Preparation method and application of a blood sugar-lowering active peptide

Abstract

The application belongs to the technical field of fermentation, and particularly relates to a preparation method and application of a blood sugar reducing active peptide, and comprises the following steps: mixing casein aqueous solution and compound lactic acid bacteria to perform fermentation, so as to obtain casein fermentation liquor; mixing the casein fermentation liquor with leucine aminopeptidase to perform enzymolysis, so as to obtain casein hydrolysate; and performing Plastein reaction on the casein hydrolysate, so as to obtain the blood sugar reducing active peptide. The specific protease and peptidase generated by Lactobacillus helveticus and Lactobacillus plantarum are used to hydrolyze casein to generate specific peptide segments, and the leucine at the N-terminal of the peptide segments is further separated out by leucine aminopeptidase, so that more target peptides are enriched. Then, amino acids are synthesized into new peptide chains through Plastein reaction modification, so that the blood sugar reducing peptide with higher activity is obtained.

Description

Technical Field

[0001] This invention belongs to the technical field of combined fermentation and enzymatic hydrolysis, specifically relating to a method and application for preparing and hydrolyzing casein with leucine aminopeptidase to obtain hypoglycemic active peptides with higher activity. Background Technology

[0002] Diabetes mellitus, as one of the most common metabolic disorders in recent years, has a high incidence and mortality rate. Synthetic insulin and insulin analogs are one method to control high blood sugar, but chemically synthesized drugs have certain side effects on the human body. Therefore, the search for safe and effective natural hypoglycemic drugs is a hot research topic.

[0003] Some milk-derived peptides possess hypoglycemic activity, controlling blood sugar levels by inhibiting the activity of enzymes that break down carbohydrates into glucose. Hypoglycemic peptides typically contain 2-15 amino acid residues and often exist in proteins as inactive precursors, requiring methods such as enzymatic hydrolysis, fermentation, or processing to release them.

[0004] In the prior art, CN 105506046A discloses a casein hypoglycemic and lipid-lowering peptide based on yak milk chyle and its preparation method. This method effectively improves the degree of casein hydrolysis by combining ultra-high pressure / ultrasound / microwave-assisted technology with a simulated gastrointestinal digestion hydrolysis mode, achieving controlled release of active amino acid sequences and improving its gastrointestinal stability. The protein-like reaction modification debittering technique used solves the bitterness problem of the hypoglycemic and lipid-lowering peptides without damaging their biological activity. It is evident that this preparation process only releases the active peptide sequences from casein through a hydrolysis reaction; therefore, how to synthesize new, more active hypoglycemic peptides is the next technical problem to be solved. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing and applying a more active hypoglycemic peptide.

[0006] The preparation method provided by this invention utilizes specific proteases and peptidases produced by the fermentation of compound lactic acid bacteria, combined with exogenous leucine aminopeptidase to enzymatically hydrolyze casein, and combines the Plastein reaction to synthesize the added exogenous amino acids into the new peptide chain, thereby obtaining a hypoglycemic peptide with higher activity.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing hypoglycemic active peptides, comprising the following steps: preparing a casein aqueous solution with casein and water, sterilizing it, and then mixing it with compound lactic acid bacteria for fermentation to obtain a casein fermentation broth; sterilizing the casein fermentation broth and then adding leucine aminopeptidase for enzymatic hydrolysis to obtain a casein hydrolysate; concentrating the casein hydrolysate under vacuum, and then performing a Plastein reaction followed by vacuum freeze-drying to obtain hypoglycemic active peptides.

[0008] Preferably, the compound lactic acid bacteria includes: Lactobacillus helveticus and Lactobacillus plantarum; the colony count ratio of Lactobacillus helveticus and Lactobacillus plantarum is 1~6:1.

[0009] Preferably, the fermentation temperature is 30~50℃, and the casein aqueous solution is fermented to a pH of 4.0~6.0.

[0010] Preferably, the weight ratio of casein fermentation broth to leucine aminopeptidase is 500~1500:1.

[0011] Preferably, the enzymatic hydrolysis is performed at a pH of 6.0 to 8.0, a temperature of 30 to 45°C, and a time of 2 to 4 hours.

[0012] Preferably, prior to the Plastein reaction, the casein hydrolysate is further concentrated, and the concentrated solids content is 40% to 60%.

[0013] Preferably, the Plastein reaction further includes adding amino acids to the casein hydrolysate, wherein the amount of added amino acids is 1.0% to 4.5%, and the amino acids include one or more of alanine, lysine, proline, and glycine.

[0014] Preferably, the pH of the Plastein reaction is 6.5-8.0, the temperature is 30-50°C, and the time is 2-8 hours.

[0015] This invention provides a hypoglycemic active peptide prepared using the above-described method.

[0016] Beneficial Effects: This invention provides a method for preparing hypoglycemic active peptides. Specific proteases and peptidases produced by fermentation with compound lactic acid bacteria are used to hydrolyze casein to generate specific peptides. These peptides are then further enriched by leucine aminopeptidase to release the N-terminal leucine residues, resulting in more target peptides. Finally, the amino acids are synthesized into new peptide chains via a Plastein reaction, yielding hypoglycemic peptides with higher activity.

[0017] This invention provides hypoglycemic active peptides prepared using the above-described preparation method. Casein is enzymatically hydrolyzed by proteases and peptidases produced by fermentation of compound lactic acid bacteria, combined with exogenous leucine aminopeptidase. Under the combined action of endopeptidase and exopeptidase, casein is first decomposed into large peptide molecules, and then further decomposed into a series of small peptides, resulting in peptides with a higher degree of protein hydrolysis and higher peptide content.

[0018] The present invention also utilizes the above-mentioned hypoglycemic active peptides to prepare fermented milk with α-glucosidase inhibitory activity of 15.78~20.14% and dipeptidyl peptidase (DPP-IV enzyme) inhibitory activity of 54.45~71.44%.

[0019] This invention uses leucine aminopeptidase as an exonuclease to release leucine residues from the N-terminus of the peptide chain, thereby obtaining more target peptides and improving the activity of hypoglycemic peptides.

[0020] This invention provides a hypoglycemic active peptide prepared by the above-described preparation method. In the presence of a protease, the condensation or transpeptidation involved in the Plastein reaction can synthesize the added exogenous amino acids into a new peptide chain, thereby generating a new peptide sequence that is not present in the original hydrolysate, resulting in a better hypoglycemic effect.

[0021] The hypoglycemic active peptide of the present invention has a good hypoglycemic ability. Attached Figure Description

[0022] Figure 1 A comparison of the α-glucosidase inhibitory activities of bovine milk-derived hypoglycemic peptides prepared using different amino acids; Figure 2 A comparison of α-glucosidase inhibitory activity in fermented milk before and after the addition of hypoglycemic active peptides; Figure 3 A comparison of the DPP-IV inhibitory activity in fermented milk before and after the addition of hypoglycemic active peptides; Figure 4 A comparison of α-glucosidase inhibitory activity before and after the addition of hypoglycemic active peptides in fermented goat milk; Figure 5 A comparison of the DPP-IV inhibitory activity of fermented goat milk before and after the addition of hypoglycemic active peptides; Figure 6 A comparison of α-glucosidase inhibitory activity before and after the addition of hypoglycemic active peptides to fermented yak milk; Figure 7 This is a comparison of the DPP-IV inhibitory activity of fermented yak milk before and after the addition of hypoglycemic active peptides. Detailed Implementation

[0023] This invention provides a method for preparing a hypoglycemic active peptide, comprising the following steps: A casein aqueous solution was mixed with compound lactic acid bacteria for fermentation to obtain casein fermentation broth; the casein fermentation broth was mixed with leucine aminopeptidase for enzymatic hydrolysis to obtain casein hydrolysate; the casein hydrolysate was subjected to Plastein reaction to obtain hypoglycemic active peptides.

[0024] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0025] In this invention, casein is preferably prepared as an aqueous solution, wherein the mass concentration of the casein aqueous solution is preferably 2% to 10%, more preferably 4% to 8%, further preferably 6% to 7%, and most preferably 7%. The casein source of this invention preferably includes cow's milk, sheep's milk, or yak milk. This invention does not have special requirements regarding the source of cow's milk casein, sheep's milk casein, and yak milk; commercially available products well known to those skilled in the art can be used. In this invention, the casein aqueous solution is preferably sterilized, wherein the sterilization temperature is preferably 80 to 100°C, more preferably 90 to 95°C, and most preferably 95°C; and the sterilization time is preferably 2 to 15 minutes, more preferably 5 to 10 minutes, and most preferably 5 minutes.

[0026] In this invention, the casein aqueous solution is preferably mixed with a compound lactic acid bacteria for fermentation to obtain a casein fermentation broth. The compound lactic acid bacteria preferably include *Lactobacillus helveticus* and *Lactobacillus plantarum*. Utilizing the specific proteases and peptidases produced by *Lactobacillus helveticus* and *Lactobacillus plantarum*, casein is hydrolyzed to generate specific peptides, facilitating the subsequent release of leucine residues at the N-terminus of these peptides and enabling the enrichment of more target peptides. The live colony count ratio of *Lactobacillus helveticus* to *Lactobacillus plantarum* is preferably 1-6:1, more preferably 2-5:1, further preferably 3-4:1, and most preferably 3:1. The live colony count of *Lactobacillus helveticus* is preferably 3 × 10⁻⁶. 7 CFU, wherein the viable colony count of *Lactobacillus plantarum* is preferably 1 × 10⁻⁶. 7 CFU. The preferred amount of the compound lactic acid bacteria, based on the mass of the casein aqueous solution, is 4 × 10⁻⁶ CFU. 7 CFU / g. The fermentation temperature of the present invention is preferably 30~50℃, more preferably 35~45℃, further preferably 37~42℃, and most preferably 40℃; the pH of the casein aqueous solution after fermentation is 4.0~6.0, more preferably 4.5~5.5, and most preferably 5.0.

[0027] In this invention, the casein fermentation broth obtained by fermentation is preferably sterilized. The sterilization temperature is preferably 80~100℃, more preferably 90~95℃, and most preferably 95℃. The sterilization time is preferably 2~15min, more preferably 5~10min, and most preferably 5min.

[0028] In this invention, sterilized casein fermentation broth is mixed with leucine aminopeptidase for enzymatic hydrolysis to obtain casein hydrolysate. The weight ratio of casein fermentation broth to leucine aminopeptidase is preferably 500-1500:1, more preferably 800-1200:1, further preferably 900-1100:1, and most preferably 1000:1. The activity of leucine aminopeptidase is preferably 10,000 U / g. The pH of the fermentation broth is preferably pre-adjusted for enzymatic hydrolysis, preferably 6.0-8.0, more preferably 6.5-7.5, further preferably 6.8-7.2, and most preferably 7.0; the hydrolysis temperature is 30-45℃, more preferably 32-42℃, further preferably 35-40℃, and most preferably 37℃; the hydrolysis time is preferably 2-4 h, more preferably 2-3.5 h, further preferably 2.5-3.5 h, and most preferably 2.5 h. This invention uses leucine aminopeptidase as an exopeptidase to release leucine residues at the N-terminus of the peptide chain, thereby obtaining more of the target peptide. The enzymatic hydrolysis of this invention preferably includes an enzyme inactivation treatment. The enzyme inactivation temperature is preferably 80-95°C, more preferably 85-90°C, and most preferably 85°C. The enzyme inactivation time is preferably 4-15 min, more preferably 6-10 min, and most preferably 10 min.

[0029] This invention involves subjecting the prepared casein hydrolysate to a Plastein reaction to obtain hypoglycemic active peptides. Preferably, the casein hydrolysate is first concentrated, with the solid content of the concentrate preferably being 40%–60%, more preferably 45%–55%, and most preferably 50%. Preferably, this invention also includes adding a neutral protease to the casein hydrolysate concentrate. The neutral protease is an endopeptidase that can break down large protein molecules into peptides and amino acids, further hydrolyzing casein to form new peptide chains, preparing for the Plastein reaction. The weight ratio of the casein hydrolysate concentrate to the neutral protease is preferably 2500–3500:1, more preferably 2800–3200:1, further preferably 2900–3100:1, and most preferably 3000:1; the activity of the neutral protease is preferably 100,000 U / g. The present invention preferably further includes adding amino acids to the casein hydrolysate concentrate. The amino acids preferably include one or more selected from alanine, lysine, proline, and glycine. The weight of the added amino acids is preferably 1.0% to 4.5% of the casein hydrolysate concentrate, more preferably 1.5% to 4.0%, further preferably 1.7% to 3.0%, and most preferably 2.0%. The pH of the Plastein reaction in the present invention is preferably 6.5 to 8.0, more preferably 7.0 to 7.5, and most preferably 7.2; the temperature of the Plastein reaction is 30 to 50°C, more preferably 35 to 45°C, and most preferably 40°C; the reaction time is preferably 2 to 8 hours, more preferably 3 to 6 hours, and most preferably 4.5 hours. The target peptide obtained by the above hydrolysis of the present invention is modified by Plastein reaction to synthesize amino acids into a new peptide chain, thereby generating a new peptide sequence that is not present in the original hydrolysate, and obtaining a hypoglycemic active peptide with higher activity; and the free leucine generated by hydrolysis can be embedded and aggregated in the Plastein reaction, thereby reducing the bitterness of the prepared hypoglycemic active peptide.

[0030] This invention provides a hypoglycemic active peptide prepared by the above-described preparation method. In the presence of a protease, the condensation or transpeptidation involved in the Plastein reaction can synthesize the added exogenous amino acids into a new peptide chain, thereby generating a new peptide sequence that is not present in the original hydrolysate, resulting in a better hypoglycemic effect.

[0031] The hypoglycemic active peptide of the present invention has a good hypoglycemic ability.

[0032] To further illustrate the present invention, the preparation method and application of a hypoglycemic active peptide provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 The preparation of fermented milk with high hypoglycemic activity using bovine casein and bovine milk involves the following steps: Preparation of casein hydrolysate Step 1: Prepare a 7% (w / w) aqueous solution of commercially available bovine casein powder and sterilize it at 95°C for 5 minutes. Add 4 × 10⁻⁶ ppm of the solution per gram of casein. 7 CFU (Lactobacillus helveticus) Lactobacillus helveticus CICC 6024 and Lactobacillus plantarum ( Lactobacillus plantarum The compound lactic acid bacteria of CICC 20263, Lactobacillus helveticus: Lactobacillus plantarum = 3:1, were stirred evenly and fermented at 37℃ to the pH 5.0 to prepare bovine milk casein fermentation broth.

[0034] Step two: The milk casein fermentation broth is stirred to break the emulsion, heated at 90℃ for 10 minutes, and then the pH of the fermentation broth is adjusted to 7.0 with alkali solution. Leucine aminopeptidase is added at a weight ratio of fermentation broth:leucine aminopeptidase = 1000:1, with an activity of 10,000 U / g protein. Enzymatic hydrolysis is carried out at 37℃ for 2.5 hours, followed by enzyme inactivation at 85℃ for 10 minutes, yielding the milk casein hydrolysate.

[0035] Comparative Example 1 The experiment was conducted in the same manner as in Example 1, using the same batch of experimental raw materials. The difference was that after the bovine milk casein fermentation broth was prepared, the hydrolysis process in step two was not performed.

[0036] Experimental Example 1 1. Methods for determining the activity of proteolytic enzymes Mix 2g of sample with 1mL of deionized water, add 5mL of 0.75mol / L trichloroacetic acid, mix well, and let stand for 10min. Centrifuge and collect the supernatant. Take 200μL of the supernatant and add 4mL of OPA reagent, mix well. Using deionized water and OPA reagent as a blank, react at room temperature for 10min and measure the absorbance at 340nm, converting it to mmol / L of the sample. Construct a standard curve using 0-10mmol / L L-leucine, and calculate the protein hydrolysis activity using the standard curve.

[0037] y = 0.3011x - 0.0145, R 2 =0.9993.

[0038] In the formula, y represents protein hydrolysis activity in mmol / L; x represents the content of L-leucine in mmol / L.

[0039] 2. Methods for determining peptide content After centrifugation, the supernatant was collected, and the pH of the supernatant was adjusted to 7.5 using 1 mol / L sodium hydroxide. The supernatant was then filtered through a 0.45 μm hydrophilic membrane. 20 μL of the supernatant sample was used for RP-HPLC analysis. The peptide was reacted with C at 30℃. 18 Separation was performed using a chromatographic column. Elution program: 0–10 min, 0.1% trifluoroacetic acid aqueous solution; 10–90 min, acetonitrile gradually increased from 0% to 48%. Flow rate: 1 mL / min, detection wavelength: 210 nm. The peptide content in the sample was calculated by summing the peak areas at 210 nm in the peptide spectra. The peptide area at retention times of 10.0–90.0 min was determined.

[0040] The bovine casein hydrolysate obtained in Example 1 and the unhydrolyzed bovine casein fermentation broth obtained in Comparative Example 1 were tested, and the results are shown in Table 1.

[0041] Table 1. Protein hydrolysis activity and peptide content of bovine milk casein hydrolysate before and after hydrolysis.

[0042] As can be seen from Table 1, the casein fermentation broth of Example 1, after being hydrolyzed by leucine aminopeptidase, had significantly higher protein hydrolysis activity and peptide content than the casein fermentation broth of Comparative Example 1 before hydrolysis. This indicates that using the compound lactic acid bacteria fermentation technology of the present invention in combination with aminopeptidase to hydrolyze casein yields a casein hydrolysate with a higher degree of protein hydrolysis and a higher content of peptides than using only the compound lactic acid bacteria fermentation technology.

[0043] Example 2 Preparation of hypoglycemic active peptides The casein hydrolysate obtained in Example 1 was concentrated under vacuum to a solids content of 50%. The pH of the bovine milk casein hydrolysate was adjusted to 7.2. Neutral protease was added at a weight ratio of 3000:1 (casein hydrolysate concentrate: neutral protease), with a neutral protease activity of 100,000 U / g. Lysine was then added at 2% of the weight of the casein hydrolysate concentrate, and the Plastein reaction was carried out at 40°C for 4.5 h. The Plastein reaction solution was then freeze-dried under vacuum to obtain bovine milk-derived hypoglycemic active peptides.

[0044] Example 3 The procedure was carried out in accordance with Example 2, except that lysine was replaced with alanine.

[0045] Example 4 The procedure was carried out in accordance with Example 2, except that lysine was replaced with proline.

[0046] Example 5 The procedure was carried out in accordance with Example 2, except that lysine was replaced with glycine.

[0047] Comparative Example 2 The procedure was carried out in accordance with Example 2, except that lysine was replaced with arginine.

[0048] Comparative Example 3 The procedure was carried out in accordance with Example 2, except that lysine was replaced with phenylalanine.

[0049] Comparative Example 4 The procedure was carried out in accordance with Example 2, except that lysine was replaced with threonine.

[0050] Experimental Example 2 1. Method for determining α-amylase inhibitory activity Take 100 μL of the sample solution and 100 μL of 1 U / mL α-amylase (dissolved in pH 6.8, 0.1 mol / L phosphate buffer), mix well, and incubate at 37 °C for 5 min. Then add 250 μL of 1% starch solution (dissolved in pH 6.8, 0.1 mol / L phosphate buffer), mix well, and incubate at 37 °C for 5 min. Add 200 μL of DNS reagent, heat in a boiling water bath for 15 min, remove and cool rapidly, then add 2 mL of deionized water, mix well, and measure the absorbance at 540 nm. Calculate the α-amylase inhibitory activity using the following formula.

[0051] α-Amylase inhibitory activity (%) = [1-(A S -A B ) / A C 100%. Among them, A S A represents the absorbance of the sample reaction solution. B The absorbance of the enzyme is replaced by phosphate buffer; A C The absorbance of the sample is replaced by phosphate buffer.

[0052] 2. Method for determining α-glucosidase inhibitory activity Take 50 μL of the sample solution and 100 μL of 1 U / mL α-glucosidase (dissolved in pH 6.8, 0.1 mol / L phosphate buffer), mix well, and incubate at 37 °C for 10 min. Add 50 μL of 5 mmol / L pNPG solution (dissolved in pH 6.8, 0.1 mol / L phosphate buffer), mix well, and incubate at 37 °C for 30 min. Then add 1 mL of 0.1 mol / L sodium carbonate solution and measure the absorbance at 400 nm. Calculate the α-glucosidase inhibitory activity using the following formula.

[0053] α-glucosidase inhibitory activity (%) = [1-(A S -A B ) / A C 100%. Among them, AS A represents the absorbance of the sample reaction solution. B The absorbance of the enzyme is replaced by phosphate buffer; A C The absorbance of the sample is replaced by phosphate buffer.

[0054] 3. Method for Assay of DPP-IV Inhibitory Activity Transfer 25 μL of 12 mmol / L Gly-Pro-ρNA (dissolved in pH 8.0, 0.1 mol / L Tris-HCl buffer) and an equal volume of sample solution (dissolved in pH 8.0, 0.1 mol / L Tris-HCl buffer) into a 96-well plate and incubate at 37 °C for 10 min. Then add 50 μL of 2 U / mL DPP-IV enzyme solution (dissolved in pH 8.0, 0.1 mol / L Tris-HCl buffer) and incubate at 37 °C for 30 min. Finally, stop the reaction by adding 100 μL of 1 mol / L acetate-sodium acetate (pH 4.0) solution and measure the absorbance at 405 nm. Calculate the DPP-IV inhibitory activity using the following formula.

[0055] DPP-IV inhibitory activity (%) = [1 - (A S -A1) / (A N -A2)]100%. Where A S A1 is the absorbance of the sample reaction solution; A2 is the absorbance of the Tris-HCl buffer solution instead of the enzyme; A N A1 represents the absorbance of the Tris-HCl buffer instead of the sample; A2 represents the absorbance of the Tris-HCl buffer instead of the enzyme and sample.

[0056] The hypoglycemic active peptides prepared in Examples 2-5 and Comparative Examples 2-4, as well as the hypoglycemic active peptide prepared without the addition of any amino acids, were used as blank controls. The inhibitory activities of α-amylase, α-glucosidase, and DPP-IV were detected. Three samples were taken, and the average value was calculated. The results are shown in […]. Figure 1 And Table 2.

[0057] Table 2. Hypoglycemic effects of bovine milk-derived hypoglycemic active peptides prepared with different amino acids.

[0058] Depend on Figure 1As shown in Table 2, lysine in Example 2 exhibited the best effect, with α-amylase inhibitory activity of 40.02%, α-glucosidase inhibitory activity of 68.22%, and DPP-IV inhibitory activity of 96.58%. Alanine in Example 3 was the next best, with α-amylase inhibitory activity of 39.23%, α-glucosidase inhibitory activity of 61.47%, and DPP-IV inhibitory activity of 92.11%. Threonine in Comparative Example 4 showed the worst effect, with α-amylase inhibitory activity of 25.78% and α-glucosidase inhibitory activity of 37.89%, but its DPP-IV inhibitory activity was not significantly different from the blank and other comparative examples. This indicates that, in the presence of neutral protease, the hypoglycemic effects of bovine milk-derived hypoglycemic peptides prepared by the Plastein reaction from different exogenous amino acids vary considerably.

[0059] Comparative Example 5 The casein hydrolysate obtained in Example 1 was concentrated under vacuum to a solids content of 50%, and the pH of the bovine milk casein hydrolysate was adjusted to 7.2. The bovine milk casein hydrolysate was then directly freeze-dried under vacuum to obtain bovine milk-derived hypoglycemic active peptides.

[0060] Experimental Example 3 Using the method of Experimental Example 2, the α-amylase inhibitory activity and α-glucosidase inhibitory activity of Example 2 and Comparative Example 5 were detected, and the results are shown in Table 3.

[0061] Table 3. Hypoglycemic effects of bovine milk-derived hypoglycemic active peptides before and after the Plastein reaction.

[0062] Table 3 shows that the α-amylase inhibitory activity of the bovine milk-derived hypoglycemic active peptide prepared in Example 2 reached 40.02%, which was 15.45% higher than that of the peptide in Comparative Example 5; the α-glucosidase inhibitory activity of Example 2 reached 68.22%, which was 27.64% higher than that of the peptide in Comparative Example 5 that had not undergone the Plastein reaction. It is evident that the peptides subjected to the Plastein reaction significantly improved the hypoglycemic effect.

[0063] Sensory evaluation methods The freeze-dried powder samples prepared in Example 2 and Comparative Example 5 were dissolved in water at a mass concentration of 5% and serially diluted at a ratio of 1:1. Sensory evaluation was performed using the three-point test method to test whether the subjects could distinguish the degree of bitterness. The bitterness value was represented by the smallest dilution factor at which no difference in bitterness could be tasted. The larger the dilution factor, the higher the bitterness value, indicating that the product was more bitter.

[0064] The sensory evaluation data (Example 2 involved the Plastein reaction, detailed conditions are shown in Example 2; Comparative Example 5 omitted the Plastein reaction) showed that the bitterness value of Example 2 was 3, while the bitterness value of Comparative Example 5 was 11.

[0065] Example 6 Add 3% of the milk-derived hypoglycemic active peptide by weight of milk. Mix the milk with the hypoglycemic active peptide prepared in Example 1 and sterilize at 90°C for 10 minutes. Add 10g of the peptide per gram of milk. 8 CFU (thermophilic streptococcus) Streptococcus thermophilus Fermented milk is obtained by fermenting bacterial solution at 37°C until curd forms.

[0066] Comparative Example 6 The experiment was conducted in accordance with Example 6, using the same batch of experimental raw materials, except that no blood sugar-lowering active peptides derived from bovine milk were added during fermentation.

[0067] Test Example 4 Using the method of Experimental Example 2, the α-glucosidase inhibitory activity and DPP-IV inhibitory activity of Example 6 and Comparative Example 6 were detected, and the results are shown in [Figure 2]. Figure 2 , 3 And Table 4.

[0068] Table 4. Inhibitory activity of fermented milk before and after the addition of hypoglycemic active peptides

[0069] Depend on Figure 2 , 3 As shown in Table 4, the fermented milk of Example 6 exhibited significantly higher α-glucosidase inhibitory activity and DPP-IV inhibitory activity than the fermented milk of Comparative Example 6 without the addition of milk-derived hypoglycemic active peptides before and after simulated gastrointestinal digestion. Furthermore, these hypoglycemic active peptides demonstrated good resistance to gastrointestinal digestion. Therefore, the fermented milk product prepared using this invention possesses good hypoglycemic ability.

[0070] Example 7 Step 1: Prepare an 8% (w / w) aqueous solution of commercially available sheep milk casein powder and sterilize it at 90°C for 10 minutes. Add 4 × 10⁻⁶ ppm of the casein solution per gram of solution. 7 CFU (Lactobacillus helveticus) Lactobacillus helveticus CICC6024, and Lactobacillus plantarum ( Lactobacillus plantarum CICC 20263 mixed compound lactic acid bacteria, Lactobacillus helveticus: Lactobacillus plantarum = 3:1, were stirred evenly and fermented at 40℃ to a casein aqueous solution until pH 5.4 to prepare sheep milk casein fermentation broth.

[0071] Step two: Stir the fermentation broth thoroughly, heat at 90℃ for 10 minutes, and then adjust the pH of the fermentation broth to 7.0 with alkaline solution. Add leucine aminopeptidase at a weight ratio of fermentation broth:leucine aminopeptidase = 1000:1, with leucine aminopeptidase activity of 10,000 U / g protein. Incubate at 37℃ for 3 hours, then heat at 85℃ for 10 minutes to inactivate the enzyme, obtaining a sheep milk casein hydrolysate.

[0072] Comparative Example 7 The experiment was conducted in accordance with Example 7, using the same batch of experimental raw materials. The difference was that after the sheep milk casein fermentation broth was prepared, the second step of hydrolysis was not performed.

[0073] Experimental Example 5 The protein hydrolysis activity and peptide content of Example 7 and Comparative Example 7 were detected using the method of Experimental Example 1, and the results are shown in Table 5.

[0074] Table 5. Protein hydrolysis activity and peptide content of sheep milk casein hydrolysate after aminopeptidase hydrolysis.

[0075] As shown in Table 5, the casein fermentation broth of Example 7, after hydrolysis with leucine aminopeptidase, exhibited significantly higher protein hydrolysis activity and peptide content than the unhydrolyzed casein fermentation broth of Comparative Example 7. This demonstrates that utilizing the compound lactic acid bacteria fermentation technology of this invention combined with aminopeptidase hydrolysis of casein yields a casein hydrolysate with a higher degree of protein hydrolysis and a higher peptide content compared to using only compound lactic acid bacteria fermentation technology.

[0076] Example 8 The casein hydrolysate obtained in Example 7 was concentrated under vacuum to a solids content of 45%. The pH of the sheep milk casein hydrolysate was adjusted to 7.5. Neutral protease was added at a weight ratio of 3000:1 (casein hydrolysate concentrate: neutral protease), with a neutral protease activity of 100,000 U / g. Lysine was then added at 3% of the weight of the casein hydrolysate concentrate, and the Plastein reaction was carried out at 40°C for 4 hours. The Plastein reaction solution was then freeze-dried under vacuum to obtain sheep milk-derived hypoglycemic active peptides.

[0077] Comparative Example 8 The experiment was conducted in the same manner as in Example 8, using the same batch of experimental materials, except that the addition of lysine for the Plastein reaction was omitted.

[0078] Experimental Example 6 Using the method of Experimental Example 2, the α-amylase inhibitory activity and α-glucosidase inhibitory activity of Example 8 and Comparative Example 8 were detected, and the results are shown in Table 6.

[0079] Table 6. Hypoglycemic effects of sheep milk-derived hypoglycemic active peptides before and after the Plastein reaction.

[0080] Table 6 shows that the α-amylase inhibitory activity of the sheep milk-derived hypoglycemic active peptide prepared in Example 8 reached 54.25%, which was 20.78% higher than that of the peptide in Comparative Example 8 that had not undergone the Plastein reaction; the α-glucosidase inhibitory activity of Example 8 reached 33.87%, which was 16.30% higher than that of the peptide in Comparative Example 8. It is evident that the peptides subjected to the Plastein reaction significantly improved the hypoglycemic effect.

[0081] Example 9 The procedure was carried out in accordance with Example 6, except that 2% of the weight of the sheep milk was added with the blood sugar-lowering active peptide prepared in Example 8.

[0082] Comparative Example 9 The experiment was conducted in accordance with Example 9, using the same batch of experimental raw materials, except that no blood sugar-lowering active peptides derived from sheep milk were added during fermentation.

[0083] Experimental Example 7 Using the method of Experimental Example 2, the α-glucosidase inhibitory activity and DPP-IV inhibitory activity of Example 9 and Comparative Example 9 were detected, and the results are shown in [Figure 2]. Figure 4 , 5 See Table 7.

[0084] Table 7. Inhibitory activity of fermented goat milk before and after the addition of hypoglycemic active peptides

[0085] Depend on Figure 4 , 5 As shown in Table 7, the fermented goat milk of Example 9 exhibited significantly higher α-glucosidase inhibitory activity and DPP-IV inhibitory activity than the fermented goat milk of Comparative Example 9 without the addition of goat milk-derived hypoglycemic active peptides before and after simulated gastrointestinal digestion. Furthermore, these hypoglycemic active peptides demonstrated good resistance to gastrointestinal digestion. Therefore, the fermented milk product prepared using this invention possesses good hypoglycemic ability.

[0086] Example 10 Step 1: Prepare a 5% (w / w) aqueous solution of commercially available yak milk casein powder and sterilize it at 90℃ for 10 minutes. Add 4×10 [units of chemical reagent] per gram of casein aqueous solution. 7 CFU (Lactobacillus helveticus) Lactobacillus helveticus CICC6024 and Lactobacillus plantarum ( Lactobacillus plantarumThe compound lactic acid bacteria of CICC 20263, Lactobacillus helveticus: Lactobacillus plantarum = 3:1, were stirred evenly and fermented at 42℃ to the pH 5.0 to prepare yak milk casein fermentation broth.

[0087] Step two: Stir the fermentation broth thoroughly, heat at 90℃ for 10 minutes, and then adjust the pH of the fermentation broth to 7.5 with alkali solution. Add leucine aminopeptidase at a weight ratio of fermentation broth:leucine aminopeptidase = 1000:1, with leucine aminopeptidase activity of 10,000 U / g protein. Incubate at 37℃ for 4 hours, then heat at 85℃ for 10 minutes to inactivate the enzyme, obtaining yak milk casein hydrolysate.

[0088] Comparative Example 10 The experiment was conducted in accordance with the method of Example 10, using the same batch of experimental raw materials. The difference was that after the yak milk casein fermentation broth was prepared, the hydrolysis process in step two was not carried out.

[0089] Experimental Example 8 The protein hydrolysis activity and peptide content of Example 10 and Comparative Example 10 were detected using the method of Experimental Example 1, and the results are shown in Table 8.

[0090] Table 8. Protein hydrolysis activity and peptide content of yak milk casein hydrolysate after aminopeptidase hydrolysis.

[0091] As shown in Table 8, the yak milk casein fermentation broth of Example 10, after hydrolysis with leucine aminopeptidase, exhibited significantly higher protein hydrolysis activity and peptide content than the unhydrolyzed yak milk casein fermentation broth of Comparative Example 10. This demonstrates that utilizing the compound lactic acid bacteria fermentation technology of this invention combined with aminopeptidase hydrolysis of casein yields a casein hydrolysate with a higher degree of protein hydrolysis and a higher peptide content compared to using only the compound lactic acid bacteria fermentation technology.

[0092] Example 11 The casein hydrolysate obtained in Example 10 was concentrated under vacuum to a solids content of 55%. The pH of the yak milk casein hydrolysate was adjusted to 7.0. Neutral protease was added at a weight ratio of 3000:1 (casein hydrolysate concentrate: neutral protease), with a neutral protease activity of 100,000 U / g. Alanine was then added at 2% of the weight of the casein hydrolysate concentrate, and the Plastein reaction was carried out at 40°C for 6.5 h. The Plastein reaction solution was then freeze-dried under vacuum to obtain yak milk-derived hypoglycemic active peptides.

[0093] Comparative Example 11 The experiment was conducted in accordance with Example 11, using the same batch of experimental materials, except that the addition of alanine for the Plastein reaction was omitted.

[0094] Experimental Example 9 Using the method of Experimental Example 2, the α-amylase inhibitory activity and α-glucosidase inhibitory activity of Example 11 and Comparative Example 11 were detected, and the results are shown in Table 9.

[0095] Table 9. Hypoglycemic effects of yak milk-derived hypoglycemic active peptides before and after the Plastein reaction.

[0096] Table 9 shows that the yak milk-derived hypoglycemic active peptide prepared in Example 11 exhibited an α-amylase inhibitory activity of 67.88%, which was 25.19% higher than that of the peptide in Comparative Example 11 that had not undergone the Plastein reaction; the α-glucosidase inhibitory activity of Example 11 reached 40.08%, which was 19.61% higher than that of the peptide in Comparative Example 11. It is evident that the peptides subjected to the Plastein reaction significantly improved the hypoglycemic effect.

[0097] Example 12 The procedure was carried out in accordance with Example 6, except that 3% of the weight of yak milk was added as a yak milk-derived hypoglycemic active peptide.

[0098] Comparative Example 12 The experiment was conducted in the same manner as in Example 12, using the same batch of experimental raw materials, except that no yak milk-derived hypoglycemic active peptides were added during fermentation.

[0099] Experimental Example 10 Using the method of Experimental Example 2, the α-glucosidase inhibitory activity and DPP-IV inhibitory activity of Example 12 and Comparative Example 12 were detected, and the results are shown in [Figure 1]. Figure 6 , 7 See Table 10.

[0100] Table 10. Inhibitory activity of fermented yak milk before and after the addition of hypoglycemic active peptides.

[0101] Depend on Figure 6 , 7 As shown in Table 10, the fermented yak milk of Example 12 exhibited significantly higher α-glucosidase inhibitory activity and DPP-IV inhibitory activity than the fermented yak milk of Comparative Example 12 without the addition of yak milk-derived hypoglycemic active peptides before and after simulated gastrointestinal digestion. Furthermore, these hypoglycemic active peptides demonstrated good resistance to gastrointestinal digestion. Therefore, the fermented milk product prepared using this invention possesses good hypoglycemic ability.

[0102] Therefore, this invention utilizes specific proteases and peptidases produced by *Lactobacillus helveticus* and *Lactobacillus plantarum* to hydrolyze casein into specific peptides. Further, leucine aminopeptidase is used to release the N-terminal leucine residues from these peptides, enriching them to obtain more target peptides. Then, through Plastein reaction modification, amino acids are synthesized into new peptide chains to obtain hypoglycemic peptides with higher activity. The fermented milk product prepared using this invention exhibits good hypoglycemic ability.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a hypoglycemic active peptide, characterized in that, Includes the following steps: Casein was prepared into an aqueous solution with water and then mixed with a compound lactic acid bacteria for fermentation to obtain a casein fermentation broth. The casein fermentation broth was then mixed with leucine aminopeptidase for enzymatic hydrolysis to obtain a casein hydrolysate. The casein hydrolysate was subjected to a Plastein reaction to obtain hypoglycemic active peptides. The compound lactic acid bacteria include: Lactobacillus helveticus and Lactobacillus plantarum; the colony count ratio of Lactobacillus helveticus and Lactobacillus plantarum is 1~6:1; the fermentation temperature is 30~50℃, and the casein aqueous solution is fermented to a pH of 4.0~6.0; The Plastein reaction further includes concentrating the casein hydrolysate to obtain a casein hydrolysate concentrate, and sequentially adding a neutral protease and amino acids to the casein hydrolysate concentrate. The amount of amino acids added is 1.0% to 4.5%, and the amino acids are one or more of alanine, lysine, proline, and glycine.

2. The preparation method according to claim 1, characterized in that, The weight ratio of casein fermentation broth to leucine aminopeptidase is 500~1500:

1.

3. The preparation method according to claim 1 or 2, characterized in that, The enzymatic hydrolysis was performed at a pH of 6.0–8.0, a temperature of 30–45°C, and a time of 2–4 hours.

4. The preparation method according to claim 1, characterized in that, When concentrating the casein hydrolysate, the solid content of the concentrated solution is 40% to 60%.

5. The preparation method according to claim 1 or 4, characterized in that, The Plastein reaction was carried out at a pH of 6.5–8.0, a temperature of 30–50 °C, and a time of 2–8 h.

6. A hypoglycemic active peptide prepared by the preparation method according to any one of claims 1 to 5.

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