CN103757070B - A kind of exocellular polysaccharide with immunoregulation effect and its preparation method and application - Google Patents
A kind of exocellular polysaccharide with immunoregulation effect and its preparation method and application Download PDFInfo
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
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Abstract
The invention discloses a kind of exocellular polysaccharide with immunoregulation effect and its preparation method and application.This exocellular polysaccharide by massfraction than be 20.70 ~ 22.74% glucosamine, 33.87 ~ 35.21% glucose, the pectinose of 20.57 ~ 22.38%, the galn of 11.85 ~ 12.74% and 8.74 ~ 11.20% semi-lactosi form, its weight-average molecular weight is 11,864 ~ 15,473 dalton.Exocellular polysaccharide of the present invention is that a kind of composition is clear and definite, possesses the novel polysaccharide of certain immunoregulation effect, and it can be used as additive application in pharmacy, the association area such as clinical, and application prospect is very wide.
Description
Technical field
The present invention relates to a kind of exocellular polysaccharide with immunoregulation effect and its preparation method and application.
Background technology
Milk-acid bacteria (Lactic acid bacteria) is that a class can utilize fermentable sugar to produce the bacterium general name of a large amount of lactic acid, and this bacterioid found at nature at present has 23 genus at least on taxonomy.Apply more milk-acid bacteria mainly contain lactobacillus, streptococcus, enterococcus spp, lactococcus, Pediococcus and leuconos toc etc. in food, medicine and other fields.Milk-acid bacteria is the topmost source of probiotic bacterium, and many milk-acid bacterias are the intrinsic probiotic bacteriums of human intestinal, have and improve human intestinal microflora, regulates immunity of organisms, inhibiting cancer, reduces serum cholesterol, regulates the physiologically active that blood pressure etc. is important.
Milk-acid bacteria plays the mechanism of action of main functional characteristics, and improve except surely growing, by main metabolites (lactic acid etc.) except intestinal environment etc., some secondary metabolites such as bacteriocin, exocellular polysaccharide etc. also play very important effect.Wherein, there is the research interest that Exopolysaccharides Produced by Lactic Acid Bacteria that is theoretical and actual application value causes domestic and international many scholars.
Exopolysaccharides Produced by Lactic Acid Bacteria is that milk-acid bacteria produces and is secreted into extracellular a kind of polysaccharide.Exopolysaccharides Produced by Lactic Acid Bacteria has important technology function, has important impact to the rheological property of yogurt, cheese and most cultured milk prod, matter structure, mouthfeel and local flavor.Exopolysaccharides Produced by Lactic Acid Bacteria can improve rheological properties and the texture characteristic of milk-product.Due to natural thickening power, produce that mouthfeel compared with yogurt that Acarasiales strain formed is lubricious, viscosity increases by producing yogurt that slimy milk bacillus and the mixed fermentation of non-product myxococcus formed with non-; Producing exocellular polysaccharide milk-acid bacteria can give yogurt stronger retention ability, thus avoids whey to separate out; In addition, the retentiveness produced by exocellular polysaccharide strengthens the yield contributing to improving the products such as cheese.Exopolysaccharides Produced by Lactic Acid Bacteria also has good physiological function, as strengthened mucosal adhesive effect, antitumor, antiulcer agent, immunomodulatory, decreasing cholesterol, hypotensive, the growth of other probiotic bacteriums in enteron aisle can also be promoted as prebiotics, optimize intestine microenvironment etc.Therefore, carry out the research of producing exocellular polysaccharide milk-acid bacteria, for the lactobacillus-fermented milk-product improving milk-product process for processing, exploitation has specific function character, there is very important Research Significance and economic worth.The Exopolysaccharides Produced by Lactic Acid Bacteria that exploitation has prebiotic function becomes the focus studied at present.
Since the sixties in 20th century, glycocalix thinks a kind of nonspecific immunopotentiating agent of wide spectrum, cellular immunization and the humoral immune function of host cell can be strengthened, as activating macrophage, T cell, B cell and NK cell etc., activating complement and induction produce Interferon, rabbit etc., its effect will be the nonspecific defense function of human activin, in antiviral, antitumor, radioprotective etc., have good curative effect.
But, at present in this area, research ripe and the kind of the Exopolysaccharides Produced by Lactic Acid Bacteria that can apply in practice few, produce and scientific research all exist the needs studied new exocellular polysaccharide, to enrich the kind of excellent property, broad-spectrum Exopolysaccharides Produced by Lactic Acid Bacteria.
Summary of the invention
Technical problem to be solved by this invention is the present situation enriched not for current Exopolysaccharides Produced by Lactic Acid Bacteria kind and product function, and a kind of new Exopolysaccharides Produced by Lactic Acid Bacteria is provided, be specially the exocellular polysaccharide of a kind of lactobacillus rhamnosus (Lactobacillus rhamnosus), the present invention also provides the preparation method and application of described exocellular polysaccharide.Exocellular polysaccharide of the present invention is proved to be has good mitogen activity and immunoregulatory activity.
The exocellular polysaccharide of lactobacillus rhamnosus of the present invention finds to be produced by the lactobacillus rhamnosus of deposit number by CGMCC No.6430 at first.Described deposit number is the ight soil that the lactobacillus rhamnosus of CGMCC No.6430 screens from normal adults, can produce exocellular polysaccharide in fermented-milk.The present inventor finds that this bacterial strain has good acid and bile salt tolerance performance under study for action, infer that it has potential prebiotic function, and In vitro cell experiment finds that its Crude polysaccharides can improve the lymphocytic activity of mouse T/B, infer that it has the function of potential raising immunity of organisms.In order to understand its structure and function better, separation and purification and functional study thereof are carried out to its Crude polysaccharides.This bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) preservation, and its deposit number is CGMCC No.6430.On this basis, the invention provides following technical proposals.
One of technical scheme provided by the invention is: the exocellular polysaccharide of a kind of lactobacillus rhamnosus, described exocellular polysaccharide by massfraction than be 20.70 ~ 22.74% glucosamine, 33.87 ~ 35.21% glucose, the pectinose of 20.57 ~ 22.38%, the galn of 11.85 ~ 12.74% and 8.74 ~ 11.20% semi-lactosi form, the average weight of this exocellular polysaccharide is 11,864 ~ 15,473 dalton.
In the present invention, lactobacillus rhamnosus (L.rhamnosus) bacterial strain that described exocellular polysaccharide can be CGMCC NO.6430 by preserving number produces, but is not limited thereto, and also can be produced by other the bacterial strain that can produce exocellular polysaccharide.Preferably, lactobacillus rhamnosus (L.rhamnosus) bacterial strain that described exocellular polysaccharide is CGMCC NO.6430 by aforementioned preserving number is produced.
Two of technical scheme provided by the invention is: the preparation method of the exocellular polysaccharide of aforementioned lactobacillus rhamnosus.
In the present invention, described preparation method can be by described lactobacillus rhamnosus conventionally in conventional cultural method cultivate and obtain fermented liquid, then adopt conventional separation of polysaccharides method to be separated fermented liquid and obtain exocellular polysaccharide.
Preferably, described preparation method comprises the steps:
(1) by deposit number be CGMCC No.6430 lactobacillus rhamnosus seed liquor with the inoculum size of volume ratio 1.0 ~ 5.0% be inoculated in sterile absorbent breast substratum, in 28 ~ 36 DEG C cultivate 24 ~ 40 hours fermented liquid;
(2) fermented liquid of step (1) gained is heated 10 ~ 30min at 95 ~ 100 DEG C, 3 ~ 16 hours are left standstill after being cooled to 15 ~ 25 DEG C, centrifuging and taking supernatant to add solution of trichloroacetic acid to final concentration in described supernatant be 5 ~ 8%, leaves standstill 8 ~ 16 hours, centrifugal acquisition fermented supernatant fluid; Described per-cent is the grams of the trichoroacetic acid(TCA) contained in every 100 milliliters, described solution of trichloroacetic acid to be mass percent be 75 ~ 85% solution of trichloroacetic acid;
(3) percent by volume adding 2 ~ 4 times of volumes in the fermented supernatant fluid of step (2) gained is the ethanol of 80 ~ 100%, centrifugal or collected by filtration thing is also water-soluble by throw out, be that 5000 ~ 14000 daltonian dialysis tubings are dialysed 48 ~ 72 hours in water with molecular weight cut-off, within every 8 ~ 12 hours, change water once, at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains the crude product of described exocellular polysaccharide.
In step of the present invention (1), described sterile absorbent breast substratum is the sterile absorbent breast substratum described in the routine of this area, be preferably and 10 ~ 12 weight part skimming milks and 0.8 ~ 1.2 weight part glucose are dissolved in the water of 85 ~ 90 weight parts, in 115 ~ 125 DEG C of sterilizings 15 ~ 20 minutes, be cooled to the sterile absorbent breast substratum of 15 ~ 25 DEG C of gained.Described inoculum size is preferably 2.0 ~ 4.0%, is more preferably 2.0 ~ 2.5%.Wherein, the time of fermentation is preferably 24 ~ 36 hours, is more preferably 28 ~ 32 hours.
In step of the present invention (2), described heating is preferably at 95 ~ 100 DEG C of heating 15 ~ 20min by the fermented liquid of step (1) gained.The mass percent of described trichoroacetic acid(TCA) is preferably 78 ~ 82%.
In step of the present invention (3), the percent by volume of described ethanol is preferably 82 ~ 90%.The molecular weight cut-off of described dialysis tubing is preferably 12000 ~ 14000 dalton.
Preparation method of the present invention more preferably also comprises step (4), namely the crude product of the exocellular polysaccharide of step (3) gained is carried out to the step of further separation and purification;
Described step (4) is: adopt the crude product of ion exchange column to the exocellular polysaccharide of step (3) gained to carry out separation and purification, with 45 ~ 55mM, the Tris-HCl damping fluid of pH7.5 ~ 7.8 carries out isocratic elution, elution speed is 2.5 ~ 3.5mL/min, merge the eluted product of collecting component peaks, loading molecular weight cut-off is that 10000 ~ 15000 daltonian dialysis tubings are dialysed 48 ~ 72 hours in deionized water, within every 8 ~ 12 hours, change water once, at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains described exocellular polysaccharide.
Preferred steps of the present invention (4) is: adopt the crude product of DEAE-Sepharose Fast Flow ion exchange column to the exocellular polysaccharide of step (3) gained to carry out separation and purification, the specification of described ion exchange column is D2.6cm × 30cm, with 50mM, the Tris-HCl damping fluid of pH7.6 carries out isocratic elution, elution speed is 3mL/min, merge the eluted product of collecting component peaks, loading molecular weight cut-off is that 14000 daltonian dialysis tubings are dialysed 72 hours in deionized water, within every 8 hours, change water once, dry or vacuum lyophilization at temperature is no more than 105 DEG C, obtain described exocellular polysaccharide.
Three of technical scheme provided by the invention is: the exocellular polysaccharide of aforementioned lactobacillus rhamnosus is in the application of food, medicine and association area.
Exocellular polysaccharide of the present invention has certain immunocompetence, has a good application prospect in food, medicine and association area.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
Exocellular polysaccharide of the present invention is a kind ofly form clear and definite, to possess certain immunoregulation effect novel polysaccharide, and it can be used as additive application in pharmacy, the association area such as clinical, and application prospect is very wide.As the polymer substance that microorganism produces, exocellular polysaccharide of the present invention has various biological effect, as immunostimulation and immunosuppressive activity, it can by stimulating the propagation of certain immune cells, the immunization of enhancing body, apply time for the determination of dosage and the scope of application also more clear and definite, there is significant technical superiority.
Accompanying drawing explanation
Fig. 1 is the gel chromatography isocratic elution graphic representation of CGMCC No.6430 exocellular polysaccharide crude product.
Fig. 2 is the gel chromatography isocratic elution graphic representation of CGMCC No.6430 exocellular polysaccharide crude product.
Fig. 3 is the gel chromatography isocratic elution graphic representation of CGMCC No.6430 exocellular polysaccharide crude product.
Fig. 4 is CGMCC No.6430 exocellular polysaccharide one-component S2 gel filtration chromatography elution curve.
Fig. 5 is the HPLC-UV detection of CGMCC No.6430 exocellular polysaccharide one-component S2.
Fig. 6 is the chromatography of ions figure of CGMCC No.6430 exocellular polysaccharide one-component S2 monose composition.
Fig. 7 is the infrared spectrogram of CGMCC No.6430 exocellular polysaccharide one-component S2.
Fig. 8 is that CGMCC No.6430 exocellular polysaccharide one-component S2 is to the result figure of Proliferation of lymphocytes.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Lactobacillus rhamnosus CGMCC No.6430 described in the present invention screens the ight soil (it is open in patent CN102994432A) from normal adults, it is at MRS agar plate (MRS substratum, Bifidobacterium selective substratum, Merck, Germany) bacterium colony that upper formation is smooth, moistening, circular, contact bacterium colony surface gently with transfering loop, can very long wire drawing be formed, there is good wire-drawing performance.
The present invention utilizes lactobacillus rhamnosus CGMCC No.6430 to produce exocellular polysaccharide in fermented-milk, extract Crude polysaccharides, and utilize chromatography of ions chromatography and gel chromatography chromatography to its separation and purification, prepare single polysaccharide component, and study partial materialization character and the biological activity of its polysaccharide.
Room temperature described in the present invention refers to the temperature of carrying out the operation room tested, and is generally 20-25 DEG C.
The preparation of embodiment 1 exocellular polysaccharide
From 37 DEG C, the lactobacillus rhamnosus CGMCC No.6430 bacterium colony of picking fresh culture the MRS agar plate of Anaerobic culturel 48h, be transferred in the sterile absorbent breast substratum of 12% (w/w) of interpolation 2% (w/v) glucose, cultivate 20h for 32 DEG C, obtain seed liquor.Then be transferred in above-mentioned sterile absorbent breast substratum by the inoculum size of 1% volume ratio, 36 DEG C of fermentation 40h, obtain the fermented-milk of lactobacillus rhamnosus CGMCC NO.6430.Fermented-milk is in 95 DEG C of heating 30min, and to be cooled to room temperature, centrifugal (4 DEG C, 14,000g, 20min) remove thalline and coagulating egg white; The trichoroacetic acid(TCA) of 85% (w/w) is added to final concentration 8.0%(w/v in supernatant liquor) hold over night, centrifugal (4 DEG C, 14,000g, 20min) remove protein precipitation; Get 80% ethanol that supernatant liquor adds 4 times of volumes, 4 DEG C of hold over night, centrifugal (4 DEG C, 14,000g, 20min) collecting precipitation, being dissolved in deionized water and loading molecular weight cut-off is in 8000 daltonian dialysis tubings, and to dialyse 48h with deionized water, every 12h changes water once, at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains the crude product A of exocellular polysaccharide.
Adopt DEAE-Sepharose Fast Flow (D2.6cm × 30cm) ion exchange column to be separated the crude product A of exocellular polysaccharide, use 45mM Tris-HCl(pH7.5) damping fluid balance pillar.The above-mentioned 45mM Tris-HCl(pH7.5 of crude product A of exocellular polysaccharide) loading after buffer solution, with Tris-HCl damping fluid (45mM, pH7.5) isocratic elution, elution speed is 2.5mL/min, often pipe 6mL fraction collection.Polysaccharide content is detected with sulfuric acid-phynol method, (in Fig. 1,0-50 manages to merge the eluted product of collecting component peaks, in Fig. 1, X-coordinate is the numbering of pipe, and ordinate zou is absorbance value), loading molecular weight cut-off is in 8000 daltonian dialysis tubings, with deionized water dialysis 48h to remove buffering salt, every 8h changes water once, and at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains the polysaccharide of one-component, namely exocellular polysaccharide of the present invention, is called S2-A.
The preparation of embodiment 2 exocellular polysaccharide
From 37 DEG C, the lactobacillus rhamnosus CGMCC No.6430 bacterium colony of picking fresh culture the MRS agar plate of Anaerobic culturel 48h, be transferred in sterile absorbent breast substratum, cultivate 20h for 32 DEG C, obtain seed liquor, described sterile absorbent breast substratum is dissolved in the water of 8 weight parts by 10 weight part skimming milks and 0.8 weight part glucose, in 115 DEG C of sterilizings 20 minutes, be cooled to the sterile absorbent breast substratum of 25 DEG C of gained.Then be transferred in above-mentioned sterile absorbent breast substratum by the inoculum size of 3% volume ratio, 32 DEG C of fermentation 30h, obtain the fermented-milk of lactobacillus rhamnosus CGMCC NO.6430.Fermented-milk is in 100 DEG C of heating 10min, and to be cooled to room temperature, centrifugal (4 DEG C, 14,000g, 20min) remove thalline and coagulating egg white; The trichoroacetic acid(TCA) of 80% (w/w) is added to final concentration 7.0%(w/v in supernatant liquor) hold over night, centrifugal (4 DEG C, 14,000g, 20min) remove protein precipitation; Get 95% ethanol that supernatant liquor adds 3 times of volumes, 4 DEG C of hold over night, centrifugal (4 DEG C, 14,000g, 20min) collecting precipitation, being dissolved in deionized water and loading molecular weight cut-off is in 14000 daltonian dialysis tubings, and to dialyse 72h with deionized water, every 12h changes water once, at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains the crude product B of exocellular polysaccharide.
Adopt DEAE-Sepharose Fast Flow (D2.6cm × 30cm) ion exchange column to be separated the crude product B of exocellular polysaccharide, use 50mM Tris-HCl(pH7.6) damping fluid balance pillar.The above-mentioned 50mM Tris-HCl(pH7.6 of crude product B of exocellular polysaccharide) loading after buffer solution, with Tris-HCl damping fluid (50mM, pH7.6) isocratic elution, elution speed is 3.0mL/min, often pipe 6mL fraction collection.Polysaccharide content is detected with sulfuric acid-phynol method, (in Fig. 2,0-50 manages to merge the eluted product of collecting component peaks, in Fig. 2, X-coordinate is the numbering of pipe, and ordinate zou is absorbance value), loading molecular weight cut-off is in 14000 daltonian dialysis tubings, with deionized water dialysis 72h to remove buffering salt, every 12h changes water once, and at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains the polysaccharide of one-component, namely exocellular polysaccharide of the present invention, is called S2-B.
The preparation of embodiment 3 exocellular polysaccharide
From 37 DEG C, the lactobacillus rhamnosus CGMCC No.6430 bacterium colony of picking fresh culture the MRS agar plate of Anaerobic culturel 48h, be transferred in sterile absorbent breast substratum, cultivate 20h for 32 DEG C, obtain seed liquor, described sterile absorbent breast substratum is dissolved in the water of 90 weight parts by 12 weight part skimming milks and 1.2 weight part glucose, in 125 DEG C of sterilizings 15 minutes, be cooled to the sterile absorbent breast substratum of 15 DEG C of gained.Then be transferred in above-mentioned sterile absorbent breast substratum by the inoculum size of 5% volume ratio, 28 DEG C of fermentation 24h, obtain the fermented-milk of lactobacillus rhamnosus CGMCC NO.6430.Fermented-milk is in 98 DEG C of heating 20min, and to be cooled to room temperature, centrifugal (4 DEG C, 14,000g, 20min) remove thalline and coagulating egg white; The trichoroacetic acid(TCA) of 75% (w/w) is added to final concentration 5.0%(w/v in supernatant liquor) hold over night, centrifugal (4 DEG C, 14,000g, 20min) remove protein precipitation; Get 100% ethanol that supernatant liquor adds 2 times of volumes, 4 DEG C of hold over night, centrifugal (4 DEG C, 14,000g, 20min) collecting precipitation, being dissolved in deionized water and loading molecular weight cut-off is in 5000 daltonian dialysis tubings, and to dialyse 56h with deionized water, every 10h changes water once, at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains the crude product C of exocellular polysaccharide.
Adopt DEAE-Sepharose Fast Flow (D2.6cm × 30cm) ion exchange column to be separated the crude product C of exocellular polysaccharide, use 55mM Tris-HCl(pH7.8) damping fluid balance pillar.The above-mentioned 55mM Tris-HCl(pH7.8 of crude product C of exocellular polysaccharide) loading after buffer solution, with Tris-HCl damping fluid (55mM, pH7.8) isocratic elution, elution speed is 3.5mL/min, often pipe 6mL fraction collection.Polysaccharide content is detected with sulfuric acid-phynol method, (in Fig. 3,0-50 manages to merge the eluted product of collecting component peaks, in Fig. 3, X-coordinate is the numbering of pipe, and ordinate zou is absorbance value), loading molecular weight cut-off is in 5000 daltonian dialysis tubings, with deionized water dialysis 56h to remove buffering salt, every 10h changes water once, and at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains the polysaccharide of one-component, namely exocellular polysaccharide of the present invention, is called S2-C.
The monistic checking of embodiment 4 exocellular polysaccharide
For verifying the unicity of exocellular polysaccharide of the present invention, embodiment 1 ~ 3 is collected polysaccharide sample S2-A, S2-B of obtaining continue to be separated with S2-C on Sepharose Cl-6B (D1.6cm × 100cm) gel chromatography column, balance pillar and wash-out with the 50mM Tris-HCl damping fluid containing 0.15M NaCl.Elution speed is 0.375mL/min, 15min/ pipe fraction collection, detects polysaccharide content with sulfuric acid-phynol method.
Result shows, S2-A, S2-B and S2-C tri-samples all only present single symmetrical peak, shows that exocellular polysaccharide of the present invention is the relatively homogeneous polysaccharide of molecular weight.Wherein, as shown in Figure 4, wherein, X-coordinate is the numbering of pipe to the gel chromatographic columns purification result of sample S2-B, and ordinate zou is light absorption value.
The molecular weight determination of embodiment 5 exocellular polysaccharide
Embodiment 1 ~ 3 is collected the exocellular polysaccharide sample of three one-components obtained, namely S2-A, S2-B, S2-C adopt high-efficient liquid phase chromatogram HPLC method to measure weight-average molecular weight (Mw).By different molecular weight (5,900,9,600,21,100,47,100,107,000,200,000,708,000 and 1,330,000Da) blue polysaccharide (the Pullulan polysaccharides calibration kit in the general Shandong of standard, Agilent technologies, USA) sample introduction in succession, record retention time TR, take TR as X-coordinate, LgMw is ordinate zou drawing standard curve, draws the regression equation of molecular weight and retention time TR.Testing sample is sample introduction in the steps below, according to gained TR, by the relative molecular weight of regression equation calculation sample.
Adopt Waters2690 high performance liquid phase system, be equipped with Waters2410 differential refraction detector and UV-detector; Select Waters UltrahydrogelTM Linear(ID7.8 × 300mm, 10 μm) chromatographic column two post series winding.Moving phase: 0.1M NaNO
3; Column temperature: 45 DEG C; Flow velocity: 0.9mL/min; Sample size: 20 μ L.
Wherein, as shown in Figure 5, it presents single symmetrical peak to the HPLC-UV detection of sample S2-B, further illustrating it is homogeneous polysaccharide, the retention time of S2-B elution curve on HPLC is 20.78min, and retention time being updated to regression equation, to extrapolate S2-B weight-average molecular weight be 13,583Da.The weight-average molecular weight extrapolating S2-A, S2-C is in the same way respectively 11,864Da, 15,473Da.
Conclusion: the weight-average molecular weight of CGMCC No.6430 exocellular polysaccharide S2 is 11,864 ~ 15,473Da.
The monose composition measuring of embodiment 6 exocellular polysaccharide
Adopt the monose composition of ion-chromatographic determination exocellular polysaccharide S2 of the present invention.
(1) hydrolysis of polysaccharide
Draw 100 μ L concentration be component S 2 sample solution of 4-5mg/mL in the tool plug scale test tube of 5mL, add the 4M trifluoroacetic acid (TFA) of 100 μ L, inflated with nitrogen tube sealing, in 110 DEG C of baking ovens, be hydrolyzed 2h; Open lid after cooling, dry up with nitrogen after adding 200 μ L methyl alcohol, so repeat to add methyl alcohol and blow 3 times with nitrogen, remove TFA, its residue water dissolution is settled to 5mL, supply sample introduction analysis with after 0.45 μm of micro-pore-film filtration.
(2) chromatography of ions condition
Chromatographic column: CarboPac PA20(ID3 × 150mm)
Moving phase: A, H
2o; B, 250mmol/L NaOH;
Gradient elution; Flow velocity: 0.5mL/min; Pulsed amperometry (PAD);
Sampling volume: 20 μ L; Column temperature: 30 DEG C
S2-A, S2-B, S2-C tri-samples of gained in embodiment 1 ~ 3 are carried out above-mentioned monose composition measuring, and wherein as shown in Figure 6, wherein, X-coordinate is retention time (min) to the color atlas of the HPAEC of S2-B monose composition, and ordinate zou is response value (nC).Result shows: exocellular polysaccharide S2-B forms primarily of glucosamine (GlcN), glucose (Glc), pectinose (Ara), galn (GalN) and semi-lactosi (Gal).In S2-A, S2-B, S2-C tri-one-component samples, the massfraction ratio of monose is in table 1.
The monose composition of table 1S2
| Sample | GlcN | Glc | Ara | GalN | Gal |
| S2-A(by mass percentage) | 22.74 | 35.21 | 20.57 | 12.74 | 8.74 |
| S2-B(by mass percentage) | 21.19 | 34.41 | 21.86 | 12.20 | 10.34 |
| S2-C(by mass percentage) | 20.70 | 33.87 | 22.38 | 11.85 | 11.20 |
Conclusion: CGMCC No.6430 exocellular polysaccharide one-component S2 by massfraction than be 20.70 ~ 22.74% glucosamine, 33.87 ~ 35.21% glucose, the pectinose of 20.57 ~ 22.38%, the galn of 11.85 ~ 12.74% and 8.74 ~ 11.20% semi-lactosi form.
The Infrared spectroscopy of embodiment 7 exocellular polysaccharide
Dry polysaccharide S2 and KBr is ground compressing tablet, at 4000 ~ 500cm
-1infrared spectrum scanning (Nicolet Nexus470, NICOLET, USA) is carried out in region.
Wherein, the FT-IR(Fourier infrared spectrum of S2-B) collection of illustrative plates demonstrates the charateristic avsorption band of polysaccharose substance.At 3419cm
-1neighbouring wide absorption peak is in molecule and intermolecular O-H stretching vibration causes.Due to the impact by other groups, more or less all some offsets in the position at the peak that C-H stretching vibration and flexural vibration absorb, and appears at 2922cm respectively
-1and 1431cm
-1near.At 1200-1000cm
-1region is the charateristic avsorption band of various polysaccharide, 1030 and 1068cm
-1the absorption peak at place is caused by the flexural vibration of C-OH stretching vibration and C-O-C, and indicate is pyranoid glucosides simultaneously.At 901cm
-1the absorption peak at place is caused by the asymmetric stretching vibration of pyranoid ring.Can infer that exocellular polysaccharide of the present invention is α-pyranoside type polysaccharide thus.
The immunocompetence of embodiment 8 exocellular polysaccharide measures
BALB/C mice (purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center) spleen is taken out in aseptic technique, makes splenocyte suspension.Be separated lymphocyte with lymphocyte separation medium (Huamei Bio-Engrg Co., provides purchased from Shanghai), (often liter containing 0.144g KH for PBS damping fluid
2pO
4, 9.0g NaCl, 0.795g Na
2hPO
47H
2o, pH7.4) wash 2 times, adjust cell concn to 1 × 10 with the RPMI1640 nutrient solution (Gibco, USA) containing 10% foetal calf serum
6the splenic lymphocyte suspension of/mL.The 96 every holes of well culture plate add the polysaccharide sample of 150 μ L splenic lymphocyte suspensions and 50 μ L different concns (10,100 and 1000 μ g/mL), with mitogen concanavalin A (ConA, 5 μ g/mL, Sigma) inducer T lymphocyte propagation, if negative control group (only containing splenic lymphocyte suspension) and positive controls (adding mitogen ConA).Every experimental group establishes 3 holes to repeat, and puts 37 DEG C, 5%CO
272h is cultivated under saturated humidity condition.
Adopt
3h-TdR incorporation methods measures lymphocytic proliferative response.Cultivate and terminate 8h, in every hole, add 20 μ L,
3h-TdR(370kBq/mL).Each tube cell is collected in 49 type glass fiber filter paper after cultivation terminates, paper is dried and is placed in scintillation solution and spends the night, measure the CPM value of each pipe with liquid scintillation instrument (Packard, USA).CPM (counts per minutes) value is the flicker number of 3H-TdR in per minute cell, and due in cell generation cycle, 3H-TdR is cell synthetic DNA, one of main raw material of RNA, and therefore CPM value directly reflects cell proliferative conditions.
3h-TdR method is the classical way measuring spleen lymphocyte proliferation, and it increases based on DNA, RNA synthesis in cell generation cycle,
3h-TdR can be taken in cell by as raw material, measures in cell
3h-TdR exit dose, reflects cell proliferative conditions.
ConA, as T lymphocyte mitogen, promotes the lymphocytic propagation of T.Therefore, select ConA as positive controls.To the result of splenic lymphocyte proliferation effect as shown in Figure 8, wherein, X-coordinate is the concentration value of exocellular polysaccharide to exocellular polysaccharide of the present invention (S2-B in embodiment 2), and unit is μ g/mL, and ordinate zou is CPM value.Compared with control group ConA, exocellular polysaccharide of the present invention can promote splenic lymphocyte proliferation (P<0.05) significantly, and has agent dependence.Result shows exocellular polysaccharide of the present invention and demonstrates good mitogen activity, has potential immunoregulatory activity.
Comparative example 1 exocellular polysaccharide of the present invention compares with the immunocompetent of crude product of this exocellular polysaccharide
Method according to embodiment 8, measure that to add ConA, the crude product B of exocellular polysaccharide and the exocellular polysaccharide S2-B of one-component be CPM value in sample and splenic lymphocyte reaction tubes, and be contrast with ConA, calculate the enhancing per-cent of crude product to splenic lymphocyte of exocellular polysaccharide of the present invention and this exocellular polysaccharide.Strengthen per-cent (%) and be expressed as (CPM
sample-CPM
contrast)/CPM
contrast× 100.
Determine the percentage to T lymphopoiesis promoter action under three kinds of concentration (10 μ g/mL, 100 μ g/mL and 1000 μ g/mL).Result is as shown in the table:
As can be seen from the result of upper table, compared with the exocellular polysaccharide crude product of complicated component, the exocellular polysaccharide S2-B of one-component is stronger to the proliferation of splenic lymphocyte, more remarkable effect.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (9)
1. the exocellular polysaccharide of a lactobacillus rhamnosus, it is characterized in that, described exocellular polysaccharide by massfraction ratio be 20.70 ~ 22.74% glucosamine, 33.87 ~ 35.21% glucose, the pectinose of 20.57 ~ 22.38%, the galn of 11.85 ~ 12.74% and 8.74 ~ 11.20% semi-lactosi form, the average weight of this exocellular polysaccharide is 11,864 ~ 15,473 dalton, described exocellular polysaccharide by preserving number be CGMCC NO.6430 lactobacillus rhamnosus (
l. rhamnosus) bacterial strain produced.
2. the preparation method of the exocellular polysaccharide of lactobacillus rhamnosus as claimed in claim 1, is characterized in that, comprise the steps:
(1) by deposit number be CGMCC No.6430 lactobacillus rhamnosus seed liquor with the inoculum size of volume ratio 1.0 ~ 5.0% be inoculated in sterile absorbent breast substratum, in 28 ~ 36 DEG C cultivate 24 ~ 40 hours fermented liquid;
(2) fermented liquid of step (1) gained is heated 10 ~ 30 min at 95 ~ 100 DEG C, 3 ~ 16 hours are left standstill after being cooled to 15 ~ 25 DEG C, centrifuging and taking supernatant to add solution of trichloroacetic acid to final concentration in described supernatant be 5 ~ 8%, leaves standstill 8 ~ 16 hours, centrifugal acquisition fermented supernatant fluid; Described per-cent is the grams of the trichoroacetic acid(TCA) contained in every 100 milliliters, described solution of trichloroacetic acid to be mass percent be 75 ~ 85% solution of trichloroacetic acid;
(3) percent by volume adding 2 ~ 4 times of volumes in the fermented supernatant fluid of step (2) gained is the ethanol of 80 ~ 100%, centrifugal or collected by filtration thing is also water-soluble by throw out, be that 5000 ~ 14000 daltonian dialysis tubings are dialysed 48 ~ 72 hours in water with molecular weight cut-off, within every 8 ~ 12 hours, change water once, at temperature is no more than 105 DEG C, dry or vacuum lyophilization, obtains the crude product of described exocellular polysaccharide.
3. preparation method as claimed in claim 2, it is characterized in that, in step (1), described sterile absorbent breast substratum is dissolved in the water of 85 ~ 90 weight parts by 10 ~ 12 weight part skimming milks and 0.8 ~ 1.2 weight part glucose, in 115 ~ 125 DEG C of sterilizings 15 ~ 20 minutes, be cooled to the sterile absorbent breast substratum of 15 ~ 25 DEG C of gained; Described inoculum size is 2.0 ~ 4.0%, and the time of fermentation is 24 ~ 36 hours.
4. preparation method as claimed in claim 2, it is characterized in that, in step (1), described inoculum size is 2.0 ~ 2.5%, and the time of fermentation is 28 ~ 32 hours.
5. preparation method as claimed in claim 2, is characterized in that, in step (2), described is heated to be the fermented liquid of step (1) gained at 95 ~ 100 DEG C of heating 15 ~ 20min; The mass percent of described trichoroacetic acid(TCA) is 78 ~ 82%.
6. preparation method as claimed in claim 2, it is characterized in that, in step (3), the percent by volume of described ethanol is 82 ~ 90%; The molecular weight cut-off of described dialysis tubing is 12000 ~ 14000 dalton.
7. preparation method as claimed in claim 2, it is characterized in that, it also comprises step (4): described step (4) is: adopt the crude product of ion exchange column to the exocellular polysaccharide of step (3) gained to carry out separation and purification, with 45 ~ 55 mM, the Tris-HCl damping fluid of pH 7.5 ~ 7.8 carries out isocratic elution, elution speed is 2.5 ~ 3.5 mL/min, merge the eluted product of collecting component peaks, loading molecular weight cut-off is that 10000 ~ 15000 daltonian dialysis tubings are dialysed 48 ~ 72 hours in deionized water, within every 8 ~ 12 hours, change water once, dry or vacuum lyophilization at temperature is no more than 105 DEG C, obtain described exocellular polysaccharide.
8. preparation method as claimed in claim 2, it is characterized in that, described step (4) is: adopt the crude product of DEAE-Sepharose Fast Flow ion exchange column to the exocellular polysaccharide of step (3) gained to carry out separation and purification, the specification of described ion exchange column is D 2.6 cm × 30 cm, with 50mM, the Tris-HCl damping fluid of pH 7.6 carries out isocratic elution, elution speed is 3 mL/min, merge the eluted product of collecting component peaks, loading molecular weight cut-off is that 14000 daltonian dialysis tubings are dialysed 72 hours in deionized water, within every 8 hours, change water once, dry or vacuum lyophilization at temperature is no more than 105 DEG C, obtain described exocellular polysaccharide.
9. exocellular polysaccharide as claimed in claim 1 is in the application of field of food.
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| PCT/CN2014/086000 WO2015109855A1 (en) | 2014-01-23 | 2014-09-05 | Extracellular polysaccharide with immunomodulatory effect and preparation method and use thereof |
| AU2014378873A AU2014378873B2 (en) | 2014-01-23 | 2014-09-05 | Extracellular polysaccharide with immunomodulatory effect and preparation method and use thereof |
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| CN106635924B (en) * | 2017-01-23 | 2020-04-24 | 中国药科大学 | Preparation and application of lactobacillus rhamnosus exopolysaccharide |
| CN109295126B (en) * | 2018-08-31 | 2021-01-08 | 四川农业大学 | Lactobacillus plantarum exopolysaccharide with immunoregulatory activity and preparation method thereof |
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| CN100375754C (en) * | 2005-09-09 | 2008-03-19 | 天津市工业微生物研究所 | Method for producing extracellular mucopoly-saccharide using rhamnose lactobacillus and product |
| KR101112648B1 (en) * | 2008-12-30 | 2012-02-16 | 고려대학교 산학협력단 | Method of producing exopolysaccharide |
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