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Method for preparing hesperetin from enzymatic hydrolysis neohesperidin or hesperidin

A new hesperidin, enzymatic hydrolysis technology, applied in the field of bioengineering, can solve the problems of complex reaction product types and components, affecting yield, difficult purification and separation, etc., to achieve high product purity, high conversion efficiency, and high application value. Effect

Inactive Publication Date: 2016-11-23
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the microbial fermentation method has mild reaction conditions, high reaction efficiency, and low environmental pollution, the variety of reaction products and complex components not only affect the yield, but also cause greater difficulties for purification and separation.

Method used

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  • Method for preparing hesperetin from enzymatic hydrolysis neohesperidin or hesperidin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Preparation of crude enzyme solution

[0022] Use 3% wheat bran extract, 0.2% yeast extract, 1% glucose, 0.5% KH2PO4, 0.2% (NH4)2SO4 as the seed medium, and distribute 50ml of the above seed medium into 100ml Erlenmeyer flasks , sterilized at 120° C. for 15 minutes, inoculated the agar plate culture of Penicillium decumbens into it, and cultured at 30° C. for 2 days as a seed solution.

[0023] 500ml Erlenmeyer culture bottle is put into 100ml containing 5% corn steep liquor, 0.2% yeast extract, 0.2% KH2PO4, the culture medium of 1% naringin, pH4.5, autoclave 121 degree 30min. Inoculate 1ml of the seed liquid into this culture medium, culture at 150rpm, shaker at 25°C for 5 days. Centrifuge to collect the supernatant, which is the crude enzyme solution.

Embodiment 2

[0025] Take a 100ml Erlenmeyer flask and add 20ml of phosphate buffer solution (pH6.0), 3.5ml of crude enzyme solution, 250ul of 20mM activator, 600ul of 80mM neohesperidin solution, seal the bottle mouth, and store at 37°C 200rpm rotary shaker reaction 4h, every 1h sampling for HPLC analysis (detection wavelength 283nm, mobile phase: acetonitrile (B) / pure water (A), gradient is, 0-10min, 25%-50%B; 10.0 -12.0min, 50%-90%B; 12.0-16.0min, 90%-90%B, flow rate 1ml / min), the conversion rate of neohesperidin was greater than 98% in 0.5h, and completely converted after 4h of reaction. The prepared product is hesperetin. Such as figure 1 shown by figure 1 It can be seen that the crude enzyme solution has a very high conversion rate to neohesperidin.

Embodiment 3

[0027] Get 100ml Erlenmeyer flask and add 20ml phosphate buffer (pH6.0) successively, 3.5ml concentration is the enzyme solution of 100mg / ml, 250ul concentration is the activator of 20mM, 600ul concentration is the hesperidin solution of 80mM, after closing the bottle mouth, Reaction on a rotary shaker at 200 rpm at 37°C for 4 hours, sampling every 1 hour for HPLC analysis (detection wavelength 283nm, mobile phase: acetonitrile (B) / pure water (A), gradient 0-10min, 25%-50 %B; 10.0-12.0min, 50%-90%B; 12.0-16.0min, 90%-90%B, flow rate 1ml / min), the conversion rate of hesperidin was greater than 98% in 0.5h, and completely converted after 4h of reaction. The prepared product is hesperetin. Such as figure 2 shown by figure 2 It can be seen that the crude enzyme solution has a significant advantage in converting hesperidin to prepare hesperetin.

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Abstract

The invention discloses a method for preparing hesperetin from enzymatic hydrolysis neohesperidin or hesperidin. Neohesperidin or hesperidin beta-D-glucoside bond can be hydrolyzed in one step through crude enzyme liquid to prepare corresponding aglycone hesperetin. The method includes the steps of preparing the crude enzyme liquid of Penicillium decumbens through fermentation, adjusting the content and reaction time of the crude enzyme liquid, directly converting neohesperidin or hesperidin into hesperetin, dissolving or extracting conversion liquid through organic solvent, and conducting chromatographic purification to obtain hesperetin. By means of the method, the defects that a traditional chemical method is strict in reaction condition, serious in pollution, low in conversion rate and the like when used for preparing hesperetin can be overcome, and the defects that when microbial fermentation is directly adopted, operation is complicated, products are not simplex, and products are difficult to purify can be overcome.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for preparing hesperetin by enzymatically hydrolyzing neohesperidin or hesperidin. Background technique [0002] Tangerine peel, also known as tangerine peel, a traditional medicinal resource, has pharmacological effects such as eliminating phlegm, relieving asthma, lowering blood pressure, antibacterial, anti-inflammatory, and antioxidant. In addition to essential oils, orange peel also contains a large amount of flavonoids. Studies have shown that flavonoids in orange peel are the main factors related to its pharmacological activities such as anti-inflammatory and anti-oxidation. The composition of flavonoids in orange peel is complex. At present, more than 60 kinds of flavonoids have been isolated from orange peel, among which the more common ones are hesperidin, neohesperidin, hesperidin, nobietin, naringin, Pomelo peel, rue sweet, etc. [0003] Neohesperi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/06
Inventor 杨凌崔攀葛广波窦同意王平
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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