CN104805152A - Enzyme method for production of lactulose - Google Patents

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

Production by Enzymes lactulose

(1) technical field

The present invention relates to lactulose, be specifically related to Production by Enzymes lactulose.

(2) background technology

Lactulose oral solution English name: the Lactulose Oral Solution Chinese phonetic alphabet: Ruguotang Koufurongye, this product major ingredient is lactulose.Chemical name: 4-O-β-D-galactopyranosyl glycosyl-D-Fructose.Proterties is the clear and bright thick liquid of light brown yellow.Have and reduce blood ammonia and laxative action, be mainly used in the illnesss such as treatment ammonemia hepatic coma, hyperammonemia and habitual constipation.

The research emphasis that lactulose is produced is concentrated on the use of different catalysts and the exploitation of separation method by domestic and international researchist, CN1093410 adopts heating for dissolving lactose, then boiling is kept, drip alkaline solution, reaction terminates rear adjustment pH to 4-4.5, decolouring, adopts anion and cation exchange resin desalination, and then vacuum concentration, crystallisation by cooling can obtain lactulose slurry.But efficiency of pcr product is low, complex procedures, high being still in the preparation of chemical method lactulose of cost has problem to be solved.By comparison, the preparation of enzyme process lactulose can avoid the problem such as product degradation and colored byproducts generation, and current research both domestic and external is still in the starting stage.Therefore, exploitation can be carried out enzymatic conversion method and prepares the Sumylact L of lactulose and microorganisms producing bacterial classification has certain economic benefit and social benefit.

2011 08 month 31, announce a kind of Arthrobacter mutant strain, utilize this mutant strain produce Sumylact L method and prepare in the method for lactulose with Sumylact L, disclose a kind of Arthrobacter mutant strain, this strain classification called after Arthrobacter sp.jnsb-2, this bacterial strain is preserved in Wuhan China typical culture collection center at present, it is referred to as CCTCC, and the numbering of registering on the books is CCTCC M 209210, and preservation date is: on September 27th, 2009.

This bacterial strain has following character:

1, morphological specificity: bacterial strain is good at lactose yeast extract paste peptone cultured on solid medium, and bacterium colony is faint yellow, and under electron microscope, showed cell is shaft-like, the blunt circle in two ends.

2, physio-biochemical characteristics:

(1) culture temperature: at 20-37 DEG C.Optimum temperuture is 30 DEG C.

(2) gelatine liquefication Starch Hydrolysis: positive.

(3) Lan Shi is removed from office: positive.

(4) lactose fermentation: positive.

(5) H2S generates: negative.

3,16S rDNA sequential analysis: length 1383bp, genus arthrobacter 16S rDNA in sequence and GenBank has higher homology, is wherein 99% with the similarity of Arthrobacter sp.CDB1, Arthrobacter sp.AGL 5, Arthrobactersp.2-12.Such scheme reaction conditions is gentle, does not have colored byproducts to generate, easy to operate, but its product purity and reaction times still have much room for improvement.

(3) summary of the invention

The present invention, in order to make up the deficiencies in the prior art, provides a kind of Production by Enzymes lactulose, easy to operate, and process is easy to control, and the reaction times is short, and product purity is high, is conducive to suitability for industrialized production.

The present invention is achieved through the following technical solutions:

A kind of Production by Enzymes lactulose, its special character is: comprise the following steps:

(1) save bacterium mutant strain and produce Sumylact L: Arthrobacter mutant strain is carried out fermentation culture again after solid slope activation, seed culture, wherein,

The substratum of fermentation culture: carbon source can select any one in glucose, lactose or glycerine, its mass volume ratio concentration is 1%-2%; Nitrogenous source can select in yeast extract paste, peptone or corn steep liquor one or more, its mass volume ratio concentration is 0.5-12%; Inorganic salt can select calcium chloride, Zn ²⁺, magnesium sulfate, dipotassium hydrogen phosphate, one or more in potassium primary phosphate or iron(ic) chloride, its concentration is 12-28mmol/L, and its concentration is; The condition of fermentation culture is: 25-30 DEG C of cultivation; Obtain thalline by centrifugal for the fermentation liquor 5000-6000rpm of generation, with after be enzyme liquid through smudge cells, 10000-12000rpm centrifuging and taking supernatant liquor;

(2) preparation of substrate solution: with phosphoric acid buffer preparation lactose and the fructose soln of pH5.5-8.0, wherein lactose concn is 300g/L, and fructose concentration is 250g/L;

(3) be hydrolyzed and turn glycosides reaction

600U/L Sumylact L is added, Zn in substrate solution ²⁺10mmol/L, iron(ic) chloride 4-6 mmol/L, at 20-25 DEG C of reaction 0.5-1.5h, is hydrolyzed and turns glycosides reaction, obtaining enzymolysis solution;

(4) aftertreatment of enzymolysis solution

In enzymolysis solution, add dehydrated alcohol, make its final concentration be 65-70%, after centrifugal, get supernatant liquor can obtain lactulose slurry through the 60-65 DEG C of concentrated aftertreatment of heating.

Beneficial effect of the present invention: the present invention is simple to operate for the production of lactulose, action condition is gentle, easily grasp, do not have colored byproducts to generate, product purity is high, easy purifying.Strain culturing condition is extensive, and the product enzyme cycle is short, easy to operate, substantially increases the output of Production by Enzymes lactulose, is the great innovation in zymotechnique, is worthy to be popularized.

(4) embodiment

Embodiment 1

Arthrobacter mutant strain Arthrobacter sp.jnsb-2 of the present invention can be applicable to fermentative production Sumylact L, specific as follows:

(1) preparation of substratum

Solid medium: peptone 1%, yeast extract 0.5%, NaCl1%, pH 7.0, agar 2%, 121 DEG C of sterilizing 20min.Seed culture medium: peptone 1%, yeast extract 0.5%, NaCl1%, pH 7.0,121 DEG C of sterilizing 20min.

Fermention medium: lactose 2%, yeast extract paste 1.4%, calcium chloride 0.5mmol/L Zn ²⁺10mmol/L, FeCl ₃1.5mmol/L, pH7.0,121 DEG C of sterilizing 20min.

(2) strain fermentation is cultivated

By Arthrobacter mutant strain Arthrobacter sp.jnsb-2 solid slope activation 2h, then connect this bacterium of a ring to seed culture medium, 30 DEG C of constant temperature culture 1h, shaking flask rotating speed 200r/min.Above-mentioned seed bacteria suspension is seeded to fermention medium with 10% (v/v), 25 DEG C of constant temperature culture 1h, shaking flask rotating speed 200r/min.

(3) by fermented liquid with the centrifugal 10min collecting cell of 5000rpm, resuspended with same volume phosphoric acid buffer (10mmol/L, pH7.0), through ultrasonic disruption cell, 12000rpm, centrifugal 10min, get supernatant and be enzyme liquid.

What utilize this enforcement produces the Sumylact L that the obtains method as catalyst lactose and fructose synthesis lactulose through Arthrobacter mutant strain, is specially:

(1) preparation of substrate solution

With phosphoric acid buffer preparation lactose and the fructose soln of pH5.5-8.0, wherein lactose concn is 300g/L, and fructose concentration is 250g/L;

(2) be hydrolyzed and turn glycosides reaction

600U/L Sumylact L is added, Zn in the solution of step (1) ²⁺10mmol/L, iron(ic) chloride 4 mmol/L, at 20 DEG C of reaction 1.5h, be hydrolyzed and turn glycosides reaction, obtaining enzymolysis solution;

(3) aftertreatment of enzymolysis solution

Add dehydrated alcohol in the enzymolysis solution obtained in step (2), make its final concentration be 65%, 12000rpm, centrifugal 10min, supernatant liquor is concentrated through 60 DEG C of heating.After testing, lactulose content is 94.1%.

Embodiment 2

Substantially identical with embodiment 1, its difference is:

In step (1), fermention medium used is glucose 1%, corn steep liquor 2.5%, calcium chloride 0.5mmol/L, Zn ²⁺10mmol/L, FeCl ₃1.5mmo l/L; In step (2), postvaccinal fermentation culture conditions is: 25 DEG C of constant temperature culture 0.8h, shaking flask rotating speed 200r/min.

In step (3), fermented liquid is with the centrifugal 15min collecting cell of 5000rpm, and resuspended with same volume phosphoric acid buffer (10mmol/L, pH7.0), through ultrasonic disruption cell, 10000rpm, centrifugal 15min, get supernatant and be enzyme liquid.

Utilize the method for producing the Sumylact L obtained through Arthrobacter mutant strain and synthesizing lactulose as catalyst lactose and fructose of the present invention, be specially:

(1) preparation of substrate solution

With phosphoric acid buffer preparation lactose and the fructose soln of pH5.5-8.0, wherein lactose concn is 300g/L, and fructose concentration is 250g/L;

(2) be hydrolyzed and turn glycosides reaction

In the solution of step (1), add 600U/L Sumylact L, at 25 DEG C of reaction 1h, be hydrolyzed and turn glycosides reaction, obtaining enzymolysis solution;

(3) aftertreatment of enzymolysis solution

Add dehydrated alcohol in the enzymolysis solution obtained in step (2), make its final concentration be 70%, 12000rpm, centrifugal 10min, supernatant liquor is concentrated through 60 DEG C of heating.After testing, lactulose content is 93.7%.

Embodiment 3

Substantially identical with embodiment 1, difference is: fermention medium is glycerine 0.2%, peptone 1%, yeast extract paste 0.5%, KH ₂pO ₄15mmol/L, MgSO ₄.7H ₂o 2.5mmol/L, calcium chloride 0.5mmol/L, Zn ²⁺10mmol/L; In step (2), postvaccinal fermentation culture conditions is:

25 DEG C of constant temperature culture 1.5h, shaking flask rotating speed 200r/min.

Utilize the method for producing the Sumylact L obtained through Arthrobacter mutant strain and synthesizing lactulose as catalyst lactose and fructose of the present invention, be specially:

(1) preparation of substrate solution

With phosphoric acid buffer preparation lactose and the fructose soln of pH 5.5-8.0, wherein lactose concn is 300g/L, and fructose concentration is 250g/L;

(2) be hydrolyzed and turn glycosides reaction

In the solution of step (1), add 600U/L Sumylact L, at 25 DEG C of reaction 1.5h, be hydrolyzed and turn glycosides reaction, obtaining enzymolysis solution;

(3) aftertreatment of enzymolysis solution

Add dehydrated alcohol in the enzymolysis solution obtained in step (2), make its final concentration be 70%, 12000rpm, centrifugal 10min, supernatant liquor is concentrated through 65 DEG C of heating.After testing, lactulose content is 90.7%.

Zn ²⁺can be ZnSO ₄, ZnCl ₂.

Claims (1)

1. a Production by Enzymes lactulose, is characterized in that: comprise the following steps:

(1) save bacterium mutant strain and produce Sumylact L: Arthrobacter mutant strain is carried out fermentation culture again after solid slope activation, seed culture, wherein,

The substratum of fermentation culture: carbon source can select any one in glucose, lactose or glycerine, its mass volume ratio concentration is 1%-2%; Nitrogenous source can select in yeast extract paste, peptone or corn steep liquor one or more, its mass volume ratio concentration is 0.5-12%; Inorganic salt can select calcium chloride, Zn ²⁺, magnesium sulfate, dipotassium hydrogen phosphate, one or more in potassium primary phosphate or iron(ic) chloride, its concentration is 12-28mmol/L, and its concentration is; The condition of fermentation culture is: 25-30 DEG C of cultivation; Obtain thalline by centrifugal for the fermentation liquor 5000-6000rpm of generation, with after be enzyme liquid through smudge cells, 10000-12000rpm centrifuging and taking supernatant liquor;

(2) preparation of substrate solution: with phosphoric acid buffer preparation lactose and the fructose soln of pH5.5-8.0, wherein lactose concn is 300g/L, and fructose concentration is 250g/L;

(3) be hydrolyzed and turn glycosides reaction

600U/L Sumylact L is added, Zn in substrate solution ²⁺10mmol/L, iron(ic) chloride 4-6 mmol/L, at 20-25 DEG C of reaction 0.5-1.5h, is hydrolyzed and turns glycosides reaction, obtaining enzymolysis solution;

(4) aftertreatment of enzymolysis solution

In enzymolysis solution, add dehydrated alcohol, make its final concentration be 65-70%, after centrifugal, get supernatant liquor can obtain lactulose slurry through the 60-65 DEG C of concentrated aftertreatment of heating.