Yeast and its application in tea fermentation

Abstract

The application belongs to the technical field of food fermentation, and particularly relates to a yeast and application of the yeast in tea fermentation. The yeast has a preservation number of CGMCC No. 34677, is preserved in the China General Microbiological Culture Collection Center, has a preservation address of No. 1, Xibaixili, Chaoyang District, Beijing, a preservation date of May 26, 2025, and a Latin name of Blastobotrys adeninivorans . The yeast has high tannase production, can be applied to tea fermentation, can be used for directional regulation of a biochemical conversion process of tea, and can significantly improve flavor quality, functional component content and antioxidant activity of tea.

Description

Technical Field

[0001] This invention belongs to the field of food fermentation technology, specifically relating to yeast and its application in tea fermentation. Background Technology

[0002] Dark tea is one of the six major tea categories in China, belonging to post-fermented tea. Classified by origin, it includes Anhua dark tea (Hunan), Chibi green brick tea (Hubei), Southern Route border tea and Ya'an Tibetan tea (Sichuan), Pu'er ripe tea (Yunnan), Liubao tea (Guangxi), and Jingyang Fu brick tea (Shaanxi), each with its own characteristics. The processing of dark tea includes fixation, initial rolling, pile fermentation, re-rolling, and drying. Among these, pile fermentation is the most crucial, a vital step in the formation of dark tea's color, aroma, and flavor. Essentially, it involves the extracellular enzyme system secreted by natural microorganisms (such as Aspergillus, Penicillium, and yeast) catalyzing the biochemical transformation of tea metabolites, thereby forming its unique flavor and functional characteristics. During pile fermentation, tannin enzymes hydrolyze the ester bonds of ester-type catechins (such as EGCG), regulating the composition of tea polyphenols (e.g., increasing gallic acid content), reducing bitterness and astringency, and promoting tea pigment formation, playing a decisive role in the clarity and flavor quality of the tea liquor.

[0003] Currently, the screening and application of tannin-producing strains mainly focus on Aspergillus (such as Aspergillus niger) and Penicillium. Although Aspergillus niger dominates in tannin secretion, its tannin activity stability is poor in fermented tea, and it generally reduces thearubigin and theaflavins content and increases theabrownin content during fermentation, causing quality defects such as dark brown tea liquor, dull dark brown tea leaves, and bland taste. Meanwhile, the metabolites of Penicillium strains pose potential food safety risks. In contrast, yeast, as a food-grade safe microorganism, is rich in amino acids, vitamins, and other nutrients. As the dominant microbial group in the later stages of fermentation, its metabolic products such as esterases and extracellular polysaccharides are closely related to the "sweet, mellow, and smooth" quality characteristics of dark tea. However, the tannin activity of yeast has not been fully explored. Therefore, screening yeast strains with high tannin activity and suitable for tea fermentation processes will become an important topic for the targeted regulation of fermented tea quality by microorganisms. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a yeast strain that produces tannins in high quantities and can be applied to tea fermentation. The yeast strain is adapted to tea fermentation and can directionally regulate the biochemical transformation process of tea, significantly improving the flavor quality, content of functional components and antioxidant activity of tea.

[0005] This invention is achieved through the following technical solution:

[0006] A yeast strain, with accession number CGMCC No. 34677, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 26, 2025. Its Latin scientific name is... Blastobotrys adeninivorans .

[0007] The 16S rDNA nucleotide sequence of the yeast strain CGMCC No. 34677 is shown in SEQ ID NO. 1:

[0008] .

[0009] The application of yeast CGMCC No. 34677 in tea fermentation, wherein yeast CGMCC No. 34677 produces tannins during tea fermentation.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] 1. Yeast CGMCC No. 34677 produces high levels of tannin enzymes during tea fermentation, which increases the content of gallic acid, a functional component. Gallic acid, as a natural antioxidant and pharmaceutical intermediate, can significantly increase the added value of tea.

[0012] 2. Yeast CGMCC No. 34677 can regulate tea quality in a targeted manner. By inhibiting the degradation of theaflavins, reducing the content of theabrownins, increasing the content of thearubigins, improving the color of the tea soup, and enhancing the sensory quality of tea, it can meet the market's demand for high-value tea drinks. At the same time, it produces antioxidants through metabolism, which have a stronger ability to scavenge free radicals, giving the tea a mellow taste and enhanced health benefits. Attached Figure Description

[0013] Figure 1 The growth of tannin-producing microorganisms on the screening medium;

[0014] Figure 2 The image shows the evaluation results of fermented tea. From left to right, the tea samples are: CK sample, green brick tea sample, mold L-②72h fermented tea, mold MB74 fermented tea, mold YFT2 tea sample, yeast ET1 fermented tea, yeast ET2 fermented tea, and yeast YFZT1 fermented tea. From bottom to top, the first row shows the tea leaves, the second row shows the first brew, and the third row shows the second brew. Detailed Implementation

[0015] To make the objectives and technical solutions of this invention clearer and easier to understand, the invention will be further described in detail below. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials are commercially available.

[0016] Example 1

[0017] The raw tea leaves from the core production area of ​​Lincang, Yunnan Province, are of high quality, resulting in a mellow, sweet, and excellent Pu-erh ripe tea. This invention specifically purchased Pu-erh ripe tea from Kaipu Tea Industry in this production area and conducted targeted isolation and screening of dominant microbial strains to analyze the microbiological basis of the superior quality of dark tea. The final result was the yeast strain CGMCC No. 34677 described in this invention. The process is as follows:

[0018] S1. Tea processing before bacterial screening: Prepare 100mL of sterile water containing 30 glass beads. Take 5g of tea leaves from the well-mixed fermented tea pile sample and put them into the sterile water. Then shake for 30min at 28℃ and 180r / min. Filter to obtain the bacterial screening stock solution. Dilute the stock solution and spread it evenly on the screening medium.

[0019] S2. Initial screening of enzyme-producing microorganisms: Tannic acid was added to the plate culture medium for screening. If the strain produces tanninase, it will hydrolyze the tannic acid in the culture medium, and a clear transparent hydrolysis zone will be formed around the colony. Based on the size of the transparent zone, i.e. the ratio of the diameter of the transparent zone to the diameter of the colony, if the diameter ratio is greater than 1 on the 4th day of culture, it will be retained. Finally, a total of 5 yeast strains and 39 mold strains producing enzymes were screened.

[0020] Subsequent repeated incubation, extending the culture time to 9 days, and measuring the ratio of the clear zone to the colony diameter, resulted in the retention of strains with a ratio greater than 1.5. A total of 9 strains were screened, including 3 yeasts and 6 molds. The 6 molds were named L-②72h, YFT15, MB74, YFT18, Single Brown No. 2, and YFT2, respectively. The 3 yeasts were named YFZT1, ET1, and ET2, respectively. Their growth on the screening medium was analyzed as follows: Figure 1 .

[0021] S3. Microbial fermentation of tea leaves for secondary screening: All strains were first cultured on an activated medium, and then inoculated into liquid seed culture medium (cultured at 30℃ and 180rpm for 24h) until the logarithmic growth phase. Seed liquid was then obtained and transferred to tea leaves for fermentation to obtain fermented tea. The tannin activity of the fermented tea was detected and shown in Table 1. It can be seen that the tannin activity of the tea fermented by the 3 yeast strains was higher than that of the tea fermented by the 6 mold strains. The yeast strain CGMCC No.34677, i.e. ET1, had the highest tannin activity, which was 20.92U / g.

[0022] The tea fermentation process is as follows: Sun-dried green tea (purchased from Hubei Dongzhuang Tea Industry Co., Ltd.) is used as the fermentation raw material. Stems are removed → the tea is portioned into culture bottles (15g / bottle) → sterilized → cooled → inoculated with spore / yeast solution, rehydrated → incubated at 28℃ for 7 days → dried at 40℃. The rehydration amount is based on a tea moisture content of 40%, and the inoculation solution concentration is uniformly adjusted to 1×10⁻⁶. 7 One CFU / mL, with an inoculation amount of 10%.

[0023] The culture medium used is as follows:

[0024] (1) Screening plate culture medium: 1L water, 2g tannic acid, 5g yeast powder, 5g glucose, 20g agar, sterilized at 121℃ for 25min;

[0025] (2) Activation medium: 1L water, 6g potato extract powder, 20g glucose, 20g agar, sterilized at 121℃ for 25min;

[0026] (3) Liquid seed culture medium: 1L water, 20g glucose, 20g peptone, 10g yeast powder, sterilized at 121℃ for 25min.

[0027] Table 1. Tanninase activity of enzyme-producing microorganisms in fermented tea.

[0028]

[0029] Example 2

[0030] The tea leaves fermented with an equal amount of sterile water after sterilization in Example 1 were used as the control group (CK group, i.e., sun-dried green tea). The fermented tea leaves containing the three yeast strains (ET1, ET2, and YFZT1) and three mold strains (MB74, YFT2, and L-②72h) from Example 1, the CK group, and the brick tea leaves fermented in a factory for 29 days (the fermentation raw material was sun-dried green tea, purchased from Hubei Dongzhuang Tea Industry Co., Ltd.) were all subjected to sensory evaluation. The results are shown in Table 2 and... Figure 2 The tea sample evaluation method refers to the evaluation steps of loose black tea in GB / T23776-2018, and a weighted scoring method is used for scoring, with liquor color accounting for 20%, aroma accounting for 35%, taste accounting for 30%, and infused leaf accounting for 15%.

[0031] The CK group's tea soup was bright orange-red, with a sweet aroma accompanied by smokiness and a slightly grassy note. The taste was astringent and slightly grassy. Yeast-fermented tea had numerous colonies on its surface, easily soluble in water, resulting in noticeable turbidity in the tea soup, but the overall color was a bright orange-red. The ET2 strain fermented tea received the highest overall score. Its dry tea surface had numerous and evenly distributed yeast colonies, a bright orange-red color, a rich ester aroma with a sweet note, and a sweet and mellow taste with an ester aroma, achieving an overall score of 82.03. The overall scores for ET1 and YFZT1 were close, second only to ET2. ET1 fermented tea had sweet, floral, and ester aromas, while YFZT1 fermented tea had a slight tomato sauce flavor and a wine-like aroma. All mold-fermented teas had varying degrees of moldy taste and a sour and astringent flavor. The L-②72h fermented tea had a more turbid tea soup due to the larger number of spores. Factory-processed brick tea has a typical musty smell and oily film in its aroma and taste, the tea leaves are liver-colored, and the tea soup is not bright.

[0032] In summary, yeast fermentation significantly improves the quality of tea, effectively mitigating undesirable flavors such as sourness, astringency, and grassy taste in sun-dried green tea compared to mold fermentation. Compared to brick tea, yeast fermentation can improve the musty smell and dark red (liver-colored) appearance of the tea leaves produced during the pile fermentation process, and also enhances the brightness of the tea liquor.

[0033] Table 2 Sensory evaluation results of fermented tea

[0034]

[0035] Example 3

[0036] The contents of seven conventional physicochemical components (water extract, free amino acids, tea polyphenols, soluble sugars, theaflavins, theabrownins, and thearubigins) in the CK group, green brick tea, and three yeast-fermented teas were determined, and the results are shown in Table 3.

[0037] Water extract is the general term for water-soluble substances in tea leaves, reflecting the richness and fullness of the tea soup. Compared with the control group, the water extract content of the fermented tea by all three yeast strains was reduced, while ET1 showed the least reduction, maintaining a content of 336.10 g / kg.

[0038] An increase in the content of free amino acids can enhance the freshness and crispness of tea. The content of free amino acids in ET1 fermented tea (6.57 g / kg) was significantly higher than that in the other two yeast-fermented teas.

[0039] Tea polyphenols play an important role in the flavor quality of tea infusion, affecting its astringency and bitterness. The tea polyphenol content of ET1 fermented tea (101.45 g / kg) was significantly higher than that of the CK group, as well as ET2 and YFZT1 fermented tea.

[0040] Compared with the control group, the theaflavins content of the three yeast-fermented teas were all increased to varying degrees, with ET1 fermented tea having the highest theaflavin content at 0.90 g / kg. The thearubigin content in ET1 fermented tea was also significantly higher than that in ET2 and YFZT1 fermented teas.

[0041] Compared with the CK group, the theabrownin content in ET2 fermented tea (52.14 g / kg) was not significantly different, the theabrownin content in YFZT1 fermented tea (56.66 g / kg) was significantly increased, and the theabrownin content in ET1 fermented tea (42.23 g / kg) was significantly decreased.

[0042] In factory production, the content of water extract, free amino acids, soluble sugars, theaflavins, and thearubigins in green brick tea decreases during the fermentation process, while the content of theabrownins increases significantly.

[0043] Based on the data in Table 3, ET1 has a good protective effect on tea polyphenols, water extracts, amino acids, soluble sugars, theaflavins and thearubigins in tea during the fermentation process, and can significantly promote the degradation of theabrownins, which has a certain positive effect on the taste and color quality of tea.

[0044] Table 3. Physicochemical components (g / kg) of different fermented teas

[0045]

[0046] Note: Different letters a, b, c, de indicate significant differences between groups. P <0.05, where the same letter indicates no significant difference between groups. P>0.05, the same applies below.

[0047] Example 4

[0048] In this embodiment, the contents of catechins, caffeine (CAF), and gallic acid (GA) in the CK group, green brick tea, and tea fermented by three yeast strains were further analyzed. The results are shown in Table 4.

[0049] Catechins are an important class of flavor compounds in tea, including ester-type catechins (EGCG, GCG, ECG, CG) and non-ester-type catechins (GC, EGC, C, EC). Ester-type catechins are the most abundant and are the main components responsible for the health benefits of tea. Compared to the control group, ET1 had a smaller impact on the total catechin content in tea, while the total catechin content of ET2 and YFZT1 fermented teas decreased slightly. The total catechin content of factory-fermented brick tea decreased more significantly.

[0050] EGCG is an important component of ester-type catechins. Compared with the control group, the EGCG content in the fermented teas from all three yeasts was reduced. However, the EGCG content in the ET1 fermented tea (32.01 g / kg) was significantly higher than that in YFZT1 and ET2, indicating that ET1 exhibited the least degradation of EGCG. ET1 also showed lower degradation of GCG, ECG, and CG. The brick tea fermented in the factory had the lowest EGCG content, only 2.23 g / kg, and GCG content was only 0.53 g / kg. ECG and CG were not detected.

[0051] Compared with the control group, the contents of non-ester catechins EGC and EC were significantly increased in all three yeast-fermented teas. The contents of GC and C in ET1 fermented tea were not significantly different from those in the control group, and the contents of GC in YFZT1 fermented tea were not significantly different from those in the control group. The contents of GC and C in ET2 fermented tea were significantly decreased. In brick tea, the contents of GC and C were higher than those in the control group and yeast-fermented tea, but the contents of EC and EGC were lower, significantly lower than those in the control group and yeast-fermented tea.

[0052] GA exhibits various biological activities. Compared to the control group (1.63 g / kg), fermentation with all three yeasts significantly increased the GA content. The ET1 fermented tea had the highest GA content at 7.01 g / kg, which was also higher than the GA content of naturally fermented brick tea samples from factory production (5.08 g / kg). EGCG is a key precursor for GA formation, and tanninases and esterases are key enzymes involved in the degradation of EGCG to form GA. While ET1 yeast produces a large amount of tanninase during fermentation, its degradation of EGCG is not significant, yet it produces a high content of GA. This indicates that the tanninases produced by ET1 yeast act on other precursors that can form GA.

[0053] Table 4. Content of catechins, gallic acid and caffeine in different fermented teas (g / kg)

[0054]

[0055] In summary, it can be seen that ET1 yeast inoculation and fermentation of tea can effectively inhibit the degradation of total catechins and significantly increase the content of gallic acid.

[0056] Example 5

[0057] The abundant internal substances in tea leaves endow them with good antioxidant capacity, making them considered natural antioxidants. Their antioxidant capacity is influenced by various factors, and is typically represented by a comprehensive evaluation of hydroxyl radical scavenging capacity (HSA), ferric reducing agent (FRAP), and free radical scavenging capacity (DPPH and ABTS). The results are shown in Table 5. Compared to the control group, the three yeast strains significantly increased FRAP, DPPH, and ABTS after fermenting tea leaves. ET2 and YFZT1 yeasts reduced HSA levels in tea leaves, while ET1 yeast had no significant effect on HSA levels. In conclusion, fermentation with the three yeast strains significantly improved the antioxidant capacity of tea leaves, with ET1 yeast exhibiting the best antioxidant efficacy.

[0058] Table 5. Analysis of antioxidant capacity of different fermented teas

[0059]

[0060] The detection method used in this embodiment is as follows:

[0061] (1) Enzyme activity assay:

[0062] Preparation of crude enzyme solution: Take 5.0g of tea sample and place it in a 100mL conical flask, add 50.0mL of ultrapure water, place it on a shaker at 25℃, and extract at 160r / min for 2h. Filter the extract with 8 layers of gauze to remove larger particles, and centrifuge at 8000r / min for 10min at 4℃. The supernatant obtained is the crude enzyme solution.

[0063] Tanninase activity assay: The experiment used propyl gallate (PG) as the substrate for the tanninase enzymatic reaction. The activity of tanninase produced by solid-state fermentation fungi was detected by the principle that the chromophore formed between gallic acid and rhodanine produced by tanninase hydrolysis of the substrate develops color under alkaline conditions.

[0064] Tanninase activity (U / g) = (C×V1×N) / (t×V2×M×ω)

[0065] C: Gallic acid concentration (mg / mL)

[0066] V1: Total volume of reaction solution (mL)

[0067] N: Dilution factor

[0068] t: reaction time (min)

[0069] V2: Volume of crude enzyme solution measured in mL

[0070] M: Sample mass (g)

[0071] ω: Sample dry matter percentage.

[0072] (2) Conventional quality index testing methods: Moisture content was determined according to GB / T 23776-2018; water extract content was determined according to GB / T 8305-2013 "Determination of Water Extract in Tea"; tea polyphenol content was determined according to GB / T8313-2018 Folin-Ciocalteu reagent method; free amino acid content was determined according to GB / T 8314-2002 "Determination of Total Free Amino Acids in Tea"; soluble sugar content was determined according to the anthrone-sulfuric acid colorimetric method (Gao Junfeng et al., Experimental Guide to Plant Physiology. 2006); tea pigments were determined according to Roberts et al. (Roberts EAH, Smith R F. Spectrophotometric Measurements of Theaflavins and Thearubigins in Black Tea Liquors in Assessments of Quality in Teas). Analyst , 1961, 86 (1019): 94~98, Huang Yihuan (Huang Yihuan et al., Tea Science Experimental Technology (for Tea Science Majors) textbook for national higher agricultural colleges. 1997) systematic analysis method.

Claims

1. A yeast strain, characterized in that: The accession number is CGMCC No. 34677, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 26, 2025. The Latin scientific name is... Blastobotrys adeninivorans .

2. The application of the yeast as described in claim 1 in tea fermentation.

3. The application as described in claim 2, characterized in that: The yeast produces tannins during tea fermentation.

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