Method for high-throughput screening of exopolysaccharide-producing lactic acid bacteria and identification of monosaccharide composition of exopolysaccharide

Abstract

The invention discloses a method for high-throughput screening of lactic acid bacteria capable of producing exopolysaccharides and identification of monosaccharide composition of the exopolysaccharides of the lactic acid bacteria. The method provided by the invention comprises the following steps: (1) culturing to-be-tested bacteria by adopting a porous deep-hole plate; (2) after the step (1) is completed, filtering the culture supernatant by adopting a porous filter plate, and collecting filtrate to a porous deep-hole plate; (3) sampling the filtrate obtained in the step (2), and screening the bacteria capable of producing the exopolysaccharide from the tested bacteria through an alcohol precipitation test, namely target bacteria; (4) taking the target bacteria, performing the step (2) to obtain a filtrate, filtering the filtrate by adopting Sephadex G-25, and collecting the filtrate; and (5) carrying out polysaccharide hydrolysis and PMP derivatization on the filtrate obtained in the step (4), and carrying out HPLC-ESI-MS / MS analysis to obtain the monosaccharide composition and proportion of the exopolysaccharide. The method has the advantages of high throughput, simplicity and convenience in operation and short time consumption, and has market prospects and popularization values.

Description

Technical Field

[0001] The invention relates to a method for high-throughput screening of lactic acid bacteria producing extracellular polysaccharides and identifying the monosaccharide composition of the extracellular polysaccharides. Background Art

[0002] Exopolysaccharides (EPSs) are macromolecular carbohydrate polymers secreted by lactic acid bacteria during their growth and metabolism. The structure of lactic acid bacteria EPS is complex and can be distinguished according to the composition of monosaccharides, the connection mode and the substituents carried. Different compositions and structures also give them different properties. Lactic acid bacteria exopolysaccharides have a safe use status and unique physical and chemical properties. In terms of food processing technology, lactic acid bacteria EPS can be used to improve the texture and rheological properties of traditional dairy products such as yogurt, cheese, ice cream, and even non-dairy products such as bread and fermented sausages due to their properties as emulsifiers, stabilizers and flocculants, thereby improving the taste and increasing consumer acceptance. In addition, recent studies have shown that lactic acid bacteria EPS has a variety of biological activities, such as antioxidant, anti-aging, immunomodulatory and anti-inflammatory, anti-tumor, cholesterol-lowering, blood pressure-lowering, and improving the structure of intestinal flora. Therefore, in-depth research on lactic acid bacteria EPS is of great significance in the fields of biology, chemistry and medicine.

[0003] People are paying more and more attention to lactic acid bacteria EPS, on the one hand because more and more strains from different habitats are being discovered, and on the other hand because the new biological activities of EPS are constantly being explored, the application potential has increased greatly, and the fields involved are becoming more extensive. The work of studying EPS mainly focuses on several aspects, including selecting suitable EPS-producing strains, analyzing the composition, structure and physicochemical properties of carbohydrates, and their biological activity and application research. The screening of polysaccharide producers and the identification of their composition has always been a tedious and complicated process. Understanding EPS from nature more quickly and fully exploring its application potential has always been the direction of researchers' unremitting efforts.

[0004] Until now, the identification and component analysis of EPS usually consists of several separation steps, including separation, purification, drying, decomposition and lengthy chromatographic analysis. From strain screening to understanding the monosaccharide composition, it takes several days or even weeks, which is a long time cycle and cannot be processed in large quantities. Therefore, it is crucial to establish a high-throughput method to determine EPS producers and identify polysaccharide identities. Summary of the invention

[0005] The purpose of the present invention is to provide a method for high-throughput screening of lactic acid bacteria producing exopolysaccharides and identifying the monosaccharide composition of the exopolysaccharides.

[0006] The present invention provides a method for high-throughput screening of bacteria producing exopolysaccharides and identifying the monosaccharide composition of the exopolysaccharides, comprising the following steps: (1) Use multi-well deep-well plates to culture the test bacteria; (2) After completing step (1), the culture supernatant is filtered using a porous filter plate and the filtrate is collected into a porous deep-well plate; (3) Sampling the filtrate obtained in step (2), and screening the bacteria producing exopolysaccharides from the test bacteria by an alcohol precipitation test, wherein the bacteria producing exopolysaccharides are the target bacteria; (4) taking the target bacteria and subjecting the filtrate obtained in step (2) to filtering using Sephadex G-25, and collecting the filtrate; (5) The filtrate obtained in step (4) is subjected to polysaccharide hydrolysis and PMP derivatization, and then the monosaccharide composition and ratio of the extracellular polysaccharide are obtained by HPLC-ESI-MS / MS analysis.

[0007] Specifically, step (1) is as follows: take a 96-well deep-well plate (referred to as a culture plate), add MRS broth culture medium (1.5 mL / well), then add the test bacterial solution (30 µL / well) to the sample well and add MRS broth culture medium (30 µL / well) to the NC well, and then culture anaerobically for 36 hours.

[0008] Specifically, the anaerobic culture is as follows: placing in a sealed box, adding an anaerobic gas-producing bag into the sealed box, sealing the box and then culturing statically.

[0009] Specifically, the test bacterial liquid is obtained by culturing the test bacteria using MRS broth medium.

[0010] Specifically, the concentration of the test bacteria in the test bacterial solution is 10 8 cfu / ml-10 10 cfu / ml.

[0011] Specifically, the concentration of the test bacteria in the test bacterial solution is 10 9 cfu / ml.

[0012] Specifically, step (2) is as follows: placing a 96-well deep-well plate (called the lower plate) and a 96-well filter plate (called the upper plate) in the inner cavity of the vacuum filtration box, with the 96 wells of the upper plate and the lower plate corresponding to each other; then adding the supernatant after centrifugation of the culture plate in step (1) to each well of the upper plate in a one-to-one manner, filtering, and the filtrate enters each well of the lower plate. Specifically, the centrifugation parameters are: 4°C, 4000×g, centrifugation for 30 minutes.

[0013] The alcohol precipitation test is performed by visual inspection or detection of OD 600nm The value can be used to judge whether the test bacteria are exopolysaccharide-producing bacteria.

[0014] The step (3) is: sampling the filtrate obtained in step (2), adding ethanol, and visually inspecting or detecting OD 600nm The value can be used to judge whether the test bacteria are exopolysaccharide-producing bacteria.

[0015] Specifically, step (3) is as follows: after completing step (2), the filtrate in the lower plate is sampled and added to a 96-well plate (referred to as a detection plate) in a one-to-one manner (100 µL / well), and then anhydrous ethanol (200 µL / well) is added and allowed to stand at 4°C for 24 hours; then the OD is visually observed and / or detected. 600nm The value can be used to judge whether the test bacteria are exopolysaccharide-producing bacteria.

[0016] When judging by visual observation, if criterion 1 and / or criterion 2 are met, the test bacteria are bacteria that produce extracellular polysaccharides.

[0017] Criterion 1: The system in the sample well is more turbid than the system in the NC well.

[0018] Criterion 2: Precipitation is observed in the sample wells.

[0019] The more turbid the system in the sample well is, the higher the production of extracellular polysaccharides is.

[0020] The more precipitates in the sample well, the higher the yield of extracellular polysaccharides.

[0021] Detection of OD 600nm When judging the value, if standard 3 is met, the test bacteria are bacteria that produce extracellular polysaccharides.

[0022] Criterion 3: OD of the system in the sample well 600nm The value is greater than the OD of the NC pore system 600nm value.

[0023] OD of the system in the sample well 600nm The larger the value, the higher the production of extracellular polysaccharides.

[0024] Specifically, the step (4) is as follows: according to the result of step (3), take the filtrate in the lower plate of step (2) corresponding to the target bacteria, add it to a 96-well gel filter plate (100 µL / well) filled with Sephadex G-25 as the filling medium, centrifuge at 4°C and 2000×g for 2 min, and collect the filtrate.

[0025] The method for polysaccharide hydrolysis and PMP derivatization is as follows: take 50 µL of the filtrate obtained in step (4), add 50 µL of 4 mol / L trifluoroacetic acid aqueous solution and seal, digest at 121°C for 90 min, then cool to room temperature, add 3.2% ammonia water to adjust the pH to 8, and obtain the hydrolyzate; take 100 µL of the hydrolyzate, add 100 µL of 14% ammonia water, vortex mix, then centrifuge at 13200 rpm for 4 min, and collect the supernatant; take 50 µL of the supernatant, add 200 µL of PMP derivatization reagent (mix 2 parts by volume of 0.1 mol / L PMP solution and 1 part by volume of 0.4% ammonia water to obtain PMP derivatization reagent; PMP, i.e., 1-phenyl-3-methyl-5-pyrazolone; the solvent of the PMP solution is methanol), vortex mix, then react at 70°C for 60 min, then cool to room temperature, then add 750 µL of distilled water, oscillate mix, then wash with chloroform, and then take the aqueous phase, which is the product solution.

[0026] The HPLC parameters for the HPLC-ESI-MS / MS analysis were as follows: Chromatographic column: MSLab50AA-C18 (150×4.6mm); Column temperature: 50°C; Injection volume: 5µL; The mobile phase is phase A, phase B, or a mixture of phase A and phase B; mobile phase flow rate: 1 mL / min; phase A: water containing 1‰ (volume percentage) formic acid; phase B: acetonitrile containing 1‰ (volume percentage) formic acid; Elution process: from 0 to 1 min, the volume fraction of phase A in the mobile phase was 95%, and the corresponding volume fraction of phase B in the mobile phase was 5%; from 1 to 1.1 min, the volume fraction of phase A in the mobile phase decreased linearly from 95% to 50%, and the corresponding volume fraction of phase B in the mobile phase increased linearly from 5% to 50%; from 1.1 to 12 min, the volume fraction of phase A in the mobile phase decreased linearly from 50% to 30%, and the corresponding volume fraction of phase B in the mobile phase increased linearly from 50% to 70%; from 12 to 12.1 min, the volume fraction of phase A in the mobile phase decreased linearly from 30% to 0%, and the corresponding volume fraction of phase B in the mobile phase increased linearly from 70% to 100%; from 12.1 to 15 min, the volume fraction of phase A in the mobile phase was 0%, and the corresponding volume fraction of phase B in the mobile phase was 100%; from 15 to 15.1 min, The volume fraction of phase A in the mobile phase increased linearly from 0% to 95%, and the corresponding volume fraction of phase B in the mobile phase decreased linearly from 100% to 5%; from 15.1 to 20 minutes, the volume fraction of phase A in the mobile phase was 95%, and the corresponding volume fraction of phase B in the mobile phase was 5%.

[0027] The mass spectrometry parameters for the HPLC-ESI-MS / MS analysis are as follows: Ion source: +ESI electrospray ion source; Scanning mode: MRM multiple reaction monitoring; Curtain gas: 20 psi; Spray voltage: +5500V; Atomizing gas: 55psi; Collision gas: Medium; Collision cell ejection voltage: 2.0; Auxiliary gas: 60 psi; Atomization temperature: 500℃.

[0028] The HPLC-ESI-MS / MS analysis further comprises the following steps: Monosaccharide standards were dissolved in distilled water and then subjected to HPLC-ESI-MS / MS using the same parameters; Based on the peak positions of the product solution and the standard solution, the monosaccharide composition of the extracellular polysaccharides of the test bacteria was determined, the target peak area of ​​the product solution was substituted into the standard curve equation, the monosaccharide content in the product solution was obtained, and the monosaccharide ratio was calculated.

[0029] The standard curve equation is a function with the content of monosaccharide standards and the peak area of ​​the target peak as variables.

[0030] Specifically, the monosaccharide standard is D-glucose, D-galactose, L-rhamnose, L-fucose, L-arabinose, D-mannose or D-xylose.

[0031] Specifically, the multi-porous deep-well plate is a deep-well plate having N1 holes.

[0032] Specifically, N1 is a natural number greater than or equal to 12.

[0033] Specifically, N1 is a natural number greater than or equal to 24.

[0034] Specifically, N1 is a natural number greater than or equal to 36.

[0035] Specifically, N1 is a natural number greater than or equal to 48.

[0036] Specifically, N1 is a natural number greater than or equal to 96.

[0037] Specifically, N1 is a natural number less than 384.

[0038] Specifically, N1 is a natural number less than 256.

[0039] Specifically, N1 is a natural number less than 128.

[0040] Specifically, N1 is 48 or 96 or 128.

[0041] Specifically, the specification of the multi-porous deep-well plate is 1.5-2.1 ml / well.

[0042] Specifically, the multi-well deep-well plate is a 96-well deep-well plate (specification: U-shaped bottom, 1.8 ml / well).

[0043] Specifically, the multi-porous deep well plate is a product of Bio-Tech Biotechnology Co., Ltd., and the product catalog number is FPT026.

[0044] Specifically, the specification of the multi-porous deep-well plate is 1.8 ml / well.

[0045] Specifically, the porous filter plate is a filter plate having N2 holes.

[0046] Specifically, N2 is a natural number greater than or equal to 12.

[0047] Specifically, N2 is a natural number greater than or equal to 24.

[0048] Specifically, N2 is a natural number greater than or equal to 36.

[0049] Specifically, N2 is a natural number greater than or equal to 48.

[0050] Specifically, N2 is a natural number greater than or equal to 96.

[0051] Specifically, N2 is a natural number less than 384.

[0052] Specifically, N2 is a natural number less than 256.

[0053] Specifically, N2 is a natural number less than 128.

[0054] Specifically, N2 is 48 or 96 or 128.

[0055] Specifically, the porous filter plate has a filter membrane with a pore size of 1.0 µm.

[0056] Specifically, the porous filter plate has a double-layer Pall filter membrane with a pore size of 1.0 μm.

[0057] Specifically, the porous filter plate is a 96-well filter plate (each well has a double-layer Pall filter membrane with a pore size of 1.0 μm). The porous filter plate is a product of Haimen Younaite Experimental Equipment Development Co., Ltd., and the product catalog number is AA0900196002.

[0058] Specifically, the filtering using Sephadex G-25 is filtering using a porous gel filter plate whose filling medium is Sephadex G-25.

[0059] The porous gel filter plate is a gel filter plate having N3 holes.

[0060] Specifically, N3 is a natural number greater than or equal to 12.

[0061] Specifically, N3 is a natural number greater than or equal to 24.

[0062] Specifically, N3 is a natural number greater than or equal to 36.

[0063] Specifically, N3 is a natural number greater than or equal to 48.

[0064] Specifically, N3 is a natural number greater than or equal to 96.

[0065] Specifically, N3 is a natural number less than 384.

[0066] Specifically, N3 is a natural number less than 256.

[0067] Specifically, N3 is a natural number less than 128.

[0068] Specifically, N3 is 48, 96 or 128.

[0069] Specifically, the porous gel filter plate is a 96-well gel filter plate (96-well Macro Spin Columns, filled with Sephadex G-25).

[0070] Specifically, the porous gel filter plate is a product of Harvard Bioscience, USA, with a product catalog number of 74-5652.

[0071] Specifically, the test bacteria are test lactic acid bacteria.

[0072] Specifically, the bacteria are lactic acid bacteria.

[0073] Specifically, the test bacteria are lactobacillus and / or bifidobacterium.

[0074] Specifically, the test bacteria are Lactobacillus paracasei, Bifidobacterium lactis or Bifidobacterium longum.

[0075] The method provided by the present invention is simple to operate and has strong applicability. It can realize the simultaneous operation of dozens of bacterial strains or even nearly a hundred bacterial strains, thereby achieving the purpose of rapid and efficient screening. Moreover, the monosaccharide composition information of the extracellular polysaccharide can be obtained directly through the liquid without obtaining a solid extracellular polysaccharide sample, thereby greatly shortening the time cycle and reducing labor costs. The present invention can be used to screen bacteria (especially lactic acid bacteria) that produce extracellular polysaccharides and identify the monosaccharide composition of their extracellular polysaccharides, and has the advantages of high throughput, simple operation and short time consumption, and has market prospects and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 This is an exemplary photograph of visual observation in Example 2.

[0077] Figure 2 Ion chromatogram showing the monosaccharide composition analysis of the exopolysaccharide of Lactobacillus paracasei K56.

[0078] Figure 3 This is the ion chromatogram of the monosaccharide composition analysis of the exopolysaccharide of Lactobacillus paracasei ET-22.

[0079] Figure 4 This is the ion chromatogram of the monosaccharide composition analysis of the extracellular polysaccharide of Lactobacillus paracasei L.CASEI 431.

[0080] Figure 5 The ion chromatogram shows the monosaccharide composition analysis of the exopolysaccharide of Bifidobacterium lactis BL-99.

[0081] Figure 6 The ion chromatogram shows the monosaccharide composition analysis of the exopolysaccharide of Bifidobacterium longum YLGB-1496.

[0082] Figure 7 Ion chromatogram showing the monosaccharide composition analysis of the exopolysaccharide of Bifidobacterium lactis BB-12.

[0083] Figure 8 Ion chromatogram showing the monosaccharide composition analysis of the exopolysaccharide of Bifidobacterium longum Bi-26. DETAILED DESCRIPTION

[0084] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0085] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are set up for three repeated experiments, and the results are averaged. MRS broth medium (pH 6.2 ± 0.2): Beijing Aoboxing Biotechnology Co., Ltd., product catalog number 02-291. The anaerobic gas production bag used in the example is the anaerobic gas production bag AnaeroPack (in a 2.5-liter sealed box, it can absorb all the O2 in the container and produce about 21% CO2 at the same time), Japan Mitsubishi Gas Chemical Co., Ltd., product catalog number C-01.

[0086] 96-well deep-well plate (specification: U-shaped bottom, 1.8 ml / well): Bio-Tech Biotechnology Co., Ltd., product catalog number FPT026. 96-well filter plate (each well has a double-layer Pall filter membrane with a pore size of 1.0 µm): Haimen Younet Laboratory Equipment Development Co., Ltd., product catalog number AA0900196002. 96-well gel filter plate (96-well MacroSpinColumns, filled with Sephadex G-25): Harvard Bioscience, USA, product catalog number 74-5652. Vacuum filtration box: Haimen Younet Laboratory Equipment Development Co., Ltd., product catalog number B03001Q001. Vacuum pump (model: SHB-NF circulating water multi-purpose vacuum pump): Zhengzhou Hengyan Instrument Co., Ltd. Gel spin column (MacroSpinColumns, filled with Sephadex G-25): Harvard Bioscience, USA, product catalog number 74-3905.

[0087] Example 1. Establishment of the method 1. Use 96-well deep-well plates to culture test bacteria 1. Take a 96-well deep-well plate and add 1.5 mL of MRS broth medium to each well.

[0088] 2. Take the 96-well deep-well plate that has completed step 1, add 30 µL of the test bacterial solution to each sample well, and add 30 µL of MRS broth medium to each NC well. Then transfer the plate to a 2.5-liter sealed box, add an anaerobic gas-generating bag, seal it, and incubate it for 36 hours.

[0089] 2. Filter using a 96-well filter plate (the main purpose of this step is to remove bacteria and impurities) 1. Use a silicone hose to connect the vacuum pump and the vacuum filtration box in advance, place a new 96-well deep-well plate (referred to as the lower plate) in the inner cavity of the vacuum filtration box, and cover the upper glass gasket with a silicone ring, then place a 96-well filter plate (referred to as the upper plate) on the glass gasket and clamp it around the silicone ring so that the 96 holes of the upper plate and the lower plate correspond one to one.

[0090] 2. After completing step 2 of step 1, take out the 96-well deep-well plate in the sealed box, centrifuge at 4°C and 4000×g for 30 min, and then add the supernatant to the upper plate in step 1 of step 2 in a one-to-one manner, start the vacuum pump for filtration, and the supernatant is filtered through the upper plate and then enters the lower plate.

[0091] 3. Identify whether the test bacteria are exopolysaccharide-producing bacteria 1. After completing step 2, add the filtrate in the lower plate to the 96-well plate in a one-to-one manner (100 µL per well), then add 200 µL of anhydrous ethanol to each well and let stand at 4°C for 24 h.

[0092] 2. Determine whether the test bacteria are extracellular polysaccharide-producing bacteria through visual observation.

[0093] If criteria 1 and / or 2 are met, the test bacterium is an exopolysaccharide-producing bacterium.

[0094] Criterion 1: After completing step 1, the system in the sample well is more turbid than the system in the NC well.

[0095] Criterion 2: After completing step 1, precipitation is observed in the sample wells.

[0096] 3. By detecting OD 600nm The value can be used to judge whether the test bacteria are exopolysaccharide-producing bacteria.

[0097] If criterion 3 is met, the test bacteria are exopolysaccharide-producing bacteria.

[0098] Criterion 3: After completing step 1, the OD of the system in the sample well 600nm The value is greater than the OD of the NC pore system 600nm value.

[0099] 4. 96-well gel filtration (the main purpose of this step is to remove monosaccharides) According to the identification results of step 3, add the filtrate in the lower plate obtained in step 2 of step 2 corresponding to the bacteria producing extracellular polysaccharides into a 96-well gel filter plate (100 µL / well), centrifuge at 4°C and 2000×g for 2 min, and collect the solution that passes through the gel filter plate, which is called the eluate.

[0100] 5. Analysis of Monosaccharide Composition of Extracellular Polysaccharides 1. Polysaccharide hydrolysis and PMP derivatization Take 50µL of the eluate obtained in step 4, add 50µL of 4mol / L trifluoroacetic acid aqueous solution and seal, digest at 121℃ for 90min, then cool to room temperature, add 3.2% ammonia water to adjust the pH to 8, which is the hydrolyzate. Take 100µL of the hydrolyzate, add 100µL of 14% ammonia water, vortex to mix, then centrifuge at 13200rpm for 4min, and collect the supernatant. Take 50 µL of the supernatant, add 200 µL of PMP derivatization reagent (mix 2 volumes of 0.1 mol / L PMP solution and 1 volume of 0.4% ammonia water to obtain PMP derivatization reagent; PMP, i.e., 1-phenyl-3-methyl-5-pyrazolone; the solvent of the PMP solution is methanol), vortex to mix, then react at 70 °C for 60 min, then cool to room temperature, then add 750 µL of distilled water, oscillate to mix, then wash with chloroform (twice, adding 500 µL of chloroform each time), and then take the aqueous phase, which is the product solution.

[0101] 2. Take the product solution obtained in step 1 and perform HPLC-ESI-MS / MS.

[0102] The HPLC parameters are as follows: Chromatographic column: MSLab50AA-C18 (150×4.6mm); Column temperature: 50°C; Injection volume: 5µL; The mobile phase is phase A, phase B, or a mixture of phase A and phase B; mobile phase flow rate: 1 mL / min; phase A: water containing 1‰ (volume percentage) formic acid; phase B: acetonitrile containing 1‰ (volume percentage) formic acid; Elution process: from 0 to 1 min, the volume fraction of phase A in the mobile phase was 95%, and the corresponding volume fraction of phase B in the mobile phase was 5%; from 1 to 1.1 min, the volume fraction of phase A in the mobile phase decreased linearly from 95% to 50%, and the corresponding volume fraction of phase B in the mobile phase increased linearly from 5% to 50%; from 1.1 to 12 min, the volume fraction of phase A in the mobile phase decreased linearly from 50% to 30%, and the corresponding volume fraction of phase B in the mobile phase increased linearly from 50% to 70%; from 12 to 12.1 min, the volume fraction of phase A in the mobile phase decreased linearly from 30% to 0%, and the corresponding volume fraction of phase B in the mobile phase increased linearly from 70% to 100%; from 12.1 to 15 min, the volume fraction of phase A in the mobile phase was 0%, and the corresponding volume fraction of phase B in the mobile phase was 100%; from 15 to 15.1 min, The volume fraction of phase A in the mobile phase increased linearly from 0% to 95%, and the corresponding volume fraction of phase B in the mobile phase decreased linearly from 100% to 5%; from 15.1 to 20 minutes, the volume fraction of phase A in the mobile phase was 95%, and the corresponding volume fraction of phase B in the mobile phase was 5%.

[0103] The mass spectrometry parameters are as follows: Ion source: +ESI electrospray ion source; Scanning mode: MRM multiple reaction monitoring; Curtain gas: 20 psi; Spray voltage: +5500V; Atomizing gas: 55psi; Collision gas: Medium; Collision cell ejection voltage: 2.0; Auxiliary gas: 60 psi; Atomization temperature: 500℃.

[0104] 3. Dissolve the monosaccharide standards in distilled water and perform HPLC-ESI-MS / MS (parameters are the same as step 2).

[0105] 4. Based on the peak positions of step 2 and step 3, determine the monosaccharide composition of the extracellular polysaccharide of the test bacteria. Then, according to the result of step 3, make a function with the content of the monosaccharide standard and the peak area of ​​the target peak as variables. Then, substitute the peak area of ​​the target peak in step 2 into the function to obtain the monosaccharide content, and further calculate the monosaccharide ratio.

[0106] Embodiment 2, application of method The test bacteria are (a total of seven species): Lactobacillus paracasei K56, Lactobacillus paracasei ET-22, Lactobacillus paracasei L.CASEI 431, Bifidobacterium lactis BL-99, Bifidobacterium longum YLGB-1496, Bifidobacterium lactis BB-12, and Bifidobacterium longum Bi-26. The method for preparing the test bacterial liquid is as follows: the test bacterial strains stored at -80°C are cultured in MRS broth medium to obtain the test bacterial liquid (bacterial concentration is 10 9 cfu / ml).

[0107] 1. Use 96-well deep-well plates to culture test bacteria Proceed according to step 1 of Example 1.

[0108] For each test bacterial culture, 8 replicate wells were set up as repeated treatments.

[0109] It can be observed that the seven tested bacteria all grew well in the 96-deep-well plate.

[0110] 2. Filter using 96-well filter plate Proceed according to step 2 of Example 1.

[0111] 3. Identify whether the test bacteria are exopolysaccharide-producing bacteria Proceed according to step 3 of Example 1.

[0112] See the photo of the visual inspection for example. Figure 1 All seven tested bacteria produce extracellular polysaccharides.

[0113] OD 600nm The test results of the values ​​are shown in Table 1. All the seven tested bacteria are bacteria that produce extracellular polysaccharides.

[0114] Table 1

[0115] IV. 96-well gel filtration The operation is performed according to step 4 of Example 1.

[0116] 5. Analysis of Monosaccharide Composition of Extracellular Polysaccharides The operation was performed according to step 5 of Example 1. The monosaccharide standards were D-glucose, D-galactose, L-rhamnose, L-fucose, L-arabinose, D-mannose, and D-xylose.

[0117] The ion flow diagram of the monosaccharide composition analysis of the extracellular polysaccharide of Lactobacillus paracasei K56 is shown in Figure 2 The monosaccharide composition of the extracellular polysaccharide of Lactobacillus paracasei K56 is mannose, glucose and galactose. The mass ratio is as follows: mannose: glucose: galactose = 1.82: 5.52: 2.97.

[0118] The ion flow diagram of the monosaccharide composition analysis of the extracellular polysaccharide of Lactobacillus paracasei ET-22 is shown in Figure 3 The monosaccharide composition of the extracellular polysaccharide of Lactobacillus paracasei ET-22 is glucose and galactose. The mass ratio is as follows: glucose: galactose = 4.10: 0.64.

[0119] The ion flow diagram of the monosaccharide composition analysis of the extracellular polysaccharide of Lactobacillus paracasei L.CASEI 431 is shown in Figure 4 The monosaccharide composition of the extracellular polysaccharide of Lactobacillus paracasei L.CASEI 431 is glucose and galactose. The mass ratio is as follows: glucose: galactose = 4.42: 0.75;.

[0120] The ion current diagram of the monosaccharide composition analysis of the exopolysaccharide of Bifidobacterium lactis BL-99 is shown in Figure 5 The monosaccharide composition of the extracellular polysaccharide of Bifidobacterium lactis BL-99 is mannose and glucose. The mass ratio is as follows: mannose: glucose = 0.48:5.41.

[0121] The ion current diagram of the monosaccharide composition analysis of the extracellular polysaccharide of Bifidobacterium longum YLGB-1496 is shown in Figure 6 The monosaccharide composition of the exopolysaccharide of Bifidobacterium longum YLGB-1496 is glucose.

[0122] The ion current diagram of the monosaccharide composition analysis of the extracellular polysaccharide of Bifidobacterium lactis BB-12 is shown in Figure 7 The monosaccharide composition of the extracellular polysaccharide of Bifidobacterium lactis BB-12 is glucose and galactose. The mass ratio is as follows: glucose: galactose = 1.08:0.68.

[0123] The ion current diagram of the monosaccharide composition analysis of the extracellular polysaccharide of Bifidobacterium longum Bi-26 is shown in Figure 8 The monosaccharide composition of the extracellular polysaccharide of Bifidobacterium longum Bi-26 is mannose, glucose and galactose. The mass ratio is as follows: mannose: glucose: galactose = 3.07: 4.62: 6.10.

[0124] Example 3: Comparison of Sephadex G-25 gel filtration and dialysis effects Test bacteria: Lactobacillus paracasei ET-22. Method for preparing test bacterial solution: Take the test bacterial strain stored at -80°C, culture it in MRS broth medium, and obtain the test bacterial solution (bacterial concentration is 10 9 cfu / ml).

[0125] 1. Use 96-well deep-well plates to culture test bacteria Proceed according to step 1 of Example 1.

[0126] 2. Filter using 96-well filter plate Proceed according to step 2 of Example 1.

[0127] 3. Comparison of the effects of different methods in removing monosaccharides The test stock solution is: the filtrate in the lower plate obtained in step 2.

[0128] Removal of glucose percentage (%) = [(C 总 -C 余 ) / C 总 ]×100%.

[0129] 1. Take the test solution and dilute it to 10 times the volume with distilled water. Then use the phenol-sulfuric acid method to determine the sugar concentration, and then convert it to the sugar content in 30 ml of the test solution, which is the total sugar content C. 总 .

[0130] 2. Dialysis to remove monosaccharides Transfer 30 ml of the test stock solution to a dialysis bag with a cutoff of 8000-14000 Da, then place the dialysis bag in distilled water for dialysis (48 hours in total, with new distilled water replaced every 8 hours), then take the solution in the dialysis bag and measure the volume, and then use the phenol-sulfuric acid method to determine the sugar concentration. The concentration multiplied by the volume is C 余 .

[0131] 3. Gel filtration to remove monosaccharides Add 100 µL of the test solution to the gel centrifuge column, and insert the centrifuge column into a 2 mL centrifuge tube. Centrifuge at 4 °C and 2000 × g for 2 min. Collect the liquid in the centrifuge tube and determine the sugar concentration using the phenol-sulfuric acid method. Then convert it to the sugar content in 30 mL of the test solution, which is C 余 . The method of step 2 was used to remove monosaccharides, and the percentage of glucose removed was 77.51±0.30%. The method of step 3 was used to remove monosaccharides, and the percentage of glucose removed was 73.33±1.89%. The amount of glucose removed by the two operations was similar. Therefore, a few minutes of Sephadex G-25 gel filler filtration operation can completely replace the 48-hour dialysis, and the filtration effect is basically the same, which greatly shortens the operation time and achieves the purpose of simplifying the glucose removal step. The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for screening bacteria producing exopolysaccharides and identifying the monosaccharide composition of the exopolysaccharides, comprising the following steps: (1) Use multi-well deep-well plates to culture the test bacteria; (2) After completing step (1), the culture supernatant is filtered using a porous filter plate and the filtrate is collected into a porous deep-well plate; (3) Sampling the filtrate obtained in step (2), and screening the bacteria producing exopolysaccharides from the test bacteria by an alcohol precipitation test, wherein the bacteria producing exopolysaccharides are the target bacteria; (4) taking the target bacteria and subjecting the filtrate obtained in step (2) to filtering using Sephadex G-25, and collecting the filtrate; (5) The filtrate obtained in step (4) is subjected to polysaccharide hydrolysis and PMP derivatization, and then the monosaccharide composition and ratio of the extracellular polysaccharide are obtained by HPLC-ESI-MS / MS analysis.

2. The method according to claim 1, characterized in that: The multi-well deep-well plate is a 96-well deep-well plate; The porous filter plate is a 96-well filter plate.

3. The method according to claim 2, characterized in that: The step (1) is as follows: take a 96-well deep-well plate, add 1.5 mL / well of MRS broth medium, then add 30 µL / well of the test bacterial solution to the sample well and 30 µL / well of the MRS broth medium to the NC well, and then culture anaerobically for 36 hours.

4. The method according to claim 2 or 3, characterized in that: The step (2) is as follows: placing a 96-well deep-well plate at the lower layer and a 96-well filter plate at the upper layer in the inner cavity of the vacuum filtration box in a corresponding manner, with the 96 wells of the upper plate and the 96 wells of the lower plate corresponding to each other one by one; Then, the supernatant obtained after centrifugation of the multi-well deep-well plate in step (1) is added to each well of the 96-well filter plate located on the upper layer in a one-to-one manner, and the filtrate is filtered and enters each well of the 96-well deep-well plate located on the lower layer.

5. The method according to any one of claims 1 to 4, characterized in that: The step (3) is: sampling the filtrate obtained in step (2), adding ethanol, and visually inspecting or detecting OD 600nm The value can be used to judge whether the test bacteria are exopolysaccharide-producing bacteria.

6. The method according to any one of claims 1 to 5, characterized in that: The step (3) is as follows: after completing step (2), sample the filtrate in the lower plate and add 100 µL / well of the 96-well plate in a one-to-one manner, then add 200 µL / well of anhydrous ethanol, and let stand at 4°C for 24 hours; then visually observe and / or detect OD 600nm The value can be used to judge whether the test bacteria are exopolysaccharide-producing bacteria.

7. The method according to claim 5 or 6, characterized in that: When judging by visual observation, if criteria 1 and / or 2 are met, the test bacteria are exopolysaccharide-producing bacteria; Detection of OD 600nm When judging the value, if standard 3 is met, the test bacteria are bacteria that produce exopolysaccharides; Criterion 1: The system in the sample well is more turbid than the system in the NC well; Criterion 2: Precipitation is observed in the sample well; Criterion 3: OD of the system in the sample well 600nm The value is greater than the OD of the NC pore system 600nm value.

8. The method according to any one of claims 1 to 7, characterized in that: The step (4) is as follows: according to the result of step (3), the filtrate in the lower plate of step (2) corresponding to the target bacteria is taken, added to a 96-well gel filter plate filled with Sephadex G-25 at 100 µL / well, centrifuged at 4°C and 2000×g for 2 min, and the filtrate is collected.

9. The method according to any one of claims 1 to 8, characterized in that: The bacteria are lactic acid bacteria.

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