Saccharomyces cerevisiae YSBT-1 and application of saccharomyces cerevisiae YSBT-1 in mixed fermentation Chinese wolfberry post-generation beverage

Abstract

The invention discloses a saccharomyces cerevisiae YSBT-1 which is suitable for fermenting Chinese wolfberry juice, is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and has the preservation number of CGMCC No.34262. The saccharomyces cerevisiae YSBT-1 can be used for fermenting Chinese wolfberry juice, and can be used for fermenting Chinese wolfberry juice. The strain is separated from a naturally-fermented yak yogurt sample in Qinghai province, has the characteristic of obvious aroma production, and grows well in Chinese wolfberry juice. The bacterium is mixed with plant lactobacillus to inoculate the fermented Chinese wolfberry, and the obtained mixed fermented Chinese wolfberry post-prebiotics beverage is orange red, strong in fruity flavor, has the unique flavor of Chinese wolfberry, and is moderate in sour and sweet taste and sweet in aftertaste. In-vitro and in-vivo experimental results show that the mixed fermentation Chinese wolfberry post-generation beverage has a remarkable anti-oxidation effect. As an excellent fermentation strain, the saccharomyces cerevisiae YSBT-1 has good development and application prospects in the field of functional foods such as fermented fruits and vegetables.

Description

Technical Field

[0001] This invention relates to the field of microbial applications, specifically to a yeast strain, and more specifically to the application of a strain of Saccharomyces cerevisiae YSBT-1 and its mixed fermentation with Lactobacillus plantarum O21 in wolfberry beverages. Background Technology

[0002] Goji berries are a widely recognized "food and medicine homology" material, with a consumption history of over two thousand years in my country. Goji berries are rich in nutrients such as polysaccharides, carotene, various organic acids, alkaloids, and vitamins. These substances endow goji berries with therapeutic effects for ailments caused by liver and kidney deficiency or lung and kidney weakness, as well as the ability to regulate the body's immune function, anti-aging, and anti-fatigue properties. Goji leaves, as a byproduct of goji berries, have had their nutritional value overlooked, leading to a waste of this resource. Research shows that goji leaves also contain abundant nutrients, with chemical compositions similar to goji berries, and even richer in some bioactive substances, such as polysaccharides, polyphenolic compounds, and alkaloids. Currently, research on goji berry fermentation focuses primarily on the microbial transformation of the fruit. However, mixed-base fermentation of different parts of the same plant may release novel functional molecules and enhance its efficacy through nutrient complementarity and synergistic microbial metabolism.

[0003] Synthetic microbial communities (SMCs) are microbial groups that are artificially designed and assembled to form a desired function in a specific environment. By utilizing the metabolic complementarity and synergistic effects between mixed microbial communities (co-fermentation), fermentation efficiency is improved, product diversity is enriched, and contaminating microorganisms are suppressed and stability is enhanced. SMCs have shown excellent performance in improving the flavor of traditional fermented foods and controlling product quality, and possess the potential for large-scale production.

[0004] Probiotic cultures that have undergone inactivation are collectively referred to as postbiotics. In a broader sense, postbiotics include both the bacterial cells and their metabolites, exhibiting biological activities similar to or even better than live bacteria, such as antibacterial, antioxidant, anti-inflammatory, and immunomodulatory effects. Since the activity of the bacterial strain does not need to be considered, postbiotic formulations can effectively extend product shelf life and reduce packaging, transportation, and storage costs. Therefore, the research and development of postbiotic products has received widespread attention.

[0005] In recent years, beverages fermented with a mixture of lactic acid bacteria and yeast have attracted much attention. Fermentation with mixed strains can improve the flavor of food, extend shelf life, and promote human health. The quality of fermented food can be affected by the synergistic or inhibitory effects of the two strains during fermentation. Based on this, the present invention uses newly isolated and purified brewer's yeast YSBT-1 and Lactobacillus plantarum 021 for mixed fermentation and sterilization to obtain a wolfberry post-biotic beverage with a unique flavor and high antioxidant activity.

[0006] A search revealed no patent publications related to this invention's patent application. Summary of the Invention

[0007] To address the above technical problems, the present invention aims to provide a strain of brewing yeast YSBT-1, which was deposited on April 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.34262 and classification name Saccharomyces cerevisiae. This strain grows well in wolfberry juice, produces a rich fruity aroma during fermentation, and has the ability to improve the aroma of fermentation liquid.

[0008] Furthermore, the Saccharomyces cerevisiae YSBT-1 (CGMCC NO.34262) of the present invention was isolated and purified from samples of naturally fermented yak yogurt in Batang Township, Yushu Prefecture, Qinghai Province, and identified as Saccharomyces cerevisiae using ITS sequencing.

[0009] Furthermore, the present invention provides a method for isolating, purifying, identifying, and preserving Saccharomyces cerevisiae YSBT-1.

[0010] Furthermore, the isolated and purified Saccharomyces cerevisiae YSBT-1 and Lactobacillus plantarum 021 were inoculated onto the base of wolfberry leaves at a total inoculation amount of 4% and a strain ratio of 1:2. The mixture was fermented in a shaker at 37°C for 12 hours to obtain a fermented wolfberry beverage.

[0011] The formula for the wolfberry fruit and leaf base is as follows: clean wolfberry fruits are juiced with purified water at a mass ratio of 1:10, and filtered through a 100-mesh filter (to remove wolfberry seeds) to obtain wolfberry juice; fresh wolfberry leaves are extracted with purified water at a mass ratio of 1:100 at 100℃ for 30 minutes to obtain an extract; wolfberry juice and wolfberry leaf water are mixed at a volume ratio of 1:1 to obtain the wolfberry fruit and leaf fermentation base.

[0012] Furthermore, the resulting fermented wolfberry beverage was subjected to physicochemical index testing.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The brewing yeast YSBT-1 of this invention has a rapid growth rate and grows well in the substrate of wolfberry leaves, exhibiting strong aroma-producing and antioxidant capabilities, and can be used for related applications. Specifically, this invention provides the application of this yeast in mixed fermentation with *Lactobacillus plantarum* 021 (preservation number CGMCC No. 22169) in wolfberry leaf pulp.

[0015] The brewing yeast YSBT-1 of this invention has certain acid resistance, fast growth and reproduction rate, strong aroma production ability, and the resulting wolfberry beverage after fermentation has high organic acid content, high total phenol content, strong antioxidant activity, more nutrients, and significantly improved sensory quality. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0017] Figure 1 This is a colony diagram of the YSBT-1 brewer's yeast plate culture medium of the present invention;

[0018] Figure 2 This invention relates to the pH changes during the mixed-culture fermentation of wolfberry beverages.

[0019] Figure 3 The total sugar and reducing sugar content of the mixed-culture fermented wolfberry post-biotic beverage of this invention;

[0020] Figure 4 The total phenol content of the post-fermentation beverage of wolfberry by mixed bacteria according to the present invention;

[0021] Figure 5 The content of organic acids in the mixed-culture fermented wolfberry post-biotic beverage of the present invention; Detailed Implementation

[0022] The present invention will now be described in detail with reference to specific embodiments. It should be noted that these embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0023] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0024] A strain of Saccharomyces cerevisiae YSBT-1 was deposited on April 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.34262.

[0025] Example 1: Isolation, purification and identification of Saccharomyces cerevisiae YSBT-1.

[0026] The yak yogurt samples were serially diluted and spread onto PDA solid medium. They were incubated at 30°C for 2–3 days. Based on the morphology of the single colonies, suspected yeast colonies were picked and inoculated onto fresh PDA solid medium. This process was repeated 5–6 times until the colonies were uniform. The colony morphology was then observed. (Colony morphology is shown in the image.) Figure 1 As shown. Select an appropriate amount of bacterial cells, resuspend them in 0.9% physiological saline, stain them, prepare slides, and observe the morphology of the bacterial cells under an oil immersion microscope.

[0027] Yeast genome extraction: Add 100 μL of ethyl acetate to a 1.5 mL centrifuge tube. Pick an appropriate amount of a single yeast colony and add it to the centrifuge tube, stirring thoroughly. Vortex for 5 min, then heat in a 100°C water bath until all the ethyl acetate evaporates, obtaining the yeast extract. Add 50 μL of sterile water to the centrifuge tube, vortex for 5 min, and heat in a 100°C water bath for 5 min. This yields a yeast cell lysate containing genomic DNA, which can be stored at -20°C for later use.

[0028] Species identification: Using yeast genomic DNA as a template, the ITS sequences were amplified by PCR (polymerase chain reaction) using primer pairs ITS1 and ITS4. The PCR products were subjected to agarose gel electrophoresis, and a single band was observed. The PCR products were then sequenced. The sequencing results were analyzed using the BLAST program and the GenBank database to identify the yeast species, which was identified as *Saccharomyces cerevisiae*, and named *Saccharomyces cerevisiae* YSBT-1.

[0029] Microbial Characteristics: As one of the dominant microbial strains in the fermentation process of goji berries, *Saccharomyces cerevisiae* possesses excellent aroma-producing ability and adaptability. During metabolism, it can decompose substrates such as sugars and amino acids in goji berries to synthesize flavor compounds such as esters (e.g., ethyl acetate), alcohols (e.g., phenylethyl alcohol), aldehydes, and ketones, imparting a rich fruity, floral, and fermentation aroma to the fermentation liquid, effectively enhancing the flavor profile and uniqueness of the product. Simultaneously, *Saccharomyces cerevisiae* exhibits good growth activity on the goji berry substrate, efficiently utilizing carbon sources such as glucose and fructose, as well as nutrients such as proteins and vitamins in goji berries. Under suitable fermentation conditions, it rapidly proliferates and maintains stable metabolic capacity, ensuring the smooth progress of the fermentation process and the formation of product quality.

[0030] Implementation Case 2: Preparation of Postbiotic Beverage from Mixed-Strain Fermentation of Goji Berries

[0031] Preparation of wolfberry fruit and leaf pulp: Clean wolfberry fruits and purified water were juiced at a mass ratio of 1:10, and filtered through a 100-mesh filter (to remove wolfberry seeds) to obtain wolfberry juice; fresh wolfberry leaves and purified water were extracted at 100℃ for 30 minutes at a mass ratio of 1:100 to obtain an extract; wolfberry juice and wolfberry leaf water were mixed at a volume ratio of 1:1 to obtain wolfberry fruit and leaf pulp.

[0032] Sterilization: Place the wolfberry leaf pulp in a 65℃ water bath for 30 minutes for sterilization, cool to room temperature, and store at -20℃ for later use.

[0033] Activation of Saccharomyces cerevisiae YSBT-1: Streaking of frozen Saccharomyces cerevisiae YSBT-1 onto PDA solid medium, followed by static incubation at 30℃ for 2–3 days to obtain single colonies. Single colonies were then picked up using an inoculation loop and incubated on YPD liquid medium at 30℃ with shaking at 220 rpm for 18–20 h. The inoculation concentration was adjusted to 1 × 10⁻⁶ using the culture medium. 8 CFU / mL.

[0034] Activation of *Lactobacillus plantarum* 021: Frozen *Lactobacillus plantarum* 021 was streaked onto MRS solid medium and incubated statically at 37°C for 24 h to obtain single colonies. Single colonies were picked up with an inoculation loop and incubated on MRS liquid medium at 37°C with shaking at 220 rpm for 12 h. The inoculum concentration was adjusted to 1 × 10⁻⁶ using the culture medium. 8 CFU / mL.

[0035] Inoculation and fermentation: The activated inoculum of Lactobacillus plantarum 021 and Saccharomyces cerevisiae YSBT-1 was simultaneously inoculated into the wolfberry leaf pulp at a ratio of 2:1, with a total inoculation amount of 4%. The mixture was fermented in a shaker at 37°C for 12 hours to obtain a mixed-culture fermented wolfberry beverage.

[0036] Pasteurization: The fermented goji berry beverage is pasteurized by holding it at 65°C for 30 minutes to terminate the fermentation process and improve the storage stability of the product, ultimately producing a fermented goji berry post-biotic beverage.

[0037] Implementation Case 3: Physicochemical Index Testing of Fermented Goji Berry Postbiotic Beverage

[0038] (1) pH value detection

[0039] The pH changes during the mixed-culture fermentation of wolfberry beverage were measured using a pH meter. The results are as follows: Figure 2As shown, the pH value of the goji berry beverage exhibits a continuous downward trend during fermentation: the pH value is relatively high in the early stages of fermentation, but as the fermentation time extends, organic acids (such as lactic acid and citric acid) produced by microbial metabolism accumulate, leading to a gradual increase in the acidity of the system, a gradual decrease in the pH value, and eventually a stabilization. This change not only creates a suitable acidic environment for the growth of probiotics but also inhibits the reproduction of putrefactive microorganisms.

[0040] (2) Determination of total sugar and reducing sugar content in wolfberry leaf beverage

[0041] Determination of total sugar content in wolfberry beverages using the phenol-sulfuric acid method

[0042] Glucose standard solutions with concentrations of 0, 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 g / 100 mL were prepared. 1 mL of glucose sample solution was placed in a test tube, and an equal volume of 6% phenol solution was added. 4 mL of concentrated sulfuric acid was rapidly added while the solution was in an ice-water bath, and the mixture was incubated at 37°C for 20 min. After cooling to room temperature, the reaction solution was transferred to a 96-well plate, and its A490 was measured using a microplate reader to plot a standard curve. 1 mL of the obtained wolfberry fermented beverage was placed in a 1.5 mL Eppendorf tube, centrifuged at 12000 r / min for 3 min, and 1 mL of the fermentation supernatant was collected. The total sugar content of the sample solution was determined using the method described above, and the standard curve was used to calculate the total sugar content.

[0043] The reducing sugar content in wolfberry beverages was determined using the DNS method.

[0044] Glucose standard solutions with concentrations of 0, 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 g / 100 mL were prepared. 400 μL of glucose sample solution was placed in a test tube, 800 μL of DNS was added, and the mixture was shaken well and heated at 100℃ for 5 min in a constant temperature mixer. After removal, the solution was placed in an ice-water mixture and cooled to room temperature. The absorbance was measured using an ELISA reader at a wavelength of 540 nm. A standard curve was plotted with glucose concentration x (μg / mL) on the x-axis and A540 nm on the y-axis. 1 mL of the obtained wolfberry fermented beverage was placed in a 1.5 mL Eppendorf tube, centrifuged at 12000 r / min for 3 min, and 400 μL of the fermentation broth supernatant was collected. The reducing sugar content of the sample solution was determined using the above method, and the standard curve was used to calculate the reducing sugar content.

[0045] The results of total sugar and reducing sugar content in fermented wolfberry leaf beverage are as follows: Figure 3 As shown, compared with before fermentation, the total sugar and reducing sugar content in the wolfberry post-fermentation beverage decreased significantly after fermentation.

[0046] (3) Determination of total phenolic content in wolfberry leaf beverages:

[0047] Accurately weigh 0.01 g of gallic acid standard, dissolve it in 70% ethanol solution, and dilute to 100 mL to obtain a standard solution. Perform serial dilutions to obtain standard solutions of different concentrations. Take 0.5 mL of the standard solution, add 2.5 mL of distilled water, 0.5 mL of Folin-Ciocalteu colorimetric reagent, and 1.5 mL of 7.5% Na₂CO₃ solution, let stand for 2 hours, and measure the absorbance at 760 nm to obtain a standard curve. Measure 0.5 mL of the supernatant from the wolfberry fermentation beverage and determine the total phenol content of the sample solution according to the above method. Calculate the total phenol content of the sample solution based on the standard curve.

[0048] The results are as follows Figure 4 As shown, compared with before fermentation, the total phenol content of the fermented wolfberry post-fermentation beverage increased significantly (P<0.01). As a natural antioxidant, the increase in total phenol content can significantly enhance the antioxidant activity of the beverage, effectively scavenge free radicals in the body, reduce oxidative damage, and further enhance the health function value of the product. On the other hand, the increase in total phenol content not only gives the beverage a richer natural color and unique flavor, enriching the sensory experience, but also works synergistically with organic acids and pasteurization process to inhibit oil oxidation and the growth of spoilage microorganisms, enhance the storage stability of the product, extend the shelf life, and add physiological activities such as regulating intestinal health and alleviating inflammatory responses to the beverage.

[0049] (4) Determination of organic acid content in wolfberry leaf beverage:

[0050] Prepare stock solutions of various organic acid standards (oxalic acid, tartaric acid, malic acid, ascorbic acid, lactic acid, citric acid, and succinic acid). Then, extract 1 mL of each stock solution to prepare a mixed standard stock solution for high-performance liquid chromatography (HPLC) analysis (at 210 nm and 30 °C, using 0.1% phosphoric acid-ultrapure water as the mobile phase, a flow rate of 0.5 mL / min, and an injection volume of 20 μL). Based on the concentration and peak area, obtain the linear regression equation for each standard. Then, substitute the peak area of ​​the sample into the equation to calculate the results. The results are as follows: Figure 5 As shown, compared with before fermentation, the organic acid content of the post-fermentation goji berry beverage was significantly increased after fermentation with mixed strains. This increased the flavor profile of the post-fermentation goji berry beverage, enhanced its preservation ability, improved its antioxidant properties, and maintained stable product quality.

[0051] (5) Determination of the antioxidant activity of fermented wolfberry bio-based beverages:

[0052] Determination of DPPH free radical scavenging rate: Take 1 mL of the obtained wolfberry fermentation beverage into a 1.5 mL Eppendorf tube, centrifuge at 12000 r / min for 3 min, take 100 μL of fermentation supernatant, add 100 μL of 0.2 mmol / L DPPH-ethanol solution, use an equal volume of deionized water as a blank control, react at room temperature in the dark for 30 min, and measure the OD value at 517 nm.

[0053] Clearance rate (%) = [1-A 517(样品) / A 517(空白) Equation (1) × 100%

[0054] Determination of ABTS free radical scavenging rate:

[0055] Configure 7mmol / LABTS + Mix with 2.45 mmol / L K2S2O8 solution in an equal proportion, react in the dark for 12–16 h, and adjust the absorbance of the diluted solution to 0.70 ± 0.01 to prepare ABTS. + Working solution. Take 1 mL of the fermented wolfberry bio-based beverage and place it in a 1.5 mL Eppendorf tube. Centrifuge at 12000 rpm for 3 min. Take 200 μL of the fermentation supernatant and add 800 μL of ... + The working solution was prepared with an equal volume of anhydrous ethanol as a blank control. The reaction was carried out at room temperature in the dark for 6 minutes, and the OD value at 734 nm was measured.

[0056] Clearance rate (%) = [1-A (样品) / A (空白) Equation (2) × 100%

[0057] The results of the free radical scavenging rate in the wolfberry fermented bio-based beverage are shown in Table 1. Compared with the wolfberry juice before fermentation, the DPPH free radical scavenging rate and ABTS of the wolfberry fermented bio-based beverage after mixed culture fermentation were significantly higher. + The free radical scavenging rate was significantly increased (P<0.001). This indicates that the wolfberry beverage after mixed fermentation can produce more antioxidants, thus significantly enhancing its antioxidant activity.

[0058] Table 1 Free radical scavenging rate before and after fermentation

[0059] Implementation Case 4: Constructing an aging mouse model to detect the antioxidant effect of a mixed-strain fermented wolfberry post-biotic beverage

[0060] Animal grouping and model establishment: Forty four-week-old SPF-grade male Kunming mice weighing 20±2g were selected and given free access to food and water during the experiment. After one week of acclimatization, the mice were randomly divided into 5 groups of 8 mice each: normal control group (NC group), D-galactose model group (MC group), vitamin C positive control group (Vc group), unfermented wolfberry beverage group (GQBJ group), and fermented wolfberry beverage group (GQMF group). Except for the normal group, the mice in the other groups were injected subcutaneously into the neck and back of the neck daily with 200 mg / (kg·d) of D-galactose to establish an aging model. Mice in the normal group were injected daily with an equal volume of physiological saline. At the same time, the experimental groups were treated by gavage with 0.2 mL of wolfberry beverage before and after fermentation, respectively. Mice in the vitamin C positive control group were administered 0.2 mL of 2 mmol / L vitamin C by gavage daily. The experiment lasted for 6 consecutive weeks, during which the mice had free access to food and water.

[0061] Sample collection for mouse testing: After the last gavage, the mice were fasted and deprived of water for 12 hours. Blood was collected from the eyeballs and the serum was separated and stored at -80℃ for testing. The mice were then euthanized by cervical dislocation.

[0062] Antioxidant capacity assay in mouse serum: To analyze whether the fermented wolfberry post-biotic beverage exerted an antioxidant effect in mice, aged mice were administered the wolfberry beverage before and after fermentation by gavage. After 6 weeks of gavage, serum levels of three antioxidant markers—T-AOC, GSH-Px, and MDA—were measured. The results are shown in Table 2.

[0063] Table 2. Effects of wolfberry beverages before and after fermentation on serum antioxidant capacity in mice. Note: Different lowercase letters in the figure indicate significant differences between groups (P<0.05), while the same letter indicates no significant differences between groups.

[0064] Compared with the normal group of mice, the model group mice showed significantly decreased T-AOC activity, significantly increased MDA content, and significantly decreased GSH-PX activity in serum, indicating that subcutaneous injection of D-gal induced oxidative aging in mice. Compared with the model group, the vitamin C group and the wolfberry beverage groups before and after fermentation showed significantly decreased MDA content, significantly increased T-AOC activity, and significantly increased GSH-PX activity in serum, demonstrating that all of these groups exerted antioxidant effects in vivo.

[0065] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A brewing yeast, characterized in that: The name of the brewing yeast strain is: Saccharomyces cerevisiae YSBT-1, its taxonomic name is: Saccharomyces cerevisiae, its accession number is CGMCC NO.34262, its accession date is April 18, 2025, and its depository institution is: China General Microbiological Culture Collection Center (CGMCC), Institute of Biology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The brewing yeast YSBT-1 according to claim 1, characterized in that, This strain was isolated from naturally fermented yak yogurt in Batang Township, Yushu Prefecture, Qinghai Province. After ITS sequencing, the sequencing results were analyzed using the BLAST program and the GenBank database to identify the yeast species, which was identified as Saccharomyces cerevisiae.

3. The brewing yeast YSBT-1 according to claim 1 is used in the preparation of a post-fermented wolfberry biosuppressant beverage, characterized in that, The beverage is prepared as follows: S1. Preparation of wolfberry fruit and leaf base: Clean wolfberry fruits and purified water are juiced at a mass ratio of 1:10, and filtered through a 100-mesh filter to obtain wolfberry juice; fresh wolfberry leaves and purified water are mixed at a mass ratio of 1:100, and extracted at 100℃ for 30 minutes to obtain an extract; wolfberry juice and extract are mixed at a volume ratio of 1:1, sterilized in a water bath at 65℃ for 30 minutes, and then cooled to room temperature; S2. Strain activation: YSBT-1 *Saccharomyces cerevisiae* was activated and cultured in PDA medium, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL; Lactobacillus plantarum O21 was activated and cultured in MRS medium, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL; S3. Inoculation and fermentation: The activated brewer's yeast YSBT-1 and plant lactobacillus 021 were mixed at an inoculation ratio of 1:2 and inoculated into the base of wolfberry fruit leaves at a total inoculation amount of 4%. The mixture was fermented in a shaker at 37°C for 12 hours to obtain fermented wolfberry beverage. S4. Pasteurization: After pasteurization at 65℃ for 30 minutes, the fermentation of fermented goji berry beverage can be terminated and the product stability can be improved, ultimately yielding fermented goji berry post-biotic beverage.

4. The preparation method according to claim 3, characterized in that, In step S2, the activation conditions for Saccharomyces cerevisiae YSBT-1 are: static culture at 30℃ for 2-3 days, followed by shaking culture at 220r / min in YPD liquid medium for 18-20h.

5. The preparation method according to claim 3, characterized in that, In step S2, the activation conditions for Lactobacillus plantarum O21 were: static culture at 37°C for 24 hours, followed by shaking culture at 220 r / min in MRS liquid medium for 12 hours.

6. The preparation method according to claim 3, characterized in that, The preservation number of the *Lactobacillus plantarum* O21 is CGMCC No. 22169.

7. A mixed-culture fermented wolfberry post-biotic beverage obtained by any of the preparation methods described in claims 3 to 6.

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