Mannan from leuconostoc as well as preparation method and application of mannan

Abstract

The invention belongs to the technical field of functional microbial polysaccharide preparation, and particularly provides leuconostoc-sourced mannan as well as a preparation method and application thereof. The preparation method of the mannan from the leuconostoc has the advantages that the culture medium is simple, the fermentation period is short, the yield is higher, the extraction and purification process is simple and convenient, a high-purity sample is easy to obtain and the like, the comprehensive production cost is low, the application safety is high, and industrial large-scale popularization and application are easy. The prepared mannan from leuconostoc has good oxidation resistance, bacteriostasis and probiotic activity, can regulate the microecology of intestinal flora, promote proliferation of probiotics such as bifidobacterium and the like, inhibit the number of flora related to generation of part of intestinal diseases, promote growth, reproduction and acid production of fermented milk strains, remarkably shorten the production cycle of the fermented milk and reduce the production cost of the fermented milk. The lactobacillus cell activity in the fermented milk is enhanced, the reduction of the viable count of the product is delayed, the shelf life of the product is prolonged, and the lactobacillus has a good prospect of being developed into multifunctional prebiotics and fermented dairy product ingredients.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of functional microbial polysaccharides, and particularly relates to a mannan derived from Leuconostoc, a preparation method thereof, and an application thereof. Background Art

[0002] Mannan is a natural macromolecular polysaccharide substance composed of mannose with a relative molecular weight range of 20 - 200 kDa. Its main chain is mainly composed of mannopyranose connected by α-1,6-glycosidic bonds, and the side chains are composed of mannose, mannotriose, and mannotetraose, which are connected by α-1,2 or α-1,3 glycosidic bonds. Mannan has a wide range of biological activities such as improving intestinal health, stimulating immunity, antioxidation, reducing blood lipid, and adsorbing mycotoxins. At the same time, as an excellent water-soluble dietary fiber with low calories and high cellulose content, it has broad application prospects in the fields of medicine, food, daily chemical industry, and feed industry.

[0003] Mannan is rich in sources, including microbial and plant sources. Among them, mannan from microbial sources has the advantages of short production cycle, being not interfered by natural environments such as climate, and strong production controllability. At present, the mannan from microbial sources is mainly the mannan, its glycoprotein, and glycopeptide extracted from yeast. Its wide range of biological activities has been proven by a large number of research materials and literature. Yeast mannan mainly exists in the outer cell wall of yeast, and most of it is tightly combined with other components of the cell wall in a covalent binding manner. Therefore, methods such as acid extraction, alkali extraction, and enzymatic extraction are required to break the yeast cells for lysis treatment, and the extraction and separation process is relatively complex. The extraction yield of mannan is low, the purity is not high, and the biological activity is easily affected by the extraction method, ultimately resulting in high production costs of mannan and great difficulties in large-scale production and preparation, which limits its industrial production and wide application. For example, the optimized yield of mannan from yeast in Chinese Patent (CN201510333610.8) is only 3.2 g / L, and Chinese Patent (CN202311411135.2) has increased the yield of yeast mannan to 3.4 g / L through the optimization of the culture medium and culture conditions.

[0004] The exopolysaccharides of lactic acid bacteria are high-molecular polysaccharide substances synthesized and secreted by lactic acid bacteria through extracellular enzymes or intracellular enzyme systems and localized outside the cells. They have higher safety compared to exopolysaccharides from other bacterial sources. At the same time, they have the advantages of fast fermentation speed, short culture cycle, and simple extraction process compared to polysaccharides from yeast cell walls. At present, there are no relevant reports on the production and preparation of bioactive mannan using lactic acid bacteria at home and abroad. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a strain of lactic acid bacteria with the ability to synthesize mannan, and to apply this strain to efficiently ferment and prepare mannan with significant probiotic activity and food industry application value, so as to achieve the efficient and low-cost production and preparation of functional mannan of microbial origin that meets the requirements of industrial scale production.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A mannan derived from Leuconostoc, which is composed of polysaccharides with a weight percentage of more than 99.8%; the monosaccharide composition of the polysaccharide is mannose; the structure of the polysaccharide is mainly composed of α-mannose residues connected by (1→2) and (1→3), and every 6 α-mannose residues form a repeating unit, in which 2 α-mannose residues are connected by (1→3), and the other residues are connected by (1→2). The head and tail mannose residues of the main chain repeating unit are connected by (1→3); in each main chain repeating unit, the O-6 position of 1 α-mannose residue is respectively substituted by branch chain one, and the O-6 position of another α-mannose residue is substituted by branch chain two; the branch chain one is α-mannose residue and terminal α-mannose connected by (1→6); the branch chain two is terminal α-mannose connected by (1→6); the Leuconostoc is Leuconostoc suionicum M1, and the preservation number is CGMCC No. 26046; the preservation institution is the China General Microbiological Culture Collection Center (abbreviated as CGMCC), the preservation date is November 4, 2022, and the taxonomic name is: Leuconostoc suionicum.

[0008] Further explanation, the structure of the repeating unit of the mannan has various different variation combinations. The (1→3) connection in the main chain can be between any 2 of the 6 α-mannose residues, and the α-mannose residues substituted by branch chain one and branch chain two at the O-6 position can be any α-mannose residue in the repeating unit, and the arrangement order of the branch chain one and the branch chain two is any order.

[0009] Further explanation, the structure of the repeating unit of the mannan has a repeating unit with the structure shown in Structural Formula I:

[0010]

[0011] Among them, in Structural Formula I, Manp is pyranose mannose; n is the number of repeating units, which can be determined by the weight average molecular weight.

[0012] Further explanation, the weight average molecular weight of the mannan derived from Leuconostoc suionicum M1 is 45KDa - 65KDa.

[0013] The present invention also provides a method for preparing the mannan derived from Leuconostoc as described above, and the preparation steps are as follows:

[0014] (1) Strain activation: Transfer the glycerol tube containing the above-mentioned strain Leuconostoc suionicum M1 to MRS liquid medium and activate it at 37 °C for 24 - 48 h;

[0015] (2) Seed liquid preparation: Inoculate the above-activated Leuconostoc suionicum M1 strain into MRS liquid medium at an inoculation amount of 1 - 5%, and culture it at 30 - 37 °C for 12 - 24 h;

[0016] (3) Fermentation culture: Inoculate the above seed liquid into the fermentation medium added with cane molasses or beet molasses at an inoculation amount of 2% - 6%, and culture it at 25 - 40 °C for 24 - 48 h; The formula of the fermentation medium: cane molasses or beet molasses 200 - 350 g / L, yeast extract 0 - 20 g / L, beef extract 0 - 20 g / L, pH 5.0 - 9.0, sterilize at 121 °C for 15 min;

[0017] (4) Crude polysaccharide extraction: Take the fermentation broth obtained in step (3), centrifuge it at 6000 - 10000 r / min for 5 - 10 min, take the centrifuged supernatant, add 3 volumes of pre-cooled 95% ethanol, stand at 4 °C for 12 h, centrifuge it at 4000 - 8000 r / min for 10 - 20 min, and collect the precipitate to obtain the crude polysaccharide sample;

[0018] (5) Dialysis for impurity removal: Dissolve the crude polysaccharide sample obtained in step (4) in distilled water, add trichloroacetic acid solution to a final concentration of 14%, centrifuge it at 6000 - 8000 r / min for 10 min, collect the supernatant and transfer it to a dialysis bag with a molecular weight cut-off range of 8000 - 14000 Da, dialyze it with deionized water for 48 - 72 h, and change the water every 12 h; Collect the dialyzed sample, freeze-dry it for 48 h to obtain the polysaccharide sample powder;

[0019] (6) Separation: Dissolve the polysaccharide sample obtained in step (5) in ultrapure water to prepare a polysaccharide solution with a concentration of 5 g / L, and filter it through a 0.45 μm filter membrane; Take 5 mL of the polysaccharide solution, load it onto a DEAE-52 cellulose anion exchange chromatography column, and perform gradient elution separation using NaCl solutions with concentrations of 0, 0.3, 0.5, and 0.7 mol / L. The eluent flow rate is 0.7 mL / min, and collect 1 tube every 7 min; Detect the polysaccharide concentration of each collected tube sample by the phenol-sulfuric acid method, draw an elution curve, combine the elution samples in the corresponding tubes under the same separation peak, freeze-dry them for 48 h, and weigh them to calculate the mannan yield per liter of fermentation broth;

[0020] (7) Purification: Dissolve the mannan obtained in step (6) in ultrapure water to prepare a polysaccharide solution with a concentration of 3 mg / mL, and filter it through a 0.45 μm filter membrane; Take 3 mL of the polysaccharide solution, load it onto a Sepharose CL-6B agarose gel chromatography column, elute with deionized water at a flow rate of 0.5 mL / min, collect the eluate every 7 min per tube, detect OD490 tube by tube using the phenol-sulfuric acid method, and draw an elution curve; According to the elution curve, combine and collect the single peak components, and perform dialysis for 36 - 48 h according to the method of step (6), collect the dialysate and vacuum freeze-dry to obtain a purified mannan sample.

[0021] Further explanation, in step (3), the cane molasses or beet molasses added to the medium is pre-treated by pectinase enzymolysis, and the enzymolysis conditions are: the dosage of pectinase is 50,000 - 100,000 U / L, the enzymolysis temperature is 35 - 45 °C, and the enzymolysis time is 30 - 120 min.

[0022] On the other hand, the present invention also provides the application of the above-mentioned mannan in the preparation of an anti-pathogenic bacterial biofilm preparation.

[0023] On the other hand, the present invention also provides the application of the above-mentioned mannan in the preparation of an intestinal prebiotic preparation, and the application is that it can promote the proliferation of probiotic bacteria and reduce the quantity of flora related to the occurrence of inflammatory bowel disease and / or colorectal cancer and / or depression and / or irritable bowel syndrome.

[0024] On the other hand, the present invention also provides the application of the above-mentioned mannan in the efficient production of high-activity fermented milk products, and the application can be a biological preservative.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention first provides a Leuconostoc species Leuconostoc suionicum M1 in a lactic acid bacterium with high mannan production. This strain can ferment to obtain mannan with a weight-average molecular weight range of 45 kDa - 65 kDa in a fermentation medium using molasses as a carbon source, and the yield can reach 4.23 - 5.34 g / L. Compared with the production preparation methods of mannan from other microbial sources such as yeast, the mannan preparation method from the Leuconostoc species in the lactic acid bacterium provided by the present invention has the advantages of simple medium, short fermentation period, higher yield, simple extraction and purification process, easy access to high-purity samples, etc., with low comprehensive production cost, high application safety, and easy industrial scale promotion and application. At the same time, it also provides a new technical approach for the value-added transformation and utilization of molasses by-products in the traditional sugar industry.

[0027] 2. The mannan derived from Leuconostoc prepared by the present invention has good antioxidant, antibacterial, and probiotic activities, and can regulate the microecology of the intestinal flora.

[0028] 3. The mannan derived from Leuconostoc prepared by the present invention can promote the growth, reproduction, and acid production of fermented milk strains, significantly shorten the production cycle of fermented milk, maintain the activity of lactic acid bacteria cells in fermented milk, delay the decrease of viable cell count of the product, extend the shelf life of the product, and has good prospects for development into a multifunctional prebiotic and fermented dairy product ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the elution curve of the mannan LSMN derived from Leuconostoc suionicum M1 prepared by the present invention through Sepharose CL-6B agarose gel filtration chromatography, where the ordinate is the absorbance at 490 nm after phenol-sulfuric acid color development, and the abscissa is the tube number of the elution solution collected during chromatography.

[0030] Figure 2 It is the ultraviolet absorption spectrum of LSMN of the present invention.

[0031] Figure 3 It is the infrared absorption spectrum of LSMN of the present invention.

[0032] Figure 4 It is the GC control spectrum (A) of various monosaccharide standard solutions after acetylation derivation and the GC spectrum (B) of the LSMN sample after acetylation derivation of the present invention; among them, the ordinate is the response value, the abscissa is the retention time (minutes), Fuc is fucose, Gal is galactose, Ara is arabinose, Rha is rhamnose, GlcN is glucosamine, Xyl is xylose, Man is mannose, GlcNAcN is N-acetyl-D-glucosamine, Fru is fructose, Rib is ribose, GalA is galacturonic acid, GulA is guluronic acid, GlcA is glucuronic acid, and ManA is mannuronic acid.

[0033] Figure 5 It is the high performance gel permeation chromatogram and molecular weight determination result of LSMN of the present invention, with the ordinate being the RI signal and the abscissa being the elution time.

[0034] Figure 6 It is the GC-MS chromatogram of the methylated derivative of LSMN of the present invention.

[0035] Figure 7 It is the 1 1H-NMR (A) 13 13C-NMR (B) spectrum of the present invention.

[0036] Figure 8 It is the two-dimensional nuclear magnetic spectrum of LSMN of the present invention.1 H 1 H-COSY (A), HSQC (B), HMBC (C) and NOESY (D) spectra.

[0037] Figure 9 For the in vitro antioxidant activities of LSMN at different concentrations.

[0038] Figure 10 For the inhibition rates of LSMN at different concentrations against Escherichia coli biofilms.

[0039] Figure 11 For the analysis results of short-chain fatty acid contents after 48 h of in vitro fermentation of blank, inulin and LSMN.

[0040] Figure 12 For the between-group difference test (A) of bacterial α-diversity and the principal coordinate analysis plot of diversity of the in vitro fermentation products of blank, inulin and LSMN.

[0041] Figure 13 For the comparison plots of the relative abundances of species at the phylum level (A) and genus level (B) of bacteria in the in vitro fermentation products of blank, inulin and LSMN.

[0042] Figure 14 For the effects of adding the LSMN of the present invention on the pH value and OD600 during the cultivation of Streptococcus thermophilus (A, B) and Lactobacillus bulgaricus (C, D).

[0043] Figure 15 For the effects of adding the LSMN of the present invention on the bacterial proliferation (A) and the pH of yogurt (B) during the yogurt fermentation process.

[0044] Figure 16 For the trend plot of the total viable count during the storage of the fermented yogurt added with the LSMN of the present invention. Detailed implementation manners

[0045] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description is made in combination with the specific implementation manners of the present invention. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementations disclosed below.

[0046] The MRS liquid medium is a conventional medium in the art, and the formula can be referred to "Experimental Techniques for Modern Research on Lactic Acid Bacteria" (Science Press, the first edition in July 2013).

[0047] Example 1: Preparation of mannan LSMN

[0048] (1) Strain activation: Transfer the glycerol tube containing the strain Leuconostoc suionicum M1 (Leuconostoc suionicum M1 was deposited on November 4, 2022, with the deposit number CGMCC No. 26046, deposited in the China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) into MRS liquid medium and activate it at 37°C for 24 h.

[0049] (2) Seed liquid preparation: Inoculate the above-activated M1 strain into MRS liquid medium at an inoculation amount of 2% and culture it at 37°C for 12 h.

[0050] (3) Fermentation culture: Inoculate the above seed liquid into the fermentation medium added with cane molasses at an inoculation amount of 2.5% and culture it at 30°C for 36 h. Fermentation medium formula: beet molasses 200 g / L, yeast extract 10 g / L, beef extract 5 g / L, pH 8.0. The beet molasses added to the fermentation medium is pre-treated with pectinase, and the enzymatic hydrolysis conditions are: pectinase dosage 50,000 U / L, enzymatic hydrolysis temperature 40°C, enzymatic hydrolysis time 50 min.

[0051] (4) Extraction of crude polysaccharide: Take 100 mL of the obtained fermentation broth and centrifuge it at 6000 r / min for 10 min. Take the centrifuged supernatant, add 3 volumes of pre-cooled 95% ethanol, let it stand at 4°C for 12 h, centrifuge it at 6000 r / min for 10 min, collect the precipitate, dissolve it in 50 mL of deionized water, then add 3 volumes of pre-cooled 95% ethanol, let it stand at 4°C for 12 h, centrifuge it at 8000 r / min for 10 min, and collect the precipitate to obtain the crude polysaccharide sample.

[0052] (5) Dialysis for impurity removal: Dissolve the crude polysaccharide sample obtained in step (4) in 50 mL of distilled water, add trichloroacetic acid solution to a final concentration of 14%, centrifuge it at 8000 r / min for 10 min, collect the supernatant and transfer it to a dialysis bag with a molecular weight cut-off range of 8000 - 14000 Da, dialyze it with deionized water for 72 h, and change the water every 12 h. Collect the dialyzed sample and freeze-dry it for 48 h to obtain the polysaccharide sample powder.

[0053] (6) Separation: Dissolve the dried polysaccharide sample obtained in step (5) in ultrapure water to prepare a polysaccharide solution with a concentration of 5 g / L, and filter it through a 0.45 μm filter membrane. Take 5 mL of the polysaccharide solution, load it onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 30 cm), and perform gradient elution separation using NaCl solutions with concentrations of 0, 0.3, 0.5, and 0.7 mol / L. The eluent flow rate is 0.7 mL / min, and 1 tube is collected every 7 min. The elution times for the 0 - 0.7 mol / L concentration NaCl solutions are 140 min, 210 min, 210 min, and 140 min respectively. Detect the polysaccharide concentration of each collected tube sample by the phenol-sulfuric acid method, and plot the elution curve. Collect and combine the elution samples from the 51st to the 70th tubes, dialyze them for 48 h according to the method in step (5), freeze-dry them for 48 h, and weigh them to calculate the yield of mannan LSMN per liter of fermentation broth.

[0054] (7) Purification: Dissolve the mannan obtained in step (6) in ultrapure water to prepare a polysaccharide solution with a concentration of 3 mg / mL, and filter it through a 0.45 μm filter membrane. Take 3 mL of the polysaccharide solution, load it onto a Sepharose CL-6B agarose gel chromatography column (1.0 cm × 70 cm), elute it with deionized water, the flow rate is 0.5 mL / min, collect the eluent every 7 min / tube, detect OD490 tube by tube by the phenol-sulfuric acid method, and plot the elution curve. As Figure 1 shown, it indicates that the LSMN elution peak is single and symmetrical, and the molecular weight distribution is uniform. Combine and collect the single peak components, and perform dialysis for 48 h according to the method in step (6), collect the dialysis solution and vacuum freeze-dry it to obtain a purified mannan sample.

[0055] (8) Yield analysis: Weigh the dried powder sample of the purified mannan LSMN obtained in step (8), and calculate that the yield of mannan LSMN is 4.23 g / L.

[0056] Example 2: Preparation of mannan LSMN

[0057] (1) Strain activation: Take a glycerol tube containing the strain Leuconostoc suionicum M1 (deposit number CGMCC No. 26046) and transfer it to MRS liquid medium, and activate it at 37 °C for 48 h.

[0058] (2) Seed solution preparation: Inoculate the above-activated M1 strain into MRS liquid medium at an inoculation amount of 4%, and culture it at 37 °C for 18 h.

[0059] (3) Fermentation culture: Inoculate the above-mentioned seed liquid into the fermentation medium added with beet molasses at an inoculation amount of 4%, and culture at 33°C for 36 h. Fermentation medium formula: 300 g / L of beet molasses, 5 g / L of yeast extract, 10 g / L of beef extract, pH 8.0. The beet molasses added to the fermentation medium is pre-treated with pectinase. The enzymatic hydrolysis conditions are: pectinase dosage of 75,000 U / L, enzymatic hydrolysis temperature of 45°C, and enzymatic hydrolysis time of 40 min.

[0060] (4) Extraction of crude polysaccharide: Take 100 mL of the obtained fermentation broth and centrifuge at 8000 r / min for 5 min. Take the centrifuged supernatant, add 3 volumes of pre-cooled 95% ethanol, stand at 4°C for 12 h, centrifuge at 8000 r / min for 6 min, collect the precipitate, dissolve it in 50 mL of deionized water, then add 3 volumes of pre-cooled 95% ethanol, stand at 4°C for 12 h, centrifuge at 10000 r / min for 5 min, and collect the precipitate to obtain the crude polysaccharide sample.

[0061] (5) Dialysis for impurity removal: Dissolve the crude polysaccharide sample obtained in step (4) in 50 mL of distilled water, add trichloroacetic acid solution to a final concentration of 14%, centrifuge at 6000 r / min for 10 min, collect the supernatant and transfer it to a dialysis bag with a molecular weight cut-off range of 8000 - 14000 Da, dialyze with deionized water for 72 h, and change the water every 12 h. Collect the dialyzed sample, freeze-dry it for 48 h to obtain the polysaccharide sample powder.

[0062] (6) Separation: Dissolve the dried polysaccharide sample obtained in step (5) in ultrapure water to prepare a polysaccharide solution with a concentration of 5 g / L, and filter it through a 0.45 μm filter membrane. Take 5 mL of the polysaccharide solution and load it onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 30 cm), and perform gradient elution separation using NaCl solutions with concentrations of 0, 0.3, 0.5, and 0.7 mol / L. The eluent flow rate is 0.7 mL / min, and 1 tube is collected every 7 min. The elution times for the 0 - 0.7 mol / L concentration NaCl solutions are 140 min, 210 min, 210 min, and 140 min respectively. Detect the polysaccharide concentration of each collected tube sample by the phenol-sulfuric acid method and plot the elution curve. Collect and combine the elution samples from the 51st to the 70th tubes, dialyze them for 48 h according to the method in step (5), freeze-dry them for 48 h, and weigh them to calculate the yield of mannan LSMN per liter of fermentation broth.

[0063] (7) Purification: Dissolve the mannan obtained in step (6) in ultrapure water to prepare a polysaccharide solution with a concentration of 3 mg / mL, and filter it through a 0.45 μm filter membrane. Take 3 mL of the polysaccharide solution and load it onto a Sepharose CL-6B agarose gel chromatography column (1.0 cm × 70 cm), elute with deionized water at a flow rate of 0.5 mL / min, collect the eluate in tubes every 7 min, detect OD490 in each tube by the phenol-sulfuric acid method, and plot the elution curve. As Figure 1 shown, it indicates that the LSMN elution peak is single and symmetrical, and the molecular weight distribution is uniform. Combine and collect the single peak components, and perform dialysis for 48 h according to the method in step (6), and collect the dialysis solution and vacuum freeze-dry to obtain the purified mannan sample.

[0064] (8) Yield analysis: Weigh the dried powder sample of the purified mannan LSMN obtained in step (8), and calculate that the yield of mannan LSMN is 4.83 g / L.

[0065] Example 3: Preparation of mannan LSMN

[0066] (1) Strain activation: Transfer the glycerol tube containing the strain Leuconostoc suionicum M1 (deposit number CGMCC No. 26046) to MRS liquid medium and activate it at 37 °C for 36 h.

[0067] (2) Seed solution preparation: Inoculate the activated M1 strain prepared above into MRS liquid medium at an inoculation amount of 6%, and culture it at 37 °C for 24 h.

[0068] (3) Fermentation culture: Inoculate the above seed solution into the fermentation medium added with beet molasses at an inoculation amount of 4%, and culture it at 37 °C for 32 h. Fermentation medium formula: Beet molasses 300 g / L, yeast extract 5 g / L, beef extract 10 g / L, pH 8.0. The beet molasses added to the fermentation medium is pre-treated with pectinase, and the enzyme digestion conditions are: pectinase dosage 100,000 U / L, enzyme digestion temperature 45 °C, enzyme digestion time 35 min.

[0069] (4) Extraction of crude polysaccharide: Take 100 mL of the obtained fermentation broth and centrifuge it at 7000 r / min for 5 min. Take the centrifuged supernatant, add 3 volumes of pre-cooled 95% ethanol, let it stand at 4 °C for 12 h, centrifuge it at 8000 r / min for 8 min, collect the precipitate, dissolve it in 50 mL of deionized water, then add 3 volumes of pre-cooled 95% ethanol, let it stand at 4 °C for 12 h, centrifuge it at 10000 r / min for 5 min, and collect the precipitate to obtain the crude polysaccharide sample.

[0070] (5) Dialysis for impurity removal: Dissolve the crude polysaccharide sample obtained in step (4) in 50 mL of distilled water, add trichloroacetic acid solution to a final concentration of 14%, centrifuge at 8000 r / min for 10 min, collect the supernatant and transfer it into a dialysis bag with a molecular weight cut-off range of 8000 - 14000 Da, dialyze with deionized water for 60 h, and change the water every 12 h. Collect the dialyzed sample and freeze-dry it for 48 h to obtain a polysaccharide sample powder.

[0071] (6) Separation: Dissolve the dried polysaccharide sample obtained in step (5) in ultrapure water to prepare a polysaccharide solution with a concentration of 5 g / L, and filter it through a 0.45 μm filter membrane. Take 5 mL of the polysaccharide solution and load it onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 30 cm), and perform gradient elution separation using NaCl solutions with concentrations of 0, 0.3, 0.5, and 0.7 mol / L. The flow rate of the eluent is 0.7 mL / min, collect 1 tube every 7 min, and the elution times of the 0 - 0.7 mol / L concentration NaCl solutions are 140 min, 210 min, 210 min, and 140 min respectively. Detect the polysaccharide concentration of each collected tube sample by the phenol-sulfuric acid method and plot an elution curve. Collect and combine the eluted samples from the 51st to the 70th tubes, dialyze them for 48 h according to the method in step (5), freeze-dry them for 48 h, and weigh them to calculate the yield of mannan LSMN per liter of fermentation broth.

[0072] (7) Purification: Dissolve the mannan obtained in step (6) in ultrapure water to prepare a polysaccharide solution with a concentration of 3 mg / mL, and filter it through a 0.45 μm filter membrane. Take 3 mL of the polysaccharide solution and load it onto a Sepharose CL-6B agarose gel chromatography column (1.0 cm × 70 cm), elute it with deionized water, with a flow rate of 0.5 mL / min, collect the eluate every 7 min / tube, detect OD490 of each tube by the phenol-sulfuric acid method, and plot an elution curve. As Figure 1 shown, it indicates that the LSMN elution peak is single and symmetric, and the molecular weight distribution is uniform. Combine and collect the single peak components, and perform dialysis for 48 h according to the method in step (6), collect the dialysate and vacuum freeze-dry it to obtain a purified mannan sample.

[0073] (8) Yield analysis: Weigh the dried powder sample of the purified mannan LSMN obtained in step (8), and calculate that the yield of mannan LSMN is 5.34 g / L.

[0074] Example 4: Structural analysis of mannan LSMN

[0075] In this example, ultraviolet spectroscopy (UV), Fourier transform infrared spectroscopy (FT-IR), ion chromatography (IC), high performance gel permeation chromatography (HPGPC), methylation derivation, and nuclear magnetic resonance spectroscopy (NMR) analysis were used to analyze the purity, monosaccharide composition, molecular weight distribution characteristics, types of sugar residues, and linkage modes of the purified mannan LSMN.

[0076] (1) Ultraviolet absorption spectroscopy analysis

[0077] The purity of polysaccharides and the presence of impurities such as proteins and nucleic acids can be preliminarily identified using ultraviolet absorption spectroscopy. The purified LSMN sample was dissolved in deionized water to prepare a polysaccharide solution with a concentration of 1 mg / mL, and ultraviolet absorption spectroscopy scanning was performed in the wavelength range of 190 - 350 nm. Figure 2 It can be seen that there are no obvious absorption peaks at 260 nm and 280 nm, indicating that the purified LMSN has no nucleic acid and protein impurities and has high purity.

[0078] (2) Infrared spectroscopy analysis

[0079] From Figure 3 the infrared spectrum, it can be seen that the purified LMSN shows a broad and strong absorption peak at 3445 cm -1 , indicating the presence of a large number of hydroxyl groups in the component. The small sharp peak near 2920 cm -1 originates from the C—H stretching vibrations in CH3, CH2, and CH. The stretching vibration peaks of C—O—C glycosidic bonds and C—O bonds may be around 1043 cm -1 . The absorption peak near 1637 cm -1 is the characteristic peak of bound water, indicating a strong interaction between the polysaccharide and water molecules. The absorption peak at 1384 cm -1 comes from the deformation vibration of C—H. The presence of functional groups such as glycosidic bonds, carbonyl groups, and hydroxyl groups in this polysaccharide component is consistent with the common structure of polysaccharides.

[0080] (3) Determination of monosaccharide composition

[0081] The polysaccharide sample was hydrolyzed with trifluoroacetic acid (TFA), and the monosaccharide composition of the hydrolysate was determined by ion chromatography. The specific method is as follows: Weigh 5 mg of the LSMN polysaccharide sample precisely and place it in an ampoule. Add 2 mL of 3 M TFA and hydrolyze at 120 °C for 3 h. Accurately pipette the acid hydrolysis solution and transfer it to a tube, then 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 12,000 rpm for 5 min, and take 25 μL of the supernatant for analysis using an ICS5000 ion chromatograph (equipped with an electrochemical detector). The chromatographic analysis conditions are as follows: Chromatographic column: Dionex CarbopacTM PA20 (3 × 150 mm); Mobile phase: A: H2O; B: 15 mmol / L NaOH; C: 15 mmol / L NaOH, 100 mmol / L NaOAc; Elution gradient: 0 min, phase A / phase B / phase C (98.8:1.2:0, V / V), 18 min, phase A / phase B / phase C (98.8:1.2:0, V / V), 20 min, phase A / phase B / phase C (50:50:0, V / V), 30 min, phase A / phase B / phase C (50:50:0, V / V), 30.1 min, phase A / phase B / phase C (0:0:100, V / V), 46 min, phase A / phase B / phase C (0:0:100, V / V), 46.1 min, phase A / phase B / phase C (0:100:0, V / V), 50 min, phase A / phase B / phase C (0:100:0, V / V), 50.1 min, phase A / phase B / phase C (98.8:1.2:0, V / V), 80 min, phase A / phase B / phase C (98.8:1.2:0, V / V). Flow rate: 0.3 mL / min; Column temperature: 30 °C. Select 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, D-galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid) to prepare a standard stock solution. Take each monosaccharide standard solution and precisely configure concentration standards as a mixed standard. According to the absolute quantification method, determine the mass of different monosaccharides contained in the polysaccharide sample to be measured, and calculate the molar ratio based on the molar mass of the monosaccharides. From Figure 4 As shown in A, the monosaccharide standards were well separated under the said chromatographic conditions. After comparison with the 16 standards, it was determined that LSBM1 EPS is a homopolysaccharide composed of mannose ( Figure 4 B), with a molar proportion of 100%.

[0082] (4) Analysis of molecular weight distribution characteristics

[0083] The molecular weight distribution characteristics of polysaccharides were analyzed by high performance gel permeation chromatography (HPGPC). The specific method is as follows: Accurately weigh 2 mg of each of 8 dextran standards with different molecular weights (P5, P10, P20, P50, P100, P200, P400, P800), dissolve them in 1 mL of 0.2 mol / L NaCl solution to prepare a 2 mg / mL solution, filter it through a 0.22 μm hydrophilic microfiltration membrane and then measure it on the machine. According to the retention time of the chromatogram, respectively plot the standard working curves of lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw weight average molecular weight), and lgMn-RT (Mn number average molecular weight). Precision weigh 5 mg of the mannan LSMN sample, dissolve it in 1.0 mL of the mobile phase solution to prepare a 5 mg / mL solution. After ultrasonic treatment for 10 min, centrifuge at 12000 r / min for 10 min, absorb the supernatant, filter it through a 0.22 μm hydrophilic microfiltration membrane, and measure it on the machine. Substitute the retention time into the standard working curve to calculate the molecular weight (Mp, Mw, Mw). Chromatographic conditions: Mobile phase: 0.2 mol / L NaCl solution; Chromatographic column: BRT105-103-101 tandem gel column (8×300 mm); Flow rate: 0.8 mL / min; Column temperature: 40 °C; Injection volume: 25 μL; Detector: Differential detector RID-10A; Analysis time: 60 min.

[0084] It can be seen from Figure 5 that LSMN shows a single symmetric peak shape, indicating that it is a homogeneous polysaccharide. The weight average molecular weight Mw is 50671 Da, Mn is 49517 Da, and the molecular weight distribution coefficient Mw / Mn value is 1.14, indicating that LSMN is a polysaccharide component with low dispersion and uniform molecular weight.

[0085] (5) Methylation analysis of polysaccharides

[0086] Take 2 mg of purified LSMN and dissolve it in 1 mL of dimethyl sulfoxide (DMSO), and quickly add anhydrous base solution of methylation reagent A for blocking treatment. Under the action of ultrasonic waves, the sample is fully dissolved. Subsequently, add methylation reagent B methyl iodide solution, and react in a magnetic stirring water bath at 30 °C for 60 min. Finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction. After methylation is completed, use 1 mL of 2 mol / L trifluoroacetic acid (TFA) to hydrolyze the methylated polysaccharide sample for 90 min, then evaporate the solution to dryness by a rotary evaporator, add 2 mL of double distilled water, reduce it with 60 mg of sodium borohydride for 8 h, neutralize it with glacial acetic acid, rotary evaporate, dry it in an oven at 101 °C, then add 1 mL of acetic anhydride and acetylate it at 100 °C for 1 h, and cool it. Then add 3 mL of toluene, concentrate it under reduced pressure and evaporate it to dryness, repeat 4 - 5 times to remove the excess acetic anhydride. Dissolve the acetylated product with 3 mL of CH 2 Cl 2Transfer it to a separatory funnel after dissolution. Add a small amount of distilled water and shake well, then remove the upper aqueous solution. Repeat this process 4 times. CH 2 Cl 2 The CHCl layer was dried over an appropriate amount of anhydrous sodium sulfate and made up to 10 mL. It was determined by an Agilent GCMS 6890-5973 gas chromatography-mass spectrometry instrument, and the obtained results were compared and analyzed with the standard mass spectrometry library. GC-MS analysis conditions: An RXI-5SILMS chromatographic column (30×0.25 mm×0.25 μm) was used. Programmed temperature rising conditions: The initial temperature was 120 °C, and then it was heated to 250 °C at a rate of 3 °C / min and held at this temperature for 5 min. The inlet temperature and the detector temperature were both set at 250 °C, and the carrier gas (helium) flow rate was 1 mL / min.

[0087] Figure 6 Figure 1 is the total ion chromatogram of the methylation derivative GC-MS. Table 1 is the analysis result table of the methylation derivative GC-MS of LSMN. As can be seen from Table 1, LSMN mainly contains 5 kinds of methylation sugar residue fragments, corresponding to 5 different mannose residues, namely Manp-(1→,→2)-Manp-(1→,→3)-Manp-(1→,→6)-Manp-(1→ and →2,6)-Manp-(1→, and their molar ratios are respectively: 28%, 18%, 26.2%, 12.1%, 15.7%. It can be speculated from this that LSMN may be mainly composed of Manp-(1→,→2)-Manp-(1→,→3)-Manp-(1→,→2,6)-Manp-(1→, with a small amount of →6)-Manp-(1→ branched structure.

[0088] Table 1 Analysis result table of the methylation derivative GC-MS of LSMN

[0089]

[0090] (6) Nuclear magnetic resonance (NMR) analysis

[0091] Take 50 mg of the polysaccharide sample and dissolve it in 0.5 mL of heavy water and freeze-dry. Then dissolve the freeze-dried powder in 0.5 mL of heavy water again and repeat the freeze-drying to fully exchange the labile hydrogen. Then dissolve the sample in 0.5 mL of heavy water and measure the 1H NMR spectrum, 13C NMR spectrum, one-dimensional spectrum and two-dimensional spectrum on a Bruker 600 MHz nuclear magnetic resonance instrument at room temperature of 25 °C.

[0092] From Figure 7 -A 1It can be seen from the 1H-NMR spectrum that the hydrogen spectrum signals are mainly concentrated between 3.0 and 5.5 ppm. The proton signals of the sugar ring are at δ 3.2 - 4.0 ppm, and the main anomeric proton peaks, δ 5.19, 5.07, 5.01, 4.96, 4.82, are concentrated in the region of 4.3 - 5.5 ppm. From Figure 7 -B 13 It can be seen from the 13C-NMR spectrum that the carbon spectrum signals are mainly concentrated between 60 and 120 ppm, and the anomeric carbon signal peaks are mainly distributed between δ 93 and 105 ppm. And δ 71.55, 79.30, 68.40, 74.75, 62.46, 71.54, 71.82, 67.87, 74.71, 62.46, 79.67, 71.62, 67.74, 74.63, 62.46, 71.42, 71.95, 68.03, 74.53, 67.20, 80.12, 71.58, 68.06, 74.52, 67.21, the main signal peaks are distributed in the region of 60 - 85 ppm. In most cases, for the α-configuration of the pyranose form, it is usually between δ 4.9 - 5.8 ppm in the 1H-NMR spectrum, and the coupling constant J-H1~H2 is between 0 and 3 Hz; for the β-configuration, it is usually between δ 4.4 - 4.8, and the coupling constant J-H1~H2 is between 6 and 8 Hz. For the α-configuration of the pyranose form, it is usually between δ 90 - 98 ppm in the 13C-NMR spectrum; for the β-configuration, it is usually between δ 98 - 110. However, in the actual analysis of polysaccharides, there will be certain differences due to different monosaccharides, structures, spaces, and interactions, etc. For mannose, if there is coupling, the configuration can be determined by the coupling constants of J1,2 (0 - 2.0 Hz) for the α-configuration and J4,5 (8 - 10 Hz) for the β-configuration. However, in most cases, the J1,2 between the positions H1 and H2 of mannose is very small, and no coupling can be found. What appears in the 1H-NMR spectrum are all bulges, and the α or β configuration of mannose cannot be determined by the coupling constant between J1 and 2. At the same time, it should be noted that affected by the spatial positions of H1 and H2 (the hydroxyl groups at this position are perpendicular to the dominant chair conformation), the α-configuration mannose often appears at a lower field (between 98 - 105 ppm) in the C1 carbon spectrum, and is basically at the same displacement as the C1 position of the β-configuration. Therefore, in most cases, it is necessary to analyze in combination with the position of the 1H-NMR spectrum. Combining the literature data and the position signals of the five residual sugars, through comprehensive judgment, the 1H-NMR signals are all greater than 4.8 ppm, and the position of the 13C-NMR spectrum is difficult to judge. Finally, it is judged that the mannose in the EPS2 molecule is in the α-configuration.

[0093] From Figure 8 It is the 2D-NMR of the polysaccharide sample ( 1 1H- 1 1H COSY, 1 1H- 13 13C HSQC, NOESY and1 H- 13 C HMBC) results. From Figure 8 -A 1 H- 1 H-COSY spectrum shows that the signals of H1-2 are 5.19 / 4.04; the signals of H2-3 are 4.04 / 3.86; the signals of H3-4 are 3.86 / 3.79; the signals of H4-5 are 3.79 / 3.65; the signals of H5-6a are 3.65 / 3.71; according to the chemical shifts, it can be inferred that H1, H2, H3, H4, H5, H6a are 5.19, 4.04, 3.86, 3.79, 3.66, 3.71 respectively, and its H6b is 3.82. The corresponding carbon spectrum is δ101.89, 79.67, 71.62, 67.74, 74.63, 62.16. Through Figure 8 -B HSQC spectrum, the anomeric carbon signal can be observed as δ101.88, and the corresponding anomeric hydrogen signal in the HSQC spectrum is 5.19. According to the chemical shift migration, it can be inferred that this signal belongs to the glycosidic bond →2-Manp-1→. According to similar rules and combined with HMBC and NOESY spectra, all glycosidic bond signals are assigned, and the results are shown in Table 2.

[0094] Table 2 Carbon and hydrogen chemical shift assignments of glycosidic bonds in LSMN

[0095]

[0096] From the HMBC spectrum ( Figure 8 -C), it can be seen that there are correlation peaks between the anomeric hydrogen of the methylated C-type residue glycosidic bond and C2 of the methylated B-type residue glycosidic bond, indicating the existence of the linkage →3-Manp-1→2-Manp-1→ in this polysaccharide. And there are also correlation peak signals between the anomeric hydrogen of the B-type residue glycosidic bond and C2 of the E-type residue glycosidic bond, indicating the existence of the linkage →2-Manp-1→2,6-Manp-1→ in this polysaccharide. There are correlation peaks between the anomeric hydrogen of the E-type residue glycosidic bond and C2 of its own E-type residue glycosidic bond, indicating the existence of

[0097] →2,6-Manp-1→2,6-Manp-1→ linkage. Therefore, it can be inferred that the main chain has:

[0098] →3-Manp-1→2-Manp-1→2,6-Manp-1→2,6-Manp-1→ sugar linkage mode. From the NOESY spectrum ( Figure 8-D), it can be seen that the anomeric hydrogen of the A-type glycosidic bond Manp-1→ has a correlation peak with H6b of the D-type glycosidic bond →6-Manp-1→, indicating the linkage relationship of these two glycosidic bonds. The anomeric hydrogen of the D-type glycosidic bond →6-Manp-1→ has a correlation peak with H6b of the D-type glycosidic bond itself, indicating the linkage of the →6-Manp-1→6-Manp-1→ glycosidic bond. The anomeric hydrogen of the D-type glycosidic bond has a correlation peak with H6b of the E-type glycosidic bond, indicating the DE glycosidic bond linkage mode in the side chain: →6-Manp-1→2,6-Manp-1→.

[0099] In summary, combining the results of methylation analysis, based on the molar ratios of several glycosidic bonds and the analysis of two-dimensional NMR spectra, the structure of LSMN is deduced as shown in Structural Formula 1.

[0100]

[0101] Example 5: In vitro biological activity evaluation of mannan LSMN

[0102] (1) In vitro antioxidant activity

[0103] To evaluate the in vitro antioxidant ability of the purified mannan LSMN obtained in Example 2, the obtained polysaccharide was dissolved in ultrapure water to prepare polysaccharide solutions with concentrations of 0.5, 1.0, 2.0, 4.0, and 9.0 mg / L. Referring to the method of Cao et al., 2020 (Cao, C., Liu, Y., Li, Y. et al. Structural characterization and antioxidant potential of a novel exopolysaccharide produced by Bacillus velezensis SN-1 from spontaneously fermented Da-Jiang. [J]. Glycoconjugate Journal, 2020, 37, 307-317.), with ascorbic acid (VC) at the same concentration as the control, the scavenging abilities against DPPH, ·OH, ABTS + 、O 2- Four kinds of free radicals were measured respectively, and the results are as Figure 9 shown. As Figure 9 can be seen, LSMN has different scavenging abilities against 4 kinds of free radicals, and with the increase of the polysaccharide concentration, the free radical scavenging rate shows a gradually increasing trend. Under the concentration condition of 8 mg / mL, the scavenging rate of LSMN against ABTS+ free radicals can be as high as 82.25%, the scavenging rate against DPPH free radicals is 58.46%, the scavenging rate against ·OH free radicals is 72.36%, and the scavenging rate against O 2-The scavenging rate of free radicals was 45.50%, which was higher than that of another component (named EPS1) in crude polysaccharide at the same concentration. Therefore, the purified mannan of the present invention has good in vitro antioxidant activity and good potential for developing products related to antioxidant functions.

[0104] (2) Bacterial biofilm inhibition activity

[0105] To evaluate the inhibitory activity of the purified mannan LSMN obtained in Example 2 against bacterial biofilms, Escherichia coli ATCC25922 was selected as the indicator strain for the study.

[0106] The specific experimental method is as follows: Weigh the purified polysaccharide component mannan LSMN powder and dissolve it in PBS solution (pH 7.2) to prepare EPS solutions with different concentrations (0.5 - 8 mg / mL). Add 100 μL of polysaccharide solutions with different concentrations and 100 μL of Escherichia coli bacterial solution (bacterial concentration is 10 5 CFU / mL) into the 96-well plate in sequence, incubate statically at 37 °C for 48 h, aspirate the bacterial solution in the well plate, and wash it twice with sterile water to remove the planktonic bacteria that are not firmly adhered. Then add 200 μL of methanol to each well to fix for 15 min, discard the methanol, and add 200 μL of crystal violet (0.1%) to stain for 20 min. Wash the excess crystal violet staining solution with distilled water and dry at room temperature. Finally, add 200 μL of 33% acetic acid to each well and measure the absorbance at 595 nm. At the same time, use the well plate without adding polysaccharide as a control to calculate the biofilm inhibition rate. The results are as Figure 10 and shown in Table 3. The inhibition rate of LSMN on the biofilm formation of Escherichia coli strains increased with the increase of the polysaccharide addition concentration (from 0.5 to 8 mg / mL). Among them, the biofilm inhibition rate under the condition of 8 mg / mL polysaccharide concentration reached 49.94%. It can be seen that the mannan obtained in the present invention has certain biofilm scavenging activity and can be used to develop and prepare a new type of green and safe anti-biofilm preparation to improve the food safety level.

[0107] Table 3

[0108] Mannan LSMN Concentration (mg / mL) Inhibitory Rate of Escherichia coli Biofilm (%) 0.5 11.61±1.01 1.0 19.94±3.97 2.0 33.51±2.54 4.0 34.16±4.21 8.0 49.94±5.37

[0109] Example 6: Evaluation of the intestinal flora regulation and probiotic activity of mannan LSMN

[0110] To further evaluate the possible flora regulation effect and health benefits brought by the mannan LSMN obtained in Example 2 when ingested into the human digestive system and entering the intestine, this study designed an in vitro simulated intestinal fermentation experiment to analyze the changes in the content of short-chain fatty acids in the simulated fermented intestinal fluid, combined with 16S rRNA high-throughput sequencing to analyze the changes in bacterial diversity, and comprehensively evaluate the intestinal flora regulation activity and potential health benefits of LSMN.

[0111] (1) Sample preparation and fermentation

[0112] In an anaerobic operating box, fresh feces collected from 6 healthy volunteers with no history of antibiotic use in the past 3 months were mixed in equal amounts. 10 g of the mixed fecal sample was added to 100 mL of sterile phosphate buffer (pH 6.8), and the mixture was vortexed to prepare a fecal dilution suspension. The suspension was filtered through 4 layers of sterile gauze, and the filtrate was used as the bacterial source solution. The collection and processing of all fresh fecal samples were completed within 1 hour. The basic fermentation broth was used as the blank control group (Blanks), 1% inulin (w / v) was added as the positive control, and 1% (w / v) LSMN was added as the test group (LSMN). The bacterial source solution was inoculated at an inoculation ratio of 1:9 (v:v), and the cultures were incubated in an anaerobic incubator at 37°C.

[0113] (2) Analysis of short-chain fatty acid content

[0114] LC-ESI-MS / MS (UHPLC-Qtrap) was used to quantitatively analyze the content of short-chain fatty acids in samples cultured in vitro for 48 h. Preparation of short-chain fatty acid standard solution and drawing of working curve: Mixed standard stock solutions of 8 short-chain fatty acids, namely acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, and isohexanoic acid, with a concentration of 20 μg / mL and an internal standard stock solution (2-ethylbutyric acid) were prepared with n-butanol (HPLC grade). The above mixed standard solution was diluted with n-butanol into 7 working solutions with different concentrations, filled into sample vials, and subjected to GC-MS detection and analysis. Sample preparation: 500 μL of extraction solution (methanol: water = 4:1) was added to the sample, followed by cryogenic grinding for 6 min (-10 °C, 50 Hz), low-temperature ultrasonic treatment for 30 min (5 °C, 40 KHz), standing at -20 °C for 30 min, and centrifugation at 4 °C, 13,000 g for 15 min. 20 μL of the supernatant was added with 20 μL of 200 mM 3NPH.HCL and 20 μL of 120 mM EDC.HCL (containing 6% pyridine) solution, reacted at 40 °C for 30 min, and then diluted to 1000 μL with 50% aqueous acetonitrile solution and injected into the instrument for analysis. LC-MS detection conditions: Chromatographic conditions: ExionLCAD system, Waters BEH C18 (150*2.1 mm, 1.7 μm) liquid chromatography column, column temperature 40 °C, injection volume 2 μL. Mobile phase A (0.1% formic acid-aqueous solution), mobile phase B (0.1% formic acid-acetonitrile). Mass spectrometry conditions: AB SCIEX QTRAP 6500+, detected in negative mode, Curtain Gas (CUR) was 35, Collision Gas (CAD) was Medium, IonSpray Voltage (IS) was -4500, Temperature (TEM) was 450, Ion Source Gas1 (GS1) was 40, Ion Source Gas2 (GS2) was 40. A linear regression standard curve was drawn with the mass spectrometry peak area of the analyte as the ordinate and the concentration of the analyte as the abscissa. Calculation of sample concentration: The mass spectrometry peak area of the sample analyte was substituted into the standard working curve equation to calculate the concentration of short-chain fatty acids in each sample.

[0115] Short-chain fatty acids are important metabolites of intestinal bacteria fermenting carbohydrates, and their concentration changes are closely related to the composition and abundance of the intestinal flora. Short-chain fatty acids can be effectively absorbed by the intestinal mucosa, serve as an energy source, and also act as gene expression regulators and signal molecules recognized by specific receptors, having an important impact on host physiology. Figure 11 It can be seen that compared with the blank group, the concentrations of 8 short-chain fatty acids in the samples of the inulin- and mannan-added groups changed significantly. Among them, the two short-chain fatty acids, propionic acid and valeric acid, were significantly increased compared with the control, indicating that the addition of mannan LSMN can promote the proliferation of bacteria related to the production of propionic acid and valeric acid.

[0116] It has been reported that propionate is usually produced by intestinal bacteria through the fermentation of carbohydrates such as L-rhamnose, mannan oligosaccharide, and laminarin. Propionate can serve as a precursor for glycogen synthesis in the liver in vivo, helping to regulate blood glucose levels. It also has the effects of reducing cholesterol and anti-lipogenesis, and has an anti-proliferative effect on colon cancer cells. Recent studies have also shown that propionate may have beneficial effects on cardiovascular risk factors such as hypertension, endothelial dysfunction, and hypercholesterolemia. In addition, valeric acid can increase the secretion of IL-10 and significantly inhibit the apoptosis of regulatory B cells, showing certain therapeutic potential for body inflammation and autoimmune diseases.

[0117] In summary, the purified mannan LSMN prepared by the present invention has the potential to further promote human health by increasing the concentrations of intestinal propionate and valeric acid.

[0118] (3) Analysis of intestinal bacterial diversity

[0119] Take the sample solution after 48 h of fermentation, centrifuge at 8000 r / min for 15 min. Treat the sample precipitate with liquid nitrogen and temporarily store it in a -80 °C refrigerator. Send it for storage with dry ice to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for intestinal flora sequencing analysis. Set 3 biological replicates for each treatment. The experimental process includes sample DNA extraction, PCR amplification of the V3-V4 variable region of the 16S rRNA gene and sequencing library construction, sequencing, and sequencing data processing and analysis.

[0120] DNA extraction: According to the soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.) instruction manual, extract the total genomic DNA of the microbial community. Use 1% agarose gel electrophoresis to detect the quality of the extracted genomic DNA, and use NanoDrop 2000 (Thermo Scientific, U.S.) to measure the DNA concentration and purity.

[0121] PCR Amplification: Using the extracted DNA mentioned above as a template, the V3-V4 variable region of the 16S rRNA gene was amplified by PCR with the upstream primer 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and the downstream primer 806R (5’-GGACTACHV GGGTWTCTAAT-3’) carrying the Barcode sequence. The amplification procedure was as follows: pre-denaturation at 95°C for 3 min, 27 cycles (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s), then stable extension at 72°C for 10 min, and finally preservation at 4°C (PCR instrument: T100 Thermal Cycler, BIO-RAD, USA). The PCR reaction system was: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and made up to 20 μL. The PCR products were recovered using a 2% agarose gel, and the recovered products were purified using a DNA gel recovery and purification kit (PCR Clean-Up Kit, Yuhua, China), and the recovered products were detected and quantified using Qubit 4.0 (Thermo Fisher Scientific, USA).

[0122] Sequencing Library Construction and Sequencing: The NEXTFLEX Rapid DNA-Seq Kit was used to construct a library for the purified PCR products, including adapter ligation; magnetic bead screening was used to remove self-ligated adapter fragments; PCR amplification was used to enrich the library template; and the PCR products were recovered by magnetic beads to obtain the final library. Sequencing was performed using the Illumina Nextseq2000 platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.).

[0123] Data Processing and Analysis: The microbial diversity QIIME2 process interactive cloud analysis platform developed by Shanghai Majorbio was used to compare the differences in bacterial community composition between the LSMN group and the control group at the phylum and genus levels.

[0124] As shown in Figure 12 -A, there were significant differences in the shannon index of bacterial species diversity at the genus level between the addition of LSMN and the blank group (Blanks) and the inulin group (Inulin) (P<0.05), indicating that the addition of the mannan of the present invention can significantly change the composition of the intestinal microbiota. As shown in Figure 12In the principal coordinate analysis (PCoA) scatter plot at the genus level of -B bacteria, it can be seen that the repeated samples in the LSMN group are far apart from the samples in the other two groups and cluster in different coordinate quadrants. The three repeats within the same group are very close, indicating that the addition of LSMN has a significant impact on the intestinal bacterial species composition, and there are also obvious differences in this impact compared with the addition of inulin.

[0125] Figure 13 These are the bar charts of the relative abundances of bacterial species at the phylum level and genus level in the three groups of treated samples. As Figure 13 shown in -A, at the phylum level, the dominant bacterial communities in the blank group (Blanks) are mainly Firmicutes, Proteobacteria, and Fusobacteria. Compared with the blank group, the proportions of Firmicutes and Actinobacteria in the inulin-added group increased significantly, and the proportion of Fusobacteria decreased significantly. At the same time, compared with the blank group, the proportions of Proteobacteria, Actinobacteria, and Bacteroidetes in the group with the addition of mannan LSMN increased significantly, and the proportion of Fusobacteria decreased significantly. The results show that the addition of mannan LSMN significantly changed the intestinal bacterial community composition. Existing research results have proved that an increase in the level of Bacteroidetes can bring benefits to the recovery of intestinal damage caused by cyclophosphamide. The beneficial bacteria in Actinobacteria - Bifidobacterium have significant probiotic effects. To further analyze the intestinal probiotic effects brought by inulin and mannan, the community structure composition of the three groups of samples was analyzed at the genus level, and the results are as Figure 13 shown in -B. As Figure 13 shown in -B, compared with the blank group, the genera with significantly increased proportions are Bifidobacterium, Megasphaera, Parabacteroides, and Megamonas. Among them, Bifidobacterium can improve digestive problems, improve blood glucose control, lower blood lipid levels, and enhance immunity. Parabacteroides is a core member of the human gut microbiota and has been proven to regulate the host mucosal immune system, reduce inflammation, participate in carbon metabolism, have more polysaccharide utilization loci (PUL), and can secrete short-chain fatty acids such as acetic acid and propionic acid. The relative abundance of Megamonas is negatively correlated with the increased risk of colorectal polyps. In addition, compared with the blank control group, the proportions of Fusobacterium, Lachnoclostridium, Hungatella, Sellimonas, and Blautia in the group with the addition of mannan LSMN decreased significantly. Among them, Fusobacterium has a strong ability to invade the human host as a major pathogen, and many virulence determinants have been described, including the expression of leukotoxin, proteolytic enzymes, lipopolysaccharide (LPS), and hemagglutinin. It is related to the occurrence of inflammatory bowel disease (IBD) and colorectal cancer; the relative abundance of Lachnoclostridium increases in enteritis lesions, intestinal tumors, liver steatosis, and metabolic diseases; the abundances of Hungatella and Sellimonas are higher in patients with depression and may be correlated with the occurrence of depression; the species level of Blautia is significantly increased in patients with irritable bowel syndrome (IBS).

[0126] Based on the above analysis, mannan can significantly improve the structural composition of the intestinal bacterial community, promote the proliferation of beneficial bacteria such as Bifidobacterium, Megasphaera, Parabacteroides, and Megamonas, and reduce the population of bacteria related to the occurrence of various diseases such as inflammatory bowel disease (IBD), colorectal cancer, depression, and irritable bowel syndrome (IBS), including Fusobacterium, Lachnoclostridium, Hungatella, Sellimonas, and Blautia.

[0127] Example 7: Application of Mannan LSMN in the Production of Fermented Milk

[0128] To further evaluate the application potential of the mannan LSMN prepared in Example 2 in the production of fermented milk, different concentrations of LSMN were added to MRS medium to investigate its effects on the proliferation and acid production ability of fermented milk strains. Further, LSMN was added as an ingredient to the raw materials for fermented milk production to evaluate its promoting effect on the growth and acid production of strains during the yogurt fermentation process. Finally, through a storage test, the effect of adding LSMN on the shelf life of fermented milk was evaluated.

[0129] (1) Proliferation-promoting and acid production-promoting effects of adding mannan LSMN on two fermented strains

[0130] Lactobacillus bulgaricus and Streptococcus thermophilus were respectively inoculated into MRS liquid medium and activated at 37 °C for 48 h, and then inoculated into MRS liquid medium supplemented with 5 g / L LSMN at a volume fraction of 1% and continuously cultured at 37 °C. The OD600 value and pH value of the culture solution were measured at 0, 6, 20, 30, and 48 h respectively. Taking MRS liquid medium supplemented with 5 g / L glucose as a control, the effects of prebiotics on the proliferation-promoting and acid production ability of probiotics during the fermentation process were analyzed. The results are as Figure 14 shown. It can be seen that the OD600 of the culture solutions of Lactobacillus bulgaricus and Streptococcus thermophilus supplemented with LSMN was significantly higher than that of the glucose control, and its pH value was significantly lower than that of the glucose control, indicating that LSMN has an obvious effect on promoting the proliferation and acid production of fermented milk strains.

[0131] (2) Effects of adding mannan LSMN on the viable count and acidity during the fermentation of fermented milk

[0132] 7% white granulated sugar was added to pure milk and stirred until completely dissolved. Then, mannan LSMN with a mass fraction of 0.3% and 0.6% was added, sterilized at 85 °C for 15 min, inoculated with 2% starter culture (composed of Lactobacillus bulgaricus and Streptococcus thermophilus), and fermented in an incubator at 37 °C with the sample without adding LSMN as a control. The pH value of the fermented sample during the fermentation process was measured using a pH meter, and the total viable count of the sample was measured by the plate counting method. The results are asFigure 15 As shown in the figure, at 6 h of fermentation, the total viable count of the sample with LSMN added was significantly higher than that of the control sample, and the total viable count of the sample with 0.6% LSMN added was higher than that of the sample with 0.3% added. At the same time, the pH of the sample with LSMN added decreased faster than that of the non-added control group. Among them, for the samples with 0.3% and 0.6% LSMN added, the pH dropped below 4.6 at 7 h and 6 h of fermentation respectively, indicating that the fermented milk reached the fermentation end point, while the pH of the control sample dropped below 4.6 after 8 h. This shows that adding LSMN for acid milk fermentation can make the fermentation bacteria multiply and produce acid faster, shortening the fermentation time by 1 - 2 h compared to the control.

[0133] (3) Analysis of the change in the total viable count during the storage of the fermented milk prepared by adding mannan LSMN

[0134] Furthermore, the change in the viable count during the storage at 4 °C of the fermented milk prepared by adding the mannan LSMN of the present invention was compared. The results are as Figure 16 shown. It can be seen that the viable count of the fermented milk with mannan LSMN added during storage was significantly higher than that of the blank control group, and the viable count of the fermented milk with a high addition level (0.6%) was significantly higher than that with a low addition level (0.3%). This indicates that the mannan of the present invention can keep a relatively high total viable count during the shelf life of the fermented milk product, extend the shelf life, and has the potential to be used as a natural source additive for preparing fermented milk products with a high viable count in the industrial production.

[0135] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A mannan derived from Leuconostoc, characterized in that: The invention is composed of polysaccharides with a weight percentage of more than 99.8%; the monosaccharide composition of the polysaccharide is mannose; the structure of the polysaccharide is based on (1→2)-linked and (1→3)-linked α-mannose residues as the main chain, and every 6 α-mannose residues form a repeating unit, wherein two α-mannose residues are linked by (1→3), the other residues are linked by (1→2), and the first and last mannose residues of the main chain repeating unit are linked by (1→3); in each main chain repeating unit, one α-mannose residue is substituted by branch chain 1 at the O-6 position, and another α-mannose residue is substituted by branch chain 2 at the O-6 position; the branch chain 1 is an α-mannose residue linked by (1→6) and a terminal α-mannose; the branch chain 2 is a terminal α-mannose linked by (1→6); the Leuconostoc suionicum M1 is a Leuconostoc suionicum M1, and the deposit number is CGMCC No. 26046.

2. The mannan derived from Leuconostoc as claimed in claim 1, characterized in that The structure of the repeating unit of the mannan has a variety of different variations and combinations. The (1→3) connection in the main chain can be between any two residues of the six α-mannose residues. The α-mannose residue substituted by branch one and branch two at the O-6 position can be any α-mannose residue in the repeating unit, and the arrangement order of branch one and branch two is arbitrary.

3. The mannan derived from Leuconostoc as claimed in claim 2, characterized in that The structure of the repeating unit of the mannan has a repeating unit with the structure shown in structural formula I: Wherein, in structural formula I, Manp is pyranose; n is the number of repeating units, which can be determined by the weight average molecular weight.

4. The mannan derived from Leuconostoc as claimed in claim 1, characterized in that The weight average molecular weight of the mannan derived from Leuconostocsuionicum M1 is 45 KDa to 65 KDa.

5. A method for preparing the mannan derived from Leuconostoc as claimed in claim 1, characterized in that: The preparation steps are as follows: (1) Activation of strains: Transfer the glycerol tube containing the above strain Leuconostoc suionicum M1 to MRS liquid culture medium and activate and culture at 37°C for 24-48 hours; (2) Seed liquid preparation: The activated Leuconostoc suionicum M1 strain was inoculated into MRS liquid culture medium at a rate of 1-5%, and cultured at 30-37° C. for 12-24 h; (3) Fermentation culture: inoculating the above seed liquid into a fermentation medium added with sugarcane molasses or beet molasses at an inoculum rate of 2% to 6%, and culturing at 25 to 40° C. for 24 to 48 hours; the fermentation medium formula: 200 to 350 g / L sugarcane molasses or beet molasses, 0 to 20 g / L yeast extract, 0 to 20 g / L beef extract, pH 5.0 to 9.0, and sterilizing at 121° C. for 15 minutes; (4) Extraction of crude polysaccharides: Take the fermentation broth obtained in step (3), centrifuge at 6000-10000 r / min for 5-10 min, take the centrifugal supernatant, add 3 times the volume of pre-cooled 95% ethanol, let stand at 4°C for 12 h, centrifuge at 4000-8000 r / min for 10-20 min, collect the precipitate, and obtain a crude polysaccharide sample; (5) Dialysis removal: The crude polysaccharide sample obtained in step (4) was dissolved in distilled water, trichloroacetic acid solution was added to a final concentration of 14%, and the mixture was centrifuged at 6000-8000 r / min for 10 min. The supernatant was collected and transferred to a dialysis bag with a molecular weight cutoff range of 8000-14000 Da, and dialyzed with deionized water for 48-72 h, with the water being changed every 12 h. The dialyzed sample was collected and freeze-dried for 48 h to obtain a polysaccharide sample powder; (6) Separation: Dissolve the polysaccharide sample obtained in step (5) in ultrapure water to prepare a polysaccharide solution with a concentration of 5 g / L, and filter it with a 0.45 μm filter membrane; take 5 mL of the polysaccharide solution, add it to a DEAE-52 cellulose anion exchange chromatography column, and use NaCl solutions with concentrations of 0, 0.3, 0.5, and 0.7 mol / L for gradient elution separation, with an eluent flow rate of 0.7 mL / min, and collect one tube every 7 minutes; use the phenol-sulfuric acid method to detect the polysaccharide concentration of the samples in each collection tube, and draw an elution curve, and combine the eluted samples in each tube corresponding to the same separation peak, freeze-dry for 48 hours, and weigh them to calculate the mannan yield per liter of fermentation broth; (7) Purification: The mannan obtained in step (6) was dissolved in ultrapure water to prepare a polysaccharide solution with a concentration of 3 mg / mL, and filtered with a 0.45 μm filter membrane; 3 mL of the polysaccharide solution was added to a Sepharose CL-6B agarose gel chromatography column, eluted with deionized water at a flow rate of 0.5 mL / min, and the eluate was collected at 7 min / tube. The OD490 of each tube was detected by the phenol-sulfuric acid method, and an elution curve was drawn; According to the elution curve, single peak components are combined and collected, and dialyzed for 36 to 48 hours according to the method in step (6), and the dialysate is collected and vacuum freeze-dried to obtain a purified mannan sample.

6. The method according to claim 5, characterized in that In step (3), the sugarcane molasses or beet molasses added to the culture medium is pre-treated with pectinase, and the enzymatic hydrolysis conditions are: pectinase dosage 50,000 to 100,000 U / L, enzymatic hydrolysis temperature 35 to 45° C., and enzymatic hydrolysis time 30 to 120 min.

7. Use of the mannan according to any one of claims 1 to 6 in the preparation of an anti-pathogenic bacterial biofilm preparation.

8. Use of the mannan according to any one of claims 1 to 6 in the preparation of an intestinal prebiotic preparation, wherein the use can promote the proliferation of probiotic bacteria and reduce the number of bacteria associated with inflammatory bowel disease and / or colorectal cancer and / or depression and / or irritable bowel syndrome.

9. Use of the mannan according to any one of claims 1 to 6 in the efficient production of highly active fermented milk products, wherein the application can be a biological preservative.

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