CN101717729B - Method for preparing polypeptide protein feed and cellulase produced by fermentation of aspergillus niger strains - Google Patents

Abstract

The invention discloses cellulase produced by fermentation of aspergillus niger strains and a method for preparing polypeptide protein feed. In the aspergillus, the aspergillus niger 2221-P, CCTCCNO:M209143 is used. The steps comprise: A, selection of bacteria; B, identification of bacteria; C, activation of bacteria; D, culture of seed liquid; E, solid fermentation; and F, post-treatment. The aspergillus niger 2221-P bacteria are inoculated onto a solid culture medium for fermentation to obtain solid culturing materials, the solid culturing materials are dried, crushed and screened, and the enzymatic activity is also detected. For the use of the aspergillus niger 2221-P bacteria in the production of the cellulase, in the CMC method, the density of the enzymatic activity unit of power is 31,591.7 to 40,916.3U/g, and in the FPA method, the density of the enzymatic activity unit of power is 1,768.3 to 2,200.7U/g; and the protein content in the produced polypeptide protein feed is over 35 percent. The cellulase has the advantages of having a function of degrading celluloses, achieving good preparation stability and easily realizing the mass production; and the cellulase can be used for fermenting palm kernels and cakes, other cakes and vegetable fiber, and producing biological feed, biological fertilizers and biological fuel, and also be used in industries of chemical engineering, medicaments and the like. The polypeptide protein feed produced by the fermentation of the bacteria can be used as a protein additive of various feed.

Description

The preparation method of a kind of cellulase produced by fermentation of aspergillus niger strains and polypeptide protein feed

Technical field

The present invention relates to the microbial product field, more specifically relate to a kind of Aspergillus niger strain of high cellulase-producing, also relate to the preparation method of cellulase produced by fermentation of aspergillus niger strains and polypeptide protein feed simultaneously.This bacterial strain is applicable to the cellulose degradation of agriculture and forestry plant, produces industries such as alcohol fuel, biological fodder, bio-feritlizer, food, environmental improvement, textile garment, chemical and medicine industry.

Background technology

Cellulase is a kind of enzyme that when decomposition of cellulose, can play katalysis, and it extensively is present in the organisms such as fungi, bacterium and animal.At present, main using fungus fermentation produces cellulase, and bacterial strain uses therefor belongs to Trichoderma (Trichoderma), Aspergillus (Aspergillus), penicillium (Penicillium).Cellulase is difficult to purifying.Mostly be to mix enzyme during practical application.It is mainly used in every field such as foodstuffs industry, environmental industry, Brewing industry, fodder industry, textile clothing industry, pharmaceutical sector, papermaking, daily-use chemical industry, industrial washing, tobacco, oil production, and its application prospect is wide (Gao Lunjiang etc., 2007) very.In December, 2007, the US President Bush has signed " the independent and safe bill of the energy in 2007 ", and this bill requires to the year two thousand twenty U.S. to produce 36000000000 gallons recyclable fuel at least per year, and this almost is present 5 times.Wherein the biofuel with plant cellulose manufacturings such as agricultural wastes, wood chip and herbages will account for 16,000,000,000 gallons, accounts for 44.44% of total amount.

Choose in the most breathtaking technology in 2008 according to U.S.'s " technology review ", the cellulase technology comes out at the top.Because cellulase can [be about 1.55 * 10 with the plant materials fiber that the whole world produces ¹¹The ton dry-matter, wherein Mierocrystalline cellulose and semicellulose are about 0.85 * 10 ¹¹Ton, there are agriculture and forestry plant fiber and industrialness fiber 1,500,000,000 tons of (Zhang Jianquan etc., 2003 in China; Zhao Lin fruit etc., 2007)] be degraded into monose, oligosaccharides etc.And can supply mikrobe to utilize, be converted into the important channel (DuffSTB.1966) that fuel, food, feed, medicine, industrial chemicals etc. have solved the human energy, grain, resource and ecocrisis undoubtedly.

Cellulase can be divided into 1 according to the difference of its catalyzed reaction function, 4-β-D-glucanophydrolase or endo-1,4-β-D-glucanase; EC3.2.1.4 (NCE5), 1,4-β-D-glucanase cellobilhydrolase or exo-1; 4-β-D-glucanase; EC3.2.1.91 (exoglucanase) and β-1,4-glucosidase, EC3.2.1.21 (beta-glucosidase).NCE5 is cut β-1,4 glycosidic link at the inner enzyme of cellulosic molecule randomly; Circumscribed dextrin can be from the reduction or the non-reducing end cutting glycosidic link of cellulosic molecule, producd fibers disaccharides; Cellobiose is cut to one glucose molecule through beta-glucosidase (cellose) enzyme.Only, finally could cellulose degraded be become glucose in the synergy of these three kinds of enzymes.Mikrobe particularly fungi can produce this type prozyme.Thereby cellulose macromolecule is degraded to the glucose small molecules utilizes (Liu Shuli etc., 2007 for own or other mikrobes; Yang Yongbin etc., 2004).

Cellulase is mainly derived from plant, animal and mikrobe.Cellulase extensively is present in the plant, is bringing into play the effect of hydrolysis cell walls in the different steps of development of plants, like processes such as fruit are ripe, base of a fruit handle comes off.But in plant, extract relatively difficulty of cellulase, and content is not high.Nineteen twenty-four, why the arthropods that cleveland proposes food wood property and a vegetarian animal be foodstuff with the plant, is to contain a large amount of causes that can cellulolytic fungal component because of its body.1963, marshall etc. detected the snail body and contain cellulase.The existence that there is cellulase mesothorax of discovery cockroaches such as Scrivener and shirtfront.1998, Smant etc. and Watanabe etc. used molecular biological method, from Plant nematode and ant, obtain β-1 respectively, the DNA of 4-glucuroide, thus further proved and had the endogenous cellulase in the animal body really.But it is extremely difficult utilizing animal production of cellulose enzyme.

According to incomplete statistics, since the sixties in 20th century, several thousand bacterial strains of nearly 53 genus of bacterial strain of cellulase-producing have been write down both at home and abroad altogether.Output by the actinomycetes cellulase-producing is minimum, so seldom research.Bacteria-produced cellulase output is not high yet, and is intracellular enzyme, and the production difficulty is very big, thus industry be seldom with bacterium as producing bacterial classification.At present, be mainly filamentous fungus at the bacterial strain of suitability for industrialized production cellulase, the bacterial strain that wherein enzyme activity is stronger is that wood is mould, aspergillus, head mold and mould.With maximum in Trichoderma and the Aspergillus, like Trichodermareesei, viride, koning trichoderma, black mold.Processed at present preparation have that viride, black mold, reaping hook are mould, cellulase (Gao Lunjiang etc., 2007) that Paecilomyces varioti and Penicillium decumbens etc. are produced.

In order to obtain the superior strain of high cellulase-producing, domestic and international scientist has spent plenty of time, wisdom and money and has carried out the screening and the research of high yield cellulase strain.1909, Dale, Eligabeth; Nineteen twenty-six, Thom, C etc. have studied the Asperillius bacterium in the Aspergillus.In China, 1921 Adherence and Dai Fanglan, held the jobs Chao, Yu Fu and other great scholars have published research papers, and in the institutions of higher learning set up mycology courses.In the Anti-Japanese War, the Zhao Xue of Wuhan University is intelligent to be born in 63 strain bacterium among sampling in 1942～1945 years, the screening Aspergillius (the Qu Pseudomonas is an Aspergillus) earlier; Studied their characteristic such as morphological structure, growth characteristics, source, product look, and classified.This possibly be China systematic study Aspergillus bacterium paper (Hua Zhong Agriculture University food microorganisms research department, 2008.) the earliest.But the thing that is still 60～seventies of last century that really mikrobe and cellulase is linked together; Just waited feed to lack and the separation screening high yield cellulase strain ensiling plant stem-leaf etc. that ferments at that time, be made into the feed that can supply pig, ox etc. to eat in order to solve to raise pigs.Wuhan Institute of Micro-biology of the Chinese Academy of Sciences (existing Wuhan virus institute) tissue people's more than ten tackling key problem group, research cellulase fermentations feed, but can not continue too greatly because of difficulty.Along with modern bio-science and development of biology; Since the nineties in last century; Screening high yield cellulase strain and selection by mutation high yield cellulase strain, and the fermentative prodn cellulase has had breakthrough, the plain enzyme sale of existing commercial fibre in nearly ten years.

In order to produce high-quality cellulase product, except that the cellulase-producing bacterial strain that screening obtains naturally, many investigators introduce the cellulase-producing bacterial strain; Carry out mutagenesis through ultraviolet ray, gamma-rays, nitrosoguanidine etc., wherein wooden mould NT-15 mutant strain solid culture detects through station, Ministry of Light Industry food quality supervision inspection center Nanjing and shows filter paper enzyme activity 3670U/g; C-1 enzyme 24460U/g alive; Cx enzyme 1800U/g alive, reach advanced world standards (Wang Jialin, 1996).Zhang Linghua etc. (1998) have obtained mutagenic strain Wu-932 strain with sulfovinic acid and UV-light complex mutation Kang Shi wood enzyme W-925 bacterium, and the work of CMC enzyme reaches 2975U/g and filter paper enzyme activity reaches 531U/g, has improved 100% and 51% respectively than starting strain.Hu Tingting, Qiu Yanlin (2008) are starting strain with the wooden enzyme of green, through the mutagenesis of protoplastis UV-light, obtain mutant strain UVT12, and its CMC enzyme work reaches 1020.24U/g, has improved 124.3% than starting strain.In China; (2008) such as Dong Zhiyang etc. (2001), Feng Peiyong, Zhang Qiumin (2008), Qu Erjun (2008), Liu Chunfang (2008), Lan Shile (2007), Qiu Xiangfeng (2006) etc. carry out physics, chemistry and the two bonded mutagenesis with the bacterial strain of former cellulase-producing, screen the mutant strain of high cellulase-producing.

In the production of cellulase, can carry out two kinds of methods of liquid fermenting and solid fermentation.But the enzyme that liquid fermenting produces is lived not as the enzyme of solid fermentation height alive.Duan Jinzhu, Cao Danjun (2000) result of study show that ((640～663U/g) is high by 25% than liquid fermenting productive rate for FPA 742～827U/g) for the solid fermentation productive rate.And liquid fermenting utilizes the comparatively small amt of vegetable fibre, influences next step strain fermentation and produces products such as biofuel ethanol, protein, fat, organic acid.Thereby present cellulase fermentations adopts the working method of solid fermentation more.China's solid fermentation is with a long history, before its source can be traced back to several thousand.Solid fermentation is had many uses; Like the biological detoxication of the biological prosthetic of hazardous compound and degraded, industrial or agricultural waste residue, enrichment protein is produced animal-feed, bio-pulping, the integrated management of farm crop and the production (microbiotic, growth hormone and enzyme) of utilization, production edible fungus and biofuel, production biotic pesticide and metabolism product.Find that in the comparison of solid fermentation and liquid fermenting production same products β-dextrin that the former produces contains 17% zytase, and the latter has only 12%, descends 30% approximately; And for example the former the galacturonic acid enzyme of production approximately contains 90% Rohapect MPE, and identical bacterial strain only produces 10% Rohapect MPE (Wu Zhenqiang, 2006 with liquid fermenting; Wang Shihua etc., 2006).

Protein feed mainly refers in the feed natural moisture content less than 45%, and crude fiber content is less than 18% in the dry-matter, and gross protein value is a protein feed more than or equal to one type of feed of 20% in the dry-matter.The protein feed that gross protein value is high more has bigger competitive power on market.

Protein fodder is divided into plant protein fodder, animal protein feed and single-cell protein (abbreviation SCP) feed again.Plant proteinoid feed mainly is meant high-protein plant raw materials such as soybean meal, cottonseed meal, rapeseed meal, peanut meal, sesame seed meal; The animal protein feed mainly refers to from fishery products, meat, breast and egg product processed side product, and slaughterhouse and the waste of tannery and the silkworm chrysalis of silk mill etc., like protein-high animal materials such as fish meal, meat meal tankage, feather meal, blood meals; Single cell protein is meant the microbial proteinous (or tropina) of large scale culturing bacterium, yeast, mould, algae and the mould acquisition of load, is the key protein source of foodstuffs industry and fodder industry.Compare with bean powder, the protein contnt of single cell protein exceeds 10%～20%, and available nitrogen exceeds 20%, and nitrogen capable of using reaches more than 95% when having methionine(Met) to add, and is rich in multivitamin.

The lasting increase of market demand and the shortage in feed source have aggravated the imbalance between supply and demand in market.According to expert calculation, at present, 3,000 ten thousand tons of China's fodder industry energy feed shortage; The protein fodder shortage is then even more serious; Take in 25 gram animal proteins per capita the moon by China and be converted into the livestock and poultry feed proteinometer; 3,700 ten thousand tons of albumen of 1 year needs of 1,300,000,000 populations; Calculate with protein fodder (crude protein content is 43%) and to be equivalent to China and to need 8,600 ten thousand tons of protein fodders the whole year approximately, but because energy class concentrated feed can provide about 10% crude protein, so China is annual, and to need plant protein fodder and animal protein feed be more than 6,000 ten thousand tons; But the present protein fodder supply of China is only less than 3,000 ten thousand tons, suitable big of its protein fodder breach.

Aspect animal protein feed; Though multiple protein sources such as fish meal, blood meal, meat meal tankage, hoof first feather meal are arranged, because the Biosafety problem, animal protein feed is main with fish meal; And 100,000 tons of self-produced fish meal less thaies of China; A large amount of fish meal lean on import, are the maximum consumption markets of world's fish meal, and actual use fish meal amount accounts for global aquatic feeds fish meal and uses nearly 50% of total amount.In recent years, receive the influence of the global fishery decline of natural resources, world's fish meal output descends year by year.And at present, the usage ratio of fish meal higher (common fresh water fish feed fish meal accounts for about 10%, and the fish meal ratio of fresh water famous-particular-excellent kind and sea water fish shrimp feed surpasses 50%) in China's aquatic feeds, and wasting phenomenon is comparatively serious.With the turbot feed is example, and fish meal amount ratio Europe is high by 20% in the homemade feed.The world's fish meal resource that descends day by day and the supply market of worsening shortages cause the rising of animal protein feed cost.

Aspect the plant protein fodder source, the YO of the domestic albumen dregs of rice surpasses 4,500 ten thousand tons, and wherein dregs of beans output surpasses 2,928 ten thousand tons, nearly 3,000,000 tons of peanut meal output.Because the amino acid composition is not good enough in the peanut meal, be prone to dye flavus and produce Toxins, afla etc., make the feeding amount of peanut meal also receive certain limitation; Contain toxic substances such as giucosinolate, erucic acid in the rapeseed meal, make the application of dregs of rapeseed cake receive very big restriction,, be used for the also less than 30% of feed mostly as fertilizer; Contain toxic substances such as gossypol in the cottonseed meal, can cause that growth of animal is obstructed, throughput decline, anaemia, expiratory dyspnea, prolificacy descends even sterile, can cause death when serious.Therefore, the addition of cottonseed cake (dregs of rice) in feed seldom is generally 3%～4%; So the plant protein fodder source is with the soybean meal master.Though China is the big producing country of dregs of beans, dregs of beans output is positioned at after the U.S., occupies the second place of the world, and along with the demand of fodder industry to the albumen dregs of rice increases, after 1996, China has become soybean meal net importer.

Brazil, Argentina are because domestic dregs of beans consumption is less, and the dregs of beans major part of producing is used for outlet.Brazil is after the early seventies replacement U.S. becomes the world No.1 dregs of beans big export country, and annual export volume raises up steadily.Since the nineties, Argentinian dregs of beans export volume is a dark horse, and export volume surpasses Brazil after 1998, occupies first place in the world.Although U.S.'s dregs of beans YO is positioned at first place, the world, because domestic dregs of beans consumption is big, export volume only accounts for about 1/5 of its gross output.Different with concentrating relatively of export State, dregs of beans importer relatively disperses, and European Union, China and the U.S. are three main consumption markets of global dregs of beans, and in 2008/09 year, the dregs of beans consumption of European Union is more stable, between ten thousand tons of 3250-3400; The U.S. is the maximum dregs of beans producing country in the whole world, also is the bigger dregs of beans country of consumption in the whole world, and annual consumption is between ten thousand tons of 2900-3100; India's total consumption of annual dregs of beans is 2,100,000 tons; The dregs of beans import volume of other countries in Asia such as Japan, Korea S and Association of South-east Asian Nations has also kept strong growth in recent years.Because the global dregs of beans market requirement is with double-digit growth, and the dregs of beans output in 2008 years has only 0.8% with respect to 2007 annual increasing amounts.According to Food and Argriculture OrganizationFAO (FAO) statistics, in 20 end of the centurys, the true protein in whole world shortage amount is about 2,500 ten thousand tons, amounts to into protein fodder (crude protein content is 40%) and is about 6,300 ten thousand tons.Whole world livestock industry is through the fast development in 10 years, and the breach of its protein fodder is bigger.(annotate: above data from State Statistics Bureau, Chinese herding industrial information net, Chinese customs, Department of Commerce, milky way futures tracking statistics, USDA, Food and Argriculture OrganizationFAO (FAO), Germany " the oily world ".)

This product polypeptide protein feed is to be base-material with PKC, and through the method for mould, yeast and bacterium co-fermentation, crude fiber content increases its palatability as animal-feed among the reduction PKC; Increase percent protein reach 40% and more than, and vegetable-protein is converted into tropina, improve its amino acid and form, increase proteinic digesting and assimilating and utilization ratio; X 1000 is polypeptide, increases animal to amino acid whose utilization in the feed and proteinic conversion.Simultaneously, contain probiotic bacterium and benefit materials such as cellulase, proteolytic enzyme such as yeast, genus bacillus in the feed.21 century is " polypeptide " century." polypeptide " is life development in science and technology product, and " polypeptide, protein fodder " Products Development is the Gospel of aquaculture, will promote the fodder industry high-efficient development." research of the fermentation palm-kernel dregs of rice (PKC) preparation polypeptide feed " project is a blank of filling up world's palm-kernel dregs of rice utilization technology, has reduced the robust fibre among the PKC, improves the content of protein and polypeptide.Protein contnt is high in the product, and particularly protein is hydrolyzed into active polypeptide, and bio-absorbable is fast, and bioavailability is high, and biological value is high, and the price of deed is high.Have the unknown factor of promotes growth, regulate effects such as immunity, disease resistance.Feeding scope is wide, and the market space is huge.

World's oil palm harvest area has more than 950 ten thousand hectares, and Malaysia has an appointment 4,000,000 hectares, and Indonesia is about 4,000,000 hectares, and China has 50,000 hectares approximately.Malaysia's oil palm really is about 7,600 ten thousand tons, accounts for more than 40% of world wide production; Indonesia's oil palm output is about 6,500 ten thousand tons, accounts for more than 35% of world wide production; China's output has only 700,000 tons, accounts for 0.4% of ultimate production.After oil palm really deoils through processing, remaining a large amount of oil palm benevolence dregs of rice (being PKC).Produce a large amount of PKC in countries in Southeast Asia such as Malaysia and Indonesia, be about every year more than 3,200,000 tons.Because PKC contains lignocellulose and fiber is long and thick, palatability is relatively poor, is difficult to be mixed with animal-feed (Xie Long, 2007; The cooperation suggestion of two plam oils industries of middle horse, 2007).Thereby need PKC be produced cellulase through thread fungi fermentation, and because cellulase carries out enzyme to it cuts and be degraded into monose or oligosaccharides, supply other mikrobes such as utilizations such as yeast and subtilis in addition, can be used to produce polypeptide protein feed.

Summary of the invention

The objective of the invention is to be to provide a kind of Aspergillus niger strain of high cellulase-producing, its bacterium number is 2221-P.This bacterium can vast vegetable fibre as unique carbon source base-material (particularly PKC) in well growth, and growth is rapidly, it is high that the productive rate of cellulase is high, enzyme is lived.

Another object of the present invention is the preparation method who has been to provide a kind of cellulase produced by fermentation of aspergillus niger strains and polypeptide protein feed, and present method is simple, is applicable to suitability for industrialized production.Adopt present method production of cellulose enzyme productive rate height, enzyme to live high.Adopt present method to produce polypeptide protein feed productive rate height, polypeptide protein content height.

In order to achieve the above object, the present invention takes following technical measures:

Cellulase system is the extracellular enzyme that black mold 2221-P bacterial strain produces, and it can the enzymolysis Mierocrystalline cellulose, semicellulose becomes monose or oligosaccharides.Being inoculated in the liquid seeds of shake-flask culture and semi-automatic fermentor cultivation with PKC is in the solid medium or liquid culture base fluid of main carbon source, and the temperature and humidity control shaking table is cultivated, or the shallow tray fermentation cultivation and fermentation is produced high-load cellulase preparation.Existing both at home and abroad report is about technology such as the generation bacterium of this enzyme, fermentative prodn, but do not see the bacterial strain of the high cellulase-producing that from PKC, screens, and is the technology of preparing that high-content cellulase and polypeptide protein feed are produced in the matrix top fermentation of main carbon source with PKC.

The bacterial classification that is used for producing cellulase of the present invention and polypeptide protein feed is that the present invention is categorized as Ascomycotina (Ascomycotina) through what repeated screening obtained in the perfect stage from palm kernel meal; Plectomycetes (Plectomycetes); Eurotiale (Eurotiales); Eurotiaceae (Eurotiaceae) or be categorized as Deuteromycotina (Deuteromycotina) in the imperfect stage; Haplomycetes (Hyphomycates); Hyphomycetales (Hyphomycatales); The bacterial strain of the Aspergillus (Aspergillus) of light color Cordycepps (Moniliaceae).It has the ability of high cellulase-producing, and in Nikkei China typical culture collection center preservation July 13 in 2009, strain number is CCTCCM209143 to this bacterial strain.

This bacterial strain is activated; Slant culture produces conidium, is inoculated in (murphy juice liquid nutrient medium) in the liquid shaking bottle substratum with conidium, cultivates 48～72 hours; Being inoculated in palm kernel meal is in the solid fermentation cultivation of main carbon source (other carbon sources comprise a spot of wheat bran); Cultivation and fermentation in temperature-controlling chamber's (26～30 ℃) makes it growth, and metabolism produces cellulase.After lyophilize, carry out aftertreatment, the enzyme of the acquisition CMC of unit method 33217U/g alive and FPA method 1378U/g.

Bacterial classification as previously mentioned, the bacterial classification 2221-P that the present invention uses was categorized as Ascomycotina (Ascomycotina), Plectomycetes (Plectomycetes), Eurotiale (Eurotiales), Eurotiaceae (Eurotiaceae) or is categorized as the Aspergillus awamori (Aspergillus awamori 2221-P) in the Aspergillus (Aspergillus) of Deuteromycotina (Deuteromycotina), Haplomycetes (Hyphomycates), hyphomycetales (Hyphomycatales), light color Cordycepps (Moniliaceae) in the imperfect stage in the perfect stage.In Nikkei China typical culture collection center preservation July 13 in 2009, strain number is CCTCCNO:M209143, is the new bacterial strain of high cellulase-producing.

A kind of preparation method of Aspergillus niger strain, its step is following:

1. bacterial screening.From rotten kind of soil sample, tree branches and leaves (cypress, pine tree, palm tree etc.), totally 70 of the palm kernel meal appearance of gathering; Separation and purification through five kinds of (PDA, CMC-Na, CMC-Na+ is Congo red, PKC+ is Congo red, beef extract-peptone) different substratum is handled with differentiating, obtains 146 strain filamentous funguss.Filter out 19 strain effective strains (screening process is seen accompanying drawing 1) through CMC-Na+ congo red method, PKC+ congo red method and filter paper strip method.Pass through the Congo red and Congo red culture medium culturing of PKC+ of further CMC-Na+ again, select 9 strains according to hydrolysis circle size and produce the higher bacterial strain of Mierocrystalline cellulose, select 3 plant heights with its CMC enzyme work of shake-flask culture fermented liquid detection and the work of FPA enzyme again and produce cellulosic bacterial strain.3～5 potato nutrient solution shake flask fermentations are measured the dynamics research of its living weight and its cellulase-producing of PKC liquid culture mensuration, finally select the bacterial strain of three plant height cellulase-producings, and strain number is respectively 1042-2,2221-P and 13005.Wherein 1042-2 and 2221-P are the bacterial strain of the new screening of applicant, and 13005 for the applicant preserves bacterial strain, belong to healthy and free from worry (family name) wood mould (trichodorma koningii).

2. strain identification.Morphologic observation through growth characteristic, hyphostoma development and opticmicroscope, electronic scanning mirror, produce look, produce enzyme kinetics and 18SrDNA molecular biology etc. identify 2221-P on the PDA flat board is; Mycelia just is white, fine hair shape; Bacterium colony median rise (24h) is to the sparse growth of radiation all around; Bacterium colony central authorities, mycelia fades to yellow-gray, then unusual thick close (48h); Back mycelia becomes black (72h); Conidium black, numbers of poles is many.(image document such as accompanying drawing 4).With 2221-P be accredited as Aspergillus (Aspergillus) black mold (Aspergillus niger, 2221-P).In Nikkei China typical culture collection center preservation July 13 in 2009, strain number is CCTCCNO:M209143, is the new bacterial strain of high cellulase-producing.

3. actication of culture.With containing 0.01～0.10% volume ratio tween-80 sterilized water spore is processed bacteria suspension, inhale the 0.5ml spore suspension with little pipettor and in the plate of PDA substratum, cultivate 24～100h, produce spore at 20～45 ℃.

4.2221-P bacterial strain culture of seed liquid.The bacterial classification that activation is good is with the aseptic spore suspension of washing into of 0.01～0.10% volume ratio tween-80, is inoculated in the liquid nutrient medium by the inoculum size of 5～20% mass ratioes, and this substratum is the PKC of 5～15% mass ratioes; 0.1～0.4% quality is than peptone, 0.5～1.5% quality is than glucose, and 0.1～0.5% quality compares ammonium sulfate; PH 4～8; The bottle that shakes of inoculation is placed the temperature control shaking table, and 20～45 ℃, 220rpm cultivates 24～100h;

5. solid fermentation.Solid medium is to be basic carbon source matrix with PKC (buying in Malaysia); Concrete prescription is through the certain humidity of 150～300 ℃ of high temperature 20～60% expanded PKC powder 75～90%, soybean meal 2～10%, wheat bran 2～10%, glucose 0.4～2.0%, peptone 0.4～2.0%, ammonium sulfate 0.1～0.6%, lime carbonate 0.1～0.5%; Material: water=1: 1.0～2.5, pH4～8.Earlier PKC powder, soybean meal, wheat bran and lime carbonate are weighed according to quantity, dried tremble even, add water tremble again even, when being sub-packed in the cloth bag in high-pressure sterilizing pot 115～126 ℃ of sterilization 15～40min, vexed again 1～2h; Other raw material in the above-mentioned solid culture based formulas (glucose, peptone, ammonium sulfate) is weighed up according to quantity, add solid substratum 10～50% water and be mixed with suspension-s, 115 ℃ of sterilization 20～30min are cooled to 40～50 ℃ and treat inoculation usefulness.The solid material that sterilization is good places sterile culture indoor, and is to be cooled during to 40～50 ℃, suspension-s such as inoculation liquid seeds and glucose; The liquid seeds inoculum size is inoculated and mixed thoroughly by 5～20% of solid culture base unit weight, and is final, the solid material of substratum: liquid water=1: 1.0～2.5; Liquid is the water of mixing siccative, the total amount of the suspension-s of bacterium seed liquor and glucose, and the solid material that inoculation is good is sub-packed in the tray; Thickness 1～10cm, tray bottom hide with aseptic cotton above the material with aseptic cotton place mat again; In the sterile culture chamber, be incubated 20～35 ℃ and cultivated 2～12 days, cultivate after 48 hours the plain enzyme work of detection fibers of taking a sample every day with the RH40～90% of preserving moisture.

6. aftertreatment.The solid-state culture of gained after black mold 2221-P bacterium is inoculated in the solid medium top fermentation, dry through 20～120 ℃ of bake dryings after 50～200 mesh sieves are crossed in pulverizing, and detect enzyme and live.Said preparation is for tawny to dark reddish brown or black, loose, lubricated, free from extraneous odour, detects enzyme work and can reach CMC method 31397.1～41826.7U/g and FPA method 1779.3～2212.1U/g.

A kind of preparation method of Aspergillus niger strain fermentative prodn polypeptide protein feed, its step is following:

A. bacterial screening: from soil sample, the tree branches and leaves of gathering rot appearance, palm kernel meal appearance; Separation and purification through five kinds of different substratum is handled with differentiating; Obtain 146 strain filamentous funguss; Filter out bacterial strain through CMC-Na congo red method, PKC congo red method and filter paper strip method, pass through the Congo red and Congo red culture medium culturing of PKC of further CMC-Na again, select 9 strains according to hydrolysis circle size and produce cellulosic bacterial strain; Select the cellulosic bacterial strain of high yield with its CMC enzyme work of shake-flask culture fermented liquid detection and the work of FPA enzyme again; 3～5 times potato nutrient solution shake flask fermentation is measured its living weight and palm-kernel dregs of rice liquid, selects the bacterial strain of three plant height cellulase-producings, and strain number is respectively 1042-2,2221-P and 13005;

B. strain identification:

Morphologic observation through growth characteristic, hyphostoma development and opticmicroscope, electronic scanning mirror, produce look, produce enzyme kinetics and 18SrDNA molecular biology etc. identify 2221-P on the PDA flat board is; Mycelia just is white, fine hair shape; The bacterium colony median rise is to the sparse growth of radiation all around; Bacterium colony central authorities, mycelia fades to yellow-gray, and is then unusual thick close; Back mycelia becomes black; Conidium black is accredited as the black mold of Aspergillus;

C. actication of culture: with containing 0.01～0.10% volume ratio tween-80 sterilized water spore is processed bacteria suspension, inhale the 0.5ml spore suspension with little pipettor and in the plate of PDA substratum, cultivate 24～100h, produce spore at 20～45 ℃;

D.2221-P bacterial strain culture of seed liquid: the bacterial classification that activation is good is with the aseptic spore suspension of washing into of 0.01～0.10% volume ratio tween-80, is inoculated in the liquid nutrient medium by the inoculum size of 5～20% mass ratioes, and this substratum is the PKC of 5～15% mass ratioes; 0.1～0.4% quality is than peptone, 0.5～1.5% quality is than glucose, and 0.1～0.5% quality compares ammonium sulfate; PH 4～8; The bottle that shakes of inoculation is placed the temperature control shaking table, and 20～45 ℃, 220rpm cultivates 24～100h;

E. solid fermentation: solid medium is to be basic carbon source matrix with PKC; Concrete prescription is through 150～300 ℃ of humidity of high temperature 20～60% expanded PKC powder 75～90%, soybean meal 2～10%, wheat bran 2～10%, glucose 0.4～2.0%, peptone 0.4～2.0%, ammonium sulfate 0.1～0.6%, lime carbonate 0.1～0.5%; Material: water=1: 1.0～2.5; PH4～8 are earlier with PKC powder, soybean meal, wheat bran and CaCO ₃Weigh according to quantity, dry mixing is even, adds water and mixes thoroughly, when being sub-packed in the cloth bag in high-pressure sterilizing pot 115～126 ℃ of sterilization 30min, and vexed again 1～2h; Above-mentioned solid medium is weighed up according to quantity, add solid substratum 10～80% water and be mixed with suspension-s, 115 ℃ of sterilization 20～30min; Be cooled to 40～50 ℃ of usefulness to be inoculated, the solid material that sterilization is good places sterile culture indoor, and is to be cooled during to 40～50 ℃; Suspension-s such as inoculation liquid seeds and glucose, the liquid seeds inoculum size is inoculated and mixed thoroughly by 5～20% of solid culture base unit weight, and is final; The solid material of substratum: liquid water=1: 1.0～2.5, liquid are the water of mixing siccative, the total amount of the suspension-s of bacterium seed liquor and glucose; The solid material that inoculation is good is sub-packed in the tray, thickness 1～10cm, and the tray bottom is with aseptic cotton place mat; Hide with aseptic cotton again above the material, in the sterile culture chamber, be incubated 20～45 ℃ and RH40～90% of preserving moisture.Cultivated 24～72 hours; Inoculate at the good yeast starter liquid of PDA culture medium culturing by 5～20% of solid culture base unit weight; Cultivated 24～72 hours; Inoculate in the cultured subtilis seed liquor of beef broth culture by 5～20% of solid culture base unit weight, continue to cultivate up to the scheduled time whole cultivation 3～12 days;

F. aftertreatment: dry after 50～200 mesh sieves are crossed in pulverizing through black mold 2221-P inoculation solid-state culture of gained after the solid medium top fermentation through 20～120 ℃ of bake dryings, and detect polypeptide protein content.

Thus; The applicant is substrate with PKC; Through the systematic research separation screening of science to a strain black mold 2221-P bacterium can high cellulase-producing bacterial strain, through being accredited as: be categorized as Ascomycotina (Ascomycotina), Plectomycetes (Plectomycetes), Eurotiale (Eurotiales), Eurotiaceae or be categorized as the black mold (Aspergillus niger2221-P) in the Aspergillus (Aspergillus) of Deuteromycotina (Deuteromycotina), Haplomycetes (Hyphomycates), hyphomycetales (Hyphomycatales), Moniliaceae in the imperfect stage in the perfect stage.And through solid fermentation, the CMC enzyme work of measuring its preparation is up to 31000～41000U/g, and the work of FPA enzyme is up to 1700～2100U/g.This haves laid a good foundation for the industrial production cellulase.

The present invention has the following advantages and effect: have the new bacterial strain of the high cellulase-producing of independent intellectual property right, be used for cultivation and fermentation, technology is simple, and cost hangs down and preparation stable performance tawny to dark reddish brown, loose, lubricated and free from extraneous odour, is suitable for producing in enormous quantities.The CMC enzyme work of said preparation up to 31591.7～40916.3U/g and the work of FPA enzyme up to (knowing the details data and see attached list 1) more than 1768.3～2200.7U/g.Through using said preparation fermentation PKC is the matrix of main carbon source, and inoculation yeast bacterium and Bacillus Subtilis can produce protein content and be higher than 40% simultaneously, and wherein content of peptides is higher than 80% polypeptide feed, and good market outlook will be arranged.

The work of CMC enzyme, filter paper enzyme activity and the reducing sugar of 5 batches of solid fermentation gained of table 1 2221-P bacterial strain zymin

Annotate: " 2221-P in the table 1 ¹" first fermentation of expression 2221-P, " 2221-P ²" expression second batch fermentation, the rest may be inferred by analogy for it.

Description of drawings

Fig. 1 strain separating screening process synoptic diagram

What Fig. 1 showed is strains separation screening process of the present invention.From different places, different samples source (soil sample, deadwood and rotten leaf, palm compel etc.), collect 70 samples altogether.After through CMC-Na, CMC-Na+ is Congo red, PKC+ is Congo red, the sample line of substratum after with enrichment separates in the beef extract-peptone, PDA etc. 5.Separation and purification through repeated multiple times obtains fungi 146 strains altogether, and enlarges the inclined-plane preservation.With three kinds of method parllel screenings such as CMC-Na congo red method, PKC+ congo red method and filter paper bar collapse methods, select effective strain 19 strains according to indexs such as hydrolysis transparent circle size and the rotten degree of filter paper bar collapse, effectively yield is 13%.Is sole carbon source with this 19 strain bacterium with the pretreated PKC of different methods in 4; In its liquid nutrient medium, ferment; The CMC enzyme of detection fermented liquid is lived and the FPA enzyme is lived, and therefrom selects 213-B, 623-2,912-2,1042-2,1633,2221-P, 6612, the 6922 and 13005 screening bacterial strains as lower whorl.With PKC is sole carbon source, with indexs such as reducing sugar content in enzyme work of fermentation cellulase-producing and the fermented liquid, from 9 strain bacterium, filters out 3 plant height cellulase-producings and leavening property good bacterial strain 1042-2,2221-P and 13005.Wherein, 13005 (koning trichodermas) are the bacterial strain of our company's preservation.To the evaluation of classifying of 1042-2 and 2221-P two bacterium; Observe, produce look, produce enzyme kinetics and 18SrDNA molecular biology identification through strain growth characteristic, colony development and morphological structure; With 2221-P classification be accredited as black mold (Apergillus niger, 2221-P).For this bacterium of better utilised; Test is studied to solid fermentation; Warp is the solid fermentation of basic carbon source with PKC; Obtaining microbial inoculum is tawny to dark reddish brown or black, and loose, lubricated, free from extraneous odour detect to such an extent that the CMC enzyme is lived 31591.7～40916.3U/g with more than FPA enzyme 1768.3～2200.7U/g alive.

Fig. 2 2221-P bacterial strain CMC-Na-congo red method hydrolysis loop graph

Fig. 2 is that the 2221-P bacterial strain is being used congo red staining behind the growth 48h on the CMC-Na substratum, again with sodium chloride solution decolouring back gained hydrolysis loop graph sheet.From figure, can know, after the decolouring of 2221-P bacterial strain tangible hydrolysis circle is arranged all, and the hydrolysis loop diameter be bigger, show that the 2221-P bacterial strain can good use CMC.

Fig. 3 2221-P bacterial strain filter paper strip method collapse figure

In Fig. 3, the right side test tube is a control samples, and the left side is a sample hose.From figure, can know that the 2221-P bacterial strain is cultivated after 7 days in filter paper bar substratum, the filter paper bar has tangible deliquescing, bending, faldstool phenomenon; Nutrient solution is muddy, and length has a large amount of thalline; Long on liquid level and the above filter paper bar thereof have a large amount of bacterium colonies, and produce a large amount of spores.

Fig. 4 2221-P bacterium electron-microscope scanning morphological structure figure

The 4-1 mycelia is long, fine and close, barrier film, hyphal diameter 10 μ m are arranged.

4-2 conidia Hui, spherical diameter of 77 ~ 91μm, an average of 84μm.Sporophore is elongated smooth.

4-3 conidium pine nut shape after shell comes off, becomes slick apple shape.Diameter 3～3.5 μ m, average out to 3.3 μ m.

Embodiment

In conjunction with embodiment and instance the present invention is made further detailed description.Technological method in the enforcement; Some is with reference to teaching material, data and the paper of microbiology, microbiology experimental technique method, biotechnology, biotechnology, industrial microbiology, mycology and taxonomy, zymetology etc.; Some is the method for formulating with reference to relative national standards; Some is the technology that we innovate ourselves, like the pre-treatment of PKC and the triage techniques of purpose bacterium.Carrying out embodiment below describes in detail.

Embodiment 1: the separation of bacterial strain, purifying and screening

A sampling and enrichment: at first be sampling, from collecting totally 70 in sample, sample comes from soil sample, deadwood and rotten leaf appearance (cypress, pine tree, palm tree etc.), PKC appearance, septic plant stem-leaf appearance (leaf of bamboo, various grass etc.) etc.Pack with sterile bag the sampling back.Place 0～4 ℃ of refrigerator to preserve.Next is enrichment, uses through extruding puffing and two kinds of pretreated PKC of method of gas explosion to be sole carbon source, is equipped with inorganic salt and is configured to enrichment medium, every cuvette cartridge 4ml, sterilization.After sterility test, insert the 1g sample again, place 20～45 ℃, 220r/min vibration enrichment culture 5～7 days.Every kind of culture medium inoculated repeats once, totally 280 culture tubes.

The separation and purification of B bacterial strain: a, separation; After treating enrichment culture; Every test tube is all Congo red at PDA, CMC-Na+, PKC, PKC+ are Congo red, beef extract-peptone three plates of totally 5 kinds of substratum line, and is provided with the contrast ware at every turn, draws plate so altogether more than 5000.And in 20～45 ℃ of incubators, cultivated 3～5 days.B, purifying, in the ware of line separation and Culture, select growth vigorous, the cellulase hydrolysis circle arranged, have special bacterium such as chromogenesis circle, form and color to carry out purifying.Through 2～6 purifying repeatedly, obtain 146 strain bacterium.And be stored on the PDA slant medium with 1 * 4 ratio.

The C bacterial strain screening: the one-level screening, the 146 strain bacterium that obtained all can grow in the substratum of PKC as sole carbon source, but differ greatly.Respectively with CMC-Na+ congo red method, PKC+ congo red method and three kinds of method parllel screenings of liquid filter paper strip method, every processing triplicate is up to obtaining reliable and stable result, shared plate more than 2000 covers with 146 strains.CMC-Na+ congo red method and PKC+ congo red method are used congo red staining after being and cultivating 48h, the salts solution decolouring, and record hydrolysis circle transparent circle is also taken pictures.Liquid filter paper strip method was cultivated 7 days, saw its bacteria growing situation and to the etch of filter paper bar, collapse and hydrolysis situation, write down and took pictures.Through above-mentioned screening, from 146 strain bacterium, select effective strain 19 strains, effectively yield is 13.0%; The secondary screening, 19 strain bacterium with the one-level screening is obtained carry out the secondary screening through indexs such as reducing sugar content in enzymatic production test and the detection fermented liquid.19 strain bacterium are being handled (115～200 ℃, 0.5～1.5Mpa), extruding puffing and the expanded 4 kinds of pretreated PKC liquid nutrient medium of the method shake flask fermentations experiment of gas through alkaline purification (1% sodium hydroxide solution), high temperature steaming; Detect its CMC enzyme work and FPA enzyme and live, therefrom select 213-B, 623-2,912-2,1042-2,1633,2221-P, 6612,6922 and 13005 9 strain bacterium as the lower whorl screening; Three level screen, the 9 strain bacterium warp that is obtained through the secondary screening is in the substratum of sole carbon source with PKC, carries out three level screen with indexs such as enzyme work of fermentation cellulase-producing and fermented liquid reducing sugar content thereof.Cellulase is lived with the CMC enzyme and the work of FPA enzyme is differentiated, and through 3～5 revision tests, therefrom selects the high bacterial strain of 3 strain cellulase-producing content, and they are numbered 1042-2,2221-P and 13005.Wherein 1042-2 and 2221-P are new screening bacterial strain, and 13005 for laboratory preservation bacterial strain, belongs to healthy and free from worry (family name) wood mould (Trichodorma koningii).9 days CMC enzyme of 1042-2 fermentation is lived and the work of FPA enzyme is respectively 2782U/ml and 142U/ml, and the CMC enzyme of 2221-P is lived and the work of FPA enzyme is respectively 1654U/ml and 76U/ml, and 13005 CMC enzyme is lived and the work of FPA enzyme is respectively 1151U/ml and 76U/ml.

Embodiment 2: strain classification is identified

In the process of separation screening, just notice the incubation growth characteristic, morphological structure of 2221-P bacterial strain and produce look etc.Except that above-mentioned research, also increased the substratum utilization, living weight and product enzyme dynamics, the molecular biology identification of electron microscope and scanning electron microscopic observation and 18SrDNA, it is following to obtain the result.

2221-P is on the PDA flat board is, mycelia just is white, fine hair shape, and bacterium colony median rise (24h) is to the sparse growth of radiation all around; Bacterium colony central authorities, mycelia fades to yellow-gray, then unusual thick close (48h); Back mycelia becomes black (72h); Conidium black, numbers of poles is many.Mycelia is long, and densification has barrier film hyphal diameter 10 μ m (Fig. 4-1).Conidia Hui, spherical diameter of 77 ~ 91μm, an average of 84μm.Sporophore elongated smooth (Fig. 4-2).Conidium pine nut shape after shell comes off, becomes slick apple shape.Diameter 3～3.5 μ m, average out to 3.3 μ m (Fig. 4-3).

Through above research and consult (the ultra posthumous work of Wei Jing, fungi identification handbook, 1979; Wear fragrant billows posthumous work, the morphological classification of fungi, 1987; Yao Yijian, Li Yuzhu translate, bacteriology outline (work such as C.J Ah Russell Borrow), 2002; The Lu Jiayun chief editor, plant pathogenic fungi is learned, and 2004; China National Academy of Food & Fermentation Industries, 2008; Chinese Academy of Sciences microbial strains storehouse, 2009; Microbial strains preservation center, Liaoning Province, 2007,2009; Southern Yangtze University China colleges and universities industrial microorganism resource database platform, 2009; Microbial strains preservation center, Guangdong Microbes Inst; Documents and materials such as 2009) identify that 2221-P was categorized as Ascomycotina (Ascomycotina), Plectomycetes (Plectomycetes), Eurotiale (Eurotiales), Eurotiaceae (Eurotiaceae) or is categorized as the black mold (Aspergillus niger 2221-P) in the Aspergillus (Aspergillus) of Deuteromycotina (Deuteromycotina), Haplomycetes (Hyphomycates), hyphomycetales (Hyphomycatales), light color Cordycepps (Moniliaceae) in the imperfect stage in the perfect stage.

Embodiment 3 solid fermentations produce enzyme test

1042-2,2221-P and 13,005 three strain bacterium that three level screen is obtained produce enzyme test through the tray solid fermentation, and solid medium contains 60～85% PKC and 1～10% compositions such as soybean meal, wheat bran and inorganic salt.Through test, CMC enzyme that 1042-2 and 2221-P two bacterium are produced is lived alive all than 13005 height that produced with the FPA enzyme, and the applicant selects 1042-2 and 2221-P two bacterium further to make shallow tray fermentation to test.The CMC enzyme of detected result average out to 1042-2 is lived and is that it is 1116.6U/g that 20331.3U/g, FPA enzyme live; The CMC enzyme of 2221-P is lived and is that it is 966.4U/g that 23431.0U/g, FPA enzyme live; 13005 CMC enzyme is lived and is that it is 679.9U/g that 6299.4U/g, FPA enzyme live, and through comprehensive evaluation and analysis, has proceeded test with the 1042-2 and the 2221-P two strain bacterium of screening newly.

Embodiment 4:PKC pre-treatment

Because the Mierocrystalline cellulose in the wood fibre structure is wrapped in by xylogen and semicellulose; For making Institute of Micro-biology's cellulase-producing have more contact site to fiber; Must carry out pre-treatment to PKC, change its crystalline structure, let Mierocrystalline cellulose and semicellulose more be exposed in the environment; Make it become monose or oligosaccharides by enzymolysis more fully, supply mikrobe to utilize.

4 kinds of methods such as alkaline purification, HTHP boiling processing, puffing and the quick-fried processing of vapour have been adopted in pre-treatment altogether.Through adopting settleability detection, granularity detection, microscopic examination, cellulase degradation test and enzymatic production test totally 5 researchs, comprehensively its result selects the pretreatment process of puffing as PKC.This not only reaches the material requirements that institute needs, and puffing technique and equipment all maturation also can be used for suitability for industrialized production.Environment is not polluted.

Embodiment 5: shake-flask seed liquid tray solid fermentation production of cellulose zymin

1 actication of culture.Sterilized water with containing 0.01～0.10% (volume ratio) tween-80 is mixed with suspension-s, and the spore suspension of inhaling 0.5ml black mold 2221-P bacterium with little pipettor 20～35 ℃ of cultivations 2～7 days, produces plentiful spore in the plate of PDA substratum.

2 culture of seed liquid.The bacterial classification that activation is good is with the aseptic spore suspension of washing into of 0.01～0.10% volume ratio tween-80; Inoculum size by 5～20% mass ratioes is inoculated in the liquid nutrient medium; This substratum is the PKC of 5～15% mass ratioes; 0.1～0.4% quality is than peptone, 0.5～1.5% quality is than glucose, 0.1～0.5% mass ratio (NH ₄) ₂SO ₄PH 4～8, and the bottle of inoculating that shakes is placed the temperature control shaking table, and 20～35 ℃, 220rpm cultivates 24～72h.

3 solid fermentations.Solid medium is to be basic carbon source matrix with PKC.Concrete prescription 1: through hot and humid expanded PKC powder 83%, soybean meal 7%, wheat bran 8%, glucose 0.9%, peptone 0.6%, ammonium sulfate 0.2%, lime carbonate 0.3%, material: water=1: 1.3, pH 6.4; Or specifically fill a prescription 2: through hot and humid expanded PKC powder 86%, soybean meal 4.0%, wheat bran 7.5%, glucose 1.3%, peptone 0.7%, ammonium sulfate 0.4%, lime carbonate 0.1%, material: water=1: 1.7, pH 6.1.Earlier with PKC powder, soybean meal, wheat bran and CaCO ₃Weigh according to quantity, dried tremble even, add water tremble again even, when being sub-packed in the cloth bag in high-pressure sterilizing pot 115～126 ℃ of sterilization 30min, vexed again 2h; 115 ℃ of sterilizations 20 or 21 22 or 25 27 or 30min get final product, be cooled to 50 ℃ of usefulness to be inoculated, the solid material that sterilization is good places sterile culture indoor, and is to be cooled during to 50 ℃, inoculates suspension-s such as liquid seeds and glucose, liquid seeds inoculum size 20% is mixed thoroughly.Like this, solid siccative: liquid (water)=1: 2.0, liquid are the water of mixing siccative, the total amount of the suspension-s of bacterium seed liquor and glucose.Be sub-packed in the tray inoculating good solid material, degree of depth 10cm, tray bottom hide with aseptic cotton above the material with aseptic cotton place mat again, and tray is placed on the discharging frame, are incubated 35 ℃ and the RH 80% that preserves moisture, and inoculation culture 7 days is got material detection enzyme and lived.This batch is dropped into solid material 40kg altogether.

4 aftertreatments.Solid fermentation gained culture, dry through 80 ℃ of vacuum-dryings after 100 mesh sieves are crossed in pulverizing, and detect enzyme and live.This bacterium is tawny to dark reddish brown, loose, lubricated, free from extraneous odour, detects that enzyme work can reach CMC method 15831.8～24864.2U/g and more than PFA method 958～1273.2U/g.This batch obtains dry preparation 30kg.

Embodiment 6:15L jar fermentation seed liquid tray solid fermentation production of cellulose zymin

1. liquid seeds.The actication of culture method is cultivated 60h with black mold 2221-P bacterial classification inoculation with embodiment 5 in the semi-natural seed fermentation jar of 15L, 25 ℃ of temperature, and seeding tank is a semi-air-lift formula stirring at low speed jar, air flow 1:1.5, stirring velocity 60～80rpm.It is sturdy to detect the seed liquor bacterial classification, does not have assorted bacterium, treats subsequent use.

2. solid fermentation.This batch of solid siccative is 100kg, specifically fills a prescription 1: through hot and humid expanded PKC powder 82%, soybean meal 7%, wheat bran 9%, glucose 0.7%, peptone 0.3%, ammonium sulfate 0.6%, lime carbonate 0.4%, and material: water=1: 1.5, pH 6.5; Or specifically fill a prescription 2: through hot and humid expanded PKC powder 80%, soybean meal 10%, wheat bran 8%, glucose 0.5%, peptone 0.5%, ammonium sulfate 0.5%, lime carbonate 0.5%, material: water=1: 6, pH 6.0.At first with PKC, soybean meal, wheat bran and CaCO ₃Weigh up according to quantity, mix through 115～126 ℃ of autoclaving 30min vexed again 1～2 hour thoroughly.Other raw material weigh up be mixed with suspension-s through 115 ℃ the sterilization 20 21 or 22 24 or 26 28 or 30min get final product, cool off subsequent use.When the solid material that sterilization is good is cooled to 45 ℃, suspension-s and bacterium seed liquor such as inoculation glucose, final siccative: liquid (water)=1: 1.5, liquid are the water of mixing siccative, the summation of the suspension-s of bacterium seed liquor and glucose.The solid medium of inoculation is sub-packed in the tray, and the tray bottom hides with aseptic cotton above the material with aseptic cotton place mat again, places on the plate rail of sterilisable chamber, is incubated 31 ℃ and the RH70% cultivation 1～9 day of preserving moisture.And the plain enzyme of detection fibers of taking a sample is lived every day after the 3rd day, and its enzyme work can reach CMC method 23431.0U/g and more than the PFA method 1286.5U/g.

3. aftertreatment.With the fermentation after the solid material 60～80 ℃ of vacuum-dryings.Through pulverizing, cross 150 mesh sieve coating-dividing sealings.This batch obtains the black mold 2221-P bacteria solid fermentation cellulase-producing zymin 76kg of institute.

Embodiment 7: shake-flask seed liquid and the shared tray solid fermentation of 15L jar fermentation seed liquid production of cellulose zymin

1. liquid seeds.Actication of culture is with embodiment 5, liquid seeds be embodiment 5 with embodiment 6 in shaking culture and the culture method co-cultivation liquid seeds of the semi-automatic fermentor tank of 15L.Used bacterial classification is a black mold 2221-P bacterium, and its cultural method is with embodiment 5 and the method described in the embodiment 6.

2. solid fermentation.This batch with solid material 140kg.Concrete prescription 1: through hot and humid expanded PKC powder 84%, soybean meal 8%, wheat bran 6%, glucose 0.9%, peptone 0.6%, ammonium sulfate 0.3%, lime carbonate 0.2%, material: water=1: 1.4, pH 6.8; Or specifically fill a prescription 2: through hot and humid expanded PKC powder 77%, soybean meal 9%, wheat bran 10%, glucose 1.5%, peptone 1.5%, ammonium sulfate 0.6%, lime carbonate 0.4%, material: water=1: 1.6, pH 7.0.With PKC, soybean meal, wheat bran, CaCO ₃Press solid: water=1: 1 poach.Pack is at 126 ℃ of autoclaving 30min, vexed again 1.5h.Other raw material weigh up be mixed with suspension-s through 115 ℃ the sterilization 20 21 or 22....30min get final product, cool off subsequent use.During cold 50 ℃ of the solid material that sterilization is good, rob the temperature inoculation, finally make siccative: liquid (water)=1: 1.4, liquid are the summation of suspension-s of water, bacterium seed liquor and the glucose of siccative.The solid culture of inoculation is sub-packed in the tray, and the tray bottom hides with aseptic cotton on the material with aseptic cotton place mat, places on the plate rail of sterilisable chamber, and 32 ℃ of temperature controls and insulation RH75% cultivated 1～6 day.Detect through CMC method and FPA method that enzyme work is respectively 31591.7～40916.3U/g and the FPA method is 1768.3～2200.7U/g.

3. aftertreatment.With the fermentation after the solid material 90 ℃ of exhausting dryings.Through pulverizing, cross 100 mesh sieve coating-dividing sealings.This batch obtains the 2221-P solid fermentation cellulase-producing zymin 110kg of institute.

Embodiment 8:15L jar fermentation seed liquid tray solid fermentation is produced the polypeptide protein feed preparation

1. liquid seeds.

1.1 2221-P seed liquor:

The actication of culture method is cultivated 60h with black mold 2221-P bacterial classification inoculation with embodiment 5 in the semi-natural seed fermentation jar of 15L, 25 ℃ of temperature, and seeding tank is a semi-air-lift formula stirring at low speed jar, air flow 1: 1.5, stirring velocity 60～80rpm.It is sturdy to detect the seed liquor bacterial classification, does not have assorted bacterium, treats subsequent use.

1.2 yeast seed liquid:

1.2.1 actication of culture.Get yeast 1-2 ring and be mixed with suspension-s, inhale 0.5ml yeast suspension-s in the plate of PDA substratum, cultivated 1～5 day at 20～45 ℃ with little pipettor with the sterilized water that contains 0.02% (volume ratio) tween-80.

1.2.2 the cultivation of yeast seed liquid.The bacterial classification that activation is good is processed suspension-s with the tween-80 sterilized water of 0.01～0.10% volume ratio with spore; Inoculum size by 5～20% mass ratioes is inoculated in the liquid nutrient medium; In the semi-automatic seed fermentation jar of 15L, cultivate 24～60h, 20～45 ℃ of temperature, seeding tank is a semi-air-lift formula stirring at low speed jar; Air flow 1: 1.5, stirring velocity 60～80rpm.It is sturdy to detect the seed liquor bacterial classification, does not have assorted bacterium, treats subsequent use.This culture medium prescription is that 5～25% yams are soaked juice, 0.5～2.5% glucose, pH nature.

1.3 subtilis seed liquor:

1.3.1 actication of culture.Get subtilis 1-2 ring and be mixed with suspension-s, inhale 0.5ml yeast suspension-s in the plate of PDA substratum, cultivated 1～5 day at 20～45 ℃ with little pipettor with the sterilized water that contains 0.02% (volume ratio) tween-80.

1.3.2 the cultivation of yeast seed liquid.The bacterial classification that activation is good is processed suspension-s with the tween-80 sterilized water of 0.01～0.10% volume ratio with spore; Inoculum size by 5～20% mass ratioes is inoculated in the liquid nutrient medium; In the semi-automatic seed fermentation jar of 15L, cultivate 24～60h, 20～45 ℃ of temperature, seeding tank is a semi-air-lift formula stirring at low speed jar; Air flow 1: 1.5, stirring velocity 60～80rpm.It is sturdy to detect the seed liquor bacterial classification, does not have assorted bacterium, treats subsequent use.This culture medium prescription is 0.2～1% Carnis Bovis seu Bubali cream, 0.5～1.5% peptone, 0.2～1%NaCl, pH nature.

2 solid fermentations.This batch of solid siccative is 100kg, specifically fills a prescription 1: through hot and humid expanded PKC powder 78%, soybean meal 8%, wheat bran 10%, glucose 1.5%, peptone 1.5%, ammonium sulfate 0.6%, lime carbonate 0.4%, and material: water=1: 1.6, pH 7.0; Or specifically fill a prescription 2: through hot and humid expanded PKC powder 84%, soybean meal 8%, wheat bran 6%, glucose 0.9%, peptone 0.6%, ammonium sulfate 0.3%, lime carbonate 0.2%, material: water=1: 1.4, pH 6.8.At first with PKC, soybean meal, wheat bran and CaCO ₃Weigh up according to quantity, mix through 125 ℃ of autoclaving 30min vexed again 1.5 hours thoroughly.Other raw material weigh up be mixed with suspension-s through 115 ℃ the sterilization 20 21 or 22 24 or 26 28 or 30min get final product, cool off subsequent use.When the solid material that sterilization is good cools off 50 ℃, suspension-s and bacterium seed liquor such as inoculation glucose, final siccative: liquid (water)=1: 1.6, liquid are the water of mixing siccative, the summation of the suspension-s of bacterium seed liquor and glucose.The solid medium of inoculation is sub-packed in the tray, and the tray bottom hides with aseptic cotton above the material with aseptic cotton place mat again, places on the plate rail of sterilisable chamber, is incubated 23 ℃ and RH 50% cultivation 2 days of preserving moisture.Press solid culture base unit weight 12% inoculation yeast seed liquor after cultivating 48h, continue to cultivate 3 days, cultivate and inoculate the subtilis seed liquor after 3 days, cultivated again at last four days.

3. aftertreatment.With the fermentation after the solid material 80 ℃ of vacuum-dryings.Through pulverizing 200 mesh sieve coating-dividing sealings.This batch obtains polypeptide protein feed preparation 80kg.

Embodiment 9: shake-flask seed liquid is produced the polypeptide protein feed preparation with the shared tray solid fermentation of jar fermentation seed liquid

1. liquid seeds.2221-P, yeast and Bacillus subtilis strain activation are with embodiment 8, and the seed liquor that activation is good is inoculated into shakes bottle and place the temperature control shaking table, and 20～45 ℃, 220rpm cultivates 24～72h.

2. solid fermentation.Use solid medium 140kg for this batch.Concrete prescription 1: through hot and humid expanded PKC powder 86%, soybean meal 4.0%, wheat bran 7.5%, glucose 1.3%, peptone 0.7%, ammonium sulfate 0.4%, lime carbonate 0.1%, material: water=1: 1.7, pH 6.1; Or specifically fill a prescription 2: through hot and humid expanded PKC powder 83%, soybean meal 7%, wheat bran 8%, glucose 0.9%, peptone 0.6%, ammonium sulfate 0.2%, lime carbonate 0.3%, material: water=1: 1.3, pH 6.4.With PKC, soybean meal, wheat bran, CaCO ₃Press solid: water=1: 3 poach.Pack is at 121～126 ℃ of autoclaving 30min, vexed again 2h.Other raw material weigh up be mixed with suspension-s through 115 ℃ the sterilization 20 or 21 or 22 or 23 25 or 27 or 30min get final product, cool off subsequent use.During 40 ℃ of solid material coolings that sterilization is good, rob the temperature inoculation, finally make siccative: liquid (water)=1: 1.3, liquid are the summation of suspension-s of water, bacterium seed liquor and the glucose of siccative.The solid culture of inoculation is sub-packed in the tray, and the tray bottom hides with aseptic cotton on the material with aseptic cotton place mat, places on the plate rail of sterilisable chamber, and 33 ℃ of temperature controls and insulation RH 80% cultivated 8 days altogether.After cultivating 72h,, continue to cultivate 3 days, cultivate and inoculate the subtilis seed liquor after 3 days, cultivated again at last 3 days by solid culture base unit weight 12% inoculation yeast seed liquor.

3. aftertreatment.With the fermentation after the solid material 70 ℃ of exhausting dryings.Through pulverizing 160 mesh sieve coating-dividing sealings.This batch obtains polypeptide protein feed preparation 120kg.

Claims (3)

1. the Aspergillus niger strain of a high cellulase-producing, it is characterized in that: this bacterial strain is black mold (Aspergillus niger) 2221-P in the Aspergillus (Aspergillus), and its preserving number is CCTCC NO:M209143.

2. the preparation method of the described a kind of cellulase produced by fermentation of aspergillus niger strains of claim 1, its step is following:

A. actication of culture: with containing 0.01～0.10% volume ratio tween-80 sterilized water spore is processed bacteria suspension, inhale the 0.5ml spore suspension with little pipettor and in the plate of PDA substratum, cultivate 24～100h, produce spore at 20～45 ℃;

B.2221-P bacterial strain culture of seed liquid: the bacterial classification that activation is good is with the aseptic spore suspension of washing into of 0.01～0.10% volume ratio tween-80; Inoculum size by 5～20% mass ratioes is inoculated in the liquid nutrient medium; This substratum is the PKC of 5～15% mass ratioes; 0.1～0.4% quality is than peptone, 0.5～1.5% quality is than glucose, and 0.1～0.5% quality compares ammonium sulfate; PH 4～8; Shake

of inoculation placed the temperature control shaking table, and 20～45 ℃, 220rpm cultivates 24～100h;

C. solid fermentation: solid medium is to be basic carbon source matrix with PKC; Through the certain humidity of 150～300 ℃ of high temperature 20～60% expanded PKC powder 75～90%, soybean meal 2～10%, wheat bran 2～10%, glucose 0.4～2.0%, peptone 0.4～2.0%, ammonium sulfate 0.1～0.6%, lime carbonate 0.1～0.5%; Material: water=1: 1.0～2.5; PH4～8 are earlier with PKC powder, soybean meal, wheat bran and CaCO ₃Weigh according to quantity, dried tremble even, add water tremble again even, when being sub-packed in the cloth bag in high-pressure sterilizing pot 115～126 ℃ of sterilization 30min, vexed again 1～2h; Glucose, peptone, ammonium sulfate in the above-mentioned solid culture based formulas are weighed up according to quantity, add solid substratum 10～50% water and be mixed with suspension-s, 115 ℃ of sterilization 20～30min; Be cooled to 40～50 ℃ of usefulness to be inoculated, the solid material that sterilization is good places sterile culture indoor, and is to be cooled during to 40～50 ℃; The suspension-s of inoculation liquid seeds and glucose, the liquid seeds inoculum size is inoculated and mixed thoroughly by 5～20% of solid culture base unit weight, and is final; The solid material of substratum: liquid water=1: 1.0～2.5, liquid are the water of mixing siccative, the total amount of the suspension-s of bacterium seed liquor and glucose; The solid material that inoculation is good is sub-packed in the tray; Thickness 1～10cm, tray bottom hide with aseptic cotton above the material with aseptic cotton place mat again; In the sterile culture chamber, be incubated 20～45 ℃ and cultivated 2～12 days, cultivate after 48 hours the plain enzyme work of detection fibers of taking a sample every day with the RH40～90% of preserving moisture;

D. aftertreatment: the solid-state culture of gained after black-koji mould 2221-P strain is inoculated in the solid medium top fermentation, dry through 20～120 ℃ of bake dryings after 50～200 mesh sieves are crossed in pulverizing, and detect enzyme and live.

3. the preparation method of the described a kind of Aspergillus niger strain fermentative prodn polypeptide protein feed of claim 1, its step is following:

A. actication of culture: with containing 0.01～0.10% volume ratio tween-80 sterilized water spore is processed bacteria suspension, inhale the 0.5ml spore suspension with little pipettor and in the plate of PDA substratum, cultivate 24～100h, produce spore at 20～45 ℃;

B.2221-P bacterial strain culture of seed liquid: the bacterial classification that activation is good is with the aseptic spore suspension of washing into of 0.01～0.10% volume ratio tween-80; Inoculum size by 5～20% mass ratioes is inoculated in the liquid nutrient medium; This substratum is the PKC of 5～15% mass ratioes; 0.1～0.4% quality is than peptone, 0.5～1.5% quality is than glucose, and 0.1～0.5% quality compares ammonium sulfate; PH 4～8; Shake

of inoculation placed the temperature control shaking table, and 20～45 ℃, 220rpm cultivates 24～100h;

C. solid fermentation: solid medium is to be basic carbon source matrix with PKC; Concrete prescription is through 150～300 ℃ of humidity of high temperature 20～60% expanded PKC powder 75～90%, soybean meal 2～10%, wheat bran 2～10%, glucose 0.4～2.0%, peptone 0.4～2.0%, ammonium sulfate 0.1～0.6%, lime carbonate 0.1～0.5%; Material: water=1: 1.0～2.5; PH4～8 are earlier with PKC powder, soybean meal, wheat bran and CaCO ₃Weigh according to quantity, dry mixing is even, adds water and mixes thoroughly, when being sub-packed in the cloth bag in high-pressure sterilizing pot 115～126 ℃ of sterilization 30min, and vexed again 1～2h; Above-mentioned solid medium is weighed up according to quantity, add solid substratum 10～80% water and be mixed with suspension-s, 115 ℃ of sterilization 20～30min; Be cooled to 40～50 ℃ of usefulness to be inoculated, the solid material that sterilization is good places sterile culture indoor, and is to be cooled during to 40～50 ℃; The suspension-s of inoculation liquid seeds and glucose, the liquid seeds inoculum size is inoculated and mixed thoroughly by 5～20% of solid culture base unit weight, and is final; The solid material of substratum: liquid water=1: 1.0～2.5, liquid are the water of mixing siccative, the total amount of the suspension-s of bacterium seed liquor and glucose; The solid material that inoculation is good is sub-packed in the tray, thickness 1～10cm, and the tray bottom is with aseptic cotton place mat; Hide with aseptic cotton again above the material, in the sterile culture chamber, be incubated 20～45 ℃ and RH40～90% of preserving moisture, cultivated 24～72 hours; Inoculate at the good yeast starter liquid of PDA culture medium culturing by 5～20% of solid culture base unit weight, cultivated 24～72 hours, inoculate in the cultured subtilis seed liquor of beef broth culture by 5～20% of solid culture base unit weight; Continue to cultivate up to the scheduled time whole cultivation 3～12 days;

D. aftertreatment: dry after 50～200 mesh sieves are crossed in pulverizing through black mold 2221-P inoculation solid-state culture of gained after the solid medium top fermentation through 20～120 ℃ of bake dryings, and detect polypeptide protein content.

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