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Method for synthesizing new hesperidin through high-flux enzyme process

A technology for neohesperidin and enzymatic synthesis, which is applied in the field of high-throughput enzymatic synthesis of neohesperidin, can solve the problems of environmental pollution and limited sources of discarded orange peels, and achieves no environmental pollution and wide source of raw materials. , the effect of saving purification costs

Active Publication Date: 2010-05-19
SHANDONG BENYUE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to address the defects of the prior art, prepare water-soluble hesperidin metal complexes through coordination modification, and then use this as a substrate to utilize immobilized hesperidin with mild reaction conditions, less side reactions, and easy industrialization Glycosidase and rhamnosyltransferase, high-throughput enzymatic preparation of the target product NH, solve the long-term technical bottleneck problem of limited source of sweetener NHDC synthesis precursor (NH), and propose a solution to the waste orange in China's citrus industry A New Method for Environmental Pollution and High Value Resource Utilization of Leather

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  • Method for synthesizing new hesperidin through high-flux enzyme process

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Embodiment 1

[0029] Such as figure 1 As shown, the new method for high-throughput enzymatic conversion of hesperidin to neohesperidin is mainly realized through the following four steps: pairing of hesperidin and metal ions (I), formation of hesperidin metal complexes Enzymatic hydrolysis of hesperidin (II), biotransformation of the hydrolyzed product of hesperidin, i.e. high-throughput enzymatic synthesis of neohesperidin metal complexes (III) and decoordination of neohesperidin metal complexes (IV).

[0030] Configure 6×10 with 80% methanol -3 mol / L hesperidin and 3×10 3 mol / L CuCl 2 Solution, airtight reaction in 40 ℃ of water baths 1h, rotary evaporation reclaims methanol and HCl, obtains hesperidin copper complex aqueous solution, concentration 1.85g / L, higher than the solubility of hesperidin more than 100 times (I). Hesperidinase was immobilized by glutaraldehyde cross-linking method, and acted on the above-mentioned hesperidin metal complex aqueous solution, the reaction temper...

Embodiment 2

[0032] Example 2 is similar to Example 1. The difference is: the concentration of methanol in step I is adjusted to 50%, by Cu(NO 3 ) 2 Provide Cu 2+ Coordinate with hesperidin, adjust the reaction temperature to 85°C; adjust the action temperature of hesperidinase in step II to 65°C, adjust the reaction pH to 2.0, and the reaction time is only 20min at this time, indicating that the enzyme activity under this condition Higher than Example 1; rhamnosyltransferase 1 in step (III), the action temperature of 2-RhaT is adjusted to 25 ℃, and this immobilized enzyme can be reused for 24 hours equally; Adjusting the pH to 11.5 can also precipitate Cu in the copper ammonium complex 2+ ’, realizing the high-throughput enzymatic conversion of hesperidin to neohesperidin.

Embodiment 3

[0034] Configure 6×10 with 70% methanol -3 mol / L hesperidin and 3×10 3 mol / L FeCl 2 The solution was airtightly reflected in a water bath at 65° C. for 1 h, and methanol and HCl were recovered by rotary evaporation to obtain an aqueous solution of hesperidin-ferrous complex with a concentration of 2.01 g / L, which was more than 110 times higher than the solubility of hesperidin (I). The hesperidinase was immobilized by glutaraldehyde cross-linking method, and acted on the above-mentioned hesperidin ferrous complex aqueous solution, the reaction temperature was 55°C, the reaction pH was 3.0, and the reaction time was 40min. In the ultraviolet-visible spectrum scanning, the characteristic absorption peak of the hesperidin ferrous complex at around 380nm no longer appears. The enzymatic hydrolysis product does not need to be separated, and is directly used as a substrate for rhamnosyltransferase (II). Rhamnosyltransferase 1,2-RhaT from tobacco or carrot plant cells was also imm...

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Abstract

The invention discloses a method for synthesizing new hesperidin through a high-flux enzyme process, which comprises the steps of performing metal coordination on hesperidin, biologically transforming a hesperidin metal complex into the new hesperidin by using hesperidinase and rhamnosyltransferase 1,2-RhaT, and de-coordinating a new hesperidin metal complex. The method is suitable for the industrial production of a low heat value sweetening agent NHDC, overcomes the technical bottleneck that the hesperidin water-solubility is poor during the biotransformation of the new hesperidin, achieves the high-flux enzyme process synthesis of the new hesperidin by using an NHDC synthesis precursor, solves the problem that an NH source is limited, and simultaneously opens up a new way for the resource utilization of industrial waste orange peels and the high added value of the hesperidin.

Description

technical field [0001] The present invention relates to the field of food additives, in particular to the enzymatic synthesis of low-calorie sweetener neohesperidin dihydrochalcone (NHDC), and specifically provides a high-throughput enzymatic method for neohesperidin (NH) resolve resolution. technical background [0002] NHDC is a safe low-calorie sweetener (1500-1800 times the sweetness of sucrose) and a bitter inhibitor. It has good thermal stability and a wide range of application pH. It is widely used in the fields of food and medicine. It is often used as fresh orange peel. Glycosides are used as precursors to be synthesized by enzymatic or chemical methods (Chinese invention patents ZL02806186, ZL200510055397 and ZL200410042658). In its synthesis, the limited source of substrate NH is the main technical bottleneck. NH can be extracted from Citrus aurantium and Citrus aurantii (Chinese invention patents ZL200710111229, ZL200810036498, ZL200410025729, ZL200710017735 an...

Claims

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Application Information

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IPC IPC(8): C12P19/60
Inventor 朱思明于淑娟何树珍
Owner SHANDONG BENYUE BIOTECH
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