CN109942428A - A kind of method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid - Google Patents
A kind of method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid Download PDFInfo
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- CN109942428A CN109942428A CN201910156361.8A CN201910156361A CN109942428A CN 109942428 A CN109942428 A CN 109942428A CN 201910156361 A CN201910156361 A CN 201910156361A CN 109942428 A CN109942428 A CN 109942428A
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- chlorogenic acid
- mulberry leaf
- complex enzyme
- catalytic activation
- purity
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- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 title claims abstract description 70
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 title claims abstract description 70
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 title claims abstract description 70
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 title claims abstract description 70
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Abstract
A kind of method that complex enzyme catalytic activation mulberry leaf obtain through refining chlorogenic acid, belongs to technical field of chlorogenic acid extraction.The present invention obtains product using mulberry leaf as raw material, through mulberry leaf pretreatment, the complex enzyme catalytic activation simple process that extracts, isolate and purify, be freeze-dried.For the complex enzyme that the method for the present invention uses at 35 DEG C of water-bath, catalytic activation 90min, chlorogenic acid dissolves out quality up to 110.08mg/L；It isolated and purified through preparative high performance liquid chromatography, be freeze-dried obtained high-purity chlorogenic acid product, extracted amount 5.5042mg/g, purity 99%.The features such as raw material sources of the method for the present invention are wide, have mulberry leaf using sufficiently, and the chlorogenic acid product recovery rate and purity is high and bioactivity of preparation are complete, and production equipment corrosion is small, and organic solvent usage amount is few, are conducive to environmental protection.It the composite can be widely applied to extract the natural products such as Salanesol, nicotine and pectin from solid tobacco waste, the recycling for effectively realizing abandoned tobacco recycles.
Description
Technical field
The invention belongs to phenolic acid class active skull cap components extractive technique fields in plant, and in particular to a variety of enzyme synergistic effects
Obtain through refining phenolic acid compound monomer in mulberry leaf --- the method for chlorogenic acid.
Background technique
Mature mulberry leaf moisture content 75%, dry matter 25%, rich in nutrition content and it is uniform, and contain multivitamin, mineral
Matter.Contain 25 g of soluble-carbohydrate in every dry mulberry leaf of 100g, 15~40 g of phytoprotein and injection Vitamin B_6,
A, the various trace elements such as B race, vitamin K and beta carotene, folic acid.Furthermore mulberry leaf also contain some distinctive functions because
Son, such as flavone compound, organic phenolic acid and the high active material of alkaloid value medical health care.Organic acid in mulberry leaf
Constituents mainly include chlorogenic acid (Chlorogenic acid), neochlorogenic acid (Neochlorogenicacid), hidden green original
Acid (Cryptochlorogenic
Acid) etc..It is proved through modern medicine and Nutritional studies, such active material has good hypoglycemic, drop blood
Rouge, it is anti-inflammatory, anti-oxidant the effects of.The health care pharmacological action of mulberry leaf, makes it obtain the favor of food industry, and some places are by mulberry leaf
It is processed into mulberry leaf slurry or mulberry leaf powder is added into food by a certain percentage, be made into mulberry leaf noodle, mulberry leaf bean curd, mulberry leaf biscuit, mulberry leaf
Soymilk powder, mulberry leaf sauce etc. can not only increase food, nutritive and health protection components, while color that food is also adjusted is fragrant and flavor.
With the continuous deepening of research, mulberry leaf be expected to as some diseases especially geriatric disease and chronic disease treatment drug and
Health product, and beauty and skin care, in terms of have broad prospects.
Existing number of patent application discloses a kind of " mulberry leaf phenolic acid extract with bacteriostatic activity for 201210255122.6
Preparation method and application " the patent is related to method and application using ultrasonic extraction, resin separation mulberry leaf polyphenol.Chinese patent
" preparation method and its microcapsules of a kind of mulberry leaf polyphenol and application " is disclosed application No. is 200810219728.8 using super
Sound-microwave coordinates the techniques such as extraction, ultrafiltration, purification with macroreticular resin and obtains mulberry leaf polyphenol and prepared with microcapsules technology more
Phenol microcapsules, applied to anti-oxidant and protect liver effect functional food, in the above method, the monitoring of polyphenol components fails have tool
The index components of body, but using major class overall composition as index, it is relatively rough.
Publication No. CN106256353 disclosed in existing on December 28th, 2016 " one kind isolates and purifies green original in mulberry leaf
The method of acrylic component ", disclosed method is: mulberry leaf crush, and are extracted with a certain amount of ethyl alcohol multi-reflow, combined extract；
It will be eluted by macroporous resin adsorption, then successively by pure water rinsing, eluted through lye and pure water after alcohol extract acid adding tune pH；It collects
Macroreticular resin is gone up after eluent acid adding tune pH again, with a certain concentration ethanol elution, collects alcohol eluen；Alcohol eluen is depressurized dense
Contracting, spray drying both obtain mulberry-leaf extract.Though the content for the chlorogenic acid ingredient that this method is extracted is stablized, there are still following
Deficiency: (1) entire extract preparation process takes a long time, and ethyl alcohol usage amount is big, and macroporous absorbent resin organic residue is high, into
And influence the safety of product；(2) desired active substance purity is bad in extract；(3) adsorption process is repeatedly added lye and washes
It is de-, it not only pollutes the environment, while also having damage to the active function of extract.
Summary of the invention
The object of the present invention is to be directed to the deficiency of existing mulberry leaf extraction process of active component, a kind of complex enzyme is provided
The extracting method of enzymatic reaction, realize extraction conditions operating procedure it is few and simple, it is mild efficiently, environmental pollution is small, production
Energy consumption and at low cost, isolates and purifies the features such as process is simple, and monomer purity is high.
Mechanism of the present invention is: the present invention using mulberry leaf as raw material, select complex enzyme (laccase, hemicellulase, cellulase,
Papain) activator as catalytic activation mulberry leaf tissue tissue, pass through laccase and degrades the wood of yoke cellulose in mulberry leaf
Quality increases cellulase to the hydrolysis of cellulose, reduces chlorogenic acid and dissolve out resistance, in ethanol water, increase green
Ortho acid recovery rate.Mulberry leaf plant tissue is handled using enzyme activation, avoids the conditions such as high temperature and long-time heating from destroying chlorogenic acid raw
Object activity, improves chlorogenic acid yield and product quality；Meanwhile mulberry leaf plant tissue can be improved in the concerted catalysis effect of complex enzyme
Hydrolysis efficiency, and then avoid chemical activating agent acid, oxygenation pretreatment and extract chlorogenic acid organic solvent usage amount, reduce
Production safety hidden danger, is conducive to environmental protection, realizes that the recycling of Sang Ziyuan recycles.
Realizing the technical solution of goal of the invention is: a kind of complex enzyme catalytic activation mulberry leaf obtain through refining the side of high-purity chlorogenic acid
Method, using Mulberry Leaf as raw material, in the system anyway of several enzymes synergistic effect, through enzymatic hydrolysis catalysis, filtering, centrifugation, constant volume, and
The fractionation purification of chromatography is tested and analyzed and prepared using efficient liquid phase.Specific step is as follows for the method:
(1) pretreatment of raw material
The new fresh mulberry leaf of picking is used in 50~70 DEG C of baking ovens and dries 4~6h, the mulberry leaf after drying are crushed through pulverizer, are sieved
20~60 mesh are packed into hermetic bag, for use.
(2) complex enzyme catalytic activation extracts
After the completion of (1) step, first according to the quality (g) of (1) step treated raw material: the quality (g) of complex enzyme: ethyl alcohol
(mL): the ratio that the ratio between water (mL) is 1: 0.1~0.15: 3~12: 17~25 adds in the pretreated raw material of (1) step
Enter complex enzyme and ethanol water, after mixing evenly, it is 3.5~5.5 that citric acid adjustment pH, which is added,.It again will be mixed after adjusting pH value
It closes liquid to be placed in shaking table, after 50~120min of enzymatic hydrolysis activation is carried out at being 25~55 DEG C in bath temperature, conical flask is taken out
It after being cooled to room temperature, is filtered with suction filter pump, collects filtrate and filter residue respectively.The filtrate of collection is mulberry leaf crude extract, to
With；The filter residue of collection is mulberry leaf residue, is centrally disposed after collection.Then the leachate of collection 100mL volumetric flask is transferred to be used in combination
After distilled water constant volume, after being transferred to centrifuge tube, is obtained after 10~20min of centrifugation at 2000~4000r/min of revolving speed and slightly propose centrifugate.
1mL is finally taken slightly to propose centrifugate in sample bottle, in 90~50 ﹕ 50 of mobile phase acetonitrile (mL) ﹕ water (mL)=10 ﹕, temperature 20~40
DEG C, 5~20 μ L of sample volume, flow velocity detects crude extract Content of Chlorogenic Acid quality under the conditions of being 0.6~1mL/min.Chlorogenic acid dissolves out matter
Amount is 90~110mg/L.The enzyme be laccase, hemicellulase, cellulase and papain, the compound enzyme
Mass ratio is laccase: hemicellulase: cellulase: papain=0.5~2: 1.5: 10~15: 0.5~2.
(3) chlorogenic acid isolates and purifies
After the completion of (2) step, first the leaching centrifugation crude extract that (2) step is collected is placed in a rotary evaporator, true
It is at 80~100 DEG C to carry out that mulberry leaf concentrate is concentrated under reduced pressure to obtain that pneumatics is 0.4~0.6 Mpa, temperature by force.Then preparative is used
High performance liquid chromatography processing mulberry leaf concentrate further isolates and purifies, in mobile phase methanol (90~50 ﹕ of mL) ﹕ water (mL)=10 ﹕
50,8~16mL of sample volume, flow velocity obtain chlorogenic acid fraction under conditions of being 15~30mL/min.Finally chlorogenic acid fraction is set
In rotary evaporator, concentrate is concentrated under reduced pressure to obtain in the case where vacuum pressure is 0.4~0.6 Mpa, temperature is 80~100 DEG C, i.e.,
Chlorogenic acid concentrate.
(4) preparation of chlorogenic acid monomer
After the completion of (3) step, first chlorogenic acid concentrate obtained by (3) step is placed in refrigerator after freezing, goes to vacuum refrigeration
In drying machine, after dry 24~36h, chlorogenic acid solid monomer product is made.Chlorogenic acid extracted amount is pure up to 4.7~5.6mg/g
Degree up to 96~99%.
The present invention is after adopting the above technical scheme, mainly there is following Xiao Guo ﹕
(1) present invention activates mulberry leaf by complex enzymes concerted catalysis such as laccase, hemicellulase, cellulase, papains,
Realize that active material is fully dissolved out in mulberry leaf in enzymolysis process, chlorogenic acid dissolves out quality up to 110.08mg/L.And extraction process
In be used only a small amount of dehydrated alcohol, reduce chlorogenic acid dissolution resistance to mass tranfer while, be conducive to the protection of environment, promote chlorogenic acid
Leaching is abundant.
(2) present invention is best at 35 DEG C using complex enzyme catalytic activation condition, and enzymolysis time is only 90min, easy to operate,
Reaction condition is mild, and organic solvent is ethyl alcohol, reduces environment treatment cost in production process, improves production safety performance,
Further decrease production cost.
(3) present invention uses mulberry leaf for raw material, is easy to get, chlorogenic acid extracting operating procedure is simple, method is environmentally protective.Using
Reverse phase preparative high-performance liquid chromatographic technology (RP-HPLC) has the characteristics that Gao Zhuxiao, high flow rate, high separating rate, easy to operate,
The natural products for obtaining a variety of high-purities can be separated simultaneously.It is that raw material obtains through refining different dry measure used in former times skin that the method for the present invention, which can be widely applied to mulberry leaf,
Glycosides, neochlorogenic acid, Cryptochlorogenic acid and other active components.
The present invention is further described With reference to embodiment.A kind of complex enzyme catalytic activation mulberry leaf are obtained through refining high-purity
Specific step is as follows for the method for degree chlorogenic acid:
Embodiment 1
(1) pretreatment of raw material
The new fresh mulberry leaf of picking is used in 60 DEG C of baking ovens and dries 5h, it is powder, sieving that the mulberry leaf after drying are crushed through pulverizer
60 mesh are packed into hermetic bag, for use.
(2) complex enzyme zymohydrolysis extraction and the detection of chlorogenic acid
After the completion of (1) step, first according to the quality (g) of (1) step treated raw material: the quality (g) of enzyme: ethyl alcohol (ml):
The ratio that the ratio between water (ml) is 1: 0.15: 5: 20 is added complex enzyme and ethyl alcohol is water-soluble in the pretreated raw material of (1) step
Liquid, after mixing evenly.It is 4.5 that citric acid adjustment pH, which is added,.The mixed liquor after adjusting pH value is placed in shaking table again, in water-bath
Temperature is after carrying out enzymatic hydrolysis activation 90min at 35 DEG C, after conical flask taking-up is cooled to room temperature, to be filtered with suction filter pump, point
It Shou Ji not filtrate and filter residue.The filtrate of collection is mulberry leaf crude extract, for use；The filter residue of collection is mulberry leaf residue, is collected after collection
Middle disposition.Then, the leachate of collection is transferred to 100mL volumetric flask and with after distilled water constant volume, after being transferred to centrifuge tube, in revolving speed
It is obtained after centrifugation 15min under 4000r/min and slightly proposes centrifugate.1mL is finally taken slightly to propose centrifugate in sample bottle, in mobile phase acetonitrile
(mL) ﹕ water (mL)=20 ﹕ 80,25 DEG C of temperature, 10 μ L of sample volume, flow velocity detects crude extract Content of Chlorogenic Acid under conditions of being 1mL/min
Quality, it is laccase, cellulase, hemicellulase and Papain that chlorogenic acid dissolution quality, which is enzyme described in 110.08mg/L,
Enzyme, the mass ratio of the compound enzyme are laccase: cellulase: hemicellulase: papain=0.5: 1.5: 12.5:
0.5。
(3) chlorogenic acid isolates and purifies
After the completion of (2) step, first the leaching centrifugation crude extract that (2) step is collected is placed in a rotary evaporator, true
It is at 90 DEG C to carry out that mulberry leaf concentrate is concentrated under reduced pressure to obtain that pneumatics is 0.5 Mpa, temperature by force.Then preparative high-efficient liquid phase color is used
Spectrum processing mulberry leaf concentrate further isolates and purifies, in mobile phase methanol (mL) ﹕ water (mL)=20 ﹕ 80, sample volume 10mL, flow velocity
For 20mL/min, under conditions of obtain chlorogenic acid fraction.Finally chlorogenic acid fraction is placed in rotary evaporator, in vacuum pressure
It is that concentrate is concentrated under reduced pressure to obtain at 90 DEG C for 0.5 Mpa, temperature, i.e. chlorogenic acid concentrate.
(4) preparation of chlorogenic acid monomer
After the completion of (3) step, first chlorogenic acid concentrate obtained by (3) step is placed in refrigerator after freezing, it is cold to be transferred to vacuum
In lyophilizer, after dry 36h, chlorogenic acid solid monomer product is made.Chlorogenic acid extracted amount is up to 5.5042mg/g, purity is high
Up to 99%.
Embodiment 2
A kind of method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid, with embodiment 1, in which:
In (1) step, oven temperature is 50 DEG C, drying time 6h, and be sieved 20 mesh.
In (2) step, the quality (g) of raw material: the quality (g) of enzyme: ethyl alcohol (mL): the ratio between water (mL) is 1: 0.15: 8: 18
Ratio, reaction temperature be 40 DEG C, pH 4.0, reaction time 60min.Centrifuge speed is 3000r/min, centrifugation time
For 20min.The ratio between mobile phase of high performance liquid chromatography acetonitrile (mL) and water (mL) are 25 ﹕ 75, and temperature is 20 DEG C, 5 μ L of sample volume, stream
Fast 0.8mL/min, it is 105.37mg/L that chlorogenic acid, which dissolves out quality, and the enzyme for participating in reaction is laccase.
In (3) step, vacuum pressure 0.4Mpa, temperature are at 80 DEG C；Prepare chromatogram flow phase methanol (mL) and water
It the ratio between (mL) is 25:75, sample volume 15mL, flow velocity 25mL/min.
In (4) step, drying time is for 24 hours.
Embodiment 3
A kind of method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid, with embodiment 1, in which:
In (1) step, oven temperature is 70 DEG C, drying time 4h, and be sieved 60 mesh.
In (2) step, the quality (g) of raw material: the quality (g) of enzyme: ethyl alcohol (mL): the ratio between water (mL) is 1: 0.15: 12: 25
Ratio, reaction temperature be 50 DEG C, pH 5.5, reaction time 120min.Revolving speed is 2500r/min, and centrifugation time is
10min.The ratio between acetonitrile (mL) and water (mL) are 15 ﹕ 85, and temperature is 30 DEG C, sample volume 20 μ L, flow velocity 0.6mL/min, chlorogenic acid
Dissolution quality is 97.78mg/L, and the enzyme for participating in reaction is hemicellulase.
In (3) step, vacuum pressure 0.6Mpa, temperature are at 100 DEG C；Prepare chromatography preparation chromatography methanol (mL) and water
It the ratio between (mL) is 15:85, sample volume 8mL, flow velocity 15mL/min.
In (4) step, drying time 48h.
Experimental result
With the pH value of same means discussion enzyme digestion reaction to the extracted amount of chlorogenic acid and other active materials
1. the extracted amount and purity of 1 ~ 3 chlorogenic acid of embodiment
The comparison of 1. embodiment of table, 1 ~ 3 chlorogenic acid extracted amount
| Title | Chlorogenic acid extracted amount (mg/g) | Purity (%) |
| Embodiment 1 | 5.5042 | 99 |
| Embodiment 2 | 5.2530 | 98.7 |
| Embodiment 3 | 4.8209 | 98.5 |
2. 60 DEG C of drying 5h of new fresh mulberry leaf, crushed 60 meshes, take 2.0g mulberry leaf powder that 0.3g complex enzyme is added, according to solid-liquid ratio 1:
5 are added the dehydrated alcohol of 10mL, and distilled water 40mL is added according to solid-liquid ratio 1:20.35 DEG C of reaction temperature, the reaction time is
90min, the different pH value of reaction system are the influence experiment to chlorogenic acid and other active material extracted amounts.
Comparison of the 2. enzymatic hydrolysis system difference pH value of table to chlorogenic acid extracted amount
| PH value | Chlorogenic acid extracted amount (mg/g) |
| 3.5 | 5.0782 |
| 4.0 | 5.3110 |
| 4.5 | 5.4602 |
| 5.0 | 4.2210 |
| 5.5 | 4.0047 |
Comparison of the 3. enzymatic hydrolysis system difference pH value of table to other active material extracted amounts
| PH value | Cryptochlorogenic acid extracted amount (mg/g) | Isoquercitrin extracted amount (mg/g) | Rutin extraction rate (mg/g) |
| 3.5 | 0.6331 | 0.2954 | 0.4219 |
| 4.0 | 0.7060 | 0.4196 | 0.4502 |
| 4.5 | 0.7792 | 0.3824 | 0.4330 |
| 5.0 | 0.6074 | 0.3092 | 0.4037 |
| 5.5 | 0.5289 | 0.2335 | 0.3706 |
Know from above-mentioned experiment: the present invention prepares active carbon using complex enzyme catalytic activation processing mulberry leaf solid waste, works as mulberry leaf
Mass ratio be 1:0.15 complex enzyme (laccase: hemicellulase: cellulase: papain=0.5: 1.5: 12.5: 0.5),
20% ethanol water of the Quality of Mulberry Leaves than 1: 35 is added, adjustment pH value is 4.5, at closely under room temperature 35 DEG C of water-bath, enzymatic hydrolysis
Time is 90min, isolates and purifies through preparative high performance liquid chromatography, is freeze-dried chlorogenic acid product can be made.The green original of this method
Sufficiently, chlorogenic acid dissolves out quality up to 110.08mg/L for acid dissolution；And enzyme replaces soda acid activator, preparation condition is mild, operation
Simply, chlorogenic acid product recovery rate and purity is high, chlorogenic acid extracted amount is up to 5.5042mg/g, and purity is up to 99%, and biology is living
Property is complete；The raw material of the method for the present invention is cheap and easy to get, easy to operate, and the usage amount of soda acid and organic solvent subtracts in production process
It is few, the corrosion production equipment and the secondary pollution caused by environment are avoided, environmental protection is conducive to, reduces production cost.
Claims (6)
1. a kind of method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid, it is characterised in that including following step
It is rapid: (1) mulberry leaf are dried, is pulverized and sieved,
(2) it in the pretreated raw material of (1) step, is added after complex enzyme and ethanol water, citric acid adjustment pH to be put in and shake
In bed, after enzymatic hydrolysis activation, is filtered with suction filter pump, collects filtrate respectively and filter residue, the filtrate of collection are mulberry leaf crude extract,
The leachate of collection is transferred to 100mL volumetric flask and with after distilled water constant volume, after being transferred to centrifuge tube, is centrifuged slightly to propose centrifugate,
1mL is finally taken slightly to propose centrifugate in sample bottle, by high performance liquid chromatography detection crude extract Content of Chlorogenic Acid quality,
(3) first the leaching centrifugation crude extract that (2) step is collected is placed in a rotary evaporator, carries out that mulberry leaf are concentrated under reduced pressure to obtain
Then concentrate is further isolated and purified with preparative high performance liquid chromatography processing mulberry leaf concentrate, obtains chlorogenic acid fraction, most
Chlorogenic acid fraction is placed in rotary evaporator afterwards, chlorogenic acid concentrate is concentrated under reduced pressure to obtain,
(4) chlorogenic acid concentrate obtained by (3) step is placed in refrigerator after freezing, is gone in vacuum freeze drier, dry system
Chlorogenic acid solid monomer product is obtained,
The method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid is stated according to claim 1, it is characterised in that step (1)
Middle oven temperature can be 50~70 DEG C, and the time can be 4~6h, and the mesh number of sieve can be 20~60.
2. stating the method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid according to claim 1, it is characterised in that step
(2) quality (g) of raw material in: the quality (g) of complex enzyme: ethyl alcohol (mL): the ratio between water (mL) is 1: 0.1~0.15: 3~12: 17
~25 ratio, it is 3.5~5.5 that citric acid, which adjusts pH, and bath temperature can be 25~55 DEG C, enzymatic hydrolysis activation time is 60~
120min, 2000~4000r/min of centrifuge speed, 10~20min of centrifugation time.
3. stating the method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid according to claim 1, it is characterised in that step
Mobile phase acetonitrile in (2) (mL) ﹕ water (mL)=10 ﹕ 90~50 ﹕ 50,20~40 DEG C of temperature, 5~20 μ L of sample volume, flow velocity 0.6
~1mL/min.
4. stating the method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid according to claim 1, it is characterised in that step
(2) enzyme described in is laccase, cellulase, hemicellulase and papain, and the mass ratio of the compound enzyme is paint
Enzyme: cellulase: hemicellulase: papain=0.5~2: 1.5: 10~15: 0.5~2.
5. stating the method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid according to claim 1, it is characterised in that step
(3) vacuum pressure of rotary evaporator is 0.4~0.6 Mpa in, temperature is 80~100 DEG C, and preparing chromatographic condition is mobile phase
(90~50 ﹕ 50 of mL) ﹕ water (mL)=10 ﹕, 8~16mL of sample volume, flow velocity is 15~30mL/min to methanol.
6. stating the method that complex enzyme catalytic activation mulberry leaf obtain through refining high-purity chlorogenic acid according to claim 1, it is characterised in that step
(4) sublimation drying can be 24~48h in.
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