A method for preparing plant-derived composite peptide trace element chelate for feed processing

Abstract

The invention discloses a method for preparing a plant-based composite peptide trace element chelate for feed processing, wherein rice bran is selected as a plant-based component, and rice bran protein is extracted therein, which is then degraded to produce small peptides, which are then further subjected to chelation reaction with an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate and manganese nitrate. The chelate obtained by the invention can be used for feed processing, has a high trace element chelation rate, is comprehensive in nutrition, has very excellent efficacy in feeding livestock and poultry, and has broad market prospects.

Description

Technical Field

[0001] The present invention relates to a peptide chelate, in particular to a method for preparing a plant-derived composite peptide trace element chelate for feed processing, belonging to the technical field of feed processing. Background Art

[0002] In addition to the common mineral elements such as calcium, phosphorus, sodium, potassium, etc., there are many other elements that are indispensable for the growth and metabolism of livestock and poultry. However, animals only need a very small amount of these elements, and their content in the animal body is less than one ten-thousandth of their body weight, so these elements are called trace elements. For example: copper, cobalt, iodine, zinc, selenium, fluorine, manganese, etc. Trace elements are important components of many enzyme systems in animal bodies, and they play a very important role in maintaining the normal physiological functions and health status of animals. Therefore, trace elements are essential for the growth and metabolism of animals, and once they are lacking, they will cause diseases.

[0003] Amino acid trace element chelate is the third generation of trace element feed additives, integrating amino acids and trace elements. It is formed by the coordination reaction of trace elements with amino acid hydroxyl groups. It has a unique chelate ring structure and is easily absorbed. It has the characteristics of stable chemical properties, high solubility, easy digestion and absorption, high biological efficacy, anti-interference, and no irritation. It has been gradually promoted and used in many foreign countries and is currently the most ideal trace element additive.

[0004] There are already trace element chelate products such as lysine iron, lysine copper, methionine iron, methionine copper, methionine cobalt, and glycine iron on the market, but the raw material cost of these products is high and the production process is relatively complicated. Studies have found that the end products of protein digestion in the digestive tract are often mostly small peptides rather than free amino acids. Small peptides can enter the systemic circulation intact through intestinal mucosal cells and directly participate in the synthesis of tissue proteins. Some small peptides with special physiological activities can participate in the physiological activities and metabolic regulation of the body and improve the immune ability of the animal body. Straight-chain peptides formed by 2 to 12 amino acid residues are usually called oligopeptides, also known as small peptides, short peptides, and micropeptides. Those with more than 12 but not more than 20 amino acid residues are called oligopeptides, and those containing more than 20 amino acid residues are called polypeptides. With the research on the absorption theory of small peptides, people have tried to use small peptides to chelate trace elements, which greatly improved the animal absorption rate of trace elements and reduced resource waste and environmental pollution. Therefore, the use of small peptides to chelate trace elements is of great significance for feed processing.

[0005] Small peptides can be obtained by hydrolyzing protein-containing raw materials with proteases, but the choice of raw materials and the combination of different protease systems have a great influence on the composition of the enzymatic hydrolysis products, which in turn affects the trace element chelation effect and the feeding effect on animals.

[0006] Patent CN114875103B discloses a small peptide chelated calcium and its preparation method and use. By selecting a combination of hemicellulase, α-galactosidase, acid protease and aminopeptidase, and controlling the order of enzyme addition and the interval time of addition, the enzymatic hydrolysis efficiency can be greatly improved, and the plant protein enzymes are hydrolyzed into small peptide molecules, so that the peptide content of the peptide molecules with a molecular weight below 1000 Daltons in the finally prepared small peptide chelated calcium reaches more than 98wt%, so that the obtained small peptide chelated calcium is 100% soluble in water, and the small peptide molecules can quickly transport calcium elements into cells to participate in life movements, greatly improving the absorption rate. However, this patented technology is mainly aimed at calcium chelation and cannot provide trace element nutritional support. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for preparing a plant-based composite peptide trace element chelate for feed processing. The plant-based protein is degraded into small peptide composite trace elements, which can be used for feed processing to provide sufficient trace element nutritional support for the growth of livestock and poultry.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for preparing a plant-derived composite peptide trace element chelate for feed processing, the specific steps are as follows:

[0010] (1) firstly ultrasonically disperse hexagonal mesoporous silica in an ethanol aqueous solution containing potassium hydroxide, slowly add chitosan gel while stirring, ultrasonically oscillate, centrifuge to obtain a precipitate, wash, and dry to obtain a hexagonal mesoporous silica-porous chitosan microsphere composite carrier; then use the composite carrier to load alkaline protease to obtain protease A;

[0011] (2) Glucose and calcium carbonate are used as raw materials to prepare hollow mesoporous carbon, and then the hollow mesoporous carbon, magnesium chloride, and selenium powder are compounded to prepare a mesoporous carbon composite carrier; and then the composite carrier is used to load flavor protease and papain to obtain protease B;

[0012] (3) extracting rice bran protein from the rice bran, ultrasonically dispersing the rice bran protein in deionized water, performing a primary enzymolysis using protease A, and then performing a secondary enzymolysis using protease B, deactivating the enzyme, and obtaining an enzymolysis product;

[0013] (4) inoculating the enzymatic hydrolysis product with a mixed seed liquid of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast, fermenting, filtering and collecting the filtrate, and finally adding an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate, and manganese nitrate to the filtrate, stirring for chelation reaction, and spray drying to obtain the product.

[0014] Preferably, in step (1), the preparation method of hexagonal mesoporous silicon is as follows, measured in parts by weight: first, 2 to 3 parts of dodecylamine, 25 to 30 parts of anhydrous ethanol, and 70 to 80 parts of deionized water are mixed evenly, then 12 to 15 parts of tetrabutyl orthosilicate are added dropwise, stirred at 300 to 400 r / min for 3 to 4 hours, allowed to stand at room temperature for 18 to 20 hours, centrifuged to obtain the precipitate, washed, and dried to obtain the product.

[0015] Preferably, in step (1), the preparation method of chitosan gel is as follows: first, chitosan is added to 0.01-0.02 mol / L ammonium acetate acetic acid solution which is 8-10 times its weight, and stirred at 200-300 r / min for 8-10 minutes.

[0016] Preferably, in step (1), the mass ratio of hexagonal mesoporous silica, ethanol aqueous solution containing potassium hydroxide, and chitosan gel is 1:6-7:0.03-0.05; the ethanol aqueous solution containing potassium hydroxide is obtained by adding potassium hydroxide to a 40-50% ethanol aqueous solution with a volume concentration of 2-3 times the weight of potassium hydroxide, and stirring and mixing.

[0017] Preferably, in step (1), the chitosan gel is added slowly and uniformly for 20 to 30 minutes.

[0018] Preferably, in step (1), the process conditions of ultrasonic oscillation are: 300-400W ultrasonic oscillation for 15-20 minutes.

[0019] Preferably, in step (1), the preparation method of protease A is as follows, based on weight: first, 1 part of alkaline protease is prepared into a 10-12 mg / mL alkaline protease solution using phosphate buffer, then 3-4 parts of the composite carrier are added to the alkaline protease solution, ultrasonically oscillated at 300-400W for 30-40 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1M, and the pH is 7.

[0020] Preferably, in step (2), the preparation method of the hollow mesoporous carbon is as follows, in parts by weight: first, 1 part of glucose is prepared into a glucose aqueous solution with a mass concentration of 20-30% using water, and then 3-4 parts of 100-200 mesh calcium carbonate powder are added, stirred and mixed, centrifuged to obtain a precipitate, dried, and heated to 850°C at 5°C / min under nitrogen protection, kept warm for 2 hours, naturally cooled to room temperature, and washed 5-7 times with 1 mol / L hydrochloric acid solution to obtain the product.

[0021] Preferably, in step (2), the preparation method of the mesoporous carbon composite carrier is as follows, in parts by weight: first, 1 part of hollow mesoporous carbon and 2 to 3 parts of magnesium chloride are ultrasonically dispersed in water, then 12 to 13 parts of urea are added, stirred sufficiently, heated to react, centrifuged to obtain a precipitate, and dried to obtain surface-modified mesoporous carbon, and then the surface-modified mesoporous carbon is mixed with 2 to 3 parts of selenium powder and heated under a nitrogen atmosphere to obtain the product.

[0022] More preferably, the process conditions of the heat treatment are: heating treatment at 400-420° C. for 2-3 hours.

[0023] Preferably, in step (2), the preparation method of protease B is as follows: first, flavor protease and papain are added together into a phosphate buffer, stirred and mixed to prepare a protease solution, then 3 to 4 times the weight of the composite carrier is added to the protease solution, ultrasonically oscillated at 300 to 400 W for 30 to 40 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1 M, pH = 7; the concentrations of flavor protease and papain contained in the protease solution are 8 to 10 mg / mL and 3 to 4 mg / mL, respectively.

[0024] Preferably, in step (3), the extraction method of rice bran protein is as follows, in parts by weight:

[0025] (A) First, 1 part of rice bran is added to 5-7 parts of normal hexane, and ultrasonic vibration is applied at 300-400W for 2-3 hours, and the precipitate is taken by centrifugation and dried to obtain defatted rice bran;

[0026] (B) Then, defatted rice bran is added to 10-12 parts of 0.2-0.3 mol / L sodium hydroxide solution, stirred at 40-50° C. for 4-5 hours, and the supernatant is collected by centrifugation. The pH is adjusted to 5.4 using 0.1-0.2 mol / L solution, and the precipitate is collected by centrifugation, washed with water, and dried to obtain rice bran protein.

[0027] Preferably, in step (3), the mass ratio of rice bran protein, deionized water, protease A and protease B is 1:15-20:3-4:3-4.

[0028] Preferably, in step (3), the process conditions for the primary enzymatic hydrolysis are: pH = 8-9, temperature 45-55°C, and time 3-4 hours; the process conditions for the secondary enzymatic hydrolysis are: pH = 6-7, temperature 50-55°C, and time 3-4 hours.

[0029] Preferably, in step (4), the bacterial contents of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast in the mixed seed solution are 2×10 9 cfu / mL, 5×10 8 cfu / mL, 2×109 cfu / mL.

[0030] Preferably, in step (4), the fermentation process conditions are: fermentation at 35-37° C. for 30-35 hours.

[0031] Preferably, in step (4), the mass ratio of the enzymatic hydrolysis product to the mixed seed liquid is 100:1-2.

[0032] Preferably, in step (4), the volume ratio of the filtrate to the aqueous solution is 1:1, and the mass concentration of each component in the aqueous solution is: copper nitrate 1-2%, cobalt nitrate 1-2%, zinc nitrate 1-2%, and manganese nitrate 1-2%.

[0033] Preferably, in step (4), the process conditions of the chelating reaction are: stirring speed 50-60 r / min, temperature 40-45° C., and reaction time 40-50 minutes.

[0034] Beneficial effects of the present invention:

[0035] The present invention selects rice bran as a plant-based component, extracts rice bran protein therein, and then degrades it to produce small peptides, which are further subjected to chelation reaction with an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate, and manganese nitrate, thereby obtaining a plant-based composite peptide trace element chelate. The chelate obtained by the present invention can be used for feed processing, has a high trace element chelation rate, is comprehensive in nutrition, has very excellent efficacy in feeding livestock and poultry, and has broad market prospects.

[0036] The invention first extracts rice bran protein from rice bran, ultrasonically disperses the rice bran protein in deionized water, performs enzymolysis once using protease A, then performs secondary enzymolysis using protease B, deactivates the enzyme, and obtains an enzymolysis product; then, a mixed seed liquid of plant lactobacillus, amyloliquefaciens, and selenium-enriched yeast is inoculated into the enzymolysis product, fermented, filtered to obtain a filtrate, and finally, an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate, and manganese nitrate is added to the filtrate, a chelate reaction is stirred, and spray-dried to obtain a final chelate product. Through two enzymolysis and one fermentation, the rice bran protein is fully degraded into small peptides, and the chelate reaction is promoted.

[0037] The technical key of the present invention is protease A and protease B, which are prepared by the following methods:

[0038] Protease A: First, ultrasonically disperse hexagonal mesoporous silica in an ethanol aqueous solution containing potassium hydroxide, slowly add chitosan gel while stirring, ultrasonically oscillate, centrifuge to obtain the precipitate, wash, and dry to obtain a hexagonal mesoporous silica-porous chitosan microsphere composite carrier; then use the composite carrier to load alkaline protease to obtain protease A.

[0039] Protease B: Glucose and calcium carbonate are used as raw materials to prepare hollow mesoporous carbon, and then the hollow mesoporous carbon, magnesium chloride and selenium powder are compounded to prepare a mesoporous carbon composite carrier; then the composite carrier is used to load flavor protease and papain to obtain protease B.

[0040] Different composite carriers of protease A and protease B load different proteases, which helps to promote the primary and secondary enzymolysis, thereby improving product performance. Different composite carriers have a better loading effect on the corresponding proteases, while promoting enzymolysis, improving the specific composition of small peptides, and improving product performance. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0042] Embodiment 1:

[0043] A method for preparing a plant-derived composite peptide trace element chelate for feed processing, the specific steps are as follows:

[0044] (1) firstly ultrasonically disperse hexagonal mesoporous silica in an ethanol aqueous solution containing potassium hydroxide, slowly add chitosan gel while stirring, ultrasonically oscillate, centrifuge to obtain a precipitate, wash, and dry to obtain a hexagonal mesoporous silica-porous chitosan microsphere composite carrier; then use the composite carrier to load alkaline protease to obtain protease A;

[0045] (2) Glucose and calcium carbonate are used as raw materials to prepare hollow mesoporous carbon, and then the hollow mesoporous carbon, magnesium chloride, and selenium powder are compounded to prepare a mesoporous carbon composite carrier; and then the composite carrier is used to load flavor protease and papain to obtain protease B;

[0046] (3) extracting rice bran protein from the rice bran, ultrasonically dispersing the rice bran protein in deionized water, performing a primary enzymolysis using protease A, and then performing a secondary enzymolysis using protease B, deactivating the enzyme, and obtaining an enzymolysis product;

[0047] (4) inoculating the enzymatic hydrolysis product with a mixed seed liquid of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast, fermenting, filtering and collecting the filtrate, and finally adding an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate, and manganese nitrate to the filtrate, stirring for chelation reaction, and spray drying to obtain the product.

[0048] Among them, in step (1), the preparation method of hexagonal mesoporous silicon is as follows: first, 0.2 kg of dodecylamine, 2.5 kg of anhydrous ethanol, and 7 kg of deionized water are mixed evenly, and then 1.2 kg of tetrabutyl orthosilicate is added dropwise, stirred at 300 r / min for 3 hours, and allowed to stand at room temperature for 18 hours. The precipitate is centrifuged, washed, and dried to obtain the product.

[0049] In step (1), the preparation method of chitosan gel is as follows: first, chitosan is added into 0.01 mol / L ammonium acetate acetic acid solution with a weight of 8 times of chitosan, and stirred at 200 r / min for 8 minutes.

[0050] In step (1), the mass ratio of hexagonal mesoporous silica, ethanol aqueous solution containing potassium hydroxide, and chitosan gel is 1:6:0.03; the ethanol aqueous solution containing potassium hydroxide is obtained by adding potassium hydroxide to a 40% ethanol aqueous solution with a volume concentration twice its weight and stirring and mixing.

[0051] In step (1), chitosan gel is added slowly and uniformly over a period of 20 minutes.

[0052] In step (1), the process conditions of ultrasonic oscillation are: 300W ultrasonic oscillation for 15 minutes.

[0053] In step (1), the preparation method of protease A is as follows: first, 0.1 kg of alkaline protease is prepared into a 10 mg / mL alkaline protease solution using phosphate buffer, then 0.3 kg of the composite carrier is added to the alkaline protease solution, ultrasonically oscillated at 300 W for 30 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1 M, and the pH is 7.

[0054] In step (2), the preparation method of the hollow mesoporous carbon is as follows: first, 0.1 kg of glucose is prepared into a glucose aqueous solution with a mass concentration of 20% using water, and then 0.3 kg of 100 mesh calcium carbonate powder is added, stirred and mixed, centrifuged to obtain a precipitate, dried, and heated to 850° C. at 5° C. / min under nitrogen protection, kept warm for 2 hours, naturally cooled to room temperature, and washed 5 times with 1 mol / L hydrochloric acid solution to obtain the obtained product.

[0055] In step (2), the preparation method of the mesoporous carbon composite carrier is as follows: first, 0.1 kg of hollow mesoporous carbon and 0.2 kg of magnesium chloride are ultrasonically dispersed in water, then 1.2 kg of urea is added, stirred sufficiently, heated for reaction, centrifuged to obtain the precipitate, and dried to obtain surface-modified mesoporous carbon, and then the surface-modified mesoporous carbon is mixed with 0.2 kg of selenium powder and heated at 400°C for 2 hours under a nitrogen atmosphere to obtain the product.

[0056] In step (2), the preparation method of protease B is as follows: first, flavor protease and papain are added to a phosphate buffer, stirred and mixed to prepare a protease solution, then 3 times the weight of the composite carrier is added to the protease solution, 300W ultrasonic oscillation is performed for 30 minutes, centrifugation is performed to obtain a precipitate, washing, and drying; the concentration of the phosphate buffer is 0.1M, pH=7; the concentrations of flavor protease and papain contained in the protease solution are 8mg / mL and 3mg / mL, respectively.

[0057] In step (3), the extraction method of rice bran protein is as follows:

[0058] (A) first 0.1 kg of rice bran was added to 0.5 kg of normal hexane, 300 W of ultrasonic vibration for 2 hours, centrifuged to obtain a precipitate, and dried to obtain defatted rice bran;

[0059] (B) Then, defatted rice bran was added to 1 kg of 0.2 mol / L sodium hydroxide solution, stirred at 40° C. for 4 hours, and the supernatant was collected by centrifugation. The pH value was adjusted to 5.4 using 0.1 mol / L solution, and the precipitate was collected by centrifugation. The precipitate was washed with water and dried to obtain rice bran protein.

[0060] In step (3), the mass ratio of rice bran protein, deionized water, protease A and protease B is 1:15:3:3.

[0061] In step (3), the process conditions for the first enzymatic hydrolysis are: pH=8, temperature 45°C, and time 3 hours; the process conditions for the second enzymatic hydrolysis are: pH=6, temperature 50°C, and time 3 hours.

[0062] In step (4), the bacterial contents of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast in the mixed seed solution are 2×10 9 cfu / mL, 5×10 8 cfu / mL, 2×10 9 cfu / mL.

[0063] In step (4), the fermentation process conditions are: fermentation at 35° C. for 30 hours.

[0064] In step (4), the mass ratio of the enzymatic hydrolysis product to the mixed seed solution is 100:1.

[0065] In step (4), the volume ratio of the filtrate to the aqueous solution is 1:1, and the mass concentration of each component in the aqueous solution is: 1% copper nitrate, 1% cobalt nitrate, 1% zinc nitrate, and 1% manganese nitrate.

[0066] In step (4), the process conditions of the chelating reaction are: stirring speed 50 r / min, temperature 40° C., and reaction time 40 minutes.

[0067] Embodiment 2:

[0068] A method for preparing a plant-derived composite peptide trace element chelate for feed processing, the specific steps are as follows:

[0069] (1) firstly ultrasonically disperse hexagonal mesoporous silica in an ethanol aqueous solution containing potassium hydroxide, slowly add chitosan gel while stirring, ultrasonically oscillate, centrifuge to obtain a precipitate, wash, and dry to obtain a hexagonal mesoporous silica-porous chitosan microsphere composite carrier; then use the composite carrier to load alkaline protease to obtain protease A;

[0070] (2) Glucose and calcium carbonate are used as raw materials to prepare hollow mesoporous carbon, and then the hollow mesoporous carbon, magnesium chloride, and selenium powder are compounded to prepare a mesoporous carbon composite carrier; and then the composite carrier is used to load flavor protease and papain to obtain protease B;

[0071] (3) extracting rice bran protein from the rice bran, ultrasonically dispersing the rice bran protein in deionized water, performing a primary enzymolysis using protease A, and then performing a secondary enzymolysis using protease B, deactivating the enzyme, and obtaining an enzymolysis product;

[0072] (4) inoculating the enzymatic hydrolysis product with a mixed seed liquid of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast, fermenting, filtering and collecting the filtrate, and finally adding an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate, and manganese nitrate to the filtrate, stirring for chelation reaction, and spray drying to obtain the product.

[0073] Among them, in step (1), the preparation method of hexagonal mesoporous silicon is as follows: first, 0.3 kg of dodecylamine, 3 kg of anhydrous ethanol, and 8 kg of deionized water are mixed evenly, and then 1.5 kg of tetrabutyl orthosilicate is added dropwise, stirred at 400 r / min for 4 hours, and allowed to stand at room temperature for 20 hours. The precipitate is centrifuged, washed, and dried to obtain the product.

[0074] In step (1), the preparation method of chitosan gel is as follows: first, chitosan is added into 0.02 mol / L ammonium acetate acetic acid solution with a weight of 10 times of chitosan, and stirred at 300 r / min for 10 minutes.

[0075] In step (1), the mass ratio of hexagonal mesoporous silica, ethanol aqueous solution containing potassium hydroxide, and chitosan gel is 1:7:0.05; the ethanol aqueous solution containing potassium hydroxide is obtained by adding potassium hydroxide to a 50% ethanol aqueous solution with a volume concentration of 3 times the weight of potassium hydroxide and stirring and mixing.

[0076] In step (1), chitosan gel is added slowly and uniformly over a period of 30 minutes.

[0077] In step (1), the process conditions of ultrasonic oscillation are: 400W ultrasonic oscillation for 20 minutes.

[0078] In step (1), the preparation method of protease A is as follows: first, 0.1 kg of alkaline protease is prepared into a 12 mg / mL alkaline protease solution using phosphate buffer, then 0.4 kg of the composite carrier is added to the alkaline protease solution, ultrasonically oscillated at 400 W for 40 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1 M, and the pH is 7.

[0079] In step (2), the preparation method of the hollow mesoporous carbon is as follows: first, 0.1 kg of glucose is prepared into a glucose aqueous solution with a mass concentration of 30% using water, and then 0.4 kg of 200 mesh calcium carbonate powder is added, stirred and mixed, centrifuged to obtain a precipitate, dried, and heated to 850° C. at 5° C. / min under nitrogen protection, kept warm for 2 hours, naturally cooled to room temperature, and washed 7 times with 1 mol / L hydrochloric acid solution to obtain the obtained product.

[0080] In step (2), the preparation method of the mesoporous carbon composite carrier is as follows: first, 0.1 kg of hollow mesoporous carbon and 0.3 kg of magnesium chloride are ultrasonically dispersed in water, then 1.3 kg of urea is added, stirred sufficiently, heated for reaction, centrifuged to obtain the precipitate, and dried to obtain surface-modified mesoporous carbon, and then the surface-modified mesoporous carbon is mixed with 0.3 kg of selenium powder and heated at 420° C. for 3 hours under a nitrogen atmosphere to obtain the product.

[0081] In step (2), the preparation method of protease B is as follows: first, flavor protease and papain are added to a phosphate buffer, stirred and mixed to prepare a protease solution, then 4 times the weight of the composite carrier is added to the protease solution, ultrasonically oscillated at 400W for 40 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1M, pH = 7; the concentrations of flavor protease and papain contained in the protease solution are 10 mg / mL and 4 mg / mL, respectively.

[0082] In step (3), the extraction method of rice bran protein is as follows:

[0083] (A) first 0.1 kg of rice bran was added to 0.7 kg of normal hexane, 400 W ultrasonic vibration was applied for 3 hours, and the precipitate was centrifuged and dried to obtain defatted rice bran;

[0084] (B) Then, defatted rice bran was added to 1.2 kg of 0.3 mol / L sodium hydroxide solution, stirred at 50° C. for 5 hours, and the supernatant was collected by centrifugation. The pH value was adjusted to 5.4 using 0.2 mol / L solution, and the precipitate was collected by centrifugation. The precipitate was washed with water and dried to obtain rice bran protein.

[0085] In step (3), the mass ratio of rice bran protein, deionized water, protease A and protease B is 1:20:4:4.

[0086] In step (3), the process conditions for the first enzymatic hydrolysis are: pH = 9, temperature 55°C, and time 4 hours; the process conditions for the second enzymatic hydrolysis are: pH = 7, temperature 55°C, and time 4 hours.

[0087] In step (4), the bacterial contents of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast in the mixed seed solution are 2×10 9 cfu / mL, 5×10 8 cfu / mL, 2×10 9 cfu / mL.

[0088] In step (4), the fermentation process conditions are: fermentation at 37° C. for 35 hours.

[0089] In step (4), the mass ratio of the enzymatic hydrolysis product to the mixed seed solution is 100:2.

[0090] In step (4), the volume ratio of the filtrate to the aqueous solution is 1:1, and the mass concentration of each component in the aqueous solution is: 1.5% copper nitrate, 1.5% cobalt nitrate, 1.5% zinc nitrate, and 1.5% manganese nitrate.

[0091] In step (4), the process conditions of the chelating reaction are: stirring speed 60 r / min, temperature 45° C., and reaction time 50 minutes.

[0092] Embodiment 3:

[0093] A method for preparing a plant-derived composite peptide trace element chelate for feed processing, the specific steps are as follows:

[0094] (1) firstly ultrasonically disperse hexagonal mesoporous silica in an ethanol aqueous solution containing potassium hydroxide, slowly add chitosan gel while stirring, ultrasonically oscillate, centrifuge to obtain a precipitate, wash, and dry to obtain a hexagonal mesoporous silica-porous chitosan microsphere composite carrier; then use the composite carrier to load alkaline protease to obtain protease A;

[0095] (2) Glucose and calcium carbonate are used as raw materials to prepare hollow mesoporous carbon, and then the hollow mesoporous carbon, magnesium chloride, and selenium powder are compounded to prepare a mesoporous carbon composite carrier; and then the composite carrier is used to load flavor protease and papain to obtain protease B;

[0096] (3) extracting rice bran protein from the rice bran, ultrasonically dispersing the rice bran protein in deionized water, performing a primary enzymolysis using protease A, and then performing a secondary enzymolysis using protease B, deactivating the enzyme, and obtaining an enzymolysis product;

[0097] (4) inoculating the enzymatic hydrolysis product with a mixed seed liquid of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast, fermenting, filtering and collecting the filtrate, and finally adding an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate, and manganese nitrate to the filtrate, stirring for chelation reaction, and spray drying to obtain the product.

[0098] Among them, in step (1), the preparation method of hexagonal mesoporous silicon is as follows: first, 0.25 kg of dodecylamine, 2.8 kg of anhydrous ethanol, and 7.5 kg of deionized water are mixed evenly, and then 1.3 kg of tetrabutyl orthosilicate is added dropwise, stirred at 400 r / min for 3.5 hours, and allowed to stand at room temperature for 19 hours. The precipitate is centrifuged, washed, and dried to obtain the product.

[0099] In step (1), the preparation method of chitosan gel is as follows: first, chitosan is added into 9 times its weight of 0.02 mol / L ammonium acetate acetic acid solution, and stirred at 300 r / min for 9 minutes to obtain the chitosan gel.

[0100] In step (1), the mass ratio of hexagonal mesoporous silica, ethanol aqueous solution containing potassium hydroxide, and chitosan gel is 1:6.5:0.04; the ethanol aqueous solution containing potassium hydroxide is obtained by adding potassium hydroxide to a 45% ethanol aqueous solution with a volume concentration of 2.5 times the weight of potassium hydroxide, and stirring and mixing.

[0101] In step (1), chitosan gel is added slowly and uniformly over a period of 25 minutes.

[0102] In step (1), the process conditions of ultrasonic oscillation are: 400W ultrasonic oscillation for 18 minutes.

[0103] In step (1), the preparation method of protease A is as follows: first, 0.1 kg of alkaline protease is prepared into an alkaline protease solution of 11 mg / mL using phosphate buffer, then 0.35 kg of the composite carrier is added to the alkaline protease solution, ultrasonically oscillated at 400 W for 35 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1 M, and the pH is 7.

[0104] In step (2), the preparation method of the hollow mesoporous carbon is as follows: first, 0.1 kg of glucose is prepared into a glucose aqueous solution with a mass concentration of 25% using water, and then 0.35 kg of 200 mesh calcium carbonate powder is added, stirred and mixed, centrifuged to obtain a precipitate, dried, and heated to 850° C. at 5° C. / min under nitrogen protection, kept warm for 2 hours, naturally cooled to room temperature, and washed 6 times with 1 mol / L hydrochloric acid solution to obtain the obtained product.

[0105] In step (2), the preparation method of the mesoporous carbon composite carrier is as follows: first, 0.1 kg of hollow mesoporous carbon and 0.25 kg of magnesium chloride are ultrasonically dispersed in water, then 1.3 kg of urea is added, stirred thoroughly, heated for reaction, centrifuged to obtain the precipitate, and dried to obtain surface-modified mesoporous carbon, and then the surface-modified mesoporous carbon is mixed with 0.25 kg of selenium powder and heated at 410°C for 2.5 hours under a nitrogen atmosphere to obtain the product.

[0106] In step (2), the preparation method of protease B is as follows: first, flavor protease and papain are added together into a phosphate buffer, stirred and mixed to prepare a protease solution, then 3.5 times the weight of the composite carrier is added into the protease solution, ultrasonically oscillated at 400W for 35 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1M, and the pH is 7; the concentrations of flavor protease and papain contained in the protease solution are 9 mg / mL and 3.5 mg / mL, respectively.

[0107] In step (3), the extraction method of rice bran protein is as follows:

[0108] (A) first 0.1 kg of rice bran was added to 0.6 kg of normal hexane, 400 W ultrasonic vibration for 2.5 hours, centrifuged to obtain a precipitate, and dried to obtain defatted rice bran;

[0109] (B) Then, defatted rice bran was added to 1.1 kg of 0.25 mol / L sodium hydroxide solution, stirred at 45° C. for 4.5 hours, and the supernatant was collected by centrifugation. The pH value was adjusted to 5.4 using 0.15 mol / L solution, and the precipitate was collected by centrifugation. The precipitate was washed with water and dried to obtain rice bran protein.

[0110] In step (3), the mass ratio of rice bran protein, deionized water, protease A and protease B is 1:18:3.5:3.5.

[0111] In step (3), the process conditions for the primary enzymolysis are: pH = 9, temperature 50°C, and time 3.5 hours; the process conditions for the secondary enzymolysis are: pH = 6, temperature 52°C, and time 3.5 hours.

[0112] In step (4), the bacterial contents of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast in the mixed seed solution are 2×10 9 cfu / mL, 5×10 8 cfu / mL, 2×10 9 cfu / mL.

[0113] In step (4), the fermentation process conditions are: fermentation at 36° C. for 33 hours.

[0114] In step (4), the mass ratio of the enzymatic hydrolysate to the mixed seed solution is 100:1.5.

[0115] In step (4), the volume ratio of the filtrate to the aqueous solution is 1:1, and the mass concentration of each component in the aqueous solution is: 2% copper nitrate, 2% cobalt nitrate, 2% zinc nitrate, and 2% manganese nitrate.

[0116] In step (4), the process conditions of the chelating reaction are: stirring speed 60 r / min, temperature 42° C., and reaction time 45 minutes.

[0117] Comparative Example

[0118] A method for preparing a plant-derived composite peptide trace element chelate for feed processing, the specific steps are as follows:

[0119] (1) firstly ultrasonically disperse hexagonal mesoporous silica in an ethanol aqueous solution containing potassium hydroxide, slowly add chitosan gel while stirring, ultrasonically oscillate, centrifuge to obtain a precipitate, wash, and dry to obtain a hexagonal mesoporous silica-porous chitosan microsphere composite carrier; then use the composite carrier to load alkaline protease to obtain protease A;

[0120] (2) using the composite carrier obtained in step (1) to load flavor protease and papain to obtain protease B;

[0121] (3) extracting rice bran protein from the rice bran, ultrasonically dispersing the rice bran protein in deionized water, performing a primary enzymolysis using protease A, and then performing a secondary enzymolysis using protease B, deactivating the enzyme, and obtaining an enzymolysis product;

[0122] (4) inoculating the enzymatic hydrolysis product with a mixed seed liquid of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast, fermenting, filtering and collecting the filtrate, and finally adding an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate, and manganese nitrate to the filtrate, stirring for chelation reaction, and spray drying to obtain the product.

[0123] Among them, in step (1), the preparation method of hexagonal mesoporous silicon is as follows: first, 0.2 kg of dodecylamine, 2.5 kg of anhydrous ethanol, and 7 kg of deionized water are mixed evenly, and then 1.2 kg of tetrabutyl orthosilicate is added dropwise, stirred at 300 r / min for 3 hours, and allowed to stand at room temperature for 18 hours. The precipitate is centrifuged, washed, and dried to obtain the product.

[0124] In step (1), the preparation method of chitosan gel is as follows: first, chitosan is added into 0.01 mol / L ammonium acetate acetic acid solution with a weight of 8 times of chitosan, and stirred at 200 r / min for 8 minutes.

[0125] In step (1), the mass ratio of hexagonal mesoporous silica, ethanol aqueous solution containing potassium hydroxide, and chitosan gel is 1:6:0.03; the ethanol aqueous solution containing potassium hydroxide is obtained by adding potassium hydroxide to a 40% ethanol aqueous solution with a volume concentration twice its weight and stirring and mixing.

[0126] In step (1), chitosan gel is added slowly and uniformly over a period of 20 minutes.

[0127] In step (1), the process conditions of ultrasonic oscillation are: 300W ultrasonic oscillation for 15 minutes.

[0128] In step (1), the preparation method of protease A is as follows: first, 0.1 kg of alkaline protease is prepared into a 10 mg / mL alkaline protease solution using phosphate buffer, then 0.3 kg of the composite carrier is added to the alkaline protease solution, ultrasonically oscillated at 300 W for 30 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1 M, and the pH is 7.

[0129] In step (2), the preparation method of protease B is as follows: first, flavor protease and papain are added to a phosphate buffer, stirred and mixed to prepare a protease solution, then 3 times the weight of the composite carrier is added to the protease solution, 300W ultrasonic oscillation is performed for 30 minutes, centrifugation is performed to obtain a precipitate, washing, and drying; the concentration of the phosphate buffer is 0.1M, pH=7; the concentrations of flavor protease and papain contained in the protease solution are 8mg / mL and 3mg / mL, respectively.

[0130] In step (3), the extraction method of rice bran protein is as follows:

[0131] (A) first 0.1 kg of rice bran was added to 0.5 kg of normal hexane, 300 W of ultrasonic vibration for 2 hours, centrifuged to obtain a precipitate, and dried to obtain defatted rice bran;

[0132] (B) Then, defatted rice bran was added to 1 kg of 0.2 mol / L sodium hydroxide solution, stirred at 40° C. for 4 hours, and the supernatant was collected by centrifugation. The pH value was adjusted to 5.4 using 0.1 mol / L solution, and the precipitate was collected by centrifugation. The precipitate was washed with water and dried to obtain rice bran protein.

[0133] In step (3), the mass ratio of rice bran protein, deionized water, protease A and protease B is 1:15:3:3.

[0134] In step (3), the process conditions for the first enzymatic hydrolysis are: pH=8, temperature 45°C, and time 3 hours; the process conditions for the second enzymatic hydrolysis are: pH=6, temperature 50°C, and time 3 hours.

[0135] In step (4), the bacterial contents of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast in the mixed seed solution are 2×10 9 cfu / mL, 5×10 8 cfu / mL, 2×10 9 cfu / mL.

[0136] In step (4), the fermentation process conditions are: fermentation at 35° C. for 30 hours.

[0137] In step (4), the mass ratio of the enzymatic hydrolysis product to the mixed seed solution is 100:1.

[0138] In step (4), the volume ratio of the filtrate to the aqueous solution is 1:1, and the mass concentration of each component in the aqueous solution is: 1% copper nitrate, 1% cobalt nitrate, 1% zinc nitrate, and 1% manganese nitrate.

[0139] In step (4), the process conditions of the chelating reaction are: stirring speed 50 r / min, temperature 40° C., and reaction time 40 minutes.

[0140] Test example

[0141] The feeding effects of Examples 1 to 3 and the comparative example were investigated respectively, and the specific method was as follows: 120 healthy AA broiler chicks of 1 day old were selected, half of which were male and half were female, and randomly divided into 4 groups. After the chicks hatched, they were kept in flat cages at a temperature of 25°C, and fed with complete chick feed (purchased from Lingyuan Qiushi Feed Co., Ltd.) + 0.1 g / kg / d of the obtained chelate, and free access to food and water.

[0142] After 28 days of feeding, the average weight gain, feed consumption and feed-to-meat ratio were calculated and the results are shown in Table 1.

[0143] Table 1. Comparison of feeding effects

[0144] Average weight gain (g) Feed consumption (g) Feed to meat ratio Example 1 1833.1 3702.5 2.02 Example 2 1833.5 3685.3 2.01 Example 3 1902.1 3671.1 1.93 Comparative Example 1469.7 3615.4 2.46

[0145] As shown in Table 1, the chelates of Examples 1 to 3, as feed additives, significantly improve the average weight gain and feed consumption of chicks, have a low feed-to-meat ratio, and have a good feeding effect.

[0146] In the comparative example, the mesoporous carbon composite carrier was replaced by a hexagonal mesoporous silica-porous chitosan microsphere composite carrier to obtain protease B. The feeding effect was significantly worse, indicating that different enzymes loaded with different carriers have a great influence on the enzymatic effect, thereby affecting the product performance.

[0147] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A method for preparing a plant-derived composite peptide trace element chelate for feed processing, characterized in that: The specific steps are as follows: (1) firstly ultrasonically disperse hexagonal mesoporous silica in an ethanol aqueous solution containing potassium hydroxide, slowly add chitosan gel while stirring, ultrasonically oscillate, centrifuge to obtain a precipitate, wash, and dry to obtain a hexagonal mesoporous silica-porous chitosan microsphere composite carrier; then use the composite carrier to load alkaline protease to obtain protease A; (2) Glucose and calcium carbonate are used as raw materials to prepare hollow mesoporous carbon, and then the hollow mesoporous carbon, magnesium chloride, and selenium powder are compounded to prepare a mesoporous carbon composite carrier; and then the composite carrier is used to load flavor protease and papain to obtain protease B; (3) extracting rice bran protein from the rice bran, ultrasonically dispersing the rice bran protein in deionized water, performing a primary enzymolysis using protease A, and then performing a secondary enzymolysis using protease B, deactivating the enzyme, and obtaining an enzymolysis product; (4) inoculating a mixed seed liquid of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast into the enzymatic hydrolyzate, fermenting, filtering and collecting the filtrate, and finally adding an aqueous solution containing copper nitrate, cobalt nitrate, zinc nitrate, and manganese nitrate to the filtrate, stirring for chelation reaction, and spray drying to obtain; In step (1), the preparation method of the hexagonal mesoporous silicon is as follows, in parts by weight: first, 2 to 3 parts of dodecylamine, 25 to 30 parts of anhydrous ethanol, and 70 to 80 parts of deionized water are mixed evenly, then 12 to 15 parts of tetrabutyl orthosilicate are added dropwise, stirred at 300 to 400 r / min for 3 to 4 hours, allowed to stand at room temperature for 18 to 20 hours, centrifuged to obtain a precipitate, washed, and dried to obtain the product; The preparation method of chitosan gel is as follows: firstly, chitosan is added into 0.01-0.02 mol / L ammonium acetate acetic acid solution of 8-10 times its weight, and stirred at 200-300 r / min for 8-10 minutes; The mass ratio of hexagonal mesoporous silica, ethanol aqueous solution containing potassium hydroxide, and chitosan gel is 1:6-7:0.03-0.05; the ethanol aqueous solution containing potassium hydroxide is obtained by adding potassium hydroxide to a 40%-50% ethanol aqueous solution with a volume concentration of 2-3 times the weight of the potassium hydroxide, and stirring and mixing. The preparation method of protease A is as follows: first, 1 part of alkaline protease is prepared into a 10-12 mg / mL alkaline protease solution using a phosphate buffer, then 3-4 parts of the composite carrier are added to the alkaline protease solution, ultrasonically oscillated at 300-400W for 30-40 minutes, centrifuged to obtain a precipitate, washed, and dried; the concentration of the phosphate buffer is 0.1M, and the pH is 7; In step (2), the preparation method of the hollow mesoporous carbon is as follows, in parts by weight: first, 1 part of glucose is prepared into a glucose aqueous solution with a mass concentration of 20% to 30% using water, then 3 to 4 parts of 100 to 200 mesh calcium carbonate powder are added, stirred and mixed, centrifuged to obtain a precipitate, dried, and heated to 850° C. at 5° C. / min under nitrogen protection, kept warm for 2 hours, naturally cooled to room temperature, and washed 5 to 7 times with 1 mol / L hydrochloric acid solution to obtain the hollow mesoporous carbon; The preparation method of the mesoporous carbon composite carrier is as follows: firstly, 1 part of hollow mesoporous carbon and 2-3 parts of magnesium chloride are ultrasonically dispersed in water, then 12-13 parts of urea are added, stirred fully, heated for reaction, centrifuged to obtain a precipitate, dried to obtain surface-modified mesoporous carbon, then the surface-modified mesoporous carbon is mixed with 2-3 parts of selenium powder and heated in a nitrogen atmosphere to obtain the composite carrier; The preparation method of protease B is as follows: first, flavor protease and papain are added to a phosphate buffer solution, stirred and mixed to prepare a protease solution, then 3 to 4 times the weight of the composite carrier is added to the protease solution, 300 to 400W ultrasonic oscillation is performed for 30 to 40 minutes, centrifuged to obtain a precipitate, washed, and dried to obtain the protease B; the concentration of the phosphate buffer solution is 0.1M, and the pH value is 7; the concentrations of flavor protease and papain contained in the protease solution are 8 to 10 mg / mL and 3 to 4 mg / mL, respectively; In step (3), the extraction method of rice bran protein is as follows, by weight: (A) First, 1 part of rice bran is added to 5-7 parts of normal hexane, and ultrasonic vibration is applied at 300-400W for 2-3 hours, and the precipitate is taken by centrifugation and dried to obtain defatted rice bran; (B) adding defatted rice bran to 10-12 parts of 0.2-0.3 mol / L sodium hydroxide solution, stirring at 40-50° C. for 4-5 hours, centrifuging to obtain supernatant, adjusting pH to 5.4 with 0.1-0.2 mol / L solution, centrifuging to obtain precipitate, washing with water, and drying to obtain rice bran protein; The mass ratio of rice bran protein, deionized water, protease A and protease B is 1:15-20:3-4:3-4; In step (3), the process conditions for the primary enzymolysis are: pH = 8-9, temperature 45-55°C, and time 3-4 hours; the process conditions for the secondary enzymolysis are: pH = 6-7, temperature 50-55°C, and time 3-4 hours.

2. The preparation method according to claim 1, characterized in that: In step (4), the bacterial contents of Lactobacillus plantarum, Bacillus amyloliquefaciens, and selenium-enriched yeast in the mixed seed solution are 2×10 9 cfu / mL, 5×10 8 cfu / mL, 2×10 9 cfu / mL; The fermentation process conditions are: fermentation at 35-37°C for 30-35 hours; The mass ratio of the enzymatic hydrolysate to the mixed seed solution is 100:1-2; The volume ratio of the filtrate to the aqueous solution is 1:1, and the mass concentration of each component in the aqueous solution is: 1% to 2% copper nitrate, 1% to 2% cobalt nitrate, 1% to 2% zinc nitrate, and 1% to 2% manganese nitrate; The process conditions of the chelating reaction are: stirring speed 50-60 r / min, temperature 40-45° C., and reaction time 40-50 minutes.