Straw solid-state fermentation of trichoderma longibrachiatum and fermentation process thereof

Abstract

The present application relates to the field of microorganism and biomass processing, and specifically discloses a solid-state fermentation of straw of Trichoderma longibrachiatum and a fermentation process thereof. The present application uses normal temperature and pressure plasma mutagenesis technology to improve the Trichoderma longibrachiatum, and obtains a high-efficiency lignocellulose-degrading mutant strain through a hierarchical screening system. Compared with the original strain, the mutant strain shows a significant improvement in lignocellulose-degrading enzyme activity and protein accumulation. Experiments show that the fermentation process can make the true protein content of corn straw reach 17.57%, and the degradation rates of cellulose, hemicellulose and lignin reach 24.89%, 23.54% and 22.28% respectively. The present application realizes the efficient bioconversion of corn straw, and provides a solution for the resource utilization of agricultural waste.

Description

Technical Field

[0001] This invention relates to the field of microbial and biomass treatment, and specifically discloses a solid-state fermentation method for Trichoderma longifolia from corn stalks and its fermentation process. Background Technology

[0002] Corn stalks are mainly composed of cellulose, hemicellulose, and lignin. This complex lignocellulose structure significantly limits their degradation and conversion efficiency. Currently, the treatment and utilization of corn stalks mainly include physical, chemical, and biological methods. Among these, physical and chemical treatment methods, while highly efficient, consume a lot of energy and cause serious environmental pollution. Biological methods, due to their environmental friendliness and low energy consumption, are considered the most sustainable technological approach.

[0003] Among biological treatment methods, microbial solid-state fermentation technology is widely used for the conversion of lignocellulose biomass due to its advantages such as simple process, low environmental impact, and diverse products. However, conventional solid-state fermentation suffers from problems such as low lignocellulose degradation efficiency, long fermentation cycle, and unstable product quality. This is mainly due to: (1) insufficient lignocellulose degradation capacity of existing strains; (2) fermentation process parameters not being systematically optimized; and (3) lack of in-depth understanding of the evolution of substrate structure during fermentation.

[0004] In recent years, ARTP (atmospheric pressure room temperature plasma), as a novel mutagenesis technology, has shown great potential in microbial breeding due to its high efficiency, broad spectrum, and environmental safety. This technology can induce high-frequency, broad-spectrum gene mutations by generating highly reactive free radicals and charged particles, potentially leading to strains with significantly enhanced lignocellulose degradation capabilities.

[0005] In previous studies, through the study of Aspergillus niger... Aspergillus niger Trichoderma longifolia Trichoderma longibrachiatum Aspergillus oryzae Aspergillus oryzae Trichoderma reesei Trichoderma reesei A systematic evaluation of four filamentous fungi revealed Trichoderma longifolia. T. longibrachiatum It exhibits the best overall performance in solid-state fermentation of corn stalks, but there is still considerable room for improvement in terms of degradation efficiency, protein synthesis capacity, and fermentation cycle.

[0006] Therefore, the present invention aims to utilize ARTP technology to study *Trichoderma longifolia*. T. longibrachiatum We conducted targeted mutagenesis to screen mutant strains with significantly enhanced lignocellulose degradation capabilities and systematically optimized their solid-state fermentation process parameters to achieve efficient bioconversion of corn straw. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a highly efficient lignocellulose-degrading Trichoderma longifolia mutant strain bred using ARTP mutagenesis technology, its preparation method, and an optimized solid-state fermentation process for corn straw based on this mutant strain, so as to achieve efficient biotransformation of corn straw.

[0008] To achieve the above objectives, the present invention provides the following technical solutions.

[0009] A type of Trichoderma ( Trichoderma longibrachiatum The accession number is CGMCC No. 41915. The accession date is April 15, 2025, which is the China General Microbiological Culture Collection Center (CGMCC).

[0010] The application of the aforementioned Trichoderma longifolia in solid-state fermentation of biomass straw.

[0011] Specifically, the biomass straw is one or more of corn straw, sorghum straw, wheat straw, rice straw, and oat straw.

[0012] The present invention further provides a method for solid-state fermentation of biomass straw, comprising the following steps: (1) crushing biomass straw, sterilizing it at high temperature and then cooling it to room temperature; (2) mixing the fermentation nutrient solution with the biomass straw treated in (1), inoculating it with the *Trichoderma longifolia*, and fermenting it.

[0013] Specifically, the fermentation nutrient solution contains ammonium sulfate, wheat bran, and Mg²⁺. + .

[0014] More specifically, the fermentation nutrient solution contains, based on the dry weight of the fermentation substrate (calculated by straw dry weight), 3.91% (w / w) ammonium sulfate, 22.06% (w / w) wheat bran, pH adjusted to 5.5, and 3 mmol / L Mg²⁺ added. + It should be noted that this method of addition based on substrate dry weight is more suitable for solid-state fermentation, helping to maintain the consistency of component ratios across different batches. Other inorganic salts in the nutrient solution are calculated based on solution volume concentration (g / L), and the amount added is based on the specified liquid-to-solid ratio.

[0015] In one specific embodiment, the fermentation nutrient solution further contains: KH2PO4 2.0 (g / L), NaCl 0.5 (g / L), CaCl2 0.3 (g / L), FeSO4•7H2O 0.005 (g / L), MnSO4•H2O 0.0016 (g / L), ZnSO4•7H2O 0.0014 (g / L), and CoCl2 0.002 (g / L).

[0016] In a specific embodiment, a spore suspension of *Trichoderma longicornis* cultured on a PDA plate (e.g., cultured for 4 days) is used as the fermentation starter; the spore concentration in the spore suspension is 10. 5 -10 7 Spores / g substrate.

[0017] Specifically, the fermentation nutrient solution and biomass straw are mixed at a liquid-solid ratio of 1-3:1;

[0018] The fermentation temperature is 26-37℃, and the fermentation culture lasts for 3 to 7 days to obtain the fermentation product. The treatment of the fermented product depends on the final application.

[0019] The present invention also provides fermentation products obtained by the method.

[0020] In this invention, the entire fermentation product (including the solid-liquid mixture) can be collected directly after fermentation. If used as feed, it is dried at an appropriate temperature to a suitable moisture content (usually ≤12%). If used to extract bioactive substances, it can be extracted with a buffer solution followed by centrifugation. Post-processing methods for different application scenarios can be tailored to product requirements.

[0021] Compared with existing technologies, the present invention has the following beneficial effects: The present invention successfully bred a *Trichoderma longicornis* mutant strain TL_MU07 with significantly enhanced lignocellulose degradation ability using ARTP mutagenesis technology. Compared with the original strain, the activities of filter paper enzyme FPase, endonuclease CMCase, and xylanase in this mutant strain increased by 22.1%, 10.1%, and 16.1%, respectively; the degradation rates of cellulose and hemicellulose increased by 14.6% and 12.9%, respectively; and the true protein content increased by 14.7%.

[0022] This invention establishes a solid-state fermentation process highly matched to the TL_MU07 strain by systematically optimizing process parameters. The optimized process achieves a true protein content of 17.57% in corn straw, an increase of 53.45% compared to the single-factor optimization stage; the degradation rates of cellulose, hemicellulose, and lignin reach 24.89%, 23.54%, and 22.28%, respectively, increases of 25.64%, 20.97%, and 30.37% compared to the single-factor optimization stage; the filter paper enzyme activity FPase increases by 30.44%, and the peptide content increases by 67.61%.

[0023] This invention combines ARTP-mutated strains with an optimized solid-state fermentation process, which significantly shortens the fermentation cycle (to only 4.10 days) and improves substrate conversion efficiency, making it of significant application value for the resource utilization of agricultural waste.

[0024] In summary, the *Trichoderma longicornis* mutant strain TL_MU07 and its solid-state fermentation process provided by this invention can achieve efficient bioconversion of corn stalks. It can be used to produce high-protein feed: the fermentation product contains 17.57% true protein and 2.38% peptides, serving as a protein feed resource to alleviate the shortage of feed protein resources. The process is simple and easy to implement, with low cost: the fermentation cycle is short (only 4.10 days), requiring no complex equipment, making it suitable for large-scale production. It is environmentally friendly: it realizes the biological resource utilization of agricultural waste, reduces environmental pollution, and meets the requirements of sustainable development. It can be widely applied to the bioconversion of other agricultural wastes and has broad market prospects.

[0025] In summary, the mutant strains and fermentation process provided by this invention offer a new technical solution for the resource utilization of agricultural waste, with significant economic, social, and ecological benefits.

[0026] Biological material deposit information: The strain TL_MU07 of this invention was deposited on April 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with accession number CGMCC No. 41915. It is classified and named *Trichoderma longifolia*. Trichoderma longibrachiatum . Attached Figure Description

[0027] Figure 1 Growth results of TL_MU07 under different temperature conditions (a~h: 15℃, 20℃, 25℃, 30℃, 33℃, 37℃, 40℃, 43℃).

[0028] Figure 2 Comparison of TL_MU07 growth results under different pH conditions a~h: pH = 3, 4, 5, 6, 7, 8, 9, 10.

[0029] Figure 3 The effect of inoculum size on the loss rate of true protein and dry matter during solid-state fermentation.

[0030] Figure 4 The effect of fermentation time on the loss rate of true protein and dry matter in solid-state fermentation.

[0031] Figure 5The effects of different nitrogen sources on lignocellulose hydrolases and protein yields were investigated. Specifically, (a) the effect of different nitrogen sources on filter paper enzyme activity; (b) the effect of different nitrogen sources on endonuclease activity; (c) the effect of different nitrogen sources on β-glucosidase activity; (d) the effect of different nitrogen sources on xylanase activity; (e) the effect of different nitrogen sources on β-xylosidase activity; (f) the effect of different nitrogen sources on laccase activity; (g) the effect of different nitrogen sources on manganese peroxidase activity; (h) the effect of different nitrogen sources on lignin peroxidase activity; (i) the effect of different nitrogen sources on true protein content; and (j) the effect of different nitrogen sources on peptide content.

[0032] Figure 6 The effects of different metal ions on biomass and enzyme activity were investigated. Specifically, (a) the effect of different metal ions on spore yield; (b) the effect of different metal ions on dry matter loss rate; (c) the effect of different metal ions on filter paper enzyme activity; (d) the effect of different metal ions on endonuclease activity; (e) the effect of different metal ions on xylanase activity; and (f) the effect of different metal ions on laccase activity.

[0033] Figure 7 Three-dimensional response surface plots are shown to illustrate the effects of ammonium sulfate, fermentation time, and bran addition on true protein content. (a) Effects of fermentation time and ammonium sulfate content on true protein content; (b) Effects of bran inoculum amount and ammonium sulfate content on true protein content; (c) Effects of fermentation time and bran inoculum amount on true protein content. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. Those skilled in the art should understand that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] Example 1: Screening of ARTP-mutated strains of Trichoderma longifolia

[0036] 1.1 Test strains and materials

[0037] This embodiment uses Trichoderma longifolia isolated from farmland soil. Trichoderma longibrachiatum The starting strain was preserved using PDA slant culture. The substrate was corn stalks collected in Nanning, Guangxi. After natural ventilation and sun-drying, it was dried at 65℃ to constant weight, pulverized, and passed through a 10-mesh sieve for later use. The main components of the corn stalks were: cellulose 30.30±0.86%, hemicellulose 24.91±0.72%, lignin 19.45±0.53%, and nitrogen content 10.22±0.31 g / kg (dry matter).

[0038] 1.2 Culture medium preparation

[0039] (1) PDA medium (g / L): 200g potato juice, 20g glucose, 20g agar, pH 6.5.

[0040] (2) CMC-Na primary screening plate medium (g / L): (NH4)2SO4 1.0, Na2HPO4 1.2, KH2PO4 0.9, MgSO4•7H2O 0.5, KCl 0.5, yeast extract powder 0.5, acid hydrolyzed casein 0.5, Congo red 0.2, cellulose powder 5.0, agar 15.0, pH 4.8.

[0041] (3) Shake flask fermentation seed culture medium (g / L): glucose 10.0, peptone 5.0, Tween-80 2mL, (NH4)2SO4 1.4, CaCl2 0.3, urea 0.3, MgSO4•7H2O 0.3, FeSO4•7H2O 0.005, MnSO4•H2O 0.0016, ZnSO4•7H2O 0.0014, CoCl2 0.002, pH 4.8.

[0042] (4) Shake-flask fermentation enzyme production medium (g / L): microcrystalline cellulose 20.0, wheat bran 10.0, corn steep liquor 17.0, K2HPO4 2.0, (NH4)2SO4 1.4, MgSO4•7H2O 0.3, urea 0.3, CaCl2 0.3, Tween-80 2mL, FeSO4•7H2O 0.005, MnSO4•H2O 0.0016, ZnSO4•7H2O 0.0014, CoCl2 0.002, pH 4.8.

[0043] All of the above culture media must be autoclaved at 121°C for 20 minutes before use.

[0044] 1.3 ARTP mutagenesis treatment

[0045] (1) Culture Trichoderma longifolia in PDA medium for 4 days, scrape off the spores with 10 mL of 0.9% physiological saline, transfer to 150 mL of conical flask, add 50 glass beads with a diameter of 4 mm, shake at 250 rpm / min for 15 min to fully disperse the spores, and adjust the spore concentration to 10^6~10^8 / mL.

[0046] (2) Take 10 μL of spore suspension and spread it evenly on a sterile metal slide. The ARTP mutagenesis conditions are: radio frequency input power 120W, irradiation distance 2mm, plasma temperature <35℃, gas flow rate 10SLM, and mutagenesis time set at 11 gradients from 0s to 400s.

[0047] (3) After the mutagenesis treatment, the metal slide was placed in a 5 mL centrifuge tube containing 1 mL of 0.9% physiological saline, shaken to mix, and allowed to stand for 2-3 h. After dilution to an appropriate factor, 100 μL was taken and spread on a PDA plate. Three replicates were made for each group. After incubation at 30℃ for 72 h, the survival rate was counted and calculated.

[0048] (4) Based on the survival rate curve analysis, 320s (approximately 85% lethality) was determined as the optimal exposure time for mutagenesis, which was then used for subsequent large-scale mutagenesis treatment.

[0049] 1.4 Screening of mutant strains

[0050] (1) Initial screening on plates: The spore suspension after 320s mutagenesis was appropriately diluted and spread on CMC-Na plates, and incubated at 30℃ for 4 days. The plates were stained with 0.1% Congo red solution for 15 min and destained with 1 mol / L NaCl solution for 30 min. The ratio of the diameter of the hydrolysis zone (D) to the colony diameter (d) was measured (HC=D / d). Using the HC value of the starting strain (2.081±0.262) as a reference, strains with an HC value higher than 35% (HC>2.800) were screened, resulting in 19 mutant strains.

[0051] (2) Shake-flask re-screening: The 19 mutant strains obtained from the screening were used to prepare spore suspensions (10 μL / 10 ... 6 (Cells / mL), 2.0 mL was inoculated into liquid seed culture medium and cultured at 30℃ and 200 rpm for 24 h to form an active bacterial culture. The culture was then transferred to enzyme-producing fermentation medium at a ratio of 5% (v / v) and fermented for another 96 h under the same conditions. The concentrations of FPase, water-soluble protein, and peptides in the culture medium were measured, and three mutant strains, TL_MU06, TL_MU07, and TL_MU15, were selected as having the best overall performance.

[0052] (3) Solid-state fermentation three-stage screening: Corn stalks were used as the substrate for solid-state fermentation to evaluate the performance of three candidate mutant strains. 10g of corn stalks were weighed into a 250mL Erlenmeyer flask, sterilized at 121℃ for 20min, and then cooled to room temperature. Following a 10... 6 The spore suspension of the strain was inoculated with a spore / g substrate ratio, and inorganic salt nutrient solution was added at a water-to-substrate ratio of 2:1. The mixture was then incubated at 30℃ for 5 days. The activities of FPase, CMCase, and xylanase, the degradation rates of cellulose, hemicellulose, and lignin, the true protein content, and the peptide content were determined.

[0053] The results showed that strain TL_MU07 exhibited comprehensively enhanced hydrolytic enzyme activities: FPase reached 6.382 U / g ds, an increase of 22.1% compared to the starting strain; CMCase reached 11.076 U / g ds, an increase of 10.1%; and xylanase activity increased by 16.1% to 2.874 U / g ds. Analysis of lignocellulose degradation components showed that the cellulose degradation rate of strain TL_MU07 was 14.6% higher than that of the starting strain, and the hemicellulose degradation rate increased by 12.9%. Regarding biomass and protein synthesis, the true protein content of the fermentation group of strain TL_MU07 reached 8.621%, an increase of 14.7% compared to the starting strain, and peptide content also showed a significant increase. Furthermore, the spore count of TL_MU07 was significantly higher than that of the starting strain and other mutant strains, reflecting that this strain has stronger substrate adaptability and reproductive potential under solid-state fermentation conditions.

[0054] Therefore, TL_MU07 was selected as the optimal mutant strain. Stability was determined after five generations of continuous subculturing, and the results showed that the excellent enzyme production performance of this strain was genetically stable.

[0055] Example 2: Optimization of solid-state fermentation process for mutant strain TL_MU07

[0056] 2.1 Single-factor optimization experiment

[0057] (1) Temperature optimization: TL_MU07 strain was inoculated on PDA plates and cultured for 4 days at 15, 20, 25, 30, 33, 37, 40, and 43℃ respectively. Colony morphology was observed, colony diameter was measured, and spore yield was calculated. Figure 1 The results showed that spore production peaked at 33℃ (8.69 × 10⁻⁶). 8 The colony diameter reached its maximum value (8.90 cm) at 37℃. Taking all factors into consideration, the optimal culture temperature was determined to be 33℃.

[0058] (2) pH optimization: TL_MU07 strain was inoculated onto PDA plates, and the pH of the culture medium was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10, respectively, and cultured at 33℃ for 4 days. The results showed that the colony diameter reached its maximum at pH 7 (8.54 cm), and spore formation reached its highest level at pH 6 (1.94 × 10⁻⁶ cm). 8 Cellulase activity / mL was determined to be optimal at 5.5, considering that cellulase activity in solid-state fermentation is more efficient under weakly acidic conditions. Figure 2 ).

[0059] (3) Inoculation volume optimization: Set 5 inoculation volume gradients: 10 4 10 5 10 6 107 10 8 Spores / g ds. Corn straw and inorganic salt nutrient solution were mixed at a 1:2 solid-liquid ratio and cultured at 33℃ for 4 days. True protein content and dry matter loss rate were then measured. Results showed that at 10... 6 The true protein content reached its highest value (8.75%) when the spores / g ds ratio was [value missing]. The optimal inoculum size was determined to be 10 [units missing]. 6 spores / g ds ( Figure 3 ).

[0060] (4) Fermentation time optimization: Using the optimal inoculum size, samples were taken on days 2, 3, 4, 5, 6, and 7 to determine the true protein content and dry matter loss rate. Results showed that the true protein content peaked on day 5 (8.80%) and then stabilized with a slight decrease. The dry matter loss rate exhibited a continuous increasing pattern, but the rate of increase gradually slowed. The optimal fermentation time was determined to be 4–5 days. Figure 4 ).

[0061] (5) Nitrogen source optimization: The effects of different inorganic nitrogen sources (ammonium sulfate, ammonium chloride, urea, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium nitrate) and organic nitrogen sources (peptone, yeast extract) on fermentation were investigated. The amount of nitrogen source added was adjusted to provide the same nitrogen content (0.5% substrate dry weight). The results showed that the activities of various enzymes (FPase, CMCase, BGL, xylanase, β-xylosidase, Lac, MnP, LiP) in the ammonium sulfate treatment group reached or approached the highest levels, while the contents of true protein (11.45%) and peptide (1.42%) were also significantly higher than those in other nitrogen source treatment groups. Ammonium sulfate was determined to be the optimal nitrogen source. Figure 5 ).

[0062] (6) Optimization of metal ions: Investigating Cu² + Mn² + Mg² + Zn² + and Ca² + The effects of five metal ions (1.0 mmol / L) on fermentation efficiency. The results showed that Mg²⁺… + It exhibits comprehensive advantages in promoting spore production, substrate degradation, and lignocellulase activity. The determination of Mg²⁺... + For optimal metal ion additives ( Figure 6 ).

[0063] 2.2 Response Surface Optimization Experiment

[0064] (1) Plackett-Burman design to screen significant factors: 10 factors that may affect the fermentation process were selected, including: (A) ammonium sulfate addition, (B) initial pH, (C) Mg²⁺. +The following factors were considered: (D) inoculum size, (E) fermentation time, (F) liquid-to-solid ratio, (G) temperature, (H) Tween-80 addition amount, (I) bran addition amount, and (J) PEG 6000 addition amount. Each factor was set with two levels: high (+1) and low (-1), with true protein content as the response value. Analysis of variance showed that fermentation time (E), ammonium sulfate addition amount (A), and bran addition amount (I) had a significant impact on true protein yield. P <0.05), with a cumulative contribution rate of 86.86%.

[0065] (2) Determining the optimal region using the steepest climbing method: Based on the standardization effect of each factor, a steepest climbing experiment was designed. By observing the trend of true protein yield changes, it was determined that the highest true protein yield of 15.73±0.91% was achieved when the ammonium sulfate concentration was 4%, the fermentation time was 4 days, and the bran addition was 23%.

[0066] (3) Box-Behnken design and response surface methodology: Seventeen experiments (including five centroids) were designed with ammonium sulfate concentration (X1), fermentation time (X2), and bran addition amount (X3) as independent variables and true protein yield (Y) as the response value. Response surface methodology determined the optimal combination of process parameters as follows: ammonium sulfate concentration 3.91%, fermentation time 4.10 days, and bran addition amount 22.06%. Under these conditions, the model predicted a true protein content of 16.77% (…). Figure 7 ).

[0067] (4) Process validation: Six parallel validation experiments were conducted under optimized conditions. The measured true protein content was 17.57%, with a relative error of 4.55% compared to the model prediction. P The relative standard deviation (RSD) of the six parallel experiments was only 3.8%, which confirmed the accuracy of the model and the stability of the process.

[0068] 2.3 Evaluation of the fermentation effect of the optimized process

[0069] Compared with the single-factor optimization stage, the final optimized process significantly improved the fermentation effect, as shown in the following results:

[0070] (1) The true protein content increased from 11.45% to 17.57%, an increase of 53.45%;

[0071] (2) The concentration of FPase increased from 8.41 U / g to 10.97 U / g, an increase of 30.44%;

[0072] (3) The degradation rate of cellulose increased to 24.89% (an increase of 25.64%), the degradation rate of hemicellulose increased to 23.54% (an increase of 20.97%), and the degradation rate of lignin reached 22.28% (an increase of 30.37%).

[0073] (4) The peptide content increased from 1.42% to 2.38%, an increase of 67.61%.

[0074] Example 3: Structural Characterization Analysis of Solid-State Fermentation Products

[0075] 3.1 Scanning Electron Microscopy (SEM) Observation

[0076] SEM observation of corn stalk samples before and after solid-state fermentation showed the following results:

[0077] (1) Unfermented corn stalks have a dense and smooth surface structure, with tightly packed fiber bundles and intact cell walls.

[0078] (2) The corn stalks after fermentation with optimized process showed significant structural damage characteristics: the surface became rough and porous, with a large number of cracks and micropores; the fiber bundles disintegrated, forming a loose network structure; a large number of mycelia were observed to adhere to the substrate surface and penetrate the tissue.

[0079] These microstructural changes indicate that the extracellular enzyme system of strain TL_MU07 effectively decomposes the complex structure of corn stalks, disrupts cell wall integrity, and improves substrate bioaccessibility.

[0080] 3.2 Fourier Transform Infrared Spectroscopy (FTIR) Analysis

[0081] FTIR analysis results showed that the characteristic peak positions and relative intensities of the samples changed significantly after fermentation:

[0082] (1) 3400cm -1 The weakening intensity of the -OH stretching vibration peak at the point indicates that the cellulose hydrogen bond network is disrupted;

[0083] (2) 1510cm -1 The relative intensity of the vibrational peaks of the lignin skeleton at the location decreased, indicating that the lignin structure was partially degraded;

[0084] (3) Lateral order index (LOI, α1437cm) -1 / α899cm -1 The decrease from 1.48 to 1.24 indicates that the regular arrangement of cellulose molecular chains has been disrupted;

[0085] (4) Total Crystallization Index (TCI, α1378cm) -1 / α2900cm -1 The decrease from 1.31 to 1.03 indicates a decrease in the crystallinity of cellulose;

[0086] (5) Cellulose / lignin ratio (α3400cm) -1 / α1510cm -1 The decrease from 1.60 to 1.30 indicates that the relative stability of the lignin structure is higher than that of cellulose.

[0087] These spectral changes confirm that the TL_MU07 strain had a profound impact on the chemical structure of corn stalks during solid-state fermentation, particularly by reducing cellulose crystallinity and disrupting the tissue structure of lignocellulose.

[0088] 3.3 X-ray diffraction (XRD) analysis

[0089] XRD analysis results showed that fermentation treatment caused significant changes in the cellulose crystal structure of corn straw:

[0090] (1) The intensity of the diffraction peak at 2θ=22° (002 crystal plane) of the sample after fermentation was significantly reduced, indicating that the crystallization area was reduced;

[0091] (2) The relative increase in scattering intensity at 2θ=18° (amorphous region) indicates that the proportion of amorphous region has increased;

[0092] (3) The relative crystallinity of cellulose (CrI) decreased from the initial 43.89% to 36.37% after the optimized fermentation process, indicating that crystalline cellulose transformed into amorphous regions during the fermentation process.

[0093] Decreased crystallinity is beneficial for enzymatic hydrolysis because enzyme molecules can more easily access and catalyze cellulose molecules in amorphous regions, thereby improving the bioaccessibility and degradation efficiency of the substrate.

[0094] The above structural characterization results confirm at the microscopic and molecular levels that solid-state fermentation of strain TL_MU07 can effectively destroy the physical structure and chemical composition of corn stalks and reduce the crystallinity of cellulose, which is consistent with the high degradation rate and bioconversion efficiency observed macroscopically.

Claims

1. A type of Trichoderma longifolia ( Trichoderma longibrachiatum ), characterized in that, Its accession number is CGMCC No.41915.

2. The application of Trichoderma longifolia as described in claim 1 in solid-state fermentation of biomass straw.

3. The application as described in claim 2, characterized in that, The biomass straw is one or more of the following: corn straw, sorghum straw, wheat straw, rice straw, and oat straw.

4. A method for solid-state fermentation of biomass straw, characterized in that, Includes the following steps: (1) Crush the biomass straw, sterilize it at high temperature, and then cool it to room temperature; (2) Mix the fermentation nutrient solution with the biomass straw treated in (1), inoculate with Trichoderma longibranchii as described in claim 1, and carry out fermentation; The fermentation nutrient solution contains ammonium sulfate, wheat bran, and magnesium. 2+ ; Fermentation nutrient solution and biomass straw are mixed at a liquid-solid ratio of 1-3:1; the fermentation temperature is 26-37℃, and the fermentation culture is carried out for 3 to 7 days to obtain the fermentation product.

5. The method as described in claim 4, characterized in that, The fermentation nutrient solution, based on the dry weight of the fermentation substrate (calculated by straw dry weight), contains 3.91% (w / w) ammonium sulfate, 22.06% (w / w) wheat bran, and the pH is adjusted to 5.

5. 3 mmol / L of Mg is added. 2+ .

6. The method as described in claim 4, characterized in that, The fermentation nutrient solution also contains: KH2PO4 2.0 g / L, NaCl 0.5 g / L, CaCl2 0.3 g / L, FeSO4•7H2O 0.005 g / L, MnSO4•H2O 0.0016 g / L, ZnSO4•7H2O 0.0014 g / L, and CoCl2 0.002 g / L.

7. The method as described in claim 4, characterized in that, The spore suspension of *Trichoderma longicornis* as described in claim 1 was cultured on a PDA plate as a fermentation starter; the spore concentration in the spore suspension was 10. 5 -10 7 Spores / g substrate.

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