CN106635924A - Preparation and application of lactobacillus rhamnosus exopolysaccharide - Google Patents
Preparation and application of lactobacillus rhamnosus exopolysaccharide Download PDFInfo
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Abstract
The invention belongs to the technical field of polysaccharide medicaments in microbiology, specifically relates to microbial exopolysaccharide, and particularly relates to preparation and application of lactobacillus rhamnosus exopolysaccharide. The lactobacillus rhamnosus exopolysaccharide (EPS) disclosed by the invention is a lactic acid bacteria strain which is screened out through a colony thread-drawing method by combining extracellular total sugar yield and is capable of producing exopolysaccharide, and belongs to lactobacillus rhamnosus subspecies. After the strain is fermented in a skimmed milk culture medium at constant temperature and in a standing mode, the final crude polysaccharide yield can reach 2.9g/L; the fermented crude polysaccharide is purified through anion exchange chromatography and gel chromatography to obtain a water-soluble polysaccharide component EPS, wherein the purity of the water-soluble polysaccharide component EPS is 95.3%, and the molecular weight of the water-soluble polysaccharide component EPS is 1.4*105Da. Experiments in vitro and vivo show that the lactobacillus rhamnosus exopolysaccharide EPS has the effects of promoting macrophage proliferation and treating colonitis, and has wide application value in an aspect of serving as an immunological enhancing and anti-inflammatory medicine.
Description
Technical field
The present invention relates to the polysaccharide medicine technical field in microbiology, and in particular to a kind of Microbial exopolysaccharides,
Preparation and purposes more particularly, to a kind of exocellular polysaccharide of rhamnose lactobacillus.
Background technology
Polysaccharide is all the time the indispensable part of living organism, is higher plant, animal cell membrane and micro-
The important composition of biological cell wall.Recently as membranization functional study, the chemical research of immune substance and novel drugs resource
Research and development, and the exploration of polysaccharide is deepened continuously, find polysaccharide not only complex structure, and show diversified
Biologically active, such as immunological regulation, anti-inflammatory, antitumor, anti-aging and antibacterial.
Exopolysaccharides Produced by Lactic Acid Bacteria is the class macromolecular carbohydrate that lactic acid bacteria is secreted into outside cell membrane during growth metabolism
Compound, the microorganism wall that depends on having forms pod membrane, referred to as capsular polysaccharide；The culture medium that then enters having forms mucus,
Referred to as glue polysaccharide, they are the metabolites that microorganism adapts to environment.Recent study find, originating in lactic acid bacterium it is extracellular
Polysaccharide is the good source of food-grade polysaccharide, and not only the quality structure and local flavor of dairy products are had a major impact, and as thickener,
Stabilizer, emulsifying agent, gelling agent and NMF are widely used in various food.Research shows that Exopolysaccharides Produced by Lactic Acid Bacteria also has
Have antitumor, serum cholesterol-lowering, strengthen the different physiological roles such as body immunity, thus succeeded in developing can property food or
Medicine has broad application prospects.The originating in lactic acid bacterium of the extracellular polysaccharide of nature is quite varied, such as dairy products, fermentation food
Product, animal intestinal etc..The lactic acid bacteria culturers of high-yield extracellular polysaccharide usually produce Acarasiales strain, such as lactobacillus lactis
(Lactobacillus lactis), Lactobacillus rhamnosus (Lactobacilus rhamnosus), streptococcus lactis
(Streptococcus lactis) etc..But the viscosity and quality structure of acidified milk not has direct related to the yield of polysaccharide, and
It is relevant with the molecular weight of polysaccharide, the length of chain, degree of branching and monose composition etc..Different, the lactic acid according to the species of monose composition
Bacterium exopolysaccharide can be divided into two big class：Homotype polysaccharide and special-shaped polysaccharide.Only same monose be polymerized to be formed be homotype it is many
Sugar, the homotype polysaccharide composition monose in Exopolysaccharides Produced by Lactic Acid Bacteria is mainly glucose or fructose.And it is different according to glucosides key type,
Four kinds can be divided into：(1) glucan (Dextrans)：Glucose residue is formed by connecting, is connected with C3 by α -1,6- glycosidic bonds
Different degrees of branch, and seldom have branch on C2 and C4；(2) beta glucan (β-glucans)：Glucose residue by β-
1,3- glycosidic bond is formed by connecting；(3) levulan (Fructans)：Residue of fructose is by β -2,6- glycosidic bonds or β -2,1- glucosides
It is bonded to form；(4) glycan (Mutans) is become：By α -1,3- glycosidic bonds are formed by connecting straight-chain polysaccharide (straight chain glucose residue
Middle glucosides linkage content>50%), containing a small amount of α -1,6 branches；(5) other kinds of polysaccharide.
The content of the invention
Lactobacillus rhamnosus CGMCC No.13497 in the present invention, are according to bacterium colony fiber elongation method from 8 kinds of commercially available yoghourts
Obtain with extracellular total reducing sugar yields screening, code name is ZLW-1.The specific name of bacterial strain：Lactobacillus rhamnosus Lactobacilus
Rhamnosus, deposit number is：CGMCC No.13497, preservation date：26 days 12 years 2016, depositary institution was：China is micro-
Biological inoculum preservation administration committee common micro-organisms center, preservation address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The Lactobacillus rhamnosus of one plant of extracellular polysaccharide that extracellular total reducing sugar yields screening is arrived is combined by bacterium colony fiber elongation method
CGMCC No.13497, learn that the bacterial strain belongs to Lactobacillus rhamnosus subspecies by 16S rDNA Sequence Identifications.
The invention also discloses a kind of Lactobacillus rhamnosus exocellular polysaccharide, it is by Lactobacillus rhamnosus strain of the invention
CGMCC No.13497 ferment and obtain.Preferred method is：By the Lactobacillus rhamnosus strain CGMCC No.13497 of the present invention
37 DEG C of standings are incubated in degreasing milk medium, and the initial gross separation of culture supernatant Jing obtains Thick many candies, and Thick many candies are passed through
The method purifying of DEAE-52 ion-exchange chromatographies and Sephacryl S-300 gel chromatographies, obtains final product.The method for more having choosing is：Will
Lactobacillus rhamnosus CGMCC No.13497 37 DEG C of constant temperature quiescent culture 18-24h in degreasing milk medium, nutrient solution Jing are centrifuged
Except thalline obtains supernatant, supernatant Jing Sevag method removing proteins, 3-5 times of volume ethanol precipitation is obtained thick after vacuum freeze drying
Polysaccharide；The Thick many candies are 1mol/L NaCl through DEAE-52 cation exchange chromatographies, mobile phase, and flow velocity is SV=1/
120；Elution fraction Jing molecular cut offs carry out ultrafiltration for the milipore filter of 5000Da, again through Sephacryl after 4 times of volumes of concentration
S-300 gel chromatographies, mobile phase is distilled water, and flow velocity is SV=1/500；Elution fraction Jing molecular cut offs are 7000Da
Bag filter flowing water dialysis 36-48h, dialyse trapped fluid Jing vacuum freeze dryings, obtain final product.
The bacterial strain carries out constant temperature quiescent culture in the degreasing milk medium of 2L, and final Thick many candies yield can reach 2.9g/
L.The method of Thick many candies Jing DEAE-52 anion-exchange chromatographies and Sephacryl S-300 gel chromatographies after culture is after purification
The exopolysaccharide component (Extracellular polysaccharide, EPS) that a kind of purity is 95.3% is obtained, its
Molecular weight is 1.4 × 105Da.Inside and outside experiment shows that Lactobacillus rhamnosus exocellular polysaccharide EPS of the present invention has promotion macrophage thin
Born of the same parents breed, and treat the effect of colitis, and as immunopotentiator, anti-inflammatory drug aspect has wide using value.
Macrophages in vitro proliferation experiment shows that the Lactobacillus rhamnosus exocellular polysaccharide EPS of the present invention has promotion macrophage
The activity of cell propagation.Described macrophage is the cells of mouse source macrophage RAW 264.7.
Anti-ulcerative colitis experiment in vivo shows that the Lactobacillus rhamnosus exocellular polysaccharide EPS of the present invention can substantially subtract
The inflammatory reaction of slow ulcerative colitis, significantly reduces the disease activity index of the colitis mice of TNBS inductions, while right
The colitis of TNBS inductions has good therapeutic action.
The Lactobacillus rhamnosus exocellular polysaccharide EPS of the present invention can also be used to prepare the oral or injection system with antiphlogistic effects
Agent, it can also be used to prepare the health products with immune enhancing function.
The Lactobacillus rhamnosus exocellular polysaccharide EPS of the present invention is preferably prepared with following method：By Lactobacillus rhamnosus CGMCC
No.13497 37 DEG C of constant temperature quiescent culture 18-24h in degreasing milk medium, nutrient solution Jing bactofugations body obtains supernatant,
Supernatant Jing Sevag method removing proteins, 3-5 times of volume ethanol precipitation, obtain Thick many candies after vacuum freeze drying；Thick many candies are passed through
DEAE-52 ion-exchange chromatographies (Φ 2.6cm × 30cm, BV=120mL) are separated, and mobile phase is 1mol/L NaCl, and flow velocity is SV
=1/120；Elution fraction Jing molecular cut offs carry out ultrafiltration for the milipore filter of 5000Da, are passing through after 4 times of volumes of concentration
Sephacryl S-300 gel chromatographies (Φ 1.6cm × 80cm, BV=120mL) are purified, and mobile phase is distilled water, and flow velocity is SV
=1/500, elution fraction Jing molecular cut off is the bag filter flowing water dialysis 36-48h of 7000Da, and dialysis trapped fluid Jing vacuum is cold
Lyophilized dry rear acquisition fine work sugar EPS.
The pharmacodynamics test and result of polysaccharide of the present invention is presented herein below：
First, macrophages in vitro proliferation experiment
Using CCK-8 detection methods.By the macrophage RAW 264.7 of in vitro culture, Jing after cell count and dilution
To 5 × 106The cell suspension of individual/mL, adds 100 μ L macrophage suspensions, in 37 DEG C, 5%CO in 96 orifice plates per hole2Cell is trained
In foster case cultivate 6h to macrophage it is adherent after, per hole add 20 μ L EPS (final concentration of 50,100,200,400 μ g/mL),
Control groups (plus physiological saline of 20 μ L) are concurrently set, supernatant is drawn and discarded to 3 multiple holes of per group of setting after culture 24h,
100 μ L CCK-8 solution (DMEM are added per hole：CCK-8=10:1), continue to cultivate after 1.5h, enzyme mark is used at 450nm wavelength
Instrument determines light absorption value.As a result accompanying drawing 6 is seen.
As seen from Figure 6, the growth shape of the cells of RAW 264.7 after the EPS 24h for adding is detected using CCK-8 methods
Condition.EPS has the significantly effect of stimulating proliferation to the cell growths of RAW 264.7, and with dose dependent, living cells quantity is bright
The aobvious blank group more than negative control.And the macrophage quantity of the EPS dosing groups of 400 μ g/mL is the 1.78 of blank control group
Times.Test result indicate that, the lower macrophage proliferation effects of RAW 264.7 of EPS effects are fairly obvious.
2nd, internal anti-ulcerative colitis experiment
1. the foundation of rat colitis model
By the SD rats chloral hydrate anesthesia of 300mg/kg dosage, TNB (TNBS) is dissolved in afterwards
In 40% ethanol, be injected in mouse Colon position, after mouse is inverted into 30s, it is ensured that TNBS is fully absorbed.
The disease index change of 2.TNBS inducing colitis mouse
Take the SD rats that body weight is 180-200g and be randomly divided into 4 groups, 10 per group, with aseptic water dissolves Lactobacillus rhamnosus
Exocellular polysaccharide EPS makes EPS solution (20mg/kg).Group is set to TNBS model groups, blank group, TNBS+EPS groups, TNBS+
SASP groups, per group of 10 animals.During successive administration, blank group and model group are administered daily physiological saline, and EPS groups are given daily
Medicine EPS.Administration SASP (100mg/kg) daily of SASP groups, each group successive administration 14 days.Rat body weight and loose and watery stool are recorded daily
Thin degree, and calculate rat disease activity index.Disease activity index is calculated according to following table.Draw disease activity index-when
Between change curve.As a result as shown in Figure 7.
Disease index
As seen from Figure 7, after EPS administrations, the symptom of ulcerative colitis is relieved in rat body, disease
Sick activity index is in the trend being decreased obviously.Compared to model group, the final disease activity index of EPS administration groups have dropped 3.1
Times, as a result show that the rat colonitis that Lactobacillus rhamnosus exocellular polysaccharide EPS of the present invention is induced TNBS have one significantly
Therapeutic action.
Description of the drawings
Fig. 1 is the polysaccharide yield of ten strains of lactic acid bacteria screened in the present invention
Fig. 2 is Lactobacillus rhamnosus CGMCC No.13497 polysaccharide accumulations during the fermentation and biomass variety
Fig. 3 is elution curve of the Thick many candies on DEAE-52 anion-exchange columns
Fig. 4 is elution curve of the Thick many candies on Sephacryl S-300 gel chromatography columns
Fig. 5 is the HPLC collection of illustrative plates of Lactobacillus rhamnosus exocellular polysaccharide EPS
Fig. 6 is that Lactobacillus rhamnosus exocellular polysaccharide EPS changes to the multiple of macrophage proliferation
Fig. 7 is that Lactobacillus rhamnosus exocellular polysaccharide EPS changes to the disease activity index of rat
Specific embodiment
Embodiment 1
First, the screening and identification of sugared bacterial strain are produced
1. yoghurt example pretreatment
Strain isolation sample is 8 kinds of commercially available yoghourts.Take Yoghourt fluid sample 5mL to add containing the sterile peptones of 50mL 0.1%
In the conical flask of water, 20min is stood after vibration, 4 DEG C save backup.
2. bacterium colony fiber elongation method primary dcreening operation
Take fluid sample 2mL and mixing, gradient dilution 10 are vibrated in the sterile peptone water of 20mL 0.1%5Times, take 100 μ L
MRS plating mediums are coated, 37 DEG C of constant temperature are inverted culture 72h, and picking amounts to 156 single bacterium colonies that are sticky and having obvious wire drawing
It is forwarded to MRS slant mediums, 37 DEG C of incubated 48h, 4 DEG C save backup.
3. bacterial classification microscopy
Smear, Grain stain are carried out to slide from a small amount of thalline of inclined-plane picking, thalline is then examined under a microscope
Form.Selection microscopy form size rule is homogeneous, and the bacterial strain without miscellaneous bacteria carries out secondary screening.
4. bacterial strain secondary screening
The separation strains obtained from flat board are inoculated in MRS fluid nutrient mediums and cultivate 24h, then connect by 3% inoculum concentration
Plant in 50mL MRS Shake flask mediums, the quiescent culture 18h in 37 DEG C of constant incubators.20mL nutrient solutions are taken, boiling water bath adds
Hot 10min, is cooled to room temperature, and 10000 × g centrifugation 10min take supernatant film and are concentrated into 5mL, add 1mL Servag examinations
Agent, 4 DEG C stand overnight.Supernatant is taken after 10000 × g centrifugation 10min, is settled to after 10mL with distilled water, Phenol sulfuric acid procedure is surveyed
Determine the content of total reducing sugar, DNS methods determine content of reducing sugar.And polyoses content=total sugar content-the content of reducing sugar in solution.Root
Wire-drawing performance is combined according to polysaccharide yield, 10 plants of wire drawing phenomenons is chosen substantially, the of a relatively high bacterial strain of total sugar content is carried out further
Research.Bacterial strain screening result is shown in Fig. 1.
As seen from Figure 1, when carrying out primary dcreening operation to the fiber elongation method that extracellular polysaccharide strains are used, according to the wire drawing of each bacterial strain
We coat the dilution of eight kinds of commercially available yoghourts on MRS flat boards to feature, filter out 156 plants of bacterium with obvious wire drawing phenomenon
Strain, determines that 156 plants of bacterium are gram positive bacteria by Grain stain then.And detached 156 plants of bacterium on flat board are forwarded to into MRS
After cultivating in fluid nutrient medium, take 18h zymotic fluids and boil inactivation, Sevag method removing proteins, alcohol precipitation dialysis is led to after distilled water constant volume
Cross Phenol sulfuric acid procedure and determine total sugar content, DNS methods determine content of reducing sugar, so as to calculate the yield of polysaccharide.List in accompanying drawing 1
Polysaccharide yield relatively large number of ten bacterial strains, and the polysaccharide yield that bacterial strain 5-5,8-4 and 6-19 compare other bacterial strains is higher,
In combination with wire-drawing performance, we choose this three plants of bacterium and carry out ensuing experimental study.
5. bacterial classification 16S rDNA identifications
Strain idenfication is completed by 16S rDNA sequence analyses.Picking bacterial strain 5-5 slant cultures are appropriate, add to 5mL
In MRS fluid nutrient mediums, 37 DEG C of overnight incubations take 2mL bacterium solutions, and 5000 × g centrifugation 5min abandon supernatant, add in precipitation
300 μ L cell pyrolysis liquids, add 10 μ L Proteinase Ks, 56 DEG C of water-bath 1h.After 100 DEG C of boiling water baths heating 10min, 12000 × g from
Heart 10min, takes supernatant and enters performing PCR reaction.Primer is synthesized by Nanjing Genscript Biotechnology Co., Ltd..Primer sequence is such as
Under：
Upstream primer：5'-CGGCTACCTTGTTACGACTT-3'
Downstream primer：5'-AGAGTTTGATCCTGGCTCAG-3'
PCR primer is delivered after purification Nanjing Genscript Biotechnology Co., Ltd. and is sequenced.Measure the 16S of bacterial strain 5-5
RDNA sequences.The bacterial 16 S reported in sequencing result blast program and US National Biotechnology Information center database
RDNA compares, and as a result shows, 16S rDNA sequences and Lactobacillus rhamnosus subspecies Lactobacilus of bacterial strain 5-5
The homology of rhamnosus strain HFI-K2 is 99.7%.
Can be obtained by above qualification result, Lactobacillus rhamnosus CGMCC No.13497 are Lactobacillus rhamnosus subspecies
(Lactobacilus rhamnosus), the bacterium is preserved in Chinese microorganism strain preservation management committee on December 26th, 2016
Member's meeting common micro-organisms center (CGMCC), preserving number：CGMCC No.13497.
Embodiment 2
It is prepared by the fermentation of Lactobacillus rhamnosus exocellular polysaccharide
1. Lactobacillus rhamnosus CGMCC No.13497 fermented and cultureds in 2L fermentation flasks
(1) preparation of seed culture medium
MRS fluid nutrient mediums：Peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, K2HPO42g/L, lemon
Acid two ammonium 2g/L, sodium acetate 5g/L, glucose 20g/L, Tween 80 1mL/L, MgSO4·7H2O 0.58g/L, MnSO4·4H2O
0.25g/L, adjusts pH to 6.4.MRS slant mediums：Add 1.8% agar on the basis of MRS solid mediums.
(2) culture of seed liquor
Bacterial strain needed for experiment is seeded to into MRS inclined-planes from the glycerol tube of -20 DEG C of preservations, is placed in 37 DEG C of incubators and is trained
After foster 24h, 5ml MRS fluid nutrient mediums are seeded to a little with oese picking, are placed in 37 DEG C of incubators and cultivate after 16h, turn
50mL MRS fluid nutrient mediums are connected to, are placed in 37 DEG C of incubators and are cultivated after 16h, 4 DEG C save backup.
(3) preparation of fermentation medium
2L skim milks are poured in 3L fermentation flasks, gauze plug sealing, in 105 DEG C, 20min carries out high pressure steam sterilization,
Normal temperature is placed in after cooling standby.
(4) 2L demulsifications culture medium fermented and cultured
16h inoculums in MRS liquid fat culture mediums are seeded in 2L degreasing milk mediums with 5% inoculum concentration.Put
37 DEG C of culture 24h in constant incubator.Biomass, total sugar content and content of reducing sugar are determined every 2h sampling 3mL.It is biological
Amount is determined by dry and wet weight method.Polyoses content=total sugar content-content of reducing sugar.Total sugar content is determined by Phenol sulfuric acid procedure,
Content of reducing sugar is determined by DNS methods.
As a result Fig. 2 is seen. as seen from Figure 2, during the fermentation polysaccharide is constantly being accumulated, and accumulation rate is bright after 8h
Aobvious to accelerate, yield tends towards stability after 18h, is finally reached 2.9g/L；Biomass rises rapidly in 4h-12h, 12h it
After tend towards stability, reach maximum 2.8g/L in 20h.As a result show to be sent out in Lactobacillus rhamnosus CGMCC No.13497 scales
During ferment, the control of fermentation total time is obtained in that into the exocellular polysaccharide of high yield in 18h, it is ensured that later separation is purified and pharmacology is lived
Property experimentation has enough Thick many candies source bases.At the same time Lactobacillus rhamnosus CGMCC No.13497 are in 2L fermentation flasks
The yield of extracellular polysaccharide that fermentation is obtained is of a relatively high compared with other lactic acid bacterias, therefore Lactobacillus rhamnosus CGMCC in the present invention
No.13497 is one plant of good polysaccharide production bacterial classification, has potential industrial scale to produce and using value.
2nd, the extraction of Lactobacillus rhamnosus crude extracellular polysaccharide
(1) preparation of purified reagent
Sevag reagents：Chloroform is mixed with n-butanol with 4: 1 volume ratio.
(2) in tunning Thick many candies extraction
By 2L zymotic fluids, 100 DEG C are boiled 5min, the remaining thalline of inactivation.Afterwards 2L is inactivated into the zymotic fluid of thalline in 10000
× g is centrifuged 10min, removes precipitation and takes supernatant；Supernatant film is concentrated into into 500mL, 1/5 times of volume Sevag reagent is added,
The 200rpm concussions 20min in shaking table；Sevag reagent reactings are centrifuged after 5000 × g, remove precipitation, collect supernatant, repeat three
It is secondary.Ethanol precipitation is carried out afterwards, 95% ethanol and supernatant are placed in into 4 DEG C of cooling 30min, add the second of 3-5 times of volume 95%
Alcohol, after being sufficiently stirred for (this process should claim flocculent deposit to separate out with polysaccharide), 4 DEG C stand overnight.Upper strata ethanol is removed afterwards, will
Precipitation distilled water is sufficiently stirred for dissolving, carries out obtaining dried pale yellow powder shape Thick many candies after vacuum freeze drying.
3rd, the preparation of the extracellular smart polysaccharide of Lactobacillus rhamnosus
(1) Thick many candies solution is prepared
Weigh Thick many candies 1g and be dissolved in 10mL distilled waters and fully dissolve, be made into the aqueous solution of 100mg/mL, in freezing from
After 8000 × g of scheming centrifugation 10min, take supernatant and be the 100mg/mL Thick many candies aqueous solution.
(2) DEAE-52 anion-exchange chromatographies
By Thick many candies aqueous solution loading to the DEAE-52 anion-exchange columns (Φ 2.6cm × 30cm, BV=for having balanced
120mL).Ion exchange columns are rinsed with 5 times of column volume distilled waters, flow velocity is SV=1/240 (0.5mL/min), then with 2 times of cylinders
The NaCl solution wash-out of product 1mol/L, flow velocity is SV=1/120 (1mL/min).Collected using automatic fraction collector, often pipe is received
Collection 5mL, total sugar content is determined with Phenol sulfuric acid procedure by pipe, makes elution curve, is merged and is collected same composition, obtains NaCl wash-outs
Polysaccharide component EPS.As a result Fig. 3 is seen.
In figure 3, Thick many candies are dissolved in after water and being centrifuged, and after removing water-fast contamination precipitation, take supernatant loading extremely
After DEAE-52 anion-exchange columns first weaker material will not be combined or combined with resin and is washed with distilled water 5 times of column volumes of wash-out
Take off.Then by the NaCl solution gradient elution with 0-1mol/L, detect and collect eluting peak.As a result show that EPS is carried
Certain negative electrical charge, can and DEAE-52 ion exchange resin have certain combination, it is therefore desirable to certain density salting liquid (NaCl)
EPS could be eluted.
(3) it is concentrated by ultrafiltration
Ultrafiltration is carried out to collection liquid EPS of the NaCl wash-outs in (2) with the milipore filter that the molecular weight that dams is 5000Da；Pressure
For 0.08MPa, being concentrated by ultrafiltration after 4 times of volumes carries out vacuum freeze drying.
(4) Sephacryl S-300 gel chromatographies
Take in (3) vacuum freeze drying gained sample EPS 150mg and be dissolved in respectively in 5mL distilled waters and be made into 30mg/mL's
The aqueous solution, takes 0.45 μm of membrane filtration excessively after supernatant degerming after 8000 × g of refrigerated centrifuge centrifugation 10min.Take on solution
Sample is to the Sephacryl S-300 gel chromatography columns (Φ 1.6cm × 80cm, BV=120mL) for having balanced.It is double with 2 times of column volumes
Water elution is steamed, flow velocity is SV=1/500 (0.25mL/min), and automatic fraction collector is collected, and often pipe collects 3mL, uses phenol sulphur
Acid system determines total sugar content by pipe, makes elution curve, merges and collects same composition.As a result Fig. 4 is seen.
As seen from Figure 4, the salt that NaCl is eluted in DEAE-52 anion-exchange columns is washed component EPS and is further passed through
Sephacryl S-300 gel chromatography columns are purified, and obtain elution curve collection of illustrative plates, and EPS starts appearance to the 28th from the 14th pipe
Pipe terminates, and containing a small amount of albumen, this polysaccharide component may be the protein sugar for combining a certain amount of albumen, needs further structure mirror
Surely constitute determining its polysaccharide.The eluting peak of two kinds of components is single symmetrical peak, illustrates Lactobacillus rhamnosus exocellular polysaccharide EPS
It is homogeneous monosaccharide component.
(5) dialyse lyophilized
Selection dam molecular weight be 7000Da bag filter, add (4) in wash-out collection liquid, flowing water dialysis 36-48h,
Vacuum freeze drying obtains fine work sugar EPS, mark is weighed, in -20 DEG C of Refrigerator stores.Final separated polysaccharide yield after purification
As shown in table 1.
The physical and chemical parameter of the Thick many candies of table 1 and smart polysaccharide EPS
4th, the composition of Lactobacillus rhamnosus exocellular polysaccharide EPS and its physical and chemical parameter are determined
Determination of polysaccharide
Glucose 20mg is accurately weighed in 105 DEG C of dryings of baking oven to constant weight, be dissolved in 100mL distilled waters and be made into 0.2mg/
The glucose standards solution of mL.Standard liquid is diluted to into respectively 20,40,80,100,120,160,200 μ g/mL according to following table,
Then add 0.4mL phenol solutions, then rapidly join the 2.0mL concentrated sulfuric acids, fully mix, 30min is stood under room temperature, after standing according to
200 μ L of secondary absorption are added in 96 orifice plates, and with ELIASA the light absorption value under wavelength 490nm is determined, and distilled water makees blank.With
Concentration of glucose is abscissa, and A490 is ordinate, makees the calibration curve of C-A490 and calculates corresponding regression equation.By polysaccharide
Sample is made into the solution of 0.1mg/mL, and according to above method 0.1mL is drawn, and adds 0.2mL phenol solutions and the 1.0mL concentrated sulfuric acids,
The light absorption value that ELIASA is surveyed under its 490nm, according to the polyoses content of regression equation calculation sample.
Determining the protein quantity
Protein content is carried out by forint- phenol law in polysaccharide component.
(1) preparation of solution
The preparation of (a) standard protein solution：Accurately weigh BSA 1.25mg and be dissolved in 5mL distilled waters and be configured to 250 μ g/mL
Solution.B () sample solution is prepared：Folin-Phenol first reagent (VA：VB=50：1)：A：10g Na2CO3, 2g NaOH, 0.25g wine
Stone acid potassium sodium (KNaC4H4O6·4H2O) it is dissolved in 500mL distilled waters.B：0.5g CuSO4·5H2O is dissolved in 100mL distilled waters.Good fortune
Woods-phenol second reagent：Sigma Co., USA's reagent.
Standard BSA solution is diluted to into respectively 0,25,50,100,150,200,250 μ g/mL according to following table, is then added
5mL reagent first, fully mixes, and 10min is stood under room temperature, then rapidly joins 0.5mL reagent second, shakes up immediately, 30 DEG C of water-baths
Middle standing 30min, No. 1 test tube (distilled water) is contrast solution, with each pipe of spectrophotometric measurement at 500nm
Light absorption value.With protein concentration as abscissa, A500 is ordinate mapping, draws C-A500 calibration curves and calculates it
C-A500 equations.Light absorption value of the sample in 500nm is measured in the same method, the protein content in polysaccharide sample is calculated.
Purity
Purity is carried out by JASCO highly effective liquid phase chromatographic systems.System configuration is connected for Shodex sugar 805
The chromatographic columns of Shodex sugar 802,2414 differential refraction detectors, mobile phase is distilled water, and sample concentration is 10.0mg/mL,
Sample size is 20 μ L, and flow velocity 1.0mL/min, 30 DEG C of column temperature records sample chromatogram curve.As a result it is as shown in Figure 5.
As seen from Figure 5, the purity of Lactobacillus rhamnosus exocellular polysaccharide EPS, rhamnose breast are detected by efficient liquid phase
The high performance liquid chromatography result of bacillus exocellular polysaccharide EPS is shown as single symmetrical peak, and appearance time is 11.7min, shows that EPS is
Homogeneous polysaccharide component, purity is 95.3%.From accompanying drawing 3, EPS is a kind of water-soluble polysaccharide component of purity higher than 95%,
Pharmacological activity experiment for after provides guarantee.
Molecular weight determination
The measure of Lactobacillus rhamnosus exocellular polysaccharide EPS molecular weight is carried out by High Performance Gel Permeation Chromatography.It is configured to
The chromatographic columns of the series connection Shodex of Shodex sugar 805 sugar 802, are detected with 2414 differential refraction detectors.It is with concentration
The sample and dextran standard solution of 5.0mg/mL sample introduction successively, sample size is 20 μ L, and mobile phase is 0.01mol/L's
NaNO3Solution, flow velocity 1.0mL/min records respective retention time Tr, paints LogMr-Tr calibration curves and calculates its recurrence side
Journey.According to the retention time of polysaccharide sample, its relative molecular mass is calculated by equation.
Calibration curve is drawn according to the dextran standard and its corresponding retention time of different relative molecular weights, its is linear
Regression equation is Log Mr=-0.00486Tr3+0.459Tr2–14.7Tr+163.The retention time of RAPS is 11.7min, is substituted into
Equation can extrapolate its relative molecular mass and be about 1.4 × 105Da。
Claims (7)
1. a kind of Lactobacillus rhamnosus (Lactobacilus rhamnosus) bacterial strain, its preserving number is：CGMCC No.13497.
2. a kind of Lactobacillus rhamnosus exocellular polysaccharide, its Lactobacillus rhamnosus strain fermentation by claim 1 and obtain.
3. the preparation method of the Lactobacillus rhamnosus exocellular polysaccharide of claim 2, including：By the rhamnose breast bar of claim 1
Bacteria strain 37 DEG C of standings in degreasing milk medium are incubated, and the initial gross separation of culture supernatant Jing obtains Thick many candies, Thick many candies
Purify through the method for DEAE-52 ion-exchange chromatographies and Sephacryl S-300 gel chromatographies, obtain final product.
4. the preparation method of claim 3, including：By Lactobacillus rhamnosus CGMCC No.13497 37 in degreasing milk medium
DEG C constant temperature quiescent culture 18-24h, nutrient solution Jing bactofugations body obtains supernatant, supernatant Jing Sevag method removing proteins, 3-5 times
Volume ethanol is precipitated, and Thick many candies are obtained after vacuum freeze drying；The Thick many candies through DEAE-52 cation exchange chromatographies,
Mobile phase is 1mol/L NaCl, and flow velocity is SV=1/120；Elution fraction Jing molecular cut offs are carried out for the milipore filter of 5000Da
Ultrafiltration, again through Sephacryl S-300 gel chromatographies after 4 times of volumes of concentration, mobile phase is distilled water, and flow velocity is SV=
1/500；Elution fraction Jing molecular cut offs are 7000 bag filter flowing water dialysis 36-48h, and dialysis trapped fluid Jing vacuum refrigerations are done
It is dry, obtain final product.
5. a kind of pharmaceutical composition, wherein the Lactobacillus rhamnosus exocellular polysaccharide containing claim 2 and pharmaceutically acceptable
Carrier.
6. the Lactobacillus rhamnosus exocellular polysaccharide of claim 2 is used for the purposes of the medicine for preparing treatment inflammation disease.
7. the purposes of claim 6, wherein inflammation disease is ulcerative colitis.
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| CN109182166A (en) * | 2018-08-27 | 2019-01-11 | 南昌大学 | One plant has effects that the Lactobacillus rhamnosus of relief of constipation and its application |
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