Germinated highland barley fermentation liquor with functions of resisting oxidation, reducing blood glucose and reducing lipid in vitro as well as preparation method and application of germinated highland barley fermentation liquor

Abstract

The invention provides germinated highland barley fermentation liquor with functions of resisting oxidation, reducing blood glucose and reducing lipid in vitro as well as a preparation method and application of the germinated highland barley fermentation liquor, and belongs to the technical field of functional foods. The method comprises the following steps: disinfecting highland barley seeds, soaking, and germinating the soaked highland barley seeds; freeze-drying, crushing and sieving the germinated highland barley seeds to obtain germinated highland barley powder; mixing the germinated highland barley powder with water, and adding bacterial powder for fermentation to obtain the germinated highland barley fermentation liquor. According to the method, the highland barley is firstly subjected to germination treatment, then probiotics are added for fermentation to obtain the germinated highland barley fermentation liquor, and through combined germination and fermentation treatment, bioactive components such as total phenols, total flavonoids and gamma-aminobutyric acid in the highland barley can be enriched; and the growth and metabolism of the probiotics further promote the release of bioactive components of the highland barley, so that the oxidation resistance and the blood sugar and lipid lowering capability of the germinated highland barley fermentation liquor are enhanced, and the additional value of the highland barley product is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of functional foods, and in particular relates to a germinated highland barley fermentation liquid with in vitro antioxidant, blood sugar and lipid lowering functions, and a preparation method and application thereof. Background Art

[0002] Highland barley (Hordeum vulgare L. var. nudum Hook. f.), also known as naked barley, belongs to the grass family. Its nutritional composition is comprehensive and unique, in line with the "three highs and two lows" dietary structure, that is, high protein, high soluble fiber, high vitamins and low fat and low sugar, and has extremely high nutritional and therapeutic value. In addition to bioactive ingredients such as β-glucan, polyphenols, flavonoids, and anthocyanins, highland barley is also rich in B vitamins and a variety of mineral elements that are beneficial to human health. The functional research of highland barley mainly focuses on bioactive ingredients such as β-glucan, phenols, and γ-aminobutyric acid. These active substances give highland barley physiological effects such as anti-oxidation, anti-cancer, anti-aging, and prevention of cardiovascular and cerebrovascular diseases. Highland barley has a long history and grows in plateau areas at an altitude of 4200 to 4500 meters. Highland barley has high nutritional value, among which highland barley β-glucan is one of the main bioactive components in highland barley, and it has been proven to have multiple biological activities. Currently, the products on the market include highland barley instant noodles, highland barley rice, highland barley biscuits, highland barley tea, highland barley oatmeal, etc. The product forms are relatively simple, and the research and development of its functional products and bioactive functions are not in-depth enough. Therefore, it is necessary to further clarify the effects of different processing methods on the bioactive components of highland barley and their health effects, combine a variety of processing methods, develop functional foods with high added value, and extend the highland barley industry chain. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a germinated highland barley fermentation liquid with in vitro antioxidant, blood sugar and lipid-lowering functions, and a preparation method and application thereof.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing germinated highland barley fermentation liquid, comprising the following steps:

[0006] 1) sterilizing highland barley seeds, soaking them, and germinating the soaked highland barley seeds;

[0007] 2) freeze-drying the germinated highland barley seeds, crushing them, and sieving them to obtain germinated highland barley powder;

[0008] 3) mixing sprouted highland barley powder and water, adding bacterial powder for fermentation, and obtaining sprouted highland barley fermentation liquid.

[0009] Preferably, in step 3), the mass volume ratio of the germinated highland barley powder and water is 1 g: 4-8 mL.

[0010] Preferably, the bacterial powder in step 3) is the composite bacteria VEGE-022, and the added amount of the bacterial powder is 0.02% to 0.04% (w / v) of the total volume of the mixed solution of germinated barley powder and water; the fermentation temperature is 30 to 45° C., and the fermentation time is 4 to 6 hours.

[0011] Preferably, the pH value of the water used for soaking in step 1) is 6.5-7.5, the mass volume ratio of the sterilized highland barley seeds and water is 1g:2-7mL, the soaking temperature is 20-30°C, and the soaking time is 5-7h;

[0012] The disinfection is carried out by immersion disinfection in a sodium hypochlorite solution, the mass fraction of the sodium hypochlorite solution is 0.5-2%, and the disinfection time is 10-20 minutes.

[0013] Preferably, in step 1), the soaked highland barley seeds are placed on gauze for germination, water is added during the germination process, the pH value of the water is 6.0-7.0, the mass volume ratio of the soaked highland barley seeds and water is 1g:2-7mL, the germination temperature is 25-30°C, and the germination time is 20-30h.

[0014] Preferably, after the germinated highland barley powder and water are mixed in step 3), a sterilization step is also included before fermentation, and the sterilization temperature is 60 to 80° C. and the sterilization time is 20 to 45 minutes;

[0015] The fermentation is followed by centrifugation, wherein the centrifugal speed is 3000-6000 r / min and the centrifugal time is 5-15 min.

[0016] The invention also provides highland barley fermentation liquid prepared by the preparation method.

[0017] The present invention also provides the germinated highland barley fermentation liquid prepared by the preparation method or the application of the highland barley fermentation liquid in the preparation of products helpful for anti-oxidation.

[0018] The present invention also provides the germinated highland barley fermentation liquid prepared by the preparation method or the application of the highland barley fermentation liquid in preparing a product that helps maintain a healthy blood lipid level.

[0019] The present invention also provides the germinated highland barley fermentation liquid prepared by the preparation method or the application of the highland barley fermentation liquid in preparing products that are helpful in maintaining healthy blood sugar levels.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The method comprises the following steps: firstly subjecting highland barley to germination treatment, and then adding probiotics for fermentation to obtain germinated highland barley fermentation liquid. The combined treatment of germination and fermentation can enrich the total phenols, total flavonoids, gamma-aminobutyric acid and other bioactive components in the highland barley. Meanwhile, during the 28-day shelf life, the growth and metabolism of the probiotics further promote the release of the bioactive components of the highland barley, thereby enhancing the antioxidant, blood sugar and lipid lowering capabilities of the highland barley fermentation liquid, and improving the added value of the highland barley product. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Effects of different processing methods on the content of bioactive components in highland barley (A is β-glucan content, B is total phenol content, C is flavonoid content, and D is GABA content);

[0023] Figure 2 is the changes in the physical and chemical properties and bioactive components of the fermentation broth during the 28-day shelf life (where A is the change in pH, B is the change in titratable acidity, C is the change in protein content, D is the change in total sugar content, E is the change in soluble solids content, F is the change in the total number of probiotic colonies, G is the change in β-glucan content, H is the change in total phenol content; I is the change in flavonoids content, and J is the change in GABA content);

[0024] Figure 3 is the change of antioxidant capacity of the fermentation broth during the 28-day shelf life (where A is the change of DPPH free radical scavenging capacity, B is the change of FRAP iron ion reducing capacity, and C is the change of nitrite scavenging capacity);

[0025] Figure 4 is the change of in vitro glucose-lowering ability of the fermentation broth during the 28-day shelf life (where A is the change of α-glucosidase inhibition ability and B is the change of α-amylase inhibition ability);

[0026] Figure 5 is the adsorption kinetic curve of the fermentation broth to different bile salts during the 28-day shelf life (wherein, A is the adsorption kinetic curve of sodium taurocholate by germination and fermentation, B is the adsorption kinetic curve of sodium taurocholate by fermentation, C is the adsorption kinetic curve of sodium glycocholate by germination and fermentation, D is the adsorption kinetic curve of sodium glycocholate by fermentation, E is the adsorption kinetic curve of sodium cholate by germination and fermentation, and F is the adsorption kinetic curve of sodium cholate by fermentation);

[0027] Figure 6is the isothermal adsorption curve of the fermentation broth for different bile salts during the 28-day shelf life (wherein, A is the isothermal adsorption curve of germination and fermentation for sodium taurocholate, B is the isothermal adsorption curve of fermentation for sodium taurocholate, C is the isothermal adsorption curve of germination and fermentation for sodium glycocholate, D is the isothermal adsorption curve of fermentation for sodium glycocholate, E is the isothermal adsorption curve of germination and fermentation for sodium cholate, and F is the isothermal adsorption curve of fermentation for sodium cholate);

[0028] Figure 7 The changes in the in vitro lipid-lowering ability of the fermentation broth during the 28-day shelf life (where A is the cholesterol esterase inhibition rate, B is the pancreatic lipase inhibition rate, C is the sodium taurocholate binding rate, D is the sodium glycocholate binding rate, and E is the sodium cholate binding rate). DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing germinated highland barley fermentation liquid, comprising the following steps:

[0030] 1) sterilizing highland barley seeds, soaking them, and germinating the soaked highland barley seeds;

[0031] 2) freeze-drying the germinated highland barley seeds, crushing them, and sieving them to obtain germinated highland barley powder;

[0032] 3) mixing sprouted highland barley powder and water, adding bacterial powder for fermentation, and obtaining sprouted highland barley fermentation liquid.

[0033] In the present invention, highland barley seeds are disinfected and then soaked, and the soaked highland barley seeds are germinated; the disinfection is carried out by soaking in a sodium hypochlorite solution, and the mass fraction of the sodium hypochlorite solution is preferably 0.5-2%, and more preferably 0.8-1.5%; the disinfection time is preferably 10-20 minutes, and more preferably 12-18 minutes; the soaking is carried out by water, and the mass volume ratio of the disinfected highland barley seeds and water is preferably 1g:2-7mL, and more preferably 1g:3-6mL; the water is preferably deionized water, and the pH value of the deionized water is preferably 6.5-7.5, and more preferably 6.8-7.2; the soaking The soaking temperature is preferably 20-30°C, more preferably 22-28°C; the soaking time is preferably 5-7h, more preferably 5.5-6.5h; the soaked highland barley seeds are placed on gauze for germination, and water is supplemented during the germination process, and the pH value of the water is 6.0-7.0, more preferably 6.2-6.8; the mass volume ratio of the germinated highland barley seeds and water is 1g:2-7mL, more preferably 1g:3-6mL; the germination temperature is preferably 25-30°C, more preferably 26-29°C; the germination time is preferably 20-30h, more preferably 22-28h.

[0034] In the present invention, the germinated highland barley seeds are freeze-dried, crushed, and sieved to obtain germinated highland barley powder. The freeze-drying temperature is preferably -20°C to -60°C, more preferably -30°C to -50°C, the freeze-drying time is preferably 15 to 30 hours, more preferably 20 to 25 hours; the mesh number of the sieve is preferably 70 to 90 meshes, more preferably 75 to 85 meshes.

[0035] In the present invention, germinated highland barley powder is mixed with water, and bacterial powder is added for fermentation to obtain germinated highland barley fermentation liquid. The mass volume ratio of the germinated highland barley powder and water is preferably 1g:4~8mL, and more preferably 1g:5~7mL; after the germinated highland barley powder and water are mixed, a sterilization step is also included before fermentation, and the sterilization temperature is preferably 60~80℃, and more preferably 65~75℃; the sterilization time is preferably 20~45min, and more preferably 25~40min; the sterilization is cooled, and bacterial powder is added for fermentation after cooling. The bacterial powder is the composite bacteria VEGE-022, and the amount of bacterial powder added is preferably 0.02%~0.04% (w / v) of the total volume of the mixed solution of germinated highland barley powder and water, and more preferably 0. .025%~0.035% (w / v); the fermentation temperature is preferably 30~45℃, more preferably 35~40℃; the fermentation time is preferably 4~6h, more preferably 4.5~5.5h; after the fermentation is completed, it is placed in a refrigerator for refrigeration, and the refrigeration temperature is preferably 2~6℃, more preferably 4℃; the shelf life is centrifuged on the 1st, 7th, 14th, 21st, and 28th days, and the centrifugal speed is preferably 3000~6000r / min, more preferably 4000~5000r / min; the centrifugal time is preferably 5~15min, more preferably 8~12min. After the centrifugation, the germinated highland barley fermentation liquid is obtained.

[0036] The invention also provides highland barley fermentation liquid prepared by the preparation method.

[0037] The present invention also provides the germinated highland barley fermentation liquid prepared by the preparation method or the application of the highland barley fermentation liquid in the preparation of products helpful for anti-oxidation.

[0038] The present invention also provides the germinated highland barley fermentation liquid prepared by the preparation method or the application of the highland barley fermentation liquid in preparing a product that helps maintain a healthy blood lipid level.

[0039] The present invention also provides the germinated highland barley fermentation liquid prepared by the preparation method or the application of the highland barley fermentation liquid in preparing products that are helpful in maintaining healthy blood sugar levels.

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] The bacterial powder of the present invention is commercial composite bacteria VEGE-022, purchased from Danisco (China) Co., Ltd.

[0042] Example 1

[0043] Highland barley seeds are cleaned, and then washed with deionized water after immersion disinfection with 1% sodium hypochlorite solution for 10min, and the highland barley seeds after disinfection and deionized water (pH is 7.0) are mixed and soaked according to mass volume ratio 1g:5mL, and the soaking temperature is 25°C, and the time is 6h, and the highland barley seeds after soaking are germinated on three layers of gauze, and the mass volume ratio of highland barley seeds and deionized water (pH is 6.5) is 1g:5mL replenishing water, and the temperature of germination is 28°C, and the time is 24h. The highland barley after germination is freeze-dried (-40°C, 24h), pulverized, and 80 mesh sieves are used to obtain germination highland barley powder. The germinated highland barley powder and deionized water are mixed in a mass volume ratio of 1g:7mL, and the mixture is pasteurized at 65°C for 30min. The mixture is cooled after sterilization, and 0.03% (w / v) of bacterial powder based on the total volume of the mixed solution of the germinated highland barley powder and deionized water is added. The mixture is fermented at 40°C for 5h, and the germinated highland barley fermentation liquid is obtained after centrifugation (4500r / min, 10min).

[0044] Example 2

[0045] Highland barley seeds are cleaned, and washed with deionized water after immersion disinfection with 0.5% sodium hypochlorite solution for 10min, and the highland barley seeds after disinfection and deionized water (pH is 6.5) are mixed and soaked according to mass volume ratio 1g:2mL, and the soaking temperature is 20 ℃, and the time is 5h, and the highland barley seeds after soaking are germinated on three layers of gauze, and the mass volume ratio according to highland barley seeds and deionized water (pH is 6.0) is 1g:2mL replenishing water, and the temperature of germination is 25 ℃, and the time is 20h. The highland barley after germination is freeze-dried (-20 ℃, 20h), pulverized, and obtained germination highland barley powder through 80 mesh sieves. The germinated highland barley powder and deionized water were mixed in a mass volume ratio of 1g:4mL, and the mixture was pasteurized at 60°C for 15min. The mixture was cooled after sterilization, and 0.02% (w / v) of bacterial powder based on the total volume of the mixed solution of germinated highland barley powder and deionized water was added. The mixture was fermented at 30°C for 4h, and the germinated highland barley fermentation liquid was obtained after centrifugation (3000r / min, 5min).

[0046] Example 3

[0047] Highland barley seeds are cleaned, and then washed with deionized water after immersion disinfection with 2% sodium hypochlorite solution for 20min, and the highland barley seeds after disinfection and deionized water (pH is 7.0) are mixed and soaked according to mass volume ratio 1g:7mL, and the soaking temperature is 30°C, and the time is 7h, and the highland barley seeds after soaking are germinated on three layers of gauze, and the mass volume ratio of highland barley seeds and deionized water (pH is 7.0) is 1g:7mL replenishing water, and the temperature of germination is 30°C, and the time is 30h. The highland barley after germination is freeze-dried (-60°C, 28h), pulverized, and 80 mesh sieves are used to obtain germination highland barley powder. The germinated highland barley powder and deionized water are mixed in a mass volume ratio of 1g:8mL, and the mixture is pasteurized at 80°C for 40min. The mixture is cooled after sterilization, and 0.04% (w / v) of bacterial powder based on the total volume of the mixed solution of the germinated highland barley powder and deionized water is added. The mixture is fermented at 45°C for 6h, and the germinated highland barley fermentation liquid is obtained after centrifugation (6000r / min, 15min).

[0048] Experimental Example 1

[0049] Processing method comparison experiment

[0050] The changes in the content of bioactive ingredients in unprocessed whole grain highland barley were compared between germination and fermentation and fermentation.

[0051] Highland barley extract after germination and fermentation treatment: highland barley seeds are cleaned, washed with deionized water after immersion disinfection with 1% sodium hypochlorite solution for 10min, highland barley seeds after disinfection and deionized water (pH is 7.0) are mixed and soaked according to mass volume ratio 1g:5mL, the temperature of soaking is 25 ℃, and the time is 6h, highland barley seeds after soaking are germinated as for three layers of gauze, according to the mass volume ratio of highland barley seeds and deionized water (pH is 6.5), 1g:5mL replenishes water, and the temperature of sprouting is 28 ℃, and the time is 24h. The highland barley after sprouting is freeze-dried (-40 ℃, 24h), pulverized, and obtained sprouting highland barley powder through 80 mesh sieves. The germinated highland barley powder and deionized water are mixed in a mass volume ratio of 1g:7mL, pasteurized at a temperature of 65°C for 30min, cooled after sterilization, and 0.03% (w / v) of bacterial powder of the total volume of the mixed solution of germinated highland barley powder and deionized water is added, and fermented at a temperature of 40°C for 5h. After the fermentation is completed, an appropriate amount of deionized water is added to adjust the mass volume ratio of the germinated highland barley powder and deionized water to 1g:20mL, and extracted twice in a 50°C water bath, each time for 1h, centrifuged at 4000r / min for 15min, and the supernatant is combined to obtain a highland barley extract after germination and fermentation treatment.

[0052] Highland barley extract after fermentation treatment: freeze-dry highland barley seeds (-40℃, 24h), crush, and pass through an 80-mesh sieve to obtain highland barley powder. Mix highland barley powder and deionized water in a mass volume ratio of 1g:7mL, sterilize at 65℃ for 30min by pasteurization, cool after sterilization, add 0.03% (w / v) bacterial powder of the total volume of the mixture, and ferment at 40℃ for 5h. After fermentation, add an appropriate amount of deionized water, adjust the mass volume ratio of germinated highland barley powder and deionized water to 1g:20mL, extract twice in a 50℃ water bath, each time for 1h, centrifuge at 4000r / min for 15min, and combine the supernatant; obtain the highland barley extract after fermentation treatment.

[0053] Whole grain extract: Freeze-dry highland barley seeds (-40°C, 24h), crush, and pass through an 80-mesh sieve to obtain highland barley powder. Mix highland barley powder and deionized water at a mass volume ratio of 1g:20mL, extract twice in a 50°C water bath, each time for 1h, centrifuge at 4000r / min for 15min, and combine the supernatants to obtain highland barley whole grain extract.

[0054] 1. Determination of β-glucan content

[0055] Pipette 2 mL of sample into a colorimetric tube, add 4 mL of 0.1 g / L Congo red solution, and let stand for 30 min. Measure the absorbance at 550 nm, draw a standard curve with 0.01-0.1 mol / mL β-glucan as the standard, and calculate the β-glucan content based on the β-glucan standard curve.

[0056] 2. Determination of total phenol content

[0057] The total phenol content in the extract sample was determined by the Folin phenol method. Take 200 μL of the extract sample and add it to a 5mL centrifuge tube, add 0.5mol / L 100μL Folin phenol reagent, mix well and react for 5 minutes, then add 800μL Na2CO3 solution (7.5%, w / v) and react for 30 minutes at room temperature in the dark. Move the sample from the 5mL centrifuge tube into a 96-well plate and measure the absorbance at 765nm. Draw a standard curve with 0.1-1μmol / mL gallic acid as the standard, and calculate the total phenol content based on the gallic acid standard curve.

[0058] 3. Determination of total flavonoid content

[0059] The total flavonoid content in the extract sample was determined by aluminum nitrate-sodium nitrite colorimetry. 20 μL NaNO2 (3%, w / v) was mixed evenly with 40 μL of appropriately diluted sample and fully reacted for 6 min, then 20 μL Al(NO3)3 (6%, w / v) was added to continue the reaction for 6 min, followed by 140 μL NaOH (4%, w / v) and 60 μL 70% methanol. After reacting at room temperature for 15 min, the absorbance was measured at 510 nm. A standard curve was drawn with 0.05-0.5 mmol / mL rutin as the standard, and the total flavonoid content was calculated based on the rutin standard curve.

[0060] 4. Determination of γ-aminobutyric acid (GABA) content

[0061] Weigh 10 mg of GABA standard and add 10 mL of distilled water to prepare a 1.0 mg / mL GABA standard solution. Use a pipette to draw 0.00, 0.05, 0.10, 0.15, 0.20 and 0.25 mL of the standard solution into a 10 mL test tube with a lid, and add 0.2 mL of 0.2 mol / L (pH = 9.0) sodium tetraborate buffer, 1.0 mL of 6% redistilled phenol, and 0.4 mL of sodium hypochlorite solution with 7.5% effective chlorine in sequence. After mixing evenly, boil in water for 10 minutes, immediately ice bath for 20 minutes and shake continuously. When the solution turns from blue-green, add 2.0 mL of 60% ethanol solution, shake evenly, let stand for 40 minutes, and measure the absorbance at a wavelength of 645 nm. Measure the GABA concentration according to the standard curve.

[0062] Experimental results: Figure 1 As shown, compared with whole grains, the β-glucan content of the fermentation group was significantly higher than that of the whole grain group (P<0.05); the total phenol content of the germination and fermentation group and the fermentation group was significantly higher than that of the whole grain group (P<0.05); the GABA content of the germination and fermentation group and the fermentation group was significantly higher than that of the whole grain group (P<0.05).

[0063] Experimental Example 2

[0064] Germinated highland barley fermentation broth: the germinated highland barley fermentation broth prepared according to the method of Example 1.

[0065] Highland barley fermentation broth: Based on the method of Example 1, the germination step is removed and the other operating conditions remain unchanged to prepare the highland barley fermentation broth.

[0066] The fermentation broths treated in two different ways were refrigerated in a refrigerator at 4°C, centrifuged (4500r / min, 10min) on the 1st, 7th, 14th, 21st and 28th days of the shelf life, and the supernatants were taken to determine the content of bioactive components.

[0067] 1. Measurement of titration acidity and pH

[0068] The titration acidity was carried out in accordance with the national standard "GB 5009.239-2016 National Food Safety Standard Determination of Titrative Acidity of Foods"; the pH value of the highland barley fermentation liquid was determined using a pH meter.

[0069] 2. Protein content determination

[0070] The protein content was determined using the Coomassie Brilliant Blue method.

[0071] 3. Determination of total sugar content

[0072] The total sugar content was determined using the phenol-sulfuric acid method.

[0073] 4. Determination of soluble solids content

[0074] The soluble solids content of the fermentation broth was measured using a sugar meter with an accuracy of 0.1.

[0075] 5. Determination of total probiotic colony count

[0076] The total probiotic colony count of each group of fermentation broth was counted using the “Determination of the total lactic acid bacteria colony count in GB 4789.35-2016”.

[0077] 6. The determination of β-glucan, total phenols, total flavonoids and γ-aminobutyric acid (GABA) contents is as described in Experimental Example 1.

[0078] Experimental results: Figure 2 As shown. Figure 2 As shown in A and B in Figure 3, pH decreased over time, while acidity increased over time over 28 days.

[0079] like Figure 2 As shown in Figure C, within 28 days, the protein content increased first and then stabilized. At the same time point, the protein content of the germination and fermentation group was higher than that of the fermentation group (P<0.05), and the protein content was highest on the 14th, 21st and 28th days of germination and fermentation, with no significant difference (P>0.05).

[0080] like Figure 2 As shown in D, within 28 days, the total sugar content in the germination and fermentation group first tended to be stable, then decreased, and then stabilized; the total sugar content in the fermentation group first increased, then decreased, and then stabilized. The germination and fermentation group had the highest content on the 1st, 7th, and 14th days. The fermentation group had the highest content on the 7th day.

[0081] like Figure 2 As shown in Figure E, the soluble solids first increased and then remained stable within 28 days.

[0082] like Figure 2 As shown in F, the total number of probiotic colonies first increased, then remained stable, and finally decreased within 28 days.

[0083] like Figure 2 As shown in G, within 28 days, the β-glucan content in the germination and fermentation group increased first and then stabilized; the β-glucan content in the fermentation group showed a downward trend. The β-glucan content in the fermentation group was higher than that in the germination and fermentation group in the same period (P<0.05). The content of the fermentation group was the highest on the first day (P<0.05).

[0084] like Figure 2 As shown in H, within 28 days, the total phenol content of the germination and fermentation group and the fermentation group increased first and then stabilized. At the same time point, the total phenol content of the germination and fermentation group was higher than that of the fermentation group (P<0.05). The content of the germination and fermentation group was the highest on the 14th, 21st and 28th days (P<0.05), and there was no significant difference (P>0.05).

[0085] like Figure 2 As shown in I, within 28 days, the flavonoid content of the germination and fermentation group and the fermentation group increased first and then stabilized. The germination and fermentation group had the highest content on the 14th, 21st and 28th days (P<0.05); the fermentation group had the highest content on the 14th, 21st and 28th days, with no significant difference (P>0.05).

[0086] like Figure 2 As shown in Figure J, within 28 days, the GABA content in the germination and fermentation group first tended to be stable, then increased, and finally decreased; the GABA content in the fermentation group first tended to be stable, then increased, and finally decreased. Among them, the content of the germination and fermentation group was the highest on the 21st day (P<0.05).

[0087] Experimental Example 3

[0088] In vitro functional assay of fermentation broth

[0089] The highland barley fermented liquor prepared by two different processing methods of germination fermentation and fermentation in Experimental Example 1 was refrigerated in a refrigerator at 4°C for 1, 7, 14, 21, and 28 days, and then freeze-dried in a freeze dryer. The obtained freeze-dried powder was prepared into 75 mg / mL and 100 mg / mL sample solutions, and the 75 mg / mL sample solution was used to determine the in vitro antioxidant capacity and in vitro hypoglycemic capacity. The 100 mg / mL sample solution was used to determine the in vitro lipid-lowering capacity.

[0090] 1. In vitro antioxidant capacity

[0091] (1) Determination of DPPH free radical scavenging ability

[0092] Accurately weigh 2.5 mg of DPPH powder using an electronic balance, dilute to 100 mL with anhydrous ethanol, and store in the dark for later use. Take 1.0 mL of sample solution from different groups into test tubes, add 1.0 mL of DPPH solution, shake well, and store in the dark at room temperature for 30 minutes. Use anhydrous ethanol as a blank control. Use vitamin C as a positive control to repeat the above operation. Use a pipette to draw 200 μL of the completely reacted solution into a 96-well plate, measure the absorbance at 517 nm, and calculate the clearance rate. The clearance rate calculation formula (1) is as follows:

[0093]

[0094] Where: D0 is the absorbance of the blank tube solution without adding sample solution;

[0095] D1 is the absorbance of the reaction solution;

[0096] D2 is the absorbance of the control tube without DPPH solution.

[0097] Experimental results: Figure 3 As shown in A, within 28 days, the DPPH radical scavenging rates of the germination and fermentation groups increased first and then stabilized. The DPPH radical scavenging rate of the germination and fermentation group was the highest on the 14th day (P<0.05), and except for the first day of the fermentation group, the DPPH radical scavenging capacity of the two groups was higher than that of the positive control group at the same concentration (P<0.05). This shows that the two groups of highland barley fermentation broth have good DPPH radical scavenging capacity.

[0098] (2) FRAP iron ion reducing ability assay

[0099] Prepare FRAP working solution: 300mmol / L sodium acetate buffer with pH=3.6, 10mmol / L TPTZ solution and 20mmol / L FeCl3 solution, mix them thoroughly in a volume ratio of 10:1:1, and preheat them in a 37℃ water bath for later use.

[0100] Take 0.3mL of sample, add 2.7mL of FRAP working solution preheated to 37℃, shake well and let stand for 10min. Use a pipette to draw 200μL of the completely reacted solution into a 96-well plate and measure its absorbance at 593nm. Use anhydrous ethanol to replace the sample and add the FRAP working solution as a blank. Use vitamin C as a positive control and repeat the above operation. Draw a standard curve with 10-100mmol / L FeSO4. According to the absorbance after the reaction, the corresponding FeSO4 concentration is obtained on the standard curve, which is defined as the FRAP value.

[0101] Experimental results: Figure 3As shown in Figure B, within 28 days, the FRAP values ​​of the germination and fermentation groups showed a trend of first increasing and then decreasing. Among them, the FRAP values ​​of the highland barley fermentation liquid in the germination and fermentation group were the highest on the 7th, 14th, and 21st days, indicating that the antioxidant capacity was the strongest, and was significantly higher than the positive control group at the same concentration (P<0.05). This shows that the germination and fermentation liquid of highland barley has a good antioxidant capacity.

[0102] (3) Determination of nitrite scavenging effect

[0103] Take 540 μL of sample, use a pipette to draw 400 μL of 5 μg / mL sodium nitrite standard solution, mix well, add 400 μL of 0.4% (w / v) p-aminobenzenesulfonic acid solution and 200 μL of 0.2% (w / v) ethylenediamine hydrochloride solution in turn, let stand for 15 minutes and then adjust the volume to 10 mL. Use vitamin C as the positive control and repeat the above operation. Use a pipette to draw 200 μL of the completely reacted solution to a 96-well plate and measure the absorbance value A1 at 538 nm. At the same time, the absorbance value measured by replacing the sodium nitrite standard solution with distilled water is recorded as A2, and the sample is replaced with distilled water as a blank, and its absorbance value is recorded as A0. Calculate the clearance rate of the sample for nitrite, formula (2) is as follows:

[0104]

[0105] Where: A1 is the absorbance at 538nm;

[0106] A2 is the absorbance measured by replacing sodium nitrite standard solution with distilled water;

[0107] A0 is the absorbance of distilled water instead of sample as blank.

[0108] Experimental results: Figure 3 As shown in Figure C, within 28 days, the nitrite clearance rates of the germination and fermentation groups increased first and then stabilized. The nitrite clearance rates of the highland barley fermentation broth in the germination and fermentation group were the highest on days 14, 21, and 28, and were significantly higher than those of the positive control group at the same concentration (P<0.05). This indicates that the germination and fermentation broth has a good nitrite clearance ability.

[0109] 2. In vitro hypoglycemic ability

[0110] (1) Determination of α-glucosidase inhibition rate

[0111] The α-glucosidase inhibition ability of the sample was determined by the 4-nitrophenyl-β-D-galactopyranoside (p-NPG) method. 110 μL PBS phosphate buffer (0.1 mol / L, pH = 6.8) was pipetted into a 96-well plate, and 20 μL sample standard solution and 20 μL α-glucosidase solution (1 U / mL) were added respectively. The plate was gently shaken to mix the solution evenly, and then placed in a 37°C incubator for incubation for 10 min. 20 μL 1.25 mmol / L p-NPG was added, and after another incubation for 20 min, 80 μL sodium carbonate solution (0.1 mol / L) was added to terminate the reaction. Acarbose was used as a positive control, and the above operation was repeated. The absorbance value was measured at 405 nm using an enzyme reader. The α-glucosidase inhibition rate of the sample was calculated according to the formula (3) as follows:

[0112]

[0113] Experimental results: Figure 4 As shown in A, within 28 days, the α-glucosidase inhibition rates of the germination and fermentation groups remained almost unchanged, and there was no difference in the inhibition rates between the two groups (P>0.05), but were significantly higher than the positive control group at the same concentration (P<0.05). This indicates that the two groups of highland barley fermentation broth have a good ability to inhibit α-glucosidase. By inhibiting the activity of α-glucosidase, highland barley fermentation broth can block the hydrolysis of 1,4-glucosidase bonds and delay the further conversion of carbohydrates into glucose, thereby effectively reducing postprandial blood sugar.

[0114] (2) Determination of α-amylase inhibition rate

[0115] The α-amylase inhibition ability of the sample was determined by 3,5-dinitrosalicylic acid (DNS) colorimetric method. 300 μL of sample standard solution and 300 μL of amylase solution (0.1 U / mL) were added to a 10 mL centrifuge tube, respectively. After being fully reacted in a 37 ° C water bath for 15 min, 200 μL of soluble starch with a volume fraction of 1% was added. After reacting for another 30 min, 500 μL of DNS reagent was added and reacted in a boiling water bath for 10 min. Cool in an ice bath, add 8600 μL of distilled water and vortex to terminate the reaction. Acarbose was used as a positive control, and the above operation was repeated. 200 μL of the reaction solution was transferred to a 96-well plate, and its absorbance was detected at 540 nm. The α-amylase inhibition rate of the sample was calculated according to the formula (4):

[0116]

[0117] Experimental results: Figure 4 As shown in Figure B, within 28 days, the α-amylase in the germination and fermentation groups showed a trend of increasing first and then decreasing. Among them, the α-amylase inhibition rate in the germination and fermentation group was the highest on the 14th day (P<0.05), and was significantly higher than that in the positive control group with the same concentration (P<0.05). This shows that the fermented broth of germinated highland barley has a good ability to inhibit α-amylase. By inhibiting the activity of α-amylase, the fermented broth of germinated highland barley can effectively block or delay the decomposition of carbohydrates such as starch, slow down the absorption and utilization of glucose in the human small intestine, and avoid a rapid increase in blood sugar after a meal.

[0118] 3. In vitro lipid-lowering ability

[0119] (1) Determination of cholesterol esterase inhibition rate

[0120] 4-nitrophenyl butyrate (PNPB) was pre-dissolved in acetonitrile to prepare a 4 mmol / L solution and stored at -20°C for later use. All reactions were carried out in a phosphate buffer solution (0.1 mol / L, pH = 7.0) containing sodium taurocholate (5.16 mmol / L), NaCl (0.1 mol / L), and PNPB (0.2 mmol / L). 1 mL of buffer, 50 μL of cholesterol esterase (500 U, 20 μg / mL), 50 μL of sample solution, and 20 μL of PNPB were added to the test tube, reacted at 25°C for 30 min, and the absorbance was measured at 405 nm. Ultrapure water was used instead of the sample solution in the control group, and the inhibition rate of cholesterol esterase was calculated as shown in formula (5):

[0121]

[0122] Experimental results: Figure 7 As shown in A, within 28 days, the cholesterol esterase inhibition rates of the germination and fermentation groups increased first and then decreased. Among them, the cholesterol esterase inhibition rate of the germination and fermentation group was the highest on the 14th day (P<0.05), and was significantly higher than the positive control group with the same concentration (P<0.05). This shows that the fermented broth of germinated highland barley has a good ability to inhibit cholesterol esterase. By inhibiting the activity of cholesterol esterase, the body's absorption of cholesterol can be reduced, thereby achieving the purpose of lipid reduction.

[0123] (2) Determination of pancreatic lipase inhibition rate

[0124] 0.4% volume fraction of 4-nitrophenyl laurate was dissolved in 5mmol / L sodium acetate solution with pH=5.0 for later use. Pancreatic lipase was dissolved in 100mmol / L Tris-HCl buffer with pH=8.2 to prepare a solution with a mass concentration of 5mg / mL, and stored at 4°C for later use.

[0125] 200 μL of pancreatic lipase, 50 μL of sample solution and 50 μL of lauric acid-4-nitrophenyl ester were added to a 96-well plate, reacted at 37°C for 45 min, and the absorbance was measured at 405 nm. The control group used PBS buffer instead of pancreatic lipase, and the inhibition rate of the sample on pancreatic lipase was calculated according to formula (6).

[0126]

[0127] Experimental results: Figure 7 As shown in Figure B, within 28 days, the pancreatic lipase inhibition rates of the germination and fermentation groups and the fermentation groups showed a trend of first increasing and then decreasing. Among them, the pancreatic lipase inhibition rate of the germination and fermentation group was the highest on the 14th day (P<0.05), and was significantly higher than the positive control group with the same concentration (P<0.05). This shows that the fermented liquid of germinated highland barley has a good ability to inhibit pancreatic lipase. By inhibiting the activity of pancreatic lipase, it can prevent dietary fat from being converted into glycerol and fatty acids, so that they are excreted from the body with the residue, thereby effectively inhibiting lipid intake and reducing the body's stored fat.

[0128] (3) Bile salt adsorption kinetics

[0129] Take 0.5mL of sample solution, add 0.5mL of 0.02mol / L hydrochloric acid solution, and digest at 37℃ with constant temperature and shaking for 1h; adjust the pH value to 6.3 with 0.1mol / L sodium hydroxide solution, then add 5mL of 10mg / mL trypsin (prepared with 0.1mol / L phosphate buffer with a pH value of 6.3), and digest at 37℃ with constant temperature and shaking for 1h; add 4mL, 0.3mmol / L sodium taurocholate, sodium glycocholate and sodium cholate respectively, and oscillate at 37℃ for 20, 40, 60, 80 and 100min respectively; centrifuge at 6000r / min for 10min, measure the absorbance of the supernatant at 387nm, and obtain the bile salt content in the supernatant.

[0130] Experimental results: Figure 5 As shown in the data, with the increase of time, the adsorption of sodium taurocholate, sodium glycocholate and sodium cholate in the germination and fermentation group first increased and then tended to be stable. After comparison, it can be seen that the two groups of highland barley fermentation broth have the strongest adsorption capacity for sodium taurocholate, followed by sodium glycocholate, and the weakest adsorption capacity for sodium cholate. After 40 minutes, the difference in adsorption amount was not significant (P>0.05), and the final adsorption time of the experiment was determined to be 60 minutes.

[0131] (4) Bile salt adsorption isotherm curve

[0132] Take 0.5mL of sample solution, add 0.5mL of 0.02mol / L hydrochloric acid solution, and digest at 37℃ with constant temperature and shaking for 1h; adjust the pH value of the digestion solution to 6.3 with 0.1mol / L sodium hydroxide, then add 5mL of 10mg / mL trypsin, and digest at 37℃ with constant temperature and shaking for 1h; add 0.8, 1.6, 2.4, 3.2, 4.0, and 4.8mL of 0.3mmol / L sodium taurocholate solution to each sample, and then add 0.3mmol / L sodium glycocholate solution and sodium cholate solution to the sample in the same way; shake at 37℃ for 1h; centrifuge at 6000r / min for 10min, measure the absorbance of the supernatant at 387nm, and obtain the bile salt content in the supernatant to determine the bile salt adsorption amount.

[0133] Experimental results: Figure 6 As shown in the figure, with the increase of the concentration of the three bile salts, the adsorption of sodium taurocholate, sodium glycocholate and sodium cholate by the germination and fermentation group and the fermentation group continued to increase. After comparison, it can be seen that the adsorption of sodium taurocholate by the two groups of highland barley fermentation broth is slightly higher than that of sodium glycocholate, but much higher than that of sodium cholate. When the concentration exceeds 0.12mmol / L, the difference in adsorption change is not significant (P>0.05). Finally, the adsorption concentration of the experiment was determined to be 0.12mmol / L.

[0134] (5) Bile salt binding rate

[0135] Take 0.5mL of sample solution, add 0.5mL of 0.02mol / L hydrochloric acid solution, and digest at 37℃ with constant temperature and shaking for 1h; adjust the pH value to 6.3 with 0.1mol / L sodium hydroxide solution, then add 5mL of 10mg / mL trypsin, and digest at 37℃ with constant temperature and shaking for 1h; add 4.0mL of 0.3mmol / L sodium taurocholate, sodium glycocholate and sodium cholate solution to each sample, and shake at 37℃ for 1h; centrifuge at 6000r / min for 10min, measure the absorbance of the supernatant at 387nm, and obtain the bile salt content in the supernatant to determine the bile salt binding rate.

[0136] Experimental results: Figure 7As shown in the CE, during the shelf life, the binding of the germination and fermentation group to sodium taurocholate, sodium glycocholate and sodium cholate first increased, then decreased, and then remained stable; the binding of the fermentation group to sodium taurocholate and sodium glycocholate first increased, then decreased, and then remained stable, and the binding rate to sodium cholate did not change significantly (P>0.05). Among them, the binding rate of the germination and fermentation group to sodium taurocholate and sodium cholate on the 14th day of the shelf life reached the highest (P<0.05), which was significantly higher than the positive control group with the same concentration (P<0.05); the binding rate to sodium glycocholate also reached the highest (P<0.05), but there was no significant difference with the positive control group (P>0.05). This shows that the germination of highland barley fermentation liquid has a good ability to bind bile salts. After the germination of highland barley fermentation liquid binds to part of the bile salts, the bile salt content decreases, which can promote the conversion of cholesterol in the liver into bile salts to maintain the dynamic balance of bile acid in the body. Therefore, the cholesterol content in the liver decreases accordingly, which plays a role in lowering blood lipids.

[0137] It can be seen from the above embodiments and experimental examples that the germination fermentation broth prepared by the present invention can enrich the bioactive components such as total phenols, total flavonoids, γ-aminobutyric acid in highland barley. At the same time, during the 28-day shelf life, the growth and metabolism of probiotics promote the release of bioactive components of highland barley, thereby enhancing the antioxidant, blood sugar and lipid-lowering capabilities of the highland barley fermentation broth, and improving the added value of the highland barley product.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing germinated highland barley fermentation liquid, characterized in that: The following steps are involved: 1) sterilizing highland barley seeds, soaking them, and germinating the soaked highland barley seeds; 2) freeze-drying the germinated highland barley seeds, crushing them, and sieving them to obtain germinated highland barley powder; 3) mixing sprouted highland barley powder and water, adding bacterial powder for fermentation, and obtaining sprouted highland barley fermentation liquid.

2. The preparation method according to claim 1, characterized in that: Step 3) The mass volume ratio of the germinated highland barley powder and water is 1g:4-8mL.

3. The preparation method according to claim 1, characterized in that: Step 3) the bacterial powder is the composite bacteria VEGE-022, and the addition amount of the bacterial powder is 0.02% to 0.04% (w / v) of the total volume of the mixed solution of germinated barley powder and water; the fermentation temperature is 30 to 45° C., and the fermentation time is 4 to 6 hours.

4. The preparation method according to claim 1, characterized in that: Step 1) The pH value of the water used for soaking is 6.5-7.5, the mass volume ratio of the sterilized highland barley seeds and water is 1g:2-7mL, the soaking temperature is 20-30°C, and the soaking time is 5-7h; the disinfection is carried out by soaking in a sodium hypochlorite solution, the mass fraction of the sodium hypochlorite solution is 0.5-2%, and the disinfection time is 10-20min.

5. The preparation method according to claim 1, characterized in that: Step 1) placing the soaked highland barley seeds on gauze for germination, supplementing water during the germination process, the pH value of the water is 6.0-7.0, the mass volume ratio of the soaked highland barley seeds and water is 1g:2-7mL, the germination temperature is 25-30°C, and the germination time is 20-30h.

6. The preparation method according to claim 1, characterized in that: Step 3) After the germinated highland barley powder is mixed with water, a sterilization step is also included before fermentation, the sterilization temperature is 60-80° C., and the sterilization time is 20-45 min; The fermentation is followed by centrifugation, wherein the centrifugal speed is 3000-6000 r / min and the centrifugal time is 5-15 min.

7. The germinated highland barley fermentation liquid prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the germinated highland barley fermentation liquid prepared by the preparation method according to any one of claims 1 to 6 or the germinated highland barley fermentation liquid according to claim 7 in the preparation of products that contribute to anti-oxidation.

9. Use of the germinated highland barley fermentation liquid prepared by the preparation method according to any one of claims 1 to 6 or the germinated highland barley fermentation liquid according to claim 7 in the preparation of products that help maintain healthy blood lipid levels.

10. Use of the germinated highland barley fermentation liquid prepared by the preparation method according to any one of claims 1 to 6 or the germinated highland barley fermentation liquid according to claim 7 in the preparation of products that help maintain healthy blood sugar levels.

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