A strain producing gentiobiose and its application

Abstract

The present invention relates to a gentiobiose-producing strain and its application. The strain is classified and named as Bacillus subtilis ( Bacillus height ) B878, whose deposit number is GDMCC No: 63265. The highland Bacillus provided by the present invention can use cheap glucose as a substrate to convert into gentiobiose with high economic value, with a conversion rate of 20% under optimal conditions, and has good genetic stability, and can be used as a candidate strain for industrial production of gentiobiose.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a gentiobiose-producing strain and application thereof. Background Art

[0002] Gentian oligosaccharides are a new type of functional oligosaccharides bonded by β-1, 6 glucoside bonds, mainly including gentiobiose and a small amount of gentiotriose and tetrasaccharide. As a functional food base, gentiooligosaccharides have a significant effect on the proliferation of beneficial bacteria in the intestine, such as bifidobacteria, and have the functions of regulating the balance of intestinal flora and improving laxative effects. They also have the functions of inhibiting tumors and promoting nutrient absorption and metabolism. In addition, the heat and acid resistance of gentiooligosaccharides make it a food additive that can be added to some health foods that cannot be added with oligosaccharides, so it has broad application prospects.

[0003] Currently, the production of gentiobiose reported in the literature is mostly the expression of β-glucosidase in Escherichia coli and Pichia pastoris, but the endotoxins produced by Escherichia coli itself will bring safety issues, and the expression of Pichia pastoris requires methanol induction, both of which are not suitable for the food field. Compared with these two expression systems, Bacillus thuringiensis, which can express β-glucosidase, is a food-safe strain that has low requirements for culture medium and can achieve large-scale production of food-grade gentiooligosaccharides. Summary of the invention

[0004] The first object of the present invention is to provide a gentiobiose-producing strain, which is classified and named as Bacillus subtilis ( Bacillus altitudinis )B878.

[0005] Another object of the present invention is to provide the use of the above-mentioned gentiobiose-producing strain in the fermentation production of gentiobiose.

[0006] Specifically, after the gentiobiose-producing strain is subjected to seed culture, the seed liquid is inoculated into a fermentation medium / fermentation tank for fermentation culture to obtain gentiobiose.

[0007] As a preferred embodiment, the culture medium for seed culture is LB medium.

[0008] As a preferred embodiment, the gentiobiose-producing strain produces gentiobiose by fermentation using glucose as a carbon source.

[0009] As a preferred embodiment, the glucose concentration is 40-100 g / L; preferably 60-80 g / L.

[0010] As a preferred embodiment, the pH of the fermentation culture is 5-11; preferably 7-11.

[0011] As a preferred embodiment, the fermentation culture temperature is 10-50°C; preferably 30-50°C.

[0012] As a preferred embodiment, the fermentation culture time is 24-48 h.

[0013] As a preferred embodiment, the culture medium for fermentation culture includes a carbon source, a nitrogen source and an inorganic salt; the nitrogen source is selected from peptone and yeast powder; the inorganic salt is selected from potassium dihydrogen phosphate, calcium chloride and magnesium sulfate.

[0014] The present invention screens and separates a strain producing gentiobiose from the environment, which belongs to Bacillus subtilis, and can convert cheap carbon source glucose to synthesize gentiobiose, and the conversion rate can reach 20%. The gentiobiose strain B878 is subcultured, and the output of synthesized gentiobiose is basically unchanged, and it has genetic stability. The strain screened and obtained by the present invention produces gentiobiose, which has the characteristics of safety, environmental protection and high efficiency, and has great application prospects in the fields of medical treatment, food and health. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the HPLC chart of gentiobiose.

[0016] Figure 2 This is the colony morphology of Bacillus subtilis B878.

[0017] Figure 3 This is the phylogenetic tree of Bacillus subtilis B878.

[0018] Figure 4 Standard curve for determination of gentiobiose by HPLC.

[0019] Figure 5 This is the growth curve of Bacillus subtilis B878.

[0020] The biological material of the present invention is classified and named as Bacillus subtilis ( Bacillus altitudinis )B878, was deposited in the Guangzhou Microbiological Culture Collection Center (GDMCC) on March 12, 2023, with the deposit number: GDMCCNo: 63265, and the deposit address: Guangdong, China. Implementation

[0021] The culture medium formula used in the embodiment is as follows

[0022] Seed culture medium (LB medium): 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder, pH 7.0.

[0023] β-Glucosidase identification medium: 10 g / L glucose, 5 g / L peptone, 15 g / L yeast extract, 15 g / L NaCl, 0.3 g / L ammonium ferric citrate, 10 g / L agar, 0.1 g / L esculin, pH 7.0.

[0024] Fermentation medium: 50 g / L glucose, 3 g / L peptone, 3 g / L yeast powder, 5 g / L KH2PO4, 0.5 g / L CaCl2, 0.5 g / L MgSO4, pH 7.0.

[0025] Example 1 Isolation and identification of gentiobiose-producing strains

[0026] Take 1 g of soil sample from the Populus euphratica forest in Xinjiang, add 10 mL of sterile water, and shake in a shaker at 30 ℃ and 200 r / min for 30 min to mix thoroughly. After standing for 2 h, take 1 mL of the supernatant and add it to 20 mL of LB liquid culture medium, and place it in a shaker at 37 ℃ and 200 r / min for 24 h.

[0027] Initial screening of strains: dilute the above activated bacterial solution to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , spread 200 μL of each dilution on a β-glucosidase identification medium plate, culture at 37 ℃ for 48 h, and pick a single colony from the plate as a re-screening excellent strain.

[0028] Rescreening of strains: Inoculate the primary screened strains from the plate culture medium into 50 g / L glucose solution, place them in a shaker at 37 °C and 200 r / min for 48 h, take the supernatant and centrifuge for 10 min, and determine the gentiobiose content by high performance liquid chromatography. Figure 1 Shown is the elution time of gentiobiose.

[0029] Colony morphology observation: The high-yield gentiobiose strain obtained by repeated screening was inoculated into LB medium for activation and cultured at 37 °C for 48 h for morphological observation. Figure 2 shown.

[0030] Molecular biological identification: The 16S rRNA sequence of the strain was sequenced in NCBI using BLAST and compared with the 16S rDNA gene sequence registered in GenBank for homology, and phylogenetic analysis was performed using MEGA 7.0.14, and a phylogenetic tree was constructed using the Neighbor-Joining method to determine the taxonomic status of the strain. Figure 3 As shown, the strain identification results showed that it was Bacillus subtilis, and the strain was named B878.

[0031] Example 2 Determination of gentiobiose standard curve by high performance liquid chromatography

[0032] Gentianobiose was detected by high performance liquid chromatography with the following specific parameters: differential refractometer, chromatographic column: Hanbon Sci.&Tech. Lichospher NH2 (250 mm ×4.6 mm, 5 μm); column temperature: 40 °C; cell temperature (detector): 40 °C; mobile phase: 80% acetonitrile; injection volume: 5 μL; flow rate: 1 mL / min.

[0033] Take different concentrations of gentiobiose standard solution for high performance liquid chromatography analysis and draw a standard curve diagram, such as Figure 4 As shown, the content of gentiobiose produced by the strain can be calculated using the standard curve.

[0034] Example 3 Effects of substrate concentration, pH and temperature on gentiobiose synthesis by Bacillus subtilis B878

[0035] (1) Growth of strains: The gentiobiose-producing strain stored in a -80°C glycerol tube was inoculated into LB medium at a 5% inoculation rate and cultured at 37°C and 200 r / min for 12 h. Then, the activated seed liquid was inoculated into the fermentation medium at a 1% inoculation rate and cultured at 37°C and 200 r / min for 24 h, and OD was measured in real time. 600 , the growth curve is as follows Figure 5 shown.

[0036] (2) Fermentation of the strain: The activated seed liquid was inoculated into the fermentation medium at a 1% inoculation rate and fermented at 37°C and 220 r / min for 48 h, during which the fermentation liquid was collected every 2 h. The fermentation liquid was centrifuged at 4°C and 12,000 r / min for 10 min, filtered through a 0.22 μm organic filter membrane, and then analyzed by HPLC. The results showed that the yield was basically stable after 24 h of cultivation.

[0037] 1) Effect of glucose concentration on gentiobiose production

[0038] The specific implementation method is the same as step (2), except that the glucose concentration in the fermentation medium is replaced, the fermentation is carried out for 48 hours, and the gentiobiose yield is determined by the method of Example 2.

[0039] Table 1 Gentianobiose production of strains under different glucose concentrations

[0040] Glucose concentration g / L 20 40 60 80 100 Gentianobiose g / L 3.65 6.41 10.96 10.32 7.34

[0041] 2) Effect of pH on gentiobiose production

[0042] The specific implementation method is the same as step (2), except that the glucose concentration in the fermentation medium is set to 60 g / L, the pH of the fermentation system is changed, the fermentation is carried out for 48 h, and the gentiobiose yield is determined by the method of Example 2.

[0043] Table 2 Gentianobiose production of strains under different pH conditions

[0044] pH 3 5 7 9 11 Gentianobiose g / L 2.87 6.73 11.12 10.67 10.35

[0045] 3) Effect of temperature on gentiobiose production

[0046] The specific implementation method is the same as step (2), except that the glucose concentration in the fermentation medium is set to 60 g / L, the pH value is set to 7, the fermentation temperature is changed, the fermentation is carried out for 48 h, and the gentiobiose yield is determined by the method of Example 2.

[0047] Table 3 Gentianobiose production of strains under different temperature conditions

[0048] Temperature ℃ 10 20 30 40 50 Gentianobiose g / L 7.95 8.36 10.79 12.04 11.51

[0049] Example 4 Synthesis of gentiobiose by Bacillus subtilis B878 under optimal conditions

[0050] A 1% inoculum was added to a 60 g / L glucose solution (pH 7), reacted at 40 °C for 48 h, and samples were taken at regular intervals to measure the readings. The changes in product concentration were determined by the standard curve. After 48 h, 12 g of gentiobiose could be converted, with a conversion rate of 20%.

[0051] Example 5 Genetic stability of Bacillus subtilis B878

[0052] Subculture on LB medium, culture at 30 ℃ for 2-3 days, then pick single colonies and inoculate them into LB medium, culture at 37 ℃, 220 r / min shaking for 12 hours as seed liquid. Transfer to fermentation medium and culture for 48 hours. From the following results, after the sixth generation, the yield can still be maintained at about 12. The strain used in industrial production is considered to have good stability if its yield is maintained at about 20% after the 5th to 6th generation. Therefore, Bacillus subtilis has industrial production value.

[0053] Table 4 Genetic stability of strains

[0054] Number of passages Generation 1 Generation 2 Generation 3 4th Generation 5th Generation 6th Generation Gentianobiose g / L 11.92 12.16 12.12 11.81 11.79 11.78

Claims

1. A gentiobiose-producing strain, characterized in that: Its classification name is Bacillus subtilis ( Bacillus altitudinis ) B878, the deposit number is GDMCC No: 63265.

2. Use of the gentiobiose-producing strain according to claim 1 in fermentation production of gentiobiose.

3. The use according to claim 2, characterized in that: After the gentiobiose-producing strain is subjected to seed culture, the seed liquid is inoculated into a fermentation medium for fermentation culture to obtain gentiobiose.

4. The use according to claim 3, characterized in that: The medium for seed culture was LB medium.

5. The use according to claim 2, characterized in that: The gentiobiose-producing strain uses glucose as a carbon source to ferment and produce gentiobiose.

6. The use according to claim 5, characterized in that The glucose concentration is 40-100 g / L.

7. The use according to claim 6, characterized in that The glucose concentration is 60-80 g / L.

8. The use according to claim 2, characterized in that: The pH of the fermentation culture is 5-11.

9. The use according to claim 8, characterized in that: The pH of the fermentation culture is 7-11.

10. The use according to claim 2, characterized in that The fermentation temperature is 10-50°C.

11. The use according to claim 10, characterized in that The fermentation temperature is 30-50°C.

12. The use according to claim 2, characterized in that: The fermentation time is 24-48 h.

13. The use according to claim 2, characterized in that: The culture medium for fermentation culture comprises a carbon source, a nitrogen source and an inorganic salt; the nitrogen source is selected from peptone and yeast powder; the inorganic salt is selected from potassium dihydrogen phosphate, calcium chloride and magnesium sulfate.

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