CN105112310B - A kind of method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA - Google Patents
A kind of method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA Download PDFInfo
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- CN105112310B CN105112310B CN201510629120.2A CN201510629120A CN105112310B CN 105112310 B CN105112310 B CN 105112310B CN 201510629120 A CN201510629120 A CN 201510629120A CN 105112310 B CN105112310 B CN 105112310B
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- aspergillus oryzae
- oryzae strain
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Abstract
The present invention provides a kind of method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, using this method, can not only be wholly absent bacterium NUCLEATION PHENOMENA of the sclerotized aspergillus oryzae strain in slant medium, sclerotized aspergillus oryzae strain mycelium is set to grow fine, moreover it is possible to sclerotized aspergillus oryzae strain enzymatic productivity is restored.After this method rejuvenation, the production transfructosylase ability of sclerotized aspergillus oryzae strain can be made to recover substantially, producing enzyme enzyme activity reaches 36300U/g (dry weight), the mycelial biomass of fermented and cultured also recovers to reach 90.8g/L, and the producing enzyme that control strain aspergillus oryzae strain B is fully achieved is horizontal.Method provided by the present invention is easy to operate, mild condition, less investment, easily grasp, effect it is good.
Description
Technical field
It is more particularly to a kind of to improve aspergillus oryzae strain sclerotium the present invention relates to a kind of method for improving mould bacterium NUCLEATION PHENOMENA
Change the method for phenomenon.
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 improvement aspergillus oryzae strain is provided it is an object of the invention to overcome the shortcomings of the prior art part
The method of bacterium NUCLEATION PHENOMENA.
To achieve the above object, the technical solution taken：A kind of method for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, its
It is characterized in that, the described method comprises the following steps：
(1) sclerotized aspergillus oryzae strain mycelium is inoculated on the first improvement slant medium, then in dark bar
Cultivated under part；
(2) aspergillus oryzae strain after culture in step (1) is inoculated on the first improvement slant medium again, Ran Hou
Cultivated under dark condition；
(3) aspergillus oryzae strain after culture in step (2) is inoculated on the second improvement slant medium, then in dark
Under the conditions of cultivated；
(4) aspergillus oryzae strain after culture in step (3) is inoculated on the second improvement slant medium again, Ran Hou
Cultivated under dark condition；
The first improvement slant medium is prepared by the component of following part by weight in the step (1) and (2)：Ma Ling
Potato 20%, glucose 2%, sucrose 3.0%, the first nonionic surface active agent 0.2%, agar 1.5%, surplus is water；
The second improvement slant medium is prepared by the component of following part by weight in the step (3) and (4)：Ma Ling
Potato 20%, glucose 2%, sucrose 5.0%, the first nonionic surface active agent 0.2%, agar 1.5%, surplus is water.
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), (2), (3) and (4), when incubation time is 72 small
To 120 it is small when.
Preferably, vaccination ways are zig-zag type line in the step (1), (2), (3) and (4).
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 hours to 120 it is small when.There is sclerotized aspergillus oryzae
Culture of the bacterial strain by above four steps, its bacterium NUCLEATION PHENOMENA have eliminated, and the aspergillus oryzae strain after culture in step (4) is inoculated with
It is to stablize the bacterial strain in carrying out culture on mould routine preservation slant medium, reduces the bacterial strain and occur sclerotium again and show
The probability of elephant.
Preferably, the mould routine preservation slant medium is prepared by the component of following part by weight：White granulated sugar
3.0%, bean cake powder 2.0%, corn flour 0.3%, agar 2.0%, surplus is water.
The beneficial effects of the present invention are：The present invention provides it is a kind of improve aspergillus oryzae strain bacterium NUCLEATION PHENOMENA method,
Using this method, it can not only be wholly absent bacterium NUCLEATION PHENOMENA of the sclerotized aspergillus oryzae strain in slant medium, make
Sclerotized aspergillus oryzae strain mycelium grows fine, moreover it is possible to sclerotized aspergillus oryzae strain enzymatic productivity is restored.Through
After crossing this method rejuvenation, the production transfructosylase ability of sclerotized aspergillus oryzae strain can be made to recover substantially, producing enzyme enzyme activity
Power reaches 36300U/g (dry weight), and the mycelial biomass of fermented and cultured also recovers to reach 90.8g/L, and control strain is fully achieved
The producing enzyme of aspergillus oryzae strain B is horizontal.Method provided by the present invention is easy to operate, mild condition, less investment, easily grasp, effect
It is good.
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 growth shape of aspergillus oryzae strain A squamous subcultures in the first improvement slant medium in the embodiment of the present invention 1
Condition；
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 military positioned at China
The China typical culture collection center of the Chinese, deposit number are CCTCC NO:M2012106；And in entitled " one kind production fruit
Bacterial strain of glycosyl transferase and preparation method thereof " is authorized disclosed in patent of invention, and patent authorization number is：
ZL201210374664.5。
Mould routine preservation inclined-plane culture based formulas employed in the present embodiment is：White granulated sugar 3.0wt%, bean cake powder
2.0wt%, corn flour 0.3wt%, agar 2.0wt%, surplus are water, and 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.
The first improvement slant medium employed in the present embodiment is prepared by the component of following part by weight：Ma Ling
Potato 20%, glucose 2%, sucrose 3.0%, the first nonionic surface active agent 0.2%, agar 1.5%, surplus is water；From
Right pH value；First nonionic surface active agent is anhydrous sorbitol lauric acid monoester Span-20.
The second improvement slant medium employed in the present embodiment is prepared by the component of following part by weight：Ma Ling
Potato 20%, glucose 2%, sucrose 5.0%, the second nonionic surface active agent 0.2%, agar 1.5%, surplus is water；From
Right pH value；Second nonionic surface active agent is anhydrous sorbitol lauric acid monoester Span-20.
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 inclined-plane squamous subculture, 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 0.2wt%；Specific method is as follows：
(1) picking is in sclerotized one ring of aspergillus oryzae strain A inclined-planes mycelia, is inoculated in the first improvement slant medium
In, take " it " font to rule, at 30 DEG C, put in incubator dark culturing 72 hours to 120 it is small when；
(2) one ring of inclined-plane bacterial strain in picking step (1) after culture, is inoculated in the first improvement slant medium again,
Take " it " font to rule, at 30 DEG C, put in incubator dark culturing 72 hours to 120 it is small when；Trained by subculture rejuvenation twice
After supporting, inclined-plane bacterial strain bacterium NUCLEATION PHENOMENA still has, and still occurs slight hickie and a small amount of black sclerotium in inclined-plane, such as Fig. 3 institutes
Show, but sclerotium degree has weakened than control group；Control group described here refers to exist aspergillus oryzae strain A described in the present embodiment
Cultivated described in the present embodiment on mould routine preservation slant medium.
(3) one ring of inclined-plane bacterial strain in picking step (2) after culture, is inoculated in the second improvement slant medium, takes
" it " font is rule, at 30 DEG C, when putting that dark culturing 72 is small in incubator；
(4) one ring of inclined-plane bacterial strain in picking step (3) after culture, is inoculated in the second improvement slant medium again,
" it " font is taken to rule, at 30 DEG C, when putting that dark culturing 72 is small in incubator；
(5) one ring of inclined-plane bacterial strain in picking step (4) after culture, turns to be inoculated in mould routine preservation slant medium
In, take " it " font to rule, when dark culturing 72 is small in incubator at 30 DEG C.
Experimental result：Bacterium NUCLEATION PHENOMENA is wholly absent in rejuvenation bacterial strain slant medium in step (5), and mycelium growing way is good
It is good, as shown in figure 4, strain enzyme-producing ability is restored.After past sclerotium rejuvenation experiment, original sets out aspergillus oryzae strain A's
Production transfructosylase ability is recovered substantially, and producing enzyme enzyme activity reaches 36300U/g (dry weight), the mycelium biology of fermented and cultured
Amount is also recovered to reach 90.8g/L, the producing enzyme level of control strain aspergillus oryzae strain B is fully achieved, 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 (5)
- A kind of 1. 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 on the first improvement slant medium, then under dark condition Cultivated；(2) aspergillus oryzae strain after culture in step (1) is inoculated on the first improvement slant medium again, then in dark Under the conditions of cultivated；(3) aspergillus oryzae strain after culture in step (2) is inoculated on the second improvement slant medium, then in dark condition Under cultivated；(4) aspergillus oryzae strain after culture in step (3) is inoculated on the second improvement slant medium again, then in dark Under the conditions of cultivated；The first improvement slant medium is prepared by the component of following part by weight in the step (1) and (2)：Potato 20%, glucose 2%, sucrose 3.0%, the first nonionic surface active agent 0.2%, agar 1.5%, surplus is water；The second improvement slant medium is prepared by the component of following part by weight in the step (3) and (4)：Potato 20%, glucose 2%, sucrose 5.0%, the second nonionic surface active agent 0.2%, agar 1.5%, surplus is water；First nonionic surface active agent is anhydrous sorbitol lauric acid monoester；Second nonionic surface active agent is anhydrous sorbitol lauric acid monoester.
- 2. the method according to claim 1 for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, it is characterised in that the step (1), cultivation temperature is 30 DEG C in (2), (3) and (4), incubation time be 72 hours to 120 it is small when.
- 3. the method according to claim 1 for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, it is characterised in that the step (1), vaccination ways are zig-zag type line in (2), (3) and (4).
- 4. the method according to claim 1 for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, it is characterised in that the method is also Including the aspergillus oryzae strain after culture in step (4) is inoculated on mould routine preservation slant medium, then 30 DEG C, it is black Carried out under dark condition culture 72 hours to 120 it is small when.
- 5. the method according to claim 4 for improving aspergillus oryzae strain bacterium NUCLEATION PHENOMENA, it is characterised in that the mould is normal Rule preservation slant medium is prepared by the component of following part by weight：White granulated sugar 3.0%, bean cake powder 2.0%, corn flour 0.3%, agar 2.0%, surplus is water.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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;S83-S89 * |
| 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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