WO2023066163A1 - Exopolysaccharide separated from lactobacillus delbrueckii and streptococcus thermophilus fermented yoghourt and application thereof - Google Patents

Exopolysaccharide separated from lactobacillus delbrueckii and streptococcus thermophilus fermented yoghourt and application thereof Download PDF

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WO2023066163A1
WO2023066163A1 PCT/CN2022/125459 CN2022125459W WO2023066163A1 WO 2023066163 A1 WO2023066163 A1 WO 2023066163A1 CN 2022125459 W CN2022125459 W CN 2022125459W WO 2023066163 A1 WO2023066163 A1 WO 2023066163A1
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exopolysaccharide
neutral
yogurt
freeze
yoghurt
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Chinese (zh)
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刘冬梅
唐俊
徐仪倩
吴佳娟
熊杰
余佳佳
肖兰芳
林瑾
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华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of food processing, and in particular relates to an exopolysaccharide isolated from fermented yoghurt by Lactobacillus delbrueckii and Streptococcus thermophilus and application thereof.
  • Colitis is an inflammatory bowel disease (IBD), divided into ulcerative colitis (UC) and Crohn's disease (CD), an autoimmune disease associated with immune disturbances, genetic predisposition, and disturbances in the microbial population disease.
  • IBD inflammatory bowel disease
  • CD Crohn's disease
  • UC ulcerative colitis
  • CD Crohn's disease
  • UC chronic continuous diffuse inflammatory disease caused by changes in the structure of the intestinal flora and the permeability of the intestinal barrier, which occurs in the colonic mucosa and is associated with the rectum.
  • the incidence in China has continued to increase, mainly affecting the colonic mucosa, and the lesions mostly occur in the distal colon.
  • UC lasts for a long time and is prone to repeated attacks. It is a global disease.
  • Ulcerative colitis is difficult to cure because it is prone to repeated attacks and has a long course of disease.
  • the treatment is still mainly based on anti-inflammation and immune regulation, supplemented by treatments such as biological agents or non-drug treatments such as hyperbaric oxygen and stem cell transplantation, and even surgery.
  • Commonly used drugs fall into the following 6 categories:
  • Aminosalicylic acids such as sulfasalazine (SASP), 5-aminosalicylic acid and mesalamine.
  • SASP sulfasalazine
  • 5-aminosalicylic acid 5-aminosalicylic acid
  • mesalamine mesalamine
  • Glucocorticoids have powerful anti-inflammatory effects, but due to side effects such as metabolic disorders and osteoporosis, they are generally only used in the acute or severe stages.
  • Immunosuppressants which are often used in patients who are not suitable for the first two drugs, can alleviate the active stage of UC, but because of their high liver and kidney toxicity, they are generally only used as an auxiliary effect in clinical practice.
  • Infliximab Infliximab
  • IFX Infliximab
  • Probiotics can improve intestinal flora imbalance and promote nutrient absorption in UC patients. However, the effect of treating UC symptoms is average, and it is often used in combination with other drugs.
  • Anti-infective drugs are suitable for bacterial infection or severe patients, and have the functions of inhibiting intestinal anaerobic bacteria, promoting fistula healing and preventing recurrence, but long-term use will also cause drug resistance and side effects.
  • Some polysaccharides can alleviate colonic mucosal erosion, reduce ulcer area, inhibit colonic mucosal congestion and edema, alleviate weight loss in UC mice, and reduce the incidence of diarrhea and bloody stools.
  • polysaccharides can effectively regulate the intestinal flora, increase the number of Lactobacilli, reduce the number of Enterobacteriaceae and cocci, increase the volatile fatty acids in the intestinal content, and regulate the intestinal microecology, thereby treating UC. Therefore, polysaccharides can treat UC by anti-inflammation, regulating intestinal immunity and improving intestinal flora. Polysaccharides have multiple targets for the treatment of UC, and because of their advantages such as less side effects and less repeated therapeutic effects, they have become a research hotspot in recent years.
  • the object of the present invention is to provide an exopolysaccharide isolated from fermented yoghurt with Lactobacillus delbrueckii and Streptococcus thermophilus and its application.
  • the invention aims at the shortcomings of the current medicines for treating ulcerative colitis, such as large side effects and high price, and provides a safe, efficient, and small side effect natural medicine for treating ulcerative colitis.
  • the preparation method and application are provided.
  • yogurt neutral exopolysaccharide the yogurt neutral exopolysaccharide is inoculated into skim milk by Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 for fermentation and culture , to obtain fermented yoghurt; the yoghurt is subjected to centrifugation, alcohol precipitation, protein removal, and ion-exchange column elution, and the eluent is water to obtain a neutral exopolysaccharide with a molecular weight of 32063Da.
  • the monosaccharide composition of the neutral exopolysaccharide is galactose and glucose, and the molar ratio is 42.04:57.96.
  • the glycosidic bond of the neutral exopolysaccharide is composed of t-Glcp, 4-Galp, 4-Glcp, 3,4-Glcp and 4,6-Glcp, and the relative molar ratio is 17.456:37.035:37.035:1.476 :6.998.
  • a method for preparing yogurt neutral exopolysaccharide characterized in that it comprises the steps of:
  • Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 were inoculated into MRS medium respectively, and cultured at 37°C for 24 hours; after that, secondary activation was carried out, and the inoculum size was 5-10 %v/v; the seed liquid is obtained after activation, and it is left to stand at 37°C for later use;
  • centrifugation the fermented yoghurt described in centrifugation step (3), collects the supernatant
  • Alcohol precipitation add absolute ethanol to the supernatant described in step (4), let it stand, centrifuge to take the precipitate, collect the precipitate and dissolve it in water to obtain a crude polysaccharide liquid;
  • step (6) The crude exopolysaccharide described in step (6) is configured into a 10-30 mg/mL solution, separated and purified by a DEAE-Cellulose 52 ion exchange column and a Sephadex G-150 gel column, concentrated under reduced pressure, and vacuum freeze-dried , to obtain neutral exopolysaccharide freeze-dried powder.
  • the volume ratio of the bacteria slime and the lyoprotectant in step (2) is 1: (1-3).
  • the fermentation conditions in step (3) are as follows: the initial inoculation amount is (2-4) ⁇ 10 6 CFU/mL, the inoculation ratio is (1-3):1, fermented at 42 ⁇ 5°C for 8-10 hours, Ripe at 4-6°C for 12-14 hours.
  • the centrifugation conditions in step (4) are: centrifugal force of 10,000-15,000 g, centrifugation temperature of 4° C., and centrifugation time of 5-10 minutes.
  • the volume ratio of supernatant to absolute ethanol in step (5) is 1:(4-6); the volume ratio of crude polysaccharide solution to Sevag reagent in step (6) is (4-5):1.
  • the present invention has the following advantages and beneficial effects:
  • the polysaccharide was found to be in a sheet-like structure by scanning electron microscopy, and the results of the Congo red experiment showed that the neutral polysaccharide may have a three-dimensional helical structure.
  • the neutral exopolysaccharide obtained in the present invention has a good therapeutic effect on colitis as confirmed by mouse experiments.
  • DEAE-Cellulose 52 anion exchange column chromatography is based on the principle of ion exchange chromatography.
  • the matrix is composed of charged resin or cellulose.
  • the anion exchange matrix cannot be combined with uncharged neutral polysaccharides, thus being deionized Water washes it off.
  • Lactobacillus delbrueckii (Lactobacillus delbrueckii) DMLD-H1
  • the preservation number is GDMCC NO.60645, which was preserved in Guangdong Microbial Culture Collection Center on April 16, 2019, referred to as GDMCC, address: 100 Xianlie Middle Road, Guangzhou Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 1 Courtyard.
  • the strain has been disclosed in Chinese patent CN110607255A.
  • Streptococcus thermophilus DMST-H2 with the preservation number GDMCC NO.60642, was preserved in the Guangdong Microbial Culture Collection Center on April 16, 2019, referred to as GDMCC, address: No. 100 Xianlie Middle Road, Guangzhou 5th Floor, Building 59, Guangdong Institute of Microbiology.
  • the strain has been disclosed in Chinese patent CN110607253A.
  • Figure 1 Sephadex G-150 gel column purification elution curve of yogurt neutral exopolysaccharide.
  • Fig. 2 GPC high performance liquid chromatogram of yogurt neutral exopolysaccharide.
  • Fig. 3 High performance liquid chromatogram of yogurt neutral exopolysaccharide monosaccharide composition.
  • Fig. 4 1 H NMR spectrum of yogurt neutral exopolysaccharide.
  • Fig. 6 IR spectrum of yogurt neutral exopolysaccharide.
  • Fig. 7 Scanning electron micrograph of yogurt neutral exopolysaccharide (A: 800 ⁇ , B: 2000 ⁇ ).
  • FIG. 9 Effect of yogurt neutral exopolysaccharides on body weight and disease activity index (DAI) of DSS-induced colitis mice (A: body weight change of mice; B: DAI score of mice). p* ⁇ 0.05, p** ⁇ 0.01, p*** ⁇ 0.001 indicate statistically significant difference, and the confidence interval is 95%.
  • Figure 10 Effect of yogurt neutral exopolysaccharides on serum cytokine levels in DSS-induced colitis mice (A: TNF- ⁇ level in mouse serum; B: IL-10 level in mouse serum; C: mice IL-1 ⁇ levels in serum). p* ⁇ 0.05, p** ⁇ 0.01, p*** ⁇ 0.001 indicate statistically significant difference, and the confidence interval is 95%.
  • Example 1 Isolation of exopolysaccharide from yoghurt fermented by Lactobacillus delbrueckii and Streptococcus thermophilus
  • the skim milk formula is (by mass fraction): 12% skim milk powder, 8% sucrose.
  • the formulation of the lyoprotectant is (in mass fraction): 10% skimmed milk powder, 10% trehalose.
  • Sevag reagent is obtained by mixing chloroform and n-butanol, the volume ratio of chloroform and n-butanol is 5:1.
  • MRS medium formula (in parts by weight): 0.9 part of casein digest, 0.4 part of yeast extract, 1.8 part of glucose, 0.15 part of triammonium citrate, 0.05 part of magnesium sulfate, 0.75 part of beef extract, 0.15 part of dipotassium hydrogen phosphate 0.45 parts of sodium acetate, 0.2 parts of Tween 80, 0.02 parts of manganese sulfate, and the rest is water.
  • Fermentation medium formula (in parts by weight): 0.9 parts of casein digest, 0.4 parts of yeast extract, 1.6 parts of glucose, 0.15 parts of triammonium citrate, 0.055 parts of magnesium sulfate, 0.8 parts of beef extract, 0.15 parts of dipotassium hydrogen phosphate 0.45 parts of sodium acetate, 0.2 parts of Tween 80, 0.9 parts of soybean protein peptide, 0.015 parts of ascorbic acid, 0.25 parts of manganese sulfate, and the rest is water.
  • Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 were inoculated into MRS medium respectively, and cultured at 37°C for 24 hours; after that, secondary activation was carried out, and the inoculum size was 5-10 %v/v; the seed liquid is obtained after activation, and it is left to stand at 37°C for later use;
  • step (3) Preparation of fermented yoghurt: 12% skim milk powder and 8% sucrose are mixed to obtain skim milk, heated in a water bath at 85°C for 15 minutes, cooled to 40°C to 42°C, added to the bacteria powder described in step (2) for fermentation,
  • the fermentation conditions are as follows: the initial inoculation amount is 2 ⁇ 10 6 CFU/mL, the inoculation ratio is 1:1, 8 hours of fermentation at 42°C, and post-ripening at 4°C for 12 hours;
  • centrifugation centrifuge the fermented yoghurt in step (3), collect the supernatant, the centrifugation conditions are: centrifugal force 10000g, centrifugation temperature 4°C, centrifugation time 10min;
  • the polysaccharide liquid in different tubes is collected, concentrated under reduced pressure, and vacuum freeze-dried to obtain a neutral exopolysaccharide freeze-dried powder.
  • DEAE-Cellulose 52 ion exchange column and Sephadex G-150 gel column are commonly used separation columns for polysaccharides.
  • the connection method is connected in series to realize one-step purification.
  • the molecular weight uniformity and molecular weight of the neutral exopolysaccharide prepared in step (6) of Example 1 were measured by HPLC high performance gel permeation chromatography (Waters 1525 gel chromatograph).
  • the chromatographic column is TSK G5000 PWXL (6 ⁇ m, 7.8 ⁇ 300mm) and TSK G3000 PWXL (6 ⁇ m, 7.8 ⁇ 300mm) used in series
  • the detector is Waters 2414 differential refractive index detector (RID)
  • the column temperature is 35°C
  • the injection volume is 10 ⁇ L.
  • the mobile phase was 0.02mol/L dipotassium hydrogen phosphate buffer solution, and the flow rate was 0.6mL/min.
  • Sample pretreatment process the yogurt neutral exopolysaccharide in step (6) of Example 1, the specific steps are as follows: take a clean chromatographic bottle, weigh 5 mg of each polysaccharide sample, add TFA acid solution, heat at 121 °C for 2 hours, blow with nitrogen Dry. Add methanol to wash and dry, repeat 2-3 times. Add sterile water to dissolve and transfer to a chromatographic bottle for testing.
  • Liquid sample extraction take an appropriate amount of supernatant, blow dry with nitrogen. The subsequent steps are consistent with solid sample extraction.
  • the chromatographic system adopts Thermo ICS5000+ ion chromatography system (ICS5000+, (Thermo Fisher Scientific, USA), adopts Dionex TM CarboPac TM PA10 (250*4.0mm, 10 ⁇ m) liquid chromatography column, and the injection volume is 20 ⁇ L.
  • Phase A H 2 O
  • mobile phase B 100mol/L NaOH
  • the column temperature is 30°C
  • the monosaccharide components are analyzed and detected by an electrochemical detector.
  • the specific gradient and data of the mobile phase are shown in the table below.
  • Derivatization of polysaccharide samples Weigh 10 mg of the purified sample, add 1 mL of deionized water to dissolve, then add 1 mL of 100 mg/mL carbodiimide, and react for 2 hours. Then add 1 mL of 2mol/L imidazole, divide it into two parts, add 1 mL of 30 mg/mL NaBH 4 and NaBD 4 of the same volume and concentration, and add 100 ⁇ L of glacial acetic acid after 3 hours to terminate the reaction. The samples were dialyzed for 48 hours, and then freeze-dried and methylated.
  • a quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) from Aiglent Corporation of the United States was used, equipped with an electron impact ion source (EI) and a Mass Hunter workstation. Using an electron impact ion source (EI), analytes were detected in full scan (SCAN) mode with a mass scan range (m/z) of 30-600.
  • EI electron impact ion source
  • the five derivatives are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol (1,5-di-O-acetyl-2 ,3,4,6-tetra-O-methylglucitol), 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol (1,4,5-tri- O-acetyl-2,3,6-tri-O-methyl galactitol), 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol (1,4 ,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol), 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl glucitol (1,3,4,5-Tetra-O-acetyl-2,6-di-O-methylglucitol), 1,4,5,6-tetra-O-O-O-methyl g
  • the glycosidic bond connection mode of the neutral exopolysaccharide includes t-Glcp, 4-Galp, 4-Glcp, 3,4-Glcp and 4,6-Glcp, and the relative molar ratio is 17.456: 37.035:37.035:1.476:6.998.
  • the branching degree (DB) value of yogurt neutral exopolysaccharide is calculated to be 25.93%, wherein NT refers to the terminal residue t-Glcp (1 ⁇ , NB refers to branched residues ⁇ 3,4)-Glcp(1 ⁇ and ⁇ 4,6)-Glcp(1 ⁇ , NL refers to linear residues ⁇ 4)-Galp(1 ⁇ and 4)-Glcp(1 ⁇ quantity.
  • the 1 H NMR spectrum of yogurt neutral exopolysaccharide is shown in FIG. 4 .
  • the distribution of ectopic hydrogen was between ⁇ 4.7 ⁇ 5.5ppm, indicating that yogurt neutral exopolysaccharides contained ⁇ - and ⁇ -glucosidic linkages.
  • 13 C NMR can reflect the residual amount of polysaccharide in the sample.
  • the number of polysaccharide residues and their related configurations can be analyzed and determined by the number of positive carbon peaks with chemical shifts between 95 and 110 ppm.
  • the 13 C NMR spectrum of yogurt neutral exopolysaccharide is shown in FIG. 5 .
  • Heretic carbons were found at ⁇ 103.65, 102.89, 96.41, 95.87 and 95.72ppm, indicating that yogurt neutral exopolysaccharides contained 5 kinds of glycosidic bonds, consistent with the methylation results. More detailed information on the location and sequence of these 5 glycosidic bonds will be elucidated in the future.
  • the vibration at 1649.14 cm may be related to the symmetric stretching of the carboxyl group, and the vibration at 1426.36 cm may be related to the bending vibration of C and H.
  • the absorption peak in the region of 1200 ⁇ 1000cm -1 may be caused by the stretching vibration of COH and COC.
  • the vibration at 1022.27 cm -1 may be related to the bending vibration of CO.
  • the absorption peak at 924.87cm -1 indicated that there might be a furan ring in the neutral exopolysaccharide structure of yogurt.
  • Scanning electron microscopy is a commonly used method to observe the morphology of polysaccharides and determine the types of polysaccharides. It has the advantages of simple operation, intuitive results and high resolution, so it is widely used in food science, chemistry, materials and biology.
  • Congo red is a kind of acid dye, and it can form complexes with the polysaccharide that has triple helix result, and the maximum absorption wavelength of complexes shifts compared with Congo red, takes by weighing 2mg embodiment 1 step (6) neutrophils
  • For the exopolysaccharide component dissolve in 2 mL of distilled water, add 2 mL of 80 ⁇ mol/L Congo red reagent, gradually add 1 mol/L of sodium hydroxide, so that the concentration of sodium hydroxide in the solution rises from 0.0 mol/L to 0.5 mol/L, and then Ultraviolet spectrum scanning was carried out to measure the maximum absorption wavelength under different NaOH concentrations.
  • mice Male, 4-5 weeks old, were collected from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. All mice were acclimated to standard diet (23-25°C and 12h light/dark cycle) for 7 days. The mouse colitis model was activated with 3.5% (w/v) DSS.
  • SASP group positive control mice oral sulfasalazine (SASP) solution 100mg/kg, give 3.5% DSS solution, on the 15th day, all mice were euthanized .
  • DAI disease activity index
  • TNF- ⁇ is a cytokine that can directly kill tumor cells without obvious toxicity to normal cells.
  • Figure 10 (A) is the TNF- ⁇ level in the serum of each group of mice.
  • TNF- ⁇ level of the model group (Model) is significantly higher than that of the blank group (Control), and after polysaccharide treatment, it can Reduces levels of TNF- ⁇ .
  • IL-10 is an important inhibitory cytokine that can suppress inflammatory responses and limit excessive immune responses. It can be seen from Figure 10(B) that the IL-10 level of the model group (Model) was significantly lower than that of the blank group (Control). EPS) IL-10 levels were significantly increased.
  • IL-1 ⁇ is one of the important pro-apoptotic and pro-inflammatory cytokines. It can be seen from Figure 10(C) that the IL-1 ⁇ level of the model group (Model) is significantly higher than that of the blank group (Control).
  • L-EPS low-dose polysaccharide group
  • H-EPS high-dose polysaccharide group

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Abstract

The present invention relates to the field of food processing. Disclosed are an exopolysaccharide separated from Lactobacillus delbrueckii and Streptococcus thermophilus fermented yoghourt and an application thereof. The neutral exopolysaccharide of the present invention is extracted from Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 fermented yoghourt for the first time, and has uniform components, a novel structure, a weight-average molecular weight of 32063 Da, and a monosaccharide composition molar ratio of galactose: glucose of 42.04: 57.96. A scanning electron microscope is adopted to find that the polysaccharide shows a sheet-shaped structure, and a Congo red experiment result shows that the neutral polysaccharide may have a three-dimensional spiral structure. Animal experiments prove that the neutral exopolysaccharide is good in colitis treatment effect and has a wide application prospect in the fields of food and medicines.

Description

一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用A kind of exopolysaccharide isolated from Lactobacillus delbrueckii and Streptococcus thermophilus fermented yogurt and its application 技术领域technical field
本发明属于食品加工领域，具体涉及一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用。The invention belongs to the field of food processing, and in particular relates to an exopolysaccharide isolated from fermented yoghurt by Lactobacillus delbrueckii and Streptococcus thermophilus and application thereof.
背景技术Background technique
结肠炎是一种炎症性肠病(IBD)，分为溃疡性结肠炎(UC)和克罗恩病(CD)，是一种与免疫紊乱、遗传易感性和微生物种群紊乱相关的自身免疫性疾病。在全球范围内，UC的患病率高于CD。UC是一种慢性连续性弥漫性炎症疾病，是由于肠道菌群结构改变与肠屏障通透性改变引起的，发生于结肠黏膜，并与直肠相关。近几年，中国的发病率持续增加，主要作用于结肠黏膜，病变多发生于结肠远端。UC持续时间长，且容易反复发作，是一种全球性疾病。Colitis is an inflammatory bowel disease (IBD), divided into ulcerative colitis (UC) and Crohn's disease (CD), an autoimmune disease associated with immune disturbances, genetic predisposition, and disturbances in the microbial population disease. Globally, the prevalence of UC is higher than that of CD. UC is a chronic continuous diffuse inflammatory disease caused by changes in the structure of the intestinal flora and the permeability of the intestinal barrier, which occurs in the colonic mucosa and is associated with the rectum. In recent years, the incidence in China has continued to increase, mainly affecting the colonic mucosa, and the lesions mostly occur in the distal colon. UC lasts for a long time and is prone to repeated attacks. It is a global disease.
溃疡性结肠炎，因易反复发作，病程长，较难治愈。目前治疗仍以抗炎和免疫调节为主，辅以生物制剂等治疗或高压氧、干细胞移植等非药物治疗，甚至借助外科手术。常用药物有以下6类：Ulcerative colitis is difficult to cure because it is prone to repeated attacks and has a long course of disease. At present, the treatment is still mainly based on anti-inflammation and immune regulation, supplemented by treatments such as biological agents or non-drug treatments such as hyperbaric oxygen and stem cell transplantation, and even surgery. Commonly used drugs fall into the following 6 categories:
(1)氨基水杨酸类，如柳氮磺吡啶(Sulfasalazine，SASP)、5-氨基水杨酸和美沙拉嗪。其中SASP具有较好的疗效和低廉的价格，是目前在UC治疗上使用最广的药物，长期服用机体会产生耐药性，且可能导致血液、肝肾、消化道等系统损伤和叶酸缺乏等不良反应。(1) Aminosalicylic acids, such as sulfasalazine (SASP), 5-aminosalicylic acid and mesalamine. Among them, SASP has good curative effect and low price, and is currently the most widely used drug in the treatment of UC. Long-term use will produce drug resistance, and may cause damage to the blood, liver, kidney, digestive tract and other systems and folic acid deficiency, etc. Adverse reactions.
(2)糖皮质激素类，具有强大的抗炎作用，但因代谢紊乱、骨质疏松等副作用，一般只用于急性期或重症期。(2) Glucocorticoids have powerful anti-inflammatory effects, but due to side effects such as metabolic disorders and osteoporosis, they are generally only used in the acute or severe stages.
(3)免疫抑制剂类，常用于不适用前两种药物的患者，可缓解UC活动期，但因其肝、肾毒性较大，临床一般只作辅助作用。(3) Immunosuppressants, which are often used in patients who are not suitable for the first two drugs, can alleviate the active stage of UC, but because of their high liver and kidney toxicity, they are generally only used as an auxiliary effect in clinical practice.
(4)生物制剂类，能直接作用于肿瘤坏死因子α(Tumor necrosis factor alpha，TNF-α)、细胞黏附分子等靶点，特别是单克隆抗体治疗剂，更是目前药物热点。常用的英夫昔单抗(Infliximab，IFX)，对重症患者十分有效，但因价格昂贵，且可能伴有白细胞、中性粒细胞减少和过敏等副作用，使用受到较大限制。(4) Biological agents, which can directly act on targets such as tumor necrosis factor alpha (TNF-α), cell adhesion molecules, etc., especially monoclonal antibody therapeutic agents, are currently hot spots in drugs. The commonly used Infliximab (Infliximab, IFX) is very effective for severe patients, but its use is greatly restricted due to its high price and possible side effects such as leukopenia, neutropenia, and allergies.
(5)益生菌类，可改善UC患者肠道菌群失调，促进营养吸收。但治疗UC症状效果一般，常和其他药物联用。(5) Probiotics can improve intestinal flora imbalance and promote nutrient absorption in UC patients. However, the effect of treating UC symptoms is average, and it is often used in combination with other drugs.
(6)抗感染药物，适用于细菌感染或重症患者，具有抑制肠道厌氧菌、促进瘘管愈合和预防复发等作用，但长期使用也会出现耐药性和副作用。(6) Anti-infective drugs are suitable for bacterial infection or severe patients, and have the functions of inhibiting intestinal anaerobic bacteria, promoting fistula healing and preventing recurrence, but long-term use will also cause drug resistance and side effects.
虽然在目前UC的治疗上，传统西药和新型生物制剂都对临床症状表现出较好的缓解作用，但也有副作用大、价格昂贵等问题。因此寻找副作用小，价格适宜，且对UC有显著治疗效果的新药物或干预作用的功能食品已成为迫切需求。Although in the current treatment of UC, both traditional western medicine and new biological agents have shown a good effect on relieving clinical symptoms, but there are also problems such as large side effects and high price. Therefore, it has become an urgent need to look for new drugs or functional foods with less side effects, affordable prices, and significant therapeutic effects on UC.
一些多糖可缓解结肠黏膜糜烂，减少溃疡的面积，抑制结肠黏膜充血水肿，缓解UC小鼠体重下降，减少泄泻及血便的发生率。研究表明，其机制可能为能下调TNF-α、IL-6，上调IL-10等炎症相关因子的表达，调节结肠中EGF、EGF-β含量促进其黏膜修复，降低结肠上皮细胞酶Caspase-3及Caspase-8的表达，有效控制上皮细胞凋亡，促进黏膜修复，提高肠道Occludin、ZO-1蛋白表达增强黏膜屏障能力。此外，多糖可有效调节肠道菌群，增加乳酸杆菌等数量，减少肠杆菌及球菌数量，增加肠内容物挥发性脂肪酸，调节肠道微生态，从而治疗UC。因此多糖可通过抗炎、调节肠道免疫及改善肠道菌群等手段来治疗UC。多糖具有多个治疗UC的靶点、又因为其副作用小与治疗效果不易反复等优势成为近年来研究的热点。Some polysaccharides can alleviate colonic mucosal erosion, reduce ulcer area, inhibit colonic mucosal congestion and edema, alleviate weight loss in UC mice, and reduce the incidence of diarrhea and bloody stools. Studies have shown that the mechanism may be down-regulating the expression of TNF-α and IL-6, up-regulating the expression of inflammatory factors such as IL-10, regulating the content of EGF and EGF-β in the colon to promote its mucosal repair, and reducing the colonic epithelial cell enzyme Caspase-3. And the expression of Caspase-8, effectively control the apoptosis of epithelial cells, promote mucosal repair, increase the expression of intestinal Occludin, ZO-1 protein and enhance the ability of mucosal barrier. In addition, polysaccharides can effectively regulate the intestinal flora, increase the number of Lactobacilli, reduce the number of Enterobacteriaceae and cocci, increase the volatile fatty acids in the intestinal content, and regulate the intestinal microecology, thereby treating UC. Therefore, polysaccharides can treat UC by anti-inflammation, regulating intestinal immunity and improving intestinal flora. Polysaccharides have multiple targets for the treatment of UC, and because of their advantages such as less side effects and less repeated therapeutic effects, they have become a research hotspot in recent years.
发明内容Contents of the invention
针对于微生物多糖应用问题，本发明的目的是提供一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用。本发明针对目前治疗溃疡性结肠炎药物的副作用大、价格昂 贵等不足，提供了一种安全、高效、副作用小的天然治疗溃疡性结肠炎药物的制备方法和应用。Aiming at the application of microbial polysaccharides, the object of the present invention is to provide an exopolysaccharide isolated from fermented yoghurt with Lactobacillus delbrueckii and Streptococcus thermophilus and its application. The invention aims at the shortcomings of the current medicines for treating ulcerative colitis, such as large side effects and high price, and provides a safe, efficient, and small side effect natural medicine for treating ulcerative colitis. The preparation method and application.
本发明的目的通过如下技术方案实现：The purpose of the present invention is achieved through the following technical solutions:
一种酸奶中性胞外多糖，所述酸奶中性胞外多糖是将德氏乳杆菌(Lactobacillus delbrueckii)DMLD-H1和嗜热链球菌(Streptococcus thermophilus)DMST-H2接种至脱脂乳中进行发酵培养，获得发酵酸奶；对所述酸奶进行离心、醇沉、除蛋白、经离子交换柱洗脱，洗脱液为水，得到分子量为32063Da的中性胞外多糖。A yogurt neutral exopolysaccharide, the yogurt neutral exopolysaccharide is inoculated into skim milk by Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 for fermentation and culture , to obtain fermented yoghurt; the yoghurt is subjected to centrifugation, alcohol precipitation, protein removal, and ion-exchange column elution, and the eluent is water to obtain a neutral exopolysaccharide with a molecular weight of 32063Da.
优选地，所述中性胞外多糖的单糖组成为半乳糖和葡萄糖，摩尔比为42.04:57.96。Preferably, the monosaccharide composition of the neutral exopolysaccharide is galactose and glucose, and the molar ratio is 42.04:57.96.
优选地，所述中性胞外多糖的糖苷键由t-Glcp，4-Galp，4-Glcp，3,4-Glcp和4,6-Glcp组成，相对摩尔比为17.456:37.035:37.035:1.476:6.998。Preferably, the glycosidic bond of the neutral exopolysaccharide is composed of t-Glcp, 4-Galp, 4-Glcp, 3,4-Glcp and 4,6-Glcp, and the relative molar ratio is 17.456:37.035:37.035:1.476 :6.998.
一种制备酸奶中性胞外多糖的方法，其特征在于，包括如下步骤：A method for preparing yogurt neutral exopolysaccharide, characterized in that it comprises the steps of:
(1)菌种活化：取德氏乳杆菌DMLD-H1和嗜热链球菌DMST-H2分别接种至MRS培养基里，37℃静置培养24h；之后进行二级活化，接种量为5～10％v/v；活化后得到种子液，于37℃条件下静置备用；(1) Strain activation: Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 were inoculated into MRS medium respectively, and cultured at 37°C for 24 hours; after that, secondary activation was carried out, and the inoculum size was 5-10 %v/v; the seed liquid is obtained after activation, and it is left to stand at 37°C for later use;
(2)菌粉的制备：将种子液接种到发酵培养基中进行扩大培养，接种浓度为5～10％v/v，37℃恒温条件下静置培养24～30h后离心，生理盐水洗2～3次，离心后收集菌泥，添加冻干粉保护剂后，经-80℃预冻4h、冷冻干燥48h，得到菌粉；(2) Preparation of bacterial powder: inoculate the seed liquid into the fermentation medium for expanded cultivation, the inoculation concentration is 5-10% v/v, and then centrifuge after standing for 24-30 hours at a constant temperature of 37°C, wash with normal saline for 2 ~3 times, collect the bacteria sludge after centrifugation, add freeze-dried powder protective agent, pre-freeze at -80°C for 4 hours, and freeze-dry for 48 hours to obtain the bacteria powder;
(3)发酵酸奶的准备：将12～15％脱脂乳粉和8～10％蔗糖混合得到脱脂乳，85℃水浴加热15min，冷却至40℃～42℃后接入步骤(2)所述菌粉进行发酵，得到发酵酸奶；(3) Preparation of fermented yogurt: Mix 12-15% skim milk powder and 8-10% sucrose to obtain skim milk, heat in a water bath at 85°C for 15 minutes, cool to 40°C-42°C, and insert the bacteria described in step (2). Flour is fermented to obtain fermented yoghurt;
(4)离心：离心步骤(3)所述发酵酸奶，收集上清液；(4) centrifugation: the fermented yoghurt described in centrifugation step (3), collects the supernatant;
(5)醇沉：向步骤(4)所述上清液中加入无水乙醇，静置，离心取沉淀，收集沉淀溶于水得到粗多糖液；(5) Alcohol precipitation: add absolute ethanol to the supernatant described in step (4), let it stand, centrifuge to take the precipitate, collect the precipitate and dissolve it in water to obtain a crude polysaccharide liquid;
(6)除蛋白：向步骤(5)中所得粗多糖液中加入Sevag试剂，室温下置于摇床震荡混匀，使蛋白充分吸附在有机相中，然后离心，保留水相，重复操作直至蛋白完全除去，将收集的水相进行透析冻干，得到粗胞外多糖；(6) Protein removal: add Sevag reagent to the crude polysaccharide solution obtained in step (5), place on a shaker at room temperature and shake to mix, so that the protein is fully adsorbed in the organic phase, then centrifuge, retain the aqueous phase, and repeat the operation until The protein is completely removed, and the collected aqueous phase is dialyzed and freeze-dried to obtain crude exopolysaccharide;
(7)将步骤(6)所述粗胞外多糖配置成10～30mg/mL溶液，经DEAE-Cellulose 52离子交换柱和Sephadex G-150凝胶柱分离纯化，减压浓缩，真空冷冻干燥后，得到中性胞外多糖冻干粉。(7) The crude exopolysaccharide described in step (6) is configured into a 10-30 mg/mL solution, separated and purified by a DEAE-Cellulose 52 ion exchange column and a Sephadex G-150 gel column, concentrated under reduced pressure, and vacuum freeze-dried , to obtain neutral exopolysaccharide freeze-dried powder.
优选地，步骤(2)所述菌泥和冻干保护剂体积比为1：(1～3)。Preferably, the volume ratio of the bacteria slime and the lyoprotectant in step (2) is 1: (1-3).
优选地，步骤(3)所述发酵条件为：初始接菌量为(2～4)×10 6CFU/mL，接种比例为(1～3):1，42±5℃发酵8～10h，4～6℃后熟12～14h。 Preferably, the fermentation conditions in step (3) are as follows: the initial inoculation amount is (2-4)×10 6 CFU/mL, the inoculation ratio is (1-3):1, fermented at 42±5°C for 8-10 hours, Ripe at 4-6°C for 12-14 hours.
优选地，步骤(4)所述离心条件为：离心力为10000～15000g，离心温度4℃，离心时间5～10min。Preferably, the centrifugation conditions in step (4) are: centrifugal force of 10,000-15,000 g, centrifugation temperature of 4° C., and centrifugation time of 5-10 minutes.
优选地，步骤(5)所述上清液与无水乙醇体积比为1：(4～6)；步骤(6)所述粗多糖液与Sevag试剂体积比为(4～5):1。Preferably, the volume ratio of supernatant to absolute ethanol in step (5) is 1:(4-6); the volume ratio of crude polysaccharide solution to Sevag reagent in step (6) is (4-5):1.
酸奶中性胞外多糖在食品中的应用。Application of yogurt neutral exopolysaccharide in food.
酸奶中性胞外多糖在治疗结肠炎药物中的应用。Application of yogurt neutral exopolysaccharides in the treatment of colitis drugs.
本发明与现有技术相比，具有如下优点和有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
(1)本发明所得到的中性胞外多糖为首次从德氏乳杆菌(Lactobacillus delbrueckii)DMLD-H1和嗜热链球菌(Streptococcus thermophilus)DMST-H2发酵酸奶中提取分离，具有新的结构，其分子量为32063Da，单糖组成摩尔比为半乳糖(Gal)：葡萄糖(Glc)＝42.04：57.96。(1) The neutral exopolysaccharide obtained by the present invention is extracted and separated from Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 fermented yogurt for the first time, and has a new structure, Its molecular weight is 32063Da, and the molar ratio of monosaccharide composition is galactose (Gal): glucose (Glc) = 42.04: 57.96.
(2)采用红外、甲基化和NMR分析多糖的结构由t-Glcp，4-Galp，4-Glcp，3,4-Glcp和4,6-Glcp(相对摩尔比＝17.456:37.035:37.035:1.476:6.998)组成。(2) Adopt infrared, methylation and NMR to analyze the structure of polysaccharide by t-Glcp, 4-Galp, 4-Glcp, 3,4-Glcp and 4,6-Glcp (relative molar ratio=17.456:37.035:37.035: 1.476:6.998) composition.
(4)采用扫描电镜发现多糖表现呈薄片状结构，刚果红实验结果表明该中性多糖可能存 在三维螺旋结构。(4) The polysaccharide was found to be in a sheet-like structure by scanning electron microscopy, and the results of the Congo red experiment showed that the neutral polysaccharide may have a three-dimensional helical structure.
(5)经小鼠动物实验证实本发明所得到的中性胞外多糖具有良好的治疗结肠炎效果。(5) The neutral exopolysaccharide obtained in the present invention has a good therapeutic effect on colitis as confirmed by mouse experiments.
DEAE-Cellulose 52阴离子交换柱层析，是基于离子交换层析的原理，基质是由带有电荷的树脂或纤维素组成，阴离子交换基质无法与不带电荷的中性多糖结合，从而被去离子水洗脱下来。DEAE-Cellulose 52 anion exchange column chromatography is based on the principle of ion exchange chromatography. The matrix is composed of charged resin or cellulose. The anion exchange matrix cannot be combined with uncharged neutral polysaccharides, thus being deionized Water washes it off.
菌体：德氏乳杆菌(Lactobacillus delbrueckii)DMLD-H1，保藏编号为GDMCC NO.60645，于2019年4月16日保藏于广东省微生物菌种保藏中心，简称GDMCC，地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所。该菌株已在中国专利CN110607255A中公开。Bacteria: Lactobacillus delbrueckii (Lactobacillus delbrueckii) DMLD-H1, the preservation number is GDMCC NO.60645, which was preserved in Guangdong Microbial Culture Collection Center on April 16, 2019, referred to as GDMCC, address: 100 Xianlie Middle Road, Guangzhou Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 1 Courtyard. The strain has been disclosed in Chinese patent CN110607255A.
嗜热链球菌(Streptococcus thermophilus)DMST-H2，保藏编号为GDMCC NO.60642，于2019年4月16日保藏于广东省微生物菌种保藏中心，简称GDMCC，地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所。该菌株已在中国专利CN110607253A中公开。Streptococcus thermophilus DMST-H2, with the preservation number GDMCC NO.60642, was preserved in the Guangdong Microbial Culture Collection Center on April 16, 2019, referred to as GDMCC, address: No. 100 Xianlie Middle Road, Guangzhou 5th Floor, Building 59, Guangdong Institute of Microbiology. The strain has been disclosed in Chinese patent CN110607253A.
附图说明Description of drawings
构成本发明的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用来解释本发明，并不构成对本发明的不当限定。The drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention.
图1酸奶中性胞外多糖的Sephadex G-150凝胶柱纯化洗脱曲线。Figure 1 Sephadex G-150 gel column purification elution curve of yogurt neutral exopolysaccharide.
图2酸奶中性胞外多糖GPC高效液相色谱图。Fig. 2 GPC high performance liquid chromatogram of yogurt neutral exopolysaccharide.
图3酸奶中性胞外多糖单糖组成高效液相色谱图。Fig. 3 High performance liquid chromatogram of yogurt neutral exopolysaccharide monosaccharide composition.
图4酸奶中性胞外多糖的 1H NMR谱图。 Fig. 4 1 H NMR spectrum of yogurt neutral exopolysaccharide.
图5酸奶中性胞外多糖的 13C NMR谱图。 Fig. 5 13 C NMR spectrum of yogurt neutral exopolysaccharide.
图6酸奶中性胞外多糖的红外光谱图。Fig. 6 IR spectrum of yogurt neutral exopolysaccharide.
图7酸奶中性胞外多糖扫描电镜图(A:800×，B:2000×)。Fig. 7 Scanning electron micrograph of yogurt neutral exopolysaccharide (A: 800×, B: 2000×).
图8酸奶中性胞外刚果红实验图。Figure 8 Experimental diagram of neutral extracellular Congo red in yogurt.
图9酸奶中性胞外多糖对DSS诱导结肠炎小鼠的体重和疾病活动指数(disease activity index,DAI)影响(A：小鼠的体重变化情况；B：小鼠的DAI评分)。p*<0.05,p**<0.01,p***<0.001表示统计学意义上的有显著差异，置信区间取95％。Figure 9 Effect of yogurt neutral exopolysaccharides on body weight and disease activity index (DAI) of DSS-induced colitis mice (A: body weight change of mice; B: DAI score of mice). p*<0.05, p**<0.01, p***<0.001 indicate statistically significant difference, and the confidence interval is 95%.
图10酸奶中性胞外多糖对DSS诱导结肠炎小鼠的血清细胞因子水平影响(A：小鼠血清中的TNF-α水平；B：小鼠血清中的IL-10水平；C：小鼠血清中的IL-1β水平)。p*<0.05,p**<0.01,p***<0.001表示统计学意义上的有显著差异，置信区间取95％。Figure 10 Effect of yogurt neutral exopolysaccharides on serum cytokine levels in DSS-induced colitis mice (A: TNF-α level in mouse serum; B: IL-10 level in mouse serum; C: mice IL-1β levels in serum). p*<0.05, p**<0.01, p***<0.001 indicate statistically significant difference, and the confidence interval is 95%.
具体实施方式Detailed ways
下面结合具体实施例对本发明作进一步具体详细描述，但本发明的实施方式不限于此，对于未特别注明的工艺参数，可参照常规技术进行。The present invention will be described in further detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto, and the process parameters not specifically indicated can be carried out with reference to conventional techniques.
实施例1：从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出胞外多糖Example 1: Isolation of exopolysaccharide from yoghurt fermented by Lactobacillus delbrueckii and Streptococcus thermophilus
脱脂乳配方为(以质量分数计)：12％脱脂乳粉，8％蔗糖。The skim milk formula is (by mass fraction): 12% skim milk powder, 8% sucrose.
冻干保护剂配方为(以质量分数计)：10％脱脂奶粉，10％海藻糖。The formulation of the lyoprotectant is (in mass fraction): 10% skimmed milk powder, 10% trehalose.
Sevag试剂是将氯仿与正丁醇混合得到，氯仿与正丁醇体积比为5：1。Sevag reagent is obtained by mixing chloroform and n-butanol, the volume ratio of chloroform and n-butanol is 5:1.
MRS培养基配方(以重量份数计)：酪蛋白消化物0.9份，酵母膏0.4份，葡萄糖1.8份，柠檬酸三铵0.15份，硫酸镁0.05份，牛肉膏0.75份，磷酸氢二钾0.15份，乙酸钠0.45份，吐温80 0.2份，硫酸锰0.02份，其余为水。MRS medium formula (in parts by weight): 0.9 part of casein digest, 0.4 part of yeast extract, 1.8 part of glucose, 0.15 part of triammonium citrate, 0.05 part of magnesium sulfate, 0.75 part of beef extract, 0.15 part of dipotassium hydrogen phosphate 0.45 parts of sodium acetate, 0.2 parts of Tween 80, 0.02 parts of manganese sulfate, and the rest is water.
发酵培养基配方(以重量份数计)：酪蛋白消化物0.9份，酵母膏0.4份，葡萄糖1.6份，柠檬酸三铵0.15份，硫酸镁0.055份，牛肉膏0.8份，磷酸氢二钾0.15份，乙酸钠0.45份，吐温80 0.2份，大豆蛋白肽0.9份，抗坏血酸0.015份，硫酸锰0.25份，其余为水。Fermentation medium formula (in parts by weight): 0.9 parts of casein digest, 0.4 parts of yeast extract, 1.6 parts of glucose, 0.15 parts of triammonium citrate, 0.055 parts of magnesium sulfate, 0.8 parts of beef extract, 0.15 parts of dipotassium hydrogen phosphate 0.45 parts of sodium acetate, 0.2 parts of Tween 80, 0.9 parts of soybean protein peptide, 0.015 parts of ascorbic acid, 0.25 parts of manganese sulfate, and the rest is water.
(1)菌种活化：取德氏乳杆菌DMLD-H1和嗜热链球菌DMST-H2分别接种至MRS培养基里，37℃静置培养24h；之后进行二级活化，接种量为5～10％v/v；活化后得到种子液，于37℃条件下静置备用；(1) Strain activation: Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 were inoculated into MRS medium respectively, and cultured at 37°C for 24 hours; after that, secondary activation was carried out, and the inoculum size was 5-10 %v/v; the seed liquid is obtained after activation, and it is left to stand at 37°C for later use;
(2)菌粉制备：将活化好的种子液接种到发酵培养基中进行扩培，接种浓度为5.0％(v/v)。在37℃恒温条件下静置培养24h后离心，并用生理盐水洗2～3次。离心后将上清液弃去并收集菌泥，添加冻干粉保护剂(德氏乳杆菌：保护剂为1：3，v/v；嗜热链球菌：保护剂为1：1，v/v)后在-80℃冰箱中预冻4h、之后冷冻干燥48h，得到菌粉；(2) Preparation of bacteria powder: Inoculate the activated seed liquid into the fermentation medium for expansion cultivation, and the inoculation concentration is 5.0% (v/v). After static culture at 37°C for 24 hours, centrifuge and wash with saline for 2-3 times. After centrifugation, the supernatant was discarded and the sludge was collected, and a freeze-dried powder protective agent was added (Lactobacillus delbrueckii: the protective agent was 1:3, v/v; Streptococcus thermophilus: the protective agent was 1:1, v/v v) Pre-freezing in a -80°C refrigerator for 4 hours, and then freeze-drying for 48 hours to obtain bacterial powder;
(3)发酵酸奶的准备：12％脱脂乳粉和8％蔗糖混合得到脱脂乳，经过85℃水浴加热15min，冷却到40℃～42℃，接入步骤(2)所述菌粉进行发酵，发酵条件为：初始接菌量为2×10 6CFU/mL，接种比例为1:1，42℃发酵8h，4℃后熟12h； (3) Preparation of fermented yoghurt: 12% skim milk powder and 8% sucrose are mixed to obtain skim milk, heated in a water bath at 85°C for 15 minutes, cooled to 40°C to 42°C, added to the bacteria powder described in step (2) for fermentation, The fermentation conditions are as follows: the initial inoculation amount is 2×10 6 CFU/mL, the inoculation ratio is 1:1, 8 hours of fermentation at 42°C, and post-ripening at 4°C for 12 hours;
(4)离心：将步骤(3)中发酵酸奶进行离心处理，收集上清液，离心条件为：离心力为10000g，离心温度4℃，离心时间10min；(4) Centrifugation: centrifuge the fermented yoghurt in step (3), collect the supernatant, the centrifugation conditions are: centrifugal force 10000g, centrifugation temperature 4°C, centrifugation time 10min;
(5)醇沉：向上清液加入无水乙醇(上清液：无水乙醇＝1：4，v/v)，静置，离心取沉淀，收集沉淀后溶于水得粗多糖液；(5) Alcohol precipitation: add absolute ethanol to the supernatant (supernatant: absolute ethanol = 1:4, v/v), let stand, centrifuge to take the precipitate, collect the precipitate and dissolve it in water to obtain the crude polysaccharide liquid;
(6)除蛋白：向步骤(5)中所得的粗多糖液加入Sevag试剂(粗多糖液：Sevag试剂＝4：1，v/v)，室温下置于摇床震荡(220rpm/min,10min)混匀，使得蛋白充分吸附在有机相中，然后离心，保留水相，重复操作，直至蛋白完全除去，将收集的水相进行透析冻干备用。(6) Protein removal: Add Sevag reagent to the crude polysaccharide solution obtained in step (5) (crude polysaccharide solution: Sevag reagent = 4:1, v/v), place on a shaking table at room temperature (220rpm/min, 10min ) and mix well to make the protein fully adsorbed in the organic phase, then centrifuge, keep the water phase, repeat the operation until the protein is completely removed, and then dialyze and freeze-dry the collected water phase for later use.
(7)DEAE-Cellulose 52离子交换柱和Sephadex G-150凝胶柱分离纯化：将上述步骤(6)后的胞外多糖，配置成10mg/mL溶液，取30mL加样于DEAE-Cellulose 52离子交换柱中，用去离子水以流速为1.0mL/min下进行洗脱。洗脱液紧接着经过Sephadex G-150凝胶柱，用去离子水以流速为0.2mL/min下进行洗脱，每管收集4mL，采用苯酚-硫酸法跟踪检测多糖含量如图1所示。收集不同管中的多糖液，减压浓缩，真空冷冻干燥，得到中性胞外多糖冻干粉。DEAE-Cellulose 52离子交换柱和Sephadex G-150凝胶柱为多糖常用分离柱，为节省中性胞外多糖纯化时间采用连接方式为串联连接，实现一步法纯化。(7) Separation and purification of DEAE-Cellulose 52 ion exchange column and Sephadex G-150 gel column: the exopolysaccharide after the above step (6) was prepared into a 10mg/mL solution, and 30mL was added to DEAE-Cellulose 52 ion In the exchange column, deionized water was used for elution at a flow rate of 1.0 mL/min. The eluate then passed through the Sephadex G-150 gel column, and was eluted with deionized water at a flow rate of 0.2mL/min. 4mL was collected in each tube, and the polysaccharide content was tracked and detected by the phenol-sulfuric acid method, as shown in Figure 1. The polysaccharide liquid in different tubes is collected, concentrated under reduced pressure, and vacuum freeze-dried to obtain a neutral exopolysaccharide freeze-dried powder. DEAE-Cellulose 52 ion exchange column and Sephadex G-150 gel column are commonly used separation columns for polysaccharides. In order to save the purification time of neutral exopolysaccharides, the connection method is connected in series to realize one-step purification.
实施例2：酸奶中性胞外多糖的单糖组成分析Example 2: Monosaccharide composition analysis of yogurt neutral exopolysaccharide
(1)分子量测定(1) Molecular weight determination
采用高相液相色谱高效凝胶渗透色谱法(Waters1525凝胶色谱仪)测定实施例1步骤(6)所制备的中性胞外多糖分子量的均一性和分子量。色谱柱为TSK G5000 PWXL(6μm，7.8×300mm)和TSK G3000 PWXL(6μm，7.8×300mm)串联使用，检测器为Waters 2414示差折光检测器(RID)，柱温35℃、进样量10μL，流动相为0.02mol/L的磷酸氢二钾缓冲溶液，流速0.6mL/min。分别将不同分子质量的葡聚糖标准品过0.45μm滤膜后上机，记录保留时间。以保留时间为横坐标、以葡聚糖分子量的对数为纵坐标绘制标准曲线。以同样的方法测定中性的多糖的出峰时间，根据标准曲线，计算出酸奶中性胞外多糖分子质量。由图2可知，酸奶中性胞外多糖的分子量为32063Da，从而说明酸奶中的中性胞外多糖较为均一，可进一步进行单糖组成成分的测定。 The molecular weight uniformity and molecular weight of the neutral exopolysaccharide prepared in step (6) of Example 1 were measured by HPLC high performance gel permeation chromatography (Waters 1525 gel chromatograph). The chromatographic column is TSK G5000 PWXL (6 μm, 7.8×300mm) and TSK G3000 PWXL (6 μm, 7.8×300mm) used in series, the detector is Waters 2414 differential refractive index detector (RID), the column temperature is 35°C, and the injection volume is 10 μL. The mobile phase was 0.02mol/L dipotassium hydrogen phosphate buffer solution, and the flow rate was 0.6mL/min. Dextran standards with different molecular weights were passed through a 0.45 μm filter membrane, and the retention time was recorded. Draw the standard curve with the retention time as the abscissa and the logarithm of the molecular weight of dextran as the ordinate. Determine the peak time of neutral polysaccharides in the same way, and calculate the molecular mass of neutral exopolysaccharides in yogurt according to the standard curve. It can be seen from Figure 2 that the molecular weight of neutral exopolysaccharides in yogurt is 32063 Da, which shows that the neutral exopolysaccharides in yogurt are relatively uniform, and further determination of monosaccharide components can be carried out.
标准品的配制：准备标准品与试剂，如表1所示。Preparation of standard products: Prepare standard products and reagents, as shown in Table 1.
表1 标准品与试剂信息Table 1 Standard and reagent information
Figure PCTCN2022125459-appb-000001
Figure PCTCN2022125459-appb-000001
Figure PCTCN2022125459-appb-000002
Figure PCTCN2022125459-appb-000002
在EP管中加入8mL无菌水，依次加入岩藻糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸、葡萄糖醛酸各100mg，溶解后定容至10mL得到10mg/mL的母液。稀释100倍，制备成100μg/mL工作液，将取上述溶液按照以下梯度稀释，装入1.5mL的EP管中。各单糖混标梯度浓度信息(μg/mL)如表2所示。Add 8 mL of sterile water to the EP tube, add fucose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid each 100 mg in sequence, dissolve and dilute to 10 mL yielded a 10 mg/mL stock solution. Dilute 100 times to prepare a 100μg/mL working solution. Dilute the above solution according to the following gradient and put it into a 1.5mL EP tube. The gradient concentration information (μg/mL) of each monosaccharide mixed standard is shown in Table 2.
表2 单糖混标梯度浓度信息(μg/mL)Table 2 Concentration information of monosaccharide mixed standard gradient (μg/mL)
Figure PCTCN2022125459-appb-000003
Figure PCTCN2022125459-appb-000003
样品前处理：处理实施例1步骤(6)的酸奶中性胞外多糖，具体步骤如下：取干净的色谱瓶，称量多糖样品各5mg，加入TFA酸溶液，121℃加热2h，通氮气吹干。加入甲醇清洗再吹干，重复2-3次。加无菌水溶解后转入色谱瓶中待测。Sample pretreatment: process the yogurt neutral exopolysaccharide in step (6) of Example 1, the specific steps are as follows: take a clean chromatographic bottle, weigh 5 mg of each polysaccharide sample, add TFA acid solution, heat at 121 °C for 2 hours, blow with nitrogen Dry. Add methanol to wash and dry, repeat 2-3 times. Add sterile water to dissolve and transfer to a chromatographic bottle for testing.
提取液体样本：取适量上清液，通氮气吹干。之后的步骤与固体样本提取一致。Liquid sample extraction: take an appropriate amount of supernatant, blow dry with nitrogen. The subsequent steps are consistent with solid sample extraction.
分析检测：色谱系统采用的是Thermo ICS5000+离子色谱系统(ICS5000+，(Thermo Fisher Scientific，USA)，采用Dionex TM CarboPac TM PA10(250*4.0mm，10μm)液相色谱柱，进样量为20μL。流动相A(H 2O)，流动相B(100mol/L NaOH)，柱温为30℃，利用电化学检测器对单糖组分进行分析检测。流动相的具体梯度与数据如下表所示。 Analysis and detection: The chromatographic system adopts Thermo ICS5000+ ion chromatography system (ICS5000+, (Thermo Fisher Scientific, USA), adopts Dionex TM CarboPac TM PA10 (250*4.0mm, 10 μm) liquid chromatography column, and the injection volume is 20 μL. Phase A (H 2 O), mobile phase B (100mol/L NaOH), the column temperature is 30°C, and the monosaccharide components are analyzed and detected by an electrochemical detector. The specific gradient and data of the mobile phase are shown in the table below.
表3 流动相梯度Table 3 Mobile phase gradient
Figure PCTCN2022125459-appb-000004
Figure PCTCN2022125459-appb-000004
表4 单糖含量及摩尔比Table 4 Monosaccharide content and molar ratio
Figure PCTCN2022125459-appb-000005
Figure PCTCN2022125459-appb-000005
由图3和表4可知，酸奶中性胞外多糖组成摩尔比为半乳糖(Gal)：葡萄糖(Glc)＝42.04:57.96。It can be known from Figure 3 and Table 4 that the molar ratio of neutral exopolysaccharides in yogurt is galactose (Gal):glucose (Glc)=42.04:57.96.
实施例3：酸奶中性胞外多糖的甲基化和核磁分析Example 3: Methylation and NMR analysis of yogurt neutral exopolysaccharide
(1)甲基化及GC-MS分析(1) Methylation and GC-MS analysis
准备标准品与试剂，如表5所示。Prepare standards and reagents, as shown in Table 5.
表5 标准品与试剂信息Table 5 Standard and reagent information
Figure PCTCN2022125459-appb-000006
Figure PCTCN2022125459-appb-000006
多糖样品衍生化：称取10mg经纯化后的样品，加入1mL去离子水溶解，再加入1mL 100mg/mL碳二亚胺，反应2h。随后加入1mL 2mol/L的咪唑，平均分为两份后分别加入1mL 30mg/mL的NaBH 4和同体积同浓度的NaBD 4，3h后加入100μL冰醋酸终止反应。透析样品48h，完成后冷冻干燥样品并进行甲基化处理。往冻干样品中加500μL DMSO溶解，加1mg NaOH孵育30min，加50μL碘甲烷溶液反应1h。加1mL水和2mL二氯甲烷混合混匀，离心弃水相。重复水洗3次，吸取下层二氯甲烷相并蒸干，加入100μL 2mol/L TFA于121℃条件下反应90min后于30℃蒸干；加50μL 2mol/L氨水、50μL 1mol/L NaBD 4混匀，室温下反应2.5h。加20μL乙酸终止反应，氮气吹干，250μL甲醇洗两次，氮气吹干，加乙酸酐250μL，涡旋混匀，100℃反应2.5h。入1mL水静置10min后，加500μL二氯甲烷，涡旋混匀，离心，弃水相。重复水洗3次后，取下层二氯甲烷相，准备上机检测。 Derivatization of polysaccharide samples: Weigh 10 mg of the purified sample, add 1 mL of deionized water to dissolve, then add 1 mL of 100 mg/mL carbodiimide, and react for 2 hours. Then add 1 mL of 2mol/L imidazole, divide it into two parts, add 1 mL of 30 mg/mL NaBH 4 and NaBD 4 of the same volume and concentration, and add 100 μL of glacial acetic acid after 3 hours to terminate the reaction. The samples were dialyzed for 48 hours, and then freeze-dried and methylated. Add 500 μL DMSO to the freeze-dried sample to dissolve, add 1 mg NaOH to incubate for 30 min, add 50 μL iodomethane solution to react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, mix well, and centrifuge to discard the aqueous phase. Repeat washing with water 3 times, absorb the lower dichloromethane phase and evaporate to dryness, add 100 μL 2mol/L TFA, react at 121℃ for 90 minutes, then evaporate to dryness at 30℃; add 50μL 2mol/L ammonia water, 50μL 1mol/L NaBD 4 and mix well , Reaction at room temperature for 2.5h. Add 20 μL of acetic acid to terminate the reaction, dry with nitrogen, wash twice with 250 μL of methanol, dry with nitrogen, add 250 μL of acetic anhydride, vortex mix, and react at 100 °C for 2.5 h. Add 1 mL of water and let stand for 10 min, add 500 μL of dichloromethane, vortex to mix, centrifuge, and discard the water phase. After repeated washing with water 3 times, the lower layer of dichloromethane phase was removed to prepare for detection on the machine.
气质联用色谱分析：采用Agilent气象色谱系统(Agilent 7890A；Agilent Technologies，USA)，根据化合物的性质，进样量为1μL，分流比10:1，载气为高纯氦气；保持柱温箱初始温度为140℃2.0min，以3℃/min速度升温至230℃保持3min。Gas chromatography-mass chromatography analysis: Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), according to the nature of the compound, the injection volume is 1 μL, the split ratio is 10:1, and the carrier gas is high-purity helium; keep the column oven The initial temperature is 140°C for 2.0 minutes, and the temperature is raised to 230°C at a rate of 3°C/min and kept for 3 minutes.
采用的是美国Aiglent公司的四极杆质谱检测系统(Agilent 5977B；Agilent Technologies，USA)，配有电子轰击离子源(EI)和Mass Hunter工作站。采用电子轰击离子源(EI)，分析物在全扫描(SCAN)模式下进行检测，质量扫描范围(m/z)为30-600。A quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) from Aiglent Corporation of the United States was used, equipped with an electron impact ion source (EI) and a Mass Hunter workstation. Using an electron impact ion source (EI), analytes were detected in full scan (SCAN) mode with a mass scan range (m/z) of 30-600.
(2)核磁共振分析(2) NMR analysis
分别称取实施例1-(6)所制备的酸奶中性胞外多糖5mg溶于0.6mL重水(D 2O)中，反复冻干复溶与0.6mL重水后加入核磁管中，于Bruker AV-600型核磁共振仪上进行 1H NMR和 13C NMR测定。 Weigh 5 mg of the yogurt neutral exopolysaccharide prepared in Example 1-(6) and dissolve it in 0.6 mL of deuterium water (D 2 O), redissolve with 0.6 mL of deuterium water after repeated freeze-drying, add to NMR tubes, and place in Bruker AV 1 H NMR and 13 C NMR were measured on -600 nuclear magnetic resonance instrument.
酸奶中性胞外多糖的甲基化分析如表6所示。The methylation analysis of yogurt neutral exopolysaccharides is shown in Table 6.
表6 中性胞外多糖的甲基化及GC-MS分析表Table 6 Methylation and GC-MS analysis of neutral exopolysaccharides
Figure PCTCN2022125459-appb-000007
Figure PCTCN2022125459-appb-000007
通过与PMAA数据库比对，5个衍生物分别是1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol(1,5-二-O-乙酰基-2,3,4,6-四-O-甲基葡萄糖醇)，1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol(1,4,5-三-O-乙酰基-2,3,6-三-O-甲基半乳糖醇)，1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol(1,4,5-三-O-乙酰基-2,3,6-三-O-甲基葡萄糖醇)，1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl glucitol(1,3,4,5-四-O-乙酰基-2,6-二-O-甲基葡萄糖醇)，1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl glucitol(1,4,5,6-四-O-乙酰基-2,3-二-O-甲基葡萄糖醇)。By comparison with the PMAA database, the five derivatives are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol (1,5-di-O-acetyl-2 ,3,4,6-tetra-O-methylglucitol), 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol (1,4,5-tri- O-acetyl-2,3,6-tri-O-methyl galactitol), 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol (1,4 ,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol), 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl glucitol (1,3,4,5-Tetra-O-acetyl-2,6-di-O-methylglucitol), 1,4,5,6-tetra-O-acetyl-2,3-di- O-methyl glucitol (1,4,5,6-tetra-O-acetyl-2,3-di-O-methylglucitol).
由表6中可得，中性胞外多糖的糖苷键的连接方式包括t-Glcp，4-Galp，4-Glcp，3,4-Glcp和4,6-Glcp组成，相对摩尔比为17.456:37.035:37.035:1.476:6.998。根据方程DB＝(NT+NB)/(NT+NB+NL)计算出酸奶中性胞外多糖的分支度(DB)值为25.93％，其中NT是指末端残基t-Glcp(1→，NB是指分支残基→3,4)-Glcp(1→和→4,6)-Glcp(1→，NL是指线性残基→4)-Galp(1→和4)-Glcp(1→的数量。It can be obtained from Table 6 that the glycosidic bond connection mode of the neutral exopolysaccharide includes t-Glcp, 4-Galp, 4-Glcp, 3,4-Glcp and 4,6-Glcp, and the relative molar ratio is 17.456: 37.035:37.035:1.476:6.998. According to the equation DB=(NT+NB)/(NT+NB+NL), the branching degree (DB) value of yogurt neutral exopolysaccharide is calculated to be 25.93%, wherein NT refers to the terminal residue t-Glcp (1→, NB refers to branched residues→3,4)-Glcp(1→and→4,6)-Glcp(1→, NL refers to linear residues→4)-Galp(1→and 4)-Glcp(1→ quantity.
酸奶中性胞外多糖的 1H NMR图谱如图4所示。在δ6～8ppm之间没有观察到共振，表明酸奶中性胞外多糖不含酚或阿魏酸等杂质。异位氢分布在δ4.7～5.5ppm之间，表明酸奶中性胞外多糖中含有α-和β-糖苷键。 13C NMR可以反映样品中多糖的残留量。此外，通过化学位移在95～110ppm之间的正头碳峰数可以分析和确定多糖残基的数量及其相关构型。酸奶中性胞外多糖的 13C NMR谱图如图5所示。在δ103.65、102.89、96.41、95.87和95.72ppm处发现了异端碳，表明酸奶中性胞外多糖中含有5种糖苷键，与甲基化结果一致。关于这5个糖苷键的位置和序列的更详细的信息将在将来被阐明。 The 1 H NMR spectrum of yogurt neutral exopolysaccharide is shown in FIG. 4 . No resonance was observed between δ6-8ppm, indicating that the yogurt neutral exopolysaccharide does not contain impurities such as phenol or ferulic acid. The distribution of ectopic hydrogen was between δ4.7～5.5ppm, indicating that yogurt neutral exopolysaccharides contained α- and β-glucosidic linkages. 13 C NMR can reflect the residual amount of polysaccharide in the sample. In addition, the number of polysaccharide residues and their related configurations can be analyzed and determined by the number of positive carbon peaks with chemical shifts between 95 and 110 ppm. The 13 C NMR spectrum of yogurt neutral exopolysaccharide is shown in FIG. 5 . Heretic carbons were found at δ103.65, 102.89, 96.41, 95.87 and 95.72ppm, indicating that yogurt neutral exopolysaccharides contained 5 kinds of glycosidic bonds, consistent with the methylation results. More detailed information on the location and sequence of these 5 glycosidic bonds will be elucidated in the future.
实施例4：酸奶中性胞外多糖红外光谱分析Example 4: Infrared spectrum analysis of yogurt neutral exopolysaccharide
采用溴化钾压片法，分别称取实施例1步骤(6)制得的中性胞外多糖10mg，加入100mg的KBr粉末，用压片机压成均匀的薄片，采用Bruker VERTEX 33型傅里叶变换红外光谱仪在4000-500cm -1范围内进行红外光谱扫描，记录谱图。图6可知，在3412.08cm -1处的宽拉伸峰属于羟基拉伸振动。在2937.59cm -1处的峰值是脂肪族CH 2基团的不对称C和H伸缩振动，表明存在糖等有机物。1649.14cm -1处的振动可能与羧基的对称拉伸有关，1426.36cm -1处的振动可能与C和H的弯曲振动有关。1200～1000cm -1区域的吸收峰可能是C-O-H和C-O-C伸缩振动引起的。1022.27cm -1处的振动可能与C-O的弯曲振动有关。在924.87cm -1处的吸收峰表明，酸奶中性胞外多糖结构中可能存在呋喃环。 Using the potassium bromide tabletting method, weigh 10 mg of the neutral exopolysaccharide prepared in step (6) of Example 1, add 100 mg of KBr powder, and press it into a uniform sheet with a tablet machine, using a Bruker VERTEX 33 type Fu The Lie transform infrared spectrometer scans the infrared spectrum in the range of 4000-500cm -1 and records the spectrum. It can be seen from Figure 6 that the broad stretching peak at 3412.08cm -1 belongs to the hydroxyl stretching vibration. The peak at 2937.59 cm is the asymmetric C and H stretching vibration of the aliphatic CH2 group, indicating the presence of organic matter such as sugars. The vibration at 1649.14 cm may be related to the symmetric stretching of the carboxyl group, and the vibration at 1426.36 cm may be related to the bending vibration of C and H. The absorption peak in the region of 1200～1000cm -1 may be caused by the stretching vibration of COH and COC. The vibration at 1022.27 cm -1 may be related to the bending vibration of CO. The absorption peak at 924.87cm -1 indicated that there might be a furan ring in the neutral exopolysaccharide structure of yogurt.
实施例5：酸奶中性胞外多糖表观形态和刚果红实验分析Example 5: Yogurt Neutral Exopolysaccharide Apparent Morphology and Congo Red Experimental Analysis
(1)扫描电镜观察酸奶中性胞外多糖表观形态(1) Observation of the appearance of neutral exopolysaccharides in yogurt by scanning electron microscope
扫描电镜是目前常用的观察多糖形貌和判定多糖种类的方法，具有操作简单、结果直观和分辨率高的优点，因此在食品科学、化学、材料和生物学上得到广泛应用。取充分干燥的实施例1步骤(6)中性胞外多糖组分，取少量涂抹于导电胶上，喷金后采用扫描电镜观察其表面形态。由图7可知，酸奶中性胞外多糖呈现薄片状结构，随着放大倍数的增加，可观察到其表面光滑。Scanning electron microscopy is a commonly used method to observe the morphology of polysaccharides and determine the types of polysaccharides. It has the advantages of simple operation, intuitive results and high resolution, so it is widely used in food science, chemistry, materials and biology. Take the fully dried neutral exopolysaccharide component in step (6) of Example 1, apply a small amount on the conductive adhesive, spray gold, and observe its surface morphology with a scanning electron microscope. It can be seen from Figure 7 that the yogurt neutral exopolysaccharide presents a sheet-like structure, and its surface can be observed to be smooth as the magnification increases.
(2)刚果红实验(2) Congo red experiment
刚果红是一种酸性染料，它可以和具有三股螺旋结果的多糖形成络合物，络合物的最大吸收波长同刚果红相比发生位移，称取2mg实施例1步骤(6)中性胞外多糖组分，溶解于2mL蒸馏水后加入2mL80μmol/L的刚果红试剂，逐步加入1mol/L的氢氧化钠，使溶液中的氢氧化钠浓度从0.0mol/L升至0.5mol/L，然后进行紫外波谱扫描，测得不同NaOH浓度下的最大吸收波长。由图8可知，低浓度氢氧化钠，紫外吸收移向长波说明多糖能与刚果红形成络合物，具有螺旋构象。高浓度氢氧化钠，最大吸收波长下降说明此时多糖螺旋结构解体。Congo red is a kind of acid dye, and it can form complexes with the polysaccharide that has triple helix result, and the maximum absorption wavelength of complexes shifts compared with Congo red, takes by weighing 2mg embodiment 1 step (6) neutrophils For the exopolysaccharide component, dissolve in 2 mL of distilled water, add 2 mL of 80 μmol/L Congo red reagent, gradually add 1 mol/L of sodium hydroxide, so that the concentration of sodium hydroxide in the solution rises from 0.0 mol/L to 0.5 mol/L, and then Ultraviolet spectrum scanning was carried out to measure the maximum absorption wavelength under different NaOH concentrations. It can be seen from Figure 8 that at low concentrations of sodium hydroxide, the ultraviolet absorption shifts to long waves, indicating that polysaccharides can form complexes with Congo red and have a helical conformation. With high concentration of sodium hydroxide, the maximum absorption wavelength decreases, indicating that the polysaccharide helical structure disintegrates at this time.
实施例6：酸奶中性胞外多糖治疗溃疡性结肠炎小鼠的应用Example 6: Application of yogurt neutral exopolysaccharide in treating ulcerative colitis mice
BALB/c小鼠，雄性，4～5周龄，采自浙江维通利华实验动物科技有限公司。所有小鼠被标准饲料适应(23～25℃和12h的光/暗周期)7天。采用3.5％(w/v)DSS激活小鼠结肠炎模型。将40只小鼠随机分为5组(n＝8/组)：第1～7天，对照组(Control)灌胃生理盐水，灌胃蒸馏水；(2)模型组(Model)小鼠口服生理盐水，给予3.5％DSS溶液；(3)L-EPS组小鼠口服80mg/kg酸奶中性胞外多糖，给予3.5％DSS溶液；(4)H-EPS组小鼠口服160mg/kg酸奶中性胞外多糖，给予3.5％DSS溶液；(5)SASP组(阳性对照)小鼠口服柳氮磺胺吡啶(SASP)溶液100mg/kg，给予3.5％DSS溶液，在第15天，所有小鼠被安乐死。BALB/c mice, male, 4-5 weeks old, were collected from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. All mice were acclimated to standard diet (23-25°C and 12h light/dark cycle) for 7 days. The mouse colitis model was activated with 3.5% (w/v) DSS. 40 mice were randomly divided into 5 groups (n=8/group): on the 1st to 7th days, the control group (Control) was fed with normal saline and distilled water; (2) the model group (Model) mice were orally administered physiological Saline, give 3.5% DSS solution; (3) L-EPS group mice oral administration 80mg/kg yogurt neutral exopolysaccharide, give 3.5% DSS solution; (4) H-EPS group mice oral administration 160mg/kg yogurt neutral exopolysaccharide Exopolysaccharide, give 3.5% DSS solution; (5) SASP group (positive control) mice oral sulfasalazine (SASP) solution 100mg/kg, give 3.5% DSS solution, on the 15th day, all mice were euthanized .
由图9(A)可知，在实验第三天时，饮用了DSS的四组小鼠体重都出现了明显下降，模型组小鼠的体重在实验过程中持续下降，试验结束时，L-EPS、H-EPS和SASP组小鼠平均体重较模型组有明显恢复。It can be seen from Figure 9(A) that on the third day of the experiment, the weight of the mice in the four groups that drank DSS all decreased significantly, and the weight of the mice in the model group continued to decrease during the experiment. At the end of the experiment, L-EPS, The average body weight of the mice in the H-EPS and SASP groups recovered significantly compared with the model group.
DAI(disease activity index,DAI)是疾病活动指数，评价标准按照以下表格打分。DAI (disease activity index, DAI) is the disease activity index, and the evaluation criteria are scored according to the following table.
表7 DAI评分标准Table 7 DAI Scoring Criteria
Figure PCTCN2022125459-appb-000008
Figure PCTCN2022125459-appb-000008
由图9(B)可知，模型组(Model)小鼠的DAI评分显著高于空白对照组(Control)，说明造模成功。It can be seen from Figure 9(B) that the DAI score of the model group (Model) mice was significantly higher than that of the blank control group (Control), indicating that the modeling was successful.
用ELISA方法检测了TNF-α、IL-10和IL-1β的血清水平，也就是抗原吸附在固相载体上，加待测抗体，再加相应酶标记抗体，生成抗原-待测抗体-酶标记抗体的复合物，再与该酶的底物反应生成有色产物，利用分光光度计测量吸光度后计算抗体的量。TNF-α是一种能够直接杀伤肿瘤细胞而对正常细胞无明显毒性的细胞因子。图10(A)是各组小鼠血清中的TNF-α水平，可以看到，模型组(Model)的TNF-α水平是显著高于空白组(Control)的，而经过多糖的治疗，能够降低TNF-α的水平。IL-10是重要的抑制细胞因子，可以抑制炎性反应、限制过度免疫反应。由图10(B)可知，模型组(Model)的IL-10水平是显著低于空白组(Control)的，经过多糖的治疗，能够提升IL-10的水平，其中高剂量多糖组(H-EPS)的IL-10水平显著提高。IL-1β是重要的促凋亡和促炎细胞因子之一，由图10(C)可知，模型组(Model)的IL-1β水平是显著高于空白组(Control)的，经过多糖的治疗，低剂量多糖组(L-EPS)和高剂量多糖组(H-EPS)都能显著降低IL-1β的水平。从血清中的细胞因子水平可知，经过多糖的治疗，可以恢复IL-10的水平，降低TNF-α和IL-1β的水平，从而达到抗炎的目的，缓解小鼠的结肠炎病症。The serum levels of TNF-α, IL-10 and IL-1β were detected by ELISA method, that is, the antigen was adsorbed on the solid phase carrier, the antibody to be tested was added, and the corresponding enzyme-labeled antibody was added to generate antigen-antibody-to-be-enzyme The complex of the labeled antibody reacts with the substrate of the enzyme to generate a colored product, and the amount of the antibody is calculated after measuring the absorbance with a spectrophotometer. TNF-α is a cytokine that can directly kill tumor cells without obvious toxicity to normal cells. Figure 10 (A) is the TNF-α level in the serum of each group of mice. It can be seen that the TNF-α level of the model group (Model) is significantly higher than that of the blank group (Control), and after polysaccharide treatment, it can Reduces levels of TNF-α. IL-10 is an important inhibitory cytokine that can suppress inflammatory responses and limit excessive immune responses. It can be seen from Figure 10(B) that the IL-10 level of the model group (Model) was significantly lower than that of the blank group (Control). EPS) IL-10 levels were significantly increased. IL-1β is one of the important pro-apoptotic and pro-inflammatory cytokines. It can be seen from Figure 10(C) that the IL-1β level of the model group (Model) is significantly higher than that of the blank group (Control). After polysaccharide treatment , low-dose polysaccharide group (L-EPS) and high-dose polysaccharide group (H-EPS) can significantly reduce the level of IL-1β. From the cytokine levels in serum, it can be known that after polysaccharide treatment, the level of IL-10 can be restored, and the level of TNF-α and IL-1β can be reduced, so as to achieve the purpose of anti-inflammation and relieve the colitis symptoms of mice.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

  1. 一种酸奶中性胞外多糖，其特征在于，所述酸奶中性胞外多糖是将德氏乳杆菌(Lactobacillus delbrueckii)DMLD-H1和嗜热链球菌(Streptococcus thermophilus)DMST-H2接种至脱脂乳中进行发酵培养，获得发酵酸奶；对所述酸奶进行离心、醇沉、除蛋白、经离子交换柱洗脱，洗脱液为水，得到分子量为32063Da的中性胞外多糖。A yogurt neutral exopolysaccharide, characterized in that the yogurt neutral exopolysaccharide is inoculated into skim milk by Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 The fermented yoghurt was obtained by fermenting and culturing in yoghurt; the yoghurt was centrifuged, alcohol-precipitated, protein removed, and eluted through an ion-exchange column, and the eluent was water to obtain a neutral exopolysaccharide with a molecular weight of 32063Da.
  2. 根据权利要求1所述的酸奶中性胞外多糖，其特征在于，所述中性胞外多糖的单糖组成为半乳糖和葡萄糖，摩尔比为42.04:57.96。The yogurt neutral exopolysaccharide according to claim 1, wherein the monosaccharide composition of the neutral exopolysaccharide is galactose and glucose, and the molar ratio is 42.04:57.96.
  3. 根据权利要求1所述的酸奶中性胞外多糖，其特征在于，所述中性胞外多糖的糖苷键由t-Glcp，4-Galp，4-Glcp，3,4-Glcp和4,6-Glcp组成，相对摩尔比为17.456:37.035:37.035:1.476:6.998。The yogurt neutral exopolysaccharide according to claim 1, wherein the glycosidic bond of the neutral exopolysaccharide consists of t-Glcp, 4-Galp, 4-Glcp, 3,4-Glcp and 4,6 -Glcp composition, the relative molar ratio is 17.456:37.035:37.035:1.476:6.998.
  4. 一种制备权利要求1～3任一项所述的酸奶中性胞外多糖的方法，其特征在于，包括如下步骤：A method for preparing the yogurt neutral exopolysaccharide according to any one of claims 1 to 3, characterized in that it comprises the following steps:
    (1)菌种活化：取德氏乳杆菌DMLD-H1和嗜热链球菌DMST-H2分别接种至MRS培养基里，37℃静置培养24h；之后进行二级活化，接种量为5～10％v/v；活化后得到种子液，于37℃条件下静置备用；(1) Strain activation: Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 were inoculated into MRS medium respectively, and cultured at 37°C for 24 hours; after that, secondary activation was carried out, and the inoculum size was 5-10 %v/v; the seed liquid is obtained after activation, and it is left to stand at 37°C for later use;
    (2)菌粉的制备：将种子液接种到发酵培养基中进行扩大培养，接种浓度为5～10％v/v，37℃恒温条件下静置培养24～30h后离心，生理盐水洗2～3次，离心后收集菌泥，添加冻干粉保护剂后，经-80℃预冻4h、冷冻干燥48h，得到菌粉；(2) Preparation of bacterial powder: inoculate the seed liquid into the fermentation medium for expanded cultivation, the inoculation concentration is 5-10% v/v, and then centrifuge after standing for 24-30 hours at a constant temperature of 37°C, wash with normal saline for 2 ~3 times, collect the bacteria sludge after centrifugation, add freeze-dried powder protective agent, pre-freeze at -80°C for 4 hours, and freeze-dry for 48 hours to obtain the bacteria powder;
    (3)发酵酸奶的准备：将12～15％脱脂乳粉和8～10％蔗糖混合得到脱脂乳，85℃水浴加热15～20min，冷却至40℃～42℃后接入步骤(2)所述菌粉进行发酵，得到发酵酸奶；(3) Preparation of fermented yoghurt: Mix 12-15% skim milk powder and 8-10% sucrose to obtain skim milk, heat in a water bath at 85°C for 15-20min, cool to 40°C-42°C, and then add it to step (2). The bacteria powder is fermented to obtain fermented yoghurt;
    (4)离心：离心步骤(3)所述发酵酸奶，收集上清液；(4) centrifugation: the fermented yoghurt described in centrifugation step (3), collects the supernatant;
    (5)醇沉：向步骤(4)所述上清液中加入无水乙醇，静置，离心取沉淀，收集沉淀溶于水得到粗多糖液；(5) Alcohol precipitation: add absolute ethanol to the supernatant described in step (4), let it stand, centrifuge to take the precipitate, collect the precipitate and dissolve it in water to obtain a crude polysaccharide liquid;
    (6)除蛋白：向步骤(5)中所得粗多糖液中加入Sevag试剂，室温下置于摇床震荡混匀，使蛋白充分吸附在有机相中，然后离心，保留水相，重复操作直至蛋白完全除去，将收集的水相进行透析冻干，得到粗胞外多糖；(6) Protein removal: add Sevag reagent to the crude polysaccharide solution obtained in step (5), place on a shaker at room temperature and shake to mix, so that the protein is fully adsorbed in the organic phase, then centrifuge, retain the aqueous phase, and repeat the operation until The protein is completely removed, and the collected aqueous phase is dialyzed and freeze-dried to obtain crude exopolysaccharide;
    (7)将步骤(6)所述粗胞外多糖配置成10～30mg/mL溶液，经DEAE-Cellulose 52离子交换柱和Sephadex G-150凝胶柱分离纯化，减压浓缩，真空冷冻干燥后，得到中性胞外多糖冻干粉。(7) The crude exopolysaccharide described in step (6) is configured into a 10-30 mg/mL solution, separated and purified by a DEAE-Cellulose 52 ion exchange column and a Sephadex G-150 gel column, concentrated under reduced pressure, and vacuum freeze-dried , to obtain neutral exopolysaccharide freeze-dried powder.
  5. 根据权利要求4所述的方法，其特征在于，步骤(2)所述菌泥和冻干保护剂体积比为1：(1～3)。The method according to claim 4, characterized in that the volume ratio of the bacteria slime and the freeze-drying protective agent in step (2) is 1: (1-3).
  6. 根据权利要求4所述的方法，其特征在于，步骤(3)所述发酵条件为：初始接菌量为(2～4)×10 6CFU/mL，接种比例为(1～3):1，42±5℃发酵8～10h，4～6℃后熟12～14h。 The method according to claim 4, characterized in that the fermentation conditions in step (3) are: the initial inoculation amount is (2-4)×10 6 CFU/mL, and the inoculation ratio is (1-3):1 , fermented at 42±5°C for 8-10 hours, and ripened at 4-6°C for 12-14 hours.
  7. 根据权利要求4所述的方法，其特征在于，步骤(4)所述离心条件为：离心力为10000～15000g，离心温度4℃，离心时间5～10min。The method according to claim 4, characterized in that the centrifugation conditions in step (4) are: centrifugal force of 10,000-15,000 g, centrifugation temperature of 4°C, and centrifugation time of 5-10 minutes.
  8. 根据权利要求4所述的方法，其特征在于，步骤(5)所述上清液与无水乙醇体积比为1：(4～6)；步骤(6)所述粗多糖液与Sevag试剂体积比为(4～5):1。The method according to claim 4, characterized in that the volume ratio of the supernatant to absolute ethanol in step (5) is 1: (4-6); the crude polysaccharide solution in step (6) and the volume of Sevag reagent The ratio is (4～5):1.
  9. 权利要求1～3任一项所述酸奶中性胞外多糖在食品中的应用。The application of the yoghurt neutral exopolysaccharide in any one of claims 1 to 3 in food.
  10. 权利要求1～3任一项所述酸奶中性胞外多糖在治疗结肠炎药物中的应用。The application of the yoghurt neutral exopolysaccharide described in any one of claims 1 to 3 in the medicine for treating colitis.
PCT/CN2022/125459 2021-10-21 2022-10-14 Exopolysaccharide separated from lactobacillus delbrueckii and streptococcus thermophilus fermented yoghourt and application thereof WO2023066163A1 (en)

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