阅读说明：本技术 一种甘露聚糖酶及半乳糖苷酶复配水解瓜尔豆胶生产半乳糖、甘露糖及甘露寡糖的方法 (Method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing guar gum through compounding of mannanase and galactosidase ) 是由 宋亚囝 马翠萍 刘朵朵 罗学刚 李中媛 张同存 于 2019-09-27 设计创作，主要内容包括：本发明涉及一种甘露聚糖酶及半乳糖苷酶复配水解瓜尔豆胶生产半乳糖、甘露糖及甘露寡糖的方法,步骤如下：⑴制备质量浓度0.5％的瓜尔豆胶底物；⑵向底物中加入甘露聚糖酶和半乳糖苷酶的复合酶,甘露聚糖酶和半乳糖苷酶的酶活力比为65:1,并调节混合物的pH为6.0；⑶搅拌均匀后,放入40℃水浴锅中保温24±1h,将反应液沸水浴加热,离心,收集上清液,即得半乳糖、甘露糖及甘露寡糖混合物。本方法首次利用半乳甘露聚糖底物瓜尔豆胶制备甘露糖,半乳糖及甘露寡糖,丰富了甘露糖,半乳糖及甘露寡糖的来源,条件温和,酶解过程无污染物产生,且无毒、耗能低、产物安全性高,24h单糖及寡糖的生成率达到88％。(The invention relates to a method for producing galactose, mannose and mannooligosaccharide by hydrolyzing guar gum through compounding mannase and galactosidase, which comprises the following steps of ⑴ preparing a guar gum substrate with the mass concentration of 0.5%, ⑵ adding a compound enzyme of mannase and galactosidase into the substrate, wherein the enzyme activity ratio of mannase and galactosidase is 65:1, adjusting the pH of the mixture to 6.0, stirring the mixture evenly at ⑶, placing the mixture into a water bath kettle at 40 ℃ for heat preservation for 24 +/-1 h, heating the reaction liquid in a boiling water bath, centrifuging the reaction liquid, and collecting supernatant to obtain a mixture of galactose, mannose and mannooligosaccharide.)

1. A method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing guar gum by compounding mannanase and galactosidase is characterized in that: the method comprises the following steps:

⑴ preparing guar gum substrate with mass concentration of 0.5%;

⑵ adding a complex enzyme of mannase and galactosidase into the substrate, wherein the volume ratio of the substrate to the complex enzyme is 4: 1, the enzyme activity ratio of mannase and galactosidase is 65:1, and the pH of the mixture is adjusted to 6.0;

⑶ stirring well, placing in 40 deg.C water bath, keeping the temperature for 24 + -1 h, heating the reaction solution in boiling water bath for 10min, centrifuging at 10,000-12,000rpm for 5-10min, and collecting the supernatant to obtain the mixture of galactose, mannose and mannooligosaccharide.

2. The method for producing galactose, mannose and mannooligosaccharide by hydrolyzing guar gum by compounding mannanase and galactosidase according to claim 1, wherein the step ⑴ comprises the following steps:

preparing guar gum substrate with the mass concentration of 0.5% by using 20mM citric acid buffer solution, performing high-pressure steam sterilization treatment at 115 ℃, cooling, and refrigerating at 4 ℃ for later use.

3. The method for producing galactose, mannose and mannooligosaccharides from guar gum hydrolyzed by compounding mannanase and galactosidase according to claim 1, wherein the galactosidase in step ⑵ is α -galactosidase Gal27A and the mannanase is mannanase ManA.

4. The method for producing galactose, mannose and mannooligosaccharides from guar gum hydrolyzed by compounding mannanase and galactosidase according to any one of claims 1 to 3, wherein the method comprises the following steps: the mixture of galactose, mannose and mannooligosaccharide is further treated as follows:

performing qualitative and quantitative analysis on the guar gum hydrolysate by using an ion chromatography technology, wherein the specific conditions are as follows:

mobile phase components: 200mmol NaOH; a chromatographic column: dionex Cabropac PA 1150 mm. times.3 mm, 6 μm, Diono. times.PA 1Guard pre-column, 30 mm. times.3 mm, 6 μm; a detector: Goldag-AgCl, pulsed amperometric detector; flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction amount: 25 μ L.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing guar gum through compounding mannanase and galactosidase.

Background

The natural plant-derived mannans can be classified into four types, namely polymannan, glucomannan, galactomannan and galactoglucomannan, if galactose exists in polymannan molecules and is connected with mannose residues in a main chain through α -1, 6-glycosidic bonds, galactomannan is formed, the guar gum is a polysaccharide compound taking galactose and mannose residues as structural units, the ratio of the mannose to the galactose is about two to one, the degradation of the mannan main chain needs to depend on the actions of endomannase, exomannase and the like, but the complete hydrolysis of the galactomannan needs to have the synergistic action of α -galactosidase.

Mannose is the only carbohydrate nutrient used clinically at present, is widely distributed in body fluid and tissues, can be directly utilized to synthesize glycoprotein, and participates in immune regulation. Galactose, often in the form of D-galactoside, is also an important component of certain glycoproteins because of its energy content and can be used as a nutritional sweetener. Oligosaccharides are a class of sugars that are linear or contain side chains and contain 2 to 10 identical or different monosaccharide residues. The mannan oligosaccharide has the characteristics of good stability, safety, no toxicity, low heat value and the like, and has high economic value. At present, the plant source of the mannan oligosaccharide is less, and the mannan oligosaccharide produced by the guar gum is beneficial to enriching the source of the mannan oligosaccharide, thereby having important significance for the follow-up research.

The mannooligosaccharides have a plurality of excellent characteristics and are widely applied at present. There are three main ways to obtain mannooligosaccharides: (1) degrading glycan by a chemical method to obtain oligosaccharide; (2) extracting from natural raw materials; (3) and (4) performing biodegradation. However, the content of natural mannooligosaccharides existing in nature is low, and the extraction process is complex and difficult; the chemical degradation method generally needs harsh conditions such as high temperature, acid and alkali, has poor reaction stability and repeatability, has serious corrosion to equipment, and has the defects of complex post-treatment, low product yield, high cost, strong pollution and the like. Compared to the first two methods, the biodegradation method, i.e., the enzymatic method, is the best choice. Because the biological degradation method is simple, the conditions required by the degradation process are mild, no pollutant is generated in the enzymolysis process, the method is non-toxic, the energy consumption is low, and the product safety is high, the method is the most ideal method for producing the mannan oligosaccharide. On the basis of saving resources and protecting the environment, the preparation of the mannan oligosaccharide by the enzymolysis method provides an excellent choice.

Through searching, the following patent publications related to the patent application of the invention are found:

a glucoside hydrolase and an application of a compound enzyme thereof in degradation of galactomannan (CN109055333A) clone a glucoside hydrolase gene Man113A from Bacillus alcalophilus N16-5 for the first time, a pET28a vector is introduced and is induced and expressed in escherichia coli, and the purified 113 family glucoside hydrolase Man113A has the activity of hydrolyzing mannan-oligosaccharide to generate mannose, and simultaneously, the enzyme can be used for synergistic action with α -galactosidase to degrade cheap galactomannan substrates such as locust bean gum, guar gum and the like, and simultaneously generate mannose and galactose, the conversion rates of mannose and galactose taking locust bean gum as the substrates respectively reach 11.6% and 8.8%, and the conversion rates of mannose and galactose taking guar gum as the substrates respectively reach 8.4% and 15.3%.

By contrast, the patent publications mainly obtain the Man113A protein and the application of the protein, the invention mainly optimizes the compounding process of two enzymes by means of a response surface method, and the response surface method is mainly used for optimizing the hydrolysis method and final process parameters of guar gum, so that the essence is different.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for producing galactose, mannose and mannooligosaccharides by hydrolyzing guar gum through compounding mannase and galactosidase, the method prepares the mannose, the galactose and the mannooligosaccharides through guar gum serving as a galactomannan substrate for the first time, the sources of the mannose, the galactose and the mannooligosaccharides are enriched, the conditions are mild, no pollutant is generated in the enzymolysis process, no toxicity is generated, the energy consumption is low, the product safety is high, and the generation rate of monosaccharide and oligosaccharide in 24 hours reaches 88%.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing guar gum by compounding mannanase and galactosidase comprises the following steps:

⑴ preparing guar gum substrate with mass concentration of 0.5%;

⑵ adding a complex enzyme of mannase and galactosidase into the substrate, wherein the volume ratio of the substrate to the complex enzyme is 4: 1, the enzyme activity ratio of mannase and galactosidase is 65:1, and the pH of the mixture is adjusted to 6.0;

⑶ stirring well, placing in 40 deg.C water bath, keeping the temperature for 24 + -1 h, heating the reaction solution in boiling water bath for 10min, centrifuging at 10,000-12,000rpm for 5-10min, and collecting the supernatant to obtain the mixture of galactose, mannose and mannooligosaccharide.

The specific steps of step ⑴ are as follows:

preparing guar gum substrate with the mass concentration of 0.5% by using 20mM citric acid buffer solution, performing high-pressure steam sterilization treatment at 115 ℃, cooling, and refrigerating at 4 ℃ for later use.

Furthermore, in the step ⑵, the galactosidase is α -galactosidase Gal27A, and the mannanase is mannanase ManA.

Furthermore, the mixture of galactose, mannose and mannooligosaccharides is further treated as follows:

performing qualitative and quantitative analysis on the guar gum hydrolysate by using an ion chromatography technology, wherein the specific conditions are as follows:

mobile phase components: 200mmol NaOH; a chromatographic column: dionex Cabropac PA 1150 mm. times.3 mm, 6 μm, Diono. times.PA 1Guard pre-column, 30 mm. times.3 mm, 6 μm; a detector: Goldag-AgCl, pulsed amperometric detector; flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction amount: 25 μ L.

The invention has the advantages and positive effects that:

the invention relates to a method for producing galactose, mannose and mannooligosaccharide by optimizing mannase, galactosidase compound mannase and galactosidase compound hydrolyzed guar gum by using a response surface method, the method for producing mannose, galactose and mannooligosaccharide by using galactomannan substrate guar gum for the first time enriches the sources of mannose, galactose and mannooligosaccharide, the conditions are mild, no pollutant is produced in the enzymolysis process, and the method is non-toxic, low in energy consumption and high in product safety, and the generation rate of monosaccharide and oligosaccharide reaches 88% in 24 h.

Drawings

FIG. 1 is a graph showing a standard curve of reducing sugars in the present invention;

FIG. 2 is a graph showing the effect of temperature on the single factor analysis of the synergistic effect of mannanase and galactosidase on guar gum in the present invention;

FIG. 3 is a graph of the effect of pH on the single factor analysis of the synergistic effect of mannanase and galactosidase on guar gum of the present invention;

FIG. 4 is a graph showing the effect of optimal enzyme activity addition ratio in single factor analysis of the synergistic effect of mannanase and galactosidase on guar gum in the present invention;

FIG. 5 is a graph comparing the predicted and actual response surface values of the synergistic action of mannanase and galactosidase on guar gum in accordance with the present invention;

FIG. 6 is a graph showing the interaction between factors in response surface optimization of the synergy of mannanase and galactosidase on guar gum according to the present invention; wherein (a) represents the interaction between the reaction temperature and the pH value, (b) represents the interaction between the enzyme activity addition ratio and the reaction temperature, and (c) represents the interaction between the enzyme activity addition ratio and the pH value;

FIG. 7 is a plot of the quantification of mannose by ion chromatography in accordance with the present invention;

FIG. 8 is a graph of the quantification of galactose by ion chromatography in accordance with the present invention;

FIG. 9 is a graph showing the quantitative determination of mannobiose by ion chromatography in the present invention;

FIG. 10 is a plot of ion chromatography for the quantification of mannotriose in the present invention;

FIG. 11 is an ion chromatogram of the end product of the synergistic effect of mannanase and galactosidase on guar gum substrate of the present invention; m1 in the figure: mannose; m2: mannobiose; m3: mannotriose; m4: mannose tetrasaccharide; g: mannose.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

A method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing guar gum by compounding mannanase and galactosidase comprises the following steps:

⑴ preparing guar gum substrate with mass concentration of 0.5%;

⑵ adding a complex enzyme of mannase and galactosidase into the substrate, wherein the volume ratio of the substrate to the complex enzyme is 4: 1, the enzyme activity ratio of mannase and galactosidase is 65:1, and the pH of the mixture is adjusted to 6.0;

⑶ stirring well, placing in 40 deg.C water bath, keeping the temperature for 24 + -1 h, heating the reaction solution in boiling water bath for 10min, centrifuging at 10,000-12,000rpm for 5-10min, and collecting the supernatant to obtain the mixture of galactose, mannose and mannooligosaccharide.

Preferably, the specific steps of step ⑴ are as follows:

preparing guar gum substrate with the mass concentration of 0.5% by using 20mM citric acid buffer solution, performing high-pressure steam sterilization treatment at 115 ℃, cooling, and refrigerating at 4 ℃ for later use.

Preferably, the galactosidase in step ⑵ is α -galactosidase Gal27A and the mannanase is mannanase ManA.

Preferably, the mixture of galactose, mannose and mannooligosaccharide is further treated as follows:

performing qualitative and quantitative analysis on the guar gum hydrolysate by using an ion chromatography technology, wherein the specific conditions are as follows:

mobile phase components: 200mmol NaOH; a chromatographic column: dionex Cabropac PA 1150 mm. times.3 mm, 6 μm, Diono. times.PA 1Guard pre-column, 30 mm. times.3 mm, 6 μm; a detector: gold Ag-AgCl, pulsed amperometric detector; flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction amount: 25 μ L.

The specific operation and verification test are as follows:

firstly, preparing a substrate and drawing a reducing sugar standard:

1. preparation of a substrate: weighing 0.5g guar gum, adding into 100mL citric acid buffer solution with pH of 7.0, stirring to obtain uniform emulsion, sterilizing at 115 deg.C for 15-20min, cooling, and storing at 4 deg.C.

2. Drawing a reducing sugar standard: 180mg of reducing sugar was dissolved in 10ml of citric acid-disodium hydrogen phosphate buffer solution to prepare a solution having a final concentration of 10 mM. The solutions were brought to different concentrations with citrate-disodium hydrogen phosphate buffer at pH 7.0, and were brought to standard gradient solutions of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.4, 1.8, 2.0, 2.4mM, respectively, for a final volume of 1 ml. 1ml of DNS reagent is added respectively, reaction liquid is cooled rapidly after boiling water bath for 10min, and the light absorption value is measured at 540 nm. A standard curve is plotted with the light absorption value as the abscissa and the reducing sugar concentration as the ordinate, as shown in FIG. 1.

3. The cooperation rate is calculated, and the cooperation rate is calculated,

a-amount of reducing sugar produced after reaction by synergistic addition of experimental groups Gal27A and ManA to substrate solution in mM;

AC-Experimental group Gal27A reducing sugar production amount after reaction added to substrate solution alone in mM;

AA-amount of reducing sugar formed after the reaction when the experimental group ManA was added alone to the substrate solution in mM.

Secondly, α -galactosidase Gal27A and mannanase ManA synergistic single-factor condition optimization:

1. effect of temperature on the synergistic Effect of mannanase and galactosidase on guar Gum substrate

Taking 0.5% guar gum substrate prepared by buffer solution with pH 7.0, preheating 800 mul of substrate at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 60 ℃ for 3-4min respectively, then adding 200 mul of mannase ManA (0.76U), galactosidase Gal27A (0.9U) and simultaneously adding mannase ManA (0.76U) and galactosidase Gal27A (0.9U), reacting at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 60 ℃ for 15min respectively, adding 1mL DNS, boiling in boiling water bath for 10min, cooling, measuring the absorbance value of the reaction solution at 540nm, substituting the absorbance value into a reducing sugar equivalent reducing sugar standard curve to calculate the reducing sugar production, and calculating the synergistic ratio, wherein the result is shown in figure 2, and the optimal synergistic reaction temperature is determined to be 40 ℃.

2. Effect of pH on the synergistic Effect of mannanase and galactosidase on mannan substrates

Preparing 0.5% guar gum by using buffer solution with pH values of 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, preheating 800 microliter of substrate at 40 ℃ for 3-4min, respectively adding 200 microliter of mannase ManA (0.76U), galactosidase Gal27A (0.9U) and mannase ManA (0.76U) and galactosidase Gal27A (0.9U), reacting for 15min, adding 1mL of DNS, boiling in boiling water bath for 10min, measuring absorbance value of reaction liquid at 540nm after cooling, calculating production amount by substituting into reducing sugar equivalent standard curve, and calculating synergistic rate, wherein the optimal synergistic reaction pH value is determined to be 7.0 as shown in FIG. 3.

3. Effect of different addition ratios (U: U) on the synergistic Effect of mannanase and galactosidase on mannan substrates

Preparing 0.5% guar gum by using a buffer solution with the pH value of 7.0, preheating 800 mu L of a substrate at 45 ℃ for 3-4min, and adding 200 mu L of a mixed enzyme solution of mannanase ManA and galactosidase Gal27A into a reaction system according to different addition ratios of 160:1(14.4U:0.09U), 80:1(7.2U:0.09U), 40:1(3.6U:0.09U), 20:1(1.8U: 0.09U), 10:1(0.9U:0.09U) and 5:1(0.45U: 0.09U). Reacting for 15min under the optimal reaction condition, adding 1mLDNS, boiling in boiling water bath for 10min, cooling, measuring the absorbance value of the reaction liquid at 540nm, substituting into a standard curve of reducing sugar equivalent to calculate the reducing sugar yield, and calculating the synergistic ratio, wherein the result is shown in figure 4, and the optimal synergistic reaction ratio is determined to be 80: 1.

Thirdly, optimizing the condition that the mannase and the galactosidase act on the guar gum substrate synergistically by using a response surface method:

on the basis of single-factor experimental results of temperature, pH and proportion of synergistic effect, three-factor three-level experimental design is carried out on the synergistic effect condition by three factors of temperature (35 ℃, 40 ℃ and 45 ℃), pH (5.0, 6.0 and 7.0) and enzyme activity addition ratio (20: 1, 40:1 and 80: 1). The design of the response surface experiment and the factor level are shown in table 1, the synergistic effect research is carried out according to different experimental combination conditions, the synergy rate is taken as a response value (Y value), the experimental response value of the synergistic action on the guar gum is shown in a table 2, the variance analysis is carried out on the experimental data obtained by the synergistic action on the guar gum by utilizing response surface analysis software, the analysis result is shown in a table 3, according to the result of a single-factor experiment, the temperature, the pH value and the enzyme activity ratio are selected as independent variables by combining the Box-Behnken design principle, establishing a multivariate quadratic equation according to low, medium and high level codes of-1, 0 and 1 and taking the cooperative rate as a response value, fitting the relationship among the temperature (A), the pH (B), the addition ratio (C) and the response value synergy (Y) to obtain a response surface multivariate quadratic regression model equation as follows:

TABLE 1 response surface test factors and levels on guar gum substrate

Table 2 experimental design and results of response surface of ManA and Gal27A synergistic on guar gum

Table 3 analysis of variance table of experimental results of response surface of synergistic effect of ManA and Gal27A on guar gum

Indicates significance; mean extreme significance

Variance analysis is carried out on each item of the regression equation obtained through experiments, and the F of the model is 46.47, and the significance level reaches P < 0.0001, so that the regression equation obtained through the experimental model achieves the significance level, can correctly reflect the change relation between each factor and the response value, and has statistical significance. The mismatching terms F in the model are 6.57, P is 0.0503 (> 0.05), the mismatching terms are not significant, the model has good fitting effect in the whole studied regression region, model residuals are all caused by random errors, and the model prediction accuracy is high. In addition, the comparison between the predicted value and the true value is shown in fig. 5, and the points are distributed at positions relatively close to the regression line, which shows that the predicted value and the experimental value of the model have good coincidence (R)²0.98), the model can be used instead of realThe test points were analyzed for results. The CV of the model is 1.30%, and the adeqpreparation is 17.95, which all meet the test principle of the response surface. The experimental model is reliable and has higher reliability and accuracy. As seen from the F and P values, the primary and secondary sequence of factors affecting the synergistic hydrolysis of guar gum by ManA and Gal27A is: reaction temperature > addition ratio > pH.

As can be seen from the F value and the P value in Table 3, the partial regression coefficient (P < 0.05) of the interaction term BC is significant, which shows that the interaction terms of pH and addition ratio have significant influence on the synergy. The partial regression coefficients (P > 0.05) of the interaction terms AB and AC are not significant, which indicates that the interaction term of temperature and pH and the interaction term of temperature and enzyme activity ratio have no significant influence on the synergistic rate. As shown in fig. 6, a response plot is presented for the effect of the interaction between each of the two factors on the synergistic hydrolysis of guar gum by ManA and Gal 27A.

As can be seen from the analysis of FIG. 6(a), the synergy rate increases with increasing temperature, while maintaining the addition ratio at a zero level (40: 1); at a certain temperature, an increase in pH leads to a decrease after an increase in synergy (the synergy increases with increasing pH at pH between 5 and 6, and gradually decreases with increasing pH at pH between 6 and 7). As can be seen from fig. 6(b), when the pH value is kept at the zero level (pH 6), the synergistic ratio also increases with increasing temperature, gradually increases with increasing addition ratio between 20:1 and 68:1, and slightly decreases between 68:1 and 80: 1. As can be seen from FIG. 6(c), when the temperature is kept at zero level (40 ℃), the addition ratio and the pH value are both too high or too low, which affects the synergy ratio between ManA and Gal27A, resulting in a decrease in the synergy ratio.

The experimental data were optimized and predicted using Design-Expert 8.06 software to further determine the optimal conditions for ManA and Gal27A to act synergistically on guar gum. The optimal process conditions obtained by optimization are that the reaction temperature is 40.0 ℃, the reaction pH is 6.2, the enzyme activity ratio of ManA and Gal27A is 65.5:1, and the synergy rate under the conditions is 2.49. Considering the reality and for the convenience of experimental operation, the hydrolysis conditions were modified as follows: the reaction temperature is 40 ℃, the reaction pH is 6.0, and the ratio of ManA to Gal27A is 65: 1. And carrying out a plurality of parallel experiments according to the conditions to verify the reliability of the response surface optimization method. The measured synergy rate is 2.48(n is 9) in practice, the difference between the value and the theoretical predicted value 2.49 of the model is small, so that the fact that the fitting performance of the predicted value and the actual value of the constructed experimental model is good is shown, the technological condition parameters of the artificial kidney bean gum cooperatively acted by the ManA and the Gal27A optimized by the response surface method are accurate and reliable, and the established model has reliability.

And fourthly, the mannase and the galactosidase act on the ion chromatography of the guar gum substrate final product in a synergistic way:

1. treatment of samples for ion chromatography

The sample was centrifuged by ultrafiltration using an ultrafiltration tube (3kDa) to remove proteins from the sample and passed through a 0.22 μm filter before loading. The operating conditions of the ion chromatography were as follows: mobile phase components: a: 500mmol NaOH, B: deionized water; mobile phase conditions: injecting sample at 40% A + 60% B isocratic; a chromatographic column: dionex Cabropac PA 1(150 mm. times.3 mm, 6 μm), Diono. times.PA 1Guard (pre-column, 30 mm. times.3 mm, 6 μm); a detector: Goldag-AgCl, pulsed amperometric detector; flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction amount: 25 μ L.

2. Drawing of ion chromatography standard curve

Preparing 5mg/L, 7.5mg/L, 10mg/L, 15mg/L, 20mg/L and 30mg/L mannose aqueous solution by deionized water, injecting by ion chromatography, calculating integral area of mannose peak, and drawing a standard curve with the integral area as abscissa and mannose concentration as ordinate as shown in FIG. 7. The standard curves of galactose, mannose trisaccharide and mannose tetrasaccharide are drawn in the same way as the above steps, and the graphs are respectively shown in FIGS. 8, 9 and 10.

3. Analysis of hydrolysate

According to the result of optimizing the synergistic action condition of ManA and Gal27A, the hydrolysate was prepared at 40 deg.C, pH 6.0 and 65:1 ratio of ManA to Gal 27A. mu.L of the sterilized substrate was added with 200. mu.L of mixed enzyme solution containing ManA (3250U) and Gal27A (50U) (the enzyme amount was increased to shorten the reaction time), sampling was performed at 12h and 24h time points during the reaction, and the hydrolysate was diluted 50 times with deionized water and analyzed by ion chromatography for 12h and 24h, and it was found that 24h hydrolysis was complete, the qualitative results of 24h ion chromatography are shown in FIG. 11, and the quantitative analysis results of 24h product are shown in Table 4.

The generation rates of galactose, mannose and mannooligosaccharides refer to the ratio of galactose, mannose and mannooligosaccharides released by hydrolysis to the total amount of guar gum, and the calculation formula is as follows:

therefore, hydrolysis of guar gum to galactose, mannose and mannooligosaccharides were produced at a total yield of 88%.

TABLE 4 guar gum 24h hydrolysate component analysis

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.