A high cellulose silkworm larva feed containing a protease and a preparation method thereof

Abstract

The application discloses a high-fiber silkworm larva feed containing protease and a preparation method thereof, and belongs to the technical field of enzyme engineering and feed for animal feeding in biological agriculture and related industries, and comprises a protease preparation-mulberry branch powder compound powder, mulberry branch powder, corn powder, soybean meal powder, citric acid, inorganic salt and guar gum. The application utilizes the easy-pulverization characteristics of fresh mulberry branches, adopts a pulverization and drying mode, shortens the drying time of the mulberry branches, and the artificial feed containing sufficient cellulose is beneficial to the feeding of silkworm larvae, promotes the peristalsis of the feed in the silkworm body, and is beneficial to meeting the feeding habits, main nutrition and physiological growth requirements of the silkworm; the proportion of the corn powder is increased to provide the silkworm with the required sugars and sugar compounds; the guar gum has good water retention performance, the silkworm larva feed after cooking is not prone to drying and hardening, and the use of the protease preparation effectively supplements the protease in the silkworm larvae, and promotes the growth and development of the silkworm larvae.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and animal feed technology in bio-agriculture and related industries. More specifically, this invention relates to a high-cellulose silkworm larvae feed containing protease and its preparation method. Background Technology

[0002] During the growth of silkworm larvae, adding a certain amount of protease to their feed can improve feed utilization, promote growth and development, resist the invasion of insect-borne pathogenic fungi, and reduce the disease rate of silkworm larvae. Currently, silkworm larvae are mainly fed mulberry leaves. Mulberry leaves have a lower cellulose content than mulberry branches, and cellulose is beneficial for improving intestinal peristalsis and feeding ability in silkworm larvae. Therefore, it is necessary to utilize the high cellulose content of mulberry branches as the main feed component for silkworm larvae. However, mulberry branches are relatively hard and must be ground into powder for the larvae to consume. Alkaline proteases are enzymes that can hydrolyze proteins in an alkaline environment. In the midgut tissue of silkworm larvae, there are alkaline proteases that can decompose casein and BAPNA (benzoylarginyl nitrosaniline). These proteases are synthesized in the midgut tissue and, after synthesis, are located at the filamentous edge, belonging to membrane-bound enzymes. These alkaline proteases play a crucial auxiliary role in the ingestion and digestion of feed by silkworm larvae.

[0003] Serine proteases are a class of proteases with serine as their active site, exhibiting broad substrate specificity and hydrolytic activity. In silkworms, the serine protease p37k displays varying activities in different locations, particularly in the midgut, where it possesses strong gelatinase activity, aiding silkworm larvae in the efficient digestion of protein in their feed. Therefore, adding serine proteases to feed can further enhance the protein digestibility of silkworm larvae.

[0004] In practice, the amount of protease added needs to take into account many factors, such as the growth stage of the silkworm larvae, the composition of the feed used, and the environmental conditions during rearing. If too much protease is added, it may lead to feed waste and environmental pollution; however, if too little is added, it will not provide sufficient support for the growth of the silkworm larvae.

[0005] Throughout the silkworm's growth cycle, the demand for proteases varies at different stages. For example, newly hatched larvae have developing digestive organs and relatively weak digestive abilities, resulting in low tolerance and demand for proteases. However, larvae nearing cocoon formation require higher protein digestion efficiency to meet the needs of accumulating large amounts of silk protein, thus increasing their protease requirements. The composition of the feed also affects the amount of protease added. If the feed is rich in high-content and complex proteins, more protease may be needed to ensure effective digestion; conversely, if the feed contains more easily digestible substances, the amount of protease added can be appropriately reduced. Furthermore, environmental factors such as temperature and humidity also affect the larvae's digestive capacity. Under suitable conditions, the larvae's metabolism and digestive function are normal, and protease utilization efficiency is high. However, under extreme environmental conditions, digestive function may be inhibited, requiring more careful adjustment of the protease dosage to avoid unnecessary resource waste and environmental pollution.

[0006] Currently, there are no reports or literature on using mulberry twig powder and protease preparations in combination as feed for silkworm larvae. How to determine the ratio and combination method of mulberry twig powder and protease, while improving the activity and storage time of the protein, is a problem that needs to be solved. Summary of the Invention

[0007] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0008] To achieve these and other advantages according to the present invention, a high-cellulose silkworm larvae feed containing protease is provided, comprising, by weight: 1-5 parts protease preparation-mulberry powder compound powder, 20-30 parts mulberry powder, 40-50 parts corn flour, 10-30 parts soybean meal, 1-6 parts citric acid, 1-3 parts inorganic salt, and 0.1-1 parts guar gum.

[0009] Preferably, the method for preparing mulberry twig powder includes: crushing hybrid mulberry twigs with leaves into fragments with a length not exceeding 3 mm using a pulverizer, then quickly drying them using a dryer, and finally pulverizing them into mulberry twig powder with a particle size not exceeding 0.15 mm using a grinding mill.

[0010] Preferably, the preparation method of the protease preparation-mulberry powder composite powder includes:

[0011] S1. Disperse mulberry twig powder in citric acid solution, stir and heat to 40-60℃, keep warm for 1-3 hours, stirring speed is 200-500 rpm; after cooling to room temperature, filter out mulberry twig powder, wash with deionized water several times, and dry to obtain citric acid treated mulberry twig powder.

[0012] S2. Add the citric acid-treated mulberry powder to the chitosan solution and ultrasonically disperse it at 40-60 kHz for 20-30 min to form a chitosan dispersion of mulberry powder.

[0013] S3. Add the protease preparation to the chitosan dispersion of mulberry twig powder, stir continuously at 200-500 rpm and add jingpinni, stir for 10-30 min, let stand for 6-12 h, freeze dry at -25--20℃ for 3 h to obtain protease preparation-mulberry twig powder composite powder.

[0014] Preferably, in step S1, the concentration of the citric acid solution is 1-5 wt%, and the ratio of mulberry twig powder to citric acid solution is 2-10 g: 200-500 mL.

[0015] In S2, the concentration of the chitosan solution is 1-3 wt%, and the ratio of citric acid-treated mulberry powder to chitosan solution is 2-10 g: 100-250 mL.

[0016] In step S3, the ratio of the amount of protease preparation to the citric acid-treated mulberry powder is 0.1-2:2-10, and the amount of ginseng is 0.1-0.5% of the mass of the protease preparation.

[0017] Preferably, the method for preparing the protease preparation includes:

[0018] S21. Prepare phosphate-buffered saline (PBS), add protease powder to PBS; add quercetin to anhydrous ethanol to obtain quercetin solution;

[0019] S22. Mix equal volumes of phosphate-buffered saline (PBS) solution and quercetin solution. Add cottonseed protein to the mixture, disperse by sonication, and let stand for 12–24 h to obtain a cottonseed protein-modified quercetin solution. Then inject deionized water and add citric acid to adjust the pH of the system to 4–5. The solubility of the cottonseed protein-modified quercetin decreases and it precipitates. Separate the lower layer solution, evaporate to remove ethanol, and obtain the quercetin phase loaded with protease.

[0020] S23. Mix the quercetin phase with the sodium alginate solution to obtain a mixed dispersion system. Add calcium chloride solution dropwise to the mixed dispersion system while stirring at 100-120 rpm. After the addition is complete, stop stirring and let stand for 5-12 hours. Separate the solidified gel and freeze-dry it to obtain the protease preparation.

[0021] Preferably, in step S21, the ratio of phosphate-buffered saline (PBS), protease powder, quercetin, and anhydrous ethanol is 1–20 mL: 10–15 mg: 10–30 mg: 50–150 mL.

[0022] In step S22, the mass ratio of cottonseed protein to quercetin is 1–5:10–15, the volume of deionized water is 2–5 times the volume of the quercetin solution, the ultrasonic dispersion frequency is 30–60 kHz, and the ultrasonic dispersion time is 10–20 min.

[0023] Preferably, in step S23, the concentration of the sodium alginate solution is 0.2–0.6 wt%, the volume ratio of the sodium alginate solution to the quercetin phase is 1–3:2–3, the concentration of the calcium chloride solution is 10–30 wt%, the freeze-drying temperature is -25–-18°C, and the freeze-drying time is 1–3 h.

[0024] Preferably, the inorganic salt is one or more of sodium chloride, potassium dihydrogen phosphate, calcium chloride, and magnesium sulfate.

[0025] A method for preparing a high-cellulose silkworm larvae feed containing protease includes the following steps:

[0026] Step 1: Mix the mulberry powder, corn flour, and soybean meal powder evenly to obtain a mixed coarse feed.

[0027] Step 2: Dissolve citric acid, inorganic salts, and guar gum in 100-150 parts by weight of water, then add the mixed coarse materials, stir and mix evenly, then cook and filter to obtain the mixed slurry;

[0028] Step 3: Add protease preparation-mulberry powder composite powder to the mixed slurry to obtain high-cellulose silkworm larvae feed containing protease.

[0029] Preferably, the cooking temperature is 100–120°C and the cooking time is 20–60 minutes.

[0030] This invention offers at least the following beneficial effects: It utilizes the easily pulverized nature of fresh mulberry branches, employing a pulverized and then dried method to shorten the drying time. Mulberry branches, being an artificial feed, contain ample cellulose, which is beneficial for silkworm larvae to consume, promoting feed movement within the silkworm's body and meeting the silkworm's feeding, nutritional, and physiological growth needs. Increasing the proportion of corn flour provides the sugars and saccharides required for silkworm growth. Guar gum has excellent water-retention properties, preventing the cooked silkworm feed from drying and hardening. The rapidly dried mulberry powder retains a large amount of vitamins, palatability-enhancing components, and other nutrients, which is beneficial for improving the growth and health of silkworm larvae.

[0031] This invention incorporates a protease preparation—mulberry bark powder compound powder—into high-cellulose silkworm larvae feed. Feeding silkworm larvae with this high-cellulose silkworm larvae feed during their growth process can effectively supplement the protease in the larvae's body, promote their growth and development, resist the invasion of insect-borne pathogenic fungi, and thus reduce the disease rate of silkworm larvae. Meanwhile, in the preparation method of the protease preparation of the present invention, an antisolvent method is used, and cottonseed protein-modified quercetin is used to load the protease. The covalent interaction between cottonseed protein, quercetin, and the protease achieves efficient protease loading, increasing the protease loading capacity. Furthermore, sodium alginate gel is used for coating, ensuring that the protease activity in the protease preparation remains at a high level within 60 days, effectively extending the storage time of the protease preparation. Finally, chitosan is used as a binder to coat the surface of the protease preparation with mulberry twig powder, resulting in a protease preparation-mulberry twig powder composite powder. The coating of mulberry twig powder provides a stable protective layer for the protease preparation, effectively protecting the protease within the preparation and further extending its storage time. The mulberry twig powder also has a controlled-release effect on the protease, improving the utilization rate of the protease in silkworm larvae, and simultaneously improving the biocompatibility of the protease preparation.

[0032] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0033] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0034] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0035] The preparation methods of the protease preparations used in Examples 1-3 and Comparative Example 1 include the following steps:

[0036] S21. Prepare 200 mL of phosphate-buffered saline (PBS) with a pH of 7.4. Add 150 mg of serine protease (proteinase K) powder to the PBS. Add 200 mg of quercetin to 500 mL of anhydrous ethanol and dissolve to obtain a quercetin solution.

[0037] S22. Mix 200 mL of phosphate buffer containing serine protease powder with 200 mL of quercetin solution to obtain a mixed solution. Add 45 mg of cottonseed protein powder to the mixed solution, sonicate at 40 kHz for 20 min, and let stand for 12 h. Then inject 500 mL of deionized water. The solubility of cottonseed protein-modified quercetin decreases and it precipitates. Separate the lower layer solution, evaporate and remove ethanol to obtain the quercetin phase loaded with serine protease.

[0038] S23. Mix 100 mL of quercetin phase with 100 mL of 0.6 wt% sodium alginate solution to obtain a mixed dispersion system. Add 25 mL of 30 wt% calcium chloride solution dropwise to the mixed dispersion system while stirring at 100 rpm. After the addition is complete, stop stirring and let stand for 12 h. Separate the solidified gel and freeze-dry at -25℃ for 3 h to obtain the protease preparation.

[0039] Example 1

[0040] This embodiment provides a method for preparing a high-cellulose silkworm larvae feed containing protease, comprising the following steps:

[0041] Step 1: Mix 400g of mulberry powder, 450g of corn flour, and 200g of soybean meal powder evenly to obtain mixed coarse feed;

[0042] Step 2: Dissolve 30g citric acid, 15g potassium dihydrogen phosphate, and 5g guar gum in 1200mL of water, then add the mixed coarse materials, stir and mix evenly, cook at 120℃ for 20min, and filter to obtain the mixed slurry;

[0043] Step 3: Add 10g of protease preparation-mulberry powder compound powder to the mixed slurry to obtain high-cellulose silkworm larvae feed containing protease.

[0044] The preparation method of the protease preparation-mulberry powder composite powder includes:

[0045] S1. Disperse 5g of mulberry twig powder in 250mL of 5wt% citric acid solution, stir and heat to 60℃, keep warm for 2h, stirring speed is 300rpm; after cooling to room temperature, filter out the mulberry twig powder, wash with deionized water several times, and dry to obtain citric acid treated mulberry twig powder.

[0046] S2. Add 5g of citric acid-treated mulberry powder to 100mL of 1wt% chitosan solution and ultrasonically disperse at 60kHz for 30min to form a chitosan dispersion of mulberry powder.

[0047] S3. Add 2g of protease preparation to the chitosan dispersion of mulberry twig powder, stir continuously at 500rpm and add 10mg of ginsenoside. After stirring for 30min, let stand for 8h, freeze dry at -25℃ for 3h to obtain protease preparation-mulberry twig powder composite powder.

[0048] Example 2

[0049] This embodiment provides a method for preparing a high-cellulose silkworm larvae feed containing protease, comprising the following steps:

[0050] Step 1: Mix 400g of mulberry powder, 450g of corn flour, and 200g of soybean meal powder evenly to obtain mixed coarse feed;

[0051] Step 2: Dissolve 30g citric acid, 15g potassium dihydrogen phosphate, and 5g guar gum in 1200mL of water, then add the mixed coarse materials, stir and mix evenly, cook at 120℃ for 20min, and filter to obtain the mixed slurry;

[0052] Step 3: Add 20g of protease preparation-mulberry powder compound powder to the mixed slurry to obtain high-cellulose silkworm larvae feed containing protease.

[0053] The preparation method of the protease preparation-mulberry powder composite powder includes:

[0054] S1. Disperse 8g of mulberry twig powder in 250mL of 5wt% citric acid solution, stir and heat to 60℃, keep warm for 2h, stirring speed is 400rpm; after cooling to room temperature, filter out the mulberry twig powder, wash with deionized water several times, and dry to obtain citric acid treated mulberry twig powder.

[0055] S2. Add 5g of citric acid-treated mulberry powder to 100mL of 2wt% chitosan solution and ultrasonically disperse at 60kHz for 30min to form a chitosan dispersion of mulberry powder.

[0056] S3. Add 1.5g of protease preparation to the chitosan dispersion of mulberry twig powder, stir continuously at 500rpm and add 7.5mg of ginsenoside. After stirring for 30min, let stand for 8h, freeze dry at -25℃ for 3h to obtain protease preparation-mulberry twig powder composite powder.

[0057] Example 3

[0058] This embodiment provides a method for preparing a high-cellulose silkworm larvae feed containing protease, comprising the following steps:

[0059] Step 1: Mix 400g of mulberry powder, 450g of corn flour, and 200g of soybean meal powder evenly to obtain mixed coarse feed;

[0060] Step 2: Dissolve 30g citric acid, 15g potassium dihydrogen phosphate, and 5g guar gum in 1200mL of water, then add the mixed coarse materials, stir and mix evenly, cook at 120℃ for 20min, and filter to obtain the mixed slurry;

[0061] Step 3: Add 50g of protease preparation-mulberry powder compound powder to the mixed slurry to obtain high-cellulose silkworm larvae feed containing protease.

[0062] The preparation method of the protease preparation-mulberry powder composite powder includes:

[0063] S1. Disperse 10g of mulberry twig powder in 300mL of 5wt% citric acid solution, stir and heat to 60℃, keep warm for 3h, stirring speed is 500rpm; after cooling to room temperature, filter out the mulberry twig powder, wash with deionized water several times, and dry to obtain citric acid treated mulberry twig powder.

[0064] S2. Add 5g of citric acid-treated mulberry powder to 100mL of 3wt% chitosan solution and ultrasonically disperse at 60kHz for 30min to form a chitosan dispersion of mulberry powder.

[0065] S3. Add 2g of protease preparation to the chitosan dispersion of mulberry twig powder, stir continuously at 500rpm and add 8mg of ginsenoside. After stirring for 30min, let stand for 8h, freeze dry at -25℃ for 3h to obtain protease preparation-mulberry twig powder composite powder.

[0066] Comparative Example 1

[0067] This comparative example provides a method for preparing a high-cellulose silkworm larvae feed containing protease. Unlike Example 1, this comparative example uses an equal mass of protease preparation to replace the protease preparation-mulberry powder composite powder, and the preparation method of the protease preparation in this comparative example is the same as that in Examples 1-3.

[0068] The proportions and preparation methods of the remaining raw materials in this comparative example are the same as those in Example 1.

[0069] Comparative Example 2

[0070] This comparative example provides a method for preparing a high-cellulose silkworm larvae feed containing protease. The difference from Example 1 is that the method for preparing the protease preparation in this comparative example's protease preparation-mulberry powder composite powder is replaced with:

[0071] S21. Prepare 200 mL of phosphate-buffered saline (PBS) with a pH of 7.4, and add 150 mg of serine protease (proteinase K) powder to the phosphate-buffered saline.

[0072] S22. Mix 200 mL of phosphate buffer containing serine protease powder with 100 mL of 0.6 wt% sodium alginate solution to obtain a mixed dispersion system. Add 25 mL of 30 wt% calcium chloride solution dropwise to the mixed dispersion system while stirring at 100 rpm. After the addition is complete, stop stirring and let stand for 12 h. Separate the solidified gel and freeze-dry at -25℃ for 3 h to obtain the protease preparation.

[0073] The protease preparation-mulberry powder composite powder and preparation method used in this embodiment, as well as the preparation methods for the remaining steps, are the same as in Example 1.

[0074] Comparative Example 3

[0075] This comparative example provides a method for preparing a high-cellulose silkworm larvae feed containing protease. The difference from Example 1 is that the method for preparing the protease preparation in this comparative example's protease preparation-mulberry powder composite powder is replaced with:

[0076] S21. Prepare 200 mL of phosphate-buffered saline (PBS) with a pH of 7.4. Add 150 mg of serine protease (proteinase K) powder to the PBS. Add 200 mg of quercetin to 500 mL of anhydrous ethanol and dissolve to obtain a quercetin solution.

[0077] S22. Mix 200 mL of phosphate buffer containing serine protease powder with 200 mL of quercetin solution to obtain a mixed solution. Add 45 mg of cottonseed protein powder to the mixed solution, sonicate at 40 kHz for 20 min, and let stand for 12 h. Then inject 500 mL of deionized water. The solubility of cottonseed protein-modified quercetin decreases and it precipitates. Separate the lower layer solution, evaporate and remove ethanol to obtain the quercetin phase loaded with serine protease.

[0078] In this comparative example, an equal mass of quercetin loaded with serine protease was used to replace the protease preparation in Example 1. The protease preparation-mulberry powder composite powder and preparation method, as well as the preparation methods of the remaining steps, in this comparative example are the same as those in Example 1.

[0079] The serine protease loading of the protease preparations used in Examples 1-3 and Comparative Examples 2-3 was determined, and the results are shown in Table 1.

[0080]

[0081]

[0082] Meanwhile, 100g of the protease preparations used in Examples 1-3, Comparative Example 1, the protease preparations-mulberry powder complexes prepared in Examples 1, 2, and 3, and the protease preparations prepared in Comparative Examples 2 and 3 were weighed and sealed at 25°C. Samples were taken at 0, 10, 30, 40, and 60 days to determine the enzyme activity of serine protease (proteinase K). The determination method was in accordance with GB / T 34800-2017, and the results are shown in Table 2.

[0083] Table 2. Changes in protease activity of each sample

[0084]

[0085] Eight hundred first-instar silkworm larvae were selected and divided into eight groups. Group 1 was fed the high-cellulose silkworm larvae feed containing protease prepared in Example 1; Group 2 was fed the high-cellulose silkworm larvae feed containing protease prepared in Example 2; Group 3 was fed the high-cellulose silkworm larvae feed containing protease prepared in Example 3; Group 4 was fed the high-cellulose silkworm larvae feed containing protease prepared in Comparative Example 1; Group 5 was fed the high-cellulose silkworm larvae feed containing protease prepared in Comparative Example 2; Group 6 was fed the high-cellulose silkworm larvae feed containing protease prepared in Comparative Example 3; and Group 7 served as a blank control, fed the high-cellulose silkworm larvae feed without protease preparation-mulberry powder composite powder, and without protease preparation. The high-cellulose silkworm larvae feed made from mulberry powder composite powder, without the addition of protease preparation, was prepared using the same method as in Example 1. Group 8 was fed the silkworm larvae feed without mulberry powder. The preparation method was as follows: 850g of corn flour and 200g of soybean meal were mixed evenly to obtain a mixed roughage; 30g of citric acid, 15g of potassium dihydrogen phosphate, and 5g of guar gum were dissolved in 1200mL of water, then added to the mixed roughage, stirred evenly, and then cooked at 120℃ for 20 minutes. After filtration, a mixed slurry was obtained; 10g of protease preparation was added to the mixed slurry to obtain the silkworm larvae feed without mulberry powder; the feeding rate of each group was measured, and the results are shown in Table 3.

[0086] Table 3. Feeding rate of silkworm larvae in each group

[0087]

[0088]

[0089] As can be seen from the table above, artificial silkworm larvae fed with the protease-containing high-cellulose silkworm larvae feed prepared in Examples 1-3 have a higher feeding rate.

[0090] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0091] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A method for preparing a high-cellulose silkworm larvae feed containing protease, characterized in that, Includes the following steps: Step 1: Mix the mulberry powder, corn flour, and soybean meal powder evenly to obtain a mixed coarse feed. Step 2: Dissolve citric acid, inorganic salts, and guar gum in 100-150 parts by weight of water, then add the mixed coarse materials, stir and mix evenly, and then cook at 100-120℃ for 20-60 minutes. After filtration, a mixed slurry is obtained. The inorganic salts are one or more of sodium chloride, potassium dihydrogen phosphate, calcium chloride, and magnesium sulfate. Step 3: Add protease preparation-mulberry powder composite powder to the mixed slurry to obtain high-cellulose silkworm larvae feed containing protease; The high-cellulose silkworm larvae feed containing protease comprises, by weight: 1-5 parts protease preparation-mulberry powder compound powder, 20-30 parts mulberry powder, 40-50 parts corn flour, 10-30 parts soybean meal powder, 1-6 parts citric acid, 1-3 parts inorganic salt, and 0.1-1 parts guar gum. The preparation method of the protease preparation-mulberry powder composite powder includes: S1. Disperse mulberry twig powder in citric acid solution, stir and heat to 40~60℃, keep warm for 1~3h, stirring speed is 200~500rpm; after cooling to room temperature, filter out mulberry twig powder, wash with deionized water several times, and dry to obtain citric acid treated mulberry twig powder; the concentration of citric acid solution is 1~5wt%, and the ratio of mulberry twig powder to citric acid solution is 2~10g:200~500mL; S2. Add citric acid-treated mulberry powder to chitosan solution and ultrasonically disperse at 40-60 kHz for 20-30 min to form chitosan dispersion of mulberry powder; the concentration of chitosan solution is 1-3 wt%, and the ratio of citric acid-treated mulberry powder to chitosan solution is 2-10 g: 100-250 mL. S3. Add the protease preparation to the chitosan dispersion of mulberry twig powder, stir continuously at 200-500 rpm, add jingpinni, stir for 10-30 min, let stand for 6-12 h, freeze dry at -25--20℃ for 3 h to obtain protease preparation-mulberry twig powder composite powder; the ratio of protease preparation to citric acid-treated mulberry twig powder is 0.1-2:2-10, and the amount of jingpinni is 0.1-0.5% of the mass of protease preparation; The method for preparing the protease preparation includes: S21. Prepare phosphate-buffered saline (PBS) by adding serine protease powder to PBS and adding quercetin to anhydrous ethanol to obtain a quercetin solution. The ratio of PBS, serine protease powder, quercetin, and anhydrous ethanol is 1-20 mL: 10-15 mg: 10-30 mg: 50-150 mL. S22. Mix equal volumes of phosphate-buffered saline (PBS) solution and quercetin solution. Add cottonseed protein to the mixture, disperse by ultrasonication, and let stand for 12-24 hours to obtain a cottonseed protein-modified quercetin solution. Then, inject deionized water and add citric acid to adjust the pH of the system to 4-5. The solubility of the cottonseed protein-modified quercetin decreases, and it precipitates. Separate the lower layer solution, evaporate to remove ethanol, and obtain the quercetin phase loaded with protease. In S22, the mass ratio of cottonseed protein to quercetin is 1-5:10-15, and the volume of deionized water is 2-5 times the volume of the quercetin solution. The ultrasonic dispersion frequency is 30-60 kHz, and the ultrasonic dispersion time is 10-20 min. S23. Mix the quercetin phase with the sodium alginate solution to obtain a mixed dispersion system. Add calcium chloride solution dropwise to the mixed dispersion system while stirring at 100-120 rpm. After the addition is complete, stop stirring and let stand for 5-12 hours. Separate the solidified gel and freeze-dry it to obtain the protease preparation. The preparation method of mulberry twig powder includes: crushing hybrid mulberry twigs with leaves into fragments with a length not exceeding 3mm using a crusher, then quickly drying them using a dryer, and then crushing them into mulberry twig powder with a particle size not exceeding 0.15mm using a grinding mill; In step S23, the concentration of sodium alginate solution is 0.2~0.6wt%, the volume ratio of sodium alginate solution to quercetin phase is 1~3:2~3; the concentration of calcium chloride solution is 10~30wt%; the freeze-drying temperature is -25~-18℃, and the freeze-drying time is 1~3h.

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