Method for preparing functional food with high gamma-aminobutyric acid content by fermenting glutenin bean sprout juice zymolyte and coix seeds through monascus

Abstract

The invention discloses a method for preparing functional food with high gamma-aminobutyric acid content by fermenting glutenin bean sprout juice zymolyte and coix seeds through monascus. The method comprises the following steps: dissolving vital gluten in 65% ethanol according to a ratio of 1: 30, stirring and extracting at 35 DEG C for 2 hours, standing and separating precipitate and supernate, continuously extracting the precipitate with 65% ethanol, and centrifuging after extraction to obtain the precipitate, namely glutenin. Soaking the mung beans at 25-35 DEG C to germinate the mung beans, and juicing the mung beans when the mung beans grow to 5-10 cm; mixing the mung bean sprout juice and glutenin, performing enzymolysis reaction, drying and crushing. Mixing coix seeds and glutenin, and sterilizing to obtain a solid culture medium to be fermented; spores on the monascus purpureus slant culture medium are dissolved out with sterile water and inoculated to a solid culture medium, and the solid culture medium is placed in a constant-temperature incubator to be fermented after being evenly mixed. And drying the fermented culture medium at 60 DEG C until the weight is constant, and crushing. The content of gamma-aminobutyric acid in the food with high gamma-aminobutyric acid content is higher than 2.5 mg / g.

Description

[0001] This invention relates to a method for preparing functional foods high in γ-aminobutyric acid (GABA) using Monascus purpureus fermentation of wheat gluten-sprout juice hydrolysate and Job's tears. Background Technology

[0002] Monascus purpureus is a filamentous fungus used in both medicine and food fermentation, with a history of over 2000 years in China, Japan, and other Southeast Asian countries. Records of red yeast rice originate from a poem by Wang Can: "Traveling west to Liang, I ate vegetarian meals, including red yeast rice from Guazhou, mixed with other ingredients, soft, smooth, and creamy, melting in the mouth." The *Compendium of Materia Medica* also contains records of red yeast rice, detailing its production process and medicinal uses. Based on various sources, Monascus purpureus produces various metabolites during its growth, including enzymes, pigments, antibacterial substances, γ-aminobutyric acid (GABA), and cholesterol biosynthesis inhibitors. In recent years, Monascus purpureus has been used extensively in food production, and its safety has been proven. In contrast, chemical synthesis of GABA has poor safety, causes serious environmental pollution, and results in lower phytoencapsulation levels; therefore, microbial fermentation for GABA production has a very promising future.

[0003] Gluten powder is a wheat protein product isolated from wheat or wheat flour. After hydration, it possesses viscoelasticity and certain tensile properties, hence it is also known as active wheat gluten powder. The main components of gluten powder are glutenin and gliadin, along with small amounts of fat, carbohydrates, and fiber. Glutenin has advantages such as wide availability, low allergenicity, and good biological value, and is widely used in food. Glutenin obtained through enzymatic hydrolysis of bean sprout juice contains gamma-aminobutyric acid (GABA), which has sleep-promoting effects and can effectively improve sleep in people with insomnia.

[0004] Gamma-aminobutyric acid (GABA) is a natural non-protein amino acid widely distributed in animals, plants, and microorganisms. As an important inhibitory neurotransmitter in the central nervous system of animals, GABA has been proven to possess various physiological functions, including calming the nerves, regulating appetite, lowering blood pressure, preventing arteriosclerosis, and improving lipid metabolism, making it important in the food and pharmaceutical industries. Currently, GABA is mainly obtained through microbial fermentation and plant enrichment methods. Domestic and international literature reviews systematically summarize the biochemical metabolic pathways, physiological functions, production processes, and applications of GABA in the food industry. GABA is formed by the decarboxylation of glutamate under the action of glutamate decarboxylase. This invention utilizes the principle that mung bean sprout juice has few contaminating bacteria and contains a large amount of proteases and glutamate decarboxylases that can decompose glutenin to produce glutamate, which is then converted into GABA. The fermentation process of Monascus purpureus can also produce GABA, thus preparing a food product high in GABA and glutenin polypeptides.

[0005] Due to increased life stress and the impact of various diseases, insomnia has become a serious health problem that has long plagued people. Although there are many types of drugs for treating insomnia, they all have varying degrees of side effects. This invention utilizes Monascus purpureus fermentation of bean sprout juice to enzymatically hydrolyze glutenin and Job's tears (which have spleen-strengthening, diuretic, heat-clearing, and cough-suppressing effects). The gaps between the Job's tears provide oxygen for Monascus purpureus fermentation in the culture medium, promoting its growth. During the enzymatic hydrolysis of glutenin in mung bean sprout juice, a large amount of γ-aminobutyric acid (GABA) is produced. Simultaneously, the sterilization treatment before Monascus purpureus fermentation reduces contamination from other bacteria during the hydrolysis process. The GABA produced during Monascus purpureus fermentation directly increases the GABA content in the product. This invention utilizes the Monascus purpureus fermentation of bean sprout juice to enzymatically hydrolyze glutenin and Job's tears to develop a Monascus purpureus fermented food with high GABA content, which can regulate sleep problems and improve people's quality of life. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies by providing a method for preparing functional foods high in γ-aminobutyric acid (GABA) using Monascus purpureus fermented with wheat gluten-sprout hydrolysate and Job's tears. The Monascus purpureus used in this patent is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified as *Monascus purpureus*, with a deposit date of January 17, 2024, and located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing functional foods high in γ-aminobutyric acid (GABA) using Monascus purpureus fermentation of wheat gluten-sprout juice hydrolysate and Job's tears includes the following steps:

[0009] Commercially available gluten powder was mixed at a mass ratio of 1:30 and slowly poured into 65% ethanol while stirring to dissolve. The mixture was stirred at 35°C for 2 hours, and then allowed to stand for 3 hours. This process was repeated. The precipitate containing a small amount of liquid at the bottom was centrifuged at 3500 rpm for 5 minutes. The precipitate was glutenin, which was dried at 40–60°C to constant weight and then pulverized. Mung beans were soaked in tap water and germinated at 25–35°C. When the sprouts reached 5–10 cm in length, they were juiced to obtain mung bean sprout juice. The sprout juice was mixed with deionized water at a ratio of 1:500 and then added to the glutenin powder. 5 mL of the mixed solution was added per gram of glutenin powder. After thorough stirring, the mixture was incubated at 20°C for 3 hours to allow for complete enzymatic hydrolysis. The mixture was then dried in an oven and pulverized to a fineness of 100–200 mesh.

[0010] Soak Job's tears for 2 days and dry them. Weigh Job's tears and wheat gluten and bean sprout juice hydrolysate at a mass ratio of 3:7 into an Erlenmeyer flask, stir well, seal and prepare a solid culture medium. Sterilize in an autoclave at 110℃ for 40 minutes. Dissolve the spores on the Monascus purpureus slant culture medium in sterile water in a laminar flow hood, ensuring they are evenly distributed in the solid culture medium. Incubate at 32℃ for 20–30 days.

[0011] After fermentation, the solid culture medium was dried and pulverized. 0.1 g of the culture medium and 1 mL of the extract were weighed and placed in an EP tube and shaken well. After being in a water bath for 2 hours, the mixture was centrifuged. The supernatant was taken and mixed with the reagents in the kit. After being in a water bath, the absorbance was measured at 640 nm. The content was calculated according to the standard regression equation.

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] This invention utilizes the enzymatic hydrolysis of glutenin in mung bean sprout juice to obtain raw materials containing γ-aminobutyric acid (GABA) and glutamic acid. During fermentation, a culture medium providing carbon and nitrogen sources is used only, consisting of a mixture of glutenin-bean sprout hydrolysate and Job's tears. Monascus purpureus directly utilizes the nutrients in the Job's tears protein for growth, secreting beneficial secondary metabolites such as GABA and lovastatin, further increasing the GABA content in the product. The final high-GABA glutenin hydrolysate product contains GABA levels exceeding 2.5 mg / g. Combined with the lipid-lowering effect of lovastatin produced by Monascus purpureus fermentation, a safe functional food product with both sleep-promoting and lipid-lowering properties is prepared. Attached Figure Description

[0015] Figure 1 This is a picture of Monascus purpureus fermenting for 30 days in a medium of bean sprout juice enzymatically hydrolyzed glutenin and Job's tears.

[0016] Figure 2 This image shows Monascus purpureus fermenting for 15 days in a medium of bean sprout juice hydrolyzed with gluten and Job's tears. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to specific embodiments.

[0018] Example 1

[0019] 200g of wheat gluten powder was mixed with 6000mL of 65% ethanol and extracted in a 35℃ water bath for 2 hours with stirring to dissolve the wheat gliadin in the ethanol. The extracted solution was centrifuged at 3500r / min for 5 minutes to obtain 318.78g of wet glutenin. The glutenin was dried to constant weight at 40℃ and then pulverized to a fineness of 100 mesh. 100g of mung beans were soaked in tap water and germinated at 25℃. When the sprouts reached 5cm in length, they were juiced to obtain 38.4mL of mung bean sprout juice. 1mL of mung bean sprout juice was diluted to 10mL with sterile water and mixed with 50g of glutenin at a volume-to-mass ratio. The mixture was enzymatically hydrolyzed at 20℃ for 7 days. The hydrolysate was dried to constant weight at 45℃ for 24 hours and then pulverized to a fineness of 100 mesh to obtain 20.7g of hydrolysate. The resulting food product containing high levels of γ-aminobutyric acid (GABA) wheat gluten hydrolysate had a GABA content of 0.41 mg / g.

[0020] Weigh 25g of Job's tears, 20g of glutenin, and 12.5mL of deionized water into an Erlenmeyer flask. Stir well and seal. Wrap the flask, deionized water, petri dishes, and spray bottle with newspaper. Place the flask in an autoclave at 110℃ for 40 minutes. Sterilize under UV light for 30 minutes in a laminar flow hood. Dissolve the purified Monascus purpureus in sterile water in the laminar flow hood. Let it stand and collect the supernatant in a spray bottle after autoclaving. Spray the bacterial solution into the Erlenmeyer flask and stir well. Place the solution evenly in petri dishes near an alcohol lamp, label and name them, and then incubate at 32℃.

[0021] After 30 days of cultivation Figure 1 As shown, the culture medium was dried and pulverized to a fineness of 100 mesh in a 60℃ oven. Following the kit instructions, 0.1g and 1mL of the extract were weighed into a 1.5m IEP tube and thoroughly mixed. The tube was incubated in a 95℃ water bath for 2 hours, cooled, and then incubated at 8000 rpm for 10 minutes. 90mL of the supernatant, 150mL of reagent one, and 120mL of reagent two were taken, mixed, and allowed to stand at room temperature for 5 minutes. Then, 180mL of reagent three was added, mixed, and incubated in a 95℃ water bath for 10 minutes. The mixture was then cooled in an ice bath, and 600mL of reagent four was added. The absorbance was measured at 640nm. The concentration was calculated to be 2.54mg / g based on the regression equation.

[0022] Example 2

[0023] 200g of wheat gluten powder was mixed with 6000mL of 65% ethanol and extracted in a 35℃ water bath for 2 hours with stirring to dissolve the wheat gliadin in the ethanol. The extracted solution was centrifuged at 3500r / min for 5 minutes to obtain 308.41g of wet glutenin. The glutenin was dried to constant weight at 60℃ and then pulverized to a fineness of 200 mesh. 100g of mung beans were soaked in tap water and germinated at 35℃. When the sprouts reached 10 minutes in length, they were juiced to obtain 40.3mL of mung bean sprout juice. 1mL of mung bean sprout juice was diluted with sterile water to 50mL and mixed with 200g of glutenin at a volume-to-mass ratio. The mixture was enzymatically hydrolyzed at 40℃ for 1 day. The hydrolysate was dried at 60℃ and pulverized to a fineness of 200 mesh to obtain 78.3g of hydrolysate. The resulting food product containing high levels of γ-aminobutyric acid (GABA) wheat gluten hydrolysate had a GABA content of 0.39 mg / g.

[0024] Weigh 25g of Job's tears, 20g of glutenin, and 12.5mL of deionized water into an Erlenmeyer flask. Stir well and seal. Wrap the flask, deionized water, petri dishes, and spray bottle with newspaper. Place the flask in an autoclave at 110℃ for 40 minutes. Sterilize under UV light for 30 minutes in a laminar flow hood. Dissolve the purified Monascus purpureus in sterile water in the laminar flow hood. Let it stand and collect the supernatant in a spray bottle after autoclaving. Spray the bacterial solution into the Erlenmeyer flask and stir well. Place the solution evenly in petri dishes near an alcohol lamp, label and name them, and then incubate at 32℃.

[0025] After 15 days of cultivation, as Figure 2 As shown, the culture medium was dried and pulverized to a fineness of 100 mesh in a 60℃ oven. Following the kit instructions, 0.1g and 1mL of the extract were weighed into a 1.5m IEP tube and thoroughly mixed. The tube was incubated in a 95℃ water bath for 2 hours, cooled, and then incubated at 8000 rpm for 10 minutes. 90mL of the supernatant, 150mL of reagent one, and 120mL of reagent two were taken, mixed, and allowed to stand at room temperature for 5 minutes. Then, 180mL of reagent three was added, mixed, and incubated in a 95℃ water bath for 10 minutes. The mixture was then cooled in an ice bath, and 600mL of reagent four was added. The absorbance was measured at 640nm. The concentration was calculated to be 3.2mg / g based on the regression equation.

Claims

1. A method for preparing functional foods high in γ-aminobutyric acid (GABA) using Monascus purpureus fermentation of wheat gluten-sprout juice hydrolysate and Job's tears, characterized in that: Includes the following steps: (1) Mix gluten powder and 65% ethanol at a mass ratio of 1:30, stir continuously at 35°C, and extract in a water bath for 2 hours. After standing at room temperature for 3 hours, extract the precipitate twice with 65% ethanol. After extraction, centrifuge the lower layer at 3500 rpm for 5 minutes. The precipitate obtained is glutenin. (2) Soak mung beans in tap water and germinate them at 25-35℃ for 2-3 days. When the sprouts are 5-10cm long, juice them with a juicer to obtain mung bean sprout juice. (3) Mix mung bean sprout juice and glutenin at a volume-to-mass ratio of 1:50 to 1:200 (v / g), stir thoroughly, and place in a constant temperature incubator at 20 to 40°C for 1 to 7 days for enzymatic hydrolysis. Then place it in an oven to dry and pulverize it. The drying temperature is 45 to 60°C and the pulverization fineness is 100 to 200 mesh to obtain glutenin bean sprout juice hydrolysate. (4) Sterilize the wheat gluten bean sprout juice hydrolysate and Job's tears under high pressure to prepare a solid culture medium. Dissolve the purified Monascus purpureus slant culture medium in sterile water and place it in a spray bottle after high pressure sterilization. Spray it into the culture medium, mix it evenly, and then place it in a 32℃ constant temperature incubator for constant temperature incubation for 20 to 30 days. (5) After the solid culture medium is dried and pulverized after fermentation, the content of γ-aminobutyric acid is determined according to the spectrophotometric method in the kit.

2. The method for preparing a functional food high in γ-aminobutyric acid (GABA) using Monascus purpureus fermented wheat gluten-sprout juice hydrolysate and Job's tears according to claim 1, characterized in that, The red mold is classified and named Monascus purpureus, with accession number 41082, and is deposited at the China General Microbiological Culture Collection Center (CGMCC).

3. The method for preparing a functional food high in γ-aminobutyric acid (GABA) using Monascus purpureus fermented wheat gluten sprout juice hydrolysate and Job's tears according to claim 1, characterized in that... The glutenin obtained in step 1(1) needs to be dried at 40-60℃ to constant weight and then pulverized to a fineness of 100-200 mesh.

4. The method for preparing a functional food high in γ-aminobutyric acid (GABA) using Monascus purpureus fermented wheat gluten sprout juice hydrolysate and Job's tears according to claim 1, characterized in that: The mung bean sprout juice in step 1(2) can be replaced by any grain or bean sprout juice.

5. The method for preparing a functional food high in γ-aminobutyric acid (GABA) using Monascus purpureus fermented wheat gluten sprout juice hydrolysate and Job's tears according to claim 1, characterized in that, The culture medium selected for Monascus fermentation was a mixture of wheat gluten and soybean sprout hydrolysate and Job's tears at a mass ratio of 7:

3.

6. The method for preparing a functional food high in γ-aminobutyric acid (GABA) using Monascus purpureus fermented wheat gluten-sprout juice hydrolysate and Job's tears according to claim 1, characterized in that, The Job's tears are soaked in warm water for 1-2 days. After draining the Job's tears, they are mixed with glutenin obtained from the enzymatic hydrolysis of bean sprout juice. Then, they are placed in a high-pressure steam sterilizer for sterilization at a temperature of 110°C for 40 minutes. After sterilization, a solid culture medium for fermentation is obtained.

Images