CN112972520B - Method for improving active ingredient yield by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid - Google Patents

Method for improving active ingredient yield by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid Download PDF

Info

Publication number
CN112972520B
CN112972520B CN202110295422.6A CN202110295422A CN112972520B CN 112972520 B CN112972520 B CN 112972520B CN 202110295422 A CN202110295422 A CN 202110295422A CN 112972520 B CN112972520 B CN 112972520B
Authority
CN
China
Prior art keywords
fermentation
liquid
strain
culture
eucommia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110295422.6A
Other languages
Chinese (zh)
Other versions
CN112972520A (en
Inventor
徐向群
朱振铎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Sci Tech University ZSTU
Original Assignee
Zhejiang Sci Tech University ZSTU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Sci Tech University ZSTU filed Critical Zhejiang Sci Tech University ZSTU
Priority to CN202110295422.6A priority Critical patent/CN112972520B/en
Publication of CN112972520A publication Critical patent/CN112972520A/en
Application granted granted Critical
Publication of CN112972520B publication Critical patent/CN112972520B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/46—Eucommiaceae (Eucommia family), e.g. hardy rubber tree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Toxicology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Epidemiology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a method for improving the yield of active ingredients by utilizing fuscoporia obliqua liquid to carry out submerged fermentation on eucommia ulmoides leaves, which comprises the following steps: (1) activating strains; (2) liquid strain culture; (3) preparing eucommia ulmoides leaf powder; (4) liquid submerged fermentation; and (5) polysaccharide removal of the fermentation liquor. The method can greatly improve the yield of the active ingredients in the eucommia ulmoides leaf extract, and has the advantages of short time, simple equipment requirement, less equipment investment, simple post-treatment, basically no pollution and low cost.

Description

Method for improving active ingredient yield by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to a method for improving the yield of active ingredients by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid.
Background
Eucommia ulmoides (Eucommia ulmoides Oliver) belongs to Eucommia family, and only one type of Eucommia ulmoides belongs to the family. Eucommia ulmoides is a rare or endangered two-class protection tree species in China and has special value. Eucommia bark, is used in ancient times. The first book of Chinese herbs, shennong Ben Cao Jing, records the drug effect of eucommia ulmoides, li Shizhen is pointed out in Ben Cao gang mu: "Xishu Du is used to get it's true, so its name is. Modern researches also find that the eucommia bark contains a large amount of effective components, and more than 80 active components in the eucommia bark are mainly lignans, phenylpropanoids, iridoids, flavonoids, phenols, triterpenes, polysaccharides, organic acids and rich nutritional components such as amino acids, fats, trace elements and the like. Has antioxidant, antiinflammatory, blood lipid reducing, antidiabetic, antitumor, and immunity enhancing effects.
However, the extraction of the effective components of eucommia bark usually needs a large amount of alkaline reagents and organic solvents at present, which is not favorable for environment-friendly development and increases the production cost. The mild and stable extraction method has great promotion effect on the realization of the value of the eucommia ulmoides. The enzyme is used as a catalyst, and by virtue of the characteristics of high efficiency, specificity and mild reaction conditions, the hydrolysis capacity of the cellulase to cellulose provides a new method for releasing and extracting the active ingredients of the natural Chinese herbal medicines, the plant cell walls are greatly damaged after enzymolysis pretreatment, the active ingredients are easier to release, and the extraction efficiency and the extraction rate are improved. However, pure cellulase enzymolysis also brings cost pressure to industrial production. Therefore, experts put attention to a biological glycolysis means, can greatly reduce the cost and is more environment-friendly.
Inonotus obliquus (Inonotus obliquus), also known as Inonotus obliquus, inonotus obliquus and Inonotus obliquus, is widely used as a specific medicine for treating various difficult and complicated diseases in folks, and the chemical components of the Inonotus obliquus are continuously researched and explored. Inonotus obliquus mainly contains lanoline triterpenes, ergosterol peroxide, sphingolipid analogue, and mannitol, and its water extract contains low molecular weight polyphenols. Inonotus obliquus also contains mycolic acid, hemitannin compound, steroid, alkaloid, etc. Polysaccharides in the hyphae and sclerotia of Inonotus obliquus are mainly beta-glucan, heteropolysaccharide and protein complex.
In order to ensure the sustainability of eucommia ulmoides resources and the stability of medicinal components, various active ingredients in the eucommia ulmoides leaves are fully released, extracted and utilized, the extraction rate of effective ingredients is improved, the production cost is reduced as much as possible, and the problem to be solved is urgently needed.
Disclosure of Invention
The invention aims to provide a method for improving the yield of active ingredients in an eucommia ulmoides leaf extract by utilizing the liquid submerged fermentation of inonotus obliquus, which can greatly improve the yield of the active ingredients in the eucommia ulmoides leaf extract and has the advantages of short time, simple equipment requirement, less equipment investment, simple post-treatment, basically no pollution and low cost.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for improving active ingredient yield by submerged fermentation of folium Eucommiae with Inonotus obliquus liquid comprises the following steps:
(1) Activating strains: inoculating inonotus obliquus as a strain to a slant strain culture medium for culture to obtain an activated strain;
(2) Liquid strain culture: transferring the activated strain into a seed culture medium to culture to obtain a liquid strain; performing shake culture at 28 deg.C and 150r/min for 3 days to obtain liquid strain;
(3) Preparing eucommia leaf powder: pulverizing dried Eucommiae cortex She Zhiyu in pulverizer, and sieving to obtain folium Eucommiae powder;
(4) Liquid submerged fermentation: inoculating the liquid strain into a liquid fermentation culture medium, and fermenting and culturing for 2-6 days at 28 + -1 deg.C and 150 + -10 r/min of shaking table rotation speed; the preparation method of the liquid fermentation medium comprises the following steps: mixing 3g folium Eucommiae powder with 100ml nutrient salt solution, adjusting pH =6.0, and performing wet heat sterilization at 121 + -1 deg.C for 30min to obtain;
(5) Polysaccharide removal of fermentation liquor: after fermentation, obtaining a fermentation stock solution by suction filtration, carrying out rotary evaporation and concentration on the fermentation stock solution to obtain a concentrated solution, then adding a 95% ethanol solution, fully stirring, standing overnight at 4 ℃, carrying out centrifugal separation on polysaccharide in the fermentation solution, and collecting supernatant as extracellular fluid containing active ingredients.
The invention utilizes inonotus obliquus and adopts a liquid submerged fermentation method to carry out liquid fermentation by taking the eucommia leaves as objects, thereby greatly improving the yield of active ingredients in the eucommia leaf extract, and having short time, simple equipment requirement, less equipment investment, simple post-treatment, basically no pollution, low cost and effectively improving the yield of various active ingredients.
Eucommia is a rare or endangered type of protective tree species in China, and the cost for extracting active ingredients from eucommia leaves is increased. The process can effectively improve the release of active ingredients in the eucommia leaves and reduce the cost. The measure also conforms to the basic national policy of saving resources and protecting the environment in China, and conforms to the concept of respecting nature, conforming to nature and protecting nature.
Preferably, the active ingredients include chlorogenic acid, iridoid, flavone, polyphenol and phenolic acid.
Preferably, the size of the eucommia ulmoides leaf powder is ≦ 60 mesh.
Preferably, the nutrient salt solution has the following formula: 3.5g of corn flour, 0.23g of peptone, 0.12g of yeast powder 2 0.048g，CoCl 2 ·6H 2 O 0.002g，KH 2 PO 4 0.1g，MgSO 4 0.02g，ZnSO 4 ·7H 2 O 0.001g，K 2 HPO 4 ·3H 2 O 0.05g，FeSO 4 ·7H 2 O 0.005g，CuSO 4 ·5H 2 O 0.002g，MnCl 2 ·4H 2 O0.009 g, made up to 100mL with distilled water, adjusted pH =6.0.
Preferably, the formula of the slant strain culture medium is as follows: 40g of malt extract, 4g of peptone and 10g of agar, wherein distilled water is used for fixing the volume to 100ml, and the pH value is 5.4-5.6.
Preferably, the seed culture medium consists of: KH (Perkin Elmer) 2 PO 4 0.03g，MgSO 4 0.02g, yeast extract 0.3g, peptone 0.05g and glucose 1g, adding distilled water to constant volume of 100mL, and performing moist heat sterilization at 121 +/-1 ℃ for 30min.
Preferably, in the step (4), the fermentation culture is carried out for 2-4 days under the conditions of 28 +/-1 ℃ and the rotation speed of a shaking table of 150 +/-10 r/min. The optimal fermentation culture time is 2 hours.
Preferably, in step (5), the 95% ethanol solution is used in an amount of 4 times the volume of the concentrate.
The invention has the beneficial effects that: can greatly improve the yield of active ingredients in the eucommia ulmoides leaf extract, and has the advantages of short time, simple equipment requirement, less equipment investment, simple post-treatment, basically no pollution and low cost. The active ingredients produced by the invention can be used for preparing antioxidant and anti-inflammatory drugs and the like.
Drawings
Fig. 1 shows a in fermentation control, eucommia leaf water extraction, and eucommia leaf fermentation (liquid fermentation) extracellular fluid/extract: extracellular polyphenols, b: phenolic acid, c: content change in flavone fermentation/extraction for 14 days.
FIG. 2 shows the content change of chlorogenic acid in fermentation/extraction 14 days in fermentation/extraction of extracellular fluid/extract of fermentation control, folium Eucommiae water extraction, and folium Eucommiae fermentation (liquid fermentation).
FIG. 3 shows the content change of iridoid in extracellular fluid/extract of fermentation control, folium Eucommiae water extraction, folium Eucommiae fermentation (liquid fermentation) for 14 days of fermentation/extraction.
FIG. 4 shows the change of the yield of gutta Percha obtained by solid fermentation and culture of folium Eucommiae for 8 days.
FIG. 5 shows the content change of phenolic acid in folium Eucommiae after solid fermentation for 8 days.
FIG. 6 shows the change of flavone content in folium Eucommiae after solid fermentation for 8 days.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1:
a method for improving the yield of active ingredients by deeply fermenting folium Eucommiae with Inonotus obliquus liquid comprises the following steps:
(1) Activating strains: inoculating inonotus obliquus (commercially available) as strain to slant strain culture medium, and culturing to obtain activated strain; the formula of the slant strain culture medium is as follows: malt extract 40g, peptone 4g, agar 10g, use distilled water constant volume to 100ml, pH value is 5.4-5.6, the parameter of culturing on the slant strain culture medium is: the temperature is 28 ℃, and the culture is carried out for 250h.
(2) Liquid strain culture: transferring the activated strain into a seed culture medium, and performing shake culture at 28 deg.C and 150r/min for 3 days to obtain liquid strain; the seed culture medium comprises the following components: KH (Perkin Elmer) 2 PO 4 0.03g，MgSO 4 0.02g, 0.3g of concentrated yeast extract, 0.05g of peptone and 1g of glucose, diluting to 100mL with distilled water, and performing moist heat sterilization at 121 ℃ for 30min.
(3) Preparing eucommia leaf powder: and (3) placing the dried eucommia leaves into a stirring and crushing machine for crushing, sieving crushed slag by using a 60-mesh molecular sieve, collecting sieved eucommia leaf powder (less than or equal to 60 meshes), and storing at room temperature for later use.
(4) Liquid submerged fermentation: mixing 2ml of liquid bacteriaInoculating the seeds into a liquid fermentation culture medium, and fermenting and culturing for 2 days at the temperature of 28 ℃ and at the speed of 150 r/min; the liquid fermentation culture medium is prepared by mixing 3g of folium Eucommiae powder and 100ml of nutrient salt solution, stirring thoroughly with pH of 6.0, and performing wet heat sterilization at 121 deg.C for 30 min; the nutrient salt solution has the following formula: 3.5g of corn flour, 0.23g of peptone, 0.12g of yeast powder, caCl 2 0.048g，CoCl 2 ·6H 2 O 0.002g，KH 2 PO 4 0.1g，MgSO 4 0.02g，ZnSO 4 ·7H 2 O 0.001g，K 2 HPO 4 ·3H 2 O 0.05g，FeSO 4 ·7H 2 O 0.005g，CuSO 4 ·5H 2 O 0.002g，MnCl 2 ·4H 2 O0.009 g, made up to 100mL with distilled water, adjusted to pH 6.0.
(5) Polysaccharide removal of fermentation liquor: after fermentation, obtaining a fermentation stock solution by suction filtration, carrying out rotary evaporation and concentration on the fermentation liquid to 20mL, then adding 80mL of 95% ethanol solution, fully stirring, standing in a refrigerator at 4 ℃ for 12h, separating polysaccharide in the fermentation liquid by using a high-speed centrifuge, wherein the parameter of the centrifuge is 8000r/min, centrifuging for 10min, and collecting supernatant as extracellular fluid containing active ingredients (polyphenol, flavone, phenolic acid, chlorogenic acid and iridoid).
Example 2
This example is different from example 1 in that fermentation culture was carried out at 150r/min for 4 days, and the other steps are the same as example 1.
Example 3
This example is different from example 1 in that fermentation culture was carried out at 150r/min for 6 days, and the other examples are the same as example 1.
Liquid submerged fermentation time test
And (3) experimental setting:
fermentation group of eucommia leaves: inoculating the liquid strain into a liquid fermentation culture medium, and fermenting and culturing at 28 ℃ and 150r/min for 14 days; fermentation control group: inoculating the liquid strain into nutrient salt solution, and fermenting and culturing at 28 deg.C and 150r/min for 14 days;
water extraction of folium cortex eucommiae: extracting 3g folium Eucommiae powder with 100mL water at 28 deg.C and 150r/min for 14 days.
And (4) respectively measuring the contents of polyphenol, flavone and phenolic acid in the fermentation liquor subjected to alcohol precipitation and polysaccharide removal every 2 days, and determining the optimal fermentation days. As shown in FIG. 1, the optimal fermentation time is 2 days, which is due to the content variation of polyphenol, flavone and phenolic acid. Compared with the eucommia leaf water extract sample, the yields of polyphenol (day 4), flavone (day 2) and phenolic acid (day 2) released into extracellular fluid after fermentation of the eucommia leaves are respectively improved by 6.54%,56.37% and 46.86% (fig. 1 and table 1).
TABLE 1
Figure BDA0002984153230000041
When fermented for 6 days, compared with an aqueous extract sample of eucommia ulmoides leaves, the yield of chlorogenic acid released into extracellular fluid after fermentation of the eucommia ulmoides leaves is improved by 404.30% (fig. 2 and table 1).
When fermented for 4 days, the yield of iridoid released into extracellular fluid after fermentation of the eucommia leaves is improved by 67.28 percent compared with that of eucommia leaf water extract samples (figure 3, table 1).
Control test by solid fermentation method
The inventor further adopts a solid fermentation method to treat the eucommia leaves: solid fermentation: mixing folium Eucommiae powder with nutrient salt solution according to the ratio of 2g folium Eucommiae powder to 1mL nutrient salt solution, sterilizing, inoculating liquid strain with the amount of 0.1mL per 1g folium Eucommiae powder, and fermenting at 28 deg.C for 8 days.
The content of the gutta-percha, the phenolic acid and the flavone is respectively measured every 2 days, and the extraction method of the gutta-percha comprises the following steps: drying the fermented eucommia leaf powder, adding petroleum ether into the dried eucommia leaf powder according to a material-liquid ratio of 1.
The dynamic analysis experiment of the eucommia ulmoides leaves after solid fermentation for 8 days shows that the gutta percha yield is increased and then gradually reduced, the gutta percha yield of the eucommia ulmoides leaves after fermentation for 2 days reaches 4.90 percent at most, is increased by 32.5 percent compared with the yield of a control group which is not fermented, and then is gradually reduced along with the extension of the fermentation time (figure 4).
With different solid fermentation time treatments, the yields of eucommia She Fensuan and flavone are all in a descending trend, and the yields are lower and lower with the time extension, the lowest yield is achieved on the sixth day, the phenolic acid yield is reduced from 12.92mg/g of an unfermented control group to 6.65mg/g (figure 5), and the flavone yield is reduced from 45.52mg/g of the unfermented control group to 24.21mg/g (figure 6).
Comprehensive analysis shows that the solid fermentation method can promote the yield of the gutta-percha, but can reduce the yield of the active substances.
The above-described embodiment is a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (6)

1. A method for improving the yield of active ingredients by utilizing fuscoporia obliqua liquid to carry out submerged fermentation on eucommia leaves is characterized by comprising the following steps:
(1) Activating strains: inoculating inonotus obliquus as a strain to a slant strain culture medium for culture to obtain an activated strain;
(2) Liquid strain culture: transferring the activated strain into a seed culture medium to culture to obtain a liquid strain; performing shake culture at 28 deg.C and rotation speed of 150r/min for 3 days to obtain liquid strain;
(3) Preparing eucommia leaf powder: pulverizing dried Eucommiae cortex She Zhiyu in pulverizer, and sieving to obtain folium Eucommiae powder;
(4) Liquid submerged fermentation: inoculating the liquid strain into a liquid fermentation culture medium, and fermenting and culturing for 2-6 days at the temperature of 28 +/-1 ℃ and the rotating speed of a shaking table of 150 +/-10 r/min; the preparation method of the liquid fermentation medium comprises the following steps: mixing folium Eucommiae powder 3g with 100ml nutrient salt solution, adjusting pH =6.0, and performing wet heat sterilization at 121 + -1 deg.C for 30 min;
(5) Polysaccharide removal of fermentation liquor: after fermentation, obtaining a fermentation stock solution by suction filtration, carrying out rotary evaporation and concentration on the fermentation stock solution to obtain a concentrated solution, then adding a 95% ethanol solution, fully stirring, standing overnight at 4 ℃, carrying out centrifugal separation on polysaccharide in the fermentation liquor, and collecting supernatant as extracellular fluid containing active ingredients;
the active ingredients comprise chlorogenic acid, iridoid, flavone, polyphenol and phenolic acid;
the size of the eucommia ulmoides leaf powder is ≦ 60 meshes.
2. The method of claim 1, wherein the nutrient salt solution is formulated as: corn flour 3.5g, peptone 0.23g, yeast powder 0.12g, caCl 2 0.048 g，CoCl 2 •6H 2 O 0.002 g，KH 2 PO 4 0.1 g，MgSO 4 0.02 g，ZnSO 4 •7H 2 O 0.001 g，K 2 HPO 4 •3H 2 O 0.05 g，FeSO 4 •7H 2 O 0.005 g，CuSO 4 •5H 2 O 0.002 g，MnCl 2 •4H 2 O0.009 g, brought to 100mL with distilled water, pH adjusted =6.0.
3. The method of claim 1, wherein the slant culture medium is formulated as: malt extract 40g, peptone 4g, agar 10g, distilled water to 100ml, pH 5.4-5.6.
4. The method of claim 1, wherein the seed medium consists of: KH (Perkin Elmer) 2 PO 4 0.03 g，MgSO 4 0.02g, yeast extract 0.3g, peptone 0.05g, glucose 1g, distilled water to 100mL, and moist heat sterilization at 121 +/-1 ℃ for 30min.
5. The method of claim 1, wherein in step (4), the fermentation culture is carried out at 28. + -.1 ℃ and 150. + -. 10r/min of shaking table rotation speed for 2-4 days.
6. The method according to claim 1, wherein in the step (5), the 95% ethanol solution is used in an amount of 4 times the volume of the concentrate.
CN202110295422.6A 2021-03-19 2021-03-19 Method for improving active ingredient yield by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid Active CN112972520B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110295422.6A CN112972520B (en) 2021-03-19 2021-03-19 Method for improving active ingredient yield by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110295422.6A CN112972520B (en) 2021-03-19 2021-03-19 Method for improving active ingredient yield by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid

Publications (2)

Publication Number Publication Date
CN112972520A CN112972520A (en) 2021-06-18
CN112972520B true CN112972520B (en) 2022-12-09

Family

ID=76333377

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110295422.6A Active CN112972520B (en) 2021-03-19 2021-03-19 Method for improving active ingredient yield by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid

Country Status (1)

Country Link
CN (1) CN112972520B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564212B (en) * 2021-07-26 2024-01-16 浙江树人学院（浙江树人大学） Method for extracting eucommia ulmoides leaf polysaccharide by utilizing microbial fermentation method
CN114292417B (en) * 2022-01-12 2024-01-19 河南省商业科学研究所有限责任公司 Method for efficiently extracting gutta-percha
CN115886152A (en) * 2022-11-03 2023-04-04 中国海洋大学 Novel eucommia ulmoides fermentation liquor and application thereof in large yellow croaker culture

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110121246A (en) * 2010-04-30 2011-11-07 오숙자 Natural functional food for anti-obesity wellbeing diet and its producing method
CN103156052B (en) * 2013-02-21 2014-06-04 徐向群 Submerged fermentation technology of producing immunological enhancement active materials of inonotus obliquus
CN108715870B (en) * 2018-04-19 2021-05-14 浙江理工大学 Method for increasing yield of triterpene produced by fermentation of inonotus obliquus

Also Published As

Publication number Publication date
CN112972520A (en) 2021-06-18

Similar Documents

Publication Publication Date Title
CN112972520B (en) Method for improving active ingredient yield by deeply fermenting eucommia ulmoides leaves with inonotus obliquus liquid
CN110386860B (en) Efficient extraction method of cannabidiol
CN104013657A (en) Post-fermentation extracting method of extracting saponin from American ginseng medicinal material
CN101353382B (en) Extraction method of antioxidative active Cordyceps sinensis polysaccharide
CN103266154A (en) Biological transformation method for preparing high-activity theasaponin
CN109504725B (en) Method for preparing high-purity hericium erinaceus polysaccharide by fermenting hericium erinaceus and fermentation culture medium
CN110093281B (en) Phellinus igniarius liquid submerged fermentation culture process
CN110684672A (en) Fermentation method of antioxidant cordyceps sobolifera mycelium
CN113897406A (en) Method for extracting and purifying salidroside from rhodiola rosea powder
CN110846354B (en) Preparation method of blackberry lily isoflavone aglycone
CN105766377B (en) A kind of cultural method improving black fungus flavones content and type
CN109364110B (en) Method for extracting flavone from embelia nasuta shell and application thereof
CN115976112A (en) Banana fruit extract and preparation method thereof
CN104069191A (en) Extraction process of total flavonoids of polygonum criopolitanum
CN112662568B (en) Penicillium chrysogenum and application thereof in preparing tannase and degrading tannin
CN112646728B (en) Aspergillus awamori and its application in preparing tannase and degrading tannin
CN111996223A (en) Method for obtaining biological polysaccharide of lucid ganoderma and isatis root by utilizing bidirectional fermentation technology
CN106923350B (en) Method for preparing water-soluble dietary fiber from corn stigma
CN112656829B (en) Method for simultaneously extracting notoginsenoside, notoginsenones and notoginsen polysaccharide and application thereof
CN104045723A (en) Method for extracting tea polysaccharide by biotechnology
CN106399397A (en) Method for increasing content of tyrosol in rhodiola rosea by microorganism fermentation
CN111440249A (en) Method for extracting Fuzhuan tea polysaccharide
CN107130005B (en) Method for simultaneously and efficiently enriching polyphenol and polysaccharide of mulberry leaves and improving α -glucosidase inhibition rate
CN112079938A (en) Highland barley polysaccharide extraction method, highland barley polysaccharide extract and application thereof
CN109593795B (en) Ferulic acid and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant