CN104926615B - Method of using crude extract of olive leaf oleuropein to prepare hydroxytyrosol - Google Patents
Method of using crude extract of olive leaf oleuropein to prepare hydroxytyrosol Download PDFInfo
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- CN104926615B CN104926615B CN201510371820.6A CN201510371820A CN104926615B CN 104926615 B CN104926615 B CN 104926615B CN 201510371820 A CN201510371820 A CN 201510371820A CN 104926615 B CN104926615 B CN 104926615B
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- 0 C*C(C*(C*)*1)C1(C(O)O[C@](C(C1CC(OCCc(cc2O)ccc2O)=O)=CC)OC=C1C(OC)=*)O Chemical compound C*C(C*(C*)*1)C1(C(O)O[C@](C(C1CC(OCCc(cc2O)ccc2O)=O)=CC)OC=C1C(OC)=*)O 0.000 description 2
- JUUBCHWRXWPFFH-UHFFFAOYSA-N OCCc(cc1)cc(O)c1O Chemical compound OCCc(cc1)cc(O)c1O JUUBCHWRXWPFFH-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- 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
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
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- C—CHEMISTRY; METALLURGY
- 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
- C07C37/001—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by modification in a side chain
- C07C37/002—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by modification in a side chain by transformation of a functional group, e.g. oxo, carboxyl
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- C—CHEMISTRY; METALLURGY
- 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 of using crude extract of olive leaf oleuropein to prepare hydroxytyrosol. Compared with acid hydrolysis and alkali hydrolysis, the enzymolysis process for crude extract of olive leaf oleuropein is mild in method condition, and consumption of acid and alkali catalysts is reduced in the whole process. Hemicellulase is selected from ten enzymes as an enzyme best in oleuropein degrading effect by taking oleuropein degrading rate and hydroxytyrosol content as indexes, optimal process conditions include 60 DEG C of temperature, 5.5 of pH value, 40mg of enzyme amount and 6h of time, obtained HT content is 6.07%, and OE degrading rate is 85.28%.
Description
First, technical field
The present invention relates to biological and chemical field, more particularly to leaf of Fructus oleae europaeae oleuropein crude extract prepare hydroxytyrosol
Method.
2nd, background technology
Fructus oleae europaeae (Olea europaea L.) category Oleaceae (Oleaceae), Olea (Olea) aiphyllium, be
World-renowned woody oil tree species.Olive oil can substantially reduce the sickness rate of cardiovascular and pulmonary carcinoma, its nutritive value and economy
Value is approved by everybody.Biologist is further discovered that the antioxidant active ingredients that Folium olive contains more are dashed forward than olive oil
Go out, mainly there are the polyphenol compounds such as oleuropein, hydroxytyrosol, flavonoid, lignans, caffeoyl phenethyl alcohol glycosides.Especially
Which is hydroxytyrosol (Hydroxytyrosol, HT；The entitled Hydroxytyrosol of chemistry), the ability for removing free radical is strong,
Show the biological activity of uniqueness, such as antioxidation, antibacterial, antiinflammatory, improve coronary blood flow of heart etc..And the mankind can also be suppressed
Prorubricyte HL-60 cells, adenocarcinoma cell HT29 and HT29 clone body 19A, women with breast cancer MCF-7 cell etc. spread,
Circulation through blocks tumor cells and its apoptosis is induced, with good active anticancer.
Native hydroxyl butyl alcohol content in Folium olive is very low, only 0.01-0.8%, and (Fructus Canarii albi is bitter with carboxylate for great majority
Glycosides) in the form of in each position of Fructus oleae europaeae.HT is mainly from olive fruit, leaf at present, and is preparing olive oil or meal
With detached in the residue and waste water produced during olive fruit, and the sugar such as oleuropein, nuezhenoside, verbascoside in waste water
Glycosides, flavonoid glycoside and polyphenols complicated component, low separation efficiency.With chemosynthesis HT it has been reported that Lucia and
At -25 DEG C, butyl alcohol Jing IBX oxidations and Reduction by Thiosulfate obtain HT to Napolitano etc., and yield is only 30%；With 3,4-
, used as raw material, with lithium aluminium hydride reduction, TMSCHNH/ sodium borohydrides, the boranated amine/iodomethane of tetra-n-butyl is for also for dihydroxyphenyl acetic acid
Former agent, yield are below 50%, and raw material and catalyst cost intensive, severe reaction conditions, it is impossible to preparation of industrialization.
Oleuropein (Oleuropein, OE) structure be driffractive ring alkene ethers polyphenolic substance, easily by air, sunlight, acid,
, there is the reactions such as oxidation, condensation, chelating and be subjected to brokenly in the impact of the factors such as alkali, oxidant, transition metal ionss, long-time high temperature
It is bad, it is especially degradable for HT and elemic acid in the presence of acid, alkali and enzyme, it is shown below.
In olea europaea fruit and leaf, OE contents are high, and 10～17% are especially reached in leaf.Make hence with Fructus oleae europaeae OE degradeds
Standby natural HT is domestic and international study hotspot.W02008136037 reports that acid catalyzed biodegrading process needs sulfuric acid catalyst,
Ghayth Rigane compared for the chemical treatment technology of OE in acid hydrolysis and basic hydrolysiss Fructus Canarii albi oil cake " alperujo ".Italy
Research worker is using immobilized thermophilic high temperature beta-glucosidase (pH=7.0,60 DEG C) degraded OE, and mixes from complicated enzymolysis
OE, the HT and elemic acid not digested is isolated in thing.The enzyme that the method is used is expensive, and reaction condition must be thermophilic height
Temperature.So the technique study of the oleuropein that is hydrolyzed is particularly important, this research it is critical only that various method for hydrolysis to olive
The comparison of olive hardship glycosides effect, while the enzyme system screening of optimal hydrolysis result and economic and practical, mild condition and Fructus oleae europaeae of degrading
Leaf oleuropein prepares the optimization of hydroxytyrosol enzymolysis process.
3rd, the content of the invention
It is an object of the invention to provide a kind of method that leaf of Fructus oleae europaeae oleuropein crude extract prepares hydroxytyrosol, this
The technical scheme of invention is realizing using following steps：
3.1 preparation method：
Acid hydrolysis process：Crude extract and acid hydrolysis liquid solid-to-liquid ratio are 1g: 10～100mL, be incorporated as 0.1mol/L～
1mol/L HCl solutions, are placed in 30 DEG C～100 DEG C of water bath with thermostatic control shaking table.After hydrolysis 1h～10h, neutrality is neutralized to, is filtered,
Filtrate Jing ethyl acetate is extracted, and ethyl acetate layer concentrate drying obtains hydroxytyrosol extract；
Basic hydrolysiss technique：Crude extract is 1g: 10～100mL with acid hydrolysis liquid solid-to-liquid ratio, adds 0.1mol/L～1mol/
L NaOH solutions, are placed in 30 DEG C～100 DEG C of water bath with thermostatic control shaking table.After hydrolysis 1h～10h, neutrality is neutralized to, is filtered, filtrate
Jing ethyl acetate is extracted, and ethyl acetate layer concentrate drying obtains hydroxytyrosol extract；
Biological enzyme technique：With oleuropein degradation rate and hydroxytyrosol content as integrated survey index, therefrom warm starch
Enzyme, saccharifying enzyme, cellulase, carase, alkaline protease, neutral protease, 1,4 beta-glucanase, hemicellulase, tannase,
Degraded oleuropein is filtered out in 10 kinds of enzymes of beta-glucosidase and prepares the best hemicellulase of hydroxytyrosol effect；By enzyme
Solution single factor test and orthogonal optimization determine optimum process condition, prepare hydroxytyrosol extract.
3.2 oleuropeins and hydroxytyrosol HPLC analysis methods：Weigh oleuropein and hydroxytyrosol reference substance is configured to
Mixed solution, with HPLC peak areas as vertical coordinate y, oleuropein and hydroxytyrosol quality (μ g) are respectively abscissa x, draw
HPLC standard curves, draw equation.Liquid-phase condition is：Chromatographic column is C18ODS chromatographic columns (250 × 5mm, 5 μm), and column temperature is 30
DEG C, methanol: water=35: 45 (0.2% phosphoric acid) are mobile phase, 10 μ L of sample size, uv absorption wavelength is 280nm, and flow velocity is
1mL/min.Calculate oleuropein linear with its peak area in 0.441～7.056 μ g, such as Fig. 1 (a).And
And equation of linear regression is y within this range1=1772846.78x1+ 89802.31, coefficient R2 1=0.9997.Hydroxyl cheese
Alcohol in 0.5586～8.9376 μ g with the linear Fig. 1 (b) of its peak area, and equation of linear regression is within this range
y2=1199063.90x2+ 119947.13, coefficient R2 2=0.9996.Liquid phase analysis oleuropein and hydroxytyrosol content
The relative standard deviation (RSD) of method is respectively 0.92% and 1.38%, illustrates that its analysis method precision is higher, repeatability
It is good.
3.3 hydroxytyrosol extract quality evaluations, weigh hydroxytyrosol extract and are dissolved in methanol, by 0.45 μm of organic membrane
Filtration, is analyzed using HPLC, hydroxytyrosol content 2～20% in the hydroxytyrosol extract of preparation.
HT contents after acid hydrolysis and basic hydrolysiss are respectively 7.41%, 4.09%.The HT obtained with enzymolysis optimum process is contained
Amount 6.07% is compared, and the effect of hydrochloric acid hydrolysis is slightly better than enzyme hydrolysiss, but the effect of NaOH hydrolysis OE is bad.
The screening of 3.4 enzymes：Choose midrange thermal stable amylase, saccharifying enzyme, cellulase, carase, alkaline protease, neutral protein
Enzyme, 1,4 beta-glucanase, hemicellulase, tannase, beta-glucosidase this 10 kinds of enzymes.Prepare the phosphorus that pH is respectively 3.0～7.0
Phthalate buffer, weighs the various enzymes of identical enzyme activity, and respectively 0.1～5g olive leaf extract is digested, and solid-to-liquid ratio is 1g:
10～100mL, and 2～10h is digested under the optimum temperature and pH of various enzymes.After enzymolysis terminates, 90 DEG C of enzyme denaturing 10min, enzymolysis
Liquid is centrifuged 10min in 5000r/min, filters, the extraction of filtrate Jing ethyl acetate, then carries out liquid phase analysis oleuropein and hydroxyl
Butyl alcohol content.Test every time is carried out 3 times, seeks its meansigma methods.(OE degradation rates=1- enzymolysis solutions OE mass/extract OE mass
(38.6%)；HT contents=enzymolysis solution HT mass/extract quality.) with oleuropein degradation rate and hydroxytyrosol content as comprehensive
Inspection target is closed, degraded oleuropein is filtered out and is prepared the best hemicellulase of hydroxytyrosol effect.
3.5 experiment of single factor：The olive leaf extract sample for taking 0.1～5g is placed in the conical flask of 100mL, add 10～
100mL buffer solution and 10～100mg hemicellulases, digest at 30～80 DEG C.After 2～10h of enzymolysis terminates, 90 DEG C of enzyme denaturing
10min, enzymolysis solution are centrifuged 10min in 5000r/min, filter, the extraction of filtrate Jing ethyl acetate, with hydroxyl in HPLC analysis filtrates
Butyl alcohol and oleuropein, and each content is calculated according to standard curve.With oleuropein degradation rate and hydroxytyrosol content as detection
Index, the impact of different factor (temperature, pH, time, enzyme concentration) the double cellulase hydrolysiss oleuropein efficiency of research.Every time
Test is carried out 3 times, seeks its meansigma methods.
3.6 orthogonal experiment：Filter out degraded oleuropein prepare a kind of best enzyme of hydroxytyrosol effect on the basis of,
Temperature, pH, time, enzyme concentration four are investigated because of the impact of not double cellulase hydrolysiss oleuropein efficiency of homoatomic.In Dan Yin
Element test on the basis of, in order to investigate impact of each factor reciprocal action to olive leaf extract enzymolysis efficiency and its affect because
The primary and secondary of element, and optimum enzymolysis condition is obtained, using four factors, three level [L9(34)] orthogonal, respectively with temperature,
PH, the time, 4 independent variables of enzyme concentration be investigation factor, the relation between goal in research value and influence factor.
According to single factor experiment result, with HT contents as index, have chosen temperature, pH, response time and enzyme amount is just carried out
Hand over experiment.Which the results are shown in Table 1.
Table 1L9(34) Orthogonal Experiment and Design and result
Table 1 Design and results of L9(34)orthogonal tests
Can be seen that by R in table 1, be temperature ＞ pH ＞ time ＞ enzyme amount to the size order of enzymolysis process influence factor, most
Good technique is A2B3C3D1, i.e. temperature 60 C, pH=5.5, time 6h, enzyme amount 40mg.In order to inspection institute obtains the repetition of optimal conditions
Property and stability, according to this optimal conditions, carry out 5 times repetition test, HT contents be respectively 6.03%, 6.14%, 6.12%,
5.98%th, 6.07, it is that 1.08%, OE degradation rates are 85.28% that meansigma methodss are 6.07%, RSD, illustrates that this technique stably may be used relatively
OK.
HT contents after acid hydrolysis and basic hydrolysiss are respectively 7.41%, 4.09%.The HT obtained with enzymolysis optimum process is contained
Amount 6.07% is compared, and the effect of hydrochloric acid hydrolysis is slightly better than enzyme hydrolysiss, but the effect of NaOH hydrolysis OE is bad.
The present invention is compared by three kinds of method for hydrolysis, and hydrochloric acid hydrolysis effect is slightly better than enzyme hydrolysiss, but NaOH hydrolysis effects are not
It is good.In contrast, method condition is gentleer, and whole process reduces acidand basecatalysts for olive leaf extract enzymolysis process
Use.
The present invention using midrange thermal stable amylase, saccharifying enzyme, cellulase, carase, alkaline protease, neutral protease, β-
Glucanase, hemicellulase, tannase, 10 kinds of enzymes of beta-glucosidase are screened, with oleuropein degradation rate and hydroxyl
Butyl alcohol content is index, is detected by HPLC, filters out degraded oleuropein and prepares the best hemicellulose of hydroxytyrosol effect
Enzyme.
The present invention on the basis of the screening of enzyme, with temperature, pH, the time, enzyme concentration as investigate factor, in order to investigate each factor
The primary and secondary of impact and its influence factor of the reciprocal action to olive leaf extract enzymolysis efficiency, and optimum enzymolysis condition is obtained,
Using four factors, three levels [L9 (34)] orthogonal, can be seen that by R in table 2, the size to enzymolysis process influence factor
For temperature ＞ pH ＞ time ＞ enzyme amount, it can be seen that optimised process is A2B3C3D1, i.e. temperature 60 C, pH=5.5, enzyme amount 40mg, when
Between 6h.In order to inspection institute obtains the repeatability and stability of optimal conditions, according to this optimal conditions, carry out 5 repetitions and test, HT contains
Amount is respectively 6.03%, 6.14%, 6.12%, 5.98%, 6.07, and meansigma methodss are 6.07%, and relative standard deviation (RSD) is
1.08%, OE degradation rate is 85.28%, illustrates that this technique is stablized relatively feasible.
Beneficial effects of the present invention show as：
1., compared with acid hydrolysis, basic hydrolysiss, method condition is gentleer for leaf of Fructus oleae europaeae oleuropein crude extract enzymolysis process,
Whole process reduces the use of acidand basecatalysts；
2. by relatively may fracture glycosidic bond various enzyme to the degradation effect and hydroxytyrosol of oleuropein
Content, draw hemicellulase degraded olive leaf extract prepare hydroxytyrosol effect it is best；
3. the technique that hemicellulase degraded leaf of Fructus oleae europaeae crude extract prepares hydroxytyrosol is optimized, and hydroxytyrosol content can
Obtain 6.07%.
4th, illustrate：
1 hydroxytyrosol of accompanying drawing and oleuropein standard curve；
Impact of the species of 2 enzyme of accompanying drawing to leaf of Fructus oleae europaeae crude extract enzymolysis efficiency
Accompanying drawing 3 digests the impact that single factor test is degraded to Fructus oleae europaeae crude extract oleuropein and hydroxytyrosol is generated；
4 hydroxytyrosol of accompanying drawing and oleuropein mixing reference substance and the HPLC collection of illustrative plates before and after olive leaf extract enzymolysis.
5th, specific embodiment
Following examples are some citings of the present invention, should not be seen as limitation of the invention.
Embodiment 1
The HPLC analyses of oleuropein and hydroxytyrosol and computational methods
Accurately weigh oleuropein and hydroxytyrosol reference substance 4.5mg and 5.7mg are placed in 10mL volumetric flasks together, use first
Alcohol dissolving shakes up, and constant volume, close plug, obtains the reference substance hybrid standard mother solution that concentration is 0.45mg/mL and 0.57mg/mL.Point
Inaccurate to draw 1,2,4,6,8,10,12,14,16 μ L of this standard solution, sample introduction, each sample repeat sample introduction 3 times, take respectively
The HPLC peak area meansigma methodss of oleuropein and hydroxytyrosol.Then, with HPLC peak areas as vertical coordinate, oleuropein and hydroxyl
Butyl alcohol quality (μ g) is respectively abscissa, draws HPLC standard curves, draws equation.
Liquid-phase condition is：Chromatographic column is C18ODS chromatographic columns (250 × 5mm, 5 μm), and column temperature is 30 DEG C, methanol: water=35:
45 (0.2% phosphoric acid) are mobile phase, and 10 μ L of sample size, uv absorption wavelength are 280nm, and flow velocity is 1mL/min.
Obtain oleuropein linear with its peak area and linear within this range in 0.441～7.056 μ g
Regression equation is y1=1772846.78x1+ 89802.31, coefficient R2 1=0.9997.Hydroxytyrosol 0.5586～
With the linear Fig. 1 (b) of its peak area in 8.9376 μ g, and equation of linear regression is y within this range2=
1199063.90x2+ 119947.13, coefficient R2 2=0.9996.Liquid phase analysis oleuropein and hydroxytyrosol content method
Relative standard deviation (RSD) be respectively 0.92% and 1.38%, illustrate that its analysis method precision is higher, it is reproducible.
Embodiment 2
Acid hydrolysis leaf of Fructus oleae europaeae oleuropein crude extract is tested
1g olive leaf extract is weighed in 250mL round-bottomed flasks, 50mL0.2mol/L HCl body lotions is added, is placed in 80 DEG C
Water bath with thermostatic control shaking table in.After hydrolysis 6h, neutrality is neutralized to, is filtered by 0.45 μm of organic membrane, carry out HPLC analyses.Try every time
Testing is carried out 3 times, seeks its meansigma methods.Calculate, HT contents available 7.41% after acid hydrolysis.(OE degradation rates=1- enzymolysis
Liquid OE mass/extract OE mass (38.6%)；HT contents=enzymolysis solution HT mass/extract quality.)
Embodiment 3
Basic hydrolysiss leaf of Fructus oleae europaeae oleuropein crude extract is tested
Base hydrolysis conditions：1g olive leaf extract is weighed in 250mL round-bottomed flasks, 50mL 0.2mol/L NaOH are added
Solution, is placed in 80 DEG C of water bath with thermostatic control shaking table.After hydrolysis 6h, neutrality is neutralized to, is filtered by 0.45 μm of organic membrane, carried out
HPLC is analyzed.Test every time is carried out 3 times, seeks its meansigma methods.Calculate, HT contents available 4.09% after basic hydrolysiss.(OE
Degradation rate=1- enzymolysis solutions OE mass/extract OE mass (38.6%)；HT contents=enzymolysis solution HT mass/extract quality.)
Embodiment 4
The screening of enzyme
Choose midrange thermal stable amylase, saccharifying enzyme, cellulase, carase, alkaline protease, neutral protease, beta glucan
Enzyme, hemicellulase, tannase, beta-glucosidase this 10 kinds of enzymes.The various enzymes of identical enzyme activity are weighed, respectively to 5g oil olives
Olive leaf extract is digested, and solid-to-liquid ratio is (liquid is to prepare phosphate buffered solution in above-mentioned a) 1: 50g/mL, and in various enzymes
8h is digested under optimum temperature and pH.After enzymolysis terminates, 90 DEG C of enzyme denaturing 10min, enzymolysis solution are centrifuged 10min in 5000r/min, pass through
0.45 μm of organic membrane filtration, then with carrying out liquid phase analysis oleuropein and hydroxytyrosol content.Test every time is carried out 3 times, is asked
Its meansigma methods.(OE degradation rates=1- enzymolysis solutions OE mass/extract OE mass (38.6%)；HT contents=enzymolysis solution HT mass/
Extract quality.)
Embodiment 5
Cellulase hydrolysiss leaf of Fructus oleae europaeae oleuropein crude extract is tested
The olive leaf extract sample for taking 1g is placed in the conical flask of 100mL, adds 10mL buffer solution and 40mg fibers
Plain enzyme, digests at 40 DEG C.After enzymolysis 3h terminates, 90 DEG C of enzyme denaturing 10min, enzymolysis solution are centrifuged 10min in 5000r/min, pass through
0.45 μm of organic membrane filtration, with hydroxytyrosol and oleuropein in HPLC analysis filtrates, testing every time is carried out 3 times, asks which average
Value.Calculate, the degradation rate of oleuropein is 75.23%, and hydroxytyrosol content is 5.34%.(OE degradation rates=1- enzymes
Solution liquid OE mass/extract OE mass (38.6%)；HT contents=enzymolysis solution HT mass/extract quality.)
Embodiment 6
Hemicellulase hydrolysis leaf of Fructus oleae europaeae oleuropein crude extract experiment
The olive leaf extract sample for taking 1g is placed in the conical flask of 100mL, adds 40mL buffer solution and 60mg half fine
The plain enzyme of dimension, digests at 50 DEG C.After enzymolysis 5h terminates, 90 DEG C of enzyme denaturing 10min, enzymolysis solution are centrifuged 10min in 5000r/min, lead to
0.45 μm of organic membrane filtration is crossed, with hydroxytyrosol and oleuropein in HPLC analysis filtrates, testing every time is carried out 3 times, asks which to put down
Average.Calculate, the degradation rate of oleuropein is 79.45%, and hydroxytyrosol content is 5.19%.(OE degradation rates=1-
Enzymolysis solution OE mass/extract OE mass (38.6%)；HT contents=enzymolysis solution HT mass/extract quality.)
Embodiment 7
Amylase hydrolysis leaf of Fructus oleae europaeae oleuropein crude extract is tested
The olive leaf extract sample for taking 1g is placed in the conical flask of 100mL, adds 30mL buffer solution and 40mg starch
Enzyme, digests at 45 DEG C.After enzymolysis 6h terminates, 90 DEG C of enzyme denaturing 10min, enzymolysis solution are centrifuged 10min in 5000r/min, pass through
0.45 μm of organic membrane filtration, with hydroxytyrosol and oleuropein in HPLC analysis filtrates, testing every time is carried out 3 times, asks which average
Value.Calculate, the degradation rate of oleuropein is 32.91%, and hydroxytyrosol content is 3.48%.(OE degradation rates=1- enzymes
Solution liquid OE mass/extract OE mass (38.6%)；HT contents=enzymolysis solution HT mass/extract quality.)
Embodiment 8
Beta-glucosidase hydrolysis leaf of Fructus oleae europaeae oleuropein crude extract experiment
The olive leaf extract sample for taking 1g is placed in the conical flask of 100mL, adds 60mL buffer solution and 80mg β-Portugal
Polyglycoside enzyme, digests at 37 DEG C.After enzymolysis 8h terminates, 90 DEG C of enzyme denaturing 10min, enzymolysis solution are centrifuged 10min in 5000r/min,
Filtered by 0.45 μm of organic membrane, with hydroxytyrosol and oleuropein in HPLC analysis filtrates, testing every time is carried out 3 times, asks which
Meansigma methodss.Calculate, the degradation rate of oleuropein is 61.91%, and hydroxytyrosol content is 5.42%.(OE degradation rates=
1- enzymolysis solutions OE mass/extract OE mass (38.6%)；HT contents=enzymolysis solution HT mass/extract quality.)
Claims (4)
1. a kind of method that leaf of Fructus oleae europaeae oleuropein crude extract prepares hydroxytyrosol, it is characterised in that comprise the following steps：
A. preparation method：
Acid hydrolysis process：Crude extract is 1g: 10～100mL with acid hydrolysis liquid solid-to-liquid ratio, is incorporated as 0.1mol/L～1mol/L
HCl solution, is placed in 30 DEG C～100 DEG C of water bath with thermostatic control shaking table, after hydrolysis 1h～10h, is neutralized to neutrality, filters, filtrate Jing
Ethyl acetate is extracted, and ethyl acetate layer concentrate drying obtains hydroxytyrosol extract；
Biological enzyme technique：With oleuropein degradation rate and hydroxytyrosol content as integrated survey index, from midrange thermal stable amylase, sugar
Change enzyme, cellulase, carase, alkaline protease, neutral protease, 1,4 beta-glucanase, hemicellulase, tannase, β-Portugal
Degraded oleuropein is filtered out in 10 kinds of enzymes of polyglycoside enzyme and prepares the best hemicellulase of hydroxytyrosol effect；It is single by digesting
Factor and orthogonal optimization determine optimum process condition, and the optimum process condition is the leaf of Fructus oleae europaeae crude extract in 0.1g～5g
In, 40mg hemicellulases are added, when it is 60 DEG C that pH is 5.5, temperature, 6h is digested, is prepared hydroxytyrosol extract；
B. oleuropein and hydroxytyrosol HPLC analysis methods：Weigh oleuropein and hydroxytyrosol reference substance is configured to mix molten
Liquid, concentration range are 0.1～10 μ g, select C18 chromatographic columns, and it is 1: 1～10 that mobile phase is first alcohol and water ratio, in wavelength 220
Sample introduction analysis under～280nm, with HPLC peak areas as vertical coordinate, oleuropein and hydroxytyrosol quality are respectively abscissa, paint
HPLC standard curves processed；
C. hydroxytyrosol extract quality evaluation：Weigh hydroxytyrosol extract and be dissolved in methanol, filtered by 0.45 μm of organic membrane,
Analyzed using HPLC, hydroxytyrosol content 2～20% in the hydroxytyrosol extract of preparation.
2. the method that leaf of Fructus oleae europaeae oleuropein crude extract according to claim 1 prepares hydroxytyrosol, its feature exist
The enzyme screening technology in step a：The phosphate buffer that pH is respectively 3.0～7.0 is prepared, the various of identical enzyme activity are weighed
Enzyme, is digested to 0.1g～5g leaf of Fructus oleae europaeae oleuropein crude extract respectively, and crude extract is 1g with buffer solid-to-liquid ratio:
10～100mL, and 2h～10h is digested under the optimum temperature and pH of various enzymes.After enzymolysis terminates, 90 DEG C of enzyme denaturing 10min, enzymolysis
Liquid is centrifuged 10min in 5000r/min, filters, then the extraction of Jing ethyl acetate, and then HPLC analysis oleuropeins and hydroxytyrosol contain
Amount.
3. the method that leaf of Fructus oleae europaeae oleuropein crude extract according to claim 1 prepares hydroxytyrosol, its feature exist
Single factor test technique is digested in step a：The leaf of Fructus oleae europaeae oleuropein crude extract sample for taking 0.1g～5g is placed in the cone of 100mL
Shape bottle, adds 10mL～100mL phosphate buffered solution and 10mg～100mg hemicellulases, digests at 30 DEG C～80 DEG C,
After enzymolysis 2h～10h terminates, 90 DEG C of enzyme denaturing 10min, enzymolysis solution are centrifuged 10min in 5000r/min, filter, then Jing ethyl acetate
Extraction, is analyzed hydroxytyrosol and oleuropein in filtrate with HPLC, and calculates each content according to standard curve, dropped with oleuropein
Solution rate and hydroxytyrosol content are Testing index, study different temperatures, difference pH, double fiber of different time and different enzyme concentrations
The impact of plain enzyme hydrolysiss oleuropein efficiency.
4. the method that leaf of Fructus oleae europaeae oleuropein crude extract according to claim 1 prepares hydroxytyrosol, its feature exist
Orthogonal optimization technique is digested in step a：On the basis of single factor experiment, using four factors, three level [L9 (34)] orthogonal reality
Design is tested, respectively with temperature, pH, time, 4 independent variables of enzyme concentration to investigate factor, hydroxytyrosol content desired value is studied
Relation between influence factor, as a result shows, is temperature ＞ pH ＞ time ＞ enzyme amount to the size of enzymolysis process influence factor, obtains
To optimum process condition, it is 5%～20% to obtain hydroxytyrosol content, and oleuropein degradation rate is 70～95%.
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| CN106187708A (en) * | 2016-07-25 | 2016-12-07 | 西安岳达生物科技股份有限公司 | A kind of preparation method of high-purity hydroxytyrosol |
| CN107217076B (en) * | 2017-05-19 | 2020-03-24 | 南京林业大学 | Method for preparing hydroxytyrosol by combining enzyme catalysis and high-temperature hydrolysis |
| CA3077611A1 (en) * | 2017-11-08 | 2019-05-16 | Societe Des Produits Nestle S.A. | Homovanillyl alcohol (hva), hva isomer, methods of making compositions comprising such compounds, and methods using such compounds |
| CN111748593B (en) * | 2019-03-28 | 2023-06-23 | 陈徉 | Preparation method of olive-source tyrosinase inhibitory peptide |
| CN111481962B (en) * | 2020-04-23 | 2021-12-07 | 兰州海关技术中心 | Method for extracting salmonella-inhibiting substance from olive pomace and application |
| CN113375989B (en) * | 2021-04-16 | 2023-12-15 | 中国热带农业科学院热带生物技术研究所 | Separation method and research method of rubber tree milk tube network |
| CN113277931A (en) * | 2021-06-04 | 2021-08-20 | 陕西富恒生物科技有限公司 | Method for extracting hydroxytyrosol from olive fruits |
| CN113860999A (en) * | 2021-10-12 | 2021-12-31 | 东莞职业技术学院 | Method for preparing hydroxytyrosol by using olive leaves |
| CN114150025B (en) * | 2021-12-06 | 2022-09-09 | 西安海斯夫生物科技有限公司 | Preparation method of high-purity hydroxytyrosol |
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| CN103709014B (en) * | 2013-12-20 | 2015-08-12 | 桂林莱茵生物科技股份有限公司 | High and the Hydroxytyrosol extracting method that the rate of recovery is high of a kind of transformation efficiency |
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