CN105177077A - Method for preparing epicatechin (EC) monomer through microorganism fermentation and oriented transformation - Google Patents
Method for preparing epicatechin (EC) monomer through microorganism fermentation and oriented transformation Download PDFInfo
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Abstract
The invention discloses a method for preparing an epicatechin (EC) monomer through microorganism fermentation and oriented transformation, wherein the method mainly comprises the following steps: (1) transforming epicatechin gallate (ECG) in a green tea extract into epicatechin (EC) through microorganism liquid deep fermentation; (2) separating and purifying the crude epicatechin (EC) through reversed phase packing chromatography so as to obtain a finished high-content epicatechin (EC is more than 90%) product; and (3) dissolving the obtained epicatechin product with ethanol with concentration being 20% and being 5-6 times (w/v) the total weight of the epicatechin product, standing and crystallizing at 4 DEG C, removing a mother liquid after a crystal grows sufficiently, washing the crystal until a washing liquid is colorless by virtue of cold water at 4 DEG C, and freeze-drying the obtained crystal so as to obtain a white or white-like solid which is the epicatechin (EC) monomer. The method disclosed by the invention is simple and convenient, short in preparation cycle and low in cost, and the method meets the requirements of environmental protection and safety and is applicable to large-scale industrial production.
Description
Technical field
The invention belongs to food technology field, be specifically related to a kind of microbial fermentation engineering orientation and transform the method with reverse phase filler column chromatography technique coupling separation and purification l-Epicatechol [(-)-Epicatechin, EC] (CAS:490-46-0) monomer from green tea extract.
Background technology
Catechin compounds is the main component of tea health-care function, both at home and abroad large quantity research report catechin has and removes interior free yl, anti-oxidant, prevention cardiovascular and cerebrovascular diseases, Tumor suppression growth and antiviral, radioprotective etc. significantly physiological and pharmacological is active, is widely used in food-drink and filed of daily-use chemical industry
[1-3].In October, 2006, U.S. FDA official approval is that the plant amedica Veregen(NDA of main component numbers with EGCG: No.21902) for the skin treating of genitalia wart and perianal wart, become united states drug amendment regulations in 1962 and carry out first plant (draft) prescription drug ratifying listing, huge repercussion is caused in plant amedica field, the world, also greatly advance the development of tea leaf extract industry, the separation and purification of other each monomer components of catechin, activity research and clinical application also become rapidly the new focus of Tea Science disciplinary study and industry development
[4-7].
L-Epicatechol [(-)-Epicatechin, EC] (CAS:490-46-0) have lowering blood-fat and reducing weight equally, prevention cardiovascular and cerebrovascular diseases, the multiple physiologically active effects such as anti-cancer anti-mutation, compared with other catechin compositions, especially spatial memory and the cognitive function of person in middle and old age's study subject can be significantly improved, thus cause the great interest of Chinese scholars, but due to its in green tea materials content well below EGCG, and and ECG, EGC and D, the chemical composition structural similitudies such as L-C, fine separation technical difficulty is high, the industrialization preparation technology of high purity EC monomer is not yet had to emerge so far
[8-12].The present invention with green tea extract (85% polyphenol) for raw material, screen suitable microorganism kind and carry out directed conversion of fermenting, first by the ECG(L-Epicatechin gallate in raw material) orientation is converted into EC, its content in green tea extract is made to improve 3-4 doubly, again by reverse phase filler column chromatography separating purification, reverse osmosis membrane concentrates, and low temperature crystallization, final lyophilize obtains 98%EC monomer.In three, (orientation fermentation increases EC concentration in substrate, eliminates ECG separation interference in substrate present invention process; reversed-phase column chromatography is separated) set about simultaneously; fundamentally breach technical bottleneck prepared by the industrialization of high-purity EC monomer, EC yield significantly improves, and is applicable to large-scale industrial and produces.
Reference is as follows:
[1]GADKARI,PravinVasantrao;BALARAMAN,Manohar.Catechins:Sources,extractionandencapsulation:Areview.FoodandBioproductsProcessing,2015,93:122-138.
[2] Li Ning. catechin. Journal of Nutrition, 2013,35.3:227-229.
[3] Zhu Guiqin; Li Jianke. the Research progress on Function of tea-polyphenol. food research and development, 2005,26.1:33-35.
[4]NAWAB,Amber;FAROOQ,Najaf.Reviewongreenteaconstituentsanditsnegativeeffects.2015.
[5]KARIM,AnkitaJain;DALAI,DeepakRanjan.Greentea:Areviewonitsnaturalanti-oxidanttherapyandcariostaticbenefits.2014.
[6] progress of Zhang Yan Lai, etal. NVP-XAA 723. herbal medicine, 2006,37.2:303-306.
[7] Wang Xia; GAO Lijuan; Lin Ping Chang. the separation and preparation of NVP-XAA 723 (EGCG). Food science, 2006,26.9:242-246.
[8]AZAM,S.,etal.Prooxidantpropertyofgreenteapolyphenolsepicatechinandepigallocatechin-3-gallate:implicationsforanticancerproperties.Toxicologyinvitro,2004,18.5:555-561.
[9]VAIDYANATHAN,JayaBharathi;WALLE,Thomas.Transportandmetabolismoftheteaflavonoid(–)-epicatechinbythehumanintestinalcelllineCaco-2.Pharmaceuticalresearch,2001,18.10:1420-1425.
[10]VAIDYANATHAN,JayaBharathi;WALLE,Thomas.Glucuronidationandsulfationoftheteaflavonoid(?)-epicatechinbythehumanandratenzymes.Drugmetabolismanddisposition,2002,30.8:897-903.
[11]PIOTRKOWSKI,Barbara,etal.(?)-Epicatechinpreventsalterationsinthemetabolismofsuperoxideanionandnitricoxideintheheartsofl-NAME-treatedrats.Food&function,2015,6.1:154-160.
[12]SHAY,Joseph,etal.MolecularMechanismsandTherapeuticEffectsof(?)-EpicatechinandOtherPolyphenolsinCancer,Inflammation,Diabetes,andNeurodegeneration.OxidativeMedicineandCellularLongevity,2015,2015。
Summary of the invention
Technical problem to be solved by this invention is separation and purification l-Epicatechol (EC from green tea extract, purity >=98%) monomer, optimize separation and purification of products technical process, significantly improve product yield, product can separately or composite usage in food, beverage, daily-use chemical industry and medicine field, the inventive method is easy, preparation cycle is short, cost is low, meets the requirement of environmental protection and safety, and suitability for mass industrializedization is produced.
The inventive method coupling biological fermentation engineering, reversed-phase polymerization thing filler column chromatography and reverse osmosis membrane concentration technique,
Technical scheme provided by the invention is: a kind of method transforming preparation table catechin (EC) monomer through fermentable orientation, mainly comprises the steps:
(1) utilize microbial liquid submerged fermentation that the L-Epicatechin gallate (ECG) in green tea extract is converted into epigallocatechin (EC):
Add pure water and dissolve green tea extract tea-polyphenol, being configured to concentration is 1%(w/v) solution, regulate pH=6.0, feed liquid, after high-temperature short-time sterilization, injects fermentor tank, keeps temperature 32
+1 DEG C and inoculating strain, stir fermentation culture 60-80 hour; After the deactivation of fermentation liquor high-temperature instantaneous, centrifuging, gets clear liquor and is concentrated into 20%(w/v) after concentration, spraying dry, product is l-Epicatechol (EC) crude product;
(2) by above-mentioned l-Epicatechol (EC) crude product through the separation and purification of reverse phase filler thin layer chromatography:
Add pure water to dissolve EC crude product to be configured to concentration be 10%(w/v) upper prop feed liquid, reversed-phase polymerization thing filler column chromatographic isolation and purification, reverse osmosis membrane concentrates desorb cut, and spraying dry obtains high-content l-Epicatechol (EC>90%) product;
(3) by above-mentioned high-content l-Epicatechol (EC>90%) product 5 ~ 6 times amount (w/v) 20% dissolve with ethanol, place crystallization for 4 DEG C, treat that crystal fully grows, remove mother liquor, 4 DEG C of cold water washing crystal are colourless to washings, by the crystal lyophilize obtained, obtain white or off-white color solid, be l-Epicatechol (EC) monomer (>98%).
Described method, in step (1), described bacterial strain is flavus Cr-1 bacterial strain, aspergillus oryzae (Asp.oryzae), red colouring agent for food, also used as a Chinese medicine belongs to (Monascus), aspergillus niger (Aspergillusniger), Zygosaccharomyces rouxii (Mucorrouxii), a kind of in iS-One monascus (Monascus.anka).
Described method, in step (1), regulates pH=6.0 with NaOH; Bacterial classification spore inoculating amount 8*10
11individual/L, stirs fermentation culture 72 hours.
Described method, in step (2), described reverse polymerization thing filler is: one of MCIGELCHP55Y, MCIGELCHP20SS, MCIGELCHP20P series, size distribution 35 ~ 150 μm.
Described method, in step (2), described reversed-phase polymerization thing filler column chromatographic isolation and purification, applied sample amount EC crude product weight (Kg)/amount of filler (L) is 2:1; Upper column flow rate: 100cm/h; Desorption solvent: 20% ethanol elution 1BV, 2BV resolved by 60% ethanol, collects 60% and resolves cut; Desorb flow velocity: 100cm/h; Maximum operating pressure <4MPa.
Described method, in step (2), reverse osmosis membrane concentrates 60% desorb cut, and service temperature controls, below 40 DEG C, to enter film pressure and control at below 15Bar.
The present invention has following beneficial effect: the present invention with green tea extract (85% polyphenol) for raw material, through lot of experiments, filter out a kind of orientation fermentation Transformed E CG(L-Epicatechin gallate) for l-Epicatechol (EC) and can the microorganism strains of enduring high-concentration catechin, be inoculated in the aqueous solution of green tea extract, through deep liquid fermentation process process, the content of EC in substrate is made to improve 3 ~ 4 times, again with reverse phase filler column chromatography separating purification, l-Epicatechol (EC) desorb cut is rich in collection, reverse osmosis membrane normal temperature concentrates, low temperature crystallization, final lyophilize obtains l-Epicatechol (EC) monomer (l-Epicatechol [(-)-Epicatechin that content is greater than 98%, EC] (CAS:490-46-0)).
The inventive method coupling biological fermentation engineering, reversed-phase polymerization thing filler column chromatography and reverse osmosis membrane concentration technique, separation and purification l-Epicatechol (EC from green tea extract, purity >=98%) monomer, optimize separation and purification of products technical process, significantly improve product yield, product can separately or composite usage in food, beverage, daily-use chemical industry and medicine field.The inventive method is easy, and preparation cycle is short, and cost is low, meets the requirement of environmental protection and safety, and suitability for mass industrializedization is produced.
Embodiment
Detailed description below by embodiment illustrates the present invention further, but is not limitation of the present invention, only does example explanation.
The present invention transforms the method for preparation table catechin [(-)-Epicatechin, EC] monomer through fermentable orientation, mainly comprises the steps:
A, utilize microbial liquid submerged fermentation that the L-Epicatechin gallate (ECG) in green tea extract is converted into epigallocatechin (EC): to add pure water and dissolve green tea extract 85% tea-polyphenol (purchased from good fortune bio tech ltd, Changsha three, wherein total catechin content is 71.44%, ECG content is 18.82%, EC content is 4.33%), being configured to concentration is 1%(w/v) solution, NaOH regulates pH=6.0, and feed liquid is after high-temperature short-time sterilization, inject fermentor tank, keep temperature 32
+1 DEG C and inoculating strain, spore inoculating amount 8*10
11individual/L, stirs fermentation culture 72 hours; After the deactivation of fermentation liquor high-temperature instantaneous, the centrifuging of 1000r/min tripod pendulum type batch centrifugal, gets clear liquor and is concentrated into 20%(w/v) after concentration, use centrifugal spray tower spraying dry, product is l-Epicatechol crude product (EC>15%.
Connecing bacterial strain is flavus Cr-1 bacterial strain, aspergillus oryzae (Asp.oryzae), red colouring agent for food, also used as a Chinese medicine belongs to (Monascus), aspergillus niger (Aspergillusniger), Zygosaccharomyces rouxii (Mucorrouxii), a kind of in iS-One monascus (Monascus.anka)
B, by above-mentioned l-Epicatechol (EC) crude product through the separation and purification of reverse phase filler thin layer chromatography:
Add pure water to dissolve EC crude product to be configured to concentration be 10%(
w/v) upper prop feed liquid, reversed-phase polymerization thing filler column chromatographic isolation and purification (applied sample amount 2:1, EC crude product weight (Kg)/amount of filler (L); Upper column flow rate: 100cm/h; Desorption solvent: 20% ethanol elution 1BV, 2BV resolved by 60% ethanol, collects 60% and resolves cut; Desorb flow velocity: 100cm/h; Maximum operating pressure <4MPa).Reverse osmosis membrane concentrates 60% desorb cut, and service temperature controls, below 40 DEG C, to enter film pressure and control at below 15Bar.Centrifugal spray drying obtains high-content l-Epicatechol (EC>90%) product.
C, by above-mentioned high-content l-Epicatechol (EC>90%) product 5 ~ 6 times amount (w/v) 20% dissolve with ethanol, place crystallization for 4 DEG C, treat that crystal fully grows, use that link-suspended basket centrifuge is centrifugal removes mother liquor, 4 DEG C of cold water washing crystal are colourless to washings, by the crystal lyophilize obtained, obtain white or off-white color solid, be l-Epicatechol (EC) monomer, with l-Epicatechol (EC) standard substance [purchased from American Sigma-Aldrich company, C
15h
14o
6, M.W.290.27, numbering: 207-710-1, purity>=98%(HPLC)] contrast qualification and be detected as EC>=98% through high-efficient liquid phase chromatogram technique analysis.
Claims (6)
1. transform a method for preparation table catechin (EC) monomer through fermentable orientation, mainly comprise the steps:
(1) utilize microbial liquid submerged fermentation that the L-Epicatechin gallate (ECG) in green tea extract is converted into epigallocatechin (EC):
Add pure water and dissolve green tea extract tea-polyphenol, being configured to concentration is 1%(
w/v) solution, regulate pH=6.0, feed liquid after high-temperature short-time sterilization, inject fermentor tank, keep temperature 32
+1 DEG C and inoculating strain, stir fermentation culture 60-80 hour; After the deactivation of fermentation liquor high-temperature instantaneous, centrifuging, gets clear liquor and is concentrated into 20%(w/v) after concentration, spraying dry, product is l-Epicatechol (EC) crude product;
(2) by above-mentioned l-Epicatechol (EC) crude product through the separation and purification of reverse phase filler thin layer chromatography:
Add pure water to dissolve EC crude product to be configured to concentration be 10%(w/v) upper prop feed liquid, reversed-phase polymerization thing filler column chromatographic isolation and purification, reverse osmosis membrane concentrates desorb cut, and spraying dry obtains high-content l-Epicatechol (EC>90%) product;
(3) by above-mentioned high-content l-Epicatechol (EC>90%) product 5 ~ 6 times amount (w/v) 20% dissolve with ethanol, place crystallization for 4 DEG C, treat that crystal fully grows, remove mother liquor, 4 DEG C of cold water washing crystal are colourless to washings, by the crystal lyophilize obtained, obtain white or off-white color solid, be l-Epicatechol (EC) monomer (>98%).
2. in accordance with the method for claim 1, it is characterized in that: in step (1), described bacterial strain is flavus Cr-1 bacterial strain, aspergillus oryzae (Asp.oryzae), red colouring agent for food, also used as a Chinese medicine belongs to (Monascus), aspergillus niger (Aspergillusniger), Zygosaccharomyces rouxii (Mucorrouxii), a kind of in iS-One monascus (Monascus.anka).
3. in accordance with the method for claim 1, it is characterized in that: in step (1), regulate pH=6.0 with NaOH; Bacterial classification spore inoculating amount 8*10
11individual/L, stirs fermentation culture 72 hours.
4. in accordance with the method for claim 1, it is characterized in that: in step (2), described reverse polymerization thing filler is: one of MCIGELCHP55Y, MCIGELCHP20SS, MCIGELCHP20P series, size distribution 35 ~ 150 μm.
5. in accordance with the method for claim 1, it is characterized in that: in step (2), described reversed-phase polymerization thing filler column chromatographic isolation and purification, applied sample amount EC crude product weight (Kg)/amount of filler (L) is 2:1; Upper column flow rate: 100cm/h; Desorption solvent: 20% ethanol elution 1BV, 2BV resolved by 60% ethanol, collects 60% and resolves cut; Desorb flow velocity: 100cm/h; Maximum operating pressure <4MPa.
6. in accordance with the method for claim 1, it is characterized in that: in step (2), reverse osmosis membrane concentrates 60% desorb cut, and service temperature controls, below 40 DEG C, to enter film pressure and control at below 15Bar.
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| CN115612551A (en) * | 2022-10-21 | 2023-01-17 | 湖南新金浩茶油股份有限公司 | Method for preparing high-oxidation-resistance camellia seed oil by fresh pressing of camellia seed green fruit |
Citations (2)
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|---|---|---|---|---|
| CN102115467A (en) * | 2009-12-30 | 2011-07-06 | 温尧林 | Method for preparing catechin monomers |
| 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 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115467A (en) * | 2009-12-30 | 2011-07-06 | 温尧林 | Method for preparing catechin monomers |
| 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 |
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| 魏志文等: "儿茶素单体分离制备方法", 《食品与发酵工业》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115612551A (en) * | 2022-10-21 | 2023-01-17 | 湖南新金浩茶油股份有限公司 | Method for preparing high-oxidation-resistance camellia seed oil by fresh pressing of camellia seed green fruit |
| CN115612551B (en) * | 2022-10-21 | 2024-01-16 | 湖南新金浩茶油股份有限公司 | Method for preparing high-oxidation-resistance camellia seed oil by freshly squeezing camellia seed and olive |
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Application publication date: 20151223 |