Preparation, separation and purification method of glucan oligosaccharide

Abstract

The invention discloses a preparation, separation and purification method of glucan oligosaccharide, and belongs to the technical field of active oligosaccharide preparation. The preparation, separation and purification method comprises the following steps: adding alcaligenes faecalis into a fermentation culture medium, fermenting to prepare beta-1, 3-glucan, adding beta-1, 3-glucanase when the viscosity of fermentation liquor reaches a certain value, and simultaneously controlling the temperature to promote enzymolysis, so that the beta-1, 3-glucan with high polymerization degree is hydrolyzed into oligosaccharide, and separating and purifying the oligosaccharide. The material liquid viscosity is reduced, and the mass transfer is improved, so that the single-batch fermentation yield is improved. Meanwhile, sugars with different polymerization degrees are separated through a graded alcohol precipitation process, part of impurities are removed, and the product purity is improved. According to the method, the enzymolysis reaction is directly carried out in the fermentation process, so that the steps of polysaccharide extraction and enzymolysis are simplified, the production energy consumption is reduced, and the yield and the production benefit of the final product are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of active oligosaccharide preparation, and more particularly to a method for preparing, separating and purifying oligosaccharides. Background Art

[0002] Curdlan (also known as curdlan, hot gel) is a microbial extracellular polysaccharide produced by the metabolism of a variant strain of Alcaligenes. This polysaccharide is composed of D-glucose residues connecting C1 and C3 through a β-glucosidic bond to form a linear β-1,3-glucan with a molecular formula of (C6H 10 O5) n , DP400-500, and molecular weight in the range of 44000-77000. Due to the large relative molecular mass and poor water solubility of curdlan, it not only seriously affects the single batch yield of microbial fermentation, but also greatly limits the application of curdlan.

[0003] β-1,3-glucoligosaccharide is a type of functional oligosaccharide with high stability, low caloric value, safety and non-toxicity. It can regulate the body's immunity and has physiological activities such as anti-inflammatory, anti-tumor, and improvement of intestinal microbial flora. It has broad application prospects in agriculture, medicine, food and other fields.

[0004] β-1,3-glucoligosaccharides can be produced by polysaccharide degradation, enzymatic synthesis, or chemical synthesis. However, enzymatic synthesis has a low conversion rate and high cost, while chemical synthesis has a long synthesis cycle, is difficult, and has low yields. Curdlan has a relatively simple molecular structure and is the only β-1,3-glucan without branches. It can be degraded to produce linear β-1,3-glucoligosaccharides. Furthermore, curdlan can be obtained in large quantities through microbial fermentation. Compared to other raw materials, it offers stable yield, high purity, and low price, making it an ideal raw material for the production of β-1,3-glucoligosaccharides.

[0005] Therefore, the production of β-1,3-glucoligosaccharides through the degradation of curdlan has become a viable production method. Current research focuses on acid hydrolysis, hydrogen peroxide oxidation, physical degradation, and bioenzymatic methods. Acid hydrolysis is simple and efficient, but the reagents are corrosive and highly hazardous. Hydrogen peroxide oxidation can achieve high yields, but the wastewater is difficult to treat, significantly impacting the environment. Physical degradation is simple, but the equipment is expensive, energy-intensive, and the product is difficult to degrade into oligosaccharides. Bioenzymatic hydrolysis does not destroy the glycosidic structure, operates under mild conditions, and is a clean process, making it an ideal route for oligosaccharide production. However, the enzymatic hydrolysis products produced by this method have a wide distribution of polymerization degrees, and the crude product obtained by direct drying has a complex composition. These factors contribute to the inability to industrialize β-1,3-glucoligosaccharides.

[0006] Currently, there is a lack of efficient production control methods for β-1,3-glucoligosaccharides in the field. During fermentation, the yield of β-1,3-glucoligosaccharides per batch is low and the composition is complex. Therefore, it is urgent to develop a method for the preparation, separation and purification of β-1,3-glucoligosaccharides with high yield, stability, high yield and low energy consumption.

[0007] Therefore, providing a method for preparing, separating and purifying oligosaccharides is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a method for preparing, separating and purifying oligosaccharides.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for preparing, separating and purifying oligosaccharides comprises the following steps:

[0011] (1) Preparation of Alcaligenes faecalis seed solution: Pick a single colony of Alcaligenes faecalis and inoculate it into the Alcaligenes faecalis seed solution culture medium. Culture it on a shaking table to obtain the Alcaligenes faecalis seed solution. OD 600 3 to 5;

[0012] (2) preparing a glucanase solution: dissolving glucanase powder and pure water in a mass ratio of 1 to 5:100 to obtain a glucanase solution; the glucanase activity is 80,000 to 100,000 U / g;

[0013] (3) Fermentation and enzymolysis: The fermentation medium was added to the fermentation tank for sterilization, cooled to 30-32°C, inoculated with a 3-5% inoculum of Alcaligenes faecalis seed solution, and the pH value of the fermentation solution was adjusted to 6.8-7.2 using 32% liquid caustic soda. The ventilation intensity was 800-1000 L / h, and the stirring speed was 300-500 rpm.

[0014] During the fermentation process, the viscosity of the fermentation broth is detected online. When the viscosity of the fermentation broth reaches 3000-12000 mPa·s, glucanase solution is added at a flow rate of 0.5 mL / min. When the viscosity of the fermentation broth increases to 15000-20000 mPa·s, the pH is controlled to be reduced to 6.0-6.5 and the temperature is increased to 37-40°C to promote glucanase hydrolysis. The glucanase solution is added at a flow rate of 1.0 mL / min. When the viscosity of the fermentation broth decreases to 1000 mPa·s, the enzyme solution is stopped from being added, the temperature is lowered to 28-32°C, and the fermentation is continued with ventilation for 36-48 hours, and the fermentation is terminated.

[0015] (4) Separation and purification:

[0016] ① After the fermentation in step (3) is completed, the fermentation liquid is centrifuged to remove the bacterial cells, and the supernatant is collected. The supernatant contains β-1,3-glucan and glucose monosaccharide of different polymerization degrees; anhydrous ethanol is added to the supernatant to a final concentration of 90% to remove the glucose monosaccharide, and after stirring, it is allowed to stand at 4°C for 12 to 24 hours, and then centrifuged or filtered on a plate and frame to collect the precipitate 1;

[0017] ② Dissolve the precipitate 1 in step ① with pure water at a mass ratio of 3 to 5:100, add anhydrous ethanol to a final concentration of 60%, stir and let stand at 4°C for 12 to 24 hours, centrifuge and separate the precipitate 2 and the supernatant; collect the precipitate 2 and dry it to obtain a glucan with a degree of polymerization greater than 18; concentrate the supernatant under reduced pressure to remove ethanol, and freeze-dry to obtain oligosaccharides with a degree of polymerization ranging from 2 to 18.

[0018] Furthermore, the formula of the Alcaligenes faecalis seed liquid culture medium in step (1) is: 30 g / L glucose, 3 g / L (NH4)2HPO4, 1.5 g / L KH2PO4, 3 g / L CaCO3, 1 g / L corn steep liquor, 1 g / L MgSO4·7H2O, the pH is adjusted to 6.8-7.0, and sterilized at 115°C for 20 min.

[0019] Furthermore, the shaking culture conditions in step (1) are: 200-300 rpm rotation speed, 30-32° C., and culture for 16-20 h.

[0020] Furthermore, the fermentation medium formula in step (3) is: glucose 35g / L, (NH4)2HPO41g / L, KH2PO41g / L, CaCO30.5g / L, NaCl 1g / L, corn steep liquor 1g / L, MgSO4·7H2O0.6g / L, pH is adjusted to 6.8-7.0; sterilized at 115°C for 20min.

[0021] Furthermore, the centrifugation condition in step (4) is 4000-6000 r / min for 10-15 min; the drying temperature of the precipitate 2 is 50-80° C. for 12-24 h; and the freeze-drying is performed at -40-50° C. for 24-48 h.

[0022] As can be seen from the above technical solutions, compared to the prior art, the present invention provides a method for preparing, isolating, and purifying glucan oligosaccharides. During the fermentation process, as the polysaccharide synthesis rate increases, fermentation products accumulate continuously, increasing the viscosity of the fermentation broth and making it difficult to dissolve oxygen, which affects bacterial growth and metabolism, resulting in poor polysaccharide yield. The present invention adds glucanase after the fermentation broth viscosity reaches a certain value, and controls the temperature to promote enzymatic hydrolysis, hydrolyzing the highly polymerized β-1,3-glucan into oligosaccharides. This reduces the viscosity of the slurry and improves mass transfer, thereby increasing the single-batch fermentation yield. Simultaneously, a graded alcohol precipitation process separates the highly polymerized β-1,3-glucan from the β-1,3-glucan oligosaccharides. A secondary alcohol precipitation step during extraction effectively removes residual culture medium components. Ethanol can be recovered and recycled by distillation. This method not only simplifies the polysaccharide extraction and enzymatic hydrolysis process, saving production energy, but also increases the yield of the final product, improving production efficiency. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0024] Glucanase powder was purchased from Weifang Kangdien Biotechnology Co., Ltd. with an enzyme activity of 80,000 to 100,000 U / g.

[0025] Alcaligenes faecalis was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with the product number BMZ140069.

[0026] Example 1

[0027] A method for preparing, separating and purifying oligosaccharides comprises the following steps:

[0028] (1) Preparation of Alcaligenes faecalis seed solution: Pick a single colony of Alcaligenes faecalis and inoculate it into the Alcaligenes faecalis seed solution culture medium. Incubate on a shaker at 300 rpm and 32°C for 16 h to obtain the Alcaligenes faecalis seed solution. OD 600 =3.67;

[0029] The formula of the Alcaligenes faecalis seed liquid culture medium is glucose 30 g / L, (NH4)2HPO4 3 g / L, KH2PO4 1.5 g / L, CaCO3 3 g / L, corn steep liquor 1 g / L, MgSO4·7H2O 1 g / L, adjusted to pH 7.0, and sterilized at 115°C for 20 min.

[0030] (2) preparing a glucanase solution: dissolving the glucanase powder and pure water in a mass ratio of 5:100 to obtain a glucanase solution;

[0031] (3) The formula of the fermentation medium is glucose 35 g / L, (NH4)2HPO41 g / L, KH2PO41 g / L, CaCO30.5 g / L, NaCl 1 g / L, corn syrup 1 g / L, MgSO4·7H2O 0.6 g / L, and the pH is adjusted to 7.0. The fermentation medium is added to the fermentation tank and sterilized at 115°C for 20 min. The temperature is reduced to 32°C and the Alcaligenes faecalis seed liquid is inoculated at a 3% inoculation amount.

[0032] The pH of the fermentation broth is adjusted to 6.8 using liquid alkali with a concentration of 32%, the ventilation intensity is 800 L / h, and the stirring speed is 500 rpm. The viscosity of the fermentation broth is detected online during the fermentation process. When the viscosity of the fermentation broth reaches 6000 mpa·s, the glucanase enzyme solution is started to be supplemented at a flow rate of 0.5 mL / min. When the viscosity of the fermentation broth reaches 18000 mpa·s, the pH is controlled to decrease to 6.0, the temperature is controlled to increase to 37°C, and the enzyme solution is supplemented at a flow rate of 1.0 mL / min. When the viscosity of the fermentation broth decreases to 1000 mpa·s, the enzyme solution is stopped to be supplemented, the temperature is reduced to 32°C, and the ventilation is continued for 48 h to complete the fermentation.

[0033] (4) Separation and purification: the fermentation broth after the fermentation of step (3) is centrifuged at 4000 r / min for 15 min to remove the bacteria, and the supernatant is collected. Anhydrous ethanol is added to the supernatant to a final concentration of 90%, stirred, and then placed at 4°C for 12 h. The precipitate 1 is collected by centrifugation at 4000 r / min for 15 min. The precipitate 1 in the above step is dissolved with pure water at a mass ratio of 5:100, anhydrous ethanol is added to a final concentration of 60%, stirred, and then placed at 4°C for 12 h. The precipitate 2 and the supernatant are separated by centrifugation. The precipitate 2 is collected and dried at 50°C for 24 h to obtain glucan with a degree of polymerization greater than 18. The supernatant is concentrated under reduced pressure to remove ethanol, and then freeze-dried at -40°C for 48 h to obtain glucan oligosaccharide with a degree of polymerization ranging from 2 to 18.

[0034] (5) The glucan and glucan oligosaccharide are ground, sieved, packaged, and obtained as finished products.

[0035] The purity of the glucan is 94.6% and the yield is 38.6 g / L, as measured by the phenol-sulfuric acid method. The purity of the glucan oligosaccharide is 96.2% and the yield is 42.8 g / L. The single batch yield is 81.4 g / L, and the effective material yield is 77.69 g / L.

[0036] Single batch yield = glucan yield + glucan oligosaccharide yield.

[0037] Effective material yield = glucan yield * glucan purity + glucan oligosaccharide yield * glucan oligosaccharide purity.

[0038] Example 2

[0039] A method for preparing, separating and purifying oligosaccharides comprises the following steps:

[0040] (1) Preparation of Alcaligenes faecalis seed solution: Pick a single colony of Alcaligenes faecalis and inoculate it into the Alcaligenes faecalis seed solution culture medium. Incubate on a shaker at 250 rpm and 31°C for 18 h to obtain the Alcaligenes faecalis seed solution. OD 600 =4.15;

[0041] The formula of the Alcaligenes faecalis seed liquid culture medium is glucose 30 g / L, (NH4)2HPO4 3 g / L, KH2PO4 1.5 g / L, CaCO3 3 g / L, corn steep liquor 1 g / L, MgSO4·7H2O 1 g / L, adjusted to pH 6.9, and sterilized at 115°C for 20 min.

[0042] (2) preparing a glucanase solution: dissolving the glucanase powder and pure water in a mass ratio of 4:100 to obtain a glucanase solution;

[0043] (3) The fermentation medium is formulated as 35 g / L glucose, 1 g / L (NH4)2HPO4, 1 g / L KH2PO4, 0.5 g / L CaCO3, 1 g / L NaCl, 1 g / L corn steep liquor, and 0.6 g / L MgSO4·7H2O, with the pH adjusted to 6.9. The fermentation medium is added to a fermentation tank and sterilized at 115°C for 20 min. The temperature is then lowered to 30°C and inoculated with a 5% inoculum of Alcaligenes faecalis seed solution.

[0044] The pH value of the fermentation liquid was adjusted to 7.2 using liquid caustic soda with a concentration of 32%, the ventilation intensity was 1000 L / h, and the stirring speed was 300 rpm; the viscosity of the fermentation liquid was detected online during the fermentation process, and when the viscosity of the fermentation liquid reached 12000 mPa·s, glucanase solution was added at a flow rate of 0.5 mL / min; when the viscosity of the fermentation liquid reached 20000 mPa·s, the pH was controlled to be reduced to 6.3, the temperature was raised to 38°C, and the enzyme solution was added at a flow rate of 1.0 mL / min; when the viscosity of the fermentation liquid was reduced to 1000 mPa·s, the enzyme solution was stopped, the temperature was lowered to 28°C, and the fermentation was continued with ventilation for 36 hours, and the fermentation was terminated.

[0045] (4) Separation and purification: After the fermentation in step (3) is completed, the fermentation liquid is centrifuged at 6000 r / min for 10 minutes to remove the bacteria, and the supernatant is collected. Anhydrous ethanol is added to the supernatant to a final concentration of 90%, stirred, and allowed to stand at 4°C for 18 hours, centrifuged at 6000 r / min for 10 minutes, and precipitate 1 is collected. The precipitate 1 in the above step is dissolved in pure water at a mass ratio of 4:100, anhydrous ethanol is added to a final concentration of 60%, stirred, allowed to stand at 4°C for 18 hours, and centrifuged to separate precipitate 2 and the supernatant. Precipitate 2 is collected and dried at 80°C for 12 hours to obtain glucan with a degree of polymerization greater than 18. The supernatant is concentrated under reduced pressure to remove ethanol, and freeze-dried at -50°C for 24 hours to obtain oligosaccharides with a degree of polymerization ranging from 2 to 18.

[0046] (5) Grinding, screening, and packaging to obtain finished glucan and oligosaccharide products.

[0047] The purity of glucan was 95.0% and the yield was 40.2 g / L by the phenol-sulfuric acid method. The purity of oligosaccharide was 95.2% and the yield was 38.1 g / L. The single batch yield was 78.3 g / L, and the effective product yield was 74.46 g / L.

[0048] In Example 2, when the viscosity of the fermentation broth was higher during online monitoring compared to Example 1, glucanase was added. When the viscosity reached a higher value, the temperature was increased to promote enzymatic hydrolysis. Under these process conditions, the degree of enzymatic hydrolysis was lower, resulting in a higher proportion of large molecular weight glucans and a decrease in small molecular weight oligosaccharides.

[0049] Example 3

[0050] A method for preparing, separating and purifying oligosaccharides comprises the following steps:

[0051] (1) Preparation of Alcaligenes faecalis seed solution: Pick a single colony of Alcaligenes faecalis and inoculate it into the Alcaligenes faecalis seed solution culture medium. Incubate on a shaker at 200 rpm and 30°C for 20 h to obtain the Alcaligenes faecalis seed solution. OD 600 =4.76;

[0052] The formula of the Alcaligenes faecalis seed liquid culture medium is glucose 30 g / L, (NH4)2HPO4 3 g / L, KH2PO4 1.5 g / L, CaCO3 3 g / L, corn steep liquor 1 g / L, MgSO4·7H2O 1 g / L, adjusted to pH 6.8, and sterilized at 115°C for 20 min.

[0053] (2) preparing a glucanase solution: dissolving the glucanase powder and pure water in a mass ratio of 3:100 to obtain a glucanase solution;

[0054] (3) The fermentation medium is formulated as 35 g / L glucose, 1 g / L (NH4)2HPO4, 1 g / L KH2PO4, 0.5 g / L CaCO3, 1 g / L NaCl, 1 g / L corn steep liquor, and 0.6 g / L MgSO4·7H2O, with the pH adjusted to 6.8. The fermentation medium is added to a fermentation tank and sterilized at 115°C for 20 min. The temperature is then lowered to 31°C and inoculated with a 4% inoculum of Alcaligenes faecalis seed solution.

[0055] The pH value of the fermentation liquid was adjusted to 7.0 using liquid caustic soda with a concentration of 32%, the ventilation intensity was 900 L / h, and the stirring speed was 400 rpm; the viscosity of the fermentation liquid was detected online during the fermentation process, and when the viscosity of the fermentation liquid reached 3000 mPa·s, glucanase solution was added at a flow rate of 0.5 mL / min; when the viscosity of the fermentation liquid reached 15000 mPa·s, the pH was controlled to be reduced to 6.5, the temperature was raised to 40°C, and the enzyme solution was added at a flow rate of 1.0 mL / min; when the viscosity of the fermentation liquid was reduced to 1000 mPa·s, the enzyme solution was stopped, the temperature was lowered to 30°C, and the fermentation was continued through ventilation for 42 hours, and the fermentation was terminated.

[0056] (4) Separation and purification: After the fermentation in step (3) is completed, the fermentation liquid is centrifuged at 5000 r / min for 13 minutes to remove the bacteria, and the supernatant is collected. Anhydrous ethanol is added to the supernatant to a final concentration of 90%, stirred, and allowed to stand at 4°C for 24 hours, centrifuged at 5000 r / min for 13 minutes, and precipitate 1 is collected. The precipitate 1 in the above step is dissolved in pure water at a mass ratio of 3:100, anhydrous ethanol is added to a final concentration of 60%, stirred, allowed to stand at 4°C for 24 hours, and centrifuged to separate precipitate 2 and supernatant. Precipitate 2 is collected and dried at 65°C for 16 hours to obtain glucan with a degree of polymerization greater than 18. The supernatant is concentrated under reduced pressure to remove ethanol, and freeze-dried at -45°C for 36 hours to obtain oligosaccharides with a degree of polymerization ranging from 2 to 18.

[0057] (5) Grinding, screening, and packaging to obtain finished glucan and oligosaccharide products.

[0058] The purity of glucan was 94.3% and the yield was 36.8 g / L by the phenol-sulfuric acid method. The purity of oligosaccharide was 95.8% and the yield was 42.3 g / L. The single batch yield was 79.1 g / L and the effective matter yield was 75.23 g / L.

[0059] In Example 3, when the viscosity of the fermentation broth was lower than that of Example 1, glucanase was added. When the viscosity was lower, increasing the temperature promoted enzymatic hydrolysis, resulting in a higher degree of enzymatic hydrolysis and a higher proportion of small-molecule glucan oligosaccharides.

[0060] Comparative Example 1

[0061] (1) Preparation of Alcaligenes faecalis seed solution: Pick a single colony of Alcaligenes faecalis and inoculate it into the Alcaligenes faecalis seed solution culture medium. Incubate on a shaker at 300 rpm and 32°C for 16 h to obtain the Alcaligenes faecalis seed solution. OD 600 =3.56;

[0062] The formula of the Alcaligenes faecalis seed liquid culture medium is glucose 30 g / L, (NH4)2HPO4 3 g / L, KH2PO4 1.5 g / L, CaCO3 3 g / L, corn steep liquor 1 g / L, MgSO4·7H2O 1 g / L, adjusted to pH 7.0, and sterilized at 115°C for 20 min.

[0063] (2) Fermentation: The fermentation medium is formulated as 35 g / L glucose, 1 g / L (NH4)2HPO4, 1 g / L KH2PO4, 0.5 g / L CaCO3, 1 g / L NaCl, 1 g / L corn steep liquor, and 0.6 g / L MgSO4·7H2O, with the pH adjusted to 7.0. The fermentation medium is added to a fermenter and sterilized at 115°C for 20 min. The fermentation medium is cooled to 32°C and inoculated with a 3% inoculum of a Bacillus faecalis seed solution.

[0064] The pH value of the fermentation liquid was adjusted to 6.8 using 32% liquid alkali, the ventilation intensity was 800 L / h, the stirring speed was 500 rpm, and the fermentation was completed after 48 hours of ventilation fermentation.

[0065] (4) Isolation and purification: After the fermentation in step (3), the fermentation broth was centrifuged at 4000 rpm for 15 minutes to remove the bacterial cells, and the supernatant was collected. Anhydrous ethanol was added to the supernatant to a final concentration of 60%, stirred, and allowed to stand at 4°C for 12 hours. The precipitate was collected and dried at 50°C for 24 hours to obtain a glucan with a degree of polymerization greater than 18.

[0066] (5) Grinding, screening, and packaging to obtain the finished glucan product.

[0067] The purity of dextran measured by phenol-sulfuric acid method was 89.9%, the yield was 32.8 g / L, the single batch yield was 32.8 g / L, and the effective matter yield was 29.49 g / L.

[0068] Comparative Example 1, compared to Examples 1-3, did not add glucanase for enzymatic hydrolysis during fermentation, nor did it require a graded alcohol precipitation process for extraction. Under these conditions, the fermentation broth had high viscosity, which affected mass transfer and inhibited bacterial growth and metabolism. After reaching a final yield of 32.8 g / L, further increases were difficult.

[0069] Comparative Example 2

[0070] (1) Preparation of Alcaligenes faecalis seed solution: Pick a single colony of Alcaligenes faecalis and inoculate it into the Alcaligenes faecalis seed solution culture medium. Incubate on a shaker at 300 rpm and 32°C for 16 h to obtain the Alcaligenes faecalis seed solution. OD 600 =3.60;

[0071] The formula of the Alcaligenes faecalis seed liquid culture medium is glucose 30 g / L, (NH4)2HPO4 3 g / L, KH2PO4 1.5 g / L, CaCO3 3 g / L, corn steep liquor 1 g / L, MgSO4·7H2O 1 g / L, adjusted to pH 7.0, and sterilized at 115°C for 20 min.

[0072] (2) preparing a glucanase solution: dissolving the glucanase powder and pure water in a mass ratio of 5:100 to obtain a glucanase solution;

[0073] (3) Fermentation: The fermentation medium is formulated as 35 g / L glucose, 1 g / L (NH4)2HPO4, 1 g / L KH2PO4, 0.5 g / L CaCO3, 1 g / L NaCl, 1 g / L corn steep liquor, and 0.6 g / L MgSO4·7H2O, with the pH adjusted to 7.0. The fermentation medium is added to a fermentation tank and sterilized at 115°C for 20 min. The temperature is then lowered to 32°C and a 3% inoculum of Alcaligenes faecalis seed solution is inoculated.

[0074] The pH value of the fermentation liquid was adjusted to 6.8 using liquid caustic soda with a concentration of 32%, the ventilation intensity was 800 L / h, and the stirring speed was 500 rpm; the viscosity of the fermentation liquid was detected online during the fermentation process, and when the viscosity of the fermentation liquid reached 6000 mPa·s, glucanase solution was added at a flow rate of 0.5 mL / min; when the viscosity of the fermentation liquid reached 18000 mPa·s, the pH was controlled to be reduced to 6.0, the temperature was raised to 37°C, and the enzyme solution was added at a flow rate of 1.0 mL / min; when the viscosity of the fermentation liquid was reduced to 1000 mPa·s, the enzyme solution was stopped, the temperature was lowered to 32°C, and the fermentation was continued with ventilation for 48 hours, and the fermentation was terminated.

[0075] (4) Isolation and Purification: After the fermentation in step (3), the fermentation broth was centrifuged at 4000 rpm for 15 minutes to remove the bacterial cells, and the supernatant was collected. Anhydrous ethanol was added to the supernatant to a final concentration of 90%, stirred, and allowed to stand at 4°C for 12 hours. The mixture was then centrifuged at 4000 rpm for 15 minutes, and the precipitate was collected. After drying at 50°C for 24 hours, a glucan product with a degree of polymerization of 2 or greater was obtained.

[0076] (5) Grinding, screening, and packaging to obtain the finished glucan product.

[0077] The purity of dextran was 76.3% and the yield was 67.3 g / L as determined by the phenol-sulfuric acid method. The single-batch yield was 67.3 g / L and the effective substance yield was 51.35 g / L.

[0078] Compared with Examples 1-3, in Comparative Example 2, no secondary alcohol precipitation was performed in the separation and purification step, resulting in a decrease in product purity and a lower yield. At the same time, low-polymerization-degree oligosaccharides (polymerization degree 2-18) were not separated from the product.

[0079] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing, separating and purifying oligosaccharides, characterized in that: The following steps are involved: (1) Preparation of Alcaligenes faecalis seed solution: Pick a single colony of Alcaligenes faecalis and inoculate it into the Alcaligenes faecalis seed solution culture medium. Culture it on a shaking table to obtain the Alcaligenes faecalis seed solution. OD 600 3 to 5; (2) preparing a glucanase solution: dissolving glucanase powder and pure water in a mass ratio of 1 to 5:100 to obtain a glucanase solution; the glucanase activity is 80,000 to 100,000 U / g; (3) Fermentation and enzymolysis: The fermentation medium was added to the fermentation tank for sterilization, cooled to 30-32°C, inoculated with a 3-5% inoculum of Alcaligenes faecalis seed solution, and the pH value of the fermentation solution was adjusted to 6.8-7.2 using 32% liquid caustic soda. The ventilation intensity was 800-1000 L / h, and the stirring speed was 300-500 rpm. During the fermentation process, the viscosity of the fermentation broth is detected online. When the viscosity of the fermentation broth reaches 3000-12000 mPa·s, glucanase solution is added at a flow rate of 0.5 mL / min. When the viscosity of the fermentation broth increases to 15000-20000 mPa·s, the pH is controlled to be reduced to 6.0-6.5, the temperature is increased to 37-40°C, and the glucanase solution is added at a flow rate of 1.0 mL / min. When the viscosity of the fermentation broth decreases to 1000 mPa·s, the enzyme solution is stopped, the temperature is lowered to 28-32°C, and the fermentation is continued with ventilation for 36-48 hours, and the fermentation is terminated. (4) Separation and purification: ① After the fermentation in step (3) is completed, the fermentation liquid is centrifuged to remove the bacterial cells, and the supernatant is collected. The supernatant contains β-1,3-glucan and glucose monosaccharide with different polymerization degrees; anhydrous ethanol is added to the supernatant to a final concentration of 90%, and after stirring, it is allowed to stand at 4°C for 12 to 24 hours, and then centrifuged or filtered on a plate and frame to collect the precipitate 1; ② Dissolve the precipitate 1 in step ① with pure water at a mass ratio of 3 to 5:100, add anhydrous ethanol to a final concentration of 60%, stir and let stand at 4°C for 12 to 24 hours, centrifuge and separate the precipitate 2 and the supernatant; collect the precipitate 2 and dry it to obtain a glucan with a degree of polymerization greater than 18; concentrate the supernatant under reduced pressure to remove ethanol, and freeze-dry to obtain oligosaccharides with a degree of polymerization ranging from 2 to 18.

2. The method for preparing, separating and purifying oligosaccharides according to claim 1, wherein: The formula of the alcaligenes faecalis seed liquid culture medium in step (1) is: 30 g / L glucose, 3 g / L (NH4)2HPO4, 1.5 g / L KH2PO4, 3 g / L CaCO3, 1 g / L corn steep liquor, and 1 g / L MgSO4·7H2O. The pH is adjusted to 6.8-7.0 and sterilized at 115°C for 20 min.

3. The method for preparing, separating and purifying oligosaccharides according to claim 1, wherein: The shaking culture conditions in step (1) are: 200-300 rpm rotation speed, 30-32° C. culture for 16-20 h.

4. The method for preparing, separating and purifying oligoglucose according to claim 1, wherein: The fermentation medium formula of step (3) is: glucose 35g / L, (NH4)2HPO4 1g / L, KH2PO4 1g / L, CaCO3 0.5g / L, NaCl 1g / L, corn steep liquor 1g / L, MgSO4·7H2O 0.6g / L, pH is adjusted to 6.8-7.0; sterilized at 115°C for 20min.

5. The method for preparing, separating and purifying oligosaccharides according to claim 1, wherein: The centrifugal conditions in step (4) are 4000-6000 r / min for 10-15 min; the drying temperature of the precipitate 2 is 50-80° C. for 12-24 h; and the freeze-drying is performed at -40-50° C. for 24-48 h.