Production method of corn oligopeptide

Abstract

The invention provides a production method of corn oligopeptide, and belongs to the technical field of corn oligopeptide. The production method comprises the steps of pretreatment, enzyme carrier preparation, functional enzyme preparation and enzymolysis. The pretreatment step comprises the following steps: adding 50-90wt% of an ethanol solution into the corn gluten meal for extraction treatment or adding 10-60wt% of a sodium bicarbonate solution for stirring treatment, and after the treatment is finished, washing and drying to obtain pretreated corn gluten meal; according to the corn oligopeptide prepared by the method disclosed by the invention, pigments and bitter taste are effectively removed, and the yield and the protein content of the corn oligopeptide are high.

Description

Technical Field

[0001] The invention belongs to the technical field of corn oligopeptides, and particularly relates to a production method of corn oligopeptides. Background Art

[0002] Corn oligopeptides are corn proteins extracted from natural food corn. They are powdered products with a relative molecular mass of less than 1000 obtained through enzymatic degradation and specific small peptide separation technology. Corn oligopeptides have reliable safety and multiple biological activities, such as antioxidant, anti-hypertensive, immune-enhancing, anti-fatigue, and liver protection. In recent years, with the gradual improvement of the understanding of corn peptides, the development of corn peptide foods has gradually attracted people's attention. The United States, Europe, Japan and other countries and regions have always been in the leading position in the development of corn peptides, and their technical research has been relatively mature. In particular, Japan has always been in the world's leading position in the field of corn peptide research.

[0003] my country started late in the research on peptides, and the related research on corn peptides has just begun to get on track; currently, the main methods for preparing corn peptides are microbial fermentation, enzymatic hydrolysis and chemical synthesis.

[0004] Chemical synthesis generally refers to protein hydrolysis, which includes acid hydrolysis and alkaline hydrolysis, that is, using strong acid, strong base or a combination of strong acid and strong base to directly hydrolyze protein to obtain hydrolyzate; usually in production, hydrochloric acid and sulfuric acid are used as strong acids, and barium hydroxide, sodium hydroxide, etc. are used as strong bases, with fast hydrolysis speed and high degree; the hydrolysis process of chemical synthesis is difficult to control, and the acid and base reagents used will cause varying degrees of damage to amino acids such as arginine, cystine, and serine.

[0005] The main principle of preparing corn peptides by microbial fermentation is to utilize the microbial enzymes produced during the fermentation and growth of microorganisms, and mainly rely on the enzyme system in the microorganisms to decompose the corn protein powder. This technology started late in my country, resulting in less research on the use of microbial fermentation to prepare corn peptides in China, especially there is almost no introduction to microbial strains. The microbial fermentation technology is immature, the conversion rate is low, and the refining process is difficult, making it impossible to carry out industrial production.

[0006] The enzymatic hydrolysis method uses zein as a substrate and uses a single alkaline protease or a composite enzymatic hydrolysis method to degrade it to achieve the purpose of producing bioactive short peptides and multifunctional mixed peptides. The enzymatic hydrolysis method has the disadvantages of difficult separation and low yield in preparing corn oligopeptides. In addition, the pigment and bitterness of corn oligopeptides cannot be effectively removed, which affects their taste and limits the application of corn peptides. In addition, the prior art adopts enzymatic hydrolysis to prepare corn oligopeptides. On the one hand, due to the complex and compact structure of corn protein, it is difficult for a single enzyme or a composite enzyme to fully act on corn protein, which weakens the enzymatic hydrolysis effect and reduces the production efficiency. On the other hand, in the enzymatic hydrolysis process, in addition to the generated product corn oligopeptides, a large amount of macromolecular proteins, incompletely enzymatically hydrolyzed intermediates and other impurities are also generated, which brings great difficulties to the subsequent purification and separation, thereby reducing the yield and purity of the product. Furthermore, the enzymatic hydrolysis conditions are harsh, and the requirements for conditions such as temperature and pH value are relatively strict, which can easily affect the activity of the enzyme and thus affect the enzymatic hydrolysis effect. Summary of the invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides a method for producing corn oligopeptides, which adopts an enzymatic hydrolysis method with high enzymatic hydrolysis efficiency, effectively removes the pigment and bitterness of corn oligopeptides, and has high product yield and purity.

[0008] In view of the above technical problems, the present invention adopts the following technical solutions: A method for producing corn oligopeptides, comprising pretreatment, preparation of enzyme carriers, preparation of functional enzymes and enzymolysis steps; 1. Preprocessing Adding the pretreatment liquid to the corn yellow powder, washing and drying after the treatment, to obtain the pretreated corn yellow powder; The mass volume ratio of the corn yellow powder to the pretreatment liquid is 100g:800-1000mL; The adding of the pretreatment liquid treatment is a leaching treatment by adding a 50-90wt% ethanol solution or a stirring treatment by adding a 10-60wt% sodium bicarbonate solution; The leaching treatment is performed at 70-73° C. and 100-120 rpm for 3.0-3.5 hours; The stirring temperature is 45°C and the stirring time is 18 min. When adding 10-60wt% sodium bicarbonate solution and stirring, after the treatment, the pH can be adjusted to 4.7, and Rifola amylase is added, and stirring is continued for 1.0h. The Rifola amylase is 1.2wt% of the mass of the corn yellow powder.

[0009] 2. Preparation of enzyme carrier (1) Activation The diatomite is placed in a hydrochloric acid solution of 6-8 times its mass, the temperature is increased to 35-40°C, and the solution is kept warm and soaked for 3.6-4.2 hours. After the soaking is completed, the solution is filtered out and washed, and dried at 78-82°C for 8-12 hours, and then naturally recovered to room temperature. The solution is then placed in a muffle furnace, the temperature is increased to 350-380°C at a rate of 2.0-4.0°C / min, and the solution is kept warm and calcined for 4.7-5.3 hours. After the calcination is completed, the temperature is naturally reduced to room temperature to obtain activated diatomite; The particle size of the diatomaceous earth is 210-240nm; The mass concentration of the hydrochloric acid solution is 20-25%; (2) One-time processing Add hydroxylamine hydrochloride into deionized water and stir evenly to obtain a hydroxylamine hydrochloride solution; add chitosan into acetic acid solution and stir evenly to obtain a chitosan solution; place activated diatomaceous earth in deionized water, increase the temperature to 37-43°C, slowly add hydroxylamine hydrochloride solution and chitosan solution, control the addition rate to 1.8-2.2g / min, and keep the stirring speed at 230-250rpm while adding. After the addition is completed, continue to increase the temperature to 68-72°C, keep warm and stir for 2.3-2.6h, and after washing and drying, obtain a primary enzyme carrier; The mass ratio of hydroxylamine hydrochloride to deionized water is 6.5-7.5:100; The mass ratio of chitosan to acetic acid solution is 4.6-5.3:100; The mass concentration of the acetic acid solution is 3.0-5.0%; The mass ratio of the activated diatomaceous earth, deionized water, hydroxylamine hydrochloride solution and chitosan solution is 18-22:200:34-38:33-37; (3) Secondary processing The primary carrier is placed in N, N-dimethylformamide, stirred evenly, and then succinic anhydride and triethylamine are added, the temperature is raised to 72-78°C, and the reaction is kept warm for 3.8-4.3 hours. After the reaction is completed, the enzyme carrier is obtained by filtering, washing, and drying. The mass ratio of the primary carrier, N,N-dimethylformamide, succinic anhydride and triethylamine is 9.5-10.5:100:3.4-3.8:0.37-0.42.

[0010] 3. Preparation of functional enzymes The enzyme carrier is mixed with the phosphate buffer, stirred evenly, the enzyme is added, and the mixture is shaken at room temperature at a speed of 130-150 rpm for 3.8-4.2 hours. Then, the cinnamaldehyde solution is added, and the shaking reaction is continued for 1.8-2.2 hours. After the reaction is completed, the mixture is washed by centrifugation and dried naturally to obtain a functional enzyme. The mass volume ratio of the enzyme carrier, phosphate buffer, enzyme and cinnamaldehyde solution is 2.8-3.2 g:48-53 mL:0.24-0.26 g:16.0-17.0 g; The enzyme is one or both of Alcase2.4L protease and Flavourzyme1000L enzyme, and the mass ratio of Alcase2.4L protease to Flavourzyme1000L enzyme is 2:1; The mass concentration of the cinnamaldehyde solution is 1.3-1.7%; The molar concentration of the phosphate buffer is 0.04-0.06 ml / L, and the pH value is 7.8-8.2.

[0011] 4. Enzymatic hydrolysis Deionized water is added to the pretreated corn yellow powder, the pH is adjusted to 7.5-8.0, a functional enzyme is added, and the reaction is carried out at 53-56° C. for 7.5-8.5 hours. After the reaction is completed, a clear liquid and a solid are obtained by suction filtration, 0.8-1.2wt% of activated carbon is added to the clear liquid, and the mixture is stirred for 1.0-1.3 hours. After the stirring is completed, the mixture is subjected to vacuum drying after rotary evaporation to obtain a corn oligopeptide product; The mass ratio of the pretreated corn yellow powder, deionized water and functional enzyme is 100:900-1000:22-40.

[0012] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. In the process of preparing corn oligopeptides, the corn yellow powder is first pretreated to remove pigments and grease, and then enzymolysis is performed using a functional enzyme. In the preparation method of the functional enzyme, diatomaceous earth is used as an enzyme carrier matrix, and the diatomaceous earth is first treated with a dilute acid solution to remove surface impurities, increase its specific surface area and the activity of the diatomaceous earth, and improve the stability of the diatomaceous earth after calcination. In a treatment step, amino groups are introduced into the surface of the diatomaceous earth by introducing hydroxylamine hydrochloride and diatomaceous earth. Chitosan molecules can also enhance the hydrophilicity and biocompatibility of the diatomaceous earth carrier. The obtained primary enzyme carrier reacts with succinic anhydride, and then carboxyl groups are introduced into the surface of the enzyme carrier. The hydrophilicity and reaction activity of the carrier are further increased; in the process of preparing the functional enzyme, a cinnamaldehyde solution is added, which can react with the enzyme molecules and the amino groups on the surface of the enzyme carrier, thereby enhancing the binding force between the enzyme and the enzyme carrier, thereby more firmly fixing the enzyme molecules on the enzyme carrier, ensuring the stability of the functional enzyme, and reducing the probability of enzyme inactivation and denaturation, so that the enzyme activity can be maintained for a long time during the enzymolysis process. Combined with the activated carbon adsorption step, it effectively removes the pigment and bitterness of the product, improves the reusability of the enzyme, and increases the contact area with the substrate, improves the enzymolysis efficiency, and promotes the enzymolysis reaction, thereby improving the product yield and protein content; 2. The corn oligopeptide product obtained by the method of the present invention has a yield of 61.2-63.8% and a protein content of 91.0-93.4wt%. DETAILED DESCRIPTION

[0013] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0014] Example 1 1. Preprocessing 1000 mL of 50 wt% ethanol solution was added to 100 g of corn yellow powder, and the mixture was extracted at 70° C. and 100 rpm for 3.5 h. After the extraction, the anhydrous ethanol was removed by filtration, and the mixture was washed and dried to obtain the pretreated corn yellow powder.

[0015] 2. Preparation of enzyme carrier (1) Activation The diatomite was placed in a 7-fold mass of 23wt% hydrochloric acid solution, the temperature was raised to 38°C, and the solution was kept warm and soaked for 4.0 hours. After the soaking, the solution was filtered out and washed, and dried at 80°C for 10 hours. After the solution was naturally restored to room temperature, the solution was placed in a muffle furnace, the temperature was raised to 370°C at a rate of 3.0°C / min, and the solution was kept warm and calcined for 5.0 hours. After the calcination, the temperature was naturally lowered to room temperature to obtain activated diatomite. The particle size of the diatomaceous earth is 220 nm; (2) One-time processing 7.0 g of hydroxylamine hydrochloride was added to 100 g of deionized water and stirred evenly to obtain a hydroxylamine hydrochloride solution; 5.0 g of chitosan was added to 100 g of 4.0 wt% acetic acid solution and stirred evenly to obtain a chitosan solution; 20 g of activated diatomaceous earth was placed in 200 g of deionized water, the temperature was raised to 40°C, 36 g of hydroxylamine hydrochloride solution and 35 g of chitosan solution were slowly added, the addition rate was controlled to be 2.0 g / min, and the stirring speed was kept at 240 rpm while adding. After the addition was completed, the temperature was continued to be raised to 70°C, and the mixture was stirred for 2.5 hours at the temperature. After washing and drying, a primary enzyme carrier was obtained; (3) Secondary processing Put 10.0g of the primary carrier into 100g of N,N-dimethylformamide, stir evenly, add 3.6g of succinic anhydride and 0.40g of triethylamine, raise the temperature to 75°C, keep the reaction for 4.0h, and after the reaction is completed, filter, wash and dry to obtain the enzyme carrier.

[0016] 3. Preparation of functional enzymes 3.0 g of enzyme carrier was mixed with 50 mL of phosphate buffer, stirred evenly, 0.25 g of enzyme was added, and the mixture was shaken at room temperature at a speed of 140 rpm for 4 h, and then 16.5 g of 1.5 wt% cinnamaldehyde solution was added, and the shaking reaction was continued for 2 h. After the reaction was completed, the mixture was centrifuged and washed, and then dried naturally to obtain a functional enzyme; The enzyme is Alcase2.4L protease; The molar concentration of the phosphate buffer is 0.05 ml / L, and the pH value is 8.

[0017] 4. Enzymatic hydrolysis Add 900 g of deionized water to 100 g of pretreated corn yellow powder, adjust the pH to 7.5, add 23 g of functional enzyme, react at 55 ° C for 8.0 h, and after the reaction is completed, obtain a clear liquid and a solid by suction filtration. Add 1 wt% of activated carbon to the clear liquid and stir for 1.0 h. After stirring, evaporate and vacuum dry to obtain a corn oligopeptide product.

[0018] The yield of the corn oligopeptide product obtained by the method of Example 1 was 61.2% and the protein content was 91.6wt%.

[0019] Example 2 1. Preprocessing Add 1000 mL of sodium bicarbonate solution to 100 g of corn yellow powder, stir and treat at 40° C. for 20 min, wash and dry to obtain pretreated corn yellow powder; The concentration of the sodium bicarbonate solution is 10%.

[0020] 2. Preparation of enzyme carrier (1) Activation The diatomite was placed in a 20wt% hydrochloric acid solution of 6 times its mass, the temperature was raised to 35°C, and the solution was kept warm and soaked for 3.6 hours. After the soaking, the solution was filtered out and washed, and dried at 78°C for 12 hours. After the solution was naturally restored to room temperature, the solution was placed in a muffle furnace, the temperature was raised to 350°C at a rate of 2.0°C / min, and the solution was kept warm and calcined for 4.7 hours. After the calcination, the temperature was naturally lowered to room temperature to obtain activated diatomite. The particle size of the diatomaceous earth is 210 nm; (2) One-time processing 6.5 g of hydroxylamine hydrochloride was added to 100 g of deionized water and stirred evenly to obtain a hydroxylamine hydrochloride solution; 4.6 g of chitosan was added to 100 g of 3.0 wt% acetic acid solution and stirred evenly to obtain a chitosan solution; 18 g of activated diatomaceous earth was placed in 200 g of deionized water, the temperature was raised to 37°C, 34 g of hydroxylamine hydrochloride solution and 33 g of chitosan solution were slowly added, the addition rate was controlled to be 1.8 g / min, and the stirring speed was maintained at 230 rpm while adding. After the addition was completed, the temperature was continued to be raised to 68°C, and the mixture was stirred for 2.3 hours at the temperature. After washing and drying, a primary enzyme carrier was obtained; (3) Secondary processing 9.5 g of the primary carrier was placed in 100 g of N,N-dimethylformamide, and after stirring evenly, 3.4 g of succinic anhydride and 0.37 g of triethylamine were added, and the temperature was raised to 72°C, and the reaction was kept warm for 3.8 hours. After the reaction was completed, the enzyme carrier was obtained by filtering, washing and drying.

[0021] 3. Preparation of functional enzymes 2.8 g of enzyme carrier was mixed with 48 mL of phosphate buffer, stirred evenly, 0.24 g of enzyme was added, and the mixture was shaken at room temperature at a speed of 130 rpm for 3.8 h, and then 16.0 g of 1.3 wt% cinnamaldehyde solution was added, and the shaking reaction was continued for 1.8 h. After the reaction was completed, the mixture was centrifuged and washed, and then dried naturally to obtain a functional enzyme; The enzyme is Alcase2.4L protease; The molar concentration of the phosphate buffer is 0.06 ml / L, and the pH value is 7.8.

[0022] 4. Enzymatic hydrolysis Add 900 g of deionized water to 100 g of pretreated corn yellow powder, adjust the pH to 7.8, add 25 g of functional enzyme, react at 53 ° C for 8.5 h, and after the reaction is completed, obtain a clear liquid and a solid by filtration. Add 1.2 wt% of activated carbon to the clear liquid and stir for 1.2 h. After stirring, rotary evaporation and vacuum drying are performed to obtain a corn oligopeptide product.

[0023] The yield of the corn oligopeptide product obtained by the method of Example 2 was 63.8% and the protein content was 91.0wt%.

[0024] Example 3 1. Preprocessing Add 800 mL of sodium bicarbonate solution to 100 g of corn yellow powder, stir for 18 min at 45 °C, wash and dry, adjust the pH to 4.7, add 1.2 g of Rifola amylase, continue stirring for 1.0 h, filter, wash and dry to obtain pretreated corn yellow powder; The concentration of the sodium bicarbonate solution is 60%.

[0025] 2. Preparation of enzyme carrier (1) Activation The diatomite was placed in a 25wt% hydrochloric acid solution of 8 times its mass, the temperature was raised to 40°C, and the solution was kept warm and soaked for 4.2 hours. After the soaking, it was filtered out and washed, and dried at 82°C for 8 hours. After it naturally recovered to room temperature, it was placed in a muffle furnace, heated to 380°C at a rate of 4.0°C / min, and calcined for 5.3 hours. After the calcination was completed, it was naturally cooled to room temperature to obtain activated diatomite. The particle size of the diatomaceous earth is 240 nm; (2) One-time processing 7.5 g of hydroxylamine hydrochloride was added to 100 g of deionized water and stirred to obtain a hydroxylamine hydrochloride solution; 5.3 g of chitosan was added to 100 g of 5.0 wt% acetic acid solution and stirred to obtain a chitosan solution; 22 g of activated diatomaceous earth was placed in 200 g of deionized water, the temperature was raised to 43°C, 38 g of hydroxylamine hydrochloride solution and 37 g of chitosan solution were slowly added, the addition rate was controlled to be 2.2 g / min, and the stirring speed was maintained at 250 rpm while adding. After the addition was completed, the temperature was continued to be raised to 72°C, and the mixture was stirred for 2.6 hours at the temperature. After washing and drying, a primary enzyme carrier was obtained; (3) Secondary processing Put 10.5g of the primary carrier into 100g of N,N-dimethylformamide, stir evenly, add 3.8g of succinic anhydride and 0.42g of triethylamine, raise the temperature to 78°C, keep the reaction for 4.3h, and after the reaction is completed, filter, wash and dry to obtain the enzyme carrier.

[0026] 3. Preparation of functional enzymes 3.2 g of enzyme carrier was mixed with 53 mL of phosphate buffer, stirred evenly, 0.26 g of enzyme was added, and the mixture was shaken at room temperature at a speed of 150 rpm for 4.2 h, and then 17.0 g of 1.7 wt % cinnamaldehyde solution was added, and the shaking reaction was continued for 2.2 h. After the reaction was completed, the mixture was centrifuged and washed, and then dried naturally to obtain a functional enzyme; The enzyme is Alcase2.4L protease; The molar concentration of the phosphate buffer is 0.04 ml / L, and the pH value is 8.2.

[0027] 4. Enzymatic hydrolysis Add 1000 g of deionized water to 100 g of pretreated corn yellow powder, adjust the pH to 8.0, add 22 g of functional enzyme, react at 53 ° C for 8.5 hours, and after the reaction is completed, obtain a clear liquid and a solid by suction filtration. Add 1.2 wt% of activated carbon to the clear liquid and stir for 1.2 hours. After stirring, evaporate and vacuum dry to obtain a corn oligopeptide product.

[0028] The yield of the corn oligopeptide product obtained by the method of Example 3 was 63.2% and the protein content was 92.5wt%.

[0029] Example 4 1. Preprocessing 800 mL of 90 wt % ethanol solution was added to 100 g of corn yellow powder, and the mixture was extracted at 73° C. and 120 rpm for 3.0 h. After the extraction, the anhydrous ethanol was removed by filtration, and the mixture was washed and dried to obtain the pretreated corn yellow powder.

[0030] 2. Preparation of enzyme carrier (1) Activation The diatomite was placed in a 22wt% hydrochloric acid solution of 8 times its mass, the temperature was raised to 37°C, and the solution was kept warm and soaked for 3.8 hours. After the soaking, the solution was filtered out and washed, and dried at 80°C for 12 hours. After the solution naturally recovered to room temperature, the solution was placed in a muffle furnace, the temperature was raised to 360°C at a rate of 3.5°C / min, and the solution was kept warm and calcined for 4.8 hours. After the calcination, the temperature was naturally lowered to room temperature to obtain activated diatomite. The particle size of the diatomaceous earth is 230 nm; (2) One-time processing 7.2 g of hydroxylamine hydrochloride was added to 100 g of deionized water and stirred to obtain a hydroxylamine hydrochloride solution; 4.8 g of chitosan was added to 100 g of 4.5 wt% acetic acid solution and stirred to obtain a chitosan solution; 20 g of activated diatomaceous earth was placed in 200 g of deionized water, the temperature was raised to 42°C, 37 g of hydroxylamine hydrochloride solution and 34 g of chitosan solution were slowly added, the addition rate was controlled to be 2.1 g / min, and the stirring speed was maintained at 245 rpm while adding. After the addition was completed, the temperature was continued to be raised to 69°C, and the mixture was stirred for 2.4 hours at the temperature. After washing and drying, a primary enzyme carrier was obtained; (3) Secondary processing Put 10.2g of primary carrier into 100g of N,N-dimethylformamide, stir evenly, add 3.7g of succinic anhydride and 0.38g of triethylamine, raise the temperature to 76°C, keep warm for 4.2h, after the reaction is finished, filter, wash and dry to obtain the enzyme carrier.

[0031] 3. Preparation of functional enzymes 3.0 g of enzyme carrier was mixed with 50 mL of phosphate buffer, stirred evenly, 0.26 g of enzyme was added, and the mixture was shaken at room temperature at a speed of 138 rpm for 3.8 h, and then 16.8 g of 1.4 wt% cinnamaldehyde solution was added, and the shaking reaction was continued for 2.1 h. After the reaction was completed, the mixture was centrifuged and washed, and then dried naturally to obtain a functional enzyme; The enzymes are Alcase 2.4L protease and Flavourzyme 1000L enzyme, and the mass ratio of the Alcase 2.4L protease to the Flavourzyme 1000L enzyme is 2:1; The molar concentration of the phosphate buffer is 0.05 ml / L, and the pH value is 8.

[0032] 4. Enzymatic hydrolysis Add 1000 g of deionized water to 100 g of pretreated corn yellow powder, adjust the pH to 8.0, add 40 g of functional enzyme, react at 56 ° C for 7.5 hours, and after the reaction is completed, obtain a clear liquid and a solid by filtration. Add 0.8 wt% of activated carbon to the clear liquid and stir for 1.3 hours. After stirring, evaporate and vacuum dry to obtain a corn oligopeptide product.

[0033] The corn oligopeptide product obtained by the method of Example 4 had a yield of 62.5% and a protein content of 93.4wt%.

[0034] The reusability of the functional enzyme prepared in Example 4 was tested. The specific method was as follows: after the enzymolysis reaction was completed, a clear liquid and a solid were obtained by filtration, the solid was washed with a phosphate buffer having a molar concentration of 0.05 ml / L and a pH of 8, washed 3 times, dried at 40° C. to constant weight, and then enzymolyzed according to the same operating steps as in the enzymolysis to obtain a corn oligopeptide product; the above operation was regarded as a cycle, and the above operation was repeated 20 times. The corn oligopeptide product obtained by testing had a yield of 58.7% and a protein content of 88.6%.

[0035] Comparative Example 1 On the basis of Example 4, the pretreatment step is omitted; in the enzymatic hydrolysis step, the pretreated corn yellow powder is replaced with an equal amount of untreated corn yellow powder; The rest of the operations are the same.

[0036] The corn oligopeptide product obtained by the method of Comparative Example 1 had a yield of 35.8% and a protein content of 68.5wt%.

[0037] Comparative Example 2 Based on Example 4, the steps of preparing the enzyme carrier and preparing the functional enzyme were omitted; The enzymolysis step comprises adding 1000 g of deionized water to 100 g of pretreated corn yellow powder, adjusting the pH to 8.0, adding 3.0 g of enzyme, reacting at 56° C. for 7.5 h, and after the reaction is completed, filtering to obtain a clear liquid, adding 0.8 wt % of activated carbon to the clear liquid, stirring for 1.3 h, and after the stirring is completed, performing vacuum drying after rotary evaporation to obtain a corn oligopeptide product; The enzymes are Alcase 2.4L protease and Flavourzyme 1000L enzyme, and the mass ratio of the Alcase 2.4L protease to the Flavourzyme 1000L enzyme is 2:1; The rest of the operations are the same.

[0038] The corn oligopeptide product obtained by the method of Comparative Example 2 had a yield of 42.3% and a protein content of 73.6wt%.

[0039] In the process of preparing corn oligopeptides, the corn yellow powder is firstly pretreated to remove pigments and grease, and then enzymolysis is performed using a functional enzyme. In the preparation method of the functional enzyme, diatomaceous earth is used as an enzyme carrier matrix, and the diatomaceous earth is firstly treated with a dilute acid solution to remove impurities on the surface, increase its specific surface area and the activity of the diatomaceous earth, and improve the stability of the diatomaceous earth after calcination. In a treatment step, amino groups are introduced on the surface of the diatomaceous earth by introducing hydroxylamine hydrochloride and diatomaceous earth, and chitosan molecules can also enhance the hydrophilicity and biocompatibility of the diatomaceous earth carrier. The obtained primary enzyme carrier reacts with succinic anhydride, and then carboxyl groups are introduced on the surface of the enzyme carrier. The hydrophilicity and reaction activity of the carrier are increased in one step; in the process of preparing the functional enzyme, a cinnamaldehyde solution is added, which can react with the enzyme molecules and the amino groups on the surface of the enzyme carrier, thereby enhancing the binding force between the enzyme and the enzyme carrier, thereby fixing the enzyme molecules on the enzyme carrier more firmly, ensuring the stability of the functional enzyme, and reducing the probability of enzyme inactivation and denaturation, so that the enzyme activity can be maintained for a long time during the enzymolysis process. Combined with the activated carbon adsorption step, it effectively removes the pigment and bitterness of the product, improves the reusability of the enzyme, and increases the contact area with the substrate, improves the enzymolysis efficiency, and promotes the enzymolysis reaction, thereby improving the product yield and protein content.

[0040] Comparative Example 1 omits the pretreatment step for corn yellow powder, which cannot effectively remove the pigment and oil of corn yellow powder, and the structure of corn protein is still relatively tight, which is not conducive to the subsequent binding and action of enzymes and protein molecules, thereby resulting in incomplete enzymatic hydrolysis, so that corn protein cannot be converted into oligopeptides, which exist in the solid matter in the form of larger molecular proteins, ultimately reducing the product yield and protein content; Comparative Example 2 does not load the enzyme, and in the enzymatic hydrolysis step, the contact area between the enzyme and the substrate is small, which affects the enzymatic hydrolysis reaction, and the reaction process is unstable, ultimately reducing the yield and protein content of corn oligopeptides.

[0041] Unless otherwise specified, all ratios and percentages described in the present invention are by mass ratios and percentages are by mass percentages.

[0042] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for producing corn oligopeptides, characterized in that: It includes pretreatment, preparation of enzyme carrier, preparation of functional enzyme and enzymolysis steps; The pretreatment step comprises adding a pretreatment liquid to the corn yellow powder to obtain pretreated corn yellow powder; The pretreatment solution is one of an ethanol solution or a sodium bicarbonate solution; The preparation of the enzyme carrier comprises activation, primary treatment and secondary treatment steps; The primary treatment step is to place the activated diatomaceous earth in deionized water, raise the temperature to 37-43° C., add hydroxylamine hydrochloride solution and chitosan solution, and then stir at 68-72° C. for 2.3-2.6 hours to obtain a primary enzyme carrier; The secondary treatment step is to put the primary carrier into N, N-dimethylformamide, stir evenly, add succinic anhydride and triethylamine, raise the temperature to 72-78°C, keep the temperature for reaction for 3.8-4.3h, and after the reaction is completed, filter, wash and dry to obtain the enzyme carrier.

2. The method for producing a corn oligopeptide according to claim 1, characterized in that: In the pretreatment step, the mass volume ratio of the corn yellow powder to the pretreatment liquid is 100g:800-1000mL; The adding of the pretreatment liquid treatment is a leaching treatment by adding a 50-90wt% ethanol solution or a stirring treatment by adding a 10-60wt% sodium bicarbonate solution; The leaching treatment is performed at 70-73° C. and 100-120 rpm for 3.0-3.5 hours; The stirring temperature is 45°C and the stirring time is 18 min. When adding 10-60wt% sodium bicarbonate solution and stirring, after the treatment, the pH can be adjusted to 4.7, and Rifola amylase is added, and stirring is continued for 1.0h. The Rifola amylase is 1.2wt% of the mass of the corn yellow powder.

3. The method for producing a corn oligopeptide according to claim 1, characterized in that: The activation step comprises placing the diatomite in a hydrochloric acid solution of 6-8 times its mass, raising the temperature to 35-40° C., soaking it at this temperature for 3.6-4.2 hours, filtering it out after soaking, washing it, drying it at 78-82° C. for 8-12 hours, allowing it to naturally return to room temperature, and then placing it in a muffle furnace, raising the temperature to 350-380° C. at a rate of 2.0-4.0° C. / min, and calcining it at this temperature for 4.7-5.3 hours. After the calcination is completed, naturally cooling it to room temperature to obtain activated diatomite; The particle size of the diatomaceous earth is 210-240nm; The mass concentration of the hydrochloric acid solution is 20-25%.

4. The method for producing a corn oligopeptide according to claim 1, characterized in that: The one-time treatment step is as follows: adding hydroxylamine hydrochloride into deionized water, stirring evenly, to obtain a hydroxylamine hydrochloride solution; adding chitosan into an acetic acid solution, stirring evenly, to obtain a chitosan solution; placing activated diatomaceous earth in deionized water, raising the temperature to 37-43° C., slowly adding the hydroxylamine hydrochloride solution and the chitosan solution, controlling the adding rate to be 1.8-2.2 g / min, and maintaining the stirring speed at 230-250 rpm while adding; after the addition is completed, continuing to raise the temperature to 68-72° C., keeping the temperature and stirring for 2.3-2.6 hours, and washing and drying to obtain a primary enzyme carrier.

5. The method for producing a corn oligopeptide according to claim 4, characterized in that: The mass ratio of hydroxylamine hydrochloride to deionized water is 6.5-7.5:100; The mass ratio of chitosan to acetic acid solution is 4.6-5.3:100; The mass concentration of the acetic acid solution is 3.0-5.0%; The mass ratio of the activated diatomaceous earth, deionized water, hydroxylamine hydrochloride solution and chitosan solution is 18-22:200:34-38:33-37.

6. The method for producing a corn oligopeptide according to claim 1, characterized in that: In the secondary treatment step, the mass ratio of the primary carrier, N,N-dimethylformamide, succinic anhydride and triethylamine is 9.5-10.5:100:3.4-3.8:0.37-0.

42.

7. The method for producing a corn oligopeptide according to claim 1, characterized in that: The steps of preparing the functional enzyme are as follows: mixing the enzyme carrier with the phosphate buffer, stirring evenly, adding the enzyme, oscillating at room temperature, the oscillation speed is 130-150 rpm, the oscillation time is 3.8-4.2 hours, then adding the cinnamaldehyde solution, continuing the oscillation reaction for 1.8-2.2 hours, and after the reaction is completed, centrifugation washing and natural drying are performed to obtain the functional enzyme.

8. The method for producing a corn oligopeptide according to claim 7, characterized in that: The mass volume ratio of the enzyme carrier, phosphate buffer, enzyme and cinnamaldehyde solution is 2.8-3.2 g:48-53 mL:0.24-0.26 g:16.0-17.0 g; The enzyme is one or both of Alcase2.4L protease and Flavourzyme1000L enzyme, and the mass ratio of Alcase2.4L protease to Flavourzyme1000L enzyme is 2:1; The mass concentration of the cinnamaldehyde solution is 1.3-1.7%; The molar concentration of the phosphate buffer is 0.04-0.06 ml / L, and the pH value is 7.8-8.

2.

9. The method for producing a corn oligopeptide according to claim 1, characterized in that: The enzymolysis step comprises adding deionized water to the pretreated corn yellow powder, adjusting the pH to 7.5-8.0, adding functional enzymes, reacting at 53-56° C. for 7.5-8.5 hours, filtering to obtain a clear liquid and a solid after the reaction is completed, adding 0.8-1.2 wt % of activated carbon to the clear liquid, stirring for 1.0-1.3 hours, and after the stirring is completed, performing vacuum drying after rotary evaporation to obtain a corn oligopeptide product; The mass ratio of the pretreated corn yellow powder, deionized water and functional enzyme is 100:900-1000:22-40.