CN110669805A - Method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing locust bean gum through compounding of mannanase and galactosidase - Google Patents

Abstract

The present invention relates to a method for producing galactose, mannose and mannose oligosaccharides by compounding and hydrolyzing locust bean gum with mannanase and galactosidase. The steps are as follows: (1) preparing a locust bean gum substrate with a mass concentration of 0.5%; (2) Add the compound enzyme of mannanase and galactosidase to the substrate, the ratio of mannanase to galactosidase enzyme activity is 25:1, adjust the pH of the mixture to 7.0; (3) Stir well, put it into 44 ℃ Heat the reaction solution in a boiling water bath for 12±1h, centrifuge, and collect the supernatant to obtain a mixture of galactose, mannose and mannose oligosaccharide. This method is the first to use galactomannan substrate locust bean gum to prepare mannose, galactose and mannose oligosaccharides, which enriches the sources of mannose, galactose and mannose oligosaccharides, and the generation rate of monosaccharides and oligosaccharides in 12h reaches 83% %, the synergy rate is 1.41, the method has mild conditions, no pollutants are produced in the enzymatic hydrolysis process, and it is non-toxic, low in energy consumption and high in product safety.

Description

A method for producing galactose, mannose and mannose oligosaccharides by compounding and hydrolyzing locust bean gum with mannanase and galactosidase

technical field

The invention belongs to the field of biotechnology, in particular to a method for producing galactose, mannose and mannose oligosaccharides by compounding mannanase and galactosidase to hydrolyze locust bean gum.

Background technique

Mannans are the most widely distributed type of hemicellulose in nature except xylan. Natural plant-derived mannans can be divided into four categories: polymannose, glucomannan, galactomannan and galactoglucomannan. Locust bean gum, also known as locust bean gum, is a vegetable gum processed from the seeds of the locust tree in the Mediterranean. If galactose exists in polymannose molecules and is connected to mannose residues in the main chain through α-1,6-glycosidic bonds, galactomannans are formed, and locust bean gum is a kind of galactomannan. A polysaccharide compound in which lactose and mannose residues are the building blocks, and the ratio of mannose to galactose is approximately four to one. The degradation of the mannan backbone relies on the action of enzymes such as endo- and exo-mannanases, mannosidases, etc., but the complete hydrolysis of galactomannan also requires the synergistic action of α-galactosidase.

Mannose is currently the only glyconutrient used in clinical practice. It is widely distributed in body fluids and tissues and can be directly utilized to synthesize glycoproteins and participate in immune regulation. Galactose is often present in the brain and nervous tissue in the form of D-galactoside, and is also an important component of certain glycoproteins, which can be used as a nutritional sweetener because of its energy. Oligosaccharides are a class of sugars that are linear or contain side chains and contain 2 to 10 identical or different monosaccharide residues. Mannan oligosaccharide has the characteristics of good stability, safety, non-toxicity and low calorific value, and has high economic value. At present, there are few mannose oligosaccharides derived from plants. The production of mannose oligosaccharides from locust bean gum is beneficial to enrich the sources of oligosaccharides, which is of great significance for subsequent research.

Mannan oligosaccharides have many excellent properties and have been widely used. There are three main ways to obtain mannose oligosaccharides: (1) chemical degradation of glycans to obtain oligosaccharides; (2) extraction from natural raw materials; (3) biodegradation. However, the content of natural mannose oligosaccharides in nature is low, and the extraction process is complicated and difficult; the chemical degradation method generally requires harsh conditions such as high temperature, acid and alkali, and the stability and repeatability of the reaction are poor, which is seriously corrosive to equipment. And the post-processing is cumbersome, the product yield is low, the cost is high, and the pollution is strong. Compared with the first two methods, the biodegradation method, namely the enzymatic hydrolysis method, is the best choice. Because the biological solution is relatively simple, the conditions required for the degradation process are mild, the enzymatic hydrolysis process has no pollutants, non-toxic, low energy consumption, and high product safety, it is the most ideal method for the production of mannose oligosaccharides. On the basis of saving resources and protecting the environment, the enzymatic hydrolysis method provides an excellent choice for the preparation of mannose oligosaccharides.

Through the search, we found the following patent publications related to the patent application of the present invention:

Application of a glycoside hydrolase and its complex enzyme in the degradation of galactomannan (CN109055333A), the glycoside hydrolase gene man113A from Bacillus alkalophila N16-5 was cloned for the first time, introduced into pET28a vector and induced in Escherichia coli Express. The purified family 113 glycoside hydrolase Man113A has the activity of hydrolyzing mannose oligosaccharides to produce mannose. At the same time, the enzyme can be used to synergize with α-galactosidase to degrade cheap galactomannan substrates such as locust bean gum and guar gum, and generate mannose and galactose at the same time. The conversion rates of mannose and galactose were 11.6% and 8.8% with locust bean gum as substrate, and 8.4% and 15.3% with guar gum as substrate.

By comparison, the above-mentioned patent publications mainly obtained the Man113A protein and the application of this protein. The present invention mainly optimizes the compounding process of the two enzymes by means of the response surface method, and mainly protects the optimization of the locust bean gum by the response surface method. The hydrolysis method and the final process parameters are essentially different.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the deficiencies in the prior art, and to provide a method for producing galactose, mannose and mannose oligosaccharides by compounding mannanase and galactosidase to hydrolyze locust bean gum to produce galactose, mannose and mannose oligosaccharides. The lactomannan substrate locust bean gum was used to prepare mannose, galactose and mannose oligosaccharides, which enriched the sources of mannose, galactose and mannose oligosaccharides. The production rate of monosaccharides and oligosaccharides in 12h reached 83%, and the synergy rate was 1.41, the method has mild conditions, no pollutants are produced in the enzymatic hydrolysis process, and it is non-toxic, low in energy consumption and high in product safety.

The technical scheme adopted by the present invention to solve its technical problems is:

A method for producing galactose, mannose and mannose oligosaccharides by compounding and hydrolyzing locust bean gum with mannanase and galactosidase, the steps are as follows:

(1) Prepare a locust bean gum substrate with a mass concentration of 0.5%;

(2) Add the complex enzyme of mannanase and galactosidase to the substrate, the volume ratio of the substrate to the complex enzyme is 4:1, and the enzymatic activity ratio of mannanase and galactosidase is 25:1 , and adjust the pH of the mixture to 7.0;

(3) After stirring evenly, put it in a water bath at 44°C for 12±1h, heat the reaction solution in a boiling water bath for 10min, centrifuge at 10,000-12,000rpm for 5-10min, and collect the supernatant to obtain galactose, mannose and mannose oligosaccharide. mixture.

And, the concrete steps of described step (1) are as follows:

A locust bean gum substrate with a mass concentration of 0.5% was prepared with 20 mM citric acid buffer, and subjected to high-pressure steam sterilization at 115°C, and then refrigerated at 4°C for use after cooling.

Moreover, in the step (2), the galactosidase is α-galactosidase Gal27A, and the mannanase is mannanase ManA.

Moreover, the galactose, mannose and mannose oligosaccharide mixture is also processed as follows:

Qualitative and quantitative analysis of guar gum hydrolyzate was carried out by using ion chromatography, and the specific conditions were as follows:

Mobile phase component: 200mmol NaOH; Column: Dionex Cabropac PA 1 150mm×3mm, 6μm, Diono×PA 1Guard pre-column, 30mm×3mm, 6μm; Detector: GoldAg-AgCl, pulsed amperometric detector; flow rate: 1mL/ min; column temperature: 30 °C; injection volume: 25 μL.

The advantages and positive effects obtained by the present invention are:

The method of the invention is a method for hydrolyzing locust bean gum to produce galactose, mannose and mannose oligosaccharide by a composite method of optimizing two glycoside hydrolases by response surface method. The method of the invention utilizes the galactomannan substrate of locust bean for the first time Gum preparation of mannose, galactose and mannose oligosaccharides enriches the sources of mannose, galactose and mannose oligosaccharides, the formation rate of monosaccharides and oligosaccharides in 12h reaches 83%, and the synergy rate is 1.41. No pollutants are produced in the solution process, and it is non-toxic, low in energy consumption and high in product safety.

Description of drawings

Fig. 1 is the standard curve diagram of reducing sugar in the present invention;

Fig. 2 is the influence diagram of temperature in the single factor analysis of the synergistic effect of mannanase and galactosidase on locust bean gum in the present invention;

Fig. 3 is the influence diagram of pH in the single factor analysis of the synergistic effect of mannanase and galactosidase on locust bean gum in the present invention;

Fig. 4 is the influence diagram of the optimal enzyme activity addition ratio in the single factor analysis of the synergistic effect of mannanase and galactosidase on locust bean gum in the present invention;

Figure 5 is a comparison diagram between the predicted value and the actual value of the response surface of the synergistic effect of mannanase and galactosidase on locust bean gum in the present invention;

Fig. 6 is a graph showing the interaction between various factors for optimizing the synergistic effect of mannanase and galactosidase on locust bean gum by response surface methodology in the present invention; wherein, (a) represents the relationship between reaction temperature and pH value. Interaction, (b) represents the interaction between enzyme activity addition ratio and reaction temperature, (c) represents the interaction between enzyme activity addition ratio and pH value;

Fig. 7 is the calibration curve diagram that ion chromatography carries out quantification to mannose in the present invention;

Fig. 8 is the calibration curve diagram that galactose is quantified by ion chromatography in the present invention;

Fig. 9 is the calibration curve diagram that ion chromatography carries out quantification to mannobiose in the present invention;

Fig. 10 is the calibration curve diagram that ion chromatography carries out quantification to mannotriose in the present invention;

Figure 11 is an ion chromatogram showing the synergistic effect of mannanase and galactosidase on the final product of locust bean gum substrate in the present invention; M1: mannose; M2: mannobiose; M3: mannotriose; M4: Mannotetraose; G: Mannose.

Detailed ways

The embodiments of the present invention will be described in detail below. It should be noted that the embodiments are descriptive, not restrictive, and cannot limit the protection scope of the present invention.

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

The quantitative tests in the following examples are all set to repeat the test three times, and the results are averaged.

A method for producing galactose, mannose and mannose oligosaccharides by compounding and hydrolyzing locust bean gum with mannanase and galactosidase, the steps are as follows:

(1) Prepare a locust bean gum substrate with a mass concentration of 0.5%;

(2) Add the complex enzyme of mannanase and galactosidase to the substrate, the volume ratio of the substrate to the complex enzyme is 4:1, and the enzymatic activity ratio of mannanase and galactosidase is 25:1 , and adjust the pH of the mixture to 7.0;

(3) After stirring evenly, put it in a water bath at 44°C for 12±1h, heat the reaction solution in a boiling water bath for 10min, centrifuge at 10,000-12,000rpm for 5-10min, and collect the supernatant to obtain galactose, mannose and mannose oligosaccharide. mixture.

Preferably, the concrete steps of described step (1) are as follows:

A locust bean gum substrate with a mass concentration of 0.5% was prepared with 20 mM citric acid buffer, and subjected to high-pressure steam sterilization at 115°C, and then refrigerated at 4°C for use after cooling.

Preferably, in the step (2), the galactosidase is α-galactosidase Gal27A, and the mannanase is mannanase ManA.

Preferably, the mixture of galactose, mannose and mannose oligosaccharide is also processed as follows:

Qualitative and quantitative analysis of guar gum hydrolyzate was carried out by using ion chromatography, and the specific conditions were as follows:

Mobile phase component: 200mmol NaOH; Column: Dionex Cabropac PA 1 150mm×3mm, 6μm, Diono×PA 1Guard pre-column, 30mm×3mm, 6μm; Detector: GoldAg-AgCl, pulsed amperometric detector; flow rate: 1mL/ min; column temperature: 30 °C; injection volume: 25 μL.

The specific operation and verification test are as follows:

1. Preparation of substrate and drawing of reducing sugar standard:

1. Substrate preparation: Weigh 0.5g of locust bean gum and add it to 100mL of citric acid buffer with pH 7.0 for several times, and keep stirring to form a uniform emulsion, sterilize at 115℃ for 15-20min, and wait for cooling Store at 4°C for later use.

2. Drawing of reducing sugar standard: Dissolve 180 mg of reducing sugar in 10 ml of citric acid-disodium hydrogen phosphate buffer to prepare a solution with a final concentration of 10 mM. The standard gradient solution of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.4, 1.8, 2.0, 2.4 mM was made up to different concentrations with pH 7.0 citric acid-disodium hydrogen phosphate buffer, and the final volume was 1ml. 1ml of DNS reagent was added respectively, the reaction solution was rapidly cooled after boiling water bath for 10min, and the absorbance value was measured at 540nm. Taking the absorbance value as the abscissa and the reducing sugar concentration as the ordinate, draw a standard curve as shown in Figure 1.

3. Calculate the synergy rate,

A – The amount of reducing sugar produced by the synergistic addition of Gal27A and ManA to the substrate solution in the experimental group, in mM;

AC – the amount of reducing sugar produced in the experimental group after Gal27A was added to the substrate solution alone, in mM;

AA - the amount of reducing sugar produced after the reaction of ManA alone in the substrate solution in the experimental group, in mM.

2. Single factor optimization of synergistic effect of α-galactosidase Gal27A and mannanase ManA:

1. The effect of temperature on the synergistic effect of mannanase and galactosidase on locust bean gum substrate

Take a buffer with a pH of 7.0 to prepare 0.5% locust bean gum, preheat 800 μL of substrate for 3-4 min, and then add 200 μL of mannanase ManA (0.76U), galactosidase Gal27A (0.9U) and At the same time, mannanase ManA (0.76U) and galactosidase Gal27A (0.9U) were added and reacted at 30°C, 35°C, 40°C, 45°C, 50°C and 60°C for 15 min, after adding 1 mL of DNS, put in boiling water Boil in the bath for 10min, after cooling, measure the absorbance value of the reaction solution at 540nm, substitute it into the reducing sugar equivalent standard curve to calculate the amount of reducing sugar produced, and calculate the synergy rate, the results are shown in Figure 2, determine the optimal synergistic reaction temperature is 45°C.

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

The locust bean gum with a mass concentration of 0.5% was prepared with a buffer with a pH of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, and 800 μL of the substrate was preheated at 45 °C for 3-4 min, and 200 μL of mannanase ManA (0.76 U), galactosidase Gal27A (0.9U) and adding mannanase ManA (0.76U) and galactosidase Gal27A (0.9U) at the same time, react for 15min, add 1mL DNS, cook in boiling water bath for 10min, wait for After cooling, measure the absorbance of the reaction solution at 540 nm, substitute it into the reducing sugar equivalent standard curve to calculate the amount of reducing sugar produced, and calculate the synergy rate.

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

Prepare 0.5% locust bean gum with a pH of 7.0 buffer, preheat 800 μL of substrate at 45°C for 3-4 min, and add 200 μL of mannanase ManA and galactosidase Gal27A mixed enzyme solution according to different addition ratios In the reaction system, the addition ratios were 80:1 (7.2U:0.09U), 40:1 (3.6U:0.09U), 20:1 (1.8U:0.09U), 10:1 (0.9U:0.09U) , 5:1 (0.45U:0.09U), 1:1 (0.09U:0.09U). The reaction was carried out under the optimal reaction conditions for 15 min, after adding 1 mL of DNS, boiled in a boiling water bath for 10 min, after cooling, the absorbance value of the reaction solution was measured at 540 nm, and substituted into the standard curve of reducing sugar equivalent to calculate the amount of reducing sugar produced, and calculated The synergy rate, the results are shown in Figure 4, the optimal synergistic reaction ratio was determined to be 20:1.

3. Response surface methodology to optimize the conditions for the synergistic effect of mannanase and galactosidase on the locust bean gum substrate:

On the basis of the single-factor experimental results of temperature, pH and ratio of synergistic effect, the conditions of synergistic effect were determined by temperature (40°C, 45°C, 50°C), pH (6.0, 7.0, 8.0) and enzyme activity addition ratio ( 10:1, 20:1, 40:1) three factors to carry out a three-factor three-level experimental design. The design and factor levels of the response surface experiment are shown in Table 1. The synergistic effect research is carried out according to different experimental combination conditions. The synergy rate is the response value (Y value), and the experimental response value of the synergistic effect on locust bean gum is shown in Table 2. Response surface analysis software was used to conduct variance analysis on the experimental data obtained by synergistic effect on locust bean gum in Table 2, and the analysis results are shown in Table 3. According to the single-factor experimental results, combined with the Box-Behnken design principle, the temperature, pH, and pH values were selected. The ratio of enzyme activity and enzyme activity is the independent variable, which is coded according to the low, medium and high levels of -1, 0, 1, and the response value is taken as the synergy rate. The relationship between the value synergy rate (Y) is fitted, and the obtained response surface multivariate quadratic regression model equation is:

Y=1.42-0.041*A+0.010*B+5.750*10 ^-3 *C-0.034*AB+0.013*AC+0.013*BC-0.14*A ² -0080*B ² -0.12*C ²

Table 1 Factors and levels of response surface experiments acting on locust bean gum substrates

Table 2 Design and results of response surface experiments for the synergistic effect of ManA and Gal27A on locust bean gum

Table 3 Analysis of variance table of response surface experiment results of the synergistic effect of ManA and Gal27A on locust bean gum

* means significant; ** means extremely significant

The variance analysis of the items of the regression equation obtained in the experiment shows that the model has F=160.24, and the significance level reaches P<0.0001, indicating that the effect of the regression equation obtained by the experimental model has reached a significant level, and can correctly reflect the relationship between various factors and factors. The change relationship between the response values is statistically significant. The Lack of Fit item in the model is F=4.48, P=0.0906 (>0.05), indicating that the Lack of Fit item is not significant, indicating that the model has a good fitting effect in the entire regression area under study, and the model residuals are all determined by random errors. cause, the model prediction accuracy is high. In addition, the comparison between the predicted value and the real value is shown in Figure 5. These points are distributed relatively close to the regression line, indicating that the predicted value of the model is in good agreement with the experimental value (R2=0.99), and the model can be used to replace the real value. The test points were used to analyze the results. If the CV value of the model is less than 10%, it is considered reasonable. The ratio of effective signal to noise is called the precision (Adeq Precision). In the response surface experiment, a reasonable precision value should be greater than 4. The CV=0.92% of the experimental model and the Adeq Precision=34.15 are all in line with the above test principles. It shows that the experimental model is reliable and has high reliability and accuracy. According to F value and P value, it can be seen that the primary and secondary order of factors affecting the synergistic hydrolysis of locust bean gum by ManA and Gal27A is: reaction temperature>pH>addition ratio.

From the F and P values in Table 3, it can be seen that the interaction term (AB) of temperature and pH, and the partial regression coefficient (P<0.05) of the interaction term (AC) of temperature and addition ratio are significant, indicating that temperature and pH are both significant. The interaction of influencing factors, temperature and addition ratio had a significant effect on the synergy rate of ManA and Gal27A on locust bean gum. The partial regression coefficient (P>0.05) of the interaction term (BC) of pH and addition ratio was not significant, indicating that the interaction between pH and addition ratio had no effect on the synergistic effect of ManA and Gal27A on locust bean gum. Significantly. A three-dimensional response surface plot was chosen to illustrate the interaction between independent and dependent variables. As shown in Figure 6, it is a response surface diagram for the effect of the interaction between each two factors on the synergistic hydrolysis of locust bean gum by ManA and Gal27A.

It can be seen from Figure 6(a) that when the addition ratio is zero (20:1), under the condition of low reaction pH, with the continuous increase of temperature, the synergy rate shows a trend of first increase and then decrease, and The lift speed is almost the same. Under high reaction pH conditions, the synergy rate of ManA and Gal27A increased slowly and then decreased rapidly with the increase of reaction temperature. It can be seen from Figure 6(b) that when the pH value of the reaction is zero (pH=7), at a certain temperature, the synergy rate also increases first and then decreases with the increase of the addition ratio, but the high temperature condition At a certain enzyme activity ratio, the synergy rate first increased and then decreased rapidly with the increase of temperature. It can be seen from Figure 6(c) that when the temperature is at the zero level (45°C), the synergy rate first increases and then decreases with the increase of enzyme activity ratio and reaction pH, but the change value of synergy rate lower.

Design-Expert 8.06 software was used to optimize and predict the experimental data, so as to further determine the optimal conditions for the synergistic effect of ManA and Gal27A on locust bean gum. The optimal process conditions obtained by optimization were the reaction temperature of 44.2℃, the reaction pH of 7.1, the ratio of the enzyme activity of ManA and Gal27A was 25.3:1, and the synergy rate was 1.42 under these conditions. Considering the actual situation and the convenience of experimental operation, the hydrolysis conditions were modified as follows: the reaction temperature was 44° C., the reaction pH was 7.0, and the ratio of ManA and Gal27A was 25:1. Several parallel experiments were carried out under this condition to verify the reliability of the response surface optimization method. In practice, the measured synergy rate is 1.41 (n=9), which is slightly different from the theoretical prediction value of the model, which is 1.42, which indicates that the predicted value of the constructed experimental model has a good fit with the actual value. The synergistic properties of ManA and Gal27A optimized by response surface methodology were accurate and reliable, and the established model was reliable.

4. Ion chromatography of the synergistic effect of mannanase and galactosidase on the final product of locust bean gum substrate:

1. Processing of samples for ion chromatography

The samples were centrifuged with ultrafiltration tubes (3kDa) to remove proteins in the samples, and passed through a 0.22 μm filter membrane before loading. The operating conditions of ion chromatography are as follows: mobile phase components: A: 500mmol NaOH, B: deionized water; mobile phase conditions: 40%A+60%B isocratic injection; chromatographic column: Dionex Cabropac PA 1 (150mm×3mm , 6μm), Diono×PA 1Guard (pre-column, 30mm×3mm, 6μm); detector: GoldAg-AgCl, pulsed amperometric detector; flow rate: 1 mL/min; column temperature: 30 °C; injection volume: 25 μL.

2. Drawing of ion chromatography standard

Prepare 5mg/L, 7.5mg/L, 10mg/L, 15mg/L, 20mg/L, 30mg/L concentration gradient mannose aqueous solutions with deionized water, inject samples by ion chromatography, calculate the integral area of mannose peak, and use the integral The area is the abscissa, the mannose concentration is the ordinate, and the standard curve is drawn as shown in Figure 7. Draw the standard curves of galactose, mannobiose and mannotriose in the same steps as above, as shown in Figures 8, 9, and 10, respectively.

3. Analysis of hydrolyzate

According to the results of optimizing the synergistic effect of ManA and Gal27A, the hydrolyzate was prepared under the conditions of reaction temperature of 44 °C, reaction pH of 7.0, and the ratio of ManA to Gal27A of 25:1. Take 800 μL of sterilized substrate and add 200 μL of mixed enzyme solution containing ManA (2050U) and Gal27A (82U) (in order to shorten the reaction time, increase the amount of enzyme), take samples at 12h and 24h during the reaction, and hydrolyze After the product was diluted 50 times with deionized water, the hydrolysis products of 12h and 24h were analyzed by ion chromatography. It was found that the hydrolysis was complete in 12h.

The generation rate of galactose, mannose and mannose oligosaccharide refers to the ratio of galactose, mannose and mannose oligosaccharide released by hydrolysis to the total amount of locust bean gum. The calculation formula is as follows:

Therefore, the hydrolysis of locust bean gum produces galactose, and the total production rate of mannose and mannose oligosaccharide is 83%.

Table 4 12h hydrolyzate component analysis of locust bean gum

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, therefore , the scope of the present invention is not limited to the contents disclosed in the embodiments and drawings.

Claims (4)

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

⑴ preparing locust bean gum substrate with mass concentration of 0.5%;

⑵ adding a complex enzyme of mannase and galactosidase to a substrate, wherein the volume ratio of the substrate to the complex enzyme is 4: 1, the enzyme activity ratio of mannase and galactosidase is 25:1, and the pH of the mixture is adjusted to 7.0;

⑶ stirring well, placing in a water bath kettle at 44 deg.C, keeping the temperature for 12 + -1 h, heating the reaction solution in boiling water bath for 10min, centrifuging at 12,000rpm for 5-10min at 10,000-.

2. The method for producing galactose, mannose and mannooligosaccharide by hydrolyzing locust bean gum through compounding mannanase and galactosidase according to claim 1, which is characterized in that the step ⑴ comprises the following steps:

preparing 0.5% locust bean gum substrate with 20mM citric acid buffer solution, performing high pressure steam sterilization at 115 deg.C, cooling, and refrigerating at 4 deg.C.

3. The method for producing galactose, mannose and mannooligosaccharides by hydrolyzing locust bean gum through compounding mannanase and galactosidase according to claim 1, wherein the galactosidase is alpha-galactosidase Gal27A and the mannanase is mannanase ManA in the step ⑵.

4. The method for producing galactose, mannose and mannooligosaccharides from the mannose and galactosidase complex hydrolysis locust bean gum 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.

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