CN102174437B - Lactobacillus delbrueckiisubsp.lactis and preparation method of bacteriocin thereof capable of resisting Listeria monocytogenes - Google Patents
Lactobacillus delbrueckiisubsp.lactis and preparation method of bacteriocin thereof capable of resisting Listeria monocytogenes Download PDFInfo
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- 108010062877 Bacteriocins Proteins 0.000 title claims abstract description 87
- 241001147746 Lactobacillus delbrueckii subsp. lactis Species 0.000 title claims abstract description 13
- 241000186779 Listeria monocytogenes Species 0.000 title abstract description 12
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 34
- 238000000746 purification Methods 0.000 claims abstract description 18
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 76
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
Abstract
The invention relates to a kind of Lactobacillus delbrueckiisubsp.lactis and a preparation method of a bacteriocin thereof capable of resisting Listeria monocytogenes, and the bacteriocin is suitable for the antisepsis and freshness of meat and meat products, milk and dairy products, fruits and vegetables, ready-to-use food, and the like. In the preparation method, the Lactobacillus delbrueckiisubsp.lactis zhang-LFCGMCC No.4393 is utilized and an adsorption-desorption method depending on the pH value and the cationic exchange chromatography are utilized to prepare the bacteriocin, thus the titer is increased by 32 times and the purity is increased by 25.23 times; and the bacteriocin has high antibacterial activity to Listeria monocytogenes and better thermal stability and acidic and alkaline stability and can be degraded by human protease, thus the bacteriocin is a natural and safe biopreservative. The invention provides a simple and effective preparation method of the Lactobacillus delbrueckiisubsp.lactis bacteriocin capable of resisting the activity of Listeria monocytogenes; and the extraction and purification methods are simple, the cost is lower, the demand on equipment is low and the method is suitable for industrial production.
Description
Technical field
The present invention relates to the preparation method of a kind of lactobacillus delbruckii lactic acid subspecies in the microorganism field and anti-Listeria monocytogenes bacteriocin thereof; This bacteriocin is applicable in meat and meat product, breast and milk-product, fruits and vegetables, the instant food as natural antiseptic agent, the security of raising food.
Background technology
Food is vulnerable to the harmful microbe pollution and causes generation putrid and deteriorated or food poisoning in process such as processing, storage, transportation, adopts the means of adding sanitas to suppress microbial growth, is one of important method of current food fresh keeping.In existing anticorrosion means, Chemical Preservative has potential hazard to human body, and physical sterilization causes certain influence to the sense organ and the nutritional quality of food.Because people pursue the food of " more natural " and " minimally processing ", thereby have excited the research of scholars to the natural biological antiseptic agent.
Bacteriocin lab is that some milk-acid bacteria has antibacterial bioactive peptide or precursor through one type of the generation of rrna synthesis mechanism in metabolic process; Having preferably to gram-positive microorganism, selectivity suppresses energy for growth; And it is better to heat and ph stability; Can be by characteristics such as human body protein enzyme liberating, so bacteriocin lab is the biological preservative of natural and safe.
Bacteriocin lab is if will importantly set up a kind of simple and effective purification process as food preservatives.The method of purification of bacterial element is a lot of, mainly contains methods such as ammonium sulfate precipitation, ion exchange chromatography, gel chromatography, hydrophobic displacement chromatography, performance liquid chromatography.
Bacteriocin is taked following three kinds of modes usually in Application in Food: (1) uses the bacteriocin of purifying directly to add in the food as food preservatives; (2) bacteriocin is produced bacterium and produce leavened food, can effectively prevent living contaminants in the fermenting process as starter; (3) in the raw material of preparation packaging material for food, add bacteriocin, exploitation has the food pack, preservative film of fungistatic effect etc.Therefore, seek strain leavening property milk-acid bacteria good and the ability bacteriocinogeny and promptly become research emphasis.Because lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.Lactis) is general office of Ministry of Health of the People's Republic of China one of explicitly provided bacterial classification (seeing Appendix) in " the bacterial classification list that can be used for food " printed and distributed on April 22nd, 2010; Therefore, as food preservatives reliable basis is provided for lactobacillus delbruckii lactic acid subspecies bacteriocin.
Relevant bacteriocin preparing method's patent report, like " a kind of working method of novel streptococcus thermophilus bacteriocin " of University Of Science and Technology Of Tianjin's application, number of patent application is: CN200910069707.7; " a kind of anti-streptococcus suis bacteriocin and working method thereof and the special strain therefore " of China Agricultural University's application, application number is: CN200910076352.4; " the Erwinia chrysanthemi small molecules bacteriocin and the fermented extracted method thereof " of Agricultural University Of Nanjing's application, application number is: CN99114367.1; " preparation method and its usage of antibacterial metabolin of Propionibacterium " of Shanghai Institute Of Technology's application, application number is: CN200810201678.0; " bacteriocin " of Societe Des Produits Nestle S.A's application, application number is: CN96112157.2.Still have nothing to do in lactobacillus delbruckii lactic acid subspecies bacteriocin preparing method's relevant report in present domestic patent of invention and the document.
Summary of the invention
First purpose of the present invention provides a kind of active lactobacillus delbruckii lactic acid subspecies of anti-Listeria monocytogenes zhang-LF that has.
Milk-acid bacteria provided by the present invention is: lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckiisubsp.Lactis) zhang-LF; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on December 1st, 2010, preserving number is: CGMCC No.4393.
Lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.lactis) zhang-LF, the dried fish product of separation screening in the market of farm produce, Fujian.
Containing on the MRS selective medium of lime carbonate, the bacterium colony size of lactobacillus delbruckii lactic acid subspecies (Lactobacillusdelbrueckii subsp.lactis) zhang-LF is 2～3mm, pearl; Translucent, circle, median rise; Neat in edge, periphery of bacterial colonies have dissolving CaCO
3Transparent circle; It is shaft-like that individual morphology is, and varying length is thread usually, single living or catenation, G
+Oxytolerant or little aerobic bacteria.
In order to confirm the antagonistic property of lactobacillus delbruckii lactic acid subspecies, the bacteriostasis property of its fermented supernatant fluid is measured.Test strain: intestinal bacteria, enterococcus faecalis, Listeria monocytogenes (abbreviation Listeria monocytogenes), lactobacterium casei cheese subspecies, Lactobacillus pentosus, lactobacillus curvatus, Salmonellas, streptococcus aureus, Shigellae, subtilis, brown rot fungus.Test-results shows that lactobacillus delbruckii lactic acid subspecies fermented supernatant fluid has restraining effect to the growth of enterococcus faecalis, Listeria monocytogenes, lactobacterium casei cheese subspecies, Lactobacillus pentosus, lactobacillus curvatus, brown rot fungus, wherein has stronger bacteriostatic activity to Listeria monocytogenes.
The active lactobacillus delbruckii lactic acid subspecies of the anti-Listeria monocytogenes zhang-LF that has that aforesaid method obtains belongs to protection domain of the present invention.
Second purpose of the present invention provides a kind of have anti-Listeria monocytogenes active lactobacillus delbruckii lactic acid subspecies bacteriocin and process for extracting thereof.
Process for extracting with anti-Listeria monocytogenes activated bacterial element provided by the present invention is the bacteriocin that is obtained by lactobacillus delbruckii lactic acid subspecies zhang-LF.
(1) streptococcus acidi lactici fermented solution is handled 20min in 80 ℃ of waters bath with thermostatic control, regulates pH4.5 with 1mol/LNaOH, room temperature vibration 30min after being cooled to room temperature;
(2) 15000r/min, 4 ℃ of centrifugal 15min collect bacterial sediment, wash bacterial sediment 2 times with the phosphate buffered saline buffer (pH4.5) of 5mmol/L;
(3) deposition is suspended in 100mmol/L NaCl (pH2.0) solution into original volume 5%, 4 ℃ are stirred 12h;
(4) 15000r/min, 4 ℃ of centrifugal 20min collect supernatant, and supernatant is put into pretreated dialysis tubing in 4 ℃ of deionized water dialysis 24h desalinations, and dialyzate is the bacteriocin extracting solution;
(5) the bacteriocin extracting solution is injected in the aseptic freeze-dried bottle of 600mL; Pre-freeze is to frozen state under-35 ℃ of conditions; On the LABCONCO vacuum freeze drier in vacuum tightness be 0.25mBar, condenser temperature for-55 ℃ of conditions under sublimation drying 36～72h, its dry thing is lactobacillus delbruckii lactic acid subspecies bacteriocin bullion.
The above-mentioned active lactobacillus delbruckii lactic acid subspecies of the anti-Listeria monocytogenes bacteriocin process for extracting that has also belongs to protection domain of the present invention.
The 3rd purpose of the present invention provides a kind of simple and effective purification process with the active lactobacillus delbruckii lactic acid subspecies of anti-Listeria monocytogenes bacteriocin.
Purification process with anti-Listeria monocytogenes activated bacterial element provided by the present invention is the bacteriocin that is obtained by lactobacillus delbruckii lactic acid subspecies zhang-LF.
(1) the bacteriocin bullion is redissolved in the citric acid-sodium citrate damping fluid of pH3.0; Adopt SPSepharose Fast Flow cation exchange chromatography purification of bacterial plain; Elution requirement is: flow velocity is 1mL/min; The citric acid-sodium citrate damping fluid that contains 0.1～0.5mol/L NaCl carries out linear gradient elution, collects light absorption value has absorption peak at the 280nm place solution;
(2) will collect liquid and put into pretreated dialysis tubing, dialysis 24h desalination in 4 ℃ of deionized waters, dialyzate is the bacteriocin purified product;
(3) the bacteriocin purified product is injected in the aseptic freeze-dried bottle of 600mL; Pre-freeze is to frozen state under-35 ℃ of conditions; On the LABCONCO vacuum freeze drier in vacuum tightness be 0.25mBar, condenser temperature for-55 ℃ of conditions under sublimation drying 36～72h, its dry thing is the pure article of lactobacillus delbruckii lactic acid subspecies bacteriocin.
The above-mentioned active lactobacillus delbruckii lactic acid subspecies of the anti-Listeria monocytogenes bacteriocin purification process that has also belongs to protection domain of the present invention.
The dried fish product of lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.lactis) zhang-LF separation screening in the market of farm produce, Fujian, the bacteriocin of its generation can be by the human body protein enzyme liberating.The absorption-desorption that the present invention utilizes the lactobacillus delbruckii lactic acid subspecies to adopt the pH value to rely on attaches method, cation exchange chromatography prepares bacteriocin, tires and has improved 32 times, and purity has improved 25.23 times, its convenient sources, and extraction process is simple; The bacteriocin purification process is simple to operate, and cost is lower, and is low for equipment requirements, is suitable for suitability for industrialized production.
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.Embodiment 1, have screening and the evaluation of the active lactic bacterium strains zhang-LF of anti-Listeria monocytogenes
1, the screening that has the active lactic bacterium strains zhang-LF of anti-Listeria monocytogenes
(1) has the primary dcreening operation of the active lactic bacterium strains zhang-LF of anti-Listeria monocytogenes
Get the dried fish product powder 1g that ground and put into the 9mL SPSS, the 20min that fully vibrates processes suspension, and 10 times of gradient dilutions become 10
-5～10
-8Diluent, each extent of dilution sample suspension is coated on the MRS selective medium that contains lime carbonate, 37 ℃ cultivate 24h after, carry out gramstaining, look its individual morphology and purity.Change to be inoculated in the MRS liquid nutrient medium, 37 ℃ increase bacterium and cultivated for 2～3 generations, and the centrifuging and taking fermented supernatant fluid adopts the Oxford agar diffusion method, judge whether bacteriocinogeny of bacterial strain through observing the inhibition zone size.
Oxford agar diffusion method: earlier the indicator bacterial strain is diluted to 10
7Cfu/mL behind the solid medium mixing of heating and melting, pours into about 15mL in plate, treat that it solidifies after, put sterilized Oxford cup gently, get 100 μ L lactic bacterium strains fermented supernatant fluids and add in the cup of Oxford.Behind 4 ℃ of refrigerator diffusion 4h, place 37 ℃ of incubators to cultivate 12h, observe the appearance of inhibition zone, measure antibacterial circle diameter with vernier callipers, reading is accurate to 0.01mm, and the result sees table 1.
The primary dcreening operation test-results of table 1 lactic bacterium strains zhang-LF bacteriostasis property
Annotate: Oxford cup diameter is 7.50mm.
Visible by table 1; The fermented supernatant fluid of lactic bacterium strains zhang-LF has restraining effect to the growth of enterococcus faecalis, Listeria monocytogenes, lactobacterium casei cheese subspecies, Lactobacillus pentosus, lactobacillus curvatus, brown rot fungus; Wherein Listeria monocytogenes there is stronger bacteriostatic activity, brown rot fungus, enterococcus faecalis effect are taken second place.
(2) has the multiple sieve of the active lactic bacterium strains zhang-LF of anti-Listeria monocytogenes
Selecting Listeria monocytogenes is that indicator is cooked multiple sieve experiment.Milk-acid bacteria can suppress spoilage organism and the pathogenic bacterium in the food, and its antibacterial substance mainly is its meta-bolites, like acid, hydrogen peroxide, bacteriocin, di-acetyl etc.Therefore, in the screening of bacteriocin generation bacterium, need to get rid of these interfering factorss, and confirm that tentatively its antibacterial substance is a protein matter.Transfer pH to neutral the fermented supernatant fluid that obtains behind the primary dcreening operation, to get rid of acid in the fermented supernatant fluid for the active interference of anti-Listeria monocytogenes with the active lactic bacterium strains zhang-LF of anti-Listeria monocytogenes; Whether fermented supernatant fluid is handled with katalase, be hydrogen peroxide with the antibacterial substance in the checking fermented supernatant fluid; Whether fermented supernatant fluid is handled with Proteinase K, can be by proteasome degradation with the antibacterial substance in the checking fermented supernatant fluid.Adopt the Oxford agar diffusion method to do bacteriostatic test, simultaneously with the fermented supernatant fluid of not doing any processing as blank, test-results is seen table 2.
The primary dcreening operation test-results of table 2 lactic bacterium strains zhang-LF bacteriostasis property
Annotate: Oxford cup diameter is 7.50mm.
Shown that by table 2 test-results lactic bacterium strains zhang-LF fermented supernatant fluid is after the interference of getting rid of acid and hydrogen peroxide, the activity of its anti-Listeria monocytogenes still exists; And through after the Proteinase K processing, its anti-Listeria monocytogenes activity completely loses.In conjunction with the definition of bacteriocin, prove that the active substance of the anti-Listeria monocytogenes in the lactic bacterium strains zhang-LF fermented supernatant fluid is a bacteriocin.
2, the part physicochemical characteristics of lactic bacterium strains zhang-LF bacteriocin
(1) enzyme susceptibility is made into AMS, pronase e and trypsinase respectively the solution of 20mg/mL; And regulate the ph optimum of each enzyme effect; Join in the fermented supernatant fluid of lactic bacterium strains zhang-LF, the final concentration that makes various enzymes is 1mg/mL, 37 ℃ of water-bath 2h; The unified again pH6.0 that recalls to; As blank, detect enzyme to the active influence of the anti-Listeria monocytogenes of lactic bacterium strains zhang-LF with the lactic bacterium strains zhang-LF fermented supernatant fluid of not enzyme-added processing, test-results is seen table 3.
Table 3 bacteriocin is to proteolytic enzyme, diastatic susceptibility
Annotate: Oxford cup diameter is 7.50mm.
Shown by table 3 test-results, compare with the blank group that lactic bacterium strains zhang-LF fermented supernatant fluid inhibition zone size after AMS is handled reduces to some extent, the bacteriostatic activity substance depilatory that shows this fermented supernatant fluid is in glycosylation modified; Lactic bacterium strains zhang-LF fermented supernatant fluid is after pronase e and trypsin solution processing, and no inhibition zone occurs, and promptly the anti-Listeria monocytogenes activity of bacteriocin completely loses, and shows that this bacteriocin can be had application security by the human body protein enzyme liberating.
(2) ph stability is 2.0,4.0,6.0,8.0,10.0,12.0 with lactic bacterium strains zhang-LF fermented supernatant fluid use 1mol/L NaOH and 1mol/L HCl adjusting pH; 37 ℃ of water-bath 2h; PH6.0 is recalled in unification again, measures its bacteriostatic activity respectively, and test-results is seen table 4.
The ph stability of table 4 bacteriocin
Annotate: Oxford cup diameter is 7.50mm.
Show by table 4 test-results; Lactic bacterium strains zhang-LF fermented supernatant fluid anti-Listeria monocytogenes in pH2～8 scopes is active to keep stable; The pH of most food 5～6.5 between, so this bacteriocin as aseptic applications in food, need not to worry the ph stability problem.
(3) thermostability is handled 10min, 30min, 90min through 60 ℃ respectively with lactic bacterium strains zhang-LF fermented supernatant fluid; Handle 10min, 30min, 90min for 100 ℃; Handle 15min for 121 ℃; With the fermented supernatant fluid of heat treated not is blank, and it is active to measure its anti-Listeria monocytogenes, and test-results is seen table 5.
The thermostability of table 5 bacteriocin
Annotate: Oxford cup diameter is 7.50mm.
Show by table 5 test-results; Compare with the blank group; Handle that the anti-Listeria monocytogenes activity of lactic bacterium strains zhang-LF fermented supernatant fluid still keeps more stable behind the 30min for 100 ℃; Therefore this bacteriocin is applied in the food, and the food processing technology can not impact its anti-Listeria monocytogenes activity.
3, the evaluation of bacteriocinogeny lactic bacterium strains zhang-LF
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; Turbidity to 65 ± 2%T with the special-purpose inoculation liquid adjustment bacteria suspension of Biolog is 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 seen table 6.
Certified variety and the result of table 6 lactic bacterium strains zhang-LF
Annotate: "+" bacterial strain more than 95% is positive; "-" bacterial strain more than 95% is negative.
Qualification result is as shown in table 6, identifies that lactic bacterium strains zhang-LF is lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.Lactis).
Embodiment 2, lactobacillus delbruckii lactic acid subspecies zhang-LF bacteriocin process for extracting
1, the technological condition for fermentation of bacteriocin
Be used to cultivate the medium component (w/v) of lactobacillus delbruckii lactic acid subspecies zhang-LF bacteriocinogeny: Tryptones 1%, Carnis Bovis seu Bubali cream 1%, yeast soak powder 0.5%; Triammonium citrate 0.2%, glucose 2%, tween-80 0.1%; Sodium acetate 0.5%, potassium hydrogenphosphate 0.2%, sal epsom 0.05%; Manganous sulfate 0.025%, the 0.07MPa 20min that sterilizes.
The technological condition for fermentation of bacteriocinogeny: 37 ℃ of leavening temperatures, fermentation time 20h, the initial pH6.0 of substratum, inoculum size 1%.
2, the process for extracting of bacteriocin and the analysis of tiring in the fermented liquid
(1) purpose of the process for extracting bacteriocin separation and purification of bacteriocin is that this target protein matter is extracted, and makes it to meet the requirements of purity through purifying.The gordian technique of extracting is to select to be fit to the effective ways that target protein matter is extracted.The streptococcus acidi lactici fermented solution that will under above-mentioned fermentation condition, obtain is handled 20min in 80 ℃ of waters bath with thermostatic control, regulates pH4.5 (being contrast with pH6.0 simultaneously) with 1mol/L NaOH after being cooled to room temperature, the room temperature 30min that vibrates; With 15000r/min, 4 ℃ of centrifugal 15min, collecting precipitation thalline; Phosphate buffered saline buffer (pH4.5) washing bacterial sediment 2 times (condition is the same) with 5mmol/L; Deposition is suspended in 100mmol/L NaCl (pH2.0) solution into original volume 5%, and 4 ℃ are stirred 12h, with 15000r/min; 4 ℃ of centrifugal 20min; Collect supernatant, the supernatant of collecting is put into pretreated dialysis tubing in 4 ℃ of deionized water dialysis 24h desalinations, dialyzate is the bacteriocin extracting solution.The absorption-desorption that above-mentioned employing pH relies on attaches effect, has solved conventional ammonium sulfate precipitation method and has extracted the substratum color interference problem that protein is introduced.
(2) bacteriocin is tired and is analyzed Listeria monocytogenes ATCC54003 as indicator, and bacteriostatic test adopts the Oxford agar diffusion method.
Confirming of bacteriocin valence value: will treat the continuous doubling dilution of test sample, and adopt the Oxford agar diffusion method to measure the antibacterial vigor of bacteriocin in the appearance liquid.Vigor is represented with every milliliter unit of activity (AU).The definition of AU is the inverse that can see the high dilution (n) of tangible inhibition zone.
The tiring of cultivation 20h centrifuged supernatant lyophilized powder of having been confirmed lactobacillus delbruckii lactic acid subspecies zhang-LF bacteriocin by two times of serial dilution methods is 80AU/mL.
The bacteriocin process for extracting is being optimized with reference on the basis of conventional extraction conditions, and the absorption pH of conventional process for extracting is 6.0, and the absorption pH after the optimization confirms as 4.5, and under this optimal conditions, bacteriocin is tired and improved 4 times, sees table 7.
Table 7 is optimized extraction conditions and common extraction conditions comparative test result
Annotate: Oxford cup diameter is 7.50mm.
(3) vacuum lyophilization of bacteriocin is injected 50mL or 100mL bacteriocin extracting solution in the aseptic freeze-dried bottle of 600mL; The plug sealing; Pre-freeze is to frozen state under-35 ℃ of conditions; On the LABCONCO vacuum freeze drier in vacuum tightness be 0.25mBar, condenser temperature for-55 ℃ of conditions under sublimation drying 36～72h, its dry thing is the pure article of lactobacillus delbruckii lactic acid subspecies bacteriocin.Freeze-drying bottle inner drying material is moved in the sterilization triangular flask, weigh.Dried bacteriocin bullion (or having a little pale yellow) pulverulence that is white in color.
Embodiment 3, a kind of simple and effective lactobacillus delbruckii lactic acid subspecies zhang-LF bacteriocin purification process
1, the purification process of lactobacillus delbruckii lactic acid subspecies zhang-LF bacteriocin
The bacteriocin bullion is redissolved in the citric acid-sodium citrate damping fluid of pH3.0, adopt SP SepharoseFast Flow cation exchange chromatography purification of bacterial plain; The cation-exchange chromatography elution requirement is: flow velocity is 1ml/min, and the citric acid-sodium citrate damping fluid that contains 0.1～0.5NaCl with pH3.0 carries out linear gradient elution, collects light absorption value has big absorption peak at the 280nm place solution; To collect liquid and put into pretreated dialysis tubing in 4 ℃ of deionized water dialysis 24h desalinations, dialyzate is the bacteriocin purified product.
2, the bacteriocin purification effect is estimated
The mensuration of total protein content adopts Xylene Brilliant Cyanine G G-250 staining:
Xylene Brilliant Cyanine G G-250 100mg is dissolved in 50mL 95% ethanol, adds 100mL 85% phosphoric acid, with distilled water diluting to 1000mL, filter paper filtering.Contain 0.01% (w/v) Xylene Brilliant Cyanine G G-250,4.7% (w/v) ethanol in the final reagent; The crystallization bovine serum albumin is measured protein nitrogen content through micro-nitrogen determination in advance, is mixed with 1mg/mL according to its purity with 0.15%NaCl, the 0.1mg/mL protein solution; Get 14 test tubes, divide two groups to operate by table 8.With A
595nm(light absorption value 595nm) is ordinate zou, and standard protein content is X-coordinate, the drawing standard curve; Measuring method is the same, gets suitable unknown sample volume, makes its measured value in the linear extent of typical curve.According to the A that is measured
595nmValue is found the amount that it is equivalent to standard protein, thereby is calculated the protein concn (mg/mL) of unknown sample on typical curve, the typical curve parameter list is seen table 8.
Table 8 Xylene Brilliant Cyanine G method is surveyed protein content typical curve parameter list
The formula of bovine serum albumin (BSA) the protein typical curve gained that employing Xylene Brilliant Cyanine G method records is: y=0.012x+0.016, R
2Value reaches 0.992, explains that the linear of this typical curve is higher, can be used as the typical curve of determination of protein concentration.
3, the freeze drying process of the vacuum lyophilization cell free fermentation supernatant of the pure article of bacteriocin, the pure article of bacteriocin is with the freeze drying process of bacteriocin bullion, and the bacteriocin purification effect is seen table 9.
The evaluation of table 9 bacteriocin purification effect
Shown that by table 9 the ratio vigor of bacteriocin is increased to 7056.67AU/mg behind the purifying, the purifying multiple is 25.23 times, tires and improves 32 times, shows thus to adopt simply fast that purification step can reach the purifying purpose effectively.
Claims (3)
1. lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.lactis) zhang-LF CGMCC No.4393.
2. method of utilizing lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.lactis) zhang-LF CGMCC No.4393 to extract bacteriocin; It is characterized in that: streptococcus acidi lactici fermented solution is handled 20min in 80 ℃ of waters bath with thermostatic control; Regulate pH4.5 with 1mol/L NaOH after being cooled to room temperature, room temperature vibration 30min; 15000r/min, 4 ℃ of centrifugal 15min, the collecting precipitation thalline is with the phosphate buffered saline buffer washing bacterial sediment of the 5mmol/L of pH4.5 2 times; Deposition is suspended in the 100mmol/L NaCl solution into the pH2.0 of original volume 5%, and 4 ℃ are stirred 12h; 15000r/min, 4 ℃ of centrifugal 20min collect supernatant, and supernatant is put into pretreated dialysis tubing in 4 ℃ of deionized water dialysis 24h desalinations, and dialyzate is the bacteriocin extracting solution; The bacteriocin extracting solution is injected in the aseptic freeze-dried bottle of 600mL; Pre-freeze is to frozen state under-35 ℃ of conditions; On the LABCONCO vacuum freeze drier in vacuum tightness be 0.25mBar, condenser temperature for-55 ℃ of conditions under sublimation drying 36～72h, its dry thing is lactobacillus delbruckii lactic acid subspecies bacteriocin bullion.
3. one kind is utilized the plain method of lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.lactis) zhang-LF CGMCC No.4393 purification of bacterial; It is characterized in that: the described bacteriocin bullion of claim 2 is redissolved in the citric acid-sodium citrate damping fluid of pH3.0; Adopt SPSepharose Fast Flow cation exchange chromatography purification of bacterial plain; Elution requirement is: flow velocity is 1mL/min; The citric acid-sodium citrate damping fluid that contains 0.1～0.5mol/LNaCl carries out linear gradient elution, collects light absorption value has absorption peak at the 280nm place solution; To collect liquid and put into pretreated dialysis tubing, dialysis 24h desalination in 4 ℃ of deionized waters, dialyzate is the bacteriocin purified product; With its lyophilize, its dry thing is the pure article of lactobacillus delbruckii lactic acid subspecies bacteriocin, the pure article of the dried bacteriocin pulverulence that is white in color.
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| CN1436238A (en) * | 2000-05-29 | 2003-08-13 | 罗狄亚化学公司 | Anti-listeria bacteriocin |
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