CN114672469B

CN114672469B - Method for producing laccase by fermenting coarse feed through dzo rumen natural co-culture

Info

Publication number: CN114672469B
Application number: CN202210293282.3A
Authority: CN
Inventors: 魏亚琴; 何国琳; 张静荣
Original assignee: Institute of Biology of Gansu Academy of Sciences
Current assignee: Institute of Biology of Gansu Academy of Sciences
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2022-10-25
Anticipated expiration: 2042-03-24
Also published as: CN114672469A

Abstract

The invention relates to the technical field of biology, in particular to a method for producing laccase by degrading coarse feed by a natural co-culture of ryegrass and methane brevibacterium on dzo rumen, wherein the natural co-culture of the ryegrass and the methane brevibacterium (Methanobrevibacterium) Cattle-YakTZ1 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23971; the activity of laccase produced by degrading the maize straws of the co-culture degradable roughage is the highest and reaches 81.3U/mL, and meanwhile, the natural co-culture (Methanobrevibacterium cellulolyae) Cattle-YakTZ1 of the neocallimastix neoformans and the methane brevibacterium is preserved in vitro for survival and passage, so that the co-culture degradable roughage is convenient to popularize and provides great convenience for production.

Description

Method for producing laccase by fermenting coarse feed through dzo rumen natural co-culture

Technical Field

The invention relates to the technical field of biology, in particular to a method for producing laccase by degrading coarse feed by a natural co-culture of rumen new Verbena and methane Brevibacterium of yaks.

Background

The number of yaks in China is more than 90% of the total number of yaks in the world, about 2000 thousands of yaks are mainly distributed in Qinghai, tibet, sichuan, gansu, xinjiang, yunnan, qinghai-Tibet plateau and peripheral countries. The cattle is a local breed in China, has the advantages of coarse feeding resistance, strong adaptability and the like, and has the meat tenderness index obviously superior to that of foreign introduced breeds and hybrid breeds. Dzos are derived from yaks and are fruits of yak population. Dzos are F1 generation hybrids of distant hybridization between yaks and cattle or improved breeds, male cattle are hybridized with female yaks, the hybrids are named as dzos, male yaks are hybridized with female cattle, and the hybrids are named as dzos. No matter the male of the yellow dzo and the yak are sterile, the production performance of the yellow dzo and the yak is obviously superior to that of the parent of the yak in terms of growth and development, meat production, working force and the like, and the capability of adapting to the ecological environment in a high-altitude area is higher than that of the parent of the yellow cattle. Investigation shows that by 2012, dzo accounts for 3.5% of the total number of cattle in Qinghai province, no less than 12 ten thousand of the total number of the cattle, and dzo grows fast, has strong disease resistance, is generally not easy to get ill, is suitable for grazing in high mountains, and has dzo meat and dzo service, good milk production performance, high milk quality and obvious heterosis. In the same breeding condition, compared with cattle and yaks, dzos show outstanding heterosis in weight, length, height and chest circumference as well as weight before slaughter, weight of head, weight of skin, weight of front hoof, weight of back hoof, weight of carcass meat and weight of carcass bone. The feed conversion rate, meat quality and milk quality of cattle, dzo and yak growing in the same region are different, and the reason for the difference is presumed to be closely related to rumen microorganisms besides genetic difference. Researches show that the gastric juice fungal abundance indexes of the cattle tumor, namely, chao and ACE, and the diversity indexes of Shannon and Simpson, are all obviously lower than dzos and yaks, and the community uniformity index Simpsoneven is obviously higher than dzos and is extremely higher than yaks. From the PCoA result, the genetic distance of the rumen fluid fungal regions of yaks and dzos is closer, and the difference between the gastric fluid fungal regions of yaks and dzos is larger than that of cattle rumen fluid fungi.

Anaerobic fungi present in the rumen of ruminants play an important role in the degradation of lignocellulose. Most species in anaerobic fungi can secrete high-activity enzymes including cellulase, hemicellulase, esterase, laccase and the like through rhizoid, and the enzymes synergistically decompose and utilize substances with complex and crystalline structures, such as cellulose, hemicellulose, pectin, lignin and the like. Some methanogens can use metabolites of anaerobic fungi, such as hydrogen, methane, formic acid, etc., to form stable co-cultures with anaerobic fungi, i.e.: the process of co-culturing anaerobic fungi and methanobacteria promotes the growth of the anaerobic fungi and the methanobacteria, and simultaneously obviously improves the activity of various lignocellulose degrading enzymes generated by the anaerobic fungi for degrading lignocellulose and the degradation capability of the lignocellulose.

Traditional energy substances face the danger of exhaustion, and the traditional energy substances generate a large amount of greenhouse gases when being used, thereby polluting the environment. The relief of the three crises of 'food, energy and environment' which are coming to people at present is one of the important ways to realize the strategy of agricultural sustainable development. The coarse fodder has natural water content below 60%,dried substanceFeed with medium crude fiber content higher than 18% and fed in air-dried form, including dried formGrass (Haw)And a cropStraw or stalkLeaves, fruit peel,VinasseAnd blighted shells and the like. The coarse fodder has high fiber content, low protein and mineral content, and poor palatability. The main components of coarse fodder such as straw, hay and chaff are lignocellulose, including lignin, cellulose and hemicellulose. Straw, hay, blighted shells and the like are beneficial to use and harmful to abandon, and can bring environmental pollution and other harmful places as wastes, and can be changed into valuables to benefit mankind by fully utilizing the straws, the hay, the blighted shells and the like as important renewable resources.

Biodegradation of lignin is of great importance in environmental and energy sciences. Microbial degradation of lignocellulose is due to the co-action of a series of enzymes secreted by it. In the degradation process, enzyme systems such as xylanase, carboxymethyl cellulose, laccase and the like are mutually cooperated to play an important role in the reaction process of degrading lignocellulose. Laccase is an important member of a lignin degradation enzyme system, is a key enzyme in the lignin degradation process, realizes the degradation of lignin by attacking phenolic compounds in the lignin, and usually extracts electrons from the lignin to reduce oxygen in the air into water.

Laccases were first discovered and isolated in sumac in japan in 1883, and studies on laccases have been continued for over 140 years to date. Laccases are widely distributed in nature in higher plants, microorganisms, algae, insects, and invertebrates, and many organisms are capable of producing laccases, the main sources of which are bacteria and various aerobic fungi in soil. Laccase is a multi-copper oxidase capable of oxidizing various phenolic compounds and non-phenolic compounds, belongs to copper blue oxidase, takes molecular oxygen as a final electron acceptor, and can catalyze polymerization, degradation and conversion of lignin and a large class of phenolic and arylamine substances. The peptide chain of laccase is mainly composed of about 500 amino acids, and the sugar aglycone accounts for 10% -45% of the whole molecule. Copper ions participate in the construction of laccase active centers, jointly form the most important part in the laccase structure, determine the activity and specificity of laccase, and are areas which are formed by combining substrates and directly participate in bond breakage. Because the laccase has wide substrate and non-substrate specificity, the product generated by catalysis does not pollute the environment, and the byproduct of catalytic reaction is only water, the laccase has huge application potential in various fields as a green biocatalyst, and especially plays an important role in the aspects of food industry, paper industry, textile industry, medicine, synthetic chemistry, cosmetics, soil bioremediation, biodegradation, biofuel cells, environmental protection and the like. The laccase as a biological enzyme can degrade pollutants which are difficult to degrade, such as phenols, amines, carboxylic acids and the like, into small molecular substances with weaker environmental toxicity under mild conditions, and has huge application potential and wide market prospect. At present, the laccase is mainly the laccase secreted by white rot fungi in basidiomycota in research, and the Trichoderma reesei is also an important production strain of industrial laccase. Patent CN201210007885.9 discloses a method for producing laccase by solid state fermentation using rice straw as raw material, and specifically discloses a strain of Clarithromyces for fermenting rice straw; patent CN201810580328.3 discloses a pretreatment method of straw biological feed raw material, and specifically discloses fermentation of neurospora for fermentation.

The superior properties of laccases in all aspects have led to an increasing interest. Although the research on laccase has been greatly advanced, and the biochemical research, the three-dimensional structure, the catalytic mechanism and the like are further researched, the research on the practical application of laccase is superficial, the difference of a laccase determination method and a determination system is large, the comparison between enzyme activity and yield is difficult, the large-scale industrial production report of the laccase is few, the synthesis cost of the laccase is high, the production level is low and the like, so that the screening of excellent high-yield laccase strains, the obtaining of laccase with stable performance and the realization of high-efficiency production of the laccase are urgent.

Dzos adapt to cold ecological conditions, and are resistant to rough feeding, severe cold and low oxygen severe natural environments. The rumen of dzos inhabits uniquely, complexly and various, and a large number of microbial communities synergistically and efficiently degrade wild pastures to provide survival energy and nutrient substances for dzos. The invention unexpectedly discovers that natural co-culture (Methanobacter olleyae) of Neocallimastix and Methanobacterium brevis, which is separated from rumen of Cattle at the grazing dzo of the Tianzhu Tibetan region in Gansu province, is adopted, the natural co-culture (Methanobacter olleyae) of the Neocallimastix and Methanobacterium brevis, the Cattlet-YakTZ 1 can ferment roughage, the fermented corn straw can produce high-activity laccase, the activity of the produced laccase is as high as 81.3U/mL, and good effect is achieved.

Disclosure of Invention

In view of the above technical problems, the primary object of the present invention is to provide a method for producing laccase by fermenting coarse feed with natural co-culture of newcastle disease bacteria and methane brevibacterium (methanobrevibacterium oleyae) Cattle-YaktTZ 1 of dzo rumen, wherein the natural co-culture of newcastle disease bacteria and methane brevibacterium (methanobrevibacterium oleyae) Cattle-YaktTZ 1 is preserved in the China general microbiological culture center of the culture Collection of microorganisms in 20 years, 11 months and 25 days, the preservation number is CGMCC No.23971, the preservation address is the institute of microbiology of China academy of sciences, no. 3 of North West Luo 1, yangyo, beijing city, and the postal code is 100101; the telephone is as follows: 010-64807355; the fax is as follows: 010-64807288;

the IT1S sequence of the new Verbena officinalis in the natural co-culture (Methanobacter olleyae) Cattle-Yak TZ1 of the new Verbena officinalis and the methane brevibacterium is shown as SEQ ID No. 1; the IT1S sequence of the methane brevibacterium in the natural co-culture (Methanobacter olleyae) Cattle-YakTZ1 of the new Verbena officinalis and the methane brevibacterium is shown as SEQ ID No. 2.

The method comprises the following steps:

(1) Preparation of natural co-culture of Neocallimastix and Methanobacterium brevis (Methanobacter olleyae) Cattle-YakT Z1 microbial inoculum: inoculating a natural co-culture (Methanobrevibacter oleyae) of new Verbena and methane Brevibacterium into an anaerobic culture medium taking wheat straws as a substrate, adding a compound antibiotic, and carrying out anaerobic culture to obtain a high-activity microbial inoculum;

(2) Producing laccase: and (2) absorbing the microbial inoculum obtained in the step (1), inoculating the microbial inoculum into an anaerobic culture medium with coarse feed as a substrate, adding compound antibiotics, and carrying out anaerobic culture to obtain the microbial inoculum.

Preferably, the roughage in the step (2) is any one of wheat straw, rice straw, corn straw, sorghum straw, oat straw, soybean straw, cotton straw, flax straw, sesame straw, soybean straw, potato straw, tomato pomace, wheat hull, rice hull, coconut shell, bran, apple peel, soybean peel, banana peel, corn peel, leymus chinensis, sisal hemp, wood dust, reed straw and bamboo straw.

Preferably, the roughage in the step (2) is any one of wheat straw, rice straw, corn straw, sorghum straw, oat straw, soybean straw, cotton straw, flax straw, soybean straw, chinese wildrye, soybean hull, corn hull, reed stem and bamboo stem.

Preferably, the inoculation amount of the natural co-culture of Verbena neoformans and Brevibacterium methanolicum (Methanobrevibacterium olleyae) Cattle-YakTZ1 in step (1) is 10% v/v; the inoculation amount of the microbial inoculum in the step (2) is 10 percent by volume v/v, and the straw content in the substrate is 1%w/v.

Preferably, the temperature of the anaerobic culture in the step (1) is 39 ℃ and the time is 72h.

Preferably, the temperature of the anaerobic culture in the step (2) is 39 ℃ and the time is 120h.

Preferably, the compound antibiotic in the step (1) consists of penicillin sodium with the final concentration of 1600IU/mL and streptomycin sulfate with the final concentration of 2000 IU/mL; the addition amount of the composite antibiotic is 1%v/v.

Preferably, the formula of the culture medium for anaerobic culture is as follows: yeast extract 1.0g, naHCO ₃ 7.0g,1.0g/L of resazurin 1.0mL, L-cysteine hydrochloride 1.7g, salt solution I82.5 mL, salt solution II 16.5mL, and distilled water to reach the constant volume of 1000m L; the preparation steps of the salt solution I are as follows: naCl 6.0g, (NH) ₄ ) ₂ SO ₄ 3.0g，KH ₂ PO ₄ 3.0g，CaCl ₂ ·2H ₂ O 0.4g，MgSO ₄ ·2H ₂ 0.6g of O and distilled water with constant volume of 1000mL; the preparation steps of the salt solution II are as follows: 4.0g K ₂ HPO ₄ And distilled water is added to the volume of 1000mL.

Preferably, in the step (2), the corn straw is inoculated, oxygen is removed, carbon dioxide is filled, and high-temperature and high-pressure sterilization is performed.

The second purpose of the invention is to provide an application of a natural co-culture of ruminal neocallimastix and methanobrevibacterium (methanobrevibacterium oleyae) Cattle-YaktTZ 1 fermented roughage for preparing laccase, wherein the natural co-culture of ruminal neocallimastix and methanobrevibacterium (methanobrevibacterium oleyae) Cattle-YaktTZ 1 is stored in China general microbiological culture collection center, and the storage numbers are as follows: CGMCC No.23971.

The invention has the beneficial effects that: the invention firstly provides a natural co-culture (Methanobrevibacter oleyae) of new Verbena and methane brevibacterium Cattle-YakTZ1, wherein the natural co-culture (Methanobrevibacter oleyae) of the new Verbena and the methane brevibacterium Cattle-YakTZ1 can ferment roughage to produce laccase, the roughage is any one of 25 of wheat straw, rice straw, corn straw, sorghum straw, oat straw, soybean straw, cotton straw, flax straw, sesame straw, soybean straw, potato straw, tomato residue, wheat hull, water rice hull, coconut shell, bran, apple peel, soybean peel, banana peel, corn peel, leymus, sisal, wood chip, reed straw and bamboo straw, the activity of the laccase produced by the fermented corn straw is the highest and reaches 81.3U/mL, and meanwhile, the natural co-culture (Methanobrevibacter oleyae) of the new Verbena and the methane brevibacterium can be conveniently popularized and produced in vitro through the natural co-culture (Methanobrevibacter oleyakTZ 1).

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The culture medium used in the following examples is a simplified formulation of the conventional anaerobic fungus culture medium prepared by the present inventors, as follows:

the formula of the culture medium for anaerobic culture comprises: yeast extract 1.0g, naHCO ₃ 7.0g,1.0g/L resazurin 1.0mL, L-cysteine hydrochloride 1.7g, salt solution I82.5 mL, salt solution II 16.5mL, distilled water constant volume to 1000mL; the preparation steps of the salt solution I are as follows: naCl 6.0g, (NH) ₄ ) ₂ SO ₄ 3.0g，KH ₂ PO ₄ 3.0g，CaCl ₂ ·2H ₂ O 0.4g， MgSO ₄ ·2H ₂ 0.6g of O and distilled water with constant volume of 1000mL; the preparation steps of the salt solution II are as follows: 4.0g K ₂ HPO ₄ And distilled water is added to the volume of 1000mL.

EXAMPLE 1 preparation of Natural coculture of Neocallimastix and Brevibacterium methanolica (Methanobacter olleyae) Cattle-YakT Z1 microbial inoculum

1mL of a natural co-culture (Methanobacter olleyae) of the Neocallimastix and the Brevibacterium methanolica is absorbed and inoculated into a 20mL Henry anaerobic tube containing 9.0mL of anaerobic culture medium taking air-dried and crushed wheat straws as a substrate, 0.1mL of compound antibiotics (penicillin and 2000IU/mL of streptomycin sulfate at the final concentration) is added at the same time, anaerobic culture is carried out for 72h at the temperature of 3 ℃, and the growth peak is reached, at the moment, the fermentation liquid is a high-activity microbial inoculum.

EXAMPLE 2 Natural coculture of Neocallimastix and Brevibacterium methanolicum (Methanobacter oleyae) Cattle-YakT Z1 anaerobic fermentation of roughage to produce laccase

In a 100mL volume anaerobic fermentation bottle, 45mL of liquid basic culture medium is filled, and 0.5g of dried and crushed wheat straw, rice straw, corn straw, sorghum straw, oat straw, soybean straw, cotton straw, flax straw, sesame straw, soybean straw, potato straw, tomato pomace, wheat hull, rice hull, coconut shell, bran, apple peel, soybean hull, banana peel, corn hull, leymus chinensis, sisal hemp, wood dust, reed straw and bamboo straw are respectively used as substrates. Deoxygenated and then autoclaved. A natural co-culture (Methanobrevibacter oleyae) of the new Verbenomyl and the Brevibacterium methanolicum which are subcultured for 72h is sucked by a sterile syringe to 5.0mL of the natural co-culture (Cattle-YakTZ 1) and is respectively inoculated into the anaerobic culture medium added with the 25 substrates, 0.5 mL of compound antibiotics (penicillin and streptomycin sulfate with the final concentration of 1600IU/mL and 2000 IU/mL) are simultaneously added, and the anaerobic culture is carried out for 5 days at 39 ℃, and 3 parallel experiments are set up in total.

Example 3 laccase content determination

The laccase activity of the fermentation broth in the anaerobic flask of example 2 was determined at 24h intervals.

Laccase activity determination method (using ABTS method):

(1) Centrifuging the fermentation liquor at 4 deg.C and 5000r/min for 15min, and collecting supernatant as crude enzyme solution;

(2) Preparing 0.5mmol/L ABTS solution: a first reagent: 0.0384g ABTS constant volume is 10mL; and a second reagent: 0.013 4g of potassium persulfate is metered to 10mL, and the volume ratio of the first reagent to the second reagent is 1:1, mixing, keeping out of the sun for 12 hours to obtain 7mmol/L ABTS solution, and diluting by 14 times to obtain 0.5mol/LABTS solution;

(3) Absorbing 1.00mL of crude enzyme solution, adding 2.00mL of acetic acid-sodium acetate buffer solution (pH 6.8), diluting for 3 times, bathing the diluted enzyme solution at 39 deg.C for 30min, taking 2.9mL of ABTS solution during determination, adding 0.1mL of preheated diluted enzyme solution, rapidly placing into ultraviolet spectrophotometer, and recording absorbance value from one point (A) ₁ ) Change to another point (A) ₂ ) Calculating the enzyme activity according to the enzyme activity definition:

A ₁ 、A ₂ two end values of the change of absorbance, t is the absorbance value from A ₁ Increase to A ₂ Elapsed time(s); enzyme activity units are defined as the amount of enzyme required to oxidize 1 μmol of substrate per minute as 1 enzyme activity unit (U); the unit of the enzyme activity is U/mL.

The experimental results show that: the highest values of the activities of the natural co-culture (meth nobrevbacter olleyae) Cattle-YakTZ1 of the ruminal neocallimastix and methanobrevibacterium of dzos in the 5-day culture period for degrading 25 roughage respectively to produce extracellular enzymes-laccase are respectively: wheat straw 38.0U/mL, rice straw 50.7U/mL, corn straw 81.3U/mL, sorghum straw 28.3U/mL, oat straw 40.1U/mL, soybean straw 30.0U/mL, cotton straw 25.6U/mL, flax straw 37.0U/mL, sesame straw 16.2U/mL, soybean straw 29.5U/mL, potato straw 19.1U/mL, tomato pomace 17.8U/mL, wheat hull 9.2U/mL, rice hull 7.1U/mL, coconut shell 2.9U/mL, bran 9.3U/mL, apple peel 7.9U/mL, soybean peel 25.0U/mL, banana peel 6.0U/mL, corn peel 20.4U/mL, leymus chinensis 30.1U/mL, hemp 9.5U/mL, sawdust 3.9U/mL, reed 30.5U/mL, bamboo straw 22.6U/mL, and bamboo straw 22.6U/mL. The laccase activity produced by fermenting the corn straws is the highest and reaches 81.3U/mL, which is obviously higher than the laccase activity produced by fermenting other substrates, and the laccase has important industrial application value. Wherein, the table 1 is the component analysis of 25 kinds of coarse fodder, and the table 2 is the laccase activity determination result.

Table 1 lignocellulosic composition of the 25 roughage substrates used

TABLE 2 Natural co-culture of Neocallimastix and Brevibacterium methanolica (Methanobrevibacterium oleyae) Cattle-YakTZ1 laccase Activity in the 5 days cultivation period for the degradation of 25 roughages respectively

Note: ^a , ^b , ^c , ^d indicates significance of difference (p)<0.05)。

Sequence listing

<110> institute of biological research of science institute of Gansu province

<120> method for producing laccase by fermenting coarse feed through natural dzo rumen co-culture

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<212> DNA

Dzo <213> (bull)

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tttgttgttt gtaataattt tttttgattt taaaaaaaaa ttatttatgg ttttgtctat 180

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ttttttttaa agtcttaaaa gaccttcttt aaaattaaac ttttgtattc atttgtctaa 300

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cctaaggatg ggtctgcggc agattaggta gttggttggg taatggctta ccaagcctat 180

gatctgtacg ggttgtgaga gcaagagccc ggagatggaa cctgagacaa ggttccaggc 240

cctacggggc gcagcaggcg cgaaacctcc gcaatgtgag aaatcgcgac ggggggatcc 300

caagtgccat tcttaacggg atggcttttc ttaagtgtaa aaagcttttg gaataagagc 360

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gaacgaacct gacggtgagg gacgaaagct aggggcgcga accggattag atacccgggt 660

agtcctagcc gtaaacgatg cggacttggt gttgggatgg ctttgagccg ctccggtgcc 720

gaagggaagc tgttaagtcc gccgcctggg aagtacggtc gcaagactga aacttaaagg 780

aattggcggg ggagcaccac aacgcgtgga gcctgcggtt taattggatt caacgccgga 840

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agaggaggtg catggccgcc gtcagctcgt accgtgaggc gtcctgttaa gtcaggcaac 960

gagcgagacc acgcccttag ttaccatcag attctttgga atgctgggca cactaagggg 1020

accgccagtg ataaattgga ggaaggagtg gacgacggta ggtccgtatg ccccgaatcc 1080

cctgggctac acgcgggcta caatggctgg gacaatgggt tccgacgccg aaaggtggag 1140

gtaatctttt aaacctagtc gtagttcgga ttgagggctg taactcgccc tcatgaagct 1200

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caccggtaac 1270

Claims

1. A method for producing laccase by fermenting coarse feed by a natural co-culture of dzo, new and virginia dzo and methane brevibacterium is characterized in that the natural co-culture of dzo, new and virginia dzuvium, cattle-YakTZ1 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.23971;

the method comprises the following steps:

(1) Preparing a natural co-culture of the new Verbena officinalis and the methane Brevibacterium Cattle-YakTZ1 microbial inoculum: inoculating a natural co-culture Cattle-YakTZ1 of new Verbena and methane Brevibacterium into an anaerobic culture medium taking wheat straws as a substrate, adding a compound antibiotic, and carrying out anaerobic culture to obtain a high-activity microbial inoculum;

(2) And (3) laccase production: and (2) absorbing the microbial inoculum obtained in the step (1), inoculating the microbial inoculum into an anaerobic culture medium with coarse feed as a substrate, adding compound antibiotics, and carrying out anaerobic culture to obtain the microbial inoculum.

2. The method of claim 1, wherein the roughage of step (2) is any one of wheat straw, rice straw, corn straw, sorghum straw, oat straw, soybean straw, cotton straw, flax straw, sesame straw, soybean straw, potato straw, tomato pomace, wheat hull, rice hull, coconut shell, bran, apple peel, soybean hull, banana peel, corn hull, sheep grass, sisal, wood chips, reed straw, and bamboo straw.

3. The method of claim 2, wherein the roughage of step (2) is any one of wheat straw, rice straw, corn straw, sorghum straw, oat straw, soybean straw, cotton straw, flax straw, soybean straw, guinea grass, soybean hull, corn hull, reed straw, and bamboo straw.

4. The method of claim 1, wherein the amount of inoculation of the natural co-culture of neocallimastix and brevibacterium methanolicum, cat-YakTZ 1 in step (1) is 10% v/v; the inoculation amount of the microbial inoculum in the step (2) is 10 percent by volume v/v, and the straw content in the substrate is 1%w/v.

5. The method of claim 1, wherein the anaerobic cultivation in step (1) is carried out at a temperature of 39 ℃ for 72 hours.

6. The method of claim 1, wherein the anaerobic cultivation in the step (2) is performed at a temperature of 39 ℃ for 120 hours.

7. The method of claim 1, wherein the antibiotic cocktail of step (1) consists of penicillin sodium at a final concentration of 1600IU/mL and streptomycin sulfate at a final concentration of 2000 IU/mL; the addition amount of the composite antibiotic is 1%v/v.

8. The method of claim 1, wherein the medium formulation for the anaerobic culture is: yeast extract 1.0g, naHCO ₃ 7.0g,1.0g/L resazurin 1.0mL, L-cysteine hydrochloride 1.7g, salt solution I82.5 mL, salt solution II 16.5mL, distilled water constant volume to 1000mL; the preparation steps of the salt solution I are as follows: naCl 6.0g, (NH) ₄ ） ₂ SO ₄ 3.0g，KH ₂ PO ₄ 3.0g，CaCl ₂ ·2H ₂ O 0.4g，MgSO ₄ ·2H ₂ 0.6g of O and distilled water with constant volume of 1000mL; the preparation steps of the salt solution II are as follows: 4.0g K ₂ HPO ₄ And distilled water is added to the volume of 1000mL.

9. The application of the natural co-culture of the new mebenomyelia dzo rumen and the methane brevibacterium, namely the Cattle-YakTZ1 fermented coarse feed for preparing the laccase is characterized in that the natural co-culture of the new mebenomyelia dzo rumen and the methane brevibacterium, namely the Cattle-YakTZ1 is preserved in the China general microbiological culture preservation management center, and the preservation numbers are as follows: CGMCC No.23971.