阅读说明：本技术 一种乳酸联合木聚糖酶水解制备低聚木糖的方法 (Method for preparing xylooligosaccharide by hydrolyzing lactic acid and xylanase ) 是由 张军华 杨倩倩 徐勇 应文俊 文沛瑶 于 2021-01-22 设计创作，主要内容包括：本发明提供了一种乳酸联合木聚糖酶水解制备低聚木糖的方法,属于生物质降解和转化技术领域。本发明将农林废弃物与体积浓度为0-10％的乳酸溶液混合,进行催化反应,过滤,获得含有低聚木糖的处理液和滤渣,将滤渣与木聚糖酶溶液混合,进行酶解,获得含低聚木糖的酶解液。本发明采用稀酸结合酶水解法,可充分的将农林废弃物催化后的处理液和固体残渣中的木聚糖降解为低聚木糖,低聚木糖得率可达到70％。(The invention provides a method for preparing xylo-oligosaccharide by hydrolyzing lactic acid and xylanase, belonging to the technical field of biomass degradation and conversion. The method comprises the steps of mixing the agricultural and forestry wastes with a lactic acid solution with the volume concentration of 0-10%, carrying out catalytic reaction, filtering to obtain a treatment solution containing xylo-oligosaccharide and filter residues, mixing the filter residues with a xylanase solution, and carrying out enzymolysis to obtain an enzymatic hydrolysate containing the xylo-oligosaccharide. The invention adopts a dilute acid combined enzyme hydrolysis method, can fully degrade xylan in treatment liquid and solid residue after the agricultural and forestry waste is catalyzed into xylo-oligosaccharide, and the yield of the xylo-oligosaccharide can reach 70 percent.)

1. The preparation method of xylo-oligosaccharide is characterized by comprising the following steps: mixing agricultural and forestry wastes with a lactic acid solution with the volume concentration of 0-10%, carrying out catalytic reaction, and filtering to obtain a treatment solution containing xylo-oligosaccharide and filter residues; mixing the filter residue with xylanase solution, and performing enzymolysis to obtain enzymatic hydrolysate containing xylo-oligosaccharide.

2. The method according to claim 1, wherein the agricultural and forestry waste includes any one or more of corn stover, wheat straw, and corn cobs.

3. The preparation method according to claim 1, wherein the mass-to-volume ratio of the agricultural and forestry waste to the lactic acid solution is 1g:5-20 ml.

4. The method as claimed in claim 1, wherein the temperature of the catalytic reaction is 145-175 ℃, and the time of the catalytic reaction is 20-70 min.

5. The preparation method according to claim 1, wherein the dosage of the xylanase is 200-1000U/g filter residue.

6. The method according to claim 1, wherein the xylanase is an endoxylanase.

7. The preparation method according to claim 1 or 5, wherein the volume-to-mass ratio of the xylanase solution to the filter residue is 1-10ml:20-200 mg.

8. The method of claim 1, wherein the enzymatic hydrolysis has a pH of 4.0 to 6.0.

9. The preparation method of claim 8, wherein the pH regulator is one or more of sodium citrate, sodium hydroxide or citric acid-disodium hydrogen phosphate.

10. The preparation method of claim 1, wherein the temperature of the enzymolysis is 48-52 ℃, and the time of the enzymolysis is 6-48 h.

Technical Field

The invention belongs to the technical field of biomass degradation and conversion, and particularly relates to a method for preparing xylo-oligosaccharide by lactic acid and xylanase hydrolysis.

Background

The quantity of agricultural and forestry wastes produced in China every year is very large, for example, about 1000 million tons of corncobs are produced. Wherein various agricultural and forestry wastes contain a large amount of xylan-type hemicellulose, such as wheat straws, corn straws, corncobs and the like. Corncobs contain about 35% hemicellulose, 35% cellulose and 15% lignin, with the major component of hemicellulose being xylan, one of the best raw materials for producing xylo-oligosaccharides. At present, the preparation method of xylo-oligosaccharide commonly used comprises an enzymolysis method, an alkaline method, a self-hydrolysis method, an acid method and the like. For example, Yoon et al (LWT-Food Science and Technology,2006,39(4): 388-; huang et al (food and fermentation industry, 2004, (5)) extracted xylan from corncobs with sodium hydroxide and then carried out xylan enzymatic hydrolysis (800IU/g DM, 40 ℃, 6h) to obtain 50% xylo-oligosaccharides, the process is complex and causes much pollution, and corncobs are easy to generate heavy pigments with alkali, which brings great pressure to the following procedures of xylo-oligosaccharide production. Nabarlatz et al (Industrial & Engineering Chemistry Research,2004,43(15):4124-4131) performed 15min self-hydrolysis on corncob at 190 ℃ to obtain 58% xylooligosaccharide (degree of polymerization ≥ 2), but the hydrothermal pretreatment reaction temperature and pressure were high and the equipment requirements were strict. According to Zhang et al (Bioresource Technology,234(2017):343-349), 45.9% xylo-oligosaccharide (polymerization degree 2-6) can be obtained after 20% (w/v) acetic acid is used for treating corncobs at 140 ℃ for 20min, the reaction time is short, the yield of the xylo-oligosaccharide is high, but concentrated acid is easy to corrode equipment and the separation of products is difficult. Xiao et al (Bioresource Technology,290(2019):121775) pretreated corn stalks with 2% (w/v) lactic acid at 170 ℃ for 40min to obtain 12.9% of xylo-oligosaccharide (polymerization degree 2-4), but still a large amount of xylan can not be fully utilized and converted.

Therefore, the method for preparing xylo-oligosaccharide in the prior art generally has the problems of insufficient hydrolysis of raw material xylan, low selectivity of xylo-oligosaccharide and the like.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preparing xylo-oligosaccharide with high efficiency and high yield of xylo-oligosaccharide, which can efficiently convert xylan in agricultural and forestry waste into xylo-oligosaccharide, and the yield of the prepared xylo-oligosaccharide (xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose) reaches 70%.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a preparation method of xylo-oligosaccharide, which comprises the following steps: mixing agricultural and forestry wastes with a lactic acid solution with the volume concentration of 0-10%, carrying out catalytic reaction, and filtering to obtain a treatment solution containing xylo-oligosaccharide and filter residues; mixing the filter residue with xylanase solution, and performing enzymolysis to obtain enzymatic hydrolysate containing xylo-oligosaccharide.

Preferably, the agricultural and forestry waste comprises any one or more of corn stalks, wheat stalks and corncobs.

Preferably, the mass volume ratio of the agricultural and forestry waste to the lactic acid solution is 1g:5-20 ml.

Preferably, the temperature of the catalytic reaction is 145-175 ℃, and the time of the catalytic reaction is 20-70 min.

Preferably, the dosage of the xylanase is 200-1000U/g of filter residue.

Preferably, the xylanase is an endoxylanase.

Preferably, the volume-mass ratio of the xylanase solution to the filter residue is 1-10ml to 20-200 mg.

Preferably, the pH value of the enzymolysis is 4.0-6.0.

Preferably, the regulator for regulating the pH value is one or more of sodium citrate, sodium hydroxide or citric acid-disodium hydrogen phosphate.

Preferably, the enzymolysis temperature is 48-52 ℃, and the enzymolysis time is 6-48 h.

The invention has the beneficial effects that:

1. according to the invention, lactic acid is selected as a catalyst, weak acid property with relatively low lactic acid proton dissociation degree is fully utilized, hydrolysis of most xylan in the agricultural and forestry waste is controlled in a stage from xylobiose to xylohexaose through mild reaction conditions, a xylan substrate achieves controlled catalytic hydrolysis effect, the defect of a large number of inhibitors under high-temperature hydrolysis conditions is overcome, a large amount of xylo-oligosaccharide is contained in a treatment solution obtained after catalysis, solid residues obtained after catalysis are fully utilized, and then xylan enzymolysis is carried out to prepare xylo-oligosaccharide, so that the full utilization of xylan in the agricultural and forestry waste is realized.

2. The lactic acid used for preparing the xylo-oligosaccharide is not only a catalyst, but also a feed additive, so the lactic acid in the xylo-oligosaccharide can be directly used as the feed additive without separation.

3. The method greatly degrades xylan into xylo-oligosaccharide, and the final yield of xylo-oligosaccharide reaches 70 percent, which is the highest level reported so far.

4. The polymerization degree of the xylo-oligosaccharide prepared by the method is mainly 2-6, and the xylo-oligosaccharide can be used as a functional feed additive and can also be used as a functional food additive after post-treatment.

5. The xylo-oligosaccharide prepared by the method has high purity, and the cost of subsequent separation and purification is saved.

Drawings

FIG. 1 is a flow chart of the method for preparing xylooligosaccharide of the invention.

Detailed Description

The invention provides a preparation method of xylo-oligosaccharide, which comprises the following steps: mixing agricultural and forestry wastes with a lactic acid solution with the volume concentration of 0-10%, carrying out catalytic reaction, and filtering to obtain a treatment solution containing xylo-oligosaccharide and filter residues; mixing the filter residue with xylanase solution, and performing enzymolysis to obtain enzymatic hydrolysate containing xylo-oligosaccharide.

The agricultural and forestry waste is not particularly limited in type, agricultural and forestry waste containing xylan type hemicellulose, which is well known in the art, can be adopted, and the agricultural and forestry waste containing a large amount of xylan type hemicellulose, such as corn stalks, wheat stalks, corn cobs and the like, is preferred, and in certain embodiments of the invention, the corn cobs are selected. In the present invention, it is preferable that the agricultural and forestry waste is pulverized and then mixed with a lactic acid solution having a volume concentration of 0 to 10%. The crushing mode is not particularly limited, the crushing mode can be any one of the conventional crushing modes in the field, the crushing and sieving are preferred, and the sieving mesh number is preferably less than or equal to 60 meshes. The source of lactic acid in the present invention is not particularly limited, and any commercially available product that is conventional in the art may be used. The preparation method of the lactic acid solution is not particularly limited in the present invention, and a method of preparing a specific concentration by a person skilled in the art may be adopted. In the present invention, the purity of lactic acid used for preparing the lactic acid solution is preferably more than 85%. In the invention, when the volume concentration of the lactic acid solution is 0%, the technical scheme of the invention is to prepare xylo-oligosaccharide by hydrolysis of the self-hydrolysis synergistic xylanase, and when the volume concentration of the lactic acid solution is more than 0% and less than or equal to 10%, the technical scheme of the invention is to prepare xylo-oligosaccharide by hydrolysis of the lactic acid synergistic xylanase. In the present invention, the volume concentration of the lactic acid solution is preferably 1 to 7%, more preferably 2 to 5%.

In the invention, the mass-to-volume ratio of the agricultural and forestry waste to the lactic acid solution is preferably 1g:5-20ml, more preferably 1g:8-15ml, and most preferably 1g:10 ml. In the present invention, the temperature of the catalytic reaction is preferably 145-175 ℃, more preferably 155-165 ℃, and most preferably 160 ℃, and the time of the catalytic reaction is preferably 20-70min, more preferably 30-60min, and further preferably 40-50 min. In the present invention, the catalytic reaction is preferably carried out in a sealed reaction vessel.

The filtration mode of the invention is not particularly limited, and the conventional solid-liquid separation mode in the field can be adopted. After filtering, the treatment liquid containing xylo-oligosaccharide and filter residue are obtained. Preferably, the filter residue is washed to be neutral by water and then mixed with the xylanase solution. And the filter residue is washed to be neutral by water, so that the pH value in the enzymolysis liquid is stable, and the activity of the enzyme is maintained, wherein the water is preferably deionized water.

The source of the xylanase is not particularly limited in the invention, and any commercially available product which is conventional in the art can be used. In the present invention, the xylanase is preferably an endo-xylanase, more preferably a xylanase derived from aspergillus oryzae; the dosage of the xylanase is preferably 200-1000U/g filter residue, more preferably 400-800U/g filter residue, and most preferably 600U/g filter residue; the volume-mass ratio of the xylanase solution to the filter residue is preferably 1-10ml:20-200mg, more preferably 3-8ml:50-170mg, and most preferably 5-7ml:100-120 mg.

In the invention, after mixing filter residue and xylanase, preferably adjusting the pH value by using an adjusting agent and then carrying out enzymolysis, wherein the adjusting agent is preferably one or more of sodium citrate, sodium hydroxide or citric acid-disodium hydrogen phosphate, and more preferably sodium citrate; the pH is preferably 4.0 to 6.0, more preferably 4.8.

In the invention, the enzymolysis is preferably carried out in a constant-temperature sealing system, the enzymolysis temperature is preferably 48-52 ℃, more preferably 49-51 ℃, and most preferably 50 ℃, and the enzymolysis time is preferably 6-48h, more preferably 20-34 h. Most preferably 24 h.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Catalyzing by lactic acid: accurately weighing 1g of corncobs in absolute dry weight, adding a2 vol% lactic acid solution according to a material-liquid ratio of 1g to 10ml, heating at 160 ℃ for 40min, cooling, and performing solid-liquid separation to obtain a treatment solution containing xylo-oligosaccharide; washing the filter residue with deionized water to neutrality to obtain solid residue, and naturally air drying.

And (3) xylan enzymolysis: taking 0.06g of filter residue with absolute dry weight in a 10ml centrifuge tube, adding 36U of xylanase and sodium citrate buffer solution to adjust the pH value to 4.8, shaking uniformly, placing in a constant temperature shaking table for enzymolysis at 50 ℃ for 24h, boiling in a boiling water bath for 10min to inactivate the enzyme, cooling to room temperature, centrifuging, and obtaining supernatant fluid to obtain the enzymatic hydrolysate containing xylo-oligosaccharide.

Detecting xylooligosaccharide components in the treatment solution and the enzymolysis solution by adopting high performance liquid ion chromatography, wherein the chromatographic conditions are as follows: a Dionex ICS-3000 ion chromatography system in USA is provided with a CarboPacM PA200(3mm × 250mm) chromatographic column and a holding column (3 × 50mm), an ED electrochemical detector, a column temperature of 30 ℃, and a sample injection volume of 10 μ L; and (3) performing binary gradient elution by using 500mmol/L sodium acetate solution (A) and 100mmol/L sodium hydroxide solution (B) as eluents at a flow rate of 0.3mL/min (elution program: 0min, 100% A; 30min, 75% A, 25% B; 38-45 min, 100% A). The detection mode of the electrochemical detector is a gold working electrode and a pH-Ag/AgCl composite reference electrode, and an integral pulse ampere detection method and a chromatographic peak area integration method are adopted to determine a content spectrum system of the sugar component.

Respectively calculating the yield of the oligomerization xylose in the treatment fluid and the enzymolysis fluid according to the following formula:

the yield (%) of xylooligosaccharide in the treated solution was equal to the mass (g) of xylooligosaccharide produced in the treated solution/the mass (g) of xylan in untreated corn cob x 100%

The yield (%) of xylooligosaccharide in the enzymolysis liquid is equal to the mass (g) of xylooligosaccharide in the enzymolysis liquid/the mass (g) multiplied by 100 percent of xylan in the untreated corncobs

The xylan content in the substrate in the formula was determined according to the american national renewable energy laboratory method.

The results are as follows:

the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the treatment solution were 18.5%, 13.5%, 13.4%, 8.5% and 6.0%, respectively, and the measured total xylooligosaccharide yield was 59.9%; the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymatic hydrolysate are 4.0%, 2.1%, 2.7%, 0.9% and 0.4%, respectively, and the measured total xylooligosaccharide yield is 10.1%. Finally, the yield of the xylo-oligosaccharide obtained by the catalysis and the enzymolysis method is 70 percent in total.

Example 2

The difference from the example 1 is that the corn cob is not subjected to lactic acid catalysis treatment, but is directly subjected to xylan enzymolysis, and the conditions of the rest enzymolysis are the same as the example 1.

The results show that the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymolysis liquid are 11.1%, 1.2%, 1.6%, 0.4% and 0.2%, respectively, and the measured total yield of xylo-oligosaccharide is 14.5%.

Example 3

The difference from example 1 is that the concentration of the aqueous solution of lactic acid was 0% by volume, and the rest was the same as example 1.

The results show that the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the treatment solution are 12.5%, 0.8%, 1.2%, 1.3% and 1.4%, respectively, and the measured total xylooligosaccharide yield is 17.2%; the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymatic hydrolysate are 10.2%, 2.1%, 1.2%, 0.5% and 0.5%, respectively, and the measured total xylooligosaccharide yield is 14.5%. Finally, the yield of the xylo-oligosaccharide obtained by the catalysis and the enzymolysis method is 31.7 percent in total.

Example 4

The difference from example 1 is that the lactic acid solution has a concentration of 1% by volume, and the rest is the same as example 1.

The results found that the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the treatment solution were 9.1%, 10.0%, 12.1%, 8.5% and 10.1%, respectively, and the total xylooligosaccharide yield found was 49.8%; the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymatic hydrolysate are respectively 6.5%, 1.9%, 2.4%, 0.7% and 0.4%, and the measured total xylooligosaccharide yield is 11.9%. Finally, the yield of the xylo-oligosaccharide obtained by the catalysis and the enzymolysis method is 61.7 percent in total.

Example 5

The difference from example 1 is that the lactic acid solution has a volume concentration of 5%, and the rest is the same as example 1.

The results show that the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the treatment solution are 17.7%, 8.9%, 7.6%, 2.8% and 1.7%, respectively, and the measured total xylooligosaccharide yield is 38.7%; the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymatic hydrolysate are 5.8%, 2.0%, 2.3%, 0.5% and 0.3%, respectively, and the measured total xylooligosaccharide yield is 10.9%. Finally, the yield of the xylo-oligosaccharide obtained by the catalysis and the enzymolysis method is 49.6 percent in total.

Example 6

The difference from example 1 is that the catalysis time is 30min, and the rest is the same as example 1.

The results show that the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the treatment solution are respectively 6.1%, 5.3%, 7.5%, 5.5% and 5.4%, and the measured total xylooligosaccharide yield is 29.8%; the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymatic hydrolysate are 16.8%, 6.9%, 8.5%, 3.1% and 2.0%, respectively, and the measured total xylooligosaccharide yield is 37.3%. Finally, the yield of the xylo-oligosaccharide obtained by the catalysis and the enzymolysis method is 67.1 percent in total.

Example 7

The difference from example 1 is that the catalysis time is 50min, and the rest is the same as example 1.

The results show that the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the treatment solution are 17.6%, 9.5%, 8.6%, 3.5% and 2.2% respectively, and the measured total xylooligosaccharide yield is 41.4%; the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymatic hydrolysate are respectively 2.0%, 0.7%, 1.0%, 0.1% and 0.1%, and the measured total xylooligosaccharide yield is 3.9%. Finally, the yield of the xylo-oligosaccharide obtained by the catalysis and the enzymolysis method is 45.3 percent in total.

Example 8

The difference from example 1 is that the temperature for catalysis was 145 ℃ and the rest is the same as example 1.

The results show that the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the treatment solution are respectively 2.1%, 1.8%, 3.1%, 2.3% and 2.8%, and the measured total xylooligosaccharide yield is 12.1%; the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymatic hydrolysate are 11.0%, 8.7%, 5.0%, 2.1% and 1.1%, respectively, and the measured total xylooligosaccharide yield is 27.9%. Finally, the yield of the xylo-oligosaccharide obtained by the catalysis and the enzymolysis method is 40.0 percent in total.

Example 9

The difference from example 1 is that the temperature for catalysis is 175 ℃ and the rest is the same as example 1.

The results show that the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the treatment solution are 10.2%, 2.7%, 2.2%, 0.1% and 0.2%, respectively, and the measured total xylooligosaccharide yield is 15.4%; the yields of xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose in the enzymatic hydrolysate are 10.0%, 8.7%, 7.5%, 5.1% and 2.1%, respectively, and the measured total xylooligosaccharide yield is 33.4%. Finally, the yield of the xylo-oligosaccharide obtained by the catalysis and the enzymolysis method is 48.8 percent in total.

From the above embodiments, it can be known that the method for preparing xylo-oligosaccharide by lactic acid hydrolysis in cooperation with enzymatic hydrolysis provided by the invention degrades a large amount of xylan in corncobs into xylo-oligosaccharide by lactic acid catalysis, and simultaneously degrades xylan in the catalyzed solid residue into xylo-oligosaccharide by xylanase, and the yield of xylo-oligosaccharide prepared by the two-step method reaches 70%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.