CN104031846A - Aspergillus oryzae bacterial strain and application thereof - Google Patents
Aspergillus oryzae bacterial strain and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of spergillus oryzae bacterial strain, in particular to spergillus oryzae bacterial strain FTST-2-6 which is already preserved in General Microbial Center of China Committee for Culture Collection of Microorganisms on February 24, 2014, and the preservation number is No.8862. The invention also relates to an application of the spergillus oryzae bacterial strain in the fermentation and production of fructo-oligosaccharide. The spergillus oryzae bacterial strain is anaerobically fermented for 10h to 20h with a carbon-source solution under an anerobic condition to produce spergillus oryzae bacterial strain in the fermented solution. The spergillus oryzae bacterial strain FTST-2-6 is stable in genetic performance, easy to propagate, the number of spores for eighth generation is 16.2 billion per gram, and the sprouting rate is 99.2 percent; the fermenting temperature is 28 to 40 DEG C, the fermenting temperature is moderate, and the energy consumption in the production process is effectively reduced; the conversion rate is 57 to 65 percent. Compared with the aspergillus oryzae disclosed in the prior art, the conversion rate of the fructo-oligosacharide is higher, the industrialized mass production of the fructo-oligosacharide is realized, and the application prospect is wide.
Description
technical field
The present invention relates to aspergillus oryzae strain technical field, particularly a kind of aspergillus oryzae strain, also relates to the application of described aspergillus oryzae strain.
background technology
Oligofructose (Fructooligosaccharides, FOS) be a very important class in oligose, claim again oligofructose, FOS or fructooligosaccharide, molecular formula is that G-F-Fn(G is glucose, F is fructose, n=1 ~ 3), refer on the residue of fructose of sucrose molecules by β-1, the general name of the kestose that 1 ~ 3 fructosyl of 2 glycosidic links connections forms, GF3, GF4 and composition thereof is a kind of good water-soluble dietary fibre.
FOS is present in natural phant more, and daily edible vegetables such as jerusalem artichoke, asparagus, witloof and fruit all also have a certain amount of FOS.But it is larger therefrom to extract difficulty, be difficult to realize suitability for industrialized production.Due to the good nourishing function of FOS and special physiologically active, it has become internationally recognized typical prebiotics representative, and extremely the food enterprises and consumers in general's likes.FOS can go directly and by probioticss such as lactobacillus acidophilus, bifidus bacilluss, be selected to utilize in large intestine, toxin-expelling intestine-cleaning, regulates human body microecological balance.Meanwhile, FOS is recognized has raising body immunity, and not digested absorption, improves lipid metabolism, and prevention decayed tooth waits excellent properties, is now used as extensively use of functional ingredient, also can directly drink.
The method of producing at present oligofructose both at home and abroad be take enzyme process as main, also has liquid submerged fermentation method.Patent ZL200810030332.9 utilizes Production by Enzymes oligofructose, first will cultivate a large amount of thalline, and thalline broken wall separation and purification enzyme are prepared to liquid enzyme formulation or immobilized enzyme preparation.Enzyme process weak point is that step is various, the separation and purification process of enzyme complicated and in this process unstable or activity decreased or inactivation of enzymic activity.In addition, enzyme easy inactivation in immobilization process, low conversion rate, the shortcoming such as mass transfer homogeneity is not good, and the price of enzyme is higher, and above all many-sides have restricted the industrial applications of enzyme process.
Patent application 201210552117.1 and 201210531671.1 utilizes respectively aspergillus oryzae and the full cell fermentation sucrose of aspergillus niger to prepare oligofructose, although omitted the complex steps of the extraction purifying of enzyme, avoids a difficult problem for the easy inactivation of enzyme simultaneously.But this technique fermentation period is long, and leavening temperature is high, and energy consumption is large, fermented liquid oligofructose component concentration is 50%, yields poorly, and industrial applications cost is high.
At present, utilize enzyme process or utilize the content of microorganism tank fermentation method production oligofructose all at 50%-60%, its Patent ZL201310083165.5 uses aspergillus oryzae hybrid anaerobic fermentation sucrose to prepare oligofructose, leavening temperature reaches 60 ℃, and fermented liquid oligofructose component concentration is up to 58%.There is equally the problem that energy consumption is large.
summary of the invention
In order to solve above enzyme process or microorganism tank fermentation method, produce the problem that in oligofructose, fermented liquid oligofructose component concentration is low, energy consumption is large, the invention provides the aspergillus oryzae strain that a kind of fermented liquid oligofructose component concentration is high, energy consumption is little (
aspergillus oryzae) FTST-2-6.Oligofructose superior strain FTST-2-6 of the present invention, according to its morphological specificity is observed and 18S rDNA-ITS sequential analysis, determine this bacterial strain be aspergillus oryzae (
aspergillus oryzae), called after FTST-2-6.
The present invention also provide described aspergillus oryzae strain (
aspergillus oryzae) application of FTST-2-6 in fermentative production oligofructose.
The present invention is achieved by the following measures:
An aspergillus oryzae strain, aspergillus oryzae strain (
aspergillusoryzae) FTST-2-6 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC No.8862 on 02 24th, 2014.
The application of described aspergillus oryzae strain FTST-2-6 in fermentative production oligofructose.
Described application, after preferably carbon source being mixed with to carbon source solution sterilization, add fermentor tank, aspergillus oryzae strain FTST-2-6 is added in fermentor tank, under anaerobic, regulate pH value 5.5-7.0,28～40 ℃ of temperature of reaction, anaerobically fermenting 10～20h, in fermented liquid, produce oligofructose, aspergillus oryzae strain FTST-2-6 is the bacterial classification solution through enlarged culturing.
Described application, preferably bacterial classification solution add-on is the 10-30% of carbon source solution weight.
Described application, preferably carbon source concentration of polymer solution is 10～60%.
Described application, preferably the bacterial classification solution through enlarged culturing obtains in the following manner: after seed culture medium sterilizing, picking slant strains one ring, be seeded in seed culture medium, after inoculation, at 30 ℃ of standing 5-10 hours, isothermal vibration 10-16 hour under 30 ℃ of conditions then, obtained the bacterial classification solution of enlarged culturing.
Described application, preferably in fermented liquid, oligofructose component concentration is 57-65%.
Described application, preferably the refining rear liquid glucose transmittance of fermented liquid is greater than 99%.
Aspergillus oryzae strain FTST-2-6(CGMCC No.8862) bacterium colony is rounded, in the middle of the bacterium colony initial stage, is white, and it is transparent radial that edge is.In bacterium colony increase process, bacterium colony middle white region expands, and develops into gradually ring-type, and bacterium colony central zone is pistac subsequently, and further become yellow-green colour, tawny expands to bacterium colony surrounding.This bacterial classification is fast in the upper growth of wort agar substratum (MEA), cultivates colony diameter after 5-7 days and can reach 55-65mm under 25 ℃ of dark conditions, and bacterium colony quality is velvet-like, conidial fructification forms in a large number, conidial head fades to oyster by white, and the initial stage is spherical, and the later stage is radial; The bacterium colony back side is light brown, without water colo(u)r.Conidiophore is tall and big, wide 6.5-12.9 μ m, and wall is obviously coarse; Top capsule is spherical, diameter 20-37.1 μ m; Conidial fructification individual layer or bilayer, bottle stalk 8.0-12.8 * 3.0-4.5 μ m; Conidium is subsphaeroidal, oyster, smooth surface, 3.0-4.6 μ m.There are no spermatium.
The available conventional aspergillus oryzae substratum of aspergillus oryzae strain FTST-2-6 of the present invention (CGMCC No.8862) and cultural method are cultivated.
The invention provides aspergillus oryzae strain FTST-2-6 in the application of fermentative production oligofructose.Sucrose take at present both at home and abroad as substrate, the content of producing oligofructose by tank fermentation method is at 50%-60%, wherein aspergillus oryzae bacterial classification provided by the invention be take sucrose and is generated oligofructose as substrate passes through deep layer anaerobically fermenting, mainly comprise kestose and GF3, really six sugared equal sizes are less for GF4, sugarcane.Leavening temperature is gentle, and 28 ℃-35 ℃, after fermentation ends, the oligofructose content in fermented liquid is at 57%-65%.
Beneficial effect of the present invention: the aspergillus oryzae strain providing (
aspergillus oryzae) FTST-2-6 heritability is stable, is easy to enlarged culturing, the spore count after 8 times that goes down to posterity is 16,200,000,000/g, percentage of germination is 99.2%; Leavening temperature is 28 ℃-40 ℃, and leavening temperature is gentle, effectively reduces the energy consumption in production process; Transformation efficiency, at 57%-65%, has higher oligofructose transformation efficiency than several disclosed aspergillus oryzae strains in prior art, has improved and has been applicable to large-scale industrial production oligofructose, has a extensive future.
preservation information
Aspergillus oryzae strain (
aspergillusoryzae) FTST-2-6 was stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 02 24th, 2014, preserving number CGMCC No. 8862, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment
For making technical scheme of the present invention more cheer and bright, below by specific embodiment describe high yield oligofructose of the present invention aspergillus oryzae strain acquisition and utilize this strain fermentation to produce the method for oligofructose.Test method used in the present invention, except special instruction, is method known in those skilled in the art.In addition, the case study on implementation in the present invention is illustrative, does not limit the scope of the invention.
In subordinate's example, if no special instructions, be ordinary method.
Percentage composition in subordinate's example, if no special instructions, is quality percentage composition.
embodiment 1:
In the present invention, according to bacterial strain screening object, the many parts of subacidity soil that organic content is high have been gathered targetedly.With isolation medium, the sample gathering is carried out to the screening of object bacterial strain, the bacterial strain screening is carried out to separation and purification, after cultivation is ripe, inoculation slant medium is cultivated, and the ripe inclined-plane of cultivation is stored in to 4 ℃ of refrigerators standby.Seed culture medium inoculation slant strains, cultured seed inoculation fermentation liquid ferments.
Nutrient media components and fermentation condition can be selected according to conventional experience voluntarily for those skilled in the art, as long as can realize cultivation and the fermentation of bacterial classification, can use in this application, first enumerate wherein a kind of medium component and fermentation condition, as follows:
Screening, separation and purification and slant culture based component: containing the potato-sucrose-nutrient agar of 2-6% sucrose, pH 5.5-7.0;
Seed culture based component: sucrose 2-6%, potassium primary phosphate 0.1-0.3%, magnesium sulfate 0.15-0.3%, yeast extract 0.5-4%;
Fermentation culture based component: sucrose 5%-60%;
Embodiment mono-
One, the screening of aspergillus oryzae strain, working method is as follows:
(1) aspergillus oryzae strain separation and purifying
Separated aspergillus oryzae strain from the soil gathering, adopts conventional dilution plate coating method.In sepn process, to turning out ripe doubtful object bacterium colony, take colony morphology characteristic to observe, the methods such as thalline microanalysis are carried out identification of strains, after the bacterial strain of determining separation and purification is aspergillus oryzae, the ripe single bacterium colony streak inoculation isolation medium of picking, carries out purifying to bacterial strain.This step repeats 4-6 time, until obtain single bacterium colony.By after the aspergillus oryzae numbering of separation and purification, be seeded in the PDA inclined-plane containing 2-6% sucrose, cultivate ripe latter 4 ℃ and save backup.
(2) bacterial strain screening
The aspergillus oryzae strain of separation and purification in upper step inclined-plane being preserved with transfering loop, be seeded in prepare and 121 ℃ of conditions under take in the liquid nutrient medium that sucrose is sole carbon source of sterilizing 30min, 30 ℃ of 120r/min secretly cultivate, and fermented liquid is measured respectively the oligofructose transformation efficiency of each aspergillus oryzae strain by high performance liquid chromatography.Finally, obtained the aspergillus oryzae strain FTST-2-6 of a strain high conversion efficiency.Screening process is as following table 1.
The table 1 screening bacterial strain transformation efficiency comparison that obtains
Two, bacterium colony and morphological features are observed
Aspergillus oryzae strain FTST-2-6 (CGMCC No.8862) bacterium colony is rounded, in the middle of the bacterium colony initial stage, is white, and it is transparent radial that edge is.In bacterium colony increase process, bacterium colony middle white region expands, and develops into gradually ring-type, and bacterium colony central zone is pistac subsequently, and further become yellow-green colour, tawny expands to bacterium colony surrounding.This bacterial classification is fast in the upper growth of wort agar substratum (MEA), cultivates colony diameter after 5-7 days and can reach 55-65mm under 25 ℃ of dark conditions, and bacterium colony quality is velvet-like, conidial fructification forms in a large number, conidial head fades to oyster by white, and the initial stage is spherical, and the later stage is radial; The bacterium colony back side is light brown, without water colo(u)r.Conidiophore is tall and big, wide 6.5-12.9 μ m, and wall is obviously coarse; Top capsule is spherical, diameter 20-37.1 μ m; Conidial fructification individual layer or bilayer, bottle stalk 8.0-12.8 * 3.0-4.5 μ m; Conidium is subsphaeroidal, oyster, smooth surface, 3.0-4.6 μ m.
Three, bacterial strain FTST-2-6 molecular biology identification
Our company entrusts institute of microbiology of the Chinese Academy of Sciences to carry out the evaluation of bacterial strain FTST-2-6 in February, 2014.It is 509nt that order-checking obtains bacterial strain FTST-2-6 18rDNA-ITS gene, and wherein nucleotide sequence is as shown in sequence in sequence table 1.
By the Blast comparison in ncbi database of gene sequencing result, result demonstration bacterial strain FTST-2-6 and aspergillus oryzae (
aspergillus oryzae) and flavus (
aspergillus flavus) in the ITS gene nucleotide series homology of some bacterial strains be 100%.According to experimental datas such as spawn culture feature, microscopic features and gene orders, comprehensively analyzing is aspergillus oryzae strain by FTST-2-6 identification of strains.This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.8862, and preservation date is on 02 24th, 2014, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment bis-
By aspergillus oryzae strain FTST-2-6 of the present invention 8 generations of continuous passage on slant medium, and detect 1st generation, the 4th generation and the 8th generation spore count and percentage of germination, judge accordingly the genetic stability of this bacterial strain, data are in Table 2.From each for spore count and percentage of germination detected result, aspergillus oryzae strain FTST-2-6 inheritance stability.
Table 2 mitotic stability detected result
The seed culture medium preparing is in proportion sterilizing 30min under 121 ℃ of conditions, with transfering loop picking slant strains one ring, inoculation seed culture medium.After inoculation, at 30 ℃ of standing 5-10 hours, isothermal vibration 10-16 hour under 30 ℃ of conditions then.Obtain the bacterial classification liquid of enlarged culturing.
Embodiment tri-
The sucrose liquid 8L of configuration 35%, pH5.5, under 121 ℃ of conditions after sterilizing 20min, fermented liquid after transfer sterilizing is to 10L fermentor tank, the seed liquor of access enlarged culturing, inoculum size is 10%, 28 ℃ of anaerobically fermenting 20 hours of sucrose liquid, stop after fermentation filtering thalline oligofructose component concentration in fermented liquid to be detected with high performance liquid chromatography be 58.5%.Liquid glucose transmittance 99.2% after refining.
Embodiment tetra-
The sucrose liquid 8L of configuration 40%, pH6.0, under 121 ℃ of conditions after sterilizing 20min, fermented liquid after transfer sterilizing, to 10L fermentor tank, accesses the seed liquor of enlarged culturing, and inoculum size is 10% of sucrose liquid, 45 ℃ of anaerobically fermentings 12 hours, stop after fermentation, filtering thalline oligofructose component concentration in fermented liquid to be detected with high performance liquid chromatography be 57%, refining after liquid glucose transmittance 99.5%.
Embodiment five
The sucrose liquid 8L of configuration 45%, pH6.5, under 121 ℃ of conditions after sterilizing 20min, fermented liquid after transfer sterilizing is to 10L fermentor tank, the seed liquor of access enlarged culturing, inoculum size is 15%, 30 ℃ of anaerobically fermenting 16 hours of sucrose liquid, stop after fermentation filtering thalline oligofructose component concentration in fermented liquid to be detected with high performance liquid chromatography be 64.7%.Liquid glucose transmittance 99.6% after refining.
Embodiment six
The sucrose liquid 8L of configuration 40%, pH6.5, under 121 ℃ of conditions after sterilizing 20min, fermented liquid after transfer sterilizing is to 10L fermentor tank, the seed liquor of access enlarged culturing, inoculum size is 30%, 35 ℃ of anaerobically fermenting 10 hours of sucrose liquid, stop after fermentation filtering thalline oligofructose component concentration in fermented liquid to be detected with high performance liquid chromatography be 61%.Liquid glucose transmittance 99.5% after refining.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not subject to the restriction of embodiment; other is any does not deviate from change, modification, the combination made under spirit of the present invention and principle, substitute, simplify and all should be equivalent substitute mode, within being included in protection scope of the present invention.
<110> Shandong Futian Medicine Industry Co., Ltd.
<120> aspergillus oryzae strain and application thereof
<160>1
<210>1
<211>509
<212>DNA
<213> aspergillus oryzae (
aspergillusoryzae)
<220>
<223>
<400>1
tacagagcgg gtgacaaagc cccatacgct cgaggatcgg acgcggtgcc gccgctgcct60
ttggggcccg tcccccccgg agaggggacg acgacccaac acacaagccg tgcttgatgg120
gcagcaatga cgctcggaca ggcatgcccc ccggaatacc agggggcgca atgtgcgttc180
aaagactcga tgattcacgg aattctgcaa ttcacactag ttatcgcatt tcgctgcgtt240
cttcatcgat gccggaacca agagatccat tgttgaaagt tttaactgat tgcgatacaa300
tcaactcaga cttcactaga tcagacagag ttcgtggtgt ctccggcggg cgcgggcccg360
gggctgagag cccccggcgg ccatgaatgg cgggcccgcc gaagcaacta aggtacagta420
aacacgggtg ggaggttggg ctcgctagga accctacact cggtaatgat ccttccgcag480
gttcacctac ggaaaccttg ttacgactt 509
Claims (9)
1. an aspergillus oryzae strain, it is characterized in that described aspergillus oryzae strain (
aspergillusoryzae) FTST-2-6 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC No.8862 on 02 24th, 2014.
2. an aspergillus oryzae strain FTST-2-6 claimed in claim 1 application in fermentative production oligofructose.
3. application according to claim 2, after it is characterized in that carbon source is mixed with to carbon source solution sterilization, add fermentor tank, aspergillus oryzae strain FTST-2-6 is added in fermentor tank, under anaerobic, regulate pH value 5.5-7.0,28～40 ℃ of temperature of reaction, anaerobically fermenting 10～20h, in fermented liquid, produce oligofructose, aspergillus oryzae strain FTST-2-6 is the bacterial classification solution through enlarged culturing.
4. application according to claim 3, is characterized in that bacterial classification solution add-on is the 10-30% of carbon source solution weight.
5. according to the application described in claim 3 or 4, it is characterized in that carbon source concentration of polymer solution is 10～60%.
6. according to the application described in claim 3 or 4, it is characterized in that obtaining in the following manner through the bacterial classification solution of enlarged culturing: after seed culture medium sterilizing, picking slant strains one ring, be seeded in seed culture medium, after inoculation, at 30 ℃ of standing 5-10 hours, isothermal vibration 10-16 hour under 30 ℃ of conditions then, obtained the bacterial classification solution of enlarged culturing.
7. according to the application described in any one in claim 3-6, it is characterized in that in fermented liquid, oligofructose component concentration is 57-64%.
8. according to the application described in any one in claim 3-7, it is characterized in that the refining rear liquid glucose transmittance of fermented liquid is greater than 99%.
9. according to the application described in any one in claim 3-7, it is characterized in that described carbon source is sucrose.
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Effective date of registration: 20161031 Address after: Three road 266000 in Shandong province Qingdao city Yanan No. 234 Building No. 1 room 1808 Applicant after: Qingdao Health Technology Co., Ltd. Address before: 266000 Shandong province Qingdao City, Shandong Road No. 177 1002 Applicant before: Shandong Mifutang Food Co., Ltd. |
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Application publication date: 20140910 |