CN109234175B

CN109234175B - Fusarium oxysporum DCLZJ-4 strain for producing paris polyphylla saponin and application thereof

Info

Publication number: CN109234175B
Application number: CN201811347659.9A
Authority: CN
Inventors: 朱晓松; 石安华; 陈文慧; 谭丽萍; 刘录
Original assignee: Yunnan University of Traditional Chinese Medicine TCM
Current assignee: Yunnan University of Traditional Chinese Medicine TCM
Priority date: 2018-11-13
Filing date: 2018-11-13
Publication date: 2021-08-13
Anticipated expiration: 2038-11-13
Also published as: CN109234175A

Abstract

The invention relates to a Fusarium oxysporum DCLZJ-4 strain for producing paris polyphylla saponin, which is obtained by separation and culture of paris polyphylla by adopting a method for separating and purifying endophytic fungi, and is identified as Fusarium sp by microorganisms. The strain is preserved in China general microbiological culture Collection center, and the address is as follows: china, Beijing, institute of sciences, China; the zip code 100101 has a preservation date of 2017, 10 months and 30 days, and the preservation number is as follows: CGMCC No. 14791; the ITS rDNA sequence of the strain is shown in SEQ ID NO. 1. The strain can be used for preparing the paris polyphylla saponin by fermentation. The invention provides a fusarium oxysporum strain with stable properties, simple culture medium and good growth for large-scale fermentation preparation of the paris polyphylla saponin, and a practical, simple and convenient fermentation and separation extraction process, and extract obtained by extracting and concentrating fermentation liquor can be used as an active ingredient of hemostatic drugs or a medicinal carrier.

Description

Fusarium oxysporum DCLZJ-4 strain for producing paris polyphylla saponin and application thereof

Technical Field

The invention relates to a fusarium oxysporum strain capable of producing paris polyphylla saponin, and belongs to the technical field of microorganisms.

Background

The plant endophyte has strong metabolic function, can produce active ingredients which are the same as or similar to those of a host, is widely present in various plants, and can be cultured and separated from the plants through proper culture conditions. At present, the research of plant endophytes is gradually paid attention, and various endophytes of different plants are discovered in succession. The diversity of the endophyte species also endows the endophyte with the diversity of metabolites, and the method of utilizing the endophyte fermentation for mining new resources has great potential. With the gradual research of medicinal plant endophytes, the endophytes capable of metabolizing to generate corresponding active ingredients are utilized to produce novel, efficient and low-cost active medicaments, the current situation that rare traditional Chinese medicine plant resources are in short supply is solved, and the method becomes the key point of medicinal plant endophyte research.

The Chinese medicine paris polyphylla has obvious pharmacological activity and wide clinical application, is one of the main raw materials of a plurality of Chinese patent medicines such as Yunnan white drug powder, Gongxuening and the like, but the paris polyphylla has long growth period and slow growth speed, the introduction and domestication have no breakthrough progress, the price is high, the damage of the genuine wild resources is serious, and the paris polyphylla resources face the danger of exhaustion. Research shows that a large amount of endophytes exist in the paris polyphylla plant and can produce active metabolites. At present, some scholars screen some endophytes producing saponin or analogues thereof, but the number and the types of the strains are not large, and some new strains producing saponin or sapogenin are continuously screened, so that the method has important significance for deeply developing the research of the way of producing saponin or sapogenin by microbial fermentation. Screening out endophyte capable of metabolizing to produce paris saponin from paris polyphylla plant body provides a new way for solving the current situation of paris polyphylla shortage.

Wang Q collects endophytic fungi in Yunnan rhizoma paridis of Songming, Baoshan, Heqing, etc., and separates 749 endophytic fungi from roots, tubers and leaves, and the Weijuan, etc. separates 44 endophytic bacteria and 40 endophytic bacteria from wild Yunnan rhizoma paridis and artificially cultivated Yunnan rhizoma paridis, respectively. Zhang Peng et al found 1 endogenous Enterobacter cloacae capable of producing Paris saponin I and 2 endogenous Escherichia coli and Bacillus amyloliquefaciens capable of producing Paris saponin VI. The endophytic bacteria of Paris polyphylla which can produce steroid saponin are also separated from Zhao Ming, Zhang Xiaojie, Cao Xiaodong, etc. The variety of the paris polyphylla endophyte is various, the paris polyphylla endophyte has strong biological activity and can produce saponin compounds.

The paris polyphylla endophyte has been paid attention to in recent years, but researches on paris polyphylla endophytes, particularly components capable of metabolizing to produce corresponding steroid saponins, are still relatively few, most of the researches are concentrated on paris polyphylla, the researches on paris polyphylla endophytes are also mainly concentrated on the aspect of biological diversity, and the researches on the paris polyphylla saponin metabolites produced by the endophytes are also few.

Disclosure of Invention

The invention solves the problems in the background technology and provides a fusarium oxysporum DCLZJ-4 strain for producing various paridis saponins.

The invention aims to provide a fusarium oxysporum DCLZJ-4 strain for producing paris polyphylla saponin, a preparation method, a fermentation culture method and application of an extract prepared from fermentation liquor of the fusarium oxysporum DCLZJ-4 strain in preparation of medicines for treating/preventing hemostasis.

The object of the invention is achieved in that,

a Fusarium oxysporum DCLZJ-4 strain with preservation number of CGMCC No.14791 is provided.

The preparation method of fusarium oxysporum DCLZJ-4 strain is characterized by comprising the steps of cleaning, disinfecting, inoculating, separating, culturing and storing, and comprises the following specific steps:

a) cleaning and disinfecting: cleaning collected fresh paris polyphylla tubers, removing necrotic hard parts on the surfaces, carrying out surface disinfection for 20min by using 75% ethanol, and then cleaning for 3-5 times by using sterile water;

b) inoculation: putting the rhizoma paridis tubers treated in the step a) into a mortar which is sterilized for later use in advance, adding 5mL of sterile water, and grinding into white suspension; taking 300 mu L of turbid liquid by a liquid transfer device, putting the turbid liquid into each PDA culture medium flat plate, and uniformly coating the turbid liquid by using a sterile coating rod; photographing and recording in 3 rd, 5 th, 7 th and 9 th days, if the fungus grows on the flat plate, separating, and sterilizing;

c) inoculation: transversely cutting the rhizoma paridis tubers treated in the step a) into small slices of 1cm2, soaking for 3-5 min by using 75% alcohol, cleaning for 2-3 times by using sterile water, washing out residual alcohol, soaking for about 5min by using sterile water, taking out, and sucking out surface water by using sterilized filter paper; attaching the processed small blocks to a PDA solid flat plate, and placing 5 pieces on each flat plate; photographing and recording in 3 rd, 5 th, 7 th and 9 th days, if the fungus grows on the flat plate, separating, and sterilizing;

d) separation: culturing the PDA culture medium plate obtained in the step b) or the step c) in a constant temperature incubator at 28 ℃ for 5-9 days, observing in time, inoculating different fungi on the PDA solid culture medium plate by using an inoculating needle, and repeatedly purifying until a single bacterial colony is obtained;

e) culturing and preserving: picking the purified fungus on the plate into a solid slant culture medium test tube filled with PDA, culturing at 28 deg.C for 5d, adding glycerol, plugging with a silica gel plug, sealing with a sealing film, and storing in an ultra-low temperature refrigerator at-80 deg.C.

A fermentation culture method of fusarium oxysporum DCLZJ-4 strain is characterized in that the strain is inoculated into 50ml of PDA liquid culture medium, activated and cultured in a constant temperature shaking table at 28 ℃ for 150r/min, 1ml of activated strain is taken to be fermented in a triangular flask filled with the PDA liquid culture medium after 24 hours, the liquid filling amount is 100ml/250ml, and shaking table culture at 28 ℃ and 150r/min is carried out for 7 d; after the fermentation culture, collecting fermentation liquor.

Every 1000ml of PDA culture medium consists of 300g of potatoes and 18g of cane sugar.

An extract containing multiple paridis saponins is characterized in that fermentation liquor of fusarium oxysporum DCLZJ-4 strain is used as a raw material, n-butyl alcohol is used for extraction for three times, and the n-butyl alcohol is volatilized to obtain the extract.

The application of extract containing multiple rhizoma paridis saponins in preparing medicine for treating/preventing hemostasis is provided.

The dosage form of the medicine is oral dosage form or intravenous injection.

The dosage forms of the medicine are tablets, hard capsules, soft capsules, powder, pills and granules.

A pharmaceutical composition contains multiple rhizoma paridis saponin extracts as active ingredients or medicinal carriers.

The inventor selects a novel strain, the strain is named as DCLZJ-4 and belongs to Fusarium oxysporum (Fusarium oxysporum), the strain is preserved in the China general microbiological culture Collection center, and the address is as follows: china, Beijing, institute of sciences, China; the zip code 100101 has a preservation date of 2017, 10 months and 30 days, and the preservation number is as follows: CGMCC No. 14791; the ITS rDNA sequence of the strain is shown in SEQ ID NO. 1.

The strain can be used for preparing the paris polyphylla saponin by fermentation. The culture medium formula adopted by microbial fermentation is a PDA culture medium: 300g of potatoes and 18g of cane sugar. The optimal culture conditions are as follows: the pH value is 7.0, the culture temperature is 28 ℃, and the rotating speed of a shaking table is 150 r/min.

Compared with the prior art, the invention has the following advantages: the fusarium oxysporum DCLZJ-4 strain provided by the invention can be metabolized to produce various paridis saponins. The strain provided by the invention can be subjected to large-scale fermentation culture in a short period, has low fermentation cost and is not limited by conditions such as time domain, season and the like, so that the current situation that the natural resources of the paris polyphylla are increasingly deficient can be solved through a microbial fermentation way, and the long-term sustainable utilization of the medicinal resources of the paris polyphylla is ensured.

Drawings

FIG. 1 is the colony front of Fusarium oxysporum strain DCLZJ-4.

FIG. 2 is the back side of a colony of Fusarium oxysporum strain DCLZJ-4.

FIG. 3 is a thin layer chromatogram of metabolites of Fusarium oxysporum strain DCLZJ-4.

FIG. 4 is a total ion diagram of mixed LC-MS chromatogram of standard substances of rhizoma paridis saponin I, II, VI and VII.

FIG. 5 is the first-order mass spectrum of the standard substance of paris polyphylla saponin I, II, VI and VII mixed liquid chromatography-mass spectrometry.

FIG. 6 is the second-order mass spectrum of the standard substance of paris polyphylla saponin I, II, VI and VII mixed liquid chromatography-mass spectrometry.

FIG. 7 is the first-order mass spectrum of the standard substance of paris polyphylla saponin II of paris polyphylla saponin I, II, VI and VII mixed liquid chromatography-mass spectrometry.

FIG. 8 is the second-order mass spectrum of the standard substance of Paris saponin II of the standard substance of Paris saponin I, II, VI, VII by mixed liquid chromatography-mass spectrometry.

FIG. 9 is the first-order mass spectrum of the standard substance of Paris saponin VI of the standard substance of Paris saponin I, II, VI, VII by mixed liquid chromatography-mass spectrometry.

FIG. 10 is the second-order mass spectrum of the standard substance of Paris saponin VI of the standard substance of Paris saponin I, II, VI, VII by mixed liquid chromatography-mass spectrometry.

FIG. 11 is the first-order mass spectrum of the paris saponin VII standard substance of paris saponin I, II, VI and VII standard substance mixed liquid chromatography-mass spectrometry.

FIG. 12 is the second-order mass spectrum of the paris saponin VII standard substance of paris saponin I, II, VI, VII standard substance mixed liquid chromatography-mass spectrometry.

FIG. 13 is the mass spectrum of the Paris polyphylla saponin I in the liquid chromatography mass spectrometry of the metabolite of the Fusarium oxysporum strain DCLZJ-4.

FIG. 14 is ion diagram of Paris saponin I ion chromatogram of metabolite of Fusarium oxysporum strain DCLZJ-4.

FIG. 15 is the mass spectrum of the Paris saponin II in the liquid chromatography-mass spectrometry of the metabolite of Fusarium oxysporum DCLZJ-4 strain.

FIG. 16 is ion diagram of the metabolite LC-MS of Fusarium oxysporum strain DCLZJ-4 showing the ion diagram of rhizoma paridis saponin II.

FIG. 17 is mass spectrum of paris polyphylla saponin VI in liquid chromatography of metabolite of fusarium oxysporum DCLZJ-4 strain.

FIG. 18 is the ion diagram of the paris polyphylla saponin VI in the liquid chromatography mass spectrometry of the metabolite of the fusarium oxysporum strain DCLZJ-4.

FIG. 19 is the mass spectrum of paris polyphylla saponin VII in the liquid chromatography mass spectrometry of the metabolite of fusarium oxysporum DCLZJ-4 strain.

FIG. 20 is ion diagram of paris polyphylla saponin VII in fusarium oxysporum DCLZJ-4 strain metabolite liquid chromatography.

Description of preservation of biological Material

Preservation of the strains: the strain is preserved in China general microbiological culture Collection center, and the address is as follows: china, Beijing, institute of sciences, China; the zip code 100101 has a preservation date of 2017, 10 months and 30 days, and the preservation number is as follows: CGMCC No. 14791; the ITS rDNA sequence of the strain is shown in SEQ ID NO. 1.

Detailed Description

The present invention will be described in detail with reference to specific examples, but the scope of the present invention is not limited to the examples.

d) separation: culturing the PDA solid plate culture medium obtained in the step b) or the step c) in a constant temperature incubator at 28 ℃ for 5-9 days, observing in time, inoculating different fungi on a PDA solid plate culture medium by using an inoculating needle, and repeatedly purifying until a single bacterial colony is obtained;

e) culturing and preserving: picking the purified fungus on the plate into a slant solid culture medium test tube filled with PDA, culturing at 28 deg.C for 5d, adding glycerol, plugging with a silica gel plug, sealing with a sealing film, and storing in an ultra-low temperature refrigerator at-80 deg.C.

The PDA culture medium consists of 300g of potatoes and 18g of cane sugar.

The dosage form of the medicine is oral dosage form or intravenous injection.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail by examples and experimental data. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of the invention.

Example 1

Separating and culturing strains:

cleaning collected fresh paris polyphylla tubers, removing necrotic hard parts on the surfaces, carrying out surface disinfection for 20min by using 75% ethanol, then washing for 3-5 times by using sterile water, putting into a mortar which is sterilized for later use in advance, adding 5mL of sterile water, and grinding into white suspension. And (3) taking 300 mu L of turbid liquid by using a pipette, uniformly coating the turbid liquid in each PDA culture medium plate by using a sterile coating rod, culturing the turbid liquid for 5-9 days in a constant-temperature incubator at 28 ℃, observing in time, inoculating different fungi on the PDA solid culture medium plate by using an inoculating needle, and repeatedly purifying until a single bacterial colony is obtained. And photographing and recording at 3 rd, 5 th, 7 th and 9 th days, if the fungus grows out of the plate, the fungus is not separated, and sterilizing.

Meanwhile, cleaning tubers with disinfected surfaces, transversely cutting the tubers into small slices of 1cm2, soaking the small slices in 75% alcohol for 3-5 min, then cleaning the small slices with sterile water for 2-3 times, washing off residual alcohol, finally soaking the small slices in the sterile water for about 5min, taking out the small slices, and sucking the surface water with sterilized filter paper. And (3) sticking the treated small blocks to a PDA solid plate, placing 5 pieces on each plate, culturing for 5-9 days in a constant-temperature incubator at 28 ℃, observing in time, inoculating different fungi on the PDA solid culture medium plate by using an inoculating needle, and repeatedly purifying until a single bacterial colony is obtained. And photographing and recording at 3 rd, 5 th, 7 th and 9 th days, if the fungus grows out of the plate, the fungus is not separated, and sterilizing.

Picking the purified fungi on the flat plate into a slant solid culture medium test tube filled with PDA, culturing at the constant temperature of 28 ℃ for 5 days, adding glycerol, sealing with a sealing film, and storing in an ultra-low temperature refrigerator of-80 ℃, wherein at least 3 parts of each strain is stored.

FIG. 1 is the colony front of Fusarium oxysporum strain DCLZJ-4.

FIG. 2 is the back side of a colony of Fusarium oxysporum strain DCLZJ-4.

Example 2

Fermentation culture of the strain:

inoculating the preserved strain into 50ml of PDA liquid culture medium, performing activation culture at a constant temperature of 28 ℃ by a shaking table at 150r/min, taking 1ml of activated strain after 24 hours, fermenting in a triangular flask filled with the PDA liquid culture medium, filling 100ml/250ml of liquid, and performing shaking culture at 28 ℃ and 150r/min for 7 d. After the fermentation culture, collecting fermentation liquor.

Example 3

Screening of strains:

extracting each strain fermentation liquor sample with n-butanol for three times, each time 100ml, volatilizing n-butanol, dissolving with methanol to obtain metabolite, and detecting rhizoma paridis saponin I, II, VI, and VII in the metabolite by silica gel thin layer chromatography. The developing agent is a (chloroform-methanol-water) system, the color developing agent is phosphomolybdic acid, and the fungi capable of producing the steroid saponins are obtained by screening.

FIG. 3 is a thin layer chromatogram of metabolites of Fusarium oxysporum strain DCLZJ-4. As can be seen from FIG. 3, the shift of many blue spots is consistent between the fermentation broth (Zuo-second) and the compound (Zuo-first) for extracting the saponins from the rhizoma paridis standard crude drug with methanol, and it is presumed that there are many saponins in the metabolite of the strain, and the identification by LC-MS is required to verify whether the saponins are true saponins.

Example 4

Detecting the fungus metabolite capable of producing steroid saponin obtained in the example 3 by using a liquid chromatography-mass spectrometry, and comparing a spectrogram and data thereof with standard data to roughly determine whether a sample contains corresponding paris saponin.

And mixing the paris polyphylla saponin I, II, VI and VII standard substances for liquid chromatography-mass spectrometry analysis. Fig. 4 is a total ion diagram of a liquid chromatography-mass spectrometry chromatogram, fig. 5 and 6 are a primary mass spectrum and a secondary mass spectrum of a paris saponin I standard product, fig. 7 and 8 are a primary mass spectrum and a secondary mass spectrum of a paris saponin II standard product, fig. 9 and 10 are a primary mass spectrum and a secondary mass spectrum of a paris saponin VI standard product, and fig. 11 and 12 are a primary mass spectrum and a secondary mass spectrum of a paris saponin VII standard product.

The metabolite of the fusarium oxysporum strain DCLZJ-4 obtained in example 3 is subjected to liquid chromatography-mass spectrometry, wherein FIGS. 13 and 14 are a mass spectrum and an ion map of the paris polyphylla saponin I, FIGS. 15 and 16 are a mass spectrum and an ion map of the paris polyphylla saponin II, FIGS. 17 and 18 are a mass spectrum and an ion map of the paris polyphylla saponin VI, and FIGS. 19 and 20 are a mass spectrum and an ion map of the paris polyphylla saponin VII.

As shown in FIGS. 3-20, metabolites of Fusarium oxysporum strain DCLZJ-4 contain parinuin I, II, VI, VII.

The preserved fusarium oxysporum DCLZJ-4 strain is subjected to fermentation culture and post-treatment according to the methods of the embodiment 2 and the embodiment 3 to obtain another 7 parts of fusarium oxysporum DCLZJ-4 strain metabolite samples, and the results of the chromatographic analysis of liquid chromatography-mass spectrometry respectively show that the samples all contain the paridis saponins I, II, VI and VII.

Example 5

The strain is subjected to molecular identification. An upstream primer: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), downstream primer: ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The sequencing result is subjected to Blast similarity analysis in a GenBank nucleic acid database, and through Blast sequence comparison and evolutionary tree analysis, the homology of the DCLZJ-4 strain and Fusarium oxysporum is 99.0 percent, and the strain is determined to be Fusarium oxysporum.

Example 6

Optimizing fermentation conditions: the components of the fermentation medium of the microorganism must meet the requirement of rapid growth and propagation of the strain, and the optimal fermentation culture conditions can ensure the large accumulation of secondary metabolites. The invention optimizes the fermentation medium components of the DCLZJ-4 strain through single factor and orthogonal test experiments, and the optimized optimal medium formula is as follows: after the components and the corresponding concentrations of the culture medium are determined, the optimum initial pH value, the culture temperature and the shaking table rotating speed of the paris polyphylla saponin produced by the metabolism of the strain are further optimized, and finally the optimum culture medium of the DCLZJ-4 strain is determined as follows: 300g of potatoes and 18g of cane sugar. The optimal culture conditions are as follows: the pH value is 7.0, the culture temperature is 28 ℃, and the rotating speed of a shaking table is 150 r/min.

SEQUENCE LISTING

<110> Yunnan college of traditional Chinese medicine

<120> fusarium oxysporum DCLZJ-4 strain for producing paris polyphylla saponin and application thereof

<130> 2018

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<170> PatentIn version 3.3

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ttttcctccg cttattgata tgcttaagtt cagcgggtat tcctacctga tccgaggtca 60

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ctactacgct atggaagctc gacgtgaccg ccaatcaatt tgaggaacgc gaattaacgc 180

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gggtcctctg gcgggccgtc ccgttttacc gggagcgggc tgatccgccg aggcaacaag 480

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cctacgga 548

<210> 2

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<212> DNA

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tccgtaggtg aacctgcgg 19

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tcctccgctt attgatatgc 20

Claims

1. A fermentation liquid obtained by fermentation culture of fusarium oxysporum DCLZJ-4 strain is characterized in that the preservation number of the fusarium oxysporum DCLZJ-4 strain is CGMCC No.14791, the strain is inoculated into 50mL of PDA liquid culture medium, activated culture is carried out by a constant temperature shaking table at 28 ℃ for 150r/min, 1mL of activated strain is taken to be fermented in a triangular flask filled with the PDA liquid culture medium after 24 hours, the liquid loading amount is 100mL/250mL, and shaking table culture is carried out at 28 ℃ for 7d at 150 r/min; after the fermentation culture, collecting fermentation liquor.

2. The fermentation broth of claim 1, wherein the PDA culture medium comprises 300g of potato, 18g of sucrose and water per 1000mL of the culture medium.

3. An extract obtained from the fermentation broth according to claim 1 or 2, wherein the extract is obtained by extracting the fermentation broth as a raw material with n-butanol three times and volatilizing the n-butanol.

4. Use of the extract according to claim 3 for the preparation of a hemostatic medicament.

5. The use of claim 4, wherein the medicament is in an oral dosage form.

6. The use according to claim 5, wherein the oral dosage form is a tablet, hard capsule, soft capsule, powder, pill or granule.

7. A pharmaceutical composition comprising the extract of claim 3 as an active ingredient or as a pharmaceutically acceptable carrier.