阅读说明：本技术 一种酿酒酵母和乳酸杆菌在普洱茶发酵中的应用 (Application of saccharomyces cerevisiae and lactobacillus in fermentation of Pu' er tea ) 是由 伍晓斌 孙曌月 李晶 吴杰 于怡 于 2021-08-12 设计创作，主要内容包括：本发明提供一种酿酒酵母和乳酸杆菌在普洱茶发酵中的应用,通过从普洱茶渥堆中期茶样中筛选出酿酒酵母和乳酸杆菌,并应用于普洱茶的发酵中,极大的加快普洱茶的发酵速度,并且在发酵结束后茶叶中生物活性物质的含量显著性增高,抗氧化能力明显增加,提升了普洱茶的品质并增强其稳定性,使得发酵后普洱茶中功能性物质的含量更高,对于提升普洱茶品质并增强其安全性和稳定性具有重要意义。(The invention provides an application of saccharomyces cerevisiae and lactobacillus in fermentation of Pu ' er tea, which is characterized in that the saccharomyces cerevisiae and the lactobacillus are screened from a tea sample at the middle stage of Pu ' er tea pile fermentation and are applied to the fermentation of the Pu ' er tea, the fermentation speed of the Pu ' er tea is greatly accelerated, the content of bioactive substances in tea leaves after the fermentation is finished is remarkably increased, the oxidation resistance is remarkably increased, the quality of the Pu ' er tea is improved, the stability of the Pu ' er tea is enhanced, the content of functional substances in the fermented Pu ' er tea is higher, and the method has important significance for improving the quality of the Pu ' er tea and enhancing the safety and the stability of the Pu ' er tea.)

1. An application of Saccharomyces cerevisiae and lactobacillus in fermentation of Pu' er tea is provided.

2. The use of claim 1, wherein the saccharomyces cerevisiae and the lactobacillus are respectively placed in a culture solution to be cultured to obtain a saccharomyces cerevisiae seed solution and a lactobacillus seed solution, and the saccharomyces cerevisiae seed solution and the lactobacillus seed solution are inoculated in a Pu 'er tea raw material to carry out Pu' er tea fermentation.

3. The use of claim 2, wherein the total volume of the saccharomyces cerevisiae seed solution and the lactobacillus seed solution is 10% of the total mass of the pu-erh tea raw material.

4. The use of claim 3, wherein the volume ratio of the Saccharomyces cerevisiae seed solution to the Lactobacillus seed solution is 1: 1.

5. the use according to claim 2, wherein the Pu' er tea raw material has a water content of 20-30%.

6. Use according to claim 2, wherein the fermentation temperature is 35-50 ℃.

7. The use according to claim 2, wherein the water content of the Pu 'er tea raw material is maintained at 20-30% during the fermentation of Pu' er tea.

8. The use according to any one of claims 1 to 7, wherein the Saccharomyces cerevisiae and Lactobacillus are prepared by:

and collecting a Pu' er tea pile fermentation middle-stage sample, crushing and grinding the sample, adding sterile water, oscillating the sample, collecting supernatant, coating the supernatant on a tea substrate culture medium, and performing purification culture to obtain the saccharomyces cerevisiae and the lactobacillus.

9. The use according to claim 8, wherein the tea-based medium is any one of YPD solid medium, PDA solid medium, LB solid medium and NA solid medium supplemented with tea powder, and the tea powder is obtained by grinding and sieving Pu' er tea raw material.

10. The use according to claim 8, wherein the incubation temperature is 30-40 ℃ for 3-4 days.

Technical Field

The invention relates to application of saccharomyces cerevisiae and lactobacillus in fermentation of Pu' er tea, and relates to the technical field of biology.

Background

In recent years, in the field of food fermentation, Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Lactobacillus (Lactobacillus) have been widely used. The saccharomyces cerevisiae is mainly applied to the fermentation industry of alcoholic beverages such as white spirit, beer, fruit wine and the like and the fermentation of flour foods such as bread, steamed bread and the like. In addition, the saccharomyces cerevisiae contains high protein, trace elements, vitamins and the like in thalli, and is also prepared into auxiliary materials to be applied to the fields of medical care and feed production. The lactobacillus is mainly applied to industrial fermentation of lactic acid beverages, fermented milk, ethanol and the like.

In the traditional fermentation process of the Pu 'er raw tea, because endophytes in tea bodies need to be propagated in a proper damp and hot environment, a large amount of enzymes are generated to catalyze and convert bioactive substances in the tea leaves, and the Pu' er ripe tea can be generated after 5-7 weeks of fermentation. However, in the modern Pu' er tea fermentation process, people pay more attention to the color, the smell and the taste of the fermented tea leaves, and neglect the nutrition, the health care function, the safety and the stability of the fermented tea leaves. Therefore, more and more attention is paid to how to provide a fermentation strain applied to the Pu ' er tea industry, improve the quality of the Pu ' er tea and enhance the safety and stability of the Pu ' er tea.

Disclosure of Invention

The invention provides an application of saccharomyces cerevisiae and lactobacillus in fermentation of Pu' er tea, which is used for overcoming the problems of long fermentation period, no attention on color, aroma, taste, nutrition, health care function, safety, stability and the like of fermented tea in the prior art.

The invention provides application of saccharomyces cerevisiae and lactobacillus in fermentation of Pu' er tea.

Further, respectively placing the saccharomyces cerevisiae and the lactobacillus into a culture solution for culturing to obtain a saccharomyces cerevisiae seed solution and a lactobacillus seed solution, and inoculating the saccharomyces cerevisiae seed solution and the lactobacillus seed solution into a Pu 'er tea raw material for Pu' er tea fermentation.

Further, the total volume of the saccharomyces cerevisiae seed solution and the lactobacillus seed solution is 10% of the total mass of the puer tea raw materials.

Further, the volume ratio of the saccharomyces cerevisiae seed liquid to the lactobacillus seed liquid is 1: 1.

further, the water content of the Pu' er tea raw material is 20-30%.

Further, the fermentation temperature is 35-50 ℃.

Further, in the fermentation process of the Pu 'er tea, the water content of the Pu' er tea raw material is maintained to be 20-30%.

Further, the saccharomyces cerevisiae and the lactobacillus are prepared by the following method:

and collecting a Pu' er tea pile fermentation middle-stage sample, crushing and grinding the sample, adding sterile water, oscillating the sample, collecting supernatant, coating the supernatant on a tea substrate culture medium, and performing purification culture to obtain the saccharomyces cerevisiae and the lactobacillus.

Further, the tea medium culture medium is any one of YPD solid culture medium, PDA solid culture medium, LB solid culture medium and NA solid culture medium added with tea powder, and the tea powder is obtained by grinding and sieving Pu' er tea raw materials.

Further, the culture temperature is 30-40 ℃ and the culture time is 3-4 days.

The saccharomyces cerevisiae strain and the lactobacillus strain are screened out and applied to fermentation of the Pu ' er tea, so that the fermentation speed of the Pu ' er tea is greatly increased, the content of bioactive substances in the tea after fermentation is obviously increased, the oxidation resistance is obviously improved, the quality of the Pu ' er tea is improved, the stability of the Pu ' er tea is enhanced, the content of functional substances in the fermented Pu ' er tea is higher, and the method has important significance for improving the quality of the Pu ' er tea and enhancing the safety and the stability of the Pu ' er tea.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The embodiment provides a preparation method of saccharomyces cerevisiae and lactobacillus, which comprises the following steps:

collecting a Pu' er tea pile-fermentation middle-stage sample according to GB/T8302-2013 standard, grinding to obtain 1g of tea powder, adding 10mL of sterile water into a clean bench, and marking the concentration as 10 after full oscillation^-1And sequentially diluted to 10^-2、10^-3、10^-4、10^-5(ii) a To a concentration of 10^-2、10^-3、10^-4、10^-5The bacterial suspension is taken out by 100 mu L respectively and coated on four tea medium culture mediums, and three groups of repeats are arranged for each concentration and each culture medium, so that the subsequent observation and comparison are convenient. After the coating liquid is dried, the coating liquid is placed in an incubator at the temperature of 30-40 ℃ for culture, and the observation is carried out once every 24 hours. After culturing for 3-4 days, placing the grown single strain on a new solid culture medium for a plurality of times until obtaining a purified strain, and identifying the strain through gene sequencing to obtain a wild Saccharomyces cerevisiae strain (WT, s.cerevisiae) and a Lactobacillus strain (Lactobacillus), wherein the diameter of the colony of the wild Saccharomyces cerevisiae strain (WT, s.cerevisiae) is 100mm, and the colony is milky white, glossy, flat and regular in edge; the cells have a width of 2.5-10 μm, a length of 4.5-21 μm, an aspect ratio of about 1-2, and are mostly circular, oval or ovoid; the Lactobacillus (Lactobacillus) colony is distributed in the whole culture dish, has the diameter of 100mm, is milk white, light yellow, semitransparent, smooth and convex, has regular edge, width of about 2 mu m, indefinite length, is usually interlaced in a filament-shaped curled shape, is a gram-positive bacterium, has no motility and does not produce spores.

The preparation method of the tea medium culture medium comprises the following steps:

YPD solid Medium: 950mL of distilled water, 20g of Bacto Peptone, 15g of Bacto Agar and 10g of Yeast Extract, 5g of fine tea powder passing through the middle stage of 100-mesh pile fermentation, and 50mL of 40% glucose solution after high-temperature and high-pressure sterilization.

PDA solid medium: taking 200g of potato small pieces, adding 500mL of distilled water, boiling the potatoes, filtering the soup, adding 20g of glucose and 15-20g of Bacto Agar into the soup, adding distilled water to 1000mL, sieving with a 100-mesh sieve, piling for middle-stage fine tea powder by 5g, and sterilizing at high temperature and high pressure.

LB solid medium: bacto Agar 15g, Bacto Tryptone 10g, NaCl 10g and Yeast Extract 5g, sieving with 100-mesh sieve to obtain fine tea powder at the middle stage, adding 950mL of distilled water, mixing well, and heating for sterilization.

NA solid medium: 3g of beef extract, 10g of peptone, 5g of NaCl and 15-20g of agarose, 5g of fine tea powder in the middle stage of pile fermentation by a 100-mesh sieve, adding 1000mL of distilled water, and sterilizing at high temperature and high pressure.

Saccharomyces cerevisiae and Lactobacillus are conventional strains in the art.

Example 2

Respectively placing the saccharomyces cerevisiae and the lactobacillus in a culture solution for culturing to obtain saccharomyces cerevisiae seed solution and lactobacillus seed solution, and inoculating the saccharomyces cerevisiae seed solution and the lactobacillus seed solution in a Pu 'er tea raw material for Pu' er tea fermentation, wherein the specific steps are as follows:

adding water into the sun-dried green raw tea until the water content reaches 20-30%, then mixing the saccharomyces cerevisiae and lactobacillus seed liquid according to the volume ratio of 1:1, inoculating the mixture into the sun-dried green raw tea, inoculating the mixture according to the volume ratio of the saccharomyces cerevisiae and lactobacillus seed liquid to the mass ratio of 10%, and fermenting for several weeks at the temperature of about 35-50 ℃. Water is added every other week until the water content is 20-30%.

The enzyme secreted by the saccharomyces cerevisiae can directly act on the tea matrix, and macromolecular water-insoluble compounds such as polysaccharide, protein, fat and the like in the tea matrix are effectively decomposed into micromolecular water-soluble carbohydrates such as monosaccharide, polypeptide, amino acid and the like under the action of amylase, protease, lipase and cellulose pectinase; in addition, a small amount of ethanol generated can increase the flavor of Pu' er tea and inhibit the growth of mixed bacteria. The lactobacillus can utilize metabolites of the saccharomyces cerevisiae, has strong carbohydrate metabolism capability, can generate ester compounds such as acetyl, ethyl acetate and the like besides lactic acid and other acid compounds, and increases the fragrance of the puer tea. The fermentation speed of the sun-dried green raw tea can be greatly increased by co-fermenting saccharomyces cerevisiae and lactobacillus, the content of bioactive substances in the tea is remarkably increased after the fermentation is finished, the oxidation resistance is obviously improved, the quality of the Pu 'er tea is improved, and the stability of the Pu' er tea is enhanced. Compared with the traditional fermentation samples, the content of active substances is increased.

Compared with the traditional fermentation sample, the Pu 'er tea fermented by inoculating the mixed strain of wild Saccharomyces cerevisiae strains (WT, s. cerevisiae) and Lactobacillus strains has the advantages that the content of tea polysaccharide is increased by about 5.3%, the content of tea polyphenol is increased by about 9.9%, the content of tea brown is increased by about 19.6%, the content of theanine is increased by about 11.5%, the Pu' er ripe tea with thick and bright red liquor color, obvious elecampane and mellow taste is obtained, the quality of the Pu 'er ripe tea is close to three years, the quality of the Pu' er ripe tea is obviously improved, the fermentation period is obviously shortened, and the Pu 'er tea has important effects on the improvement and the stability of the quality of the Pu' er tea.

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.