CN109735599A - A method of improving staphylococcus aureus detection limit in food liquid easy to foaming - Google Patents
A method of improving staphylococcus aureus detection limit in food liquid easy to foaming Download PDFInfo
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- CN109735599A CN109735599A CN201910049279.5A CN201910049279A CN109735599A CN 109735599 A CN109735599 A CN 109735599A CN 201910049279 A CN201910049279 A CN 201910049279A CN 109735599 A CN109735599 A CN 109735599A
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- staphylococcus aureus
- miillpore filter
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Abstract
The present invention provides a kind of method for improving staphylococcus aureus detection limit in food liquid easy to foaming, increase sampling amount, it solves the problems, such as to blister in sample pre-treatments enrichment process using effective defoaming agent, it is filtered using vacuum filter membrane on the microorganism to miillpore filter in enriched sample, improves detection limit and the sensitivity of Staphylococcus aureus in food.After handling liquid food samples using this method, the detection sensitivity of staphylococcus aureus, detection limit and Detection accuracy have all obtained large increase in food liquid, and testing result false negative substantially reduces.
Description
Technical field
The invention belongs to detection technique fields, and in particular to a kind of to improve staphylococcus aureus in food liquid easy to foaming
The method of detection limit.
Background technique
Staphylococcus aureus (Staphylococcus aureus) is a kind of common food-borne pathogens, is deposited extensively
In nature.Staphylococcus aureus is the gram-positive cocci that fat-soluble yellow pigment is generated in staphylococcus, directly
About 0.8-1.0 μm of diameter, it is under microscopy conditions or spreads or agglomerate botryoidalis, no gemma, flagellum, majority is without pod membrane.
Staphylococcus aureus easily contaminated food products, American Centers for Disease Control and Prevention's report display, by golden yellow Portugal
The coccigenic food poisoning of grape accounts for the 2nd, is only second to Escherichia coli, accounts for the 33% of entire food posioning.In recent years,
China poisons by food as caused by staphylococcus aureus every year has occupied the 4th, seriously endangers human health and life peace
Entirely.It is the enterotoxin that it is generated that staphylococcus aureus, which causes an important factor for food poisoning, when S. aureus L-forms pollution reaches certain
The enterotoxin accumulation generated after quantity can cause to poison by food.Therefore the detection limit of staphylococcus aureus is improved, it is accurate to detect
Staphylococcus aureus quantity in food is particularly important.
The detection method of staphylococcus aureus maturation still continues to use traditional national standard method GB 4789.10- at present
2016, sampling amount is 25g (mL), therefore can only detect the part viable bacteria in 25g (mL) sample, and recall rate is compared in actual sample
It pollutes low, is easily easy to appear false negative result, endanger the majority of consumers' safety, cause food safety hazards.Therefore, compel to be essential
The pre-treating method for improving Staphylococcus aureus in food detection carries out the microorganism in sample efficiently rich
Collection, improves detection limit and the sensitivity of staphylococcus aureus.
Summary of the invention
The purpose of the present invention is to provide a kind of sides for improving staphylococcus aureus detection limit in food liquid easy to foaming
Method increases sampling amount, solves the problems, such as to blister in sample pre-treatments enrichment process using effective defoaming agent, using vacuum filter membrane
It filters on the microorganism to miillpore filter in enriched sample, improves detection limit and the sensitivity of Staphylococcus aureus in food.
After handling liquid food samples using this method, the detection sensitivity of staphylococcus aureus, detection limit and inspection in food liquid
It surveys accuracy rate and has all obtained large increase, testing result false negative substantially reduces.
Based on above-mentioned purpose, the present invention is adopted the following technical scheme that:
A method of improving staphylococcus aureus detection limit in food liquid easy to foaming, comprising the following steps:
1) 10mL dimethyl silicone polymer (PMX) defoaming is added in the food liquid sample for taking 500mL easy to foaming, in 20 DEG C of nothings
8000rmp/min is centrifuged 6min under the conditions of bacterium, collects clear liquid；
2) gained supernatant is filtered with polyethers PES miillpore filter, and Enrichment by Microorganisms is combined on miillpore filter；
3) miillpore filter is placed in Φ 55mm 50mL tool plug flat bottom glass cup, is rinsed and is soaked with 10mLHanks eluent
Bubble shakes 2min in microoscillator, and Microbiological release in micropore is made to obtain sample concentration liquid into solution；This operation will be in 3min
Interior completion；
4) Gold Samples staphylococcus aureus detects
1mL sample concentration liquid and 9mL phosphate buffer is taken to be mixed to get dilution bacteria suspension, 10 times of gradient dilutions, every gradient
Bacteria suspension 1mL, be coated on Baird-Parker agar plate；36 DEG C of culture 24-48h count staphylococcus aureus typical case bacterium
It falls, calculated clump count is the quantity of Gold Samples staphylococcus aureus.
In step 2), polyethers PES miillpore filter uses 0.45 μm of polyethers PES miillpore filter of Φ 50mm.
It in step 4), is shaken using whirlpool when mixing, the concussion time is 2min.
The present invention increases the sampling amount of detection first, carries out defoaming treatment to food liquid sample easy to foaming, and 20 DEG C
Under the conditions of high speed centrifugation, remove the macromolecular substances such as fat, albumen in food samples, then use filtering with microporous membrane, will
Staphylococcus aureus in sample is enriched on miillpore filter, the target on the protective elution filter membrane of apparatus
Bacterium, the bacterium solution after collecting elution are sample prepare liquid, and prepare liquid is taken to carry out the detection of Baird-Parker plate count.This method makes
With the object bacteria on the elution filter membrane for being conducive to staphylococcus aureus growth and protective effect and in filter sizes, solution
Standard method of having determined detects the low problem of food liquid staphylococcus aureus detection limit, reaches golden yellow in enrichment food liquid
Staphylococcic purpose improves the detection limit of staphylococcus aureus and sensitivity in food liquid.
Particularly, it shows in the following areas:
1) sample volume that the present invention increases detection can increase to 500g/ by the sampling 25g/mL of standard method
mL.The increase of sampling amount greatly increases the recall rate of objective microbe staphylococcus aureus, the Staphylococcus aureus avoided
The low problem of bacterium missing inspection, few inspection, false negative, the detection practical soiling value of numerical value；
2) be directed to sample easy to foaming, be added defoaming agent dimethyl silicone polymer (PMX) pre-process, can avoid it is subsequent from
The heart, miillpore filter filter excessive problem of blistering in the process；
3) present invention uses low-temperature and high-speed centrifugal treating sample, can effectively remove the fat being rich in sample and albumen etc.
Macromolecular components chaff interferent is conducive to the filtering with microporous membrane enrichment procedure of next step；
4) present invention is rich using the suction filtration even liquid of food samples under 0.45 μm polyether sulfone (PES) miillpore filter vacuum condition
Collect on the bacteriums to miillpore filter such as the staphylococcus aureus in sample, achievees the purpose that be enriched with object bacteria, well solve
The test problems of low-level pathogenic microorganism increase the recall rate of staphylococcus aureus.Both using 0.45 μm of aperture
Objective microbe can effectively be retained to be unlikely to block filter membrane micropore again, material is the miillpore filter of polyether sulfone (PES) compared with other
Less cracky when the filtering with microporous membrane and washing operation of material；
5) present invention elutes the object bacteria in miillpore filter using special Φ 55mm 50mL tool plug flat bottom glass cup.Glass
Glass cup diameter of phi 55mm is slightly larger than filter membrane diameter of phi 50mm, that is, facilitates pick-and-place filter membrane, and can elute solution in active set；Using flat
Bottom glass can make miillpore filter maximum area contact eluent, help to improve the eluting rate of object bacteria；Glass uses
Tool plug sealing when earthquake elutes object bacteria, effectively prevents the splashing evolution of eluent and object bacteria, both can guarantee data
Validity, and can ensure the safety of experiment operator；
6) for the miillpore filter eluent that the present invention uses for balanced salt solution containing 10mLHanks, ingredient can be one
Bacterial cell is effectively protected in fixing time not to be damaged, and is conducive to staphylococcus aureus and is eluted rear certain time few
It measures and saves and grow in Hanks buffer solution；
7) after the present invention is enriched with processing sample, the detection limit of staphylococcus aureus and sensitivity is significantly in food liquid
It improves.
And the studies above achievement of the present invention has no similar application study at home at present, has powerful market competition
Power.
Specific embodiment
Below by specific embodiment, the present invention will be further described.
Embodiment 1
A method of improving staphylococcus aureus detection limit in food liquid easy to foaming, comprising the following steps:
1) 10mL dimethyl silicone polymer (PMX) defoaming is added in the food liquid sample for taking 500mL easy to foaming, in 20 DEG C of nothings
8000rmp/min is centrifuged 6min under the conditions of bacterium, collects clear liquid；
2) gained supernatant is filtered with 0.45 μm of polyethers PES miillpore filter of Φ 50mm, and Enrichment by Microorganisms is combined in miillpore filter
On；
3) miillpore filter is placed in Φ 55mm 50mL tool plug flat bottom glass cup, is rinsed and is soaked with 10mLHanks eluent
Bubble shakes 2min in microoscillator, and Microbiological release in micropore is made to obtain sample concentration liquid into solution；This operation will be in 3min
Interior completion；
4) Gold Samples staphylococcus aureus detects
It taking 1mL sample concentration liquid and 9mL phosphate buffer is mixed to get dilution bacteria suspension, when mixing, is shaken using whirlpool,
The concussion time is 2min, and 10 times of gradient dilutions, the bacteria suspension 1mL of every gradient is coated on Baird-Parker agar plate；36℃
It cultivates 24-48h and counts staphylococcus aureus colonies typical, calculated clump count is Gold Samples staphylococcus aureus
Quantity.
Effect experiment:
1, the selection of debubbling method
For some fluid samples, such as: beer, beverage, soya-bean milk fermented food feature easy to foaming have carried out defoaming effect
The research of fruit.After handling sample using different methods, whether there is or not excessive situation of blistering when observation miillpore filter filters sample, test
Demonstrate,prove defoaming effect.Test specimen the results are shown in Table 1 by taking 500mL beer as an example.
Test result is shown: being handled sample using the dimethyl silicone polymer (PMX) of 10mL, can be reached preferable defoaming effect
Fruit will not influence subsequent miillpore filter and filter operation.
The research of 1. debubbling method of table
2, the selection of centrifugation time
It will be centrifuged under 20 DEG C of aseptic conditions of fluid sample after defoaming treatment, divided greatly with removing fat, albumen etc. in sample
Sub- substance.0min, 2min, 4min, 6min, 8min, 10min are centrifuged respectively with revolving speed 8000rmp/min in test, removal is heavy
It forms sediment, collects centrifuged supernatant.The miillpore filter that 0.45 μm of supernatant, negative pressure of vacuum filter sample, and record filters used in sample
Time situation, influence of the verifying centrifugation time to macromolecular substances in removal sample, determines best centrifugation time.It the results are shown in Table 2.
It is operated, is centrifuged within 4min, Wu Fayou as it can be seen that not being centrifuged and being unable to complete subsequent membrane filtration by 2 test data of table
Effect removes most macromolecular substances, and causing subsequent filter membrane to filter, time-consuming, and centrifugation can be such that the suction filtration time foreshortens to for 6 minutes
Within 2min, the centrifugation longer time has no larger difference with centrifugation 6min.Therefore at 20 DEG C of selection, 8000rmp/min 6min effect
Fruit is best.
The selection of 2. centrifugation time of table
3, the selection of miillpore filter material and aperture
The present invention needs that object bacteria is enriched on miillpore filter to food liquid by way of vacuum filtration, and vacuum is taken out
When filter, there are certain pressure, therefore Material Strength to miillpore filter and toughness have certain requirement.Mixing is chosen in test
Cellulose (CN-CA) filter membrane, polyamide-based filter membrane Nylon (PA-66), polyether sulfone (PES) filter membrane, polypropylene (PP) fibrous filter membrane,
It is compared through filter negative pressure leaching, polyether sulfone (PES) filter membrane breakage rate is minimum.
Test choose respectively aperture be 0.1 μm, 0.22 μm, 0.45 μm, 0.8 μm, 1.2 μm of miillpore filter carry out enrichment effect
Fruit verifying.500mL sample containing bacterium is taken under aseptic condition, after centrifugation removes macromolecular substances, supernatant is taken to pass through different pore size
Miillpore filter, filtered miillpore filter is washed, the solution after washing takes 1mL in Baird-Parker plate, 36 DEG C points
From culture 48h, positive clump count is counted.
The selection in 3. miillpore filter aperture of table
Experiment discovery, because aperture is too small when the filtering with microporous membrane that 0.1 μm and 0.22 μm of diameter, the rate of filtration is especially delayed
Slowly, can not continue to filter after 10s.0.45 μm, 0.8 μm, 1.2 μm of miillpore filter can meet rate of filtration requirement.But
Be 0.8 μm and 1.2 μm of miillpore filter object bacteria accumulation rate it is relatively low, reason may be aperture be greater than object bacteria diameter it is big,
Through filter membrane micro porous filtration into bottle,suction, failing to intercept can be enriched in filter membrane surface, therefore only object bacteria when causing to filter
A small amount of object bacteria causes testing result relatively low.Therefore, the factors such as the rate of filtration, bioaccumulation efficiency are comprehensively considered, using diameter 0.45
μm miillpore filter enrichment staphylococcus aureus effect it is preferable.
4, the selection of maximum sampling amount
When microorganism detection, sampling amount number be directly related to detection object bacteria numerical values recited, sampling amount is bigger, mesh
The recall rate for marking bacterium is bigger, and numerical value is bigger.Therefore, sampling amount is increased as far as possible, could effectively avoid testing result practical
The problem of level of pollution is low, false negative.
Experiment has carried out experimental study to sampling amount, and different amounts of sample is taken to be detected respectively, comparative experiments process
The accuracy of feasibility and testing result.
By taking beer as an example, take respectively 25mL, 50mL, 100mL, 150mL, 200mL, 250mL, 300mL, 350mL, 400mL,
The liquid sample of 450mL, 500mL, 550mL, 600mL containing object bacteria, using 0.45 μm of miillpore filter of Φ 50mm, vacuum is negative
Pressure filters sample, and record filters time situation used in sample, after filtering after filter membrane elution, removes eluent 1mL and is coated on
Baird-Parker agar plate, 36 DEG C of culture 24-48h count staphylococcus aureus colonies typical.It the results are shown in Table 4.
As seen from the data in Table 4, the target bacterium number that filter membrane intercepts increases as sampling amount increases, but sampling amount increase causes
Filtering the time increases, and filters that the time is longer, and it is more to get lodged in the media such as the bacterium in filter sizes, needed for filtering after 550mL liquid
Time increases severely, and needs about 5min, and when 600mL can not filter sample liquid completely.Therefore, selection sampling amount 500mL is best.
The selection of the different sampling amounts of table 4.
5, the selection of miillpore filter eluent
After miillpore filter is enriched with object bacteria, the elution efficiency of filter membrane directly affects the accuracy of result, therefore selects properly
Type of elution and eluent it is particularly important.It selects different solution to elute filtered miillpore filter in test, takes 1mL respectively
Bacterium solution after filter membrane elution does the counting of S. aureus-positive bacterium in 36 DEG C of culture 48h of Baird-Parker plate.
Respectively to Nacl buffer, phosphate buffer PBS, Hanks balanced salt solution, 7.5% sodium chloride meat in test
Soup elution effect is studied.Detailed results are shown in Table 5.
It can be seen that by 5 test data of table, imitated using NaCl buffer, phosphate buffer PBS and 7.5% sodium chloride broth
Fruit is unsatisfactory, and Hanks balanced salt solution can achieve the purpose that elution again and can achieve protection staphylococcus aureus
Purpose, it is best using the effect of 10mL Hanks balanced salt solution elution through Experimental Comparison.
The Selection experiment of 5. eluent of table
6, the selection of miillpore filter elution concussion time
Using the miillpore filter after the elution object bacteria pollution of Hanks balanced salt solution, 5mLHanks solution is first taken to impregnate micro-
Hole filter membrane, microoscillator shake 0s, 30s, 60s, 90s, 120s, 150s, 180s, are taken out filter membrane with aseptic nipper, use
5mLHanks balanced salt solution repeated flushing miillpore filter, elution solution are collected as prepare liquid, are coated on Baird-Parker fine jade
Rouge plate, 36 DEG C of culture 24-48h count staphylococcus aureus colonies typical.It the results are shown in Table 6.
The selection of the concussion time of table 6.
As seen from the data in Table 6, miillpore filter is lower without the bacteria containing amount of concussion elution measured, and increases filter with the concussion time
Bacterium in film surface and micropore is detached from filter membrane into solution, and 120s reaches optimum efficiency, and the viable bacteria amount measured after 120s is gradually
It reduces.Therefore selection concussion 120s object bacteria result of extraction is best.
7, the comparison of this method and national standard method
Under aseptic condition, food liquid sample 500mL is taken, 10mL dimethyl silicone polymer (PMX) pretreatment sample is added,
20 DEG C of 8000rmp/min are centrifuged 6min, remove the macromolecular substances such as fat, albumen in sample, collect centrifuged supernatant, supernatant
Liquid is filtered with 0.45 μm of polyethers PES miillpore filter of Φ 50mm, and Enrichment by Microorganisms is combined on miillpore filter, filtered filter is taken out
Film is placed in Φ 55mm 50mL tool plug flat bottom glass cup, is rinsed and impregnated with 10mLHanks eluent, shaken in microoscillator
2min is swung, Microbiological release in micropore is made to be prepared into sample concentration liquid into solution.Sample concentration prepare liquid is taken to carry out 10 times of ladders
Degree dilution, takes 1mL 10 respectively0、10-1、10-2、10-3、10-4、10-5、10-6The sample solution of concentration gradient, is coated on Baird-
Parker agar plate, 36 DEG C of culture 24-48h count staphylococcus aureus colonies typical.
Same sample 25mL is taken according to the second method of GB4789.10-2016 simultaneously, 10 times of gradient dilutions take 1mL 100、
10-1、10-2、10-3、10-4、10-5、10-6The sample solution of concentration gradient is coated on Baird-Parker agar plate, 36 DEG C of trainings
It supports 24-48h and counts staphylococcus aureus colonies typical.
Detailed results are shown in Table 7.
The comparing result of table 7. this method and national standard method
By experimental result as it can be seen that using the method for the present invention detection food liquid in staphylococcus aureus, it is countable extremely
10-6, and examined according to national standard method only countable to 10-3, the method for the present invention detection limit and sensitivity are significantly higher than national standard side
Method.
8, the compliance test result comparative test of the method for the present invention
With 1 × 102The staphylococcus aureus bacterium solution of CFU/mL pollutes beer sample, and sample is according to described in embodiment 1
Method is detected, while carrying out detection comparison, contrasting detection result such as 8 institute of table using " GB 4789.10-2016 " second method
Show.
The contrasting detection result of the detection method of the invention of table 8 and national standard method
By analysis of experimental data as it can be seen that the present invention improves the sensitivity of detection compared with national standard method, liquid is improved
The recall rate and detection limit of staphylococcus aureus in food samples reduce food liquid due to polluting staphylococcus aureus
Caused food safety risk.
Claims (3)
1. a kind of method for improving staphylococcus aureus detection limit in food liquid easy to foaming, which is characterized in that including following
Step:
1) defoaming of 10 mL dimethyl silicone polymers (PMX) is added in the food liquid sample for taking 500mL easy to foaming, sterile in 20 DEG C
Under the conditions of 8000rmp/min be centrifuged 6min, collect clear liquid；
2) gained supernatant is filtered with polyethers PES miillpore filter, and Enrichment by Microorganisms is combined on miillpore filter；
3) miillpore filter is placed in Φ 55mm 50mL tool plug flat bottom glass cup, is rinsed and is impregnated with 10mL Hanks eluent, in
Microoscillator shakes 2min, and Microbiological release in micropore is made to obtain sample concentration liquid into solution；This operation is complete in 3min
At；
4) Gold Samples staphylococcus aureus detects
1mL sample concentration liquid and 9mL phosphate buffer is taken to be mixed to get dilution bacteria suspension, 10 times of gradient dilutions, the bacterium of every gradient
Suspension 1mL is coated on Baird-Parker agar plate；36 DEG C of culture 24-48h count staphylococcus aureus colonies typical,
Calculated clump count is the quantity of Gold Samples staphylococcus aureus.
2. the pre-treating method of staphylococcus aureus detection limit in food liquid easy to foaming is improved as described in claim 1,
It is characterized in that, polyethers PES miillpore filter uses 0.45 μm of polyethers PES miillpore filter of Φ 50mm.
3. the pre-treating method of staphylococcus aureus detection limit in food liquid easy to foaming is improved as described in claim 1,
It is characterized in that, being shaken when mixing in step 4) using whirlpool, the concussion time is 2min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112301140A (en) * | 2020-11-23 | 2021-02-02 | 浙江省食品药品检验研究院 | Method for detecting staphylococcus aureus in microecological live bacteria product |
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| CN112301140A (en) * | 2020-11-23 | 2021-02-02 | 浙江省食品药品检验研究院 | Method for detecting staphylococcus aureus in microecological live bacteria product |
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