CN110305921A - A method of xylo-oligosaccharide being made by raw material of stalk - Google Patents
A method of xylo-oligosaccharide being made by raw material of stalk Download PDFInfo
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Abstract
The invention discloses a kind of method that xylo-oligosaccharide is made as raw material using stalk, stalk is by impregnating, the xylan the amount of dissolution in stalk can be improved in soda acid processing；Xylan, cellulose, hemicellulose degradation in treatment fluid can be improved the yield of xylo-oligosaccharide by cellulase, hemicellulase and zytase；By using molecular cut off be 1000~1500Da, the nanofiltration membrane of 600~800Da, 200~400Da carry out separating and filtering to enzymolysis liquid, macroporous absorbent resin decoloration, ion exchange column elutes desalination, can effectively improve the purity of xylo-oligosaccharide, and product homogeneity is good, quality is stablized.Preparation process of the present invention is simple, and reaction condition is mild, is convenient for industrialized production.
Description
Technical field
The present invention relates to xylo-oligosaccharide field, especially a kind of method that xylo-oligosaccharide is made as raw material using stalk.
Background technique
Human body intestinal canal and body surface inhabit hundreds of millions of bacteriums, and type up to more than 400 weighs two kilograms, this works as
In have and be pernicious to people, be known as harmful bacteria, have, referred to as probiotics beneficial to people, also have therebetween
Conditioned pathogen, i.e., will lead to the sick bacterium of human body under certain condition, and the intracorporal probiotics of people mainly has lactic acid bacteria, bifid
Bacillus etc..
Scientific research confirms that probiotics can treat in the mass propagation of enteron aisle because largely using caused by antibiotic
Pseudomembranous enteritis；Treat constipation and chronic diarrhea；Protect liver；It prevents and treats hypertension and artery sclerosis and anti-aging, reduce blood
Clearing gallbladder sterol, pre- anti-cancer and the effect for inhibiting tumour growth.
Xylo-oligosaccharide is that one of strongest kind of Bifidobacterium function is proliferated in polymerization carbohydrate, its effect of property is that other are poly-
Nearly 20 times of carbohydrate are closed, does not have to hydrolyze the enzyme of xylo-oligosaccharide in human gastrointestinal tract, so it can be directly entered big enteral is preferably
Bifidobacterium is utilized, and is promoted Bifidobacterium proliferation while being generated a variety of organic acids.Enteron aisle pH value is reduced, inhibits harmful bacteria raw
It is long, it is proliferated probiotics largely in enteron aisle, reaches above-mentioned health-care efficacy, here it is where the health care secret of xylo-oligosaccharide.
The common preparation method of xylo-oligosaccharide has enzymatic isolation method, acid-hydrolysis method and hydro-thermal method at present.Hydro-thermal method is oligomeric at present
A kind of most common method in xylose production.Will the agricultural fibre waste material containing hemicellulose hydrolyze at high temperature under high pressure, water
Solution liquid further filter, be concentrated, purifying, crystallizing, dry after obtain xylo-oligosaccharide product.But the method needs heatproof, pressure-resistant equipment,
Energy consumption is big, and efficiency of pcr product is low.Acid-hydrolysis method be by the agricultural fibre waste material acidolysis containing hemicellulose, common acid have hydrochloric acid,
Sulfuric acid, trifluoroacetic acid etc..Acid hydrolysis solution further neutralizes, filters, being concentrated, being centrifuged, crystallizing, dries and obtains xylo-oligosaccharide product.But
This method process flow is long, and the auxiliary materials such as consumption water, electricity, vapour, acid, alkali are more, and production cost is higher；And to equipment, technical requirements
It is higher；It is serious that difficulty, environmental pollution are separated after reaction.Enzymatic isolation method is that the internal cutting type xylanase for using microorganism to generate decomposes
Then xylan is isolated to xylo-oligosaccharide.The problems such as that there are conversion ratios is low for this method, and subsequent separation is difficult.
Summary of the invention
In view of this, there is height the object of the present invention is to provide a kind of method that xylo-oligosaccharide is made as raw material using stalk
Purity, and product homogeneity is good, quality stablize.
To achieve the goals above, the invention provides the following technical scheme:
A method of xylo-oligosaccharide being made by raw material of stalk, comprising the following steps:
1) after stalk being crushed be added water 3~4h of immersion treatment at 50~80 DEG C, add acid or alkali process 15~
20h obtains treatment fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6~7, it is poly- that cellulase, hemicellulase and wood is added
Carbohydrase, 12~15h of enzymolysis processing, obtains enzymolysis liquid at 35~50 DEG C；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) it will successively use molecular cut off for 1000~1500Da, 600 in the enzymolysis liquid after inactivation that step 3) obtains
The isolated filtered fluid of nanofiltration membrane of~800Da, 200~400Da；
5) filtered fluid that step 4) obtains is decolourized through macroporous absorbent resin, then elutes desalination through ion exchange column, obtained
Eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
Preferably, in step 1), the acid is sulfuric acid or hydrochloric acid, and the alkali is sodium hydroxide or potassium hydroxide；The mistake
The concentration of acid or alkali is 0.1~0.15mol/L in filtrate.
Preferably, the additional amount of the cellulase is that the fiber that active unit is 50~70U is added in every 100g stalk
Plain enzyme, the additional amount of the hemicellulase are that the hemicellulase that active unit is 60~80U, institute are added in every 100g stalk
The additional amount for stating zytase is that the zytase that active unit is 80~100U is added in every 100g stalk.
Preferably, dilution is obtained after step 4) is specifically, dilute 10~15 times for enzymolysis liquid, dilution is passed through retention point
The nanofiltration membrane that son amount is 1000~1500Da, isolated by-pass filtration liquid；
It is passed through the nanofiltration membrane that molecular cut off is 600~800Da after deionized water is added into by-pass filtration liquid, is separated
To secondary filtration liquid；
It is passed through the nanofiltration membrane that molecular cut off is 200~300Da after deionized water is added into secondary filtration liquid, is separated
To filtered fluid.
Preferably, step 5) is specially and is eluted to take off with the flow velocity of 0.5~2.5ml/min at room temperature through macroporous absorbent resin
Color, mobile phase are water, collect eluent；By the eluent of macroporous absorbent resin elution processing successively through cation exchange column and yin
Ion exchange column elutes desalination, and elution flow rate is 0.5~2.5ml/min, and mobile phase is water, obtains eluent.
Preferably, model D-101, D3520, DM301 or DM130 of the macroporous absorbent resin.
Preferably, model the MAbPac SCX-10 or HPD200B of the cation exchange column.
Preferably, the model D301 of the anion-exchange column.
A kind of method that xylo-oligosaccharide being made as raw material using stalk provided by the invention, stalk is by impregnating, soda acid is handled
The xylan the amount of dissolution in stalk can be improved；Cellulase, hemicellulase and zytase can gather the wood in treatment fluid
Sugar, cellulose, hemicellulose degradation improve the yield of xylo-oligosaccharide；Be 1000~1500Da by using molecular cut off,
600~800Da, 200~400Da nanofiltration membrane to enzymolysis liquid carry out separating and filtering, macroporous absorbent resin decoloration, ion exchange column
Desalination is eluted, the purity of xylo-oligosaccharide can be effectively improved, and product homogeneity is good, quality is stablized.Preparation process of the present invention is simple,
Reaction condition is mild, is convenient for industrialized production.
Specific embodiment
A kind of method that xylo-oligosaccharide is made as raw material using stalk provided by the invention, comprising the following steps:
1) after stalk being crushed be added water 3~4h of immersion treatment at 50~80 DEG C, add acid or alkali process 15~
20h obtains treatment fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6~7, it is poly- that cellulase, hemicellulase and wood is added
Carbohydrase, 12~15h of enzymolysis processing, obtains enzymolysis liquid at 35~50 DEG C；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) it will successively use molecular cut off for 1000~1500Da, 600 in the enzymolysis liquid after inactivation that step 3) obtains
The isolated filtered fluid of nanofiltration membrane of~800Da, 200~400Da；
5) filtered fluid that step 4) obtains is decolourized through macroporous absorbent resin, then elutes desalination through ion exchange column, obtained
Eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
In the present invention, water 3~4h of immersion treatment at 50~80 DEG C is added after stalk is crushed, adds acid or alkali process
15~20h obtains treatment fluid；In an embodiment of the present invention, stem shatter granularity is 1~2cm.In above-mentioned steps, after flour
Stalk impregnate 3~4h in 50~80 DEG C of water the bating effect of stalk can be improved, conducive to the dissolution of xylo-oligosaccharide, while
Conducive to its hydrolysis of subsequent acid-base pair.
It should be noted that acid is sulfuric acid or hydrochloric acid, alkali is sodium hydroxide or potassium hydroxide；It is sour or alkali dense in filtered fluid
Degree is 0.1~0.15mol/L.
The pH value of obtained filtered fluid is adjusted to 6~7, cellulase, hemicellulase and zytase is added, 35
12~15h of enzymolysis processing, obtains enzymolysis liquid at~50 DEG C；In above-mentioned steps, using cellulase, hemicellulase and xylan
Enzyme digests filtered fluid, and the yield and purity of xylo-oligosaccharide can be improved.
In an embodiment of the present invention, the additional amount of cellulase be added in every 100g stalk active unit be 50~
The cellulase of 70U, the additional amount of hemicellulase are that the hemicellulose that active unit is 60~80U is added in every 100g stalk
Enzyme, the additional amount of zytase are that the zytase that active unit is 80~100U is added in every 100g stalk.
Obtained enzymolysis liquid is put out a fire 15min at 100 DEG C；In order to avoid cellulase, hemicellulase and xylan
Enzyme influences subsequent step, needs to inactivate cellulase, hemicellulase and zytase, improves the product of xylo-oligosaccharide
Matter.
To successively be used in enzymolysis liquid after inactivation molecular cut off for 1000~1500Da, 600~800Da, 200~
The isolated filtered fluid of the nanofiltration membrane of 400Da；Above-mentioned steps can efficiently separate purifying oligo-xylose, improve the pure of xylo-oligosaccharide
Degree.
In an embodiment of the present invention, dilution is obtained after enzymolysis liquid being diluted 10~15 times, dilution is passed through retention point
The nanofiltration membrane that son amount is 1000~1500Da, isolated by-pass filtration liquid；Lead to after deionized water is added into by-pass filtration liquid
Enter the nanofiltration membrane that molecular cut off is 600~800Da, isolated secondary filtration liquid；Deionization is added into secondary filtration liquid
The nanofiltration membrane that molecular cut off is 200~300Da, isolated filtered fluid are passed through after water.It should be noted that in order to improve point
From effect, the purity of xylo-oligosaccharide is improved, before the nanofiltration UF membrane using different molecular weight cut off, the volume phase of dilution
Together, that is to say, that the deionized water of by-pass filtration liquid and the total volume of the deionized water of addition, secondary filtration liquid and addition it is total
Volume is equal to the volume of dilution.
Obtained filtered fluid is decolourized through macroporous absorbent resin, then elutes desalination through ion exchange column, obtains eluent；On
Stating step can be improved the quality of xylo-oligosaccharide.In an embodiment of the present invention, filtered fluid through macroporous absorbent resin at room temperature
Decoloration is eluted with the flow velocity of 0.5~2.5ml/min, mobile phase is water, collects eluent；By macroporous absorbent resin elution processing
Eluent successively elutes desalination through cation exchange column and anion-exchange column, and elution flow rate is 0.5~2.5ml/min, stream
Dynamic Xiang Weishui, obtains eluent.
In an embodiment of the present invention, model D-101, D3520, DM301 or DM130 of macroporous absorbent resin；Sun from
Model the MAbPac SCX-10 or HPD200B of sub- exchange column；The model D301 of anion-exchange column.
In above scheme, stalk is by impregnating, the xylan the amount of dissolution in stalk can be improved in soda acid processing；Cellulase,
Xylan, cellulose, hemicellulose degradation in treatment fluid can be improved xylo-oligosaccharide by hemicellulase and zytase
Yield；By using molecular cut off be 1000~1500Da, 600~800Da, 200~400Da nanofiltration membrane to enzymolysis liquid into
Row separating and filtering, macroporous absorbent resin decoloration, ion exchange column elute desalination, can effectively improve the purity of xylo-oligosaccharide, and produce
Product homogeneity is good, quality is stablized.Preparation process of the present invention is simple, and reaction condition is mild, is convenient for industrialized production.
In order to further illustrate the present invention, below with reference to embodiment to provided by the invention a kind of obtained by raw material of stalk
The method of xylo-oligosaccharide is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1) water immersion treatment 3h at 50 DEG C is added after crushing stalk, adds acid or alkali process 15h, is handled
Liquid；Acid is hydrochloric acid, and sour concentration is 0.1mol/L in filtered fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6, cellulase, hemicellulase and xylan is added
Enzyme, enzymolysis processing 15h, obtains enzymolysis liquid at 35 DEG C；The additional amount of cellulase is that active unit is added in every 100g stalk
For the cellulase of 50U, the additional amount of hemicellulase is that the hemicellulose that active unit is 60U is added in every 100g stalk
Enzyme, the additional amount of zytase are that the zytase that active unit is 80U is added in every 100g stalk；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) dilution is obtained after the enzymolysis liquid after inactivation that step 3) obtains being diluted 10~15 times, dilution is passed through retention
Molecular weight is the nanofiltration membrane of 1000Da, isolated by-pass filtration liquid；Be added deionized water into by-pass filtration liquid, addition go from
Volume after sub- water is identical as the volume of dilution, then is passed through the nanofiltration membrane that molecular cut off is 600Da, isolated second level mistake
Filtrate；Deionized water is added into secondary filtration liquid, the volume after deionized water is added is identical as the volume of dilution, then is passed through
Molecular cut off is the nanofiltration membrane of 200Da, isolated filtered fluid；
5) macroporous absorbent resin of the filtered fluid for obtaining step 4) through model D-101 is at room temperature with 0.5ml/min
Flow velocity elute decoloration, mobile phase is water, collect eluent；By the eluent of macroporous absorbent resin elution processing successively through model
Desalination is eluted for the cation exchange column and model D301 anion-exchange column of MAbPac SCX-10, elution flow rate is
0.5ml/min, mobile phase are water, obtain eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
Embodiment 2
1) water immersion treatment 4h at 80 DEG C is added after crushing stalk, adds acid or alkali process 20h, is handled
Liquid；Alkali is potassium hydroxide；The concentration of alkali is 0.15mol/L in filtered fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 7, cellulase, hemicellulase and xylan is added
Enzyme, enzymolysis processing 15h, obtains enzymolysis liquid at 50 DEG C；The additional amount of cellulase is that active unit is added in every 100g stalk
For the cellulase of 70U, the additional amount of hemicellulase is that the hemicellulose that active unit is 80U is added in every 100g stalk
Enzyme, the additional amount of zytase are that the zytase that active unit is 100U is added in every 100g stalk；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) dilution is obtained after the enzymolysis liquid after inactivation that step 3) obtains being diluted 15 times, dilution is passed through retention molecule
Amount is the nanofiltration membrane of 1500Da, isolated by-pass filtration liquid；Deionized water is added into by-pass filtration liquid, deionized water is added
Volume afterwards is identical as the volume of dilution, then is passed through the nanofiltration membrane that molecular cut off is 800Da, isolated secondary filtration
Liquid；Deionized water is added into secondary filtration liquid, the volume after deionized water is added is identical as the volume of dilution, then is passed through and cuts
Staying molecular weight is the nanofiltration membrane of 300Da, isolated filtered fluid；
5) macroporous absorbent resin of the filtered fluid for obtaining step 4) through model DM301 is at room temperature with 2.5ml/min
Flow velocity elute decoloration, mobile phase is water, collect eluent；By the eluent of macroporous absorbent resin elution processing successively through model
Desalination is eluted for the cation exchange column of HPD200B and the anion-exchange column of model D301, elution flow rate is 2.5ml/
Min, mobile phase are water, obtain eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
Embodiment 3
1) water immersion treatment 3.5h at 60 DEG C is added after crushing stalk, adds acid or alkali process 18h, is handled
Liquid；Alkali is sodium hydroxide；The concentration of alkali is 0.12mol/L in filtered fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6, cellulase, hemicellulase and xylan is added
Enzyme, enzymolysis processing 14h, obtains enzymolysis liquid at 40 DEG C；The additional amount of cellulase is that active unit is added in every 100g stalk
For the cellulase of 60U, the additional amount of hemicellulase is that the hemicellulose that active unit is 70U is added in every 100g stalk
Enzyme, the additional amount of zytase are that the zytase that active unit is 90U is added in every 100g stalk；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) dilution is obtained after the enzymolysis liquid after inactivation that step 3) obtains being diluted 12 times, dilution is passed through retention molecule
Amount is the nanofiltration membrane of 1200Da, isolated by-pass filtration liquid；Deionized water is added into by-pass filtration liquid, deionized water is added
Volume afterwards is identical as the volume of dilution, then is passed through the nanofiltration membrane that molecular cut off is 700Da, isolated secondary filtration
Liquid；Deionized water is added into secondary filtration liquid, the volume after deionized water is added is identical as the volume of dilution, then is passed through and cuts
Staying molecular weight is the nanofiltration membrane of 200Da, isolated filtered fluid；
5) macroporous absorbent resin of the filtered fluid for obtaining step 4) through model DM130 is at room temperature with 1.5ml/min
Flow velocity elute decoloration, mobile phase is water, collect eluent；By the eluent of macroporous absorbent resin elution processing successively through model
Desalination is eluted for the cation exchange column of HPD200B and the anion-exchange column of model D301, elution flow rate is 1.5ml/
Min, mobile phase are water, obtain eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
Comparative example 1
1) water immersion treatment 3.5h at 60 DEG C is added after crushing stalk, adds acid or alkali process 18h, is handled
Liquid；Alkali is sodium hydroxide；The concentration of alkali is 0.12mol/L in filtered fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6, cellulase, hemicellulase and xylan is added
Enzyme, enzymolysis processing 14h, obtains enzymolysis liquid at 40 DEG C；The additional amount of cellulase is that active unit is added in every 100g stalk
For the cellulase of 60U, the additional amount of hemicellulase is that the hemicellulose that active unit is 70U is added in every 100g stalk
Enzyme, the additional amount of zytase are that the zytase that active unit is 90U is added in every 100g stalk；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) dilution is obtained after the enzymolysis liquid after inactivation that step 3) obtains being diluted 12 times, dilution is successively passed through retention
The nanofiltration membrane that nanofiltration membrane that nanofiltration membrane that molecular weight is 1200Da, molecular cut off are 700Da, molecular cut off are 200Da, point
From obtaining filtered fluid；
5) macroporous absorbent resin of the filtered fluid for obtaining step 4) through model DM130 is at room temperature with 1.5ml/min
Flow velocity elute decoloration, mobile phase is water, collect eluent；By the eluent of macroporous absorbent resin elution processing successively through model
Desalination is eluted for the cation exchange column of HPD200B and the anion-exchange column of model D301, elution flow rate is 1.5ml/
Min, mobile phase are water, obtain eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
Comparative example 2
1) water immersion treatment 3.5h at 60 DEG C is added after crushing stalk, adds acid or alkali process 18h, is handled
Liquid；Alkali is sodium hydroxide；The concentration of alkali is 0.12mol/L in filtered fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6, cellulase, hemicellulase and xylan is added
Enzyme, enzymolysis processing 14h, obtains enzymolysis liquid at 40 DEG C；The additional amount of cellulase is that active unit is added in every 100g stalk
For the cellulase of 40U, the additional amount of hemicellulase is that the hemicellulose that active unit is 70U is added in every 100g stalk
Enzyme, the additional amount of zytase are that the zytase that active unit is 90U is added in every 100g stalk；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) dilution is obtained after the enzymolysis liquid after inactivation that step 3) obtains being diluted 12 times, dilution is passed through retention molecule
Amount is the nanofiltration membrane of 1200Da, isolated by-pass filtration liquid；Deionized water is added into by-pass filtration liquid, deionized water is added
Volume afterwards is identical as the volume of dilution, then is passed through the nanofiltration membrane that molecular cut off is 700Da, isolated secondary filtration
Liquid；Deionized water is added into secondary filtration liquid, the volume after deionized water is added is identical as the volume of dilution, then is passed through and cuts
Staying molecular weight is the nanofiltration membrane of 200Da, isolated filtered fluid；
5) macroporous absorbent resin of the filtered fluid for obtaining step 4) through model DM130 is at room temperature with 1.5ml/min
Flow velocity elute decoloration, mobile phase is water, collect eluent；By the eluent of macroporous absorbent resin elution processing successively through model
Desalination is eluted for the cation exchange column of HPD200B and the anion-exchange column of model D301, elution flow rate is 1.5ml/
Min, mobile phase are water, obtain eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
Comparative example 3
1) water immersion treatment 3.5h at 60 DEG C is added after crushing stalk, adds acid or alkali process 18h, is handled
Liquid；Alkali is sodium hydroxide；The concentration of alkali is 0.12mol/L in filtered fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6, cellulase, hemicellulase and xylan is added
Enzyme, enzymolysis processing 14h, obtains enzymolysis liquid at 40 DEG C；The additional amount of cellulase is that active unit is added in every 100g stalk
For the cellulase of 60U, the additional amount of hemicellulase is that the hemicellulose that active unit is 50U is added in every 100g stalk
Enzyme, the additional amount of zytase are that the zytase that active unit is 90U is added in every 100g stalk；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) dilution is obtained after the enzymolysis liquid after inactivation that step 3) obtains being diluted 12 times, dilution is passed through retention molecule
Amount is the nanofiltration membrane of 1200Da, isolated by-pass filtration liquid；Deionized water is added into by-pass filtration liquid, deionized water is added
Volume afterwards is identical as the volume of dilution, then is passed through the nanofiltration membrane that molecular cut off is 700Da, isolated secondary filtration
Liquid；Deionized water is added into secondary filtration liquid, the volume after deionized water is added is identical as the volume of dilution, then is passed through and cuts
Staying molecular weight is the nanofiltration membrane of 200Da, isolated filtered fluid；
5) macroporous absorbent resin of the filtered fluid for obtaining step 4) through model DM130 is at room temperature with 1.5ml/min
Flow velocity elute decoloration, mobile phase is water, collect eluent；By the eluent of macroporous absorbent resin elution processing successively through model
Desalination is eluted for the cation exchange column of HPD200B and the anion-exchange column of model D301, elution flow rate is 1.5ml/
Min, mobile phase are water, obtain eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
Comparative example 4
1) water immersion treatment 3.5h at 60 DEG C is added after crushing stalk, adds acid or alkali process 18h, is handled
Liquid；Alkali is sodium hydroxide；The concentration of alkali is 0.12mol/L in filtered fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6, cellulase, hemicellulase and xylan is added
Enzyme, enzymolysis processing 14h, obtains enzymolysis liquid at 40 DEG C；The additional amount of cellulase is that active unit is added in every 100g stalk
For the cellulase of 60U, the additional amount of hemicellulase is that the hemicellulose that active unit is 70U is added in every 100g stalk
Enzyme, the additional amount of zytase are that the zytase that active unit is 60U is added in every 100g stalk；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) dilution is obtained after the enzymolysis liquid after inactivation that step 3) obtains being diluted 12 times, dilution is passed through retention molecule
Amount is the nanofiltration membrane of 1200Da, isolated by-pass filtration liquid；Deionized water is added into by-pass filtration liquid, deionized water is added
Volume afterwards is identical as the volume of dilution, then is passed through the nanofiltration membrane that molecular cut off is 700Da, isolated secondary filtration
Liquid；Deionized water is added into secondary filtration liquid, the volume after deionized water is added is identical as the volume of dilution, then is passed through and cuts
Staying molecular weight is the nanofiltration membrane of 200Da, isolated filtered fluid；
5) macroporous absorbent resin of the filtered fluid for obtaining step 4) through model DM130 is at room temperature with 1.5ml/min
Flow velocity elute decoloration, mobile phase is water, collect eluent；By the eluent of macroporous absorbent resin elution processing successively through model
Desalination is eluted for the cation exchange column of HPD200B and the anion-exchange column of model D301, elution flow rate is 1.5ml/
Min, mobile phase are water, obtain eluent；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
The yield and purity for the xylo-oligosaccharide that measurement Examples 1 to 3 and comparative example 1~4 obtain, the results are shown in Table 1.
The experimental result of table 1 Examples 1 to 3 and comparative example 1~4
As it can be seen from table 1 Examples 1 to 3, compared to comparative example 1~4, preparation method provided by the invention can mention simultaneously
The yield and purity of high xylo-oligosaccharide；And according to embodiment 3 compared with comparative example 1, in the nanofiltration using different molecular weight cut off
First is diluted before film filtering, can significant xylo-oligosaccharide yield；Embodiment 3 is compared with comparative example 2, the addition of cellulase
It measures low, the yield of xylo-oligosaccharide can be significantly reduced；For embodiment 3 compared with comparative example 3, the additional amount of hemicellulase is low, can show
Write the yield for reducing xylo-oligosaccharide；For embodiment 3 compared with comparative example 4, the additional amount of zytase is low, can be to the receipts of xylo-oligosaccharide
Rate has an impact.
Above description can be realized professional and technical personnel in the field or using this to stating in the disclosed embodiments
It is bright, it enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments are to this field
It will be apparent for professional technician, the general principles defined herein can not depart from spirit of the invention
Or in the case where range, realize in other embodiments.Therefore, the present invention is not intended to be limited to these implementations shown in this article
Example, and it is to fit to the widest scope consistent with principles disclosed herein and novel features.
Claims (8)
1. a kind of method that xylo-oligosaccharide is made as raw material using stalk, which comprises the following steps:
1) water 3~4h of immersion treatment at 50~80 DEG C is added after crushing stalk, adds acid or 15~20h of alkali process, obtains
To treatment fluid；
2) pH value for the filtered fluid that step 1) obtains is adjusted to 6~7, cellulase, hemicellulase and zytase is added,
12~15h of enzymolysis processing, obtains enzymolysis liquid at 35~50 DEG C；
3) enzymolysis liquid that step 2) obtains is put out a fire 15min at 100 DEG C；
4) will successively be used in the enzymolysis liquid after inactivation that step 3) obtains molecular cut off for 1000~1500Da, 600~
The isolated filtered fluid of nanofiltration membrane of 800Da, 200~400Da；
5) filtered fluid that step 4) obtains is decolourized through macroporous absorbent resin, then elutes desalination through ion exchange column, eluted
Liquid；
6) eluent for obtaining step 5) is concentrated, is dry to get xylo-oligosaccharide.
2. the method that xylo-oligosaccharide is made as raw material using stalk as described in claim 1, which is characterized in that in step 1), acid
For sulfuric acid or hydrochloric acid, alkali is sodium hydroxide or potassium hydroxide；The concentration of acid or alkali is 0.1~0.15mol/L in filtered fluid.
3. the method that xylo-oligosaccharide is made as raw material using stalk as described in claim 1, which is characterized in that cellulase adds
Entering amount is that the cellulase that active unit is 50~70U is added in every 100g stalk, and the additional amount of hemicellulase is every 100g
The hemicellulase that active unit is 60~80U is added in stalk, the additional amount of zytase is to be added to live in every 100g stalk
Property unit be 80~100U zytase.
4. the method that xylo-oligosaccharide is made as raw material using stalk as described in claim 1, which is characterized in that step 4) is specific
To obtain dilution after enzymolysis liquid is diluted 10~15 times, dilution is passed through the nanofiltration that molecular cut off is 1000~1500Da
Film, isolated by-pass filtration liquid；
Into by-pass filtration liquid be added deionized water after be passed through molecular cut off be 600~800Da nanofiltration membrane, isolated two
Grade filtered fluid；
The nanofiltration membrane that molecular cut off is 200~300Da, isolated mistake are passed through after deionized water is added into secondary filtration liquid
Filtrate.
5. the method that xylo-oligosaccharide is made as raw material using stalk as described in claim 1, which is characterized in that step 5) is specially
Decoloration is eluted with the flow velocity of 0.5~2.5ml/min at room temperature through macroporous absorbent resin, mobile phase is water, collects eluent；It will
The eluent of macroporous absorbent resin elution processing successively elutes desalination, elution flow rate through cation exchange column and anion-exchange column
It is 0.5~2.5ml/min, mobile phase is water, obtains eluent.
6. the method that xylo-oligosaccharide is made as raw material using stalk as claimed in claim 5, which is characterized in that macroporous absorbent resin
Model D-101, D3520, DM301 or DM130.
7. the method that xylo-oligosaccharide is made as raw material using stalk as claimed in claim 5, which is characterized in that cation exchange column
Model MAbPac SCX-10 or HPD200B.
8. the method that xylo-oligosaccharide is made as raw material using stalk as claimed in claim 5, which is characterized in that anion-exchange column
Model D301.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608352A (en) * | 2020-11-30 | 2021-04-06 | 济南茂腾生物科技有限公司 | Method for preparing high-purity xylo-oligosaccharide |
| WO2021082440A1 (en) * | 2019-10-28 | 2021-05-06 | 南京林业大学 | Method for producing xylooligosaccharide under catalysis of xylonic acid |
-
2019
- 2019-06-19 CN CN201910533763.5A patent/CN110305921A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021082440A1 (en) * | 2019-10-28 | 2021-05-06 | 南京林业大学 | Method for producing xylooligosaccharide under catalysis of xylonic acid |
| CN112608352A (en) * | 2020-11-30 | 2021-04-06 | 济南茂腾生物科技有限公司 | Method for preparing high-purity xylo-oligosaccharide |
| CN112608352B (en) * | 2020-11-30 | 2022-05-03 | 济南茂腾生物科技有限公司 | Method for preparing high-purity xylo-oligosaccharide |
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