CN103983626B - A kind of lipase quick determination method of lipase detection reagent - Google Patents
A kind of lipase quick determination method of lipase detection reagent Download PDFInfo
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Abstract
The invention discloses lipase quick detection reagent in a kind of dairy products, including A liquid, B liquid and standard items, wherein standard items are methoxyl group resorufins, and A liquid is PBS, and B liquid contains fluorogenic substrate, buffer solution, emulsifying agent and preservative.The invention also discloses the method using lipase in the detection reagent quick detection dairy products, including Specification Curve of Increasing, sample fluorescence value are read and lipase activity is calculated.Sensitivity of the present invention is high, and simple to operate, workload is small, and systematic error is small, and test of many times is reproducible, convenient and swift, and testing result can be obtained in 20~60min.
Description
Technical field
The present invention relates to Food Monitoring field is belonged to, it is related to a kind of lipase detection reagent and detects lipase using it
The quick determination method of lipase in quick determination method, more particularly to dairy products such as milk.
Background technology
Dairy products are nutritious, are the nutraceutical of ideals of human being, but are also the good culture medium of microorganism simultaneously, and category is high
Dangerous raw-food material.In numerous microorganism pollutions, psychrophile pollution is to influence the principal element of dairy products shelf-life.It is thermophilic cold
Bacterium can amount reproduction in the newborn low-temperature storage of milk raw material, although these bacterium can be by by pasteurize and UHT sterilization phases
Kill, but some of which Pseudomonas such as pseudomonas can produce extremely heat-resisting lipase, even across 140 DEG C of superelevation
Warm sterilization treatment, still has a small amount of residual.The thermostable lipase of these residuals is activated during dairy products are stored, water
The fat globule in dairy products is solved, so as to cause product quality to change, bitter taste, stale flavor, fat occurs in such as fat floating layering
Fat activated flavour, has a strong impact on dairy products quality.When consumer buys problems product, also often Products quality can be complained to ask
Topic.Every year because of the product quality accident that problems trigger, huge economic loss is not only brought to company, while to enterprise
Brand brings negative effect.Lipase activity in rapid screening raw milk, the raw milk of control high enzymatic activity enters dairy products
Especially UHT breast production links, can be effectively reduced the product quality hidden danger that psychrophile amount reproduction is brought, and improve overall
Product quality, lifting corporate economy benefit.
The detection method to lipase mainly titrates (QB/T 1803-1993) using standard solution of sodium hydroxide at present,
Using the emulsion of olive oil and polyvinyl alcohol as substrate, detection method is sufficiently complex, time-consuming longer, but results change is big,
Easily produce error.In addition the common detection method of lipase also includes Copoloid method and para-nitrophenol method.Copoloid method is foundation
Aliphatic acid and Cu2+Green complex compound is formed, absorbance is determined at 710nm wavelength and is quantified to carry out enzyme activity, Copoloid method accuracy
It is higher, but cumbersome, stability is not high, and because this method carries out the extraction of Copoloid using substantial amounts of benzene, easily make
Into pollution and human body is damaged；Para-nitrophenol method as substrate, is produced using p-nitrophenyl phenolic ester after lipase hydrolysis
Coloured p-nitrophenol, under 420nm wavelength determine absorbance carry out enzyme activity quantify, para-nitrophenol method determine enzyme activity compared with
Deviation is larger during low lipase, and p-nitrophenol is poisonous, is unsuitable for food inspection.
The content of the invention
The present invention be directed to the deficiency of art methods, it is desirable to provide it is a kind of easily and fast, it is safe and highly sensitive
Lipase quick detection reagent and its detection method, particularly for the lipase in detection dairy products such as milk.
In order to realize the purpose of the present invention, the invention provides a kind of detection reagent of the lipase based on fluorescent method,
It includes A liquid, B liquid and standard items, wherein
Standard items are methoxyl group resorufins；
A liquid is PBS, is formulated and is：137mM NaCl, 2.7mM KCl, 10mM Na2HPO4、2mM KH2PO4With
Water, pH is 7.4；
B liquid contains fluorogenic substrate, buffer solution, emulsifying agent and preservative.
Wherein, the fluorogenic substrate is 1,2- oxygen-dilauryl-(±) glycerine base-glutaric acid -6 '-methyl-resorufin
Ester (No. CAS is 110033-82-4), concentration can be 10 μM~200 μM, preferably 50 μM~100 μM.
Buffer solution in the B liquid is selected from tartaric acid buffer, sodium-acetate buffer, sodium citrate buffer solution and glycine
The one or more of buffer solution；Emulsifying agent is selected from Tween (tween) 20, Tween 40, Tween 60 and Tween 80 one kind
Or it is a variety of；Preservative is selected from the one or more of Proclin 150, Proclin 200, Proclin 300 and sodium azide.
In the detection reagent, the volume ratio of A liquid and B liquid can preferably be 2:1~4:1.
In the present invention, the amount of the detection reagent can be determined by those skilled in the art according to according to common technical knowledge,
When using liquid form, the standard items methoxyl group resorufin of various concentrations, such as 0.1~4mM, such as 100 μM can be used.
In one embodiment, the buffer solution in the B liquid is sodium-acetate buffer, and emulsifying agent is Tween (tween)
20, preservative is Proclin 300.
The concentration of buffer solution is 1mM~20mM in the B liquid, and concentration preferably is 1.5mM~10mM；PH be 3.0~
6.0, pH preferably is 4.0~5.0.The concentration of described emulsifying agent is 1/5000~1/500, preferred concentration for 1/2000~
1/800.The concentration of the preservative is 1/10000~1/1000, and concentration preferably is 1/5000~1/2000.
In one embodiment, the concentration of buffer solution is 5mM in the B liquid, and pH is 4.0, and the concentration of emulsifying agent is 1/
1000, the concentration of preservative is 1/5000.
The present invention also provides a kind of kit, and it includes the detection reagent described in any of the above-described kind of scheme.
Detection reagent of the present invention or kit, for detecting the lipase in dairy products, the dairy products can be selected from
Raw milk, Pasteur's breast, UHT breasts, reconstituted milk and milk powder.
In the present invention, raw milk refer to extrude from the healthy dairy stock breast for meeting the relevant requirement of country without it is any into
Divide the normal breast changed.Pasteur's breast refers to only with raw ox (sheep) breast for raw material, through fluid product made from the processes such as pasteurize.
UHT breasts refer to raw ox (sheep) breast for raw material, add or without reconstituted milk, in the state of continuous flowing, are heated at least
132 DEG C and the sterilizing of very short time is kept, then the fluid product being made through processes such as sterile fillings.Reconstituted milk refer to condensed milk or/
The raw milk blent into whole-fat milk powder and water.Milk powder refers to, the processed powder product that is made newborn for raw material with raw ox (sheep).
Present invention also offers it is a kind of using foregoing detection reagent detect dairy products in lipase activity method, it include with
Lower step：
1) A liquid and B liquid are pressed 2:1~4:1 volume ratio mixing, is configured to working solution C；
2) 5,10,15,20 μ L standard items are separately added into 96 hole elisa Plates, 100~200 μ L are separately added into per Kong Zhongzai
Working solution C, is well mixed, and blank control is the working solution C of corresponding volume；
3) ELISA Plate is put into 25~37 DEG C of preheated fluorescence microplate readers, with 510~550nm of excitation wavelength, preferably
520~540nm is excited, and is detected at 580~620nm of launch wavelength, preferably 590nm~610nm, is read fluorescent value, is recorded as
AMark, blank control is recorded as AIt is empty, with standard items fluorescent value (AMark-AIt is empty) be ordinate (unit is RFU), using the volume of standard items as
Abscissa (unit is μ L), draws standard curve；
4) 2~20 μ L dairy food samples are added into 96 hole elisa Plates, 100~200 μ L working solutions are separately added into per Kong Zhongzai
C, is well mixed, and blank control is the working solution C of corresponding volume；
5) ELISA Plate is put into 25~37 DEG C of preheated fluorescence microplate readers, with 510~550nm of excitation wavelength, preferably
520~540nm is excited, and is detected at 580~620nm of launch wavelength, preferably 590nm~610nm, is read fluorescent value, is recorded as
A1Sample, blank control is recorded as A1It is empty, 20~60min is incubated, fluorescent value is again read off after preferably 30~60min, is recorded as
A2Sample, blank control is recorded as A2It is empty；
6) by the fluorescent value [A of sampleSample=(A2Sample-A2It is empty)-(A1Sample-A1It is empty)] substitute into standard curve, from standard curve
The standard items volume corresponding to the sample is read, following calculation formula is brought into and obtains sample lipase activity；
Calculation formula is：
Wherein B is sample standard items volume (μ L) corresponding on standard curve
N is the concentration (μM) of standard items
V is the volume (μ L) for adding sample
T is the time (min) of sample incubation
In the present invention, enzyme activity is defined：Hydrolysis substrate per minute obtains 1 μm of ol fluorescent material, definition under certain temperature
For 1 lipase activity unit of force.
In the detection method of the present invention, when dairy food sample is milk powder, 8 times of dissolvings are carried out to milk powder, that is, weigh 100g
Milk powder, adds 700ml ultra-pure waters and is dissolved.
The invention has the advantages that：
1) present invention is based on fluorescence detection method, and fluorescence selects methoxyl group resorufin, and methoxyl group resorufin is fluorescence
The Methyl ether derivatives of resorufin, compared with resorufin, are all significantly increased in terms of stability and sensitivity；
2) launch wavelength of methoxyl group resorufin excitation wavelength under 580~650nm, acid condition is 450~550nm,
Excitation wavelength is 550~580nm under alkalescence condition, it is contemplated that the excitation wavelength and transmitted wave of methoxyl group resorufin under alkalescence condition
Length is got too close to, and selection is excited to methoxyl group resorufin in acid condition, at the same in order to avoid dairy products 450~
Interference at 500nm, the excitation wavelength of selection methoxyl group resorufin is 510~550nm, preferably 520~540nm；
3) under fluorogenic substrate is the coupled product of aliphatic acid and methoxyl group resorufin, coupling state, methoxyl group resorufin
Fluorescence is quenched, after fatty cleavage fluorogenic substrate, discharges free methoxyl group resorufin, so as to send fluorescence, is led to
The enzyme activity of lipase can more accurately be calculated by crossing the amount for the methoxyl group resorufin that detection is discharged；
4) sensitivity of the invention is high, and the fluorescence intensity of display is strong, lipase that can accurately in Rapid Detection dairy products；
5) detection of the dairy products to the present invention is noiseless, without carrying out pre-treatment to dairy products；
6) simple to operate, workload is small, and testing result is accurate, and systematic error is small, and test of many times is reproducible；
7) convenient and swift, detection speed is fast, and whole detection process can be completed in 20~60min, greatly save detection
Time；
Brief description of the drawings
In order that present disclosure is more likely to be clearly understood, specific embodiment and combination below according to the present invention
Accompanying drawing, the present invention is further detailed explanation, wherein：
Fig. 1 is the standard curve drawn according to embodiment 2
Fig. 2 is the standard curve drawn according to embodiment 5
Embodiment
It is following that technical scheme is further elaborated using embodiment, it is to be understood that this
Inventive technique scheme is not limited to act embodiment set forth below, change or replacement that any principle according to the present invention is made
It should all be within the scope of the invention.
The specification of the present invention, material, reagent, device especially used in embodiment etc., do not refer to especially such as
Go out, be commercially available or commonly used in the art from business.
Embodiment 1：Working solution C preparation
1) NaCl 8g, KCl 0.2g, Na are weighed2HPO41.42g and KH2PO40.27g, plus the dissolving of 800ml ultra-pure waters,
It is settled to ultra-pure water after 1L, high-temperature sterilization and is formulated as A liquid, 4 DEG C of preservations；
2) 2.5ml 1.5mM 1,2- oxygen-dilauryl-(±) glycerine base-glutaric acid -6 is taken '-methyl-resorufin
Ester, the 150 μ l0.5M buffer solutions of sodium acetate pH 4.0,25 μ l Tween 20 and 10 μ l Proclin 300, add water and are settled to
50ml, that is, be formulated as B liquid, and 4 DEG C are kept in dark place；
3) A liquid and B liquid are pressed 2:1 volume ratio mixing, is configured to working solution C.
Embodiment 2：Specification Curve of Increasing
1) be separately added into 5,10,15,20 μ L concentration be 100 μM of standard items into 96 hole elisa Plates, the difference per Kong Zhongzai
The working solution C that 200 μ L are prepared according to embodiment 1 is added, is well mixed, blank control is the working solution C of corresponding volume；
2) ELISA Plate is put into 37 DEG C of preheated fluorescence microplate readers, excited with excitation wavelength 520nm, in launch wavelength
Detected at 610nm, reading fluorescent value is AMark, blank control is AIt is empty, with standard items fluorescent value (AMark-AIt is empty) it is that (unit is ordinate
RFU), using the volume of standard items as abscissa (unit is μ L), standard curve is drawn.
Obtained standard curve is as shown in figure 1, standard curve is linearly fine, and linear equation is y=2.58x, coefficient correlation
R2 is 0.998.
Embodiment 3：Raw milk and Pasteur's butter oil enzyme enzyme activity determination
1) 2 raw milk samples are taken, 1# and 2# is named as, Pasteur's milk sample product is taken, is named as 3#；
2) 10 μ L samples are separately added into 96 hole elisa Plates, 200 μ L working solution C are separately added into per Kong Zhongzai, mixing is equal
Even, blank control is the working solution C of corresponding volume；
3) ELISA Plate is put into 37 DEG C of preheated fluorescence microplate readers, excited with excitation wavelength 520nm, in launch wavelength
Detected at 610nm, read fluorescent value, be recorded as A1Sample, blank control is recorded as A1It is empty；Fluorescence is again read off after incubation 40min
Value, is recorded as A2Sample, blank control is recorded as A2It is empty；
4) by the fluorescent value [A of sampleSample=(A2Sample-A2It is empty)-(A1Sample-A1It is empty)] substitute into standard curve, from standard curve
The standard items volume (B) corresponding to the sample is read, following calculation formula is brought into and obtains sample lipase activity：
Wherein B is sample standard items volume (μ L) corresponding on standard curve
N is the concentration (100 μm of ol/L) of standard items
V is the volume (μ L) for adding sample
T is the time (min) of sample incubation
Enzyme activity is defined：Hydrolysis substrate per minute obtains 1 μm of ol fluorescent material at 37 DEG C, is defined as 1 lipase activity
Unit.
The obtained following table of fluorescence Value Data is detected, fluorescent value unit is RFU
| Blank control | 1# | 2# | 3# | |
| 0min | 140 | 152 | 132 | 124 |
| 40min | 199 | 587 | 516 | 340 |
The standard curve y=2.58x obtained according to embodiment 1, the corresponding standard items volume of three samples is respectively 146 μ
L, 126 μ L and 61 μ L.Calculated according to formula, the lipase activity of 1# raw milks is 36U/L, and the lipase activity of 2# raw milks is
The lipase activity of 31U/L, 3# Pasteur breast is 15U/L.
Embodiment 4：Working solution C preparation
1) NaCl 8g, KCl 0.2g, Na2HPO4 1.42g and KH2PO4 0.27g, plus the dissolving of 800ml ultra-pure waters are weighed,
It is settled to ultra-pure water after 1L, high-temperature sterilization and is formulated as A liquid, 4 DEG C of preservations；
2) 3.3ml 1.5mM 1,2- oxygen-dilauryl-(±) glycerine base-glutaric acid -6 is taken '-methyl-resorufin
Ester, the 1000 μ l0.5M buffer solutions of sodium citrate pH 5.0,50 μ l Tween 20 and 25 μ l Proclin 300, add water constant volume
To 50ml, that is, B liquid is formulated as, 4 DEG C are kept in dark place；
3) A liquid and B liquid are pressed 4:1 volume ratio mixing, is configured to working solution C.
Embodiment 5：Specification Curve of Increasing
1) be separately added into 5,10,15,20 μ L concentration be 100 μM of standard items into 96 hole elisa Plates, the difference per Kong Zhongzai
The working solution C that 200 μ L are prepared according to embodiment 1 is added, is well mixed, blank control is the working solution C of corresponding volume；
2) ELISA Plate is put into 37 DEG C of preheated fluorescence microplate readers, excited with excitation wavelength 540nm, in launch wavelength
Detected at 590nm, reading fluorescent value is AMark, blank control is AIt is empty, with standard items fluorescent value (AMark-AIt is empty) it is that (unit is ordinate
RFU), using the volume of standard items as abscissa (unit is μ L), standard curve is drawn.
Obtained standard curve is as shown in Fig. 2 standard curve is linearly fine, and linear equation is y=2.346x, coefficient correlation
R2 is 0.999.
Obviously, above-described embodiment is used for the purpose of exemplary elaboration the solution of the present invention, without limiting this in any way
The scheme of invention.For those of ordinary skill in the field, under without departing from the spirit and scope of the present invention, upper
State it is bright on the basis of can also make other changes in different forms, and the obvious change thus extended out
Among changing or changing still in the protection domain of the invention.
Claims (1)
1. a kind of method of lipase activity in lipase detection reagent detection dairy products, it is characterised in that：
Lipase detection reagent includes A liquid, B liquid and standard items, wherein：
Standard items are methoxyl group resorufins；
A liquid is PBS, is formulated and is：137mM NaCl, 2.7mM KCl, 10mM Na2HPO4、2mM KH2PO4And water, pH is
7.4；
B liquid contains fluorogenic substrate, buffer solution, emulsifying agent and preservative；Wherein：
Fluorogenic substrate is 1,2- oxygen-dilauryl-(±) glycerine base-glutaric acid -6 '-methyl-resorufin ester；The fluorescence bottom
The concentration of thing is 50 μM~100 μM；
Buffer solution in the B liquid is selected from：Tartaric acid buffer, sodium-acetate buffer, sodium citrate buffer solution and glycine are slow
One or more in fliud flushing；The concentration of buffer solution is 1.5mM~10mM；PH is 4.0~5.0；
Emulsifying agent is selected from：One or more in Tween 20, Tween 40, Tween 60 and Tween 80；
One or more of the preservative in Proclin 150, Proclin 200, Proclin 300 and sodium azide；
Described lipase detection reagent is used to detect the lipase in dairy products, and the dairy products are selected from raw milk, Pasteur's breast, UHT
Breast, reconstituted milk and milk powder；
The method of lipase activity in described lipase detection reagent detection dairy products, including：
1) A liquid and B liquid are pressed 2:1~4:1 volume ratio mixing, is configured to working solution C；
2) 5 μ L are separately added into, 10 μ L, 15 μ L, 20 μ L standard items are separately added into 100~200 into 96 hole elisa Plates per Kong Zhongzai
μ L working solution C, are well mixed, and blank control is the working solution C of corresponding volume；
3) ELISA Plate is put into 25~37 DEG C of preheated fluorescence microplate readers, excited with 520~540nm of excitation wavelength, in hair
Detected at the long 590nm~610nm of ejected wave, read fluorescent value, be recorded as AMark, blank control is recorded as AIt is empty, with the fluorescence of standard items
Value AMark-AIt is emptyFor ordinate：Unit is RFU, using the volume of standard items as abscissa：Unit is μ L, draws standard curve；
4) 2~20 μ L dairy food samples are added into 96 hole elisa Plates, 100~200 μ L working solution C is separately added into per Kong Zhongzai, mixes
Close uniform；
5) ELISA Plate is put into 25~37 DEG C of preheated fluorescence microplate readers, excited with 520~540nm of excitation wavelength, in hair
Detected at the long 590nm~610nm of ejected wave, read fluorescent value, be recorded as A1Sample, blank control is recorded as A1It is empty, it is incubated 30~60min
Fluorescent value is again read off afterwards, is recorded as A2Sample, blank control is recorded as A2It is empty；
6) by the fluorescent value [A of sampleSample=(A2Sample-A2It is empty)-(A1Sample-A1It is empty)] substitute into standard curve, read from standard curve
Standard items volume corresponding to the sample, brings following calculation formula into and obtains sample lipase activity；
Calculation formula is：
Lipase activity
Wherein B is sample standard items volume corresponding on standard curve：Unit is μ L
N is the concentration of standard items：Unit for μM
V is the volume for adding sample：Unit is μ L
T is the time of sample incubation：Unit is min.
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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 |
| 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 |
| CN105651715B (en) * | 2016-01-25 | 2019-04-09 | 中国农业科学院农产品加工研究所 | A method for high-throughput detection of lipase enzyme activity in liquid dairy products and its kit |
| CN113640262A (en) * | 2021-07-30 | 2021-11-12 | 安徽科技学院 | A technical method for rapid identification of compost maturity by fluorescence method |
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| CN101131389A (en) * | 2006-08-22 | 2008-02-27 | 浙江养生堂天然药物研究所有限公司 | Method for screening pancreatic lipase restrainer from traditional Chinese medicine |
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| DE3516001A1 (en) * | 1985-05-03 | 1986-11-06 | Boehringer Mannheim Gmbh, 6800 Mannheim | LIPASE COLOR TEST |
| JPH09215500A (en) * | 1996-02-13 | 1997-08-19 | Towns:Kk | Substrate solution for lipase analysis and reagent solution kid for lipase analysis |
| JP2008306942A (en) * | 2007-06-12 | 2008-12-25 | Fujifilm Corp | Dry analytical element for lipase measurement |
| CN102621138B (en) * | 2012-04-06 | 2014-06-18 | 上海蓝怡科技有限公司 | Preparation method of micro-emulsion kit |
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| CN101131389A (en) * | 2006-08-22 | 2008-02-27 | 浙江养生堂天然药物研究所有限公司 | Method for screening pancreatic lipase restrainer from traditional Chinese medicine |
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| 原料乳中嗜冷菌及其主要热稳定性酶类的研究进展;郭德军等;《中国乳品工业》;20061231;第34卷(第8期);第48页右栏第4段 * |
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Effective date of registration: 20190517 Address after: 223201 A4-5 Floor, Huai'an Wisdom Valley, 19 Meigao Road, Huai'an Economic and Technological Development Zone, Jiangsu Province Patentee after: Huaian Anfu Technology Co. Ltd. Address before: Room D304, D District, 3rd Floor, 12 Shangdi Information Road, Haidian District, Beijing 100085 Patentee before: Beijing peaceful Lv Yuan Science and Technology Ltd. |