CN112972520A - 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 PDFInfo
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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) culturing liquid strains; (3) preparing eucommia leaf powder; (4) liquid submerged fermentation; (5) and (3) polysaccharide removal of 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
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 effective components of the natural Chinese herbal medicines, the plant cell walls are greatly damaged after enzymolysis pretreatment, the effective components 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 in folks as a specific medicine for treating various difficult and complicated diseases, and the chemical components of 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 folium Eucommiae in a 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 to 6.0, and wet-heat sterilizing at 121 + -1 deg.C for 30min to obtain the final product;
(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 ulmoides is a rare or endangered two-class protective tree species in China, and the cost for extracting active ingredients from eucommia ulmoides leaves is increased along with the eucommia ulmoides. 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 and CaCl2 0.048g，CoCl2·6H2O 0.002g，KH2PO4 0.1g，MgSO4 0.02g，ZnSO4·7H2O 0.001g，K2HPO4·3H2O 0.05g，FeSO4·7H2O 0.005g，CuSO4·5H2O 0.002g，MnCl2·4H2O0.009 g, made 100mL with distilled water and adjusted to 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, and the volume is adjusted to 100ml by distilled water, and the pH value is 5.4-5.6.
Preferably, the seed culture medium consists of: KH (Perkin Elmer)2PO4 0.03g，MgSO40.02g, yeast extract 0.3g, peptone 0.05g and glucose 1g, adding distilled water to a constant volume of 100mL, and performing moist heat sterilization at 121 +/-1 ℃ for 30 min.
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: 40g of malt extract, 4g of peptone and 10g of agar, wherein the volume is fixed to 100ml by using distilled water, the pH value is 5.4-5.6, and the parameters of culture on a slant strain culture medium are as follows: the temperature is 28 ℃, and the culture is carried out for 250 h.
(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)2PO4 0.03g，MgSO40.02g, 0.3g of concentrated yeast extract, 0.05g of peptone and 1g of glucose, wherein distilled water is used for fixing the volume to 100mL, and the wet heat sterilization is carried out at 121 ℃ for 30 min.
(3) Preparing eucommia leaf powder: and (3) placing the dried eucommia leaves into a stirring pulverizer for pulverizing, 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: inoculating 2ml of liquid strain into a liquid fermentation culture medium, and fermenting and culturing for 2 days at 28 ℃ and 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 and CaCl2 0.048g，CoCl2·6H2O 0.002g，KH2PO4 0.1g，MgSO4 0.02g，ZnSO4·7H2O 0.001g，K2HPO4·3H2O 0.05g，FeSO4·7H2O 0.005g，CuSO4·5H2O 0.002g，MnCl2·4H2O0.009 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 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
When fermented for 6 days, compared with the eucommia ulmoides leaf water extract sample, the yield of chlorogenic acid released into extracellular fluid after fermentation of the eucommia ulmoides leaves is improved by 404.30% (figure 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 and 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 the material-liquid ratio of 1:16(g/mL), heating and refluxing for 1h at 70 ℃, filtering while hot to obtain filtrate, cooling to room temperature, placing in a refrigerator at-20 ℃ for 7h to separate out gutta-percha, filtering, freeze-drying to obtain gutta-percha, and weighing.
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).
The yields of the phenolic acid and the flavone of the eucommia ulmoides leaves are in a descending trend by different solid fermentation time treatments, the yield is lower and lower along with the time, the lowest yield is achieved on the sixth day, the yield of the phenolic acid is reduced from 12.92mg/g of an unfermented control group to 6.65mg/g (figure 5), and the yield of the flavone 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 embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (8)
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 150r/min for 3 days to obtain liquid strain;
(3) preparing eucommia leaf powder: pulverizing dried folium Eucommiae in a 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 to 6.0, and wet-heat sterilizing at 121 + -1 deg.C for 30min to obtain the final product;
(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.
2. The method of claim 1, wherein the active ingredients comprise chlorogenic acid, iridoids, flavones, polyphenols and phenolic acids.
3. The method of claim 1, wherein the folium Eucommiae powder has a size ≦ 60 mesh.
4. The method of claim 1, wherein the nutrient salt solution is formulated as: 3.5g of corn flour, 0.23g of peptone, 0.12g of yeast powder and CaCl2 0.048g，CoCl2·6H2O 0.002g，KH2PO4 0.1g，MgSO4 0.02g，ZnSO4·7H2O 0.001g，K2HPO4·3H2O 0.05g，FeSO4·7H2O 0.005g，CuSO4·5H2O 0.002g，MnCl2·4H2O0.009 g, made 100mL with distilled water and adjusted to pH 6.0.
5. The method of claim 1, wherein the slant culture medium is formulated as: 40g of malt extract, 4g of peptone and 10g of agar, and the volume is adjusted to 100ml by distilled water, and the pH value is 5.4-5.6.
6. The method of claim 1, wherein the seed medium consists of: KH (Perkin Elmer)2PO4 0.03g，MgSO40.02g, yeast extract 0.3g, peptone 0.05g and glucose 1g, adding distilled water to a constant volume of 100mL, and performing moist heat sterilization at 121 +/-1 ℃ for 30 min.
7. The method of claim 1, wherein in step (4), the fermentation culture is carried out at a temperature of 28. + -. 1 ℃ and a shaker rotation speed of 150. + -.10 r/min for 2-4 days.
8. 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.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113564212A (en) * | 2021-07-26 | 2021-10-29 | 浙江树人学院（浙江树人大学） | Method for extracting eucommia ulmoides leaf polysaccharide by using microbial fermentation method |
| CN114292417A (en) * | 2022-01-12 | 2022-04-08 | 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 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113564212A (en) * | 2021-07-26 | 2021-10-29 | 浙江树人学院（浙江树人大学） | Method for extracting eucommia ulmoides leaf polysaccharide by using microbial fermentation method |
| 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 |
| CN114292417A (en) * | 2022-01-12 | 2022-04-08 | 河南省商业科学研究所有限责任公司 | Method for efficiently extracting gutta-percha |
| 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 |
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| CN112972520B (en) | 2022-12-09 |
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