CN100413957C - Apergillus niger strain and application thereof - Google Patents
Apergillus niger strain and application thereof Download PDFInfo
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- CN100413957C CN100413957C CNB2006100963546A CN200610096354A CN100413957C CN 100413957 C CN100413957 C CN 100413957C CN B2006100963546 A CNB2006100963546 A CN B2006100963546A CN 200610096354 A CN200610096354 A CN 200610096354A CN 100413957 C CN100413957 C CN 100413957C
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Abstract
The invention discloses Aspergillus niger ML-0016 strain and application, which is characterized by the following: the reserving number is CCTCC.NO.M206034; the soy isoflavone glycoside is converted by strain to manufacture soy isoflavone glycosyl and aglycone.
Description
Technical field
The present invention relates to a kind of microorganism and application thereof, especially a kind of aspergillus niger (Aspergillusniger) ML-0016 bacterial strain and application thereof.
Background technology
Soybean isoflavones is the isoflavones components that belongs in the flavonoid compound.The basic parent nucleus of flavonoid compound is a series of compounds of 2-phenyl chromone, at present two a series of compounds that phenyl ring interconnects and forms by three carbochains of flavonoid compound general reference.
Soybean isoflavones is a kind of mixture; mainly contain 3 classes; be soybean glucoside, young fustic glucoside, soya bean glucoside; there be (free type generally is called glucoside unit, and back three classes then are summed up as the glucoside form of mating type) in every class with four kinds of forms such as free type, glycoside type, ethanoyl glycoside type and malonyl glycoside types.Isoflavones in the soybean is divided into the glucoside unit of free type and glucoside two classes of mating type, and the glucoside unit of free type accounts for the 2%-3% of total amount, comprises genistein, big legumin and glycitein.The glucoside of mating type accounts for the 97%-98% of total amount, mainly exists with Genistin, Daidzin, soya bean glucoside and malonyl-Genistin, malonyl-Daidzin, malonyl-soya bean glucoside form.
The down purples that show of the micro-yellow of isoflavonoid, greyish white or colourless, ultraviolet ray more.Genistein in the soybean isoflavones is the canescence crystallization, and ultraviolet lamp is no fluorescence down, and big legumin is the micro white crystallization, and ultraviolet lamp is no fluorescence down.The glucoside unit of soybean isoflavones does not have opticity, but for the glucoside structure of mating type, owing to introduced glycosyl in the structure, thereby have opticity.The general indissoluble or water insoluble of glucoside unit of soybean isoflavones, dissolve in and reach in the diluted alkaline in the organic solvents such as methyl alcohol, ethanol, ethyl acetate, ether, the combined type glucoside of soybean isoflavones is soluble in methyl alcohol, ethanol, pyridine, ethyl acetate and the sig water, be insoluble in organic solvents such as benzene, ether, chloroform, sherwood oil, water solubility is increased, dissolve in hot water.Owing to phenolic hydroxyl group is arranged in the isoflavones molecule,, dissolve in the alkaline aqueous solution and reach in the pyridine so it shows acid.
Soybean isoflavones has weak estrogen activity, be equivalent to 100,000 approximately/ the estradiol activity, be called phytoestrogen, be considered to estrogenic natural substitute for, caused people's very big concern, 1986, U.S. scientist found the isoflavones of anticancer in the soybean.In June nineteen ninety, American National cancer research institute organizes the famous scholar in the whole America to discuss the anticancer effect of soybean, has affirmed that soybean isoflavones is best crude substance.After this confirm again that soybean isoflavones can prevent osteoporosis, alleviate the uncomfortable disease of Woman climacteric and reduce evidence of coronary heart diseases.For this reason, the benefit that soybean food is eaten in United States Government and public opinion a whoop and a holler, especially for the food that contains soybean isoflavones etc., personal value training increases, and soybean isoflavones is considered as treasure.
In a word, soybean isoflavones plays certain preventing cancer effect by different mechanism, particularly weak estrogen effect, and female hormone can play the class estrogenic effect when not enough, and female hormone plays the hormone antagonist effect when superfluous.
The effect of soybean isoflavone female hormone just had report as far back as 1962, and the preventing osteoporosis effect is still just making progress in the recent period.Soybean isoflavones also has regulating blood fat, reduces blood cholesterol in addition, prevents and treat atherosclerotic effect.
Raising along with the human living standard, people more and more pay attention to the health care of health, soybean isoflavones has useful regulating effect to the human physiological metabolism, it can play estrogen-like effects for low hormonal readiness person, can produce the hormone antagonist effect for high hormonal readiness person, particularly soybean isoflavones helps the generation of diseases such as preventing cardiovascular disease and tumour, for the mankind provide the prevention of chronic disease may.China's soybean resource is abundant, and the bean product kind is various, and the value of soybean isoflavones is considerable, has good society and economic worth, for bright prospects have been showed in its development and use.
For soybean isoflavones, the molecular structure of natural nucleoside is not the optimum regime of its active performance, generally believes that aglycon is only the optimum regime of active performance, is absorbed by the body because of aglycon is easier.Soybean isoflavone glucoside can not pass through small bowel, but the hydrolysis by the beta-glucosidase enzyme of microorganism in the colon, hydrolysate is further generated aglycon by cell degradation.Therefore, if can be aglycon, just can significantly strengthen the biological activity of soybean isoflavones at the external glucoside of hydrolysis effectively.Though glycosidic link can rupture under acidity or alkaline condition, and percent hydrolysis is also very high, and many people doubt for the stability of isoflavone genin under acid or the alkaline condition.In addition, under strong acid or highly basic condition, also be difficult for carrying out suitability for industrialized production.And enzymic hydrolysis has its special advantages, hydrolysising condition gentleness not only, and adopt slightly acidic buffered soln, and isoflavone genin volatility not more.Obtain highly active soybean isoflavone glucoside hydrolase at present and mainly contain two kinds of approach, the one, derive from plant, the 2nd, derive from microorganism.
Be to find that soybean isoflavone glucoside constantly changes into the aglycon form of biologically active under the beta-glucosidase enzyme effect of microorganisms in the food of raw material or the antibiotic fermentation process with the soybean.As Rhizopus oligosporus (Rhizopus oligosporus) in red shellfish production process, aspergillus in fermented soya bean fermentations, red saccharopolyspora (Saccharopolyspora erythaea ATCC 11635) in erythromycin is produced, subtilis (Bacillus subtilis) in the natto fermentation, bifidus bacillus (Bifidobacterium) can both make aglycon content increase at the ferment beta-glucosidase enzyme of medium generation of soymilk.In addition, can the enzymatic hydrolysis of soybean iso-flavone glucoside especially genistein and the Daidezin microorganism that generates aglycon also have Streptomyces roseolus (Streptomyces roseolus ATCC 31047), have a liking for the halogen micromonospora and have a liking for salt subspecies (Micomonospora halophytica subsp.halophytica ATCC27596), pseudomonas (Pseudomonas ZD-8), streptomycete (Streptomyces sp.), graceful radiation Mucor (Actinomucorelegans AS3.227), lactobacillus bulgaricus (Lactobacillus bulgaricus), De Bilishi breast genus bacillus (Lactobacillus debr ü ckii KCTC 1047), Rhizopus stolonifer bacterium (R.stolonifervuill) etc.
The beta-glucosidase enzyme of microorganisms belongs to a component in the cellulase system, can cellulolytic beta-glucosidase enzyme the hydrolyzed soy bean isoflavone glucosides, perhaps can the beta-glucosidase enzyme of hydrolyzed soy bean isoflavone glucosides hydrocellulose, and the systematic study of relevant this respect does not appear in the newspapers as yet.Soybean isoflavones structurally has similarity with ginsenoside, all is that glycosyl links to each other with the oxygen glycosidic bond with aglycon, belongs to the small molecular sugar conjugate.The specific activity that Chen Guanxiong etc. have carried out beta-glucosidase enzyme to Mierocrystalline cellulose and ginsenoside, the result shows that circumscribed beta-glucosidase enzyme of Mierocrystalline cellulose and ginsenoside beta-glucosidase enzyme are not the same enzymes, it has substrate selective, is the subclass of beta-glucosidase enzyme.And the small molecular sugar conjugate is very wide in the occurring in nature distribution, and its aglycon has diversity.The beta-glucosidase enzyme that links to each other with glycosyl about glycosyl such as the research of cellulase are more, and the research of the beta-glucosidase enzyme of relevant hydrolysis small molecular sugar conjugate is less.
Summary of the invention
The objective of the invention is to: a kind of aspergillus niger (Aspergillus niger) ML-0016 bacterial strain is provided,, produces soybean isoflavones glycosyl and aglycon by this bacterial strain soybean transformation iso-flavone glucoside.
The object of the present invention is achieved like this: with preserving number is that aspergillus niger (Aspergillus niger) the ML-0016 inoculation of CCTCC.NO.M206034 is on 9cm potato solid agar plate, 28 ℃ of cultivations, the mycelium canescence, quality is loose, the mycelia at edge sparse and to around extend, bacterium colony central authorities mycelia is cotton-shaped, on have a small amount of brown spore to produce; The bacterium colony reverse side is radial, and substratum is colourless, and spore quantity increases; After bacterium colony covered with flat board, spore gradually was black; The top capsule of conidial head is subsphaeroidal, radial, brown to brownish black, be made up of top capsule, conidium and individual layer stigma, conidium is concatenated on stigma, and amount is many, the band spinule, spherical in shape or subsphaeroidal under the simple microscope, ovalize or subsphaeroidal under the scanning electron microscope, the conidiophore wall is smooth, colourless.
A kind of preserving number is the application of aspergillus niger (Aspergillus niger) the ML-0016 bacterial strain of CCTCC.NO.M206034, it is characterized in that: by this bacterial strain soybean transformation iso-flavone glucoside, produce soybean isoflavones glycosyl and aglycon.
In an application of the invention: by this bacterial strain soybean transformation iso-flavone glucoside, the method for producing soybean isoflavones glycosyl and aglycon is: this bacterium is fermented, obtain enzyme liquid or crude enzyme liquid; The enzyme liquid or the crude enzyme liquid that obtain are joined in the substrate soybean isoflavones solution, utilize enzyme liquid or crude enzyme liquid to decompose substrate, then the soybean isoflavones glycosyl in the hydrolyzed solution is separated with aglycon, and purify.
In an application of the invention: described this bacterium is fermented is: this bacterium is seeded to substratum ferments, the acquisition seed liquor is seeded to the inclined-plane with this bacterium again or seed liquor is fermented, and cultivates thalline and obtains enzyme liquid or crude enzyme liquid.
In an application of the invention: described substratum is the per-cent of kg/ volume L by weight, for: soybean isoflavones: 0.5%～12%, wheat bran: 0.1%～2.0%, peptone 0.1%～2.0%, sodium-acetate: 0.1%～1.5%, NaH
2PO
42H
2O:0.1%～0.8%, MgSO
4: 0.01%～0.1%, xitix: 0.1%～0.4%, saligenin: 0.05～0.1%, with tap water preparation, pH value 4.0～7.0.
In an application of the invention: describedly this bacterium is seeded to the culture condition that inclined-plane or seed liquor ferment is: 20 ℃～40 ℃ of temperature, initial pH is 4.0～7.0, incubation time is 24h～120h.
In an application of the invention: in the culturing process, after the thalli growth logarithmic phase, obtain enzyme liquid, or after the thalli growth logarithmic phase, carry out centrifugation and obtain crude enzyme liquid, the crude enzyme liquid that obtains is joined in the substrate soybean isoflavones solution, the soybean isoflavones glycosyl in the hydrolyzed solution is separated with aglycon.
In an application of the invention: described substrate soybean isoflavones solution is: use 0.02M, the HAc-NaAc damping fluid of pH5.0 is made into the substrate solution that concentration is 1～10mg/ml with soybean isoflavones, utilize crude enzyme liquid to decompose substrate, concentration of substrate is 0.5%～15.0%, reaction times 1h～100h.
In an application of the invention: the described reaction times is 24h.
The invention has the advantages that: the Aspergillus niger strain that the present invention screens can be produced the higher relatively soy bean isoflavone glycosidase of vigor, and this aspergillus niger can utilize the waste of industrial and agricultural productions such as corn cob, soybean cake powder to produce soy bean isoflavone glycosidase as nutraceutical matrix, helps making biological process to prepare the soybean isoflavone aglycones industrialization.Enzymolysis process has the reaction conditions gentleness, and isoflavone genin is advantage such as volatility not, therefore is the optimal path of preparation isoflavone genin.
Embodiment:
Embodiment 1
The seed selection of aspergillus niger ML-0016 bacterial strain
Screen starting strain in the beans sauce sample, then slant culture (28 ℃, 2～3d), obtain spore suspension; Utilize that NTG handles 2,4,6,8 respectively, behind the 10min, stop mutagenesis, the spore suspension dilution of handling is coated with behind the ware under 28 ℃, cultivate 4～5d.Bacterium colony on the plate is chosen, and purebred separation is carried out in line, utilize then shake bottle carry out primary dcreening operation (28 ℃, 50h).A screening bacterial strain and a selected strain are the starting strain of further mutagenesis, and the preparation spore suspension utilizes
60Co gamma-ray irradiation irradiation dose is respectively 0rad, 30,000 rad, 50,000 rad, 7.5 ten thousand rad, 100,000 rad, 150,000 rad.With the spore suspension behind the irradiation be coated with ware cultivate (28 ℃, 4～5d) choose the bacterium colony on the plate, purebred separation is carried out in line, utilize then shake bottle carry out primary dcreening operation (28 ℃, 50h).Utilize again and shake bottle and carry out multiple sieve (28 ℃ 50h), finally obtain good bacterium producing multi enzyme preparation, and utilize sand tube preservation.
The feature of aspergillus niger ML-0016 bacterial strain:
Inoculation on 9cm potato solid agar plate, 28 ℃ of cultivations, the mycelium canescence, quality is loose, the mycelia at edge sparse and to around extend, bacterium colony central authorities mycelia is cotton-shaped, on have a small amount of brown spore to produce; The bacterium colony reverse side is radial, and substratum is colourless, and spore quantity increases; After bacterium colony covered with flat board, spore gradually was black; The top capsule of conidial head is subsphaeroidal, radial, brown to brownish black, be made up of top capsule, conidium and individual layer stigma, conidium is concatenated on stigma, and amount is many, the band spinule, spherical in shape or subsphaeroidal under the simple microscope, ovalize or subsphaeroidal under the scanning electron microscope, the conidiophore wall is smooth, colourless.
18s rDNA sequential analysis: with the total DNA of the cell that extracts is template, utilize the 18S rDNA gene of primer P1:5 '-TCC GTA GGT GAA CCT GCG G-3 ' and P2:5 '-TCC TCC GCT TAT TGATAT GC-3 ' amplification bacterial strain, gene product is connected with the T carrier, confirm that through order-checking this fragment physical length is 382bp, (submitted this sequence to GenBank, the GenBank registration number is DQ223426) with GenBank in related data carry out similarity analysis and find, the highest (the homology of homology of this bacterium and aspergillus niger (Aspergillus niger), 98.43%/382bps, based on 18s rDNA).Physiology and biochemistry is identified the binding molecule evaluation, can confirm that this bacterial strain belongs to Eurotium (Aspergillu), classification called after Aspergillus niger.Oneself is preserved in Chinese typical culture collection center (CCTCC) on April 10th, 2006 this bacterial strain, and deposit number is CCTCC M206034.
Embodiment 2
Culture medium prescription (weight/volume percent, as follows): defatted soybean meal 4%, wheat bran 2%, peptone 0.1%, (NH
4)
2SO
40.1%, KH
2PO
40.1%, sodium-acetate 0.1%, xitix 0.1%, MgSO
40.1%, NaH2PO42H2O:0.4%, saligenin 0.05%, pH 6.0, and with the tap water preparation, 121 ℃ of sterilization 20min are standby, defatted soybean meal can utilize the dregs of beans after industry is extracted oil in the present embodiment, and when specifically implementing, its consumption can be controlled in 0.5%～12% scope.
Get the above-mentioned substratum of 100mL, average mark is contained in 2 250mL triangular flasks, sterilization.Insert deposit number and be the cultivation thalline of slant strains of aspergillus niger (Aspergillus niger) the ML-0016 bacterial strain of CCTCC M 206034, shaking speed 150r/min cultivates 24 hours as seed liquor at 28 ℃ of shaking tables, and is standby.
Get the above-mentioned substratum of 2L, average mark 40 500mL that pack into shake in the bottle, sterilization.Insert seed liquor, inoculum size is 1.5% (v/v), cultivates, and culture temperature is 20 ℃, and incubation time is 72h, shaking speed 150r/min.Collect above-mentioned fermented liquid 1.5L, process is centrifugal, and (10,000 * g 10min) after the degerming, adds 70% ammonium sulfate precipitated protein, collects albumen and adds 500ml pH 5.0HAc-NaAc damping fluid, as crude enzyme liquid.
Embodiment 3
Use 0.02M, pH 5.0HAc-NaAc damping fluid is made into the substrate solution that concentration is 1～10mg/ml with soybean isoflavones, adds an amount of ethanol as the substrate auxiliary agent.Get the preparation crude enzyme liquid 50ml among the embodiment 1, crude enzyme liquid and substrate solution (concentration of substrate is 0.5%～15.0%) are carried out according to 1: 1 mixed, under 20 ℃～50 ℃, carry out enzyme digestion reaction, behind 1～10h, obtain enzymolysis solution 100ml.
Embodiment 4
The enzymolysis solution that embodiment 3 is obtained filters the clear liquid 85ml that obtains, and the ethyl acetate that adds 2 times of volumes extracts, and evaporated under reduced pressure obtains the soybean isoflavones crude product, separates with silicagel column.
Embodiment 5
Press the method for embodiment 1, utilize the 10L fermentor tank to produce enzyme, the method for pressing embodiment 2, with the reaction that is hydrolyzed of the retort of 10L, the pH value is regulated automatically with mineral acid or organic acid, bases, and the pH value is controlled at 5.0, carries out the separation and the extraction of product by the method for embodiment 3.The result is: fermentation time 2 days, and enzyme work reaches 162.75U, and enzymolysis after product extraction yield is 70%.
Claims (6)
1. an aspergillus niger (Aspergillus niger) bacterial strain, it is characterized in that: the preserving number of Aspergillus niger strain is CCTCC.NO.M206034, with this inoculation on 9cm potato solid agar plate, 28 ℃ of cultivations, the mycelium canescence, quality is loose, the mycelia at edge sparse and to around extend, bacterium colony central authorities mycelia is cotton-shaped, on have a small amount of brown spore to produce; The bacterium colony reverse side is radial, and substratum is colourless, and spore quantity increases; After bacterium colony covered with flat board, spore gradually was black; The top capsule of conidial head is subsphaeroidal, radial, brown to brownish black, be made up of top capsule, conidium and individual layer stigma, conidium is concatenated on stigma, and amount is many, the band spinule, spherical in shape or subsphaeroidal under the simple microscope, ovalize or subsphaeroidal under the scanning electron microscope, the conidiophore wall is smooth, colourless.
2. application that preserving number is the Aspergillus niger strain of CCTCC.NO.M206034, it is characterized in that: this bacterium is fermented, fermented liquid is carried out after the centrifugation degerming, add 70% ammonium sulfate precipitated protein, collect albumen and add damping fluid, obtain crude enzyme liquid; The crude enzyme liquid that obtains is joined in the substrate soybean isoflavones solution, utilize crude enzyme liquid to decompose substrate, then the soybean isoflavones glycosyl in the hydrolyzed solution is separated with aglycon, and purify.
3. preserving number according to claim 2 is the application of the Aspergillus niger strain of CCTCC.NO.M206034, it is characterized in that: to this bacterium used substratum per-cent of kg/ volume L by weight that ferments, for: soybean isoflavones: 0.5%～12%, wheat bran: 0.1%～2.0%, peptone 0.1%～2.0%, sodium-acetate: 0.1%～1.5%, NaH
2PO
42H
2O:0.1%～0.8%, MgSO
4: 0.01%～0.1%, xitix: 0.1%～0.4%, saligenin: 0.05～0.1%, with tap water preparation, pH value 4.0～7.0.
4. preserving number according to claim 2 is the application of the Aspergillus niger strain of CCTCC.NO.M206034, it is characterized in that: the culture condition that this bacterium is fermented is: 20 ℃～40 ℃ of temperature, and initial pH is 4.0～7.0, incubation time is 24h～120h.
5. preserving number according to claim 2 is the application of the Aspergillus niger strain of CCTCC.NO.M206034, it is characterized in that: described substrate soybean isoflavones solution is: use 0.02M, the HAc-NaAc damping fluid of pH 5.0 is made into the substrate solution that concentration is 1～10mg/ml with soybean isoflavones, utilize crude enzyme liquid to decompose substrate, the concentration of soybean isoflavones in substrate reactions liquid is 0.5%～15.0%, reaction times 1h～100h.
6. preserving number according to claim 5 is the application of the Aspergillus niger strain of CCTCC.NO.M206034, it is characterized in that: the described reaction times is 24h.
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Families Citing this family (5)
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| KR100992800B1 (en) * | 2010-05-14 | 2010-11-08 | 주식회사 지씨에이치앤피 | A process for preparing novel processed ginseng or extract thereof showing increased amount of minor ginsenosides |
| CN107022586B (en) * | 2017-04-24 | 2021-03-02 | 安徽工程大学 | Application of cerium nitrate in preparation of flavonoid compound by fermentation of aspergillus niger |
| CN107699498B (en) * | 2017-10-04 | 2020-09-25 | 北京康缘益生生物科技有限公司 | Composite microbial preparation for degrading organophosphorus pesticide and preparation method thereof |
| CN108642102A (en) * | 2018-04-27 | 2018-10-12 | 佛山科学技术学院 | A kind of method of isoflavone genin using microwave abstracting extraction microorganism production and its obtained isoflavone genin |
| CN115044628B (en) * | 2022-06-17 | 2023-09-01 | 华南农业大学 | Method for producing soybean isoflavone aglycone by whole cell transformation |
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