阅读说明：本技术 一株温驯驹形杆菌sw-1及其发酵应用 (Wenzhi colt-shaped bacillus SW-1 and fermentation application thereof ) 是由 王大红 王晓彤 曹璐纬 王梦洋 焦时阳 王贺敏 孙建瑞 原江锋 刘小娟 姚继云 于 2021-06-28 设计创作，主要内容包括：本发明涉及一株温驯驹形杆菌SW-1及其发酵应用,属于生物工程技术领域。所述温驯驹形杆菌SW-1是从天然发酵醋醅中分离并筛选得到的,是一种醋酸菌,已进行保藏,保藏编号为CGMCC No.22548。本发明通过温驯驹形杆菌SW-1的使用,可以与酿酒酵母及嗜酸乳杆菌的复配,形成纯化红茶菌。以红茶为原料发酵的红茶菌饮料共检测出84种风味物质,以绿茶为原料发酵的红茶菌饮料共检测出59种挥发性风味物质,且通过发酵,其抗氧化性和总黄酮含量都比未发酵的茶水有很大的提升。本发明主要使用具有自主知识产权的菌种,发酵红茶或绿茶,显著提高红茶菌饮料的风味成分和营养价值,提高了茶叶的附加值。(The invention relates to a Citrobacter wentamini SW-1 and a fermentation application thereof, belonging to the technical field of bioengineering. The Vibrio eutrophus SW-1 is obtained by separating and screening fermented grains of natural fermented vinegar, is an acetic acid bacterium, and is preserved with the preservation number of CGMCC No. 22548. The invention can be compounded with saccharomyces cerevisiae and lactobacillus acidophilus by using the halofoenus halodurans SW-1 to form the purified black tea fungus. The black tea fungus beverage fermented by taking black tea as a raw material is detected with 84 flavor substances, the black tea fungus beverage fermented by taking green tea as a raw material is detected with 59 volatile flavor substances, and the inoxidizability and the total flavone content of the black tea fungus beverage fermented by taking green tea as a raw material are greatly improved compared with unfermented tea water through fermentation. The invention mainly uses the strains with independent intellectual property rights to ferment the black tea or the green tea, obviously improves the flavor components and the nutritive value of the black tea fungus beverage, and improves the added value of the tea.)

1. The Wentanei colt-shaped bacillus SW-1 is classified and named as Wentanei colt-shaped bacillus (Vittanei colt-shaped bacillus)Komagataeibacter oboediens) The biological preservative has been preserved in the China general microbiological culture Collection center, the preservation address is in Beijing, China, the preservation number is CGMCC NO.22548, and the preservation date is 2021, 05 and 17 days.

2. A microbial agent comprising the Vibrio eutrophus SW-1 according to claim 1.

3. The method for producing the black tea fungus beverage by utilizing the SW-1 fermentation of the Vietnamese foal according to claim 1, is characterized in that: the method comprises the following steps:

(1) boiling water, adding tea leaves into the water according to the proportion of 10-20g/L, adding 80-100g/L of cane sugar, dissolving, cooling to room temperature, and preparing tea sugar water;

(2) inoculating halothrin coltsfoot SW-1, saccharomyces cerevisiae and lactobacillus acidophilus into the tea sugar water obtained in the step (1), and fermenting for 2-4 days at 25-30 ℃ to obtain fermentation liquor;

(3) and (3) separating and sterilizing the fermentation liquor obtained in the step (2), standing and after-ripening for 5-10d to obtain the black tea fungus beverage.

4. The method of claim 3, wherein: in the step (1), the tea leaves are at least one of black tea, green tea and Pu' er tea.

5. The method of claim 3, wherein: in the step (2), the Vibrio eutrophus SW-1 is activated and expanded to be cultured before inoculation, and the specific steps are as follows:

step one, slant solid culture:

inoculating the strain on a solid slant culture medium, and culturing at 25-32 ℃ for 2-3 days to obtain an activated strain for slant solid culture for later use;

step two, shake flask strain expansion culture:

taking the activated strain obtained in the step one, adding sterile water to prepare a bacterial suspension, wherein the adding amount of the sterile water is 3-5 mL according to a test tube; inoculating the bacterial suspension into a liquid seed amplification culture medium, wherein the inoculation amount is 5-10% of the volume of the liquid seed amplification culture medium, the culture temperature is 28-32 ℃, the shaking table speed is 70-150r/min, and the culture time is 1-2 days, so as to obtain seed liquid serving as seeds of fermented black tea fungus;

the formula of the liquid seed amplification culture medium is as follows: every 1000mL of culture medium contains 40-70 g of glucose, 15-20 g of yeast powder, 8-12 g of trehalose and Na₂HPO₄2-5 g, lactic acid 1-3 g, MgSO₄0.2-0.5 g, 15-30 mL of absolute ethyl alcohol and the balance of water.

6. The method according to claim 3 or 5, characterized in that: the inoculation amount of the Citrobacter wentamini SW-1 is 3% -5%, the inoculation amount of the saccharomyces cerevisiae is 1% -3%, and the inoculation amount of the Lactobacillus acidophilus is 1% -3%.

7. The use of the bacterium trematobacter wenyunnanensis SW-1 according to claim 1 or the microbial agent according to claim 2 in fermented beverages.

8. Use according to claim 7, characterized in that: the fermented beverage is a black tea fungus beverage.

9. The use of the microorganism bacterium agent SW-1 according to claim 1 or the use of the microorganism bacterium agent according to claim 2 for brewing vinegar, wherein the vinegar is solid-brewed vinegar or liquid-brewed vinegar.

Technical Field

The invention belongs to the fields of microbial application and food brewing, relates to an acetic acid bacterium strain and application thereof, and particularly relates to a Wenzhi colt-shaped bacillus which has good fermentation characteristic and more flavor substances and is suitable for brewing black tea fungus beverage and vinegar.

Background

Colt-shaped bacilli are mainly present in fermented foods, such as vinegar, black tea fungus beverages, wine, etc., and most colt-shaped bacilli, which belong to the family acetobacter in taxonomic terms, can produce cellulose, such as european colt-shaped bacilli, intermediate colt-shaped bacilli, persimmon vinegar colt-shaped bacilli, and wentamin colt-shaped bacilli, etc. Moreover, research shows that the foal shaped bacillus can promote the formation of aromatic substances including acid, alcohol and ester, and the flavor of the foal shaped bacillus can be greatly enhanced by adding the foal shaped bacillus in a small proportion to produce the traditional alcohol fermented food.

The black tea fungus is widely welcomed as a traditional fermented beverage with a long history, also called as 'Haibao' and 'Weibao', because the black tea fungus has sour and sweet taste and tea fragrance and has better probiotic effect on human bodies. Researches find that predominant strains playing a main role in fermentation in the black tea fungus are acetic acid bacteria, saccharomycetes and lactic acid bacteria. However, the natural black tea fungus contains a large amount of mixed fungi besides the three fungi, the fungi do not all contribute to the flavor of the black tea fungus beverage, and some of the fungi even generate substances harmful to human bodies. And the fermentation characteristic and the probiotic characteristic of the black tea fungus strain cultured traditionally in folk are greatly different, and the nutrient components in the tea water cannot be fully utilized by the naturally fermented black tea fungus drink. When the black tea fungus beverage is fermented, the quality of a finished product is difficult to control, so that mixed fungi are easily polluted, the sanitation is not up to the standard, and meanwhile, the flavor and the taste are poor.

The purified and cultured black tea fungus is formed by artificially compounding acetic acid bacteria, saccharomycetes and lactic acid bacteria in a certain proportion, and is added into tea sugar water for fermentation, so that the purified black tea fungus beverage which is controllable in fermentation process, short in fermentation period, high in product quality, good in flavor and high in economic value can be obtained. Researches show that in the black tea fungus beverage, the acetic acid bacteria which have the greatest contribution to the flavor are selected, so that the screening and fermentation characteristics are good, and the method has important significance for artificially compounding the acetic acid bacteria.

Disclosure of Invention

Aiming at the defect that the existing artificial purified black tea fungus is less in acetic acid bacteria, the colt-shaped bacillus suitable for preparing the artificial purified black tea fungus is obtained by separating and screening from the vinegar culture, and the purified black tea fungus beverage prepared by fermenting and aging saccharomyces cerevisiae and lactobacillus acidophilus mainly contains the colt-shaped bacillus and has mellow flavor, moderate sour and sweet taste, higher oxidation resistance and higher total flavone and total polyphenol content.

The invention aims to provide a Wen taming colt shaped bacillus SW-1, wherein the Wen taming colt shaped bacillus SW-1 is colt shaped bacillus, is classified and named as Wen taming colt shaped bacillus (Komagataeibacter oboedemans), is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of Beijing, China, the preservation number of CGMCC NO.22548 and the preservation date of 2021 year, 05 month and 17 days.

The invention also aims to provide a microbial agent which comprises the Vibrio eutrophus SW-1.

The third purpose of the invention is to provide a method for producing a black tea fungus beverage by utilizing the SW-1 fermentation of the Vibrio eutrophus, which comprises the following steps:

(1) boiling water, adding tea leaves into the water according to the proportion of 10-20g/L, adding 80-100g/L of cane sugar, dissolving, cooling to room temperature, and preparing tea sugar water;

(2) inoculating halothrin coltsfoot SW-1, saccharomyces cerevisiae and lactobacillus acidophilus into the tea sugar water obtained in the step (1), and fermenting for 2-4 days at 25-30 ℃ to obtain fermentation liquor;

(3) and (3) separating and sterilizing the fermentation liquor obtained in the step (2), standing and after-ripening for 5-10d to obtain the black tea fungus beverage.

Wherein in the step (1), the tea leaves are at least one of black tea, green tea and Pu' er tea.

Wherein in the step (2), the Vibrio eutrophus SW-1 is activated and expanded to be cultured before inoculation, and the specific steps are as follows:

step one, slant solid culture:

inoculating the strain on a solid slant culture medium, and culturing at 25-32 ℃ for 2-3 days to obtain an activated strain for slant solid culture for later use;

step two, shake flask strain expansion culture:

taking the activated strain obtained in the step one, adding sterile water to prepare a bacterial suspension, wherein the adding amount of the sterile water is 3-5 mL according to a test tube; inoculating the bacterial suspension into a liquid seed amplification culture medium, wherein the inoculation amount is 5-10% of the volume of the liquid seed amplification culture medium, the culture temperature is 28-32 ℃, the shaking table speed is 70-150r/min, and the culture time is 1-2 days, so as to obtain seed liquid serving as seeds of fermented black tea fungus;

the formula of the liquid seed amplification culture medium is as follows: every 1000mL of culture medium contains glucose 40-70 g, 15-20 g of yeast powder, 8-12 g of trehalose and Na₂HPO₄2-5 g, lactic acid 1-3 g, MgSO₄0.2-0.5 g, 15-30 mL of absolute ethyl alcohol and the balance of water.

The inoculation amount of the Vietnamese foal-shaped bacillus SW-1 is 3% -5%, the inoculation amount of the saccharomyces cerevisiae is 1% -3%, and the inoculation amount of the Lactobacillus acidophilus is 1% -3%.

The fourth purpose of the invention is to provide the application of the Vibrio eutrophus SW-1 or the microbial agent in the fermented beverage. Further, the fermented beverage is a black tea fungus beverage.

The fifth purpose of the invention is to provide the application of the Vibrio eutrophus SW-1 or the microbial agent in vinegar brewing, wherein the vinegar is solid-state brewed vinegar or liquid-state brewed vinegar.

Has the advantages that: compared with the traditional black tea fungus fermented beverage, the invention has the following advantages: the purified black tea fungus beverage prepared by adding the Wenrongcolal shaped bacillus SW-1 and adding the saccharomyces cerevisiae and the lactobacillus acidophilus is excellent in flavor, has pleasant acid aroma, fruity aroma and honey aroma and has light tea aroma. Through fermentation, the inoxidizability and the total flavone content of the tea are greatly improved compared with those of unfermented tea, and the tea has great probiotic effect on human bodies. Different from the traditional fermentation method, the method has the advantages of short fermentation period, high product quality, easy control of the fermentation process, improved utilization rate of the strains and certain industrial application value.

Drawings

FIG. 1 is a photograph of SW-1 colonies;

FIG. 2 is a gram-stained micrograph (1600 Xmagnification) of SW-1 cells;

FIG. 3 shows a phylogenetic tree of SW-1 constructed by the NJ method.

Detailed Description

The method comprises the following specific steps of:

10g of fermented vinegar grains from Luoyang vinegar industry Co., Ltd are added into 90mL of sterile water, and the mixture is shaken up and vibrated. Shaking up, adding 1mL of sample into 9mL of sterile water, mixing uniformly on a vortex mixer, adding 1mL of sample into 9mL of sterile water, and so on. Selecting three appropriate concentration gradients, sequentially coating a solid culture plate (10 g of glucose, 10g of yeast powder, 10g of calcium carbonate and 1000mL of distilled water, adjusting the pH value to 6.5, adding 20g/l of agar powder), culturing at 28 ℃ for 48-72h, and selecting a strain with a large transparent ring on the plate for later use. Inoculating the primarily screened strain to a re-screening solid culture medium plate (10 g of glucose, 3g of yeast powder, 30mL of acetic acid, 30mL of ethanol, 20g of agar and 940mL of distilled water), and culturing at 28 ℃ for 48h to select the strain with better growth. The selected strains are inoculated into a re-screening liquid culture medium (5 g of glucose, 4g of peptone, 3g of yeast extract, 30mL of acetic acid, 60mL of ethanol and 910mL of distilled water), shake-flask culture is carried out at 28 ℃ and 60r/min for 15h, and the content of total acid (calculated by acetic acid) is titrated by sodium hydroxide. After two rounds of rescreening, a strain producing acetic acid SW-1 (shown in figure 1) is finally obtained, the gram stain of the strain is negative (shown in figure 2), the yield of the acetic acid is 5.6g/100mL, and the strain can tolerate 10% of ethanol.

The acetogenic microorganism SW-1 obtained by screening is sent to the Huada gene company for sequencing, the sequencing result is submitted to an NCBI database for comparison, and the result shows that the homology between the 16S rDNA sequence of the strain SW-1 and the Komagataibacter ecoediens BPZTR01 is as high as 99%. A phylogenetic tree of a bacterium SW-1 is constructed by MEGA7.0 software, and is combined with physiological and biochemical identification of Bojie's bacteria identification handbook (eighth edition), wherein the bacterium is Rhodospiriales acetobacter (Acetobacter) Vietnamese (Komagataeibacter) Wenna Vietnamei (Komagataeibacter) Vietnamei. The strain is rod-shaped, gram-negative, small in colony during separation, smooth in surface, heterotrophic, glucose and aerobic bacteria are used for growth, the optimal temperature is 25-32 ℃, and the optimal pH value for growth is 5.5-8.0.

Secondly, a fermentation process method for fermenting the black tea fungus beverage comprises the following specific steps:

(1) slant solid culture:

inoculating the Vibrio tamarensis SW-1 on a solid slant culture medium, and culturing at 25-32 ℃ for 2-3 days to obtain an activated strain for slant solid culture for later use;

the formula of the slant solid culture mediumComprises the following steps: each 1000mL of culture medium contains 10-30 g of glucose, 10-20g of yeast powder and K₂HPO₄2-5 g of agar, 15-30 mL of absolute ethyl alcohol and the balance of water;

(2) shake flask strain expansion culture:

taking the obtained Wenchun colt SW-1 in the step (1), adding sterile water to prepare a bacterial suspension, adding 3-5 mL of the sterile water according to a test tube, inoculating the bacterial suspension into an expanded culture medium, wherein the inoculation amount is 5-10% of the volume of a liquid culture medium, the culture temperature is 28-32 ℃, the shaking table speed is 150-250 r/min, and the culture time is 1-2 days, so as to obtain a seed solution which is used as a seed of fermented black tea fungus;

the formula of the liquid seed amplification culture medium is as follows: every 1000mL of culture medium contains 40-70 g of glucose, 15-20 g of yeast powder, 8-12 g of trehalose and Na₂HPO₄2-5 g lactic acid 1-3 g, MgSO₄0.2-0.5 g, 15-30 mL of absolute ethyl alcohol and the balance of water;

(3) fermentation of black tea fungus beverage

Boiling water, adding tea leaves according to the proportion of 10-20g/L, adding 80-100g/L of cane sugar, inoculating Vibrio thermosiphon SW-1, saccharomyces cerevisiae and lactobacillus acidophilus according to the proportion, fermenting for 2-4 days at 25-30 ℃, separating out fermentation liquor, standing and maturing for 5-10 days.

And thirdly, testing the scavenging capability of DPPH free radicals:

sample treatment: 2.5mL of the sample was added to a 50mL volumetric flask and water was added to a constant volume. Then, 1mL of the sample solution was diluted by a 200-fold dilution with 9mL of distilled water. Taking 2mL of diluted sample solution and 2mL of diluted sample solution as experimental groups, replacing the DPPH solution with 2mL of absolute ethyl alcohol for a control group, taking 2mL of absolute ethyl alcohol and 2mL of absolute ethyl alcohol as standard groups, reacting for 30min in a dark place, measuring absorbance at 517nm, and calculating DPPH clearance.

Fourthly, volatile flavor substance determination:

adopting a method of combining headspace-solid phase microextraction-gas chromatography-mass spectrometry to carry out the following steps:

(1) extraction conditions are as follows: firstly, an extraction head is aged at 250 ℃ at a sample inlet of a gas chromatograph until no impurity peak exists, 6mL of sample (10 muL of 2-octanol with the concentration of 100mg/L is added as an internal standard) is placed into a 15mL solid phase microextraction sample bottle, 1g of sodium chloride is added, a magnetic stirrer is placed, and a cover is covered. Placing the sample in a constant-temperature water bath at 40 ℃, balancing for 10min, inserting an SPME extraction head into a headspace part of the sample through a bottle cap, pushing out a fiber head, adsorbing for 40min by the headspace, then withdrawing the fiber head, pulling out the extraction head from a sample bottle, inserting the extraction head into a gas chromatography sample inlet of a GC-MS instrument, pushing out the fiber head, desorbing for 5min at 250 ℃, pulling out the extraction head after withdrawing the fiber head, and starting the instrument to acquire data;

(2) the instrument conditions were as follows: chromatographic conditions are as follows: TG-WAXMS capillary chromatography column, column length 30 μm, inner diameter 0.25mm, liquid film thickness 0.25 μm, carrier gas: he gas, flow rate 1mL/min, split flow rate: 50mL/min, purge flow: 3mL/min, injection port temperature: 250 ℃;

temperature rising procedure: the initial temperature is 35 ℃, and the temperature is kept for 5 min; heating to 180 deg.C at 4 deg.C/min, and maintaining for 2 min; heating to 220 deg.C at a rate of 5 deg.C/min, and maintaining for 5min, 56.25 min;

mass spectrum conditions: transmission line temperature: 250 ℃, ion source temperature: 280 ℃, ionization mode: EI ionization source, electron energy: 70eV, mass scan range: 33-450 amu;

(3) data processing and analysis: the search spectrum library is replib and mainlab, the detected compounds with the matching degree of volatile components larger than 800 have the highest matching degree of 1000. And calculating the relative peak area ratio of all the components by adopting a peak area normalization method. The concentration of each component flavor substance in the sample is calculated according to the formula: ci ═ Ai/As × Cs;

in the formula: ci: the concentration of each volatile flavor compound in the sample, μ g/L;

cs: 2-octanol concentration, μ g/L;

ai: the area of a chromatographic peak corresponding to a substance to be detected in a sample;

as: chromatographic peak area of internal standard.

Fifthly, measuring the content of the total flavonoids:

taking 1mL of sample in a 10mL colorimetric tube, adding 60% ethanol to ensure that the volume of all the colorimetric tubes is 5mL, adding 0.3mL of 5% sodium nitrite solution, shaking uniformly, standing for 5 minutes, then adding 0.3mL of 10% aluminum nitrate solution, shaking uniformly by using an oscillator, standing for 6 minutes, adding 4mL of 1mol/L sodium hydroxide solution, finally completing volume fixing by using 60% ethanol, standing for 15 minutes, measuring absorbance at the wavelength of 510nm, and calculating the content of total flavonoids according to a standard curve.

Sixthly, measuring the total polyphenol content:

taking 1.00mL of sample, putting 0.65mL of 0.80mol/L forskolin reagent into a 25mL volumetric flask, adding 4mL of 20% sodium carbonate solution after reacting in a dark place, diluting 2 times with distilled water, carrying out water bath at 60 ℃ for 60min, taking 2mL of the reacted sample, adding 2mL of distilled water, measuring the absorbance at 720nm, and calculating the total polyphenol content.

The following detailed description of the preparation process and application process of the product of the present invention will be provided by the applicant in conjunction with specific examples to facilitate the clear understanding of the present invention by those skilled in the art. It should be understood that the following examples should not be construed as limiting the scope of the claims of the present application in any way.

Example 1: the screening method is explained to obtain the Vibrio eutrophus SW-1.

(1) Primary screening by a flat plate: 10g of fermented vinegar grains from a vinegar factory are added into 90mL of sterile water, and the mixture is shaken up. Shaking up, adding 1mL of sample into 9mL of sterile water, mixing uniformly on a vortex mixer, adding 1mL of sample into 9mL of sterile water, and so on. Three appropriate concentration gradients are selected and sequentially coated on a solid culture plate (10 g of glucose, 10g of yeast powder, 10g of calcium carbonate, 20g of agar powder, 1000mL of distilled water and pH 6.5), the solid culture plate is cultured for 72 hours at 28 ℃, and a strain with a large transparent ring is selected on the plate. Inoculating the primarily screened strain to a double-screen solid culture medium plate (10 g of glucose, 3g of yeast powder, 30mL of acetic acid, 30mL of ethanol, 20g of agar powder and 940mL of distilled water), and culturing at 28 ℃ for 48h to select a strain with better growth.

(2) And (3) shaking a flask for re-screening: the selected strain is inoculated into a re-screening liquid culture medium (5 g of glucose, 4g of peptone, 3g of yeast extract, 30mL of acetic acid, 60mL of ethanol and 910mL of distilled water), shake-flask culture is carried out at 28 ℃ and 60r/min for 48h, and the content of total acid (calculated by acetic acid) is titrated by sodium hydroxide.

(3) After two rounds of re-screening, the acetic acid resistant SW-1 strain (shown in figure 1) is finally obtained, and the strain SW-1 is negative in gram stain (shown in figure 2), good in ethanol resistance and strong in acid production capacity.

Example 2: it is explained how to identify Vibrio eutrophus SW-1.

(1) A single colony inclined plane is separated and purified, the single colony inclined plane is sent to Wuhan Hua big gene company for sequencing, the sequencing result is submitted to an NCBI database for comparison, and the result shows that the homology of the 16S rDNA sequence (SEQ ID NO: 01) of the strain SW-1 and the Komagataibacteriter degrees BPZTR01 is as high as 99 percent. The phylogenetic tree of SW-1 was constructed using MEGA7.0 software, selecting the NJ method, and the bootstrap value of 3000 (see FIG. 3).

(2) The SW-1 is determined to be the Cinobacter hygrophicus (Komagataeibacter oboedins) through the morphological characteristics, physiological and biochemical characteristics and the 16S rDNA sequence of the strain SW-1.

(3) The strain SW-1 identified as the Bacteroides atrophaeus is preserved, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation place is the microorganism institute of China academy of sciences, No. 3, West Lu No. 1 Hospital, North Jing, Chaoyang, the preservation number is CGMCC N0.22548, and the preservation date is 2021, 5 and 17 days.

(4) The base sequence of 16S rDNA of the Wen taming foal bacterium SW-1 with the serial number of CGMCC 22548 is shown as SEQ ID NO: 01, shown in the figure.

Example 3 illustrates how black tea fungus beverages can be fermented from black tea.

(1) Preparing a seed solution: the SW-1 slant stored at 4 ℃ in the refrigerator was inoculated into a 250mL Erlenmeyer flask containing 100mL of liquid (1L liquid medium formulation: glucose 70g, yeast powder 15g, trehalose 10g, Na)₂HPO₄3g, lactic acid 2g, MgSO₄0.4g, 20mL of absolute ethyl alcohol and the balance of water), and culturing at 28 ℃ and 80r/min for 36 h; saccharomyces cerevisiae was inoculated into a 250mL Erlenmeyer flask containing 100mL of YPD medium at 30 ℃. Culturing at 180r/min for 24 h; inoculating Lactobacillus acidophilus slant into 250mL triangular flask containing 100mL MRS culture medium, and culturing at 37 deg.C and 180r/min for 36 h.

(2) Preparing sugar tea water: 1000mL of water is boiled for sterilization, and tea leaves (black tea) are weighed and added into boiling water, wherein the dosage of the tea leaves is 15 g. Then 80g of white granulated sugar was added and dissolved completely. Preparing a sterilized container, adding the tea sugar water into the container, and cooling to room temperature.

(3) Fermenting the black tea fungus: culturing a Wenchun colt-shaped bacillus SW-1: and (3) saccharomyces cerevisiae: lactobacillus acidophilus was inoculated in 3%: 1%: inoculating 2% of the extract into tea sugar water, and standing and fermenting at 30 deg.C for 3 d.

(4) After-ripening after filtration: and (4) taking the black tea bacterium liquid fermented for 3d, filtering and carrying out pasteurization. And (4) after the sterilized black tea fungus fermentation liquor is subjected to light-shielding and normal-temperature after-ripening for 5-10 d.

The measurement of the oxidation resistance, the total flavone content and the total polyphenol content of the fermented black tea fungus liquid is carried out, and the unfermented tea water is used as a control, and the result is shown in the table I.

Table one: example 3 nutritional indicators.

Example 4: the black tea fungus beverage fermentation is carried out by taking black tea as raw material

(1) Preparing a seed solution: the SW-1 slant stored at 4 ℃ in the refrigerator was inoculated into a 250mL Erlenmeyer flask containing 100mL of liquid (1L liquid medium formulation: 80g of glucose, 20g of yeast powder, 11g of trehalose, Na)₂HPO₄3g, lactic acid 3g, MgSO₄0.5g of absolute ethyl alcohol 20mL), culturing at 30 ℃ and 90r/min for 36 h; inoculating a saccharomyces cerevisiae slant to a 250mL triangular flask with 100mL YPD medium liquid loading volume, and culturing at 30 ℃ for 24h at 180 r/min; inoculating Lactobacillus acidophilus slant into 250mL triangular flask containing 100mL MRS culture medium, and culturing at 37 deg.C and 180r/min for 36 h.

(2) Preparing sugar tea water: 1000mL of water is boiled for sterilization, and tea leaves (black tea) are weighed and added into boiling water, wherein the dosage of the tea leaves is 20 g. Then 100g of white granulated sugar is added and dissolved completely. Preparing a sterilized container, adding the tea sugar water into the container, and cooling to room temperature.

(3) Fermenting the black tea fungus: culturing a Wenchun colt-shaped bacillus SW-1: and (3) saccharomyces cerevisiae: lactobacillus acidophilus was added at a ratio of 5%: 3%: inoculating 2% of the extract into tea sugar water, and standing and fermenting at 30 deg.C for 4 days.

(4) After-ripening after filtration: and (3) filtering the black tea fungus liquid fermented for 4 days, performing pasteurization, and after the sterilized black tea fungus fermentation liquid is subjected to after-ripening for 5-10 days in the dark at normal temperature, the black tea fungus can be drunk.

The measurement of the oxidation resistance, the total flavone content and the total polyphenol content of the fermented black tea fungus liquid is carried out, and the unfermented tea water is used as a control, and the result is shown in the second table.

Table two: example 4 nutritional indicators.

The volatile flavor substances in the example were analyzed, and after data processing, 29 alcohol compounds, 17 aldehyde/ketone compounds, 17 organic acids, 21 ester compounds, and 84 volatile flavor substances in total were detected, and specific substances are shown in table five.

Example 5: the black tea fungus beverage fermentation is performed by using green tea as a raw material.

(1) Preparing a seed solution: the SW-1 slant stored at 4 ℃ in the refrigerator was inoculated into a 250mL Erlenmeyer flask containing 100mL of liquid (1L liquid medium formulation: glucose 80g, yeast powder 20g, trehalose 10g, Na)₂HPO₄3g, lactic acid 2g, MgSO₄0.4g, 20mL of absolute ethyl alcohol), culturing at 28 ℃ and 90r/min for 36 h; inoculating a saccharomyces cerevisiae slant to a 250mL triangular flask with 100mL YPD medium liquid loading volume, and culturing at 30 ℃ for 24h at 180 r/min; inoculating Lactobacillus acidophilus slant into 250mL triangular flask containing 100mL MRS culture medium, and culturing at 37 deg.C and 180r/min for 36 h.

(2) Preparing sugar tea water: boiling 1000mL of water for sterilization, weighing 15g of tea (green tea), cooling the water to below 40 ℃, adding the water, and then adding 100g of white granulated sugar for complete dissolution. The sterilized container is prepared and the tea sugar water is added to the container.

(3) Fermenting the black tea fungus: culturing a Wenchun colt-shaped bacillus SW-1: and (3) saccharomyces cerevisiae: inoculating lactobacillus acidophilus into tea sugar water cooled to room temperature according to the proportion of 4% to 2% to 3%, and standing and fermenting at 30 ℃ for 4 days.

(4) After-ripening after filtration: and (4) taking the black tea fungus liquid fermented for 4d, filtering and carrying out pasteurization. The sterilized black tea fungus fermentation liquor can be drunk after being subjected to light-proof normal-temperature after-ripening for 5-10 d.

The measurement of the oxidation resistance, the total flavone content and the total polyphenol content of the fermented black tea fungus liquid is carried out, and the unfermented tea water is used as a control, and the result is shown in the table III.

Table three: example 5 nutritional indicators.

After the analysis of the volatile flavor substances in this example and data processing, 16 kinds of alcohol compounds, 8 kinds of aldehyde/ketone compounds, 15 kinds of organic acids, 20 kinds of ester compounds were detected altogether, and 59 kinds of volatile flavor substances were determined in total, which is specifically shown in table five.

Example 6: the black tea fungus beverage fermentation using green tea as raw material is explained

(1) Preparing a seed solution: the SW-1 slant stored at 4 ℃ in the refrigerator was inoculated into a 250mL Erlenmeyer flask containing 100mL of liquid (1L liquid medium formulation: glucose 70g, yeast powder 15g, trehalose 10g, Na)₂HPO₄3g, lactic acid 2g, MgSO₄0.4g, 20mL of absolute ethyl alcohol), culturing at 28 ℃ and 70r/min for 36 h; inoculating a saccharomyces cerevisiae slant to a 250mL triangular flask with 100mL YPD medium liquid loading volume, and culturing at 30 ℃ for 24h at 180 r/min; inoculating Lactobacillus acidophilus slant into 250mL triangular flask containing 100mL MRS culture medium, and culturing at 37 deg.C and 180r/min for 36 h.

(2) Preparing sugar tea water: boiling 1000mL of water for sterilization, weighing tea (green tea), cooling to below 40 deg.C, and adding into water at a tea amount of 10 g. Then 90g of white granulated sugar was added and dissolved completely. The sterilized container is prepared and the tea sugar water is added to the container.

(3) Fermenting the black tea fungus: culturing a Wenchun colt-shaped bacillus SW-1: and (3) saccharomyces cerevisiae: lactobacillus acidophilus was added at 3%: 2%: inoculating 1% of the extract into tea sugar water cooled to room temperature, and standing at 30 deg.C for fermentation for 3 days.

(4) After-ripening after filtration: and (4) taking the black tea bacterium liquid fermented for 3d, filtering and carrying out pasteurization. The sterilized black tea fungus fermentation liquor can be drunk after being subjected to light-proof normal-temperature after-ripening for 5-10 d.

The measurement of the oxidation resistance, the total flavone content and the total polyphenol content of the fermented black tea fungus liquid is carried out, and the unfermented tea water is used as a control, and the result is shown in the fourth table.

Table four: example 6 nutritional indicators.

Through the measurement of three nutritional indexes, namely DPPH free radical clearance rate, total flavone content, total polyphenol content and total polyphenol content, of the examples 3, 4, 5 and 6, the content of the tea is greatly improved compared with unfermented tea water, and the fact that natural ingredients in tea can be well utilized and the additional value of the tea can be improved through the fermentation of purified black tea fungus is shown.

Table five: volatile flavors in examples 4 and 5.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> university of Henan science and technology

<120> Wentanflam foal shaped bacillus SW-1 and fermentation application thereof

<130> 1

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1420

<212> DNA

<213> Salmonella choleraesuis SW-1

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tggctcagag cgaacgctgg cggcatgctt aacacatgca agtcgcacga acctttcggg 60

gttagtggcg gacgggtgag taacgcgtag ggatctatcc acgggtgggg gataactttg 120

ggaaactgaa gctaataccg catgacacct gagggtcaaa ggcgcaagtc gcctgtggag 180

gaacctgcgt tcgattagct agttggtggg gtaaaggcct accaaggcga tgatcgatag 240

ctggtctgag aggatgatca gccacactgg gactgagaca cggcccagac tcctacggga 300

ggcagcagtg gggaatattg gacaatgggc gcaagcctga tccagcaatg ccgcgtgtgt 360

gaagaaggtt ttcggattgt aaagcacttt cagcggggac gatgatgacg gtacccgcag 420

aagaagcccc ggctaacttc gtgccagcag ccgcggtaat acgaaggggg caagcgttgc 480

tcggaatgac tgggcgtaaa gggcgcgtag gcggttgaca cagtcagatg tgaaattccc 540

gggcttaacc tgggggctgc atttgatacg tggcgactag agtgtgagag agggttgtgg 600

aattcccagt gtagaggtga aattcgtaga tattgggaag aacaccggtg gcgaaggcgg 660

caacctggct catgactgac gctgaggcgc gaaagcgtgg ggagcaaaca ggattagata 720

ccctggtagt ccacgctgta aacgatgtgt gctggatgtt gggtgacttt gtcattcagt 780

gtcgtagtta acgcgataag cacaccgcct ggggagtacg gccgcaaggt tgaaactcaa 840

aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcgc 900

agaaccttac cagggcttga catgcggagg ccgtgtccag agatgggcat ttctcgcaag 960

agacctccag cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 1020

agtcccgcaa cgagcgcaac cctcgccttt agttgccatc acgtctgggt gggcactcta 1080

aaggaactgc cggtgacaag ccggaggaag gtggggatga cgtcaagtcc tcatggccct 1140

tatgtcctgg gctacacacg tgctacaatg gcggtgacag tgggaagcca ggtggtgaca 1200

ccgagccgat ctcaaaaagc cgtctcagtt cggattgcac tctgcaactc gagtgcatga 1260

aggtggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg 1320

tacacaccgc ccgtcacacc atgggagttg gtttgacctt aagccggtga gcgaaccgca 1380

aggacgcagc cgaccacggt cgggtcagcg actggggtga 1420