Bifidobacterium breve H4-2 capsular polysaccharide with oxidation resistance

Abstract

The invention discloses a bifidobacterium breve H4-2 capsular polysaccharide with oxidation resistance and a preparation method of the bifidobacterium breve H4-2 capsular polysaccharide. The uniform capsular polysaccharide CPS1 is obtained by extracting and purifying the bifidobacterium breve H4-2. The weight-average molecular weight of CPS1 is 11047 Da, and full spectrum scanning shows that the content of protein and nucleic acid in a sample is low. The CPS1 comprises mannose, rhamnose, glucose and galactose, and the molar ratio of the mannose to the rhamnose to the glucose to the galactose is 1: 0.3: 3.77: 0.85. Infrared spectroscopic analysis shows that CPS1 has a typical polysaccharide characteristic absorption peak. A methylation result shows that CPS1 is composed of seven glucosidic bonds. And further combining with a nuclear magnetic resonance spectrum to speculate that the skeleton of the CPS1 is composed of residues of-4)-alpha-D-Glcp-(1->,-4)-alpha-D-Manp-(1->,-3, 4)-alpha-D-Glcp-(1->,-4, 6)-beta-D-Galp-(1->,-2)-L-Rhap-(1-> and-2, 4)-alpha-L-Rhap-(1->, and the tail end of the CPS1 is composed of residues of alpha-D-Glcp-(1->. A scanning electron microscope observes that the apparent form of CPS1 is irregular particles. Thermal performance analysis shows that CPS1 has certain thermal stability. The polysaccharide has an antioxidant effect and is expected to be developed into a natural antioxidant.

Description

Technical Field

[0001] The invention belongs to the technical field of food microorganisms and mainly relates to a bifidobacterium breve H4-2 capsular polysaccharide with antioxidant ability and application thereof. Background Art

[0002] As a gram-positive bacteria, Bifidobacterium is the first colonizer in the human intestine and exhibits powerful prebiotic functions in maintaining intestinal homeostasis, anti-inflammatory, anti-oxidation and lowering blood sugar and lipids. The prebiotic effects are closely related to its metabolites such as exopolysaccharides, short-chain fatty acids, bacteriocins, γ-aminobutyric acid and biotin. Among them, exopolysaccharides (EPSs), as macromolecular carbohydrate polymers secreted by bifidobacteria, have multiple biological response regulation activities, including antioxidant, anti-tumor, anti-inflammatory, antibacterial, antihypertensive activities and cholesterol-lowering activities. Intestinal epithelial cells are the first line of defense in the intestine, and they are constantly at risk of oxidative stress due to exposure to foreign bodies and microorganisms. Studies have shown that EPSs can play an antioxidant role by scavenging free radicals or improving the defense capacity of cells. As a safe and efficient natural antioxidant, the development of bifidobacterium EPSs has received increasing attention.

[0003] EPSs can be divided into capsular polysaccharides (CPSs) and mucus polysaccharides (SPSs), of which SPSs are released into the surrounding environment, while CPSs are tightly bound to the cell surface to form a protective capsule structure. Due to the different secretion locations of the two, SPSs are easily extracted together with other carbohydrates in the culture medium, increasing the difficulty of separation and purification. In addition, compared with SPSs, CPSs have unique structures and functions. At present, the structure and function of probiotic SPSs are gradually being recognized by people, while the research on CPSs is mainly focused on the development of pathogenic microorganism CPSs vaccines. There is still a lack of understanding of the structural and functional diversity of probiotic CPSs, especially for Bifidobacterium, which is often used as a probiotic. In-depth research on its CPSs has important guiding significance for revealing the probiotic function and application of the bacteria. Summary of the invention

[0004] The present invention uses a simple and effective process and method for extracting and purifying capsular polysaccharides, and obtains uniform capsular polysaccharide CPS1 from Bifidobacterium breve H4-2 through extraction and purification. In vitro and in vivo tests have shown that the capsular polysaccharide has certain antioxidant activity. It is characterized in that Bifidobacterium breve H4-2 is inoculated into an mMRS broth medium containing 2% (v / v) L-cysteine ​​hydrochloride, and the capsular polysaccharide is extracted after fermentation. The Bifidobacterium breve H4-2 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, Beijing, with a deposit number of CGMCC No. 31450 and a deposit date of July 25, 2024.

[0005] The weight average molecular weight of the CPS1 is 11047 Da.

[0006] The CPS1 contains mannose, rhamnose, glucose and galactose in a molar ratio of 1:0.3:3.77:0.85.

[0007] The CPS1 is composed of 7 glycosidic bonds.

[0008] The backbone of CPS1 is composed of →4)-α-D-Glcp-(1→, →4)-α-D-Manp-(1→, →3,4)-α-D-Glcp-(1→, →4,6)-β-D-Galp-(1→, →2)-L-Rhap-(1→ and →2,4)-α-L-Rhap-(1→ residues, and the tail end is composed of α-D-Glcp-(1→ residues.

[0009] The CPS1 was observed under a scanning electron microscope and its apparent morphology was irregular granules.

[0010] The CPS1 has certain thermal stability.

[0011] The CPS1 can be used to prepare antioxidant foods or antioxidant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 is the UV-visible spectrum of CPS1;

[0014] Figure 2 For determination of molecular weight of CPS1;

[0015] Figure 3 is the FTIR spectrum of CPS1;

[0016] Figure 4 is the HPLC chromatogram of CPS1;

[0017] Figure 5 is the total ion current of CPS1 (A); the fragment ion peak of CPS1 (BH);

[0018] Figure 6 For CPS1 1 H NMR spectra (A) and 13 C NMR spectrum (B);

[0019] Figure 7 Microscopic morphology of CPS1 (A, ×10.0k; B, ×20.0k);

[0020] Figure 8 TG curve and DTG curve of CPS1;

[0021] Fig. 9 Superoxide anion radical (A) of CPS1, ABTS + Free radical (B) and hydroxyl radical (C) scavenging activity; DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0023] Example 1

[0024] Extraction of crude CPSs from H4-2. The cells from the five cultures were collected by centrifugation (8000 rpm, 4 ℃, 10 min) and washed with PBS buffer (pH 7.2) at 4 ℃. The cells after centrifugation were resuspended in 30 mL of NaCl solution (1 mol / L) and sonicated at 4 ℃ for 5 min (power 65 W, working 3 s, stopping 2 s). The sonicated cell suspension was centrifuged at 9000 rpm and 4 ℃ for 30 min, and the supernatant was collected and concentrated under reduced pressure at 55 ℃. Subsequently, three times the volume of anhydrous ethanol was added to the concentrate and allowed to stand at 4 ℃ overnight. The ethanol mixture obtained above was centrifuged at 8000 rpm and 4 ℃ for 15 min to collect the precipitated polysaccharide, and the precipitated polysaccharide was resuspended in double distilled water, and the suspension was dialyzed at 4 ℃ for 72 h using a dialysis bag with a cutoff of 8-14 kDa. The final polysaccharide solution was lyophilized to obtain crude CPSs.

[0025] Crude CPSs were purified by DEAE-52 anion exchange column. First, the expanded and activated DEAE-52 filler was wet packed into a column (26×300 mm), requiring no bubbles and no stratification in the column. The 15 mg / mL crude CPSs solution was centrifuged at 4 ℃ and 8000 rpm for 10 min to remove impurities, and then loaded onto the DEAE cellulose column. Then, deionized water, 0.1 mol / L and 0.3 mol / L NaCl solutions were used for elution at 1.0 mL / min, respectively, and the collected sample eluate (10 mL / tube) was monitored using the sulfuric acid-phenol method. Finally, the polysaccharide solution of the main component was concentrated under reduced pressure, dialyzed and freeze-dried to obtain purified CPS1.

[0026] Example 2

[0027] CPS1 was dissolved in double distilled water, and the ultraviolet absorption spectrum of the polysaccharide solution in the wavelength range of 190-600 nm was recorded using a UV-1500C ultraviolet spectrophotometer, with deionized water as a blank control.

[0028] The results showed that the protein and nucleic acid contents in CPS1 were low. Figure 1 .

[0029] Example 3

[0030] FT-IR was used to analyze the main functional groups of CPS1. A mixture of CPSs powder and KBr in a ratio of 1:100 was pressed onto a transparent sheet using a hydraulic press, and then scanned from 400 to 4000 cm -1 Infrared wavelength spectrum.

[0031] The results showed that CPS1 has a wavelength of 400-4000 cm -1 It has typical absorption peaks of polysaccharides. Figure 2 .

[0032] Example 4

[0033] The molecular weight of CPSs was determined using high performance gel permeation chromatography (HPGPC). First, a 5 mg / mL CPS1 solution was prepared, centrifuged at 12000rpm for 10min, the supernatant was taken, filtered with a 0.22μm water system microporous filter membrane, and then the polysaccharide solution was transferred to a 1.8 mL injection bottle. The instrument used was a Shimadzu LC-10A high performance liquid chromatograph equipped with a BRT105-103-101 gel column (8×300mm) and a RID-20A differential detector. The mobile phase was 0.05M NaCl, the flow rate was 0.8mL / min, the column temperature was 40ºC; the injection volume was 25 μL. Standards (dextran) selected P5, P10, P20, P50, P100, P200, P400 and P800.

[0034] The results showed that the chromatograms of CPS1 showed a relatively symmetrical single peak, indicating that it was a homogeneous polysaccharide; the Mw of CPS1 was 11047 Da. Figure 3 .

[0035] Example 5

[0036] First, prepare a 5 mg / mL CPS1 solution and take 200 μL in a high temperature resistant test tube. Add 200 μL of 4 mol / L trifluoroacetic acid and hydrolyze at 120 ℃ for 2 h. After cooling, take 200 μL of the hydrolyzed solution and spin dry it in a distillation flask, add 200 μL of methanol to re-dissolve and spin dry, and repeat this operation 3 times. Then add 100 μL of deionized water to obtain a 1 mg / mL sample hydrolyzed solution. Take 500 μL of sample hydrolyzate or mixed monosaccharide standard solution (glucose, galactose, mannose, rhamnose, galacturonic acid, glucuronic acid and arabinose) in a test tube, add 500 μL of 0.3 M NaOH solution, vortex for 30 s, add 500 μL of 0.5 mol / L PMP-methanol solution, vortex again for 30 s and place in a 70 ℃ water bath for 60 min. After the solution was cooled, 500 μL of 0.3 mol / L HCl solution was added, followed by 1 mL of chloroform, vortexed and centrifuged to extract the aqueous phase, and repeated three times. Finally, the extracted solution was filtered through a 0.22 μm filter membrane and placed in a 1.8 mL injection bottle. The chromatographic column was a cosmosil 5C18-MS-II column (250 mm×4.6 mm, 5μm; Puyu Science and Trade Co., Ltd., Shanghai, China), the mobile phase was 0.1 mol / L phosphate buffer (pH 6.7) and acetonitrile (20:80, v / v), the flow rate was 1.0 mL / min, the column temperature was 25 °C, and the detection wavelength was 245 nm.

[0037] The results showed that the CPS1 sample mainly contained mannose, rhamnose, glucose and galactose with a molar ratio of 1:0.3:3.77:0.85. Figure 4 .

[0038] Example 6

[0039] The type of glycosidic bonds in CPS1 was determined by methylation analysis. First, 3 mg of polysaccharide was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) and treated with NaOH for 30 min, followed by the addition of 50 μL of iodomethane solution for methylation reaction for 1 h. 1 ml of water and 2 ml of dichloromethane were added, and the aqueous phase was removed by vortex centrifugation, which was repeated three times, and the lower dichloromethane was blown dry with nitrogen. Then, 100 μl of TFA (2 mol / L) was added for hydrolysis for 90 min and evaporated to dryness. 50 μL of ammonia (2 mol / L) and 50 μL of NaBD4 (1 mol / L) were added for reduction treatment for 2.5 h, followed by the addition of acetic acid to terminate the reaction, and 250 μL of methanol was added for spin drying, which was repeated twice. Acetic anhydride was added for acetylation at 100°C for 2.5 h, and 1 mL of water was added to terminate the reaction. Finally, dichloromethane was added to extract the discarded aqueous phase, and gas chromatography-mass spectrometry (GC-MS) analysis was performed. The analysis was performed using an Agilent 7890A-5977B GC-MS instrument equipped with a BPX70 column (30 m × 0.25 mm × 0.25 μm). The injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was high purity helium. The column temperature was started at an initial temperature of 140 °C for 2 min and then increased at a rate of 3 °C / min until it reached 230 °C. The results are shown in Tables 1 and Figure 5 .

[0040] Table 1 Glycosidic bond composition of CPS1

[0041] Tab.1 Glycosidic linkage composition of CPS1

[0042] Glycosidic bond type Methylated sugar residues Retention time (min) Fragment ion peak (m / z) Molar ratio (%) →2)-Rha-(1→ <![CDATA[3,4-Me 2 Rap]]> 8.786 89,100,115,130,131,175,190 1.82 Glc-(1→ <![CDATA[2,3,4,6-Me 3 Glcp]]> 8.940 87,102,118,129,145,161,162,205 27.29 →2,3)-Rha-(1→ <![CDATA[4-Me 1 Rap]]> 11.833 71,89,101,131,262 2.24 →4)-Man-(1→ <![CDATA[2,3,6-Me 3 Manp]]> 13.756 87,102,113,118,129,162,233 15.02 →4)-Glc-(1→ <![CDATA[2,3,6-Me 3 Glcp]]> 14.162 87,102,113,118,129,162,233 36.25 →3,4)-Glc-(1→ <![CDATA[2,6-Me 2 Glcp]]> 16.254 87,118,129,143,185,203,305 3.70 →4,6)-Gal-(1→ <![CDATA[2,3-Me 2 Galp]]> 18.368 85,102,118,127,159,162,201,261 13.68

[0043] Example 7

[0044] The CPS1 structure was further analyzed using a Bruker Avance AV-600 NMR spectrometer. 40 mg of CPS1 sample was weighed and dissolved in 1 mL of D 2 O. Then, the dissolved solution was transferred to an NMR tube and placed in a 600 MHz NMR spectrometer at room temperature (25 °C). 1 H NMR and 13 C NMR spectroscopy measurements.

[0045] The results showed that the backbone of CPS1 was mainly composed of →4)-α-D-Glcp-(1→, →4)-α-D-Manp-(1→, →3,4)-α-D-Glcp-(1→, →4,6)-β-D-Galp-(1→, →2)-α-L-Rhap-(1→, and →2,4)-α-L-Rhap-(1→ residues, and the end of the branch chain was composed of α-D-Glcp-(1→ residues. The results are shown in Figure 6 .

[0046] Example 8

[0047] The microstructure and surface morphology of CPS1 powder were observed using field emission scanning electron microscopy (SEM). The CPS1 samples were fixed on aluminum bars, coated with a thin layer of metal, and then observed using a SU8010 scanning electron microscope (FE-SEM) at a voltage of 10 kV and a magnification of 10.0 k and 20.0 k.

[0048] The results show that under 10.0 k magnification, CPS1 is a dense and compact sheet. When further magnified to 20.0 k, the surface of CPS1 is rough and presents an irregular granular structure. Figure 7 .

[0049] Example 9

[0050] Take 10 mg of polysaccharide sample (CPS1) and place it in Al 2 O 3 Thermodynamic analysis was performed using a SDT-650 Synchronous Thermal Analyzer (STA) in a platinum crucible. The temperature range was 30-800 °C, with a linear heating rate of 10 °C / min. The experimental gas was nitrogen with a flow rate of 20 mL / min.

[0051] The results showed that the TG curve showed that CPS1 lost about 9% of its weight in the first degradation stage at 25-100 °C, which was due to the evaporation of bound water in the polysaccharide. CPS1 caused a loss of 52.21% in the second degradation stage, mainly due to the depolymerization and fission of the sugar rings in the polysaccharide structure. As the temperature increased, CPS1 gradually degraded to a constant weight. The final residual capsule polysaccharide mass percentage was 32.56%. The DTG curve showed that CPS1 reached its peak at 254.93 °C. Results are shown in Figure 8 .

[0052] Example 10

[0053] 50 μL of a mixture of 0.4 mmol / L xanthine and 0.24 mmol / L NBT, 100 μL of xanthine oxidase (0.049 U / mL), 50 μL of CPS1 sample solution and ascorbic acid (Vc) solution (concentrations of 0.5, 1, 2, and 4 mg / mL, respectively) were added to a 96-well plate. After mixing, the samples were incubated at 30 °C for 30 min and the OD was measured using an enzyme reader. 560 Ascorbic acid was used as a positive control. The group without sample was the blank control, and its OD 560 Value is A 0 The absorbance value of the sample group is A 1 The clearance rate was calculated according to the following formula:

[0054]

[0055] Mix 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution in a ratio of 1:1, place in the dark for 14 h, and dilute 30 times before use. Take 0.5 mL of the sample to be tested and add 4.5 mL of ABTS working solution to mix, incubate at 30 °C for 6 min, adjust the UV spectrophotometer to 734 nm, and detect the absorbance of 0.5, 1, 2, and 4 mg / mL CPS1 samples and ascorbic acid (Vc) solution, respectively, and record it as A 1 The mixed solution of double distilled water and working solution was used as the control group, and its absorbance value was determined as A 0 The calculation formula is:

[0056]

[0057] CPS1 samples and ascorbic acid (Vc) solutions at concentrations of 0.1, 0.2, 0.4, and 0.8 mg / mL were prepared, respectively, and the hydroxyl radical scavenging activity of CPS1 samples and ascorbic acid (Vc) was detected using a hydroxyl radical test kit according to the manufacturer's instructions.

[0058] The results showed that Vc had the highest superoxide anion radical scavenging activity, with a scavenging rate of 99.06% at 4 mg / mL; when the concentration was 4 mg / mL, the superoxide anion radical scavenging activity of CPS1 was 58.87%. CPS1 has the function of scavenging ABTS+ free radicals, and its scavenging ability is dose-dependent with the polysaccharide concentration. At a concentration of 4.0 mg / mL, the scavenging rate of CPS1 on ABTS+ free radicals was 35.39%. V C The ability of CPS1 to scavenge hydroxyl radicals also increased with the increase of concentration, showing a concentration-dependent relationship. At a concentration of 0.8 mg / mL, the scavenging rate of CPS1 for hydroxyl radicals was 93.33%. Fig. 9 .

Claims

1. A bifidobacterium breve H4-2 capsular polysaccharide having antioxidant capacity, It is characterized in that The Bifidobacterium breve H4-2 was inoculated into an mMRS broth medium containing 2% (v / v) L-cysteine ​​hydrochloride, and the capsular polysaccharide was extracted after fermentation. The Bifidobacterium breve H4-2 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, Beijing, with a deposit number of CGMCC No.31450 and a deposit date of July 25, 2024.

2. The capsular polysaccharide with antioxidant capacity obtained by the method of claim 1, It is characterized in that It is obtained by fermenting Bifidobacterium breve H4-2, and the weight average molecular weight of CPS1 is 11047 Da.

3. The capsular polysaccharide with antioxidant capacity obtained by the method of claim 1, It is characterized in that Obtained by fermenting Bifidobacterium breve H4-2, CPS1 contains mannose, rhamnose, glucose and galactose in a molar ratio of 1:0.3:3.77:0.

85.

4. The capsular polysaccharide with antioxidant capacity obtained by the method of claim 1, It is characterized in that It is obtained by fermenting Bifidobacterium breve H4-2. CPS1 is composed of 7 glycosidic bonds.

5. The capsular polysaccharide with antioxidant capacity obtained by the method of claim 1, It is characterized in that It was obtained by fermenting Bifidobacterium breve H4-2. The backbone of CPS1 is composed of →4)-α-D-Glcp-(1→, →4)-α-D-Manp-(1→, →3,4)-α-D-Glcp-(1→, →4,6)-β-D-Galp-(1→, →2)-L-Rhap-(1→ and →2,4)-α-L-Rhap-(1→ residues, and the tail is composed of α-D-Glcp-(1→ residues.

6. The capsular polysaccharide with antioxidant capacity obtained by the method of claim 1, It is characterized in that It is obtained by fermenting Bifidobacterium breve H4-2. The apparent morphology of CPS1 is irregular granules as observed by scanning electron microscopy.

7. The capsular polysaccharide with antioxidant capacity obtained by the method of claim 1, It is characterized in that It is obtained by fermenting Bifidobacterium breve H4-2, and CPS1 has a certain degree of thermal stability.

8. Use of the capsular polysaccharide according to claim 1 in the preparation of antioxidant foods or antioxidant drugs.

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