Black tea pile fermentation method based on functional flora regulation and control

Abstract

The invention provides an efficient pile fermentation method for Tianjian dark tea based on functional flora regulation and control. According to the method disclosed by the invention, key functional microbial florae, including the saccharomycetes and the klebsiella, which have positive effects on pile fermentation in the Tianjian dark tea are inoculated, and the Tianjian dark tea is subjected to efficient pile fermentation based on regulation and control of the functional florae, so that the pile fermentation effect of the Tianjian dark tea is improved. The functional flora respectively grows well in a culture medium which takes tea polyphenol as a main carbon source under the condition of 45 DEG C, and the conversion rate of the tea polyphenol reaches 40.98% when Tianjian dark tea is fermented for 24 hours. The functional flora is applied to Tianjian dark tea pile fermentation, polyphenol substances in tea leaves can be effectively converted, beneficial substances such as theabrownin and flavonoids in the tea leaves are increased, and efficient fermentation and quality improvement of the tea leaves are achieved. The functional flora has huge application value in the aspect of regulating and controlling dark tea fermentation.

Description

Technical field:

[0001] The invention belongs to the technical field of tea processing, and particularly relates to a Tianjian dark tea efficient pile fermentation method based on functional bacterial flora regulation. Background technology:

[0002] Tianjian black tea is a high-quality post-fermented tea made from first-grade black tea raw materials. It has a dark and moist appearance, bright red and yellow soup color, and a mellow taste. The older the tea is, the thicker the soup color is and the more durable it is. The unique pile fermentation process is a key step in the formation of the quality of Tianjian black tea. Pile fermentation is a biochemical process that relies on the participation of microorganisms. Through the metabolic activities of microorganisms, it promotes the transformation of the chemical components inside the tea leaves, thus giving Tianjian black tea its unique color, aroma and taste. However, the traditional pile fermentation process mainly relies on natural fermentation by microorganisms in the natural environment. This method has the problems of low efficiency and unstable quality. Due to the lack of in-depth understanding of the structure and function of the microbial community involved in fermentation, the potential of the flavor and health benefits of Tianjian black tea has not been fully realized.

[0003] With the development of modern biotechnology, especially the application of high-throughput sequencing technology, powerful tools are provided for the accurate identification and functional analysis of microbial communities. Through these technologies, key microbial flora that have a positive effect on the transformation of tea components can be screened out, thereby providing a new strategy for optimizing the fermentation process. The efficient fermentation method for Tianjian black tea based on functional flora regulation proposed in the present invention, through scientific screening and cultivation, accurately utilizes the dominant functional flora to regulate the fermentation process of Tianjian black tea, significantly promotes the transformation of phenolic substances and the generation of beneficial substances in Tianjian black tea, improves the quality of Tianjian black tea, and meets the market's growing demand for high-quality tea. The efficient fermentation method of the present invention can promote technological innovation in the black tea industry, and also provides a model for reference for the microbial fermentation process of other teas, and has broad commercial value and application prospects. Summary of the invention:

[0004] The main purpose of the present invention is to provide an efficient Tianjian black tea pile-fermentation method regulated by functional bacteria. The method uses scientific screening to accurately utilize dominant functional bacteria to regulate the Tianjian black tea pile-fermentation process, thereby significantly promoting the transformation of phenolic substances and the generation of beneficial substances in the Tianjian black tea, and improving the quality of the Tianjian black tea.

[0005] In order to solve the above technical problems, the following technical solutions are adopted:

[0006] Dark tea pile fermentation method based on functional bacterial flora regulation,

[0007] The cultured functional bacteria are mixed with dark tea leaves, and pile fermentation is performed, and fermented tea leaves are obtained after cooling; the functional bacteria are composed of yeast and Klebsiella.

[0008] The functional bacterial flora is a mixture of yeast and Klebsiella liquid in a volume ratio of 1:1-1:10, and the number of viable bacteria of each bacterium is ≥10 ^7 CFU / mL.

[0009] Further,

[0010] The functional bacteria in the logarithmic growth phase were centrifuged and then added with sterile water to make a bacterial suspension. The bacterial count reached 10 ^7 CFU / mL to 10 ^9 CFU / mL, and the functional bacteria are mixed according to the volume ratio, and then added in an amount of 1-20% (V / W) with the first-grade dark tea fresh leaves after withering and rolling for pile fermentation.

[0011] Further,

[0012] The fermentation time ranges from 20 to 30 hours, the temperature ranges from 40 to 55°C, and the air humidity ranges from 60% to 80%.

[0013] Furthermore,

[0014] The yeast was inoculated into a modified YPD liquid medium with tea polyphenols as the main carbon source and cultured to the logarithmic growth phase, and the Klebsiella was inoculated into an LB liquid medium and cultured to the logarithmic growth phase before being used for heap fermentation.

[0015] The functional bacterial flora of the present invention is preferably used for Tianjian dark tea fermentation.

[0016] The present invention also provides the use of the functional bacterial flora in improving the water extract content, tea polyphenol conversion capacity, amino acid conversion capacity, flavonoid content, and tea pigment content of pile-fermented dark tea leaves; and reducing the degree of decrease in soluble sugar content.

[0017] The present invention firstly screens out the microbial flora having a positive effect on the fermentation of Tianjian dark tea according to the high-throughput sequencing results, which mainly includes yeast and Klebsiella.

[0018] The high-throughput sequencing results for screening functional bacterial communities include analysis of the composition, abundance, and diversity of the bacterial communities.

[0019] Furthermore, the functional bacterial flora in step (2) is composed of yeast and Klebsiella, the Saccharomyces cerevisiae has a preservation number of CICC 31111, and the Klebsiella sp. has a preservation number of CICC 10702, both of which can be obtained from commercial channels.

[0020] The growth curves of the functional bacteria and the conversion rate of tea polyphenols were both at 45℃ and 200r min. -1 Measured under conditions.

[0021] The flavor quality components of the fermented black tea of ​​the present invention include but are not limited to the water extract content of tea leaves, the tea polyphenol content, the amino acid content, the soluble sugar content, the flavonoid content, and the tea pigment content.

[0022] The present invention has the following beneficial effects:

[0023] The functional microbial flora provided by the present invention can grow well in a culture medium with tea polyphenols as the main carbon source at 45°C, and the tea polyphenol conversion rate can reach 40.98% within 24 hours. The functional flora is applied to Tianjian dark tea fermentation pile, can effectively convert polyphenols and amino acids in tea leaves, increase the beneficial substances such as theabrownin and flavonoids in tea leaves, reduce the degree of decrease in soluble sugar content, and achieve efficient fermentation and quality improvement of tea leaves. The functional flora has great application value in regulating dark tea fermentation. Description of the drawings:

[0024] Figure 1 Figure 1. The microbial community structure at the genus level of Tianjian dark tea at different fermentation times: bacteria (A); fungi (B); among them, the fermentation times of 0h, 6h, 12h, 18h, and 24h were marked as W0, W6, W12, W18, and W24, respectively.

[0025] Figure 2 is the growth curve of the strain; among them, Klebsiella: KH; yeast: JH; Bacillus subtilis: BH.

[0026] Figure 3 It is the ability of the strain to transform tea polyphenols; among them, Klebsiella: KH; Yeast: JH; Bacillus subtilis: BH.

[0027] Figure 4 The changes of various physical and chemical parameters during the fermentation process of functional bacteria group and common bacteria group: (A) water extract content; (B) tea polyphenol content; (C) flavonoids content; (D) amino acid content; (E) soluble sugar content; (F) tea pigment content. Among them, the common bacteria group: CY, the functional bacteria group: GN, and the control group: CK. Specific implementation method:

[0028] The following examples are provided to further illustrate the above contents of the present invention in detail, but are not intended to limit the present invention.

[0029] Example 1: Analysis and screening of functional bacterial flora

[0030] Tea leaves from Hunan Anhua Yuntaishan Bajiao Tea Co., Ltd. were taken for high-throughput sequencing at 0h, 6h, 12h, 18h, and 24h of fermentation. Total DNA was extracted from each sample by CTAB method, the quality of DNA extraction was detected by agarose gel electrophoresis, and then the DNA was quantified by UV spectrophotometer. The bacterial V3-V4 region was amplified using universal forward primer 341F (5'-CCTACGGGNGGCWGCAG-3') and reverse primer 805R (5'-GACTAC HVGGGTATCTAATCC-3'). The fungal ITS region was amplified using universal forward primer ITS1FI2 (5'-GTGARTCATCGAATCTTTG-3') and reverse primer ITS2 (5'-TCCTCCGCTTATTGATATGC-3'). PCR products were verified by 2% agarose gel electrophoresis. Ultrapure water was used throughout the DNA extraction process instead of sample solution, so the possibility of false positive PCR results could be excluded as a negative control. PCR products were purified with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA), quantified with Qubit (Invitrogen, USA), and recovered using the AMPure XT beads recovery kit. Finally, high-throughput sequencing of bacterial 16S rRNA gene sequences and fungal ITS sequences was performed on the Illumina NovaSeq PE250 platform by Hangzhou Lianchuan Biotechnology Co., Ltd. (Zhejiang, China).

[0031] Experimental results: Figure 1 A reflects the changes in the community structure at the genus level during the fermentation of Tianjian dark tea. The top five genera with the highest relative abundance in the fermentation system are Klebsiella (24.87-68.66%), Mixta (3.91-31.77%), Pantoea (1.07-6.88%), Pectobacterium (0.23-3.51%), and Methylobacterium-Methylorubrum (0.03-5.23%). Figure 1B reflects the changes in the community structure of fungi at the genus level during the fermentation of Tianjian dark tea. The top five genera with high relative abundance in the fermentation system are Saccharomyces_Incertae_sedis_unclassified (4.68-93.62%), Debaryomyces (0.89-43.08%), Ascomycota_unclassified (0.77-36.57%), Se tophoma (0.58-15%) and Alternaria (0.41-6.57%). The results show that Klebsiella and Saccharomyces are the dominant genera in the fermentation process.

[0032] Example 2: Application of strains in the conversion of tea polyphenols

[0033] YPD liquid medium containing 1% tea polyphenols: 2g peptone, 1g yeast extract, 2g glucose, 1g tea polyphenols, distilled water to 100mL for yeast culture;

[0034] LB liquid medium containing 1% tea polyphenols: 1g peptone, 0.5g yeast extract, 1g NaCl, 1g tea polyphenols, distilled water to 100mL, used for culturing Klebsiella or Bacillus subtilis;

[0035] In this example, Saccharomyces cerevisiae with a preservation number of CICC 31111 and Klebsiella sp. with a preservation number of CICC 10702 were used. Meanwhile, Bacillus subtilis with a preservation number of CICC 21322, which is commonly used for pile fermentation, was used as a control.

[0036] Yeast, Klebsiella, and Bacillus subtilis were inoculated into the liquid culture medium and incubated at 45°C and 200 rmin. -1 The culture was cultured under the conditions of 4% paraformaldehyde and 1% paraformaldehyde, 200 μl of the culture was taken into a 96-well plate every 1 h, the OD600 value was measured with an ELISA reader, and the growth curve was drawn.

[0037] The strain was inoculated into 100 mL of medium containing tea polyphenols as the main carbon source at a rate of 5%, and incubated at 45°C for 200 rpm. -1 After 24 hours of culture, 1 mL of bacterial solution was taken every 4 hours, centrifuged at 12000 rpm for 10 minutes, and the supernatant was taken. The supernatant was then diluted and the absorbance was measured at 765 nm. Figure 2The delay period of yeast, Klebsiella, and Bacillus subtilis was 1 hour, followed by the logarithmic period, and the stable period at 8 hours, reaching the highest peak of growth. The highest OD600 of yeast was 0.618, the highest OD600 of Klebsiella was 0.6277, and the highest OD600 of Bacillus subtilis was 0.6167, indicating that the three bacteria grew well at 45°C.

[0038] Figure 3 The results show that yeast, Klebsiella and Bacillus subtilis have the ability to transform tea polyphenols. Within 24 hours, the conversion rate of tea polyphenols by the three bacteria gradually increased with time. At the 24th hour, the conversion rate of tea polyphenols by yeast reached 17.17%, the conversion rate of tea polyphenols by Klebsiella reached 24.89%, and the conversion rate of tea polyphenols by Bacillus subtilis reached 22.44%. Klebsiella has a stronger ability to transform tea polyphenols.

[0039] Example 3: Application of functional flora in Tianjian dark tea fermentation

[0040] Using the strains, culture medium and method of Example 2, yeast, Klebsiella and Bacillus subtilis were inoculated into 100 mL YPD and LB culture medium at a 5% inoculum, respectively, at 45°C, 200 r min -1 After culturing for 5 hours, collect the bacterial suspension. Mix yeast and Klebsiella in a volume ratio of 1:1, centrifuge at 10000rpm for 10min to collect the bacterial cells, wash them twice with sterile water, and then resuspend them in sterile water to obtain a bacterial suspension with a bacterial count concentration of ≥10 ^7 CFU / mL. Then the bacterial solution was added to the tea leaves after withering and rolling at a ratio of 10% (V / M). This group was named the functional bacterial group (GN). Yeast and Bacillus subtilis were added to the tea leaves in the same steps and named the common bacterial group (CY). An equal amount of sterile water was added to the tea leaves and named the control group (CK). The fermentation cycle was 24 hours. Samples were taken from the two fermentation systems at 0, 4, 8, 12, 16, 20, and 24 hours to determine the contents of water extract, tea polyphenols, amino acids, soluble sugars, flavonoids, and tea pigments. Detection method: The water extract content was determined according to the national standard GB / T 8305-2013 (Determination of the content of tea water extract). The tea polyphenol content was determined according to the national standard GB / T 8313-2018 (Detection method for the content of tea polyphenols and catechins in tea leaves). The contents of flavonoids, theaflavins, thearubigins, and theabrownins were determined by colorimetry. The soluble sugar content was determined by the phenol-sulfuric acid method. The free amino acid content was determined according to the national standard GB / T8314-2013 (Determination of the total amount of free amino acids in tea).

[0041] Experimental results: Figure 4A shows the change of water extract content. Water extract refers to some water-soluble substances, including polyphenols, amino acids, caffeine, polysaccharides, etc., which are related to the thickness and flavor intensity of tea soup. In the first 4 hours of the start of pile fermentation, the content of water extract increased rapidly, and after 20 hours of fermentation, the content of water extract decreased. In the early stage of fermentation, the microbial flora will produce a series of enzymes, such as polyphenol oxidase and cellulase, through metabolic activities. These enzymes can catalyze the decomposition of macromolecules such as polyphenols and cellulose in tea leaves to generate more water-soluble substances, resulting in an increase in water extract content. As fermentation proceeds, some water-soluble substances in tea leaves may further undergo polymerization or condensation reactions to form macromolecules, and be consumed by microorganisms as nutrients, resulting in a decrease in water extract content. At the end of pile fermentation, the water extract content of the functional bacterial group increased by 16.37%, the commonly used bacterial group increased by 12.98%, and the control group increased by 9.4%, indicating that inoculation of functional bacterial flora can increase the extraction rate of effective ingredients in tea leaves.

[0042] Tea polyphenols are generally divided into four categories: catechins, flavonoids, anthocyanins and phenolic acids. They are the main components that determine the color, aroma, taste and efficacy of tea. At the same time, tea polyphenols are the main factor that causes the astringency of tea. Figure 4 B shows that during the fermentation process, the content of tea polyphenols decreases with time, which is mainly attributed to the exoenzymes secreted by microorganisms and a series of reactions such as enzymatic and non-enzymatic oxidation, polymerization and transformation under the action of moisture and heat. At the end of the fermentation, the tea polyphenol content of the functional bacteria group decreased by 40.98%, the common bacteria group decreased by 23.36%, and the control group decreased by 16.67%. This shows that the metabolic activity of the bacteria in the functional bacteria group is higher, and the conversion of tea polyphenols is more significant, which reduces the astringency of the tea and makes the taste more mellow.

[0043] Flavonoids are widely found in plant-derived foods and have many benefits for human health. Figure 4 C shows that the flavonoid content fluctuates during the fermentation process. This may be because flavonoid compounds undergo glycosylation and methylation, resulting in a decrease in their content, while microbial activity promotes the formation of flavonoids. At the end of the fermentation, the flavonoid content of the functional bacteria group increased by 24.13%, the common bacteria group increased by 6.04%, and the control group increased by 4.52%. This shows that the addition of functional bacteria plays a positive role in the fermentation process of tea, which helps to improve the nutritional value and health function of tea.

[0044] Amino acids are the main source of tea's refreshing taste. Figure 4D shows that the amino acid content fluctuates during the fermentation process. This may be because the growth and reproduction of microorganisms require the use of amino acids as a nitrogen source, and Maillard reaction with soluble sugars, resulting in a decrease in amino acid content, while the exoenzymes secreted by microorganisms hydrolyze the protein in tea into amino acids, causing their content to increase. At the end of the fermentation, the amino acid content of the functional bacteria group decreased by 10.54%, the common bacteria group decreased by 8.41%, and the control group decreased by 5.84%. This shows that the addition of functional bacteria promotes the conversion of amino acids.

[0045] Soluble sugar can increase the sweetness and viscosity of tea soup and reduce the bitterness to a certain extent. Figure 4 E shows that during the fermentation process, the soluble sugar content decreased within 0 to 4 hours, increased within 4 to 8 hours, and continued to decrease after the 8th hour. In the early stage of fermentation, the metabolic activity of microorganisms may lead to the consumption of soluble sugars, especially simple sugars such as monosaccharides and disaccharides are absorbed by microorganisms for growth and reproduction, resulting in a decrease in sugar content. As microbial activity increases, enzyme activity increases, and the rate of sugar production may exceed consumption, resulting in an increase in soluble sugar content. After the number of microorganisms reaches a peak, the rate of sugar consumption may accelerate, and Maillard reaction with amino acids may occur, causing its content to decrease again. At the end of the fermentation, the soluble sugar content of the functional bacteria group decreased by 9.38%, the commonly used bacteria group decreased by 11.57%, and the control group decreased by 14.97%. This shows that the tea with the addition of functional bacteria is sweeter and has a better flavor.

[0046] Tea pigment is a general term for a series of natural pigments with biological activity in tea, including theaflavins, thearubigins and theabrownins. Theabrownins are a health-care ingredient that has a significant effect on lowering blood sugar, blood lipids, blood pressure and uric acid. Theabrownins can also improve the body's overall metabolic balance and are produced in large quantities during microbial fermentation. Figure 4 F shows that during the fermentation process, the contents of theaflavins, thearubigins and theabrownins all increased overall. In the later stage of the fermentation, the increase in theaflavin content tended to be gentle, and the content of thearubigins and theabrownins showed an obvious upward trend. This can be explained by the fact that the components mainly composed of phenolic substances formed tea pigments through a series of complex chemical and microbial changes under the action of moisture, heat and microorganisms. At the end of the fermentation, the theaflavins content of the functional bacteria group was 0.621%, the common bacteria group was 0.59%, and the control group was 0.55%; the thearubigins content of the functional bacteria group was 2.639%, the common bacteria group was 2.094%, and the control group was 1.834%; the theabrownin content of the functional bacteria group was 5.843%, the common bacteria group was 5.470%, and the control group was 5.171%. The contents of theaflavins, thearubigins and theabrownins in the functional bacteria group were all higher than those in the common bacteria group, which indicates that the addition of functional bacteria can help improve the color of tea soup and increase the nutritional value of tea.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dark tea pile fermentation method based on functional flora regulation, characterized in that: The cultured functional bacteria are mixed with dark tea leaves, and pile fermentation is performed, and fermented tea leaves are obtained after cooling; the functional bacteria are composed of yeast and Klebsiella.

2. The method according to claim 2, characterized in that: The functional bacterial flora is a mixture of yeast and Klebsiella liquid in a volume ratio of 1:1-1:10, and the number of viable bacteria of each bacterium is ≥10 ^7 CFU / mL.

3. The method according to claim 1 or 2, characterized in that: The functional bacteria in the logarithmic growth phase were centrifuged and then added with sterile water to make a bacterial suspension. The bacterial count reached 10 ^7 CFU / mL to 10 ^9 CFU / mL, and the functional bacteria are mixed according to the volume ratio, and then added in an amount of 1-20% (V / W) with the first-grade dark tea fresh leaves after withering and rolling for pile fermentation.

4. The method according to any one of claims 1 to 3, characterized in that: The fermentation time ranges from 20 to 30 hours, the temperature ranges from 40 to 55°C, and the air humidity ranges from 60% to 80%.

5. The method according to any one of claims 1 to 4, characterized in that: The yeast was inoculated into a modified YPD liquid medium with tea polyphenols as the main carbon source and cultured to the logarithmic growth phase, and the Klebsiella was inoculated into an LB liquid medium with tea polyphenols as the main carbon source and cultured to the logarithmic growth phase before being used for heap fermentation.

6. The method according to claim 1, characterized in that The functional bacterial flora is used for Tianjian dark tea fermentation.

7. Use of the functional bacterial flora described in any one of claims 1 to 6 in improving the water extract content, tea polyphenol conversion capacity, amino acid conversion capacity, flavonoid content, and tea pigment content of pile-fermented dark tea leaves; and reducing the degree of decrease in soluble sugar content; the functional bacterial flora is composed of yeast and Klebsiella.

8. The use according to claim 7, characterized in that: The dark tea is Tianjian dark tea.

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