Saccharomycetes and application thereof in tea fermentation

Abstract

The invention belongs to the technical field of food fermentation, and particularly relates to saccharomycetes and application thereof in tea fermentation. The preservation number of the saccharomycetes is CGMCC (China General Microbiological Culture Collection Center) No.34677, the saccharomycetes are preserved in the China General Microbiological Culture Collection Center, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is May 26, 2025, and the Latin literature name is Blastobotrys adeninivorans. The saccharomycetes can produce tannase at high yield, can be applied to fermentation of tea leaves, can directionally regulate and control the biochemical conversion process of the tea leaves, and remarkably improves the flavor quality, the functional component content and the antioxidant activity of the tea leaves.

Description

Technical Field

[0001] The invention belongs to the technical field of food fermentation, and particularly relates to yeast and application thereof in tea fermentation. Background Art

[0002] Dark tea, one of China's six major tea categories, is a post-fermented tea. Categorized by origin, it includes Anhua dark tea (Hunan), Chibi green brick tea (Hubei), Nanlubian tea and Ya'an Tibetan tea (Sichuan), Pu'er ripe tea (Yunnan), Liubao tea (Guangxi), and Jingyang Fuzhuan tea (Shaanxi), each with its own distinct characteristics. The dark tea production process includes withering, initial rolling, pile fermentation, re-rolling, and drying. Pile fermentation is the most critical step, crucial for developing dark tea's color, aroma, and flavor. Essentially, it involves the biochemical transformation of tea metabolites catalyzed by extracellular enzymes secreted by natural bacterial flora (such as Aspergillus, Penicillium, and Saccharomyces), resulting in its unique flavor and functional properties. During the pile fermentation process, tannins 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 promoting the formation of tea pigments. This process plays a decisive role in the clarity and flavor quality of the tea.

[0003] Currently, the screening and application of tannase-producing strains primarily focus on Aspergillus (such as Aspergillus niger) and Penicillium. Although Aspergillus niger plays a dominant role in tannase secretion, its tannase activity in fermented tea is poorly stable. Furthermore, during fermentation, it generally decreases thearubigins and theaflavins and increases theabrownins, resulting in quality defects such as a dark brown tea soup, a dark brown, dull leaf base, and a bland flavor. Furthermore, the metabolites of Penicillium strains pose potential food safety risks. In contrast, yeasts are food-grade, safe microorganisms rich in nutrients such as amino acids and vitamins. As the dominant microbial community in the middle and late stages of fermentation, their metabolic production of esterases and exopolysaccharides is closely associated with the sweet, mellow, and smooth quality characteristic of dark tea. However, the tannase activity of yeasts has been largely unexplored. Therefore, the identification of yeasts with high tannase activity and adaptability to tea fermentation processes will be a crucial area for microbial-directed control of fermented tea quality. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a yeast that can produce high tannase and can be used in tea fermentation. The yeast is adapted to tea fermentation and can directionally regulate the biochemical transformation process of tea, thereby significantly improving the flavor quality, functional component content and antioxidant activity of tea.

[0005] The present invention is achieved through the following technical solutions: A yeast, with the deposit number CGMCC No.34677, is deposited in the General Microbiology Center of China Culture Collection Administration Committee, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 26, 2025. Its Latin name is Blastobotrys adeninivorans .

[0006] The 16S rDNA nucleotide sequence of the yeast CGMCC No.34677 is shown in SEQ ID NO.1: .

[0007] The yeast CGMCC No. 34677 is used in tea fermentation, and the yeast CGMCC No. 34677 produces tannase during tea fermentation.

[0008] Compared with the prior art, the present invention has the following beneficial effects: 1. Yeast CGMCC No. 34677 produces high levels of tannase during tea fermentation, increasing the content of the functional ingredient gallic acid. Gallic acid, as a natural antioxidant and pharmaceutical intermediate, can significantly increase the added value of tea. 2. Yeast CGMCC No. 34677 can regulate tea quality by inhibiting the degradation of theaflavins, reducing theabrownin content, increasing thearubigins content, improving the color of tea soup, and enhancing the sensory quality of tea, meeting the market demand for high-quality tea drinks. At the same time, it metabolizes and produces antioxidants with stronger free radical scavenging ability, giving tea a mellow taste and enhanced health benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The growth of tannase-producing microorganisms in the screening culture medium; Figure 2 This is the evaluation result of fermented tea. From left to right in the picture 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, yeast YFZT1 fermented tea. From bottom to top, the first row is the leaf base, the second row is the first brew of tea, and the third row is the second brew of tea. DETAILED DESCRIPTION

[0010] In order to make the purpose and technical solution of the present invention clearer and easier to understand, the present invention is further described in detail below. The experimental methods described in the following examples are all conventional methods unless otherwise specified. If no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The reagents and materials described are all commercially available unless otherwise specified.

[0011] Example 1 High-quality raw tea leaves originate from the core production area of ​​Lincang, Yunnan Province, producing Pu'er cooked tea with a mellow, sweet, and excellent quality. This study specifically purchased Pu'er cooked tea produced by Kaipu Tea in this production area and conducted targeted isolation and screening of dominant strains to elucidate the microbiological basis for the excellent quality of dark tea. Ultimately, the yeast strain CGMCC No. 34677 described in this invention was obtained. The process is as follows: S1. Tea treatment before screening: prepare 100 mL of sterile water containing 30 glass beads, take 5 g of tea leaves from the fully mixed ripe tea pile sample and put it into the sterile water, then shake at 28°C and 180 rpm for 30 minutes, filter to obtain the strain screening stock solution, dilute the stock solution and evenly apply it to the screening culture medium.

[0012] S2. Initial screening of enzyme-producing microorganisms: Tannic acid was added to the screening plate culture medium. If the strain produced tannase, it would hydrolyze the tannic acid in the culture medium, and an obvious transparent hydrolysis zone would form around the colony. The size of the transparent zone, that is, the ratio of the transparent zone diameter to the colony diameter, was determined. If the diameter ratio was greater than 1 on the 4th day of culture, the strain was retained. Finally, a total of 5 enzyme-producing yeast strains and 39 mold strains were screened.

[0013] Subsequently, the culture was repeated and the time was extended to 9 days. The ratio of the transparent zone to the colony diameter was measured, and those with a diameter ratio greater than 1.5 were retained. A total of 9 strains were screened, including 3 yeasts and 6 molds. The 6 molds were named mold L-②72h, YFT15, MB74, YFT18, Single No. 2 Brown and YFT2, and the 3 yeasts were named yeast YFZT1, ET1 and ET2. Their growth on the screening medium was as follows: Figure 1 .

[0014] S3. Rescreening of microbial fermented tea leaves: All strains were first cultured in an activated medium and then inoculated into a liquid seed medium (30°C, 180 rpm for 24 h) until the logarithmic growth phase. Seed solution was then obtained and transferred to tea leaves for fermentation. Fermented teas were then tested for tannase activity. Table 1 shows that the tannase activity of the three yeast-fermented teas was higher than that of the six mold-fermented teas. The yeast CGMCC No. 34677, also known as ET1, had the highest tannase activity, at 20.92 U / g.

[0015] The tea fermentation process is as follows: sun-dried green tea (purchased from Hubei Dongzhuang Tea Co., Ltd.) is used as the fermentation raw material, the stems are picked → divided into culture bottles (15g / can) → sterilized → cooled → inoculated with spores / yeast solution, rehydrated → incubated at 28°C for 7 days → dried at 40°C. The rehydration amount is based on the water content of the tea reaching 40%, and the inoculation solution concentration is uniformly adjusted to 1×10 7 CFU / mL, and the inoculum size was 10%.

[0016] The culture medium used was as follows: (1) Screening plate culture medium: 1L water, 2g tannic acid, 5g yeast powder, 5g glucose, 20g agar, sterilize at 121℃ for 25min; (2) Activation culture medium: 1 L water, 6 g potato extract powder, 20 g glucose, 20 g agar, sterilize at 121°C for 25 min; (3) Liquid seed culture medium: 1 L water, 20 g glucose, 20 g peptone, 10 g yeast powder, sterilized at 121°C for 25 min.

[0017] Table 1 Tannase activity of enzyme-producing microorganisms in fermented tea leaves

[0018] Example 2 The tea leaves obtained by sterilizing the sun-dried green tea in Example 1 and then adding an equal amount of sterile water for fermentation were used as the control group (CK group, i.e., sun-dried green tea). The tea leaves fermented with the three yeast strains (ET1, ET2, and YFZT1) and three mold strains (MB74, YFT2, and L-②72h) in Example 1, the CK group, and green brick tea fermented in a factory-based pile for 29 days (the fermentation raw material was sun-dried green tea purchased from Hubei Dongzhuang Tea Industry Co., Ltd.) were subjected to sensory evaluation. The results are shown in Tables 2 and 3. Figure 2 The tea sample evaluation method refers to the evaluation steps of black tea loose tea in GB / T23776-2018, and adopts weighted scoring method for scoring, with soup color accounting for 20%, aroma accounting for 35%, taste accounting for 30%, and leaf bottom accounting for 15%.

[0019] The tea infusion of the CK group was bright orange-red, with a sweet aroma, smoky, and greasy green notes, and a green, astringent flavor. Yeast-fermented tea leaves, which have numerous bacterial colonies on their surfaces and are easily soluble in water, exhibit a noticeable turbidity in the tea infusion, but the overall color is bright orange-red. Tea fermented with the ET2 strain had the highest overall score, with numerous and evenly distributed yeast colonies on the dry tea leaves, a bright orange-red tea infusion, a rich ester aroma with a sweet undertone, and a sweet, mellow flavor with an ester aroma. The overall score was 82.03. ET1 and YFZT1 had similar overall scores, ranking second to ET2-fermented tea. ET1-fermented tea had a sweet, floral, and ester aroma, while YFZT1-fermented tea had a slightly ketchup-like and winey flavor. The mold-fermented teas all had varying degrees of musty odor and a sour and astringent taste. The L-② 72h-fermented tea had a more turbid tea infusion due to the high number of spores. The aroma and taste of factory-fermented green brick tea have a typical musty and oily smell, the leaf bottom is liver-colored and the brightness of the tea soup is poor.

[0020] In summary, yeast fermentation significantly improves tea quality, and is more effective than mold fermentation in reducing undesirable flavors such as sourness and greasy greenness in sun-dried green tea. Compared to green brick tea, yeast fermentation can improve the musty odor and dark red (liver-colored) leaf bottom that develop during pile fermentation, and can also brighten the tea's tea soup.

[0021] Table 2 Sensory evaluation results of fermented tea

[0022] Example 3 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. The results are shown in Table 3.

[0023] Water extract, the collective term for water-soluble substances in tea leaves, reflects the richness of the tea's flavor. Compared to the CK group, all three yeast strains showed reduced water extract content in tea fermented with tea leaves. ET1 produced the least reduction, maintaining a high extract content of 336.10 g / kg.

[0024] The increase in free amino acid content can enhance the freshness of tea. The free amino acid content in ET1 fermented tea (6.57g / kg) was significantly higher than that in tea fermented by the other two yeast strains.

[0025] Tea polyphenols play an important role in the flavor quality of tea, 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 teas.

[0026] Compared with the CK group, the theaflavins content of teas fermented with the three yeast strains increased to varying degrees, with ET1-fermented tea having the highest theaflavins 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.

[0027] Compared with the CK group, the theabrownin content in ET2 fermented tea (52.14 g / kg) had no significant difference, 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.

[0028] In factory production, the content of water extract, free amino acids, soluble sugar, theaflavins and thearubigins in green brick tea decreases during the fermentation process, while the content of theabrownins increases significantly.

[0029] 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.

[0030] Table 3 Physical and chemical composition content of different fermented teas (g / kg)

[0031] Note: Different letters abcde represent significant differences among the groups. P <0.05, the same letters indicate no significant difference between the groups. P >0.05, the same below.

[0032] Example 4 In this example, the contents of catechins, caffeine (CAF), and gallic acid (GA) in the CK group, green brick tea, and three yeast fermented teas were further analyzed. The results are shown in Table 4.

[0033] Catechins are an important class of flavor compounds in tea, including ester catechins (EGCG, GCG, ECG, CG) and non-ester catechins (GC, EGC, C, EC). Ester catechins are the most abundant and are the primary contributor to tea's health benefits. Compared to the CK group, ET1 had a minimal effect on the total catechin content of tea. ET2 and YFZT1 fermented teas showed a slight decrease in total catechin content, while factory-fermented green brick tea showed a significant decrease.

[0034] EGCG, a key component of ester-type catechins, decreased in all three yeast-fermented teas compared to the CK group. However, the EGCG content in tea fermented with ET1 (32.01 g / kg) was significantly higher than that in YFZT1 and ET2, indicating that ET1 had the least degradation of EGCG. ET1 also had low degradation of GCG, ECG, and CG. Factory-fermented green brick tea had the lowest EGCG content, at only 2.23 g / kg, and GCG at only 0.53 g / kg. ECG and CG were undetectable.

[0035] Compared with the CK group, the contents of non-ester catechins EGC and EC were significantly increased in all three yeast-fermented teas. The GC and C contents in ET1-fermented tea were not significantly different from those in the CK group, nor were the GC contents in YFZT1-fermented tea. However, the GC and C contents in ET2-fermented tea were significantly decreased. In green brick tea, the GC and C contents were higher than those in the CK and yeast-fermented teas, but the EC and EGC contents were low, significantly lower than those in the CK and yeast-fermented teas.

[0036] GA has multiple biological activities. Compared to the CK group (1.63 g / kg), all three yeast fermentations significantly increased GA content. ET1-fermented tea had the highest GA content, at 7.01 g / kg, which was also higher than the GA content of green brick tea samples fermented naturally in factory production (5.08 g / kg). EGCG is a key precursor for GA formation, and tannases and esterases are key enzymes that degrade EGCG to GA. ET1 yeast produces significant amounts of tannas during fermentation, but its ability to degrade EGCG is minimal, while it produces high levels of GA. This suggests that the tannas produced by ET1 yeast act on other precursors that can form GA.

[0037] Table 4 Contents of catechins, gallic acid, and caffeine in different fermented teas (g / kg)

[0038] 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.

[0039] Example 5 The rich content of tea leaves gives them a strong antioxidant capacity, making them considered natural antioxidants. This antioxidant capacity is influenced by various factors, and is typically measured through the comprehensive evaluation of hydroxyl radical scavenging ability (HSA), ferric ion reducing ability (FRAP), and free radical scavenging abilities (DPPH and ABTS). The results are shown in Table 5. Compared to the CK group, the three yeast strains significantly increased FRAP, DPPH, and ABTS levels after fermentation. ET2 and YFZT1 yeasts decreased HSA levels in tea leaves, while ET1 yeast had no significant effect. In summary, fermentation with the three yeast strains significantly enhanced the antioxidant capacity of tea leaves, with ET1 yeast demonstrating the greatest antioxidant activity.

[0040] Table 5 Analysis of antioxidant capacity of different fermented teas

[0041] The detection method adopted in the embodiment is as follows: (1) Enzyme activity assay: Preparation of crude enzyme solution: Take 5.0g tea sample and place it in a 100mL conical flask, add 50.0mL ultrapure water, place it in a 25℃ shaker, 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.

[0042] Tannase activity determination: The experiment uses propyl gallate (PG) as the substrate for the tannase enzymatic reaction. The activity of tannase produced by solid fermentation fungi is detected by the principle that the chromophore generated by the reaction of gallic acid produced by tannase hydrolysis of the substrate and rhodanine develops color under alkaline conditions.

[0043] Tannase activity (U / g) = (C×V1×N) / (t×V2×M×ω) C: Gallic acid concentration mg / mL V1: total volume of reaction solution mL N: dilution multiple t: reaction time min V2: Crude enzyme solution volume mL M: sample mass g ω: sample dry matter rate (%).

[0044] (2) Conventional quality index testing methods: Determination of moisture content refers to GB / T 23776-2018 for the determination of moisture content of tea; determination of water extract content refers to GB / T 8305-2013 "Determination of water extract of tea"; determination of tea polyphenols content refers to GB / T8313-2018 Folin phenol reagent method; determination of free amino acid content refers to GB / T 8314-2002 "Determination of total free amino acids in tea"; soluble sugar content refers to the anthrone-sulfuric acid colorimetric method (Gao Junfeng et al., Plant Physiology Experimental Guide. 2006); tea pigments refer to Roberts et al. (Roberts EAH, Smith RF. 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 Major), a textbook for national higher agricultural colleges. 1997). System analysis method.

Claims

1. A yeast, characterized in that: The deposit number is CGMCC No.34677, and it is deposited in the General Microbiology Center of China Culture Collection Administration Committee. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is May 26, 2025. The Latin name is Blastobotrys adeninivorans .

2. Use of the yeast according to claim 1 in tea fermentation.

3. The use according to claim 2, characterized in that: The yeast produces tannase during tea fermentation.

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