CN106153613A - The detection method of sulbactam in milk - Google Patents
The detection method of sulbactam in milk Download PDFInfo
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- CN106153613A CN106153613A CN201610448378.7A CN201610448378A CN106153613A CN 106153613 A CN106153613 A CN 106153613A CN 201610448378 A CN201610448378 A CN 201610448378A CN 106153613 A CN106153613 A CN 106153613A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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Abstract
The invention discloses the detection method of sulbactam in milk.Described method includes: the antibiotic in antibiotic positive controls, beta lactamase positive controls and sample sets is detected by (1) respectively, wherein, described antibiotic positive controls is obtained by carrying out the milk without antibiotic, beta lactamase and sulbactam with antibiotic mixing；Described beta lactamase positive controls is obtained by carrying out the milk without antibiotic, beta lactamase and sulbactam, antibiotic and beta lactamase mixing；Milk to be measured, antibiotic and beta lactamase are carried out mixing obtained by described sample sets；(2) based on testing result, sulbactam in described milk to be measured is qualitatively judged.Sulbactam in milk can be detected by the method for the present invention exactly, and easy and simple to handle.
Description
Technical field
The present invention relates to field of food.In particular it relates to the detection method of sulbactam in milk.
Background technology
Along with the development of raising dairy cattle industry, antibiotic is widely used in terms of prevention and treatment cow disease.So
And, the antibiotic in Contents in Cows can flow in Lac Bovis seu Bubali along with mammary gland, and in Lac Bovis seu Bubali, health can be caused not by the antibiotic of residual
Profit impact.Thus, the antibiotic remains in Lac Bovis seu Bubali is limited the quantity and is made explicit provisions by current China.But, some bad businessman profit
The feature of antibiotic can be decomposed with beta-lactamase, in milk, add beta-lactamase, to cover and to connive antibiotic
Abuse, additionally, after the edible milk containing beta-lactamase, can make the drug effect of the antibiotic that people takes in reduce, it is impossible to realize controlling
Treat the effect of disease.Therefore, what Ministry of Public Health was announced " may the non-edible material from soybeans of illegal interpolation and the food additive of easy abuse in food
Add agent kind list " in specifically mentioned beta-lactamase.
But, at present the detection method of antibiotic in Lac Bovis seu Bubali and beta-lactamase is still required study.
Summary of the invention
It is contemplated that at least solve one of technical problem present in prior art.
It should be noted that the present invention is following discovery based on inventor and completes:
Inventor when the milk detected without beta-lactamase is detected it was unexpectedly observed that this milk not
Without beta-lactamase, but beta-lactamase is inhibited enzyme to live by the beta-lactamase inhibitor of entitled " sulbactam ", and then
Cannot be detected.
In view of this, inventor furthers investigate discovery, method based on existing mensuration antibiotic, can be by measuring cattle
Whether milk exists antibiotic such that it is able to infer and whether there is beta-lactamase, can speculate further whether exist easypro
Batan medicine.
To this end, the present invention proposes the detection method of sulbactam in a kind of milk.According to embodiments of the invention, described side
Method includes: the antibiotic in antibiotic positive controls, beta-lactamase positive controls and sample sets is examined by (1) respectively
Surveying, wherein, described antibiotic positive controls is by by the milk without antibiotic, beta-lactamase and sulbactam and antibiosis
Element carries out mixing obtained；Described beta-lactamase positive controls be by will without antibiotic, beta-lactamase and relax
The milk of Batan, antibiotic and beta-lactamase carry out mixing obtained；Described sample sets be by milk to be measured, antibiotic and
Beta-lactamase carries out mixing obtained；(2) based on following standard, carry out sulbactam in described milk to be measured qualitative sentencing
Calmly: 1) described antibiotic positive controls does not detects antibiotic in detection antibiotic, described beta-lactamase positive controls
And described sample sets detects antibiotic, containing sulbactam in the most described milk to be measured；2) in described antibiotic positive controls
Detection antibiotic, described beta-lactamase positive controls do not detect in antibiotic and described sample sets and do not detect antibiotic,
The most described milk to be measured does not contains sulbactam；3) described antibiotic positive controls does not detects antibiotic, then testing result
Invalid；And 4) described antibiotic positive controls detects in antibiotic and described beta-lactamase positive controls detect
Antibiotic, then testing result is invalid.Thus, can detect exactly according to the detection method of sulbactam in the milk of the present invention
Whether milk exists sulbactam, and easy and simple to handle.
Inventor finds, sulbactam can suppress beta-lactam enzymatic activity and beta-lactamase can decompose antibiotic, logical
Cross in milk to be measured, add antibiotic and beta-lactamase, then antibiotic is detected, and then reversely infer milk
In whether there is sulbactam.Specifically, if not detecting and milk existing antibiotic, then there is beta-lactam in explanation milk
Enzyme, and then show in milk without sulbactam.Otherwise, if detecting and milk existing antibiotic, then explanation milk in without β-
Lactamase, and then show milk exists sulbactam.
Further, inventor finds, antibiotic and beta-lactamase in order to verify addition are the most active (drug effect), needs
Will be only containing antibiotic and contain antibiotic and beta-lactamase as a control group simultaneously, antibiotic be measured.Specifically
Ground, adds antibiotic, as antibiotic positive control in the known milk without antibiotic, beta-lactamase and sulbactam
Group, adds antibiotic and beta-lactamase in the known milk without antibiotic, beta-lactamase and sulbactam, as β-
Lactamase positive matched group, measures in two kinds of matched groups whether there is antibiotic respectively.If detecting antibiotic positive control
There is antibiotic in group, then prove that antibiotic drug effect is normal, experimental result is effective, otherwise then without drug effect, experimental result is invalid.If
Determine and beta-lactamase positive controls exist antibiotic, and determine in antibiotic positive controls and there is antibiotic,
Then prove beta-lactamase inactivation, otherwise, if detecting antibiotic-free in beta-lactamase positive controls, and detect antibiosis
There is antibiotic in element positive controls, then beta-lactam enzymatic activity is normal.Thereby, it is possible to ensure the accuracy of testing result.
It should be noted that the method for antibiotic detection is not made considered critical, as long as antibiotic can be detected effectively
Whether exist.Specifically, can use the method such as test kit or inhibition zone that antibiotic is detected.
During actually detected, antibiotic positive controls, beta-lactamase positive control can be determined as required
The antibiotic concentration of mixing in group and sample sets, the application does not make considered critical, as long as concentration sufficiently achieves known detection antibiosis
The detection limit of element method.According to a particular embodiment of the invention, the end of antibiotic in the mixed liquor obtained by described mixing
Concentration is 4～6 μ g/L.If excessive concentration, being easily caused beta-lactamase cannot decompose it completely, thus the bar that relaxes occurs
Smooth false positive.
Also, it should be noted the kind of antibiotic is not made considered critical by the present invention, as long as can be by beta-lactamase
Decompose.According to embodiments of the invention, described antibiotic includes penicillin, penicillin sodium, ampicillin.
During actually detected, mixing in beta-lactamase positive controls and sample sets can be determined as required
Beta-lactam enzyme concentration, the application does not make considered critical.According to a particular embodiment of the invention, at described beta-lactam enzyme positive
In matched group and sample sets, in the mixed liquor obtained by described mixing final concentration of the 3～4U/mL of beta-lactamase.If concentration
Too high, it is easily caused the sulbactam in milk to be measured and cannot completely inhibit its activity, thus sulbactam false negative occurs.
It should be noted that in beta-lactamase positive controls and sample sets, in mixed liquor, the end of antibiotic is dense
Degree refers to be present in mixed liquor, and does not starts by the concentration of the zymolytic antibiotic of beta-lactam, if containing in milk to be measured
Antibiotic, then final concentration does not contains this part antibiotic.In like manner, for the explanation of the final concentration of beta-lactamase, at this no longer
Repeat.
According to embodiments of the invention, in described antibiotic positive controls and beta-lactamase positive controls, institute
Stating milk is plain chocolate or reconstituted milk, preferably reconstituted milk.According to a preferred embodiment of the invention, described reconstituted milk is by by perseverance
Natural milk powder is dissolved in the water obtained.Inventor it was unexpectedly observed that dairy company Fonterra of New Zealand produce milk powder impurity relatively
Few, disturb relatively low.
According to embodiments of the invention, detection is limited to 5mg/L.Inventor finds, sulbactam minimal detectable concentration (i.e. detects
Limit) it is 5mg/mL, during less than detection limit, it is impossible to completely inhibit the activity of beta-lactamase, thus be sulbactam false negative.
It addition, the present invention proposes the detection method of sulbactam in another kind of milk.According to embodiments of the invention, described
Method includes:
Take 1mL and do not contain antibiotic, beta-lactamase and the reconstituted milk of sulbactam, add 1mg/L penicillin sodium solution 6 μ L,
Making the final concentration of 6 μ g/L of penicillin sodium, mixing, as antibiotic positive controls；
Take 1mL without antibiotic, beta-lactamase and the reconstituted milk of sulbactam, be separately added into penicillin sodium solution and β-interior
Amidase standard solution, makes the final concentration of 6 μ g/L of penicillin sodium, and the final concentration of 4U/mL of beta-lactamase, as beta-lactam
Enzyme positive matched group；
Take 1mL milk to be measured, be separately added into penicillin sodium solution and beta-lactamase standard solution, make penicillin sodium eventually
Concentration is 6 μ g/L, and the final concentration of 4U/mL of beta-lactamase, as sample sets；
Respectively described antibiotic positive controls, beta-lactamase positive controls and sample sets are placed in 40 DEG C ± 1 DEG C
Water bath is hatched 1 hour 20 minutes；
Utilize antibiotic detection kit respectively to the described antibiotic positive controls after hatching, beta-lactam enzyme positive
Antibiotic in matched group and sample sets detects,
Based on following standard, sulbactam in described milk to be measured is qualitatively judged:
Described antibiotic positive controls detects in antibiotic, described beta-lactamase positive controls and does not detects antibiosis
Plain and described sample sets detects antibiotic, containing sulbactam in the most described milk to be measured；
Described antibiotic positive controls detects in antibiotic, described beta-lactamase positive controls and does not detects antibiosis
Plain and described sample sets does not detects antibiotic, the most described milk to be measured does not contains sulbactam；
Do not detect antibiotic in described antibiotic positive controls, then testing result is invalid；And
Described antibiotic positive controls detects antibiotic and described beta-lactamase positive controls detect anti-
Raw element, then testing result is invalid.
Thus, can detect in milk whether exist exactly according to the detection method of sulbactam in the milk of the present invention
Sulbactam, and easy and simple to handle.
The additional aspect of the present invention and advantage will part be given in the following description, and part will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Detailed description of the invention
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this
Bright, and be not considered as limiting the invention.
Embodiment 1
In this embodiment, in following manner sulbactam in milk is detected:
1, reagent and solution
Spain ECLIPSE50 (being called for short E50) antibiotic detection kit, penicillin sodium standard substance, beta-lactamase mark
Quasi-product (Nat'l Pharmaceutical & Biological Products Control Institute produces, and concentration is 4,460,000 U/mL), sulbactam standard substance,
Reconstituted milk: by permanent natural milk powder is dissolved in the water obtained, without antibiotic, beta-lactamase and easypro bar
Smooth.
2, equipment and instrument
Whirlpool mixed instrument, constant water bath box (40 DEG C ± 1 DEG C), the couveuse (65 DEG C ± 1 DEG C) needed for antibiotic detection, moves
Liquid device (10uL, 100uL and 1mL), centrifuge tube (1.5mL, 2mL and 15mL).
3, operating procedure
(1) taking 1mL reconstituted milk, add 1mg/L penicillin sodium solution 6 μ L, final concentration of 6 μ g/L, whirlpool mixes, as anti-
Raw element positive controls；
(2) take 1mL reconstituted milk, be separately added into penicillin sodium solution and beta-lactamase standard solution, make penicillin sodium
Final concentration of 6 μ g/L, the final concentration of 4U/mL of beta-lactamase, as beta-lactamase positive controls；
(3) take 1mL milk to be checked, be separately added into penicillin sodium solution and beta-lactamase standard solution, make penicillin
The final concentration of 6 μ g/L of sodium, the final concentration of 4U/mL of beta-lactamase, as sample sets；
(4) respectively antibiotic positive controls, beta-lactamase positive controls and sample sets are placed in 40 DEG C ± 1 DEG C water
Bath cabinet is hatched 1 hour 20 minutes；
(5) the antibiotic positive controls after hatching with the detection of E50 antibiotic detection kit, beta-lactam enzyme positive pair
According to whether group and sample sets exist antibiotic.
The antibiotic result of requirement antibiotic positive controls is positive, the antibiotic inspection of beta-lactamase positive controls
Survey result effective by the negative penicillin sodium added with proof and beta-lactamase, otherwise must reform.
Table 1 gives E50 result judgement table.Wherein purple represents the antibiotic testing result positive, and yellow represents antibiotic
Testing result is negative.It can be seen that utilize the method can determine in milk to be measured whether contain sulbactam effectively.
Table 1E50 result judges table
| Detection sample | E50 | Antibiotic result | Beta-lactamase result | Sulbactam result |
| Antibiotic positive controls | Purple | Positive (+) | Negative (-) | / |
| Beta-lactamase positive controls | Yellow | Negative (-) | Positive (+) | / |
| Treat sample | Yellow | Negative (-) | Positive (+) | Negative (-) |
| Treat sample | Purple | Positive (+) | Negative (-) | Positive (+) |
Note: negative expression does not exists or concentration is less than method detection limit, positive expression exists and concentration is not less than method and examines
Rising limit.
Embodiment 2
Accuracy detects
Concrete operations:
(1) take three parts of New Zealand's reconstituted milks, be separately added into 1mg/L, 5mg/L and 10mg/L sulbactam, obtain mark-on sample
1, mark-on sample 2 and mark-on sample 3,
Take two parts of reconstituted milks, be separately added into 10mg/L and 100mg/L sulbactam, obtain mark-on sample 4 and mark-on sample 5；
(2) according to the method for embodiment 1, sulbactam in reconstituted milk and mark-on sample 1～3 is carried out 2 parallel inspections respectively
Survey；
(3) according to the method for embodiment 1, sulbactam in mark-on sample 4 and 5 is carried out 2 Parallel testings respectively, distinguish
In, without step (1)～(3), directly carry out hatching process by mark-on sample 4 and 5.
Result is as shown in table 2.By mark-on sample 1～3 it can be seen that when sulbactam concentration is 1mg/L, beta-lactamase
The most suppressed, there is false negative in result, and sulbactam is undetected.When sulbactam concentration is 5mg/L, it is possible to suppression β-
Internal amidase activity, and then sulbactam can be detected.Thus, the sulbactam detection of the inventive method is limited to 5mg/L.
By mark-on sample 4 and 5 it can be seen that be individually added in milk sample respectively sulbactam standard substance 10mg/L,
100mg/L, antibiotic testing result is negative, illustrates that added sulbactam concentration can not cause the reagent display positive, card
Bright sulbactam is because inhibiting the activity of beta-lactamase to make the reagent display positive, and then shows the accurate of testing result
Property.
Table 2 Accuracy Verification
Embodiment 3
Stability is verified
Concrete steps:
(1) choose the milk sample without sulbactam of 20 different batches, respectively add in the milk of 10 different batches
Enter the sulbactam of 10mg/L, be denoted as sample 1～10；The milk of other 10 different batches, without sulbactam, is denoted as sample 11
～20；
(2) respectively sulbactam in sample 1～20 is detected according to the method for embodiment 1.
Result is as shown in table 3.It can be seen that the method for the present invention can detect whether sulbactam exists exactly, and
Stability is preferable.
Table 3 stability is verified
Below in conjunction with embodiment, the solution of the present invention is explained.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted concrete technology or bar in embodiment
Part, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or instrument
Device unreceipted production firm person, be can by city available from conventional products.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
Example " or the description of " some examples " etc. means to combine this embodiment or example describes specific features, structure, material or spy
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be in office
One or more embodiments or example combine in an appropriate manner.Additionally, in the case of the most conflicting, the skill of this area
The feature of the different embodiments described in this specification or example and different embodiment or example can be tied by art personnel
Close and combination.
Although above it has been shown and described that embodiments of the invention, it is to be understood that above-described embodiment is example
Property, it is impossible to being interpreted as limitation of the present invention, those of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, revises, replaces and modification.
Claims (8)
1. the detection method of sulbactam in a milk, it is characterised in that including:
Take the reconstituted milk without antibiotic, beta-lactamase and sulbactam, add penicillin sodium solution, make penicillin sodium final concentration
Being 6 μ g/L, mixing, as antibiotic positive controls；
Take the reconstituted milk without antibiotic, beta-lactamase and sulbactam, be separately added into penicillin sodium solution and beta-lactamase
Standard solution, makes the final concentration of 6 μ g/L of penicillin sodium, and the final concentration of 4U/mL of beta-lactamase, as beta-lactam enzyme positive
Matched group；
Take milk to be measured, be separately added into penicillin sodium solution and beta-lactamase standard solution, make penicillin sodium final concentration of 6
μ g/L, the final concentration of 4U/mL of beta-lactamase, as sample sets；
Respectively described antibiotic positive controls, beta-lactamase positive controls and sample sets are placed in 40 DEG C ± 1 DEG C water-bath
Case is hatched 1 hour 20 minutes；
Utilize antibiotic detection kit respectively to the described antibiotic positive controls after hatching, beta-lactamase positive control
Antibiotic in group and sample sets detects,
Based on following standard, sulbactam in described milk to be measured is qualitatively judged:
Described antibiotic positive controls detects in antibiotic, described beta-lactamase positive controls do not detect antibiotic and
Described sample sets detects antibiotic, containing sulbactam in the most described milk to be measured；
Described antibiotic positive controls detects in antibiotic, described beta-lactamase positive controls do not detect antibiotic and
Described sample sets does not detects antibiotic, the most described milk to be measured does not contains sulbactam；
Do not detect antibiotic in described antibiotic positive controls, then testing result is invalid；And
Described antibiotic positive controls detects in antibiotic and described beta-lactamase positive controls and detects antibiotic,
Then testing result is invalid.
2. the detection method of sulbactam in a milk, it is characterised in that including:
(1) respectively the antibiotic in antibiotic positive controls, beta-lactamase positive controls and sample sets is detected,
Wherein,
Described antibiotic positive controls is by entering with antibiotic without the milk of antibiotic, beta-lactamase and sulbactam
Obtained by row mixing；
Described beta-lactamase positive controls is by by the milk without antibiotic, beta-lactamase and sulbactam, antibiosis
Element and beta-lactamase carry out mixing obtained；
Milk to be measured, antibiotic and beta-lactamase are carried out mixing obtained by described sample sets；
(2) based on following standard, sulbactam in described milk to be measured is qualitatively judged:
1) described antibiotic positive controls does not detects antibiotic in detection antibiotic, described beta-lactamase positive controls
And described sample sets detects antibiotic, containing sulbactam in the most described milk to be measured；
2) described antibiotic positive controls does not detects antibiotic in detection antibiotic, described beta-lactamase positive controls
And described sample sets does not detects antibiotic, the most described milk to be measured does not contains sulbactam；
3) do not detect antibiotic in described antibiotic positive controls, then testing result is invalid；And
4) described antibiotic positive controls detects antibiosis in detection antibiotic and described beta-lactamase positive controls
Element, then testing result is invalid.
Method the most according to claim 2, it is characterised in that in the mixed liquor obtained by described mixing, the end of antibiotic is dense
Degree is 4～6 μ g/L.
Method the most according to claim 2, it is characterised in that in described beta-lactamase positive controls and sample sets
In, in the mixed liquor obtained by described mixing final concentration of the 3～4U/mL of beta-lactamase.
Method the most according to claim 2, it is characterised in that at described antibiotic positive controls and beta-lactamase sun
In property matched group, described milk is plain chocolate or reconstituted milk, preferably reconstituted milk.
Method the most according to claim 5, it is characterised in that described reconstituted milk is by permanent natural milk powder is dissolved in water
Obtained by.
Method the most according to claim 2, it is characterised in that detection is limited to 5mg/L.
Method the most according to claim 2, it is characterised in that described antibiotic includes penicillin, penicillin sodium, ammonia benzyl west
Woods.
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