CN102628018B - Strain of endophytic fungus of fermenting schisandra chinensis to improve main active components of schisandra chinensis - Google Patents
Strain of endophytic fungus of fermenting schisandra chinensis to improve main active components of schisandra chinensis Download PDFInfo
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- CN102628018B CN102628018B CN 201210126231 CN201210126231A CN102628018B CN 102628018 B CN102628018 B CN 102628018B CN 201210126231 CN201210126231 CN 201210126231 CN 201210126231 A CN201210126231 A CN 201210126231A CN 102628018 B CN102628018 B CN 102628018B
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Abstract
The invention relates to a strain of endophytic fungus, in particular to a strain of endophytic fungus of fermenting schisandra chinensis to improve main active components of schisandra chinensis. The strain of endophytic fungus of fermenting schisandra chinensis to improve main active components of schisandra chinensis is aspergillus niger WJ1, which is preserved in China Center for Type CultureCollection on Feb. 29th, 2012, the preservation number is CCTCC No. M2012044, and the preservation address is Wuhan University in Wuhan. The aspergillus niger WJ1 provided in the invention can substantially improve main active components including schisadrol A, schisantherin A, deoxyschizandrin, schisandrin B, and the like of schisandra chinensis. Thus, in raw material productions aiming at obtaining schisadrol A, schisantherin A, deoxyschizandrin, and schisandrin B, the schisandra chinensis endophytic fungus WJ1 can be selected to ferment schisandra chinensis, thereby improving yield and saving medicine resources.
Description
Technical field
The present invention relates to a strain endogenetic fungus.
Background technology
Schisandra chinensis [Schisandra chinensis (Turcz.) Baill.], the dry mature fruit of magnoliaceae schisandra is not only to have eaten but also the medicine functional health-care food.The activeconstituents of Schisandra chinensis is the lignan component take schisandrin, Wuweizi ester A, deoxyschizandrin, Wuweizisu B etc. as representative; this constituents has significantly anticancer, anti-inflammatory, calmness; anxiety; the protection liver cell; anti-chemical damage, the effect such as antibiotic now are widely used in medicine and food service industry.Because the Schisandra chinensis demand goes up, area of woods reduces and excavating blindly gradually, makes the Schisandra chinensis resource face test, and Schisandra chinensis is the Endangered Medicinal Herb of three grades of focused protections of country at present.
Fermentation technique can change or strengthen some activity of food, some activeconstituents in protection food, and saving resource is for the acquisition of food, pharmaceutical active ingredient provides novel method.Correlative study about the Schisandra chinensis fermentation has no report at present.
Summary of the invention
The invention provides the endogenetic fungus that a strain fermentation Schisandra chinensis improves Schisandra chinensis main active ingredient content.
The present invention's Schisandra chinensis that ferments improves the endogenetic fungus of Schisandra chinensis main active ingredient content, it is aspergillus niger (Aspergillus niger) WJ1, in the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC No:M2012044, the preservation address is Wuhan City Wuhan University, and preservation date is on February 29th, 2012; It is cultivated on the PDA solid medium; mycelia is just white; after become black heavy fleece shape; the back side is colourless, and spherical top capsule is formed on the bacterial strain top, covers one deck metulae and one deck stigma on it comprehensively; upper length has bunchiness chocolate spheric conidium; spore diameter 3.5 μ m, conidial head is spherical, brown-black.
In the present invention, the fermentation Schisandra chinensis improves the endogenetic fungus of Schisandra chinensis main active ingredient content, it is aspergillus niger (Aspergillus niger) WJ1, according to " classification of fungi ", " fungi identification handbook " and " Dendrochium diagram ", colony characteristics and the spore shape of visible WJ1 bacterial strain are very close with the aspergillus tubigensis feature.
In the present invention, the fermentation Schisandra chinensis improves the endogenetic fungus of Schisandra chinensis main active ingredient content, it is aspergillus niger (Aspergillus niger) WJ1, its ITS sequence is committed to the NCBI webpage, search for the sequence high with resulting sequence similarity by Blast, and by MEGA5.03 software building systematic evolution tree, the similarity of the ITS sequence of WJ1 bacterial strain and Aspergillus bacterium aspergillus niger (Aspergillus niger) bacterial strain has reached more than 99%, combining form is learned observations, determines that the WJ1 bacterial strain is aspergillus niger (Aspergillus niger).
The present invention's Schisandra chinensis that ferments improves the endogenetic fungus of Schisandra chinensis main active ingredient content, it is aspergillus niger (Aspergillus niger) WJ1, in the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC No:M2012044, the preservation address is Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
The present invention adopts and separates from the endogenetic fungus WJ1 of Schisandra chinensis fermentation Schisandra chinensis, 4 kinds of main lignans are as index in the Schisandra chinensis, the relatively dynamic change of these 4 kinds of main active ingredient before and after its fermentation, be intended to strengthen the activity of Schisandra chinensis and improve lignans content, for efficiently utilizing Schisandra chinensis, save the medicine source, the development of new functional health-care food provides foundation.
The present invention's Schisandra chinensis that ferments improves the endogenetic fungus of Schisandra chinensis main active ingredient content, it is aspergillus niger (Aspergillus niger) WJ1, it can make the main active ingredient such as schisandrin in Schisandra chinensis, Wuweizi ester A, deoxyschizandrin and Wuweizisu B significantly improve, therefore can select shizandra berry endogenetic fungus WJ1 fermentation shizandra berry to improve yield to obtain schisandrin, Wuweizi ester A, deoxyschizandrin and Wuweizisu B in the raw material production of purpose, save the medicine source.
Description of drawings
Fig. 1 is the colonial morphology figure of aspergillus niger in embodiment one (Aspergillus niger) WJ1;
Fig. 2 is the colonial morphology figure of aspergillus niger in embodiment one (Aspergillus niger) WJ1;
Fig. 3 is the colonial morphology figure of aspergillus niger in embodiment one (Aspergillus niger) WJ1;
Fig. 4 is the electrophorogram of the pcr amplification of aspergillus niger in embodiment one (Aspergillus niger) WJ1, and wherein the M swimming lane represents Marker, and the WJ1 swimming lane represents WJ1;
Fig. 5 is the phyletic evolution tree graph of aspergillus niger in embodiment one (Aspergillus niger) WJ1;
Fig. 6 mixes the HPLC color atlas of reference substance in embodiment one, wherein 1 represents schisandrin, 2 expression Wuweizi ester As, 3 expression deoxyschizandrins and 4 expression Wuweizisu Bs;
Fig. 7 is the HPLC color atlas of Schisandra chinensis in embodiment one, and wherein 1 represents schisandrin, 2 expression Wuweizi ester As, 3 expression deoxyschizandrins and 4 expression Wuweizisu Bs;
Fig. 8 is the shizandra berry HPLC color atlas after the WJ1 fermentation in embodiment one, wherein 1 expression schisandrin, 2 expression Wuweizi ester As, 3 expression deoxyschizandrins and 4 expression Wuweizisu Bs.
Embodiment
Embodiment one: present embodiment fermentation Schisandra chinensis improves the endogenetic fungus of Schisandra chinensis main active ingredient content, it is aspergillus niger (Aspergillus niger) WJ1, in the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC No:M2012044, the preservation address is Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
Aspergillus niger in present embodiment (Aspergillus niger) WJ1; it is cultivated on the PDA solid medium; mycelia be just white, after become black heavy fleece shape, the back side is colourless; spherical top capsule is formed on the bacterial strain top; cover one deck metulae and one deck stigma on it, upper length has bunchiness chocolate spheric conidium, spore diameter 3.5 μ m comprehensively; conidial head is spherical, brown-black (as shown in Fig. 1,2 and 3).
Aspergillus niger in present embodiment (Aspergillus niger) WJ1, its growth temperature is 28 ℃, growth pH value nature.
Aspergillus niger in present embodiment (Aspergillus niger) WJ1, this strains separation is from the rhizome of healthy medicinal plant Schisandra chinensis plant, pick up from Maoershan Mountaindistrict In Heilongjiang Province, and being accredited as through the Fu Kezhi researcher of Heilongjiang Province Academy of Traditional Chinese Medicine Pharmaceutical Manufacturing Plant the plant that magnoliaceae schisandra belongs to Schisandra Chinensis (Turcz.) Baill., sample is existing in Heilongjiang University's Life Science College bio-pharmaceuticals laboratory; It carries out separation and Culture according to the following steps:
(1) pretreatment: after rinsing well with tap water, the rhizome of choosing healthy Schisandra chinensis with the aseptic filter paper suck dry moisture, is cut into the 0.5cm segment, and standby;
(2) surface sterilization: the rhizome with healthy Schisandra chinensis in aseptic super clean bench carries out surface sterilization as follows: 75% alcohol-pickled 3～5min-2% clorox soaks the 15min-aseptic water washing 4 times.With the aseptic operation cutter, sample is cut from the centre, be placed in respectively potato solid separation culture medium and potato liquid separation culture medium, cultivate in 28 ℃ of constant incubators and shaking table, after its edge and top grew mycelia, in time picking colony was transferred in corresponding substratum;
(3) negative control: get the sterilized water coating culture plate (potato solid separation culture medium) of last flushing Schisandra chinensis or get appropriate this washing fluid and pour in the potato liquid separation culture medium and compare, experiment material after separately sterilizing compares in a week dull and stereotyped (potato solid separation culture medium) upper rolling, cultivates under identical condition;
(4) yeast culture: the experiment material after sterilizing is placed in respectively potato solid separation culture medium and potato liquid separation culture medium, cultivate 3-7d in 28 ℃ of incubators, treat to grow thalline around experiment material, the picking thalline changes the separation and purification of repeatedly ruling on flat board (potato solid separation culture medium) over to, bacterial strain after purifying is cultivated on the inclined-plane solid medium, and 4 ℃ save backup.
The potato liquid separation culture medium:
Potato culture (PDA): every L is comprised of the potato of 200g, the sucrose of 20g and the water of surplus, pH nature, 121 ℃ of autoclaving 30min; Wherein peeling potatoes, be cut into small pieces and boil 30min, with 6 layers of filtered through gauze.
The potato solid separation culture medium:
Potato culture (PDA): every L is by the potato of 200g, the sucrose of 20g, and the agar powder of 16g and the water of surplus form, pH nature, 121 ℃ of autoclaving 30min; Wherein peeling potatoes, be cut into small pieces and boil 30min, with 6 layers of filtered through gauze.
Inclined-plane solid medium: get the 5mL solid potato culture medium and be loaded in test tube, after 121 ℃ of autoclaving 30min, be paved into the inclined-plane cooling, in order to preserving bacterial classification.
Test sees Table 1 with key instrument equipment and reagent;
Table 1
| Instrument | Model | Production unit |
| The airbath vibrator | HZQ-C | Dongming, Harbin City Medical Instruments factory |
| The precise electronic balance | PL303 | Plum Teller-Tuo benefit Instr Ltd. |
| Electro-heating standing-temperature cultivator | DNP9082 | Shanghai accurate experimental installation company limited |
| Electric heating constant-temperature blowing drying box | DHG-9140 | Shanghai accurate experimental installation company limited |
| The Portable pressure steam sterilizing pot | YXQ-SG46 | Zhenghai prosperous medicine equipment of gold company limited |
| Bechtop | SZX | Nantong scientific instrument factory |
| Electrothermal oven | 800W | Yongfeng, Harbin City electrical apparatus factory |
| Agar powder | Analytical pure | Haiyang Chemical Plant, Qingdao |
| NaCl | Analytical pure | Tianjin recovery fine chemistry industry institute |
| Sucrose | Analytical pure | Tianjin recovery fine chemistry industry institute |
| 95% ethanol | Analytical pure | Tianjin Jin Dong days positive fine chemistry chemical reagent works |
Result: isolate endogenetic fungus WJ1 from the Schisandra chinensis rhizome, and all grow without any bacterium in the dull and stereotyped and contrast liquid nutrient medium of contrast in negative control, repeatedly repeat all so, prove that the bacterium of assigning to is plant endogenesis epiphyte, rather than surperficial epiphyte.
Molecular Identification: WJ1 fermented liquid suction filtration gets mycelium with 25% ethanol rinsing 2 times, the deionized water rinsing of sterilization 2 times, the centrifugal supernatant liquor that goes, extract the pcr amplification that carries out the purpose fragment after total DNA, the PCR product that will contain target stripe all carries out point sample again, agarose gel electrophoresis with 2%, with scalper, the purpose band is downcut under ultraviolet lamp, reclaiming test kit with DNA glue reclaims, preparation competence Bacillus coli cells, with the PCR product with after pMD 18-T carrier is connected, connecting product joins in competent cell, carry out bacterium colony PCR checking.The PCR that picking list bacterium colony carries out the intestinal bacteria transformant detects, prove the purpose fragment successfully transforms enter in competent cell after, will clone positive strain and serve extra large life work biotechnology Services Co., Ltd and check order.The nucleotides sequence of measuring is listed in ncbi database to be used BLAST and analyzes and carry out homology relatively, and select the 18S rDNA sequence application software MEGA5.03 phylogenetic tree construction of corresponding kind representative strain according to the ultimate principle of molecular systematics research, determine the classification position of aimed strain.
The WJ1 genome is after the extraction of work UNIQ-10 pillar fungal gene group extraction agent box is given birth in Shanghai, after adopting pcr amplification, the gel imaging result as shown in Figure 4, obtain an amplified fragments near 1300bp (base sequence is seen SEQ ID NO:1), prove successfully to extract its genomic dna from the WJ1 bacterial strain.
Be committed to the NCBI webpage according to the ITS sequence of WJ1 bacterial strain, search for the sequence high with resulting sequence similarity by Blast, and by MEGA5.03 software building systematic evolution tree (as shown in Figure 5), the similarity of the ITS sequence of WJ1 bacterial strain and Aspergillus bacterium aspergillus niger (Aspergillus niger) bacterial strain has reached more than 99%, combining form is learned observations, determines that the WJ1 bacterial strain is aspergillus niger (Aspergillus niger).
The dynamic change of HPLC method mensuration endogenetic fungus WJ1 to its effective constituent before and after the Schisandra chinensis fermentation:
1. the preparation of trial-product and reference substance solution
(1) chromatographic condition chromatographic column: Venusil XBP-C
18Post (post 4.6mm * 250mm, 5 μ m, USA); Moving phase: methyl alcohol-acetonitrile-water (33: 33: 34); Flow velocity: 1mLmin
-1Column temperature: 25 ℃; Detect wavelength: 250nm; Sample size: 10 μ L;
(2) preparation of reference substance solution
The preparation of reference substance solution: precision takes schisandrin, Wuweizi ester A, deoxyschizandrin and Wuweizisu B standard substance, and each is appropriate, makes respectively mass concentration take methyl alcohol as solvent and is the reference substance storing solution of 1.0mg/mL.Above-mentioned each 1mL of reference substance storing solution of accurate measuring, making mass concentration is the mixing reference substance solution that is 0.25mg/mL;
(3) need testing solution preparation
The preparation of fermented sample: get dry Schisandra chinensis and pulverize, cross 60 mesh sieves.Accurately take the Schisandra chinensis 15g after pulverizing, be placed in Erlenmeyer flask, add water according to the solid-liquid ratio of 1: 5, after sealing, in 121 ℃ of autoclaving 30min, take out, as substrate;
Endogenetic fungus WJ1 is being activated in the PDA substratum separately, adding sterilized water in the endogenetic fungus WJ1 that has activated, making 1 * 10
7The bacteria suspension of CFU/mL.The 5mL bacteria suspension is joined in substrate after sterilization; Separately get substrate and add the 5mL sterilized water as the blank sample, each 10 parts of parallel sample are put into shaking table in 28 ℃ with all samples, cultivate 15 days, take out, and freeze-drying, standby.
The preparation of positive reference substance solution: get dry Schisandra chinensis fruit, pulverize, cross 40 mesh sieves.Precision takes 3 parts of each 1.0g of Schisandra chinensis powder, accurately adds 10mL methyl alcohol, weighs, and supersound extraction 60min takes out, and lets cool, and supplies the solvent of less loss with methyl alcohol, with after 0.45 μ m filtering with microporous membrane as positive reference substance solution.
The preparation of fermented sample need testing solution: precision takes the Schisandra chinensis sample 1.0g after endogenetic fungus WJ1 fermentation, precision adds 10mL methyl alcohol, supersound extraction 60min, take out, let cool, supply the solvent of less loss with methyl alcohol, with 0.45 μ m filtering with microporous membrane, i.e. Schisandra chinensis fermented sample need testing solution.
The preparation of blank product solution: get blank sample 1.0g, precision adds 10mL methyl alcohol, and supersound extraction 60min takes out, and lets cool, and supplies the solvent of less loss with methyl alcohol, with 0.45 μ m filtering with microporous membrane, i.e. blank product solution.
The preparation of fermented liquid reference substance solution: precision measures each 100mL of Schisandra chinensis endogenetic fungus WJ1 fermented liquid, and rotary evaporation is concentrated into 10mL, with 0.45 μ m filtering with microporous membrane, is the fermented liquid reference substance solution.
(4) sample determination is under " (1) " chromatographic condition, and precision is drawn 10 μ L (three times parallel) reference substance solution and each need testing solution respectively, and sample introduction records peak area, calculates content by external standard method.
2. detected result
1. the separation of shizandra berry effective constituent under chromatographic condition
Under " (1) " chromatographic condition, in trial-product, schisandrin, Wuweizi ester A, deoxyschizandrin and Wuweizisu B are all separated preferably, not only the retention time of its chromatographic peak is consistent with reference substance, and each chromatographic peak has also all obtained confirming preferably through standard addition method, the results are shown in Figure 6, Fig. 7, Fig. 8.
2. the dynamic change of endogenetic fungus WJ1 to its effective constituent before and after the Schisandra chinensis fermentation
The content analytical results of Schisandra chinensis main 4 kinds of lignanoid's activeconstituentss before and after endogenetic fungus WJ1 fermentation sees Table 2.
Table 2
Annotate: compare * P＜0.05, * * P＜0.01 with positive (crude drug) control group;
Compare △ P＜0.05 with the blank group, △ △ P＜0.01.
Conclusion: adopt Schisandra chinensis endogenetic fungus WJ1 that Schisandra chinensis is fermented, the content of 4 kinds of main active ingredient in Schisandra chinensis before and after the fermentation of HPLC method mensuration Schisandra chinensis, its result shows, shizandra berry endogenetic fungus WJ1 can make in Schisandra chinensis that in 4, main lignanoid activeconstituents is significantly improved to a certain extent, illustrate to adopt lignans in endogenetic fungus WJ1 fermentation Schisandra chinensis High-efficient Production Schisandra chinensis, saving medicine source to have directive significance.
Claims (1)
1. a strain fermentation Schisandra chinensis improves aspergillus niger (Aspergillus niger) the WJ1 bacterial strain of Schisandra chinensis main active ingredient content, its preserving number is CCTCC No:M2012044, in the center preservation of Chinese Typical Representative culture collection, the preservation address is Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
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