CN116574664A - Application of bifidobacterium longum extracellular polysaccharide in promoting proliferation of lactic acid bacteria - Google Patents
Application of bifidobacterium longum extracellular polysaccharide in promoting proliferation of lactic acid bacteria Download PDFInfo
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- CN116574664A CN116574664A CN202310466419.5A CN202310466419A CN116574664A CN 116574664 A CN116574664 A CN 116574664A CN 202310466419 A CN202310466419 A CN 202310466419A CN 116574664 A CN116574664 A CN 116574664A
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- Prior art keywords
- bifidobacterium longum
- lactobacillus
- extracellular polysaccharide
- eps
- proliferation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/225—Lactobacillus
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Abstract
The invention discloses an application of bifidobacterium longum extracellular polysaccharide in promoting lactobacillus proliferation. The experiment shows that the extracellular polysaccharide of the bifidobacterium longum XZ01 strain has a promoting effect on the proliferation of lactobacillus such as lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus reuteri, lactobacillus rhamnosus and the like, can promote the proliferation of the beneficial lactobacillus in vivo and in vitro, can be used for promoting the growth of the lactobacillus or preparing a preparation for promoting the growth of the lactobacillus, and can be further used for regulating intestinal flora and promoting the proliferation of the beneficial lactobacillus. The invention not only enriches the application range of the extracellular polysaccharide of bifidobacterium longum, but also is beneficial to the regulation of the probiotic bacterial strain and the development of the metazoan products.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly, it relates to the use of bifidobacterium longum exopolysaccharides for promoting the proliferation of lactic acid bacteria.
Background
Probiotics are living microorganisms that are intended to be implanted in the human body to produce beneficial effects by modulating intestinal flora, immunity, etc. Although probiotics have beneficial health effects, as a microorganism, the growth state and physical and chemical activities of the microorganism are all affected by the culture environment, and the probiotic effect produced by the microorganism is unstable in the intestinal tract of a human body under the influence of different hosts and different living habits. Therefore, the research targets are turned to large molecules, small molecules, secretion or the like which play a role on probiotics, and the research targets have important market value.
The processed probiotic metabolite components (such as extracellular polysaccharide) are collectively called metazoan, and have activity and physiological function superior to that of the original bacteria. The extracellular polysaccharide of the probiotics refers to a natural secondary metabolite secreted in the growth and development of the probiotics, is a water-soluble polysaccharide secreted outside the cell wall in the growth and metabolism process of the probiotics, and is an important component of the physiological function of the bacteria. Exopolysaccharides (EPS) and capsular polysaccharides (Capsular polysaccharide, CPS) are classified according to their ability to bind to cell walls.
The bifidobacterium longum is separated from normal human intestinal tracts, is an anaerobic gram-negative bacterium, has the effects of relieving intestinal inflammation, improving diabetes and other probiotics, researches the functions of extracellular polysaccharide of the bifidobacterium longum, and has important significance in enriching the application fields of the bifidobacterium longum and developing metazoan products.
Disclosure of Invention
The invention aims to provide support for enriching the application of bifidobacterium longum extracellular polysaccharide and the development of metaplasia products, and provides the application of bifidobacterium longum extracellular polysaccharide in the aspect of promoting the proliferation of lactic acid bacteria.
It is a first object of the present invention to provide the use of bifidobacterium longum exopolysaccharide in promoting proliferation of lactic acid bacteria.
A second object of the present invention is to provide the use of bifidobacterium longum exopolysaccharide in the preparation of a formulation for promoting proliferation of lactic acid bacteria.
A third object of the present invention is to provide the use of bifidobacterium longum exopolysaccharide in modulating the intestinal flora or in the preparation of a formulation for modulating the intestinal flora.
A fourth object of the present invention is to provide a formulation for promoting proliferation of lactic acid bacteria or regulating intestinal flora.
The above object of the present invention is achieved by the following technical scheme:
experiments show that extracellular polysaccharide of bifidobacterium longum XZ01 strain has a promoting effect on proliferation of lactobacillus such as lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus reuteri, lactobacillus rhamnosus and the like, and can promote proliferation of beneficial lactobacillus in vivo and in vitro. Thus, the present application protects the following applications of bifidobacterium longum exopolysaccharide.
The invention claims the application of bifidobacterium longum exopolysaccharide in promoting the proliferation of lactic acid bacteria.
The invention also claims the use of bifidobacterium longum exopolysaccharide in the preparation of a formulation for promoting proliferation of lactic acid bacteria.
The invention also claims the use of bifidobacterium longum exopolysaccharide in modulating intestinal flora or in the preparation of a formulation for modulating intestinal flora.
Specifically, the lactobacillus is one or more of lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus reuteri and lactobacillus rhamnosus.
Specifically, the bifidobacterium longum extracellular polysaccharide is extracted from bifidobacterium longum XZ01 strain which is deposited in the cantonese province microorganism strain collection with the deposit number of GDMCC No:61618.
specifically, the polysaccharide content of the bifidobacterium longum extracellular polysaccharide is 99.20 +/-1.21%, and the relative molecular mass is 6.38X10 5 Da。
Specifically, the bifidobacterium longum extracellular polysaccharide includes mannose, glucose, rhamnose and galactose; the molar ratio of mannose, glucose, rhamnose and galactose was 11.85:5.60:0.46:0.685.
specifically, the preparation method of the bifidobacterium longum extracellular polysaccharide comprises the following steps:
s1, collecting a culture solution of bifidobacterium longum XZ01 strain;
s2, removing thalli in the culture bacterial liquid by centrifugation, inactivating enzymes in the obtained culture supernatant, precipitating polysaccharide and removing protein, and purifying to obtain the bifidobacterium longum extracellular polysaccharide.
Specifically, the culture medium for culturing the bifidobacterium longum XZ01 strain is MRS liquid culture medium, the culture temperature is 37 ℃, and the culture is carried out in an anaerobic environment for 48 hours.
Specifically, the centrifugation conditions for removing the thalli in the culture bacterial liquid by centrifugation are as follows: centrifuge at 8000rpm for 30min at 4 ℃.
Specifically, the method for inactivating the enzyme in the obtained culture supernatant is as follows: the sterile culture supernatant was collected and water-bath was performed at 100℃for 15min to inactivate the enzymes.
Specifically, the method for precipitating polysaccharide comprises the following steps: concentrating the culture supernatant after enzyme inactivation under reduced pressure to 1/10 of the original volume, adding 3 times of ice ethanol into the concentrated solution, and standing at 4 ℃ overnight; the next day, the alcohol precipitation part is centrifuged for 30min at 4 ℃ and 8000rpm, the precipitate is collected, and the precipitate is redissolved by a proper amount of ultrapure water to obtain XZ01 extracellular polysaccharide crude extract.
Specifically, the method for removing protein comprises the following steps: deproteinizing the XZ01 extracellular polysaccharide crude extract by adopting a Sevage reagent; the specific operation is as follows: adding 1/5 volume of Sevage reagent (chloroform: n-butanol=4:1, v/v) into the crude extract, shaking vigorously for 15min, centrifuging at 4deg.C and 4500rpm for 30min, and collecting upper polysaccharide solution; repeating the steps until the protein layer completely disappears; transferring the deproteinized solution into a dialysis bag with a molecular weight cut-off of 8000Da, and dialyzing with purified water for 48h to remove small molecular impurities; changing water once every 4 hours during the period; after the completion of dialysis, the crude extract sample of extracellular polysaccharide was freeze-dried to obtain a crude extract sample, which was sealed and stored in a desiccator.
Specifically, the purification comprises DEAE cellulose-52 ion exchange column purification and Sephacryl S-300HR dextran gel purification.
The invention also provides a preparation for promoting the proliferation of lactobacillus or regulating intestinal flora, which contains bifidobacterium longum extracellular polysaccharide.
In particular, the bifidobacterium longum extracellular polysaccharide is extracted from bifidobacterium longum XZ01 strain.
The invention has the following beneficial effects:
the experiment shows that the extracellular polysaccharide of the bifidobacterium longum XZ01 strain has a promoting effect on the proliferation of lactobacillus such as lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus reuteri, lactobacillus rhamnosus and the like, can promote the proliferation of beneficial lactobacillus in vivo or in vitro, can be used for promoting the proliferation of lactobacillus or preparing a preparation for promoting the proliferation of lactobacillus, and can be further used for regulating intestinal flora and promoting the proliferation of beneficial lactobacillus.
The invention not only enriches the application range of the extracellular polysaccharide of bifidobacterium longum, but also is beneficial to the regulation of the probiotic bacterial strain and the development of the metazoan products.
Drawings
FIG. 1 is a liquid chromatogram of the monosaccharide derivatization of the extracellular polysaccharide S-EPS-1 of Bifidobacterium longum.
FIG. 2 is a liquid chromatogram of PMP derivatization of a mixed monosaccharide standard; 1: mannose; 2: rhamnose; 3: glucuronic acid; 4: galacturonic acid; 5: glucose; 6: galactose; 7: xylose; 8: arabinose; 9: fucose.
FIG. 3 shows the extracellular polysaccharide S-EPS-1 of Bifidobacterium longum 1 H NMR spectrum.
FIG. 4 is a HSQC spectrum of the extracellular polysaccharide S-EPS-1 of Bifidobacterium longum.
FIG. 5 is a COSY spectrum of the extracellular polysaccharide S-EPS-1 of Bifidobacterium longum.
FIG. 6 shows the effect of inulin on the growth of different lactic acid bacteria strains; wherein, graph A is the effect of inulin on Lactobacillus acidophilus Lactobacillus acidophilus CIP 76.13 growth; panel B shows the effect of inulin on the growth of Lactobacillus casei Lacticaseibacillus casei ATCC 393; panel C shows the effect of inulin on the growth of Lactobacillus plantarum Lactobacillus plantarum DSM 10667; panel D shows the effect of inulin on the growth of Lactobacillus reuteri Lactobacillus reuteri JCM 1112; panel E shows the effect of inulin on the growth of Lactobacillus rhamnosus Lactobacillus rhamnosus JSM 1136.
FIG. 7 shows the effect of the extracellular polysaccharide S-EPS-1 of Bifidobacterium longum on the growth of different lactic acid bacteria species; wherein, the graph A is the effect result of S-EPS-1 on the growth of lactobacillus acidophilus Lactobacillus acidophilus CIP 76.13; FIG. B is a graph showing the effect of S-EPS-1 on the growth of Lactobacillus casei Lacticaseibacillus casei ATCC 393; panel C shows the effect of S-EPS-1 on the growth of Lactobacillus plantarum Lactobacillus plantarum DSM 10667; panel D shows the effect of S-EPS-1 on the growth of Lactobacillus reuteri Lactobacillus reuteri JCM 1112; panel E shows the effect of S-EPS-1 on the growth of Lactobacillus rhamnosus Lactobacillus rhamnosus JSM 1136.
FIG. 8 is the effect of Bifidobacterium longum extracellular polysaccharide S-EPS-1 on mouse body weight; in the figure, P < 0.01 is shown.
FIG. 9 is the effect of Bifidobacterium longum extracellular polysaccharide S-EPS-1 on the growth of lactic acid bacteria in the intestinal tract of mice; wherein, graph A is the effect of S-EPS-1 on Lactobacillus acidophilus growth; panel B shows the effect of S-EPS-1 on Lacticaseibacillus casei growth; panel C shows the effect of S-EPS-1 on Lactiplantibacillus plantarum growth; panel D shows the effect of S-EPS-1 on Lactobacillus reuteri growth; panel E shows the effect of S-EPS-1 on Lactobacillus rhamnosus growth; FIG. F is the result of the effect of S-EPS-1 on Lactobacillus growth; in the figure, P < 0.01, P < 0.001, and P < 0.0001 are shown.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The bifidobacterium longum XZ01 in the embodiment is bifidobacterium longum subspecies Bifidobacterium longum subsp.longum, which is preserved by pharmaceutical analysis laboratory of university of Zhongshan (Shenzhen); the strain is also deposited in the Guangdong province microorganism strain collection with the deposit number of GDMCC No:61618.
the strains Lactobacillus acidophilus CIP 76.13, lacticaseibacillus casei ATCC 393, lactobacillus plantarum DSM 10667, lactobacillus rhamnosus JSM1136 described in the examples were all deposited by the university of Zhen pharmaceutical analysis laboratory.
The strain Lactobacillus reuteri JCM 1112 described in the examples was deposited by the sugar industry technology center of the institute of biological and medical engineering, academy of sciences, guangdong.
MRS medium was purchased from Qingdao sea Bo biotechnology Co., ltd, under the trade designation HB0384-5; inulin was purchased from Guangdong Long-range medical biotechnology Co., ltd, under the product number 10644011102555; columbia platelets were purchased from Guangdong Cryptographic microorganisms Inc. under the product number CP0160; BHI medium was purchased from Guangzhou Xiangbo biotechnology Co., ltd, under the brand name Ruifer extract; the bacterial genome DNA extraction kit is purchased from Tiangen Biochemical technology (Beijing) limited company, and the product number is DP302-02; taq PCR Mix premix was purchased from Shanghai Biotechnology Co., ltd, cat# B639295-0001; c57BL/6 mice were purchased from Guangdong Yaokang and fed to the SPF environment of the laboratory animal center in the east school of the university of Zhongshan; PBS was purchased from the company bioinstrumentation limited, sense ltd, guangzhou, brand: GIBCO, cat No. C10010500BT; the real-time fluorescent quantitative PCR premixing reagent and the fecal genomic DNA extraction kit are all purchased from Guangzhou special research biotechnology Co-Ltd, and the brand is Norpraise; isopropyl Alcohol (AR) was purchased from guangzhou institute of biotechnology limited under the brand name national drug group; RNase-free water was purchased from Guangzhou institute of Biotechnology Inc. under the brand name SRB.
EXAMPLE 1 resuscitation culture and identification of Bifidobacterium longum XZ01 and lactic acid bacteria strains
1. Resuscitating culture of strains
Wiping a frozen tube of bifidobacterium longum XZ01, lactobacillus acidophilus Lactobacillus acidophilus CIP 76.13, lactobacillus casei Lacticaseibacillus casei ATCC 393, lactobacillus plantarum Lactobacillus plantarum DSM 10667, lactobacillus reuteri Lactobacillus reuteri JCM 1112 and lactobacillus rhamnosus Lactobacillus rhamnosus JSM1136 strains by using absorbent cotton soaked with 75% alcohol in an ultra-clean workbench, airing the alcohol, unwinding a wound sealing film, heating the tube around a tube opening by using an alcohol lamp, opening the frozen tube, respectively adding 500 mu L of sterile water into the freeze-dried powder of the strains, after the freeze-dried powder is dissolved and mixed uniformly, picking a bacterial solution by using a sterile inoculating loop, and culturing for 48 hours at 37 ℃ under anaerobic conditions. After 48h, individual colonies were picked from streak plates and cultured in new Columbia blood plates at 37℃under anaerobic conditions for 48h.
2. Identification of strains
Extraction of bacterial genomic DNA: genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit, and specific procedures are described in the specification. After obtaining the genome DNA of the strain, detecting the DNA concentration and purity, and taking a DNA sample with the OD260/OD280 ratio of 1.7-1.9 for identifying the strain.
The invention identifies the strain by PCR amplification of the 16S rRNA post-sequencing alignment of the strain. PCR amplification the primers were as follows:
27F：AGAGTTTGATCCTGGCTCAG
1492R：GGTTACCTTGTTACGACTT
the reaction system for PCR amplification is shown in Table 1 below:
TABLE 1PCR amplification reaction System
The reaction procedure for PCR amplification is shown in Table 2 below:
TABLE 2PCR amplification reaction procedure
The PCR reaction products were subjected to 16S rRNA sequencing by the attorney docket biological engineering (Shanghai). Sequencing shows that the strain recovered by the method has no problem.
EXAMPLE 2 extraction, purification and characterization of Bifidobacterium longum extracellular polysaccharide
The invention obtains the crude extract sample of the extracellular polysaccharide of the bifidobacterium longum through the steps of fermenting, centrifugally removing thalli, inactivating enzymes by boiling water bath, precipitating by ethanol, removing proteins, dialyzing, freeze-drying and the like. After the sample is decolorized, the decolorized crude polysaccharide is fractionated and purified by adopting a DEAE cellulose DE-52 anion exchange column and Sephacryl S-300HR sephadex, and neutral polysaccharide components with higher sugar content are collected and analyzed by combining ultraviolet spectrum (UV) and High Performance Liquid Chromatography (HPLC) for purity, relative molecular mass, monosaccharide composition and proportion.
1. Extraction of extracellular polysaccharide of bifidobacterium longum
The bifidobacterium longum XZ01 strain preserved at the temperature of minus 80 ℃ is taken out and inoculated into an MRS liquid culture medium, and is cultured for 24 hours at the temperature of 37 ℃ for activation, and after the step of activating for two generations is repeated, the bifidobacterium longum XZ01 strain is inoculated into the MRS liquid culture medium for expansion culture at the inoculum size of 2% (v/v), and the culture temperature is 37 ℃ and the culture time is 48 hours.
After 48h of incubation, the broth was centrifuged (8000 rpm,30min,4 ℃) to remove the cells, the sterile supernatant was collected and the enzyme in the broth was inactivated by water bath at 100℃for 15 min; concentrating the supernatant after water bath to 1/5 volume under reduced pressure, adding 3 times volume of glacial ethanol into the concentrated solution, and standing at 4 ℃ overnight; the precipitate was collected by centrifugation (8000 rpm,30min,4 ℃) the next day and redissolved with an appropriate amount of ultrapure water to obtain a crude extract of XZ01 extracellular polysaccharide.
Deproteinizing the obtained XZ01 extracellular polysaccharide crude extract by using a Sevage reagent, wherein the specific operation is as follows: adding 1/5 volume of Sevage reagent (chloroform: n-butanol=4:1, v/v) into the crude extract, shaking vigorously for 15min, centrifuging (4000 rpm,30min, 4deg.C), and collecting upper polysaccharide solution; repeating the steps until the protein layer completely disappears; transferring the deproteinized solution into a dialysis bag (molecular weight cut-off: 8000 Da), dialyzing with purified water for 48 hr to remove small molecule impurities, and changing water every 4 hr; after dialysis, it is freeze-dried to obtain a crude extract sample of extracellular polysaccharide, abbreviated as crude polysaccharide, which is sealed and stored in a desiccator. And (3) decoloring the obtained crude polysaccharide to obtain decolored crude polysaccharide.
2. Purification of bifidobacterium longum extracellular polysaccharide
(1) DEAE cellulose-52 ion exchange column purification
Pretreatment of ion exchange cellulose: 20g of DEAE cellulose DE-52 ion exchange cellulose is weighed into a glass beaker, a sufficient amount of ultrapure water is added to swell until the volume is unchanged, the solution is treated with 0.5mol/L NaOH solution for 1h, then the solution is repeatedly washed with ultrapure water to be neutral, and then the solution is treated with 0.5mol/L HCl solution for 1h, so that the solution is washed with ultrapure water to be neutral.
And (3) column loading: the specification of the chromatographic column is 2.5 multiplied by 30cm, the chromatographic column is vertically fixed on an iron stand after being cleaned, 1/3 column volume of ultrapure water is added, and a liquid outlet is opened; then slowly pouring the filler into a chromatographic column along a glass rod, allowing the chromatographic column to naturally settle, lightly beating the column by using a soft rod to remove bubbles, repeatedly adding the filler to the position of 5cm at the top end of the chromatographic column, stopping filling the column, connecting a constant flow pump, and balancing the column by using ultrapure water at a flow rate of 1.0 mL/min.
Loading: weighing 500mg of decolored crude polysaccharide, fully dissolving in 10mL of ultrapure water, centrifuging at 4500rpm for 15min, taking supernatant and filtering with a 0.45 μm filter membrane; and (5) loading, and starting the constant flow pump to elute.
Eluting: the eluent is NaCl solution with concentration of 0, 0.05, 0.1, 0.3 and 0.5mol/L, the flow rate is 1.0mL/min, 8mL is collected for each tube, and 25 tube fractions are collected for each concentration gradient.
And (3) detection: the collected sample was subjected to absorbance detection at 490nm by phenol sulfate method, and an elution curve was drawn.
And (3) collecting: and according to the elution curve, combining the components at different peak sections, respectively dialyzing and freeze-drying to obtain polysaccharides with different components, and placing the polysaccharides in a dryer for standby.
After the decolored crude polysaccharide is eluted by NaCl with different concentration gradients, 3 obvious elution peaks are obtained, and the elution peaks are named EPS-1, EPS-2 and EPS-3 in sequence; wherein EPS-1 is uncharged neutral polysaccharide, and EPS-2 and EPS-3 are acidic polysaccharide with a certain negative charge. Further purification was performed by gel chromatography.
(2) Sephacryl S-300HR dextran gel purification
Sephacryl S-300HR is a pretreatment filler, and is stored in 20% ethanol, and cleaned with ultrapure water before use.
And (3) column loading: the specification of the chromatographic column is 1.6X90 cm, the chromatographic column is vertically fixed on an iron stand after being cleaned, 1/3 column volume of ultrapure water is added, and a liquid outlet is opened; and then slowly pouring the liquid into a chromatographic column along a glass rod, allowing the liquid to naturally subside, lightly beating the column by using a soft rod to remove bubbles, repeatedly adding the filler to a proper height, connecting a constant flow pump after the liquid level of the filler is kept calm, flushing the sephadex column by using a 0.1mol/L NaCl solution with 5 times of the column volume so as to remove residual ethanol in the filler, and further compacting the filler of the gel column.
Loading: 50mg of each component purified by DEAE 52 was weighed and fully dissolved in 10mL of ultrapure water, centrifuged at 4500rpm for 15min, and the supernatant was collected and filtered with a 0.45 μm filter membrane; and (5) loading, and starting the constant flow pump to elute.
Eluting: the eluent was 0.1mol/L NaCl solution at a flow rate of 0.5mL/min, and 4mL was collected per tube.
And (3) detection: the collected sample liquid is detected by a sulfuric acid phenol method in a separation tube, and an elution curve is drawn.
And (3) collecting: collecting eluent under an elution peak according to an elution curve; concentrating, dialyzing and freeze-drying the eluent to obtain the purified extracellular polysaccharide.
Sephacryl S-300HR dextran gel purification to obtain 4 components, S-EPS-1-S-EPS-4 respectively; wherein, the S-EPS-1 component has higher yield and sugar content, and takes the S-EPS-1 component as a research object to carry out chemical composition measurement, molecular weight measurement, monosaccharide composition analysis and the like on the component.
3. Analysis of Bifidobacterium longum extracellular polysaccharide S-EPS-1
The invention constructs standard yeast (y=6.5226x+0.0496, R) with glucose by a phenol sulfate method 2 = 0.9932), the percentage content of S-EPS-1 was 99.20 ±1.21%.
The invention adopts HPGPC method to determine the relative molecular mass of the bifidobacterium longum extracellular polysaccharide, and according to a linear regression equation (lgMw= -0.7789TR+10.417, R 2 = 0.9937), the retention time tr= 5.921min is taken to the calculation to obtain a relative molecular mass of S-EPS-1 of 6.38x10ζ5da.
The invention adopts High Performance Liquid Chromatography (HPLC) to analyze the monosaccharide composition of the bifidobacterium longum extracellular polysaccharide S-EPS-1. The liquid chromatogram of the extracellular polysaccharide S-EPS-1 of Bifidobacterium longum is shown in figure 1, and the peak diagram of the derivatization product of the mixed monosaccharide standard is shown in figure 2. Comparing the liquid chromatogram of the extracellular polysaccharide (figure 1) with the peak diagram of the derivatization product of the mixed monosaccharide standard (figure 2), according to the retention time, the extracellular polysaccharide S-EPS-1 of the bifidobacterium longum is mainly composed of mannose, glucose and a small amount of rhamnose and galactose, and according to the peak area, the molar ratio of the monosaccharides is mannose: rhamnose: glucose: galactose=11.85:0.46:5.60:0.68.
Taking 30mg of freeze-dried neutral polysaccharide S-EPS-1 sample, adding 0.55mL of D2O, vortex-dissolving, freezing, freeze-drying, and repeatedly performing freeze-drying exchange for 3 times to remove protons in the sample; the sample was completely dissolved in 0.55mL D 2 In O, after centrifugation at 10000rpm for 5min, the supernatant was filtered through a 0.45 μm filter and transferred to a 5mm nuclear magnetic tube, and its 1D NMR signals (1H NMR and 13C NMR) and two-dimensional NMR (HSQC, HMBC and COSY) spectra were detected using 600MHz superconducting nuclear magnetic. As shown in FIG. 3, the 1H NMR spectrum of the Bifidobacterium longum extracellular polysaccharide S-EPS-1 shows that 7 proton signals exist in the heterocephalic subregion of the Bifidobacterium longum extracellular polysaccharide S-EPS-1, which indicates that the S-EPS-1 contains 7 types of sugar residues, and the seven types of sugar residues are respectively named as A, B, C, D, E, F, G.
The HSQC spectrum and COSY spectrum of extracellular polysaccharide S-EPS-1 of Bifidobacterium longum are shown in figures 4 and 5, respectively, and the long double spectrum is obtained according to the HSQC spectrum (figure 4) and COSY spectrum (figure 5)Other signals of 1H and 13C in each sugar residue in the extracellular polysaccharide of the bifidobacterium are attributed, and the results are shown in Table 3, which suggest that 7 sugar residues possibly exist in the S-EPS-1 sugar chain and are all alpha-configuration glycosidic bonds. At the position of 1 In the H NMR spectrum, delta 3.0-5.3 ppm is a characteristic signal region of polysaccharide, and the 5.22ppm is more likely to correspond to the hetero-head proton signal peak in alpha-D-mannose respectively, which further supports the results of monosaccharide composition analysis. At the position of 13 In the C NMR spectrum, the chemical shift of the alpha-type glycosidic bond ranged from 97 to 103ppm, and as can be seen from Table 3, the chemical shift of the 7 signal peaks were all within the range, indicating that 7 sugar residues were present in the S-EPS-1 sugar chain and all were in the alpha configuration, which is consistent with the results in 1H NMR.
TABLE 3 chemical shifts (ppm) of 1H and 13C of Bifidobacterium longum XZ01 exopolysaccharide S-EPS-1
Example 3 in vitro cell experiments
1. Preparation of extracellular polysaccharide S-EPS-1 solution of bifidobacterium longum and inulin solution in-vitro bacterial proliferation experiment
Preparation of Bifidobacterium longum exopolysaccharide S-EPS-1 solution: taking 100mg of bifidobacterium longum extracellular polysaccharide S-EPS-1 in an MRS culture medium to prepare a culture solution containing 11% of bifidobacterium longum extracellular polysaccharide S-EPS-and 2% of MRS culture medium, and filtering and split charging the culture solution with a microporous filter membrane of 0.22 mu m for later use. 100mg of inulin is additionally taken in MRS culture medium to prepare culture solution containing 1% and 2% of inulin, and the culture solution is filtered and split-packed by a microporous filter membrane with the diameter of 0.22 mu m for standby.
2. In vitro bacterial proliferation experimental procedure
Diluting lactobacillus strain (Lactobacillus acidophilus Lactobacillus acidophilus CIP 76.13, lactobacillus casei Lacticaseibacillus casei ATCC 393, lactobacillus plantarum Lactobacillus plantarum DSM 10667, lactobacillus reuteri Lactobacillus reuteri JCM 1112 and Lactobacillus rhamnosus Lactobacillus rhamnosus JSM 1136) to OD 600 After =0.2, the mixture was diluted 5 times again, the plates were divided into two portions, and the mixture was added in equal volumesA solution of inulin and exopolysaccharide in the same concentration; placing in anaerobic incubator, detecting OD of bacteria at different time points 600 And a growth curve is drawn.
In order to explore the influence of bifidobacterium longum exopolysaccharide S-EPS-1 on the growth of lactic acid bacteria, 5 strains of lactic acid bacteria which are most widely used are selected, lactobacillus acidophilus CIP 76.13,Lacticaseibacillus casei ATCC 393,Lactiplantibacillus plantarum DSM 10667,Lactobacillus reuteri JCM 1112 and Lactobacillus rhamnosus JSM1136, inulin is used as a positive control, and the influence of the addition of 0, 0.5% and 1% concentration inulin and the addition of 0, 0.5% and 1% concentration bifidobacterium longum exopolysaccharide S-EPS-1 on the growth of different lactic acid bacteria strains is observed under anaerobic conditions. The effect of inulin on the growth of different lactobacillus strains is shown in figure 6, and the effect of Bifidobacterium longum extracellular polysaccharide S-EPS-1 on the growth of different lactobacillus strains is shown in figure 7. As can be seen from fig. 6 and 7, inulin has a small effect on the proliferation of Lactobacillus acidophilus CIP 76.13,Lactobacillus reuteri JCM 1112 and Lactobacillus rhamnosus JSM1136 with increasing concentration, has a weak stimulating effect on the growth of Lactiplantibacillus plantarum DSM 10667, and does not promote the growth of Lacticaseibacillus casei ATCC 393, but rather has an inhibiting effect. While bifidobacterium longum exopolysaccharide S-EPS-1 has a stimulatory effect on the growth of Lactobacillus acidophilus CIP 76.13,Lacticaseibacillus casei ATCC 393 and Lactiplantibacillus plantarum DSM 10667, with less incremental impact on Lactobacillus reuteri JCM 1112 and Lactobacillus rhamnosus JSM 1136. The results show that the bifidobacterium longum extracellular polysaccharide S-EPS-1 can promote the growth of different lactic acid bacteria strains in vitro, and the effect is superior to that of fructose inulin.
Example 4 in vivo animal experiments
1. In vivo lactic acid bacterial strain qRT-PCR assay
C57B167 mouse faeces were collected, total DNA was obtained using a faecal genomic DNA extraction kit and then assayed using a qPCR system. Primer sequences are shown in table 4 below:
TABLE 4qRT-PCR primer sequences
The RT-PCR reaction system is shown in Table 5 below:
TABLE 5RT-PCR reaction System
The procedure for the RT-PCR reaction is shown in Table 6 below:
TABLE 6RT-PCR reaction procedure
2. Animal experiment flow
15 female six-week-old C57BL/6 mice were taken and randomly divided into 2 groups (7-8 per group) according to body weight after one week of adaptive feeding: normal Control group (Control) and Bifidobacterium longum exopolysaccharide S-EPS-1 group (BL-EPS), normal group lavage 200. Mu.L PBS, BL-EPS group lavage 200. Mu.g/mL of Bifidobacterium longum exopolysaccharide S-EPS-1 solution daily, and mouse sampling after 9 days.
In order to explore the influence of the extracellular polysaccharide S-EPS-1 of the bifidobacterium longum in the stomach on the safety of mice, the invention detects the change of the body weight of the mice after 9 days of stomach filling, and the result is shown in figure 8, and the figure 8 shows that the body weight of the mice after the extracellular polysaccharide S-EPS-1 of the bifidobacterium longum in the stomach is stable and has a tendency to rise, and the extracellular polysaccharide S-EPS-1 of the bifidobacterium longum has no influence on the safety of the mice on the ninth day.
In order to explore the influence of bifidobacterium longum exopolysaccharide S-EPS-1 on lactobacillus growth in vivo, the invention uses RT-PCR experiment to detect the influence of bifidobacterium longum exopolysaccharide S-EPS-1 on lactobacillus genus and lactobacillus relative abundance in mice, and the result is shown in figure 9. As can be seen from fig. 9, the bifidobacterium longum exopolysaccharide S-EPS-1 stimulated the growth of Lactobacillus in mice, and from the strain level, the bifidobacterium longum exopolysaccharide S-EPS-1 stimulated the growth of Lactobacillus acidophilus and Lacticaseibacillus casei without significant effect on Lactobacillus reuteri and Lactobacillus rhamnosus, and no detection of Lactiplantibacillus plantarum.
The results show that the bifidobacterium longum extracellular polysaccharide S-EPS-1 can promote the growth of lactobacillus in vivo or in vitro, can be used for regulating intestinal flora and can improve the growth of lactobacillus of probiotics.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The application of Bifidobacterium longum exopolysaccharide in promoting lactobacillus proliferation is provided.
2. Use of bifidobacterium longum exopolysaccharide in the preparation of a formulation for promoting proliferation of lactic acid bacteria.
3. Use of bifidobacterium longum exopolysaccharide for modulating intestinal flora or for the preparation of a formulation for modulating intestinal flora.
4. The use according to claim 1 or 2, wherein the lactic acid bacteria are one or more of lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus reuteri, lactobacillus rhamnosus.
5. The use according to any one of claims 1 to 4, wherein the extracellular polysaccharide of bifidobacterium longum is extracted from a strain XZ01 of bifidobacterium longum deposited with the collection of microbial strains in the cantonese province under the accession number GDMCC No:61618.
6. the use according to claim 5, wherein the extracellular polysaccharide of Bifidobacterium longum has a sugar content of 99.20 + -1.21% and a relative molecular mass of 6.38X10 5 Da。
7. The use according to claim 5, wherein the bifidobacterium longum extracellular polysaccharide comprises mannose, glucose, rhamnose and galactose; the molar ratio of mannose, glucose, rhamnose and galactose was 11.85:5.60:0.46:0.685.
8. the use according to claim 5, wherein the preparation method of the bifidobacterium longum extracellular polysaccharide comprises the following steps:
s1, collecting a culture solution of bifidobacterium longum XZ01 strain;
s2, removing thalli in the culture bacterial liquid by centrifugation, inactivating enzymes in the obtained culture supernatant, precipitating polysaccharide and removing protein, and purifying to obtain the bifidobacterium longum extracellular polysaccharide.
9. The use according to claim 8, wherein the purification comprises DEAE cellulose-52 ion exchange column purification and Sephacryl S-300HR dextran gel purification.
10. A formulation for promoting the proliferation of lactic acid bacteria or for regulating the intestinal flora, characterized in that it comprises an extracellular polysaccharide of bifidobacterium longum.
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