A preparation method of high-yield anti-hypertensive rice bran peptides based on mixed strain fermentation

Abstract

The application discloses a preparation method of high-yield anti-hypertensive rice bran peptides based on mixed strain fermentation. The method comprises the following steps: crushing and screening selenium-rich rice bran powder, and then performing defatting on the selenium-rich rice bran powder by using n-hexane to obtain defatted selenium-rich rice bran powder; mixing coryneform glutamic acid bacteria and bacillus licheniformis according to a volume ratio of 2:1 to prepare mixed seed liquid; taking 5% defatted selenium-rich rice bran powder and 1.8% glucose as a fermentation medium, adjusting the initial pH to 7.9, inoculating the mixed seed liquid according to a 1% inoculation amount, and then performing fermentation at 35 DEG C and 200 r / min for 3.5 days; performing centrifugation on the fermentation liquid, and then performing equal-volume 20% trichloroacetic acid precipitation treatment on the supernatant, and collecting the supernatant by centrifugation to obtain a rice bran peptide mixed solution. The specific mixed strain ratio and the optimized fermentation process are used in the application, so that the yield of rice bran peptides is significantly improved, and the predicted yield reaches 21.9 mg / mL. The obtained rice bran peptide mixed solution has excellent ACE inhibitory activity and DPPH free radical scavenging capacity, and can play a vascular protection role by adjusting the NO / ET-1 balance in HUVEC cells. Moreover, the method is simple in operation, low in cost and high in yield.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and bioactive peptide preparation technology, and particularly relates to a method for preparing a high-yield rice bran peptide mixed solution with antihypertensive activity by mixed fermentation of selenium-enriched rice bran using Corynebacterium glutamicum and Bacillus licheniformis. Specifically, it relates to a method for preparing high-yield antihypertensive rice bran peptides based on mixed strain fermentation. Background Technology

[0002] Rice bran is a major byproduct of rice processing. my country produces over 10 million tons of rice bran annually, but most of it is used as animal feed or low-value-added products, resulting in significant resource waste. Rice bran contains approximately 12%-20% protein, and after enzymatic hydrolysis or microbial fermentation, it can release various physiologically active peptides, such as antioxidant peptides, antihypertensive peptides (ACE inhibitory peptides), and hypoglycemic peptides, which have extremely high development value.

[0003] Currently, the main methods for preparing rice bran peptides are enzymatic hydrolysis and microbial fermentation. Enzymatic hydrolysis is costly and the product often has a bitter taste; microbial fermentation is attracting attention due to its low cost, mild conditions, and good product flavor. However, the efficiency of single-strain fermentation of rice bran for peptide production is generally low, with peptide yields typically below 15 mg / mL, and there is a lack of systematic optimization of fermentation conditions. Furthermore, selenium-enriched rice bran, in addition to common nutrients, is rich in organic selenium, offering higher health value, but efficient fermentation technology for peptide production from selenium-enriched rice bran has not yet been reported.

[0004] Antihypertensive peptides primarily exert their antihypertensive effect by inhibiting angiotensin-converting enzyme (ACE). Existing ACE-inhibiting peptides derived from rice bran are mostly prepared via enzymatic hydrolysis, resulting in low yields, unstable activity, and a lack of systematic research on their protective effects on vascular endothelial cells (such as regulating the NO / ET-1 balance). Therefore, developing a high-yield fermentation method for preparing rice bran peptides with clearly defined antihypertensive activity has significant industrial and clinical value. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing high-yield antihypertensive rice bran peptides based on mixed strain fermentation, so as to solve the problems of low yield, unclear antihypertensive activity, and unstable fermentation process of rice bran peptides in the prior art.

[0006] This invention achieves the above objective through the following technical solution: a method for preparing high-yield antihypertensive rice bran peptides based on mixed strain fermentation, comprising the following steps: (1) After the selenium-enriched rice bran is crushed and passed through a 100-mesh sieve, it is degreased to obtain degreased selenium-enriched rice bran powder; (2) Corynebacterium glutamicum and Bacillus licheniformis were cultured in LB medium for 12-24 h and the OD value was adjusted to 1.0 to obtain Corynebacterium glutamicum seed solution and Bacillus licheniformis seed solution for later use. (3) Prepare a fermentation medium by mixing defatted selenium-enriched rice bran powder obtained in step (1) with water at a ratio of 2%-10% of material to liquid, adjust the initial pH to 5-9, autoclave, add 0-2% (w / v) glucose, and set aside. (4) Inoculate the Corynebacterium glutamicum seed liquid and Bacillus licheniformis seed liquid from step (2) into the fermentation medium from step (3) at a total inoculation amount of 1% (v / v) and an inoculation ratio of 2:1. Ferment at 25-45℃ and 150-200 r / min for 1-5 days to obtain the fermentation broth. (5) Centrifuge the fermentation broth from step (4), collect the supernatant, add an equal volume of 20% (w / v) trichloroacetic acid to the supernatant, mix well, let stand for a period of time, centrifuge again, collect the supernatant, and the obtained supernatant is the rice bran peptide mixed solution with antihypertensive activity.

[0007] Further, in step (1), the defatting process is specifically as follows: after crushing the selenium-enriched rice bran through a 100-mesh sieve, add n-hexane at a material-to-liquid ratio of 1:3 (W / V), and extract with magnetic stirring at 1000 r / min for 1 h, then centrifuge at 8000 r / min for 20 min and discard the supernatant; add n-hexane to the precipitate at a material-to-liquid ratio of 1:1 (W / V), extract with magnetic stirring at 1000 r / min for 1 h, centrifuge at 8000 r / min for 20 min, discard the supernatant, and dry the precipitate at room temperature to obtain defatted selenium-enriched rice bran powder.

[0008] Further, in step (2), the preparation method of the Corynebacterium glutamicum seed liquid and Bacillus licheniformis seed liquid is as follows: Corynebacterium glutamicum and Bacillus licheniformis are respectively inoculated into LB liquid medium, activated and cultured at 30℃ and 200 r / min for 12h, and the concentration is adjusted to OD600=1.0.

[0009] Further, in step (3), the defatted selenium-enriched rice bran powder and water are mixed at a ratio of 5% to prepare a fermentation culture medium. The initial pH of the fermentation culture medium is adjusted to 7.9 with 0.1 mol / L NaOH or HCl. After high-pressure sterilization, 1.8% (w / v) of glucose is added.

[0010] Furthermore, in step (4), the fermentation culture is based on shaking fermentation at 35℃ / 200 r / min for 3.5 days.

[0011] Further, in step (5), the centrifugation conditions of the fermentation broth are 4°C, 10000 r / min for 10 min; after standing, the centrifugation conditions are 10000 r / min for 10 min.

[0012] Further, step (5) includes: separating the rice bran peptide mixture solution by ultrafiltration membrane (with molecular weight cutoffs of 3kD, 10kD, and 100kD), collecting the filtrate with a molecular weight less than 3kD, and obtaining a rice bran peptide mixture solution enriched with antihypertensive activity.

[0013] Furthermore, step (5) also includes: freeze-drying the rice bran peptide mixture and storing it at -80°C.

[0014] Furthermore, the present invention evaluates the antihypertensive activity of the obtained rice bran peptide mixed solution: ACE inhibitory activity: The colorimetric method showed that the ACE inhibition rate was significant, reaching 78.3% at a 10-fold dilution.

[0015] DPPH free radical scavenging activity: This indicates that it has antioxidant capacity; at a concentration diluted 20 times, the DPPH free radical scavenging activity can reach 70.7%.

[0016] HUVEC cell protective effects: The non-cytotoxicity of rice bran peptides was determined by the CCK-8 assay within the concentration range of 31.25-2000 μg / mL.

[0017] An Ang II-induced HUVEC cell damage model was established. The results showed that rice bran peptide mixed solutions (low, medium and high concentrations) could significantly reduce the content of ET-1 (endothelin-1) and increase the content of NO (nitric oxide) in the cell supernatant in a dose-dependent manner, and the effect was not significantly different from that of the positive control captopril.

[0018] The rice bran peptide mixed solution prepared by the above method has the following related activities: ACE inhibition rate ≥50%; DPPH free radical scavenging activity ≥50%; in the Ang II-induced HUVEC cell model, it can reduce ET-1 content and increase NO content.

[0019] Furthermore, the present invention also provides the application of rice bran peptide mixed solution in the preparation of functional foods or ACE inhibitors.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) High yield: Using a specific mixed strain (Corynebacterium glutamicum: Bacillus licheniformis = 2:1) and optimized fermentation process, the yield of rice bran peptide is as high as 21.9 mg / mL, which is more than 80% higher than that of single strain fermentation and more than 50% higher than that of unoptimized mixed fermentation.

[0021] (2) Clear antihypertensive activity: The obtained rice bran peptide mixed solution has significant ACE inhibitory activity and can exert vascular protection by regulating the NO / ET-1 balance of vascular endothelial cells, with a clear antihypertensive mechanism.

[0022] (3) Low raw material cost: Using selenium-enriched rice bran as raw material is a comprehensive utilization of agricultural by-products, which is in line with the concept of green manufacturing.

[0023] (4) Simple process and easy to scale up: mild fermentation conditions (35℃, natural pH fine adjustment), low equipment requirements, and easy to industrial production. Attached Figure Description

[0024] Figure 1 This is a flowchart of the preparation method of the present invention; Figure 2 This is a graph showing the effect of different initial pH values ​​on the yield of rice bran peptides in a single-factor experiment according to an embodiment of the present invention. Figure 3 This is a graph showing the effect of different sugar addition amounts on the yield of rice bran peptides in a single-factor experiment according to an embodiment of the present invention. Figure 4 This is a graph showing the effect of different fermentation times on the yield of rice bran peptides in a single-factor experiment according to an embodiment of the present invention. Figure 5 These are contour plots and 3D surface plots showing the effects of sugar addition, initial pH, and fermentation time on peptide yield in response surface optimization of this invention; wherein, (a) is a contour plot and 3D surface plot showing the effects of sugar addition and initial pH on peptide yield, (b) is a contour plot and 3D surface plot showing the effects of fermentation time and sugar addition on peptide yield, and (c) is a contour plot and 3D surface plot showing the effects of initial pH and fermentation time on peptide yield. Figure 6 This is a comparison chart of the ACE inhibitory activities of ultrafiltration components with different molecular weights (<3 kD, 3-10 kD, 10-100 kD, >100 kD) in embodiments of the present invention. Figure 7 This is a schematic diagram illustrating the effect of different concentrations of rice bran peptide on cell activity in an embodiment of the present invention; Figure 8 This is a schematic diagram illustrating the effect of rice bran peptide on Ang II-induced NO secretion in HUVEC cells according to an embodiment of the present invention. Figure 9 This is a schematic diagram illustrating the effect of rice bran peptide on Ang II-induced ET-1 secretion in HUVEC cells according to an embodiment of the present invention. Detailed Implementation

[0025] The present invention will be further illustrated below with specific embodiments. The present invention includes, but is not limited to, the following specific embodiments, which are for illustrative purposes only and are not intended to limit the present invention.

[0026] like Figure 1 As shown, this invention provides a method for preparing high-yield antihypertensive rice bran peptides based on mixed strain fermentation, comprising the following steps: (1) After the selenium-enriched rice bran is crushed and passed through a 100-mesh sieve, it is defatted with hexane to obtain defatted selenium-enriched rice bran powder.

[0027] (2) Culturing Corynebacterium glutamicum and Bacillus licheniformis in LB medium for 12-24 h and adjusting the OD value to 1.0 to obtain Corynebacterium glutamicum seed solution and Bacillus licheniformis seed solution for later use.

[0028] (3) Prepare a fermentation medium by mixing defatted selenium-enriched rice bran powder obtained in step (1) with water at a ratio of 2%-10% of material to liquid, adjust the initial pH to 5-9, autoclave, add 0-2% (w / v) glucose, and set aside.

[0029] (4) The *Corynebacterium glutamicum* seed culture and *Bacillus licheniformis* seed culture from step (2) were inoculated into the fermentation medium from step (3) at a total inoculation amount of 1% (v / v) and an inoculation ratio of 2:1. The mixture was fermented at 25-45℃ and 150-200 r / min for 1-5 days to obtain the fermentation broth. Under these conditions, the predicted yield of rice bran peptides reached 21.9 mg / mL.

[0030] (5) Centrifuge the fermentation broth from step (4), collect the supernatant, add an equal volume of 20% (w / v) trichloroacetic acid to the supernatant, mix well, let stand for 30 min, centrifuge again, collect the supernatant, and obtain a rice bran peptide mixed solution.

[0031] Example 1: Determining the optimal process parameters This invention determined the above-mentioned optimal process parameters through systematic single-factor experiments and response surface methodology: Single-factor experiments were conducted to investigate the effects of initial pH (5-9), material-to-liquid ratio (2%-10%), sugar addition (0-2%), fermentation temperature (25-45℃), and fermentation time (1-5 d) on peptide yield and DPPH scavenging rate, and the optimal range was preliminarily determined.

[0032] In this embodiment, Figure 2 This is a graph showing the effect of different initial pH values ​​on the yield of rice bran peptides in a single-factor experiment. Figure 2As shown, under the condition of initial pH of 5-9, it can be seen that the yield of rice bran peptides is optimal when the pH is around 8.

[0033] In this embodiment, Figure 3 This is a graph showing the effect of different sugar addition amounts on the yield of rice bran peptides in a single-factor experiment. Figure 3 As shown, under the condition of glucose addition of 0-2% (w / v), it can be seen that the yield of rice bran peptide is optimal when the glucose addition is about 1.75 (w / v).

[0034] In this embodiment, Figure 4 The graph shows the effect of different fermentation times on the yield of rice bran peptides in a single-factor experiment. Figure 4 As shown, under the condition of fermentation time of 1-5 days, it can be seen that the yield of rice bran peptides is optimal when the fermentation time is between 3-4 days.

[0035] To better verify the effects of sugar addition, initial pH, and fermentation time on rice bran peptide yield, such as Figure 5 As shown in Figures (a)-(c), response surface methodology optimization used sugar addition (A), initial pH (B), and fermentation time (C) as three factors, with peptide yield as the response value. A Box-Behnken design was used to establish a quadratic regression model. The optimal conditions were obtained by analysis using Design-Expert software: sugar addition 1.8%, initial pH 7.9, fermentation time 3.5 d, and predicted peptide yield 21.9 mg / mL.

[0036] Verification experiment: Three repeated fermentations were carried out under optimal conditions. The average measured peptide yield was 21.9 ± 1.47 mg / mL, which was consistent with the predicted value. The results are shown in Table 1.

[0037] Table 1: Statistical table of the results of three repeatable experiments verifying the fitting effect under optimal fermentation conditions. Example 2: Preparation of defatted selenium-enriched rice bran powder Take 5 kg of selenium-enriched rice bran raw material, pulverize it using a high-speed universal pulverizer, and pass it through a 100-mesh standard sieve to obtain 4.2 kg of fine rice bran powder. Take 1 kg of fine rice bran powder, add 3 L of n-hexane, and extract with magnetic stirring at 1000 r / min for 1 h, followed by centrifugation at 8000 r / min for 20 min, and discard the supernatant. Add another 1 L of n-hexane to the precipitate, and extract again under the same conditions for 1 h, centrifuge and discard the supernatant. Spread the defatted fine rice bran powder evenly on an enamel dish, air dry at room temperature in a fume hood for 24 h, weigh to obtain approximately 0.8 kg of defatted powder, pack it into a sealed bag, and refrigerate at 4℃ for later use.

[0038] Example 3: Activation of microbial strains and preparation of seed culture Cryopreserved *Corynebacterium glutamicum* and *Bacillus licheniformis* were streaked onto LB agar plates and incubated at 30°C for 24 h. Single colonies were picked and inoculated into 5 mL LB liquid culture tubes and incubated at 30°C and 200 rpm for 12 h. Then, 1% inoculum was transferred to 100 mL LB liquid medium (250 mL Erlenmeyer flask) and incubated at 30°C and 200 rpm for 12 h. The culture was then diluted to OD600 = 1.0 to obtain the seed culture.

[0039] Example 4: Optimal process for preparing rice bran peptide mixed solution Preparation of rice bran fermentation medium: Weigh 7.5 g of defatted selenium-enriched rice bran powder, add distilled water to 150 mL, and adjust the pH to 7.9 with 0.1 mol / L NaOH. Dispense into 250 mL Erlenmeyer flasks, 50 mL per flask, for a total of 3 flasks. Sterilize at 115℃ for 20 min. After cooling, add 3.6 mL of 50% glucose solution to each flask. Inoculate each flask with 0.33 mL of *Corynebacterium glutamicum* seed culture and 0.17 mL of *Bacillus licheniformis* seed culture (total inoculum 1%). Ferment on a shaker at 35℃ and 200 r / min for 3.5 days.

[0040] After fermentation, the supernatant was obtained by centrifugation at 10,000 r / min for 10 min at 4℃. 1 mL of the supernatant was added to 1 mL of 20% TCA, allowed to stand at room temperature for 30 min, and then centrifuged. The peptide content of the supernatant was determined by the BCA method. The average peptide yield was 21.99 mg / mL (22.18, 21.85, and 21.95 mg / mL in three batches, respectively).

[0041] Example 5: Determination of ACE inhibitory activity of rice bran peptide mixed solution The rice bran peptide mixture obtained in Example 4 (peptide concentration approximately 21.99 mg / mL) was diluted to 1.0 mg / mL with PBS buffer. ACE inhibitory activity was determined colorimetrically: 40 µL of sample, 50 µL of FAPGG buffer (1.0 mmol / L), and 10 µL of ACE solution (0.1 U / mL) were mixed, and the absorbance was immediately measured at 340 nm. After incubation at 37°C in the dark for 30 min, the absorbance was measured again. PBS buffer was used as a control instead of the sample. Figure 6 The figure shows a comparison of the ACE inhibitory activities of ultrafiltration fractions with different molecular weights (<3 kD, 3-10 kD, 10-100 kD, >100 kD). Among them, the <3 kD fraction had the highest ACE inhibition rate, which was 78.33% ± 2.97% (n=3), indicating that it has strong ACE inhibitory activity.

[0042] Example 6: Protective effect of rice bran peptide mixture on HUVEC cells The rice bran peptide mixture obtained in Example 4 was freeze-dried and then redissolved in endothelial cell culture medium to prepare different concentrations of 31.25, 62.5, 125, 250, 500, 1000, 2000, and 4000 µg / mL. Figure 7 As shown, CCK-8 assay results indicated that cell viability was >95% at concentrations below 2000, with no cytotoxicity.

[0043] Establishing an Ang II damage model: using 10 -4 HUVEC cells were treated with 1 mol / L Ang II for 24 h to induce endothelial damage. Control groups, Ang II model groups, low / medium / high dose rice bran peptide groups (250, 500, 1000 µg / mL), and captopril positive control group (100 µg / mL) were set up. Cell supernatants were collected, and NO and ET-1 levels were measured using a NO detection kit and an ELISA kit, respectively.

[0044] like Figure 8 As shown, the results indicate that compared with the Ang II model group, each dose group of rice bran peptide significantly increased NO content and significantly decreased ET-1 content in a dose-dependent manner, and the effect of the high-dose group was not significantly different from that of the captopril group.

[0045] Example 7: Ultrafiltration Fractionation and Activity Comparison The rice bran peptide mixed solution (Sp) obtained in Example 4 was sequentially passed through 100 kD, 10 kD, and 3 kD ultrafiltration membranes to obtain four molecular weight ranges: >100 kD, 10-100 kD, 3-10 kD, and <3 kD. The ACE inhibitory activity (1.0 mg / mL) of each range was measured.

[0046] like Figure 9 As shown, the results indicated that the <3 kD fraction exhibited the highest ACE inhibition rate, reaching 78.3% ± 2.9%; the 3-10 kD fraction was second, at 69.4% ± 4.0%; and the >10 kD fraction showed significantly reduced activity. This suggests that the antihypertensive activity is mainly concentrated in low molecular weight peptides (<10 kD, especially <3 kD).

[0047] Example 8: Control Experiment The following control group was set up (all other conditions were the same as in Example 4): Comparative Example 1: Inoculation with only Corynebacterium glutamicum (single bacterium, 1% inoculation amount) Comparative Example 2: Inoculation with Bacillus licheniformis only (single strain, 1% inoculation amount) Comparative Example 3: Mixed bacteria ratio 1:1 (total inoculum 1%) Comparative Example 4: Mixed bacterial ratio 1:2 (total inoculum 1%) Comparative Example 5: Unoptimized fermentation parameters (based on basic conditions: 5% solid-liquid ratio, 1% glucose, natural pH of approximately 7.2, no pH adjustment, inoculated with mixed bacteria at a ratio of 2:1, fermented for 3 days) Results: At a 10-fold dilution, Comparative Example 1 showed a peptide yield of 0.82 mg / mL and an ACE inhibition rate of 38.2%; Comparative Example 2 showed a yield of 0.78 mg / mL and an ACE inhibition rate of 39.1%; Comparative Example 3 showed a yield of 0.93 mg / mL and an ACE inhibition rate of 42.3%; Comparative Example 4 showed a yield of 0.91 mg / mL and an ACE inhibition rate of 43.1%; and Comparative Example 5 showed a yield of 0.97 mg / mL and an ACE inhibition rate of 45.6%. All results were significantly lower than those of Example 3 (yield 2.19 mg / mL, ACE inhibition rate 78.3%). This demonstrates that the specific strain ratio and optimized parameters of this invention have a significant synergistic effect.

[0048] The preparation method provided by this invention is simple to operate, highly reproducible, and yields high output. The resulting rice bran peptide mixed solution exhibits clear antihypertensive activity (ACE inhibition, NO / ET-1 regulation) and can be used as a raw material for functional foods or health products. Simultaneously, this method achieves high-value utilization of selenium-enriched rice bran, resulting in significant economic and social benefits.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing high-yield antihypertensive rice bran peptides based on mixed strain fermentation, characterized in that, Includes the following steps: (1) After the selenium-enriched rice bran is crushed and passed through a 100-mesh sieve, it is degreased to obtain degreased selenium-enriched rice bran powder; (2) Incubate Corynebacterium glutamicum and Bacillus licheniformis in LB medium for 12-24 h, adjust the OD value to 1.0, and prepare Corynebacterium glutamicum seed solution and Bacillus licheniformis seed solution for later use; (3) Prepare a fermentation medium by mixing defatted selenium-enriched rice bran powder obtained in step (1) with water at a ratio of 2%-10% of material to liquid, adjust the initial pH to 5-9, autoclave, add 0-2% (w / v) glucose, and set aside. (4) Inoculate the Corynebacterium glutamicum seed liquid and Bacillus licheniformis seed liquid from step (2) into the fermentation medium from step (3) at a total inoculation amount of 1% (v / v) and an inoculation ratio of 2:

1. Ferment at 25-45℃ and 150-200r / min for 1-5 days to obtain fermentation broth. (5) Centrifuge the fermentation broth from step (4), collect the supernatant, add an equal volume of 20% (w / v) trichloroacetic acid to the supernatant, mix well, let stand and then centrifuge again, collect the supernatant to obtain a rice bran peptide mixed solution.

2. The preparation method according to claim 1, characterized in that, In step (1), the defatting process is as follows: after crushing the selenium-enriched rice bran through a 100-mesh sieve, add n-hexane at a material-to-liquid ratio of 1:3 (W / V), and extract with magnetic stirring at 1000 r / min for 1 h. Then, centrifuge at 8000 r / min for 20 min and discard the supernatant. Add n-hexane to the precipitate at a material-to-liquid ratio of 1:1 (W / V), extract with magnetic stirring at 1000 r / min for 1 h, centrifuge at 8000 r / min for 20 min, discard the supernatant, and dry the precipitate at room temperature to obtain defatted selenium-enriched rice bran powder.

3. The preparation method according to claim 1, characterized in that, In step (2), the preparation methods of the Corynebacterium glutamicum seed liquid and Bacillus licheniformis seed liquid are as follows: Corynebacterium glutamicum and Bacillus licheniformis are inoculated into LB liquid medium, activated and cultured at 30℃ and 200 r / min for 12h, and the concentration is adjusted to OD600=1.

0.

4. The preparation method according to claim 1, characterized in that, In step (3), the defatted selenium-enriched rice bran powder and water are mixed at a ratio of 5% to prepare a fermentation culture medium. The fermentation culture medium is prepared by using 0.1 mol / L NaOH or HCl, adjusting the initial pH to 7.9, and adding 1.8% (w / v) glucose after high-pressure sterilization.

5. The preparation method according to claim 1, characterized in that, In step (4), the fermentation culture is based on 35℃ / 200r / min shaking fermentation for 3.5 days.

6. The preparation method according to claim 1, characterized in that, In step (5), the centrifugation conditions of the fermentation broth are 4℃, 10000 r / min for 10 min; after standing, the centrifugation conditions are 10000 r / min for 10 min.

7. The preparation method according to claim 1, characterized in that, Step (5) includes: separating the rice bran peptide mixture solution by ultrafiltration membrane fractionation, collecting the filtrate with a molecular weight of less than 3 kD, and obtaining a rice bran peptide mixture solution enriched with antihypertensive activity.

8. The preparation method according to claim 1, characterized in that, Step (5) further includes: freeze-drying the rice bran peptide mixture and storing it at -80°C.

9. The rice bran peptide mixed solution prepared by the preparation method according to any one of claims 1-8, characterized in that, The rice bran peptide mixture solution has the following related activities: ACE inhibition rate ≥50%; DPPH free radical scavenging activity ≥50%; and in the Ang II-induced HUVEC cell model, it can reduce ET-1 content and increase NO content.

10. The application of the rice bran peptide mixture according to claim 9 in the preparation of functional foods or ACE inhibitors.

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