CN114088626B - Method and detection system for identifying pasteurized milk - Google Patents

Method and detection system for identifying pasteurized milk Download PDF

Info

Publication number
CN114088626B
CN114088626B CN202111353273.0A CN202111353273A CN114088626B CN 114088626 B CN114088626 B CN 114088626B CN 202111353273 A CN202111353273 A CN 202111353273A CN 114088626 B CN114088626 B CN 114088626B
Authority
CN
China
Prior art keywords
alkaline phosphatase
activity
pasteurized milk
content
fluorescence intensity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111353273.0A
Other languages
Chinese (zh)
Other versions
CN114088626A (en
Inventor
杨大进
刘龙飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Zhongjian Baotai Biotechnology Co ltd
China National Center For Food Safety Risk Assessment
Original Assignee
Beijing Zhongjian Baotai Biotechnology Co ltd
China National Center For Food Safety Risk Assessment
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Zhongjian Baotai Biotechnology Co ltd, China National Center For Food Safety Risk Assessment filed Critical Beijing Zhongjian Baotai Biotechnology Co ltd
Priority to CN202111353273.0A priority Critical patent/CN114088626B/en
Publication of CN114088626A publication Critical patent/CN114088626A/en
Application granted granted Critical
Publication of CN114088626B publication Critical patent/CN114088626B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Abstract

The application provides a method and a detection system for identifying pasteurized milk. The method and the system judge the killing degree of the microorganisms by detecting the activity of the alkaline phosphatase in the pasteurized milk, and not only have high sensitivity, but also have accurate detection result and good stability.

Description

Method and detection system for identifying pasteurized milk
Technical Field
The application relates to a method and a detection system for identifying pasteurized milk, and belongs to the field of biochemical detection.
Background
Milk, known as "white blood", contains water, fat, protein, lactose and abundant minerals, and can provide a great deal of nutrients for human body, but there are many pathogenic microorganisms in it, and milk can be used as food and drinking source for human only if the pathogenic microorganisms are lost by heat treatment. The milk sterilization mode mainly comprises pasteurization and ultrahigh temperature sterilization, wherein the pasteurization is usually carried out at 72 ℃ for 15 seconds, and active substances in the milk can be kept as much as possible while pathogenic bacteria are killed, so that the pasteurized milk is favored by more and more consumers. National standards for pasteurized milk specify microbial limits and therefore require the detection of microbial content in pasteurized milk. The traditional microorganism detection method is a plate culture method, the operation is complex, the requirement on the technical level of operators is high, the detection speed is slow, the result is obtained within 48 hours, the quality guarantee period of pasteurized milk is usually 7 days, and for products with short quality guarantee period, the long detection time means that the quality guarantee period of the products is greatly shortened; the microorganism detection sheet on the market at present greatly reduces the operation difficulty, but the detection time is still very long, usually 24-48 hours, the detection speed is still very slow, and the microorganism detection sheet is not suitable for products with short quality guarantee period. In view of the above, it is necessary to develop a method capable of rapidly detecting the microbial kill level of pasteurized milk.
Alkaline phosphatase (ALP) is an enzyme naturally existing in milk, is an enzyme with stronger heat stability in milk, has slightly higher temperature stability than pathogenic bacteria existing in milk, and when the content of microorganisms in pasteurized milk is lower than 20000CFU/ml, the activity of the alkaline phosphatase is positively correlated with the content of the microorganisms, so that the degree of killing of the microorganisms can be judged by the activity of the alkaline phosphatase.
At present, the method for detecting alkaline phosphatase activity mainly adopts the steps of reacting a fluorogenic substrate compound with alkaline phosphatase to generate fluorescence, and detecting the activity of the alkaline phosphatase by a fluorescence detector. However, this approach suffers from at least two limitations: first, the sensitivity of fluorogenic substrate compounds; secondly, the accuracy of the alkaline phosphatase activity measuring and calculating method.
Disclosure of Invention
In order to be able to quickly detect the degree of microbial kill in pasteurized milk, the present application provides a method and a detection system for identifying pasteurized milk.
An object of the present invention is to provide a method for converting alkaline phosphatase to fluorescence intensity.
The relationship between alkaline phosphatase activity and fluorescence intensity is expressed as a function F (x),
Figure BDA0003356571860000021
Figure BDA0003356571860000022
the kit is related to A, B, C, D, E five parameters, wherein A is the median of fluorescence intensity obtained by 100 positive quality control samples, the activity of alkaline phosphatase of the positive quality control is 350mU/L, and the degree of variation is 30%; b is a calibration factor; c is the median of fluorescence intensity obtained by 100 parts of pasteurized milk which is subjected to heat treatment at 72 ℃ for 15s and has the microorganism content lower than 20000 CFU/ml; d is the median of the fluorescence intensities obtained for 100 negative pasteurized milk samples; e is the median of the fluorescence intensities obtained for 100 parts of milk heat-treated at 85 ℃ for 1 s.
The B value was determined by the activity of alkaline phosphatase of the standard (mU/L) and the fluorescence intensity of the Reaction (RLU).
When the microbial content in pasteurized milk is below 20,000cfu/ml, the fluorescence intensity (RLU) and the activity of alkaline phosphatase conform to the function F (x), and the microbial content and the activity of alkaline phosphatase are in positive correlation.
It is another object of the present invention to provide a highly sensitive alkaline phosphatase substrate compound (formula I). The compounds are more sensitive than conventional alkaline phosphatase substrate compounds (formula II).
Figure BDA0003356571860000031
In order to achieve the above object, the present application employs a detection system, which is composed of a kit and a detector; the kit consists of a detection tube and a calibration sheet; the detector mainly comprises a high-sensitivity photomultiplier, a built-in temperature probe stabilizer, a signal collector and calculation software.
The detector directly converts the read fluorescence intensity (RLU) into the activity of alkaline phosphatase (mU/L) according to the measurement method.
When the alkaline phosphatase activity is < 350mU/L, the amount of microorganisms is < 20,000 CFU/mL.
When the alkaline phosphatase activity is 50-150mU/L, the amount of the microorganism is about 1000-10000 CFU/mL.
When the alkaline phosphatase activity is less than 50mU/L, it indicates that overheating may occur, and it is preliminarily judged that the milk is not pasteurized.
The criteria for a preliminary determination as pasteurized milk may also include: the content of lactoferrin is more than or equal to 25 mg/L; the content of alpha-lactalbumin is more than 500mg/L, and/or the content of beta-lactoglobulin is more than or equal to 2200 mg/L.
The detection system of the application also adopts a high-sensitivity photomultiplier, and compared with a common photomultiplier, the sensitivity is higher; meanwhile, a built-in temperature probe stabilizer is adopted, which can ensure that the temperature of the reaction liquid meets the requirement, thereby ensuring the accuracy and stability of the detection result.
Drawings
FIG. 1 relationship of alkaline phosphatase activity to microbial content.
FIG. 2 is a graph showing the relationship between the fluorescence intensity and the alkaline phosphatase activity.
Figure 3 range of lactoferrin content in pasteurized milk.
FIG. 4 range of alpha-lactalbumin and beta-lactoglobulin contents in pasteurized milk.
Detailed Description
Embodiments of the present application are described below with reference to the drawings. Elements and features described in one drawing or one embodiment of the present application may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that the figures and descriptions omit representation and description of components or processes that are not relevant to the present application and that are known to those of ordinary skill in the art for the sake of clarity. The present application is further described below with reference to the accompanying drawings.
In a first aspect, the present application provides a method for identifying pasteurized milk, comprising determining the extent of microbial kill by quantitatively detecting the activity of alkaline phosphatase, the fluorescence of which is produced by the reaction of alkaline phosphatase with a substrate compound, the relationship between alkaline phosphatase activity and fluorescence intensity being represented by the function F (x),
Figure BDA0003356571860000051
the gene is related to A, B, C, D, E five parameters, wherein A is the median of fluorescence intensity obtained by 100 positive quality control samples, the activity of the positive quality control alkaline phosphatase is 350mU/L, and the degree of variation is 30 percent; b is a calibration factor; c is the median of fluorescence intensity obtained by 100 portions of pasteurized milk which is subjected to heat treatment at 72 ℃ for 15s and has the microorganism content lower than 20000 CFU/ml; d is the median of the fluorescence intensities obtained for 100 negative pasteurized milk samples; e is the median of the fluorescence intensities obtained for 100 parts of cow milk heat-treated at 85 ℃ for 1 s; determining a B value by the activity of alkaline phosphatase of the standard (mU/L) and the fluorescence intensity of the Reaction (RLU);
when the content of microorganisms in pasteurized milk is lower than 20,000cfu/ml, the fluorescence intensity and the activity of alkaline phosphatase conform to a function F (x), and the content of the microorganisms is in positive correlation with the activity of the alkaline phosphatase; higher fluorescence intensity indicates higher alkaline phosphatase activity and correspondingly higher microbial content.
In some embodiments, the substrate compound has the structure shown in formula I:
Figure BDA0003356571860000052
in some embodiments, non-pasteurized milk is initially judged when alkaline phosphatase activity < 50mU/L, indicating the possible presence of overheating.
In some embodiments, the above methods further comprise detecting the level of lactoferrin, alpha-lactalbumin and/or beta-lactoglobulin.
In some embodiments, the criteria for a preliminary determination to be pasteurized milk are: the content of lactoferrin is more than or equal to 25 mg/L; the content of alpha-lactalbumin is more than 500mg/L, and/or the content of beta-lactoglobulin is more than or equal to 2200 mg/L.
In a second aspect, the present application provides a test system for carrying out the aforementioned method, said system comprising a kit and a test meter; the kit consists of a detection tube and a calibration sheet; the detector mainly comprises a high-sensitivity photomultiplier, a built-in temperature probe stabilizer, a signal collector and calculation software.
In some embodiments, the calibration tablet is prepared by tableting raw milk with known alkaline phosphatase activity to achieve an alkaline phosphatase activity of 875 μ U ± 26 μ U.
In some embodiments, a method of making a detection tube comprises:
1) 69.1125g of fluorogenic substrate compound, 23.438g of diethylaminoethanol,
10g of 2 '-fluoro-2' -deoxyinosine was added to about 400ml of deionized water and dissolved by stirring;
2) 20.33g of MgCl were weighed 2 ·6H 2 O, adding into 100ml deionized water, and stirringDissolving to obtain 1mol/L magnesium chloride solution;
3) transferring 0.5ml of 1mol/L magnesium chloride solution into the solution in the step 1);
4) adding deionized water to constant volume of 1L, and adjusting pH to 9.7-10.3 with HCl solution;
5) 0.5ml of the solution was dispensed into a plastic test tube and sealed with aluminum foil.
In some embodiments, the wavelength of the detector is 540 nm.
In a third aspect, the present application provides a method for quantitatively detecting alkaline phosphatase using the aforementioned system, comprising the steps of:
1) adding the dairy product to be detected into a detection tube;
2) mixing the contents of the test tube and then connecting the adapter;
3) and vertically inserting the detection tube into the detector, and reading the result.
Example 1 investigation of the relationship between alkaline phosphatase and microorganism
1. Preparation of alkaline phosphatase detection reagent (detection tube)
1) 69.1125g of the fluorogenic substrate compound (formula I), 23.438g of diethylaminoethanol and 10g of 2 '-fluoro-2' -deoxyinosine were weighed into about 400ml of deionized water and dissolved with stirring.
2) 20.33g of MgCl were weighed 2 ·6H 2 And O, adding the mixture into 100ml of deionized water, and stirring to dissolve the mixture to obtain a 1mol/L magnesium chloride solution.
3) 0.5ml of a 1mol/L magnesium chloride solution was transferred into the 1) solution.
4) The volume is adjusted to 1L by deionized water, and the pH is adjusted to 9.7-10.3 by HCl solution.
0.5ml of the solution was dispensed into a plastic test tube and sealed with aluminum foil.
2. Pasteurized milk alkaline phosphatase detection
100 parts of pasteurized milk samples are selected, the alkaline phosphatase activity of the pasteurized milk samples is detected by the method disclosed by the application, and the microbial content of the pasteurized milk samples is detected by a national standard method. The alkaline phosphatase activity was plotted on the abscissa and the microbial content on the ordinate. The results are shown in FIG. 1.
As can be seen from FIG. 1, the activity of alkaline phosphatase in pasteurized milk is positively correlated with the microbial content, the correlation coefficient R 2 0.877. Therefore, the extent of microbial kill of pasteurized milk can be judged by detecting alkaline phosphatase activity.
Example 2 method for the conversion of the functional relationship between the alkaline phosphatase Activity and the fluorescence intensity of the reaction
Although the activity of alkaline phosphatase (mU/L) and the fluorescence intensity of the Reaction (RLU) have a correlation, they are not simple y ═ ax + b relationships, and it is difficult to synthesize both measurement results into a scientifically fitted curve in actual measurement. To solve the above problems, the inventors have proposed the following formula, which can more accurately convert the activity of alkaline phosphatase based on the fluorescence intensity:
Figure BDA0003356571860000081
in the above formula, the value of A, B, C, D, E can be determined by referring to the method of the present embodiment.
The method is adopted to detect 100 parts of positive quality control samples (the activity of alkaline phosphatase is 350mU/L), 100 parts of pasteurized milk with the microorganism content lower than 20000CFU/ml after being subjected to heat treatment at 72 ℃ for 15s, 100 parts of negative pasteurized milk samples and 100 parts of cow milk subjected to heat treatment at 85 ℃ for 1s respectively, and the detection results are shown in tables 1-5.
TABLE 1 Positive quality control test Results (RLU)
10261 9476 10507 9657 8495 10471 10451 9027 8613 10711
9969 8162 9959 9556 8837 10346 8762 10491 8711 10971
9699 8861 8408 10914 8788 8033 8237 10489 8478 10677
9040 9547 8261 9770 8899 10207 10185 8838 10851 9473
8409 10021 10566 9667 8620 8957 10429 10665 10096 9035
9408 10193 9548 10079 10122 9484 9304 9299 8190 10258
10839 9646 9911 10995 9011 9768 10807 10838 9586 8550
8334 9706 9063 9100 8636 8318 10416 8712 10915 8553
9219 10756 9126 10835 10899 9145 9136 8111 8344 9090
10199 10366 9982 9472 8714 8220 10937 8099 9770 10718
As can be seen from Table 1, 100 positive quality control test results are 8033-10995 with a mean value of 9563, and all test results are within. + -. 30% of the mean value with a median of 9552. Therefore, the value of A was 9552.
TABLE 272 ℃ 15s Heat treatment pasteurized milk test Results (RLU)
2204 4352 5872 4490 5529 4676 1980 5748 3874 2624
2813 3532 3204 2983 4583 5525 2044 3419 5177 4020
3052 3879 1069 1953 458 5667 5842 2982 587 487
4067 968 3855 4917 1979 1414 4311 1408 2623 1603
1164 2879 433 5994 4502 4969 4697 1092 4222 266
1639 5115 4626 4927 4224 4691 5710 5328 4452 1165
5561 4432 349 1388 5668 3445 661 5449 4565 3856
757 4970 978 4270 3511 4388 5375 5154 5742 5791
2926 5934 735 1024 1268 2512 3834 3184 2575 210
3530 2504 4210 4707 419 1216 583 4380 960 5701
As can be seen from Table 2, 100 parts of pasteurized milk having a microbial content of less than 20000CFU/ml after heat treatment at 72 ℃ for 15s had a median value of 3683. Therefore, the C value was 3683.
TABLE 3 detection Results (RLU) of negative pasteurized milk samples
138 87 91 121 156 91 103 110 148 121
141 109 148 139 144 152 93 144 110 146
144 91 109 100 84 152 151 105 128 135
146 137 121 114 138 148 128 116 116 147
122 98 98 111 140 127 106 126 133 120
142 105 143 126 150 104 98 90 119 120
110 85 155 149 109 106 146 151 133 140
90 154 102 138 111 115 121 117 155 134
89 134 120 156 97 156 124 104 132 104
92 155 90 116 153 128 153 115 111 114
As can be seen from table 3, the median of the results of the detection of 100 negative pasteurized milk samples was 121, and therefore, the D value was 121.
TABLE 485 test Results (RLU) of milk heat-treated at 1s
2641 3459 2950 1569 5382 2112 4693 2282 236 5246
5092 3818 5530 5715 2756 1510 1908 998 5763 1924
1899 5861 2460 2398 3725 1057 390 4203 4374 4073
5388 5340 319 2601 2556 5903 3888 3287 4154 713
784 4527 1988 2012 3129 3390 849 2524 2677 4083
3570 2482 5891 5587 1542 3101 4429 1900 4444 2424
753 4768 3454 3349 1599 737 5053 309 4449 2479
5214 2196 2429 3428 2513 1880 546 2354 3843 541
839 5353 5712 3180 5522 5218 5103 279 2286 4818
4216 4737 226 1744 2643 4981 5005 751 5697 1814
As can be seen from Table 4, the median of the results of 100 portions of cow's milk heat-treated at 85 ℃ for 1s was 3025.5, and thus, the E value was 3025.5.
The B value was determined by the activity of alkaline phosphatase of the standard (mU/L) and the fluorescence intensity of the Reaction (RLU). Before each detection, firstly detecting a negative pasteurized milk and a standard sample of the negative pasteurized milk, wherein the alkaline phosphatase activity of the standard sample is respectively 175mU/L and 350mU/L, each concentration detection is carried out for 3 parallels, and the average value is taken as the result. According to the detection results of 0mU/L, 175mU/L and 350mU/L respectively in the formula
Figure BDA0003356571860000101
Figure BDA0003356571860000102
The calibration factor B is calculated by substituting the values of the parameters, wherein the value B is within +/-20% of the average value, and the average value of 3 concentration values B is taken as the calibration factor. The results are shown in Table 5.
TABLE 5B value calculation Table
Figure BDA0003356571860000103
Figure BDA0003356571860000111
As can be seen from Table 5, the average values of B values obtained according to 0mU/L, 175mU/L and 350mU/L were 0.747. Therefore, the B value was 0.747.
Example 3 correlation of luminescence intensity (RLU) with alkaline phosphatase Activity
The method for detecting the alkaline phosphatase of the negative milk with the microorganism content of less than 20000cfu/ml is adopted to detect the alkaline phosphatase standard sample, the standard concentration is respectively 0, 22, 44, 66, 88, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 and 375mU/L, 3 parallels are detected in each concentration, the alkaline phosphatase activity is taken as an abscissa, the fluorescence intensity is taken as an ordinate to draw a curve, and the result is shown in figure 2.
As can be seen from FIG. 2, when the content of microorganisms in the sample is less than 20000CFU/ml, the fluorescence intensity has a positive correlation with the alkaline phosphatase activity.
Example 4 Effect of highly sensitive Fluorogenic substrate Compounds on sensitivity with common Fluorogenic substrates
20 negative samples were tested using the high sensitivity fluorogenic substrate compound (formula I) and the alkaline phosphatase conventional fluorogenic substrate (formula II) described herein, respectively, and the mean (AVG) and Standard Deviation (SD) were calculated, and the limit of detection (LOD) was calculated according to AVG plus 3 times SD, with the results shown in Table 6.
TABLE 6 sensitivity of fluorogenic substrate compounds of the present application to conventional fluorogenic substrates
Figure BDA0003356571860000121
As can be seen from Table 6, the detection limit of the method is 20.2mU/L, and the detection limit using a conventional fluorogenic substrate is 35.0mU/L, so that the sensitivity using the fluorogenic substrate compound described herein is significantly higher than that using a conventional fluorogenic substrate.
Example 5 Effect of highly sensitive photomultiplier tube and photodiode on sensitivity
The detector described in the present application and the detector containing a photodiode were used to detect 20 negative samples, the mean (AVG) and Standard Deviation (SD) were calculated, and the limit of detection (LOD) was calculated according to AVG plus 3 times SD, with the results shown in table 7.
TABLE 7 photomultiplier tube to diode sensitivity comparison
Figure BDA0003356571860000122
As can be seen from Table 7, the detection limit of the method is 20.0mU/L, and the detection limit of the detector adopting the diode is 50.1mU/L, so that the sensitivity of the detector adopting the photomultiplier is obviously higher than that of the detector adopting the diode.
Example 6 study of the accuracy of the determination of alkaline phosphatase by the method of the present application
The method is adopted to detect the standard sample of pasteurized milk, so that the activity of alkaline phosphatase in the sample is five concentration levels of 20mU/L, 60mU/L, 100mU/L, 350mU/L and 500mU/L, each concentration level is subjected to 6 times of repeated tests, and the detection result is shown in Table 8.
TABLE 8 accuracy of alkaline phosphatase detection
Figure BDA0003356571860000131
As can be seen from Table 8, the accuracy of the alkaline phosphatase assay was 96.50% to 103.43% with a coefficient of variation of 3.57% to 8.54%.
EXAMPLE 7 study of the precision of alkaline phosphatase determination by the method of the present application
The 3 batches of kit prepared by the method are used for detecting the standard sample of pasteurized milk, so that the concentrations of alkaline phosphatase are 60mU/L, 100mU/L and 350mU/L respectively, 6 times of repeated tests are carried out on each concentration level, the results are averaged, and the detection results are shown in table 9.
TABLE 9 results of alkaline phosphatase assay in different batches
Figure BDA0003356571860000132
Figure BDA0003356571860000141
As can be seen from Table 9, the coefficient of variation of alkaline phosphatase measured at 3 concentrations by 3 batches of the kit prepared by the present application was 3.03-5.21%, and both were less than 10%, indicating high precision.
EXAMPLE 8 investigation of the Effect of built-in temperature probe stabilizer on the stability of results
By adopting the kit, 5 pasteurized milk samples are respectively detected by the detector and the detector without the built-in temperature probe stabilizer, each sample is parallel to 5 samples, and the detection results are shown in a table 10.
TABLE 10 test results of the test device and the test device without the temperature controller
Figure BDA0003356571860000142
As can be seen from Table 10, the coefficient of variation of the detector of the present application is 2.96-6.32%, and the average coefficient of variation is 4.67%; the detector without the built-in temperature probe stabilizer has a coefficient of variation of 17.76-28.15% and an average coefficient of variation of 24.52%. Thus, the stability of the results obtained with the test meter described herein including the built-in temperature probe stabilizer is significantly higher than the test meter without the temperature controller.
Example 9 investigation of the content of active protein in pasteurized milk
80 pasteurized milk samples which are strictly produced by a pasteurization process are selected, the contents of lactoferrin, alpha-lactalbumin and beta-lactoglobulin in the pasteurized milk samples are detected by an ELISA method, 3 samples are paralleled, and the results are averaged. The results are shown in FIGS. 3 and 4.
As can be seen from figure 3, the lactoferrin content in the pasteurized milk is greater than or equal to 25 mg/L; as can be seen from FIG. 4, the contents of alpha-lactalbumin and beta-lactoglobulin in pasteurized milk are both more than 500mg/L and 2200mg/L respectively.
The method and the system adopt a highly sensitive fluorescent substrate compound which reacts with alkaline phosphatase to emit fluorescence, the software of the detector converts the fluorescence intensity [ expressed in Relative Luminescence Units (RLU) ] into the activity (mU/L) of the alkaline phosphatase by using an accurate algorithm, and the detection sensitivity and the detection accuracy are improved by combining a highly sensitive photomultiplier in the detector; moreover, the whole detection process does not need sample preparation, the operation is very simple, and the detection can be finished within 45 seconds; the standard substance (calibration sheet) takes raw milk as a matrix, so that the accuracy of a detection result is improved, and the calibration sheet is solid and easy to store, so that the enzyme activity is kept stable; the temperature probe stabilizer arranged in the detector can ensure that the temperature of the reaction liquid meets the requirement, thereby ensuring the accuracy and stability of the detection result.
Although the present application and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present application, processes, machines, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present application. Accordingly, the appended claims are intended to include within their scope such processes, devices, means, methods, or steps.

Claims (4)

1. A method for identifying pasteurized milk, characterized in that it consists in determining the degree of microbial kill by quantitatively determining the activity of alkaline phosphatase which reacts with a substrate compound to produce fluorescence, the relationship between alkaline phosphatase activity and fluorescence intensity being expressed as a function F (x),
Figure FDA0003736576100000011
is related to A, B, C, D, E five parameters, whereinA is the median of fluorescence intensity obtained by 100 positive quality control samples, the activity of alkaline phosphatase of the positive quality control is 350mU/L, and the degree of variation is 30 percent; b is a calibration factor; c is the median of fluorescence intensity obtained by 100 portions of pasteurized milk which is subjected to heat treatment at 72 ℃ for 15s and has the microorganism content lower than 20000 CFU/ml; d is the median of the fluorescence intensities obtained for 100 negative pasteurized milk samples; e is the median of the fluorescence intensities obtained for 100 parts of milk heat-treated at 85 ℃ for 1 s; determining a B value by the activity of the alkaline phosphatase of the standard and the fluorescence intensity of the reaction;
when the content of microorganisms in pasteurized milk is lower than 20,000cfu/ml, the fluorescence intensity and the activity of alkaline phosphatase conform to a function F (x), and the content of the microorganisms is in positive correlation with the activity of the alkaline phosphatase; a higher fluorescence intensity indicates a higher alkaline phosphatase activity and, correspondingly, a higher microbial content;
the substrate compound has a structure shown in formula I:
Figure FDA0003736576100000012
2. the method of claim 1, wherein non-pasteurized milk is initially judged when alkaline phosphatase activity < 50mU/L indicates the possible presence of overheating.
3. A method according to claim 1 or claim 2, further comprising measuring the level of lactoferrin, alpha-lactalbumin and/or beta-lactoglobulin.
4. The method according to claim 3, wherein pasteurized milk is preliminarily determined when the lactoferrin content is 25mg/L or more, the alpha-lactalbumin content is 500mg/L or more, and/or the beta-lactoglobulin content is 2200mg/L or more.
CN202111353273.0A 2021-11-16 2021-11-16 Method and detection system for identifying pasteurized milk Active CN114088626B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111353273.0A CN114088626B (en) 2021-11-16 2021-11-16 Method and detection system for identifying pasteurized milk

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111353273.0A CN114088626B (en) 2021-11-16 2021-11-16 Method and detection system for identifying pasteurized milk

Publications (2)

Publication Number Publication Date
CN114088626A CN114088626A (en) 2022-02-25
CN114088626B true CN114088626B (en) 2022-08-26

Family

ID=80300791

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111353273.0A Active CN114088626B (en) 2021-11-16 2021-11-16 Method and detection system for identifying pasteurized milk

Country Status (1)

Country Link
CN (1) CN114088626B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116858974B (en) * 2023-09-05 2023-12-01 中国农业科学院北京畜牧兽医研究所 Identification method and application of pasteurized milk sterilization temperature

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4978614A (en) * 1988-10-26 1990-12-18 Tropix, Inc. Method of detecting a substance using enzymatically-induced decomposition of dioxetanes
US5330900A (en) * 1987-12-31 1994-07-19 Tropix, Inc. Chemiluminescent 3-(substituted adamant-2'-ylidene) 1,2-dioxetanes
US5525473A (en) * 1989-01-31 1996-06-11 Hybritech Incorporated Assay for bone alkaline phosphatase
CN104263831A (en) * 2014-09-28 2015-01-07 南京诺唯赞生物科技有限公司 Method for simply, conveniently and quickly determining activity of alkaline phosphatase
CN105445393A (en) * 2015-11-17 2016-03-30 中国农业科学院北京畜牧兽医研究所 Identification method for reconstituted milk in pasteurized milk
CN108254560A (en) * 2018-04-19 2018-07-06 国家食品安全风险评估中心 Aflatoxins M1 high-throughput enzyme linked immunoassay reagent kit and its detection method in milk
CN110501317A (en) * 2019-08-27 2019-11-26 中国科学院长春应用化学研究所 A kind of fluorescence detection method of alkaline phosphatase activities
CN111272726A (en) * 2020-05-08 2020-06-12 北京中检葆泰生物技术有限公司 Method for detecting pesticide residue in food
CN112067601A (en) * 2020-08-05 2020-12-11 武汉生之源生物科技股份有限公司 Alkaline phosphate enzymatic chemiluminescence substrate reinforcing agent and application thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5004565A (en) * 1986-07-17 1991-04-02 The Board Of Governors Of Wayne State University Method and compositions providing enhanced chemiluminescence from 1,2-dioxetanes
EP0853678A4 (en) * 1995-07-31 1999-01-27 Charm Sciences Inc Chemiluminescent method of monitoring products after heat treatment
WO2011003054A2 (en) * 2009-07-03 2011-01-06 Dow Corning Corporation Film forming, silicone containing compositions
US20140308661A1 (en) * 2011-09-25 2014-10-16 Theranos, Inc. Systems and methods for multi-analysis
FR3082310B1 (en) * 2018-06-08 2020-09-04 Spectralys Innovation PROCESS FOR DETERMINATION OF CASEINS AND SERUM PROTEINS IN RAW OR LOW PASTEURIZED DAIRY PRODUCTS.
CN113310930A (en) * 2021-05-10 2021-08-27 华中农业大学 Spectral identification method of high-temperature sterilized milk, pasteurized milk and pasteurized milk mixed with high-temperature sterilized milk

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5330900A (en) * 1987-12-31 1994-07-19 Tropix, Inc. Chemiluminescent 3-(substituted adamant-2'-ylidene) 1,2-dioxetanes
US4978614A (en) * 1988-10-26 1990-12-18 Tropix, Inc. Method of detecting a substance using enzymatically-induced decomposition of dioxetanes
US5525473A (en) * 1989-01-31 1996-06-11 Hybritech Incorporated Assay for bone alkaline phosphatase
CN104263831A (en) * 2014-09-28 2015-01-07 南京诺唯赞生物科技有限公司 Method for simply, conveniently and quickly determining activity of alkaline phosphatase
CN105445393A (en) * 2015-11-17 2016-03-30 中国农业科学院北京畜牧兽医研究所 Identification method for reconstituted milk in pasteurized milk
CN108254560A (en) * 2018-04-19 2018-07-06 国家食品安全风险评估中心 Aflatoxins M1 high-throughput enzyme linked immunoassay reagent kit and its detection method in milk
CN110501317A (en) * 2019-08-27 2019-11-26 中国科学院长春应用化学研究所 A kind of fluorescence detection method of alkaline phosphatase activities
CN111272726A (en) * 2020-05-08 2020-06-12 北京中检葆泰生物技术有限公司 Method for detecting pesticide residue in food
CN112067601A (en) * 2020-08-05 2020-12-11 武汉生之源生物科技股份有限公司 Alkaline phosphate enzymatic chemiluminescence substrate reinforcing agent and application thereof

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
A portable chemiluminescence assay of alkaline phosphatase activity to monitor pasteurization of milk products;Sarver R, 等;《Journal of food protection》;20191115;第82卷(第12期);第2119-2125页 *
A survey of some commercially available kits and reagents which include bioluminescence or chemiluminescence for their operation: Including immunoassays, hybridization, labels, probes, blots and ATP-based rapid microbiology;Philip E. Stanley.;《Products from more than forty companies》;19930430;第8卷(第2期);第51-63页 *
Alkaline phosphatase activity in pasteurized milk: A quantitative comparison of Fluorophos and colourimetric procedures;Payne C 等;《International journal of dairy technology》;20090713;第62卷(第3期);第308-314页 *
Commercially available luminometers and imaging devices for low‐light level measurements and kits and reagents utilizing bioluminescence or chemiluminescence: Survey update 5;Stanley P E.;《Journal of bioluminescence and chemiluminescence》;19981204;第12卷(第2期);第61-78页 *
Determination of alkaline phosphatase activity in milk and milk products by fluorimetric method;Rola J G 等;《Bull Vet Inst Pulawy》;20100131;第54卷;第537-542页 *
Evaluation of Spectrophotometric and Fluorometric Methods for Alkaline Phosphatase Activity Determination in Ewe"s Milk;SCINTU, M. F. 等;《Journal of Food Protection》;20000901;第63卷(第9期);第1258-1261页 *
Fluorimetric assay of alkaline phosphatase in milk products;Jong, E. A. M. de 等;《Voedingsmiddelen Technologie》;19930101;第26卷(第23期);第11-16页 *
乳中ALP、LPO、γ-GGT检测方法的建立;惠永华;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》;20081015(第10期);第B024-37页 *
乳中碱性磷酸酶活性测定方法研究进展;申军士等;《中国乳品工业》;20090625;第37卷(第06期);第31-34页 *
乳及乳制品中碱性磷酸酶和乳过氧化物酶活性检测方法的评估及应用;张昊;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》;20190915(第9期);第B024-213页 *
交链孢毒素在Caco-2细胞中的转运与吸收;杨大进 等;《山东大学学报(医学版)》;20150630;第53卷(第6期);第18-22页 *
酶免疫分析技术研究进展;刘连生;《湖北省卫生职工医学院学报》;20000331(第1期);第53-54页 *

Also Published As

Publication number Publication date
CN114088626A (en) 2022-02-25

Similar Documents

Publication Publication Date Title
Rankin et al. Invited review: The application of alkaline phosphatase assays for the validation of milk product pasteurization
De Roos et al. Screening for subclinical ketosis in dairy cattle by Fourier transform infrared spectrometry
Pellegrino et al. Coupling of lactulose and furosine indices for quality evaluation of sterilized milk
Zhang et al. Detection of Subclinical Ketosis in Dairy Cows.
CN114088626B (en) Method and detection system for identifying pasteurized milk
CN106442453A (en) Method for rapidly and quantitatively measuring spore concentration
EP2401355A1 (en) Method for the bacteriological investigation of a biological sample and relative device
CN110082347B (en) Simple urine glucose quantitative detection method and urine glucose quantitative detection kit
CN105132519A (en) Selective medium used for quantitative detection of escherichia coli and escherichia coli quantitative detection method
Choudhary et al. Development of an embedded system for real-time milk spoilage monitoring and adulteration detection
Rathert-Williams et al. Colorimetric methods for accurate determination of nutrient composition in beef cow colostrum and milk
Hanuš et al. Milk acetone determination by the photometrical method after microdiffusion and via FT infra-red spectroscopy.
Luzzana et al. Milk lactose and lactulose determination by the differential pH technique
Petrović et al. Comparative examination of the analysis of β-lactam antibiotic residues in milk by enzyme, receptor–enzyme, and inhibition procedures
Kato et al. Enzymatic assay for D-aspartic acid using D-aspartate oxidase and oxaloacetate decarboxylase
CN112063683B (en) Method and kit for detecting antibiotic residues in food and production and circulation processes thereof
CN102183664A (en) Biotin content measuring method based on cylinder-plate method
CN108181245B (en) Method for quantitatively determining lactoperoxidase activity in milk by enzyme method and kit thereof
CN111721756A (en) Alkaline phosphatase activity detection kit and detection method
JP5397991B2 (en) Determination method of vitamin B12 using lactic acid bacteria
Mirecki et al. Influence of preservative concentration, pH Value and fat content in raw milk at detection limit of microbial inhibitor tests (Delvotest® Accelerator) for amoxicillin and oxytetracycline
CN109680035B (en) Screening method and application of inositol-deficient strain
AU770458B2 (en) Mastitis detection method
Wu et al. ATP bioluminescence-based method for screening of multiclass antibiotic residues in animal derived foods
Mazurenko et al. Biomarkers of subclinical mastitis in the mammary gland of cows

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant