CN111575328A - Method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis - Google Patents
Method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis Download PDFInfo
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Abstract
The invention provides a method for preparing xylo-oligosaccharide by coupling high-temperature organic acid pretreatment and an enzymatic method; the method for preparing the xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis comprises the steps of mixing a xylan raw material with acid, heating and stirring at high temperature, cooling and adjusting the pH value; adding endo-carbohydrase, mixing, heating at medium temperature, stirring, and immediately inactivating enzyme after reaction; separating to obtain xylo-oligosaccharide solution; the high-temperature heating and stirring are carried out, wherein the stirring speed is 50-120 r/min, the temperature is 130-180 ℃, and the time is 5-30 min. The invention adopts a two-step method of coupling acid hydrolysis and enzyme hydrolysis to prepare the xylo-oligosaccharide, can directly produce the xylo-oligosaccharide by taking the wood fiber rich in xylan as the raw material, and simplifies the production process; and the yield of xylo-oligosaccharide is high and the byproduct xylose is less.
Description
Technical Field
The invention belongs to the technical field of preparation of xylo-oligosaccharide, and particularly relates to a method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis.
Background
The xylo-oligosaccharide is used as a functional food or feed additive, has the characteristic of being incapable of being absorbed by a digestive system (low calorie), has high selective proliferation effect on bifidobacteria in intestinal tracts, and can indirectly activate various immune cell activities; driven by the high-speed development of the industries such as ecological big health, food safety, nonreactive animal breeding and the like, the xylo-oligosaccharide product derived from the agriculture and forestry biomass resources as a 'strongest bifidus factor/superstrong prebiotic' can remarkably promote the development of the recycling, high-valued and high-end processing and utilization industrial system of the agriculture and forestry wastes including the straws, and further promote the implementation of the 'countryside happiness' strategy in China. The current xylo-oligosaccharide production mainly adopts an endo-xylanase preparation to catalyze and hydrolyze alkali-extracted xylan, so that the purity of xylo-oligosaccharide is high, but the alkali extraction treatment process involved in the method is complex, and the wastewater pollution is serious; in addition, the acid hydrolysis method can be adopted to prepare the xylo-oligosaccharide, but mainly faces the problems of the byproduct xylose and low product quality.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, as one aspect of the present invention, the present invention overcomes the disadvantages in the prior art, and provides a method for preparing xylooligosaccharide by coupling high temperature organic acid pretreatment and enzymatic method.
In order to solve the technical problems, the invention provides the following technical scheme: a method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis comprises mixing xylan raw material and acid, heating at high temperature under stirring, cooling, and adjusting pH; adding endo-carbohydrase, mixing, heating at medium temperature, stirring, and immediately inactivating enzyme after reaction; and separating to obtain xylo-oligosaccharide solution.
As a preferred embodiment of the method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis, the method comprises the following steps: the high-temperature heating and stirring are carried out, wherein the stirring speed is 50-120 r/min, the temperature is 130-180 ℃, and the time is 5-30 min.
As a preferred embodiment of the method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis, the method comprises the following steps: the medium-temperature heating and stirring are carried out, the stirring speed is 50-120 r/min, the temperature is 50-70 ℃, and the time is 3-12 hours.
As a preferred embodiment of the method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis, the method comprises the following steps: and the enzyme deactivation is to mix the materials after heating and enzymolysis to 90-100 ℃, and the enzyme deactivation time is 10-30 min.
As a preferred embodiment of the method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis, the method comprises the following steps: the acid comprises one or more of sulfuric acid, oxalic acid, maleic acid, furoic acid, xylonic acid and gluconic acid-acetic acid; the xylan raw material is a wood fiber raw material containing xylan components, and is preferably one or more of corncobs, bagasse and birch.
As a preferred embodiment of the method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis, the method comprises the following steps: according to the mass parts, the xylan raw material accounts for 1 part, and the dilute acid solution accounts for 3.5-10 parts; the mass fraction of acid concentration in the dilute acid solution is 0.1-5.0%, the endo-type carbohydrase comprises endo-xylanase, and the amount of the endo-type carbohydrase is 0.0005-0.005 part by mass relative to 1 part of xylan raw material.
As a preferred embodiment of the method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis, the method comprises the following steps: and cooling to room temperature, adjusting the pH to 4.0-6.0, and using one or more pH regulators selected from calcium carbonate, calcium hydroxide and/or sodium hydroxide.
As a preferred embodiment of the method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis, the method comprises the following steps: the heating and stirring reactions are all completed in the same heating reaction hydrolysis tank; the separation method is microfiltration or extrusion filtration.
As another aspect of the present invention, the present invention provides a xylo-oligosaccharide: the xylo-oligosaccharide comprises one or more of xylobiose (X2) and xylotriose (X3); wherein the yield of the xylobiose (X2) and the yield of the xylotriose (X3) are respectively 20-50% and 10-30%.
As a preferred embodiment of the present invention, there is provided a process for preparing xylooligosaccharide, wherein: the yields of the xylobiose (X2) and the xylotriose (X3) are respectively 30-50% and 15-30%.
The invention has the beneficial effects that:
the invention adopts a two-step method of coupling acid hydrolysis and enzyme hydrolysis to prepare the xylo-oligosaccharide, can directly produce the xylo-oligosaccharide by taking the wood fiber rich in xylan as the raw material, and simplifies the production process; and the yield of xylo-oligosaccharide is high and the byproduct xylose is less.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a flow chart of the production process in example 1.
FIG. 2 is a high performance anion exchange chromatography chromatogram of the feed solution obtained in the two hydrolysis steps of example 1.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
adding 50g of corncob powder and 500mL of 2.5% (mass fraction) xylonic acid solution into a 1L mechanical stirring type stainless steel high-pressure reaction tank, sealing, stirring (60r/min), heating to 170 ℃, and keeping the temperature for 15 min; after the reaction is finished, adding sodium hydroxide to adjust the pH value to 5.0 after the temperature of the reaction body tank is reduced to 60 ℃; after the pH is adjusted, 0.05g of endo-xylanase is added, the mixture is sealed and stirred (60r/min), and the temperature of 60 ℃ is maintained for reaction for 6 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature of 30 ℃, sample injection volume of 20 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase and flow rate of 0.3mL/min, analysis spectra thereof are shown in figure 2, Xylose (Xylose), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6), xyloheptaose (X7), xylooctaose (X8), xylononaose (X9) and xylooligosaccharide components with polymerization degree of more than or equal to 10(X10) are simultaneously detected, finally, the main xylooligosaccharide component of enzymatic hydrolysis is xylooligosaccharide, the yield is from xylobiose to xylotriose40.1 percent and 21.2 percent respectively, and the total yield of xylo-oligosaccharide is 61.3 percent; in addition, the xylose yield is 8.2%.
In addition, the specific operation of the binary gradient elution with 100mmol/L sodium hydroxide (NaOH) and 500mmol/L sodium acetate (NaAc) as mobile phases is shown in the following table:
| Time(min) | 100mmol/L NaOH(％) | 500mmol/L NaAc(％) |
| 0 | 100 | 0 |
| 9 | 100 | 0 |
| 26 | 92 | 8 |
| 26 | 50 | 50 |
| 40 | 50 | 50 |
| 40 | 100 | 0 |
| 50 | 100 | 0 |
example 2:
adding 50g of corncob powder and 500mL of 5.0% (mass fraction) gluconic acid solution into a 1L mechanical stirring type stainless steel high-pressure reaction tank, sealing, stirring (60r/min), heating to 170 ℃, and keeping the temperature for 30 min; and after the reaction is finished, cooling the reaction body tank to room temperature, putting the solid-liquid mixture after the reaction into a vacuum pulp washer, and separating unhydrolyzed solid matters and xylan acid hydrolysis liquid by extrusion and filtration. The obtained xylan hydrolysate sample is subjected to high-efficiency anion exchange chromatography to analyze sugar components, and the chromatographic conditions are as follows: american Saimer fly ICS5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature 30 ℃, sample injection volume 10 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase, flow rate 0.3mL/min, main components of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose, yield of 15.4%, 13.2%, 10.2%, 7.6%, 5.4% and 3.3% respectively, total xylo-oligosaccharide yield of 55.1%, and xylose yield of 31.7%.
Example 3:
adding 50g of corncob powder and 0.25g of endo-xylanase into a 1L mechanical stirring type stainless steel high-pressure reaction tank, sealing, starting stirring (60r/min), heating to 60 ℃, and carrying out heat preservation reaction for 24 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and after the reaction is finished, cooling the reaction body tank to room temperature, putting the solid-liquid mixture after the reaction into a vacuum pulp washer, and separating unhydrolyzed solid matters and xylanase hydrolysate by extrusion and filtration. The obtained xylan hydrolysate sample is subjected to high-efficiency anion exchange chromatography to analyze sugar components, and the chromatographic conditions are as follows: american Saimer fly ICS5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatography column, PAD integrated Ampere detector, column temperature 30 deg.C, sample volume 10 μ L, 100 mmol/ml-And carrying out binary gradient leaching on the L sodium hydroxide and 500mmol/L sodium acetate serving as mobile phases at the flow rate of 0.3 mL/min. The main components are xylobiose and xylotriose, the yield is 11.1 percent and 6.8 percent respectively, and the yield of the total xylo-oligosaccharide is 17.9 percent; in addition, the xylose yield was 4.6%.
Example 4:
adding 50g of bagasse powder and 500mL of 2.0% (mass fraction) of maleic acid solution into a 1L mechanically-stirred stainless steel high-pressure reaction tank, sealing, stirring (60r/min), heating to 170 ℃, and keeping the temperature for 10 min; after the reaction is finished, adding calcium carbonate to adjust the pH value to 5.0 after the temperature of the reaction body tank is reduced to 60 ℃; after the pH is adjusted, 0.05g of endo-xylanase is added, the mixture is sealed and stirred (60r/min), and the temperature of 60 ℃ is maintained for reaction for 6 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature 30 ℃, sample injection volume 20 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase, flow rate 0.3mL/min, final enzymatic hydrolysis xylo-oligosaccharide mainly comprises xylobiose to xylotriose, yield is 39.3% and 18.6% respectively, total xylo-oligosaccharide yield is 57.9%, and xylose yield is 9.6%.
Example 5:
adding 50g of bagasse powder and 500mL of 5.0% (mass fraction) gluconic acid solution into a 1L mechanically-stirred stainless steel high-pressure reaction tank, sealing, stirring (60r/min), heating to 170 ℃, and keeping the temperature for 30 min; after the reaction is finished, adding calcium carbonate to adjust the pH value to 5.0 after the temperature of the reaction body tank is reduced to 60 ℃; after the pH is adjusted, 0.05g of endo-xylanase is added, the mixture is sealed and stirred (60r/min), and the temperature of 60 ℃ is maintained for reaction for 6 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimerfii ICS-5000 type ion chromatography, configured with CarboPacTM PA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature 30 ℃, sample injection volume 20 μ L; and (3) carrying out binary gradient elution by using 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phases at the flow rate of 0.3 mL/min. Finally, the enzymatic hydrolysis xylo-oligosaccharide mainly comprises xylobiose to xylotriose, the yield is respectively 28.2 percent and 13.6 percent, and the total xylo-oligosaccharide yield is 41.8 percent; in addition, the xylose yield was 39.6%.
Example 6:
in order to be more beneficial to industrial production, 50g of bagasse powder and 500mL of 5.0% (mass fraction) gluconic acid solution are added into a 1L mechanical stirring type stainless steel high-pressure reaction tank, the stirring is started (60r/min) after sealing, and the temperature is heated to 160 ℃ and kept for 25 min; after the reaction is finished, adding calcium carbonate to adjust the pH value to 5.0 after the temperature of the reaction body tank is reduced to 60 ℃; after the pH is adjusted, 0.05g of endo-xylanase is added, the mixture is sealed and stirred (60r/min), and the temperature of 60 ℃ is maintained for reaction for 6 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature 30 ℃, sample injection volume 20 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase, flow rate 0.3mL/min, final enzymatic hydrolysis xylo-oligosaccharide mainly comprises xylobiose to xylotriose, yield is 39.1% and 21.1%, total xylo-oligosaccharide yield is 60.2%, and xylose yield is 12.6%.
Example 7:
adding 50g of birch wood powder and 500mL of 5.0% (mass fraction) gluconic acid solution into a 1L mechanically-stirred stainless steel high-pressure reaction tank, sealing, stirring (60r/min), heating to 170 ℃, and keeping the temperature for 10 min; after the reaction is finished, adding strong sodium oxide to adjust the pH to 5.0 after the temperature of a reaction body tank is reduced to 60 ℃; adding 0.2g of endo-xylanase after pH adjustment is finished, sealing, stirring at 60r/min, and maintaining at 60 deg.CKeeping the temperature and reacting for 24 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature of 30 ℃, sample injection volume of 20 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase, flow rate of 0.3mL/min, final enzymatic hydrolysis of xylo-oligosaccharide mainly comprising xylobiose to xylotriose, yield of 30.1% and 12.6% respectively, total xylo-oligosaccharide yield of 42.7%, and xylose yield of 25.1%.
Example 8:
adding 50g of corncob powder and 500mL of 4.0 percent (mass fraction) acetic acid solution into a 1L mechanical stirring type stainless steel high-pressure reaction tank, sealing, starting stirring (60r/min), heating to 170 ℃, and keeping the temperature for 12 min; after the reaction is finished, adding strong sodium oxide to adjust the pH to 4.5 after the temperature of a reaction body tank is reduced to 60 ℃; after the pH is adjusted, 0.05g of endo-xylanase is added, the mixture is sealed and stirred (80r/min), and the temperature is maintained at 60 ℃ for 24 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature 30 ℃, sample injection volume 20 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase, flow rate 0.3mL/min, final enzymatic hydrolysis xylo-oligosaccharide mainly comprising xylobiose to xylotriose, yield of 38.1% and 17.0% respectively, total xylo-oligosaccharide yield of 55.1%, and xylose yield of 12.1%.
Example 9:
adding 50g of corncob powder and 500mL of 5.0 percent (mass fraction) gluconic acid solution into a 1L mechanical stirring type stainless steel high-pressure reaction tank, sealing, and then starting stirringStirring (60r/min), heating to 200 deg.C, and maintaining the temperature for 10 min; after the reaction is finished, adding strong sodium oxide to adjust the pH to 4.5 after the temperature of a reaction body tank is reduced to 60 ℃; after the pH is adjusted, 0.05g of endo-xylanase is added, the mixture is sealed and stirred (60r/min), and the temperature of 60 ℃ is maintained for reaction for 24 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature 30 ℃, sample injection volume 20 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase, flow rate 0.3mL/min, final enzymatic hydrolysis xylo-oligosaccharide mainly comprises xylobiose to xylotriose, yield is 20.5% and 12.3%, total xylo-oligosaccharide yield is 32.8%, and xylose yield is 43.0%.
Example 10:
adding 50g of corncob powder and 500mL of 5.0% (mass fraction) gluconic acid solution into a 1L mechanical stirring type stainless steel high-pressure reaction tank, sealing, starting stirring (60r/min), heating to 120 ℃, and keeping the temperature for 30 min; after the reaction is finished, adding strong sodium oxide to adjust the pH to 4.5 after the temperature of a reaction body tank is reduced to 60 ℃; after the pH is adjusted, 0.05g of endo-xylanase is added, the mixture is sealed and stirred (60r/min), and the temperature of 60 ℃ is maintained for reaction for 24 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature of 30 ℃, sample injection volume of 20 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase, flow rate of 0.3mL/min, final enzymatic hydrolysis of xylo-oligosaccharide mainly comprising xylobiose to xylotriose, yield of 20.1% and 9.3%, total xylo-oligosaccharide yield of 29.4%, and xylose yield of 6.3%。
Example 11:
adding 50g of corncob powder and 500mL of 5.0% (mass fraction) gluconic acid solution into a 1L mechanical stirring type stainless steel high-pressure reaction tank, sealing, starting stirring (60r/min), and heating to 170 ℃ for heat preservation for 15 min; after the reaction is finished, adding strong sodium oxide to adjust the pH to 4.5 after the temperature of a reaction body tank is reduced to 30 ℃; after the pH is adjusted, 0.05g of endo-xylanase is added, the mixture is sealed and stirred (60r/min), and the temperature is maintained at 30 ℃ for 24 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature 30 ℃, sample injection volume 20 mu L, binary gradient elution with 100mmol/L sodium hydroxide and 500mmol/L sodium acetate as mobile phase, flow rate 0.3mL/min, final enzymatic hydrolysis xylo-oligosaccharide mainly comprises xylobiose to xylotriose, yield is 23.6% and 14.1%, total xylo-oligosaccharide yield is 37.7%, and xylose yield is 9.3%.
Example 12:
adding 50g of corncob powder and 500mL of 2.5% (mass fraction) xylonic acid solution into a 1L mechanical stirring type stainless steel high-pressure reaction tank, sealing, stirring (60rpm), heating to 170 ℃, and keeping the temperature for 15 min; after the reaction is finished, adding sodium hydroxide to adjust the pH value to 5.0 after the reaction body tank is cooled to room temperature; after the pH adjustment is finished, 0.05g of endo-xylanase is added, the mixture is sealed and stirred at 60rpm, and the mixture is heated to 60 ℃ for heat preservation reaction for 6 hours; adjusting the temperature to 95 ℃ after the enzymolysis reaction is finished, and inactivating the enzyme for 15 min; and separating solid matters and xylan hydrolysate by extrusion and filtration to obtain crude xylo-oligosaccharide liquid. Analyzing sugar components of acid hydrolysis and enzyme hydrolysis samples by adopting high-efficiency anion exchange chromatography, wherein the chromatographic conditions are as follows: american Saimer fly ICS-5000 type ion chromatography, configured CarboPacTMPA200(3mm × 250mm) chromatographic column, PAD integrated ampere detector, column temperature 30 deg.C, sample injection volume 20 μ L, 100mmol/L NaOH and 500mmol/L NaOHAnd (3) performing binary gradient elution by using sodium acetate as a mobile phase at a flow rate of 0.3 mL/min. The analysis map of the kit is shown in figure 2, and Xylose (Xylose), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6), xyloheptaose (X7), xylooctaose (X8), xylononaose (X9) and xylooligosaccharide with the polymerization degree of more than or equal to 10(X10) are simultaneously detected. Finally, the enzymatic hydrolysis xylo-oligosaccharide mainly comprises xylobiose to xylotriose, the yield is 42.1 percent and 26.2 percent respectively, and the total xylo-oligosaccharide yield is 68.3 percent; in addition, the xylose yield is 8.2%.
The invention utilizes a two-step method of high-temperature acid hydrolysis and enzyme hydrolysis to directly take wood fiber rich in xylan components as raw materials to produce crude xylo-oligosaccharide solution; the method has technical universality and can be used for various wood fiber raw materials (straws, corncobs, bagasse, birch and the like); in the invention, the concentration, time and temperature of the dilute acid are strictly controlled, so that excessive degradation of xylan into xylose is avoided while the xylan is dissolved out; the implementation scheme takes high temperature, low acid concentration and short time as the best; under the action of dilute acid, a large amount of xylan in the wood fiber raw material is dissolved out, so that conditions are provided for xylanase hydrolysis reaction. The method for producing the xylo-oligosaccharide by adopting the two-step hydrolysis method has simpler process and higher yield of the xylo-oligosaccharide, and is very beneficial to industrial production.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis is characterized in that: the method comprises the following steps of,
mixing xylan raw materials with acid, heating and stirring at high temperature, cooling and adjusting pH;
adding endo-carbohydrase, mixing, heating at medium temperature, stirring, and immediately inactivating enzyme after reaction;
and separating to obtain xylo-oligosaccharide solution.
2. The method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis according to claim 1, wherein: the high-temperature heating and stirring are carried out, wherein the stirring speed is 50-120 r/min, the temperature is 130-180 ℃, and the time is 5-30 min.
3. The method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis according to claim 1, wherein: the medium-temperature heating and stirring are carried out, the stirring speed is 50-120 r/min, the temperature is 50-70 ℃, and the time is 3-12 hours.
4. The method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis according to any one of claims 1 to 3, wherein: and the enzyme deactivation is to mix the materials after heating and enzymolysis to 90-100 ℃, and the enzyme deactivation time is 10-30 min.
5. The method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis according to claim 4, wherein: the acid comprises one or more of sulfuric acid, oxalic acid, maleic acid, furoic acid, xylonic acid and gluconic acid-acetic acid; the xylan raw material is a wood fiber raw material containing xylan components, and is preferably one or more of corncobs, bagasse and birch.
6. The method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis according to claim 4, wherein: according to the mass parts, the xylan raw material accounts for 1 part, and the dilute acid solution accounts for 3.5-10 parts; the mass fraction of acid concentration in the dilute acid solution is 0.1-5.0%, the endo-type carbohydrase comprises endo-xylanase, and the amount of the endo-type carbohydrase is 0.0005-0.005 part by mass relative to 1 part of xylan raw material.
7. The method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis according to claim 4, wherein: and cooling to room temperature, adjusting the pH to 4.0-6.0, and using one or more pH regulators selected from calcium carbonate, calcium hydroxide and/or sodium hydroxide.
8. The method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis according to any one of claims 5 to 7, wherein the method comprises the following steps: the heating and stirring reactions are all completed in the same heating reaction hydrolysis tank; the separation method is microfiltration or extrusion filtration.
9. Xylo-oligosaccharides produced by the process according to any one of claims 1 to 8, characterized in that: the xylo-oligosaccharide comprises one or more of xylobiose (X2) and xylotriose (X3); wherein the yield of the xylobiose (X2) and the yield of the xylotriose (X3) are respectively 20-50% and 10-30%.
10. The method for preparing xylo-oligosaccharide by coupling acid hydrolysis and enzyme hydrolysis according to claim 9, wherein: the yields of the xylobiose (X2) and the xylotriose (X3) are respectively 30-50% and 15-30%.
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