An artificial feed for silkworm larvae containing protease, its preparation method and application

Abstract

This invention discloses an artificial feed for silkworm larvae containing protease, its preparation method, and its application. It belongs to the field of enzyme engineering technology in bio-agriculture and related industries. The feed comprises: 0.2-1 parts protease preparation, 60-70 parts fresh mulberry leaves, 12-18 parts soybean meal powder, 15-20 parts corn flour, 120-130 parts calcium hydroxide solution, 50-60 parts sodium bicarbonate solution, and 1-3 parts parabens. Fresh mulberry leaves are crushed into powder and mixed with calcium hydroxide solution to obtain a mulberry leaf homogenate. Soybean meal powder, corn flour, parabens, and sodium bicarbonate solution are mixed and cooked. After cooling, the mixture is mixed with the mulberry leaf homogenate, and the protease preparation is added to obtain the artificial feed for silkworm larvae containing protease. This artificial feed for silkworm larvae uses fresh mulberry leaves, soybean meal powder, and corn flour as main raw materials. The alkaline solution environment maximizes the retention of feeding and swallowing factors from mulberry leaves, while destroying the resistant protein characteristics in soybean meal powder and corn flour. The formula is nutritionally rich, has a high feed absorption rate, and the production process is simple and easy to promote.

Description

Technical Field

[0001] This invention belongs to the technical field of bio-agriculture and enzyme engineering. More specifically, this invention relates to an artificial feed for silkworm larvae containing protease, its preparation method, and its application. Background Technology

[0002] During the growth of silkworm larvae, adding a certain amount of protease to their feed can improve feed utilization, promote larval growth and development, resist the invasion of entomopathogenic fungi, and reduce the disease incidence of silkworm larvae. For example, alkaline proteases are a class of enzymes capable of hydrolyzing proteins under alkaline conditions. In the midgut tissue of silkworm larvae, there are two alkaline proteases: one that breaks down casein and the other that breaks down BAPNA (benzoylarginyl nitrosaniline). These are synthesized in the midgut tissue and located on the fine filament margin, exhibiting membrane-bound activity. These alkaline proteases aid in the digestion and absorption of feed by silkworm larvae. Serine proteases are a class of proteases with serine as their active center, possessing broad substrate specificity and hydrolytic activity. In silkworms, serine protease p37k exhibits different activities in the midgut and molting fluid. In the midgut, serine protease p37k exhibits strong gelatinase activity, which helps silkworm larvae digest feed. Therefore, adding serine proteases to feed can further improve the protein digestibility of silkworm larvae. The amount of protease added should be adjusted according to factors such as the growth stage of silkworm larvae, feed composition, and rearing environment. Excessive protease may lead to feed waste and environmental pollution, while insufficient protease may not meet the growth needs of silkworm larvae.

[0003] Currently, effectively loading the protease into silkworm feed is a technical challenge in preparing protease-containing feed. Commonly used loading methods include direct addition, encapsulation, and adsorption. Direct addition is simple and low-cost, requiring only the addition of the protease to the silkworm feed without complex processes or equipment; however, its disadvantage is poor stability, as the protease is easily deactivated by factors such as temperature and humidity. Encapsulation effectively protects the activity of the protease, preventing deactivation during preparation and storage. Encapsulation improves the stability of the protease in the feed, resisting adverse environmental factors. Furthermore, various carriers can be used for encapsulation, such as agarose and sodium alginate, allowing for selection based on needs. However, encapsulation carriers are relatively expensive, and after prolonged use, the encapsulation material may break down, leading to protease leakage and affecting feed efficacy. While adsorption is relatively simple to operate, the enzyme loading capacity is low, making it unsuitable for long-term storage. Summary of the Invention

[0004] 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.

[0005] To achieve these and other advantages according to the present invention, an artificial feed for silkworm larvae containing protease is provided, comprising, by weight: 0.2-1 parts protease preparation, 60-70 parts fresh mulberry leaves, 12-18 parts soybean meal powder, 15-20 parts corn flour, 120-130 parts calcium hydroxide solution, 50-60 parts sodium bicarbonate solution, and 1-3 parts parabens.

[0006] Preferably, the protease preparation contains one of serine protease, subtilisin, and serase.

[0007] Preferably, the concentration of the calcium hydroxide solution is 0.2–0.5 wt%; and the concentration of the sodium bicarbonate is 0.5–1 wt%.

[0008] Preferably, the method for preparing the protease preparation includes the following steps:

[0009] S1. Prepare phosphate-buffered saline (PBS), add protease powder to PBS; add flavonol to anhydrous ethanol to obtain flavonol solution;

[0010] S2. Mix equal volumes of phosphate buffer and flavonol solution, disperse by sonication, and let stand for 12-24 hours. Then inject pure water. The flavonol solubility decreases and it precipitates. Separate the lower layer solution, evaporate to remove ethanol, and obtain the flavonol phase loaded with protease.

[0011] S3. Mix the flavonol phase with the sodium alginate solution to obtain a mixed dispersion system. Add the mixed dispersion system dropwise to the calcium chloride solution while stirring at 100-120 rpm. After the addition is complete, stop stirring and let it stand for 5-12 hours. Separate the solidified gel and freeze-dry it to obtain the protease preparation.

[0012] Preferably, in S1, the pH value of phosphate-buffered saline (PBS) is 7-7.4; the ratio of PBS, protein powder, flavonol, and anhydrous ethanol is 1-10 mL: 5-8 mg: 10-30 mg: 1-10 mL.

[0013] Preferably, in step S2, the volume of deionized water is 2 to 5 times the volume of the flavonol phase; the ultrasonic dispersion frequency is 30 to 60 kHz; and the ultrasonic dispersion time is 10 to 20 min.

[0014] Preferably, in step S3, the concentration of sodium alginate solution is 0.2–0.6 wt%, the volume ratio of sodium alginate solution to flavonol solution is 1–3:2–3, the concentration of calcium chloride solution is 10–30 wt%, the freeze-drying temperature is -25–-18°C, and the freeze-drying time is 1–3 h.

[0015] A method for preparing artificial feed for silkworm larvae includes the following steps:

[0016] Step 1: Crush fresh mulberry leaves into strips 1-3 cm long, then mix them with calcium hydroxide solution to obtain a mulberry leaf homogenate;

[0017] Step 2: Mix soybean meal, corn flour, parabens and sodium bicarbonate solution, then steam and cook. After cooling, mix with mulberry leaf slurry, then add protease preparation to obtain artificial feed for silkworm larvae containing protease.

[0018] Preferably, in step two, the cooking temperature is 100–120°C and the cooking time is 10–30 minutes.

[0019] Application of an artificial feed for silkworm larvae containing protease, wherein the artificial feed for silkworm larvae containing protease is used to feed 1st to 3rd instar silkworm larvae at a feeding frequency of 4 to 5 times per day.

[0020] The present invention has at least the following beneficial effects:

[0021] The artificial feed for silkworm larvae containing protease provided by this invention uses fresh mulberry leaves, soybean meal, and corn flour as the main raw materials. The alkaline solution environment can retain the feeding and swallowing factors of mulberry leaves to the greatest extent and destroy the resistant protein characteristics in soybean meal and corn flour. The feed formula is rich in nutrients, simple in formulation, high in feeding rate, and uniform in development. The production process is simple and easy to promote.

[0022] The artificial feed for silkworm larvae prepared by this invention contains a protease preparation. Feeding silkworm larvae with this protease-containing artificial feed during their growth process effectively replenishes the protease in their bodies, promotes their growth and development, resists the invasion of invasive insect fungi, and thus reduces the disease rate of silkworm larvae. Furthermore, the preparation method of the protease preparation in this invention uses an antisolvent method and flavonols to load the protease. Utilizing the covalent interaction between flavonols and proteases, efficient protease loading is achieved, increasing the protease loading capacity. The use of sodium alginate gel for coating ensures that the protease activity in the protease preparation remains at a high level for 60 days, effectively extending the shelf life of the protease preparation.

[0023] 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

[0024] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0025] 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.

[0026] Example 1:

[0027] This embodiment provides a method for preparing an artificial feed for silkworm larvae containing protease, comprising the following steps:

[0028] Step 1: Crush fresh mulberry leaves into strips of 1-3 cm, then weigh 600g of fresh mulberry leaf strips and mix them with 0.5wt% 1200g of calcium hydroxide solution to obtain mulberry leaf homogenate;

[0029] Step 2: Mix 120g soybean meal powder, 150g corn flour, 10g parabens with 1wt% 500g sodium bicarbonate solution and cook at 115℃ for 15 minutes. After cooling, mix with mulberry leaf homogenate and then add 5g protease preparation to obtain artificial feed for silkworm larvae containing protease.

[0030] The preparation method of the protease preparation used in step two of this embodiment includes:

[0031] S1. Prepare 100 mL of phosphate-buffered saline (PBS) with a pH of 7.4. Add 50 mg of serine protease (proteinase K) powder to the PBS. Add 100 mg of flavonol to 100 mL of anhydrous ethanol and dissolve to obtain a flavonol solution.

[0032] S2. Mix 100 mL of phosphate buffer containing serine protease powder with 100 mL of flavonol solution, sonicate at 30 kHz for 15 min, and let stand for 12 h. Then inject 400 mL of deionized water. The flavonol solubility decreases and precipitates out. Separate the lower layer solution, evaporate and remove ethanol to obtain the flavonol phase loaded with serine protease.

[0033] S3. Mix 50 mL of flavonol phase with 25 mL of 0.5 wt% sodium alginate solution to obtain a mixed dispersion system. Add the mixed dispersion system dropwise to 20 wt% calcium chloride solution while stirring at 120 rpm. After the addition is complete, stop stirring and let stand for 6 hours. Separate the solidified gel and freeze-dry at -25℃ for 2 hours to obtain the protease preparation.

[0034] Example 2:

[0035] This embodiment provides a method for preparing an artificial feed for silkworm larvae containing protease, comprising the following steps:

[0036] Step 1: Crush fresh mulberry leaves into strips of 1-3 cm, then weigh 650g of fresh mulberry leaf strips and mix them with 0.5wt% 1250g of calcium hydroxide solution to obtain mulberry leaf homogenate;

[0037] Step 2: Mix 150g soybean meal powder, 180g corn flour, 20g parabens with 1wt% 550g sodium bicarbonate solution and cook at 115℃ for 15 minutes. After cooling, mix with mulberry leaf homogenate and then add 8g protease preparation to obtain artificial feed for silkworm larvae containing protease.

[0038] The preparation method of the protease preparation used in step two of this embodiment includes:

[0039] S1. Prepare 100 mL of phosphate-buffered saline (PBS) with a pH of 7.4. Add 50 mg of serine protease (proteinase K) powder to the PBS. Add 100 mg of flavonol to 100 mL of anhydrous ethanol and dissolve to obtain a flavonol solution.

[0040] S2. Mix 100 mL of phosphate buffer containing serine protease with 100 mL of flavonol solution, sonicate at 30 kHz for 15 min, and let stand for 12 h. Then inject 500 mL of deionized water. The flavonol solubility decreases and precipitates out. Separate the lower layer solution, evaporate and remove ethanol to obtain the flavonol phase loaded with serine protease.

[0041] S3. Mix 60 mL of flavonol phase with 0.5 wt% sodium alginate solution in 25 mL to obtain a mixed dispersion system. Add the mixed dispersion system dropwise to 25 wt% calcium chloride solution while stirring at 120 rpm. After the addition is complete, stop stirring and let stand for 6 h. Separate the solidified gel and freeze dry at -25℃ for 2 h to obtain the protease preparation.

[0042] Example 3:

[0043] This embodiment provides a method for preparing an artificial feed for silkworm larvae containing protease, comprising the following steps:

[0044] Step 1: Crush fresh mulberry leaves into strips of 1-3 cm in length, then weigh 700 g of fresh mulberry leaf strips and mix them with 0.5 wt% 1300 g of calcium hydroxide solution to obtain mulberry leaf homogenate;

[0045] Step 2: Mix 180g soybean meal powder, 200g corn flour, 30g parabens with 1wt% 600g sodium bicarbonate solution and cook at 115℃ for 15 minutes. After cooling, mix with mulberry leaf homogenate and then add 10g protease preparation to obtain artificial feed for silkworm larvae containing protease.

[0046] The preparation method of the protease preparation used in step two of this embodiment includes:

[0047] S1. Prepare 100 mL of phosphate-buffered saline (PBS) with a pH of 7.5. Add 50 mg of serine protease (proteinase K) powder to the phosphate-buffered saline. Add 100 mg of flavonol to 100 mL of anhydrous ethanol and dissolve to obtain a flavonol solution.

[0048] S2. Mix 100 mL of phosphate buffer containing serine protease with 100 mL of flavonol solution, sonicate at 30 kHz for 15 min, and let stand for 12 h. Then inject 500 mL of deionized water. The flavonol solubility decreases and precipitates out. Separate the lower layer solution, evaporate and remove ethanol to obtain the flavonol phase loaded with serine protease.

[0049] S3. Mix 50 mL of flavonol phase with 25 mL of 0.5 wt% sodium alginate solution to obtain a mixed dispersion system. Add the mixed dispersion system dropwise to 20 wt% calcium chloride solution while stirring at 100 rpm. After the addition is complete, stop stirring and let stand for 6 hours. Separate the solidified gel and freeze-dry at -20℃ for 2 hours to obtain the protease preparation.

[0050] Comparative Example 1:

[0051] This embodiment provides a method for preparing an artificial feed for silkworm larvae containing protease. The difference from other embodiments 1 is that the method for preparing the protease preparation used in step two of this comparative example is as follows:

[0052] S1. Prepare 100 mL of phosphate-buffered saline (PBS) with a pH of 7.4, and add 50 mg of serine protease (proteinase K) powder to the phosphate-buffered saline.

[0053] S2. Mix 50 mL of phosphate buffer containing serine protease powder with 25 mL of 0.5 wt% sodium alginate solution to obtain a mixed dispersion system. Add the mixed dispersion system dropwise to 20 wt% calcium chloride solution while stirring at 120 rpm. After the addition is complete, stop stirring, let stand for 6 h, and then separate the solidified gel. Freeze-dry at -25℃ for 2 h to obtain the protease preparation.

[0054] The methods and process parameters for the remaining steps in this comparative example are the same as those in Example 1.

[0055] Comparative Example 2:

[0056] This embodiment provides a method for preparing an artificial feed for silkworm larvae containing protease. The difference from other embodiments 1 is that the protease preparation used in step two of this comparative example is prepared as follows:

[0057] S1. Prepare 100 mL of phosphate-buffered saline (PBS) with a pH of 7.4. Add 50 mg of serine protease (proteinase K) powder to the PBS. Add 100 mg of flavonol to 100 mL of anhydrous ethanol and dissolve to obtain a flavonol solution.

[0058] S2. Mix 100 mL of phosphate buffer containing serine protease powder with 100 mL of flavonol solution, sonicate at 30 kHz for 15 min, and let stand for 12 h. Then inject 400 mL of deionized water. The flavonol solubility decreases and precipitates out. Separate the lower layer solution, evaporate and remove ethanol to obtain the flavonol phase loaded with serine protease.

[0059] In this comparative example, an equal mass of flavonol phase loaded with serine protease was used to replace the protease preparation in Example 1. The remaining steps and process parameters of this comparative example were the same as those in Example 1.

[0060] The serine protease loading of the protease preparations in Examples 1-3 and Comparative Examples 1-2 was determined, and the results are shown in Table 1.

[0061] Table 1. Serine protease loading of the protease preparations in Examples 1-3 and Comparative Examples 1-2

[0062] Serine protease loading (mg / g) Example 1 4.5 Example 2 3.9 Example 3 4.2 Comparative Example 1 1.2 Comparative Example 2 2.8

[0063] Meanwhile, 100g of the protease preparations prepared in Examples 1-3 and Comparative Examples 1-2 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 / T34800-2017, and the results are shown in Table 2.

[0064] Table 2. Changes in serine protease activity of the protease preparations in Examples 1-3 and Comparative Examples 1-2.

[0065] 0 days (U / mg) 10 days (U / mg) 30 days (U / mg) 40 days (U / mg) 60 days (U / mg) Example 1 46.8 44.2 40.7 38.3 36.0 Example 2 44.9 42.1 38.0 35.7 33.4 Example 3 45.3 42.8 38.2 35.9 33.7 Comparative Example 1 46.3 37.4 30.5 26.8 19.2 Comparative Example 2 44.5 33.5 20.9 15.4 9.4

[0066] As can be seen from Tables 1 and 2, the artificial feed for silkworm larvae containing protease in Examples 1-3 showed significantly better loading and enzyme activity of serine protease in the protease preparations compared to Comparative Examples 1 and 2.

[0067] Seven hundred first-instar silkworm larvae were selected and divided into seven groups. Group 1 was fed the artificial feed containing protease prepared in Example 1; Group 2 was fed the artificial feed containing protease prepared in Example 2; Group 3 was fed the artificial feed containing protease prepared in Example 3; Group 4 was fed the artificial feed containing protease prepared in Comparative Example 1; Group 5 was fed the artificial feed containing protease prepared in Comparative Example 2; and Group 6 served as a blank control, fed a mixed artificial feed. The mixed artificial feed was prepared by breaking fresh mulberry leaves into strips of 1-3 cm. Then, 600g of fresh mulberry leaf strips were weighed; 120g of soybean meal powder, 150g of corn flour, 10g of parabens and 500g of water were mixed and steamed at 115℃ for 15 minutes. After cooling, the mixture was mixed with the fresh mulberry leaf strips, and then 5g of protease preparation was added to obtain artificial feed for silkworm larvae containing protease. Group 7 served as blank control 2, which was artificial feed for silkworm larvae without protease preparation. The preparation method of the artificial mixed feed without protease preparation was the same as in Example 1. The feeding rate of each group was measured and the results are shown in Table 3.

[0068] Table 3. Feeding rate of silkworm larvae in each group

[0069]

[0070] As can be seen from the table above, the artificial silkworm larvae fed with the protease-containing artificial feed prepared in Examples 1-3 have a higher feeding rate.

[0071] 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.

[0072] 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. Use of a proteinase-containing artificial diet for silkworm larvae, characterized in that, The artificial silkworm larva feed containing protease is applied to feeding of 1-3 instar silkworm larvae, and the feeding frequency is 4-5 times per day; The artificial silkworm larva feed containing protease comprises, by weight, 0.2-1 parts of protease preparation, 60-70 parts of fresh mulberry leaves, 12-18 parts of soybean meal powder, 15-20 parts of corn powder, 120-130 parts of calcium hydroxide solution, 50-60 parts of sodium bicarbonate solution, and 1-3 parts of nipagin ester; The preparation method of the protease preparation comprises the following steps: S1, configure phosphate buffered saline PBS, and add serine protease powder into the phosphate buffered saline; add flavonols into anhydrous ethanol to obtain a flavonol solution; the pH value of the phosphate buffered saline PBS is 7-7.4; the dosage ratio of the phosphate buffered saline PBS, the serine protease powder, the flavonols and the anhydrous ethanol is 1-10 mL:5-8 mg:10-30 mg:1-10 mL; S2, mix the phosphate buffered saline and the flavonol solution in equal volume, ultrasonically disperse, and then stand for 12-24 hours; then inject pure water, and the solubility of the flavonols decreases to precipitate, so that the lower solution is separated, and the ethanol is removed by evaporation to obtain a flavonol phase loaded with protease; S3, mix the flavonol phase and a sodium alginate solution uniformly to obtain a mixed dispersion system, and drop the mixed dispersion system into a calcium chloride solution drop by drop while stirring at a speed of 100-120 rpm, stop stirring after the dropping is completed, and separate the solidified gel after standing for 5-12 hours, and freeze-dry to obtain the protease preparation; the concentration of the sodium alginate solution is 0.2-0.6 wt%, the volume ratio of the sodium alginate solution to the flavonol phase is 1-3:2-3, the concentration of the calcium chloride solution is 10-30 wt%, the freeze-drying temperature is-25--18℃, and the freeze-drying time is 1-3 hours; The preparation method of the artificial silkworm larva feed containing protease comprises the following steps: Step one, crush the fresh mulberry leaves into 1-3 cm long strips, and then mix the crushed leaves with the calcium hydroxide solution to obtain mulberry leaf homogenate; Step two, mix the soybean meal powder, the corn powder, the nipagin ester and the sodium bicarbonate solution uniformly, and then cook, the cooking temperature is 100-120℃, the cooking time is 10-30 minutes, and then mix the cooked mixture with the mulberry leaf homogenate, and then add the protease preparation to obtain the artificial silkworm larva feed containing protease; The concentration of the calcium hydroxide solution is 0.2-0.5 wt%, and the concentration of the sodium bicarbonate is 0.5-1 wt%.

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