CN104046585A - Bifidobacterium animal bacteriocin, production method thereof and specific production strain - Google Patents
Bifidobacterium animal bacteriocin, production method thereof and specific production strain Download PDFInfo
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Abstract
The invention discloses bifidobacterium animal bacteriocin, a production method thereof and a specific production strain, the bifidobacterium bacteriocin provided by the invention is a bacteriocin by fermenting bifidobacterium animals BB04 CGMCC NO. 7790. The bifidobacterium animals BB04 CGMCC NO. 7790 is the optimal culture condition of bacteriocin metabolic production. The invention also provides a method for extracting and purifying bifidobacterium animal bacteriocin, which comprises the following steps: performing coarse extraction by a pH value adsorption and desorption method, and purifying by cation exchange resin chromatography and gel filtration chromatography. The bifidobacterium bacteriocin with molecular weight of 1198.6832Da is a novel bifidobacterium bacteriocin, has excellent food processing characteristics, is wide in antimicrobial spectrum and high in safety, and can be applied to development of natural food biological preservatives.
Description
Technical field
The present invention relates to a kind of animal bifidobacteria bacteriocin and production method and special preparing strain, belong to technical field of food biotechnology.
Background technology
Milk-acid bacteria and active metabolite thereof and human health are closely related, are usually naturally present in food or are introduced in foods prods consciously, thereby making food have local flavor, nutrition and security that people thirst for.Lactic bacteria activity meta-bolites relates generally to lactic acid, bacteriocin, exocellular polysaccharide, conjugated linolic acid etc.Bacteriocin lab is that milk-acid bacteria is synthesized by rrna mechanism and is secreted into polypeptide or the protein matter that a class in environment has bacteriostatic activity in metabolic process, it can be degraded in human body, have efficient, without advantages such as resistance, nontoxic, noresidues, become the focus of whole food biological preservative research and development.
Almost each genus lactubacillus bacterial strain can produce bacteriocin, and the bacteriocin lab existing more than 40 of having reported is at present planted.Bifidus bacillus (Bifidobacterium spp.) is the typical lactic bacteria useful colonizing in humans and animals enteron aisle, and it can bring into play biological barrier, nutrition, immunity to host, delays senility, the physiological action such as antitumor.Have found that, only a few bifidobacterium strains also can produce bacteriocin, as bifidumbacterium bifidum (B.bifidum) NCFB1454, bifidobacterium thermophilum (B.thermophilum) RBL67, bifidobacteria infantis (B.infants) BCRC14602 etc.
Bifidus bacillus is the important indicator of human intestinal health, also occupies critical role in foodstuffs industry, and its bacteriocinogeny is also more and more subject to people's attention, and will become the exploitation focus of natural biological antiseptic agent.
Summary of the invention
The object of this invention is to provide the bacteriocinogeny bifidus bacillus that a strain has broad-spectrum antibacterial effect.
Bifidus bacillus provided by the present invention, animal bifidobacteria (Bifidobacterium animals) BB04, in on 06 21st, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is: CGMCC № .7790.
Second object of the present invention is to provide a kind of bifidus bacillus bacteriocin and production method thereof.
The method of production bifidus bacillus bacteriocin provided by the present invention is the bacteriocin that fermentation animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790 obtains.
In described method, for cultivating the incubation time of animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790, be preferably 40.4h, substratum Initial pH is preferably 6.94, and culture temperature is preferably 34.5 ℃.
In described method, the bacteriocin obtaining can extract purifying according to following steps: adopt pH adsorption-desorption method from fermented liquid, to extract bacteriocin, absorption pH value is 2.0-3.0, and desorb pH value is 5.0-7.0, obtains bacteriocin crude product; Further by cation exchange resin layer, analyse and gel permeation chromatography purifying, and obtain bacteriocin sterling through lyophilize.
Wherein, the optimal adsorption pH value of above-mentioned pH adsorption-desorption method is 3.0, and best desorb pH value is 6.0.
Above-mentioned cation exchange resin layer analyses that to take SP Sepharose Fast Flow be filler, with pH5.5,0.02M acetate buffer solution containing 0-1M NaCl carries out linear gradient elution in 130min, flow velocity 1.0mL/min, and the protein peak that collection retention time is 40-48min is bacteriocin Peak Activity.
Above-mentioned gel permeation chromatography be take Sephadex G-10 as filler, and with pH6.0,0.02mol/L phosphoric acid buffer carries out wash-out, flow velocity 0.5mL/min, and the protein peak that collection retention time is 34-48min is bacteriocin Peak Activity.
Said extracted purifying gained bacteriocin molecular weight is 1198.6832Da.
The bifidus bacillus bacteriocin that aforesaid method obtains also belongs to protection scope of the present invention.
Animal bifidobacteria of the present invention (Bifidobacterium animals) BB04 CGMCC № .7790 is the animal bifidobacteria of a kind of bacteriocinogeny, this bacteriocin is a kind of novel bifidobacterium bacteriocin, it has very good food-processing characteristic, antimicrobial spectrum is wide, safe, can be used for the exploitation of whole food biological preservative or probiotics.
Accompanying drawing explanation
Fig. 1 is the adsorption rate figure of bacteriocin to generation mycetocyte under different pH condition.
Fig. 2 is that the cation exchange resin layer of bacteriocin is analysed elution curve.
Fig. 3 is the gel permeation chromatography elution curve of bacteriocin.
Fig. 4 is MALDI-TOF MS (substance assistant laser desorpted ionized flight time mass spectrum) analysis chart of bacteriocin.
Embodiment
Experimental technique described in following embodiment, if no special instructions, is ordinary method.Should be understood that these enforcements are only not limited only to the present invention for the present invention is described.
Separated and the evaluation of embodiment 1, animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790
1, the screening of bacterial strain
The aseptic breast milk infant faeces 25g that takes, puts into rapidly 225mL physiological saline, then by its 10 times of gradient dilutions to viable count 10
-6-10
-10cfu/mL, then drawing 0.2mL carries out Heng Gaite anaerobism and rolls pipe, be put in 37 ℃ of thermostat containers and cultivate 48-72h, then choosing colony is characterized as smooth, dome, neat in edge, oyster white or micro-band are yellow, quality is soft medium and small colony inoculation in MRS liquid culture medium, anaerobism is cultivated 24-48h, carries out gramstaining.Select gramstaining observe have bifidus bacillus morphological specificity, have that transparent circle, the smooth of the edge are neat around, white or yellowish colony inoculation be in modified MRS culture medium, under aerobic and anaerobic environment, cultivate 24h in 37 ℃ respectively, carry out the tests such as KOH, catalase, indoles, metabolizable glucose simultaneously.Choose that anaerobic condition growth, aerobic conditions are not grown, KOH negative, catalase test is negative, indole test is negative, energy metabolism glucose but anaerogenic bacterium is tentatively defined as the bifidus bacillus filtering out.Adopt Oxford cup lysoplate assay, take Salmonella enteritidis (Salmonella enteritidis) 13076 and Listeria monocytogenes (Listeria monocytogenes) 54002 (1/2a) carries out bacteriostatic experiment as indicator, and screening obtains having suspicious bacterial strain 5 strains that the bacterial strain of stronger bacteriostatic activity is bacteriocinogeny.
Because bifidus bacillus can produce bacteriocin, lactic acid, H during the fermentation
2o
2etc. the multiple material with bacteriostatic activity, some bifidus bacillus somatic cells also may have antagonistic action to other bacteriums, therefore need to get rid of after these disturb the suspicious bacterial strain of bacteriocinogeny is further identified.Mainly comprise: (1) is got rid of organic acid and disturbed: in suspicious strain fermentation supernatant liquor, add 1mol/LNaOH, adjusting its pH value is 6.5～7 left and right, carries out bacteriostatic experiment, relatively the antibacterial circle diameter before and after acid discharge.(2) get rid of H
2o
2interference: with catalase, process fermented liquid 2h in 37 ℃, with untreated fermented liquid, compare and carry out bacteriostatic experiment, relatively antibacterial circle diameter difference.(3) get rid of the interference of somatic cells: in 4 ℃, the centrifugal 10min of 8000rpm, gets supernatant liquor and cross the biofilter of millipore filtration (size Φ 25, aperture 0.22 μ m) and remove by filter the impact of somatic cells on bacteriostatic experiment by fermented liquid.(4) with ammonium sulfate precipitation method, slightly carry the desalination of dialysing after bacteriocin, add Proteinase K reaction, take and determine that bacteriocin is as protein matter.The fermented liquid of the suspicious bacterial strain of 5 strain is got rid of to organic acid, H
2o
2and somatic cells interfering factors impact detect bacteriostatic activity after ammonium sulfate precipitation, dialysis treatment, finding only has bacterial strain BB04 still to have compared with high bacteriostatic activity.In dialyzate, add Proteinase K (20U/mL), after 37 ℃ of effect 1h, find that bacteriostatic activity disappears, concrete outcome is in Table 1.More than comprehensive, select bacterial strain BB04 as aimed strain.
The impact of table 1 different treatment on bacterial strain BB04 fermented liquid bacteriostatic activity
2, the evaluation of bacterial strain
Adopt morphological observation, API20A biochemical reactions and 16S rDNA sequential analysis to carry out taxonomy evaluation to aimed strain BB04.Bacterial strain BB04 is well-grown in improvement solid MRS anaerobism pipe, and bacterium colony is white in color, circle, and edge is more smooth neat, the obvious bacterium colony of nothing under aerobic conditions; By gramstaining after picking colony dilution, micro-Microscopic observation is found: BB04 somatic cells form is G
+, present shaft-like, part be the bifurcation structures such as Y or V font, without gemma, do not move, irregular alignment.According to API Bacteria Identification standard, use API20A anaerobism identification systems to carry out 20 kinds of sugar alcohol fermentation tests to bacterial strain BB04.The analytical reagent bar reaction result of tabling look-up, tentatively determines that BB04 belongs to genus bifidobacterium (Bifodobacterium spp.).The DNA genome that extracts bacterial strain BB04, obtains the DNA sequencing fragment (seeing sequence table) of 1437bp, and is reclaimed purifying through pcr amplification, transfer to the order-checking of Beijing six directions Hua Da Gene Tech. Company Limited.In Genbank by sequencing result on NCBI, carry out sequence analysis, use Mega3.1 software building phylogenetic tree.From phylogenetic tree, bacterial strain BB04 and Bifidobacterium animalis are in same subbranch, and sibship is nearest, and similarity is up to 99.1%; Combining form is learned and is observed and API20A biochemical identification result, determines that bacterial strain BB04 is animal bifidobacteria (Bifidobacterium animalis).
Animal bifidobacteria (Bifidobacterium animalis) BB04 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 06 21st, 2013, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is: CGMCC № .7790.
Embodiment 2, utilize animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790 to produce bacteriocin
1, bacteriocin activity test method
(1) choice criteria curve ranges: by 500mg nisin (nisin) standard substance (10
6aU/g) be dissolved in the hydrochloric acid diluting soln of the aseptic 0.02mol/L of 50mL, be made into 10
4the reference liquid of AU/mL is standby.With 0.02mol/L HCl successively by 10
4the nisin reference liquid of AU/mL is diluted to the standardized solution of 5000,2500,1000,750,500,250,100,75,50,25,10AU/mL.Get respectively 100 μ L and be added on successively and prepared in dull and stereotyped Oxford cup, each concentration repeats 3 flat boards, does bacteriostatic test, and the correction value of antibacterial circle diameter of take is X-coordinate, and the logarithm that nisin tires is ordinate zou, drawing standard curve.Determine antibacterial circle diameter and the nisin of logarithmic value while having good linear relationship that the tire scope of tiring.Wherein, Salmonella enteritidis (S.enteritidis) 13076 of take is indicator, and nisin standard substance are purchased from Sigma company.
(2) make valence value typical curve: according to the definite nisin of the previous step scope of tiring, Salmonella enteritidis (S.enteritidis) 13076 of take be indicator, does bacteriostatic test, the making nisin typical curve of tiring.With 0.02mol/L HCl successively by 10
4the nisin reference liquid of AU/mL is diluted to 1000,750,500,250,100,75 and 50AU/mL, get successively in 3 Oxford cups that 100 μ L add respectively space in flat board (preparing), the nisin solution of 100 μ L centre concentration is added in 3 of other interval simultaneously, each gradient repeats 3 flat boards, by corresponding valence value logarithm, be ordinate zou, the difference of corresponding antibacterial circle diameter is X-coordinate, carries out the drafting of typical curve.
(3) determine the valence value of sample: by after different diluted sample suitable multiple, get in 3 Oxford cups that 100 μ L add respectively space, the intermediate concentration of curve (nisin tire) adds in 3 Oxford cups at other interval by contrasting, and repeats 3 tests.By antibacterial circle diameter with contrast the difference substitution typical curve of antibacterial circle diameter in calculate, can obtain the valence value of sample
Result shows, when nisin tires at 50～1000AU/mL, the logarithmic value of tiring and antibacterial circle diameter difference are good linear relationship, the linear degree of matched curve is higher, illustrate that this typical curve has higher tolerance range and sensitivity for titration, therefore determine its typical curve of tiring as working sample, obtaining its regression equation is y=0.2165x+2.7633 (R
2=0.9939), wherein y represents the logarithmic value of relative inhibitory potency; X represents antibacterial circle diameter/mm, the antibacterial circle diameter substitution of tested bacteria element sample can be obtained to the inhibitory potency of this bacteriocin.
2, the optimization of bacteriocin working condition
Selection is suitable for the modified MRS culture medium of bifidobacterium growth and (fills a prescription as peptone 1%, extractum carnis 1%, yeast extract paste 0.5%, dipotassium hydrogen phosphate 0.2%, citric acid diamines 0.2%, sodium acetate 0.5%, glucose 2%, tween 80 0.1%, bitter salt 0.058%, manganous sulfate 0.025%, corn steep liquor 0.35%, Cys hydrochloric acid 0.035%, distilled water 1000mL, pH6.5, described percentage ratio is mass percent), adopt 10
4cfu/mL inoculum size, substratum Initial pH 6.5, at 37 ℃, anaerobism is cultivated 100h, respectively at 0,4,8,12,16,20,24,28,32,36,40,44,48,60,100h sampling and measuring animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790 increment and bacteriocin output.Result shows that this animal bifidobacteria enters logarithmic phase after cultivating 7h, and 20h enters stationary phase, at 24h, starts to produce bacteriocin, and 40-48h bacteriocin output is the highest, and remains relatively stable, declines to some extent afterwards.Therefore choosing the suitableeest incubation time is 40h, and bacteriocin is tired as 255.43AU/mL.Contrast respectively afterwards different vaccination amount (10
3, 10
4, 10
5, 10
6, 10
7cfu/mL), substratum Initial pH (4.5,5.0,5.5,6.0,6.5,7.0,8.0), the impact of culture temperature (25 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃) on bacteriocin output, choose the bacteriocin significant cultivation factor that exerts an influence is carried out to response surface optimization, to determine the optimal production condition of bacteriocin, the results are shown in Table 2-4.
From single factor experiment result, in incubation time 40h, inoculum size 10
5under cfu/mL, cultivation Initial pH 7 and 37 ℃ of conditions of culture temperature, bacteriocin activity is higher; Choose wherein to bacteriocin exert an influence significant incubation time, cultivate Initial pH and three factors of culture temperature are independent variable(s), take relative inhibitory potency as response value, carry out center combination test design, set up regression model checking, regression model analysis of variance table is as following table 5.
The impact of table 2 different vaccination amount on bacterial strain BB04 metabolism bacteriocinogeny
The impact of table 3 different culture media Initial pH on bacterial strain BB04 metabolism bacteriocinogeny
The impact of the different culture temperature of table 4 on bacterial strain BB04 metabolism bacteriocinogeny
Table 5 regression model analysis of variance table
Data in regression model analysis of variance table are carried out to multiple regression matching, obtain bacteriocinogeny inhibitory potency (Y) and to incubation time (A), the multinomial regression equation of cultivating Initial pH (B), culture temperature (C) be: Y=4620.509-177.263A-850.27B-132.626C+546.3965AB-229.421A C+76.5085BC-1455.55A
2-1950.68B
2-1014.88C
2.In formula: Y is the predictor of tiring; A, B, C is respectively incubation time, cultivates initial pH and encoded radio corresponding to culture temperature.
As shown in Table 5, this model p=0.0004 < 0.05, regression equation fitting degree is good; Lose plan (p=0.0668 > 0.05) not remarkable, show that model accuracy is high, suitability is good.By equation expression formula, the coefficient of learning its quadratic term is all negative value, and Open Side Down for the parabola that mapping shows, so equation has maximum point.Analyze as calculated and can obtain the optimum generation condition of bacteriocin and be: incubation time 40.4h, cultivate 34.5 ℃ of initial pH6.94, culture temperature, bacteriocin inhibitory potency after optimization reaches 886.34AU/mL, than the inhibitory potency 255.43AU/mL before optimizing, 2.47 times have been improved, reach 98.9% with predictor qualified rates of fitting, show that predictor and actual value have good fitness, Optimized model is reliable.
The extraction purifying of embodiment 3, animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790 institute bacteriocinogeny
1, pH value adsorption-desorption method is extracted
Determining of optimal adsorption desorb pH value: (1) centrifugal collecting cell, with the PBS (5mM pH6.0) of sterilizing, rinse several times and be resuspended in 1.0M NaCl the phosphoric acid tune pH to 3.0 with 5%, at 4 ℃, mix the centrifugal collection of 1h and be resuspended in the sterilized water of 20 times of original volumes, to guarantee that cell surface does not have the bacteriocin of absorption.(2) determine the impact of pH on bacteriocin absorption, respectively get 0.1mL cell, 0.1mL bacteriocin sample (tiring as 886.34AU/mL), be added to respectively in 1.8mL PBS (pH value is respectively 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0), mix 30min, the bacteriostatic activity of bacteriocin in centrifugal detection supernatant liquor.Contrast 1: the bacteriocin that replaces 0.1mL with the water of 0.1mL; Contrast 2: replace with the water of 0.1mL the cell adding: adsorption rate=100% of bacteriocin * (tiring in supernatant liquor-contrast 1)/contrast 2.Bacteriostatic activity detect to adopt lysoplate assay, and Salmonella enteritidis (S.enteritidis) 13076 of take carries out as indicator.Concrete outcome is shown in Fig. 1.
Result shows, is adsorbed onto the bacteriocin producing on mycetocyte less when pH value is adjusted to 5-7, and adsorption rate is lower than 30%, and the bacteriocin content not adsorbing is higher; And pH value while being adjusted to 2-3 bacteriocin be adsorbed onto the adsorption rate higher (more than 80%) producing on mycetocyte.Therefore choose pH value 3 for bacteriocin optimal adsorption pH value, pH value 6 is respectively the best desorb pH of bacteriocin value.
Select above-mentioned definite optimal adsorption and desorb pH value, the bacteriocin in fermented liquid is slightly carried.Be specially: by bacterial strain BB04 with 10
5cfu/mL inoculum size is inoculated in 100mL modified MRS culture medium, and anaerobically fermenting 40h at 34.5 ℃ controls pH and is stabilized in 7 left and right in fermenting process.Temperature regulating to 70 ℃ after fermentation ends, enzyme 25min goes out.After being cooled to room temperature, fermented liquid is adjusted to optimal adsorption pH value 3,4 ℃ are stirred 2h, centrifugal collecting cell, with the phosphoric acid buffer identical with absorption pH value, wash cell 1-2 time, be resuspended in sterile purified water, be adjusted to best desorb pH value 6, more than 4 ℃ of magnetic agitation 12h, 8000rpm, centrifugal 15min, gets supernatant liquor and is bacteriocin crude extract and carries out purification step below.This step can obtain 30mL through pH adsorption-desorption method by 100mL fermented liquid (than vigor 66.79AU/mg), the bacteriocin crude product that is 76.74AU/mg than vigor, and purification has improved 1.15 times.
2, the purifying of bacteriocin
The purifying of bacteriocin adopts cation exchange resin layer to analyse and gel permeation chromatography, and Salmonella enteritidis (S.enteritidis) 13076 of take in purge process is indicator, and adopts lysoplate assay to analyze every step purification of samples bacteriostatic activity.Concrete grammar is as follows: above-mentioned bacteriocin crude extract is analysed and is further purified by SP Sepharose Fast Flow cation exchange resin layer, loading 1.0mL, to contain 0-1M NaCl, the 0.02mol/L acetate buffer solution linear gradient elution of pH5.5, the time of linear gradient elution is 130min, in described linear gradient elution, the concentration of NaCl rises to 1M by 0M linearity in 130min, flow velocity is 1.0mL/min, every 2min receives 1 pipe, detect elutriant at the ultraviolet absorption value at 280nm place simultaneously, and according to A
280the situation of value figure Wave crest and wave trough, detects respectively the bacteriostatic activity of every pipe elutriant, and result as shown in Figure 2.Collection retention time is that the protein peak of 40-48min (i.e. 60-64 pipe) is bacteriocin Peak Activity, concentrates and redissolves in pH7.0 phosphoric acid buffer, as bacteriocin half sterling through lyophilize.Obtain altogether 10mL, tire as the bacteriocin half pure sample product of 3674.67AU/mL.Then, by Sephadex G-10 gel permeation chromatography, carry out purifying, loading 1mL bacteriocin half sterling, adopts pH6.0,0.02mol/L phosphoric acid buffer wash-out, and flow velocity is 0.5mL/min, and every 2min receives 1 pipe, and the A of elutriant is respectively managed in monitoring simultaneously
280value.According to A
280the situation of value figure Wave crest and wave trough, carries out bacteriostatic activity detection after the elutriant at all peaks is merged respectively, the results are shown in Figure 3.Collection retention time is that the protein peak of 34-48min (i.e. 17-24 pipe) is bacteriocin Peak Activity, through lyophilize, concentrates and redissolves in pH7.0 phosphoric acid buffer, and as bacteriocin sterling, 4 ℃ of freezer storages are standby.The final 2mL that obtains, tires as 5542.12AU/mL bacteriocin sterling (table 6).
Table 6 different steps bacteriocin purification effect analytical table
As can be seen from Table 6, by above extraction purifying procedure (pH value adsorption-desorption, cation exchange resin layer is analysed and gel filtration chromatography), every 100mL animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790 fermented liquid can finally obtain 2mL, the bacteriocin sterling than vigor up to 2067.91AU/mg.
Total protein: employing BCA determination of protein concentration test kit (purchased from green skies biotechnology research institute, product article No.: P0012) total protein content in each sample is measured;
Total activity is defined as: total Antibacterial Activity units that bacteriocin sample has;
Than vigor, be defined as: every milligram of Antibacterial Activity units that bacteriocin albumen has;
Total activity (AU)=bacteriocin sample valence value (AU/mL) * population of samples amasss (mL);
Than vigor (AU/mg)=bacteriocin sample total activity (AU)/sample total protein content (mg);
After purification=purifying bacteriocin sample than the front bacteriocin sample of vigor (AU/mg)/purifying than vigor (AU/mg);
Bacteriocin sample total activity (AU) * 100% before bacteriocin sample total activity (AU)/purifying after the rate of recovery (%)=purifying.
3, the molecular weight of purification of bacterial element is determined
In order to determine the purity of bacteriocin and molecular weight accurately, with MALDI-TOF MS (substance assistant laser desorpted ionized flight time mass spectrum), purifying gained bacteriocin sample is analyzed, determine that purifying gained sample is single component material, the molecular weight that obtains this bacteriocin is 1198.6832Da, as shown in Figure 4.
The application characteristic of embodiment 4, animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790 institute bacteriocinogeny
1, the physicochemical property of bacteriocin
(1) thermostability: bacteriocin sample (5542.12AU/mL tires) after preparation 7 pipe purifying, 1mL/ pipe, heating in water bath is processed 50 ℃, 30min respectively; 60 ℃, 30min; 70 ℃, 30min; 80 ℃, 30min; 90 ℃, 30min; 121 ℃, 20min.Take without heat treated sample is contrast, carries out bacteriostatic test, bacterial detection element activity change.Result shows: bacterial strain BB04 institute bacteriocinogeny, through 50～80 ℃, is processed 30min, can keep more than 85% activity residual; Process its bacteriostatic activity for 90 ℃ and only lose approximately half; 121 ℃, after the pyroprocessing of 20min, still have the bacteriostatic activity of 10% left and right, illustrate that this bacteriocin has stronger thermostability.
(2) soda acid tolerance: choose 2～11 processing of pH scope and carry out bacteriocin soda acid tolerance test.Get respectively the bacteriocin sample (pH7.0 of 0.5mL, 5542.12AU/mL tires) in 10 test tubes, with 1mol/L HCl and 1mol/L NaOH, adjust respectively it to the pH that requires, after 37 ℃ of incubation 4h, adjust pH to neutral (7.0 left and right) in all test tubes, take and do not regulate bacteriocin sample (pH=7.0) that the sample adding distil water of pH dilutes on year-on-year basis as contrast, carry out bacteriostatic test, bacterial detection element activity change.Result shows: the bacteriostatic activity of bacterial strain BB04 institute bacteriocinogeny substantially remains unchanged within the scope of pH3-9, and residual potency ratio is all more than 93%; And pH is 2 and 10 o'clock, bacteriostatic activity decreases, but still keeps 50% vigor.PH is greater than at 10 o'clock, and bacteriostatic activity reduces rapidly, and residual potency ratio is the highest only has 19%.These results show that this bacteriocin has more wide in range pH value field of activity, can in acid and neutral food, use.
(3) proteolytic enzyme susceptibility: the solution that different proteolytic enzyme (trypsinase, stomach en-, aspartic protease, neutral protease, N,O-Diacetylmuramidase, papoid) is made into 5mg/mL, get respectively 0.1mL and be placed in centrifuge tube, add respectively bacteriocin sample (5542.12AU/mL tires) 0.4mL after purifying, the final concentration that makes enzyme is 1mg/mL, and regulate within the scope of the optimal pH that pH value is each enzyme, with 0.1mL sterilized water, add 0.4mL bacteriocin in contrast simultaneously, 37 ℃ of incubation 4h, bacterial detection element is active.As can be seen from Table 7, bacterial strain BB04 institute bacteriocinogeny is very responsive to stomach en-, trypsinase and aspartic protease, and after processing, inhibition zone disappears, complete deactivation; Neutral protease and papoid make its part inactivation, N,O-Diacetylmuramidase to it without effect.Result shows that this bacteriocin can be decomposed digestion by Partial Protein enzyme in human body, can be digested in human body and noresidue is used and had very high security as food preservatives.
The albumen susceptibility of table 7 bacterial strain BB04 institute bacteriocinogeny
Note :+represent complete deactivation, (+) represents part inactivation ,-complete non-inactivation represented.
2, the antimicrobial spectrum of bacteriocin
Adopt Oxford cup double-layer plate agar diffusion method to make different indicators flat boards, indicator bacterium final concentration is 10
7cfu/mL, fungi, yeast cell or spore concentration are 10
4individual/mL, adds in the cup of Oxford after sample and cultivates in indicator optimum condition, measures antibacterial circle diameter.As shown in table 8: bacterial strain BB04 institute bacteriocinogeny has wider scope of restraining fungi, not only the gram-positive microorganisms such as listeria bacteria, staphylococcus, genus bacillus are had to obvious restraining effect, the negative bacterium such as intestinal bacteria, pseudomonas, Salmonellas are also shown to very strong bacteriostatic activity, the tested fungi of two strains (Candida albicans bacterium and saccharomyces cerevisiae) is also shown to bacteriostatic activity simultaneously, can be widely used in food antiseptic germicidal treatment.
The antimicrobial spectrum of table 8 bacterial strain BB04 institute bacteriocinogeny
Note: NICPBP is the calibrating of Ministry of Health pharmaceutical biological product, and ATCC is USS culture presevation institute, and CVCC is Chinese veterinary microorganism DSMZ, and CGMCC is common micro-organisms culture presevation administrative center;-represent not suppress, + expression antibacterial circle diameter < 13mm, ++ expression antibacterial circle diameter is 13～20mm, +++ represent antibacterial circle diameter > 20～25mm, ++++expression antibacterial circle diameter > 25mm.
Claims (9)
1. animal bifidobacteria (Bifidobacterium animals) BB04 CGMCC № .7790.
2. producing a method for bifidus bacillus bacteriocin, is fermentation animal bifidobacteria claimed in claim 1 (Bifidobacterium animals) BB04 CGMCC № .7790, the bacteriocin obtaining.
3. method according to claim 2, is characterized in that: described incubation time is preferably 40.4h, and substratum Initial pH is preferably 6.94, and culture temperature is preferably 34.5 ℃.
4. method according to claim 2, it is characterized in that: the bacteriocin that described method obtains carries out purifying according to following steps: adopt pH adsorption-desorption method from fermented liquid, to extract bacteriocin, absorption pH value is 2.0-3.0, and desorb pH value is 5.0-7.0, obtains bacteriocin crude product; Further by cation exchange resin layer, analyse and gel permeation chromatography purifying, and obtain bacteriocin sterling through lyophilize.
5. method according to claim 4, is characterized in that: the optimal adsorption pH value of described pH adsorption-desorption method is 3.0, and best desorb pH value is 6.0.
6. method according to claim 4, it is characterized in that: described cation exchange resin layer analyses that to take SP Sepharose Fast Flow be filler, with pH5.5,0.02M acetate buffer solution containing 0-1M NaCl carries out linear gradient elution in 130min, flow velocity 1.0mL/min, the protein peak that collection retention time is 40-48min is bacteriocin Peak Activity.
7. method according to claim 4, it is characterized in that: described gel permeation chromatography be take Sephadex G-10 as filler, with pH6.0,0.02mol/L phosphoric acid buffer carries out wash-out, flow velocity 0.5mL/min, the protein peak that collection retention time is 34-48min is bacteriocin Peak Activity.
8. method according to claim 4, is characterized in that: extracting the bacteriocin molecular weight that purifying obtains is 1198.6832Da.
9. the bifidus bacillus bacteriocin being obtained by method described in claim 2 or 3 or 4.
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| CN109924386A (en) * | 2019-03-19 | 2019-06-25 | 浙江工商大学 | A kind of biology composite preservative, preparation method and its application in sturgeon caviar |
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| CN109924386A (en) * | 2019-03-19 | 2019-06-25 | 浙江工商大学 | A kind of biology composite preservative, preparation method and its application in sturgeon caviar |
| CN110063360A (en) * | 2019-04-18 | 2019-07-30 | 浙江工商大学 | A kind of biology composite preservative, preparation method and its application in sturgeon surimi product |
| CN110063360B (en) * | 2019-04-18 | 2021-11-16 | 浙江工商大学 | Biological composite preservative, preparation method thereof and application thereof in sturgeon minced fillet products |
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