CN101134941B - Method for preparing EGCG hydrolytic enzyme and producing EGC and gallic acid by aspergillus oryzae - Google Patents
Method for preparing EGCG hydrolytic enzyme and producing EGC and gallic acid by aspergillus oryzae Download PDFInfo
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- CN101134941B CN101134941B CN2007100443141A CN200710044314A CN101134941B CN 101134941 B CN101134941 B CN 101134941B CN 2007100443141 A CN2007100443141 A CN 2007100443141A CN 200710044314 A CN200710044314 A CN 200710044314A CN 101134941 B CN101134941 B CN 101134941B
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Abstract
The process of inducing aspergillus oryzae with epigallocatechin gallate (EGCG) to produce EGCG hydrolase in high efficiency includes the following steps: 1. culturing aspergillus oryzae seed in the culture medium at 25-30 deg.c and 100-250 rpm for 36 hr; 2. inoculating to EGCG fermenting culture medium in the inoculated amount of 4-10 %, culturing at 25-30 deg.c and 100-250 rpm for 48-120 hr, filtering and centrifuging to obtain EGCG hydrolase. The process of utilizing EGCG hydrolase in hydrolyzing EGCG to produce epigallocatechin (EGC) and gallic acid includes the steps of constructing EGCG enzyme hydrolyzing reaction system, blank enzyme system and blank substrate system; reaction and substrate and product analysis. The present invention has EGC converting rate and yield, stable and easy-to-separate product, high safety, environment friendship and other advantages, and is suitable for industrial production.
Description
Technical field
The invention belongs to the preparation of enzyme and the catalyzed conversion field of enzyme, the method that particularly relates to a kind of EGCG hydrolysis Enzyme induced formation preparation and produce EGC and gallic acid with this enzyme.
Background technology
Tea catechin is physiologically active ingredient main in the tealeaves, tea catechin is by NVP-XAA 723 (epi-gallocatechin-3-gallate, EGCG), L-Epicatechin gallate (epicatechin-3-gallate, ECG), nutgall catechin gallic acid ester (gallocatechin-3-gallate, GCG), l-Epicatechol (epicatechin, have another name called cis-catechin, EC), epigallocatechin (epigallocatechin, have another name called cis-gallocatechin, EGC) etc. composition.Catechin monomers anti-oxidant, remove the aspects such as free radical, antitumor, anti-mutation, anti-ageing, antiviral, antianaphylaxis, antisepsis and anti-inflammation, enhancing immunologic function, radioprotective, hypotensive, reducing blood-fat and all have very high activity, prevention and treatment malignant tumour, hyperlipidemia, arteriosclerosis, cerebral thrombosis, diabetes, obesity etc. are all had good action.
The physiologically active of different tea catechin monomers has certain difference, separates the physiologically active that the various monomers of preparation can take full advantage of various catechin tool advantages.In addition, exploitation tea catechin new drug also requires the tea catechin of monomer.Thereby separation and purification or directed synthetic catechin monomers are to enlarging application approach and the mode of catechin, for catechin is of great significance at industrial tools of tapping new markets such as medicine, health care, food, makeup.
The method of present existing acquisition catechin monomers is the technological method by separation and purification generally.And with regard to EGC, content is lower in tealeaves, even the separation and purification yield is higher, output is still very low, causes unusual short and expensive.Therefore, the method for seeking a kind of raw material production EGC that can be large from those amounts and is easy to get just seems very urgent and necessary.EGCG is the highest component of content in the catechin, is the ester that EGC and gallic acid form, and therefore if EGCG is hydrolyzed into EGC and gallic acid just can achieve the above object by hydrolysis reaction, has realistic meaning.But the chemical processes such as conventional acid or basic hydrolysis exist percent hydrolysis extremely low, the shortcoming such as very easily oxidized in the extremely unstable or reaction process of hydrolysate.In addition, according to the literature, Pig Liver Esterase can not be hydrolyzed EGCG, does not find that also other commercially available esterase or lipase can be hydrolyzed EGCG.Only have tannase (Tannase) hydrolyzable EGCG to become non-ester catechin EGC, but this production of enzyme is few and price is high.
Aspergillus oryzae (Aspergillus oryzae) is the fungi of a kind of aerobic property, easily cultivation, the multiple prozyme of product, belongs to Deuteromycotina, Aspergillus.Aspergillus oryzae can be secreted multiple lytic enzyme, such as proteolytic enzyme, amylase and esterase etc.The zymin of aspergillus oryzae production has that consumption is large, applied range, characteristics that security is good, more and more is subject to people's attention, and has a vital role industrial.Aspergillus oryzae is long at the industrial applicating history such as fermentative production soy sauce, sauce processed, wine brewing.1987, Food and Agricultural Organization of the United Nations (the FAO)/World Health Organization (WHO)/foodstuff additive and the pollutant combined Committee of Experts (JECFA) approval aspergillus oryzae can be used for the production that zymin is used in foodstuffs industry.USEPA shows the harm of human health and environment and the risk assessment result of industrial application aspergillus oryzae, and aspergillus oryzae is to the animals and plants no pathogenicity.Product and food that the aspergillus oryzae fermentation is processed have certain health-care effect to the mankind or animal, so utilizing aspergillus oryzae to research and develop high-quality biological product has vast potential for future development.
Summary of the invention
The purpose of this invention is to provide a kind of enzymatic hydrolysis process that changes into the less and expensive EGC of content for the EGCG with the tealeaves rich content, the method is simple to operate, transformation efficiency is high, product is stable, environmental protection, can produce the EGC monomer and gallic acid provides new thinking and approach for large-scale industrialization.
A kind of aspergillus oryzae of efficiently inducing of the present invention prepares EGCG lytic enzyme method, comprises the following steps:
(1) with the aspergillus oryzae seed culture medium at 25-30 ℃, cultivated under 100-250r/min condition 36 hours;
(2) be forwarded in the fermention medium that contains EGCG with 4-10% inoculum size, at 25-30 ℃, cultivated 48-120 hours under 100-250r/min condition, filter, the centrifugal enzyme liquid that gets.
Described aspergillus oryzae is the safe bacterial classification of foodstuffs industry;
Described EGCG raw material adopts different purity;
It is step, fed-batch type or cultured continuously formula that enzyme process is produced in described cultivation.
A kind of method of utilizing the EGCG lytic enzyme to produce EGC and gallic acid of the present invention comprises the following steps:
(1) makes up EGCG enzymatic hydrolysis reaction system, enzyme liquid air lean type system and the blank system of substrate;
(2) reaction system and blank system be at 25-60 ℃, reacted 0-48 hours or until hydrolysis reaction finish just to stop;
(3) utilize high performance liquid chromatography and thin layer chromatography analysis substrate and product.
Described EGCG enzymatic hydrolysis reaction system refers to EGCG substrate, EGCG lytic enzyme and pH3-7 buffer solution system;
Described enzyme liquid air lean type is to refer to EGCG lytic enzyme and pH3-7 buffer solution system;
The blank system of described substrate refers to EGCG substrate and pH3-7 buffer solution system;
Described EGCG substrate adopts different purity;
It is purification on normal-phase silica gel plate analysis or reverse phase silica gel plate analysis that described TLC analyzes.
Beneficial effect of the present invention:
(1) utilizes foodstuffs industry aspergillus oryzae commonly used (the industrial safety bacterial classification of U.S. FDA, Food and Agricultural Organization of the United Nations (the FAO)/World Health Organization (WHO)/foodstuff additive and the pollutant combined Committee of Experts (JECFA) approval) enzymatic production, have advantages of safety and environmental protection;
(2) present method is simple to operate, product is stable, transformation efficiency is high and controllability is strong;
(3) utilize the EGCG lytic enzyme, can produce this medicine intermediate of EGC monomer for large-scale industrialization and provide new thinking and approach with the gallic acid with extensive industrial use.
Accompanying drawing is illustrated
Fig. 1 is EGCG enzymatic hydrolysis reaction formula;
Fig. 2 is the HPLC spectrogram of EGCG and EGC standard substance, shows among the figure that the retention time of EGC and EGCG is respectively 1.770min and 2.211min;
Fig. 3 is the HPLC spectrogram of diluted enzymolysis solution behind the enzyme digestion reaction 2h, shows among the figure that the 2nd peak (retention time 1.793min) is EGC, and the 3rd peak (retention time 2.165min) is EGCG, almost by complete hydrolysis.
Fig. 4 utilizes TLC to analyze the result of EGCG substrate and EGC product, and 1 refers to product EGC standard substance, and 2 refer to the sample of EGCG after the lytic enzyme hydrolysis, and 3 refer to lytic enzyme blank (containing a small amount of EGC product), and 4 refer to substrate EGCG standard substance.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
1. the seed culture of aspergillus oryzae ATCC20719
Aspergillus oryzae seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s medium N; Take aspergillus oryzae Aspergillus oryzae ATCC20719 as producing bacterial classification, at pH5.0,30 ℃, cultivated 36 hours under the 200r/min condition.
2. aspergillus oryzae enzymatic production
The aspergillus oryzae culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s medium N adds EGCG to final concentration 2.5g/L, pH5.0; With 4% inoculum size, at 30 ℃, cultivate 72h under the 160r/min condition and induce the product enzyme.The fermentation clear liquid of filtration or centrifugal collection is exactly EGCG lytic enzyme liquid.In the experiment take the fermentation culture that do not add EGCG as contrast.
3.EGCG enzymic hydrolysis
Reaction system: the substrate 2mL of 10g/L EGCG, EGCG lytic enzyme liquid 2mL, the acetate buffer solution 2mL of pH5.2; EGCG lytic enzyme liquid air lean type system: the acetate buffer solution 2mL of pH5.2, EGCG lytic enzyme liquid 2mL, the acetate buffer solution 2mL of pH5.2; The blank system of substrate: 10g/L EGCG substrate 2mL, deionized water 2mL, the acetate buffer solution 2mL of pH5.2.40 ℃, oscillatory reaction 24h, per 6-12h get 1 sample and analyze.
4. adopt thin-layer chromatography and HPLC (high performance liquid chromatography) to come the concentration of qualitative and quantitative analysis EGCG, EGC and gallic acid.
Thin-layer chromatography condition: use ethyl acetate to extract enzyme hydrolyzate 3 times with the ratio of 1:1 (v/v); Acetic acid ethyl acetate extract is reduced to and uses dissolve with methanol after doing again through revolving inspissation; Chromatographic solution (10mL) adopts 80% methylene dichloride+20% methyl alcohol+2 to drip+1 Glacial acetic acid; After launching, sample uses the cerous sulfate chromogenic reagent.
High-efficient liquid phase chromatogram condition: be equipped with chromatographic column Phenomenex Luna C18 (150mm * 4.60mm, 5 μ m) or on the liquid chromatograph of C8 post analyze, analysis condition: moving phase is acetonitrile-water-phosphoric acid buffer (pH3.5) (30:70:0.5, v/v), the detection wavelength is 270nm, and flow velocity is 1.0mL/min.
1. the seed culture of aspergillus oryzae NRRL692
Aspergillus oryzae seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s mediumN; Take aspergillus oryzae Aspergillus oryzae NRRL692 as producing bacterial classification, pH5.0,25-30 ℃, 200rpm cultivation 36 hours.
2. aspergillus oryzae enzymatic production
The aspergillus oryzae culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s medium N adds EGCG to final concentration 5.0g/L, pH5.0; With 8% inoculum size, at 30 ℃, cultivate 72h under the 160r/min condition and induce the product enzyme.The fermentation clear liquid of filtration or centrifugal collection is exactly EGCG lytic enzyme liquid.In the experiment take the fermentation culture that do not add EGCG as contrast.
3.EGCG enzymic hydrolysis
Reaction system: 15g/L EGCG substrate 2mL, EGCG lytic enzyme liquid 2mL, the acetate buffer solution 2mL of pH5.2; EGCG lytic enzyme liquid air lean type system: the acetate buffer solution 2mL of pH5.2, EGCG lytic enzyme liquid 2mL, the acetate buffer solution 2mL of pH5.2; The blank system of substrate: 15g/L EGCG substrate 2mL, deionized water 2mL, the acetate buffer solution 2mL of pH5.2.40 ℃, oscillatory reaction 24h, per 6-12h get 1 sample and analyze.
4. adopt thin-layer chromatography and HPLC (high performance liquid chromatography) to come the concentration of qualitative and quantitative analysis EGCG, EGC and gallic acid, analysis condition sees embodiment 1 for details.
1. the seed culture of aspergillus oryzae ATCC201873
Aspergillus oryzae seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween80,2%Voge1 ' s medium N; Take aspergillus oryzae Aspergillus oryzae ATCC201873 as producing bacterial classification, at pH5.0,30 ℃, cultivated 36 hours under the 200r/min condition.
2. aspergillus oryzae enzymatic production
The aspergillus oryzae culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s medium N adds EGCG to final concentration 10.0g/L, pH5.0; With 8% inoculum size, at 30 ℃, cultivate 72h under the 130r/min condition and induce the product enzyme.The fermentation clear liquid of filtration or centrifugal collection is exactly EGCG lytic enzyme liquid.In the experiment take the fermentation culture that do not add EGCG as contrast.
3.EGCG enzymic hydrolysis
Reaction system: 30g/L EGCG substrate 2mL, EGCG lytic enzyme liquid 2mL, the acetate buffer solution 2mL of pH5.2; EGCG lytic enzyme liquid air lean type system: the acetate buffer solution 2mL of pH5.2, EGCG lytic enzyme liquid 2mL, the acetate buffer solution 2mL of pH5.2; The blank system of substrate: 30g/L EGCG substrate 2mL, deionized water 2mL, the acetate buffer solution 2mL of pH5.2.40 ℃, oscillatory reaction 24h, per 6-12h get 1 sample and analyze.
4. adopt thin-layer chromatography and HPLC (high performance liquid chromatography) to come the concentration of qualitative and quantitative analysis EGCG, EGC and gallic acid, analysis condition sees embodiment 1 for details.
1. the seed culture of aspergillus oryzae ATCC76080
Aspergillus oryzae seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s medium N; Take aspergillus oryzae Aspergillus oryze ATCC76080 as producing bacterial classification, pH5.0,25-30 ℃, 200rpm cultivation 36 hours.
2. aspergillus oryzae enzymatic production
The aspergillus oryzae culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s medium N adds EGCG to final concentration 12.0g/L, pH5.0; With 8% inoculum size, at 30 ℃, cultivate 72h under the 130r/min condition and induce the product enzyme.The fermentation clear liquid of filtration or centrifugal collection is exactly EGCG lytic enzyme liquid.In the experiment take the fermentation culture that do not add EGCG as contrast.
3.EGCG enzymic hydrolysis
Reaction system: 20g/LEGCG substrate 2mL, EGCG lytic enzyme liquid 2mL, the acetate buffer solution 2mL of pH5.2; EGCG lytic enzyme liquid air lean type system: the acetate buffer solution 2mL of pH5.2, EGCG lytic enzyme liquid 2mL, the acetate buffer solution 2mL of pH5.2; The blank system of substrate: 20g/L EGCG substrate 2mL, deionized water 2mL, the acetate buffer solution 2mL of pH5.2.40 ℃, oscillatory reaction 24h, per 6-12h get 1 sample and analyze.
4. adopt thin-layer chromatography and HPLC (high performance liquid chromatography) to come the concentration of qualitative and quantitative analysis EGCG, EGC and gallic acid, analysis condition sees embodiment 1 for details.
More as can be known, aspergillus oryzae can be induced the generation lytic enzyme by add EGCG in substratum, and EGCG is converted into EGC and gallic acid according to the high performance liquid chromatography of embodiment 1 to embodiment 4 and thin layer chromatography analysis result; And the fermented liquid that does not add the EGCG inductor is not hydrolyzed the lytic enzyme vigor of EGCG.In addition, adding sweet oil in the substratum induces the lipase of preparation also not to be hydrolyzed the vigor of EGCG.
Claims (8)
1. a method of efficiently inducing aspergillus oryzae to prepare the EGCG lytic enzyme comprises the following steps:
(1) the aspergillus oryzae seed is inoculated in seed culture medium after, at 25-30 ℃, cultivated under the 100-250 r/min condition 36 hours;
Described aspergillus oryzae seed is aspergillus oryzae ATCC 20719;
(2) be forwarded in the fermention medium that contains EGCG with the aspergillus oryzae seed liquor of 4-10% inoculum size with step (1) preparation, at 25-30 ℃, cultivated 48-120 hour under the 100-250 r/min condition, filter, the centrifugal enzyme liquid that gets.
2. a kind of induction preparation of EGCG lytic enzyme method according to claim 1, it is characterized in that: described aspergillus oryzae is the safe bacterial classification of foodstuffs industry.
3. a kind of induction preparation of EGCG lytic enzyme method according to claim 1 is characterized in that: the different purity of described EGCG raw material employing.
4. a kind of induction preparation of EGCG lytic enzyme method according to claim 1 is characterized in that: it is step, fed-batch type or cultured continuously formula that enzyme process is produced in the cultivation of described step (2).
5. the EGCG lytic enzyme of a utilization such as the claim 1 preparation method of producing EGC and gallic acid comprises the following steps:
(1) makes up EGCG enzymatic hydrolysis reaction system, enzyme liquid air lean type system and the blank system of substrate;
(2) reaction system and blank system be at 40 ℃, reacted 24 hours or until hydrolysis reaction finish just to stop;
(3) utilize high performance liquid chromatography and thin layer chromatography analysis substrate and product.
6. a kind of method of utilizing the EGCG lytic enzyme to produce EGC and gallic acid according to claim 5, it is characterized in that: described EGCG enzymatic hydrolysis reaction system is EGCG substrate, EGCG lytic enzyme and pH5.2 buffer solution system, enzyme liquid air lean type system is EGCG lytic enzyme and pH5.2 buffer solution system, and the blank system of substrate is EGCG substrate and pH5.2 buffer solution system.
7. a kind of method of utilizing the EGCG lytic enzyme to produce EGC and gallic acid according to claim 5 is characterized in that: the different purity of described EGCG substrate employing.
8. a kind of method of utilizing the EGCG lytic enzyme to produce EGC and gallic acid according to claim 5, it is characterized in that: described thin layer chromatography analysis is purification on normal-phase silica gel plate analysis or reverse phase silica gel plate analysis.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1286252A (en) * | 1999-08-16 | 2001-03-07 | 弗·哈夫曼-拉罗切有限公司 | Process for preparing epigallocatechin gallate |
| CN1319597A (en) * | 2000-11-15 | 2001-10-31 | 中国科学院福建物质结构研究所 | Method for separating catechin compound from theopolyphenols |
| CN1367171A (en) * | 2001-12-21 | 2002-09-04 | 浙江一新制药股份有限公司 | Extraction method of high-content EGCg catechin |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1286252A (en) * | 1999-08-16 | 2001-03-07 | 弗·哈夫曼-拉罗切有限公司 | Process for preparing epigallocatechin gallate |
| CN1319597A (en) * | 2000-11-15 | 2001-10-31 | 中国科学院福建物质结构研究所 | Method for separating catechin compound from theopolyphenols |
| CN1367171A (en) * | 2001-12-21 | 2002-09-04 | 浙江一新制药股份有限公司 | Extraction method of high-content EGCg catechin |
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