阅读说明：本技术 一种将鲜老茶叶中茶多酚生物转化为茶褐素的方法 (Method for biologically converting tea polyphenol in fresh old tea leaves into theabrownin ) 是由 修志龙 郑梦娇 董悦生 虞诗强 王银波 张建娟 于 2021-07-30 设计创作，主要内容包括：本发明公开了一种将鲜老茶叶中茶多酚生物转化为茶褐素的方法,属于生物工程技术领域。通过从普洱熟茶中分离出能够转化茶多酚为茶褐素的优势菌株烟曲霉(Aspergillus fumigatus)、布朗克假丝酵母(Candida blankii)以及塔宾曲霉(Aspergillus tubingensis),将其中的一种或两种以上的混合菌接种于以鲜老茶叶为原料的发酵培养基,利用内源酶和微生物结合的方式,生物强化发酵转化茶多酚为茶褐素。本发明以鲜老茶叶为发酵基质,进行悬浮发酵或半固态发酵,均能有效利用茶叶内源酶,加速茶多酚的氧化,缩短发酵周期,大幅提升茶褐素生成效率,从而增加茶树的附加值,具有良好的工业应用前景。(The invention discloses a method for biologically converting tea polyphenol in fresh old tea into theabrownin, belonging to the technical field of biological engineering. The method comprises the steps of separating dominant bacteria Aspergillus fumigatus (Aspergillus fumigatus), Candida blankii (Candida blankii) and Aspergillus tubingensis (Aspergillus tubingensis) which can convert tea polyphenol into theabrownin from Pu-erh ripe tea, inoculating one or more mixed bacteria in a fermentation culture medium which takes fresh and old tea leaves as raw materials, and performing biologically-enhanced fermentation to convert the tea polyphenol into the theabrownin by utilizing a mode of combining endogenous enzyme and microorganisms. According to the invention, fresh old tea leaves are used as a fermentation substrate to carry out suspension fermentation or semi-solid fermentation, so that endogenous enzymes in the tea leaves can be effectively utilized, the oxidation of tea polyphenol is accelerated, the fermentation period is shortened, and the generation efficiency of theabrownin is greatly improved, so that the added value of tea trees is increased, and the tea tree fermentation method has a good industrial application prospect.)

1. A method for biologically converting tea polyphenol in fresh and old tea leaves into theabrownin is characterized in that dominant microorganisms capable of converting tea polyphenol into theabrownin are inoculated into a fermentation culture medium taking the fresh and old tea leaves as raw materials for fermentation, and the biological conversion of the tea polyphenol into the theabrownin is enhanced by utilizing a method of combining tea endogenous enzyme and the dominant microorganisms.

2. The method of claim 1, wherein the dominant microorganisms comprise one or a combination of more than two of Aspergillus fumigatus, Candida blankie and Aspergillus tubingensis.

3. The method as claimed in claim 1, wherein the fresh old tea leaves are fresh mature leaves below the top young leaves of the tea plant.

4. The method according to claim 1, wherein the fermentation is liquid fermentation, suspension fermentation or semi-solid fermentation.

5. The method according to any one of claims 1 to 4, wherein the conditions of the fermentation are: aerobic fermentation, inoculating 1-10% (v/v) seed culture solution, wherein the number of spore or viable bacteria cells is 1 × 10⁶～1×10⁹CFU/mL; the culture temperature is 28-45 ℃, the pH is 5-8, the rotation speed is 150-300 rpm, and the fermentation time is 72-120 h.

6. The method according to claim 5, wherein the preparation of the fermentation medium for liquid fermentation comprises the steps of: adding 10-50 times of water by mass into fresh old tea leaves, leaching for 0.1-12h at 50-100 ℃, filtering, and sterilizing the obtained tea soup.

7. The method according to claim 5, wherein the preparation of the fermentation medium for suspension fermentation comprises the steps of: adding 10-50 times of water by mass into fresh old tea leaves, homogenizing and crushing to obtain the tea leaves, or sterilizing after crushing to obtain the tea leaves, or performing enzymatic reaction on the crushed tea leaves by using tea leaf autoxidase at the temperature of 30-40 ℃ for 2-4 h, and then sterilizing to obtain the tea leaves.

8. The method according to claim 5, wherein the preparation of the fermentation medium for semi-solid fermentation comprises the steps of: adding water with the mass of 1-8 times of that of the fresh old tea leaves, homogenizing and crushing the mixture to obtain the tea leaves, or crushing the homogenate and then sterilizing the homogenate, or crushing the homogenate and then performing enzymatic reaction on the tea leaves by using the oxidase of the tea leaves at the temperature of 30-40 ℃ for 2-4 h, and then sterilizing the tea leaves to obtain the tea leaves.

9. The method of claim 5, wherein the preparation of the seed culture comprises the steps of: respectively inoculating the aspergillus fumigatus and aspergillus tubingensis strains to a PDA (PDA dextrose agar) plate culture medium, culturing for 3-5 days at 30 ℃, washing off fungal spores by using sterilized normal saline, transferring the fungal spores into a container filled with sterilized glass beads, carrying out vortex oscillation and filtering to respectively obtain seed culture solutions of the aspergillus fumigatus and the aspergillus tubingensis; inoculating Candida blankii into a YPD culture medium for activation, inoculating the Candida blankii into a fresh YPD culture medium according to the inoculation amount of 1-10% (v/v), and culturing for 24-30h to obtain a Candida blankii seed culture solution.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for biologically converting tea polyphenol in fresh and old tea leaves into theabrownin.

Background

Theabrownines are the main effective components of Pu-erh ripe tea, and have the effects of reducing blood sugar, reducing blood fat, inhibiting bacteria, resisting cancer, resisting oxidation and the like. The theabrownin is a composite pigment substance formed by polymerizing tea polyphenol, tea polysaccharide, protein and other substances under the action of moist heat and extracellular enzyme secreted by microorganisms in the pile fermentation process of Pu' er tea. The natural fermentation process is long in period (usually 50-60 days), the conversion rate of active ingredients is low, meanwhile, the precise control is difficult, the content difference of the theabrownins is large, the product quality is unstable, and the further development and utilization of the theabrownins are limited. In addition, microorganisms participating in natural pile fermentation mainly come from leaves, air and the surrounding environment, the flora composition is complex, more mixed bacteria easily cause unstable product quality, mycotoxin is easily generated, and the quality of the Pu' er tea is seriously influenced.

In order to overcome the defect of natural fermentation, the production efficiency and the product quality of the theabrownin can be improved by artificially adding microorganisms or enzymes. Exogenous enzyme preparations such as laccase, peroxidase, cellulase and the like are added in the Pu 'er tea pile fermentation process, so that the oxidation of substances such as tea polyphenol in Pu' er tea can be accelerated to different degrees, the fermentation time is shortened, and the content of theabrownin in the product is improved. However, the addition of the enzyme preparation increases additional cost, and the requirement on the enzymatic reaction condition is harsh, which is not favorable for popularization and application. In the process of producing theabrownin by using an enzyme preparation or artificially inoculating microorganism for fermentation, the green-removed sun-dried raw tea is usually adopted, and enzymes such as polyphenol oxidase and peroxidase contained in fresh tea leaves for promoting the conversion of tea polyphenol into theabrownin are not fully utilized.

The existing method for improving the production efficiency of theabrownin is an improvement on the traditional tea making process, and the used tea leaves or extracts are tender leaves of tea making, but not mature old tea leaves, namely mature leaves below tender leaves at the top end of tea trees. Old tea leaves are usually discarded and not used, and naturally wither and fall off, but the old tea leaves contain more starch, chlorophyll and tea polyphenol than tender leaves, and the content of the tea polyphenol is 7-10 percent higher. Given the availability of these old tea leaves, it would undoubtedly add additional revenue to the tea grower. For example, the tea picking area of Yunnan province in 2018 is 600 ten thousand mu, the average yield of tea per mu is 66 kg, and the profit of a tea garden per mu is about 5000-10000 yuan. If 500kg of old tea leaves can be collected in each mu of tea garden, even if only the tea polyphenol is extracted, the new yield value of 2 ten thousand yuan per mu is added. Under the condition of not influencing the normal tea making income, the additional income can be increased by 2 to 3 times for tea farmers. If the tea polyphenol in the old tea leaves is converted into thearubigins and theabrownins through biocatalysis to provide raw materials for tea drinks or health care products, the yield value of the tea industry can be further improved, and the income of tea farmers is increased.

In conclusion, the method of combining the endogenous enzyme in the fresh old tea leaves and the dominant bacteria of the Pu' er tea fermentation is utilized to convert the tea polyphenol in the fresh old tea leaves into the theabrownin, so that the tea resources are fully utilized, the production efficiency and quality of the theabrownin are improved, the production cost is reduced, and the method has important economic value and social benefit for industrial production of the theabrownin and development and utilization of the theabrownin.

Disclosure of Invention

The invention aims to provide a method for biologically converting tea polyphenol in fresh old tea leaves into theabrownin, so that the utilization rate of the old tea leaves is improved, and the additional value of tea trees is increased.

The technical scheme of the invention is as follows:

a method for biologically converting tea polyphenol in fresh old tea leaves into theabrownin is characterized in that dominant microorganisms which are screened from Pu' er ripe tea and can convert tea polyphenol into theabrownin are inoculated into a fermentation culture medium which takes the fresh old tea leaves as raw materials for fermentation, and the biological conversion of the tea polyphenol into the theabrownin is enhanced by utilizing a method of combining tea endogenous enzyme and the dominant microorganisms in the fermentation process.

Further, in the above technical solution, the dominant microorganism has the following properties: the strain is cultured on a solid tea soup screening culture medium for 2-5 days, the growth state of the strain is good, and brown plaques can be generated around colonies.

Further, in the above technical scheme, the preparation of the solid tea soup screening medium comprises the following steps: weighing 20-60g of sun-dried green raw tea, adding into 1000mL of water, boiling in a water bath for 5-20 min, filtering, adjusting the pH value of the obtained tea soup to 5.0-7.0, diluting to 1000mL, adding 10-30g of agar, and sterilizing to obtain the tea soup.

Further, in the above technical solution, the dominant microorganism includes one or a combination of two or more of Aspergillus fumigatus, Candida blankie and Aspergillus tubingensis.

Furthermore, in the technical scheme, the fresh old tea leaves refer to fresh mature leaves below tender leaves at the top end of the tea trees.

Further, in the above technical scheme, the fermentation mode adopted is liquid fermentation, suspension fermentation or semi-solid fermentation.

Further, in the above technical scheme, the fermentation conditions are as follows: aerobic fermentation; the inoculation amount of the seed culture solution is 1-10% (v/v), wherein the number of spores or viable bacteria cells is 1 multiplied by 10⁶～1×10⁹CFU/mL; the culture temperature is 28-45 ℃, the pH is 5-8, the rotating speed is 150-300 rpm, and the fermentation time is72h～120h。

Further, in the above technical scheme, the preparation of the seed culture solution comprises the following steps: respectively inoculating the aspergillus fumigatus and the aspergillus tubingensis strains to a PDA (PDA dextrose agar) plate culture medium, culturing for 3-5 days at 30 ℃, washing off fungal spores by using a proper amount of sterilized normal saline, transferring the fungal spores to a centrifuge tube filled with sterilized glass beads, carrying out vortex oscillation and filtering to respectively obtain seed culture solutions of the aspergillus fumigatus and the aspergillus tubingensis; inoculating Candida blankii into a YPD culture medium, activating for 10-12h, inoculating to a fresh YPD culture medium according to the inoculation amount of 1-10% (v/v), and culturing for 24-30h to obtain a Candida blankii seed culture solution.

Further, in the above technical solution, the preparation of the fermentation medium for liquid fermentation comprises the following steps: adding 10-50 times of water into fresh old tea leaves, leaching for 0.1-12h at 50-100 ℃, filtering, and sterilizing the obtained tea soup.

Further, in the above technical solution, the preparation of the fermentation medium for suspension fermentation comprises the following steps: adding 10-50 times of water by mass into fresh old tea leaves, homogenizing and crushing to obtain the tea leaves, or sterilizing after crushing to obtain the tea leaves, or performing enzymatic reaction on the crushed tea leaves by using tea leaf autoxidase at the temperature of 30-40 ℃ for 2-4 h, and then sterilizing to obtain the tea leaves.

Further, in the above technical solution, the preparation of the fermentation medium for semi-solid fermentation comprises the following steps: adding water with the mass of 1-8 times of that of the fresh old tea leaves, homogenizing and crushing the mixture to obtain the tea leaves, or crushing the homogenate and then sterilizing the homogenate, or carrying out enzymatic reaction on the homogenate and the crushed tea leaves by using the oxidase of the tea leaves for 2-4 hours at the temperature of 30-40 ℃, and then sterilizing the product.

The invention has the beneficial effects that:

(1) the strains Aspergillus fumigatus (Aspergillus fumigatus), Candida blankii and Aspergillus tubingensis (Aspergillus tubingensis) used in the invention are all screened from Pu-erh ripe tea samples, belong to natural strains, have the capability of efficiently converting tea polyphenol into theabrownin, are easy to culture, have high genetic stability and the like, and can be applied to industrial production.

(2) The invention takes fresh old tea leaves as raw materials, adopts a suspension fermentation or semi-solid state fermentation mode, improves the utilization rate of endogenous enzyme in the tea leaves, increases the contact between the enzyme and a substrate, and promotes the conversion of tea polyphenol into theabrownin. The method simplifies theabrownin production process, shortens fermentation period, increases additional value of tea tree, and reduces environmental stress.

Drawings

FIG. 1 shows the tea polyphenol content in fresh old tea leaves of example 1.

FIG. 2 shows the growth state of Aspergillus fumigatus DL-01, Candida blankii DL-02 and Aspergillus tubingensis DL-03 on tea soup selection medium in example 2.

FIG. 3 is a graph showing the change in the yield of theabrownin in the single fermentation broth in example 3.

FIG. 4 is a graph showing the change in the concentration of tea polyphenols and theabrownins in suspension fermentation of a single bacterium in example 4.

FIG. 5 is a graph showing the change of the concentrations of tea polyphenols and theabrownins in the semi-solid fermentation of a single bacterium in example 5.

FIG. 6 is a graph showing the change of the concentrations of tea polyphenols and theabrownins in the mixed fermentation semi-solid fermentation of example 6 (left: 30 ℃ C.; right: 37 ℃ C.).

FIG. 7 is a graph showing the change in the concentrations of tea polyphenols and theabrownins in the single-strain semi-solid fermentation catalyzed by endogenous enzymes in example 7.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, the reagents used were all available from chemical or biological reagents, and the tea leaves were collected from the large leaf tea garden in Longling county, Baoshan, Yunnan province.

The following describes the embodiments of the present invention in detail with reference to the technical solutions.

1. The media used in the following examples:

1) PDA culture medium: cutting 200g peeled potato into pieces, boiling in water bath for 20min, filtering with two layers of gauze, adding 20g glucose into the filtrate to reach a constant volume of 1000mL, adding 20g agar, autoclaving at 115 deg.C for 20min, and subpackaging into solid culture medium.

2) YPD medium: 20g of glucose, 20g of peptone, 10g of yeast extract powder, and water to 1000mL, and autoclaving at 115 ℃ for 20 min.

3) Liquid tea soup screening culture medium: weighing 30g of sun-dried green raw tea, adding into 1000mL of water, boiling in a water bath for 5-10 min, filtering under reduced pressure to remove tea residue, adjusting the pH value of the obtained tea soup to 6.0, diluting to 1000mL, and autoclaving at 115 ℃ for 20min for later use.

4) Solid tea soup screening culture medium: the preparation method comprises screening culture medium with liquid tea soup, adjusting the content of sun-dried green tea to 40g, and adding agar 20g before autoclaving.

5) Liquid fermentation medium: adding 30 times of water into fresh old tea, leaching at 100 deg.C for 15min, vacuum filtering to remove tea residue, adjusting pH to 6.0, and pasteurizing (80 deg.C, 30 min). The raw material of the fermentation medium is sun-dried raw tea, and the preparation method is the same.

6) Suspension fermentation culture medium: experimental groups: adding 30 times of water into fresh old tea, homogenizing and crushing for 2 min. Ventilating and stirring the homogenate at a constant temperature of 37 ℃ at a speed of 200rpm, fully oxidizing tea polyphenol by using tea endogenous enzyme, heating for 2 hours at a high temperature for 3-5 minutes by using microwave, stopping enzymatic reaction, adjusting the pH value to 6.0, and pasteurizing (80 ℃, 30 minutes). Control group: adding 30 times of water into fresh old tea, homogenizing and crushing for 2 min. Heating the homogenate liquid at high temperature by microwave for 3-5 min, adjusting pH to 6.0, and pasteurizing (80 deg.C, 30 min).

7) Adding 5 times of water into fresh old tea, homogenizing and crushing for 2 min. Ventilating and stirring the homogenate liquid in a water bath at a constant temperature of 37 ℃ at a speed of 200rpm, fully oxidizing tea polyphenol by using tea endogenous enzyme, heating for 3-5 min at a high temperature by using microwaves after 2h, stopping enzymatic reaction, adjusting the pH value to 6.0, and carrying out pasteurization (80 ℃, 30 min). The fermentation medium is prepared by adding 5 times of water into fresh old tea, homogenizing and crushing for 2 min.

2 analytical method

(1) Pretreatment of liquid fermentation liquor samples: centrifuging the fermentation liquid with small centrifuge at 10000rpm for 10min, discarding thallus, collecting supernatant, and performing water bath at 75 deg.C for 5min to terminate fermentation.

(2) Pre-treating a suspension fermentation liquid sample: centrifuging the fermentation liquid with small centrifuge at 10000rpm for 10min, discarding thallus and folium Camelliae sinensis suspended matter, centrifuging the supernatant at 10000rpm for 10min, collecting supernatant, water bathing at 75 deg.C for 5min, and terminating fermentation.

(3) The semi-solid fermentation liquid sample pretreatment method is the same as the suspension fermentation liquid sample pretreatment.

(4) And (3) measuring the content of tea polyphenol: referring to GB/T8313-2018 tea content detection method, gallic acid is used as a standard reference substance, and the tea polyphenol is quantitatively determined by a colorimetric standard curve method.

(5) And (3) determining the theabrownin concentration: reference system analysis.

Taking 2mL of the pretreated fermentation liquid sample, adding n-butanol with the same volume, vortex and shake for 3min, and standing for layering. Taking 0.4mL of lower water phase, adding 0.4mL of saturated oxalic acid, 1.2mL of distilled water and 3mL of 95% ethanol, uniformly mixing, and detecting the absorbance at 380 nm. 95% ethanol was used as a blank. And (3) calculating the yield and the concentration of the theabrownin in the fermentation liquor according to the formula (1) and the formula (2) respectively.

In the formula: x_TB: the yield of theabrownin in the fermentation liquor (g TB/100g dry weight of tea used for preparing the culture medium); c_TB: the theabrownin concentration (g/L) in the fermentation liquor; e_B: absorbance of the sample; ω: preparing a culture medium by using tea leaf water mass fraction; f: preparing correction coefficients of the culture medium at different material-liquid ratios, wherein f is 1 when the ratio of tea leaves to water in the culture medium is 3: 125; m: preparing a culture medium by using dry weight (g) of tea leaves; v: total volume of fermentation broth (mL); d: and (5) dilution times of fermentation liquor.

Example 1 tea leaf tea Polyphenol content

The content of tea polyphenol in fresh old tea leaves and green tea, sun-dried green tea and black tea which are purchased from the same region and made of tender leaves is measured. The tea polyphenol is a substrate for generating theabrownin through oxidation, and the content of the tea polyphenol directly influences the generation amount of the theabrownin. As shown in FIG. 1, the tea polyphenol content in fresh and old tea leaves is 28.07% (w/w, dry weight, the same below), which is significantly higher than that of green tea (19.62%), sun-dried raw tea (18.28%) and black tea (9.49%) prepared from young leaves of the same variety. Thearubigin and theaflavin in black tea are converted from tea polyphenol, and the retention rate of the tea polyphenol of green tea and sun-dried green tea is higher, so that the tea polyphenol in fresh and old tea is obviously higher than that of tender tea.

Example 2 separation and identification of advantageous strains of Pu' er tea

The strain of the invention is derived from a Pu-Er ripe tea sample. The specific separation and identification method comprises the following steps:

(1) primary screening: adding 18mL of sterilized 0.9% physiological saline into 2g of cooked Pu-Er tea, placing in a constant temperature shaking incubator at 37 ℃ for shaking for 30min, taking out, standing for 5min, and making into bacterial suspension for use. Taking 100 mu L of the solution for 10^-1、10^-2、10^-3And (3) diluting in three concentration gradients, coating 100 mu L of diluted bacterial suspension on a solid tea soup screening culture medium, inverting the culture medium in an incubator at 37 ℃ for constant-temperature culture for 1-5 days, screening bacterial colonies according to the size of the brown spots, inoculating the bacterial colonies to a new solid screening culture medium, and performing streaking separation and purification for multiple times to obtain the bacterial strains with single bacterial colony morphology. Inoculating the strain into PDA slant culture medium, and storing for use.

(2) Re-screening: inoculating the strains preserved in the primary screening to a new PDA culture medium for activation, culturing at 30 deg.C for 3-5 days, selecting single colony, respectively inoculating to liquid tea soup screening culture medium, and shake-culturing at 30 deg.C and 200rpm for 4 days. The concentrations of tea polyphenol and theabrownin in the fermentation liquor are measured to obtain 3 dominant strains with stronger capability of converting tea polyphenol into theabrownin, which are respectively named as DL-01, DL-02 and DL-03. Storing by conventional method such as slant passage method and low temperature glycerol method.

(3) Identification of the strains: by determining the ITS rDNA sequences of DL-01 and DL-03 strains and the 26S rDNA sequence of DL-02 strain, and comparing the sequence Information with the GenBank database of the National Center for Biotechnology Information (NCBI), it was revealed that DL-01, DL-02 and DL-03 have 100% homology similarity with known strains Aspergillus fumigatus (NCBI accession No. MT558940.1), Candida blankii (NCBI accession No. MF940140.1) and Aspergillus tubingensis (NCBI accession No. MN818620.1), respectively, and they were determined to be Aspergillus fumigatus (Aspergillus fumigatus), Candida blankii (Candida blankii) and Aspergillus tubingensis (Aspergillus tubingensis), respectively. The raw data obtained from sequencing the DL-01, DL-02 and DL-03 genes have been submitted to NCBI GenBank under accession numbers MZ203542.1, MZ206165.1 and MZ206179.1, respectively. The three strains have similar sources with the strains with the strain preservation numbers of CICC 41620, CICC 33217 and CICC 41400 of the China Industrial microbial culture Collection respectively.

EXAMPLE 3 liquid fermentation of three Individual bacteria

Preparing a seed culture solution: the Aspergillus fumigatus and Aspergillus tubingensis strains obtained in example 2 were inoculated into a PDA plate medium, cultured at 30 ℃ for 3-5 days, washed off the fungal spores with an appropriate amount of sterilized normal saline, transferred to a 50mL centrifuge tube equipped with sterilized glass beads, vortexed and shaken for 3min, and filtered through two layers of sterilized absorbent cotton to obtain two seed culture solutions, respectively. The Candida blankii obtained in the example 2 is inoculated into a YPD culture medium to be activated for 10-12h, and inoculated into a fresh YPD culture medium according to the inoculation amount of 1% -10% to be cultured for 24-30h, so as to obtain a Candida blankii seed culture solution.

Counting the number of spores (Aspergillus) or viable cells (Yeast) in the three seed culture solutions with a blood counting plate, and adjusting the concentration to 1 × 10 with sterilized normal saline⁷And (3) respectively inoculating the CFU/mL into conical flasks filled with liquid fermentation culture media taking fresh old tea or sun-cured raw tea leaching liquor as the only fermentation substrate by using an inoculation amount of 5% (v/v), sealing the bottle mouths by using a bidirectional breathable film, performing shake flask fermentation at the temperature of 30 ℃ and at the speed of 200rpm, periodically sampling to determine the concentrations of tea polyphenol and theabrownin fermentation liquor, and calculating the yield of theabrownin. The fermentation process is shown in FIG. 3, and the starting point tea polyphenol content (dry weight) and the end point theabrownin yield of the fermentation are shown in Table 1. The content of tea polyphenol in the fresh old tea is obviously higher than that of sun-dried green tea, and the content of theabrownin in fermentation liquor of the fresh old tea in the fermentation processThe yield is higher than that of sun-dried green raw tea, which shows that the biological conversion of fresh old tea leaves as raw materials into theabrownin has obvious advantages.

TABLE 1 starting and ending theapolyphenol content and theabrownin yield for single-strain liquid fermentation

Note: x_TP，0: starting point tea polyphenol content (w/w, dry weight) of fermentation; x_TB，f: the fermentation end point theabrownin yield.

EXAMPLE 4 suspension fermentation of three Individual bacteria

The seed culture solution was prepared as in example 3, and inoculated to fresh and old tea suspension fermentation medium pretreated with endogenous enzyme for 2h (experimental group) and not treated with endogenous enzyme for enzymolysis (control group) under the same conditions, respectively, for shake flask fermentation, periodically sampling to determine the concentrations of tea polyphenol and theabrownin in the fermentation broth, and calculating the yield of theabrownin. The fermentation process is shown in figure 4: the tea polyphenol concentration reaches an equilibrium state basically within 48 hours, and the theabrownin concentration basically tends to be stable within 72 hours. The fermentation results are shown in table 2, and the concentrations of theabrownins in the fermentation liquid of the experimental group (endogenous enzyme + bacteria) are higher than those of the control group (single bacteria) in the fermentation process, which indicates that the oxidase (endogenous enzyme) of the fresh and old tea leaves is used for carrying out the enzymatic reaction, so that more tea polyphenols can be promoted to be converted into theabrownins.

TABLE 2 Single-fungus suspension fermentation end-point tea polyphenols and Theabrownines concentrations

Note: c_TP，0: starting point of fermentation tea polyphenol concentration; c_TP，f: the tea polyphenol concentration at the end of fermentation; c_TB，0: the fermentation starting point theabrownin concentration; c_TB，f: the fermentation end point theabrownin concentration; x_TB，f(%): the fermentation end point theabrownin yield.

EXAMPLE 5 semi-solid fermentation of three individual bacteria

The preparation of the seed culture solution is carried outExample 3 adjustment of the number of spores or viable cells to 1X 10⁸And (3) inoculating the mixture to a fresh old tea leaf semi-solid fermentation medium pretreated by tea leaf endogenous enzyme for 2 hours under the same condition for shaking flask fermentation, periodically sampling to determine the concentrations of tea polyphenol and theabrownin in a fermentation liquid, and calculating the yield of the theabrownin. The fermentation process is shown in figure 5: the theabrownin concentrations in the fermentation liquid inoculated with the aspergillus tubingensis, the aspergillus fumigatus and the candida blankii at the end point are respectively 8.13, 7.38 and 13.95g/L, and the yield is respectively as follows: 17.49%, 15.87%, 30.00%. The semi-solid state fermentation theabrownin concentration is obviously higher than that of liquid state fermentation and suspension fermentation, and the theabrownin concentration in the semi-solid state fermentation liquid of Candida blankii is 4.08 times of that in suspension fermentation. Although the yield of theabrownin in the semi-solid fermentation liquid is lower than that of the liquid fermentation and the suspension fermentation, the yield of theabrownin in the suspension fermentation liquid of Candida blankii is 1.37 times of that of the semi-solid fermentation, but the concentration increase can reduce the subsequent concentration cost.

EXAMPLE 6 semi-solid fermentation with three bacteria mixture

Seed culture was prepared in the same manner as in example 3 except that the number of spores or viable cells was adjusted to 1X 10⁸CFU/mL is respectively mixed according to the proportion of 1: 1 of the seed liquid volume of the aspergillus tubingensis and the aspergillus fumigatus (combination A), 1: 1 of the seed liquid volume of the aspergillus tubingensis and the candida blankii (combination B), 1: 1 of the seed liquid volume of the aspergillus fumigatus and the candida blankii (combination C) and 1: 1 of the aspergillus fumigatus, the aspergillus tubingensis and the candida blankii (combination D), the mixed seed culture solution is inoculated to a semi-solid fermentation culture medium pretreated by tea endogenous enzyme for 2 hours in the same inoculation amount, shaking flask fermentation is respectively carried out at 30 ℃ and 37 ℃, the concentrations of tea polyphenol and theabrownin fermentation liquid are periodically sampled and measured, and the theabrownin yield is calculated. The experimental process is shown in fig. 6, and the tea polyphenol concentration, theabrownin concentration and theabrownin yield at the end of fermentation are shown in table 3. With the increase of the temperature, the concentration and the yield of the theabrownin at the end point of the fermentation of the four combinations are increased to different degrees, wherein the combination (B) of the aspergillus tubingensis and the candida blankii has the most obvious change with the temperature compared with other combinations, the theabrownin concentration is increased by 2.13g/L, and the theabrownin yield is increased by 3.28%. Similarly, under the same temperature condition, the Aspergillus tubingensis and the Candida blankii are fermentedThe parent combination (B) also has significant advantages over other combinations.

TABLE 3 semi-solid Mixed fermentation end-point tea polyphenols and Theabrownines concentrations

Note: c_TP，f: the tea polyphenol concentration at the end of fermentation; c_TB，f: the fermentation end point theabrownin concentration; x_TB，f(%): the fermentation end point theabrownin yield.

Example 7 combination of endogenous enzyme catalysis and single-bacterium semi-solid fermentation of fresh old tea leaves

Seed culture was prepared in the same manner as in example 3 except that the number of spores or viable cells was adjusted to 5X 10⁷And (3) directly inoculating the mixture to a non-sterilized semi-solid fermentation medium subjected to homogenization treatment under the same conditions for shaking flask fermentation, periodically sampling to determine the concentrations of tea polyphenol and theabrownin in the fermentation liquor, and calculating the yield of the theabrownin. The fermentation process is shown in fig. 7, the concentration of tea polyphenol is obviously reduced within 24-48 h, and the tea polyphenol basically reaches an equilibrium state after 48 h. The theabrownin concentration gradually rises after 48 hours, and the theabrownin concentration in the fermentation liquor inoculated with the Candida blankii is obviously higher than that in other two groups after 48 hours. The theabrownin concentrations in the fermentation liquor inoculated with the aspergillus tubingensis, the aspergillus fumigatus and the candida blankii at the end point are respectively 7.32, 6.40 and 13.57g/L, and the theabrownin yields are respectively as follows: 15.74%, 11.33%, 29.18%.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.