CN102199548A

CN102199548A - Polygonum cuspidatum root endophytic strain G3 for efficiently transforming polydatin into resveratrol

Info

Publication number: CN102199548A
Application number: CN2011101301271A
Authority: CN
Inventors: 易有金; 刘华金; 夏菠
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2011-05-18
Filing date: 2011-05-18
Publication date: 2011-09-28
Anticipated expiration: 2031-05-18
Also published as: CN102199548B

Abstract

The invention discloses a Polygonum cuspidatum root endophytic strain. The strain is named Penicillium oxalicum G3 in a classified way, the strain was collected in China General Microbiological Culture Collection Center on January 17, 2011, and the collection number is CGMCC NO.4558. Polydatin in Polygonum cuspidatum can be transformed into resveratrol by inoculating the strain into a culture medium containing Polygonum cuspidatum boiling extracting solution, so that the polydatin can be directly transformed into the resveratrol through microbial fermentation, the extraction of the polydatin is not required, the cost can be saved, and the pollution to the environment can be reduced.

Description

Efficient converting giant knotweed glycosides is the polygoni cuspidati,radix endophyte G3 of trans-resveratrol

Technical field:

The present invention relates to a kind of giant knotweed endophyte, refer to that especially a kind of common fermentation condition of and giant knotweed can be converted into polygonin the polygoni cuspidati,radix endophyte G3 of trans-resveratrol down.

Background technology:

Resveratrol is a kind of natural radioactivity polyphenol substance in the giant knotweed, has effects such as suppressing tumour, anti-oxidant, Green Tea Extract, and it has been listed in one of anti-cardiovascular, anticancer the most promising medicine, is the later another green cancer-resisting substance of taxol that continues.Resveratrol often exists with the form of polygonin in the giant knotweed, and resveratrol content only is 0.1%-0.2% in the dry giant knotweed rhizome, and polygonin content is about 2%, and polygonin is decomposed into resveratrol under the Glycosylase effect in enteron aisle, bring into play its pharmacological action.

Domestic about more by polygonin in the giant knotweed being transformed with the report that improves resveratrol content, as natural enzymolysis method, complex enzyme zymohydrolysis method, and thick extraction polygonin is carried out enzymolysis with cellulase; There is the scholar to adopt the glucuroide of purification that polygonin is transformed abroad; Also useful acid and alkali hydrolysis method is decomposed polygonin respectively.Above method specificity is low, the operation is more loaded down with trivial details, cost height, transformation efficiency low (1.23%-1.68%), and environment polluted.And, take from natural resource that method extracting effective components such as chemistry is time-consuming, effort, waste resource, therefore, microbe transformation method is applied in the research of natural product gradually.But do not see the relevant report that bacterium transforms giant knotweed in endophyte of plant or the people's enteron aisle that utilizes so far.Therefore bacterium transforms giant knotweed and has very important significance in screening giant knotweed endophyte or the people's enteron aisle.

Summary of the invention:

Technical problem to be solved by this invention is: at above-mentioned the deficiencies in the prior art, provide a kind of polygoni cuspidati,radix endophyte G3, under the common fermentation of this bacterium and giant knotweed, polygonin can be converted into trans-resveratrol.

Polygoni cuspidati,radix endophyte G3 of the present invention, its classification called after penicillium oxalicum (Penicillium oxalicum) G3, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 17th, 2011, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC NO.4558.

The colonial morphology of bacterial strain G3 of the present invention:

Under naked eyes, observe:

Bacterial strain G3 of the present invention on the CYA substratum 28 ℃ cultivate 7d after, about colony diameter 45mm; The peripheral white of bacterium colony; Form a large amount of spores, it is dark green producing spore district color; Colony edge is irregular, no transudate, and no special odor, bacterium colony back side yellow is seen Fig. 1.

Bacterial strain G3 of the present invention on the MEA substratum 28 ℃ cultivate 7d after, diameter reaches 10mm-15mm, the bacterium colony colony edge is irregular, no transudate, no special odor, bacterium colony back side yellow is seen Fig. 2.

The bacterial strain G3 of the present invention limitation of on the G25N substratum, growing, 28 ℃ cultivate 7d after, diameter 7mm-10mm.The peripheral white of bacterium colony, there is a spot of Steel Gray spore the centre, and the bacterium colony back side is light yellow, sees Fig. 3.

Bacterial strain G3 of the present invention is 28 ℃ of cultivation 7d on the CA substratum, diameter 45mm, and colony edge is irregular; White mycelium, central authorities are light green to the edge to deepen greenly, and bacterium colony back side yellow is seen Fig. 4.

With the DNA of SDS method extraction bacterial strain of the present invention, adopt universal primer ITS1/ITS4 to carry out pcr amplification, obtain amplified fragments, the amplified fragments order-checking.Sequence is the comparison of BLAST homology in GenBank, the result shows that the bacterial strain of the present invention and the number of landing are the highest for penicillium oxalicum (Penicillium oxalicum) homology of FJ977097.1, similarity is 100%, in conjunction with colonial morphology and microscopic features result, can judge that bacterial strain of the present invention is a novel species of penicillium oxalicum (Penicillium oxalicum), called after penicillium oxalicum (Penicillium oxalicum) G3.

Bacterial strain G3 of the present invention is inserted in the 10% giant knotweed leach cooking liquid (pH nature), but the polygonin in the common fermentation converting giant knotweed of bacterial strain of the present invention and giant knotweed is a trans-resveratrol that fermentation condition is: bacterial strain spore suspension concentration 1 * 10 ⁶CFU/mL, inoculum size is 10%, and temperature is 27-29 ℃, and the 160-200rpm shaker fermentation is cultivated 84-96h.Preferable fermentation condition is: bacterial strain spore suspension concentration 1 * 10 ⁶CFU/mL, inoculum size is 10%, and temperature is 28 ℃, and rotating speed is 180rpm, and fermentation time is 96h.

So, can directly the polygonin in the giant knotweed be converted into trans-resveratrol, not need to carry out the extraction of polygonin by microbial fermentation, cost-saved, reduce pollution to environment.

Description of drawings:

Fig. 1 is the bacterium colony figure of bacterial strain of the present invention after cultivating 7d on the CYA.

Fig. 2 is the bacterium colony figure of bacterial strain of the present invention after cultivating 7d on the MEA.

Fig. 3 is the bacterium colony figure of bacterial strain of the present invention after cultivating 7d on the G25N.

Fig. 4 is the bacterium colony figure of bacterial strain of the present invention after cultivating 7d on the CA.

Fig. 5 is polygonin standard specimen (40 μ g/ml) HPLC figure (1 refers to the polygonin typical curve among the figure).

Fig. 6 is trans-resveratrol standard specimen (40 μ g/ml) HPLC figure (2 refer to the trans-resveratrol typical curve among the figure).

Fig. 7 is that polygonin, Resveratrol content HPLC detect collection of illustrative plates in the preceding nutrient solution of fermentation.

Wherein: curve 1 refers to polygonin, and curve 2 refers to trans-resveratrol.

Fig. 8 is that polygonin, Resveratrol content HPLC detect collection of illustrative plates in the fermented liquid of fermentation behind the 96h.

Wherein: curve 1 refers to polygonin, and curve 2 refers to trans-resveratrol.

Embodiment:

Embodiment 1

The wild giant knotweed that to gather from Tian Xinping village, Lianyuan, Hunan Province is got root, and is clean with aseptic water washing, then with sterilization filter paper suck dry moisture.(following aseptic technique) 75% alcohol immersion sterilization 3～5min, aseptic water washing 3～4 times, 0.1% mercuric chloride sterilization 30s uses 75% ethanol disinfection 1min again.Root, stem stalk phloem are cut, get xylem is cut into 0.5cm * 0.5cm with sterile scissors fritter.Aseptic water washing 3～4 times grinds to form slurries in the mortar of sterilization.After 10 times of the grinding milk sterilized water dilutions, pipette 1ml and grind diluent, conventional coating on the primary dcreening operation substratum.Parallel 3 times of every kind of material, cultivate 72～120h at 28 ℃ respectively, select the different shape bacterium colony that the grows fine separation of on the PDA flat board, rule, behind 28 ℃ of cultivation 72h, get single bacterium colony and repeat line for 3 times and separate, it is standby that the bacterial strain of the present invention that obtains purifying is transferred to the PDA slant preservation.

Embodiment 2

Adopt the HPLC method to detect polygonin and Resveratrol content in the fermented liquid:

The preparation of reference substance solution and typical curve are drawn

Precision takes by weighing trans-resveratrol and polygonin standard substance 0.2mg in 1mL methyl alcohol, and mixing must contain trans-resveratrol, polygonin 0.2mg/mL reference substance storing solution, and is standby.Accurate reference substance solution 100,200,300,400, the 500 μ L that draw are diluted among the 1mL with methyl alcohol, and mixing is made the reference substance of different concns.Pressing chromatographic condition and measure the peak area integrated value, is ordinate zou with peak area Y, and sample introduction concentration X is an X-coordinate, and the drawing standard curve is seen Fig. 5 and Fig. 6, and calculates regression equation.

The sample solution preparation

Bacterial strain G3 of the present invention is inserted (spore suspension concentration 1 * 10 in the 10% giant knotweed leach cooking liquid (pH nature) ⁶CFU/mL, inoculum size is 10%, liquid amount 100mL/250mL triangular flask), 28 ℃, fermentation culture 96h in the 180rpm shaking table.Behind the centrifugal 10min of fermented liquid 10000rpm, get supernatant liquor 100 μ L, after the 0.45 μ m millipore filtration, add the dilution of 900 μ L methyl alcohol, mixing, standby.

Assay

Precision takes by weighing sample solution, presses the chromatographic condition sample introduction, parallel 3 times, according to polygonin, Resveratrol content in the calculated by peak area sample solution, sees Fig. 8.The HPLC of polygonin, Resveratrol content detects and sees Fig. 7 in the preceding nutrient solution of fermentation, and wherein, polygonin concentration is 44.164 μ g/ml, and resveratrol concentration is 6.364 μ g/ml.

Chromatographic condition: moving phase: methyl alcohol: water (40: 60), chromatographic column: ODS-C ₁₈Post (150mm * 4.6mm, 5 μ m), flow velocity 0.6mL/min, sample size 10 μ L, ultraviolet detection wavelength 306nm, column temperature are 25 ℃.

The result shows: 3 parallel controls are set at every turn, the contrasting colour spectrogram, the absorption peak area of polygonin obviously reduces as can be seen, the absorption peak area of resveratrol obviously increases, this is consistent with the standard substance color atlas for contrast, and by calculating as can be known, polygonin concentration is that 8.375 μ g/ml, resveratrol concentration are 28.879 μ g/ml in the fermented liquid, transformation efficiency is 87.2%, but this common fermentation converting giant knotweed glycosides that has fully proved bacterial strain of the present invention and giant knotweed is a trans-resveratrol.

The culture medium preparation of mentioning herein:

Primary dcreening operation substratum: MgSO ₄0.5g, NaNO ₃5g, FeSO ₄0.01g, KH ₂PO ₄1g, agar 18g adds 10% giant knotweed leach cooking liquid to 1L, the pH nature.

Cha Shi mother liquor (Czapek concentrate): NaNO ₃30g, KCl 5g, MgSO ₄7H ₂O 5g, FeSO ₄7H ₂O 0.1g, ZnSO ₄7H ₂O 0.1g, CuSO ₄5H ₂O 0.05g, distilled water 100mL.

Cha Shi yeast extract paste agar (Czapek Yeast Extract Agar, CYA): K ₂HPO ₄1.0g, Cha Shi mother liquor 10mL, yeast powder 5g, sucrose 30g, agar 15g, distilled water 1L.

Wort agar (Malt Extract Agar, MEA): malt extract 20g, peptone 1.0g, glucose 20g, agar 20g, distilled water 1L.

25% glycerine nitrate agar (25%Glycerol Nitrate Agar, G25N): K ₂HPO ₄0.75g, Cha Shi mother liquor 7.5mL, yeast powder 3.7g, glycerine 250g, agar 12g, distilled water 750mL.

Cha Shi (Czapek Agar, CA): NaNO ₃3g, K ₂HPO ₄1g, MgSO ₄7H ₂O 0.5g, KCl 0.5g, FeSO ₄7H ₂O 0.01g, sucrose 30g, agar 15g, distilled water 1L.

Claims

1. polygoni cuspidati,radix endophyte strain, its classification called after penicillium oxalicum (Penicillium oxalicum) G3, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on January 17th, 2011, and deposit number is CGMCC NO.4558.

2. a fermentation method prepares the method that the converting giant knotweed glycosides is the fermented liquid of trans-resveratrol, it is characterized in that: the fermentation bacterial strain uses therefor is the described bacterial strain of claim 1; The used substratum that ferments is 10% giant knotweed leach cooking liquid, the pH nature; Fermentation condition is: bacterial strain spore suspension concentration 1 * 10 ⁶CFU/mL, inoculum size is 10%, and temperature is 27-29 ℃, and rotating speed is 160-200rpm, and fermentation time is 84-96h.

3. method as claimed in claim 2 is characterized in that: described fermentation condition is: bacterial strain spore suspension concentration 1 * 10 ⁶CFU/mL, inoculum size is 10%, and temperature is 28 ℃, and rotating speed is 180rpm, and fermentation time is 96h.

4. the converting giant knotweed glycosides that obtains as method as described in claim 2 or 3 is the fermented liquid of trans-resveratrol.