CN106046066B - A method of purifying prepares high-purity xylobiose - Google Patents
A method of purifying prepares high-purity xylobiose Download PDFInfo
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- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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Abstract
The invention discloses the methods that purifying prepares high-purity xylobiose.This method comprises the following steps：(1) xylan enzymolysis liquid is subjected to ultrafiltration, collects filtrate；(2) filtrate is flowed through into ion exchange resin column, obtains the scavenging solution of xylan enzymolysis liquid；(3) gel permeation chromatography is carried out to the scavenging solution, you can obtain the xylobiose.The xylan enzymolysis liquid of alkali process bagasse preparation is isolated and purified using the present invention, the xylobiose of high-purity, coproduction xylotriose and xylose can be prepared, purity is respectively 95.74 ± 1.43%, 85.11 ± 2.27% and 86.61 ± 3.57%, and xylobiose loss is minimum, is 6.38%.The method of the present invention is easy, flexible practicality, quick, at low cost, is conducive to promotion development and application.
Description
Technical field
The present invention relates to the field that isolates and purifies of oligosaccharides, especially a kind of method that purifying prepares high-purity xylobiose.
Background technology
Xylo-oligosaccharide is that have low-heat, stabilization, nothing by 2~7 xyloses with oligosaccharide made of β-Isosorbide-5-Nitrae glucosides key connection
The characteristics such as poison and unique physiological function.Xylo-oligosaccharide can promote the proliferation of Bifidobacterium in humans and animals enteron aisle, inhibit to have
The growth of evil bacterium, it is xylobiose and xylotriose (Carvalho AFA, de Oliva to promote the absorption of calcium, principle active component
Neto P,da Silva DF,et al.Xylo-oligosaccharides from lignocellulosic
materials:Chemical structure,health benefits and production by chemical and
enzymatic hydrolysis[J].Food Research International,2013,51:75-85).The sweet tea of xylobiose
Degree is about the 40% of sucrose, and the sugariness that content reaches 50% xylo-oligosaccharide product is about the 30% of sucrose, and sweet taste is pure, similar
Sucrose.Currently, xylo-oligosaccharide is as novel functional additive, application is more and more extensive.For example, functional food adds
Agent：It is mainly used for low sugar, Sugarless type food and health-care medical food；Feed addictive：It is main to improve micro- life in animal intestinal tract
The ecological balance of object, absorption and utilization of the enhancing animal to feed nutrition substance；And agricultural additives：Battalion as crops
Object is supported, the speed of growth and disease resistance (Zhang Ling, fourth long river, the application study and production of the refined xylo-oligosaccharides of Ruan Wen of crops are improved
Product development progress grains and grease, 2015,28 (2):9-12).
Currently, xylo-oligosaccharide is mainly derived from lignocellulosic, the preparation and purification method of one pack system mainly passes through enzyme
Method hydrolyzed xylan, and one-component is obtained after chromatographic purifying, as (Zhang Junhua, Xu Yong bravely by force wait wood two to gel chromatography
The separation of sugar and xylotriose and its in vitro culture [J] the chemistry of forest product for Bifidobacterium and industry, 2005,25 (1):15-
18.).Patent CN1847254A uses polyacrylamide gel chromatographic column separating oligomeric xylose each component, due to polyacrylamide
Gel is expensive, and regeneration is difficult.Currently, being only difficult to the large-scale production of xylo-oligosaccharide in laboratory research.Adsorption layer
Analysis method (Tan SS, Li DY, Jiang ZQ, et al.Production of xylobiose from the
autohydrolysis explosion liquor of corncob using Thermotoga maritima xylanase
B(XynB)immobilized on nickel-chelated Eupergit C.[J].Bioresource Technology,
2008,99(1):200-204).Patent CN101632877A carries out point of each component in xylo-oligosaccharide using activated carbon chromatography method
From.The method is eluant, eluent using ethyl alcohol, of high cost；Activated carbon service life is short, and dress charcoal unloads charcoal heavy workload, wastes time,
Largely useless charcoal is discarded as production rubbish；In addition, Activated carbon separation effect is poor, obtained product purity is low, is unfavorable for green production.
(model is beautiful, Xu Yong, Lian Zhina, and High Performance Anion Exchange Chromatography Coupled with Pulsed Amperometric Detection is waited to quantitative determine for ion exchange chromatography
Xylo-oligosaccharide [J] chromatographies in xylo-oligosaccharide sample, 2011,29 (1):75-78), wherein gel chromatography is most widely used.Specially
Sharp CN10830936B uses xylobiose and miscellaneous sugar component in Simulation moving bed purifying oligo-xylose, although xylobiose component
Content is higher, is 94% or more, but purification efficiency is relatively low, only 30-46%, of high cost, and can not achieve other one-components
Separation.
There is document to prepare xylobiose using the aqueous two-phase xylanase hydrolysis system of Macrogol 6000/sodium citrate, then
Isolating and purifying xylobiose using sephadex lh-20, (Li Xin, Gu Ximei, restrict Ao Nan, waits aqueous two-phase xylanase hydrolysis bodies
System prepares xylobiose and its purifying [J] chemistry of forest product and industry, 2015,35 (5):93-97.), though isolating high-purity
(98.5%) xylobiose, but it is cumbersome, and of high cost, experimental size is small.Nanofiltration separation xylo-oligosaccharide (Zhao Hefei, Yang Ruijin,
Zhao Wei waits stalk xylo-oligosaccharide solution nanofiltration separation characteristics and diafiltration technique [J] Journal of Agricultural Engineering, 2009,25 (4):
253-259.), though the separation of one pack system can be can not achieve, and the loss of xylobiose is larger with efficiently purifying xylo-oligosaccharide,
It is 12.45%.
In view of above it is found that the report both at home and abroad in terms of xylobiose isolates and purifies is few.On domestic and international market only
Its standard items is sold, expensive.Therefore, develop simple and convenient, product purity is high, and can detach xylobiose and xylotriose simultaneously
Isolation and purification method be necessary.
Invention content
The object of the present invention is to provide a kind of method that purifying prepares high-purity xylobiose, this method can realize oligomeric wood
One-component isolates and purifies in sugar, and the purity and the rate of recovery of gained xylobiose are high, can coproduction isolate xylotriose and xylose, grasp
Make simple, quick and at low cost.
The method that purifying provided by the invention prepares high-purity xylobiose, includes the following steps：(1) by xylan enzymolysis liquid
Ultrafiltration is carried out, filtrate is collected；(2) filtrate is flowed through into ion exchange resin column, obtains the scavenging solution of xylan enzymolysis liquid；
(3) gel permeation chromatography is carried out to the scavenging solution, you can obtain the xylobiose.
Above-mentioned method, in step (1), the xylan enzymolysis liquid refers to being carried out to xylan raw material using zytase
Product after enzymolysis, the xylan raw material can be bagasse, corncob, straw or manioc waste；The xylan enzymolysis liquid can
For the product obtained as follows：The pH value of a concentration of 20~60g/L xylans raw material and the mixed system of water is adjusted
To 5.0~6.0, by the amount of every gram of 800 units of raw material solid content after zytase enzyme solution is added, the enzyme at 35~45 DEG C
Solution 24 hours, obtains the xylan enzymolysis liquid.
Further include the steps that being pre-processed to obtain thick xylan to raw material using alkali before the enzymolysis, the alkali is with alkali
Aqueous solution form exist, mass concentration can be 8.0%~10.0%；May also include in the alkali mass concentration be 0~
3.0% H202, but be not 0；Processing time can be 12~for 24 hours；Feed liquid such as 1g is concretely pressed in the processing:The ratio of 10mL
Example is by bagasse and the NaOH for being 8.0% such as mass concentration, the H that mass concentration is 2.48%202Lye is blended in 40 DEG C, impregnates
Processing is as 24 hours, filtered through gauze, and filtrate is concentrated by ultrafiltration through ultrafiltration (molecular cut off 3000Da) again, then with 4 times of volumes 95%
Ethanol precipitation centrifuges, and precipitates the thick xylan of drying.
Above-mentioned method, in step (1), the aperture of the ultrafiltration membrane used in the ultrafiltration can be 0.001~0.01 μm, tool
Body can be 0.001 μm, and operating pressure can be 0.1~0.2MPa, concretely 0.15MPa.
Above-mentioned method, in step (2), the filler of the ion exchange resin column can be anion exchange resin D201
(strongly acidic styrene's cation exchanges by (strong-basicity styrene series anion exchange resin) and cation exchange resin 001-7
Resin)；Series sequence is preferably cation exchange resin 001-7- anion exchange resin D201, the cation exchange resin
The volume ratio of 001-7 and the anion exchange resin D201 can be (0.5~2.5)：1, preferably (1.0~2.0)：1, it is more excellent
It is selected as 1.5：1；Sample introduction flow velocity can be (0.5~3.0) mL/min, preferably (1.5~2.5), more preferably 2.0mL/min；Most
Good condition is：The volume ratio of the cation exchange resin 001-7 and the anion exchange resin D201 are 1.67：1 and into
Sample flow velocity is 1.76mL/min, and gained percent of decolourization is that 96.85 ± 1.43%, salt rejection rate is 77.59 ± 3.79% and sugared retention rate
It is 96.64 ± 2.59%.
In above-mentioned method, in step (3), the chromatography of sephadex G -10 specifically can be used in the gel permeation chromatography
The ratio of height to diameter of column, gel chromatography column can be (15~35)：1, preferably 30：1；Sampling volume can be 1~9% bed volume,
Such as 5%；The gel permeation chromatography is eluted using water, the water can be deionised degassed water, elution speed can be 0.3~
1.2mL/min, preferably 0.6mL/min, elution volume can be 1.0~3.0 times of bed volumes, such as 1.5 times；Optimum condition is：
Elution speed is 0.73mL/min, gel column ratio of height to diameter is 34.03：1, the purity of gained xylobiose is 95.74 ± 1.43%, returns
Yield is 93.61 ± 3.50%.
The each component in the scavenging solution of the xylan enzymolysis liquid can be detached by the gel permeation chromatography；
The method further includes the steps that obtaining xylose and xylotriose after the gel permeation chromatography, that is, is obtaining the xylobiose
Further include simultaneously obtaining xylose and xylotriose, therefore, the method that purifying of the present invention prepares xylobiose can coproduction xylotriose and xylose.
The method determines each component as follows after the gel permeation chromatography：It is detected and is received using HPLC
Ingredient in liquid collecting determines each component, such as by comparing collection liquid and wood two by the appearance time of contrast sample and standard items
The appearance time of saccharide determines that the group is divided into xylobiose.
Specifically, according to the elution order of eluent, the eluent for the 0.8th~1.2 times of bed volume collected is
The eluent of xylobiose, the 1.2nd~1.4 times of bed volume is xylose, and the eluent of the 0.5th~0.7 times of bed volume is wood three
Sugar.
The present invention has the advantages that：
(1) the xylan enzymolysis liquid that the preparation of alkali process bagasse is isolated and purified using the present invention, can prepare high-purity
Xylobiose, coproduction xylotriose and xylose, purity are respectively 95.74 ± 1.43%, 85.11 ± 2.27% and 86.61 ± 3.57%,
And xylobiose loss is minimum, is 6.38%.
(2) the method for the present invention is easy, flexible practicality, quick, at low cost, is conducive to promotion development and application.
Description of the drawings
Fig. 1 is the process flow chart of the present invention.
Fig. 2 is the influence for the xylan enzymolysis liquid decoloration desalting effect that different fillings prepare oxygenation pretreatment bagasse,
Wherein, A is percent of decolourization in Fig. 2；B is salt rejection rate in Fig. 2；C is sugared retention rate in Fig. 2.
Fig. 3 is the xylan enzymolysis liquid decoloration desalination effect that different zwitterion series sequences prepare oxygenation pretreatment bagasse
The influence of fruit.
Fig. 4 is that different zwitterion packing volume ratios decolourize to take off to xylan enzymolysis liquid prepared by oxygenation pretreatment bagasse
The influence of salt effect, wherein left ordinate corresponds to percent of decolourization, sugared retention rate, and right ordinate corresponds to salt rejection rate.
Fig. 5 is the shadow for the xylan enzymolysis liquid decoloration desalting effect that different sample introduction flow velocitys prepare oxygenation pretreatment bagasse
It rings, wherein left ordinate corresponds to percent of decolourization, sugared retention rate, and right ordinate corresponds to salt rejection rate.
Fig. 6 is elution flow velocity, zwitterion resin columns in series zwitterion packing volume ratio to oxygenation pretreatment
The 3D figures of xylan enzymolysis liquid decoloration desalting effect prepared by bagasse, A is percent of decolourization in wherein Fig. 6, and B is salt rejection rate in Fig. 6,
C is sugared retention rate in Fig. 6.
Fig. 7 is the shadow that different elution speeds isolate and purify xylan enzymolysis liquid prepared by oxygenation pretreatment bagasse effect
It rings.
Fig. 8 is the xylan enzymolysis liquid separation that -10 pillar height diameter of different sephadex Gs compares prepared by oxygenation pretreatment bagasse
The influence of purification effect.
Fig. 9 elution speeds, gel column ratio of height to diameter isolate and purify effect to xylan enzymolysis liquid prepared by oxygenation pretreatment bagasse
The 3D of fruit schemes, wherein A is xylobiose purity in Fig. 9, and B is the xylobiose rate of recovery in Fig. 9.
Figure 10 HPLC detection sephadex Gs -10 isolate and purify each component component-part diagram, wherein and Figure 10 A are standard sample,
Wherein X1 represents xylose, and X2 represents xylobiose, and X3 represents xylotriose；Xylan enzymolysis prepared by Figure 10 B oxygenation pretreatment bagasse
Liquid；C xyloses component in Figure 10；D is xylobiose component in Figure 10；E is xylotriose component in Figure 10.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Determine that the operating procedure for the time for starting and terminating collection filtrate is as follows in following embodiments using DNS methods：It takes
0.5mL effluxes be added 2 times of volumes DNS, boiling water bath 5min, with microplate reader detect light absorption value, so that it is determined that efflux whether
Containing sugar, when sugared content is more than 0, starting to collect to can't detect sugar in efflux, stopping collecting.
The method that purifying prepares high-purity xylobiose in following embodiments, includes the following steps：(1) by xylan enzymolysis liquid
Ultrafiltration is carried out, filtrate is collected；The aperture of ultrafiltration membrane used can be 0.001~0.01 μm, and operating pressure can be 0.1~0.2MPa；
(2) filtrate is flowed through into ion exchange resin column, obtains the scavenging solution of xylan enzymolysis liquid；(3) gel filtration is carried out to scavenging solution
Chromatography, you can obtain xylobiose.
Embodiment 1, purifying prepare the optimization of the condition of high-purity xylobiose
One, the selection of decoloration desalination column packing
Purifying prepares high-purity xylobiose in accordance with the following steps：
(1) ultrafiltration：By xylan enzymolysis liquid prepared by oxygenation pretreatment bagasse, with ultrafiltration membrane, (molecular cut off is
10000Da, aperture are 0.001 μm) it is filtered (operating pressure 0.15MPa), filtrate is collected, macromolecular substances and portion are removed
Divide non-glucide.Wherein, oxygenation pretreatment bagasse prepare xylan enzymolysis liquid step reference literature " Xue, JL,
Zhao,S,et al.A biotechnological process efficiently co-produces two high
value-added products,glucose and xylooligosaccharides,from sugarcane
Bagasse.Bioresourse Technology, 2016,204, method disclosed in 130-138 " is specific as follows：By feed liquid
Such as 1g:The ratio of 10mL by bagasse with containing mass concentration be 8.0% NaOH, mass concentration be 2.48% H202Solution
After mixing, for 24 hours, filtered through gauze, filtrate concentrates through ultrafiltration (molecular cut off 3000Da) immersion treatment again, then uses at 40 DEG C
It is centrifuged after the ethanol precipitation of 4 times of volumes 95%, drying precipitates up to thick xylan；By the thick xylans of a concentration of 35g/L and water
The pH value of mixed system is adjusted to 5.5, after zytase enzyme solution is added by the enzyme amount of every gram of 800 units of raw material solid content,
It is digested 24 hours at 36 DEG C, obtains xylan enzymolysis liquid.
(2) selection of decoloration desalination column packing：Respectively taking anion exchange resin SA10AP, (the green hundred grass science and technology in Beijing is public
Department), D900 and macroporous absorbent resin D101, D201 (Hebei Cangzhou Bao En resin processing plants) and (Beijing cation exchange resin SK1B
Green hundred careless scientific ＆ technical corporation) and 001-7 (Hebei Cangzhou Bao En resin processing plants) 20g, excellent suitable board activated carbon (the excellent suitable activated carbon of Ningbo of Zhejiang
Factory), craboraffin, boda 1#, 2# activated carbon (Henan boda water-purifying material Co., Ltd), non-phosphorus active carbon (holds Yu in Changsha
Chemical Co., Ltd.) 1g, it is separately added into the filtrate 100mL obtained by step (1), Static Adsorption 2h, separates a period of time concussion one
Secondary, filter paper filters after 2h, collection filtrate, uses ultraviolet-uisible spectrophotometer (the silent winged generation that of U.S.'s match), conductivity gauge (Shanghai respectively
Thunder magnetic), HPLC (U.S. waters) surveys the light absorption value of ultrafiltrate obtained by each filtrate and step (1), conductivity (ionic strength) and
(chromatographic condition is as follows for sugared content：Dalian Yi Lite nh 2 columns (Hypersil NH2 250*4.6/5um) mobile phase：76% second
Nitrile, flow velocity 1.0mL/min, 30 DEG C of column temperature.
Contained according to light absorption value before and after absorption (A in Fig. 2) and Conductivity Calculation percent of decolourization (B in Fig. 2) and salt rejection rate and sugar
The variation (C in Fig. 2) of amount.Filtering out anion exchange resin D201 and cation exchange resin 001-7 has highest decoloration
Rate, salt rejection rate and sugared retention rate.Calculation formula is as follows：
Percent of decolourization=(A0-A1)/A0 × 100；In formula, A0 was that light absorption value, the A1 of xylan enzymolysis liquid before ion column are
Cross the light absorption value (being measured with ultraviolet-visible spectrophotometer) of xylan enzymolysis liquid after ion column；
Salt rejection rate=(K0-K1)/K0×100；In formula, K0 was that the conductivity of xylan enzymolysis liquid, K1 were before ion column
The conductivity (being measured with conductivity gauge) of xylan enzymolysis liquid after ion column；
Sugared retention rate=X1/X0×100；In formula, X0 was that total sugar content, X1 were in xylan enzymolysis liquid before ion column
Total sugar content (being measured with HPLC) in xylan enzymolysis liquid after ion column.
Two, the determination of anion-cation exchange resin column series sequence
(1) ultrafiltration：With the operation of step (1) in above-mentioned one.
(2) determination of anion-cation exchange resin column series sequence：D201--001-7 is exchanged respectively using wet packing method
Resin (V (D201):V (001-7)=1：1), 001-7--D201 exchanger resins (V (001-7):V (D201)=1：1) it is encased inEmpty chromatographic column in, under conditions of flow velocity 2.0mL/min, filter obtained by the step of being pumped into 200mL respectively (1)
Liquid determines with DNS methods and starts to collect and terminate the time for collecting filtrate, the method for recycling step (2) in one surveys the de- of filtrate
Color rate, salt rejection rate, sugared retention rate (Fig. 3).Compare influence of the different zwitterion series sequences to desalination of decolourizing, determines and press 001-
Percent of decolourization, salt rejection rate and sugared retention rate when 7--D201 sequential series is larger.
Three, influence of the different anion-cation exchange resin loadings to desalination of decolourizing
(1) ultrafiltration：With the operation of step (1) in above-mentioned one.
(2) determination of different anion-cation exchange resin loadings：By the amberlite of 001-7--D201 sequential series
Fat is respectively with V (001-7):V (D201) is 0.5：1、1：1、1.5：1、2：1 and 2.5：1 ratio wet method dress post, in flow velocity
Under conditions of 2.0mL/min, liquid obtained by the step of being pumped into 200mL respectively (1) starts to collect and terminate to receive with the determination of DNS methods
Collect the time of filtrate, the method for recycling step (2) in one measures percent of decolourization, salt rejection rate and the sugared retention rate (Fig. 4) of filtrate.Than
Influence of the more different anion-cation exchange resin filling proportions to desalination of decolourizing, determines V (001-7):V (D201)=1.5：When 1,
Percent of decolourization, salt rejection rate and sugared retention rate highest.
Four, influence of the sample introduction flow velocity to desalination of decolourizing
(1) ultrafiltration：With the operation of step (1) in above-mentioned one.
(2) determination of sample introduction flow velocity：Under conditions of above-mentioned determination, respectively with 0.5mL/min, 1.0mL/min, 1.5mL/
Filtrate obtained by 200mL steps (1) is pumped into V (001-7) by min, 2.0mL/min, 2.5mL/min and 3.0mL/min sample introduction speed：
V (D201)=1.5：In 1 concatenated ion column, is determined with DNS methods and start to collect and terminate the time for collecting filtrate, recycle step
Suddenly the method for (2) measures percent of decolourization, salt rejection rate and the sugared retention rate (Fig. 5) of filtrate.Compare different sample introduction flow velocitys to desalination of decolourizing
Influence, determine 2.0mL/min be decolourize desalination best sample introduction flow velocity.
Five, response surface optimization xylo-oligosaccharide decoloration desalination condition
(1) ultrafiltration：With the operation of step (1) in above-mentioned one.
(2) response surface optimization xylo-oligosaccharide decoloration desalination condition：According to experiment of single factor data, sample introduction flow velocity and the moon are selected
For cation exchange resin filling proportion as independent variable, percent of decolourization, salt rejection rate and sugared retention rate are dependent variable, carry out two factors three
The optimum condition of horizontal response surface design experiment, the decoloration desalination of optimization xylo-oligosaccharide is sample introduction flow velocity 1.76mL/min, V
(001-7):V (D201)=1.67：1, gained percent of decolourization is that 96.85 ± 1.43%, salt rejection rate is 77.59 ± 3.79% and sugar is protected
It is 96.64 ± 2.59% (Fig. 6, table 1-3) to stay rate.
The enzymolysis liquid decolorizing effect for the xylan that 1 zwitterion resin columns in series of table prepares oxygenation pretreatment bagasse responds
Face quadratic equation model analysis of variance table
The enzymolysis liquid desalting effect for the xylan that 2 zwitterion resin columns in series of table prepares oxygenation pretreatment bagasse responds
Face quadratic equation model analysis of variance table
The enzymolysis liquid retention for the xylan that 3 zwitterion resin columns in series of table prepares oxygenation pretreatment bagasse responds
Face quadratic equation model analysis of variance table
Six, elution speed isolates and purifies sephadex G -10 influence of xylobiose
(1) ultrafiltration：With the operation of step (1) in above-mentioned one.
(2) filtrate is flowed through into ion exchange resin column (ion exchange resin column condition：Filler series sequence is 001-7-
D201, loading V (001-7):V (D201)=1.67：1, sample introduction flow velocity is 1.76mL/min), DNS methods, which determine, to be started and terminates
Collect the time of filtrate, the scavenging solution of collection liquid, that is, xylan enzymolysis liquid.
(3) determination of elution speed：Liquid is pumped into the glucan balanced obtained by the step of 5% bed volume (2)
Gel G-10 chromatographic columns, after liquid fully enters column bed, respectively with 0.3mL/min, 0.6mL/min, 0.9mL/min and
The elution speed of 1.2mL/min is eluted with the deionised degassed water of 1.5 times of bed volumes, and 1 pipe is collected per 2min.By collection
(chromatographic condition is as follows with high performance liquid chromatography for liquid：Dalian Yi Lite nh 2 columns (Hypersil NH2 250*4.6/5um)
Mobile phase：76% acetonitrile, flow velocity 1.0mL/min, 30 DEG C of column temperature) measure the rate of recovery of its constituent content and each component, optimization
Best elution speed is 0.6mL/min (Fig. 7).
Seven, gel column ratio of height to diameter isolates and purifies sephadex G -10 influence of xylobiose
(1) ultrafiltration：With the operation of step (1) in above-mentioned one.
(2) with the operation of step (2) in above-mentioned six.
(3) determination of gel column ratio of height to diameter：Under the best elution speed determined in above-mentioned six, by 5% bed volume
It is 15 that liquid obtained by step (6), which is pumped into gel column ratio of height to diameter,:1、20:1、25:1、30:1 and 35:The gel of 1 glucan G-10
Column collects 1 pipe per 2min, by the liquid of the collection rate of recovery of its constituent content of high effective liquid chromatography for measuring and each component
(chromatographic condition is with six), the best gel column ratio of height to diameter of optimization are 30:1 (Fig. 8).
Eight, response phase method optimization sephadex G -10 isolates and purifies the optimum condition of xylobiose
(1) ultrafiltration：With the operation of step (1) in above-mentioned one.
(2) with the operation of step (2) in above-mentioned six.
(3) response phase method optimization sephadex G -10 isolates and purifies the optimum condition optimization of xylobiose：According to single factor test
Experiment conclusion, using elution speed and G-10 gel columns ratio of height to diameter as independent variable, the purity and the rate of recovery of xylobiose are dependent variable, into
Two factor of row, three horizontal respone face is tested, and the optimum condition that optimization isolates and purifies is elution speed 0.73mL/min, gel column
Ratio of height to diameter 34.03:1, it is 93.61 ± 3.50% (Fig. 9, table 4- that the purity of gained xylobiose, which is 95.74 ± 1.43%, rate of recovery,
5)。
4 sephadex G -10 of table isolates and purifies xylobiose purity response surface quadratic equation model analysis of variance table
5 sephadex G -10 of table isolates and purifies xylobiose rate of recovery response surface quadratic equation model analysis of variance table
Embodiment 2, purifying prepare high-purity xylobiose
Purifying prepares high-purity xylobiose under conditions of after optimizing in embodiment 1, and concrete operations are as follows：
(1) ultrafiltration：With the operation of step (1) in above-mentioned one.
(2) filtrate is flowed through into ion exchange resin column (ion exchange resin column condition：Filler series sequence is 001-
7--D201, loading V (001-7):V (D201)=1.67：1, sample introduction flow velocity is 1.76mL/min), started with the determination of DNS methods
Collect and terminate the time for collecting filtrate, the scavenging solution of collection liquid, that is, xylan enzymolysis liquid.
(3) liquid obtained by the step of taking 5% bed volume (2) is pumped into the gel chromatography that has balanced, and (ratio of height to diameter is
34.03：1-10 column of sephadex G), after liquid fully enters column bed, using the degassing deionization of 1.5 times of bed volumes
Water elution, elution speed 0.73mL/min collect 1 pipe per 2min.By the liquid of collection with high effective liquid chromatography for measuring its
The rate of recovery of constituent content and each component (chromatographic condition is with six in embodiment 1).
Experimental result is as shown in Figure 10, wherein standard items are purchased from the Shanghai biotech inc Suo Laibao, purity
It is 99% or more.The purity of gained xylobiose is that 95.74 ± 1.43%, rate of recovery is 93.61 ± 3.50%.Sephadex G-
During 10 isolate and purify xylobiose, xylotriose and xylose are purified out simultaneously, and the purity of gained xylotriose is 85.11 ±
2.27%, the rate of recovery is 52.93 ± 12.92%；The purity of xylose be 86.61 ± 3.57%, rate of recovery be 44.16 ±
8.99%.The calculation formula of each component is：Purity=M (xylobiose content)/M (total sugar content), the rate of recovery=M (cross gel column
Preceding xylobiose content)/M (crossing xylobiose content after gel column).
Claims (9)
1. a kind of method that purifying prepares high-purity xylobiose, includes the following steps：
(1) xylan enzymolysis liquid is subjected to ultrafiltration, collects filtrate；
The xylan enzymolysis liquid refers to the product after being digested to xylan raw material using zytase, and the xylan is former
Material is bagasse；
(2) filtrate is flowed through into ion exchange resin column, obtains the scavenging solution of xylan enzymolysis liquid；
The filler of the ion exchange resin column is anion exchange resin D201 and cation exchange resin 001-7；
(3) gel permeation chromatography is carried out to get to the xylobiose to the scavenging solution；
The gel permeation chromatography uses -10 chromatographic column of sephadex G.
2. according to the method described in claim 1, it is characterised in that：In step (1), the ultrafiltration membrane that is used in the ultrafiltration
Aperture is 0.001~0.01 μm, and operating pressure is 0.1~0.2MPa.
3. according to the method described in claim 1, it is characterized in that：In step (2), the filler of the ion exchange resin column
Series sequence be cation exchange resin 001-7- anion exchange resin D201；And/or the cation exchange resin
The volume ratio of 001-7 and the anion exchange resin D201 are (0.5~2.5)：1.
4. method according to any one of claim 1-3, it is characterised in that：In step (2), sample introduction flow velocity be (0.5~
3.0)mL/min。
5. method according to any one of claim 1-3, it is characterised in that：In step (3), the gel permeation chromatography
Condition be following 1) -4) at least one of：1) ratio of height to diameter of gel chromatography column is (15~35)：1；2) sampling volume is 1
~9% bed volume；3) it is eluted using water；4) elution speed be 0.3~1.2mL/min, elution volume be 1.0~
3.0 times of bed volumes.
6. method according to any one of claim 1-3, it is characterised in that：The method is in the gel permeation chromatography
After further include the steps that obtaining xylose and xylotriose.
7. method according to any one of claim 1-3, it is characterised in that：The method is in the gel permeation chromatography
Later, each component is determined as follows：Ingredient in collection liquid is detected using HPLC, passes through contrast sample and standard items
Appearance time determines each component.
8. according to the method described in claim 5, it is characterized in that：The eluent of 0.8th~1.2 times of bed volume is wood two
Sugar.
9. according to the method described in claim 5, it is characterized in that：The eluent of 1.2nd~1.4 times of bed volume is xylose；
The eluent of 0.5th~0.7 times of bed volume is xylotriose.
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| CN1847254A (en) * | 2005-08-30 | 2006-10-18 | 南京林业大学 | Prepn and separation process of components for xylose oligomer of polymerization degree 2-8 |
| CN101096376A (en) * | 2006-06-26 | 2008-01-02 | 黄广民 | Method for purifying D-xylose and xylooligosacchrides from coconut hard shell acid hydrolysate |
| CN101830936A (en) * | 2010-05-18 | 2010-09-15 | 禹城绿健生物技术有限公司 | Method for preparing solid xylobiose |
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| CN1847254A (en) * | 2005-08-30 | 2006-10-18 | 南京林业大学 | Prepn and separation process of components for xylose oligomer of polymerization degree 2-8 |
| CN101096376A (en) * | 2006-06-26 | 2008-01-02 | 黄广民 | Method for purifying D-xylose and xylooligosacchrides from coconut hard shell acid hydrolysate |
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