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Enzyme method for production of lactulose

A lactulose and enzymatic technology, applied in the field of enzymatic production of lactulose, can solve problems such as product purity and reaction time need to be improved, and achieve the effects of extensive strain culture conditions, simple operation and mild action conditions

Inactive Publication Date: 2015-07-29
SHANDONG BAILONG CHUANGYUAN BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reaction conditions of the above scheme are mild, no colored by-products are generated, and the operation is convenient, but the product purity and reaction time still need to be improved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The Arthrobacter sp.jnsb-2 mutant strain of Arthrobacter sp.jnsb-2 of the present invention can be applied to ferment and produce lactase, specifically as follows:

[0027] (1) Preparation of medium

[0028] Solid medium: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0, 2% agar, sterilized at 121°C for 20min. Seed medium: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0, sterilized at 121°C for 20min.

[0029] Fermentation medium: lactose 2%, yeast extract 1.4%, calcium chloride 0.5mmol / L Zn 2+ 10mmol / L, FeCl 3 1.5mmol / L, pH7.0, sterilized at 121°C for 20min.

[0030] (2) Strain fermentation culture

[0031] The Arthrobacter sp.jnsb-2 mutant strain Arthrobacter sp.jnsb-2 was activated on a solid slope for 2 hours, and then a loop of the bacteria was added to the seed medium, and incubated at a constant temperature of 30°C for 1 hour, and the shaker flask rotated at 200r / min. The above-mentioned seed bacteria suspension was inoculated into the fermentation medium a...

Embodiment 2

[0041] Basically the same as Example 1, the difference is:

[0042] The fermentation medium used in step (1) was glucose 1%, corn steep liquor 2.5%, calcium chloride 0.5mmol / L, Zn 2+ 10mmol / L, FeCl 3 1.5mmo l / L; the fermentation culture conditions after inoculation in step (2) are: 25°C constant temperature culture for 0.8h, shake flask rotation speed 200r / min.

[0043] In step (3), the fermentation broth was centrifuged at 5000rpm for 15min to collect the cells, resuspended with the same volume of phosphate buffer (10mmol / L, pH7.0), and the cells were disrupted by ultrasonic waves, centrifuged at 10000rpm for 15min, and the supernatant was obtained as the enzyme solution.

[0044] The method of using the lactase produced by the Arthrobacterium mutant strain of the present invention as a catalyst to catalyze the synthesis of lactulose from lactose and fructose is specifically:

[0045] (1) Preparation of substrate solution

[0046] Prepare lactose and fructose solutions w...

Embodiment 3

[0052]Basically the same as Example 1, the difference is: the fermentation medium is glycerol 0.2%, peptone 1%, yeast extract 0.5%, KH 2 PO 4 15mmol / L, MgSO 4 .7H 2 O 2.5mmol / L, calcium chloride 0.5mmol / L, Zn 2+ 10mmol / L; The fermentation culture condition after the inoculation in the step (2) is:

[0053] Incubate at a constant temperature of 25°C for 1.5h, and shake the flask at a speed of 200r / min.

[0054] The method of using the lactase produced by the Arthrobacterium mutant strain of the present invention as a catalyst to catalyze the synthesis of lactulose from lactose and fructose is specifically:

[0055] (1) Preparation of substrate solution

[0056] Prepare lactose and fructose solution with phosphate buffer solution of pH 5.5-8.0, wherein the concentration of lactose is 300g / L, and the concentration of fructose is 250g / L;

[0057] (2) Hydrolysis and transglycoside reaction

[0058] Add 600U / L lactase to the solution in step (1), react at 25°C for 1.5h, per...

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Abstract

The invention relates to an enzyme method for production of lactulose. The method comprises the following steps: producing lactase by using an arthrobacter mutant strain; preparing a lactose and fructose solution by using a phosphate buffer of pH 5.5-8.0, wherein concentration of lactose is 300g / L and concentration of fructose is 250g / L; adding 600U / L of lactase, 10 mmol / L of Zn<2+> and 4-6mmol / L of iron chloride into the substrate solution to react at 20-25 DEG C for 0.5-1.5h, and carrying out hydrolysis and glucoside conversion reaction so as to obtain an enzymatic hydrolysate; and adding anhydrous ethanol into the enzymatic hydrolysate, and centrifuging to prepare the lactulose syrup. The method for production of lactulose is simple to operate, has mild reaction conditions and is easy to grasp. By the method, no colored by-product is generated, and the product has high purity and is easy to purify. Strain culture conditions are extensive; enzyme production period is short; and operation is convenient. By the method, yield of lactulose produced by the enzyme method is greatly raised. The method provided by the invention is a great innovation in fermentation technologies and is worth of popularization.

Description

(1) Technical field [0001] The present invention relates to lactulose, in particular to enzymatic production of lactulose. (2) Background technology [0002] Lactulose Oral Solution English name: Lactulose Oral Solution Pinyin: Ruguotang Koufurongye, the main ingredient of this product is lactulose. Chemical name: 4-O-β-D-galactopyranosyl-D-fructose. Appearance is light brown yellow clear viscous liquid. It has the effect of lowering blood ammonia and laxative, and is mainly used to treat diseases such as ammoniacal hepatic coma, hyperammonemia and habitual constipation. [0003] Researchers at home and abroad focus on the use of different catalysts and the development of separation methods for the production of lactulose. CN1093410 uses heating to dissolve lactose, then keeps it boiling, and adds alkaline solution dropwise. After the reaction, adjust the pH to 4-4.5 to decolorize. Desalting with anion and cation exchange resins, followed by vacuum concentration, cooling ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/14C12P19/12C12R1/06
Inventor 窦宝德张继红窦光朋
Owner SHANDONG BAILONG CHUANGYUAN BIO TECH
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