CN106011196A - Method for purifying lactobacillus rhamnosus extracellular polysaccharides - Google Patents
Method for purifying lactobacillus rhamnosus extracellular polysaccharides Download PDFInfo
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Abstract
The invention discloses a method for purifying lactobacillus rhamnosus extracellular polysaccharides and relates to the extracellular polysaccharides. The method includes: inoculating a single colony on the MRS solid culture medium of seed conservation to the activated fermentation liquid cultured in the MRS liquid culture medium; adding CaCl2 with the final concentration being 10mM into the MRS liquid culture medium, using the activated fermentation liquid for inoculation to obtain inoculated fermentation liquid, and measuring the OD600 and pH values of 1ml of inoculated liquid which is cultured for 0, 2, 4, 6, 12 and 24 hours; removing the cells and protein of the inoculated fermentation liquid, adding glacial acetic acid, placing into a refrigerator for one night, centrifuging, removing supernatant, performing freeze-drying on sediment, taking out, dialyzing, performing freeze-drying to obtain crude extracellular polysaccharides, dissolving into pure water, dissolving the extracellular polysaccharides sample after dialyzing and freeze-drying into distilled water to prepare a 20mg/mL solution, centrifuging, and performing ion column chromatography on supernatant to obtain the lactobacillus rhamnosus extracellular polysaccharides.
Description
Technical field
The present invention relates to extracellular polysaccharide, especially relate to the method for purification of a kind of lactobacillus rhamnosus extracellular polysaccharide.
Background technology
Probiotic bacteria application has long history.Numerous studies show, lactic acid bacteria can regulate body gastrointestinal tract normal bacteria
The balance of group, reduces serum cholesterol, controls endotoxin, putrefaction bacteria and the generation of spoilage product in suppression intestinal, manufactures nutrition
Material, thus to generation effects such as the nutritional status of body, physiological function, immunoreation[1].Lactic acid, is the main of lactic acid bacteria
Tunning, lactic acid not only local flavor is treated very, and is had extremely strong sterilizing power, outshines othersOne branch of the tree is particularly thriving in organic acid.Its sterilizing ability is more than
Citric acid, tartaric acid, several times of succinic acid[2].The chemical industry precursor of lactic acid or a very attractive, it can conduct
Produce biodegradable polylactide precursor, and substitute the potentiality of the polymer derived from petrochemical industry.Lactic acid bacteria can also be by useless
Biomass are converted into industry and have the product of industrial value.
Active lactic acid bacteria drink is a kind of leben containing live lactobacillus.Compared with solidification type yoghourt, Lactic acid bacteria drink
Acid bacteria beverage not only has the health care of Yoghourt, and concentration is lower, and mobility is more preferable, and mouthfeel is more refreshing, and cost is lower, so
Once appearance, dominate the market the most rapidly[3-7].The leben market in the whole world in 2006 increases 9.3%, greatly exceed it
The rate of increase of his milk product.The taste flavor of active lactic acid bacteria drink and the quantity of lactic acid bacteria be constitute product quality main because of
Element.Research shows[8], probiotic bacteria survival in human body and the multiplication capacity prebiotic effect important to probiotic bacteria,
Obtaining the health-care effect of desired probiotic bacteria, probiotic bacteria must reach sufficient amount.Lactobacillary milk is drunk by China's national standard
Viable count regulation in material: lactic acid bacteria number >=106Individual/ml.Japan's fermentation milk specifies with lactobacillus beverage association: Ruzhong probiotic bacteria
Number at least 107Individual/ml, to guarantee that viable bacteria enough after entering human body plays health care.But, the most many researchs are sent out
Show, enter the quantity of probiotic bacteria in the product on shelf, understand the growth characteristics due to self and the impact of ambient environmental factors, in
Certain downward trend.Therefore, desired health-care effect to be obtained, it is necessary to improve the quantity of lactic acid bacteria in food, to guarantee
Lactic acid bacteria, by still large number of viable after digestive tract, plays prebiotic effect after entering intestinal.But means currently mainly are all passed through
The optimization of Lactobacillus milk beverage fermentation technology, complex steps.
Exopolysaccharides Produced by Lactic Acid Bacteria (EPS) is that lactic acid bacteria is secreted into extracellular mucus or pod in its growth, metabolic process
Film polysaccharide, has important physicochemical characteristics, such as good rheological properties, can improve the viscosity of fermentation milk, matter structure and mouth
Sense, prevents from shrinking and milk surum precipitation, makes product thickening, stablizes, and exquisite quality is uniform, taste lubrication；And EPS also has well
Physiologically active, such as antitumor, immunomodulating, antiviral, antiulcer, antioxidation, cholesterol reducing, blood pressure lowering etc., be alternatively arranged as
Prebiotics promotes the growth of intestinal other probiotic bacterias interior, regulating intestinal canal flora[9-11].In recent years about lactic acid bacteria and lactic acid mycetocyte
The research of exo polysaccharides becomes focus.
List of references:
[1]E,Alvarez S,Medina M,et al.Gut mucosal immunostimulation by
lactic acid bacteria[J].Biocell,2000,24(3):223-232.
[2] Zhen Guangming, Zhang Jie. biodegradable polylactic acid and commercial Application [J] thereof. new material industry, 2009, (8):
36-41.
[3] Wang Fangan. lactic acid bacteria fermentation nourishing healthy and food development [J] thereof. western science and technology of grain and oil, 1995, (3): 36-
37.
[4] Zhang Yanli, Tang Xiumin. the nutritive value of Yoghourt and health care [J]. food science and technology, 1997, (4): 38-40.
[5] Dong Kaifa, Xu Mingsheng. the nutrition health-care functions [J] of Yoghourt. Chinese food and nutrition, 2000, (2): 33-34
[6]Ahme S,Nobaek S,Jeppsson B,et al.The normalLactibacillus flora of
healthy human rectal and oral mucosa[J].Journal of Applied Microbiology,1998,
85(1):88-94.
[7]Hosoda M,Hashimoto H,He F,et al.Effect of administration of milk
Fermented with Lactobacillus acidophilus LA-2 on fecal mutagenicity and
microflora in the human Intestine[J].Journal of Dairy Science,1995,79(5):45-
749.
[8] Song Kungang. lactobacillus milk beverage Development Trends [J]. food industry science and technology, 2005, (7): 10-11.
[9]Wei C,Zhao Z,Shi-Fei C,et al.Optimization for the production of
exopolysaccharide from Fomes fomentarius in submerged culture and its
antitumor effect in vitro[J].Bioresource Technology,2008,99(8):3187-3194.
[10]Xu R,Nan S,Li P.Invitro and invivo antioxidant activity of
exopolysaccharide fractions from Bifidobacterium animalis RH[J].Anaerobe,
2011,17(5):226-231.
[11]Matés J M.Effects of antioxidant enzymes in the molecular control
of reactive oxygen species toxicology[J].Toxicology,2000,153(1-3):83-104.
Summary of the invention
It is an object of the invention to provide the method for purification of a kind of lactobacillus rhamnosus extracellular polysaccharide.
The present invention comprises the following steps:
1) from the MRS solid medium of conservation to be, choose single bacterium colony connected in MRS fluid medium cultivate, must activate
Fermentation liquid；
In step 1) in, described strain is lactobacillus rhamnosus Lactobacillus rhamnosus, during this bacterial strain is purchased from
State's Microbiological Culture Collection administration committee common micro-organisms center；Consisting of of described MRS solid medium: glucose 20g/
L, Carnis Bovis seu Bubali cream 10g/L,Peptone 10g/L, yeast powder 5g/L, ammonium citrate 1g/L, sodium acetate 5g/L, KH2PO4 2g/L,
MgSO4·7H2O 0.05g/L,MnSO4·H2O 0.05g/L, agar powder 15g/L, pH 6～6.5；Described MRS fluid medium
Consist of: glucose 20g/L, Carnis Bovis seu Bubali cream 10g/L,Peptone 10g/L, yeast powder 5g/L, ammonium citrate 1g/L, sodium acetate
5g/L, KH2PO4 2g/L,MgSO4·7H2O 0.05g/L,MnSO4·H2O 0.05g/L, pH 6～6.5, sterilising conditions is 118
DEG C, 15min；The condition of described cultivation can at 37 DEG C constant temperature quiescent culture 12h；
2) in MRS fluid medium, add the CaCl of final concentration of 10mM2, take step 1) in the fermentation liquid that activated enter
Row switching, obtains the fermentation liquid after switching, takes the fermentation liquid after the 1ml switching of cultivation 0,2,4,6,12,24h and measures OD600, pH
Value；
In step 2) in, described switching by volume percentage ratio 4% is transferred.
3) take step 2) in fermentation liquid after switching, after removing cell and albumen, add ice ethanol at 1: 3 by volume,
, it is then centrifuged in refrigerator overnight, removes supernatant and stay precipitation, pellet frozen is taken out after drying, dialysis, then after lyophilization, i.e.
Obtain thick extracellular polysaccharide (c-EPS)；
In step 3) in, the ice ethanol that described ice ethanol can use mass percentage concentration to be 95%；The temperature of described refrigerator
Can be 4 DEG C；Described centrifugal condition can be at 4 DEG C, and 8000g is centrifuged 10min；The described cryodesiccated time can be 8h；Described
The condition of analysis can be dialysed 3d at 4 DEG C.
4) by step 3) the thick extracellular polysaccharide of gained is dissolved in pure water, the thick extracellular polysaccharide sample after dialysis lyophilizing of learning from else's experience
Product, are dissolved in distilled water, the solution of preparation 20mg/mL, centrifugal, take supernatant ion column chromatography, obtain outside lactobacillus rhamnosus born of the same parents
Polysaccharide, is designated as extracellular polysaccharide component 2 (EPS-2).
In step 4) in, described centrifugal condition can be that 8000g is centrifuged 10min；Described ion column chromatography can use DEAE-
Sepharose Fast Flow (GE company) ion column chromatographs.
The lactobacillus rhamnosus extracellular polysaccharide of gained can carry out purity analysis with GPC method；By 25mg lactobacillus rhamnosus born of the same parents
Exo polysaccharides is dissolved in D2In O, carry out the analysis of structure by Bruker 600MZ nuclear magnetic resonance analyser.
The purposes of lactic acid constantly extends, and active lactic acid bacteria drink market is the most ripe, and the practical value of extracellular polysaccharide obtains
To extensive concern, but it is limited to yield and cost.The present invention adds final concentration of 10mM CaCl in liquid MRS culture medium2,
Simplicity is easy to get, low cost, great lactic acid bacteria industrial value.Containing CaCl2MRS fluid medium in, rhamnose breast bar
The growth of bacterium is promoted, and fermentation 12h viable count can promote 140%, and acid producing ability, the yield of extracellular polysaccharide the most significantly carry simultaneously
Rise, the extracellular polysaccharide purified under the method is analyzed, be found to be a kind of novel lactic bacterium exopolysaccharide-lactobacillus rhamnosus
Extracellular polysaccharide.The present invention utilizes fermentation liquid that e. coli bl21, Bacillus cercus, staphylococcus aureus are carried out suppression in fact
Testing, result shows that the fermentation liquid of high viable count can strengthen the streptococcus acidi lactici fermented solution suppression to harmful bacteria, by straight for full cell bacterium solution
Connect the suppression for harmful bacteria.
Accompanying drawing explanation
Fig. 1 is the impact that lactobacillus rhamnosus is grown by calcium ion.
Fig. 2 is the calcium ion impact on lactobacillus rhamnosus viable count.
Fig. 3 is DEAE-Sepharose anion-exchange column NaCl solution elution curve.
Fig. 4 is the purity that GPC measures EPS.
Fig. 5 is the 600MHz of EPS-21H NMR spectra.
Fig. 6 is the 600MHz of EPS-213C NMR spectra.
Fig. 7 is the 600MHz Edit-HSQC NMR spectra of EPS-2.
Fig. 8 is the 600MHz COSY NMR spectra of EPS-2.
Fig. 9 is the 600MHz HSQC-TOCSY NMR spectra of EPS-2.
Figure 10 is the 600MHz gHMBC NMR spectra of EPS-2.
Figure 11 is the 600MHz NOESY NMR spectra of EPS-2.
Figure 12 is the 600MHz H2BC NMR spectra of EPS-2.
Detailed description of the invention
For illustrating the purpose of the present invention and technique effect thereof, below in conjunction with accompanying drawing, the present invention is done the most specifically
Bright.
Embodiment of the present invention step is as follows:
Embodiment 1:CaCl2Promote lactobacillus rhamnosus growth
During the fermentation in line between viable count and the absorbance of the Lactic Acid from Fermentation Broth bacterium with skimmed milk as culture medium
Property dependency relation.Therefore, the viable count of lactic acid bacteria in fermented dairy product can be evaluated by the absorbance of mensuration fermentation liquid.Simultaneously
The pH value of detection fermentation liquid.
1) culture medium:
1, MRS fluid medium (g/L): glucose 20, Carnis Bovis seu Bubali cream 10,Peptone 10, yeast powder 5, ammonium citrate 1,
Sodium acetate 5, KH2PO4 2,MgSO4·7H2O 0.05,MnSO4·H2O 0.05, pH 6～6.5.
2, MRS solid medium (g/L): glucose 20, Carnis Bovis seu Bubali cream 10,Peptone 10, yeast powder 5, ammonium citrate 1,
Sodium acetate 5, KH2PO4 2,MgSO4·7H2O 0.05,MnSO4·H2O 0.05, agar powder 15, pH 6-6.5.
2) aseptic CaCl2The preparation of solution
CaCl2The preparation of solution: accurately weigh 1.500g 80 DEG C and dry 6h anhydrous calcium chloride powder in 4ml centrifuge tube
In, add 3ml sterile deionized water, be formulated as the solution of concentration 0.5g/ml.After ultrasonic 10min, put into super-clean bench standby.
Aseptic CaCl2Prepared by solution: draw the above-mentioned CaCl prepared of 2ml with the asepsis injector of 2ml2Solution, 0.22 μm
After nylon filter membrane filtration, standby in loading 4ml sterile centrifugation tube.
3) the lactobacillus rhamnosus fermentation liquid of activation 12h is inoculated in containing 10mM CaCl by 4% (v/v)2MRS liquid
In culture medium and MRS fluid medium.
The method of described activation is: by inoculating loop alcohol burner burn red and cool down after, with it from conservation MRS of three rides
Picking list bacterium colony on flat board, accesses in the 100mL shaking flask equipped with 20mL MRS fluid medium, is placed in 37 DEG C of quiescent culture casees
Cultivate 12h.
4) cultural method
Culture medium prepares: in the shaking flask of 250ml, loads the MRS fluid medium of 50ml.118 DEG C of sterilizing 15min.?
The MRS fluid medium of 50ml adds 110 μ l steps 2) the aseptic CaCl that prepared2Solution, now CaCl2Final concentration i.e.
For 10mM.
Described cultural method: the bacterium solution of front cultivation is equipped with 50ml MRS or has added 10mM by the inoculum concentration by 4%
CaCl2MRS fluid medium, operation is equipped with 2～3 parallel, is placed in the quiescent culture case of 37 DEG C cultivation.
Add CaCl2Impact on lactobacillus rhamnosus growth and lactic acid producing ability characterizes, by the following method:
5) detection of growth curve
1. sampling: the separately sampled 1mL of 0,2,4,6,12,24h is for detecting OD600 value and the pH of fermentation liquid after inoculation
Value.MRS liquid fermentation liquid is as a control group.
2. the mensuration of bacterial concentration: after bacterium solution is diluted certain gradient multiple (1～30 times), at 600nm ripple
Long lower its light absorption value that detects, the supernatant after wherein blank is bacterium solution centrifugal (14000rpm, 5min).
The detection of 3.pH value: with the pH of pH meter detection fermented liquid supernatant liquid.
4. data process:
OD600,Lac=(OD600-OD600,blank) × extension rate
Calculate meansigma methods and the standard deviation of each Duplicate Samples with actual measurement data, draw growth curve.
It can be seen from figure 1 that compared with normal MRS culture medium, add 10mM CaCl2MRS culture medium light absorption value more
Height, when 12h, light absorption value OD600 reaches 4.24, and normal MRS culture medium OD600 is 3.40.When arriving fermentation termination 24h,
10mM CaCl2The OD600 of MRS culture medium reach 5.10, and normal MRS culture medium OD600 is 4.60.Now, the product of the two
Acid ability also has larger difference.10mM CaCl2The pH of MRS culture medium reach 3.64, and normal MRS medium pH is 3.51.
Illustrate that calcium ion not only promotes the growth of lactobacillus rhamnosus, more strengthen its acid producing ability.
Embodiment 2:CaCl2Promote lactobacillus rhamnosus viable count
Calculate the sum of antibacterial since colony counting method, the most do not contain the number of dead bacterium, thus can accurately reflect
Viable count in fermentation liquid.
1) cultivate antibacterial according to the training method of example 1, cultivate after 12h or 24h, take 1ml fermentation broth sample, with sterilized water by
Level is diluted to 10-7Times.Take 10 of fermentation culture sample under the conditions of 2 respectively-6With 10-7Two dilution factors again, draw dilution
After bacterium solution 50 μ l be spread evenly across MRS solid medium plate, cultivate 48h for 37 DEG C.Take out culture dish flat board, the process of counting
In on plate it is noted that distinguish small bacterium colony, insoluble little granule or precipitate, carefully investigate and prosecute suspicious item, use magnifier
Observe, in order to from other materials, distinguish bacterium colony.Note the clump count to be used plate count between 10～300.
2) being calculated as follows of lactobacillus rhamnosus total plate count:
Clump count × 20 × 10 of CFU/ml)=grand total6
As can be known from Fig. 2, normal MRS culture medium is added 10mM CaCl2The viable bacteria of lactobacillus rhamnosus can be promoted
Number, when 12h, viable count enhancing rate is 140%.And to 24h, cell enters decline phase, with normal MRS liquid culture condi phase
Relatively, 10mM CaCl2Viable count 167% can be promoted.
Embodiment 3: application example
Select e. coli bl21 (E.coli BL21), staphylococcus aureus (B.cereus), Bacillus cercus
(S.aureus) bacteriostatic experiment is carried out.Concrete operations are as follows:
1) e. coli bl21, Bacillus cercus being inoculated in LB fluid medium cultivation 12h, rotating speed is
200rpm, standby.S. aureus Inoculate to TSB fluid medium is cultivated 12h, and rotating speed is 150rpm, standby.
2) with the card punch punching filter paper of internal diameter 5mm, it is thus achieved that the circular filter paper sheet that some internal diameters are homogeneous, sterilizing.
3) a certain amount of LB and TSB liquid and agar culture medium, sterilizing are prepared.
4) lactobacillus rhamnosus at MRS fluid medium and is added 10mM CaCl2MRS cultivate (37 DEG C, stand)
12h, standby.
5), after the heating of LB and TSB agar culture medium is dissolved, treat that it is cooled to about 50 DEG C, be rapidly added at super-clean bench
4.1.1 cultivate the cause of disease bacterium solution (2%, v/v) of 12h in, after mixing, pour sterilized flat board into.
6) after above-mentioned flat board solidifies, with tweezers, the filter paper of sterilizing is placed on the agar culture medium adding pathogen,
Draw 20 μ l and cultivate the MRS liquid lactobacillus rhamnosus drop under the normal of 12h and stress conditions on filter paper.By flat board just
Be placed in 4 DEG C of refrigerators and fix 1h, after be inverted in 37 DEG C of incubators cultivation 12h, take out flat board observe and use Image J software
(Japan) average diameter (LZD, cm) of inhibition zone is measured.
Table 1 lactobacillus rhamnosus is the inhibition zone of 37 DEG C of fermentation 12h under 2 condition of culture
Note: a diameter of 8.00mm of punching circle.
According to experimental result, normal MRS culture medium is added 10mM CaCl2Pressing down of lactobacillus rhamnosus can be strengthened
Bacterium effect.
Embodiment 4:CaCl2Promote purification and the purity testing of lactobacillus rhamnosus secretion EPS
1) removing protein is removed
1, cultivate cell: cultivate lactobacillus rhamnosus according to the mode of example 1, be statically placed in 37 DEG C of constant incubators standing training
Support 12h.
2, cell is removed: by bacterium solution supersound process 20min, amplitude is 20%.4 DEG C, 3000g removes thin after being centrifuged 10min
Born of the same parents' precipitation stays supernatant.
3, removing removing protein: add final concentration 4% trichloroacetic acid (v/w), 4 DEG C, 3000g is centrifuged in 4 DEG C of refrigerators overnight
Remove albumen precipitation after 10min and take supernatant.
2) precipitate polysaccharides
Add 95% ice ethanol, 4 DEG C refrigerators in overnight, 4 DEG C at 1: 3 by volume, and 8000g removes supernatant after being centrifuged 10min
Stay precipitation.Take out after pellet frozen is dried 8h, 4 DEG C of dialysis 3d, after lyophilization, obtain thick extracellular polysaccharide (c-EPS).
3) DEAE-Sepharose anion-exchange column purifies EPS
Learnt from else's experience dialysis lyophilizing after crude polysaccharides sample, be dissolved in respectively in distilled water, preparation 20mg/mL solution, 8000g
Centrifugal 10min, takes supernatant and carries out DEAE-Sepharose Fast Flow (GE company) ion column chromatography, and tomographic system uses
Amersham Bioscience'sprime.First with ultra-pure water eluting, then the NaCl gradient elution with 0～1mol/L,
Flow velocity 1.0mL/min, takes a pipe every 5min, and eluent detects every 1 effective Phenol sulfuric acid procedure.Collect each eluting peak portion respectively
Point, dialyse 3d, lyophilization.Draw elution curve.
4) purity testing of EPS
Carry out the detection of purity with high performance liquid chromatograph (Agilent company), after taking above-mentioned purification, sample 3mg is dissolved in
1mL distilled water, 10000g is centrifuged 10min, automatic loading 20 μ L, and record elution curve is for judging the purity of this polysaccharide component.
The concrete test condition of the HPLC used by experiment is as follows:
Instrument: Agilent 1200 high performance liquid chromatograph (joins differential refraction detector and Chemical work station)；
Chromatographic condition: chromatographic column is TOSOHTMG4000PW XL 300mm×7.8mm；
Flowing phase: ultra-pure water；
Flow velocity: 0.6ml/min；
Column temperature: 50 DEG C；
Prepared by sample: sample is dissolved in flowing mutually, with the laggard sample of microporous filter membrane filtration.
As it is shown on figure 3, add 10mM CaCl2After, the crude polysaccharides that lactobacillus rhamnosus produces is separated rear containing 2 EPS
Component.Extract main EPS-2 component, carry out the analysis of purity.As shown in Figure 4, through gpc analysis, appearance time is
9.231min, chromatogram is the peak of single symmetry, it is known that EPS-2 component is pure component.
Embodiment 5:NMR means analysis EPS-2 structure
NMR analytical procedure: 25mg polysaccharide EPS-2 is dissolved in 0.7mL D2In O, by 600MHz Bruker nuclear magnetic resonance analyser
It is analyzed.Make internal standard with four deuterated trimethylsilane sodium propionate (T.S.P), test temperature 25 DEG C.1D NMR measures:13C NMR
With1H NMR.2D NMR measures: H-H COSY, TOCSY, HSQC, HMBC and NOESY.
Such as Fig. 5～12, comprehensive above spectrum analysis, EPS-2 saccharide residue1H and13The chemical shift ownership result of C NMR
It is shown in Table 2.IC analysis monosaccharide composition according to EPS-2, determines and wherein contains mannose and glucose.It is hydrolyzed into according to EPS-2 again
NMR spectra after monosaccharide determining, (NMR spectra does not provide in paper) E, F and F' points out and point out into mannose, B, B' and D
For glucose.According to the chemical shift of anomer signal, above-mentioned (A, F, F') and (B, B', D) residue pointed out respectively into β-
Glcp and β-Manp.Cause3JH1, H2(6.8HZ) chemical shift with anomeric proton is less than 5.0ppm, and this shows that E remaining is that α is different
Head.
This NOESY and HMBC collection of illustrative plates provides the information about glycosidic bond.5.16/4.24 (A-H1/B-is composed according to NOESY
H2), 5.07/4.13 (B'-H1/D-H2), 4.91/3.79 (E-H1/A-H4) and 5.31/4.03 (D-H1/F-H4), and HMBC
The coherent signal 5.31/81.58 (D-H1/F-C 4) of spectrogram, 5.06/81.32 (B-H1/B'-C 2), 4.91/75.77 (E-H1/
A-4), and the correlation peak of F-H1/F'-H4 and F'-H1/E-H4 that cannot definitely point out due to serious spectra overlapping, and
Monosaccharide result according to polysaccharide hydrolysis can get table 2, belongs to hydrocarbon atom, and the structure obtaining a kind of novel holosaccharide is:
[-4)-α-Manp-(1→4)-β-Manp-(1→2)-β-Manp-(1→2)-β-Glcp-(1→4)β-Manp-(1
→4)β-Manp-(1→]
Table 2 EPS-2 saccharide residue1H and13Chemical shift (the D of C NMR2O,25℃)
The present invention uses using lactobacillus rhamnosus as experimental strain, by cultivating at the MRS that normal glucose is carbon source
Base adds 10mM CaCl2, to increase the viable count of lactobacillus rhamnosus in fermentation liquid, detection matched group and the product of normal group
Acid ability compares, and with fermentation liquid, harmful bacteria is carried out Inhibition test simultaneously.By checking, MRS culture medium is added 10mM
CaCl2, fermentation 12h can promote the viable count 140% of lactobacillus rhamnosus, for escherichia coli, Bacillus cercus, golden yellow
The inhibition zone of color glucose coccus respectively from 0.85 ± 0.02,1.11 ± 0.06,1.29 ± 0.03cm increase to 1.00 ± 0.02,
1.29±0.03、1.41±0.08cm.At 10mM CaCl2Coerce down, from culture medium, extract a kind of novel polysaccharide, through NMR
Analyzing, structure is:
[-4)-α-Manp-(1→4)-β-Manp-(1→2)-β-Manp-(1→2)-β-Glcp-(1→4)β-Manp-(1
→4)β-Manp-(1→]
A kind of method that the invention discloses simple low cost increases viable count of lactobacillus and the yield of lactic acid, the use of lactic acid
Way constantly extension, but application is still limited by yield and cost.The carbon source of culture medium, nitrogen source, cultivate pH, bacterial strain, trace element
Content all influences whether the yield of lactic acid.The present invention uses a kind of simple, method of low cost, compared with traditional zymotic, sends out
Ferment 12h maximum can promote viable count of lactobacillus 140%, the suppression circle of pathogenic bacterium is also had significantly increase, produces simultaneously
Acid capability improving, and from adding 10mM CaCl2Culture medium in, isolated and purified go out many outside a kind of novel lactobacillus rhamnosus born of the same parents
Sugar.
Claims (10)
1. the method for purification of a lactobacillus rhamnosus extracellular polysaccharide, it is characterised in that comprise the following steps:
1) from the MRS solid medium of conservation to be, choose single bacterium colony connected in MRS fluid medium cultivate, obtain the fermentation activated
Liquid；
2) in MRS fluid medium, add the CaCl of final concentration of 10mM2, take step 1) in the fermentation liquid that activated carry out turning
Connect, obtain the fermentation liquid after switching, take the fermentation liquid after the 1ml switching of cultivation 0,2,4,6,12,24h and measure OD600, pH value；
3) take step 2) in fermentation liquid after switching, after removing cell and albumen, add ice ethanol, refrigerator at 1: 3 by volume
In overnight, be then centrifuged for, remove supernatant and stay precipitation, pellet frozen is taken out after drying, dialysis, then after lyophilization, obtain slightly
Extracellular polysaccharide；
4) by step 3) the thick extracellular polysaccharide of gained is dissolved in pure water, the thick extracellular polysaccharide sample after dialysis lyophilizing of learning from else's experience,
It is dissolved in distilled water, the solution of preparation 20mg/mL, centrifugal, take supernatant ion column chromatography, obtain outside lactobacillus rhamnosus born of the same parents many
Sugar.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 1) in,
Consisting of of described MRS solid medium: glucose 20g/L, Carnis Bovis seu Bubali cream 10g/L,Peptone 10g/L, yeast powder 5g/L, lemon
Lemon acid ammonium 1g/L, sodium acetate 5g/L, KH2PO4 2g/L,MgSO4·7H2O 0.05g/L,MnSO4·H2O 0.05g/L, agar powder
15g/L, pH 6～6.5.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 1) in,
Consisting of of described MRS fluid medium: glucose 20g/L, Carnis Bovis seu Bubali cream 10g/L,Peptone 10g/L, yeast powder 5g/L, lemon
Lemon acid ammonium 1g/L, sodium acetate 5g/L, KH2PO4 2g/L,MgSO4·7H2O 0.05g/L,MnSO4·H2O 0.05g/L, pH 6～
6.5, sterilising conditions is 118 DEG C, 15min.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 1) in,
The condition of described cultivation is constant temperature quiescent culture 12h at 37 DEG C.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 2) in,
Described switching by volume percentage ratio 4% is transferred.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 3) in,
The ice ethanol that described ice ethanol uses mass percentage concentration to be 95%.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 3) in,
The temperature of described refrigerator is 4 DEG C.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 3) in,
Described centrifugal condition is that 8000g is centrifuged 10min at 4 DEG C；The described cryodesiccated time can be 8h；The condition of described dialysis
Can be dialysed at 4 DEG C 3d.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 4) in,
Described centrifugal condition is that 8000g is centrifuged 10min.
The method of purification of a kind of lactobacillus rhamnosus extracellular polysaccharide the most as claimed in claim 1, it is characterised in that in step 4)
In, described ion column chromatography uses the DEAE-Sepharose Fast Flow ion column chromatography of GE company.
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