Method for efficiently saccharifying brewing sorghum straws

Abstract

The invention discloses a method for efficiently saccharifying brewing sorghum straws, which comprises the following steps: pretreating the brewing sorghum straws by adopting steam explosion under the conditions that the explosion pressure is 0.9-1.5 MPa, the pressure maintaining time is 4-8 minutes, the particle size of a material is 3-9 cm and the water content of the material is 30-70%; carrying out enzymolysis saccharification on the brewing sorghum straws subjected to steam explosion treatment, wherein the initial solid content is 10-20%, the enzyme addition amount is 20-50 FPU / g, the enzymolysis temperature is 45-55 DEG C, and the material particle size is 10-30 meshes; in the enzymolysis saccharification process, materials are supplemented in stages to regulate and control the solid content, the materials are supplemented at least once, and the final solid content is 35-40%. According to the method disclosed by the invention, the biomass conversion efficiency is remarkably improved through system optimization pretreatment and an enzymolysis process, and the solid content is improved and the fermentable sugar concentration is remarkably improved in combination with stepped material supplementation.

Description

TECHNICAL FIELD

[0001] The application relates to a saccharification method, in particular to a method for efficiently saccharifying high-cereal-sugar sorghum straw. BACKGROUND

[0002] There are significant obstacles to the direct utilization of lignocellulosic biomass, and its complex structure needs to be deconstructed through a pretreatment process to release fermentable sugars and remove inhibitory components. This biomass is mainly composed of cellulose (35-45%), hemicellulose (25-30%), lignin (15-30%), and 15-20% of minor components, among which cellulose contains both crystalline and amorphous forms and is the main structural polysaccharide that constitutes the plant cell wall. Hemicellulose, as a heterogeneous polymer, is composed of pentose, hexose, and uronic acid monosaccharides, and its amorphous characteristics make it more prone to hydrolysis. Lignin is a heteropolymer composed of three phenylpropanol units: coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol. Its hydrophobicity and complex three-dimensional structure not only hinder the enzymatic hydrolysis process but also produce toxic metabolites. The rigid network structure formed by the close cross-linking of these components significantly reduces the degradability of the biomass, so pretreatment technology is needed to break down its stubborn structure, separate the components, and improve the accessibility of cellulose.

[0003] Pretreatment processes can be divided into four categories according to the mechanism of action: physical, chemical, physical-chemical, and biological. Physical methods mainly reduce particle size through mechanical crushing, while chemical methods use acid or base reagents to achieve component dissolution. Steam explosion, as a typical physical-chemical method, uses high-temperature and high-pressure steam (0.7-4.8 MPa, 160-290℃) to treat biomass and then implements rapid pressure relief. The acetic acid produced in this process can promote hemicellulose hydrolysis, has advantages such as improving enzymatic hydrolysis efficiency and expanding the range of raw materials. Biological pretreatment relies on fungi (such as filamentous fungi that produce lignin peroxidase) or enzyme preparations (such as Trichoderma cellulase) for selective degradation, which has the advantage of specificity but is less economical. Pretreatment processes need to balance multiple objectives: maximizing carbohydrate recovery while avoiding sugar degradation and inhibitory substance generation. Key control parameters include temperature, pH, reaction time, and catalyst concentration, and their optimization needs to consider the characteristics of the raw material and the requirements of the subsequent process. Enzymatic hydrolysis, as the core conversion link, its efficiency depends on the synergistic action of multi-enzyme system and the precise regulation of process conditions, and through the depolymerization of polysaccharides into fermentable monosaccharides, it lays the foundation for subsequent biological refining. The current technical bottlenecks mainly focus on energy cost control, inhibitory substance elimination, and process integration optimization, which directly affect the commercialization process of lignocellulosic bio-refining.

[0004] As a typical lignocellulosic biomass resource, the structural components of brewing sorghum straw include a ternary composite system of cellulose, hemicellulose and lignin, which forms a highly crystallized three-dimensional network superstructure through intermolecular cross-linking. This structure, as a natural anti-degradation barrier formed by plant evolution, not only maintains the mechanical strength of the plant, but also constitutes a significant structural resistance in the process of biological refining. Studies have shown that effectively breaking the covalent bonding network of lignin-carbohydrate complex is a prerequisite for the high-value utilization of biomass resources. Only by directed deconstruction of its stubborn structural barrier can subsequent enzymatic saccharification and biological conversion be efficiently carried out. In view of this, a multi-preprocessing technology system including mechanical crushing, solvent deconstruction and enzymatic decomposition has been developed at the present stage. Through the differential mechanisms of physical energy field intervention, selective chemical bond rupture and biological catalytic modification, the controllable dissociation of lignocellulosic structure is realized. However, these methods are single preprocessing processes, and the efficiency of single mechanical crushing and single enzymatic reaction treatment process is low, and solvent deconstruction has environmental pollution problems.

[0005] It should be noted that the information disclosed in the above background section is only used to strengthen the understanding of the background of the present disclosure, and therefore can include information that does not constitute prior art known to those of ordinary skill in the art. SUMMARY

[0006] The purpose of the present application is to provide a method for efficient saccharification of brewing sorghum straw, which can significantly improve the biomass conversion efficiency by optimizing the pretreatment and enzymatic hydrolysis process, and can also improve the solid content by stepwise feeding, significantly increase the fermentable sugar concentration, and also does not have the problem of environmental pollution.

[0007] In order to achieve the above purpose, the present application provides a method for efficient saccharification of brewing sorghum straw, which comprises: pretreating brewing sorghum straw by steam explosion, the explosion pressure is 0.9-1.5 MPa, the pressure holding time is 4-8 min, the material particle size of the brewing sorghum straw after steam explosion treatment is controlled to be 3-9 cm, and the moisture content of the material is 30-70%; enzymatic saccharification of the brewing sorghum straw after steam explosion treatment, the initial solid content is 10-20%, the enzyme addition amount is 20-50 FPU / g, the enzymatic hydrolysis temperature is 45-55°C, and the material particle size is 10-30 mesh; during the enzymatic saccharification process, the brewing sorghum straw after steam explosion treatment is added in stages after liquefaction to regulate the solid content, at least once, and the final solid content is 35-40%.

[0008] Preferably, the solid content is increased by 5-15% in the first feeding. More preferably, the solid content is 20-35% after the first feeding.

[0009] More preferably, the n+1th feeding increases the solid content by 5-10%, n≥1.

[0010] Specifically, two feedings are performed, the initial solid content is 10-20% (such as 10, 15, 20%), after the 1st feeding, the solid content is 25-30% (such as 25, 30%), and after the 2nd feeding, the solid content is 35%. Five feedings are performed, the initial solid content is 15%, and after the 1st-5th feedings, the solid content is 20, 25, 30, 35, and 40%, respectively.

[0011] Preferably, the blasting pressure is 1.5 MPa, the pressure maintaining time is 4-8 min, the material particle size is 3-9 cm, and the material moisture content is 50-70%.

[0012] More preferably, the blasting pressure is 1.5 MPa, the pressure maintaining time is 8 min, the material particle size is 5-7 cm, and the material moisture content is 70%.

[0013] Preferably, the enzyme addition amount is 20-30 FPU / g, the enzymolysis temperature is 45-50℃, and the material particle size is 20-30 mesh.

[0014] More preferably, the enzyme addition amount is 30 FPU / g, the enzymolysis temperature is 50℃, and the material particle size is 30 mesh.

[0015] The method for efficiently saccharifying brewing highland millet straw of the application has the following advantages: The application establishes a pretreatment and enzymatic saccharification process for brewing highland millet straw, and significantly improves the biomass conversion efficiency by optimizing the pretreatment and enzymatic saccharification process. Single-factor experiments show that the blasting pressure (0.9-1.5 MPa), pressure maintaining time (4-8 min), material particle size (3-9 cm), and moisture content (30-70%) have a significant effect on the pretreatment effect. After optimization by the response surface method, the optimal pretreatment conditions are determined as 1.5 MPa / 8 min / 5-7 cm / 70% moisture, at which the fermentable sugar concentration is 43.602 g / L, and the verification result (43.589±0.14 g / L) confirms the reliability of the model. In the optimization of the enzymatic saccharification process, the enzyme addition amount (20-40 FPU / g), temperature (45-55℃), and particle size (10-30 mesh) are determined as the key parameters through orthogonal experiments, and the final optimized conditions are 30 FPU / g / 50℃ / 30 mesh. Combined with the step feeding, the solid content is increased to 35%, and the fermentable sugar concentration is 168.63±2.46 g / L. This process system realizes efficient saccharification of lignocellulose through multi-stage parameter optimization, and provides a reliable technical solution for the biological refining process. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The effect of steam explosion on the content of enzymatically hydrolyzed sugar in Experimental Example 1 of this invention: a) explosion pressure; b) holding time; c) particle size; d) moisture content.

[0017] Figure 2 The effects of steam explosion on cellulose, hemicellulose, and pH in Experimental Example 1 of this invention are shown below: a) Explosion pressure; b) Holding time; c) Particle size; d) Moisture content.

[0018] Figure 3 The results show the effects of the interaction between pretreatment moisture, pressure, and particle size on the pretreatment effect in Experimental Example 2 of this invention; a) interaction between moisture and particle size; b) interaction between moisture and pressure.

[0019] Figure 4 The images shown are SEM images before and after the steam explosion in Experiment Example 4 of this invention.

[0020] Figure 5 The results of the determination of the three elements (cellulose, hemicellulose and lignin) before and after steam explosion in Experiment Example 4 of this invention are shown.

[0021] Figure 6 The figure shows the results of the single-factor enzymatic hydrolysis experiment in Experiment Example 4 of the present invention; a) TS content; b) enzyme addition amount; c) temperature; d) particle size.

[0022] Figure 7 The sugar content under different treatments in the fed-batch enzymatic hydrolysis optimization experiment of Experiment Example 6 of this invention is shown. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] It should be noted that: Unless otherwise specified in the examples, standard conditions or conditions recommended by the manufacturer should be followed. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0025] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0026] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0027] Experiment Example 1: Single-factor experiment to investigate the effects of pretreatment conditions on sorghum straw for brewing. This invention employs a single-factor experimental design to systematically investigate the effects of steam explosion parameters (pressure, holding time, particle size, and moisture content) on the pretreatment effect of sorghum straw for brewing. The key evaluation indicators are the cellulose retention rate, hemicellulose degradation rate, system pH, and total fermentable sugar concentration of the enzymatic hydrolysate. Specifically:

[0028] (1) Single-factor experiment of bursting pressure: Under the conditions of fixed pressure maintenance time of 6 min, material particle size of 1~3 cm and moisture content of 50%, the effects of five pressure gradients of 0.6, 0.9, 1.2, 1.5 and 1.8 MPa on the pretreatment effect were investigated.

[0029] (2) Single-factor experiment on pressure maintenance time: Under the conditions of fixed burst pressure of 1.5 MPa, material particle size of 1-3 cm and moisture content of 50%, the effects of five pressure maintenance times (2, 4, 6, 8 and 10 min) on the pretreatment effect were investigated.

[0030] (3) Single-factor experiment on particle size: Under the conditions of fixed burst pressure of 1.5 MPa, pressure maintenance time of 6 min and moisture content of 50%, the effects of six particle size gradients (<0.5 cm, 0.5 cm≤particle size<1 cm, 1 cm≤particle size<3 cm, 3 cm≤particle size<5 cm, 5 cm≤particle size<7 cm, 7 cm≤particle size<9 cm) on the pretreatment effect were investigated.

[0031] (4) Single-factor experiment on material moisture content: Under the conditions of fixed burst pressure of 1.5 MPa, pressure maintenance time of 6 min, and material particle size of 1~3 cm, the effect of moisture content on pretreatment effect was systematically studied. The moisture content range studied was 10%~90%, and the moisture content gradient was 10%.

[0032] The methods for determining key performance indicators are as follows: (1) pH value of the system: The pH value of the system was measured immediately after pretreatment using an FE28 pH meter (Mettler Toledo, Switzerland); (2) Cellulose retention rate and hemicellulose degradation rate: The cellulose and hemicellulose contents were determined according to the standard method of NREL / TP-510-42618. SN2 cellulase (Baiyin Sainuo, Ningxia, China) was used for saccharification with an enzyme loading of 30 FPU / g substrate. The reaction was carried out at 50℃ and 180 rpm for 48 h.

[0033] (3) Total fermentable sugar concentration in the enzymatic hydrolysate: The total fermentable sugar concentration was determined by HPLC. Chromatographic conditions: Aminex HPX-87H column, mobile phase 5 mM H2SO4, flow rate 0.6 mL / min, column temperature 35℃, differential detector (RID), detector temperature 40℃, injection volume 20 μL.

[0034] All experiments were performed with three independent replicates, and the data were analyzed using SPSS 22.0 ANOVA (P<0.05).

[0035] like Figure 1 a) and Figure 2 As shown in a), the concentration of fermentable sugars increases with increasing pressure, while the xylose content initially increases and then decreases with increasing pressure. When the steam explosion pressure reaches 1.8 MPa, the total sugar concentration reaches 45.37 g / L, the cellulose content increases to 38.14%, and the hemicellulose content decreases to 15.42%. However, due to cost considerations, the steam explosion pressure in industrial applications generally does not exceed 1.5 MPa, and at 1.2 MPa, hemicellulose loss is significant, while at 1.5 MPa, the xylose content in the hydrolysate also begins to decrease. Considering economic benefits and the changes in hemicellulose and xylose content, three pressure gradients of 0.9 MPa, 1.2 MPa, and 1.5 MPa were selected for further condition optimization.

[0036] The duration of pressure regulation, as a key process parameter, not only directly affects the depolymerization and dissolution efficiency of hemicellulose components in the lignocellulose matrix, but also synergistically influences the plasticization and recombination process of lignin macromolecules, and has a decisive impact on the diffusion and permeation efficiency of the vapor medium within the material's pore structure. For example... Figure 1 b) and Figure 2As shown in b), the concentration of fermentable sugars increased with increasing pressure time, as did the xylose content. Simultaneously, the pH of the system decreased with increasing pressure time. The total sugar concentration reached a maximum of 42.09 g / L at 8 min, and decreased to 41.98 g / L at 10 min, showing no significant difference. Therefore, three pressure time gradients of 4 min, 6 min, and 8 min were selected for further condition optimization.

[0037] Particle size distribution plays a crucial role in regulating the efficiency of steam explosion processes. Smaller particle sizes exhibit superior steam permeation kinetics and heat transfer uniformity under the same process parameters. However, excessive particle size reduction leads to excessive thermal effects, causing non-selective thermal degradation of hemicellulose-derived sugars (such as xylose), which in turn induces the formation of furfural inhibitors and exacerbates the thermomechanical losses of the fiber matrix. Furthermore, finer particle size reduction significantly increases the energy consumption load of mechanical grinding in the pretreatment stage. Figure 1 c) and Figure 2 As shown in c), the concentration of fermentable sugars decreases with increasing particle size, while the contents of cellulose and hemicellulose increase. This suggests that particle size affects the enzymatic hydrolysis efficiency, and the loss of cellulose and hemicellulose becomes more severe as the particle size decreases. When the particle size is <0.5 cm, the total sugar concentration reaches 57.79 g / L, but the cellulose content decreases from 43.05±0.64% for particles with a diameter of 7 cm ≤ <9 cm to 36.7±0.58%. Therefore, three particle size gradients—3 cm ≤ <5 cm, 5 cm ≤ <7 cm, and 7 cm ≤ <9 cm—were selected for further optimization of the conditions.

[0038] Hydration pretreatment, as a core control step in the steam explosion process, operates primarily through two mechanisms: firstly, it reduces material brittleness by plasticizing the fiber matrix, achieving gentle dissociation of fiber bundles (avoiding mechanical structural damage); secondly, it expands material porosity through the fiber swelling effect, significantly enhancing steam diffusion kinetics, thereby improving the permeability and homogeneity of the steam phase within the fiber network, ultimately synergistically optimizing component separation efficiency and product yield during the explosion process. Figure 1 d) and Figure 2 As shown in d), the concentration of fermentable sugars increases with increasing moisture content, reaching a peak of 47.72 ± 0.34 g / L at a moisture content of 40%. Hemicellulose content also increases with increasing moisture content, while cellulose content shows no significant change, but both reach their lowest values ​​at a moisture content of 50% (cellulose content 32.54 ± 0.24%, hemicellulose content 15.12 ± 0.56%). Therefore, three moisture gradients (30%, 50%, and 70%) are selected for further optimization of the conditions.

[0039] Experiment Example 2: Optimization of Steam Explosion Pretreatment Conditions using Response Surface Methodology Based on the results of single-factor experiments, the Box-Behnken experimental design principle was used to systematically optimize key process parameters. Four independent variables with significant impact on pretreatment effects were selected: burst pressure (A), holding time (B), particle size (C), and moisture content (D). The critical level ranges of each variable were determined based on previous single-factor experiments. The gradient ranges of each key independent variable in the experimental design were scientifically configured according to the interaction laws of the parameters. The specific parameter combination matrix is ​​shown in Table 1. Using the total fermentable sugar concentration in the enzymatic hydrolysate (Y1) as the response value, a four-factor, three-level response surface experimental matrix was constructed using Design-Expert 13.0 software. The results are summarized in Tables 2 and 3.

[0040] Table 1. Experimental Factor Levels for Pretreatment Response Surface Methodology

[0041] A steam explosion model was established by designing 29 sets of parameters using Design-Expert 13 software and then performing enzymatic hydrolysis.

[0042] Table 2 Response Surface Design

[0043] Based on 29 sets of orthogonal experimental data, a quadratic polynomial regression model was performed on the experimental matrix in Table 2 using Design-Expert 13 statistical software (independent variables A: burst pressure, B: holding time, C: material particle size, D: moisture content; response variable Y: total sugar yield from enzymatic hydrolysis). Statistical analysis showed that the final regression model can quantitatively characterize the nonlinear response relationship between key process parameters and enzymatic hydrolysis efficiency. Its mathematical expression is as follows:

[0044] Enzymatic hydrolysis total sugar = 74.86593455175 - 64.244183041389A - 1.976939336375B - 0.41279118352777C - 0.40847444158333D + 0.18684752916664AB - 1.0026652916667AC + 0.50872216625AD + 0.20008659333333BC - 0.0241837204375BD + 0.026564981375CD + 25.823842637037A 2 +0.19642967652083B 2 -0.10148251432407C²-0.0017799738816667D² Table 3. Box-Behnken Design Variance Analysis

[0045] Note: p <0.05 indicates a significant difference. p <0.001 indicates a highly significant difference.

[0046] The statistical analysis in Table 3 shows that the quadratic polynomial regression model exhibits highly significant characteristics (p<0.0001), and the model's lack-of-fit term did not reach a significant level (p=0.5917), proving that the model has a good fit with the experimental data. The model's coefficient of determination R²=0.9479 indicates that 94.79% of the variation in the response variable can be explained by the model, with unexplained variation accounting for only 5.21%. The goodness of fit between the theoretical prediction and the actual values ​​was further verified by adjusting the coefficient of determination R²adj=0.8958 (highly close to R²=0.9476), confirming the reliability of the model's prediction of the total sugar content in enzymatic hydrolysis. F-value analysis shows that the weighting of the process parameters on the enzymatic hydrolysis efficiency is ranked as follows: A (burst pressure) > C (particle size) > B (holding time) > D (moisture content). In particular, the quadratic term A² (p<0.001) and the interaction terms AD and CD (p<0.05) have a significant regulatory effect on sugar production, indicating that the nonlinear effect of burst pressure and its synergistic effect with water content, particle size and water content have a key impact on the sugar metabolism pathway.

[0047] Visualization via 3D response surface (see...) Figure 3 The parameter interaction mechanism can be quantitatively analyzed: the enzymatic sugar content increases monotonically with the synergistic increase of burst pressure and moisture content, while exhibiting a parabolic characteristic of first increasing and then decreasing with changes in the combination of particle size and moisture content, with the pressure-moisture content interaction being particularly prominent (the slope of the surface is steep). The model signal-to-noise ratio AdeqPrecision = 16.7414 (>4 threshold) fully verifies the resolution of the experimental design and the accuracy of model prediction, ensuring the reliability of the optimization results.

[0048] Using DesignExpert13 software, with the enzymatic hydrolysis sugar content as the response value, the experimental data were optimized and analyzed. The optimal culture conditions were found to be: burst pressure 1.5 MPa, pressure holding time 7.995 min, material particle size 5.59 cm, material moisture 69.844%, and sugar content of the enzymatic hydrolysate under these conditions was 43.602 g / L.

[0049] Based on the optimal process conditions obtained above, and considering the feasibility of actual production conditions, the process parameters were adjusted to: burst pressure 1.5 MPa, holding time 8 min, material particle size 5 cm ≤ particle size < 7 cm, and material moisture content 70%. Three sets of parallel verification experiments were conducted. The average LA yield obtained using this method was 43.589 ± 0.14 g / L, which is very close to the predicted value of 43.602 g / L, indicating that this regression model is reliable.

[0050] Experiment Example 3: Determination of Components in Sorghum Straw for Brewing To accurately analyze the pretreatment effects of pretreatment conditions on brewing sorghum straw, SEM (scanning electron microscopy) images were taken of the straw before and after the blasting process, and the components of cellulose, hemicellulose, and lignin were determined. This experiment established a lignocellulose raw material component analysis system according to the standard procedure of the National Renewable Energy Laboratory (NREL) (SLUITER, 2008). The specific procedures are as follows:

[0051] (1) The pretreated sorghum straw samples were crushed to 80 mesh (pore size 180 μm) and dried in an oven at 105℃ to constant weight (Δm ≤ 0.2 mg / 24 h). The following samples were weighed precisely using an analytical balance: experimental group: 0.3000±0.0001 g sample; calibration group: 0.3000±0.0001 g standard (D-glucose and D-xylose, Sigma-Aldrich, purity ≥99.9%). Each group was replicated in 3 places. The samples were transferred to 50 mL pressure-resistant hydrolysis tubes (Allihn type, borosilicate glass).

[0052] (2) Add 3.00±0.05 mL of 72% (w / w) sulfuric acid (pre-cooled to 4℃) to each hydrolysis tube quantitatively, and immediately vortex mix for 2 min to ensure sufficient solid-liquid contact. Then place the hydrolysis tube in a constant temperature water bath shaker (30.0±0.5℃, 120 rpm) for 60 min, and manually vortex for 5 s every 10 min during the process (keep the tube submerged below the water bath liquid level throughout the operation) to avoid sample particles adhering to the tube wall;

[0053] (3) Slowly add 84.00±0.50 mL of ultrapure water (18.2 MΩ·cm) to each hydrolysis tube to reduce the final concentration of sulfuric acid to 4% (w / w); transfer the hydrolysis tube to an autoclave and treat it at 121℃ for 1 h with a pressurization rate of 2℃ / min. After depressurization, allow it to cool naturally to room temperature. (4) After the reaction was completed, the hydrolysate was vacuum filtered through a G4 sand core funnel (pre-calcined in a muffle furnace at 550℃ for 4 h), and the filtrate was filtered through a 0.22 μm nylon filter membrane and stored at -20℃ for analysis. The residual solid was washed with ultrapure water until neutral (pH 6.8-7.2), dried at 105℃ to constant weight, and the acid-insoluble lignin content was calculated by weighing.

[0054] (5) HPLC conditions: Column: Aminex HPX-87H (300×7.8 mm, Bio-Rad); Mobile phase: 5 mM H2SO4, flow rate: 0.6 mL / min; Column temperature: 65℃; Detector: Differential refractive index detector (RID-20A); Injection volume: 20 μL; Run time: 30 min. Glucose and xylose concentrations were calculated based on the standard curve, combined with a hydrolysis efficiency correction factor (based on the recovery rate of the correction group).

[0055] (6) Data correction and calculation: (Recovery rate ≥ 95%)

[0056]

[0057]

[0058] In the formula, C glu , C xyl These represent the corrected glucose and xylose concentrations (g / L), respectively. V Indicates the volume of the hydrolysate (L); m dry This indicates the initial dry weight of the sample (g). m residue The formula represents the dry weight (g) of the solid residue after hydrolysis; where 0.9 and 0.88 are correction factors for the conversion of carbohydrates into polysaccharides.

[0059] The microstructure of steam-explosion pretreated sorghum straw was characterized by scanning electron microscopy (SEM), such as... Figure 4As shown, significant topological reconstruction of the raw material surface morphology is observed. The untreated sample exhibits a dense and smooth surface with regularly arranged fiber bundles and low porosity. After blasting treatment, the fiber matrix undergoes mechanical fracture and delamination, forming a multi-scale fracture network on the surface accompanied by irregular topological deformation, while still retaining some oriented, intact fiber units. This structural evolution is attributed to the preferential dissociation of amorphous components (hemicellulose-lignin complex) triggered by intense thermomechanical coupling during blasting, leading to interfacial delamination between them and the crystalline cellulose skeleton. Further analysis shows that the treated sample surface exhibits a typical honeycomb pore structure and nanoscale particle deposits, confirming that lignin undergoes a dynamic phase transition process of depolymerization-migration-repolymerization. The synergistic effect of increased fiber surface roughness and partial lignin removal significantly improves the diffusion kinetics of cellulase molecules in the fiber supramolecular structure, increasing the surface area accessible for enzymatic hydrolysis. It can be observed that the waxy coating on the straw surface is effectively removed, the straw arrangement structure is completely destroyed, and the detectable content of total sugar and cellulose from enzymatic hydrolysis is significantly increased.

[0060] like Figure 5 As shown, the results of the three elements determination before and after steam explosion in Experiment Example 3 of the present invention can be seen. It can be seen that, compared with the untreated group, the cellulose content increased significantly after steam explosion treatment.

[0061] Experiment Example 4: Optimization of Single-Factor Conditions for Enzymatic Hydrolysis of Exploded Straw A single-factor experimental design was used to systematically investigate the effects of enzymatic hydrolysis parameters (solid content, enzyme loading, hydrolysis temperature, and particle size sieve size) on the enzymatic hydrolysis efficiency of sorghum straw for brewing after steam explosion pretreatment. Cellulase was used as the enzyme. The total fermentable sugar concentration in the hydrolysate was used as the key evaluation index.

[0062] (1) Single-factor experiment on solid content (TS, i.e. straw content): Under the conditions of fixed enzyme loading of 30 FPU / g substrate, enzymatic hydrolysis temperature of 50℃ and material particle size of 20 mesh, the effects of six groups of solid content (5%~30%, gradient 5%) on enzymatic hydrolysis effect were investigated. (2) Single-factor experiment on enzyme loading: Under the conditions of TS 20%, enzymatic hydrolysis temperature 50℃ and material particle size 20 mesh, the effects of six groups of enzyme loading (10~50 FPU / g substrate, gradient 10 FPU / g substrate) on the enzymatic hydrolysis effect were investigated. (3) Single-factor experiment of enzymatic hydrolysis temperature: Under the conditions of TS 20%, fixed enzyme loading of 30 FPU / g substrate and material particle size of 20 mesh, the effects of six groups of enzymatic hydrolysis temperatures (40~60℃, gradient 5℃) on the enzymatic hydrolysis effect were investigated. (4) Single-factor experiment on material particle size: Under the conditions of TS 20%, fixed enzyme loading of 30 FPU / g substrate and enzymatic hydrolysis temperature of 50℃, the effects of six groups of material particle sizes (non-sieved ck, 6 mesh, 10 mesh, 20 mesh, 30 mesh and 40 mesh) on the enzymatic hydrolysis effect were systematically studied.

[0063] The total fermentable sugar concentration after enzymatic hydrolysis was determined by HPLC. The chromatographic conditions were: HPX-87H column, column temperature 35℃, detector 40℃ (RID), mobile phase 5 mM H₂SO₄, flow rate 0.6 mL / min, injection volume 20 μL. All experiments were performed in triplicate, and data were analyzed using SPSS 22.0 ANOVA (P < 0.05).

[0064] like Figure 6 The figure shown is a graph of the results of the single-factor enzymatic hydrolysis experiment in Experiment Example 4 of this invention. It can be seen that the concentration of fermentable sugars in the enzymatic hydrolysis increases significantly with the increase of the solid content (TS), and there is no significant difference after TS reaches 25%. p <0.05) is approximately 106.9±0.48278 g / L, which is insufficient to provide enough carbon source to achieve LA yield at the cell factory level (100 g / L) even under theoretical conversion conditions. Furthermore, since the straw enzymatic hydrolysis liquefaction rate is faster at TS 20%, approximately half that at TS 25%, a solids content of 20% was selected for further condition optimization. Solids content supplementation optimization will be conducted after the enzymatic hydrolysis process is finalized. The concentration of fermentable sugars also increased with increasing enzyme addition, but no significant difference was observed after the enzyme addition reached 30 FPU / g. p <0.05), therefore, enzyme addition amounts of 20 FPU / g, 30 FPU / g, and 40 FPU / g were selected for further condition optimization. As the enzymatic hydrolysis temperature increased, the total sugar concentration showed a trend of increasing from low to high and then decreasing. p >0.05), the total sugar concentration reached a peak of 75.32±0.11688 g / L at 50℃. Therefore, three temperatures of 45℃, 50℃, and 55℃ were selected for further condition optimization. As the straw particle size increased through the sieve mesh, the total sugar concentration continued to rise, and there was no significant difference in the total sugar concentration after 30 mesh. p Since the particle size is <0.05, three particle sizes of straw were selected for further optimization: 10, 20, and 30 mesh sizes.

[0065] Experiment Example 5: Orthogonal optimization of enzymatic hydrolysis process for blasted straw Based on the results of previous single-factor experiments, a three-factor, three-level orthogonal experimental design was used to systematically optimize key process parameters. Three independent variables that significantly affected the total fermentable sugar concentration were identified: enzyme loading (20, 30, 40 FPU / g substrate), hydrolysis temperature (45, 50, 55℃), and particle size (10, 20, 30 mesh). An L9(3)2 model was constructed. 4 The orthogonal experimental matrix is ​​shown in Table 4. Experiments were conducted with a fixed solids content of 20%, and each group was replicated three times. Total fermentable sugar concentration was quantitatively analyzed by HPLC (as described above). Experimental data were analyzed using SPSS 22.0 software for multifactor ANOVA.

[0066] Table 4. Factor Level Table for Orthogonal Experiment (n=3)

[0067] From the k and R values ​​in Table 5, it can be seen that the influence of each factor on the enzymatic hydrolysis effect is in the order of C > B > A, that is, enzymatic hydrolysis temperature > particle size > enzymatic hydrolysis temperature. Table 6 shows that the three levels of factors B and C have significant differences in their effects on the enzymatically hydrolyzed sugar content, while A has no significant difference. From the three k values ​​of group A in Table 5, it can be seen that k2 is less than k3 but their values ​​are similar and significantly greater than k1. Therefore, through orthogonal experimental optimization, the final enzymatic hydrolysis process is obtained as A2B3C2, namely: TS 20%, temperature 50℃, particle size 30 mesh, enzyme amount 30 FPU / g.

[0068] Table 5. Results of the orthogonal experiment

[0069] Note: The blank group does not involve any experimental factors, and its data variation can be considered as a manifestation of random error. In analysis of variance (ANOVA), the variation (sum of squared errors) of the blank group is used as a baseline to calculate the significance (F-value) of each factor.

[0070] Table 6 Analysis of Variance Table

[0071] Experiment Example 6: Optimization Experiment of Fed Enzymatic Hydrolysis The enzymatic hydrolysis of lignocellulose biomass into fermentable glycosyl platform compounds is a key conversion pathway for the biorefining of lactic acid (LA). This process requires the preparation of high-density glycosides to meet the industrial-grade concentration threshold (>100 g / L) of LA required in subsequent fermentation stages.

[0072] To address the bottleneck issue that the sugar concentration of the enzymatic hydrolysate obtained from the previous single-factor optimized process (TS=20%) was insufficient to meet the requirements of industrial fermentation, a multi-stage fed-batch enzymatic hydrolysis operation strategy was adopted to improve the total fermentable sugar concentration in the system. This study systematically investigated the effect of a gradient feeding pattern at high solids concentrations (TS=35%, 40%) on improving enzymatic hydrolysis efficiency. Based on the interaction between substrate mass transfer limitation and enzymatic hydrolysis kinetics, nine fed-batch processes (BL1~BL9) were designed, with feeding after each liquefaction step, and the parameters are as follows:

[0073] (1) Single-stage feeding group BL1: Total TS 30%, Initial TS 30% (No feed addition, control group) BL2: Total TS 35%, Initial TS 35% (single-step loading) BL7: Total TS 40%, Initial TS 40% (single-step loading) (2) Multi-stage progressive feeding group TS=35% series: BL3: Feed in one batch (initial TS 20% → after liquefaction, feed to 35%) BL4: Feed in two stages (initial TS 10% → 25% → 35%) BL5: Feed in two stages (initial TS 15% → 25% → 35%) BL6: Feed in two stages (initial TS 20% → 30% → 35%) TS=40% series: BL8: Feed in 5 stages (initial TS 15% → 20% → 25% → 30% → 35% → 40%) BL9: Feed in 3 stages (initial TS 15% → 25% → 35% → 40%) Cellulase (30 FPU / g substrate) was pre-loaded in one step based on the total TS content to avoid introducing enzyme activity loss variables due to multiple additions. The liquefaction endpoint was used as the feed execution threshold. Enzymatic hydrolysis parameters: temperature 50℃, 180 rpm, 48 h.

[0074] The results are shown in Table 7. When the total solids content of the system was optimized to 35%, the BL6 experimental group exhibited the best liquefaction characteristics and saccharification efficiency, with glucose and xylose yields reaching 105.85±0.18 g / L and 57.05±0.34 g / L, respectively, and a total fermentable sugar concentration of 168.63 g / L. This index significantly exceeded the industrial fermentation substrate concentration benchmark (≥100 g / L), fully verifying the technical feasibility of this pretreatment system in large-scale LA biosynthesis. Although BL8 had a higher fermentable sugar content, its liquefaction was poor, making it unsuitable for fermentation. Therefore, by establishing a batch feeding strategy to systematically optimize the enzymatic hydrolysis process, a breakthrough was achieved with a fermentable sugar concentration of 166±3 g / L in the liquid enzymatic hydrolysate.

[0075] Table 7 Enzymatic hydrolysis under different feed additions

[0076] Note: "-" in the table indicates that no detection was performed.

[0077] In summary, this invention combines the pretreatment of sorghum straw for brewing with enzymatic hydrolysis and saccharification processes, and increases the solid content through a step-by-step feeding process. By optimizing multi-stage parameters, it achieves efficient saccharification of lignocellulose, providing a reliable technical solution for biorefining processes.

[0078] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for efficient saccharification of sorghum straw for brewing, characterized in that, The method includes: The sorghum straw for brewing was pretreated by steam explosion, with an explosion pressure of 0.9~1.5 MPa, a holding time of 4~8 min, a particle size of 3~9 cm, and a moisture content of 30~70%. The sorghum straw for brewing, which has undergone steam explosion treatment, is enzymatically hydrolyzed and saccharified. The initial solid content is 10-20%, the enzyme addition is 20-50 FPU / g, the enzymatic hydrolysis temperature is 45-55℃, and the particle size of the sorghum straw for brewing after steam explosion treatment is controlled to be 10-30 mesh. During the enzymatic hydrolysis and saccharification process, after liquefaction, brewing sorghum straw that has undergone steam explosion treatment is added in stages to regulate the solid content. The addition is done at least once, and the final solid content is 35-40%.

2. The method for efficient saccharification of sorghum straw for brewing according to claim 1, characterized in that, The first feeding increases the solids content by 5-15%.

3. The method for efficient saccharification of sorghum straw for brewing according to claim 2, characterized in that, For the (n+1)th feeding, the solid content increases by 5-10%, where n≥1.

4. The method for efficient saccharification of sorghum straw for brewing according to claim 1, characterized in that, The bursting pressure is 1.5 MPa, the pressure holding time is 4-8 min, the material particle size is 3-9 cm, and the material moisture content is 50-70%.

5. The method for efficient saccharification of sorghum straw for brewing according to claim 4, characterized in that, The bursting pressure is 1.5 MPa, the pressure holding time is 8 min, the material particle size is 5~7 cm, and the material moisture content is 70%.

6. The method for efficient saccharification of sorghum straw for brewing according to claim 1, characterized in that, The enzyme addition amount is 20~30 FPU / g, the enzymatic hydrolysis temperature is 45~50℃, and the material particle size is 20~30 mesh.

7. The method for efficient saccharification of sorghum straw for brewing according to claim 6, characterized in that, The enzyme addition amount is 30 FPU / g, the enzymatic hydrolysis temperature is 50℃, and the material particle size is 30 mesh.

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