CN104611255A - High cohesion pediococcus pentosaceus and use thereof in purifying of vibrio parahaemolyticus in water body - Google Patents

High cohesion pediococcus pentosaceus and use thereof in purifying of vibrio parahaemolyticus in water body Download PDF

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CN104611255A
CN104611255A CN201410780421.0A CN201410780421A CN104611255A CN 104611255 A CN104611255 A CN 104611255A CN 201410780421 A CN201410780421 A CN 201410780421A CN 104611255 A CN104611255 A CN 104611255A
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pediococcus pentosaceus
vibrio parahemolyticus
water body
vibrio parahaemolyticus
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杨振泉
高璐
饶胜其
靳彩娟
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Yangzhou University
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Abstract

The invention belongs to the technical field of microorganism, and in particular relates to high cohesion pediococcus pentosaceus screened from fermented bean products and use thereof in purifying of vibrio parahaemolyticus in water body. The pediococcus pentosaceus F28-8 is preserved in China General Microbiological Culture Collection Center in 2014 November 13, and the preservation number is CGMCC No.9956. The pediococcus pentosaceus has high cohesion activity, can be used for bacteria-reducing and purifying treatment of vibrio parahaemolyticus in water body to avoid use of a chemical antibacterial agent and antibiotics for water bacteria-reducing and purifying treatment, provides new means for breeding and biological control of vibrio parahaemolyticus in temporary rearing water, and is conducive to the prevention of the spread of aquatic animal disease and food borne diseases caused by the vibrio parahaemolyticus.

Description

One plant height coherency Pediococcus pentosaceus and the application in purifying water body in Vibrio parahemolyticus thereof
Technical field
The invention belongs to microbial technology field, be specifically related to the application had in the active Pediococcus pentosaceus of high cohesion and the Vibrio parahemolyticus in purifying water body thereof that a strain filters out from fermented bean products.
Background technology
Vibrio parahemolyticus (Vibrio parahaemolyticus), is distributed widely in coastal ocean and river entrance, and being the important pathogen that sea-food causes food poisoning, is also the important virulence factor of bacteriosis of aquatic livestock.At present, in control hydrocoles vibriosis, reduction water body, the important means of Vibrio parahemolyticus carrying capacity uses microbiotic and chemicals, but cause the problems such as drug tolerant bacteria generation and antibiotic remains thereupon, great effect is caused to food safety, therefore needs the new means of exploitation badly to carry out water body bacteria reducing and purifying treatment.
Summary of the invention
The technical problem to be solved in the present invention is to provide a strain and has the active Pediococcus pentosaceus of high cohesion, can be used for bacteria reducing and the purifying treatment of the Vibrio parahemolyticus in water body, and avoid using chemical fungistat and antibiosis usually to carry out water body bacteria reducing and purifying treatment, for the biological control of cultivation and the temporary Vibrio parahemolyticus supported in water body provides new means, be conducive to the propagation of hydrocoles disease and the food origin disease preventing Vibrio parahemolyticus to cause simultaneously.
The present invention isolates a strain Pediococcus pentosaceus F28-8(Pediococcus pentosaceus from fermented bean products bittern), be deposited in Chinese microorganism strain preservation conservator's common micro-organisms center (CGMCC), address: No. 1, BeiChen West Road, Chaoyang District, BeiJing City No. 3 Institute of Microorganism, Academia Sinica of institute, its culture presevation is numbered: CGMCC No.9956, preservation date is on November 13rd, 2014, the Classification And Nomenclature of bacterial classification is Pediococcus pentosaceus, Pediococcus pentosaceus.
Pediococcus pentosaceus F28-8 in the present invention has following biological characteristics:
(1) morphological specificity: Pediococcus pentosaceus F28-8 gramstaining result is positive, and cell is spheroidal, paired or tetrad arrangement, without brood cell, without pod membrane, atrichia.
(2) colony characteristics: Pediococcus pentosaceus F28-8 is good at MRS cultured on solid medium, colonial morphology is circle, oyster white, smooth, protruding, and neat in edge is opaque.
(3) physiological and biochemical property: negative catalase, edwardsiella hoshinae, hydrogen sulfide and ammonia, do not reduce nitrate, be not hydrolyzed arginine, can glucose fermentation, nonfermented sorbose, melibiose, Xylitol, sucrose, lactose, melizitose, pectinose, wood sugar, rhamnosyl, raffinose, sorbyl alcohol, N.F,USP MANNITOL; Can from cellobiose, Vitamin C2, Tan sugar, ribose, glucose, semi-lactosi, fructose, seminose fermentation and acid.
(4) to Vibrio parahemolyticus, there is strong coherency and biocidal property.
The present invention isolates Pediococcus pentosaceus strain isolated from the Pediococcus pentosaceus fermented bean products, by measuring it to the antagonistic property of Vibrio parahemolyticus with condense ability altogether, filter out the Pediococcus pentosaceus with high cohesion, and the surface microstructure of the Pediococcus pentosaceus bacterial strain of different cohesive force is observed by Electronic Speculum, measure it to the impact of Vibrio parahemolyticus survival rate pathogenic in water body to investigate it to Vibrio parahemolyticus clean-up effect in water body, specifically comprise the following steps:
(1) bacterial strain recovery is cultivated Pediococcus pentosaceus F28-8 and is inoculated 5 mL MRS liquid nutrient mediums, and the pathogenic Vibrio parahemolyticus ATCC33847 of tdh gene masculine inoculates LBS liquid nutrient medium, for subsequent use.
(2) bacteriostasis measures by agar hole diffusion process, detects its bacteriostasis by measuring the size of Pediococcus pentosaceus bacterial strain cell-free extract to the inhibition zone of Vibrio parahemolyticus on LBS solid plate.
(3) concentration that ability measures the bacteria suspension regulating Pediococcus pentosaceus is condensed altogether, the Pediococcus pentosaceus suspension mixing up concentration is respectively got with corresponding Vibrio parahemolyticus bacteria suspension and mixes, measure light absorption value, be denoted as Amix, with the Vibrio parahemolyticus bacteria suspension of unmixed Pediococcus pentosaceus in contrast, according to following formula: { [(Ap+Av)/2]-Amix}/(Ap+Av)/2*100 calculates bacterium cohesion rate altogether, and wherein Ap and Av is respectively the A value that Pediococcus pentosaceus and Vibrio parahemolyticus bacteria suspension are surveyed at 600 nm places.
(4) the bacteria suspension sample condensing the unmixed Pediococcus pentosaceus of electron microscopic observation absorption self coagulation 2 h of cell carries out scanning electron microscope, the plastc ring sample drawing the Pediococcus pentosaceus and Vibrio parahemolyticus condensing 2 h altogether carries out scanning electron microscope, the structure of difference observation of cell and cohesion feature.
(5) in agglomeration process, Vibrio parahemolyticus survivaling cell measures by Pediococcus pentosaceus and Vibrio parahemolyticus plastc ring after 37 DEG C of mixing quiescent culture 2 h, 12h, 24h, 60h, 96h, respectively pipette samples at the middle and upper levels with lower floor solution by the bacterial concentration in selectivity TCBS agar plate mensuration system.
The invention has the beneficial effects as follows:
Pediococcus pentosaceus (Pediococcus pentosaceus), be a kind in milk-acid bacteria, being distributed widely in the traditional fermented food such as vegetables, cheese, sausage, is the generally acknowledged microorganism with security.This bacterium metabolize sugars produces lactic acid, and produce IIa class pediocin, strong retarding effect is produced to pathogenic bacteria and spoilage microorganisms, some of them bacterial strain has the probiotic properties such as invasion and attack improving natural animal immunological competence, promotion health and opposing pathogenic bacteria, to control and the biological control of vibriosis has potential value and bright prospects with Pediococcus pentosaceus and meta-bolites research and development new bacteriostatic agent thereof and probiotics to vibrios purification in food and environment.
Pediococcus pentosaceus F28-8 of the present invention has stronger bacteriostasis property to pathogenic Vibrio parahemolyticus, condenses ability altogether, and its bacteriostatic activity is all insensitive to hydrogen oxide enzyme, proteolytic enzyme and hot conditions, therefore can applied range.Electronic Speculum shows this bacterium and has unique structure of cell surface and cell aggregation mode, is easy to self coagulation and forms large cluster, and is formed with Vibrio parahemolyticus and be total to condensation product closely, thus to the bacteria reducing of the Vibrio parahemolyticus in water body and purifying treatment.Vibrio parahemolyticus in water body after the common cohesion of Pediococcus pentosaceus F28-8, can all significantly reduce by culturing cell in upper strata suspension and condensation product.
Accompanying drawing explanation
Fig. 1: the scanning electron microscope result of different coherent Pediococcus pentosaceus cell.
Fig. 2: Pediococcus pentosaceus condenses altogether can the impact of culturing cell number on Vibrio parahemolyticus in water body.
Embodiment
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, embodiment of the present invention used medium and test conditions are this area conventional medium and test.Unless stated otherwise, embodiment of the present invention agents useful for same is commercial.
the recovery of embodiment 1 bacterial strain is cultivated
Pediococcus pentosaceus conservation liquid is thawed in 0 DEG C, aseptic absorption 200 μ L conservation liquid is inoculated in the MRS liquid nutrient medium of 10 mL, 24 h are cultivated under 37 DEG C of anaerobic conditions, get culture streak inoculation on MRS solid plate, at 37 DEG C of Anaerobic culturel 24 h, then picking list colony inoculation is in the MRS liquid nutrient medium of 5 mL, cultivates 24 h under 37 DEG C of anaerobic conditions, for subsequent use.
The pathogenic Vibrio parahemolyticus ATCC33847 of tdh gene masculine is inoculated in LBS liquid nutrient medium, at 37 DEG C of shaking culture 16 h; Get culture and line TCBS flat board, cultivate 20 h, picking list colony inoculation 5 mL LBS liquid nutrient medium at 37 DEG C, under 37 DEG C of constant temperature oscillation conditions, cultivate 12 h, for subsequent use.
embodiment 2 bacteriostasis measures
Pediococcus pentosaceus F28-8, Y27-4, Y45-30, H13, the bacteriostasis comparative determination of H6, H10 and F3:
Respectively by centrifugal for Pediococcus pentosaceus culture 12000g 15 min under 70 DEG C, 100 DEG C, 121 DEG C differing tempss, get supernatant liquor, degerming by the millipore filtration of 0.22 μm, obtain Pediococcus pentosaceus culture cell-free extract.The bacteriostasis of agar hole diffusion process to bacterial strain cell-free extract is used to measure: by stroke-physiological saline solution, the bacteria suspension of Vibrio parahemolyticus to be diluted to 10 8cFU/mL, pipette 1 mL bacteria suspension in LBS solid plate, coating evenly, 15 min are dried in Bechtop, in flat board, evenly beat the aperture that diameter is 10 mm with punch tool, every hole adds 200 μ L Pediococcus pentosaceus culture cell-free extracts and by 9 mg/mL catalases, 1 mg/mL Proteinase K and 1 mg/mL trypsin treatment.The bacterial strain of process at three different conditions 70 DEG C, 100 DEG C, 121 DEG C is respectively done respectively 3 holes parallel, room temperature spreads 5 h, overnight incubation at being then positioned over 37 DEG C, carries out measuring and recording antibacterial circle diameter with vernier callipers.By MRS liquid nutrient medium with newborn acid for adjusting pH to identical with the pH of strain subject cell-free extract in contrast, and compare the fungistatic effect of different treatment group.
The meta-bolites of 7 strain Pediococcus pentosaceuss in this research trial all show strong retarding effect to pathogenic Vibrio parahemolyticus ATCC33847, antibacterial circle diameter is between 21.0 mm to 25.5mm, be 1.1 to 1.3 times of the contrast of same pH lactic acid, wherein F28-8 has the strongest bacteriostatic action.Culture supernatant fungistatic effect after catalase, trypsinase, Proteinase K and heat treated has reduction in various degree, but residual fungistatic effect is still between 75-96%, result shows that organic acid that Pediococcus pentosaceus F28-8 metabolism produces is the principal element to the effect of Vibrio parahemolyticus growth-inhibiting, hydrogen peroxide produce and the protein-based antipathogenic composition effect such as bacteriocin relatively little.
embodiment 3 is condensed ability altogether and is measured
Pediococcus pentosaceus F28-8, Y27-4, Y45-30, H13, the common cohesion ability comparative determination of H6, H10 and F3:
By centrifugal for Pediococcus pentosaceus strain culture 5000g 10 min, collect thalline, with brine twice, by physiological saline adjustment strain subject suspension concentration, make its light absorption value under 600 nm wavelength be about 0.4, i.e. A600=0.4.The Pediococcus pentosaceus suspension and corresponding Vibrio parahemolyticus bacteria suspension that mix up concentration are respectively got 2 mL to be mixed in 15 mL test tube with a scale, vortex 120s, 37 DEG C leave standstill 2 h, draw 1 mL upper solution and measure light absorption value under 600 nm, be denoted as Amix, parallel 3 pipes that do of each mixed solution repeat, and not add the Vibrio parahemolyticus bacteria suspension of Pediococcus pentosaceus in contrast, calculate bacterium cohesion rate altogether.Bacterium cohesion rate is altogether according to following formula: { [(Ap+Av)/2]-Amix}/(Ap+Av)/2*100 calculates, and wherein Ap and Av is respectively the A value that Pediococcus pentosaceus and Vibrio parahemolyticus bacteria suspension are surveyed at 600 nm places.
Test-results shows that Pediococcus pentosaceus has the ability that pathogenic Vibrio parahemolyticus assembles that obviously promotes, but ability of condensing altogether has significant strain differences.The common cohesion rate of different Pediococcus pentosaceus and Vibrio parahemolyticus ATCC33847 is between 14.8-37.6%, the size order of cohesion rate is altogether: F28-8, Y27-4 > Y45-30>H13, H6, H10> F3, all bacterial strains be all significantly higher than do not add Pediococcus pentosaceus control group ( p<0.01), wherein F28-8 and Y27-4 cohesion rate is altogether the highest, be respectively 37.5% and 36.9% be significantly higher than other bacterial strain ( p<0.01), thus for the purification of Vibrio parahemolyticus in water body and bacteria reducing process provide candidate strain.
embodiment 4 condenses the electron microscopic observation of cell
Pediococcus pentosaceus bacterial strain F28-8 and H13 is to the common cohesion comparative determination of Vibrio parahemolyticus ATCC33847:
The Vibrio parahemolyticus bacteria suspension sample drawing the unmixed Pediococcus pentosaceus of self coagulation 2 h carries out scanning electron microscope, the plastc ring sample drawing Pediococcus pentosaceus F28-8 or H13 and Vibrio parahemolyticus condensing 2 h altogether carries out scanning electron microscope, the structure of difference observation of cell and cohesion feature, step is as follows: the lower floor solution 1mL of absorption self coagulation or altogether cohesion bacteria suspension sample, and the glutaraldehyde solution adding equal-volume 5% fixedly spends the night; Distilled water washs 3 times, each 15min; With 50%, 70%, 80%, 90%, 100% Gradient elution using ethanol; Ethyl acetate and dehydrated alcohol equal proportion mixing effect 30 min, centrifugally abandon supernatant; Ethyl acetate effect 30 min, centrifugally abandons supernatant; Precipitation is resuspended in ethyl acetate solution, and sample adds in point sample dish, dries; Plated film, upper sem observation.
Test-results as shown in Figure 1, the somatic cells of high coherency bacterial strain F28-8 is bonded together firmly, be easy to form large cenobium, see accompanying drawing 1(A), and low coherency bacterial strain H13 cell compares dispersion, only have a small amount of cell adhesion at one piece, see accompanying drawing 1(B), cenobium large in the visual field is comparatively rare.In cell aggregation mode, H13 cell surface smoother, without raised structures, iuntercellular connects mainly through tetrad or diad four end, easily forms chain or sheet, and F28-8 cell surface is more coarse, be attached with raised structures, cell aggregation is not cohered by means of only four ends, and is linked by side superposition, form large three-dimensional cluster, see accompanying drawing 1(C), (D).The common condensation product scanning result of bacterial strain F28-8 and H13 to Vibrio parahemolyticus ATCC33847 is shown in accompanying drawing 1(E), (F).In common condensation product, F28-8 makes ATCC33847 closely be deposited in together, and the agglutinator compacted mass of formation, is shown in accompanying drawing 1(E), it is then more open that H13 condenses ATCC33847 cell, sees accompanying drawing 1(F), be easy to Eddy diffusion.Result shows that the common cohesion ability of Pediococcus pentosaceus to Vibrio parahemolyticus is relevant with the surface tissue of bacterial strain and the tightness degree of cell condensation.Early stage report think that different genera milk-acid bacteria urgees cohesion to pathogenic bacterium may be relevant with albumen with the polysaccharide of cell surface, but the difference mechanism of planting short cohesion ability between interior bacterial strain is still not clear at present, this research, by contrast Pediococcus pentosaceus high coherency bacterial strain and low coherency strain cell form, shows that bacterial strain surface microstructure and adhesive aggregation mode diversity may cause the short cohesion ability opposite sex.
in embodiment 5 agglomeration process, Vibrio parahemolyticus survivaling cell measures
Vibrio parahemolyticus survivaling cell comparative determination in Pediococcus pentosaceus bacterial strain F28-8 and H13 agglomeration process:
Condensing measuring method altogether according to milk-acid bacteria and pathogenic bacterium, to prepare initial concentration be Pediococcus pentosaceus suspension 9.1Log 10cFU/mL and Vibrio parahemolyticus 5.5 Log 10the Pediococcus pentosaceus of CFU/mL and Vibrio parahemolyticus plastc ring, not add the Vibrio parahemolyticus bacteria suspension of Pediococcus pentosaceus in contrast, after Pediococcus pentosaceus and Vibrio parahemolyticus plastc ring leave standstill 2 h, 12h, 24h, 60h, 96h at 37 DEG C, respectively pipette samples at the middle and upper levels with each 1 mL of lower floor's solution, dull and stereotyped with coating TCBS after stroke-physiological saline solution 10 times dilution, each extent of dilution is got 200 μ L and is coated with 3 flat boards, cultivates 24 h by dull and stereotyped for TCBS in 37 DEG C.Cultivate and terminate the extent of dilution of rear selection colony number between 30-300, with bacterial concentration in the mean value calculation system of colony number on 3 flat boards.
Test-results as shown in Figure 2, can slowly decline with the prolongation of storage period by culturing cell number in control group, and through bacterial strain F28-8 and H13 altogether agglomeration process all significantly exacerbate in system can the decline of culturing cell number, see accompanying drawing 2, accompanying drawing 2(A), (B) to be respectively initial concentration be 9.1 Log 10cFU mL -1vibrio parahemolyticus condense altogether in rear upper strata suspension and lower floor's condensation product can the change of culturing cell number; Accompanying drawing 2(C), (D) to be respectively initial concentration be 5.5 Log 10cFU mL -1vibrio parahemolyticus upper strata suspension and lower floor's condensation product can the change of culturing cell number; In figure, data point is 3 reproducible results, is expressed as mean+SD.
Initial concentration is 9.1 Log 10cFU mL -1vibrio parahemolyticus bacteria suspension through F28-8 cohesion 2 h, in the suspension of upper strata can culturing cell 1.8 Logs lower than control group 10cFU mL -1, at 24 h lower than control group 2.7 Log 10cFU mL -1, decline at most, lower than contrast 3.1 Log at effect 60 h 10cFU mL -1; And low cohesion bacterial strain H13 acts on 2 h, 24 h, and respectively lower than control group 0.9,2.0 and 2.5 Log after 60 h 10cFU mL -1, bacteria reducing Be very effective is lower than F28-8( p<0.05), but after 96 h the bacteria reducing effect of F28-8 and H13 do not have significant difference ( p>0.05), all lower than contrast 2.6 Log 10cFU mL -1, see accompanying drawing 2(A).Initial concentration is 5.5 Log 10cFU mL -1vibrio parahemolyticus bacteria suspension result also in through Pediococcus pentosaceus process can the remarkable minimizing of culturing cell, downtrending is consistent with high density suspension, but bacteria reducing effect is more weak, and after effect 24h, F28-8 treatment group reduces 1.3 Log than control group 10cFU mL -1, and H13 process reduces 0.8 Log 10cFU mL -1, see accompanying drawing 2(C).Also remarkable in control group to the residual cell number in the analytical results display agglutinator of the Vibrio parahemolyticus that (comprises agglutinator) in lower floor's suspension, high density suspension significantly lower than contrast, is shown in accompanying drawing 2(B after 12 h); And lower concentration suspension is remarkable in control group after 60 h, see accompanying drawing 2(D).
Result to show that in common coacervated system Pediococcus pentosaceus can not only make the Vibrio parahemolyticus cell be suspended in water body be bonded together to form large flocs unit to be removed by sedimentation by cohesion altogether, and in flocs unit, Pediococcus pentosaceus also can produce antibacterial substance and causes Vibrio parahemolyticus inactivation, reaches the bacteria reducing effect of Vibrio parahemolyticus in water body.Although the antibacterial and germicidal action of the meta-bolites that milk-acid bacteria is formed in the medium has been reported in early-stage Study, but due to nutraceutical restriction in water surrounding, the meta-bolites kind that milk-acid bacteria produces and limited amount, the antibacterial substance being beneficial to milk-acid bacteria generation that is formed of agglutinator closely plays a role, and Pediococcus pentosaceus may be the important channel playing germicidal action in water body environment by cohesion reduces intercellular operating distance altogether.

Claims (2)

1. a strain Pediococcus pentosaceus F28-8(Pediococcus pentosaceus), be deposited in Chinese microorganism strain preservation conservator's common micro-organisms center on November 13rd, 2014, deposit number is CGMCC No.9956.
2. the application in the Vibrio parahemolyticus of bacterial strain described in claim 1 in purifying water body.
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CN106190894A (en) * 2016-07-12 2016-12-07 汕头大学 One strain Pediococcus pentosaceus G11 and screening with application
CN108373983A (en) * 2018-03-15 2018-08-07 江南大学 Pediococcus pentosaceus CCFM1012, its fermented food and its application in preparing antagonism C. jejuni infec-tion drug
CN109504637A (en) * 2018-12-29 2019-03-22 福建省农业科学院农业工程技术研究所 One plant of Pediococcus pentosaceus and its application

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Cited By (7)

* Cited by examiner, † Cited by third party
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CN106148231A (en) * 2016-07-12 2016-11-23 汕头大学 One strain enterococcus faecalis Y17 and screening and culturing thereof and application
CN106190894A (en) * 2016-07-12 2016-12-07 汕头大学 One strain Pediococcus pentosaceus G11 and screening with application
CN106148231B (en) * 2016-07-12 2019-09-13 汕头大学 One plant of enterococcus faecalis Y17 and its screening and culturing and application
CN106190894B (en) * 2016-07-12 2019-09-13 汕头大学 One plant of Pediococcus pentosaceus G11 and its screening and application
CN108373983A (en) * 2018-03-15 2018-08-07 江南大学 Pediococcus pentosaceus CCFM1012, its fermented food and its application in preparing antagonism C. jejuni infec-tion drug
CN109504637A (en) * 2018-12-29 2019-03-22 福建省农业科学院农业工程技术研究所 One plant of Pediococcus pentosaceus and its application
CN109504637B (en) * 2018-12-29 2021-10-08 福建省农业科学院农业工程技术研究所 Pediococcus pentosaceus and application thereof

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