阅读说明：本技术 一种高茶褐素茶珍及其制备工艺 (High-tea-brown-element tea extract and preparation process thereof ) 是由 胥伟 廖思雨 赵以桥 于 2021-08-21 设计创作，主要内容包括：一种利用绿茶副茶生产高茶褐素速溶茶珍的方法。将绿茶副茶投入60倍量室温水中沸水浴浸提30分钟,过滤、定容获得茶汤。茶汤经2次纱布过滤、1次滤纸过滤,冷却至室温,随后过0.22um除菌膜除菌得初始无菌茶汤。适量刮取发酵真菌菌株塔宾曲霉菌株M-2的孢子于无菌水中,不断调整至孢子悬浮液浓度为1.0×10～(6)CFU/mL,即获得接种液。按2％的体积比将接种液移入初始无菌茶汤中,室温下以80r/min的速度振荡培养5天获得发酵茶汤,将发酵茶汤过滤除菌后经真空冷冻干燥,干燥后的固形物经干冰研磨即可获得成品。本速溶茶珍具有溶解迅速彻底、茶褐素含量高、汤色明艳透亮等优点,是一种具有生物活性的新型便携茶叶固体饮料,具有良好的保健功效。(A method for producing instant tea powder with high theabrownin content from side tea of green tea is provided. Putting the auxiliary green tea into water with the volume 60 times of the room temperature, leaching in a boiling water bath for 30 minutes, filtering, and fixing the volume to obtain the tea soup. Filtering the tea soup with gauze for 2 times, filtering with filter paper for 1 time, cooling to room temperature, and sterilizing with 0.22um sterilizing membrane to obtain the final product. Scraping spores of fermentation fungus strain Aspergillus tubingensis strain M-2 into sterile water, and continuously adjusting to spore suspension concentration of 1.0 × 10 6 CFU/mL, namely obtaining inoculation liquid. Transferring the inoculation liquid into the initial sterile tea soup according to the volume ratio of 2%, performing shaking culture at the speed of 80r/min for 5 days at room temperature to obtain fermented tea soup, filtering and sterilizing the fermented tea soup, performing vacuum freeze drying, and grinding the dried solid matter with dry ice to obtain the finished product. The instant tea has the advantages of rapid and thorough dissolution, high theabrownin content, and bright and transparent soup colorThe tea solid beverage has the advantages of brightness, and the like, is a novel portable tea solid beverage with bioactivity, and has good health care effect.)

1. A preparation process of high theabrownin tea extract comprises the following steps:

extracting the auxiliary green tea with water for 30min, filtering, cooling, and sterilizing to obtain sterile tea soup;

inoculating bacteria liquid to the initial sterile tea soup, and fermenting to obtain fermented tea soup; and

the fermented tea soup is pre-frozen, then is subjected to vacuum freeze drying, and is ground by dry ice to obtain the high-theabrownin tea delicacies.

2. The production process according to claim 1, wherein:

the secondary tea of green tea includes, but is not limited to, tea flakes, tea dust and tea capitata.

3. The production process according to claim 1, wherein:

the mass volume ratio of the auxiliary green tea to the room temperature water is 1:60, boiling water bath is carried out for 30min, and initial tea soup is obtained by filtering.

4. The production process according to claim 1, wherein:

the filtration comprises three times of filtration, which are respectively as follows: gauze filtration, and filter paper filtration.

5. The production process according to claim 1, wherein:

the sterilization treatment comprises the following steps: the initial tea broth was sterilized through a 0.22um filter.

6. The production process according to claim 1, wherein:

the inoculation liquid is prepared by the following method:

the Yaan dark green tea pile fermentation sample is taken as a raw material, the tea leaves are soaked in sterile water and then spread on a PDA flat plate, the constant temperature culture is carried out at 28 ℃, and the fungus strain is obtained through separation and purification;

inoculating the purified fungus strain into PDB culture medium, performing shake culture at 28 deg.C and 120r/min for 2 days, freezing mycelium pellet in liquid nitrogen, and rapidly grinding into powder;

extracting genome DNA of the strain, amplifying ITS and beta-tubulin sequences by PCR, storing and sequencing the amplification product at 4 ℃, and determining the species name of the fungus strain by combining the two sequence information;

screening out strains which have the prior toxin production and generate adverse reaction reports to human bodies, fermenting the initial tea soup by using the residual purified strains, detecting ochratoxin and citrinin in the fermented tea soup, and screening out the toxin production strains;

measuring the tea pigment in the fermented tea soup of the strains obtained after the second screening, analyzing the capability of each strain for converting the tea pigment, and reserving the strains with high yield of the tea pigment, namely the fermentation strain Aspergillus tubingensis strain M-2;

the A.tubingensis strain M-2 was activated and prepared in a spore suspension concentration of 1.0X 10⁶CFU/mL inoculation solution.

7. The production process according to claim 1, wherein:

the inoculation amount of the inoculation liquid is 2% of the volume of the sterilized tea soup.

8. The production process according to claim 1, wherein:

the fermentation treatment comprises the following steps:

culturing at room temperature at 80r/min under shaking for 5 days, and filtering to remove mycelium pellet.

9. The production process according to claim 1, wherein:

the low-temperature freeze drying conditions are as follows:

pre-freezing the fermented tea soup at-20 deg.C, transferring into vacuum freeze-drying machine, and drying at-40 deg.C under vacuum condition until water in the fermented tea soup is completely lost;

fully grinding the dried solid matter by using dry ice until fine powder is obtained; and

collecting the powder to obtain the high-theabrownin tea.

10. A high theabrownin tea delicacy prepared according to the preparation process of any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of preparation of instant tea delicacies, and relates to a preparation process of high-theabrownin tea delicacies and high-theabrownin tea delicacies prepared by the preparation process.

Background

The instant tea is a powdered tea material prepared by using black tea, green tea, scented tea or oolong tea as raw materials and processing the raw materials through the technical processes of extraction, filtration, concentration (and drying) and the like, and can be widely applied to foods and tea beverages in different forms. A portable solid tea beverage having tea flavor contains tea polyphenols, theanine, caffeine, etc. The instant tea can adapt to the fast-paced life of the modern times, has strong plasticity and convenient allocation, and is more and more popular in the market.

The secondary green tea is a general term of tea flakes, tea dust, tea stem and the like formed in the processing process of green tea, accounts for about 10% of the finished tea, has the characteristics of poor content ratio, low fragrance, light and thin taste, peculiar smell and the like, is difficult to sell through the traditional tea consumption channel, and is a main byproduct generated in the processing process of the tea. The secondary green tea is one of the main raw materials for deep processing of tea due to the characteristics of similar content to that of the traditional finished tea, low price, easy obtainment and the like. If the secondary green tea is used as a raw material, the deep processing and utilization approaches of the secondary green tea are expanded, which has great significance for the sustainable development of the tea industry.

The tea pigment is a water-soluble pigment mixture derived from oxidation of polyphenol compounds mainly comprising catechin in tea, and comprises theaflavin, thearubigin and theabrownin. The studies show that theabrownin is the most stable and the bioactivity is the greatest. Particularly, theabrownin has the function of remarkably inhibiting protein tyrosine phosphatase, and can be used for treating and preventing type II diabetes, obese diabetes and obesity; has effects in regulating blood lipid, resisting atherosclerosis, removing speckle, caring skin, preventing cancer, and preventing ultraviolet irradiation; has strong free radical scavenging activity and antioxidant effect; has no toxic and side effects, and is safe and reliable to take.

The existing theabrownin extraction method mainly comprises an extraction method and a microbial fermentation method. Wherein CN102334574 discloses an extraction method, and the optimal yield of theabrownin extracted by an organic solvent can reach 22.92%. CN101455257 discloses a microbial fermentation method, which states that the extract of the method has a theabrownin content of 14.50-18.28% by chemical composition analysis. CN102258099 also discloses a microbial fermentation process. The patent states that the theabrownin content of the extract obtained by the method is (60.45 +/-1.5)%, by chemical composition analysis. The method has the defects of low theabrownin extraction rate, high extraction cost, long preparation time, easy pollution by aspergillus, excessive use of organic solvents and the like.

Based on the analysis, an instant tea beverage which can make full use of the side tea of green tea and has high theabrownin content is urgently needed in the industry at present. Not only can fully utilize the byproducts of the tea, but also can reduce the production cost.

Disclosure of Invention

Theabrownines are generally mainly extracted from dark tea, but most of the dark tea is prepared by adopting the traditional solid-state' pile fermentation process at present, and the fermentation process has the problems of low production efficiency, serious restriction by natural conditions, long production period, uneven theaabrownines content, easy pollution of mixed bacteria in the production process and the like. In view of the above disadvantages, the present invention aims to provide a method for preparing an instant tea beverage with high theabrownin content, which uses the secondary green tea as the main raw material, has the advantages of simple production process, easily controllable production conditions, low fermentation cost, and capability of significantly increasing the theabrownin content, and is suitable for industrial scale production.

The invention is realized by the following means:

a preparation process of high theabrownin tea extract comprises the following steps:

leaching the secondary green tea in boiling water bath for 30min, filtering, cooling and sterilizing to obtain initial sterile tea soup;

inoculating bacteria liquid to the initial sterile tea soup, and fermenting to obtain fermented tea soup; and

and pre-freezing the fermented tea soup, performing low-temperature vacuum drying, and grinding by dry ice to obtain the high-theabrownin tea delicacies.

Further, the secondary green tea includes, but is not limited to, tea flakes, tea dust, and tea capitata.

Further, the mass volume ratio of the auxiliary green tea to the room temperature water is 1:60, boiling water bath is carried out for 30min, and initial tea soup is obtained by filtering; the pH value of the initial tea soup is natural.

Further, the filtering comprises three times of filtering, which are respectively: gauze filtration, and filter paper filtration.

Further, the sterilization treatment comprises the following steps: the initial tea broth was sterilized by filtration through a 0.22 μm filter.

Further, the inoculation liquid is prepared by the following method:

the Yaan dark green tea pile fermentation sample is taken as a raw material, the tea leaves are soaked in sterile water and then spread on a PDA flat plate, the constant temperature culture is carried out at 28 ℃, and the fungus strain is obtained through separation and purification;

inoculating the purified fungus strain into a PDB culture medium, carrying out shaking culture at 28 ℃ and 120r/min for 2 days, taking mycelium pellets, freezing in liquid nitrogen, quickly grinding into powder, extracting the genomic DNA of the strain according to a fungus genomic DNA extraction kit provided by Beijing Solebao science and technology Co., Ltd, and carrying out PCR amplification on ITS and beta-tubulin sequences. Storing the amplified product at 4 ℃, sending the amplified product to a biological engineering (Shanghai) corporation for sequencing, carrying out homology comparison (http:// www.ncbi.nlm.nihgov/blast.cgi) on the sequencing result and sequences in a GenBank database, selecting the sequences with the homology of more than 99 percent, constructing a phylogenetic tree by using MEGA 7.0 software, and determining the category of the fungal strain;

on the basis, strains are screened, and the specific process is as follows: screening out strains which have the prior toxin production and produce adverse reaction reports on human bodies; fermenting the initial tea soup by using the residual purified strains, respectively detecting ochratoxin and citrinin in the fermented tea soup by referring to national standard GB 5009.96-2016 (determination of ochratoxin A in national food safety standards) and national standard GB 5009.222-2016 (determination of citrinin in national food safety standards), and screening out toxigenic strains; measuring the tea pigment in the fermented tea soup of the strains obtained after the second screening, analyzing the capability of each strain for converting the tea pigment, and reserving the strains with high yield of the theabrownin, namely the fermented strains;

determining polyphenol oxidase activity of the fermentation strain by measuring the content of theabrownin in the fermented tea soup, and sequentially measuring the activities of starch hydrolase, protein hydrolase and tannin hydrolase of the fermentation strain by adopting an amylase screening culture medium, a protease screening culture medium and a tannin enzyme screening culture medium;

the Aspergillus tubingensis strain M-2 is obtained by screening, has the characteristics of stable heredity, no toxicity, safety, reliability, high activity of hydrolase and oxidase and the like, and is an excellent fermentation strain;

the A.tubingensis strain M-2 was activated and prepared in a spore suspension concentration of 1.0X 10⁶CFU/mL inoculation solution.

Further, the inoculation amount of the inoculation liquid is 2% of the volume of the sterilized tea soup.

Further, the fermentation treatment comprises: culturing at room temperature at 80r/min under shaking for 5 days, and filtering to remove mycelium pellet.

Further, the low-temperature freeze drying conditions are as follows: freezing the fermented tea soup in a refrigerator at-20 deg.C, transferring into a vacuum freeze-drying machine, drying at vacuum state (about 1pa) and-40 deg.C until water in the fermented tea soup is completely lost, rapidly and fully grinding the dried solid with dry ice to obtain fine powder, and collecting the powder to obtain the high-theabrownin tea beverage.

The invention also discloses the high-theabrownin tea delicacies prepared by any one of the preparation processes.

The invention has the beneficial effects that:

1. the fermentation fungus Aspergillus tubingensis strain M-2 is obtained by separating, purifying and screening a big pile sample produced by the traditional black tea, has the characteristics of no toxicity production, safety, reliability, high hydrolase and oxidase activity and the like, can fully utilize the tea content substances for metabolism, can quickly convert the tea content substances and the like, is the best fermentation strain which is screened from hundreds of fungi and has the capability of preparing instant tea juice by fermenting the side tea soup of the green tea, and the catechin of the fermented tea soup is fully converted into theabrownin.

2. The tea soup prepared by fermenting the green tea side tea by using the fermentation fungus Aspergillus tubingensis strain M-2 is quickly converted to obtain the beneficial nutrient component of theabrownin, and the tea soup is treated by a vacuum freeze drying technology, so that the tea soup contains primary and secondary metabolic active products of Aspergillus tubingensis, and has good health care effects on the aspects of digestion elimination, greasiness removal and gastrointestinal function conditioning. The fermented instant tea beverage has rich theabrownin content, and has health promotion effects of regulating blood lipid and preventing cardiovascular diseases.

3. The green tea raw materials in the invention have wide sources and low price, and the prepared instant tea powder has high theabrownin yield and strong biological activity. The fermentation process flow is simple, the operation is controllable, no harmful solvent is left, the preparation technology is green and energy-saving, and the sanitation index and the physical and chemical index of the product are high.

Detailed description of the preferred embodiments

Example 1

Adding tea auxiliary tea (including tea tablet, tea powder, tea of fructus Toosendan, etc.) into water, wherein the ratio of tea to water is 1:60 (g/mL), natural pH value, 30 minutes of boiling water bath extraction, and obtaining the initial tea soup by three times of filtration (gauze filtration, gauze filtration and filter paper filtration) when the extraction is finished. Sterilizing the initial tea soup cooled to room temperature with 0.22um filter membrane to obtain the initial sterilized tea soup. Activating the selected Aspergillus tubingensis strain M-2 with high hydrolase and oxidase activity, and making into spore suspension with concentration of 1.0 × 10⁶CFU/mL inoculation solution. Inoculating the inoculation liquid into the initial sterilized tea soup according to 2% of the volume of the initial sterilized tea soup, carrying out oscillation culture at the speed of 80r/min for 5 days at room temperature, carrying out suction filtration to remove mycelium clusters, thus obtaining the high-theabrownin fermented tea soup, completely freezing the high-theabrownin fermented tea soup in a refrigerator at the temperature of-20 ℃, transferring the high-theabrownin fermented tea soup into a vacuum freeze dryer, drying the high-theabrownin fermented tea soup at the temperature of vacuum state (about 1pa) and-40 ℃ until the water in the fermented tea soup is completely lost, rapidly and fully grinding the dried solid matter with dry ice to obtain fine powder, and collecting the powder, thus obtaining the high-theabrownin tea soup.

Specifically, the inoculation liquid used in the embodiment is prepared by the following method:

the Yaan dark green tea pile fermentation sample is taken as a raw material, the tea leaves are soaked in sterile water and then spread on a PDA flat plate, the constant temperature culture is carried out at 28 ℃, and the fungus strain is obtained through separation and purification;

inoculating the purified fungus strain into a PDB culture medium, carrying out shaking culture at 28 ℃ and 120r/min for 2 days, taking mycelium pellets, freezing in liquid nitrogen, quickly grinding into powder, extracting the genomic DNA of the strain according to a fungus genomic DNA extraction kit provided by Beijing Solebao science and technology Co., Ltd, and carrying out PCR amplification on ITS and beta-tubulin sequences. Storing the amplified product at 4 ℃, sending the amplified product to a biological engineering (Shanghai) corporation for sequencing, carrying out homology comparison (http:// www.ncbi.nlm.nihgov/blast.cgi) on the sequencing result and sequences in a GenBank database, selecting the sequences with the homology of more than 99 percent, constructing a phylogenetic tree by using MEGA 7.0 software, and determining the category of the fungal strain;

on the basis, strains are screened, and the specific process is as follows: screening out strains which have the prior toxin production and produce adverse reaction reports on human bodies; fermenting the initial tea soup by using the residual purified strains, respectively detecting ochratoxin and citrinin in the fermented tea soup by referring to national standard GB 5009.96-2016 (determination of ochratoxin A in national food safety standards) and national standard GB 5009.222-2016 (determination of citrinin in national food safety standards), and screening out toxigenic strains; measuring the tea pigment in the fermented tea soup of the strains obtained after the second screening, analyzing the capability of each strain for converting the tea pigment, and reserving the strains with high yield of the theabrownin, namely the fermented strains;

determining polyphenol oxidase activity of the fermentation strain by measuring the content of theabrownin in the fermented tea soup, and sequentially measuring the activities of starch hydrolase, protein hydrolase and tannin hydrolase of the fermentation strain by adopting an amylase screening culture medium, a protease screening culture medium and a tannin enzyme screening culture medium;

the Aspergillus tubingensis strain M-2 is obtained by screening, has the characteristics of stable heredity, no toxicity, safety, reliability, high activity of hydrolase and oxidase and the like, and is an excellent fermentation strain;

the A.tubingensis strain M-2 was activated and prepared in a spore suspension concentration of 1.0X 10⁶CFU/mL inoculation solution.

Comparative example 1

Compared with the example 1, the fermentation step is eliminated, and the method specifically comprises the following steps:

adding tea auxiliary tea (including tea tablet, tea powder, tea of fructus Toosendan, etc.) into water at a tea-water ratio of 1:60 (g/mL), pH 7.0, leaching in a boiling water bath for 30 minutes, and filtering for three times (gauze filtration, gauze filtration and filter paper filtration) when the leaching is finished to obtain the initial tea soup. Sterilizing the initial tea soup cooled to room temperature with 0.22um filter membrane to obtain the initial sterilized tea soup. Completely freezing the initial sterilized tea soup in a refrigerator at-20 deg.C, transferring into a vacuum freeze-drying machine, drying at vacuum state (about 1pa) and-40 deg.C until the water in the fermented tea soup is completely lost, rapidly and fully grinding the dried solid with dry ice to obtain fine powder, and collecting the powder to obtain the high-theabrownin tea.

Test example 1

Comparing the theabrownin content and sensory evaluation of the two fermented tea soups

TABLE 1 Theabrownine content determination and sensory evaluation results in fermented tea soup

Grouping	Theabrownin (%)	Fragrance	Color and luster of tea soup	Taste of the product
					Example 1	18.98	Sweet, old and tea fragrance	Brownish red and bright	Sweet and tasty
Comparative example 1	7.16	Tea fragrance with slight sweet smell	Deep apricot yellow and lack of brightness	Sweet and tasty

As can be seen from the data in Table 1, the theabrownin content of example 1 is significantly higher than that of the control group, and meanwhile, the instant tea extract prepared by using the Aspergillus tubingensis strain M-2 shows instant property after being brewed in cold and hot water, and has better fragrance and taste than the control group, obvious old fragrance, sweet fragrance and tea fragrance, and no odor and mildew taste. The vacuum freeze drying does not damage the substances in the fermented tea soup, and the finished product of the tea treasure contains the primary and secondary metabolic active products of the aspergillus tubingensis, thus the tea treasure is a novel solid tea drink with health care efficacy.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.