Method for extracting sugarcane leaf xylan through ultrasonic-assisted enzymolysis method and application

Abstract

The invention provides a method for extracting sugarcane leaf xylan through an ultrasonic-assisted enzymolysis method. The method comprises the steps of sugarcane leaf pretreatment, enzyme treatment, ultrasonic-assisted enzymolysis and the like. After sugarcane leaves are crushed, soaked in a sodium hydroxide solution and dried, cellulase and xylanase are added for enzymolysis, ultrasonic treatment is assisted, the enzymolysis efficiency is improved, and the sugarcane leaf xylan solution can be obtained. The method is simple and convenient to operate and high in enzymolysis efficiency, and an agricultural waste resource, namely the sugarcane leaves, is effectively utilized. In addition, the extracted xylan has biological activity of inhibiting escherichia coli and staphylococcus aureus, and the application range of the xylan is expanded. The invention not only provides a new way for comprehensive utilization of the sugarcane leaves, but also provides a new raw material source and a new product development idea for the fields of food preservation, medicine health and the like, and has relatively high practical value and market prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for extracting sugarcane leaf xylan by ultrasonic-assisted enzymatic hydrolysis and application thereof. BACKGROUND

[0002] Sugarcane, as the main crop in Guangxi Zhuang Autonomous Region, not only occupies an important position in the economy, but also has a wide range of utilization value. The stems of sugarcane can be used for juicing and sugar production, medicinal injection of sucrose, etc., the skin of sugarcane can be processed into activated carbon, and the residue of sugarcane can be used as biomass fuel, which effectively improves the comprehensive utilization efficiency of sugarcane. However, as an important component of sugarcane plants, sugarcane leaves are often directly burned during the leaf stripping process before the sugarcane is harvested by the sugarcane farmers, which not only causes great waste of resources, but also may have a negative impact on the environment.

[0003] As a rich agricultural waste resource, sugarcane leaves have diverse chemical components, including reducing sugars, polysaccharides, amino acids, etc. Among them, sugarcane leaf polysaccharides have attracted much attention due to their significant biological activities. Sugarcane leaf polysaccharides have various biological functions such as antioxidant, antibacterial, anti-inflammatory, and hypoglycemic effects, which make them have broad application prospects in the field of biological medicine. However, despite the potential application value of sugarcane leaf polysaccharides, there are relatively few reports on their extraction process and application, which limits the effective development and utilization of sugarcane leaf resources.

[0004] Xylan, as an important component of sugarcane polysaccharides, is a functional prebiotic with various physiological functions such as regulating intestinal flora, enhancing immune function, regulating lipid metabolism, improving antioxidant capacity, preventing dental caries, regulating blood sugar, and anti-tumor. These characteristics of xylan make it have broad application potential in food, medicine and other fields, and have high market added value. Therefore, developing an efficient and environmentally friendly xylan extraction process is of great significance to improve the comprehensive utilization value of sugarcane leaves.

[0005] Currently, the treatment of sugarcane residue has formed a relatively mature system, but the efficient and reasonable utilization of sugarcane leaves is still in the exploratory stage. Traditional xylan extraction methods may have low extraction efficiency and produce many by-products.

[0006] In addition, with the increasing problem of antibiotic resistance, functional oligosaccharides, as a substitute for antibiotics, have attracted widespread attention due to their similar physiological effects. Xylan, as a functional oligosaccharide, has certain research significance for inhibiting the growth of foodborne pathogenic microorganisms such as Escherichia coli and Staphylococcus aureus.

[0007] In summary, in view of the important position of sugarcane in the agriculture of Guangxi Zhuang Autonomous Region, and the multiple biological activities and wide application potential of sugarcane leaf xylan, developing an efficient and environmentally friendly ultrasonic-assisted enzymatic hydrolysis method for extracting sugarcane leaf xylan is not only of great significance for optimizing the utilization of sugarcane leaf resources, but also is expected to provide new raw material sources and product development ideas for the fields of biological medicine, food, etc. SUMMARY

[0008] The present application aims to overcome the deficiencies in the prior art, and provides a method for extracting sugarcane leaf xylan by ultrasonic-assisted enzymatic hydrolysis.

[0009] To achieve the above-mentioned object, the technical solution provided by the present application is as follows: A method for extracting sugarcane leaf xylan by ultrasonic-assisted enzymatic hydrolysis, comprising the following steps: Step S1, pretreatment of sugarcane leaves: crushing the sugarcane leaves, soaking them in a sodium hydroxide solution for 10-14 hours, washing them and drying them to a constant weight; Step S2, enzyme treatment: taking the pretreated sugarcane leaf powder, adding cellulase and xylanase, and then adding distilled water, and simultaneously performing ultrasonic treatment during the enzymatic hydrolysis; after the enzymatic hydrolysis is completed, boiling the enzyme solution in a water bath to inactivate the enzyme, and then centrifuging to obtain the supernatant, which is the sugarcane leaf xylan solution.

[0010] Preferably, in step S1, the sugarcane leaves are crushed to 200 mesh.

[0011] Preferably, in step S1, the molar concentration of the sodium hydroxide solution is 2 mol / L, and the soaking time is 12 hours.

[0012] Preferably, in step S1, after washing with distilled water, the drying is performed at 60°C to a constant weight.

[0013] Preferably, in step S2, the ratio of cellulase to xylanase is (0.5-4):1; more preferably, the ratio of cellulase to xylanase is 1.5:1.

[0014] Preferably, in step S2, the addition amount of cellulase and xylanase is 1%-2.5% of the sugarcane leaf powder; more preferably, the addition amount of cellulase and xylanase is 2% of the sugarcane leaf powder.

[0015] Preferably, in step S2, the mass ratio of distilled water to sugarcane leaf powder is (15-30):1; more preferably, the mass ratio of distilled water to sugarcane leaf powder is 15:1.

[0016] Preferably, in step S2, the ultrasonic treatment time is 10 minutes-25 minutes; more preferably, the ultrasonic treatment time is 15 minutes.

[0017] The application also provides the use of the xylan extracted according to the above method for inhibiting Escherichia coli and Staphylococcus aureus.

[0018] Compared with the prior art, the application has the following beneficial effects: The application provides an innovative ultrasonic-assisted enzymatic hydrolysis method for efficiently extracting xylan from sugarcane leaves. The method ingeniously combines the complex enzymatic hydrolysis of cellulase and xylanase, and is assisted by ultrasonic technology, which significantly improves the enzymatic hydrolysis efficiency, so that the previously low-utilization agricultural waste of sugarcane leaves is effectively developed and utilized. This technology not only realizes the rational utilization of resources and reduces waste, but also opens up a new way for the value-added utilization of sugarcane leaves. In addition, the application also finds that the extracted xylan has the biological activity of inhibiting Escherichia coli and Staphylococcus aureus, which further expands the application range of xylan and provides a scientific basis for its potential application in food preservation, medicine and health, etc. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 D-xylose standard curve Figure 2 Effect of enzyme addition amount on xylan extraction rate Figure 3 Effect of liquid-to-material ratio on xylan extraction rate Figure 4 Effect of ultrasonic treatment time on xylan extraction rate Figure 5 Effect of complex enzyme ratio on xylan extraction rate Figure 6 Effect of complex enzyme on xylan extraction Figure 7 Inhibition of four different concentrations of sample solutions on Escherichia coli and Staphylococcus aureus DETAILED DESCRIPTION

[0020] The application will be described in detail below with specific embodiments, but it should be understood that the protection scope of the application is not limited by the specific embodiments. The raw materials used in the examples are commercially available unless otherwise specified.

[0021] Example 1: Ultrasonic-assisted enzymatic hydrolysis method for extracting xylan from sugarcane leaves Step S1, pretreatment of sugarcane leaves: crush the sugarcane leaves to 200 mesh, soak in 2 mol / L sodium hydroxide solution at room temperature for 12 h, rinse with distilled water to remove sucrose, dry at 60℃ to constant weight, take out the dried solid and crush again; Step S2, enzyme treatment: take the pretreated sugarcane leaf powder, add cellulase and xylanase, and then add distilled water. Ultrasonic treatment is performed simultaneously with enzyme hydrolysis. After enzyme hydrolysis, the enzyme solution is boiled in a water bath to inactivate the enzyme. The supernatant is obtained by centrifugation. The obtained supernatant is the xylan solution of sugarcane leaves. The ratio of cellulase to xylanase is 1.5:1. The addition amount of cellulase and xylanase is 2% of the sugarcane leaf powder. The mass ratio of distilled water to sugarcane leaf powder is 15:1. The ultrasonic treatment time is 15 min.

[0022] Example 2: Ultrasonic-assisted enzymatic hydrolysis method for extracting xylan from sugarcane leaves Step S1, pretreatment of sugarcane leaves: The sugarcane leaves are crushed to 200 mesh, soaked in 2 mol / L sodium hydroxide solution at room temperature for 10 h, washed with distilled water to remove sucrose, and dried at 60°C to constant weight. The dried solid is taken out and crushed again. Step S2, enzyme treatment: take the pretreated sugarcane leaf powder, add cellulase and xylanase, and then add distilled water. Ultrasonic treatment is performed simultaneously with enzyme hydrolysis. After enzyme hydrolysis, the enzyme solution is boiled in a water bath to inactivate the enzyme. The supernatant is obtained by centrifugation. The obtained supernatant is the xylan solution of sugarcane leaves. The ratio of cellulase to xylanase is 1.5:1. The addition amount of cellulase and xylanase is 2% of the sugarcane leaf powder. The mass ratio of distilled water to sugarcane leaf powder is 15:1. The ultrasonic treatment time is 15 min.

[0023] Example 2: Ultrasonic-assisted enzymatic hydrolysis method for extracting xylan from sugarcane leaves Step S1, pretreatment of sugarcane leaves: The sugarcane leaves are crushed to 200 mesh, soaked in 2 mol / L sodium hydroxide solution at room temperature for 10 h, washed with distilled water to remove sucrose, and dried at 60°C to constant weight. The dried solid is taken out and crushed again. Step S2, enzyme treatment: take the pretreated sugarcane leaf powder, add cellulase and xylanase, and then add distilled water. Ultrasonic treatment is performed simultaneously with enzyme hydrolysis. After enzyme hydrolysis, the enzyme solution is boiled in a water bath to inactivate the enzyme. The supernatant is obtained by centrifugation. The obtained supernatant is the xylan solution of sugarcane leaves. The ratio of cellulase to xylanase is 1.5:1. The addition amount of cellulase and xylanase is 2% of the sugarcane leaf powder. The mass ratio of distilled water to sugarcane leaf powder is 15:1. The ultrasonic treatment time is 15 min.

[0024] Example 3: Ultrasonic-assisted enzymatic hydrolysis method for extracting xylan from sugarcane leaves Step S1, pretreatment of sugarcane leaves: The sugarcane leaves are crushed to 200 mesh, soaked in 2 mol / L sodium hydroxide solution at room temperature for 10 h, washed with distilled water to remove sucrose, and dried at 60°C to constant weight. The dried solid is taken out and crushed again. Step S2, enzyme treatment: taking the pretreated sugarcane leaf powder, adding cellulase and xylanase, and then adding distilled water, and performing ultrasonic treatment while performing enzyme hydrolysis, and after the enzyme hydrolysis is completed, boiling the enzyme hydrolysis liquid in a boiling water bath to inactivate the enzyme activity, and centrifuging to obtain the supernatant, wherein the cellulase and xylanase are in a ratio of 4:1, the cellulase and xylanase are added in an amount of 2.5% of the sugarcane leaf powder, the mass ratio of distilled water to sugarcane leaf powder is 30:1, and the ultrasonic treatment time is 25 min.

[0025] Example 4: Establishment of determination method for reducing sugar content and total soluble sugar 1. Determination of the maximum absorption peak of xylan 0.05 g of D-xylose standard powder was precisely weighed and dissolved in 50 mL of pure water to prepare a standard D-xylose solution with a concentration of 1 mg / mL. Then, the ultraviolet spectrum was determined by full wavelength scanning with a UV-visible spectrophotometer to determine the maximum absorption peak.

[0026] 2. Preparation of xylan standard curve 0.05 g of D-xylose standard powder was precisely weighed and dissolved in 50 mL of distilled water to prepare a standard D-xylose solution with a concentration of 1 mg / mL. 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the 1 mg / mL D-xylose standard solution were accurately measured and then injected into seven different 25 mL volumetric flasks. Then, 2 mL of distilled water and 1.5 mL of DNS solution were accurately added to each volumetric flask. To ensure thorough mixing, the volumetric flasks were shaken thoroughly. Then, the volumetric flasks were placed in a boiling water bath for 5 minutes, and after heating was completed, they were quickly cooled to room temperature. Finally, the absorbance values of the solutions in each volumetric flask were measured at the determined maximum absorption wavelength. The standard curve of the D-xylose solution was established with the solution concentration c (mg / mL) as the horizontal coordinate and the absorbance value A as the vertical coordinate.

[0027] After full wavelength scanning, the maximum absorption peak of xylan is located at 520 nm. To carry out subsequent determination and analysis, the following steps are taken: first, 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of 1 mg / mL D-xylose standard solution are respectively taken; then, 1.5 mL of DNS standard solution is added to each sample; then, these mixtures are placed in a boiling water bath for reaction, and the duration is 5 minutes; after the reaction is completed, the reaction solution is quickly cooled, and accurately diluted to 25 mL in a volumetric flask. It is worth noting that since the sugar content of the DNS reaction solution is fixed at 40 mg / L, after the above operation, the sugar content of each sample corresponds to 0 mg / L, 8 mg / L, 16 mg / L, 24 mg / L, 32 mg / L and 40 mg / L respectively. The accurate execution of this step is crucial for the accuracy of the subsequent experiment. The standard curve of the measured absorbance at 520 nm with 0 as the reference is plotted as shown in Figure 1 The relationship between the obtained absorbance and the concentration of xylose solution is: y = 18.069x + 0.014, R² = 0.9973.

[0028] 3. DNS method for determining the content of reducing sugar In a volumetric flask, 7 mL of the measured sugarcane leaf xylan sample solution is accurately taken, 1.5 mL of 3, 5-dinitrosalicylic acid (DNS) standard solution is mixed with 2 mL of distilled water, and is placed in a boiling water bath for heating for 5 minutes. After it is fully colored, it is quickly cooled and immediately diluted to 25 mL of capacity. At the same time, another 25 mL volumetric flask is taken, the same amount of DNS solution is added, and is diluted to the mark with distilled water as a blank control solution. Then, using a 1 cm cuvette, the absorbance is measured at the maximum absorption wavelength of the D-xylose standard, and the content of xylan is calculated accordingly. According to the standard curve, the concentration of xylan and reducing sugar is calculated.

[0029] 4. Determination of soluble total sugar 1.5 mL of concentrated sulfuric acid with a mass concentration of 98% is added to the enzyme hydrolyzed sugarcane leaf supernatant, and is boiled in a water bath for 20 minutes. After it is cooled, it is neutralized with 6 mol / L sodium hydroxide solution to neutral, and the content of soluble total sugar is determined by DNS method.

[0030] 5. Formula of the degree of polymerization of xylan DP = total sugar content / reducing sugar content Example 5: Investigation of the influence of different factors on the extraction rate of xylan 1. The influence of different enzyme addition amounts on the extraction rate of xylan The experiment was divided into 4 groups, 2.0 g of pretreated sugarcane leaf powder was weighed by an analytical balance, 1.0% (0.02 g), 1.5% (0.03 g), 2.0% (0.04 g) and 2.5% (0.05 g) of complex enzyme (mass ratio of cellulase and xylanase 1:1) was added according to the mass of the weighed sugarcane leaf powder, distilled water was added according to the liquid-solid ratio of 25:1, the ultrasonic time was 20 min, after enzyme hydrolysis, the enzyme activity was inactivated by boiling water bath for 10 min. The extract was placed in a centrifuge for 15 minutes of centrifugation. After centrifugation, the supernatant was determined for reducing sugar content and soluble total sugar content in order to further calculate the average degree of polymerization of xylan. The change of xylan extraction rate with enzyme addition amount and the degree of polymerization of xylan were investigated. The test results are shown in Figure 2 , in Figure 2 , the vertical axis represents the xylan extraction rate, it can be seen that under the condition that other conditions are unchanged, the xylan extraction rate is the highest when the enzyme addition amount is 1.5% (0.03 g).

[0031] 2, the effect of different liquid-solid ratios on xylan extraction rate The experiment was divided into 4 groups, 2.0 g of pretreated sugarcane leaf powder was weighed by an analytical balance, 2.0% (0.04 g) of complex enzyme (mass ratio of cellulase and xylanase 1:1) was added according to the mass of the weighed sugarcane leaf powder, distilled water was added according to the liquid-solid ratio of 15:1, 20:1, 25:1 and 30:1, the ultrasonic time was 20 min, after enzyme hydrolysis, the enzyme activity was inactivated by boiling water bath for 10 min. The extract was placed in a centrifuge for 15 minutes of centrifugation. After centrifugation, the supernatant was determined for reducing sugar content and soluble total sugar content in order to further calculate the average degree of polymerization of xylan. The change of xylan extraction rate with enzyme addition amount and the degree of polymerization of xylan were investigated. The test results are shown in Figure 3 , in Figure 3 , the vertical axis represents the xylan extraction rate, it can be seen that under the condition that other conditions are unchanged, the xylan extraction rate is the highest when the liquid-solid ratio is 1:25.

[0032] 3, the effect of different ultrasonic treatment times on xylan extraction rate The experiment was divided into 4 groups, 2.0 g of pretreated sugarcane leaf powder was weighed by an analytical balance, 2.0% (0.04 g) of composite enzyme (cellulase and xylanase mass ratio 1:1) was added according to the mass of the weighed sugarcane leaf powder, distilled water was added according to the liquid-solid ratio 25:1, and the ultrasonic treatment time was 10, 15, 20 and 25 min respectively. After enzyme hydrolysis, the enzyme activity was inactivated by boiling water bath for 10 min. The extract was placed in a centrifuge for 15 minutes of centrifugation. After centrifugation, the supernatant was determined for reducing sugar content and soluble total sugar content in order to further calculate the average degree of polymerization of xylan. The change of xylan extraction rate with enzyme addition amount and the degree of polymerization of xylan were investigated. The test results are shown in Figure 4 , in Figure 4 , the vertical axis represents the xylan extraction rate, and it can be seen that the xylan extraction rate is the highest when the ultrasonic treatment time is 15 min under the condition that other conditions remain unchanged.

[0033] 4, the influence of different composite enzyme ratios on the extraction rate of xylan The experiment was divided into 4 groups, 2.0 g of pretreated sugarcane leaf powder was weighed by an analytical balance, 2.0% (0.04 g) of composite enzyme (cellulase and xylanase mass ratio respectively 4:1, 2:1, 1:1 and 1:2) was added according to the mass of the weighed sugarcane leaf powder, distilled water was added according to the liquid-solid ratio 25:1, and the ultrasonic treatment time was 20 min. After enzyme hydrolysis, the enzyme activity was inactivated by boiling water bath for 10 min. The extract was placed in a centrifuge for 15 minutes of centrifugation. After centrifugation, the supernatant was determined for reducing sugar content and soluble total sugar content in order to further calculate the average degree of polymerization of xylan. The change of xylan extraction rate with enzyme addition amount and the degree of polymerization of xylan were investigated. The test results are shown in Figure 5 , in Figure 5 , the vertical axis represents the xylan extraction rate, and it can be seen that the xylan extraction rate is the highest when the mass ratio of cellulase to xylanase is 2:1 under the condition that other conditions remain unchanged.

[0034] Example 6 orthogonal experiment Based on the above single factor experiment results, the single factor experiment scheme and results were combined, the three factors with greater influence were selected as the orthogonal optimization experiment, namely enzyme addition amount, liquid-solid ratio and composite enzyme ratio, the extraction rate was taken as the index, and the operation was carried out according to the designed experiment, and the obtained results were analyzed. The scheme with the highest extraction rate was repeated for three parallel samples to verify the accuracy of the orthogonal experiment. The orthogonal scheme is shown in Table 1, and the data of the orthogonal experiment is shown in Table 2: Table 1 orthogonal experiment scheme

[0035] Table 2 Orthogonal experimental scheme results

[0036] The experiment 7 scheme with the highest extraction rate was repeated three times in parallel, and the obtained data are shown in Table 3.

[0037] Table 3 Parallel experiment results

[0038] Example 7 Effect of separate enzymolysis on extraction Three groups of 2.0 g of pretreated sugarcane leaf powder were weighed with an analytical balance, 2.0% (0.04 g) of cellulase, xylanase, xylanase and composite enzyme (cellulase:xylanase, mass ratio 1:1) were added according to the mass of the weighed sugarcane leaf powder, distilled water was added according to a liquid-to-material ratio of 25:1, and ultrasonic treatment was performed for 20 min. After the enzymolysis was completed, the enzyme activity was inactivated by boiling in a water bath for 10 min. The extraction liquid was placed in a centrifuge and centrifuged for 15 min. After centrifugation, the supernatant was subjected to determination of the content of reducing sugar and the content of soluble total sugar, so as to further calculate the average degree of polymerization of xylan. The change of xylan extraction rate with the amount of enzyme added was investigated, and the degree of polymerization of xylan was investigated. The test results are shown in Table 4. Figure 6 Figure 6 In Table 4, the vertical axis represents the xylan extraction rate, and it can be seen that the extraction effect of the composite enzyme is better than that of the enzyme alone under the condition that other conditions remain unchanged.

[0039] Example 8 Antibacterial performance experiment 1. Sample preparation The xylan solution prepared in Example 1 was taken as the sample 2. Sterilization The liquid medium to be used, the pipette gun head, distilled water and other materials needed were placed in a vertical pressure steam sterilizer for high-temperature and high-pressure sterilization, 3. Preparation of culture medium ​The bacteria inhibition experiment adopts the double dilution method. The culture medium is divided into solid medium and liquid medium. The solid medium is prepared by weighing 1.2 g of agar powder and 1.8 g of broth powder, diluting with 100 mL of pure water, placing in a sterilization pot for high-temperature and high-pressure sterilization, then pouring into a culture dish. The culture dish should not be shaken during pouring. Place it in a biochemical incubator for 10 hours until it solidifies. Then it is the solid medium ready for use. The strains of E. coli and S. aureus are transferred to the solid medium with a loop to culture a new generation of E. coli and S. aureus, which can be used for strain culture in liquid medium. The liquid medium is prepared by weighing 1.8 g of broth powder and diluting with 100 mL of pure water, then placing it in a sterilization pot for high-temperature and high-pressure sterilization to obtain the blank liquid medium ready for use. Take 10 mL of the blank liquid medium into two glass bottles, then use a loop to transfer the two prepared strains in the solid medium into the glass bottles respectively. Culture them in a 37°C shaking bed for 10 hours to obtain the culture medium containing E. coli and S. aureus ready for use. Seal the culture medium with plastic wrap and store it properly in the refrigerator.

[0040] 4. Determination of minimum inhibitory concentration: (1) Preparation of different concentrations of antibacterial drugs: The sugarcane leaf xylan solution of Example 1 to 3 is concentrated by rotary evaporation, and 2 mL of pure water is added as standby drug solution. In a 96-well polyethylene microplate, dilute according to the double dilution method to obtain different concentrations of antibacterial drug solution.

[0041] (2) Preparation of bacteria to be tested: Pick single colonies from the incubated overnight plate and inoculate in liquid medium. Culture to the logarithmic growth phase.

[0042] (3) Inoculation of bacteria to be tested: Add 100 μL of different concentrations of antibacterial drugs to each well of the 96-well polyethylene microplate, then add 100 μL of diluted bacteria to be tested in the logarithmic growth phase (106 CFU / mL) to each well. The final concentration is 5*105 CFU / mL. At the same time, set up negative (without bacteria and antibiotics) and positive (without antibiotics) controls.

[0043] (4) Incubation: Mix the microplate by shaking for 1 min, seal with plastic wrap, and incubate at 37°C for 16-24 h.

[0044] (5) Determination of the results of the bacteria inhibition experiment: Observe with the naked eye. The drug concentration without visible bacterial growth (clear broth) in the well is the inhibitory concentration of the test bacteria.

[0045] (6) Determination of the minimum inhibitory concentration: Dry the sample solutions extracted under the same conditions by rotary evaporation, respectively. Add 1 mL, 2 mL, 4 mL, and 6 mL of sterilized distilled water to dissolve them. Add different concentrations of sample solutions to the 96-well plate with liquid medium, and observe the growth of the two bacterial populations.

[0046] 5. Experimental Results By observing the bacterial populations and growth status in 96-well plates containing sample solutions of different concentrations and in blank culture medium, it was found that both bacterial populations could still grow at the maximum concentration in sample solutions diluted with 6 mL and 4 mL of distilled water. However, neither bacterial population could grow at the maximum concentration in sample solutions with 2 mL and 1 mL of distilled water added. Furthermore, as the sample concentration increased, the xylan content in the wells increased, and the antibacterial effect became more significant.

[0047] It can be seen that xylan extracted from sugarcane leaves has an inhibitory effect on the growth of Escherichia coli and Staphylococcus aureus. The maximum concentrations of the four solutions are shown in Table 4, and the antibacterial properties of the four concentrations of sample solutions are as follows: Figure 7 As shown. The calculated minimum inhibitory concentration (MIC) for both Escherichia coli and Staphylococcus aureus in the sugarcane xylan sample solution was 1.5 mg / mL.

[0048] Table 4. Antibacterial status of sample solutions at different concentrations

[0049] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for extracting xylan from sugarcane leaves using ultrasound-assisted enzymatic hydrolysis, characterized in that, Includes the following steps: Step S1, Sugarcane leaf pretreatment: Crush sugarcane leaves, soak them in sodium hydroxide solution for 10-14 hours, wash them and dry them to constant weight; Step S2, Enzyme treatment: Take the pretreated sugarcane leaf powder, add cellulase and xylanase, then add distilled water, and perform ultrasonic treatment while enzymatically hydrolyzing. After the enzymatic hydrolysis is completed, boil the hydrolysate in a water bath to inactivate the enzyme activity, centrifuge and take the supernatant. The supernatant contains the extracted xylan.

2. The method according to claim 1, characterized in that: In step S1, sugarcane leaves are crushed to 200 mesh, the molar concentration of sodium hydroxide solution is 2 mol / L, the soaking time is 12 h, and after washing with distilled water, they are dried at 60℃ to constant weight.

3. The method according to claim 1, characterized in that: In step S2, the ratio of cellulase to xylanase is 0.5 to 4:

1.

4. The method according to claim 1, characterized in that: In step S2, the ratio of cellulase to xylanase is 1.5:

1.

5. The method according to claim 1, characterized in that: In step S2, the amount of cellulase and xylanase added is 1% to 2.5% of the sugarcane leaf powder.

6. The method according to claim 1, characterized in that: In step S2, the amount of cellulase and xylanase added is 2% of the sugarcane leaf powder.

7. The method according to claim 1, characterized in that: In step S2, the mass ratio of distilled water to sugarcane leaf powder is 15~30:

1.

8. The method according to claim 1, characterized in that: In step S2, the mass ratio of distilled water to sugarcane leaf powder is 15:

1.

9. The method according to claim 1, characterized in that: In step S2, the ultrasonic treatment time is 10 min to 25 min.

10. Use of the sugarcane leaf xylan extracted by the method of claim 1, wherein the use is for inhibiting Escherichia coli and Staphylococcus aureus.

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