阅读说明：本技术 一种长双歧杆菌、长双歧杆菌胞外多糖及其提取方法和应用 (Bifidobacterium longum and bifidobacterium longum exopolysaccharide as well as extraction method and application thereof ) 是由 谢智勇 刘雯 于 2021-05-28 设计创作，主要内容包括：本申请属于生物医药技术领域,尤其涉及一种长双歧杆菌、长双歧杆菌胞外多糖及其提取方法和应用。本申请提供了一种长双歧杆菌,其为长双歧杆菌亚种Bifidobacteriumlongumsubsp.longum,保藏号为GDMCCNo：61618。本申请提供了长双歧杆菌胞外多糖,包括从长双歧杆菌提取的胞外多糖。本申请提供的长双歧杆菌胞外多糖的提取方法,包括：将长双歧杆菌在培养基中培养,收集发酵液；将发酵液去除菌体,使发酵液的酶失活,沉淀多糖和除去蛋白后,纯化得长双歧杆菌胞外多糖。本申请提供了一种新的长双歧杆菌,以及具有提高免疫活性以及改善胰岛素抵抗效果的长双歧杆菌胞外多糖。(The application belongs to the technical field of biomedicine, and particularly relates to bifidobacterium longum and bifidobacterium longum exopolysaccharide as well as an extraction method and application thereof. The application provides bifidobacterium longum which is bifidobacterium longum subspecies bifidum longum subsp. 61618. The present application provides bifidobacterium longum exopolysaccharides, including exopolysaccharides extracted from bifidobacterium longum. The application provides an extraction method of bifidobacterium longum exopolysaccharide, which comprises the following steps: culturing Bifidobacterium longum in culture medium, and collecting fermentation broth; removing thallus from the fermentation liquid, inactivating enzyme of the fermentation liquid, precipitating polysaccharide and removing protein, and purifying to obtain Bifidobacterium longum extracellular polysaccharide. The present application provides a novel bifidobacterium longum, and bifidobacterium longum exopolysaccharide having an effect of improving immune activity and improving insulin resistance.)

1. Bifidobacterium longum which is Bifidobacterium longum subsp. 61618.

2. A bifidobacterium longum exopolysaccharide comprising exopolysaccharide extracted from bifidobacterium longum as claimed in claim 1.

3. Bifidobacterium longum exopolysaccharide according to claim 2, characterized in that it has a sugar content of 99.20 ± 1.21%.

4. Bifidobacterium longum exopolysaccharide according to claim 2, characterized in that it has a relative molecular mass of 6.38 x 10⁵Da。

5. A bifidobacterium longum exopolysaccharide as claimed in claim 2 wherein the bifidobacterium longum exopolysaccharide includes mannose, glucose, rhamnose and galactose; the molar ratio of the mannose, the glucose, the rhamnose and the galactose is 11.85:5.60:0.46: 0.68.

6. a method for extracting bifidobacterium longum exopolysaccharide is characterized by comprising the following steps:

step 1, culturing the bifidobacterium longum of claim 1 in a culture medium, and collecting a fermentation broth;

and 2, removing thalli from the fermentation liquor, inactivating enzyme of the fermentation liquor, precipitating polysaccharide and removing protein, and purifying to obtain bifidobacterium longum extracellular polysaccharide.

7. The extraction process according to claim 6, wherein the purification comprises DEAE cellulose-52 ion exchange column purification and Sephacryl S-300HR sephadex purification.

8. Use of a bifidobacterium longum exopolysaccharide as claimed in any of claims 2 to 5 or obtainable by the extraction process as claimed in claim 6 or 7 for increasing immunomodulatory activity.

9. The use according to claim 8, wherein the enhancing immunomodulatory activity is specifically activating murine macrophages to produce NO, enhancing phagocytic activity of murine macrophages, and up-regulating cytokine expression of murine macrophages.

10. Use of a bifidobacterium longum exopolysaccharide as claimed in any of claims 2 to 5 or obtainable by the extraction process as claimed in claim 6 or 7 for improving insulin resistance.

Technical Field

The application belongs to the technical field of biomedicine, and particularly relates to bifidobacterium longum and bifidobacterium longum exopolysaccharide as well as an extraction method and application thereof.

Background

Polysaccharides, also known as polysaccharides, are carbohydrate compounds formed by polymerization of more than 20 monosaccharide molecules, and can be divided into three types according to sources, namely animal polysaccharides, plant polysaccharides and microbial polysaccharides, and are divided into homopolysaccharides and heteropolysaccharides according to carbohydrate components, so that the polysaccharides have various biological activities and wide sources, and therefore, infinite possibilities are provided for the development of new drugs. At present, the research mainly focuses on fungal polysaccharides and plant polysaccharides, such as ganoderan, lentinan, lycium barbarum polysaccharide and the like, which have important effects on the aspects of tumor resistance, oxidation resistance, inflammation resistance and the like. As the intestinal flora becomes the focus of attention of researchers, it is found that intestinal microorganisms play a crucial role in maintaining the nutritional metabolism, ecological niche competition, immune development and pathophysiological processes in human body, and the mechanism of certain specific flora to exert these physiological actions is realized by active polysaccharides generated by bacteria themselves.

Exopolysaccharides (EPS) are long-chain polysaccharides secreted extracellularly during bacterial growth and are the first contact point of bacteria with the immune system. In recent years, with the development of genomics, proteomics, glycobiology and glycomics, researches on exopolysaccharides of bacteria have been made, and a variety of biological activities of exopolysaccharides of bacteria have been discovered. Therefore, the development of certain intestinal flora with specific functions and active substances derived from the intestinal flora into health-care food and even medicines has very wide prospect.

Disclosure of Invention

In view of the above, the present application provides a bifidobacterium longum, bifidobacterium longum exopolysaccharide, and extraction methods and applications thereof, and provides a novel bifidobacterium longum, and bifidobacterium longum exopolysaccharide having effects of improving immune activity and improving insulin resistance.

The application provides a Bifidobacterium longum in a first aspect, which is Bifidobacterium longum subsp. 61618.

In a second aspect, the present application provides a bifidobacterium longum exopolysaccharide comprising exopolysaccharides extracted from said bifidobacterium longum.

In another embodiment, the bifidobacterium longum exopolysaccharide has a sugar content of 99.20 ± 1.21%.

In another embodiment, the bifidobacterium longum exopolysaccharide has a relative molecular mass of 6.38 x 10⁵Da。

In another embodiment, the bifidobacterium longum exopolysaccharide comprises mannose, glucose, rhamnose and galactose; the molar ratio of the mannose, the glucose, the rhamnose and the galactose is 11.85:5.60:0.46: 0.68.

the third aspect of the application provides a method for extracting bifidobacterium longum exopolysaccharide, which comprises the following steps:

step 1, culturing the bifidobacterium longum of claim 1 in a culture medium, and collecting a fermentation broth;

and 2, removing thalli from the fermentation liquor, inactivating enzyme of the fermentation liquor, precipitating polysaccharide and removing protein, and purifying to obtain bifidobacterium longum extracellular polysaccharide.

Specifically, the culture medium is MRS liquid culture medium, the culture temperature is 37 ℃, and the culture is carried out for 48 hours in an anaerobic environment.

Specifically, the removing of the cells includes: the fermentation broth was centrifuged to remove the bacteria at 8000rpm for 30min at 4 ℃.

Specifically, enzyme inactivation includes: the sterile supernatant was collected and placed in a water bath at 100 ℃ for 15min to inactivate the enzymes in the fermentation broth.

Specifically, precipitating the polysaccharide includes: the supernatant after the enzyme inactivation was concentrated under reduced pressure to 1/10 in the original volume, and 3 times the volume of glacial ethanol was added to the concentrate, and the mixture was allowed to stand at 4 ℃ overnight. Centrifuging the ethanol precipitation part the next day (8000rpm,30min,4 deg.C), collecting precipitate, and re-dissolving with appropriate amount of ultrapure water to obtain XZ01 extracellular polysaccharide crude extract.

Specifically, protein removal includes: adopting Sevage reagent to carry out deproteinization treatment on the crude XZ01 extracellular polysaccharide extract, and specifically comprising the following steps: the crude extract was added with 1/5 volumes of Sevage reagent (chloroform: n-butanol: 4: 1, v/v), shaken vigorously for 15min and centrifuged (4500rpm,30min,4 ℃) to collect the upper layer polysaccharide solution. The above steps were repeated until the protein layer completely disappeared. The protein-depleted solution was transferred to a dialysis bag (molecular weight cut-off: 8000Da) and dialyzed against purified water for 48h to remove small molecular impurities, during which water was changed every 4 h. After dialysis, freeze-drying is carried out to obtain a crude exopolysaccharide extract sample, and the crude exopolysaccharide extract sample is sealed and stored in a dryer.

In another embodiment, the purification comprises DEAE cellulose-52 ion exchange column purification and Sephacryl S-300HR sephadex purification.

The fourth aspect of the application provides the application of the bifidobacterium longum exopolysaccharide or the bifidobacterium longum exopolysaccharide obtained by the extraction method in improving the immunoregulation activity.

In another embodiment, the enhancing immunomodulatory activity is specifically activating murine macrophages to produce NO, enhancing phagocytic activity of murine macrophages, and up-regulating cytokine expression of murine macrophages.

In a fifth aspect, the application provides the application of the bifidobacterium longum exopolysaccharide or the bifidobacterium longum exopolysaccharide obtained by the extraction method in improving the insulin resistance activity.

It should be noted that although originating from the same genus, the extracellular polysaccharides expressed by different bifidobacteria are distinct in structure and thus exhibit different functional properties.

The application provides a novel bifidobacterium longum, and extracellular polysaccharide extracted from the bifidobacterium longum has the effects of improving the immunoregulation activity, activating murine macrophage cells to generate NO, enhancing the phagocytic activity of the murine macrophage cells, up-regulating the cytokine expression of the murine macrophage cells and improving the insulin resistance activity. Specifically, the experimental data of the application show that extracellular polysaccharides C-EPS and S-EPS-1 extracted from Bifidobacterium longum disclosed by the application can activate macrophage RAW264.7, and can enhance the phagocytic activity and improve the immune function while activating the macrophage, C-EPS and S-EPS-1 with different concentrations can up-regulate the expression of IL-1 beta, IL-6 and TNF-alpha of RAW264.7 cells at the gene level, and meanwhile, the C-EPS and S-EPS-1 are proved to have good immunoregulatory activity; in addition, the bifidobacterium longum-extracted exopolysaccharide disclosed in the application has the effect of improving the insulin resistance activity. The bifidobacterium longum and the extracellular polysaccharide C-EPS and S-EPS-1 extracted from the bifidobacterium longum disclosed by the application have certain application potential and commercial value in the aspects of development of functional foods, health-care products and immunologic adjuvants.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a graph of the gram stain results of strain XZ01 provided in the examples herein;

FIG. 2 is an electrophoretogram of the product of the strain XZ0116S rRNAPCR provided in the examples of the present application;

FIG. 3 is a phylogenetic tree of strain XZ01 as provided in the examples herein;

FIG. 4 is a UV spectrum of C-EPS, S-EPS-1 polysaccharide of strain XZ01 provided in the examples herein;

FIG. 5 is a permeation gel chromatogram of the S-EPS-1 polysaccharide of strain XZ01 provided in the examples herein;

FIG. 6 is an analysis of the monosaccharide composition of S-EPS-1 of strain XZ01 provided in the examples herein; wherein, A is a liquid chromatogram of mixed monosaccharide standard substance derivatized by PMP; b is a liquid chromatogram of derivatized S-EPS-1 PMP; 1: mannose; 2: rhamnose; 3: glucuronic acid; 4: galacturonic acid; 5: glucose; 6: galactose; 7: xylose; 8: arabinose; 9: fucose;

FIG. 7 shows the effect of the C-EPS, S-EPS-1 polysaccharide of strain XZ01 on the morphology of RAW264.7 (scale length 200 μm) provided in the examples of the present application;

FIG. 8 shows the effect of C-EPS, S-EPS-1 of strain XZ01 on the release of RAW264.7 NO (p < 0.05, p < 0.01 compared to control);

FIG. 9 shows the phagocytosis of neutral red by RAW264.7 cells in different treatment groups according to the present application, wherein the red arrows represent the phagocytosis of neutral red by RAW264.7 cells;

FIG. 10 shows the effect of C-EPS and S-EPS-1 of strain XZ01 on the phagocytic activity of RAW264.7 cells, compared to control group,. p < 0.05,. p < 0.01;

FIG. 11 shows the effect of C-EPS, S-EPS-1 of strain XZ01 on the cytokine expression of RAW264.7 (p < 0.05, p < 0.01 compared to control);

figure 12 is a graph showing the effect of different concentrations of C-EPS polysaccharides of strain XZ01 on HepG2 cell activity (p < 0.05, p < 0.01 compared to control) provided in the examples herein;

fig. 13 shows the improvement of TNF α -induced insulin resistance of HepG2 cells by C-EPS polysaccharides of strain XZ01 at different concentrations (p < 0.05, p < 0.01, compared to control group) provided in the examples;

fig. 14 shows the expression of PI3K genes from HepG2 cells after TNF α induction by various concentrations of C-EPS polysaccharides of strain XZ01 provided in the examples of the present application (p < 0.05, p < 0.01 compared to control);

fig. 15 shows the expression of IRS1 genes in HepG2 cells after induction of TNF α by various concentrations of C-EPS polysaccharides of strain XZ01 provided in the examples of the present application (p < 0.05, p < 0.01 compared to the control group).

Detailed Description

The application provides bifidobacterium longum and bifidobacterium longum exopolysaccharide as well as an extraction method and application thereof, and provides novel bifidobacterium longum and bifidobacterium longum exopolysaccharide with the effects of improving immunocompetence and improving insulin resistance.

The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The reagents and raw materials of the following examples are all commercially available or self-made.

The application has a deposit number GDMCC No: 61618 bifidobacterium longum is named: bifidobacterium longum sp. longum XZ01, hereinafter referred to as XZ 01.

RAW264.7 of the following examples is mouse mononuclear macrophage.

The recovery and culture of strain XZ01 include: taking out the strain XZ01 freezing tube from a refrigerator at-80 ℃, thawing in a water bath at 37 ℃, and transferring the freezing tube to a sterile ultra-clean workbench. Then, a cotton ball dipped with 75% ethanol is wiped around the cryopreservation tube for a circle, the sealing film is uncovered and then heated slightly on an alcohol lamp, the cover of the cryopreservation tube is unscrewed, a sterile inoculating ring is stretched into the cryopreservation tube to dip a proper amount of bacteria liquid, the proper amount of bacteria liquid is inoculated on an MRS plate by adopting a three-region scribing method, and the MRS plate is cultured for 48 hours at 37 ℃ in an anaerobic environment. After the strain is activated, screening single colonies in a streak plate, transferring the single colonies to a liquid culture medium, and carrying out amplification culture according to the same culture conditions.

Example 1

The examples herein provide assays for identifying strain XZ01, comprising:

1. activated strain XZ01 was gram stained in a clean bench. Dyeing is carried out in accordance with conventional process steps. After dyeing is finished, performing microscopic examination and photographing under a microscope, and if the dyeing result is red, determining that the bacteria are gram-negative bacteria; on the contrary, blue is a gram-positive bacterium.

After being activated, the strain XZ01 grows on an MRS plate in a round dot shape with regular edges, smooth white and opaque. The gram staining results are shown in fig. 1, and the strain XZ01 is a gram-positive bacterium, and is typically a rod-shaped or bifurcated (Y-shaped) microscopic form, which is a typical morphological feature of bifidobacterium.

2. Physiological and biochemical indexes of strain XZ01 were identified according to conventional methods, including raffinose, sorbitol, melezitose, D-cellobiose, D-arabinose, glucose, D-galactose, D-sucrose, D-fructose, starch, hydrogen sulfide, catalase, oxidase, kinetic experiments, nitrate reduction, indole production, urease, ornithine decarboxylase, arginine double hydrolase, phenylalanine deaminase, and growth pH. The results are shown in Table 1.

TABLE 1

Identification index	GDMCC1.248	XZ01	Identification index	GDMCC1.248	XZ01
						Cotton seed candy	+	+	Hydrogen sulfide	—	—
Sorbitol	—	—	Catalase enzyme	—	—
						Melezitose	+	+	Oxidase enzyme	—	—
D-Cellobiose	—	—	Dynamic experiment	—	—
						D-arabinose	—	—	Nitrate reduction	—	—
Glucose	+	+	Indole production	—	—
						D-galactose	+	+	Urease	—	—
Galactose	+	+	Ornithine decarboxylase	+	+
						D-sucrose	—	—	Arginine double hydrolase	—	—
D-fructose	—	—	Phenylalanine deaminase	—	—
						Starch	+	+	Growth pH	4-7	3-9

As can be seen from the table, the carbon source utilization characteristics of strain XZ01 were: can utilize raffinose, melezitose, glucose, galactose, D-galactose and starch in the culture medium, but can not utilize sorbitol, D-cellobiose, D-arabinose, D-sucrose and D-fructose. In addition, the results of the enzymology experiments show that the ornithine decarboxylase is positive, and the rest ornithine decarboxylase is negative. Strain XZ01 can grow at a slightly broader pH range than the control strain GDMCC1.248 for growth pH conditions, i.e. is more pH tolerant than the control strain. In conclusion, the physiological and biochemical properties of strain XZ01 were substantially identical to those of Bifidobacterium longum.

3. 16S rRNA identification assay for strain XZ01, comprising:

(1) extracting bacterial genome DNA:

the genomic DNA of the strain XZ01 was extracted using a bacterial genomic DNA extraction kit, and the specific procedures were strictly performed according to the instructions. After obtaining the DNA of strain XZ01, the DNA concentration and purity were checked, OD₂₆₀/OD₂₈₀Ratios in the range of 1.7-1.9 were considered acceptable.

(2) PCR amplification system and conditions:

placing the upstream and downstream primers, Taq PCRMaster Mix and sterile water on ice for melting, blowing and uniformly mixing each reagent by using a pipette before use, sequentially preparing a PCR reaction system in a PCR tube with sterile enzyme inactivation according to the table 2, centrifuging the PCR tube for a short time after preparation so as to throw off reaction liquid stained with the wall, and then carrying out a PCR amplification procedure. The primer sequences and amplification conditions are shown in tables 2 to 4.

TABLE 2 PCR amplification System

TABLE 316S rRNA amplification primer sequences

TABLE 4 PCR amplification conditions

Procedure	Temperature/. degree.C	Time	Number of cycles
				Pre-denaturation	94	4min	1
Denaturation of the material	94	1min	30
				Annealing	55	1min	30
Extension	72	1.5min	30
				Extension of	72	10min	1
Heat preservation	4	-	-

(3) And (3) detecting a PCR amplification product:

detecting the PCR amplification result by agarose gel electrophoresis, wherein the specific conditions are as follows: agarose (0.8%, 1 XTAE buffer, w/V), PCR amplification product (loading 5. mu.L), Marker D (loading 2.5. mu.L), voltage (110V), electrophoresis time (30 min). And (3) carrying out electrophoresis according to the conditions, and imaging and observing whether a target band appears at 1500bp under a gel imager after the electrophoresis is finished.

(4) And (3) recovering PCR amplification products:

and (3) screening a target band in the agarose gel according to the position indicated by the Marker, and recovering a target amplification product by using a gel recovery kit, wherein the operation is strictly carried out according to the steps in the specification.

(5) And (3) connecting the PCR amplification product with a T vector:

the target amplification product thus recovered was ligated to the T vector pUCm-TVector under the ligation conditions shown in Table 5.

TABLE 5T Carrier ligation systems

After the reaction system is prepared, the ligation is carried out at 22 ℃ for 10 min. Meanwhile, the competent cells are unfrozen on ice, and then the reaction solution which is well bathed is added, and after uniform mixing, the mixture is kept stand on ice for 30 min. Then, after heat shock at 42 ℃ for 90 seconds, the reaction mixture was allowed to stand on ice for 5 minutes and 900. mu.L of a sterile LB liquid medium was added to the reaction mixture, followed by shaking culture at 37 ℃ for 1 hour. The bacterial solution was centrifuged at 4000rpm for 3min, 750. mu.L of the upper medium was discarded, the bacteria were suspended in the remaining liquid in the centrifuge tube, the bacterial suspension was spread evenly on LB plate containing IPTG and X-gal, the plate was incubated at 37 ℃ for 1h and then inverted and incubated overnight.

And on the next day, picking white single colonies around the blue colonies by using a suction head on a sterile super-clean bench, and obtaining the positive single colonies. Subsequently, the single colony was inoculated into a liquid LB medium containing ampicillin and cultured overnight at 37 ℃ and the cultured bacterial solution was assigned to the Shanghai organism to complete 16S rRNA sequencing.

(6) Analysis of 16S rRNA sequencing results:

and checking a sequencing peak map by using Chromas software, judging a sequencing result, removing vector sequences positioned at two ends by using DNAStar software, and splicing the sequences.

Logging in an NCBI database, selecting a Blast function, comparing the sequence of the strain XZ01 with the 16S rRNA sequence of other bacteria in a similarity manner, performing phylogenetic analysis and construction of phylogenetic trees by using a Neighbor-Joining method (Meighbor-Joining method) in MEGAX software, and confirming the species of the strain XZ01 by combining morphological characteristics and physiological and biochemical results.

After extracting and PCR amplifying the genome DNA of the strain XZ01, identifying the amplification product by agarose gel electrophoresis, and obtaining the gel imaging result as shown in FIG. 2, wherein the first column is the Marker, the second column is the DNA of the control strain GDMCC1.248, and the third column is the DNA of the strain XZ01, when the FIG. 2 is viewed from left to right. FIG. 2 shows that a specific band appears at 1500bp, and the two single bands are the bands of the target fragment which are successfully amplified. Subsequently, the position of the target band is subjected to subsequent operations such as gel recovery and the like, and positive clones are screened for 16S rRNA sequencing identification.

After sequencing is completed, the obtained sequencing peak map of XZ01 is analyzed, the waveform of the XZ0116S rRNA sequencing peak map is symmetrical and clear, the distance between the peak and the peak is uniform, and the bottom of the peak is free from the interference of miscellaneous peaks, so that the sequencing success and the result are reliable.

The XZ0116S rRNA sequence was aligned for similarity by BLAST function to the 16S rRNA sequence homology of other bacteria in the NCBI database as shown in tables 2-5 (strains shown in tables are those with similarity greater than 97% to strain XZ 01). It can be seen from the table that all strains having a sequence similarity of more than 97% to strain XZ01 belong to the genus Bifidobacterium, and that all four strains have a similarity of more than 99% to strain XZ01, which is the highest in Bifidobacterium longum subsp. species (Bifidobacterium longum subsp. longum) of 99.86%, indicating that XZ01 is highly likely to belong to the Bifidobacterium longum subsp.

TABLE 6

Strain name	Degree of similarity
		Bifidobacterium longum subsp.longum	99.86％
Bifidobacterium longum subsp.suis	99.52％
		Bifidobacterium longum subsp.infantis	99.17％
Bifidobacterium longum subsp.suillum	99.17％
		Bifidobacterium felsineum	97.51％
Bifidobacterium scaligerum	97.17％

And selecting an alignment sequence with higher similarity, and drawing a phylogenetic tree by utilizing MEGAX software, wherein Escherichia coli is an introduced exogenous gene and is used as a control. According to the phylogenetic tree (fig. 3), the strain XZ01 is located in the same branch as Bifidobacterium longum subsp. In summary, in combination with morphological characteristics, biochemical characteristics, sequencing results of 16S rRNA, the present application identified strain XZ01 as Bifidobacterium longum subsp.

Example 2

The embodiment of the application provides an extraction test of exopolysaccharide of XZ01 strain, which comprises the following steps:

taking out the XZ01 strain preserved at-80 deg.C, inoculating in MRS liquid culture medium, culturing at 37 deg.C for 24 hr, activating two generations according to the above steps, transferring into MRS liquid culture medium with inoculum size of 2% (v/v) for amplification culture, and culturing at 37 deg.C for 48 hr.

After 48h of incubation, the broth was centrifuged (8000rpm,30min,4 ℃) to remove the bacteria, and the sterile supernatant was collected and placed in a 100 ℃ water bath for 15min to inactivate the enzymes in the broth. The supernatant was then concentrated under reduced pressure to 1/10 in its original volume, and 3 volumes of glacial ethanol were added to the concentrate and left to stand overnight at 4 ℃. Centrifuging the ethanol precipitation part the next day (8000rpm,30min,4 deg.C), collecting precipitate, and re-dissolving with appropriate amount of ultrapure water to obtain XZ01 extracellular polysaccharide crude extract.

Adopting Sevage reagent to carry out deproteinization treatment on the polysaccharide crude extract, and specifically operating as follows: the crude extract was added with 1/5 volumes of Sevage reagent (chloroform: n-butanol: 4: 1, v/v), shaken vigorously for 15min and centrifuged (4500rpm,30min,4 ℃) to collect the upper layer polysaccharide solution. The above steps were repeated until the protein layer completely disappeared. The protein-depleted solution was transferred to a dialysis bag (molecular weight cut-off: 8000Da) and dialyzed against purified water for 48h to remove small molecular impurities, during which water was changed every 4 h. After dialysis, freeze-drying is carried out to obtain a crude exopolysaccharide extract sample, and the crude exopolysaccharide extract sample is sealed and stored in a dryer.

The fermentation liquor of the bifidobacterium longum subspecies XZ01 cultured for 48 hours at 37 ℃ is centrifuged to remove thalli, enzyme is inactivated by boiling water bath, polysaccharide is precipitated by using ice ethanol overnight, Sevage reagent is repeatedly subjected to protein removal, dialysis, freeze drying and other steps to obtain brown fluffy bifidobacterium longum subspecies XZ01 crude polysaccharide, the yield of the brown fluffy bifidobacterium longum subspecies XZ01 crude polysaccharide is 747.86mg/L, and the extracellular crude polysaccharide of the bifidobacterium longum subspecies XZ01 strain is subjected to decolorization treatment to obtain the decolorized bifidobacterium longum subspecies XZ01 crude polysaccharide, which is called C-EPS for short.

Example 3

The embodiment of the application provides an extracellular polysaccharide separation and purification test of an XZ01 strain, which comprises the following steps:

in this example, a DEAE cellulose DE-52 anion exchange column and Sephacryl S-300HR sephadex were used to fractionate and purify the crude Bifidobacterium longum subspecies XZ01 polysaccharide (C-EPS) obtained from example 2 after decolorization, and the S-EPS-1 neutral polysaccharide fraction with higher sugar content was collected, followed by analysis of the purity and relative molecular mass of S-EPS-1, monosaccharide composition and ratio by ultraviolet spectroscopy (UV) and High Performance Liquid Chromatography (HPLC).

1. A DEAE cellulose-52 ion exchange column purification assay comprising:

1.1, pretreatment of cellulose: weighing a proper amount of DEAE Cellulose 52 ion exchange Cellulose in a glass beaker, adding sufficient ultrapure water to swell, removing suspended matters and insoluble large particles, treating for 1h by using 0.5mol/L NaOH solution, then repeatedly washing by using the ultrapure water to be neutral, treating for 1h by using 0.5mol/L HCl solution, and washing by using the ultrapure water to be neutral.

1.2, column filling: the specification of the chromatographic column is 2.6 multiplied by 50cm, the cellulose is slowly poured into the chromatographic column and naturally settled, the outer wall is tapped by an aurilave to remove air bubbles, then the filler is repeatedly added to a proper height and connected with a constant flow pump, and the flow rate is balanced by ultrapure water at 1.0 mL/min.

1.3, loading: weighing 400mg of C-EPS, dissolving in 10mL of ultrapure water, centrifuging at 4500rpm for 15min, taking supernatant, and filtering with a 0.45-micron filter membrane; after loading, the constant flow pump was started and elution was started.

1.4, elution: the eluent is 0, 0.05, 0.1, 0.3 and 0.5mol/L NaCl solution in sequence, the flow rate is 1.0mL/min, and 10mL is collected in each tube.

1.5, detection: and detecting the sugar content of the eluent by using a sulfuric acid phenol method separation tube, and drawing a corresponding elution curve.

1.6, collecting: according to the elution curve, the same components are combined, and after decompression and concentration, dialysis and freeze-drying are carried out to obtain polysaccharide samples with different components.

DEAE-Cellulose 52 is an anion exchange Cellulose, and is mainly used for separation and purification of macromolecular polysaccharide. The separation principle is based on ion exchange reaction, when polysaccharide solution passes through the filler, the acidic polysaccharide with negative charges is adsorbed by the filler, the neutral polysaccharide without the charges directly flows out, and then the eluent with different ionic strengths is adopted for gradient elution, so that different acidic polysaccharide components in the polysaccharide mixture can be eluted out to realize the purposes of separation and purification.

In this example, anion exchange cellulose was used to further separate and purify the decolorized crude polysaccharide of Bifidobacterium longum subspecies XZ 01. After the crude polysaccharide is eluted by NaCl with different concentration gradients, 3 obvious elution peaks are obtained, and the crude polysaccharide is sequentially named as EPS-1, EPS-2 and EPS-3 according to the peak appearance. As can be seen from the elution profile, EPS-1 is a fraction eluted with ultrapure water, indicating that EPS-1 is an uncharged neutral polysaccharide. While EPS-2 and EPS-3 were eluted with 0.05mol/L and 0.3mol/L NaCl solutions, respectively, indicating that the polysaccharide polymers of these two components are acidic polysaccharides with a certain amount of negative charge. However, the elution peak obtained by DEAE-Cellulose 52 is not necessarily a single component, and may be a mixture of components having the same charge amount but different molecular weights, and thus further purification by gel chromatography is required.

2. Sephacryl S-300HR Sephadex purification assay comprising:

2.1, pretreatment of cellulose: ethanol in Sephacryl S-300HR was washed with ultrapure water, and then the filler was packed in a chromatography column (1.6X 90cm) in the same manner as in steps 1.1 and 1.2, and after the filler was settled, a constant flow pump was connected and equilibrated with 0.1mol/LNaCl solution at a flow rate of 0.5 mL/min.

2.2, loading: 50mg of each component purified by an ion exchange column was weighed out, dissolved in 10mL of ultrapure water, and loaded in the same manner as in step 1.3.

2.3, elution: the eluent was 0.1mol/L NaCl solution at a flow rate of 0.5mL/min, 4mL collected per tube.

2.4, the detection and collection method is the same as the methods 1.5 and 1.6 of the step 1.

In addition, after the purification of the step, 4 components are obtained in total, namely S-EPS-1 to S-EPS-4. The yield and the sugar content of the S-EPS-1 component are high, and the chemical composition determination, the ultraviolet spectrum analysis, the molecular weight determination and the monosaccharide composition analysis are carried out on the S-EPS-1 component.

Sephacryl S-300HR is an allyl dextran cross-linked copolymer with good rigidity, chemical stability and a wide separation range, and is commonly used for separation between polysaccharide samples with different molecular weights.

Thus, the three fractions obtained above were subjected to further purification using allyl dextran gel in the examples of the present application. After the EPS-1 and the EPS-2 are eluted by a Sephacryl S-300HR sephadex column, single symmetric elution peaks S-EPS-1 and S-EPS-2 are obtained, namely, the components EPS-1 and EPS-2 are components with uniform molecular weight. The EPS-3 component is purified to obtain 2 mutually separated elution peaks, which shows that the EPS-3 component may be composed of two polysaccharide components with similar charge and different molecular weights. Considering that the sugar content and yield of the S-EPS-1 fraction were higher than those of the other fractions, the fraction was collected, dialyzed and lyophilized for subsequent analysis.

3. And (3) analyzing the S-EPS-1 polysaccharide purified in the step (2), wherein the analysis comprises the following steps:

3.1, determining the total sugar content and the protein content in the S-EPS-1 polysaccharide by adopting the conventional method.

3.2, preparing the components of C-EPS and S-EPS-1 into a solution of 1mg/mL by using ultrapure water, scanning in the range of 190-400nm, and checking whether characteristic absorption occurs at 260nm and 280 nm.

3.3, determination of molecular weight: the molecular weight of S-EPS-1 was determined by Gel Permeation Chromatography (GPC) while checking its purity. Dextran standards of different molecular weights were injected sequentially while recording their retention times (T)_R) And the T of each sample is measured_RAs abscissa, log lg of the corresponding molecular weight (Mw) as ordinate to plot a standard curve, giving lg (Mw) and T_RHas a regression equation of lg (Mw) ═ 0.7789T_R+10.417, coefficient of correlation R²＝0.9937。

Chromatographic conditions are as follows: shimadzu LC-20AT high performance liquid chromatograph, PolySep-GFC-P4000 chromatographic column (Phenomenex, 300 x 7.8mm), evaporative light scattering detector, the detector temperature is 60 deg.C, the gain value is 10, the column temperature is 35 deg.C, ultra pure water is used as mobile phase, the flow rate is 1.0mL/min, and the sample introduction amount is 20 μ L.

And (3) injecting and detecting the S-EPS-1 component according to the steps, substituting the S-EPS-1 component into a regression equation to calculate the relative molecular weight of the corresponding S-EPS-1 according to the corresponding retention time, and judging the purity of the polysaccharide according to the peak shape of the S-EPS-1 on a chromatogram.

In this example, the relative molecular mass of the S-EPS-1 component in bifidobacterium longum subspecies XZ01 exopolysaccharide was determined by HPGPC, and as shown in fig. 5, the permeation gel chromatogram of S-EPS-1 exhibited a single and symmetrical chromatographic peak, which indicates that the relative molecular mass distribution of the component was relatively uniform and the component was homogeneous polysaccharide with high purity. According to the linear regression equation, retaining the retention time T_RThe relative molecular mass of S-EPS-1 was calculated to be 6.38 × 10 by substitution at 5.921min⁵Da. Meanwhile, Sephacryl S-300HR allyl dextran gel is adopted for separating and purifying XZ01 EPS in the earlier stage of the embodiment, and the separation range is 10⁴～1.5×10⁶And the molecular weight of the S-EPS-1 component is within the range, which shows that the measurement result of GPC on the molecular weight of S-EPS-1 is consistent with the previous separation and purification result by gel chromatography.

3.4, monosaccharide composition analysis: PMP pre-column derivatization-high performance liquid chromatography is adopted to determine the monosaccharide composition of the S-EPS-1 component.

Hydrolysis of polysaccharide samples: weighing 5.0mg of S-EPS-1 sample into a closed reaction tube, adding 2mL of trifluoroacetic acid (TFA, 3M), placing the reaction tube at 120 ℃ after confirming that the reaction tube is sealed, hydrolyzing for 6h, cooling at room temperature after the hydrolysis is finished, then adding methanol, spin-drying (repeating for 3 times), adding 800 mu L of ultrapure water for dissolving, and transferring the polysaccharide solution which is completely hydrolyzed into a centrifuge tube for standby.

Derivatization reaction of polysaccharide samples: taking 100 mu L of completely hydrolyzed polysaccharide sample solution, adding 100 mu L of each of 0.5M PMP methanol solution and 0.3M NaOH solution into a closed reaction tube, uniformly mixing, placing in 70 ℃ water bath for reaction for 30min, then taking out, cooling at room temperature, sequentially adding 105 mu L0.3M HCl, 200 mu L of ultrapure water and 600 mu L of chloroform, whirling, uniformly mixing, centrifuging (10000rpm,15min), removing the lower layer of chloroform, repeating for 3 times to remove excessive PMP, finally taking the upper layer of aqueous solution, filtering through a 0.45 mu M filter membrane, and detecting by HPLC. The monosaccharide standards and the mixed standard are subjected to derivatization reaction according to the steps.

Chromatographic conditions are as follows: shimadzu LC-20AT high performance liquid chromatograph, Symmetry C18(Waters, 4.6X 250mm) chromatographic column, ultraviolet detector for detection, detection wavelength of 250nm, mobile phase of 0.05M phosphate buffer (pH 6.7) -acetonitrile (volume ratio 83:17), flow rate of 1.0mL/min, and sample injection of 20 μ L.

And comparing and calculating the peak emergence time and peak area of the sample S-EPS-1 after derivatization with a monosaccharide standard substance to obtain the monosaccharide composition and molar ratio of the S-EPS-1.

In this example, the type and the corresponding ratio of monosaccharides in S-EPS-1 samples were determined by high performance liquid chromatography. Comparing the liquid chromatogram (figure 6-B) of the S-EPS-1 component with the peak diagram (figure 6-A) of the derivative product of the standard product of the mixed monosaccharide, knowing that S-EPS-1 mainly comprises mannose, glucose and a small amount of rhamnose and galactose according to the retention time, and obtaining the molar ratio of the monosaccharides as mannose according to the peak area: rhamnose: glucose: galactose ═ 11.85: 0.46: 5.60: 0.68. from the above results, it can be seen that the component S-EPS-1 is mainly heteropolysaccharide (Heps) composed of 4 monosaccharides in different ratios.

Example 4

The present application provides the immunomodulatory activity test of C-EPS and S-EPS-1 polysaccharide in example 3, specifically comprising:

the activities of the C-EPS and purified S-EPS-1 polysaccharide fractions of example 3 were tested in sequence using RAW264.7 cells as test subjects, and mainly included measurement of cellular NO release amount, cellular phagocytic activity, mRNA expression level of cytokines after C-EPS and S-EPS-1 intervention, and examination of immunomodulatory activities of C-EPS and S-EPS-1.

1. Recovering and culturing RAW264.7 cells, inoculating RAW264.7 cells in a logarithmic growth phase to a 96-well plate, adding 100 mu L of cell suspension into each well, after the cells adhere to the wall overnight, removing culture solution, adding 100 mu L of C-EPS or S-EPS-1 with different concentrations diluted by using culture medium, wherein the concentrations are respectively 25, 50, 100, 150, 200 and 300 mu g/mL, only adding the culture medium with the same volume in a blank control group, and correspondingly adding an LPS solution (LPS, 1 mu g/mL) with the same volume in a positive control group. After the cells were treated as described above and transferred to an incubator for 24 hours, the cells were observed under a microscope for morphology and photographed and recorded, and the results are shown in FIG. 7. Subsequently, 20. mu.L of MTT solution (5mg/mL) was added to each well and incubated in an incubator at 37 ℃ for 4 hours in the absence of light, the supernatant was discarded, 200. mu.L of DMSO was further added to each well, followed by shaking for 10min to completely dissolve the crystals, and the absorbance was measured at a wavelength of 490nm, and the control group was regarded as 100% cell activity.

Morphological characteristics of C-EPS and S-EPS-1 treated RAW264.7 (figure 7) were observed under a microscope, cells in the blank control group were plump and round, attached to the wall in a monolayer and distributed in a tight cluster, and the edges of the cells were clear without morphological changes. And RAW264.7 cells treated by 100 mu g/mL C-EPS and S-EPS-1 for 24 hours are bright, a large number of pseudopodia are extended out, the cell volume is obviously increased and distributed in a fusiform or irregular polygon shape, and the cell shape is basically consistent with that of the positive control group treated by LPS. Typically, changes in the morphology of RAW264.7 cells often indicate activation of macrophages.

2. A Griess method test for detecting the influence of C-EPS and S-EPS-1 on NO secretion of RAW264.7 comprises the following steps:

2.1, establishing a standard curve: NaNO is added to the cell complete medium₂Diluting (1M) standard substance into standard solutions with concentration of 0, 1, 2, 5, 10, 40, 60, 100 μ M, respectively, placing 50 μ L of the above standard solutions in 96-well plate, respectively adding 50 μ L of Griess reagent 1 and Griess reagent 2 in the kit, respectively, measuring absorbance at 540nm wavelength after full color development, and measuring with NaNO₂The molar concentration of (A) is an abscissa, and a standard curve is drawn by taking a corresponding light absorption value under each concentration as an ordinate. The regression equation of the standard curve is that y is 0.0064x +0.0493, and the correlation coefficient R²0.9994. Wherein, Griess reagent 1: 0.5g of sulfanilic acid and 150mL of dilute acetic acid (10%); griess reagent 2: alpha naphthylamine 0.1g, distilled water 20mL and dilute acetic acid (10%) 150 mL.

2.2, determination of NO secreted by RAW 264.7: the inoculation, grouping and administration modes of the cells are synchronous with the method of the step 1 and the step 2. After the cells are treated according to the method, the cells are cultured in an incubator at 37 ℃ for 24h, 50 mu L of supernatant is taken from each well and put into a new 96-well plate, Griess reagent 1 and Griess reagent 2 with equal volumes are sequentially added, the absorbance value is measured at the wavelength of 540nm after shaking up, and the absorbance value is substituted into a standard curve to calculate the NO concentration in the supernatant of each group of cells. The results are shown in FIG. 8. Wherein, Griess reagent 1: 0.5g of sulfanilic acid and 150mL of dilute acetic acid (10%); griess reagent 2: alpha naphthylamine 0.1g, distilled water 20mL and dilute acetic acid (10%) 150 mL.

As shown in FIG. 8, the amount of NO released from RAW264.7 after stimulation by C-EPS and S-EPS-1 was significantly increased compared to the control group, while the amount of NO released from RAW264.7 stimulated by C-EPS and S-EPS-1 did not exceed LPS even at the highest concentration compared to the LPS positive control group, which indicates that the effects of C-EPS and S-EPS-1 on RAW264.7 cells are milder, and thus apoptosis caused by excessive NO production can be avoided. Based on the above results, it was found that both C-EPS and S-EPS-1 can activate RAW264.7 cells, promote their secretion, and produce NO.

2.3, C-EPS and S-EPS-1 are used for testing the phagocytic activity of RAW 264.7: the inoculation, grouping and administration of the cells are the same as in step 1. Treating the cells according to the method, culturing the cells in an incubator at 37 ℃ for 24h, adding preheated PBS buffer solution to gently wash the cells for 3 times, adding 100 mu L of 0.75mg/mL neutral red-PBS solution, continuously placing the cells in the incubator at 37 ℃ for 45min in the dark place, taking the cells out, placing the cells under a microscope for microscopic examination, photographing and recording, removing supernatant, washing the cells for 3 times by using the PBS buffer solution to wash out neutral red crystals which are not phagocytosed on the surfaces of the cells, adding 100 mu L of lysate (ethanol: glacial acetic acid ═ 1:1, v/v) into each hole, standing the cells in the dark place for 2h at room temperature, measuring the light absorption value at the wavelength of 540nm after the cells are dissolved, and taking a blank control group as 100% of the phagocytosis rate of the cells. The results are shown in FIGS. 9 to 10.

FIG. 9 is a microscopic image of phagocytosis of RAW264.7 by C-EPS and S-EPS-1 for 24h, and it can be seen from the image that RAW264.7 cells in the control group are mainly in round aggregation and hardly take up neutral red, while RAW264.7 cells treated by C-EPS, S-EPS-1 or LPS are expanded in size, protrude a large amount of pseudopodia and remarkably increase the amount of uptake of neutral red, which indicates that C-EPS and S-EPS-1 can enhance the phagocytosis activity of RAW264.7 cells to different degrees. As shown in the quantitative chart (figure 10), both C-EPS and S-EPS-1 can enhance the phagocytosis of neutral red by RAW264.7 cells in the concentration interval of 25-300 mug/mL, and the phagocytosis activity of the cells is stronger along with the increase of the concentration, so that certain dose dependence is presented. In addition, compared with C-EPS, the enhancement effect of S-EPS-1 on the phagocytic activity of RAW264.7 is more obvious. The results are combined to show that C-EPS and S-EPS-1 can enhance the phagocytic activity of RAW264.7 cells.

3. C-EPS and S-EPS-1 are tested for RAW264.7 cytokine expression, and the method comprises the following steps:

3.1, extracting total RNA of cells: RAW264.7 cells in logarithmic growth phase were treated in accordance with 1.5X 10⁶And (3) inoculating the cells/well in a 6-well plate, adding 2mL of cell suspension into each well, after overnight adherence, removing the culture solution, adding C-EPS or S-EPS-1 solutions with the concentrations of 50, 100 and 200 mu g/mL respectively, and performing blank and positive control group treatment in the same way as the method in the step 1. After the cells are cultured in an incubator at 37 ℃ for 24 hours, the 6-well plate is taken out to extract the total RNA of the cells.

The culture medium in the 6-well plate was aspirated, the cells were thoroughly washed 2 times with pre-cooled PBS, 500 μ L TRIzol reagent was added to each well, and the 6-well plate was gently shaken to completely cover the cell surface with TRIzol. And repeatedly blowing by using a pipette gun to enable cells to fall off from the pore plate, transferring the cell lysate to a marked enzyme-free centrifuge tube, standing for 5min at room temperature, adding 200 mu L of chloroform, uniformly mixing by turning upside down, vortexing for 10s, standing for 10min at room temperature again, and centrifuging for 15min at 12000rpm4 ℃. Subsequently, the upper layer colorless liquid was transferred to a new centrifuge tube, an equal volume of pre-cooled isopropanol was added, mixed by inversion from top to bottom and left standing at room temperature for 10min, and then centrifuged at 12000rpm at 4 ℃ for 10 min. The supernatant was discarded, and precooled 75% ethanol was added to the centrifuge tube to resuspend the RNA pellet at the bottom of the centrifuge tube, centrifuged at 12000rpm for 3min at 4 deg.C, and the supernatant was discarded. The RNA was dried to translucency at room temperature, dissolved and diluted by adding 40 μ l epc water, and purity and concentration of RNA were determined and recorded.

3.2, reverse transcription reaction: taking out the RNA template extracted in the step 3.1, the reaction liquid in the FSQ-301 kit and DEPC water, blowing and uniformly mixing each reaction liquid by using a liquid transfer gun after the RNA template, the reaction liquid and the DEPC water are melted, preparing a reaction system according to the table 7, and sequentially carrying out reaction I and reaction II after the preparation is finished, wherein the conditions of the reverse transcription reaction are set as shown in the table 8.

TABLE 7 preparation of reverse transcription reaction solution

Reaction solution	Volume of addition
		Reaction I: 4 XDN Master Mix	2.0μL
RNA	0.5μg
		DEPC water	5.5μL
Total volume	8.0μL
		Reaction II: the reaction solution obtained in the last step	8.0μL
5×RT Master Mix II	2.0μL
		Total volume	10.0μL

TABLE 8

Reaction temperature	Reaction time
		Reaction I: 37 deg.C	5min
Reaction II: 37 deg.C	15min
		50℃	5min
98℃	5min

3.3, real-time quantitative PCR reaction: separately taking out the cDNA obtained by reverse transcription reaction and corresponding primers (the primer sequences are shown in a table 9), QPK-201 reaction liquid in a kit, taking DEPC water out, placing on ice, preparing the reaction liquid in an 8-tube sterile and enzyme-killed way in a dark place according to the table 10 after the DEPC water is melted, centrifuging for a short time after the preparation is finished to prevent partial reaction liquid from being stained on the wall, then transferring the 8-tube to a fluorescence quantitative PCR instrument, setting according to the program in the table 11, and starting the reaction. After completion of the reaction, GAPDH was used as an internal reference gene, 2^-△△CtThe relative expression levels of IL-1. beta. IL-6 and TNF-. alpha.were calculated.

TABLE 9 PCR primer sequences

TABLE 10 real-time fluorescent quantitative PCR reaction system

Composition of matter	Adding amount of
		cDNA	2.0μL
SYBR Green Realtime PCR Mater Mix	10.0μL
		Forward primer	0.8μL
Reverse primer	0.8μL
		DEPC water	6.4μL
Total volume	20.0μL

TABLE 11 real-time fluorescent quantitative PCR reaction procedure

The data in each group of experiments were expressed as mean ± standard deviation (mean ± SD) and statistically processed using IBM SPSS 23.0 using One-way analysis of variance (One-way anova), and when the data satisfied homogeneity of variance, using Tukey HSD method for pairwise comparison, otherwise using Dunnett's T3 method for processing. When p < 0.05, the difference is considered statistically significant. The results are shown in FIG. 11.

IL-1 beta, IL-6 and TNF-alpha are important cytokines expressed by activated macrophages and are responsible for regulating the immune response in the host organism. In this example, gene transcription levels of IL-1 beta, IL-6 and TNF-alpha in RAW264.7 cells after 24h of stimulation by different concentrations of C-EPS and S-EPS-1 were determined, and the results are shown in FIG. 11. As can be seen, both C-EPS and S-EPS-1 can promote the expression of cytokines IL-1 beta, IL-6 and TNF-alpha on mRNA level of RAW264.7 cells in a concentration-dependent manner in a concentration range of 50-200. mu.g/mL compared with the blank control group, and the promotion effect of S-EPS-1 at a high concentration (200. mu.g/mL) on the expression of cytokines by RAW264.7 cells is almost the same as that of LPS in the positive control group. The results are combined, and the C-EPS and S-EPS-1 can not only activate macrophage RAW264.7, but also can regulate the expression of cytokines IL-1 beta, IL-6 and TNF-alpha in RAW264.7 cells on the mRNA level, so that the C-EPS and S-EPS-1 expressed by Bifidobacterium longum subspecies XZ01 have very considerable immunoregulation activity.

Example 5

The embodiment of the application provides an insulin resistance improving activity test of the decolored C-EPS crude polysaccharide in embodiment 2, which specifically comprises the following steps:

1. a blank control group and a C-EPS treatment group are set, only a cell culture medium is added into the control group, the C-EPS treatment group is that HepG2 cells are added with C-EPS crude polysaccharide in example 2 with different concentrations, after 24 hours of co-culture, the MTT method is used for detecting the cell activity, the influence of the C-EPS polysaccharide on the HepG2 cell activity is examined, and the result is shown in figure 12. As shown in FIG. 12, it was revealed that the C-EPS polysaccharide produced by strain XZ01 did not exert an influence on the cell activity when HepG2 cells were treated at a concentration of 50-600. mu.g/mL for 24 hours.

2. A blank control group, a C-EPS treatment group and a model group (TNF alpha) were set, the C-EPS treatment group is that HepG2 cells are treated by TNF alpha and C-EPS crude polysaccharide in example 2 with different concentrations, the model group is treated by TNF alpha to induce an insulin resistance model, and glucose concentration in cell supernatant is measured by using a glucose oxidase kit after 24 hours, and the result is shown in FIG. 13. As shown in FIG. 13, 25-100. mu.g/mL of C-EPS can increase the utilization and consumption of glucose by HepG2 cells after TNF alpha induction, i.e., the C-EPS produced by strain XZ01 can improve the insulin resistance of TNF alpha-induced HepG2 cells.

A blank control group, a C-EPS treatment group and a model group (TNF alpha) are set, the C-EPS treatment group is that HepG2 cells are treated by TNF alpha and C-EPS crude polysaccharide in example 2 at different concentrations, the model group is treated by TNF alpha to induce an insulin resistance model, the transcription levels of PI3K and IRS1 genes in the HepG2 cells are measured by qPCR after 24 hours, and the PI3K and IRS1 primer sequences are shown in Table 9. The results are shown in FIGS. 14 and 15. As shown in FIGS. 14 and 15, the expression of PI3K and IRS1 genes of HepG2 cells after TNF alpha induction can be up-regulated by C-EPS in the concentration range of 25-200 mug/mL, namely, the C-EPS generated by the strain XZ01 can improve the insulin resistance of HepG2 cells induced by TNF alpha.

In summary, in the embodiments of the present application, the strain XZ01 is first identified by morphological examination, determination of physiological and biochemical indicators, 16S rRNA sequencing and phylogenetic tree, the staining result indicates that the strain XZ01 is a gram-positive bacterium, the microscopic morphology of the gram-positive bacterium is typically rod-shaped or Y-shaped, the physiological and biochemical characteristics of the gram-positive bacterium are basically the same as those of the control strain bifidobacterium longum subspecies GDMCC1.248, the 16S rRNA sequencing sequence is highly similar (99.86%) to that of the bifidobacterium longum subspecies, and the phylogenetic tree indicates that the gram-positive bacterium is located in the same branch as that of the bifidobacterium longum subspecies JCM1217 and has very similar genetic relationship, so that the strain XZ01 is identified as the bifidobacterium longum subspecies.

Secondly, in the embodiment of the application, a DEAE-Cellulose 52 ion exchange column and a Sephacryl S-300HR allyl sephadex column are adopted to further fractionate and purify C-EPS to obtain a neutral polysaccharide component S-EPS-1 with high yield and sugar content, and then the chemical composition, purity, relative molecular mass, monosaccharide composition and proportion of the S-EPS-1 are characterized by combining with analysis methods such as ultraviolet spectrum, gel permeation chromatography, high performance liquid chromatography and the like, and the results are as follows:

1) after the C-EPS is purified by a DEAE-Cellulose 52 ion exchange column, 3 components are obtained in total, namely neutral polysaccharide EPS-1, acidic polysaccharide EPS-2 and EPS-3. And further purifying the 3 obtained components by using a Sephacryl S-300HR allyl sephadex column to obtain 4 components including S-EPS-1, S-EPS-2, S-EPS-and S-EPS-4.

2) The ultraviolet scanning result shows that the S-EPS-1 does not contain impurities such as nucleic acid, protein and the like, and meanwhile, the measurement results of a sulfuric acid phenol method and a BCA protein quantitative method also show that the S-EPS-1 component does not contain protein, and the sugar content is 99.20 +/-1.21%.

3) The molecular weight and monosaccharide composition of S-EPS-1 are analyzed by adopting a high performance liquid chromatography, and the S-EPS-1 is found to be homogeneous polysaccharide with the relative molecular mass of 6.38 multiplied by 10^5 Da; the analysis result of monosaccharide composition shows that S-EPS-1 mainly comprises mannose, glucose and a small amount of rhamnose and galactose, and the molar ratio is 11.85:5.60:0.46: 0.68.

Thirdly, the examples of the present application found that C-EPS and S-EPS-1 have immunomodulatory activity, the main results are summarized below:

1) C-EPS and S-EPS-1 can affect the morphology of RAW264.7 cells.

2) C-EPS and S-EPS-1 can activate RAW264.7 cells and can promote NO production in a concentration-dependent manner within a concentration range of 25-300 mu g/mL.

3) The C-EPS and S-EPS-1 can enhance the phagocytic activity of RAW264.7 cells, and particularly enhance the phagocytic capacity of RAW264.7 to neutral red.

4) The C-EPS and S-EPS-1 can obviously up-regulate the expression of RAW264.7 cell factors IL-1 beta, IL-6 and TNF-alpha at mRNA level, and have ideal immunoregulation activity.

Fourth, exopolysaccharides produced by strain XZ01 provided herein can improve TNF α -induced insulin resistance in HepG2 cells.

The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.