CN103134716A - Pretreatment method and detection method for detecting progesterone content in lactobacillis beverage - Google Patents
Pretreatment method and detection method for detecting progesterone content in lactobacillis beverage Download PDFInfo
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- CN103134716A CN103134716A CN2011103893156A CN201110389315A CN103134716A CN 103134716 A CN103134716 A CN 103134716A CN 2011103893156 A CN2011103893156 A CN 2011103893156A CN 201110389315 A CN201110389315 A CN 201110389315A CN 103134716 A CN103134716 A CN 103134716A
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Abstract
The invention provides a pretreatment method for detecting progesterone content in a lactobacillis beverage. The pretreatment method for detecting the progesterone content in the lactobacillis beverage comprises the following steps: taking out a to-be-detected lactobacillis beverage, heating the to-be-detected lactobacillis beverage in boiling water bath for 30-90 seconds, centrifuging for 5-15 minutes in 2000g-4000g, taking out supernatant liquor after removing a fat layer which floats on the upper layer, and regulating a potential of hydrogen (PH) value of the supernatant liquor to 6.7-7.0 to acquire the to-be-detected liquor. The pretreatment method for detecting the progesterone content in the lactobacillis beverage can effectively remove interfering substances in the lactobacillis beverage, especially bacteria, and treated lactobacillis beverage is suitable for quickly detecting a kit of progesterone. The invention further provides a detection method for detecting the progesterone content in the lactobacillis beverage. A progesterone enzyme-linked immunosorbent assay kit is utilized for detecting after the to-be-detected lactobacillis beverage is detected according to the pretreatment method for detecting the progesterone content in the lactobacillis beverage. Through the detection method for detecting the progesterone content in the lactobacillis beverage, the enzyme-linked immunosorbent assay kit with low cost is applied to detecting the lactobacillis beverage, the detection method for detecting the progesterone content in the lactobacillis beverage is quick, simple and convenient, and high in accuracy. Adding standard recovery rate of the detection method is 84%-110%, and relative standard deviation of a detection result is 2.3%-4.5%.
Description
Technical field
The present invention relates to a kind of pre-treating method that detects progesterone content, be particularly useful for the detection of progesterone content in sour milk beverage, the present invention also provides a kind of detection method that adopts this pre-treating method.
Background technology
Food Sex Hormones residual contamination problem has caused global extensive concern.Milk and milk products is nutritious, but has the phenomenon of abuse sex hormone (as progesterone) in the animal feeding process, causes milk food Sex Hormones residual contamination problem.Progesterone content in sour milk beverage is detected, can effectively control the quality of sour milk beverage, improve the security of product.
At present, can be by progesterone content in enzyme linked immunosorbent assay fast detecting sour milk beverage, wherein pre-treating method is: sour milk beverage to be measured is carried out centrifugal treating, remove after the fat deposit of upper strata remainder for detection of.Adopt this pre-treating method can't effectively remove interfering material in sample, when the sample after processing detected with the progesterone enzyme-linked immunologic detecting kit, the accuracy of its detection was undesirable.
Existing progesterone enzyme-linked immunologic detecting kit is usually used in the progesterone in fast detecting blood or urine, if still directly use the progesterone in this kit detection sour milk beverage, accuracy is not high, is difficult to realize the purpose of fast detecting.
Summary of the invention
The purpose of this invention is to provide a kind of pre-treating method that detects progesterone content in sour milk beverage, can effectively remove the interfering material in sour milk beverage.
Another object of the present invention is to provide the detection method of progesterone content in a kind of sour milk beverage, and the interfering material by in specific pre-treating method removal sour milk beverage is used further to enzyme-linked immunologic detecting kit and detects, and improves the accuracy that detects.
The invention provides a kind of pre-treating method that detects progesterone content in sour milk beverage, comprise: get sour milk beverage to be measured, prior to heating 30-90 second in boiling water bath, under the centrifugal condition of 2000g-4000g centrifugal 5-15 minute again, after removing the floating fat deposit in upper strata, get supernatant, and the pH value of regulating supernatant obtains liquid to be measured to 6.7-7.0.Wherein, centrifugal temperature can be 2-8 ℃.Carry out the pH value when regulating, can select concentration is that the sodium hydroxide solution of 1mol/L is regulated the pH value of supernatant.
The present invention also provides a kind of method that detects progesterone content in sour milk beverage, comprises the steps: according to above-mentioned pre-treating method, sour milk beverage to be measured to be processed, and obtains liquid to be measured; Utilize the progesterone enzyme-linked immunologic detecting kit that the progesterone content in liquid to be measured is detected, obtain the progesterone content of liquid to be measured.
A kind of pre-treating method that detects progesterone content in sour milk beverage of the present invention, regulate by boiling water bath heating, centrifugal treating and pH, removed the interfering material in the sour milk beverage, especially can remove to a certain extent in sour milk beverage the noisy bacterium of testing result.Make the sour milk beverage after pre-treatment in this way be specially adapted to use kit fast detecting progesterone wherein.
The detection method of progesterone content in a kind of sour milk beverage of the present invention after by said method, sour milk beverage being carried out pre-treatment, then detects wherein progesterone content with enzyme-linked immunologic detecting kit, obtains the content of progesterone in sour milk beverage.This detection method applies to detect sour milk beverage with lower-cost enzyme-linked immunologic detecting kit, and is fast and convenient, and accuracy is higher.
Embodiment
Understand for technical characterictic, purpose and effect to invention have more clearly, now in conjunction with each embodiment, the specific embodiment of the present invention is described.
The first embodiment: a kind of pre-treating method of sour milk beverage.
The present embodiment selects the excellent beneficial C of product as sour milk beverage to be measured.
Get sour milk beverage to be measured in centrifuge tube, prior to heating in boiling water bath 30 seconds, again at the temperature of 4 ℃, under the centrifugal condition of 2000g centrifugal 8 minutes, after removing the floating fat deposit in upper strata, get supernatant, and the pH value to 6.7 that is the sodium hydroxide solution adjusting supernatant of 1mol/L with concentration obtains liquid to be measured.
The second embodiment: a kind of pre-treating method of sour milk beverage.
The present embodiment selects the product chlorella yakult as sour milk beverage to be measured.
Get sour milk beverage to be measured in centrifuge tube, prior to heating in boiling water bath 90 seconds, again at the temperature of 2 ℃, under the centrifugal condition of 3000g centrifugal 5 minutes, after removing the floating fat deposit in upper strata, get supernatant, and the pH value to 7.0 that is the sodium hydroxide solution adjusting supernatant of 1mol/L with concentration obtains liquid to be measured.
The 3rd embodiment: a kind of pre-treating method of sour milk beverage.
The present embodiment selects the product happiness happy as sour milk beverage to be measured.
Get sour milk beverage to be measured in centrifuge tube, prior to heating in boiling water bath 60 seconds, again at the temperature of 8 ℃, under the centrifugal condition of 4000g centrifugal 15 minutes, after removing the floating fat deposit in upper strata, get supernatant, and the pH value to 6.8 that is the sodium hydroxide solution adjusting supernatant of 1mol/L with concentration obtains liquid to be measured.
The 4th embodiment: the detection method of progesterone content in a kind of sour milk beverage.
1, kit and microplate reader.
The progesterone enzyme-linked immunologic detecting kit: select the Progesterone Elisa kit of Biostest company, article No. is DS314301.
Microplate reader: the Elx808 microplate reader of selecting BioTek company.
2, detecting step.
The present embodiment selects the excellent beneficial C of product as sour milk beverage to be measured.
1) according to the first described pre-treating method of embodiment, sour milk beverage to be measured is carried out pre-treatment, obtain liquid to be measured.
2) take out microwell plate, get respectively 25 μ L standard items (progesterone content is respectively 0 ng/mL, 0.3 ng/mL, 1.25 ng/mL, 2.5 ng/mL, 5 ng/mL and 15 ng/mL) and 25 μ L liquid to be measured in micropore separately, microwell plate was hatched 5 minutes in 20 ℃.
3) every hole adds 200 μ L enzyme labeling things, moves mixing 60 seconds with the have gentle hands jog, shrouding, then microwell plate was hatched 1 hour in 20 ℃ of lucifuges.
4) liquid in micropore is dried, every hole adds 300 μ L washing lotions, and rocker 10 seconds discards washing lotion and microwell plate is patted dry on thieving paper.
5) repeating step 4) 3 times.
6) every hole adds 200 μ L colour formers, with the moving mixing of have gentle hands jog, then microwell plate is hatched 15 minutes in 20 ℃ of lucifuges.
7) every hole adds 100 μ L reaction terminating liquids, with the moving mixing of have gentle hands jog.Set microplate reader in the 450nm place, measure the absorbance in every hole.
8) take the absorbance of standard items corresponding aperture as ordinate, standard items concentration builds typical curve as horizontal ordinate, the match mode is four parameter logic matches.In absorbance substitution typical curve with the supernatant corresponding aperture, read corresponding concentration from typical curve, be the progesterone content of liquid to be measured, be progesterone content in sour milk beverage.
3, the Accuracy Verification of detection method.
Get sour milk beverage to be measured, the interpolation of progesterone shown according to the form below level adds the progesterone standard items (commercial) of different amounts wherein, obtain the mark-on sour milk beverage, detection method with the present embodiment is carried out Parallel testing three times to the mark-on sour milk beverage, according to the average recovery rate under the different interpolation levels of testing result calculating and the relative standard deviation of testing result, result of calculation is as shown in the table.
| Sample number | Progesterone adds level | Average recovery rate | The relative standard deviation of testing result |
| Mark-on sour milk beverage 1-1 | 0.5 ng/mL | 92% | 2.3% |
| Mark-on sour milk beverage 1-2 | 1.0 ng/mL | 89% | 3.7% |
| Mark-on sour milk beverage 1-3 | 2.5 ng/mL | 105% | 4.5% |
4, contrast experiment.
Operation steps: get above-mentioned mark-on sour milk beverage, in at the temperature of 4 ℃, under the centrifugal condition of 2000g centrifugal 8 minutes, after removing the floating fat deposit in upper strata, get supernatant, and the pH value to 6.7 that is the sodium hydroxide solution adjusting supernatant of 1mol/L with concentration obtains contrasting liquid to be measured.Step 2 according to the present embodiment detecting step) to 8) in the detection method of liquid to be measured is detected contrasting liquid to be measured, obtain contrasting the progesterone content of liquid to be measured.Parallel testing is three times as stated above, and according to the average recovery rate under the different interpolation levels of testing result calculating and the relative standard deviation of testing result, result of calculation is as shown in the table.
| Sample number | Progesterone adds level | Average recovery rate | The relative standard deviation of testing result |
| Mark-on sour milk beverage 1-1 | 0.5ng/mL | 56% | 7.6% |
| Mark-on sour milk beverage 1-2 | 1.0ng/mL | 43% | 6.1% |
| Mark-on sour milk beverage 1-3 | 1.5ng/mL | 40% | 8.9% |
The 5th embodiment: the detection method of progesterone content in a kind of sour milk beverage.
1, kit and microplate reader.
Described identical with the 4th embodiment.
2, detecting step.
The present embodiment selects the product chlorella yakult as sour milk beverage to be measured.
According to the second described pre-treating method of embodiment, sour milk beverage to be measured is carried out pre-treatment, obtain liquid to be measured.The step 2 of all the other steps and the 4th embodiment detecting step) described identical to step 8).
3, the Accuracy Verification of detection method.
Get sour milk beverage to be measured, the interpolation of progesterone shown according to the form below level adds the progesterone standard items (commercial) of different amounts wherein, obtain the mark-on sour milk beverage, detection method with the present embodiment is carried out Parallel testing three times to the mark-on sour milk beverage, according to the average recovery rate under the different interpolation levels of testing result calculating and the relative standard deviation of testing result, result of calculation is as shown in the table.
| Sample number | Progesterone adds level | Average recovery rate | The relative standard deviation of testing result |
| Mark-on sour milk beverage 2-1 | 0.5 ng/mL | 95% | 2.3% |
| Mark-on sour milk beverage 2-2 | 1.0 ng/mL | 110% | 3.7% |
| Mark-on sour milk beverage 2-3 | 2.5 ng/mL | 92% | 4.5% |
4, contrast experiment.
Operation steps: get above-mentioned mark-on sour milk beverage, in at the temperature of 2 ℃, under the centrifugal condition of 3000g centrifugal 5 minutes, after removing the floating fat deposit in upper strata, get supernatant, and the pH value to 7.0 that is the sodium hydroxide solution adjusting supernatant of 1mol/L with concentration obtains contrasting liquid to be measured.Step 2 according to the present embodiment detecting step) to 8) in the detection method of liquid to be measured is detected contrasting liquid to be measured, obtain contrasting the progesterone content of liquid to be measured.Parallel testing is three times as stated above, and according to the average recovery rate under the different interpolation levels of testing result calculating and the relative standard deviation of testing result, result of calculation is as shown in the table.
| Sample number | Progesterone adds level | Average recovery rate | The relative standard deviation of testing result |
| Mark-on sour milk beverage 2-1 | 0.5ng/mL | 42% | 10.3% |
| Mark-on sour milk beverage 2-2 | 1.0ng/mL | 50% | 9.7% |
| Mark-on sour milk beverage 2-3 | 1.5ng/mL | 48% | 8.7% |
The 6th embodiment: the detection method of progesterone content in a kind of sour milk beverage.
1, kit and microplate reader.
Described identical with the 4th embodiment.
2, detecting step.
The present embodiment selects the product happiness happy as sour milk beverage to be measured.
According to the 3rd described pre-treating method of embodiment, sour milk beverage to be measured is carried out pre-treatment, obtain liquid to be measured.The step 2 of all the other steps and the 4th embodiment detecting step) described identical to step 8).
3, the Accuracy Verification of detection method.
Get sour milk beverage to be measured, the interpolation of progesterone shown according to the form below level adds the progesterone standard items (commercial) of different amounts wherein, obtain the mark-on sour milk beverage, detection method with the present embodiment is carried out Parallel testing three times to the mark-on sour milk beverage, according to the average recovery rate under the different interpolation levels of testing result calculating and the relative standard deviation of testing result, result of calculation is as shown in the table.
| Sample number | Progesterone adds level | Average recovery rate | The relative standard deviation of testing result |
| Mark-on sour milk beverage 3-1 | 0.5 ng/mL | 84% | 4.3% |
| Mark-on sour milk beverage 3-2 | 1.0 ng/mL | 95% | 3.6% |
| Mark-on sour milk beverage 3-3 | 2.5 ng/mL | 103% | 2.4% |
4, contrast experiment.
Operation steps: get above-mentioned mark-on sour milk beverage, in at the temperature of 8 ℃, under the centrifugal condition of 4000g centrifugal 15 minutes, after removing the floating fat deposit in upper strata, get supernatant, and the pH value to 6.8 that is the sodium hydroxide solution adjusting supernatant of 1mol/L with concentration obtains contrasting liquid to be measured.Step 2 according to the present embodiment detecting step) to 8) in the detection method of liquid to be measured is detected contrasting liquid to be measured, obtain contrasting the progesterone content of liquid to be measured.Parallel testing is three times as stated above, and according to the average recovery rate under the different interpolation levels of testing result calculating and the relative standard deviation of testing result, result of calculation is as shown in the table.
| Sample number | Progesterone adds level | Average recovery rate | The relative standard deviation of testing result |
| Mark-on sour milk beverage 3-1 | 0.5ng/mL | 59% | 12.5% |
| Mark-on sour milk beverage 3-2 | 1.0ng/mL | 38% | 7.2% |
| Mark-on sour milk beverage 3-3 | 1.5ng/mL | 41% | 8.1% |
According to the experimental result of above-described embodiment as can be known, the recovery of standard addition of detection method of the present invention is 84%-110%, and the relative standard deviation of testing result is 2.3%-4.5%, and the recovery is higher, and relative standard deviation is lower.With reference to the contrast experiment of above-described embodiment, can also find that heating steps of the present invention has improved the accuracy of testing result greatly, has reduced the relative standard deviation of testing result.
Be to be understood that, although this instructions is described according to each embodiment, but be not that each embodiment only comprises an independently technical scheme, this narrating mode of instructions is only for clarity sake, those skilled in the art should make instructions as a whole, technical scheme in each embodiment also can through appropriate combination, form other embodiments that it will be appreciated by those skilled in the art that.
Above listed a series of detailed description is only illustrating for feasibility embodiment of the present invention; they are not to limit protection scope of the present invention, all disengaging within equivalent embodiment that skill spirit of the present invention does or change all should be included in protection scope of the present invention.
Claims (4)
1. pre-treating method that detects progesterone content in sour milk beverage, it is characterized in that, comprise: get sour milk beverage to be measured, prior to heating 30-90 second in boiling water bath, under the centrifugal condition of 2000g-4000g centrifugal 5-15 minute again, after removing the floating fat deposit in upper strata, get supernatant, and the pH value of regulating described supernatant obtains liquid to be measured to 6.7-7.0.
2. pre-treating method as claimed in claim 1, wherein, described centrifugal temperature is 2-8 ℃.
3. pre-treating method as claimed in claim 1, wherein, be that the sodium hydroxide solution of 1mol/L carries out described adjusting to the pH value of described supernatant with concentration.
4. a method that detects progesterone content in sour milk beverage, is characterized in that, comprises the steps:
according to claim 1 to 3, the described pre-treating method of any one is processed sour milk beverage to be measured, obtains described liquid to be measured;
Utilize the progesterone enzyme-linked immunologic detecting kit that the progesterone content in described liquid to be measured is detected, obtain the progesterone content of described liquid to be measured.
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Application publication date: 20130605 |