Method for preparing a fermentation tea product enriched in exopolysaccharides and use thereof

Abstract

The application belongs to the technical field of food fermentation and functional beverage product, and discloses a preparation method of a fermented tea product rich in exopolysaccharide. The method uses the water extract of tea leaves or its processing products as the fermentation substrate of tea beverage, adopts a multi-bacterial synergistic step-by-step fermentation process, inoculates Bacillus amyloliquefaciens in the initial fermentation stage to promote the generation of exopolysaccharide, and then inoculates exopolysaccharide-producing yeast and acetic acid bacteria in the re-fermentation stage to perform subsequent fermentation, so that the directional enrichment of exopolysaccharide is realized and the flavor quality of the tea beverage is considered. Through the regulation of the combination and fermentation sequence of the fermentation bacterial flora, the method can reduce the sugar content while improving the level of functional ingredients in the fermented tea beverage, and the obtained fermented tea beverage has good stability and functional characteristics. The method has mild process conditions and strong adaptability, is suitable for different tea fermentation substrates, and is convenient for industrial production and popularization and application.

Description

Technical Field

[0001] This invention belongs to the technical field of microorganisms, food fermentation and functional beverage products, and more specifically, relates to a method for preparing fermented tea products rich in extracellular polysaccharides and its application. Background Technology

[0002] With the rise of health-conscious consumption and the rapid development of the functional beverage market, fermented tea beverages have gained widespread attention due to their unique flavor characteristics and potential health benefits. Existing fermented tea beverages are mostly represented by kombucha, typically using tea extract as a base and fermentation to create their distinctive flavor and acidity.

[0003] However, most existing fermented tea beverages employ multi-strain simultaneous fermentation, which involves complex interactions among microbial communities, easily leading to low efficiency or insufficient stability in the generation of functional metabolites. For example, in simultaneous fermentation systems, the growth and metabolism of extracellular polysaccharide-producing microorganisms are often affected by acidified environments or competition from other microbial communities, making it difficult to effectively enrich functional macromolecules such as extracellular polysaccharides.

[0004] On the other hand, while pursuing flavor development, existing fermented tea beverages often face the problem of balancing sugar consumption and functional component enhancement. Some products are prone to over-acidification, monotonous flavor, or insufficient stability during fermentation, which limits their further application in the functional beverage field.

[0005] Furthermore, for dark tea resources, especially tea raw materials with post-fermentation characteristics, their deep processing and utilization in fermented tea beverages are still mainly based on traditional brewing or simple fermentation. There is insufficient systematic exploration of their functional potential, and a stable fermentation process that can take into account both the enrichment of functional components and the control of sensory quality is lacking.

[0006] Therefore, there is an urgent need to develop a new method for preparing fermented tea beverages. By rationally controlling the combination of fermentation microorganisms and the fermentation sequence, a method can be developed to achieve efficient generation and stable enrichment of functional components such as extracellular polysaccharides while maintaining good flavor quality, so as to meet the research and development and industrialization needs of functional fermented tea beverages. Summary of the Invention

[0007] The first objective of this invention is to provide a novel Bayer conjugating yeast strain.

[0008] A second objective of this invention is to provide the application of the Bayer conjugating yeast strain in fermented tea products rich in extracellular polysaccharides.

[0009] The third objective of this invention is to provide a method for preparing a fermented tea product rich in extracellular polysaccharides.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A strain of Bayer zygosaccharomyces, namely Zygosaccharomyces bailii S392, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 26, 2026, with accession number GDMCC NO: 67743.

[0012] This invention also claims protection for the use of Bayer conjugating yeast S392 in the preparation of hypoglycemic, lipid-lowering, and antioxidant products.

[0013] This invention provides a method for preparing a fermented tea product rich in extracellular polysaccharides, comprising the following steps:

[0014] S1. Primary fermentation: Bacillus amyloliquefaciens D189 is inoculated into the tea fermentation substrate and primary fermentation is carried out at 28~37 ℃ for 3~7 days to obtain primary fermented tea liquid;

[0015] The Bacillus amyloliquefaciens D189 was deposited at the China Center for Type Culture Collection on April 29, 2021, with accession number CCTCC NO: M2021484.

[0016] S2. First stage of re-fermentation: Inoculate the tea fermentation substrate with yeast that produces extracellular polysaccharides; the yeast strain is Bayer Zygosaccharomyces bailii S392, which was deposited at Guangdong Provincial Microbial Culture Collection Center on January 26, 2026, with accession number GDMCC NO: 67743.

[0017] S3. Second stage of re-fermentation: Acetic acid bacteria are inoculated into the fermented tea liquid described in step S2 to continue fermentation and obtain a fermented tea product rich in extracellular polysaccharides.

[0018] Preferably, in step S1, the inoculation amount of Bacillus amyloliquefaciens is 2%-5% and the inoculation amount of yeast is 1%-8%; in step S2, the inoculation amount of acetic acid bacteria is 0.5%-5%.

[0019] Preferably, during the fermentation process in step S1, an exogenous carbon source and / or an exogenous nitrogen source is added to the tea fermentation substrate; the exogenous carbon source includes at least one of sucrose, glucose, fructose or lactose; the exogenous nitrogen source includes at least one of soybean peptide powder, peptone, corn oligopeptide powder, whey protein powder or casein phosphopeptide.

[0020] Preferably, the tea used in the tea fermentation substrate in step S1 is one or more of black tea, red tea, oolong tea, and green tea.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. Achieving targeted enrichment of extracellular polysaccharides and increasing the content of functional substances: This invention constructs a multi-strain synergistic stepwise fermentation process, combining extracellular polysaccharide-producing yeasts and Bacillus amyloliquefaciens with acetic acid bacteria according to their functional divisions and fermentation sequence. This ensures that extracellular polysaccharides are efficiently generated mainly in the initial fermentation stage and stably retained in the secondary fermentation stage, thereby significantly increasing the content of extracellular polysaccharides in fermented tea beverages. This method overcomes the problems of low extracellular polysaccharide generation efficiency and poor stability in traditional simultaneous fermentation.

[0023] 2. While reducing sugar content, it promotes the enrichment of active ingredients and improves nutritional composition: Using the method of this invention, the soluble solids and total sugar content in tea beverages can be effectively reduced during fermentation, while promoting the release and enrichment of tea polyphenols and flavonoids, improving the nutritional structure of fermented tea beverages, and making them more in line with the development needs of low sugar and functionalization.

[0024] 3. Imparting good antioxidant activity and functional properties to fermented tea beverages: Fermented tea beverages prepared by the multi-strain synergistic stepwise fermentation process exhibit strong antioxidant activity and good scavenging ability against free radicals; at the same time, the fermented tea beverages have a certain binding ability to cholesterol micelles and bile acids, showing good functional characteristics and are suitable for use as functional tea beverages.

[0025] 4. Balancing flavor quality and fermentation stability to enhance the drinking experience: This invention implements the primary and secondary fermentation processes in steps, avoiding excessive acidification and flavor imbalance. This is conducive to the formation and preservation of aroma substances, thereby improving the aroma complexity and sensory harmony of fermented tea beverages, so that the resulting product maintains good flavor quality while enhancing functionality.

[0026] 5. Strong process adaptability, conducive to standardized production and industrialization: The method described in this invention has mild process conditions and adjustable parameters, which are suitable for different tea fermentation substrates, especially for the deep processing development of black tea beverages. It has good repeatability and stability, which is convenient for industrial scale-up and standardized production, and has positive significance for the functional development and industrial upgrading of fermented tea beverages. Attached Figure Description

[0027] Figure 1 Colony morphology and cell morphology of strain S392.

[0028] Figure 2 Phylogenetic tree of functional yeast strain S392.

[0029] Figure 3 The effects of different fermentation methods on the yield of extracellular polysaccharides and sensory scores of Liubao tea soup.

[0030] Figure 4 Effects of different carbon sources on the yield of extracellular polysaccharides and sensory scores of Liubao tea soup.

[0031] Figure 5 Effects of different nitrogen sources on the yield of extracellular polysaccharides and sensory scores of Liubao tea soup.

[0032] Figure 6 Effects of Z. bailii S392 inoculation amount on the extracellular polysaccharide yield and sensory score of Liubao tea.

[0033] Figure 7 The effect of S1 fermentation time during the re-fermentation stage on the extracellular polysaccharide yield and sensory score of Liubao tea soup.

[0034] Figure 8 Effects of acetic acid bacteria inoculum amount on the extracellular polysaccharide yield and sensory score of Liubao tea soup.

[0035] Figure 9 The effect of S2 time during the re-fermentation stage on the extracellular polysaccharide yield and sensory score of Liubao tea soup.

[0036] Figure 10 Response surface methodology: (A) Z. balilii S392 and acetic acid bacteria inoculum size; (B) acetic acid bacteria inoculum size and S1 fermentation time; (C) Z. balilii S392 inoculum size and S1 fermentation time; (D) acetic acid bacteria inoculum size and S2 fermentation time; (E) Z. balilii S392 inoculum size and S2 fermentation time; (F) S1 fermentation time and S2 fermentation time.

[0037] Figure 11 Contour plot (A) Z. balilii S392 and acetic acid bacteria inoculum; (B) acetic acid bacteria inoculum and S1 fermentation time; (C) Z. balilii S392 inoculum and S1 fermentation time; (D) acetic acid bacteria inoculum and S2 fermentation time; (E) Z. balilii S392 inoculum and S2 fermentation time; (F) S1 fermentation time and S2 fermentation time.

[0038] Figure 12DPPH free radical scavenging rate of Liubao tea beverage (A) Different fermentation times (B) Different sample types. Note: Different letters for the same indicator indicate significant differences (P<0.05). P0: Tea extract (unfermented); P1-5: B. amyloliquefaciens D189 fermented for 5 days (initial fermentation stage P); S1-5: Z. balilii fermented for 5 days each (re-fermentation stage S1); S2-2: Z. balilii and acetic acid bacteria co-fermented for 2 days (re-fermentation stage S2). Natural fermentation (SCOBY): Natural fermentation with commercially available bacterial films; Commercially available brand (KOM): Purchased commercially available kombucha products.

[0039] Figure 13 ABTS free radical scavenging rate of Liubao tea beverage (A) Different fermentation times (B) Different sample types. Note: Different letters for the same indicator indicate significant differences (P<0.05). P0: Tea extract (unfermented); P1-5: B. amyloliquefaciens D189 fermented for 5 days (initial fermentation stage P); S1-5: Z. balilii fermented for 5 days each (re-fermentation stage S1); S2-2: Z. balilii and acetic acid bacteria co-fermented for 2 days (re-fermentation stage S2). Natural fermentation (SCOBY): Natural fermentation with commercially available bacterial films; Commercially available brand (KOM): Purchased commercially available kombucha products.

[0040] Figure 14 Cholesterol binding capacity of Liubao tea beverages (A) Different fermentation times (B) Different sample types.

[0041] Figure 15 Bile acid binding capacity of Liubao tea beverage (A) Different fermentation times (B) Different sample types. Detailed Implementation

[0042] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the equipment used are conventional experimental equipment unless otherwise specified.

[0044] The acetic acid bacteria used in the following examples are commercially available strains, which only need to be capable of oxidizing ethanol to acetic acid. Specifically, this invention uses commercially available *Acetobacter pasteurianus*. All strains were activated and cultured to obtain inoculum solutions, which were then stored for later use.

[0045] Example 1: Yeast screening and identification

[0046] 1. Source: Forty-six yeast strains isolated from kombucha were initially screened, and strains with polysaccharide-producing potential were preliminarily selected based on colony color depth (blue-purple / reddish-brown) and viscosity. Maximum value normalization was used to perform dimensionless and weighted processing on EPS yield, cholesterol adsorption capacity, and sensory scores to construct a comprehensive scoring model, resulting in strain S392. S392 was subsequently used as the functional yeast strain for fermentation, and morphological observation and molecular biological identification were performed.

[0047] 2. Identification

[0048] (1) Colony characteristics

[0049] Colony morphology and cell morphology of microorganisms are fundamental criteria for species identification. For example... Figure 1 As shown, after culturing on YEPD solid medium for 48 h, strain S392 formed round colonies with a diameter of 2-3 mm. The surface was smooth and moist, with neat edges, and the overall color was milky white and glossy. The colonies were dome-shaped, with a uniform and viscous texture, making them easy to pick up. After staining with iodine solution and microscopic examination, it could be observed that the cell ends were blunt and rounded oval, and budding was observed. These characteristics are consistent with the typical morphological characteristics of yeast.

[0050] (2) ITS sequencing of strains and construction of phylogenetic tree

[0051] The obtained S392 gene sequence was BLASTed using the NCBI database, and a phylogenetic tree was constructed using the Neighbor Joining (NJ) method in MEGA7 software. Figure 2As shown, the gene sequence of strain S392 showed 100% similarity to the type strain Zygosaccharomyces bailii ATCC 58445, thus preliminarily identifying strain S392 as a Bayer conjugated yeast. Bayer conjugated yeast is an important non-Saccharomyces yeast in food fermentation, characterized by its tolerance to high osmotic pressure, acidity, and aroma production. This characteristic is highly compatible with the fermentation environment of Liubao tea (containing 10% sucrose) and a weakly acidic pH (initial pH 5.5-6.0) required in this invention. The screening of this strain provides a key species for the subsequent construction of the mixed-culture system; its synergistic potential for aroma production and extracellular polysaccharide production needs further verification in process optimization. For ease of subsequent research, this strain is named Z. bailii S392.

[0052] Furthermore, the strain was preserved, and the preservation information is as follows:

[0053] Preservation period: January 26, 2026;

[0054] Name of depositary institution: Guangdong Provincial Center for the Preservation of Microbial Cultures;

[0055] Accession number: GDMCC NO: 67743;

[0056] Address of the repository: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0057] Classification name: Zygosaccharomyces bailii S392.

[0058] Comparative Example 1: Preparation of Fermented Liubao Tea Beverage by Simultaneous Mixed Fermentation

[0059] 1) Preparation of tea fermentation substrate: Take Liubao tea raw material, add water to extract, filter to remove residue, and obtain Liubao tea extract as fermentation substrate.

[0060] 2) Simultaneous fermentation: Bacillus amyloliquefaciens D189, Zygosaccharomyces bailii S392, and acetic acid bacteria are simultaneously inoculated into the aforementioned fermentation substrate and fermented to the endpoint under conventional and suitable conditions to obtain fermented tea beverage.

[0061] 3) Results: The obtained product yields a certain amount of extracellular polysaccharides, which contribute to the formation of sourness and flavor. However, in the simultaneous fermentation system, due to factors such as early acidification and microbial competition, the extracellular polysaccharide synthesis potential of strain D189 was inhibited in the early stage of fermentation, resulting in insufficient accumulation of extracellular polysaccharides, and its harmony with flavor still needs to be improved (e.g., Figure 3(As shown). The experimental results showed that the polysaccharide content of the synchronously fermented tea infusion was 249.45 ± 13.39 mg / L.

[0062] Example 1: Stepwise mixed-culture fermentation for the preparation of Liubao tea beverage rich in extracellular polysaccharides (basic implementation method)

[0063] 1) Preparation of tea fermentation substrate: Same as comparative example 1.

[0064] 2) Primary fermentation (P) - D189 inoculation only: After adjusting the Liubao tea extract to suitable fermentation conditions, only Bacillus amyloliquefaciens D189 (inoculation amount 4%, v / v) is inoculated, and primary fermentation is carried out at 37℃ to obtain primary fermented tea liquid.

[0065] 3) Re-fermentation (S) - Stepwise inoculation with yeast and acetic acid bacteria: Inoculate the primary fermented tea liquid with yeast that produces extracellular polysaccharides to carry out the first stage of re-fermentation (S1); after S1, inoculate with acetic acid bacteria to carry out the second stage of re-fermentation (S2) to obtain fermented Liubao tea beverage.

[0066] 4) Results and Effects: Compared with the simultaneous fermentation of Comparative Example 1, the stepwise fermentation method described in this embodiment can significantly improve the enrichment level of extracellular polysaccharides in tea beverages while maintaining good sensory harmony (e.g., Figure 3 (As shown). Experimental results show that different fermentation methods have a significant impact on the polysaccharide content and sensory quality of Liubao tea soup (P<0.05): the polysaccharide content of the tea soup fermented simultaneously was 249.45±13.39 mg / L, while the stepwise fermentation process significantly increased the polysaccharide content to 694.62±26.31 mg / L (reaching levels above 1.2 g / L), and the sensory score also reached the highest value of 87.33±2.53 points. This fully demonstrates that the process of "primary fermentation inoculated only with D189 + secondary fermentation stepwise inoculated with yeast and acetic acid bacteria" is more conducive to the targeted enrichment of extracellular polysaccharides and the synergistic optimization of flavor quality. The fermented Liubao tea beverage prepared using this optimized condition not only has a high extracellular polysaccharide content and superior sensory performance, but also exhibits good process repeatability, proving that the stepwise fermentation process parameters described in this invention are stable and feasible, and suitable for industrial application.

[0067] Example 2: Screening of carbon sources during the initial fermentation stage (preferably sucrose)

[0068] Based on Example 1, different exogenous carbon source conditions (including sucrose, glucose, fructose, lactose, etc.) were set in the initial fermentation stage (P), while the other conditions remained the same. In the initial fermentation stage, only D189 was inoculated and fermented at 37°C. The secondary fermentation stage was completed in steps S1 and S2 as in Example 1.

[0069] Results and effects: Different carbon sources had a significant impact on the enrichment of extracellular polysaccharides and flavor quality during the fermentation of Liubao tea beverages (P<0.05). Figure 4 As shown, the sucrose group exhibited superior performance in both polysaccharide production and sensory characteristics: its extracellular polysaccharide yield reached 998.38±49.44 mg / L, which was 40.8%, 14.2%, and 67.6% higher than that of the glucose, fructose, and lactose groups, respectively (no statistical difference compared to the fructose group, P>0.05). Simultaneously, the sensory score of the sucrose group was 91.20±1.30 points, both higher than 91 points and not significantly different from the fructose group (91.80±1.10 points), but significantly better than the glucose group (86.00±1.22 points) and the lactose group (82.20±1.44 points), whose scores decreased by 5.70% and 9.87% respectively compared to the sucrose group. In summary, sucrose demonstrates a more balanced advantage in terms of extracellular polysaccharide yield and sensory quality, achieving both high polysaccharide enrichment and good flavor performance, thus making it a suitable preferred carbon source for the method of this invention.

[0070] Example 3: Nitrogen source screening during the initial fermentation stage (preferably soybean peptide powder)

[0071] Based on Example 1, different nitrogen source conditions (including soybean peptide powder, peptone, corn oligopeptide powder, whey protein powder, casein phosphopeptide, etc.) were set in the initial fermentation stage (P), while the other conditions remained the same. In the initial fermentation stage, only D189 was inoculated and fermented at 37°C. The re-fermentation stage was completed in steps S1 and S2 as in Example 1.

[0072] Results and effects: Different nitrogen sources had a significant impact on the accumulation of extracellular polysaccharides and sensory quality during the fermentation process of Liubao tea beverages (P<0.05). Figure 5 As shown, the soybean peptide powder group exhibited a good overall balance: its extracellular polysaccharide content reached 1235.16±40.97 mg / L, significantly higher than the peptone group by 30.8%, demonstrating a strong ability to promote polysaccharide synthesis. In terms of sensory evaluation, the soybean peptide powder group scored 90.60±1.82 points, with no significant difference from the peptone group. Although lower than the corn oligopeptide powder group (96.2±2.39 points), it was significantly better than the whey protein powder group and the casein phosphopeptide group, avoiding the sensory defects caused by the latter two due to fishy / off-flavors. In summary, soybean peptide powder promoted extracellular polysaccharide production without introducing significant flavor degradation, achieving a good balance between function and sensory benefits. Therefore, it is suitable as a preferred nitrogen source for the method of this invention.

[0073] Example 4: Optimization and Validation of Key Process Parameters in the Re-fermentation Stage (Preferred Implementation)

[0074] Based on Example 1, sucrose was used as the preferred carbon source and soybean peptide powder as the preferred nitrogen source to further optimize the key parameters of the re-fermentation stage. The optimal factors were selected as yeast inoculation amount, acetic acid bacteria inoculation amount, S1 fermentation time, and S2 fermentation time, and the optimal combination of conditions was verified.

[0075] Results and Effects: During the fermentation of Liubao tea beverages, the inoculum size of *Z. bailii* S392 significantly affected the polysaccharide content and sensory quality of the fermentation products (P<0.05). Figure 6 As shown, in terms of polysaccharide content, polysaccharide yield initially increased and then stabilized with increasing Z. bailii S392 inoculum amount. When the Z. bailii S392 inoculum amount was 5%, the polysaccharide yield reached 1082.90±59.30 mg / L, significantly higher than that of 2% and 3% inoculum amounts (P<0.05), but not significantly different from that of 4% and 6% inoculum amounts (P>0.05). This indicates that polysaccharide yield tends to stabilize within the 4% to 6% inoculum amount range. Regarding sensory evaluation, the sensory score showed a similar trend to polysaccharide content, initially increasing and then stabilizing. When the Z. bailii S392 inoculum amount was 5%, the sensory score was 92.40±1.14 points, which was at a relatively high level, indicating that the fermentation product exhibited the best flavor and texture at this stage. Sensory scores were relatively low at inoculum levels of 2% and 3%, scoring 71.40±1.95 and 80.80±1.92 respectively, indicating that excessively low inoculum levels are detrimental to sensory quality improvement. Sensory scores were 88.80±2.28 at 4% and 91.80±1.64 at 6%, with no significant difference compared to 5% (P>0.05). This suggests that palatability is good within the 4% to 6% inoculum range, at which point *Z. bailii* S392, as the primary fermentation strain, can perform normal and efficient metabolism. Below 3% inoculum, insufficient microbial metabolism leads to residual sugar accumulation, significantly reducing sensory acceptability. Overall, a 5% *Z. bailii* S392 inoculum level demonstrates a more balanced advantage in polysaccharide content and sensory score, achieving a good balance between polysaccharide synthesis and product quality, and can be considered a preferred inoculum level.

[0076] In the fermentation process of Liubao tea beverages, the fermentation time of the re-fermentation stage S1 (yeast-only fermentation stage) has a significant impact on the polysaccharide content and sensory quality of the fermentation product (P<0.05). For example... Figure 7As shown, the polysaccharide content initially increased and then decreased with prolonged fermentation time. When the S1 fermentation time was 5 days, the polysaccharide yield reached 984.79±46.23 mg / L, significantly higher than that of 2-4 days (P<0.05). There was no significant difference in polysaccharide content when the S1 fermentation time was 6 days (P>0.05). Regarding sensory evaluation, the sensory score initially increased and then decreased with prolonged S1 fermentation time. The sensory score for S1 fermentation time of 5 days was 95.80±1.48 points, significantly higher than other times (P<0.05). This may be because before reaching the optimal S1 fermentation time, the sensory quality of the fermented Liubao tea juice from *Z. bailii* S392 improves with prolonged fermentation time, undergoing sufficient metabolic fermentation. However, when the S1 fermentation time is too long, the bacterial concentration is too high, leading to turbidity in the fermentation liquid, poorer sensory quality, and a more pronounced mellow taste. It is worth noting that while extending fermentation to 6 days maintained a relatively high polysaccharide level of 966.60±45.02 mg / L, the sensory score decreased by 5.6% compared to 5 days, indicating that over-fermentation may lead to flavor deterioration. Therefore, properly controlling the fermentation time and avoiding premature introduction of acetic acid bacteria is a key factor in ensuring the fermentation quality and flavor of Liubao tea beverages. Overall, a 5-day fermentation time in the S1 stage showed a relatively balanced advantage in both polysaccharide content and sensory score, achieving a good balance between polysaccharide synthesis and product quality. Therefore, this fermentation time condition was the preferred choice in the experiment.

[0077] During the fermentation of Liubao tea beverages, the inoculum size of acetic acid bacteria had a significant impact on the polysaccharide content and sensory quality of the fermentation products (P<0.05). For example... Figure 8 As shown, in terms of polysaccharide content, polysaccharide yield initially increased and then decreased with increasing acetic acid bacteria inoculum amount. When the acetic acid bacteria inoculum amount was 2%, the polysaccharide yield reached 1033.60±55.70 mg / L, significantly higher than that of 1% and 5% inoculum amounts (P<0.05), but not significantly different from that of 3% and 4% inoculum amounts (P>0.05). Regarding sensory evaluation, the sensory score initially increased and then decreased with increasing acetic acid bacteria inoculum amount. When the inoculum amount was 3%, the sensory score was 90.60±1.41 points, which was relatively high, indicating that the fermentation product performed best in terms of flavor and taste at this level. The sensory scores for 1% and 5% inoculum amounts were relatively low, at 81.60±1.52 points and 79.80±1.86 points respectively, indicating that both excessively low and excessively high inoculum amounts were detrimental to improving sensory quality. In summary, a 2% acetic acid bacteria inoculum amount shows a relatively balanced advantage in terms of polysaccharide content and sensory score, achieving a good balance between polysaccharide synthesis and product quality, and can be used as a preferred inoculum amount.

[0078] In the fermentation process of Liubao tea beverages, the S2 fermentation time during the re-fermentation stage has a significant impact on the polysaccharide content and sensory quality of the fermentation products (P<0.05). For example... Figure 9 As shown, in terms of polysaccharide content, the polysaccharide yield reached 973.92±47.86 mg / L when the S2 fermentation time in the re-fermentation stage was 2 days, significantly higher than that of other S2 fermentation stages (P<0.05). With the extension of the S2 fermentation time in the re-fermentation stage, the polysaccharide content gradually decreased, reaching 834.77±49.02 mg / L when the S2 fermentation time was 5 days. Regarding sensory evaluation, the sensory scores for the S2 fermentation stages of 2-3 days were relatively high, at 90.00±1.87 and 88.80±1.92 points respectively. The sensory score for the S2 fermentation stage of 5 days was significantly lower than that of other times (P<0.05). Overall, the S2 fermentation time of 2 days showed the best performance. At this point, it showed a relatively balanced advantage in terms of both polysaccharide content and sensory score, achieving a good balance between polysaccharide synthesis and product quality, and thus became the preferred fermentation time condition in the experiment.

[0079] RSM is an important method for optimizing microbial fermentation parameters. Based on the results of single-factor experiments, four factors that significantly affected the yield and sensory score of extracellular polysaccharides (EPS) were selected: Z. balilii S392 inoculum size (A), acetic acid bacteria inoculum size (B), fermentation time in the S1 stage of re-fermentation (C), and fermentation time in the S2 stage of re-fermentation (D). A four-factor, three-level experimental design was implemented, with the comprehensive score Y3 as the response value. The comprehensive score Y3 was calculated using the formula Y3 = 0.55U6 + 0.45U7. Table 1 shows the experimental design and results. Multiple regression analysis was performed on the experimental results using Design-Export 13 software, and the model equation is as follows:

[0080] Y3=94.23+1.61×A+1.09×B-1.31×C+1.16×D+2.35×AB+0.72×AC+1.27×AD-1.45×BC+0.80×BD-3.11×CD-4.29×A 2 -4.73×B 2 -4.45×C 2 -9.65×D 2 R 2 =0.9608, R 2 adj =0.9216

[0081] Table 1 Response Surface Experimental Design and Results

[0082] The results of the analysis of variance for the above regression model are shown in Table 2. The p-value < 0.0001 indicates that the model is significant, while the lack-of-fit term p = 0.669 > 0.05 is not significant, indicating a good model fit. The model's coefficient of determination R0 is also shown. 2 =0.9608 and correction factor R 2 Adj =0.9216 is close, indicating that the model has good correlation. The coefficient of variation (CV) of the model represents the precision of the experiment; the larger the value, the lower the reliability of the experimental results. CV = 1.89% < 10%, indicating that the experimental results have high precision and reliability, and this model can be used for prediction. The regression equation can reflect the relationship between each factor and the overall score well. From the ANOVA table, we can see that A, B, C, D, AB, CD, A 2 B 2 C 2 D 2 The significance of all factors was high (P<0.05), indicating that the influence of each factor on the overall score is not a simple linear relationship.

[0083] Table 2. Analysis of variance of the quadratic model

[0084] Note: * Significant difference (P<0.05), ** Extremely significant difference (P<0.01), "-" indicates no significant effect on the result (P>0.05).

[0085] Response surface methodology and contour plots reflect the interactions between pairs of factors. A steeper response surface slope indicates a greater impact of the factors on EPS yield; a more elliptical contour plot indicates a greater impact of the interactions between pairs of factors on EPS yield. Based on the response surface slope and the F-values ​​in Table 2, the order of influence of the four factors on the overall score is: Z. balilii S392 inoculum size (A) > S1 fermentation time (C) > S2 fermentation time (D) > acetic acid bacteria inoculum size (B). Analysis using Design-Expert 13 software yielded... Figure 10 and Figure 11 As shown in the figure, the contour plots of AB and CD are elliptical, indicating a significant interaction between the two factors. This suggests that the interaction has a significant impact on the overall score. The corresponding 3D surface plots also show obvious color changes and steep peaks. Meanwhile, the response surfaces of AC, AD, BC, and BD are relatively flat, and their contour plots are approximately circular, indicating that the interaction between the two factors has no significant impact on the overall score, consistent with the results of the analysis of variance in Table 2.

[0086] The optimal process conditions for comprehensive score obtained through response surface methodology optimization were: yeast inoculum of 5.2%, acetic acid bacteria inoculum of 2.2%, fermentation time of S1 stage of re-fermentation of 4.8 days, and fermentation time of S2 stage of re-fermentation of 2.1 days. Under these conditions, the predicted comprehensive score was 94.75, with a polysaccharide content of 1328.60 mg / L and a sensory score of 91.20. For experimental convenience, the above conditions were optimized to: yeast inoculum of 5.2%, acetic acid bacteria inoculum of 2.2%, fermentation time of S1 stage of re-fermentation of 5 days, and fermentation time of S2 stage of re-fermentation of 2 days. Three parallel validation experiments were conducted. The resulting comprehensive score was 92.40, with a polysaccharide content of 1291.91±63.65 mg / L and a sensory score of 92.40±1.34. There was no significant difference from the predicted values ​​(P>0.05), indicating high model reliability and that the optimized process is stable and feasible.

[0087] The antioxidant activity of Liubao tea beverage was evaluated using the DPPH and ABTS free radical scavenging methods. The scavenging abilities of both methods increased with fermentation. Figure 12 As shown, the DPPH scavenging rate increased from 55.56±1.72% in the initial tea infusion (P0) to 71.48±1.96% after 5 days of initial fermentation with Bacillus amyloliquefaciens D189 (P1-5), and finally reached a peak of 88.12±1.45% in the co-fermentation stage with Z. balilii S392 and acetic acid bacteria (S2-2), an increase of 58.60% compared to P0. The ABTS scavenging rate increased from 55.16±2.62% in P0 to 63.66±3.58% in P1-5, and finally reached 83.23±1.61% in the S2-2 stage, an increase of 50.9% compared to P0. Compared with commercially available products (KOM) and naturally fermented kombucha (SCOBY), the Liubao tea beverage prepared by this method showed better performance in both free radical scavenging rates. The results showed that targeted microbial fermentation effectively promoted the release and transformation of antioxidants such as tea polyphenols and flavonoids, thereby significantly enhancing the antioxidant activity of the product. This process is superior to natural fermentation and existing commercially available products in regulating functional components, providing an effective method for the industrial production of Liubao tea beverages with high antioxidant activity.

[0088] ABTS is a stable cationic free radical. The addition of antioxidants can reduce its absorbance, indicating that electron transfer occurs and ABTS is scavenged. The reaction amount and rate increase and accelerate with the enhancement of the sample's antioxidant capacity. Figure 13As shown in Figure A, the ABTS free radical scavenging capacity generally showed an upward trend as fermentation progressed. The initial tea infusion (P0) scavenging rate was 55.16±2.62%, increasing to 63.66±3.58% after the initial fermentation stage (P1-5), an increase of 15.4%. During the re-fermentation stage, the ABTS free radical scavenging rate continued to rise, increasing to 78.93±1.71% after 5 days of fermentation with Z. balilii S392 alone (S1-5), while reaching 83.23±1.61% after 2 days of co-fermentation with Z. balilii S392 and acetic acid bacteria (S2-2), an increase of 50.9% compared to P0. Figure 13 As shown in B, the ABTS free radical scavenging rates of commercially available products (KOM) and naturally fermented kombucha (SCOBY) were 72.22±1.88% and 77.33±2.02%, respectively. The ABTS free radical scavenging rate of Liubao tea beverage was significantly better than that of naturally fermented kombucha (SCOBY) and commercially available brands (KOM).

[0089] Polysaccharides in the fermentation broth exert their lipid-lowering effect by binding to bile acids, inhibiting their reabsorption, and promoting cholesterol conversion. Bile acid binding capacity is a key indicator for evaluating lipid-lowering function. Figure 14 As shown in Figure A, the cholesterol micelle binding rate increased with fermentation: the initial tea infusion (P) binding rate was 8.25±0.41%, increasing to 18.60±1.08% in the primary fermentation stage (P1-5), and reaching a peak of 32.42±1.03% in the secondary fermentation stage (S1-5), an increase of 74.3% compared to P. Figure 14 As shown in Figure B, the cholesterol binding rates of commercially available products (KOM) and naturally fermented kombucha (SCOBY) were 12.99±0.93% and 16.65±0.94%, respectively. The cholesterol binding rate of the Liubao tea beverage of this invention was significantly higher than both. The data indicate that targeted microbial fermentation (especially Z. balilii S392 alone and co-fermentation with acetic acid bacteria) significantly improved the cholesterol binding capacity of the system through microbial metabolism, with effects superior to natural fermentation and commercially available products. This demonstrates that artificially controlled fermentation processes have significant advantages in terms of active ingredient accumulation and enhanced lipid-lowering function.

[0090] like Figure 15 As shown in Figure A, the bile acid binding rate generally increased with fermentation. The initial tea infusion (P0) had a binding rate of 6.37±0.81%, which increased to 15.79±1.77% after the primary fermentation stage (P1-5), an increase of 148.0%. The bile acid binding rate continued to increase during the secondary fermentation stage, reaching 25.28±1.48% in S1-5 and further increasing to 26.70±1.32% in S2-2. Figure 15As shown in B, the bile acid binding rates of the commercially available brand product (KOM) and the naturally fermented sample (SCOBY) were 8.56±0.65% and 10.39±1.08%, respectively. Compared with the naturally fermented (SCOBY) and commercially available brand (KOM), the Liubao tea beverage had a significantly higher bile acid binding rate. This result is consistent with the trend of cholesterol data, indicating that the artificially controlled fermentation process is superior to the naturally fermented and commercially available products in terms of enrichment of active ingredients.

[0091] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A Bayer conjugating yeast strain, characterized in that, The strain is Zygosaccharomyces bailii S392, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 26, 2026, with accession number GDMCCNO: 67743.

2. The application of Bayer conjugated yeast S392 as described in claim 1 in the preparation of hypoglycemic, lipid-lowering, and antioxidant products.

3. A method for preparing a fermented tea product rich in extracellular polysaccharides, characterized in that, Includes the following steps: S1. Primary fermentation: Bacillus amyloliquefaciens D189 is inoculated into the tea fermentation substrate for primary fermentation to obtain primary fermented tea liquid; The Bacillus amyloliquefaciens D189 was deposited at the China Center for Type Culture Collection on April 29, 2021, with accession number CCTCC NO: M2021484. S2. First stage of re-fermentation: Inoculate the tea fermentation substrate with yeast that produces extracellular polysaccharides; the yeast strain is Bayer Zygosaccharomyces bailii S392, which was deposited at Guangdong Provincial Microbial Culture Collection Center on January 26, 2026, with accession number GDMCC NO: 67743. S3. Second stage of re-fermentation: Acetic acid bacteria are inoculated into the fermented tea liquid described in step S2 to continue fermentation and obtain a fermented tea product rich in extracellular polysaccharides.

4. The method for preparing the fermented tea product rich in extracellular polysaccharides according to claim 3, characterized in that, In step S1, the inoculation amount of Bacillus amyloliquefaciens is 2%-5%, and primary fermentation is carried out at 28-37 ℃ for 3-7 days to obtain primary fermented tea liquid.

5. The method for preparing the fermented tea product rich in extracellular polysaccharides according to claim 4, characterized in that, In step S2, the inoculation amount of yeast is 4%-6%, and the fermentation time is 4 to 6 days; in step S3, the inoculation amount of acetic acid bacteria is 2%-4%, and the fermentation time is 1 to 3 days.

6. The method for preparing the fermented tea product rich in extracellular polysaccharides according to claim 3, characterized in that, In the fermentation process of step S1, an exogenous carbon source and / or an exogenous nitrogen source is added to the tea fermentation substrate; the exogenous carbon source includes at least one of sucrose, glucose, fructose or lactose; the exogenous nitrogen source includes at least one of soybean peptide powder, peptone, corn oligopeptide powder, whey protein powder or casein phosphopeptide.

7. The method for preparing the fermented tea product rich in extracellular polysaccharides according to claim 6, characterized in that, The exogenous carbon source is sucrose; the exogenous nitrogen source is soybean peptide powder.

8. The method for preparing the fermented tea product rich in extracellular polysaccharides according to claim 3, characterized in that, The tea used in the tea fermentation substrate in step S1 is one or more of the following: black tea, red tea, oolong tea, and green tea.

9. A fermented tea product rich in extracellular polysaccharides prepared by any one of claims 3 to 8.

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