Lactobacillus delbrueckii AWT1-12, method for extracting exopolysaccharides from lactobacillus delbrueckii AWT1-12 and application of exopolysaccharides

Abstract

The invention discloses lactobacillus delbrueckii AWT1-12, the preservation number of the lactobacillus delbrueckii AWT1-12 is CCTCC No: M20242865, the preservation date of the lactobacillus delbrueckii AWT1-12 is December 19, 2024, the Latin name of the lactobacillus delbrueckii AWT1-12 is Lactobacillus delbrueckii sp.bulgaricus, the lactobacillus delbrueckii AWT1-12 is preserved in the China Center for Type Culture Collection, and the preservation unit is Wuhan University, Wuhan University, No. 299, Eight Road, Wuchang District, Wuhan City, Hubei Province. The strain is high in yield of exopolysaccharide, and the polysaccharide is applied to low-fat yoghourt, so that the curdling of the yoghourt can be promoted, the viscosity and the water-holding capacity of the yoghourt are remarkably improved, and the texture of the yoghourt is improved.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to a Lactobacillus delbrueckii AWT1-12, a method for extracting its exopolysaccharide, and the application of the exopolysaccharide. Background Art

[0002] Lactic acid bacteria (LAB) are a group of non-spore-forming, Gram-positive bacteria that produce a large amount of lactic acid by fermenting carbohydrates in the surrounding environment. LAB are widely present in natural environments, the intestines of humans and animals, and certain foods, and exhibit extremely high application value in the fields of industry, agriculture, food processing, and medicine. Lactic acid bacteria exopolysaccharide (LAB EPS) refers to the general term for mucopolysaccharides secreted outside the cell wall during the fermentation of LAB or capsular polysaccharides attached to the microbial cell wall. LAB are recognized as green and safe (GRAS) food-grade microorganisms, and their metabolite EPS can be directly applied to the production of fermented foods, such as a natural alternative to commercial stabilizers, which can effectively improve the physical and chemical properties of the fermentation broth, such as viscosity, stability, emulsifying property, and gel property, and further improve the rheological properties, texture, and flavor of the fermentation broth. In addition, extensive research has shown that some EPS produced by LAB have effects such as enhancing immunity, immunomodulation, anti-cancer, anti-biofilm, antioxidant, and cholesterol-lowering. Therefore, the development and utilization of lactic acid bacteria exopolysaccharide in foods have great prospects, but currently, the application research on the antioxidant aspect and in dairy products of Lactobacillus delbrueckii exopolysaccharide is still relatively lacking. Summary of the Invention

[0003] Technical problem to be solved: In view of the above problems, the purpose of the present invention is to provide a Lactobacillus delbrueckii AWT1-12, a method for extracting its exopolysaccharide, and the application of the exopolysaccharide. A strain of Lactobacillus delbrueckii was isolated from a naturally fermented milk sample collected from Kashgar Prefecture, Xinjiang Uygur Autonomous Region. This strain has a high yield of exopolysaccharide. Applying its exopolysaccharide to low-fat yogurt can promote yogurt coagulation, significantly improve the viscosity and water-holding capacity of yogurt, and improve the texture of yogurt.

[0004] Technical solution: A Lactobacillus delbrueckii AWT1-12, the preservation number of the Lactobacillus delbrueckii AWT1-12 is CCTCC No: M20242865, the preservation date is December 19, 2024, and the Latin name is Lactobacillus delbrueckii subsp. bulgaricus. It has been preserved in the China Center for Type Culture Collection, and the preservation unit is inside Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Further, the Lactobacillus delbrueckii is derived from a naturally fermented cow milk sample from Kashgar Prefecture, Xinjiang Uygur Autonomous Region. An extraction method for extracellular polysaccharide produced by Lactobacillus delbrueckii AWT1-12 described above, comprising the following steps: S1. Fermentation: Inoculate the activated Lactobacillus delbrueckii AWT1-12 into F-MRS medium, culture at 37 °C for 36 h. After obtaining the fermentation broth, perform a boiling water bath for 10 min, cool to room temperature, centrifuge to collect the supernatant, and rotary evaporate and concentrate to obtain a concentrated solution; S2. Protein removal: Add 80% trichloroacetic acid to the concentrated solution to a final concentration of 4%, stand at 4 °C for 12 h, centrifuge, and collect the supernatant; S3. Alcohol precipitation: Add 3 volumes of absolute ethanol to the supernatant, stand at 4 °C for 12 h, centrifuge, and collect the precipitate; S4. Dialysis and freeze-drying: Dissolve the precipitate in distilled water, dialyze, and vacuum freeze-dry to obtain crude extracellular polysaccharide; S5. Crude product separation: Dissolve the crude extracellular polysaccharide in an aqueous solution of pure water to prepare a polysaccharide solution with a concentration of 10 mg / mL, perform DEAE-52 anion exchange chromatography, and perform gradient elution in sequence with a 0-0.5 M gradient concentration of NaCl solution from small to large. Collect the eluate at a NaCl concentration of 0.1 M, and after vacuum freeze-drying, obtain the crude product; S6. Purification: Dissolve the crude product in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL, perform G-100 gel column chromatography, collect the main component of the eluate, and after vacuum freeze-drying, obtain the extracellular polysaccharide. Further, the inoculation amount in step S1 is 3%; the centrifugation conditions are centrifugation at 4 °C and 8000 xg for 10 min; the rotary evaporation conditions are rotary evaporation and concentration at 60 °C and 40 rpm to 1 / 5 of the original volume. Further, the components of the F-MRS medium in step S1 are glucose, beef extract powder, dipotassium hydrogen phosphate, ammonium citrate hydrogen, anhydrous sodium acetate, manganese sulfate, magnesium sulfate, and Tween-80 in a ratio of (41-43 parts):(19-21 parts):(1-3 parts):(1-3 parts):(4-6 parts):(0.04-0.06 parts):(0.1-0.3 parts):(0.9-1.1 parts), and the pH value is 6.62. Further, the centrifugation conditions in step S2 are centrifugation at 4 °C and 8000 xg for 20 min. Further, the centrifugation conditions in step S3 are centrifugation at 4 °C and 8000 xg for 20 min. Further, the cut-off molecular weight for dialysis in step S4 is 8000-14000 Da, dialysis is performed at 4 °C for 72 h, and distilled water is changed every 8 h. Further, the exopolysaccharide is a heteropolysaccharide composed of rhamnose, arabinose, galactose, glucose, mannose and glucuronic acid, and the molar ratio is 0.041:0.037:0.079:0.608:0.181:0.053. Application of the exopolysaccharide extracted by the above-mentioned extraction method in the processing of low-fat yogurt. Beneficial effects: 1. In the present invention, a strain of Lactobacillus delbrueckii subsp. bulgaricus AWT-12 with high exopolysaccharide production is screened from natural fermented milk samples in Kashgar Prefecture, Xinjiang Uygur Autonomous Region. The exopolysaccharide produced by the fermentation of this strain is higher than that of commercial strains commonly used in fermented milk production, and the yield can reach 1.79 g / L. 2. The exopolysaccharide produced by Lactobacillus delbrueckii in the present invention has good antioxidant properties, and with the increase of the exopolysaccharide concentration, the antioxidant ability of the exopolysaccharide also increases. 3. In the present invention, the exopolysaccharide and a commercial starter are combined to prepare low-fat yogurt, which can promote yogurt coagulation, significantly improve the viscosity value, water holding capacity and adhesion characteristics of yogurt. The prepared yogurt has a delicate texture, is viscous, has a strong flavor, and has a stable texture during storage without whey separation. Description of the drawings Figure 1 It is the colony characteristics and microscopic characteristics diagram of Lactobacillus delbrueckii AWT1-12. Among them, a is the colony of Lactobacillus growing on MRS solid medium; b is the microscopic morphology of Lactobacillus at 400 times magnification. Figure 2 It is the growth curve and sugar production process curve diagram of Lactobacillus delbrueckii AWT1-12 in MRS medium; Figure 3 It is the separation and purification diagram of the exopolysaccharide of Lactobacillus delbrueckii AWT1-12. Among them, a is the DEAE-52 elution curve, and b is the G-100 elution curve; the abscissa represents the numbering of the collected exopolysaccharide eluate tubes, and the ordinate represents the absorbance value of each tube of eluate detected by the microplate reader at 490 nm. Figure 4 It is the monosaccharide composition diagram of the exopolysaccharide of Lactobacillus delbrueckii AWT1-12; Figure 5 It is the DPPH and ABTS free radical scavenging ability diagram of the exopolysaccharide of Lactobacillus delbrueckii AWT1-12. Specific embodiments The present invention will be further described below in conjunction with the drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 Isolation, purification and identification of the strain (1) Isolation and purification of the strain Using yogurt from Kashi region as a sample, dilute it 10-fold with sterile normal saline in gradient to 10 -6 , take 10 -4 , 10 -5 , 10 -6 Three gradients of 100 μL dilution solutions are respectively spread on MRS solid plates, cultured at 37 °C for 48 h, pick colonies with different morphologies for streak isolation until pure single colonies with consistent colony morphology are obtained. Pick single colonies and inoculate them into 3 mL of MRS liquid medium, culture for 24 h, take 1 mL of the bacterial solution, add 500 μL of 75% glycerol and store at -80 °C; (2) Identification of strains Perform PCR amplification of 16S rDNA on the isolated strains, send the PCR products to Genewiz for sequencing, and perform nucleic acid sequence alignment of the sequencing results in NCBI to obtain 1 strain of Lactobacillus delbrueckii, named Lactobacillus delbrueckii AWT1-12; (3) Cultivation After inoculating Lactobacillus delbrueckii into MRS solid medium and culturing anaerobically at 37 °C for 48 h, observe that the colony morphology is milky white, the colonies are small, semi-transparent, relatively moist, and the edges are neat, as Figure 1 shown. Example 2 Determination of the growth curve and exopolysaccharide content of the strain Pick a single colony of Lactobacillus delbrueckii with good growth state from the above and inoculate it into MRS liquid medium for activation, then inoculate it into MRS liquid medium at an inoculation amount of 2%, passage 2 times, statically culture for 48 h, take the bacterial suspension every 4 h to measure OD600 and EPS production, and draw the growth curve and sugar production process curve of Lactobacillus delbrueckii. Use the phenol-sulfuric acid method to determine the polysaccharide content: Weigh 1 g of glucose dried to constant weight into a 1 L volumetric flask and dissolve it with water; respectively pipette 0, 0.2, 0.4, 0.6, 0.8, 1 mL of 1 mg / mL glucose standard solution into test tubes, add distilled water to 2 mL, add 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid, shake well, let stand at room temperature for 30 min, then measure OD490 with an enzyme-labeled instrument, using water as a blank control to prepare a standard curve, with the abscissa being the milligram number of polysaccharide and the ordinate being the absorbance value of OD490. Measure the OD490 value of the sample, and obtain the polysaccharide content according to the standard curve. The standard curve equation is: y = 1.3791x + 0.1385, R 2 = 0.9992. As Figure 2 shown, 4 - 24 hours is the logarithmic growth phase of this bacterium, and after 24 hours, it enters the stationary phase. The exopolysaccharide production reaches the highest at 36 hours, which is 753.86 ± 13.91 mg / L. Example 3 Determination of the sugar production conditions of Lactobacillus delbrueckii AWT1-12 On the basis of culturing in MRS medium, the effects of carbon source and its concentration, nitrogen source and its concentration, initial pH value of the medium, fermentation temperature, and inoculum size on the extracellular polysaccharide production of Lactobacillus delbrueckii AWT1-12 were investigated. The medium for producing extracellular polysaccharide by Lactobacillus delbrueckii AWT1-12 was named F-MRS: the ratio of glucose, beef extract powder, dipotassium hydrogen phosphate, ammonium citrate dibasic, anhydrous sodium acetate, manganese sulfate, magnesium sulfate, and Tween-80 was (41 - 43 parts): (19 - 21 parts): (1 - 3 parts): (1 - 3 parts): (4 - 6 parts): (0.04 - 0.06 parts): (0.1 - 0.3 parts): (0.9 - 1.1 parts), and the pH value was 6.62. The optimal fermentation conditions for Lactobacillus delbrueckii AWT1-12 to produce extracellular polysaccharide were: inoculum size 3%, culture temperature 37°C, culture time 36 h. Under these conditions, the yield of extracellular polysaccharide of Lactobacillus delbrueckii AWT1-12 was 1.79 ± 0.01 g / L. Example 4 Extraction and Purification of Extracellular Polysaccharide from Lactobacillus delbrueckii AWT-12 S1. Fermentation: Inoculate the activated Lactobacillus delbrueckii AWT1-12 into the F-MRS medium at an inoculum size of 2%, culture at 37°C for 36 h. After obtaining the fermentation broth, perform a boiling water bath for 10 min, cool to room temperature, centrifuge at 4°C and 8000 xg for 10 min, collect the supernatant, and rotary evaporate and concentrate it to 1 / 5 of the original volume at 60°C and 40 rpm to obtain a concentrated solution. S2. Protein removal: Add 80% trichloroacetic acid to the concentrated solution to a final concentration of 4%, let it stand at 4°C for 12 h, centrifuge at 4°C and 8000 xg for 20 min, and collect the supernatant. S3. Alcohol precipitation: Add 3 volumes of absolute ethanol to the supernatant, let it stand at 4°C for 12 h, centrifuge at 4°C and 8000 xg for 20 min, and collect the precipitate. S4. Dialysis and freeze-drying: Dissolve the precipitate in distilled water, then dialyze it at 4°C for 72 h using a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da, change the distilled water every 8 h. After the dialysis solution is vacuum freeze-dried, the crude extracellular polysaccharide is obtained. S5. Crude product separation: Dissolve the crude extracellular polysaccharide in an aqueous solution of pure water to prepare a polysaccharide solution with a concentration of 10 mg / mL, perform DEAE-52 anion exchange chromatography, and use 0, 0.1, 0.3, and 0.5 M gradient concentrations of NaCl solution for gradient elution in ascending order, collect 8 mL per tube, the elution rate is 1 mL / min, detect the polysaccharide content in each tube by the phenol-sulfuric acid method, collect the eluate under the concentration of 0.1 M NaCl, and obtain the crude product after vacuum freeze-drying, as Figure 3 a). S6. Purification: Dissolve the crude product in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL, perform G-100 gel column chromatography, elute with distilled water, collect in 5 mL / tube, detect the polysaccharide content in each tube by the phenol-sulfuric acid method, collect and combine the single peak components according to the polysaccharide content value, dialyze with deionized water (100 Da) until no Cl- is detected, and freeze-dry to obtain a purified polysaccharide with a uniform molecular weight (such as Figure 3 b). Example 5 Determination of the monosaccharide composition of the exopolysaccharide of Lactobacillus delbrueckii AWT-12 Use an ion chromatograph to determine the monosaccharide composition. Take appropriate amounts of 15 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, D-galactosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid) and add 2 mL of 3M TFA, hydrolyze at 120 °C for 3 h, blow dry with nitrogen, add deionized water and vortex to mix evenly to prepare a standard stock solution; accurately weigh and prepare concentration standards of each monosaccharide stock solution as a mixed standard. According to the absolute quantification method, determine the mass of different monosaccharides, and calculate the molar ratio based on the molar mass of the monosaccharides. Accurately weigh 5 mg of the exopolysaccharide sample and place it in an ampoule, add 2 mL of 3M TFA, and hydrolyze at 120 °C for 3 h; accurately pipette the acid hydrolysis solution and transfer it to a tube, blow dry with nitrogen, add 5 mL of water and vortex to mix evenly; pipette 50 μL and add 950 μL of deionized water, centrifuge at 12000 rpm for 5 min, and take the supernatant for IC analysis. Chromatographic column: Dionex CarbopacTM PA20 (3 * 150 mm); Mobile phase: A - H 2 O; B - 250 mM NaOH; C - 500 mM NaOH & 50 mM NaAc; Flow rate: 0.3 mL / min; Injection volume: 25 μL; Column temperature: 30 °C; Elution gradient: 0 min A phase / B phase / C phase (98:2:0, V / V), 23 min A phase / B phase / C phase (98:2:0, V / V), 23.1 min A phase / B phase / C phase (80:20:0, V / V), 33 min A phase / B phase / C phase (80:20:0, V / V), 33.1 min A phase / B phase / C phase (80:0:20, V / V), 46 min A phase / B phase / C phase (80:0:20, V / V), 46.1 min A phase / B phase / C phase (20:0:80, V / V), 66 min A phase / B phase / C phase (20:0:80, V / V), 66.1 min A phase / B phase / C phase (98:2:0, V / V), 80 min A phase / B phase / C phase (98:2:0, V / V); Detector: Electrochemical detector. Such as Figure 4As shown, the extracellular polysaccharide of Lactobacillus delbrueckii AWT1-12 is a heteropolysaccharide composed of rhamnose, arabinose, galactose, glucose, mannose, and glucuronic acid, and the molar ratio of monosaccharides is 0.041:0.037:0.079:0.608:0.181:0.053. Example 6 Determination of DPPH and ABTS radical scavenging abilities of extracellular polysaccharide of Lactobacillus delbrueckii AWT-12 1. Determination of DPPH radical scavenging ability Prepare extracellular polysaccharide solutions with different concentrations. Take 1 mL of each solution into a test tube, add 0.25 mL of 1 mmol / L DPPH ethanol solution, mix well, and let it stand in the dark for 30 min. Measure the absorbance at 517 nm (A i ); Take 1 mL of the sample solution and mix it with 0.25 mL of absolute ethanol, let it stand in the dark for 30 min, and then measure the absorbance (A j ); Take 1 mL of absolute ethanol and mix it with 0.25 mL of DPPH ethanol solution, let it stand in the dark for 30 min, and then measure the absorbance (A 0 ). 2. Determination of ABTS radical scavenging ability Weigh 6 mg of ABTS, add 1.47 mL of distilled water to prepare a 7.4 mmol / L ABTS stock solution; weigh 2 mg of K 2 S 2 O 8 and add 2.86 mL of distilled water to prepare a 2.6 mmol / L ABTS stock solution; Mix the above two solutions in a ratio of 1:1, place them in the dark at room temperature for 12 h, and then dilute them 40 - 50 times with PBS buffer to make the absorbance within the range of 0.7 ± 0.02 at OD734 nm to obtain an ABTS mixed solution; Take 0.8 mL of the ABTS mixed solution and mix it with 0.2 mL of PBS buffer for 10 s, let it stand for 6 min, and measure the absorbance at 734 nm, denoted as A 0 ; Take 0.8 mL of the ABTS mixed solution and mix it with 0.2 mL of the sample solution for 10 s, let it stand for 6 min, and measure the absorbance at 734 nm, denoted as A. As Figure 5 shown, the DPPH and ABTS radical scavenging abilities of the extracellular polysaccharide of Lactobacillus delbrueckii increase with the increase in the concentration of the extracellular polysaccharide, indicating that the antioxidant ability increases with the increase in concentration. Example 7 Application of extracellular polysaccharide of Lactobacillus delbrueckii AWT-12 in the processing of low-fat yogurt Using the commonly used commercial direct vat set (Angel two-strain starter) as a control (addition amount 0.1% (w / w)), exopolysaccharide of Lactobacillus delbrueckii was used in combination with the commercial starter to produce low-fat yogurt. The dosage of exopolysaccharide was 0.1% (w / w), the dosage of the commercial starter was 0.1% (w / w), the sucrose addition amount was 6% (w / w), the fermentation time was 4 - 5.5 h, and the fermentation temperature was 42°C. The fermented yogurt was placed in a 4°C refrigerator for 24 h of after-ripening, and then the conventional physicochemical indexes and texture indexes of the yogurt were measured. Table 1 Physicochemical indexes and texture characteristics of yogurt As can be seen from Table 2, the water-holding capacity, viscosity value and hardness of the yogurt added with exopolysaccharide are all higher than those of the control yogurt, indicating that adding exopolysaccharide can improve the texture of yogurt. As described above, it is only a preferred embodiment of the present invention, and there is no any formal limitation to the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A Lactobacillus delbrueckii AWT1-12, characterized in that: The preservation number of the Lactobacillus delbrueckii AWT1-12 is CCTCCNo:M20242865, the preservation date is December 19, 2024, the Latin name is Lactobacillusdelbrueckii subsp.bulgaricus, and it has been preserved in the China Center for Type Culture Collection, and the preservation unit is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A method for extracting the extracellular polysaccharide produced by the Lactobacillus delbrueckii AWT1-12 according to claim 1, characterized in that: The following steps are involved: S1. Fermentation: The activated Lactobacillus delbrueckii AWT1-12 was inoculated into F-MRS medium and cultured at 37°C for 36 hours to obtain the fermentation broth, which was then placed in a boiling water bath for 10 minutes, cooled to room temperature, and the supernatant was collected by centrifugation and concentrated by rotary evaporation to obtain a concentrate; S2. Protein removal: add 80% trichloroacetic acid to the concentrate to a final concentration of 4%, incubate at 4°C for 12 h, centrifuge, and collect the supernatant; S3. Alcohol precipitation: add 3 volumes of anhydrous ethanol to the supernatant, let stand at 4°C for 12 h, centrifuge, and collect the precipitate; S4. Dialysis and freeze drying: dissolve the precipitate with distilled water, dialyze, and vacuum freeze dry to obtain crude extracellular polysaccharide; S5. Crude product separation: The crude extracellular polysaccharide was dissolved in a pure water solution to prepare a 10 mg / mL polysaccharide solution, and DEAE-52 anion exchange chromatography was performed, and a 0-0.5 M NaCl solution was used for gradient elution from small to large, and the eluate at a concentration of 0.1 M NaCl was collected and vacuum freeze-dried to obtain a crude product; S6. Purification: The crude product was dissolved in distilled water to prepare a 10 mg / mL polysaccharide solution, subjected to G-100 gel column chromatography, the main component of the eluate was collected, and vacuum freeze-dried to obtain extracellular polysaccharides.

3. The extraction method according to claim 2, characterized in that: The inoculation amount in step S1 is 3%; the centrifugation conditions are 4° C., 8000×g for 10 min; and the rotary evaporation conditions are 60° C., 40 rpm, and rotary evaporation concentration to 1 / 5 of the original volume.

4. The extraction method according to claim 2, characterized in that: The centrifugation conditions in step S2 are 4° C. and 8000×g for 20 min.

5. The extraction method according to claim 2, characterized in that: The centrifugation conditions in step S3 are 4° C. and 8000×g for 20 min.

6. The extraction method according to claim 2, characterized in that: The molecular weight cut-off of the dialysis in step S4 is 8000-14000Da, the dialysis is performed at 4°C for 72h, and the distilled water is replaced every 8h.

7. The extraction method according to claim 2, characterized in that The extracellular polysaccharide is a heteropolysaccharide composed of rhamnose, arabinose, galactose, glucose, mannose and glucuronic acid, with a molar ratio of 0.041:0.037:0.079:0.608:0.181:0.

053.

8. Use of the extracellular polysaccharide extracted by the extraction method according to any one of claims 2 to 7 in the processing of low-fat yogurt.

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