CN114088626B

CN114088626B - Method and detection system for identifying pasteurized milk

Info

Publication number: CN114088626B
Application number: CN202111353273.0A
Authority: CN
Inventors: 杨大进; 刘龙飞
Original assignee: Beijing Zhongjian Baotai Biotechnology Co ltd; China National Center For Food Safety Risk Assessment
Current assignee: Beijing Zhongjian Baotai Biotechnology Co ltd; China National Center For Food Safety Risk Assessment
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2022-08-26
Anticipated expiration: 2041-11-16
Also published as: CN114088626A

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),

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).

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),

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:

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:

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

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

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

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

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

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

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

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

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),

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:

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.