A preparation method of oligosaccharide

Abstract

The present invention discloses a method for preparing β-glucose oligosaccharides, and relates to the field of fermentation technology. The method comprises the following steps: (1) preparing a Bacillus faecalis seed solution; (2) preparing a Trichoderma LE02 seed solution; (3) fermenting: inoculating the Bacillus faecalis seed solution into a fermentation medium, and inoculating the Trichoderma LE02 seed solution once at 8-12 hours, 45-55 hours, and 85-95 hours after the inoculation of the Bacillus faecalis seed solution, respectively. The method is ventilated and fermented for 120 hours to obtain a β-1,3-glucose oligosaccharide fermentation liquid; and (4) purifying. The present invention, by extending the fermentation time or adding the Trichoderma LE02 seed solution in batches, ultimately obtains a thermogelling oligosaccharide β-1,3-glucose oligosaccharide with a lower degree of polymerization and a higher concentration, with a yield of up to 89-93%, which can be industrialized.

Description

Technical Field

[0001] The present invention relates to the field of fermentation technology, and more particularly to a method for preparing oligosaccharides. Background Art

[0002] β-1,3-glucosidic oligosaccharides are oligosaccharides composed of multiple glucosides linked by β-1,3 glycosidic bonds. Their degree of polymerization (DP) generally ranges from 3 to 20 dp. They possess physical and chemical properties such as low caloric value, stability, safety, non-toxicity, and resistance to gastrointestinal digestion. Research has shown that β-1,3-glucosidic oligosaccharides can target human, animal, and plant cells through various recognition functions, including recognition, aggregation, and receptors. Furthermore, studies have shown that β-1,3-glucosidic oligosaccharides have immunostimulatory properties, enhancing resistance to infectious diseases caused by bacteria, fungi, viruses, and parasites, and also possess anti-tumor activity. In food applications, β-1,3-glucosidic oligosaccharides can provide dietary fiber, promote the growth of probiotics, inhibit the growth of harmful intestinal bacteria, maintain normal peristaltic muscle function, and lower serum cholesterol and triglyceride levels, thereby reducing the incidence of intestinal cancer and digestive system diseases. Therefore, as a functional oligosaccharide, β-1,3-glucosidic oligosaccharides have a wide range of applications in food, pharmaceuticals, and biomedicine.

[0003] Compared with other polysaccharides, curdlan has a relatively simple molecular structure and is the only β-1,3-glucoligosaccharide in nature that does not contain branches. It can be obtained in large quantities through microbial fermentation, with stable yield, high purity, and relatively low price. The preparation of β-1,3-glucoligosaccharides through thermal gel hydrolysis can reduce pollution to the environment and remove impurities in the raw materials, effectively reducing the cost of oligosaccharide production. Existing methods for producing β-1,3-glucan by degrading β-1,3-glucan mainly include enzymatic, chemical, and physical methods. The purification process is difficult during extraction and the content of the target product is low. β-1,3-glucan itself is not highly water-soluble, which increases the cost of producing β-1,3-glucoligosaccharides and is one of the main reasons why the quality of β-1,3-glucan industrial production cannot be improved.

[0004] Therefore, providing a method for producing β-1,3-glucoligosaccharides with high yield and low cost is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing oligoglucose.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing oligoglucose comprises the following steps:

[0008] (1) Preparation of Alcaligenes faecalis seed liquid: Pick a single colony of Alcaligenes faecalis and inoculate it into a culture medium for Alcaligenes faecalis seed liquid. Cultivate it on a shaking table to obtain Alcaligenes faecalis seed liquid. The viable bacterial count of the seed liquid is 2-4×10 9 CFU / ml;

[0009] (2) Preparation of Trichoderma LE02 seed liquid: Pick a single colony of Trichoderma LE02, inoculate it into Trichoderma LE02 seed culture medium, and culture it on a shaking table to obtain Trichoderma LE02 seed liquid; the number of viable bacteria in the seed liquid is 1-1.5×10 8 CFU / mL;

[0010] (3) Fermentation: Inoculate the fermentation medium with a Bacillus faecalis seed solution, and inoculate the Trichoderma LE02 seed solution 8-12 hours, 45-55 hours, and 85-95 hours after the inoculation of the Bacillus faecalis seed solution. Ferment the medium with ventilation for 120 hours to obtain a β-1,3-glucopyranose fermentation solution.

[0011] (4) Purification: The β-1,3-glucoligosaccharide fermentation broth was centrifuged, and the supernatant was collected, concentrated, and dried to obtain a crude β-1,3-glucoligosaccharide product.

[0012] Furthermore, the Alcaligenes faecalis is capable of producing curdlan.

[0013] Furthermore, the Trichoderma LE02 is capable of producing β-1,3-endoglucanase.

[0014] Furthermore, the formula of the Alcaligenes faecalis seed liquid culture medium in step (1) is: carbon source 22-25 g / L, (NH4)2HPO4 1.2-1.4 g / L, KH2PO4 1.1-1.2 g / L, CaCO3 0.4-0.6 g / L, NaCl 0.9-1.1 g / L, corn steep liquor 1 g / L, MgSO4 0.4-0.6 g / L, and the pH is adjusted to 6.8-7.2;

[0015] The inoculation amount of the Alcaligenes faecalis is one inoculation loop of a single colony of the Alcaligenes faecalis inoculated into 100 ml of the Alcaligenes faecalis seed liquid culture medium.

[0016] Furthermore, the shaking culture conditions in step (1) are: 200 rpm, 32° C., and 18 h.

[0017] Furthermore, the formula of the Trichoderma LE02 seed culture medium in step (2) is: yeast powder 3g, NaNO30.3g, KCL0.05g, MgSO4·7H2O0.05g, FeSO4·7H2O0.001g, distilled water 100mL, natural pH value;

[0018] The inoculation amount of Trichoderma LE02 is one inoculation loop of a single colony of Trichoderma LE02 inoculated into 50 ml of Trichoderma LE02 seed culture medium.

[0019] Furthermore, the shaking culture conditions in step (2) are: 150 rpm, 30-32° C., and 4 days of culture.

[0020] Furthermore, the formula of the fermentation medium in step (3) is: carbon source 25-30 g / L, (NH4)2HPO4 1.5-1.8 g / L, KH2PO4 0.9-1.1 g / L, CaCO3 0.4-0.6 g / L, NaCl 1-1.2 g / L, corn steep liquor 1 g / L, (NH4)2SO4 0.8-1 g / L, MgSO4 0.4-0.6 g / L, and the pH is adjusted to 5.8-7.2;

[0021] The volume ratio of the Alcaligenes faecalis seed solution to the fermentation medium is 7-9:100;

[0022] The volume ratio of the single inoculation amount of the Trichoderma LE02 seed liquid to the fermentation medium is 1:700;

[0023] Furthermore, the conditions for aeration fermentation in step (3) are as follows: the pH value of the fermentation liquid is adjusted to 7.0 with a concentration of 32% liquid caustic soda, the fermentation temperature is 32°C, the aeration intensity is 800-1000 L / h, and the stirring speed is 500 rpm; the pH drops to 6.5 after 24 hours of fermentation and drops to 6.25 after 48 hours of fermentation, and the residual sugar is controlled at 1.5-2.5 g / 100 ml during the fermentation process;

[0024] After 120 h of fermentation with ventilation, the fermentation liquid temperature was heated to 80°C and maintained for 30 min to inactivate the enzyme.

[0025] Furthermore, the centrifugation conditions in step (4) are: 5000 r / min, 15 min; and the concentration and drying temperature does not exceed 110°C.

[0026] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0027] Fungal fermentation culture is difficult to scale up, is shear-sensitive, and has an inhibitory effect on the growth of Alcaligenes faecalis. The process of the present invention directly adds the curdlan fermentation broth from the fermentation process to the seed liquid of Trichoderma LE02 cultured in a shake flask for mixed fermentation. The curdlan particles do not form a core-shell double-layer structure, allowing the substrate to be thoroughly hydrolyzed, resulting in a high substrate conversion rate. At the same time, the curdlan fermentation broth is one of the main raw materials for the ventilation fermentation of Trichoderma LE02. Curdlan serves as both a carbon source for the growth of Trichoderma LE02 and an inducer of β-1,3-glucanase biosynthesis, facilitating the efficient synthesis of β-1,3-glucanase. By extending the fermentation time or adding Trichoderma LE02 seed liquid in batches, a lower degree of polymerization and higher concentration of thermogel oligosaccharides, β-1,3-glucan oligosaccharides, are ultimately obtained, with a yield of 89-93%, enabling industrial production. DETAILED DESCRIPTION

[0028] 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.

[0029] Source of strains: Alcaligenes faecalis and Trichoderma LE02 are both commercially available varieties.

[0030] Example 1

[0031] A method for preparing oligosaccharide:

[0032] Bacterial strain selection: Alcaligenes faecalis can produce curdlan; Trichoderma LE02 can produce β-1,3-endoglucanase.

[0033] Culture medium formula:

[0034] The formula of the Alcaligenes faecalis seed culture medium is as follows: carbon source 23 g / L, (NH4)2HPO4 1.3 g / L, KH2PO4 1.1 g / L, CaCO3 0.5 g / L, NaCl 1.0 g / L, corn steep liquor 1 g / L, MgSO4 0.5 g / L, and the pH is adjusted to 7.0;

[0035] The formula of the Trichoderma LE02 seed culture medium is: yeast powder 3g, NaNO3 0.3g, KCL 0.05g, MgSO4·7H2O 0.05g, FeSO4·7H2O 0.001g, distilled water 100mL, natural pH value;

[0036] The formula of the fermentation medium is as follows: carbon source 28 g / L, (NH4)2HPO4 1.7 g / L, KH2PO4 1.0 g / L, CaCO3 0.5 g / L, NaCl 1.1 g / L, corn steep liquor 1 g / L, (NH4)2SO4 0.9 g / L, MgSO4 0.5 g / L, and the pH is adjusted to 6.5;

[0037] Preparation steps:

[0038] (1) Preparation of Alcaligenes faecalis seed solution: a single colony of Alcaligenes faecalis was picked from an inoculation loop and inoculated into 100 ml of Alcaligenes faecalis seed solution culture medium. The culture was shaken at 200 rpm and 32°C for 18 h to obtain Alcaligenes faecalis seed solution. The viable bacterial count of the seed solution was 3.1 × 10 9 CFU / ml;

[0039] (2) Preparation of Trichoderma LE02 seed liquid: a single colony of Trichoderma LE02 was picked from an inoculation loop and inoculated into 50 ml of Trichoderma LE02 seed medium. The culture was shaken at 150 rpm and 31 °C for 4 days to obtain Trichoderma LE02 seed liquid. The viable bacterial count of the seed liquid was 1 × 10 8 CFU / mL;

[0040] (3) Fermentation: Alcaligenes faecalis seed liquid was inoculated into the fermentation medium, and the volume ratio of the Alcaligenes faecalis seed liquid to the fermentation medium was 8:100. At 10 h, 50 h, and 90 h after the inoculation of the Alcaligenes faecalis seed liquid, the Trichoderma LE02 seed liquid was inoculated once, and the volume ratio of the Trichoderma LE02 seed liquid to the fermentation medium was 1:700. The fermentation was carried out under ventilation for 120 h (the pH value of the fermentation liquid was adjusted to 7.0 with 32% liquid caustic soda, the fermentation temperature was 32°C, the ventilation intensity was 900 L / h, and the stirring speed was 500 rpm. The pH dropped to 6.5 after 24 h of fermentation and to 6.25 after 48 h of fermentation. The residual sugar content was controlled at 2.0 g / 100 ml during the fermentation process). After 120 h of fermentation under ventilation, the fermentation liquid temperature was heated to 80°C and maintained for 30 min to inactivate the enzyme to obtain β-1,3-glucoligosaccharide fermentation liquid.

[0041] (4) Purification: The β-1,3-glucoligosaccharide fermentation broth was centrifuged at 5000 r / min for 15 min, the supernatant was collected, and the temperature of the concentrated drying was not more than 110°C to obtain the crude β-1,3-glucoligosaccharide product.

[0042] Example 2

[0043] A method for preparing oligosaccharide:

[0044] Bacterial strain selection: Alcaligenes faecalis can produce curdlan; Trichoderma LE02 can produce β-1,3-endoglucanase.

[0045] Culture medium formula:

[0046] The formula of the Alcaligenes faecalis seed culture medium is as follows: carbon source 22 g / L, (NH4)2HPO4 1.2 g / L, KH2PO4 1.1 g / L, CaCO3 0.4 g / L, NaCl 0.9 g / L, corn steep liquor 1 g / L, MgSO4 0.4 g / L, and the pH is adjusted to 6.8;

[0047] The formula of the Trichoderma LE02 seed culture medium is: yeast powder 3g, NaNO3 0.3g, KCL 0.05g, MgSO4·7H2O 0.05g, FeSO4·7H2O 0.001g, distilled water 100mL, natural pH value;

[0048] The formula of the fermentation medium is as follows: carbon source 25 g / L, (NH4)2HPO4 1.5 g / L, KH2PO4 0.9 g / L, CaCO3 0.4 g / L, NaCl 1 g / L, corn steep liquor 1 g / L, (NH4)2SO4 0.8 g / L, MgSO4 0.4 g / L, and the pH is adjusted to 5.8;

[0049] Preparation steps:

[0050] (1) Preparation of Alcaligenes faecalis seed solution: a single colony of Alcaligenes faecalis was picked from an inoculation loop and inoculated into 100 ml of Alcaligenes faecalis seed solution culture medium. The culture was shaken at 200 rpm and 32°C for 18 h to obtain Alcaligenes faecalis seed solution. The viable bacterial count of the seed solution was 2 × 10 9 CFU / ml;

[0051] (2) Preparation of Trichoderma LE02 seed liquid: a single colony of Trichoderma LE02 was picked from an inoculation loop and inoculated into 50 ml of Trichoderma LE02 seed medium. The culture was shaken at 150 rpm and 30 °C for 4 days to obtain Trichoderma LE02 seed liquid. The viable bacterial count of the seed liquid was 1.2 × 10 8 CFU / mL;

[0052] (3) Fermentation: Alcaligenes faecalis seed liquid was inoculated into the fermentation medium, and the volume ratio of the Alcaligenes faecalis seed liquid to the fermentation medium was 7:100. At 8 h, 45 h, and 85 h after the inoculation of the Alcaligenes faecalis seed liquid, the Trichoderma LE02 seed liquid was inoculated once, and the volume ratio of the Trichoderma LE02 seed liquid to the fermentation medium was 1:700. The fermentation was carried out under ventilation for 120 h (the pH value of the fermentation liquid was adjusted to 7.0 with 32% liquid caustic soda, the fermentation temperature was 32°C, the ventilation intensity was 800 L / h, and the stirring speed was 500 rpm. The pH dropped to 6.5 after 24 h of fermentation and to 6.25 after 48 h of fermentation. The residual sugar content was controlled at 1.5 g / 100 ml during the fermentation process). After 120 h of fermentation under ventilation, the fermentation liquid temperature was heated to 80°C and maintained for 30 min to inactivate the enzyme to obtain β-1,3-glucoligosaccharide fermentation liquid.

[0053] (4) Purification: The β-1,3-glucoligosaccharide fermentation broth was centrifuged at 5000 r / min for 15 min, the supernatant was collected, and the temperature of the concentrated drying was not more than 110°C to obtain the crude β-1,3-glucoligosaccharide product.

[0054] Example 3

[0055] A method for preparing oligosaccharide:

[0056] Bacterial strain selection: Alcaligenes faecalis can produce curdlan; Trichoderma LE02 can produce β-1,3-endoglucanase.

[0057] Culture medium formula:

[0058] The formula of the Alcaligenes faecalis seed culture medium is as follows: carbon source 25 g / L, (NH4)2HPO4 1.4 g / L, KH2PO4 1.2 g / L, CaCO3 0.6 g / L, NaCl 1.1 g / L, corn steep liquor 1 g / L, MgSO4 0.6 g / L, and the pH is adjusted to 7.2;

[0059] The formula of the Trichoderma LE02 seed culture medium is: yeast powder 3g, NaNO3 0.3g, KCL 0.05g, MgSO4·7H2O 0.05g, FeSO4·7H2O 0.001g, distilled water 100mL, natural pH value;

[0060] The formula of the fermentation medium is as follows: carbon source 30 g / L, (NH4)2HPO4 1.8 g / L, KH2PO4 1.1 g / L, CaCO3 0.6 g / L, NaCl 1.2 g / L, corn steep liquor 1 g / L, (NH4)2SO4 1 g / L, MgSO4 0.6 g / L, and the pH is adjusted to 7.2;

[0061] Preparation steps:

[0062] (1) Preparation of Alcaligenes faecalis seed solution: a single colony of Alcaligenes faecalis was picked from an inoculation loop and inoculated into 100 ml of Alcaligenes faecalis seed solution culture medium. The culture was shaken at 200 rpm and 32°C for 18 h to obtain Alcaligenes faecalis seed solution. The number of viable bacteria in the seed solution was 4 × 10 9 CFU / ml;

[0063] (2) Preparation of Trichoderma LE02 seed liquid: a single colony of Trichoderma LE02 was picked from an inoculation loop and inoculated into 50 ml of Trichoderma LE02 seed medium. The culture was shaken at 150 rpm and 32 °C for 4 days to obtain Trichoderma LE02 seed liquid. The viable bacterial count of the seed liquid was 1.5 × 10 8 CFU / mL;

[0064] (3) Fermentation: Alcaligenes faecalis seed liquid was inoculated into the fermentation medium, and the volume ratio of the Alcaligenes faecalis seed liquid to the fermentation medium was 9:100. At 12h, 55h, and 95h after the inoculation of the Alcaligenes faecalis seed liquid, the Trichoderma LE02 seed liquid was inoculated once, and the volume ratio of the Trichoderma LE02 seed liquid to the fermentation medium was 1:700. The fermentation was carried out under ventilation for 120h (the pH value of the fermentation liquid was adjusted to 7.0 with 32% liquid caustic soda, the fermentation temperature was 32°C, the ventilation intensity was 1000L / h, and the stirring speed was 500rpm. The pH dropped to 6.5 after 24h of fermentation and to 6.25 after 48h of fermentation. The residual sugar content was controlled at 2.5g / 100ml during the fermentation process. After 120h of fermentation under ventilation, the fermentation liquid temperature was heated to 80°C and maintained for 30min to inactivate the enzyme to obtain β-1,3-glucoligosaccharide fermentation liquid.

[0065] (4) Purification: The β-1,3-glucoligosaccharide fermentation broth was centrifuged at 5000 r / min for 15 min, the supernatant was collected, and the temperature of the concentrated drying was not more than 110°C to obtain the crude β-1,3-glucoligosaccharide product.

[0066] Comparative Example 1

[0067] Steps (1)-(2) are the same as in Example 1;

[0068] Step (3) inoculating the Trichoderma LE02 seed solution once at 5h, 45h, and 85h after the inoculation of the Alcaligenes faecalis seed solution, respectively, with the remaining steps being the same as in Example 1;

[0069] Comparative Example 2

[0070] Steps (1)-(2) are the same as in Example 1;

[0071] Step (3) inoculating the Trichoderma LE02 seed solution once at 20h, 60h, and 90h after the inoculation of the Alcaligenes faecalis seed solution, respectively, with the remaining steps being the same as in Example 1;

[0072] β-1,3-glucoligosaccharide production determination:

[0073] The fermentation broth prepared in step (3) of Example 1-3 and Comparative Example 1-2 was centrifuged at 9000 rpm for 10 minutes, and the supernatant was collected. The total sugar content in the supernatant was determined using the phenol-sulfuric acid method, and the residual sugar content was determined using a biosensor analyzer. The β-1,3-glucoligosaccharide yield was calculated as the total sugar content minus the residual sugar content.

[0074] The yields of β-1,3-glucopyranose in Examples 1-3 and Comparative Examples 1-2 were statistically analyzed. The results are shown in Table 1.

[0075] Table 1: β-1,3-glucoligosaccharide production statistics

[0076] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Yield 93％ 90％ 89％ 8％ 35％

[0077] Compared with Examples 1-3, in Comparative Example 1, Trichoderma LE02 was added after 5 hours to inhibit the growth of Bacillus faecalis, and insufficient polysaccharides were produced to be hydrolyzed into oligosaccharides, resulting in low oligosaccharide yield. In Comparative Example 2, the biomass of Bacillus faecalis was already large at 20 hours, and a large amount of polysaccharides were produced at this time. The high viscosity caused insufficient oxygen supply, which affected the growth of Trichoderma LE02, making it impossible for Trichoderma LE02 to produce sufficient endo-β-1,3-glucanase to hydrolyze polysaccharides. Finally, the oligosaccharide yield after drying was 35%.

[0078] The results showed that the window period for successfully producing β-1,3-glucoligosaccharides by directly adding Trichoderma LE02 seed liquid is very narrow. Therefore, in industrial production, Trichoderma LE02 should still be fermented separately and then batch-inoculated into the curd polysaccharide fermentation broth for co-cultivation. In this way, the two strains can coexist for a longer period of time, which is conducive to obtaining higher yields. When Trichoderma LE02 is inoculated too early, the growth of Alcaligenes faecalis is inhibited, and not enough polysaccharides are produced to be hydrolyzed into oligosaccharides, resulting in low oligosaccharide yields. When Trichoderma LE02 is inoculated too late, the biomass of Alcaligenes faecalis is already large, and a large amount of polysaccharides are produced at this time. The high viscosity causes insufficient oxygen supply, which affects the growth of Trichoderma LE02, making it unable to produce enough endo-β-1,3-glucanase to hydrolyze polysaccharides, also resulting in very low oligosaccharide yields.

[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 oligoglucose, characterized in that: The following steps are involved: (1) Preparation of Alcaligenes faecalis seed liquid: Pick a single colony of Alcaligenes faecalis and inoculate it into a culture medium for Alcaligenes faecalis seed liquid. Cultivate it on a shaking table to obtain Alcaligenes faecalis seed liquid. The viable bacterial count of the seed liquid is 2-4×10 9 CFU / ml; (2) Preparation of Trichoderma LE02 seed liquid: Pick a single colony of Trichoderma LE02, inoculate it into Trichoderma LE02 seed culture medium, and culture it on a shaking table to obtain Trichoderma LE02 seed liquid; the number of viable bacteria in the seed liquid is 1-1.5×10 8 CFU / mL; (3) Fermentation: Inoculate the fermentation medium with a Bacillus faecalis seed solution, and inoculate the Trichoderma LE02 seed solution 8-12 hours, 45-55 hours, and 85-95 hours after the inoculation of the Bacillus faecalis seed solution. Ferment the medium with ventilation for 120 hours to obtain a β-1,3-glucopyranose fermentation solution. The formula of the fermentation medium in step (3) is: carbon source 25-30 g / L, (NH4)2HPO4 1.5-1.8 g / L, KH2PO4 0.9-1.1 g / L, CaCO3 0.4-0.6 g / L, NaCl 1-1.2 g / L, corn steep liquor 1 g / L, (NH4)2SO4 0.8-1 g / L, MgSO4 0.4-0.6 g / L, and the pH is adjusted to 5.8-7.2; The volume ratio of the Alcaligenes faecalis seed solution to the fermentation medium is 7-9:100; The volume ratio of the single inoculation amount of the Trichoderma LE02 seed liquid to the fermentation medium is 1:700; (4) Purification: The β-1,3-glucoligosaccharide fermentation broth was centrifuged, and the supernatant was collected, concentrated, and dried to obtain a crude β-1,3-glucoligosaccharide product.

2. The method for preparing oligoglucose according to claim 1, wherein: The Alcaligenes faecalis is capable of producing curdlan.

3. The method for preparing oligoglucose according to claim 1, wherein: The Trichoderma LE02 can produce β-1,3-endoglucanase.

4. The method for preparing oligoglucose according to claim 1, wherein: The formula of the Alcaligenes faecalis seed liquid culture medium described in step (1) is: carbon source 22-25 g / L, (NH4)2HPO4 1.2-1.4 g / L, KH2PO4 1.1-1.2 g / L, CaCO3 0.4-0.6 g / L, NaCl 0.9-1.1 g / L, corn steep liquor 1 g / L, MgSO4 0.4-0.6 g / L, and the pH is adjusted to 6.8-7.2; The inoculation amount of the Alcaligenes faecalis is one inoculation loop of a single colony of the Alcaligenes faecalis inoculated into 100 ml of the Alcaligenes faecalis seed liquid culture medium.

5. The method for preparing oligoglucose according to claim 1, wherein: The shaking culture conditions in step (1) are: 200 rpm, 32° C., and 18 h.

6. The method for preparing oligoglucose according to claim 1, characterized in that: The formula of the Trichoderma LE02 seed culture medium described in step (2) is: yeast powder 3g, NaNO3 0.3g, KCL 0.05g, MgSO4·7H2O 0.05g, FeSO4·7H2O 0.001g, distilled water 100mL, natural pH value; The inoculation amount of Trichoderma LE02 is one inoculation loop of a single colony of Trichoderma LE02 inoculated into 50 ml of Trichoderma LE02 seed culture medium.

7. The method for preparing oligoglucose according to claim 1, characterized in that: The shaking culture conditions in step (2) are: 150 rpm, 30-32° C., and 4 days of culture.

8. The method for preparing oligoglucose according to claim 1, wherein: The conditions for the ventilation fermentation in step (3) are as follows: the pH value of the fermentation liquid is adjusted to 7.0 with a 32% concentration of liquid caustic soda at the initial and early stages of fermentation, the fermentation temperature is 32° C., the ventilation intensity is 800-1000 L / h, and the stirring speed is 500 rpm; the pH drops to 6.5 after 24 hours of fermentation and to 6.25 after 48 hours of fermentation, and the residual sugar is controlled at 1.5-2.5 g / 100 ml during the fermentation process; After 120 h of fermentation under ventilation, the fermentation liquid temperature was heated to 80°C and maintained for 30 min to inactivate the enzyme.

9. The method for preparing oligoglucose according to claim 1, wherein: The centrifugal conditions in step (4) are: 5000 r / min, 15 min; the concentration and drying temperature does not exceed 110°C.