CN103983626A - Reagent for detecting lipase and method for rapidly detecting lipase - Google Patents
Reagent for detecting lipase and method for rapidly detecting lipase Download PDFInfo
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- CN103983626A CN103983626A CN201410215727.1A CN201410215727A CN103983626A CN 103983626 A CN103983626 A CN 103983626A CN 201410215727 A CN201410215727 A CN 201410215727A CN 103983626 A CN103983626 A CN 103983626A
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Abstract
The invention discloses a reagent for rapidly detecting lipase in a dairy food. The reagent comprises a solution A, a solution B and a standard substance, wherein the standard substance is methoxyl resorufin, the solution A is a PBS, and the solution B contains a fluorogenic substrate, a buffer solution, an emulsifying agent and a preservative. The invention further discloses a method for rapidly detecting lipase in the dairy food by using the reagent. The method comprises the steps: drawing a standard curve, reading a sample fluorescence value and calculating the lipase enzyme. According to the method, the sensitivity is high, the operation is simple, the workload is small, the system errors are small, multiple test repeatability is good, rapidness and convenience are achieved, and a detection result can be obtained within 20-60min.
Description
Technical field
The present invention relates to belong to Food Monitoring field, relate to a kind of lipase and detect reagent and use it to detect the method for quick of lipase, particularly dairy products are as the method for quick of lipase in milk.
Background technology
Dairy products are nutritious, are the nutraceutical of ideals of human being, but are also the good nutrient culture media of microorganism simultaneously, belong to the raw-food material of high risk.In numerous microbial contamination, it is the principal element that affects the dairy products shelf-life that psychrophile pollutes.Psychrophile is meeting amount reproduction when milk raw material breast low-temperature storage, although these bacterium can be killed through pasteurize and UHT sterilization phase, but some Pseudomonas in them can produce extremely heat-resisting lipase as pseudomonas, even process through the ultra high temperature sterilization (UHTS) of 140 ℃, still have a small amount of residual.These residual thermostable lipases are activated in the process of dairy products storage, the fat globule in hydrolysis dairy products, thus cause product quality to change, as fat floating layering, there is bitter taste, stale flavor, fat oxidation taste, have a strong impact on dairy products quality.When consumer buys problems product, also often can complain company's product quality problem.The product quality accident causing because of problems every year, has not only brought huge economic loss to company, brand names has been brought to negative effect simultaneously.Lipase activity in rapid screening raw milk, the raw milk of controlling high enzymatic activity enters especially UHT breast production link of dairy products, can effectively reduce the product quality hidden danger that psychrophile amount reproduction brings, improve integral product quality, promote corporate economy's benefit.
To the detection method of lipase, be mainly to adopt standard solution of sodium hydroxide titration (QB/T 1803-1993) at present, adopt the emulsion of olive oil and polyvinyl alcohol (PVA) as substrate, detection method is very complicated, consuming time longer, but results change is large, easily produce error.In addition the common detection method of lipase also comprises Copoloid method and para-nitrophenol method.Copoloid method is according to fatty acid and Cu
2+form green complex compound, at 710nm wavelength place, measure absorbance and carry out enzyme and live quantitatively, Copoloid method degree of accuracy is higher, but complex operation, stability are not high, and due to the method, use a large amount of benzene and carry out the extraction of Copoloid, easily pollute and human body is damaged; Para-nitrophenol method adopts p-nitrophenyl phenolic ester as substrate, after lipase hydrolysis, produce coloured p-nitrophenol, under 420nm wavelength, measure absorbance and carry out enzyme work quantitatively, when para-nitrophenol method is measured enzyme lower lipase alive, deviation is larger, and p-nitrophenol is poisonous, be unsuitable for food inspection.
Summary of the invention
The present invention be directed to the deficiency of art methods, aim to provide a kind of easily and fast, safety and highly sensitive lipase quick detection reagent and detection method thereof, be particularly useful for detecting dairy products as the lipase in milk.
In order to realize object of the present invention, the invention provides a kind of detection reagent of the lipase based on fluorescent method, it comprises A liquid, B liquid and standard items, wherein
Standard items are methoxyl resorufins;
A liquid is PBS damping fluid, fills a prescription to be: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4and water, pH is 7.4;
B liquid contains fluorogenic substrate, damping fluid, emulsifying agent and antiseptic.
Wherein, described fluorogenic substrate is 1,2-oxygen-dilauryl-(±) glycerine base-glutaric acid-6 '-methyl-resorufin ester (being for No. CAS 110033-82-4), and concentration can be 10 μ M～200 μ M, preferably 50 μ M～100 μ M.
Damping fluid in described B liquid is selected from one or more of tartaric acid buffer, sodium-acetate buffer, sodium citrate buffer solution and glycine buffer; Emulsifying agent is selected from one or more of Tween (tween) 20, Tween 40, Tween 60 and Tween 80; Antiseptic is selected from one or more of Proclin 150, Proclin 200, Proclin 300 and sodium azide.
In described detection reagent, the volume ratio of A liquid and B liquid can be preferably 2:1～4:1.
In the present invention, the amount of described detection reagent can, by those skilled in the art according to determining according to technology general knowledge, when using liquid form, can adopt the standard items methoxyl resorufin of variable concentrations, such as 0.1～4mM, such as 100 μ M.
In one embodiment, the damping fluid in described B liquid is sodium-acetate buffer, and emulsifying agent is Tween (tween) 20, and antiseptic is Proclin 300.
In described B liquid, the concentration of damping fluid is 1mM～20mM, and preferred concentration is 1.5mM～10mM; PH is 3.0～6.0, and preferred pH is 4.0～5.0.The concentration of described emulsifying agent is 1/5000～1/500, and preferred concentration is 1/2000～1/800.The concentration of described antiseptic is 1/10000～1/1000, and preferred concentration is 1/5000～1/2000.
In one embodiment, in described B liquid, the concentration of damping fluid is 5mM, and pH is 4.0, and the concentration of emulsifying agent is 1/1000, and the concentration of antiseptic is 1/5000.
The present invention also provides a kind of kit, and it comprises the detection reagent described in above-mentioned any scheme.
Detection reagent of the present invention or kit, for detection of the lipase in dairy products, described dairy products can be selected from that raw milk, Pasteur breast, UHT are newborn, reconstituted milk and milk powder.
In the present invention, raw milk refers to the normal breast changing without any composition of extruding from meet the healthy dairy stock breast of national relevant requirements.Pasteur's breast refers to that only take raw ox (sheep) breast is raw material, the fluid product making through operations such as pasteurizes.UHT breast refers to raw ox (sheep) breast for raw material, adds or does not add reconstituted milk, under the state of continuous flow, is heated at least 132 ℃ and keep the sterilizing of very short time, then the fluid product of making through operations such as sterile fillings.Reconstituted milk refers to condensed milk or/and the raw milk that whole-fat milk powder and water blend into.Milk powder refers to raw ox (sheep) breast for raw material, the powder product through being processed into.
The present invention also provides a kind of method of using aforementioned detection reagent to detect lipase activity in dairy products, and it comprises the following steps:
1) A liquid is mixed by the volume ratio of 2:1～4:1 with B liquid, be mixed with working fluid C;
2) add respectively 5,10,15,20 μ L standard items in 96 hole ELISA Plate, every Kong Zhongzai adds respectively 100～200 μ L working fluid C, mixes the working fluid C that blank is corresponding volume;
3) ELISA Plate is put into 25～37 ℃ of preheated fluorescence microplate readers, with excitation wavelength 510～550nm, preferably 520～540nm excites, and at emission wavelength 580～620nm, preferably 590nm～610nm place is detected, and reads fluorescent value, is recorded as A
mark, blank is recorded as A
empty, with standard items fluorescent value (A
mark-A
empty) for ordinate (unit is RFU), the volume of standard items of take is horizontal ordinate (unit is μ L), drawing standard curve;
4) add 2～20 μ L dairy products samples in 96 hole ELISA Plate, every Kong Zhongzai adds respectively 100～200 μ L working fluid C, mixes the working fluid C that blank is corresponding volume;
5) ELISA Plate is put into 25～37 ℃ of preheated fluorescence microplate readers, with excitation wavelength 510～550nm, preferably 520～540nm excites, and at emission wavelength 580～620nm, preferably 590nm～610nm place is detected, and reads fluorescent value, is recorded as A1
sample, blank is recorded as A1
empty, hatch 20～60min, preferably after 30～60min, again read fluorescent value, be recorded as A2
sample, blank is recorded as A2
empty;
6) by the fluorescent value [A of sample
sample=(A2
sample-A2
empty)-(A1
sample-A1
empty)] in substitution typical curve, from typical curve, read the corresponding standard items volume of this sample, bring following computing formula into and obtain sample lipase activity;
Computing formula is:
Wherein B is sample corresponding standard items volume (μ L) on typical curve
N is the concentration (μ M) of standard items
V is the volume (μ L) that adds sample
T is the time (min) that sample is hatched
In the present invention, enzyme is lived and is defined: under uniform temperature, per minute hydrolysis substrate obtains the fluorescent material of 1 μ mol, is defined as 1 lipase activity unit of force.
In detection method of the present invention, when dairy products sample is milk powder, milk powder is carried out to 8 times of dissolvings, take 100g milk powder, add 700ml ultrapure water to dissolve.
Tool of the present invention has the following advantages:
1) the present invention is based on fluorescence detection method, fluorescence is selected methoxyl resorufin, and methoxyl resorufin is the methyl ether derivant of fluorescence resorufin, compares with resorufin, aspect stability and sensitivity, is all being significantly increased;
2) emission wavelength of methoxyl resorufin is at 580～650nm, under acid condition, excitation wavelength is 450～550nm, under alkali condition, excitation wavelength is 550～580nm, excitation wavelength and the emission wavelength of considering methoxyl resorufin under alkali condition are too approaching, be chosen under acid condition methoxyl resorufin is excited, in order to avoid dairy products in the interference at 450～500nm place, selecting the excitation wavelength of methoxyl resorufin is 510～550nm, preferably 520～540nm simultaneously;
3) fluorogenic substrate is the coupled product of fatty acid and methoxyl resorufin, under coupling state, the fluorescence of methoxyl resorufin is quenched, after lipase cutting fluorogenic substrate, discharge methoxyl resorufin freely, thereby send fluorescence, the amount of the methoxyl resorufin discharging by detection can more accurately calculate the enzyme of lipase and live;
4) of the present invention highly sensitive, the fluorescence intensity of demonstration is strong, accurately the lipase in Rapid Detection dairy products;
5) dairy products are noiseless to detection of the present invention, without dairy products are carried out to pre-treatment;
6) simple to operate, workload is little, and testing result is accurate, and systematic error is little, and test of many times is reproducible;
7) convenient and swift, detection speed is fast, and whole testing process can complete in 20～60min, has greatly saved detection time;
Accompanying drawing explanation
For content of the present invention is more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein:
Fig. 1 is the typical curve of drawing according to embodiment 2
Fig. 2 is the typical curve of drawing according to embodiment 5
Embodiment
The following embodiment that adopts is further elaborated to technical scheme of the present invention, but be to be understood that technical solution of the present invention is not limited to following cited embodiment, the change that any principle according to the present invention is made or substitute and all should fall into scope of the present invention within.
The material using in instructions of the present invention, especially embodiment, reagent, device etc., as do not pointed out especially, all or this area conventional uses commercially available from business.
Embodiment 1: the preparation of working fluid C
1) take NaCl 8g, KCl 0.2g, Na
2hPO
41.42g and KH
2pO
40.27g, adds 800ml ultrapure water and dissolves, and with ultrapure water, is settled to 1L, is formulated as A liquid, 4 ℃ of preservations after high-temperature sterilization;
2) get 1 of 2.5ml 1.5mM, 2-oxygen-dilauryl-(±) glycerine base-glutaric acid-6 '-methyl-resorufin ester, sodium acetate pH 4.0 damping fluids of 150 μ l 0.5M, 25 μ l Tween 20 and 10 μ l Proclin 300, add water and be settled to 50ml, be formulated as B liquid, 4 ℃ keep in Dark Place;
3) A liquid is mixed by the volume ratio of 2:1 with B liquid, be mixed with working fluid C.
Embodiment 2: Specification Curve of Increasing
1) add respectively 5,10,15,20 μ L concentration be the standard items of 100 μ M in 96 hole ELISA Plate, every Kong Zhongzai adds respectively 200 μ L according to the working fluid C of embodiment 1 preparation, mixes the working fluid C that blank is corresponding volume;
2) ELISA Plate is put into 37 ℃ of preheated fluorescence microplate readers, by excitation wavelength, 520nm excites, and at emission wavelength 610nm place, detects, and reading fluorescent value is A
mark, blank is A
empty, with standard items fluorescent value (A
mark-A
empty) for ordinate (unit is RFU), the volume of standard items of take is horizontal ordinate (unit is μ L), drawing standard curve.
As shown in Figure 1, typical curve linearity is fine for the typical curve obtaining, and linear equation is y=2.58x, and coefficient R 2 is 0.998.
Embodiment 3: raw milk and Pasteur's butter oil enzyme enzyme activity determination
1) get 2 raw milk samples, called after 1# and 2#, get Pasteur's breast sample, called after 3#;
2) add respectively 10 μ L samples in 96 hole ELISA Plate, every Kong Zhongzai adds respectively 200 μ L working fluid C, mixes the working fluid C that blank is corresponding volume;
3) ELISA Plate is put into 37 ℃ of preheated fluorescence microplate readers, by excitation wavelength, 520nm excites, and at emission wavelength 610nm place, detects, and reads fluorescent value, is recorded as A1
sample, blank is recorded as A1
empty; After hatching 40min, again read fluorescent value, be recorded as A2
sample, blank is recorded as A2
empty;
4) by the fluorescent value [A of sample
sample=(A2
sample-A2
empty)-(A1
sample-A1
empty)] in substitution typical curve, from typical curve, read the corresponding standard items volume of this sample (B), bring following computing formula into and obtain sample lipase activity:
Wherein B is sample corresponding standard items volume (μ L) on typical curve
N is the concentration (100 μ mol/L) of standard items
V is the volume (μ L) that adds sample
T is the time (min) that sample is hatched
Enzyme is lived and is defined: at 37 ℃, per minute hydrolysis substrate obtains the fluorescent material of 1 μ mol, is defined as 1 lipase activity unit of force.
The fluorescent value data that detection obtains are as following table, and fluorescent value unit is RFU
| ? | Blank | 1# | 2# | 3# |
| 0min | 140 | 152 | 132 | 124 |
| 40min | 199 | 587 | 516 | 340 |
The typical curve y=2.58x obtaining according to embodiment 1, three standard items volumes corresponding to sample are respectively 146 μ L, 126 μ L and 61 μ L.According to formula, calculate, the lipase activity of 1# raw milk is 36U/L, and the lipase activity of 2# raw milk is 31U/L, and the lipase activity of 3# Pasteur breast is 15U/L.
Embodiment 4: the preparation of working fluid C
1) take NaCl 8g, KCl 0.2g, Na2HPO4 1.42g and KH2PO4 0.27g, add 800ml ultrapure water and dissolve, and with ultrapure water, is settled to 1L, is formulated as A liquid, 4 ℃ of preservations after high-temperature sterilization;
2) get 1 of 3.3ml 1.5mM, 2-oxygen-dilauryl-(±) glycerine base-glutaric acid-6 '-methyl-resorufin ester, sodium citrate pH 5.0 damping fluids of 1000 μ l 0.5M, 50 μ l Tween 20 and 25 μ l Proclin 300, add water and be settled to 50ml, be formulated as B liquid, 4 ℃ keep in Dark Place;
3) A liquid is mixed by the volume ratio of 4:1 with B liquid, be mixed with working fluid C.
Embodiment 5: Specification Curve of Increasing
1) add respectively 5,10,15,20 μ L concentration be the standard items of 100 μ M in 96 hole ELISA Plate, every Kong Zhongzai adds respectively 200 μ L according to the working fluid C of embodiment 1 preparation, mixes the working fluid C that blank is corresponding volume;
2) ELISA Plate is put into 37 ℃ of preheated fluorescence microplate readers, by excitation wavelength, 540nm excites, and at emission wavelength 590nm place, detects, and reading fluorescent value is A
mark, blank is A
empty, with standard items fluorescent value (A
mark-A
empty) for ordinate (unit is RFU), the volume of standard items of take is horizontal ordinate (unit is μ L), drawing standard curve.
As shown in Figure 2, typical curve linearity is fine for the typical curve obtaining, and linear equation is y=2.346x, and coefficient R 2 is 0.999.
Obviously, above-described embodiment is only used to exemplary elaboration the solution of the present invention, and the scheme not limiting the present invention in any way.For those of ordinary skill in the field; do not deviating under the spirit and scope of the present invention; can also make other changes in different forms on the basis of the above description, and the apparent variation of being extended out thus or change are still among the protection domain in the invention.
Claims (7)
1. lipase detects a reagent, and it comprises A liquid, B liquid and standard items, wherein
Standard items are methoxyl resorufins;
A liquid is PBS damping fluid, fills a prescription to be: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4and water, pH is 7.4;
B liquid contains fluorogenic substrate, damping fluid, emulsifying agent and antiseptic, and wherein fluorogenic substrate is 1,2-oxygen-dilauryl-(±) glycerine base-glutaric acid-6 '-methyl-resorufin ester.
2. detection reagent according to claim 1, the damping fluid in wherein said B liquid is selected from: one or more in tartaric acid buffer, sodium-acetate buffer, sodium citrate buffer solution and glycine buffer; Emulsifying agent is selected from: one or more in Tween 20, Tween 40, Tween 60 and Tween 80; Antiseptic is selected from one or more in Proclin 150, Proclin 200, Proclin 300 and sodium azide.
3. according to the detection reagent described in any one in claim 1-2, the concentration of wherein said fluorogenic substrate is 10 μ M～200 μ M, preferably 50 μ M～100 μ M; The concentration of the damping fluid in B liquid is 1mM～20mM, preferably 1.5mM～10mM; The concentration of emulsifying agent is 1/5000～1/500, preferably 1/2000～1/800.
4. according to the detection reagent described in any one in claim 1-3, the pH of the damping fluid in wherein said B liquid is 3.0～6.0, and preferred pH is 4.0～5.0; The concentration of antiseptic is 1/10000～1/1000, preferably 1/5000～1/2000.
5. according to the detection reagent described in any one in claim 1-4, for detection of the lipase in dairy products, described dairy products are selected from that raw milk, Pasteur's breast, UHT are newborn, reconstituted milk and milk powder.
6. right to use requires the lipase quick detection reagent described in any one in 1-5 to detect a method for lipase activity in dairy products, comprising:
A liquid is mixed by the volume ratio of 2:1～4:1 with B liquid, be mixed with working fluid C;
Add respectively 5,10,15,20 μ L standard items in 96 hole ELISA Plate, every Kong Zhongzai adds respectively 100～200 μ L working fluid C, mixes the working fluid C that blank is corresponding volume;
ELISA Plate is put into 25～37 ℃ of preheated fluorescence microplate readers, and with excitation wavelength 510～550nm, preferably 520～540nm excites, and at emission wavelength 580～620nm, preferably 590nm～610nm place is detected, and reads fluorescent value, is recorded as A
mark, blank is recorded as A
empty, with the fluorescent value (A of standard items
mark-A
empty) for ordinate: unit is RFU, and the volume of standard items of take is horizontal ordinate: unit is μ L, drawing standard curve;
Add 2～20 μ L dairy products samples in 96 hole ELISA Plate, every Kong Zhongzai adds respectively 100～200 μ L working fluid C, mixes;
ELISA Plate is put into 25～37 ℃ of preheated fluorescence microplate readers, and with excitation wavelength 510～550nm, preferably 520～540nm excites, and at emission wavelength 580～620nm, preferably 590nm～610nm place is detected, and reads fluorescent value, is recorded as A1
sample, blank is recorded as A1
empty, hatch 20～60min, preferably after 30～60min, again read fluorescent value, be recorded as A2
sample, blank is recorded as A2
empty;
By the fluorescent value [A of sample
sample=(A2
sample-A2
empty)-(A1
sample-A1
empty)] in substitution typical curve, from typical curve, read the corresponding standard items volume of this sample, bring following computing formula into and obtain sample lipase activity;
Computing formula is:
Wherein B is sample corresponding standard items volume (μ L) on typical curve
N is the concentration (μ M) of standard items
V is the volume (μ L) that adds sample
T is the time (min) that sample is hatched.
7. kit, it comprises the detection reagent described in any one in claim 1-5.
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Cited By (4)
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| CN104198474A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Detection kit for measuring content of lipase in serum by colorimetric method |
| CN105651715A (en) * | 2016-01-25 | 2016-06-08 | 中国农业科学院农产品加工研究所 | Method and kit for high throughput testing of enzymatic activity in lipase in liquid milk product |
| CN104730014B (en) * | 2015-03-27 | 2017-08-04 | 宁波博泰生物技术有限公司 | A kind of serum lipase determination kit and preparation method thereof |
| CN113640262A (en) * | 2021-07-30 | 2021-11-12 | 安徽科技学院 | Technical method for rapidly identifying compost maturity degree by using fluorescence method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198474A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Detection kit for measuring content of lipase in serum by colorimetric method |
| CN104730014B (en) * | 2015-03-27 | 2017-08-04 | 宁波博泰生物技术有限公司 | A kind of serum lipase determination kit and preparation method thereof |
| CN105651715A (en) * | 2016-01-25 | 2016-06-08 | 中国农业科学院农产品加工研究所 | Method and kit for high throughput testing of enzymatic activity in lipase in liquid milk product |
| CN105651715B (en) * | 2016-01-25 | 2019-04-09 | 中国农业科学院农产品加工研究所 | A kind of high-throughput method and its kit for detecting lipase enzymatic activity in liquid diary product |
| CN113640262A (en) * | 2021-07-30 | 2021-11-12 | 安徽科技学院 | Technical method for rapidly identifying compost maturity degree by using fluorescence method |
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| CN103983626B (en) | 2017-08-29 |
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