CN107312720B - Cochinchinensis endophytic fungus for efficiently converting ginsenoside Rb1 into Rd and application thereof - Google Patents
Cochinchinensis endophytic fungus for efficiently converting ginsenoside Rb1 into Rd and application thereof Download PDFInfo
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Abstract
The invention discloses a pigeon pea endophytic fungi strain Fusarium proliferatum G11-7 for efficiently converting ginsenoside Rb1 in panax notoginseng into Rd, a separation method thereof and application of the strain in fermenting and converting ginsenoside. The strain (G11-7) is derived from pigeon pea plants and is obtained by solid culture, separation and purification. The strain can efficiently and specifically convert ginsenoside Rb1 into Rd, and the content of the converted ginsenoside Rd is about 4 times of that before conversion. The Fusarium delavayi strain (G11-7) is an endophytic fungus obtained from pigeon pea for the first time, and the strain is used for converting ginsenoside Rb1 into Rd, so that the time is short, the conversion rate is high, the strain is green and pollution-free, the industrial production is easy to realize, and the application prospect is wide.
Description
Technical Field
The invention relates to an endophytic fungus for efficiently converting ginsenoside Rb1 into Rd and application thereof, belonging to the field of application of microbial technology.
Technical Field
Endophytic fungi (Endophytic fungi) refer to fungi that live in plant tissues at some stage of their life history, but do not cause significant disease symptoms in plants. Certain endophytes have the ability to produce the same or similar bioactive substances as the host plant by "co-evolution" with medicinal plants (Chen et al, 2006). The endophytic fungi have abundant species diversity, and secondary metabolites are also abundant. A series of enzymes can be generated through nutrient culture and fermentation culture of endophytic fungi, and the enzymes from microorganisms can react with substrates to change the structures of the enzymes, so that the microbial transformation of the endophytic fungi has the characteristics of fast biomass accumulation, short transformation time, high transformation efficiency, convenience for industrial production and the like (Guo De Liang et al, 2016).
Notoginseng [ Panax notogeng (Burk.) F.H.Chen ]. Commonly known as Shangeng, jin Bu Shi and Tian Qi, are plants of Araliaceae of Umbelliferae and mainly distributed in Yunnan, Guangxi and Sichuan provinces. Pseudo-ginseng is a traditional and rare Chinese medicinal material in China, has an application history of more than 400 years from now on, is famous for a long time at home and abroad, and has a name of a hemostatic magical medicine from ancient times. The complex chemical components of panax notoginseng are the basis of good efficacy, the chemical components found in panax notoginseng at present mainly comprise saponins and dencichine, and the saponins can be divided into ginsenoside, notoginsenoside, escin and the like (Chinese pharmacopoeia, one part), wherein the content of ginsenoside Rg1 and Rb1 is the highest. The pharmacological actions of notoginseng are inseparable from its own chemical components, mainly reflected in the effects on the blood system, cardiovascular system, cerebrovascular system, nervous system, metabolism, immunoregulatory system, etc. (von luobiao et al, 2008).
Ginsenoside Rd (ginsenoside Rd) with molecular formula of C48H82O18The panaxadiol type ginsenoside belongs to diol type in dammarane type ginsenoside, and is rare saponin in pseudo-ginseng. Researches show that the pharmacological activity of ginsenoside Rd is superior to that of other main saponins, the Rd is one of the main forms of the main saponins absorbed and utilized in intestinal tracts after metabolism, can protect cardiovascular and renal functions, plays various pharmacological roles of tumor resistance, immunity regulation and the like, and also shows good neuroprotective effect on the central nervous system (Zhengel and the like, 2011). Because the content of ginsenoside Rd in the plant is low, the cost is high due to the direct extraction from the original plant medicinal material, the loss of a target compound is large, the collection of the raw material is limited by time and space, and large-scale production is difficult to form, and the enzyme generated by the metabolism of endophytic fungi is used for carrying out biotransformation on the panax notoginseng saponins, so that the method has the characteristics of mild reaction conditions, no damage to saponin structures, strong specificity, high transformation rate, no pollution and the like, and is expected to implement large-scale industrial production.
Disclosure of Invention
The invention provides a pigeon pea endophytic fungus (Fusarium proliferatum) G11-7 capable of efficiently converting panax notoginseng ginsenoside Rb1 into Rd and application thereof. The invention overcomes the defects of low content, high energy consumption, environmental pollution and the like in the traditional acquisition mode, can realize large-scale production and has higher application value.
The invention provides a pigeon pea endophytic fungus capable of efficiently converting panax notoginseng ginsenoside Rb1 into Rd and application thereof, and is characterized in that when the pigeon pea endophytic fungus is cultured in a PDA solid culture medium, aerial hyphae are white and slightly purple, sclerotium is mostly blue-purple, when a large number of sclerotium exist, the lower surface of a bacterial colony is black-purple, conidia are yellow brown to orange, and the conidia are scattered or gather by myxospore groups, are monospora and have no chlamydospore.
The pigeon pea endophytic fungus strain is obtained by submitting a gene sequence of the pigeon pea endophytic fungus strain to NCBI, obtaining a gene accession number KY303906 through BLAST comparison, constructing a phylogenetic evolution tree by utilizing MEGA6 software, and determining that the endophytic fungus G11-7 is Fusarium delayer (Fusarium proliferatum) by combining a morphological observation result.
The invention relates to a pigeon pea endophytic fungi layer Fusarium (Fusarium proliferatum) G11-7 which is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of West Lu No. 1 of Beijing Korean district, the preservation number is CGMCC No. 13195, and the preservation date is 2016, 12 and 7 days. The strain has the capability of efficiently converting panax notoginseng ginsenoside Rb1 into Rd, and experiments show that the ginsenoside Rd has good conversion effect.
Drawings
FIG. 1 shows the structural formulas of ginsenoside Rb1 and ginsenoside Rd;
FIG. 2 is a colony morphology of cajan endophytic fungus G11-7;
FIG. 3 is a spore morphology of cajan endophytic fungus G11-7 under a light microscope;
FIG. 4 is a morphological diagram of a colony and a transparent circle of the pigeon pea endophytic fungus G11-7 strain on a cellulose-Congo red medium;
FIG. 5 is a phylogenetic tree of the strain Fusarium proliferatum G11-7;
FIG. 6 is an HPLC chart before and after the conversion of Panax notoginsenosides Rb1 and Rd by endophytic fungi G11-7;
Detailed Description
Example 1: the isolation of Fusarium proliferatum G11-7 in the present invention.
Fusarium graminearum (Fusarium proliferatum) G11-7 in the embodiment, the strain is separated from healthy pigeon pea roots, the pigeon pea roots are collected from a botanical garden of northeast forestry university, the plant age is 3 months, and a sample of the pigeon pea roots is currently stored in a key laboratory of the department of forest plant ecology education of the northeast forestry university; it is isolated and cultured according to the following steps:
(1) preparation of potato dextrose agar medium (PDA): selecting ripe potatoes with smooth surfaces and no bud or fester, cleaning and peeling, weighing 200g of the potatoes, cutting into blocks, boiling for 30min, filtering by 4 layers of gauze to obtain potato liquid, adding 20g of glucose and 20g of agar powder, adding distilled water to a constant volume of 1L, keeping the pH natural, and sterilizing at 121 ℃ for 15min under high temperature and high pressure;
(2) cleaning the surface of a healthy pigeon pea plant with distilled water, sequentially rinsing with 75% ethanol for 1min, 5% NaClO solution for 5min and 10% hydrogen peroxide for 3min for surface disinfection, and washing with sterile water for 3-4 times;
(3) cutting the sterilized root, stem and leaf tissues of pigeon pea into small tissues with the diameter of about 1cm by using a sterilized scalpel, inoculating the small tissues on the surface of a PDA solid culture medium by using sterile forceps, placing the PDA solid culture medium in a constant temperature box, and carrying out inverted culture at the constant temperature of 28 ℃ for 7-10 days;
(4) observing hyphae growing from the interior of each plant tissue block to the periphery on the culture medium, adopting a tip hypha picking method, picking hyphae growing from the edge cuts of different parts of a sample by using a sterile toothpick, transferring the hyphae to a freshly configured PDA culture medium flat plate, culturing at a constant temperature of 28 ℃, picking the tip hyphae again after a new bacterial colony grows out, transferring the tip hyphae to the PDA culture medium flat plate, and repeating the operation for more than 4 times until a purified single bacterial strain is obtained (as shown in figures 2 and 3);
(5) numbering the separated fungus strains, respectively inoculating the fungus strains on a PDA inclined plane, and storing at 4 ℃;
(6) and (3) performing molecular identification, namely filtering the pigeon pea endophytic fungus fermentation liquor by four layers of gauze to obtain mycelia, extracting total DNA of the mycelia, performing PCR amplification on a target fragment, sequencing by Shanghai bio-engineering technology service company, performing BLAST homology comparison on the determined nucleic acid sequence in an NCBI database, selecting an ITS sequence of a corresponding species representative strain according to the basic principle of molecular biology research, constructing a phylogenetic tree by applying a software MEGA6 and a Neighbor binding method (Neighbor-join), and determining the classification status of a target strain.
Example 2: the invention relates to screening of Fusarium delavayi (Fusarium proliferatum) G11-7 strain with directionally transformed panax notoginseng ginsenoside Rb1 as Rd.
(1) Preparation of a screening culture medium: the cellulose-Congo red solid culture medium contains 0.2% of sodium carboxymethylcellulose and 0.2% (NH)4)2S04，0.05％MgSO4·7H2O，0.1％KH2P041.5 percent of agar, adjusting the pH value to 7.0, and sterilizing for 15min at 121 ℃ under high temperature and high pressure;
(2) inoculating the pigeon pea endophytic fungi strain into a cellulose-congo red solid culture medium, culturing until a regular bacterial colony grows, covering a congo red solution with the mass concentration of 1mg/mL on the bacterial colony, pouring the congo red solution after 10-15 min, adding a NaCI solution with the mass concentration of lmol/L, and pouring a NaCl solution after 15min, wherein a transparent ring appears around the bacterial colony generating cellulase, and the larger the ratio of the diameter of the transparent ring to the diameter of the bacterial colony is, the higher the activity of the cellulase is;
(3) the experimental results show that: a pigeon pea endophytic fungus strain named G11-7 is determined by screening a cellulose-congo red solid culture medium, the ratio of the transparent circle to the colony diameter of the strain is 1.23 (see figure 4), and a G11-7 strain is selected for subsequent transformation experiments.
Example 3: the G11-7 strain of Fusarium proliferatum of the invention directionally converts notoginsenoside Rb1 into Rd.
(1) Activating strains: inoculating the trichoderma viride (Fusarium proliferatum) G11-7 to a PDA solid slant culture medium for culturing for 7-10 days;
(2) preparation of a transformation medium: selecting ripe potatoes with smooth surfaces and no bud or fester, cleaning, peeling, weighing 200g of the potatoes, cutting into blocks, boiling in boiling water for 30min, filtering by 4 layers of gauze to obtain potato liquid, fixing the volume to 1L, and keeping the pH value natural;
(3) pretreating raw powder of pseudo-ginseng: pulverizing Notoginseng radix, sieving with 40 mesh sieve to obtain Notoginseng radix powder, adding 2g Notoginseng radix powder into 50ml transformation culture medium, and sterilizing at 121 deg.C under high temperature and high pressure for 40 min;
(4) fermentation and transformation: taking the slant strain G11-7 grown in the step 1, scraping off spores on the surface of the strain to prepare spore suspension with a certain concentration, adding 10ml of spore suspension into the pretreated crude powder of the panax notoginseng medicine for biotransformation, wherein the transformation temperature is 30 ℃, the rotation speed is 120r/min, and the transformation time is 48 h;
(5) extracting ginsenoside: centrifuging to remove mycelium and Notoginseng radix crude powder to obtain supernatant, extracting n-butanol and supernatant with equal volume, repeating for three times, mixing organic phases, and concentrating under reduced pressure to obtain ginsenoside crude extract.
Example 4: the strain Fusarium proliferatum G11-7 of the invention directionally converts panax notoginseng ginsenoside Rb1 into Rd for detection.
The detection of the converted product by high performance liquid chromatography comprises diluting the crude extract of ginsenoside with chromatographic methanol, and analyzing and detecting by high performance liquid chromatography. The chromatographic conditions are Agilent 1100 chromatograph, HiQ sil C18W ((II))
5 μm) chromatography column, mobile phase: water (A) -acetonitrile (B), column temperature 30 ℃; the detection wavelength is 203nm, the flow rate is 0.8mL/min, the sample injection amount is 10 mu L, and the gradient elution condition is as follows: 0-5min, 25-30% (B); 5-15min, 30-40% (B); 15-16min, 40-41% (B); 16-20min, 41-42% (B); 20-30min, 42-50% (B); 30-35min, 50-60% (B); 35-60min, 60-40% (B).
The detection result shows that: the pigeon pea endophytic fungus G11-7 strain is adopted for fermentation biotransformation, and the content of ginsenoside Rd in each liter of fermentation liquid is increased from 118.36mg to 478.44mg (as shown in figure 6). Meanwhile, the time for converting the panax notoginseng ginsenoside Rb1 into Rd is short, the conversion rate is high, the method is green and pollution-free, large-scale production can be realized, and the method has high application value.
Claims (3)
1. A pigeon pea endophytic fungus capable of efficiently converting panax notoginseng ginsenoside Rb1 into Rd is characterized in that: the described pigeon pea endophytic fungi is Fusarium proliferatum (Fusarium proliferatum), and is fungi Imperfecti (Imperfecti fungi), Moniliales (Moniliales), Cinobospermaceae (Tuberculataceae), Fusarium (Fusarium);
the pigeon pea endophytic fungi is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of No. 1 Xilu on North Chen of the Korean district in Beijing, the preservation number is CGMCC No. 13195, and the preservation date is 2016, 12 and 7 days.
2. The pigeon pea endophytic fungus for efficiently converting panax notoginseng saponins Rb1 to Rd according to claim 1, wherein colonies of the pigeon pea endophytic fungus are white and slightly purple, conidiospore is oval or corynebomonas, and are scattered or aggregated by mucoconidiospore groups when the pigeon pea endophytic fungus is cultured at 28 ℃ in PDA solid culture.
3. The use of the pigeon pea endophytic fungi according to any one of claims 1-2, wherein the cellulase produced by metabolism of the pigeon pea endophytic fungi has the activity of biologically converting ginsenoside Rb1 in panax notoginseng into ginsenoside Rd;
when the pigeon pea endophytic fungi is used for biologically converting ginsenoside Rb1 in the panax notoginseng into ginsenoside Rd, the conversion temperature is 30 ℃, the rotation speed is 120r/min, and the conversion time is 48 h.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101481725A (en) * | 2009-01-21 | 2009-07-15 | 华东理工大学 | Method for preparing ginseng saponin F2 by enzymatic hydrolysis of ginseng saponin Rb1 |
| CN103255193A (en) * | 2013-03-05 | 2013-08-21 | 吉林农业大学 | Ginsenoside conversion method by use of ginseng endophytic Paenibacillus polymyxa |
| CN105838613A (en) * | 2015-01-14 | 2016-08-10 | 东北林业大学 | Pigeon pea endophytic fungi high in yield of flavipin and application of pigeon pea endophytic fungi |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101481725A (en) * | 2009-01-21 | 2009-07-15 | 华东理工大学 | Method for preparing ginseng saponin F2 by enzymatic hydrolysis of ginseng saponin Rb1 |
| CN103255193A (en) * | 2013-03-05 | 2013-08-21 | 吉林农业大学 | Ginsenoside conversion method by use of ginseng endophytic Paenibacillus polymyxa |
| CN105838613A (en) * | 2015-01-14 | 2016-08-10 | 东北林业大学 | Pigeon pea endophytic fungi high in yield of flavipin and application of pigeon pea endophytic fungi |
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