CN102703571B - Method for detecting salmonellas in dehydrated garlic products - Google Patents
Method for detecting salmonellas in dehydrated garlic products Download PDFInfo
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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 isolation technique to detect Salmonellas in the dehydration garlic goods.
Background technology
Food sanitation and safety problem are healthy closely bound up with everyone, are always paid close attention to by countries in the world government and main flow medium.In recent years along with people are more and more higher to the attention rate of food safety, 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 the garlic goods have yield unique product characteristics because dewater, and expose a large amount of problems in the actual detected, show significantly to be not suitable for.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 carry out the research and development of safety and sanitation detection technique of dehydration garlic goods 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.Studies show that 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 wherein all there is the possibility of microbial contamination and breeding in 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 the ability of its sterilization almost is 100 times of penicillin! 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 isolation 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 isolation technique is the pathogenic micro-organism in the separation and purification environmental water sample effectively, pathogenic bacteria and two worms (giardia lamblia stiles, Cryptosporidium) such as enterohemorrhagic Escherichia coli 0157:H7, and this technology also has the applications well prospect in virus detects in addition.Because the food sample often is the heterogeneous mixture 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, tested pathogenic microorganism generation specificity is combined in sample, be loaded with the magnetic-particle of pathogenic microorganism under the effect of externally-applied magnetic field, 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 can with specific antigens (bacterium to be measured) in conjunction with and magnetic microballon, i.e. immunomagnetic beads.Mix under the magnetic sheet background, 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, as traditional plating method, PCR, ELISA, optical pickocff, electrochemical method etc.
The advantage that the immunomagnetic beads isolation technique is the most outstanding is high specificity, can obviously improve the accuracy that detects target bacteria, in addition, and at the incubation time that can save 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
Purpose of the present invention aims to provide a kind of method that detects Salmonellas in the dehydration garlic goods quickly and efficiently.
The contriver thinks: at 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 as follows:
(1) gets garlic powder 25g, add 225mL sterilization diluent homogeneous, through the homogeneous bag filtration of filter membrane is arranged, draw filtrate, move in the 500 mL centrifugal bottles, and get filter residue;
(2) filter residue is carried out the washing first time, namely adding 50mL sterilization diluent in filter residue washs while vibrating, oscillation rate is 70 times/minute, duration of oscillation is 20 minutes, obtain washings and filter residue for the first time for the first time, to the first time filter residue by the first time wash conditions wash, obtain washings and filter residue for the second time 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) adding concentration in amalgamation liquid 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, gets the vibration thing;
(4) the thing centrifugation 5 minutes under 1800 rev/mins of conditions of rotating speed of will vibrating, supernatant liquor is removed in then centrifugation 10 minutes under 10000 rev/mins of conditions of rotating speed, obtains centrifugal sediment;
(5) add 500 mL buffered peptone water enrichment liquids (with reference to GB GB4789 series) in centrifugal sediment, 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 described sterilization diluent: the potassium primary phosphate that takes by weighing 34.0g is dissolved in the 500mL distilled water, regulates pH7.2 with the 1mol/L sodium hydroxide solution of 175mL, and is standby as stock solution to the 1000mL constant volume with distilled water diluting.Get stock solution 1.25mL,, be sub-packed in the appropriate vessel to 1000mL with distilled water diluting, 121 ℃ of autoclavings 15 minutes.
Described 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 the sample liquid by immunomagnetic beads, it is separated from dehydration garlic product sample homogenizing fluid, to solve this detection difficult problem again.The immunomagnetic beads isolation technique has solved the influence that the garlicin bacteriostatic action detects Salmonellas in the dehydration garlic goods well;
(2) the immunomagnetic beads isolation 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 further described.
1 material and instrument
1.1 material
The Salmonellas bacterial classification (
Salmonella) provided 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 filtrate, moves in the 500 mL centrifugal bottles.Filter residue is carried out the washing first time, namely adding 50mL sterilization diluent in filter residue washs while vibrating, oscillation rate is 70 times/minute, duration of oscillation is 20 minutes, obtain washings and filter residue for the first time for the first time, to the first time filter residue by the first time wash conditions wash, obtain washings and filter residue for the second time 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;
Adding concentration in amalgamation liquid 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, gets the vibration thing; The thing centrifugation 5 minutes under 1800 rev/mins of conditions of rotating speed of will vibrating, supernatant liquor is removed in then centrifugation 10 minutes under 10000 rev/mins of conditions of rotating speed, obtains centrifugal sediment;
Add 500 mL buffered peptone water enrichment liquids in centrifugal sediment, 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 is drawn filtrate by the homogeneous bag filtration (the filter membrane aperture is 330 μ m, diameter 50mm) of band filter membrane, moves in the 500 mL centrifugal bottles.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 washings and filter residue for the second time 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 liquor, obtain centrifugal sediment; Add 500 mL buffered peptone water enrichment liquids in centrifugal sediment, temperature was cultivated 24 hours for 36 ℃, obtained nutrient solution;
Add 10 mL selenite cystine broths in the 1mL nutrient solution, 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 studies show that consumption is that 40 μ L, incubation time reach 20 minutes, both can satisfy the detection requirement, saves reagent and time relatively again when using the Salmonellas immunomagnetic beads.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, through the homogeneous bag filtration of filter membrane is arranged, draw filtrate, and obtain filter residue; The collocation method of described sterilization diluent: the potassium primary phosphate that takes by weighing 34.0g is dissolved in the 500mL distilled water, regulates pH7.2 with the 1mol/L sodium hydroxide solution of 175mL, and is standby as stock solution to the 1000mL constant volume with distilled water diluting;
(2) filter residue is carried out the washing first time, namely adding 50mL sterilization diluent in filter residue washs while vibrating, oscillation rate is 70 times/minute, duration of oscillation is 20 minutes, obtain washings and filter residue for the first time for the first time, to the first time filter residue by the first time wash conditions wash, obtain washings and filter residue for the second time 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) adding concentration in amalgamation liquid 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, gets the vibration thing;
(4) the thing centrifugation 5 minutes under 1800 rev/mins of conditions of rotating speed of will vibrating, supernatant liquor is removed in then centrifugation 10 minutes under 10000 rev/mins of conditions of rotating speed, obtains centrifugal sediment;
(5) add 500mL buffered peptone water enrichment liquid in centrifugal sediment, with reference to GB GB4789 series, temperature was cultivated 24 hours for 36 ℃, obtained nutrient solution;
(6) in the 1mL nutrient solution, add the 10mL selenite cystine broth, 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 method that detects Salmonellas in the dehydration garlic goods according to claim 1, it is characterized in that: the collocation method of described sterilization diluent: the potassium primary phosphate that takes by weighing 34.0g is dissolved in the 500mL distilled water, 1mol/L sodium hydroxide solution with 175mL is regulated pH7.2, and is standby as stock solution to the 1000mL constant volume with distilled water diluting;
Get stock solution 1.25mL,, be sub-packed in the appropriate vessel to 1000mL with distilled water diluting, 121 ℃ of autoclavings 15 minutes.
3. a kind of method that detects Salmonellas in the dehydration garlic goods according to claim 1, it is characterized in that: described filter membrane aperture is 330 μ m, diameter 50mm.
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| 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 |
| 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 |
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| 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 | Method using quantum dot to detect bacterium |
| KR20090066126A (en) * | 2007-12-18 | 2009-06-23 | 영남대학교 산학협력단 | Detection method of salmonella spp. using immunomagnetic separation and immunoliposomes |
| GB0806147D0 (en) * | 2008-04-04 | 2008-05-14 | Alaska Food Diagnostics | Assay system |
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