Preparation method of highland barley liquid rich in GABA (gamma-aminobutyric acid), highland barley fermented beverage rich in GABA as well as preparation method and application of highland barley fermented beverage rich in GABA

Abstract

The invention belongs to the technical field of functional food processing, and particularly relates to a preparation method of a highland barley liquid rich in GABA, a highland barley fermented beverage rich in GABA, and a preparation method and application of the highland barley fermented beverage rich in GABA. The invention provides a preparation method of a highland barley liquid rich in GABA (gamma-aminobutyric acid) and a highland barley fermented beverage. According to the preparation method, endogenous enzyme glutamate decarboxylase in highland barley is activated through germination treatment, the content of active substances such as polyphenol and flavone in the highland barley is increased, the oxidation resistance is enhanced, protein is promoted to be degraded into free amino acid, and a substrate is provided for enrichment of GABA; meanwhile, glutamic acid is promoted to be decarboxylated to generate GABA through germination treatment. A rich glutamic acid substrate is provided for enrichment of GABA through enzymolysis, the obtained highland barley enzymatic hydrolysate is mixed with highland barley powder, re-enrichment of GABA is achieved, and the highland barley liquid is obtained. The antioxidant activity of the GABA fermented beverage in the highland barley fermented beverage is improved through fermentation, and meanwhile, the taste and flavor of the product are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of functional food processing, and particularly relates to a method for preparing a highland barley liquid rich in GABA, a highland barley fermented beverage rich in GABA, and a preparation method and application thereof. Background Art

[0002] Cereal beverages are made from cereals, using bioactive ingredients such as carbohydrates, dietary fiber, protein, etc. in the cereals. They are produced through processes such as enzymatic hydrolysis, fermentation, and blending. They are convenient and healthy.

[0003] As a staple food crop, highland barley is rich in polyphenols, dietary fiber and other nutritional functional ingredients. The highland barley drinks reported in the prior art are usually beer. The mass concentration of GABA in highland barley red yeast beer is 91.3±6.9 mg / L, and the GABA content is low. GABA is an important neurotransmitter in the human body, which has the effects of assisting in lowering blood pressure, promoting sleep, and relieving anxiety. Therefore, how to retain and improve the nutritional value of highland barley whole grains to the greatest extent, increase the GABA content, and develop a highland barley liquid with a special highland barley flavor plays an important role in increasing the added value of highland barley. Summary of the invention

[0004] The invention provides a preparation method of highland barley liquid rich in GABA, a fermented highland barley beverage rich in GABA, a preparation method and an application thereof. The prepared highland barley liquid has a high GABA content.

[0005] In order to solve the above technical problems, the following technical solutions are proposed:

[0006] The present invention provides a method for preparing a highland barley liquor rich in GABA, comprising the following steps:

[0007] The highland barley is processed after being germinated to obtain highland barley pulp;

[0008] The highland barley slurry is subjected to a first-stage enzymolysis and a second-stage enzymolysis in sequence to obtain an enzymolysis solution;

[0009] The enzymatic hydrolysis solution is mixed with highland barley powder to obtain highland barley solution rich in GABA; the enzyme used in the first stage of enzymatic hydrolysis includes alpha-amylase; the enzyme used in the second stage of enzymatic hydrolysis includes one or more of saccharifying enzyme, flavor protease and cellulase.

[0010] Preferably, the germination treatment includes low-temperature germination treatment and high-temperature germination treatment; the temperature of the low-temperature germination treatment is 13-18°C and the time is 24-48 hours; the temperature of the high-temperature germination treatment is 35-55°C and the time is 1-2 hours.

[0011] Preferably, the temperature of the first stage enzymolysis is 80-90°C, the time of the first stage enzymolysis is 0.5-3.0h; and the amount of α-amylase added is 0.5‰-0.7‰ of the mass of highland barley.

[0012] Preferably, the temperature of the second stage enzymatic hydrolysis is 35-60°C, and the time of the second stage enzymatic hydrolysis is 0.5-2.0h;

[0013] The amount of saccharifying enzyme added is 1.2‰ to 1.8‰ of the mass of highland barley;

[0014] The amount of flavor protease added is 2.0‰ to 8.0‰ of the mass of highland barley;

[0015] The amount of cellulase added is 0.5‰ to 1.5‰ of the mass of highland barley.

[0016] Preferably, the amount of highland barley powder added is 0.5% to 2.0% of the mass of the enzymatic hydrolyzate, and the mixing further includes incubation, the incubation temperature is 40 to 60° C., and the time is 60 to 120 minutes.

[0017] Preferably, the first stage of enzymatic hydrolysis further includes aging; the aging temperature is 80-90° C. and the time is 15-30 min.

[0018] The present invention provides a method for preparing a fermented highland barley beverage rich in GABA, comprising: mixing highland barley liquid obtained by the preparation method described in any one of the above technical solutions with microorganisms and fermenting the mixture to obtain a fermented highland barley beverage;

[0019] The microorganisms include Lactobacillus plantarum and / or sweet wine koji.

[0020] Preferably, when the microorganism includes Lactobacillus plantarum, the amount of Lactobacillus plantarum added is 0.01‰ to 0.1‰ of the mass of highland barley liquid;

[0021] When the microorganism contains sweet wine yeast, the amount of sweet wine yeast added is 0.5‰ to 5.0‰ of the mass of highland barley liquid;

[0022] The fermentation temperature is 25-40° C., and the fermentation time is 6-48 hours.

[0023] The present invention provides a fermented highland barley beverage prepared by the preparation method described in the above technical solution, wherein the GABA content in the fermented highland barley beverage is 70-80 mg / L.

[0024] The present invention provides the application of the preparation method described in the above technical solution in one or more of the following 1) to 3),

[0025] 1) Improve the antioxidant activity of fermented highland barley beverages;

[0026] 2) Enrich the types of volatile flavor substances in fermented highland barley beverages;

[0027] 3) Increase the content of volatile flavor substances in fermented highland barley beverages.

[0028] Beneficial effects of the present invention:

[0029] The present invention provides a method for preparing a highland barley liquid rich in GABA, comprising the following steps: processing highland barley after germination treatment to obtain highland barley pulp; performing first-stage enzymolysis and second-stage enzymolysis on the highland barley pulp in sequence to obtain enzymolysis solution; mixing the enzymolysis solution with highland barley powder to obtain highland barley liquid rich in GABA; the enzyme used in the first-stage enzymolysis includes α-amylase; the enzyme used in the second-stage enzymolysis includes one or more of saccharifying enzyme, flavor protease and cellulase. The present invention increases the content of active substances such as polyphenols and flavonoids in highland barley through germination treatment, enhances antioxidant properties, promotes protein degradation into free amino acids, and provides substrates for GABA enrichment; at the same time, activates the endogenous enzyme glutamate decarboxylase in highland barley, and GABA is generated by glutamate decarboxylase catalyzing glutamic acid to undergo decarboxylation reaction, thereby achieving GABA enrichment.

[0030] In addition, enzymatic hydrolysis provides abundant glutamate substrate for GABA enrichment, the obtained highland barley enzymatic hydrolysate is mixed with highland barley powder, highland barley powder provides glutamate decarboxylase, glutamate decarboxylase is a synthase of GABA, and GABA is synthesized using glutamate in the highland barley enzymatic hydrolysate as a raw material, thereby achieving the re-enrichment of GABA.

[0031] The present invention also provides a method for preparing a GABA-rich highland barley fermented beverage, which can significantly increase the GABA, polyphenol and flavonoid contents in the highland barley fermented beverage and the antioxidant activity of the fermented beverage, while improving the taste and flavor of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the effect of the amount of α-amylase added on the total solid content of highland barley hydrolysate;

[0033] Figure 2 This is the effect of the amount of saccharifying enzyme added on the total solid content of highland barley hydrolysate;

[0034] Figure 3 This is the effect of flavor protease addition on the total solid content of highland barley hydrolysate;

[0035] Figure 4 This is the effect of the amount of cellulase added on the total solid content of highland barley hydrolysate;

[0036] Figure 5 This is the effect of the amount of saccharifying enzyme added on the reducing sugar content of highland barley enzymatic hydrolysate;

[0037] Figure 6 This is the effect of flavor protease addition on the soluble protein content of highland barley enzymatic hydrolysate;

[0038] Figure 7 This is the effect of the amount of cellulase added on the β-glucan content of highland barley hydrolysate;

[0039] Figure 8 This is the effect of highland barley powder addition on the GABA content of highland barley enzymatic hydrolysate;

[0040] Fig. 9 This is the effect of incubation temperature on the GABA content of highland barley enzymatic hydrolysate;

[0041] Fig.10 This is the effect of incubation time on the GABA content of highland barley enzymatic hydrolysate;

[0042] Fig.11 The results of taste evaluation of fermented highland barley beverages of Example 3 and Comparative Examples 14 to 17 are shown;

[0043] Fig.12 This is a graph showing the flavor evaluation results of fermented highland barley beverages of Example 3 and Comparative Examples 14 to 17;

[0044] Fig.13 The results of the evaluation of volatile flavor substances in fermented highland barley beverages of Example 3 and Comparative Examples 14 to 17 are shown;

[0045] Fig.14 The present invention is a process flow chart for preparing fermented highland barley beverage;

[0046] Figures 1 to 10 Different lowercase letters represent significant differences (P<0.05). DETAILED DESCRIPTION

[0047] The present invention provides a method for preparing a highland barley liquor rich in GABA, comprising the following steps:

[0048] The highland barley is processed after being subjected to germination treatment to obtain highland barley slurry;

[0049] The highland barley slurry is subjected to a first-stage enzymolysis and a second-stage enzymolysis in sequence to obtain an enzymolysis solution;

[0050] After the enzymatic hydrolyzate is mixed with highland barley powder, highland barley liquid rich in GABA is obtained.

[0051] As an optional implementation mode, the present invention processes highland barley into highland barley pulp after germination treatment. As an optional implementation mode, the highland barley of the present invention includes highland barley grains. The present invention has no special limitation on the source of the highland barley grains, and conventional products can be used.

[0052] As an optional implementation mode, the present invention places the highland barley grains in a germination box for germination treatment. As an optional implementation mode, the germination treatment of the present invention includes low-temperature germination treatment and high-temperature germination treatment. As an optional implementation mode, the temperature of the low-temperature germination treatment of the present invention is 13-18°C, or 14-17°C. As an optional implementation mode, the time of the low-temperature germination treatment of the present invention is 24-48h, or 36-40h. In a specific embodiment of the present invention, the temperature of the low-temperature germination treatment is 13, 14, 15, 16, 17 or 18°C; the time of the low-temperature germination treatment is 24, 28, 32, 38, 42, 44 or 48h. The present invention activates the endogenous enzyme glutamate decarboxylase (GAD) in highland barley through low-temperature germination treatment, and increases the content of active substances such as polyphenols and flavonoids, enhances antioxidant properties, promotes protein degradation into free amino acids, increases the content of free amino acids, and provides a substrate for GABA enrichment. As an optional implementation mode, the temperature of the high temperature germination treatment of the present invention is 35-55°C, or 40-50°C. As an optional implementation mode, the time of the high temperature germination treatment is 1-2h, or 1.3-1.8h. In a specific embodiment of the present invention, the temperature of the high temperature germination treatment is 35°C, 38°C, 41°C, 46°C, 49°C, 53°C or 55°C, and the time of the high temperature germination treatment is 1, 1.3, 1.5, 1.8 or 2h. The present invention raises the germination temperature and continues the germination treatment, which can enhance the activity of glutamate decarboxylase, and glutamate decarboxylase can convert glutamate into GABA, thereby achieving preliminary enrichment of GABA.

[0053] After the germination treatment, the present invention processes the highland barley grains obtained by the germination treatment to obtain highland barley slurry. As an optional implementation method, the processing method includes grinding. The present invention has no special limitation on the operation of the grinding, and a conventional operation method can be used. During the grinding of the present invention, the highland barley grains are mixed with water, and the mass ratio of the highland barley grains to water of the present invention is 1: (5-15), or can also be 1: (8-13). In a specific embodiment of the present invention, the mass ratio of the highland barley grains to water is 1:5, 1:8, 1:10, 1:13 or 1:15.

[0054] As an optional implementation mode, the present invention first ripens the highland barley slurry, and then sequentially performs the first stage enzymolysis and the second stage enzymolysis to obtain an enzymolysis solution. As an optional implementation mode, the ripening temperature can be 80-90°C, or 83-88°C; in a specific embodiment of the present invention, the ripening temperature can be 80, 83, 85, 88 or 90°C; the ripening time can be 15-30min, or 18-25min; in a specific embodiment of the present invention, the ripening time can be 15, 18, 20, 25 or 30min. The ripening can promote the swelling and collapse of starch granules, the disappearance of the crystalline structure, promote the binding of α-amylase to starch, and improve the starch hydrolysis rate and efficiency.

[0055] As an optional implementation mode, the enzyme used in the first stage of enzymolysis of the present invention includes α-amylase; the temperature of the first stage of enzymolysis is 80-90°C, or 84-87°C; in a specific embodiment of the present invention, the temperature of the first stage of enzymolysis is 80, 84, 87 or 90°C; the time of the first stage of enzymolysis of the present invention is 0.5-3.0h, or 1-2h; in a specific embodiment of the present invention, the time of the first stage of enzymolysis is 0.5, 1, 1.5, 2, 2.5 or 3h. Based on the mass of highland barley processed into highland barley slurry, the amount of α-amylase added in the present invention is 0.3‰-0.7‰ of the mass of highland barley, or 0.4‰-0.6‰. In a specific embodiment of the present invention, the amount of α-amylase added is 0.3‰, 0.4‰, 0.5‰, 0.6‰ or 0.7‰ of the mass of highland barley. The present invention adds α-amylase to hydrolyze highland barley starch into dextrin and reducing sugar. The present invention has no particular limitation on the source of the α-amylase, and conventional commercially available products may be used.

[0056] As an optional implementation mode, the present invention reduces the temperature of the enzyme solution obtained by the first stage enzymolysis to 35-60°C and then performs the second stage enzymolysis to obtain an enzymolysis solution. As an optional implementation mode, the enzyme used in the second stage enzymolysis of the present invention includes one or more of saccharifying enzymes, flavor proteases and cellulases, and may also include saccharifying enzymes, flavor proteases and cellulases. As an optional implementation mode, the present invention simultaneously adds saccharifying enzymes, flavor proteases and cellulases to the first stage enzyme solution for the second stage enzymolysis. The present invention adds α-amylase to hydrolyze highland barley starch into dextrin and reducing sugar, and then hydrolyzes dextrin into reducing sugar glucose by adding saccharifying enzymes, thereby improving the taste of highland barley liquid and providing a carbon source for plant lactobacillus and oryzae used during fermentation; by adding cellulase to hydrolyze cellulose in highland barley, the solid content in highland barley liquid is increased; by adding flavor proteases to hydrolyze proteins in highland barley to generate small molecule peptides and free amino acids, a rich glutamate substrate is provided for GABA enrichment. Based on the mass of highland barley processed into highland barley slurry, the amount of saccharifying enzyme added in the present invention is 1.2‰ to 1.8‰ of the mass of highland barley, or 1.4‰ to 1.6‰. In a specific embodiment of the present invention, the amount of saccharifying enzyme added is 1.2‰, 1.4‰, 1.5‰, 1.6‰ or 1.8‰ of the mass of highland barley. Based on the mass of highland barley processed into highland barley slurry, the amount of flavor protease added in the present invention is 2‰ to 8‰ of the mass of highland barley, or 2‰ to 6‰, more preferably 4‰ to 5‰. In a specific embodiment of the present invention, the amount of flavor protease added is 2‰, 3‰, 4‰, 5‰ or 6‰ of the mass of highland barley. Based on the mass of highland barley processed into highland barley slurry, the amount of cellulase added in the present invention is 0.5‰ to 1.5‰ of the mass of highland barley, or 1.0‰ to 1.3‰. In a specific embodiment of the present invention, the amount of cellulase added is 0.5‰, 1.0‰, 1.3‰ or 1.5‰ of the mass of highland barley. As an optional implementation method, when the second stage enzymolysis is performed, when one or two enzymes are added, the amount of enzyme added is the same as when three enzymes are added simultaneously. The temperature of the second stage enzymolysis of the present invention is 35-60°C, or 40-55°C; in a specific embodiment of the present invention, the temperature of the second stage enzymolysis is 35, 40, 45, 50 or 55°C; the time of the second stage enzymolysis of the present invention is 0.5-2.0h, or 0.8-1.5h; in a specific embodiment of the present invention, the time of the second stage enzymolysis is 0.5, 1, 1.5 or 2h. As an optional implementation method, when the second stage enzymolysis is performed, when one or two enzymes are added, the temperature and time of enzymolysis are the same as the temperature and time of enzymolysis when three enzymes are added simultaneously. The present invention has no special limitation on the sources of the saccharifying enzyme, flavor protease and cellulase, and conventional commercial products can be used.

[0057] As an optional implementation mode, the present invention mixes the enzymolysis solution with highland barley powder to obtain highland barley solution rich in GABA. The amount of highland barley powder added in the present invention is 0.5% to 2.0% of the mass of the enzymolysis solution, and can also be 1.0% to 1.5%; in a specific embodiment of the present invention, the amount of highland barley powder added is 0.5%, 1.0%, 1.5% or 2% of the mass of the enzymolysis solution. Highland barley powder provides a donor for glutamate decarboxylase, which is a synthase of GABA, and glutamate decarboxylase synthesizes GABA using glutamic acid in highland barley as a raw material.

[0058] As an optional implementation mode, the mixing of the present invention further includes incubation, and the incubation temperature is 40-60°C, or 45-55°C; in a specific embodiment of the present invention, the incubation temperature is 40, 45, 50, 55 or 60°C. The incubation time of the present invention is 60-120min, or 80-100min; in a specific embodiment of the present invention, the incubation time is 60, 70, 80, 90, 100, 110 or 120min. The incubation temperature and time of the present invention promote the synthesis of GABA.

[0059] The present invention provides a method for preparing a fermented highland barley beverage rich in GABA, comprising: mixing highland barley liquid obtained by the preparation method described in the above technology with microorganisms and then fermenting to obtain the fermented highland barley beverage; the microorganisms include Lactobacillus plantarum and / or Rhizopus oryzae, more preferably Lactobacillus plantarum and Rhizopus oryzae, and the Rhizopus oryzae is a microorganism contained in sweet wine koji.

[0060] As an optional implementation mode, when the microorganism has plant lactobacillus, the plant lactobacillus addition amount of the present invention is 0.01‰~0.1‰ of the mass of the highland barley liquid, and can also be 0.04‰~0.08‰; In a specific embodiment of the present invention, the plant lactobacillus addition amount is 0.01‰, 0.03‰, 0.04‰, 0.06‰, 0.08‰ or 0.1‰ of the mass of the highland barley liquid. The present invention has no special limitation on the source of the plant lactobacillus, and conventional commercial products can be used. In a specific embodiment of the present invention, the plant lactobacillus powder produced by Hongkang Biotechnology is verified, and the powder is purchased from Taobao store Hongkang Biology. When the microorganism has sweet wine koji, the amount of sweet wine koji added in the present invention is 0.5‰ to 5.0‰ of the mass of the highland barley liquid, and can also be 2‰ to 4.5‰; in a specific embodiment of the present invention, the amount of plant lactobacillus added is 0.01‰, 0.5‰, 1‰, 2‰, 3‰, 4‰, 4.5‰ or 5‰ of the mass of the highland barley liquid. The present invention does not specifically limit the source of the sweet wine koji, and conventional commercial products can be used. In a specific embodiment of the present invention, Angel sweet wine koji (sweet type) powder purchased from Angel Yeast Co., Ltd. is used as an example for verification.

[0061] As an optional implementation mode, the fermentation temperature of the present invention is 25-40°C, or 30-38°C. In a specific embodiment of the present invention, the fermentation temperature is 25, 30, 36, 38 or 40°C. The fermentation time of the present invention is 6-48h, or 18-32h; in a specific embodiment of the present invention, the fermentation time is 6, 12, 18, 26, 32, 36, 40, 44 or 48h. The present invention co-fermented with plant lactobacillus and sweet wine koji, and plant lactobacillus and sweet wine koji further decomposed the macromolecules in highland barley into soluble small molecules, further increasing the solid content in the beverage, and fermentation can also improve the taste, increase the sourness, and reduce the bitterness, making it sweet and sour. The increase in sourness is mainly due to the formation of acidic substances such as acetic acid during the fermentation process, and the decrease in bitterness is due to the further hydrolysis of small molecule peptides to generate amino acids during the fermentation process, and the content of bitter peptides is reduced.

[0062] As an optional implementation mode, the present invention obtains highland barley fermented beverage after centrifuging and sterilizing the fermented liquid. The present invention does not specifically limit the parameters of the centrifugation and sterilization and enzyme inactivation, and conventional parameters can be used. In a specific embodiment of the present invention, the centrifugal speed can be 4000r / min, and the centrifugal time can be 5min; the sterilization and enzyme inactivation temperature can be 90°C, and the sterilization and enzyme inactivation temperature can be 30min.

[0063] The present invention provides a fermented highland barley beverage prepared by the preparation method described in the above technical solution, wherein the GABA content in the fermented highland barley beverage is 70-80 mg / L. In a specific embodiment of the present invention, the GABA content in the fermented highland barley beverage is 75.21±0.96 mg / L, 75.26±3.61 mg / L, 76.29±4.12 mg / L or 78.87±3.09 mg / L. The present invention significantly improves the GABA, polyphenol and flavonoid content and antioxidant activity in the fermented highland barley beverage by integrating germination, enzymolysis, incubation and fermentation processes, while improving the taste and flavor of the product.

[0064] The present invention provides the application of the preparation method described in the above technical solution in improving the antioxidant activity and / or flavor and volatile flavor substance content of fermented highland barley beverage.

[0065] As an optional implementation mode, the antioxidant property of the present invention may include one or more of total antioxidant capacity, DPPH, superoxide anion free radical and hydroxyl free radical scavenging capacity. The fermented highland barley beverage prepared by the present invention can significantly increase the total antioxidant capacity, DPPH, superoxide anion free radical, hydroxyl free radical scavenging capacity, and enhance the antioxidant activity of the fermented highland barley beverage.

[0066] The present invention provides the application of the preparation method described in the above technical solution in enriching the types of volatile flavor substances in fermented highland barley beverages.

[0067] The present invention provides application of the preparation method described in the above technical solution in improving the content of volatile flavor substances in fermented highland barley beverages.

[0068] As an optional implementation mode, the volatile flavor substances of the present invention may include one or more of alcohols, esters, phenols, aldehydes and acids. The acids of the present invention may include one or more of acetic acid, octanoic acid, hexanoic acid and guanidinopropionic acid; the esters include linalyl butyrate and / or hexyl formate; the alcohols may include one or more of 3-methyl-3-butene-1-ol, styraxol, tetrahydrolavandulol, benzyl alcohol and n-heptanol. The phenols include one or more of 2-methoxy-4-methylphenol, 2-methoxy-4-vinylphenol, 2,4-di-tert-butylphenol and 2,3-dihydrobenzofuran. The aldehydes of the present invention include furfural.

[0069] The technical solution of the present invention can increase the types of flavor substances in fermented highland barley beverages, increase the content of flavor substances, and improve the flavor of fermented highland barley beverages.

[0070] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0071] In the embodiments and comparative examples of the present invention, the brand of the α-amylase is Novozymes, the product type is α-amylase SC-DS type, and the enzyme activity is 240KNU-S / g; the brand of the saccharifying enzyme is Novozymes, the product type is saccharifying enzyme AMG-300L, and the enzyme activity is 300AGU / mL; the brand of the flavor protease is Novozymes, the product type is flavor protease 500MG, and the enzyme activity is 500LAPU / g; the cellulase is purchased from Ningxia Heshibi Biotechnology Co., Ltd., and the enzyme activity is 100000U / g.

[0072] The highland barley grains and highland barley flour used in the following embodiments and comparative examples are all products from the same batch and have no difference.

[0073] Example 1 Staged enzymolysis

[0074] (1) Pretreatment: After cleaning and removing impurities from highland barley grains, the grains were placed in a germination box at 16° C., and germinated for 24 h. After the temperature of the germination box was adjusted to 50° C. and continued to germinate for 2 h, water was added at a mass ratio of 1:10 and ground into slurry to obtain highland barley slurry;

[0075] (2) First stage enzymatic hydrolysis: After the highland barley slurry obtained in step (1) is matured at 85°C for 20 min, 0.5‰ of the mass of highland barley grains of α-amylase is added, and the enzymatic hydrolysis temperature is 85°C for 2 h to obtain a first stage enzymatic hydrolyzate.

[0076] (3) Second stage enzymolysis: After the temperature of the first stage enzymolysis solution dropped to 50°C, saccharifying enzyme, flavor protease and cellulase were added to the first stage enzymolysis solution. The amount of saccharifying enzyme added was 1.8‰ of the mass of highland barley grains, the amount of flavor protease added was 6.00‰ of the mass of highland barley grains, and the amount of cellulase added was 1.5‰ of the mass of highland barley grains. After addition, enzymolysis was carried out at 50°C for 1.0 h to obtain highland barley enzymolysis solution.

[0077] Example 1-1

[0078] Same as Example 1, the only difference is that the amount of α-amylase added in step (2) is 0.6‰ of the mass of highland barley grains.

[0079] Example 1-2

[0080] Same as Example 1, the only difference is that the amount of α-amylase added in step (2) is 0.7‰ of the mass of highland barley grains.

[0081] Examples 1-3

[0082] The same as Example 1, the only difference is that the amount of saccharifying enzyme added in step (3) is 1.2‰ of the mass of highland barley grains.

[0083] Examples 1-4

[0084] The same as Example 1, the only difference is that the amount of saccharifying enzyme added in step (3) is 1.5‰ of the mass of highland barley grains.

[0085] Examples 1-5

[0086] The same as Example 1, the only difference is that the amount of flavor protease added in step (3) is 2.0‰ of the mass of highland barley grains.

[0087] Examples 1-6

[0088] The same as Example 1, the only difference is that the amount of flavor protease added in step (3) is 4.0‰ of the mass of highland barley grains.

[0089] Examples 1-7

[0090] The same as Example 1, the only difference is that the amount of flavor protease added in step (3) is 8.0‰ of the mass of highland barley grains.

[0091] Examples 1-8

[0092] Same as Example 1, the only difference is that the amount of cellulase added in step (3) is 0.5‰ of the mass of highland barley grains.

[0093] Examples 1-9

[0094] Same as Example 1, the only difference is that the amount of cellulase added in step (3) is 1.0‰ of the mass of highland barley grains.

[0095] Comparative Example 1

[0096] Same as Example 1, the only difference is that the amount of α-amylase added in step (2) is 0.3‰ of the mass of highland barley grains.

[0097] Comparative Example 2

[0098] Same as Example 1, the only difference is that the amount of α-amylase added in step (2) is 0.4‰ of the mass of highland barley grains.

[0099] Comparative Example 3

[0100] The same as Example 1, the only difference is that the amount of saccharifying enzyme added in step (3) is 2.1‰ of the mass of highland barley grains.

[0101] Comparative Example 4

[0102] The same as Example 1, the only difference is that the amount of saccharifying enzyme added in step (3) is 2.4‰ of the mass of highland barley grains.

[0103] Comparative Example 5

[0104] The same as Example 1, the only difference is that the amount of flavor protease added in step (3) is 10.0‰ of the mass of highland barley grains.

[0105] Comparative Example 6

[0106] Same as Example 1, the only difference is that the amount of cellulase added in step (3) is 2.0‰ of the mass of highland barley grains.

[0107] Comparative Example 7

[0108] Same as Example 1, the only difference is that the amount of cellulase added in step (3) is 2.5‰ of the mass of highland barley grains.

[0109] Experimental Example 1 Total solid content analysis

[0110] The enzymatic hydrolysates prepared in Examples 1 to 1-9 and Comparative Examples 1 to 7 were centrifuged at 4000 r / min for 10 min, and the supernatant was taken, and the total solid content in the supernatant was determined by constant weight method. The specific determination was as follows: 10.0 g of the supernatant was weighed in a weighing dish with a known constant weight, and then placed in a constant temperature drying oven again, and dried at 105°C to a constant weight. The results were calculated as follows:

[0111] X=(m2-m1) / m×100

[0112] X—total solid content in the sample (g / 100g);

[0113] m2—the mass of the sample plus the weighing dish after drying (g);

[0114] m1—the mass of the weighing dish after drying (g);

[0115] m—mass of the sample (g).

[0116] Specific results such as Figure 1 to Figure 4 .

[0117] Depend on Figure 1 It can be seen that with the increase of α-amylase addition, the total solid content showed an upward trend and then tended to be flat. When the α-amylase addition amount was 0.3‰, 0.4‰, 0.5‰, 0.6‰, and 0.7‰, the total solid content of highland barley hydrolysate was 7.15±0.07, respectively. a g / 100g, 7.21±0.08 a g / 100g, 7.62±0.08 b g / 100g, 7.74±0.17 b g / 100g, 7.70±0.21 b g / 100g, different letters represent significant differences at the P<0.05 level. When the addition amount of α-amylase was 0.5‰~0.7‰, the total solid content was higher and there was no significant change. Figure 2 It can be seen that when the amount of saccharifying enzyme added was 1.2‰, 1.5‰, 1.8‰, 2.1‰, and 2.4‰, the total solid content of highland barley hydrolysate was 8.61±0.44, respectively. a g / 100g, 8.53±0.27 ab g / 100g, 8.93±0.25 bc g / 100g, 9.10±0.10 c g / 100g, 9.34±0.23 c g / 100g. Different letters represent significant differences at the P<0.05 level.

[0118] It can be seen that with the increase of the amount of saccharifying enzyme added, the reducing sugar content showed an increasing trend. When the amount of enzyme added reached 1.8‰, with the increase of the amount of saccharifying enzyme added, the total solid content did not change significantly. Figure 3 It can be seen that

[0119] When the addition amount of flavor protease was 2.0‰, 4.0‰, 6.0‰, 8.0‰, and 10.0‰, the total solid content of highland barley enzymatic hydrolysate was 9.11±0.25, b g / 100g, 8.88±0.09 b g / 100g, 8.88±0.09 b g / 100g, 8.53±0.21 a g / 100g, 8.38±0.09 a g / 100g. Different letters represent significant differences at the P<0.05 level. With the increase of flavor protease addition, the total solid content showed a decreasing trend. When the flavor protease addition reached 6.0‰, the total solid content decreased significantly with the increase of enzyme addition. Figure 4 It can be seen that when the addition amount of cellulase is 0.5‰, 1.0‰, 1.5‰, 2.0‰, and 2.5‰, the total solid content of highland barley hydrolysate is 9.03±0.38, respectively. b g / 100g, 9.20±0.45 c g / 100g, 8.93±0.25 b g / 100g, 8.15±0.07 a g / 100g, 8.17±0.25 a g / 100g. Different letters represent significant differences at the P<0.05 level. With the increase of cellulase addition, the total solid content of the enzymatic hydrolysate showed a trend of first increasing and then decreasing. When the cellulase addition was 1.0‰, the total solid content was the highest.

[0120] Experimental Example 2 Analysis of reducing sugar content

[0121] The enzymatic hydrolysates prepared in Examples 1, 1-3, 1-4 and Comparative Examples 3-4 were centrifuged at 4000 r / min for 10 min, and the supernatant was taken. The reducing sugar content in the supernatant was determined by the DNS method. The reducing sugar content determination process is as follows: the supernatant was diluted to obtain a dilution, 1.0 mL of the dilution was added to 3.0 mL of the DNS reagent, mixed well, and placed in a boiling water bath for reaction for 7 min, and then immediately placed in cold water to cool to room temperature, and the absorbance was measured at 540 nm. The results are as follows: Figure 5 and Table 1.

[0122] Depend on Figure 5 As shown in Table 1, with the increase of the amount of saccharifying enzyme added, the reducing sugar content showed an increasing trend. When the enzyme addition amount reached 1.5‰, the reducing sugar content did not change significantly with the increase of the enzyme addition amount.

[0123] Table 1 Reducing sugar content of highland barley hydrolysate at different saccharifying enzyme addition amounts

[0124] Glucoamylase addition amount (‰) Reducing sugar content (mg / ml) 1.2 <![CDATA[55.65±1.06 a ]]> 1.5 <![CDATA[60.03±0.35 bc ]]> 1.8 <![CDATA[59.44±1.43 bc ]]> 2.1 <![CDATA[58.61±2.43 b ]]> 2.4 <![CDATA[61.97±0.40 c ]]>

[0125] Note: Different letters represent significant differences at the P<0.05 level, the same below.

[0126] Experimental Example 3 Analysis of soluble protein content

[0127] The enzymatic hydrolysates prepared in Examples 1, 1-5, 1-6, 1-7 and Comparative Example 5 were centrifuged at 4000 r / min for 10 min, the supernatant was taken, and the soluble protein content in the supernatant was determined by the Coomassie Brilliant Blue method. The soluble protein content determination process is as follows: the supernatant is diluted to obtain a dilution, 0.1 mL of the dilution is added to 3.0 mL of Coomassie Brilliant Blue solution for reaction for 2 min, and then the absorbance is measured at 595 nm. The specific results are shown in FIG. Figure 6 and Table 2.

[0128] Depend on Figure 6 As shown in Table 2, with the increase of the amount of flavor protease added, the soluble protein content showed a trend of first increasing and then decreasing, reaching the maximum when the amount of enzyme added reached 6.0‰.

[0129] Table 2 Soluble protein content of highland barley hydrolysate with different addition amounts of flavor protease

[0130] Flavor protease addition amount (‰) Soluble protein content (mg / ml) 2.0 <![CDATA[4.93±0.30 a ]]> 4.0 <![CDATA[4.78±0.57 a ]]> 6.0 <![CDATA[5.25±0.23 b ]]> 8.0 <![CDATA[5.25±0.33 ab ]]> 10.0 <![CDATA[4.93±0.11 ab ]]>

[0131] Experimental Example 4 Analysis of β-glucan content

[0132] The enzymatic hydrolysates prepared in Examples 1, 1-8, 1-9, Comparative Examples 6 and 7 were centrifuged at 4000 r / min for 10 min, and the supernatant was taken and the β-glucan content in the enzymatic hydrolysates was determined according to the method in NY / T 2006-2011. The specific results are shown in FIG. Figure 7 and Table 3.

[0133] Depend on Figure 7 As shown in Table 3, with the increase of enzyme dosage, the β-glucan content showed a trend of first increasing and then decreasing. When the enzyme dosage reached 1.0‰, it reached a peak value. As the enzyme dosage continued to increase, the β-glucan content showed a downward trend. The reason is that with the increase of cellulase addition, the content of soluble polysaccharides gradually increased, and cross-linking polymerization occurred between polysaccharides and polysaccharides or between polysaccharides and polypeptides, and sedimentation occurred, resulting in a gradual decrease in the β-glucan content.

[0134] Table 3 β-glucan content of highland barley hydrolysate at different cellulase addition amounts

[0135] Cellulase addition amount (‰) β-Glucan content (μg / ml) 0.5 <![CDATA[0.50±0.03 ab ]]> 1.0 <![CDATA[0.58±0.04 b ]]> 1.5 <![CDATA[0.51±0.06 ab ]]> 2.0 <![CDATA[0.46±0.14 a ]]> 2.5 <![CDATA[0.44±0.04 a ]]>

[0136] Example 2 GABA incubation enrichment

[0137] The highland barley hydrolysate prepared in Example 1 was used as a raw material, and raw highland barley powder was used as a glutamate decarboxylase supplier. Highland barley powder was added to the highland barley hydrolysate, and the amount of highland barley powder added was 1.0% of the mass of the highland barley hydrolysate, and then incubated at 50° C. for 60 minutes to obtain a highland barley incubation solution rich in GABA. The incubation solution was sterilized by high pressure to obtain a sterile incubation solution.

[0138] Example 2-1

[0139] Same as Example 2, the only difference is that the amount of highland barley powder added is 1.5% of the mass of highland barley hydrolysate.

[0140] Example 2-2

[0141] Same as Example 2, the only difference is that the addition amount of highland barley powder is 2.0% of the mass of highland barley enzymatic hydrolyzate.

[0142] Example 2-3

[0143] Same as Example 2, the only difference being that the incubation temperature was 40°C.

[0144] Embodiment 2-4

[0145] Same as Example 2, the only difference being that the incubation temperature was 45°C.

[0146] Embodiment 2-5

[0147] Same as Example 2, the only difference is that the incubation time is 90 min.

[0148] Embodiment 2-6

[0149] Same as Example 2, the only difference is that the incubation time is 120 min.

[0150] Comparative Example 8

[0151] The highland barley enzymolysis solution prepared in Example 1 was used as a raw material, and the highland barley enzymolysis solution was incubated at 50° C. for 60 minutes to obtain a highland barley-rich incubation solution. The incubation solution was sterilized by high pressure to obtain a sterile incubation solution.

[0152] Comparative Example 9

[0153] Same as Example 2, the only difference is that the amount of highland barley powder added is 0.5% of the mass of highland barley enzymatic hydrolyzate.

[0154] Comparative Example 10

[0155] Same as Example 2, the only difference being that the incubation temperature was 55°C.

[0156] Comparative Example 11

[0157] Same as Example 2, the only difference being that the incubation temperature was 60°C.

[0158] Comparative Example 12

[0159] Same as Example 2, the only difference is that the incubation time is 10 min.

[0160] Comparative Example 13

[0161] Same as Example 2, the only difference is that the incubation time is 30 min.

[0162] Experimental Example 5 GABA content analysis

[0163] The incubation solutions prepared in Examples 2 to 2-6 and Comparative Examples 8 to 13 were centrifuged at 4000 r / min for 10 min, and the supernatant was taken and the GABA content in the supernatant was determined by liquid chromatography.

[0164] The specific determination process is: the supernatant is derivatized with 4-dimethylaminophenylazobenzenesulfonyl chloride derivatizing agent, and the derivatization process is: take 1 mL of supernatant sample, add 0.2 mL of sodium bicarbonate solution, add 0.4 mL of 4-dimethylaminophenylazobenzenesulfonyl chloride, mix well, and then derivatize in a 70°C water bath for 20 minutes, cool and then pass through a membrane for use.

[0165] The HPLC detection conditions are as follows: phase A is sodium acetate solution, phase B is chromatographic acetonitrile; the chromatographic column is Agilent20RBAX SB-C18, the flow rate is 1.0mL / min, the detection wavelength is 436nm, the column temperature is 30℃, and the sample volume is 10μL; the elution program is 0~20min, 69% phase A, 31% phase B; 20.01~30min, 31% phase A, 69% phase B; 30.01~35min, 69% phase A, 31% phase B. The specific results are as follows Figures 8 to 10 As shown in Tables 4 to 6.

[0166] Depend on Figure 8 As shown in Table 4, with the increase of highland barley flour addition, the GABA content showed an upward trend, and then tended to be flat. When the addition amount of highland barley flour was 1.0% to 2.0%, the GABA content was high and there was no significant change. When the addition amount was 1.0%, the GABA content was 76.29±4.12mg / L.

[0167] Depend on Fig. 9 As shown in Table 5, with the increase of incubation temperature, the GABA content showed a trend of first increasing and then decreasing. When the incubation temperature was 50°C, the GABA content was the highest, which was 75.26±3.61 mg / L.

[0168] Depend on Fig.10As shown in Table 6, as the incubation time increases, the GABA content shows an upward trend and then tends to be flat. When the incubation time is 60min to 120min, the GABA content is high and there is no significant change. When the incubation time is 60min, the GABA content is 78.87±3.09mg / L.

[0169] Table 4 GABA content of highland barley hydrolysate with different addition amounts of highland barley flour

[0170] Addition amount of highland barley flour (%) GABA content (mg / L) 0.0 <![CDATA[44.33±2.06 a ]]> 0.5 <![CDATA[53.61±1.03 b ]]> 1.0 <![CDATA[76.29±4.12 c ]]> 1.5 <![CDATA[74.23±1.03 c <!-- 10 -->]]> 2.0 <![CDATA[75.26±2.06 c ]]>

[0171] Table 5 GABA content of highland barley hydrolysate at different incubation temperatures

[0172] Incubation temperature (℃) GABA content (mg / L) 40.0 <![CDATA[54.64±2.06 bc ]]> 45.0 <![CDATA[59.80±1.03 c ]]> 50.0 <![CDATA[75.26±3.61 d ]]> 55.0 <![CDATA[48.46±3.09 ab ]]> 60.0 <![CDATA[44.33±5.16 a ]]>

[0173] Table 6 GABA content of highland barley hydrolysate at different incubation times

[0174] Incubation time (min) GABA content (mg / L) 10.0 <![CDATA[52.58±2.06 a ]]> 30.0 <![CDATA[57.74±1.03 b ]]> 60.0 <![CDATA[78.87±3.09 c ]]> 90.0 <![CDATA[77.84±3.09 bc ]]> 120.0 <![CDATA[79.39±2.06 bc ]]>

[0175] Example 3

[0176] The process flow chart of the preparation of fermented highland barley beverage is shown in Fig.14 .

[0177] (1) After cleaning and removing impurities from highland barley grains, they were placed in a germination box at 16°C. After germination for 24 hours, the temperature of the germination box was adjusted to 50°C and continued to germinate for 2 hours. Water was then added at a mass ratio of 1:10 to grind into slurry.

[0178] (2) After the above-mentioned highland barley slurry is matured at 85°C for 10 min, α-amylase accounting for 0.5‰ of the mass of highland barley grains is added, and enzymolysis is carried out at 85°C for 2.0 h. After the temperature of the first-stage enzymatic hydrolysis liquid drops to 50°C, saccharifying enzyme accounting for 1.8‰ of the mass of highland barley grains, flavor protease accounting for 6.0‰ of the mass of highland barley grains, and cellulase accounting for 1.0‰ of the mass of highland barley grains are added, and enzymolysis is carried out at 50°C for 1.0 h to obtain highland barley enzymatic hydrolyzate.

[0179] (3) adding 1.0% by weight of highland barley flour to the above enzymatic hydrolyzate, incubating at 50° C. for 60 min to obtain a highland barley incubation solution (i.e., highland barley solution) rich in GABA.

[0180] (4) After the highland barley liquid is sterilized and cooled by high pressure, 0.02‰ of the mass of the highland barley liquid is inoculated with plant lactobacillus powder and 4‰ of the mass of the highland barley liquid is inoculated on a sterile workbench, and then the highland barley fermentation liquid is obtained by culturing in a constant temperature incubator at 36°C for 24 hours.

[0181] (5) The highland barley fermented liquid was centrifuged at 4000 r / min for 5 min, the supernatant was collected and sealed, and sterilized and enzyme-inactivating in a 90° C. water bath for 30 min to obtain a highland barley fermented beverage, which was cooled and refrigerated for later use.

[0182] Comparative Example 14

[0183] (1) After the highland barley grains are cleaned and impurities removed, water is added according to a mass ratio of highland barley grains to water of 1:10 to grind the highland barley grains into a slurry.

[0184] (2) After the highland barley slurry is matured at 85°C for 10 min, 0.5‰ α-amylase is added, and enzymolysis is carried out at 85°C for 2.0 h to obtain the first-stage enzymolysis solution. After the temperature of the first-stage enzymolysis solution drops to 50°C, 1.8‰ saccharifying enzyme, 6.0‰ flavor protease and 1.0‰ cellulase are added, and enzymolysis is carried out at 50°C for 2.0 h to obtain highland barley enzymolysis solution.

[0185] (3) The enzymatic hydrolysate was centrifuged at 4000 r / min for 5 min, the supernatant was collected and sealed, and sterilized and enzyme-inactivating in a 90° C. water bath for 30 min to obtain a highland barley fermented beverage, which was cooled and refrigerated for later use.

[0186] Comparative Example 15

[0187] (1) After cleaning and removing impurities from highland barley grains, place them in a germination box at 16°C. After germination for 24 hours, adjust the temperature of the germination box to 50°C and continue germination for 2 hours. Then, add water at a mass ratio of highland barley grains to water of 1:10 and grind the highland barley grains into slurry.

[0188] (2) After the highland barley slurry is matured at 85°C for 10 min, 0.5‰ α-amylase is added and enzymatic hydrolysis is carried out at 85°C for 2.0 h. After the temperature of the first-stage enzymatic hydrolyzate drops to 50°C, 1.8‰ saccharifying enzyme, 6.0‰ flavor protease and 1.0‰ cellulase are added and enzymatic hydrolysis is carried out at 50°C for 2.0 h to obtain highland barley enzymatic hydrolyzate.

[0189] (3) The enzymatic hydrolysate was centrifuged at 4000 r / min for 5 min, the supernatant was collected and sealed, and sterilized and enzyme-inactivating in a 90° C. water bath for 30 min to obtain a highland barley fermented beverage, which was cooled and refrigerated for later use.

[0190] Comparative Example 16

[0191] (1) After cleaning and removing impurities from highland barley grains, water is added according to a mass ratio of highland barley grains to water of 1:10 to grind into slurry.

[0192] (2) After the highland barley slurry is matured at 85°C for 10 min, 0.5‰ α-amylase is added and enzymatic hydrolysis is carried out at 85°C for 2.0 h. After the temperature of the first-stage hydrolyzate drops to 50°C, 1.8‰ saccharifying enzyme, 6.0‰ flavor protease and 1.0‰ cellulase are added and enzymatic hydrolysis is carried out at 50°C for 1.0 h to obtain highland barley hydrolyzate.

[0193] (3) Add 1.0% highland barley powder to the above enzymatic hydrolyzate, incubate at 50° C. for 60 min, and prepare a highland barley incubation solution rich in GABA.

[0194] (4) The incubation solution was centrifuged at 4000 r / min for 5 min, the supernatant was collected and sealed, and sterilized and enzyme-inactivated in a 90° C. water bath for 30 min to obtain a highland barley fermented beverage, which was cooled and refrigerated for later use.

[0195] Comparative Example 17

[0196] (1) After cleaning and removing impurities from highland barley grains, place them in a germination box at 16°C. After germination for 24 hours, adjust the temperature of the germination box to 50°C and continue germination for 2 hours. Then, add water at a mass ratio of highland barley grains to water of 1:10 and grind them into slurry.

[0197] (2) After the highland barley slurry is matured at 85°C for 10 min, 0.5‰ α-amylase is added and enzymatic hydrolysis is carried out at 85°C for 2.0 h. After the temperature of the first-stage hydrolyzate drops to 50°C, 1.8‰ saccharifying enzyme, 6.0‰ flavor protease and 1.0‰ cellulase are added and enzymatic hydrolysis is carried out at 50°C for 1.0 h to obtain highland barley hydrolyzate.

[0198] (3) Add 1.0% highland barley powder to the above enzymatic hydrolyzate, incubate at 50° C. for 60 min, and prepare a highland barley incubation solution rich in GABA.

[0199] (4) The incubation solution was centrifuged at 4000 r / min for 5 min, the supernatant was collected and sealed, and sterilized and enzyme-inactivated in a 90° C. water bath for 30 min to obtain a highland barley fermented beverage, which was cooled and refrigerated for later use.

[0200] Experimental Example 6

[0201] 1. Determine the total solids (determination method is the same as Experimental Example 1), amino acids, GABA (determination method is the same as Experimental Example 5), total phenols and flavonoids in the fermented barley drinks (i.e., beverages) obtained in Example 3 and Comparative Examples 14 to 17, and analyze their antioxidant activity and volatile substances.

[0202] Amino acids were determined according to the instructions of the amino acid content detection kit (BC1575) produced by Beijing Solebow Technology Co., Ltd. Weigh 0.1g of beverage; add 2mL of 60% ethanol solution, crush and extract by ultrasonic wave for 30min (300W, 60℃), and centrifuge at 4000rpm and 25℃ for 10min; take the supernatant and determine the total phenol content according to the instructions of the total polyphenol detection kit (A143-1-1) of Nanjing Jiancheng Bioengineering Institute. Weigh 0.1g of beverage, add 1mL of 60% ethanol solution, extract by ultrasonic wave for 30min (300W, 60℃) at 12000rpm and 25℃, centrifuge for 10min, take the supernatant and determine the flavonoid content according to the instructions of Solebow flavonoid content determination kit (BC1335). The total antioxidant capacity, superoxide anion free radical scavenging capacity, and hydroxyl free radical scavenging capacity were determined according to the instructions of the total antioxidant capacity (T-AOC) test kit (A015-1), the inhibition and generation of superoxide anion free radical test kit (A052-1), and the hydroxyl free radical test kit (A018-1) of Nanjing Jiancheng Bioengineering Institute. The DPPH free radical scavenging capacity was determined according to the instructions of the DPPH free radical scavenging capacity test kit produced by Beijing Solebow Technology Co., Ltd.

[0203] 2. Use an electronic tongue to test the taste. The samples are respectively the fermented highland barley drinks obtained in Example 3 and Comparative Examples 14 to 17. The electronic tongue is activated, calibrated, and diagnosed, and the measurement is carried out under the condition that the collected data is reliable and stable. During the test, the instrument, reagents, and electrodes are all at room temperature of 25°C, the injection volume is 35mL, the data collection time is 120s, the collection cycle is 1s, and the cleaning time is 10s. All data obtained by the electronic tongue are absolute output values ​​based on artificial saliva (reference solution) as the standard. The electronic tongue tests the state of artificial saliva to simulate the state when there is only saliva in the human mouth. The output value of the reference solution is the tasteless point (Tasteless), and the reference solution (reference) is composed of potassium chloride and tartaric acid to form a taste value.

[0204] 3. Use electronic nose to test flavor. The samples are respectively the fermented highland barley drinks obtained in Example 3 and Comparative Examples 14-17. Accurately measure 10mL of sample, add 3g of sodium chloride and place it in a 50mL bottle to seal it, place it in a 50℃ water bath for 30min and wait for detection. First, initialize, calibrate and diagnose the electronic nose equipment. Sample preparation time 5s; sensor cleaning time 120s; sensor zeroing time 10s; injection volume 400mL / min; analysis sampling time 90s; feature value extraction time 57-59s. The characteristics of the electronic nose sensor are shown in Table 7 below.

[0205] Table 7 Electronic nose sensor array characteristics

[0206] Array number Sensor Name Performance characteristics R1 W1C Sensitive to aromatic ingredients R2 W5S Very sensitive to nitrogen oxides R3 W3C Sensitive to ammonia aroma components R4 W6S Selective for hydrogen R5 W5C Sensitive to aromatic components of alkanes R6 W1S Sensitive to methane R7 W1W Sensitive to sulfide R8 W2S Sensitive to ethanol R9 W2W Sensitive to aromatic components and organic sulfides R10 W3S Sensitive to alkanes

[0207] 4. Determination of volatile substances in fermented highland barley beverages by GC-MS. The samples are respectively the fermented highland barley beverages obtained in Example 3 and Comparative Examples 14 to 17. Add 3g of sodium chloride to 5mL of the sample and shake it to fully dissolve it. Equilibrate in a 50℃ constant temperature water bath for 5min, adsorb in the extraction head space for 40min, and analyze for 5min during injection. GC conditions: chromatographic column Elite-5MS; heating program: the starting temperature of the column oven is 40℃, maintained for 2min, heated to 90℃ at 2℃ / min, and then heated to 250℃ at 10℃ / min, maintained for 5min; carrier gas flow rate is 1mL / min; injection port temperature is 250℃. MS conditions: electron impact (EI) ion source, electron energy 70eV; transmission line temperature 250℃; ion source temperature 230℃; solvent delay 0.5min; mass scanning range 35-400m / z. Use NIST spectral library search, the matching degree is greater than 800, and qualitative analysis is performed in combination with the retention index. The relative content of each component was analyzed using the peak area normalization method.

[0208] 5. Analysis of the results of the fermented highland barley beverages obtained in Example 3 and Comparative Examples 14 to 17

[0209] The results of the total solid content (determination method is the same as that of Experimental Example 1), amino acids (determination method is the same as that of Experimental Example 6), GABA (determination method is the same as that of Experimental Example 5), total phenols and flavonoids (determination method is the same as that of Experimental Example 6) in the fermented highland barley beverages obtained in Example 3 and Comparative Examples 14 to 17 are shown in Table 8. As can be seen from Table 8, compared with Comparative Example 14, there is no significant change in the soluble solids of Comparative Examples 15 to 17, but after fermentation, the soluble solids in Example 3 increased significantly. It may be that during the fermentation process, plant lactobacillus and sweet wine koji further decompose the macromolecular substances in highland barley into soluble small molecular substances.

[0210] As shown in Table 8, compared with Comparative Example 14, the amino acid and GABA contents of Example 3 and Comparative Examples 15-17 all significantly increase, with the amino acid content ranging from 7.68 to 12.29 μmol / mL, and the GABA content ranging from 34.46 to 83.28 mg / L, and the trend of both increases is consistent. The amino acid and GABA contents of Comparative Example 17, which is germinated and added with highland barley powder during enzymolysis, are the highest, and the contents of amino acid and GABA of Comparative Example 15 are increased by 47% and 80% respectively, because germination activates glutamate decarboxylase (GAD) and converts glutamate into GABA. Adding highland barley powder (Comparative Example 16) increases the contents of amino acid and GABA by 34% and 60% respectively, because the GAD enzyme activity of highland barley powder is higher and glutamate can be synthesized into GABA. Compared with the sample of Comparative Example 17, the content of amino acids and GABA in Example 3 after fermentation treatment decreased significantly, but was still significantly higher than that in Comparative Examples 15 and 16. The reason may be that microorganisms consume part of the amino acids and GABA during the growth process.

[0211] As shown in Table 8, compared with Comparative Example 14, the total phenol content of Example 3 and Comparative Examples 15-17 is improved. The total phenol content of Example 3 is increased from 0.59mmoL / L in Comparative Example 14 to 0.94mmoL / L, an increase of 60%. The total phenol content of Comparative Example 16 is higher than that of Comparative Example 15, indicating that the added highland barley powder releases more phenolic substances during the enzymatic hydrolysis process. The total phenol content of Comparative Example 15 is also slightly increased, and the reason may be that during the germination period, essential enzymes in the biosynthesis of phenols, such as phenylalanine ammonia lyase, can be activated. Compared with Comparative Example 14, the flavonoid content of Comparative Examples 15-17 and Example 3 increased by 37%, 62%, 76%, and 40%, respectively. The flavonoid content in the fermented sample of Example 3 is significantly reduced compared with the unfermented sample of Comparative Example 17, and the reason may be that the fermentation of Lactobacillus plantarum metabolizes complex flavonoids into simple compounds.

[0212] Table 8 Total solids, GABA, amino acid total phenols and flavonoids content in highland barley enzymatic beverage

[0213]

[0214] As can be seen from Table 8, the technical solution of the present invention can significantly increase the content of total solids, amino acids, GABA, total phenols and flavonoids through germination, enzymolysis, incubation and fermentation processes, and improve the nutritional value of highland barley fermented beverages.

[0215] The results of the total antioxidant capacity, DPPH free radical, superoxide anion free radical, and hydroxyl free radical scavenging capacity of the fermented highland barley beverage obtained in Example 3 and Comparative Examples 14 to 17 are shown in Table 9. It can be seen that compared with Comparative Example 14, Comparative Example 15 significantly improved the superoxide anion and hydroxyl free radical scavenging capacity. In addition to the significant decrease in the DPPH free radical scavenging capacity of the sample in Comparative Example 16, the other three antioxidant activities were significantly enhanced, and the superoxide anion scavenging capacity was increased by 126%. The sample in Comparative Example 17 significantly improved the DPPH free radical, superoxide anion, and hydroxyl free radical scavenging capacity. The four antioxidant capacities of Example 3 were significantly improved, and the total antioxidant capacity was increased by 96%. Except for the superoxide anion scavenging capacity of Example 3, which ranked second, which was lower than Comparative Example 16, the other three antioxidant activity capacities ranked first. It is illustrated that the technical solution of the present invention integrates germination, enzymolysis, incubation and fermentation processes, which can significantly increase the total antioxidant capacity of highland barley fermented beverages, and significantly increase the DPPH, superoxide anion radical and hydroxyl radical scavenging capacity, thereby improving the antioxidant activity of highland barley fermented beverages.

[0216] Table 9 Contents of total solids, GABA, amino acid total phenols and flavonoids in highland barley enzymatic beverages

[0217]

[0218] The taste analysis of the fermented highland barley beverages obtained in Example 3 and Comparative Examples 14 to 17 is shown in Fig.11 . Flavor analysis see Fig.12 .Depend on Fig.11 and 12 It can be seen that the taste and olfactory profiles of comparative examples 14 to 17 overlap, and the taste characteristics and olfactory sense are basically the same. The umami, salty taste and richness are higher than those of Example 3, and there is a clear lack of sourness. The sourness of Example 3 increased through fermentation, the bitterness decreased, and the olfactory profile increased significantly. Among them, the response values ​​of the olfactory sensors W1S (sensitive to methane), W2S (sensitive to ethanol), and W5S (very sensitive to nitrogen oxides), and the sensors W1W (sensitive to sulfides) and W2W (sensitive to organic sulfides and aromatic compounds) increased significantly. It shows that fermentation can not only improve the taste of highland barley enzymatic beverages, but also produce more volatile substances, thereby improving the flavor of highland barley enzymatic beverages.

[0219] The results of volatile flavor substances of the fermented highland barley beverages obtained in Example 3 and Comparative Examples 14 to 17 are shown in Fig.13 .Depend on Fig.13It can be seen that Example 3 is also significantly different from Comparative Examples 14 to 17 in terms of volatile flavor substances. Comparative Examples 14 to 17 and Example 3 contain 37, 32, 42, 47 and 43 volatile compounds, respectively, of which n-pentanol, 1-octen-3-ol, n-heptanol, 2-ethylhexanol, n-valeraldehyde, 2,4-di-tert-butylphenol, n-hexanal, trans-2-heptanal, n-nonanal, benzaldehyde, 2-phenylacetaldehyde, acetone, acetophenone, hexyl formate, spironolactone, benzyl acetate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, and methoxyphenyl oxime are common, and it is speculated that these components are mainly derived from highland barley raw materials. Compared with Comparative Example 14, Comparative Example 15 added one lauryl alcohol through germination, Comparative Example 16 added one isobutyl acetate through the addition of active highland barley powder, and Comparative Example 17 added four compounds through germination and the addition of active highland barley powder, namely 4-ethylcyclohexanol, 2-butylcyclohexanone, octyl formate, and 2-ethylhexanoic anhydride.

[0220] In Example 3, 17 new substances were added, involving alcohols, esters, phenols, aldehydes, and acids. Among them, the most new substances were acids, including acetic acid, octanoic acid, n-hexanoic acid, and guanidinopropionic acid. The aldehyde substance was furfural. The ester substances were linalyl butyrate and hexyl formate, 2,4-di-tert-butylphenol, and n-butyl isooctyl phthalate. The alcohols were styraxol, tetrahydrolavandulol, benzyl alcohol, n-heptanol, and 3-methyl-3-butene-1-ol. The phenolic substances were 2-methoxy-4-methylphenol, 2-methoxy-4-vinylphenol, and 2,3-dihydrobenzofuran.

[0221] The contents of the above 17 substances in Example 3 are higher than those in Comparative Examples 14 to 17, wherein n-heptanol and benzyl alcohol both have aromatic flavors. The technical solution of the present invention prepares highland barley fermented beverages through germination, enzymolysis, incubation and fermentation processes, which can increase the types of flavor substances in highland barley fermented beverages, increase the content of flavor substances, and improve the flavor of the product.

[0222] In summary, considering the nutrient content, antioxidant activity, taste, flavor and volatile flavor substance content, the present invention provides an integrated germination, enzymatic hydrolysis, incubation and fermentation process, which can improve the GABA, polyphenol and flavonoid content and antioxidant activity in highland barley fermented beverages, while improving the taste and flavor of the product.

[0223] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing highland barley liquor rich in GABA, characterized in that: The following steps are involved: The highland barley is processed after being germinated to obtain highland barley pulp; The highland barley slurry is subjected to a first-stage enzymolysis and a second-stage enzymolysis in sequence to obtain an enzymolysis solution; The enzymatic hydrolysis solution is mixed with highland barley powder to obtain highland barley solution rich in GABA; the enzyme used in the first stage of enzymatic hydrolysis includes alpha-amylase; the enzyme used in the second stage of enzymatic hydrolysis includes one or more of saccharifying enzyme, flavor protease and cellulase.

2. The preparation method according to claim 1, characterized in that: The germination treatment includes low-temperature germination treatment and high-temperature germination treatment; the temperature of the low-temperature germination treatment is 13-18°C and the time is 24-48 hours; the temperature of the high-temperature germination treatment is 35-55°C and the time is 1-2 hours.

3. The preparation method according to claim 1, characterized in that: The temperature of the first stage enzymolysis is 80-90°C, and the time of the first stage enzymolysis is 0.5-3.0h; the amount of α-amylase added is 0.5‰-0.7‰ of the mass of highland barley.

4. The preparation method according to claim 1, characterized in that: The temperature of the second stage enzymatic hydrolysis is 35-60°C, and the time of the second stage enzymatic hydrolysis is 0.5-2.0h; The amount of saccharifying enzyme added is 1.2‰ to 1.8‰ of the mass of highland barley; The amount of flavor protease added is 2.0‰ to 8.0‰ of the mass of highland barley; The amount of cellulase added is 0.5‰ to 1.5‰ of the mass of highland barley.

5. The preparation method according to claim 1, characterized in that: The addition amount of highland barley powder is 0.5% to 2.0% of the mass of the enzymatic hydrolyzate. The mixing further comprises incubation, the incubation temperature is 40 to 60° C., and the time is 60 to 120 minutes.

6. The preparation method according to claim 1, characterized in that: Before the first stage of enzymolysis, the process also includes aging; the aging temperature is 80-90° C. and the time is 15-30 minutes.

7. A method for preparing a fermented highland barley beverage rich in GABA, characterized in that: include: The highland barley liquid obtained by the preparation method according to any one of claims 1 to 6 is mixed with microorganisms and fermented to obtain a highland barley fermented beverage; The microorganisms include Lactobacillus plantarum and / or sweet wine koji.

8. The preparation method according to claim 7, characterized in that: When the microorganism includes plant lactobacillus, the amount of plant lactobacillus added is 0.01‰ to 0.1‰ of the mass of highland barley liquid; When the microorganism contains sweet wine yeast, the amount of sweet wine yeast added is 0.5‰ to 5.0‰ of the mass of highland barley liquid; The fermentation temperature is 25-40° C., and the fermentation time is 6-48 hours.

9. The fermented highland barley beverage prepared by the preparation method according to claim 7 or 8, characterized in that: The GABA content in the fermented highland barley beverage is 70-80 mg / L.

10. Use of the preparation method according to claim 7 or 8 in one or more of the following 1) to 3), 1) Improve the antioxidant activity of fermented highland barley beverages; 2) Enrich the types of volatile flavor substances in fermented highland barley beverages; 3) Increase the content of volatile flavor substances in fermented highland barley beverages.

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