CN102304482B - Cifidobacterium longum strain - Google Patents
Cifidobacterium longum strain Download PDFInfo
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- CN102304482B CN102304482B CN 201110188038 CN201110188038A CN102304482B CN 102304482 B CN102304482 B CN 102304482B CN 201110188038 CN201110188038 CN 201110188038 CN 201110188038 A CN201110188038 A CN 201110188038A CN 102304482 B CN102304482 B CN 102304482B
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Abstract
The invention provides a bifidobacterium longum LJM002 strain, the preservation No. of which is CGMCC No. 4900. The LJM002 strain is ultra-sensitive to acephate and is a work strain for detecting the residue of the acephate in foods. The invention also provides a microorganism method for screening the residue of the acephate in the foods, which comprises the step of judging whether the residue limit of acephate in food samples to be detected exceeds the standard according to the size of a bacteriostatic ring on a screening medium. The method for screening the residue of the acephate in the foods has the characteristics that the method is simple to operate, has low cost and high sensitivity and is suitable for sample screening. The method conforms to the screening requirements of China's pesticide residue detection department for a large quantity of samples.
Description
Technical field
The present invention relates to a strain bifidus longum bb bacterial strain and the residual application of acephate in screening food of conduct work bacterial strain thereof.
Background technology
Acephate, chemical name are O, the S-dimethyl, and N-ethanoyl thio-phosphamide is the N-acetyl derivative of insecticide methamidophos (methmaidophos).International English is called Acephate, molecular formula C
4H
10NO
3PS, molecular weight 183.16, structural formula is:
Acephate is a kind of organic phosphorous insecticide of efficient, the lasting period long, interior absorption is strong broad spectrum, have stomach toxicity and action of contace poison, can kill ovum, and certain fumigation action is arranged, be applicable to crops such as vegetables, tea tree, paddy rice, wheat, can effectively prevent and treat multiple chewing type and sucking pest.
But the excessive use of acephate makes its high residue in vegetable and fruit, brings huge threat for people's health, so China's use of having limited the quantity of.In January, 2005 Ministry of Health of the People's Republic of China and Standardization Administration of China issue GB2763-2005 is defined in maximum residue limit (the maximum residue limits of vegetables, MRL) be 1mg/kg, the MRL of fruit is 0.5mg/kg, and will detect in the food acephate residual quantity and classify residual monitoring emphasis as.
At present, the method for mensuration acephate mainly contains vapor-phase chromatography (GC), high performance liquid chromatography (HPLC), chemoluminescence method, enzyme inhibition method, capillary electrophoresis technique (CE) etc.There are plant and instrument complexity, sample pre-treatment and the loaded down with trivial details defective of measurement operation with the aforesaid method detection, are not suitable for the great amount of samples screening, and costly, it is promoted the use of be restricted.
The outstanding advantage of microbial method is easy and economical, does not need complicated plant and instrument, just can carry out under general experiment condition, can carry out quick primary dcreening operation to great amount of samples.But the work bacterial strain just is the most critical issue that influences microbial method accuracy and precision to susceptibility and the specificity of test substance.Up to now, still do not have and filter out the high bifidus bacillus work bacterial strain of acephate susceptibility height, specificity and the residual screening of acephate is used in food relevant report thereof.
Summary of the invention
The object of the invention provides the strain bifidus bacillus high to acephate susceptibility.
Bifidus bacillus of the present invention is: bifidus longum bb (Bifidobacterium longum) LJM002CGMCC No.4900.
Bifidus longum bb of the present invention (Bifidobacterium longum) LJM002, in on May 25th, 2011 at China Committee for Culture Collection of Microorganisms common micro-organisms center, it abbreviates CGMCC (address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City as, postcode 100101) preservation, classification called after bifidus longum bb (Bifidobacterium longum), deposit number is CGMCCNo.4900.
Bifidus longum bb LJM002 of the present invention is located away from Zhejiang Province's Healthy Youth human faecal mass.
Bifidus longum bb LJM002 bacterial strain of the present invention has following microbial characteristic:
(1) colonial morphology: LJM002 bacterial strain bacterium colony on flat board is canescence or oyster white, opaque, glossy, smooth surface, projection, soft, the neat in edge of quality, diameter 1-1.5mm.
(2) individual morphology: be irregular G
+Sporeless bacterium has straight-bar, knee, match head, dumbbell shape and wild goose shape, wherein based on the wild goose shape.
(3) physiological and biochemical property: D-ribose (+); L-arabinose (+); Lactose (+); Cellobiose (-); Melizitose (+); Raffinose (+); Sorbyl alcohol (-); Starch (-); Sunmorl N 60S (-); Wood sugar (+); Seminose (-); Fructose (+); Semi-lactosi (+); Sucrose (+); Maltose (+); Trehalose (-); Close disaccharides (+); N.F,USP MANNITOL (+); Synanthrin (-); Salicin (-); F6PPK enzyme (+); Reaction (on good gas-solid body substratum, not growing) to oxygen; Nitrate reduction (-); Catalase (-); Indole reaction (-).
Well-grown under the anaerobism is not grown in the aerobic environment.Optimum growth temperature 37-41 ℃; Minimum growth temperature 25-28 ℃; The highest 43-45 ℃; Growth optimal pH 6.5-7.0; Do not grow at pH4.5-5.0 or 8.0-8.5.
The LJM002 bacterial strain with belong to reference culture of the same race together and compare acephate susceptibility and improved more than 100 times.
Another object of the present invention is to set up a kind of bifidus longum bb LJM002 that uses as the residual microbial method of work bacterial strain screening food acephate.
The residual principle of screening food acephate of the present invention is based on acephate its responsive bifidus longum bb LJM002 is had the obvious suppression effect, on food samples point sample bifidus bacillus bacterium layer screening substratum, anaerobism was cultivated after 18-24 hour, measure the inhibition zone of its formation, can judge according to the size of inhibition zone whether the acephate residual quantity is qualified in the food samples to be measured.
The present invention sets up a kind of bifidus longum bb LJM002 that uses as the residual microbial method of work bacterial strain screening food acephate, comprises step:
1, screening medium preparation: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, sodium acetate 5g, K
2HPO
42g, MgSO
47 H
2O 0.5g, MnSO
44H
2O 0.2g, oligofructose 3g, dibasic ammonium citrate 2g, tween-80 1ml, agar 15g, developer 0.1g, distilled water 1L proofreaies and correct pH6.5 with above composition heating for dissolving, 115 ℃ autoclaving 15-20 minute.
Developer is 5-bromo-4-chloro-3-indoles-β-D galactoside (X-Gal).
2, bacteria suspension preparation: bifidus longum bb LJM002 is inoculated in the slant medium of nutrient agar medium, and 35 ± 2 ℃ of anaerobism were cultivated 24 hours, with sterile saline lawn were washed, and made suspension, put in the 2-8 ℃ of refrigerator and preserved.
3, the dull and stereotyped preparation of bacterium layer: cut-off is the flat board of about 90mm, high 16～17mm directly, injects the screening substratum 20mL of heating and melting, makes evenly to spread out cloth at the bottom of the dish, places to make on the horizontal stand and solidifies, as bottom; Add bacteria suspension 0.5mL at the bottom substratum that has solidified (put and be chilled to 48～50 ℃), fully mixing makes and evenly spread out cloth on bottom, as the bacterium layer.
4, sample pre-treatments: get food samples homogenate to be checked and organize 5g, with aseptic 1% phosphate buffered saline buffer (pH6.0) 20ml dilution, the whirlpool mixing is in 80 ℃ of heating in water bath 5min, supernatant liquor is got as supplying test agent liquid in the centrifugal 15min of 5000r/min in the cooling back.
5, sample determination: (diameter 13 ± 1mm) with the light paper-pressing sheet of tweezers, makes the scraps of paper closely contact with substratum, drips for test agent liquid 90 μ l at the circle filter paper with micropipet to place the circle filter paper in the culture dish of bacterium layer flat board.Each repeats 2 culture dish at least for sample, and 35 ± 2 ℃ of anaerobism were cultivated 18～24 hours, and antibacterial circle diameter is measured by inhibition zone computer Measurement and analysis instrument.
6, the result judges:
As inhibition zone 〉=15mm, judge the residual positive of acephate (+) in the food samples.
Work bacterial strain of the present invention is bifidus longum bb LJM002, on May 25th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.4900.
Step 2 bacteria suspension of the present invention is that light transmission rate is controlled to be 0.8 under the 721 spectrophotometer 580nm wavelength.
Food samples of the present invention comprises: vegetable and fruits such as Chinese cabbage, eggplant, cucumber, pumpkin, wax gourd, apple, pear, peach.
Advantage of the present invention and effect:
(1) the acephate susceptibility of bifidus longum bb LJM002 of the present invention height, specificity are good, can be used as the residual work bacterial classification of acephate in the microbial method screening food;
(2) added the X-Gal indicator in the screening substratum, made the inhibition zone of generation more clear;
(3) sample pre-treatments of the present invention is simple, a large amount of food samples of screening simultaneously;
(4) detection method of the present invention is simple and easy to do, have highly sensitive, characteristics such as tolerance range height, lowest detection is limited to 0.1mg/kg in vegetables, be lower than the maximum residue limit of national standard, the rate of recovery is all greater than 70%, and the variation coefficient is in 10%, the accuracy and the precision requirement that meet the residual screening of food acephate are fit to the residual screening of acephate in the food fully.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but illustrated embodiment is not as a limitation of the invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Separation and the evaluation of embodiment 1 bifidus longum bb LJM002 bacterial strain
With the ight soil of Zhejiang Province's healthy young people as sample separation, on the modified MRS nutrient agar, under 37 ℃ of anaerobic conditions, 48h is coated with separation and Culture, obtain bifidus longum bb LJM002 of the present invention, be accredited as bifidus longum bb, this bacterial strain on May 25th, 2011 at China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, classification called after bifidus longum bb (Bifidobacterium longum), preserving number is CGMCC No.4900.
The prescription of modified MRS nutrient agar: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, sodium acetate 5g, K
2HPO
42g, MgSO
47 H
2O 0.5g, MnSO
44H
2O 0.2g, oligofructose 3g, dibasic ammonium citrate 2g, tween-80 1ml, agar 15g, 5-bromo-4-chloro-3-indoles-β-D galactoside (X-Gal) 0.1g, distilled water 1L transfers pH to 7.0, sterilizes 15 minutes for 115 ℃.
LJM002 bacterial strain of the present invention has following microbial characteristic:
(1) colonial morphology: LJM002 bacterial strain bacterium colony on flat board is canescence or oyster white, opaque, glossy, smooth surface, projection, soft, the neat in edge of quality, diameter 1-1.5mm.
(2) individual morphology: be irregular G
+Sporeless bacterium has straight-bar, knee, match head, dumbbell shape and wild goose shape, wherein based on the wild goose shape.
(3) physiological and biochemical property: D-ribose (+); L-arabinose (+); Lactose (+); Cellobiose (-); Melizitose (+); Raffinose (+); Sorbyl alcohol (-); Starch (-); Sunmorl N 60S (-); Wood sugar (+); Seminose (-); Fructose (+); Semi-lactosi (+); Sucrose (+); Maltose (+); Trehalose (-); Close disaccharides (+); N.F,USP MANNITOL (+); Synanthrin (-); Salicin (-); F6PPK enzyme (+); Reaction (on good gas-solid body substratum, not growing) to oxygen; Nitrate reduction (-); Catalase (-); Indole reaction (-).
Well-grown under the anaerobism is not grown in the aerobic environment.Optimum growth temperature 37-41 ℃; Minimum growth temperature 25-28 ℃; The highest 43-45 ℃; Growth optimal pH 6.5-7.0; Do not grow at pH4.5-5.0 or 8.0-8.5.
Test 1 sensitivity test
Adopt scraps of paper agar diffusion (K-B) method, choose the acephate reference liquid of different concns, with the modified MRS nutrient agar, culture medium prescription is seen embodiment 1, with bifidus longum bb LJM002 of the present invention serve as the test bacterium, place 37 ℃ of incubator anaerobism to cultivate the growing state of observing the LJM002 bacterial strain behind the 24h, do the acephate sensitivity test with agar diffusion method of the paper, measure acephate to the bacteriostasis property of LJM002 bacterial strain.
3 repetitions are done in above-mentioned test, are contrast to belong to bifidus longum bb reference culture of the same race together.
Table 1 sensitivity test
Test-results: the acephate of bifidus longum bb LJM002 of the present invention has the susceptibility of height, its with belong to reference culture together and compare, susceptibility to acephate has improved more than 100 times, acephate susceptibility≤0.1mg/kg of bifidus longum bb LJM002, be lower than the maximum residue limit (MRL) of national standard, can be used as the residual work bacterial strain of screening acephate fully.
Test the test of 2 specificitys
On flat board, drip acephate in the alternate Oxford cup respectively with reference to working standard liquid such as concentration (0.1mg/kg) reference liquid and Rogor, acephatemet, acephate, omethoate, SD-1750, thiophos.The susceptibility that compares the Rogor of bifidus longum bb LJM002 of the present invention, acephatemet, acephate, omethoate, SD-1750,6 kinds of agricultural chemicals of thiophos.
Calculate the cross reacting rate of acephate and other 5 kinds of agricultural chemicals with following formula.
Test-results: the acephate of bifidus longum bb LJM002 of the present invention is the most responsive, and is insensitive to other 5 kinds of agricultural chemicals, and in other 5 kinds of agricultural chemicals, the LJM002 bacterial strain is all insensitive to each agricultural chemicals of 2 times of MRL and following dosage level thereof.
The residual screening of acephate in the embodiment 2 Chinese cabbage samples
1, screening medium preparation:
Peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, sodium acetate 5g, K
2HPO
42g, MgSO
47 H
2O 0.5g, MnSO
44H
2O 0.2g, oligofructose 3g, dibasic ammonium citrate 2g, tween-80 1ml, agar 15g, 5-bromo-4-chloro-3-indoles-β-D galactoside (X-Gal) 0.1g, distilled water 1L proofreaies and correct pH6.5 with above composition heating for dissolving, 115 ℃ autoclaving 15-20 minute.
2, bacteria suspension preparation:
Bifidus longum bb LJM002 is inoculated in the slant medium of nutrient agar medium, 35 ± 2 ℃ of anaerobism were cultivated 24 hours, with sterile saline lawn were washed, and made suspension, under 721 spectrophotometer 580nm wavelength, light transmission rate is controlled to be 0.8, puts in 2～8 ℃ of refrigerators and preserve.
3, the dull and stereotyped preparation of bacterium layer:
Cut-off is the flat board of about 90mm, high 16～17mm directly, injects the screening substratum 20mL of heating and melting, makes evenly to spread out cloth at the bottom of the dish, places to make on the horizontal stand and solidifies, as bottom; Add bacteria suspension 0.5mL at the bottom substratum that has solidified (put and be chilled to 48～50 ℃), fully mixing makes and evenly spread out cloth on bottom, as the bacterium layer.
4, sample pre-treatments:
Get Chinese cabbage sample homogenization to be checked and organize 5g, with aseptic 1% phosphate buffered saline buffer (pH6.0) 20ml dilution, whirlpool mixing, in 80 ℃ of heating in water bath 5min, supernatant liquor is got as supplying test agent liquid in the centrifugal 15min of 5000r/min in the cooling back.
5, sample determination:
(diameter 13 ± 1mm) with the light paper-pressing sheet of tweezers, makes the scraps of paper closely contact with substratum, drips for test agent liquid 90 μ l at the circle filter paper with micropipet to place the circle filter paper in the culture dish of bacterium layer flat board.Each repeats 2 culture dish at least for sample, and 35 ± 2 ℃ of anaerobism were cultivated 18～24 hours, and antibacterial circle diameter is measured by inhibition zone computer Measurement and analysis instrument.
6, the result judges:
As inhibition zone 〉=15mm, judge the residual positive of acephate (+) in the food samples.
Test the mensuration of 1 standard substance precision
Getting concentration is 0.1mg/kg, the acephate reference liquid of 1mg/kg, pipette 90 μ l with pipettor respectively and add bacterium layer screening media surface, four round filter papers that the bacterium laminar surface is placed are (on the diameter 13 ± 1mm), each concentration drips a round filter paper, repeat 5 culture dish, 35 ± 2 ℃ of anaerobism were cultivated 18-24 hour.
The mensuration of table 2 standard substance precision
Annotate: circle filter paper diameter is 13 ± 1mm, the not long bacterium of circle filter paper inferior segment
Test-results: the variation coefficient of 0.1mg/kg acephate reference liquid is that the variation coefficient of 2.6%, 1mg/kg acephate reference liquid is 2.5%.The variation coefficient all in 10%, meets the screening precision requirement.
Test the mensuration of 2 food samples precision
Get the blank homogenate Chinese cabbage of agricultural chemicals sample 5g, in triplicate, make that acephate concentration is respectively 0.1mg/L in the food, 1mg/L, pipetting 90 μ l with pipettor adds on four round filter papers of bacterium layer media surface placement, each concentration drips 2 round filter papers, repeats 3 culture dish, cultivates 18～24 hours for 35 ± 2 ℃.
The test of table 3 food samples precision
Test-results: the variation coefficient between 0.1mg/kg concentration sample plate is 1.83%, and total variation coefficient is 2.43%; The variation coefficient is 1.77% between the dish of 1mg/kg concentration sample, and total variation coefficient is 2.97%.
The inventive method variation coefficient all in 10%, meets the precision requirement of agricultural chemicals screening fully.
Test 3 food samples accuracy tests
Get each 5g of the blank homogenate food samples of agricultural chemicals, in quintuplicate, make in the food that acephate concentration is respectively 0.1,0.5,0.75,1,1.25mg/kg, pipetting 90 μ l with pipettor adds on four round filter papers of bacterium layer media surface placement, each concentration drips 2 round filter papers, and 3 splash into reference liquid with reference to concentration in addition.Repeat 5 culture dish, put in 35 ± 2 ℃ the incubator 18～24 hours, measure antibacterial circle diameter.
Calculate the rate of recovery of acephate in food samples with following formula.
Table 4 food samples accuracy test
Test-results: the acephate interpolation rate of recovery is 83.2%～98.6% in the food samples, in the daytime the variation coefficient is 1.32%～8.89%, the rate of recovery is all greater than 70%, the variation coefficient is in 10%, show that present method has higher accuracy and repeatability, meet the requirement of the existing food pesticide residue screening of China fully.
Test the test of 4 false negative rates
Take by weighing blank each 5g of food samples of agricultural chemicals of homogeneous, add the acephate reference liquid, make acephate concentration 1mg/kg in the food, measuring method is with embodiment 2, and replication 100 times is observed the probability that negative findings occurs, and calculates false negative rate.
Test-results: acephate is residual in the cucumber when being 1mg/kg, and false negative rate is 0; Acephate is residual in the wax gourd when being 1mg/kg, and false negative rate is 2%.
The test of table 5 false negative rate
Claims (1)
1. a strain bifidus longum bb (Bifidobacterium longum) LJM002, preserving number is CGMCC No.4900.
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| CN103789268B (en) * | 2014-02-21 | 2016-04-27 | 刘洛贤 | A kind of method of separation and Culture hippocampal neurons and special culture solution thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1641015A (en) * | 2004-01-18 | 2005-07-20 | 北京三元食品股份有限公司 | Bifidobacterium and its use |
| CN101649303A (en) * | 2009-05-08 | 2010-02-17 | 内蒙古蒙牛乳业（集团）股份有限公司 | Bifidobacterium, application thereof and food composition containing same |
| CN101671638A (en) * | 2009-09-28 | 2010-03-17 | 中国农业大学 | New strain of bifidobacterium and fermentative preparation method and application thereof |
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2011
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1641015A (en) * | 2004-01-18 | 2005-07-20 | 北京三元食品股份有限公司 | Bifidobacterium and its use |
| CN101649303A (en) * | 2009-05-08 | 2010-02-17 | 内蒙古蒙牛乳业（集团）股份有限公司 | Bifidobacterium, application thereof and food composition containing same |
| CN101671638A (en) * | 2009-09-28 | 2010-03-17 | 中国农业大学 | New strain of bifidobacterium and fermentative preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 双歧杆菌的生物学特性及对人体的生理功能;徐营等;《微生物学通报》;20010630;第28卷(第6期);94-96 * |
| 徐营等.双歧杆菌的生物学特性及对人体的生理功能.《微生物学通报》.2001,第28卷(第6期),94-96. |
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Inventor after: Liu Jiaming Inventor after: Wang Fangyan Inventor after: Yu Xichong Inventor after: Lu Zhongqiu Inventor after: Hong Guangliang Inventor before: Liu Jiaming |
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