CN114672469A

CN114672469A - Method for producing laccase by fermenting coarse feed through dzo rumen natural co-culture

Info

Publication number: CN114672469A
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: 2022-06-28
Anticipated expiration: 2042-03-24
Also published as: CN114672469B

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 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. Dzo is a derivative cattle species of yaks and is a fruit which is created by the yak population continuously. Dzos are F1 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 finds that by 2012, dzos account for 3.5 percent of the total number of cattle, not less than 12 ten thousand of the total number of the cattle, and dzos grow fast and have strong disease resistance, are generally not easy to get ill, are suitable for grazing at high mountains, are used together with meat, have good milk production performance and high milk quality, and show obvious heterosis. Under the same breeding conditions, compared with cattle and yaks, dzos show outstanding heterosis in weight, length, height and chest circumference, weight before slaughter, weight of head, weight of skin, weight of front second hoof, weight of back second hoof, weight of carcass meat and weight of carcass bone. The feed conversion rate, meat quality and milk quality of cattle, dzos and yaks 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 and 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, chaff and the like are beneficial to use and harmful to abandon, the straw, the hay and the chaff and hull as wastes can bring environmental pollution and other harmful places, and the straw, the hay and the chaff and hull as important renewable resources can be fully utilized to change waste into valuable, thereby benefiting mankind.

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 cellulase and laccase and the like are mutually synergistic, and 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 copper-rich 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 the active center of laccase, together form the most important part in the laccase structure, determine the activity and specificity of laccase, and are the areas which are combined with substrates and directly participate in bond cleavage formation. 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 as a green biocatalyst has great application potential in various fields, 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 research mainly comprises the laccase secreted by white rot fungi in basidiomycota, 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 the natural co-culture (Methanobacter olleyae) of the Neocallimastix and the Brevibacterium methanolica separated from the rumen of the Cattle on the grazing dzo of the Tianzhu Tibetan region in Gansu province can be used for fermenting roughage, the natural co-culture (Methanobacter olleraya) of the Neocallimastix and the Brevibacterium methanolica can be used for fermenting roughage, the fermented corn straw can be used for producing high-activity laccase, the activity of the produced laccase is as high as 81.3U/mL, and a 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 ruminal neocallimastix and methanobrevibacterium (Methanobrevibacter oleyae) cat-YakTZ 1, wherein the natural co-culture of the neocallimastix and the methanobrevibacterium (Methanobrevibacter oleyae) cat-YakTZ 1 is stored in the common microorganism center of the china microorganism culture preservation management committee at 11 months and 25 days 2021, the preservation number is CGMC No.23971, the preservation address is the microbial research institute of china academy of sciences No. 3, west way 1, north kingdom city, and the post code is 100101; the telephone is as follows: 010-; the fax is as follows: 010-;

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 methanobrevibacterium in the natural co-culture (Methanobrevibacter olleyae) Cattle-YakTZ1 of the neocallimastix and methanobrevibacterium is shown as SEQ ID No. 2.

The method comprises the following steps:

(1) preparation of natural co-culture of neocallimastix and methanobrevibacterium (Methanobrevibacter oleyae) 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 the new Verbena and the methane brevibacterium (Methanobacter oleyae) Cattle-YakTZ1 in the step (1) is 10% v/v; the inoculation amount of the microbial inoculum in the step (2) is 10% 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 72 h.

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

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 complex antibiotic was 1% v/v.

Preferably, the formula of the culture medium for anaerobic culture is as follows: yeast extract 1.0g, NaHCO₃7.0g, 1.0g/L of resazurin, 1.0mL, 1.7g of L-cysteine hydrochloride, 82.5mL of salt solution I, 16.5mL of salt solution II and distilled water with constant volume of 1000m L; the preparation steps of the salt solution I are as follows: NaCl 6.0g, (NH)₄)₂SO₄3.0g，KH₂PO₄3.0g，CaCl₂·2H₂ O 0.4g，MgSO₄·2H₂0.6g of O and distilled water with constant volume of 1000 mL; the preparation steps of the salt solution II are as follows: 4.0g K₂HPO₄And distilled water is added to the volume of 1000 mL.

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 application of natural co-culture of ruminal neocallimastix and methanobacterium dzo (Methanobrevibacter olleyae) Cattle-YaktZ1 fermented roughage to prepare laccase, wherein the natural co-culture of ruminal neocallimastix and methanobacterium dzo (methanobacterium olleyae) Cattle-YaktZ1 is stored in China general microbiological culture collection and management 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 of new Verbena officinalis and methane brevibacterium (Methanobrevibacter oleyae) Cattle-YakTZ1, wherein the natural co-culture of the new Verbena officinalis and the methane brevibacterium (Methanobrevibacter oleyae) Cattle-YakTZ1 can ferment roughage to produce laccase, the roughage is any one of 25 types 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 husk, rice hull, coconut shell, bran, apple peel, soybean peel, banana peel, corn peel, leymus chinensis, sisal, sawdust, reed straw and bamboo straw, the activity of the laccase produced by the fermented corn straw is highest and reaches 81.3U/mL, and meanwhile, the natural co-culture of the new Verbena bacterium and the methane brevibacterium (Methanolbenzyme oleyae) is preserved in vitro by a Canobile-YakTZ 1, is convenient to popularize and provides great convenience for production.

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 conventional anaerobic bacteria culture medium prepared by the present inventors, as follows:

the formula of the culture medium for anaerobic culture is as follows: yeast extract 1.0g, NaHCO₃7.0g, 1.0g/L resazurin 1.0mL, L-cysteine hydrochloride 1.7g, salt solution I82.5 mL, salt solution II16.5mL, distilled water constant volume to 1000 mL; the preparation steps of the salt solution I are as follows: NaCl 6.0g, (NH)₄)₂SO₄3.0g，KH₂PO₄3.0g，CaCl₂·2H₂O 0.4g， MgSO₄·2H₂0.6g of O and distilled water with constant volume of 1000 mL; the preparation steps of the salt solution II are as follows: 4.0g K₂HPO₄And distilled water is added to the volume of 1000 mL.

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 ollyyae) 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 streptomycin sulfate with the final concentration of 1600IU/mL and 2000 IU/mL) are added at the same time, anaerobic culture is carried out for 72h at 39 ℃, 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 olleyae) Cattle-YakT Z1 production of laccase by anaerobic fermentation of the roughage

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) Cattle-Ya kTZ1 of neocallimastix and Brevibacterium methanolica subcultured for 72h was inoculated into the anaerobic medium added with 25 substrates by sucking 5.0mL with a sterile syringe, and simultaneously added with 0.5 mL of complex antibiotics (final concentration: 1600IU/mL penicillin and 2000IU/mL streptomycin sulfate), anaerobically cultured at 39 ℃ for 5 days, and 3 parallel experiments were performed 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 10 mL; and a second reagent: 0.0134 g of potassium persulfate is metered to 10mL, and the volume of the first reagent and 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 (pH6.8) to dilute for 3 times, bathing the diluted enzyme solution in water at 39 ℃ for 30min, taking 2.9mL of ABTS solution during measurement, adding 0.1mL of preheated diluted enzyme solution, quickly placing into an ultraviolet spectrophotometer, and recording the absorbance value from one point (A)₁) Change to another point (A)₂) Calculating the enzyme activity according to the enzyme activity definition:

A₁、A₂two end values of the change of absorbance, t is the absorbance value from A₁Increase to A₂Elapsed 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 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 Methanobacterium tritici of dzo in the 5-day culture period for degrading 25 kinds of roughage respectively to produce extracellular enzyme-laccase are respectively: 38.0U/mL wheat straw, 50.7U/mL rice straw, 81.3U/mL corn straw, 28.3U/mL sorghum straw, 40.1U/mL oat straw, 30.0U/mL soybean straw, 25.6U/mL cotton straw, 37.0U/mL flax straw, 16.2U/mL sesame straw, 29.5U/mL soybean straw, 19.1U/mL potato straw, 17.8U/mL tomato pomace, 9.2U/mL wheat hull, 7.1U/mL of rice hull, 2.9U/mL of coconut shell, 9.3U/mL of bran, 7.9U/mL of apple peel, 25.0U/mL of soybean peel, 6.0U/mL of banana peel, 20.4U/mL of corn peel, 30.1U/mL of leymus chinensis, 9.5U/mL of sisal, 3.9U/mL of sawdust, 30.5U/mL of reed stalk and 22.6U/mL of bamboo stalk. The activity of the laccase produced by fermenting the corn straws is the highest and reaches 81.3U/mL, and is obviously higher than that of the laccase produced by fermenting other substrates, so that the laccase-fermented corn straw 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 coculture of Neocallimastix and Brevibacterium methanolica (Methanobacter oleyae) Cattle-YakTZ1 laccase activity produced by the respective degradation of 25 roughages during the 5-day cultivation period

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

Sequence listing

<110> institute of biological research of academy of sciences of Gansu province

<120> method for producing laccase from crude feed fermented by natural coculture of rumen of dzo

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 395

<212> DNA

Dzo <213> (bull)

<400> 1

cttggtcatt tagaggaagt aaaagtcgta acaaggtttc cgtaggtgaa cctgcggaag 60

gatcattaaa aaatatcgct cgattgagag tgatttaata atcatcctac cctttgtgaa 120

tttgttgttt gtaataattt tttttgattt taaaaaaaaa ttatttatgg ttttgtctat 180

ccctaaaatt ggtttgttgt gaaatgaatc aaatttaggg aataggcttt cataaataag 240

ttttttttaa agtcttaaaa gaccttcttt aaaattaaac ttttgtattc atttgtctaa 300

atttttttta aaataattta aaaacaactt ttgacaatgg atctcttggt tctcgcaacg 360

atgaagaacg cagcaaaatg cgataagtaa ttcat 395

<210> 2

<211> 1270

<212> DNA

Dzo <213> (bull)

<400> 2

tgctcagtaa cacgtggata acctaccctt aggactggga taaccctggg aaactggggc 60

taataccgga tagatgattt ttttcctgta atggtgtttt gtttaaatgt ttttttttcg 120

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

tgggcaagac cggtgccagc cgccgcggta acaccggcag ctctagtggt agctgttttt 420

attgggccta aagcgttcgt agccggtttg gtaagtcact ggtgaaatcc tgtagcttaa 480

ctatgggaat tgctggtgat actgttgaac ttgaggtcgg gagaggttag cggtactccc 540

agggtagagg tgaaattctg taatcctggg aggaccacct gtggcgaagg cggctaactg 600

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

catctcacca ggagcgacag ctgtatgatt accaggctga tgaccttgtt tgactagctg 900

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

ggaatgcgta gtaatcgcgt gtcacaatcg cgcggtgaat acgtccctgc tccttgcaca 1260

caccggtaac 1270

Claims

1. A method for producing laccase by fermenting coarse feed by a natural co-culture (Methanobrevibacter oleyae) Cattle-YakT Z1 of dzo and Pilus ozogae is characterized in that the natural co-culture (Methanobrevibacter oleyae) Cattle-YakTZ1 of the Pianobacter neoformans and the Brevibacter methanolica 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 method comprises the following steps:

(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 taking 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, guinea 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 neoformans and Brevibacterium methanolica (Methanobacter olleyae) Cattle-YakTZ1 in step (1) is 10% v/v; the inoculation amount of the microbial inoculum in the step (2) is 10% 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 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 complex antibiotic was 1% v/v.

8. The method of claim 1, wherein the anaerobic culture medium is formulated as: yeast extract 1.0g, Na HCO₃7.0g, 1.0g/L resazurin 1.0mL, L-cysteine hydrochloride 1.7g, salt solution I82.5 mL, salt solution II16.5mL, distilled water constant volume to 1000 mL; the preparation steps of the salt solution I are as follows: NaCl 6.0g, (NH)₄)₂SO₄3.0g，KH₂PO₄3.0g，CaCl₂·2H₂O 0.4g，MgSO₄·2H₂0.6g of O and distilled water with constant volume of 1000 mL; the preparation steps of the salt solution II are as follows: 4.0g K₂HPO₄And distilled water is added to the volume of 1000 mL.

9. The method of claim 1, wherein in step (2), the corn stalks are inoculated, deoxygenated, aerated with carbon dioxide, and sterilized at high temperature and high pressure.

10. An application of a natural co-culture of ruminal neocallimastix and methanobrevibacterium (methanobacterolyae) Cattle-Yak TZ1 fermented roughage for preparing laccase is characterized in that the natural co-culture of ruminal neocallimastix and methanobrevibacterium (methanobacterolyae) Cattle-YaktZ1 is preserved in China general microbiological culture collection center with the preservation numbers as follows: CGMCC No. 23971.