CN105837699A - Exopolysaccharide of Lactobacillus plantarum, and application of same in antagonism against toxicity of Bacillus cereus enterotoxins - Google Patents
Exopolysaccharide of Lactobacillus plantarum, and application of same in antagonism against toxicity of Bacillus cereus enterotoxins Download PDFInfo
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- CN105837699A CN105837699A CN201610202595.8A CN201610202595A CN105837699A CN 105837699 A CN105837699 A CN 105837699A CN 201610202595 A CN201610202595 A CN 201610202595A CN 105837699 A CN105837699 A CN 105837699A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
Abstract
The invention provides an exopolysaccharide of Lactobacillus plantarum, and application of the same in antagonism of toxicity against Bacillus cereus enterotoxins. The exopolysaccharide is characterized by using a preparation method; since mild purifying conditions are used, the molecular structure of the exopolysaccharide is retained, and the produced exopolysaccharide is simple in structural constitution, clear in structure and high in purity and yield. Moreover, it is accidentally discovered in experimental means that in the presence of the exopolysaccharide of Lactobacillus plantarum, toxicity of the Bacillus cereus enterotoxins to epithelial cells is obviously inhibited; and specifically, the vitality of the epithelial cells of small intestines is effectively maintained and the protection effect has broad spectrum activity to a certain extent. Based on the novel property of the exopolysaccharide of Lactobacillus plantarum, the exopolysaccharide is applied as an antagonist against the Bacillus cereus enterotoxins or even applied to a drug used for treating poisoning by the Bacillus cereus enterotoxins in the invention, and the exopolysaccharide has definite therapeutic effect, rapid action and wide application prospects.
Description
Technical field
The present invention relates to microbial technology field, further to the isolated and purified of microbial metabolic products and medical usage thereof, be specifically related to a kind of Lactobacillus plantarum extracellular polysaccharide and the purposes for antagonism bacillus cereus enterotoxin toxicity thereof.
Background technology
Exopolysaccharides Produced by Lactic Acid Bacteria (Exopolysaccharide, EPS), refers to and is secreted into extracellular saccharide compound in lactic acid bacteria metabolic process.Owing to Exopolysaccharides Produced by Lactic Acid Bacteria has good rheological characteristic and the taste and flavor that fermented food is good can be given, therefore the production of the food such as milk product, beverage, cake, confection, ice cream can be widely used at present as the gellant of food industry, stabilizer, antistaling agent and emulsifying agent etc..Additionally, there are some researches show that EPS has antitumor, reduces the effects such as cholesterol, slow down aging, regulating intestinal canal colony balance, therefore EPS is probably one of material base of lactic acid bacteria prebiotic effect.
Lactobacillus plantarum (Lactobacillus plantarum), genus homofermentative lactic bacteria, Gram-positive, facultative aerobic.This bacterial strain is proved to have acidproof, bile tolerance, anti-pathogenic bacterium, the effect of regulating intestinal canal colony balance, there are some researches show that above-mentioned acid-fast ability and prebiotic effect may be relevant with its extracellular polysaccharide simultaneously.Lactobacillus plantarum extracellular polysaccharide is that hybrid form exists, the single compound that not character is homogeneous；And polysaccharide is as the complicated and huge glucide of molecular structure, the primary structure change no matter determined by sugar unit kind and order, or the secondary structure change determined by hydrogen bond and other noncovalent interactions, all can cause the difference of its physicochemical property, therefore for the character of research Lactobacillus plantarum polysaccharide, first should specify its material typing, obtain the sterling of different classes of Lactobacillus plantarum extracellular polysaccharide, thus clear and definite its material base, and then inquire into the character of different Lactobacillus plantarum extracellular polysaccharide.
Bacillus cereus is the common pathogen causing alimentary toxicosis, the alimentary toxicosis type that can cause is broadly divided into diarrhoea and symptoms of emesis, wherein diarrhoea is the most relevant to the enterotoxin of strain secretes, and vomit and mainly caused by bacterial strain generation vomitoxin, having document to report, bacillus cereus enterotoxin can cause eukaryotic cell dead.In prior art, the treatment to bacillus cereus enterotoxin poisoning case is broadly divided into many by antibiotherapy and symptomatic treatment, and wherein symptomatic treatment essentially consists in relief of symptoms, maintains body electrolyte balance；Although and antibiotherapy can direct killing microorganisms, but owing to the direct antigenic substance of this disease is the metabolite (enterotoxin) of bacillus cereus rather than bacillus cereus itself, therefore this therapy onset is relatively slow, and therapeutic effect is undesirable.In prior art, the therapy or the medicine that directly reduce bacillus cereus enterotoxin pathogenicity have no report.
Summary of the invention
It is contemplated that for the technological deficiency of prior art, it is provided that a kind of Lactobacillus plantarum extracellular polysaccharide, to solve prior art lacks the technical problem of above-mentioned Lactobacillus plantarum extracellular polysaccharide.
Another that the invention solves the problems that technical problem is that in prior art, and Lactobacillus plantarum extracellular polysaccharide is as a kind of polysaccharide mixture, and wherein composition is indefinite.
The invention solves the problems that further technical problem is that this Lactobacillus plantarum extracellular polysaccharide is difficult to prepare.
Another this Lactobacillus plantarum extracellular polysaccharide that technical problem is that prepared by conventional method that the invention solves the problems that, its purity is relatively low.
Invention also provides the above-mentioned Lactobacillus plantarum extracellular polysaccharide purposes for antagonism bacillus cereus enterotoxin toxicity, to solve the technical problem being difficult to directly reduce bacillus cereus enterotoxin pathogenicity in prior art.
The another purposes that technical problem is that this Lactobacillus plantarum extracellular polysaccharide that the invention solves the problems that more is limited to.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of Lactobacillus plantarum extracellular polysaccharide, described Lactobacillus plantarum extracellular polysaccharide is prepared by the following method:
1) taking Lactobacillus plantarum fermentation liquid, solid-liquid separation takes supernatant；
2) to step 1) described supernatant adds ethanol, precipitate with ethanol, solid-liquid separation takes precipitation, dialyses with the bag filter of 8～14KDa molecular cut offs, takes bag filter content and be dried, be crude polysaccharides；
3) with containing 40～60mM Tris-HCl, 5～15mM MgSO4·7H2The solution of O is as crude polysaccharides lysate, dissolving step 2) described crude polysaccharides, then add nuclease DNAse type-I to final concentration 2～3 μ g/mL, hydrolysis；
4) protease P ronase E is then added to final concentration 40～60 μ g/mL, enzymolysis；
5) then addition trichloroacetic acid is to final concentration 10～15% (w/v), and after 20～40min, solid-liquid separation takes supernatant, and dialysis takes bag filter content and is described Lactobacillus plantarum extracellular polysaccharide.
As preferably, step 1) described in fermentation liquid be fermentation liquid during Lactobacillus plantarum anaerobic fermentation 22～26h；More excellent, fermentation time is 24h.
As preferably, step 2) in the addition of ethanol be 1.5～2.5 times of described supernatant volume；More excellent, amount of alcohol added is 2 times of described supernatant volume.
As preferably, step 3) in crude polysaccharides final concentration of 4～6mg/L；More excellent, it is 5mg/L.
As preferably, step 3) described hydrolysis is to continue 4～8h under the conditions of 35～39 DEG C；More excellent, it is under the conditions of 37 DEG C, continue 6h.
As preferably, step 4) described enzymolysis is to continue 16～20h under the conditions of 35～39 DEG C；More excellent, it is under the conditions of 37 DEG C, continue 18h.
As preferably, step 5) before dialysis, first regulation supernatant pH to 4～5, then dialyse；More excellent, regulate pH to 4.5；Optimum, described regulation is that the NaOH solution utilizing 10mol/L realizes.
On the basis of any of the above technical scheme preferably, step 1) described in Lactobacillus plantarum, be deposit number be the Lactobacillus plantarum of CCTCC M 2014170.Now this polysaccharide has definite safety assurance for human body
On the basis of any of the above technical scheme preferably, described solid-liquid separation, is to be centrifuged 15～25min with the rotating speed of 10000～14000rpm.
On the basis of any of the above technical scheme preferably, the persistent period of dialysis is 60～84h；More excellent, the persistent period of dialysis is 72h.On this basis it is further preferred that during Tou Xi every day change ultra-pure water outside bag filter 2 times.
As preferably, step 1) described in fermentation liquid, obtain with the fermentation of MRS culture medium.
As preferably, step 1) described in fermentation liquid, under the conditions of 35～39 DEG C, fermentation obtains；More excellent fermentation temperature is 37 DEG C.
As preferably, step 2) described in dry be lyophilization.
As preferably, step 3) pH value of described crude polysaccharides lysate is 7.2～7.8；More excellent, its pH is 7.5.
As preferably, step 5) in trichloroacetic acid add after final concentration of 12% (w/v), continue 30min after adding trichloroacetic acid and carry out solid-liquid separation again.
Invention also provides above-mentioned Lactobacillus plantarum extracellular polysaccharide for suppressing the application to intestinal epithelial cell toxicity of the bacillus cereus enterotoxin.
On this application foundation preferably, described bacillus cereus enterotoxin is cytotoxin K.Described cytotoxin K refers to toxin that produced, that be normally referred to as CytK by Bacillus cereus.
On this application foundation preferably, described bacillus cereus enterotoxin is hemolytic enterotoxin HBL.
On this application foundation preferably, described bacillus cereus enterotoxin is non-hemolytic enterotoxin N he.
On this application foundation preferably, described suppression bacillus cereus enterotoxin is to alleviate intestinal epithelial cell vigor under bacillus cereus enterotoxin existence condition to reduce trend to intestinal epithelial cell toxicity.
On this application foundation preferably, described application is directly to be contacted with intestinal epithelial cell under bacillus cereus enterotoxin existence condition by Lactobacillus plantarum extracellular polysaccharide solution；More excellent, the concentration of described Lactobacillus plantarum extracellular polysaccharide solution is 0.5～1.5mg/mL；Optimum, the concentration of described Lactobacillus plantarum extracellular polysaccharide solution is 1mg/mL.
Invention also provides above-mentioned Lactobacillus plantarum extracellular polysaccharide for preparing the application of bacillus cereus enterotoxin poisoning treatment medicine.
As preferably, the dosage form of described medicine is oral agents.
In above technical scheme, deposit number be the depositary institution of the biomaterial of CCTCC M 2014170 be " China typical culture collection center ", its address is " No. 299 Wuhan Universitys of Wuchang District, Wuhan City, Hubei Province Bayi Road are in the school ", the preservation date of this biomaterial is on April 28th, 2014, and its Classification And Nomenclature is Lactobacillus plantarum (Lactobacillus plantarum).
In above technical scheme, suppression bacillus cereus enterotoxin, to intestinal epithelial cell toxicity, can be that bacillus cereus enterotoxin hatches intestinal epithelial cell altogether with Lactobacillus plantarum extracellular polysaccharide of the present invention；Certainly can also be under bacillus cereus enterotoxin existence condition, be added thereto to Lactobacillus plantarum extracellular polysaccharide of the present invention, thus suppress its toxicity to intestinal epithelial cell.In above technical scheme, described cell viability refers to the quantity levels of survivaling cell, might not characterize with the specific number of living cells or cell density, and can be by arbitrary parameter for reacting cells vigor in the art and be evaluated；In the present invention, mtt assay can be selected for evaluating the index of cell viability.
Lactobacillus plantarum extracellular polysaccharide provided by the present invention, the direct precipitate polysaccharides in Lactobacillus plantarum fermentation liquid with ethanol precipitation, small molecular weight impurity is removed through dialysis after and, and successively utilize enzyme solution to remove nucleic acid and protein impurities, recycling trichloroacetic acid precipitation foreign protein, solid-liquid separation performs dialysis after taking supernatant, thus obtains the desired polysaccharide of purification.The polysaccharide characterized by method made above, is effectively retained owing to have matched purification condition therefore its molecular structure of gentleness, and products therefrom constituent is single, structure clear and definite, has higher purity and yield simultaneously.
In addition; the present invention is had been surprisingly found that by laboratory facilities; under this Lactobacillus plantarum extracellular polysaccharide existence condition; the toxicity of intestinal epithelial cell is substantially suppressed by bacillus cereus enterotoxin; it is embodied in intestinal epithelial cell vigor to be positively maintained, and this protective effect has certain broad spectrum activity.Above new property based on this Lactobacillus plantarum extracellular polysaccharide, present invention determine that it is as bacillus cereus enterotoxin antagonist or even the new application of bacillus cereus enterotoxin poisoning treatment medicine, its determined curative effect, onset is quick, has broad application prospects.
Accompanying drawing explanation
Fig. 1 is in the embodiment of the present invention 1, the colour developing figure of the intestinal epithelial cell after hatching with mtt assay three kinds of bacillus cereus supernatant of detection on orifice plate.
Fig. 2 is in the embodiment of the present invention 1, under extracellular polysaccharide not existence condition, after three strain bacillus cereus supernatant hatch intestinal epithelial cell respectively, and the mtt assay testing result of cell viability.
Fig. 3 is in the embodiment of the present invention 1, under extracellular polysaccharide existence condition, after three strain bacillus cereus supernatant hatch intestinal epithelial cell respectively, and the mtt assay testing result of cell viability.
In figure 1 above～Fig. 3: Nhe/HBL/CytK part is strains A TCC14579 simultaneously producing tri-kinds of toxin of Nhe, HBL, CytK；HBL/CytK part is strains A TCC10987 simultaneously producing two kinds of toxin of HBL, CytK；Nhe part is to produce a strain bacillus cereus of Nhe toxin, and its internal number is JX0061LY.
Fig. 4 is in the embodiment of the present invention 1, after hatching intestinal epithelial cell with the bacillus cereus ATCC14579 supernatant of variable concentrations, and the mtt assay testing result of cell viability.Wherein experimental group is the experimental result that bacillus cereus ATCC14579 supernatant, both Lactobacillus plantarum extracellular polysaccharide of the present invention hatch intestinal epithelial cell altogether；Matched group is the experimental result that bacillus cereus ATCC14579 supernatant individually hatches intestinal epithelial cell.
Fig. 5 is in the embodiment of the present invention 1, after hatching intestinal epithelial cell with the bacillus cereus ATCC10987 supernatant of variable concentrations, and the mtt assay testing result of cell viability.Wherein experimental group is the experimental result that bacillus cereus ATCC10987 supernatant, both Lactobacillus plantarum extracellular polysaccharide of the present invention hatch intestinal epithelial cell altogether；Matched group is the experimental result that bacillus cereus ATCC10987 supernatant individually hatches intestinal epithelial cell.
Fig. 6 is in the embodiment of the present invention 1, after hatching intestinal epithelial cell with the bacillus cereus JX0061LY supernatant of variable concentrations, and the mtt assay testing result of cell viability.Wherein experimental group is the experimental result that bacillus cereus JX0061LY supernatant, both Lactobacillus plantarum extracellular polysaccharide of the present invention hatch intestinal epithelial cell altogether；Matched group is the experimental result that bacillus cereus JX0061LY supernatant individually hatches intestinal epithelial cell.
Detailed description of the invention
The detailed description of the invention of the present invention will be described in detail below.In order to avoid the most unnecessary details, in the examples below to belonging to known structure or function will not be described in detail.
Approximating language used in following example can be used for quantitative expression, shows to allow quantity to have certain variation in the case of not changing basic function.Therefore, the numerical value revised with the language such as " about ", " left and right " is not limited to this exact value itself.In certain embodiments, " about " represents that the numerical value allowing its correction changes in the range of positive and negative 10 (10%), and such as, what " about 100 " represented can be any numerical value between 90 to 110.Additionally, in the statement of " the about first numerical value is to second value ", at about revise two numerical value of the first and second numerical value.In some cases, approximating language may be relevant with the precision of measuring instrument.
In addition to being defined, technology used in following example and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.
Test reagent consumptive material used in following example, if no special instructions, is routine biochemistry reagent；Described experimental technique, if no special instructions, is conventional method；Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged；% in following example, if no special instructions, is weight/mass percentage composition.
Embodiment 1
The extraction of 1.1 Lactobacillus plantarum extracellular polysaccharide, purification
Lactobacillus plantarum is 37 DEG C of Anaerobic culturel 24h in MRS culture medium, and 12000 × g is centrifuged 20min, abandon precipitation, supernatant molecular cut off be 8000～14000Da bag filter dialyse 3 days, change ultra-pure water every day 2 times.After lyophilization, with 50mM Tris-HCl, 10mM MgSO4·7H2Crude polysaccharides is dissolved (final concentration of 5mg/mL) by O (pH7.5), uses nuclease DNAse type-I (D5025, Sigma) of final concentration of 2.5 μ g/mL at 37 DEG C of 6h that are hydrolyzed nucleic acid.Crude polysaccharides after hydrolysis uses the protease P ronase E (165921, Roche) of final concentration of 50 μ g/mL, at 37 DEG C, protein is carried out enzymolysis 18h.After Protease Treatment, the supernatant being centrifuged 20min acquisition with trichloroacetic acid the process 30min, 12000 × g of final concentration of 12% need to regulate pH to 4.0～5.0, continues dialysis 3 days, changes water every day 2 times.
1.2 different bacillus cereus the produced enterotoxin impacts on Caco-2 cell viability
Take 100 μ L Caco-2 cell (104) to 96 porocyte culture plates, 5%CO2Under the conditions of 37 DEG C cultivate to formed monolayer adherence cell (about 12h).The enterotoxin of three strain bacillus cereuss being carried out 2 times of seven gradient dilutions (diluting by culture medium DMEM), processes cell monolayer respectively with the enterotoxin of the same concentration of three strain bacterium, each concentration is in triplicate.Wherein experimental group is further continued for adding 20 μ L extracellular polysaccharide solution (final concentration of 1mg/mL).5%CO2Under the conditions of 37 DEG C hatch 6h.Add the MTT (0.5%) of final concentration of 1mg/mL, at 5%CO2Under the conditions of 37 DEG C hatch 4h, sop up supernatant gently, then every hole adds 150 μ L dimethyl sulfoxide, and vibrate at a slow speed 10min, measures the enterotoxin of three strain bacillus cereuss by microplate reader and processes after cell the light absorption value at 490nm, and result is as shown in Figure 2 and Figure 3.
By Fig. 2,3 all it appeared that, along with the reduction of toxin concentration, cell viability raises, illustrates that cell viability is relevant with the concentration of toxin, and Fig. 3 raises trend and becomes apparent from, embody the above Lactobacillus plantarum extracellular polysaccharide antagonism to bacillus cereus enterotoxin toxicity.
1.3 Lactobacillus plantarum extracellular polysaccharide made above are to Caco-2 cell viability protective effect
Take 100 μ L Caco-2 cell (104) to 96 porocyte culture plates, 5%CO2Under the conditions of 37 DEG C cultivate to formed monolayer adherence cell (about 12h).Experiment is divided into matched group and experimental group, and matched group carries out induction process with the enterotoxin of different gradient dilutions to cell respectively, and experimental group adds 20 μ L final concentration of 1mg/mL extracellular polysaccharide solution while processing with the enterotoxin of different gradient dilutions respectively.5%CO2Under the conditions of 37 DEG C hatch 6h.Add the MTT (0.5%) of final concentration of 1mg/mL, at 5%CO2Under the conditions of 37 DEG C hatch 4h, sop up supernatant gently, then every hole adds 150 μ L dimethyl sulfoxide, and vibrate at a slow speed 10min, measures the light absorption value change of cell 490nm under the process of variable concentrations toxin by microplate reader, and result is as shown in figures 4-6.
By Fig. 4～6 all it is found that the cell viability that there is the experimental group of extracellular polysaccharide is significantly higher than there is not the matched group of extracellular polysaccharide.This shows that Lactobacillus plantarum extracellular polysaccharide of the present invention has definite inhibitory action to the toxicity of bacillus cereus enterotoxin, and this inhibitory action has certain broad spectrum activity.
Embodiment 2
A kind of Lactobacillus plantarum extracellular polysaccharide, this Lactobacillus plantarum extracellular polysaccharide is prepared by the following method:
1) taking Lactobacillus plantarum fermentation liquid, solid-liquid separation takes supernatant；
2) to step 1) described supernatant adds ethanol, precipitate with ethanol, solid-liquid separation takes precipitation, dialyses with the bag filter of 8KDa molecular cut off, takes bag filter content and be dried, be crude polysaccharides；
3) with containing 40mM Tris-HCl, 5mM MgSO4·7H2The solution of O is as crude polysaccharides lysate, dissolving step 2) described crude polysaccharides, then add nuclease DNAse type-I to final concentration 2 μ g/mL, hydrolysis；
4) protease P ronase E is then added to final concentration 40 μ g/mL, enzymolysis；
5) then addition trichloroacetic acid is to final concentration 10% (w/v), and after 20min, solid-liquid separation takes supernatant, and dialysis takes bag filter content and is described Lactobacillus plantarum extracellular polysaccharide.
On the basis of above technical scheme, meet following condition:
Step 1) described in fermentation liquid be fermentation liquid during Lactobacillus plantarum anaerobic fermentation 22h.Step 2) in the addition of ethanol be 1.5 times of described supernatant volume.Step 3) in the final concentration of 4mg/L of crude polysaccharides.Step 3) described hydrolysis be under the conditions of 35 DEG C continue 4h.Step 4) described enzymolysis be under the conditions of 35 DEG C continue 16h.Step 5) before dialysis, first regulation supernatant pH to 4, then dialyses.
Embodiment 3
A kind of Lactobacillus plantarum extracellular polysaccharide, this Lactobacillus plantarum extracellular polysaccharide is prepared by the following method:
1) taking Lactobacillus plantarum fermentation liquid, solid-liquid separation takes supernatant；
2) to step 1) described supernatant adds ethanol, precipitate with ethanol, solid-liquid separation takes precipitation, dialyses with the bag filter of 14KDa molecular cut off, takes bag filter content and be dried, be crude polysaccharides；
3) with containing 60mM Tris-HCl, 15mM MgSO4·7H2The solution of O is as crude polysaccharides lysate, dissolving step 2) described crude polysaccharides, then add nuclease DNAse type-I to final concentration 3 μ g/mL, hydrolysis；
4) protease P ronase E is then added to final concentration 60 μ g/mL, enzymolysis；
5) then addition trichloroacetic acid is to final concentration 15% (w/v), and after 40min, solid-liquid separation takes supernatant, and dialysis takes bag filter content and is described Lactobacillus plantarum extracellular polysaccharide.
On the basis of above technical scheme, meet following condition:
Step 1) described in fermentation liquid be fermentation liquid during Lactobacillus plantarum anaerobic fermentation 26h.Step 2) in the addition of ethanol be 2.5 times of described supernatant volume.Step 3) in the final concentration of 6mg/L of crude polysaccharides.Step 3) described hydrolysis be under the conditions of 39 DEG C continue 8h.Step 4) described enzymolysis be under the conditions of 39 DEG C continue 20h.Step 5) before dialysis, first regulation supernatant pH to 5, then dialyses.
Embodiment 4
A kind of Lactobacillus plantarum extracellular polysaccharide, this Lactobacillus plantarum extracellular polysaccharide is prepared by the following method:
1) taking Lactobacillus plantarum fermentation liquid, solid-liquid separation takes supernatant；
2) to step 1) described supernatant adds ethanol, precipitate with ethanol, solid-liquid separation takes precipitation, dialyses with the bag filter of 10～12KDa molecular cut offs, takes bag filter content and be dried, be crude polysaccharides；
3) with containing 45mM Tris-HCl, 8mM MgSO4·7H2The solution of O is as crude polysaccharides lysate, dissolving step 2) described crude polysaccharides, then add nuclease DNAse type-I to final concentration 2.5 μ g/mL, hydrolysis；
4) protease P ronase E is then added to final concentration 45 μ g/mL, enzymolysis；
5) then addition trichloroacetic acid is to final concentration 13% (w/v), and after 25min, solid-liquid separation takes supernatant, and dialysis takes bag filter content and is described Lactobacillus plantarum extracellular polysaccharide.
On the basis of above technical scheme, meet following condition:
Step 2) in the addition of ethanol be 2.2 times of described supernatant volume.Step 3) described hydrolysis be under the conditions of 38 DEG C continue 5h.Step 5) before dialysis, first regulation supernatant pH to 4.7, then dialyses.Step 1) described in Lactobacillus plantarum, be deposit number be the Lactobacillus plantarum of CCTCC M 2014170.
Embodiment 5
A kind of Lactobacillus plantarum extracellular polysaccharide, this Lactobacillus plantarum extracellular polysaccharide is prepared by the following method:
1) taking Lactobacillus plantarum fermentation liquid, solid-liquid separation takes supernatant；
2) to step 1) described supernatant adds ethanol, precipitate with ethanol, solid-liquid separation takes precipitation, dialyses with the bag filter of 10KDa molecular cut off, takes bag filter content and be dried, be crude polysaccharides；
3) with containing 55mM Tris-HCl, 13mM MgSO4·7H2The solution of O is as crude polysaccharides lysate, dissolving step 2) described crude polysaccharides, then add nuclease DNAse type-I to final concentration 2.8 μ g/mL, hydrolysis；
4) protease P ronase E is then added to final concentration 55 μ g/mL, enzymolysis；
5) then addition trichloroacetic acid is to final concentration 14% (w/v), and after 35min, solid-liquid separation takes supernatant, and dialysis takes bag filter content and is described Lactobacillus plantarum extracellular polysaccharide.
Above embodiments of the invention are described in detail, but described content has been only presently preferred embodiments of the present invention, not in order to limit the present invention.All any amendment, equivalent and improvement etc. made in the application range of the present invention, should be included within the scope of the present invention.
Claims (10)
1. a Lactobacillus plantarum extracellular polysaccharide, it is characterised in that described Lactobacillus plantarum extracellular polysaccharide is to pass through
Prepared by following methods:
1) taking Lactobacillus plantarum fermentation liquid, solid-liquid separation takes supernatant；
2) to step 1) described supernatant adds ethanol, precipitate with ethanol, solid-liquid separation takes precipitation, with 8～14KDa
The bag filter dialysis of molecular cut off, takes bag filter content and is dried, be crude polysaccharides；
3) with containing 40～60mM Tris-HCl, 5～15mM MgSO4·7H2The solution of O is molten as crude polysaccharides
Solve liquid, dissolving step 2) described crude polysaccharides, then addition nuclease DNAse type-I is to final concentration 2～3 μ g/mL,
Hydrolysis；
4) protease P ronase E is then added to final concentration 40～60 μ g/mL, enzymolysis；
5) then addition trichloroacetic acid is to final concentration 10～15% (w/v), and after 20～40min, solid-liquid separation takes supernatant,
Dialysis, takes bag filter content and is described Lactobacillus plantarum extracellular polysaccharide.
Preparation method the most according to claim 1, it is characterised in that step 2) in the addition of ethanol
It it is 1.5～2.5 times of described supernatant volume.
Preparation method the most according to claim 1, it is characterised in that step 3) in end of crude polysaccharides dense
Degree is 4～6mg/L.
Preparation method the most according to claim 1, it is characterised in that step 5) dialysis before, first regulate
Supernatant pH to 4～5, then dialyse.
5. according to the preparation method described in any one of Claims 1 to 4, it is characterised in that step 1) described in
Lactobacillus plantarum, be deposit number be the Lactobacillus plantarum of CCTCC M 2014170.
6. Lactobacillus plantarum extracellular polysaccharide described in claim 1 is used for suppressing bacillus cereus enterotoxin to little
The application of enterocyte toxicity.
Application the most according to claim 6, it is characterised in that described bacillus cereus enterotoxin is thin
Born of the same parents toxin K.
Application the most according to claim 6, it is characterised in that described bacillus cereus enterotoxin is molten
Courageous and upright enterotoxin HBL.
Application the most according to claim 6, it is characterised in that described bacillus cereus enterotoxin right and wrong
Hemolytic enterotoxin N he.
10. Lactobacillus plantarum extracellular polysaccharide described in claim 1 is used for preparing the poisoning of bacillus cereus enterotoxin
The application of medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107243645A (en) * | 2017-06-06 | 2017-10-13 | 东南大学 | A kind of method of utilization Lactobacillus plantarum transposon mutagenesis noble metal nano particles |
| CN107243645B (en) * | 2017-06-06 | 2019-12-10 | 东南大学 | Method for synthesizing precious metal nanoparticles by using lactobacillus plantarum exopolysaccharides |
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