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Method for producing alternan sucrase by fermenting Leuconostoccitreum and its application

A technology of alternan sucrase and Leuconostoc citrea, applied in the field of Leuconostoc citrea SK24.002 to ferment and produce alternan sucrase, which can solve the problems of rare research and achieve short fermentation time, high yield and low cost Effect

Inactive Publication Date: 2012-06-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research institutions of alternan sucrase are mainly concentrated in European and American countries, and the relevant reports are limited to the research on the nucleic acid sequence and biological properties of the enzyme molecule, and domestic research in this field is rare

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Fermentation of Leuconostoc citrea to produce enzymes

[0024] Slant culture: glucose 1 g, yeast powder 5 g, anhydrous calcium chloride 0.025 g, dipotassium hydrogen phosphate 10 g, magnesium sulfate heptahydrate 0.3 g, manganese sulfate monohydrate 0.01 g, Vc 0.05 g, agar 20 g, distilled water to volume 1000mL, adjust the pH to 6.9. Incubate at 30°C for 12h.

[0025] Seed culture: glucose 10 g, yeast powder 15 g, anhydrous calcium chloride 0.025 g, dipotassium hydrogen phosphate 10 g, magnesium sulfate heptahydrate 0.3 g, manganese sulfate monohydrate 0.01 g, Vc 0.05 g, distilled water to 1000 mL, adjusted pH to 6.9. 40mL of liquid in 100mL Erlenmeyer flask, slant medium at 5% (v / v) into seed medium, wrap with kraft paper, shake at 160rpm at 30°C for 12h.

[0026] Fermentation culture: 20 g of sucrose, 15 g of yeast powder, 0.025 g of anhydrous calcium chloride, 10 g of dipotassium hydrogen phosphate, 0.3 g of magnesium sulfate heptahydrate, Tween 80 10 mL / ...

Embodiment 2

[0027] Embodiment 2 Preparation of alternan sucrase enzyme powder

[0028] Centrifuge the fermentation broth at 10,000g at 4°C for 10 minutes to collect the supernatant and bacterial cells. The supernatant is ultrafiltered with an ultrafiltration membrane with a molecular cut-off of 30KDa to remove salt and impurities, and then filtered with 50% polyethylene glycol Alcohol precipitation, centrifugation to obtain precipitate 1; bacterial cells were washed twice with pH 5.4 sodium acetate buffer solution, added 10mL pH 5.4, 8M urea-sodium acetate buffer solution and shaken at 25°C for 3 hours, the molecular cut-off was 10KDa dialysis bag to remove residual urea, precipitate with 50% polyethylene glycol to make the final concentration of polyethylene glycol reach 20%, and centrifuge to obtain precipitate 2; precipitate 1 and precipitate 2 are directly cooled to dryness to obtain alternan sucrose Enzyme powder, the enzyme activity reaches 18U / mg.

Embodiment 3

[0029] Example 3 Application of alternan sucrase

[0030] Dissolve the obtained alternan sucrase enzyme powder in sodium acetate buffer solution with pH 5.4 to make its activity reach 8U / mL, use 10% sucrose as the substrate, carry out the enzyme reaction at 30°C for 48h, and boil to kill the enzyme , centrifuged to obtain the supernatant, freeze-dried to obtain a new type of dextran, the yield reached 32 mg / mL, and its structure was determined by NMR as -[α-Glcp(1→3)-α-Glcp(1→6)]- n , can be efficiently multiplied by intestinal probiotics such as Bifidobacterium and Lactobacillus.

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Abstract

The invention relates to a method for producing alternan sucrase by fermenting Leuconostoccitreum and its application, which belongs to the food biology technical field. According to the invention, an alternan sucrase is used for producing Leuconostoccitreum SK24.002 for three-stage cultivation of slant cultivation, seed cultivation and fermentation cultivation, and the alternan sucrase is obtained by separating and purifying a broth and mycelium cells. The method for producing alternan sucrase by using the Leuconostoccitreum SK24.002 possesses the advantages of short fermentation time and low cost, the safety performance is reliable, and the deep fermentation of liquid and cell cultivation with high density can be carried out, thereby the method of the invention is suitable for large scale production. The activity of the produced alternan sucrase can reach more than 10U / mg. A fermented product is non-toxic, and can be directly used for producing a plurality of novel functional carbohydrates possessing prebiotics efficacy.

Description

technical field [0001] The invention relates to a method for fermenting Leuconostoc citrea SK24.002 to produce alternan sucrase and its application, belonging to the field of food biotechnology. Background technique [0002] In today's society, the accelerated pace of human life, fierce social competition, changes in diet structure, and environmental pollution are all serious threats to human health, making most people in a sub-health state. At present, the general public is paying more and more attention to their own health status, and more and more people choose to use food to change this situation, shifting from "treating existing diseases" to "preventing diseases" and health care, from "passive Medical” to “Active Health”. In order to conform to the development direction of modern food science and technology and meet the health needs of consumers, functional carbohydrates with low blood sugar and low calories have become the development trend of healthy food in the 21st...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P19/18C12R1/01
Inventor 江波缪铭白爱娟张涛周榴明贾敏
Owner JIANGNAN UNIV
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