A strain of heterostem mold and a method for degrading cellulose in Chinese medicine residues and a composite fungus and a method for degrading cellulose in Chinese medicine residues

Abstract

The present invention relates to the field of microbial fermentation technology, and in particular to a strain of Paraphoma chlamydocopiosa and a method for degrading cellulose in Chinese medicine residues, and a composite bacteria and a method for degrading cellulose in Chinese medicine residues. The present invention provides a strain of Paraphoma chlamydocopiosa AM‑PC, which is deposited in the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of October 9, 2023 and a deposit number of CGMCC No. 40893. The Paraphoma chlamydocopiosa AM‑PC of the present invention is combined with Trichoderma viride to produce cellulase using Chinese medicine residues as a culture medium, has high activity, and can degrade cellulose in Chinese medicine residues.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, in particular to a strain of Heterostyphus heterostemonus and a method for degrading cellulose in traditional Chinese medicine residues and a composite bacteria and a method for degrading cellulose in traditional Chinese medicine residues. Background Art

[0002] Cellulosic waste, including Chinese medicine residue, is extremely difficult to degrade in the natural environment. Its degradation process mainly relies on the cellulase system composed of exoglucanase, endoglucanase and β-glucosidase produced by microorganisms. Biofermentation technology, especially solid-state fermentation technology, is to directly inoculate cellulose-degrading strains isolated from the natural environment into cellulosic waste for proliferation and enzyme production. Due to its economic, efficient and environmentally friendly characteristics, it has been widely used in the field of recycling of cellulose residues. In addition to cellulose, Chinese medicine residue also contains nutrients such as soluble sugars, plant proteins, fats, and effective ingredients such as flavonoids, saponins, polysaccharides and amino acid compounds, which are suitable for microbial growth and have high utilization value in the field of biofermentation to produce cellulase.

[0003] Cellulose-degrading strains in nature include bacteria, fungi, and actinomycetes. Among them, Trichoderma spp., especially Trichoderma viride, has become one of the most widely studied filamentous fungi due to its rapid growth, simple culture conditions, complete composition of extracellular cellulase system, and ability to efficiently degrade cellulose. However, single-species fermentation has problems such as easy degradation of strains and low substrate utilization. Mixed fermentation is faster and more thorough than single pure strain culture fermentation. Microorganisms in mixed bacterial communities usually select different components of the matrix as nutrients, which can improve the utilization rate of raw materials. There are often beneficial interactions between mixed multi-species, which helps to promote growth and metabolism. In addition, the complex interactions between mixed fermentations can also help inhibit contamination by foreign bacteria. Therefore, further exploring new cellulase-producing microbial resources and screening strains with high cellulase activity that can be co-cultured with Trichoderma viride have great application prospects. Summary of the invention

[0004] The invention aims to develop a strain for co-fermenting with viride to produce cellulase to degrade cellulose in Chinese medicine residues, and particularly relates to a strain of heterostem mold and a method for degrading cellulose in Chinese medicine residues and a composite bacteria and a method for degrading cellulose in Chinese medicine residues.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a strain of Paraphomachlamydocopiosa AM-PC, which is deposited in the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of October 9, 2023 and a deposit number of CGMCC No.40893.

[0007] The invention also provides the use of the polypore AM-PC in preparing products for degrading cellulose in traditional Chinese medicine residues.

[0008] The present invention also provides a method for degrading cellulose in traditional Chinese medicine residues by using the AM-PC fungus, comprising the following steps:

[0009] The fungus blocks of AM-PC were inoculated into the Chinese medicine residue culture medium and cultured for 8-10 days.

[0010] The mass ratio of the fungus blocks of the heterostemona AM-PC to the Chinese medicine residue culture medium is 9 to 11 blocks: 10 g;

[0011] The diameter of the fungus block of the AM-PC fungus is 6 to 10 mm.

[0012] Preferably, the Chinese medicinal residue culture medium comprises Chinese medicinal residue and water;

[0013] The mass volume ratio of the Chinese medicine residue and water is 1g:1-3mL;

[0014] The culture temperature is 26-30°C.

[0015] The invention also provides a composite fungus, comprising the heterostemonas AM-PC and trichoderma viride.

[0016] The invention also provides application of the composite bacteria in preparing products for degrading cellulose in traditional Chinese medicine residues.

[0017] The present invention also provides a method for degrading cellulose in traditional Chinese medicine residues using the composite bacteria, comprising the following steps:

[0018] (1) Inoculating the fungus block of AM-PC into a Chinese medicine residue culture medium and culturing for 3 to 6 days to obtain an intermediate;

[0019] (2) Inoculate the intermediate with Trichoderma viride and culture for 3 to 6 days.

[0020] Preferably, in step (1), the mass ratio of the fungus blocks of the heterostemonas AM-PC to the Chinese medicinal residue culture medium is 9 to 11 blocks: 10 g;

[0021] The diameter of the fungus block of the AM-PC fungus is 6 to 10 mm;

[0022] The Chinese medicinal residue culture medium in step (1) comprises Chinese medicinal residue and water;

[0023] The mass volume ratio of the traditional Chinese medicine residue and water is 1g:1-3mL.

[0024] Preferably, the mass volume ratio of the Chinese medicinal residue culture medium to the green Trichoderma is 10 g: 1-3 mL;

[0025] The initial spore concentration of the green mold is 9 to 11×10 6 cfu / mL.

[0026] Preferably, the culture temperatures in step (1) and step (2) are independently 26 to 30°C.

[0027] The present invention provides a strain of Heterostyphus heterostemonus and a method for degrading cellulose in Chinese medicine residues and a composite fungus and a method for degrading cellulose in Chinese medicine residues. The scheme of the present invention has the following advantages:

[0028] The invention provides a strain of heterostem mold AM-PC, which can be co-cultured with green Trichoderma. After the heterostem mold AM-PC is first inoculated in a Chinese medicine residue culture medium for 3 to 6 days, the double fermentation system inoculated with green Trichoderma can produce high-activity cellulase. Using astragalus residue as a fermentation medium, the AM-PC is first inoculated for 3 days and then the green Trichoderma is inoculated to obtain a fermentation system in which the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme are respectively increased by 225.07% and 72.03% compared with the single fermentation of green Trichoderma; and are respectively increased by 56.53% and 41.17% compared with the single fermentation of AM-PC. The filter paper enzyme and sodium carboxymethyl cellulose enzyme activities in the fermentation system obtained by first inoculating AM-PC for 6 days and then inoculating green Trichoderma are respectively increased by 164.93% and 39.64% compared with the single fermentation of green Trichoderma; and are respectively increased by 27.57% and 14.59% compared with the single fermentation of AM-PC. In the fermentation system obtained by inoculating AM-PC for 3 days and then inoculating Trichoderma viride with mixed residues of Panax notoginseng and Salvia miltiorrhiza as fermentation medium, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme increased by 69.77% and 34.42% respectively compared with Trichoderma viride fermentation alone; and increased by 37.32% and 36.09% respectively compared with AM-PC fermentation alone. In the fermentation system obtained by inoculating AM-PC for 6 days and then inoculating Trichoderma viride, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme increased by 37.42% and 9.37% respectively compared with Trichoderma viride fermentation alone; and increased by 11.15% and 10.73% respectively compared with AM-PC fermentation alone. After 6 days of fermentation, the cellulose degradation rate of Astragalus residue treated by double fermentation was 54.43%-58.38%, which was 73.36%-85.93% higher than that of single fermentation of Trichoderma viride, and 19.68%-28.35% higher than that of single fermentation of AM-PC; the cellulose degradation rate of mixed residue of Panax notoginseng and Salvia miltiorrhiza treated by double fermentation was 65.54%-66.28%, which was 72.48%-74.42% higher than that of single fermentation of Trichoderma viride, and 30.37%-31.83% higher than that of single fermentation of AM-PC. It has the advantages of higher cellulase activity, lower cost and higher environmental protection in the rational resource utilization of Chinese medicine residue. It can be used to degrade cellulose in Chinese medicine residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The colony structure and hyphae morphology of AM-PC in PDA medium (Figure A shows the colony, and Figure B shows the hyphae).

[0030] Figure 2 These are the degradation circles of AM-PC in the limited carbon source medium (Figure A is the degradation circle of AM-PC in the limited carbon source medium of sodium carboxymethyl cellulose, Figure B is the degradation circle of AM-PC in the limited carbon source medium of xylan, and Figure C is the degradation circle of AM-PC in the limited carbon source medium of citrus pectin).

[0031] Figure 3 These are the results of co-cultivation of AM-PC and Trichoderma viride (A1, B1 and C1 represent the results of AM-PC being cultured for 3d, 7d and 15d, respectively; A2, B2 and C2 represent the results of co-cultivation of AM-PC and Trichoderma viride for 3d, 7d and 15d, respectively).

[0032] Figure 4 Cellulase activity of Astragalus residue in single fermentation and double fermentation of AM-PC and Trichoderma viride (TV, PC, PC-3d-TV and PC-6d-TV represent single fermentation of Trichoderma viride, single fermentation of AM-PC, double fermentation of AM-PC inoculated with Trichoderma viride after 3 days of fermentation and double fermentation of AM-PC inoculated with Trichoderma viride after 6 days of fermentation, respectively).

[0033] Figure 5 Cellulase activity of mixed medicinal residues of Panax notoginseng and Salvia miltiorrhiza in single fermentation and double fermentation of AM-PC and Trichoderma viride (TV, PC, PC-3d-TV and PC-6d-TV represent single fermentation of Trichoderma viride, single fermentation of AM-PC, double fermentation of AM-PC inoculated with Trichoderma viride after 3 days of fermentation and double fermentation of AM-PC inoculated with Trichoderma viride after 6 days of fermentation, respectively).

[0034] Figure 6 These are pictures of Astragalus residues fermented with AM-PC and inoculated with green Trichoderma after 3 and 6 days of fermentation (A is the case where AM-PC was fermented for 3 days and then inoculated with green Trichoderma for another 6 days, and B is the case where AM-PC was fermented for 6 days and then inoculated with green Trichoderma for another 3 days).

[0035] Figure 7 This is the microscopic structure of the Astragalus residue after AM-PC fermentation for 3 days and inoculation with green Trichoderma for double fermentation.

[0036] Figure 8 These are pictures of dual fermentation of Panax notoginseng and Salvia miltiorrhiza residues by inoculation of green Trichoderma after AM-PC fermentation for 3d and 6d (A shows the case where AM-PC was fermented for 3d and then inoculated with green Trichoderma for another 6d of fermentation, and B shows the case where AM-PC was fermented for 6d and then inoculated with green Trichoderma for another 3d of fermentation).

[0037] Fig. 9 This is the microscopic structure of the residues after AM-PC fermentation for 3 days after inoculation with green Trichoderma to double ferment Panax notoginseng and Salvia miltiorrhiza.

[0038] Fig.10 Cellulose degradation rates of Astragalus residues and mixed residues of Panax notoginseng and Salvia miltiorrhiza by single fermentation and dual fermentation of AM-PC and Trichoderma viride (A is the degradation rate of Astragalus residues, B is the degradation rate of mixed residues of Panax notoginseng and Salvia miltiorrhiza).

[0039] Collection Instructions

[0040] The Paraphoma chlamydocopiosa AM-PC is deposited in the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 9, 2023, and the deposit number is CGMCC No.40893.

[0041] Trichoderma viride TV-AG is deposited in the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 9, 2023, and the deposit number is CGMCC No.40841. DETAILED DESCRIPTION

[0042] The present invention provides a strain of Paraphomachlamydocopiosa AM-PC, which is deposited in the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of October 9, 2023 and a deposit number of CGMCC No.40893.

[0043] The AM-PC was isolated from the root system of Atractylodes macrocephala, which was planted in a traditional Chinese medicine planting base in Anguo City, Hebei Province. After separation, DNA was extracted using a DNA extraction kit, purified by PCR and sequenced, and identified as AM-PC by BLAST comparison, and named AM-PC.

[0044] The invention also provides the use of the polypore AM-PC in preparing products for degrading cellulose in traditional Chinese medicine residues.

[0045] The present invention also provides a method for degrading cellulose in traditional Chinese medicine residues by using the AM-PC fungus, comprising the following steps:

[0046] The fungus blocks of AM-PC of Heterostemma sp. are inoculated into a Chinese medicine residue culture medium and cultured for 8 to 10 days; the mass ratio of the fungus blocks of AM-PC of Heterostemma sp. to the Chinese medicine residue culture medium is 9 to 11:10 g, preferably 10:10 g; the diameter of the fungus blocks of AM-PC of Heterostemma sp. is 6 to 10 mm, preferably 8 mm. The culture time is preferably 9 days. In the present invention, the Chinese medicine residue culture medium includes Chinese medicine residue and water; the mass volume ratio of the Chinese medicine residue and water is 1 g:1 to 3 mL, preferably 1 g:2 mL; the Chinese medicine residue is the residue of Astragalus membranaceus and the mixed residue of Panax notoginseng and Salvia miltiorrhiza; the Chinese medicine residue is the plant residue remaining after the active ingredients are extracted by water extraction. The culture temperature is 26 to 30°C, preferably 28°C.

[0047] The invention also provides a composite fungus, comprising the heterostemonas AM-PC and trichoderma viride.

[0048] In the present invention, the green Trichoderma is green Trichoderma TV-AG, and the green Trichoderma Trichoderma virideTV-AG is deposited in the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of October 9, 2023 and a deposit number of CGMCC No.40841.

[0049] The invention also provides application of the composite bacteria in preparing products for degrading cellulose in traditional Chinese medicine residues.

[0050] The present invention also provides a method for degrading cellulose in traditional Chinese medicine residues using the composite bacteria, comprising the following steps:

[0051] (1) inoculating the fungus block of AM-PC into a Chinese medicine residue culture medium and culturing for 3 to 6 days to obtain an intermediate; (2) inoculating green Trichoderma into the intermediate and culturing for 3 to 6 days.

[0052] In the present invention, the mass ratio of the number of the fungus blocks of the heterostem mold AM-PC in step (1) to the Chinese medicine residue culture medium is 9 to 11:10g, preferably 10:10g; the diameter of the fungus blocks of the heterostem mold AM-PC is 6 to 10mm, preferably 8mm; the Chinese medicine residue culture medium in step (1) includes Chinese medicine residue and water; the mass volume ratio of the Chinese medicine residue and water is 1g:1 to 3mL, preferably 1g:2mL. The Chinese medicine residue is the residue of Astragalus and the mixed residue of Panax notoginseng and Salvia miltiorrhiza; the Chinese medicine residue is the plant residue remaining after the medicinal components are extracted with water. The culturing time in step (1) is preferably 4.5d. In the present invention, the mass volume ratio of the Chinese medicine residue culture medium to Trichoderma viride is 10g:1 to 3mL, preferably 10g:2mL; the initial spore concentration of Trichoderma viride is 9 to 11×10 6 cfu / mL, preferably 10×10 6 cfu / mL.

[0053] In the present invention, the culture temperature in step (1) and step (2) is independently 26-30° C., preferably 28° C. The culture time in step (2) is preferably 4.5 days.

[0054] The solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0055] The green Trichoderma described in the embodiment of the present invention is green Trichoderma Trichoderma viride TV-AG.

[0056] Example 1

[0057] Isolation and identification of strains

[0058] The roots of Atractylodes macrocephala were taken from the Chinese herbal medicine planting base in Anguo City, Hebei Province, washed with sterile water for 6 times, disinfected with 75% ethanol for 4 minutes, disinfected with sodium hypochlorite for 2 minutes, and then washed with sterile water for 3 times. They were cultured in PDA plate culture medium, colonies were picked, and the colonies were purified. Figure 1 A. The mycelial morphology was observed under a 400x optical microscope. Figure 1 As shown in B.

[0059] Colony DNA was extracted using a genomic DNA extraction kit (SolarBio). Colony DNA was amplified using primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3'), and the amplified products were sequenced. NCBI's BLAST tool was used for comparison, and the phylogenetic tree was constructed using MEGA software. The results showed that the strain was Paraphoma chlamydocopiosa, named AM-PC.

[0060] Example 2

[0061] Determination of the ability of strains to degrade cellulose

[0062] Colonies of AM-PC strains were selected and inoculated on restricted carbon source medium (sodium carboxymethylcellulose, xylan and citrus pectin) respectively, and the ratio of the transparent degradation zone formed after culture to the colony diameter was used to evaluate the strain's ability to degrade cellulose, hemicellulose and pectin. The restricted carbon source medium was configured as follows: 1L sterile water contained 1g NH4NO3, 0.5g MgSO4·7H2O, 1g KH2PO4, 0.5g NaCl, and 20g agar. The sodium carboxymethylcellulose restricted carbon source medium also included 15g sodium carboxymethylcellulose; the xylan restricted carbon source medium also included 5g xylan; and the citrus pectin restricted carbon source medium also included 2g citrus pectin.

[0063] The AM-PC strain was inoculated into a limited carbon source sodium carboxymethyl cellulose medium, cultured at 28°C in the dark for 7 days, stained with 1% Congo red dye for 20 minutes, the dye was discarded, and then decolorized with 1 mol / L NaCl solution for 15 minutes to produce a transparent degradation zone. The results are as follows: Figure 2 As shown in A, the ratio of the transparent zone to the colony diameter is 1.031.

[0064] The AM-PC strain was inoculated into the limited carbon source xylan medium, cultured at 28°C in the dark for 5 days, stained with 2 mg / mL Congo red dye for 2 hours, the dye was discarded, and then decolorized with 1 mol / L NaCl solution for 15 minutes to produce a transparent degradation zone. The results are as follows Figure 2 As shown in B, the ratio of the transparent zone to the colony diameter is 0.972.

[0065] The AM-PC strain was inoculated into the limited carbon source citrus pectin medium, cultured at 28°C in the dark for 5 days, and stained with 1% hexadecyltrimethylammonium bromide solution for 4 hours to produce a transparent degradation zone. Figure 2 As shown in C, the ratio of the transparent zone to the colony diameter is 0.988.

[0066] Figure 2 The results showed that the AM-PC strain had the ability to degrade cellulose, hemicellulose and pectin.

[0067] Example 3

[0068] Co-culture of AM-PC strain with Trichoderma viride TV-AG

[0069] AM-PC strains and Trichoderma viride TV-AG solid strains were inoculated on both sides of the PDA medium and co-cultured for 15 days. Photos were taken at 3 days, 7 days, and 15 days to record the co-culture of the two strains. The results are as follows: Figure 3 shown.

[0070] Figure 3 The results showed that the two strains did not show inhibition zones when co-cultured in PDA medium, and they had the characteristics of co-culture. The growth rate of Trichoderma viride was higher than that of AM-PC, so when conducting double fermentation, AM-PC strains were inoculated first and then Trichoderma viride.

[0071] Example 4

[0072] Degradation of cellulose in traditional Chinese medicine residues by AM-PC strain and Trichoderma viride TV-AG alone

[0073] The astragalus residue and water were prepared in a mass volume ratio of 1g:2mL to obtain the astragalus residue culture medium.

[0074] The notoginseng residues and the salvia miltiorrhiza residues were mixed in a mass ratio of 1:5.1 to obtain mixed residues; the mixed residues were mixed with water in a mass volume ratio of 1 g:2 mL to obtain a mixed Chinese medicine residue culture medium.

[0075] Use a blue pipette tip (8mm in diameter) to take 10 AM-PC strains in 10g of astragalus residue culture medium or mixed Chinese medicine residue culture medium, place it in a constant temperature and humidity incubator, avoid light, and ferment it at 28℃ for 9 days. Take the fermentation sample, weigh it, add sterile water to the fermentation matrix at a ratio of 1:10, extract it at 28℃ and 140rpm for 1h, centrifuge the extract at 4000rpm for 15min, take the supernatant as the crude enzyme solution, and measure the activity of filter paper enzyme and sodium carboxymethyl cellulose enzyme in the crude enzyme solution. The results are as follows Figures 4-5 shown.

[0076] Take Trichoderma viride TV-AG (viable spore count is 1.0×10 7 cfu / mL) were inoculated into 10g of astragalus residue culture medium or mixed Chinese medicine residue culture medium, and placed in a constant temperature and humidity incubator in the dark at 28°C for 9 days. The fermentation sample was weighed and sterile water was added to the fermented substrate at a ratio of 1:10. The fermentation was performed at 28°C and 140rpm for 1h. The extract was centrifuged at 4000rpm for 15min. The supernatant was taken as the crude enzyme solution, and the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme in the crude enzyme solution were determined. The results are shown in Figures 4-5 shown.

[0077] Using the residue of Astragalus as the culture medium, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme fermented by Trichoderma viride alone were 1.73 and 2.74 U / g, respectively; the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme fermented by AM-PC alone were 3.59 and 3.34 U / g, respectively. Using the mixed residue of Panax notoginseng and Salvia miltiorrhiza as the culture medium, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme fermented by Trichoderma viride alone were 1.96 and 2.68 U / g, respectively; the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme fermented by AM-PC alone were 2.43 and 2.64 U / g, respectively. The strain AM-PC of the present invention can produce higher cellulase activity than Trichoderma viride for Astragalus residue, and can produce higher filter paper enzyme activity than Trichoderma viride for the mixed residue of Panax notoginseng and Salvia miltiorrhiza, and has strong application value in the field of preparing cellulase by biological fermentation.

[0078] Example 5

[0079] Co-culture of AM-PC strain and Trichoderma viride TV-AG to degrade cellulose in traditional Chinese medicine residues

[0080] Ten AM-PC strains were taken with a blue pipette tip (8 mm in diameter) and placed in 10 g of the astragalus residue culture medium or the mixed Chinese medicine residue culture medium prepared in Example 4. The mixture was placed in a constant temperature and humidity incubator and kept away from light at 28 °C for 3 and 6 days of fermentation. On the 4th and 7th days of fermentation, 2 mL of green Trichoderma (the number of viable spores was 1.0 × 10 7cfu / mL), and continued to ferment at 28℃ for 6d and 3d. Take the fermentation sample and weigh it, add sterile water to the fermentation substrate at a ratio of 1:10, extract at 28℃ and 140rpm for 1h, centrifuge the extract at 4000rpm for 15min, take the supernatant as the crude enzyme solution, and determine the activity of filter paper enzyme and sodium carboxymethyl cellulose enzyme in the crude enzyme solution. The results are as follows Figures 4-5 As shown. Using the Astragalus residue as the culture medium, AM-PC strains were fermented for 3 days and 6 days before inoculating green Trichoderma for double fermentation. After the fermentation, the fermentation of the Astragalus residue was as shown in Figure 6 As shown in the figure, the microstructure of the double-fermented Astragalus residue observed under a 400x microscope is as follows Figure 7 The AM-PC strain was fermented with the residues of Panax notoginseng and Salvia miltiorrhiza as the culture medium for 3 and 6 days before inoculation with Trichoderma viride for double fermentation. The fermentation of the mixed residues of Panax notoginseng and Salvia miltiorrhiza after fermentation was completed was shown in Figure 2. Figure 8 As shown in Figure 2, the microstructure of the double-fermented mixed residue of Panax notoginseng and Salvia miltiorrhiza was observed under a 400x microscope. Fig. 9 shown.

[0081] Using astragalus residue as culture medium, after AM-PC fermentation for 3 days, inoculated with viride, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme were 5.61 and 4.72 U / g, respectively; after AM-PC fermentation for 6 days, inoculated with viride, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme were 4.57 and 3.83 U / g, respectively. Inoculation of viride after AM-PC fermentation for 3 days was the best inoculation method for AM-PC and viride dual fermentation of astragalus residue. Under this inoculation condition, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme were significantly increased by 225.07% and 72.03% respectively compared with viride single fermentation, and significantly increased by 56.53% and 41.17% respectively compared with AM-PC single fermentation.

[0082] Using the mixed residues of Panax notoginseng and Salvia miltiorrhiza as the culture medium, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme inoculated with Trichoderma viride after 3 days of AM-PC fermentation were 3.33 and 3.60 U / g, respectively; the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme inoculated with Trichoderma viride after 6 days of AM-PC fermentation were 2.70 and 2.93 U / g, respectively. Inoculation of Trichoderma viride after 3 days of AM-PC fermentation was the best inoculation method for AM-PC and Trichoderma viride dual fermentation of Panax notoginseng and Salvia miltiorrhiza. Under this inoculation condition, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme increased by 69.77% and 34.42% respectively compared with Trichoderma viride single fermentation; and increased by 37.32% and 36.09% respectively compared with AM-PC single fermentation.

[0083] Figures 6 to 9 The results showed that the fermentation matrix and microscopic observations revealed the presence of AM-PC hyphae and Trichoderma viride hyphae and conidia in the fermentation matrix, indicating that the two strains coexisted well in the dual fermentation system.

[0084] Example 6

[0085] Cellulose degradation rate test

[0086] The cellulose content in the residues of Astragalus, Panax notoginseng and Salvia miltiorrhiza, which were fermented for 6 days after the fermentation and co-fermentation of Example 4 and Example 5, was determined using a cellulose (CLL) content detection kit from Boxbio, and the degradation rate of cellulose after fermentation was calculated. Cellulose degradation rate (%) = (cellulose content of the matrix before fermentation - cellulose content of the matrix after fermentation) / cellulose content of the matrix before fermentation

[0087] The results are as follows Fig.10 shown.

[0088] Using the residue of Astragalus membranaceus as the culture medium, the degradation rate of cellulose in the residue was 31.40% when Trichoderma viride was fermented alone; the degradation rate of cellulose in the residue was 45.48% when AM-PC was fermented alone; the degradation rate of cellulose in the residue was 58.38% when AM-PC was inoculated 3 days later and Trichoderma viride was inoculated 6 days later, and the degradation rate of cellulose in the residue was 54.43%. The degradation rate of cellulose in the residue treated with double fermentation was 73.36% to 85.93% higher than that of Trichoderma viride alone, and 19.68% to 28.35% higher than that of AM-PC alone.

[0089] Using the residues of Panax notoginseng and Salvia miltiorrhiza as culture medium, the cellulose degradation rate in the residues was 38.00% when Trichoderma viride was fermented alone; the cellulose degradation rate in the residues was 50.27% when AM-PC was fermented alone; the cellulose degradation rate in the residues was 66.28% when AM-PC was inoculated 3 days later and Trichoderma viride was inoculated 6 days later, and the cellulose degradation rate in the residues was 65.54%. The cellulose degradation rate in the residues treated with double fermentation was 72.48% to 74.42% higher than that of Trichoderma viride alone, and 30.37% to 31.83% higher than that of AM-PC alone.

[0090] It can be seen from the above embodiments that the present invention provides a strain of Heterostysoma and a method for degrading cellulose in Chinese medicine residues, as well as a composite fungus and a method for degrading cellulose in Chinese medicine residues. The Heterostysoma AM-PC of the present invention is co-cultured with Trichoderma viride, and cellulase is produced using Chinese medicine residues as a carbon source, with high activity. With Astragalus residues as a culture medium, Heterostysoma AM-PC was fermented alone for 9 days, and the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme reached 3.59 and 3.34 U / g, respectively; with a mixture of Panax notoginseng and Salvia miltiorrhiza residues as a culture medium, Heterostysoma AM-PC was fermented alone for 9 days, and the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme were 2.43 and 2.64 U / g, respectively. The dual fermentation inoculation system of AM-PC and green Trichoderma was inoculated with green Trichoderma 3 days after AM-PC. When Astragalus residues were used as the culture medium, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme were 5.61 and 4.72 U / g, respectively, which were significantly increased by 225.07% and 72.03% compared with single fermentation of green Trichoderma, and by 56.53% and 41.17% compared with single fermentation of AM-PC; when Panax notoginseng and Salvia miltiorrhiza residues were used as the culture medium, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme were 3.33 and 3.60 U / g, respectively, which were increased by 69.77% and 34.42% compared with single fermentation of green Trichoderma, and by 37.32% and 36.09% compared with single fermentation of AM-PC. When Trichoderma viride was inoculated 6 days after AM-PC was inoculated, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme were 4.57 and 3.83 U / g, respectively, when Astragalus residues were used as culture medium, which were significantly increased by 164.93% and 39.64% respectively compared with the single fermentation of Trichoderma viride, and significantly increased by 27.57% and 14.59% respectively compared with the single fermentation of AM-PC; when Panax notoginseng and Salvia miltiorrhiza residues were used as culture medium, the activities of filter paper enzyme and sodium carboxymethyl cellulose enzyme were 2.70 and 2.93 U / g, respectively, which were increased by 37.42% and 9.37% respectively compared with the single fermentation of Trichoderma viride, and increased by 11.15% and 10.73% respectively compared with the single fermentation of AM-PC.

[0091] After the double fermentation, the cellulose content in the residue was fermented for another 6 days, and the cellulose degradation rate was calculated. It was finally concluded that the cellulose degradation rate of the Astragalus residue treated with double fermentation was 54.43%~58.38%, which was 73.36%~85.93% higher than that of single fermentation with Trichoderma green, and 19.68%~28.35% higher than that of single fermentation with AM-PC; the cellulose degradation rate of the mixed residue of Panax notoginseng and Salvia miltiorrhiza treated with double fermentation was 65.54%~66.28%, which was 72.48%~74.42% higher than that of single fermentation with Trichoderma green, and 30.37%~31.83% higher than that of single fermentation with AM-PC.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Heterostysoma Paraphoma chlamydocopiosa ) AM-PC, characterized in that Heterostyma Paraphoma chlamydocopiosa AM-PC is deposited in the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 9, 2023, and the deposit number is CGMCC No.40893.

2. Use of the AM-PC of claim 1 in preparing products that degrade cellulose in traditional Chinese medicine residues.

3. A method for degrading cellulose in traditional Chinese medicine residues using the AM-PC of claim 1, characterized in that: The steps include: The fungus blocks of AM-PC were inoculated into the Chinese medicine residue culture medium and cultured for 8-10 days. The mass ratio of the fungus blocks of the heterostemona AM-PC to the Chinese medicine residue culture medium is 9-11:10g; The diameter of the fungus block of the AM-PC fungus is 6-10 mm.

4. The method according to claim 3, characterized in that The Chinese medicine residue culture medium comprises Chinese medicine residue and water; The mass volume ratio of the Chinese medicine residue and water is 1g:1~3mL; The culture temperature is 26-30°C.

5. A composite bacteria, characterized in that: It comprises the heterostemonas AM-PC and Trichoderma viride described in claim 1; The green Trichoderma is green Trichoderma ( Trichoderma viride ) TV-AG, deposited in the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is October 9, 2023, and the deposit number is CGMCC No.40841.

6. Use of the composite bacteria according to claim 5 in preparing products for degrading cellulose in traditional Chinese medicine residues.

7. A method for degrading cellulose in Chinese medicine residues using the composite bacteria according to claim 5, characterized in that: The steps include: (1) Inoculate the fungus block of AM-PC into the Chinese medicine residue culture medium and culture for 3 to 6 days to obtain the intermediate; (2) Inoculate the intermediate with Trichoderma viride and culture for 3 to 6 days.

8. The method according to claim 7, characterized in that The mass ratio of the fungus blocks of the AM-PC of step (1) to the Chinese medicine residue culture medium is 9-11 blocks: 10 g; The diameter of the fungus block of the AM-PC fungus is 6-10 mm; The Chinese medicinal residue culture medium in step (1) comprises Chinese medicinal residue and water; The mass volume ratio of the traditional Chinese medicine residue and water is 1g:1~3mL.

9. The method according to claim 8, characterized in that The mass volume ratio of Chinese medicine residue culture medium and Trichoderma viride is 10g:1~3mL; The initial spore concentration of the green mold is 9-11×10 6 cfu / mL.

10. The method according to any one of claims 7 to 9, characterized in that: The culture temperatures in step (1) and step (2) are independently 26 to 30°C.

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