CN102031226A - Aspergillus niger strain and application thereof - Google Patents
Aspergillus niger strain and application thereof Download PDFInfo
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- CN102031226A CN102031226A CN2009101796239A CN200910179623A CN102031226A CN 102031226 A CN102031226 A CN 102031226A CN 2009101796239 A CN2009101796239 A CN 2009101796239A CN 200910179623 A CN200910179623 A CN 200910179623A CN 102031226 A CN102031226 A CN 102031226A
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Abstract
The invention discloses an Aspergillus niger strain which has higher cellulase activity and can be used for producing food protein and feed protein. The strain is Aspergillus niger ZM-8 strain the preservation number of which is CCTCC NO: M209125. The application of the Aspergillus niger CCTCC NO: M209125 strain has the following steps: preparing the Aspergillus niger into a suspension containing 1.0*10<9>-3.0*10<10> spores/L, and then inoculating the suspension with the inoculum size of 50-100mL/L into a culture medium containing a carbon source, bran and inorganic salt, and culturing at the constant temperature of 20-35 DEG C in a tray for 2-7 days, wherein a pure oxygen culture mode is adopted, and the carbon source comprises apple pomace, or corn stalk powder, or wheat straw powder.
Description
Technical field
The present invention relates to a kind of microorganism and application thereof, especially a kind of aspergillus niger (Aspergillus niger) bacterial strain and application thereof.
Background technology
Utilizing the microorganism of avirulence to carry out large scale culturing and obtain protein on matrix such as agricultural crop straw, agricultural and sideline converted products and organic waste, is the key protein source of foodstuffs industry and fodder industry; Though people have carried out a large amount of research to it for a long time; but up to now; also wide material sources and cheap agricultural crop straw and various agricultural byproducts are not converted into protein formation mass-producing, industrialization, one of its root problem is the strain excellent of not turning out the high yield cellulase activity of widely applying on producing.Big quantity research has confirmed that the space special conditions is the good method of microorganism mutation breeding, though the bacterial strain that the high-cellulose enzyme that utilizes various approach to filter out is lived has report at present, but because tiring of test strain itself is lower, even living, its enzyme improves tens percent, no more than the height of tiring of producing bacterial classification, so can't on producing, play a role.
Utilizing microorganism to carry out high-density culture is that cellulosic is converted into a proteinic important way, it is meant growth conditions or culture technique when cell colony density is cultivated more than 10 times above routine in the microorganism liquid medium within, if can improve the culture density of microorganism, improve the specific production rate of product, not only can reduce the middle efficient of separating, extracting of consumption, raising " downstream engineering " of the volume of culture vessel and substratum, but also can shorten the production cycle, minimizing equipment input and reducing production costs, therefore have important production practice valency meaning.But, because people are shorter to its search time, theoretical investigation awaits deeply, the microbe species that was studied at present is also very limited, mainly be confined to not see so far and utilize aerobic aspergillus niger to carry out the report of high-density culture on intestinal bacteria (E.coli) and the cereuisiae fermentum minority facultative anaerobes such as (S.cerevisiae).
Aspergillus niger is the safe bacterial strain of generally acknowledging at present, the cellulase activity that it produces is higher and enzyme system is more complete, especially can secrete high beta-glucosidase enzyme and live, be the strain excellent of degraded cellulose, but utilize the space flight induced-mutation technique that the strain excellent that it screens high potency ratio was not also appeared in the newspapers.
Summary of the invention
Technical problem to be solved by this invention provides a kind of Aspergillus niger strain, and this bacterial strain has higher cellulase activity, and produces food protein and feedstuff protein by this bacterial strain.
For solving the problems of the technologies described above, the present invention adopts the basic design of technical scheme to be:
A kind of Aspergillus niger strain is provided:
Aspergillus niger ZM-8 (Aspergillus niger ZM-8) bacterial strain, by the depositary institution's preservation that is defined in State Intellectual Property Office approval of the 25 of patent law detailed rules for the implementation, preservation date: on June 10th, 2009; Depositary institution's title and abbreviation: Chinese typical culture collection center, CCTCC; Deposit number: CCTCC NO:M209125.
The Microbiological Characteristics of this bacterial strain shows as: this bacterial strain ratio of transparent circle diameter and colony diameter on Congo red-carboxymethyl cellulose nutrient agar is 1.8～2.2; On the Cha Shi plate culture medium, cultivate, the white mycelium on bacterium colony surface, the heavy fleece shape, quality is loose, and the spore grey black in the intensive growth in whole surface, is evenly distributed, and bacterium colony reverse side white is smooth; Examine under a microscope, its conidiophore bears from matrix, and length is homogeneous comparatively; great majority are 1.5mm～2mm, top capsule sphere, and diameter is 35 μ m～50 μ m; the stigma bilayer, certainly the top capsule comprehensive give birth to, metulae length is 15 μ m～20 μ m; wide is 3 μ m～4 μ m; no tabula, stigma length is 5 μ m～7 μ m, wide is 2 μ m～3 μ m; conidium sphere, diameter are 4.5 μ m～5 μ m.
The application of aspergillus niger CCTCC NO:M209125 bacterial strain:
Described aspergillus niger is made 1.0 * 10
9～3.0 * 10
10The suspension of individual spore/L, again this suspension is inserted 20 ℃～35 ℃ tray constant temperature culture of substratum 2 days～7 days that comprise carbon source, wheat bran and inorganic salt with the inoculum size of 50～100mL/L, the pure oxygen mode is adopted in described cultivation, described carbon source comprises apple residue, or corn stalk powder, or wheat stalk powder.
Optionally, described medium component is by the per-cent of quality kg/ volume L, for: apple residue: 5%～8%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%.
Optionally, described medium component is by the per-cent of quality kg/ volume L, for: corn stalk powder: 3%～7%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%.
Optionally, described medium component is by the per-cent of quality kg/ volume L, for: wheat stalk powder: 2%～5%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%.
Wherein, described substratum is prepared with tap water, initial pH value 4.0～7.0.
The invention has the beneficial effects as follows that the aspergillus niger CCTCC NO:M209125 bacterial strain that the present invention's screening obtains is produced FPU (filter paper enzyme activity), CMC (cellobiohydrolase vigor), C on the solid medium by carbon source with the corn stalk powder
1(endoglucanase vigor) and β-Glase (beta-glucoside enzyme activity) have reached 58U/g, 1176U/g, 96U/g and 72486U/g (under reaction conditionss such as certain pH, temperature, per hour generate the required enzyme amount of 1 μ mol glucose by substrate and be defined as 1U) respectively; Up to now, the high enzymatic activity of the FPU that once reported is 19U/g, and the high enzymatic activity of CMC is 908.2U/g, and the high enzymatic activity of β-Glase is 141649U/g; Degradation rate to Mierocrystalline cellulose, hemicellulose and xylogen in the corn stalk is respectively 20.92%, 23.99% and 1.04%; It is 19.30% that enzymolysis gets sugared rate; Simultaneously, be in the liquid nutrient medium of main carbon source with apple residue, corn stalk powder and wheat stalk powder, its film-forming properties has reached 32mm, 25mm and 19mm respectively, and wherein the tray degree of depth is 40mm, and the degree of depth of the nutrient solution of adorning is 35mm; Its high intensity values g (weight in wet base)/L is respectively 406.4,358.2 and 307.5, and the actual highest record of the high-density growth of having reported so far is that 174g (the weight in wet base)/L of E.coli W330 and E.coli are used to produce 175.4g (the weight in wet base)/L of PHB (poly butyric ester) " engineering bacteria ".
Described aspergillus niger ZM-8 (Aspergillus niger ZM-8) bacterial strain, by the depositary institution's preservation that is defined in State Intellectual Property Office approval of the 25 of patent law detailed rules for the implementation, preservation date: on June 10th, 2009; Depositary institution's title and abbreviation: Chinese typical culture collection center, CCTCC; Deposit number: CCTCC NO:M209125.
Embodiment
Embodiment 1
The seed selection of aspergillus niger CCTCC NO:M209125 bacterial strain:
1, space flight mutagenesis
According to cultivating target, the aspergillus niger that selection itself has the more efficient price ratio is equipped on the recoverable spacecraft, and it is morphed under the special environmental conditions mutagenesis of space.After spacecraft returns ground, take out the aspergillus niger that sets out that carries, be stored in potato slant medium, with the screening of pending Microbiological Characteristics research and strain excellent.
2, Microbiological Characteristics
The Microbiological Characteristics of space flight mutagenesis aspergillus niger CCTCC NO:M209125 bacterial strain shows as: this bacterial strain ratio of transparent circle diameter and colony diameter on Congo red-carboxymethyl cellulose nutrient agar is 1.8～2.2; On the Cha Shi plate culture medium, cultivate, the white mycelium on bacterium colony surface, the heavy fleece shape, quality is loose, and the spore grey black in the intensive growth in whole surface, is evenly distributed, and bacterium colony reverse side white is smooth; Examine under a microscope, its conidiophore bears from matrix, and length is homogeneous comparatively; great majority are 1.5mm～2mm, top capsule sphere, and diameter is 35 μ m～50 μ m; the stigma bilayer, certainly the top capsule comprehensive give birth to, metulae length is 15 μ m～20 μ m; wide is 3 μ m～4 μ m; no tabula, stigma length is 5 μ m～7 μ m, wide is 2 μ m～3 μ m; conidium sphere, diameter are 4.5 μ m～5 μ m.
3, the screening of aspergillus niger CCTCC NO:M209125 bacterial strain
Utilize ordinary method to this actication of culture, plate isolation earlier, picking eugonic single bacterium colony on plate culture medium is inoculated in respectively in the test tube of containing filter paper bar substratum then, in 20 ℃～35 ℃ constant temperature culture 24h～72h, the bacterial strain that will show the filter paper degraded is then made serial dilution, on Congo red-carboxymethyl cellulose nutrient agar, make plate isolation, chosen the single colony inoculation in HC＞1 in the preservation of Cha Shi slant medium, in order to multiple sieve.When sieving again, with test strain about 3mm of picking slant culture on slant medium of primary dcreening operation
2～5mm
2Be inoculated on the seed culture medium, with aseptic glass stick crushing, and stir evenly with substratum, 20 ℃～35 ℃ constant temperature culture 24h～72h, produce on the enzyme substratum and cultivate than inserting the basis by 1: 5～1: 10 inoculation of solid koji mass ratio, each enzyme of surveying cellulase system behind 20 ℃～35 ℃ constant temperature culture 24h～72h lives, sugared rate and cultured products composition.
Described seed culture based component is pressed the per-cent of quality kg/ volume L, for: corn stalk powder: 20%～50%, ammonium sulfate: 0.5%～2%, sal epsom: 0.01%～0.05%, potassium primary phosphate: 0.002%～0.012%, prepare with distilled water.
The per-cent of enzyme medium component by quality kg/ volume L is produced on described basis, for: corn stalk powder: 40%～85%, wheat bran: 15%～60%, ammonium phosphate: 0.5%～2%, sal epsom: 0.01%～0.05%, potassium primary phosphate: 0.002%～0.012%, prepare with tap water.
Measurement result shows, each enzyme work of a few plant mutant bacterial strains of primary dcreening operation is compared with starting strain, raising is in various degree all arranged, the increase rate maximum of aspergillus niger ZM-8 bacterial strain cellulase activity wherein, FPU (filter paper enzyme activity), CMC (cellobiohydrolase vigor), C
1(endoglucanase vigor) and β-Glase (beta-glucoside enzyme activity) have improved 2.1 times, 3.5 times, 1.7 times and 1.8 times successively than starting strain, each component enzyme activity that bacterial strain is sieved in several strains again all utmost point is significantly higher than or is significantly higher than starting strain, wherein each component enzyme activity of aspergillus niger ZM-8 bacterial strain all the utmost point be significantly higher than or be significantly higher than other each sieves bacterial strain again, therefore determine that aspergillus niger ZM-8 is the final high yield cellulase live strain that screens, i.e. aspergillus niger CCTCC NO:M209125 bacterial strain.
Embodiment 2
The application of aspergillus niger CCTCC NO:M209125 bacterial strain:
With the slant pore of described aspergillus niger stroke-physiological saline solution wash-out, make 1.0 * 10
9～3.0 * 10
10The suspension of individual spore/L, with the inoculum size access substratum of this suspension with 50～100mL/L, 20 ℃～35 ℃ tray constant temperature pure oxygens were cultivated 2 days～7 days again; Described medium component is by the per-cent of quality kg/ volume L, for: apple residue: 5%～8%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%, with tap water preparation, initial pH value 4.0～7.0.
Embodiment 3
The application of aspergillus niger CCTCC NO:M209125 bacterial strain:
Compare with embodiment 2 and only to be that substratum is different.The medium component of present embodiment is by the per-cent of quality kg/ volume L, for: corn stalk powder: 3%～7%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%.
Embodiment 4
The application of aspergillus niger CCTCC NO:M209125 bacterial strain:
Compare with embodiment 2 and only to be that substratum is different.The medium component of present embodiment is by the per-cent of quality kg/ volume L, for: wheat stalk powder: 2%～5%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%.
Claims (6)
1. an aspergillus niger ZM-8 (Aspergillus niger ZM-8) bacterial strain, it is characterized in that, the deposit number of this bacterial strain is CCTCC NO:M209125, and this bacterial strain ratio of transparent circle diameter and colony diameter on Congo red-carboxymethyl cellulose nutrient agar is 1.8～2.2; On the Cha Shi plate culture medium, cultivate, the white mycelium on bacterium colony surface, the heavy fleece shape, quality is loose, and the spore grey black in the intensive growth in whole surface, is evenly distributed, and bacterium colony reverse side white is smooth; Examine under a microscope, its conidiophore bears from matrix, and length is homogeneous comparatively; great majority are 1.5mm～2mm, top capsule sphere, and diameter is 35 μ m～50 μ m; the stigma bilayer, certainly the top capsule comprehensive give birth to, metulae length is 15 μ m～20 μ m; wide is 3 μ m～4 μ m; no tabula, stigma length is 5 μ m～7 μ m, wide is 2 μ m～3 μ m; conidium sphere, diameter are 4.5 μ m～5 μ m.
2. the application of an aspergillus niger CCTCC NO:M209125 bacterial strain is characterized in that, this aspergillus niger is made 1.0 * 10
9～3.0 * 10
10The suspension of individual spore/L, again this suspension is inserted 20 ℃～35 ℃ tray constant temperature culture of substratum 2～7 days that comprise carbon source, wheat bran and inorganic salt with the inoculum size of 50mL/L～100mL/L, the pure oxygen mode is adopted in described cultivation, described carbon source comprises apple residue, or corn stalk powder, or wheat stalk powder.
3. the application of aspergillus niger CCTCC NO:M209125 bacterial strain according to claim 2, it is characterized in that, described medium component is by the per-cent of quality kg/ volume L, for: apple residue: 5%～8%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%.
4. the application of aspergillus niger CCTCC NO:M209125 bacterial strain according to claim 2, it is characterized in that, described medium component is by the per-cent of quality kg/ volume L, for: corn stalk powder: 3%～7%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%.
5. the application of aspergillus niger CCTCC NO:M209125 bacterial strain according to claim 2, it is characterized in that, described medium component is by the per-cent of quality kg/ volume L, for: wheat stalk powder: 2%～5%, wheat bran: 1%～5%, ammonium phosphate: 0.1%～1.0%, potassium primary phosphate: 0.05%～0.2%, sal epsom: 0.01%～0.05%, ferric sulfate: 0.001%～0.005%.
6. according to the application of claim 2 or 3 or 4 or 5 described aspergillus niger CCTCC NO:M209125 bacterial strains, it is characterized in that described substratum is prepared with tap water, initial pH value 4.0～7.0.
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Cited By (7)
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| CN102417884A (en) * | 2011-06-21 | 2012-04-18 | 江苏科技大学 | Aspergillus niger strain and application thereof to enrichment and extraction of 1-deoxynojirimycin (1-DNJ) from white mulberry root-bark |
| CN102911875A (en) * | 2012-04-27 | 2013-02-06 | 河南省科学院生物研究所有限责任公司 | Preparation method for multifunctional soil remediation microbial inoculum |
| CN103289903A (en) * | 2013-05-07 | 2013-09-11 | 山西省农业科学院农业环境与资源研究所 | Aspergillus niger H active decomposition accelerator with bacteriostasis function and preparation method thereof |
| CN104774772A (en) * | 2015-04-16 | 2015-07-15 | 乐山师范学院 | Straw decomposing fungicide with fertility increasing effect and application of fungicide |
| CN105586295A (en) * | 2016-01-22 | 2016-05-18 | 竹山县鑫源皂素有限责任公司 | Bacillus coagulans, culture method thereof and application of Bacillus coagulans in preparation of bio-organic fertilizer |
| CN106591140A (en) * | 2015-10-19 | 2017-04-26 | 粮华生物科技（北京）有限公司 | Aspergillus niger, culture method and applications thereof, and bacterial agent |
| CN107058122A (en) * | 2017-03-01 | 2017-08-18 | 北京禾和润生科技有限公司 | One plant of aspergillus versicolor and its tunning and purposes |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102417884A (en) * | 2011-06-21 | 2012-04-18 | 江苏科技大学 | Aspergillus niger strain and application thereof to enrichment and extraction of 1-deoxynojirimycin (1-DNJ) from white mulberry root-bark |
| CN102911875A (en) * | 2012-04-27 | 2013-02-06 | 河南省科学院生物研究所有限责任公司 | Preparation method for multifunctional soil remediation microbial inoculum |
| CN103289903A (en) * | 2013-05-07 | 2013-09-11 | 山西省农业科学院农业环境与资源研究所 | Aspergillus niger H active decomposition accelerator with bacteriostasis function and preparation method thereof |
| CN103289903B (en) * | 2013-05-07 | 2015-02-04 | 山西省农业科学院农业环境与资源研究所 | Aspergillus niger H active decomposition accelerator with bacteriostasis function and preparation method thereof |
| CN104774772A (en) * | 2015-04-16 | 2015-07-15 | 乐山师范学院 | Straw decomposing fungicide with fertility increasing effect and application of fungicide |
| CN104774772B (en) * | 2015-04-16 | 2019-07-09 | 乐山师范学院 | It is a kind of with the straw decomposition microbial inoculum of getting fat effect and its application |
| CN106591140A (en) * | 2015-10-19 | 2017-04-26 | 粮华生物科技（北京）有限公司 | Aspergillus niger, culture method and applications thereof, and bacterial agent |
| CN106591140B (en) * | 2015-10-19 | 2019-09-03 | 粮华生物科技（北京）有限公司 | Aspergillus niger and its cultural method and microbial inoculum and application |
| CN105586295A (en) * | 2016-01-22 | 2016-05-18 | 竹山县鑫源皂素有限责任公司 | Bacillus coagulans, culture method thereof and application of Bacillus coagulans in preparation of bio-organic fertilizer |
| CN107058122A (en) * | 2017-03-01 | 2017-08-18 | 北京禾和润生科技有限公司 | One plant of aspergillus versicolor and its tunning and purposes |
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Application publication date: 20110427 |