Gluconobacter oxydans, saccharomycetes and application thereof in tea enzyme product

Abstract

The invention discloses a gluconobacter oxydans strain, a saccharomycetes strain and application of the gluconobacter oxydans strain and the saccharomycetes strain in tea enzyme products. The invention provides a Gluconobacter oxydans (Gluconobacter oxydans) strain A21, a Starmeria davenportii (Starmeria davenportii) strain YW1 and a microbial agent containing the Gluconobacter oxydans (Gluconobacter oxydans) strain A21 and the Starmeria davenportii strain YW1. The bacterial strain and the microbial agent do not generate pellicle when the tea enzyme is produced by fermenting sugar water, the pellicle does not need to be removed subsequently, the content of polyphenol and organic acid in the obtained tea enzyme is high, and the product yield is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of food fermentation and brewing, and particularly relates to Gluconobacter oxydans, yeast, and their uses in tea enzyme products. Background Art

[0002] Tea enzyme, also known as kombucha, kefir tea, sea treasure, or stomach treasure, is a functional beverage produced by the combined fermentation of tea sugar water by acetic acid bacteria and yeast. It has a sweet and sour taste and a fragrant smell, and its acidity inhibits the growth of harmful bacteria. Kombucha is rich in nutrients such as vitamin C and vitamin B, so it can regulate the physiological functions of the human body, promote metabolism, and aid digestion.

[0003] The kombucha pellicle is a thin film that grows on the surface of tea water after inoculating acetic acid bacteria and yeast strains in tea sugar water. As a fermentation by-product, the pellicle is not required in the final products of tea enzyme or tea beverages, so generally post-treatment such as filtration and clarification is required after fermentation to remove the pellicle. In addition, during the preparation of kombucha, the production of the pellicle consumes a large amount of substrates and reduces the sucrose utilization rate.

[0004] Tea leaves contain various active substances such as tea polyphenols, thearubigins, theaflavins, caffeine, and tea polysaccharides. Tea polyphenols are the main components that form the taste of tea and are also important components for the good physiological and health care functions of tea. Tea polyphenols have extremely strong free radical scavenging and antioxidant properties, so that tea has health care effects such as anti-aging, anti-inflammatory, cancer prevention, improvement of insulin resistance, and reduction of the risk of type 2 diabetes.

[0005] As a traditional famous tea in China, Wuyi rock tea has a fragrant, elegant, long-lasting, and layered aroma. It has a strong texture, a thick and mellow taste, rich connotations, a layered feeling, smoothness, sweetness, a long aftertaste, and a comfortable and lasting taste, so it is famous at home and abroad for its "rocky bones and floral fragrance". However, the regional characteristics of Wuyi rock tea are obvious. From the Zheng Yan area to the semi-rock, Zhou tea, and outer mountain tea, the quality of rock tea gradually decreases. The output in the Zheng Yan area is limited, only accounting for 5% of the total output; the output of Zhou tea and outer mountain tea is large, but due to poor quality and low price, there is an inventory backlog of 7,000 tons / year. There is a large amount of low-end rock tea being abandoned in production, resulting in a large amount of waste.

[0006] Therefore, the use of low-end rock tea fermentation to prepare tea enzymes helps to remove the inventory pressure of low-end rock tea and increase the economic added value of deep processing of rock tea, which has important economic benefits. However, the traditional natural red tea fungus strains use biofilm as the source of strains. In addition to the acetic acid bacteria and yeasts required for fermentation, there are some miscellaneous bacteria in the biofilm. Different regions, differences in raw materials, environmental changes and different fermentation methods may change the flora composition of red tea fungi, and may also cause strain contamination, mutation and decline, leading to product differentiation, thereby affecting the stability (polyphenol content and organic acid content) and quality (yield) of bioactive substances in the fermentation broth. Therefore, its separation and purification is necessary. At the same time, as a fermentation by-product, the biofilm is not required in the final product of tea enzymes. The production of biofilm consumes a large amount of substrate and creates an additional burden on the filtration and clarification processes during the treatment process.

[0007] CN100429302 discloses that four yeasts, namely, Pachysaccharomyces cerevisiae, Saccharomyces delbrueckii, and Pichia pastoris, were isolated from the bacterial film of kombucha, and then Saccharomyces cerevisiae was screened to obtain Saccharomyces cerevisiae; CN100445368 discloses that four acetic acid bacteria strains, namely, Acetobacter xylinum, Acetobacter pasteurianus, Acetobacter liquefaction, and Acetobacter oxidans, were isolated from the bacterial film of kombucha, and then the Acetobacter xylinum strain was screened; CN110791516 discloses a kombucha fermentation bacterial agent, which comprises a recombinant engineered bacterium, at least one selected from yeast and Acetobacter, and at least one selected from Lactobacillus plantarum and Lactobacillus casei. CN114410523 discloses a strain combination for preparing black tea fungus and its application, and screens out Gluconacetobacter intermedius, which does not produce biofilm and has the highest sugar-acid conversion rate, and then cooperates with strain Starmerella davenportii KB17 for fermentation, greatly improving the acid production efficiency and quickly obtaining high-quality black tea fungus. CN105779297 discloses that yeast (Arxula adeninivorans) ferments Pu'er tea, and the tea polyphenol content in the obtained tea soup is 14.2-15%. CN115105452 discloses that yeast is inoculated into black tea extract and fermentation medium for primary fermentation, and then lactic acid bacteria seed liquid and acetic acid seed liquid are inoculated for secondary fermentation, and filtered to obtain a black tea fermentation filtrate, and the solid content in the black tea fermentation filtrate is (≥7.5%).

[0008] Therefore, there is an urgent need for a strain of fermented tea enzyme that does not produce biofilm and has high flavor quality, and the tea enzyme obtained by fermentation. Summary of the invention

[0009] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide Gluconobacter oxidans, yeast and their use in tea enzyme products, in order to solve the above-mentioned problems.

[0010] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0011] The first aspect of the present invention protects Gluconobacter oxydans strain A21, whose deposit number is CCTCC NO:M 20232673.

[0012] The second aspect of the present invention protects yeast (Starmerella davenportii) strain YW1, whose deposit number is CCTCCNO:M20232674.

[0013] The third aspect of the present invention protects the use of the aforementioned Gluconobacter oxydans strain or the aforementioned yeast strain in the preparation of a microbial agent.

[0014] The fourth aspect of the present invention protects a microbial agent comprising the aforementioned Gluconobacter oxydans strain and / or the aforementioned yeast strain.

[0015] The fifth aspect of the present invention protects the use of the aforementioned Gluconobacter oxidans strain or the aforementioned yeast strain and / or the aforementioned microbial agent in the preparation of a tea enzyme product.

[0016] The sixth aspect of the present invention protects a method for preparing tea enzyme, characterized in that it comprises the following steps:

[0017] 1) preparing tea syrup;

[0018] 2) inoculating bacterial liquid into the tea sugar water, fermenting, and obtaining the tea enzyme;

[0019] The bacterial solution comprises the aforementioned Gluconobacter oxidans strain or the aforementioned yeast strain or the aforementioned microbial agent.

[0020] The seventh aspect of the present invention protects the tea enzyme obtained by the preparation method as described above.

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

[0022] 1) The present invention selects and cultivates Gluconobacter acidobacillus strains and yeast strains, and no biofilm is produced during the fermentation to produce tea enzymes. The present invention selects excellent strains to avoid biofilm formation, and through a specific combination of strains, high-quality rock tea enzyme products are obtained quickly and stably. The technical solution provided by the present invention makes the production process of black tea fungus simple and efficient, the product quality is excellent, and it is suitable for large-scale preparation of rock tea enzymes.

[0023] 2) The tea enzyme obtained by fermentation of the Gluconobacter acidobacillus strain and the yeast strain of the present invention has a polyphenol content increased by 59.8% and a titratable acidity increased by 10% compared with the traditional kombucha from which it is isolated, and the content of glucuronic acid is higher than 3.9 g / L. It not only has a richer and stronger flavor, but also significantly increases the content of functional ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown are microscopic images of the Gluconobacter oxydans strain A21 and the yeast strain YW1 in Example 1 of the present invention.

[0025] Figure 2 Shown are actual pictures of the experimental group and the control group in Example 4 of the present invention.

[0026] Figure 3 Shown is a graph of polyphenol content in the fermentation products of the experimental group and the control group in Example 4 of the present invention. DETAILED DESCRIPTION

[0027] During the fermentation process of tea enzyme, a layer of powdery or mesh-like substance will be produced, which is white, milky white or slightly brown, called white film or biofilm. Biofilm is mainly formed by the massive growth of yeast or acetobacter under aerobic conditions. Enzymes contain yeast or acetobacter. When the seal is not tight, the liquid comes into contact with oxygen in the air. Since yeast or acetobacter are aerobic bacteria, they will grow in large numbers under aerobic conditions, and a biofilm of yeast or acetobacter will form on the surface.

[0028] At present, tea enzymes are mainly produced by natural multi-strain co-fermentation, which not only has a long production cycle, but also has a low content of functional component glucuronic acid, which is about 0.003-0.01g / L. In addition, the strain is also a key factor affecting the flavor quality of tea enzymes. The present invention accidentally discovered that a strain of acetic acid bacteria and a strain of yeast were isolated from the traditional natural black tea fungus on the market, and it was found that they would not produce biofilm when fermented alone or co-fermented with tea syrup. After applying them to tea enzymes, it was found that they could increase the content of glucuronic acid, the main functional component in tea enzymes. In addition, the titratable acidity can be increased, the production of acetic acid can be reduced, and the production of other flavor organic acids can be increased. The present invention was completed on this basis.

[0029] The first aspect of the present invention protects Gluconobacter oxydans strain A21, whose deposit number is CCTCC NO:M 20232673.

[0030] The morphology of the Gluconobacter oxydans strain A21 was observed to be round, cheese-colored colonies with smooth and opaque surfaces and neat edges.

[0031] The Gluconobacter oxydans strain A21 has a 16Sr DNA sequence as shown in SEQ ID NO:1.

[0032] The Gluconobacter oxydansi strain A21 protected by the present invention is obtained by screening and separation from the culture product of commercially available kombucha fungus, and belongs to the genus Gluconobacteroxydansi through morphological observation, 16Sr DNA gene sequencing and Blast comparison.

[0033] The liquid culture medium for culturing Gluconobacter oxydans is: mannitol (10-30) g / L, yeast extract (1-10) g / L, and peptone (1-5) g / L. The culture method is: spread the Gluconobacter oxydans strain A21 on a solid plate culture medium, then place it in an incubator for culture, and then transfer it to a liquid culture medium for subculture after it grows into a single colony. The solid plate culture medium is a liquid culture medium with agar added.

[0034] The Gluconobacter oxydans strain A21 of the present invention has been deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on December 25, 2023, and the deposit registration number is CCTCC NO:M 20232673.

[0035] Another aspect of the present invention protects yeast (Starmerella davenportii) strain YW1, whose deposit number is CCTCCNO:M20232674.

[0036] The yeast strain YW1 is observed to be cream-colored (milky white), smooth on the surface, with spherical protrusions, and is opaque and creamy.

[0037] Wherein, the yeast strain YW1 has an ITS rDNA sequence as shown in SEQ ID NO:2.

[0038] The yeast strain YW1 protected by the present invention is obtained by screening and separation from the culture product of commercially available kombucha fungus, and belongs to the genus Starmerella davenportii through morphological observation, ITS rDNA gene sequencing and Blast comparison.

[0039] The liquid culture medium for culturing yeast is: glucose (10-30) g / L, yeast extract (5-15) / L, and peptone (10-30) g / L. The culture method is: spread the yeast strain YW1 on a solid plate culture medium, then place it in an incubator for culture, and then transfer it to a liquid culture medium for subculture after it grows into a single colony. The solid plate culture medium is a liquid culture medium with agar added.

[0040] The yeast (Starmerella davenportii) strain YW1 of the present invention has been deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on December 25, 2023, and the deposit registration number is CCTCC NO:M 20232674.

[0041] Another aspect of the present invention protects a microbial agent comprising the aforementioned Gluconobacter oxydans strain and / or the aforementioned yeast strain.

[0042] In some embodiments, the effective viable count of the microbial agent is at least (1-5)×10 8 CFU / mL, the effective viable bacteria count of Gluconobacter oxydans contained in the microbial agent is at least (1-5)×10 8 CFU / mL or the effective viable count of yeast is at least (1-5)×10 7 CFU / mL. Preferably, the effective viable count of the microbial agent is at least 1×10 8 CFU / mL, the effective viable count of Gluconobacter oxidans contained in the microbial agent is at least 1×10 8 CFU / mL or the effective viable count of yeast is at least 1×10 7 CFU / mL.

[0043] In certain embodiments, the microbial agent is in the form of one or more of dry powder, bacterial suspension, and fermentation product.

[0044] In certain embodiments, the microbial agent is further added with auxiliary materials for prolonging the activity time of the strain, or other auxiliary materials acceptable to the microbial agent, and the obtained microbial agent product also falls within the protection scope of the present invention.

[0045] Another aspect of the present invention is to protect a method for preparing the aforementioned microbial agent, comprising:

[0046] In certain embodiments, the Gluconobacter oxidans strain is fermented and cultured, and solid-liquid separation is performed to obtain Gluconobacter oxidans cells.

[0047] Preferably, the fermentation temperature is usually 25-35° C., 25-29° C., 28-32° C., or 31-35° C. In some embodiments, it is 30° C.

[0048] Preferably, the fermentation culture time can be generally 24 to 48 hours, or 24 to 32 hours, or 28 to 38 hours, or 32 to 48 hours. In some specific embodiments, the fermentation culture time is 30°C.

[0049] Preferably, the culture medium for fermentation culture comprises mannitol, yeast extract and peptone.

[0050] More preferably, based on the total volume of the fermentation medium, the fermentation medium comprises the following raw materials: mannitol (10-30) g / L, yeast extract (1-10) g / L, and peptone (1-5) g / L. In certain specific embodiments, it is 25 g / L mannitol, 5 g / L yeast extract, and 3 g / L peptone.

[0051] In certain embodiments, the yeast strain is fermented and cultured, and solid-liquid separation is performed to obtain yeast cells.

[0052] Preferably, the fermentation temperature is usually 25-35° C., 25-29° C., 28-32° C., or 31-35° C. In some embodiments, it is 30° C.

[0053] Preferably, the fermentation culture time can be generally 24 to 48 hours, or 24 to 32 hours, or 28 to 38 hours, or 32 to 48 hours. In some specific embodiments, the fermentation culture time is 30°C.

[0054] Preferably, the culture medium for fermentation culture contains glucose, yeast extract and peptone.

[0055] More preferably, based on the total volume of the fermentation medium, the fermentation medium comprises the following: glucose (10-30) g / L, yeast extract (5-15) / L, and peptone (10-30) g / L. In certain embodiments, glucose 20 g / L, yeast extract 10 g / L, and peptone 20 g / L.

[0056] In certain specific embodiments, the Gluconobacter oxidans strain is fermented to obtain Gluconobacter oxidans cells, which are diluted with water to an OD of 600 The yeast strain is fermented to obtain yeast cells, and then diluted with water to an OD 600is 0.50 to 0.06; and mixing to obtain the microbial agent. During mixing, the volume ratio can be selected according to actual needs. In a specific embodiment, the volume ratio is 1:1.

[0057] Another aspect of the present invention protects the use of the aforementioned Gluconobacter oxidans strain, the aforementioned yeast strain, or the aforementioned microbial agent in the preparation of a tea enzyme product.

[0058] In certain embodiments, the tea enzyme product comprises one or more of a direct drinking liquid, a concentrated liquid, and a freeze-dried powder.

[0059] Another aspect of the present invention is to provide a method for preparing tea enzyme, comprising the following steps:

[0060] 1) preparing tea syrup;

[0061] 2) inoculating bacterial liquid into the tea sugar water, fermenting, and obtaining the tea enzyme;

[0062] The bacterial solution comprises the aforementioned Gluconobacter oxidans strain or the aforementioned yeast strain or the aforementioned microbial agent.

[0063] When the Gluconobacter oxidans strain, yeast strain and strain combination thereof and microbial agent of the present invention are used to ferment tea syrup, no biofilm will be generated even if fermented for 15 days in an aerobic environment.

[0064] In certain embodiments, the raw materials for preparing the tea syrup include tea leaves, water and sugar.

[0065] Preferably, the tea leaves are selected from at least one of green tea, black tea or oolong tea. The oolong tea is selected from Wuyi rock tea.

[0066] Preferably, the mass ratio of tea leaves to water can generally be (0.5-1.3) : 100, or (0.5-0.9) : 100, or (0.8-1.2) : 100, or (1.1-1.3) : 100, such as 0.8:100.

[0067] Preferably, based on the total mass of tea leaves and water, the added amount of sugar can generally be (7-13) wt%, (7-9) wt%, (8-11) wt%, or (9-13) wt%, such as 10:100.8.

[0068] In certain embodiments, the preparation of tea syrup comprises: boiling tea leaves and water for 10 to 20 minutes, then adding sugar and continuing to boil for 5 to 15 minutes, filtering, sterilizing, and obtaining tea syrup. For example, the tea leaves and water can be boiled for 15 minutes and then continued to boil for 10 minutes. The tea leaves and water are mixed and boiled, and the boiling time after the sugar is added cannot be too long, because too long will cause Maillard reaction, and too short will result in less dissolution of organic acid, which is not conducive to fermentation.

[0069] Preferably, the sterilization condition is 115-120°C, and the sterilization time is 10-20 minutes, specifically, sterilization at 115°C for 15 minutes.

[0070] In certain embodiments, based on the total volume of the tea syrup, the inoculation amount of the bacterial solution can generally be (5-15) v / v%, (5-9) v / v%, (8-11) v / v%, or (10-15) v / v%, such as 10 v / v%.

[0071] Preferably, in the bacterial solution, the number of effective live bacteria of the Gluconobacter oxidans is at least (1-5)×10 8 CFU / mL and / or the effective viable count of yeast is at least (1-5)×10 7 CFU / mL.

[0072] In certain embodiments, the fermentation temperature may generally be 20-37°C, 10-25°C, 20-35°C, or 30-37°C, such as 30°C, 37°C, or 35°C.

[0073] In certain embodiments, the fermentation time may generally be 10 to 25 days, or 10 to 15 days, or 13 to 18 days, or 17 to 25 days, such as 15 days, 14 days, 16 days, or 18 days.

[0074] In some embodiments, post-treatment is further included, wherein the post-treatment is solid-liquid separation, and the bacterial cells are removed to terminate the fermentation. The solid-liquid separation is centrifugation, the centrifugation speed is 3000-13000 rpm, and the centrifugation time is 1-20 min.

[0075] In certain embodiments, the bacterial liquid is a bacterial suspension formed by fermenting the bacterial bodies obtained by the Gluconobacter oxidans strain or the bacterial bodies obtained by fermenting the yeast strain.

[0076] Preferably, the Gluconobacter oxidans strain is fermented and cultured, and the solid and liquid are separated to obtain a bacterial suspension of Gluconobacter oxidans.

[0077] More preferably, the fermentation temperature may be generally 25-35° C., 25-29° C., 28-32° C., or 31-35° C. In some specific embodiments, it is 30° C.

[0078] More preferably, the fermentation culture time can be generally 24 to 48 hours, or 24 to 32 hours, or 28 to 38 hours, or 32 to 48 hours. In some specific embodiments, it is 30°C.

[0079] More preferably, the culture medium for fermentation culture contains mannitol, yeast extract and peptone.

[0080] Further preferably, based on the total volume of the fermentation medium, the fermentation medium comprises the following raw materials: mannitol (10-30) g / L, yeast extract (1-10) g / L, and peptone (1-5) g / L. In certain specific embodiments, mannitol 25 g / L, yeast extract 5 g / L, and peptone 3 g / L.

[0081] Preferably, the yeast strain is fermented and cultured, and solid-liquid separation is performed to obtain a yeast cell suspension.

[0082] More preferably, the fermentation temperature may be generally 25-35° C., 25-29° C., 28-32° C., or 31-35° C. In some specific embodiments, it is 30° C.

[0083] More preferably, the fermentation culture time can be generally 24 to 48 hours, or 24 to 32 hours, or 28 to 38 hours, or 32 to 48 hours. In some specific embodiments, it is 30°C.

[0084] More preferably, the medium for fermentation culture contains glucose, yeast extract and peptone.

[0085] Further preferably, based on the total volume of the fermentation medium, the fermentation medium comprises the following: glucose (10-30) g / L, yeast extract (5-15) / L, and peptone (10-30) g / L. In certain specific embodiments, glucose 20 g / L, yeast extract 10 g / L, and peptone 20 g / L.

[0086] Preferably, the bacterial liquid is a bacterial suspension formed by a first bacterial body obtained by fermentation of a Gluconobacter oxidans strain and a second bacterial body obtained by fermentation of a yeast strain.

[0087] The first bacterial cell was diluted with water to OD 600 is 0.50-0.60, and the effective viable count is (1-3)×10 8CFU / mL; dilute the second bacteria with water to OD 600 is 0.50-0.60, and the effective viable count is (1-3)×10 8 CFU / mL; mix to obtain the bacterial suspension. During mixing, the volume ratio can be selected according to actual needs. In a specific embodiment, the volume ratio is 1:1, and the effective viable count of the obtained bacterial suspension is (1-3)×10 8 CFU / mL. In a specific embodiment, they are 2.7×10 8 CFU / mL, 1.6×10 7 CFU / mL and 1.44×10 8 CFU / mL.

[0088] The tea enzyme obtained by fermentation of the Gluconobacter acidobacter strain and the yeast strain of the present invention has a polyphenol content increased by 59.8% and a titratable acidity increased by 10% compared with the polyphenol content of the source thereof, and the content of glucuronic acid is higher than 4.3 g / L, which not only has a richer and stronger flavor, but also can significantly increase the content of functional ingredients. However, the maximum content of glucuronic acid in the production of kombucha using Pichia guilliermong reported in the literature is only 2.75 g / L.

[0089] The purpose of the present invention is to provide a method for producing rock tea enzyme by fermentation using isolated and purified Gluconobacter oxidans strains and yeast, aiming to improve the quality of rock tea enzyme while utilizing low-end rock tea. When the selected Gluconobacter oxidans strains and yeast are mixed and used in the present invention, the rock tea enzyme produced after fermentation has a higher polyphenol content and contains more abundant organic acids and product yield than the original bacteria.

[0090] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0091] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0092] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0093] Example 1 Isolation of Gluconobacter oxidans strains

[0094] In this embodiment, the strains of Gluconobacter oxidans were isolated from natural kombucha fungus, including the following:

[0095] Take the traditional natural kombucha strain obtained from Shanghai, take the activated traditional natural kombucha fermentation liquid and shake it evenly, draw the liquid under sterile conditions and streak it on a plate containing 0.1g / L natamycin MAD solid medium, place it in a 30℃ incubator and culture it for 2-3 days. Pick the round, protruding, opaque, cheese-colored single colony on the plate, such as Figure 1 As shown, add to a test tube containing 5 mL of MAD liquid culture medium, and then continue to streak separation, streak separation for more than 2 times, observe under a microscope, and select strains with short rod-shaped cells. Then inoculate the selected strains in the screening liquid culture medium and culture at 30°C for 3 days. Screen strains that do not produce cellulose film (biofilm).

[0096] MAD liquid medium: mannitol 25 g / L, yeast extract 5 g / L and peptone 3 g / L.

[0097] MAD solid culture medium: Add 15g / L agar to MAD liquid culture medium.

[0098] Screening liquid culture medium: yeast extract 10 g / L, glucose 10 g / L and anhydrous ethanol 30 mL / L.

[0099] Shanghai ShengBiological Co., Ltd. was commissioned to perform 16Sr DNA sequencing on the screened strains, and the PCR amplified sequence is shown in SEQID NO.1.

[0100] cgggggaagg gcggctgggg ggggttagca accactgtga accgctaaggcttaggctttagcaattgct tgggtggttcttagagctg cccaaacttt aaaacttatcatattgttttctgaaaccac gataatcaaa actttcaaca acggatctcttggc gtaatgcgaa ttgcagacgt gagtcattgaatttttgaacgcaaattgcg ccattaggttttcctaatgg catgcttgtt ggagcgtcgatctctctctcaaccatcttg gtatgaggta ttgctccttt taggagttaaaacatggaagtgcacacgttaatc aatcaagcaagattacccgctgaacttaag catatcaataagcggaggaa gactggcatc ttgcttgattgggggtcaaaagtgtagcgt agctgcacaa agttgattaa cgtgtgcacttccatgtttaactcctaaaggagcaat acctcatacc aagatggttg agaggaagat cgacctaccacac acctaatggcgcaatttgcg ttcaaaaatt caatgactcacgtctgcaattcgcattacc tatcgcgctt tgctgcgttc ttcatcgatacgagaaccaagagatccgttgttgaaagtt ttgattatcg tggtttcaga aaacaatatg ataagttttaaagggctgctcctc aagcctaagc cttagcggttcacagtggttttggctaaac gccttcagaa atgatccttc cgcaggtccc cttccgaaaggg(SEQ IDNO.1)

[0101] After removing the primer sequences from the sequencing results, a Blast comparison was performed in the GenBank database of the National Center for Biotechnology Information (NCBI).

[0102] A strain of Gluconobacter oxydans was screened and identified as Gluconobacter oxydans A21.

[0103] The Gluconobacter oxydans strain A21 of the present invention has been deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on December 25, 2023, and the deposit registration number is CCTCC NO:M 20232673.

[0104] Example 2 Isolation of yeast strains

[0105] In this embodiment, yeast strains were isolated from natural kombucha fungus species, including the following:

[0106] Under sterile conditions, 0.5 mL of the bacterial solution of Example 1 (i.e., the bacterial solution after sufficient shaking) was taken into a centrifuge tube containing 4.5 mL of physiological saline and mixed evenly. Then, the mixture was streaked onto a YPD solid medium containing 0.1 g / L chloramphenicol and cultured in a 28°C incubator for 1 to 2 days. The colonies on the plate were selected to be cream (milky white) in color. Figure 1 As shown, the strain with smooth surface, spherical convexity, opaque, creamy texture was separated by streaking more than 2 times.

[0107] YPD solid medium: Add 15g / L agar to YPD liquid medium.

[0108] Shanghai ShengBiological Co., Ltd. was commissioned to perform ITS rDNA sequencing on the yeast strain selected in the example, and the PCR amplified sequence is shown as SEQ ID NO.2.

[0109] cggcttaagg tcgaaccaac tcccatggtg tgacgggcgg tgtgtacaaggcccgggaacgtattcaccg cggcatgctgatccgcgatt actagcgatt ccaccttcatgtactcgagttgcagagtac aatccgaact gagacggctt ttagagatcagcaccatgtcaccatgtagcttcccactgt caccgccatt gtagcacgtg tgtagcccag gacataagggccatgaggacttgacgtcat ccccaccttcctccggcttg tcaccggcag tctctctagagtgcccacctgaaagtgctg gcaactaaag acaagggttg cgctcgttgcgggacttaacccaacatctcacgacacgag ctgacgacag ccatgcagca cctgtgcggg aggtccgaagaaaggtccat ctctgaaccggtcctcccca tgcaagccct ggtaaggttc tgcgcgttgcttcgaattaaaccacatgct ccaccgcttg tgcgggcccc cgtcaattcctttgagtttcaaccttgcggccgtactccc caggcggtgt gcttagcgcg ttagcttcga cactgaaaaa ctaagtttcccaacatccagcacacatcgt ttacagcgtg gactaccagg gtatctaatc ctgtttgctccccacgctttcgcgcctcag cgtcagtatc gagccaggttgccgccttcg ccaccggtgttcttcccaat atctacgaatttcacctcta cactgggaat tccacaaccc tctctcgaactctagtcgtcacgtatcaaa tgcagttcccaggttaagcc cggggatttc acatctgactgtaacaaccg cctacgcgcc ctttacgcccagtcattccgagcaacgcta gcccccttcg tattaccgcg gctgctggca cgaagttcggccttagtggcggacgggtga gtaacgcgtagggatctatc cacgggtggg ggacaacttc gggaaactgg agctaataccgcatgatacctgagggtcaa aggcgcaagtcgcctgtgga ggaacctgcg ttcgattagc tagttggtggggtaaaggcc taccaaggcg atgatcgata gctggtttga gaggatgatcagccacactgggactgagacacggcccaga ctcctacggg aggcagcagt ggggaatatt ggacaatgggcgcaagcctgatccagcaatgccgcgtgtg tgaagaaggt cttcggattg taaagcactttcgacgggga cgatgatgacggtacccgtagaagaagccc cggctaactt cgtgccagcagccgcggtaa tacgaagggg gctagcgttgctcggaatga ctgggcgtaaagggcgcgtaggcggttgtt acagtcagat gtgaaatccc cgggcttaacctgggaactg catttgatacgtgacgacta gagttcgagagagggttgtg gaattcccag tgtagaggtgaaattcgtagatattgggaa gaacaccggt ggcgaaggcg gcaacctggctcgatactgacgctgaggcgcgaaagcgtg gggagcaaac aggattagat accctggtag tccacgctgtaaacgatgtgtgctggatgttgggaaactt agtttttcag tgtcgaagct aacgcgctaagcacaccgcctggggagtac ggccgcaagg ttgaaactca aaggaattgacgggggcccg cacaagcggtggagcatgtg gtttaattcg aagcaacgcg cagaacctta ccaggcttgcatggggaggaccggttcagagatggacctt tcttcggacc tcccgcacag gtgctgcatg gc(SEQ ID NO.2)

[0110] After removing the primer sequences from the sequencing results, Blast alignment was performed in the GenBank database of the National Center for Biotechnology Information (NCBI).

[0111] A yeast strain identified as belonging to the genus Starmerella davenportii was screened out and named Starmerella davenportii YW1.

[0112] The yeast (Starmerella davenportii) strain YW1 of the present invention was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on December 25, 2023, and the deposit registration number is CCTCC NO: M 20232674.

[0113] Example 3 Preparation of microbial inoculum

[0114] In this example, a microbial inoculum formed by Gluconobacter oxydans and yeast includes the following:

[0115] 3.1 Preparation of Gluconobacter oxydans bacterial liquid

[0116] 1) Take the activated Gluconobacter oxydans A21, and then inoculate it into the MAD liquid medium at an inoculation amount of 5 v / v%, and culture it with shaking at 30°C and 180 rpm for 24 - 48 h.

[0117] 2) After cultivation, take the fermentation broth into a sterile centrifuge tube, centrifuge at 8000 rpm for 5 min, and then discard the supernatant. Add sterile water to the centrifuge tube, mix the cells and sterile water with a vortex mixer, centrifuge again and discard the supernatant, repeat washing the cells with sterile water twice, and adjust the OD of the bacterial liquid with sterile water 600 to 0.550 ± 0.050.

[0118] 3.2 Preparation of yeast bacterial liquid

[0119] 1) Take Starmerella davenportii YW1, inoculate it into the YPD liquid medium at an inoculation amount of 5 v / v%, and culture it with shaking at 30°C and 180 rpm for 24 - 48 h.

[0120] 2) After cultivation, take the fermentation broth and place it in a sterile centrifuge tube. Centrifuge at 8000 rpm for 5 min, then discard the supernatant. Add sterile water to the centrifuge tube, mix the bacteria and sterile water with a vortex mixer, centrifuge again, and discard the supernatant. Repeat the process of washing the bacteria with sterile water twice, and adjust the OD of the bacterial solution with sterile water. 600 To be 0.550 ± 0.050.

[0121] YPD liquid medium: Glucose 20 g / L, yeast extract 10 g / L, and peptone 20 g / L.

[0122] 3.3. Mixing

[0123] Mix the bacterial solution in step 3.1 and the bacterial solution in step 3.2 at a volume ratio of 1:1 to obtain the seed solution. Among them, the effective viable count in the Gluconobacter oxydans bacterial solution obtained in step 3.1 is 2.7×10 8 CFU / mL, the effective viable count in the yeast bacterial solution obtained in step 3.2 is 1.6×10 7 CFU / mL, and the effective viable count in the seed solution is 1.44×10 8 CFU / mL.

[0124] Example 4 Preparation of tea enzyme

[0125] In this example, a microbial inoculant formed by Gluconobacter oxydans and yeast is used to prepare tea enzyme. It includes the following:

[0126] 4.1. Preparation of tea sugar water

[0127] Preparation of rock tea sugar water: Prepare low - end rock tea leaves, granulated sugar, and water according to a mass ratio of 0.8﹕10﹕100. First, add the tea leaves to the water and boil for 15 min, then add granulated sugar and continue to boil for 10 min. Then filter, and sterilize the filtered tea sugar water at 115℃ for 15 min.

[0128] 4.2. Fermentation of rock tea enzyme

[0129] Take the seed solution obtained in Example 3 and inoculate it into the rock tea sugar water obtained in step 4.1 at an inoculation amount of 10 v / v% (that is, inoculate 10 mL of the seed solution into 100 mL of the medium), and statically culture at 30℃ for 15 days.

[0130] At the same time, set up a control group. Use the traditional natural black tea fungus in Example 1, that is, the Gluconobacter oxydans A21 and yeast YW1 strains separated from it, as the seed solution, inoculate it into the rock tea sugar water at an inoculation amount of 10 v / v%, and statically culture at 30℃ for 15 days.

[0131] Meanwhile, a Gluconobacter oxydans A21 group was established. The Gluconobacter oxydans A21 bacterial liquid obtained in step 3.1 of Example 3 was inoculated into the rock tea sugar water obtained in step 4.1 of this example at an inoculation amount of 10 v / v%, and statically cultured at 30°C for 15 days.

[0132] Meanwhile, a Saccharomyces cerevisiae YW1 group was established. The Saccharomyces cerevisiae YW1 bacterial liquid obtained in step 3.2 of Example 3 was inoculated into the rock tea sugar water obtained in step 4.1 of this example at an inoculation amount of 10 v / v%, and statically cultured at 30°C for 15 days.

[0133] The actual picture after 15 days of fermentation is shown in Figure 2 .

[0134] From Figure 2 it can be seen that after fermentation, no bacterial film grew in the experimental group, while a thick bacterial film floated on the liquid surface of the control group.

[0135] Take the tea enzyme solutions of each group, centrifuge at 8000 rpm for 5 min, take the supernatant, and use the Folin-Ciocalteu colorimetric method to detect the polyphenol content in the tea enzyme. Use the titration method to detect the titratable acidity of the tea enzyme. Use the HPLC method to analyze the contents of gluconic acid, glucuronic acid, malic acid, tartaric acid and acetic acid in the tea enzyme. Use the HPLC method to analyze the glucose and sucrose contents of the tea enzyme.

[0136] Polyphenols have functions such as antioxidant, anti-inflammatory, and lipid-lowering, which can help promote fat metabolism and inhibit fat absorption and accumulation. In addition, tea enzyme tea polyphenols also contain various vitamins and minerals and other beneficial components to the human body, which helps to improve human immunity and maintain physical health. Acidity is the main indicator to measure the maturity of tea enzyme, and it is also an important physical and chemical indicator reflecting the quality of tea enzyme. Like sugar, it is an important factor affecting flavor quality. High acidity means strong flavor and excellent quality. Glucuronic acid is the result of glucose oxidation and has a detoxifying effect. It can combine with endogenous and exogenous toxins to form water-soluble glucuronides and excrete them from the body, thus timely removing toxins that may cause organ lesions.

[0137] Determination of polyphenols by Folin-Ciocalteu colorimetric method: Respectively pipette 1.0 mL of gallic acid standard solution (10, 20, 40, 60, 80, 100 μg / mL), water, and the test solution (the test solution is 100 μL of the supernatant dissolved in 900 μL of water) into test tubes. In each test tube, add 5.0 mL of 10% Folin-Ciocalteu reagent, shake well, react for 8 min, add 4.0 mL of 7.5% sodium carbonate solution, and make up the volume to 25 mL with water. Let it stand at room temperature for 60 min. Measure the absorbance at a wavelength of 756 nm. Calculate the polyphenol content in the tea enzyme based on the absorbance of the gallic acid standard solution. The test results are shown in Figure 3 .

[0138] Determination of titratable acidity by titration method: Take 10 mL of the supernatant and add it to a 100 mL conical flask containing 20 mL of distilled water. Add two drops of 1% phenolphthalein reagent and titrate with 0.1 M NaOH solution until the solution turns red and does not fade within 30 s. Record the volume of NaOH solution consumed. The titratable acidity is represented by the mass of NaOH consumed at the end of the titration. The results are shown in Table 1.

[0139] Determination of organic acids by HPLC method: Take the supernatant, filter it through a 0.45 μm filter membrane, and use it for the determination of organic acid content by HPLC. The HPLC conditions are as follows:

[0140] Chromatographic column: C18 column

[0141] Mobile phase: Phase A: 0.02 M KH2PO4 solution (adjusted to pH 2.4), Phase B: methanol, Phase A:Phase B = 97:3

[0142] Flow rate: 0.8 mL / min

[0143] Temperature: 25 °C

[0144] Detection wavelength: 210 nm.

[0145] After the detection, compare with the organic acid standard product and calculate the organic acid content in the sample. The results are shown in Table 1.

[0146] Determination of sugar content by HPLC method: Take the supernatant, filter it through a 0.45 μm filter membrane, and use it for the determination of sugar content by HPLC. The HPLC conditions are as follows:

[0147] Chromatographic column: NH2 column

[0148] Mobile phase: Phase A: acetonitrile, Phase B: water, Phase A:Phase B = 70:30

[0149] Flow rate: 1 mL / min

[0150] Temperature: 40 °C

[0151] Injection volume: 20 μm

[0152] Detection: Refractive index differential detector.

[0153] After the detection, compare with the glucose and sucrose standard products and calculate the sugar content in the sample. The results are shown in Table 2.

[0154] From Figure 3 it can be seen that the polyphenol content in the tea enzyme of the experimental group is significantly higher than that of the control group, with a 59.8% increase compared to the control group.

[0155] The titratable acidity test results showed that 7.34 g of sodium hydroxide was used to titrate per liter of the tea enzyme in the experimental group, while 6.88 g of sodium hydroxide was used to titrate per liter of the tea enzyme in the control group, indicating that the sugar-acid conversion rate of the tea enzyme of the present invention is higher. That is, the tea enzyme of the present invention has a strong flavor and good taste.

[0156] Table 1

[0157] Experimental group Control group Gluconobacter oxydans A21 group Saccharomyces cerevisiae YW1 group Gluconic acid content (g / L) 13.6 8.5 13.9 9.748 Acetic acid (g / L) 6.8 11.1 7.1 7.8 Glucuronic acid (g / L) 4.3 0 3.9 9.82 Tartaric acid (g / L) 0 0 0 0 α-Ketoglutaric acid (g / L) 0.6 0.56 0.62 0.35 Malic acid (g / L) 0 0 0 0

[0158] As can be seen from Table 1, the acetic acid content in the control group was almost twice that of the experimental group, Gluconobacter oxydans A21 group, and yeast YW1, indicating that the content of other organic acids in the tea enzyme obtained by the single strain and its combination fermentation of the present invention is higher than that of acetic acid; glucuronic acid was not detected in the control group, while the glucuronic acid content of the single strain and its combination of the present application was above 3.9 g / L and could reach 9.82 g / L; in addition, the contents of gluconic acid and α-ketoglutaric acid in the single strain and its combination of the present application were also significantly higher than those in the control group. Generally speaking, the organic acids in the tea enzyme of the present invention are rich in variety, with a low acetic acid content and a high content of other organic acids, making the sour taste of the tea enzyme richer.

[0159] The detection results of the contents of fructose, sucrose, and glucose are shown in Table 2.

[0160] Table 2

[0161]

[0162]

[0163] As can be seen from Table 2, after 15 days of fermentation, the glucose in the tea enzyme produced by the experimental group was higher than that of Gluconobacter oxydans A21 alone and yeast YW alone, indicating that the white sugar in the experimental group was converted into glucose that is more easily digested and absorbed by the human body; in addition, the sucrose content in the tea enzyme of the single strain and its combination was much lower than that in the control group, and the sucrose content in the tea enzyme produced by the experimental group was the lowest, indicating that the conversion rate of the single strain and its combination of the present invention to sucrose is very high.

[0164] Therefore, compared with traditional natural kombucha, using the single strain and its combination provided by the present invention to produce tea enzyme not only significantly improves the production efficiency, but also significantly improves the quality of the obtained tea enzyme.

[0165] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. Gluconobacter oxydans strain A21, with the deposit number CCTCC NO: M20232673.

2. Starmerella davenportii strain YW1, with the deposit number CCTCC NO: M 20232674.

3. Use of the Gluconobacter oxydans strain according to claim 1 or the Starmerella davenportii strain according to claim 2 in the preparation of a microbial inoculant.

4. A microbial inoculant, characterized in that, Comprising the Gluconobacter oxydans strain according to claim 1 and / or the Starmerella davenportii strain according to claim 2.

5. The microbial inoculant according to claim 4, wherein The effective viable count of the microbial inoculum is at least (1-5)×10 8 CFU / mL, and the effective viable count of Gluconobacter oxydans contained in the microbial inoculum is at least (1-5)×10 8 CFU / mL and / or the effective viable count of yeast is at least (1-5)×10 7 CFU / mL.

6. Use of the Gluconobacter oxydans strain according to claim 1 or the Starmerella davenportii strain according to claim 2 or the microbial inoculant according to claim 4 or 5 in the preparation of a tea enzyme product.

7. The use according to claim 6, wherein, The tea enzyme product comprises one or more of a direct drink liquid, a concentrated liquid, and a freeze-dried powder.

8. A method for preparing tea enzyme, characterized in that, Including the following steps: 1) Prepare tea sugar water; 2) Inoculate the tea sugar water with a bacterial liquid and ferment to obtain the tea enzyme; The bacterial liquid comprises the Gluconobacter oxydans strain according to claim 1 or the Starmerella davenportii strain according to claim 2 or the microbial inoculant according to claim 4 or 5.

9. The preparation method according to claim 8, characterized in that, Based on the total volume of the tea sugar water, the inoculation amount of the bacterial liquid is 5 - 15 v / v%; and / or, the fermentation temperature is 20 - 37 °C; and / or, the fermentation time is 10 - 25 d; and / or, post-treatment is further included, and the post-treatment is solid-liquid separation to remove the bacterial cells; And / or, in the bacterial liquid, the effective viable count of Gluconobacter oxydans is at least (1-5)×10 8 CFU / mL and / or the effective viable count of yeast is at least (1-5)×10 7 CFU / mL; and / or, the preparation raw materials of the tea sugar water include tea leaves, water, and sugar; Preferably, the tea leaves are selected from one or more of green tea, black tea, and oolong tea; Preferably, the mass ratio of the tea leaves to the water is (0.5 - 1.3):100; Preferably, based on the total mass of the tea leaves and the water, the addition amount of the sugar is (7 - 13) wt%.

10. The tea enzyme obtained by the preparation method according to claim 8 or 9.

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