Streptomyces griseorubidus with cutin layer degradation ability, microbial inoculum and application thereof

Abstract

This invention relates to a *Streptomyces griseorubens* strain with cuticle-degrading capabilities, its inoculum, and its applications. The strain is named *Streptomyces griseorubens* F-1, with accession number CCTCC NO: M 2024899. *Streptomyces griseorubens* F-1 can grow rapidly and stably using inexpensive culture media, has a wide temperature tolerance range, and is particularly suitable for applications under ambient temperature (15-40℃) conditions. This fungus can be used for the biological degradation of plant residues, specifically degrading the cuticle, cellulose, hemicellulose, and lignin, thereby effectively promoting the high-value utilization of plant residues and possessing significant economic and ecological value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a griseoerythromyces blight-like fungus with cuticle degradation ability, its fungal agent, and its application. Background Technology

[0002] As a major agricultural country, China had a theoretical straw resource of 977 million tons in 2022. Lignocellulose biomass is a key resource for achieving sustainable development by replacing petroleum production systems. Plants use complex structures and chemical properties to resist damage to their tissues from the natural environment and other organisms; the cuticle is the first barrier. Although the composition of the cuticle varies among different species, it is generally composed of the following main components: cuticle, polyesters naturally polymerized from C16 or C18 hydroxy fatty acids, and waxes. The cuticle exhibits significant hydrophobicity during interactions with the external environment. Solvent extraction experiments have shown that waxes are key to this hydrophobic effect. The physical barrier and surface hydrophobicity of recalcitrant polyesters give plants strong resilience but also hinder the natural degradation efficiency of agricultural waste. Decomposing or removing the cuticle is a crucial step in utilizing plant cellulose and hemicellulose. Therefore, finding a method that is both efficient and energy-saving to fully degrade the straw cuticle is very important, providing a possible direction for future applications and research on straw biodegradation.

[0003] Because the cuticle covers the surface of plant fibers, the effective contact between natural microorganisms and enzymes and cellulose and lignin is reduced, resulting in a decrease in the utilization level of polysaccharides and lignin located inside. Currently, commonly used cuticle treatment methods include chemical and physical methods. Chemical methods have problems such as potential harm from toxic chemicals and the need to purchase expensive equipment, leading to high costs. Physical methods have problems such as high energy consumption, high equipment maintenance costs, and environmental and safety hazards.

[0004] Northeast my country, as a major grain production base, generates a large amount of agricultural waste such as crop straw every year. After harvesting, the surface of the straw is covered with a difficult-to-degrade cuticle layer, which significantly impacts the subsequent resource utilization of the straw. Screening for straw cuticle-degrading bacteria can enrich the straw cuticle-degrading bacteria resource bank, provide a basis for the research and development of cuticle-degrading bacteria, and may provide new resources for the decomposition and destruction of plant residue cuticles.

[0005] In China's "five-fold" utilization of crop straw, fertilization is the most prevalent method and an effective way to process large quantities of surplus straw. The primary method of crop straw fertilization is direct return of straw to the field. This protective utilization of straw is beneficial to soil and environmental needs. However, in-situ return presents several problems. Slow decomposition of straw in the field is a primary concern for annual farming, leading to issues such as nutrient imbalances and crop pests and diseases. Compared to in-situ straw return, composting is more efficient, promotes more microbial growth, and reduces the abundance of pathogenic microorganisms. However, the collection, storage, crushing, and fermentation processes increase costs. Whether in-situ or composting, the straw cuticle layer hinders the decomposition process. Straw return often employs crushing and compaction methods; the depth of compaction affects the decomposition rate, meaning the abundance of relevant functional microorganisms in the soil varies at different depths. The decomposition of straw after it is returned to the field involves nutrient release, organic carbon mineralization, and soil organic carbon balance. This process is mediated by different soil microorganisms with specific functions. Therefore, selecting suitable microorganisms is of great significance for the efficient degradation of plant residue cuticle and the promotion of straw return to the field. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a Streptomyces strain with cuticle degradation ability, which can effectively degrade the cuticle under normal temperature conditions.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] This invention provides a strain of *Streptomyces griseorubens*, named *Streptomyces griseorubens* F-1, with accession number CCTCCNO: M 2024899. This strain was deposited on May 10, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China.

[0009] This invention screened a strain F-1 from soil returned to the field in Heilongjiang Province that can effectively degrade the cuticle. This strain can grow rapidly and stably using inexpensive culture media, has a wide temperature tolerance range, and is particularly suitable for applications under ambient temperature (15-40℃) conditions, adapting to various treatment scenarios. This strain can be used for the biological degradation of plant residues, degrading the cuticle, cellulose, hemicellulose, and lignin, thereby effectively promoting the high-value utilization of plant residues and possessing high economic and ecological value.

[0010] Strain F-1 grew relatively small on Gao's No. 1 solid medium, with colonies measuring 2 mm in size. The colony protrusions were indistinct, and the colonies were opaque, whitish-gray with neat edges and a rough surface. It then developed into a powdery, actinomycete pattern, showing differentiation of both intracellular and aerial hyphae. After 72 hours of cultivation on Gao's No. 1 solid medium, electron microscopy revealed that individual conidia were approximately 1 μm long and 0.7-0.9 μm wide, with spiral-shaped hyphae.

[0011] After culturing *Streptomyces griseus* F-1 on Gao's No. 1 medium supplemented with treated corn stalk epidermis for 5 days, the weight loss rate of the corn stalk epidermis was 28.87%. After culturing *Streptomyces griseus* F-1 on a carbon-free mineral salt medium supplemented with treated corn stalk epidermis for 5 days, the weight loss rate of the corn stalk epidermis was 22.57%.

[0012] The 16S rRNA gene sequence of *Streptomyces griseus* F-1 includes the nucleotide sequence shown in SEQ ID NO: 3.

[0013] The present invention provides a straw decomposing microbial agent, comprising the above-mentioned Streptomyces griseus and / or the fermentation product of the above-mentioned Streptomyces griseus.

[0014] In addition to *Streptomyces griseus* and its fermentation products, the inoculant may also include excipients. Other microorganisms may also be added to the inoculant, and these other microorganisms can be compounded with the strain F-1 provided in this invention. This invention does not have special requirements for the formulation of the straw-decomposing inoculant; for example, it can be a liquid formulation, a solid formulation, or other formulations.

[0015] Furthermore, the cuticle-degrading bacterial agent may also include a culture medium for culturing strain F-1.

[0016] This invention provides a method for preparing a straw-decomposing microbial agent, comprising the following steps: culturing the above-mentioned Streptomyces griseus F-1 in a culture medium.

[0017] The present invention provides a fermentation culture method for the above-mentioned Streptomyces griseus, comprising the following steps: inoculating Streptomyces griseus F-1 into a culture medium and fermenting.

[0018] Furthermore, the fermentation temperature can be 15-40℃.

[0019] The present invention provides the use of the above-mentioned Streptomyces griseus in any one or more of (1) to (5);

[0020] (1) Degradation of the cuticle of plant debris;

[0021] (2) Degradation of plant residues;

[0022] (3) Degradation of cellulose;

[0023] (4) Degradation of hemicellulose;

[0024] (5) Degradation of lignin.

[0025] The present invention provides the application of the above-mentioned microbial agent in any one or more of (1) to (5);

[0026] (1) Degradation of the cuticle of plant debris;

[0027] (2) Degradation of plant residues;

[0028] (3) Degradation of cellulose;

[0029] (4) Degradation of hemicellulose;

[0030] (5) Degradation of lignin.

[0031] The *Streptomyces griseus* F-1 fungal agent provided by this invention has advantages such as rapid and stable growth using inexpensive culture media and a wide temperature adaptability range. It is suitable for applications under different temperature conditions and exhibits good degradation effect on plant residue cuticles. It can be used for the biological degradation of plant residue cuticles, as well as the degradation of cuticle-containing substances (e.g., straw), thereby improving the utilization rate of cuticle-containing substances and possessing high economic and ecological value.

[0032] This invention provides a method for degrading the cuticle using the aforementioned *Streptomyces griseus*, comprising the following steps: inoculating the *Streptomyces griseus* into a culture medium containing the cuticle, and then fermenting and culturing it. The method of this invention has advantages such as not generating secondary pollution and being environmentally friendly.

[0033] The fermentation temperature can be 15-40℃.

[0034] The present invention provides a method for degrading the cuticle using the above-mentioned microbial agent, comprising the following steps: inoculating the above-mentioned microbial agent into a culture medium containing the cuticle and fermenting it.

[0035] The fermentation temperature can be 15-40℃.

[0036] In addition to the cuticle (e.g., straw epidermis), the above-mentioned culture medium may also contain water-soluble starch, inorganic salts, etc.

[0037] This invention isolates Streptomyces griseus F-1 from soil. This strain and the inoculum prepared using this strain can effectively degrade the cuticle and can grow normally in the temperature range of 15℃ to 40℃, thus completely destroying the cuticle on the surface of straw in the fermentation liquid. Attached Figure Description

[0038] Figure 1The colony morphology of strain F-1 (left image) and electron micrographs of conidial filaments (right image) are shown.

[0039] Figure 2 Phylogenetic trees were constructed for strain F-1 and related strains.

[0040] Figure 3 The results show the experimental results of detecting weight loss rate and degradation rate in different culture media.

[0041] Figure 4 The results show the experimental results of detecting the dry weight and pH of the strains in different culture media.

[0042] Figure 5 Scanning electron microscope images of corn stalk epidermis after different treatments.

[0043] Figure 6 The water contact angle of the corn stalk epidermis after different treatments. Detailed Implementation

[0044] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0045] To better understand the present invention, the following detailed description is provided in conjunction with embodiments; however, the scope of protection of the present invention is not limited to the scope represented by the embodiments. The present invention provides a general and / or specific description of the materials and test methods used in the experiments. Although many materials and methods of operation used to achieve the objectives of the present invention are well known in the art, the present invention is still described in as much detail as possible herein.

[0046] All quantities of substances added to the following culture media are in g / L. All culture media are used after sterilization.

[0047] Carbon-free mineral salt culture medium is prepared according to the following proportions: K2HPO4·3H2O 0.92g / L, KH2PO4 0.7g / L, MgSO4·7H2O 0.7g / L, (NH4)2SO4 1g / L, NaCl 0.005g / L, FeSO4·7H2O 0.002g / L, ZnSO4·7H2O 0.002g / L, MnSO4·H2O 0.001g / L, with distilled water, pH 7.2-7.6.

[0048] Carbon-free mineral salt solid culture medium is prepared according to the following proportions: K2HPO4·3H2O 0.92g / L, KH2PO4 0.7g / L, MgSO4·7H2O 0.7g / L, (NH4)2SO4 1.0g / L, NaCl 0.005g / L, FeSO4·7H2O 0.002g / L, ZnSO4·7H2O 0.002g / L, MnSO4·H2O 0.001g / L, agar powder 18-20g / L, prepared with distilled water, pH 7.2-7.6.

[0049] Gao's No. 1 culture medium is prepared according to the following proportions: soluble starch 20g / L, KNO3 1g / L, NaCl 0.5g / L, K2HPO4·3H2O 0.5g / L, MgSO4·7H2O 0.5g / L, FeSO4·7H2O 0.01g / L, prepared with distilled water, pH 7.4-7.6.

[0050] The preparation method of Gao's No. 1 solid culture medium includes the following steps: add 18-20 g / L of agar powder to Gao's No. 1 culture medium.

[0051] The corn stalk cuticle solid culture medium was prepared according to the following proportions: A carbon-free mineral salt solid culture medium was prepared, and three 1cm x 1cm pieces of treated corn stalk cuticle were added to agar plates. The preparation method of the treated corn stalk cuticle included the following steps: Corn stalks were harvested and air-dried until a gap formed between the cuticle and the stalk. The cuticle was peeled off and boiled in a solution containing 4g / L oxalic acid and 16g / L ammonium oxalate for 4 hours to remove carbohydrates. After washing with sterile deionized water, the cuticle was dried and cut into 1cm x 1cm square slices. The slices were then sterilized before use.

[0052] Polycaprolactone (PCL) plates are prepared as follows: Dissolve 1 g of PCL in 25 mL of acetone, then mix with 25 mL of distilled water to obtain 50 mL of flocculent solution. Add this solution to 950 mL of carbon-free mineral salt medium. If preparing solid plates, add an additional 18-20 g / L of agar powder.

[0053] For preparing paraffin-based plates, use the following ratio: Take 4g of paraffin (melting point 56-58℃) and add it to 1L of carbon-free mineral salt culture medium. Handle the liquid culture medium gently when removing it from the container and allow it to cool completely to prevent the paraffin from adhering to the glass bottle surface and solidifying. If preparing solid plates, add an additional 18-20g / L of agar powder to the liquid culture medium. Pour the plates at a temperature above 60℃, shaking well before each pour.

[0054] In this invention, the terms "straw cuticle" and "cuticle layer" have the same meaning.

[0055] Primers 27F and 1492R were both synthesized by Shanghai Paisenno Biotechnology Co., Ltd. The sequence of primer 27F is 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO:1); the sequence of primer 1492R is 5′-CTACGGCTACCTTGTTACGA-3′ (SEQ ID NO:2).

[0056] Bacterial genomic DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number DP302-02. AxyPrep DNA gel extraction kit was purchased from AxyPrep, catalog number AP-GX-50. 10×Buffer (containing 2.5 mM Mg) 2+ The following were purchased from Shanghai Yuanmu Biotechnology Co., Ltd., catalog number YM-MY162J. Taq polymerase was purchased from TaKaRa, catalog number DRR20AM. dNTPs were purchased from TaKaRa, catalog number D4030A.

[0057] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0058] The following is a description through specific embodiments.

[0059] Example 1

[0060] In July 2021, soil samples were collected from the oilfield square in Daqing City, Heilongjiang Province (124.92°N, 46.61°E), Chahayang Farm in Qiqihar City (124.24°N, 48.23°E), and Longyou Lake artificial lake in Nantong City, Jiangsu Province (32.42°N, 129.16°E). The collection method included the following steps: multiple sampling points were collected using a sterile shovel, with 50g of sample taken from each location. The collected samples were quickly placed into sterile polyethylene sealed bags and brought back to the laboratory under low temperature conditions. The samples were then stored in a 4°C refrigerator for subsequent screening.

[0061] Weigh 5g of soil sample and add it to a 100mL Erlenmeyer flask containing 60mL of sterile distilled water. Incubate at 30℃ and 150rpm on a shaker for 1 hour to enrich the culture. Let it stand for 30 minutes, and collect the supernatant as the original bacterial culture. Dilute the original bacterial culture 10... -5 10 -6 and 10 -7The diluted bacterial suspension was spread onto polycaprolactone-selective solid plates, paraffin-selective solid plates, and straw cuticle solid agar plates, with 50 μL of diluted bacterial suspension spread on each plate. All plates were incubated at 30°C for 3 days. A single colony was picked and streaked onto the plate. The type of plate used for streaking was the same as that used for spreading the bacterial suspension. The plates were incubated at 30°C for 3 days. The above method was repeated to isolate and purify the bacterial strain until a pure culture strain was obtained. The strain was then stored at -80°C for later use.

[0062] Weigh 5g of soil sample and add it to a 100mL Erlenmeyer flask containing 60mL of sterile distilled water. Incubate at 35℃ and 150rpm on a shaker for 1 hour to enrich the culture. Let it stand for 30 minutes, and collect the supernatant as the original bacterial culture. Dilute the original bacterial culture 10... -5 10 -6 and 10 -7 The diluted bacterial suspension was spread onto polycaprolactone-selected solid plates, paraffin-selected solid plates, and straw cuticle solid agar plates, with 50 μL of diluted bacterial suspension spread on each plate. All plates were incubated at 35°C for 3 days. A single colony was picked and streaked onto the plate. The type of plate used for streaking was the same as that used for spreading the bacterial suspension. The plates were incubated at 35°C for 3 days. The above method was repeated to isolate and purify the bacterial strain until a pure culture strain was obtained. The strain was then stored at -80°C for later use.

[0063] Weigh 5g of soil sample and add it to a 100mL Erlenmeyer flask containing 60mL of sterile distilled water. Incubate at 40℃ and 150rpm on a shaker for 1 hour to enrich the culture. Let it stand for 30 minutes, and collect the supernatant as the original bacterial culture. Dilute the original bacterial culture 10... -5 10 -6 and 10 -7 The diluted bacterial suspension was spread onto polycaprolactone-selective solid plates, paraffin-selective solid plates, and straw cuticle solid agar plates, with 50 μL of diluted bacterial suspension spread on each plate. All plates were incubated at 40°C for 3 days. A single colony was picked and streaked onto the plate. The type of plate used for streaking was the same as that used for spreading the bacterial suspension. The plates were incubated at 40°C for 3 days. The above method was repeated to isolate and purify the bacterial strain until a pure culture strain was obtained. The strain was then stored at -80°C for later use.

[0064] The above method yielded the initial screening strains. The initial screening strains were then further screened, including the following steps: after stabilizing the initial screening strains through subculturing, they were inoculated onto straw cuticle solid culture medium plates and incubated at 40℃ for 3-5 days. Samples that could grow colonies on the straw cuticle solid culture medium were selected, and the degradation effect on the corn straw epidermal cuticle was observed using scanning electron microscopy. The degradation effect on the corn straw epidermal cuticle was used as the decisive factor for the selection of subsequent experimental strains.

[0065] Strain F-1 was obtained using the above method. Strain F-1 was screened from samples from the Chahayang Farm. Strain F-1 was cultured on Gao's No. 1 solid medium at 35℃ for 3 days. The colony morphology is as follows: Figure 1 The left image shows that strain F-1 grows relatively small, with colonies measuring 2 mm. The colony protrusions are indistinct, the colonies are opaque (white-gray), with neat edges and a rough surface. It then develops into a powdery, filamentous form, showing differentiation of intracellular and aerial hyphae. Electron micrographs of conidial hyphae are shown below. Figure 1 As shown in the right image, a single conidium is about 1 μm long and 0.7-0.9 μm wide, with the hyphae being spiral-shaped.

[0066] Regarding growth characteristics, strain F-1 was cultured under different temperature conditions, and it was verified that strain F-1 can grow in the range of 15-40℃. Among them, strain F-1 grew faster at 30℃-40℃ within 5 days of culture.

[0067] Example 2

[0068] Bacterial DNA was extracted using a bacterial genomic DNA extraction kit and amplified by PCR using universal primers 27F / 1492R.

[0069] The PCR amplification reaction system included: 1 μL of genomic DNA (20 ng / μL), 10× Buffer (containing 2.5 mM Mg) 2+ 5 μL, Taq polymerase (5 u / μL) 1 μL, dNTP (10 mM) 1 μL, primer 27F (10 μM) 1.5 μL, primer 1492R (10 μM) 1.5 μL, ddH2O to make up to 50.0 μL.

[0070] The PCR amplification reaction program included: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1.5 min, for 35 cycles; and final extension at 72℃ for 7 min.

[0071] PCR products were recovered using the AxyPrep DNA gel extraction kit. The PCR amplification products were sent to Shanghai Paisennong Biotechnology Co., Ltd. for sequencing. The obtained sequences were compared with the GenBank database using BLAST software for homology analysis.

[0072] After amplification and sequencing of strain F-1 using universal bacterial primers, the sequence was compared with the NCBI BLAST database, and a phylogenetic tree was constructed using MAGE 11.0 software. Figure 2 As shown, strain F-1 is most closely related to Streptomyces griseus, reaching 100%.

[0073] The 16S rRNA gene sequence of strain F-1 is shown in SEQ ID NO:3.

[0074]

[0075] Strain F-1 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on May 10, 2024, with accession number CCTCC NO:M2024899 and accession name Streptomyces griseorubens F-1.

[0076] When using the waxy cuticle of straw as a degradation indicator, conventional methods are insufficient to verify its degradation effect. This invention uses straw epidermis as a culture medium and employs multiple methods to analyze and verify surface changes to validate the degradation effect.

[0077] Example 3

[0078] (1) Preparation of a culture medium containing treated corn stalk epidermis

[0079] Treated corn stalk epidermis was added to Gao's No. 1 medium and carbon-free mineral salt medium, respectively, with a final concentration of 2 g / L, to obtain a medium containing treated corn stalk epidermis.

[0080] The preparation method of the above-mentioned treated corn stalk skin includes the following steps: harvesting corn stalks, air-drying them naturally to form a gap between the skin and the stalk, peeling off the surface skin, boiling it in a solution containing 4 g / L oxalic acid and 16 g / L ammonium oxalate for 4 hours to remove carbohydrates, washing it with sterile deionized water and drying it, cutting it into square thin slices ≥0.5cm×0.5cm in size, sterilizing it before use.

[0081] (2) The strain F-1 was activated at 35°C for 24 hours in a carbon-free mineral salt medium to obtain the activated F-1 bacterial solution, which was then used in the following experiments.

[0082] (3) The activated F-1 bacterial culture was inoculated into the culture medium containing treated corn stalk epidermis prepared in step (1) at an inoculation rate of 6% (volume percentage). The volume of the culture medium containing the treated corn stalk epidermis was 50% (i.e., the ratio of culture medium volume to Erlenmeyer flask volume was 50%). The medium was cultured on a shaker at 35°C and 120 rpm. On day 5, the corn stalk epidermis was removed, the mycelium attached to the surface was washed off, and then dried in an oven at 80°C for 10 hours. The dry weight of the corn stalk epidermis was then weighed using a microbalance. The changes in cellulose, hemicellulose, and lignin in the corn stalk epidermis were measured using a CXC-06 crude fiber analyzer. Six parallel treatments were set up for each group.

[0083] Weight loss rate = (initial weight of corn stalk husk dry weight - final weight of corn stalk husk dry weight) / initial weight of corn stalk husk dry weight * 100%.

[0084] Cellulose degradation rate = (initial weight of cellulose - final mass of cellulose) / initial weight of cellulose * 100%.

[0085] The degradation rate of hemicellulose = (initial weight of hemicellulose - final weight of hemicellulose) / initial weight of hemicellulose * 100%.

[0086] The degradation rate of lignin = (initial weight of lignin - final mass of lignin) / initial weight of lignin * 100%.

[0087] The results of the weight loss rate test are as follows Figure 3 As shown in Figure A, the weight loss of corn stalk epidermis was 28.87% after culturing with Gao's No. 1 medium containing treated corn stalk epidermis for 5 days. After culturing with carbon-free mineral salt medium containing treated corn stalk epidermis for 5 days, the weight loss was 22.57%. These results indicate that strain F-1 can degrade corn stalk epidermis in both Gao's No. 1 medium and inorganic salt medium. The effect of culturing with Gao's No. 1 medium was superior to that with inorganic salt medium.

[0088] The results of the degradation rates of cellulose, hemicellulose, and lignin are as follows: Figure 3 As shown in Figure B, after 5 days of fermentation, strain F-1 significantly degraded cellulose, hemicellulose, and lignin, with strain F-1 showing the best degradation effect on lignin. Compared to carbon-free mineral salt medium, strain F-1 exhibited better degradation performance when cultured on Gao's No. 1 medium.

[0089] Example 4

[0090] The effects of different culture media on the growth of strain F-1 were investigated using the following experimental methods:

[0091] (1) Activate strain F-1 to obtain activated F-1 bacterial solution. The activation method is the same as in Example 3.

[0092] (2) The activated F-1 bacterial solution from step (1) was inoculated into Gao's No. 1 medium at 6% (volume percentage) and cultured on a shaker at 35°C and 120 rpm for 24 hours to obtain the bacterial solution; the bacterial solution was inoculated into Gao's No. 1 medium at 6% (volume percentage) and cultured on a shaker at 35°C and 120 rpm for 100 hours.

[0093] (3) The activated strain F-1 bacterial solution from step (1) was inoculated into carbon-free mineral salt medium at a rate of 6% (volume percentage) and cultured on a shaker at 35°C and 120 rpm for 24 h to obtain the bacterial solution; the bacterial solution was inoculated into carbon-free mineral salt medium at a rate of 6% (volume percentage) and cultured on a shaker at 35°C and 120 rpm for 100 h.

[0094] (4) Under aseptic conditions, the fermentation broth was taken once every 10 hours, with 2 mL of fermentation broth taken each time, and three replicates were set up for each sample. The pH of the fermentation broth was measured using a pH meter and the dry weight of the strain was determined.

[0095] Experimental results are as follows Figure 4 As shown. From Figure 4 A shows that when F-1 was cultured in Gao's No. 1 medium, the logarithmic growth phase lasted from 20 to 60 hours, and then stabilized after 70 hours. When F-1 was cultured in carbon-free mineral salt medium, it grew rapidly from 30 to 40 hours, and then stabilized after 60 hours. The logarithmic growth phase of F-1 was shorter under carbon-free mineral salt medium conditions. Figure 4 As can be seen from B, the pH of F-1 showed a partial decreasing trend within 10-40 hours of culture. In the later stage of culture, the pH of F-1 showed a significant increasing trend regardless of whether it was cultured in carbon-free mineral salt medium or Gao's No. 1 medium.

[0096] Example 5

[0097] The morphology of the stratum corneum under different treatments was analyzed using scanning electron microscopy (SEM), and the samples were divided into four groups: A, B, C, and D.

[0098] Group A consists of straw outer skins after sterilization and autoclaving. The preparation method includes the following steps: Harvesting corn stalks, allowing them to air dry naturally until a gap forms between the outer skin and the stalk, peeling off the outer skin, boiling it for 4 hours in a solution containing 4 g / L oxalic acid and 16 g / L ammonium oxalate to remove carbohydrates, washing with sterile deionized water, drying, cutting into 0.8 cm × 0.8 cm square sheets, and sterilizing. The outer skins are then dried in a 60℃ oven for 8 hours, followed by gold coating. The straw outer skins are observed using a scanning electron microscope.

[0099] Group B consisted of straw epidermis soaked in a chloroform-methanol (1:1 volume ratio) mixed solution for 24 hours. The preparation method included the following steps: Corn stalks were harvested and air-dried until a gap formed between the epidermis and the stalk. The epidermis was peeled off, soaked in a chloroform-methanol (1:1 volume ratio) mixed solution for 24 hours, washed with sterile deionized water, dried, and cut into 0.8cm × 0.8cm square slices, which were then sterilized. The epidermis was dried in a 60℃ oven for 8 hours, then sprayed with gold coating. The straw epidermis was observed using a scanning electron microscope.

[0100] Group C consisted of straw epidermis from F-1 fermentation culture for 3 days. The preparation method included the following steps: F-1 was cultured using Gao's No. 1 medium. The straw epidermis was prepared as a 0.8cm × 0.8cm square sheet. The experimental method followed the procedure in Example 3. After 3 days of culture, the straw epidermis was removed. The epidermis was then dried in a 60℃ oven for 8 hours, followed by gold coating. The straw epidermis was observed using a scanning electron microscope.

[0101] Group D consisted of straw epidermis fermented from F-1 cells for 5 days. The preparation method included the following steps: F-1 cells were cultured using Gao's No. 1 medium. The straw epidermis was prepared as a 0.8cm × 0.8cm square sheet. The experimental method followed the procedure in Example 3. After 5 days of culture, the straw epidermis was removed, and the mycelium was wiped off. The epidermis was then dried in a 60℃ oven for 8 hours, followed by gold coating. The straw epidermis was observed using a scanning electron microscope.

[0102] Experimental results are as follows Figure 5 As shown, A is the straw skin after sterilization and high-pressure sterilization, B is the straw skin after soaking in a chloroform-methanol (volume ratio 1:1) mixed solution for 24 hours, C is the straw skin after fermentation culture of strain F-1 for 3 days, and D is the straw skin after fermentation culture of strain F-1 for 5 days.

[0103] After sterilization and high-pressure sterilization, there were no obvious changes on the surface of the straw, but a few wrinkles appeared in the cuticle wax. Figure 5 A). After soaking in a chloroform-methanol (1:1 volume ratio) solution for 24 hours, some changes occurred in the epidermis. The smoothness of the keratin wax surface was altered, and the surface became rougher, but the dissolved keratin wax and keratinocytes were not destroyed. Figure 5 B).

[0104] The corn stalk epidermis treated with strain F-1 showed significant differences compared to other treatments. After 3 days of cultivation, the mycelium of strain F-1 adhered to the epidermis, some epidermal cells were exposed, some intercellular spaces were blocked, and the stomata on the epidermis were also closed. Figure 5 C). After culturing strain F-1 for 5 days and wiping away the hyphae, it can be seen that the keratin wax disappears, the epidermal hairs and epidermal cells are completely exposed, gaps appear between some epidermal cells, the grid pattern between epidermal cells is exposed, the guard cells in the stomata are compressed to the center, the stomata are opened, and the keratin and keratin wax are degraded. Figure 5 D).

[0105] Example 6

[0106] The water contact angle of the stratum corneum under different treatments was measured and designated as group A, group B, and group C.

[0107] Group A consists of untreated corn stalk skins. The preparation method includes the following steps: harvesting corn stalks, air-drying them naturally until a gap forms between the skin and the stalk, peeling off the surface skin, washing with sterile deionized water, drying, cutting into 0.8cm × 0.8cm square slices, sterilizing, and then using.

[0108] Group B consists of pretreated corn stalk outer skin. The preparation method includes the following steps: harvesting corn stalks, allowing them to air dry naturally until a gap forms between the outer skin and the stalk, peeling off the outer skin, boiling it in a solution containing 4 g / L oxalic acid and 16 g / L ammonium oxalate for 4 hours to remove carbohydrates, washing with sterile deionized water, drying, and cutting into 0.8 cm × 0.8 cm square sheets. Sterilize before use.

[0109] Group C consists of corn stalk epidermis treated with strain F-1. The preparation method includes the following steps: referring to the method in Example 3, F-1 is inoculated into Gao's No. 1 medium containing the treated corn stalk epidermis and cultured at an inoculation amount of 6%. The size of the corn stalk epidermis is 0.8×0.8cm. Fermentation culture is carried out at 35℃ for 3 days. The corn stalk epidermis with mycelium attached is taken and the mycelium is wiped off.

[0110] The surface of each group was dried in a 60℃ oven for 8 hours. The test was conducted at a constant temperature of 20℃, and the test liquid was deionized water (1.5μL). The seat drop method was used, that is, deionized water was dropped onto the coating surface. The average contact angle of the left and right sides of each test was taken as the contact angle of the tested area. Three different areas of each sample were tested to calculate the average value.

[0111] Water contact angle of corn stalk epidermis after different treatments, such as Figure 6 As shown in the figure, A represents the untreated straw epidermis, B represents the pretreated epidermis, and C represents the straw epidermis treated with strain F-1. It can be seen that after treating the straw epidermis with strain F-1, the average contact angle is 60.59°, indicating a decrease in hydrophobicity. This means that after treatment with the strain, the cuticle wax on the straw epidermis is degraded, reducing hydrophobicity and making the straw epidermis more easily wetted by water.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of *Streptomyces griseus* with cuticle-degrading ability, characterized in that, The strain name is Streptomyces griseorubens F-1, and the preservation number is CCTCC NO:M2024899.

2. A microbial agent, characterized in that, Includes the *Streptomyces griseus* of claim 1 and / or the fermentation product of the *Streptomyces griseus*.

3. A fermentation culture method for *Streptomyces griseus* as described in claim 1, characterized in that, Includes the following steps: The *Streptomyces griseus* was inoculated into the culture medium and fermented.

4. The fermentation culture method of *Streptomyces griseus* according to claim 3, characterized in that, The fermentation temperature is 15-40℃.

5. The use of the *Streptomyces griseus* according to claim 1 in any one or more of (1) to (5); (1) Degradation of the cuticle of plant debris; (2) Degradation of plant residues; (3) Degradation of cellulose; (4) Degradation of hemicellulose; (5) Degradation of lignin.

6. The use of the microbial agent according to claim 2 in any one or more of (1) to (5); (1) Degradation of the cuticle of plant debris; (2) Degradation of plant residues; (3) Degradation of cellulose; (4) Degradation of hemicellulose; (5) Degradation of lignin.

7. A method for degrading the cuticle of plant debris using *Streptomyces griseus* as described in claim 1, characterized in that, The process includes the following steps: inoculating the *Streptomyces glaucusi* of claim 1 onto a culture medium containing the cuticle of plant debris, and then fermenting and culturing it.

8. The method according to claim 7, characterized in that, The fermentation temperature is 15-40℃.

9. A method for degrading the cuticle of plant residues using the microbial agent according to claim 2, characterized in that, The process includes the following steps: inoculating the microbial agent described in claim 2 into a culture medium containing the cuticle of plant residues, and then fermenting and culturing it.

10. The method according to claim 9, characterized in that, The fermentation temperature is 15-40℃.

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