CN106316797A - Method for preparing hydroxytyrosol by using immobilized enzymic method coupled film technique - Google Patents
Method for preparing hydroxytyrosol by using immobilized enzymic method coupled film technique Download PDFInfo
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- CN106316797A CN106316797A CN201510384388.4A CN201510384388A CN106316797A CN 106316797 A CN106316797 A CN 106316797A CN 201510384388 A CN201510384388 A CN 201510384388A CN 106316797 A CN106316797 A CN 106316797A
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- hydroxytyrosol
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/004—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by obtaining phenols from plant material or from animal material
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Abstract
The invention discloses a method for preparing hydroxytyrosol by using an immobilized enzymic method coupled film technique, relates to a preparation method for effective components in plants, and in particular to a method for extracting and preparing hydroxytyrosol. The method for preparing the hydroxytyrosol comprises the following steps: A, performing extraction; B, performing immobilized enzyme enzymolysis; C, performing film filtration; and D, performing spray drying. Compared with the prior art, the method adopts an immobilized enzyme hydrolysis extract; in comparison with acid hydrolysate, the result shows that a relatively large amount of byproducts can be generated in acid hydrolysis, and moreover, oleuropein is not completely converted; immobilized enzyme hydrolysis is good in specificity, the oleuropein can be completely converted into the hydroxytyrosol, the enzymolysis conditions are gentle, and the yield of the hydroxytyrosol can be increased. In addition, immobilization of enzyme plays a role of enzyme protection, and possibility of repeated use of the enzyme is provided. The production cycle of the whole preparation process is shortened, and the production cost is reduced. The method comprises a relatively small number of process steps, is easy to operate and is capable of achieving large-scale industrial production, moreover, the product yield can be up to 85-89%, and the product purity can be up to 40-45%.
Description
Technical field
The present invention relates to the preparation method of effective components in plants, the extraction preparation method of specific design hydroxytyrosol.
Background technology
The molecular formula Vc of 3,4-dihydroxyphenyl ethanol (hydroxytyrosol)8H10O3, structural formula
Being a kind of natural polyphenol compound, it is mainly presented in carboxylate cleupin in each position of Fructus Canarii albi, and cleupin can obtain the hydroxytyrosol dissociated after hydrolysis.Research finds that hydroxytyrosol has the bio-pharmacology activity being much of value to health, is deeply paid attention to by biological and medical circle in recent years.
Hydroxytyrosol is in Fructus Canarii albi mainly presented in cleupin, and cleupin all occupies considerable proportion at each position of Fructus Canarii albi, accounts for 6% ~ 9% (mass fraction) in dry Fructus Canarii albi leaf, is also the olive fruits reason with strong warehouse compartment.In the olive oil course of processing, for going out the sour and astringent taste in olive oil, obtaining polyphenol compound after hydrolysis cleupin, hydroxytyrosol is the main component of these phenol mixtures.Hydroxytyrosol is mainly separated from olive fruit, leaf and the residue produced during preparing olive oil and waste water at present.And the method that the purification of hydroxytyrosol mainly uses chromatographic isolation, such as silicagel column, ion exchange column.Such as the patent of invention of Publication No. CN1665764, disclose a kind of method preparing the compositions rich in hydroxytyrosol, including 1. preparing vegetation water with Fructus Canarii albi；2. add an acid in vegetation water, thus prepare the vegetation water calculating words；3. the vegetation water of acidifying being cultivated a period of time until the Fructus oleae europaeae hardship glycoside (iridoids glucoside Fructus oleae europaeae hardship glycoside) being originally present in vegetation water has at least 50% to change into hydroxytyrosol, wherein described a period of time is at least 2 months, usually 2 ~ 12 months.Although the method can make the 90% of the Fructus oleae europaeae hardship glycoside being originally present in vegetation water be converted into hydroxytyrosol, but complete this conversion and need the time spent oversize, cause the production cycle long, be unsuitable for industrialized production.The and for example patent of invention of Publication No. CN10129811, discloses a kind of method extracting hydroxytyrosol from Fructus Canrii Albi extract liquid, comprises the steps: 1. olive extract adds deionized water dilution；2. cross polythylene resin chromatographic column, with deionized water rinsing, discard the effluent of front 300mL ~ 500mL, then start to collect transparent eluent；3. concentrate described transparent eluent, be extracted with ethyl acetate, by ethyl acetate layer concentrate, reclaim ethyl acetate, concentrated juice can not reconcentration, obtain shallow orange jelly；4. shallow orange jelly is carried out molecular distillation, obtain hydroxytyrosol crude product.The purity of this invention products obtained therefrom is higher, maintain original quality of natural goods the most largely, but the method does not has the step for that in olive extract, Fructus oleae europaeae hardship glycoside changing into hydroxytyrosol, simply being extracted by the hydroxytyrosol of individualism in extracting solution and separate, therefore the yield ratio of this application is relatively low.The patent of invention of publication number CN101973853, disclose the extracting method of the hydroxytyrosol of a kind of with short production cycle, low cost and diversification of feedstock, comprise the steps: that 1. Scrophulariaceae, Oleaceae, Aquifoliaceae, Hypericaceae, Boraginaceae or Verenaceae and Burseraceae Canarium plant are raw material, prepare extracting solution；2. taking said extracted liquid, add concentrated hydrochloric acid, boil 2 ~ 4h hydrolysis, hydrolyzed solution crosses macroporous adsorbent resin, with 10 ~ 30% ethanol elutions, collects eluent, concentrates, is dried, obtains hydroxytyrosol.Although extracting solution is calculated the conversion realizing hydroxytyrosol after talking about by boiling operation by this invention, and shortens transformation time, but high-temperature boiling destroys the molecular structure of hydroxytyrosol, is therefore that purity and the yield of hydroxytyrosol is on the low side.Hydroxytyrosol can't substantial amounts of from the Wastewater by Solvent Extraction of the polyphenol compound containing different molecular weight and be separated by current technology.
Hydroxytyrosol is because of its special biology and pharmacologically active, and purposes is quite varied.In the food industry, the oxidation deterioration of food can be prevented as natural preservative；In nourishing healthy field, can add in various food as supplementary, antibacterial；Can also add in cosmetics, utilize it to catch the ability of free radical, reduce the ultraviolet damaging action to skin, delay skin aging.
The most in succession have at present abroad the capsule of hydroxytyrosol, tablet and powder-product to come out: Life Extension(lengthens the life mechanism) registered trade mark product be Polyphen-oilTM(granule)；The product of Indena company is OleaselectTM(tablet)；The product of the Genosa of company of Spain is HytoliveTM(granule)；Product P rolivols of Seppic company of FranceTM(tablet)；CreAgri company and DSM (DSM) company of Holland have developed HIDROX cooperativelyTM(dry powder).In addition to directly being used as a kind of trophism, functional material by hydroxytyrosol, for some of derivant, such as cleupin, the acetyl derivatives of hydroxytyrosol, the applied research with the derivant etc. of polyunsaturated fat chain is the most constantly carried out.This has critically important facilitation for the application being continuously increased hydroxytyrosol.
Summary of the invention
The present invention is the method using enzyme process coupled film technology to prepare hydroxytyrosol.Using the method to prepare hydroxytyrosol, conversion ratio is high, yield is high.The present invention is by the following technical solutions:
1. preparing olive extract, described raw material is the sarcocarp of Burseraceae Canarium plant, stem or leaf；
2. take any of the above-described kind of extracting solution, add immobilization glycosidase, add the Acetic acid-sodium acetate buffer of pH4.8,50 ~ 55 DEG C of water-bath 2 ~ 4h.
3. membrane filtration hydrolyzed solution.
4. concentrate, be spray-dried, obtain hydroxytyrosol.
In the preparation method of hydroxytyrosol of the present invention, the described step 2. middle immobilization glycosidase added, after selecting sodium alginate to be carrier, with glutaraldehyde cross-linking, glycosidase is embedded glomeration immobilized enzyme.
In the preparation method of hydroxytyrosol of the present invention, 3. described step farther includes to use extracting solution described in micro-filtration membrane, ultrafilter membrane and nanofiltration membrane.
Compared with prior art, the present invention uses immobilized enzyme hydrolysis extracting solution, compares with acid hydrolysis liquid, and result shows that acid hydrolysis can produce more by-product, and cleupin is not completely converted；And immobilized enzyme hydrolysis, specificity is strong, and cleupin can be completely converted as hydroxytyrosol, and enzymatic hydrolysis condition is gentle, improves the yield of hydroxytyrosol.Additionally, enzyme immobilizatio plays the effect of protective enzyme, the recycling for enzyme provides possibility.From whole preparation process, shorten the production cycle, save production cost.
Detailed description of the invention
With embodiment, the invention will be further described below, but the invention is not limited in these embodiments.
Embodiment 1:
1. with Folium olive as raw material, it is ground into coarse powder, adds in 50% ethanol water of 6 times of weight, multi-function extractor carries out dynamic circulation extraction, extracts twice, extract 1h, it is thus achieved that extracting solution every time；
2. concentrated extracting solution, stirs and carries out water precipitating 12 hours；
3. by above-mentioned water precipitating liquid plate-and-frame filtration, separate and obtain limpid filtrate；
4. adding 10U immobilized enzyme in filtrate, add the NaAc_HAc buffer solution of pH4.8,50 DEG C are reacted 3 hours；
5. by said hydrolyzed liquid, join in the feed liquid bucket of film p-type UF-19 membrane ultrafiltration device, with retaining the organic membrane that relative molecular weight is 30,000, carrying out the ultrafiltration of continuous way, during ultrafiltration, temperature of charge controls at about 30 DEG C, regulates reflux inlet, the pressure controlling material liquid outlet is 100PSI, collects and filters liquid；
6. it is concentrated into extractum, is spray-dried, obtains hydroxytyrosol, purity 40%, yield 85%.
Embodiment 2:
1. with Folium olive as raw material, it is ground into coarse powder, adds in 30% ethanol water of 6 times of weight, multi-function extractor carries out dynamic circulation extraction, extracts twice, extract 1h, it is thus achieved that extracting solution every time；
2. concentrated extracting solution, stirs and carries out water precipitating 12 hours；
3. by above-mentioned water precipitating liquid plate-and-frame filtration, separate and obtain limpid filtrate；
4. adding 10U immobilized enzyme in filtrate, add the NaAc_HAc buffer solution of pH4.8,55 DEG C are reacted 2 hours；
5. by said hydrolyzed liquid, join in the feed liquid bucket of film p-type UF-19 membrane ultrafiltration device, with retaining the organic membrane that relative molecular weight is 10,000, carrying out the ultrafiltration of continuous way, during ultrafiltration, temperature of charge controls at about 30 DEG C, regulates reflux inlet, the pressure controlling material liquid outlet is 100PSI, collects and filters liquid；
6. it is concentrated into extractum, is spray-dried, obtains hydroxytyrosol, purity 42%, yield 88%.
Embodiment 3:
1. with Folium olive as raw material, it is ground into coarse powder, adds in 70% ethanol water of 6 times of weight, multi-function extractor carries out dynamic circulation extraction, extracts twice, extract 1h, it is thus achieved that extracting solution every time；
2. concentrated extracting solution, stirs and carries out water precipitating 12 hours；
3. by above-mentioned water precipitating liquid plate-and-frame filtration, separate and obtain limpid filtrate；
4. adding 10U immobilized enzyme in filtrate, add the NaAc_HAc buffer solution of pH4.8,52 DEG C are reacted 2.5 hours；
5. by said hydrolyzed liquid, join in the feed liquid bucket of film p-type UF-19 membrane ultrafiltration device, with retaining the organic membrane that relative molecular weight is 10,000, carrying out the ultrafiltration of continuous way, during ultrafiltration, temperature of charge controls at about 30 DEG C, regulates reflux inlet, the pressure controlling material liquid outlet is 100PSI, collects and filters liquid；
6. it is concentrated into extractum, is spray-dried, obtains hydroxytyrosol, purity 45%, yield 86%.
Claims (4)
1. immobilized enzyme method coupled film technology prepares a preparation method for hydroxytyrosol, comprises the steps:
1. the extraction of hydroxytyrosol
Described raw material is the sarcocarp of Burseraceae Canarium plant, stem or leaf, extracts than for 1:2 ~ 10 with extraction volumetric quality, extract twice time, merge twice extracting solution after pulverizing；
2. enzymolysis
Take said extracted liquid, add immobilization glycosidase, add the Acetic acid-sodium acetate buffer of pH4.8,50 ~ 55 DEG C of water-bath 2 ~ 4h；
3. filter
Enzymolysis solution being cooled to room temperature, is that 48 ~ 0.8 μm flame filter presses filter with filter cloth aperture, wherein pressure is 0.1 ~ 0.8Mpa；Liquid after plate-and-frame filtration carries out lower reaches through UF-19 membrane ultrafiltration device, and wherein the pressure of micro-filtration is 80 ~ 200PSL；
4. concentrate, be spray-dried
Obtaining not alcoholic solution after decompression recycling ethanol, this solution is concentrated further by nanofiltration or reverse osmosis and puts proportion is 1.02 ~ 1.20, and wherein NF membrane aperture is 200 ~ 500Da；Liquid after concentration is spray-dried makes powder, obtains hydroxytyrosol.
Method the most according to claim 1, it is characterised in that what 1. step was selected prepares the raw material of hydroxytyrosol is the sarcocarp of Burseraceae Canarium plant, stem or leaf.
Method the most according to claim 1, it is characterised in that the immobilized enzyme that 2. step adds is glycosidase, hydrolysis temperature is 50 ~ 55 DEG C, and the response time is 2 ~ 4h.
Method the most according to claim 1, it is characterised in that 3. step selects membrane technology to carry out isolated and purified to hydroxytyrosol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108314607A (en) * | 2018-05-08 | 2018-07-24 | 广西大学 | A kind of purification process of high-purity solid hydroxytyrosol |
| CN114150025A (en) * | 2021-12-06 | 2022-03-08 | 西安海斯夫生物科技有限公司 | Preparation method of high-purity hydroxytyrosol |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004009206A1 (en) * | 2002-07-17 | 2004-01-29 | Centro De Investigaciones Energéticas, Medioambientales Y Tecnológicas (C.I.E.M.A.T.) | Method of extracting phenolic compounds from a residual plant material using a hydrothermal process |
| CN104725197A (en) * | 2015-03-19 | 2015-06-24 | 黑龙江八一农垦大学 | Method for preparing hydroxytyrosol by converting oleuropein through ion exchange resin |
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2015
- 2015-06-30 CN CN201510384388.4A patent/CN106316797A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004009206A1 (en) * | 2002-07-17 | 2004-01-29 | Centro De Investigaciones Energéticas, Medioambientales Y Tecnológicas (C.I.E.M.A.T.) | Method of extracting phenolic compounds from a residual plant material using a hydrothermal process |
| CN104725197A (en) * | 2015-03-19 | 2015-06-24 | 黑龙江八一农垦大学 | Method for preparing hydroxytyrosol by converting oleuropein through ion exchange resin |
Non-Patent Citations (2)
| Title |
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| 卜文文: "油橄榄叶中羟基酪醇的提取纯化及抗氧化性研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 郑宝东 等: "《食品酶学》", 31 October 2006, 东南大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108314607A (en) * | 2018-05-08 | 2018-07-24 | 广西大学 | A kind of purification process of high-purity solid hydroxytyrosol |
| CN114150025A (en) * | 2021-12-06 | 2022-03-08 | 西安海斯夫生物科技有限公司 | Preparation method of high-purity hydroxytyrosol |
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