Preparation process of trichoderma reesei cell protein feed fermented by steam exploded rice straw

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a preparation process of Trichoderma reesei cell protein feed produced by steam explosion rice straw fermentation. The present application first applies sophorolipid biosurfactant in a slow-release manner to a solid-state fermentation system with steam explosion straw as a substrate, breaking through the limitation that sophorolipid biosurfactant is traditionally mainly applied to liquid fermentation. Through carefully designed pH-responsive slow-release particles, the three core problems of poor substrate wettability, uneven nutrient distribution and inhibitor toxicity in solid-state fermentation are accurately solved, and the fermentation efficiency is significantly improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a preparation process of Trichoderma reesei cell protein feed produced by steam explosion rice straw fermentation. BACKGROUND

[0002] As a major agricultural waste, rice straw has a huge yield, and its resource utilization is of great significance to sustainable development. Rice straw is rich in organic components such as cellulose and hemicellulose, and can theoretically be used as a high-quality carbon source for microbial fermentation to produce high-value cell protein (single-cell protein), replacing traditional feed protein sources and alleviating the conflict between human and livestock for grain.

[0003] However, rice straw has a complex structure and hard texture, and the lignin in its cell wall is tightly cross-linked with cellulose and hemicellulose, forming a natural biomass anti-degradation barrier, which leads to poor accessibility and low enzymatic efficiency, directly limiting the microbial conversion and utilization of rice straw. At present, steam explosion pretreatment is one of the effective means to solve this problem. It uses high-temperature and high-pressure steam to penetrate into the inside of the straw, and instantaneously releases pressure to blast, which can effectively destroy the physical structure of the straw, separate lignin from carbohydrates, and significantly improve the accessibility of the substrate. However, steam explosion treatment also produces some by-products such as furan and phenolic acid that inhibit microbial growth, and the treated material still has problems such as strong hydrophobicity and easy to form clumps, which affect the adhesion and growth of microorganisms.

[0004] Trichoderma reesei is a recognized, high-efficiency cellulase-producing fungus, which is widely used in cellulose degradation and biological transformation processes. Using it as a fermentation strain, it can utilize the rich enzyme system secreted by it to decompose cellulose and hemicellulose in steam-exploded straw, and convert sugars into its own cell protein. However, in the actual fermentation process, the hydrophobicity of the substrate and the presence of inhibitors still restrict the growth efficiency and enzymatic action of Trichoderma reesei, resulting in low final cell protein yield.

[0005] To improve fermentation efficiency, surfactants such as biosurfactant sophorolipid are often used to improve the hydrophilicity and dispersibility of the substrate, and can reduce the toxicity of inhibitors by binding with them. However, the traditional method of direct addition has obvious defects: one-time addition in the early stage of fermentation may inhibit the growth of mycelium, and surfactants are easily degraded or adsorbed, which cannot continuously play a role in the whole fermentation period, making it difficult to match the dynamic process requirements of Trichoderma reesei, i.e. producing enzymes first, then enzymatic hydrolysis, and then assimilation.

[0006] Therefore, it is of urgent need and great significance to develop an intelligent assistant agent addition technology that can match the fermentation process of Trichoderma reesei, in order to break through the technical bottleneck of high-efficiency biological conversion of rice straw to produce cell protein. SUMMARY

[0007] In view of the deficiencies in the prior art, the present application provides a preparation process of Trichoderma reesei biomass protein feed fermented from steam-exploded rice straw.

[0008] A preparation process of Trichoderma reesei biomass protein feed fermented from steam-exploded rice straw, comprising the following steps:

[0009] (1) 2400-2600 parts of rice straw powder with a moisture content of 25-35 wt% is crushed to a particle size of 0.1-0.5 cm, and then steam-exploded at 180-200℃ and 2-3 Mpa for 4-6 min to obtain steam-exploded straw;

[0010] (2) The carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) is adjusted to 25: (1-2) by urea, then potassium dihydrogen phosphate and magnesium sulfate are added, sterilized at 120-122℃ for 19-21 min, and finally pH-responsive sophorolipid slow-release particles are added to obtain a fermentation medium, in which Trichoderma reesei is inoculated and subjected to aerobic fermentation at 25-35℃ and 4.8-5.2 pH for 36-72 h, and after fermentation, drying is performed to obtain the Trichoderma reesei biomass protein feed.

[0011] In step (2), the addition amount of potassium dihydrogen phosphate is 0.08-0.12 wt% of the steam-exploded straw, and the addition amount of magnesium sulfate is 0.03-0.07 wt% of the steam-exploded straw.

[0012] In step (2), the inoculation amount of Trichoderma reesei is 0.8×10 7 -1.2×10 7 spores / g of fermentation medium.

[0013] In step (2), the 4.8-5.2 pH is adjusted by 1-2 mol / L sulfuric acid.

[0014] In step (2), the addition amount of pH-responsive sophorolipid slow-release particles is 0.5-1 wt% of the mass of the steam-exploded straw.

[0015] The preparation method of the pH-responsive sophorolipid slow-release particles in step (2) comprises the following steps:

[0016] S1 Dissolve 2-3 parts of sodium alginate in 90-100 parts of water by mass fraction, stir until completely dissolved, and obtain a sodium alginate aqueous solution;

[0017] S2 Add 0.1-0.2 parts of sophorolipid to the sodium alginate aqueous solution obtained in step S1 by mass fraction, stir uniformly, and obtain a sophorolipid-sodium alginate aqueous solution;

[0018] S3 adds the sophorolipid-sodium alginate aqueous solution obtained in step S2 into the calcium chloride aqueous solution with a concentration of 1.8-2.2 wt% at a rate of 1-2 ml / min, stands for 30-40 min, filters, washes, and dries to obtain the pH-responsive sophorolipid sustained-release particles.

[0019] Further preferably, the preparation method of the pH-responsive sophorolipid sustained-release particles comprises the following steps:

[0020] S1 dissolves 2-3 parts of sodium alginate in 90-100 parts of water by mass fraction, stirs until completely dissolved to obtain a sodium alginate aqueous solution;

[0021] S2 adds 0.1-0.2 parts of sophorolipid to the sodium alginate aqueous solution obtained in step S1 by mass fraction, stirs uniformly to obtain a sophorolipid-sodium alginate aqueous solution;

[0022] S3 mixes 40-60 parts of corn oil and 0.3-0.8 parts of Span 80 by mass fraction to obtain an emulsion; mixes the sophorolipid-sodium alginate aqueous solution obtained in step S2 and the emulsion and homogenizes at a speed of 10000-15000 rpm for 5-10 min to obtain a sophorolipid-sodium alginate emulsion;

[0023] S4 adds the sophorolipid-sodium alginate emulsion obtained in step S3 into the calcium chloride aqueous solution with a concentration of 1.8-2.2 wt% at a rate of 1-2 ml / min, stands for 30-40 min, filters, washes, and dries to obtain the pH-responsive sophorolipid sustained-release particles.

[0024] Biological surfactants are surface-active molecules produced by microorganisms (such as yeast, bacteria), with a hydrophilic head and a hydrophobic tail structure, which can reduce interfacial tension, improve emulsification, dispersion and wetting properties. Common types include sophorolipids, rhamnolipids, etc. Sophorolipids are a kind of glycolipid biological surfactant produced by yeast, which is widely used in biological processes due to its biodegradability, low toxicity and high surface activity. As a surfactant, sophorolipids adsorb on the substrate-water interface, reducing surface tension and promoting emulsification and dispersion. This increases the available surface area of the substrate, allowing sugar molecules to be more evenly dispersed in the fermentation broth, thereby increasing the sugar uptake rate of the fermentation bacteria. Therefore, it is widely used in liquid fermentation.

[0025] However, whether it can promote the reproduction of fermentation bacteria in solid-state fermentation has not been reported. The present application takes sophorolipid as the core, designs and synthesizes a pH-responsive sophorolipid slow-release particle, and applies it to the solid-state fermentation of steam-exploded rice straw, and finds that after a certain treatment, it can greatly improve the yield of Trichoderma reesei cell protein in the solid-state fermentation of steam-exploded rice straw. The present application speculates that in the process of liquid fermentation, sophorolipid as a biosurfactant mainly improves the dispersibility and emulsification performance in the liquid, and the mechanism of action in solid-state fermentation needs to be changed to improve the substrate wettability, nutrient release and inhibitor management. In solid-state fermentation, the substrate may be dry or agglomerated, resulting in uneven distribution of water and nutrients. The hydrophilic-hydrophobic structure of sophorolipid can help water molecules better infiltrate the hydrophobic solid surface, increase the accessible surface area of the substrate, and thus improve the wettability of the substrate. Better wettability can promote the growth and enzyme secretion of Trichoderma reesei and improve sugar utilization. In addition, sugars and other nutrients in the solid substrate may be bound by lignocellulose structures. Sophorolipid can destroy the hydrophobic barrier in the substrate, making the embedded sugar molecules more easily accessible to microbial enzymes. This may accelerate the hydrolysis process and improve sugar conversion. Finally, inhibitors such as furfural produced by steam-exploded straw may be adsorbed on the surface of the solid substrate, and sophorolipid forms micelles or adsorbs on the surface of the inhibitors, reducing their direct contact with Trichoderma reesei, thereby reducing the toxicity of the inhibitors and improving the growth of the cells and the synthesis of the product.

[0026] The beneficial effects of the present application are:

[0027] The present application first applies sophorolipid biosurfactant in a slow-release manner to the solid-state fermentation system with steam-exploded straw as the substrate, breaking through the limitation of its traditional application mainly in liquid fermentation. Through the carefully designed pH-responsive slow-release particles, the three core problems of poor substrate wettability, uneven nutrient distribution and inhibitor toxicity in solid-state fermentation are accurately solved, and the fermentation efficiency is significantly improved.

[0028] In the early stage of fermentation, the slow-release particles remain intact, avoiding the potential inhibition of high-concentration sophorolipid on mycelial growth; as Trichoderma reesei grows and metabolizes acids, the environmental pH gradually decreases, triggering the decomposition of the sodium alginate gel in the core of the particles, thereby slowly and continuously releasing sophorolipid. This release mode is highly matched with the fermentation process of Trichoderma reesei "growing and producing enzymes first, then enzymatic assimilation", ensuring that sophorolipid plays a key role at the most needed stage and maximizes its efficacy.

[0029] The released sophorolipids significantly improve the wettability of the hydrophobic steam-exploded straw through its amphiphilic structure, making the moisture and mycelium distribution more uniform; it can also destroy the hydrophobic barrier of lignocellulose, release the bound fermentable sugars, and improve the accessibility and saccharification efficiency of the substrate; at the same time, sophorolipids can effectively embed or adsorb fermentation inhibitors such as furfural, reducing their toxic effects on Trichoderma reesei. Under the synergistic effect of the three mechanisms, the growth environment and metabolic efficiency of Trichoderma reesei are fundamentally improved, and the yield of Trichoderma reesei protein is significantly increased.

[0030] The sophorolipids and sodium alginate used in the present application are both biodegradable materials and environmentally friendly; the entire preparation process is simple, easy to integrate with the existing solid state fermentation process, and does not require complex equipment; the use amount of surfactant is reduced through the slow-release technology, waste is avoided, cost-effectiveness is improved, and a new technical path with great application potential is provided for the high-value resource utilization of agricultural waste. DETAILED DESCRIPTION

[0031] Sophorolipids, item number: HBWS-L429, Hubei Weideli Chemical Technology Co., Ltd.

[0032] Sodium alginate, item number: S11053, Shanghai Yuanye Biotechnology Co., Ltd.

[0033] Trichoderma reesei, preservation number: CGMCC 3.3711, purchased from China General Microbiological Culture Collection Center.

[0034] Aspergillus niger, preservation number: CICC 40273, purchased from China Industrial Microbial Culture Collection Center.

[0035] Example 1

[0036] A preparation process of steam-exploded rice straw fermented to produce Trichoderma reesei protein feed, comprising the following steps:

[0037] (1) 2500 parts of rice straw with a water content of 30wt% are crushed to a particle size of 0.5 cm, and then steam-exploded at 190℃ and 2.5Mpa for 5min to obtain steam-exploded straw;

[0038] (2) The carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) is adjusted to 25:1 by urea, then potassium dihydrogen phosphate and magnesium sulfate are added, sterilized at 121℃ for 20min, and finally pH-responsive sophorolipid slow-release particles are added to obtain a fermentation medium, in which Trichoderma reesei is inoculated, and then subjected to aerobic fermentation at 30℃ and 5.0pH for 72h, and dried to obtain the Trichoderma reesei protein feed.

[0039] The adding amount of potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the adding amount of magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0040] The inoculation amount of Trichoderma reesei in step (2) is 10 7 Spores / g of fermentation medium.

[0041] The 5.0 pH in step (2) is adjusted by 2 mol / L sulfuric acid.

[0042] The adding amount of the pH-responsive sophorolipid slow-release particles in step (2) is 0.8 wt% of the mass of the steam-exploded straw.

[0043] The preparation method of the pH-responsive sophorolipid slow-release particles in step (2) comprises the following steps:

[0044] S1, 2 parts of sodium alginate are dissolved in 100 parts of water by mass fraction, and stirred until completely dissolved to obtain a sodium alginate aqueous solution;

[0045] S2, 0.1 parts of sophorolipid are added to the sodium alginate aqueous solution obtained in step S1 by mass fraction, and stirred uniformly to obtain a sophorolipid-sodium alginate aqueous solution;

[0046] S3, 50 parts of corn oil and 0.5 parts of Span 80 are mixed to obtain an emulsion; the sophorolipid-sodium alginate aqueous solution obtained in step S2 and the emulsion are mixed and homogenized at a speed of 12000 rpm for 10 min to obtain a sophorolipid-sodium alginate emulsion;

[0047] S4, the sophorolipid-sodium alginate emulsion obtained in step S3 is added dropwise into a 2 wt% calcium chloride aqueous solution at a rate of 1 ml / min, and is placed for 30 min, then is filtered, washed and dried to obtain the pH-responsive sophorolipid slow-release particles.

[0048] Example 2

[0049] A preparation process of steam-exploded rice straw fermented to produce Trichoderma reesei mycelial protein feed, comprising the following steps:

[0050] (1) 2500 parts of rice straw with a water content of 30 wt% are crushed to a particle size of 0.5 cm, and are steam-exploded at 190℃ and 2.5Mpa for 5 min to obtain steam-exploded straw;

[0051] (2) urea is used to adjust the carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) to 25:1, then potassium dihydrogen phosphate and magnesium sulfate are added, sterilized at 121℃ for 20 min, and finally pH-responsive sophorolipid slow-release particles are added to obtain a fermentation medium, in which Trichoderma reesei is inoculated, and aerobic fermentation is carried out at 30℃ and 5.0 pH for 72 h, and then dried to obtain the Trichoderma reesei cell protein feed.

[0052] The addition amount of the potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the addition amount of the magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0053] The inoculation amount of the Trichoderma reesei in step (2) is 10 7 spores / g of the fermentation medium.

[0054] The 5.0 pH in step (2) is adjusted by 2 mol / L sulfuric acid.

[0055] The addition amount of the pH-responsive sophorolipid slow-release particles in step (2) is 0.8 wt% of the mass of the steam-exploded straw.

[0056] The preparation method of the pH-responsive sophorolipid slow-release particles in step (2) comprises the following steps:

[0057] S1, 2 parts of sodium alginate are dissolved in 100 parts of water by mass fraction, and stirred until completely dissolved to obtain a sodium alginate aqueous solution;

[0058] S2, 0.1 parts of sophorolipid are added to the sodium alginate aqueous solution obtained in step S1 by mass fraction, and stirred uniformly to obtain a sophorolipid-sodium alginate aqueous solution;

[0059] S3, the sophorolipid-sodium alginate aqueous solution obtained in step S2 is added dropwise to a 2 wt% calcium chloride aqueous solution at a rate of 1 ml / min, and then placed for 30 min, filtered, washed, and dried to obtain the pH-responsive sophorolipid slow-release particles.

[0060] Example 3

[0061] A preparation process of a steam-exploded rice straw fermented Trichoderma reesei cell protein feed comprises the following steps:

[0062] (1) 2500 parts of rice straw with a water content of 30 wt% are crushed to a particle size of 0.5 cm, and then steam-exploded at 190℃ and 2.5 Mpa for 5 min to obtain steam-exploded straw;

[0063] (2) urea is used to adjust the carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) to 25:1, then potassium dihydrogen phosphate and magnesium sulfate are added, sterilized at 121℃ for 20 min, and finally fermentation aids are added to obtain a fermentation medium, in which Trichoderma reesei is inoculated, aerobic fermentation is carried out at 30℃ and 5.0 pH for 72 h, and drying is performed to obtain the Trichoderma reesei mycelial protein feed.

[0064] The addition amount of the potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the addition amount of the magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0065] The inoculation amount of the Trichoderma reesei in step (2) is 10 7 spores / g of the fermentation medium.

[0066] The 5.0 pH in step (2) is adjusted by 2 mol / L sulfuric acid.

[0067] The addition amount of the fermentation aids in step (2) is 0.8 wt% of the mass of the steam-exploded straw, and the fermentation aids are a mixture of sophorolipids and sodium alginate in a mass ratio of 2:0.1.

[0068] Comparative Example 1

[0069] A preparation process of a Trichoderma reesei mycelial protein feed fermented from steam-exploded rice straw, comprising the following steps:

[0070] (1) 2500 parts of rice straw with a water content of 30 wt% are crushed to a particle size of 0.5 cm, steam-exploded at 190℃ and 2.5 Mpa for 5 min to obtain steam-exploded straw;

[0071] (2) urea is used to adjust the carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) to 25:1, then potassium dihydrogen phosphate and magnesium sulfate are added, sterilized at 121℃ for 20 min, and finally fermentation aids are added to obtain a fermentation medium, in which Trichoderma reesei is inoculated, aerobic fermentation is carried out at 30℃ and 5.0 pH for 72 h, and drying is performed to obtain the Trichoderma reesei mycelial protein feed.

[0072] The addition amount of the potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the addition amount of the magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0073] The inoculation amount of the Trichoderma reesei in step (2) is 10 7 spores / g of the fermentation medium.

[0074] The 5.0 pH in step (2) is adjusted by 2 mol / L sulfuric acid.

[0075] The addition amount of the fermentation aids in step (2) is 0.8 wt% of the mass of the steam-exploded straw, and the fermentation aids are sophorolipids.

[0076] Comparative Example 2

[0077] A preparation process of Trichoderma reesei biomass protein feed fermented from steam-exploded rice straw, comprising the following steps:

[0078] (1) 2500 parts of rice straw with a water content of 30 wt% is crushed to a particle size of 0.5 cm, and then steam-exploded at 190℃ and 2.5Mpa for 5 min to obtain steam-exploded straw;

[0079] (2) The carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) is adjusted to 25:1 by urea, then potassium dihydrogen phosphate and magnesium sulfate are added, sterilized at 121℃ for 20 min, and finally a fermentation aid is added to obtain a fermentation medium, in which Trichoderma reesei is inoculated, and aerobic fermentation is carried out at 30℃ and 5.0pH for 72h, and then dried to obtain the Trichoderma reesei biomass protein feed.

[0080] The addition amount of potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the addition amount of magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0081] The inoculation amount of Trichoderma reesei in step (2) is 10 7 spores / g of fermentation medium.

[0082] The 5.0pH in step (2) is adjusted by 2 mol / L sulfuric acid.

[0083] The addition amount of the fermentation aid in step (2) is 0.8 wt% of the mass of the steam-exploded straw, and the fermentation aid is sodium alginate.

[0084] Comparative Example 3

[0085] A preparation process of Trichoderma reesei biomass protein feed fermented from steam-exploded rice straw, comprising the following steps:

[0086] (1) 2500 parts of rice straw with a water content of 30 wt% is crushed to a particle size of 0.5 cm, and then steam-exploded at 190℃ and 2.5Mpa for 5 min to obtain steam-exploded straw;

[0087] (2) The carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) is adjusted to 25:1 by urea, then potassium dihydrogen phosphate and magnesium sulfate are added, sterilized at 121℃ for 20 min, and finally a fermentation aid is added to obtain a fermentation medium, in which Trichoderma reesei is inoculated, and aerobic fermentation is carried out at 30℃ and 5.0pH for 72h, and then dried to obtain the Trichoderma reesei biomass protein feed.

[0088] The addition amount of potassium dihydrogen phosphate in step (2) is 0.1 wt% of the steam-exploded straw, and the addition amount of magnesium sulfate is 0.05 wt% of the steam-exploded straw.

[0089] The inoculation amount of Trichoderma reesei in step (2) is 10 7 spores / g of fermentation medium.

[0090] The 5.0 pH in step (2) is adjusted by 2 mol / L sulfuric acid.

[0091] Comparative Example 4

[0092] A preparation process for Aspergillus niger mycelial protein based on steam explosion of rice straw fermentation, comprising the following steps:

[0093] (1) 2500 parts of rice straw powder with a moisture content of 30wt% is crushed to a particle size of 0.5 cm, and then subjected to steam explosion at 190℃ and 2.5Mpa for 5min to obtain steam exploded straw;

[0094] (2) The carbon-nitrogen ratio of the steam exploded straw obtained in step (1) is adjusted to 25:1 by urea, then potassium dihydrogen phosphate and magnesium sulfate are added, sterilized at 121℃ for 20min, and finally pH-responsive sophorolipid slow-release particles are added to obtain a fermentation medium, in which Aspergillus niger is inoculated, subjected to aerobic fermentation at 30℃ and 5.0 pH for 72h, and then dried to obtain the Aspergillus niger mycelial protein.

[0095] The addition amount of potassium dihydrogen phosphate in step (2) is 0.1wt% of the steam exploded straw, and the addition amount of magnesium sulfate is 0.05wt% of the steam exploded straw.

[0096] The inoculation amount of Aspergillus niger in step (2) is 10 7 spores / g of fermentation medium.

[0097] The 5.0 pH in step (2) is adjusted by 2 mol / L sulfuric acid.

[0098] The addition amount of pH-responsive sophorolipid slow-release particles in step (2) is 0.8wt% of the mass of the steam exploded straw.

[0099] The preparation method of the pH-responsive sophorolipid slow-release particles in step (2) comprises the following steps:

[0100] S1, 2 parts of sodium alginate are dissolved in 100 parts of water by mass fraction, and stirred until completely dissolved to obtain a sodium alginate aqueous solution;

[0101] S2, 0.1 parts of sophorolipid are added to the sodium alginate aqueous solution obtained in step S1 by mass fraction, and stirred uniformly to obtain a sophorolipid-sodium alginate aqueous solution;

[0102] S3: 50 parts of corn oil and 0.5 parts of Span 80 were mixed to obtain an emulsion by mass fraction; the sophorolipid-sodium alginate aqueous solution obtained in step S2 and the emulsion were mixed and homogenized at a speed of 12000 rpm for 10 min to obtain a sophorolipid-sodium alginate emulsion;

[0103] S4: The sophorolipid-sodium alginate emulsion obtained in step S3 was added dropwise into a 2wt% calcium chloride aqueous solution at a rate of 1 ml / min, and was allowed to stand for 30 min, and then was filtered, washed and dried to obtain the pH-responsive sophorolipid slow-release particles.

[0104] Comparative Example 5

[0105] A preparation process of a Trichoderma reesei cell protein feed fermented from steam-exploded rice straw, comprising the following steps:

[0106] (1) 2500 parts of rice straw with a water content of 30wt% were crushed to a particle size of 0.5 cm, and were steam-exploded at 190℃ and 2.5Mpa for 5 min to obtain steam-exploded straw;

[0107] (2) The carbon-nitrogen ratio of the steam-exploded straw obtained in step (1) was adjusted to 25:1 by urea, then potassium dihydrogen phosphate and magnesium sulfate were added, and sterilized at 121℃ for 20 min, and finally the pH-responsive rhamnolipid slow-release particles were added to obtain a fermentation medium, and Trichoderma reesei was inoculated in the above fermentation medium, and was subjected to aerobic fermentation at 30℃ and 5.0pH for 72h, and then was dried to obtain the Trichoderma reesei cell protein feed.

[0108] In step (2), the addition amount of potassium dihydrogen phosphate was 0.1wt% of the steam-exploded straw, and the addition amount of magnesium sulfate was 0.05wt% of the steam-exploded straw.

[0109] In step (2), the inoculation amount of Trichoderma reesei was 10 7 spores / g of the fermentation medium.

[0110] In step (2), the 5.0pH was adjusted by 2mol / L sulfuric acid.

[0111] In step (2), the addition amount of the pH-responsive rhamnolipid slow-release particles was 0.8wt% of the mass of the steam-exploded straw.

[0112] The preparation method of the pH-responsive rhamnolipid slow-release particles in step (2) comprises the following steps:

[0113] S1: 2 parts of sodium alginate were dissolved in 100 parts of water by mass fraction, and were stirred until completely dissolved to obtain a sodium alginate aqueous solution;

[0114] S2, 0.1 parts of rhamnolipid was added into the sodium alginate aqueous solution obtained in step S1 by mass fraction, and stirred uniformly to obtain a rhamnolipid-sodium alginate aqueous solution;

[0115] S3, 50 parts of corn oil and 0.5 parts of Span 80 were mixed by mass fraction to obtain an emulsion; the rhamnolipid-sodium alginate aqueous solution obtained in step S2 and the emulsion were mixed and homogenized at a speed of 12000 rpm for 10 min to obtain a rhamnolipid-sodium alginate emulsion;

[0116] S4, the rhamnolipid-sodium alginate emulsion obtained in step S3 was added dropwise into a 2wt% calcium chloride aqueous solution at a rate of 1 ml / min, and stood for 30 min, then filtered, washed and dried to obtain the pH-responsive rhamnolipid slow-release particles.

[0117] Test Example 1

[0118] The Trichoderma reesei mycelial protein feed obtained in Examples 1-3 and Comparative Examples 1-5 was prepared using 1500g of water rice straw as raw material, and the mycelial protein content was determined, and the results are shown in Table 1.

[0119] After fermentation, 3 times the mass of water of the Trichoderma reesei mycelial protein feed was added, and stirred and extracted for 1h, then the solid residue was separated from the liquid containing mycelia by coarse filtration, and the mycelia in the liquid containing mycelia was collected by plate and frame filter, and the mycelia were washed with clean water, and dried to obtain the mycelial protein.

[0120] Table 1: Mycelial protein content

[0121]

[0122] Test Example 2

[0123] The test was carried out in accordance with three standards of GB / T 20806-2022 "Determination of neutral detergent fiber (NDF) in feed", NY / T 1459-2022 "Determination of acid detergent fiber in feed", and GB / T 20805-2006 "Determination of acid detergent lignin (ADL) in feed".

[0124] Sample preparation: The water rice straw samples before and after fermentation were dried in an oven at 105℃ to a constant weight, and then ground through a 1mm sieve. The dry weight of each sample was recorded. 0.5g of dried sample (denoted as W1) was accurately weighed and placed in a crucible of known weight (the weight of the crucible was recorded as Wc). At least 3 replicates were performed for each sample to improve accuracy.

[0125] Neutral detergent fiber (NDF) determination: Put the sample and crucible into a fiber analyzer or a reflux device, add 100 ml of neutral detergent solution, heat and reflux for 60 min; after cooling, filter the sample with a suction filtration device, and wash the crucible and residue with hot distilled water (90-100°C) for 3 times to remove soluble substances; drying and weighing: dry the crucible and residue in an oven at 105°C for 4 hours, and weigh after cooling (recorded as W NDF );

[0126] The neutral detergent consists of the following components in mass parts: 30 parts of sodium dodecyl sulfate, 18.61 parts of ethylenediaminetetraacetic acid disodium salt, 6.81 parts of sodium tetraborate, 4.65 parts of anhydrous sodium hydrogen phosphate, 10 parts of ethylene glycol ether, 1000 parts of water, and the final pH of the solution should be adjusted to 7.0 ± 0.1, and 1 mol / L NaOH aqueous solution or 1 mol / L HCl aqueous solution is used for fine adjustment;

[0127] Calculate NDF: NDF content (%) = (W NDF −W C ) / W1x100%;

[0128] Acid detergent fiber (ADF) determination: use the residue after NDF determination, add 100 ml of acid detergent solution. Heat and reflux for 60 min, cool and filter, and wash with hot distilled water until neutral. Dry at 105°C for 4h, and weigh after cooling (recorded as W ADF );

[0129] The acid detergent consists of the following components in mass parts: 20 parts of cetyltrimethylammonium bromide, 1000 parts of 0.5 mol / L sulfuric acid aqueous solution;

[0130] Calculate ADF: ADF content (%) = (W ADF −W C ) / W1x100%;

[0131] Acid detergent lignin (ADL) determination: cool the residue after ADF determination in the crucible, add 15 ml of 72wt% sulfuric acid aqueous solution, react at 20°C for 3h, filter, wash with hot distilled water until neutral, dry at 105°C for 4h, and weigh after cooling (recorded as W ADL ), put the crucible and residue into a muffle furnace, and ash at 550°C for 2h, and weigh after cooling (recorded as W ash );

[0132] Calculate ADL: ADL content (%) = (W ADL −W ash ) / W1x100%;

[0133] Calculate the contents of cellulose, hemicellulose and lignin:

[0134] Hemicellulose content (%) = NDF - ADF;

[0135] Cellulose content (%) = ADF - ADL;

[0136] Lignin content (%) = ADL;

[0137] Degradation efficiency (%) = (content before fermentation - content after fermentation) / content before fermentation x 100%;

[0138] Table 2: Results of main components degradation efficiency determination of straw

[0139]

[0140] As can be seen from Table 1 and Table 2, the pH-responsive sophorolipid slow-release particles prepared by the emulsification method in Example 1 have the greatest effect on the production of Trichoderma reesei mycelial protein from steam-exploded rice straw, and the emulsification step is omitted in the preparation method of pH-responsive sophorolipid slow-release particles in Example 2, that is, the sophorolipid-sodium alginate aqueous solution is directly added to the calcium chloride solution in Example 2 without being mixed with the corn oil, Span80 emulsion and homogenized first. The emulsification step in Example 1 is used to form a water-oil emulsion, and smaller and more uniform microcapsule particles are created by homogenization. After omitting, Example 2 may generate larger and irregular calcium alginate gel particles, which may have poor encapsulation efficiency and slow-release characteristics, and the pH-responsive release of the particles may not be accurate, and sophorolipid may be released too early in the early stage of fermentation, which cannot match the acid production process of Trichoderma reesei, thereby reducing the optimization effect on substrate wettability and inhibitor management.

[0141] Example 3 uses a physical mixture of fermentation aids sophorolipid and sodium alginate, which means there is no slow-release mechanism, and the aids are simply mixed and added directly. After sophorolipid and sodium alginate are mixed, sophorolipid may immediately dissolve or release without pH-triggered slow release. This may result in too high a concentration of sophorolipid in the early stage of fermentation, potentially inhibiting microbial growth, or a lack of auxiliary effect in the later stage. The comparison of Examples 1-3 shows that sophorolipid has the effect of assisting Trichoderma reesei in fermenting rice straw to increase the yield of mycelial protein, but direct addition of sophorolipid may inhibit the yield of mycelial protein in the early stage, but by coating with sodium alginate to form slow-release particles with pH response, as the fermentation process proceeds, Trichoderma reesei produces acid, reducing the pH of the fermentation medium, causing the sodium alginate coating to decompose, slowly releasing the sophorolipid contained therein, so that sophorolipid can gradually exert its effect, better promoting the reproduction of Trichoderma reesei and producing more mycelial protein.

[0142] Example 1 uses sophorolipids as fermentation aid without sodium alginate or any slow-release carrier. Sophorolipids are directly added, which will be completely released at the beginning of fermentation, possibly improving initial wetting, but cannot sustain the effect, after Trichoderma reesei produces acid, sophorolipids can have been degraded or depleted, or high initial concentration can inhibit Aspergillus niger growth, or cause foam problems. Example 2 uses sodium alginate as fermentation aid without sophorolipids. Sodium alginate is a thickening or gelling agent, which can improve the viscosity or moisture of the medium, but has no surfactant function. Therefore, it cannot enhance the substrate wetting or reduce inhibitors, and cannot replace the role of sophorolipids. Example 3 does not use any fermentation aid, and the fermentation completely relies on the substrate itself. The steam-exploded straw still has good accessibility, but lacks the assistance of surfactants, and the hydrophobicity of the substrate and inhibitors will limit the growth of Trichoderma reesei and protein yield. Example 4 replaces Trichoderma reesei with Aspergillus niger. As can be seen from the data of the test examples, the effect of sophorolipids on Aspergillus niger fermentation of rice straw to produce cell protein is significantly lower than that of Trichoderma reesei. The present application speculates that this is because the fermentation core of Trichoderma reesei is cellulase, sophorolipids can directly improve the efficiency of enzyme and substrate degradation, and the pH-responsive release matches the pH change pattern of Trichoderma reesei better, ensuring that sophorolipids are released at the key stage. Example 5 uses pH-responsive rhamnolipid slow-release particles instead of sophorolipid slow-release particles. Rhamnolipids are another biosurfactant with similar surface activity properties, but their effect is significantly inferior to sophorolipids, which indicates that sophorolipids have an effect on promoting Trichoderma reesei fermentation of rice straw to produce cell protein that goes beyond that of general surfactants.

[0143] In summary, from the comparison data of the examples and comparative examples, it can be fully judged that the technical solution of preparing pH-responsive sophorolipid slow-release particles by emulsification method proposed by the present application has produced significant and unexpected technical effects, has outstanding substantial features and significant progress, compared with conventional technical means in the art such as direct addition of surfactants or use of non-slow-release mixtures. This scheme solves the contradiction that direct addition of sophorolipids may inhibit at the beginning and fail at the end, and ultimately achieves a cell protein yield far exceeding any single component or simple combination. In addition, this effect has significant specificity for Trichoderma reesei, indicating that it has successfully matched the unique fermentation mode of this strain with cellulase as the core.

Claims

1. A process for the preparation of Trichoderma reesei cell protein feed from steam exploded rice straw fermentation, characterized by, The method comprises the following steps: (1) crushing 2400-2600 parts of rice straw powder with a water content of 25-35 wt% to a particle size of 0.1-0.5 cm, and steam-explosion at 180-200 ℃ and 2-3 MPa for 4-6 min to obtain steam-explosion straw; (2) adjusting the carbon-nitrogen ratio of the steam-explosion straw obtained in step (1) to 25: (1-2) by urea, then adding potassium dihydrogen phosphate and magnesium sulfate, sterilizing at 120-122 ℃ for 19-21 min, and finally adding pH-responsive sophorolipid slow-release particles to obtain a fermentation medium, inoculating Trichoderma reesei in the fermentation medium, and aerobically fermenting at 25-35 ℃ and 4.8-5.2 pH for 36-72 h, and drying after fermentation to obtain the Trichoderma reesei cell protein feed; The preservation number of the Trichoderma reesei in step (2) is CGMCC 3.3711; The preparation method of the pH-responsive sophorolipid slow-release particles in step (2) comprises the following steps: S1 dissolving sodium alginate in water and stirring until completely dissolved to obtain a sodium alginate aqueous solution; S2 adding sophorolipid to the sodium alginate aqueous solution obtained in step S1 and stirring uniformly to obtain a sophorolipid-sodium alginate aqueous solution; S3 mixing corn oil and Span 80 to obtain an emulsion; mixing the sophorolipid-sodium alginate aqueous solution obtained in step S2 and the emulsion and homogenizing to obtain a sophorolipid-sodium alginate emulsion; S4 adding the sophorolipid-sodium alginate emulsion obtained in step S3 dropwise into a calcium chloride aqueous solution with a concentration, standing, filtering, washing, and drying to obtain the pH-responsive sophorolipid slow-release particles.

2. The process for the preparation of Trichoderma reesei biomass protein feed from steam-exploded rice straw as claimed in claim 1 wherein, The addition amount of the potassium dihydrogen phosphate in step (2) is 0.08-0.12 wt% of the steam-explosion straw, and the addition amount of the magnesium sulfate is 0.03-0.07 wt% of the steam-explosion straw. ​ 3. The process for the preparation of Trichoderma reesei biomass protein feed from steam-exploded rice straw as claimed in claim 1 wherein, The inoculation amount of Trichoderma reesei in step (2) is 0.8 x 10 7 -1.2 x 10 7 spores / g of fermentation medium. ​ 4. The process for the preparation of Trichoderma reesei biomass protein feed from steam-exploded rice straw as claimed in claim 1 wherein, The 4.8-5.2 pH in step (2) is adjusted by 1-2 mol / L sulfuric acid. ​ 5. The process for the preparation of Trichoderma reesei biomass protein feed from steam-exploded rice straw as claimed in claim 1 wherein, The addition amount of the pH-responsive sophorolipid slow-release particles in step (2) is 0.5-1 wt% of the mass of the steam-explosion straw. ​ 6. The process for the preparation of Trichoderma reesei biomass protein feed from steam-exploded rice straw as claimed in claim 1 wherein, The mass ratio of sodium alginate to sophorolipid is (2-3):(0.1-0.2). ​ 7. The process for the preparation of Trichoderma reesei biomass protein feed from steam-exploded rice straw as claimed in claim 1 wherein, The mass ratio of corn oil to Span 80 is (40-60):(0.3-0.8). ​

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