CN105199967A - Method for quickly improving coring phenomena of aspergillus oryzae - Google Patents
Method for quickly improving coring phenomena of aspergillus oryzae Download PDFInfo
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- CN105199967A CN105199967A CN201510623553.7A CN201510623553A CN105199967A CN 105199967 A CN105199967 A CN 105199967A CN 201510623553 A CN201510623553 A CN 201510623553A CN 105199967 A CN105199967 A CN 105199967A
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Abstract
The invention provides a method for quickly improving coring phenomena of aspergillus oryzae strains. The method has the advantages that liquid and solid alternate cultivation modes are adopted on the basis of improving composition of cultivation media, so that the operation time can be shortened and is a week from the conventional operation time of a month approximately, and the coring phenomena of aspergillus oryzae can be eliminated as quickly as possible; the method is easy to implement and master and low in input, and good effects can be realized; the coring phenomena of the slant cultivation media for the aspergillus oryzae strains can completely disappear by the aid of the method, mycelia are excellent in growth vigor, and the enzyme producing ability of the strains can be recovered; the fructosyl transferase producing ability of the original aspergillus oryzae strains can be basically recovered and reaches the enzyme producing level of aspergillus oryzae strains B which are comparison strains after de-coring rejuvenation experiments are carried out, the enzyme activity can reach 35000 U/g at least, and the biomass weights of the fermented and cultivated mycelia can be recovered and reach 85 g/L at least.
Description
Technical field
The present invention relates to a kind of method improving mould bacterium NUCLEATION PHENOMENA, particularly a kind of quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA.
Background technology
Aspergillus oryzae strain is propagated in culturing process on inclined-plane; through the inoculation that algebraically does not wait; often there will be that mycelia is short, spore is few; media surface occur little in a large number and justify, the solid hard particulate material of quality; color or white or black or occur hickie, and along with the phenomenon that bacterial strain enzyme activity declines.The appearance of this phenomenon, is referred to as " sclerotium " on inclined-plane, as shown in Figure 1 usually.
In the process of slant culture, bacterial strain particularly derives from artificial mutagenic fungi all can there is this phenomenon usually.Sclerotized inclined-plane is difficult to go down to posterity seed with bacterium as production, and be also difficult to proceed preservation as bacterial classification and use, this situation must be well solved.
At present, the concrete reason that inclined-plane sclerotiumization is formed, it be unclear that, but estimates to fail with the energy for growth of bacterial strain, draws and utilizes the deteriorated of nutritive substance relevant with genovariation.Someone says, and " sclerotium " is mycelial aggregate, examines under a microscope, and sclerotium cuts the tight heap state all occurring mycelia inside rear sclerotium outward appearance and core, in " honeycomb " structural state.We can inference learn thus: if in cell growth process, and mycelium can be disperseed well, separately grow equably, bacterium NUCLEATION PHENOMENA will well be improved.
Tensio-active agent is the material that a class has very strong surfactivity, can make liquid, solid surface tension significantly declines.Because its structure itself has the building stone of very strong wetting ability and lipophilicity and parents, it is made to be widely used in the industries such as chemical industry, food, weaving, medicament, mining, usually be used as solubilizing agent, emulsifying agent, wetting agent, suspending agent, diffuse agent, pore forming material, defoamer, stain remover, sterilizing agent and sterilant etc., particularly anti-cohere with anti-caking etc. in, show gratifying diversified utility function.
In the growing environment of microorganism cells, add appropriate surfactant in the medium, particularly add the nonionogenic tenside deriving from good, water-soluble large, the readily biodegradable of nature, dispersing property, biology is inscribed to nontoxic polyol type, for slow down produce between mycelium in cell growth process cohere, become block, thus improve the bacterium NUCLEATION PHENOMENA that organoid matter is formed, there is very large promoter action.
Summary of the invention
The object of the invention is to overcome weak point that prior art exists and provide a kind of quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA.
For achieving the above object, the technical scheme taked: a kind of quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, said method comprising the steps of:
(1) sclerotized aspergillus oryzae strain mycelium is inoculated in improvement liquid nutrient medium and carries out shaking culture under dark condition;
(2) aspergillus oryzae strain after cultivation in step (1) is inoculated on improvement slant medium, then cultivates under dark condition;
(3) aspergillus oryzae strain after cultivating in step (2) is inoculated in improvement liquid nutrient medium under dark condition, carries out shaking culture;
(4) aspergillus oryzae strain after cultivation in step (3) is inoculated on improvement slant medium, then cultivates under dark condition;
Described step (1) and (3) middle improvement liquid culture based formulas are: potato 20%, glucose 2%, sucrose 3.0%, the first nonionic surface active agent 0.2%, and surplus is water, pH nature;
Described step (2) and (4) middle improvement slant medium are made up of the component of following part by weight: potato 20%, glucose 2%, sucrose 3.0%, second nonionic surface active agent 0.2%, agar 1.5%, surplus is water, pH nature.
Preferably, described first nonionic surface active agent is anhydrous sorbitol lauric acid monoester.
Preferably, described second nonionic surface active agent is anhydrous sorbitol lauric acid monoester.
Preferably, described step (1) and (3) middle culture temperature are 30 DEG C, and incubation time is 24 hours.
Preferably, described step (1) and (3) middle hunting speed are 150r/min.
Preferably, described step (2) and (4) middle culture temperature are 30 DEG C, and incubation time is 48 hours.
Preferably, described step (2), 4) in vaccination ways be zig-zag type line.
Preferably, described method also comprises and is inoculated on the conventional preservation slant medium of mould by the aspergillus oryzae strain after cultivating in step (4), then 30 DEG C, carry out cultivation 72 hours under dark condition.There is the cultivation of sclerotized aspergillus oryzae strain through four steps above, its bacterium NUCLEATION PHENOMENA is eliminated, aspergillus oryzae strain after cultivating in step (4) being inoculated in the conventional preservation slant medium of mould carries out cultivation is to stablize this bacterial strain, reducing the probability that bacterium NUCLEATION PHENOMENA appears in this bacterial strain again.
Preferably, the conventional preservation slant medium of described mould is made up of the component of following part by weight: white sugar 3.0wt%; Bean cake powder 2.0wt%; Semen Maydis powder 0.3wt%; Agar 2.0wt%; Surplus is water.
Beneficial effect of the present invention is: the invention provides a kind of quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, its concrete following advantage:
(1) the present invention is on the basis that improved culture medium forms, adopt liquid and solid alternate culture mode, make the operating time within about one month, shorten to one week from routine, eliminate the sclerotized phenomenon of mould as quickly as possible, and method is simple to operate, less investment, easily grasp, effective.
(2) adopt method of the present invention, can make bacterium NUCLEATION PHENOMENA completely dissolve in aspergillus oryzae strain slant medium, mycelium grows fine, and strain enzyme-producing ability is restored.After past sclerotium rejuvenation experiment, the product fructosyl transferase ability of the former aspergillus oryzae strain that sets out returns to the product enzyme level of control strain aspergillus oryzae strain B substantially, enzyme activity all reaches more than 35000U/g, and the mycelial biomass of fermentation culture also recovers to reach more than 85g/L.
Accompanying drawing explanation
Fig. 1 is the bacterium NUCLEATION PHENOMENA occurred in aspergillus oryzae strain slant culture process in the embodiment of the present invention 1; Wherein, A: black, white sclerotium particle; B: mycelia Desertification phenomenon; C: mycelia hickie phenomenon;
Fig. 2 is the mycelium morphology of aspergillus oryzae strain A and B when liquid culture in the embodiment of the present invention 1;
Fig. 3 is the upgrowth situation of aspergillus oryzae strain A succeeding transfer culture in improvement slant medium in the embodiment of the present invention 1;
Fig. 4 is the mycelium morphology of aspergillus oryzae strain A after going sclerotium rejuvenation in the embodiment of the present invention 1;
Fig. 5 is the enzymatic productivity of aspergillus oryzae strain A after going sclerotium rejuvenation and biomass level in the embodiment of the present invention 1.
Embodiment
For better the object, technical solutions and advantages of the present invention being described, below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
The aspergillus oryzae strain adopted in the present embodiment is:
Aspergillus oryzae strain A: sclerotized fructosyl transferase produces strain Aspergillus oryzae scut209 (Aspergillusoryzaescut209), as shown in Figure 1;
Aspergillus oryzae strain B: the normal fructosyl transferase of this Laboratories Accession produces bacterial strain, aspergillus oryzae strain A is the aspergillus oryzae strain that bacterium NUCLEATION PHENOMENA appears in aspergillus oryzae strain B after several pickup kind, this aspergillus oryzae strain B is preserved in the China typical culture collection center being positioned at Wuhan, China, and deposit number is CCTCCNO:M2012106; And being called that " a kind of bacterial strain producing fructosyl transferase and preparation method thereof " authorizes in patent of invention open in name, license number is: ZL201210374664.5.
The conventional preservation slant medium of the mould adopted in the present embodiment is made up of the component of following part by weight: white sugar 3.0wt%, bean cake powder 2.0wt%, Semen Maydis powder 0.3wt%, agar 2.0wt%; PH nature.
The liquid culture based formulas adopted in embodiment is: white sugar 3.0wt%, bean cake powder 2.0wt%, Semen Maydis powder 0.3wt%, and surplus is water, pH nature.
The improvement liquid nutrient medium adopted in embodiment is: potato 20%, glucose 2%, sucrose 3.0%, first nonionic surface active agent 0.2%, surplus is water, and pH nature, described first nonionic surface active agent is anhydrous sorbitol lauric acid monoester Span-2.
The improvement slant medium adopted in embodiment is made up of the component of following part by weight: potato 20%, glucose 2%, sucrose 3.0%, the second nonionic surface active agent 0.2%, agar 1.5%, and surplus is water; PH nature, described second nonionic surface active agent is anhydrous sorbitol lauric acid monoester Span-2.
The fructosyl transferase vigour-testing method adopted in the present embodiment is: fructosyl transferase or zymogenic bacteria body act on sucrose, first generates kestose (1-kestose); Kestose content assaying method adopts HPLC method, and test method is pressed GB/T23528-20096.5 and performed.
Enzyme activity unit defines: under the condition of the best enzymeization reaction of enzyme supplier mark, be oligofructose by sucrose inversion, it is an enzyme activity unit (U) that per minute produces enzyme amount needed for 1 μm of ol kestose;
In formula:
Sucrose total amount in 10----10% (w/w) sucrose solution, g;
The percentage composition of GF2----kestose, %;
0.504----1 μm of ol kestose=0.504mg;
The t----reaction times, 60min;
W----mycelia quality, g.
High performance liquid chromatography (HPLC) detect parameters is: Cosmosil chromatogram sugar post; Moving phase: acetonitrile-water (75%, v/v); Flow velocity: 1.0mL/min; Differential refraction detector (RI): Waters2410; Monitor sensitivity: 4; Column temperature: 30 DEG C; Sampling volume: 10 μ L.
The experimentation that the sclerotium bacterial strain biological pollution possibility adopted in embodiment is got rid of:
(1) operate: mycelia one ring on picking aspergillus oryzae strain A and B inclined-plane, is inoculated in aseptic liquid nutrient medium (PDA), shaking culture 48 hours at 30 DEG C respectively, sampling is observed.
(2) shaking flask is observed: shaking flask A (corresponding aspergillus oryzae strain A) is normal with mycelial growth in shaking flask B (corresponding aspergillus oryzae strain B), and in liquid nutrient medium, liquid is limpid, transparent, and nose hears free from extraneous odour, presents the peculiar rice fragrance of aspergillus oryzae.Mycelium is spherical shape, and the mycelium of wall built-up presents long mycelioid, as shown in Figure 2.
(3) microscopy is observed: sampling basis of microscopic observation, does not find that there is bacteriological infection.
Result shows that white, black particle and the hickie occurred in inclined-plane should be the problem of self variation, eliminates the possibility that miscellaneous bacteria infects.
The present embodiment adopt the method improving aspergillus oryzae bacterium NUCLEATION PHENOMENA to be the working method of solid-liquid alternate culture, wherein nonionic surface active agent selects dispersing property good, water-soluble large, readily biodegradable, hypotoxic EPE polyol EPE anhydrous sorbitol lauric acid monoester Span-20, and working concentration is 0.2%v/v; Concrete grammar is as follows:
(1) picking is in sclerotized aspergillus oryzae strain A inclined-plane mycelia one ring, is inoculated in improvement liquid nutrient medium, in 150rmp/m, and 30 DEG C, shaking culture 24 hours under dark condition;
(2) in picking step (1) liquid cultivate after mycelium one ring, get the line of " it " font, be inoculated in improvement slant medium, at 30 DEG C, to put in incubator dark culturing 48 hours, cultivation results is as shown in Figure 3;
(3) inclined-plane bacterial strain one ring after cultivating in picking step (2), is inoculated in improvement liquid nutrient medium again, and in 150rmp/m, 30 DEG C, shaking culture 24 hours under dark condition, observes process suspension cell degree of scatter;
(4) again in picking step (3) liquid cultivate after suspended bacteria filament one ring, get the line of " it " font, be inoculated in improvement slant medium, at 30 DEG C, to put in incubator dark culturing 48 hours, then be inoculated on the conventional storage medium flat board of mould and observe inclined-plane sclerotium disappearance degree, as shown in Figure 4, the bacterium NUCLEATION PHENOMENA of bacterial strain obviously disappears;
(5) inclined-plane bacterial strain one ring after cultivating in picking step (4), turns and is inoculated on the conventional storage medium of mould, cultivate 72 hours at 30 DEG C in incubator under dark condition.
Experimental result: after aforesaid method, bacterium NUCLEATION PHENOMENA completely dissolve in sclerotized aspergillus oryzae strain A slant medium, mycelium grows fine, and strain enzyme-producing ability is restored.After past sclerotium rejuvenation experiment, the product fructosyl transferase ability of the former aspergillus oryzae strain that sets out returns to the product enzyme level of control strain aspergillus oryzae scut209 substantially, enzyme activity all reaches more than 35000U/g, and the mycelial biomass of fermentation culture also recovers to reach more than 85g/L, as shown in Figure 5.
Finally to should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although be explained in detail the present invention with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to technical scheme of the present invention or equivalent replacement, and not depart from essence and the scope of technical solution of the present invention.
Claims (9)
1. improve a method for aspergillus oryzae strain bacterium NUCLEATION PHENOMENA fast, it is characterized in that, said method comprising the steps of:
(1) sclerotized aspergillus oryzae strain mycelium is inoculated in improvement liquid nutrient medium and carries out shaking culture under dark condition;
(2) aspergillus oryzae strain after cultivation in step (1) is inoculated on improvement slant medium, then cultivates under dark condition;
(3) aspergillus oryzae strain after cultivating in step (2) is inoculated in improvement liquid nutrient medium under dark condition, carries out shaking culture;
(4) aspergillus oryzae strain after cultivation in step (3) is inoculated on improvement slant medium, then cultivates under dark condition;
Described step (1) and (3) middle improvement liquid culture based formulas are: potato 20%, glucose 2%, sucrose 3.0%, the first nonionic surface active agent 0.2%, and surplus is water, pH nature;
Described step (2) and (4) middle improvement slant medium are made up of the component of following part by weight: potato 20%, glucose 2%, sucrose 3.0%, second nonionic surface active agent 0.2%, agar 1.5%, surplus is water, pH nature.
2. the quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, is characterized in that, described first nonionic surface active agent is anhydrous sorbitol lauric acid monoester.
3. the quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, is characterized in that, described second nonionic surface active agent is anhydrous sorbitol lauric acid monoester.
4. the quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, is characterized in that, described step (1) and (3) middle culture temperature are 30 DEG C, and incubation time is 24 hours.
5. the quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, is characterized in that, described step (1) and (3) middle hunting speed are 150r/min.
6. the quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, is characterized in that, described step (2) and (4) middle culture temperature are 30 DEG C, and incubation time is 48 hours.
7. the quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, is characterized in that, described step (2), 4) in vaccination ways be zig-zag type line.
8. the quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, it is characterized in that, described method also comprises and is inoculated on the conventional preservation slant medium of mould by the aspergillus oryzae strain after cultivating in step (4), then 30 DEG C, carry out cultivation 72 hours under dark condition.
9. the quick method improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 8, is characterized in that, the conventional preservation slant medium of described mould is made up of the component of following part by weight: white sugar 3.0wt%; Bean cake powder 2.0wt%; Semen Maydis powder 0.3wt%; Agar 2.0wt%; Surplus is water.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1212834A (en) * | 1997-09-26 | 1999-04-07 | 化学工业部沈阳化工研究院 | Dimetachlone-chlorothalonil bactericidal agent |
| CN102899256A (en) * | 2012-09-27 | 2013-01-30 | 量子高科(中国)生物股份有限公司 | Bacterial strain for producing fructosyl transferase and preparation method thereof |
| CN103205364A (en) * | 2013-03-15 | 2013-07-17 | 保龄宝生物股份有限公司 | Aspergillus oryzae strain and application thereof to fructose-oligosaccharide fermentation production |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1212834A (en) * | 1997-09-26 | 1999-04-07 | 化学工业部沈阳化工研究院 | Dimetachlone-chlorothalonil bactericidal agent |
| CN102899256A (en) * | 2012-09-27 | 2013-01-30 | 量子高科(中国)生物股份有限公司 | Bacterial strain for producing fructosyl transferase and preparation method thereof |
| CN103205364A (en) * | 2013-03-15 | 2013-07-17 | 保龄宝生物股份有限公司 | Aspergillus oryzae strain and application thereof to fructose-oligosaccharide fermentation production |
Non-Patent Citations (3)
| Title |
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| JAN W. M. VAN DER LINDEN, ET AL.: "Aspergillus species intrinsically resistant to antifungal agents", 《MEDICAL MYCOLOGY》 * |
| 何小妮 等: "非对称灭活双亲原生质体融合法选育产果糖基转移酶的米曲霉新菌株的研究", 《现代食品科技》 * |
| 何小妮 等: "高产果糖基转移酶米曲霉菌株的选育", 《食品工业科技》 * |
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