CN101892205B - Extraction method of bile salt hydrolase in Lactococcus lactis and Streptococcus thermophilus - Google Patents
Extraction method of bile salt hydrolase in Lactococcus lactis and Streptococcus thermophilus Download PDFInfo
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- CN101892205B CN101892205B CN2010101859508A CN201010185950A CN101892205B CN 101892205 B CN101892205 B CN 101892205B CN 2010101859508 A CN2010101859508 A CN 2010101859508A CN 201010185950 A CN201010185950 A CN 201010185950A CN 101892205 B CN101892205 B CN 101892205B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to an extraction method of bile salt hydrolase (BSH) and extracellular polysaccharide (EPS) in Lactococcus lactis and Streptococcus thermophilus, which is suitable for developing and producing novel functional foods by using BSH and EPS as functional factors and using EPS as a natural stabilizer in functional dairy products. In the invention, Lactococcus lactis subsp. lactisKS4 (CGMCC No.1807) and Streptococcus thermophilus Tx (CGMCC No.1810) of high-yield BSH and EPS are utilized, and the fermentation conditions and the extraction method are optimized to obtain crude products of BSH and EPS of lactic acid bacteria. A proper ultrasonic cell breakage extraction method is adopted to solve the problem that intracellular BSH can not be extracted easily; and the extraction conditions of the EPS are optimized to improve the extraction ratio of the EPS. The invention has simple fermentation process and extraction method for preparing BSH and EPS, shorter fermentation period, low requirements for equipment and lower cost, and is suitable for industrialized production.
Description
Technical field
The present invention relates to (the Bile Salt Hydrolase of bile salt hydrolase in Lactococcus lactis and the thermophilus streptococcus, BSH) and exocellular polysaccharide (exopolysaccharide, EPS) extracting method, be applicable to BSH in the functional dairy product, EPS as functional factor and EPS as natural stabiliser, the functional food that Development and Production is novel.
Background technology
Probiotic bacterium (Probiotics) is meant that a class grows improved effect host privileged site microecological balance surely by enteron aisle and have the microorganism of some other useful physiological functions concurrently.They are by improving the enteric microorganism eubiosis, and the control intestinal tract infections reduces serum cholesterol level, improve the utilization of lactose intolerance people to lactose, and can stimulate specificity or nonspecific immune reaction, have the effect of immunostimulant.The viable bacteria of probiotic composition should metabolic stability, and activity is stronger, by large number of viable behind the digestive tube, brings into play probiotic action after entering enteron aisle.The excellent species that is used for producing probiotic agent at present has Lactococcus lactis subsp.lactis of genus bifidobacterium, lactobacillus, lactococcus etc.
Just confirm that absorbing the sour milk that contains certain Bacterium lacticum or bifidobacterium fermentation has the content that reduces serum cholesterol as far back as people in 1963.So far, the investigator remains in different viewpoints for the mechanism of action of milk-acid bacteria reducing cholesterol both at home and abroad, mainly concentrates on following 3 points: the direct assimilation cholesterol of (1) milk-acid bacteria somatic cells; (2) the bile salt hydrolase activity of milk-acid bacteria makes the combined cholate be degraded to the free state cholate, and latter's solubleness descends and cholesterol generation coprecipitation effect; (3) other theories are as assimilation and co-precipitation combined action.Bile salt hydrolase (BSH) is the meta-bolites that milk-acid bacteria produces.This enzyme can produce amino acid and the lower free state cholate of solubleness with the degraded of combined cholate in the circulation of human body liver sausage.The latter can combine the formation sediment composite with cholesterol and excrete, thereby reduces the content of serum total cholesterol.
There is the bibliographical information milk-acid bacteria to optimize under the growth conditions,, can secretes exocellular polysaccharide (EPS) for provide protection to self in the Asia.Whitfield etc. studies show that being formed with of exocellular polysaccharide is beneficial to microorganism opposing poor environment factor, as the invasion and attack of drying, osmotic pressure condition, microbiotic or poisonous substance effect, scavenger cell or phage etc.Since people such as nineteen eighty-two Japan scholar Shiomi report that milk-acid bacteria EPS has antitumor action, caused many scholars' attention.From the result of study of report, milk-acid bacteria EPS mainly contains the function of the following aspects: improve the cultured milk prod structural state, the synersis of control sour milk prevents that whey from separating out; Improve the non-specific adhesion of bacterial strain, strengthen decide the grow effect of bacterial strain enteron aisle to intestinal mucosa; Antitumor action; Regulate the immunity system effect; The pair cell body has provide protection; Suppress the breeding of enteron aisle spoilage organism, prevent and treat the effect of colorectal carcinoma.Think that at present the mechanism of milk-acid bacteria EPS antitumor action has the following aspects: (1) influences blood supply, and in view of bacteriocin can cause tumor cell tissue's ischemic necrosis, the antitumor action of once imagining polysaccharide may influence the blood supply of tumour; (2) stimulate certain organ or tissue, secrete a kind of material and attack tumour cell; (3) cytolemma contact inhibition.Tumor cell surface has very strong negative charge, and some polysaccharide can be in conjunction with these electric charges, make cell surface by " neutralization " thus helping the cell received signal stops dividing.
Domestic research about lactobacterium casei mainly is on the health-care effecies such as its reducing blood-fat, hypertension, treatment anaphylactic disease, as " the lactobacterium casei Bd-II bacterial strain and in the application aspect the blood fat reducing " of Shanghai Bright Dairy ﹠ Food Co., Ltd application, number of patent application is: CN03128995.9; Lactobacterium casei LC2W bacterial strain and the application number of patent application aspect hypertension thereof are: two patents of CN03129450.2; Jingyue Biological Science and Technology Co., Ltd. application: new microbial strain lactobacillus paracasei GM-080 and treat the purposes of irritated relative disease, number of patent application is: CN200410038566.X.
Though abroad the research about lactobacterium casei reduction serum cholesterol effect also has a patent report: Lactobacillus casei bd-II stain and used to reduce blood cholesterol (20060127380), but, abroad almost have nothing to do at present and produce the patent report of bile salt hydrolase and exocellular polysaccharide in bacterial strains such as Lactococcus lactis subsp.lactis, thermophilus streptococcuses.Though domestic have " a kind of lactobacterium casei bile salt hydrolase and production method thereof and special preparing strain (200610112696.2) ", " a kind of Bifodobacterium Exopolysaccharides and production method thereof and special preparing strain (200510105465.4) ", " lactobacillus casei exocellular polysaccharide and crude product thereof, preparation method and application (200510028970.3) " three patent reports, and domestic related article report and the patent report that does not still have the extracting method of bile salt hydrolase and exocellular polysaccharide in Lactococcus lactis subsp.lactis, the thermophilus streptococcus.
Summary of the invention
First purpose of the present invention provides two kinds of milk-acid bacterias with efficient decreasing cholesterol and high yield bile salt hydrolase and exocellular polysaccharide.
Milk-acid bacteria provided by the present invention is: Lactococcus lactis subsp.lactis (Lactococcus lactissubsp.lactis) KS4, thermophilus streptococcus (Streptococcus thermophilus) Tx has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on September 11st, 2006.Preserving number is respectively: CGMCC № 1807 (bacterial strain KS4), CGMCC № 1810 (bacterial strain Tx).
Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4, thermophilus streptococcus (Streptococcus thermophilus) Tx, separation screening from Jilin, the Tibetan of Huhehaote City's average family spirit mushroom (claiming the Kai Feier grain again).
Containing on the MRS selective medium of lime carbonate and tennecetin, the bacterium colony size of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4 and thermophilus streptococcus (Streptococcus thermophilus) Tx bacterial strain is 1～2mm, smooth surface, neat in edge, rounded, tarnish, projection, canescence, translucent, periphery of bacterial colonies has dissolving CaCO
3Transparent circle, bacterium colony has than high viscosity.Spherical in shape or the oval of individual morphology, general paired or catenation, G
+Facultative anaerobe.
Aforesaid method obtains is used to produce two kinds of genus lactubacillus with efficient reducing cholesterol and high yield bile salt hydrolase and exocellular polysaccharide of bile salt hydrolase and exocellular polysaccharide in protection domain of the present invention.
Second purpose of the present invention provides two kinds of Conjugated bile salt hydrolases and extracting method thereof.
The extracting method of Conjugated bile salt hydrolase provided by the present invention is by Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4, the bile salt hydrolase that thermophilus streptococcus (Streptococcus thermophilus) Tx obtains.
(1) streptococcus acidi lactici fermented solution is with 10000r/min, 4 ℃ of centrifugal 10min, collecting precipitation bacterium mud;
(2) with physiological saline centrifuge washing bacterial sediment 2 times (condition is the same);
(3) phosphate buffered saline buffer (pH6.5) that adds 0.1mol/L is to the fermented liquid original volume, and cell suspending liquid carries out ultrasonic disruption 10min (power 400～450w, work 5s, intermittently 3s) in ice bath;
(4) with 10000r/min, 4 ℃ of centrifugal 10min remove cell debris, collect to contain the enzyme supernatant liquor.
(5) supernatant liquor with the ammonium sulfate of 70%～75% saturation concentration in 4 ℃ of 24h that saltout, with 10000r/min, 4 ℃ of centrifugal 15～20min, the proteic substance of collecting precipitation;
(6) with phosphate buffered saline buffer (pH6.5) the soluble protein material of 0.1mol/L, again with pretreated dialysis tubing in 4 ℃ dialyse to dialyzate through BaCl
2Check does not have till the precipitation, and dialyzate is the bile salt hydrolase extracting solution.
(7) the bile salt hydrolase extracting solution with 50mL or 100mL injects in the aseptic freeze-dried bottle of 500mL, pre-freeze is to frozen state (16～24h) under-25 ℃ of conditions, going up in 0.22～0.24mBar vacuum tightness, condenser temperature at LABCONCO frozen vacuum dryer (U.S.'s product) is sublimation drying 36～72h under-55 ℃ of conditions, and its dry thing is the Conjugated bile salt hydrolase crude product.
Aforesaid method obtains Conjugated bile salt hydrolase and special preparing strain also belongs to protection domain of the present invention.
The 3rd purpose of the present invention provides the extracting method of two kinds of milk-acid bacteria exocellular polysaccharides.
The extracting method of exocellular polysaccharide provided by the present invention is by thermophilus streptococcus (Streptococcusthermophilus) Tx, the exocellular polysaccharide that Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4 obtains.
(1) streptococcus acidi lactici fermented solution is with 8000r/min, and 4 ℃ of centrifugal 10min collect supernatant liquor;
(2) supernatant liquor adds 10% trichoroacetic acid(TCA) of 1/5 volume pH5.0 in 4 ℃ of oscillation treatment 30min;
(3) treatment solution is with 10000r/min, and 4 ℃ of centrifugal 10～15min collect supernatant liquor;
(4) supernatant liquor adds 95% cold ethanol of 3 times of volumes in 4 ℃ of processing 11～12h, centrifugal (condition is the same), the polysaccharide material of collecting precipitation;
(5) polysaccharide material is with dissolved in distilled water, and absorbent cotton removes by filter insolubles, again with pretreated dialysis tubing in 4 ℃ of dialysed overnight, dialyzate is polysaccharide extraction liquid.
(6) 50mL or 100mL polysaccharide extraction liquid are injected in the aseptic freeze-dried bottle of 500mL, pre-freeze is to frozen state under-25 ℃ of conditions, going up in 0.16～0.18mBar vacuum tightness, condenser temperature at LABCONCO frozen vacuum dryer (U.S.'s product) is sublimation drying 36～72h under-55 ℃ of conditions, and its dry thing is milk-acid bacteria exocellular polysaccharide crude product.Dried exocellular polysaccharide (or the slightly pale yellow) crystalline state that is white in color.
Aforesaid method obtains milk-acid bacteria exocellular polysaccharide and special preparing strain and optimization extracting method thereof and also belongs to protection domain of the present invention.
Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4, thermophilus streptococcus (Streptococcus thermophilus) Tx separation screening has reliable security in the Tibetan of northeast average family spirit mushroom (claiming the Kai Feier grain again).The product of the bile salt hydrolase hydrolysis combined cholate of its generation is free state cholate and amino acid, and is harmless.Bile salt hydrolase and exocellular polysaccharide that the present invention extracts with these two kinds of lactobacillus strains, its raw material sources are convenient, and extraction process is simple, and fermentation period is short, and simple to operate, cost is lower, and is low for equipment requirements, is suitable for suitability for industrialized production.
Description of drawings
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is volumn concentration.
Lactococcus lactis subsp.lactis (the Lactococcus lactis subsp.lactis) KS4 of embodiment 1, efficient decreasing cholesterol and high yield bile salt hydrolase and exocellular polysaccharide, screening and the evaluation of thermophilus streptococcus (Streptococcus thermophilus) Tx
1, the screening of efficient decreasing cholesterol and high yield bile salt hydrolase bacterial strain
Get and hide the 45mL stroke-physiological saline solution that clever mushroom filtrate 5mL injects the band granulated glass sphere, the 30min that fully vibrates makes bacteria suspension, and then 10 times of gradient dilutions become 10
-4～10
-6Dilution bacterium liquid.Dull and stereotyped with the inoculation of dilution tilt-pour process, pour the MRS selective medium that contains lime carbonate and tennecetin into, cultivate 24h for 37 ℃, picking has single bacterium colony streak inoculation of dissolving circle in the MRS slant medium, cultivates 24h in 37 ℃.Gramstaining is looked its individual morphology and purity.Change and be inoculated in the MRS liquid nutrient medium, 37 ℃ increase bacterium and cultivated for 2～3 generations, transfer into the I substratum that contains cholesterol with 1%～2% inoculum size again, behind 37 ℃ of cultivation 20h, with content of cholesterol before and after the fermentation in the o-phthalaldehyde(OPA) colorimetric method for determining I substratum, and calculate cholesterol reduced rate (%).Determine the bacterial strain of efficient decreasing cholesterol according to the cholesterol reduced rate.Concrete measuring method: get 0.4mL and cultivate the fermented liquid of front and back in colorimetric cylinder, add 0.2mL o-phthalaldehyde(OPA) (1mg/mL) and 4mL mixing acid (Glacial acetic acid, vitriol oil balanced mix), room temperature leaves standstill 10min behind the thorough mixing, with the I substratum that does not contain cholesterol is blank accent 0, measure light absorption value (A) in 550nm, on cholesterol-light absorption value typical curve, check in fermentation front and back content of cholesterol (the results are shown in Table 1).
The primary dcreening operation test-results of the different lactobacillus strain reducing cholesterol of table 1
Studies show that the milk-acid bacteria of adopting high flux screening technology and o-phthalaldehyde method to obtain efficient reducing cholesterol is KS4 and Tx bacterial strain.Wherein, KS
4Bacterial strain reducing cholesterol ability is the strongest.They belong to the strong bacterial strain of reducing cholesterol ability in relevant bibliographical information.
With the bacterial strain that obtains behind the primary dcreening operation with efficient decreasing cholesterol; on the MRS culture medium flat plate that contains 0.1% cholesterol and 0.3% Bile Salts (respectively in contrast) with the MRS substratum of the MRS substratum that contains 0.1% cholesterol, 0.3% Bile Salts; symmetry is placed the Oxford cup; add 0.1mL MRS and cultivate bacterium liquid; after 37 ℃ of anaerobism are cultivated 3～4d; around observing the Oxford cup whether the silver color deposited phenomenon is arranged, it the results are shown in Table 2.Positive person detects the hydrolysate whether cholate is arranged in the silver color throw out with ninhydrin method---the amino acid existence, determine according to the bluish voilet reaction is preliminary whether bacterial strain produces bile salt hydrolase.The streak inoculation of ninhydrin reaction positive strain in the MRS slant tube, is cultivated the back and preserved standby.
Show that by table 1 and table 2 result the size of precipitation circle is directly proportional with the vigor of bile salt hydrolase and the percentage of reducing cholesterol.Multiple sieve test is relatively overall, and bacterial strain KS4 and Tx can the high yield bile salt hydrolases, and wherein bacterial strain KS4 enzymatic productivity is the strongest.The lactobacillus strain of efficient reducing cholesterol comes from its bile salt hydrolase that produces volume or high vigor.So can utilize the KS4 bacterial strain to extract bile salt hydrolase in a large number, and utilize the further functional yoghourt of developing efficient decreasing cholesterol of lactobacillus strain of these high yield bile salt hydrolases.
Table 2 produces the bile salt hydrolase lactobacillus strain and sieves test-results again
Annotate: silver color deposited phenomenon is in various degree arranged around the cup of "+" expression Oxford; The no deposited phenomenon of "-" expression.
2, the screening of high-yield extracellular polysaccharide strains
Get and hide the 45mL stroke-physiological saline solution that clever mushroom filtrate 5mL injects the band granulated glass sphere, the 30min that fully vibrates makes bacteria suspension, and then 10 times of gradient dilutions become 10
-4～10
-6Dull and stereotyped with dilution tilt-pour process inoculation, pour the MRS selective medium that contains lime carbonate and tennecetin into, cultivate 24h for 37 ℃, picking has the higher single bacterium colony streak inoculation of dissolving circle, stickiness in the MRS slant medium, cultivates 24h in 37 ℃.Gramstaining is looked its individual morphology and purity.Change to be inoculated in the MRS liquid nutrient medium, 37 ℃ increase bacterium and cultivated for 2～3 generations, transfer in the MRS liquid nutrient medium with 1%～2% inoculum size again, 37 ℃ cultivate 12h after, with the content of EPS in the phenolsulfuric acid colorimetric method for determining nutrient solution.To record sample absorbancy y value substitution typical curve formula y=0.8649x (R
2=0.9991) promptly obtain EPS concentration x value (100mg/L), it the results are shown in Table 3.According to the EPS content in the MRS substratum, determine to produce the bacterial strain of exocellular polysaccharide.The bacterial strain streak inoculation of producing exocellular polysaccharide in the MRS slant tube, is cultivated the back and preserved standby.
Table 3 isolated strains produces the exocellular polysaccharide test result
Studies show that the milk-acid bacteria of adopting high flux screening technology and phenolsulfuric acid method to obtain higher synthetic EPS is KS4 and Tx bacterial strain.Wherein the output of KS4 bacterial strain EPS is the highest.They all belong in relevant bibliographical information produces the stronger bacterial strain of EPS ability.
3, screening strain fermentation Performance Testing
The KS4 of screening and Tx strain culture are transferred in the sterilization skimming milk 2～3 generations of continuous passage activation, and 37 ℃ of overnight incubation to cow's milk solidify.Transfer with 1% inoculum size again and contain in the triangular flask of skimming milk, after 37 ℃ of overnight incubation to cow's milk solidify, refrigerate after-ripening again and spend the night, measure the vigor (comprising curdled milk time, acidity, viscosity, tough reddish black time of reduction, viable lactic acid bacteria quantity) of starter.After solidifying respectively at 37 ℃, 40 ℃, 43 ℃ overnight incubation to cow's milk, carry out the sense organ Comprehensive Assessment, it the results are shown in Table 4.
The leavening property test result of table 4 screening lactobacillus strain
Studies show that bacterial strain KS4 and Tx all have good leavening property.It is the highest wherein to prepare the viscosity of starter by the Tx bacterial strain, and the curdled milk time is the shortest, and the sense organ comprehensive grading is the highest, secondly is the KS4 bacterial strain.So can utilize the Tx bacterial strain to extract EPS in a large number, and utilize these lactobacillus strains that produce EPS further to develop functional yoghourt with antitumous effect.
According to the vigor and the sense organ Comprehensive Assessment mark height of starter, determine the bacterial strain that leavening property is good.Bile salt hydrolase, exocellular polysaccharide and the good MRS inclined-plane bacterial strain of leavening property are produced in screening, be inoculated in various physics and chemistry and identify that 37 ℃ of cultivations are identified in the substratum.
4, the evaluation of efficient decreasing cholesterol and high yield bile salt hydrolase and extracellular polysaccharide strains
Conventional physiological and biochemical test is identified: glycitols fermentation test, salt tolerant test (7.5%NaCl), differing temps test (10 ℃, 40 ℃).Biolog full automatic microorganism assessing instrument is identified: the single bacterium colony streak inoculation of the purpose on the picking MRS flat board (contains 5% aseptic defiber sheep blood) on the BUA+B flat board, after 37 ℃ of cultivations obtain single bacterium colony, adjust turbidity to the 65 ± 2%T of bacteria suspension with the special-purpose inoculation liquid of Biolog, be inoculated in the AN trace identification plate, after 35～37 ℃ of anaerobism are cultivated 24～48h, on assessing instrument, read qualification result.Qualification result is as shown in table 5.Identify that bacterial strain KS4 is Lactococcus lactis subsp.lactis (Lactococcus lactissubsp.lactis), bacterial strain Tx is thermophilus streptococcus (Streptococcus thermophilus).
Certified variety and the result of table 5 bacterial strain KS4, Tx
Annotate: "+" bacterial strain more than 95% is positive; "-" bacterial strain more than 95% is negative.
Embodiment 2, utilize Lactococcus lactis subsp.lactis KS4, thermophilus streptococcus Tx to extract bile salt hydrolase
1, the technological condition for fermentation of high yield bile salt hydrolase
Be used to cultivate the medium component (w/v) of Lactococcus lactis subsp.lactis KS4, thermophilus streptococcus Tx high yield bile salt hydrolase: glucose 2%, soy peptone 2%, MnSO
40.025%, extractum carnis 1%, yeast extract paste 0.5%, tween 80 0.1%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, dibasic ammonium citrate 0.2%, sal epsom 0.058%, distilled water 1000mL, the 0.07MPa 20min that sterilizes.
The technological condition for fermentation of high yield bile salt hydrolase: 37 ℃ of leavening temperatures, fermentation time 12h, the initial pH value 6.0 of substratum, inoculum size 2%.
Under this fermentation condition, the enzyme activity of Lactococcus lactis subsp.lactis KS4 is 11.84 times before optimizing.
2, the extracting method of bile salt hydrolase and enzyme activity determination in the fermented liquid
(1) the separation and purification purpose of the extracting method bile salt hydrolase of bile salt hydrolase is that this purpose intracellular enzyme is extracted, and makes it to meet the requirements of purity through purifying.The gordian technique of extracting is to select the effective ways of smudge cells.The streptococcus acidi lactici fermented solution that will obtain under above-mentioned fermentation condition is with 10000r/min, 4 ℃ of centrifugal 10min, abandon supernatant liquor, thalline stroke-physiological saline solution centrifuge washing 2 times (condition is the same), abandon supernatant liquor, the phosphate buffered saline buffer (pH6.5) that adds 0.1mol/L is to the fermented liquid original volume, cell suspending liquid carries out ultrasonic disruption 10min (power 400～450w in ice bath, work 5s, 3s intermittently), with 10000r/min, 4 ℃ of centrifugal 10min, remove cell debris, collect the supernatant liquor that contains bile salt hydrolase; Supernatant liquor with the ammonium sulfate of 70%～75% saturation concentration in 4 ℃ of 24h that saltout, with 10000r/min, 4 ℃ of centrifugal 15～20min, the proteic substance of collecting precipitation; With phosphate buffered saline buffer (pH6.5) the soluble protein material of 0.1mol/L (be with the amount of damping fluid initial fermentating liquid volume half), with pretreated dialysis tubing in 4 ℃ dialyse to dialyzate through BaCl
2Check does not have till the precipitation, i.e. dialysis is (during change the sterile phosphate damping fluid one time every 12h) fully, and dialyzate is the bile salt hydrolase extracting solution.The extracting method of the ultrasonic disruption cell that above-mentioned employing is suitable has solved the difficult problem of extracting of bile salt hydrolase in the born of the same parents.
(2) enzyme activity determination is got 0.1mL bile salt hydrolase extracting solution; add 1.8mL0.1mol/L phosphate buffered saline buffer (pH6.0) and 0.1mL in conjunction with cholate (6mmol/L Bile Salts); vibration mixing 1min on vortex mixer, with mixed solution in 37 ℃ of vibration enzymolysis 40min.Enzyme reaction finishes, and adds 80% trichoroacetic acid(TCA) (dosage and enzyme liquid equal-volume) immediately, mixing, and behind the termination enzyme reaction 1min centrifugal (condition is the same), supernatant liquor is enzyme reaction solution.
The mensuration of bile salt hydrolase vigor is how much weighing by the amount of amino acid that discharges in conjunction with cholate degraded back.Promptly this enzyme activity is directly proportional with amino acid whose growing amount.Can adopt ninhydrin to measure amino acid whose content.Get the 2mL enzyme reaction solution, add triketohydrindene hydrate developer 1mL, in boiling water bath, heat 16min behind the mixing, the tap water cooling, add the 5mL diluent, shake up, the same operation substitutes enzyme reaction solution with 2mL distilled water and transfers 0 as blank, survey light absorption value A (color of generation is stable in 60min, is preferably within the 30min and finishes) in 570nm.Bile salt hydrolase is defined as an enzyme activity unit in the amino acid that per minute generates 1 μ mol.
(3) vacuum lyophilization of bile salt hydrolase is injected the bile salt hydrolase extracting solution of 50mL or 100mL in the aseptic freeze-dried bottle of 500mL, pre-freeze is to frozen state (16～24h) under-25 ℃ of conditions, going up in 0.22～0.24mBar vacuum tightness, condenser temperature at LABCONCO frozen vacuum dryer (U.S.'s product) is sublimation drying 36～72h under-55 ℃ of conditions, and its dry thing is the Conjugated bile salt hydrolase crude product.
Embodiment 3, utilize thermophilus streptococcus Tx, Lactococcus lactis subsp.lactis KS4 to extract exocellular polysaccharide
1, the technological condition for fermentation of high-yield extracellular polysaccharide
Be used to cultivate the medium component (w/v) of thermophilus streptococcus Tx, Lactococcus lactis subsp.lactis KS4 high-yield extracellular polysaccharide: sucrose 6%, Tryptones 3%, manganous sulfate 0.025%, extractum carnis 1.0%, yeast powder 0.50%, tween 80 0.10%, dipotassium hydrogen phosphate 0.20%, sodium acetate 0.50%, dibasic ammonium citrate 0.20%, sal epsom 0.058%, distilled water 1000mL, the 0.07MPa 20min that sterilizes.
The fermentation condition of high-yield extracellular polysaccharide: 32 ℃ of leavening temperatures, fermentation time 16h, the initial pH value 6.5 of substratum, inoculum size 3%.
Under this fermentation condition, the output of thermophilus streptococcus Tx exocellular polysaccharide is 2.6 times before optimizing.
2, the extracting method of exocellular polysaccharide and concentration determination in the fermented liquid
(1) the extracting method streptococcus acidi lactici fermented solution of exocellular polysaccharide is with 8000r/min, 4 ℃ of centrifugal 10min, collect supernatant liquor, 10% trichoroacetic acid(TCA) that adds 1/5 volume pH5.0 is in 4 ℃ of oscillation treatment 30min (optimization extraction conditions), again with 10000r/min, and 4 ℃ of centrifugal 10～15min, collect supernatant liquor, the 95% cold ethanol that adds 3 times of volumes is handled 11～12h, centrifugal (condition is the same), the polysaccharide material of collecting precipitation in 4 ℃.Polysaccharide material is with sterile distilled water dissolving (amount of adding distil water is half of initial fermentating liquid volume), aseptic absorbent cotton removes by filter insolubles, (boil 10min with 50% ethanol earlier with pretreated dialysis tubing again, then boil 10min with the 1mmol/L EDTA solution that contains 2% sodium bicarbonate, boil 3 times with distilled water again, change water boil 1min at every turn) in 4 ℃ of dialysed overnight (during change sterile distilled water 3～4 times), dialyzate is polysaccharide extraction liquid.Simultaneously being treated to common extraction conditions with 10% trichoroacetic acid(TCA) of pH0.5 compares.
(2) the exocellular polysaccharide concentration determination takes a morsel polysaccharide extraction liquid with its light absorption value at the 490nm place of phenolsulfuric acid colorimetric method for determining, calculates EPS concentration according to typical curve.If the concentration of EPS is higher, can suitably dilute 2 times.Under the calculation optimization extraction conditions, during EPS concentration, on typical curve, check in the result and multiply by 2.
Accurately take by weighing standard glucose 100mg in the 500mL volumetric flask, add water to scale.All ingredients is pressed the uniform mixing operation in colorimetric cylinder of the amount shown in the table 6, and room temperature is measured EPS light absorption value (serves as blank accent 0 by same color operation with 2.0ml distilled water) in 490nm after placing 20min.With glucose content (100mg/L) is X-coordinate, absorbancy (A
490nm) be ordinate zou drawing standard curve.To record sample absorbancy y value substitution typical curve formula y=0.380x (R
2=0.9555) promptly obtains EPS concentration x value (100mg/L).It optimizes extraction conditions and common extraction conditions EPS output comparative test result sees Table 7.
Table 6 phenolsulfuric acid method is surveyed polysaccharide typical curve parameter list
(3) vacuum lyophilization of exocellular polysaccharide is injected 50mL or 100mL polysaccharide extraction liquid in the aseptic freeze-dried bottle of 500mL, the plug sealing, and pre-freeze is to frozen state (24h) under-25 ℃ of conditions.Freeze drier preoperation inspection inside has or not ponding and checks closure, opens after determining to have or not ponding and gas leakage point and closing breather valve.Vacuum pump is started working when treating that LABCONCO frozen vacuum dryer (U.S.'s product) internal temperature is reduced to below-40 ℃, and its internal vacuum is reduced to 0.200mBar and begins to go up sample when following.Should rapidly the freeze-drying bottle be taken out from low temperature refrigerator during last sample, plug and Freeze Drying Equipment interface are locked to guarantee closure, open breather valve.Freeze-drying begins when whole closed system vacuum tightness is reduced to 0.180mBar.In vacuum tightness is that 0.16～0.18mBar, condenser temperature are sublimation drying under-55 ℃ of conditions.The initial liquid of 50ml needs freeze-drying 36h, and the initial liquid of 100ml needs freeze-drying 72h.After the internal moisture of freeze-drying bottle distils fully, close breather valve, the freeze-drying bottle is taken off and slowly discharge negative pressure in the Freeze Drying Equipment, treat that it recovers normal pressure after, close vacuum pump earlier, close freeze drier again.Freeze-drying bottle inner drying material is moved in the sterilization triangular flask, weigh.Dried exocellular polysaccharide crude product (or the slightly pale yellow) crystalline state that is white in color.
Table 7 is optimized extraction conditions and common extraction conditions comparative test result
Studies show that the optimization extraction conditions improves 5.2 times than the polysaccharide concentration of common extraction conditions.Obtaining the 1.76g Crude polysaccharides with the 1600mL fermented liquid after lyophilize is example, and its exocellular polysaccharide extraction yield is 91.3%.Illustrate and adopt the extraction conditions of optimizing, can obviously improve the extraction yield of exocellular polysaccharide.
Project under this patent: Beijing's natural science fund " is hidden the research that clever mushroom probiotic bacterium produces active substances and physiological function "
Item number: 5062006
The project beginning and ending time: 2006.01-2008.12
Project leader: Liu Hui
Claims (1)
1. one kind is utilized Lactococcus lactis breast subspecies (Lactococcus lactis subsp.lactis) KS4 CGMCC No.1807, thermophilus streptococcus (Streptococcus thermophilus) Tx CGMCCNo.1810 extracts the method for bile salt hydrolase, it is characterized in that: the fermented liquid of described milk-acid bacteria is with 10000r/min, 4 ℃ of centrifugal 10min, collecting precipitation bacterium mud; With physiological saline centrifuge washing bacterial sediment 2 times, condition is the same; The phosphate buffered saline buffer of the pH6.5 of adding 0.1mol/L is to the fermented liquid original volume, and cell suspending liquid carries out ultrasonic disruption 10min in ice bath, power 400～450w, work 5s, intermittently 3s; With 10000r/min, 4 ℃ of centrifugal 10min remove cell debris, collect the supernatant liquor that contains bile salt hydrolase; Supernatant liquor with the ammonium sulfate of 70%～75% saturation concentration in 4 ℃ of 24h that saltout, with 10000r/min, 4 ℃ of centrifugal 15～20min, the proteic substance of collecting precipitation; With the phosphate buffered saline buffer soluble protein material of the pH6.5 of 0.1mol/L, with pretreated dialysis tubing in 4 ℃ dialyse to dialyzate till the BaCl2 check does not have precipitation, dialyzate is the bile salt hydrolase extracting solution; The bile salt hydrolase extracting solution of 50mL or 100mL is injected in the aseptic freeze-dried bottle of 500mL, 16～24h pre-freeze is to frozen state under-25 ℃ of conditions, producing on the LABCONCO frozen vacuum dryer in 0.22～0.24mBar vacuum tightness, condenser temperature in the U.S. is sublimation drying 36～72h under-55 ℃ of conditions, and its dry thing is the Conjugated bile salt hydrolase raw product.
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