CN101434974B - Method for preparing soluble beta-1,3-oligoglucoside by using yeast - Google Patents

Abstract

The invention relates to a method for preparing soluble beta-1,3-glucan oligosaccharides by using yeast. The method includes alkali pretreatment, acid hydrolysis and separation and purification, wherein the separation and purification is to dissolve the soluble β-1,3-glucan oligosaccharide obtained in the acid hydrolysis, and pass through the AG50W-X4 chromatographic column and A molecular sieve chromatography system composed of Bio-Gel P4 chromatography columns connected in series for separation and purification. The method for preparing β-1,3-glucan oligosaccharides provided by the present invention is relatively simple to operate, and the obtained oligosaccharides are of more types, the preparation amount is larger, and the operation can be repeated, so it is suitable for large-scale industrial production; and because the raw materials of the method It comes from yeast, and no toxic chemical reagents are added in the production process, so the products produced by this method can be used in food, medicine and other fields.

Description

A method for preparing soluble β-1,3-glucan oligosaccharides by using yeast

technical field

The invention relates to a preparation method of β-1, 3-glucan oligosaccharides, in particular to a method of using yeast to prepare soluble β-1, 3-glucan oligosaccharides, which belongs to the technical field of biotechnology.

Background technique

β-1,3-glucan can enhance the ability of organisms to resist infectious diseases caused by bacteria, fungi, viruses and parasites, and also has anti-tumor activity. β-glucan is used as a food additive in the food industry to provide fiber and short-chain fatty acids, which can inhibit intestinal bacteria, ensure the normal work of peristaltic muscles, reduce serum cholesterol and triglyceride levels, thereby reducing intestinal The role of cancer and digestive disease incidence.

The main component of the yeast cell wall is β-1,3-glucan, and the insoluble β-1,3-glucan (Zymosan) can be obtained from the yeast cell wall by alkaline extraction. This glucan has the above-mentioned biological activity, but studies have found that insoluble β-1, 3-glucan can produce certain toxicity to organisms and easily cause inflammation, while soluble β-1, 3-glucan maintains It is relatively safer while being biologically active. So soluble β-1,3-glucan has a good application prospect.

The Chinese patent with the publication number CN1373134 discloses β-1,3 oligoglucan and its preparation method and application. It uses glucose as a raw material to prepare β-1,3 oligoglucan through chemical synthesis steps. However, since a variety of chemical synthesis reagents need to be added in the chemical synthesis process, the prepared product contains some toxic and harmful components to organisms, which cannot be applied to fields such as medicine and food.

Jamas et al. used partial acid hydrolysis to prepare soluble β-1,3-glucan from yeast cell walls, and then separated it by membrane separation technology to obtain soluble glucans in different molecular weight ranges; Micheal et al. also used partial acid water Soluble oligosaccharides were prepared from yeast cell walls and separated in two steps by Bio-Gel P4 and P2 chromatography media at different temperatures. However, the above-mentioned separation and purification method for soluble sugars is relatively complicated to operate, and the types of oligosaccharides obtained are relatively small, and the preparation amount is low, so it is difficult to apply in large-scale production.

Contents of the invention

Aiming at the deficiencies of the prior art, the invention provides a method for preparing soluble β-1,3-glucan oligosaccharides by using yeast.

Summary of the invention

The present invention prepares soluble glucan from insoluble glucan by partial acid hydrolysis, the main component is β-1,3-dextran oligosaccharide, and then realizes a one-step process by connecting AG50W-X4 and Bio-Gel P4 chromatography columns in series Isolation and Purification.

Detailed description of the invention

A method for preparing soluble β-1,3-glucan oligosaccharides by using yeast, comprising alkali pretreatment of yeast sludge, acid hydrolysis of alkali-insoluble β-1,3-glucan and soluble β-1,3-glucan oligosaccharides Sugar separation and purification. It is characterized in that the separation and purification is to dissolve the dried soluble β-1,3-glucan oligosaccharides, and then carry out separation and purification through a molecular sieve chromatography system. The molecular sieve chromatography system is mainly composed of an AG50W-X4 chromatography column It is connected in series with the Bio-Gel P4 chromatography column. The soluble β-1,3-dextran oligosaccharide solution first enters the AG50W-X4 chromatography column, and is carried into the Bio-Gel P4 chromatography column by the mobile phase. From the Bio-Gel Fractions were collected from the top of the P4 chromatography column.

The AG50W-X4 chromatographic column and the Bio-Gel P4 chromatographic column both have jackets, and circulating hot water is introduced into the jackets to maintain the column temperature at 65-75°C to ensure the separation effect. The mobile phase is distilled water, which first enters from the top of the AG50W-X4 chromatography column, flows out from the bottom of the AG50W-X4 chromatography column, then enters the Bio-Gel P4 chromatography column from the bottom of the Bio-Gel P4 chromatography column, and flows out from the bottom of the Bio-Gel P4 chromatography column. The top of the chromatography column flows out. The temperature of the mobile phase is 65-75°C, the flow rate of the mobile phase is 1-2ml/min, and a tube of components is collected from the top of the Bio-GelP4 chromatography column every 5-8 minutes, and the components with a retention time of 3-24 hours are collected.

Described alkali pretreatment step concrete operation is as follows:

(1) After filtering the yeast sludge to remove impurities and washing it with water, suspend it in 4wt% NaOH solution, stir vigorously in a water bath at 90-100°C for 0.75-1h, centrifuge at 2000-5000 rpm, remove the supernatant, and take the precipitate;

(2) Suspend the precipitate obtained in step (1) in 3wt% NaOH solution, stir in a water bath at 75 to 80°C for 2.5 to 3.5 hours, adjust the pH of the solution to 3.5 to 5.5 with concentrated hydrochloric acid, and stir in a water bath at 75 to 80°C for 1 hour ~2h, cooled to room temperature, continued to stir and extract for 14~16h, centrifuged to obtain precipitate;

(3) The precipitate obtained in step (2) is washed with water and absolute ethanol respectively, and dried to obtain powdered alkali-insoluble β-1,3-glucan.

Described acid hydrolysis step concrete operation is as follows:

(1) Dissolve the alkali-insoluble β-1,3-glucan obtained from the alkali pretreatment step in anhydrous formic acid, place it in a water bath at 80-90°C, stir for 20-60 minutes, and dissolve it with 1-3 times the volume of absolute ethanol The sugar dissolved in formic acid precipitates and is centrifuged to obtain the precipitate;

(2) Dissolving the precipitate obtained in step (1) in distilled water, centrifuging to obtain a supernatant to obtain a soluble β-1,3-glucan oligosaccharide solution, and drying to obtain a soluble β-1,3-glucan oligosaccharide.

Preferably, the drying in the acid hydrolysis step (2) is to concentrate the soluble β-1,3-glucan oligosaccharide solution by a vacuum rotary evaporator, and then obtain the soluble β-1,3-glucan oligosaccharide through a freeze-drying step.

The above-mentioned raw materials and chemical reagents are all commercially available products unless otherwise specified. All the operations of the present invention that are not limited in detail are performed according to existing routines in the art.

The beneficial effects of the present invention are as follows:

The method for preparing β-1,3-glucan oligosaccharides provided by the present invention is relatively simple to operate, and the obtained oligosaccharides have more types, a larger amount of preparation, and can be repeatedly operated, so it is suitable for large-scale industrial production; and because the raw materials of the method It comes from yeast, and no toxic chemical reagents are added in the production process, so the products produced by this method can be used in food, medicine and other fields. The invention has application prospect in preparing high-value oligosaccharide products by using waste yeast in breweries.

Description of drawings

Fig. 1 is the structure diagram of molecular sieve chromatography system; Wherein: 1, chromatography column (AG50W-X4), 2, chromatography column (Bio-Gel P4), 3, jacket, 4, constant temperature water bath, 5, flow Phase, 6. Peristaltic pump, 7. Circulating hot water pipeline, 8. Mobile phase pipeline, 9. Automatic collector

Fig. 2 is the composition of oligosaccharide mixture detected by high performance liquid chromatography; wherein: curve 1 is the β-1,3-glucan oligosaccharide standard obtained by partial acid hydrolysis of laminarin (Laminarin, purchased from Sigma); curve 2 is the yeast Soluble β-1,3-glucan oligosaccharide samples prepared from cell walls.

Figure 3 is the detection of the total sugar content in the collected fractions by the phenol-sulfuric acid method.

Fig. 4 is the purity of some components obtained by high performance liquid chromatography detection; wherein: curves 1 to 5 are molecular sieve chromatography separation components (corresponding to peaks 1 to 5 in Fig. 3); curve 6 is the soluble β - 1,3-Oligodextran sample.

Figure 5 shows the types of glycosidic bonds in oligosaccharides separated by infrared spectroscopic analysis.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited thereto.

Reagents and instruments:

Laminarin (Laminarin, purchased from Sigma)

High performance liquid chromatography column (Aminex HPX-42A Carbohydrate Column, purchased from Bio-Rad company)

AG50W-X4 chromatographic column (chromatographic column model: XK50/30, purchased from Pharmacia Company; filler model: AG50W-X4, purchased from Bio-Rad Company)

Bio-Gel P4 chromatographic column (column model: XK50/30, purchased from Pharmacia Company; filling material model: Bio-GelP4, purchased from Bio-Rad Company)

Example 1

The method of the present invention utilizes yeast to prepare soluble β-1,3-glucan oligosaccharides, the waste yeast mud of the brewery is filtered through 6 to 8 layers of gauze to remove impurities and washed with water, suspended in 4wt% NaOH solution, and placed in a water bath at 100°C Stir vigorously in the medium for 1 h, centrifuge to remove the supernatant, suspend the precipitate in 3wt% NaOH solution, stir in a 75°C water bath for 3 h, adjust the pH to 4.5 with concentrated hydrochloric acid, stir in a 75°C water bath for 1 h, cool to room temperature, continue stirring and extracting for 15 h, The precipitate was obtained by centrifugation, washed with water and absolute ethanol respectively, and dried to obtain powdered alkali-insoluble β-1,3-glucan.

Dissolve the above-mentioned alkali-insoluble β-1,3-glucan in anhydrous formic acid, place in a water bath at 80°C, stir for 30 minutes, use 1.5 times the volume of absolute ethanol to precipitate the sugar dissolved in formic acid, centrifuge to obtain the precipitate, add Appropriate amount of distilled water, fully dissolve soluble oligosaccharides, centrifuge and take the supernatant to obtain soluble β-1,3-glucan oligosaccharide solution, concentrate the oligosaccharide solution by vacuum rotary evaporator and freeze-dry to obtain soluble β-1,3-glucan oligosaccharides sugar. It is known that laminarin is β-1,3-glucan, and the soluble β-1,3-glucan oligosaccharide standard substance is prepared from laminarin by the above-mentioned partial acid hydrolysis method. Carbohydrate Column, Bio-Rad) analyzed the standard substance and the prepared yeast cell wall oligosaccharides (as shown in Figure 2), and found that the components contained in the two had the same peak time, indicating that the two components were the same.

The prepared β-1,3-glucan oligosaccharide was formulated into an aqueous solution with a certain concentration, and the molecular sieve chromatography system shown in Figure 1 was used for separation and purification. The system consists of an AG50W-X4 chromatography column (XK50/30) with a height of 30 cm and a diameter of 5 cm and a Bio-Gel P4 (XK50/100) chromatography column with a height of 100 cm and a diameter of 5 cm in series. The jacket, the circulating hot water in the jacket is used to maintain the proper column temperature to ensure the separation effect. The sample first enters from the top of the AG50W-X4 chromatography column, flows out from the bottom of the AG50W-X4 chromatography column, then enters the Bio-Gel P4 chromatography column from the bottom of the Bio-Gel P4 chromatography column, and flows out from the top of the Bio-Gel P4 chromatography column flow out. The mobile phase is distilled water, the temperature of the mobile phase and the column are both 70°C, the flow rate of the mobile phase is 1.5ml/min, and a tube of components is collected from the top of the Bio-Gel P4 chromatography column every 6 minutes, and the collection and retention chromatography time is 3~ 24h composition. Adopt phenol sulfuric acid method to detect the total sugar content in the collected fraction (as shown in Figure 3), the yield of gained disaccharide to hexasaccharide single pure fraction is respectively 2.9%, 6.3%, 4.4%, 5.3%, 3.8% %. Part of the separated components were analyzed by high performance liquid chromatography, and a single peak corresponding to the mixed oligosaccharide sample (as shown in Figure 4 ) could be obtained, indicating that the mixed sample had been successfully separated. The average degree of polymerization of some components was determined by chemical method (Table 1), and the results showed that there was a difference of one glucose unit between these adjacent components. The separated oligosaccharide components were analyzed by infrared spectroscopy (as shown in Figure 5), and it was found that the sample had an obvious characteristic absorption peak at 890cm-1, indicating that it was a β configuration; at 2920cm-1 and 1370cm-1 A characteristic absorption peak indicates that the sample is glucan with β-1,3-glucosidic bonds.

Table 1

Note: degree of polymerization (DP) = total sugar concentration (μM) / reducing end concentration (μM),

The total sugar concentration was determined by the phenol-sulfuric acid method; the reducing end concentration was determined by the biquinolinecarboxylic acid (BCA) detection method.

Claims (4)

1. one kind is utilized yeast to prepare solubility β-1, the method of 3-ligoglucoside, comprise the pre-treatment of yeast slurry alkali, alkali insoluble β-1, acid hydrolysis of 3-dextran and solubility β-1, the separation and purification of 3-ligoglucoside, it is characterized in that described separation and purification is with exsiccant solubility β-1, after the dissolving of 3-ligoglucoside, carry out separation and purification through the molecular sieve layer analysis system; Described molecular sieve layer analysis system mainly is in series by AG50W-X4 chromatography column and Bio-Gel P4 chromatography column, and described AG50W-X4 chromatography column and Bio-Gel P4 chromatography column all have chuck, feeds circulating hot water in the chuck and is used for keeping column temperature 65-75 ℃; Solubility β-1,3-ligoglucoside solution is introduced into the AG50W-X4 chromatography column, brings Bio-Gel P4 chromatography column into by moving phase, by Bio-Gel P4 chromatography column, collects from Bio-Gel P4 chromatography column top from lower to upper; Described moving phase is distilled water, enters from AG50W-X4 chromatography column top earlier, flows out from AG50W-X4 chromatography column bottom, enters Bio-Gel P4 chromatography column from Bio-Gel P4 chromatography column bottom again, flows out from Bio-Gel P4 chromatography column top; The moving phase temperature is 65-75 ℃, and flow rate of mobile phase is 1～2ml/min, and per 5～8min collects a pipe component from Bio-Gel P4 chromatography column top, and collecting retention time is the component of 3～24h.

2. preparation solubility β-1 as claimed in claim 1, the method for 3-ligoglucoside is characterized in that, the concrete operations of described alkali pre-treatment step are as follows:

(1) with yeast slurry filtering and impurity removing washing back , be suspended from the NaOH solution of 4wt%, vigorous stirring 0.75～1h in 90～100 ℃ of water-baths, 2000～5000 rev/mins are centrifugal, remove supernatant liquor, get precipitation;

(2) precipitation that step (1) is made is suspended from the 3wt%NaOH solution, and 75～80 ℃ of stirring in water bath 2.5～3.5h transfer to 3.5～5.5 with concentrated hydrochloric acid with pH value of solution, 75～80 ℃ of stirring in water bath 1～2h, be cooled to room temperature, continue to stir extracting 14～16h, the centrifugal precipitation that obtains;

(3) precipitation that step (2) is obtained is water and absolute ethanol washing respectively, obtains powdery alkali insoluble β-1 after the drying, the 3-dextran.

3. preparation solubility β-1 as claimed in claim 1, the method for 3-ligoglucoside is characterized in that, described acid hydrolysis step concrete operations are as follows:

(1) will be by the alkali insoluble β-1 of alkali pre-treatment acquisition, the 3-dextran is dissolved in anhydrous formic acid, places 80～90 ℃ of water-baths, stirs 20～60min, and the sugar that will be dissolved in the formic acid with 1～3 times of volume dehydrated alcohol precipitates, the centrifugal precipitation that obtains;

(2) resolution of precipitate that step (1) is obtained is in distilled water, and the centrifuging and taking supernatant obtains solubility β-1,3-ligoglucoside solution, and drying obtains solubility β-1, the 3-ligoglucoside.

4. preparation solubility β-1 as claimed in claim 3, the method for 3-ligoglucoside is characterized in that, acid hydrolysis step

(2) drying in is to concentrate solubility β-1 with the rotary evaporation in vacuo instrument earlier, behind the 3-ligoglucoside solution, and freeze-drying again.

Images