Method for enzymatic synthesis of theaflavin TF by virtue of tea polyphenol oxidase isoenzyme PPO1

A tea polyphenol oxidase isoenzyme and enzymatic synthesis technology, which is applied in the field of theaflavin TF preparation, can solve the problems of large differences in biological activities of theaflavin components, diverse enzymatic synthesis products, and theaflavin components. The problem of high cost is to avoid the tedious separation and purification process, improve the utilization rate of the substrate, and achieve the effect of a single product.

Inactive Publication Date: 2015-05-27
HUNAN AGRICULTURAL UNIV
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Problems solved by technology

[0008] The technical problem to be solved by the present invention is: to directly extract and prepare theaflavins from tea leaves and prepare theaflavins by chemical oxidation to form the theaflavins mixture according to the above-mentioned prior art, polyphenol oxidase isozymes have complex composition and various enzymatic synthesis products, tea Due to the large differences in biological activity of flavin components, high cost of purification and preparation of theaflavin components, low degree of industrialization, and cumbersome operations, a method for enzymatically synthesizing single theaflavins using tea polyphenol oxidase isoenzyme PPO1 is provided. The method of the flavin component TF, using the specific tea polyphenol oxidase isoenzyme PPO1, to form the theaflavin component TF by catalyzing the oxidation of tea polyphenol solution conversion

Method used

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  • Method for enzymatic synthesis of theaflavin TF by virtue of tea polyphenol oxidase isoenzyme PPO1
  • Method for enzymatic synthesis of theaflavin TF by virtue of tea polyphenol oxidase isoenzyme PPO1
  • Method for enzymatic synthesis of theaflavin TF by virtue of tea polyphenol oxidase isoenzyme PPO1

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Experimental program
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Embodiment 1

[0029] Tea polyphenols are dissolved in citric acid-disodium hydrogen phosphate buffer solution with a pH value of 5.5 to prepare a tea polyphenol solution with a mass volume concentration of 1.2%.

[0030] Take 1ml of tea polyphenol oxidase isoenzyme PPO1 enzyme solution with an enzyme activity of 4340U / ml, add it to 500ml tea polyphenol solution with a mass volume concentration of 1.2%, use air as the source of oxygen for enzymatic synthesis, at 36°C After oscillating on a shaker at 60r / min for 60min, immediately use ethyl acetate equal to the volume of the reaction solution to stand for extraction for 5min, and the extract was centrifuged at 1500r / min for 3min, and the ethyl ester layer solution of the organic phase was taken for the inorganic phase The solution part was extracted once again by repeating the above operation, centrifuged, and the organic phase ethyl acetate solution obtained twice was combined, and the ethyl acetate solution was vacuum concentrated and recove...

Embodiment 2

[0032] Tea polyphenols are dissolved in sodium acetate-acetic acid buffer solution with a pH of 4.5 to prepare a tea polyphenol solution with a mass volume concentration of 3.0%.

[0033] Add 1ml of tea polyphenol oxidase isoenzyme monomer PPO1 enzyme solution with an enzyme activity of 4340U / ml to 650ml tea polyphenol solution with a mass volume concentration of 3%, and use air as the oxygen source for enzymatic synthesis at 20°C After oscillating on a shaker at 90r / min for 45min under certain conditions, immediately use ethyl acetate equal to the volume of the reaction solution to stand for extraction for 20min, and the extract is centrifuged at 3000r / min for 1min, and the ethyl ester layer solution of the organic phase is taken for the inorganic For the phase solution part, repeat the above operation to extract and centrifuge once again, combine the organic phase ethyl acetate solution obtained twice, concentrate and recover ethyl acetate under the condition of vacuum degree...

Embodiment 3

[0035] Tea polyphenols were dissolved in Tris-HCl buffer solution with a pH of 6.5 to prepare a tea polyphenol solution with a mass volume concentration of 2.1%.

[0036] Add 1ml of tea polyphenol oxidase isoenzyme monomer PPO1 enzyme solution with an enzyme activity of 4340U / ml into 800ml tea polyphenol solution with a mass volume concentration of 2.1%, and use air as the source of oxygen for enzymatic synthesis. After oscillating on a shaker at 150 rpm at ℃ for 30 min, immediately use ethyl acetate equal to the volume of the reaction solution to stand for extraction for 30 min, then centrifuge the extract at 500 r / min for 5 min, take the ethyl ester layer solution of the organic phase, and For the inorganic phase solution, repeat the above operation to extract and centrifuge once again, combine the organic phase ethyl acetate solution obtained twice, and concentrate and recover ethyl acetate under the condition of vacuum degree of -0.075Mpa and temperature of 55°C, and finall...

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Abstract

The invention discloses a method for enzymatic synthesis of theaflavin TF by virtue of tea polyphenol oxidase isoenzyme PPO1. The method is the optimization of a technique and a technology, which is capable of achieving the oriented enzymatic synthesis of a single theaflavin component TF through an enzymatic reaction with a shaking table as well as subsequent steps of extracting, centrifuging, concentrating, freeze-drying and the like by taking tea polyphenol oxidase isoenzyme PPO1 as an enzyme source, catechins EC and EGC components in tea polyphenol a major reaction substrates and a buffer solution at a certain pH value as a reaction system. The method overcomes shortcomings that multiple theaflavin components are generated simultaneously, a technology for separating and purifying the theaflavin components is complex, the preparation of a high-purity theaflavin component is quite difficult and the like in a conventional process which carries out the enzymatic synthesis of theaflavin by virtue of crude enzyme fluid of tea polyphenol oxidase; the method achieves a technological innovation that a single product is generated from enzymatic synthesis and a side product is avoided at reaction end, and the method avoids a technological bottleneck in subsequent separation and purification of the theaflavin component, so as to lay the theoretical and practical foundation for the green, safe and efficient large-scale industrial production and utilization of the theaflavin component.

Description

technical field [0001] The invention belongs to the field of bioengineering preparation, and relates to a preparation method of theaflavin TF, in particular to a tea polyphenol oxidase isoenzyme PPO1 enzyme liquid in a tea polyphenol solution reaction system to synthesize theaflavin TF directional enzymatically Methods. Background technique [0002] Theaflavins (theaflavins, TFs) are found in black tea with polyhydroxy benzotropolone structure mixture, accounting for 0.3%-1.5% of the dry weight of black tea, and play a key role in the color, aroma and quality of black tea. Current studies have shown that theaflavins have a variety of pharmacological effects related to human health, and they all play a role in preventing cardiovascular and cerebrovascular diseases, delaying aging, enhancing immune function, preventing dental caries, anti-tumor, antibacterial and virus-killing, etc. Known as "brain gold", it is one of the natural products with the most obvious efficacy, the m...

Claims

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Application Information

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IPC IPC(8): C12P17/16
Inventor 肖文军滕杰龚志华陈栋邓燕莉
Owner HUNAN AGRICULTURAL UNIV
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