CN108441432B

CN108441432B - Fast decomposing microbial inoculum and preparation method and application thereof

Info

Publication number: CN108441432B
Application number: CN201810274944.6A
Authority: CN
Inventors: 李玲娣; 李爽; 赵敬敬; 王灵敏; 周天惠; 严立恩; 李学辉
Original assignee: Shaanxi Fengdan Baili Biotechnology Co ltd
Current assignee: Shaanxi Fengdan Baili Biotechnology Co ltd
Priority date: 2018-03-30
Filing date: 2018-03-30
Publication date: 2020-07-24
Anticipated expiration: 2038-03-30
Also published as: CN108441432A

Abstract

The invention belongs to the technical field of decomposed fermentation, and particularly relates to a fast decomposing microbial inoculum, and a preparation method and application thereof. The preparation method of the fast rotting microbial inoculum comprises the following steps: respectively carrying out solid state fermentation on the ascochyta sporotrichum, aspergillus oryzae, aspergillus niger and white rot fungi to prepare a single fungus agent; according to the weight portion, 30-60 portions of saccharomyces fibuligera, 10-20 portions of aspergillus oryzae, 10-20 portions of aspergillus niger and 10-30 portions of white rot fungus are mixed by taking 100 portions of light calcium carbonate and 500 portions of grass carbon powder as dilution carriers, and are crushed and sieved by a 40-mesh sieve to obtain the fast rot fungus agent. Preferably, the Saccharomycopsis fibuligera is a preserved strain CGMCC No. 15255. The microbial inoculum can be used for vinasse decomposition and vinasse bio-organic fertilizer.

Description

Fast decomposing microbial inoculum and preparation method and application thereof

Technical Field

The invention belongs to the technical field of decomposed fermentation, and particularly relates to a fast decomposing microbial inoculum, and a preparation method and application thereof.

Background

The distiller's grains are residual solid matters obtained by adding fermenting agents such as distiller's yeast and saccharifying enzyme into sorghum, wheat, corn, rice, rye and the like serving as raw materials in the production process of the white spirit and performing the procedures of stewing, saccharification fermentation, distillation and the like, and have the advantages of complex components, high water content and high acidity, and can be rotten and deteriorated if discharged in time, thus seriously polluting the environment.

At present, many large-scale white spirit enterprises select secondary utilization of vinasse, namely, a steaming car room is built, and heat energy generated by vinasse combustion is used as a source of heat power required to be consumed by white spirit production. But needs to solve the problem of secondary pollution caused by vinasse combustion, namely vinasse ash.

According to statistics, the annual yield of the formed dry white spirit grains reaches more than 400 ten thousand t in 1563 white spirit enterprises with the Chinese scale of 2015. At present, the reutilization of the distiller's grains of the white spirit is mainly the research of animal feed, the research of ethanol fermentation of cellulose resources in the distiller's grains and the research of functional components with higher added values in the distiller's grains besides the combustion capacity. However, these applications are inefficient, costly to add, and not suitable for large-scale applications.

The vinasse is rich in crude protein, crude starch, crude fat, crude fiber, amino acid, various vitamins, nitrogen, phosphorus, calcium and other substances for promoting growth, and meanwhile, the vinasse contains high-temperature-resistant strains domesticated at high temperature for a long time, such as probiotics including bacillus, actinomycetes and the like, so that the utilization value of the vinasse is greatly improved. The vinasse ash is a secondary pollutant for vinasse combustion, but the pH value of the vinasse ash is about 7.4, is rich in phosphorus and potassium, and can also be used as a production raw material of an organic fertilizer. Therefore, the vinasse and the vinasse ash are compounded according to a certain proportion and then fermented by the functional microbial agent to prepare the vinasse bio-organic fertilizer, so that the resource utilization is realized, the waste is changed into the valuable, and the environmental protection problem is solved.

However, the precondition of using vinasse as the raw material of organic fertilizer is that the vinasse can be thoroughly decomposed, most vinasse decomposing agents mainly comprising bacteria are sold in the market at present, the temperature raising speed is slow, the fermentation time is long, most strains disclosed by related patents of the existing vinasse decomposing agents are bacteria, and the characteristics and function disclosures of the decomposing strains are few.

Fresh vinasse is high in water content (about 65%) and acidity (about pH value of 3.5), and the characteristics of high water content and high acidity make the decomposed bacteria difficult to grow and propagate, so that most of decomposed agents are slowly heated and the water is not easy to dissipate, therefore, most researchers in the prior patents can spread and ventilate the vinasse for a plurality of days to reduce the water content of the vinasse, and simultaneously, quicklime is added to reduce the acidity of the vinasse for fermentation, so that the time and the raw material cost are greatly increased.

Most of the vinasse decomposing agents in the prior patent comprise bacillus, lactic acid bacteria, streptomyces and the like, the bacteria are aerobic bacteria, the aerobic bacteria cannot survive due to oxygen deficiency in a stacking state, and frequent turning is needed, so that the labor and mechanical cost is greatly increased, in addition, the long decomposing time can cause deamination to lose nitrogen, so that the fertilizer efficiency is reduced, and organic fertilizers produced by the decomposing agents mainly comprising the bacteria have ammonia smell and fishy smell, which is the defect of most of the vinasse decomposing agents in the current market.

Although the nutrition of the vinasse is rich, most of the vinasse is macromolecular substances and cannot be directly absorbed and utilized by plants, decomposition functional enzymes are needed to decompose the vinasse into small molecular substances which can be easily absorbed and utilized by the plants, the macromolecular substances in the vinasse mainly comprise crude protein, crude starch, crude fat, cellulose, lignin and the like, the enzymes needed for decomposing the substances comprise decomposition functional enzymes such as protease, α -amylase, saccharifying enzyme, lipase, cellulase, laccase and the like, and if the macromolecular nutrient substances in the vinasse cannot be decomposed, the vinasse cannot be made into high-quality organic fertilizers, so the enzyme production function of decomposed vinasse decomposition agent strains is crucial.

Chinese patent CN 105296378A discloses a strain for quickly decomposing vinasse and application thereof, the preservation number of the strain is CGMCC No.8268, and the name is: the method comprises the following steps of (1) adjusting the pH value of the vinasse to be 6-6.5 by using quicklime after the temperature of Bacillus mojavensis rises to 60-65 ℃ for 4-5 days; chinese patent CN 104151042B discloses a vinasse organic fertilizer and a production method thereof, wherein the decomposed microbial inoculum mainly comprises bacillus amyloliquefaciens with the preservation number of CGMCC No.8143 and bacillus mojavensis with the preservation number of CGMCC No. 8268; the fermentation period is 20-25 days.

The main strains of the vinasse decomposing inoculant disclosed in the patent are aerobic bacteria, the fermentation temperature is low, the fermentation period is long, and the acid and alkali resistance, the enzyme production performance and the like of the decomposing inoculant strain are not disclosed in the patent.

The Cao Jianlan, Wang Xiao Dan, Long Rubia, Zhonghong, Yuan, Qiu Zhi Ying and the like published a 'two-step fermentation method for preparing solid-state white spirit vinasse-losing bio-organic fertilizer' (total 236 th stage 2 of 2014 in brewing science) and disclosed a group of fermented white spirit vinasse strains, wherein Aspergillus fumigatus (Aspergillus fumigatus) CICC2343, lactic acid bacteria (L actinobacillus plantarum) GIMCCAS1.3, white rot fungi (Phanerochaete chrysosporium) CICC40299, Bacillus megaterium CICC 32, Aspergillus niger and the like are mainly used for carrying out a vinasse-decomposing experiment in a laboratory stage, inoculating Aspergillus fumigatus firstly, inoculating other 4 strains after 6-7 days, and measuring and analyzing the pH, available phosphorus, organic matter, total nitrogen content and performance of vinasse in a fermentation process, thereby proving that the functional microorganisms can carry out secondary inoculation on vinasse-producing fermentation and researching the functional enzymes and the like.

Chinese patent CN200710064203 discloses a fertilizer leavening agent and a preparation method and application thereof, wherein the raw material formula of the fertilizer leavening agent comprises 10-80 parts of fermentation industrial waste residues, 5-30 parts of straws, 5-30 parts of cakes, 10-30 parts of bran and rice bran, 1.5-2 parts of urea, 0.5-1 part of NaCl and/or glucose, and 1-30 parts of azotobacter chroococcum, Bacillus megaterium, Bacillus licheniformis, L obacillus plantium and 1-5 strains of saccharomyces mulberryi, wherein the saccharomyces mulberryi has the characteristics of fast soil hardening relieving function, wide material utilization range, fast growth and high enzyme activity, and high enzyme activity and high yield.

Chinese patent CN 104909854A discloses a Maotai-flavor distiller's grains organic fertilizer and a preparation method thereof, wherein the decomposed microbial inoculum is formed by compounding bacillus, saccharomycetes, actinomycetes and bacillus aceticus, and fermentation raw materials of the organic fertilizer comprise the following components: 80-90 parts of sauce-flavor distiller's grains, 5-10 parts of rapeseed cakes, 4-8 parts of bone meal, 3-4 parts of straws, 3-4 parts of sawmilling powder, 5-10 parts of corn cob cores, 2-4 parts of egg shells, 1-2 parts of quick lime, 0.1-0.2 part of decomposed microbial inoculum and 2-4 parts of kitchen waste, and the organic fertilizer is low in heavy metal content and sufficient in nutrients.

The strains disclosed in the patent documents and the technical documents have low acid resistance, can be normally decomposed and fermented only by performing acid reduction pretreatment on vinasse, have slow starting temperature, and do not disclose enzyme production performance, but the enzyme production function of the strains is directly related to the degradation of vinasse nutrient substances and the decomposition degree of the vinasse, and further related to whether crops can grow by using organic fertilizers taking the vinasse as main raw materials.

In conclusion, although the disclosed white spirit vinasse decomposing fungi are more, the acid resistance of the strains is not high, the temperature rise time is longer, the high-temperature period is short, and the thorough decomposition is influenced; the aerobic bacteria are abundant, the nitrogen is frequently stacked and turned over, the fermentation time is long, the ammonia is deaminated and nitrogen lost, the ammonia odor is heavy, the process is complex, the raw material cost, the mechanical cost and the labor cost are increased, more importantly, the functional characteristics of the strain used by the vinasse decomposing bacterium and the enzyme production function of the decomposing bacterium are not disclosed, and the enzyme production function is the key of decomposing the vinasse.

Therefore, if a decomposed strain which can adapt to the high-acid and high-humidity environment of the vinasse, can quickly raise the temperature, has multiple enzyme production types and high enzyme production activity and can directly decompose the rich macromolecular substances of the vinasse into the small molecular substances which can be directly absorbed by plants can be found, the method is an urgent need in the field of vinasse decomposition.

The saccharomyces cerevisiae belongs to the order of saccharomyces (saccharomyces cerevisiae), the family of saccharomyces cerevisiae and the genus of saccharomyces cerevisiae, and the bacterial enzyme system is rich and can secrete α -amylase, saccharifying enzyme, acid protease and other enzymes, so the bacterial enzyme system is very suitable for being applied to the fermentation industry;

chinese patent CN 107189952A discloses an enveloped saccharomycete CICC33077 with amylase activity and its application in high-temperature Daqu in sesame-flavor liquor. The strain has developed mycelium and high amylase activity, the amylase activity can reach 7425.7U/g after being cultured in a bran culture medium for 4 days, and the strain is prepared into a microbial inoculum for producing the sesame flavor type medium-high temperature yeast.

Chinese patent CN 107475030A discloses a production process of pure-breed Xiaoqu, belonging to the technical field of biological wine brewing by using microorganisms such as fungi, the pure-breed Xiaoqu is prepared by mixing pure-breed rhizopus moldy bran with pure-breed yeast bran and pure-breed sacculus Fulviculus Fulvus bran, the sacculus Fulvus high-yield ester and amylase activity are obtained, and the pure-breed Xiaoqu has the advantages of pure fragrance, excellent quality and the like when being applied to wine brewing.

Chinese patent CN 103749967A discloses a sweet potato residue fermented feed and a preparation method thereof, relating to the field of animal feed. The patent leaven comprises the following components: the feed additive comprises bacillus subtilis, saccharomycetes-coated saccharomycetes, trichoderma viride and lactobacillus plantarum, wherein the saccharomycetes-coated saccharomycetes increases the content of feed protein.

The saccharomyces cerevisiae is applied to the brewing or feed fermentation industry, is different from the technical field of the invention, and does not disclose the application in the field of vinasse decomposition, and the saccharomyces cerevisiae used in the patent does not disclose whether other enzymes are produced or not and the enzyme activity except α -amylase activity and protease activity, and does not disclose a microbial inoculum prepared by the saccharomyces cerevisiae and the application of the microbial inoculum.

Disclosure of Invention

In order to solve the technical problems, the invention provides a rapid corrosion inoculant, and a preparation method of the rapid corrosion inoculant comprises the following steps:

the single-bacterium microbial inoculum is prepared by respectively carrying out solid state fermentation on the ascochyta Fuciformis, aspergillus oryzae, aspergillus niger and white rot fungi, and the bacterium activity can respectively reach 7.8 × 10¹¹cfu/g、3.6×10⁹cfu/g、4.4×10⁹cfu/g and 5.2 × 10⁹cfu/g；

According to the weight portion, 30-60 portions of saccharomyces fibuligera, 10-20 portions of aspergillus oryzae, 10-20 portions of aspergillus niger and 10-30 portions of white rot fungus are mixed by taking 100 portions of light calcium carbonate and 500 portions of grass carbon powder as dilution carriers, and are crushed and sieved by a 40-mesh sieve to obtain the fast rot fungus agent.

The fast rotting microbial inoculum has the activity of 78.90 × 10⁸The cfu/g, the cellulase activity 74.6U/g and the protease activity 95.2U/g are obviously superior to the national standard of the rotten microbial inoculum, and the test shows that the fast rotten microbial inoculum of the invention is stored for 6 months at room temperature, and the bacterial activity is 75.26 × 10⁸cfu/g, the survival rate is up to 95.39 percent, the storage period is 9 months, and the bacterial activity is 70.05 × 10⁸cfu/g, the survival rate is as high as 88.78%, and the bacterial activity is 64.31 × 10 after 1 year of storage⁸cfu/g, the survival rate is 81.51%, which shows that the fast-rotting microbial inoculum has high stability and long shelf life;

preferably, the saccharomyces cerevisiae is saccharomyces cerevisiae (Saccharomyces fibuligera) Y-1; the strain is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of 100101, postal code, of institute of China academy of sciences, No. 3, West Lu 1, Beijing, Chaoyang, and the like) in 2018, 24.1.2018, and the preservation number is CGMCC No. 15255.

The saccharomycete Fujianfecti yeast strain has the capability of starch decomposition, protein decomposition, fat decomposition, cellulose decomposition and lignin decomposition, and has better capability of adapting to a high-humidity and high-acid environment of vinasse;

the Saccharopolyspora fibuligera Y-1 can grow in the pH value range of 2.5-6.5, the optimal pH value is 3.0-5.0, and the Saccharopolyspora fibuligera has higher low pH tolerance;

the saccharomycete Fujianfectio yeast Y-1 has multiple enzyme producing types and high enzyme activity, wherein the produced saccharifying enzyme reaches 55247.00U/g, the produced α -amylase reaches 19030.00U/g, the produced CMC enzyme reaches 659.38U/g, the produced FPA enzyme reaches 285.20U/g, the produced neutral protease reaches 1178.67U/g, the produced acidic protease reaches 986.20U/g, the produced lipase reaches 8.40U/g, and the produced laccase reaches 8.64U/g.

The application of the rapid-decomposing inoculant can be used for decomposing vinasse, can quickly decompose vinasse, and can also be used for preparing vinasse bio-organic fertilizer.

In some embodiments of the present invention, the amount of the fast-rotting microbial inoculum is: 3kg of fast decomposing inoculant is inoculated into each ton of vinasse;

the application of the rapid-decomposing inoculant in decomposing vinasse is recommended to be carried out according to the following steps:

mixing the vinasse and the vinasse ash according to the weight ratio of 3:1, adding 1 per mill of urea, inoculating 3kg of the fast-decomposing microbial inoculum into each ton of vinasse, uniformly mixing and stacking, wherein the length and the width of a vinasse stack body are 40m × 1.8.8 m × 1.5.5 m;

stacking and turning over the vinasse stacks every other day in a high-temperature period of more than 80 ℃, fermenting until the temperature is no longer higher than 80 ℃ in 3d when the highest temperature is reached to 89.8 ℃ for 14 days, performing middle-high temperature period, stacking and turning over once a week, fermenting until the stacking temperature is reduced to below 70 ℃ in 22 days, and performing a cooling stage; fermenting for 45 days, wherein the stacking temperature of the vinasse is lower than 25 ℃, the vinasse does not rise any more, the vinasse is loose and dark brown, the water content is 21.2%, the pH value is 8.16, the C/N is 14.13, and the germination index of the seeds is 86.34%, thus obtaining the decomposed vinasse.

The fast-decomposing microbial inoculum is applied to a vinasse bio-organic fertilizer and is recommended to be carried out according to the following steps:

adding 1 ‰ biochemical fulvic acid potassium into decomposed distiller's grains, mixing, pulverizing with semi-wet material pulverizer, sieving with 3-4mm rotary sieve, and adding 5% peptoid like Bacillus (bacteria activity: 10)⁹cfu/g), 5% bacillus megaterium powder (viable bacteria amount: 10¹⁰cfu/g) and 5% o of bacillus amyloliquefaciens powder (viable bacteria amount: 10¹¹cfu/g) to obtain the vinasse bio-organic fertilizer.

The vinasse bio-organic fertilizer is rich in peptoid type bacillus with phosphate and potassium dissolving functions and bacillus megaterium, is rich in bacillus amyloliquefaciens with biocontrol function and a preserved strain cyst-coated sporotrichium Y-1, has effective bacterial activity of 2.99 hundred million/g, organic matters of 60.12 percent, pH 7.90 and water of 12.36 percent, does not detect ascarid eggs and fecal coliform groups, and has heavy metal obviously lower than the national standard.

Advantageous effects

The activity of the α -amylase produced by the saccharomycete ascomycete is 2.56 times of the highest enzyme activity (7425.4U/g) known at present, and the saccharomycete yeast also has the capability of producing saccharifying enzyme, cellulase (CMC enzyme), cellulase (FPA enzyme), neutral protease, acid protease, lipase and laccase as well as other 7 enzymes, and has multiple enzyme producing types and high enzyme activity;

the saccharomyces cerevisiae is a facultative bacterium, can normally grow and propagate under aerobic and anaerobic conditions, is suitable for the high-humidity environment of fresh vinasse, does not need to be aired or other raw materials to adjust the water content, has the optimal pH of 3-5, is completely suitable for the acid environment of the fresh vinasse, does not need to adjust the pH of the vinasse, has multiple enzyme production types and high enzyme activity, wherein the produced diastase reaches 55247.00U/g, the produced α -amylase reaches 19030.00U/g, the produced CMC enzyme reaches 659.38U/g, the produced FPA enzyme reaches 285.20U/g, the neutral protease 1178.67U/g, the acid protease 986.20U/g, the lipase 8.40U/g and the laccase 8.64U/g, can fully degrade rich macromolecular substances in the vinasse, and is beneficial to the absorption of crops;

the fast-decomposing inoculant provided by the invention is a decomposing inoculant mainly composed of fungi, and compared with a decomposing inoculant mainly composed of bacteria, the fast-decomposing inoculant has no ammonia odor in a fermentation process, does not cause ammonia and nitrogen loss, and has high fertilizer efficiency; the fast decomposing microbial inoculum can quickly heat the vinasse, simultaneously activates the original high-temperature-resistant strains subjected to long-term domestication in the vinasse to jointly play a role, reaches 80.3 ℃ the next day (19h), enters a high-temperature period, reaches the highest temperature of 89.8 ℃ at the 3 rd, and approaches to the high temperature of 90 ℃; in the fermentation process, the high temperature period of more than 80 ℃ is maintained for 13 days, the medium-high temperature period of more than 70 ℃ is maintained for 21 days, the high temperature time is long, pathogenic microorganisms and worm eggs are thoroughly killed, and the harmless standard of compost is achieved; when the fermentation time is 45 days, the vinasse is loose and dark brown, the water content is 21.2 percent, the pH value is 8.16, the C/N is 14.13, and the germination index of the seeds is 86.34 percent, which shows that the rapid-rotting microbial inoculum can directly and rapidly thoroughly rotte the vinasse;

the functional decomposing bacteria in the fast decomposing microbial inoculum not only degrade the undecomposed macromolecular substances such as crude protein, crude starch, crude fat, crude fiber and the like in the vinasse into micromolecular nutrient substances which can be directly absorbed and utilized by plants, but also metabolize to generate various vitamins, phytohormones, active enzymes and other substances, thereby playing an important role in promoting and regulating the growth and development processes of various crops and fruits and vegetables.

When the vinasse bio-organic fertilizer is applied to the litchi winter jujube, the fruit rot rate is the lowest and is only 3.44%, the maximum single fruit weight reaches 30.80 g/fruit, the average single fruit weight is the largest and reaches 25.12 g/fruit, the average single fruit weight is obviously higher than that of the other fertilizers, and the average single fruit weight is 8.79% higher than that of the CK (without any fertilizer) in a control group; the single plant yield is the highest and reaches 10.69 kg/plant, which is improved by 17.34 percent compared with the CK of a control group;

the Vc content of the winter jujube using the vinasse bio-organic fertilizer is increased by 11.98 percent compared with that of a CK group; the content of soluble sugar is increased by 23.07 percent compared with CK; the content of soluble protein is obviously higher than that of CK of a control group, and is increased by 16.55 percent compared with the CK of the control group; the content of soluble solid is higher, and is increased by 13.21 percent compared with the CK of a control group; the content of organic acid is reduced by 11.34 percent compared with the CK of a control group, and the nutritional and health-care value and the taste of the litchi and winter jujube are greatly improved; the vinasse bio-organic fertilizer can obviously improve the size, yield and quality of the jujube fruits of the litchi chinensis jujube.

Drawings

FIG. 1-colony morphology on PDA of Y-1;

FIG. 2-colony morphology on YPD of Y-1;

FIG. 3-Y-1 cell morphology map;

FIG. 4-sub-cyst-map Y-1;

FIG. 5-Y-1 phylogenetic tree of ITS region sequences of related strains;

FIG. 6 is a graph showing temperature changes during vinasse decomposition;

FIG. 7 is a graph showing changes in moisture content during the decomposition of distiller's grains;

FIG. 8 is a graph showing pH changes during the decomposition of distiller's grains;

FIG. 9 is a graph of total organic carbon content changes during the decomposition of distiller's grains;

FIG. 10 is a graph showing the total nitrogen content change during the decomposition of distiller's grains;

FIG. 11 is a graph showing the variation of carbon-nitrogen ratio during the decomposition of distiller's grains;

FIG. 12 is a graph showing the effect of maturing whole stillage on the germination index of seeds at different times.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

Example 1 isolation, purification, screening and identification of Yeast Y-1

1) Separating and purifying, wherein 10g distiller's grains sample obtained from natural stacking fermentation of the group of five-grain liquid in Yibin of Sichuan province is accurately weighed, added into a sterilized triangular flask with glass beads containing 100m L cooled sterile water, shaken and mixed uniformly for 30min by a shaking table at 200r/min, kept stand for 5min, and then supernatant is sucked for 1m L, and the 10g distiller's grains sample is sequentially diluted in a gradient manner to obtain 10^-1-10^-5Concentration, selected from 10^-1、10^-2、10^-3、10^-4、10^-5The concentration of the diluted yeast strain is diluted, 100 mu L of each gradient suspension is respectively sucked by a pipette, the gradient suspension is evenly coated into a YPD plate added with streptomycin sulfate by an applicator, the final concentration of streptomycin sulfate in a culture medium is 30 mu g/m L, the plate is inversely placed in an incubator by contrast of sterile water, the culture is carried out for 7 days at the temperature of 30 ℃, 3 groups are horizontally repeated in each treatment setting, 5 strains of yeast are obtained by separation according to the characteristics of the shape, the size, the surface structure, the edge structure, the texture, the taste, the color and the like of a microbial colony, and the 5 strains of yeast are repeatedly purified on the YPD culture medium to obtain pure strains with the serial numbers of Y-1, Y-2, Y-3, Y-4 and Y-5;

the YPD culture medium comprises 20g of glucose, 20g of peptone, 10g of yeast powder, 20g of agar and 1L of distilled water, and has a natural pH value;

2) the method comprises the following steps of (1) primarily screening strains with better decomposed functional indexes, namely strains capable of effectively decomposing starch, protein, fat, cellulose, lignocellulose and the like, respectively dotting Y-1, Y-2, Y-3, Y-4 and Y-5 to a starch decomposing culture medium, a protein decomposing culture medium, a fat decomposing culture medium, cellulose and a lignin decomposing culture medium, using plates without bacteria inoculation as blank controls, repeating each bacteria with 3 decomposed functional indexes, placing the bacteria at 30 ℃ for culturing for 5 days, using a Lugol iodine solution and Congo red to dye the starch decomposing and cellulose decomposing plates respectively to observe whether transparent circles exist around the bacterial colonies, using the fat decomposing plate to directly observe whether a halo exists around the bacterial colonies, using the protein decomposing plate to directly observe whether a transparent circle exists around the bacterial colonies, using the lignin decomposing plate to directly observe whether a aniline blue degrading circle exists or not to judge whether the strains have corresponding decomposed functions and simultaneously record the diameters (D) of the bacterial colonies and the transparent circles or the diameters (D), calculating the ratio D/D, and screening strains with the functions of decomposing starch, decomposing protein, cellulose, laccase and the strains capable of producing various enzymes capable of producing various types, namely, capable of producing various primary screening enzymes capable of producing various types, such as Y-1, Y-2, Y-3 and lignin-5;

table 1: qualitative detection result of rotten function of different strains

Note: *: weakly positive-: negative of

It can be seen that Y-1, Y-3 and Y-5 can simultaneously produce a plurality of decomposed functional enzymes, so that the 3 strains are subjected to the next step of re-screening;

the preparation method of the amylolysis culture medium plate comprises the following steps: cutting potato 200g into 1cm³Boiling for 30min, filtering with 3 layers of gauze, adding distilled water to the filtrate to 1L, glucose 20g, soluble starch 10g, agar powder 20g, and adjusting pH to natural value;

after inoculating and culturing at 30 ℃ for 5 days, adding Luogoni iodine solution (diluted by 50 times of the stock solution) to cover the culture medium, and obtaining a constant blue hydrolytic ring around the colony as positive.

The preparation method of the protein-decomposing culture medium plate comprises the following steps: 3g of beef extract, 5g of NaCl, 2g of casein and CaCl₂0.1g of agar powder, 20g of agar powder and 1L of distilled water, wherein the pH value is 7.6-8.0;

after inoculating and culturing for 5 days at 30 ℃, directly observing, and determining that a transparent ring appears around a bacterial colony as positive;

the preparation method of the fat lysis culture medium plate comprises the following steps: peptone 10.0g, NaCl 5.0g, CaCl₂·2H₂O0.1g, pH 7.4, distilled water 1L, agar powder 20.0g, sterilizing at 121 deg.C for 30min, cooling to 40-50 deg.C, adding separately sterilized Tween40, Tween60, Tween80 to final concentration of Tween 1%, shaking and pouring into plate.

After 5 days of incubation at 30 ℃ when fat had been dissolved, an opaque halo formed around the colony on at least one plate. The halo is formed by characteristic saponification crystals formed by thalli by Tween;

the preparation method of the cellulolytic culture medium plate comprises the following steps: sodium carboxymethylcellulose 3g, ammonium sulfate 1.2g, K₂HPO₄0.6g，MgSO₄·7H₂0.15g of O, 0.3g of peptone, 6g of agar powder, natural pH value and 300m of distilled water L;

after inoculating and culturing at 30 ℃ for 5 days, staining with 0.2% Congo red for 10-30min, and rinsing with 1% NaCl aqueous solution for 10 min. And observing whether the transparent ring exists or not to judge whether the strain has the capability of defibering.

The preparation method of the lignin-decomposing culture medium plate comprises the steps of preparing 10g of yeast powder, 20g of glucose and 20g of agar powder, sterilizing and cooling to about 50 ℃ with distilled water of 1L, adding separately sterilized aniline blue with the concentration of 1%, and adding 1m L mother liquor into every 100m L of culture medium.

After inoculating and culturing for 5 days at 30 ℃, observing whether aniline blue degradation rings appear or not and judging whether the strain has the lignin dissolving capacity or not;

3) and (2) secondary screening, namely marking Y-1, Y-3 and Y-5 obtained by primary screening on a PDA (personal digital assistant) plate culture medium for activation, then respectively selecting a loop by using an inoculating loop, inoculating the loop into a triangular flask filled with 100m L sterilized secondary screening culture medium, placing the triangular flask in a shaking table at 30 ℃, carrying out shaking culture at 180r/min, numbering 24 bottles of each strain, wherein a control group CK does not inoculate the strain, a treatment group is respectively bacterial solutions for culturing for different times (8h, 16h, 24h, 32h, 40h, 48h, 56h and 64h), carrying out three repetitions of each strain at different culturing times, immediately placing the bacterial solutions into a 4 ℃ refrigerator for storage after taking out the bacterial solutions, simultaneously measuring bacterial suspension bacterial activity in different time periods, finally screening to obtain saccharomycetes capable of simultaneously generating α -amylase, protease, lipase, cellulase, laccase and other decomposed function-related enzymes, adapting to the acidic environment of fresh vinasse, and fully utilizing the nutrition of the fresh vinasse for growth, and preserving the strains on an inclined plane for later use.

Weighing 5g of fresh vinasse, adding the fresh vinasse into a triangular flask with glass beads containing 200m L sterile water, shaking and uniformly mixing the mixture for 30min by a shaking table at 200r/min, filtering the mixture by 4 layers of gauze to obtain vinasse filtrate, subpackaging the vinasse filtrate into 500m L triangular flasks at 100m L/flask, adjusting the pH value, and sterilizing the mixture for 30min at 121 ℃;

the preparation method of the PDA plate culture medium comprises the following steps: cutting potato 200g into 1cm³Boiling for 30min, filtering with 3 layers of gauze, adding distilled water to 1L, glucose 20g, agar powder 20g, and adjusting pH to natural value;

TABLE 2 growth Capacity of different Yeast strains Using vinasse Nutrition (viable amount)

It can be seen that Y-1 has stronger growth ability in the vinasse filtrate than Y-3 and Y-5, which indicates that the vinasse filtrate can fully utilize the vinasse nutrient for growth and is more suitable for vinasse fermentation;

4) identification of Y-1 Strain

The method is characterized by identifying the saccharomycete Y-1 according to morphological, physiological, biochemical and hereditary characteristics and comprises the following specific steps:

(1) morphological level identification

Streaking Y-1 onto two culture media of PDA and YPD, culturing at 30 deg.C for 48h, observing colony morphology, odor and cell morphology, inoculating activated Y-1 with inoculating loop, shake culturing at 30 deg.C and 150rpm for 4 days, and observing produced ascospore by microscopic examination; as shown in figure 1: culturing Y-1 in PDA culture medium for 48h, wherein the colony is gray, round, and has a raised center, rough and villous surface and ester fragrance; as shown in fig. 2: culturing in YPD medium for 48h to obtain white brown colony with round shape and central bulge with obvious concentric circle center; ester fragrance was less intense than PDA; as shown in fig. 3: the Y-1 cell is elliptical, approximately circular and oval in shape, and has mycelium formation similar to that of the mold; as shown in fig. 4: carrying out spore production culture, wherein Y-1 ascomycetes is rectangular or oval, and each ascospore contains 1-4 ascospores;

the spore-forming culture medium comprises 1g of yeast extract, 10g of potassium acetate, 0.5g of glucose and 1L of distilled water, and has natural pH value;

(2) physiological and biochemical characteristics

In the sugar fermentation test, Y-1 can utilize glucose, maltose, sucrose, but cannot utilize lactose, galactose, starch, cellobiose, raffinose, trehalose, and the like; in the tests of assimilation reaction and other characteristics, Y-1 can assimilate glucose, cellobiose, erythritol, maltose, succinic acid, soluble starch, raffinose, trehalose, sucrose, heterogenous ribitol, citric acid, mannitol, D-arabinose, galactose, ribose, rhamnose, lactose, xylose and the like, is resistant to actinomycin ketone, does not produce starch substances, cannot hydrolyze urea, can tolerate 50% of glucose and 60% of glucose, and combines the physiological and biochemical characteristics of Y-1 and S.fibuligera, YEASTS: the comparison of Characteridiics and Identification confirmed that yeast Y-1 was Saccharomyces cerevisiae (Saccharomyces fibuligera).

TABLE 3 test results of the main physiological and biochemical characteristics of yeast Y-1

Note: "+": represents positive; "-" represents negativity

(3) Genetic traits

Inoculating yeast Y-1 into YPD liquid culture medium, shake culturing at 30 deg.C for 3 days until turbid thallus appears in the culture solution, adding thallus into centrifuge tube (1.5m L), and extracting genome strictly according to the genome extraction kit process;

PCR is carried out by taking extracted DNA genome as a template and ITS1/ITS4 as primers (ITS 1: 5'-TCCGTAGGTGAACCTGCGG-3'; ITS 4: 5'-TCCTCCGCTTATTGATATGC-3') to amplify ITS sequence fragments, PCR reaction conditions are 95 ℃ for 5min, 95 ℃ for 30S, 56 ℃ for 30S, 72 ℃ for 40S, 30 cycles, 72 ℃ final extension for 10min, electrophoresis is carried out, PCR products are recovered by cutting gel (gel recovery steps are strictly operated according to a full-scale gold gel recovery kit method), pEASYT3 carriers are connected, then Top competence is transformed by a hot-shock transformation method, white-blue-spot screening is adopted, white-spot colonies are selected and added into L B culture medium added with Amp, culture is carried out at 37 ℃ for 200rpm for 8-12h, detection of positive clone is carried out by adopting T7/SP6 primers, bacterium liquid of positive clone is sent to Beijing Liuhua large gene science and technology limited company for sequencing, obtained sequence alignment is subjected to gene bank database application, Cluster analysis, Neugal analysis is carried out by using a strain homology analysis software, and a Jolga 0.7. meigy tree strain generation system is established.

The YPD liquid culture medium comprises, by mass, 20g of glucose, 20g of peptone, 10g of yeast powder and 1L of distilled water, and has a natural pH value;

as shown in FIG. 5, the ITS gene sequence phylogenetic tree analysis of yeast Y-1 shows that Y-1 and Saccharomyces fibuligera KJJ81 are gathered in one branch, the similarity reaches 100%, and the yeast Y-1 is further identified as Saccharomyces cerevisiae (Saccharomyces fibuligera).

In conclusion, according to the morphological level identification, the physiological and biochemical characteristic identification and the genetic characteristic comprehensive analysis, the yeast Y-1 is finally determined to be the saccharomyces cerevisiae (Saccharomyces fibuligera).

Example 2 Low pH tolerance test of Saccharopolyspora Fungiensis Y-1

(1) The method comprises the following steps: selecting a proper amount of saccharomycete-coated saccharomycete Y-1 thalli, inoculating into a YPD liquid culture medium, performing shake culture at 30 ℃ and 180rpm for 48 hours to obtain a seed solution, inoculating the seed solution into triangular flasks containing sterilized YPD liquid culture media with different pH values (1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5) according to an inoculation proportion of 0.5%, repeating the steps for three pH values, taking no inoculation as a blank control, performing shake culture at 30 ℃ and 180r/min for 48 hours, observing whether the bacterial liquid in the triangular flasks is turbid or not at each pH value and turbidity, and simultaneously determining the bacterial activity of the turbid bacterial liquid;

(2) and (3) test results: except that the pH values of 1.0, 1.5 and 2.0 and the CK group bacterial liquid are still clear, other YPD liquid culture media with different pH values (2.5-6.5) are turbid, which indicates that the Saccharopolyspora fibuligera Y-1 can grow in the pH value range of 2.5-6.5, bacterial suspensions with different pH values are coated on YPD plates, and the bacterial activity can reach 10 when the pH value is 2.5⁶cfu/m L, pH value 3.0-5.0, and bacterial activity can reach 10⁸cfu/m L, pH value 5.5-6.5, and bacterial activity can reach 10⁷cfu/m L, the above experimental results show that the Saccharopolyspora fibuligera Y-1 can grow in the pH value range of 2.5-6.5, the optimum pH value is 3.0-5.0, and has higher low pH tolerance, and the fresh vinasse has the pH value of about 3.5, and is the optimum pH value of Y-1, so that the Y-1 can be well adapted to the acid environment of the vinasse, and the well decomposed vinasse can be obtained.

EXAMPLE 3 solid fermentation of Saccharopolyspora fibuligera Y-1

(1) Activating strains: selecting a proper amount of Saccharopolyspora fibuligera Y-1 by using an inoculating loop, streaking the Saccharopolyspora fibuligera Y-1 to a PDA plate culture medium, and culturing for 48h at 30 ℃;

(2) preparing a first-level seed solution: selecting a proper amount of activated saccharomyces fibuligera Y-1 by using an inoculating loop, inoculating the activated saccharomyces fibuligera Y-1 into a YPD liquid culture medium, and performing shake culture at 30 ℃ and 200rpm for 24 hours to obtain a first-level seed solution;

(3) preparing a secondary seed liquid: inoculating the primary seed solution to a YPD liquid culture medium again according to the inoculation amount of 1% (v/v), and performing shake culture at 30 ℃ and 180rpm for 36h to obtain a secondary seed solution;

(4) solid state fermentation study:

① mixing materials, according to the formula of the solid fermentation culture medium A, firstly mixing glucose, peptone and (NH) with 500m L tap water₄)₂SO₄、NaH₂PO₄、MgSO₄·7H₂O、ZnSO₄·7H₂O、MnSO₄·H₂O、CaCl₂·2H₂Dissolving O, etc., mixing with other materials at a ratio of 1:0.8, packaging into sterilization bags, sterilizing at 121 deg.C for 45min, and cooling to 35 deg.C;

②, loading into a tray, inoculating, and fermenting, namely sterilizing a tray with the length of 50cm, the width of 30cm and the depth of 5cm at 121 ℃ for 45min, loading the sterilized solid fermentation medium A obtained in the step ① into the tray to the thickness of 1cm, inoculating a secondary seed solution according to the inoculation amount of 10% by volume and mass, covering the bottom layer with sterilized newspaper, covering the upper layer with sterilized bale, transferring into a tray house, culturing at the temperature of 30 ℃ and the humidity of 65% for 60h, transferring into a drying room, drying at the temperature of 35 ℃ until the water content is lower than 5%, crushing, and sieving with a 40-mesh sieve to obtain the Y-1 microbial inoculum.

(5) Y-1 microbial inoculum activity determination, accurately weighing 10g Y-1 microbial inoculum, adding into sterilized glass bead triangular flask containing 100m L of cooled sterile water, shaking and mixing uniformly for 30min at 200rpm, standing for 5min, absorbing 1m L of supernatant, and sequentially diluting in gradient to obtain 10^-3-10^-10Concentration, selected from 10^-8、10^-9、10^-10The diluted concentration of (1), sucking 100 μ L of each gradient suspension by a pipette, uniformly coating the suspension on a PDA culture medium plate by an applicator, inverting the plate in an incubator by using sterile water control, and culturing for 48h at 30 ℃, wherein each treatment setting is horizontally repeated by 3 groups;

as a result, the final activity of the bacterial agent of Saccharopolyspora fibuligera Y-1 was 7.8 × 10¹¹cfu/g, the viable count of the bacteria is far higher than that disclosed in the prior art;

the solid fermentation culture medium A comprises the following components in parts by mass: 160g of bran, 480g of corn flour, 320g of soybean meal, 10g of glucose, (NH)₄)₂SO₄9g,NaH₂PO₄9g, peptone 10g, MgSO₄·7H₂O 2.4g，ZnSO₄·7H₂O 0.4g，MnSO₄·H₂O0.02g，CaCl₂·2H₂0.12g of O, and the ratio of material to water is 1: 0.8;

EXAMPLE 4 study of the enzyme-producing Activity of Saccharopolyspora fibuligera Y-1

Research on the activity of cellulase (CMC enzyme), cellulase (FPA enzyme), saccharifying enzyme, α -amylase, neutral protease, acid protease, lipase and laccase produced by Saccharopolyspora sinensis Y-1 specifically comprises the following steps:

1. activity measurement of cellulase (CMC enzyme and FPA enzyme) produced by Saccharopolyspora fibuligera Y-1

The activity of Y-1 cellulase (CMC) production is detected according to a method of NY2321-2013 microbial fertilizer product inspection regulation, the enzyme activity is defined as that the enzyme amount required by 1g (m L) of a sample to degrade sodium carboxymethylcellulose (CMC) for 1min to generate 1 mu g of glucose is defined as1 enzyme activity unit under the conditions of 50 ℃ and pH 5.0, and is expressed as U/g, the activity of Y-1 cellulase (FPA) production is detected according to a method of QB 2583 and 2003 cellulase preparation, the enzyme activity is defined as that the enzyme amount required by 1g of the sample to degrade filter paper for 1min to generate 1 mu g of glucose is defined as1 enzyme activity unit under the conditions of 50 ℃ and pH 5.0, and is expressed as U/g.

(1) Preparation of crude enzyme solution

Weighing 5.0g (accurate to 0.001g) of a Saccharopolyspora sinensis Y-1 microbial inoculum sample, adding the sample into a 150m L conical flask which is filled with 50m L citric acid buffer solution with pH 5.0 and is provided with glass beads, oscillating for 30min at 200r/min on an oscillator, absorbing a proper amount of suspension, and centrifuging for 5min by a centrifugal force of 3000 × g, wherein the centrifuged supernatant is crude enzyme solution;

(2) CMC method for determining cellulase activity

Placing the crude enzyme solution into a 50 ℃ constant-temperature water bath, preheating for 5min, absorbing 1.5m L0.51% sodium carboxymethylcellulose (CMC) solution, placing the solution into a 20m L test tube with a plug scale, adding 1.5m L DNS solution, preheating for 5min in a 50 ℃ water bath, adding 0.5m L enzyme solution to be tested, fully shaking, reacting for 30min in a 50 ℃ water bath, immediately taking out the solution to obtain a blank tube, absorbing 1.5m L0.51.51% sodium carboxymethylcellulose (CMC) solution, placing the solution into a 20m L test tube with a plug scale, preheating for 5min in a 50 ℃ water bath, adding 0.5m L enzyme solution to be tested, fully shaking, reacting for 30min in a 50 ℃ water bath, immediately taking out, adding 1.5m L solution, uniformly mixing to obtain a blank tube, simultaneously placing the blank tube and the blank tube in a water bath for 5min, taking out, quickly cooling, metering the volume to 20.0m L by using distilled water, fully shaking, adjusting the wavelength of a sample tube under a zero point, using a boiling point light photometer, adjusting the wavelength of the DNS solution under 540nm, and measuring the absorbance of the instrument.

(3) Cellulase activity determination-FPA method

Cutting filter paper into small paper sheets of 1cm x 1cm for later use, taking a test tube of 25m L with a plug, adding 50mg of filter paper, 1.5m L buffer solution and 0.5m L crude enzyme solution as test groups, adding crude enzyme solution as inactivated enzyme solution as a control group, carrying out water bath at 50 ℃ for 30min, adding 1.5m L DN S solution, carrying out water bath for 5min, taking out, rapidly cooling, fixing the volume to 20.0m L by using distilled water, adjusting the zero point of an instrument by using a blank tube solution under the wavelength of 540nm of a spectrophotometer, and measuring the absorbance of a sample tube solution.

2. Determination of activity of neutral and acidic protease produced by Saccharopolyspora fibuligera Y-1

Detecting the activity of the protease produced in Y-1 according to a method of NY2321-2013 microbial fertilizer product inspection regulation, wherein the enzyme activity is defined as follows: the amount of enzyme required to hydrolyze casein to 1. mu.g of tyrosine in 1min for 1g of sample at 40 ℃ and pH7.5 is defined as1 enzyme activity unit in U/g.

(1) Preparation method of crude enzyme solution

Weighing 5.0g (accurate to 0.001g) of a Saccharopolyspora fibuligera Y-1 microbial inoculum sample, adding the sample into a 150m L conical flask which is filled with 50m L phosphate buffer solution with pH7.5 or acetic acid buffer solution with pH3.6 and is provided with glass beads, oscillating for 30min at 200r/min on an oscillator, absorbing a proper amount of suspension, centrifuging for 5min by a centrifugal force of 3000 × g, and obtaining a supernatant after centrifugation as a crude enzyme solution.

(2) Protease activity determination-Fulin phenol method

Placing casein solution into 40 deg.C water bath, preheating for 5min, sucking 1.0m L crude enzyme solution, placing into 10m L centrifuge tube, adding 2.0m L0 trichloroacetic acid, shaking, reacting in 40 deg.C water bath for 30min, adding 1.0m L casein solution, shaking, centrifuging at 3000L g for 5min, taking 1.0m L supernatant, adding into test tube, sequentially adding 5.0m L sodium carbonate solution, 1.0m L Forklin reagent solution, placing in 40 deg.C water bath for developing color for 20min, taking out to obtain control group, sucking 1.0m L enzyme solution to be tested, placing into 10m L centrifuge tube, placing in 40 deg.C water bath for 2min, adding 1.0m L preheated casein solution, shaking, reacting in 40 deg.C water bath for 30min, taking out, immediately adding 2m L trichloroacetic acid, shaking, taking out 3000 g × min, centrifuging at 3000 g, taking out 5m 1.0m, adding into supernatant, adding into 8280 min, adding 890 m 890.8 g supernatant, adding in 40 deg.8 m neutral density test tube, adding in sample solution, measuring absorbance measuring at room temperature, and measuring.

3. Determination of Activity of α -Amylase produced by Saccharomycopsis fibuligera Y-1

Detecting the activity of α -amylase produced by Y-1 according to the method of QB/T1803-1993 general test method for industrial enzyme preparations, wherein the enzyme activity is defined as that the enzyme amount required by hydrolyzing soluble starch to produce 1mg of maltose per min is defined as1 enzyme activity unit (U) under the environment that the temperature is 40 ℃ and the pH value is 5.6;

(1) preparation of crude enzyme solution

Weighing 5.0g (accurate to 0.001g) of a Saccharopolyspora sinensis Y-1 microbial inoculum sample, adding the sample into a 150m L conical flask which is filled with 50m L citric acid buffer solution with pH5.6 and is provided with glass beads, oscillating for 30min at 200r/min on an oscillator, absorbing a proper amount of suspension, and centrifuging for 5min by a centrifugal force of 3000 × g, wherein the centrifuged supernatant is crude enzyme solution;

(2) α determination of Amylase Activity

Diluting the crude enzyme solution, pouring into a triangular flask, performing water bath at 40 deg.C for 5min, adding 1% starch solution 4.5m L into 10m L centrifuge tube, performing water bath at 40 deg.C for 5min, adding diluted enzyme solution 0.5m L into preheated starch solution, shaking, mixing, placing in 40 deg.C water bath for 20min, using control group to inactivate enzyme solution instead of enzyme solution, pouring reaction solution 1m L into a test tube, adding 1.5m L DNS solution, boiling for 5min, cooling, diluting to 20m L, adjusting to zero, measuring OD, and mixing₅₄₀。

4. Measurement of the Activity of saccharogenic enzyme produced by Saccharopolyspora fibuligera Y-1

The activity of the Y-1 saccharifying enzyme is detected according to the method of QB/T1803-1993 general test method for industrial enzyme preparations, and the enzyme activity is defined as: the enzyme amount required for hydrolyzing the soluble starch to produce 1 mug of glucose per min at 40 ℃ and pH4.6 is an enzyme activity unit expressed in U/g.

(1) Preparation of crude enzyme solution

Weighing 5.0g (accurate to 0.001g) of a Saccharopolyspora sinensis Y-1 microbial inoculum sample, adding the sample into a 150m L conical flask which is filled with 50m L pH4.6 acetic acid buffer solution and is provided with glass beads, oscillating for 30min at 200r/min on an oscillator, absorbing a proper amount of suspension, and centrifuging for 5min at 3000 × g, wherein the centrifuged supernatant is crude enzyme solution;

(2) assay for glucoamylase activity

Taking a clean 10m L centrifuge tube, adding acetic acid buffer solution with pH4.6 of 2m L, crude enzyme solution of 1m L as a test group, adding inactivated crude enzyme solution as a control group, preheating for 5min at 40 ℃, adding 2% starch solution of 5m L, carrying out water bath for 20min at 40 ℃, then taking reaction solution of 1m L in a test tube, then adding 1.5m L DNS solution, rapidly boiling for 5min, cooling, fixing the volume to 20m L, uniformly mixing, measuring OD (optical density)₅₄₀；

5. Activity measurement of Saccharopolyspora fibuligera Y-1 for producing lipase

Detecting the activity of cellulase (FPA enzyme) produced by Y-1 according to a method of GB/T23535-: the amount of enzyme required to hydrolyze fats and oils to 1. mu. mol of fatty acids per gram of sample per minute at 40 ℃ was defined as1 activity unit (U)

(1) Preparing a crude enzyme solution: crude enzyme solution preparation method in homoprotease method

(2) Lipase activity assay (indicator titration)

Taking a 100m L triangular flask, adding a PVA emulsion substrate 4m L and a phosphate buffer solution 5m L to serve as a test group, adding 95% ethanol of the PVA emulsion substrate 4m L, the phosphate buffer solution 5m L and 15m L to serve as a control group, preheating for 5min at 40 ℃, adding a crude enzyme solution 1m L, reacting for 15min at 40 ℃, then adding 95% ethanol of 15m L to the test group, adding 2 drops of phenolphthalein indicator to the triangular flasks of the control group and the test group respectively, titrating with a sodium hydroxide solution until the solution turns reddish and keeps 30s constant, and recording the volume of the consumed sodium hydroxide standard solution.

6. Activity determination of Saccharopolyspora fibuligera Y-1 laccase production

(1) Preparing a crude enzyme solution:

weighing 5.0g (accurate to 0.001g) of a Saccharopolyspora fibuligera Y-1 microbial inoculum sample, adding the sample into a 150m L conical flask which is filled with 50m L pH 4.5 acetic acid-sodium acetate buffer solution and is provided with glass beads, oscillating for 30min at 200r/min on an oscillator, absorbing a proper amount of suspension, centrifuging for 5min by a centrifugal force of 3000 × g, and obtaining a supernatant after centrifugation as a crude enzyme solution.

(2) Laccase activity assay

At room temperature, 0.5 mmol/L ABTS 2m L was added to the tube, 2m L enzyme solution was added to start the reaction, and the increase of absorbance in the first 3min at 420nm was measured using inactivated bacteria solution as a blank.

7. Results of enzyme production Activity test of Saccharopolyspora fibuligera Y-1

In summary, the production of various enzymes by Saccharopolyspora fibuligera Y-1 is shown in Table 4

TABLE 4 production of 8 enzyme activities by Saccharopolyspora fibuligera Y-1

As can be seen from Table 4, the Saccharopolyspora fibuligera Y-1 produces many kinds of enzymes and has high enzyme activity, wherein the produced glucoamylase reaches 55247.00U/g, the α -amylase reaches 19030.00U/g, the CMC enzyme reaches 659.38U/g, the FPA enzyme reaches 285.20U/g, the neutral protease 1178.67U/g, the acid protease 986.20U/g, the lipase 8.40U/g and the laccase 8.64U/g, and the Saccharopolyspora fibuligera Y-1 with rich enzyme system and high enzyme activity can fully decompose abundant macromolecular substances in vinasse, such as crude starch, crude fat, crude protein, crude fiber, lignocellulose and the like, into small molecules which are beneficial to being absorbed by crops, thereby improving the decomposition efficiency of the vinasse.

EXAMPLE 5 preparation of fast-decomposing inoculant

The preparation of the fast rotting microbial inoculum comprises the following steps:

preparation of Saccharomycopsis fibuligera Y-1 microbial inoculum

Inoculating the strain of the Saccharopolyspora fibuligera Y-1 slant to a YPD liquid culture medium by using an inoculating loop, and performing shake culture at 30 ℃ and 200rpm for 24 hours to obtain a first-level seed solution; inoculating the primary seed solution to a YPD liquid culture medium again according to the inoculation amount of 1% (v/v), and performing shake culture at 30 ℃ and 180rpm for 36h to obtain a secondary seed solution; inoculating the second-stage seed liquid to a solid fermentation culture medium A according to the inoculation amount of 10% by volume and mass, standing and culturing at the temperature of 30 ℃ for 60h, drying at the temperature of 35 ℃ until the water content is lower than 5%, crushing and sieving by a 40-mesh sieve to obtain the Y-1 microbial inoculum, wherein the capsule is buckled and coated with a filmThe viable bacteria content of the spore yeast can reach 7.8 × 10¹¹cfu/g。

Preparation of Aspergillus oryzae microbial inoculum

Inoculating Aspergillus oryzae slant strain into PDA liquid culture medium with inoculating needle, shake culturing at 28 deg.C and 200rpm for 48h, inoculating into solid fermentation culture medium B according to inoculum size of 5% by volume and mass ratio, culturing at 28 deg.C for 3d, drying at 37 deg.C until water content is less than 5%, pulverizing, and sieving with 40 mesh sieve to obtain Aspergillus oryzae microbial inoculum, wherein the content of viable Aspergillus oryzae can reach 3.6 × 10⁹cfu/g；

The PDA liquid culture medium comprises potato extract 1L, glucose 20g, and natural pH value;

the solid fermentation medium B comprises the following components in percentage by mass: 100g of bran, 9g of bean cake powder, (NH)₄)₂SO₄1g, the ratio of material to water is 1.1:1, and the pH value is natural;

preparation of Aspergillus niger microbial inoculum

Inoculating Aspergillus niger slant strain into PDA liquid culture medium with inoculating needle, shake culturing at 28 deg.C and 200rpm for 48h, inoculating into solid fermentation culture medium C according to inoculum size of 5% by volume and mass ratio, culturing at 28 deg.C for 4d, drying at 37 deg.C until water content is less than 5%, pulverizing, and sieving with 40 mesh sieve to obtain Aspergillus niger preparation, wherein the content of Aspergillus niger viable bacteria can reach 4.4 × 10⁹cfu/g；

The solid fermentation medium C comprises the following components in percentage by mass: 100g of bran, 10g of corn flour, 5g of soybean meal and CaCl₂0.4g，MnSO₄·H₂O 0.04g，KH₂PO₄0.2g of distilled water (110 m L), and natural pH value;

preparation of white rot fungus agent

The preparation method of white rot fungus agent comprises inoculating white rot fungus to PDA culture medium slant with inoculating needle, culturing at 33 deg.C for 7-10 days, scraping spores to obtain a product with a concentration of 2.0 × 10⁷Absorbing 100m L spore suspension of m L, inoculating into 1kg solid culture medium D, culturing at 33 deg.C for 8D, drying at 40 deg.C until the water content is less than 5%, pulverizing with a pulverizer, and sieving with 40 mesh sieve to obtain white rot fungus preparation, wherein the content of viable white rot fungus can reach 5.2 × 10⁹cfu/g；

The solid fermentation culture medium D comprises the following components in massComprises the following steps: 250g of straw powder, 1g of starch, (NH)₄)₂SO₄1g of Tween 801 m L, the ratio of materials to water is 1:0.9, and the pH value is natural;

compounding of fast-decomposing microbial inoculum

According to the weight portion, 30-60 portions of Saccharopolyspora fibuligera Y-1 microbial inoculum, 10-20 portions of Aspergillus oryzae microbial inoculum, 10-20 portions of Aspergillus niger microbial inoculum and 10-30 portions of white rot fungus microbial inoculum are mixed, crushed and sieved by a 40-mesh sieve by taking 100 portions of light calcium carbonate and 500 portions of grass carbon powder as dilution carriers.

Detection of technical indexes of fast-rotting microbial inoculum

The rapid corrosion inoculant provided by the invention is subjected to detection of technical indexes according to GB 20287. supplement 2006 and NY/T2321-2013 of inspection regulations for microbial fertilizer products, and the results are as follows:

TABLE 5 technical index test results of the fast-decomposing inoculant of the present invention

The fast rotting microbial inoculum has the activity of 78.90 × 10⁸The cfu/g, the cellulase activity 74.6U/g and the protease activity 95.2U/g are all higher than the national standard, and tests show that the fast-rotting microbial inoculum of the invention is stored for 6 months at room temperature, and the microbial activity is 75.26 × 10⁸cfu/g, the survival rate is up to 95.39 percent, the storage period is 9 months, and the bacterial activity is 70.05 × 10⁸cfu/g, the survival rate is as high as 88.78%, and the bacterial activity is 64.31 × 10 after 1 year of storage⁸cfu/g, the survival rate is 81.51%, which shows that the fast-rotting microbial inoculum has high stability and long shelf life;

in conclusion, the fast decomposing microbial inoculum provided by the invention is obviously superior to the national standard of decomposing microbial inoculum, and can be used for fast decomposing vinasse.

Example 6 rotten pot test:

1. process for decomposing vinasse

Uniformly mixing vinasse and vinasse ash according to a weight ratio of 3:1, adding 1 ‰ urea to obtain a vinasse mixture, inoculating the rapid-rotting microbial inoculum prepared in example 5 into the vinasse mixture in an inoculation amount of 3 kg/square, stacking after uniformly mixing, setting the length and width of a vinasse stack body to be 40m × 1.8.8 m × 1.5.5 m, setting the vinasse stack body to be a test group Y, simultaneously setting two control stack bodies CK and CK1, wherein CK is a blank control stack body which is made of raw materials and has the same scale but is not inoculated with any rotting agent, and CK1 is a raw material, and is inoculated with the same scale and is a commercially available white spirit vinasse rotting agent stack body;

the stack is turned over once every other day in the high temperature period, and turned over once every week in the medium and high temperature period. The temperature of test group Y and control group CK, CK1 was monitored by inserting a thermometer every 4m in the east, west, south, north and middle of the stack at 9:00 am and 16:00 pm, respectively, and the average was taken as the stacking temperature of the distillers grains. Sampling at 5 points regularly to measure moisture, pH, organic carbon, carbon-nitrogen ratio and germination index, and observing physical properties of the vinasse stack;

2. and (3) comparing the test results of vinasse decomposition:

2.1 physical property change of vinasse stacking:

the volumes of the test group Y, the control group CK and the control group CK1 are reduced after fermentation is finished, wherein the volume of the stack of the test group Y is reduced by about 29.36%, the volume of the control group CK1 is reduced by about 21.58%, and the volume of the control group CK is reduced at least; the color of the test group Y vinasse is dark brown after the fermentation is finished, the texture is loose, the color of the control group CK1 vinasse is brown, and the color of the CK vinasse is still yellow brown; in the initial stage of composting, the alcohol taste and sour taste of the test group and the control group heaps were severe, but as the vinasse was rotten, the alcohol taste and sour taste of the test group Y heaps were first reduced and disappeared, and in the second of the control group CK1, the taste of the control group CK was always present and generated a large amount of fly maggots; the vinasse of the CK group which is not inoculated with the decomposition agent can not be decomposed naturally and has serious rotten taste, and the vinasse of the Y group which is inoculated with the rapid decomposition microbial inoculum of the invention is decomposed more thoroughly than the vinasse of the CK1 which is inoculated with the commercial decomposition agent;

2.2 change of temperature in the vinasse decomposing process:

as shown in figure 6, the vinasse stacking of the test group Y can reach 80.3 ℃ the next day (19h), and enters a high temperature period, wherein the vinasse stacking is turned over every other day in the high temperature period, and the highest temperature of 89.8 ℃ is reached in the 3 rd period, and the temperature is close to 90 ℃; fermenting until the temperature is no more than 80 ℃ at day 14, and entering a medium-high temperature period; turning over once per week in medium-high temperature period, fermenting until stacking temperature is reduced to below 70 deg.C in 22 days, cooling, fermenting for 45 days, and stopping fermentation when temperature is below 25 deg.C; in the whole fermentation process, the high temperature period of more than 80 ℃ is maintained for 13 days, the medium-high temperature period of more than 70 ℃ is maintained for 21 days, the high temperature time is long, pathogenic microorganisms and worm eggs are thoroughly killed, and the harmless standard of compost is achieved; the control CK1 stack slowly heated, reaching a maximum temperature of 77.8 ℃ at 22d, which is 12 ℃ lower than the maximum temperature of the test group. The whole fermentation period does not reach the high temperature of more than 80 ℃, and the temperature is maintained for 10 days at more than 70 ℃; the fermentation time of the test group Y is 45 days, and the decomposition is finished, which is 20 days earlier than that of the CK1 control group; the CK in the control group basically does not start temperature, the water is slowly lost and the CK cannot become thoroughly decomposed; the results show that the vinasse can be decomposed only by inoculating the decomposition agent, and compared with the vinasse decomposition agent of the commercial white spirit, the stacking and temperature rising speed is high when the fast decomposition agent vinasse is inoculated for decomposing, the time for entering the high-temperature period is short and only 19 hours are needed, the time for maintaining the high-temperature period is long, pathogenic microorganisms and worm eggs are thoroughly killed, organic substances and other nutrients of the vinasse are fully degraded, and the vinasse is thoroughly decomposed;

2.3 change of water content in the process of decomposing the vinasse:

as shown in the attached figure 7, the water content of the stack of the test group Y and the control group CK1 is in a descending trend, the water content is reduced most rapidly in a high-temperature period, when the fermentation of the test group Y is finished, the water content of the test group Y and the water content of the control group CK1 are respectively 21.2%, 53.1% and 27.9%, and the control group CK basically does not rise in temperature, so that the water is slowly lost and has a decayed taste, which indicates that the vinasse can not be naturally decayed and is easy to decay without adding a decay agent; compared with a commercially available vinasse decomposition agent CK1, the test group Y vinasse stack body has faster water loss, can effectively shorten the water volatilization time and quickens the decomposition of vinasse;

2.4 pH value change in the process of decomposing the vinasse:

as shown in the attached figure 8, the pH values of the test group Y and the control group CK1 vinasse stacks are both decreased and then increased, and then decreased to a stable trend, and the control group CK has no temperature, almost no microbial decomposition effect, slow water loss and no obvious change of pH; after the fermentation is finished, the pH values of the test group Y and the control group CK1 are stabilized at a higher level, the difference is small, the pH values are 8.16 and 8.01 respectively, and the pH values meet the national standard of the pH value range of 5.5-8.5 of the organic fertilizer;

2.5 the total organic carbon content change in the vinasse decomposing process:

the change of the total organic carbon content reflects the decomposition degree of the vinasse to a certain extent, and the composting process is to degrade organic matters into mineral nutrients and CO under the action of microorganisms₂、H₂O and heat, etc., with CO₂And H₂The loss of O reduces the content of organic matters; on the other hand, in the mineralization process of the organic matters, the organic matters are converted into stable humus along with humification, so that the organic matters in the heap body tend to be stable. As shown in FIG. 9, after the fermentation was completed, the organic carbon contents of CK1 in test group Y and control group Y were 33.48% and 40.92%, respectively, which were 16.61% and 9.17% lower than the initial organic carbon content (50.09%), respectively, while the difference between CK 48.27% in control group and the initial content of the distiller's grains was small. The degradation effect of the fast decomposing microbial inoculum on the total organic matters of the vinasse is obviously higher than that of a commercial decomposing agent CK1 in a control group, which shows that the fast decomposing microbial inoculum strain can fully degrade the organic matters of the vinasse and thoroughly decompose the vinasse.

2.6 the total nitrogen content change in the vinasse decomposing process:

as shown in fig. 10, the final test group Y had a total nitrogen content of 2.37% which was 0.31% higher than the initial total nitrogen content (2.06%), the control group Y1 had a total nitrogen content of 2.27% which was 0.21% higher than the initial total nitrogen content (2.06%), and the control group CK had a total nitrogen content which was slightly lower than the initial total nitrogen content; the full nitrogen content of the test group Y and the control group CK1 has consistent change trend, but the nitrogen content is always higher than that of the control group CK 1. Further, the rapid-decomposing inoculant provided by the invention can degrade the organic matters in the vinasse more fully and thoroughly.

2.7 the carbon-nitrogen ratio changes in the vinasse decomposition process:

the carbon-nitrogen ratio is an important factor influencing the composting effect, and the compost is generally considered to be decomposed when the carbon-nitrogen ratio of the compost is less than 20. As shown in fig. 11, the initial carbon-nitrogen ratio of the pile is 24.31, the carbon-nitrogen ratio of the test group Y is gradually reduced, the control group CK1 has a tendency of ascending first and then descending, and the carbon-nitrogen ratios of the test group Y, the control group CK1 and the CK after the decomposition are respectively 14.13, 18.02 and 24.50, so that the test group Y and the control group CK1 are decomposed basically, and the carbon-nitrogen ratio of the test group Y is reduced more rapidly than that of the control group CK1, which indicates that the inoculation of the fast decomposing inoculant of the invention has higher efficiency in decomposing vinasse than that of a commercial decomposing inoculant;

2.8 germination index of vinasse treated seeds in different periods in the decomposition process:

the seed germination index is one of the most convenient and reliable parameters for evaluating the degree of decomposition of the tolerant toxicity of plants, the level of toxicity which can be tolerated by the plants is usually 50%, and the degree of decomposition can be basically judged to be finished when GI is more than 80%; as shown in fig. 12: after fermentation, the GI of the seeds treated by the test group Y vinasse reaches 86.34%, the GI of the seeds treated by the control group CK1 vinasse reaches 82.23%, and the vinasse is completely decomposed. The GI of the CK vinasse treated seeds of the control group is only 30.46 percent, and the vinasse is not decomposed; the test results show that the inoculation of the rapid-decomposing inoculant can obviously accelerate the decomposing process of the vinasse and improve the decomposing efficiency of the vinasse;

example 7 preparation and application of distiller's grains bio-organic fertilizer

1. Preparation of vinasse bio-organic fertilizer

1.1 the preparation method comprises the following steps:

the completely decomposed vinasse in example 6 was mixed with 1 ‰ biochemical fulvic acid potassium, further crushed with a semi-wet material crusher, sieved with a 3-4mm roller sieve, and then 5% of Paenibacillus mucilaginosus (bacterial activity: 10) was added⁹cfu/g), 5% bacillus megaterium powder (viable bacteria amount: 10¹⁰cfu/g) and 5% o of bacillus amyloliquefaciens powder (viable bacteria amount: 10¹¹cfu/g), and obtaining the vinasse bio-organic fertilizer;

1.2 detection of organic fertilizer from distillers' grains

The vinasse bio-organic fertilizer is detected according to NY 884 detection standard of bio-organic fertilizer and NY/T2321-2013 inspection regulation of microbial fertilizer products, and the detection results are as follows:

TABLE 6 technical indexes of the distiller's grains biological organic fertilizer

TABLE 7 technical requirement for 5 heavy metals limit of the distiller's grains biological organic fertilizer

Remarking: unit mg/kg

The vinasse bio-organic fertilizer is rich in peptoid type bacillus with phosphate and potassium dissolving functions and bacillus megatherium and is rich in bacillus amyloliquefaciens powder with biocontrol function and other functional bacteria, the effective bacteria activity reaches 2.99 hundred million/g, the organic matter reaches 60.12%, the pH value is 7.90, the water content is 12.36%, the death rate of ascarid eggs and faecal coliform groups are not detected, and the heavy metal is obviously lower than the national standard, so the quality of the vinasse bio-organic fertilizer is obviously better than the national bio-organic fertilizer standard, the fertilizer effect and the drug effect are realized, the growth of fruits and vegetables can be promoted, the diseases can be prevented and controlled, and the quality of the fruits and the vegetables can be obviously improved;

2. the field application test of the vinasse bio-organic fertilizer to the litchi winter jujube

2.1 test sites are: shanxi Weinan city Dali county wood-beautiful local winter jujube test cold shed

2.2 test tree species: six-year-old litchi winter jujubes with regular growth are processed into 2 rows of 18 jujube trees in each row, and 36 jujube trees are processed in each test;

2.3 test methods:

treatment group I: applying the vinasse bio-organic fertilizer;

treatment group II: applying a commercial bio-organic fertilizer;

treatment group III: applying a traditional organic fertilizer;

treatment group IV: applying a compound fertilizer;

control CK: no fertilizer is applied;

and (3) applying the fertilizer for each plant in five times, wherein except for applying 0.5kg of urea per plant in the germination stage and applying 0.5kg of diammonium phosphate and 0.2kg of potassium sulfate in the fruit expansion stage, the fertilizer is applied in 3 stages such as applying a base fertilizer in autumn, applying a fertilizer in the flowering stage, applying a fertilizer in the fruit setting stage and the like, and applying the fertilizer in a circular ditch at the vertical projection of the crown, wherein the depth of the ditch is about 20-30 cm. And (4) uniformly mixing the fertilizer and the soil around the root system and then burying. Other management is similar to conventional measures;

2.4 influence of different treatments on the quality of the fruits of the litchi chinensis and the winter jujubes:

2.4.1 influence of different treatments on appearance quality and yield of the litchi and winter jujube fruits:

randomly selecting 10 plants in the crisp and mature period of the Chinese date fruits of the litchi chinensis, picking the Chinese date fruits, weighing, and calculating the yield of each plant; randomly selecting 300 winter jujube fruits and 100 winter jujube fruits per group, observing the appearance characters of the fruits, and then measuring longitudinal and transverse channels, fruit shape indexes, single fruit weight and the like; wherein the longitudinal and transverse diameters of the fruit are measured by a vernier caliper; measuring the yield and the single fruit by using an electronic balance; the results are shown in Table 8:

TABLE 8 influence of different fertilization treatments on the appearance quality and yield of the litchi and winter jujube fruits

Note: different lower case letters in the same column indicate that the difference reaches a significant level (P <0.05)

As can be seen from Table 8, the longitudinal diameter of the winter jujubes to which the distiller's grain bio-organic fertilizer of the present invention was applied was the largest, followed by the treatment group to which the compound fertilizer was applied, and the longitudinal diameter of the control group CK winter jujubes was the smallest; the transverse diameter of the winter jujube applying the vinasse bio-organic fertilizer is obviously larger than that of the winter jujube applying other fertilizer groups and control groups CK; the fruit shape index of winter jujube applying the distiller's grain biological organic fertilizer is 1.07, then the fruit shape index of other three treated fruits is not greatly different by applying the traditional organic fertilizer; the weight of each single winter jujube applying the vinasse bio-organic fertilizer is the largest, and reaches 30.80 g/jujube; the average single fruit weight is the largest and reaches 25.12 g/fruit, which is respectively 8.70%, 8.32% and 4.62% higher than that of the treatment groups II, III and IV, and is 8.79% higher than that of the control group CK; the fruit failure rate is shown in the way that the fruit failure rate of the winter jujube applying the vinasse bio-organic fertilizer is obviously lower than that of other treatment groups; the yield of the winter jujube single plant using the vinasse bio-organic fertilizer is the highest, and reaches 10.69kg per plant, which is improved by 17.34 percent compared with the CK of a control group; the application of the vinasse bio-organic fertilizer can obviously improve the appearance quality and the yield of the litchi winter jujubes.

2.4.2 influence of different treatments on the intrinsic quality of the litchi and winter jujube fruits:

measuring the fruit quality, including indexes such as Vc content, soluble sugar content, soluble protein content, organic acid content, soluble solid content and the like in the fruit; the Vc content is determined by adopting a 2-6 dichlorophenol indophenol method, the soluble sugar content is determined by adopting an anthrone method, the soluble protein content is determined by adopting a Coomassie brilliant blue method, the organic acid content is determined by adopting a NaOH titration method, the soluble solid content is determined by adopting a handheld sugar meter, and the results are shown in Table 9:

TABLE 9 Effect of different fertilization treatments on the intrinsic quality of the litchi and winter jujube fruits

As can be seen from table 8, the Vc content is expressed as: the winter jujube applying the vinasse bio-organic fertilizer increases by 11.98% compared with CK, has little difference with the Vc content of winter jujube applying commercial bio-organic fertilizer, but is obviously higher than winter jujube applying traditional organic fertilizer and compound fertilizer; the soluble sugar content is shown as: the CK of the control group is obviously lower than that of other treatment groups, the content of the soluble sugar of the winter jujube applied with the vinasse bio-organic fertilizer is increased by 23.07 percent compared with that of the CK, and then the control group is a treatment group II, a treatment group IV and a treatment group III; the content of soluble protein of winter jujube applying the vinasse bio-organic fertilizer and the compound fertilizer is obviously higher than that of CK of a control group, and is respectively increased by 16.55% and 16.18% compared with the CK of the control group; the soluble solids are expressed as: the content of the vinasse bio-organic fertilizer and the content of the traditional organic fertilizer are higher, and are respectively increased by 13.21% and 13.16% compared with the CK of a control group; the organic acid content of the winter jujube applying the vinasse bio-organic fertilizer is reduced by 11.34 percent compared with that of a control group CK, and the organic acid content of the winter jujube applying the traditional organic fertilizer is highest; the results show that the Vc content, the soluble protein content, the soluble sugar content and the soluble solid content of the litchi winter jujube can be obviously improved by applying the vinasse bio-organic fertilizer, the organic acid content is obviously reduced, the nutritional and health-care value and the mouthfeel of the winter jujube are greatly improved, and the internal quality of the winter jujube is generally improved;

in conclusion, the vinasse bio-organic fertilizer can obviously improve the size, the yield and the quality of the jujube fruits of the litchi and the winter jujubes;

the above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, the above embodiments can be combined and modified, and can be applied to other fruits and vegetables and crops without departing from the concept of the present invention, and these embodiments are all within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Sequence listing

<110> Shanxi Feng Dan Baili Biotech Co Ltd

<120> quick-rotting microbial inoculum and preparation method and application thereof

<130>2018

<141>2018-03-30

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>655

<212>DNA

<213> Saccharomyces fibuligera CGMCC No.15255)

<400>1

tccgtaggtg aacctgcgga aggatcatta atgttatttg tttttagacc tgcgcttaac 60

tgcgcggttt aataaactct tatacacagt gtttttgttt gcgaatttgg tttagtttgt 120

tggttttcat tcgaaaggat gaagattgat tgctaaatct tattcagctt tttaaactca 180

gatctctttt taagagaaat gtattttttt aattacaact agtcgatttt acaaactaaa 240

agtttaaaac tttcagcaac ggatctcttg gttctcgcat cgatgaagaa cgcagcgaat 300

tgcgataagt aatgtgaatt gcagattttc gtgaatcatc gaatctttga acgcatattg 360

cgctctatag tattctatag agcatgcctg tttgagcgtc atttctctct taaacctttg 420

ggtttagtat tgaaggttgt gttagcttct gctaactcct ttgaaatgac ttggcaattg 480

attgagtttt ccatatattt gcttaaggat ttaatattag gttctaccaa cttattaaat 540

acccttttgc gaaggactta ctcgtgtatc aaggccttat aactttgtca ttaattttga 600

cctcaaatca ggtaaggata cccgctgaac ttaagcatat caataagcgg aggaa 655

Claims

1. A preparation method of a rapid decay microbial inoculum comprises the following steps:

respectively carrying out solid state fermentation on the ascochyta sporotrichum, aspergillus oryzae, aspergillus niger and white rot fungi to prepare a single fungus agent;

according to the weight portion, 30-60 portions of saccharomyces fibuligera, 10-20 portions of aspergillus oryzae, 10-20 portions of aspergillus niger and 10-30 portions of white rot fungus are mixed by taking 100 portions of light calcium carbonate and 500 portions of grass carbon powder as dilution carriers, and are crushed and sieved by a 40-mesh sieve to obtain the fast rot fungus; the strain is characterized in that the saccharomyces cerevisiae is saccharomyces cerevisiae Y-1 with the preservation number of CGMCC No. 15255.

2. The preparation method of the fast-decomposing microbial inoculum according to claim 1, which is characterized in that the preparation method of the single microbial inoculum of the saccharomycete, namely the saccharomycete, comprises the following steps:

activating strains: selecting Saccharopolyspora fibuligera with inoculating loop, streaking to PDA plate culture medium, and culturing at 30 deg.C for 48 hr;

preparing a first-level seed solution: inoculating activated Saccharomycopsis fibuligera in YPD liquid culture medium, and shake culturing at 30 deg.C and 200rpm for 24 hr to obtain first-stage seed liquid;

preparing a secondary seed liquid: inoculating the primary seed solution to a YPD liquid culture medium again according to the inoculation amount of 1% (v/v), and performing shake culture at 30 ℃ and 180rpm for 36h to obtain a secondary seed solution;

solid state fermentation:

mixing materials, according to the formula of the solid fermentation culture medium A, firstly using 500m L tap water to mix glucose, peptone and (NH)₄)₂SO₄、NaH₂PO₄、MgSO₄·7H₂O、ZnSO₄·7H₂O、MnSO₄·H₂O、CaCl₂·2H₂Dissolving O, and then uniformly mixing with other raw materials, wherein the ratio of the raw materials to water is 1:0.8, subpackaging into sterilization bags, sterilizing at 121 ℃ for 45min, and cooling to 35 ℃ for later use;

tray loading, inoculation and fermentation:

filling the sterilized solid fermentation culture medium A into a sterilized curved plate to the thickness of 1cm, and inoculating a secondary seed solution according to the inoculation amount of 10% of the volume mass ratio; covering the bottom layer with sterilized newspaper, covering the upper layer with sterilized bale of hemp, and transferring into a koji room; culturing at 30 deg.C and humidity of 65% for 60 hr, drying at 35 deg.C until water content is less than 5%, pulverizing, and sieving with 40 mesh sieve;

the solid fermentation culture medium A comprises the following components in parts by mass: 160g of bran, 480g of corn flour, 320g of soybean meal, 10g of glucose, (NH)₄)₂SO₄9g， NaH₂PO₄9g, peptone 10g, MgSO₄·7H₂O 2.4g，ZnSO₄·7H₂O 0.4g，MnSO₄·H₂O0.02g，CaCl₂·2H₂0.12g of O, and the ratio of material to water is 1: 0.8.

3. the method for preparing the fast-decomposing inoculant according to claim 1, wherein the activities of the saccharomycesella Fungiensis inoculant, the Aspergillus oryzae inoculant, the Aspergillus niger inoculant and the white-rot inoculant reach 7.8 × 10¹¹cfu/g、3.6×10⁹cfu/g、4.4×10⁹cfu/g and 5.2 × 10⁹cfu/g。

4. An agent for rapid decay of microorganisms obtained by the method according to any one of claims 1 to 3.

5. The fast decomposing agent as claimed in claim 4, wherein the viable count is 78.90 × 10⁸cfu/g, cellulase activity of 74.6U/g and protease activity of 95.2U/g.

6. The use of the rapid-decomposing inoculant of claim 4 or 5 in vinasse decomposition and vinasse bio-organic fertilizer.

7. The application of the rapid-decomposing inoculant according to claim 6, wherein the vinasse is decomposed, and the dosage of the rapid-decomposing inoculant is as follows: 3kg of fast rotting microbial inoculum is inoculated into each ton of vinasse.

8. The application of the rapid-decomposing inoculant according to claim 7, wherein the vinasse decomposition comprises the following steps:

mixing vinasse and vinasse ash according to the weight ratio of 3:1, blending, adding 1 per mill of urea, inoculating 3kg of the fast-rotting microbial inoculum to each ton of vinasse, uniformly mixing and stacking;

stacking and turning over the vinasse stacks every other day in a high-temperature period of more than 80 ℃, fermenting until the temperature is no longer higher than 80 ℃ in 3d when the highest temperature is reached to 89.8 ℃ for 14 days, performing middle-high temperature period, stacking and turning over once a week, fermenting until the stacking temperature is reduced to below 70 ℃ in 22 days, and performing a cooling stage; fermenting for 45 days, and keeping the stacking temperature of the distiller's grains below 25 ℃ to obtain the decomposed distiller's grains.