermentation temperature, sugar content, and inoculum size, indicating that the model has good regression. Therefore, the model can analyze the content of sea buckthorn flavonoids (Y). Through variance analysis, the primary and secondary order of the effects of the four factors on the content of sea buckthorn flavonoids in the fermentation of Tibetan sea buckthorn juice by abnormal Wickerhamomyces Hta-3 is D > C > B > A, i.e. inoculum size > sugar content > fermentation temperature > fermentation time.

[0080] According to the regression equation, when the fermentation time is 5 days, the fermentation temperature is 27°C, the sugar content is 14%, and the inoculum size is 1.6%, the predicted value of sea buckthorn flavonoids in Tibetan sea buckthorn juice is as high as 4037.68 μg / mL, which is 28.79% higher than the content of sea buckthorn flavonoids in unfermented sea buckthorn juice.

[0081] Example Eight, Characterization of the Biological Activity of Fermented Tibetan Sea Buckthorn Juice

[0082] The fermented Tibetan sea buckthorn juice obtained under the optimal conditions according to Example Seven is used for subsequent experiments.

[0083] Repair Effect of Fermented Tibetan Sea Buckthorn Juice on Alcoholic Liver Injury by Gavage

[0084] 1. Mouse Experimental Grouping

[0085] Take 64 C57 male mice weighing about 18-25 g and randomly divide them into 8 groups, 8 in each group:

[0086] Blank control group (CK): 9:00 gavage with PBS, 0.05 mL / 10 g, 15:00 gavage with PBS 0.2 mL / 10 g;

[0087] Positive drug control group (BIF + PBS): 9:00 gavage with PBS, 0.05 mL / 10 g, 15:00 gavage with bifendate (1.5 mg / 10 g);

[0088] Sea buckthorn juice control group (SJ+PBS): 9:00 gavage PBS, 0.05 mL / 10 g, 15:00 gavage sea buckthorn juice 0.2 mL / 10 g;

[0089] Fermented sea buckthorn juice control group (FSJ+PBS): 9:00 gavage PBS, 0.05 mL / 10 g, 15:00 gavage fermented sea buckthorn juice 0.2 mL / 10 g;

[0090] Model group (M): 9:00 gavage 56° Hongxing Erguotao, 0.05 mL / 10 g, 15:00 gavage PBS 0.2 mL / 10 g;

[0091] Positive drug experimental group (BIF+Et): 9:00 gavage 56° Hongxing Erguotao, 0.05 mL / 10 g, 15:00 gavage bifendate 1.5 mg / 10 g;

[0092] Sea buckthorn juice experimental group (SJ+Et): 9:00 gavage 56° Hongxing Erguotao, 0.05 mL / 10 g, 15:00 gavage sea buckthorn juice 0.2 mL / 10 g;

[0093] Fermented sea buckthorn juice experimental group (FSJ+Et): 9:00 gavage 56° Hongxing Erguotao, 0.05 mL / 10 g, 15:00 gavage fermented sea buckthorn juice 0.2 mL / 10 g.

[0094] 2. Modeling and drug administration

[0095] After 5 days of adaptive feeding of the mice, the body weight of each mouse was weighed at 9:00 every day, and the control groups (blank control group, positive drug control group (bifendate), sea buckthorn juice control group, fermented sea buckthorn juice control group) were treated by gavage with PBS at 0.05 mL per 10 g of body weight. Except for the four control groups, the mice in the remaining groups were gavaged with 56° baijiu at 0.05 mL / 10 g, and 6 hours later, each group was gavaged with the corresponding drug. The control groups and the model group were gavaged with PBS (0.2 mL / 10 g), sea buckthorn juice (0.2 mL / 10 g), fermented sea buckthorn juice (0.2 mL / 10 g), and positive drug (bifendate 1.5 mg / 10 g), respectively. The modeling was continued for 14 days. During the entire experiment, the mice were normally fed.

[0096] After the last administration, the mice were fasted for 16 hours without water, the body weight of each mouse was weighed, all the mice were anesthetized, and the eyeball blood was taken. After blood collection, the mice were executed by decapitation, the mouse liver was obtained by dissection, the excess blood stains were removed by rinsing in physiological saline, the excess water was absorbed by filter paper, and then the weight was measured. The liver was stored in a -80°C refrigerator.

[0097] 3. Index determination

[0098] 3.1 Liver Index

[0099] The mice and their liver weight were weighed and the liver index was calculated.

[0100] Liver index = mouse liver mass (g) / mouse body weight (g) × 100%

[0101] 3.2 Proinflammatory factors

[0102] 30g of liver tissue from each mouse group was minced with scissors at 4°C or on ice. 600μL of RIPA lysis buffer was added, mixed, and lysed for 10-20 minutes. The liver tissue was then disrupted using a tissue disruptor until the liver tissue was no longer visible to the naked eye. The homogenate was then sonicated using a cell sonicator. The sonicated homogenate was centrifuged at 12,000g for 10 minutes at 4°C. The supernatant was used as the test sample. IL-6 and TNF-α levels in the samples were assayed using ELISA kits.

[0103] 3.3 Oxidative stress

[0104] 10g of mouse liver tissue from each group was added to 100μL of SOD sample preparation solution and ground at 4°C or on ice. After grinding, the homogenate was sonicated using a cell sonicator. The sonicated homogenate was centrifuged at 12000g for 5 minutes at 4°C. The supernatant was used as the test sample. SOD activity in the samples was assayed using a SOD kit.

[0105] 3.4 Alcohol metabolism in the liver

[0106] Total protein was extracted from liver tissue of each group of mice, quantified, and separated by electrophoresis. After transfer to a PVDF membrane and blocking for 3 hours at room temperature, the membranes were incubated with primary antibodies against ADH1 and ALDH2, respectively, at 4°C overnight. The membranes were washed five times with PBST, each for 6 minutes, for a total of 30 minutes. Secondary antibodies were added and incubated for 1 hour at room temperature. The membranes were washed five times with PBST and exposed to light after washing. Finally, the grayscale values ​​of the protein bands were analyzed using Image J software, and the relative expression levels of each protein were calculated.

[0107] 3.5 Apoptosis of liver tissue cells

[0108] Total protein was extracted from liver tissues of mice in each group, quantified, and separated by electrophoresis. After transfer to a PVDF membrane and blocking for 3 hours at room temperature, the membranes were incubated with primary antibodies against Bax and Bcl-2, respectively, at 4°C overnight. The membranes were washed five times with PBST, each for 6 minutes, for a total of 30 minutes. Secondary antibodies were added and incubated for 1 hour at room temperature. The membranes were washed five times with PBST and exposed to light after washing. Finally, the grayscale values ​​of the protein bands were analyzed using Image J software, and the relative expression levels of each protein were calculated.

[0109] 4. Results Analysis

[0110] Figure 6 The effect of fermented sea buckthorn juice on the liver index of mice with alcohol-induced damage by gavage. As can be seen from the figure, the liver index of the model group mice after alcohol treatment was significantly higher than that of the blank group (P<0.5), increasing by 17.5%, indicating that the modeling of acute alcoholic mice was successful. Compared with the model group, the liver index of the fermented sea buckthorn juice experimental group was significantly reduced (P<0.5), by 11.7% lower than the model group, indicating that fermented sea buckthorn juice can effectively alleviate the damage to the mouse liver caused by alcohol.

[0111] Figure 7 The effect of fermented sea buckthorn juice on the content of pro-inflammatory factors IL-6 and TNF-α in the liver tissue of mice after alcohol-induced damage by gavage. As can be seen from the figure, the content of IL-6 and TNF-α in the model group mice after alcohol treatment was significantly increased (P<0.001), indicating that excessive alcohol can increase the inflammatory response of the mouse liver and promote liver damage, while after treatment with positive drug bifendate, sea buckthorn juice and fermented sea buckthorn juice, the content of IL-6 and TNF-α was significantly reduced (P<0.001)

[0112] The inflammatory response caused by alcohol in mice was reduced, and the effect of fermented sea buckthorn juice was the best, with IL-6 and TNF-α reduced by 49.6% and 21.2% respectively compared with the model group.

[0113] Figure 8 The effect of fermented sea buckthorn juice on the oxidative stress of the liver of mice with alcohol-induced damage by gavage. As can be seen from the figure, the content of SOD in the liver tissue of the model group mice after alcohol treatment was significantly lower than that of the blank control group (P<0.01), decreasing by 70.3%, while the content of SOD after treatment with positive drug bifendate, sea buckthorn juice and fermented sea buckthorn juice increased, among which the content of SOD in the liver of mice treated with fermented sea buckthorn juice was only decreased by 24.6% compared with the blank control group, and significantly increased compared with the model group (P<0.001), indicating that fermented sea buckthorn juice has good antioxidant activity.

[0114] Figure 9 The effect of fermented sea buckthorn juice on alcohol metabolism in mice with alcohol-induced damage by gavage. As can be seen from the figure, the expression levels of ADH1 and ALDH2 in the liver tissue of the model group mice after alcohol treatment were significantly higher than those of the blank group, indicating that excessive alcohol intake can affect the metabolism of alcohol in the liver in a short period of time, and the expression of ALDH2 in the liver of mice treated with fermented sea buckthorn juice was significantly increased, thus accelerating the metabolism of alcohol in the liver.

[0115] Figure 10The effect of fermented sea buckthorn juice administered orally on apoptosis in alcohol-induced liver cells in mice is shown in the figure. As shown in the figure, the expression of the pro-apoptotic protein Bax in liver cells of mice treated with alcohol significantly increased compared to the blank control group. Bax protein decreased after treatment with sea buckthorn juice and fermented sea buckthorn juice. The expression of the anti-apoptotic protein Bcl-2 was significantly reduced compared to the blank group, while Bcl-2 levels increased significantly after treatment with sea buckthorn juice and fermented sea buckthorn juice, with fermented sea buckthorn juice showing the greater effect.

[0116] In summary, the present invention provides an abnormal Wickham yeast Hta-3, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on June 6, 2025, with a deposit number of CGMCC No. 34786; and specifically discloses its application in fermented Tibetan sea buckthorn products. The flavonoid content in Tibetan sea buckthorn juice after fermentation with the abnormal Wickham yeast Hta-3 can be significantly increased by 28.79%, and it has a certain repair effect on acute alcoholic damage, reduces the content of pro-inflammatory factors IL-6 and TNF-α, alleviates the inflammatory response of the liver, increases the activity of liver SOD, the expression of ALDH2 and Bcl-2 proteins, enhances the oxidative stress capacity of the liver, reduces liver damage caused by excessive drinking, and alleviates acute alcoholic damage in mice; in addition, fermentation can improve the taste of Tibetan sea buckthorn juice, extend the shelf life of sea buckthorn juice, and is more conducive to the transformation of traditional fermented products and improve the economic benefits of the product.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Wickerhamomyces anomalus Hta-3, characterized in that The abnormal Wickham yeast Hta-3 was deposited in the General Microbiology Center of the China Culture Collection Administration on June 6, 2025, with the deposit number CGMCC No.34786.

2. A bacterial agent, characterized in that The bacterial agent comprises the abnormal Wickham yeast Hta-3 according to claim 1.

3. Use of the abnormal Wickham yeast Hta-3 according to claim 1 or the bacterial agent according to claim 2 in fermenting sea buckthorn.

4. A seabuckthorn product, characterized in that: The seabuckthorn product is obtained by fermenting seabuckthorn with the abnormal Wickham yeast Hta-3 described in claim 1.

5. Use of the seabuckthorn product according to claim 4 in preparing a hangover relief product.

6. Use of the seabuckthorn product according to claim 4 in the preparation of a medicament for treating liver damage.

7. Use of the seabuckthorn product according to claim 4 in the preparation of antioxidant products.

8. Use of the seabuckthorn product according to claim 4 in the preparation of liver protection products.

9. Use of the seabuckthorn product according to claim 4 in the preparation of anti-inflammatory products.

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