A marine bacterium bacillus sp. SQ3 and a method and application of producing dextranase thereof

Abstract

The application relates to the field of microbial technology, and particularly discloses a marine bacterium Bacillus sp. SQ3, a method for producing dextranase by the marine bacterium Bacillus sp. SQ3 and application, the bacterium is preserved in the China General Microbiological Culture Collection Center on January 20, 2025, the preservation number is CGMCC No. 33458, and the address of the preservation unit is No. 3, Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing. The dextranase produced by the strain SQ3 has the advantage of preparing high-polymerization isomaltodextrin, in the hydrolysis product, the proportion of tetrasaccharide is 9.9%, the proportion of pentasaccharide is 15.5%, the proportion of hexasaccharide is 32.7%, the proportion of heptasaccharide is 33.9%, and the total proportion is 92%, the bacterium and the enzyme produced by the bacterium have the significant advantage of being applied to the preparation of high-polymerization oligosaccharide.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a marine bacterium, Bacillus sp. SQ3, and its method and application for producing dextranase. Background Technology

[0002] The ocean's ecological environment experiences minimal temperature fluctuations and boasts a rich diversity of life, with microorganisms adapted to varying temperatures, pH levels, and salinity. Marine microorganisms predominantly exist in slightly alkaline environments, and their secreted enzymes exhibit low optimal reaction temperatures and slightly alkaline optimal pH levels. Fungi, yeasts, and bacteria have all been found to produce dextranase, and the dextranase produced by marine bacteria also exhibits salt and alkali tolerance, as well as low reaction temperatures.

[0003] Dextran is a poly-D-glucose with a main chain linked by α-1,6 glycosidic bonds. Dextranase (EC 3.2.1.11) is a hydrolase that specifically hydrolyzes the α-1,6 glycosidic bonds in dextran, producing oligosaccharides and isomaltooligosaccharides (IMO). Dextranase is mainly used in medicine, food, health products, sugar refining, and biotechnology. It can hydrolyze high-molecular-weight dextran to prepare functional isomaltooligosaccharides; dextran with varying degrees of hydrolysis can be used as a food additive to improve the softness of food products.

[0004] IMO (Inorganic Oligosaccharide) is a non-digestible oligosaccharide with a low glycemic index, which helps maintain blood glucose homeostasis. It also has functions such as lowering cholesterol levels, promoting gut microbiota, and alleviating intestinal inflammation, and is widely used in the food and health food industry. Studies have shown that the effects of oligosaccharides are closely related to their degree of polymerization. High-polymerization oligosaccharides (polymerization degree greater than 4) can significantly promote the growth of beneficial gut bacteria such as Bifidobacteria and Lactobacillus, regulating the balance of gut microbiota. Simultaneously, it can enhance intestinal barrier function and immunity, improving anti-infection and anti-inflammatory capabilities. Furthermore, high-polymerization oligosaccharides help improve metabolism and have certain hypoglycemic and hypolipidemic effects. In contrast, low-polymerization oligosaccharides are easily digested and absorbed by the body, resulting in lower biological activity. Testing has revealed that commercially available prebiotic oligosaccharide products are mainly composed of low-polymerization components, which to some extent weakens their effect on promoting probiotic growth. Therefore, increasing the proportion of high-polymerization oligosaccharides in products is of great significance for enhancing the function of prebiotics. However, current oligosaccharide production and processing technologies lack key enzymes for the efficient preparation of high-polymerization oligosaccharides. This provides a method and application for the production of dextranase from the marine bacterium Bacillus sp. SQ3. Summary of the Invention

[0005] The purpose of this invention is to address the deficiencies of the prior art by providing a marine bacterium, Bacillus sp. SQ3, and its method and application for producing dextranase, thereby solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a marine bacterium, Bacillus sp. SQ3, which was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33458. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0007] A method for producing dextranase from the marine bacterium Bacillus sp. SQ3 as described above, comprising the following specific steps:

[0008] Step 1: Strain SQ3 slant culture was stored at 4℃; it was inoculated into growth medium with a liquid volume of 10%, and cultured at 180 r / min and 40℃ for 12 h to obtain a bacterial suspension;

[0009] Step 2: Inoculate the bacterial suspension into the enzyme-producing medium at an inoculation rate of 2% and a liquid volume of 10%, and incubate at 180 r / min and 30℃ for 60 h to obtain the fermentation broth;

[0010] Step 3: Centrifuge the fermentation broth at 4℃ and 8000rpm for 10min. Use a 0.22μm filter membrane to sterilize the supernatant. Centrifuge the filtrate at 4℃ and 4500rpm for 10min using an ultrafiltration tube with a molecular weight of 30kDa. The portion with a molecular weight greater than 30kDa is the enzyme solution.

[0011] As a preferred embodiment of the present invention, the growth culture medium in step 1 is prepared by mixing 1 g / L potato starch, 5 g / L soybean peptone, and pure water, with a pH of 9.0, and sterilized at 121°C for 20 min.

[0012] As a preferred embodiment of the present invention, the enzyme-producing culture medium in step 2 is prepared by sterilizing at 121°C for 20 minutes with 1 g / L cassava starch, 5 g / L fish meal peptone, 4 g / L dextran T20, and pH 7.5.

[0013] One application of the dextranase produced by the method described above is to hydrolyze dextran T20 with enzyme solution to produce isomalt oligosaccharide.

[0014] The specific steps are as follows: Mix the enzyme solution with the substrate at a ratio of 1:3, react at 35°C for 12 hours, boil in water for 5 minutes, centrifuge at 8000 r / min for 5 minutes, take the supernatant and filter it through a 0.22 μm filter membrane. After drying the filtrate, it is isomaltooligosaccharide, in which the content of oligosaccharides with a degree of polymerization greater than 4 is greater than 90%.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] This invention provides a novel Bacillus sp. SQ3 strain capable of producing dextranase, enriching the variety of dextranase-producing marine bacteria. This strain has the following characteristics: a growth temperature range of 20-45℃, an initial growth pH range of 5.0-11.0, an optimal growth temperature of 40℃, and an optimal initial growth pH of 9.0; the optimal operating temperature for the dextranase produced by strain SQ3 is 40℃, which is relatively low.

[0017] The dextranase produced by strain SQ3 of this invention has the advantage of preparing high-polymerization-degree isomalt oligosaccharides. In the hydrolysis products of this enzyme, the proportion of tetrasaccharides is 9.9%, pentasaccharides is 15.5%, hexasaccharides is 32.7%, and heptasaccharides is 33.9%, with the total proportion of high-polymerization-degree oligosaccharides reaching 92%. This strain and its enzyme have significant advantages in the preparation of high-polymerization-degree oligosaccharides. Attached Figure Description

[0018] Figure 1 This is a diagram of the transparent circles formed on the screening plate of strain SQ3 of the present invention;

[0019] Figure 2 Gram staining image of strain SQ3 of this invention;

[0020] Figure 3 This is a scanning electron microscope image of strain SQ3 of the present invention;

[0021] Figure 4 This is a phylogenetic tree diagram of the rDNA of strain SQ316S of this invention;

[0022] Figure 5 This is a graph showing the effect of time on the growth of strain SQ3 in this invention;

[0023] Figure 6 This is a graph showing the effect of temperature on the growth of strain SQ3 in this invention;

[0024] Figure 7 This is a graph showing the effect of the initial pH value of the culture medium of this invention on the growth of strain SQ3;

[0025] Figure 8 This is a graph showing the effect of NaCl concentration in the culture medium of this invention on the growth of strain SQ3;

[0026] Figure 9 This is a diagram showing the effect of the carbon source of this invention on the growth of strain SQ3;

[0027] Figure 10 This is a diagram showing the effect of the nitrogen source of this invention on the growth of strain SQ3;

[0028] Figure 11 This is a graph showing the effect of time on enzyme production by strain SQ3 in this invention;

[0029] Figure 12 This is a graph showing the effect of temperature on enzyme production by strain SQ3 in this invention;

[0030] Figure 13 This is a graph showing the effect of the initial pH of the culture medium on enzyme production by strain SQ3.

[0031] Figure 14 This is a graph showing the effect of NaCl concentration in the culture medium of this invention on enzyme production by strain SQ3;

[0032] Figure 15 This is a diagram showing the effect of the carbon source of this invention on enzyme production by strain SQ3;

[0033] Figure 16 This is a diagram showing the effect of the nitrogen source of the present invention on enzyme production by strain SQ3;

[0034] Figure 17 This is a graph showing the effect of different molecular weight inducers of the present invention on enzyme production by strain SQ3;

[0035] Figure 18 This is a graph showing the effect of T20 concentration on enzyme production by strain SQ3 in this invention;

[0036] Figure 19 This is a graph showing the effect of the enzyme's reaction temperature on its activity.

[0037] Figure 20 This is a graph showing the thermal stability of the enzyme of this invention;

[0038] Figure 21 This is a graph showing the effect of pH on enzyme activity in this invention.

[0039] Figure 22 This is a pH stability diagram of the enzyme of the present invention;

[0040] Figure 23 The figure shows the HPLC detection results of the dextran T20 product hydrolyzed by dextranase in this invention.

[0041] (A): HPLC chromatogram of sugar standard; (B): HPLC chromatogram of dextranase hydrolysis of T2012h by strain SQ3. Detailed Implementation

[0042] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0043] A marine bacterium, Bacillus sp. SQ3, was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33458. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0044] The colonies of this bacterium are milky white, viscous, with serrated edges, a smooth surface, and a slightly raised center, indicating that it is a Gram-positive bacillus. Phylogenetic analysis of the 16S rDNA of strain SQ3 and comprehensive morphological characteristics identified strain SQ3 as belonging to the genus Bacillus sp. The optimal growth medium for strain SQ3 is: potato starch: 1 g / L, soybean peptone: 5 g / L, pH 9.0, and a culture temperature of 40℃.

[0045] A method for producing dextranase from the marine bacterium Bacillus sp. SQ3, the specific steps of which are as follows:

[0046] S1: The strain SQ3, which was screened from sea mud and preserved in a 4℃ refrigerator, was inoculated into a growth medium (1 g / L potato starch, 5 g / L soybean peptone, pH 9.0, sterilized at 121℃ for 20 min before use), with a liquid volume of 10%, 180 r / min, and 40℃ for 12 h to obtain a bacterial suspension;

[0047] S2: The bacterial suspension was inoculated into the enzyme-producing medium (1 g / L cassava starch, 5 g / L fish meal peptone, 4 g / L dextran T204, pH 7.5, sterilized at 121℃ for 20 min before use) with an inoculation amount of 2% and a liquid volume of 10%. The culture was carried out at 180 r / min and 30℃ for 60 h to obtain the fermentation broth.

[0048] S3: The fermentation broth was centrifuged at 8000 rpm for 10 min at 4℃. The supernatant was then filtered through a 0.22 μm filter membrane for sterilization. The filtrate was purified by ultrafiltration using a 30 kDa ultrafiltration tube at 4℃ and 4500 rpm for 10 min to obtain the enzyme solution.

[0049] The application of dextranase produced by the marine bacterium Bacillus sp. SQ3 in the preparation of prebiotics: High-performance liquid chromatography (HPLC) analysis was performed on the product of dextranase hydrolysis of dextran T20.

[0050] The specific steps are as follows:

[0051] The enzyme solution and substrate were mixed at a 1:3 ratio and reacted at 35°C for 12 h. The mixture was then boiled in water for 5 min, centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and stored at 4°C for subsequent high-performance liquid chromatography (HPLC) analysis. The chromatographic column was a Waters Sugar-PAK1 (6.5 × 300 mm), the mobile phase was deionized water, the flow rate was 0.4 mL / min, and the column temperature was 75°C.

[0052] Example: The features of this invention include the marine strain Bacillus sp. SQ3, which is a marine bacterium isolated from marine mud in the waters off Qingdao, Shandong, China. This strain was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33458. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. This invention provides the strain and a method for producing dextran using the strain, as well as the product obtained by hydrolyzing dextran T20 using dextranase produced by the strain. The product contains 90% oligosaccharides with a high degree of polymerization.

[0053] 1. Morphological characteristics of strain SQ3 of the present invention

[0054] 1.1 Colony morphology characteristics

[0055] The dextranase produced by strain SQ3 has milky white, viscous colonies with serrated edges, a smooth surface, and a slightly convex center. Figure 1 ), is a Gram-positive bacillus ( Figure 2 ), electron microscopic morphology as ( Figure 3 ).

[0056] 1.2 Molecular biological identification of strain SQ3

[0057] Genes from strain SQ3 were extracted using a kit, and PCR amplification was performed using universal primers for prokaryotes: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTACGACTT-3'). The PCR amplification system consisted of 25 μL (1 μL DNA template, 12.5 μL PCR Mix, 1 μL 27F, 1 μL 1492R, and 9.5 μL dd H2O). Amplification conditions were: 94℃ denaturation for 4 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 2 min, for 35 cycles; final extension at 72℃ for 10 min; and storage at 4℃. The PCR products were sent to Shanghai Sangon Biotech for sequencing. The sequencing results were submitted to the National Center for Biotechnology Information (NCBI) database for comparison, and a phylogenetic tree was constructed using MEGA 7.0 software via neighbor-joining. The 16S rDNA length was measured to be 1444 bp. BLAST analysis on NCBI showed that the 16S rDNA of strain SQ3 shared 98% homology with *Bacillus subtilis*. Based on morphological characteristics, strain SQ3 was identified as belonging to the genus *Bacillus*. A phylogenetic tree was constructed using the NJ method in MEGA 7.0 software. The results are shown below. Figure 4 .

[0058] 2. Growth characteristics of strain SQ3 of the present invention

[0059] The growth characteristics of strain SQ3 provided by this invention were studied, and the growth conditions of this strain were obtained.

[0060] 2.1 Preparation of Seed Liquid

[0061] Strain SQ3 was inoculated into 2216E medium at pH 8.0, 35℃, 180 rpm, with a liquid volume of 20%, and cultured for 12 h.

[0062] 2.2 Effect of time on the growth of strain SQ3

[0063] The seed culture was inoculated into 2216E medium at a 2% inoculum, and cultured at pH 8.0, 35℃, and 180 r / min. OD was measured every 2 hours. 600nm Value. Strain SQ3 exhibits the most active growth during the 0-18h period, being in the logarithmic growth phase; after 18h, the growth rate slows down, entering the stationary growth phase. Figure 5 ).

[0064] 2.3 Effect of temperature on the growth of strain SQ3

[0065] The seed culture was inoculated at a rate of 2% into 2216E medium at pH 8.0 and cultured at 180 rpm for 12 h at different temperatures. Strain SQ3 could grow in the range of 20–45℃, with an optimal growth temperature of 40℃. Figure 6 ).

[0066] 2.4 Effect of initial pH of culture medium on the growth of strain SQ3

[0067] The seed culture was inoculated at a rate of 2% into 2216E medium at different pH values ​​and cultured at 40℃ and 180 rpm for 12 h. The optimal pH range for strain SQ3 was 8–10, with the best growth observed at pH 9. Figure 7 ).

[0068] 2.5 Effect of NaCl concentration in culture medium on the growth of strain SQ3

[0069] The seed culture was inoculated at a rate of 2% into 2216E medium with different NaCl concentrations, and cultured at pH 9.0, 40℃, and 180 rpm for 12 h. Strain SQ3 could grow at NaCl concentrations ranging from 0% to 8%, with the best growth observed under conditions without NaCl. Figure 8 ).

[0070] 2.6 Effects of carbon and nitrogen sources on the growth of strain SQ3

[0071] Maltose, sucrose, lactose, dextrin, yeast, soluble starch, potato starch, cassava starch, and corn starch were used as carbon sources to replace yeast. The cultures were incubated at 40℃ and 180 r / min for 12 h, and the absorbance (OD600 nm) of the bacterial suspension was measured. The optimal carbon source was selected, and fish meal peptone, ammonium sulfate, soybean peptone, ammonium chloride, beef extract, and urea were used as nitrogen sources to replace fish meal peptone. The effect of nitrogen source on the growth of strain SQ3 was studied. Strain SQ3 grew well under various carbon source conditions, with potato starch being the optimal carbon source. Figure 9 The optimal nitrogen source is soybean peptone ( ), Figure 10 ).

[0072] 3. Conditions for dextranase production by strain SQ3 of the present invention

[0073] 3.1 Effect of fermentation time on enzyme production by strain SQ3

[0074] The seed culture was inoculated at a rate of 2% into the enzyme-producing medium, cultured at pH 8.0, 35℃, and 180 rpm, and enzyme activity was measured at regular intervals. The highest enzyme activity was observed at a fermentation time of 60 hours. Figure 11 ).

[0075] 3.2 Effect of fermentation temperature on enzyme production by strain SQ3

[0076] The seed culture was inoculated at a rate of 2% into the enzyme-producing medium, pH 8.0, and cultured at 20-40℃ and 180 rpm for 60 h. The optimal enzyme production temperature for the strain was 30℃. Figure 12 ).

[0077] 3.3 Effect of initial pH of culture medium on enzyme production by strain SQ3

[0078] The seed culture was inoculated at a rate of 2% into enzyme-producing media with different pH values ​​and cultured at 30℃ and 180 rpm for 60 h. The optimal pH for enzyme production of the strain was 7.5. Figure 13 ).

[0079] 3.4 Effect of NaCl concentration in culture medium on enzyme production by strain SQ3

[0080] The seed culture was inoculated at a rate of 2% into enzyme-producing media with different NaCl concentrations and cultured at pH 7.5, 30℃, and 180 rpm for 60 h. The optimal NaCl concentration for enzyme production by the strain was 0% ( Figure 14 ).

[0081] 3.5 Effects of carbon and nitrogen sources on enzyme production by strain SQ3

[0082] The basal culture medium was an enzyme-producing medium with a formulation of 0.1% yeast, 0.5% fish meal peptone, and 1% dextran T20, pH 9.0, and a volume of 40%. Maltose, sucrose, lactose, dextrin, yeast, soluble starch, potato starch, cassava starch, and corn starch were used as carbon sources to replace yeast, respectively. The inoculum size was 2%, the culture temperature was 30℃, the speed was 180 rpm, and the culture time was 60 h. Samples were taken to determine enzyme activity. The optimal carbon source was selected, and then fish meal peptone, ammonium sulfate, soybean peptone, ammonium chloride, beef extract, and urea were used as nitrogen sources to replace fish meal peptone, respectively, to study the effects of these factors on enzyme production by the strain. Among them, the optimal carbon source for enzyme production in strain SQ3 was cassava starch. Figure 15 The optimal nitrogen source is fishmeal peptone. Figure 16 ).

[0083] 3.6 Effects of different molecular weight inducers on enzyme production by strain SQ3

[0084] Equal amounts of dextran T40, T70, and T500 were used to replace T20 as inducers. Seed culture was inoculated at a 2% inoculum on enzyme-producing medium and cultured for 60 h at pH 7.5, 30℃, and 180 rpm. The optimal inducer for the strain was dextran T20. Figure 17 ).

[0085] 3.7 Effect of dextran T20 concentration on enzyme production by strain SQ3

[0086] The seed culture was inoculated at a rate of 2% into enzyme-producing medium with T20 concentrations ranging from 0% to 1.6%, and cultured at pH 7.5, 30℃, and 180 rpm for 60 h. The optimal dextran T20 concentration for enzyme production by the strain was 0.4%. Figure 18 ).

[0087] 4. Enzymatic properties of dextranase produced by strain SQ3 of this invention

[0088] 4.1 Enzyme activity assay

[0089] Enzyme activity was determined using the DNS method. 150 μL of substrate (75 μL Tris-HCl buffer 8.0 + 75 μL 6% dextran T20) and 50 μL of enzyme solution were incubated in a 40°C water bath for 20 min. Then, 200 μL of DNS was added, and the mixture was boiled in boiling water for 5 min. After the reaction, 3 mL of distilled water was added to the resulting mixture. The enzyme activity was then measured using the OD method. 540 nm The absorbance of the samples was measured and enzyme activity was calculated under the specified conditions. In the control group, DNS was added first, followed by the enzyme solution, and all other procedures were the same. The dextranase activity was calculated using the following formula:

[0090]

[0091] (a is the slope of the DNS curve; b is the intercept of the curve)

[0092] 4.2 Effect of temperature on dextranase activity and the thermostability of the enzyme

[0093] The optimal operating temperature for this enzyme is 40℃, and it maintains high catalytic activity in the temperature range of 35~40℃, with a relative enzyme activity of not less than 80%. Figure 19 After incubation at 30℃ for 5 hours, the enzyme activity decreased by nearly 60%; after storage at 40℃ for 5 hours, the enzyme activity decreased by nearly 80%. Figure 20 ).

[0094] 4.3 Effect of pH on dextranase activity and stability

[0095] The enzyme maintains high activity levels within a pH range of 6.0–9.0, with its optimal pH being 8.0 of Tris-HCl. Figure 21 After being incubated in a pH 6.0-9.0 buffer solution at 30°C for 1 hour, the enzyme still retained more than 60% of its activity. Figure 22 ).

[0096] 4.4 Effects of Metal Ions on Enzyme Activity

[0097] The effects of metal ions on dextranase activity are shown in Table 1. 2+ It can increase enzyme activity, Cu2+ Fe 3+ Mn 2+ Co 2+ Zn 2+ K + Na + Mg 2+ Ni 2+ Inhibit enzyme activity, 1 mmol / L 5 mmol / L Ca 2+ It promotes enzyme activity.

[0098] Table 1: Effects of metal ions on dextranase activity

[0099]

[0100]

[0101] 4.5 Effects of Organic Solvents on Enzyme Activity

[0102] Hexane and Tween 20 have a certain promoting effect on this enzyme, while ethyl acetate and petroleum ether have little effect on its efficacy. All other organic solvents have a certain inhibitory effect on it.

[0103] Table 2: Effects of organic solvents on dextranase activity

[0104] Comparison 100.0±6.73 ethanol 70.48±0.45 Ethylene glycol 66.03±6.22 methanol 75.51±5.50 Ethyl acetate 100.53±3.33 glycerin 70.77±8.97 Acetonitrile 59.33±3.31 n-Propanol 56.34±7.01 n-Butanol 29.66±8.80 n-Hexane 115.72±1.75 Twain 20 115.50±7.04 Twain 80 58.09±7.90 petroleum ether 102.82±7.97

[0105] 4.6 Substrate specificity of enzymes

[0106] As shown in Table 3, the optimal substrate for this enzyme is dextran T20. This enzyme can specifically hydrolyze dextran of different molecular weights composed of α-1,6 glycosidic bonds. It still retains 10.98% catalytic activity for soluble starch composed of α-1,4 and α-1,6 glycosidic bonds, but cannot hydrolyze pullulan composed of α-1,4 and α-1,6 glycosidic bonds or sucrose composed of β-1,2 glycosidic bonds.

[0107] Table 3: Substrate specificity of dextranase

[0108]

[0109]

[0110] 5. Application of dextranase from strain SQ3

[0111] 5.1 High-performance liquid chromatography analysis of dextran T20 hydrolyzed by dextranase from strain SQ3

[0112] The enzyme solution and substrate were mixed at a 1:3 ratio and reacted at 35℃ for 12 h. The mixture was then boiled in water for 5 min, centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and stored at 4℃ for subsequent high-performance liquid chromatography (HPLC) analysis. The chromatographic column was a Waters Sugar-PAK1 (6.5 × 300 mm), the mobile phase was deionized water, the flow rate was 0.4 mL / min, and the column temperature was 75℃. Figure 23 The proportion of oligosaccharides in the product is calculated based on the peak area.

[0113] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A type of Bacillus ( Bacillus sp.)SQ3, characterized in that: This strain was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 33458, address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The products of dextran T20 hydrolyzed by dextranase produced by Bacillus SQ3 contain 9.9% tetrasaccharides, 15.5% pentasaccharides, 32.7% hexasaccharides, and 33.9% heptasaccharides. The total proportion of high-polymerization-degree isomalt oligosaccharides with a degree of polymerization greater than 4 is 92%.

2. A method for producing dextranase solution from Bacillus SQ3 as described in claim 1, characterized in that: The specific steps are as follows: Step 1: Bacillus SQ3 slant culture was stored at 4℃; it was inoculated into growth medium at a volume of 10%, and cultured at 180 r / min and 40℃ for 12 h to obtain a bacterial suspension; Step 2: Inoculate the bacterial suspension into the enzyme-producing medium at an inoculation rate of 2% and a liquid volume of 10%, and incubate at 180 r / min and 30℃ for 60 h to obtain the fermentation broth; Step 3: Centrifuge the fermentation broth at 4°C and 8000 rpm for 10 min. Use a 0.22 μm filter membrane to sterilize the supernatant. Centrifuge the filtrate at 4°C and 4500 rpm for 10 min using an ultrafiltration tube with a molecular weight of 30 kDa. The portion with a molecular weight greater than 30 kDa is the dextran enzyme solution.

3. The method for producing dextranase solution from Bacillus SQ3 according to claim 2, characterized in that: The growth medium in step 1 was prepared by mixing 1 g / L potato starch, 5 g / L soybean peptone, and pure water, with a pH of 9.0, and sterilized at 121°C for 20 min.

4. The method for producing dextranase solution from Bacillus SQ3 according to claim 3, characterized in that: The enzyme-producing culture medium in step 2 was prepared by sterilizing at 121°C for 20 min with 1 g / L tapioca starch, 5 g / L fish meal peptone, 4 g / L dextran T20, and pH 7.5.

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