Method for producing biomass protein from straw biomass by steam explosion combined with deep eutectic solvent efficient pretreatment

Abstract

The present application relates to the technical field of agricultural waste regeneration, and discloses a method for producing biomass protein by steam explosion combined with deep eutectic solvent efficient pretreatment of straw biomass, which comprises the following steps: drying, crushing and pre-extracting straw at room temperature to obtain a straw sample for standby; adjusting the straw sample to a target moisture content, fully absorbing water at room temperature, loading into a steam explosion reaction device, and performing steam explosion reaction; performing deep eutectic solvent treatment; performing enzymatic saccharification and fermentation treatment. By using the above method, the lignin removal rate is as high as 40.8%, while more than 43.2% of hemicellulose is retained; meanwhile, the specific surface area and pore volume are improved, so that the structure of the straw is more loose and curled, which is beneficial to the adsorption of enzymes, and the total sugar conversion rate reaches 50.4%. The content of crude protein in the fermentation product is more than 25%, and the content of true protein is 24.5%. The method overcomes the obstacles of straw biomass conversion, and lays a foundation for the value-added processing of straw biomass into protein feed.

Description

Technical Field

[0001] This invention relates to the field of agricultural waste recycling technology, and in particular to a method for producing biomass protein from straw biomass through efficient pretreatment using a combination of steam explosion and deep eutectic solvent. Background Technology

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

[0003] As a major cotton producer in the world, my country generates tens of thousands of tons (dry weight) of cotton stalks (CS) annually. However, a large proportion of CS is carelessly discarded in the fields or burned, causing serious environmental pollution and biomass waste. In fact, CS and other lignocellulosic biomass, as a widely available and inexpensive renewable resource, can be converted into "biomass protein" through pretreatment, enzymatic hydrolysis, saccharification, and microbial fermentation. However, like other biomass resources, the compact physical and chemical structure of CS lignocellulosic cellulose poses a challenge to its further processing and utilization. The integration of pretreatment processes has shown significant advantages in addressing this challenge. In fact, pretreatment is considered the core unit in lignocellulosic biorefining schemes.

[0004] Over the past few decades, various physical, chemical, physicochemical, and biological pretreatment methods have been developed. For example, steam explosion (SE) is a highly efficient physicochemical pretreatment method that synergistically disrupts the polymer structure of lignocellulose through the autocatalytic hydrolysis of hemicellulose's acetyl groups under high temperature and pressure, combined with the mechanical shear force of instantaneous pressure release. It can increase the porosity and bulk of the substrate, expand the contact area between cellulose and / or hemicellulose and enzymes, and is also environmentally friendly. Although SE can effectively broaden the spatial structure of lignocellulose and increase enzyme accessibility, its weak effect on lignin may allow lignin-carbohydrate complexes (which hinder enzyme entry into cellulose) to persist. Furthermore, the non-productive adsorption of lignin by enzymes caused by hydrogen bonding, hydrophobicity, and electrostatic interactions can severely reduce the efficiency of ligninase. In addition, the steric hindrance of lignin derivatives generated during pretreatment can also negatively impact enzymatic hydrolysis. Fortunately, these negative effects of lignin can be eliminated by introducing chemical reagents, highlighting the necessity of combining SE with chemical treatment.

[0005] Deep eutectic solvents (DES) typically consist of a hydrogen bond acceptor (HBA) and at least one hydrogen bond donor (HBD). The HBA and HBD can self-bond via 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 through different combinations of HBA and HBD. Thanks to its high selectivity for lignin extraction, DES can selectively remove lignin while retaining fibrous components, providing a substrate basis for their conversion into fermentable sugars. However, different DES systems exhibit varying degrees of fiber retention, particularly since hemicellulose is readily degraded into low-molecular-weight compounds and easily reduced to monosaccharides (e.g., oligosaccharides, xylose) in acidic DES. This makes xylan recovery via solid fractions nearly impossible. Therefore, designing and customizing DES to maximize lignin removal and retain more hemicellulose is essential, as this helps reduce the complexity and cost of xylose recovery processes, provides more saccharifiable substrates, and eliminates the negative impact of lignin on enzymatic saccharification.

[0006] Therefore, this invention aims to design a highly efficient and novel combination of SE and DES physicochemical pretreatments, simultaneously achieving efficient conversion of CS biomass into biomass protein through enzymatic hydrolysis and yeast fermentation. First, the optimal SE parameters for CS were obtained through single-factor optimization. Then, a typical Type III DES system was formulated, using quaternary ammonium choline chloride as HBA and amide, carboxylic acid, and polyol as HBD, respectively. The fiber retention and delignification effects of different types of DES combined with SE pretreatment were investigated. Furthermore, we attempted to analyze the mechanisms by which the combined pretreatment efficiently delignifies and leads to efficient enzymatic saccharification of lignocellulose using 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). Finally, the CS with the best pretreatment effect was efficiently converted into biomass protein through enzymatic hydrolysis and yeast fermentation. This paves the way for cutting-edge innovation in the value-added processing of straw biomass into protein feed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the efficient pretreatment of straw biomass to produce biomass protein using a combination of steam explosion and deep eutectic solvent. This method achieves a lignin removal rate of up to 40.8% while retaining over 43.2% of hemicellulose. Simultaneously, it increases the specific surface area and pore volume, making the straw structure more loose and curled, which is beneficial for enzyme adsorption. Furthermore, the total sugar conversion rate reaches 50.4%, and the crude protein content in the fermentation product reaches over 25%, with the highest true protein content reaching 24.5%. This overcomes the obstacles in 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 objectives, a method for producing biomass protein from straw biomass through efficient pretreatment using a combination of steam explosion and deep eutectic solvent includes the following steps:

[0009] Step 1, straw pretreatment: The straw is air-dried and crushed at room temperature, and then pre-extracted 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, allow it to fully absorb water at room temperature for 12 hours, then load it into the steam explosion reaction device, adjust the target pressure and holding time to carry out the steam explosion reaction;

[0011] Step 3: Treat the steam-exploded straw with a deep eutectic solvent.

[0012] Step 4: Perform enzymatic hydrolysis and saccharification on the straw treated with the deep eutectic solvent in Step 3, and calculate the sugar yield;

[0013] Step 5: Ferment 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 to ethanol is 2:1, and the pre-extraction time is 6 h.

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

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

[0017] Furthermore, when choline chloride is used as the 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] Further, in step 4, during enzymatic hydrolysis, CS is mixed with 0.05M citrate buffer at pH 4.8, and cellulase is added. Then, 10 mg / ml tetracycline hydrochloride solution is added at 0.5% of the hydrolysate volume, and the total mixture is incubated at 50°C and 200 rpm for 72 hours.

[0019] Further, in step 5, the straw treated with deep eutectic solvent is enzymatically hydrolyzed in a buffer system for 72 hours. Then, yeast extract and tryptone are added to the hydrolysate as supplementary nutrients. Yeast culture is inoculated at 4% of the volume of the hydrolysate, 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, the product comprising lignocellulose and yeast biomass protein.

[0021] Furthermore, the present invention also provides the application of the above-mentioned product in the preparation of protein feed.

[0022] Furthermore, the present invention also provides the application of the above-mentioned product in the preparation of protein foods.

[0023] The advantages and positive effects of the method for producing biomass protein from straw biomass through efficient pretreatment using a combination of steam explosion and deep eutectic solvent as described in this invention are:

[0024] 1. The method for producing biomass protein from straw biomass through efficient pretreatment using steam explosion combined with deep eutectic solvent as described in this invention is an effective, economical, and environmentally friendly pretreatment method used for the graded separation and high-value utilization of the three components of lignocellulose, paving the way for cutting-edge innovations in biomass processing value-added.

[0025] 2. This invention provides a highly efficient physicochemical combined pretreatment using steam explosion (SE) and deep eutectic solvent (DES). It maximizes the yield of cotton stalk (CS) biomass while being mild, economical, and environmentally friendly. The synergistic effect of SE and DES yields ideal results, with the final results showing that SE combined with ternary DES pretreatment (ELG; choline chloride: lactic acid: glycerol) achieves a lignin removal rate of up to 40.8% while retaining over 43.2% of hemicellulose. More importantly, the specific surface area of ​​CS after ELG pretreatment reaches 27.7 m². 2 / g, pore volume reaches 0.07cm³ 3 The total sugar content of the pretreated CS was increased by 19.8 times and 16.1 times compared to the untreated CS, respectively. Scanning electron microscopy (SEM) results also showed that the CS structure was more loose and coiled, which was more conducive to enzyme adsorption. These factors laid the foundation for the ELG-pretreated CS to achieve a total sugar conversion rate of up to 50.4% at 72 h. The crude protein content in the fermentation product reached over 25%, and the true protein content reached a maximum of 24.5%. This overcame the obstacles in straw biomass conversion and laid the foundation for research on the value-added processing of straw biomass into protein feed.

[0026] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0027] Figure 1 The conversion rates of dextran, xylan, and total sugar (dextran + xylan) under different SE parameters in the embodiments of the present invention are shown. A represents different explosion pressures at 4 min and 50% moisture content, B represents different holding times at 1.5 MPa and 50% moisture content, and C represents different moisture contents at 1.5 MPa and 4 min.

[0028] Figure 2 The effect of SE combined with DES pretreatment on the composition and removal of CS lignocellulose in this embodiment of the invention is shown in A, where A is the composition of cellulose, hemicellulose and lignin after CS pretreatment, 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 3The enzymatic saccharification rate of CS in this embodiment of the invention is measured every 12 hours before and after the combined pretreatment for 0-72 hours. A is the dextran yield, B is the xylan yield, C is the total sugar (dextran + 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 The images shown are SEM images of unprocessed and preprocessed CS in this embodiment of the 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; and E is ELG, SE + lactic acid and glycerol type DES.

[0031] Figure 5 In this embodiment, A represents the FT-IR spectra of untreated and pretreated CS, B represents the XRD pattern, C represents the BET surface area, D represents the pore volume, E represents the pore size, F represents the N2 adsorption-desorption isotherm, G represents the pore size distribution, CON represents the control group, EN represents SE+urea type DES, EL represents SE+lactic acid type DES, EG represents SE+glycerol type DES, and ELG represents SE+lactic acid and glycerol type DES.

[0032] Figure 6 The structural characterization of lignin extracted by different pretreatment methods in the embodiments of the present invention is shown in the figure. A is the infrared spectrum of lignin, B is the basic structure and connecting unit of lignin, C is the 2D HSQC spectrum of lignin side chain and aromatic region, 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 In this embodiment of the invention, CS is used for fermentation pretreatment to produce biomass protein, where 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 Implementation

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

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

[0036] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0037] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0038] Example 1: Materials and Methods

[0039] 1.1 Raw materials and reagents:

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

[0041] 1.2 Gas Explosion Handling:

[0042] The steam explosion parameters were optimized using a single-factor optimization method, 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%). CS (50 g, DM) was adjusted to the target moisture content and allowed to fully absorb water for 12 h at room temperature before being loaded into a 0.4 L steam explosion reactor (QBS-80, Suzhou, China). The steam explosion reaction was carried out 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 BHA:BHD = 1:2) were prepared by using choline chloride as hydrogen bond acceptor (HBA) and urea, lactic acid, and glycerol as hydrogen bond donors (HBD). Meanwhile, ternary DES (molar ratio choline chloride:lactic acid:glycerol = 1:2:2) were prepared by using both lactic acid and glycerol as hydrogen bond donors.

[0045] The specific procedure is as follows: Weigh the corresponding amounts of HBA and HBD into a glass beaker, and heat and stir continuously at 80°C until the solution becomes a uniform transparent liquid. Then, cool the liquid to room temperature and use it immediately.

[0046] Cotton stalks treated with the optimal steam explosion parameters (1.5 MPa, 4 min, 50%) were subjected to DES pretreatment. Based on the different hydrogen bond donors in the DES, the treatment groups were named CON (no treatment), EN (SE + urea-type DES), EL (SE + lactic acid-type DES), EG (SE + glycerol-type DES), and ELG (SE + lactic acid and glycerol-type DES). The CS sample was mixed with the DES solution at a solid-liquid ratio of 1:10 and heated and stirred at 100 °C for 6 h. The solid and liquid were then separated by vacuum filtration, and the solid was washed repeatedly with anhydrous ethanol to remove residual DES until the filtrate was clear and neutral. The washed solid was then dried in an oven at 55 °C 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, allowed to stand, centrifuged, and the precipitate was washed with ethanol to remove impurities. The resulting pure precipitate is lignin, which is freeze-dried (48 h) and stored under dry conditions for further analysis.

[0047] 1.4CS Lignocellulose Composition and Removal Analysis:

[0048] The cellulose and hemicellulose components of 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, 222:119626.). The lignin content was determined using a two-step acid hydrolysis method according to the standard analytical procedures of the National Renewable Energy Laboratory (NREL). 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 hydrolysis and saccharification:

[0051] 1 g of CS was mixed with 50 mL of citrate buffer (0.05 M, pH 4.8), and cellulase was added at a concentration of 30 FPU / g CS (DM). Then, 0.5% of the hydrolysate volume was added with 10 mg / mL tetracycline hydrochloride solution 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 hydrolysate was aspirated, heated in a boiling water bath for 5 min to remove enzyme activity, and then centrifuged at 4000 rpm for 10 min to obtain the supernatant. The determination of glucose and xylose in the hydrolysate was performed 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. The mixture was degassed after filtration through a 0.22 μm filter membrane. The flow rate of the mobile phase was set to 0.6 μmL / min. The enzymatic saccharification rate was calculated according to formulas (5)-(7):

[0052]

[0053] 1.6 Structural characterization of CS after pretreatment:

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

[0055] The microscopic morphological changes of CS before and after pretreatment were observed using a SEM (SU3500, Hitachi Co., Ltd., Matsuda, Japan). First, the sample to be tested was evenly spread on a sample stage coated with conductive adhesive. Then, gold sputtering was performed on the surface. Finally, the sample was removed and placed on the SEM stage for observation and analysis. The SEM resolution was 3.0 nm, the accelerating voltage was 5–30 kV (continuously variable), and the imaging mode was secondary electron imaging (magnification 3–300,000).

[0056] 1.6.2 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis:

[0057] The functional group composition of CS samples before and after pretreatment was analyzed using a Fourier transform infrared spectroscopy (Spectrum 400, PerkinElmer, USA). CS samples were directly packed onto a ZnSe-diamond crystal substrate without further processing. A constant pressure was applied to the sample under software monitoring to ensure good contact between the sample and the infrared beam and to prevent loss of infrared radiation. Before recording each spectrum, the ZnSe-diamond crystal substrate was washed with deionized water, and a clean background scan was collected. The spectral range was 4000–400 cm⁻¹. -1 The resolution is 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). The sample was dried at 45°C for 72 hours before testing. Test conditions: Co target X-ray tube, Ni filter to eliminate CuKα radiation, tube voltage and current of 36 kV and 20 mA respectively, scanning range of 5°–40°, scanning speed of 2° / min, and scanning step size of 0.02°. The crystallinity index (CrI) was calculated using the following formula (8).

[0060]

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

[0062] 1.6.4 N2 Adsorption-Desorption Desorption Measurement:

[0063] The specific surface area (SSA) of CS cellulose was determined using a fully automated specific surface area and pore size distribution analyzer (Nova Touch LX1, Quantachrome Instruments, USA). In brief, 0.2 g of sample powder was weighed and degassed at 100 °C for 6 h to remove moisture and other adsorbates. Adsorption-desorption was then performed using high-purity N2. The SSA of the sample was calculated using the multi-point 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 determine its functional group composition. The 2D HSQC nuclear magnetic resonance spectrum of the recovered lignin was analyzed using an AVANCE III HD 600MHz spectrometer (Bruker, Germany). 50 mg of dried lignin was dissolved in 0.5 mL of DMSO solution. 1 H and 13 The spectral widths in dimension C are set to 5000Hz and 20000Hz, respectively. 1 The transient parameters in dimension H are 64, with 1024 sampling points; 13 The transient parameters in dimension C 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 an internal standard. The collected data were analyzed using Bruker Topspin 4.4.0.

[0066] 1.8 Pretreatment of CS fermentation for the production of biomass protein (SCP):

[0067] Yeast inoculum was prepared using yeast extract peptone glucose medium (YPD, 1% yeast extract, 2% peptone, 2% glucose). The culture was incubated at 30°C and 150 rpm for 24 h before inoculation. CS treated with EN and ELG, which had 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 citrate buffer (0.05 M, pH 4.8) for 72 h. Then, 0.5 g of yeast extract and 1 g of tryptone were added to 100 mL of the hydrolysate as supplementary nutrients. Yeast culture was inoculated at 4% (v / v) of the hydrolysate volume and fermented at 30°C and 200 rpm for 72 h to produce SCP. After fermentation, the fermentation broth was freeze-dried, and the crude protein content of the obtained solid product was analyzed using the Kjeldahl method. The true protein content was determined, and the non-protein nitrogen content was calculated by subtracting the true protein from the crude protein content. The amino acid composition of the solid product was determined using an automated amino acid analyzer (Hitachi L-8900, Japan).

[0068] 1.8 Data Analysis:

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

[0070] Results and Discussion of Example 2

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

[0072] SE, or thermomechanical-chemical process, is a typical physicochemical pretreatment method. Pressure, pressure holding time, and biomass characteristics (e.g., moisture content or particle size) are the most important parameters affecting the steam explosion reaction. The conversion rates of dextran, xylan, and total sugars obtained from CS enzymatic hydrolysis after pretreatment were used as response values. One-factor optimization (OFAT) was employed to screen for the optimal conditions for each parameter, and the results are as follows: Figure 1 As shown, under the same pressure holding 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 force, which can break the hydrogen bonds and covalent bonds connecting cellulose, hemicellulose, and lignin, transforming the original biomass into loose and porous 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 basically unchanged (from 44.9% to 45.0%). In addition, under high temperature and high pressure conditions, the pentose released by the hydrolysis of hemicellulose is easily dehydrated and converted into inhibitors such as furfural and 5-hydroxymethylfurfural, and excessively high pressure requires more saturated steam, which is not economical. Therefore, 1.5 MPa was selected as the optimal steam explosion pressure. Under the same pressure (1.5 MPa) and moisture content (50%), the yields of glucose, xylose, and total sugars generally increased as the pressure holding time increased from 1 min to 7 min. With prolonged exposure of CS biomass to high-pressure saturated steam, the thermally unstable acetyl groups in hemicellulose are cleaved, releasing acetic acid. This released acid disrupts glycosidic bonds, ether bonds, and other acid-labile bonds between lignocellulose molecules, weakening the fiber's rigid structure to some extent and increasing enzyme accessibility and saccharification efficiency. At 4 min, the dextran yield was 46.3%, decreasing to 45.3% at 7 min, which may be related to the loss of cellulose due to prolonged exposure to high-pressure steam. Considering the limited increase in total sugar yield with prolonged steam explosion time, 4 min was chosen as the optimal pressure holding time. Moisture content is a key factor determining SE efficiency, influencing it by regulating the permeability of steam to biomass (which determines mass transfer efficiency). Therefore, the initial moisture content of CS was optimized, adjusted to 20%, 50%, and 80%, respectively, and then subjected to steam explosion reaction after 12 hours of full water absorption at room temperature. The results showed that the conversion rates of dextran and total sugars reached their highest at a moisture content of 50%, at 44.1% and 42.8%, respectively. Therefore, 50% was selected as the optimal moisture content for SE (Selenium Extraction). Overall, a pressure of 1.5 MPa, a pressure holding time of 4 min, and a moisture content of 50% were the optimal conditions for SE pretreatment of CS biomass, which can fully break down the polymer structure of lignocellulose, providing a solid foundation for downstream combined pretreatment and enzymatic saccharification.

[0073] 2.2 Analysis of Lignocellulose Composition and Removal:

[0074] A typical type III DES system was prepared, using quaternary ammonium salts (choline chloride) as hydrogen bond acceptors and various organic compounds (urea, lactic acid, glycerol) as hydrogen bond donors. Pretreatment with a deep eutectic solvent composed of different hydrogen bond donors, combined with steam explosion, aimed to retain more of the enzymatically hydrolyzable saccharification substrates cellulose and hemicellulose, while maximizing lignin removal and reducing non-productive enzyme adsorption. Untreated CS biomass consisted of 38.35% cellulose, 16.69% hemicellulose, and 22.26% lignin. Figure 2 (A). Compared to CON, the lignocellulose composition of CS changed accordingly due to the removal of lignin and hemicellulose after pretreatment. After different DES coupled with SE pretreatment, the cellulose retention rate of CS exceeded 90% ( Figure 2 In the combined pretreatment (B), the EN pretreatment showed a 95% retention rate, indicating good protection of cellulose and providing sufficient saccharifiable substrate for subsequent enzymatic hydrolysis. Different binary DES combined with SE pretreatment showed significant differences in the removal of hemicellulose and lignin. EL exhibited the lowest hemicellulose recovery rate (33.12%) and the highest lignin removal rate (36.55%). Conversely, EG showed the highest hemicellulose recovery rate (51.27%) and the lowest lignin removal rate (27.86%). EN was in the middle range, 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 the easy hydrolysis of acetyl groups. The SE step in the combined pretreatment easily induces acidic autocatalytic hydrolysis of hemicellulose acetyl groups through high temperature and pressure, and synergistically breaks glycosidic bonds through mechanical shear force, 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, DES using polyols, carboxylic acids, and amides as hydrogen bond donors achieved a hemicellulose removal rate of 16%–90%, which is also related to the ratio of hydrogen bond acceptors and the reaction temperature. However, numerous studies have shown that acidic DES removes more hemicellulose, while neutral or basic DES reduces hemicellulose loss. Our results are consistent with this, showing that neutral or basic EN and EG DES have higher hemicellulose recovery rates, while acidic EL DES has the lowest recovery rate at only 33.12%. The high deligninization rate of EL (36.55%) may be related to its DES using lactic acid as a hydrogen bond donor. Studies have shown that the dissociation of H protons from hydrogen bond donors accelerates the breaking of ether and ester bonds between polysaccharide moieties (cellulose and hemicellulose) and lignin, leading to lignin removal. Furthermore, in acidic DES, the β-O-4 bonds connecting monomers within lignin are more easily broken, making lignin more soluble.

[0075] Because the binary DES systems EL and EG exhibited opposing effects in retaining hemicellulose and removing lignin, a ternary DES system was prepared at a molar ratio of 1:2:2 (choline chloride: lactic acid: glycerol) to combine the advantages of both systems, aiming to remove more lignin while retaining more hemicellulose. The results showed that ELG reduced hemicellulose loss, achieving a recovery rate of 43.24%; while the lignin removal rate was the highest among all treatment groups, at 40.84%. The addition of polyols such as glycerol effectively optimized 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 was reduced, lignin could still be selectively removed while mitigating excessive degradation of xylan.

[0076] 2.3CS pretreatment followed by enzymatic hydrolysis and saccharification:

[0077] Lignocellulose is a high-molecular-weight polymer formed by the cross-linking of cellulose, hemicellulose, and lignin. The enzymatic hydrolysis efficiency of structural polysaccharides largely depends 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 down the polymer structure of lignocellulose and improve enzyme accessibility, but also achieve high recovery of carbohydrates (especially hemicellulose) while removing a large amount of lignin. All these approaches lead to the ultimate goal: increasing monosaccharide yield. After combined pretreatment, the enzymatic digestibility of residual solids fractions of CS was determined from 0 to 72 h. In the initial stage of enzymatic hydrolysis (12 h), compared with the original CS, the dextran yield of CS pretreated with different DES coupled with SE increased rapidly, but they all maintained similar saccharification values, namely EN 30.4%, EL 32.8%, EG 34.7%, and ELG 32.8%. Figure 3 (A). As the most abundant component of CS lignocellulose, dextran rapidly releases glucose in the first stage of hydrolysis, meaning that the content of amorphous fraction in the pretreated solid fraction is high, promoting faster hydrolysis. Essentially, the coupling of the thermomechanical destructive action of SE and the bond breaking action of DES causes structural changes in lignocellulose, including the formation of vacancies, dislocations, and phase transitions, which is beneficial for the digestion of fractionated polysaccharides. With the extension of enzymatic hydrolysis time, the dextran yield of ELG gradually increased, reaching 42.8% at 72h, which was the highest level, while the dextran yield of other treatment groups was limited to around 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(See section B). The superior enzymatic hydrolysis efficiency of ELG can be attributed to the combined effect of the highest lignin removal rate, large specific surface area, and pore volume. These findings are further supported by changes in CS lignocellulose composition and subsequent characterization. First, carbohydrate hydrolysis efficiency is closely related to enzymatic adsorption; biomass exposed to ELG and EN has a larger surface area and larger pore volume, thus accelerating the enzymatic adsorption process. Second, hydrolysis is essentially initiated by the adsorption of cellulase and xylanase onto polysaccharides (cellulose and hemicellulose), where they undergo a continuous adsorption / desorption cycle to form the next polysaccharide. Therefore, high surface area and good porosity enhance polysaccharide accessibility, ultimately improving enzymatic hydrolysis. Furthermore, the ELG-enhanced enzymatic hydrolysis can be partly attributed to the high lignin removal rate, as the large presence of lignin reduces enzymatic hydrolysis by forming steric hindrance and / or non-specific adsorption, resulting in a reduction in the amount of enzyme available for digestion. Total sugar yield is the most direct parameter for evaluating pretreatment efficiency. 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 greatly limited enzymatic digestion. Thanks to the high yields of dextran and xylan, the total sugar yield of ELG reached 50.4% and EN reached 47.1% after 72 hours, which were 2.5 times and 2.3 times higher than those of the original CS, respectively.

[0078] 2.4 Changes in the microstructure of CS:

[0079] A thorough understanding of the structural transformations of lignocellulose (CS) before and after pretreatment is crucial for elucidating the dissociation process of the three components of lignocellulose and for providing information for the functionalization of different downstream components. The morphological changes of CS before and after pretreatment were analyzed using SEM. Micrographs at different magnifications are shown below. Figure 4 As shown. Unprocessed CS( Figure 4 A) The surface is smooth and flat, with a dense and orderly fibrous structure. This non-destructive and orderly nature is due to the inherent network structure of lignin-carbohydrates, which is stubborn and severely hinders the entry of pretreatment solvents and the adsorption of enzymes. Figure 4As shown in Figures B, C, D, and E, after pretreatment, the CS surface is rough, with obvious wrinkles and broken fiber bundles, indicating significant deformation and degradation of the cell wall structure. This structural and morphological change in CS can be attributed to the combined effect of SE and DES. SE tears the cell wall structure by generating shear force through instantaneous decompression under high temperature and pressure, transforming the initial biomass into smaller, dispersed fibers. DES, on the other hand, accelerates the breaking of chemical bonds between polysaccharides and lignin, resulting in looser connections between fibers and a more fragmented structure, especially for acidic EL and ELG (which have more coiled and folded structures). Therefore, the complete disintegration of the overall CS structure after combined pretreatment enhances the accessibility of carbohydrate streams and provides a favorable morphology for subsequent enzymatic hydrolysis and adsorption.

[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 signals at this location are related to the CO stretching vibrations in cellulose, hemicellulose, and lignin, or the COC stretching vibrations in cellulose and hemicellulose. Different DES pretreatments can cause the breaking of glycosidic or ether bonds between polysaccharides and lignin, potentially leading to shifts in peak position and changes in peak intensity. (892 cm⁻¹) -1 The peak at 1731 cm⁻¹ is formed by β-glycosidic groups between the monomeric sugars that make up cellulose and hemicellulose. -1 Bands formed by the stretching of hemicellulose acetyl C=O groups were observed nearby. The peak variation here is likely due to the removal of hemicellulose by DES, particularly acidic EL. (1616 and 1501 cm⁻¹) -1 The peak at 3400 cm⁻¹ corresponds to the aromatic skeleton vibration of lignin C=C extension. -1 The peak at 3400 cm⁻¹ corresponds to the extension of the lignin hydroxyl group -OH. Compared with the original CS, the treated CS shows an increase in peak length at 3400 cm⁻¹. -1 The peak value is relatively low. This may be because DES has a strong delignination ability, which leads to the shift and reduction of the lignin peak in the CS structure.

[0081] XRD analysis was used to evaluate the change in crystallinity before and after CS pretreatment. The resulting diffraction patterns are shown below. 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 peak shapes at this point among different pretreatments (CS) indicates that a large amount of the original cellulose structure was preserved during pretreatment. Compared with the original CS (29.46%), the decrease in CrI after EN (25.91%) pretreatment suggests that natural cellulose I may be transforming into amorphous cellulose and cellulose II. It is also noteworthy that the CrI in other pretreatment groups increased from 29.46% in the original CS to 29.50–40.45%. This change does not seem to be conducive to enzyme accessibility during subsequent saccharification, but it needs to be analyzed in conjunction with other structural changes. The increase in biomass CrI after pretreatment may be due to the removal of a large amount of amorphous hemicellulose and lignin during pretreatment.

[0082] In the conversion of lignocellulose into other value-added products, surface accessibility and pore structure are considered important factors for pretreatment solvents and enzymatic adsorption. For example... Figure 5 As shown in C and D, compared with the original CS, the SSA of the CS increased by 12-28 times and the pore volume increased by 11-33 times after different DES coupled SE pretreatments. Among them, EN pretreatment showed the largest increase, with SSA increasing from 1.40m. 2 / g increased to 38.57m 2 / g, pore volume from 0.0045cm³ 3 / g increased to 0.15cm 3 / g. Generally, the overall surface area of ​​biomass pellets consists of an outer surface and an 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 accessible pore volume. The shear force generated by the SE step in the combined pretreatment system can reduce the particle size of biomass and increase its bulkiness by tearing fibers, thereby inducing a significant increase in the outer surface area. Meanwhile, DES composed of different hydrogen bond donors expands the inner surface area by removing lignin, reducing crystallinity (especially EN), and adjusting porosity during bond breakdown. Furthermore, the average pore size of CS was analyzed ( Figure 5 In the study of CS (e.g., the average pore size of both the original CS and CS after different pretreatments exceeded 10 nm), suggesting that mesopore volume may play an important role in the adsorption process. Furthermore, as... Figure 5 As shown in Figure F, the pretreated CS samples all exhibited an exponential increase in N2 absorption curves with pressure, a characteristic inherent to Type III (IUPAC nomenclature) isotherms and typical of many straw biomass samples. At higher relative pressures (P / P0 > 0.5), the N2 adsorption capacity increased sharply, indicating the presence of numerous mesopores in the samples. The pore size distribution calculated using Barrett-Joyner-Halenda (BJH) is shown in Figure F. Figure 5The findings (G) further support this observation that the pretreated CS pore size is mainly distributed in the mesoporous range (2-50 nm), which is crucial for enzymatic hydrolysis because enzymes can theoretically pass through pores with diameters greater than 5 nm, playing a vital role in improving the accessibility of lignocellulose. In short, the combined pretreatment demonstrates multiple potentials to alter the pore structure of CS biomass and increase both external and internal surface areas, resulting in greater contact between lignocellulose and the solvent, ultimately making the cellulose highly suitable for enzymatic hydrolysis.

[0083] 2.5 Characterization of extracted lignin:

[0084] A thorough analysis of the structural changes in lignin is crucial for understanding the impact of this pretreatment technology platform on lignin, and can also provide valuable information for the downstream functional applications of lignin. Information on lignin functional groups obtained by FT-IR is as follows: Figure 6 As shown in Figures A and B, lignin extracted by SE in combination with different types of DES exhibits similar characteristic absorption bands, including 15-15 cm⁻¹. -1 The aromatic bone vibration zone at 1656cm -1 The absorption peak originates from the stretching vibration of the conjugated carbonyl group, and the characteristic band of the hydroxyl stretching is at 3440 cm⁻¹. -1 CH bond stretching (2935 and 2840 cm) -1 In addition, 1274cm -1 The signal at this location corresponds to the guaiac wood-based unit (G), 1121cm -1 Corresponding to the syringyl unit (S). Peak value at 835 cm⁻¹ -1 The peaks are attributed to out-of-plane vibrations in the p-hydroxyphenyl unit (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, suggesting that SE combined with different types of DES can specifically extract lignin without altering its chemical structure.

[0085] The lignin side chain (δC / δH 50–90 / 2.5–6.0) and aromatic (δC / δH 90–150 / 6.0–8.0) region spectra obtained by 2D-HSQC NMR analysis are shown below. Figure 6As shown in Figure C. Cross-signal peaks of lignin were identified, and their specific assignments are shown in Table S1. Obvious methoxy (OMe) signals were detected in the side-chain regions of all four lignins, further validating 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 the four lignins. After acidic EL pretreatment, the signal of Aβ (S) weakened, indicating severe cleavage of β-O-4 ether bonds. The calculated content of major lignin bonds also showed a significant reduction in the total number of β-O-4 ether bonds in EL lignin, ranking lowest among all treatments (17.08 / 100Ar). This indicates that acidic EL pretreatment can effectively cleave β-O-4 ether bonds to obtain more lignin fragments. When glycerol was introduced into the EL to form ELG, the β-O-4 content in the lignin increased to 26.28 / 100Ar. This indicates that polyols, as effective stabilizers for lignin functionalization, can protect β-aryl ether bonds from cleavage and inhibit subsequent condensation. In the aromatic region, G, S, and H unit signals were clearly observed in all four lignin samples. The ferulate (FA2) and p-coumarate (PCAβ / FAβ, PCAα / FAα) signals of EL and ELG were weakened to varying degrees, mainly attributed to the hydrolysis of ester bonds and acid-catalyzed condensation forming new carbon-carbon bonds under acidic conditions. Lignin extraction typically involves two common reactions: fragmentation and condensation. The S / G ratio can be used to evaluate the degree of lignin condensation. Generally, G units are more prone to depolymerization than S units during DES extraction. However, the results showed little difference in the S / G ratio among the four lignins, indicating that different DES combined with SE pretreatment can induce similar degrees of fragmentation and condensation in lignin, which may modulate lignin properties for downstream applications.

[0086] 2.6 Pre-treatment of straw for fermentation to produce biomass protein:

[0087] CS, as an important biomass resource, can produce abundant 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 a safe microorganism applicable in the food and feed industries. This study utilized physicochemically pretreated CS as a substrate, followed by enzymatic hydrolysis and co-fermentation with K. marxianus to produce biomass protein. The fermentation products mainly included unutilized lignocellulose and yeast biomass protein. The entire composition did not require further separation and could be used as a high-quality protein feed. 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 7(A). Crude protein consists of non-protein nitrogen and true protein, with the true protein portion being the most important for animal growth and health. After fermentation, the true protein contents of CON, EN, and ELG were 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 higher concentration of fermentable sugars produced during enzymatic hydrolysis after optimal pretreatment, which the yeast utilizes to produce more cell protein. Since carbon is the most abundant element in cells, carbon sources are crucial for the formation and regulation of cellular biomass. Additionally, the non-protein nitrogen content of EN and ELG is 5.4% and 4.3%, respectively. Figure 7 The concentration of C was slightly higher than that of CON (2.9%), mainly due to residual choline chloride and urea from the deep eutectic solvent pretreatment process. The essential amino acid profile of the fermentation products was also evaluated, and the results are shown in Table 1.

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

[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 glutamic acid 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] Amino acid profiles showed that the fermented EN and ELG were balanced in terms of essential amino acid content, containing all the essential amino acids required for animal feed as 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 a ternary deep eutectic solvent (ELG; choline chloride: lactic acid: glycerol) in this invention synergistically enhances the pretreatment of cotton stalks (CS), achieving preferential removal of lignin (40.8%) while retaining 43.2% of hemicellulose. This combined method significantly increases the specific surface area (27.7 m²). 2 / g, 19.8 times) and pore volume (0.07cm³) 3 / g (16.1 times), SEM revealed a looser, coiled structure, which is beneficial for enzyme adsorption. Therefore, ELG-pretreated CS 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 to overcome lignocellulose conversion barriers through selective lignin separation and structural disruption, advancing a sustainable biorefining route for biomass protein conversion.

[0092] Therefore, this invention employs the aforementioned method of high-efficiency pretreatment of straw biomass using a combination of steam explosion and deep eutectic solvent to produce biomass protein. This method achieves a lignin removal rate as high as 40.8% while retaining over 43.2% of hemicellulose. Simultaneously, it increases the specific surface area and pore volume, making the straw structure more loose and curled, which is beneficial for enzyme adsorption. Furthermore, the total sugar conversion rate reaches 50.4%, and the crude protein content in the fermentation product exceeds 25%, with the highest true protein content reaching 24.5%. This approach overcomes the obstacles in straw biomass conversion and paves the way for cutting-edge innovation 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 and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions 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 from straw biomass through efficient pretreatment using a combination of steam explosion and deep eutectic solvent, characterized in that, Includes the following steps: Step 1, straw pretreatment: The straw is air-dried and crushed at room temperature, and then pre-extracted 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, allow it to fully absorb water at room temperature for 12 hours, then load it into the steam explosion reaction device, adjust the target pressure and holding time to carry out the steam explosion reaction; The target moisture content is 20%-80%, the target pressure is 1.1MPa-1.9MPa, and the pressure holding time is 1min-7min. Step 3: Treat the steam-exploded straw with a deep eutectic solvent. In the eutectic solvent, choline chloride is used as a hydrogen bond acceptor, and urea and glycerol are used as hydrogen bond donors. The molar ratio of choline chloride: lactic acid: glycerol is 1:2:

2. The solid-liquid ratio of the straw after steam explosion treatment to the eutectic solvent is 1:

10. The reaction temperature is 100℃ and the reaction time is 6h. The solid and liquid are separated by vacuum filtration. Step 4: Perform enzymatic hydrolysis and saccharification on the straw treated with the deep eutectic solvent in Step 3, and calculate the sugar yield; During enzymatic hydrolysis, CS was mixed with 0.05M citrate buffer at pH 4.8, and cellulase was added. Then, 10 mg / ml tetracycline hydrochloride solution was added at 0.5% of the hydrolysate volume, and the total mixture was incubated at 50°C and 200 rpm for 72 hours. Step 5: Ferment the straw treated with the deep eutectic solvent in Step 3 to produce biomass protein and determine the protein content; Straw treated with eutectic solvent was enzymatically hydrolyzed in a buffer system for 72 hours. Then, yeast extract and tryptone were added to the hydrolysate as supplementary nutrients. Yeast culture was inoculated at 4% of the hydrolysate volume, and fermentation was carried out at 30°C and 200 rpm for 72 hours to produce biomass protein.

2. The method for producing biomass protein from straw biomass through efficient pretreatment 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 to ethanol is 2:1, and the pre-extraction time is 6 h.

3. The product obtained by the method according to any one of claims 1-2, characterized in that: The products include lignocellulose and yeast single-cell protein.

4. The use of the product as described in claim 3 in the preparation of protein feed.

5. The application of the product as described in claim 3 in the preparation of protein foods.

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