CN110999703B - Method for improving secondary metabolite content of vaccinium bracteatum by infecting with megasporocyst mould - Google Patents
Method for improving secondary metabolite content of vaccinium bracteatum by infecting with megasporocyst mould Download PDFInfo
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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Abstract
The invention discloses a method for improving the content of secondary metabolites in vaccinium bracteatum by using giant cystospora. The method adopts giant cystospora to infect the root system of the oriental blueberry to form a symbiotic system of the oriental blueberry and the giant cystora G. The content of secondary metabolites in the oriental blueberry plant is promoted to be obviously increased through the mutual influence and mutual benefit and mutual assistance relationship formed between the giant cystospora G. The invention can obviously improve the content of secondary metabolites in vaccinium bracteatum plants, wherein the content of flavonoids compounds is improved by not less than 0.01 percent, the content of iridoid glycosides is improved by not less than 0.01 percent, the content of procyanidine components is improved by not less than 0.01 percent, and the content of other secondary metabolites is improved by not less than 0.01 percent. The method can improve the content of iridoid glycosides which are effective components of oriental blueberry branch and leaf dyeing, and improve the coloring capability of oriental blueberry branches and leaves; can also improve the content of flavonoid and procyanidine secondary metabolites, thereby increasing the nutritional ingredients of the black rice.
Description
Technical Field
The invention belongs to the field of soil microorganisms and environment, also belongs to the field of food additives, and relates to a method for improving the content of secondary metabolites of vaccinium bracteatum plants by adopting megasporangium infection.
Technical Field
Gigaspora gigantea (hereinafter, abbreviated as g.gigantea) is an arbuscule mycorrhizal fungus (arbuscula mycorrhiza fungi). The arbuscular mycorrhizal fungi can form a symbiotic relationship with plants, but the infection of the arbuscular mycorrhizal fungi has specificity, and the self life history can be completed only by establishing symbiosis with host plants. The existence of the arbuscular mycorrhizal fungi plays an important role in the survival of host plants, and can obviously increase the adaptability of the host plants to the adverse environment and enhance the competitiveness of the survival of the host plants.
Oriental blueberry, also called as Vaccinium bracteatum (L.) DC, is a evergreen shrub or arbor of Ericaceae and Vaccinium, also called as vaccinium bracteatum (L.) DC, and is mainly distributed in northeast and southeast Asia, and China mainly has 11 provinces, such as Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, Taiwan, Hunan, Guangdong, Guangxi, Sichuan and Yunnan provinces. Fresh branches and leaves of oriental blueberry are edible parts in oriental blueberry and are main raw materials for preparing oriental blueberry rice by impregnating glutinous rice in south China. The chemical components in the leaves are complex, and the leaves contain various fat-soluble active ingredients such as triundecane, friedelin alcohol, quercetin, isoorientin and the like. The vaccinium bracteatum thunb leaves have the functions of resisting oxidation, delaying senility, resisting fatigue, reducing blood sugar, resisting and preventing cancer, improving retina function and the like. However, the content of secondary metabolites in oriental blueberry leaves growing in a natural state is low, and the secondary metabolites are expected to be further improved.
Disclosure of Invention
The invention aims to provide a method for improving the content of secondary metabolites of oriental blueberry plants by adopting gigantism cyst mould G.
In order to achieve the above purpose, the invention specifically adopts the following technical scheme:
a method for improving the content of secondary metabolites of vaccinium bracteatum by using giant cystospora gigas infection comprises the following steps: before or during the culture of the vaccinium bracteatum plant, the root system of the vaccinium bracteatum giganteum is infected and inoculated by utilizing Gigaspora giganteum.
Normally culturing infected vaccinium bracteatum, and measuring the content change of secondary metabolites in the vaccinium bracteatum plant by using High Performance Liquid Chromatography (HPLC). The result shows that the method can improve the content of iridoid glycoside which is an effective component for dyeing the branches and leaves of the oriental blueberry, enhance the coloring capability of the oriental blueberry, and improve the content of secondary metabolites such as flavonoids and procyanidine, thereby increasing the nutrient content of the oriental blueberry.
Preferably, the roots of the vaccinium bracteatum plant are sterilized and washed in advance before the vaccinium bracteatum plant is cultured.
Further, the effective components in the disinfectant adopted by the disinfection are alcohol, mercuric chloride, mop body fluid, chlorothalonil and KMnO4One or more of the components are mixed according to any proportion. Furthermore, the mass percentages of the active ingredients in the disinfectant are preferably as follows: alcohol (1-100%), mercuric chloride (0.1-1.0%), tractor-trazine (0.1-1.0%), chlorothalonil (0.1-1.0%), KMnO4(0.01％-1.0％)。
Further, the sterilization time may be 0.01 to 24 hours.
Further, the washing after root system disinfection is carried out by using sterile water, wherein the sterile water is one or more of distilled water, filtered water and sterilized water.
Preferably, the strain infecting the roots of the vaccinium bracteatum adopts a single giant cystospora strain, and the concentration is 1-500 spores/ml; or mixed strains containing giant megasporangium are adopted, the total concentration of the mixed strains is 1-500 spores/ml, and the proportion of the giant megasporangium in the mixed strains is 1.0% -99.9%.
Preferably, the method for infecting the roots of vaccinium bracteatum is any one of 1), 2) and 3):
1) directly soaking the oriental blueberry root system in a culture medium containing G.gigantea spores for 0.01-24 h;
2) mixing a culture medium containing G.gigantea spores with a soil matrix for cultivating oriental blueberry, wherein the mass ratio of the culture medium to the soil matrix is 1:10-10: 1;
3) directly irrigating the culture solution containing G.gigantea spores to the root of the soil around the planted Oriental blueberry tree, wherein the irrigation volume is 0.1-10 ml per Oriental blueberry tree.
Preferably, the secondary metabolites are flavonoid secondary metabolites, iridoid glycoside secondary metabolites, procyanidine secondary metabolites or other secondary metabolites.
In the invention, the content of flavonoid secondary metabolites in the oriental blueberry branches and leaves is improved by not less than 0.01 percent after the G.gigantea strain infects the oriental blueberry root system for 1 day until the whole survival period of the oriental blueberry. The improvement is the percentage improvement of one flavonoid monomer component determined by High Performance Liquid Chromatography (HPLC), or the sum of the percentage improvement of any several flavonoid monomer components determined by HPLC.
In the invention, the content of iridoid glycoside secondary metabolite in oriental blueberry branches and leaves is improved by not less than 0.01 percent after the G.gigantea strain infects oriental blueberry root system for 1 day until the whole survival period of oriental blueberry. The improvement is the percentage improvement of one iridoid glycoside monomer component content determined by an HPLC method, or the sum of the percentage improvement of any several iridoid glycoside monomer components determined by the HPLC method.
In the invention, the content of procyanidine secondary metabolites in the bracteatum branches and leaves is improved by not less than 0.01 percent after the G.gigantea strain infects the bracteatum root system for 1 day until the whole survival period of the bracteatum. The improvement is the percentage improvement of one procyanidin monomer component determined by an HPLC method or the sum of the percentage improvement of any several procyanidin monomer components determined by the HPLC method.
In the invention, the content of other secondary metabolites in the oriental blueberry branches and leaves is improved by not less than 0.01 percent after the G.gigantea strain infects the oriental blueberry root system for 1 day until the whole survival period of the oriental blueberry. The improvement is the percentage improvement of the content of other monomer components determined by an HPLC method, and can also be the sum of the percentage improvement of the content of any of several other monomer components determined by the HPLC method.
Further, the flavonoid secondary metabolites include, but are not limited to, luteolin, quercetin, coercidin-7-O-beta-D-glucoside, ouvisosidin B, quercetin-3-O-alpha-L-rhamnoside, quercetin-3-O-alpha-L-arabinoside, vitexin, coercidin-7-O- (6' -O-p-hydroxycinnamoyl) -beta-D-glucoside, quercetin-3-O-beta-D-glucuronide methyl ester glycoside, quercetin-3-O-beta-D-galactoside, isorhamnetin-3-O-beta-D-glucoside, isorhamnetin-D-glucoside, and the like which have been found in leaves of Vaccinium bracteatum L Isoorientin, orientin, and quercetin-3-O-beta-D-glucuronide.
Further, the iridoid glycoside secondary metabolites include, but are not limited to, the six iridoid glycosides which are (trans) 10-O-p-hydroxycinnamoyl-dihydrokamisoside, (cis) 10-O-p-hydroxycinnamoyl-dihydrokamisoside, (trans) 10-O-p-hydroxycinnamoyl-paederoside, (cis) 10-O-p-hydroxycinnamoyl-paederoside, (trans) 10-O-p-hydroxycinnamoyl-kamisoside, (cis) 10-O-p-hydroxycinnamoyl-kamisoside, and glycoside components of the six aglycones with any number of five-carbon sugars or six-carbon sugars, which have been found in the leaves of Vaccinium bracteatum.
Further, the secondary metabolites of procyanidins include, but are not limited to, (+) -catechin, (-) -epicatechin monomers and dimeric and trimeric compounds of catechin and epicatechin, which have been found in Vaccinium bracteatum L.
Further, the other classes of secondary metabolites include, but are not limited to, beta-sitosterol, ethyl p-hydroxycinnamate, ethyl caffeate, scopoletin, p-hydroxycinnamic acid, esculetin, caffeic acid, and isolariciresin-9-O-beta-D-xyloside, which have been found in leaves of Vaccinium bracteatum.
The invention overcomes the technical defect of low content of secondary metabolites in the original vaccinium bracteatum plant, and provides a method for remarkably improving the content of the secondary metabolites in the vaccinium bracteatum plant. The giant cystospora G.gigantea provided by the invention can successfully infect the root system of the oriental blueberry and establish a symbiotic relationship, and the absorption of nutrient elements by the oriental blueberry root system is promoted, the absorption of moisture by the oriental blueberry root system is improved, and the stability of an ecological system is assisted to be maintained; the method can improve the survival capability of vaccinium bracteatum, can obviously improve the content of various secondary metabolites in vaccinium bracteatum plants, and especially can obviously improve the content of secondary metabolites such as flavonoids, iridoid glycosides and procyanidine. Wherein the content of flavonoids is increased by not less than 0.01%, the content of iridoid glycosides is increased by not less than 0.01%, the content of procyanidins is increased by not less than 0.01%, and the content of other secondary metabolites is increased by not less than 0.01%.
Detailed Description
The invention will be further illustrated and described by means of specific examples.
The reagent materials used in the present invention may be any commercially available ones unless otherwise specified. Wherein the giant cystospora gigantea G.gigantea is purchased from the genetic resource bank of the department of agriculture, forestry and fisheries of Japan, and the strain number is Gigaspora gigantean MAFF 520054. Of course, the effect of the present invention can be achieved by other commercially available bacterial species, and is not limited.
Example 1
The root system of the oriental blueberry is disinfected by 0.01 percent mercuric chloride, washed by distilled water, soaked for 0.01h by using 1 spore/ml G.gigantea strain culture solution, and the content of the flavonoid secondary metabolite orientin in leaves of the oriental blueberry is measured by an HPLC method after the infected oriental blueberry is cultured in normal soil for 1 day, and the result shows that the content of the orientin is increased by 0.01 percent compared with the content of the orientin before the G.gigantea strain is infected.
Example 2
The root system of the vaccinium bracteatum is disinfected by 0.25 percent of KMnO4, washed by sterilized water, soaked in a G.giganeta strain culture solution for 0.5h for infection, and the content of a secondary metabolite 10-O-p-hydroxycinnamoyl-dihydrocrystal orcein in the plant is measured by an HPLC method after the vaccinium bracteatum is infected and cultured in normal soil for 3 months, and the result shows that the content of the 10-O-p-hydroxycinnamoyl-dihydrocrystal orcein is increased by 0.5 percent compared with that before the G.giganta strain infection.
Example 3
Using 10 ml of G.gigantea strain culture solution containing 500 spores/ml, irrigating the soil around the roots of the soil for infection, after 1 month of infection, measuring contents of secondary metabolites vitexin, isoorientin, orientin, quercetin-3-O-beta-D-glucuronide, (trans) 10-O-p-hydroxycinnamoyl-dihydrozeatin, (cis) 10-O-p-hydroxycinnamoyl-dihydrozeatin, (trans) 10-O-p-hydroxycinnamoyl-dihydrozeatin, (cis) 10-O-p-hydroxycinnamoyl-paederoside, and (cis) 10-O-p-hydroxycinnamoyl-paederoside) in the oriental blueberry plants by an HPLC method, and the results show that the content of vitexin is increased by 0.1%, the content of isoorientin is increased by 0.5%, the orientin content is increased by 0.3 percent, the quercetin-3-O-beta-D-glucuronide content is increased by 0.01 percent, the (trans) 10-O-p-hydroxycinnamoyl-dihydrocrystal orchid aglycone content is increased by 0.02 percent, the (cis) 10-O-p-hydroxycinnamoyl-dihydrocrystal orchid aglycone content is increased by 0.01 percent, the (trans) 10-O-p-hydroxycinnamoyl-paederia scandens aglycone content is increased by 0.01 percent, and the (cis) 10-O-p-hydroxycinnamoyl-paederia scandens aglycone content is increased by 0.01 percent.
Example 4
Mixing culture solution of G.gigantea strain with 300 spores/ml and soil matrix, wherein the ratio of the culture solution to the soil is 1: 10. The mixed soil is used for cultivating the vaccinium bracteatum thunb. After 3 years of cultivation, HPLC method is adopted to determine the content of secondary metabolites including vitexin, isoorientin, orientin, quercetin-3-O-beta-D-glucuronide, (trans) 10-O-p-hydroxycinnamoyl-dihydroisovaleryl, cis) 10-O-p-hydroxycinnamoyl-dihydroisovaleryl, trans) 10-O-p-hydroxycinnamoyl-paederosissiosigenin, and cis-10-O-p-hydroxycinnamoyl-paederosigenin, the result shows that the content of vitexin is increased by 1%, the content of isoorientin is increased by 5%, the content of orientin is increased by 3%, the content of quercetin-3-O-beta-D-glucuronide is increased by 1%, the content of (trans) 10-O-p-hydroxycinnamoyl-dihydrocrystal aglycone is improved by 2 percent, the content of (cis) 10-O-p-hydroxycinnamoyl-dihydrocrystal aglycone is improved by 1 percent, the content of (trans) 10-O-p-hydroxycinnamoyl-paederia scandens aglycone is improved by 1 percent, and the content of (cis) 10-O-p-hydroxycinnamoyl-paederia scandens aglycone is improved by 1 percent.
The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.
Claims (7)
1. A method for improving the content of secondary metabolites of vaccinium bracteatum by using giant cystospora megalosa infection is characterized in that: before or during the culture of vaccinium bracteatum plant, utilizing megasporangium giganteumGigaspora giganteaCarrying out infection inoculation on the root system;
the method for infecting the roots of vaccinium bracteatum is any one of 1), 2) and 3):
1) soaking root system of Vaccinium bracteatum directly in the solution containingG. giganteaSoaking in spore culture medium for 0.01-24 hr;
2) will containG. giganteaThe culture medium of the spores is mixed with a soil matrix and then used for cultivating the vaccinium bracteatum, and the mass ratio of the culture medium to the soil matrix is 1:10-10: 1;
3) will containG. giganteaDirectly irrigating the soil around the roots of the planted oriental blueberry with the spore culture solution, wherein the irrigation volume is 0.1-10 ml per oriental blueberry;
the secondary metabolite is flavonoid secondary metabolite, iridoid glycoside secondary metabolite, procyanidine secondary metabolite orOther classes of secondary metabolites; the other classes of secondary metabolites includeβ-sitosterol, ethyl p-hydroxycinnamate, ethyl caffeate, scopoletin, p-hydroxycinnamic acid, esculetin, caffeic acid, and isolariciresinol-9-O- β -D-xyloside.
2. The method for increasing the content of secondary metabolites of vaccinium bracteatum by infecting with megaspora giganteum according to claim 1, wherein: before the vaccinium bracteatum plant is cultured, the root system of the vaccinium bracteatum plant needs to be disinfected and washed in advance.
3. The method for increasing the content of secondary metabolites of vaccinium bracteatum by infecting with megaspora giganteum according to claim 2, wherein: the effective components in the disinfectant used for disinfection are alcohol, mercuric chloride, thiophanate, chlorothalonil and KMnO4One or more of the above, the disinfection time is 0.01-24 h; and (3) washing after root system disinfection is carried out by adopting sterile water, wherein the sterile water is one or more of distilled water, filtered water and sterilized water.
4. The method for increasing the content of secondary metabolites of vaccinium bracteatum by infecting with megaspora giganteum according to claim 1, wherein: the strain infecting the roots of the vaccinium bracteatum adopts a single giant cystospora strain, and the concentration is 1-500 spores/ml; or mixed strains containing giant megasporangium are adopted, the total concentration of the mixed strains is 1-500 spores/ml, and the proportion of the giant megasporangium in the mixed strains is 1.0% -99.9%.
5. The method for increasing the content of secondary metabolites of vaccinium bracteatum by infecting with megaspora giganteum according to claim 1, wherein: the flavonoid secondary metabolite comprises luteolin, quercetin, chrysoeriol-7-O-β-D-glucoside, oudemansinoside B, quercetin-3-O-α-L-rhamnoside, Quercetin-3-O-α-L-arabinoside, vitexin, chrysoeriol-7-O-(6’’-OP-hydroxycinnamoyl) -β-D-glucoside, quercus acutissimaDermatan-3-O-β-D-glucuronic acid methyl ester glycoside, quercetin-3-O-β-D-galactoside, isorhamnetin-3-O-β-D-glucoside, isoorientin, orientin, quercetin-3-O-β-D-glucuronide.
6. The method for increasing the content of secondary metabolites of vaccinium bracteatum by infecting with megaspora giganteum according to claim 1, wherein: the secondary metabolites of iridoid glycosides comprise (trans) 10-O-p-hydroxycinnamoyl-dihydrochloranthus glycosides, (cis) 10-O-p-hydroxycinnamoyl-dihydrochloranthus glycosides, (trans) 10-O-p-hydroxycinnamoyl-paederia scandens aglycone, (cis) 10-O-p-hydroxycinnamoyl-paederia scandens aglycone, (trans) 10-O-p-hydroxycinnamoyl-chloranthus glycosides, (cis) 10-O-p-hydroxycinnamoyl-chloranthus glycosides and glycoside components formed by the six aglycones and any number of pentoses or hexoses.
7. The method for increasing the content of secondary metabolites of vaccinium bracteatum by infecting with megaspora giganteum according to claim 1, wherein: the proanthocyanidin secondary metabolite comprises (+) -catechin, (-) -epicatechin monomer and dimer and trimer compound formed by catechin and epicatechin.
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