CN109287784B

CN109287784B - Method for quick pile fermentation of green brick tea

Info

Publication number: CN109287784B
Application number: CN201811049306.0A
Authority: CN
Inventors: 付瑞燕; 徐倩; 宁井铭; 陈军海
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2018-09-10
Filing date: 2018-09-10
Publication date: 2022-02-15
Anticipated expiration: 2038-09-10
Also published as: CN109287784A

Abstract

The invention provides a quick pile fermentation method for green brick tea, belonging to the technical field of green brick tea fermentation, and the pile fermentation method for green brick tea comprises the following steps: mixing the sun-dried green raw tea and water according to the proportion of 2: 1-4: 1(W/V), then filling the mixture into a pile fermentation container, and carrying out sterilization treatment; inoculating the pile fermentation broth to a sterilized pile fermentation container in an inoculation amount of 5-20% (V/W), and carrying out aerobic culture at 45 +/-3 ℃. According to the invention, pure pile fermentation is carried out by adopting two strains of bacillus subtilis and aspergillus fumigatus, so that the product components are the same as those of the traditional open pile fermentation, the flavor is the same, compared with the traditional open microbial natural pile fermentation process in the utilization environment, the time can be shortened by about 4 weeks, the microbial natural pile fermentation process is not easily polluted by other bacteria, and the microbial natural pile fermentation process is beneficial to increasing the enterprise benefit and improving and stabilizing the product quality.

Description

Method for quick pile fermentation of green brick tea

Technical Field

The invention belongs to the technical field of green brick tea fermentation, and particularly relates to a method for quick pile fermentation of green brick tea.

Background

Pile fermentation is a key process for forming the quality of the green brick tea, and the traditional green brick tea pile fermentation is mainly controlled by experience, so that the guarantee of the quality of the tea and the application of the modern fermentation process are greatly limited. Therefore, the community structure of microorganisms in the green brick tea pile fermentation process is known, so that potential leavening agents are selected and developed, the pile fermentation time is shortened, and clean and standardized production is realized.

The key procedures of green brick tea quality formation are pile fermentation and storage aging, wherein the pile fermentation is characterized in that microbes suspended in the air naturally live on a tea blank to generate extracellular enzymes to initiate a series of violent chemical reactions, the temperature of leaves is gradually increased, and the action of moist heat and the action of microbes are alternately carried out, so that the green brick tea with reddish brown soup color, mellow taste and full-bodied aroma is formed. In the important process of 'pile fermentation' for forming the unique flavor of the green brick tea, the key for ensuring the good quality of the green brick tea is how to promote the beneficial microorganisms to play a role and control the propagation of the unfavorable microorganisms. According to the invention, the separated high-temperature strains which are beneficial to the quality of the green brick tea are utilized, and a pile fermentation method is adopted for manual inoculation, so that the pile fermentation period can be shortened, the interference of mixed bacteria which are not beneficial to the quality can be avoided, and the stability and the improvement of the quality of the tea are facilitated.

Through retrieval, a plurality of research papers report the pile fermentation of the green brick tea, and the Aspergillus niger is most reported. At present, the method has no report of separating aspergillus fumigatus from Pu' er tea pile fermentation and applying the aspergillus fumigatus to fermentation processing of green brick tea, and the method has no report of fermenting the contents of main quality components in sun-dried green raw tea to an ideal pile tea sample level within one week by only using two strains.

Disclosure of Invention

The invention discloses a bacillus subtilis, which has a preservation number of: CCTCC M2018288, wherein the colony characteristics of the bacillus subtilis are as follows: the bacterial colony is medium in size, the surface of the bacterial colony is light yellow, the surface of the bacterial colony is rough and has wrinkles, the edge of the bacterial colony is irregular, the surface of the bacterial colony is dry, opaque and lackluster, gram reaction is positive, no capsule exists, a single bacterial cell is rod-shaped, two ends of the single bacterial cell are blunt and round, and cysts do not expand obviously.

The Bacillus subtilis belongs to Mycomonera of the bacterial kingdom, Protophyta of the phylum Protophyta, Schizomycetes, Eubacterioles, Bacillaceae, Bacillus.

The invention preserves aspergillus fumigatus, and the preservation number of the strains of the aspergillus fumigatus is as follows: CCTCC M2018287. The colony characteristics of the aspergillus fumigatus are as follows: the bacterial colony is circular, the diameter is large, the surface of the bacterial colony is villous, the center is light cyan, the edge is grey white, the surface is dry and non-transparent, hyphae have partitions, the conidium is spherical, and the top of the conidium peduncle forms an expanded spherical top sac.

The Aspergillus fumigatus belongs to Mycota of the kingdom fungi, Eumycophyta of the phylum Eumycophyta, Deuteromycotina of the subdivision Deuteromycotina, Hyphomycetes of the class Hyphomycetales, Hyphomycetales of the order Hyphomycetales, Moniliaceae, Aspergillus.

Based on the two preserved strains, the invention provides a method for quick pile fermentation of green brick tea, which comprises the following steps:

mixing the sun-dried green raw tea and water according to the proportion of 2: 1-4: 1(W/V), then filling the mixture into a pile fermentation container, and carrying out sterilization treatment;

inoculating the pile fermentation broth to a sterilized pile fermentation container in an inoculation amount of 5-20% (V/W), and carrying out aerobic culture at 45 +/-3 ℃;

the preparation method of the pile fermentation broth comprises the following steps:

placing bacillus subtilis with CCTCC of M2018288 in a NA liquid culture medium, culturing at 45 +/-3 ℃, dispersing somatic cells, and performing suspension treatment to obtain bacillus subtilis liquid;

placing aspergillus fumigatus with CCTCC (China center for culture) of M2018287 in a PDA (potato dextrose agar) liquid culture medium, culturing at the temperature of 45 +/-3 ℃, dispersing thallus cells, and performing suspension treatment to obtain aspergillus fumigatus liquid;

and mixing the aspergillus fumigatus liquid and the bacillus subtilis liquid to obtain the pile-fermentation mixed bacteria fermentation liquid.

Further, the sterilization treatment is performed for 20-40 min at the temperature of 100-121 ℃.

Further, the culture time of the bacillus subtilis in the NA liquid culture medium is 2-36 h.

Furthermore, in the bacillus subtilis liquid, the number of somatic cells is 10⁶～10⁷one/mL.

Further, the culture time of the aspergillus fumigatus in the PDA liquid culture medium is 2-36 h, and the mycelium segments are dispersed to be 0.2-0.4 cm.

Further, in the aspergillus fumigatus liquid, the mycelium section is 10⁶～10⁷one/mL.

Further, the NA culture medium comprises the following components: beef extract: 3-5 g/L, peptone: 10-15 g/L, NaCl: 3-9 g/L, and the balance being sterile water.

Further, the PDA culture medium comprises the following components: potato leaching powder: 4-15 g/L, 15-25 g/L glucose and the balance of sterile water.

Further, the mixing ratio of the aspergillus fumigatus liquid and the bacillus subtilis liquid is as follows: 2:1 to 1: 3.

Advantageous effects

Cellulase, pectinase, protease and polyphenol oxidase are key enzymes participating in green brick tea production and are important factors determining the quality of green brick tea. In the process of pile fermentation of the dark tea, the twisted leaves are coarse, old, hard, crisp and extremely poor in sticky feeling, and can be gradually softened under the action of cellulase in the pile fermentation; in the processing of the green brick tea, pectinase can hydrolyze pectin into micromolecular carbohydrate substances, so that 'silk network' of tea leaves is reduced, the alcohol degree of tea soup is enhanced, and the quality and flavor of the green brick tea are improved; the protease is one of hydrolase, can catalyze the protein in the tea to hydrolyze to form various amino acids, can reduce the generation of insoluble compounds, can improve the aroma and freshness of the tea, and can improve the quality of tea soup; the catechin, the main component of tea polyphenol, is oxidized and condensed into theabrownin under the action of polyphenol oxidase.

The high-temperature dominant strains of bacillus subtilis X4 and M1 aspergillus fumigatus obtained by separation and purification of the invention have unique enzymological characteristics respectively. As shown in table 1, bacillus subtilis X4 was able to produce more cellulase, pectinase and protease simultaneously, but not polyphenol oxidase, whereas mould strain M1 was able to produce cellulase, pectinase, protease and polyphenol oxidase simultaneously.

In the present invention, the measurement method of each data is as follows:

1. determination of enzyme Activity

And (3) cellulase activity determination: 3, 5-dinitrosalicylic acid (DNS) sugar determination method. 1mL of crude enzyme solution is put into a control tube and a sample tube, 2mL of DNS solution is firstly added into the control tube to destroy the enzyme activity, then preheated pH4.8 is respectively added, and 2mL of carboxymethyl cellulose (CMC) solution with the mass fraction of 1.0 percent is put into the two tubes. The mixture is placed in a constant temperature water bath at 40 ℃ for reaction for 30min, and immediately taken out, a sample tube is added with 2mL of DNS solution to stop the reaction. Boiling in boiling water for 5min, taking out running water, cooling, diluting to 25mL, and measuring absorbance at 540nm wavelength.

m₁Quality of released glucose/ug as determined by the Standard Curve

time/min for t-enzymolysis

m₂Sample mass/g

And (3) measuring the activity of the pectinase: the substrate is changed into preheated pectin liquid with the mass fraction of 0.25%, and other steps are the same as the cellulase activity determination method.

Protease activity assay: the Folin phenol (Folin) method was used. Preheating 1mL of enzyme solution in 40 deg.C water bath for 3min, adding 1mL of preheated casein, rapidly adding 2mL of trichloroacetic acid solution after 40 deg.C water bath for 15min, precipitating for 10min, centrifuging at 5000r/min for 10min, adding 2mL of trichloroacetic acid solution before adding casein to inactivate enzyme activity, and the rest is the same as above. 1mL of the supernatant was taken, 5mL of Na2CO3 solution and 1mL of Folin reagent were added, and the mixture was taken out and subjected to color comparison at 660nm in a thermostatic water bath at 40 ℃ for 20 min.

C-is the mass of tyrosine/ug found from the standard curve

n-is the dilution multiple

V-volume of enzyme solution participating in reaction/mL

time/min for t-enzymolysis

M₀Sample mass/g

Measurement of Polyphenol oxidase Activity: 1.5mL of enzyme solution is respectively put in a blank tube and a sample tube, and 3mL of reaction mixed solution is added (the reaction mixed solution is prepared by citric acid phosphate buffer solution (pH5.6) with the volume ratio of 10:2:3, proline with the mass fraction of 0.1 percent and catechol with the mass fraction of 2 percent, and the catechol in the blank tube is replaced by the buffer solution). Then, the temperature is kept in a constant-temperature water bath kettle at 37 ℃ for 10min, and 1mL of 20% trichloroacetic acid is immediately added to stop the reaction. The absorbance was measured at a wavelength of 460 nm.

time/min for t-enzymolysis

m-sample mass/g

E₄₆₀-absorbance value

2. Determination of tea polyphenol content

Referring to a second method in the detection method for the content of tea polyphenol and catechins in tea (GB/T8313-2008), the specific operation method is as follows:

(1) gallic acid standard stock solution (in situ)

Accurately weighing 0.110g of gallic acid, placing the gallic acid in a 100mL volumetric flask, dissolving the gallic acid in redistilled water, fixing the volume to a scale, shaking up, and storing the gallic acid in a dark place.

(2) Gallic acid working solution

Respectively measuring 1.0, 2.0, 3.0, 4.0 and 5.0mL of gallic acid standard stock solution by using a pipette, placing the gallic acid standard stock solution into a 100mL volumetric flask, respectively metering to a scale with redistilled water, shaking up, and preparing into working solutions with the concentrations of 10, 20, 30, 40 and 50 mu g/mL.

(3) Preparation of mother liquor

Accurately weighing 0.2g (accurate to 0.0001g) of ground tea sample, placing the ground tea sample into a 10mL centrifuge tube, adding 5mL of 70% methanol preheated at 70 ℃, uniformly stirring the mixture by a glass rod, transferring the mixture into a 70 ℃ water bath for leaching for 10min (stirring once every 5 min), cooling the mixture to room temperature after leaching, transferring the mixture into a centrifuge at 3500r/min for centrifugation for 10min, transferring the supernatant into a 10mL volumetric flask, extracting the residue once by 5mL of preheated 70% methanol, and repeating the steps. Mixing the extractive solutions, diluting with 70% methanol to 10mL, and shaking.

(4) Preparation of test solutions

Transferring 1mL of mother liquor into a 100mL volumetric flask, adding water to a constant volume to a scale, and shaking up to be tested.

(5) Measurement of

Transferring 1mL gallic acid working solution, water and test solution into graduated test tubes, adding 5mL Folin phenol reagent into each test tube, shaking, reacting for 3-8min, adding 4mL of 7.5% Na₂CO₃Shaking the solution, and standing at room temperature for 60 min. The absorbance (A) was measured with a spectrophotometer in a 10mm cuvette at a wavelength of 765 nm.

A-absorbance of sample test solution

V-volume of sample extract, 10mL

d-dilution factor (usually 1mL to 100mL, then 100)

Slope of the slopes-gallic acid standard curve

m-sample dry matter content%

m₁Sample mass, g

3. Determination of catechin content

The method adopts a Vanillin colorimetric method, and comprises the following specific operation methods:

(1) preparation of test solutions

Accurately weighing 2g of pulverized tea sample, placing into 100mL conical flask, adding 20mL of 95% ethanol, extracting in 80-85 deg.C water bath for 30min, filtering, and diluting to 25mL with 95% ethanol.

(2) Measurement of

20uL of the test solution was aspirated, the solution was poured into a stoppered test tube containing 1mL of 95% ethanol, shaken well, then 5mL of 1% vanillin hydrochloric acid solution was added, shaken well after stoppering, left to stand for 40min, and then the absorbance (A) was measured with a spectrophotometer in a 10mm cuvette at a wavelength of 500 nm.

L₁Total amount of sample test solution (mL)

L₂Amount of liquid used in measurement (mL)

M-Mass (g) of sample

W-dry matter content of sample (%)

Absorbance of A-sample

4. Determination of amino acid content

Referring to the total amount of free amino acids in tea (GB/T8314-:

(1) drawing of standard curve of glutamic acid

100mg of glutamic acid was accurately weighed, dissolved in water, and then sequentially diluted to the following concentrations: 40ug/mL, 80ug/mL, 160ug/mL, 240ug/mL, 320 ug/mL. Transferring 1mL of each concentration solution into a 25mL volumetric flask, adding 0.5mL of buffer solution and 0.5mL of ninhydrin developer, heating in boiling water for 15min, cooling, adding water to a constant volume of 25mL, standing for 10-15min, and measuring the absorbance (A) with a spectrophotometer in a 10mm cuvette at a wavelength of 570 nm. A is used as the ordinate, and the amino acid concentration is used as the abscissa for plotting, so that a standard curve can be drawn.

(2) Preparation and measurement of test solutions

Accurately weighing 3g of crushed tea sample, placing in a 500mL conical flask, adding 450mL of boiling water, leaching in a boiling water bath for 45min, shaking once every 10min, filtering under reduced pressure while hot, cooling, and diluting to 500 mL. 1mL of the test solution was placed in a 25mL volumetric flask, and then 0.5mL of the buffer and 0.5mL of the ninhydrin reagent were added in the same manner as above.

L₁Total amount of test solution, mL

L₂Amount of test solution for determination, mL

M₀Sample mass, g

C-number of milligrams of glutamic acid found on the standard curve according to absorbance

m-dry matter content of the sample,%

5. Determination of soluble sugar content

(1) Drawing of glucose standard curve

Weighing anhydrous glucose to prepare glucose standard solutions of 25, 50, 100, 150 and 200ug/mL respectively, sucking 1mL of glucose solutions with different solubilities respectively, dropping the glucose solutions into a volumetric flask in which 8mL of anthrone reagent is added in advance, using water as blank control, heating in boiling water for 7min, immediately taking out and carrying out ice bath to room temperature, and measuring absorbance by using a 10mm cuvette under the condition of a wavelength of 620 nm. And drawing a standard curve by taking the absorbance as the ordinate and the concentration as the abscissa.

(2) Preparation and measurement of test solution

Accurately weighing 1g of tea sample, placing in a 250mL conical flask, adding 80mL of boiling water, placing in a boiling water bath for leaching for 30min, immediately filtering, washing residues for several times by the boiling water, combining filtrates, adding water to 500mL, and shaking uniformly for later use. Taking 4 dry 25mL volumetric flasks, adding 8mL anthrone reagent into each volumetric flask, respectively dropwise adding 1mL test solution into three volumetric flasks, adding 1mL distilled water into the other flask, shaking uniformly, heating in a boiling water bath for 7min, immediately taking out, cooling in the ice bath to room temperature, and measuring the absorbance A by using a 10mm cuvette under the condition of a wavelength of 620 nm.

C-sugar content found on the Standard Curve (ug/mL)

V_{General assembly}Total volume of extract, mL

m-sample mass, mg

w-sample dry matter content (%)

6. Determination of theabrownin content

Accurately weighing 3g of ground tea sample, placing in a 250mL conical flask, adding 125mL of boiling water, placing in a boiling water bath, leaching for 10min, stirring for 2-3 times, filtering under reduced pressure when the solution is hot, and rapidly cooling to room temperature for use.

Placing 25mL of test solution and 25mL of n-butanol in a 100mL separating funnel, accurately shaking for 3min, standing for layering, placing the lower layer (water layer) in a 50mL conical flask, placing 2mL of water layer liquid in a 25mL volumetric flask, adding 2mL of saturated oxalic acid solution and 6mL of distilled water, and diluting to a constant volume with 95% ethanol to obtain a standard volume. The absorbance A was measured at a wavelength of 380nm using 95% ethanol as a control.

M: quality (g) of sample

w: dry matter content of sample (%)

Ab: absorbance of solution b

In order to prove the action mechanism of the strain of the invention, the inventor makes corresponding control experiments, wherein, CK groups are as follows: the blank group of the non-inoculated strains, group 1 was inoculated with only bacillus subtilis (CCTCC M2018288) X4, group 2 was inoculated with only aspergillus fumigatus (CCTCC M2018287) M1, and group 3 was the group of example 1 of the present invention, and the enzyme activities thereof were measured as shown in table 1.

TABLE 1 determination of enzyme activity content of semi-finished tea sample of sun-dried green tea by artificial pile fermentation

The results of enzyme activity data show that the enzyme activities of cellulase, pectinase and polyphenol oxidase are obviously lower than those of a mixed fermentation group during single-strain fermentation of groups 1 and 2, and thus a certain synergistic enhancement effect exists between bacillus subtilis X4 and aspergillus fumigatus M1 in the aspect of secreting the enzymes, for example, the yield of the polyphenol oxidase in the embodiment group is 1.72 times that of the single-strain fermentation of a strain M1, and the yield of the pectinase is increased by 0.52 times after the mixed fermentation of bacillus subtilis X4 and the strain M1. The exception is that mixed fermentation is unfavorable for the production of protease by the bacillus subtilis X4, because the protease content in the tea sample is obviously lower than that in single-strain fermentation of the bacillus subtilis X4 when the bacillus subtilis X4 and the strain M1 are fermented together, and the strain M1 can possibly inhibit the secretion of protease by the bacillus subtilis X4.

In order to study the influence of the artificial fermentation on the conversion of the components of the aged green tea, the contents of tea polyphenols, catechins, amino acids, soluble sugars and theabrownins in the tea under different fermentation conditions were measured. Tea polyphenols, also known as tea tannins or tea tannins, are a mixture of polyphenols present in tea plant and are a generic name for various phenolic derivatives in tea. The tea polyphenol has bitter taste in the tea soup and strong irritation; catechin is the main component of tea polyphenol, accounts for about 70% of total amount of tea polyphenol, and is one of the main substances forming color, aroma and taste of six kinds of tea; the astringency and the bitterness of the tea soup are obviously reduced in the pile solid state fermentation process, and the reason is mainly that the tea polyphenol and the catechin in the raw material sun-dried green raw tea are oxidized and condensed into the theabrownin. The amino acid is a main substance for making the tea fresh and cool in taste, and is also the most direct nitrogen source for the growth and the propagation of microorganisms on the tea. The theabrownin is brown pigment which can be dissolved in water but not dissolved in ethanol, ethyl acetate, n-butyl alcohol and chloroform, and is also a very complex non-dialyzing high polymer compound, and a certain amount of theabrownin content plays an important role in forming the unique quality of the green brick tea soup reddish brown.

TABLE 2 determination of the quality and composition of the semi-finished tea of the sun-dried green tea by manual pile-fermentation

Bit: %)

Wherein, CK group is: the blank group of the non-inoculated strains, group 1, group 2, group 3 and group 1 are groups of the invention, wherein the group is inoculated with only bacillus subtilis (CCTCC M2018288) X4, the group is inoculated with only aspergillus fumigatus (CCTCC M2018287) M1, and the group is inoculated with the strain of the invention.

After the completion of the artificial pile fermentation, the measurement results of the main quality components of the tea sample are shown in table 2, and compared with the CK of a control group which is not inoculated with any strain, the contents of tea polyphenol, catechin, amino acid and soluble sugar are reduced to different degrees except that the content of theabrownin is increased. In general, the change range of each index is small when the first group of bacillus subtilis X4 acts on the old green tea alone, which indicates that the influence on the biotransformation of the tea is small. The aspergillus fumigatus M1 for high yield of polyphenol oxidase can obviously reduce the content of tea polyphenol and catechin of the tea sample and improve the content of theabrownin, and the influence on the content of amino acid and soluble sugar of the tea sample is obviously higher than that of bacillus subtilis X4, which shows that the aspergillus fumigatus M1 has an important effect on the conversion of components of old green tea.

When the bacillus subtilis X4 and the aspergillus fumigatus M1 are fermented in a mixed manner, the contents of tea polyphenol, catechin and soluble sugar are obviously lower than those of single-bacterium fermentation; the content of tea-like amino acid is slightly higher than that of single-strain fermentation of aspergillus fumigatus M1; especially, the theabrownin content is 1.34 times of that of single-strain fermentation of the strain M1. Therefore, the mixed fermentation mode is more beneficial to the transformation of the main biochemical components of the green brick tea than single-bacterium fermentation, and the bacillus subtilis X4 can enhance the biotransformation effect of the strain M1 on old green tea. Compared with the pile tea samples in the Zhao Li Qiao tea factory in Hubei province, the results are shown in Table 3, and the main quality indexes of the tea samples in the manual pile fermentation 6d are basically equivalent to those of the tea samples in the natural pile fermentation for 35 days. In conclusion, the bacillus subtilis X4 and the aspergillus fumigatus M1 are core flora for quick pile fermentation of the green brick tea.

Compared with the natural tea sample of the natural pile fermentation 35d in the Zhao Li Qiao tea factory in Hubei province, the result shows that the main quality indexes of the tea sample of the artificial pile fermentation 6d carried out by using the bacillus subtilis X4 and the aspergillus fumigatus M1 (the number 3) are the closest to the main quality indexes. Particularly, the tea sample obtained by the co-pile fermentation of the bacillus subtilis X4 and the bacillus subtilis M1 has higher theabrownin content than a natural pile semi-finished product tea sample in a tea factory, and the theabrownin is an important physiologically active substance in the green brick tea, so that the high theabrownin content is beneficial to further improvement of the quality of the green brick tea.

TABLE 3 determination of the content of the semi-finished tea components of the sun-dried raw tea by the natural pile fermentation (unit:%)

According to the invention, pure-strain pile fermentation is carried out by adopting two strains of bacillus subtilis and aspergillus fumigatus, compared with the traditional open microbial natural pile fermentation process in the utilization environment, the time can be shortened by about 4 weeks, and the method is not easily polluted by other bacteria, and is beneficial to increasing enterprise benefits and improving and stabilizing product quality. The two microorganisms preserved by the method are dominant microorganisms in the green brick tea fermentation, so that the fermentation speed is improved, the components and the flavor of the traditional green brick tea are highly simulated, and favorable conditions are provided for the industrial production of the green brick tea.

The inventor also used other aspergillus fumigatus and bacillus subtilis to mix simultaneously, all can't reach the effect of quick fermentation, and the general fermentation is very slow, can't reach the effect of speeding up. The screened bacterial strains have strong adaptability and synergistic effect on the mixture of the sun-dried green raw tea and water, and if a single bacterial strain cannot realize the invention, other similar bacterial strains cannot realize the quick, stable and high-quality fermentation sun-dried green raw tea of the invention.

Description of the drawings:

FIG. 1: the invention deposits the colony characteristic diagram of bacillus subtilis.

FIG. 2: the invention provides a colony characteristic map of the preserved aspergillus fumigatus.

Detailed Description

The invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to limit its scope, which after reading the present invention, is susceptible of modification in various equivalent forms by those skilled in the art, all falling within the scope of the invention as defined in the appended claims.

Culturing at 45 +/-3 ℃ by adopting a plate separation method, separating and purifying two core strains which are respectively X4 Bacillus subtilis and M1 Aspergillus fumigatus from pile tea samples of a Leqiao tea factory in Hubei Zhao province, wherein the strains are preserved in the China center for type culture collection in 5 and 17 months in 2018, and the preservation number of the M1 Aspergillus fumigatus is as follows: CCTCC M2018287, wherein the preservation number of the X4 Bacillus subtilis is as follows: CCTCC M2018288. And (4) storage address: wuhan university in Wuhan city, Hubei province, zip code: 430072, telephone: 027-68754052.

The colony characteristics of the bacillus subtilis (CCTCC M2018288) preserved by the invention are as follows: the bacterial colony is medium in size, the surface of the bacterial colony is light yellow, the surface of the bacterial colony is rough and has wrinkles, the edge of the bacterial colony is irregular, the surface of the bacterial colony is dry, opaque and lackluster, gram reaction is positive, no capsule exists, a single bacterial cell is rod-shaped, two ends of the single bacterial cell are blunt and round, and cysts do not expand obviously.

The colony characteristics of the aspergillus fumigatus (CCTCC M2018287) preserved by the invention are as follows: the bacterial colony is circular, the diameter is large, the surface of the bacterial colony is villous, the center is light cyan, the edge is grey white, the surface is dry and non-transparent, hyphae have partitions, the conidium is spherical, and the top of the conidium peduncle forms an expanded spherical top sac.

Other reagents used in the embodiment of the invention are analytically pure.

The present invention is described in detail below with reference to examples.

Example 1

The embodiment provides a method for quick pile fermentation of green brick tea, which can quickly ferment green brick tea, and has the same main quality and chemical components as the traditional green brick tea, and the time is shortened by 80%. The pile fermentation method comprises the following steps:

1. preparing bacillus subtilis liquid: the bacillus subtilis (CCTCC M2018288) preserved by the method is placed in an NA culture medium for culture, after the bacillus subtilis is cultured for 2-36 hours at the temperature of 45 +/-3 ℃, the somatic cells of the bacillus subtilis are dispersed, and then the bacillus subtilis is suspended until the bacterial cells reach 10 DEG C^6-10⁷And (4) obtaining bacillus subtilis liquid per mL. Wherein the NA culture medium is: 3g/L beef extract and 10g/L, NaCl 5g/L peptone.

2. Preparing an aspergillus fumigatus liquid: placing the preserved aspergillus fumigatus (CCTCC M2018287) in a PDA culture medium, culturing for 2-36 h at 45 +/-3 ℃, dispersing into 0.2-0.4cm of mycelium segments, then suspending, and 10^6-10⁷The individual hypha segments/mL to obtain the cigaretteAspergillus liquid. Wherein, the PDA culture medium is: 10g/L of potato extract powder and 20g/L of glucose.

3. Preparing a pile fermentation broth: and mixing the aspergillus fumigatus liquid and the bacillus subtilis liquid according to the ratio of 1:1 to obtain the pile-fermentation mixed bacteria fermentation liquid.

4. And (3) sterilizing the sun-dried raw tea: mixing sun-dried green tea and water at a ratio of 5:2(W/V), placing into a pile fermentation container (500 ml triangular flask in this example), and sealing and sterilizing at 105 deg.C for 40 min.

5. Pile fermentation: inoculating the pile fermentation broth prepared by the invention with an inoculation amount of 10% (V/W), and aerobically culturing for 6 days at 45 +/-3 ℃, wherein 5-10% (V/W) of sterile water is added when the time is 2-5 days, and a sun-dried crude tea sample is turned over, and is taken out after 6 days to obtain a green brick tea semi-finished product.

And piling, copying, steaming, pressing, cooling and drying the green brick tea semi-finished product by a large traditional method to obtain a green brick tea finished product.

Example 2

1. preparing bacillus subtilis liquid: the bacillus subtilis (CCTCC M2018288) preserved by the method is placed in an NA culture medium for culture, after the bacillus subtilis is cultured for 2-36 hours at the temperature of 45 +/-3 ℃, the somatic cells of the bacillus subtilis are dispersed, and then the bacillus subtilis is suspended until the bacterial cells reach 10 DEG C^6-10⁷And (4) obtaining bacillus subtilis liquid per mL. Wherein the NA culture medium is: a mixed solution of 4g/L beef extract and 15g/L, NaCl 3g/L peptone.

2. Preparing an aspergillus fumigatus liquid: placing the preserved aspergillus fumigatus (CCTCC M2018287) in a PDA culture medium, culturing for 2-36 h at 45 +/-3 ℃, dispersing into 0.2-0.4cm of mycelium segments, and then suspending until the length is 10%^6-10⁷And (4) obtaining aspergillus fumigatus liquid by each mycelium section/mL. Wherein said PDA cultureThe base is as follows: 15g/L of potato extract powder and 25g/L of glucose.

3. Preparing a pile fermentation broth: and mixing the aspergillus fumigatus liquid and the bacillus subtilis liquid according to the proportion of 1:0.5 to obtain the pile fermentation liquid.

4. And (3) sterilizing the sun-dried raw tea: mixing sun-dried green tea and water at a ratio of 4:1(W/V), placing into a pile fermentation container (500 ml triangular flask), and sealing and sterilizing at 100 deg.C for 40 min.

5. Pile fermentation: inoculating the pile fermentation broth prepared by the invention with an inoculation amount of 5% (V/W), and carrying out aerobic culture for 5 days at 45 +/-3 ℃, wherein 5-10% (V/W) of sterile water is added when the time is 2-4 days, and a sun-dried crude tea sample is turned over, and is taken out after 6 days, so as to obtain a green brick tea semi-finished product.

Example 3

1. preparing bacillus subtilis liquid: the bacillus subtilis (CCTCC M2018288) preserved by the method is placed in an NA culture medium for culture, after the bacillus subtilis is cultured for 2-36 hours at the temperature of 45 +/-3 ℃, the somatic cells of the bacillus subtilis are dispersed, and then the bacillus subtilis is suspended until the bacterial cells reach 10 DEG C^6-10⁷And (4) obtaining bacillus subtilis liquid per mL. Wherein the NA culture medium is: 5g/L beef extract and 12g/L, NaCl 9g/L peptone.

2. Preparing an aspergillus fumigatus liquid: placing the preserved aspergillus fumigatus (CCTCC M2018287) in a PDA culture medium, culturing for 2-36 h at 45 +/-3 ℃, dispersing into 0.2-0.4cm of mycelium segments, and then suspending until the length is 10%^6-10⁷And (4) obtaining aspergillus fumigatus liquid by each mycelium section/mL. Wherein, the PDA culture medium is: 4g/L potato extract powder and grapeSugar 15 g/L.

3. Preparing a pile fermentation broth: and mixing the aspergillus fumigatus liquid and the bacillus subtilis liquid according to the proportion of 1:3, and mixing to obtain the pile-fermentation mixed bacteria fermentation liquid.

4. And (3) sterilizing the sun-dried raw tea: mixing the sun-dried green tea and water at a ratio of 2:1(W/V), placing into a pile fermentation container (500 ml triangular flask), and sealing and sterilizing at 121 deg.C for 20 min.

5. Pile fermentation: inoculating the pile fermentation broth prepared by the invention with an inoculation amount of 20% (V/W), and carrying out aerobic culture for 7 days at 45 +/-3 ℃, wherein 5-10% (V/W) of sterile water is added when the fermentation broth is cultured for 2-6 days, and a sun-dried crude green tea sample is turned over and taken out after 6 days, so as to obtain a green brick tea semi-finished product.

Claims

1. A method for quick pile fermentation of green brick tea is characterized by comprising the following steps: the green brick tea pile fermentation method comprises the following steps:

mixing the sun-dried green raw tea and water according to the mass-volume ratio of 2: 1-4: 1, then filling the mixture into a pile fermentation container, and sterilizing;

inoculating the pile fermentation broth into a sterilized pile fermentation container according to the inoculation amount of 5-20% V/W, and carrying out aerobic culture for 5-7 d at the temperature of 45 +/-3 ℃;

placing Bacillus subtilis with CCTCC M2018288 in NA liquid culture medium, culturing at 45 + -3 deg.C, dispersing thallus cells, and suspending to obtain Bacillus subtilis liquid, wherein the thallus cells in the Bacillus subtilis liquid are 10⁶~10⁷Per mL;

putting Aspergillus fumigatus of CCTCC M2018287 in a PDA liquid culture medium, culturing at 45 +/-3 ℃, dispersing thallus cells, and performing suspension treatment to obtain Aspergillus fumigatus liquid, wherein a mycelium section in the Aspergillus fumigatus liquid is10⁶~10⁷Per mL;

and mixing the aspergillus fumigatus liquid and the bacillus subtilis liquid according to the volume ratio of 2: 1-1: 3, and mixing to obtain the pile-fermentation mixed bacteria fermentation liquid.

2. The method for rapid pile fermentation of green brick tea according to claim 1, characterized in that: the sterilization treatment is performed for 20-40 min at the temperature of 100-121 ℃.

3. The method for rapid pile fermentation of green brick tea according to claim 1, characterized in that: the culture time of the bacillus subtilis in the NA liquid culture medium is 2-36 h.

4. The method for rapid pile fermentation of green brick tea according to claim 1, characterized in that: the culture time of the aspergillus fumigatus in the PDA liquid culture medium is 2-36 h, and the mycelium segments are dispersed to be 0.2-0.4 cm.

5. The method for rapid pile fermentation of green brick tea according to claim 1, characterized in that: the NA culture medium comprises the following components: beef extract: 3-5 g/L, peptone: 10-15 g/L, NaCl: 3-9 g/L, and the balance being sterile water.

6. The method for rapid pile fermentation of green brick tea according to claim 1, characterized in that: the PDA culture medium comprises the following components: potato leaching powder: 4-15 g/L, 15-25 g/L glucose and the balance of sterile water.