Preparation method of compound soybean oligopeptide with wide application

Abstract

The invention discloses a preparation method of compound soybean oligopeptide with wide application, and belongs to the technical field of soybean processing. The preparation method comprises the following steps: mixing and stirring soybean powder and deionized water to obtain a mixed solution, performing first enzymolysis on the mixed solution, inactivating, adjusting the pH value, and performing centrifugal separation to obtain a soybean protein isolate precipitate and a supernatant containing water-soluble polysaccharide; oxidizing the water-soluble polysaccharide to obtain oxidized water-soluble polysaccharide; carrying out secondary enzymolysis on the soybean protein isolate precipitate, inactivating, and carrying out chromatographic separation to obtain a soybean oligopeptide mixed solution; the preparation method comprises the following steps: mixing and homogenizing oxidized water-soluble polysaccharide, a soybean oligopeptide mixed solution, a carboxymethyl chitosan solution, absolute ethyl alcohol and an emulsifier to obtain a mixed emulsion, drying and dewatering the mixed emulsion, then cross-linking to obtain a cross-linked solution, mixing the cross-linked solution with water to obtain a feed liquid, and carrying out spray drying on the feed liquid to obtain the composite soybean oligopeptide.

Description

Technical Field

[0001] The invention belongs to the technical field of soybean processing, and in particular relates to a method for preparing a composite soybean oligopeptide with wide applications. Background Art

[0002] As an important crop, soybean is rich in protein, fat and a variety of bioactive ingredients. In recent years, as people pay more attention to healthy diet, the nutritional value and functional ingredients of soybean have received widespread attention. Among them, soybean oligopeptides have become a research hotspot in the fields of food, health care products and medicine due to their unique biological activity. Soybean oligopeptides are a class of small molecule peptides hydrolyzed from soybean protein. They are obtained by separating and extracting soybean protein from soybeans using protein engineering separation technology, and then catalyzing soybean protein isolate into low soybean oligopeptides using bioengineering enzyme catalysis technology. They are the products of protein hydrolysis, with small molecular weight (mostly in the range of 150-500Da), good water solubility and easy absorption by the human body.

[0003] Soybean oligopeptides have multiple biological activities, including: (1) promoting the recovery of mental and physical strength after excessive fatigue; (2) Antioxidant effect: Soybean oligopeptides have significant antioxidant capacity, which can remove free radicals in the body and slow down oxidative damage to cells; (3) Lipid-lowering effect: Soybean oligopeptides can significantly reduce the levels of total cholesterol and low-density lipoprotein cholesterol in serum, which is beneficial to cardiovascular health. Its mechanism may be related to the regulation of lipid metabolism and inhibition of liver cholesterol synthesis by soy oligopeptides; (4) Antihypertensive effect: Some studies have shown that soy oligopeptides can lower blood pressure by inhibiting the activity of angiotensin-converting enzyme. This effect is closely related to the peptide chain structure and amino acid composition of soy oligopeptides; (5) Active functions such as easy digestion and promotion of energy metabolism.

[0004] Patent CN105614899A discloses a low-molecular soybean peptide and a preparation method thereof. The invention adds soybean protein isolate to RO pure water at 20°C to 45°C, adjusts the pH to be in the range of 5 to 9, then heats the temperature to 40°C to 70°C, adds a composite protease and mixes and stirs for enzymolysis, and then obtains the low-molecular soybean peptide by filtering and spray drying.

[0005] Patent CN101974591A discloses a method for extracting soybean peptides by enzymatic method. The invention uses soybean protein powder as the main raw material, and is subjected to water preparation, alcohol and sodium sulfite addition, heating and steaming, filtering, and then adding alkaline protease and flavor protease for enzymatic hydrolysis, and then filtering and drying to obtain soybean peptides.

[0006] Patent CN105907826A discloses a clean preparation method for plant polypeptides / proteins, wherein alkali is added to soybean meal and other materials containing plant polypeptides to form a slurry, the slurry is heated and centrifuged to obtain a supernatant, the supernatant is then acid-adjusted and precipitated to obtain protein, the protein is enzymatically hydrolyzed by protease, filtered, separated and dried to obtain a plant polypeptide product.

[0007] Patent CN108586581A discloses a soybean polypeptide and a preparation method and application thereof. The invention uses fermented bean curd as a raw material, extracts fermented bean curd, freeze-dried fermented bean curd or defatted fermented bean curd with water, collects the filtrate by centrifugation or filtration, and obtains a crude extract by evaporation, concentration and drying of the filtrate; after the crude extract is redissolved, water-soluble soybean polypeptides with a molecular weight of 500-1000Da and a molecular weight of less than 500Da are obtained by chromatography filtration.

[0008] The soy oligopeptide hydrolysate obtained after enzymatic hydrolysis of soy protein needs to be dried and collected to obtain the finished product. The existing technology mainly uses spray drying as the drying method. However, due to the high temperature of spray drying, the hydrolysate containing soy oligopeptides is not pretreated, which easily leads to the loss of activity of the soy oligopeptides.

[0009] Therefore, it is of great significance to design a preparation method for soybean oligopeptides that can reduce the inactivation of soybean oligopeptides caused by high-temperature extraction. Summary of the invention

[0010] According to the shortcomings of the prior art, the present invention is used to solve the problems in the background technology. Specifically, the technical solution of the present invention includes the following contents:

[0011] A method for preparing a widely used composite soybean oligopeptide, the method comprising the following steps:

[0012] The soybean powder and deionized water are mixed and stirred to obtain a mixed solution, the mixed solution is mixed with a cell wall decomposing enzyme for a first enzymatic hydrolysis, and then inactivated at 80° C. for 5 to 10 minutes to obtain a first inactivated solution; the first inactivated solution is adjusted to a pH of 4.0 to 4.5, mixed and stirred for 10 to 20 minutes, and then allowed to stand for 20 to 30 minutes, and then centrifuged at a centrifugal speed of 6000 r / min to 8000 r / min for 10 to 20 minutes to obtain a soybean protein isolate precipitate and a supernatant containing water-soluble polysaccharides;

[0013] The supernatant containing the water-soluble polysaccharide and sodium periodate are mixed in a mass ratio of 1:0.01-0.05 and reacted in the dark for 5h-6h. After the reaction is completed, ethylene glycol is added to quench to obtain a product liquid. The product liquid is dialyzed to obtain an oxidized water-soluble polysaccharide.

[0014] The soy protein isolate precipitate is redissolved to obtain a soy protein isolate solution, the soy protein isolate solution is mixed with a protein complex enzyme for a second enzymolysis, and then inactivated at 80° C. for 10 min to 15 min to obtain a second inactivated solution, and the second inactivated solution is chromatographically separated to obtain a soy oligopeptide mixed solution;

[0015] Oxidized water-soluble polysaccharides, soybean oligopeptide mixed solution, carboxymethyl chitosan solution, anhydrous ethanol and an emulsifier are mixed and homogenized to obtain a mixed emulsion. After the mixed emulsion is dried to remove water, it is placed at 30°C to 40°C for 18h to 20h to obtain a cross-linked liquid. The cross-linked liquid and water are mixed in a weight ratio of 2 to 2.5:7 to 8 to obtain a feed liquid. The feed liquid is spray-dried to obtain the composite soybean oligopeptide.

[0016] Preferably, the mass ratio of the soybean powder to deionized water is 1:20-30.

[0017] Preferably, the cell wall decomposing enzyme is obtained by mixing cellulase, pectinase and xylanase in a mass ratio of 1:1:0.5.

[0018] Preferably, the mass ratio of the mixed solution:cell wall decomposing enzyme is 1:0.01-0.03.

[0019] Preferably, the conditions of the first enzymatic hydrolysis include an enzymatic hydrolysis pH of 5.0 to 5.5, an enzymatic hydrolysis temperature of 50° C. to 55° C., and an enzymatic hydrolysis time of 3 h to 4 h.

[0020] Preferably, the mass concentration of the soy protein isolate solution is 3% to 5%.

[0021] Preferably, the mass ratio of the soy protein isolate solution to the protein complex enzyme is 1:0.05-0.08.

[0022] Preferably, the protein complex enzyme is obtained by mixing trypsin and neutral protease in a mass ratio of 1:1.

[0023] Preferably, the conditions for the second enzymatic hydrolysis include an enzymatic hydrolysis pH of 7.0 to 7.5, an enzymatic hydrolysis temperature of 45° C. to 50° C., and an enzymatic hydrolysis time of 5 h to 6 h.

[0024] Preferably, the mass concentration of the carboxymethyl chitosan solution is 1% to 2%.

[0025] Preferably, the emulsifier comprises Tween 40 or Tween 60.

[0026] Preferably, the weight ratio of the oxidized water-soluble polysaccharide: soybean oligopeptide mixed solution: carboxymethyl chitosan solution: anhydrous ethanol: emulsifier is 30-45: 10-15: 1-5: 200-300: 4-8.

[0027] Preferably, the desiccant used for the drying and dehydration is an inorganic desiccant anhydrous sodium sulfate.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention uses soybean as a direct raw material, and pre-treats and decomposes soybean powder using cell wall decomposing enzymes that can enzymatically decompose plant cell walls to release polysaccharides, proteins and other active substances. The soybean powder is then inactivated to make the cell wall decomposing enzymes lose their activity to obtain an inactivated solution. Acid is then added to adjust the pH value so that the soybean protein isolate in the inactivated solution precipitates in an acidic environment, and is separated from the water-soluble polysaccharides by high-speed centrifugation to obtain a soybean protein isolate precipitate and a supernatant containing water-soluble polysaccharides. The supernatant containing the water-soluble polysaccharides is subjected to an oxidation treatment to obtain an oxidized water-soluble polysaccharide having an aldehyde functional group for standby use; the soybean protein isolate precipitate is re-dissolved in water, and the soybean protein isolate is enzymatically decomposed using a protein complex enzyme to obtain an aqueous solution containing a composite soybean oligopeptide with a high degree of hydrolysis. After the aqueous solution is inactivated, the oxidized water-soluble polysaccharide obtained by the previous oxidation treatment is added to the inactivated aqueous solution containing the soybean oligopeptide with a high degree of hydrolysis, and the mixture is mixed and stirred. A water-soluble amino polysaccharide derivative is then added and continued to be mixed, heated and stirred to oxidize the water. Soluble polysaccharides act as cross-linking agents, and achieve encapsulation and high-temperature protection of soybean oligopeptides through imine bond cross-linking and hydrogen bond cross-linking with amino groups on soybean oligopeptides and amino groups on carboxymethyl chitosan. After spray drying, a microcapsule powder structure containing soybean oligopeptides is obtained. After microencapsulation, the activity of the composite soybean oligopeptides obtained by the high-temperature spray drying process is improved. After the microcapsule powder enters the body after consumption, the imine bond can be hydrolyzed in the acidic gastric acid environment, thereby releasing the soybean oligopeptides and achieving absorption and utilization of the soybean oligopeptides.

[0030] (2) Compared with the single cross-linking effect of hydrogen bonding protection generated by carboxymethyl chitosan and sodium alginate, the present invention oxidizes the water-soluble polysaccharides after soybean enzymatic hydrolysis to obtain aldehyde groups, and forms imine bonds through covalent cross-linking between the aldehyde groups and the amino groups. This not only improves the high temperature resistance of the cross-linking structure and can achieve high temperature protection for soybean oligopeptides, but also further utilizes the water-soluble polysaccharide waste liquid obtained after enzymatic hydrolysis, thereby realizing the rational utilization of soybean resources and reducing waste. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below through the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0033] Example 1

[0034] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0035] The soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Weigh 300g of soybean powder and dissolve it in 6kg of deionized water and stir evenly to obtain a mixed solution. Hydrochloric acid was added to the mixed solution to adjust the pH to 5.0-5.5, and then the temperature was raised to 50°C for insulation. In this temperature environment, cell wall decomposing enzymes (cellulase, pectinase and xylanase were mixed in an EP tube at a mass ratio of 1:1:0.5, and then carefully blown and mixed with a pipette gun for use, or prepared in advance, but it needs to be stored at low temperature after preparation in advance to prevent the enzyme activity from decreasing and affecting the catalytic function) were added according to 0.01 times the mass of the mixed solution for the first enzymolysis, and the first enzymolysis time was controlled to be 3h. After the enzymolysis was completed, the enzymolysis solution was placed in a water bath at 80°C for inactivation for 5min and cooled to room temperature to obtain an inactivated solution. The inactivated liquid was adjusted to a pH of 4.0-4.5 with hydrochloric acid, then stirred at a stirring speed of 50 r / min for 10 minutes, then allowed to stand for 20 minutes, and then placed in a centrifuge at 6000 r / min for separation for 10 minutes to obtain a soy protein isolate precipitate and a supernatant containing water-soluble polysaccharides.

[0036] 5 kg of the supernatant containing the water-soluble polysaccharide and 50 g of sodium periodate were mixed and stirred evenly, and then immediately shielded from light, and then oxidized at room temperature for 5 hours. After the oxidation treatment, ethylene glycol was added to quench the oxidation reaction to obtain a product mixture, and then the product mixture was poured into a dialysis bag with a molecular weight cutoff of 1000 kDa, dialyzed with distilled water, and dried to obtain an oxidized water-soluble polysaccharide.

[0037] Soy protein isolate precipitate was mixed with deionized water and dissolved until the mass concentration of the soy protein isolate precipitate was 3% to obtain a soy protein isolate solution. Subsequently, the pH value of the soy protein isolate solution was adjusted to 7.0-7.5, and then 0.05 times the mass of the soy protein isolate solution was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 45°C water bath for the second enzymolysis, and the enzymolysis time was controlled to 5h. After the enzymolysis was completed, the inactivated solution was inactivated for 10min at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the remaining soy protein isolate from the inactivated solution, and then the soy oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of soy protein isolate was determined to be 25.6% by the pH stat method.

[0038] 30 parts by weight of oxidized water-soluble polysaccharide, 10 parts by weight of soybean oligopeptide mixed solution and 1 part by weight of carboxymethyl chitosan solution with a mass concentration of 1% were mixed and stirred, and then 200 parts by weight of anhydrous ethanol and 4 parts by weight of Tween 40 were added for homogenization and emulsification to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixture was placed in a temperature environment of 30°C for 18 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2:8 to obtain a feed liquid, and the feed liquid was sprayed in a 160°C spray dryer at a liquid feed rate of 300 mL / h to obtain a composite soybean oligopeptide.

[0039] Example 2

[0040] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0041] The soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Weigh 300g of soybean powder and dissolve it in 6.5kg of deionized water and stir evenly to obtain a mixed solution. Hydrochloric acid was added to the mixed solution to adjust the pH to 5.0-5.5, and then the temperature was raised to 51°C for insulation. In this temperature environment, cell wall decomposing enzymes (cellulase, pectinase and xylanase were mixed in an EP tube at a mass ratio of 1:1:0.5, and then carefully blown and mixed with a pipette to be used, or prepared in advance, but it needs to be stored at low temperature after preparation in advance to prevent the enzyme activity from decreasing and affecting the catalytic function) were added according to 0.01 times the mass of the mixed solution for the first enzymolysis, and the first enzymolysis time was controlled to 3hh. After the enzymolysis was completed, the enzymolysis solution was placed in a water bath at 80°C for inactivation for 6min and cooled to room temperature to obtain an inactivated solution. The inactivated liquid was adjusted to a pH of 4.0-4.5 with hydrochloric acid, then stirred at a stirring speed of 60 r / min for 12 minutes, then allowed to stand for 20 minutes, and then placed in a centrifuge at 6000 r / min for separation for 12 minutes to obtain a soy protein isolate precipitate and a supernatant containing water-soluble polysaccharides.

[0042] 5 kg of the supernatant containing the water-soluble polysaccharide and 100 g of sodium periodate were mixed and stirred evenly, and then immediately shielded from light, and then oxidized for 5 hours at room temperature. After the oxidation treatment, ethylene glycol was added to quench the oxidation reaction to obtain a product mixture, and then the product mixture was poured into a dialysis bag with a molecular weight cutoff of 1000 kDa, dialyzed with distilled water, and dried to obtain an oxidized water-soluble polysaccharide.

[0043] Soy protein isolate precipitate was mixed with deionized water and dissolved until the mass concentration of the soy protein isolate precipitate was 3% to obtain a soy protein isolate solution. Then the pH value of the soy protein isolate solution was adjusted to 7.0-7.5, and then 0.0 times the mass of the soy protein isolate solution was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 46°C water bath for the second enzymolysis, and the enzymolysis time was controlled to 5h. After the enzymolysis was completed, the inactivated solution was inactivated for 11min at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the remaining soy protein isolate from the inactivated solution, and then the soy oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of soy protein isolate was determined to be 26.4% by the pH stat method.

[0044] 33 parts by weight of oxidized water-soluble polysaccharide, 11 parts by weight of soybean oligopeptide mixed solution and 2 parts by weight of carboxymethyl chitosan solution with a mass concentration of 1% were mixed and stirred, and then 220 parts by weight of anhydrous ethanol and 5 parts by weight of Tween 40 were added for homogenization and emulsification to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixed emulsion was placed in a temperature environment of 30°C for 18 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2.1:7.9 to obtain a feed liquid, and the feed liquid was sprayed in a 160°C spray dryer at a liquid feed rate of 310 mL / h to obtain a composite soybean oligopeptide.

[0045] Example 3

[0046] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0047] The soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Weigh 300g of soybean powder and dissolve it in 7kg of deionized water and stir evenly to obtain a mixed solution. Hydrochloric acid was added to the mixed solution to adjust the pH to 5.0-5.5, and then the temperature was raised to 52°C for insulation. In this temperature environment, cell wall decomposing enzymes (cellulase, pectinase and xylanase were mixed in an EP tube at a mass ratio of 1:1:0.5, and then carefully blown and mixed with a pipette to be used, or prepared in advance, but it needs to be stored at low temperature after preparation in advance to prevent the enzyme activity from decreasing and affecting the catalytic function) were added according to 0.02 times the mass of the mixed solution for the first enzymolysis, and the first enzymolysis time was controlled to 3.5h. After the enzymolysis was completed, the enzymolysis solution was placed in an 80°C water bath for inactivation for 7 minutes and cooled to room temperature to obtain an inactivated solution. The inactivated liquid was adjusted to a pH of 4.0-4.5 with hydrochloric acid, then stirred at a stirring speed of 70 r / min for 14 minutes, then allowed to stand for 25 minutes, and then placed in a centrifuge at 7000 r / min for separation for 14 minutes to obtain a soy protein isolate precipitate and a supernatant containing water-soluble polysaccharides.

[0048] 5 kg of the supernatant containing the water-soluble polysaccharide and 100 g of sodium periodate were mixed and stirred evenly, and then immediately shielded from light, and then oxidized for 5.5 hours at room temperature. After the oxidation treatment, ethylene glycol was added to quench the oxidation reaction to obtain a product mixture, and then the product mixture was poured into a dialysis bag with a molecular weight cutoff of 1000 kDa, dialyzed with distilled water, and dried to obtain an oxidized water-soluble polysaccharide.

[0049] Soy protein isolate precipitate was mixed with deionized water and dissolved until the mass concentration of the soy protein isolate precipitate was 4% to obtain a soy protein isolate solution. Then the pH value of the soy protein isolate solution was adjusted to 7.0-7.5, and then 0.06 times the mass of the soy protein isolate solution was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 47°C water bath for the second enzymolysis, and the enzymolysis time was controlled to be 5.5h. After the enzymolysis was completed, the inactivated solution was inactivated for 12 minutes at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the remaining soy protein isolate from the inactivated solution, and then the soy oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of soy protein isolate was determined by the pH stat method to be 28.2%.

[0050] 35 parts by weight of oxidized water-soluble polysaccharide, 12 parts by weight of soybean oligopeptide mixed solution and 13 parts by weight of carboxymethyl chitosan solution with a mass concentration of 1.5% were mixed and stirred, and then 240 parts by weight of anhydrous ethanol and 6 parts by weight of Tween 40 were added for homogenization and emulsification to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixed emulsion was placed in a temperature environment of 35°C for 19 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2.2:7.8 to obtain a feed liquid, and the feed liquid was sprayed in a 165°C spray dryer at a liquid feed rate of 320 mL / h to obtain a composite soybean oligopeptide.

[0051] Example 4

[0052] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0053] The soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Weigh 300g of soybean powder and dissolve it in 8kg of deionized water and stir evenly to obtain a mixed solution. Hydrochloric acid was added to the mixed solution to adjust the pH to 5.0-5.5, and then the temperature was raised to 53°C for insulation. In this temperature environment, cell wall decomposing enzymes (cellulase, pectinase and xylanase were mixed in an EP tube at a mass ratio of 1:1:0.5, and then carefully blown and mixed with a pipette gun for use, or prepared in advance, but it needs to be stored at low temperature after preparation in advance to prevent the enzyme activity from decreasing and affecting the catalytic function) were added according to 0.02 times the mass of the mixed solution for the first enzymolysis, and the first enzymolysis time was controlled to 3.5h. After the enzymolysis was completed, the enzymolysis solution was placed in a water bath at 80°C for inactivation for 8min, and cooled to room temperature to obtain an inactivated solution. The inactivated liquid was adjusted to a pH of 4.0-4.5 with hydrochloric acid, then stirred at a stirring speed of 80 r / min for 16 minutes, then allowed to stand for 25 minutes, and then placed in a centrifuge at 7000 r / min for separation for 16 minutes to obtain a soy protein isolate precipitate and a supernatant containing water-soluble polysaccharides.

[0054] 5 kg of the supernatant containing the water-soluble polysaccharide and 150 g of sodium periodate were mixed and stirred evenly, and then immediately shielded from light, and then oxidized for 5.5 h at room temperature. After the oxidation treatment, ethylene glycol was added to quench the oxidation reaction to obtain a product mixture, and then the product mixture was poured into a dialysis bag with a molecular weight cutoff of 1000 kDa, dialyzed with distilled water, and dried to obtain an oxidized water-soluble polysaccharide.

[0055] Soy protein isolate precipitate was mixed with deionized water and dissolved until the mass concentration of the soy protein isolate precipitate was 4% to obtain a soy protein isolate solution. The pH value of the soy protein isolate solution was then adjusted to 7.0-7.5, and then 0.07 times the mass of the soy protein isolate solution was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 48°C water bath for the second enzymolysis, and the enzymolysis time was controlled to be 5.5h. After the enzymolysis was completed, the inactivated solution was inactivated for 13 minutes at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the remaining soy protein isolate from the inactivated solution, and then the soy oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of soy protein isolate was determined to be 28.9% by the pH stat method.

[0056] 38 parts by weight of oxidized water-soluble polysaccharide, 13 parts by weight of soybean oligopeptide mixed solution and 4 parts by weight of carboxymethyl chitosan solution with a mass concentration of 1.5% were mixed and stirred, and then 260 parts by weight of anhydrous ethanol and 7 parts by weight of Tween 60 were added and homogenized to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixed emulsion was placed in a temperature environment of 35°C for 19 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2.3:7.7 to obtain a feed liquid, and the feed liquid was sprayed in a 165°C spray dryer at a liquid feed rate of 330 mL / h to obtain a composite soybean oligopeptide.

[0057] Example 5

[0058] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0059] The soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Weigh 300g of soybean powder and dissolve it in 8kg of deionized water and stir evenly to obtain a mixed solution. Hydrochloric acid was added to the mixed solution to adjust the pH to 5.0-5.5, and then the temperature was raised to 54°C for insulation. In this temperature environment, cell wall decomposing enzymes (cellulase, pectinase and xylanase were mixed in an EP tube at a mass ratio of 1:1:0.5, and then carefully blown and mixed with a pipette gun for use, or prepared in advance, but it needs to be stored at low temperature after preparation in advance to prevent the enzyme activity from decreasing and affecting the catalytic function) were added according to 0.03 times the mass of the mixed solution for the first enzymolysis, and the first enzymolysis time was controlled to 4h. After the enzymolysis was completed, the enzymolysis solution was placed in an 80°C water bath for inactivation for 9 minutes and cooled to room temperature to obtain an inactivated solution. The inactivated liquid was adjusted to a pH of 4.0-4.5 with hydrochloric acid, then stirred at a stirring speed of 90 r / min for 18 minutes, then allowed to stand for 30 minutes, and then placed in a centrifuge at 8000 r / min for separation for 18 minutes to obtain a soy protein isolate precipitate and a supernatant containing water-soluble polysaccharides.

[0060] 5 kg of the supernatant containing the water-soluble polysaccharide and 200 g of sodium periodate were mixed and stirred evenly, and then immediately shielded from light, and then oxidized for 6 hours at room temperature. After the oxidation treatment, ethylene glycol was added to quench the oxidation reaction to obtain a product mixture, and then the product mixture was poured into a dialysis bag with a molecular weight cutoff of 1000 kDa, dialyzed with distilled water, and dried to obtain an oxidized water-soluble polysaccharide.

[0061] Soy protein isolate precipitate was mixed with deionized water and dissolved until the mass concentration of the soy protein isolate precipitate was 5% to obtain a soy protein isolate solution. Then the pH value of the soy protein isolate solution was adjusted to 7.0-7.5, and then 0.07 times the mass of the soy protein isolate solution was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 49°C water bath for the second enzymolysis, and the enzymolysis time was controlled to 6h. After the enzymolysis was completed, the inactivated solution was inactivated for 14min at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the remaining soy protein isolate from the inactivated solution, and then the soy oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of soy protein isolate was determined to be 30.1% by the pH stat method.

[0062] 42 parts by weight of oxidized water-soluble polysaccharide, 14 parts by weight of soybean oligopeptide mixed solution and 5 parts by weight of carboxymethyl chitosan solution with a mass concentration of 2% were mixed and stirred, and then 280 parts by weight of anhydrous ethanol and 8 parts by weight of Tween 60 were added for homogenization and emulsification to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixed emulsion was placed in a temperature environment of 40°C for 20 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2.4:7.6 to obtain a feed liquid, and the feed liquid was sprayed in a 170°C spray dryer at a liquid feed rate of 340 mL / h to obtain a composite soybean oligopeptide.

[0063] Example 6

[0064] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0065] The soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Weigh 300g of soybean powder and dissolve it in 9kg of deionized water and stir evenly to obtain a mixed solution. Hydrochloric acid was added to the mixed solution to adjust the pH to 5.0-5.5, and then the temperature was raised to 55°C for insulation. In this temperature environment, cell wall decomposing enzymes (cellulase, pectinase and xylanase were mixed in an EP tube at a mass ratio of 1:1:0.5, and then carefully blown and mixed with a pipette gun for use, or prepared in advance, but it needs to be stored at low temperature after preparation in advance to prevent the enzyme activity from decreasing and affecting the catalytic function) were added according to 0.03 times the mass of the mixed solution for the first enzymolysis, and the first enzymolysis time was controlled to 4h. After the enzymolysis was completed, the enzymolysis solution was placed in a water bath at 80°C for inactivation for 10min and cooled to room temperature to obtain an inactivated solution. The inactivated liquid was adjusted to a pH of 4.0-4.5 with hydrochloric acid, then stirred at a stirring speed of 100 r / min for 20 minutes, then allowed to stand for 30 minutes, and then placed in a centrifuge at 8000 r / min for separation for 20 minutes to obtain a soy protein isolate precipitate and a supernatant containing water-soluble polysaccharides.

[0066] 5 kg of the supernatant containing the water-soluble polysaccharide and 250 g of sodium periodate were mixed and stirred evenly, and then immediately shielded from light, and then oxidized at room temperature for 6 hours. After the oxidation treatment, ethylene glycol was added to quench the oxidation reaction to obtain a product mixture, and then the product mixture was poured into a dialysis bag with a molecular weight cutoff of 1000 kDa, dialyzed with distilled water, and dried to obtain oxidized water-soluble polysaccharides.

[0067] Soy protein isolate precipitate was mixed with deionized water and dissolved until the mass concentration of the soy protein isolate precipitate was 5% to obtain a soy protein isolate solution. Subsequently, the pH value of the soy protein isolate solution was adjusted to 7.0-7.5, and then 0.08 times the mass of the soy protein isolate solution was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 50°C water bath for the second enzymolysis, and the enzymolysis time was controlled to 6h. After the enzymolysis was completed, the inactivated solution was inactivated for 15min at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the remaining soy protein isolate from the inactivated solution, and then the soy oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of soy protein isolate was determined to be 30.3% by the pH stat method.

[0068] 45 parts by weight of oxidized water-soluble polysaccharide, 15 parts by weight of soybean oligopeptide mixed solution and 5 parts by weight of carboxymethyl chitosan solution with a mass concentration of 2% were mixed and stirred, and then 300 parts by weight of anhydrous ethanol and 8 parts by weight of Tween 60 were added for homogenization and emulsification to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixed emulsion was placed in a temperature environment of 40°C for 20 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2.5:7.5 to obtain a feed liquid, and the feed liquid was sprayed in a 170°C spray dryer at a liquid feed rate of 350 mL / h to obtain a composite soybean oligopeptide.

[0069] Comparative Example 1

[0070] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0071] Soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Soybean powder was added to deionized water and dispersed to a mass concentration of 5% to obtain a soybean powder mixed dispersion. Subsequently, the pH value of the soybean powder mixed dispersion was adjusted to 7.0-7.5, and then 0.08 times the mass of the soybean powder mixed dispersion was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 50°C water bath for enzymolysis, and the enzymolysis time was controlled to 6h. After the enzymolysis was completed, the inactivated solution was inactivated for 15min at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the undecomposed soybean protein isolate from the inactivated solution, and then the soybean oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of the soybean protein isolate was determined by the pH stat method to be 9.37%.

[0072] 15 parts by weight of the soybean oligopeptide mixture, 5 parts by weight of a carboxymethyl chitosan solution with a mass concentration of 2%, and 45 parts by weight of the oxidized water-soluble polysaccharide obtained in Example 6 were mixed and stirred, and then 300 parts by weight of anhydrous ethanol and 8 parts by weight of Tween 60 were added for homogenization and emulsification to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixed emulsion was placed in a temperature environment of 40°C for 20 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2.5:7.5 to obtain a feed liquid, and the feed liquid was sprayed in a 170°C spray dryer at a liquid feeding rate of 350 mL / h to obtain a composite soybean oligopeptide.

[0073] Comparative Example 2

[0074] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0075] Soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Soybean powder was added to deionized water and dispersed to a mass concentration of 5% to obtain a soybean powder mixed dispersion. Subsequently, the pH value of the soybean powder mixed dispersion was adjusted to 7.0-7.5, and then 0.08 times the mass of the soybean powder mixed dispersion was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 50°C water bath for enzymolysis, and the enzymolysis time was controlled to 6h. After the enzymolysis was completed, the inactivated solution was inactivated for 15min at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the undecomposed soybean protein isolate from the inactivated solution, and then the soybean oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of the soybean protein isolate was determined by the pH stat method to be 8.96%.

[0076] 15 parts by weight of the soybean oligopeptide mixture and 5 parts by weight of a 2% carboxymethyl chitosan solution were mixed and stirred, and then 300 parts by weight of anhydrous ethanol and 8 parts by weight of Tween 60 were added for homogenization and emulsification to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixed emulsion was placed in a temperature environment of 40°C for 20 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2.5:7.5 to obtain a feed liquid, and the feed liquid was sprayed in a 170°C spray dryer at a liquid feed rate of 350 mL / h to obtain a composite soybean oligopeptide.

[0077] Comparative Example 3

[0078] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0079] Soybeans were crushed by a grinder and passed through a 50-mesh sieve to obtain soybean powder. Soybean powder was added to deionized water and dispersed to a mass concentration of 5% to obtain a soybean powder mixed dispersion. Subsequently, the pH value of the soybean powder mixed dispersion was adjusted to 7.0-7.5, and then 0.08 times the mass of the soybean powder mixed dispersion was added to mix the protein complex enzyme (mixed in an EP tube with a mass ratio of 1:1 by trypsin and neutral protease, and then carefully blown and mixed with a pipette) and then placed in a 50°C water bath for enzymolysis, and the enzymolysis time was controlled to 6h. After the enzymolysis was completed, the inactivated solution was inactivated for 15min at a temperature of 80°C. The inactivated solution was adjusted to a pH of 4.5 to separate and precipitate the undecomposed soybean protein isolate from the inactivated solution, and then the soybean oligopeptide mixture was obtained by chromatography separation; the degree of hydrolysis (DH) of the soybean protein isolate was determined by the pH stat method to be 9.11%.

[0080] 15 parts by weight of a soybean oligopeptide mixture, 5 parts by weight of a carboxymethyl chitosan solution with a mass concentration of 2%, and 5 parts by weight of a calcium chloride solution with a mass concentration of 2% were mixed and stirred, and then 300 parts by weight of anhydrous ethanol and 8 parts by weight of Tween 60 were added for homogenization and emulsification to obtain a mixed emulsion, and the mixed emulsion was dried and dehydrated with anhydrous sodium sulfate until the weight remained unchanged, and then the anhydrous sodium sulfate was removed. Then, the mixed emulsion was placed in a temperature environment of 40°C for 20 hours to obtain a cross-linking liquid, and the cross-linking liquid and water were mixed and diluted in a weight ratio of 2.5:7.5 to obtain a feed liquid, and the feed liquid was sprayed in a 170°C spray dryer at a liquid feed rate of 350 mL / h to obtain a composite soybean oligopeptide.

[0081] Comparative Example 4

[0082] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0083] The oxidized water-soluble polysaccharide in Example 6 was replaced with sodium alginate, and the other conditions remained the same as in Example 6.

[0084] Comparative Example 5

[0085] A preparation method of a widely used composite soybean oligopeptide specifically includes the following processes:

[0086] The soybean oligopeptide mixture obtained in Example 6 and water were mixed and diluted in a weight ratio of 2.5:7.5 to form a feed liquid, and the feed liquid was spray-dried in a spray dryer at 170° C. at a liquid feeding rate of 350 mL / h to obtain a composite soybean oligopeptide.

[0087] The composite soybean oligopeptides obtained in Examples 1 to 6 and Comparative Examples 1 to 5 were subjected to anti-fatigue tests:

[0088] 120 experimental mice in good health were randomly divided into 12 groups according to Examples 1 to 6, Comparative Examples 1 to 5 and blank controls (gavage with purified water). The mice were subjected to 60-min swimming training once a day. After the swimming training, the corresponding composite soybean oligopeptides and purified water were gavaged, and the gavage dose was 3.0 g per 1 kg of body weight for 20 consecutive days. The tail blood was taken at 20 minutes and 60 minutes after the end of the last day to measure the lactic acid content. The results are shown in Table 1.

[0089] Then, on the 21st day, mice from each group that had undergone lactate measurement the day before were taken, and 5% of their body weight of lead was loaded on the base of their tails. They were placed in a water tank with a water temperature of 25±1°C and a water depth of 50 cm to swim. The total swimming time of the mice was recorded from the time they entered the water to the time they sank into the water and could not float to the surface for 10 seconds (at this time they were in a state of exhaustion). The results are shown in Table 2.

[0090] Table 1 Lactic acid content levels

[0091]

[0092]

[0093] Table 2 Statistics of total weight-bearing swimming time of mice

[0094]

[0095]

[0096] The following conclusions can be drawn from Tables 1 and 2 of the above mouse experimental results:

[0097] (1) The experimental results of Examples 1 to 6 show that by removing polysaccharides such as starch contained in soybeans in advance and then oxidizing the removed water-soluble polysaccharides to obtain oxidized water-soluble polysaccharides as cross-linking agents, the encapsulation and high-temperature protection of soybean oligopeptides can be achieved through imine bond cross-linking and hydrogen bond cross-linking with the amino groups on soybean oligopeptides and amino groups on carboxymethyl chitosan, which has a good effect on improving the hydrolysis degree of soybean protein isolate, and the high-temperature resistant protective structure improves the activity of the composite soybean oligopeptides after being treated by a high-temperature spray drying process.

[0098] (2) Comparative Example 1 shows that soybeans contain a high content of polysaccharides such as starch. If the polysaccharides are not separated and then separated from the protein solution, the polysaccharides may affect the solubility and stability of the protein by interacting with the protein molecules. This interaction may cause protein precipitation or denaturation, thereby affecting the hydrolysis degree of the soybean protein isolate, resulting in a low content of soybean oligopeptides in the obtained composite soybean oligopeptides, thereby resulting in low biological activity of the composite soybean oligopeptides.

[0099] (3) It can be found from Comparative Example 2 that due to the loss of the high-temperature resistant dynamic imine bond that can be formed by cross-linking the aldehyde group and the amino group on the oxidized water-soluble polysaccharide, and the low degree of hydrolysis of the soy protein isolate, the soy oligopeptide is only wrapped by the hydrogen bonding effect of carboxymethyl chitosan, resulting in the low biological activity of the composite soy oligopeptide prepared by the high-temperature spray drying process.

[0100] (4) It can be found from Comparative Example 3 that although metal ions and calcium ions can achieve the encapsulation of soybean oligopeptides through metal coordination, the metal coordination effect in the encapsulation and high temperature protection constructed by the present invention may lead to poor improvement in high temperature resistance and low degree of hydrolysis of soybean protein isolate, resulting in low biological activity of the composite soybean oligopeptide.

[0101] (5) It can be found from Comparative Example 4 that although the encapsulation of soybean oligopeptides can be achieved by utilizing the hydrogen bonding between polysaccharide structures and between polysaccharides and soybean oligopeptides, compared with the high-temperature resistant structure of chemical covalent cross-linking, the high-temperature resistance effect brought about by the single hydrogen bonding effect may be poor, resulting in lower biological activity of the composite soybean oligopeptide.

[0102] (6) It can be found from Comparative Example 5 that the bioactivity of the composite soybean oligopeptide obtained by high-temperature spray drying of the soybean oligopeptide mixture is relatively low due to the lack of any high-temperature protection treatment.

[0103] The above-described embodiments are compared with the technical solutions and beneficial effects of the present invention, and it should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected.

Claims

1. A method for preparing a widely used composite soybean oligopeptide, characterized in that: The preparation method comprises the following steps: The soybean powder and deionized water are mixed and stirred to obtain a mixed solution, the mixed solution is mixed with a cell wall decomposing enzyme for a first enzymatic hydrolysis, and then inactivated at 80° C. for 5 to 10 minutes to obtain a first inactivated solution; the first inactivated solution is adjusted to a pH of 4.0 to 4.5, mixed and stirred for 10 to 20 minutes, and then allowed to stand for 20 to 30 minutes, and then centrifuged at a centrifugal speed of 6000 r / min to 8000 r / min for 10 to 20 minutes to obtain a soybean protein isolate precipitate and a supernatant containing water-soluble polysaccharides; The supernatant containing the water-soluble polysaccharide and sodium periodate are mixed in a mass ratio of 1:0.01-0.05 and reacted in the dark for 5h-6h. After the reaction is completed, ethylene glycol is added to quench to obtain a product liquid. The product liquid is dialyzed to obtain an oxidized water-soluble polysaccharide. The soy protein isolate precipitate is redissolved to obtain a soy protein isolate solution, the soy protein isolate solution is mixed with a protein complex enzyme for a second enzymolysis, and then inactivated at 80° C. for 10 min to 15 min to obtain a second inactivated solution, and the second inactivated solution is chromatographically separated to obtain a soy oligopeptide mixed solution; Oxidized water-soluble polysaccharides, soybean oligopeptide mixed solution, carboxymethyl chitosan solution, anhydrous ethanol and an emulsifier are mixed and homogenized to obtain a mixed emulsion. After the mixed emulsion is dried to remove water, it is placed at 30°C to 40°C for 18h to 20h to obtain a cross-linked liquid. The cross-linked liquid and water are mixed in a weight ratio of 2 to 2.5:7.5 to 8 to obtain a feed liquid. The feed liquid is spray-dried to obtain the composite soybean oligopeptide.

2. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The mass ratio of the soybean powder to deionized water is 1:20-30.

3. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The cell wall decomposing enzyme is obtained by mixing cellulase, pectinase and xylanase in a mass ratio of 1:1:0.

5.

4. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The mass ratio of the mixed solution to the cell wall decomposing enzyme is 1:0.01-0.

03.

5. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The mass ratio of the soy protein isolate solution to the protein complex enzyme is 1:0.05-0.

08.

6. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The protein complex enzyme is obtained by mixing trypsin and neutral protease in a mass ratio of 1:

1.

7. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The mass concentration of the carboxymethyl chitosan solution is 1% to 2%.

8. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The emulsifier includes Tween 40 or Tween 60.

9. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The weight ratio of the oxidized water-soluble polysaccharide: soybean oligopeptide mixed solution: carboxymethyl chitosan solution: anhydrous ethanol: emulsifier is 30-45: 10-15: 1-5: 200-300: 4-8.

10. The method for preparing a widely used composite soybean oligopeptide according to claim 1, characterized in that: The desiccant used for the drying and dehydration is an inorganic desiccant anhydrous sodium sulfate.

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