CN101935641A - Coproduction and fermentation method for high-activity cellulase and feed protein - Google Patents
Coproduction and fermentation method for high-activity cellulase and feed protein Download PDFInfo
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Abstract
The invention discloses a method for preparing high-activity cellulase and feed protein through fermentation and coproduction technology. The production technology comprises the following steps of: inoculating bred aspergillus niger FL26 (Aspergillus niger FL26) into agricultural cellulose wastes (corn cobs and rice stalks) serving as main raw materials, performing liquid state fermentation on high-activity cellulose, inoculating domesticated candida utilis LW05 (Candida utilis LW05) on the fourth day of the fermentation, and continuously fermenting; after fermentation, performing ultrafiltration to obtain liquid supernatant and solid residue, wherein the liquid supernatant is solution of the high-activity cellulose from which high-activity endoglucanase (265.7U/mL) and beta-glucuroide (191.4U/mL) can be separated and purified, and the solid residue is dried and crushed to obtain high-quality feed protein. The crude protein accounts for over 39 percent of the feed protein. Test results from National Quality Supervision and Inspection Center (Beijing) prove that the feed protein has the advantages of no toxin, no harm and rich nutrition.
Description
Technical field:
The invention belongs to biological technical field, relate to a kind of method of utilizing agricultural wastes (corn cob, rice straw) fermentation coproduction high active cellulase and feedstuff protein particularly.
Background technology:
China is large agricultural country, and the wide material sources of the plain waste of agricultural fibre mainly contain maize straw, straw, straw, corn cob etc., and wherein the overwhelming majority is directly burnt, and not only wastes resource, and causes environmental pollution.Utilize the plain waste material of biotechnology exploitation agricultural fibre, produce high value added product, not only have important economic implications, have environmental benefit simultaneously
[1]
Utilize cellulase with the plain waste of agricultural fibre be transformed into produce and life in required material be very effective means.Cellulase is the enzyme system of a class complexity, their synergies, and catalyse cellulose is converted into glucose.Product according to substrate, action site and the release of cellulase is divided three classes cellulase endoglucanase, exoglucanase and beta-glucosidase
[2]Cellulase has a wide range of applications in the many fields of industrial or agricultural.In foodstuffs industry, cellulase can be applicable to the production of fruit vegetable drink, and the raising of brewers malt sugar concentration is in the raising of the nutritive value of feed; At pulp and paper industry, utilize cellulase to carry out the bio-modification of rough paper pulp, can increase the toughness and the whiteness of paper; In washing industry, cellulase also is widely used as detergent additive, strengthens washing effect; In the bioenergy exploitation, cellulase has play a part crucial, utilizes highly active cellulase that cellulose hydrolysis is generated fermentable sugars, further is converted into ethanol again
[3]At present, domestic production about cellulase fermentations had a large amount of research and application, but also has some bottleneck problems:
(1) solid fermentation method is mainly adopted in the production of domestic cellulase.Though this method has low, the advantage of simple technology of input, also there are limitation such as level of automation is low, labour intensity big, unstable product quality
[4]Therefore, along with the continuous progress of liquid fermentation process, adopt the solution fermentation production of cellulose enzyme trend that is inevitable.
(2) cellulase activity that utilizes existing technology to produce is still waiting to improve, and enzyme system composition is still waiting perfect.The bacterial strain of general production usefulness is a fungus Trichoderma, and the cellulase system that this class fungi produces is formed not exclusively, though have very high endoglucanase activity, often the beta-glucoside enzyme activity is on the low side, and such characteristics can make its application be subjected to certain restriction
[5]
(3) in the production fermentation and enzymatic hydrolysis system of cellulase, often have the accumulation of a large amount of glucose.The glucose of these accumulation can produce catastatic checking and feedback inhibition to the fermentation and the enzymolysis process of cellulase, thereby the output and the enzyme that influence cellulase are lived.Accumulation how in time to eliminate a large amount of glucose also is the bottleneck that faces in the enzyme production
[6]
Feedstuff protein is a single cell protein, claims microbial proteinous or tropina again, be meant yeast, fungi, mould, and unicellular organism body such as non-characteristic of disease bacterium in contained protein
[7]It is higher generally to have protein content, the collocation of each amino acid rationally, A wide selection of colours and designs, VITAMIN and trace element are abundant, do not contain advantages such as cholesterol, and these advantages all be general farm crop feed can not compare.Along with the fast development and the insufficient contradiction of feed resource of aquaculture is on the rise, the demand of single-cell protein feed additive increases just day by day
[8]Produce feedstuff protein and can utilize multiple industrial effluent, agricultural byproducts processing tankage and plant cellulose.People such as Ma Xuguang utilize maize straw to produce yeast feed for base-material, and screening has obtained, and a strain can be grown in the maize straw hydrolyzed solution and the Wine brewing yeast strain of high yield single cell protein
[9]It is main raw material that people such as Zhuan Gui have studied with the acid-sludge, produces protein fodder through the yeast solid state fermentation, has improved the acid-sludge protein content
[10]The cellulose resource of utilizing all kinds of straws, wood chip the like waste mainly is to produce the mould tropina.Generally be to utilize fermentation such as aspergillus, mould, the mould of wood, head mold, Mucor or carry out mixed fungus fermentation with yeast.Li Ri waits the people to utilize aspergillus niger to carry out the solid state fermentation maize straw by force and produces feedstuff protein, and its SCP transformation efficiency reaches 23%
[11]People such as Wu Ping are sole carbon source with bagasse, brewer's yeast, Penicllium chrysogenum are mixed the cultivation of bacterium combination and produce feedstuff protein, and its crude protein content can reach 22.4%
[12]People such as Yang Guonong are raw material with the beet pulp, solid state fermentation is prepared animal feeding-stuff containing somatic protein study.The cellulase hydrolysis method is substituted conventional fermentation of Aspergillus niger method carry out the pre-treatment of raw material, its crude protein massfraction reaches 21%
[13]Though China carries out a large amount of work in the exploitation of feedstuff protein with in producing.But in the production application process, also have some problems, as independent fermentation method have that seed culture medium consumption is many, inoculum size is big, fermentation time is long, investment and the more high deficiency of production cost; In the mixed fungus fermentation technology, because cellulose enzyme is imperfect, output of sugar is relatively low, has suppressed saccharomycetic growth, thereby influences the quality of feedstuff protein; The solid state fermentation level of automation of domestic employing is low, labour intensity is big, the content of crude protein is not very high in the unstable product quality, feedstuff protein.These restrictive factors are restricting China's feedstuff protein industrial expansion, and the zymotechnique of traditional feedstuff protein is compared, and advantage of the present invention is as follows:
(1) cellulase solution of coproduction gained possesses the high enzyme vigor, and its endoglucanase vigor can reach 265.7U/mL; And having more complete enzyme system composition, its beta-glucoside enzyme activity can reach 191.4U/mL, can effectively improve the hydrolysis and saccharification ability of this cellulase solution;
(2) gained feedstuff protein protein content height not only, its crude protein content can reach more than 39%; And detected result shows that this feedstuff protein is nontoxic, and is nutritious through national feeding quality supervision and inspection center.
(3) present method adopts coproduction fermentation technique by stages, inserts through the product gastral cavity candiyeast LW05 of domestication in the middle and later periods of producing enzymic fermentation and can not only utilize the glucose that produces in fermentation and the enzymolysis process to grow, and accumulates a large amount of tropinas; Eliminated also simultaneously that glucose has improved the output and the enzyme activity of cellulase to the feedback inhibition effect of cellulase production and enzymolysis process in the fermented liquid.
Reference:
[1] Li Riqiang, Xi Yuying. complex use of wastes containing cellulose [J]. China Environmental Science .2002,22 (001): 24-27.
[2]Lynd?L?R，Weimer?P?J，van?Zyl?W?H，et?al.Microbial?Cellulose?Utilization：Fundamentals?andBiotechnology[J].Microbiol.Mol.Biol.Rev.2002，66(3)：506-577.
[3]Sukumaran?R?K，Singhania?R?R，Pandey?A.Microbial?cellulases-Production，applications?andchallenges[J].
[4] Liu Kai, Lin Jianqiang, Qu Yinbo. cellulase production technology [J]. biological industry technology .2008,4.
[5]Wen?Z，Liao?W，Chen?S.Production?of?cellulase/[beta]-glucosidase?by?the?mixed?fungi?cultureTrichoderma?reesei?and?Aspergillus?phoenicis?on?dairy?manure[J].Process?Biochemistry.2005，40(9)：3087-3094.
[6]Kang?S?W，Ko?E?H，Lee?J?S，et?al.Over-production?of?β-glucosidase?by?Aspergillus?nigermutant?from?lignocellulosic?biomass[J].Biotechnology?Letters.1999，21(8)：647-650.
[7]Chae?S?R，Hwang?E?J，Shin?H?S.Single?cell?protein?production?of?Euglena?gracilis?and?carbondioxide?fixation?in?an?innovative?photo-bioreactor[J].Bioresource?technology.2006，97(2)：322-329.
[8] Zhang Ji, Wu Guangpeng, Wang Wenqiang. single-cell protein feed progress [J]. fodder industry .2006,27 (019): 50-52.
[9] Ma Xuguang, Zhang Zongzhou. the screening [J] of space flight mutagenicity high-yield single cell protein cereuisiae fermentum YB-6 bacterial strain. Chinese feed .2008 (004): 42-44.
[10] Zhuan Gui. saccharification Vinegar Dregs for Producing yeast single cell protein material Study on brewing soy [J]. .2005 (011): 26-29. brewages in China
[11] Li Riqiang, Wang Aiying, Ge Zhian. the solid state fermentation maize straw is produced fodder protein fermentation substratum research [J]. agricultural environment science journal .2008,27 (006): 2484-2488.
[12] Wu Ping, Li Zhengpeng. bagasse produces the research [J] of single cell protein strain excellent screening. secondary special product .2007 (004): the 19-21. of Chinese woods
[13] Tian Ping, Wang Haoju, Wang Ni. the research [J] of beet pulp fermentative preparation protein fodder. amino acid and Biological resources .2009,31 (4).
Summary of the invention:
The object of the present invention is to provide that a kind of production technique is simple, with low cost, the method for utilizing plain waste fermentation coproduction high active cellulase of agricultural fibre and feedstuff protein of economic environmental protection.In order to achieve the above object, the fermentation strain that the present invention adopts is the aspergillus niger FL26 (Aspergillus niger FL26) of seed selection and the product gastral cavity candiyeast LW05 (Candida utilisLW05) of domestication, with the plain waste of agricultural fibre is raw material, fermentation high vigor cellulase of coproduction and high-quality feed albumen may further comprise the steps:
1. actication of culture: the bacterial strain that 2 strains are preferably obtained is inoculated in PDA solid activation medium respectively, 30 ℃ of constant temperature culture 3 days.To and produce gastral cavity candiyeast LW05 and insert among liquid activation medium I and the liquid activation medium II 30 ℃, 180r/min aerated culture 48h respectively through the aspergillus niger FL26 after the activation of PDA solid medium.
PDA solid activation medium: potato juice (with the stripping and slicing of 200g peeling potatoes, add an amount of distilled water digestion, use filtered through gauze), glucose 20g, agar 20g adds water to 1000mL, natural pH.
Seed activation substratum I: glucose 10g, peptone 1g, mineral nutrition liquid 1L.
Seed activation medium ii: sucrose 30g, NaNO
32g, mineral nutrition liquid 1L.
Mineral nutrition liquid: (NH
4)
2SO
46g, KH
2PO
41g, MgSO
47H
2O 5g, CaCl
20.1g, NaCl 0.1g, FeSO
47H
2O 0.05g, MnSO
4H
2O 0.016g, ZnSO
47H
2O 0.014g, CoCl
20.02g, distilled water 1L.
2. zymotechnique: earlier will be through seed activation substratum I activatory aspergillus niger FL26 liquid seeds nutrient solution, insert liquid fermentation medium according to the inoculum size of liquid fermentation medium volume 10%, 30 ℃, 180r/min aerated culture 4 days.Insert with the inoculum size of primary liquid fermention medium volume 10% and produce gastral cavity candiyeast LW05 liquid seeds nutrient solution, 30 ℃, 180rpm continued aerated culture 2 days.
Liquid fermentation medium: corn cob (pulverizing back 60 orders sieves) 20g, rice straw (pulverizing back 60 orders sieves) 10g, yeast powder 20g, mineral nutrition liquid 1L, natural pH.
3. product separates: resulting fermentation end product is carried out uf processing, and molecular weight cut-off is 20kD, ultrafiltration time 24h, and the filtrate that obtains is cellulase solution, and solid residue dehydrates with drying machine or pressure filter, makes feedstuff protein after the pulverizing.
4. the production of cellulase preparation
(1) the enzyme liquid that ultrafiltration obtains of learning from else's experience adds the ammonium sulfate of 50% saturation ratio earlier, and 4 ℃ leave standstill 12h, and the centrifugal 30min of 11000rpm discards precipitation, adds ammonium sulfate then and leaves standstill 12h for 80%, 4 ℃ to saturation ratio, the centrifugal 60min of 11000rpm, collecting precipitation.Acetate buffer solution with pH4.8 dissolves this precipitation again, obtains concentrating enzyme liquid.
(2) adopt Sephadex G200 gel chromatography to remove the salt in the enzyme liquid and enzyme liquid carried out separation and purification, elution speed is 0.5mL/min, can obtain the endoglucanase and the beta-glucosidase of high vigor respectively.
Description of drawings:
Fig. 1. mix bacterium fermentation in 6 days co-production of cellulose and feedstuff protein curve
Fig. 2. the coproduction zymotechnique schema of high vigor cellulase and feedstuff protein
Embodiment:
The invention will be further described below with reference to accompanying drawing and specific examples.
Embodiment 1: fermentation high vigor cellulase of coproduction and feedstuff protein
1. the preparation of the pre-treatment of fermentation raw material and fermention medium
With pulverizer corn cob, rice straw are pulverized, sieved dry for standby with 60 orders.
The obtaining liq fermention medium, its composition is: corn cob (pulverizing back 60 orders sieves) 20g, rice straw (pulverizing back 60 orders sieves) 10g, yeast powder 20g, mineral nutrition liquid 1L.121 ℃, the 0.1Mpa 30min that sterilizes.
2. bacterial classification and activation
(1) bacterial classification: produce high vigor cellulase strain Aspergillus niger FL26
High yield single cell protein bacterial strain Candida.utilis LW05
(2) actication of culture:
The fermentation strain that 2 strains are preferably obtained is inoculated in PDA solid activation medium respectively, 30 ℃ of constant temperature culture 3 days.To and produce gastral cavity candiyeast LW05 bacterial strain and insert among liquid activation medium I and the liquid activation medium II 30 ℃, 180rpm aerated culture 48h respectively through the aspergillus niger FL26 after the solid plate activation.
PDA solid activation medium: potato juice (with the stripping and slicing of 200g peeling potatoes, add an amount of distilled water digestion, use filtered through gauze), glucose 20g, agar 20g adds water to 1000mL, natural pH.
Seed activation substratum I: glucose 10g, peptone 1g, mineral nutrition liquid 1L.
Seed activation medium ii: sucrose 30g, NaNO
32g, mineral nutrition liquid 1L.
Mineral nutrition liquid: (NH
4)
2SO
46g, KH
2PO
41g, MgSO
47H
2O 5g, CaCl
20.1g, NaCl 0.1g, FeSO
47H
2O 0.05g, MnSO
4H
2O 0.016g, ZnSO
47H
2O 0.014g, CoCl
20.02g, distilled water 1L.
3. zymotechnique
Earlier with activated aspergillus niger FL26 liquid seeds, according to the inoculum size access liquid fermentation medium of liquid fermentation medium volume 10%, 30 ℃, 180rpm aerated culture 4 days.Insert with the inoculum size of primary liquid fermention medium volume 10% and produce gastral cavity candiyeast LW05 liquid seeds, 30 ℃, 180rpm are aerated culture 2 days again.
Liquid fermentation medium: corn cob (pulverizing back 60 orders sieves) 20g, rice straw (pulverizing back 60 orders sieves) 10g, yeast powder 20g, mineral nutrition liquid 1L.
The content of endoglucanase, beta-glucosidase and fermentation residue crude protein in the fermenting process, as shown in Figure 1.Fermentation to the is in the time of 6 days, and the enzyme activity of gained endoglucanase, beta-glucosidase and the crude protein content of feedstuff protein all are in higher level, are respectively 265.7U/mL, 191.4U/mL and 39.1%.The technical process of high vigor cellulase of mixed fungus fermentation coproduction and feedstuff protein, as shown in Figure 2.
Embodiment 2: the production of cellulase preparation
1. product separates: will carry out uf processing according to 6 days end products of embodiment 1 resulting fermentation, molecular weight cut-off is 20kD, ultrafiltration time 24h, and the filtrate that obtains is cellulase solution.
2. concentrate enzyme liquid: the enzyme liquid that the ultrafiltration of learning from else's experience obtains, add the ammonium sulfate of 50% saturation ratio earlier, 4 ℃ leave standstill 12h, and the centrifugal 30min of 11000rpm discards precipitation, adds ammonium sulfate then and leaves standstill 12h for 80%, 4 ℃ to saturation ratio, the centrifugal 60min of 11000rpm, collecting precipitation.Acetate buffer solution with an amount of pH4.8 dissolves this precipitation, obtains concentrating enzyme liquid.
3. sieve chromatography: collect gained and concentrate enzyme liquid, adopt Sephadex G200 gel filtration chromatography, elution speed is 0.5mL/min.By the molecular sieve gel chromatography, both can remove the salt (ammonium sulfate) in the enzyme concentrated solution, also can from concentrate enzyme liquid, separate obtaining high vigor endoglucanase and beta-glucosidase simultaneously.Through the research of zymologic property, wherein the optimal reactive temperature of endoglucanase is 60 ℃, and optimal reaction pH is 4.0; The optimal reactive temperature of beta-glucosidase is 60 ℃, and optimal reaction pH is 5.0, and molecular weight is about 130kD.
Embodiment 3: the production of feedstuff protein
To carry out uf processing according to 6 days end products of embodiment 1 resulting fermentation, the gained solid residue dehydrates with drying machine or pressure filter, makes feedstuff protein after the pulverizing.
Detected result shows through State Fodder Quality Supervision ﹠ Examination Center (Beijing): with the plain waste of agricultural fibre is raw material, and through the feedstuff protein that the coproduction fermentation obtains, its crude protein, crude fat, ash oontent are improved, and robust fibre then has obvious reduction; All kinds of useful aminoacids contents improve, enrich, and nutritive value increases; Each biostearin such as A, D, E, B1, B2, folic acid etc. are improved; Contents of heavy metal elements meets the requirement of national similar feed base-material hygienic standard; Salmonellas does not detect, and mould, total plate count meet national regulation, and flavacin content is far smaller than the requirement of national forage health standards such as similar feed base-material.Product belongs to nontoxic level, can be used as feedstuff protein and is widely used in the breed field.
Claims (10)
1. the coproduction fermentation process of one kind high vigor cellulase and feedstuff protein, it is characterized in that aspergillus niger FL26 (A.nigerFL26) and produce two kinds of fermentation strains of gastral cavity candiyeast LW05 (C.utilis LW05) to be inoculated into the plain waste of agricultural fibre (corn cob and rice straw) at the different times of fermentation respectively be in the substratum of raw material, use liquid-state fermentation technology, produce high vigor cellulase and feedstuff protein simultaneously.
2. the coproduction fermentation process of a kind of high vigor cellulase according to claim 1 and feedstuff protein is characterized in that may further comprise the steps:
(1) actication of culture: aspergillus niger FL26 and product gastral cavity candiyeast LW05 are inoculated in the PDA solid activation medium 30 ℃ of constant temperature culture 3d respectively.Aspergillus niger FL26 after will activating then and product gastral cavity candiyeast LW05 insert in seed activation substratum I and the seed activation medium ii 30 ℃, 180rpm aerated culture 48h respectively.
(2) zymotechnique: earlier with activated aspergillus niger FL26 liquid seeds, according to the inoculum size access liquid fermentation medium of liquid fermentation medium volume 10%, 30 ℃, 180rpm aerated culture 4 days.Insert with the inoculum size of primary liquid fermention medium volume 10% and produce gastral cavity candiyeast LW05 liquid seeds, 30 ℃, 180rpm are aerated culture 2 days again.
(3) product isolation and purification: resulting fermentation end product is carried out uf processing, the filtrate that obtains is the cellulase crude enzyme liquid, and utilization is saltoutd, Sephadex G200 gel chromatography carries out the zymin that purifying can obtain endoglucanase and beta-glucosidase respectively; Solid residue dehydrates with drying machine or pressure filter, makes feedstuff protein after the pulverizing.
3. the method for high vigor cellulase of fermentation coproduction according to claim 2 and feedstuff protein, it is characterized in that: the fermentation strain aspergillus niger FL26 (A.niger FL26) that preferably obtains, possesses the ability of utilizing the high vigor cellulase of the plain waste fermentative production of agricultural fibre, institute's cellulase-producing possesses complete cellulase system to be formed, and contains higher activity of beta-glucosidase.
4. the method for high vigor cellulase of fermentation coproduction according to claim 2 and feedstuff protein, it is characterized in that: the high yield SCP bacterial strain that obtains through preferred domestication produces gastral cavity candiyeast LW05 (C.utilis LW05), can utilize the plain waste of agricultural fibre to decompose the glucose that produces and be carbon source, growth fast, and and the mutual compatibility of aspergillus niger FL26, effectively improve the enzyme activity and the output of cellulase.
5. the method for high vigor cellulase of fermentation coproduction according to claim 2 and feedstuff protein is characterized in that, the seed activation substratum I component in the above-mentioned steps 2 (1) is as follows:
Glucose 10g, peptone 1g, mineral nutrition liquid 1L.
Mineral nutrition liquid consists of: (NH
4)
2SO
46g, KH
2PO
41g, MgSO
47H
2O 5g, CaCl
20.1g, NaCl 0.1g, FeSO
47H
2O 0.05g, MnSO
4H
2O 0.016g, ZnSO
47H
2O 0.014g, CoCl
20.02g, distilled water 1L.
6. the method for high vigor cellulase of fermentation coproduction according to claim 2 and feedstuff protein is characterized in that, the seed activation medium ii component in the above-mentioned steps 2 (1) is as follows:
Sucrose 30g, NaNO
32g, mineral nutrition liquid 1L.
7. the method for high vigor cellulase of fermentation coproduction according to claim 2 and feedstuff protein is characterized in that, the liquid fermentation medium component in the above-mentioned steps 2 (2) is as follows:
Corn cob (pulverizing back 60 orders sieves) 20g, rice straw (pulverizing back 60 orders sieves) 10g, yeast powder 20g, mineral nutrition liquid 1L.
8. the method for high vigor cellulase of fermentation coproduction according to claim 2 and feedstuff protein is characterized in that, in the above-mentioned steps 2 (2), aspergillus niger FL26 separately the time of fermentation be 4 days, with the time of producing gastral cavity candiyeast LW05 mixed fermentation be 2 days.
9. the method for high vigor cellulase of fermentation coproduction according to claim 2 and feedstuff protein is characterized in that, used ammonium sulfate saturation ratio interval is 50-80% in above-mentioned steps 2 (3) the middle salting-out process.
10. the method for high vigor cellulase of fermentation coproduction according to claim 2 and feedstuff protein is characterized in that, used elution speed is 0.5mL/min in above-mentioned steps 2 (4) the middle Sephadex G200 gel chromatography processes.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102652532A (en) * | 2011-03-02 | 2012-09-05 | 盐城工学院 | Manufacturing method for producing biological protein feed by corncob |
| CN102726597A (en) * | 2012-06-22 | 2012-10-17 | 湖南省天金科技有限公司 | Method for preparing feed additive by using citrus waste residues |
| CN103555694A (en) * | 2013-11-11 | 2014-02-05 | 中国科学院天津工业生物技术研究所 | Liquid-state fermentation production method of cellulase |
| CN103907744A (en) * | 2013-01-04 | 2014-07-09 | 中粮营养健康研究院有限公司 | Bacterial strain and protein feed and preparation method thereof |
| CN104886337A (en) * | 2015-07-03 | 2015-09-09 | 四川理工学院 | Method for preparing sauce-and-vinegar-residue protein feed |
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2010
- 2010-03-30 CN CN2010101347533A patent/CN101935641A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102652532A (en) * | 2011-03-02 | 2012-09-05 | 盐城工学院 | Manufacturing method for producing biological protein feed by corncob |
| CN102726597A (en) * | 2012-06-22 | 2012-10-17 | 湖南省天金科技有限公司 | Method for preparing feed additive by using citrus waste residues |
| CN103907744A (en) * | 2013-01-04 | 2014-07-09 | 中粮营养健康研究院有限公司 | Bacterial strain and protein feed and preparation method thereof |
| CN103555694A (en) * | 2013-11-11 | 2014-02-05 | 中国科学院天津工业生物技术研究所 | Liquid-state fermentation production method of cellulase |
| CN103555694B (en) * | 2013-11-11 | 2016-08-24 | 中国科学院天津工业生物技术研究所 | A kind of Liquid-state fermentation production method of cellulase |
| CN104886337A (en) * | 2015-07-03 | 2015-09-09 | 四川理工学院 | Method for preparing sauce-and-vinegar-residue protein feed |
| CN104886337B (en) * | 2015-07-03 | 2018-10-09 | 四川理工学院 | A method of preparing sauce vinegar grain protein feed |
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