CN101113411B - Induction preparation of EGCG esterase and method for producing EGC and gallic acid by using the enzyme - Google Patents
Induction preparation of EGCG esterase and method for producing EGC and gallic acid by using the enzyme Download PDFInfo
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- CN101113411B CN101113411B CN2007100428298A CN200710042829A CN101113411B CN 101113411 B CN101113411 B CN 101113411B CN 2007100428298 A CN2007100428298 A CN 2007100428298A CN 200710042829 A CN200710042829 A CN 200710042829A CN 101113411 B CN101113411 B CN 101113411B
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Abstract
The invention relates to a method for inducing aspergillus niger to generate EGCG (epigallocatechin-3-gallate) esterase with EGCG, comprising the steps that: (1) an aspergillus niger seed culture medium is cultivated for 36 hours at 25-30 DEG C and 100-250r/min; (2) the aspergillus niger seed culture medium with a inoculated dose of 4-10 percent is transferred to a fermented culture medium that contains the EGCG and cultivated for 72 hours at 25-30 DEG C and 100-250r/min, and filtered and centrifugated to get enzyme liquid; then the enzyme is used for hydrolyzing the EGCG so as to transfer the EGCG into EGC (epigallocatechin) and ellagic acid, comprising: (1) an EGCG enzyme hydrolysis reaction system, an enzyme liquid blank system and a substrate blank system are constructed; (2) the reaction system and the blank systems are reacted for 0-48 hours at 25-60 DEG C or until the hydrolysis reaction is almost over; (3) high performance liquid and thin layer chromatography are used for analyzing the substrate and the products. The invention has high EGC transfer ratio and yield and the products are stable and easy to be separated, besides, the invention has the advantages of safety, environment protective, simple operation and strong controllability, thereby being applicable to mass scale production.
Description
Technical field
The invention belongs to the preparation of enzyme and the catalyzed conversion field of enzyme, particularly relate to a kind of method of inducing preparation and producing EGC and gallic acid with this enzyme of EGCG esterase.
Background technology
Tea catechin is a 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) wait composition.Catechin monomers anti-oxidant, remove aspects such as free radical, antitumor, anti-mutation, anti-ageing, antiviral, antianaphylaxis, antisepsis and anti-inflammation, raise immunity, 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 monomeric physiologically active of different tea catechins has certain difference, separates the physiologically active that the various monomers of preparation can make full use of various catechin tool advantages.In addition, exploitation tea catechin new drug also requires monomeric tea catechin.Thereby separation and purification or directed synthetic catechin monomers are to enlarging the application approach and the mode of catechin, for catechin has crucial meaning industrial 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 cost an arm and a leg.Therefore, the method for seeking a kind of raw material production EGC that can be big 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 can EGCG be hydrolyzed into EGC and gallic acid just can achieve the above object by hydrolysis reaction, has realistic meaning.But chemical processes such as conventional acid or basic hydrolysis exist percent hydrolysis extremely low, and hydrolysate is shortcoming such as very easily oxidized in instability or the reaction process extremely.In addition, according to the literature, Pig Liver Esterase can not hydrolysis EGCG, does not find that also other commercially available esterase or lipase can hydrolysis EGCG.Have only tannase (Tannase) hydrolyzable EGCG to become non-ester catechin EGC, but this production of enzyme is few and price is high.
Aspergillus niger (Aspergillus niger) is the fungi of a kind of good gas, easily cultivation, the multiple prozyme of product, belongs to Deuteromycotina, Aspergillus.Aspergillus niger can be secreted multiple lytic enzyme, as proteolytic enzyme, amylase and esterase etc.The zymin of aspergillus niger production has that consumption is big, applied range, characteristics that security is good, more and more is subject to people's attention, and has a vital role industrial.1987, Food and Agricultural Organization of the United Nations (FAO)/The World Health Organization (WHO)/foodstuff additive and the pollutant combined Committee of Experts (JECFA) approval aspergillus niger can be used for the foodstuffs industry production of enzyme preparation.
Summary of the invention
The purpose of this invention is to provide a kind of enzymatic hydrolysis process that the EGCG that tealeaves content is abundant changes into the less and expensive EGC of content that is used for, this 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 induction preparation of EGCG esterase method of the present invention comprises the following steps:
(1) with the aspergillus niger seed culture medium at 25-30 ℃, cultivated under the 100-250r/min condition 36 hours;
(2) be forwarded in the fermention medium that contains EGCG with the 4-10% inoculum size,, cultivated 72 hours under the 100-250r/min condition, filter, the centrifugal enzyme liquid that gets at 25-30 ℃.
Described aspergillus niger is the safe bacterial classification of universally acknowledged foodstuffs industry;
Described EGCG raw material can adopt different purity;
It is step, batch feeding formula or cultured continuously formula that enzyme process is produced in described cultivation.
A kind of method of utilizing the EGCG esterase 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 hour or finish substantially just to stop until hydrolysis reaction;
(3) utilize high performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) to analyze substrate and product.
Described EGCG enzymatic hydrolysis reaction system is meant EGCG substrate, EGCG lytic enzyme and pH3-7 buffer solution system;
Described enzyme liquid air lean type is to be meant EGCG lytic enzyme and pH3-7 buffer solution system;
The blank system of described substrate is meant EGCG substrate and pH3-7 buffer solution system;
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 niger commonly used (the industrial safety bacterial classification of U.S. FDA approval) enzymatic production, have the advantage 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 esterase, can produce this medicine intermediate of EGC monomer for large-scale industrialization and provide new thinking and approach with gallic acid with extensive industrial use.
Accompanying drawing is illustrated
Fig. 1 is an 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 12h, shows among the figure that the 2nd peak (retention time 1.773min) is EGC, and the 3rd peak (retention time 2.216min) is EGCG;
Fig. 4 is the HPLC spectrogram of diluted enzymolysis solution behind the enzyme digestion reaction 24h, show among the figure that the 2nd peak is EGC, and EGCG is almost by complete hydrolysis.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described 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 institute restricted portion equally.
Embodiment 1
1. the seed culture of aspergillus niger ATCC 46890
The aspergillus niger seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N; With aspergillus niger AspergillusnigerATCC 46890 serves as to produce bacterial classification, at pH5.0,30 ℃, cultivates 36 hours under the 200r/min condition.
2. fermentation of Aspergillus niger produces enzyme
Aspergillus niger produces the enzyme substratum: 1% glucose, and 0.1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds EGCG to final concentration 2.5g/L, pH5.0; With 4% inoculum size,, cultivate 72h under the 160r/min condition and induce the product enzyme at 30 ℃.The fermentation clear liquid of filtration or centrifugal collection is exactly an EGCG lytic enzyme liquid.Be contrast with the fermentation culture that does not add EGCG in the experiment.
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, every 6-12h get 1 sample and analyze.
4. the concentration that adopts thin-layer chromatography and HPLC (high performance liquid chromatography) to come 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 used dissolve with methanol through revolving to steam to be concentrated into after doing again; Chromatographic solution (10mL) adopts 80% methylene dichloride+20% methyl alcohol+2 to drip+1 Glacial acetic acid; Sample launches back 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 niger MC 57
The aspergillus niger seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N; With aspergillus niger Aspergillus nigerMC 57 serves as to produce bacterial classification, pH5.0, and 30 ℃, 200rpm cultivated 36 hours.
2. fermentation of Aspergillus niger produces enzyme
Aspergillus niger produces the enzyme substratum: 1% glucose, and 0.1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds EGCG to final concentration 5.0g/L, pH5.0; With 8% inoculum size,, cultivate 72h under the 160r/min condition and induce the product enzyme at 30 ℃.The fermentation clear liquid of filtration or centrifugal collection is exactly an EGCG lytic enzyme liquid.Be contrast with the fermentation culture that does not add EGCG in the experiment.
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/LEGCG substrate 2mL, deionized water 2mL, the acetate buffer solution 2mL of pH5.2.40 ℃, oscillatory reaction 24h, every 6-12h get 1 sample and analyze.
4. the concentration that adopts thin-layer chromatography and HPLC (high performance liquid chromatography) to come qualitative and quantitative analysis EGCG, EGC and gallic acid, analysis condition sees embodiment 1 for details.
1. the seed culture of aspergillus niger ATCC 46890
The aspergillus niger seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N; With aspergillus niger Aspergillus nigerATCC 46890 serves as to produce bacterial classification, at pH5.0,30 ℃, cultivates 36 hours under the 200r/min condition.
2. fermentation of Aspergillus niger produces enzyme
Aspergillus niger produces the enzyme substratum: 1% glucose, and 0.1% peptone, 0.05% citric acid, 0.015 %Tween 80, and 2%Vogel ' s medium N adds EGCG to final concentration 10.0g/L, pH5.0; With 8% inoculum size,, cultivate 72h under the 130r/min condition and induce the product enzyme at 30 ℃.The fermentation clear liquid of filtration or centrifugal collection is exactly an EGCG lytic enzyme liquid.Be contrast with the fermentation culture that does not add EGCG in the experiment.
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, every 6-12h get 1 sample and analyze.
4. the concentration that adopts thin-layer chromatography and HPLC (high performance liquid chromatography) to come qualitative and quantitative analysis EGCG, EGC and gallic acid, analysis condition sees embodiment 1 for details.
Embodiment 4
1. the seed culture of aspergillus niger MC57
The aspergillus niger seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015 %Tween 80,2%Vogel ' s medium N; With aspergillus niger AspergillusnigerMC 57 serves as to produce bacterial classification, pH5.0, and 30 ℃, 200rpm cultivated 36 hours.
2. fermentation of Aspergillus niger produces enzyme
Aspergillus niger produces the enzyme substratum: 1% glucose, and 0.1% peptone, 0.05% citric acid, 0.015 %Tween 80, and 2%Vogel ' s medium N adds EGCG to final concentration 12.0g/L, pH5.0; With 8% inoculum size,, cultivate 72h under the 130r/min condition and induce the product enzyme at 30 ℃.The fermentation clear liquid of filtration or centrifugal collection is exactly an EGCG lytic enzyme liquid.Be contrast with the fermentation culture that does not add EGCG in the experiment.
3.EGCG enzymic hydrolysis
Reaction system: 20g/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: 20g/L EGCG substrate 2mL, deionized water 2mL, the acetate buffer solution 2mL of pH5.2.40 ℃, oscillatory reaction 24h, every 6-12h get 1 sample and analyze.
4. the concentration that adopts thin-layer chromatography and HPLC (high performance liquid chromatography) to come qualitative and quantitative analysis EGCG, EGC and gallic acid, analysis condition sees embodiment 1 for details.
More as can be known, aspergillus niger can be induced the generation esterase 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 does not have the esterase activity of hydrolysis EGCG.
Claims (4)
1. method of utilizing the EGCG esterase to produce EGC and gallic acid comprises:
(1) with aspergillus niger ATCC46890 seed culture medium at 25-30 ℃, cultivated under the 100-250r/min condition 36 hours;
(2) be forwarded in the fermention medium that contains EGCG with the 4-10% inoculum size,, cultivated 72 hours under the 100-250r/min condition, filter, the centrifugal enzyme liquid that gets at 25-30 ℃;
(3) make up EGCG enzymatic hydrolysis reaction system, enzyme liquid air lean type system and the blank system of substrate;
(4) reaction system and blank system be at 25-60 ℃, reacted 0-48 hour or finish just to stop until hydrolysis reaction;
(5) utilize high performance liquid chromatography and thin layer chromatography analysis substrate and product.
2. a kind of method of utilizing the EGCG esterase to produce EGC and gallic acid according to claim 1 is characterized in that: it is step, batch feeding formula or cultured continuously formula that described step (2) is produced enzyme process.
3. a kind of method of utilizing the EGCG esterase to produce EGC and gallic acid according to claim 1, it is characterized in that: described step (3) EGCG enzymatic hydrolysis reaction system is EGCG substrate, EGCG lytic enzyme and pH3-7 buffer solution system, enzyme liquid air lean type system is EGCG lytic enzyme and pH3-7 buffer solution system, and the blank system of substrate is EGCG substrate and pH3-7 buffer solution system.
4. a kind of method of utilizing the EGCG esterase to produce EGC and gallic acid according to claim 1, it is characterized in that: described step (5) thin layer chromatography analysis is purification on normal-phase silica gel plate analysis or reverse phase silica gel plate analysis.
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| CN102329739B (en) * | 2011-08-31 | 2012-12-05 | 云南大学 | Aspergillus versicolor for fermenting Jatropha cake to manufacture biological bacterial manure |
| CN102424799A (en) * | 2011-12-27 | 2012-04-25 | 云南大学 | Aspergillus versicolor strain for manufacturing detoxicated feedstuff through fermentation of Jatropha curcas cakes |
| CN104673844B (en) * | 2015-02-15 | 2016-08-17 | 华南农业大学 | Prevent the method that epigallo catechin and gallic acid are converted |
| CN104673845B (en) * | 2015-02-15 | 2016-07-20 | 华南农业大学 | Utilize the method that aspergillus niger resting cell produces epigallo catechin and gallic acid |
| CN110879269B (en) * | 2019-12-10 | 2022-04-08 | 浙江尖峰健康科技有限公司 | Combined identification method of cranberry extract |
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