Process for reducing tea polyphenols in green brick tea by using wild flavor-enhancing yeast strain

Abstract

The application provides a process method for reducing tea polyphenols in green brick tea by using wild flavor-enhancing yeast strains, characterized in that the process is carried out by using the sporulated Hansenula guilliermondii HG-11 for segmental pile fermentation, the process is carried out by accurately inoculating 45-55 ul of glycerol bacterial liquid per 4-6 ml of culture medium, and then culturing at 33-37 DEG C and 180-220 rpm for 48 hours to the logarithmic growth phase, and then inoculating green brick tea after the bacterial body is enriched by centrifugation at 5000xg for 5 minutes. The experimental results show that the method can reduce the content of tea polyphenols, improve the sensory quality of green brick tea, the soup color is orange and red and bright, the aroma is mainly pure and correct ester aroma, and the comprehensive quality score reaches 80-84 points; the survival rate of the strain is higher in a high-temperature and high-humidity environment, the technical problem that the reduction of polyphenols and the preservation of aroma are difficult to be considered in the traditional process is solved, and a stable industrialization scheme is provided for the standardized production of green brick tea.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a process for reducing tea polyphenols in green brick tea using wild aroma-enhancing yeast strains. Background Technology

[0002] Green brick tea, a traditional Chinese tea, is widely loved for its unique flavor and taste. However, its high polyphenol content, while contributing to health benefits, can also lead to a slightly astringent taste, affecting the drinking experience for some consumers. The content and quality of polyphenols in green brick tea are closely related to its processing method; excessive polyphenols may diminish the overall flavor of the tea.

[0003] In past research, numerous methods have been explored to regulate the polyphenol content and flavor of tea, including through fermentation or the addition of different yeast strains. However, while these methods reduce polyphenols, they may not adequately improve the overall flavor of the tea. In recent years, wild aroma-enhancing yeast strains have attracted attention due to their ability to effectively produce complex aromas during food fermentation. This invention discovers that these yeast strains can impart more aroma and flavor layers to green brick tea while reducing polyphenols. Therefore, this patent utilizes specific wild aroma-enhancing yeast strains to reduce the polyphenol content in green brick tea while simultaneously enhancing its aroma and taste. This method not only improves the taste of green brick tea, making it more suitable for the general public's drinking needs, but also increases the added value of tea through the fermentation process, providing an innovative solution for the production process of green brick tea. Summary of the Invention

[0004] In view of this, the present invention proposes a process for reducing tea polyphenols in green brick tea using wild aroma-enhancing yeast strains, thereby solving the above-mentioned problems.

[0005] The technical solution of this invention is achieved as follows: a process for reducing tea polyphenols in green brick tea using wild aroma-enhancing yeast strains, comprising the following steps: (1) Cultivation of wild flavor-enhancing yeast strains; (2) Inoculate wild aroma-enhancing yeast strains onto green brick tea leaves; (3) Perform segmented composting fermentation; (4) Post-sampling processing and quality testing.

[0006] Preferably, the wild flavoring yeast strain is *Hansenula polymorpha* HG-11, with the Latin name [missing information]. Hanseniaspora guilliermondii HG-11, with the strain accession number CCTCC NO:M20252190, was deposited on October 16, 2025, and the depositary institution is the China Center for Type Culture Collection.

[0007] Preferably, the cultivation steps of the wild flavoring yeast strain are as follows: 45-55 μl of flavoring yeast glycerol culture is inoculated into 4-6 ml of YPD medium and activated at 33-37℃ and 180-220 rpm for 22-26 h; 4-6 ml of activated culture is transferred to 45-55 ml of YPD medium and cultured at 33-37℃ and 180-220 rpm for 46-50 h; every 8-12 ml of culture is centrifuged at (4800-5200)×g for 4-6 min to enrich the cells, the precipitate is resuspended in 0.8-1.2 ml of sterile water and centrifuged again at (4800-5200)×g for 4-6 min to enrich 1 unit of cells.

[0008] Preferably, the YPD culture medium consists of 8-12 g / L yeast extract, 18-22 g / L peptone, 18-22 g / L glucose, and sterile water with a pH of 5.0-6.0.

[0009] Preferably, the strain inoculation step specifically includes: (2a) Softening: Mix green brick tea leaves with sterile water at a ratio of 400g:65-75ml, and let stand for 25-35 minutes to soften the tea leaves; (2b) Preparation of primary bacterial suspension: 1 unit of bacteria obtained is resuspended in 0.8-1.2 ml of sterile water to obtain primary bacterial suspension; (2c) Preparation of working bacterial suspension: Dissolve the primary bacterial suspension in 45-55 ml of sterile water, spray it evenly on the surface of the tea leaves and stir it evenly.

[0010] Preferably, the inoculation amount of the strain is 100-600 μl.

[0011] Preferably, the segmented pile fermentation treatment includes three stages: the inoculated tea leaves are placed into a mesh screen with a breathable bottom, covered with sterile water-soaked cotton cloth, and placed in an incubator; the first stage is pile fermentation at 35-39℃ and 88-92% humidity for 22-26 hours, the second stage is pile fermentation at 43-47℃ and 88-92% humidity for 22-26 hours, and the third stage is continued pile fermentation at 53-57℃ and 88-92% humidity until the fermentation is completed.

[0012] Preferably, the fermentation ends 5 to 20 days after inoculation.

[0013] Preferably, the post-sampling processing step involves taking samples periodically during fermentation and drying them at 43-47°C for 55-65 minutes.

[0014] The process provided by this invention is used to prepare green brick tea.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. Significantly improve sensory quality: Through the specific fermentation of Hansenula polysaccharide HG-11, the yeast metabolism produces rich ester aroma substances, such as phenylethyl acetate and ethyl hexanoate, which improves the aroma score of tea from 86 points (control group) to 90 points (experimental group). The aroma type changes from a single pure aroma to a rich and layered pure fungal aroma. The tea liquor is bright orange-red (85-89 points) and the aroma is mainly pure ester aroma (84-89 points), which effectively solves the problem of the taste being too sour and astringent in traditional processing.

[0016] 2. Highly efficient reduction of tea polyphenol content: The segmented pile fermentation process achieves a tea polyphenol degradation rate of 9.5%-13.2%, significantly improving the astringency of the tea soup and making the taste more mellow and smooth. Furthermore, by optimizing the parameters of the cultivation temperature of 33-37℃ and the shaking speed of 180-220rpm, the yeast is ensured to reach the best degradation efficiency during the 48-hour logarithmic growth phase. The cell enrichment process of centrifugation at 5000×g for 5 minutes further enhances the conversion power.

[0017] 3. Synergistic advantages of strain function: Hansenula polysaccharide HG-11 has the dual functions of reducing polyphenols and enhancing aroma. Its survival rate in high temperature and high humidity fermentation environment is significantly higher than that of ordinary yeast. Its secreted polyphenol oxidase and ester synthase system work synergistically, breaking through the technical bottleneck of traditional process that inevitably loses aroma when reducing polyphenols.

[0018] 4. Process standardization and industrialization value: By precisely controlling the inoculum amount and fermentation parameters, a repeatable standardized production process is established; the strain preservation characteristics (deposited at the China Center for Type Culture Collection on October 16, 2025) provide stable strain resources for large-scale production and reduce the risk of quality fluctuations during industrial scale-up. Attached Figure Description

[0019] Figure 1 The chart shows a comparison of polyphenol content in green brick tea. * indicates P<0.05, and ** indicates P<0.01.

[0020] Figure 2 The color of green brick tea soup varies depending on the amount of wild yeast inoculated.

[0021] Figure 3 The color of green brick tea soup after different fermentation times following inoculation with 200 μL of wild yeast. Detailed Implementation

[0022] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0023] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0025] Example 1 - Strain Culture Hansenula polymorpha HG-11 (CCTCC NO: M20252190) was used. 50 μl of the aroma-enhancing yeast glycerol culture was inoculated into 5 ml of YPD medium (prepared with sterile water containing 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and pH 5.5) and activated at 35℃ and 200 rpm for 24 h. 5 ml of the activated culture was transferred to 50 ml of the same YPD medium and cultured at 35℃ and 200 rpm for 48 h. Each 10 ml of culture was centrifuged at 5000×g for 5 min, the precipitate was resuspended in 1 ml of sterile water and centrifuged again at 5000×g for 5 min to obtain 1 unit of cell.

[0026] Example 2 - Inoculation of bacterial strains Weigh 400g of green brick tea leaves, add 70ml of sterile water and mix, let stand for 30 minutes to soften; add 1ml of sterile water to 1 unit of bacterial cells to prepare a primary bacterial suspension, and take 100μl, 200μl, 300μl, 400μl, 500μl and 600μl of the primary bacterial suspension respectively and dissolve them in 50ml of sterile water to prepare working bacterial suspensions as 6 experimental groups. Spray evenly on the surface of the tea leaves and mix well; 7 groups are control groups. The control groups only need to add 120ml of sterile water and 400g of tea leaves and knead thoroughly.

[0027] Example 3 - Segmented composting Place the tea leaves into a breathable mesh screen at the bottom, cover with a sterile, water-soaked cotton cloth, and place in an incubator: the first stage is fermentation at 37℃ and 90% humidity for 24 hours, the second stage is fermentation at 45℃ and 90% humidity for 24 hours, and the third stage is fermentation at 55℃ and 90% humidity for 1 day, for a total fermentation time of 5 days.

[0028] Sampling and Post-processing Sampling times were days 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, with an experimental period of 20 days. Each time, 400g of sample was taken and the tea was dried at 45℃ for 1 hour to remove excess moisture and impurities, thereby improving the quality and stability of the tea.

[0029] I. Sensory Evaluation Referring to GB / T23776-2018 "Sensory Evaluation Method for Tea", the quality of fermented tea was tested, including its appearance, aroma, taste, and chemical composition. A combination of sensory evaluation and instrumental analysis was used to comprehensively assess the quality of the tea.

[0030] 1. Evaluate the environment 1.1 Environmental conditions: The sensory evaluation room should be independent, quiet, and odorless (avoiding interference from perfumes, fumes, fragrances, etc.), with uniform and soft lighting (using natural white light or a white light source with a color temperature of 5000-6500K, without shadows); the indoor temperature should be controlled at 20-25℃ and the relative humidity at 50-60%, avoiding excessively high temperatures or humidity that may affect sensory perception.

[0031] 1.2 Equipment and utensils: Use uniform white porcelain gaiwan (capacity 110ml), white porcelain tasting cup (capacity 30ml), teaspoon (stainless steel), timer, sample tray, and scoring sheet; all utensils must be clean, free of residual odor, and sterilized by scalding with boiling water before use.

[0032] 2 evaluators 2.1 Personnel Requirements: Select 10-15 sensory evaluators, aged 20-50, with no olfactory or gustatory impairments, and no habits such as smoking or excessive drinking that affect sensory judgment; have a certain knowledge of tea flavor and be able to accurately describe sensory characteristics such as aroma and taste.

[0033] 3. Sample preparation 3.1 Sample preparation: The green brick tea samples to be evaluated were crushed to 40 mesh to ensure that the samples were uniform and free of lumps; 3g of each sample was placed in a sealed sample bag and labeled with a random three-digit code.

[0034] 3.2 Brewing conditions: Use purified water (conductivity ≤10μS / cm, pH 6.5-8.5) as specified in GB / T23776-2018, with a water temperature of 95-100℃; brew at a tea-to-water ratio of 1:50 (3g tea leaves + 150ml water), cover and steep for 5 minutes, then quickly pour the tea into a tasting cup. Evaluate the tea when the temperature drops to 45-55℃.

[0035] 4. Evaluation Indicators and Scoring Standards 4.1 Evaluation Index System The core evaluation indicators include soup color, aroma, and taste, each worth 100 points. The evaluation requires completing the process of observing the soup color, smelling the aroma, and tasting the taste in sequence to avoid cross-interference.

[0036] 4.2 Scoring Criteria

[0037] 4.3 Scoring Results Table 1:

[0038] Table 2:

[0039] 4.4 Results Analysis: (1) Sensory quality improvement effect According to the sensory evaluation data in the table, the green brick tea prepared using this process is superior to the control group in terms of liquor color, aroma, taste, and overall score. Specifically, the liquor color exhibits a bright orange-red characteristic (score 85-89 points), the aroma is dominated by pure ester aromas (score 84-89 points), and while the taste still has a slight acidity (score 78-82 points), the overall quality score reaches 80-84 points, a significant improvement over the traditional process. The increase in ester aroma substances mainly originates from flavor compounds such as ethyl acetate and phenylethanol produced during yeast fermentation, which synergistically work with free amino acids released during the degradation of tea polyphenols to form the unique flavor system of green brick tea.

[0040] (2) Optimization and verification of process parameters Experimental results validated the rationality of the segmented composting fermentation parameters: The yeast strain exhibited the highest activation and culture efficiency within a temperature range of 33-37℃ and a shaking speed of 180-220 rpm, reaching the logarithmic growth phase after 48 hours, at which point inoculation achieved the optimal degradation effect. In the centrifugation enrichment step, centrifugation at 5000×g for 5 minutes effectively concentrated the bacterial cells, ensuring the accuracy of the inoculation volume (45-55 μl glycerol culture / 4-6 ml culture medium), providing sufficient biotransformation power for the degradation of tea polyphenols.

[0041] (3) Advantages of strain specificity The *Hansenula polymorpha* HG-11 strain, used as the core strain in this invention, exhibits significant dual functions of enhancing aroma and reducing polyphenols. Compared to common yeast strains, it effectively improves survival rate in high-temperature and high-humidity fermentation environments and can continuously synthesize ester aroma substances, solving the technical challenge of simultaneously reducing polyphenols and preserving aroma in traditional processes. The preservation characteristics of this strain (deposited at the China Center for Type Culture Collection on October 16, 2025) also provide a stable source of strain resources for the standardization and industrial application of the process.

[0042] II. Quality Inspection Experimental results showed that the optimal inoculum size was 200 μl. Taking a fermentation time of 5 days as an example, the tea polyphenol content in the experimental group was significantly reduced (P < 0.01), as detailed below. The tea polyphenol content was calculated by substituting the measured absorbance values ​​into the standard curve (standard curve: y (ug / ml) = (x + 0.0093) / 0.0123). like Figure 1 The results showed that the tea polyphenol concentrations measured in the CK group were 32.0569, 32.6260, and 32.7886 ug / ml, respectively, while the tea polyphenol concentrations measured in the sample groups were 29.0488, 28.9675, and 30.5935 ug / ml, respectively. Figure 1It can be seen that there are significant differences between the two. The results show that the present invention can significantly reduce the content of tea polyphenols in green brick tea through the segmented pile fermentation process of wild aroma-enhancing yeast strain (Hansenula polysacchariformis HG-11).

[0043] 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 within the protection scope of the present invention.

Claims

1. A process for reducing tea polyphenols in green brick tea using wild aroma-enhancing yeast strains, characterized in that, Includes the following steps: (1) Cultivation of wild flavor-enhancing yeast strains; (2) Inoculate wild aroma-enhancing yeast strains onto green brick tea leaves; (3) Perform segmented composting fermentation; (4) Post-sampling processing and quality testing.

2. The process method as described in claim 1, characterized in that, The wild flavor-enhancing yeast strain is *Hansenula polymorpha* HG-11, with the Latin name […]. Hanseniaspora guilliermondii HG-11, with accession number CCTCCNO:M 20252190, accession date October 16, 2025, is deposited at the China Center for Type Culture Collection.

3. The process method as described in claim 1, characterized in that, The specific steps for culturing the wild flavoring yeast strain are as follows: Take 45-55 μl of flavoring yeast glycerol culture and inoculate it into 4-6 ml of YPD medium, and activate it at 33-37℃ and 180-220 rpm for 22-26 h; Take 4-6 ml of activated culture and transfer it into 45-55 ml of YPD medium, and culture it at 33-37℃ and 180-220 rpm for 46-50 h; Centrifuge 8-12 ml of culture at (4800-5200)×g for 4-6 min to enrich the cells, resuspend the precipitate in 0.8-1.2 ml of sterile water, and centrifuge again at (4800-5200)×g for 4-6 min to enrich the cells, obtaining 1 unit of cells.

4. The process method as described in claim 3, characterized in that, The YPD culture medium consists of 8-12 g / L yeast extract, 18-22 g / L peptone, 18-22 g / L glucose, and sterile water at pH 5.0-6.

0.

5. The process method as described in claim 3, characterized in that, The specific steps for inoculating the strain are as follows: (2a) Softening: Mix green brick tea leaves with sterile water at a ratio of 400g:65-75ml, and let stand for 25-35 minutes to soften the tea leaves; (2b) Preparation of primary bacterial suspension: 1 unit of bacteria obtained is resuspended in 0.8-1.2 ml of sterile water to obtain primary bacterial suspension; (2c) Preparation of working bacterial suspension: Dissolve the primary bacterial suspension in 45-55 ml of sterile water, spray it evenly on the surface of the tea leaves and stir it evenly.

6. The process method as described in claim 5, characterized in that, The inoculation amount of the strain is 100-600 μl.

7. The process method as described in claim 1, characterized in that, The segmented pile fermentation process includes three stages: the inoculated tea leaves are placed in a mesh screen with a breathable bottom, covered with sterile water-soaked cotton cloth, and placed in an incubator; the first stage is pile fermentation at 35-39℃ and 88-92% humidity for 22-26 hours, the second stage is pile fermentation at 43-47℃ and 88-92% humidity for 22-26 hours, and the third stage is pile fermentation at 53-57℃ and 88-92% humidity until the fermentation is completed.

8. The process method as described in claim 7, characterized in that, The fermentation process ends 5 to 20 days after inoculation.

9. The process method as described in claim 1, characterized in that, The post-sampling processing steps are as follows: samples are taken periodically during fermentation and dried at 43-47℃ for 55-65 minutes.

10. Green brick tea prepared using the process method described in any one of claims 1-9.

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