CN103937691A - Aspergillus oryzae strain capable of producing beta-fructofuranosidase, as well as culture method and application thereof - Google Patents
Aspergillus oryzae strain capable of producing beta-fructofuranosidase, as well as culture method and application thereof Download PDFInfo
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Abstract
The invention relates to an aspergillus oryzae strain capable of producing beta-fructofuranosidase, as well as a culture method and application thereof. An aspergillus oryzae FS4 strain is collected in China General Microbiological Culture Collection Center (CGMCC) on April 24, 2014, with the collection number of CGMCC No. 9087 and the address of Institute of Microbiology, Chinese Academy of Sciences, Building 3, NO.1 Beichen West Road, Chaoyang District, Beijing. The invention further relates to the culture method and application of the aspergillus oryzae strain. The aspergillus oryzae FS4 strain obtained by screening has the advantages of simple culture, stable genetic nature, high synthesis efficiency and stable enzyme activity, can be applied to the field of synthesis of levan type oligofructoses and the like by a modern bio-engineering technical enzyme method, and further has broad application prospects.
Description
Technical field
The present invention relates to a strain and produce aspergillus oryzae strain and cultural method and the application of beta-fructosidase enzyme, relate in particular to one and can produce aspergillus oryzae strain and the cultural method thereof of beta-fructosidase enzyme (EC3.2.1.26) and apply in synthetic levan type oligofructose, belong to microbial technology field.
Technical background
Oligofructose is fructosyl is connected to the fructose oligomer on sucrose (GF) molecule general designation with β (2-1) key (synanthrin type) and β (2-6) key (levan type).Oligofructose is a kind of novel sweetener with nourishing functions such as regulating intestinal canal flora, propagation bifidus bacillus, the absorption that promotes calcium, adjusting blood fat, anti-dental caries.The production of oligofructose at present is mainly divided into plant extraction method and enzyme process.
Plant extraction method, mainly taking witloof as starting material, can only obtain the single of bonding oligofructose of β (2-1) key, far can not meet the demand to its different physiological roles exploitation.Enzymatic conversion method sucrose, can produce multiple of bonding oligofructose, and wherein the more conventional synanthrin type oligofructose of levan type oligofructose has higher physiologically active, receives more and more many concerns.Aspergillus oryzae is engineering bacteria conventional in fermentation industry, derives from some beta-fructosidase enzymes of aspergillus oryzae, has the function of synthesis of oligonucleotides fructose, can be used for industrial mass production.
Existing known aspergillus oryzae source beta-fructosidase enzyme, only has the synthetic report of synanthrin type oligofructose, therefore finds the aspergillus oryzae bacterial classification of the beta-fructosidase enzyme of the new levan type that can produce oligofructose, to enzyme process synthesis of oligonucleotides fructose important in inhibiting.
Summary of the invention
The present invention is directed to that in existing microbial enzyme method industrial application, to produce the aspergillus oryzae strain of oligofructose limited, particularly lack the aspergillus oryzae that produces levan type oligofructose, provide a kind of aspergillus oryzae strain of beta-fructosidase enzyme of good stability of produced levan type oligofructose of new separation, the generation that the beta-fructosidase enzyme that this bacterium produces can efficient catalytic levan type oligofructose.
Summary of the invention
Main points of the present invention be to provide a kind of aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain that produces beta-fructosidase enzyme with and feature, cultural method and be applied to the synthetic method of levan type oligofructose taking sucrose as substrate.By literature search, do not have the beta-fructosidase enzyme in aspergillus oryzae source to be applied to taking sucrose as the synthetic report of substrate levan type oligofructose at present both at home and abroad.
Detailed Description Of The Invention
One Aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2014, deposit number is CGMCC No.9087, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Described aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain is grown rapidly on potato culture, within 2～3 days, bacterium colony is sprawled, produce a large amount of mycelium, bacterium colony is just white, part is yellow, when 7 days left and right, become green, follow the prolongation of incubation time to fade to brown to dun, substrate mycelium produces water-soluble brown pigment.Conidial head is radial, and the subsphaeroidal or flask shape of conidiophore top capsule, above covers double-deck stigma.When conidium children, ocean pyriform or oblong, be spherical or subsphaeroidal after maturation, and surface irregularity has fold.The 18S rDNA of this bacterial strain is EU680477 in the accession number of GenBank, with 18S rDNA sequence (GenBank No.HM064501) homology of an Aspergillus oryzae be 99%.
The cultural method of above-mentioned aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain, step is as follows:
Picking aspergillus oryzae FS4 inoculation was to slant medium activation 48～60 hours, and activation temperature is 25～30 DEG C, and the aspergillus oryzae FS4 getting after activation is inoculated in seed culture fluid, cultivates 48～60 hours, and culture temperature is 25～30 DEG C, to obtain final product;
Described slant medium component is as follows: potato 200g/L, glucose 20g/L, agar 15g/L;
Described seed culture fluid component is as follows, is mass percent: sucrose 0.5～4%, yeast powder 1～4%, Xylo-Mucine (CMC) 0.01～0.7%, excess water.
Preferred according to the present invention, described seed culture fluid component is as follows, is mass percent: sucrose 2%, yeast powder 3.5%, Xylo-Mucine (CMC) 0.5%, excess water.
Preferred according to the present invention, described culture temperature is 28 DEG C.
Above-mentioned aspergillus oryzae (Aspergillus oryzae) FS4 is in the application of producing in levan type oligofructose.
Above-mentioned application, step is as follows:
(1) the aspergillus oryzae FS4 learning from else's experience after above-mentioned cultivation, by volume the inoculum size of per-cent 1～5% is transferred in producing enzymic fermentation nutrient solution, and shaking table is cultivated 48～72 hours, and culture temperature is 25～30 DEG C, and shaking speed is 150 revs/min, makes fermented liquid;
Described product enzymic fermentation nutrient solution component is as follows, is all weight percentage: sucrose 0.5～4%, yeast powder 1～4%, Xylo-Mucine (CMC) 0.01～0.7%, excess water;
(2) fermented liquid step (1) being made, through solid-liquid separation, is got precipitation, makes mycelium;
(3) mycelium step (2) being made is after fragmentation, by mass volume ratio 1:(1～10) be suspended in reaction buffer, the g/mL of unit, 50～100r/min stirs 1～3min, makes crude enzyme liquid;
(4) sucrose solution 1:(1～4 by volume that crude enzyme liquid step (3) being made is 20～70% with mass concentration) mix, be under the condition of 30～60 DEG C in temperature, react 10～400 minutes, make the solution that contains levan type oligofructose.
Preferred according to the present invention, the shaking table incubation time in described step (1) is 48 hours.
Preferred according to the present invention, the product enzymic fermentation nutrient solution component in described step (1) is as follows: sucrose 2%, yeast powder 3.5%, Xylo-Mucine (CMC) 0.5%, excess water.
Preferred according to the present invention, the solid-liquid separation in described step (2) is suction filtration or centrifugal.
Preferred according to the present invention, being broken in described step (3) used liquid nitrogen grinding fragmentation.
Preferred according to the present invention, in described step (4), reaction buffer is the potassium phosphate buffer of pH7.0, concentration 50mM; In described step (4), sucrose solution mass concentration is 60%.
Preferred according to the present invention, in described step (5), temperature of reaction is 45 DEG C, and the reaction times is 300 minutes.
Above-mentioned slant medium, seed culture fluid and product enzymic fermentation nutrient solution pH need not adjust, all sterilizings 30 minutes under 115 DEG C of high temperature before use.
Beneficial effect
Aspergillus oryzae (Aspergillus oryzae) FS4 that the present invention screens acquisition cultivates simple, stable hereditary property, combined coefficient is high, and enzyme is lived stable, can be applicable to the fields such as the synthetic levan type oligofructose of modern biotechnology enzyme process, have broad application prospects.
Brief description of the drawings
Fig. 1 is the TLC separation graph of the synthetic levan type oligofructose sample of beta-fructosidase enzyme of aspergillus oryzae (Aspergillus oryzae) FS4 generation;
Fig. 2 is the nucleus magnetic resonance heteronuclear Multiple-Quantum Coherences spectrogram of the synthetic levan type oligofructose trisaccharide sample of beta-fructosidase enzyme of aspergillus oryzae FS4 (Aspergillus oryzae FS4) generation.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is further elaborated, but institute of the present invention protection domain is not limited only to this.
Embodiment 1
One Aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2014, deposit number is CGMCC No.9087, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
(1) Morphological observation
With transfering loop picking aspergillus oryzae (Aspergillus oryzae), FS4 receives on potato culture flat board, 28 DEG C of cultivations.On potato culture, rapidly, within 2～3 days, bacterium colony is sprawled, and produces a large amount of mycelium in growth, and bacterium colony is just white, and part is yellow, when 7 days left and right, becomes green, follows the prolongation of incubation time to fade to brown to dun, and substrate mycelium produces water-soluble brown pigment.Conidial head is radial, and the subsphaeroidal or flask shape of conidiophore top capsule, above covers double-deck stigma.When conidium children, ocean pyriform or oblong, be spherical or subsphaeroidal after maturation, and surface irregularity has fold.
Above-mentioned potato culture component is as follows: potato 200g/L, glucose 20g/L, agar 15g/L.Peeling potatoes, is cut into piece and boils 30 minutes, then gets juice by filtered through gauze, then sugaring and agar, after dissolving, supplies water to 1L, 115 DEG C of sterilizings 30 minutes.
(2) 18S rDNA sequencing and analysis:
Extract test kit (BioFlux) with genome and extract aspergillus oryzae (Aspergillus oryzae) FS4 genomic dna, taking genomic dna as template, the general upstream primer NS1 of employing fungi 18S rDNA (5 '-GTAGTCATATGCTTGTCTC-3 ') and downstream primer FS2 (5 '-TAGGNATTCCTCGTTGAAGA-3 ') pcr amplification 18S rDNA sequence.
PCR reaction system 50 μ L:10 × PCR buffer5 μ L, 2.5mmol/L dNTP mixture 4 μ L, the each 2 μ L of 20 μ mol/L primer, template 2 μ L (10ng), LATaq enzyme 1 μ L, sterilized water 34 μ L.Pcr amplification condition: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 30 seconds, 50 DEG C of annealing 30 seconds, 72 DEG C are extended 1.5 minutes, 30 circulations; 72 DEG C are extended 5 minutes.The amplified production obtaining carries out sequencing by Shanghai Sheng Gong biotechnology company limited, size is 1563 bases, this sequence is carried out to BLAST Nucleotide compare of analysis in NCBI website, 18S rDNA sequence (GenBank No.HM064501) homology of aspergillus oryzae is 99%.。
The GenBank accession number of aspergillus oryzae (Aspergillus oryzae) FS418S rDNA is EU680477.
Embodiment 2
The cultural method of aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain, step is as follows:
Get aspergillus oryzae (Aspergillus oryzae) FS4 inoculation to fresh slant medium, under the condition of 28 DEG C, activate 48 hours, aspergillus oryzae (Aspergillus oryzae) FS4 getting after activation is inoculated in 100mL seed culture fluid, cultivate 48 hours, make aspergillus oryzae seed liquor;
This slant medium component is as follows: potato 200g/L, glucose 20g/L, agar 15g/L.115 DEG C of sterilizings 30 minutes.
This seed culture fluid component is as follows, is all weight percentage: sucrose 2%, yeast powder 3.5%, Xylo-Mucine (CMC) 0.5%, excess water.115 DEG C of sterilizings 30 minutes.
Aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain biography is carried out to 50 and go down to posterity, opticmicroscope and electron microscopic examination, the bacterial strain through going down to posterity is consistent with original strain form.The output of the synthetic levan type oligofructose of crude enzyme liquid of the bacterial strain after 50 generations went down to posterity is consistent with original strain.Stability experiment shows, this bacterial strain has very high stability, and number rear enzymatic productivity of generation does not reduce.
Embodiment 3
The synthetic levan type oligofructose of beta-fructosidase enzyme that utilizes aspergillus oryzae FS4 bacterial strain to produce, concrete steps are as follows:
(1) the aspergillus oryzae FS4 learning from else's experience after above-mentioned cultivation, by volume the inoculum size of per-cent 1% is transferred and is produced in enzymic fermentation nutrient solution in 500mL, and shaking table is cultivated 48 hours, and culture temperature is 28 DEG C, and shaking speed is 150 revs/min, makes fermented liquid;
Described product enzymic fermentation nutrient solution component is as follows, is all weight percentage: sucrose 2%, yeast powder 3.5%, Xylo-Mucine (CMC) 0.5%, excess water;
(2) fermented liquid step (1) being made, after suction filtration, makes mycelium;
(3) mycelium step (2) being made, after liquid nitrogen grinds fragmentation, is suspended in by mass volume ratio 1:5 in the potassium phosphate buffer of pH7.0, concentration 50mM, the g/mL of unit, and 50r/min stirs 1min, makes crude enzyme liquid;
(4) the sucrose solution 100 μ L that the crude enzyme liquid 25 μ L that step (3) made are 60% with mass concentration mix, mass concentration is that 60% sucrose solution is the potassium phosphate buffer preparation with pH7.0, concentration 50mM, after 5 hours, make the solution that contains levan type oligofructose 45 DEG C of shaking bath reactions.
TLC analysis is carried out in sampling, and result as shown in Figure 1.
Above-mentioned TLC analyzes equipment used and condition: reaction product point sample is in TLC thin plate, exhibition layer in developing agent (propyl carbinol: ethanol: water=5:3:2), spray painting developer (3 of 20% sulphuric acid soln+0.5%, 5-orcin), in 120 DEG C of bakings 3～5 minutes, sugared spot colour developing.
To above-mentioned product carry out quantitatively, purifying and nucleus magnetic resonance qualification chemical structure.Wherein, the nucleus magnetic resonance qualification result of kestose as shown in Figure 2.
Above-mentioned nuclear magnetic resonance spectroscopy equipment used and condition: the reaction product of purifying is dissolved in deuterated water, use Brooker DRX600MHz nuclear magnetic resonance spectrometer to compose (heteronuclear multiple-bond correlation (HMBC) spectra) in 26 DEG C of heteronuclear Multiple-Quantum Coherences that detect product, the chemical structure of determining product is the levan type oligofructose of β (2-6) key.
Through quantitative experiment, in this reaction system, to account for the content of total reducing sugar be 56% to oligofructose.
Claims (10)
1. an Aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2014, deposit number is CGMCC No.9087, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
2. the cultural method of aspergillus oryzae (Aspergillus oryzae) FS4 bacterial strain described in claim 1, is characterized in that, step is as follows:
Picking aspergillus oryzae FS4 inoculation was to slant medium activation 48～60 hours, and activation temperature is 25～30 DEG C, and the aspergillus oryzae FS4 getting after activation is inoculated in seed culture fluid, cultivates 48～60 hours, and culture temperature is 25～30 DEG C, to obtain final product;
Described slant medium component is as follows: potato 200g/L, glucose 20g/L, agar 15g/L;
Described seed culture fluid component is as follows, is mass percent: sucrose 0.5～4%, yeast powder 1～4%, Xylo-Mucine 0.01～0.7%, excess water.
3. cultural method as claimed in claim 2, is characterized in that, described seed culture fluid component is as follows, is mass percent: sucrose 2%, yeast powder 3.5%, Xylo-Mucine 0.5%, excess water.
4. the application of aspergillus oryzae (Aspergillus oryzae) FS4 in production levan type oligofructose described in claim 1.
5. application as claimed in claim 4, is characterized in that, step is as follows:
(1) the aspergillus oryzae FS4 learning from else's experience after above-mentioned cultivation, by volume the inoculum size of per-cent 1～5% is transferred in producing enzymic fermentation nutrient solution, and shaking table is cultivated 48～72 hours, and culture temperature is 25～30 DEG C, and shaking speed is 150 revs/min, makes fermented liquid;
Described product enzymic fermentation nutrient solution component is as follows, is all weight percentage: sucrose 0.5～4%, yeast powder 1～4%, Xylo-Mucine 0.01～0.7%, excess water;
(2) fermented liquid step (1) being made, through solid-liquid separation, is got precipitation, makes mycelium;
(3) mycelium step (2) being made is after fragmentation, by mass volume ratio 1:(1～10) be suspended in reaction buffer, the g/mL of unit, 50～100r/min stirs 1～3min, makes crude enzyme liquid;
(4) sucrose solution 1:(1～4 by volume that crude enzyme liquid step (3) being made is 20～70% with mass concentration) mix, be under the condition of 30～60 DEG C in temperature, react 10～400 minutes, make the solution that contains levan type oligofructose.
6. application as claimed in claim 5, is characterized in that, the product enzymic fermentation nutrient solution component in described step (1) is as follows: sucrose 2%, yeast powder 3.5%, Xylo-Mucine 0.5%, excess water.
7. application as claimed in claim 5, is characterized in that, the solid-liquid separation in described step (2) is suction filtration or centrifugal.
8. application as claimed in claim 5, is characterized in that, being broken in described step (3) used liquid nitrogen grinding fragmentation.
9. application as claimed in claim 5, is characterized in that, in described step (4), reaction buffer is the potassium phosphate buffer of pH7.0, concentration 50mM; In described step (4), sucrose solution mass concentration is 60%.
10. application as claimed in claim 5, is characterized in that, in described step (5), temperature of reaction is 45 DEG C, and the reaction times is 300 minutes.
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| CN110172407A (en) * | 2018-12-11 | 2019-08-27 | 青岛蔚蓝生物集团有限公司 | One plant of aspergillus oryzae for producing transfructosylase and its application |
| CN110564626A (en) * | 2019-09-17 | 2019-12-13 | 山东百龙创园生物科技股份有限公司 | aspergillus oryzae strain, culture method thereof and method for preparing kestose |
| WO2020135893A1 (en) * | 2018-12-26 | 2020-07-02 | 山东百龙创园生物科技股份有限公司 | Aspergillus oryzae blcy-006 strain and application thereof in preparation of galactooligosaccharide |
| CN111487332A (en) * | 2019-11-08 | 2020-08-04 | 天津科技大学 | Evaluation method and application of prebiotics effect of fructo-oligosaccharide with single polymerization degree |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483027A (en) * | 2016-01-04 | 2016-04-13 | 山东星光生物科技有限公司 | Aspergillus tubingensis and method for producing fructooligosaccharides through whole-cell catalysis of aspergillus tubingensis |
| CN110172407A (en) * | 2018-12-11 | 2019-08-27 | 青岛蔚蓝生物集团有限公司 | One plant of aspergillus oryzae for producing transfructosylase and its application |
| WO2020135893A1 (en) * | 2018-12-26 | 2020-07-02 | 山东百龙创园生物科技股份有限公司 | Aspergillus oryzae blcy-006 strain and application thereof in preparation of galactooligosaccharide |
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| CN110564626A (en) * | 2019-09-17 | 2019-12-13 | 山东百龙创园生物科技股份有限公司 | aspergillus oryzae strain, culture method thereof and method for preparing kestose |
| CN110564626B (en) * | 2019-09-17 | 2021-12-07 | 山东百龙创园生物科技股份有限公司 | Aspergillus oryzae strain, culture method thereof and method for preparing kestose |
| CN111487332A (en) * | 2019-11-08 | 2020-08-04 | 天津科技大学 | Evaluation method and application of prebiotics effect of fructo-oligosaccharide with single polymerization degree |
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