CN109593736A - A kind of theaflavin production industrial production process of immobile polyphenol oxidase - Google Patents
A kind of theaflavin production industrial production process of immobile polyphenol oxidase Download PDFInfo
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- CN109593736A CN109593736A CN201811557810.1A CN201811557810A CN109593736A CN 109593736 A CN109593736 A CN 109593736A CN 201811557810 A CN201811557810 A CN 201811557810A CN 109593736 A CN109593736 A CN 109593736A
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- theaflavin
- polyphenol oxidase
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
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Abstract
The invention discloses a kind of industrial production process of theaflavin production immobile polyphenol oxidase, separating-purifying is carried out to the polyphenol oxidase in fresh tea passes, absorption fixation is carried out to polyphenol oxidase using macroreticular resin, and related optimization is carried out to the process conditions, sufficiently eliminate the small-molecule substances such as tea polyphenols and the caffeine in tealeaves, influence of the other impurities to theaflavin product purity in fresh tea passes is effectively reduced, the quality of theaflavin product is improved；Polyphenol oxidase by immobilization may be reused repeatedly, thus the utilization rate of polyphenol oxidase in theaflavin production process is not only increased, the industrial production cost of theaflavin is reduced, while reducing the generation of tea grounds waste, has reached energy-saving and environment-friendly purpose；Present invention process is easy to operate, and reaction condition is mild, pollution-free, is suitble to large-scale industrial production.
Description
Technical field
The present invention relates to theaflavin production technical fields, and in particular to immobile polyphenol oxidase is used in a kind of production of theaflavin
Industrial production process.
Background technique
Theaflavin is important quality ingredient and functional component in black tea, has reducing blood lipid, blood pressure lowering, anti-oxidant, anti-spoke
It penetrates, multiple biological activities and the pharmacological effect such as antibacterial, anticancer, thus has the function of good medicines and health protection.Theaflavin is dark brown
Plain series product middle-molecular-weihydroxyethyl is minimum, structure is most simple, the most specific chemical component of bioactivity, is only one pharmacological action
Specific tea active constituent.
Theaflavin is that tea polyphenols substance and oxygen generate o-quinone under the catalytic action of polyphenol oxidase, and o-quinone is further
It is oxidized to xenol quinones intermediate product, which is reduced to bisflavanol class, and another part exists
Further oxidation obtains theaflavin under polyphenol oxidase catalyzed effect.Theaflavin be earliest directly extracted from black tea, however
The theaflavin that black tea itself contains only accounts for 0.5-2.0%, thus causes its extract yield lower.Industrial production theaflavin at present
Tea polyphenols enzymatic oxidation method is mainly used, reaction kettle is put into after fresh tea passes are crushed, solvent and tea polyphenols are added, blowing air carries out
Reaction obtains theaflavin using polyphenol oxidase catalyzed synthesis naturally occurring in fresh tea passes.But due to the polyphenol oxygen in tealeaves
Change enzyme content is lower, causes to use the industrial cost of this method higher, and polyphenol oxidase utilization efficiency is lower, deposits in fresh tea passes
The small-molecule substances such as caffeine adverse effect will also result in the quality of theaflavin product；In addition, can produce after reaction
Raw a large amount of tealeaf residue wastes need to carry out it special environmentally friendlyization processing.
Summary of the invention
For the above technical problems, the present invention provides a kind of work of theaflavin production immobile polyphenol oxidase
Industry preparation method, and related optimization has been carried out to the process conditions, sufficiently to improve polyphenol oxidase in theaflavin production process
Utilization rate, promoted theaflavin product quality, reduce the industrial production cost of theaflavin, while reaching energy-saving and environment-friendly mesh
's.
The technical solution adopted by the present invention to solve the technical problems is that a kind of theaflavin production is aoxidized with immobile polyphenol
The industrial production process of enzyme, comprising the following steps:
A. break process: fresh tea passes are taken, are tentatively crushed using pulverizer, then further milled with colloid mill；
B. it extracting: being added pure water into tea-leaf power obtained in above-mentioned steps, the ratio of tealeaves and pure water is 1:2-10,
Control temperature is maintained at 0-30 DEG C, and phosphate buffer solution is added and adjusts pH between 4-6, in the item that revolving speed is 60-150r/min
Leaching liquor is obtained after stirring 1-5h under part；
C. separating-purifying: coarse filtration is carried out to above-mentioned leaching liquor using filter cloth, collects filtrate, and filtrate is concentrated by ultrafiltration
2-8 times, obtain crude enzyme liquid；
D. immobilization handle: immobilized using macroreticular resin as carrier, by after activation processing macroreticular resin with
Crude enzyme liquid mixing, wherein the content of macroreticular resin is 0.05-0.5g/mL, controls pH between 4-6, and temperature is 25-40 DEG C, with
The revolving speed of 100-300r/min immobilizes processing in water bath with thermostatic control shaking table, and wherein the immobilization processing time is 2-4h, most
Theaflavin production immobile polyphenol oxidase is obtained eventually.
Preferably, in step b, the ratio of tealeaves and pure water is 1:5, and extraction temperature is 10 DEG C hereinafter, pH value control exists
Between 4.5-5.5, revolving speed is preferably 80r/min, mixing time 3h；In step c, 5 times are concentrated by ultrafiltration to filtrate；In step d,
The content of macroreticular resin is 0.1g/mL, and pH is controlled between 4.5-5.5, and shaking table temperature is 30 DEG C, shaking speed 180r/
Min, immobilization handle the time as 3h.
Preferably, in step d, the activation processing of the macroreticular resin is the following steps are included: by the anhydrous second of macroreticular resin
Alcohol impregnates for 24 hours, after resin is sufficiently swollen, is washed till eluate with dehydrated alcohol and is diluted with water and muddiness do not occur, then washed with pure water
To no alcohol taste, filter, drying, it is spare.
The beneficial effects of the present invention are:
1. the present invention in fresh tea passes polyphenol oxidase carry out separating-purifying, using macroreticular resin to polyphenol oxidase into
Row absorption is fixed, and has carried out related optimization to the process conditions, is successively carried out to tealeaves using pulverizer and colloid mill abundant
It grinds, the polyphenol oxidase in tealeaves is extracted with pure water, coarse filtration and ultrafiltration are successively carried out to leaching liquor, sufficiently gone
In addition to the tea polyphenols and the small-molecule substances such as caffeine in tealeaves, other impurities are effectively reduced in fresh tea passes to theaflavin product
The influence of purity improves the quality of theaflavin product；
2. in the present invention, the polyphenol oxidase by immobilization may be reused repeatedly, fresh tea passes are greatly reduced
Thus usage amount not only increases the utilization rate of polyphenol oxidase in theaflavin production process, the industry for reducing theaflavin is raw
Cost is produced, while reducing the generation of tea grounds waste, has reached energy-saving and environment-friendly purpose；
3. present invention process is easy to operate, reaction condition is mild, pollution-free, is suitble to large-scale industrial production.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
Embodiment 1
A kind of theaflavin production industrial production process of immobile polyphenol oxidase, comprising the following steps:
A. break process: 10g fresh tea passes are taken, are tentatively crushed using pulverizer, then further milled with colloid mill；
B. it extracts: being added pure water into tea-leaf power obtained in above-mentioned steps, the ratio of tealeaves and pure water is 1:2, control
Temperature processed is 0 DEG C, and phosphate buffer solution is added and adjusts pH to 4, is extracted after stirring 1h under conditions of revolving speed is 60r/min
Liquid；
C. separating-purifying: coarse filtration is carried out to above-mentioned leaching liquor using filter cloth, collects filtrate, and filtrate is concentrated by ultrafiltration
2 times, obtain crude enzyme liquid；
D. immobilization is handled: being immobilized using macroreticular resin as carrier, is taken 1g macroreticular resin, soaked with dehydrated alcohol
Bubble for 24 hours, after resin is sufficiently swollen, is washed till eluate and is diluted with water and muddiness do not occur, then be washed till nothing with pure water with dehydrated alcohol
Alcohol taste filters, drying, the macroreticular resin after activation processing is mixed with 20mL crude enzyme liquid, and control pH is 4, and temperature is 25 DEG C, with
The revolving speed of 100r/min immobilizes processing in water bath with thermostatic control shaking table, and wherein the immobilization processing time is 2h, finally obtains
Immobile polyphenol oxidase is used in theaflavin production.
Embodiment 2
A kind of theaflavin production industrial production process of immobile polyphenol oxidase, comprising the following steps:
A. break process: 10g fresh tea passes are taken, are tentatively crushed using pulverizer, then further milled with colloid mill；
B. it extracts: being added pure water into tea-leaf power obtained in above-mentioned steps, the ratio of tealeaves and pure water is 1:10, control
Temperature processed is 30 DEG C, and phosphate buffer solution is added and adjusts pH to 6, is soaked after stirring 5h under conditions of revolving speed is 150r/min
Extract；
C. separating-purifying: coarse filtration is carried out to above-mentioned leaching liquor using filter cloth, collects filtrate, and filtrate is concentrated by ultrafiltration
8 times, obtain crude enzyme liquid；
D. immobilization is handled: being immobilized using macroreticular resin as carrier, is taken 5g macroreticular resin, soaked with dehydrated alcohol
Bubble for 24 hours, after resin is sufficiently swollen, is washed till eluate and is diluted with water and muddiness do not occur, then be washed till nothing with pure water with dehydrated alcohol
Alcohol taste filters, drying, the macroreticular resin after activation processing is mixed with 10mL crude enzyme liquid, and control pH is 6, and temperature is 40 DEG C, with
The revolving speed of 300r/min immobilizes processing in water bath with thermostatic control shaking table, and wherein the immobilization processing time is 4h, finally obtains
Immobile polyphenol oxidase is used in theaflavin production.
Embodiment 3
A kind of theaflavin production industrial production process of immobile polyphenol oxidase, comprising the following steps:
A. break process: 10g fresh tea passes are taken, are tentatively crushed using pulverizer, then further milled with colloid mill；
B. it extracts: being added pure water into tea-leaf power obtained in above-mentioned steps, the ratio of tealeaves and pure water is 1:5, control
Temperature processed is 6 DEG C, and phosphate buffer solution is added and adjusts pH to 5, is extracted after stirring 3h under conditions of revolving speed is 80r/min
Liquid；
C. separating-purifying: coarse filtration is carried out to above-mentioned leaching liquor using filter cloth, collects filtrate, and filtrate is concentrated by ultrafiltration
5 times, obtain crude enzyme liquid；
D. immobilization is handled: being immobilized using macroreticular resin as carrier, is taken 2g macroreticular resin, soaked with dehydrated alcohol
Bubble for 24 hours, after resin is sufficiently swollen, is washed till eluate and is diluted with water and muddiness do not occur, then be washed till nothing with pure water with dehydrated alcohol
Alcohol taste filters, drying, the macroreticular resin after activation processing is mixed with 20mL crude enzyme liquid, and control pH is 5, and temperature is 30 DEG C, with
The revolving speed of 180r/min immobilizes processing in water bath with thermostatic control shaking table, and wherein the immobilization processing time is 3h, finally obtains
Immobile polyphenol oxidase is used in theaflavin production.
Immobile polyphenol oxidase obtained in embodiment 1-3 is applied to the production of theaflavin, as a result, it has been found that, through weight
Again using after 50 times, the enzyme activity of immobile polyphenol oxidase is not decreased obviously.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not limited to this hair the foregoing is merely a specific embodiment of the invention
Bright, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be included in the present invention
Protection scope within.
Claims (3)
1. the industrial production process that immobile polyphenol oxidase is used in a kind of theaflavin production, which comprises the following steps:
A. break process: fresh tea passes are taken, are tentatively crushed using pulverizer, then further milled with colloid mill；
B. it extracts: being added pure water into tea-leaf power obtained in above-mentioned steps, the ratio of tealeaves and pure water is 1:2-10, control
Temperature is maintained at 0-30 DEG C, and phosphate buffer solution is added and adjusts pH between 4-6, under conditions of revolving speed is 60-150r/min
Leaching liquor is obtained after stirring 1-5h；
C. separating-purifying: coarse filtration is carried out to above-mentioned leaching liquor using filter cloth, collects filtrate, and ultrafiltration concentration 2-8 is carried out to filtrate
Times, obtain crude enzyme liquid；
D. immobilization is handled: being immobilized using macroreticular resin as carrier, by the macroreticular resin and thick enzyme after activation processing
Liquid mixing, wherein the content of macroreticular resin is 0.05-0.5g/mL, controls pH between 4-6, and temperature is 25-40 DEG C, with 100-
The revolving speed of 300r/min immobilizes processing in water bath with thermostatic control shaking table, and wherein the immobilization processing time is 2-4h, final
To theaflavin production immobile polyphenol oxidase.
2. a kind of theaflavin production as described in claim 1 industrial production process of immobile polyphenol oxidase, feature
Be: preferably, in step b, the ratio of tealeaves and pure water is 1:5, and extraction temperature is 10 DEG C hereinafter, pH value control is in 4.5-
Between 5.5, revolving speed is preferably 80r/min, mixing time 3h；In step c, 5 times are concentrated by ultrafiltration to filtrate；In step d, macropore
The content of resin is 0.1g/mL, and pH is controlled between 4.5-5.5, and shaking table temperature is 30 DEG C, shaking speed 180r/min, Gu
Fixedization handles the time as 3h.
3. a kind of theaflavin production as claimed in claim 1 or 2 industrial production process of immobile polyphenol oxidase, special
Sign is: in step d, the activation processing of the macroreticular resin is the following steps are included: by macroreticular resin soaked in absolute ethyl alcohol
For 24 hours, it after resin is sufficiently swollen, is washed till eluate with dehydrated alcohol and is diluted with water and muddiness do not occur, then be washed till no alcohol with pure water
Taste filters, drying, spare.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811557810.1A CN109593736A (en) | 2018-12-19 | 2018-12-19 | A kind of theaflavin production industrial production process of immobile polyphenol oxidase |
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| CN201811557810.1A CN109593736A (en) | 2018-12-19 | 2018-12-19 | A kind of theaflavin production industrial production process of immobile polyphenol oxidase |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029326A (en) * | 2022-05-27 | 2022-09-09 | 湖南福来格生物技术有限公司 | Enzyme cross-linked aggregate and preparation method and application thereof |
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| WO2007090384A2 (en) * | 2006-02-09 | 2007-08-16 | Ernst-Moritz-Arndt-Universität Greifswald | Adhesives and adhesive methods |
| CN101492666A (en) * | 2008-10-16 | 2009-07-29 | 中国农业科学院茶叶研究所 | Method for fixing polyphenol oxidase with ion exchange resin support |
| CN102071232A (en) * | 2009-11-23 | 2011-05-25 | 湖州来色生物基因工程有限公司 | Method for synthesizing theaflavin by immobilized enzyme method |
| CN102226211A (en) * | 2011-04-25 | 2011-10-26 | 中国农业科学院茶叶研究所 | Method for preparing theaflavin by dynamic continuous oxidation on immobilized polyphenol oxidase |
-
2018
- 2018-12-19 CN CN201811557810.1A patent/CN109593736A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007090384A2 (en) * | 2006-02-09 | 2007-08-16 | Ernst-Moritz-Arndt-Universität Greifswald | Adhesives and adhesive methods |
| CN101492666A (en) * | 2008-10-16 | 2009-07-29 | 中国农业科学院茶叶研究所 | Method for fixing polyphenol oxidase with ion exchange resin support |
| CN102071232A (en) * | 2009-11-23 | 2011-05-25 | 湖州来色生物基因工程有限公司 | Method for synthesizing theaflavin by immobilized enzyme method |
| CN102226211A (en) * | 2011-04-25 | 2011-10-26 | 中国农业科学院茶叶研究所 | Method for preparing theaflavin by dynamic continuous oxidation on immobilized polyphenol oxidase |
Non-Patent Citations (1)
| Title |
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| 赵淑娟等: "茶多酚氧化酶酶学性质研究", 《湖南农业大学学报（自然科学版）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029326A (en) * | 2022-05-27 | 2022-09-09 | 湖南福来格生物技术有限公司 | Enzyme cross-linked aggregate and preparation method and application thereof |
| CN115029326B (en) * | 2022-05-27 | 2024-01-02 | 湖南福来格生物技术有限公司 | Enzyme cross-linked aggregate and preparation method and application thereof |
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Application publication date: 20190409 |