CN106243238A - A kind of method extracting probiotic cell wall skeleton polysaccharide - Google Patents
A kind of method extracting probiotic cell wall skeleton polysaccharide Download PDFInfo
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Abstract
The present invention relates to a kind of method extracting probiotic cell wall skeleton polysaccharide.Take probiotic bacteria, utilize trichloroacetic acid (TCA) to remove the teichoic acid in probiotic bacteria thalline, obtain precipitation；Gained precipitation continues with chloroform methanol mixed solution and removes lipid component in probiotic bacteria thalline, obtains precipitation；Gained precipitation continues with trypsin neutral protease mixed enzyme solution and removes the protein in probiotic bacteria thalline.The present invention ensureing that purity is high, integrity good while, save time, low cost, easily operate.
Description
Technical field
The invention belongs to field of biological extraction, more particularly to a kind of method extracting probiotic cell wall skeleton polysaccharide,
It is applicable to the multiple breast bar such as lactobacillus rhamnosus, Lactobacillus reuteri, bacillus acidophilus, animal bifidobacteria, Bifidobacterium lactis
Bacterium and bacillus bifidus.
Background technology
Lactobacillus is a section of antibacterial, and for Gram-positive, sporeless bacterium, the major end products decomposing sugar is breast
Acid, azymic lactate.Lactobacillus has multiple probiotic effects, and its intestinal Stickiness is high, and colonization ability is strong, and has efficiently fall
Cholesterol, promotes cell division, can play regulating intestinal canal flora, prevents and treat diarrhoea, get rid of toxin, pre-anti-caries, raising
Immunity of organisms and the important physiological hygiene function such as anticancer, be a big class important in probiotic bacteria.
Bacillus bifidus be 1899 by France scholar Tissier isolated a kind of anaerobism from the feces of breast milk nutrition youngster
Gram-positive bacillus, end usually bifurcated, therefore named bacillus bifidus.Bacillus bifidus is deposited in breast-feeding infant intestinal in a large number
, infant there are many benefits, such as nutrition, immunity and anti-infectious function.Meanwhile, bacillus bifidus also has antiallergic, resists and swell
Tumor, adjust function of intestinal canal and improve the effect such as nutrition.Function of intestinal canal aspect, bacillus bifidus can pre-anti-diarrhea, reduce constipation, i.e.
Two-ways regulation, this regulation can be played prevention and treat the effect of various intestinal tract disease.
Currently, with respect to the activity in terms of probiotic bacteria immunomodulating, mainly it is considered to be caused by the cell wall structure of bacterial strain.
The macromole of cell wall is the key factor of probiotic bacteria and the interphase interaction of host.Probiotic cell wall framing structure and
The main function material of physicochemical property is Peptidoglycan, the most mucopeptide, and it is connected into network structure by peptide bridging, forms cryptomere big
Molecule, surrounds whole antibacterial, has the multiple prebiotic effects such as immune enhancing function, infection, antitumor, antiallergic.Grind at present
Studying carefully confirmation, cell wall skeleton polysaccharide has mediated the multiple important physiological function of bacillus bifidus, such as antitumor and immunocompetence etc., is
The main target molecule of body immune system identification antibacterial
1985, Sekine etc. established the separation method of cell wall integrated peptidoglycan, and hereafter, the method is studied at this type of
The unique separation method of middle conduct is widely used, and whole process needs about 15 days.The cell wall skeleton obtained by the method
Purity of polysaccharide is high, integrity is good, but with duration, cost is high, operation is complicated.If the extraction of a kind of cell wall skeleton polysaccharide can be found
Method, ensureing that purity is high, integrity good while, save time again, low cost, easily operate, it will help from now on to entering in this respect
Row deeper into research, it is also possible to advance cell wall skeleton polysaccharide the aspect such as food, medicine actual application on development.
Summary of the invention
For the problems referred to above, it is an object of the invention to provide a kind of while ensureing that purity height, integrity are good, save
Time, low cost, the method for easy-operating extraction probiotic cell wall skeleton polysaccharide.
The technical solution used in the present invention is: a kind of method extracting probiotic cell wall skeleton polysaccharide, takes probiotic bacteria, profit
Remove the teichoic acid in probiotic bacteria thalline with trichloroacetic acid (TCA), obtain precipitation；Gained precipitation continues with chloroform-methanol mixing
Solution removes lipid component in probiotic bacteria thalline, obtains precipitation；Gained precipitation continues with trypsin-neutral protease mixed enzyme
Liquid removes the protein in probiotic bacteria thalline.
The above-mentioned method extracting probiotic cell wall skeleton polysaccharide, probiotic bacteria thalline removed by the described trichloroacetic acid that utilizes
In teichoic acid, method is as follows: joined by solution of trichloroacetic acid in probiotic bacteria thalline, and mix homogeneously, in the water-bath of 65-75 DEG C
Middle process 1-1.5h, centrifugal, take precipitation, washing.
Preferably, every gram of probiotic bacteria thalline adds the solution of trichloroacetic acid 5-15ml that concentration is 5-15%.
The above-mentioned method extracting probiotic cell wall skeleton polysaccharide, the described chloroform-methanol mixed solution that utilizes is removed
Lipid component in probiotic bacteria thalline, method is as follows: in the ratio of solid-to-liquid ratio 1:15-25, and trichloroacetic acid of learning from else's experience is remaining after processing
Precipitation and chloroform-methanol mixed solution, mixing, stand, be centrifuged and discard supernatant, take precipitation.
Preferably, the preparation method of described chloroform-methanol mixed solution is as follows: 1:1.5-2.5 by volume, takes chloroform
And methanol, adding acetic acid-sodium acetate buffer solution that appropriate pH is 4.6, mix homogeneously, gained solution is chloroform-methanol and mixes
Close solution.
The above-mentioned method extracting probiotic cell wall skeleton polysaccharide, the described trypsin-neutral protease that utilizes mixes
Synthase liquid removes the protein in probiotic bacteria thalline, and method is as follows: in the ratio of solid-to-liquid ratio 1:4-6, and chloroform-methanol of learning from else's experience mixes
Remaining precipitation and trypsin-neutral protease mixed enzyme solution after solution process, mixing, it is subsequently placed in isothermal vibration incubator
In, shaken cultivation 35-40h at a temperature of 38 DEG C, after taking-up, it is centrifuged 15-20min in 12000r/min, discards supernatant, precipitation
Being divided into two-layer, retain upper strata precipitation, be centrifuged repeatedly cleaning by proper amount of methanol, until supernatant achromaticity and clarification is transparent, it is heavy to take
Shallow lake is probiotic cell wall skeleton polysaccharide.
Preferably, the preparation method of described trypsin-neutral protease mixed enzyme solution: by weight 1:4-5, take pancreas
Protease and neutral protease, add the phosphate buffered solution that appropriate pH is 7.4, it is ensured that every kind of enzyme activity is not less than 250U/
Ml, stirs, and gained solution is required trypsin-neutral protease mixed enzyme solution.
The above-mentioned method extracting probiotic cell wall skeleton polysaccharide, described probiotic bacteria is lactobacillus and bacillus bifidus.
Preferably, described lactobacillus is lactobacillus rhamnosus, Lactobacillus reuteri or bacillus acidophilus；Described bifid
Bacillus is animal bifidobacteria or Bifidobacterium lactis.
The invention has the beneficial effects as follows:
1) present invention, utilizes TCA to remove teichoic acid in lactobacillus or bacillus bifidus thalline, and lactobacillus and bacillus bifidus are all
Gram positive bacteria, and teichoic acid is the special component of gram-positive bacteria cell wall, it be by ribitol or glycerol residue via
The polymer that di(2-ethylhexyl)phosphate key is interconnected together.Teichoic acid is permeability-related with cell wall, therefore determines first to remove dephosphorization wall
Acid, makes cell permeability increase, and is so conducive to external substance preferably to act on cell, and the containing substance of cell can also be more
Easily from intracellular discharge.
2) present invention, utilizes chloroform-methanol mixed solution to remove lipid in lactobacillus or bacillus bifidus thalline, and conventional carries
Take the soxhlet extraction methods of oils and fats, the fat of free state can only be extracted, then can not be by the lipid of the combined state such as lipoprotein, phospholipid
Extracting completely, another kind of conventional acid-hydrolysis method can make again phospholipid hydrolysis lose, and the condition existed at certain moisture
Under, the methanol of polarity and nonpolar chloroform mixed solution, combined state lipid can be efficiently extracted.Lactobacillus cell is contained within
A large amount of combined state lipids such as phospholipid, lipoprotein, therefore select chloroform-methanol mixed solution to remove lactobacillus or bacillus bifidus thalline
In lipid.
3) present invention, utilizes trypsin-neutral protease mixed enzyme solution to remove egg in lactobacillus or bacillus bifidus thalline
White matter, trypsin is a kind of proteolytic enzyme, can selectively in aminosal by lysine or arginic carboxyl institute
The peptide chain constituted, can digest dissolving denatured protein, and neutral protease belongs to a kind of restriction endonuclease, can be used for various proteolysis
Process.
4) the inventive method is simple, it is easy to operation, and the cell wall skeleton purity of polysaccharide of preparation is high, integrity is good.5 days
Obtain product, substantially reduce preparation time.Raw material is easy to get, and significantly reduces cost.Averagely carrying of cell wall skeleton polysaccharide
The rate of taking is 0.183g/L bacterium solution.
Accompanying drawing explanation
Fig. 1 is occluded corrosion cell standard curve.
Fig. 2 is vanillin method standard curve.
Detailed description of the invention
Embodiment 1 one kinds extracts the method for probiotic cell wall skeleton polysaccharide
(1) preparation of thalline:
MRS culture medium prescription: peptone 10g, beef powder 10g, yeast leaching powder 5g, K2HPO42g, anhydrous sodium acetate 5g, Portugal
Grape sugar 20g, MnSO4·7H2O 0.58g, MnSO40.25g, Fructus Citri Limoniae diammonium hydrogen 2g, distilled water 1000ml.
Preparation method: 1) prepare 2800ml MRS culture medium by above-mentioned formula proportion, it is placed in high-pressure sterilizing pot, 115
Sterilizing 20min at a temperature of DEG C, standby；
2) lactobacillus rhamnosus of picking Laboratories Accession is in 50ml MRS culture medium, is subsequently placed in isothermal vibration and cultivates
In case, shaken cultivation 12h under the conditions of 37 DEG C；
3) then by this bacterium solution, by the inoculum concentration of 2%, it is inoculated in 2750ml MRS culture medium, is subsequently placed in constant temperature shake
Swing in incubator, shaken cultivation 12h under the conditions of 37 DEG C；
4) cultured lactobacillus rhamnosus bacterium solution is centrifuged 20min with the rotating speed of 8000r/min, discards supernatant, will
Precipitation physiological saline solution is centrifuged repeatedly cleaning with same rotating speed and centrifugation time, until supernatant is colourless, for the last time
Centrifugal gained precipitation is lactobacillus rhamnosus thalline；
5) through weighing, gained lactobacillus rhamnosus thalline weight in wet base is 23.3g.
(2) extraction of probiotic cell wall skeleton polysaccharide
1, TCA is utilized to remove teichoic acid in lactobacillus rhamnosus thalline
The preparation of 1.1TCA solution
Accurately weighing 25g TCA in 250ml volumetric flask, add water and be settled to graduation mark, shake up, gained solution is concentration
It is the TCA solution of 10%.
The removal of teichoic acid in 1.2 lactobacillus rhamnosus thalline
Accurately measure the TCA solution that 233ml concentration is 10%, join in 23.3g lactobacillus rhamnosus thalline, use glue head
Dropper is blown and beaten repeatedly until mixing, then processes 1h under the water bath condition of 70 DEG C.After water-bath terminates, with High speed refrigerated centrifuge with
The rotating speed of 8000r/min is centrifuged 20min, discards supernatant, the most again with same rotating speed and centrifugation time, by precipitation with appropriate
Methanol eccentric cleaning 3 times.
1.3 through weighing, and last centrifugal gained precipitation quality is 7.2g.
2, chloroform-methanol mixed solution is utilized to remove in lactobacillus rhamnosus thalline the compositions such as lipid
The preparation of 2.1 acetic acid-sodium acetate buffer solution
Accurately measuring 0.3ml glacial acetic acid in 100ml volumetric flask, add water and be settled to graduation mark, shake up, gained solution is
Concentration is the acetic acid solution of 0.05M；Accurately weigh 0.164g sodium acetate in 100ml volumetric flask, add water and be settled to graduation mark, shake
Even, gained solution is the sodium acetate solution that concentration is 0.02M.Measure acetic acid solution and concentration that 15m concentration is 0.05M respectively
For the sodium acetate solution of 0.02M, stir with Glass rod, pH value determination, and the pH of mixed solution is regulated with both solution
To 4.6, standby.Gained solution is required acetic acid-sodium acetate buffer solution.
The preparation of 2.2 chloroform-methanol mixed solutions
Accurately measure acetic acid-sodium acetate buffer solution, 37.9ml chloroform and 75.8ml methanol that 30.3ml prepares, use glass
The stirring of glass rod makes its three's mix homogeneously, and gained solution is required chloroform-methanol mixed solution.
The removal of the composition such as lipid in 2.3 lactobacillus rhamnosus thalline
Accurately measure the chloroform-methanol mixed solution that 144ml prepares, in the ratio of solid-to-liquid ratio 1:20 (w/v), join
In 7.2g precipitation remaining after TCA processes, repeatedly blow and beat until mixing with glue head dropper, stand 24 hours, then with at a high speed
Refrigerated centrifuger is centrifuged 20min with the rotating speed of 8000r/min, discards supernatant, then precipitation proper amount of methanol is turned with identical
Speed and centrifugation time eccentric cleaning twice.
2.4 through weighing, and last centrifugal gained precipitation quality is 6.917g.
3, trypsin-neutral protease mixed enzyme solution is utilized to remove protein in lactobacillus rhamnosus thalline
The preparation of 3.1 phosphate buffered solution
Accurately weigh 1.36g KH2PO4In 100ml volumetric flask, adding water and be settled to graduation mark, shake up, gained solution is
Concentration is the KH of 0.1M2PO4Solution；Accurately weigh 0.4g NaOH in 100ml volumetric flask, add water and be settled to graduation mark, shake up,
Gained solution is the NaOH solution that concentration is 0.1M.Measure the KH that 20ml concentration is 0.1M respectively2PO4Solution and concentration are
The NaOH solution of 0.1M, stirs with Glass rod, pH value determination, and regulates the pH to 7.4 of mixed solution with both solution,
Standby.Gained solution is required phosphate buffered solution.
The preparation of 3.2 trypsin-neutral protease mixed enzyme solution
Accurately weigh 34.6mg trypsin and 144mg neutral protease, join in 34.6ml phosphate buffered solution,
Every kind of enzyme activity is all not less than 250U/ml.Then stirring with Glass rod, gained solution is required trypsin-neutrality
Protease mixed enzyme solution.
The removal of protein in 3.3 lactobacillus thalline
Accurately measure trypsin-neutral protease mixed enzyme solution that 34.6ml prepares, by solid-to-liquid ratio 1:5 (w/v)
Ratio, joins after chloroform-methanol mixed solution processes in remaining 6.917g precipitation, repeatedly blow and beat with glue head dropper until
Mixing, is subsequently placed in isothermal vibration incubator, shaken cultivation 36h at a temperature of 38 DEG C.After taking-up, use High speed refrigerated centrifuge
Being centrifuged 15min with the rotating speed of 12000r/min, discard supernatant, precipitation now be can be observed and be divided into two-layer, upper strata is milky
Cell wall skeleton polysaccharide, lower floor is the undissolved neutral protease of light brown, discards the neutral protease of lower floor, in reservation
The cell wall skeleton saccharide portion of layer, by proper amount of methanol, still rotating speed and centrifugation time with 12000r/min, 15min is centrifuged repeatedly
Clean, until supernatant achromaticity and clarification is transparent.Gained precipitation vacuum freeze drier lyophilizing will be centrifuged for the last time, i.e.
The lactobacillus rhamnosus cell wall skeleton polysaccharide powder being dried can be obtained.
3.4 through weighing, and after lyophilizing, gained lactobacillus rhamnosus cell wall skeleton polysaccharide quality is 0.478g, and productivity is
0.177g/L bacterium solution.
(3) probiotic cell wall skeleton purity of polysaccharide detection
1, the preparation of lysozyme soln
1.1 the preparation of phosphate buffered solution
Accurately weigh 1.36g KH2PO4In 100ml volumetric flask, adding water and be settled to graduation mark, shake up, gained solution is
Concentration is the KH of 0.1M2PO4Solution；Accurately weigh 0.4g NaOH in 100ml volumetric flask, add water and be settled to graduation mark, shake up,
Gained solution is the NaOH solution that concentration is 0.1M.Measure the KH that 20ml concentration is 0.1M respectively2PO4Solution and concentration are
The NaOH solution of 0.1M, stirs with Glass rod, pH value determination, and regulates the pH to 6.2 of mixed solution with both solution,
Standby.Gained solution is required phosphate buffered solution.
The preparation of 1.2 lysozyme solns
Accurately weigh 20mg lysozyme in 100ml volumetric flask, add the above-mentioned phosphate buffered solution prepared to carving
Degree line, mixing, gained solution is the lysozyme soln that required concentration is 200 μ g/ml.
2, sample purity detection
Accurately weighing appropriate amount of sample in lysozyme soln, making sample concentration is 1mg/ml, be subsequently placed into temperature be 37 DEG C,
In rotating speed is the isothermal vibration incubator of 120r/min, take out after 12h, with High speed refrigerated centrifuge, with the rotating speed of 8000r/min
Centrifugal 20min, abandons supernatant, and precipitation distilled water cleans repeatedly, then dries, weighs.It addition, accurately measure isopyknic, no
Add the lysozyme soln of sample, make process same as described above, as blank.It is calculated as follows sample purity:
Computing formula:
A=[1-(m1-m0)/m] × 100%=[1-(4-0)/20] × 100%=80%
In formula: A-sample purity
m1-sample final insoluble matter quality (mg)
m0-blank group final insoluble matter quality (mg)
M-initial sample mass (mg)
The product obtained is solvable in lysozyme soln, and what acquisition was described is exactly that lactobacillus rhamnosus cell wall skeleton is many
Sugar.
Embodiment 2 one kinds extracts the method for probiotic cell wall skeleton polysaccharide
(1) preparation of thalline:
MRS culture medium prescription: peptone 10g, beef powder 10g, yeast leaching powder 5g, K2HPO42g, anhydrous sodium acetate 5g, Portugal
Grape sugar 20g, MnSO4·7H2O 0.58g, MnSO40.25g, Fructus Citri Limoniae diammonium hydrogen 2g, distilled water 1L.
Preparation method: 1) prepare MRS culture medium by above-mentioned formula proportion, it is placed in high-pressure sterilizing pot, the temperature of 115 DEG C
Lower sterilizing 20min, standby；
2) lactobacillus rhamnosus of picking Laboratories Accession is in 10ml MRS culture medium, is subsequently placed in isothermal vibration and cultivates
In case, shaken cultivation 12h under the conditions of 37 DEG C；
3) by this bacterium solution, by the inoculum concentration of 2%, it is inoculated in 100ml MRS culture medium, is subsequently placed in isothermal vibration and cultivates
In case, shaken cultivation 12h under the conditions of 37 DEG C；
4) again by this bacterium solution, by the inoculum concentration of 2%, it is inoculated in 3L MRS culture medium, is subsequently placed in isothermal vibration and cultivates
In case, shaken cultivation 12h under the conditions of 37 DEG C；
5) cultured lactobacillus rhamnosus bacterium solution is centrifuged 20min with the rotating speed of 8000r/min, discards supernatant, will
Precipitation physiological saline solution is centrifuged repeatedly cleaning with same rotating speed and centrifugation time, until supernatant is colourless, for the last time
Centrifugal gained precipitation is lactobacillus rhamnosus thalline.
(2) selection of teichoic acid method is removed
Above-mentioned centrifugal gained thalline is equally divided into 4 parts by weight, processes the most using the following method.Finally, whole sample is used
In phosphorus content determine the removal degree of teichoic acid.
1, cold TCA method
The preparation of 1.1TCA solution
Accurately weighing 25g TCA in 250ml volumetric flask, add water and be settled to graduation mark, shake up, gained solution is concentration
The TCA solution of 10%.
1.2 sample treatment
Take a above-mentioned sample, by solid-to-liquid ratio 1:10 add be cooled in advance 4 DEG C, concentration be 10% TCA solution, 4 DEG C of stirrings
Process 24h, be then centrifuged 20 minutes with the rotating speed of 8000r/min, discard supernatant.Remaining precipitation sterilized water is with same
Rotating speed and centrifugation time eccentric cleaning 5 times, then clean with ethanol, acetone, ether continuous centrifugal successively, then phosphorus in detection sample
Content.
2, hot TCA method
The preparation of 2.1TCA solution
Accurately weighing 25g TCA in 250ml volumetric flask, add water and be settled to graduation mark, shake up, gained solution is concentration
It is the TCA solution of 10%.
2.2 sample treatment
Take a above-mentioned sample, by solid-to-liquid ratio 1:10 add be preheated to 70 DEG C, concentration be 10% TCA solution, 70 DEG C of water
Bath stir process 1h, is then centrifuged 20 minutes with the rotating speed of 8000r/min, discards supernatant.Remaining precipitation sterilized water with
Same rotating speed and centrifugation time eccentric cleaning 5 times, then clean with ethanol, acetone, ether continuous centrifugal successively, then detect sample
Phosphorus content in product.
3, cold phenol method
The preparation of 3.1 phenol solution
Accurately weighing 80g phenol in 100ml volumetric flask, add water and be settled to graduation mark, shake up, gained solution is concentration
It it is the phenol solution of 80%.
3.2 sample treatment
Take a above-mentioned sample, add equal-volume be cooled in advance 4 DEG C, concentration be the phenol of 80%, 4 DEG C of stir process 24h, so
After be centrifuged 20 minutes with the rotating speed of 8000r/min.Supernatant is layered, removal upper strata aqueous phase, phenol phase and insoluble sludge addition etc.
Volumetric concentration is the sodium carbonate liquor of 0.02mol/L.Remaining precipitation sterilized water is centrifuged with same rotating speed and centrifugation time
Clean 5 times, then clean with ethanol, acetone, ether continuous centrifugal successively, then phosphorus content in detection sample.
4, hot phenol method
The preparation of 4.1 phenol solution
Accurately weighing 80g phenol in 100ml volumetric flask, add water and be settled to graduation mark, shake up, gained solution is concentration
It it is the phenol solution of 80%.
4.2 sample treatment
Take a above-mentioned sample, add equal-volume be preheated to 65 DEG C, concentration be the phenol of 80%, 65 DEG C of stir process 1h,
Then it is centrifuged 20 minutes with the rotating speed of 8000r/min.Supernatant is layered, removal upper strata aqueous phase, and phenol phase and insoluble sludge add
Equal-volume concentration is the sodium carbonate liquor of 0.02mol/L.Remaining precipitation sterilized water with same rotating speed and centrifugation time from
The heart cleans 5 times, then cleans with ethanol, acetone, ether continuous centrifugal successively, then phosphorus content in detection sample.
5, the mensuration of phosphorus content
Using molybdenum blue colorimetric method that above-mentioned 4 groups of thalline processed carry out the mensuration of phosphorus content respectively, concrete grammar is as follows:
The preparation of 5.1 various solution
Accurately weighing 0.143g KH2PO4 in 1L volumetric flask, add water and be settled to graduation mark, shake up, obtaining concentration is 100
The phosphate radical standard solution of μ g/ml；Accurately measure 30ml nitric acid and 70ml water, mixing, obtain the salpeter solution that concentration is 30%；
Accurately weigh 0.5g ammonium metavanadate in 100ml volumetric flask, add water and be settled to graduation mark, shake up, obtain concentration be 0.5% inclined
Ammonium Vanadate Solution；Accurately weighing 10g ammonium molybdate in 100ml volumetric flask, add water and be settled to graduation mark, shake up, obtaining concentration is
The ammonium molybdate solution of 10%；Accurately weigh 1.5g ascorbic acid in 100ml volumetric flask, add water and be settled to graduation mark, shake up, i.e.
Obtaining concentration is the ascorbic acid solution of 1.5%.
The drafting of 5.2 standard curves
In 50ml color comparison tube, be separately added into 0,0.5,1.0,1.5,2.0,2.5, the phosphate radical standard solution of 3.0ml, so
After be sequentially added into salpeter solution 10ml, ammonium metavanadate solution 5ml, ammonium molybdate solution 5ml, ascorbic acid solution 5ml, add water constant volume
To 50ml, shake up, after horizontal positioned 10min, with ultraviolet spectrophotometer, under conditions of wavelength 660nm, measure each pipe extinction
Value, and draw phosphate radical standard curve according to this value.
5.3 sample pretreatment
Sample is put in evaporating dish, after little fire slight fever is to remove moisture, then this sample is placed in muffle furnace, 600 DEG C
Ashing processes 12h, is finally settled in the volumetric flask of 10ml by ash.
The mensuration of phosphorus content in 5.4 samples
Take the above-mentioned sample liquid prepared of 1ml, join in 50ml color comparison tube, be then sequentially added into salpeter solution 10ml, partially
Ammonium Vanadate Solution 5ml, ammonium molybdate solution 5ml, ascorbic acid solution 5ml, add water and be settled to 50ml, shake up, horizontal positioned 10min
After, with ultraviolet spectrophotometer, under conditions of wavelength 660nm, measure light absorption value, finally according to standard curve, calculate sample
Middle phosphorus content.After testing, result such as table 1:
Table 1
| Processing method | Cold TCA method | Hot TCA method | Cold phenol method | Hot phenol method |
| Phosphorus content (μ g) | 92.32 | 82.73 | 150 | 154.7 |
As shown in Table 1, the sample after hot TCA method processes, phosphorus content is minimum, therefore selects hot TCA method as going dephosphorization
The method of Teichaic acid.
(3) selection of degreasing method
1, the process of sample
Respectively take 5g lactobacillus rhamnosus bacterium mud, through above-mentioned hot TCA method process after, respectively by solid-to-liquid ratio 1:20 add chloroform-
Methanol mixed solution, ether, hexane and acetone, stand 24h, be then centrifuged 20min with the rotating speed of 8000r/min, abandon supernatant, then
With identical rotating speed methanol eccentric cleaning 2 times.After precipitation volatile organic solvent, dry.
2, fat content in vanillin method test sample product
The preparation of 2.1 each solution
Accurately weighing 1g cholesterol in 100ml volumetric flask, ice acetic acid is settled to graduation mark, shakes up, gained i.e. concentration
Cholesterol titer for 10mg/ml；Accurately weigh 15g vanillin, add 250ml ethanol and 2.5ml concentrated sulphuric acid, mixing, institute
Vanillin-sulfuric acid developer must be.
The drafting of 2.2 standard curves
The according to the form below each solution of addition:
| Numbering | 1 | 2 | 3 | 4 |
| Cholesterol titer (ml) | 0 | 0.1 | 0.2 | 0.3 |
| Dehydrated alcohol (ml) | 1 | 0.9 | 0.8 | 0.7 |
| Cholesterol concentration (mg/ml) | 0 | 1 | 2 | 3 |
After above-mentioned cholesterol titer and ethanol being shaken up, take 0.02ml respectively, add 1ml concentrated sulphuric acid, mix, then
100 DEG C of water-bath 10min, are cooled to after room temperature add 4ml developer, left at room temperature 20min, with ultraviolet spectrophotometer, in
Survey absorbance at 525nm, and draw standard curve.
Determination of fat in 2.3 samples
Accurately weigh 100mg sample, dissolve with 1ml glacial acetic acid, take 0.02ml, add 1ml concentrated sulphuric acid, mixing, then 100
DEG C water-bath 10min, adds 4ml developer after being cooled to room temperature, and room temperature stands 20min, with ultraviolet spectrophotometer, in 525nm
Place surveys absorbance, and according to standard curve, calculates fat content.Result such as table 2:
Table 2
| Degreasing agent | Chloroform-methanol mixed solution | Ether | Hexane | Acetone |
| Sample fat content (mg/g) | 34.1 | 46.3 | 54.8 | 44.1 |
As shown in Table 2, through chloroform-methanol mixed solution process after sample in fat content minimum, therefore select chloroform-
Methanol solution carrys out defat.
(4) selection of method of removing protein
1, the preparation of 4 kinds of mixed enzyme solutions
1.1 the preparation of phosphate buffered solution
Accurately weigh 1.36g KH2PO4In 100ml volumetric flask, adding water and be settled to graduation mark, shake up, gained solution is
Concentration is the KH of 0.1M2PO4Solution；Accurately weigh 0.4g NaOH in 100ml volumetric flask, add water and be settled to graduation mark, shake up,
Gained solution is the NaOH solution that concentration is 0.1M.Measure the KH that 20m concentration is 0.1M respectively2PO4Solution and concentration are 0.1M
NaOH solution, stir with Glass rod, pH value determination, and with both solution regulation mixed solution pH to 7.4, standby
With.Gained solution is required phosphate buffered solution.
The preparation of 1.2 4 kinds of mixed enzyme solutions
Accurately weigh appropriate trypsin, trypsin and neutral protease, trypsin and alkaline protease, Trypsin
Enzyme and papain, be dissolved in appropriate phosphate buffered solution respectively, it is ensured that every kind of enzyme activity, all not less than 250U/ml, stirs
Mixing after uniformly, gained solution is i.e. respectively required trypsin solution, trypsin-neutral protease mixed enzyme solution, Trypsin
Enzyme-alkaline protease mixed enzyme solution, trypsin-papain mixed enzyme solution.
2, the process of sample
By lactobacillus rhamnosus bacterium mud, after above-mentioned hot TCA method and chloroform-methanol mixed solution process, it is equally divided into 4
Part, respectively by solid-to-liquid ratio 1:5 add 4 kinds of enzyme liquid, put in isothermal vibration incubator simultaneously, 38 DEG C of hydrolysis process 36h, then with
The rotating speed of 12000r/min is centrifuged 15min, retains supernatant.
3, free amino nitrogen content during formaldehyde potentiometric titration surveys supernatant
The preparation of 3.1 various solution
Accurately weigh 0.4g NaOH in 100ml volumetric flask, add water and be settled to graduation mark, shake up, gained solution i.e. concentration
NaOH standard solution for 0.1mol/L；In 60-80 DEG C of hot water, it is continuously added potassium oxalate, till saturated, uses
The salt acid for adjusting pH of 0.1mol/L is to 8.4, and gained is required saturated oxalic acid potassium solution；It is the formalin of 37% by concentration,
PH to 8.4 is regulated, the formalin that gained is the most required by the NaOH solution of 0.1mol/L.
The detection of 3.2 amino nitrogens
By sample supernatant distilled water diluting to 100ml, add 2ml saturated oxalic acid potassium solution, stir evenly, stand 2min,
Regulate pH to 8.4 with the NaOH standard solution of 0.1mol/L, be subsequently adding 10ml formalin, stir evenly, stand 2min, use
The NaOH standard solution of 0.1mol/L is titrated to pH 8.4, and record consumes NaOH solution volume V1ml.Repeat with 100ml distilled water
Above-mentioned steps, as blank, finally consuming NaOH solution volume is V0ml。
Computing formula: C=(V1-V0)×1.4/(VSample×1000)
In formula: C-free amine group nitrogen content (mg/L)
V1-sample supernatant consumes NaOH solution volume (ml)
V0-blank consumes NaOH solution volume (ml)
VSample-sample supernatant volume (ml)
Result is as shown in table 3.
Table 3
| Enzyme class | Supernatant amino nitrogen content (mg/L) |
| Trypsin | 508 |
| Trypsin-alkaline protease | 683 |
| Trypsin-neutral protease | 726 |
| Trypsin-papain | 635 |
As shown in Table 3, the free amine group nitrogen content after trypsin-neutral protease mixed solution hydrolysis is the highest, explanation
Its proteolysis degree is best, therefore selects trypsin-neutral protease mixed enzyme solution to make a return journey isolating protein.
Embodiment 3 one kinds extracts the method for probiotic cell wall skeleton polysaccharide
Lactobacillus rhamnosus, with embodiment 1, is simply replaced with Lactobacillus reuteri, bacillus acidophilus, moves by method respectively
Thing bacillus bifidus and Bifidobacterium lactis, respectively obtain dry four kind probiotic cell wall skeleton polysaccharide powder.Through weighing, lyophilizing
After, gained each thalline purity is as shown in table 4 with productivity.
Table 4
Claims (10)
1. the method extracting probiotic cell wall skeleton polysaccharide, it is characterised in that: take probiotic bacteria thalline, utilize trichloroacetic acid
Remove the teichoic acid in probiotic bacteria thalline, obtain precipitation；Gained precipitation continues with chloroform-methanol mixed solution and removes probiotics bacterial
Lipid component in body, obtains precipitation；Gained precipitation continues with trypsin-neutral protease mixed enzyme solution and removes probiotic bacteria thalline
In protein.
The method of extraction probiotic cell wall skeleton polysaccharide the most according to claim 1, it is characterised in that: described utilization
The teichoic acid in probiotic bacteria thalline removed by trichloroacetic acid, and method is as follows: joined by solution of trichloroacetic acid in probiotic bacteria thalline, mixed
Close uniformly, in the water-bath of 65-75 DEG C, process 1-1.5h, centrifugal, take precipitation, washing.
The method of extraction probiotic cell wall skeleton polysaccharide the most according to claim 2, it is characterised in that: every gram of probiotic bacteria
Thalline adds the solution of trichloroacetic acid 5-15ml that concentration is 5-15%.
The method of extraction probiotic cell wall skeleton polysaccharide the most according to claim 1, it is characterised in that: described utilization
Chloroform-methanol mixed solution removes lipid component in probiotic bacteria thalline, and method is as follows: in the ratio of solid-to-liquid ratio 1:15-25, learn from else's experience
Remaining precipitation and chloroform-methanol mixed solution after trichloroacetic acid process, mixing, stand, be centrifuged and discard supernatant, take precipitation.
The method of extraction probiotic cell wall skeleton polysaccharide the most according to claim 4, it is characterised in that: described chlorine
The preparation method of imitation-carbinol mixed solution is as follows: 1:1.5-2.5 by volume, takes chloroform and methanol, and adding appropriate pH is 4.6
Acetic acid-sodium acetate buffer solution, mix homogeneously, gained solution is chloroform-methanol mixed solution.
The method of extraction probiotic cell wall skeleton polysaccharide the most according to claim 1, it is characterised in that: described utilization
Trypsin-neutral protease mixed enzyme solution removes the protein in probiotic bacteria thalline, and method is as follows: by solid-to-liquid ratio 1:4-6
Ratio, remaining precipitation and trypsin-neutral protease mixed enzyme solution after chloroform-methanol mixed solution of learning from else's experience process, mixing,
It is subsequently placed in isothermal vibration incubator, shaken cultivation 35-40h at a temperature of 38 DEG C, after taking-up, is centrifuged in 12000r/min
15-20min, discards supernatant, and precipitation is divided into two-layer, retains upper strata precipitation, is centrifuged repeatedly cleaning by proper amount of methanol, until supernatant
Till liquid achromaticity and clarification is transparent, takes precipitation and be probiotic cell wall skeleton polysaccharide.
The method of extraction probiotic cell wall skeleton polysaccharide the most according to claim 6, it is characterised in that: described pancreas egg
The preparation method of white enzyme-neutral protease mixed enzyme solution: by weight 1:4-5, takes trypsin and neutral protease, adds suitable
Amount pH is the phosphate buffered solution of 7.4, stirs, and gained solution is trypsin-neutral protease mixed enzyme solution.
The method of extraction probiotic cell wall skeleton polysaccharide the most according to claim 7, it is characterised in that: described pancreas egg
In white enzyme-neutral protease mixed enzyme solution, the enzyme activity of every kind of enzyme is all not less than 250U/ml.
9. according to the arbitrary described method extracting probiotic cell wall skeleton polysaccharide of claim 1-8, it is characterised in that: described
Probiotic bacteria be lactobacillus and bacillus bifidus.
The method of extraction probiotic cell wall skeleton polysaccharide the most according to claim 9, it is characterised in that: described breast
Bacillus is lactobacillus rhamnosus, Lactobacillus reuteri or bacillus acidophilus；Described bacillus bifidus is animal bifidobacteria or breast
Bacillus bifidus.
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| CN102321187A (en) * | 2011-08-15 | 2012-01-18 | 内蒙古双奇药业股份有限公司 | Method for extracting whole peptidoglycan from bifidobacterium longum NQ-1501 |
| CN102335413A (en) * | 2011-08-16 | 2012-02-01 | 东北农业大学 | Preparation method, product and application of lactobacillus whole peptidoglycan with effect for resisting beta-lactoglobulin hypersusceptibility |
| CN105219817A (en) * | 2015-11-02 | 2016-01-06 | 辽宁大学 | A kind of preparation method and application of Exopolysaccharides Produced by Lactic Acid Bacteria |
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| CN102321187A (en) * | 2011-08-15 | 2012-01-18 | 内蒙古双奇药业股份有限公司 | Method for extracting whole peptidoglycan from bifidobacterium longum NQ-1501 |
| CN102335413A (en) * | 2011-08-16 | 2012-02-01 | 东北农业大学 | Preparation method, product and application of lactobacillus whole peptidoglycan with effect for resisting beta-lactoglobulin hypersusceptibility |
| CN105219817A (en) * | 2015-11-02 | 2016-01-06 | 辽宁大学 | A kind of preparation method and application of Exopolysaccharides Produced by Lactic Acid Bacteria |
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