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Purification method of nojirimycin

A purification method, the technology of deoxynojirimycin, applied in the field of microbial fermentation and purification, can solve the problems of unusable detection means and difficult purification, and achieve the effect of simple and controllable purification process, low energy consumption, safety and environmental protection

Active Publication Date: 2022-01-14
ZHEJIANG HUIDA BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

1-DNJ fermentation broth contains a large amount of protein, polysaccharide and pigment impurities; microbial metabolism produces nojirimycin, and also produces nojirimycin analogues; meanwhile, nojirimycin has no ultraviolet absorption, and conventional detection methods cannot be used, making Purification of 1-DNJ from microbial fermentation broth faces great difficulties

Method used

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  • Purification method of nojirimycin
  • Purification method of nojirimycin
  • Purification method of nojirimycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Recovery and activation of strains: take HDCC00252 original strains, thaw at room temperature, inoculate 0.1ml of bacterial suspension onto LB solid plate, spread evenly, and culture in 37°C incubator for 24 hours to obtain activated and recovered strains moss.

[0030] (2) Preparation of liquid seeds: Take the activated and revived bacterial lawn, scrape a loop with an inoculation loop, inoculate it into a 500ml triangular flask containing 100ml liquid seed medium, wrap it up and place it on a shaking table at 37°C and 220rpm for shaking culture 16h, control the OD value of the seed solution ≥ 5.0.

[0031] The liquid seed culture medium consists of 1.5% glucose, 0.5% yeast extract powder, 1% peptone and 1% sodium chloride. Adjust the pH to 7.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.

[0032] (3) Fermentation culture: Get qualified liquid seeds for cultivation, inoculate in the 50L tank that 27L liquid fermentation mediu...

Embodiment 2

[0035] Ferment 8L of the fermented broth obtained by the Nojirimycin-producing strains, add 0.5N HCL to the fermented broth to adjust the pH to 3.0, add 0.3kg of diatomaceous earth to the acidified fermented broth, stir well, pour it into a plate frame and filter , collect filtrate 6.5L. Add 65 g of activated carbon to the filtrate, stir for 30 minutes and start to filter. The filter membrane uses a 0.22 μm aqueous membrane. After three times of filtration, wash the membrane with 1 L of water, and collect 7.5 L of decolorized filtrate. Use LX-67 anion exchange resin to further decolorize and remove impurities from the feed liquid, load the sample according to the ratio of the volume of the column liquid to the volume of the resin 1:5, repeat the column for 3-5 times, and finally use 2 times the column volume of purified water for top washing Resin, collect combined effluent and top wash. Use LX-712 cation exchange resin to remove related substances and impurities from the fee...

Embodiment 3

[0037]Ferment 15L of the fermented broth obtained by the Nojirimycin-producing strain, add 0.5N HCL to the fermented broth to adjust the pH to 3.5, add 0.5 kg of diatomaceous earth to the acidified fermented broth, stir well, pour it into a plate frame and filter , Collect filtrate 12L. Add 180 g of activated carbon to the filtrate, stir for 30 minutes and start to filter. The filter membrane uses a 0.22 μm aqueous membrane. After three cycles of filtration, wash the membrane with 1.8 L of water, and collect 13.8 L of decolorized filtrate. Use LX-67 anion exchange resin to further decolorize and remove impurities, load the sample according to the ratio of the volume of the column liquid to the volume of the resin 1:4, repeat the column for 3-5 times, and finally use 3 times the column volume of purified water for top washing Resin, collect combined effluent and top wash. Use LX-712 cation exchange resin to remove related substances and impurities from the feed liquid treated ...

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Abstract

The invention discloses a purification method of 1-deoxynojirimycin, which comprises the steps of by taking bacillus amyloliquefaciens fermentation liquor for producing 1-deoxynojirimycin as a raw material, carrying out solid-liquid separation, decolorizing with a decolorizing agent, carrying out anion resin chromatography, carrying out cation resin chromatography and crystallizing to obtain high-purity 1-deoxynojirimycin with the yield higher than 50%. The method has the characteristics of simplicity in operation, short preparation time, small solvent dosage and the like.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation purification, in particular to a purification method of 1-deoxynojirimycin. Background technique [0002] 1-deoxynojirimycin (1-DNJ) is a naturally occurring polyhydroxypiperidine alkaloid with a chemical name of 3,4,5-trihydroxy-2-hydroxymethyltetrahydropyridine. It exists in the branches, leaves and roots of mulberry trees, as well as in silkworm larvae and silkworm chrysalis. 1-DNJ has a strong inhibitory effect on α-glucosidase and α-amylase, can delay the absorption of sugar in the small intestine after a meal, and achieve the purpose of inhibiting the rise of blood sugar after a meal in diabetics. Based on its strong glycosidase inhibitory activity, Nojirimycin can be used to treat diabetes and diabetic complications, obesity and related dysfunction, etc. In addition, 1-DNJ can also be used in the field of food, food with mulberry leaves as raw materials, As a functional hyp...

Claims

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Application Information

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IPC IPC(8): C07D211/46
CPCC07D211/46
Inventor 张敏郑玲辉石磊彭湘屏高祥朱进伟陈世敏汪超
Owner ZHEJIANG HUIDA BIOTECHNOLOGY CO LTD