CN105754887B - Lactobacillus pentosus for producing gamma-aminobutyric acid and application of lactobacillus pentosus in production of Pu' er tea - Google Patents
Lactobacillus pentosus for producing gamma-aminobutyric acid and application of lactobacillus pentosus in production of Pu' er tea Download PDFInfo
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Abstract
The invention discloses a Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 capable of generating gamma-aminobutyric acid. Experiments prove that when the strain is applied to the production of Pu 'er tea, on one hand, the fermentation period can be obviously shortened to 3-4 weeks, and on the other hand, the strain has the effect of improving the quality of the Pu' er tea, the tea soup of the Pu 'er ripe tea produced by the strain through fermentation has fresh flower fragrance, mellow taste and rich layers, not only makes up the defects of flat taste and unobvious fragrance of the traditional Pu' er ripe tea, and ensures that the quality of the Pu 'er ripe tea is obviously improved in fragrance, liquor color and taste, but also ensures that the content of gamma-aminobutyric acid reaches 1.5-2.2% (mass/mass percentage concentration), so that the Pu' er ripe tea not only has the effects of reducing blood pressure, improving brain function, enhancing long-term memory, improving liver and kidney functions and the like, but also avoids the exciting and stimulating effect of caffeine due to higher content of gamma-aminobutyric acid, is beneficial for tranquilizing and sleeping. The invention provides an important application basis for the development of new Pu 'er tea products and provides a new microbial resource for the development of the Pu' er tea industry.
Description
Technical Field
The invention relates to Lactobacillus pentosus (Lactobacillus pentosus) capable of generating gamma-aminobutyric acid and application thereof in production of Pu' er tea in the field of microorganisms.
Background
Gamma-aminobutyric acid (GABA) is a naturally occurring functional amino acid. The research shows that the gamma-aminobutyric acid has the physiological activities of reducing blood pressure, improving brain function, enhancing long-term memory, improving liver and kidney functions and the like. Gamma-aminobutyric acid is widely present in animals and plants in nature, is present only in nerve tissues in animals, is an important inhibitory neurotransmitter in the central nerve of mammals, and is involved in various metabolisms. Gamma-aminobutyric acid is formed by decarboxylation of L-glutamic acid by glutamate dehydrogenase (GAD).
Many Pu 'er tea not only contains caffeine, but also has indexes far higher than those of green tea and oolong tea, and in the test of tea detection center of department of agriculture, the caffeine content in Pu' er tea is as high as 4.41% (mass/mass percentage concentration). The caffeine content of the Pu 'er tea is the highest in various known tea leaves at present, but the normal sleep of people is not influenced after the Pu' er ripe tea is drunk. Many scholars think that Pu ' er tea affects the main ' fierce ' of human sleep in the repeated processing process, which is caused by gradual disappearance of caffeine. However, the conclusion is not valid after the Pu' er tea is chemically analyzed. The answer is gamma-aminobutyric acid (GABA), because the basic physiological function of GABA is to reduce neuronal activity, prevent neuronal overheating, and act as a nerve-calming and thus antagonizing receptor for caffeine. In other words, it is the "effect" of GABA on caffeine to excite the central nervous system of the human body that is counteracted.
The preparation method of gamma-aminobutyric acid (GABA) mainly comprises a chemical synthesis method and a biological synthesis method. The chemical synthesis method is reported in patent documents, has high cost and low yield, and commonly uses dangerous solvents, even toxic solvents in the production process. Therefore, GABA prepared by a chemical synthesis method cannot be used in the food field and cannot be considered as a natural food additive. Compared with the biosynthesis method, the method is safe and low in cost. According to the recent research reports and patent literature reports, some high-safety microorganisms such as lactic acid bacteria, yeast and aspergillus have been applied to the preparation of GABA foods. In 2009, 9/27 days, the ministry of health of China officially approved gamma-aminobutyric acid, colostrum basic protein, conjugated linoleic acid, glyceryl oleate, lactobacillus plantarum (strain number ST-III) and eucommia seed oil as new resource food. From this time, a new era has been reached for gamma-aminobutyric acid.
Lactic acid bacteria are gram-positive bacteria, and many strains have high GAD (glutamic acid decarboxylase) activity and can produce GABA (gamma-aminobutyric acid) in an enriched manner, such as Lactobacillus plantarum M-10, Lactobacillus acidophilus, Lactobacillus brevis hjxj-01 and the like. As an auxiliary starter, lactobacillus pentosus has been widely used in the production of yogurt, cheese, ice cream, fermented meat products, chocolate, bean products, and the like. Due to the problems of harsh reaction conditions, expensive natural raw materials and poor safety of chemical methods for synthesizing GABA, the GABA is difficult to be widely applied. Among microorganisms, GAD is currently found in a variety of lactic acid bacteria, yeasts and molds.
In the research field of Pu' er tea, the acquisition of probiotics is relatively difficult in the early stage due to the lack of systematic research from the aspect of microbial specialty. With the maturity of a microorganism separation means and the high-speed development of modern bioinformatics, it becomes possible to separate and purify probiotic strains from a traditional pile fermentation sample and apply the probiotic strains to a production process of Pu ' er tea, and the probiotic strains play an important role in improving the quality of the Pu ' er tea and the development of the Pu ' er tea industry.
Disclosure of Invention
The first purpose of the invention is to provide a lactobacillus pentosus strain capable of producing gamma-aminobutyric acid (GABA).
The lactobacillus pentosus (Lactobacillus pentosus) capable of producing gamma-aminobutyric acid provided by the invention is named as TMCC70009, and the strain is preserved in China general microbiological culture Collection center (address: Beijing city Shanghai district Beichen Xilu No. 1 Hospital No. 3) in 06 days 2014, and the preservation number is CGMCC No. 8685.
The 16S rRNA gene sequence of the TMCC70009 strain is shown as a sequence 1 in a sequence table, and is analyzed and identified as Lactobacillus pentosus (Lactobacillus pentosus) capable of producing gamma-aminobutyric acid.
The strain is cultured in an ISP 2 culture medium at 35 ℃ for 3 days in a liquid state, and the content of gamma-aminobutyric acid of the strain is measured to reach 1.6 mu g/mL.
The invention can generate the morphological characteristics and the optimal growth conditions of the Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 of gamma-aminobutyric acid:
culturing on ISP 2 culture medium at 35 deg.C for 72 hr to form milky colony with smooth surface, uniform colony, no wrinkle at edge, and diameter of about 1 mm; observed under a microscope, the cells are in an oblong shape and exist in a single or chain shape;
can grow under anaerobic condition, is facultative anaerobe, and can not grow at the temperature of 20-50 ℃ and above 55 ℃.
The Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 capable of generating the gamma-aminobutyric acid has the effect of improving the quality of the Pu 'er tea, and can be applied to the production process of the Pu' er tea.
The invention also provides a method for producing Pu' er tea by using Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 which can generate gamma-aminobutyric acid, and the specific method comprises the following steps;
1) carrying out moistening on the Pu' er raw tea: adding water into Pu' er tea until the water content reaches 20% -50% (volume/volume percentage concentration, preferably 45%), and sterilizing;
2) fermentation: inoculating seed liquid of Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 in a growth index period into wet Pu 'er raw tea at an inoculation amount of 2-10% (weight/volume percentage concentration), fermenting at 20-50 ℃ for 3-4 weeks, and supplementing water and turning over piles every other week during fermentation to obtain the Pu' er ripe tea.
In the production method of the puer tea, the growth exponential phase in the step 2) refers to that the puer tea is cultured in an ISP 2 liquid culture medium for 16-18h under anaerobic or facultative anaerobic conditions; the inoculation amount of the seed liquid is preferably 5% (weight/volume percentage concentration); the fermentation temperature is preferably 45 ℃.
The Pu-Er ripe tea soup produced by the method has fresh flower fragrance, mellow taste and rich layers; through detection, the content of the gamma-aminobutyric acid is 1.5-2.2% (mass/mass percentage concentration, the same is shown below), while the content of the gamma-aminobutyric acid in the Pu' er raw tea is very low, namely 4.3mg/100g (0.0043%). The content of gamma-aminobutyric acid in the ordinary Pu' er ripe tea is generally 0.32-0.9%, which shows that Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 can generate high-concentration gamma-aminobutyric acid, on one hand, the fermentation period can be obviously shortened, the fermentation period is only 3-4 weeks, and the fermentation period of the ordinary method is generally 6-8 weeks, because the optimal growth environment of the strain is created under artificial conditions, the microorganism is single, no other strains compete, and the enzyme amount is sufficiently secreted. On the other hand, the strain makes up the defects of flat taste and unobvious aroma of the traditional Pu-Er ripe tea, plays an important role in improving the quality of the Pu-Er tea, because the strain takes the tea as a substrate to secrete aroma substances (such as cedrol, trans-caryophyllene, 1-methyl-4- (1-methyl-vinylidene) -cyclic ethylene and the like) and adds special aroma components; in terms of taste, the taste is purer as the influence of mixed bacteria is avoided.
The Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 capable of generating gamma-aminobutyric acid is obtained from a traditional separation and purification method of plate dilution coating, primary screening and secondary screening in a self-stacked Pu' er tea fermentation sample. The strain is cultured in an ISP 2 culture medium at 35 ℃ for 3 days in a liquid state, and the content of gamma-aminobutyric acid of the strain is measured to reach 1.6 mu g/mL. Experiments prove that when the strain is applied to the production of Pu 'er tea, on one hand, the fermentation period can be obviously shortened to 3-4 weeks, and on the other hand, the strain has the effect of improving the quality of the Pu' er tea, the tea soup of the Pu 'er ripe tea produced by the strain through fermentation has fresh flower fragrance, mellow taste and rich layers, not only makes up the defects of flat taste and unobvious fragrance of the traditional Pu' er ripe tea, and ensures that the quality of the Pu 'er ripe tea is obviously improved in fragrance, liquor color and taste, but also ensures that the content of gamma-aminobutyric acid reaches 1.5-2.2% (mass/mass percentage concentration), so that the Pu' er ripe tea not only has the effects of reducing blood pressure, improving brain function, enhancing long-term memory, improving liver and kidney functions and the like, but also avoids the exciting and stimulating effect of caffeine due to higher content of gamma-aminobutyric acid, is beneficial for tranquilizing and sleeping. In addition, with the mass propagation of lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685, the amount of enzyme secreted by the lactobacillus pentosus is increased, under the action of cellulase and saccharifying enzyme, the content substances in the tea are oxidized, decomposed and polymerized, the macromolecular insoluble substances can be converted into soluble micromolecular substances, the taste of the Pu 'er tea is enriched, the enrichment enzyme system also inhibits the breeding of other mixed bacteria, particularly a few harmful microorganisms, and the failure risk in the fermentation production of the Pu' er tea is reduced. The invention provides an important application basis for the development of new Pu 'er tea products and provides a new microbial resource for the development of the Pu' er tea industry.
The present invention will be described in further detail with reference to specific examples.
Detailed Description
The percentage concentration is a mass/mass (W/W, unit g/100g) percentage concentration, a mass/volume (W/V, unit g/100mL) percentage concentration, or a volume/volume (V/V, unit mL/100mL) percentage concentration, unless otherwise specified.
The various biomaterials described in the examples are obtained by way of experimental acquisition for the specific purpose disclosed and should not be construed as limiting the source of the biomaterials of the present invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
The embodiments are provided in order to provide detailed embodiments and specific procedures, which will help understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.
Example 1 isolation, identification and preservation of Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 that produces gamma-aminobutyric acid
Separation and purification of TMCC70009 strain
KMB isolation medium formula: peptone 20g, glycerol 10g, K2HPO41.5g，MgSO4.7H21.5g of O, 15g of agarose and 1000mL of distilled water, wherein the pH value is 5.6, and the mixture is sterilized at 121 ℃ for 40min for later use;
the KMB culture medium can be used for separating and culturing lactobacillus pentosus.
ISP 2 purification medium formula: 4g of peptone, 4g of glucose, 5g of yeast extract, 2g of agarose and 1000mL of distilled water, wherein the pH is natural (6.8-7.3), and the peptone is sterilized at 121 ℃ for 40min for later use;
ISP 2 culture medium can be used for purifying bacteria, actinomycetes and yeast.
Collecting fresh Pu' er tea fermented tea samples from traditional pile fermentation, separating by a KMB plate to obtain 300 strains of lactobacillus pentosus, and purifying by using an ISP 2 culture medium purification culture medium. The separation and purification method comprises the following steps:
1) heating and melting the prepared solid culture medium, cooling to about 45 ℃, pouring into sterile culture dishes with about 20mL of each dish, and cooling for later use.
2) Grinding and pulverizing Pu her tea sample to be separated, collecting 1g, adding into a test tube containing 9mL sterile water in a super clean bench, and oscillating to obtain bacterial suspension with concentration of 100。
3) Diluting step by step according to ten-fold dilution method until 7 times. Ten-fold dilution means that the concentration of the bacterial suspension in the next tube is one tenth of the concentration in the previous tube.
4) Will be marked with 10-2、10-3、10-4、10-5、10-6、10-7500 mul of bacterial suspension with concentration is respectively injected into a prepared culture dish and evenly coated by a coating rod, and each concentration is repeated for three times, thereby facilitating comparison. Culturing at 20-50 deg.C, and observing for 1 time every 24 hr;
5) after culturing for 3-7 days, selecting a single colony, transferring the single colony to a fresh ISP 2 culture medium for culturing, and streaking and purifying for many times until a purified strain is obtained, wherein the strain is named as TMCC 70009.
II, molecular biological identification of TMCC70009 strain
The 16S rRNA gene sequence of the TMCC70009 strain is shown as a sequence 1 in a sequence table, and is analyzed and identified as Lactobacillus pentosus (Lactobacillus pentosus) capable of producing gamma-aminobutyric acid.
Thirdly, measuring the enzyme activity of polyphenol oxidase produced by TMCC70009 strain
The strain is cultured in an ISP 2 culture medium at 35 ℃ for 3 days in a liquid state, and the content of the gamma-aminobutyric acid is measured to reach 1.6 mu g/mL, while the content of the gamma-aminobutyric acid produced by other lactobacillus pentosus strains is generally only 0.1 mu g/mL-1.8 mu g/mL.
Fourthly, morphological characteristics and optimal growth conditions of TMCC70009 strain
Culturing on KMB culture medium at 35 deg.C for 72 hr to form milky colony with smooth surface, uniform colony, no wrinkle at edge, and diameter of about 1 mm; observed under a microscope, the cells are in an oblong shape and exist in a single or chain shape;
can grow under anaerobic condition, is facultative anaerobe, and can not grow at the temperature of 20-50 ℃ and above 55 ℃.
Five, preservation of TMCC70009 Strain
The Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009 strain capable of generating gamma-aminobutyric acid is preserved in China general microbiological culture Collection center (address: Beijing area rising district, West Lu No. 1 Hospital No. 3) in 06 months in 2014, and the preservation number is CGMCC No. 8685.
Example 2: application of Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 capable of generating gamma-aminobutyric acid in production of Pu' er tea
The method for producing the Pu' er tea by using Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 which can produce gamma-aminobutyric acid comprises the following steps;
1) carrying out moistening on the Pu' er raw tea: adding water into Pu' er tea until the water content reaches 45% (volume/volume percentage concentration, 25% -50%), and sterilizing;
2) fermentation: inoculating seed liquid of Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 in a seed liquid of 5 percent (2 to 10 percent, weight/volume percent concentration) into Pu 'er raw tea with moist water in a growth index period (the growth index period refers to 16 to 18 hours of culture in an ISP 2 liquid culture medium under an anaerobic or facultative anaerobic condition), fermenting for 3 to 4 weeks at the temperature of 45 ℃ (20 to 50 ℃), and replenishing water and turning over piles every other week during the fermentation period to obtain the Pu' er ripe tea.
The content of gamma-aminobutyric acid in 7572 Pu 'er ripe tea in 2012 and the Pu' er ripe tea of the invention are detected by a Bertholt colorimetric method.
The results are shown in Table 1.
TABLE 1 quality test results (mass/mass percentage concentration) of Pu-Er ripe tea
| Detecting items | 7572 Pu' er ripe tea of Dayi 2012 | The invention relates to Pu-Er ripe tea |
| Content of gamma-aminobutyric acid | 0.63％-0.86％ | 1.5-2.2％ |
Through detection, the content of the gamma-aminobutyric acid is 1.5-2.2% (mass/mass percentage concentration, the same is shown below), while the content of the gamma-aminobutyric acid in the Pu' er raw tea is very low, namely 4.3mg/100g (0.0043%). The content of gamma-aminobutyric acid in the ordinary Pu' er ripe tea is generally 0.32-0.9%, which shows that Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 can generate high-concentration gamma-aminobutyric acid, on one hand, the fermentation period can be obviously shortened, the fermentation period is only 3-4 weeks, and the fermentation period of the ordinary method is generally 6-8 weeks, because the optimal growth environment of the strain is created under artificial conditions, the microorganism is single, no other strains compete, and the enzyme amount is sufficiently secreted. On the other hand, the Pu-Er ripe tea soup produced by the method has fresh flower fragrance, mellow taste and rich levels, makes up the defects of flat taste and unobvious fragrance of the traditional Pu-Er ripe tea, and plays an important role in improving the quality of the Pu-Er tea, because the strain takes the tea leaves as a substrate to secrete certain fragrant substances and increases special fragrant components; in terms of taste, the taste is purer as the influence of mixed bacteria is avoided. The gamma-aminobutyric acid enables the Pu-Er ripe tea to have the effects of reducing blood pressure, improving brain function, enhancing long-term memory, improving liver and kidney functions and the like, and simultaneously, due to the high content of the gamma-aminobutyric acid, the excitation stimulation effect of caffeine is avoided, and the nerve calming and sleeping are facilitated. In addition, with the mass propagation of Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685, the amount of enzyme secreted by the Lactobacillus pentosus is increased, under the action of cellulase and saccharifying enzyme, the content substances in the tea are oxidized, decomposed and polymerized, the macromolecular insoluble substances can be converted into soluble micromolecular substances, the taste of the Pu 'er tea is enriched, the enrichment enzyme system also inhibits the breeding of other mixed bacteria, particularly a few harmful microorganisms, and the failure risk in the fermentation production of the Pu' er tea is reduced. The invention provides an important application basis for the development of new Pu 'er tea products and provides a new microbial resource for the development of the Pu' er tea industry.
Claims (8)
1. Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 used in Pu 'er tea production and capable of generating gamma-aminobutyric acid is obtained by collecting fresh Pu' er tea fermented tea samples from traditional pile fermentation, separating by a KMB plate to obtain Lactobacillus pentosus, and purifying by using an ISP 2 culture medium;
the culture medium formula for KMB plate separation is as follows: peptone 20g, glycerol 10g, K2HPO41.5g，MgSO4.7H2O1.5g, 15g of agarose, 1000mL of distilled water, pH 5.6, sterilization at 121 ℃ for 40 min;
the ISP 2 culture medium comprises the following components in parts by weight: 4g of peptone, 4g of glucose, 5g of yeast extract, 2g of agarose and 1000mL of distilled water, wherein the pH value is 6.8-7.3, and the sterilization is carried out at 121 ℃ for 40 min.
2. Lactobacillus pentosus according to claim 1, characterized in that: the 16S rRNA gene sequence of the strain is shown as a sequence 1 in a sequence table.
3. Lactobacillus pentosus according to claim 1, characterized in that: the strain is cultured in an ISP 2 culture medium at 35 ℃ for 3 days in a liquid state, and the content of gamma-aminobutyric acid of the strain is measured to reach 1.6 mu g/mL.
4. Lactobacillus pentosus according to claim 1, characterized in that: the morphological characteristics and the optimal growth conditions of the strain are as follows:
culturing on KMB culture medium at 35 deg.C for 72 hr to form milky colony with smooth surface, uniform colony, no wrinkle at edge, and diameter of about 1 mm; observed under a microscope, the cells are in an oblong shape and exist in a single or chain shape;
can grow under anaerobic condition, is facultative anaerobe, and can not grow at the temperature of 20-50 ℃ and above 55 ℃.
5. Use of lactobacillus pentosus (lactobacillus pentosus) TMCC70009CGMCC No.8685 capable of producing gamma-aminobutyric acid according to any one of claims 1 to 4 in the production of puer tea.
6. Use according to claim 5, characterized in that: the method for producing the Pu' er tea by using the Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 which can produce gamma-aminobutyric acid comprises the following steps;
1) carrying out moistening on the Pu' er raw tea: adding water into Pu' er raw tea until the water content reaches 45% by volume, and sterilizing;
2) fermentation: inoculating seed liquid of Lactobacillus pentosus (Lactobacillus pentosus) TMCC70009CGMCC No.8685 in a growth index period into moisturized Pu 'er raw tea in an inoculation amount of 2-10% of weight/volume percentage concentration, fermenting at 20-50 ℃ for 3-4 weeks, and supplementing water and turning over piles every other week during fermentation to obtain the Pu' er ripe tea.
7. Use according to claim 6, characterized in that: the growth exponential phase in the step 2) refers to culturing for 16-18h in an ISP 2 liquid culture medium under anaerobic or facultative anaerobic conditions; the inoculation amount of the seed liquid is 5%; the fermentation temperature was 45 ℃.
8. Use according to claim 6 or 7, characterized in that: the Pu-Er ripe tea soup produced by the method has fresh flower fragrance, mellow taste and rich layers; through detection, the content of the gamma-aminobutyric acid in the Pu-Er ripe tea produced by the method is 1.5-2.2% by mass percent.
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| CN106434779B (en) * | 2016-11-15 | 2019-10-29 | 华南农业大学 | Lactobacillus pentosus SS6 is producing the application in γ-aminobutyric acid |
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| CN107197966B (en) * | 2017-05-18 | 2020-07-31 | 华南农业大学 | Method for preparing GABA tea through microbial fermentation |
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