CN116814460B - High-yield tannase lactobacillus and application thereof in preparation of fermented tea beverage - Google Patents

High-yield tannase lactobacillus and application thereof in preparation of fermented tea beverage Download PDF

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CN116814460B
CN116814460B CN202310041341.2A CN202310041341A CN116814460B CN 116814460 B CN116814460 B CN 116814460B CN 202310041341 A CN202310041341 A CN 202310041341A CN 116814460 B CN116814460 B CN 116814460B
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lactobacillus
tea
tannase
fermented tea
tea beverage
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CN116814460A (en
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许勇泉
邹纯
陈德权
尹军峰
黄奕宾
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Tea Research Institute Chinese Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A23F3/06—Treating tea before extraction; Preparations produced thereby
    • A23F3/08—Oxidation; Fermentation
    • A23F3/10—Fermentation with addition of microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A23F3/16—Tea extraction; Tea extracts; Treating tea extract; Making instant tea
    • A23F3/166—Addition of, or treatment with, enzymes or microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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Abstract

A lactobacillus with high tannase yield and an application thereof in the preparation of fermented tea beverage belong to the technical field of food fermentation and brewing. The invention provides a lactobacillus Lactiplantibacillus plantarum AT with high tannase yield and a preservation number of CCTCC M20221899 on the one hand and an application of the strain in preparation of fermented tea drinks on the other hand. The lactobacillus provided by the invention can be well adapted to a tea juice system, and has high fermentation efficiency and good flavor quality; in addition, the strain can form a large amount of tannase in fermentation, can efficiently hydrolyze tea polyphenol, avoid complexing of polyphenol and protein, and the produced lactobacillus fermented tea beverage has good stability. The technical scheme provided by the invention ensures that the production process of the lactobacillus fermented tea beverage is simple and efficient, and the product has the advantages of excellent quality, high stability and the like.

Description

High-yield tannase lactobacillus and application thereof in preparation of fermented tea beverage
Technical Field
The invention belongs to the technical field of food fermentation and brewing, and particularly relates to a high-yield tannase lactobacillus and application thereof in preparation of fermented tea drinks.
Background
Lactic acid bacteria are an important class of probiotics that can colonize the host body to exert beneficial effects. The tea is a traditional healthy drink, combines lactobacillus and tea to form lactobacillus fermented tea drink with both flavor and health attribute, and has wide market prospect. However, there are two major technical bottlenecks in the current preparation of lactic acid bacteria tea beverages: 1. few excellent strains are specially used for preparing lactobacillus fermented tea drinks; 2. lactic acid bacteria fermentation is prone to produce large amounts of proteins and polyphenols in tea juice are prone to complex into precipitates, resulting in poor stability. These have greatly limited the development of the industry.
Sour tea is tea fermented by microorganisms and is deeply favored by Yunnan minority nationalities such as De-ang and Brownian. In the process of fermenting the sour tea, a large amount of lactobacillus and yeast act, so that the quality characteristics of 'sour flavor showing, body fluid production promoting and sweet returning' are formed. Under the action of microorganisms, the content of caffeine and catechin in the sour tea is obviously reduced, and the content of water-soluble total sugar, free amino acid and other components are increased, so that the bitter taste of the sour tea is reduced, and the taste is soft and mellow. Therefore, it is possible to select excellent lactic acid bacteria from sour tea for producing a lactic acid bacteria fermented tea drink.
The prior art discloses some technical schemes for producing lactobacillus fermented tea drinks. The reference CN201710931254.9 discloses a method for processing novel lactobacillus fermented milk tea beverage by using lactobacillus plantarum ST, wherein the lactobacillus plantarum is selected from acid tea, and although the lactobacillus plantarum can be well adapted to a tea juice system, the problem of complexation of lactobacillus forming proteins and tea juice polyphenols is not solved. The tannase can hydrolyze tea polyphenol efficiently and effectively avoid complexing with protein, so that the problem that lactobacillus tea beverage is easy to form sediment can be solved. These technical problems are well addressed if a strain is obtained which is adapted to the tea juice system and which produces tannase.
Based on the problems, the invention screens out the lactobacillus with high tannase yield from the sour tea as the production strain of the fermented tea beverage. On one hand, the strain can be well adapted to a tea juice system, and has high fermentation efficiency and good flavor quality; on the other hand, the strain can form a large amount of tannase in fermentation, can hydrolyze tea polyphenol efficiently, and effectively avoid complexing with protein, so that the produced lactobacillus fermented tea beverage has good stability. The technical scheme provided by the invention ensures that the production process of the lactobacillus fermented tea beverage is simple and efficient, and the product has the advantages of excellent quality, high stability and the like, and is suitable for large-scale preparation of the lactobacillus fermented tea beverage.
Disclosure of Invention
Aiming at the problems that the lactobacillus fermented tea beverage in the prior art has few excellent strains, protein formed by lactobacillus and tea polyphenol are easy to complex into precipitate, the stability is poor and the like, the invention provides the lactobacillus Lactiplantibacillus plantarum AT with high tannase yield. The method is applied to the preparation of the lactobacillus fermented tea beverage, and the lactobacillus fermented tea beverage with excellent quality and high stability can be simply and quickly obtained.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a lactobacillus Lactiplantibacillus plantarum AT, a preservation unit: china center for type culture Collection, address: eight-way university of Wuhan in Wuhan district of Hubei province, date of preservation: 2022, 12, 08, deposit No.: cctccc M20221899.
Furthermore, the invention provides an application of the lactobacillus in preparation of fermented tea beverage.
Further, the fermented tea beverage obtained by the lactic acid bacteria comprises a lactic acid bacteria tea beverage, a lactic acid bacteria solid tea beverage and tea-containing yoghourt.
Further, the fermented tea beverage obtained by the lactic acid bacteria comprises an active lactic acid bacteria beverage and an inactivated lactic acid bacteria beverage.
Compared with the prior art, the invention has the following advantages:
on the one hand, the lactobacillus Lactiplantibacillus plantarum AT29 provided by the method can be well adapted to a tea juice system, and has high fermentation efficiency and good flavor quality; on the other hand, the strain can form a large amount of tannase in fermentation, can efficiently hydrolyze tea polyphenol, and effectively avoid complexing of polyphenol and protein, so that the produced lactobacillus fermented tea beverage has good stability. The technical scheme provided by the invention ensures that the production process of the lactobacillus fermented tea beverage is simple and efficient, and the product has the advantages of excellent quality, high stability and the like.
Drawings
FIG. 1 is a diagram showing the isolation and purification of lactic acid bacteria in example 1;
FIG. 2 is a graph showing the growth of lactic acid bacteria on a primary screening medium in example 1;
FIG. 3 is an electrophoretogram of the 16S rDNA sequencing PCR amplification sequence of lactic acid bacteria in example 2.
Detailed Description
In order that the invention may be more readily understood, the invention will be further described in connection with specific examples. These examples are only for the purpose of illustrating the invention and are not intended to limit the scope of the invention.
Example 1
Separation and purification of lactic acid bacteria: adding 10g of acid tea containing active bacteria into shake flask containing 90mL of sterile physiological saline, and shaking thoroughly to obtain dilution of 10 -1 Sequentially and gradually diluting to 10 -5 . 0.2mL of each gradient of diluent is coated on MRS separation culture medium and placed in a 37 ℃ incubator for 1-2 d. The bacterial colony (figure 1) with the typical characteristics of round, protruding, opaque and smooth surface on the flat plate is picked, streaked and separated for more than 3 times, and is observed under a microscope, and the bacterial strain with the rod-shaped cells is screened out, so that 125 bacterial strains are obtained.
Lactobacillus primary screening: the strain separated out is inoculated into a primary screening culture medium which takes tannic acid as the only carbon source and takes bromophenol blue as an indicator, and if the strain can decompose and utilize tannic acid in the culture medium, the gallic acid produced by decomposing tannic acid can change bromophenol blue in the culture medium from blue-violet to yellow, and the gallic acid can be oxidized to yellow-green in the air. The culture was inverted at 37℃in an incubator for 72 hours. When the selected strain is incapable of producing tannase or can only produce very low tannase, it cannot grow on the primary culture medium and the culture medium hardly changes color (left side of fig. 2); strains with obvious bacterial colony and obvious color change (right side of fig. 2) can grow on the primary screening culture medium, and tannic acid can be used as a sole carbon source for growth, so that the strains with the ability of producing tannase can be screened out, and 56 strains can be screened out for further screening.
Re-screening lactic acid bacteria: activating lactobacillus obtained by primary screening, inoculating the activated lactobacillus liquid into an enzyme-producing culture medium according to the inoculation amount of 2%, and culturing in a 37 ℃ incubator for 48 hours. And centrifuging to collect fermentation supernatant, and measuring tannase activity to obtain strain 1 strain with highest tannase production capacity, wherein the enzyme yield is 266U/L.
MRS Medium (g/L): peptone 10, yeast extract 5, glucose 20, beef extract 10, K 2 HPO 4 2, diammonium citrate 2, sodium acetate 5, tween-80 5, mgSO 4 ·7H 2 O 0.58，MnSO 4 ·4H 2 O0.25, pH value 6.2-6.4.
Primary screening media (g/L): tannic acid 10, naNO 3 3；K 2 HPO 4 1，MgSO 4 ·7H 2 O 0.5，KCl 0.5，FeSO 4 ·7H 2 O0.01, bromophenol blue 0.04, agar 15; the pH was adjusted to 5.0.
Enzyme production medium (g/L): tannic acid 20, sucrose 10, naNO 3 3，K 2 HPO 4 1，KCl 0.5，MgSO 4 ·7H 2 O 0.5。
Example 2
The strain selected in example 1, which has the highest ability to produce tannin enzyme, was subjected to 16S rDNA sequencing by the Shanghai Pair-North Bio Inc. PCR amplification was performed using the sequences 5-AGAGTTTGATCCTGGGCTCAG-3 and 5-CTACGGCTACCTTGTTACGA-3 as primers, and the amplified sequences were analyzed by electrophoresis as shown in FIG. 3. The PCR amplified sequence was analyzed and the result was shown as SEQ ID NO. 1. Sequencing results were removed from the primer sequences and Blast aligned in the GenBank database of the national center for biotechnology information (national center for biotechnologyinformation, NCBI). Identified as Lactiplantibacillus plantarum and named Lactiplantibacillus plantarum AT. It is deposited in China center for type culture Collection, address: eight-way university of Wuhan in Wuhan district of Hubei province, date of preservation: 2022, 12, 08, deposit No.: cctccc M20221899.
Example 3
The Lactiplantibacillus plantarum AT selected above was used as an experimental group for the preparation of lactobacillus tea beverages by using commercial lactobacillus (Lactobacillus plantarum GDMCC 1.2685) as a control, and was compared.
Firstly, seed liquid culture is carried out: commercial lactic acid bacteria and Lactiplantibacillus plantarum AT29 were inoculated into MRS medium at an inoculum size of 0.1%, respectively, and cultured at 37℃for 24 hours.
And then preparing a fermentation medium: the tea leaves, glucose and water are subjected to heat preservation for 20min at 90 ℃ according to the mass ratio of 0.6:10:100, and cooled to room temperature to obtain the fermentation medium. The tea is green tea.
Then, the obtained seed liquid commercial lactic acid bacteria and Lactiplantibacillus plantarum AT29 are respectively inoculated into a fermentation medium according to the inoculation amount of 4 percent, and are subjected to static culture for 30 hours at 37 ℃. The pH and organic acid composition were determined. Commercial lactic acid bacteria broth pH was found to be 4.31, while Lactiplantibacillus plantarum AT broth pH was found to be 3.25, indicating a higher degree of fermentation of Lactiplantibacillus plantarum AT29. The possible reasons are that commercial lactic acid bacteria cannot adapt to the tea juice system, the fermentation speed is slow, and Lactiplantibacillus plantarum AT29 is selected from sour tea and can adapt to the tea juice system well.
In the aspect of flavor, the commercial lactobacillus fermentation liquor has higher sweetness, less obvious sour taste and heavier bitter and astringent taste, and has a sensory score of 61 minutes; while Lactiplantibacillus plantarum AT and Lactiplantibacillus plantarum AT are coordinated in sweetness and sourness of the fermentation broth, and the tea taste is obvious, and the sensory score is 87 minutes. In addition, commercial lactic acid bacteria fermentation broths have a large amount of sediment and new sediment is formed during storage (30 days); while Lactiplantibacillus plantarum AT29 fermented tea beverages have better clarity and do not form significant precipitates during storage (30 days).
Example 4
The commercial lactobacillus is used as a control, the screened Lactiplantibacillus plantarum AT is used as an experimental group and applied to the preparation of the yogurt containing tea for comparison.
Firstly, seed liquid culture is carried out: commercial lactic acid bacteria and Lactiplantibacillus plantarum AT29 were inoculated into MRS medium at an inoculum size of 0.1%, respectively, and cultured at 37℃for 24 hours.
And then preparing a fermentation medium: leaching at 90deg.C for 20min at tea water ratio of 1:10 to obtain tea juice; adding 5% of tea juice and 5% of glucose into pure milk, sterilizing at 85deg.C for 30min, and cooling to obtain fermentation medium. The tea is green tea.
Then, the obtained seed liquid commercial lactobacillus and Lactiplantibacillus plantarum AT are respectively inoculated into a fermentation culture medium according to the inoculation amount of 2 percent, and are cultured for 6 hours at 37 ℃ to obtain the tea-containing yoghourt. The tea-containing yoghourt fermented by the commercial lactic acid bacteria has lighter fermentation degree, heavier sweetness, obvious bitter and astringent taste, uncoordinated flavor and 67 sensory scores; the tea-containing yoghurt fermented by Lactiplantibacillus plantarum AT is rich in tea aroma, good in compatibility between tea taste and milk taste, moderate in sweetness and sourness and has a sensory score of 89 minutes.
There are a large number of precipitations in commercial lactic acid bacteria fermented tea-containing yoghurt and new precipitations are also formed during storage (15 d); while Lactiplantibacillus plantarum AT29 fermented tea yogurt is more homogeneous and does not form significant precipitates during storage (15 d).

Claims (4)

1. The high-yield tannase lactobacillus (Lactiplantibacillus plantarum) AT29 has a preservation number of CCTCC M20221899.
2. Use of the lactic acid bacteria according to claim 1 for the preparation of a fermented tea beverage.
3. Use according to claim 2, characterized in that the fermented tea beverage comprises a lactic acid bacteria tea beverage, a lactic acid bacteria solid tea beverage and a tea-containing yoghurt.
4. Use according to claim 2, characterized in that the fermented tea beverage comprises an active lactic acid bacteria beverage and an inactivated lactic acid bacteria beverage.
CN202310041341.2A 2023-01-13 2023-01-13 High-yield tannase lactobacillus and application thereof in preparation of fermented tea beverage Active CN116814460B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112175845A (en) * 2020-10-19 2021-01-05 中国农业科学院茶叶研究所 Saccharomyces cerevisiae suitable for brewing tea wine and application thereof
CN113068782A (en) * 2021-04-01 2021-07-06 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) Brown active lactobacillus beverage and preparation method thereof
CN113980858A (en) * 2021-11-18 2022-01-28 上海植酵盛生物科技有限公司 Lactobacillus plantarum YL399 for producing high-activity tannase and application thereof in preparation of codonopsis pilosula fermented feed
CN114410523A (en) * 2022-01-20 2022-04-29 中国农业科学院茶叶研究所 Strain combination for efficiently preparing black tea fungus and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112175845A (en) * 2020-10-19 2021-01-05 中国农业科学院茶叶研究所 Saccharomyces cerevisiae suitable for brewing tea wine and application thereof
CN113068782A (en) * 2021-04-01 2021-07-06 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) Brown active lactobacillus beverage and preparation method thereof
CN113980858A (en) * 2021-11-18 2022-01-28 上海植酵盛生物科技有限公司 Lactobacillus plantarum YL399 for producing high-activity tannase and application thereof in preparation of codonopsis pilosula fermented feed
CN114410523A (en) * 2022-01-20 2022-04-29 中国农业科学院茶叶研究所 Strain combination for efficiently preparing black tea fungus and application thereof

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