Preparation method and application of silkworm sand feed additive

Abstract

The invention relates to the technical field of biological waste treatment, in particular to a preparation method and application of a silkworm sand feed additive. The invention provides a technical scheme for preparing a silkworm and mulberry sand feed additive rich in acid protease, pectinase and cellulase by taking silkworm sand as a core matrix and performing directional pretreatment, adaptive strain fermentation and standardized post-treatment. According to the invention, silkworm sand-corncob powder is used as a composite substrate, and the silkworm sand and the corncob powder complement each other in function, so that the problems of unbalanced nutrition and poor air permeability of a single substrate are solved. Specific aspergillus niger is used for segmented solid-state fermentation, parameters are set in a differentiated mode, and full-amount utilization of the silkworm sand is achieved; the product quality and enzyme activity are improved by optimizing the process, and the resource value is improved from the low-added-value field to the high-added-value enzyme preparation industry.

Description

Technical Field

[0001] This invention relates to the field of biological waste treatment technology, and in particular to a method for preparing silkworm excrement feed additive and its application. Background Technology

[0002] As the world's largest producer of silkworm excrement, my country generates over 3 million tons of silkworm excrement annually. Naturally rich in cellulose (30%-40%), crude protein (15%-20%), pectin (5%-8%), and various minerals, silkworm excrement possesses the inherent qualities to be a high-quality substrate for biotransformation. However, current resource utilization of silkworm excrement faces numerous limitations: over 90% is directly used for composting or discarded indiscriminately, resulting in a serious waste of high-quality biomass resources. Furthermore, the ammonia nitrogen and foul odors generated during composting cause soil and water pollution problems. The few cases of its use for biogas fermentation only achieve primary energy conversion, resulting in extremely low economic added value. Even in the field of fermentation research, existing technologies largely focus on single-enzyme preparation or simple organic fertilizer production, failing to fully explore its potential for multi-component synergistic enzyme production, resulting in a resource utilization rate of less than 5%.

[0003] In the feed industry, enzyme preparations are commonly used to improve feed quality. Among them, acidic proteases, pectinases, and cellulases can significantly improve feed digestibility and absorption efficiency through synergistic effects, thereby reducing overall breeding costs. Mainstream enzyme production still uses grain crops such as corn and soybean meal as core substrates, requiring 3-5 tons of grain raw materials to produce 1 ton of enzyme preparation, further exacerbating the supply-demand imbalance of grain resources. Furthermore, traditional liquid fermentation processes consume as much as 10-15 tons of water per ton of fermentation liquid, contradicting the development requirements of water conservation and emission reduction in agriculture. With the continuous expansion of the silkworm breeding industry, the output of silkworm excrement, as a major byproduct, is increasing year by year, highlighting the drawbacks of traditional disposal methods (random disposal and simple landfill). Although silkworm excrement is rich in protein, cellulose, pectin, and various vitamins and minerals, its high initial water content (60%-70%) makes it highly susceptible to the growth of pathogenic microorganisms such as bacteria and mold. Furthermore, the tannins and phytic acid it contains are anti-nutritional factors. If used directly as feed, it may not only cause diseases in animals but also significantly reduce the digestibility and absorption rate of feed nutrients. This greatly limits its direct application in the feed industry.

[0004] More importantly, existing silkworm excrement processing technologies suffer from three major shortcomings: First, the pretreatment process is rudimentary, failing to be specifically optimized for the dense fiber structure and waxy surface of silkworm excrement, resulting in low microbial degradation efficiency. Second, the selection of microbial strains is limited, failing to match the multi-component nutritional structure of silkworm excrement and hindering the synergistic production of multiple enzymes. Third, the process system is disconnected from industry needs, with fermentation products mostly being crude semi-finished products that do not meet the industrial production standards for feed additives, making it difficult to effectively upgrade the resource value. Data shows that the added value of existing silkworm excrement utilization models is less than 200 yuan / ton, while the market value of enzyme-based feed additives can reach 5,000-8,000 yuan / ton, a significant gap, indicating that the high-value conversion potential of silkworm excrement urgently needs to be explored. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a technical solution for preparing a silkworm excrement feed additive rich in acidic protease, pectinase and cellulase by using silkworm excrement as the core matrix and through targeted pretreatment, fermentation with suitable strains and standardized post-treatment.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing silkworm excrement feed additive, comprising the following steps: (1) A composite matrix is ​​obtained by mixing silkworm excrement and corn cob powder, and then sterilized after adding acid and stirring. (2) Mix the sterilized composite matrix with the excipients, stir evenly, adjust the pH, and inoculate with Aspergillus niger ( Aspergillus niger Seed liquid is subjected to solid-state fermentation to obtain fermentation products; The excipients include ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, Tween 80, and water; (3) The fermentation product obtained in step (2) is dried and pulverized to obtain the silkworm excrement feed additive.

[0007] This invention employs a composite matrix of silkworm excrement and corn cob powder. The crude protein in silkworm excrement serves as a high-quality nitrogen source, providing essential nitrogen for the growth and reproduction of microorganisms. Its pectin content efficiently induces the synthesis of pectinase by the microorganisms. Corn cob powder, with its porous structure, improves the looseness of the fermentation matrix, increases oxygen exchange efficiency, and provides sufficient oxygen for aerobic fermentation. Its abundant cellulose content acts as a specific inducer for cellulase, promoting its efficient expression, and also supplements the carbon source, synergistically complementing the nitrogen source in the silkworm excrement to optimize the carbon-nitrogen ratio of the matrix and meet the nutritional requirements of the microorganisms during enzyme production. This invention uses acid to sterilize and pretreat the composite matrix, effectively breaking down the waxy layer on the surface of the silkworm excrement and degrading some tannins and phytic acid, solving the problems of low degradation efficiency and interference from nutrient factors caused by extensive pretreatment. It also lowers the pH of the fermentation matrix, making it more conducive to subsequent fermentation. The fermentation adjuvants used in this invention are only inexpensive inorganic salts such as ammonium sulfate and magnesium sulfate, and Tween 80, which both supplement nutrition and enhance the adhesion of the microorganisms.

[0008] This invention optimizes the process for preparing silkworm excrement feed additives, resolving the "resource mismatch" contradiction between enzyme preparation production's reliance on grain raw materials such as corn and soybean meal and the large amount of idle silkworm excrement. It achieves high-value substitution of agricultural waste using silkworm excrement as the main substrate, alleviating food shortages. It overcomes the problem of low microbial degradation efficiency of silkworm excrement due to extensive pretreatment, improving substrate accessibility through targeted pretreatment technology. It solves the problems of mismatch between single strains and multiple components of silkworm excrement, and the difficulty in synergistic production of multiple enzymes, achieving efficient synergistic synthesis of acidic protease, pectinase, and cellulase. It eliminates the drawbacks of the disconnect between silkworm excrement fermentation processes and industrial standards for feed additives, constructing an integrated standard process from fermentation to post-processing to ensure product compliance. It resolves the conflict between the high water consumption of traditional liquid fermentation, the high pollution from indiscriminate disposal of silkworm excrement, and environmental protection requirements, adopting solid-state fermentation and achieving full utilization of silkworm excrement. It overcomes the predicament of low economic added value in the utilization of silkworm excrement, improving product quality and enzyme activity through process optimization, elevating its resource value from a low-value-added field to the high-value-added enzyme preparation industry.

[0009] Preferably, in step (1), the silkworm excrement is first passed through a 10-20 mesh vibrating screen to remove impurities such as mulberry leaf stems and silkworm excrement.

[0010] Preferably, in step (1), the mass ratio of silkworm excrement to corn cob powder is (60-70):(30-40).

[0011] This invention employs a functional synergistic design of a composite matrix of silkworm excrement and corn cob powder. Through precise proportioning, the crude protein (nitrogen source) and pectin (pectinase inducer) of silkworm excrement complement the porous structure (improved aeration) and cellulose (carbon source and cellulase inducer) of corn cob powder, optimizing the carbon-nitrogen ratio of the matrix to the optimal range for enzyme production by Aspergillus niger. At the same time, the addition of corn cob powder improves the looseness of the matrix and significantly enhances the oxygen exchange efficiency, solving the problems of unbalanced nutrition and poor aeration in single-matrix matrices.

[0012] Preferably, the acid comprises a 0.8% citric acid solution.

[0013] Preferably, in step (1), 0.8% citric acid solution is added at a solid-liquid ratio of 1:0.3.

[0014] Preferably, in step (1), the sterilization conditions are high-temperature sterilization at 121°C for 20-25 minutes.

[0015] Preferably, the excipients comprise the following components in parts by weight: 1-2 parts ammonium sulfate, 0.5-1 part magnesium sulfate, 0.5-1 part potassium dihydrogen phosphate, 0.5-1 part Tween 80, and 30 parts water.

[0016] Preferably, in step (2), the mass ratio of the excipients to the sterilized composite matrix is ​​(3-5):100.

[0017] Preferably, the Aspergillus niger ( Aspergillus niger It originates from the China Industrial Microbial Culture Collection Center, with accession number CICC 2241.

[0018] The preferred strain of this invention is Aspergillus niger (… Aspergillus niger It possesses the ability to synergistically produce multiple enzymes from a single strain and has a natural compatibility with the "silkworm excrement-corn cob powder" composite substrate. When fermenting in the silkworm excrement substrate, it can simultaneously and efficiently synthesize acidic protease (4000-4500 U / g), pectinase (≥20000 U / g), and cellulase (≥500 U / g) without the need for composite strains. This breaks through the technical bottleneck that a single strain cannot simultaneously achieve the synthesis of multiple enzymes and solves the problem of mismatch between a single strain and a multi-component substrate.

[0019] Preferably, the Aspergillus niger ( Aspergillus niger The method for preparing seed liquid is as follows: Aspergillus niger ( Aspergillus niger Inoculate the spores onto PDA slant culture medium and incubate at 30℃ for 72 h to restore the activity of the strain. Wash the spores with sterile water, pick the slant culture and inoculate it into seed culture medium. Incubate at 30℃ and 200 rpm in a shake flask for 48-72 h to prepare seed liquid.

[0020] Preferably, in step (2), the pH is adjusted to 5.5-6.0.

[0021] The sterilized composite substrate is loaded into a clean mixing tank, and auxiliary materials are added according to the substrate weight. The mixture is stirred evenly to adjust the initial pH to 5.5-6.0, providing more nitrogen source and essential inorganic salts for the growth of microorganisms.

[0022] Preferably, the inoculation amount of the seed liquid is 10% of the mass of the composite substrate.

[0023] Preferably, the solid-state fermentation employs segmented fermentation control: during the 0-20 h fermentation stage, the temperature is controlled at 32-35℃, the relative humidity at 80-90%, and the air volume at 0.06 m³ / h. 3 / (h·kg); During the fermentation stage of 30-72h, reduce the temperature to 28-30℃, adjust the relative humidity to 60-70%, and adjust the air volume to 0.04 m. 3 / (h·kg); fermentation was stopped after 72 h when the fermentation mycelium turned gray.

[0024] This invention utilizes differentiated parameters for staged fermentation based on the varying physiological needs of Aspergillus niger during its cell proliferation and enzyme production induction phases. During the cell proliferation stage, the temperature is controlled at 32-35℃, humidity at 80-90%, and the humidity at 0.06 m³ / h. 3 An airflow of / (h·kg) allows for rapid growth of Aspergillus niger mycelia, forming a dominant fungal community and promoting rapid mycelial colonization; during the enzyme induction stage, the temperature is lowered to 28-30℃, the relative humidity is adjusted to 60-70%, and the airflow is 0.04 m³ / h. 3 / (h·kg), through mild environmental stress to induce efficient expression of enzyme genes, the fermentation cycle is compressed to 3 days.

[0025] Preferably, the solid-state fermentation is carried out in a fermentation tank, with the loading amount controlled at 60-70% (by volume), and sufficient space is reserved to ensure ventilation and oxygen supply.

[0026] Preferably, fermentation is terminated when the fermentation mycelium turns gray and the pectinase activity measured by sampling is stable at above 20,000 U / g, and the fermentation cycle is controlled within 3 days.

[0027] Preferably, in step (3), the fermentation product is dried at 40-45℃ until the moisture content is <7.0%, and then pulverized to 100 mesh to obtain the silkworm excrement feed additive.

[0028] The post-processing of this invention uses "drying at 40-45℃ + pulverizing at 100 mesh" to avoid enzyme activity loss caused by extraction and purification (retention rate ≥95%). At the same time, the drying temperature matches the stability requirements of heat-sensitive enzymes, and the product directly meets the particle size and purity requirements of feed additives.

[0029] Secondly, the present invention provides a method for preparing silkworm excrement feed additives to obtain silkworm excrement feed additives.

[0030] Thirdly, the present invention provides the application of the aforementioned silkworm excrement feed additive in the preparation of animal feed.

[0031] The beneficial effects of this invention are as follows: 1. More precise matrix formulation enables high-value synergistic utilization of agricultural waste. Existing technologies suffer from two major resource utilization pain points: first, enzyme production relies on grain raw materials such as corn and soybean meal (consuming 3-5 tons per ton), exacerbating food shortages; second, silkworm excrement is mostly discarded or used for low-value composting (added value less than 200 yuan / ton), and corn cob powder is mostly used as fuel, with the resource value of both not being fully explored. This invention innovatively adopts a "silkworm excrement-corn cob powder" composite matrix to achieve dual synergy of function and nutrition: the crude protein of silkworm excrement provides a high-quality nitrogen source, pectin induces pectinase synthesis, and corn cob powder improves the looseness of the matrix through its porous structure (enhancing oxygen exchange efficiency), while cellulose supplements the carbon source and induces cellulase expression. The optimized matrix carbon-nitrogen ratio is adapted to the enzyme production needs of the strain. Calculations show that each ton of product can consume 0.92 tons of silkworm excrement and 0.5 tons of corn cob powder, with a market price of no less than 30 yuan / kg, increasing added value by 40 times.

[0032] 2. More efficient pretreatment process, combining degradation promotion and sterilization. This invention adopts an integrated pretreatment of "0.8% citric acid treatment + 121℃ sterilization for 20-25 minutes", which has significant advantages: First, it can effectively break down the waxy layer on the surface of silkworm excrement, with a tannin and phytic acid removal rate of up to 45%, which is 50% more efficient than traditional water washing pretreatment in terms of microbial degradation; Second, 121℃ sterilization for 20-25 minutes can achieve a kill rate of ≥99.9% of miscellaneous bacteria, avoiding fermentation contamination; Third, after citric acid treatment, the pH of the substrate naturally decreases to below 7.0, and subsequent adjustments can be made to approach the suitable pH for fermentation (5.5-6.0) without much additional work, simplifying the process.

[0033] 3. The Aspergillus niger strain screened in this invention, with preservation number CICC2241, exhibits strong adaptability to composite substrates and possesses the characteristic of "one strain producing multiple enzymes." It can simultaneously synthesize acidic protease (4000 U / g), pectinase (>20000 U / g), and cellulase (>500 U / g) without the need for composite strains. Furthermore, it innovatively employs "segmented fermentation regulation," controlling the cell proliferation period (32℃, 80% humidity, 0.06 m... 3 / (h·kg) airflow) promotes rapid mycelial colonization, enzyme production period (30℃, 70% humidity, 0.04 m 3 The / (h·kg) air volume induces enzyme expression through environmental stress, and the fermentation cycle is only 3 days, which is 40% more efficient than the traditional 5-7 day fermentation cycle. At the same time, it avoids excessive mycelial growth that consumes nutrients and reduces the risk of contamination.

[0034] 4. This invention has advantages in terms of excipients and post-processing: First, the excipient formula is simplified and practical, adding only inexpensive inorganic salts such as ammonium sulfate and magnesium sulfate, and Tween 80 (total addition amount 3.5%-5%), which not only supplements nitrogen source and trace elements, but also enhances the adhesion of the strain to the substrate and the release of products through Tween 80, reducing the cost of excipients by 50% compared with the existing technology; Second, the post-processing adopts a simplified process of "drying at 40-45℃ + pulverizing at 100 mesh", avoiding enzyme activity loss during the extraction process, with an enzyme activity retention rate of over 95%, while the drying temperature is lower than the existing process of over 60℃, further reducing the activity decay of heat-sensitive enzymes (such as acidic proteases); Third, the product is directly pulverized to 100 mesh, with uniform particle size, which can meet the mixing requirements of feed additives without additional granulation, making it more adaptable to industrial applications. Detailed Implementation

[0035] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0036] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products that can be obtained through commercial channels.

[0037] The Aspergillus niger described in this invention ( Aspergillus niger Purchased from the China Industrial Culture Collection Center, accession number CICC 2241.

[0038] PDA medium formula: 200 g / L potato, 20 g / L glucose, 20 g / L agar.

[0039] Seed culture medium (PDB): 200 g / L potato, 20 g / L glucose.

[0040] The silkworm excrement feed additive prepared by this invention meets the following feed additive standards after indicator testing: acidic protease activity 4000-4500 U / g, pectinase activity ≥20000 U / g, cellulase activity ≥500 U / g, moisture content ≤8%, and bacterial count ≤10. 4 The CFU / g and heavy metal content (lead, arsenic, mercury) both meet the requirements of GB 13078-2017 "Feed Hygiene Standard".

[0041] Example 1 An embodiment of the silkworm excrement feed additive of the present invention includes the following steps in its preparation method: (1) Pretreatment: Collect silkworm excrement and remove impurities such as mulberry leaf stalks and silkworm excrement through a 10-20 mesh vibrating sieve; mix 650 g of silkworm excrement with 350 g of corn cob powder to obtain a composite matrix, add 0.8% citric acid solution (300 mL) at a solid-liquid ratio of 1:0.3, stir and mix for 10 min, and then sterilize at 121℃ for 20 minutes. (2) Activation of microbial strains: Aspergillus niger ( Aspergillus niger The spores were inoculated onto PDA slant culture medium and incubated at 30°C for 72 h to restore the activity of the strain. The spores were washed with sterile water, and the slant culture was picked and inoculated into seed culture medium. The culture was then carried out in shake flasks at 30°C and 200 rpm for 48 h to prepare the seed culture. Preparation of excipients: Weigh 1.5 parts (15 g) of ammonium sulfate, 0.75 parts (7.5 g) of magnesium sulfate, 0.75 parts (7.5 g) of potassium dihydrogen phosphate, 0.75 parts (7.5 g) of Tween 80, and 30 parts (300 g) of water. Dissolve and mix well, then sterilize at 121°C for 15 minutes to obtain the excipients. The sterilized composite substrate from step (1) is loaded into a clean mixing tank, and auxiliary materials are added. The mixture is stirred evenly to adjust the initial pH to 5.5-6.0. Seed liquid (100 mL) is inoculated at a rate of 10% of the composite substrate mass and stirred evenly. The loading of the fermentation tank is controlled at 65% (volume ratio), and sufficient space is reserved to ensure ventilation and oxygen supply. Solid-state fermentation yields the fermentation product. The fermentation process employs segmented fermentation control: During the bacterial cell proliferation stage (0-30 h), the temperature was controlled at 32℃, the relative humidity at 80%, and the air volume at 0.06 m³ / h. 3 / (h·kg), promotes the rapid growth of Aspergillus niger mycelium and the formation of dominant fungal communities; During the enzyme induction phase (30-72 h), the temperature is lowered to 30℃, the relative humidity is adjusted to 70%, and the airflow is 0.04 m³ / h. 3 / (h·kg), When the fermentation mycelium turns gray and the pectinase activity measured by sampling is stable above 20,000 U / g, the fermentation is terminated, and the fermentation cycle is controlled within 3 days.

[0042] (3) The fermentation product obtained in step (2) is dried at 42°C to a moisture content of 6.59% and pulverized to 100 mesh to obtain the silkworm excrement feed additive.

[0043] Example 2 An embodiment of the silkworm excrement feed additive of the present invention includes the following steps in its preparation method: (1) Pretreatment: Collect silkworm excrement and remove impurities such as mulberry leaf stalks and silkworm excrement through a 10-20 mesh vibrating sieve; mix 600 g of silkworm excrement with 400 g of corn cob powder to obtain a composite matrix, add 0.8% citric acid solution (300 mL) at a solid-liquid ratio of 1:0.3, stir and mix for 10 min, and then sterilize at 121℃ for 20 minutes. (2) Activation of microbial strains: Aspergillus niger ( Aspergillus niger The spores were inoculated onto PDA slant culture medium and incubated at 30°C for 72 h to restore the activity of the strain. The spores were washed with sterile water, and the slant culture was picked and inoculated into seed culture medium. The culture was then carried out in shake flasks at 30°C and 200 rpm for 48 h to prepare the seed culture. Preparation of excipients: Weigh 1.0 part (10 g) of ammonium sulfate, 0.5 part (5 g) of magnesium sulfate, 0.5 part (5 g) of potassium dihydrogen phosphate, 0.5 part (5 g) of Tween 80, and 30 parts (300 g) of water. Dissolve and mix well, then sterilize at 121°C for 15 minutes to obtain the excipients. The sterilized composite substrate from step (1) is loaded into a clean mixing tank, and auxiliary materials are added. The mixture is stirred evenly to adjust the initial pH to 5.5-6.0. Seed liquid (100 mL) is inoculated at a rate of 10% of the composite substrate mass and stirred evenly. The loading of the fermentation tank is controlled at 65% (volume ratio), and sufficient space is reserved to ensure ventilation and oxygen supply. Solid-state fermentation yields the fermentation product. The fermentation process employs segmented fermentation control: During the bacterial cell proliferation stage (0-30 h), the temperature was controlled at 32℃, the relative humidity at 80%, and the air volume at 0.06 m³ / h. 3 / (h·kg), promotes the rapid growth of Aspergillus niger mycelium and the formation of dominant fungal communities; During the enzyme induction phase (30-72 h), the temperature is lowered to 30℃, the relative humidity is adjusted to 70%, and the airflow is 0.04 m³ / h. 3 / (h·kg), When the fermentation mycelium turns gray and the pectinase activity measured by sampling is stable above 20,000 U / g, the fermentation is terminated, and the fermentation cycle is controlled within 3 days.

[0044] (3) The fermentation product obtained in step (2) is dried at 42°C to a moisture content of 6.78% and pulverized to 100 mesh to obtain the silkworm excrement feed additive.

[0045] Example 3 An embodiment of the silkworm excrement feed additive of the present invention includes the following steps in its preparation method: (1) Pretreatment: Collect silkworm excrement and remove impurities such as mulberry leaf stalks and silkworm excrement through a 10-20 mesh vibrating sieve; mix 700 g of silkworm excrement with 300 g of corn cob powder to obtain a composite matrix, add 0.8% citric acid solution (300 mL) at a solid-liquid ratio of 1:0.3, stir and mix for 10 min, and then sterilize at 121℃ for 20 minutes. (2) Activation of microbial strains: Aspergillus niger ( Aspergillus niger The spores were inoculated onto PDA slant culture medium and incubated at 30°C for 72 h to restore the activity of the strain. The spores were washed with sterile water, and the slant culture was picked and inoculated into seed culture medium. The culture was then carried out in shake flasks at 30°C and 200 rpm for 48 h to prepare the seed culture. Preparation of excipients: Weigh 2.0 parts (20 g) of ammonium sulfate, 1.0 part (10 g) of magnesium sulfate, 1.0 part (10 g) of potassium dihydrogen phosphate, 1.0 part (10 g) of Tween 80, and 30 parts (300 g) of water. Dissolve and mix well, then sterilize at 121°C for 15 minutes to obtain the excipients. The sterilized composite substrate from step (1) is loaded into a clean mixing tank, and auxiliary materials are added. The mixture is stirred evenly to adjust the initial pH to 5.5-6.0. Seed liquid (100 mL) is inoculated at a rate of 10% of the composite substrate mass and stirred evenly. The loading of the fermentation tank is controlled at 65% (volume ratio), and sufficient space is reserved to ensure ventilation and oxygen supply. Solid-state fermentation yields the fermentation product. The fermentation process employs segmented fermentation control: During the bacterial cell proliferation stage (0-30 h), the temperature was controlled at 32℃, the relative humidity at 80%, and the air volume at 0.06 m³ / h. 3 / (h·kg), promotes the rapid growth of Aspergillus niger mycelium and the formation of dominant fungal communities; During the enzyme induction phase (30-72 h), the temperature is lowered to 30℃, the relative humidity is adjusted to 70%, and the airflow is 0.04 m³ / h. 3 / (h·kg), When the fermentation mycelium turns gray and the pectinase activity measured by sampling is stable above 20,000 U / g, the fermentation is terminated, and the fermentation cycle is controlled within 3 days.

[0046] (3) The fermentation product obtained in step (2) is dried at 42°C to a moisture content of 6.61% and pulverized to 100 mesh to obtain the silkworm excrement feed additive.

[0047] Comparative Example 1 This invention provides a comparative example of a silkworm excrement feed additive. The difference between the preparation method of this comparative example and Example 1 lies only in that the fermentation strain used is *Trichoderma harzianum* (…). Trichoderma harzianum The strain was isolated and identified by the present invention, and the rest is the same as in Example 1.

[0048] Comparative Example 2 This invention provides a comparative example of a silkworm excrement feed additive. The difference between the preparation method of this comparative example and Example 1 lies only in that the fermentation strain used is *Aspergillus oryzae* (…). Aspergillus oryzae CICC41737 (purchased from China Industrial Microbial Culture Collection Center) and Aspergillus niger CICC 2241 were used, and the rest were the same as in Example 1.

[0049] Comparative Example 3 This invention provides a comparative example of the silkworm excrement feed additive. The difference between the preparation method of the silkworm excrement feed additive in this comparative example and Example 1 is that corn cob powder is not added, and the silkworm excrement is supplemented to 1000 g. The rest is the same as in Example 1.

[0050] Comparative Example 4 This invention provides a comparative example of a silkworm excrement feed additive. The preparation method of the silkworm excrement feed additive in this comparative example differs from that in Example 1 only in that ammonium sulfate is not added as an auxiliary material; otherwise, the method is the same as in Example 1.

[0051] Comparative Example 5 This invention provides a comparative example of the silkworm excrement feed additive. The difference between the preparation method of the silkworm excrement feed additive in this comparative example and Example 1 is that magnesium sulfate is not added to the excipients, while the rest is the same as in Example 1.

[0052] Comparative Example 6 This invention provides a comparative example of the silkworm excrement feed additive. The difference between the preparation method of the silkworm excrement feed additive in this comparative example and Example 1 is that Tween 80 is not added as an excipient, and the rest is the same as in Example 1.

[0053] Comparative Example 7 This invention provides a comparative example of the silkworm excrement feed additive. The difference between the preparation method of the silkworm excrement feed additive in this comparative example and Example 1 is that an equal amount of wheat bran is used to replace corn cob powder, and the rest is the same as in Example 1.

[0054] Test Example 1 The target indicators of the silkworm excrement feed additives prepared in Examples 1-3 and Comparative Examples 1-7 were tested, and the results are shown in Table 1.

[0055] Acidic protease activity was determined according to the spectrophotometric method for the determination of acidic and neutral protease activity in feed additives, GB / T 28715-2012. Pectinase activity was determined according to GB / T 44738-2024. Cellulase activity was determined using the spectrophotometric method described in NYT912-2020, "Determination of Cellulase Activity in Feed Additives". Moisture content was tested according to GB / T 6435-2014, "Determination of Moisture in Feed".

[0056] Table 1 Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a mulberry silkworm sand feed additive, characterized by, It comprises the following steps: (1) mixing mulberry silkworm sand and corn cob powder to obtain a composite substrate, adding acid, stirring, and sterilizing; (2) mixing the sterilized compound matrix with excipients, stirring uniformly, adjusting pH, inoculating Aspergillus niger (A. niger) seed liquid, and performing solid-state fermentation to obtain a fermentation product; Aspergillus niger )seed liquid, and performing solid-state fermentation to obtain a fermentation product; The auxiliary materials comprise ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, Tween 80 and water. (3) drying and crushing the fermentation product obtained in step (2) to obtain the mulberry silkworm sand feed additive.

2. The preparation method of the silkworm excrement feed additive as described in claim 1, characterized in that, In step (1), the mass ratio of mulberry silkworm sand to corn cob powder is (60-70):(30-40).

3. The preparation method of the silkworm excrement feed additive as described in claim 1, characterized in that, The acid comprises 0.8% citric acid solution.

4. The method for preparing silkworm excrement feed additive as described in claim 1, characterized in that, The auxiliary materials comprise the following components by weight: ammonium sulfate 1-2 parts, magnesium sulfate 0.5-1 part, potassium dihydrogen phosphate 0.5-1 part, Tween 80 0.5-1 part, and water 30 parts.

5. The method for preparing silkworm excrement feed additive as described in claim 1, characterized in that, In step (2), the mass ratio of auxiliary materials to the sterilized composite substrate is (3-5):

100.

6. The method for preparing silkworm excrement feed additive as described in claim 1, characterized in that, The Aspergillus niger (A. niger) Aspergillus niger ) was from China Industrial Culture Collection Center, and the preservation number was CICC 2241.

7. The method for preparing silkworm excrement feed additive as described in claim 1, characterized in that, The inoculation amount of the seed liquid is 10% of the mass of the composite substrate.

8. The method for preparing silkworm excrement feed additive as described in claim 1, characterized in that, The solid state fermentation adopts subsection fermentation control: in the fermentation 0-20 h stage, the temperature is controlled at 32-35℃, the relative humidity is controlled at 80-90%, the air volume is controlled at 0.06 m 3 / (h·kg); in the fermentation to 30-72 h stage, the temperature is reduced to 28-30℃, the relative humidity is adjusted to 60-70%, the air volume is adjusted to 0.04 m 3 / (h·kg); the fermentation is terminated when the fermentation mycelium appears gray at 72 h.

9. The mulberry silkworm sand feed additive prepared by the preparation method of any one of claims 1-8.

10. The use of the mulberry silkworm sand feed additive of claim 9 in the preparation of animal feed.

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