Protease-containing artificial feed for silkworm larvae as well as preparation method and application of protease-containing artificial feed

Abstract

The present invention discloses a protease-containing silkworm larva artificial feed and a preparation method and application thereof, and belongs to the technical field of enzyme engineering of biological agriculture and related industries, the protease-containing silkworm larva artificial feed comprises 0.2-1 part of a protease preparation, 60-70 parts of fresh mulberry leaves, 12-18 parts of soybean meal powder, 15-20 parts of corn flour, 120-130 parts of a calcium hydroxide solution, 50-60 parts of a sodium bicarbonate solution, and 1-3 parts of nipagin ester. Crushing fresh mulberry leaves into powder, and mixing with a calcium hydroxide solution to obtain mulberry leaf homogenate; uniformly mixing soybean meal, corn flour, nipagin ester and a sodium bicarbonate solution, cooking, spreading for cooling, uniformly mixing with the mulberry leaf homogenate, and adding a protease preparation to obtain the protease-containing silkworm larva artificial feed. According to the artificial feed for the silkworm larvae, the fresh mulberry leaves, the bean pulp powder and the corn flour are used as main raw materials, the alkaline solution environment retains food calling factors and swallowing factors of the mulberry leaves to the maximum extent, the characteristics of resistant protein in the bean pulp powder and the corn flour are damaged, and the formula is rich in nutrition, high in feeding rate, simple in manufacturing process and convenient to popularize.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological agriculture and enzyme engineering related industries, and more specifically, relates to an artificial feed for silkworm larvae containing protease, and a preparation method and application thereof. Background Art

[0002] During the growth of silkworm larvae, adding a certain amount of protease to the feed can improve the utilization rate of feed, promote the growth and development of silkworm larvae, resist the invasion of insect pathogenic fungi, and reduce the morbidity of silkworm larvae. For example, alkaline protease is a class of enzymes that have the ability to hydrolyze protein under alkaline conditions. In the midgut tissue of silkworm larvae, there are two alkaline proteases that decompose casein and BAPNA (benzoylarginine for p-nitroaniline). After synthesis in the midgut tissue, they are located on the fine line edge and are membrane-bound. These alkaline proteases help silkworm larvae digest and absorb feed. Serine protease is a class of proteases with serine as the active center, which has a wide range of substrate specificity and hydrolytic activity. In silkworms, serine protease p37k shows different activities in the midgut and molting fluid. In the midgut, serine protease p37k shows strong gelatinase activity, which helps silkworm larvae digest feed. Therefore, adding serine protease to feed can further improve the digestion ability of silkworm larvae to protein. The amount of protease added should be adjusted according to the growth stage of the silkworm larvae, feed composition, feeding environment and other factors. Excessive protease may cause feed waste and environmental pollution, while too little protease may not meet the growth needs of the silkworm larvae.

[0003] At present, when preparing silkworm feed containing protease, how to effectively load protease into silkworm feed is a technical problem. The loading methods that can be used for reference include direct addition, embedding, adsorption, etc. The direct addition method is simple to operate and low in cost. It only needs to directly add protease to silkworm feed without complicated processes and equipment; but the disadvantage is poor stability. Protease is easily inactivated by factors such as temperature and humidity. The embedding method can better protect the activity of protease and prevent it from being inactivated during preparation and storage. Through embedding, the stability of protease in feed is improved and can resist the influence of adverse environmental factors. At the same time, the embedding method uses a variety of carriers, such as agarose, sodium alginate, etc., and a suitable carrier can be selected according to needs. However, the cost of the carrier of the embedding method is relatively high, and after long-term use, the embedding material may be damaged, resulting in protease leakage, affecting the feed effect. Although the adsorption loading method is relatively simple to operate, the enzyme loading amount is low and it is not suitable for long-term storage. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] In order to achieve these purposes and other advantages according to the present invention, a protease-containing artificial feed for silkworm larvae is provided, which 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 flour, 120-130 parts of calcium hydroxide solution, 50-60 parts of sodium bicarbonate solution, and 1-3 parts of paraben.

[0006] Preferably, the protease contained in the protease preparation is one of serine protease, subtilisin and serrapeptase.

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

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

[0009] S1. Prepare phosphate buffer saline (PBS), add protease powder into the phosphate buffer; add flavonol into anhydrous ethanol to obtain a flavonol solution;

[0010] S2, mixing equal volumes of phosphate buffer and flavonol solution, dispersing by ultrasonication, and standing for 12 to 24 hours; then injecting pure water, the solubility of flavonol decreases and precipitates, separating the lower layer solution, evaporating and removing ethanol, and obtaining a flavonol phase loaded with protease;

[0011] S3. Evenly mix the flavonol phase and the sodium alginate solution to obtain a mixed dispersion system, and drop the mixed dispersion system into the calcium chloride solution drop by drop while stirring at a speed of 100-120 rpm. After the addition is completed, stop stirring, let it stand for 5-12 hours, separate the solidified gel, and freeze-dry to obtain a protease preparation.

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

[0013] Preferably, in 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 S3, the concentration of the sodium alginate solution is 0.2-0.6wt%, the volume ratio of the sodium alginate solution to the flavonol solution is 1-3:2-3; the concentration of the calcium chloride solution is 10-30wt%; the freeze-drying temperature is -25--18°C, and the freeze-drying time is 1-3h.

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

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

[0017] Step 2: Mix soybean meal powder, corn flour, paraben and sodium bicarbonate solution and then steam them, spread them to cool and mix them with mulberry leaf slurry, then add protease preparation to obtain artificial feed for silkworm larvae containing protease.

[0018] Preferably, in the step 2, the cooking temperature is 100-120° C., and the cooking time is 10-30 min.

[0019] The invention discloses an application of an artificial feed for silkworm larvae containing protease. The artificial feed for silkworm larvae containing protease is applied to feeding 1-3 instar silkworm larvae, and the feeding frequency is 4-5 times a day.

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

[0021] The artificial feed for silkworm larvae containing protease provided by the present invention uses fresh mulberry leaves, soybean meal powder and corn flour as main raw materials. The alkaline solution environment has the characteristics of retaining the mulberry leaf feeding attractant factor and swallowing factor to the greatest extent and destroying the resistance protein in soybean meal powder and corn flour. The feed formula is rich in nutrition, simple in formula, high in feeding rate, and uniform in growth. The production process is simple and easy to promote.

[0022] The artificial feed for silkworm larvae containing protease prepared by the present invention contains a protease preparation. During the growth of silkworm larvae, the artificial feed for silkworm larvae containing protease of the present invention can effectively supplement the protease in the silkworm larvae, promote the growth and development of the silkworm larvae, resist the invasion of insect pathogenic fungi, and thus reduce the morbidity of the silkworm larvae. At the same time, in the preparation method of the protease preparation of the present invention, an anti-solvent method is used, flavonol is used to load the protease, and the covalent action between the flavonol and the protease is utilized to achieve efficient loading of the protease, increase the loading amount of the protease, and use sodium alginate gel to coat it, thereby ensuring that the activity of the protease in the protease preparation is maintained at a high level within 60 days, and effectively prolonging the storage time of the protease preparation.

[0023] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0024] The present invention is described in further detail below so that those skilled in the art can implement it according to the description.

[0025] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0026] Embodiment 1:

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

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

[0029] Step 2: Mix 120g soybean meal powder, 150g corn flour, 10g paraben and 1wt% 500g sodium bicarbonate solution and steam them at a temperature of 115°C for 15min. After cooling, mix them with mulberry leaf slurry, 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 2 of this embodiment includes:

[0031] S1. Prepare 100 mL of phosphate buffer solution (PBS) with a pH value of 7.4, add 50 mg of serine protease (proteinase K) powder into the phosphate buffer solution; add 100 mg of flavonol into 100 mL of anhydrous ethanol to dissolve and obtain a flavonol solution;

[0032] S2, 100 mL of phosphate buffer containing serine protease powder was mixed with 100 mL of flavonol solution, and after 30 kHz ultrasonic dispersion for 15 min, the mixture was allowed to stand for 12 h; then 400 mL of deionized water was injected, and the solubility of flavonol decreased and precipitated, and the lower layer of solution was separated, and ethanol was evaporated to obtain a flavonol phase loaded with serine protease;

[0033] S3. Evenly mix 50 mL of flavonol phase and 25 mL of 0.5 wt% sodium alginate solution to obtain a mixed dispersion system, and drop the mixed dispersion system into a 20 wt% calcium chloride solution drop by drop while stirring at 120 rpm. After the addition is completed, stop stirring, let it stand for 6 hours, separate the solidified gel, and freeze-dry it at -25°C for 2 hours to obtain a protease preparation.

[0034] Embodiment 2:

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

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

[0037] Step 2: Mix 150g soybean meal powder, 180g corn flour, 20g paraben and 550g 1wt% sodium bicarbonate solution and steam them at a temperature of 115°C for 15min. After cooling, mix them with mulberry leaf slurry, 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 2 of this embodiment includes:

[0039] S1. Prepare 100 mL of phosphate buffer solution (PBS) with a pH value of 7.4, add 50 mg of serine protease (proteinase K) powder into the phosphate buffer solution; add 100 mg of flavonol into 100 mL of anhydrous ethanol to dissolve and obtain a flavonol solution;

[0040] S2, 100 mL of phosphate buffer containing serine protease was mixed with 100 mL of flavonol solution, and after 30 kHz ultrasonic dispersion for 15 min, the mixture was allowed to stand for 12 h; then 500 mL of deionized water was injected, and the solubility of flavonol decreased and precipitated, and the lower layer solution was separated, and ethanol was evaporated to obtain a flavonol phase loaded with serine protease;

[0041] S3. Evenly mix 60 mL of flavonol phase and 25 mL of 0.5 wt% sodium alginate solution to obtain a mixed dispersion system, and drop the mixed dispersion system into a 25 wt% calcium chloride solution drop by drop while stirring at 120 rpm. After the addition is completed, stop stirring, let it stand for 6 hours, separate the solidified gel, and freeze-dry it at -25°C for 2 hours to obtain a protease preparation.

[0042] Embodiment 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 the fresh mulberry leaves into strips of 1 to 3 cm, then weigh 700 g of the fresh mulberry leaf strips and mix them with 1300 g of 0.5 wt % calcium hydroxide solution to obtain a mulberry leaf homogenate;

[0045] Step 2: Mix 180g soybean meal powder, 200g corn flour, 30g paraben and 1wt% 600g sodium bicarbonate solution and cook them at a cooking temperature of 115°C for 15min. After cooling, mix them with mulberry leaf slurry, 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 2 of this embodiment includes:

[0047] S1. Prepare 100 mL of phosphate buffer solution (PBS) with a pH of 7.5, add 50 mg of serine protease (proteinase K) powder into the phosphate buffer solution; add 100 mg of flavonol into 100 mL of anhydrous ethanol to dissolve and obtain a flavonol solution;

[0048] S2, 100 mL of phosphate buffer containing serine protease was mixed with 100 mL of flavonol solution, and after 30 kHz ultrasonic dispersion for 15 min, the mixture was allowed to stand for 12 h; then 500 mL of deionized water was injected, and the solubility of flavonol decreased and precipitated, and the lower layer solution was separated, and ethanol was evaporated to obtain a flavonol phase loaded with serine protease;

[0049] S3. Evenly mix 50 mL of flavonol phase and 25 mL of 0.5 wt% sodium alginate solution to obtain a mixed dispersion system, and drop the mixed dispersion system into a 20 wt% calcium chloride solution drop by drop while stirring at 100 rpm. After the addition is completed, stop stirring, let it stand for 6 hours, separate the solidified gel, and freeze-dry it at -20°C for 2 hours to obtain a protease preparation.

[0050] Comparative Example 1:

[0051] This example provides a method for preparing an artificial feed for silkworm larvae containing protease. The difference from the other examples 1 is that the preparation method of the protease preparation used in step 2 of this comparative example is:

[0052] S1. Prepare 100 mL of phosphate buffer (PBS) with a pH value of 7.4, and add 50 mg of serine protease (proteinase K) powder into the phosphate buffer;

[0053] S2. Evenly mix 50 mL of phosphate buffer containing serine protease powder and 25 mL of 0.5 wt% sodium alginate solution to obtain a mixed dispersion system. Drop the mixed dispersion system into a 20 wt% calcium chloride solution drop by drop while stirring at 120 rpm. After the addition is completed, stop stirring, let stand for 6 hours, separate the solidified gel, and freeze-dry at -25°C for 2 hours to obtain a protease preparation.

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

[0055] Comparative Example 2:

[0056] This example provides a method for preparing an artificial feed for silkworm larvae containing protease. The difference from the other examples 1 is that the preparation method of the protease preparation used in step 2 of this comparative example is:

[0057] S1. Prepare 100 mL of phosphate buffer solution (PBS) with a pH value of 7.4, add 50 mg of serine protease (proteinase K) powder into the phosphate buffer solution; add 100 mg of flavonol into 100 mL of anhydrous ethanol to dissolve and obtain a flavonol solution;

[0058] S2, 100 mL of phosphate buffer containing serine protease powder was mixed with 100 mL of flavonol solution, and after 30 kHz ultrasonic dispersion for 15 min, the mixture was allowed to stand for 12 h; then 400 mL of deionized water was injected, and the solubility of flavonol decreased and precipitated, and the lower layer of solution was separated, and ethanol was evaporated to obtain a flavonol phase loaded with serine protease;

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

[0060] The serine protease loading of the protease preparations in Examples 1 to 3 and Comparative Examples 1 to 2 was measured respectively, and the results are shown in Table 1:

[0061] Table 1 Serine protease loading of 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] At the same time, 100 g of the protease preparations prepared in Examples 1 to 3 and Comparative Examples 1 to 2 were weighed, sealed and placed at 25 ° C. Samples were taken at 0 days, 10 days, 30 days, 40 days, and 60 days, and the enzyme activity of serine protease (proteinase K) was determined according to GB / T34800-2017. Table 2 was obtained:

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

[0065] Day 0 (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] It can be seen from Table 1 and Table 2 that the loading amount and enzyme activity of the serine protease in the protease preparation used in the artificial feeds for silkworm larvae containing protease in Examples 1 to 3 are significantly better than those in Comparative Examples 1 and 2.

[0067] 700 1-year-old silkworm larvae were selected and divided into 7 groups. The first group was fed with the artificial feed for silkworm larvae containing protease prepared in Example 1, the second group was fed with the artificial feed for silkworm larvae containing protease prepared in Example 2, the third group was fed with the artificial feed for silkworm larvae containing protease prepared in Example 3, the fourth group was fed with the artificial feed for silkworm larvae containing protease prepared in Comparative Example 1, the fifth group was fed with the artificial feed for silkworm larvae containing protease prepared in Comparative Example 2, and the sixth group was used as a blank control 1 and fed with an artificial mixed feed, wherein the preparation method of the artificial mixed feed is as follows: fresh mulberry leaves are crushed into strips of 1 to 3 cm , then weigh 600g of fresh mulberry leaf strips; mix 120g of soybean meal powder, 150g of corn flour, 10g of paraben and 500g of clean water and then steam them at a cooking temperature of 115°C for 15min, spread them out to cool and mix them with fresh mulberry leaf strips, then add 5g of protease preparation to obtain artificial feed for silkworm larvae containing protease; Group 7 is used as blank control 2, artificial feed for silkworm larvae without adding protease preparation, and the artificial mixed feed without adding protease preparation is prepared in the same way as in Example 1 without adding protease preparation, and the feeding rate of each group is measured to obtain Table 3:

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

[0069]

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

[0071] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0072] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. An artificial feed for silkworm larvae containing protease, characterized in that: The composition comprises, by weight: 0.2-1 part of protease preparation, 60-70 parts of fresh mulberry leaves, 12-18 parts of soybean meal powder, 15-20 parts of corn flour, 120-130 parts of calcium hydroxide solution, 50-60 parts of sodium bicarbonate solution and 1-3 parts of paraben.

2. The artificial feed for silkworm larvae containing protease according to claim 1, characterized in that: The protease contained in the protease preparation is one of serine protease, subtilisin and serrapeptase.

3. The artificial feed for silkworm larvae containing protease according to claim 1, characterized in that: The concentration of the calcium hydroxide solution is 0.2-0.5wt%; the concentration of the sodium bicarbonate is 0.5-1wt%.

4. The artificial feed for silkworm larvae containing protease according to claim 1, characterized in that: The preparation method of the protease preparation comprises the following steps: S1. Prepare phosphate buffer solution (PBS), add protease powder into the phosphate buffer solution; add flavonol into anhydrous ethanol to obtain a flavonol solution; S2, mixing equal volumes of phosphate buffer and flavonol solution, dispersing by ultrasonication, and standing for 12 to 24 hours; then injecting pure water, the solubility of flavonol decreases and precipitates, separating the lower layer solution, evaporating and removing ethanol, and obtaining a flavonol phase loaded with protease; S3. Evenly mix the flavonol phase and the sodium alginate solution to obtain a mixed dispersion system, and drop the mixed dispersion system into the calcium chloride solution drop by drop while stirring at a speed of 100-120 rpm. After the addition is completed, stop stirring, let it stand for 5-12 hours, separate the solidified gel, and freeze-dry to obtain a protease preparation.

5. The artificial feed for silkworm larvae containing protease according to claim 4, characterized in that: In the S1, the pH value of the phosphate buffer solution PBS is 7-7.4; the dosage ratio of the phosphate buffer solution PBS, the protein powder, the flavonol, and the anhydrous ethanol is 1-10 mL: 5-8 mg: 10-30 mg: 1-10 mL.

6. The artificial feed for silkworm larvae containing protease according to claim 4, characterized in that: In S2, the volume of deionized water is 2 to 5 times the volume of the flavonol solution; the ultrasonic dispersion frequency is 30 to 60 kHz, and the ultrasonic dispersion time is 10 to 20 min.

7. The artificial feed for silkworm larvae containing protease according to claim 4, characterized in that: In S3, the concentration of the sodium alginate solution is 0.2-0.6wt%, and 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 10-30wt%; the freeze-drying temperature is -25--18°C, and the freeze-drying time is 1-3h.

8. A method for preparing the artificial feed for silkworm larvae containing protease according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step 1: crush the fresh mulberry leaves into strips of 1 to 3 cm, and then mix them with a calcium hydroxide solution to obtain a mulberry leaf homogenate; Step 2: Mix soybean meal powder, corn flour, paraben and sodium bicarbonate solution and then steam them, spread them to cool and mix them with mulberry leaf slurry, then add protease preparation to obtain artificial feed for silkworm larvae containing protease.

9. The method for preparing the artificial feed for silkworm larvae containing protease according to claim 8, characterized in that: In the step 2, the cooking temperature is 100-120° C., and the cooking time is 10-30 min.

10. Use of the artificial feed for silkworm larvae containing protease according to any one of claims 1 to 7, characterized in that: The artificial feed for silkworm larvae containing protease is used for feeding 1-3 instar silkworm larvae, and the feeding frequency is 4-5 times a day.

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