A method for preparing milk-derived angiotensin-converting enzyme inhibitory peptides by fermentation with composite bacteria

Abstract

The present invention discloses a method for preparing milk-derived angiotensin-converting enzyme inhibitory peptides by fermentation with composite strains, which belongs to the fields of microbial technology and fermentation. The ratio of whey protein hydrolysate with a molecular weight below 1000 Da and the angiotensin-converting enzyme inhibition rate in the milk-based fermentation broth are used as evaluation indicators. The composite strain Lactobacillus helveticus ( Lactobacillus helveticus , CICC: 20289), composite strain Pediococcus dilactici ( Pediococcus acidilactici , CICC:20719) were rescreened to obtain strains with excellent whey protein hydrolysis performance and high angiotensin-converting enzyme inhibitory peptide production capacity. The two composite strains obtained from the rescreening were enriched and cultured separately, and then inoculated into skim milk powder culture medium at a specific compound ratio. Through composite fermentation, the angiotensin-converting enzyme inhibitory peptide content in the milk-based fermentation product was increased. The process of this invention is rationally designed and highly operable. It provides a reference for further increasing the content of small molecule functional peptides in fermented dairy products, efficiently preparing milk-derived angiotensin-converting enzyme inhibitory peptides, and improving the nutritional value of dairy products, and has great application prospects.

Description

Technical Field

[0001] The invention relates to a method for preparing milk-derived angiotensin-converting enzyme inhibitory peptide by fermentation with composite bacteria, and belongs to the fields of microbial technology and fermentation. Background Art

[0002] Angiotensin-converting enzyme (ACE) is a zinc-containing dipeptide carboxypeptidase primarily found in mammalian somatic cells and male germ cells. It exerts its effects through the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS), leading to elevated blood pressure. ACE inhibitory peptides competitively inhibit ACE activity by binding to the ACE active site, thereby regulating the RAS and KKS to lower blood pressure.

[0003] Modern research indicates that bioactive peptides possess greater biological activity and nutritional value than proteins and single amino acids. Due to their strong specificity, high biological activity, minimal toxic side effects, ease of digestion and absorption, and diverse functions, bioactive peptides hold enormous potential for development as preventative and therapeutic pharmaceuticals. Food-derived ACE inhibitors, with their rapid onset, potent effects, and high safety profile, have already played a crucial role in the clinical treatment of hypertension and cardiovascular disease.

[0004] Milk and dairy products are the most complete animal protein source for humans. Research has shown that milk and dairy products not only provide rich nutrition but also serve as a significant source of numerous bioactive peptides, of which antihypertensive peptides are of particular interest for research, development, and application. Since the discovery of small peptides with ACE inhibitory activity isolated from casein hydrolysates, the study of antihypertensive peptides derived from milk and dairy products has become a new hotspot in dairy research. Numerous antihypertensive peptides have been discovered from the microbial fermentation broths used in the production of fermented dairy products, demonstrating their diverse potential and lack of side effects.

[0005] The present invention discloses a method for preparing milk-derived angiotensin-converting enzyme inhibitory peptides by fermenting a composite bacterial strain. The method has a reasonable process design and strong operability, can provide a reference for further increasing the content of small molecule functional peptides in fermented milk products, developing milk-derived ACE inhibitory peptides, and improving the nutritional value of dairy products, and has good application prospects. Summary of the Invention

[0006] The present invention provides a method for preparing milk-derived angiotensin-converting enzyme inhibitory peptide by fermentation of a composite bacterial strain, comprising the following steps:

[0007] Step 1: Inoculate a 3% inoculum of a bacterial suspension (approximately 106 colonies / mL) of each of the composite strains Lactobacillus helveticus (CICC:20289) and Pediococcus acidilactici (CICC:20719) into 50 ml of 12% skim milk powder medium, mix thoroughly, and incubate at 38°C for 12 hours. Using the proportion of whey protein hydrolysates with a molecular weight below 1000 Da and angiotensin-converting enzyme (ACE) inhibition rate in the milk-based fermentation broth as evaluation indicators, rescreen the composite strains. These strains were screened for excellent whey protein hydrolysis and high ACE inhibitory peptide production.

[0008] Step 2: After the composite strain 1 and the composite strain 2 re-screened strains obtained in step 1 are enriched and cultured separately, they are inoculated into skim milk powder culture medium at a certain compound ratio (3:1), the total amount of strain added is 3%, and the initial fermentation pH is 8.0; after the fermentation cycle, the fermentation products in the composite strain fermentation broth are subjected to protein hydrolyzate ratio analysis, angiotensin-converting enzyme inhibition rate IC50 analysis, and dipeptide characteristic peptide segment identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Mass spectrometric identification of ACE inhibitory peptides in fermented dairy products of composite strains.

[0010] Figure 2 : Chromatographic analysis of ACE inhibition rate in composite bacterial strain fermented milk products. DETAILED DESCRIPTION

[0011] The present invention is further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0012] Example 1

[0013] Suspensions of the composite bacterial strains Lactobacillus helveticus (CICC: 20289) and Pediococcus acidilactici (CICC: 20719) (approximately 106 colonies / mL) were prepared and inoculated at a 3% concentration into 50 mL of 12% skim milk powder. Fermentation was performed for 12 hours at 37°C and a pH of 6.5. Protein hydrolysate molecular weight analysis was performed using high-performance gel filtration chromatography (HPLC) according to Appendix A of GB 22729-2008, National Food Safety Standard for Marine Fish Oligopeptide Powder. This method uses a porous packing as the stationary phase, separating sample components based on their molecular size. The mobile phase consisted of acetonitrile:water:trifluoroacetic acid (45:55:0.1), with UV detection at 220 nm, a flow rate of 0.5 mL / min, and a column temperature of 30°C. Weigh 100 μL of sample and dilute to 2 mL with mobile phase. After ultrasonic vibration for 10 minutes, filter through a polytetrafluoroethylene membrane with a pore size of 0.2 μm to 0.5 μm. Sample injection and analysis are performed on the instrument. The gel chromatogram and its data are processed to determine the relative molecular weight and distribution range of the protein hydrolysates. The sample chromatographic data are incorporated into the calibration curve equation to calculate the relative molecular weight and distribution range of the peptides in the sample. Peak area normalization is used to calculate the sum of the relative percentages of the peak areas of protein hydrolysates (including oligopeptides and a small amount of free amino acids) with different relative molecular weights.

[0014] Example 2

[0015] A suspension of the composite bacterial strains Lactobacillus helveticus (CICC: 20289) and Pediococcus acidilactici (CICC: 20719) (approximately 10<6 > colonies / mL) was prepared and inoculated into 50 mL of 12% skim milk powder medium at a 3% bacterial addition rate. Fermentation was performed for 12 hours at a temperature of 37°C and a pH of 6.5. The fermentation broth was then assayed for ACE inhibition using RP-HPLC. The method is as follows:

[0016] (1) Chromatographic conditions: Mobile phase A: 70% water, 30% methanol (containing 0.1% trifluoroacetic acid, 0.05% acetic acid); Mobile phase B: 20% water, 80% methanol (containing 0.1% trifluoroacetic acid, 0.05% acetic acid); Flow rate: 1.0 mL / min; Column temperature: 30°C; Injection volume: 100 μL; Detection wavelength: UV 227 nm; Gradient program: 0-2.50 min, mobile phase B: 0%-0%; 2.51-4.50 min, mobile phase B: 100%-100%; 4.51-15 min, mobile phase B: 0%-0%;

[0017] (2) Sample preparation: Prepare 0.05 mol / L boric acid buffer (containing 0.3 mol / L NaCl) with a pH of 8.3. Use boric acid buffer to prepare 50 mU / mL ACE solution, and use boric acid buffer to prepare 7.6 mmol / L HHL solution. Mix 20 μL of the sample to be tested (the blank control is boric acid buffer) with 30 μL ACE solution and preheat in a 37°C water bath for 5 min. Add 50 μL substrate HHL solution and incubate in a 37°C water bath for 30 min. After the reaction is completed, add 100 μL 1M HCl to terminate the reaction, and finally add 400 μL boric acid buffer, mix well, and then perform HPLC detection. Repeat 3 times for each sample. Filter the sample solution with a 0.2 μm pore size polytetrafluoroethylene filter membrane and inject it into the machine;

[0018] (3) Calculation of ACE inhibition rate: Based on the linear relationship between the elution peak area of ​​hippuric acid, the enzymatic hydrolysis product of ACE, and the concentration of hippuric acid in the RP-HPLC spectrum, the ACE inhibition rate of the inhibitory peptide can be determined by measuring the difference in hippuric acid peak area before and after the addition of the antihypertensive peptide. The inhibition rate calculation formula is as follows:

[0019] ACE inhibition rate =

[0020] Where: M is the peak area of ​​hippuric acid in the blank control group (mAU·s); N is the peak area of ​​hippuric acid in the inhibitor-added group (mAU·s).

[0021] Example 3

[0022] A bacterial suspension (approximately 106 colonies / mL) was prepared using the selected composite bacterial strains Lactobacillus helveticus (CICC:20289) and Pediococcus acidilactici (CICC:20719). The suspension was inoculated into 50 ml of 12% skim milk powder medium at a ratio of 3:1 (Lactobacillus helveticus:Pediococcus acidilactici). Fermentation was performed for 12 hours at 37°C and a pH of 6.5. The fermentation broth was analyzed for dipeptide detection using ultra-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry. The method is as follows:

[0023] (1) Sample treatment: Dilute the sample 100 times with pure water, centrifuge it (10000 r / min for 10 min), take the supernatant, filter it with a nylon filter membrane with a pore size of 0.22 μm, and then pre-treat the sample to be tested. First, the fermented mare's milk was separated by liquid chromatography using a C18 column. Subsequently, the separated products were scanned by electrospray tandem triple quadrupole mass spectrometry. The characteristic ions were used to qualitatively infer the peptide sequences present in the sample. Combined with the qualitative results, the peptide structure was quantitatively analyzed;

[0024] (2) Liquid chromatography conditions: Column: Inertsil ODS-3 (5 μm, 2.1×250 mm); Mobile phase: A is 0.1% formic acid in water, B is 0.1% formic acid in acetonitrile; Gradient elution program: 0–15 min, B (0%–50%); 15–20 min, B (50%–100%); 20–25 min, B (100%); 25.1–35 min, B (0%); Flow rate: 0.2 mL / min; Injection volume: 10 μL; Column temperature: 40 °C;

[0025] (3) Mass spectrometry conditions: Ionization mode: ESI, positive ion mode; ion spray voltage: +4.5 kV; nebulizer gas flow rate: nitrogen 3.0 L / min; heating gas flow rate: nitrogen 10 L / min; drying gas flow rate: nitrogen 10 L / min; DL temperature: 250 °C; heating module temperature: 400 °C; ion source temperature: 300 °C; scan mode: multiple reaction monitoring (MRM); dwell time: 100 ms; delay time: 3 ms;

[0026] The results of the determination of the proportion of protein hydrolysates with a molecular weight below 1000 Da, the angiotensin-converting enzyme inhibition rate, and the angiotensin-converting enzyme inhibitory peptide of the fermentation product are shown in Table 1 ;

[0027] Table 1 The proportion of protein hydrolysates with molecular weight below 1000 Da, angiotensin converting enzyme inhibition rate and angiotensin converting enzyme inhibitory peptide results of fermentation products

[0028]

Claims

1. A method for preparing milk-derived angiotensin-converting enzyme inhibitory peptide by fermentation of a composite bacterial strain, characterized in that: The steps include: Step 1: The bacterial suspensions of the composite strain Lactobacillus helveticus, CICC: 20289 and the composite strain Pediococcus acidilactici, CICC: 20719 were respectively added, with the number of colonies being about 10 6 The strains were inoculated with 50 ml of 12% skim milk powder medium at a rate of 3%, mixed well, and cultured at 38°C for 12 h. The proportion of whey protein hydrolysates with a molecular weight below 1000 Da and the angiotensin-converting enzyme inhibition rate in the milk-based fermentation broth were used as evaluation indicators. The composite strains 1 and 2 were rescreened, and the composite strains 1 and 2 with excellent whey protein hydrolysis performance and high angiotensin-converting enzyme inhibitory peptide production ability were obtained. Step 2: After the composite strain 1 and the composite strain 2 re-screened strains obtained in step 1 are enriched and cultured respectively, they are inoculated into skim milk powder culture medium at a ratio of 3:1 between composite strain 1 and composite strain 2, with a total strain addition amount of 3%, a fermentation time of 11 h, a fermentation temperature of 40°C, and an initial fermentation pH of 8.0; after the fermentation cycle, the fermented mare's milk is subjected to liquid chromatography separation using a C18 chromatographic column to obtain a protein hydrolyzate with a molecular weight below 1000 Da.

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