Method for producing biomass protein by efficiently pretreating straw biomass through combination of steam explosion and deep eutectic solvent

Abstract

The invention relates to the technical field of agricultural waste regeneration, and discloses a method for producing biomass protein by efficiently pretreating straw biomass through combination of steam explosion and a deep eutectic solvent, and the method comprises the following steps: air-drying straw at room temperature, crushing, and pre-extracting to obtain a straw sample for later use; adjusting the water content of the straw sample to a target water content, fully absorbing water at room temperature, filling the straw sample into a steam explosion reaction device, and carrying out steam explosion reaction; carrying out deep eutectic solvent treatment; and carrying out enzymolysis saccharification and fermentation treatment. By adopting the method disclosed by the invention, the removal rate of lignin reaches up to 40.8%, and meanwhile, more than 43.2% of hemicellulose is reserved; meanwhile, the specific surface area and the pore volume are increased, the straw structure is looser and curler, enzyme adsorption is facilitated, and the total sugar conversion rate reaches 50.4%. The crude protein content in the fermentation product reaches 25% or above, and the true protein content reaches 24.5%. The obstacle of straw biomass conversion is overcome, and a foundation is laid for processing the straw biomass into protein feed in a value-added mode.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural waste regeneration, and in particular to a method for producing biomass protein by efficiently pretreating straw biomass using steam explosion combined with a deep eutectic solvent. Background Art

[0002] The gradual depletion of fossil fuels and the resulting environmental problems have prompted the vigorous development of lignocellulosic biomass to produce biofuels and value-added chemicals as an alternative resource. Lignocellulose is the most abundant renewable resource on Earth. Unlike the production of fuel ethanol from starchy grains such as corn and wheat, the production of downstream value-added products after saccharification of lignocellulose does not compete with food sources, thus offering great potential for utilization. However, the primary structure of the three biopolymers that make up lignocellulose—cellulose, hemicellulose, and lignin—(tightly connected by hydrogen bonds, ester bonds, and ether bonds) and the secondary structure within each polymer (connected by glycosidic bonds, ether bonds, and carbon-carbon bonds) severely hinder their conversion into downstream products.

[0003] As the world's major cotton producer, my country produces tens of thousands of tons (dry weight) of cotton straw (CS) every year. However, a large proportion of CS is discarded in the fields or burned, causing serious environmental pollution and biomass waste. In fact, lignocellulosic biomass such as CS is a widely available and inexpensive renewable resource that can be converted into "biomass protein" through pretreatment, enzymatic saccharification, and microbial fermentation. However, like other biomass resources, the compact physical and chemical structure of CS lignocellulose poses a challenge to its further processing and utilization. The integration of pretreatment processes shows great advantages in solving its challenges. In fact, pretreatment is considered to be the most core unit in the lignocellulosic biorefining scheme.

[0004] Over the past few decades, a variety of physical, chemical, physicochemical, and biological pretreatment methods have been developed. For example, steam explosion (SE) is a highly effective physicochemical pretreatment method that synergistically disrupts the polymeric structure of lignocellulose through the autocatalytic hydrolysis of acetyl groups in hemicellulose at high temperature and pressure, combined with the mechanical shear force of instantaneous pressure release. This method can increase the pore structure and bulk of the substrate, broaden the contact area between cellulose and / or hemicellulose and enzymes, and is environmentally friendly. Although SE effectively widens the spatial structure of lignocellulose and increases enzyme accessibility, its weak effect on lignin may lead to the persistence of lignin-carbohydrate complexes (which hinder enzyme access to cellulose). Furthermore, unproductive enzyme adsorption by lignin due to hydrogen bonding, hydrophobic, and electrostatic interactions can significantly reduce the efficiency of cellulose enzymes. Furthermore, steric hindrance from lignin derivatives produced during pretreatment can negatively impact enzymatic hydrolysis. Fortunately, these negative effects of lignin can be eliminated through the introduction of chemical reagents, highlighting the necessity of combining SE with chemical treatment.

[0005] Deep eutectic solvents (DES) are typically composed of a hydrogen bond acceptor (HBA) and at least one hydrogen bond donor (HBD). The HBA and HBD can self-associate through hydrogen bonds, forming a mixture with a melting point lower than that of each individual component. This modularity allows the chemical properties and functionality of the DES to be customized by varying the HBA and HBD combinations. Due to their high selectivity for lignin extraction, DES can selectively remove lignin while retaining fiber components, providing a substrate for their conversion to fermentable sugars. However, the degree of fiber retention varies among different DES systems. In particular, hemicellulose is easily degraded into low-molecular-weight compounds and readily reduced to monosaccharides (e.g., oligosaccharides, xylose) in acidic DESs. This makes xylan recovery from the solid fraction nearly impossible. Therefore, designing and customizing DES to maximize lignin removal and retain more hemicellulose is highly desirable. This can reduce the complexity and cost of xylose recovery processes, provide more saccharifiable substrates, and mitigate the negative effects of lignin on enzymatic saccharification.

[0006] Therefore, the present invention aims to design a highly efficient and novel combination of SE and DES physicochemical pretreatment, enabling efficient conversion of CS biomass to biomass protein through enzymatic hydrolysis and yeast fermentation. First, the optimal SE parameters for CS were determined by single-factor optimization. Next, a typical Type III DES system was formulated, using the quaternary ammonium salt choline chloride as the HBA and amides, carboxylic acids, and polyols as the HBDs. The fiber retention and delignification effects of combined DES and SE pretreatment were investigated. Furthermore, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), nitrogen adsorption / desorption, and two-dimensional nuclear magnetic resonance spectroscopy (2D HSQC) were used to analyze the mechanism by which the combined pretreatment enables efficient delignification and enzymatic saccharification of lignocellulose. Finally, the CS with the optimal pretreatment efficiency was efficiently converted to biomass protein through enzymatic hydrolysis and yeast fermentation. This approach paves the way for cutting-edge innovation in the value-added processing of straw biomass into protein feed. Summary of the Invention

[0007] The present invention aims to provide a method for producing biomass protein by efficiently pretreating straw biomass using steam explosion combined with a deep eutectic solvent. This method achieves a lignin removal rate of up to 40.8% while retaining over 43.2% of hemicellulose. The method also increases the specific surface area and pore volume, making the straw structure looser and more curly, which is beneficial for enzyme adsorption. The method also achieves a total sugar conversion rate of 50.4%, a crude protein content of over 25% in the fermentation product, and a true protein content of up to 24.5%. This method overcomes the barriers to straw biomass conversion and lays the foundation for research on the value-added processing of straw biomass into protein feed.

[0008] To achieve the above-mentioned purpose, a method for efficiently pretreating straw biomass by steam explosion combined with a deep eutectic solvent to produce biomass protein comprises the following steps:

[0009] Step 1, straw pretreatment: air-dry and crush the straw at room temperature, and then pre-extract it with a toluene / ethanol mixed solution to obtain a straw sample for later use;

[0010] Step 2: Adjust the straw sample to the target moisture content, fully absorb water at room temperature for 12 hours, and then place it in a steam explosion reaction device, adjust the target pressure and pressure holding time to perform a steam explosion reaction;

[0011] Step 3, treating the steam-exploded straw with a deep eutectic solvent;

[0012] Step 4, performing enzymatic saccharification on the straw treated with the deep eutectic solvent in step 3, and calculating the sugar yield;

[0013] Step 5: fermenting the straw treated with the deep eutectic solvent in step 3 to produce biomass protein and determine the protein content.

[0014] Furthermore, in step 1, the straw is air-dried at room temperature until the dry matter content is greater than 90%, and the particle size of the crushed straw is less than 1 cm; the volume ratio of toluene / ethanol is 2:1, and the pre-extraction time is 6 hours.

[0015] Furthermore, in step 2, the target moisture content is 20%-80%, the target pressure is 1.1 MPa-1.9 MPa, and the pressure holding time is 1 min-7 min.

[0016] Furthermore, in the deep eutectic solvent of step 3, choline chloride is used as a hydrogen bond acceptor, and one or more of urea, lactic acid or glycerol is used as a hydrogen bond donor; the solid-liquid ratio of the steam-exploded straw to the deep eutectic solvent is 1:10, the reaction temperature is 100°C, the reaction time is 6h, and the solid and liquid are separated by vacuum filtration.

[0017] Furthermore, when choline chloride is used as a hydrogen bond acceptor and lactic acid and glycerol are used as hydrogen bond donors, the molar ratio of choline chloride: lactic acid: glycerol is 1:2:2.

[0018] Furthermore, in step 4, during enzymatic hydrolysis, CS was mixed with 0.05 M citric acid buffer at pH 4.8, and cellulase was added, and then 10 mg / ml tetracycline hydrochloride solution was added at 0.5% of the volume of the enzymatic hydrolysis solution, and the total mixture was incubated at 50°C and 200 rpm for 72 h.

[0019] Furthermore, in step 5, the straw treated with the deep eutectic solvent is enzymatically hydrolyzed in a buffer system for 72 hours, and then yeast extract and trypsin are added to the hydrolyzate as supplementary nutrients, yeast culture is inoculated at 4% of the volume of the hydrolyzate, and then fermented at 30°C and 200 rpm for 72 hours to produce biomass protein.

[0020] Furthermore, the present invention also provides a product obtained by the above method, wherein the product comprises lignocellulose and yeast biomass protein.

[0021] Furthermore, the present invention also provides the use of the above product in preparing protein feed.

[0022] Furthermore, the present invention also provides the use of the above product in preparing protein food.

[0023] The advantages and positive effects of the method for efficiently pretreating straw biomass to produce biomass protein by combining steam explosion with deep eutectic solvent are as follows:

[0024] 1. The method of the present invention for efficiently pretreating straw biomass using steam explosion combined with deep eutectic solvent to produce biomass protein is an effective, economical and environmentally friendly pretreatment method for the fractionation and high-value utilization of the three components of lignocellulose, which can pave the way for cutting-edge innovations in value-added biomass processing.

[0025] 2. The present invention provides an efficient physical and chemical combined pretreatment of steam explosion (SE) combined with deep eutectic solvent (DES). The yield of cotton straw (CS) biomass is maximized on a mild, economical and environmentally friendly basis. The synergistic effect of SE and DES brings about ideal results. The final results show that SE combined with ternary DES pretreatment (ELG; choline chloride: lactic acid: glycerol) can achieve a lignin removal rate of up to 40.8% while retaining more than 43.2% of hemicellulose. More importantly, the specific surface area of ​​CS after ELG pretreatment reaches 27.7m 2 / g, the pore volume reaches 0.07cm 3 / g, representing increases of 19.8 and 16.1 times, respectively, compared to untreated CS. Scanning electron microscopy (SEM) results also revealed a looser and more coiled structure, making it more conducive to enzyme adsorption. This laid the foundation for the ELG-pretreated CS to achieve a maximum total sugar conversion rate of 50.4% after 72 hours. The crude protein content of the fermentation product exceeded 25%, with the true protein content reaching a maximum of 24.5%. This overcomes the barriers to straw biomass conversion and lays the foundation for research on the value-added processing of straw biomass into protein feed.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the conversion rate of glucan, xylan and total sugar (glucan + xylan) under different SE parameters in the embodiment of the present invention, where A is the different explosion pressures at 4 min and 50% moisture content, B is the different holding time at 1.5 MPa and 50% moisture content, and C is the different moisture content at 1.5 MPa and 4 min;

[0028] Figure 2 Figure 3 is the effect of SE combined with DES pretreatment on the composition and removal of CS lignocellulose in the examples of the present invention, where A is the composition of cellulose, hemicellulose, and lignin after CS pretreatment, and B is the solid recovery rate, cellulose and hemicellulose recovery rate, and lignin removal rate of CS after pretreatment. CON is the control group, EN is SE + urea type DES, EL is SE + lactic acid type DES, EG is SE + glycerol type DES, and ELG is SE + lactic acid and glycerol type DES.

[0029] Figure 3is the enzymatic saccharification rate of CS before and after combined pretreatment for 0-72 hours in the embodiment of the present invention (measured every 12 hours), where A is the glucan yield, B is the xylan yield, C is the total sugar (glucan + xylan) yield, CON is the control group, EN is SE + urea type DES, EL is SE + lactic acid type DES, EG is SE + glycerol type DES, and ELG is SE + lactic acid and glycerol type DES;

[0030] Figure 4 These are SEM images of untreated and pretreated CS in the examples of the present invention, where A is CON, the control group; B is EN, SE + urea-type DES; C is EL, SE + lactic acid-type DES; D is EG, SE + glycerol-type DES; E is ELG, SE + lactic acid and glycerol-type DES;

[0031] Figure 5 A is the FT-IR spectrum of the untreated and pretreated CS in the examples of the present invention, B is the XRD pattern, C is the BET surface area, D is the pore volume, E is the pore size, F is the N2 adsorption-desorption isotherm, G is the pore size distribution, CON is the control group, EN is SE + urea type DES, EL is SE + lactic acid type DES, EG is SE + glycerol type DES, and ELG is SE + lactic acid and glycerol type DES;

[0032] Figure 6 Figure 1 is the structural characterization of lignin extracted by different pretreatment methods in the examples of the present invention, where A is the infrared spectrum of lignin, B is the basic structure and connecting unit of lignin, C is the 2D HSQC spectrum of the side chain and aromatic region of lignin, EN is SE + urea type DES, EL is SE + lactic acid type DES, EG is SE + glycerol type DES, and ELG is SE + lactic acid and glycerol type DES;

[0033] Figure 7 The CS pretreated by fermentation in the embodiment of the present invention is used to produce biomass protein, wherein A is crude protein, B is true protein, C is non-protein nitrogen, CON is the control group, EN is SE+urea type DES, and ELG is SE+lactic acid and glycerol type DES. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0035] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0036] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. Experimental instruments, equipment, and reagents in the following examples where the sources are not specified are all commercially available raw materials.

[0037] Unless otherwise defined or indicated, all technical and scientific terms used in this invention have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention. It should be noted that, unless there is a conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0038] Example 1 Materials and Methods

[0039] 1.1 Raw materials and reagents:

[0040] The cotton straw used in this experiment was collected from the experimental farmland in Aksu Prefecture (Xinjiang, China). The collected cotton straw was air-dried at room temperature to a dry matter content greater than 90%, and then crushed (particle size <1 cm) using a hammer mill (SM-100, Zhengzhou, China) and pre-extracted with toluene / ethanol (2:1, v / v) at 110°C for 6 h. The obtained samples were stored in a dry container for further use. Unless otherwise specified, all chemical reagents and sugars used in this experiment were purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China) and were of analytical grade. Cellulase ( CTec3HS, 300 FPU / mL) was purchased from Novozymes Biotechnology Co., Ltd. (Tianjin, China). Kluyveromyces marxianus (CGMCC2.3959) was purchased from China General Microbiological Culture Collection Center.

[0041] 1.2 Steam explosion treatment:

[0042] The steam explosion parameters, including pressure (1.1 MPa, 1.5 MPa, 1.9 MPa), holding time (1 min, 4 min, 7 min), and moisture content (20%, 50%, 80%), were optimized using a single-factor optimization method. CS (50 g, DM) was adjusted to the target moisture content and fully absorbed for 12 h at room temperature before being loaded into a 0.4 L steam explosion reactor (QBS-80, Suzhou, China). Steam explosion reactions were performed according to the target pressure and holding time. The resulting samples were dried at 45°C and then enzymatically hydrolyzed. The optimal steam explosion parameters were selected based on the maximum sugar conversion rate of CS as the response value.

[0043] 1.3 Preparation and pretreatment of deep eutectic solvent (DES):

[0044] Binary DES (molar ratio of BHA:BHD=1:2) was prepared using choline chloride as hydrogen bond acceptor (HBA) and urea, lactic acid and glycerol as hydrogen bond donors (HBD). Ternary DES (molar ratio of choline chloride:lactic acid:glycerol=1:2:2) was prepared using lactic acid and glycerol as hydrogen bond donors.

[0045] The specific operation is as follows: weigh the corresponding mass of HBA and HBD into a glass beaker, and heat and stir continuously at 80°C until the solution becomes a uniform transparent liquid, and then use it immediately after cooling the liquid to room temperature.

[0046] Cotton straw treated with the optimal steam explosion parameters (1.5 MPa, 4 min, 50%) was pretreated with DES. The treatment groups were designated CON (no treatment), EN (SE + urea DES), EL (SE + lactic acid DES), EG (SE + glycerol DES), and ELG (SE + lactic acid and glycerol DES) based on the hydrogen bond donors of the DES. CS samples were mixed with DES solution at a solid-to-liquid ratio of 1:10 and heated and stirred at 100°C for 6 h. The solid and liquid were separated by vacuum filtration and washed multiple times with anhydrous ethanol to remove residual DES in the solid until the filtrate was transparent and neutral. The washed solid was then dried in a 55°C oven and recovered for further analysis. The anhydrous ethanol in the filtrate was then evaporated and recovered using a rotary evaporator at 60°C. The remaining dark brown liquid was diluted with deionized water and allowed to stand before centrifugation and washing with ethanol to remove impurities. The obtained pure precipitate was lignin, which was freeze-dried (48 h) and stored in dry conditions for further analysis.

[0047] 1.4CS lignocellulose composition and removal analysis:

[0048] The cellulose and hemicellulose compositions of the raw CS and pretreated CS were determined according to the method of Jose et al. (Jose D, Vasudevan S, Venkatachalam P, et al. Effective deep eutectic solvent pretreatment in one-pot lignocellulose biorefinery for ethanol production [J]. Industrial Crops and Products, 2024, 222: 1196-26.). The lignin content was determined by a two-step acid hydrolysis method according to the standard analytical procedure of the National Renewable Energy Laboratory (NREL). The solid recovery rate, cellulose recovery rate, hemicellulose recovery rate, and lignin removal rate were calculated according to formulas (1)-(4):

[0049]

[0050] 1.5 Enzymatic saccharification:

[0051] 1 g of CS was mixed with 50 mL of citric acid buffer (0.05 M, pH 4.8), and cellulase was added at 30 FPU / g of CS (DM). A 10 mg / mL tetracycline hydrochloride solution was then added at 0.5% of the volume of the hydrolyzate to prevent microbial contamination. Finally, the total mixture was incubated at 50°C and 200 rpm for 72 hours. Every 12 hours, 1 mL of the hydrolyzate was aspirated, heated in a boiling water bath for 5 minutes to inactivate the enzyme, and then centrifuged at 4000 rpm for 10 minutes to obtain the supernatant. Glucose and xylose in the hydrolyzate were determined using a high-performance liquid chromatograph (HPLC, 1260 Infinity II, Agilent, Santa Clara, CA, USA) equipped with an Aminex HPX-87H column (Biorad Laboratories Inc., Hercules, California, USA). The injection volume was 25 μL, and 5 mmol / L H2SO4 was used as the mobile phase, which was filtered through a 0.22 μm filter membrane and then degassed. The flow rate of the mobile phase was set at 0.6 μmL / min. The enzymatic saccharification rate was calculated according to formulas (5)-(7):

[0052]

[0053] 1.6 Structural characterization of CS after preprocessing:

[0054] 1.6.1 Scanning electron microscope (SEM) observation:

[0055] The micromorphological changes of CS before and after pretreatment were observed using a scanning electron microscope (SEM) (SU3500, Hitachi Co., Ltd., Matsuda, Japan). The sample was evenly spread on a sample stage covered with conductive adhesive, then gold-sprayed. Finally, the sample was removed and placed on the SEM stage for observation and analysis. The SEM had a resolution of 3.0 nm, a continuously variable acceleration voltage of 5–30 kV, and secondary electron imaging (magnification of 3–300,000).

[0056] 1.6.2 Fourier transform infrared spectroscopy FT-IR analysis:

[0057] The functional group composition of CS before and after pretreatment was analyzed using a Fourier transform infrared spectrometer (Spectrum 400, PerkinElmer, USA). CS samples were directly loaded onto ZnSe-diamond crystal substrates without further treatment. Constant pressure was applied to the sample under software monitoring to ensure good contact between the sample and the infrared beam and prevent loss of infrared radiation. Before recording each spectrum, the ZnSe-diamond crystal substrate was rinsed with deionized water, and a clean background scan was collected. The spectral range was 4000–400 cm -1 , with a resolution of 4cm -1 , scanned 32 times.

[0058] 1.6.3 X-ray diffraction (XRD) analysis:

[0059] The crystallinity of cellulose in CS was determined using an X-ray diffractometer (D8 Advance). Samples were dried at 45°C for 72 h before testing. The test conditions were: a Co target, a Ni filter to eliminate CuKα radiation, a tube voltage of 36 kV and a tube current of 20 mA, respectively, with a scanning range of 5°–40°, a scanning speed of 2° / min, and a scanning step size of 0.02°. The crystallinity index (CrI) was calculated according to the following formula (8).

[0060]

[0061] Where, I 002 is the diffraction intensity at 2θ = 22.5° in the crystalline region; I am It is the diffraction intensity of the amorphous region at 2θ=18°.

[0062] 1.6.4 N2 adsorption-desorption determination:

[0063] The specific surface area (SSA) of CS cellulose was measured using a fully automated surface area and pore size distribution analyzer (Nova touch LX1, Quantachrome Instruments, USA). Briefly, 0.2 g of sample powder was weighed and degassed at 100°C for 6 h to remove moisture and other adsorbates. The adsorption-desorption analysis was then performed using high-purity nitrogen. The SSA of the samples was calculated using the multipoint Brunauer-Emmett-Teller (BET) method.

[0064] 1.7 Structural characterization of extracted lignin:

[0065] The recovered lignin was analyzed using an FT-IR spectrometer (Spectrum 400, PerkinElmer, USA) to clarify the composition characteristics of its functional groups. The 2D HSQC nuclear magnetic resonance spectrum of the recovered lignin was analyzed using an AVANCE III HD 600 MHz spectrometer (Bruker, Germany). 50 mg of dry lignin was dissolved in 0.5 mL of DMSO solution. 1 H and 13 The spectrum widths of the C dimension are set to 5000 Hz and 20000 Hz, respectively. 1 The transient parameter of the H dimension is 64,1024 sampling points; 13 The transient parameters for the C dimension were 32,256 sampling points. The pulse delay (d1) was set to 5 s. The solvent center peak δC / δH = 39.5 / 2.49 ppm was used as the internal standard. The collected data were analyzed using Bruker Topspin 4.4.0.

[0066] 1.8 Pretreatment of CS fermentation to produce biomass protein (SCP):

[0067] Yeast inoculum was prepared using yeast extract peptone dextrose medium (YPD; 1% yeast extract, 2% peptone, 2% glucose). The culture was incubated at 30°C and 150 rpm for 24 hours before use as an inoculum. CS treated with EN and ELG with the highest total sugar yield was selected for fermentation to produce SCP. 5 g of pretreated CS was first enzymatically hydrolyzed in 100 mL of citric acid buffer (0.05 M, pH 4.8) for 72 hours. 0.5 g of yeast extract and 1 g of tryptone were then added to the 100 mL hydrolyzate as supplemental nutrients. The yeast culture was inoculated at 4% (v / v) of the hydrolyzate volume and fermented at 30°C and 200 rpm for 72 hours to produce SCP. After fermentation, the fermentation broth was freeze-dried, and the resulting solid product was analyzed for crude protein content using the Kjeldahl method. True protein was also determined, and non-protein nitrogen content was calculated by subtracting true protein from crude protein. The amino acid composition of the solid product was determined using an automatic amino acid analyzer (Hitachi L-8900, Japan).

[0068] 1.8 Data Analysis:

[0069] The experimental results are expressed as mean ± standard deviation (SD), and each treatment was performed in triplicate.

[0070] Example 2 Results and Discussion

[0071] 2.1 Steam Explosion (SE) Pretreatment Optimization:

[0072] SE is known as a thermomechanical chemical process and is a typical physical-chemical pretreatment method. Pressure, pressure maintenance time, and biomass characteristics (such as moisture content or particle size) are the most important parameters affecting the steam explosion reaction effect. The conversion rates of glucan, xylan, and total sugars in CS enzymatic hydrolysis after pretreatment were used as response values, and single factor optimization (OFAT) was used to screen the optimal conditions for each parameter. The results are shown in Figure 2. Figure 1 As shown in Figure 2 . Under the same pressure maintenance time (4 min) and moisture content (50%), when the SE pressure increased from 1.1 MPa to 1.9 MPa, the total sugar conversion rate increased from 39.3% to a maximum of 45.0%. The rapid decompression process of SE generates mechanical shear forces, which can break the hydrogen bonds and covalent bonds between cellulose, hemicellulose, and lignin, converting the raw biomass into loose, porous, and dispersed fibers. The higher the pressure, the stronger the tearing effect on the fibers during decompression, which is more conducive to enzyme adsorption and saccharification. At a pressure of 1.5 MPa, the xylose yield was 36.7%, and at 1.9 MPa, the xylose yield increased to 42.7%, but the total sugar yield remained essentially unchanged (from 44.9% to 45.0%). In addition, under high temperature and high pressure conditions, the pentoses released by hemicellulose hydrolysis are easily dehydrated and converted into inhibitors such as furfural and 5-hydroxymethylfurfural. Excessively high pressures require more saturated steam, which is not conducive to economic efficiency. Therefore, 1.5 MPa was selected as the optimal steam explosion pressure. At the same pressure (1.5 MPa) and moisture content (50%), the yields of glucose, xylose, and total sugars generally increased when the pressure maintenance time increased from 1 to 7 minutes. As the exposure of CS biomass to high-pressure saturated steam increases, the thermally labile acetyl groups in the hemicellulose are cleaved and release acetic acid. This released acid breaks down the glycosidic bonds, ether bonds, and other acid-labile bonds between the lignocellulose, weakening the fiber's rigid structure to a certain extent, increasing enzyme accessibility and saccharification efficiency. At 4 minutes, the glucan yield was 46.3%, decreasing to 45.3% at 7 minutes. This may be related to the loss of cellulose caused by prolonged exposure to high-pressure steam. Furthermore, considering that the total sugar yield increased only slightly with increasing steam explosion time, 4 minutes was selected as the optimal pressure maintenance time. Moisture content is a key factor in determining the effectiveness of SE, and can influence SE efficiency by regulating the permeability of steam to the biomass (which determines mass transfer efficiency). Therefore, the initial moisture content of CS was optimized, adjusting it to 20%, 50%, and 80%. After fully absorbing water at room temperature for 12 hours, the steam explosion reaction was performed. Results showed that at a moisture content of 50%, the conversion rates of glucan and total sugars reached the highest levels, reaching 44.1% and 42.8%, respectively. Therefore, 50% was selected as the optimal SE moisture content. Overall, a pressure of 1.5 MPa, a pressure holding time of 4 minutes, and a moisture content of 50% were the optimal conditions for SE pretreatment of CS biomass, fully breaking down the polymeric structure of lignocellulose and providing a solid foundation for downstream combined pretreatment and enzymatic saccharification.

[0073] 2.2 Lignocellulose composition and removal analysis:

[0074] A typical type III DES system was prepared, which consisted of a quaternary ammonium salt (choline chloride) as a hydrogen bond acceptor and various organic compounds (urea, lactic acid, glycerol) as hydrogen bond donors. The use of deep eutectic solvents composed of different hydrogen bond donors combined with steam explosion pretreatment is to retain more enzymatically saccharifiable substrates cellulose and hemicellulose, while maximizing the removal of lignin and reducing non-productive adsorption of enzymes. Untreated CS biomass consists of 38.35% cellulose, 16.69% hemicellulose and 22.26% lignin ( Figure 2 Compared with CON, the cellulose composition of CS after pretreatment changed due to the removal of lignin and hemicellulose. After different DES coupled SE pretreatments, the cellulose retention rate of CS exceeded 90% ( Figure 2 Figure B) shows that the EN pretreatment retention rate was 95%, indicating that this pretreatment system effectively protects cellulose and provides sufficient saccharifiable substrate for subsequent enzymatic hydrolysis. Hemicellulose and lignin removal varied significantly among different binary DES combined with SE pretreatment. EL exhibited the lowest hemicellulose recovery rate of 33.12% and the highest lignin removal rate of 36.55%. In contrast, EG exhibited the highest hemicellulose recovery rate of 51.27% and the lowest lignin removal rate of 27.86%. EN fell in between, with a hemicellulose recovery rate of 38.09% and a lignin removal rate of 34.87%. Hemicellulose is structurally unstable due to its highly branched structure and readily hydrolyzed acetyl groups. The SE step in the combined pretreatment process, through high temperature and high pressure, triggers the acidic autocatalytic hydrolysis of hemicellulose acetyl groups and the synergistic breakdown of glycosidic bonds through mechanical shear, ultimately leading to hemicellulose degradation and loss. The type of hydrogen bond donor compound plays a crucial role in determining the performance of type III quaternary ammonium salt-based DES. Specifically, hemicellulose removal rates for DES using polyols, carboxylic acids, and amides as hydrogen bond donors range from 16% to 90%, depending on the ratio of hydrogen bond acceptors and reaction temperature. However, numerous studies have shown that acidic DES achieve greater hemicellulose removal, while neutral or alkaline DES reduce hemicellulose loss. The results of this study are consistent with these findings, indicating that neutral or alkaline EN and EG yielded higher hemicellulose recovery rates, while acidic EL had the lowest recovery rate, at only 33.12%. The high delignification rate of EL (36.55%) may be related to the fact that lactic acid is the hydrogen bond donor in this DES. Studies have shown that the dissociation of H protons from the hydrogen bond donor accelerates the cleavage of ether and ester bonds between polysaccharides (cellulose and hemicellulose) and lignin, leading to lignin removal. Furthermore, in acidic DES, the β-O-4 bonds connecting lignin monomers are more easily cleaved, making lignin more soluble.

[0075] Because the binary DES systems EL and EG were observed to have opposite effects on hemicellulose retention and lignin removal, a ternary DES system was formulated with a molar ratio of 1:2:2 (choline chloride:lactic acid:glycerol). This was done in the hope of combining the advantages of both, namely, removing more lignin while retaining more hemicellulose. The results showed that ELG reduced hemicellulose loss, with a recovery rate of 43.24%, while the lignin removal rate reached the highest of all treatment groups, at 40.84%. The addition of polyols such as glycerol can effectively optimize the H-bond acidity of DES. Since the ether bonds of lignin are more easily broken than the glycosidic bonds of xylan, although the H-bond acidity of ELG is reduced, it can still selectively remove lignin while reducing excessive xylan degradation.

[0076] 2.3 Enzymatic saccharification after CS pretreatment:

[0077] Lignocellulose is a high molecular weight polymer formed by cross-linking of cellulose, hemicellulose and lignin. The enzymatic hydrolysis efficiency of structural polysaccharides depends largely on the robustness (high recovery of carbohydrates and removal of lignin) and characteristics of the pretreatment method. In fact, an ideal pretreatment can not only break the polymer structure of lignocellulose and improve the accessibility of enzymes, but also obtain high recovery of carbohydrates (especially hemicellulose) while removing a large amount of lignin. All of these approaches lead to the ultimate goal: to increase the yield of monosaccharides. After CS was pretreated with a combination of methods, the enzymatic digestibility of the residual solid fraction was measured from 0 to 72 hours. In the early stage of enzymatic hydrolysis (12 hours), the glucan yield of CS after different DES coupled SE pretreatments increased rapidly compared with the original CS, but they all maintained similar saccharification values, i.e., EN was 30.4%, EL was 32.8%, EG was 34.7%, and ELG was 32.8% ( Figure 3 A). As the most abundant component in CS lignocellulose, the rapid release of glucose by glucan in the first stage of hydrolysis means that the content of amorphous part in the pretreated solid fraction is very high, which promotes faster hydrolysis. In essence, the coupling of the thermomechanical chemical deconstruction of SE and the bond breaking effect of DES will cause changes in the structure of lignocellulose, including the formation of vacancies, dislocations and phase transitions, which is beneficial to the digestion of graded polysaccharides. With the extension of enzymatic hydrolysis time, the glucan yield of ELG gradually increased, reaching 42.8% at 72h, which was the highest level. The glucan yield of other treatment groups was limited to about 39%. At the same time, the xylan yield of ELG at 72h was also at a high level (85.1%), second only to EN's 92.0% ( Figure 3(B) The superior enzymatic efficiency of ELG can be attributed to the combined effects of the highest removal rate of lignin, large specific surface area and pore volume. These findings were further supported by the changes in the CS lignocellulose composition and subsequent characterization. First, the hydrolysis efficiency of carbohydrates is closely related to enzymatic adsorption. The biomass exposed to ELG and EN has a larger surface area and larger pore volume, which accelerates the enzyme adsorption process. Second, in essence, hydrolysis is initiated by the adsorption of cellulases and xylanases onto polysaccharides (cellulose and hemicellulose), where they undergo continuous adsorption / desorption cycles to form the next polysaccharide. Therefore, high surface area and good porosity enhance the accessibility of polysaccharides and ultimately improve enzymatic hydrolysis. In addition, the enhanced enzymatic hydrolysis of ELG can be partly attributed to the high removal rate of lignin, because the large presence of lignin will reduce enzymatic hydrolysis by forming steric hindrance and / or nonspecific adsorption of the enzyme, resulting in a reduction in the enzyme available for digestion. Total sugar yield is the most direct parameter for evaluating pretreatment efficiency. As Figure 3 As shown in Figure C, after 72 hours of enzymatic hydrolysis, the total sugar yield of untreated CS was only 21%. The original stubborn structure of lignocellulose significantly limited enzymatic digestion. Thanks to the high yields of glucans and xylans, the total sugar yields of ELG and EN reached 50.4% and 47.1% after 72 hours, respectively, representing 2.5-fold and 2.3-fold increases over the original CS.

[0078] 2.4CS microstructure changes:

[0079] A deep understanding of the structural transformation of CS before and after pretreatment is crucial for analyzing the dissociation process of the three components of lignocellulose and providing information for the functional application of different components in downstream. The changes in the morphological characteristics of CS before and after pretreatment were analyzed by SEM. Micrographs at different magnifications are shown in Figure 2. Figure 4 Untreated CS ( Figure 4 The surface of A) is smooth and flat, with dense fiber structure and orderly arrangement. This non-destructive and orderly nature is due to the inherent network structure of lignin-carbohydrate, which is stubborn and will seriously hinder the entry of pretreatment solvent and enzyme adsorption. Figure 4As shown in Figures B, C, D, and E, after pretreatment, the surface of CS was rough and wrinkled, and the fiber bundles were broken and separated, indicating significant deformation and degradation of the cell wall structure. These structural and morphological changes in CS can be attributed to the combined effects of SE and DES. Specifically, SE generates shear forces through instantaneous pressure relief under high temperature and high pressure, tearing the cell wall structure and converting the initial biomass into smaller, dispersed fibers. DES, on the other hand, accelerates the breakage of chemical bonds between polysaccharides and lignin, resulting in looser connections between fibers and a more fragmented structure, especially in the acidic EL and ELG (which have a more curled and folded structure). Therefore, after combined pretreatment, the complete disintegration of the overall CS structure enhances the accessibility of carbohydrate flux and provides a favorable morphology for subsequent enzymatic desorption.

[0080] The effect of pretreatment on the functional groups of CS lignocellulose was obtained by FT-IR characterization ( Figure 5 Middle A). 1026cm -1 The signal at 892 cm is related to the CO stretching vibration in cellulose, hemicellulose and lignin or the COC stretching vibration in cellulose and hemicellulose. The cleavage of the glycosidic bond or ether bond between polysaccharides and lignin caused by different DES pretreatments may lead to the shift of the corresponding peak position and the change of peak intensity. -1 The peak at 1731 cm is composed of β-glycosidic groups between monomeric sugars that constitute cellulose and hemicellulose. -1 Bands formed by the C=O stretching of the acetyl groups of hemicellulose were observed nearby. The peak shift here may be due to the removal of hemicellulose by DES, especially the acidic EL. 1616 and 1501 cm -1 The peak at 3400 cm corresponds to the aromatic skeleton vibration of lignin C=C extension. -1 The peak at 3400 cm-1 corresponds to the extension of lignin hydroxyl group -OH. Compared with the original CS, the treated CS has a peak at 3400 cm-1. -1 This may be because DES has a strong delignification ability, which leads to the shift and reduction of the lignin peak in the CS structure.

[0081] The change in crystallinity before and after CS pretreatment was evaluated by XRD analysis, and the obtained diffraction patterns were as follows: Figure 5As shown in Figure B. The diffraction peak at 2θ = 22.5° (002 plane) belongs to the typical structure of cellulose I. The high similarity of the peak types of different pretreated CS at this location indicates that the original structure of cellulose was largely retained during the pretreatment process. Compared with the original CS (29.46%), the decrease in CrI after EN (25.91%) pretreatment indicates that natural cellulose I may be transformed into amorphous cellulose and cellulose II. It is also worth noting that the CrI of other pretreatment groups increased from 29.46% of the original CS to 29.50–40.45%. This change is apparently not conducive to the accessibility of enzymes in the subsequent saccharification process, but it also needs to be comprehensively analyzed in conjunction with other structural changes. The increase in CrI of pretreated biomass may be due to the removal of a large amount of amorphous hemicellulose and lignin during the pretreatment process.

[0082] Surface accessibility and pore structure are considered to be important factors for pretreatment solvents and enzymatic adsorption during the conversion of lignocellulose to other value-added products. Figure 5 As shown in C and D, compared with the original CS, the SSA of CS after different DES coupled SE pretreatments increased by 12-28 times, and the pore volume increased by 11-33 times. Among them, the EN pretreatment had the largest increase, with SSA increasing from 1.40m 2 / g increased to 38.57m 2 / g, pore volume from 0.0045cm 3 / g increased to 0.15cm 3 / g. In general, the overall surface area of ​​biomass particles consists of the outer surface and the inner surface. The outer surface area depends on the particle size and shape, while the inner surface area is determined by the pore size distribution and the accessible pore volume. The shear force generated by the SE step in the combined pretreatment system can reduce the particle size of the biomass and increase the fluffiness by tearing the fibers, thereby inducing a significant increase in the outer surface area. The DES composed of different hydrogen bond donors expands the inner surface area by removing lignin, reducing crystallinity (especially EN) and adjusting the porosity during bond breakdown. In addition, the average pore size of CS ( Figure 5 In Figure E), the average pore size of the original CS and the CS after different pretreatments is more than 10 nm, so the mesopore volume may play an important role in the adsorption process. Figure 5 As shown in Figure F, the pretreated CS exhibited an exponential increase in N2 uptake with pressure, an inherent characteristic of a Type III (IUPAC nomenclature) isotherm, typical of many straw biomasses. At high relative pressures (P / P0 > 0.5), the N2 adsorption capacity increased dramatically, indicating the presence of a large number of mesopores in the sample. The pore size distribution ( Figure 5This observation is further supported by the observation that the pore size distribution of pretreated CS is primarily in the mesoporous range (2–50 nm), which is important for enzymatic hydrolysis because enzymes can theoretically pass through pores larger than 5 nm in diameter, playing a significant role in enhancing the accessibility of lignocellulose. In short, the combined pretreatments showed diverse potential to alter the pore structure of CS biomass and increase both the external and internal surface areas, leading to greater contact between lignocellulose and the solvent and ultimately making the cellulose highly amenable to enzymatic hydrolysis.

[0083] 2.5 Characterization of extracted lignin:

[0084] In-depth analysis of the structural changes of lignin is crucial for understanding the impact of the pretreatment technology platform on lignin, and can also provide valuable information for the downstream functional applications of lignin. Figure 6 As shown in A and B, lignin extracted by SE combined with different types of DES showed similar characteristic absorption bands, including 1515 cm -1 Aromatic skeletal vibration band at 1656 cm -1 The absorption peak originating from the stretching vibration of conjugated carbonyl groups and the characteristic band of hydroxyl stretching (3440 cm -1 ), CH bond stretching (2935 and 2840 cm -1 ). In addition, 1274cm -1 The signal at 1121 cm corresponds to the guaiacyl unit (G), -1 Corresponding to the syringyl unit (S). The peak is at 835cm -1 These peaks are attributed to the out-of-plane vibrations of the CH groups in the p-hydroxyphenyl units (H). These characteristic peaks indicate that CS lignin is a herbaceous lignin with typical SGH units. Similar absorption bands were identified in the four lignins, indicating that SE combined with different types of DES can specifically extract lignin without changing its chemical structure.

[0085] The spectra of the lignin side chain (δC / δH 50–90 / 2.5–6.0) and aromatic (δC / δH 90–150 / 6.0–8.0) regions obtained by 2D-HSQC NMR analysis are shown in Figure 2. Figure 6As shown in Figure C. Lignin cross-signal peaks were identified, and the specific signal assignments are shown in Table S1. Clear methoxyl (OMe) signals were identified in the side chain regions of all four lignins, further confirming the FT-IR spectroscopy results that these lignin samples are rich in S and G units. Signals for β-O-4 ether bonds (A), resin alcohols (β-β, B), and phenylcoumarins (β-5, C) were observed in the side chain regions of all four lignins. After acidic EL pretreatment, the Aβ(S) signal weakened, indicating severe cleavage of the β-O-4 ether bond. Calculation of the content of major lignin bonds also showed a significant decrease in the total number of β-O-4 ether bonds in EL lignin, reaching the lowest level among all treatments (17.08 / 100Ar). This indicates that acidic EL pretreatment effectively cleaves β-O-4 ether bonds to obtain more lignin fragments. When glycerol was introduced into EL to form ELG, the β-O-4 content in the lignin increased to 26.28 / 100Ar. This suggests that polyols act as effective stabilizers for lignin functionalization, protecting β-aryl ether bonds from cleavage and inhibiting subsequent condensation. Within the aromatic region, signals for G, S, and H units were clearly observed in all four lignin samples. The ferulate (FA2) and p-coumarate (PCAβ / FAβ, PCAα / FAα) signals for EL and ELG were attenuated to varying degrees, primarily due to the hydrolysis of ester bonds and acid-catalyzed condensation under acidic conditions to form new carbon-carbon bonds. The lignin extraction process typically involves two general reactions: fragmentation and condensation. The S / G ratio can be used to assess the degree of lignin condensation. Generally, G units are more susceptible to depolymerization than S units during DES extraction. However, the observed S / G ratios for the four lignins were not significantly different, indicating that different DES combined with SE pretreatments all induce similar degrees of fragmentation and condensation in lignin, potentially modulating lignin properties for downstream applications.

[0086] 2.6 Pre-treated straw fermentation to produce biomass protein:

[0087] As an important biomass resource, CS can produce rich fermentable sugars through pretreatment and enzymatic hydrolysis, making it an ideal carbon source for yeast to produce biomass protein. K.marxianus (CGMCC 2.3959) is generally considered to be a safe microorganism that can be used in the food and feed industry. In this study, CS after efficient physical and chemical pretreatment was used as a substrate, and after enzymatic hydrolysis, it was fermented with K.marxianus to produce biomass protein. The fermentation products mainly include unused lignocellulose and yeast biomass protein. The entire component does not need to be further separated to be used as a high-quality protein feed. Because feeds with crude protein ≥20% (DM) are classified as protein feeds. In this study, the crude protein content of the fermented EN and ELG groups reached 25.3% and 28.7%, respectively ( Figure 7Crude protein consists of non-protein nitrogen and true protein, of which the true protein is the most important for animal growth and health. After fermentation, the true protein content of CON, EN and ELG are 15.6%, 20.0% and 24.5% respectively. Figure 7 (B) The 4.5% higher true protein content of ELG compared to EN can be attributed to the fact that after optimal pretreatment, enzymatic hydrolysis produces a higher concentration of fermentable sugars, which the yeast uses to produce more bacterial protein. Because carbon is the most abundant element in cells, the carbon source is crucial for the formation and regulation of cellular biomass. In addition, the non-protein nitrogen content of EN and ELG is 5.4% and 4.3%, respectively. Figure 7 The content of essential amino acids in the fermentation products was also evaluated, and the results are shown in Table 1.

[0088] Table 1 Amino acid composition of pretreated straw after fermentation (DM%)

[0089] Amino acid composition CON EN ELG Aspartic acid 1.93±0.08 1.95±0.05 1.97±0.02 Threonine 0.95±0.05 0.98±0.03 0.96±0.06 Serine 1.04±0.12 1.05±0.03 1.08±0.08 glutamate 3.24±0.13 3.82±0.18 4.32±0.05 Proline 1.37±0.01 1.34±0.07 1.51±0.11 Glycine 0.76±0.03 0.90±0.02 0.84±0.04 Alanine 0.81±0.06 1.03±0.02 0.88±0.05 Cystine 0.21±0 0.23±0.01 0.24±0.02 Valine 1.29±0.03 1.12±0 1.09±0.07 Methionine 0.25±0.02 0.23±0 0.24±0.02 Isoleucine 0.86±0.05 0.87±0.01 0.90±0.06 Leucine 1.14±0.01 1.22±0.01 1.20±0.07 Tyrosine 0.47±0.03 0.48±0.02 0.47±0.01 Phenylalanine 0.67±0.03 0.74±0.02 0.70±0.03 Histidine 0.51±0.04 0.54±0 0.52±0.03 Lysine 1.30±0.02 1.41±0.03 1.59±0.13 Arginine 0.52±0.03 0.57±0.04 0.56±0.03 Tryptophan 0.24±0.06 0.20±0 0.19±0.02

[0090] The amino acid profile showed that the EN and ELG after fermentation were balanced in terms of essential amino acid content, containing all the essential amino acids required for animal feed published by the Food and Agriculture Organization of the United Nations (FAO). This indicates that the biomass protein produced by this process is a suitable protein feed resource for the feed industry.

[0091] Therefore, the combination of steam explosion (SE) and ternary deep eutectic solvent (ELG; choline chloride: lactic acid: glycerol) synergistically enhanced the pretreatment of cotton straw (CS), achieving preferential removal of lignin (40.8%) while retaining 43.2% of hemicellulose. This combined approach significantly increased the specific surface area (27.7 m 2 / g, 19.8 times) and pore volume (0.07 cm 3 / g, 16.1 times), and SEM revealed a looser, curled structure that facilitates enzyme adsorption. Consequently, CS pretreated with ELG achieved a total sugar conversion rate of 50.4% within 72 hours, with crude protein content exceeding 25% and true protein content reaching a maximum of 24.5% in the fermentation product. This demonstrates an effective and environmentally friendly strategy for overcoming barriers to lignocellulose conversion through selective lignin separation and structural disruption, advancing sustainable biorefining routes for biomass-to-protein conversion.

[0092] Therefore, the present invention uses the above-mentioned method of steam explosion combined with deep eutectic solvent to efficiently pretreat straw biomass to produce biomass protein. This method achieves a lignin removal rate of up to 40.8% while retaining more than 43.2% of hemicellulose. It also increases the specific surface area and pore volume, making the straw structure looser and more curled, which is conducive to enzyme adsorption. The total sugar conversion rate reaches 50.4%, the crude protein content in the fermentation product reaches more than 25%, and the true protein content reaches a maximum of 24.5%. This route can overcome the obstacles to straw biomass conversion and pave the way for cutting-edge innovations in the value-added processing of straw biomass into protein feed.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for producing biomass protein by efficiently pretreating straw biomass using steam explosion combined with deep eutectic solvent, characterized in that: The following steps are involved: Step 1, straw pretreatment: air-dry and crush the straw at room temperature, and then pre-extract it with a toluene / ethanol mixed solution to obtain a straw sample for later use; Step 2: Adjust the straw sample to the target moisture content, fully absorb water at room temperature for 12 hours, and then place it in a steam explosion reaction device, adjust the target pressure and pressure holding time to perform a steam explosion reaction; Step 3, treating the steam-exploded straw with a deep eutectic solvent; Step 4, performing enzymatic saccharification on the straw treated with the deep eutectic solvent in step 3, and calculating the sugar yield; Step 5: fermenting the straw treated with the deep eutectic solvent in step 3 to produce biomass protein and determine the protein content.

2. The method for efficiently pretreating straw biomass to produce biomass protein using steam explosion combined with deep eutectic solvent according to claim 1, characterized in that: In step 1, the straw is air-dried at room temperature until the dry matter content is greater than 90%, and the particle size of the crushed straw is less than 1 cm; the volume ratio of toluene / ethanol is 2:1, and the pre-extraction time is 6 hours.

3. The method for producing biomass protein by efficiently pretreating straw biomass using steam explosion combined with deep eutectic solvent according to claim 1, characterized in that: In step 2, the target moisture content is 20%-80%, the target pressure is 1.1 MPa-1.9 MPa, and the pressure holding time is 1 min-7 min.

4. The method for producing biomass protein by efficiently pretreating straw biomass using steam explosion combined with deep eutectic solvent according to claim 1, characterized in that: In step 3, in the deep eutectic solvent, choline chloride is used as a hydrogen bond acceptor, and one or more of urea, lactic acid or glycerol is used as a hydrogen bond donor; the solid-liquid ratio of the steam-exploded straw to the deep eutectic solvent is 1:10, the reaction temperature is 100° C., the reaction time is 6 h, and the solid and liquid are separated by vacuum filtration.

5. The method for producing biomass protein by efficiently pretreating straw biomass using steam explosion combined with deep eutectic solvent according to claim 4, characterized in that: When choline chloride is used as a hydrogen bond acceptor and lactic acid and glycerol are used as hydrogen bond donors, the molar ratio of choline chloride: lactic acid: glycerol is 1:2:

2.

6. The method for producing biomass protein by efficiently pretreating straw biomass using steam explosion combined with deep eutectic solvent according to claim 1, characterized in that: In step 4, during enzymatic hydrolysis, CS was mixed with 0.05 M citric acid buffer at pH 4.8, and cellulase was added. Then, 10 mg / ml tetracycline hydrochloride solution was added at 0.5% of the volume of the enzymatic hydrolysis solution, and the total mixture was incubated at 50°C and 200 rpm for 72 h.

7. The method for producing biomass protein by efficiently pretreating straw biomass using steam explosion combined with deep eutectic solvent according to claim 1, characterized in that: In step 5, the straw treated with the deep eutectic solvent is enzymatically hydrolyzed in a buffer system for 72 hours, and then yeast extract and trypsin are added to the hydrolyzate as supplementary nutrients. Yeast culture is inoculated at 4% of the volume of the hydrolyzate, and then fermented at 30°C and 200 rpm for 72 hours to produce biomass protein.

8. The product obtained by the method according to any one of claims 1 to 7, characterized in that: The products include lignocellulose and yeast single cell protein.

9. Use of the product as claimed in claim 8 in preparing protein feed.

10. Use of the product according to claim 8 in preparing protein foods.

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