CN113969242B - Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and application thereof in preparation of gamma-aminobutyric acid products - Google Patents
Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and application thereof in preparation of gamma-aminobutyric acid products Download PDFInfo
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Abstract
The invention provides a saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and application thereof in preparing gamma-aminobutyric acid products, and relates to the technical field of food processing. The method specifically comprises the step of classifying and naming Saccharomyces cerevisiae with high yield of gamma-aminobutyric acid as Saccharomyces cerevisiae Saccharomyces cerevisiae CLNJ1, and preserving the Saccharomyces cerevisiae in China general microbiological culture collection center (CGMCC) No.23423 in 2021, 9 and 16 days. The invention also discloses an application of the saccharomyces cerevisiae CLNJ in preparing gamma-aminobutyric acid and a method for improving the yield of gamma-aminobutyric acid by adding tea powder into a fermentation medium. In addition, the invention discloses a method for preparing the Kangpu tea rich in gamma-aminobutyric acid by utilizing acetic acid bacteria and saccharomyces cerevisiae CLNJ, which has simple preparation process and is particularly suitable for large-scale industrial production. The prepared Kangpu tea has high nutritive value, is rich in gamma-aminobutyric acid, increases the nutrition and health care functions of the beverage, and has wide market demands and economic benefits.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and application thereof in preparation of gamma-aminobutyric acid products.
Background
Gamma-aminobutyric acid is a new resource food approved by the national ministry of health. Gamma-aminobutyric acid can lower blood pressure by inhibiting angiotensin peptide converting enzyme; gamma-aminobutyric acid can inhibit the shuttle removing reaction of glutamic acid, promote more glutamic acid to be combined with ammonia to generate urea to be discharged out of the body, and achieve the functions of invigorating liver and benefiting kidney; gamma-aminobutyric acid can inhibit nerve-induced airway smooth muscle contraction, capillary leakage and anaphylactic reaction, so as to control asthma; gamma-aminobutyric acid is involved in hormone secretion regulation as an inhibitory neurotransmitter, promoting release of growth hormone, corticotropin, glucocorticoid and gonadotropin; gamma-aminobutyric acid can promote energy metabolism of brain, activate cerebral blood flow, increase oxygen supply, finally recover brain cell function, improve nerve function, and achieve the functions of tranquilizing and allaying excitement and enhancing memory; gamma-aminobutyric acid can promote secretion of insulin in pancreas, and diabetes mellitus is effectively prevented; gamma-aminobutyric acid has effects of promoting alcohol metabolism, preventing obesity, preventing arteriosclerosis, regulating arrhythmia and preventing skin aging; gamma-aminobutyric acid can also be used as a therapeutic agent for uremia and CO poisoning. The development of GABA-enriched foods has become a hot spot of research nowadays, and the application prospect is very wide.
The Kangpu tea is a traditional natural fermentation beverage, and is prepared by fermenting tea syrup with microorganismssuch as saccharomycetes and acetic acid bacteria. The Kangpu tea contains partial tea extract, active microorganisms and metabolites thereof, and is a fermented beverage with multiple nutrition and health care functions. The main functional nutritional ingredients comprise tea polyphenol, gluconic acid, glucuronic acid, caffeine, saccharides, organic acid, protein, amino acid, vitamins, microelements, acetic acid, lactic acid, citric acid and the like, and contain various microorganisms beneficial to human bodies, so that the natural healthy beverage has the advantages of regulating physiological functions of the human bodies, improving immunity of the human bodies, regulating intestines and stomach, eliminating fatigue, promoting metabolism and enhancing vitality of the human bodies, and is a natural healthy beverage with great development potential.
The tea beverage rich in gamma-aminobutyric acid is prepared by fermenting tea syrup with saccharomycetes and acetic acid bacteria, and the beverage contains functional factor nutrients in the tea and gamma-aminobutyric acid, so that the tea beverage has the functions of nutrition and health care, is a health beverage suitable for people of all ages, and has wide development space and huge market demand.
Disclosure of Invention
The invention aims to provide a saccharomyces cerevisiae strain with high yield of gamma-aminobutyric acid, which is obtained through separation, purification and mutation breeding, wherein the classification of the saccharomyces cerevisiae strain is named as saccharomyces cerevisiae Saccharomyces cerevisiae CLNJ1, and the saccharomyces cerevisiae strain is preserved in China general microbiological culture collection center for 9 months and 16 months in 2021, and the preservation address is North Chen West Lu No. 1 and No. 3 in the Korean region of Beijing city, and the preservation number is CGMCC No.23423.
The invention further aims to provide an application of the saccharomyces cerevisiae strain CLNJ in preparing gamma-aminobutyric acid, wherein the saccharomyces cerevisiae strain CLNJ is fermented in a culture medium, and the content of gamma-aminobutyric acid in fermentation liquid is detected to be more than 5.6 g/L.
In a preferred embodiment, the culture medium comprises 18-22g/L peptone, 8-12g/L yeast extract powder and 18-22g/L glucose, and the fermentation condition is 28-32 ℃ and the shaking culture is carried out for 24-48 hours;
more preferably, the culture medium comprises 20g/L of peptone, 10g/L of yeast extract powder and 20g/L of glucose, and the fermentation condition is 30 ℃ and the shaking culture is carried out for 36 hours.
In a preferred embodiment, the culture medium further comprises 8-12g/L tea powder, and under the same fermentation condition, the yield of gamma-aminobutyric acid is up to more than 8.7g/L, and is increased by 11-55% compared with the culture medium without the tea powder;
preferably, the culture medium component also comprises 10g/L tea powder, more preferably, the 10g/L tea powder in the culture medium component is oolong tea powder.
The invention further aims to provide an application of the saccharomyces cerevisiae strain CLNJ in preparing a high-yield gamma-aminobutyric acid product.
In a preferred embodiment, the Saccharomyces cerevisiae strain CLNJ is used to prepare a fermented beverage.
More preferably, kang Pucha enriched in gamma-aminobutyric acid is prepared using acetic acid bacteria and said Saccharomyces cerevisiae CLNJ.
The invention also aims to provide a preparation method of Kang Pucha rich in gamma-aminobutyric acid, which specifically comprises the following steps:
(1) Preparing a fermentation base solution: taking tea leaves for brewing by boiling water, collecting brewed tea, and adding a carbon source when the temperature of the tea is reduced to 60 ℃ to obtain fermentation base liquid after the tea is fully dissolved;
(2) Culturing strains: respectively culturing in a proper culture medium to obtain acetobacter xylinum bacterial liquid and saccharomyces cerevisiae bacterial strain CLNJ1 bacterial liquid for later use;
(3) Preparing a fermented beverage: inoculating the bacterial liquid in the step (2) into the fermentation base liquid according to the set inoculation amount of 8-12wt% of the fermentation base liquid in the step (1), and fermenting and culturing to obtain a fermentation base liquid;
wherein, in the inoculation bacterial liquid, the mass ratio of the acetobacter xylosojae bacterial liquid to the saccharomyces cerevisiae bacterial strain CLNJ1 bacterial liquid is (0.1-10): 1, a step of;
(4) Preparing a food additive: adding acceptable food additives into the fermentation stock solution according to taste requirements for blending to obtain Kang Pucha rich in gamma-aminobutyric acid.
In a preferred embodiment, in the step (1), the tea leaves include any one or more tea varieties of black tea, oolong tea, white tea, green tea and the like;
the boiling water infusing times are 2-4 times, and the mass ratio of tea to boiling water is 1 (100-500); more preferably, the mass ratio of tea to boiling water is 1:100;
the carbon source comprises white granulated sugar or sucrose;
the mass ratio of the carbon source to the tea water is 1 (8-12), and more preferably, the mass ratio of the carbon source to the tea water is 1:10.
In a preferred embodiment, in the step (2), the medium for culturing the acetobacter xylinum comprises the following components in mass content: glucose 20-30g/L, soytone 8-12g/L, yeast extract powder 8-12g/L, disodium hydrogen phosphate 3-8g/L, ammonium citrate 0.3-0.8g/L, magnesium sulfate 0.1-0.3g/L, pH4.5-5.5, 121 ℃, sterilizing for 20min;
preferably, the culture medium for culturing the acetobacter xylosojae comprises the following components in mass content: 25g/L of glucose, 10g/L of peptone, 10g/L of yeast extract powder, 5g/L of disodium hydrogen phosphate, 0.5g/L of citric acid, 0.2g/L of magnesium sulfate, pH5.0 and sterilization for 20min for standby;
fermentation conditions for culturing the acetobacter xylosojae include: shake culturing at 28-32deg.C at rotation speed of 120-180 rpm for 36-60 hr; preferably, the culture is carried out under shaking at 30℃and at a rotation speed of 150 rpm for 48 hours.
In a preferred embodiment, the medium for culturing the saccharomyces cerevisiae strain CLNJ1 comprises the following components in mass content: dissolving 8-12g of peptone and 8-12g of yeast extract powder in 900mL of water, and sterilizing at 121 ℃ for 20min to obtain 900mL of culture medium for later use; dissolving 18-22g of glucose in 100mL of water, sterilizing at 115 ℃ for 15min, and adding into the 900mL of culture medium for later use;
preferably, the culture medium for culturing the saccharomyces cerevisiae strain CLNJ1 comprises the following components in mass content: dissolving 10g of peptone and 10g of yeast extract powder in 900mL of water, and sterilizing at 121 ℃ for 20min to obtain 900mL of culture medium for later use; 20g of glucose is dissolved in 100mL of water, sterilized for 15min at 115 ℃, and then added into the 900mL of culture medium for standby;
the fermentation conditions for culturing the saccharomyces cerevisiae strain CLNJ1 strain include: shake culturing at 28-32deg.C at rotation speed of 120-180 rpm for 24-48 hr; preferably, the culture is carried out at 30 ℃ under shaking at a rotation speed of 150 revolutions per minute for 36 hours.
In a preferred embodiment, in the step (3), the bacterial liquid in the step (2) is inoculated into the fermentation base liquid in a set inoculation amount of 10wt% of the fermentation base liquid in the step (1), and fermentation culture is carried out to obtain a fermentation base liquid;
in the inoculation bacterial liquid, the mass ratio of the acetobacter xylosojae bacterial liquid to the saccharomyces cerevisiae bacterial strain CLNJ1 bacterial liquid is 1:1, a step of;
the fermentation culture conditions are as follows: and (3) standing and fermenting for 6-14 days at 26-30 ℃.
In a preferred embodiment, in the step (4), the acceptable food auxiliary agents include food auxiliary agents capable of adjusting taste, such as fruit juice, honey, pectin, sucrose and/or citric acid, and the types of the auxiliary agents and the addition amount of the auxiliary agents are adjusted according to the taste requirements.
In a preferred embodiment, after obtaining the conpu tea rich in gamma-aminobutyric acid, the method further comprises the steps of homogenizing by a homogenizer, sterilizing, filling and the like.
Compared with the prior art, the saccharomyces cerevisiae for high-yield gamma-aminobutyric acid and the application thereof in preparing gamma-aminobutyric acid products have the following advantages:
1) The invention provides saccharomyces cerevisiae CLNJ1, which has the characteristic of high yield of gamma-aminobutyric acid, and through practical verification, the yield of gamma-aminobutyric acid is stable above 5.6g/L and is obviously higher than that of common saccharomyces cerevisiae gamma-aminobutyric acid. The genetic property of the mutant strain is stable, the capability of the strain for producing gamma-aminobutyric acid is measured after 10 times of passage genetic stability comparison, the problem of back mutation does not occur,
2) The invention provides the saccharomyces cerevisiae CLNJ1, which is used for preparing Kang Pucha rich in gamma-aminobutyric acid by combined fermentation with acetic acid bacteria and saccharomycetes, so that the cost is saved, the culture medium components of the saccharomyces cerevisiae are reduced, the preparation process is simple, and the method is particularly suitable for large-scale industrial production. The prepared Kangpu tea has high nutritive value, contains various nutritive substances such as tea polyphenol, D-glucaric acid-1, 4 lactone, glucuronic acid, caffeine, vitamins and the like, is rich in gamma-aminobutyric acid, increases the nutrition and health care functions of the beverage, and has wide market demands and economic benefits.
3) The Kang Pucha rich in gamma-aminobutyric acid prepared by using the saccharomyces cerevisiae CLNJ1 and the acetic acid bacteria has the gamma-aminobutyric acid content of 280mg/L, and the conpu tea prepared by using the acetic acid bacteria and the commercially available saccharomyces cerevisiae in the prior art does not contain the gamma-aminobutyric acid; the content of gamma-aminobutyric acid in the Kangpu tea which is fermented by combining acetic acid bacteria, saccharomyces cerevisiae and lactobacillus plantarum is only 78mg/L. The invention has the beneficial effects of reducing the cost and enhancing the efficiency under the condition of reducing the consumption of raw materials, and also improves the nutritive value of the product.
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These and/or other aspects and advantages of the present invention will become more apparent and more readily appreciated from the following detailed description of the embodiments of the invention, taken in conjunction with the accompanying drawings, wherein:
FIG. 1 is a phylogenetic tree of Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain CLNJ1 according to an embodiment of the present invention;
FIG. 2 is a diagram showing colony morphology before and after mutagenesis of Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain CLNJ1, with the left side being colony morphology before mutagenesis and the right side being colony morphology after mutagenesis according to the present invention;
FIG. 3 is a diagram showing a bacterial microscope morphology of Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain CLNJ1 according to an embodiment of the present invention.
Detailed Description
Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Example 1 selection and identification of Yeast strains
1) Preparation of culture medium
YPD medium: 20g/L of peptone, 10g/L of yeast extract powder and 20g/L of glucose;
in the case of YPD solid medium, 20g/L agar powder was added to the YPD medium.
2) Isolation and purification of strains
Mixing laboratory self-brewed wine, diluting to obtain diluted solutions with different concentrations, coating into YPD solid culture medium, and culturing in an incubator at 30deg.C for 2-3 days. And then, picking individual colonies formed on the culture medium, repeatedly scribing and purifying on a YPD solid culture medium flat plate to obtain pure bacterial colonies, respectively transferring the pure bacterial colonies to YPD solid slant culture medium, marking, standing and culturing at 30 ℃ for 1 day, and then, preserving in a refrigerator at 4 ℃ for every 2 cycles for later use. And (3) inoculating the separated and purified strain into YPD liquid culture medium, putting into an incubator at 30 ℃ for shaking culture for 36 hours for fermentation experiment, and detecting that the content of gamma-aminobutyric acid in fermentation liquid is 1.65g/L.
3) Strain mutation breeding
The screened yeast strain (starting strain) is subjected to shaking culture for 24 hours at the temperature of 30 ℃ in a YPD liquid culture medium to activate, a lithium chloride YPD liquid culture medium is prepared and added, the activated yeast strain is transferred into lithium chloride YPD liquid culture mediums with different concentrations, shaking culture is performed for 24 hours at the temperature of 30 ℃, and then the yeast suspension is coated on a YPD solid flat-plate culture medium, and a bacterial suspension coated plate without lithium chloride mutagenesis is used as a control.
The strain after mutation breeding is inoculated into YPD liquid culture medium, placed into an incubator at 30 ℃, subjected to shaking culture for 36h at the rotating speed of 150 rpm, subjected to fermentation experiment, and detected that the content of gamma-aminobutyric acid in fermentation broth is 5.64g/L. The original strain and the mutant strain are respectively transferred for 10 times for carrying out passaging genetic stability comparison, the capability of the strain for producing gamma-aminobutyric acid is measured, the result shows that the mutant strain is stable, the problem of back mutation does not occur, the gamma-aminobutyric acid yield is stable at 5.6g/L, the mutant strain is named CLNJ1 and is preserved in China general microbiological culture Collection center (China center) for 9 months and 16 days in 2021, the preservation address is North Chen West road No. 1, no. 3 in the Korean region of Beijing city, and the preservation number is CGMCC No.23423. The strain is stored in 60% glycerol pipe and kept at-80 ℃ for standby.
4) Identification of strains
4.1 colony Strain morphology
The morphology and physiological and biochemical identification of the strain are carried out by referring to fungus identification handbook.
Through morphological identification and biological characteristic research, the saccharomyces cerevisiae is white, large and thick in colony after being cultured in YPD solid culture medium for 48 hours at 30 ℃, smooth, moist and sticky in surface, uniform in colony texture and uniform in color, and the thallus is in an oval shape when observed under a microscope. The colony morphology and the fungus morphology are shown in fig. 2 and 3.
4.2 molecular characterization
The molecular identification is carried out according to 26S rDNA sequence analysis method, and the identification is Saccharomyces cerevisiae (Saccharomyces cerevisiae). China general microbiological culture collection center (CGMCC) with collection number of CGMCC 23423 and collection address of Beijing city, 9 and 16 days of 2021.
Example 2 Effect of tea powder addition to fermentation Medium on the production of gamma-aminobutyric acid by Saccharomyces cerevisiae strain CLNJ1
Culture medium and culture conditions: dissolving 10g of peptone, 10g of yeast extract powder and 10g of tea powder in 900mL of water, and sterilizing at 121 ℃ for 20min for later use; 20g of glucose was dissolved in 100mL of water, sterilized at 115℃for 15min, and then added to the above 900mL of medium for further use.
Inoculating Saccharomyces cerevisiae strain CLNJ1 in culture medium, shake culturing at 30deg.C at 150 rpm for 36 hr.
Wherein the tea powder is selected from black tea, oolong tea, white tea, and green tea.
The content of gamma-aminobutyric acid in the fermentation broth was measured, and the results are shown in Table 1:
TABLE 1 addition of different tea powder strains CLNJ1 to produce gamma-aminobutyric acid content in the culture medium
| Black tea | Black tea | Oolong tea | White tea | Green tea | |
| Gamma-aminobutyric acid (g/L) | 7.3 | 6.5 | 8.7 | 8.1 | 6.2 |
EXAMPLE 3 preparation of Kang Pucha Using Acetobacter and Saccharomyces cerevisiae Strain CLNJ1
1. Kang Pucha preparation of fermentation base solution
Commercial oolong tea was brewed three times with boiling water, each tea brewing ratio 1:100, placing the brewed tea into a sterilized glass container, and after the temperature of the tea is reduced to 60 ℃, adding white granulated sugar, wherein the mass ratio of the white granulated sugar to the tea is 1:10, and the dissolved tea sugar is Kangpu tea beverage fermentation base liquid.
2. Kang Pucha strain culture
Fermenting with acetobacter xylinum and Saccharomyces cerevisiae strain CLNJ 1.
Acetobacter xylinum medium and culture conditions: 25g/L of glucose, 10g/L of peptone, 10g/L of yeast extract powder, 5g/L of disodium hydrogen phosphate, 0.5g/L of citric acid, 0.2g/L of magnesium sulfate, pH5.0 and sterilization for 20min for standby; culturing at 30deg.C under shaking at 150 rpm for 48 hr.
Saccharomyces cerevisiae strain CLNJ1 medium and culture conditions: dissolving 10g of peptone and 10g of yeast extract powder in 900mL of water, and sterilizing at 121 ℃ for 20min for later use; 20g of glucose is dissolved in 100mL of water, sterilized for 15min at 115 ℃ and then added into 900mL of culture medium for standby; shake culturing at 30deg.C at 150 rpm for 36 hr.
3. Kang Pucha fermented beverage preparation
Fermenting bacteria liquid of acetobacter xylinum and CLNJ1 strain according to the proportion of 1; adding the mixture into Kang Pucha fermentation base liquid in a proportion of 1, and pouring the mixture into a glass container after the mixture is uniform to obtain the Kangpu tea fermentation liquid. And (3) standing and culturing for 10 days at 30 ℃ to obtain Kang Pucha fermentation stock solution. Adding fruit juice, honey, sucrose, citric acid and the like into the fermentation stock solution to prepare the taste, homogenizing the prepared fermentation solution by using a homogenizer, sterilizing and filling to obtain the Kang Pucha fermented beverage with stable properties and proper taste.
The detection of the gamma-aminobutyric acid and the content in Kang Pucha shows that: the Kang Pucha gamma-aminobutyric acid content in example 3 was 280mg/L.
Comparative example 1 preparation of Kang Pucha Using Acetobacter and Saccharomyces cerevisiae (commercially available)
1. Kang Pucha preparation of fermentation base solution
Commercial oolong tea was brewed three times with boiling water, each tea brewing ratio 1:100, placing the brewed tea into a sterilized glass container, and after the temperature of the tea is reduced to 60 ℃, adding white granulated sugar, wherein the mass ratio of the white granulated sugar to the tea is 1:10, and the dissolved tea sugar is Kangpu tea beverage fermentation base liquid.
2. Kang Pucha strain culture
Fermentation was performed using acetobacter xylinum and Saccharomyces cerevisiae (commercially available).
Acetobacter xylinum medium and culture conditions: 25g/L of glucose, 10g/L of peptone, 10g/L of yeast extract powder, 5g/L of disodium hydrogen phosphate, 0.5g/L of citric acid, 0.2g/L of magnesium sulfate, pH5.0 and sterilization for 20min for standby; culturing at 30deg.C under shaking at 150 rpm for 48 hr.
Saccharomyces cerevisiae medium and culture conditions: dissolving 10g of peptone and 10g of yeast extract powder in 900mL of water, and sterilizing at 121 ℃ for 20min for later use; glucose 20g was dissolved in 100mL of water, sterilized at 115℃for 15min, and then added to the above 900mL of medium for further use. Culturing at 30deg.C under shaking at 150 rpm for 48 hr.
3. Kang Pucha fermented beverage preparation
The acetobacter xylinum and saccharomycetes (commercially available) are added into Kang Pucha fermentation base liquid in proportion, and are poured into a glass container after being uniformly mixed, so that the Kangpu tea fermentation liquid is obtained. And (3) standing and culturing for 10 days at 30 ℃ to obtain Kang Pucha fermentation stock solution. Adding fruit juice, honey, sucrose, citric acid and the like into the fermentation stock solution to prepare the taste, homogenizing the prepared fermentation solution by using a homogenizer, sterilizing and filling to obtain the Kang Pucha fermented beverage with stable properties and proper taste.
The detection of the gamma-aminobutyric acid and the content in Kang Pucha shows that: the comp tea of comparative example 1 does not contain gamma-aminobutyric acid.
Comparative example 2 preparation of Kang Pucha Using Acetobacter (commercially available) and Lactobacillus plantarum
1 Kangpu tea fermentation base liquid preparation
Commercial oolong tea was brewed three times with boiling water, each tea brewing ratio 1:100, placing the brewed tea into a sterilized glass container, and after the temperature of the tea is reduced to 60 ℃, adding white granulated sugar, wherein the mass ratio of the white granulated sugar to the tea is 1:10, and the dissolved tea sugar is Kangpu tea beverage fermentation base liquid.
2 Kang Pucha Strain culture
Fermenting with acetobacter xylinum, saccharomyces cerevisiae and Lactobacillus plantarum.
Acetobacter xylinum medium and culture conditions: 25g/L of glucose, 10g/L of peptone, 10g/L of yeast extract powder, 5g/L of disodium hydrogen phosphate, 0.5g/L of citric acid, 0.2g/L of magnesium sulfate, pH5.0 and sterilization for 20min for standby; culturing at 30deg.C under shaking at 150 rpm for 48 hr.
Saccharomyces cerevisiae medium and culture conditions: dissolving 10g of peptone and 10g of yeast extract powder in 900mL of water, and sterilizing at 121 ℃ for 20min for later use; 20g of glucose is dissolved in 100mL of water, sterilized for 15min at 115 ℃ and then added into 900mL of culture medium for standby; culturing at 30deg.C under shaking at 150 rpm for 48 hr.
Lactobacillus plantarum medium and culture conditions: 10g/L of glucose, 10g/L of casein peptone, 10g/L of beef extract, 5g/L of yeast extract powder, 5g/L of sodium acetate, 10mL of tween-80, 2g/L of ammonium citrate, 2g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate, pH6.8, sterilizing for 20min for standby use at 37 ℃ and standing for 48h.
3. Kang Pucha fermented beverage preparation
Fermenting bacteria liquid of acetobacter xylinum, saccharomyces cerevisiae and lactobacillus plantarum according to the following ratio of 1:1: adding the mixture into Kang Pucha fermentation base liquid in a proportion of 1, and pouring the mixture into a glass container after the mixture is uniform to obtain the Kangpu tea fermentation liquid. And (3) standing and culturing for 10 days at 30 ℃ to obtain Kang Pucha fermentation stock solution. Adding fruit juice, honey, sucrose, citric acid and the like into the fermentation stock solution to prepare the taste, homogenizing the prepared fermentation solution by using a homogenizer, sterilizing and filling to obtain the Kang Pucha fermented beverage with stable properties and proper taste.
The detection of the gamma-aminobutyric acid and the content in Kang Pucha shows that: the content of gamma-aminobutyric acid in Kang Pucha in comparative example 2 was 78mg/L.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (4)
1. The application of the saccharomyces cerevisiae with high yield of gamma-aminobutyric acid in preparing a product with high yield of gamma-aminobutyric acid is characterized in that the saccharomyces cerevisiae CLNJ is utilized to prepare Kang Pucha rich in gamma-aminobutyric acid, and the method specifically comprises the following steps:
(1) Preparing a fermentation base solution: taking tea leaves for brewing by boiling water, collecting brewed tea, and adding a carbon source when the temperature of the tea is reduced to 60 ℃ to obtain fermentation base liquid after the tea is fully dissolved;
(2) Culturing strains: respectively culturing in a proper culture medium to obtain acetobacter xylinum bacterial liquid and saccharomyces cerevisiae bacterial strain CLNJ1 bacterial liquid for later use;
(3) Preparing a fermented beverage: inoculating the bacterial liquid in the step (2) into the fermentation base liquid according to the set inoculation amount of 8-12wt% of the fermentation base liquid in the step (1), and fermenting and culturing to obtain a fermentation base liquid;
wherein, in the inoculation bacterial liquid, the mass ratio of the acetobacter xylosojae bacterial liquid to the saccharomyces cerevisiae bacterial strain CLNJ1 bacterial liquid is (0.1-10): 1, a step of;
(4) Preparing a food additive: adding acceptable food additives into the fermentation stock solution according to taste requirements for blending to obtain Kang Pucha rich in gamma-aminobutyric acid;
the classification of the saccharomyces cerevisiae is named as saccharomyces cerevisiae Saccharomyces cerevisiae CLNJ1, and the saccharomyces cerevisiae is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 9 months and 16 days in 2021, wherein the preservation address is 1 # 3 of North Xishu in the Guangxi area of Beijing city, and the preservation number is CGMCC No.23423.
2. The use of claim 1, wherein in step (1), the tea leaves comprise any one or more tea varieties selected from black tea, oolong tea, white tea, green tea, and the like;
the boiling water infusing times are 2-4 times, and the mass ratio of tea to boiling water is 1: (100-500);
the carbon source comprises white granulated sugar or sucrose;
the mass ratio of the carbon source to the tea water is 1 (8-12).
3. The use of claim 2 for preparing a high-yield gamma-aminobutyric acid product, wherein in step (2), the medium for culturing acetobacter xylosojae comprises the following components in mass content: glucose 20-30g/L, soytone 8-12g/L, yeast extract powder 8-12g/L, disodium hydrogen phosphate 3-8g/L, ammonium citrate 0.3-0.8g/L, magnesium sulfate 0.1-0.3g/L, pH4.5-5.5, 121 ℃, sterilizing for 20min;
fermentation conditions for culturing the acetobacter xylosojae include: shake culturing at 28-32deg.C at rotation speed of 120-180 rpm for 36-60 hr;
the culture medium for culturing the saccharomyces cerevisiae strain CLNJ1 comprises the following components in mass content: dissolving 8-12g of peptone and 8-12g of yeast extract powder in 900mL of water, and sterilizing at 121 ℃ for 20min to obtain 900mL of culture medium for later use; dissolving 18-22g of glucose in 100mL of water, sterilizing at 115 ℃ for 15min, and adding into the 900mL of culture medium for later use;
the fermentation conditions for culturing the saccharomyces cerevisiae strain CLNJ1 strain include: shaking culture at 28-32deg.C at rotation speed of 120-180 rpm for 24-48 hr.
4. The use of claim 2 for preparing a high-yield gamma-aminobutyric acid product, wherein in the step (3), the bacterial liquid is inoculated into the fermentation base liquid in a set inoculation amount of 10wt% of the fermentation base liquid, and fermentation culture is carried out to obtain a fermentation base liquid;
in the inoculation bacterial liquid, the mass ratio of the acetobacter xylosojae bacterial liquid to the saccharomyces cerevisiae bacterial strain CLNJ1 bacterial liquid is 1:1, a step of;
the fermentation culture conditions are as follows: and (3) standing and fermenting for 6-14 days at 26-30 ℃.
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