CN101560491B - Luminous bacteria and application thereof in detecting general biological toxicity in food or water sample - Google Patents
Luminous bacteria and application thereof in detecting general biological toxicity in food or water sample Download PDFInfo
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Abstract
The invention discloses luminous bacillus CCTCC M 208027 and application thereof in detecting general biological toxicity in a food or water sample. The invention also discloses a kit that contains luminous bacillus CCTCC M 208027 and is capable of detecting general biological toxicity in the food or water sample.
Description
Technical field
Poisonous substance detection range of the present invention relates in particular to a kind of photogenic bacterium and the application in food or the detection of water sample general biological toxicity thereof.
Background technology
Food-safety problem is a problem that the mankind possibly ignore never.Along with improving constantly of living standards of the people; The food origin disease that hazardous and noxious substances contaminated food productss such as the toxin that heavy metal, pesticide residue, microbiotic and hormone, rotten food produce, wrapping material migration thing cause has become the emphasis that current society is concerned about food-safety problem.Cause the reason of venomous injurant confrontation food contamination to have a lot, existing foeign element is as spraying insecticide, add in the food processing process auxiliary material etc. in the agricultural-food production process; Internal factor is arranged, as chemical reaction takes place in the food processing process again; Also have production environment pollution factors such as water, air, soil.
Traditional food safety monitoring method is main with instrumental analysis such as gas phase, liquid chromatography and spectrophotometers; These methods can detect the contaminant trace species in the sample; Have characteristics such as detectability is low, tolerance range height; But exist complicated operation, long, cost problem of higher of time, especially unsuitable to the field monitoring in the foodstuff production process of circulation, also can't carry out the assessment of biological hazard property to pollution substance.Some traditional method for quick that are the basis with biological chemistry, immunology, molecular biology principle generally all are to detect to known contaminants in food, for principal component is then not powerless mostly.Need the kind and the component of detection a lot of in the food, molecular structure is complicated, often is difficult to set about.The Preliminary detection method sensitivity of total hazardous and noxious substances is low not strong with operability in some food of having set up.The for example acute and long term toxicity test of rat, if the detection that is used for the food hazardous and noxious substances not only the cycle longer relatively, and it is also too high to detect cost.
The biosensor analysis technology is compared advantages such as having selectivity is good, highly sensitive, analysis speed is fast, cost is low, the online detection of ability with traditional detection method, it has become the important development trend of food safety detection as a kind of detection means.Common Biological Detection system to hazardous and noxious substances total in the food comprises Mammalss such as rat, mouse at present, and fish etc. hang down waits vertebrates, mikrobes such as zooblast and bacterium, yeast etc.To use unicellular microorganism bacterium or yeast method the easiest, be easy to realize high flux screening in all these detection architecture, and the detection cost also is minimum usually to a large amount of samples.But present bacterium or yeast detection system iff detect the influence of hazardous and noxious substances for its speed of growth, can omit a large amount of poisonous and harmful information.Though microbiological test method is because of it is quick, sensitive, the economic dispatch characteristics are able to develop rapidly; But its defective is to satisfy the needs that environmental quality real-time prediction early warning, burst poisonous substance leaked emergency disposal, the on-line monitoring of pollution treatment facility etc., and microbiological sensor meets above-mentioned requirements.
The typical case represents photogenic bacterium application in bio-toxicity detects the most extensive in the microbiological sensor.Photogenic bacterium be one type can visible emitting under normal physiological condition heterotrophic organism, its principle of luminosity is that cell utilizes FMNH2 (FMNH under the normal physiological situation
2), long-chain fat aldehyde (RCHO) is substrate, in the presence of oxygen and bacterial luciferase, can send the visible light that wavelength is 420-670nm.When bad or Toxic existed when envrionment conditions, because bacteriofluorescein enzymic activity or cellular respiration are suppressed, luminous power was affected and weakens, and it weakens the big or small and concentration proportion relation of toxicity of degree and poisonous substance.Utilize this characteristics of luminescence, photogenic bacterium can produce luminous receiving to multiple poisonous substance simultaneously and press down reaction, and pre-treatment is simple, and is with low cost, in qualitative, semiquantitative field quick detection, shown its advantage gradually.Along with the development of foodstuffs industry, adopt the photogenic bacterium method to detect food safety as a kind of prescreening method fast, get more and more people's extensive concerning gradually.
When carrying out the hazardous and noxious substances detection with photogenic bacterium usually, need to add certain density salt concn in the detection architecture to guarantee the luminous power of photogenic bacterium.The result of study of domestic and international existing ocean photogenic bacterium shows: when detecting with it, the final concentration that needs in the sample to add salt ion is at least 2-3%, just can make photogenic bacterium that hazardous and noxious substances in the sample is made response preferably.But consider from application point of view, when detecting with the photogenic bacterium method, the adding to join and influence the character of detected sample itself and change of high salt concentration like this, thus verity and safety that its toxicity is assessed throw doubt upon.Generally speaking, comparatively harsh to the requirement of temperature when utilizing photogenic bacterium to carry out the hazardous and noxious substances detection, all will be near the optimum growth temperature of bacterium, obviously, this will reduce its scope of application.
Therefore this area presses for a kind of new photogenic bacterium, not only can utilize it to carry out the hazardous and noxious substances detection under the high salt concentration condition, and under low salt concn, also can detect sample, and have higher susceptibility; And can under awide temperature range, play a role, thereby assess the toxicity of testing sample more effectively, truly, and widen the scope of application of test sample, have more profound application prospect.
Summary of the invention
The present invention aims to provide a kind of photogenic bacterium.
Another object of the present invention provides the detection method of general biological toxicity in a kind of food or the water sample.
A further object of the present invention provides a kind of test kit that detects general biological toxicity in food or the water sample.
In first aspect of the present invention, a kind of mikrobe CCTCC M 208027 (Photobacteriumsp.) is provided, it has following characteristic:
(i) thalline is rod-short;
(ii) bacterium colony be creamy white, translucent;
(iii) bacterium colony and bacterium liquid all can send blue green light.
In second aspect of the present invention, the purposes of a kind of aforesaid mikrobe is provided, described mikrobe is used for detecting food or water sample general biological toxicity.
In the third aspect of the invention, the detection method of general biological toxicity in a kind of food or the water sample is provided, described method comprises step:
(1) aforesaid mikrobe is mixed with food to be measured and blank article respectively, obtain testing mixture and negative control mixture respectively;
(2) detect the testing mixture luminous value, with negative control mixture luminous value relatively, the acquisition luminous intensity inhibition ratio (Inhibition Rate, IR);
(3) if luminous intensity inhibition ratio >=50% explains that then product to be tested is poisonous.
In another preference, described IR can through (but being not limited to) following formula calculate luminous intensity inhibition ratio (Inhibition Rate, IR):
In another preference, contain salt in described testing mixture and/or the negative control mixture, said salt concn is 0.1-6.0w/v%; Described salt particular certain cancers.
In another preference, said testing mixture and/or negative control mixture pH6-8.
In another preference; It also uses the positive control mixture; Contain positive reference substance, aforesaid mikrobe and salt in the said positive control mixture; Salt concn is 0.1-6.0w/v% in the said positive control mixture, and the luminous intensity inhibition ratio of said positive control mixture equals 50%; Described salt particular certain cancers.
In another preference, described blank article are selected from deionized water or nontoxic food.
In another preference, described positive reference substance is the toxic substance standard substance, includes but not limited to mercury chloride, SRM 935a, copper sulfate etc.
In another preference, described food is selected from cereal, fruit, vegetables, meat poultry or marine food; Described water sample is selected from tap water, sewage or irrigation water.
In another preference, said detection can be carried out at 1-37 ℃; More preferably 5-30 ℃.
In fourth aspect of the present invention, general biological toxicity detection kit in a kind of food or the water sample is provided, described test kit comprises aforesaid mikrobe, blank article, positive reference substance, salts solution and damping fluid; The preferred luminous bacillus CCTCC of described mikrobe M 208027 lyophilized powders.
In another preference, described blank article are selected from deionized water or nontoxic food.
In another preference, described positive reference substance is the toxic substance standard substance, includes but not limited to mercury chloride, SRM 935a, copper sulfate etc.
In another preference, salt concn is 0.05-50w/v% in the described salts solution, and preferred concentration is 0.1-25w/v%; Salt particular certain cancers in the described salts solution.
In another preference, described damping fluid is the damping fluid of pH5-9, and the damping fluid of preferred pH6-8 includes but not limited to: Tris damping fluid, phosphoric acid buffer etc.
In view of the above, the invention provides a kind of new photogenic bacterium, not only can utilize it to carry out the hazardous and noxious substances detection under the high salt concentration condition, and under low salt concn, also can detect, and have higher susceptibility sample; And can under awide temperature range, play a role, thereby assess the toxicity of testing sample more effectively, truly, and widen the scope of application of test sample, have more profound application prospect.
Description of drawings
Fig. 1 has shown the difference of the different photogenic bacterium luminous intensities that obtained among the embodiment 1.
Fig. 2 has shown luminous bacillus CCTCC M 208027 bacterium colony luminous photos.
Fig. 3 has shown luminous bacillus CCTCC M 208027 nutrient solution luminous photos.
Fig. 4 has shown the micro-thalline photo of luminous bacillus CCTCC M 208027.
Fig. 5 has shown that 208027 pairs of simulations of luminous bacillus CCTCC M mix the toxic rapid detection effect of toxicity population of samples.
Fig. 6 has shown the response of heavy metal Hg in 208027 pairs of different samples of luminous bacillus CCTCC M (water and apple); Wherein " ppm " is the concentration unit of the heavy metal Hg that added; Sucus Mali pumilae-CK representes nontoxic apple.
Fig. 7 has shown the response of fenvalerate in 208027 pairs of different samples of luminous bacillus CCTCC M (water and apple); Wherein " ppm " is the concentration unit of the fenvalerate that added; Sucus Mali pumilae-CK representes nontoxic apple.
Fig. 8 has shown the response of agricultural chemicals 1605 in 208027 pairs of different samples of luminous bacillus CCTCC M (water and green vegetables); Wherein " ppm " is the concentration unit of the agricultural chemicals 1605 that added; Green vegetables-CK representes nontoxic green vegetables.
Fig. 9 has shown the response of heavy metal Hg in 208027 pairs of different samples of luminous bacillus CCTCC M (water and flour); Wherein " ppm " is the concentration unit of the heavy metal Hg that added; Flour-CK representes nontoxic flour.
Figure 10 has shown the response of agricultural chemicals 1605 in 208027 pairs of different samples of luminous bacillus CCTCC M (water and flour); Wherein " ppm " is the concentration unit of the agricultural chemicals 1605 that added; Flour-CK representes nontoxic flour.
Figure 11 has shown NO in 208027 pairs of different samples of luminous bacillus CCTCC M (water and pork)
2-Response; Wherein " ppm " is the NO that is added
2-Concentration unit; Pork-CK representes nontoxic pork.
Figure 12 has shown the comparable situation of 208027 pairs of heavy metal Hg responses of different detected temperatures luminous bacillus CCTCC M; Wherein " ppm " is the concentration unit of the heavy metal Hg that added, and 10 ℃, 15 ℃, 25 ℃ and 30 ℃ is bath temperature, and 20 ℃ is room temperature.
Embodiment
The contriver is through extensive and deep research; Be surprised to find a kind of photogenic bacterium; Be a kind of of Photobacterium Photobacterium sp.; This bacterial strain is preserved in Chinese typical culture collection center (China Center for Type Culture Collection is called for short CCTCC), numbering M 208027 on February 29th, 2008.
Further, the contriver find to use this bacterial strain in a wide in range salt concn scope to sample in the sensitivity of general biological toxicity when detecting all very high; And utilize bacterial strain provided by the invention in comparatively wide in range TR, to obtain good detection effect.
Particularly, described salt concn scope is meant that the final concentration of salt in detection architecture is 0.1-6.0w/v%, preferably is 0.5-3.5w/v%, more preferably is 0.8-3.0w/v%; Described detected temperatures scope is 1-37 ℃, preferably is 5-30 ℃.
As used herein; " acute toxicity (acute toxicity) " be meant people or laboratory animal (like mouse, rat, rabbit etc.) once or in 24 hours repeatedly contact tried the poisonous effect that causes behind the thing; Even it is dead; The result is many with half-inhibition concentration (50%inhibition concentration, IC
50) expression.
As used herein, " bio-toxicity (biotoxicity) " is meant a certain or several physical signs with mikrobe as indication (like cell growth, breathing, enzymic activity etc.), and the degree that influences these indications according to determinand is judged toxic intensity.
As used herein, " medium effective concentration (50%effective concentration, EC
50) " or " half-inhibition concentration (50%inhibition concentration, IC
50) " all be evaluation index or the value that adopts during bio-toxicity in assessment, promptly calculate influence or suppress the required testing concentration of certain normal physiological desired value of mikrobe 50%.
As used herein; " luminous bacillus CCTCC M 208027 detected temperatures " are meant when utilizing 208027 pairs of hazardous and noxious substances of luminous bacillus CCTCCM to detect; The temperature of detection system contains determinand and luminous bacillus CCTCC M 208027 when measuring luminous value in the said detection system.
Luminous bacillus CCTCC M 208027 provided by the invention has following characteristic:
1) thalline is rod-short;
2) bacterium colony be creamy white, translucent, be prone to picking;
3) bacterium colony and the bacterium liquid blue green light that all can send;
4) be in the bacterium of logarithmic phase, its luminous value can reach 2000-3000 ten thousand RLU (relative luminous intensity)/100ul.
5) 0.1-6.0w/v% (preferably being 0.5-3.5w/v%, more preferably is 0.8-3.0w/v%) in wide in range salt concn scope is comparatively sensitive (like the IC to various heavy (Hg, Cu, and/or Pb) to the response of multiple hazardous and noxious substances
50Can reach below the 4ppm).
A preferred method that obtains luminous bacillus CCTCC M 208027 provided by the invention is:
To take from abyssal ooze, and place basic medium, and in 25-35 ℃, cultivate 2-4 days for 100-400 rev/min, the inoculum size with 1% is linked in the identical substratum, goes down to posterity 3-5 time.Get 0.5ml nutrient solution gradient dilution then, get 10
-4-10
-7Each 0.1ml of dilution liquid coats on the basic medium flat board, cultivates after 2 days for 25-35 ℃, carries out the darkroom and observes, and picking sends the bacterium colony of fluorescence.And respectively it is inoculated in the basic salt culture medium test tube, in 25-35 ℃, 100-400 rev/min of shaking table cultivated 10-20 hour, detected its illumination effect with Chemiluminescence Apparatus.
In sepn process, detect the photogenic bacterium that 7 strains have different luminous powers, wherein a strain luminous power is superpower, and detected luminous value just can reach 2,200 ten thousand RLU/s when cultivating for the first time, and with its called after LuB-1.Genomic dna with this bacterial strain is a template; Utilize bacterial 16 S rRNA universal primer to carry out pcr amplification, obtain the amplified production that length is about 1.5kb, sequencing result is comparison result on GenBank; And combine its physio-biochemical characteristics; According to uncle Jie Shi handbook, various features of this bacterium and luminous bacillus Photobacterium sp. degree of agreement are higher, tentatively it are accredited as Photobacterium sp..
Luminous bacillus CCTCC M 208027 provided by the invention can process lyophilized powder through ordinary method, and a kind of preferable methods is with being cultured to logarithmic phase luminous bacillus CCTCC M 208027 nutrient solutions in mid-term, 4 ℃; 10000-15000g/ minute, centrifugal 5-10 minute, abandoning supernatant; With 10-30ml basic culture solution resuspended once (even) with micropipet or the light and slow piping and druming of dropper; Again in 4 ℃, 15000g/ minute, centrifugal 5 minutes; Abandon supernatant, utilize freeze drier to be made into luminous bacillus CCTCC M 208027 freeze-dried vaccine powder.
Described basic medium or basic culture solution can use this area conventional basic medium or basic culture solution, preferred prescription as follows: yeast powder 2-7 grams per liter, peptone 2-7 grams per liter, sodium-chlor 7-25 grams per liter, Repone K 0.2-0.7 grams per liter, calcium chloride 0.5-2 grams per liter, magnesium chloride 1-5 grams per liter, sodium hydrogencarbonate 0.05-0.2 grams per liter, sal epsom 1-4 grams per liter, glycerine 10-30 milliliter/liter.
In the cultivation, the suitable growth temperature of luminous bacillus CCTCC M 208027 is 5-37 ℃, preferably is 10-30 ℃; Its suitable growth pH is 5-9, preferably is 6-8.
When luminous bacillus CCTCC M 208027 was in logarithmic phase, its luminous value can reach 2000-3000 ten thousand RLU (relative luminous intensity)/100ul.
The invention provides a kind of method that detects general biological toxicity in the food; Luminous bacillus CCTCC M208027 is obtained negative control mixture and testing mixture with blank article and food to be measured mixing respectively; Detect the luminous value of negative control mixture and testing mixture then respectively, receive the inhibition situation to judge general biological toxicity situation in the food to be measured through the testing mixture luminous intensity at last.
In a preference of the present invention, the detection method of general biological toxicity is following in the described food:
A, get a pipe luminous bacillus CCTCC M 208027 lyophilized powders, dissolve with the salts solution of 0.1-3.0w/v%, and the mixing that turns upside down, place 5-30 ℃ of water-bath to hatch 5 minutes;
B, with the even bacterium liquid of the soft piping and druming of micropipet, divide with every pipe 0.1ml to install to each detector tube, place immediately to detect on the Chemiluminescence Apparatus and respectively manage the background luminescence value;
C, be but that the sample solution 0.4ml of 0.1-3.0w/v% adds in the detector tube with salt concn; Mixing turns upside down; Place the reaction of 5-30 ℃ of water-bath after 10-30 minute again, detect and each detector tube luminous value of comparing with control group receives the situation that presses down, with the judgement sample bio-toxicity.
Can by following formula calculate luminous intensity inhibition ratio (Inhibition Rate, IR):
In this preferred method, set up 1 blank (feminine gender) contrast (being selected from deionized water or known non-toxic sample) and 1 positive control (mercury chloride) simultaneously; Wherein said salt is selected from sodium salt.
Wherein the luminous intensity inhibition ratio of positive control should be 50%; The result judges that according to the form below carries out:
| IR％ | 30-50 | 50-80 | ＞80 |
| Toxicity | Suspicious | Poisonous | Strong poison |
The present invention provides a kind of test kit that detects general biological toxicity in the food, comprising luminous bacillus CCTCCM 208027 freeze-dried vaccine powder, feminine gender, positive reference substance and damping fluid.
In another preference, also contain salts solution in the described test kit, salt concn is 0.1-6.0w/v% (preferably being 0.5-3.5w/v%, more preferably is 0.8-3.0w/v%); Described salt is selected from sodium salt.
In another preference, the blank article in the said test kit are selected from deionized water or nontoxic food; Positive reference substance is selected from mercury chloride, SRM 935a, copper sulfate etc.
The acute toxicity that test kit provided by the invention adopts luminous bacillus CCTCC M 208027 to carry out sample detects.According to the change situation that adds testing sample front and back bacterial luminescence intensity, judge the level of safety of sample.
Test kit provided by the invention is applicable to that mainly (but being not limited to) carry out the toxic rapid evaluation of overall biology to securities such as food, water quality.Whether contain the hazardous and noxious substances that exceeds standard on the aggregate level in can disposable rapid detection sample, even unknown sample is not needed its toxic ingredient composition, structure, character etc. are made a concrete analysis of yet, individual event detects index, just can carry out result's judgement; It all has higher response to multiclass hazardous and noxious substances such as common heavy metal, agricultural chemicals, multiclass carcinogenss; Lack easy and simple to handle, detection time, can accomplish the detection of a collection of sample in 10-30 minute; Can carry out field quick detection to batch samples, be particularly suitable for the primary dcreening operation of (but being not limited to) water sample and food safety; Also can be used for containing the mensuration of the complex sample of insoluble substance; Equipment is simple, and cost value is low, only needs a Chemiluminescence Apparatus, just can accomplish whole detection demands.
The above-mentioned characteristic that the present invention mentions, or the characteristic that embodiment mentions can arbitrary combination.All characteristics that this case specification sheets is disclosed can with any composition forms and usefulness, each characteristic that is disclosed in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, the characteristic that is disclosed to be merely the general example of equalization or similar features.
Major advantage of the present invention is:
1, CCTCC M 208027 bacterial strains can detect the population of samples bio-toxicity in 0.1-6.0w/v% salt concn scope, particularly under the environment of low salt concn (0.1-3.0w/v%), still can guarantee the sensitivity and the repeatability that detect;
2, CCTCC M 208027 bacterial strains can require more broadly to the salt concn of detected sample, and the Application Areas and the sensing range of detection have been widened in the detection of the several samples that does not wait applicable to salt concn;
3, rapid detection sample, whether assessment contains the hazardous and noxious substances that exceeds standard on the aggregate level; Can improve detection efficiency in batches and hazardous and noxious substances recurrent item greatly; Also can be used as to substitute utilizes Mammals and fish to carry out the method for quick that the hazardous and noxious substances general biological toxicity is judged;
4, unknown sample need not its toxic ingredient composition, structure, character etc. are made a concrete analysis of, individual event detects index, just can carry out result's judgement, time saving and energy savingly significantly reduces cost;
5,, also include, but is not limited to the overall biology toxicity in water quality, the environment is detected except food;
6, the range of application of test kit provided by the invention is wider, and is gentle to the requirement of detected temperatures.
To combine specific embodiment below, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; People such as Sambrook for example; Molecular cloning: the condition described in the laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all per-cent and umber by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Obtain luminous bacillus CCTCC M 208027
To take from marine site, Ganyu, Hai Zhou gulf abyssal ooze 5.00g, and place the 100ml basic medium, and in 30 ℃, cultivate 2 days for 200 rev/mins, the inoculum size with 1% is linked in the identical substratum, goes down to posterity 3 times.Get 0.5ml nutrient solution gradient dilution then, get 10
-4-10
-7Each 0.1ml of dilution liquid coats on the basic medium flat board, cultivates after 2 days for 30 ℃, carries out the darkroom and observes, and picking sends the bacterium colony of fluorescence.And respectively it is inoculated in the basic salt culture medium test tube, in 30 ℃, 200 rev/mins of shaking tables were cultivated 24 hours, detected its illumination effect with Chemiluminescence Apparatus.
Culture medium prescription is:
Yeast powder Yeast extract 5.00g
Peptone Tryptone 5.00g
Sodium chloride nacl 9.00g
Potassium chloride (KCl) 0.50g
Calcium chloride CaCl
2* 2H
2O 1.00g
Magnesium chloride Mg Cl
2* 6H
2O 3.00g
Sodium hydrogencarbonate NaHCO
30.10g
Sal epsom MgSO
4* 7H
2O 2.00g
Glycerine Glycerol 20.00ml
In sepn process, detect the photogenic bacterium that 7 strains have different luminous powers, wherein a strain luminous power is superpower, and detected luminous value just can reach 2,200 ten thousand RLU/s when cultivating for the first time, sees Fig. 1, and with its called after LuB-1.On behalf of the bacterium colony of the growth of photogenic bacterium on culture medium flat plate and nutrient solution thereof, Fig. 2,3 all can send macroscopic bright blue green light respectively.Fig. 4 is the microscopically thalline photo of photogenic bacterium LuB-1, and thalline is rod-short.
Genomic dna with this bacterial strain is a template, utilizes bacterial 16 S rRNA universal primer to carry out pcr amplification, obtains the amplified production that length is about 1.5kb.Sequencing result is comparison result on GenBank, and combines its physio-biochemical characteristics, and according to uncle Jie Shi handbook, various features of this bacterium and luminous bacillus Photobacteriumsp. degree of agreement are higher, tentatively it are identified that it is Photobacterium sp..
Photogenic bacterium LuB-1 is preserved in Chinese typical culture collection center (ChinaCenter for Type Culture Collection is called for short CCTCC), numbering M 208027 on February 29th, 2008.
This luminous bacillus CCTCC M 208027 has following characteristic:
(i) thalline is rod-short;
(ii) bacterium colony be creamy white, translucent;
(iii) bacterium colony and bacterium liquid all can send blue green light.
The preparation of luminous bacillus CCTCC M 208027 lyophilized powders
With being cultured to logarithmic phase luminous bacillus CCTCC M 208027 nutrient solutions in mid-term among the embodiment 1, placing immediately and carry out following operation on the ice-water bath:
4 ℃, 15000g/ minute, centrifugal 5 minutes; Abandoning supernatant is with 10ml basic culture solution resuspended once (even with micropipet or the light and slow piping and druming of dropper), again in 4 ℃; 15000g/ minute; Centrifugal 5 minutes, abandon supernatant, use freeze drier to be made into luminous bacillus CCTCC M 208027 freeze-dried vaccine powder.
The solid state powder of gained is luminous bacillus CCTCC M 208027 lyophilized powder finished products.
Luminous bacillus CCTCC M 208027 is used to mix the bio-toxicity assessment of toxicity analog sample
Test simulation has prepared and has contained multiple hazardous and noxious substances (concentration of every kind of Toxic is IC
50) mixed sample H1, H2, H3; The kind of the hazardous and noxious substances that contains in every kind of sample is respectively 2 kinds, 4 kinds, 6 kinds and (presses table 1 preparation; Kind is added in "+" representative); Simultaneously water sample H1, H2, H3 are carried out concentration gradient dilution (2 times, 4 times, 6 times, 8 times, 10 times), the response condition that detects different samples, different concns sample with the luminous bacillus CCTCC M that obtains among the embodiment 1 208027 respectively then.Suppressing 50% with luminous value is toxicity criterion.The result sees Fig. 6.
Table 1 mixed sample adds the kind of hazardous and noxious substances
| Hg | As | Cr 6+ | Cd | Pb | Cu | |
| H1 | + | + | ||||
| H2 | + | + | + | + | ||
| H3 | + | + | + | + | + | + |
The result shows, is mixed with the toxic substance response of multiple different concns in 208027 pairs of samples of luminous bacillus CCTCC M, has certain adding up or synergistic effect.Along with the kind increase of heavy metal in the biased sample, the diluted sample multiple also increases gradually, can make the luminous value inhibiting rate reach 50% equally.
When the kind of hazardous and noxious substances in the sample was increased to 6 kinds (H3), 10 times of sample solution dilutions can reach 50% luminous inhibiting rate equally.
It is thus clear that this method not only makes the detection sensitivity of single component in 208027 pairs of biased samples of luminous bacillus CCTCC M improve, also can carry out the fast qualitative and the sxemiquantitative assessment of general biological toxicity to containing the sample of different hazardous and noxious substances.
Application-the apple of luminous bacillus CCTCC M 208027 in food safety detection
Take by weighing fresh apple 100g to be detected, be cut into the square fritter of 2cm, with the about fruit juice that can squeeze 50ml of domestic type juicer, initial pH is 5.0.With redistilled water suitably after the dilution, the pH of sample is transferred to 6.0-8.0 with a certain amount of Tris damping fluid.
When apple during as the food safety detection object; Often to occur exceeding standard or residual hazardous and noxious substances (as: heavy metal Hg, draw up worm pyrethrin fenvalerate etc.) is a research object; External source is added a certain amount of heavy metal Hg (Fig. 7) or fenvalerate (Fig. 8) in the good fruit juice of fresh press for extracting juice; Make its final concentration respectively shown in Fig. 7 and 8, intend the preparation of polluting the apple sample, simultaneously with nontoxic apple sample as negative control (being that Sucus Mali pumilae-CK represents nontoxic apple).Detected result comparing when detecting identical toxic substance in the blank water sample with luminous bacillus CCTCC M208027 lyophilized powder.
Luminous bacillus CCTCC M 208027 lyophilized powders that obtain with embodiment 2 detect:
A, get a pipe luminous bacillus CCTCC M 208027 lyophilized powders, dissolve with the NaCl solution of 2.5w/v%, and the mixing that turns upside down, place 10-30 ℃ of water-bath to hatch 5 minutes;
B, with the even bacterium liquid of the soft piping and druming of micropipet, divide with every pipe 0.1ml to install to each detector tube, place immediately to detect on the Chemiluminescence Apparatus and respectively manage the background luminescence value;
C, be but that the sample solution 0.4ml of 2.5w/v% adds in the detector tube with NaCl concentration; Mixing turns upside down; Place 15 ℃ of water-bath reactions after 15 minutes again; Detect and receive but situation, with the judgement sample bio-toxicity with control group (with nontoxic apple through same treatment process) each detector tube luminous value of comparing.
Detected result is seen Fig. 7 and 8.
The result shows no matter the hazardous and noxious substances in the food (apple) is heavy metal or agricultural chemicals, can detect the IC of the hazardous and noxious substances that can in water sample, respond with photogenic bacterium through luminous bacillus CCTCC M 208027 provided by the invention
50Compare, the detection of agricultural chemicals is had similar detection sensitivity, and the response value of heavy metal is wanted more lower slightly, maybe be relevant to the chelate effect of heavy metal ion with food samples.
Application-the green vegetables of luminous bacillus CCTCC M 208027 in food safety detection
Take by weighing fresh last Ulva lactuca L. 100g to be detected (for fear of the interference of pigment to detected result, as far as possible choosing dish stalk part), with the about green vegetables juice that can squeeze 70ml of domestic type juicer, initial pH is 5.5.With redistilled water suitably after the dilution, the pH of sample is transferred to 6.0-8.0 with a certain amount of Tris damping fluid.
External source is added and a certain amount ofly, often to be occurred exceeding standard or residual hazardous and noxious substances (heavy metal Hg, organophosphorus insecticide 1605 (Fig. 9)) during as food safety detection with green vegetables, intends the preparation of polluting the green vegetables sample.
The method that luminous bacillus CCTCC M 208027 lyophilized powders that obtain with embodiment 2 and embodiment 4 provide detects, and when detecting luminous value and receiving to press down situation, NaCl concentration is 0.5w/v%, detected result such as Fig. 9.
Application-the flour of luminous bacillus CCTCC M 208027 in food safety detection
Take by weighing fresh flour 1.00g; Be dissolved in the 10ml redistilled water, concuss is placed on room temperature and left standstill 30 minutes, gets its supernatant respectively at adding certain density hazardous and noxious substances (heavy metal Hg (Figure 10) or agricultural chemicals 1605 (Figure 11)) etc. in the different samples; The method that luminous bacillus CCTCC M 208027 lyophilized powders that obtain with embodiment 2 and embodiment 4 provide detects; When the detection luminous value received to press down situation, the NaCl concentration in the system was 1.5w/v%, detected result such as Figure 10 and Figure 11.
Application-the pork of luminous bacillus CCTCC M 208027 in food safety detection
Take by weighing fresh pork 250g, place clean pallet,, put into-20 ℃ of quick-frozen numbers hour, treat under room temperature, to thaw fully again after sample freezes fully, intend the preparation of contaminated samples with gravy leach liquor (about 10ml) with behind the preservative film parcel.When external source was added a certain amount of pork as food safety detection, the hazardous and noxious substances (heavy metal Hg, the nitrite NO that exceed standard often appearred
2-(Figure 12)).
The method that luminous bacillus CCTCC M 208027 lyophilized powders that obtain with embodiment 2 and embodiment 4 provide detects, and when detecting luminous value and receiving to press down situation, NaCl concentration is 4.8w/v% in the system, detected result such as Figure 12.
The result of embodiment 4-7 shows that luminous bacillus CCTCC M 208027 can be used for detecting the toxicant that the different foods sample exists.
In several foods sample to be detected, the agricultural chemicals that exists in 208027 pairs of apples of luminous bacillus CCTCC M, green vegetables, the flour foods 1605 detects also comparatively responsive, the IC that in water sample, can respond with luminous bacillus CCTCC M 208027
50Concentration difference slightly.
To the detection of flour sample, the response of heavy metal Hg and agricultural chemicals 1605 in 208027 pairs of flour of luminous bacillus CCTCC M, the IC that in water sample, can respond with luminous bacillus CCTCC M 208027
50The concentration basically identical.
The composition that flour sample is described is than fruit or vegetable type food, and microenvironment is simple relatively, detects comparatively sensitive with luminous bacillus CCTCC M208027.And showing that for the detected result of charcuterie pork is as typical animal derived food, the microenvironment of its food is than all complicated many of fruits and vegetables class, cereals, hazardous and noxious substances NO in 208027 pairs of these samples of luminous bacillus CCTCCM
2-Response want more lower slightly.
Comparative Examples 1
Material and method
The T3 microspecies, according to document (photogenic bacterium to the prominent effect of causing of compound with detect research. ACTA Scientiae Circumstantiae, 1993,13 (4): 435-441; The mutagenic effect of the industrial residue that the dark mutation of photogenic bacterium is handled the soil. ACTA Scientiae Circumstantiae, 1994,14 (3): 349-354)) relevant report.It is the sensitiveest (in general, the photogenic bacterium high salt concentration during than low salt concn detection sensitivity high) detection architecture in salt concn be 3w/v%, to the IC of heavy metal Hg, Cu, Pb response
50As shown in table 2.
Luminous bacillus CCTCC M 208027 lyophilized powders that embodiment 2 obtains according to the detection method among the embodiment 4, are respectively 0.6 and 3.0w/v% with the final concentration of salt in the detection architecture (NaCl) respectively.The IC of the response of the 208027 pairs of part hazardous and noxious substances of luminous bacillus CCTCC M that obtain
50The result compares with the result of above-mentioned bibliographical information, and the result sees table 2.
The IC of table 2 luminous bacillus CCTCC M 208027 and T3
50Relatively
The result shows, the toxic action order of 3 heavy metal species pair ion luminous bacillus CCTCC M 208027 with to T3 luminescent bacteria toxicity sequence consensus.Except that Hg, adopt luminous bacillus CCTCC M 208027, the sensitivity of toxotest is significantly improved.The more important thing is, for the part sample relatively responsive as far as salt concn, when using luminous bacillus CCTCC M 208027 to detect, in the detection architecture to the requirement of salt concn well below the T3 microspecies, more help the accuracy that detects; Even and under same salt concn (3w/v%), the detection sensitivity of luminous bacillus CCTCC M 208027 is also more preponderated.
Embodiment 8
The test of luminous bacillus CCTCC M 208027 detected temperatures
Material and method
Luminous bacillus CCTCC M 208027 lyophilized powders that embodiment 2 obtains, according to the detection method among the embodiment 4, the final concentration of NaCl is 1.0w/v% in the detection architecture, carries out hatching and detecting of lyophilized powder at 10 ℃, 20 ℃, 25 ℃, 30 ℃ respectively.
The result sees Figure 13
The result shows; Though the righttest detected temperatures of photogenic bacterium is 15 ℃; But under 10 ℃, 20 ℃, 25 ℃ condition; Even its luminous value all will be lower than 15 ℃, but this does not influence the detection sensitivity of this photogenic bacterium to the hazardous and noxious substances response, and it is at 10-25 ℃ (the being room temperature condition) IC to heavy metal Hg response
50Be 0.5ppm.
Embodiment 9
The preparation test kit
Provide in the test kit:
Luminous bacillus CCTCC M 208027 lyophilized powders that embodiment 2 obtains;
Blank (feminine gender) contrast: deionized water;
Positive control (mercury chloride/or other standard substance);
The sodium salt solution of salts solution: 0.6w/v-6.0w/v%;
Tris damping fluid, testing tube, product description.
Embodiment 10
Luminous bacillus CCTCC M 208027 is used to detect heavy metal cation
Detect the method for half effective concentration (EC50):
A, cultivate the luminous bacillus CCTCC M 208027 of logarithmic phase, OD
600≈ 1.0, and luminous value ≈ 2,000 ten thousand-3,000 ten thousand RLU/s/100ul get 10ml bacterium liquid, and 4 ℃, 7000rpm/ minute, centrifugal 3 minutes, abandoning supernatant, with the dissolving of 10ml 0.2%NaCl salts solution, and the mixing that turns upside down, place 5-30 ℃ of water-bath, subsequent use;
B, different heavy metallic salts are configured to the 10mg/ml mother liquor with sterilized water; Preparation is during test sample, is diluted to each 1ml of detection liquid that contains heavy metal cation of different concns gradient with sterilized water, adds NaCl again; Adjusting its salt concn is 0.9% and 2.0%, supplies to detect to use;
C, with the even bacterium liquid of the soft piping and druming of micropipet, divide with every pipe 0.1ml to install to each detector tube, place immediately to detect on the Chemiluminescence Apparatus and respectively manage the background luminescence value;
D, but the sample solution 0.9ml for preparing is added in the detector tube; Mixing turns upside down; Place the reaction of 5-30 ℃ of water-bath after 10-30 minute again, detect and compare with control group that each detector tube luminous value receives but situation, and calculating is compared with blank; Make luminous value suppress 50% o'clock sample concentration, be the EC that this photogenic bacterium CCTCC M208027 detects this heavy metal cation
50(mg/L).
The result sees table 3.
The EC that 208027 pairs of heavy metal cations of table 3 photogenic bacterium CCTCC M detect
50(mg/L)
| Reagent | The EC of photogenic bacterium CCTCC M 208027 among the 0.9%NaCl 50 | The EC of photogenic bacterium CCTCC M 208027 among the 2%NaCl 50 | Microtox among the 2%NaCl TMEC 50 |
| Mercury | 0.04 | 0.039 | 0.065 a |
| Arsenic | 0.1 | 0.19 | NF |
| Zinc | 2.5 | 1.07 | 2.5 a，17 b，2-49 d |
| Copper | 0.65 | 2.12 | 8 a |
| Plumbous | 0.6 | 0.82 | 11 a，0.6 b |
| Cadmium | 7.5 | 14.37 | 41.4 d，106 e |
| |
20 | 49.89 | 356 b，135-177 e |
NF: do not have
The EC of report or compilation
50: a Bulich and Isenberg, 1981; B Kahru, 1993; C Kaiser and Palabrica, 1991; DBlondin et al., 1987; E Munkittrick et al., 1991.
Embodiment 11
Luminous bacillus CCTCC M 208027 is used to detect organic poison
The method that detects half effective concentration (EC50) is with embodiment 10, wherein b, when test sample prepares, various organic reagents all directly are diluted to the liquid to be detected of different concns gradient with sterilized water; Various agricultural chemicals are configured to the 10mg/ml mother liquor with sterilized water, during the preparation test sample, are diluted to each 1ml of detection liquid that contains agricultural chemicals of different concns gradient again with sterilized water, add NaCl again, and adjusting its salt concn is 0.9% and 2.0%, supplies to detect to use;
The result sees table 4.
The EC that 208027 pairs of organic poisons of table 4 photogenic bacterium CCTCC M detect
50(mg/L)
| Reagent | The EC of photogenic bacterium CCTCC M 208027 among the 0.9%NaCl 50 | The EC of photogenic bacterium CCTCC M 208027 among the 2%NaCl 50 | Microtox among the 2%NaCl TMEC 50 |
| Chloroform | 435 | 1471 | 435 e，736 b |
| Ethanol | 60000 | 66876 | 39285cc |
| DMSO | 200 | 63 | 98360 c |
| Superlysoform | 15 | 44.07 | 8 a |
| Acetone | 20000 | 40369 | 17141 c |
| 1605 (thiophos) | 2.5 | 19.29 | 68.3 c，8.4 c |
| DDT | 50 | 100.91 | 7 c，13.8 c |
| Chlorpyrifos 94 | 46.2 | 94.48 | 46.2 c |
| PP-383 | 0.5 | 7.5 | NF |
NF: do not have
The EC of report or compilation
50: a Bulich and Isenberg, 1981; B Kahru, 1993; C Kaiser and Palabrica, 1991; DBlondin et al., 1987; E Munkittrick et al., 1991.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (8)
1. a luminous bacillus CCTCC M 208027 (Photobacterium sp.), it has following characteristic:
(i) thalline is rod-short;
(ii) bacterium colony be creamy white, translucent;
(iii) bacterium colony and bacterium liquid all can send blue green light.
2. the purposes of a luminous bacillus as claimed in claim 1 is characterized in that, described mikrobe is used for detecting food or water sample general biological toxicity.
3. the detection method of general biological toxicity in food or the water sample is characterized in that it comprises step:
(1) luminous bacillus as claimed in claim 1 is mixed with food to be measured and blank article respectively, obtain testing mixture and negative control mixture respectively;
(2) detect the testing mixture luminous value, with negative control mixture luminous value relatively, obtain luminous intensity inhibition ratio;
(3) if luminous intensity inhibition ratio >=50% explains that then product to be tested is poisonous;
Contain salt in described testing mixture and/or the negative control mixture, said salt concn is 0.1-6.0w/v%; Described salt is sodium-chlor;
Said testing mixture and/or negative control mixture pH6-8;
Said detection is carried out at 1-37 ℃.
4. detection method as claimed in claim 3; It is characterized in that; It also uses the positive control mixture; Contain positive reference substance, mikrobe as claimed in claim 1 and salt in the said positive control mixture, salt concn is 0.1-6.0w/v% in the said positive control mixture, and the luminous intensity inhibition ratio of said positive control mixture equals 50%; Described salt is sodium-chlor.
5. like claim 3 or 4 described detection methods, it is characterized in that described blank article are selected from deionized water or nontoxic food.
6. detection method as claimed in claim 3 is characterized in that described food is selected from cereal, fruit, vegetables, meat poultry or marine food; Described water sample is selected from tap water, sewage or irrigation water.
7. detection method as claimed in claim 3 is characterized in that said detection is carried out at 5-30 ℃.
8. general biological toxicity detection kit in food or the water sample is characterized in that it comprises luminous bacillus as claimed in claim 1, blank article, positive reference substance, salts solution and damping fluid; Described luminous bacillus is luminous bacillus CCTCC M 208027 lyophilized powders.
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| CN107287132A (en) * | 2017-06-02 | 2017-10-24 | 烟台出入境检验检疫局检验检疫技术中心 | One plant of photobacteria, isolation and identification method and its application |
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| CN101936910B (en) * | 2010-08-04 | 2013-02-20 | 聚光科技(杭州)股份有限公司 | Method for analyzing water toxicity |
| CN102127588B (en) * | 2010-12-09 | 2012-08-22 | 济南市供排水监测中心 | Method for quantitatively detecting toxicity of water quality |
| CN102213721B (en) * | 2011-04-11 | 2013-10-23 | 同济大学 | Method for detecting toxicity of luminescent bacteria |
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| CN104215479B (en) * | 2013-05-30 | 2016-12-28 | 四川省中医药科学院 | A kind of biological test method of quick detection Chinese medicine comprehensive toxicity |
| CN103868915B (en) * | 2013-11-15 | 2016-08-17 | 四川省中医药科学院 | A kind of biological test method of quick detection Flos Carthami injection comprehensive toxicity |
| CN108872520A (en) * | 2018-08-14 | 2018-11-23 | 杭州绿洁水务科技股份有限公司 | A kind of water-quality test method, system and equipment |
| CN115184567A (en) * | 2022-06-02 | 2022-10-14 | 上海海恒机电仪表股份有限公司 | Device and method for detecting water toxicity by microorganisms |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991001366A1 (en) * | 1989-07-14 | 1991-02-07 | Biosphere Corporation S.A. | A method for stabilising bacteria by centrifuging to a non-aqueous paste |
| CN101131384A (en) * | 2007-09-27 | 2008-02-27 | 上海交通大学 | Method for detecting subaqueous acute biological toxicity using photobacteria |
| CN101339134A (en) * | 2008-08-14 | 2009-01-07 | 上海市农业科学院 | Method for quickly detecting vegetable integrated toxicity |
-
2009
- 2009-04-15 CN CN2009101350162A patent/CN101560491B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991001366A1 (en) * | 1989-07-14 | 1991-02-07 | Biosphere Corporation S.A. | A method for stabilising bacteria by centrifuging to a non-aqueous paste |
| CN101131384A (en) * | 2007-09-27 | 2008-02-27 | 上海交通大学 | Method for detecting subaqueous acute biological toxicity using photobacteria |
| CN101339134A (en) * | 2008-08-14 | 2009-01-07 | 上海市农业科学院 | Method for quickly detecting vegetable integrated toxicity |
Non-Patent Citations (4)
| Title |
|---|
| I. Villaescusa · M. Martinez · M. Pilar · J. C. Murat ·.Toxicity of cadmium species on luminescent bacteria.《Fresenius J Anal Chem》.1996,566－570. * |
| M. Devarea and M. Bahadir.Biological monitoring of landfill leachate using plants and luminescent bacteria.《Chemosphere》.1994,第28卷261-271. * |
| 于海等.检测水中急性毒性污染物的发光细菌光纤传感器的研究.《环境科学》.2008,第29卷(第2期),375－379. * |
| 蒋园芳.发光细菌检测水中综合毒性.《科技资讯》.2008,(第30期),215-217. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287132A (en) * | 2017-06-02 | 2017-10-24 | 烟台出入境检验检疫局检验检疫技术中心 | One plant of photobacteria, isolation and identification method and its application |
| CN107287132B (en) * | 2017-06-02 | 2020-07-07 | 烟台出入境检验检疫局检验检疫技术中心 | Luminescent bacterium, separation and identification method and application thereof |
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