CN103981232B - The preparation method of a kind of functional bacillus acidophilus's Peptidoglycan and anti-inflammatory activity application thereof - Google Patents
The preparation method of a kind of functional bacillus acidophilus's Peptidoglycan and anti-inflammatory activity application thereof Download PDFInfo
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Abstract
The invention discloses preparation method and the anti-inflammatory activity application thereof of a kind of functional bacillus acidophilus's Peptidoglycan, feature is to comprise the following steps: be that CGMCC No.8920 by volume percentage ratio 2.0 4.0% is seeded in MRS culture medium by bacillus acidophilus's preserving number, at 37 DEG C after Anaerobic culturel 18 24h, it is centrifuged 15 20min in 4000 8000g and removes fermentation liquid, precipitation thalline will add the SDS solution of 4wt% again, at 100 DEG C, Pintsch process 30min removes protein ingredient, in precipitate, add the solution of trichloroacetic acid of 10wt%, remove teichoic acid；Taking precipitate is after 4 DEG C of dialyzed overnight desalinations, by precipitate after 80 DEG C of pre-freeze 6h, putting into rapidly in 80 120Pa dried 24h in vacuum freeze, the powder obtained is product, and advantage is that the inflammatory reaction causing LPS has and preferably presses down scorching regulatory function.
Description
Technical field
The present invention relates to the preparation method of Peptidoglycan, especially relate to the preparation of a kind of functional bacillus acidophilus's Peptidoglycan
Method and anti-inflammatory activity application thereof.
Background technology
Lactic acid bacteria, as the important field planting type beneficial bacteria of intestinal tract of a class, is widely used in fermented food processing, especially
It is yogurt, cheese, Pickles etc..Enteral lactic acid bacteria and the substantial connection of host health, more and more by domestic and international microorganism
Scholar illustrates, especially carrys out stimulating immune system in induction about the release of immune regulatory factor and plays its infection, antitumor etc.
Biological effect aspect.In terms of lactic acid bacteria is to the immunoregulatory activity of host, recently between research some viable bacteria of discovery and dead bacterium also
It is not significantly different from, thus it is speculated that Peptidoglycan (PGN) that one of reason playing its immunoregulation effect is probably in its cell wall, phosphorus
The materials such as Teichaic acid (LTA) and S-layer proteins.Probiotic bacteria Peptidoglycan, owing to what it acted on played in immunomodulating probes into relatively
Lacking, the most subtle is paid close attention to by increasing researchers.
Bacillus acidophilus (Lactobacillus acidophilus) is as a kind of important beneficial bacteria of intestinal tract, its cell
Wall Peptidoglycan is as a kind of immunostimulant, by regulating intestinal canal immune system, can enhancing body non-specific immunity, right
Body health has important function.But, the preparation method of bacillus acidophilus's Peptidoglycan and anti-inflammatory activity research have no relevant report
Road, also for there is the functional food relevant with bacillus acidophilus's Peptidoglycan on market.
Summary of the invention
The technical problem to be solved is to provide a kind of inflammatory reaction causing LPS to be had and preferably presses down scorching tune
The preparation method of functional bacillus acidophilus's Peptidoglycan of joint function and anti-inflammatory activity application thereof.
The present invention solves the technical scheme that above-mentioned technical problem used: a kind of functional bacillus acidophilus's Peptidoglycan
Preparation method, specifically includes following steps: by bacillus acidophilus (Lactobacillus acidophilus) preservation in source, ocean
Number it is CGMCC No.8920, the by volume inoculum concentration of percentage ratio 2.0-4.0%, it is seeded in MRS culture medium, at 37 DEG C, detests
After oxygen cultivates 18-24h, it is centrifuged 15-20min in 4000-8000g, removes thalline fermentation liquid, then precipitation thalline will add matter
Amount mark is the SDS solution of 4%, and Pintsch process 30min at 100 DEG C removes protein ingredient；Taking precipitate, adds in precipitate
Enter the solution of trichloroacetic acid that mass fraction is 10%, remove teichoic acid；Taking precipitate, by precipitate in 4 DEG C of dialyzed overnight desalinations
After, by precipitate after-80 DEG C of pre-freeze 6h, put into rapidly in 80-120Pa dried 24h in vacuum freeze,
To powder be bacillus acidophilus's Peptidoglycan.
Described precipitation thalline is 1g:100ml with the ratio of described SDS solution, and described precipitation thalline is with described
The ratio of solution of trichloroacetic acid is 1g:100ml.
The anti-inflammatory activity application of above-mentioned functions bacillus acidophilus's Peptidoglycan, this functional bacillus acidophilus's Peptidoglycan is to greatly
The inflammatory reaction that enterobacterial lipopolysaccharide (LPS) causes has and preferably presses down scorching regulatory function, i.e. bacillus acidophilus's Peptidoglycan pair
INOS and COX-2 protein expression is inhibited, and effectively pressing down scorching concentration on Raw264.7 Macrophage Model is 100-200
μg/mL。
This functional bacillus acidophilus's Peptidoglycan finds through immunity section, and this functional bacillus acidophilus's Peptidoglycan is effective
Reducing the iNOS expression at small intestinal intestinal wall cell, the scorching dosage that effectively presses down on mouse model is 100mg/kg/d.
Compared with prior art, it is an advantage of the current invention that:
The preparation method of a kind of functional bacillus acidophilus's Peptidoglycan of the present invention and anti-inflammatory activity application thereof, proving first should
Bacillus acidophilus's Peptidoglycan also exists and reduces the inflammatory reaction that e. coli lipopolysaccharide causes, and lives for lactic acid bacteria regulating intestinal canal immunity
Property, the release of the regulation and control related immune factor carrys out stimulating immune system and plays its infection, develops the lactic acid bacteria new type of health factor, merit
Can property food or medicine offer theoretical foundation.
Bacillus acidophilus (Lactobacillus acidophilus), bacterial strain is WZ-D1, and preserving number is CGMCC
No.8920, was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation on 03 14th, 2014
Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Accompanying drawing explanation
Fig. 1 is bacillus acidophilus's thalline scanning electron microscope (SEM) photograph in embodiment 1；
Fig. 2 is bacillus acidophilus's Peptidoglycan scanning electron microscope (SEM) photograph of preparation in embodiment 1；
Fig. 3 is that the HPLC of bacillus acidophilus's Peptidoglycan in embodiment 1 analyzes collection of illustrative plates；
Fig. 4 is bacillus acidophilus's Peptidoglycan infrared scan spectrogram in embodiment 2；
Fig. 5 is pressing down of the RAW264.7 macrophage inflammatory factor that in embodiment 3, LPS is induced by bacillus acidophilus's Peptidoglycan
The Experimental Comparison figure made；
Fig. 6 is the test of the expression difference of iNOS in the enterocyte in different disposal group in embodiment 3ICR mouse experiment
Comparison diagram；From left to right it is followed successively by Normal group, Escherichia coli infected group, Peptidoglycan treatment group.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment 1
The preparation of bacillus acidophilus's Peptidoglycan
It is CGMCC No.8920 by bacillus acidophilus (Lactobacillus acidophilus) preserving number in source, ocean
The by volume inoculum concentration of percentage ratio 2.0-4.0%, is seeded in MRS culture medium, at 37 DEG C, after Anaerobic culturel 18-24h, in
4000-8000g is centrifuged 15-20min, removes thalline fermentation liquid, and then take precipitation thalline 1g adding mass fraction is the SDS of 4%
Solution, Pintsch process 30min at 100 DEG C, remove protein ingredient；Taking precipitate, adding mass fraction in precipitate is 10%
Solution of trichloroacetic acid, remove teichoic acid；Taking precipitate, by precipitate after 4 DEG C of dialyzed overnight desalinations, by precipitate in-80
After DEG C pre-freeze 6h, putting into rapidly in 80-120Pa dried 24h in vacuum freeze, the powder obtained is addicted to acid
Lactobacillus peptidoglycan.Sample Storage in-40 DEG C of refrigerators with standby.Wherein Fig. 1 is bacillus acidophilus's thalline scanning electron microscope (SEM) photograph, figure
Bacillus acidophilus's Peptidoglycan scanning electron microscope (SEM) photograph that 2 is preparation.Bacillus acidophilus's Peptidoglycan compares bacillus acidophilus to be had the most not
With, the form of ellipse bar is destroyed, and Fig. 2 is the Peptidoglycan composition extracted.
The extraction ratio of Peptidoglycan is calculated by following equation, and the Peptidoglycan extraction ratio obtained by this example is 26.5%.
Extraction ratio (%)=(Peptidoglycan dry weight/dry cell weight) × 100
The purity of Peptidoglycan pass through HPLC method validation, Agilent Agilent1200Infinity equipment, Bole company
Aminex HPX-87H sugar post (300 × 7.8mm), sample size 20ul, sample introduction concentration 1mg/ml, column temperature 37 DEG C, eluent is 5mM
Sulfuric acid solution, flow velocity 0.5ml/min, time 20min, detection method 225nm ultraviolet spectrometry.Result is as it is shown on figure 3, derive from
The HPLC appearance time of lactobacillus acidophilus cells's wall Peptidoglycan is 7.618min.Peptidoglycan purity obtained by this example is
99.9%.
Purity (%)=(Peptidoglycan go out peak area/detection sample always go out peak area) × 100
Above-mentioned bacillus acidophilus (Lactobacillus acidophilus), bacterial strain is WZ-D1, and preserving number is CGMCC
No.8920, was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation on 03 14th, 2014
Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Specific embodiment 2
The bacillus acidophilus's Peptidoglycan prepared by above-described embodiment 1 carries out mass spectrum (MALDI-TOF/TOF MS) detection
Analyze；Bacillus acidophilus's Peptidoglycan powder carries out amino acid analysis and infrared (RI) detection.
1, the molecular weight of Peptidoglycan and substructure
By bacillus acidophilus's Peptidoglycan solution that 1 lli is 1mg/ml and the 2,5-dihydroxy benzenes of 1 microlitre 50mg/ml
Formic acid mixes, and after mix homogeneously, takes 1 microlitre mixed liquor loading, carries out mass spectral analysis.INSTRUMENT MODEL FLEX-PC, type is
ultraflex TOF/TOF.The molecular weight of the Peptidoglycan detected by the program is 875.260Da, and concrete analysis is such as table 1 below institute
Show.
Table 1 Peptidoglycan ion fragment table
2, analysis of amino acids
Utilize A200Amino Nova amino-acid analyzer, analyze the amino acid whose composition of Peptidoglycan and relative amount height,
Thus confirm amino acid whose composition in Peptidoglycan.Specifically comprise the following steps that and the hydrochloric acid of 10mg Peptidoglycan 6M is hydrolyzed at 110 DEG C
24h, the product after the hydrolysis composition of the aminoacid in amino-acid analyzer analyzes hydrolysate and relative amount again.Result such as table
Shown in 2,
The relative amount of primary amino acid (concentration >=80mg/g) in table 2 Peptidoglycan
3, the characteristic group in IR spectrum scanning Peptidoglycan sample
Peptidoglycan powder sample is placed in agate mortar uniform with KBr powder mixed grinding, is pressed into thin slice through tablet machine,
In wave number 4,000-400cm-1Carry out IR spectrum scanning, to determine characteristic group.Infrared scan spectrogram as shown in Figure 4,
1070cm-1, there is C-O-C glycosidic bond absworption peak in point bands of a spectrum region.1650cm-1, there is C=O stretching vibration in point bands of a spectrum region,
Carbonyl peak and the superposition peak of COC；1540cm-1There is N-H bending vibration, amino peak (protonated amino) in point bands of a spectrum region, shows
Peptidoglycan exists amido link (peptide bond) functional group.Comprehensive above detection method, draws the knot of bacillus acidophilus's muramyl peptide polysaccharide
Structure model is as follows,
Specific embodiment 3
The anti-inflammatory activity analysis of bacillus acidophilus's Peptidoglycan of above-described embodiment 1 preparation
(1) the inflammatory model regulation activity of the macrophage Raw264.7 that LPS induces by bacillus acidophilus's Peptidoglycan is utilized,
The expression analyzing iNOS and COX-2 in end-product changes, and whether analysis Peptidoglycan has good suppression Inflammatory substances is released
The property let live.
Six orifice plates cultivate Raw264.7 cell, and normal group is that adherent rate reaches 60-80% normal macrophages RAW264.7
Cell, extracts cytoplasm protein after continuing to cultivate 18h；LPS stimulation group is to reach in adherent rate that the RAW264.7 of 60-80% is huge to be bitten
The LPS adding final concentration of 100ng/ml in cell induces, and extracts cytoplasm protein after continuing to cultivate 18h；Peptidoglycan is repaiied
Multiple group reaches to be simultaneously introduced in the macrophage of 60-80% the LPS of final concentration of 100ng/ml and final concentration successively for adherent rate
Be 25 μ g/ml, the Peptidoglycan of the Concentraton gradient of 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, continue to cultivate and extract Cytoplasm after 18h
Albumen.
The albumen of different groups is carried out Western blot analysis (Western Blot), to destination protein iNOS and
COX-2 expressing quantity is analyzed.The RAW264.7 macrophage inflammatory factor that LPS is induced by bacillus acidophilus's Peptidoglycan
Inhibitory action result is as it is shown in figure 5, for the cell producing inflammatory reaction, the expression of itself iNOS and COX-2 can be significantly larger than
Blank group (left side first row in diagram), and in Peptidoglycan process group, find its macrophage Raw264.7 to LPS induction
Inflammatory model have and significantly regulate activity, its activity is, when PNG concentration is at 100-200 μ g/ml, can significantly inhibit
Inflammatory factor iNOS and the expression of COX-2.Wherein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is house keeping protein, at all cells
In expression stable, use as internal reference.
(2) ICR mouse experiment
Experiment select 60 neonatal rats of 25-28g ICR mice (each 30 of male and female), be divided into 4 groups: Normal group, cause a disease
Property coli-infection group, Peptidoglycan treatment group, often group 15.After raising 5 days, infected group every the syringe per os of improvement
Gavage 0.50mL Escherichia coli liquid (5 × 108CFU/ml)；Treatment group gavages 0.50mL Escherichia coli on the 5th day
Liquid (5 × 108CFU/ml), after 1h, gavage bacillus acidophilus's Peptidoglycan suspension with the dosage of 100mg/kg/d, play every day next day
Gavage the Peptidoglycan of same dose；Normal control gavages 0.50mL normal saline after raising 5 days.After 25 days, mouse is implemented peace
Happy dead, the immunity section carrying out intestinal cell detects iNOS expression in different groups.Result as shown in Figure 6, institute in figure
The speckle of display, is inflammatory factor iNOS confirmed by immunization method expression in mouse intestinal cell, at gavage peptide
In the mouse intestinal cell of polysaccharide, the expression of iNOS is significantly lower than Escherichia coli process group.Visible, real by mice
Test the effect also having absolutely proved Peptidoglycan played in intestinal antiinflammatory.
Certainly, described above not limitation of the present invention, the present invention is also not limited to the example above.The art
Change that those of ordinary skill is made in the essential scope of the present invention, retrofit, add or replace, also should belong to present invention protection
Scope.
Claims (2)
1. the preparation method of functional bacillus acidophilus's Peptidoglycan, it is characterised in that specifically include following steps: by ocean
Bacillus acidophilus (Lactobacillus acidophilus) preserving number in source is CGMCC No.8920, by volume percentage ratio
The inoculum concentration of 2.0-4.0%, is seeded in MRS culture medium, at 37 DEG C, after Anaerobic culturel 18-24h, in 4000-8000g from
Heart 15-20min, removes thalline fermentation liquid, then will add the SDS solution that mass fraction is 4% in precipitation thalline, at 100 DEG C
Pintsch process 30min, removes protein ingredient；Taking precipitate, adds the trichloroacetic acid that mass fraction is 10% molten in precipitate
Liquid, removes teichoic acid；Taking precipitate, by precipitate after 4 DEG C of dialyzed overnight desalinations, by precipitate after-80 DEG C of pre-freeze 6h, fast
Speed is put in 80-120Pa dried 24h in vacuum freeze, and the powder obtained is bacillus acidophilus's Peptidoglycan,
Described precipitation thalline is 1g:100ml with the ratio of described SDS solution, described precipitation thalline and described trichloroacetic acid
The ratio of solution is 1g:100ml.
2. functional bacillus acidophilus's Peptidoglycan according to claim 1 answering in terms of preparation anti-inflammatory activity medicine
With, it is characterised in that: the inflammatory reaction that this functional bacillus acidophilus's Peptidoglycan causes at preparation suppression e. coli lipopolysaccharide
Application in terms of medicine, the i.e. bacillus acidophilus's Peptidoglycan application in terms of preparation suppression iNOS and COX-2 protein expression medicine.
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| CN115068510A (en) * | 2022-05-24 | 2022-09-20 | 宁波大学 | Extraction method of lactobacillus lipoteichoic acid and anti-inflammatory activity application thereof |
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| CN101144062A (en) * | 2006-09-15 | 2008-03-19 | 内蒙古农业大学 | Lactobacillus casei strain and application for products thereof in bird immunity |
| CN101717806A (en) * | 2009-12-03 | 2010-06-02 | 内蒙古双奇药业股份有限公司 | Integrated peptidoglycan and preparation method thereof |
| CN103396496A (en) * | 2013-07-16 | 2013-11-20 | 宁波大学 | Preparation method of modified lactobacillus acidophilus peptidoglycan |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101144062A (en) * | 2006-09-15 | 2008-03-19 | 内蒙古农业大学 | Lactobacillus casei strain and application for products thereof in bird immunity |
| CN101717806A (en) * | 2009-12-03 | 2010-06-02 | 内蒙古双奇药业股份有限公司 | Integrated peptidoglycan and preparation method thereof |
| CN103396496A (en) * | 2013-07-16 | 2013-11-20 | 宁波大学 | Preparation method of modified lactobacillus acidophilus peptidoglycan |
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