CN103642695A - Strain of Aspergillus oryzae and applications in preparation of feed additives through microbial fermentation - Google Patents
Strain of Aspergillus oryzae and applications in preparation of feed additives through microbial fermentation Download PDFInfo
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- CN103642695A CN103642695A CN201310381698.1A CN201310381698A CN103642695A CN 103642695 A CN103642695 A CN 103642695A CN 201310381698 A CN201310381698 A CN 201310381698A CN 103642695 A CN103642695 A CN 103642695A
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Abstract
The invention provides a new bacterial strain-Aspergillus oryzae 2013-DP1, and applications in preparation of feed additives through microbial fermentation. The strain is preserved in China Center for Type Culture Collection. The address is Wuhan University, Wuhan, China. The postcode is 430072. The preservation date is June 21th, 2013. The preservation number is CCTCC No: M2013276. Through fermentation and enzymatic hydrolysis conversion of low-value cellulose substances of straws and the like, the strain raises the protein content properly, raises nutritive values, can promote development of feed sources, raises the utilization rate of waste biological resources, and has obvious economic and ecological significances.
Description
(1) technical field
The present invention relates to the new bacterial strain of a strain---aspergillus oryzae (Aspergillus oryzae) 2013-DP1 obtaining through Screening, Mutation, and prepare the application in fodder additives in microorganism fermentation.
(2) background technology
China has a large population and a few land, and in recent years, along with the fast development of livestock industry, the contradiction that people and animals strive grain is quite outstanding, and it is very deficient that China has become the country that feed resource is very nervous, especially a protein feed resource.Because protein is meet animal growth and development and improve one of main nutrient composition of nutrition and quality, and energy ingredient has formed the main body of feed, protein content and the good and bad nutritive value that has determined feed in feed.By best cultural technique, the protein resource amount in China's feed is over half by breach, and along with the Sustainable development of livestock industry, protein feed continues shortage and seriously restricting further developing of aquaculture.The main feedstuff protein of China has fish meal and dregs of beans etc. at present, every year all a large amount of imports.Due to running down and excessive the fishing for of the mankind of oceanic resources, the supply of fish meal is more and more not enough, and price constantly increases.Relevant expert according to Institute of Feeds,China Academy of Agriculture Sciences analyzes, and to the year two thousand twenty, China's protein feed insufficiency of supply-demand is 0.48 hundred million ton.Therefore, depend merely on and utilize existing conventional protein fodder, far can not meet the needs of China's fodder industry, be badly in need of exploitation more novel protein feed source.
Microorganism has the advantages such as kind is many, growth is quick, nutritional requirement is simple, can utilize multiple organic waste growth and breeding and can be converted into the available organic nutrient substance of animal.For example, utilize microorganism fermentation to contain organism such as enriching cellulosic stalk and the macromole cellulose degradation of difficulty utilization can be converted into available small organic molecule, simultaneously, a large amount of microbial cells that breeding obtains, bacterium protein content is higher than the organic substance of cultivating, and contains abundant amino acid, VITAMIN etc.Therefore, utilizing microorganism fermented organic matter, is the approach that has potentiality of the additive of protein feed of development of new.
The dregs of rice class materials such as dregs of beans are that soybean etc. is extracted the by product after lipoid material, dregs of beans class material successfully exploitation become plant protein fodder raw material that can part Peru Fish Dietary, widespread use in aquaculture of dregs of beans in recent years.Yet, in dregs of beans, contain a large amount of antinutritional factor, as soybean agglutinin, trypsin ihhibitor, soy oligosaccharide, phytic acid, gossypol oxalate, glucosinolates, tannin etc.In dregs of beans, also contain the antigen proteins such as glycinin.The existence meeting of these antinutritional factor materials is by disturbing animal digesting and assimilating, destroying normal metabolism and causing that the bad modes such as physiological response affect the health of animal, have affected the utilization of dregs of beans to a great extent nutritive substance.Research shows to utilize microbe fermentation method to process after dregs of beans, can utilize and the proteolytic enzyme system of secreting carries out degraded to a certain degree to antinutritional factor and antigen protein by microorganism growth, reduce the level of these undesirable substances in dregs of beans, significantly improve the nutritional quality of dregs of beans, realize the efficient utilization of dregs of beans.
In addition, in the processing of the agriculture production of China, agricultural-food and light industry and food, all can produce the organism of the materials such as a large amount of rich cellulose, albumen, as vinasse, stalk, cotton dregs, industrial waste organism, rice bran etc., these organism are all good feedstuff raw materials, for producing bacterium protein, provide sufficient raw material, as long as can filter out the microbe species that utilizes these raw materials, be expected to develop protein feed additive.By Mierocrystalline cellulose, there is abundant cellulose resource in China, only the annual output of stalk is approximately 5.7 hundred million tons, yet only less than 10% amount due to fodder production, because straw feed crude fiber content is high, protein, content of mineral substances are low, and livestock and poultry digestibility is low, palatability is poor, thereby has limited its application.After microbiological treatment processing, can promote its quality, comprise the small-molecule substance that the materials such as degraded macromole Mierocrystalline cellulose are readily digested absorption, increase percent protein, improve nutritive value, produce a large amount of beneficial microorganisms simultaneously, improve animal intestinal healthy and improve the quality of animal product.Therefore, utilize microbial fermentation technology, develop unconventional feed resource and produce additive of protein feed, can alleviate effectively the wretched insufficiency of protein feed resources.
(3) summary of the invention
The object of the invention is to provide the new bacterial strain of a strain---aspergillus oryzae (Aspergillus oryzae) 2013-DP1 that a strain obtains through Screening, Mutation, and prepares the application in fodder additives in microorganism fermentation.This bacterium can utilize the organism such as dregs of beans, wheat bran, rice straw to grow for substratum, and the organism after fermentation can be used as fodder additives.
The technical solution used in the present invention is:
Aspergillus oryzae (Aspergillus oryzae) 2013-DP1, is preserved in Chinese Typical Representative culture collection center, address: China, and Wuhan, Wuhan University, postcode: 430072, preservation date: on June 21st, 2013, deposit number: CCTCC No:M2013276.
Bacterial strain of the present invention is that screening obtains from natural condition at first, produces prozyme, comprises proteolytic enzyme, cellulase, amylase etc.In the long-term organic process of fermentation culture, constantly purification and rejuvenation, improve.In addition, this bacterium has been carried out to the breeding of many wheels ultraviolet mutagenesis, screening has obtained the bacterial strain 2013-DP1 of cellulase-producing and the raising of proteolytic enzyme ability, and this strain fermentation transforms the organic production performances such as dregs of beans, stalk and greatly improves.
Aspergillus oryzae strain 2013-DP1 cultivates and propagation method:
Aspergillus oryzae can, in the upper cultivation of PDA substratum (potato, nutrient agar), also can be cultivated on Cha Shi substratum.The Growth and reproduction that can be used for Candida utilis KF1-1.30 ℃ of culture temperature.On this substratum, aspergillus oryzae growth is vigorous, produces abundant mycelia and a large amount of spores.
PDA substratum preparation: 200 grams of potatos (fresh potato peeling, is cut into bulk, boils after 15min, crosses leaching juice and uses), 20 grams of glucose, 15～20 grams, agar, tap water 1000mL, natural pH.
Cha Shi substratum preparation: NaNO
3, 2g; K
2hPO
4, 1g; KCl, 0.5g; MgSO
4, 0.5g; FeSO
4, 0.01g; Sucrose, 30g; Agar, 15～20g; Water, 1000mL, pH nature.
The sterilizing 20 minutes at 121 ℃ of above-mentioned two kinds of substratum.
Bacterium colony, morphological feature: aspergillus oryzae bacterium colony quality is loose, just white is to faint yellow, and along with the prolongation of incubation time, bacterium colony middle portion starts to produce a large amount of conidiums, and bacterium colony front becomes pale green brown, and the back side is colourless.Bacterium colony surface villous shape mycelia, easily provokes.Conidiophore raw on the capsule of subglobose top, top capsule diameter 150～300 μ m.Conidium oval, ellipse, diameter 3.5～5 μ m.When liquid shaking bottle is cultivated, the easy balling of mycelia, produces a large amount of little mycelium pellets.
Physiological property and metabolic characteristic: aspergillus oryzae strain 2013-DP1 can utilize common monose, as glucose, semi-lactosi, fructose etc.Also can ferment and utilize disaccharides, as sucrose, maltose.The mixtures such as molasses are also the good carbon source materials that aspergillus oryzae growth utilizes.The organic substance that can also ferment and utilize stalk, sugared slag, dregs of beans, wheat bran, Semen Maydis powder, sawdust etc. to contain a large amount of polysaccharide and protein, transforms and produces tropina, fodder additives, cellulase, amylase etc.
The invention still further relates to described aspergillus oryzae 2013-DP1 and prepare the application in fodder additives in microorganism fermentation.
Concrete, described is applied as: get one or more in dregs of beans, wheat bran, stalk, adding water to water content is 40～50%(w/w), through high-temperature sterilization, make fermention medium again, inoculation aspergillus oryzae 2013-DP1, carry out aerobic fermentation and cultivate 48～64h, after fermentation ends, tunning is made fodder additives after dried, crushed.
Conventionally, bacterial strain is before carrying out fermentation culture, generally also need through seed enlarged culturing, the bacterial classification that very low temperature is preserved or lyophilized powder is preserved is after inoculation activation on PDA culture medium flat plate, be inoculated into seed shaking flask and cultivate acquisition seed liquor, then be inoculated into fermentation shake flask or fermentor tank and carry out thalline amplification culture, cultivate and obtain after a large amount of thalline, the organism matrix such as inoculum size according to a certain percentage (being generally 10% volume ratio) and dregs of beans, wheat bran, straw powder mix after cultivation and fermentation, fermented substrate is containing the moisture (40～50%) of appropriate amount.Through the fermentation of certain hour, microorganism is degraded and transforms macromolecular substance such as polysaccharide, protein.In fermenting process, through measures such as stirring, temperature control, permeability adjustment, process, guarantee normally carrying out of fermentation.Matrix after fermentation is made fodder additives finished product after drying, pulverizing.
Aspergillus oryzae is prepared the organism such as fermented bean dregs, and to prepare the technical process of fodder additives as follows:
---------stirring is ventilative---dries---pulverizing---finished product to solid state fermentation for material water proportioning, bacterial classification inoculation for the sterilizing of substratum material, maturation process.
Concrete, described bacterial classification aspergillus oryzae strain 2013-DP1 can activate upper cultivation of PDA substratum (every liter containing 200 grams of potatos, 20 grams of glucose, 15～20 grams, agar, 1000 milliliters, tap water, natural pH).The inoculation of cryopreservation, to PDA substratum, is placed in 30 ℃ of incubators, carries out activation culture 72h.3～5 single bacterium colonies of inoculation of activation, to seed shaking flask (PDA substratum, containing agar), are 200rpm at rotating speed, carry out the preparation of seed liquor on the shaking table of 30 ℃.Seed shaking flask is cultivated after 24h, according to 10% bacterial classification inoculum size, is inoculated into fermentation shake flask or fermentor tank (substratum is the same) carries out thalline amplification culture, cultivates after 24h, inoculates fermentation according to 10% of weight of material.
In fermenting process, every 24h stirring once, fully to breathe freely, be convenient to microorganism and carry out sufficient aerobic fermentation.Meanwhile, the temperature variation of monitoring fermentation materials, during the internal temperature too high (being greater than 50 ℃) of bank, carries out stirring cooling.When there is no heating installation, when natural environmental condition bottom fermentation, if when envrionment temperature is lower, can proper extension fermentation time.In fermenting process, also need to observe the changed condition of fermentation materials, comprise in the variation, fermented product of color outward appearance, smell whether turning black, go mouldy, the phenomenon such as caking.
After strain fermentation dregs of beans of the present invention, can produce the additive of protein feed that is rich in sour molten albumen (little peptide) isoreactivity peptide.This bacterial strain is secreted a large amount of high active cellulases, utilizes this bacterial strain can high-efficiency fermenting stalk, and improves the content of its protein.The Agro-industry by-product volume discarded due to China is larger, utilize the microorganism strains in the present invention, develop the new protein feed resource that can partly substitute and supplement existing hay varieties, can reduce costs again, fodder industry is extremely important, and feasibility is higher.
Beneficial effect of the present invention is mainly reflected in: in the present invention, by screening and culturing and seed selection, obtained an Aspergillus oryzae bacterial strain 2013-DP1, the ability that this bacterial strain produces the enzyme systems such as proteolytic enzyme, cellulase is strong, can carry out fermentative degradation and conversion to the feedstuff raw material material of dregs of beans, cellulose family.Utilize this bacterial strain to carry out fermentative processing to dregs of beans, can significantly improve protein content, the crude protein content of raw material dregs of beans is improved more than 6%, produce the little peptide of a large amount of solubilities (sour molten protein content reaches more than 12%) simultaneously.In addition, antinutritional factor and antigen protein that the dregs of beans of this bacterium fermentation can be degraded in dregs of beans effectively, significantly improve the nutritional quality of dregs of beans, thereby improve animal, the utilising efficiency of dregs of beans improved to animal health.This bacterial strain can transform with enzymolysis by fermentation to the cellulose substances of the low values such as straw, suitably improve protein content, improve its nutritive value, can promote the exploitation in feed source, improve the utilization ratio of discarded Biological resources, had obvious economy, ecological significance.
(4) accompanying drawing explanation
Fig. 1 is the colonial morphology of aspergillus oryzae 2013-DP1 on PDA substratum;
Fig. 2 is that the cellulase activity of aspergillus oryzae fermented liquid crude enzyme liquid is identified;
Fig. 3 is the hydrolysis circle of aspergillus oryzae on dregs of beans substratum.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the screening of bacterial strain and seed selection
Separated cellulase-producing, the aspergillus oryzae that protease activity is higher
Separated aspergillus oryzae from the discarded plant samples such as stalk.Preparation PDA substratum (200 grams of potatos, 20 grams of glucose, 15～20 grams, agar, tap water 1000mL, natural pH, sterilizing 20min at 121 ℃).Get the sample that may contain aspergillus oryzae, get 1 gram and join in 99mL water after grinding, fully vibration mixes and gets 100 μ L after 10min and coat PDA flat board.After being positioned over 25 ℃ of cultivation 48h, observe bacterium colony, pick out candidate's aspergillus oryzae strain, carry out monospore separation and purification, further microscopic examination identifies that the rear bacterial strain of preserving carries out further experiment.
The bacterial strain of primary dcreening operation through repeatedly cultivate and selection by mutation after, screening acquisition is to the fermentive bacterial strain 2013-DP1 of the tools such as dregs of beans.The cellulase-producing of this bacterial strain and protease activity are identified as follows:
Congo red development process is identified cellulase.Congo red is a kind of dyestuff, it can form red complex with the polysaccharide material as Mierocrystalline cellulose, but can not with hydrolysis after cellobiose and glucose there is this reaction, in containing cellulosic substratum, add when Congo red, Congo red will combination with Mierocrystalline cellulose forms Congo red-Mierocrystalline cellulose red complex, after Mierocrystalline cellulose in substratum is decomposed, Congo red-cellulose composite just cannot form, with sodium-chlor, wash away and be combined not firm Congo redly with Mierocrystalline cellulose, in substratum, there will be like this transparent circle centered by cellulose degrading enzyme or bacterium.
Aspergillus oryzae is cultivated the rear centrifugal mycelia of removing in liquid medium (above-mentioned potato culture does not add agar, adds the rice straw powder of 10g/L), gets supernatant liquor (crude enzyme liquid) and identifies on the culture dish flat board that contains cellulosic substrate.Be specially: with making solid culture ware containing the aqueous solution of 0.5%CMC-Na substrate and 20g/L agar, the crude enzyme liquid of 10 μ L is dropped on culture dish, cover lid, 37 ℃ of incubator reaction 3～4h, by 0.1% congo red staining immersion, dye 30min, water rinses, then with the NaCl solution of the 1M 15min that decolours.There is obvious yellow transparent circle, illustrate that the fermented liquid of aspergillus oryzae has cellulase activity.Fig. 2 is shown in by congo red staining flat board.
Aspergillus oryzae strain 2013-DP1 protease production is identified.Hydrolysis by aspergillus oryzae on casein food grade flat board and dregs of beans flat board is enclosed, and judges whether to produce proteolytic enzyme and active height.
Casein substrate is dull and stereotyped, extractum carnis: 3g, casein food grade: 10g, agar: 20g, Nacl:5g, water: 1000mL, Sodium phosphate dibasic: 2g, final pH=7.4.According to be dissolved in 50mL damping fluid (0.1mol/l Sodium phosphate dibasic-Sodium phosphate dibasic pH=8) low-grade fever with 2g casein food grade, dissolve.Dregs of beans culture medium flat plate, take 1000mL substratum as standard, adds the bean cake powder of 20g left and right, then boils 1h, filters dregs of beans granulated slag, adds 20g agar powder after it is cooling, and mix and blend, at 121 ℃ of high pressure moist heat sterilization 15min, is down flat plate after cooling.Inoculation is cultivated after 48 hours on above substratum, observe its hydrolysis circle size, found that colony edge has hydrolysis circle to produce (Fig. 3).
Above result demonstration, aspergillus oryzae strain 2013-DP1 generation proteolytic enzyme of the present invention and cellulase ability are stronger.
Embodiment 2:
PDA substratum preparation: 200 grams of potatos, 20 grams of glucose, 15～20 grams, agar, 1000 milliliters, tap water, natural pH, sterilizing is 20 minutes at 121 ℃.
The aspergillus oryzae strain CCTCC No:M2013276 of cryopreservation is inoculated on PDA substratum, is placed in 30 ℃ of incubators, carries out activation culture 72h.
3～5 single bacterium colonies of inoculation of activation, to seed shaking flask (PDA substratum, containing agar), are 200rpm at rotating speed, on the shaking table of 30 ℃, cultivate after 24h, obtain seed liquor.
Get dregs of beans, adding water to water content is 45%, at 121 ℃, fermention medium is made in sterilizing for 20 minutes, according to 10% volume ratio inoculation aspergillus oryzae seed liquor, carry out aerobic fermentation and cultivate 64h(every 24h stirring once), after fermentation ends, get a little tunning through drying to about 10% left and right of moisture, after pulverizing, cross 90 mesh sieves, detect each index content.
The crude protein content 45.93% of raw material dregs of beans, sour molten protein content 4.58%.
In tunning: moisture 11.00%, crude protein 53.63%, sour molten protein 12 .93%, total volatile basic nitrogen 35.23mg/100g.
Embodiment 3:
The activation of aspergillus oryzae strain CCTCC No:M2013276 and seed culture are with embodiment 2.
Get dregs of beans: wheat bran: the mixture of rice straw powder mass ratio 1:1:1, adding water to water content is 40%, at 121 ℃, fermention medium is made in sterilizing for 20 minutes, according to 10% volume ratio inoculation aspergillus oryzae seed liquor, carry out aerobic fermentation and cultivate 48h(every 24h stirring once), after fermentation ends, get a little tunning through drying to about 10% left and right of moisture, after pulverizing, cross 90 mesh sieves, detect each index content.
The crude protein content of each raw material: dregs of beans, 45.93%; Wheat bran, 14.57%; Rice straw powder, 2.89%.
In tunning: moisture 11.00%, crude protein 25.63%, sour molten albumen 4.89%.
Claims (3)
1. an Aspergillus oryzae (Aspergillus oryzae) 2013-DP1, is preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, preservation date: on June 21st, 2013, deposit number: CCTCC No:M2013276.
2. aspergillus oryzae 2013-DP1 as claimed in claim 1 prepares the application in fodder additives in microorganism fermentation.
3. application as claimed in claim 2, it is characterized in that described being applied as: get one or more in dregs of beans, wheat bran, stalk, adding water to water content is 40～50%, through high-temperature sterilization, make fermention medium again, inoculation aspergillus oryzae 2013-DP1, carry out aerobic fermentation and cultivate 48～64h, after fermentation ends, tunning is made fodder additives after dried, crushed.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104726348A (en) * | 2015-03-27 | 2015-06-24 | 贵州省轻工业科学研究所 | Slant test-tube culture medium for aspergillus candidus strain |
| CN105754876A (en) * | 2016-04-10 | 2016-07-13 | 西藏藏真堂藏药产业有限公司 | Highland barley starter, highland barley starter product and preparation method thereof |
| CN107022493A (en) * | 2017-03-24 | 2017-08-08 | 江苏天种牧业股份有限公司 | A kind of aspergillus oryzae strain of high yield complex enzyme for feed and its application |
| CN107929693A (en) * | 2017-12-11 | 2018-04-20 | 重庆盈捷科技有限公司 | A kind of Chinese medicine microorganism formulation for treating nan yang yellow cattle inflatable and preparation method |
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| CN101144060A (en) * | 2007-04-25 | 2008-03-19 | 浙江省农业科学院 | Aspergillus zryzae strain capable of highly producing neutral proteinase and fermentation method thereof on solid state substrate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1422957A (en) * | 2001-12-07 | 2003-06-11 | 北京锐思嘉业饲料应用技术研究中心 | Method for preparing bioactive micro peptide of plant protein |
| CN101144060A (en) * | 2007-04-25 | 2008-03-19 | 浙江省农业科学院 | Aspergillus zryzae strain capable of highly producing neutral proteinase and fermentation method thereof on solid state substrate |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726348A (en) * | 2015-03-27 | 2015-06-24 | 贵州省轻工业科学研究所 | Slant test-tube culture medium for aspergillus candidus strain |
| CN105754876A (en) * | 2016-04-10 | 2016-07-13 | 西藏藏真堂藏药产业有限公司 | Highland barley starter, highland barley starter product and preparation method thereof |
| CN105754876B (en) * | 2016-04-10 | 2020-03-17 | 蒋川湘 | Highland barley rice koji, highland barley rice koji product and preparation method thereof |
| CN107022493A (en) * | 2017-03-24 | 2017-08-08 | 江苏天种牧业股份有限公司 | A kind of aspergillus oryzae strain of high yield complex enzyme for feed and its application |
| CN107929693A (en) * | 2017-12-11 | 2018-04-20 | 重庆盈捷科技有限公司 | A kind of Chinese medicine microorganism formulation for treating nan yang yellow cattle inflatable and preparation method |
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