CN105483177B

CN105483177B - Preparation method of water-insoluble exopolysaccharide of leuconostoc mesenteroides

Info

Publication number: CN105483177B
Application number: CN201510971582.2A
Authority: CN
Inventors: 吴正钧; 韩瑨; 徐晓芬; 高彩霞; 吴江
Original assignee: Bright Dairy and Food Co Ltd
Current assignee: Bright Dairy and Food Co Ltd
Priority date: 2015-12-21
Filing date: 2015-12-21
Publication date: 2020-11-27
Anticipated expiration: 2035-12-21
Also published as: CN105483177A

Abstract

The invention discloses a preparation method of water-insoluble exopolysaccharide of leuconostoc mesenteroides. The preparation method comprises the following steps: 1) inoculating Leuconostoc mesenteroides BD3749 in tomato juice sucrose culture medium, and fermenting to obtain fermentation liquid; 2) freeze drying the fermentation liquor obtained in the step 1), extracting the powder with alkali liquor, taking the supernatant, adjusting the pH value of the supernatant to 7, taking the precipitate, and freeze drying to obtain the product. The tomato juice and sucrose culture medium used in the preparation method is mainly prepared from tomatoes, has wide sources, low cost, is natural and safe, improves the food safety while reducing the material cost, and the fermentation strain is a single Leuconostoc mesenteroides strain, so that the combination is beneficial to the standardization of product quality and the cost control of industrial large-scale production, and has very wide application prospect.

Description

Preparation method of water-insoluble exopolysaccharide of leuconostoc mesenteroides

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of water-insoluble exopolysaccharide of leuconostoc mesenteroides.

Background

water-Insoluble Glucan (IG) is a kind of carbohydrate polymer that is Insoluble in water and polymerized with glucose as a single component, and such carbohydrate with special physicochemical properties is not only widely available, but also widely available in metabolic products of plants, fungi and bacteria. Most IG reported at present comprises three types, namely dextran, curdlan and cellulose, and from the structural characteristics, the IG branches are reported to be connected with a main chain by only one of alpha- (1,2), alpha- (1,3) or alpha- (1,4) glycosidic bonds, and the branched structure of 2 or more glycosidic bonds is rarely involved. Therefore, the screening and discovery of new IG configuration not only adds new members to the IG family, but also greatly promotes the frontier field of IG structure-related research.

In addition, the IG is obtained from a synthetic culture medium at present, the culture medium has various components and complex formula, the preparation process is complicated, potential food safety risks are caused, the preparation cost is relatively high, the IG source is relatively limited, and the development and utilization of the IG are limited to a great extent.

Disclosure of Invention

The invention provides a preparation method of water-insoluble exopolysaccharide of leuconostoc mesenteroides, aiming at solving the problems that the preparation of the existing water-insoluble glucan uses various components, the synthetic culture medium with a complex formula has higher cost, the preparation of the culture medium is complicated, the food safety risk exists and the like. The tomato juice sucrose culture medium used in the preparation method is safe and purely natural, is simple to prepare, has wide raw material sources and is low in cost.

The inventor finds that the water-insoluble extracellular polysaccharide with a special structure can be obtained by freeze-drying, alkali liquor leaching and centrifuging fermentation liquor of Leuconostoc mesenteroides BD3749 with the preservation number of CGMCC No.100064 after shaking culture in a tomato juice sucrose culture medium, then adjusting the pH to 7 by acid liquor, centrifuging to obtain precipitate, and freeze-drying, thereby completing the invention.

In order to solve the technical problems, the invention adopts the technical scheme that: a method for preparing water-insoluble exopolysaccharide of leuconostoc mesenteroides, which comprises the following steps:

1) inoculating Leuconostoc mesenteroides BD3749 in tomato juice sucrose culture medium, and fermenting to obtain fermentation liquid;

2) freeze drying the fermentation liquor obtained in the step 1), extracting the powder with alkali liquor, taking the supernatant, adjusting the pH value of the supernatant to 7, taking the precipitate, and freeze drying to obtain the product.

The preparation method comprises the following steps 1): inoculating Leuconostoc mesenteroides (Leuconostoc mesenteroides) BD3749 in tomato juice sucrose culture medium, and fermenting and culturing to obtain fermentation liquid.

Step 1) of the invention, leuconostoc mesenteroides BD3749 has been preserved in China general microbiological culture Collection center (CGMCC) in 11 months and 26 months in 2014, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101. the proposed classification of this strain was named: leuconostoc mesenteroides, the preservation number of the strain is as follows: CGMCC No.10064, the name of the preserved culture is: BD 3749.

The preparation method of the leuconostoc mesenteroides BD3749 is conventional, and preferably further comprises the step of activating the leuconostoc mesenteroides BD3749 to obtain leuconostoc mesenteroides seeds. The leuconostoc mesenteroides seed is obtained by a conventional method, and preferably the method comprises the following steps: dissolving lyophilized powder of Leuconostoc mesenteroides (Leuconostoc mesenteroides) (preservation number of CGMCC No.10064) in a small amount of sterile distilled water, taking a loop by using an inoculating loop, streaking the loop on M17 solid culture medium (purchased from OXOID Co., UK) containing 5% of sucrose, aerobically culturing at 30 ℃ for 48h, taking a single colony by using the inoculating loop, putting the single colony into 10mL M17 liquid culture medium (purchased from OXOID Co., UK) containing 5% of sucrose, uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, performing shaking culture at 30 ℃ and 180rpm for 24h, inoculating the single colony on M17 liquid culture medium (purchased from OXOID Co., UK) containing 5% of sucrose in an inoculation amount of 2% (v/v), performing shaking culture at 30 ℃ and 180rpm for 24h, centrifuging the culture at 15,000rpm for 10min, discarding supernatant, washing the supernatant with sterile distilled water, and culturing the sterile suspension in a distilled water volume, obtaining seeds for fermentation. The freeze-dried powder of leuconostoc mesenteroides BD3749 is prepared by the following method: 2 colonies were picked and inoculated into 300mL of 10% (w/w) skim milk solution at 30 ℃ for 4h, pre-frozen at-80 ℃ overnight, and freeze-dried at-20 ℃.

The preparation method of the tomato juice and sucrose culture medium in the step 1) of the invention is conventional in the field, and is preferably prepared by a method comprising the following steps: squeezing fructus Lycopersici Esculenti, filtering, boiling, centrifuging to obtain supernatant, adding sucrose, heating to dissolve, cooling, adjusting pH, sterilizing, and cooling. Wherein the filtering method is conventional, and preferably, filtering and juice extracting are carried out by using gauze of 80-120 meshes; the boiling time is conventional, preferably 1 to 10 minutes (min), more preferably 3 to 8 minutes, most preferably 5 minutes; the centrifugation is conventional, the speed of centrifugation is preferably 4,000-12,000 g, more preferably 6,000-10,000g, preferably 8,000g, the centrifugation time is preferably 8 to 12 minutes, more preferably 9 to 11 minutes, most preferably 10 minutes; the addition amount of the sucrose is preferably 5 to 30%, more preferably 10 to 20%, and most preferably 10%; the sterilization is conventional, the temperature of the sterilization is preferably 110 to 135 ℃, more preferably 115 to 125 ℃, most preferably 121 ℃, and the time of the sterilization is preferably 10 to 30 minutes, more preferably 15 to 25 minutes, most preferably 20 minutes; the pH is adjusted by methods conventional in the art, preferably by adding a food grade base, which is conventional in the art, preferably: na (Na)₂CO₃、NaHCO₃And NaOH; the pH is conventional, preferably 5 to 8, more preferably 6 to 7, and most preferably 7.

The fermentation culture in step 1) of the invention is conventional, the inoculum size of the fermentation strain is preferably 1-5%, more preferably 2-4%, and most preferably 3%, and the percentage is volume percentage. The fermentation culture is preferably shaking fermentation culture, and the shaking speed is preferably 100-300 rpm, more preferably 150-250 rpm, and most preferably 200 rpm; the temperature of the fermentation culture is preferably 15 to 37 ℃, more preferably 25 to 33 ℃, and most preferably 30 ℃; the fermentation time is preferably 24 to 96 hours (h), more preferably 48 to 84 hours, and most preferably 72 hours.

The preparation method comprises the following steps 2): freeze drying the fermentation liquor obtained in the step 1), extracting the powder with alkali liquor, taking the supernatant, adjusting the pH value of the supernatant to 7, taking the precipitate, and freeze drying to obtain the product.

The freeze drying in step 2) of the present invention is a conventional freeze drying technique in the art, and only needs to achieve the purpose of removing moisture and obtaining solid. The alkali liquor is conventional, preferably NaOH solution, and the concentration of the NaOH solution is 1-5M, preferably 2-4M, and more preferably 3M. The leaching is conventional, and the ratio of freeze-dried powder to alkali liquor in the leaching is 1 g: 1 mL-1 g: 5mL, preferably 1 g: 2 mL-1 g: 4mL, more preferably 1 g: 3 mL. The supernatant is obtained by a conventional method, preferably centrifugation, the centrifugation is conventional, the centrifugation speed is preferably 8,000-15,000 g, more preferably 10,000-12,000 g, and the centrifugation time is preferably 5-15 minutes, more preferably 8-12 minutes. The pH is adjusted using conventional methods, preferably with an acid solution, which is conventional, preferably an HCl solution, having a concentration of 1-3M, preferably 1.5-2.5M, more preferably 2M. The precipitation is obtained by conventional means, preferably centrifugation, under centrifugation conditions as preferred for obtaining the supernatant by centrifugation.

The water-insoluble exopolysaccharide of Leuconostoc mesenteroides obtained by the above preparation method is a water-insoluble glucan having both alpha- (1,3) and alpha- (1,4) glycosidic bonds, and is in the form of white powder.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

1. the invention solves the current situation that the source of the water-insoluble polysaccharide (especially the water-insoluble glucan) is narrow at present, and provides another choice for the source of the water-insoluble polysaccharide;

2. the water-insoluble polysaccharide prepared by the method has an unusual coexistence form of two branched chain connecting bonds, is a polysaccharide with a novel structure, and has certain development significance according to the theory that the more complex the structure, the more functions of the polysaccharide are relatively more.

3. The preparation steps of the conventional polysaccharide usually involve a deproteinization step (such as the deproteinization by a SAVAGE method), but in the preparation method, the fermentation liquor is directly frozen and dried without adjusting the pH, when the acidic fermentation liquor is gradually frozen and dehydrated, the pH is continuously reduced, so that local peracid is caused, free protein in the fermentation liquor is rapidly denatured, and the part of protein is centrifugally removed during alkali liquor extraction, so that the purpose of deproteinization is achieved.

4. Leuconostoc mesenteroides belongs to the category of lactic acid bacteria, so compared with insoluble polysaccharide produced by other source strains, the polysaccharide of leuconostoc mesenteroides has higher food safety. The exopolysaccharide prepared by the method can be used as a functional additive to replace water-insoluble polysaccharides from other sources to be applied to food, pharmacy and related fields, and has a very wide application prospect.

5. At present, the preparation of IG depends on a synthetic culture medium, but the research of preparing IG by using a tomato juice sucrose culture medium is not reported, and the invention firstly uses the tomato juice sucrose culture medium as a natural fermentation culture medium to prepare IG, so that the product has higher food safety.

6. The main raw material for preparing IG is tomato, the source is wide, the cost is low, the fermentation strain is a single Leuconostoc mesenteroides strain, and the combination is favorable for standardization of product quality and cost control of industrial large-scale production.

Biological material preservation information

The leuconostoc mesenteroides BD3749 of the invention is preserved in China general microbiological culture Collection center (CGMCC) in 11 months and 26 days in 2014, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101, accession number: CGMCC No.10064, the name of the culture is BD3749, and the classification name is Leuconostoc mesenteroides.

Drawings

FIG. 1 shows the monosaccharide composition measurement results of Leuconostoc mesenteroides BD3749 water-insoluble exopolysaccharide.

FIG. 2 shows the results of IR spectroscopy of water-insoluble exopolysaccharides of Leuconostoc mesenteroides BD 3749.

FIG. 3 shows the results of NMR measurement of water-insoluble exopolysaccharides of Leuconostoc mesenteroides BD 3749.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The "room temperature" used herein means a temperature between operations for carrying out the test, and is generally 15 to 25 ℃, and reagents used in examples are analytically pure reagents and purchased from the national pharmaceutical group unless otherwise specified.

Example 1 preparation and production of Leuconostoc mesenteroides Water-insoluble exopolysaccharide

1. Materials and methods

Preparation of seeds (fermentation strain): dissolving lyophilized powder of Leuconostoc mesenteroides (Leuconostoc mesenteroides) (preservation number of CGMCC No.10064) in a small amount of sterile distilled water, taking a loop by using an inoculating loop, streaking the loop on M17 solid culture medium (purchased from OXOID Co., UK) containing 5% of sucrose, aerobically culturing at 30 ℃ for 48h, taking a single colony by using the inoculating loop, putting the single colony into 10mL M17 liquid culture medium (purchased from OXOID Co., UK) containing 5% of sucrose, uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, performing shaking culture at 30 ℃ and 180rpm for 24h, inoculating the single colony on M17 liquid culture medium (purchased from OXOID Co., UK) containing 5% of sucrose in an inoculation amount of 2% (v/v), performing shaking culture at 30 ℃ and 180rpm for 24h, centrifuging the culture at 15,000rpm for 10min, discarding supernatant, washing the supernatant with sterile distilled water, and culturing the sterile suspension in a distilled water volume, obtaining seeds for fermentation, namely leuconostoc mesenteroides seed liquid. The freeze-dried powder of leuconostoc mesenteroides BD3749 is prepared by the following method: 2 colonies were picked and inoculated into 300mL of 10% (w/w) skim milk solution at 30 ℃ for 4h, pre-frozen at-80 ℃ overnight, and freeze-dried at-20 ℃.

Preparing a tomato juice sucrose culture medium: cleaning ripe fructus Lycopersici Esculenti, peeling, squeezing with juicer, filtering with 100 mesh gauze to obtain juice, boiling for 5min, centrifuging at 8000g for 10min to obtain supernatant, adding sucrose 10 wt%, heating to dissolve, cooling to room temperature, adding Na₂CO₃Adjusting pH to 7, sterilizing at 121 deg.C for 20min, and cooling to room temperature to obtain sterile tomato juice sucrose culture medium.

2. Preparation of leuconostoc mesenteroides water-insoluble exopolysaccharide

Inoculating the obtained leuconostoc mesenteroides seed solution into a tomato juice sucrose culture medium with 10 mass percent of sucrose and pH of 7 according to the inoculation amount of 3% (v/v), performing shaking fermentation culture at 30 ℃ and 200rpm for 72 hours to obtain a fermentation solution, performing freeze drying (namely freeze drying) on the fermentation solution, and mixing the freeze-dried powder with a 3M NaOH solution according to the ratio of 1 g: mixing the materials in a ratio of 3mL, leaching at room temperature for 2h, centrifuging at 15,000rpm for 5min, taking supernatant, adjusting the pH to 7 with 2M HCl solution, centrifuging at 15,000rpm for 5min, taking precipitate, and freeze-drying to obtain the water-insoluble extracellular polysaccharide A.

3. Yield of Leuconostoc mesenteroides water-insoluble exopolysaccharide

The yield of BD3749 water-insoluble exopolysaccharide A is 9.2g/L by weighing and calculation.

Example 2 preparation and production of Leuconostoc mesenteroides Water-insoluble exopolysaccharide

1. Materials and methods

Preparation of seeds (fermentation strain): the same as in example 1.

Preparing a tomato juice sucrose culture medium: cleaning ripe fructus Lycopersici Esculenti, peeling, squeezing with juicer, filtering with 120 mesh gauze to obtain juice, boiling for 10min, centrifuging at 4000g for 12min to obtain supernatant, adding sucrose 30 wt%, heating to dissolve, cooling to room temperature, and adding NaHCO₃Adjusting pH to 8, sterilizing at 135 deg.C for 10min, and cooling to room temperature to obtain sterile tomato juice sucrose culture medium.

2. Preparation of leuconostoc mesenteroides water-insoluble exopolysaccharide

Inoculating the obtained leuconostoc mesenteroides seed liquid into a tomato juice and sucrose culture medium with the mass percentage of sucrose of 30 percent and the pH of 8 according to the inoculation amount of 5 percent (v/v), performing shaking fermentation culture at 15 ℃ and 300rpm for 24 hours to obtain a fermentation liquid, performing freeze-drying on the fermentation liquid, and mixing freeze-dried powder with a 5M NaOH solution according to the ratio of 1 g: mixing the materials in a ratio of 1mL, leaching at room temperature for 2h, centrifuging at 8,000rpm for 15min, taking supernatant, adjusting the pH to 7 with 3M HCl solution, centrifuging at 8,000rpm for 15min, taking precipitate, and freeze-drying to obtain the water-insoluble extracellular polysaccharide B.

3. Yield of Leuconostoc mesenteroides water-insoluble exopolysaccharide

The yield of BD3749 water-insoluble exopolysaccharide B is 5.49g/L by weighing and calculation.

Example 3 preparation and yield of Leuconostoc mesenteroides Water insoluble exopolysaccharide

1. Materials and methods

Preparation of seeds (fermentation strain): the same as in example 1.

Preparing a tomato juice sucrose culture medium: cleaning ripe tomatoes, peeling, squeezing by a juicer, filtering by 80-mesh gauze to obtain juice, boiling for 1min, centrifuging for 8min at 12000g, taking supernatant, adding sucrose with the mass percentage content of 5%, heating to dissolve, cooling to room temperature, adjusting the pH to 5 by NaOH, sterilizing at 110 ℃ for 30min, and cooling to room temperature to obtain the sterile tomato juice sucrose culture medium.

2. Preparation of leuconostoc mesenteroides water-insoluble exopolysaccharide

Inoculating the obtained leuconostoc mesenteroides seed liquid into a tomato juice sucrose culture medium with 5 mass percent of sucrose and 5 pH according to the inoculation amount of 1% (v/v), performing shaking fermentation culture at 37 ℃ and 100rpm for 96 hours to obtain a fermentation liquid, performing freeze-drying on the fermentation liquid, and mixing freeze-dried powder with a 1M NaOH solution according to the ratio of 1 g: mixing the materials in a ratio of 5mL, leaching at room temperature for 2h, centrifuging at 10,000rpm for 12min, taking supernatant, adjusting the pH to 7 with 1M HCl solution, centrifuging at 10,000rpm for 12min, taking precipitate, and freeze-drying to obtain the water-insoluble extracellular polysaccharide C.

3. Yield of Leuconostoc mesenteroides water-insoluble exopolysaccharide

The yield of BD3749 water-insoluble exopolysaccharide C is 3.79g/L by weighing and calculation.

Example 4 preparation and yield of Leuconostoc mesenteroides Water-insoluble exopolysaccharide

1. Materials and methods

Preparation of seeds (fermentation strain): the same as in example 1.

Preparing a tomato juice sucrose culture medium: cleaning ripe fructus Lycopersici Esculenti, peeling, squeezing with juicer, filtering with 100 mesh gauze to obtain juice, boiling for 8min, centrifuging for 9min at 10000g, collecting supernatant, adding sucrose with mass percent of 20%, heating to dissolve, cooling to room temperature, adding Na₂CO₃And NaHCO₃Adjusting pH to 6, sterilizing at 115 deg.C for 25min, and cooling to room temperature to obtain sterile tomato juice sucrose culture medium.

2. Preparation of leuconostoc mesenteroides water-insoluble exopolysaccharide

Inoculating the obtained leuconostoc mesenteroides seed liquid into a tomato juice sucrose culture medium with 20 mass percent of sucrose and 6 pH according to the inoculation amount of 2% (v/v), performing shaking fermentation culture at 25 ℃ and 250rpm for 48 hours to obtain a fermentation liquid, performing freeze-drying on the fermentation liquid, and mixing freeze-dried powder with 2M NaOH solution according to the ratio of 1 g: mixing the materials in a ratio of 4mL, leaching at room temperature for 2h, centrifuging at 12,000rpm for 8min, taking supernatant, adjusting the pH to 7 with 1.5M HCl solution, centrifuging at 12,000rpm for 8min, taking precipitate, and freeze-drying to obtain the water-insoluble extracellular polysaccharide D.

3. Yield of Leuconostoc mesenteroides water-insoluble exopolysaccharide

The yield of the BD3749 water-insoluble exopolysaccharide D is 6.01g/L by weighing and calculation.

Example 5 preparation and yield of Leuconostoc mesenteroides Water-insoluble exopolysaccharide

1. Materials and methods

Preparation of seeds (fermentation strain): the same as in example 1.

Preparing a tomato juice sucrose culture medium: cleaning ripe fructus Lycopersici Esculenti, peeling, squeezing with juicer, filtering with 80 mesh gauze to obtain juice, boiling for 3min, centrifuging at 6000g for 11min to obtain supernatant, adding sucrose 15 wt%, heating to dissolve, cooling to room temperature, adding Na₂CO₃、NaHCO₃Adjusting pH to 6.5 with NaOH, sterilizing at 125 deg.C for 15min, and cooling to room temperature to obtain sterile culture medium.

2. Preparation of leuconostoc mesenteroides water-insoluble exopolysaccharide

Inoculating the obtained leuconostoc mesenteroides seed liquid into a tomato juice and sucrose culture medium with 15 percent of sucrose by mass and 6.5 of pH according to the inoculation amount of 4 percent (v/v), carrying out shaking fermentation culture at 33 ℃ and 150rpm for 84 hours to obtain a fermentation liquid, freeze-drying the fermentation liquid, mixing the freeze-dried powder with a 4M NaOH solution according to the ratio of 1 g: mixing the materials in a ratio of 2mL, leaching at room temperature for 2h, centrifuging at 14,000rpm for 7min, taking supernatant, adjusting pH to 7 with 2.5M HCl solution, centrifuging at 14,000rpm for 7min, taking precipitate, and freeze-drying to obtain water-insoluble extracellular polysaccharide E.

3. Yield of Leuconostoc mesenteroides water-insoluble exopolysaccharide

The yield of the BD3749 water-insoluble exopolysaccharide E is 7.88g/L by weighing and calculation.

Example 6 monosaccharide composition determination of Leuconostoc mesenteroides Water-insoluble exopolysaccharide

The monosaccharide composition of the polysaccharide is determined by high-efficiency anion chromatography on the extracellular polysaccharide A prepared by the method.

(1) Polysaccharide hydrolysis

Weighing 3mg exopolysaccharide A and 2mL of 4moL/L TFA (trifluoroacetic acid, from Sigma-Aldrich Co. LLC, USA), mixing well, charging N₂Sealing the tube, and hydrolyzing in an oven at 110 ℃ for 20 hours; after cooling, the lid was opened, 200. mu.L of methanol was added and the mixture was washed with N₂Blow drying, repeating the steps of adding methanol and using N₂And blowing for 3 times to remove TFA, dissolving the residue with water to a constant volume of 5mL, filtering with a 0.45-micron microporous membrane, and feeding for sample analysis.

(2) Chromatographic conditions

A chromatographic column: CarboPac PA203mm i.d. x 150 mm;

mobile phase: a, H₂O；B，250mmol/L NaOH；C，1mol/L CH₃COONa；

Ternary gradient elution: flow rate: 0.5 mL/min; an integrating pulse ampere detector;

au working electrode: an Ag/AgCl reference electrode is arranged on the electrode,

sample introduction volume: 20 mu L of the solution; column temperature: at 30 ℃.

The chromatographic analysis result of the glycosyl composition of the Leuconostoc mesenteroides BD3749 water-insoluble exopolysaccharide A is shown in figure 1, the polysaccharide has a single absorption peak at 5.875min, and the retention time of the absorption peak is consistent with that of a glucose standard.

And (4) conclusion: leuconostoc mesenteroides BD3749 water insoluble exopolysaccharide is composed of glucose single glycosyl, and is dextran.

Example 7 structural determination of Leuconostoc mesenteroides Water-insoluble exopolysaccharide

(1) Fourier Infrared Spectroscopy (FTIR) measurements

The water-insoluble extracellular polysaccharide A prepared by the method is fully mixed with potassium bromide powder, pressed into a sheet shape, and measured by Fourier infrared spectroscopy (FTIR) by a Fourier infrared spectrometer (Nicolet 6700, Thermo Fisher company, USA).

The results are shown in FIG. 2, 919cm^-1The absorption peak at (A) proves that the carbon skeleton of the polysaccharide is composed of alpha-glycoside, 1004cm^-1The absorption peak at (A) indicates that a large number of alpha- (1,6) glycosidic linkages, 1102cm, are present in the polysaccharide^-1Is the absorption peak of C-O bond vibration in glucose residue at 1147cm^-1The absorption peak at (A) is the result of the stretching vibration of C-O-C on the glycosidic bond bridge. 3281cm^-1The strong broad peak is intermolecular and intramolecular hydroxyl absorption peak, 2922cm^-1Is the absorption peak of C-H bond stretching vibration, 1645cm^-1The absorption peak at (a) is due to bound water.

And (4) conclusion: BD3749 water-insoluble exopolysaccharide is mainly composed of α - (1,6) glycosidic linkages.

(2) Nuclear Magnetic Resonance (NMR) measurement

The water-insoluble exopolysaccharide A prepared by the above method was completely dissolved in 1M deuterated sodium hydroxide solution at a concentration of 5mg/mL, and subjected to Nuclear Magnetic Resonance (NMR) measurement using a nuclear magnetic resonance spectrometer (Avance III 400MHz, Bruker, Germany).

In FIG. 3, A is NMR-¹H spectrum, showing that the absorption peaks at 7 protons of the sample were 4.945(H-1), 3.970(H-6), 3.887(H-5), 3.787 (H-6'), 3.705(H-3), 3.554(H-2), 3.477(H-4) pm, 5.223 and 5.315ppm, respectively, as a result of glycosidic bond vibration, and B is NMR-¹³C spectrum, showing that the sample has resonances at 101.252(C-1), 77.245(C-3), 74.843(C-2), 73.449(C-5), 72.850(C-4) and 68.554(C-6) ppm.

And (4) conclusion: FTIR and¹H、¹³the data of the C spectrum are combined and analyzed, the BD3749 water-insoluble exopolysaccharide is glucan with a main chain connected by alpha- (1,6) glycosidic bonds and a branched chain connected by alpha- (1,3) and alpha- (1,4) glycosidic bonds.

Example 8 determination of the linkage proportion of Leuconostoc Water-insoluble exopolysaccharide

BD3749 water-insoluble polysaccharide prepared in examples 1 to 5 was used¹Detection of H-NMR was confirmed by comparing the integrated areas at the absorption peaks for H-1 (. alpha. - (1,6)), alpha. - (1,3) and alpha. - (1,4) on the chromatogram (see FIG. 3A)The results of the measurement of each sample are shown in Table 1, with the molar ratio of each connecting bond being determined.

TABLE 1 molar ratio relationships between α - (1,6), α - (1,3) and α - (1,4)

As can be seen from table 1, the molar ratio of each linkage in the BD3749 water-insoluble polysaccharide is α - (1, 6): α - (1, 3): α - (1,4) ═ 1: 0.2-0.3: 0.3 to 0.4.

Comparative example 1

The inoculation amount, the pH of the synthetic medium, the culture temperature, the fermentation time, the speed of fermentation oscillation, the sucrose concentration and the alkali solution concentration during extraction in example 2 were adjusted one by one to obtain the following group of water-insoluble extracellular polysaccharides prepared by different methods, and the yields of each group are shown in Table 2.

TABLE 2 yields of water-insoluble exopolysaccharides prepared by different methods

From the results shown in table 2, it can be seen that when the inoculation amount, the pH of the synthetic medium, the culture temperature, the fermentation time, the speed of fermentation oscillation, the sucrose concentration and the alkali solution concentration during extraction in the preparation method of extracellular polysaccharide of the present invention are adjusted to be out of the protection range of the present invention, the yield of extracellular polysaccharide is significantly reduced. All of the above starting materials and reagents are commercially available.

It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

Claims

1. A preparation method of water-insoluble exopolysaccharide of Leuconostoc mesenteroides is characterized by comprising the following steps:

1) inoculating Leuconostoc mesenteroides BD3749 with the preservation number of CGMCC No.100064 into a tomato juice sucrose culture medium, and performing fermentation culture to obtain a fermentation liquid; the tomato juice sucrose culture medium is prepared by the method comprising the following steps: squeezing fructus Lycopersici Esculenti, filtering, boiling, centrifuging to obtain supernatant, adding sucrose, heating to dissolve, cooling, adjusting pH, sterilizing, and cooling;

wherein the addition amount of the sucrose is 5-30%;

the pH value is 5-8;

the fermentation temperature of the fermentation culture is 15-37 ℃;

2) freeze drying the fermentation liquor obtained in the step 1), taking powder, leaching with alkali liquor, taking supernatant, adjusting the pH value of the supernatant to 7, taking precipitate, and freeze drying to obtain the product;

the water-insoluble exopolysaccharide is glucan with a main chain connected by alpha- (1,6) glycosidic bonds and a branched chain connected by alpha- (1,3) and alpha- (1,4) glycosidic bonds, wherein the molar ratio of the glycosidic bonds alpha- (1,6) to alpha- (1,3) to alpha- (1,4) is 1: 0.2-0.3: 0.3-0.4.

2. The method according to claim 1, wherein the method of preparing Leuconostoc mesenteroides of step 1) further comprises a step of activating Leuconostoc mesenteroides BD3749 to obtain Leuconostoc mesenteroides seeds.

3. The preparation method according to claim 1, wherein the filtering in step 1) is performed by filtering the juice with 80-120 mesh gauze; the boiling time is 1-10 minutes; the centrifugation speed is 4,000-12,000 g, and the centrifugation time is 8-12 minutes; the sterilization temperature is 110-135 ℃, and the sterilization time is 10-30 minutes; the pH value adjusting method comprises the following steps of adding food-grade alkali to adjust the pH value, wherein the food-grade alkali is: na (Na)₂CO₃、NaHCO₃And NaOH.

4. The preparation method according to claim 3, wherein the sucrose is added in the step 1) in an amount of 10-20%; the pH value is 6-7; the fermentation temperature of the fermentation culture is 25-33 ℃.

5. The method of claim 4, wherein the sucrose is added in step 1) in an amount of 10%; the pH value is 7; the fermentation temperature of the fermentation culture is 30 ℃.

6. The method according to claim 5, wherein the boiling time in step 1) is 3 to 8 minutes; the centrifugation speed is 6,000-10,000 g, and the centrifugation time is 9-11 minutes; the sterilization temperature is 115-125 ℃, and the sterilization time is 15-25 minutes.

7. The method of claim 6, wherein the boiling time of step 1) is 5 minutes; the speed of the centrifugation is 8,000g, and the time of the centrifugation is 10 minutes; the temperature of the sterilization is 121 ℃, and the time of the sterilization is 20 minutes.

8. The method according to claim 1, wherein the amount of the Leuconostoc mesenteroides of step 1) is 1 to 5% by volume.

9. The method according to claim 8, wherein the amount of the Leuconostoc mesenteroides of step 1) is 2-4%.

10. The method according to claim 9, wherein the amount of the Leuconostoc mesenteroides of step 1) is 3%.

11. The method according to claim 1, wherein the fermentation culture in step 1) is a shaking culture at a speed of 100 to 300 rpm.

12. The method according to claim 1, wherein the fermentation time in step 1) is 24 to 96 hours.

13. The method of claim 11, wherein the oscillating speed in step 1) is 150 to 250 rpm.

14. The method according to claim 12, wherein the fermentation time in step 1) is 48 to 84 hours.

15. The method of claim 13, wherein the speed of the oscillation in step 1) is 200 rpm.

16. The method of claim 14, wherein the fermentation time of step 1) is 72 hours.

17. The preparation method of claim 1, wherein the alkali solution in the step 2) is NaOH solution, and the concentration of the NaOH solution is 1-5M.

18. The preparation method according to claim 1, wherein the ratio of the freeze-dried powder to the alkali liquor in the leaching in the step 2) is 1 g: 1 mL-1 g: 5 mL.

19. The method of claim 17, wherein the NaOH solution has a concentration of 2 to 4M.

20. The method of claim 18, wherein the ratio of lyophilized powder to lye in the extraction is 1 g: 2 mL-1 g: 4 mL.

21. The method of claim 19, wherein the NaOH solution has a concentration of 3M.

22. The method of claim 20, wherein the ratio of lyophilized powder to lye in the extraction is 1 g: 3 mL.

23. The method according to claim 1, wherein the supernatant obtained in step 2) is obtained by centrifugation.

24. The method according to claim 1, wherein the pH in step 2) is adjusted with an acid solution.

25. The method of claim 23, wherein the centrifugation is at a speed of 8,000 to 15,000g for 5 to 15 minutes.

26. The method according to claim 24, wherein the acid solution is an HCl solution, and the concentration of the HCl solution is 1 to 3M.

27. The method of claim 25, wherein the centrifugation is at a speed of 10,000 to 12,000g for 8 to 10 minutes.

28. The method of claim 26, wherein the HCl solution is at a concentration of 1.5 to 2.5M.

29. The method of claim 28, wherein the HCl solution is at a concentration of 2M.