WO2017210815A1 - Microbial strain and application thereof in production of pu'er tea - Google Patents

Abstract

Provided are a yeast strain (Arxula adeninivorans, TMCC 70007 CGMCC No. 8683) capable of producing highly active polyphenol oxidase and a Lactobacillus pentosus capable of producing γ-aminobutyric acid (TMCC 70009 CGMCC No. 8685). The invention further provides a method for using the microbes for producing Pu'er tea, and a Pu'er tea produced using the method.

Description

Microbial strains and their application in Pu'er tea production Technical field

The invention relates to the field of microorganisms, in particular to a yeast (Arxula adeninivorans) capable of producing high activity polyphenol oxidase, Lactobacillus pentosus which can produce γ-aminobutyric acid and its production in Pu'er tea Application in .

Background technique

Polyphenoloxidase (PPO) is a widely distributed metalloproteinase in nature. It is ubiquitous in the plastids of plants, fungi, insects, and even polyphenols can be detected on decaying plant residues in soil. Enzyme activity. Because of its convenient detection, it is one of the first few enzymes studied. Polyphenol oxidase, also known as catechol oxidase, tyrosinase, phenolase, cresolase, and catechol oxidoreductase, is the first major class of oxidoreductases among the six major classes of enzymes. Among all the chemical components in tea, tea polyphenols and polyphenol oxidase are particularly important. Tea polyphenols in tea are oxidized and polymerized by polyphenol oxidase to form the oxidation product of the tea polyphenols. Thearubigin and the tea brown pigment. The fermentation in the processing of Pu'er cooked tea is through the combination of microorganisms, moist heat and enzyme catalysis, which promotes the oxidative polymerization of tea polyphenols under the catalysis of polyphenol oxidase to produce theaflavins, thearubigins and tea brown. The oxidation products such as sulphur form the characteristic characteristics of Pu'er tea soup with red and bright color.

Tea brown pigment is one of the most important active ingredients in Pu'er cooked tea. It is known as the gold in Pu'er tea. The formation and increase of tea brown pigment is the main value of Pu'er tea. The content of tea brown pigment in Pu'er hair tea is very low, about 0.3% (mass/mass percentage, the same below), the content of tea brown pigment in Pu'er cooked tea fermented by glutinous rice can reach 8% or more, generally 8%- 15%. The role of theaflavin in softening blood vessels, blood pressure lowering, lipid lowering, weight loss, and uric acid lowering has been known.

Gamma-aminobutyric acid (GABA) is a naturally occurring functional amino acid. Studies have shown that γ-aminobutyric acid has the physiological activities of lowering blood pressure, improving brain function, enhancing long-term memory and improving liver and kidney function. Γ-aminobutyric acid is widely found in animals and plants in nature. It is only found in nervous tissues in animals. It is an important inhibitory neurotransmitter in mammalian central nervous system and participates in various metabolisms. Γ-aminobutyric acid is obtained by decarboxylation of L-glutamic acid by glutamate dehydrogenase (GAD).

Many Pu'er teas contain not only caffeine, but also the index is much higher than that of green tea and oolong tea. In the test of the “Agricultural Tea Testing Center”, the caffeine content in Pu'er tea is as high as 4.41% (mass/mass concentration). Among the various teas known to date, Pu'er tea has the highest caffeine content, but it does not affect people's normal sleep after drinking Pu'er tea. Many scholars believe that Pu'er tea affects the main "murderer" of human sleep during repeated processing. Caffeine gradually disappeared. However, after chemical analysis of Pu'er tea, this conclusion is not valid. The answer lies in gamma-aminobutyric acid (GABA), because the most basic physiological function of GABA is to reduce the activity of neurons, prevent nerve cells from overheating, and play a role in sedating nerves, thereby exerting an antagonistic effect on the receptors of caffeine. In other words, GABA offsets the “action” of caffeine excited by the central nervous system.

The preparation methods of γ-aminobutyric acid (GABA) mainly include chemical synthesis method and biosynthesis method. Chemical synthesis is more common in the patent literature, which has higher cost, lower yield, and often uses dangerous solvents or even toxic solvents in the production process. Therefore, GABA prepared by chemical synthesis cannot be used in the food field or as a natural food additive. Biosynthesis is a relatively safe and cost-effective method. According to the latest research reports and patent literature reports, some highly safe microorganisms such as lactic acid bacteria, yeast, and Aspergillus have been used in the preparation of GABA foods. On September 27, 2009, the Ministry of Health of China officially approved γ-aminobutyric acid, colostrum basic protein, conjugated linoleic acid, conjugated sub, oleic acid glyceride, Lactobacillus plantarum (strain number ST-III), Eucommia seed oil is a new resource food. Since then, for γ-aminobutyric acid, it has entered a new era.

Lactic acid bacteria are Gram-positive bacteria. Many strains have high GAD (glutamate decarboxylase) activity and can be enriched to produce GABA, such as Lactobacillus plantarum M-10, Lactobacillus acidophilus, Lactobacillus brevis hjxj-01. As an auxiliary starter, Lactobacillus pentosus has been widely used in the production of products such as yogurt, cheese, ice cream, fermented meat products, chocolate, and soy products. Due to the harsh reaction conditions of the synthetic GABA chemical method, the high cost of natural raw materials and the poor safety, it is difficult to be widely used. Among microorganisms, the presence of GAD is currently found in various lactic acid bacteria, bacteria, yeasts, and molds.

In the field of Pu'er tea research, the early stage of the study of probiotics is relatively difficult due to the lack of systematic research from the perspective of microbiology. With the maturity of microbial separation methods and the rapid development of modern bioinformatics, probiotic strains were isolated and purified from traditional plutonium fermentation samples and applied to Pu'er tea production process to improve the quality of Pu'er tea and the development of Pu'er tea industry. Will play an important role.

Summary of the invention

A first object of the present invention is to provide a yeast which produces a highly active polyphenol oxidase.

The present invention provides a yeast (Arxula adeninivorans or Blastobotrys adeninivorans, homologous) which can produce high activity polyphenol oxidase, and is named TMCC 70007, which has been preserved in Chinese microbial strains on January 6, 2014. General Microbiology Center of the Deposit Management Committee (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the preservation number CGMCC No.8683.

The 16S rRNA gene sequence of the TMCC 70007 strain is shown in SEQ ID NO: 1 in the sequence listing, and the ITS sequence is shown in SEQ ID NO: 2 in the sequence listing, and is identified as a yeast capable of producing polyphenol oxidase (Arxula adeninivorans or Blastobotrys adeninivorans, the same species). Synonym).

The strain was cultured in an ISP 2 medium at 35 ° C for 5 days, and the enzyme activity of the polyphenol oxidase was measured to be 1800 U/mL.

The present invention can produce morphological characteristics and optimum growth conditions of the highly active polyphenol oxidase yeast (Arxula adeninivorans) TMCC 70007CGMCC No.8683:

Growing in the range of pH 4-9, the optimum growth pH is 5-7;

Growing in the temperature range of 4-50 ° C, the optimum growth temperature is 35-45 ° C;

After culturing on ISP 2 medium for 48 hours at 35 ° C, the cells were observed under a microscope: the colonies were neat and milky white, the surface was moist and lustrous, the hyphae penetrated into the medium, and the diameter was about 0.8 mm; the growth temperature was 25-45 ° C. Observed under the microscope: the cells are oval and have pseudofilaria;

After culturing on PDA medium for 3 days at 35 ° C, it was observed under a microscope: the colonies were milky white, 3-4 mm in diameter, rounded at the edges, mounded, slightly wet, non-smooth, with aroma of wine, slightly yellow on the back of the colon; After microscopic examination, the cells were observed to be elliptical or nearly circular, with a large volume, and budded on the opposite side.

The present invention can produce high activity polyphenol oxidase yeast (Arxula adeninivorans) TMCC 70007CGMCC No.8683 has the effect of improving the quality of Pu'er tea, and can be applied to the production process of Pu'er tea.

The invention also provides a method for producing Pu'er tea by using a yeast (Arxula adeninivorans) TMCC 70007CGMCC No.8683 which can produce high activity polyphenol oxidase, and the specific method comprises the following steps;

1) Tide the Pu'er hair tea: add Pu'er hair tea to water to a moisture content of 20%-40% (volume/volume percent concentration), and sterilize;

2) Fermentation: seeding the seed solution of the yeast (Arxula adeninivorans) TMCC 70007CGMCC No. 8683 in the growth index period in an inoculated amount of 0.5% to 15% (weight/volume percentage) into the tided Puer tea. After 2-6 weeks of fermentation at a temperature of 35-45 ° C, the water is replenished every other week during the fermentation and turned over to obtain Pu'er cooked tea.

In the above-mentioned production method of Pu'er tea, the growth index period in the step 2) means that it is cultured in an ISP 2 liquid medium under aerobic conditions for 8 to 10 hours; the inoculum amount of the seed liquid is preferably 8% ( Weight/volume percent concentration); the fermentation temperature is preferably 45 °C.

The Pu'er cooked tea tea soup produced by the above method is bright red, bright and fragrant, smooth and slightly sweet; after testing, the content of theanine is 1.8% (mass/mass percentage, the same below), tea The polyphenol content is 10-14%, the water extract content is 39%, the soluble sugar content is 2.1%, the tea brown pigment content is as high as 16-18%, and the tea brown pigment content in Pu'er hair tea is very low, about 0.3%. In the ordinary Pu'er tea, the content of the tea brown pigment is generally only 8-15%, indicating that the yeast (Arxula adeninivorans) TMCC 70007CGMCC No.8683 can produce highly active polyphenol oxidase, promoting the polyphenol oxidase in the tea polyphenols. Oxidative polymerization occurs under catalysis to produce high concentrations An oxidation product such as tea brown pigment, theaflavin and thearubigin, thereby forming a high quality Pu'er cooked tea.

The invention obtains a yeast (Arxula adeninivorans) TMCC 70007CGMCC No which can produce high activity polyphenol oxidase from the conventional sputum Pu'er tea fermentation sample by the traditional plate dilution coating, primary screening and rescreening separation and purification methods. .8683. The strain was cultured in ISP 2 medium at 35 ° C for 5 days, and the enzyme activity of polyphenol oxidase was determined to be 1800 U/mL. The highly active polyphenol oxidase can significantly increase the content of tea in the fermentation process. The conversion efficiency of the ingredients increases the content of the tea brown pigment, and the high content of the tea brown pigment helps to improve the health care quality of the tea. The experiment proves that the application of this strain to the production of Pu'er tea does have the effect of improving the quality of Pu'er tea. The tea soup of Pu'er cooked tea fermented by this strain is bright red, bright and fragrant, smooth and slightly sweet. It not only makes the quality of Pu'er cooked tea significantly improved in aroma, soup color and taste, but also makes the content of tea brown pigment as high as 16-18% (mass/mass percentage). In addition, with the proliferation of the yeast (Arxula adeninivorans) TMCC 70007CGMCC No.8683, the amount of enzyme secreted by the yeast (Arxula adeninivorans) is also increased. Under the action of enzymes such as protease, cellulase, pectinase and glucoamylase, the reducing sugar The content of amino acids, soluble carbohydrates and hydrated pectin is increasing, which enriches the taste of Pu'er tea. Its rich enzyme system also inhibits the growth of other bacteria, especially a few harmful microorganisms, and reduces the production of Pu'er tea. The risk of failure. The invention provides an important application basis for the development of Pu'er tea new products and provides new microbial resources for the development of Pu'er tea industry.

A second object of the present invention is to provide a strain of Lactobacillus pentosus which can produce gamma-aminobutyric acid (GABA).

Lactobacillus pentosus, which can produce gamma-aminobutyric acid, is named TMCC 70009, which was deposited with the General Microbiology Center of the China Microbial Culture Collection Committee on January 6, 2014 (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with the preservation number CGMCC No.8685.

The 16S rRNA gene sequence of the TMCC 70009 strain is shown in SEQ ID NO: 1 in the sequence listing, and is identified as Lactobacillus pentosus which can produce γ-aminobutyric acid.

The strain was cultured in an ISP 2 medium at 35 ° C for 3 days, and its γ-aminobutyric acid content was measured to be 1.6 μg / mL.

The present invention can produce morphological characteristics and optimum growth conditions of Lactobacillus pentosus TMCC70009CGMCC No.8685 of γ-aminobutyric acid:

After incubation on ISP 2 medium for 72 hours at 35 ° C, milky white colonies were formed with smooth surface, neat colonies, no wrinkles at the edges, and a diameter of about 1 mm. Under microscope, the cells were oblong and existed in a single or chain shape. ;

It can also grow under anaerobic conditions. It is a facultative anaerobic bacteria with a growth temperature between 20 and 50 ° C. It can not grow above 55 ° C.

The invention can produce γ-aminobutyric acid Lactobacillus pentosus TMCC70009CGMCC No.8685 has the effect of improving the quality of Pu'er tea, and can be applied to the production process of Pu'er tea.

The present invention also provides a method for producing Pu'er tea by using Lactobacillus pentosus TMCC 70009CGMCC No.8685 which can produce γ-aminobutyric acid, and the specific method comprises the following steps;

1) tidal water of Pu'er hair tea: adding Pu'er hair tea to water to a moisture content of 20%-50% (volume/volume percentage concentration, preferably 45%), sterilization;

2) Fermentation: Seed solution of Lactobacillus pentosus TMCC 70009CGMCC No. 8685 in the growth index period is inoculated into the tidal Pu'er tea with an inoculation amount of 2%-10% (weight/volume percentage). After 3-4 weeks of fermentation at a temperature of 20-50 ° C, the water is replenished every other week during the fermentation and turned over to obtain Pu'er cooked tea.

In the above method for producing Pu'er tea, the growth index period in the step 2) means culturing in an ISP 2 liquid medium under anaerobic or facultative anaerobic conditions for 16-18 h; the inoculum amount of the seed liquid It is preferably 5% (weight/volume percent concentration); the fermentation temperature is preferably 45 °C.

The Pu'er cooked tea tea soup produced by the above method has fresh floral fragrance, good taste and richness; and the content of γ-aminobutyric acid is 1.5-2.2% (mass/mass concentration, the same below), and γ in Pu'er hair tea. The aminobutyric acid content is very low, being 4.3 mg/100 g (0.0043%). The content of γ-aminobutyric acid in ordinary Pu'er tea is generally 0.32%-0.9%, indicating that Lactobacillus pentosus TMCC 70009CGMCC No.8685 can produce high concentration of γ-aminobutyric acid, which can be significantly shortened on the one hand. In the fermentation cycle, the fermentation cycle is only 3-4 weeks, and the fermentation cycle of the common method is generally 6-8 weeks. This is because the optimal growth environment of the strain is created under artificial conditions, and the microorganisms are single and no other strains compete. Adequate secretion. On the other hand, it makes up for the lack of traditional Pu'er tea taste and the lack of aroma, which plays an important role in improving the quality of Pu'er tea. This is because the strain secretes aroma substances (such as cedar enol, anti- Syringene, 1-methyl-4-(1-methyl-vinylidene)-cycloethylene, etc., add special aroma components; in terms of taste, the taste is more pure because it avoids the influence of bacteria.

The invention obtains a Lactobacillus pentosus TMCC 70009CGMCC which can produce γ-aminobutyric acid by the traditional method of separating and purifying, sieving and rescreening by conventional plate dilution coating, preliminary screening and rescreening. No.8685. The strain was cultured in an ISP 2 medium at 35 ° C for 3 days, and its γ-aminobutyric acid content was measured to be 1.6 μg / mL. The experiment proves that the strain can be applied to the production of Pu'er tea. On the one hand, the fermentation cycle can be shortened significantly, and the fermentation cycle can be shortened to 3-4 weeks. On the other hand, it has the effect of improving the quality of Pu'er tea. The tea soup of Pu'er cooked tea has fresh floral fragrance, rich taste and richness. It not only makes up for the lack of traditional Pu'er cooked tea taste, but also has obvious aroma. The quality of Pu'er cooked tea is obviously improved in aroma, soup color and taste. Moreover, the content of γ-aminobutyric acid is as high as 1.5-2.2%. (mass/mass concentration), so that Pu'er cooked tea not only has the effect of lowering blood pressure, improving brain function, enhancing long-term memory and improving liver and kidney function, but also avoiding caffeine due to high content of γ-aminobutyric acid. Exciting and stimulating, it helps to calm the nerves. In addition, with the proliferation of Lactobacillus pentosus TMCC 70009CGMCC No.8685, the amount of enzyme secreted by it is also increased. Under the action of cellulase and glucoamylase, the contents of tea leaves are oxidized and decomposed. Polymerization, macromolecular insoluble matter can be converted into soluble small molecular substances, which enriches the taste of Pu'er tea. Its rich enzyme system also inhibits the growth of other bacteria, especially a few harmful microorganisms, and reduces the fermentation production of Pu'er tea. The risk of failure in . The invention provides an important application basis for the development of Pu'er tea new products and provides new microbial resources for the development of Pu'er tea industry.

The present invention will be further described in detail below in conjunction with specific embodiments.

detailed description

The percentage concentration is mass/mass (W/W, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, unless otherwise specified). Percent concentration in units of mL/100 mL).

The various biological materials described in the examples are obtained by merely providing an experimentally obtained route for the purpose of specific disclosure and should not be a limitation on the source of the biological material of the present invention. In fact, the source of the biological material used is extensive, and any biological material that can be obtained without violating laws and ethics can be replaced by the instructions in the examples.

The embodiments are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given. The embodiments will be helpful for understanding the present invention, but the scope of protection of the present invention is not limited to the following embodiments. .

Example 1. Isolation, Identification and Preservation of Yeast (Arxula adeninivorans) TMCC70007CGMCC No.8683, which Produces Highly Active Polyphenol Oxidase

I. Isolation and purification of TMCC 70007 strain

PDA separation medium formula: glucose 20g, potato 200g, agarose 2g, distilled water 1000mL, pH natural (6.8-7.3);

PDA medium can be used for the isolation and culture of fungi.

ISP 2 purification medium formula: peptone 4g, glucose 4g, yeast extract 5g, agarose 2g, distilled water 1000mL, pH natural (6.8-7.3), sterilization at 121 °C for 40min, standby;

ISP 2 medium can be used for the purification of bacteria, actinomycetes, and yeast.

Fresh Pu'er tea fermented tea samples were collected from traditional sputum piles, and one strain was isolated from PDA plates, named TMCC 70007, and purified by ISP 2 purification medium. The separation and purification method comprises the following steps:

1) The prepared solid medium is heated and melted, cooled to about 45 ° C, poured into a sterile Petri dish, about 20 mL per dish, and cooled for use.

2) The Pu'er tea sample to be separated is taken, ground and pulverized, and 1 g is taken, added to a test tube containing 9 mL of sterile water in a clean bench, and shaken to prepare a bacterial suspension, and the concentration is recorded as 10 ⁰ .

3) Gradually dilute by ten-fold dilution method and dilute 7 times. The ten-fold dilution method means that the concentration of the bacterial suspension in the next tube is one tenth of that of the previous tube.

4) Inject 500 μL of the bacterial suspensions labeled with concentrations of 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , and 10 ^-7 into the prepared culture dish and use a coating stick Uniform coating, three repetitions per concentration for easy comparison. After completion, the cells were cultured at 35-45 ° C and observed once every 24 hours.

5) After 2-5 days of culture, single colonies were picked and transferred to fresh ISP 2 medium, and purified by streaking multiple times until a purified strain was obtained.

2. Molecular biological identification of TMCC 70007 strain

The 16S rRNA gene sequence of the TMCC 70007 strain is shown in SEQ ID NO: 1 in the sequence listing, and the ITS sequence is shown in SEQ ID NO: 2 in the sequence listing, and is identified as a yeast capable of producing polyphenol oxidase (Arxula adeninivorans or Blastobotrys adeninivorans, the same species). Synonym).

3. Determination of the enzyme activity of polyphenol oxidase produced by TMCC 70007 strain

The strain was cultured in an ISP 2 medium at 35 ° C for 5 days, and the enzyme activity of the polyphenol oxidase was measured to be 1800 U/mL, while the enzyme activities of other strains were generally only 100-1600 U/mL.

4. Morphological characteristics and optimum growth conditions of TMCC 70007 strain

Growing in the range of pH 4-9, the optimum growth pH is 5-7;

Growing in the temperature range of 4-50 ° C, the optimum growth temperature is 35-45 ° C;

After culturing on ISP 2 medium for 48 hours at 35 ° C, the cells were observed under the microscope: the colonies were neat and milky white, the surface was moist and lustrous, the hyphae penetrated into the medium, and the diameter was about 0.8 mm; the growth temperature was 35-45 ° C. Observed under the microscope: the cells are oval and have pseudofilaria;

After culturing on PDA medium for 3 days at 35 ° C, it was observed under a microscope: the colonies were milky white, 3-4 mm in diameter, rounded at the edges, mounded, slightly wet, non-smooth, with aroma of wine, slightly yellow on the back of the colon; After microscopic examination, the cells were observed to be elliptical or nearly circular, with a large volume, and budded on the opposite side.

V. Deposit of TMCC 70007 strain

The yeast (Arxula adeninivorans or Blastobotrys adeninivorans, homologous) TMCC 70007 strain which can produce polyphenol oxidase of the present invention has been deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 6, 2014 (Address: Beijing No. 3, No. 1 Beichen West Road, Chaoyang District, the city, the deposit number is CGMCC No.8683.

Example 2: Application of polyphenol oxidase-producing yeast (Arxula adeninivorans) TMCC 70007CGMCC No.8683 in Pu'er tea production

The Pu'er tea is produced by using a yeast (Arxula adeninivorans) TMCC 70007CGMCC No. 8683 which can produce polyphenol oxidase, and the specific method comprises the following steps;

1) Tidal water of Pu'er hair tea: first add Pu'er hair tea to water to a moisture content of 20%-40% (volume/volume percentage concentration), and then sterilize at 121 °C for 30 min.

2) Fermentation: seed solution of yeast (Arxula adeninivorans) TMCC 70007CGMCC No. 8683 in the growth index phase (growth index period refers to culture in ISP 2 liquid medium under aerobic conditions for 8-10 h) at 8% Inoculation amount (0.5%-15%, weight/volume percentage concentration) is inoculated into the tidal Pu'er tea, and the fermentation is carried out at a temperature of 45 ° C (35-45 ° C) for 2-6 weeks. Make water every other week and turn it over to get Pu'er cooked tea.

For the quality test of 7572 Pu'er cooked tea in 2012 and Pu'er cooked tea of the present invention, the test items and methods are referred to the national standard (GB) method, and the content of the tea brown pigment is detected by systematic analysis.

The test results are shown in Table 1.

Table 1 Quality test results of Pu'er cooked tea (quality / mass percentage)

Test items	Da Yi 2012 7572 Pu'er cooked tea	Pu'er cooked tea of the present invention
Theanine	2.3%	3.8%
Tea polyphenol	15.4%	14.2-15%
Water extract	34.5%	39%
Soluble sugar	3.2%	4.6%
Tea brown pigment	10.2%	16-18%

After testing, the Pu'er cooked tea tea soup produced by the above method is bright red, bright and fragrant, smooth and slightly sweet. After testing, the content of theanine is 3.8% (mass/mass percentage, the same below) ), the tea polyphenol content is 14.2-15%, the water extract content is 39%, the soluble sugar content is 4.6%, the tea brown pigment content is as high as 16-18%, and the tea brown pigment content in Pu'er hair tea is very low, about 0.3%, the content of tea brown pigment in ordinary Pu'er cooked tea is generally only 8-15%, indicating that the yeast (Arxula adeninivorans) TMCC 70007CGMCC No.8683 can produce highly active polyphenol oxidase, which promotes tea polyphenols. Oxidative polymerization occurs under the catalysis of phenol oxidase to produce high concentrations of oxidation products such as theaflavins, theaflavins and thearubigins, thereby forming a high quality Pu'er cooked tea. In addition, with the proliferation of the yeast (Arxula adeninivorans) TMCC 70007CGMCC No.8683, the amount of enzyme secreted by the yeast (Arxula adeninivorans) is also increased, under the action of enzymes such as protease, cellulase, pectinase and glucoamylase. The content of reducing sugar, amino acid, soluble carbohydrate and hydrated pectin is increasing, which enriches the taste of Pu'er tea. Its rich enzyme system also inhibits the growth of other bacteria, especially a few harmful microorganisms, and reduces Pu'er. The risk of failure in tea fermentation production.

Example 3, isolation, identification and preservation of Lactobacillus pentosus TMCC70009CGMCC No.8685 which can produce γ-aminobutyric acid

I. Isolation and purification of TMCC 70009 strain

KMB separation medium formula: peptone 20g, glycerol 10g, K ₂ HPO ₄ 1.5g, MgSO ₄ .7H ₂ O 1.5g, agarose 15g, distilled water 1000mL, pH 5.6, sterilized at 121 ° C for 40min, standby;

KMB medium can be used for the isolation and culture of Lactobacillus pentosus.

ISP 2 purification medium formula: peptone 4g, glucose 4g, yeast extract 5g, agarose 2g, distilled water 1000mL, pH natural (6.8-7.3), sterilization at 121 °C for 40min, standby;

ISP 2 medium can be used for the purification of bacteria, actinomycetes, and yeast.

First, the fresh Pu'er tea fermented tea sample was collected from the traditional pile, and 300 strains of Lactobacillus pentosus were isolated by KMB plate, and then purified by ISP 2 medium purification medium. The separation and purification method comprises the following steps:

1) The prepared solid medium is heated and melted, cooled to about 45 ° C, poured into a sterile Petri dish, about 20 mL per dish, and cooled for use.

2) The Pu'er tea sample to be separated is taken, ground and pulverized, and 1 g is taken, added to a test tube containing 9 mL of sterile water in a clean bench, and shaken to prepare a bacterial suspension, and the concentration is recorded as 10 ⁰ .

3) Gradually dilute by ten-fold dilution method and dilute 7 times. The ten-fold dilution method means that the concentration of the bacterial suspension in the next tube is one tenth of that of the previous tube.

4) Inject 500 μL of the bacterial suspensions labeled with concentrations of 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , and 10 ^-7 into the prepared culture dish and use a coating stick Uniform coating, three repetitions per concentration for easy comparison. After completion, the cells were cultured at 20-50 ° C, and observed once every 24 hours;

5) After 3-7 days of culture, single colonies were picked and transferred to fresh ISP 2 medium, and purified by streaking multiple times until a purified strain was obtained, which was named TMCC 70009.

2. Molecular biological identification of TMCC 70009 strain

The 16S rRNA gene sequence of the TMCC 70009 strain is shown in SEQ ID NO: 3 in the Sequence Listing, and is identified as Lactobacillus pentosus which can produce γ-aminobutyric acid.

3. Determination of the enzyme activity of polyphenol oxidase produced by TMCC 70009 strain

The strain was cultured in the ISP 2 medium at 35 ° C for 3 days, and the γ-aminobutyric acid content was found to be 1.6 μg / mL, while the other γ - aminobutyric acid produced by the Lactobacillus pentasaccharide strain was generally only 0.1μg/mL-1.8μ g/mL.

4. Morphological characteristics and optimal growth conditions of TMCC 70009 strain

After incubation on KMB medium for 72 hours at 35 ° C, milky white colonies were formed with smooth surface, neat colonies, no wrinkles at the edges, and a diameter of about 1 mm. Observed under a microscope, the cells were oblong and existed in a single or chain shape;

It can also grow under anaerobic conditions. It is a facultative anaerobic bacteria with a growth temperature between 20 and 50 ° C. It can not grow above 55 ° C.

V. Deposit of TMCC 70009 strain

The Lactobacillus pentosus TMCC70009 strain which can produce γ-aminobutyric acid has been deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 6, 2014 (Address: Beichen West Road, Chaoyang District, Beijing) No. 3, No. 1), the deposit number is CGMCC No.8685.

Example 4: Application of Lactobacillus pentosus TMCC70009CGMCC No.8685, which can produce γ-aminobutyric acid, in the production of Pu'er tea

Production of Pu'er tea by Lactobacillus pentosus TMCC 70009CGMCC No. 8685 which can produce γ-aminobutyric acid, and the specific method comprises the following steps;

1) Tide the Pu'er hair tea: add Pu'er hair tea to water to a water content of 45% (volume/volume percentage concentration, 25%-50%), and sterilize;

2) Fermentation: Lactobacillus pentosus TMCC 70009CGMCC No.8685, which is in the growth index phase (growth index period refers to culture in ISP 2 liquid medium under anaerobic or facultative anaerobic conditions for 16-18 h). The seed solution is inoculated into the tidal Pu'er tea with a 5% (2%-10%, weight/volume percentage) inoculum, and passed through 3-4 at 45 ° C (20-50 ° C). Weekly fermentation, hydrating and turning over every other week during the fermentation to obtain Pu'er cooked tea.

For the detection of γ-aminobutyric acid content of Dayi 2012 7572 Pu'er cooked tea and the Pu'er cooked tea of the present invention, the method is described by Berthelot colorimetry.

The results are shown in Table 2.

Table 2 Quality test results of Pu'er cooked tea (mass/mass concentration)

Test items	Dayi 2012 7572 Pu'er cooked tea	Pu'er cooked tea of the present invention
Γ-aminobutyric acid content	0.63%-0.86%	1.5-2.2%

The content of γ-aminobutyric acid was determined to be 1.5-2.2% (mass/mass percent concentration, the same below), while the content of γ-aminobutyric acid in Pu'er hair tea was very low, being 4.3 mg/100 g (0.0043%). The content of γ-aminobutyric acid in ordinary Pu'er tea is generally 0.32%-0.9%, indicating Lactobacillus pentosus (Lactobacillus Pentosus) TMCC 70009CGMCC No.8685 can produce high concentration of γ-aminobutyric acid, which can shorten the fermentation cycle significantly, and the fermentation cycle is only 3-4 weeks. The fermentation cycle of common methods is generally 6-8 weeks, because Under the artificial conditions, the optimal growth environment of the strain was created. The microorganisms were single and no other strains competed, and the amount of enzymes was sufficient. On the other hand, the Pu'er cooked tea tea soup produced by the above method has fresh floral fragrance, rich taste and richness, and makes up for the deficiency of the traditional Pu'er cooked tea taste and the aroma is not obvious, which plays an important role in improving the quality of Pu'er tea. The strain secretes a certain aroma substance with tea as a substrate, and adds a special aroma component; in the taste, the taste is more pure because the influence of the bacteria is avoided. γ-Aminobutyric acid makes Pu'er cooked tea not only have the effects of lowering blood pressure, improving brain function, enhancing long-term memory and improving liver and kidney function, but also avoiding the excitatory stimulation of caffeine due to the high content of γ-aminobutyric acid. It is good for sleeping in peace. In addition, with the proliferation of Lactobacillus pentosus TMCC 70009CGMCC No.8685, the amount of enzyme secreted by it is also increased. Under the action of cellulase and glucoamylase, the contents of tea leaves are oxidized and decomposed. Polymerization, macromolecular insoluble matter can be converted into soluble small molecular substances, which enriches the taste of Pu'er tea. Its rich enzyme system also inhibits the growth of other bacteria, especially a few harmful microorganisms, and reduces the fermentation production of Pu'er tea. The risk of failure in .

Industrial applicability

The invention separates and purifies the probiotic strain from the traditional plutonium fermentation sample, and applies it to the production process of Pu'er tea, which provides a new microbial resource for the development of Pu'er tea industry, and improves the quality of Pu'er tea and the new Pu'er tea product. The development provides an application foundation.

Claims (18)

1. A highly active polyphenol oxidase-producing yeast (Arxula adeninivorans) TMCC 70007 CGMCC No. 8683 can be produced.
2. The yeast according to claim 1, wherein the 16S rRNA gene sequence of the strain is as shown in SEQ ID NO: 1 in the sequence listing, and the ITS sequence is as shown in SEQ ID NO: 2 in the Sequence Listing.
3. The yeast according to claim 1, wherein the strain is cultured in an ISP 2 medium at 35 ° C for 5 days, and the enzyme activity of the polyphenol oxidase is determined to be 1800 U/mL.
4. The yeast according to any one of claims 1 to 3, characterized in that the morphological characteristics and optimum growth conditions of the strain are:

   Growing in the range of pH 4-9, the optimum growth pH is 5-7;

   Growing in the temperature range of 4-50 ° C, the optimum growth temperature is 35-45 ° C;

   After culturing on ISP 2 medium for 48 hours at 35 ° C, the cells were observed under the microscope: the colonies were neat and milky white, the surface was moist and lustrous, the hyphae penetrated into the medium, and the diameter was about 0.8 mm; the growth temperature was 35-45 ° C. Observed under the microscope: the cells are oval and have pseudofilaria;

   After culturing on PDA medium for 3 days at 35 ° C, it was observed under a microscope: the colonies were milky white, 3-4 mm in diameter, rounded at the edges, mounded, slightly wet, non-smooth, with aroma of wine, slightly yellow on the back of the colon; After microscopic examination, the cells were observed to be elliptical or nearly circular, with a large volume, and budded on the opposite side.
5. The use of a highly active polyphenol oxidase-producing yeast (Arxula adeninivorans) TMCC 70007 CGMCC No. 8683 in the production of Pu'er tea.
6. The use according to claim 5, characterized in that the Pu'er tea is produced by using the yeast (Arxula adeninivorans) TMCC 70007 CGMCC No. 8683 which can produce high activity polyphenol oxidase, comprising the following steps;

   1) Tide the Pu'er hair tea: add Pu'er hair tea to water to a moisture content of 20%-40% (volume/volume percent concentration), and sterilize;

   2) Fermentation: The seed solution of the yeast (Arxula adeninivorans) TMCC 70007 CGMCC No. 8683 in the growth index period is inoculated into the tided Puer tea with an inoculum of 0.5%-15% (weight/volume percent concentration). After 2-6 weeks of fermentation at a temperature of 35-45 ° C, the water is hydrated and turned over every other week during the fermentation to obtain Pu'er cooked tea.
7. The use according to claim 6, characterized in that the growth index period in the step 2) means culturing in an ISP 2 liquid medium under aerobic conditions for 8-10 h; the inoculum amount of the seed liquid is preferably It is 8% (weight/volume percent concentration); the fermentation temperature is preferably 45 °C.
8. The Pu'er cooked tea produced by the application of claim 6 or 7 has a theanine content of 3.8%, a tea polyphenol content of 14.2-15%, a water extract content of 39%, and a soluble sugar. The content is 4.6%, and the content of the tea brown pigment is 16-18%.
9. The Pu'er tea according to claim 8, wherein the tea soup has a bright red color, a distinct white scent, a smooth taste, and a slightly sweet taste.
10. Lactobacillus pentosus TMCC 70009 CGMCC No. 8685 can produce gamma-aminobutyric acid.
11. The Lactobacillus pentosus according to claim 10, wherein the 16S rRNA gene sequence of the strain is as shown in SEQ ID NO:3 in the Sequence Listing.
12. The Lactobacillus pentosus according to claim 10 or 11, wherein the strain is cultured in an ISP 2 medium at 35 for 3 days, and the γ-aminobutyric acid content is measured to be 1.6 μg/mL.
13. The Lactobacillus pentosus according to claim 10 or 11 or 12, characterized in that the morphological characteristics and optimum growth conditions of the strain are:

    After incubation on KMB medium for 72 hours at 35 ° C, milky white colonies were formed with smooth surface, neat colonies, no wrinkles at the edges, and a diameter of about 1 mm. Observed under a microscope, the cells were oblong and existed in a single or chain shape;

    It can also grow under anaerobic conditions. It is a facultative anaerobic bacteria with a growth temperature between 20 and 50 ° C. It can not grow above 55 ° C.
14. The use of Lactobacillus pentosus TMCC 70009 CGMCC No. 8685 which can produce γ-aminobutyric acid in the production of Pu'er tea.
15. The use according to claim 14, wherein the production of Pu'er tea by the Lactobacillus pentosus TMCC 70009 CGMCC No. 8685 which can produce γ-aminobutyric acid comprises the following steps;

    1) tidal water of Pu'er hair tea: adding Pu'er hair tea to water to a moisture content of 20%-50% (volume/volume percentage concentration, preferably 45%), sterilization;

    2) Fermentation: seeding solution of Lactobacillus pentosus TMCC 70009 CGMCC No. 8685 in the growth index period is inoculated with 2%-10% (weight/volume percentage) inoculation amount to the tided Pu'er tea. In the fermentation at 2-4 weeks at a temperature of 20-50 ° C, the water is replenished every other week during the fermentation and turned over to obtain Pu'er cooked tea.
16. The use according to claim 15, wherein the growth index period in the step 2) means culturing in an ISP 2 liquid medium under anaerobic or facultative anaerobic conditions for 16-18 h; the seed liquid The inoculum amount is preferably 5% (weight/volume percent concentration); the fermentation temperature is preferably 45 °C.
17. A Pu-erh tea produced by the application of claim 15 or 16, wherein the γ-aminobutyric acid content is from 1.5 to 2.2% (mass/mass concentration).
18. The Pu'er tea according to claim 17, the tea soup has the characteristics of fresh floral fragrance, mellow taste and rich layers.