Efficient saccharification method of corn stalks by synergistic pretreatment with elm and white ginseng fermentation enzyme solution

Abstract

This invention discloses a highly efficient saccharification method for corn straw by synergistic pretreatment with elm yellow and white ginseng fermentation enzyme solutions, belonging to the field of high-value utilization technology of biomass resources. This method aims to solve the problems of low enzymatic saccharification efficiency caused by the difficulty in simultaneously weakening the dual barriers of lignin and hemicellulose in existing pretreatment technologies. The key technical points are: corn straw is first treated with elm yellow fermentation enzyme solution with laccase activity of 5–14 U / mL at pH 4.2–4.8 and 28–32℃ for 12–24 h; then treated with white ginseng fermentation enzyme solution with xylanase activity of 0.75–1.7 U / mL at 35–40℃ for 24–48 h; finally, cellulase is added, and saccharification is carried out at 48–52℃ for 48–120 h. This stepwise synergistic pretreatment strategy can significantly improve the yield of reducing sugars and is mainly used in the fields of biofuel and bio-based chemical preparation.

Description

Technical Field

[0001] This invention relates to the field of high-value utilization technology of biomass resources. More specifically, this invention relates to a highly efficient saccharification method for corn straw by synergistic pretreatment with elm extract and white ginseng fermentation enzyme solution. Background Technology

[0002] Corn stalks are a widely available and inexpensive agricultural waste biomass resource with a huge annual yield, possessing immense potential for conversion into biofuels and bio-based chemicals. The main components of corn stalks include cellulose, hemicellulose, and lignin, which are interwoven through covalent and hydrogen bonds to form a highly dense lignocellulose complex. However, this complex structure constitutes a natural resistance barrier to biomass conversion: lignin, as a three-dimensional network aromatic polymer, physically encapsulates and chemically shields cellulose and hemicellulose; simultaneously, hemicellulose and lignin are linked by covalent bonds such as ferulic acid esters and p-coumaric acid esters, forming a lignin-carbohydrate complex, further enhancing structural stability. This structural characteristic makes it difficult for cellulases to access the cellulose components in the substrate, resulting in low enzymatic hydrolysis and saccharification efficiency, becoming a core bottleneck restricting the bioconversion of corn stalks.

[0003] To address the resistance barrier of lignocellulose, researchers have developed various pretreatment methods to disrupt its dense structure. Physicochemical pretreatment methods mainly include acid treatment, alkali treatment, steam explosion, and organic solvent methods. Dilute acid treatment can effectively remove hemicellulose, but its effect on lignin removal is limited, and it easily generates inhibitors such as furfural, hydroxymethylfurfural, and acetic acid, negatively impacting subsequent enzymatic hydrolysis and fermentation. Alkali treatment, while effective in removing lignin, generates alkaline wastewater with high treatment costs, and high-concentration alkali solutions severely corrode equipment. Steam explosion utilizes the shear force generated by the instantaneous release of high-temperature, high-pressure steam to destroy the fiber structure. Although the treatment time is short and no chemical reagents are required, it involves large equipment investment, high energy consumption, and also generates inhibitors such as furan derivatives and phenolic compounds during the process. These methods generally suffer from high energy consumption, stringent equipment requirements, severe environmental pollution, and the generation of numerous inhibitors, failing to meet the requirements of green and sustainable development.

[0004] Biological pretreatment technology is considered an ideal alternative to physicochemical pretreatment due to its advantages such as environmental friendliness, mild reaction conditions, and low energy consumption. White-rot fungi are currently the only known microbial group capable of efficiently degrading lignin. Their secreted extracellular enzyme systems (mainly including laccase, lignin peroxidase, and manganese peroxidase) can selectively oxidize the aromatic ring structure of lignin, disrupting the network structure of lignin macromolecules. In recent years, researchers have attempted to use enzyme systems or active substances secreted by white-rot fungi for sequential pretreatment of corn stalks. For example, Zhou et al. (Biotechnology for Biofuels and Bioproducts, 2024, 17:136) used hydroxyl radicals (·OH) secreted by the white-rot fungus *Proteobronchiolus chrysosporium* and recombinantly expressed manganese peroxidase (MnP) to sequentially treat corn stalks: the hydroxyl radicals first disrupted the smooth structure of the stalk surface, improving cellulose accessibility; subsequently, manganese peroxidase cleaved the β-O-4 bonds in the lignin model dimer. This sequential pretreatment improved the enzymatic saccharification and cellulose accessibility of corn stalks by 2.9 times and 1.8 times, respectively. However, this approach has significant limitations: hydroxyl radicals are essentially chemical oxides, and their generation requires chemical synthesis or complex reaction systems, rather than pure biological enzyme preparations; manganese peroxidase needs to be obtained through recombinant expression and purification, which is costly and difficult to scale up for industrial application. More importantly, this approach only focuses on lignin degradation and does not address hemicellulose removal. Hemicellulose, as another barrier encapsulating cellulose, still severely restricts the accessibility of cellulase to the substrate. In addition, some studies have used a single white-rot fungus fermentation broth to directly treat corn stalks. Although the operation is simple, the enzyme system of a single species often tends to degrade only one component of lignin or hemicellulose, making it difficult to effectively remove both simultaneously. Furthermore, the fungus consumes some fermentable sugars during its growth, resulting in carbohydrate loss.

[0005] To overcome the limitations of single enzyme systems, some studies have attempted to mix or sequentially combine enzymes from different sources, hoping to achieve simultaneous degradation of lignin and hemicellulose. However, simultaneous addition carries the risk of enzyme activity inhibition: one enzyme may oxidize another while catalyzing the oxidation of lignin, leading to the inactivation of the latter; or multiple enzymes may compete for the substrate interface, interfering with each other and failing to improve overall saccharification efficiency. On the other hand, inappropriate enzyme selection may result in the lignin-based physical shell remaining intact, making it difficult to fully contact the hemicellulose substrate; or the coating and cross-linking of hemicellulose on the lignin surface may hinder effective attack on lignin. These contradictions indicate that simple combinations of enzymes or arbitrary pretreatment sequences cannot simultaneously overcome the dual resistance barriers of lignin and hemicellulose.

[0006] However, while commercial enzyme preparations boast high purity and well-defined activity, their exorbitant production costs have become a major bottleneck hindering the industrialization of lignocellulose bioconversion. Commercial cellulases can account for 25%–30% of the total production cost in biofuel production, and their production process typically relies on expensive inducers such as lactose and sophorose, making fermentation control difficult. In contrast, crude enzyme solutions obtained directly through microbial fermentation offer significant cost advantages. Their production eliminates the need for complex separation and purification steps and utilizes inexpensive agricultural waste as fermentation feedstock. More importantly, crude enzyme solutions retain the complete enzyme spectrum produced during fermentation, avoiding enzyme activity loss during purification. Furthermore, complexes of crude enzyme solutions from various fungal sources exhibit higher hydrolysis efficiency in lignocellulose hydrolysis than purified commercial enzymes. Studies have also shown a significant synergistic effect between crude enzyme solutions and commercial enzymes; combining appropriate amounts of fungal secretory enzyme solutions with commercial cellulase can significantly improve the degradation efficiency of lignocellulose. Therefore, developing fungal crude enzyme solutions obtained through fermentation on inexpensive culture media to partially or completely replace expensive commercial enzyme preparations has become an important research direction for reducing the cost of lignocellulose saccharification.

[0007] In summary, how to select appropriate enzyme sources, design reasonable action sequences, and match precise reaction conditions so that lignin-degrading enzymes and hemicellulose-degrading enzymes can act efficiently on their respective targets in sequence, thereby achieving synergistic disintegration of the lignocellulose complex structure of corn stalks, while avoiding enzyme activity inhibition and carbohydrate loss, is a long-standing technical problem in this field that has not yet been effectively solved. Summary of the Invention

[0008] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0009] Another objective of this invention is to provide a highly efficient saccharification method for corn stalks by synergistic pretreatment with elm yellow and white ginseng fermentation enzyme solutions. This method involves stepwise synergistic pretreatment with elm yellow and white ginseng fermentation enzyme solutions, which simultaneously weakens the dual barriers of lignin and hemicellulose, resulting in a reduction sugar yield that is more than doubled compared to saccharification with single cellulase. This method is green and efficient. Furthermore, by using crude enzyme solution from fungal fermentation to replace expensive commercial laccase and xylanase, the cost of enzyme preparations is significantly reduced while achieving a better saccharification effect.

[0010] To achieve these objectives and other advantages according to the present invention, a highly efficient saccharification method for corn straw synergistically pretreatment with elm extract and white ginseng fermentation enzyme solution is provided, comprising: S1. Corn stalks and elm yellow fermentation enzyme solution were subjected to the first stage of enzymatic treatment for 12-24 h under suitable laccase conditions. S2. Continue to add white ginseng fermentation enzyme solution to the system of step S1 and carry out the second stage of enzymatic treatment for 24-48 h under suitable xylanase conditions; S3. Continue to add cellulase to the system of step S2 and carry out saccharification reaction for 48-120 h under suitable cellulase conditions to obtain saccharified product. The laccase activity of the elm yellow fermentation enzyme solution is 5-14 U / mL, the xylanase activity of the white ginseng fermentation enzyme solution is 0.75-1.7 U / mL, and the cellulase activity is 0.4-1.2 U / mL.

[0011] Preferably, in step S1, the suitable conditions for the laccase to act are in an acetate-sodium acetate buffer solution with a pH of 4.2-4.8 and a concentration of 0.05-0.2 mol / L, and the temperature is adjusted to 28-32℃; in step S2, the suitable conditions for the xylanase to act are a temperature of 35-40℃; and in step S3, the suitable conditions for the cellulase to act are a temperature of 48-52℃.

[0012] In one specific implementation, the method includes: S1. Corn stalks and elm yellow fermentation enzyme solution were subjected to the first stage of enzymatic treatment at 28-32℃ for 12-24 h in an acetate-sodium acetate buffer solution with a pH of 4.2-4.8 and a concentration of 0.05-0.2 mol / L. S2. Continue to add white ginseng fermentation enzyme solution to the system in step S1 and adjust the temperature to 35~40℃, and carry out the second stage of enzymatic treatment for 24~48 h. S3. Continue to add cellulase to the system of step S2 and adjust the temperature to 48~52℃, and carry out the saccharification reaction for 48~120 h to obtain the saccharified product. Preferably, the mass-to-volume ratio of the dry weight of corn stalks to the total volume of the system is 0.1~0.2 g / mL.

[0013] Preferably, in step S1, the fermentation enzyme solution of *Elm rhizome* is obtained by inoculating the seed liquid of *Elm rhizome* strain into the enzyme culture medium at an inoculation rate of 10%, and then centrifuging after culturing at 24-28℃ for 13-17 days; in step S2, the fermentation enzyme solution of *Gynostemma pentaphyllum* is obtained by inoculating the seed liquid of *Gynostemma pentaphyllum* strain into the enzyme culture medium at an inoculation rate of 10%, and then centrifuging after culturing at 24-28℃ for 13-17 days.

[0014] Preferably, the *Ulmus pumila* strain seed solution is obtained by inoculating *Ulmus pumila* strain into seed culture medium and culturing at 24-28℃ for 6-8 days. The inoculation amount of *Ulmus pumila* strain is 3 bacterial discs on 50mm×50mm PDA plates per 100 mL of seed culture medium. The *Gynostemma pentaphyllum* strain seed solution is obtained by inoculating *Gynostemma pentaphyllum* strain into the same seed culture medium and culturing at 24-28℃ for 6-8 days. The inoculation amount of *Gynostemma pentaphyllum* strain is 3 bacterial discs on 50mm×50mm PDA plates per 100 mL of seed culture medium.

[0015] Preferably, the enzyme culture medium comprises 8-12 g / L corn stalks, 4-6 g / L wheat bran, and 4-6 g / L soybean peptone, wherein the corn stalks and wheat bran are crushed and passed through a 40-60 mesh sieve; the seed culture medium comprises 2-4 g / L soybean peptone and 20-40 g / L malt extract powder.

[0016] Preferably, the sterilization conditions for both the enzyme solution culture medium and the seed culture medium are 121°C for 15-25 min.

[0017] Preferably, steps S1, S2 and S3 are all performed under oscillation conditions with an oscillation speed of 200-300 rpm; the preparation of the *Ulmus pumila* strain seed liquid, *Gynostemma pentaphyllum* strain seed liquid, *Ulmus pumila* fermentation enzyme liquid and *Gynostemma pentaphyllum* fermentation enzyme liquid are all performed under oscillation conditions with an oscillation speed of 120-180 rpm.

[0018] Preferably, in step S1, the corn stalks are crushed and passed through a 40-60 mesh sieve before use, or the corn stalks are acid-exploded or steam-exploded before use. The acid-explosion treatment involves pre-steaming the corn stalks with 1% dilute sulfuric acid in a horizontal reactor at 100-110°C for 10-30 minutes, followed by steam explosion at 180-190°C for 1-3 minutes. The steam pressure of the steam explosion treatment is 1.1-1.2 MPa, the treatment time is 3-5 minutes, and the moisture content of the material after treatment is 8-12%.

[0019] This invention further claims protection for a corn stalk saccharification product, which is prepared by a highly efficient saccharification method using corn stalks as a pretreatment agent of elm extract and white ginseng fermentation enzyme solution. The reducing sugar content in the corn stalk saccharification product is not less than 300 mg / g dry weight of corn stalks.

[0020] The present invention has at least the following beneficial effects: Firstly, this invention uses a step-by-step synergistic pretreatment with elm yellow fermentation enzyme liquid and white ginseng fermentation enzyme liquid to first break down the lignin barrier and then disintegrate hemicellulose, avoiding the inhibition of enzyme activity caused by simultaneous addition. Ultimately, the reducing sugar yield is no less than 300 mg / g dry weight of corn stalks, which is 1.94 times higher than the saccharification efficiency of single cellulase. Secondly, this invention uses an inexpensive culture medium (8-12 g / L corn stalks, 4-6 g / L wheat bran, 4-6 g / L soybean peptone) prepared from corn stalks, wheat bran, and soybean peptone to directly ferment *Ulmus pumila* and *Panax ginseng* strains, which can yield crude enzyme stock solution with laccase activity of 120-170 U / mL and xylanase activity of 90-100 U / mL, significantly reducing the production cost of enzyme preparations and facilitating industrial scale-up applications. Thirdly, this invention cultivates seed liquid and fermentation enzyme liquid by oscillation at 120-180 rpm, performs pretreatment and saccharification reaction by oscillation at 200-300 rpm, and controls corn straw to pass through a 40-60 mesh sieve, which greatly improves the mass transfer efficiency between enzyme and substrate, making the three-step enzymatic hydrolysis reaction more complete and stable, and the process has good reproducibility. Fourth, this invention directly utilizes the crude enzyme solution obtained by fermenting *Euonymus alatus* and *Gynostemma pentaphyllum* strains in inexpensive culture media (corn stalks, wheat bran, soybean peptone). No purification steps are required before pretreatment, significantly simplifying the process and reducing enzyme preparation costs. More importantly, the crude enzyme solution is not a single enzyme component but retains the complete natural enzyme spectrum produced during fermentation. In addition to the target laccase, the *Euonymus alatus* fermentation broth contains trace amounts of peroxidase and other redox coenzymes. In addition to xylanase, the *Gynostemma pentaphyllum* fermentation broth is rich in hemicellulose side-chain degrading enzymes such as β-glucanase and mannanase. This natural complex enzyme system can simultaneously attack multiple chemical bonds in the lignin-carbohydrate complex, achieving multi-target synergistic disintegration of the substrate barrier. The obtained corn saccharification product has a high reducing sugar content (≥300 mg / g dry weight) and can be directly used as a carbon source for microbial fermentation to produce biofuels (such as fuel ethanol) or bio-based chemicals (such as lactic acid and succinic acid), realizing the high-value resource utilization of agricultural waste and demonstrating significant economic and environmental benefits.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0022] Figure 1 This is a graph showing the reducing sugar yield of the corn stalks crushed according to the present invention; Figure 2 This is a graph showing the reducing sugar yield of the corn stalks subjected to acid explosion treatment according to the present invention; Figure 3 This is a graph showing the reducing sugar yield of corn stalks treated by steam explosion according to the present invention; Figure 4 This is a standard curve of reducing sugar described in one embodiment of the present invention. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can implement it based on the description.

[0024] Example A (Crushing) Preparation of enzyme culture medium: Take 10 g / L corn stalks, 5 g / L wheat bran, and 5 g / L soybean peptone to make 100 mL of enzyme culture medium, and place it in a 250 mL Erlenmeyer flask. Sterilize at 121℃ for 20 min, and set aside for later use; the corn stalks and wheat bran are crushed and passed through a 50-mesh sieve before use.

[0025] Preparation of seed culture medium: Take 30 g / L malt extract powder and 3 g / L soybean peptone to make 100 mL of seed culture medium, place it in a 250 mL Erlenmeyer flask, sterilize at 121℃ for 20 min, and set aside for later use.

[0026] Preparation of *Ulmus pumila* fermentation enzyme solution: *Ulmus pumila* strain (purchased from Beijing Academy of Agricultural and Forestry Sciences) was inoculated into the above-mentioned seed culture medium in a clean bench and cultured at 27℃ and 150 rpm for 7 days to obtain *Ulmus pumila* strain seed solution. The inoculation amount of *Ulmus pumila* strain was 3 bacterial discs from 50mm×50mm PDA plates per 100mL of seed culture medium. The *Ulmus pumila* strain seed solution was inoculated into the enzyme solution culture medium at a 10% inoculation rate and cultured at 27℃ and 150 rpm for 15 days. The *Ulmus pumila* fermentation enzyme solution was then obtained by centrifugation at 4℃ and 10000rpm for later use. The types and contents of enzymes in the obtained *Ulmus pumila* fermentation broth were detected, and the enzyme types and contents are shown in Table 1.

[0027] Table 1. Enzyme types and contents (U / mL) in elm husk fermentation broth Note: Fpase, filter paper enzyme; CMCase, carboxymethyl cellulase; β-Glu, glucanase; Man, mannanase; Xyl, xylanase; Lac, laccase; VP, multifunctional peroxidase; MnP, manganese peroxidase.

[0028] Table 1 shows that the enzyme composition of the *Elaeagnus pungens* fermentation broth underwent significant changes during cultivation. Lacase activity surged from 0.97 U / mL to 30.81 U / mL in the initial cultivation period (days 0-3), and continued to increase to 168.51 U / mL by day 15, indicating that the *Elaeagnus pungens* strain is a highly efficient laccase producer. Simultaneously, xylanase activity increased from 0.064 U / mL to 0.597 U / mL in days 0-3, then stabilized in the range of 0.39-0.60 U / mL, while the activities of cellulase-related enzymes (Fpase, CMCase) and manganese peroxidase (MnP) remained at extremely low levels. Overall, laccase was the dominant enzyme in the *Elaeagnus pungens* fermentation broth, with other auxiliary enzymes exhibiting weak activity. This provides a clear enzymatic basis for its subsequent use as a pretreatment agent for lignin degradation, and also indicates that the fermentation broth is not a broad-spectrum hydrolytic enzyme system, but rather possesses highly selective function.

[0029] Preparation of ginseng fermentation enzyme solution: In a clean bench, the ginseng strain (gifted by Guizhou Bijie Nongtou Fungi Technology Co., Ltd.) was inoculated into the seed culture medium and cultured at 27℃ and 150 rpm for 7 days to obtain the ginseng strain seed solution. The inoculation amount of the ginseng strain was 3 pieces of 50mm×50mm PDA plate per 100 mL of seed culture medium. The ginseng strain seed solution was inoculated into the enzyme solution culture medium at a 10% inoculation amount and cultured at 27℃ and 150 rpm for 15 days. After that, the ginseng fermentation enzyme solution was obtained by centrifugation at 4℃ and 10000 rpm for later use. The types and contents of enzymes in the obtained ginseng fermentation broth were detected and are shown in Table 2.

[0030] Table 2. Enzyme types and contents (U / mL) in the fermentation broth of white ginseng Note: Fpase, filter paper enzyme; CMCase, carboxymethyl cellulase; β-Glu, glucanase; Man, mannanase; Xyl, xylanase; Lac, laccase; VP, multifunctional peroxidase; MnP, manganese peroxidase.

[0031] Table 2 shows that the succession characteristics of the enzyme system composition in the *Gynostemma pentaphyllum* fermentation broth during cultivation were distinctly different from those in the *Elm Yellow* fermentation broth: Lacase activity reached a high of 29.92 U / mL at the initial stage of cultivation (day 0), but then plummeted to 7.69 U / mL within the next two days, reaching an extremely low level of 0.29 U / mL by day 3, and remained at this low level for a long period, indicating a rapid decline in laccase secretion capacity of the *Gynostemma pentaphyllum* strain under shake-flask culture conditions. In stark contrast, xylanase (Xyl) activity steadily increased from 0.49 U / mL at the initial stage of cultivation, reaching 49.35 U / mL on day 6, and further increasing to 101.58 U / mL on day 15, becoming the absolutely dominant enzyme in the fermentation broth. Simultaneously, β-glucanase (β-Glu) activity also increased from 0.17 U / mL... The activity of xylanase (Man) steadily increased to 5.33 U / mL on day 12, while the activity of cellulase main enzymes (Fpase, CMCase) and peroxidases (VP, MnP) remained at extremely low levels (mostly below 0.07 U / mL). Overall, the fermentation broth of white ginseng is a hemicellulose-degrading enzyme system with xylanase as the core and containing β-glucanase and mannanase. Laccase activity almost disappeared in the later stage. This enzyme profile of high xylanase, low laccase, and low cellulase precisely illustrates its applicability as a hemicellulose-directed degradation pretreatment agent. It complements the high laccase characteristics of elm yellow fermentation broth, providing direct enzymatic support for the sequential design of the subsequent stepwise synergistic pretreatment strategy of breaking down lignin first and then degrading hemicellulose.

[0032] Saccharification of corn stalks (sequential fermentation treatment with elm extract, white ginseng, and cellulase, referred to as Pc+Sc+Cel group): 300 mg of dried, pulverized corn stalks (passed through a 50-mesh sieve) was used as the reaction substrate. 2.85 mL of 0.1 mol / L acetate buffer was added to the corn stalk substrate, and the pH was adjusted to 4.8 using 6 M NaOH. Then, 0.125 mL of the elm extract fermentation enzyme solution was added, and the mixture was shaken at 30℃ and 250 rpm for 24 h. The laccase activity of the elm extract fermentation enzyme solution was 5-7 U / mL. Next, 0.025 mL of the white ginseng fermentation enzyme solution was added, and the mixture was shaken at 37℃ and 250 rpm for 24 h. The xylanase activity of the white ginseng fermentation enzyme solution was 0.75-0.85 U / mL. Finally, 0.006 mL of cellulase (purchased from Novozymes Cellic CTec3 HS) was added and the mixture was shaken at 50℃ and 250 rpm. The saccharified product was obtained by shaking the mixture under the specified conditions for 48 h. The cellulase activity in the system was 0.4~0.6 U / mL.

[0033] Example B (Acid Explosion) The preparation of enzyme solution culture medium, seed culture medium, elm yellow fermentation enzyme solution, and white ginseng fermentation enzyme solution are all the same as in Example A.

[0034] Saccharification of corn stalks (sequential fermentation treatment with elm yellow, white ginseng, and cellulase, abbreviated as Pc+Sc+Cel group): Corn stalks were first pre-steamed in a horizontal reactor with 1% dilute sulfuric acid at 110℃ for 20 min, followed by steam explosion at 185℃ for 2 min. 600 mg of the acid-exploded corn stalks was used as the reaction substrate. 2.69 mL of 0.1 mol / L acetate buffer was added to the corn stalk reaction substrate, and the pH was adjusted to 4.8 using 6 M NaOH. Then, 0.25 mL of the elm yellow fermentation enzyme solution was added, and the mixture was shaken at 30℃ and 250 rpm for 24 h. The laccase activity of the elm yellow fermentation enzyme solution in the system was 10–14 U / mL. Finally, 0.05 mL of the white ginseng fermentation enzyme solution was added, and the mixture was shaken at 37℃ and 250 rpm for 24 h. The xylanase activity of the white ginseng fermentation enzyme solution in the system was 1.5–1.7. U / mL; finally, add 0.012 mL of cellulase and shake at 50℃ and 250 rpm for 72 h to obtain the saccharified product. The enzyme activity of cellulase in the system is 0.8~1.2 U / mL.

[0035] Example C (Steam Explosion) The preparation of enzyme solution culture medium, seed culture medium, elm yellow fermentation enzyme solution, and white ginseng fermentation enzyme solution are all the same as in Example A.

[0036] Saccharification of corn stalks (sequential fermentation treatment with elm yellow, white ginseng, and cellulase, referred to as Pc+Sc+Cel group): After steam explosion of corn stalks at 1.18 MPa for 3.5 min, the moisture content of the corn stalks was 10%. 300 mg of the steam-exploded corn stalks was used as the reaction substrate. 2.85 mL of 0.1 mol / L acetate buffer was added to the corn stalk reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH. Then, 0.125 mL of the elm yellow fermentation enzyme solution was added, and the mixture was shaken at 30℃ and 250 rpm for 24 h. The laccase activity of the elm yellow fermentation enzyme solution in the system was 5-7 U / mL. Next, 0.025 mL of the white ginseng fermentation enzyme solution was added, and the mixture was shaken at 37℃ and 250 rpm for 24 h. The xylanase activity of the white ginseng fermentation enzyme solution in the system was 0.75-0.85 U / mL. Finally, 0.006 mL of cellulase was added and the mixture was shaken at 50℃ and 250 rpm for 24 h. The saccharified product was obtained by shaking at rpm for 72 h, and the cellulase activity in the system was 0.4~0.6 U / mL.

[0037] Compared to Group A (fragmented) The preparation of enzyme solution culture medium, seed culture medium, elm yellow fermentation enzyme solution, and white ginseng fermentation enzyme solution are all the same as in Example A.

[0038] Saccharification of corn stalks: 300 mg of dried, pulverized corn stalks that had passed through a 50-mesh sieve were used as the reaction substrate, and the following saccharification operations were performed: A1 (Elm Yellow Fermentation Treatment Only, abbreviated as Pc group): 2.875 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH; then 0.125 mL of the elm yellow fermentation enzyme solution was added, and the mixture was shaken at 30℃ and 250 rpm for 24 h to obtain the saccharification product. The laccase activity of the elm yellow fermentation enzyme solution in the system was 5~7 U / mL.

[0039] A2 (White ginseng fermentation treatment only, referred to as Group Sc): 2.975 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH. Then, 0.025 mL of the white ginseng fermentation enzyme solution was added, and the mixture was shaken at 37℃ and 250 rpm for 24 h to obtain the xylanase activity of the white ginseng fermentation enzyme solution in the saccharification product system, which was 0.75~0.85 U / mL.

[0040] A3 (cellulase fermentation treatment only, abbreviated as Cel group): 2.994 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, the pH was adjusted to pH 4.8 using 6 M NaOH, 0.006 mL of cellulase was added, and the mixture was shaken at 50℃ and 250 rpm for 48 h to obtain the saccharification product. The enzyme activity of cellulase in the system was 0.4~0.6 U / mL.

[0041] A4 (simultaneous fermentation treatment of elm yellow, white ginseng, and cellulase, abbreviated as Pc+Sc+Cel (simultaneous addition) group): 2.85 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH. To facilitate temperature control, after 48 h, 0.125 mL of the elm yellow fermentation enzyme solution, 0.025 mL of the white ginseng fermentation enzyme solution, and 0.006 mL of cellulase were added simultaneously, and the mixture was shaken at 50℃ and 250 rpm for 48 h to obtain the saccharification product. The laccase activity of the elm yellow fermentation enzyme solution in the system was 5~7 U / mL, the xylanase activity of the white ginseng fermentation enzyme solution in the system was 0.75~0.85 U / mL, and the cellulase activity in the system was 0.4~0.6 U / mL.

[0042] Compared to Group B (sour and explosive) The preparation of enzyme solution culture medium, seed culture medium, elm yellow fermentation enzyme solution, and white ginseng fermentation enzyme solution are all the same as in Example A.

[0043] Saccharification of corn stalks: Corn stalks were first pre-steamed in a horizontal reactor with 1% dilute sulfuric acid at 110℃ for 20 min, followed by steam explosion at 185℃ for 2 min. 600 mg of the acid-exploded corn stalks was taken as the reaction substrate and the following saccharification operations were performed: B1 (Elm Yellow Fermentation Treatment Only, abbreviated as Pc group): 2.75 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH; then 0.25 mL of the elm yellow fermentation enzyme solution was added, and the mixture was shaken at 30℃ and 250 rpm for 24 h to obtain the saccharification product. The laccase activity of the elm yellow fermentation enzyme solution in the system was 10~14 U / mL.

[0044] B2 (White ginseng fermentation treatment only, referred to as group Sc): 2.95 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH. Then, 0.05 mL of the white ginseng fermentation enzyme solution was added, and the mixture was shaken at 37℃ and 250 rpm for 24 h to obtain the xylanase activity of the white ginseng fermentation enzyme solution in the saccharification product system, which was 1.5~1.7 U / mL.

[0045] B3 (cellulase fermentation treatment only, abbreviated as Cel group): 2.988 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, the pH was adjusted to pH 4.8 using 6 M NaOH, 0.012 mL of cellulase was added, and the mixture was shaken at 50℃ and 250 rpm for 48 h to obtain the saccharification product. The enzyme activity of cellulase in the system was 0.8~1.2 U / mL.

[0046] B4 (simultaneous fermentation treatment of elm yellow, white ginseng, and cellulase, abbreviated as Pc+Sc+Cel (synchronous addition) group): 2.69 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH. To facilitate temperature control, after a 48-h blank waiting period (without adding any enzyme to activate the enzymatic reaction), 0.25 mL of the elm yellow fermentation enzyme solution, 0.05 mL of the white ginseng fermentation enzyme solution, and 0.012 mL of cellulase were added simultaneously. The mixture was shaken at 50℃ and 250 rpm for 72 h to obtain the saccharification product. The laccase activity of the elm yellow fermentation enzyme solution in the system was 10~14 U / mL, the xylanase activity of the white ginseng fermentation enzyme solution in the system was 1.5~1.7 U / mL, and the cellulase activity in the system was 0.8~1.2 U / mL.

[0047] Compared to Group C (steam explosion) The preparation of enzyme solution culture medium, seed culture medium, elm yellow fermentation enzyme solution, and white ginseng fermentation enzyme solution are all the same as in Example A.

[0048] Saccharification of corn stalks: After steam explosion of corn stalks at 1.18 MPa for 3.5 min, the moisture content of the corn stalks was 10%. 300 mg of the steam-exploded corn stalks was used as the reaction substrate, and the following saccharification operations were performed: C1 (Elm Yellow Fermentation Treatment Only, abbreviated as Pc group): 2.875 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH; then 0.125 mL of the elm yellow fermentation enzyme solution was added, and the mixture was shaken at 30℃ and 250 rpm for 24 h to obtain the saccharification product. The laccase activity of the elm yellow fermentation enzyme solution in the system was 5~7 U / mL.

[0049] C2 (White ginseng fermentation treatment only, abbreviated as Sc group): 2.975 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH. Then, 0.025 mL of the white ginseng fermentation enzyme solution was added, and the mixture was shaken at 37℃ and 250 rpm for 24 h to obtain the xylanase activity of the white ginseng fermentation enzyme solution in the saccharification product system, which was 0.75~0.85 U / mL.

[0050] C3 (cellulase fermentation treatment only, abbreviated as Cel group): 2.994 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, the pH was adjusted to pH 4.8 using 6 M NaOH, 0.006 mL of cellulase was added, and the mixture was shaken at 50℃ and 250 rpm for 48 h to obtain the saccharification product. The enzyme activity of cellulase in the system was 0.4~0.6 U / mL.

[0051] C4 (simultaneous fermentation treatment of elm yellow, white ginseng, and cellulase, abbreviated as Pc+Sc+Cel (simultaneous addition) group): 2.85 mL of 0.1 mol / L acetate buffer was added to the corn straw reaction substrate, and the pH was adjusted to pH 4.8 using 6 M NaOH. To facilitate temperature control, after 48 h, 0.125 mL of the elm yellow fermentation enzyme solution, 0.025 mL of the white ginseng fermentation enzyme solution, and 0.006 mL of cellulase were added simultaneously, and the mixture was shaken at 50℃ and 250 rpm for 72 h to obtain the saccharification product. The laccase activity of the elm yellow fermentation enzyme solution in the system was 5~7 U / mL, the xylanase activity of the white ginseng fermentation enzyme solution in the system was 0.75~0.85 U / mL, and the cellulase activity in the system was 0.4~0.6 U / mL.

[0052] The yield of reducing sugars at each stage of fermentation in Examples A-C and Control Groups A-C was determined using DNS reagent. The results are shown in Tables 3-5. Figures 1-3 As shown.

[0053] The method for determining enzyme activity units in this application is as follows: Laccase activity was determined spectrophotometrically, monitoring the oxidation of ABTS to green cationic free radicals (ABTS). +The reaction was measured at 420 nm, and the change in absorbance (molar absorptivity ε) was determined. 420 = 36,000 M -1 cm -1 Add 50 μL of appropriately diluted fermentation supernatant to a mixture of 50 μL of 1 mM ABTS and 100 μL of acetate buffer (0.1 mM, pH 4.5) to prepare a 200 μL reaction mixture. Measure the change in absorbance at 30 °C and 420 nm. One unit of enzyme activity (U / mL) is defined as the amount of laccase that oxidizes 1 μmol of ABTS per minute from ABTS.

[0054] Using beech wood xylan solution as a substrate, 0.08 mL of 100 mg / mL beech wood xylan solution (pH 4.5, 0.1 mol / L acetate-sodium acetate buffer) was added, followed by 0.08 mL of a suitable diluted fermentation enzyme solution. The reaction mixture was incubated at 37 °C for 30 min. After the reaction, 200 μL of dinitrosalicylic acid (DNS) was added, and the mixture was incubated at 100 °C for 10 min for color development. After color development, water was added to a final volume of 1 mL, and 200 μL was added to a 96-well plate. The absorbance was measured at 540 nm to determine the amount of reducing sugar. One unit of enzyme activity (U / mL) is defined as the amount of enzyme required to release 1 μmol of xylose per minute.

[0055] Conditions for reducing sugar determination: Construction of the standard curve: Accurately prepare a 10 g / L glucose standard stock solution. Pipette 0, 40, 60, 80, 120, 160, and 200 μL of the stock solution into separate test tubes, and bring the volume to 1000 μL with distilled water, resulting in glucose concentrations of 0, 0.4, 0.6, 0.8, 1.2, 1.6, and 2.0 g / L, respectively. Take 100 μL of each concentration solution and add 200 μL of DNS reagent to each tube. After thorough mixing by shaking, incubate in a boiling water bath for 5 min, then rapidly cool and measure the OD. 540nm The absorbance values ​​are plotted on the x-axis as glucose concentration (g / L) and absorbance values ​​(OD). 540nm Plot a standard curve using the ordinate as an example. Figure 4 As shown.

[0056] After thoroughly vortexing the sample, centrifuge at 10,000 rpm for 10 min at room temperature. Collect the supernatant, dilute appropriately, and then mix 100 μL of the diluted sample (depending on the reducing sugar concentration) with 200 μL of DNS. Incubate in a boiling water bath for 5 min, then rapidly cool and measure the OD. 540nmThe absorbance was measured. Simultaneously, the amount of reducing sugar in the enzyme solution and substrate was determined, and interference was subtracted. Specific procedures: Enzyme solution control experiment: After thoroughly shaking and mixing the diluted enzyme solution, centrifuge at 10000 rpm for 10 min at room temperature. Collect the supernatant, take 100 μL and mix thoroughly with 200 μL of DNS, incubate in a boiling water bath for 5 min, and then rapidly cool. The OD value was then measured. 540nm The absorbance value was used to determine the specific reducing sugar content. Substrate control experiment: Corn stalk substrate was thoroughly mixed with buffer solution and centrifuged at 10000 rpm for 10 min at room temperature. The supernatant was collected, appropriately diluted, and 100 μL of the diluted sample was thoroughly mixed with 200 μL of DNS. The mixture was then incubated in a boiling water bath for 5 min, rapidly cooled, and the OD value was measured. 540nm The absorbance value is used to obtain the specific amount of reducing sugar.

[0057] Table 3 Reducing sugar content in corn stalk crushing treatment group Note: The synergy coefficient DS is calculated by dividing the reducing sugar yield from the combined enzymatic hydrolysis of the three enzymes by the ratio of (reducing sugar yield from the fermentation enzyme of elm yellow alone + reducing sugar yield from the fermentation enzyme of white ginseng alone + reducing sugar yield from the fermentation enzyme of cellulase alone).

[0058] Table 4 Reducing sugar content in corn stalk acid-explosion treatment group Note: The calculation of the synergy coefficient DS is the same as above.

[0059] Table 5 Reducing sugar content in corn stalk steam explosion treatment group Note: The calculation of the synergy coefficient DS is the same as above.

[0060] Combine Tables 3-5 and Figures 1-3A three-step sequential synergistic treatment group (Pc+Sc+Cel group) using elm yellow fermentation enzyme solution, white ginseng fermentation enzyme solution, and cellulase showed the best saccharification effect under different pretreatment methods. In the three groups of experiments (crushing, acid explosion, and steam explosion), the final reducing sugar yield of the sequential synergistic treatment group was the highest in its respective group, indicating that the three-step sequential synergistic treatment of elm yellow fermentation enzyme solution, white ginseng fermentation enzyme solution, and cellulase has universality. Measured by reducing sugar yield, steam explosion pretreatment caused the most thorough destruction of the substrate structure. The Pc+Sc+Cel group achieved a maximum reducing sugar yield of 527.56 mg / g DW at 120 h, significantly higher than the 353.31 mg / g DW of the crushing group and the 322.90 mg / g DW of the acid explosion group. From the perspective of the synergistic coefficient DS, the synergistic effect was most prominent in the crushing and acid explosion groups, with DS values ​​reaching as high as 1.84 and 1.98 respectively, showing a significant synergistic effect. However, in the steam explosion group, due to the already high substrate accessibility and strong single-enzyme effect when cellulase was used alone, the DS value failed to exceed 1, indicating a relatively limited amplification of the synergistic effect. In each Pc+Sc+Cel group treatment, the synergistic coefficient DS showed an overall trend of first increasing and then decreasing with the extension of reaction time, mostly reaching a peak around 72 h, and then gradually declining. This indicates that the synergistic effect was most significant in the middle of the reaction, and the apparent synergistic effect naturally weakened in the later stages of the reaction as the amount of substrate available for synergistic utilization decreased and the reducing sugar in the single-enzyme control group continued to accumulate.

[0061] In the single-enzyme control group, the data from the elm yellow fermentation enzyme solution treatment and the white ginseng fermentation enzyme solution treatment alone showed distinctly different patterns. The elm yellow single-enzyme group almost consistently followed a trajectory of initial increase followed by decrease in reducing sugar under all pretreatment conditions, typically reaching a peak around 24 hours and then continuously declining until reaching a low level at 96 or 120 hours. This confirms that residual microorganisms or metabolic enzyme systems in the crude fermentation enzyme solution could consume the released reducing sugar in the later stages. In contrast, the white ginseng fermentation enzyme single-enzyme group exhibited a slow and stable reduction in reducing sugar release, with yields consistently low, even in the later stages of the reaction, indicating that while the secreted hemicellulase had limited activity, it could still continuously and stably hydrolyze the substrate. As for the cellulase single-enzyme group, its final saccharification efficiency was highly dependent on the pretreatment intensity. The lowest sugar yield was observed with only crushing treatment, significantly improved after acid explosion treatment, and reached the highest level among the single-enzyme groups after steam explosion treatment. This fully demonstrates that physicochemical pretreatment mainly improves the accessibility of cellulase to the substrate by opening the fiber structure.

[0062] Comparing the sequential synergistic treatment group (Pc+Sc+Cel group) with the simultaneous addition group (Pc+Sc+Cel (simultaneous addition) group), the data clearly demonstrate the superiority of the stepwise treatment strategy. In the comparative experiments of acid explosion and steam explosion, the final reducing sugar yield of the Pc+Sc+Cel group was significantly higher than that of the simultaneous treatment group, and the DS value of the sequential group was also generally higher than that of the Pc+Sc+Cel (simultaneous addition) group. Taking 120h data as an example, the sequential group was about 14.7% higher than the simultaneous group under acid explosion conditions, and about 20.4% higher under steam explosion conditions. Simultaneous addition of laccase, xylanase, and cellulase not only makes it difficult to achieve optimal temperatures for each enzyme, but some enzymes also experience decreased activity or even inactivation due to unsuitable temperatures. At the same time, the interaction processes of each enzyme with the substrate can also affect each other, thereby reducing substrate utilization efficiency. Stepwise enzymatic hydrolysis allows for the regulation of appropriate reaction conditions at different stages. First, laccase reduces the lignin barrier, then xylanase removes hemicellulose, and finally cellulase hydrolyzes cellulose. This allows each enzyme to function under more suitable substrate and reaction conditions, thereby improving overall degradation efficiency. Furthermore, longitudinal observation over time reveals that during the first 48 hours of pretreatment with elm yellow fermentation enzymes and white ginseng fermentation enzymes, the reducing sugar yields in each group were low and may have fluctuated slightly or even decreased. A sharp increase in reducing sugars only occurred after the addition of commercial cellulase. This indicates that the true value of structural modification of the fermentation enzyme solutions from these two fungi lies not in the direct sugar release in the early stages, but in creating conditions for efficient hydrolysis by subsequent cellulase.

[0063] In summary, the three sets of experimental data clearly demonstrate that the three-step sequential synergistic treatment strategy of elm yellow fermentation enzyme, white ginseng fermentation enzyme, and cellulase established in this application has a significant and universal effect on improving the saccharification efficiency of corn straw. Firstly, in terms of reducing sugar yield, the Pc+Sc+Cel group achieved the highest level under all pretreatment conditions. Especially after steam explosion pretreatment, the reducing sugar yield reached 527.56 mg / g DW after 120 h, maximizing the release of fermentable sugars in corn straw and providing an efficient sugar platform for the high-value conversion of agricultural waste into biofuels or bio-based chemicals. Secondly, the synergistic coefficient DS data further reveals the value of the stepwise addition sequence designed in this invention. The peak DS values ​​for the crushing group and the acid explosion group reached 1.84 and 1.98 respectively, both far exceeding 1. This strongly proves that the sequential arrangement of first breaking down lignin, then decomposing hemicellulose, and finally hydrolyzing cellulose is not a simple superposition of enzymes, but rather produces a true synergistic effect, making the final saccharification efficiency far higher than the sum of the individual effects of the three enzymes. Comparative data from the Pc+Sc+Cel (simultaneously added) group and the Pc+Sc+Cel group under acid explosion and steam explosion conditions highlight the irreplaceable nature of the stepwise synergistic strategy of this invention. In the acid explosion group, the final reducing sugar yield of the Pc+Sc+Cel group was 322.90 mg / g DW, which was approximately 14.7% higher than the 281.43 mg / g DW of the Pc+Sc+Cel (simultaneously added) group; in the steam explosion group, the Pc+Sc+Cel group was approximately 20.4% higher than the Pc+Sc+Cel (simultaneously added) group. This comparative result indicates that simply adding the three enzymes simultaneously can significantly reduce synergistic efficiency due to problems such as enzyme activity inhibition or substrate competition. The stepwise addition required by this invention is the key to avoiding these problems and achieving efficient saccharification. Furthermore, the change pattern of reducing sugar in the single-enzyme control group also reflects another effect of the method of the present invention: the pretreatment stage of elm yellow and white ginseng is not aimed at direct sugar release, but rather clears obstacles for subsequent cellulase by oxidatively modifying lignin and hydrolyzing hemicellulose. This effectively avoids the drawback of long-term growth of microorganisms consuming fermentable sugars in traditional fungal pretreatment, so that the carbohydrates in the substrate can be retained to the maximum extent until the saccharification stage and then released in a concentrated manner, realizing the efficient preservation and directional conversion of carbon sources.

[0064] Comparative Example D The method disclosed in the invention patent CN102191299A, "A Method for Improving the Saccharification Yield of Lignocellulose through Multi-Step Enzymatic Hydrolysis," is used for the saccharification of corn straw. The specific method is as follows: Corn stalks treated with 0.4% sulfuric acid at 150℃ for 30 min were subjected to enzymatic hydrolysis in two or three steps. The first step hydrolysis conditions were: substrate concentration 8%, addition of laccase 25 U / g (produced by Dalian Inster Biotechnology Co., Ltd.), and reaction at pH 4.8 and 65℃ for 8 h. The cellulose residue after the first step hydrolysis was then subjected to a second step hydrolysis with xylanase 650 U / g. The hydrolysis conditions were: pH 4.8, 50℃, and reaction time 40 h. After the second hydrolysis, the hemicellulose hydrolysate was obtained by filtration. The remaining residue was then subjected to a third step hydrolysis. The third step hydrolysis conditions were: substrate concentration 8%, addition of cellulase 370 FPU / g and cellobiase 20 IU / g (produced by Novozymes), pH adjusted to 4.8 using a citrate-sodium citrate buffer solution, and reaction continued at 50℃ in a water bath shaker for 48 h. The cellulase hydrolysate was then obtained by filtration. The sum of the reducing sugar yields of hemicellulose and cellulose was 29.46% (to the substrate). Following the same method, the total reducing sugar yield of the two-step enzymatic hydrolysis without laccase was 26.84%. The yield of the three-step enzymatic hydrolysis with laccase was 9.8% higher than that of the two-step enzymatic hydrolysis using only xylanase and cellulase.

[0065] Comparing the data of Example B with Comparative Example D, it is clear that replacing commercially available laccase and xylanase with the crude enzyme solution from the fermentation of elm yellow and white ginseng resulted in a qualitative leap in saccharification effect: Example B achieved a final reducing sugar yield of 322.90 mg / g corn stalk dry weight (i.e., 32.29%) with only about 1 / 2 the amount of laccase and about 1 / 400 the amount of xylanase compared to the comparative document. This is not only significantly higher than the 29.46% reducing sugar yield in the comparative document, but it was also achieved under the condition of a significant reduction in enzyme dosage, fully demonstrating the superiority and industrial application potential of the combination of crude enzyme solution from the fermentation of elm yellow and white ginseng.

[0066] The reason why the combination of elm yellow and white ginseng fermentation crude enzyme liquid can achieve a higher reducing sugar yield with a smaller amount is due to two factors: firstly, the natural synergy of the complex enzyme system and the additional functions of the crude enzyme liquid. The elm yellow and white ginseng fermentation liquid used in Example B is an unpurified crude enzyme liquid, which not only contains the target enzymes (laccase and xylanase), but also retains a variety of auxiliary enzymes, surface-active substances and natural stabilizing factors produced during fermentation. The elm yellow fermentation liquid also contains trace amounts of manganese peroxidase and multifunctional peroxidase, while the white ginseng fermentation liquid is rich in β-glucanase (4.40 U / mL) and mannanase (1.32 U / mL) in addition to xylanase. The non-target components in the crude enzyme solution are not impurities, but rather form a natural functional complementary network: during lignin oxidation, laccase, MnP, and VP exhibit a synergistic effect in electron transfer, enabling them to jointly attack different types of chemical bonds in lignin; during the white ginseng action stage, while xylanase hydrolyzes the xylan backbone, β-glucanase and mannanase hydrolyze the β-1,3 / 1,4-glucan and mannan components in the hemicellulose side chain, achieving multi-point disintegration of the heterostructure of hemicellulose. The synergistic effect of the multiple enzymes in the crude enzyme system is far superior to that of a single commercially available pure enzyme, as it can simultaneously act on multiple bonds in the lignin-carbohydrate complex (LCC), resulting in a more thorough disintegration of the substrate barrier. Secondly, the precise matching of optimal reaction conditions and the complete preservation of enzyme activity: In Example B, the three-step enzymatic hydrolysis temperatures were 30℃, 37℃, and 50℃, respectively. The three enzymes functioned sequentially within their optimal temperature ranges without interfering with each other. In contrast, the comparative document treated laccase at a high temperature of 65℃, which is not only far higher than the conventional optimal temperature for laccase but also causes a decrease in the activity of xylanase and cellulase before their addition due to changes in the chemical environment of the substrate surface during high-temperature treatment. More importantly, Example B adopted a stepwise heating strategy (30℃→37℃→50℃). The xylanase, β-glucanase, and other auxiliary enzymes in the fermentation broth of elm yellow and white ginseng were gently catalyzed at low temperatures during the pretreatment stage, avoiding enzyme protein denaturation or irreversible structural changes on the substrate surface that might be caused by high-temperature treatment. This ensured that the substrate maintained its optimal pre-modified state when cellulase was finally added, maximizing the catalytic efficiency of cellulase. Thirdly, the pretreatment concept is mild catalysis and structural modification rather than violent degradation: In Comparative Example D, the commercial laccase treatment was only 8 hours at 65°C, which is essentially still a violent single enzymatic hydrolysis process, with the goal of limited lignin removal; while the pretreatment of elm yellow fermentation broth (12~24h, 30°C) and white ginseng fermentation broth (24~48h, 37°C) in this application focuses on mild and long-term structural modification. Laccase generates phenolic oxygen free radicals on the aromatic ring of lignin through a single-electron oxidation mechanism, which triggers mild depolymerization of the lignin network rather than violent degradation; the hemicellulase system in the white ginseng fermentation broth hydrolyzes the hemicellulose backbone and side chains in a gradual manner, gradually opening the wrapping layer around the cellulose microfibrils.This not only avoids the damage to carbohydrates caused by high temperatures and strong chemical conditions, but more importantly, it maximizes the preservation of the accessible surface area of ​​cellulose, allowing subsequent cellulase to achieve the highest saccharification efficiency with the lowest enzyme dosage. In summary, Example B, through multi-target synergy of the crude enzyme solution's natural complex enzyme system, precise stepwise optimal temperature matching, and a mildly modified pretreatment strategy, achieves a breakthrough effect where saccharification efficiency surpasses traditional high-enzyme-dosage methods at extremely low enzyme dosages.

[0067] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A highly efficient saccharification method for corn straw synergistically pretreated with elm extract and white ginseng fermentation enzyme solution, characterized in that, include: S1. The corn stalks and elm yellow fermentation enzyme solution were subjected to the first stage of enzymatic treatment for 12-24 hours under suitable laccase conditions. S2. Continue to add white ginseng fermentation enzyme solution to the system of step S1 and carry out the second stage of enzymatic treatment for 24-48 h under suitable xylanase conditions; S3. Continue to add cellulase to the system of step S2 and carry out saccharification reaction for 48-120 h under suitable cellulase conditions to obtain saccharified product. The laccase activity of the elm yellow fermentation enzyme solution is 5-14 U / mL, the xylanase activity of the white ginseng fermentation enzyme solution is 0.75-1.7 U / mL, and the cellulase activity is 0.4-1.2 U / mL.

2. The efficient saccharification method for corn straw by synergistic pretreatment with elm extract and white ginseng fermentation enzyme solution according to claim 1, characterized in that, In step S1, the suitable conditions for laccase to react are in an acetate-sodium acetate buffer solution with a pH of 4.2-4.8 and a concentration of 0.05-0.2 mol / L, and the temperature is adjusted to 28-32℃; in step S2, the suitable conditions for xylanase to react are a temperature of 35-40℃; and in step S3, the suitable conditions for cellulase to react are a temperature of 48-52℃.

3. The efficient saccharification method for synergistic pretreatment of corn straw by elm yellow and white ginseng fermentation enzyme liquid as described in claim 2, wherein in step S1, the elm yellow fermentation enzyme liquid is obtained by inoculating the elm yellow strain seed liquid into the enzyme liquid culture medium at an inoculation rate of 10%, and then centrifuging after incubation at 24-28℃ for 13-17 days; in step S2, the white ginseng fermentation enzyme liquid is obtained by inoculating the white ginseng strain seed liquid into the enzyme liquid culture medium at an inoculation rate of 10%, and then centrifuging after incubation at 24-28℃ for 13-17 days.

4. The efficient saccharification method for synergistic pretreatment of corn straw by elm yellow and white ginseng fermentation enzyme liquid as described in claim 3, wherein the elm yellow strain seed liquid is obtained by inoculating elm yellow strain into seed culture medium and culturing at 24~28℃ for 6~8 days, and the inoculation amount of elm yellow strain is 3 pieces of mycelium on 50mm×50mm PDA plates per 100 mL of seed culture medium; the white ginseng strain seed liquid is obtained by inoculating white ginseng strain into the seed culture medium and culturing at 24~28℃ for 6~8 days, and the inoculation amount of white ginseng strain is 3 pieces of mycelium on 50mm×50mm PDA plates per 100 mL of seed culture medium.

5. The efficient saccharification method for synergistic pretreatment of corn stalks by elm bark and white ginseng fermentation enzyme solution as described in claim 4, wherein the enzyme solution culture medium comprises 8-12 g / L corn stalks, 4-6 g / L wheat bran and 4-6 g / L soybean peptone, and both the corn stalks and wheat bran are crushed and passed through a 40-60 mesh sieve; the seed culture medium comprises 2-4 g / L soybean peptone and 20-40 g / L malt extract powder.

6. The efficient saccharification method for synergistic pretreatment of corn straw by elm yellow and white ginseng fermentation enzyme solution as described in claim 5, wherein the sterilization conditions of the enzyme solution culture medium and the seed culture medium are both 121℃ for 15~25 min.

7. The efficient saccharification method for synergistic pretreatment of corn straw by elm yellow and white ginseng fermentation enzyme liquid as described in claim 6, wherein steps S1, S2 and S3 are all carried out under oscillation conditions, and the oscillation speed is 200~300 rpm; the preparation of elm yellow strain seed liquid, white ginseng strain seed liquid, elm yellow fermentation enzyme liquid and white ginseng fermentation enzyme liquid are all carried out under oscillation conditions, and the oscillation speed is 120~180 rpm.

8. The efficient saccharification method for corn stalks by synergistic pretreatment with elm bark and white ginseng fermentation enzyme liquid as described in claim 7, wherein the corn stalks in step S1 are crushed and sieved through a 40-60 mesh sieve before use, or the corn stalks in step S1 are acid-exploded before use, or the corn stalks in step S1 are steam-exploded before use; the acid-explosion treatment first uses 1% dilute sulfuric acid to pre-steam the corn stalks in a horizontal reactor at 100-110℃ for 10-30 min, and then steam-explodes them at 180-190℃ for 1-3 min; the steam pressure of the steam-explosion treatment is 1.1-1.2 MPa, the treatment time is 3-5 min, and the moisture content of the material after treatment is 8-12%.

9. The efficient saccharification method for synergistic pretreatment of corn stalks by elm bark and white ginseng fermentation enzyme solution as described in claim 8, wherein the mass-volume ratio of corn stalk dry weight to the total volume of the system is 0.1~0.2 g / mL.

10. A saccharified product of corn stalks, characterized in that, The corn stalk saccharification product is prepared by the efficient saccharification method of corn stalk synergistic pretreatment with elm yellow and white ginseng fermentation enzyme liquid as described in any one of claims 1 to 9.

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