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Method for preparing gentio-oligosaccharide by using immobilized beta-glucosidase

A technology of glucosidase and gentian oligosaccharide, which is applied in the field of preparing gentian oligosaccharide, can solve the problems of low utilization rate, difficult recovery of enzymes and high production cost, so as to improve the utilization rate, reduce the difficulty of product separation, and reduce the The effect of production costs

Active Publication Date: 2014-04-16
SHAANXI UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of free enzymes to prepare gentian oligosaccharides is difficult to recycle the enzymes, the utilization rate is low, and it is not conducive to the separation of product oligosaccharides, and the production cost is high.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1) Prepare Aspergillus niger solid-state fermentation medium, add Aspergillus niger according to the mass percentage, the inoculation amount is 5%, and culture at 30° C. for 3 days to form a fermented product. The composition of the culture medium is: bran 120g, ammonium sulfate 2.7g, potassium dihydrogen phosphate 0.72g, magnesium sulfate 1.08g, distilled water 240mL. Enzyme preparation: take the fermented product, leaching three times at 40°C with pH 3.4 citric acid-sodium citrate buffer solution, filter, salt out with 20% saturated ammonium sulfate, remove impurities, take the clear liquid, and use Secondary salting out of ammonium sulfate with 70% saturation, centrifuged at 4°C for 12 min with a centrifugal force of 12400×g, discarded the solution to obtain β-glucosidase.

[0023] (2) The process of immobilizing β-glucosidase by cross-linking-embedding method is as follows: take 7.2 mL of 3.0% sodium alginate and 2 mL of β-glucosidase solution with an enzyme activit...

Embodiment 2

[0026] 1) Prepare Aspergillus niger solid-state fermentation medium, add Aspergillus niger according to the mass percentage, the inoculum size is 8.5%, and culture at 30° C. for 4 days to form a fermented product. The composition of the medium is: bran 1200g, ammonium sulfate 27g, potassium dihydrogen phosphate 7.2g, magnesium sulfate 10.8g, distilled water 2400mL. Enzyme preparation: take the fermented product, leaching 3 times with citric acid-sodium citrate buffer solution with pH 3.8 at 45°C, filter, salt out with ammonium sulfate with 30% saturation, remove impurities, take the clear liquid, and use Secondary salting out of ammonium sulfate with 75% saturation, centrifuged at 4°C for 15 minutes with a centrifugal force of 13400×g, discarded the solution to obtain β-glucosidase.

[0027] 2) Take 7.2mL of sodium alginate with a mass concentration of 3.6% and 2mL of β-glucosidase solution with an enzyme activity of 46U. After fully mixing, add 0.8mL of glutaraldehyde with a ...

Embodiment 3

[0030] 1) Prepare Aspergillus niger solid-state fermentation medium, add Aspergillus niger according to mass percentage, the inoculum size is 10%, and culture at 30° C. for 7 days to form a fermented product. The composition of the medium is: bran 12000g, ammonium sulfate 270g, potassium dihydrogen phosphate 72g, magnesium sulfate 108g, distilled water 24000mL. Enzyme preparation: take the fermented product, leaching 3 times with citric acid-sodium citrate buffer solution with pH 4.8 at 60°C, filter, salt out with 35% saturated ammonium sulfate, remove impurities, take the clear liquid, and use Secondary salting out of ammonium sulfate with 80% saturation, centrifuged at 4°C for 18 minutes with a centrifugal force of 25900×g, discarded the solution to obtain β-glucosidase.

[0031] 2) Take 7.2mL of sodium alginate with a mass concentration of 5.0% and 2mL of β-glucosidase solution with an enzyme activity of 70U. After mixing thoroughly, add 0.8mL of glutaraldehyde with a volum...

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Abstract

The invention discloses a method for preparing gentio-oligosaccharide by using immobilized beta-glucosidase. The method for preparing the gentio-oligosaccharide by using the immobilized beta-glucosidase comprises the following steps of: 1) preparing beta-glucosidase through solid-state fermentation of Aspergillus niger; 2) immobilizing the beta-glucosidase by a crosslinking-embedding method; and 3) producing the gentio-oligosaccharide by transforming glucose by using the immobilized beta-glucosidase. The recovery rate of enzyme activity of an immobilized enzyme prepared by the method is up to 60 percent, and the conversion rate of a substrate, namely the glucose converted by the immobilized enzyme is 45 percent; and the stability and pH tolerance of the immobilized enzyme are remarkably improved, the mechanical strength is high, the immobilized enzyme can be continuously and repeatedly used for 4 to 6 bathes, and the enzyme activity is kept unchanged. The utilization rate of the gentio-oligosaccharide produced by an enzyme method is improved, the production cost and difficulty in separation and purification of the gentio-oligosaccharide are reduced, and the method is suitable for large-scale continuous production and has a great significance for improving the technical level of the production of the gentio-oligosaccharide in China.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a method for preparing gentian oligosaccharides by using immobilized β-glucosidase. Background technique [0002] Gentian oligosaccharides are a class of oligosaccharides bound by glucose with β-1,6 glycosidic bonds. It is not easy to digest, low calorific value; relatively stable to pH and heat, has the functions of regulating the environment of intestinal flora, proliferating intestinal beneficial flora, and improving the effect of laxative, etc. It can produce good results in products such as ice cream and ice cream, so it is widely used in health food and other fields. [0003] At present, there are many methods for preparing gentian oligosaccharides. The first ones were extracted from the stems and roots of Gentiana. Due to the limitation of raw materials, it is difficult to mass-produce them. In recent years, with the rise of enzymatic preparation of oligosacch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/14C12N9/24C12N11/10C12R1/685
Inventor 齐香君王钏王艳丽张雯
Owner SHAANXI UNIV OF SCI & TECH
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