Protein-decomposing enzyme for plant forage and method for preparing the same

Abstract

This invention belongs to the field of feed additive technology, specifically relating to a plant-based protein-degrading enzyme and its preparation method. The protein-degrading enzyme is composed of feed-grade protease, plant protease, tannic acid-crosslinked chitosan stabilized microparticles, Sophora japonica extract calcium alginate slow-release microparticles, a plant carrier, a protectant, a buffer salt, and a flow aid. In preparation, a mixture of porous plant carriers, a pre-stabilized enzyme mixture, a pre-slow-release mixture, and a complex protease protectant mixture are first prepared, then mixed, spray-dried, granulated, dried, and sieved to obtain the product. This invention can improve the stability and slow-release properties of the protease, promote the decomposition of proteins in plant-based feed into small peptides and amino acids, improve feed utilization, reduce fecal nitrogen emissions, and facilitate the continuous degradation of proteins in uneaten feed, feces, and organic matter.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a protein-degrading enzyme for plant feed and its preparation method. Background Technology

[0002] With the development of ecological farming, forage resource utilization, and circular agriculture, the proportion of plant-based feed used in livestock, poultry, and aquaculture is constantly increasing. Plant-based feed typically consists of forage powder, soybean meal, wheat bran, rice bran, agricultural by-product protein feed, and various herbal raw materials, offering advantages such as wide availability, low cost, good palatability, and strong ecological attributes. However, the proteins in plant-based feeds are often bound to fiber, polyphenols, phytic acid, starch, and cell wall structures. Some proteins are encapsulated, entangled, or cross-linked, making them difficult for animals to fully release and digest after direct ingestion, resulting in insufficient protein nutrient utilization in the feed. Undigested and unabsorbed protein substances are excreted in feces and continue to enter the pen, pond, bottom mud, or soil environment, not only wasting nitrogen nutrients but also easily converting into ammonia nitrogen and other nitrogenous metabolites under the action of microorganisms, affecting the farming environment and water stability.

[0003] Currently, proteases are often added to feed to improve protein digestibility and utilization. However, most common protease products focus on short-range digestion in the animal's gastrointestinal tract, and do not adequately consider continuous application scenarios such as pretreatment of plant feed, sustained intestinal decomposition after feeding, degradation of residual protein in feces, decomposition of uneaten feed in water, and release of organic protein from soil. Due to significant variations in moisture content, salinity, pH, and microbial environment within the forage system, single free proteases are prone to activity decline during storage, mixing, granulation, drying, transportation, and use. Once in water or soil, they are easily diluted, adsorbed, or inactivated, making it difficult to maintain continuous decomposition capabilities. Furthermore, some immobilized or encapsulated enzyme preparations use synthetic polymers, complex cross-linking agents, or high-cost carriers, resulting in high costs, complex processes, insufficient ecological compatibility, and difficulties in large-scale promotion, failing to meet the practical needs of low-cost, high-volume use of plant feed and its circular use in ecological agriculture.

[0004] Therefore, there is an urgent need to develop a compound protein-degrading enzyme suitable for plant-based feeds. This enzyme should not only promote the conversion of large protein molecules into small peptides and amino acids during feed processing and early feeding, but also improve protein absorption and utilization after animal consumption, and further reduce the emission of undigested protein and nitrogenous organic matter in feces. Such products should also address the need for continuous degradation of proteinaceous organic matter in aquaculture ponds, including uneaten feed, feces, and bottom sediment, as well as the nitrogen release requirements during pond water reuse, manure application to fields, and organic matter maturation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a protein-degrading enzyme for plant feed and its preparation method.

[0006] In a first aspect, the present invention provides a proteolytic enzyme for plant feed, comprising the following components in parts by weight: Feed-grade alkaline protease 18-28 parts, feed-grade neutral protease 12-22 parts, feed-grade acidic protease 5-10 parts, food-grade papain 3-8 parts, food-grade bromelain 1-4 parts, tannic acid cross-linked chitosan enzyme stabilizers 3-8 parts, Sophora japonica extract calcium alginate slow-release microparticles 2-6 parts, feed-grade wheat bran 15-25 parts, feed-grade corn cob powder 12-22 parts, feed-grade alfalfa meal 6-12 parts, feed-grade rice bran meal 8-16 parts, feed-grade soybean peptide powder 2-6 parts, food-grade maltodextrin 3-8 parts, food-grade trehalose 1-4 parts, food-grade glycerol 1-3 parts, food-grade light calcium carbonate 2-5 parts, food-grade anhydrous calcium chloride 0.3-1.0 parts, feed-grade magnesium sulfate heptahydrate 0.2-0.8 parts, food-grade sodium chloride 0.2-0.8 parts, food-grade silicon dioxide 0.5-2.0 parts.

[0007] A second aspect of the present invention provides a method for preparing the aforementioned plant protein-degrading enzyme, comprising the following steps: S1. By weight, under stirring, mix 15-25 parts of feed-grade wheat bran, 12-22 parts of feed-grade corn cob powder, 6-12 parts of feed-grade alfalfa meal, 8-16 parts of feed-grade rice bran meal, 2-5 parts of food-grade light calcium carbonate, 0.5-2.0 parts of food-grade silica, 0.2-0.8 parts of food-grade sodium chloride, and 0.2-0.8 parts of feed-grade magnesium sulfate heptahydrate to obtain a plant porous carrier mixture; mix 3-8 parts of tannic acid cross-linked chitosan enzyme-stabilizing microparticles with 0.5-1.5 parts of feed-grade soybean peptide powder and 0.8-2 parts of food-grade maltodextrin to obtain a first pre-stabilized enzyme mixture; mix 2-6 parts of Sophora japonica extract calcium alginate slow-release microparticles with 0.3-1... 0.0 parts of feed-grade soybean peptide powder and 0.5-1.5 parts of food-grade maltodextrin are mixed to obtain a second pre-sustaining mixture; 18-28 parts of feed-grade alkaline protease, 12-22 parts of feed-grade neutral protease, 5-10 parts of feed-grade acidic protease, 3-8 parts of food-grade papain and 1-4 parts of food-grade bromelain are mixed, and 1.2-3.5 parts of feed-grade soybean peptide powder, 1.7-4.5 parts of food-grade maltodextrin, 1-4 parts of food-grade trehalose and 1-3 parts of food-grade glycerol are added and stirred to obtain a complex protease protected mixture; under stirring, the first pre-stabilized enzyme mixture and the second pre-sustaining mixture are added to the complex protease protected mixture to obtain a stabilized enzyme mixture; S2. Add the enzyme-stabilizing mixture to the plant porous carrier mixture, and spray in a deionized aqueous solution containing 0.3-1.0 parts of food-grade anhydrous calcium chloride while stirring to granulate and obtain wet granules; dry and sieve the wet granules.

[0008] In this invention, during the preparation of the plant-based protein-degrading enzyme, feed-grade wheat bran, feed-grade corn cob powder, feed-grade alfalfa hay powder, and feed-grade rice bran meal together constitute a porous plant carrier. The fibrous pores can adsorb powder and moisture, providing a supporting framework for enzyme granulation. Food-grade light calcium carbonate provides a mild buffering environment, food-grade silica reduces powder adhesion, and food-grade sodium chloride and feed-grade magnesium sulfate heptahydrate regulate the ionic environment of the system, jointly maintaining the conformational stability of the protease. After tannic acid-crosslinked chitosan enzyme-stabilizing microparticles are premixed with feed-grade soybean peptide powder and food-grade maltodextrin, the feed-grade soybean peptide powder provides substrate competitive protection for the enzyme protein, and the food-grade maltodextrin improves the dispersibility of the enzyme-stabilizing microparticles. After Sophora japonica extract calcium alginate slow-release microparticles are premixed with feed-grade soybean peptide powder and food-grade maltodextrin, the aggregation of the slow-release microparticles is inhibited, and they are uniformly distributed in the composite system. Feed-grade alkaline protease, feed-grade neutral protease, feed-grade acidic protease, food-grade papain, and food-grade bromelain collectively form a complex protein hydrolysis system adapted to different acidic and alkaline environments. Food-grade trehalose and food-grade glycerol reduce enzyme activity loss during mixing, granulation, and drying through water retention and conformational protection. After the stable enzyme mixture is mixed with a porous plant carrier mixture, a deionized aqueous solution containing food-grade anhydrous calcium chloride is sprayed in to wet the material and promote granulation. At the same time, it allows the local alginate structure to continue to calcify and solidify. After low-temperature granulation, low-temperature drying, and sieving, a plant-based protein hydrolyzing enzyme that can be stably dispersed and gradually releases protease is obtained.

[0009] According to a preferred embodiment of the present invention, in step S1, the stirring temperature is 15-25°C and the stirring time is 10-20 min.

[0010] According to a preferred embodiment of the present invention, in step S2, the drying temperature is 35-45°C.

[0011] According to a preferred embodiment of the present invention, the method for preparing the tannic acid-crosslinked chitosan enzyme-stabilizing microparticles includes: A1. By weight, add 8-12 parts of food-grade chitosan and 3-6 parts of food-grade lactic acid to 90-120 parts of deionized water and stir to obtain a chitosan lactate solution; add 3-6 parts of food-grade tannic acid, 1-2 parts of food-grade glycerol and 2-4 parts of food-grade maltodextrin to 30-50 parts of deionized water and stir to obtain a tannic acid protective solution; under stirring, add the tannic acid protective solution to the chitosan lactate solution and stir to obtain a tannic acid-chitosan composite solution; dissolve 1.0-2.0 parts of food-grade sodium tripolyphosphate in 20-30 parts of deionized water to obtain an ion-crosslinking solution; under stirring, dropwise add the ion-crosslinking solution to the tannic acid-chitosan composite solution to perform ion crosslinking and obtain a wet enzyme-stabilized gel; A2. Add 0.5-1.0 parts of food-grade silica to the wet enzyme stabilized gel, stir, and obtain an enzyme stabilized gel slurry; dry the enzyme stabilized gel slurry to obtain the dried product; grind the dried product and vibrate sieve.

[0012] In this invention, during the preparation of tannic acid-crosslinked chitosan enzyme-stabilizing microparticles, the amino groups on the molecular chain of food-grade chitosan undergo protonation under the combined action of deionized water and food-grade lactic acid. These amino groups combine with the food-grade lactic acid to form a chitosan lactate solution. The food-grade chitosan, originally insoluble in water, transforms into a uniformly dispersed, positively charged polysaccharide solution, providing molecular-level active sites for subsequent reactions. After mixing food-grade tannic acid with food-grade glycerol and food-grade maltodextrin in deionized water, hydrogen bonds form between the polyphenolic hydroxyl groups of the food-grade tannic acid and the hydroxyl groups of the food-grade glycerol and maltodextrin. This stabilizes the food-grade tannic acid in the aqueous phase and reduces its oxidation tendency, forming a tannic acid protective solution. After adding the tannic acid protective solution to the chitosan lactate solution, multi-point hydrogen bonds are formed between the polyphenolic hydroxyl groups of food-grade tannic acid and the amino and hydroxyl groups on the food-grade chitosan molecular chain. Simultaneously, hydrophobic association occurs between the aromatic backbone of food-grade tannic acid and the hydrophobic segments of food-grade chitosan, enhancing the bonding strength of the composite network and yielding a tannic acid-chitosan composite solution. Food-grade sodium tripolyphosphate dissolved in deionized water forms an ionic cross-linking solution. When added dropwise to the tannic acid-chitosan composite solution, the polyphosphate groups in the sodium tripolyphosphate attract electrostatically with the protonated amino groups of the food-grade chitosan, forming ionic bonds. This causes the composite system to gradually solidify from a solution state into a wet enzyme-stabilized gel. After adding food-grade silica to the wet enzyme-stabilized gel, it disperses between the gel particles, reducing particle adhesion and agglomeration during drying and improving the flowability of the powder obtained from subsequent grinding and vibratory sieving. This yields tannic acid-crosslinked chitosan enzyme-stabilized microparticles with a combination of polyphenol protection, chitosan coating, and ionic cross-linking structure.

[0013] According to a preferred embodiment of the present invention, in step A1, the dropping time of the ionic crosslinking liquid into the tannic acid-chitosan composite liquid is 30-50 min.

[0014] According to a preferred embodiment of the present invention, in step A2, the drying temperature is 45-55°C.

[0015] According to a preferred embodiment of the present invention, the method for preparing the Sophora japonica extract calcium alginate sustained-release microparticles includes: B1. By weight, add 10-14 parts of food-grade sodium alginate to 120-160 parts of deionized water and stir to obtain a sodium alginate solution; mix 0.8-1.5 parts of Sophora japonica extract and 30-45 parts of edible ethanol and stir to obtain an ethanol dispersion of Sophora japonica extract; while stirring, add the ethanol dispersion of Sophora japonica extract dropwise to the sodium alginate solution and stir to combine to obtain a Sophora japonica extract-sodium alginate composite solution; B2. Under stirring, add 1.2-2.0 parts of a deionized aqueous solution containing food-grade anhydrous calcium chloride to the sodium alginate complex solution of Sophora japonica extract to carry out ion gelation. Add 1-2 parts of food-grade trehalose and 1-3 parts of food-grade maltodextrin, stir, and obtain a slow-release gel slurry. Dry the slow-release gel slurry to obtain the dried product. Grind the dried product and vibrate sieve it.

[0016] In this invention, during the preparation of the Sophora japonica extract calcium alginate sustained-release microparticles, food-grade sodium alginate is hydrated and swollen in deionized water. The carboxyl groups and hydroxyl groups on the molecular chain fully combine with water molecules to form a viscoelastic sodium alginate solution. After mixing and stirring the Sophora japonica extract with edible ethanol, the flavonoid components, represented by rutin and quercetin, dissolve and are uniformly dispersed in the edible ethanol, resulting in an ethanol dispersion of the Sophora japonica extract. When the ethanol dispersion of the Sophora japonica extract is added dropwise to the sodium alginate solution, the edible ethanol is gradually diluted with deionized water to avoid excessively high local organic phase concentrations that could cause precipitation of food-grade sodium alginate. Simultaneously, the phenolic hydroxyl groups of the flavonoid components in the Sophora japonica extract undergo hydrogen bonding association with the carboxyl groups and hydroxyl groups on the molecular chain of the food-grade sodium alginate, and through hydrophobic association between the aromatic rings of flavonoids and the segments of the food-grade sodium alginate chain, the Sophora japonica extract is embedded in the food-grade sodium alginate dispersion system, resulting in a Sophora japonica extract sodium alginate composite solution. When a deionized aqueous solution containing food-grade anhydrous calcium chloride is added dropwise to a composite solution of Sophora japonica extract and sodium alginate, calcium ions coordinate and crosslink with the carboxylate groups on the sodium alginate molecular chains. This crosslinks the originally linear or entangled sodium alginate segments to form a calcium alginate gel network, encapsulating the Sophora japonica extract within the gel structure. After ionogelation, food-grade trehalose and maltodextrin are added. During drying, trehalose provides a glassy protective effect, mitigating the damage to the gel structure caused by moisture removal. Food-grade maltodextrin enhances the film-forming properties and supporting strength of the dried particles. After drying, grinding, and vibrating sieving, sustained-release microparticles of Sophora japonica extract and calcium alginate with sustained-release encapsulation are obtained.

[0017] According to a preferred embodiment of the present invention, in step B1, the temperature for stirring and compounding is 45-55°C, and the stirring and compounding time is 40-60 min.

[0018] According to a preferred embodiment of the present invention, in step B2, the drying temperature is 45-55°C.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention utilizes a combination of feed-grade alkaline protease, feed-grade neutral protease, feed-grade acidic protease, food-grade papain, and food-grade bromelain to enable the protein-degrading enzyme for plant feed to adapt to different acid-base environments during feed pretreatment, animal digestion, and organic matter decomposition. The feed-grade alkaline protease, feed-grade neutral protease, feed-grade acidic protease, food-grade papain, and food-grade bromelain act on proteins in different binding states within the plant feed, promoting the gradual decomposition of large protein molecules into small peptides and amino acids, thereby increasing the release of protein nutrients from the plant feed. Feed-grade soybean peptide powder, food-grade maltodextrin, food-grade trehalose, and food-grade glycerol participate in the formation of a complex protease-protected mixture, reducing enzyme activity loss during mixing, granulation, and drying, thus maintaining good protein-degrading capacity of the protein-degrading enzyme for plant feed during storage and use.

[0020] (2) This invention uses tannic acid-crosslinked chitosan-stabilized enzyme microparticles and Sophora japonica extract calcium alginate slow-release microparticles as protective and slow-release components. The tannic acid-crosslinked chitosan-stabilized enzyme microparticles are made from food-grade chitosan, food-grade lactic acid, food-grade tannic acid, food-grade glycerol, food-grade maltodextrin, food-grade sodium tripolyphosphate, and food-grade silica. Chitosan coating, polyphenol protection, and ionic crosslinking can enhance the enzyme preparation's resistance to inactivation and reduce the impact of moisture, salt, and drying processes on the enzyme protein structure. The Sophora japonica extract calcium alginate slow-release microparticles are made from food-grade sodium alginate, Sophora japonica extract, edible ethanol, food-grade anhydrous calcium chloride, food-grade trehalose, and food-grade maltodextrin. They can form a loaded slow-release structure, allowing the active components to be slowly released in forage, water, and organic matter environments, thus prolonging the protein decomposition time.

[0021] (3) This invention uses feed-grade wheat bran, feed-grade corn cob powder, feed-grade alfalfa hay powder, and feed-grade rice bran meal as porous plant carriers, which can improve the compatibility and dispersion uniformity of the plant feed protein-degrading enzyme with the plant feed system. Food-grade light calcium carbonate, food-grade silicon dioxide, food-grade sodium chloride, and feed-grade magnesium sulfate heptahydrate can improve the flowability, buffering, and granulation stability of the powder, making the resulting product easier to mix, granulate, and use. After the plant feed protein-degrading enzyme is used in the plant feed, it can improve the degree of pre-decomposition of feed protein and the absorption and utilization rate of animal feed, and reduce the excretion of undigested protein with feces; it continues to play a protein decomposition role in the residual feed, feces, and organic matter environment, which is conducive to reducing the accumulation of nitrogen-containing organic matter, improving the breeding environment, and the utilization effect of circular agriculture. Detailed Implementation

[0022] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0023] Example 1

[0024] This embodiment provides a method for preparing a proteolytic enzyme for plant feed, the steps of which include: S1. Under stirring, mix 20g of feed-grade wheat bran, 17g of feed-grade corn cob powder, 9g of feed-grade alfalfa meal, 12g of feed-grade rice bran meal, 3.5g of food-grade light calcium carbonate, 1.25g of food-grade silica, 0.5g of food-grade sodium chloride, and 0.5g of feed-grade magnesium sulfate heptahydrate for 20 minutes to obtain a plant porous carrier mixture; mix 5.5g of tannic acid cross-linked chitosan enzyme-stabilizing microparticles with 1g of feed-grade soybean peptide powder and 1.4g of food-grade maltodextrin for 15 minutes to obtain the first pre-stabilized enzyme mixture; mix 4g of Sophora japonica extract calcium alginate slow-release microparticles with 0.65g of feed-grade soybean peptide powder and 1g of food-grade maltodextrin Mix for 15 min to obtain the second pre-sustaining mixture; mix 23 g of feed-grade alkaline protease, 17 g of feed-grade neutral protease, 7.5 g of feed-grade acidic protease, 5.5 g of food-grade papain and 2.5 g of food-grade bromelain for 10 min, add 2.35 g of feed-grade soybean peptide powder, 3.1 g of food-grade maltodextrin, 2.5 g of food-grade trehalose and 2 g of food-grade glycerol, stir at 20 °C for 15 min to obtain the complex protease protected mixture; while stirring, add the first pre-stabilized enzyme mixture and the second pre-sustaining mixture to the complex protease protected mixture, and continue stirring for 15 min to obtain the stabilized enzyme mixture; S2. Add the enzyme-stabilizing mixture to the plant porous carrier mixture and stir for 15 min. Add 0.65 g of food-grade anhydrous calcium chloride to 20 g of deionized water and stir until completely dissolved to obtain a deionized aqueous solution containing food-grade anhydrous calcium chloride. While stirring, spray the deionized aqueous solution containing food-grade anhydrous calcium chloride evenly into the material. After spraying, continue stirring for 10 min and granulate to obtain wet granules. Dry the wet granules at 40℃ until the moisture content is no higher than 10%, and sieve to obtain plant protein-degrading enzyme with a particle size of 0.3 mm-1.2 mm.

[0025] Preparation steps of tannic acid cross-linked chitosan stabilized enzyme microparticles: A1. Add 105g of deionized water to a container equipped with a stirrer, maintain the water temperature at 40℃, add 10g of food-grade chitosan and 4.5g of food-grade lactic acid, and stir mechanically for 60 minutes until the food-grade chitosan is evenly dispersed and forms a chitosan lactate solution without obvious agglomerates; Separately, take 40g of deionized water, maintain the water temperature at 30℃, add 4.5g of food-grade tannic acid, 1.5g of food-grade glycerol and 3g of food-grade maltodextrin, and stir for 30 minutes to obtain a tannic acid protective solution. Under stirring, tannic acid protective solution was added to chitosan lactate solution over 10 minutes. After addition, stirring was continued for 40 minutes to obtain tannic acid-chitosan composite solution. 1.5g of food-grade sodium tripolyphosphate was added to 25g of deionized water and stirred until completely dissolved to obtain ionic crosslinking solution. Under stirring, ionic crosslinking solution was added dropwise to tannic acid-chitosan composite solution over 40 minutes. After addition, stirring was continued for 20 minutes to carry out ionic crosslinking and obtain wet enzyme-stabilized gel. A2. Add 0.75g of food-grade silica to the wet enzyme-stabilizing gel and continue stirring for 20min to ensure uniform dispersion of the food-grade silica, thus obtaining an enzyme-stabilizing gel slurry. Spread the enzyme-stabilizing gel slurry into a 3mm thick layer and dry it at 50℃ until the moisture content is no higher than 8%, thus obtaining the dried product. Grind the dried product and vibrate it through a 180μm sieve to obtain tannic acid cross-linked chitosan enzyme-stabilizing microparticles, and take 5.5g for later use.

[0026] Preparation steps of Sophora japonica flower bud extract calcium alginate sustained-release microparticles: B1. Add 140g of deionized water to a container equipped with a stirrer, control the water temperature to 50℃, add 12g of food-grade sodium alginate, stir for 60min until the food-grade sodium alginate is fully hydrated to obtain a sodium alginate solution; mix 1.15g of Sophora japonica extract and 37.5g of edible ethanol, stir at 50℃ for 30min to obtain an ethanol dispersion of Sophora japonica extract; while stirring, add the ethanol dispersion of Sophora japonica extract dropwise to the sodium alginate solution over a period of 20min, and after the addition is complete, stir at 50℃ for 50min to obtain a composite solution of Sophora japonica extract and sodium alginate; B2. Add 1.6g of food-grade anhydrous calcium chloride to 30g of deionized water and stir until completely dissolved to obtain a food-grade anhydrous calcium chloride deionized water solution. While stirring, add the food-grade anhydrous calcium chloride deionized water solution dropwise to the Sophora japonica extract sodium alginate composite solution over 30 minutes. After the addition is complete, continue stirring for 20 minutes to perform ion gelation. Add 1.5g of food-grade trehalose and 2g of food-grade maltodextrin and continue stirring for 20 minutes to obtain a slow-release gel slurry. Spread the slow-release gel slurry into a 3mm thick layer and dry it at 50℃ until the moisture content is no higher than 8% to obtain the dried product. Grind the dried product and vibrate it through a 180μm sieve to obtain Sophora japonica extract calcium alginate slow-release microparticles. Take 4g for later use.

[0027] Example 2

[0028] The difference between this embodiment and Embodiment 1 is that this embodiment provides a method for preparing a proteolytic enzyme for plant feed, the steps of which include: S1. Under stirring, mix 15g of feed-grade wheat bran, 12g of feed-grade corn cob powder, 6g of feed-grade alfalfa meal, 8g of feed-grade rice bran meal, 2g of food-grade light calcium carbonate, 0.5g of food-grade silica, 0.2g of food-grade sodium chloride, and 0.2g of feed-grade magnesium sulfate heptahydrate to obtain a plant porous carrier mixture; mix 3g of tannic acid cross-linked chitosan enzyme-stabilizing microparticles with 0.5g of feed-grade soybean peptide powder and 0.8g of food-grade maltodextrin to obtain a first pre-stabilized enzyme mixture; mix 2g of Sophora japonica extract calcium alginate slow-release microparticles with 0.3g of feed-grade soybean peptide powder and 0.5g of food-grade magnesium sulfate heptahydrate. A second pre-sustaining mixture was obtained by mixing 18g of feed-grade alkaline protease, 12g of feed-grade neutral protease, 5g of feed-grade acidic protease, 3g of food-grade papain, and 1g of food-grade bromelain. Then, 1.2g of feed-grade soybean peptide powder, 1.7g of food-grade maltodextrin, 1g of food-grade trehalose, and 1g of food-grade glycerol were added, and the mixture was stirred at 15°C for 10 minutes to obtain a complex protease-protected mixture. While stirring, the first pre-stabilized enzyme mixture and the second pre-sustaining mixture were added to the complex protease-protected mixture to obtain a stabilized enzyme mixture. S2. Add the enzyme stabilizer mixture to the plant porous carrier mixture, and spray in a deionized aqueous solution containing 0.3g of food-grade anhydrous calcium chloride while stirring to granulate and obtain wet granules; dry and sieve the wet granules at 35℃ to obtain plant protein degrading enzyme.

[0029] Preparation steps of tannic acid cross-linked chitosan stabilized enzyme microparticles: A1. Add 8g of food-grade chitosan and 3g of food-grade lactic acid to 90g of deionized water, and stir until the food-grade chitosan is evenly dispersed to obtain a chitosan lactate solution; add 3g of food-grade tannic acid, 1g of food-grade glycerol and 2g of food-grade maltodextrin to 30g of deionized water, and stir until the material is uniform to obtain a tannic acid protective solution; while stirring, add the tannic acid protective solution to the chitosan lactate solution, and continue stirring to fully combine the food-grade tannic acid and food-grade chitosan to obtain a tannic acid-chitosan composite solution; dissolve 1g of food-grade sodium tripolyphosphate in 20g of deionized water to obtain an ion-crosslinking solution; while stirring, add the ion-crosslinking solution dropwise to the tannic acid-chitosan composite solution over a period of 30 minutes to carry out ion crosslinking and obtain a wet-state enzyme-stabilized gel; A2. Add 0.5g of food-grade silica to the wet enzyme-stabilizing gel and stir until the food-grade silica is evenly dispersed to obtain an enzyme-stabilizing gel slurry. Dry the enzyme-stabilizing gel slurry at 45℃ to obtain the dried product. Grind the dried product and vibrate it to obtain tannic acid cross-linked chitosan enzyme-stabilizing microparticles. Take 3g for later use.

[0030] Preparation steps of Sophora japonica flower bud extract calcium alginate sustained-release microparticles: B1. Add 10g of food-grade sodium alginate to 120g of deionized water and stir until the sodium alginate is fully hydrated to obtain a sodium alginate solution; mix 0.8g of Sophora japonica extract and 30g of edible ethanol and stir to obtain an ethanol dispersion of Sophora japonica extract; while stirring, add the ethanol dispersion of Sophora japonica extract dropwise to the sodium alginate solution and stir and compound at 45℃ for 40min to obtain a Sophora japonica extract-sodium alginate composite solution; B2. Under stirring, add a deionized aqueous solution containing 1.2g of food-grade anhydrous calcium chloride to the Sophora japonica extract sodium alginate composite solution to carry out ion gelation. Add 1g of food-grade trehalose and 1g of food-grade maltodextrin, and continue stirring until the material is uniform to obtain a slow-release gel slurry. Dry the slow-release gel slurry at 45℃ to obtain the dried product. Grind the dried product and vibrate and sieve to obtain Sophora japonica extract calcium alginate slow-release microparticles. Take 2g for later use.

[0031] Example 3

[0032] The difference between this embodiment and Embodiment 1 is that this embodiment provides a method for preparing a proteolytic enzyme for plant feed, the steps of which include: S1. Under stirring, mix 25g of feed-grade wheat bran, 22g of feed-grade corn cob powder, 12g of feed-grade alfalfa meal, 16g of feed-grade rice bran meal, 5g of food-grade light calcium carbonate, 2g of food-grade silica, 0.8g of food-grade sodium chloride, and 0.8g of feed-grade magnesium sulfate heptahydrate to obtain a plant porous carrier mixture; mix 8g of tannic acid cross-linked chitosan enzyme-stabilizing microparticles with 1.5g of feed-grade soybean peptide powder and 2g of food-grade maltodextrin to obtain a first pre-stabilized enzyme mixture; mix 6g of Sophora japonica extract calcium alginate slow-release microparticles with 1g of feed-grade soybean peptide powder and 1.5g of food-grade maltodextrin... The feed-grade maltodextrin was mixed to obtain a second pre-sustaining mixture; 28g of feed-grade alkaline protease, 22g of feed-grade neutral protease, 10g of feed-grade acidic protease, 8g of food-grade papain, and 4g of food-grade bromelain were mixed, and 3.5g of feed-grade soybean peptide powder, 4.5g of food-grade maltodextrin, 4g of food-grade trehalose, and 3g of food-grade glycerol were added. The mixture was stirred at 25°C for 20 minutes to obtain a complex protease-protected mixture; while stirring, the first pre-stabilized enzyme mixture and the second pre-sustaining mixture were added to the complex protease-protected mixture to obtain a stabilized enzyme mixture; S2. Add the enzyme stabilizer mixture to the plant porous carrier mixture, and spray in a deionized aqueous solution containing 1g of food-grade anhydrous calcium chloride while stirring to granulate and obtain wet granules; dry and sieve the wet granules at 45℃ to obtain plant protein degrading enzyme.

[0033] Preparation steps of tannic acid cross-linked chitosan stabilized enzyme microparticles: A1. Add 12g of food-grade chitosan and 6g of food-grade lactic acid to 120g of deionized water, and stir until the food-grade chitosan is evenly dispersed to obtain a chitosan lactate solution; add 6g of food-grade tannic acid, 2g of food-grade glycerol and 4g of food-grade maltodextrin to 50g of deionized water, and stir until the material is uniform to obtain a tannic acid protective solution; while stirring, add the tannic acid protective solution to the chitosan lactate solution, and continue stirring to fully combine the food-grade tannic acid and food-grade chitosan to obtain a tannic acid-chitosan composite solution; dissolve 2g of food-grade sodium tripolyphosphate in 30g of deionized water to obtain an ion-crosslinking solution; while stirring, add the ion-crosslinking solution dropwise to the tannic acid-chitosan composite solution over a time of 50min to carry out ion crosslinking and obtain a wet-state enzyme-stabilized gel; A2. Add 1g of food-grade silica to the wet enzyme-stabilizing gel and stir until the food-grade silica is evenly dispersed to obtain an enzyme-stabilizing gel slurry. Dry the enzyme-stabilizing gel slurry at 55℃ to obtain the dried product. Grind the dried product and vibrate and sieve to obtain tannic acid cross-linked chitosan enzyme-stabilizing microparticles. Take 8g for later use.

[0034] Preparation steps of Sophora japonica flower bud extract calcium alginate sustained-release microparticles: B1. Add 14g of food-grade sodium alginate to 160g of deionized water and stir until the sodium alginate is fully hydrated to obtain a sodium alginate solution; mix 1.5g of Sophora japonica extract and 45g of edible ethanol and stir to obtain an ethanol dispersion of Sophora japonica extract; while stirring, add the ethanol dispersion of Sophora japonica extract dropwise to the sodium alginate solution and stir and compound at 55℃ for 60min to obtain a Sophora japonica extract-sodium alginate composite solution; B2. Under stirring, add a deionized aqueous solution containing 2g of food-grade anhydrous calcium chloride to the Sophora japonica extract sodium alginate composite solution to carry out ion gelation. Add 2g of food-grade trehalose and 3g of food-grade maltodextrin, and continue stirring until the material is uniform to obtain a slow-release gel slurry. Dry the slow-release gel slurry at 55℃ to obtain the dried product. Grind the dried product and vibrate it to obtain Sophora japonica extract calcium alginate slow-release microparticles. Take 6g for later use.

[0035] Comparative Example 1 The difference between this comparative example and Example 1 is that 5.5g of tannic acid cross-linked chitosan enzyme-stabilizing microparticles are not added, and 5.5g of feed-grade wheat bran is used instead of 5.5g of tannic acid cross-linked chitosan enzyme-stabilizing microparticles. The rest is the same as in Example 1.

[0036] Comparative Example 2 The difference between this comparative example and Example 1 is that 4g of Sophora japonica extract calcium alginate slow-release microparticles are not added, and 4g of feed-grade wheat bran is used instead of 4g of Sophora japonica extract calcium alginate slow-release microparticles. The rest is the same as in Example 1.

[0037] Comparative Example 3 The difference between this comparative example and Example 1 is that 5.5g of tannic acid cross-linked chitosan enzyme-stabilizing microparticles and 4g of Sophora japonica extract calcium alginate slow-release microparticles are not added, and 9.5g of feed-grade wheat bran is used to replace 5.5g of tannic acid cross-linked chitosan enzyme-stabilizing microparticles and 4g of Sophora japonica extract calcium alginate slow-release microparticles. The rest is the same as in Example 1.

[0038] The performance of the proteolytic enzymes for plant feed obtained in Examples 1-3 and Comparative Examples 1-3 was tested.

[0039] Plant feed prepared in Examples 1, 2, 3, 1, 2, and 3 were used as test samples with proteolytic enzymes. Three parallel samples were set up for each sample. Before the test, the samples were placed in an environment of 25°C and 50% relative humidity for 12 hours to equilibrate.

[0040] For the enzyme activity retention rate test after drying, 1.000g of the pre-granulation complex protease protection mixture was first added to 100mL of phosphate buffer at pH 7.0 and extracted by shaking at 25℃ for 30min. After centrifugation, the supernatant was used as the pre-granulation enzyme solution. Then, 1.000g of the dried and sieved product was taken and the enzyme solution was extracted in the same way. The enzyme solution was then reacted with a 1% casein solution at 40℃ for 10min. The reaction was terminated by adding trichloroacetic acid. After centrifugation, the amount of tyrosine released in the supernatant was measured. The percentage of the enzyme activity of the finished product relative to the enzyme activity of the pre-granulation complex protease protection mixture was used as the enzyme activity retention rate after drying.

[0041] When testing the enzyme activity retention rate after 30 days of storage at 40℃, the finished product sample was sealed and stored in a constant temperature and humidity environment of 40℃ and 75% relative humidity for 30 days. After taking it out, the residual enzyme activity was determined according to the casein substrate method described above, and the percentage of enzyme activity after storage to enzyme activity before storage was taken as the enzyme activity retention rate after 30 days of storage at 40℃.

[0042] For the test of forage protein hydrolysis rate, soybean meal, feed-grade wheat bran, feed-grade alfalfa meal, and feed-grade rice bran meal were mixed in a mass ratio of 4:3:2:1 to prepare simulated plant feed. 100g of simulated plant feed was added to 200g of deionized water, and then 1.0g of the sample to be tested was added. The mixture was kept at 37℃ for 6h. After the reaction was completed, the mixture was centrifuged, and the acid-soluble protein content in the supernatant was measured. The forage protein hydrolysis rate was calculated by combining the original total protein content of the simulated plant feed.

[0043] During the small peptide release test, the supernatant obtained from the protein hydrolysis rate test of the forage was taken, trichloroacetic acid was added to precipitate the macromolecular protein, and after centrifugation again, the small peptide content in the supernatant was measured. The results were calculated based on the mass of small peptides released per 1g of simulated plant forage.

[0044] For the 72-hour water slow-release residual enzyme activity test, 1.0 g of the sample to be tested was added to 1000 mL of deionized water and stirred at low speed at 28 °C for 72 hours. After centrifugation, the residual protease activity was determined according to the casein substrate method. The percentage of residual protease activity after 72 hours to the initial released protease activity was taken as the 72-hour water slow-release residual enzyme activity.

[0045] For ammonia nitrogen concentration testing, fish meal, soybean meal powder, and dried fecal powder were mixed in a mass ratio of 3:5:2 to prepare a simulated uneaten feed fecal substrate. 50g of the simulated uneaten feed fecal substrate was added to 500mL of deionized water, followed by 0.5g of the sample to be tested. The mixture was reacted at 28℃ for 72h. After the reaction was completed, the mixture was filtered, and the ammonia nitrogen concentration of the filtrate was measured. The results are expressed in mg / L.

[0046] The performance test data above are shown in Table 1.

[0047] Table 1: Performance Test Results

[0048] The test results in Table 1 above clearly show that Examples 1-3 effectively solved the technical problems of large enzyme activity loss, insufficient storage stability, low protein hydrolysis efficiency, short duration of action in aquatic environments, and insufficient ammonia nitrogen control after degradation of residual feed and feces for protein-degrading enzymes used in ordinary plant feeds, compared with Comparative Examples 1-3.

[0049] The enzyme activity retention rates after drying in Examples 1-3 were 91.8%, 88.6%, and 93.5%, respectively, which were significantly higher than those in Comparative Example 1 (76.4%), Comparative Example 2 (84.7%), and Comparative Example 3 (68.9%). This indicates that the combined use of tannic acid-crosslinked chitosan enzyme-stabilizing microparticles and Sophora japonica extract calcium alginate sustained-release microparticles can more effectively protect against protease formation during granulation and drying, reducing enzyme activity loss caused by hot and humid environments and the granulation process.

[0050] The enzyme activity retention rates of Examples 1-3 after 30 days of storage at 40℃ were 86.5%, 82.1%, and 88.9%, respectively, which were significantly higher than those of Comparative Example 1 (62.8%), Comparative Example 2 (77.3%), and Comparative Example 3 (54.2%). Among them, the storage retention rate of Comparative Example 1 decreased significantly after the absence of tannic acid cross-linked chitosan enzyme-stabilizing microparticles, indicating that the enzyme-stabilizing microparticles play a key role in improving long-term storage stability.

[0051] The forage protein hydrolysis rates in Examples 1-3 reached 42.6%, 38.1%, and 48.8%, respectively, and the small peptide release amounts reached 96.8 mg / g, 94.2 mg / g, and 102.4 mg / g, respectively. These were all higher than those of Comparative Example 3 (28.9% and 62.7 mg / g), and also better than Comparative Example 1 (lacking tannic acid-crosslinked chitosan stabilizing microparticles) and Comparative Example 2 (lacking Sophora japonica extract calcium alginate slow-release microparticles). This indicates that the synergistic use of tannic acid-crosslinked chitosan stabilizing microparticles and Sophora japonica extract calcium alginate slow-release microparticles can improve the dispersibility and effective action time of proteases in the plant-based forage system, enabling the large protein molecules in the forage to be more fully converted into small peptides and absorbable amino acids, thereby solving the problems of insufficient release of plant-based forage protein and low animal utilization.

[0052] The residual enzyme activities of the slow-release enzymes in water after 72 hours in Examples 1-3 were 68.4%, 61.2%, and 72.6%, respectively, which were significantly higher than those in Comparative Example 2 (38.6%) and Comparative Example 3 (31.4%). This indicates that the slow-release microparticles of calcium alginate from Sophora japonica extract can significantly prolong the residual activity and continuous release time of the protease in water.

[0053] The ammonia nitrogen concentrations after treating simulated uneaten feed and feces in Examples 1-3 were 1.18 mg / L, 1.46 mg / L, and 0.96 mg / L, respectively, which were lower than those in Comparative Example 1 (1.63 mg / L), Comparative Example 2 (1.92 mg / L), and Comparative Example 3 (2.38 mg / L). This indicates that the present invention can promote the continuous decomposition of protein substances in uneaten feed, feces, and organic matter, and reduce the accumulation of nitrogenous organic matter and the subsequent ammonia nitrogen release pressure.

[0054] In summary, Examples 1-3 improve the enzyme preparation's resistance to inactivation and storage stability by cross-linking chitosan with tannic acid to stabilize the enzyme microparticles, improve the residual enzyme activity in water by using calcium alginate slow-release microparticles from Sophora japonica extract, and improve the hydrolysis rate of forage protein and the release of small peptides by combining a complex protease system. Compared with Comparative Examples 1-3, these examples simultaneously solve the problems of poor stability, short-lasting release, insufficient pre-decomposition of forage protein, and poor control of nitrogen pollution in the aquaculture environment of existing plant-based forage enzyme preparations.

Claims

1. A protein-degrading enzyme for plant feed, characterized in that, Includes the following components in parts by weight: Feed-grade alkaline protease 18-28 parts, feed-grade neutral protease 12-22 parts, feed-grade acidic protease 5-10 parts, food-grade papain 3-8 parts, food-grade bromelain 1-4 parts, tannic acid cross-linked chitosan enzyme stabilizers 3-8 parts, Sophora japonica extract calcium alginate slow-release microparticles 2-6 parts, feed-grade wheat bran 15-25 parts, feed-grade corn cob powder 12-22 parts, feed-grade alfalfa meal 6-12 parts, feed-grade rice bran meal 8-16 parts, feed-grade soybean peptide powder 2-6 parts, food-grade maltodextrin 3-8 parts, food-grade trehalose 1-4 parts, food-grade glycerol 1-3 parts, food-grade light calcium carbonate 2-5 parts, food-grade anhydrous calcium chloride 0.3-1.0 parts, feed-grade magnesium sulfate heptahydrate 0.2-0.8 parts, food-grade sodium chloride 0.2-0.8 parts, food-grade silicon dioxide 0.5-2.0 parts.

2. A method for preparing a proteolytic enzyme for plant feed according to claim 1, characterized in that the step... include: S1. By weight, under stirring, mix 15-25 parts of feed-grade wheat bran, 12-22 parts of feed-grade corn cob powder, 6-12 parts of feed-grade alfalfa meal, 8-16 parts of feed-grade rice bran meal, 2-5 parts of food-grade light calcium carbonate, 0.5-2.0 parts of food-grade silica, 0.2-0.8 parts of food-grade sodium chloride, and 0.2-0.8 parts of feed-grade magnesium sulfate heptahydrate to obtain a plant porous carrier mixture; mix 3-8 parts of tannic acid cross-linked chitosan enzyme-stabilizing microparticles with 0.5-1.5 parts of feed-grade soybean peptide powder and 0.8-2 parts of food-grade maltodextrin to obtain a first pre-stabilized enzyme mixture; mix 2-6 parts of Sophora japonica extract calcium alginate slow-release microparticles with 0.3-1... 0.0 parts of feed-grade soybean peptide powder and 0.5-1.5 parts of food-grade maltodextrin are mixed to obtain a second pre-sustaining mixture; 18-28 parts of feed-grade alkaline protease, 12-22 parts of feed-grade neutral protease, 5-10 parts of feed-grade acidic protease, 3-8 parts of food-grade papain and 1-4 parts of food-grade bromelain are mixed, and 1.2-3.5 parts of feed-grade soybean peptide powder, 1.7-4.5 parts of food-grade maltodextrin, 1-4 parts of food-grade trehalose and 1-3 parts of food-grade glycerol are added and stirred to obtain a complex protease protected mixture; under stirring, the first pre-stabilized enzyme mixture and the second pre-sustaining mixture are added to the complex protease protected mixture to obtain a stabilized enzyme mixture; S2. Add the enzyme-stabilizing mixture to the plant porous carrier mixture, and spray in a deionized aqueous solution containing 0.3-1.0 parts of food-grade anhydrous calcium chloride while stirring to granulate and obtain wet granules; dry and sieve the wet granules.

3. The method for preparing the plant protein-degrading enzyme according to claim 2, characterized in that, In step S1, the stirring temperature is 15-25℃ and the stirring time is 10-20 minutes.

4. The method for preparing the plant protein-degrading enzyme according to claim 2, characterized in that, In step S2, the drying temperature is 35-45℃.

5. The plant protein-degrading enzyme according to claim 1, characterized in that, The preparation method of the tannic acid cross-linked chitosan enzyme-stabilizing microparticles includes: A1. By weight, add 8-12 parts of food-grade chitosan and 3-6 parts of food-grade lactic acid to 90-120 parts of deionized water and stir to obtain a chitosan lactate solution; add 3-6 parts of food-grade tannic acid, 1-2 parts of food-grade glycerol and 2-4 parts of food-grade maltodextrin to 30-50 parts of deionized water and stir to obtain a tannic acid protective solution; under stirring, add the tannic acid protective solution to the chitosan lactate solution and stir to obtain a tannic acid-chitosan composite solution; dissolve 1.0-2.0 parts of food-grade sodium tripolyphosphate in 20-30 parts of deionized water to obtain an ion-crosslinking solution; under stirring, dropwise add the ion-crosslinking solution to the tannic acid-chitosan composite solution to perform ion crosslinking and obtain a wet enzyme-stabilized gel; A2. Add 0.5-1.0 parts of food-grade silica to the wet enzyme stabilized gel, stir, and obtain an enzyme stabilized gel slurry; dry the enzyme stabilized gel slurry to obtain the dried product; grind the dried product and vibrate sieve.

6. The plant protein-degrading enzyme according to claim 5, characterized in that, In step A1, the dropping time for adding the ionic crosslinking liquid to the tannic acid-chitosan composite liquid is 30-50 minutes.

7. The plant protein-degrading enzyme according to claim 5, characterized in that, In step A2, the drying temperature is 45-55℃.

8. The plant protein-degrading enzyme according to claim 1, characterized in that, The preparation method of the Sophora japonica extract calcium alginate sustained-release microparticles includes: B1. By weight, add 10-14 parts of food-grade sodium alginate to 120-160 parts of deionized water and stir to obtain a sodium alginate solution; mix 0.8-1.5 parts of Sophora japonica extract and 30-45 parts of edible ethanol and stir to obtain an ethanol dispersion of Sophora japonica extract; while stirring, add the ethanol dispersion of Sophora japonica extract dropwise to the sodium alginate solution and stir to combine to obtain a Sophora japonica extract-sodium alginate composite solution; B2. Under stirring, add 1.2-2.0 parts of a deionized aqueous solution containing food-grade anhydrous calcium chloride to the sodium alginate complex solution of Sophora japonica extract to carry out ion gelation. Add 1-2 parts of food-grade trehalose and 1-3 parts of food-grade maltodextrin, stir, and obtain a slow-release gel slurry. Dry the slow-release gel slurry to obtain the dried product. Grind the dried product and vibrate sieve it.

9. The plant protein-degrading enzyme according to claim 8, characterized in that, In step B1, the mixing temperature is 45-55℃, and the mixing time is 40-60 minutes.

10. The plant protein-degrading enzyme according to claim 8, characterized in that, In step B2, the drying temperature is 45-55℃.

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