CN108426879A - The detection method of l-cn content in a kind of milk powder - Google Patents
The detection method of l-cn content in a kind of milk powder Download PDFInfo
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- CN108426879A CN108426879A CN201810498612.6A CN201810498612A CN108426879A CN 108426879 A CN108426879 A CN 108426879A CN 201810498612 A CN201810498612 A CN 201810498612A CN 108426879 A CN108426879 A CN 108426879A
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- milk powder
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Abstract
The embodiment of the invention discloses a kind of detection methods of l-cn content in milk powder, include the following steps：Gauge orifice and sample well are set on ELISA Plate, 200ul standard items are taken to be added in gauge orifice, 200ul samples are taken to be added in sample well, 80ul colour developing working solutions and 10ul acetyl coenzyme As are sequentially added in gauge orifice and each hole of sample well, 5min is reacted under room temperature, and absorbance OD1 is read in 405nm；Gauge orifice and sample well are added 10ul acetylcarnitines per hole and shift enzyme solutions mixing, react 10min under room temperature, in 405nm test absorbances OD2；L-cn content in sample is calculated using absorbance OD1 and OD2 combination l-cn standard curve.This l-cn assay method is substantially reduced relative to National Standard Method, testing cost, while it can carry out batch detection, accordingly save detection time, greatly promote detection efficiency so that l-cn measures more efficient and convenient.
Description
Technical field
The present invention relates to a kind of detection methods of l-cn content in field of detection of food safety more particularly to milk powder.
Background technology
L-cn (L-carnitine), it is that one kind promotes adipose conversion for energy to be also l-carnitine, transliteration Carnitine
Amino acid, be a kind of nutritional ingredient in milk powder, because it is a kind of biostearin nutrient, be also named as vitamin
Bt.L-cn cannot be below 30mg/kg in national standard GB14880-2012 regulation children's milk powder, left-handed meat in other modulation milk powder
Alkali cannot be below 300mg/kg.National Standard Method detects l-cn content at present, using ultraviolet spectrophotometry, on the one hand due to second
On the other hand acyl coenzyme A and carnitine transacetylase somewhat expensive spend the time longer in the colorimetric stage.Therefore, national standard at present
The method of method detection l-cn content is significantly limited by the defect of itself in use.
Therefore, the prior art need further to develop.
Invention content
In view of the above technical problems, an embodiment of the present invention provides a kind of detection method of l-cn content in milk powder,
To solve the problems, such as that existing detection method testing cost is high, one-time detection amount is few, detection time is long.
The detection method of l-cn content in a kind of milk powder, wherein include the following steps：
Gauge orifice and sample well are set on ELISA Plate, takes 200ul standard items to be added in gauge orifice, takes 200ul samples
It is added in sample well, 80ul colour developing working solutions and 10ul acetyl coenzyme As, room temperature is sequentially added in gauge orifice and each hole of sample well
Under the conditions of react 5min, read absorbance OD1 in 405nm；
Gauge orifice and sample well are added 10ul acetylcarnitines per hole and shift enzyme solutions mixing, react 10min under room temperature,
In 405nm test absorbances OD2；
L-cn content in sample is calculated using absorbance OD1 and OD2 combination l-cn standard curve.
The detection method of l-cn content in the milk powder, wherein the gauge orifice is set as 5,5 gauge orifices
The standard concentration of addition is 1.6ug/ml, 3.2ug/ml, 6.4ug/ml, 9.6ug/ml, 16ug/ml.
The detection method of l-cn content in the milk powder, wherein pre-processed before sample detection：Accurately weigh
In 50ml centrifuge tubes with a scale, with 40 DEG C of warm water dissolvings of 15mL, 13% high chlorine of 5mL is added in sample uniformly mixed 2.5g
Acid solution, static 20min, is settled to scale, mixing is filtered with quantitative filter paper with distilled water after mixing；
Filtrate 20mL is taken, after being 12.5~13.0 with 4mol/L potassium hydroxide solution tune pH, is placed in 40 DEG C of water-bath 60min.
After cooling with 13% perchloric acid tune pH be 7.0~7.5.Sample liquid is transferred in 50mL volumetric flasks, with distilled water constant volume；Mixing postposition
It is for use to room temperature environment that placement is taken out after 4 DEG C of refrigerator overnights.
The detection method of l-cn content in the milk powder, wherein l-cn standard curve is surveyed by standard items
Fixed number is according to being drawn.
The detection method of l-cn content in the milk powder, wherein make ordinate with OD2-OD1 absorbance differences, it is dense
Angle value is abscissa, concentration value is read from l-cn standard curve and is multiplied by extension rate and obtains l-cn in sample contains
Amount.
The detection method of l-cn content in the milk powder, wherein the colour developing working solution is made by the following method
It is standby：50mg 2- nitrobenzoic acids, 5.96g n-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 185mg ethylenediamine tetraacetics are weighed respectively
Acetic acid disodium is dissolved in 30mL deionized waters, with 10mol/L NaOH solution tune pH to 7.4~7.6, is then settled to water
50mL is made storing solution, and when use draws 5.0mL storing solutions and is settled to 25mL with water.
The detection method of l-cn content in the milk powder, wherein acetylcarnitine transfer enzyme solutions pass through with lower section
Method configures：10 μ L acetylcarnitine transferase suspension are drawn, 10min is centrifuged through 1500r/min, discard supernatant liquor, precipitation is used
0.2mL water dissolutions.
The detection method of l-cn content in the milk powder, wherein in the milk powder that the detection method can be detected out
L-cn content minimum value is 80ppm.
This l-cn assay method is substantially reduced relative to National Standard Method, testing cost, while it can carry out batch detection,
Detection time accordingly is saved, greatly promotes detection efficiency so that l-cn measures more efficient and convenient.
Description of the drawings
Fig. 1 is the l-cn mark being calculated using the measured value of l-cn standard items in the specific embodiment of the invention
Directrix curve.
Specific implementation mode
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, art technology
The every other embodiment that personnel are obtained without creative efforts, shall fall within the protection scope of the present invention.
The assay method the principle on which of l-cn content of the present invention is：L-cn is with acetyl coenzyme A in acetyl
Reaction generates acetylcarnitine and free coacetylase under the catalysis of carnitine transferase.Free coacetylase and the reaction of 2- nitrobenzoic acids
Yellow substance is generated, shade is directly proportional to free CoA contents.Because free coacetylase and l-cn are etc. to rub
You can find out l-cn content in sample at reaction relation by making standard curve.This is expanded on further below by embodiment
The scheme of invention.
Embodiment 1
1, reagent prepares
Perchloric acid solution (13%)：13mL perchloric acid is diluted to 100mL.
Sodium hydroxide solution (10mol/L)：40g sodium hydroxide water dissolutions are weighed, 100mL is diluted to after cooling.
Potassium hydroxide solution (4.0mol/L)：22.4g potassium hydroxide water dissolutions are weighed, 100mL is diluted to after cooling.
Develop the color storing solution：50mg 2- nitrobenzoic acids, 5.96g N-2- hydroxyethyl piperazine-N-2- ethane sulphurs are weighed respectively
Acid, 185mg disodium ethylene diamine tetraacetates are dissolved in 30mL deionized waters, with 10mol/L NaOH solution tune pH to 7.4~7.6,
Then it is settled to 50mL with water.This liquid is placed in 4 DEG C of refrigerators and can save 3 months.
Develop the color working solution：It draws 5.0mL colour developing storing solutions and is settled to 25mL with water.
Acetyl coenzyme A (AcetylCoA) solution：20.0mg acetyl coenzyme As are dissolved in 2.0mL water.Face used time preparation.
Acetylcarnitine transferase (CAT) solution：10 μ L acetylcarnitine transferase suspension are drawn, are centrifuged through 1500r/min
10min discards supernatant liquor, precipitation 0.2mL water dissolutions.Face used time preparation.
L-cn standard items, sigma, purity 99%.It is spare to be configured to 5 concentration, it is now with the current, it is 1.6 respectively,
3.2,6.4,9.6,16ug/ml.
2, instrument
Assay balance：A ten thousandth.
PH meter：Precision 0.01.
Centrifuge：Rotating speed >=1500r/min.
Thermostat water bath：Temperature control is at 40 DEG C ± 2 DEG C.
Microplate reader 405nm and 96 mating hole elisa Plates.
3, sample treatment
The accurate uniformly mixed samples of 2.5g that weigh are dissolved in 50ml centrifuge tubes with a scale with 15mL40 DEG C of warm water.
5mL13% perchloric acid solutions are added, after mixing static 20min.It is settled to scale with distilled water, mixing uses quantitative filter paper
Filtering.
Filtrate 20mL is taken, after being 12.5~13.0 with 4mol/L potassium hydroxide solution tune pH, is placed in 40 DEG C of water-bath 60min.
After cooling with 13% perchloric acid tune pH be 7.0~7.5.Sample liquid is transferred in 50mL volumetric flasks, with distilled water constant volume.Mixing postposition
In 4 DEG C of refrigerator overnights.Sample treatment solution is taken out into placement to room temperature from refrigerator, supernatant is taken to measure.
4, analysis measures
Gauge orifice 5 (1.6,3.2,6.4,9.6,16) and sample well, best diplopore parallel testing are set on ELISA Plate；
200ul mark product or sample are taken to be added in corresponding micropore；
80ul substrates are added to each hole, 10ul coacetylases are added to each hole.Gently mixing gets started timing 5 and divides
Clock, room temperature；
Absorbance OD1 is read in 405nm；
10ul is added per hole and shifts enzyme solutions, mixing (it is good to be gently mixed effect), timing 10 minutes, room temperature；
With microplate reader 405nm test absorbances OD2.
5, result calculates
Make ordinate with absorbance poor (OD2-OD1), concentration value is abscissa, mapping, standard curve show as shown in Figure 1,
Sample concentration is read from figure, is multiplied by extension rate (extension rate 50) and is obtained final result.
This method improves National Standard Method so that l-cn measures more efficient and convenient.First, used in this method
To reagent belong to enzyme preparation, price is relatively high, with ELISA Plate hole react required amount of reagent be original ten/
One, the dosage and cost of key reagents can be greatlyd save.Second is that the method for the present invention can with the absorbance in 96 hole of disposable test,
Many times are saved, and spectrophotometer can only survey the absorbance value of a sample every time, thus a mesh quickly detected can be reached
Mark, such as when 10 samples of measurement, the method for the present invention can save 210 minutes with respect to National Standard Method.
Embodiment 2
The l-cn content in a milk powder is tested using this method, and carries out recovery testu verification.
1, milk powder is handled：
2.5g milk powder accurately is weighed, is put into 50ml centrifuge tubes, is dissolved with 15mL40 DEG C of warm water, mixing.
13% perchloric acid solutions of 5mL are added, after mixing, places 20 minutes, 50ml scales is settled to distilled water,
Mixing filters (having to clear filtrate) with filter paper.
Filtrate 20ml is taken, with 4mol/L potassium hydroxide solution tune pH to 12.5-13, is placed in 40 | 60min is shaken in C water-baths.It is cold
But afterwards with 13% perchloric acid tune pH to 7.0~7.5,
Add water to 50ml.Refrigeration is stood overnight after mixing, takes supernatant to detect within second day.(extension rate 50)
Mark-on sample：2.5g milk powder is weighed, 100ul4000ppm l-cn mark product liquid is added, is shaken 10 seconds.By upper
Milk powder processing step is stated to be handled.
2, analysis measurement is carried out within second day
(1), gauge orifice 5 (1.6,3.2,6.4,9.6,16) and 2 sample wells on ELISA Plate are set, all set parallel；
(2) 200ul mark product or sample, is taken to be added in corresponding micropore；
(3) 80ul, is added and develops the color working solution to each hole,
(4), 10ul coacetylases are added to each hole.Mixing is gently vibrated, timing 5min, room temperature are got started；
(5), absorbance OD1 is read in 405nm；
(6) 10ul, is added per hole and shifts enzyme solutions, mixing (it is good to be gently mixed effect), timing 10min, room temperature；
(7), with microplate reader 405nm test absorbances OD2.
3, result calculates
Make ordinate with absorbance poor (OD2-OD1), concentration value is abscissa, standard curve is made, from standard curve
Sample concentration is read, extension rate is multiplied by and obtains final result.Following table is data processing table
| Mark product | 1.6 | 3.2 | 6.4 | 9.6 | 16 | Sample | Mark-on |
| Average OD1 | 0.326 | 0.401 | 0.501 | 0.655 | 0.850 | 0.41 | 0.565 |
| Average OD2 | 0.248 | 0.262 | 0.248 | 0.265 | 0.249 | 0.32 | 0.367 |
| OD is poor | 0.078 | 0.139 | 0.253 | 0.39 | 0.601 | 0.09 | 0.198 |
| As a result ppm | 91.5 | 239 |
It is 91.5mg/kg that this measurement, which obtains its l-cn result, and obtaining this rate of recovery by mark-on reclaims is
92.2%.This time the result is that believable in terms of the rate of recovery, also within curve quantification range.
It, can according to the technique and scheme of the present invention and this hair it is understood that for those of ordinary skills
Bright design is subject to equivalent substitution or change, and all these changes or replacement should all belong to the guarantor of appended claims of the invention
Protect range.
Claims (8)
1. the detection method of l-cn content in a kind of milk powder, which is characterized in that include the following steps：
Gauge orifice and sample well are set on ELISA Plate, 200ul standard items is taken to be added in gauge orifice, 200ul samples is taken to be added
Into sample well, 80ul colour developing working solutions and 10ul acetyl coenzyme As, room temperature condition are sequentially added in gauge orifice and each hole of sample well
Lower reaction 5min reads absorbance OD1 in 405nm；
Gauge orifice and sample well are added 10ul acetylcarnitines per hole and shift enzyme solutions mixing, react 10min under room temperature,
405nm tests absorbance OD2；
L-cn content in sample is calculated using absorbance OD1 and OD2 combination l-cn standard curve.
2. the detection method of l-cn content in milk powder according to claim 1, which is characterized in that the gauge orifice is set
5 are set to, the standard concentration of 5 gauge orifices addition is 1.6ug/ml, 3.2ug/ml, 6.4ug/ml, 9.6ug/ml, 16ug/
ml。
3. the detection method of l-cn content in milk powder according to claim 1, which is characterized in that sample detection is advanced
Row pretreatment：The accurate uniformly mixed samples of 2.5g that weigh are molten with 40 DEG C of warm water of 15mL in 50ml centrifuge tubes with a scale
13% perchloric acid solutions of 5mL are added, after mixing static 20min in solution, and scale, mixing, with quantitatively are settled to distilled water
Filter paper filters；
Filtrate 20mL is taken, after being 12.5~13.0 with 4mol/L potassium hydroxide solution tune pH, is placed in 40 DEG C of water-bath 60min.It is cooling
It is afterwards 7.0~7.5 with 13% perchloric acid tune pH.Sample liquid is transferred in 50mL volumetric flasks, with distilled water constant volume；Mixing is placed on 4
Taken out after DEG C refrigerator overnight place it is for use to room temperature environment.
4. the detection method of l-cn content in milk powder according to claim 2, which is characterized in that l-cn standard
Curve negotiating standard items determination data is drawn.
5. the detection method of l-cn content in milk powder according to claim 4, which is characterized in that inhaled with OD2-OD1
Luminosity difference makees ordinate, and concentration value is abscissa, and concentration value is read from l-cn standard curve and is multiplied by extension rate and obtains
To l-cn content in sample.
6. the detection method of l-cn content in milk powder according to claim 1, which is characterized in that the colour developing work
Liquid is prepared by the following method：50mg 2- nitrobenzoic acids, 5.96g N-2- hydroxyethyl piperazine-N-2- ethane sulphurs are weighed respectively
Acid, 185mg disodium ethylene diamine tetraacetates are dissolved in 30mL deionized waters, with 10mol/L NaOH solution tune pH to 7.4~7.6,
Then it is settled to 50mL with water and storing solution is made, 5.0mL storing solutions are drawn when use and are settled to 25mL with water.
7. the detection method of l-cn content in milk powder according to claim 1, which is characterized in that acetylcarnitine shifts
Enzyme solutions configure by the following method：10 μ L acetylcarnitine transferase suspension are drawn, 10min is centrifuged through 1500r/min, discards
Supernatant liquor, precipitation 0.2mL water dissolutions.
8. the detection method of l-cn content in milk powder according to claim 1, which is characterized in that the detection method institute
L-cn content minimum value is 80ppm in the milk powder that can be detected.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175673A (en) * | 2011-01-18 | 2011-09-07 | 安徽泰格生物技术股份有限公司 | Method for detecting total selenium content |
| CN106153773A (en) * | 2016-08-22 | 2016-11-23 | 河北三元食品有限公司 | Utilize the method for L carnitine in Ultra Performance Liquid Chromatography tandem mass spectrum quantitative determination baby formula milk powder |
| CN106198531A (en) * | 2016-08-30 | 2016-12-07 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method of detection L-carnitine content |
| CN106644992A (en) * | 2017-03-10 | 2017-05-10 | 深圳市瑞赛生物技术有限公司 | Method for rapid detection of L-carnitine and kit thereof |
-
2018
- 2018-05-23 CN CN201810498612.6A patent/CN108426879A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175673A (en) * | 2011-01-18 | 2011-09-07 | 安徽泰格生物技术股份有限公司 | Method for detecting total selenium content |
| CN106153773A (en) * | 2016-08-22 | 2016-11-23 | 河北三元食品有限公司 | Utilize the method for L carnitine in Ultra Performance Liquid Chromatography tandem mass spectrum quantitative determination baby formula milk powder |
| CN106198531A (en) * | 2016-08-30 | 2016-12-07 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method of detection L-carnitine content |
| CN106644992A (en) * | 2017-03-10 | 2017-05-10 | 深圳市瑞赛生物技术有限公司 | Method for rapid detection of L-carnitine and kit thereof |
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Application publication date: 20180821 |