CN101792781B

CN101792781B - Fermenting method for improving xylitol yield

Info

Publication number: CN101792781B
Application number: CN2009102592120A
Authority: CN
Inventors: 李荣杰; 薛培俭; 尚海涛; 徐斌; 段绪果; 薛亮; 胡长浩
Original assignee: Anhui BBCA Fermentation Technology Engineering Research Co Ltd
Current assignee: Anhui BBCA Fermentation Technology Engineering Research Co Ltd
Priority date: 2009-12-16
Filing date: 2009-12-16
Publication date: 2012-05-23
Anticipated expiration: 2029-12-16
Also published as: CN101792781A

Abstract

The invention provides a fermenting method for improving the xylitol yield. The method is to add a proper amount of Zn2+ in a fermentation medium, and uses the activating characteristics of the Zn2+ on xylose reductase, thereby improving the conversion rate of the xylose-xylitol and improving the fermentation yield of the xylitol. Experiments prove that the conversion rate of xylose-xylitol is improved from 68 percent to 79 percent on the basis of not increasing the production cost by the method. The method is simple and practical, and reduces the production cost of xylitol production process in the currently used fermenting method.

Description

A kind of fermentation process that improves xylitol yield

Technical field

The present invention relates to fermentation technique, be specifically related to a kind of fermentation process that improves xylitol yield.

Background technology

Xylitol is a kind of important function property multi-sugar alcohol, and Xylitol metabolism does not in vivo need the participation of Regular Insulin, can not improve blood glucose value after edible, can be used in the dieletic foodstuff.Xylitol can not utilized by mikrobe in the oral cavity, can prevent that carious tooth from taking place.Xylitol also can be used as the energy derive of non-intestinal feeding etc.Exactly because Xylitol has the above-mentioned functions characteristic, industry such as food and medicine have been widely used in.

The working method of Xylitol has: extraction, chemosynthesis, three kinds of methods of biosynthesizing.Wood sugar shortening method is the main method of present industrial production Xylitol, but this production technique needs independent hydrogen manufacturing, and will under HTHP, react complex process, poor stability.Because the no specificity of reaction must be a raw material with the higher pure wood sugar of price, otherwise it is easily separated and influence quality to have multiple fusel in the product, cost is higher.

Biological synthesis process is to utilize mikrobe to produce Xylitol, can reduce the production cost of Xylitol effectively.Fermentation method not only might save the wood sugar purification step, can also simplify the separating step of Xylitol, is a kind of up-and-coming working method.Enzyme process synthesizes Xylitol, then is to realize continuous high-efficient production through the metabolic balance of Xylose reductase cofactors, and this method does not also realize suitability for industrialized production.

In the natural mikrobe, the yeast ratio is easier to wood sugar is transformed the generation Xylitol.Wherein, The yeast strain that produces the Xylitol superior performance mainly concentrates on mycocandida (Candida), like Ji Lemeng ground candiyeast (C.guillier mondii), candida tropicalis (C.tropicalis), not basic candiyeast (C.mogii), level and smooth candiyeast (C.parasilosis) or the like.Suitability for industrialized production is mainly used candida tropicalis.Yeast at first is that Xylose reductase is coenzyme (mainly being NADPH) with NADPH or NADH to the path for transformation of D-wood sugar; The D-wood sugar is reduced to Xylitol, and Xylitol both can be used as primary product and had got in the substratum, also can be by with NAD ⁺For the xylitol dehydrogenase Catalytic Oxygen of coenzyme changes into the D-xylulose, get into the phosphopentose pathways metabolism at last.Can pass through D-xylulose approach during the yeast metabolism wood sugar, consume the Xylitol that a part has generated, so the Xylitol transformation efficiency generally can only reach 65%～90% of theoretical value.But in the actual industrial production process, wood sugar-Xylitol transformation efficiency has only 50%～70%.The activity of the Xylose reductase in the yeast receives the influence of temperature, pH, NADPH concentration and ionic concn, and the activity of this enzyme directly has influence on the transformation efficiency of wood sugar-Xylitol.

How to improve wood sugar in the suitability for industrialized production-Xylitol transformation efficiency, be this area problem demanding prompt solution always.So far, also there is not the relevant fermentation process that can effectively improve xylitol yield in the industrial production.

Summary of the invention

The present invention is directed to above-mentioned deficiency a kind of method that improves the xylitol fermentation yield is provided.

The present invention finds when studying this enzyme characteristic through the Xylose reductase of separation and purification candida tropicalis: zine ion is the activator of this enzyme, keeps suitable Zn ²⁺Concentration can make Xylose reductase maintain the highest activity level, can obtain the highest wood sugar-Xylitol transformation efficiency with this understanding.

And then the present invention provides a kind of method that improves the xylitol fermentation yield, and this method is in fermention medium, to add an amount of Zn ²⁺Discover, in fermention medium, Zn ²⁺Concentration control can significantly increase the fermentation yield of Xylitol within the specific limits, preferred Zn ²⁺Concentration is 0.3～0.6mmol/L, more preferably 0.5mmol/L.

The raw material sources of producing Xylitol at present are in corn cob.With the corn cob is raw material, uses acid hydrolysis, through neutralization, evaporation, decolouring, IX, hydrogenation, concentrate, crystallization and mother liquor handled again can produce Xylitol.In above-mentioned ion-exchange step, strong acidic ion resin and highly basic porous anion resin complete use, and can make liquid glucose be the water white transparency shape, and purity reaches 95%～97%.But in wood sugar separation and purification process, owing to use the reinforcing yin essence cation-exchange chromatography, the ion in the hydrolyzed solution will be adsorbed and remove.Zn in the general fermention medium ²⁺Be mainly derived from yeast powder or yeast extract paste, but, can at utmost reduce the cost that making of organic nitrogen source is used for reducing substratum in order to improve the competitive power of fermentative Production Xylitol.The organic nitrogen source that uses in the at present common substratum mainly is a yeast powder, and its concentration is the 10g/L fermented liquid, at this moment Zn in the substratum ²⁺Concentration 8～11mg/L (0.12～0.17mmol/L), well below the righttest Zn ²⁺Concentration 32.5mg/L (0.5mmol/L).Therefore, add Zn in addition at fermention medium ²⁺To 32.5mg/L, thereby the vigor of raising Xylose reductase reaches the purpose of raising wood sugar-Xylitol transformation efficiency with this.

The present invention utilizes Zn ²⁺As the characteristic of the activator of Xylose reductase, in fermention medium, add Zn ²⁺To the righttest concentration 32.5mg/L.On the basis that does not increase fermentation method explained hereafter cost, use the transformation efficiency of wood sugar-Xylitol to bring up to 79% from 68%.The present invention is simple and practical, has reduced the production cost of fermentative Production Xylitol.

Description of drawings

Fig. 1 shows is the SDS-PAGE electrophorogram of purified Xylose reductase, and wherein, swimming lane 1 is the Xylose reductase behind the purifying, and swimming lane 2 is the standard protein molecular weight.

That Fig. 2 shows is Zn ²⁺Concentration is to the active influence of Xylose reductase.

That Fig. 3 shows is Zn ²⁺Concentration is to the influence of wood sugar-Xylitol transformation efficiency.

Embodiment

Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.

The percentage sign that relates among the present invention " % " if do not specify, is meant mass percent; But the per-cent of solution except as otherwise herein provided, is meant and contains the some grams of solute among the solution 100ml; Per-cent between the liquid is meant the ratio of capacity in the time of 20 ℃.

Embodiment 1 strain fermentation

With candida tropicalis (Candida tropicalis ACCC 20148; Purchase in Chinese agriculture microbial strains preservation administrative center) be inoculated in YPD (Y:yeast extract; P:peptone; D:glucose) on the plate culture medium 30 ℃ cultivate activation, grow in the liquid nutrient medium of 100mL with aseptic toothpick single bacterium colony of transferring then, be inoculated in the liquid nutrient medium that 1000mL is sole carbon source with 3% wood sugar according to 2% inoculum size again and ferment.Fermentation condition is 30 ℃, 200rpm, 24h.

The purifying and the enzyme activity determination of embodiment 2 Xylose reductases

1. purifying: the fermented liquid low-temperature centrifugation that fermentation among the embodiment 1 finishes is also collected thalline, get supernatant behind the ultrasonic disruption, pH is transferred to 7.0 with supernatant.2. under 4 ℃ of conditions, add 40% saturation ratio ammonium sulfate; Leave standstill 4～6h, low-temperature centrifugation is removed foreign protein, and then adding ammonium sulfate, to make the saturation ratio of solution be 80%; 4 ℃ leave standstill 20h; Centrifugal collecting precipitation, deposition is partially soluble in the Tris-HCl damping fluid of a small amount of 20mmol/L pH7.0, and fully dialyses with the Tris-HCl damping fluid.3. low-temperature centrifugation is got supernatant.With on the spissated enzyme liquid in the step 3 appearance in the DEAE-Sepharose column chromatography; Wash-out behind abundant flush away foreign protein, flow velocity is 1.0mL/min, elutriant is the Tris-HCl of 20mmol/L pH7.0; The elutriant that active part is contained 0.5MNaCl elutes, and collects protein peak.With the YM ultra-filtration membrane of 10KD α and Amicon ultrafiltration cup by N ₂Press (pressure is 0.4Mpa) that the active part of collecting is carried out ultrafiltration and concentration, desalination acquisition purified product, and carry out the SDS-PAGE electrophoresis and identify (Fig. 1).

2. enzyme activity determination: detect the changing down of the special absorption value of reduced coenzyme NADH, indirect detection enzyme activity size in the 340nm wavelength with spectrophotometer.Concrete parameter is: Xylose reductase liquid 50 μ L, NADH 0.15mmol/L, wood sugar 0.1mol/L, damping fluid be PBS (0.05mol/L, pH7.0), temperature of reaction is 30 ℃, TV is 2.5mL.Enzyme activity unit is defined as PM, and to consume the required enzyme amount of 1 μ mol NADH be 1 enzyme unit that lives.The protein determination method adopts Xylene Brilliant Cyanine G G-250 method.The ratio vigor of the Xylose reductase after measuring purifying reaches 12U/mg.

Embodiment 3 Zn ²⁺Concentration is to the active influence of Xylose reductase

Get the purifying enzyme liquid of 50 μ L respectively, join the abundant mixing of 20mMTris-HCl damping fluid of 300 μ L pH7.0, the xylose solution that adds 100 μ L again is as substrate, and makes the Zn of reaction system ²⁺Final concentration reaches 0.1,0.2,0.3,0.4,0.5,0.6 respectively, 0.7mmol/L, and the final volume of enzymatic reaction is 500 μ L.Measuring the enzyme of Xylose reductase then respectively lives.As a result, work as Zn ²⁺When concentration is 0.5mmol/L, the active relative reactivity the highest (Fig. 2) of Xylose reductase.

Embodiment 4 Zn ²⁺Concentration is to the influence of wood sugar-Xylitol transformation efficiency

1. Zn in the substratum ²⁺Concentration determination: choose the yeast extract paste of 10g different manufacturers respectively, configuration 1L solution detects wherein Zn with HI93731 zinc concentration determinator ²⁺Concentration is through detecting its Zn ²⁺Concentration is between 8～11mg/L.

2. seed culture: 10g/L wood sugar, 5g/L yeast extract paste, 0.2g/L MgSO ₄7H ₂O, 2.5g/L KH ₂PO ₄Liquid amount is 50mL/250mL, and medium pH value nature inserts the inclined-plane seed, 30 ℃, cultivates 20h on the 180rpm shaking table.

3. xylose-fermenting produces Xylitol: the inoculum size by 2% inserts liquid seeds in two groups of fermentation flasks.Media components is (g/L) wood sugar 100, yeast extract paste 10.0, NaCl 6.0, MgSO ₄7H ₂O 0.4, KH ₂PO ₄3, (NH ₄) ₂HPO ₄, liquid amount is 120mL/250mL, medium pH value nature.To wherein adding Zn in one group ²⁺, make it reach 32.5mg/L, and another group is not added Zn ²⁺, two groups all in 30 ℃, and 200rpm stops behind the 250mL shake-flask culture 50h.

4. Xylitol Determination on content: with above-mentioned fermented liquid broken cell homogenate, the centrifugal deposition of going is got the 20mL supernatant and is boiled 5min, centrifugal Deproteinization, and supernatant detects as chromatogram.Chromatographic column is SS-100Ca ²⁺The sugar post, 60 ℃ of column temperatures, moving phase is deionized water, flow velocity 1mL/min, sample size are 3.0 μ L.As a result, add Zn ²⁺Make the transformation efficiency of wood sugar-Xylitol bring up to 79% during for 32.5mg/L from 68%.(Fig. 3)

Claims

1.Zn ²⁺Zn is added in application in improving the xylitol fermentation yield in substratum ²⁺To final concentration 0.3～0.6mmol/L, said zymophyte is a candida tropicalis.

2. application as claimed in claim 1 is characterized in that in substratum, adding Zn ²⁺To final concentration 0.5mmol/L.