CN103146775A - Method for preparing epigallocatechin-3-gallate and epigallocatechin gallate by liquid-submerged fermentation - Google Patents
Method for preparing epigallocatechin-3-gallate and epigallocatechin gallate by liquid-submerged fermentation Download PDFInfo
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Abstract
The invention relates to a method for increasing yield of epigallocatechin-3-gallate (EGCG) and epigallocatechin gallate (ECG) by liquid-submerged fermentation of white-rot fungi by adding cellulose, hemi-cellulose and lignin, fatty acids, organic solvents and surfactants. Fermentation in a liquid fermentation tank is carried out with preparation of catechins by fermentation white-rot fungi such as inonotus obliquus as an object and Holland CBS culture collection inonotus obliquus as initial strains. Fermentation conditions are as follows: a temperature is 27-28 DEG C; an air flow is 0.5-1.5 vvm; a stirring speed is 80-160 rounds per minute; an internal pressure is 0.15-0.35 kilograms per cubic centimeter; and a fermentation cycle is 10-14 days. The method can increase the yield of epigallocatechin-3-gallate (EGCG) and epigallocatechin gallate (ECG) by liquid-submerged fermentation of the inonotus obliquus.
Description
Technical field
The present invention relates to a kind of technique of utilizing white-rot fungi liquid submerged fermentation preparation table nutgall catechin gallic acid ester (EGCG) and L-Epicatechin gallate (ECG), belong to biotechnology or bioengineering field.
Technical background
NVP-XAA 723 (EGCG) and L-Epicatechin gallate (ECG) are the strongest tea catechins of anti-oxidant activity, are widely used in medicine, foods and cosmetics industry.Mainly obtain from tealeaves at present, have following problem yet extract to separate from tealeaves: need organic solvent extraction, the mixture of acquisition needs loaded down with trivial details purge process to cause the high and environmental influence of cost, and yield is low.The preparation method exists harmful organic solvent (chloroform, methylene dichloride) residue problem; The purification cost that obtains high-purity epigallocatechin-3-gallate (EGCG), L-Epicatechin gallate (ECG) is high, expensive; The color of extract affects the problem such as use more deeply in beverage, high-grade skin care product.And the uneconomical problem with being difficult to large scale culturing that faces is cultivated by plant tissue cell, so scientific circles still do not have large-scale commercially through effort in 10 years, and the system of plant tissue cell's product tea catechin occurs.
Fungi has been had very long history by mankind's discovery, understanding, utilization, and people are known the most is large-scale edible and medicinal fungi.They are not only nutritious, and the many compositions that contain have stronger physiology and pharmacologically active.Modern study shows that senior basidiomycetes has treatment and prophylactic function widely.The pore fungus fungi is the large monoid of one in basidiomycetes, manyly in the mushroom medicine of China belongs to this class fungi, as famous traditional Chinese medicine glossy ganoderma, umbellate pore furgus, Poria cocos etc.
Adopt the method for liquid submerged fermentation, compare with traditional fungi method of cultivation, have with short production cyclely, cost is low, and output and quality is stable, and product such as can control at the superiority.The biological effective components that utilizes biotechnology the to produce fungi trend that is inevitable, the existing commercial employing liquid submerged fermentation of many medicinal fungis obtains effective constituent in the world at present.Successfully mushroom has been carried out since submerged fermentation and nineteen fifty-seven cultivate the deep liquid of morel with fermentor tank from the U.S. in 1948, the liquid submerged fermentation technology is increasingly extensive in the application of the aspects such as the edible medicinal fungus production of hybrid seeds, comprehensive utilization.At present, existing more than 50 kind of edible medicinal fungus carried out the liquid fermenting experiment in the world.The domestic edible medicinal fungus that has carried out the application of submerged fermentation and suitability for industrialized production has Cordyceps sinensis, rainbow conk, glossy ganoderma, white fungus, hedgehog hydnum, dictyophora phalloidea, umbellate pore furgus, honey mushroom, mushroom, ergot, cuvette goatsbeard, Hericium caput-medusae etc.
When liquid state fermentation was cultivated, various chemical factors all can affect the speed of growth, biomass and the pathways metabolism of fungi, so exploitation suitable medium and fermentation condition are to improve edible and medicinal fungi active substance output, obtained the effective way of high reactivity product.
The present invention utilizes white-rot fungi liquid submerged fermentation preparation table nutgall catechin gallic acid ester (EGCG) and L-Epicatechin gallate (ECG), and in order to improve the biosynthesizing of white-rot fungi EGCG and ECG in liquid submerged fermentation, add bagasse, maize straw, rice straw, straw and wood chip in the liquid submerged fermentation substratum, and the promotor such as lipid acid, organic solvent, tensio-active agent, improve the output of EGCG and ECG.
The present invention has another name called Chaga as example take the rare medicinal fungi Phaeopoms obliquus (Inonotus obliquus) that belongs to white-rot fungi.Phaeopoms obliquus belongs to Basidiomycotina, Hymenomycetes, and non-brown Zoopagales, polyporaceae, brown transverse hole fungus belongs to.Be born on dried-up under the barks of white birch, silvery birch, elm, alder etc. or after felling.Since 16th century, Phaeopoms obliquus is as a kind of Medicinal fungi, in Russia and siberian, eastern Europe is used to prevent and treat difficult and complicated cases, comprise diabetes, infective virus and kinds cancer, be considered to have significant pharmacologically active and to a kind of medicinal fungi of human body without any side effect.
The present invention utilizes the degraded of Mierocrystalline cellulose, hemicellulose, xylogen and the EGCG and the biosynthetic efficient of ECG that are used for improving the Phaeopoms obliquus liquid fermenting of promotor, has important scientific meaning and application prospect.
Summary of the invention
The present invention produces Phaeopoms obliquus liquid fermenting catechins EGCG and ECG is target, has set up a kind of method that Phaeopoms obliquus liquid fermenting is efficient, high yield prepares EGCG and ECG.
Technical scheme of the present invention: the Phaeopoms obliquus fungi is the bacterial classification that sets out, and adopts slant strains to carry out liquid seeds and cultivates.Be on the basis of fermenting carbon source at the W-Gum hydrolysis sugar, add a certain amount of stalk or wood chip and lipid acid, organic solvent, tensio-active agent in liquid medium within, carry out liquid submerged fermentation.Fermentation condition is: 27-28 ℃, air flow quantity 0.5-1.0vvm, mixing speed are 70-150 rev/min, internal pressure 0.1-0.25 kilogram/cubic centimetre, fermentation period 10-14 days.The output of the outer EGCG of born of the same parents and ECG improves more than 200%.
Realize that concrete steps of the present invention are as follows:
(1) take out in the slant medium of the fresh configuration of inclined-plane bacterial strain access, the slant strains culture temperature is 25-30 ℃, cultivates 200-250 hour.
(2) slant strains in step 1 is changed in the shaking flask that liquid seed culture medium is housed over to the liquid seeds culture condition: temperature is 27-28 ℃, shaking speed 70-150 rev/min.Cultivated 96-120 hour.
(3) liquid spawn in step 2 is changed in the fermentor tank that liquid fermentation medium is housed, carry out liquid submerged fermentation.Fermentation condition: temperature is 27-28 ℃, shaking speed 70-150 rev/min.Cultivated 288 hours.
In the present invention, the slant strains substratum forms (g/100mL): malt extract 30, peptone 3, agar 15.The pH value is 5.4-5.6.
In the present invention, the shake-flask seed substratum forms (g/100mL): glucose 2, and peptone 0.3, yeast extract 0.2, KH2PO4 0.1, and MgSO4 0.15, and CaCl2 0.01.
In the present invention, stalk liquid submerged fermentation substratum forms (g/100mL): Semen Maydis powder 5, peptone 0.3, KH2PO4 0.1, ZnSO42H2O 0.001, and K2HPO4 0.04, and FeSO47H2O 0.005, MgSO47H2O 0.05, CuSO45H2O0.002, CoCl2 0.001, and MnSO4H2O 0.008.PH value 6.0.
The standard that fermentation termination of the present invention is established be reducing sugar content lower than 2%, microscopy finds that mycelium senesces.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described in detail
Embodiment 1:
The Phaeopoms obliquus mycelium in the Dutch CBS culture presevation of inoculation storehouse in slant medium.28 ℃ of culture temperature were cultivated 240 hours.Fresh slant strains access is equipped with in the 500mL shaking flask of 200mL liquid seed culture medium, cultivated 96 hours 150 rev/mins of shaking speed for 28 ℃.Cultured liquid seeds is equipped with in the fermentor tank of rice straw fermentation substratum according to 10% (v/v) access.Cultivated approximately 288 hours for 28 ℃.Air flow quantity 1.0vvm, 0.2 kilogram/cubic centimetre of internal pressure, mixing speed is 140 rev/mins.
In the present embodiment 1, slant strains is cultivated, and it is (g/100mL) that substratum forms: malt extract 25, and peptone 4, agar 15, the pH value is 5.4-5.6.
In the present embodiment 1, shake-flask seed is cultivated, and substratum forms (g/100mL): glucose 3, and peptone 0.4, yeast extract 0.1, KH2PO4 0.1, and MgSO4 0.15, and CaCl2 0.01.
In the present embodiment 1, the maize straw liquid submerged fermentation is cultivated, and substratum forms (g/100mL): Semen Maydis powder 3.5, wood chip powder 4, oleic acid 0.1, peptone 0.2, yeast extract 0.1, KH
2PO
40.1, ZnSO
42H
2O 0.001, K
2HPO
40.04, FeSO
47H
2O 0.005, MgSO
47H
2O 0.05, CuSO
45H
2O 0.002.The pH value is 6.0.
The standard that in the present embodiment 1, fermentation stops be reducing sugar content lower than 2%, senile symptom appears in the microscopy mycelium.
The extraction of the outer EGCG of fermentation born of the same parents and ECG: isolate fermented liquid with the vacuum filtration method after fermentation ends, or the Fermented Condensed liquid after ethanol precipitate and separate polysaccharide, with 1: 1 ethyl acetate extraction of volume ratio, extraction liquid concentrating under reduced pressure, vacuum or lyophilize obtain total catechins.The methods such as employing chromatogram are further purified.
Fig. 1 be in the liquid submerged fermentation process the outer EGCG of born of the same parents and ECG content with the Changing Pattern of fermentation time.Phaeopoms obliquus produces cell free fermentation EGCG and ECG at stalk fermentation substratum of the present invention output improves with the prolongation of fermentation time, reaches the highest on the 13rd day in fermentation.In the yield increased substratum the cell free fermentation EGCG that produces and ECG improve 400%.
Embodiment 2:
The Phaeopoms obliquus mycelium in the Dutch CBS culture presevation of inoculation storehouse in slant medium.28 ℃ of culture temperature were cultivated 240 hours.Fresh slant strains access is equipped with in the 500mL shaking flask of 200mL liquid seed culture medium, cultivated 96 hours 140 rev/mins of shaking speed for 28 ℃.Cultured liquid seeds is equipped with in the fermentor tank of wood chip fermention medium according to 10% (v/v) access.Cultivated approximately 302 hours for 28 ℃.Air flow quantity 0.5vvm, 0.1 kilogram/cubic centimetre of internal pressure, mixing speed is 140 rev/mins.
In the present embodiment 2, slant strains is cultivated, and it is (g/100mL) that substratum forms: malt extract 25, and peptone 4, agar 15, the pH value is 5.4-5.6.
In the present embodiment 2, shake-flask seed is cultivated, and substratum forms (g/100mL): glucose 3, and peptone 0.4, yeast extract 0.1, KH2PO4 0.1, and MgSO4 0.15, and CaCl2 0.01.Shaking speed is 150 rev/mins.
In the present embodiment 2, liquid submerged fermentation is cultivated, and substratum forms (g/100mL): Semen Maydis powder 2.5, corn/paddy rice/Wheat Straw powder 3.5, tween 80 0.1, peptone 0.2, yeast extract 0.1, KH
2PO
40.1, ZnSO
42H
2O 0.001, K
2HPO
40.04, FeSO
47H
2O 0.005, MgSO
47H2O 0.05, CuSO
45H
2O 0.002.PH value 6.0.
The standard that in the present embodiment 2, fermentation stops be reducing sugar content lower than 2%, senile symptom appears in the microscopy mycelium.Phaeopoms obliquus produces cell free fermentation EGCG and ECG at stalk fermentation substratum of the present invention output improves with the prolongation of fermentation time, reaches the highest (accompanying drawing 1, blue curve) on the 9th day in fermentation.In the yield increased substratum the cell free fermentation EGCG that produces and ECG improve 300% (accompanying drawing 1, red curve).
Description of drawings
Fig. 1 is the outer EGCG of born of the same parents and ECG output figure in Phaeopoms obliquus liquid submerged fermentation process, and fermentation basic medium (red curve) adds stalk substratum (blue curve), adds sawdust medium (black curve).
Claims (5)
1. one kind is utilized white-rot fungi, the method for liquid submerged fermentation production NVP-XAA 723 (EGCG) and L-Epicatechin gallate (ECG).It is characterized in that with white-rot fungi for the bacterial classification that sets out, adopt slant strains to carry out liquid shaking bottle seed culture, ferment tank.Be fermenting carbon source except adopting traditional W-Gum hydrolysis sugar or glucose in fermenting process, separately replenish a certain amount of Mierocrystalline cellulose, hemicellulose and xylogen, make bacterial classification during the fermentation except utilizing the W-Gum hydrolysis sugar, decompose and utilize these materials.Add simultaneously a certain amount of lipid acid, organic solvent, tensio-active agent in liquid fermentation medium.This method has improved the output of liquid submerged fermentation NVP-XAA 723 (EGCG) and L-Epicatechin gallate (ECG) greatly.
2. the method for utilizing fermented by white rot fungus preparation table nutgall catechin gallic acid ester (EGCG) and L-Epicatechin gallate (ECG) according to claim 1 is characterized in that with white-rot fungi as Phaeopoms obliquus for the bacterial classification that sets out.
3. utilize according to claim 1 the maize straw liquid submerged fermentation to produce the technique of white-rot fungi such as Phaeopoms obliquus fermentation preparation table nutgall catechin gallic acid ester (EGCG) and L-Epicatechin gallate (ECG), it is characterized in that adding the waste of cellulose, hemicellulose and the lignins such as maize straw, rice straw, Wheat And Barley stalk, bagasse, wood chip, waste paper.
4. utilize according to claim 1 the maize straw liquid submerged fermentation to produce the technique of white-rot fungi such as Phaeopoms obliquus fermentation preparation table nutgall catechin gallic acid ester (EGCG) and L-Epicatechin gallate (ECG), it is characterized in that adding lipid acid (linolic acid, oleic acid, palmitinic acid, stearic acid), organic solvent (methyl alcohol, ethanol, normal hexane, toluene), tensio-active agent (tween 80, polysorbas20, Span80, Triton X-100).
5. utilize according to claim 1 the method for white-rot fungi such as Phaeopoms obliquus fermentation preparation table nutgall catechin gallic acid ester (EGCG) and L-Epicatechin gallate (ECG), it is characterized in that adding in the liquid fermentation tank substratum Mierocrystalline cellulose, hemicellulose and xylogen, and lipid acid, organic solvent, tensio-active agent, substratum forms (g/100mL): Semen Maydis powder 3-5, agricultural crop straw powder or wood chip 2-10%, lipid acid, organic solvent, tensio-active agent 0.1-0.5%, peptone 0.2, yeast extract 0.1, KH
2PO
40.1, ZnSO
42H
2O 0.001, K
2HPO
40.04, FeSO
47H
2O 0.005, MgSO
47H
2O 0.05, CuSO
45H
2O 0.002.PH value 6.0.Liquid fermentation condition: 27-28 ℃, air flow quantity 0.5-1.0vvm, mixing speed 70-150 rev/min, internal pressure 0.1-0.25 kilogram/cubic centimetre, fermentation period 12-14 days.
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| CN104673845A (en) * | 2015-02-15 | 2015-06-03 | 华南农业大学 | Method for producing epigallocatechin and gallic acid through transformation of aspergillus niger whole cell |
| CN104673844A (en) * | 2015-02-15 | 2015-06-03 | 华南农业大学 | Method for preventing transformation of epigallocatechin and gallic acid |
| CN106119333A (en) * | 2016-06-30 | 2016-11-16 | 浙江大学 | A kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus |
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| CN106119333A (en) * | 2016-06-30 | 2016-11-16 | 浙江大学 | A kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus |
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Application publication date: 20130612 |