A method for preparing a blood pressure lowering peptide complex by fermentation synergistic enzymolysis and a blood pressure lowering peptide complex

Abstract

The application discloses a method for preparing a blood pressure reducing peptide complex through fermentation and synergistic enzymolysis and the blood pressure reducing peptide complex, and belongs to the technical field of biological peptide preparation, in particular relates to a method for preparing a blood pressure reducing peptide complex through fermentation and synergistic enzymolysis, which comprises the following steps: filtering yak milk, preheating, and centrifuging to obtain skimmed milk; mixing Swiss lactobacillus CICC 6024 and lactococcus lactis subsp cremoris CICC 20406 to prepare a composite microbial inoculum; inoculating the composite microbial inoculum into the skimmed milk to ferment to obtain fermented milk; compounding alkaline protease and papain to obtain a compounded enzyme preparation; adding the compounded enzyme preparation into the fermented milk to perform enzymolysis to obtain an enzymolysis liquid; centrifuging to obtain supernatant, filtering through a microporous filter membrane to obtain a filtrate, ultrafiltering the filtrate through an ultrafilter membrane to obtain a permeate, re-ultrafiltering, taking a cut-off liquid, and freeze-drying to obtain the blood pressure reducing peptide complex. The application utilizes the fermentation and synergistic enzymolysis mode of Swiss lactobacillus and lactococcus lactis subsp cremoris to prepare the blood pressure reducing peptide complex with high ACE inhibitory activity.

Description

Technical Field

[0001] This invention belongs to the field of biopeptide preparation technology, and particularly relates to a method for preparing a blood pressure-lowering peptide complex by fermentation and synergistic enzymatic hydrolysis, and the blood pressure-lowering peptide complex. Background Technology

[0002] Hypertension is one of the most common chronic diseases worldwide and a major risk factor for cardiovascular and cerebrovascular diseases. Therefore, developing safe and effective antihypertensive functional ingredients has significant social and economic value. In recent years, food-derived antihypertensive peptides have received widespread attention due to their advantages such as being natural, safe, easily absorbed, and having few side effects. These bioactive peptides are usually derived from food proteins and exert their blood pressure regulating effect by specifically inhibiting the activity of angiotensin-converting enzyme (ACE).

[0003] Currently, the main methods for preparing antihypertensive peptides include chemical synthesis, direct enzymatic hydrolysis, and microbial fermentation. Chemical synthesis is cumbersome, costly, and may introduce harmful chemical residues, limiting its application. Direct enzymatic hydrolysis involves adding single or complex proteases, such as alkaline proteases, trypsin, or flavor proteases, to hydrolyze the substrate protein in vitro, offering advantages such as controllable conditions and high efficiency. However, this method has a relatively simple enzymatic process, and the types and biological activities of the resulting peptides are sometimes limited.

[0004] In contrast, microbial fermentation, which utilizes the abundant endogenous enzyme systems produced by microorganisms during their growth to hydrolyze substrates, is considered a more promising green preparation method. Fermentation is not only less costly, but microbial metabolism can also produce a variety of beneficial substances, potentially leading to new bioactivities or improving product flavor. However, traditional fermentation methods also have significant drawbacks: firstly, the controllability of the fermentation process is relatively poor, and the yield of bioactive peptides and batch-to-batch stability of the composition are difficult to guarantee; secondly, relying solely on endogenous microbial enzyme systems may not be efficient or targeted enough to fully release and enrich highly active antihypertensive peptides.

[0005] Therefore, there is an urgent need in this field to develop a new method for preparing antihypertensive peptides to overcome the shortcomings of existing technologies and thus efficiently prepare antihypertensive peptides with high ACE inhibitory activity. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a method for preparing a blood pressure-lowering peptide complex through fermentation and synergistic enzymatic hydrolysis, and the blood pressure-lowering peptide complex itself, utilizing *Lactobacillus helveticus* (…). Lactobacillus helveticus CICC 6024 and Lactococcus lactis subsp. milk fat ( Lactococcus lactis subsp. cremoris After fermentation with CICC 20406, a hypotensive peptide complex with high ACE inhibitory activity was prepared by synergistic enzymatic hydrolysis.

[0007] To achieve the above objectives, the present invention provides a method for preparing a blood pressure-lowering peptide complex by fermentation and synergistic enzymatic hydrolysis, comprising the following steps: 1) filtration of yak milk, preheating treatment, and centrifugation to obtain skim milk; 2) A compound bacterial agent was prepared by mixing Lactobacillus helveticus CICC 6024 bacterial suspension and Lactococcus lactis subsp. milk fat CICC 20406 bacterial suspension; 3) The compound microbial agent obtained in step 2) is inoculated into the skim milk obtained in step 1) and fermented to obtain fermented milk; 4) Alkaline protease and papain are combined to obtain a compound enzyme preparation; 5) Adjust the pH of the fermented milk obtained in step 3) to 7.0~8.0, add the compound enzyme preparation obtained in step 4), perform enzymatic hydrolysis, and inactivate the enzyme to obtain the enzymatic hydrolysate; 6) Centrifuge the enzymatic hydrolysate obtained in step 5) and collect the supernatant. Filter the supernatant through a microporous membrane to obtain the filtrate. Ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 3-5 kDa to obtain the permeate. Ultrafilter the permeate again through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Collect the retentate and freeze-dry it to obtain the antihypertensive peptide complex.

[0008] Preferably, the preheating temperature in step 1) is 35~40℃; the centrifugation temperature in step 1) is 4℃, the centrifugation speed is 4000~8000rpm, and the centrifugation time is 15~30min.

[0009] Preferably, the effective viable count of Lactobacillus helveticus CICC 6024 bacterial suspension in step 2) is 1×10⁻⁶. 6 cfu / mL; the effective viable count of the *Lactococcus lactis* subsp. *milk fat* CICC 20406 bacterial suspension mentioned in step 2) is 1×10⁻⁶. 6 cfu / mL; the volume ratio of the Lactobacillus helveticus CICC 6024 bacterial solution and Lactococcus lactis subsp. milk fat CICC 20406 bacterial solution in step 2) is 1:1~2.

[0010] Preferably, the inoculation amount of the compound microbial agent in step 3) is 2%~4%v / v.

[0011] Preferably, the fermentation temperature in step 3) is 30~40℃, the fermentation pH is 4.3~4.7, and the fermentation time is 12~24h.

[0012] Preferably, the mass ratio of alkaline protease to papain in step 4) is 1:1~2.

[0013] Preferably, the amount of compound enzyme preparation added in step 5) is calculated as 1000~4000U / g; the enzymatic hydrolysis temperature in step 5) is 50~55℃, and the enzymatic hydrolysis time is 2~4h.

[0014] Preferably, the enzyme inactivation treatment in step 5) is performed at a temperature of 85-90°C for 10-15 minutes.

[0015] Preferably, the pore size of the microporous filter membrane used in step 6) is 0.45 μm.

[0016] The present invention also provides a method for preparing the blood pressure-lowering peptide complex to obtain the obtained blood pressure-lowering peptide complex.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: The fermentation compound microbial agent used in this invention consists of *Lactobacillus helveticus* CICC 6024 and *Lactococcus lactis* subsp. *milkfat* CICC20406. *Lactococcus lactis* subsp. *milkfat* rapidly initiates acid and aroma production at the beginning of fermentation, while *Lactobacillus helveticus* lowers the pH further in the later stages of fermentation, ensuring fermentation stability and safety. This avoids the sharp sourness and insufficient flavor that might result from using only *Lactobacillus helveticus*, while providing a full and rich characteristic flavor. The growth and metabolism of *Lactococcus lactis* subsp. *milkfat* provides abundant metabolites and a favorable microenvironment, indirectly promoting the activity of *Lactobacillus helveticus* proteolytic enzymes. Under favorable symbiotic conditions, *Lactobacillus helveticus* can hydrolyze proteins more efficiently, producing a richer peptide spectrum. The compound system of *Lactococcus lactis* subsp. *milkfat* and *Lactobacillus helveticus* can more quickly form a dominant bacterial community and inhibit other bacteria. Fermentation treatment utilizes a compound bacterial agent to break down large protein molecules into medium- and long-chain peptides and a small amount of amino acids. These peptides provide more accessible sites for the subsequently added alkaline protease and papain, enabling the enzymes to generate target small peptides more efficiently and specifically. Alkaline protease can widely hydrolyze the peptide bonds of proteins, producing a large number of soluble peptides. Papain can specifically cleave hydrophobic amino acids. The C-terminus of many ACE-inhibiting peptides is often a hydrophobic amino acid, which helps to release potential antihypertensive peptides with specific terminal structures. The complementary cleavage sites of the two enable more thorough and diverse hydrolysis, increasing the diversity of active peptides, thereby giving the prepared antihypertensive peptide complex higher ACE inhibitory activity. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results of ACE inhibition rate determination of the retentate solutions prepared in Examples 1-3 and Comparative Examples 1-3; Figure 2The results show the ACE inhibition rate of the retentate obtained from Examples 1-3 and Comparative Examples 1-3 after digestion with simulated gastrointestinal fluid. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The materials used in this invention are sourced from: yak milk from the Gannan Hezuo Ecological Industrial Park, Hezuo City, Gannan Tibetan Autonomous Prefecture, Gansu Province; and Lactobacillus helveticus CICC 6024 and Lactobacillus helveticus (…). Lactobacillus helveticus CICC 20540 and Lactococcus lactis subsp. lactis CICC 20406 were both purchased from the China Industrial Microbial Culture Collection Center; materials without specified sources were commercially available products.

[0025] Example 1 1) Yak milk is filtered to remove large impurities such as hair and grass clippings. It is preheated at 37°C to reduce the viscosity of the yak milk, making the fat globules easier to move and aggregate, thus improving the separation efficiency. It is then centrifuged at 4°C and 6000 rpm for 20 minutes to obtain skim milk. 2) Lactobacillus helveticus CICC 6024 bacterial suspension (effective viable count is 1×10⁻⁶) 6 (cfu / mL) and Lactococcus lactis subsp. milk fat CICC 20406 bacterial suspension (effective viable count of 1×10⁻⁶) 6 A compound bacterial agent was prepared by mixing (cfu / mL) at a volume ratio of 1:1; 3) The compound microbial agent was inoculated into skim milk at an inoculation rate of 3% v / v, and fermented at 37°C and pH 4.5 for 18 hours to obtain fermented milk; 4) Alkaline protease and papain are mixed at a mass ratio of 1:1 to obtain a compound enzyme preparation; 5) Stir the fermented milk evenly, adjust the pH to 7.5 with 1M NaOH, add the compound enzyme preparation at 2500U / g, enzymatically hydrolyze at 52℃ for 3h, inactivate the enzyme at 90℃ for 10min, adjust the pH to 7.0, and obtain the enzymatic hydrolysate. 6) Centrifuge the enzymatic hydrolysate at 4°C and 7000 rpm for 15 min, and collect the supernatant. Filter the supernatant through a 0.45 μm microporous membrane to obtain the filtrate. Ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and ultrafilter the permeate again through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Collect the retentate and freeze-dry it to obtain the antihypertensive peptide complex.

[0026] Example 2 1) Yak milk is filtered to remove large impurities such as hair and grass clippings. It is preheated at 35℃ to reduce the viscosity of the yak milk, making the fat globules easier to move and aggregate, thus improving the separation efficiency. It is then centrifuged at 4℃ and 4000rpm for 30 minutes to obtain skim milk. 2) Lactobacillus helveticus CICC 6024 bacterial suspension (effective viable count is 1×10⁻⁶) 6 (cfu / mL) and Lactococcus lactis subsp. milk fat CICC 20406 bacterial suspension (effective viable count of 1×10⁻⁶) 6 A compound bacterial agent was prepared by mixing (cfu / mL) at a volume ratio of 1:2; 3) Inoculate the compound microbial agent into skim milk at an inoculation rate of 2% v / v, ferment at 30℃ and pH 4.3 for 12 hours to obtain fermented milk; 4) Alkaline protease and papain are mixed at a mass ratio of 1:2 to obtain a compound enzyme preparation; 5) Stir the fermented milk evenly, adjust the pH to 7.0 with 1M NaOH, add the compound enzyme preparation at 1000U / g, enzymatically hydrolyze at 50℃ for 2h, inactivate the enzyme at 85℃ for 10min, adjust the pH to 7.0, and obtain the enzymatic hydrolysate. 6) Centrifuge the enzymatic hydrolysate at 4°C and 6000 rpm for 15 min, and collect the supernatant. Filter the supernatant through a 0.45 μm microporous membrane to obtain the filtrate. Ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and ultrafilter the permeate again through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Collect the retentate and freeze-dry it to obtain the antihypertensive peptide complex.

[0027] Example 3 1) Yak milk is filtered to remove large impurities such as hair and grass clippings. It is preheated at 40℃ to reduce the viscosity of yak milk, making fat globules easier to move and aggregate, thus improving separation efficiency. It is then centrifuged at 4℃ and 8000rpm for 30 minutes to obtain skim milk. 2) Lactobacillus helveticus CICC 6024 bacterial suspension (effective viable count is 1×10⁻⁶) 6 (cfu / mL) and Lactococcus lactis subsp. milk fat CICC 20406 bacterial suspension (effective viable count of 1×10⁻⁶) 6 A compound bacterial agent was prepared by mixing (cfu / mL) at a volume ratio of 1:2; 3) The compound microbial agent was inoculated into skim milk at an inoculation rate of 4% v / v, and fermented at 40℃ and pH 4.7 for 24 hours to obtain fermented milk; 4) Alkaline protease and papain are mixed at a mass ratio of 1:2 to obtain a compound enzyme preparation; 5) Stir the fermented milk evenly, adjust the pH to 8.0 with 1M NaOH, add the compound enzyme preparation at 4000U / g, enzymatically hydrolyze at 55℃ for 4h, inactivate the enzyme at 90℃ for 15min, adjust the pH to 7.0, and obtain the enzymatic hydrolysate. 6) Centrifuge the enzymatic hydrolysate at 4°C and 8500 rpm for 20 min, and collect the supernatant. Filter the supernatant through a 0.45 μm microporous membrane to obtain the filtrate. Ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and ultrafilter the permeate again through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Collect the retentate and freeze-dry it to obtain the antihypertensive peptide complex.

[0028] Comparative Example 1 1) Yak milk is filtered to remove large impurities such as hair and grass clippings. It is preheated at 37°C to reduce the viscosity of the yak milk, making the fat globules easier to move and aggregate, thus improving the separation efficiency. It is then centrifuged at 4°C and 6000 rpm for 20 minutes to obtain skim milk. 2) Lactobacillus helveticus CICC 20540 bacterial suspension (effective viable count is 1×10⁻⁶) 6 (cfu / mL) and Lactococcus lactis subsp. milk fat CICC 20406 bacterial suspension (effective viable count of 1×10⁻⁶) 6 The compound bacterial agent was prepared by mixing (cfu / mL) at a volume ratio of 1:1; 3) The compound microbial agent was inoculated into skim milk at an inoculation rate of 3% v / v, and fermented at 37°C and pH 4.5 for 18 hours to obtain fermented milk; 4) Alkaline protease and papain are mixed at a mass ratio of 1:1 to obtain a compound enzyme preparation; 5) Stir the fermented milk evenly, adjust the pH to 7.5 with 1M NaOH, add the compound enzyme preparation at 2500U / g, enzymatically hydrolyze at 52℃ for 3h, inactivate the enzyme at 90℃ for 10min, adjust the pH to 7.0, and obtain the enzymatic hydrolysate. 6) Centrifuge the enzymatic hydrolysate at 4°C and 7000 rpm for 15 min, and collect the supernatant. Filter the supernatant through a 0.45 μm microporous membrane to obtain the filtrate. Ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and ultrafilter the permeate again through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Collect the retentate and freeze-dry it to obtain the antihypertensive peptide complex.

[0029] Comparative Example 2 1) Yak milk is filtered to remove large impurities such as hair and grass clippings. It is preheated at 37°C to reduce the viscosity of the yak milk, making the fat globules easier to move and aggregate, thus improving the separation efficiency. It is then centrifuged at 4°C and 6000 rpm for 20 minutes to obtain skim milk. 2) Lactobacillus helveticus CICC 6024 bacterial suspension (effective viable count is 1×10⁻⁶) 6 (cfu / mL) and Lactococcus lactis subsp. milk fat CICC 20406 bacterial suspension (effective viable count of 1×10⁻⁶) 6 A compound bacterial agent was prepared by mixing (cfu / mL) at a volume ratio of 2:1; 3) The compound microbial agent was inoculated into skim milk at an inoculation rate of 3% v / v, and fermented at 37°C and pH 4.5 for 18 hours to obtain fermented milk; 4) Alkaline protease and papain are mixed at a mass ratio of 1:1 to obtain a compound enzyme preparation; 5) Stir the fermented milk evenly, adjust the pH to 7.5 with 1M NaOH, add the compound enzyme preparation at 2500U / g, enzymatically hydrolyze at 52℃ for 3h, inactivate the enzyme at 90℃ for 10min, adjust the pH to 7.0, and obtain the enzymatic hydrolysate. 6) Centrifuge the enzymatic hydrolysate at 4°C and 7000 rpm for 15 min, and collect the supernatant. Filter the supernatant through a 0.45 μm microporous membrane to obtain the filtrate. Ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and ultrafilter the permeate again through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Collect the retentate and freeze-dry it to obtain the antihypertensive peptide complex.

[0030] Comparative Example 3 1) Yak milk is filtered to remove large impurities such as hair and grass clippings. It is preheated at 37°C to reduce the viscosity of the yak milk, making the fat globules easier to move and aggregate, thus improving the separation efficiency. It is then centrifuged at 4°C and 6000 rpm for 20 minutes to obtain skim milk. 2) Lactobacillus helveticus CICC 6024 bacterial suspension (effective viable count is 1×10⁻⁶) 6 (cfu / mL) and Lactococcus lactis subsp. milk fat CICC 20406 bacterial suspension (effective viable count of 1×10⁻⁶) 6 The compound bacterial agent was prepared by mixing (cfu / mL) at a volume ratio of 1:1; 3) The compound microbial agent was inoculated into skim milk at an inoculation rate of 3% v / v, and fermented at 37°C and pH 4.5 for 18 hours to obtain fermented milk; 4) Alkaline protease and papain are mixed at a mass ratio of 2:1 to obtain a compound enzyme preparation; 5) Stir the fermented milk evenly, adjust the pH to 7.5 with 1M NaOH, add the compound enzyme preparation at 2500U / g, enzymatically hydrolyze at 52℃ for 3h, inactivate the enzyme at 90℃ for 10min, adjust the pH to 7.0, and obtain the enzymatic hydrolysate. 6) Centrifuge the enzymatic hydrolysate at 4°C and 7000 rpm for 15 min, and collect the supernatant. Filter the supernatant through a 0.45 μm microporous membrane to obtain the filtrate. Ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and ultrafilter the permeate again through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Collect the retentate and freeze-dry it to obtain the antihypertensive peptide complex.

[0031] Experimental Example 1 In vitro determination of ACE inhibition rate: Hippuryl histidine (HHL) and angiotensin-converting enzyme (ACE) were dissolved in buffer solutions containing 0.3 mol / L NaCl and 0.1 mol / L sodium borate (pH 8.3), respectively. 50 μL of HHL (0.010 mol / L) and 50 μL of the test sample (the retentate obtained in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, or Comparative Example 3) were vortexed and incubated at 37°C for 2 min. Then, 50 μL of ACE (0.010 U / mL) solution was added, and the mixture was thoroughly vortexed and incubated at 37°C for 40 min. The reaction was then terminated by heating in an 85°C water bath for 10 min, and 200 μL of 0.1 mol / L EDTA solution was added. In the control group, 50 μL of sodium borate buffer solution was used instead of the test sample. The content of hippuric acid, the reaction product in the enzymatic hydrolysis reaction, was determined by RP-HPLC.

[0032] ACE inhibition rate is calculated using the following formula: ACE inhibition rate (%) = (C 对照 –C 待测 ) / (C 对照–C 标准 ) × 100%; where, C 对照 The concentration of hippuric acid in the control sample; C 待测 C represents the hippuric acid concentration in the sample to be tested. 标准 This refers to the concentration of hippuric acid contained in the HHL standard.

[0033] Table 1. Results of ACE inhibition rate determination in different groups of retentate solutions.

[0034] As shown in Table 1 and Figure 1 As shown, the ACE inhibition rate of the retentate prepared in Examples 1-3 of this invention is significantly higher than that of Comparative Examples 1-3. The different strains of *Lactobacillus helveticus* used in Comparative Example 1 resulted in a less effective fermentation treatment of the compound bacterial agent compared to the bacterial agent composed of *Lactobacillus helveticus* CICC 6024 and *Lactococcus lactis* subsp. *lactofat* CICC 20406, which affected the ACE inhibition rate of the peptide complex obtained by further enzymatic hydrolysis. In Comparative Example 2, the compound ratio of *Lactobacillus helveticus* CICC 6024 and *Lactococcus lactis* subsp. *lactofat* CICC 20406 was adjusted. In Comparative Example 3, the compound ratio of alkaline protease and papain in the enzyme preparation was adjusted, and the ACE inhibition rate of the resulting peptide complex was also lower than that of the peptide complex prepared in Examples 1-3 of this invention.

[0035] Experiment Example 2 The retentate solutions prepared in Examples 1-3 and Comparative Examples 1-3 were digested with pepsin (21000 U / mL) for 4 h at pH 2.0 and 37°C, then digested with trypsin (2500 U / mL) for 4 h at pH 8.0 and 37°C. Finally, the enzymes were inactivated at 90°C for 10 min. After cooling the digest to room temperature, the ACE inhibition rate was measured. The method for measuring the ACE inhibition rate was the same as in Experimental Example 1.

[0036] Table 2. Results of ACE inhibition rate determination of different groups of retentate after simulated gastrointestinal digestion.

[0037] As shown in Table 2 and Figure 2 As shown, in order to further prove whether the prepared polypeptide complex can reach the cardiovascular system in an active form through the digestion and absorption of human gastrointestinal fluid, the present invention simulated the physiological conditions of human gastrointestinal fluid. It was found that the ACE inhibition rate of the retentate prepared in Examples 1-3 and Comparative Examples 1-3 was improved after simulated gastrointestinal digestion compared with that before digestion, which proved that the antihypertensive peptide complex prepared in Examples 1-3 can play a role in the human body in an active form, and the ACE inhibition rate of the retentate prepared in Example 1 was still the highest after simulated gastrointestinal digestion.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a blood pressure-lowering peptide complex through fermentation and synergistic enzymatic hydrolysis, characterized in that, Includes the following steps: 1) Yak milk is filtered, preheated, and centrifuged to obtain skim milk; 2) Lactobacillus helveticus ( Lactobacillus helveticus CICC 6024 bacterial culture and Lactococcus lactis subsp. milk fat ( Lactococcus lactis subsp. cremoris A compound bacterial agent was prepared by mixing CICC 20406 bacterial solution; 3) The compound microbial agent obtained in step 2) is inoculated into the skim milk obtained in step 1) and fermented to obtain fermented milk; 4) Alkaline protease and papain are combined to obtain a compound enzyme preparation; 5) Adjust the pH of the fermented milk obtained in step 3) to 7.0~8.0, add the compound enzyme preparation obtained in step 4), perform enzymatic hydrolysis, and inactivate the enzyme to obtain the enzymatic hydrolysate; 6) Centrifuge the enzymatic hydrolysate obtained in step 5) and collect the supernatant. Filter the supernatant through a microporous membrane to obtain the filtrate. Ultrafilter the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 3-5 kDa to obtain the permeate. Ultrafilter the permeate again through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Collect the retentate and freeze-dry it to obtain the antihypertensive peptide complex.

2. The method for preparing the antihypertensive peptide complex according to claim 1, characterized in that, The preheating temperature in step 1) is 35~40℃; the centrifugation temperature in step 1) is 4℃, the centrifugation speed is 4000~8000rpm, and the centrifugation time is 15~30min.

3. The method for preparing the antihypertensive peptide complex according to claim 1, characterized in that, The effective viable count of Lactobacillus helveticus CICC 6024 bacterial suspension mentioned in step 2) is 1×10⁻⁶. 6 cfu / mL; the effective viable count of the *Lactococcus lactis* subsp. *milk fat* CICC 20406 bacterial suspension mentioned in step 2) is 1×10⁻⁶. 6 cfu / mL; the volume ratio of the Lactobacillus helveticus CICC 6024 bacterial solution and Lactococcus lactis subsp. milk fat CICC 20406 bacterial solution in step 2) is 1:1~2.

4. The method for preparing the antihypertensive peptide complex according to claim 1, characterized in that, The inoculation amount of the compound microbial agent mentioned in step 3) is 2%~4% v / v.

5. The method for preparing the antihypertensive peptide complex according to claim 1, characterized in that, The fermentation temperature in step 3) is 30~40℃, the fermentation pH is 4.3~4.7, and the fermentation time is 12~24h.

6. The method for preparing the antihypertensive peptide complex according to claim 1, characterized in that, The mass ratio of alkaline protease to papain in step 4) is 1:1~2.

7. The method for preparing the antihypertensive peptide complex according to claim 1, characterized in that, The amount of compound enzyme preparation added in step 5) is calculated as 1000~4000U / g; the enzymatic hydrolysis temperature in step 5) is 50~55℃, and the enzymatic hydrolysis time is 2~4h.

8. The method for preparing the antihypertensive peptide complex according to claim 1, characterized in that, The enzyme inactivation treatment in step 5) is performed at a temperature of 85-90°C for 10-15 minutes.

9. The method for preparing the antihypertensive peptide complex according to claim 1, characterized in that, The pore size of the microporous filter membrane used in step 6) is 0.45 μm.

10. The antihypertensive peptide complex is prepared by the method for preparing antihypertensive peptide complex according to any one of claims 1 to 9.

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