CN102703571A - Method for detecting salmonellas in dehydrated garlic products - Google Patents
Method for detecting salmonellas in dehydrated garlic products Download PDFInfo
- Publication number
- CN102703571A CN102703571A CN2012101810685A CN201210181068A CN102703571A CN 102703571 A CN102703571 A CN 102703571A CN 2012101810685 A CN2012101810685 A CN 2012101810685A CN 201210181068 A CN201210181068 A CN 201210181068A CN 102703571 A CN102703571 A CN 102703571A
- Authority
- CN
- China
- Prior art keywords
- time
- salmonellas
- filter residue
- washings
- detects
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000607142 Salmonella Species 0.000 title claims abstract description 37
- 235000004611 garlic Nutrition 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 30
- 244000245420 ail Species 0.000 title 1
- 240000002234 Allium sativum Species 0.000 claims abstract description 36
- 238000005406 washing Methods 0.000 claims abstract description 30
- 239000011324 bead Substances 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 230000001954 sterilising effect Effects 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000003085 diluting agent Substances 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 12
- 239000013049 sediment Substances 0.000 claims abstract description 10
- 229940029982 garlic powder Drugs 0.000 claims abstract description 7
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims abstract description 6
- 239000001888 Peptone Substances 0.000 claims abstract description 6
- 108010080698 Peptones Proteins 0.000 claims abstract description 6
- 229960003067 cystine Drugs 0.000 claims abstract description 6
- 235000019319 peptone Nutrition 0.000 claims abstract description 6
- 229940082569 selenite Drugs 0.000 claims abstract description 6
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 claims abstract description 6
- 238000001914 filtration Methods 0.000 claims abstract description 5
- 230000018044 dehydration Effects 0.000 claims description 22
- 238000006297 dehydration reaction Methods 0.000 claims description 22
- 230000010355 oscillation Effects 0.000 claims description 16
- 238000005267 amalgamation Methods 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 238000009987 spinning Methods 0.000 claims description 6
- 235000010633 broth Nutrition 0.000 claims description 5
- 238000013016 damping Methods 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 9
- 238000000926 separation method Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000005119 centrifugation Methods 0.000 abstract 2
- 239000000872 buffer Substances 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 238000000265 homogenisation Methods 0.000 abstract 1
- 238000002156 mixing Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 10
- 235000013305 food Nutrition 0.000 description 10
- 239000000047 product Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 6
- 244000000010 microbial pathogen Species 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 238000002955 isolation Methods 0.000 description 4
- 238000007885 magnetic separation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000036039 immunity Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- JDLKFOPOAOFWQN-VIFPVBQESA-N Allicin Natural products C=CCS[S@](=O)CC=C JDLKFOPOAOFWQN-VIFPVBQESA-N 0.000 description 1
- 241000143060 Americamysis bahia Species 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241000223935 Cryptosporidium Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 241000224467 Giardia intestinalis Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- JDLKFOPOAOFWQN-UHFFFAOYSA-N allicin Chemical compound C=CCSS(=O)CC=C JDLKFOPOAOFWQN-UHFFFAOYSA-N 0.000 description 1
- 235000010081 allicin Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000002070 germicidal effect Effects 0.000 description 1
- 229940085435 giardia lamblia Drugs 0.000 description 1
- 235000001497 healthy food Nutrition 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 201000007094 prostatitis Diseases 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting salmonellas in dehydrated garlic products by adopting the immunomagnetic separation technology. The method comprises the following steps: (1) adding garlic powder into sterilization diluent, obtaining filter residues through filtering by a film filter bag after homogenization; (2) oscillating and washing the residues by the sterilization diluent for three times, mixing washing liquid of the three times; (3) oscillating salmonella immunomagnetic bead buffer solution and the mixed washing liquid; (4) performing centrifugal separation to obtain centrifugation sediment; (5) adding buffer peptone water enrichment broth into the centrifugation sediment and culturing for 24 hours; and (6) taking the culture solution, adding selenite cystine enrichment broth for enrichment culture, and using the VIDAS system to detect the salmonellas in the dehydrated garlic products. According to the invention, the immunomagnetic separation technology is utilized to realize fast detection of the salmonellas in the dehydrated garlic products, and the technology has the advantages of high specificity and high sensitivity.
Description
Technical field
The invention belongs to the microorganism detection technical field, be specifically related to a kind of method that adopts the immunomagnetic beads stripping technique to detect Salmonellas in the dehydration garlic goods.
Background technology
Food sanitation and safety-problems are healthy closely bound up with everyone, are always paid close attention to by countries in the world government and main flow medium.Along with people's is increasingly high to the attention rate of food safety in recent years; Particularly take place frequently in China's food safety accident in recent years; Under the overall background that the food quality security incident relevant with the Chinese exports product is reported in media often, importer is more and more stricter to the food inspection from China.China is maximum in the world garlic (Allium sativum L.) and production of articles and export State, has become China first place and has exported the vegetables of exchanging, and its outlet increases year by year.The garlic goods that wherein dewater are exactly the large agricultural exports of Anhui Province's tradition, product main exit Japan and Korea S, American-European market, and export volume is sure to occupy national prostatitis always.Strengthen the supervision of outlet dehydration garlic goods sanitary inspection and not only be related to the single product outlet, protect the trade of the agricultural-food whole industry normally to carry out especially.Yet dehydration garlic goods do not have the related microorganisms inspecting standard to put into effect so far; Use the check of food microorganisms GB4789 series standard for a long time; But because dehydration garlic goods have yield unique product characteristics, expose a large amount of problems in the actual detected, it is significantly unsuitable to show.There is not suitable inspecting standard to guaranteeing quality product, consumer safety, the very big hidden danger of the guarantee smooth and easy formation of export of farm produce trade.Therefore the research and development of safety and sanitation detection technique of carrying out dehydration garlic goods are for guaranteeing that Agricultural Products Trade safety and human consumer's edible safety all have great significance.
Dehydration garlic goods are mainly garlic powder, garlic grain and garlic sheet, are first-class seasonings and healthy food materials, and this product is selected the high-quality garlic for use, process through multiple working procedures such as peeling, cleaning, oven dry, selected, abrasive dust, packings.Moisture content is lower than 6%, does not add any foodstuff additive, does not add sanitas.Research shows as protective foods,, arteriosclerosis higher for maldigestion, flu, respiratory tract infection, digestive tract ulcer, blood fat, cancered adult personage and middle-aged and old heart trouble, cardiovascular and cerebrovascular diseases person; The immunocompromised person all has the effect of assisting therapy.The garlic powder has broad-spectrum antimicrobial and antibacterial, germicidal action: can effectively suppress growth, the breeding of harmful mushrooms such as intestinal bacteria, Salmonella, livestock and poultry are infected respiratory tract, digestive tract diseases and the complication significant curative effect thereof that causes because of Gram-negative and positive bacteria.But produce the amino acid whose formation of fragrance in the garlicin effective stimulus animal body, increase the flavour ingredient in the meat egg, make meat egg delicate flavour more beautiful.Add in the feed and can improve livestock and poultry and fishes and shrimps resistance against diseases, accelerate the speed of growth.Pest clearly, detoxifcation, function promoting blood circulation and removing blood stasis are arranged, can significantly reduce the toxicity of active substance in the feed.
Dehydration garlic goods are garlicky strong, pure, do not develop immunity to drugs, and are nontoxic, noresidue in the body.Exactly because the good moral character of dehydration garlic goods by utilization and consumption more and more widely, is strengthened its hygienic safety research is just seemed important all the more.The production of dehydration garlic goods will be passed through a plurality of links, and all there is the possibility of microbial contamination and breeding in wherein a lot of links.Do so why, be not suitable for the dewatering safety and sanitation of garlic goods of present GB 4789 series standards detect? Because contain a kind of natural phant fungistat---garlicin in the garlic; Allicin is the general name of sulfocompound in garlic bulblet and the garlic essential oil; Has (pressing down) bacterium effect of killing in various degree; Be described as natural bactericidal agent, have and report that its germ-resistant ability almost is 100 times of penicillium mould! 1/80000 garlicin can suppress or kill staphylococcus, suis, typhoid fever, dysentery bacterium etc.The garlic bulb extracting solution is to mould, head mold, aspergillus, the wooden mould obvious suppression effect that has.
Immunomagnetic beads stripping technique (Immunomagnetic Separation; IMS) integrated immunoreactive high degree of specificity and magnetic resolution fast, simple advantage, be widely used in the separation purification, amplification in vitro, HLA somatotype, targeting drug release, microorganism detection of cell etc.The immunomagnetic isolation technology not only is widely used in the every field of medical science but also the also first meeting clue of application aspect food hygiene detection.The immunomagnetic beads stripping technique is the pathogenic micro-organism in the separation and purification environmental water sample effectively, and pathogenic bacteria and two worms (giardia lamblia stiles, Cryptosporidium) such as EHEC 0157:H7 should technology also have the applications well prospect in addition in virus detects.Because the food sample often is the heterogeneous mixtinite of solid-liquid, adopt ordinary method to be difficult to a spot of pathogenic microorganism is separated.By the immunomagnetic isolation technology, can reach the purpose of sharp separation.The immunomagnetic isolation method; Be that specific antibody is coupled at the magnetic-particle surface, combine that the magnetic-particle that is loaded with pathogenic microorganism is under the effect of externally-applied magnetic field with seized pathogenic microorganism generation specificity in the sample; Assemble to pole orientation; Discard the sample mixed solution, make pathogenic microorganism not only obtain separating, and concentrated.The immunomagnetic isolation technology has obtained result preferably with its distinctive performance in food hygiene detection and research.
The immunity magnetic separation technique be exactly with diameter be the surface of the particulate of 0.02～5 μ m with superparamagnetism through chemically modified, make it and the specific antibody mortise, become and can combine and magnetic microballon, i.e. immunomagnetic beads with specific antigens (bacterium to be measured).Under the magnetic sheet background, mix, if there is corresponding antigens to exist, immunomagnetic beads will be caught it, forms thalline one immunomagnetic beads mixture.Then under suitable magnetic field condition, thalline one immunomagnetic beads mixture can be separated, and measures with additive method at last, like traditional plating method, PCR, ELISA, optical pickocff, electrochemical method etc.
The advantage that the immunomagnetic beads stripping technique is the most outstanding is a high specificity, can obviously improve the accuracy that detects target bacteria, in addition, and at the incubation time that can practice thrift 12～18 h in some cases.The immunity magnetic separation technique is one of technology of at present tool promotional value.
Summary of the invention
The object of the invention aims to provide a kind of method that detects Salmonellas in the dehydration garlic goods quickly and efficiently.
The contriver thinks: to the detection of Salmonellas in the dehydration garlic goods, the interference that should solve antibacterial substance in the sample.
So the present invention proposes following technical scheme:
The concrete operations step that detects Salmonellas method in the dehydration garlic goods is following:
(1) get garlic powder 25g, adding 225mL sterilization diluent homogeneous through the homogeneous bag filtration of filter membrane is arranged, is drawn filtrating, moves in the 500 mL centrifugal bottles, and gets filter residue;
(2) filter residue is carried out the washing first time, promptly in filter residue, add 50mL sterilization diluent and wash while vibrating, oscillation rate is 70 times/minute; Duration of oscillation is 20 minutes; Obtain for the first time washings and filter residue for the first time, to the first time filter residue by the first time wash conditions wash, obtain for the second time washings and filter residue for the second time; To the second time filter residue by the first time wash conditions wash; Obtain washings and filter residue for the third time for the third time, merge for the first time washings, washings and washings for the third time for the second time, obtain amalgamation liquid;
(3) in amalgamation liquid, adding concentration is the Salmonellas immunomagnetic beads damping fluid 10-40 μ L of 10mg/mL, and in 25 ℃ of vibrations down of temperature, oscillation rate is 70 times/minute, and duration of oscillation is 30 minutes, and thing must vibrate;
(4) the thing spinning 5 minutes under 1800 rev/mins of conditions of rotating speed of will vibrating, supernatant is removed in then spinning 10 minutes under 10000 rev/mins of conditions of rotating speed, obtains centrifugal sediment;
(5) in centrifugal sediment, add 500 mL buffered peptone water enrichment liquids (with reference to GB GB4789 series), temperature was cultivated 24 hours for 36 ℃, obtained nutrient solution;
(6) in the 1mL nutrient solution, add 10 mL selenite cystine broths (with reference to GB GB4789 series); Temperature was cultivated 24 hours down for 36 ℃; The gained culture detects the wherein content of Salmonellas with automatic bacterial detection system (VIDAS), detects the content that the Salmonellas content that obtains is Salmonellas in the dehydration garlic goods.
The collocation method of said sterilization diluent: the potassium primary phosphate that takes by weighing 34.0g is dissolved in the 500mL zero(ppm) water, regulates pH7.2 with the 1mol/L sodium hydroxide solution of 175mL, and is subsequent use as stock solution to the 1000mL constant volume with distilled water diluting.Get stock solution 1.25mL, to 1000mL, be sub-packed in the appropriate vessel, 121 ℃ of autoclavings 15 minutes with distilled water diluting.
Said filter membrane aperture is 330 μ m, diameter 50mm.
Useful technique effect of the present invention:
(1) the present invention catches a spot of object bacteria in kind liquid through immunomagnetic beads, it is separated from dehydration garlic product sample homogenizing fluid again, should detect a difficult problem to solve.The immunomagnetic beads stripping technique has solved the influence that the garlicin bacteriostatic action detects Salmonellas in the dehydration garlic goods well;
(2) the immunomagnetic beads stripping technique detects target bacteria high specificity, highly sensitive, can obviously improve the accuracy that detects target bacteria;
(3) the present invention has realized the rapid detection of Salmonellas in the dehydration garlic goods, and only need 48 hours detection time.
Embodiment
Below in conjunction with embodiment, the present invention is done explanation further.
1 material and instrument
1.1 material
The Salmonellas bacterial classification (
Salmonella) provide by Beijing Lan Borui company, Salmonellas immunomagnetic beads damping fluid is available from Jiahe, Shanghai bio tech ltd, and various substratum are provided by Beijing road and bridge company, and sample keeps sample from east, Anhui universe Food Co., Ltd submitted sample; Buffered peptone water enrichment liquid and selenite cystine broth configuration are with reference to GB GB4789 series;
1.2 instrument
Desk type high speed refrigerated centrifuge (BECKMAN 64R), vertical and high-speed refrigerated centrifuge (BECKMAN J30I), micro sample adding appliance: 0.5 μ L～10 μ L, 10 μ L～100 μ L, high-pressure sterilizing pot, VIDAS 30 automatic bacterial detection systems
BAX Q7 microorganism detection system, the constant temperature shaking table.
2 methods and result
2.1 immune magnetic separation technique condition optimizing
Get garlic powder 25g, artificial (a Maxwell unit) the Salmonellas bacterial classification that quantitatively adds adds 225mL sterilization diluent, and through the homogeneous bag filtration of band filter membrane, the filter membrane aperture is 330 μ m, diameter 50mm behind the homogeneous, draws filtrating, moves in the 500 mL centrifugal bottles.Filter residue is carried out the washing first time, promptly in filter residue, add 50mL sterilization diluent and wash while vibrating, oscillation rate is 70 times/minute; Duration of oscillation is 20 minutes; Obtain for the first time washings and filter residue for the first time, to the first time filter residue by the first time wash conditions wash, obtain for the second time washings and filter residue for the second time; To the second time filter residue by the first time wash conditions wash; Obtain washings and filter residue for the third time for the third time, merge for the first time washings, washings and washings for the third time for the second time, obtain amalgamation liquid;
In amalgamation liquid, adding concentration is 10mg/mL Salmonellas immunomagnetic beads damping fluid (Dynabeads) 10-40 μ L, and in 25 ℃ of soft vibrations down of temperature, oscillation rate is 70 times/minute, and duration of oscillation is 30 minutes, and thing must vibrate; The thing spinning 5 minutes under 1800 rev/mins of conditions of rotating speed of will vibrating, supernatant is removed in then spinning 10 minutes under 10000 rev/mins of conditions of rotating speed, obtains centrifugal sediment;
In centrifugal sediment, add 500 mL buffered peptone water enrichment liquids, temperature was cultivated 24 hours for 36 ℃, obtained nutrient solution;
In the 1mL nutrient solution, add 10 mL selenite cystine broths; Temperature was cultivated 24 hours down for 36 ℃; The gained culture detects the wherein content of Salmonellas with automatic bacterial detection system (VIDAS), detects the content that the Salmonellas content that obtains is Salmonellas in the dehydration garlic goods.
The immune magnetic separation technique of table 1 is caught the Salmonellas condition optimizing
2.2 detection method sensitivity determination
Get garlic powder 25g, artificial (a Maxwell unit) the Salmonellas bacterial classification that quantitatively adds adds 225mL sterilization diluent, and homogeneous moves in the 500 mL centrifugal bottles after the homogeneous bag filtration (the filter membrane aperture is 330 μ m, diameter 50mm) of band filter membrane is drawn filtrating.In filter residue, add 50mL sterilization diluent and wash while vibrating that (oscillation rate is 70 times/minute; Duration of oscillation is 20 minutes) obtain for the first time washings and filter residue for the first time; To the first time filter residue by the first time wash conditions wash, obtain for the second time washings and filter residue for the second time, to the second time filter residue by the first time wash conditions wash; Obtain washings and filter residue for the third time for the third time, for the first time washings, for the second time washings and for the third time washings merge into amalgamation liquid;
Adding concentration in the amalgamation liquid is 10mg/mL Salmonellas immunomagnetic beads damping fluid 40 μ L, and vibrating down 25 ℃ of temperature, thing must vibrate (oscillation rate is 70 times/minute, and duration of oscillation is 30 minutes);
To vibrate thing under 1800 rev/mins of conditions of rotating speed centrifugal 5 minutes, then under 10000 rev/mins of conditions of rotating speed centrifugal 10 minutes, remove supernatant, obtain centrifugal sediment; In centrifugal sediment, add 500 mL buffered peptone water enrichment liquids, temperature was cultivated 24 hours for 36 ℃, obtained nutrient solution;
In the 1mL nutrient solution, add 10 mL selenite cystine broths, temperature was cultivated 24 hours down for 36 ℃, and the gained culture detects the wherein content of Salmonellas with automatic bacterial detection system (VIDAS).
Table 2 immunomagnetic beads method detection method sensitivity determination
2.3 research is presented at when using the Salmonellas immunomagnetic beads, consumption is that 40 μ L, incubation time reach 20 minutes, both can satisfy the detection requirement, saves reagent and time relatively again.Proper extension incubation time when the bacterium amount is rare increases the immunomagnetic beads consumption and has positive meaning.
Claims (3)
1. method that detects Salmonellas in the dehydration garlic goods is characterized in that:
(1) gets garlic powder 25g, add 225mL sterilization diluent homogeneous,, draw filtrating, and obtain filter residue through the homogeneous bag filtration of filter membrane is arranged;
(2) filter residue is carried out the washing first time, promptly in filter residue, add 50mL sterilization diluent and wash while vibrating, oscillation rate is 70 times/minute; Duration of oscillation is 20 minutes; Obtain for the first time washings and filter residue for the first time, to the first time filter residue by the first time wash conditions wash, obtain for the second time washings and filter residue for the second time; To the second time filter residue by the first time wash conditions wash; Obtain washings and filter residue for the third time for the third time, merge for the first time washings, washings and washings for the third time for the second time, obtain amalgamation liquid;
(3) in amalgamation liquid, adding concentration is the Salmonellas immunomagnetic beads damping fluid 10-40 μ L of 10mg/mL, and in 25 ℃ of vibrations down of temperature, oscillation rate is 70 times/minute, and duration of oscillation is 30 minutes, and thing must vibrate;
(4) the thing spinning 5 minutes under 1800 rev/mins of conditions of rotating speed of will vibrating, supernatant is removed in then spinning 10 minutes under 10000 rev/mins of conditions of rotating speed, obtains centrifugal sediment;
(5) in centrifugal sediment, add 500 mL buffered peptone water enrichment liquids (with reference to GB GB4789 series), temperature was cultivated 24 hours for 36 ℃, obtained nutrient solution;
(6) in the 1mL nutrient solution, add 10 mL selenite cystine broths (with reference to GB GB4789 series); Temperature was cultivated 24 hours down for 36 ℃; The gained culture detects the wherein content of Salmonellas with automatic bacterial detection system (VIDAS), detects the content that the Salmonellas content that obtains is Salmonellas in the dehydration garlic goods.
2. a kind of according to claim 1 method that detects Salmonellas in the dehydration garlic goods; It is characterized in that: the collocation method of said sterilization diluent: the potassium primary phosphate that takes by weighing 34.0g is dissolved in the 500mL zero(ppm) water; 1mol/L sodium hydroxide solution with 175mL is regulated pH7.2, and is subsequent use as stock solution to the 1000mL constant volume with distilled water diluting;
Get stock solution 1.25mL, to 1000mL, be sub-packed in the appropriate vessel, 121 ℃ of autoclavings 15 minutes with distilled water diluting.
3. a kind of according to claim 1 method that detects Salmonellas in the dehydration garlic goods, it is characterized in that: said filter membrane aperture is 330 μ m, diameter 50mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101810685A CN102703571B (en) | 2011-06-29 | 2012-06-05 | Method for detecting salmonellas in dehydrated garlic products |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110177977 | 2011-06-29 | ||
| CN201110177977.7 | 2011-06-29 | ||
| CN2012101810685A CN102703571B (en) | 2011-06-29 | 2012-06-05 | Method for detecting salmonellas in dehydrated garlic products |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102703571A true CN102703571A (en) | 2012-10-03 |
| CN102703571B CN102703571B (en) | 2013-08-28 |
Family
ID=46896617
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101810685A Expired - Fee Related CN102703571B (en) | 2011-06-29 | 2012-06-05 | Method for detecting salmonellas in dehydrated garlic products |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102703571B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104133065A (en) * | 2013-05-02 | 2014-11-05 | 北京市科学器材公司 | Nano immune magnetic sphere, preparation method and application thereof |
| CN104312939A (en) * | 2014-09-23 | 2015-01-28 | 青岛康和食品有限公司 | Selenite cystine enrichment broth and application thereof |
| CN104459126A (en) * | 2014-12-16 | 2015-03-25 | 四川大学 | Method of gathering salmonella based on immunomagnetic beads |
| CN104651483A (en) * | 2013-11-19 | 2015-05-27 | 北京市理化分析测试中心 | Method for detecting living bacteria body of salmonella in sample |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020192676A1 (en) * | 2001-06-18 | 2002-12-19 | Madonna Angelo J. | Method for determining if a type of bacteria is present in a mixture |
| CN101158635A (en) * | 2007-11-19 | 2008-04-09 | 上海师范大学 | A method to detect bacteria using quantum dots |
| KR20090066126A (en) * | 2007-12-18 | 2009-06-23 | 영남대학교 산학협력단 | Detection method of Salmonella using immunomagnetic separation and immunoliposomes |
| WO2009122216A1 (en) * | 2008-04-04 | 2009-10-08 | Alaska Food Diagnostics Limited | Assay system based on immunomagnetic separation for detecting microorganisms in a food sample |
-
2012
- 2012-06-05 CN CN2012101810685A patent/CN102703571B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020192676A1 (en) * | 2001-06-18 | 2002-12-19 | Madonna Angelo J. | Method for determining if a type of bacteria is present in a mixture |
| CN101158635A (en) * | 2007-11-19 | 2008-04-09 | 上海师范大学 | A method to detect bacteria using quantum dots |
| KR20090066126A (en) * | 2007-12-18 | 2009-06-23 | 영남대학교 산학협력단 | Detection method of Salmonella using immunomagnetic separation and immunoliposomes |
| WO2009122216A1 (en) * | 2008-04-04 | 2009-10-08 | Alaska Food Diagnostics Limited | Assay system based on immunomagnetic separation for detecting microorganisms in a food sample |
Non-Patent Citations (3)
| Title |
|---|
| S.KUMAR ET AL: "Rapid dectction of Salmonella typhi in foods by combination of iｍｍｕｎｏｍａｇｎｅｔｉｃ sｅｐａｒａｔｉｏｎ and plomerase chain rection", 《WORLD JOURNAL OF MICROBILOGY》 * |
| 叶明强等: "一种快速检测沙门氏菌的新方法研究", 《食品科技》 * |
| 王海明等: "应用磁免疫技术快速检测食源性沙门氏菌方法研究", 《中国卫生监督杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104133065A (en) * | 2013-05-02 | 2014-11-05 | 北京市科学器材公司 | Nano immune magnetic sphere, preparation method and application thereof |
| CN104133065B (en) * | 2013-05-02 | 2016-08-03 | 北京市科学器材公司 | Nano immune magnetic ball, Preparation Method And The Use |
| CN104651483A (en) * | 2013-11-19 | 2015-05-27 | 北京市理化分析测试中心 | Method for detecting living bacteria body of salmonella in sample |
| CN104651483B (en) * | 2013-11-19 | 2017-01-18 | 北京市理化分析测试中心 | Method for detecting living bacteria body of salmonella in sample |
| CN104312939A (en) * | 2014-09-23 | 2015-01-28 | 青岛康和食品有限公司 | Selenite cystine enrichment broth and application thereof |
| CN104459126A (en) * | 2014-12-16 | 2015-03-25 | 四川大学 | Method of gathering salmonella based on immunomagnetic beads |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102703571B (en) | 2013-08-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Valenzuela et al. | Isolation and identification of Enterococcus faecium from seafoods: antimicrobial resistance and production of bacteriocin-like substances | |
| CN107686832B (en) | Novel vibrio parahaemolyticus bacteriophage, and composition, preparation method and application thereof | |
| CN105567647B (en) | A methicillin-resistant Staphylococcus aureus phage and its antibacterial application | |
| CN103320365B (en) | Fish-sourced aeromonas hydrophila disease antagonistic strain and application thereof | |
| CN105331587B (en) | A kind of vibrio parahaemolyticus phage and its preparation method and application | |
| CN102703571B (en) | Method for detecting salmonellas in dehydrated garlic products | |
| CN107723280B (en) | Baltic Sea Shewanella bacteriophage SppYZU01 and application thereof | |
| Gunasegaran et al. | Isolation and identification of Salmonella from curry samples and its sensitivity to commercial antibiotics and aqueous extracts of Camelia sinensis (L.) and Trachyspermum ammi (L.) | |
| Shahin et al. | Effective control of Shigella contamination in different foods using a novel six-phage cocktail | |
| CN115786279A (en) | A kind of high temperature-resistant pigeon-derived Salmonella typhimurium phage, its phage composition and its application | |
| CN117070472A (en) | Vibrio parahaemolyticus phage aiming at highly pathogenic vibrio and drug-resistant vibrio and application thereof | |
| Ajibola et al. | Toxicity assessment of Lactococcus lactis IO-1 used in coconut beverages against artemia salina using brine shrimp lethality test | |
| CN101649299B (en) | Bdellovibrio bacteriovorus bacterial strain for eliminating Listeria monocytogenes and application thereof | |
| CN107828742A (en) | A kind of Shewanella putrefaciens bacteriophage and its application | |
| CN114940977A (en) | A Duck Salmonella phage, its phage composition and its application in the prevention and treatment of Duck Salmonella infection | |
| CN102172248A (en) | Bacteriophage for preventing and treating apostichopus japonicus skin ulcer syndrome and applications thereof | |
| CN114097927A (en) | Preparation method of biological feed additive based on bacteriophage | |
| Jones et al. | Development of an optimized method for the recovery of infectious F-RNA coliphage MS2 from meat | |
| Pramono et al. | Detection of antibiotic-resistant Salmonella sp. in the seafood products of Surabaya local market | |
| CN115851617B (en) | Escherichia coli phage LHE83 and its application | |
| CN117070471A (en) | Multivalent salmonella phage capable of entering blood orally and application thereof | |
| CN102586391B (en) | Method for determining colony count of pathogenic microbes in dehydrated garlic product | |
| CN101849608B (en) | Rape-seed meal antibacterial peptide | |
| CN116121203B (en) | A strain of Clostridium perfringens phage PM11, its phage composition and its application | |
| CN102093473A (en) | Preparation method of bacteriostatic active protein of Sinonovacula constricta Lamarck epipsymbiosis strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130828 Termination date: 20160605 |