CN104450528A - Method for isolating and screening endophytic fungi from gardenia jasminoides - Google Patents
Method for isolating and screening endophytic fungi from gardenia jasminoides Download PDFInfo
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- CN104450528A CN104450528A CN201310415842.9A CN201310415842A CN104450528A CN 104450528 A CN104450528 A CN 104450528A CN 201310415842 A CN201310415842 A CN 201310415842A CN 104450528 A CN104450528 A CN 104450528A
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- cape jasmine
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- endogenetic fungus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
Abstract
The invention discloses a method for isolating and screening endophytic fungi from gardenia jasminoides. The method comprises the steps of gardenia jasminoides tissue preparation, surface sterilization and sterility detection, isolated culture of endophytic fungi of gardenia jasminoides, preparation of a liquid fermentation culture medium suitable for endophytic fungus of gardenia jasminoides, endophytic fungus culture and screening of functional strains. The invention provides an effective strain isolating and screening method for large-scale preparation of high purity genipin.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to one and separate from cape jasmine tissue, through fermentation energy, Geniposide is changed into the method for the endogenetic fungus of genipin.
Background technology
Rubiaceae Gardenia Ellis plant mountain Cape jasmine (
gardenia jasminoidesellis) abounding with the province mountain areas such as Yu Jiang, Zhejiang, Fujian, Jiangxi, emblem, Hunan, is the large traditional Chinese medicine material of China, belongs to the first batch of medicine-food two-purpose resources of medicinal plant that the Ministry of Health promulgates.Common Cultivar and mutation are mountain Cape jasmine, yellow Cape jasmine and water Cape jasmine.Its mature fruit is used as medicine, and has effects such as protecting liver, cholagogic, step-down, calmness, hemostasis, detumescence; Tcm clinical practice is used for the treatment of the diseases such as Jaundice Jaundice, sprain and contusion, hypertension, diabetes.Main containing two large constituents in cape jasmine, iridoid and diterpenes, the representative composition of iridoids is Geniposide (geniposide), and account for more than 50% of iridoid total amount, its content can reach 1.9 ~ 5.5 %.
In iridoid molecule, C
1-OH is very active, and easily combine with sugar, naturally occurring iridoid mostly is the form of glycosides, and mostly is D-Glucose glycosides.This kind of material has multiple physiologically active, thus receives much concern, and develops also very rapid.Genipin (genipin) is the product of Geniposide after beta-glucoside enzymic hydrolysis, it is a kind of excellent natural biological linking agent, can with the crosslinked making biomaterials such as protein, collagen, gelatin and chitosan, as artificial skelecton, wound dressing materials etc., its toxicity is far below glutaraldehyde and other conventional chemical cross-linking agents.Also can be used for treatment hepatic diseases, step-down, defaecation etc.The preparation method of genipin generally adopts and extract Geniposide from cape jasmine, then uses beta-glucoside enzymic hydrolysis, then obtains with extracted with diethyl ether, vacuum concentration, recrystallization, also can adopt the microbe transformation method preparation of producing beta-glucosidase.
Summary of the invention
The object of the invention is to provide one and separates from cape jasmine tissue, through fermentation energy, Geniposide is changed into the method for the endogenetic fungus of genipin, to reach the object being prepared genipin by cape jasmine endogenetic fungus fermentation method.
Object of the present invention is achieved by the following technical programs.
According to cape jasmine without the feature of the easy blackening that completes and " endosymbiotic theory ", we are separation screening endogenetic fungus from cape jasmine tissue first, and by fermentation acquisition, there is cape jasmine endogenetic fungus Geniposide being changed into genipin function, this is the extensive preparation realizing high purity genipin, provides effective strain separating screening method.
The preparation that the present invention is organized by cape jasmine, surface sterilization and Sterility testing, cape jasmine are organized the separation and Culture of endogenetic fungus, are applicable to the preparation of the liquid fermentation medium of cape jasmine endogenetic fungus, endogenetic fungus is cultivated and function stem the step such as to be screened and obtains fungi.
A separating screening method for cape jasmine endogenetic fungus, the method comprises the following steps:
The preparation of A, cape jasmine tissue: gather madder wort cape jasmine
gardenia jasminoidesthe fresh fruit without insect pest of Ellis and leaf;
The surface sterilization of B, cape jasmine tissue: by steps A cape jasmine tissue through surface sterilization, is directly inoculated on potato glucose (PDA) substratum plate, puts 26-28 ° of C thermostat container and cultivates, and therefrom selects the tissue of surface without any growth;
C, cape jasmine organize the separation and Culture of endogenetic fungus: the sterile tissue of step B is aseptically cut into patch-graft on potato glucose (PDA) substratum or Ma Dingshi substratum, put 26-28 ° of C thermostat container to cultivate, when growing mycelia around sample, adopt Tip Splitting picking method, the bacterium colony that picking form is different, be transferred to PDA substratum after purified after 4-10 days, obtain the Endophytic Fungal Hyphae of separation and purification;
The preparation of D, endogenetic fungus liquid fermentation medium: in often liter of substratum, the quality of each component is peptone 0.6-6 g, glucose 1-10 g, potassium primary phosphate 0.2-2 g, magnesium sulfate 0.2-2 g, is settled to 1L with pure water;
E, endogenetic fungus fermentation culture: get step D liquid fermentation medium 100 mL and join in the culturing bottle of 250 mL, picking through the Endophytic Fungal Hyphae of step C separation and purification in culturing bottle, add the Geniposide that 3 mg purity are greater than 98% simultaneously, put the shaking table of 26-28 ° of C, 100-180r/min, after shaking culture 5-10 days, fermentation liquor 3000-4500 r/min is centrifugal, get fermented supernatant fluid for subsequent use;
F, function stem screening: get each supernatant liquor of step e and add 100mL n-butanol extraction for several times, combining extraction liquid, reclaim under reduced pressure propyl carbinol, by methanol constant volume to 5mL, detect Geniposide and genipin through high performance liquid chromatography (HPLC), calculate Geniposide and genipin transformation efficiency by both peak area ratios;
The preservation of G, bacterial classification: the endogenetic fungal bacterial strain with Efficient Conversion rate step F filtered out is placed in liquid Ma Dingshi substratum/glycerine (V/V 3:1) and carries out preservation.
Endogenetic fungus liquid fermentation medium of the present invention is preferably, and in often liter of substratum, the quality of each component is peptone 5 g, and glucose 10 g, potassium primary phosphate 1 g, magnesium sulfate 0.5 g, be settled to 1L with pure water.
Potato glucose (PDA) nutrient media components described in step B of the present invention and C and content are: potato 300g, glucose 20g, agar 16g, is settled to 1L with pure water.
Ma Dingshi nutrient media components described in step C of the present invention and content are: peptone 5 g, glucose 10 g, agar 12 g, and potassium primary phosphate 1 g, magnesium sulfate 0.5 g, is settled to 1L with pure water.
The invention has the advantages that:
(1) from cape jasmine, the endogenetic fungus with high conversion Geniposide being changed into genipin is filtered out according to cape jasmine without the feature of the easy blackening that completes and " endosymbiotic theory ", this is the extensive preparation realizing high purity genipin, provides effective strain separating screening method;
(2) the present invention adopts high performance liquid chromatography chromatogram (HPLC) method to measure the content of substrate Geniposide and product genipin respectively, and compare the transformation efficiency of different strains, thus acquisition effective strain, prepare genipin for fermentable and provide a collection of starting strain;
(3) the present invention have developed the liquid fermentation medium that cape jasmine endogenetic fungus is suitable for, and improves the success ratio to cape jasmine endogenetic fungus separation screening and efficiency.
Accompanying drawing explanation
Fig. 1 is the color atlas transforming Geniposide.
Wherein: 1-Geniposide 2-genipin.
Embodiment
In order to understand the present invention more clearly, below in conjunction with embodiment, the present invention is further illustrated, but the invention is not restricted to following examples.
Embodiment 1:
Cape jasmine fruit tissue is gathered from Nanjing Lishui County, ultrapure water, 75% ethanol rinse 5min, aseptic water washing 6 times, then soak 3min with mercuric chloride, aseptic water washing 6 times, sterilizing filter paper sucks moisture, fruit tissue after surface sterilization is directly planted on PDA substratum plate, puts 25 ° of C thermostat containers and cultivates, and therefrom selects the fruit tissue of surface without any growth; The small pieces aseptic fruit tissue aseptically being cut into about 0.3cm are transplanted respectively on PDA or Ma Dingshi substratum, put 28 ° of C thermostat containers to cultivate, solid medium is PDA substratum (potato dextrose medium) and Ma Dingshi substratum, PDA nutrient media components and content are: potato 300g, glucose 20g, agar 16g, pure water (being settled to 1L); Ma Dingshi nutrient media components and content are: peptone 5 g, glucose 10 g, agar 12 g, potassium primary phosphate 1 g, magnesium sulfate 0.5 g, pure water (being settled to 1L).When solid medium is cultured to and grows mycelia around sample, adopt Tip Splitting picking method, the bacterium colony that picking form is different, is transferred to Ma Dingshi culture medium culturing after 7 days, purifying again, until endogenetic fungus is separated completely, cultivates for subsequent use, according to peptone 5 g, the proportions substratum of glucose 10 g, potassium primary phosphate 1 g, magnesium sulfate 0.5 g, pure water (being settled to 1L), getting liquid fermentation medium 100 mL joins in the culturing bottle of 250 mL, the A1 Endophytic Fungal Hyphae of a little separation and purification of picking is in culturing bottle, add the Geniposide that 3 mg purity are greater than 98% simultaneously, put the shaking table of 26-28 ° of C, 122 r/min, shaking culture 8 days, after the centrifugal 15min of fermentation liquor 4500 r/min, get fermented supernatant fluid, add 100mL n-butanol extraction 2 times, combining extraction liquid, reclaim under reduced pressure propyl carbinol, by methanol constant volume to 5mL, Geniposide and genipin is detected through high performance liquid chromatography (HPLC), Geniposides are calculated and genipin transformation efficiency is 96.3% by both peak area ratios.
Embodiment 2:
The separation of bacterial classification is with embodiment 1.The A2 Endophytic Fungal Hyphae of a little separation and purification of picking is in culturing bottle, add the Geniposide that 3 mg purity are greater than 98% simultaneously, put the shaking table of 26-28 ° of C, 122 r/min, shaking culture 8 days, after the centrifugal 15min of fermentation liquor 4500 r/min, get fermented supernatant fluid, add 100mL n-butanol extraction 2 times, combining extraction liquid, reclaim under reduced pressure propyl carbinol, by methanol constant volume to 5mL, detecting Geniposide and genipin through high performance liquid chromatography (HPLC), is 41.8% by both peak area ratios calculating Geniposides and genipin transformation efficiency.
Embodiment 3:
Cape jasmine leaf tissue is gathered from Nanjing Lishui County, ultrapure water, 75% ethanol rinse 5min, aseptic water washing 6 times, soak 3min with mercuric chloride again, aseptic water washing 6 times, the leaf tissue after surface sterilization is directly planted on PDA substratum plate, put 25 ° of C thermostat containers to cultivate, therefrom select the leaf tissue of surface without any growth; The small pieces aseptic leaf tissue aseptically being cut into about 0.3cm are transplanted respectively on PDA or Ma Dingshi substratum, put 28 ° of C thermostat containers to cultivate, solid medium is PDA substratum (potato dextrose medium) and Ma Dingshi substratum, PDA nutrient media components and content are: potato 300g, glucose 20g, agar 16g, pure water (being settled to 1L); Ma Dingshi nutrient media components and content are: peptone 5 g, glucose 10 g, agar 12 g, potassium primary phosphate 1 g, magnesium sulfate 0.5 g, pure water (being settled to 1L).When solid medium is cultured to and grows mycelia around sample, adopt Tip Splitting picking method, the bacterium colony that picking form is different, is transferred to Ma Dingshi culture medium culturing after 7 days, purifying again, until endogenetic fungus is separated completely, cultivates for subsequent use, according to peptone 5 g, the proportions substratum of glucose 10 g, potassium primary phosphate 1 g, magnesium sulfate 0.5 g, pure water (being settled to 1L), getting liquid fermentation medium 100 mL joins in the culturing bottle of 250 mL, the Endophytic Fungal Hyphae of a little separation and purification A3 of picking is in culturing bottle, add the Geniposide that 3 mg purity are greater than 98% simultaneously, put the shaking table of 26-28 ° of C, 122r/min, shaking culture 8 days, after the centrifugal 15min of fermentation liquor 4500 r/min, get fermented supernatant fluid, add 100mL n-butanol extraction 2 times, combining extraction liquid, reclaim under reduced pressure propyl carbinol, by methanol constant volume to 5mL, Geniposide and genipin is detected through high performance liquid chromatography (HPLC), Geniposides are calculated and genipin transformation efficiency is 97.2% by both peak area ratios.
Embodiment 4:
The separation of bacterial classification is with embodiment 3.The Endophytic Fungal Hyphae of a little separation and purification A4 of picking is in culturing bottle, add the Geniposide that 3 mg purity are greater than 98% simultaneously, put the shaking table of 26-28 ° of C, 122r/min, shaking culture 8 days, after the centrifugal 15min of fermentation liquor 4500 r/min, get fermented supernatant fluid, add 100mL n-butanol extraction 2 times, combining extraction liquid, reclaim under reduced pressure propyl carbinol, by methanol constant volume to 5mL, detecting Geniposide and genipin through high performance liquid chromatography (HPLC), is 72.4% by both peak area ratios calculating Geniposides and genipin transformation efficiency.
Embodiment 5:
Adopt HPLC method to measure the transformation efficiency of Geniposide and genipin in cape jasmine endogenetic fungus fermented liquid, see Fig. 1 and table 1.
Laboratory apparatus and reagent: Agilent high performance liquid chromatograph (quaternary gradient pump, DAD detector, manual injector).Acetonitrile (analytical pure, Tianjin chemical reagent company limited), water is secondary redistilled water.Reference substance Geniposide and genipin self-control, purity detects through high performance liquid chromatography and is all greater than 98% by areas of peak normalization method mensuration purity.
Chromatographic condition: chromatographic column: Kromasil C18 post (5 μm, 4.6mm × 250mm, Dalian Inst of Chemicophysics, Chinese Academy of Sciences); Moving phase: acetonitrile (A)-water (B), 0 min 6 % A, 40 min 28 % A; Flow velocity: 1mL/min; Determined wavelength: 238nm; Column temperature: room temperature; Sample size: 10 μ L.Helium is degassed online.Qualitative according to retention time, the peak area recording Geniposide and genipin is respectively used for transformation efficiency and calculates.
。
Claims (4)
1. a separating screening method for cape jasmine endogenetic fungus, the method comprises the following steps:
The preparation of A, cape jasmine tissue: the fresh fruit without insect pest and the leaf that gather madder wort cape jasmine;
The surface sterilization of B, cape jasmine tissue: by steps A cape jasmine tissue through surface sterilization, be directly inoculated on potato dextrose medium plate, puts 26-28 ° of C thermostat container and cultivates, and therefrom selects the tissue of surface without any growth;
C, cape jasmine organize the separation and Culture of endogenetic fungus: the sterile tissue of step B is aseptically cut into patch-graft on potato dextrose medium or Ma Dingshi substratum, put 26-28 ° of C thermostat container to cultivate, when growing mycelia around sample, adopt Tip Splitting picking method, the bacterium colony that picking form is different, be transferred to PDA substratum after purified after 4-10 days, obtain the Endophytic Fungal Hyphae of separation and purification;
The preparation of D, endogenetic fungus liquid fermentation medium: in often liter of substratum, the quality of each component is peptone 0.6-6 g, glucose 1-10 g, potassium primary phosphate 0.2-2 g, magnesium sulfate 0.2-2 g, is settled to 1L with pure water;
E, endogenetic fungus fermentation culture: get step D liquid fermentation medium 100 mL and join in the culturing bottle of 250 mL, picking through the Endophytic Fungal Hyphae of step C separation and purification in culturing bottle, add the Geniposide that 3 mg purity are greater than 98% simultaneously, put the shaking table of 26-28 ° of C, 100-180r/min, after shaking culture 5-10 days, fermentation liquor 3000-4500 r/min is centrifugal, get fermented supernatant fluid for subsequent use;
F, function stem screening: get each supernatant liquor of step e and add 100mL n-butanol extraction for several times, combining extraction liquid, reclaim under reduced pressure propyl carbinol, by methanol constant volume to 5mL, detect Geniposide and genipin through high performance liquid chromatography, calculate Geniposide and genipin transformation efficiency by both peak area ratios;
The preservation of G, bacterial classification: the volume ratio that the endogenetic fungal bacterial strain with Efficient Conversion rate step F filtered out is placed in liquid Ma Dingshi substratum and glycerine is that 3:1 carries out preservation.
2. the method for claim 1, is characterized in that, endogenetic fungus liquid fermentation medium, and in often liter of substratum, the quality of each component is peptone 5 g, and glucose 10 g, potassium primary phosphate 1 g, magnesium sulfate 0.5 g, be settled to 1L with pure water.
3. the method for claim 1, is characterized in that, the potato dextrose medium component described in step B and C and content are: potato 300g, glucose 20g, and agar 16g, is settled to 1L with pure water.
4. the method for claim 1, is characterized in that, the Ma Dingshi nutrient media components described in step C and content are: peptone 5 g, glucose 10 g, agar 12 g, and potassium primary phosphate 1 g, magnesium sulfate 0.5 g, is settled to 1L with pure water.
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| CN201310415842.9A CN104450528A (en) | 2013-09-13 | 2013-09-13 | Method for isolating and screening endophytic fungi from gardenia jasminoides |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944696A (en) * | 2020-06-05 | 2020-11-17 | 浙江农林大学 | Radix tetrastigme endophytic fungus, and screening method and application thereof |
| CN113546117A (en) * | 2020-04-23 | 2021-10-26 | 百岳特生物技术（上海）有限公司 | Preparation method for improving geniposide content and application of regulating gene expression level |
-
2013
- 2013-09-13 CN CN201310415842.9A patent/CN104450528A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 王永吉: "京尼平的微生物制备及其作为交联剂的应用研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113546117A (en) * | 2020-04-23 | 2021-10-26 | 百岳特生物技术（上海）有限公司 | Preparation method for improving geniposide content and application of regulating gene expression level |
| CN111944696A (en) * | 2020-06-05 | 2020-11-17 | 浙江农林大学 | Radix tetrastigme endophytic fungus, and screening method and application thereof |
| CN111944696B (en) * | 2020-06-05 | 2022-05-17 | 浙江农林大学 | Radix tetrastigme endophytic fungus, and screening method and application thereof |
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