A high-yield antihypertensive peptide direct-vat starter and its application in preparing antihypertensive yogurt

Abstract

This invention relates to the field of health food, and discloses a high-yield direct-inoculation starter culture for antihypertensive peptides and its application in the preparation of antihypertensive yogurt. The starter culture is prepared by mixing *Streptococcus thermophilus* WHH1590, *Lactobacillus delbrueckii* subsp. bulgaricus WHH699, and *Lactobacillus helveticus* WHH2580 in a ratio of 90-100:15-25:1. This starter culture, after mixing, exhibits a significant synergistic effect, significantly enhancing the ability of microorganisms to decompose proteins and significantly increasing the content of antihypertensive peptides in yogurt. When using the starter culture of this application to ferment yogurt, it achieves the technical advantages of a short fermentation cycle, high yield of antihypertensive peptides, and high ACE inhibitory activity. Yogurt prepared with the starter culture provided by this application not only ensures a high yield of antihypertensive peptides but also possesses excellent sensory qualities, with a thick texture, delicate structure, and no unpleasant flavor. Yogurt prepared with the starter culture provided by this application exhibits higher thermal stability and safety compared to existing technologies.

Description

Technical Field

[0001] This invention relates to the field of health food, and in particular to a high-yield, direct-inoculation fermentation agent for lowering blood pressure peptides and its application in the preparation of blood pressure-lowering yogurt. Background Technology

[0002] Hypertension is a common disease among middle-aged and elderly people, characterized by elevated systemic blood pressure, specifically systolic blood pressure ≥140 mmHg and diastolic blood pressure ≥90 mmHg. In recent years, the prevalence of hypertension has been increasing year by year, and patients are becoming younger. It has now been listed by the World Health Organization as the number one risk factor for human health. Therefore, the prevention and control of hypertension and related diseases has become a major health problem facing humanity. Human blood pressure is regulated by a variety of factors, the most important of which are the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). An important factor affecting these two systems is angiotensin-converting enzyme (ACE). ACE can convert angiotensin I (AngI) in the RAS system into angiotensin II (AngII), which can cause elevated blood pressure. ACE can also inactivate bradykinin, a blood pressure-lowering substance in the KKS system, thereby inhibiting the KKS system and causing elevated blood pressure. Therefore, ACE inhibitors have become an important class of substances for the treatment of hypertension.

[0003] Currently, there are many ACE inhibitor products available. Among them, lactic acid bacteria fermented antihypertensive peptides have attracted much attention from people in the field due to their advantages such as ease of operation, ease of promotion, and low cost. For example, CN114097921A discloses a method for preparing and applying a milk protein-derived angiotensin-converting enzyme inhibitory peptide. This application uses desalted whey powder as a fermentation substrate, which is fermented by Lactobacillus fermentum and Streptococcus thermophilus, and then undergoes a protein-like reaction to prepare a milk protein-derived angiotensin-converting enzyme inhibitory peptide with an ACE inhibition rate as high as 47.87%. At the same time, it also increases the content of free amino acids and flavor peptides, giving the product a better flavor. The prepared milk protein-derived angiotensin-converting enzyme inhibitory peptide is used to prepare milk cake, which can significantly improve the ACE inhibition rate of milk cake, thereby improving the effect of blood pressure reduction.

[0004] For example, CN103141575A discloses a method for preparing hawthorn yogurt rich in angiotensin-converting enzyme inhibitory peptides. The technical solution of this application includes: adding 5%-6% white sugar to raw milk, homogenizing it, sterilizing it at 90℃ for 5 minutes, and cooling it to 42-43℃; inoculating the sterilized and cooled raw milk with Lactobacillus helveticus starter and yogurt starter at a ratio of 3% each; fermenting it at 41℃ for 3.5 hours to obtain fermented milk; adding hawthorn jam to the fermented milk, and allowing it to mature for 24 hours to produce the product. The product prepared by this application is rich in angiotensin-converting enzyme inhibitory peptides, has a unique flavor, and has certain antihypertensive effects.

[0005] In the prior art, research on antihypertensive peptides from fermented milk sources focuses on fermentation processes using single strains such as Lactobacillus helveticus, Lactobacillus reuteri, and Lactobacillus plantarum. However, this fermentation method suffers from problems such as long fermentation time, low ACE inhibitory activity, and poor stability of ACE inhibitory peptides in fermented products. Therefore, in order to solve the above problems, this application provides a high-yield direct-inoculation starter culture for antihypertensive peptides and its application in the preparation of antihypertensive yogurt. Summary of the Invention

[0006] To overcome the problems in the prior art, this invention provides a high-yield direct-inoculation starter culture for antihypertensive peptides and its application in the preparation of antihypertensive yogurt. The direct-inoculation starter culture in this application is a compound starter culture composed of *Streptococcus thermophilus* WHH1590, *Lactobacillus delbrueckii* subsp. bulgaricus WHH699, and *Lactobacillus helveticus* WHH2580 in a ratio of 90-100:15-25:1. This compound starter culture is a direct-inoculation product that can perform one-step yogurt fermentation. It is simple to operate, has a wide range of applications, and is easy to industrialize. When using the direct-inoculation starter culture of this application for yogurt fermentation, the fermentation cycle is short, and the ACE inhibitory peptides in the fermentation products have high activity and high stability. Products made using the direct-inoculation starter culture of this application have high sensory quality and a long shelf life.

[0007] The specific technical solution of this invention is as follows:

[0008] A direct-inoculation fermentation agent for producing high-yield antihypertensive peptides, wherein the raw materials of the fermentation agent include: freeze-dried Streptococcus thermophilus powder, freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder, and freeze-dried Lactobacillus helveticus powder;

[0009] The thermophilic streptococcus lyophilic powder was obtained from Streptococcus thermophilus and / or its mutants; the thermophilic streptococcus was named WHH1590 and was deposited on July 27, 2022 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25413, and its microbiological classification was Streptococcus thermophilus, deposited in Beijing, China.

[0010] The freeze-dried powder of *Lactobacillus delbrueckii* subsp. bulgaricus was obtained from *Lactobacillus delbrueckii* subsp. bulgaricus and / or its mutants; the *Lactobacillus delbrueckii* subsp. bulgaricus was named WHH699 and was deposited on July 27, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25414. Its microbiological classification is *Lactobacillus delbrueckii* subsp. bulgaricus, and its deposit address is Beijing, China.

[0011] The lyophilized powder of *Lactobacillus helveticus* is obtained from *Lactobacillus helveticus* and / or its mutants; the *Lactobacillus helveticus* is named WHH2580 and was deposited on October 23, 2019, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 18730, and its microbiological classification is *Lactobacillus helveticus*, deposited in Beijing, China.

[0012] The starter culture in this application is prepared by compounding freeze-dried powders of Streptococcus thermophilus WHH1590, Lactobacillus delbrueckii subsp. bulgaricus WHH699, and Lactobacillus helveticus WHH2580 in a specific ratio. These three strains exhibit a significant synergistic effect after compounding, significantly enhancing the ability of microorganisms to decompose proteins and significantly increasing the content of antihypertensive peptides in yogurt. Yogurt prepared using the starter culture of this application shows an ACE inhibition rate of 99.6% after 4–8 hours of fermentation, while existing starter cultures require 12–24 hours of fermentation to achieve an ACE inhibition rate of 70%. Therefore, compared with existing technologies, this application has the advantages of a shorter fermentation cycle, higher yield of antihypertensive peptides, and higher ACE inhibitory activity.

[0013] This application found that not all combinations of Streptococcus thermophilus WHH1590 with any Lactobacillus delbrueckii subsp. bulgaricus or any Lactobacillus helveticus produce a significant synergistic effect. Through experiments, this application found that only when Streptococcus thermophilus WHH1590, Lactobacillus delbrueckii subsp. bulgaricus WHH699, and Lactobacillus helveticus WHH2580 are combined at a ratio of 90-100:15-25:1 can a significant synergistic effect be achieved. Yogurt prepared using this application's starter culture can achieve levels of 62.7 mg / L and 36.8 mg / L of the two antihypertensive peptides, VPP and IPP, respectively, indicating a high content of antihypertensive peptides in the yogurt. Furthermore, yogurt prepared using this application's starter culture, while ensuring a high yield of antihypertensive peptides, also possesses excellent sensory qualities, with a thick texture, delicate structure, and no unpleasant flavor.

[0014] Yogurt prepared using the starter culture provided in this application exhibits higher thermal stability and safety compared to existing technologies. After being placed at 65°C, 95°C, 105°C, a simulated digestive tract environment, and a simulated gastric juice environment for a period of time, the activity of antihypertensive peptides in the yogurt was not significantly affected. Furthermore, after being placed at 4°C and room temperature for six consecutive months, the activity of antihypertensive peptides in the yogurt was also not significantly affected.

[0015] Preferably, the ratio of the freeze-dried Streptococcus thermophilus powder, the freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder, and the freeze-dried Lactobacillus helveticus powder is 90-100:15-25:1.

[0016] Preferably, the compound fermentation agent is freeze-dried bacterial powder.

[0017] Preferably, the preparation method of the compound fermentation agent includes the following steps:

[0018] a. Preparation of Streptococcus thermophilus lyophilized powder: Streptococcus thermophilus strain was activated to prepare Streptococcus thermophilus seed liquid, which was then inoculated into expansion medium for expansion culture. After expansion culture, the culture medium was centrifuged to remove the supernatant and the precipitate was collected. Finally, the precipitate was mixed with a protectant and then freeze-dried to prepare Streptococcus thermophilus lyophilized powder. The activation medium was M17 medium, the inoculum size for expansion culture was 1-4%, the temperature was 37-43℃, the pH value was 5.5-6.5, the time was 5-8h, and nitrogen gas was introduced.

[0019] b. Preparation of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder: Lactobacillus delbrueckii subsp. bulgaricus strain was activated to prepare Lactobacillus delbrueckii seed culture. This seed culture was then inoculated into an expansion medium for expansion culture. After expansion culture, the culture medium was centrifuged to remove the supernatant and the precipitate was collected. Finally, the precipitate was mixed with a protectant and freeze-dried to prepare freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder. The activation medium was MRS + lactose + tomato juice liquid medium. The inoculum size for expansion culture was 1–4%, the temperature was 37–43℃, the pH was 4.5–5.5, the time was 9–12 h, and nitrogen gas was introduced.

[0020] c. Preparation of Lactobacillus helveticus freeze-dried powder: Lactobacillus helveticus strain was activated to prepare Lactobacillus helveticus seed solution, which was then inoculated into an expansion medium for expansion culture. After the expansion culture was completed, the culture medium was centrifuged and the precipitate was collected. Finally, the precipitate was mixed with a protectant and freeze-dried to prepare Lactobacillus helveticus freeze-dried powder. The activation medium was MRS + lactose + tomato juice liquid medium. The inoculum size for expansion culture was 1-4%, the temperature was 37-43℃, the pH was 5.0-6.5, the time was 9-12h, and nitrogen gas was introduced.

[0021] d. Preparation of direct-inoculation starter for high-yield antihypertensive peptides: The above-prepared freeze-dried powders of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus were mixed evenly in proportion and packaged to prepare a direct-inoculation starter for high-yield antihypertensive peptides.

[0022] Preferably, the protective agent comprises skim milk, sucrose, and monosodium glutamate in a mass ratio of 0.25–0.75:0.1–0.5:0.05–0.2.

[0023] Preferably, the viable count of the freeze-dried Streptococcus thermophilus powder is 1×107~1×1012 CFU / g; the viable count of the freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder is 1×107~1×1012 CFU / g; and the viable count of the freeze-dried Lactobacillus helveticus powder is 1×107~1×1012 CFU / g.

[0024] Application of a direct-inoculation starter for high-yield hypotensive peptides, wherein the high-yield hypotensive peptide direct-inoculation starter is used in the preparation of hypotensive yogurt, the preparation method includes the following steps: adding milk powder to ultrapure water and mixing to form an emulsion, then heating the emulsion to form a sterilized emulsion, the sterilization temperature of the emulsion is 90-105℃, the sterilization time is 5-15 min, after sterilization, the sterilized emulsion is cooled to 37-44℃, then the above-prepared high-yield hypotensive peptide direct-inoculation starter is inoculated into the sterilized emulsion, the inoculation amount is 0.01-0.1%, after inoculation, the mixture is stirred evenly and fermented, the fermentation temperature is 37-44℃, the fermentation time is 4-10 h, after fermentation, the emulsion is sheared and demulsified to obtain hypotensive yogurt, the shearing and demulsification speed is 2000-3000 r / min, the shearing and demulsification time is 20-30 min.

[0025] Preferably, the milk powder is selected from one or more of cow's milk, mare's milk, and goat's milk.

[0026] Preferably, the mass of the milk powder is 10% to 15% of the total mass of the emulsion.

[0027] Compared with the prior art, this application has the following technical effects:

[0028] (1) The starter culture of this application is prepared by compounding Streptococcus thermophilus WHH1590, Lactobacillus delbrueckii subsp. bulgaricus WHH699 and Lactobacillus helveticus WHH2580, which can produce a significant synergistic effect, significantly enhance the ability of microorganisms to decompose proteins, and significantly increase the content of antihypertensive peptides in yogurt.

[0029] (2) When using the starter culture of this application to ferment yogurt, it has the technical effects of short fermentation cycle, high yield of antihypertensive peptides and high ACE inhibitory activity.

[0030] (3) The yogurt produced by the fermentation agent provided in this application not only ensures high production of antihypertensive peptides, but also has good sensory quality. The yogurt has a thick texture, delicate structure, and no unpleasant flavor.

[0031] (4) Yogurt prepared by the starter provided in this application has higher thermal stability and safety compared with the prior art. Attached Figure Description

[0032] Figure 1 The figure shows the acid production kinetic curve of the fermentation agent.

[0033] Figure 2 This is a diagram showing the stringy texture of yogurt.

[0034] Figure 3 This is a bar graph showing the thermal stability of antihypertensive peptides in yogurt.

[0035] Figure 4 This is a bar graph showing the simulated digestive fluid thermal stability of the antihypertensive peptides in yogurt.

[0036] Figure 5 A bar graph showing the room temperature storage stability of antihypertensive peptides in yogurt.

[0037] Figure 6 A bar graph showing the 4°C storage stability of antihypertensive peptides in yogurt. Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments.

[0039] Example 1:

[0040] A direct-inoculation fermentation agent for high-yield antihypertensive peptides, comprising freeze-dried powders of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus mixed in a ratio of 90-100:15-25:1; the preparation method includes the following steps:

[0041] a. Preparation of Streptococcus thermophilus freeze-dried powder: Streptococcus thermophilus strain was activated to prepare Streptococcus thermophilus seed liquid, and then the Streptococcus thermophilus seed liquid was inoculated into an expansion medium for expansion culture. The activation medium was M17 medium, the inoculum size was 1-4%, the temperature was 37-43℃, the pH value was 5.5-6.5, the time was 5-8h, and nitrogen gas was introduced. After the expansion culture was completed, the culture medium was centrifuged to remove the supernatant and the precipitate was collected. Finally, the precipitate was mixed with a protectant and freeze-dried to prepare Streptococcus thermophilus freeze-dried powder. The protectant included skim milk, sucrose and monosodium glutamate in a mass ratio of 0.25-0.75:0.1-0.5g:0.05-0.2. The viable count in the Streptococcus thermophilus freeze-dried powder was 1×107-1×1012 CFU / g.

[0042] b. Preparation of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder: Lactobacillus delbrueckii subsp. bulgaricus strain was activated to prepare Lactobacillus delbrueckii seed culture. The seed culture was then inoculated into an expansion culture medium for expansion culture. The activation medium was MRS + lactose + tomato juice liquid medium. The inoculum size for expansion culture was 1–4%, the temperature was 37–43℃, the pH was 4.5–5.5, and the time was 9–12 h. Nitrogen gas was introduced. After expansion culture, the culture medium was centrifuged to remove the supernatant and the precipitate was collected. Finally, the precipitate was mixed with a preservative and freeze-dried to prepare freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder. The viable count of the freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder was 1×10⁷–1×10¹² CFU / g.

[0043] c. Preparation of Lactobacillus helveticus freeze-dried powder: Lactobacillus helveticus strain was activated to prepare Lactobacillus helveticus seed liquid, and then the Lactobacillus helveticus seed liquid was inoculated into an expansion medium for expansion culture. The activation medium was MRS + lactose + tomato juice liquid medium. The inoculum size for expansion culture was 1-4%, the temperature was 37-43℃, the pH value was 5.0-6.5, the time was 9-12h, and nitrogen gas was introduced. After the expansion culture was completed, the culture medium was centrifuged and the precipitate was collected. Finally, the precipitate was mixed with a protective agent and then freeze-dried to prepare Lactobacillus helveticus freeze-dried powder. The viable count of Lactobacillus helveticus freeze-dried powder was 1×107-1×1012 CFU / g.

[0044] d. Preparation of direct-inoculation starter for high-yield antihypertensive peptides: The above-prepared freeze-dried powders of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus were mixed evenly in proportion and packaged to prepare a direct-inoculation starter for high-yield antihypertensive peptides.

[0045] The application of a direct-inoculation starter with high-yield antihypertensive peptides in the preparation of antihypertensive yogurt includes the following steps: Milk powder is added to ultrapure water and mixed to form an emulsion. The milk powder is selected from one or more types of cow's milk, mare's milk, and goat's milk, and the mass of the milk powder is 10%–15% of the total mass of the emulsion. The temperature of the ultrapure water is 40–60℃, the shear mixing speed is 2000–3000 r / min, and the shear mixing time is 20–30 min. The emulsion is then heated to form a sterilized emulsion. The sterilization temperature of the emulsion is 90–105℃, and the sterilization time is 5–15 minutes. After sterilization, the sterilized emulsion is cooled to 37–44°C. The high-yield antihypertensive peptide direct-inoculation starter culture prepared above is then inoculated into the sterilized emulsion at an inoculation rate of 0.01–0.1%. After inoculation, the mixture is stirred thoroughly and fermented at 37–44°C for 4–10 hours. After fermentation, the emulsion is broken down by shearing to obtain antihypertensive yogurt. The shearing speed is 2000–3000 r / min, and the shearing time is 20–30 min. The pH is continuously monitored during fermentation using an acidity tracker. The pH changes during the above process are as follows: Figure 1As shown in the figure, the yogurt produced by the above process has a stringy consistency as shown in the figure. Figure 2 As shown.

[0046] Comparative Example 1: (The starter culture was Streptococcus thermophilus WHH1590)

[0047] Compared with Example 1, freeze-dried powder of Streptococcus thermophilus WHH1590 was used as the starter culture, and all other conditions were the same as in Example 1.

[0048] Comparative Example 2: (The starter culture was Lactobacillus delbrueckii subsp. bulgaricus WHH699)

[0049] Compared with Example 1, the freeze-dried powder of Lactobacillus delbrueckii subsp. bulgaricus WHH699 was used as the starter culture, and all other conditions were the same as in Example 1.

[0050] Comparative Example 3: (The starter culture was Lactobacillus helveticus WHH2580)

[0051] Compared with Example 1, the freeze-dried powder of Lactobacillus helveticus WHH2580 was used as the starter culture, and all other conditions were the same as in Example 1.

[0052] Comparative Example 4: (The fermentation agent is a compound inoculum made from other microbial strains)

[0053] Compared with Example 1, a compound bacterial agent prepared by mixing freeze-dried bacterial powders of Streptococcus thermophilus WHH449, Lactobacillus delbrueckii subsp. bulgaricus WHH3887 and Lactobacillus helveticus WHH1889 was used as the fermenting agent, and all other conditions were the same as in Example 1.

[0054] Test Example 1: ACE Inhibition Rate Measurement

[0055] The yogurt prepared in Example 1 and Comparative Examples 1-4 was used to treat the ACE hydrolysis of urylhistylleucine (HHL). The ACE inhibition rate was calculated by detecting the hippuric acid content in the hydrolysis products using high performance liquid chromatography. The specific steps are as follows:

[0056] (1) Centrifuge the yogurt sample to be tested at 12000r / min for 10min, and take the supernatant for later use;

[0057] (2) First, add HHL to the NaCl borate buffer (pH 8.3, Cl... -Prepare a 5.0 mmol / L HHL solution by mixing 0.3 mol / L HHL solution with 0.1 mol / L borate. Then, mix 200 μL of the HHL solution with 80 μL of the sample to be tested in a centrifuge tube and incubate at 37 °C for 5 minutes. Then, add 20 μL of ACE solution (enzyme activity 0.1 U / mL), mix well, and incubate at 37 °C for 30 minutes. Finally, add 250 μL of 1.0 mol / L hydrochloric acid solution to terminate the reaction.

[0058] (3) The hippuric acid content in the reaction substrate was detected using high performance liquid chromatography (HPLC), and the ACE inhibition rate was calculated (calculation formula). The formula for calculating the ACE inhibition rate is:

[0059] ACE inhibition rate (%) = (B - A) / (B - C) × 100

[0060] In the formula: A is the absorbance of the ACE inhibitor and ACE solution reacting with HHL simultaneously; B is the absorbance of ACE reacting completely with HHL without sample, i.e., control; C is the absorbance of sample and ACE not present, i.e. blank.

[0061] (4) Test results

[0062] The inhibition rate of ACE by the yogurts prepared in Example 1 and Comparative Examples 1-4 is shown in Table 1.

[0063] Table 1. ACE inhibition rate of antihypertensive peptides in yogurt

[0064]

[0065] Test Example 2: Thermal Stability Determination

[0066] The yogurt prepared in Example 1 was subjected to heat treatment, and the ACE inhibition rate of the treated sample was detected using the method in Test Example 1. The specific steps included: placing the yogurt sample in a water bath and heating it at 65°C for 30 min, 90°C for 10 min, and 105°C for 10 min, respectively. Then, the ACE inhibition rate of the treated yogurt sample was measured, and the results are as follows: Figure 3 As shown, the results indicate that the ACE inhibition rate in yogurt did not change significantly after treatment under different heating conditions, suggesting that the antihypertensive peptides obtained by this starter culture have excellent heat resistance.

[0067] Test Example 3: Stability Determination of Simulated Digestive Tract Environment

[0068] The yogurt prepared in Example 1 was subjected to heat treatment, and the ACE inhibition rate of the treated sample was detected using the method in Test Example 1. The specific operation steps are as follows:

[0069] (1) Simulate gastric juice environment: Mix the yogurt sample to be tested (pH 2.5) with pepsin (Sigma, P7000-100G, 800-2500 units / mg) at a final concentration of 3 g / L. After mixing evenly, place it in a water bath at 37°C for 3 h. After treatment, centrifuge the treated sample at 12000 r / min for 10 min and take the supernatant to measure the ACE inhibition rate.

[0070] (2) Simulating intestinal fluid environment: The yogurt sample to be tested (pH 8.0) was mixed with pepsin (Sigma, T4799-100G, 1000-2000 BAEE units / mg) at a final concentration of 1 g / L and bile salts (Sigma, 48305-50G-F). After mixing evenly, the sample was placed in a water bath at 37°C for 3 h. After treatment, the sample was centrifuged at 12000 r / min for 10 min, and the supernatant was taken to measure the ACE inhibition rate.

[0071] (3) Storage stability test

[0072] The yogurt samples to be tested were aliquoted into centrifuge tubes in a sterile environment with a volume of 15 ml. The aliquoted yogurt samples were then stored at room temperature and 4°C. The ACE inhibition rate of the yogurt was measured every 0.5 months and monitored continuously for 6 months.

[0073] (4) Test results

[0074] The measurement results are as follows Figure 4 , Figure 5 and Figure 6 As shown, after treatment with simulated gastric juice, the ACE inhibition rate in yogurt did not change significantly; after treatment with simulated intestinal juice, the ACE inhibition rate decreased slightly, but there was no significant difference. The results indicate that the antihypertensive peptides obtained by fermentation with this starter culture have excellent resistance to acid, alkali, bile salts and enzymatic hydrolysis. After 6 months of storage at room temperature and 4°C, the ACE inhibition rate in yogurt did not change significantly, indicating that the antihypertensive peptides obtained by fermentation with the starter culture provided in this application have a long activity maintenance time and excellent shelf-life stability.

[0075] Unless otherwise specified, the raw materials and equipment used in this invention are common in the field; unless otherwise specified, the methods used in this invention are conventional methods in the field.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. The application of a direct-inoculation starter culture with high yield of antihypertensive peptides in the preparation of antihypertensive yogurt, characterized in that, Includes the following steps: Milk powder is mixed with ultrapure water to form an emulsion. The emulsion is then heated to form a sterilized emulsion. After sterilization, the sterilized emulsion is cooled to 37-44°C. A high-yield antihypertensive peptide direct-inoculation starter culture is then inoculated into the sterilized emulsion at an inoculation rate of 0.01-0.1%. After inoculation, the mixture is stirred evenly and fermented at a temperature of 37-44°C for 4-10 hours. After fermentation, the emulsion is cut and broken to obtain antihypertensive yogurt. The raw materials for the high-yield antihypertensive peptide direct-inoculation fermentation agent include: *Streptococcus thermophilus* freeze-dried powder, *Lactobacillus delbrueckii* subsp. bulgaricus freeze-dried powder, and *Lactobacillus helveticus* freeze-dried powder; the *Streptococcus thermophilus* freeze-dried powder is prepared from *Streptococcus thermophilus*; the *Streptococcus thermophilus* WHH1590 has the accession number CGMCC No. 25413; the *Lactobacillus delbrueckii* subsp. bulgaricus freeze-dried powder is prepared from *Lactobacillus delbrueckii* subsp. bulgaricus; the *Lactobacillus delbrueckii* subsp. bulgaricus WHH699 has the accession number CGMCC No. 25414; the *Lactobacillus helveticus* freeze-dried powder is prepared from *Lactobacillus helveticus*; the *Lactobacillus helveticus* WHH2580 has the accession number CGMCC No. 18730.

2. The application according to claim 1, characterized in that, The ratio of the freeze-dried Streptococcus thermophilus powder, the freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder, and the freeze-dried Lactobacillus helveticus powder is 90-100:15-25:

1.

3. The application according to claim 1, characterized in that, The high-yield antihypertensive peptide direct-inoculation fermentation agent is a freeze-dried bacterial powder.

4. The application according to claim 1, characterized in that, The preparation method of the high-yield antihypertensive peptide direct-inoculation fermentation agent includes the following steps: a. Preparation of Streptococcus thermophilus lyophilized powder: Streptococcus thermophilus strain was activated to prepare Streptococcus thermophilus seed liquid, which was then inoculated into expansion medium for expansion culture. After expansion culture, the culture medium was centrifuged to remove the supernatant and the precipitate was collected. Finally, the precipitate was mixed with a protectant and then freeze-dried to prepare Streptococcus thermophilus lyophilized powder. The activation medium was M17 medium, the inoculum size for expansion culture was 1-4%, the temperature was 37-43℃, the pH value was 5.5-6.5, the time was 5-8h, and nitrogen gas was introduced. b. Preparation of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder: Lactobacillus delbrueckii subsp. bulgaricus strain was activated to prepare Lactobacillus delbrueckii seed culture. This seed culture was then inoculated into an expansion medium for expansion culture. After expansion culture, the culture medium was centrifuged to remove the supernatant and the precipitate was collected. Finally, the precipitate was mixed with a protectant and freeze-dried to prepare freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder. The activation medium was MRS + lactose + tomato juice liquid medium. The inoculum size for expansion culture was 1-4%, the temperature was 37-43℃, the pH was 4.5-5.5, the time was 9-12 hours, and nitrogen gas was introduced. c. Preparation of Lactobacillus helveticus freeze-dried powder: Lactobacillus helveticus strain was activated to prepare Lactobacillus helveticus seed solution, which was then inoculated into an expansion medium for expansion culture. After the expansion culture was completed, the culture medium was centrifuged and the precipitate was collected. Finally, the precipitate was mixed with a protectant and freeze-dried to prepare Lactobacillus helveticus freeze-dried powder. The activation medium was MRS + lactose + tomato juice liquid medium. The inoculum size for expansion culture was 1-4%, the temperature was 37-43℃, the pH value was 5.0-6.5, the time was 9-12h, and nitrogen gas was introduced. d. Preparation of direct-inoculation starter for high-yield antihypertensive peptides: The above-prepared freeze-dried powders of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus were mixed evenly in proportion and packaged to prepare a direct-inoculation starter for high-yield antihypertensive peptides.

5. The application according to claim 4, characterized in that, The protective agent comprises skim milk, sucrose, and monosodium glutamate in a mass ratio of 0.25–0.75: 0.1–0.5 g: 0.05–0.

2.

6. The application according to claim 1, characterized in that, The viable count of Streptococcus thermophilus lyophilized powder is 1×10⁻⁶. 7 ~1×10 12 CFU / g; The viable count of the freeze-dried Lactobacillus delbrueckii subsp. bulgaricus powder is 1×10⁻⁶. 7 ~1×10 12 CFU / g; The viable count of the Lactobacillus helveticus freeze-dried powder is 1×10⁻⁶. 7 ~1×10 12 CFU / g.

7. The application according to claim 1, characterized in that, The sterilization temperature of the emulsion is 90~105℃, and the sterilization time is 5~15min.

8. The application according to claim 1, characterized in that, The shearing demulsification speed is 2000~3000 r / min, and the shearing demulsification time is 20~30 min.

9. The application according to claim 1, characterized in that, The milk powder is selected from one or more of cow's milk, mare's milk, and goat's milk.

10. The application according to claim 1, characterized in that, The mass of the milk powder is 10% to 15% of the total mass of the emulsion.

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