Bacillus flosfluvii Zjut HJ2101 and its application in the production of galactooligosaccharides

Abstract

The present invention discloses a Bacillus floricola Zjut HJ2101 and its application in the production of galactooligosaccharides. The wet cells obtained by fermenting and culturing Bacillus floricola Zjut HJ2101 are used as catalysts, lactose is used as a substrate, and a buffer solution with a pH of 6.0 - 8.0 is used as a reaction medium. The conversion reaction is carried out under the conditions of 180 - 200 rpm and 30 - 35 °C to obtain a conversion solution containing galactooligosaccharides, which is then separated and purified to obtain galactooligosaccharides. The high-salt environment for the growth of the strain of the present invention can effectively prevent the contamination of other microorganisms, saving the energy consumed by sterilization, etc. In addition, the β-galactosidase produced by this strain has salt tolerance and can still maintain 50% of the initial enzyme activity within the salinity range of 3 - 15%. It can produce β-galactosidase under high-salt concentration conditions (the yield reaches 4.5 U / g 菌体 ), and can also be used for the synthesis of galactooligosaccharides.

Description

(1) Technical Field

[0001] The present invention relates to the technical field of microbial isolation and application, and particularly to a halophilic microorganism - Bacillus hwajinpoensis and its application in catalyzing lactose to prepare galacto-oligosaccharides. (2) Background Art

[0002] Galacto-oligosaccharides (GOS) is an important prebiotic, which can promote the growth of Bifidobacterium and Lactobacillus, and at the same time can improve mineral absorption and regulate the immune system. As a functional oligosaccharide, GOS can be widely used in various fields, especially infant formula foods and baked foods. Currently, the production of galacto-oligosaccharides mainly uses enzymatic synthesis, that is, using the hydrolytic activity of β-galactosidase (also known as lactase) to decompose the substrate lactose into galactosyl and glucosyl groups, and then using the transglycosylation activity of this enzyme to transfer galactosyl groups to different glycosyl acceptors to form a mixture of galacto-oligosaccharides.

[0003] The sources of β-galactosidase are very extensive, existing in animals, plants and microorganisms. Industrially, the main sources are microorganisms, such as Bacillus circulans, Klebsiella oxytoca, Escherichia coli, Aspergillus oryzae, etc. Most microorganisms cannot survive under extreme conditions (such as high salt concentration, high temperature, strong acid, etc.) and cannot produce β-galactosidase, and most β-galactosidases from microorganisms do not have transglycosylation activity and are difficult to be applied to the synthesis of GOS. The present invention not only provides a strain that produces β-galactosidase under high salt concentration conditions, but also examines the ability of using this strain for whole-cell catalysis of lactose to produce galacto-oligosaccharides, providing a new reference for the enzymatic production of galacto-oligosaccharides. (3) Summary of the Invention

[0004] The object of the present invention is to provide a new halophilic strain - Bacillus hwajinpoensis Zjut HJ2101 that can grow under high salt concentration conditions and produce β-galactosidase, and its application in the production of galacto-oligosaccharides, providing a new reference for the enzymatic production of galacto-oligosaccharides.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a novel halophilic strain, Bacillus hwajinpoensis Zjut HJ2101, which can produce β-galactosidase and grow under high salt concentration conditions. It is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2022196, the preservation date being March 4, 2022, and the address: Wuhan University, Wuhan, China, Zip Code: 430072.

[0007] The present invention also provides an application of the Bacillus hwajinpoensis Zjut HJ2101 in the production of galactooligosaccharides. The application is as follows: using the wet cells obtained by fermenting and culturing Bacillus hwajinpoensis Zjut HJ2101 as a catalyst, lactose as a substrate, and a buffer solution with a pH of 6.0 - 8.0 as a reaction medium, carrying out a conversion reaction at 180 - 200 rpm and 30 - 35 °C (preferably for 24 - 48 h) to obtain a conversion solution containing galactooligosaccharides, and then separating and purifying to obtain galactooligosaccharides.

[0008] Preferably, the addition amount of lactose is 100 - 300 g / L based on the volume of the buffer solution, preferably 300 g / L; the dosage of the wet cells is 100 - 300 g / L based on the volume of the buffer solution, preferably 200 g / L.

[0009] Preferably, the buffer solution is preferably a sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with a pH of 7.0.

[0010] Preferably, the catalyst is prepared as follows:

[0011] (1) Plate culture: Inoculate Bacillus hwajinpoensis Zjut HJ2101 on a plate medium and culture it at 30 °C for 48 - 72 h to obtain plate - cultured strains. The composition of the plate medium is: lactose 15 g / L, anhydrous magnesium sulfate 20 g / L, trisodium citrate dihydrate 3 g / L, tryptone 10 g / L, KCl 2 g / L, NaCl 50 g / L, agar 20 g / L, pH 7.0, and the solvent is deionized water;

[0012] (2) Proliferation culture: Pick one loop of the strains after plate culture and inoculate them into a proliferation medium, and culture them at a constant temperature of 30 °C in an incubator at a rotation speed of 200 rpm for 24 h to obtain a proliferated bacterial liquid. The composition of the proliferation medium is: lactose 15 g / L, anhydrous magnesium sulfate 20 g / L, trisodium citrate dihydrate 3 g / L, tryptone 10 g / L, KCl 2 g / L, NaCl 50 g / L, pH 7.0, and the solvent is deionized water;

[0013] (3) Fermentation culture: Inoculate the proliferated bacterial liquid in step (2) into the enzyme-producing fermentation medium at an inoculation amount of 2% (v / v), culture at 200 rpm and 30 °C for 36 h, centrifuge the culture solution at 8000 rpm and 4 °C for 10 min, and collect the wet bacterial cells; The composition of the enzyme-producing fermentation medium is: lactose 15 g / L, tryptone 5 g / L, yeast extract powder 10 g / L, anhydrous magnesium sulfate 0.3 g / L, KH2PO4 0.05 g / L, NaCl 50 g / L, pH is 7.0, and the solvent is deionized water.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) The present invention provides a new strain for producing β-galactosidase - Bacillus huajintanensis Zjut HJ2101; the high-salt environment for the growth of this strain can effectively prevent the contamination of other microorganisms, save the energy consumed by sterilization, etc. In addition, the β-galactosidase produced by this strain has salt tolerance and can still maintain 50% of the initial enzyme activity within the salt concentration range of 3-15%.

[0016] (2) Bacillus huajintanensis Zjut HJ2101 of the present invention can produce β-galactosidase (the yield reaches 4.5 U / g 菌体 ) under high-salt concentration conditions (3%-15%, preferably 5%), and can also be used for the synthesis of galactooligosaccharides. (IV) Description of the Drawings

[0017] Figure 1 TLC diagram of the transformation product of Bacillus huajintanensis Zjut HJ2101. (In the figure, sample 1 is the galactose standard sample; sample 2 is the glucose standard sample; sample 3 is the lactose standard sample; sample 4 is the mixed standard sample of glucose, galactose and lactose; sample 5 is the commercially available galactooligosaccharide product (G909325, Macklin); sample 6 is the sample of lactose conversion catalyzed by the wet bacterial cells of Bacillus huajintanensis Zjut HJ2101; the concentration of the standard samples in the figure is 10 mg / mL).

[0018] Figure 2 Colony morphology of Bacillus huajintanensis Zjut HJ2101.

[0019] Figure 3 Gram staining of Bacillus huajintanensis Zjut HJ2101.

[0020] Figure 4 Phylogenetic tree of Bacillus huajintanensis Zjut HJ2101.

[0021] Figure 5HPLC analysis of the transformation products of Bacillus flosfluvii strain Zjut HJ2101 and the mixed standard sample; 1 is the liquid chromatogram of the mixed standard sample of glucose, galactose and lactose; 2 is the liquid chromatogram of the transformation products of Bacillus flosfluvii strain Zjut HJ2101. (V) Specific implementation manners

[0022] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0023] The culture media used in the embodiments of the present invention are as follows:

[0024] The enrichment culture medium composition is (g / L): magnesium sulfate anhydrous 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, NaCl 50, pH 7.0, and the solvent is deionized water.

[0025] The screening plate culture medium composition is (g / L): magnesium sulfate anhydrous 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, NaCl 50, X-gal 0.024, agar 20 is added to the solid medium, pH 7.0, and the solvent is deionized water.

[0026] The screening fermentation culture medium composition is (g / L): lactose 10, magnesium sulfate anhydrous 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, NaCl 50, pH 7.0, and the solvent is deionized water.

[0027] The slant culture medium composition is (g / L): lactose 15, magnesium sulfate anhydrous 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, NaCl 50, agar 20 is added to the solid medium, pH 7.0, and the solvent is deionized water.

[0028] The plate culture medium composition is (g / L): lactose 15, magnesium sulfate anhydrous 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, NaCl 50, agar 20 is added to the solid medium, pH 7.0, and the solvent is deionized water.

[0029] The proliferation culture medium composition is (g / L): lactose 15, magnesium sulfate anhydrous 20, trisodium citrate dihydrate 3, tryptone 10, KCl 2, NaCl 50, pH 7.0, and the solvent is deionized water.

[0030] The enzyme-producing fermentation culture medium composition is (g / L): lactose 15, tryptone 5, yeast extract powder 10, magnesium sulfate anhydrous 0.3, KH2PO4 0.05, NaCl 50, pH 7.0, and the solvent is deionized water.

[0031] Example 1. Isolation of Halophilic Strain Zjut HJ2101 Producing β-Galactosidase

[0032] 1. Primary Screening

[0033] Take 1 g of soil around Paryu Co Salt Lake in Tibet and place it in 10 mL of sterilized NaCl aqueous solutions with different concentrations (5%, 10%, 15%, 25%). Shake it in a shaker at 30 °C and 200 rpm for 30 min to mix evenly. After standing for 15 min, pipette 1 mL of the soil suspension into 50 mL of enrichment medium and enrich it at 30 °C and 200 rpm for 3 - 5 days. Pipette 100 μL of the above-enriched culture solution respectively, and dilute it stepwise with sterile saline to 10 -4 ,10 -5 ,10 -6 respectively. Pipette 200 μL of each dilution and spread it evenly on the screening plate medium. Invert it and culture it in an incubator at 30 °C. After the colonies grow, pick the blue single colonies, streak and purify them on the plate medium, and culture them in an incubator at 30 °C for 2 - 3 days to obtain pure cultures. Perform Gram staining and microscopic examination on the isolated strains, and then transfer the pure cultures with different morphologies after microscopic examination to the slant medium for preservation.

[0034] 2. Detection of Wet Bacteria

[0035] Pick one loop of the strain preserved on the slant in step 1 and inoculate it into 50 mL of proliferation medium. Activate it at 30 °C and 200 rpm for 24 h, and then inoculate it into 100 mL of screening fermentation medium at an inoculation amount of 2% (v / v). Culture it at 30 °C and 200 rpm for 24 h. Centrifuge the fermentation broth at 8000 rpm and 4 °C for 10 min to obtain wet bacteria. Take 100 mg of wet bacteria and add 400 μL of 1 mg / mL oNPG solution (colorless, 2-nitrophenyl-β-D-galactopyranoside) prepared with 0.1 M sodium phosphate dibasic-sodium phosphate buffer solution at pH 7.0. React at 37 °C for 10 min, and then add 0.5 mL of 0.15 M Na2CO3 aqueous solution to terminate the reaction. In the control tube, add Na2CO3 aqueous solution first, and then add the same amount of wet bacteria. Centrifuge it at 8000 rpm at room temperature for 10 min. If the supernatant of the sample tube is yellow, it indicates that the original colorless oNPG (2-nitrophenyl-β-D-galactopyranoside) is hydrolyzed into yellow oNP (o-nitrophenol), proving the presence of β-galactosidase in the wet bacteria.

[0036] Example 2. Screening of Strains Producing Transglycosylation-Active β-Galactosidase

[0037] Pick a loop of the slant-preserved strain containing β-galactosidase that has been proven to have β-galactosidase in step 2 of Example 1 and inoculate it into 50 mL of the proliferation medium. After activating at 30 °C and 200 rpm for 24 h, inoculate it into 100 mL of the screening fermentation medium at an inoculation amount of 2% (v / v). Culture under the same conditions for 36 h. Centrifuge the fermentation broth at 8000 rpm and 4 °C for 10 min to collect the cells. Wash and centrifuge 3 times with sodium hydrogen phosphate-sodium dihydrogen phosphate buffer with the same volume as the fermentation broth and a pH of 7.0 to obtain wet bacterial cells. Add lactose to 10 mL of sodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH of 7.0 to make the added concentration 0.3 g / mL, then add 2 g of wet bacterial cells, and carry out the transformation at 200 rpm and 30 °C for 36 h. Centrifuge at 8000 rpm at room temperature for 10 min, and the supernatant is the product of the transglycosylation reaction.

[0038] Perform thin-layer chromatography (TLC) analysis on the reaction products of different strains. The TLC analysis uses activated silica gel aluminum plates (Silica gel 60F254 Merck). Spot with a 10 μL capillary tube, use n-butanol:ethanol:water = 5:3:2 (v / v / v) as the developing agent, chromatograph until 1 cm from the top, take out, dry with a hair dryer, and then spray 10% (v / v) sulfuric acid as the color developing agent, and bake in an oven at 120 °C for 10 min, as Figure 1 . If new oligosaccharide spots appear at the lower part of the lactose spot on the TLC plate of the reaction product of the strain, it indicates that the β-galactosidase produced by the strain has transglycosylation activity; otherwise, there is no transglycosylation activity. According to the size of the corresponding oligosaccharide product spots on the TLC plate, select the β-galactosidase-producing strain with high transglycosylation activity and label it as strain Zjut HJ2101 (as shown in Figure 1 Sample 6).

[0039] Example 3. Identification of Strain Zjut HJ2101

[0040] 1) Morphological observation

[0041] Inoculate strain Zjut HJ2101 on the plate medium. After culturing at 30 °C for 48 - 72 h, observe the colony and cell morphology. The colony is round, with a smooth surface, neat edges, and a milky white color. The results are shown in Figure 2 . The result of Gram staining is purple, and the cell morphology is short rod-shaped. The results are shown in Figure 3 , and it is a positive bacillus.

[0042] 2) Physiological and biochemical tests

[0043] The VITEK physiological and biochemical identification of strain Zjut HJ2101 was carried out (see Table 1), and its physiological and biochemical characteristics were consistent with those of Bacillus hwajinpoensis.

[0044] Table 1 Physiological and Biochemical Identification Table

[0045]

[0046]

[0047] (Note: “(-), (+)” in the table indicate weak negative and weak positive.)

[0048] 3) 16S rRNA sequence analysis

[0049] The 16S rRNA gene sequence of strain Zjut HJ2101 was completed by Sangon Biotech (Shanghai) Co., Ltd. The length of the 16S rRNA sequence of strain Zjut HJ2101 was measured to be 1444 bp, and the specific nucleotide sequence is shown as SQE ID NO.1. The obtained 16S rRNA sequence of the strain was aligned with the Genebank sequence, and a phylogenetic tree was established using MEGA-X. The results are shown in Figure 4 , and strain Zjut HJ2101 was identified as Bacillus hwajinpoensis ZjutHJ2101, which was deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2022196, the deposit date being March 4, 2022, and the address: Wuhan University, Wuhan, China, Zip Code 430072.

[0050] SQE ID NO.1

[0051]

[0052] Example 4: Preparation of wet cells of Bacillus huajintanensis Zjut HJ2101

[0053] (1) Plate culture

[0054] Inoculate Bacillus huajintanensis Zjut2101 on a plate medium and culture it at 30 °C for 48 - 72 h to obtain a plate-cultured strain.

[0055] (2) Proliferation culture

[0056] Pick a loop of the strain after plate culture and inoculate it into a proliferation medium. Incubate it at a constant temperature in an incubator at 30 °C with a rotation speed of 200 rpm for 24 h to obtain a proliferated bacterial liquid.

[0057] (3) Fermentation culture

[0058] Inoculate the above-mentioned proliferated bacterial liquid into 100 mL of an enzyme-producing fermentation medium at an inoculation amount of 2% (v / v). Incubate it at 200 rpm and 30 °C for 36 h. Centrifuge the culture solution at 8000 rpm and 4 °C for 10 min to collect wet cells.

[0059] Example 5: Production of galactooligosaccharides by whole-cell catalysis of Bacillus huajintanensis Zjut HJ2101

[0060] (1) Add lactose to 10 mL of a disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with a pH of 7.0 to a concentration of 0.3 g / mL. Then add 2 g of the wet cells after fermentation in Example 4. Incubate at 200 rpm and 30 °C for 36 h to obtain a conversion solution containing galactooligosaccharides.

[0061] (2) Take 1 mL of the conversion solution and centrifuge it at 8000 rpm and 4 °C for 10 min. Dilute the supernatant with the mobile phase to 50 times. After filtering through a 0.22 μm microporous filter, detect the concentration of galactooligosaccharides in the filtrate by HPLC to determine that the yield of galactooligosaccharides in the transglycosylation reaction can reach 15% (as Figure 5 shown). Centrifuge the remaining conversion solution containing galactooligosaccharides at 8000 rpm and 4 °C for 10 min. Add absolute ethanol to the supernatant to an ethanol concentration of 70%. Mix well and centrifuge at 8000 rpm for 10 min. Discard the supernatant to obtain galactooligosaccharides.

[0062] The yield of galactooligosaccharides is calculated by the following formula:

[0063] [GOS] = [Lactose] i - [Lactose] r - [Glicose] - [Galactose]

[0064]

[0065] wherein: [GOS] is the concentration of galactooligosaccharides in the reaction product; [Lactose] i is the initial lactose concentration in the reaction product; [Lactose] r is the residual lactose concentration of the reaction solution product; [Glucose] is the concentration of glucose in the reaction product; [Galactose] is the concentration of galactose in the reaction product; Y GOS is the yield of galactooligosaccharides.

[0066] Chromatographic determination conditions: Agilent 1290 high performance liquid chromatography - evaporative light scattering detector (ELSD), chromatographic column: NH2P - 50 4E 4.6 mm i.D.×250 mm amino column; mobile phase: acetonitrile: water = 70:30 (v / v), flow rate: 1.0 mL / min; temperature: 30 °C; injection volume: 10 μL.

[0067] The technical solution of the present invention is not limited to the limitations of the above - specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. Bacillus huajintanensis ( Bacillus hwajinpoensis ) Zjut HJ2101, which is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M 2022196, the deposit date is March 4, 2022, and the deposit address is: Wuhan University, Wuhan, China.

2. The application of the Bacillus huajintanensis Zjut HJ2101 according to claim 1 in the production of galactooligosaccharides, characterized in that The application is as follows: using the wet cells obtained by fermenting and culturing Bacillus hwajinpoensis Zjut HJ2101 as a catalyst, lactose as a substrate, and a buffer solution with a pH of 6.0 - 8.0 as a reaction medium, carrying out a conversion reaction at 180 - 200 rpm and 30 - 35 °C to obtain a conversion solution containing galactooligosaccharides, and separating and purifying to obtain galactooligosaccharides.

3. The application according to claim 2, characterized in that The addition amount of the lactose is 100 - 300 g / L based on the volume of the buffer solution; the dosage of the wet cells is 100 - 300 g / L based on the volume of the buffer solution.

4. The application according to claim 2, characterized in that The buffer solution is a sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with a pH of 7.

0.

5. The application according to claim 2, characterized in that The catalyst is prepared by the following method: (1) Plate culture: Inoculate Bacillus hwajinpoensis Zjut HJ2101 on a plate medium and culture at 30 °C for 48 - 72 h to obtain plate - cultured strains; the composition of the plate medium is: lactose 15 g / L, anhydrous magnesium sulfate 20 g / L, trisodium citrate dihydrate 3 g / L, tryptone 10 g / L, KCl 2 g / L, NaCl 50 g / L, agar 20 g / L, pH 7.0, and the solvent is deionized water; (2) Proliferation culture: Pick one loop of the strains after plate culture and inoculate them into a proliferation medium, and culture at a constant temperature of 30 °C in an incubator at a rotation speed of 200 rpm for 24 h to obtain a proliferated bacterial solution; the composition of the proliferation medium is: lactose 15 g / L, anhydrous magnesium sulfate 20 g / L, trisodium citrate dihydrate 3 g / L, tryptone 10 g / L, KCl 2 g / L, NaCl 50 g / L, pH 7.0, and the solvent is deionized water; (3) Fermentation culture: Inoculate the proliferated bacterial solution in step (2) into an enzyme - producing fermentation medium at an inoculation amount of 2% by volume concentration, culture at 200 rpm and 30 °C for 36 h, centrifuge the culture solution at 8000 rpm and 4 °C for 10 min, and collect the wet cells; the composition of the enzyme - producing fermentation medium is: lactose 15 g / L, tryptone 5 g / L, yeast extract powder 10 g / L, anhydrous magnesium sulfate 0.3 g / L, KH2PO4 0.05 g / L, NaCl 50 g / L, pH 7.0, and the solvent is deionized water.

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