CN105199967B - A kind of quick method for improving aspergillus oryzae bacterium NUCLEATION PHENOMENA - Google Patents
A kind of quick method for improving aspergillus oryzae bacterium NUCLEATION PHENOMENA Download PDFInfo
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- CN105199967B CN105199967B CN201510623553.7A CN201510623553A CN105199967B CN 105199967 B CN105199967 B CN 105199967B CN 201510623553 A CN201510623553 A CN 201510623553A CN 105199967 B CN105199967 B CN 105199967B
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Abstract
The present invention provides a kind of quick method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, the present invention is on the basis of improved culture medium composition, using liquid and solid alternate culture mode, so that the operating time shorten to one week for one month or so from conventional, as quickly as possible eliminate the sclerotized phenomenon of mould, and method is easy to operate, less investment, easily grasp, effect it is good.Method using the present invention, enables to bacterium NUCLEATION PHENOMENA in aspergillus oryzae strain slant medium to be wholly absent, and mycelium grows fine, and strain enzyme-producing ability is restored.After past sclerotium rejuvenation experiment, original set out aspergillus oryzae strain production transfructosylase ability substantially return to control strain aspergillus oryzae strain B producing enzyme it is horizontal, enzyme activity all reaches more than 35000U/g, and the mycelial biomass of fermented and cultured also recovers to reach more than 85g/L.
Description
Technical field
The present invention relates to a kind of method for improving mould bacterium NUCLEATION PHENOMENA, more particularly to a kind of quick improvement aspergillus oryzae strain
The method of bacterium NUCLEATION PHENOMENA.
Background technology
Aspergillus oryzae strain is propagated on inclined-plane in incubation, the inoculation not waited by algebraically, often occurs that mycelia is short, spore
Son is few, and media surface occurs a large amount of small and justifies, the solid hard particulate material of quality, and color or white or black or appearance are white
Spot, and the phenomenon declined with bacterial strain enzyme activity." sclerotium " of the appearance of this phenomenon, normally referred to as inclined-plane, it is such as attached
Shown in Fig. 1.
During inclined-plane culture, bacterial strain particularly this phenomenon usually can all occur from artificial mutagenic fungi.
Sclerotized inclined-plane is difficult to pass on seed with bacterium as production, it is also difficult to and continue preservation as strain and use, this situation
It must be well solved.
At present, the concrete reason that inclined-plane sclerotiumization is formed, it is unclear, but estimate to fail with the growth ability of bacterial strain, draw
Take related with using the deteriorated and genetic mutation of nutriment.Someone says that " sclerotium " is mycelial aggregate, micro-
, all there is the close heap state of mycelia, in " honeycomb " configuration state after sclerotium is cut inside sclerotium appearance and core in Microscopic observation.
Thus we can be learnt with inference：If in cell growth process, mycelium can be disperseed well, equably separately given birth to
Long, bacterium NUCLEATION PHENOMENA will be improved well.
Surfactant is a kind of material for having very strong surface-active, being remarkably decreased liquid, solid surface tension.By
There is very strong hydrophily and the building stone of lipophile, that is, parents in itself in its structure, make it in chemical industry, food, weaving, medicine
Be widely used in the industries such as agent, mining, be commonly used as solubilizer, emulsifying agent, wetting agent, suspending agent, infiltration dispersant,
Foaming agent, defoamer, detergent, disinfectant and fungicide etc., particularly cohere with anti-caking etc. anti-, it is to show
Satisfied diversified utility function.
In the growing environment of microbial cell, suitable surfactant is added in the medium, and particularly addition comes
Come from the non-ionic surface work of nature, dispersion performance good, water-soluble big, easily biological-degradable, the polyol type nontoxic to biology topic
Property agent, for slow down produced in cell growth process between mycelium cohere, it is blocking, so as to improve the bacterium formed in organelle matter
NUCLEATION PHENOMENA, there is very big facilitation.
The content of the invention
A kind of quick improvement aspergillus oryzae is provided it is an object of the invention to overcome the shortcomings of the prior art part
The method of bacterial strain bacterium NUCLEATION PHENOMENA.
To achieve the above object, the technical solution taken：A kind of quick side for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA
Method, the described method comprises the following steps：
(1) sclerotized aspergillus oryzae strain mycelium is inoculated in improvement fluid nutrient medium and shaken under dark condition
Swing culture；
(2) aspergillus oryzae strain after culture in step (1) is inoculated on improvement slant medium, then in dark condition
Under cultivated；
(3) by the aspergillus oryzae strain after culture in step (2) be inoculated in improvement fluid nutrient medium under dark condition into
Row shaken cultivation；
(4) aspergillus oryzae strain after culture in step (3) is inoculated on improvement slant medium, then in dark condition
Under cultivated；
Improvement Liquid Culture based formulas is in the step (1) and (3)：Potato 20%, glucose 2%, sucrose
3.0%, the first nonionic surface active agent 0.2%, surplus is water, and pH is natural；
Improvement slant medium is made of the component of following part by weight in the step (2) and (4)：Potato 20%,
Glucose 2%, sucrose 3.0%, the second nonionic surface active agent 0.2%, agar 1.5%, surplus are water, and pH is natural.
Preferably, first nonionic surface active agent is anhydrous sorbitol lauric acid monoester.
Preferably, second nonionic surface active agent is anhydrous sorbitol lauric acid monoester.
Preferably, cultivation temperature is 30 DEG C in the step (1) and (3), when incubation time is 24 small.
Preferably, hunting speed is 150r/min in the step (1) and (3).
Preferably, cultivation temperature is 30 DEG C in the step (2) and (4), when incubation time is 48 small.
Preferably, the step (2), 4) in vaccination ways be zig-zag type line.
Preferably, the method is further included is inoculated in mould routine preservation by the aspergillus oryzae strain after culture in step (4)
On slant medium, then carried out under 30 DEG C, dark condition culture 72 it is small when.Before there is sclerotized aspergillus oryzae strain process
The culture of four step of face, its bacterium NUCLEATION PHENOMENA have eliminated, and the aspergillus oryzae strain after culture in step (4) is inoculated in mould routine
It is to stablize the bacterial strain that culture is carried out on preservation slant medium, reduces the probability that bacterium NUCLEATION PHENOMENA occurs again in the bacterial strain.
Preferably, the mould routine preservation slant medium is made of the component of following part by weight：White granulated sugar
3.0wt%；Bean cake powder 2.0wt%；Corn flour 0.3wt%；Agar 2.0wt%；Surplus is water.
The beneficial effects of the present invention are：The present invention provides a kind of quick side for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA
Method, its specific advantages below：
(1) present invention is on the basis of improved culture medium composition, using liquid and solid alternate culture mode so that operation
Time shorten to one week for one month or so from conventional, eliminates the sclerotized phenomenon of mould, and method operation letter as quickly as possible
List, less investment, easily grasp, effect are good.
(2) method using the present invention, enables to bacterium NUCLEATION PHENOMENA in aspergillus oryzae strain slant medium to be wholly absent,
Mycelium grows fine, and strain enzyme-producing ability is restored.After past sclerotium rejuvenation experiment, the former aspergillus oryzae strain that sets out
The producing enzyme that production transfructosylase ability substantially returns to control strain aspergillus oryzae strain B is horizontal, and enzyme activity all reaches 35000U/
More than g, the mycelial biomass of fermented and cultured also recover to reach more than 85g/L.
Brief description of the drawings
Fig. 1 is the bacterium NUCLEATION PHENOMENA occurred in the embodiment of the present invention 1 during aspergillus oryzae strain inclined-plane culture；Wherein, A：
Black, white sclerotium particle；B：Mycelia desertification phenomenon；C：Mycelia hickie phenomenon；
Fig. 2 is mycelium morphologies of the aspergillus oryzae strain A and B in Liquid Culture in the embodiment of the present invention 1；
Fig. 3 is the upgrowth situation of aspergillus oryzae strain A squamous subcultures in slant medium is improved in the embodiment of the present invention 1；
Fig. 4 is mycelium morphologies of the aspergillus oryzae strain A after sclerotium rejuvenation is gone in the embodiment of the present invention 1；
Fig. 5 is enzymatic productivities of the aspergillus oryzae strain A after sclerotium rejuvenation is gone and biomass water in the embodiment of the present invention 1
It is flat.
Embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
Aspergillus oryzae strain employed in the present embodiment is：
Aspergillus oryzae strain A：Sclerotized transfructosylase production strain Aspergillus oryzae scut209 (Aspergillus
Oryzae scut209), as shown in Figure 1；
Aspergillus oryzae strain B：The normal transfructosylase production bacterial strain of this Laboratories Accession, aspergillus oryzae strain A is meter Qu
There is the aspergillus oryzae strain of bacterium NUCLEATION PHENOMENA after several pickup kinds in trichoderma strain B, and aspergillus oryzae strain B is preserved in positioned at China
The China typical culture collection center in Wuhan, deposit number are CCTCC NO:M2012106；And in entitled " one kind production
Bacterial strain of transfructosylase and preparation method thereof " is authorized disclosed in patent of invention, and patent authorization number is：
ZL201210374664.5。
Mould routine preservation slant medium employed in the present embodiment is made of the component of following part by weight：White sand
Sugared 3.0wt%, bean cake powder 2.0wt%, corn flour 0.3wt%, agar 2.0wt%；PH is natural.
Liquid Culture based formulas employed in embodiment is：White granulated sugar 3.0wt%, bean cake powder 2.0wt%, corn flour
0.3wt%, surplus are water, and pH is natural.
Improvement fluid nutrient medium employed in embodiment is：Potato 20%, glucose 2%, sucrose 3.0%, first
Nonionic surface active agent 0.2%, surplus are water, and pH is naturally, first nonionic surface active agent is Sorbitan
Alcohol lauric acid monoester Span-2.
Improvement slant medium employed in embodiment is made of the component of following part by weight：Potato 20%, Portugal
Grape sugar 2%, sucrose 3.0%, the second nonionic surface active agent 0.2%, agar 1.5%, surplus is water；PH is naturally, described
Second nonionic surface active agent is anhydrous sorbitol lauric acid monoester Span-2.
Transfructosylase vigour-testing method employed in the present embodiment is：Transfructosylase or producing enzyme thalline are made
For sucrose, ketose (1-kestose) is firstly generated；Ketose content assaying method uses HPLC methods, test method
Performed by GB/T 23528-2009 6.5.
Enzyme activity unit defines：It is oligomeric by sucrose inversion under conditions of the optimal enzymeization reaction of enzyme supplier mark
Fructose, enzyme amount needed for 1 μm of ol ketose of generation per minute is an enzyme activity unit (U)；
In formula：
Sucrose total amount in 10----10% (w/w) sucrose solution, g；
The percentage composition of GF2---- ketoses, %；
0.504----1 μm of ol ketoses=0.504mg；
T---- reaction time, 60min；
W---- mycelia quality, g.
High performance liquid chromatography (HPLC) detection parameters are：Cosmosil chromatography sugar columns；Mobile phase：Acetonitrile-water (75%, v/
v)；Flow velocity:1.0mL/min；Differential refraction detector (RI):Waters2410；Monitor sensitivity:4；Column temperature:30℃；Sample introduction
Volume:10μL.
The experimentation that sclerotium bacterial strain biological pollution possibility employed in embodiment excludes：
(1) operate：One ring of mycelia on picking aspergillus oryzae strain A and B inclined-planes, is inoculated in sterile fluid nutrient medium respectively
(PDA) in, when shaken cultivation 48 is small at 30 DEG C, sampling observation.
(2) shaking flask is observed：Shaking flask A (corresponding aspergillus oryzae strain A) and mycelial growth in shaking flask B (corresponding aspergillus oryzae strain B)
Normally, liquid is limpid, transparent in fluid nutrient medium, and nose hears free from extraneous odour, and the peculiar rice fragrance of aspergillus oryzae is presented.Mycelium is in ball
Long mycelioid is presented in shape, the mycelium of wall built-up, as shown in Figure 2.
(3) microscopy is observed：Micro- Microscopic observation is sampled, has not found bacterium infection.
The result shows that the problem of white occurred in inclined-plane, black particle and hickie should be itself variations, eliminates miscellaneous
The possibility of bacterium infection.
The present embodiment uses the operating method that the method for improvement aspergillus oryzae bacterium NUCLEATION PHENOMENA is solid-liquid alternate culture, its
Middle nonionic surface active agent selects that dispersion performance is good, water-soluble big, easily biological-degradable, hypotoxicity polyol type surface is lived
Property agent anhydrous sorbitol lauric acid monoester Span-20, concentration 0.2%v/v；Specific method is as follows：
(1) picking is in sclerotized one ring of aspergillus oryzae strain A inclined-planes mycelia, is inoculated in improvement fluid nutrient medium, in
150rmp/m, 30 DEG C, when shaken cultivation 24 is small under dark condition；
(2) one ring of mycelium in picking step (1) after liquid culture, takes " it " font to rule, is inoculated in improvement inclined-plane
In culture medium, at 30 DEG C, when putting that dark culturing 48 is small in incubator, cultivation results are as shown in Figure 3；
(3) one ring of inclined-plane bacterial strain in picking step (2) after culture, is inoculated in improvement fluid nutrient medium again, in
150rmp/m, when shaken cultivation 24 is small under dark condition, observes process suspension cell degree of scatter by 30 DEG C；
(4) one ring of suspension mycelium in picking step (3) after liquid culture again, takes " it " font to rule, is inoculated in
Improve in slant medium, at 30 DEG C, when putting that dark culturing 48 is small in incubator, be then inoculated in mould routine preservation culture
Inclined-plane sclerotium disappearance degree is observed on base tablet, as shown in figure 4, the bacterium NUCLEATION PHENOMENA of bacterial strain substantially disappears；
(5) one ring of inclined-plane bacterial strain in picking step (4) after culture, turns to be inoculated on mould routine storage medium, in
When culture 72 is small under dark condition in incubator at 30 DEG C.
Experimental result：After the above method, bacterium NUCLEATION PHENOMENA is complete in sclerotized aspergillus oryzae strain A slant mediums
Disappear, mycelium grows fine, and strain enzyme-producing ability is restored.After past sclerotium rejuvenation experiment, original is set out aspergillus oryzae
The producing enzyme that the production transfructosylase ability of bacterial strain substantially returns to control strain aspergillus oryzae scut209 is horizontal, and enzyme activity all reaches
To more than 35000U/g, the mycelial biomass of fermented and cultured also recovers to reach more than 85g/L, as shown in Figure 5.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, can be to technical scheme technical scheme is modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And scope.
Claims (7)
- A kind of 1. quick method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, it is characterised in that the described method comprises the following steps：(1) sclerotized aspergillus oryzae strain mycelium is inoculated in improvement fluid nutrient medium and vibration training is carried out under dark condition Support；(2) aspergillus oryzae strain after culture in step (1) is inoculated on improvement slant medium, then under dark condition into Row culture；(3) aspergillus oryzae strain after culture in step (2) is inoculated in improvement fluid nutrient medium and shaken under dark condition Swing culture；(4) aspergillus oryzae strain after culture in step (3) is inoculated on improvement slant medium, then under dark condition into Row culture；Improvement Liquid Culture based formulas is in the step (1) and (3)：Potato 20wt%, glucose 2wt%, sucrose 3.0wt%, the first nonionic surface active agent 0.2wt%, surplus are water, and pH is natural；Improvement slant medium is made of the component of following part by weight in the step (2) and (4)：Potato 20%, grape Sugar 2%, sucrose 3.0%, the second nonionic surface active agent 0.2%, agar 1.5%, surplus are water, and pH is natural；First nonionic surface active agent is anhydrous sorbitol lauric acid monoester；Second nonionic surface active agent is anhydrous sorbitol lauric acid monoester.
- 2. the quick method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, it is characterised in that the step Suddenly cultivation temperature is 30 DEG C in (1) and (3), when incubation time is 24 small.
- 3. the quick method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, it is characterised in that the step Suddenly hunting speed is 150r/min in (1) and (3).
- 4. the quick method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, it is characterised in that the step Suddenly cultivation temperature is 30 DEG C in (2) and (4), when incubation time is 48 small.
- 5. the quick method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, it is characterised in that the step Suddenly (2), 4) in vaccination ways be zig-zag type line.
- 6. the quick method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 1, it is characterised in that the side Method, which further includes, is inoculated in the aspergillus oryzae strain after culture in step (4) on mould routine preservation slant medium, then 30 DEG C, carry out under dark condition culture 72 it is small when.
- 7. the quick method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA according to claim 6, it is characterised in that described mould Bacterium routine preservation slant medium is made of the component of following part by weight：White granulated sugar 3.0wt%；Bean cake powder 2.0wt%；Corn Powder 0.3wt%；Agar 2.0wt%；Surplus is water.
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Citations (3)
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|---|---|---|---|---|
| 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 |
-
2015
- 2015-09-25 CN CN201510623553.7A patent/CN105199967B/en not_active Expired - Fee Related
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 |
|---|
| Aspergillus species intrinsically resistant to antifungal agents;JAN W. M. VAN DER LINDEN, et al.;《Medical Mycology》;20110430;第49卷;S82-89 * |
| 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;何小妮 等;《现代食品科技》;20131231;第29卷(第5期);993-997 * |
| 高产果糖基转移酶米曲霉菌株的选育;何小妮 等;《食品工业科技》;20131231;第34卷(第22期);135-140 * |
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