Aspergillus oryzae TJTSW001 and the application in the fermentation of Chinese medicine Semen Sojae Preparatum thereof
Technical field
The present invention relates to aspergillus oryzae, especially relate to a kind of aspergillus oryzae TJTSW001 and the application in the fermentation of Chinese medicine Semen Sojae Preparatum thereof.
Background technology
Semen Sojae Preparatum is the traditional Chinese medicine of commonly using simply, < < Mingyi Bielu > > begins to be loaded in, successive dynasties book on Chinese herbal medicine is all on the books, it is one of main flavour of a drug of conventional Chinese patent medicine " YINQIAO JIEDU PIAN " prescription, is also the kind of including of successive dynasties < < Chinese Pharmacopoeia > >.This product is the fermentation processed goods of the mature seed of legume soybean, except the soybean of black kind skin strain is main raw material, also with medicinal materials such as mulberry leaf, sweet wormwoods, is equipped with wherein and ferments.Traditional Chinese medical science tradition application this product in clinical effect is: induce sweat, relieving restlessness, a surname send out hot and suffocating.For flu, fever and chills headaches, irritated uncomfortable in chest, restlessness of asrhenia type and insomnia etc.
Soybean isoflavones is the major physiological activeconstituents of soybean, and modern study shows that it has following function:
(1) anti-cancer, tumour and anticancer function of tumor.
(2) preventing osteoporosis.
(3) decreasing cholesterol and cardioprotection.
(4) neuroprotective and anti-nerve degenerative diseases etc.
Soybean isoflavones, in soybean, mainly contains two kinds of existence forms, and a kind of is Aglycons, comprises genistein (claiming again daidzein, Sophoricol) and daidzin, and another kind is the glucosides of mating type.Glucosides is mainly with malonyl-genistein, malonyl-daidzin, and Genistoside, the form of daidzin exists.In soybean, mainly take the glucosides of mating type as main, and free aglycon accounting is quite few, accounts for the 2-3% of total amount.The activity that it is pointed out that aglycon will be far above glucosides.The glucosides of mating type need to be hydrolyzed into the effect of aglycon competence exertion.Therefore,, if glycoside hydrolysis can be become to the form of aglycon, higher physiologically active will be obtained.Guo Wen bravely found through experiments isoflavones in (quality evaluating method of Chinese medicine Semen Sojae Preparatum and " relieving restlessness of inducing sweat " study on mechanism thereof, master thesis, 2004) Semen Sojae Preparatum and its " relieving restlessness of inducing sweat " and effect there is certain contact.Therefore, obtain more highly active isoflavone genin, to improving the drug effect of Semen Sojae Preparatum, have very significant realistic meaning.
The microorganism of Chinese medicine Semen Sojae Preparatum fermentation use, most modes that are all recited as spontaneous fermentation, as < < Pharmacopoeia of People's Republic of China > > 2010 editions, about the record of Semen Sojae Preparatum " with the mulberry leaf of decocting, sweet wormwood slag, cover; vexed make to ferment on yellow clothes all over time; take out, remove the dregs of a decoction.Clean, put in container vexed 15-20 days again, to fully fermentation, when fragrance overflows, take out, slightly steam, dry, obtain." in addition; the same < < Hunan Province Chinese medicine preparation standard > > 1983 editions that is expressed in, < < Shanghai City Chinese medicine preparation standard > > 2008 editions is medium all to be occurred.Undoubtedly, in the process of above-mentioned fermentation, there is the acting in conjunction of numerous microorganisms.But from length " yellow clothes ", and correlative study can be clear and definite, aspergillus oryzae in the process of its fermentation in occupation of the status of advantage very.The Chinese medicine preparation standard > > of < < Zhejiang Province 2005 editions very clear and definite statement inoculation aspergillus oryzae spore ferment.
Summary of the invention
Object of the present invention is exactly, in order to address the above problem, to provide a kind of aspergillus oryzae TJTSW001, and this aspergillus oryzae strain can high yield Glycosylase, can be efficiently in connection with the soybean isoflavones of type, be hydrolyzed into aglycon and sugar.
Another object of the present invention is application aspergillus oryzae strain TJTSW001 fermentative production Chinese medicine Semen Sojae Preparatum, obtains the high Chinese medicine Semen Sojae Preparatum containing aglycon.
Aspergillus oryzae strain provided by the invention is (Aspergillus oryzae) TJTSW001, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 20th, 2014, culture presevation is numbered CGMCC NO.8945, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
This bacterial strain is to obtain through 60Co and ultraviolet complex mutation.This bacterial strain has very high product glycosidase activity, utilize this bacterial strain produce Chinese medicine Semen Sojae Preparatum can obtain high containing aglycon, significantly higher than aglycon content in bibliographical information Semen Sojae Preparatum at present.
Aspergillus oryzae strain provided by the present invention, be by
60co and ultraviolet mutagenesis complex mutation obtain.
First, the aspergillus oryzae spore of having grown, with under aseptic washing, is made to spore suspension, then this spore suspension being placed in to irradiation dose is 20ky's
60co carries out mutagenesis under irradiating.The spore suspension that mutagenesis finishes, pours on plate, under the irradiation of ultraviolet lamp, carries out secondary mutagenesis.After mutagenesis completes, static a moment in dark environment, to prevent photoreactivation.
Then, at the enterprising row filter of screening culture medium, cultivate, screening culture medium be take soybean isoflavones as only carbon source, with (NH
4)
2sO
4for nitrogenous source, take and in screening culture medium, form single bacterium colony as suitable extent of dilution.With bacterium colony size, for selecting foundation, select object bacteria.
The aspergillus oryzae that the present invention obtains (Aspergillus oryzae) TJTSW001, can take soybean isoflavones as only carbon source, with (NH
4)
2sO
4for well-grown on the substratum of nitrogenous source, to cultivate three days, its colony diameter can reach 3 centimetres.Further utilize its fermentation Semen Sojae Preparatum, measure its Glycosylase.
The present invention has following beneficial effect:
Aspergillus oryzae that the present invention obtains (Aspergillus oryzae) TJTSW001, is used in the production of Chinese medicine Semen Sojae Preparatum, can obtain the high Semen Sojae Preparatum containing daidzein, thereby can increase its effect, has prospect widely.
Embodiment
For technique means, creation characteristic that the present invention is realized, reach object and effect is easy to understand, below in conjunction with specific embodiment, further set forth the present invention.
Aspergillus oryzae strain provided by the present invention, be by
60co and ultraviolet mutagenesis complex mutation obtain.Preparation process is as follows:
First, the aspergillus oryzae spore of having grown, with under aseptic washing, is made to spore suspension, then this spore suspension being placed in to irradiation dose is 20ky's
60co carries out mutagenesis under irradiating.The spore suspension that mutagenesis finishes, pours on plate, under the irradiation of ultraviolet lamp, carries out secondary mutagenesis.After mutagenesis completes, static a moment in dark environment, to prevent photoreactivation.
Then, at the enterprising row filter of screening culture medium, cultivate, screening culture medium be take soybean isoflavones as only carbon source, with (NH
4)
2sO
4for nitrogenous source, take and in screening culture medium, form single bacterium colony as suitable extent of dilution.With bacterium colony size, for selecting foundation, select object bacteria.
The aspergillus oryzae that the present invention obtains (Aspergillus oryzae) TJTSW001, can take soybean isoflavones as only carbon source, with (NH
4)
2sO
4for well-grown on the substratum of nitrogenous source, to cultivate three days, its colony diameter can reach 3 centimetres.Further utilize its fermentation Semen Sojae Preparatum, measure its Glycosylase.
Embodiment 1:
Aspergillus oryzae (Aspergillus oryzae) TJTSW001 and the growing state comparison in screening culture medium of its starting strain.
Aspergillus oryzae (Aspergillus oryzae) TJTSW001 and its starting strain are seeded in simultaneously in screening culture medium, (soybean isoflavones is only carbon source, (NH
4)
2sO
4for only nitrogenous source), be placed on the mould medium of 35 ℃ and cultivate, observe its bacterium colony size (referring to table 1).
Table 1
? |
24h |
48h |
72h |
96h |
Aspergillus oryzae TJTSW001 |
1.5cm |
2.0cm |
2.7cm |
3.0cm |
Aspergillus oryzae starting strain |
0.5cm |
0.6cm |
0.9cm |
1.2cm |
Embodiment 2:
Aspergillus oryzae (Aspergillus oryzae) TJTSW001 and starting strain produce glycosidase activity comparison.
Get aspergillus oryzae TJTSW001 and starting strain, they are inoculated in slant tube simultaneously, at 35 ℃, cultivate 5d, after length is good, then use aseptic washing lower surface spore, be inoculated in liquid culture medium the analysis for soybean powder that liquid culture medium is 6%.At 35 ℃, under 180rpm, cultivate 3 days.Be inoculated in solid medium, solid culture consists of corn cob 50, analysis for soybean powder 50 (weight part), and water content is 50%.At 35 ℃, cultivate 3 days, measure its glycosidase activity (referring to table 2).
Table 2
? |
For the first time |
For the second time |
For the third time |
Aspergillus oryzae TJTSW001 |
638U/g |
723U/g |
774U/g |
Starting strain |
289U/g |
276U/g |
323U/g |
Embodiment 3:
Aspergillus oryzae (Aspergillus oryzae) TJTSW001 and starting strain are produced Semen Sojae Preparatum experiment relatively.
Get aspergillus oryzae TJTSW001 and starting strain, they are inoculated in slant tube simultaneously, at 35 ℃, cultivate 5d, after length is good, then use aseptic washing lower surface spore, be inoculated in liquid culture medium the analysis for soybean powder that liquid culture medium is 6%.At 35 ℃, under 180rpm, cultivate 3 days.Using it as liquid state kind.
Get individual 100 grams of mulberry leaf, sweet wormwood, boiling, decocts 3 times, and merging filtrate soaks black soya bean wherein, until concoction blots completely.Then Semen sojae atricolor cooks, and after its cool to room temperature, above-mentioned liquid kind is admixed wherein, covers 4 layers of gauze thereon, to keep moisture.After its surface is covered with " yellow clothes ", take out and be placed in encloses container, at 50 ℃, fermentation 15-20 days.Take out, steam 30 minutes, then dry.Finally measure the content of daidzein in finished product.This experiment is carried out three batches altogether.Its last measurement result is as shown in table 3 below:
Table 3
Embodiment 4:
The test of aspergillus oryzae (Aspergillus oryzae) TJTSW001 mitotic stability.
Aspergillus oryzae (Aspergillus oryzae) TJTSW001 is gone down to posterity 5 times, respectively they are inoculated in slant tube, simultaneously at 35 ℃, cultivate 5d. after length is good, then use aseptic washing lower surface spore, be inoculated in liquid culture medium the analysis for soybean powder that liquid culture medium is 6%.At 35 ℃, under 180rpm, cultivate 3 days.Be inoculated in solid medium, solid culture consists of corn cob 50, analysis for soybean powder 50 (weight part), and water content is 50%.At 35 ℃, cultivate 3 days, measure its glycosidase activity (referring to table 4).
Table 4
? |
The first-generation |
The s-generation |
The third generation |
The 4th generation |
The 5th generation |
Glycosylase U/g |
734 |
746 |
763 |
721 |
706 |
The foregoing is only the preferred embodiment of the present invention, protection scope of the present invention is not limited in above-mentioned embodiment, and every technical scheme that belongs to the principle of the invention all belongs to protection scope of the present invention.For a person skilled in the art, some improvement of carrying out under the prerequisite that does not depart from principle of the present invention, these improvement also should be considered as protection scope of the present invention.