CN112480286A

CN112480286A - Method for preparing ganoderma lucidum beta-glucooligosaccharide by degrading ganoderma lucidum beta-glucan

Info

Publication number: CN112480286A
Application number: CN202011383417.2A
Authority: CN
Inventors: 刘艳芳; 张劲松; 秦秀; 周帅; 唐庆九; 王晨光; 冯杰; 颜梦秋; 张忠; 周靖
Original assignee: SHANGHAI BAIXIN BIO-TECH CO LTD; Shanghai Academy of Agricultural Sciences
Current assignee: SHANGHAI BAIXIN BIO-TECH CO LTD; Shanghai Academy of Agricultural Sciences
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2021-03-12

Abstract

The invention provides a method for preparing ganoderma lucidum beta-gluco-oligosaccharide by degrading ganoderma lucidum beta-glucan, which comprises the following steps: preparing ganoderma lucidum beta-glucan solution; homogenizing the ganoderma lucidum beta-glucan solution to uniformly disperse the ganoderma lucidum beta-glucan in the solution; placing the ganoderma lucidum beta-glucan solution in a microwave digestion instrument, setting the microwave power at 800-; and centrifuging to remove insoluble impurities after degradation to obtain the ganoderma lucidum beta-gluco-oligosaccharide. Compared with the traditional degradation method, the molecular weight of the product is obviously reduced, the degradation efficiency is high, the oligosaccharide composition in the product is rich, the post-treatment of the product is simple, and the pollution to the environment is less.

Description

Method for preparing ganoderma lucidum beta-glucooligosaccharide by degrading ganoderma lucidum beta-glucan

Technical Field

The application relates to the field of chemistry, in particular to a method for preparing ganoderma lucidum beta-glucooligosaccharide by degrading ganoderma lucidum beta-glucan.

Background

Ganoderma lucidum has the names of 'immortal grass' and 'Ruilao' from ancient times, the ganoderma lucidum is considered by people to prolong life in ancient times, the ganoderma lucidum is rich in nutrition and high in medicinal value, and has more than 4000 years of traditional Chinese medicine application history in China. Ganoderma beta-glucan is one of the most abundant components in Ganoderma, and has wide pharmacological effects such as immunoregulation, anticancer, antioxidant and antiinflammatory effects. Ganoderma beta-glucan is a biological macromolecule, the main chain structure of the Ganoderma beta-glucan is beta-1, 3-glucan, the side chain is beta-1, 6-glucopyranose group, usually adopt the method of water extraction and alcohol precipitation to get, as the high molecular weight polysaccharide, the Ganoderma beta-glucan has poor water solubility and high viscosity, this has greatly hindered its clinical wide application. The literature indicates that the biological activity of the oligosaccharide obtained by degrading polysaccharide is improved, so that the ganoderma lucidum beta-glucan is degraded into oligosaccharide, and the oligosaccharide is considered to have higher clinical application potential. The commonly used degradation methods at present mainly comprise a chemical method, a physical method, an enzymatic method and the like, and the methods are widely applied to microorganisms, grains and yeast beta-glucan, but the degradation effect of ganoderma lucidum beta-glucan is still rarely reported. Because different beta-glucans have differences in molecular weight, structure, physical characteristics and the like, the degradation effects of the beta-glucans also have obvious differences, and the research on the degradation of the beta-glucan of the ganoderma lucidum is less at present, the search for a method for effectively degrading the beta-glucan of the ganoderma lucidum is very important.

Disclosure of Invention

The invention obtains the ganoderma lucidum beta-glucooligosaccharides by degrading from the ganoderma lucidum beta-glucan through a simple and efficient microwave degradation method, the degradation rate is determined by adopting a chemical method, and the molecular weight and the polymerization degree distribution after degradation are determined by a method combining chromatographic analysis and mass spectrometry. The method has high degradation rate, and can obtain multiple oligosaccharide fragments with specific molecular weight sections.

The invention aims to provide a simple and efficient microwave degradation method of ganoderma lucidum beta-glucan with less pollution, and the product obtained by the method has high degradation rate and rich oligosaccharide composition.

The invention provides a degradation method of ganoderma lucidum beta-glucan, which comprises the following steps:

1. preparing ganoderma lucidum beta-glucan with the concentration of 5-30 mg/mL;

2. homogenizing the solution to uniformly disperse the Ganoderma beta-glucan in the solution;

3. placing the solution in a microwave digestion instrument, setting the microwave power at 800-;

4. and centrifuging to remove insoluble impurities after degradation, thus obtaining the ganoderma lucidum beta-gluco-oligosaccharide.

The invention has the following beneficial effects:

1) the beta-glucan degraded by the microwave method has good reproducibility and stable product quality;

2) the invention has simple process, short flow, low cost and greatly shortened degradation time;

3) compared with the traditional degradation method, the molecular weight of the product is obviously reduced, the degradation efficiency is high, the oligosaccharide composition in the product is rich, the post-treatment of the product is simple, and the pollution to the environment is less.

Drawings

FIG. 1 shows a high performance gel exclusion chromatogram of microwave degradation products of Ganoderma lucidum beta-glucan of example 1.

FIG. 2 shows a high performance anion chromatogram of microwave degradation products of Ganoderma lucidum beta-glucan of example 1.

FIG. 3 shows the mass spectrum of the microwave degradation product of Ganoderma lucidum beta-glucan of example 1.

FIG. 4 shows a high performance anion chromatogram of microwave degradation products of Ganoderma lucidum beta-glucan of example 2.

FIG. 5 shows a high performance gel exclusion chromatogram of microwave degradation products of Ganoderma lucidum beta-glucan of example 3.

Detailed Description

The following examples are presented to enable those skilled in the art to more fully understand the present application and are not intended to limit the present application in any way. Unless otherwise indicated, all reagents used in this application are commonly available commercial reagents.

Example 1: degradation of ganoderma lucidum beta-glucan

Weighing Ganoderma beta-glucan GLP206 g, adding 700mL distilled water, and preparing into 8.57mg/mL Ganoderma beta-glucan solution. Heating in water bath at 100 deg.C for 30min, cooling to room temperature, and homogenizing for 3min to disperse Ganoderma beta-dextran in the solution. The solution is placed in a microwave digestion instrument, the microwave power is adjusted to 1000W, and the solution is degraded for 50min at 140 ℃. Centrifuging the degradation product with a centrifuge at 8000rpm for 20min, taking out supernatant, and freeze drying to obtain light yellow solid product.

Example 2: degradation of ganoderma lucidum beta-glucan

Weighing Ganoderma beta-glucan GLP206 g, adding 1200mL of distilled water, and preparing into 5mg/mL Ganoderma beta-glucan solution. Heating in water bath at 100 deg.C for 30min, cooling to room temperature, and homogenizing for 3min to disperse Ganoderma beta-dextran in the solution. The solution is placed in a microwave digestion instrument, the microwave power is adjusted to 1000W, and the solution is degraded for 60min at 120 ℃. Centrifuging the degradation product with a centrifuge at 8000rpm for 20min, taking out supernatant, and freeze drying to obtain light yellow solid product.

Example 3: degradation of ganoderma lucidum beta-glucan

Weighing Ganoderma beta-glucan GLP206 g, adding 200mL distilled water, and preparing into 30mg/mL Ganoderma beta-glucan solution. Heating in water bath at 100 deg.C for 30min, cooling to room temperature, and homogenizing for 3min to disperse Ganoderma beta-dextran in the solution. The solution is placed in a microwave digestion instrument, the microwave power is adjusted to 1000W, and degradation is carried out for 40min at 180 ℃. Centrifuging the degradation product with a centrifuge at 8000rpm for 20min, taking out supernatant, and freeze drying to obtain light yellow solid product.

The degradation rate is measured by combining alcohol precipitation separation with a phenol-sulfuric acid method, and the calculation formula is as follows:

in the formula: a. the₁The supernatant sugar content is obtained after 50 percent ethanol precipitation; a. the₂The sugar content was precipitated after 50% ethanol precipitation.

Alcohol precipitation separation: adding absolute ethanol into the degradation product while stirring until the alcohol concentration is 50%, standing at 4 deg.C overnight, centrifuging at 8000rpm for 20min, respectively collecting supernatant and precipitate, concentrating under reduced pressure to remove ethanol, and measuring the sugar content in the supernatant and precipitate by phenol-sulfuric acid method.

Determination of sugar content by phenol-sulfuric acid method:

measurement of a standard curve: accurately sucking 0.1mg/mL glucose standard solution 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mL into a test tube, adding water to complement to 1mL, respectively adding 0.5mL 5% phenol and 2.5mL concentrated sulfuric acid, uniformly mixing, heating in a water bath at 100 ℃ for 15min, cooling to room temperature, and measuring the absorbance value at 490 nm. And (5) plotting the ultraviolet absorbance to the concentration of the glucose standard solution to obtain a glucose concentration standard curve and a regression equation.

Measuring the sugar content of the sample: accurately sucking 1mL of sample solution of about 0.1mg/mL, adding 0.5mL of 5% phenol and 2.5mL of concentrated sulfuric acid, mixing uniformly, heating in a water bath at 100 ℃ for 15min, cooling to room temperature, and measuring the absorbance value at 490 nm. And calculating the sugar content of the sample according to a regression equation of a glucose standard curve.

The degradation rate of microwave degradation of ganoderma lucidum beta-glucan is higher than 94% by adopting an alcohol precipitation separation and phenol-sulfuric acid method.

Separating by high performance GEL chromatography (Waters 2695 pump separation system) series TSK-GEL series G6000PWXL and G4000PWXL chromatographic column (7.8mm × 300mm, TOSOH, Japan) and analyzing by Waters 2414 differential detector (RI), the molecular weight of Ganoderma beta-glucan as it is 2.28 × 10⁶g/mol, the molecular weight of the degraded product is obviously reduced, and the result is consistent with the degradation rate result.

The degradation products were separated by high performance anion chromatography (ICS-5000 + ion chromatography system of Dionex corporation) using a CarbopacTM PA-100 column (4 mm. times.250 mm) and analyzed by a pulsed amperometric detector, and the results are shown in FIGS. 2 and 4, in which the degradation products had a high oligosaccharide content.

The polymerization degree of the product was measured by matrix-assisted laser desorption tandem flight time mass spectrometry (AB SCIEX 5800MALDI-TOF/TOF system), and the result is shown in FIG. 3, where the polymerization degree of the product obtained by microwave degradation was 3-19.

As shown in FIGS. 1 to 5, the microwave degradation product has a chromatographic peak at the original position which is basically disappeared and the molecular weight is obviously reduced by the high performance gel exclusion chromatography, which shows that the microwave method has obvious degradation effect on the ganoderma lucidum beta-glucan, and the degradation rate of the product is higher than 94% by the phenol-sulfuric acid method, which is consistent with the liquid phase result. The degradation products are analyzed by high performance anion chromatography and mass spectrometry (as shown in figure 2 and figure 3), and the results show that the types of oligosaccharides in the microwave degradation products are rich, and the polymerization degree of the products is 3-19.

The results obtained in examples 2 and 3 were similar to those obtained in example 1, except that the respective measurements were carried out.

Those skilled in the art will appreciate that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the application.

Claims

1. A method for preparing ganoderma lucidum beta-glucooligosaccharide by degrading ganoderma lucidum beta-glucan is characterized by comprising the following steps:

preparing ganoderma lucidum beta-glucan solution;

homogenizing the ganoderma lucidum beta-glucan solution to uniformly disperse the ganoderma lucidum beta-glucan in the solution;

placing the ganoderma lucidum beta-glucan solution in a microwave digestion instrument, setting the microwave power at 800-;

and centrifuging to remove insoluble impurities after degradation to obtain the ganoderma lucidum beta-gluco-oligosaccharide.

2. The method according to claim 1, wherein the concentration of the ganoderma lucidum beta-glucan solution is 5-30 mg/mL.