Preparation method of high-F-value protein oligopeptide

Abstract

The invention discloses a preparation method of high-F-value protein oligopeptide, and belongs to the technical field of special food processing. Alkaline protease, PepS enzyme and malic acid acidolysis are adopted to construct a three-stage synergistic reaction system, and meanwhile, beta-cyclodextrin, phenylethylamine and chitosan are introduced as composite adsorbents to synergistically enhance the selective adsorption capacity to a target product, so that the loss of branched chain amino acid (BCAA) in the separation and purification process is effectively prevented. The prepared protein oligopeptide has excellent nutritional indexes, the Fisher ratio of the protein oligopeptide is greater than 36, and the protein oligopeptide shows good protein quality and bioavailability. Besides, the final product is uniform in particle size distribution, active ingredients are completely reserved, and freeze drying and sieving process treatment are combined, so that the physical stability and storage performance of the product are greatly improved, the damage of a high-temperature link to the active ingredients is effectively avoided, and the nutritional value and physiological function characteristics of the protein oligopeptide are completely reserved.

Description

Technical Field

[0001] This invention relates to a method for preparing high-F-value protein oligopeptides, belonging to the field of special food processing technology. Background Technology

[0002] High-F-value oligopeptides (branched-chain amino acid BCAA / aromatic amino acid AAA > 20, composed of 3-9 amino acid residues) are a key protein source for people with aromatic amino acid metabolism disorders (such as phenylketonuria PKU, patients with liver and kidney dysfunction). They can not only provide essential amino acids to meet growth needs, but also alleviate hepatic encephalopathy by competing with BCAA for the blood-brain barrier transport system, control blood phenylalanine levels to assist in the treatment of PKU, and reduce tryptophan entering the brain during exercise to achieve anti-fatigue effects. They are of great significance for improving the nutritional status of special populations.

[0003] Currently, research on high-F-value oligopeptides primarily focuses on plant-based proteins such as rice protein, corn protein, and soy protein, while exploration of mammalian proteins, which have higher nutrient density and better absorption and utilization rates by the human body, remains relatively scarce. Among these, whey protein isolate (WPI) possesses both excellent nutritional properties (such as aiding in lowering blood pressure, improving blood lipids, promoting probiotic proliferation, and regulating insulin secretion) and outstanding functional value, making it an ideal material for the development of functional foods in the food industry. Casein, as a high-quality source of mammalian protein, not only has extremely high nutritional value but also has wide applications in the food industry. It is rich in various essential amino acids required by the human body, has high bioavailability, and can effectively promote muscle synthesis and maintain bodily health.

[0004] In the preparation process of high-F-value oligopeptides, the sufficient exposure and efficient removal of aromatic amino acids (AAA, including phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp)) are the core key steps. Currently, the industry mostly uses the combined hydrolysis of proteins with alkaline proteases and flavor enzymes to release aromatic amino acids. Although this method is simple to operate, it has the limitation of a single system. In the adsorption stage, activated carbon, single β-cyclodextrin, and synthetic adsorbents are widely used, achieving certain results due to their advantages of low cost and convenient operation. However, these methods still have the following problems in the field of aromatic amino acid exposure and adsorption: (1) Insufficient exposure of aromatic amino acids. Existing hydrolysis methods can only achieve secondary exposure of aromatic amino acids, which is far from maximizing the exposure effect. At the same time, flavor enzymes do not have the specific recognition function of aromatic amino acids and cannot directionally hydrolyze peptide chains containing aromatic amino acids, thus limiting the subsequent adsorption efficiency. For example, CN120966939A discloses a method for preparing high F-value oligopeptides based on whey protein, which uses alkaline protease, chymotrypsin and flavor protease for enzymatic hydrolysis, followed by dearomatization with activated carbon, and the F value of the hydrolysate is 26.46; (2) Activated carbon has poor adsorption specificity and easily adsorbs essential branched-chain amino acids simultaneously. This will lead to a decrease in the BCAA content in the final oligopeptide product, resulting in the loss of essential amino acids and failing to provide sufficient nutrition for special populations such as phenylketonuria (PKU) and liver and kidney dysfunction, thus disrupting the amino acid composition balance of the product. For example, in CN108517004A, activated carbon chromatography was used to obtain cuttlefish high F-value oligopeptides with an F value of 26.31. (3) The adsorption mechanism of a single β-cyclodextrin is singular, with no synergistic effect and limited adsorption performance. A single β-cyclodextrin relies solely on hydrophobic cavity inclusion, resulting in low adsorption capacity and poor binding stability; (4) Synthetic adsorbents, such as polystyrene-based adsorbent resins, have complex chemical structures and poor biocompatibility, making them unsuitable for use in human-related food and medical fields and only suitable for industrial separation. Some natural adsorbent materials have weak adsorption capacity and no biological activity, and even after adsorption, they cannot improve the functional properties of the products (such as desensitization and hypoglycemia), making it difficult to meet the needs of special populations.

[0005] In summary, there is an urgent need to develop a specific aromatic amino acid (AAA) removal technology that maximizes the exposure and efficiency of aromatic amino acids while incorporating multiple synergistic mechanisms, ultimately achieving the precise preparation of high-F-value protein oligopeptides. This technology can effectively fill the gap in the field of foods for special medical purposes, providing a safe, suitable, and highly nutritious solution for individuals facing aromatic amino acid metabolic disorders. Summary of the Invention

[0006] [Technical Issues] In existing technologies, the preparation of high F-value oligopeptides suffers from poor adsorption material specificity, a single mechanism, nutrient loss, and insufficient biocompatibility.

[0007] [Technical Solution] This invention provides a high Fischer ratio (Fr) oligopeptide preparation technology, specifically achieved through the following core process: First, a combined enzymatic and acidic hydrolysis treatment maximizes the exposure of aromatic amino acids (AAA) in the target protein. Then, the unique "triple synergistic adsorption mechanism" of the chitosan-phenylethylamine-β-cyclodextrin composite material is utilized to target and adsorb AAA. Combined with the construction of porous structures from natural raw materials and precise process control, protein oligopeptides with a Fröhl ratio > 36 are finally obtained. The method proposed in this invention is not only applicable to animal protein systems but also performs excellently in plant protein systems, stably and efficiently preparing high Fröhl ratio oligopeptide products, demonstrating outstanding versatility and wide applicability.

[0008] The first objective of this invention is to provide a method for preparing high-F-value protein oligopeptides, comprising the following steps: (1) Enzymatic hydrolysis: Using protein as a substrate, alkaline protease and peptidase are used sequentially to hydrolyze the protein to obtain protein hydrolysate; (2) Acid hydrolysis: Malic acid is added to the protein hydrolysate in step (1) and acid hydrolysis is performed to obtain the acid-hydrolyzed product; (3) Adsorption: The composite material is added to the hydrolysate from step (2) after acid hydrolysis, adsorption occurs, solid-liquid separation occurs, and the liquid phase is collected; (4) Drying: The liquid phase in step (3) is dried to obtain high F-value oligopeptides.

[0009] In one embodiment of the present invention, in step (1), the protein is dissolved in water at a mass-volume ratio of (10~20) g: 200 mL to obtain a protein solution.

[0010] In one embodiment of the present invention, in step (1), the amount of alkaline protease added is 400~1000 U / g, and the amount of peptidase added is 400~1000 U / g.

[0011] In one embodiment of the present invention, the peptidase includes aminopeptidase.

[0012] Preferably, the aminopeptidase is aminopeptidase S (PepS) derived from Streptococcus thermophilus.

[0013] In one embodiment of the present invention, in step (1), alkaline protease is hydrolyzed at pH 9.0~11.0 and temperature 50~60℃ for 60~150 min; peptidase is hydrolyzed at pH 7.0~9.0 and temperature 50~60℃ for 60~150 min.

[0014] In one embodiment of the present invention, in step (2), the amount of malic acid added is 5 to 8% of the volume of the protein hydrolysate.

[0015] In one embodiment of the present invention, in step (2), the acid is stirred at 200-240 rpm for 30-60 min at room temperature.

[0016] In one embodiment of the present invention, in step (3), the solid-liquid ratio of the composite material and the acid-hydrolyzed product is (0.5~1.5) g: 100 mL.

[0017] In one embodiment of the present invention, in step (3), the adsorption conditions are: adsorption by shaking at 20~30℃ and 150~200 rpm for 1~2 h.

[0018] In one embodiment of the present invention, in step (3), the method for preparing the composite material is as follows: (a) Chitosan was dissolved in an acidic solution, and then phenylethylamine, β-cyclodextrin and a crosslinking agent were added to carry out a preliminary crosslinking reaction; (b) Add a pore-forming agent, an auxiliary crosslinking agent, an emulsifier, and an oil phase to the reaction solution to carry out a secondary crosslinking reaction; (c) The reaction product is washed with ethanol, acid-soaked, and dried to obtain the composite material.

[0019] In one embodiment of the present invention, the protein includes plant protein or animal protein.

[0020] Preferably, the plant protein includes soybean protein, rice protein, corn protein, and pea protein.

[0021] Preferably, the animal protein includes whey protein, casein, ovalbumin, and fish protein.

[0022] In one embodiment of the present invention, in step (a), the feeding ratio of chitosan, phenylethylamine and β-cyclodextrin is (0.4~1 g): (1~3 mL): (1~2 g).

[0023] In one embodiment of the present invention, in step (a), the crosslinking agent is selected from glutaraldehyde and epichlorohydrin.

[0024] In one embodiment of the present invention, in step (a), the acidic solution is selected from hydrochloric acid solution and sulfuric acid solution.

[0025] In one embodiment of the present invention, in step (a), a preliminary crosslinking reaction is carried out at pH 3.0, 37°C, and 240 rpm.

[0026] In one embodiment of the present invention, in step (b), the pore-forming agent is selected from one or more of calcium carbonate, sodium carbonate, or sodium bicarbonate; the auxiliary crosslinking agent is selected from ethylene glycol diglycidyl ether; the emulsifier is selected from Tween-80 or Span-80; and the oil phase is selected from liquid paraffin.

[0027] In one embodiment of the present invention, in step (b), a secondary crosslinking reaction is carried out at a pH of 8.8 to 9.2, a temperature of 55 to 65°C, and a speed of 150 to 200 rpm.

[0028] A second objective of this invention is to provide a high-F-value protein oligopeptide prepared by the method described above.

[0029] A third objective of this invention is to provide a method for reducing aromatic amino acids in proteins, the method comprising: (1) Enzymatic hydrolysis: Using protein as a substrate, alkaline protease and peptidase are used sequentially to hydrolyze the protein to obtain protein hydrolysate; (2) Acid hydrolysis: Malic acid is added to the protein hydrolysate in step (1) and acid hydrolysis is performed to obtain the acid-hydrolyzed product; (3) Adsorption: The composite material is added to the hydrolysate from step (2) after acid hydrolysis, adsorption occurs, solid-liquid separation occurs, and the liquid phase is collected; (4) Drying: The liquid phase in step (3) is dried to obtain high F-value oligopeptides.

[0030] In one embodiment of the present invention, in step (1), the protein is dissolved in water at a mass-volume ratio of (10~20) g: 200 mL to obtain a protein solution.

[0031] In one embodiment of the present invention, in step (1), the amount of alkaline protease added is 400~1000 U / g, and the amount of peptidase added is 400~1000 U / g.

[0032] In one embodiment of the present invention, in step (1), alkaline protease is hydrolyzed at pH 9.0~11.0 and temperature 50~60℃ for 60~150 min; peptidase is hydrolyzed at pH 7.0~9.0 and temperature 50~60℃ for 60~150 min.

[0033] In one embodiment of the present invention, in step (2), the amount of malic acid added is 5 to 8% of the volume of the protein hydrolysate.

[0034] In one embodiment of the present invention, in step (2), the acid is stirred at 200-240 rpm for 30-60 min at room temperature.

[0035] In one embodiment of the present invention, in step (3), the solid-liquid ratio of the composite material and the acid-hydrolyzed product is (0.5~1.5) g: 100 mL.

[0036] In one embodiment of the present invention, in step (3), the adsorption conditions are: adsorption by shaking at 20~30℃ and 150~200 rpm for 1~2 h.

[0037] In one embodiment of the present invention, in step (3), the method for preparing the composite material is as follows: (a) Chitosan was dissolved in an acidic solution, and then phenylethylamine, β-cyclodextrin and a crosslinking agent were added to carry out a preliminary crosslinking reaction; (b) Add a pore-forming agent, an auxiliary crosslinking agent, an emulsifier, and an oil phase to the reaction solution to carry out a secondary crosslinking reaction; (c) The reaction product is washed with ethanol, acid-soaked, and dried to obtain the composite material.

[0038] In one embodiment of the present invention, the protein includes plant protein or animal protein.

[0039] Preferably, the plant protein includes soybean protein, rice protein, corn protein, and pea protein.

[0040] Preferably, the animal protein includes whey protein, casein, ovalbumin, and fish protein.

[0041] In one embodiment of the present invention, in step (a), the feeding ratio of chitosan, phenylethylamine and β-cyclodextrin is (0.4~1 g): (1~3 mL): (1~2 g).

[0042] In one embodiment of the present invention, in step (a), the crosslinking agent is selected from glutaraldehyde and epichlorohydrin.

[0043] In one embodiment of the present invention, in step (a), the acidic solution is selected from hydrochloric acid solution and sulfuric acid solution.

[0044] In one embodiment of the present invention, in step (a), a preliminary crosslinking reaction is carried out at pH 3.0, 37°C, and 240 rpm.

[0045] In one embodiment of the present invention, in step (b), the pore-forming agent is selected from one or more of calcium carbonate, sodium carbonate, or sodium bicarbonate; the auxiliary crosslinking agent is selected from ethylene glycol diglycidyl ether; the emulsifier is selected from Tween-80 or Span-80; and the oil phase is selected from liquid paraffin.

[0046] In one embodiment of the present invention, in step (b), a secondary crosslinking reaction is carried out under the conditions of pH 8.8~9.2, 55~65°C, and 150~200 rpm.

[0047] A fourth objective of this invention is to provide the application of the high-F-value protein oligopeptide in the preparation of food.

[0048] Preferably, the food includes health products, functional foods, nutritional supplements, or special dietary foods.

[0049] Preferably, the food has functions such as anti-oxidation, relieving physical fatigue, regulating intestinal flora, or assisting in the protection against chemical liver damage.

[0050] A fifth objective of this invention is to provide the application of the high-F-value protein oligopeptide in the preparation of pharmaceuticals.

[0051] Preferably, the high-F-value protein oligopeptide is used in the preparation of a medicament for the treatment or adjunctive treatment of phenylketonuria.

[0052] Preferably, the high-F-value protein oligopeptide is used in the preparation of a medicament for the treatment or adjuvant treatment of hepatic encephalopathy.

[0053] [Beneficial Effects] This invention first uses a dual-enzymatic digestion and acid-assisted approach to induce changes in protein structure, maximizing the exposure of aromatic amino acids. With the aid of a triple-synergistic adsorption material, aromatic amino acids are precisely identified, their content is reduced, and high-F-value oligopeptides are prepared.

[0054] This invention utilizes a three-tiered synergistic system of non-directional hydrolysis by alkaline protease, specific directional hydrolysis by aminopeptidase PepS, and acid hydrolysis by malic acid to gradually break down the spatial conformation and secondary bond interactions of proteins. This effectively increases the exposure of aromatic amino acid residues embedded within proteins, enabling the directional release of small molecule aromatic amino acid peptides. This provides sufficient target sites for subsequent specific adsorption, overcoming the technical limitation of traditional hydrolysis that only allows for secondary exposure.

[0055] This invention utilizes a "triple synergistic adsorption mechanism" of the CD-CS-PEA composite material to achieve targeted adsorption of aromatic amino acids. β-cyclodextrin, phenylethylamine, and chitosan synergistically enhance the adsorption selectivity for aromatic amino acids. The adsorption capacity for AAA is significantly higher than that for non-aromatic amino acids (especially branched-chain amino acids BCAA), effectively avoiding the BCAA loss caused by the "broad-spectrum adsorption" of traditional adsorbent materials (such as activated carbon), ensuring a balanced amino acid ratio in the product, and ultimately achieving a Fisher ratio (BCAA / AAA) > 36 for the protein oligopeptide. This result can be fully achieved in both plant and animal proteins, demonstrating its broad applicability and high efficiency across various protein sources.

[0056] This invention uses natural biomass such as chitosan and β-cyclodextrin as raw materials, combined with calcium carbonate pore-forming and freeze-drying processes, to construct a stable hierarchical porous structure. The material not only exhibits excellent biocompatibility and environmental degradability, making it suitable for the food and specialized medical industries, but also possesses strong mechanical strength and abundant adsorption active sites, solving the problems of poor biocompatibility in traditional synthetic adsorbents and weak adsorption in natural materials.

[0057] This invention yields a product with uniform particle size and intact active ingredients. Freeze-drying and sieving processes prevent product aggregation and activity loss, improving storage stability. The entire preparation process avoids high-temperature degradation, balancing the nutritional characteristics of the protein itself with the physiological functions of the oligopeptides, making it suitable for the specific nutritional needs of individuals with aromatic amino acid metabolism disorders. Attached Figure Description

[0058] Figure 1 This is a comparison chart of the absorbance values ​​of aromatic amino acids at 280 nm in Example 1 and Comparative Examples 1-10 of the present invention.

[0059] Figure 2 This is a comparison chart of the adsorption rates of the composite materials of Example 1 and Comparative Examples 1-10 for phenylalanine, an aromatic amino acid.

[0060] Figure 3 This is a comparison chart of the Fisher ratios of the composite materials of Example 1 and Comparative Examples 1-10 after adsorption of aromatic amino acids. Detailed Implementation

[0061] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0062] Materials involved in the following embodiments: Whey protein isolate: CAS: 84082 51 9; Casein: CAS: 9000-71-9; Soy protein: CAS: 9010-10-0; Ovalbumin: CAS: 9006-59-1; Chitosan: CAS: 9012-76-4; Phenylethylamine: CAS: 64-04-0; β-Cyclodextrin: CAS: 7585-39-9; Malic acid: CAS: 6915-15-7; Alkaline protease: purchased from Shanghai Yuanye Company; Pure enzyme PepS: Commercially available Streptococcus thermophilus (PepS) Streptococcus thermophilus Published in Proteolytic System of Streptococcus thermophilus (As described in the article) The cells were cultured to the logarithmic growth phase, collected by centrifugation, and washed twice. The cells were then sonicated, centrifuged again, and the supernatant containing the peptidase was collected. The collected supernatant was purified by ion exchange chromatography and hydrophobic interaction chromatography to obtain the pure enzyme PepS.

[0063] The detection methods involved in the following embodiments: (1) Absorbance of total aromatic amino acids at 280 nm: Accurately transfer an appropriate amount of the supernatant after the adsorption reaction and perform a 100-fold serial dilution with deionized water (ensuring the absorbance of the diluted solution falls within the instrument's optimal response range of 0.2–0.8). Using deionized water as a blank control, set the wavelength of the UV spectrophotometer to 280 nm. After the instrument baseline stabilizes, measure the absorbance of the diluted sample solution three times in parallel, record the data, and calculate the average value, denoted as A. 280 .

[0064] (2) Determination of phenylalanine content: Accurately weigh 100.0 mg of dried L-phenylalanine standard to constant weight, place it in a 100 mL volumetric flask, add deionized water, sonicate to dissolve, and dilute to the mark. Shake well to prepare a solution with a concentration of 1.00 mg. mL -1 The standard stock solution was prepared by accurately transferring 0.025 mL, 0.05 mL, 0.10 mL, 0.20 mL, 0.30 mL, and 0.40 mL of the above stock solution into 10 mL volumetric flasks, respectively. The flasks were then diluted to the mark with deionized water and mixed well to obtain standard stock solutions with concentrations of 0.0025 mg. mL -1 0.005 mg mL -1 0.01 mg mL -1 0.02 mg mL -1 0.03 mg mL -1 0.04 mg mL -1 A series of standard working solutions.

[0065] After adjusting the wavelength of the UV spectrophotometer to 257 nm and calibrating the instrument, the absorbance values ​​of the standard working solutions at various concentrations were measured sequentially.

[0066] Using phenylalanine concentration (c, unit: mg) mL -1 Using the x-axis as the abscissa and the corresponding absorbance value (A) as the ordinate, a standard curve was calculated. The supernatant after adsorption was diluted with deionized water to the range characteristic of the standard curve (denoted as a dilution factor of 100 times), and the absorbance value (A) of the diluted sample was measured in parallel at a wavelength of 257 nm. Substituting sample A into the standard curve regression equation, the concentration of phenylalanine in the diluted sample (C0, unit: mg) was calculated. mL -1 The adsorption capacity and removal rate of the adsorbent for phenylalanine in the original solution are calculated using the following formula:

[0067] In the formula: q e The adsorption capacity per unit mass of adsorbent (mg / g); C e C0 represents the initial concentration of phenylalanine in the solution before the adsorption reaction (unit: mg / mL); C0 represents the concentration of phenylalanine in the supernatant after adsorption (unit: mg / mL); V is the solution volume (mL); and m is the mass of the adsorbent (g).

[0068] (3) Determination of amino acid composition and F value: Add 4 mL of the test sample to 10 mL of 6 mol / L hydrochloric acid, seal, and heat at 120°C for 22 h. After cooling, filter through Whatman No. 4 paper and collect the filtrate. Dilute the filtrate to 4 mL with ultrapure water and filter through a 1 mL membrane (Millipore® 0.45 μm regenerated cellulose membrane). Then, determine the filtrate using a high-performance liquid chromatography system (HPLC, Agilent 1260, USA). The F value is calculated based on the molar ratio of BCAAs to AAAs.

[0069] (4) Calculation of degree of hydrolysis: The degree of hydrolysis (DH) of the hydrolysate was determined by the o-o-phthalaldehyde (OPA) method. The hydrolysate (400 μL) was mixed with 3 mL of OPA reagent and incubated at room temperature for 2 min. The absorbance of the mixture was then measured using a UV-Vis spectrophotometer at a wavelength of 340 nm. The degree of hydrolysis (DH%) of the hydrolysate was calculated using the following formula:

[0070] In the formula: B represents the volume of sodium hydroxide solution consumed during the reaction, i.e., mL; N represents the concentration of sodium hydroxide solution M; α represents the average degree of dissociation of α-NH; pK represents the dissociation constant of the amino group of the peptide bond; pH is 8; T represents the hydrolysis temperature in Kelvin units; m is the mass of the substrate used, g; h_hour is the total content of peptide bonds in the protein sample. h tot It was 8.1 mmol / g.

[0071] Example 1: Preparation of high-Fischer ratio whey protein oligopeptides 1. Preparation of composite materials, (1) Dissolve 1 g of chitosan in 100 mL of hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 3 mL of phenylethylamine, 2 g of β-cyclodextrin solution that has been dissolved in 50 mL of ethanol (30%) and 10 mL of 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 37 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0072] 2. Hydrolysis (1) Dissolve 15 g of whey protein isolate in 200 mL of distilled water and stir until completely dissolved to obtain whey protein isolate solution; (2) Add 1000 U / g alkaline protease to the whey protein separation solution from step (1), adjust the pH to 10.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant. (3) Add 1000 U / g of pure enzyme PepS to the supernatant of step (2), adjust the pH to 8.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, heat in a boiling water bath for 10 min to inactivate the enzyme, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 8% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 30 min at room temperature to obtain the acid-hydrolysate.

[0073] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 1 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 2 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0074] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain the high-F-value whey protein oligopeptide.

[0075] Example 2: Preparation of high-Fischer ratio whey protein oligopeptides 1. Preparation of composite materials, (1) Dissolve 0.8 g of chitosan in 100 mL of hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 2 mL of phenylethylamine, 1.5 g of β-cyclodextrin solution that has been dissolved in 50 mL of ethanol (30%) and 8 mL of 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 30 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0076] 2. Hydrolysis (1) Dissolve 15 g of whey protein isolate in 200 mL of distilled water and stir until completely dissolved to obtain whey protein isolate solution; (2) Add 800 U / g alkaline protease to the whey protein separation solution from step (1), adjust the pH to 10.0, and hydrolyze for 90 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant; (3) Add 800 U / g of pure enzyme PepS to the supernatant of step (2), adjust the pH to 8.0, and hydrolyze for 90 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 7% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 40 min at room temperature to obtain the acid-hydrolysate.

[0077] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 2 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 1 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0078] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain the high-F-value whey protein oligopeptide.

[0079] Example 3: Preparation of high-Fischer ratio whey protein oligopeptides 1. Preparation of composite materials, (1) Dissolve 0.6 g of chitosan in 100 mL of hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 1.5 mL of phenylethylamine, 1.2 g of β-cyclodextrin solution that has been dissolved in 50 mL of ethanol (30%) and 6 mL of 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 35 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0080] 2. Hydrolysis (1) Dissolve 15 g of whey protein isolate in 200 mL of distilled water and stir until completely dissolved to obtain whey protein isolate solution; (2) Add 600 U / g alkaline protease to the whey protein separation solution from step (1), adjust the pH to 10.0, and hydrolyze for 120 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant. (3) Add 600 U / g of pure enzyme PepS to the supernatant from step (2), adjust the pH to 8.0, and hydrolyze for 120 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 6% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 50 min at room temperature to obtain the acid-hydrolysate.

[0081] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 0.5 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 2 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0082] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain the high-F-value whey protein oligopeptide.

[0083] Example 4: Preparation of high-Fischer ratio whey protein oligopeptides 1. Preparation of composite materials, (1) Dissolve 0.4 g chitosan in 100 mL hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 1 mL phenylethylamine, 1 g β-cyclodextrin solution dissolved in 50 mL ethanol (30%) and 4 mL 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 40 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0084] 2. Hydrolysis (1) Dissolve 15 g of whey protein isolate in 200 mL of distilled water and stir until completely dissolved to obtain whey protein isolate solution; (2) Add 400 U / g alkaline protease to the whey protein separation solution from step (1), adjust the pH to 10.0, and hydrolyze for 150 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant; (3) Add 400 U / g of pure enzyme PepS to the supernatant from step (2), adjust the pH to 8.0, and hydrolyze for 150 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 5% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 60 min at room temperature to obtain the acid-hydrolysate.

[0085] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 1 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 1 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0086] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain the high-F-value whey protein oligopeptide.

[0087] Example 5: Preparation of high-Fischer ratio whey protein oligopeptides 1. Preparation of composite materials, (1) Dissolve 1 g of chitosan in 100 mL of hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 3 mL of phenylethylamine, 2 g of β-cyclodextrin solution dissolved in 50 mL of ethanol (30%) and 10 mL of 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 45 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0088] 2. Hydrolysis (1) Dissolve 10 g of whey protein isolate in 200 mL of distilled water and stir until completely dissolved to obtain whey protein isolate solution; (2) Add 1000 U / g alkaline protease to the whey protein separation solution from step (1), adjust the pH to 10.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant. (3) Add 1000 U / g of pure enzyme PepS to the supernatant of step (2), adjust the pH to 8.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, heat in a boiling water bath for 10 min to inactivate the enzyme, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 9% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 30 min at room temperature to obtain the acid-hydrolysate.

[0089] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 1 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 2 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0090] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain the high-F-value whey protein oligopeptide.

[0091] Example 6 Preparation of high-Fischer ratio whey protein oligopeptides 1. Preparation of composite materials, (1) Dissolve 1 g of chitosan in 100 mL of hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 3 mL of phenylethylamine, 2 g of β-cyclodextrin solution that has been dissolved in 50 mL of ethanol (30%) and 10 mL of 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 50 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0092] 2. Hydrolysis (1) Dissolve 20 g of whey protein isolate in 200 mL of distilled water and stir until completely dissolved to obtain whey protein isolate solution; (2) Add 1000 U / g alkaline protease to the whey protein separation solution from step (1), adjust the pH to 10.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant. (3) Add 1000 U / g of pure enzyme PepS to the supernatant of step (2), adjust the pH to 8.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, heat in a boiling water bath for 10 min to inactivate the enzyme, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 10% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 30 min at room temperature to obtain the acid-hydrolysate.

[0093] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 1 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 2 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0094] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain the high-F-value whey protein oligopeptide.

[0095] Comparative Example 1 The specific implementation method is the same as in Example 1, except that the preparation of the composite material in step 1 is adjusted: (1) 1 g chitosan, 3 mL phenylethylamine, 4 g β-cyclodextrin; (2) 1 g chitosan, 3 mL phenylethylamine, 0 g β-cyclodextrin; (3) 0 g chitosan, 3 mL phenylethylamine, 2 g β-cyclodextrin; (4) 0 g chitosan, 0 mL phenylethylamine, 2 g β-cyclodextrin.

[0096] Comparative Example 2 The specific implementation method is the same as in Example 1, except that the hydrolysis of pure enzyme PepS is omitted in the hydrolysis step 2.

[0097] The results showed that, after quantitative calculation of the absorbance value of aromatic amino acids at 280 nm, the exposure of aromatic amino acids was found to be lower than that in Example 1. Comparative Example 3 The specific implementation method is the same as in Example 1, except that the alkaline protease and PepS in step 2 are replaced with alkaline protease, chymotrypsin, and flavor protease: (1) Dissolve 15 g of whey protein isolate in 200 mL of distilled water and stir until completely dissolved to obtain whey protein isolate solution; (2) Add 1000 U / g alkaline protease to the whey protein separation solution from step (1), adjust the pH to 10.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant. (3) Add 1000 U / g chymotrypsin to the supernatant from step (2), adjust the pH to 8.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge and collect the supernatant; (4) Add 1000 U / g of flavor protease to the supernatant of step (3), adjust the pH to 8.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, heat in a boiling water bath for 10 min to inactivate the enzyme, centrifuge and collect the supernatant to obtain the hydrolysate.

[0098] Comparative Example 4 The specific implementation method is the same as in Example 1, except that step 3 is omitted and acid hydrolysis is not performed.

[0099] The results showed that after quantitative calculation of the absorbance value of aromatic amino acids at 280 nm, the exposure of aromatic amino acids was found to be lower than that in Example 1.

[0100] Comparative Example 5 The specific implementation method is the same as in Example 1, except that the malic acid in step 3 is replaced with tartaric acid, oxalic acid, and succinic acid, respectively.

[0101] Comparative Example 6 The specific implementation method is the same as in Example 1, except that the amount of malic acid added in step 3 is adjusted to 1%, 2%, and 3%.

[0102] Comparative Example 7 The specific implementation method is the same as in Example 1, except that the adsorption conditions in step 4 are: adsorption is carried out at a dosage ratio of 1 g / 100 mL, with shaking at 40°C and 180 rpm for 1 h to obtain the supernatant after adsorption.

[0103] Comparative Example 8 The specific implementation method is the same as in Example 1, except that the composite material in step 1 is omitted and the composite material in step 4 is replaced with activated carbon: according to the carbon-liquid ratio of 1:10, powdered activated carbon is added to the hydrolysate after acid hydrolysis in step 3, and adsorption is carried out by shaking at 25°C and 180 rpm for 2 h.

[0104] Comparative Example 9 The specific implementation method is the same as in Example 1, except that ethylene glycol diglycidyl ether in step 1 is omitted.

[0105] Comparative Example 10 The specific implementation method is the same as in Example 1, except that the freeze-drying of the composite material in step 1 is replaced by hot air drying.

[0106] Comparative Example 11 The specific implementation method is the same as in Example 1, except that phenylethylamine in step 1 is omitted.

[0107] Table 1 Results of high F-value oligopeptides prepared in Examples 1-6 and Comparative Examples 1-11

[0108] From Table 1 and Figures 1-3 It can be seen that the high F-value whey protein oligopeptides prepared in Examples 1 to 6 all exhibited good overall performance in terms of F-value, absorbance at 280 nm, and degree of hydrolysis. Example 1 showed the highest F-value, reaching 40.2, while its absorbance at 280 nm decreased significantly after adsorption, indicating less exposure of aromatic amino acids and higher product purity. Although the F-values ​​of Examples 2 to 6 fluctuated slightly, they remained at a high level overall, and the degree of hydrolysis remained consistently above 22%.

[0109] In Comparative Example 1, by adjusting the preparation formula of the composite material, it was found that when chitosan and phenylethylamine were omitted, the F value of the product decreased significantly, especially in Comparative Examples 1-(3) and 1-(4), where the F value dropped to single digits, indicating that phenylethylamine and chitosan play a key role in the composite material. Comparative Example 2 omitted the pure enzyme PepS hydrolysis step, resulting in a reduction in the exposure of aromatic amino acids. In Comparative Example 3, by replacing the enzyme type, although the degree of hydrolysis did not change significantly, the F value decreased significantly, indicating that the combination of alkaline protease and PepS is more conducive to the preparation of high-F-value oligopeptides.

[0110] Comparative Example 4 omitted the acidolysis step, resulting in reduced exposure of aromatic amino acids and a lower F value, indicating that the acidolysis step is crucial for increasing aromatic amino acid exposure and improving product purity. Comparative Example 5, by changing the type of acid used in acidolysis, found that tartaric acid, oxalic acid, and succinic acid were all less effective than malic acid. Comparative Example 6 adjusted the amount of malic acid added, finding that with increasing addition, the F value increased slightly, but the increase was not significant, indicating that when the malic acid concentration is ≤3%, it affects the exposure of aromatic amino acids.

[0111] Comparative Example 7 adjusted the adsorption conditions and found that increasing the temperature and shortening the adsorption time led to a significant decrease in the F value. Comparative Example 8 replaced the composite material with activated carbon for adsorption and found that both the F value and the degree of hydrolysis decreased significantly.

[0112] Example 7 This embodiment provides the application of the high-F-value whey protein oligopeptide prepared in Example 1 in the preparation of products that improve gut microbiota, as detailed below: Table 2. A solid beverage that improves gut microbiota

[0113] Weigh the raw materials according to the proportions, and pass the high F-value whey protein oligopeptide, resistant dextrin, and erythritol prepared in Example 1 through an 80-mesh sieve. Premix the silica with a small amount of resistant dextrin for 5 min, and then add the remaining resistant dextrin, fructooligosaccharides, compound fruit and vegetable powder, as well as the sieved high F-value whey protein oligopeptide, resistant dextrin, and erythritol. Mix for 30 min to finally obtain a solid beverage.

[0114] Example 8 Preparation of high-Fischer ratio casein oligopeptides 1. Preparation of composite materials, (1) Dissolve 1 g of chitosan in 100 mL of hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 3 mL of phenylethylamine, 2 g of β-cyclodextrin solution that has been dissolved in 50 mL of ethanol (30%) and 10 mL of 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 37 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0115] 2. Hydrolysis, (1) Dissolve 15 g of casein in 200 mL of distilled water and stir until completely dissolved to obtain a casein solution; (2) Add 1000 U / g alkaline protease to the casein solution from step (1), adjust the pH to 10.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant. (3) Add 1000 U / g of pure enzyme PepS to the supernatant of step (2), adjust the pH to 8.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, heat in a boiling water bath for 10 min to inactivate the enzyme, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 8% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 30 min at room temperature to obtain the acid-hydrolysate.

[0116] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 1 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 2 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0117] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain casein oligopeptides with a Fischer ratio > 36.

[0118] Example 9 Preparation of high-Fischer ratio soybean protein oligopeptides 1. Preparation of composite materials, (1) Dissolve 1 g of chitosan in 100 mL of hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 3 mL of phenylethylamine, 2 g of β-cyclodextrin solution that has been dissolved in 50 mL of ethanol (30%) and 10 mL of 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 37 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0119] 2. Hydrolysis (1) Dissolve 15 g of soybean protein in 200 mL of distilled water and stir until completely dissolved to obtain a soybean protein solution; (2) Add 1000 U / g alkaline protease to the soybean protein solution from step (1), adjust the pH to 10.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant. (3) Add 1000 U / g of pure enzyme PepS to the supernatant of step (2), adjust the pH to 8.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, heat in a boiling water bath for 10 min to inactivate the enzyme, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 8% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 30 min at room temperature to obtain the acid-hydrolysate.

[0120] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 1 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 2 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0121] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain soybean protein oligopeptides with a Fischer ratio > 36.

[0122] Example 10 Preparation of high Firth-Ratio Ovalbumin Oligopeptides 1. Preparation of composite materials, (1) Dissolve 1 g of chitosan in 100 mL of hydrochloric acid solution (0.1 mol / L) to prepare a homogeneous chitosan solution; then add 3 mL of phenylethylamine, 2 g of β-cyclodextrin solution that has been dissolved in 50 mL of ethanol (30%) and 10 mL of 25% glutaraldehyde to the chitosan solution in sequence, and crosslink at 37 °C for 30 min to obtain a preliminarily crosslinked mixed solution; (2) Add 0.5 g calcium carbonate, 1 mL ethylene glycol diglycidyl ether, 0.5 mL Tween-80 and 300 mL liquid paraffin to the pre-crosslinked mixed solution, adjust the pH to 9.0, and crosslink at 60℃ and 180 rpm for 1 h; (3) After the crosslinking reaction was completed, the product was washed with ethanol to remove the oil phase, and the precipitate was collected by centrifugation. The precipitate was then soaked in 1 mol / L hydrochloric acid solution and stirred for 2 h until no bubbles were generated. Finally, it was washed with deionized water until neutral, vacuum filtered, and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain the composite material product.

[0123] 2. Hydrolysis, (1) Dissolve 15 g of ovalbumin in 200 mL of distilled water and stir until completely dissolved to obtain an ovalbumin solution; (2) Add 1000 U / g alkaline protease to the ovalbumin solution from step (1), adjust the pH to 10.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, inactivate the enzyme by heating in a boiling water bath for 10 min, centrifuge, and collect the supernatant. (3) Add 1000 U / g of pure enzyme PepS to the supernatant of step (2), adjust the pH to 8.0, and hydrolyze for 60 min at 55℃ and 180 rpm. Then, heat in a boiling water bath for 10 min to inactivate the enzyme, centrifuge and collect the supernatant to obtain the hydrolysate; 3. Acid hydrolysis, Add 8% malic acid (by volume of the hydrolysate solution) to the hydrolysate obtained in step 2, and stir at 240 rpm for 30 min at room temperature to obtain the acid-hydrolysate.

[0124] 4. Adsorption, The composite material prepared in step 1 was added to the acid-hydrolyzed product in step 3 at a solid-liquid ratio of 1 g / 100 mL, and the mixture was shaken and adsorbed at 30 °C and 180 rpm for 2 h. After adsorption, the mixture was filtered through a 0.22 μm mixed cellulose membrane, and the supernatant was collected.

[0125] 5. Product preparation, The supernatant from step 4 was freeze-dried at -50°C for 48 h and then sieved through a 30-mesh sieve to obtain ovalbumin oligopeptides with a Fischer ratio > 36.

[0126] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a high-F-value protein oligopeptide, characterized in that, Includes the following steps: (1) Enzymatic hydrolysis: Using protein as a substrate, alkaline protease and peptidase are used sequentially to hydrolyze the protein to obtain protein hydrolysate; (2) Acid hydrolysis: Malic acid is added to the protein hydrolysate in step (1) and acid hydrolysis is performed to obtain the acid-hydrolyzed product; (3) Adsorption: The composite material is added to the hydrolysate from step (2) after acid hydrolysis, adsorption occurs, solid-liquid separation occurs, and the liquid phase is collected; (4) Drying: The liquid phase from step (3) is dried to obtain high F-value oligopeptides; wherein, In step (3), the preparation method of the composite material is as follows: (a) Chitosan was dissolved in an acidic solution, and then phenylethylamine, β-cyclodextrin and a crosslinking agent were added to carry out a preliminary crosslinking reaction; (b) Add a pore-forming agent, an auxiliary crosslinking agent, an emulsifier, and an oil phase to the reaction solution to carry out a secondary crosslinking reaction; (c) The reaction product is washed with ethanol, acid-soaked, and dried to obtain the composite material.

2. The preparation method according to claim 1, characterized in that, In step (1), the protein includes plant protein or animal protein; the amount of alkaline protease added is 400~1000 U / g, and the amount of peptidase added is 400~1000 U / g; Preferably, the alkaline protease is hydrolyzed at pH 9.0~11.0 and temperature 50~60℃ for 60~150 min; the peptidase is hydrolyzed at pH 7.0~9.0 and temperature 50~60℃ for 60~150 min; preferably, the peptidase is aminopeptidase.

3. The preparation method according to claim 1, characterized in that, In step (2), the amount of malic acid added is 5-8% of the volume of the protein hydrolysate; Optionally, acid hydrolysis can be carried out at 200-240 rpm for 30-60 min at room temperature.

4. The preparation method according to claim 1, characterized in that, In step (3), the solid-liquid ratio of the composite material and the acid-hydrolyzed product is (0.5~1.5) g: 100 mL; the adsorption conditions are oscillation adsorption at 20~30℃ and 150~200 rpm for 1~2 h.

5. The preparation method according to claim 1, characterized in that, In step (a), the ratio of chitosan, phenylethylamine, and β-cyclodextrin is (0.4~1 g):(1~3 mL):(1~2 g); the initial cross-linking reaction is carried out at pH 3.0, 37℃, and 240 rpm; in step (b), the secondary cross-linking reaction is carried out at pH 8.8~9.2, 55~65℃, and 150~200 rpm.

6. The preparation method according to claim 1, characterized in that, In step (a), the crosslinking agent is selected from glutaraldehyde and epichlorohydrin; the acidic solution is selected from hydrochloric acid solution and sulfuric acid solution; in step (b), the pore-forming agent is selected from one or more of calcium carbonate, sodium carbonate or sodium bicarbonate; the auxiliary crosslinking agent is selected from ethylene glycol diglycidyl ether; the emulsifier is selected from Tween-80 and Span-80; and the oil phase is selected from liquid paraffin.

7. A high-F-value protein oligopeptide, characterized in that, Prepared by the method described in any one of claims 1 to 6.

8. A method for reducing aromatic amino acids in proteins, characterized in that, The method is as follows: (1) Enzymatic hydrolysis: Using protein as a substrate, alkaline protease and peptidase are used sequentially to hydrolyze the protein to obtain protein hydrolysate; (2) Acid hydrolysis: Malic acid is added to the protein hydrolysate in step (1) and acid hydrolysis is performed to obtain the acid-hydrolyzed product; (3) Adsorption: The composite material is added to the hydrolysate from step (2) after acid hydrolysis, adsorption occurs, solid-liquid separation occurs, and the liquid phase is collected; (4) Drying: The liquid phase from step (3) is dried to obtain high F-value oligopeptides; wherein, In step (3), the preparation method of the composite material is as follows: (a) Chitosan was dissolved in an acidic solution, and then phenylethylamine, β-cyclodextrin and a crosslinking agent were added to carry out a preliminary crosslinking reaction; (b) Add a pore-forming agent, an auxiliary crosslinking agent, an emulsifier, and an oil phase to the reaction solution to carry out a secondary crosslinking reaction; (c) The reaction product is washed with ethanol, acid-soaked, and dried to obtain the composite material.

9. The application of the high F-value protein oligopeptide according to claim 7 in the preparation of food; Preferably, the food includes health products, functional foods, nutritional supplements, or special dietary foods; Preferably, the health product has functions such as anti-oxidation, relieving physical fatigue, regulating intestinal flora, or assisting in the protection against chemical liver damage.

10. The use of the high F-value protein oligopeptide according to claim 7 in the preparation of pharmaceuticals; Preferably, the high-F-value protein oligopeptide is used in the preparation of a medicament for the treatment or adjunctive treatment of phenylketonuria; Preferably, the high-F-value protein oligopeptide is used in the preparation of a medicament for the treatment or adjuvant treatment of hepatic encephalopathy.

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