CN116858974B - Identification method and application of pasteurized milk sterilization temperature - Google Patents

Identification method and application of pasteurized milk sterilization temperature Download PDF

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CN116858974B
CN116858974B CN202311133273.9A CN202311133273A CN116858974B CN 116858974 B CN116858974 B CN 116858974B CN 202311133273 A CN202311133273 A CN 202311133273A CN 116858974 B CN116858974 B CN 116858974B
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content
milk
lactoglobulin
beta
pasteurized milk
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CN116858974A (en
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张养东
郑楠
王加启
李宁
孟璐
刘慧敏
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Institute of Animal Science of CAAS
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The invention discloses a method for identifying the sterilization temperature of pasteurized milk and application thereof, wherein the method comprises the following steps: (1) Determining the content of beta-lactoglobulin and furoic acid in fresh milk; (2) Detecting the content of beta-lactoglobulin and furfuryl amino acid in pasteurized milk and calculating the content difference of the beta-lactoglobulin and furfuryl amino acid from the corresponding substances in raw and fresh milk; (3) According to the predictive modelCalculating a sterilization temperature of the pasteurized milk, wherein:X 1 representing the content difference of beta-lactoglobulin in pasteurized milk and beta-lactoglobulin in fresh milk, and the unit is mg/L;X 2 the difference between the content of furoic acid in pasteurized milk and the content of furoic acid in fresh milk is expressed in mg/100g protein. The method can identify the sterilization temperature of the pasteurized milk, is used for guiding the production of the pasteurized milk, ensures the quality of the pasteurized milk, ensures the rights and interests of consumers and promotes the healthy development of the dairy industry.

Description

Identification method and application of pasteurized milk sterilization temperature
Technical Field
The invention relates to the technical field of dairy products, in particular to a method for identifying pasteurized milk sterilization temperature and application thereof.
Background
Pasteurized milk processing conditions are milder than UHT sterilized milk and better retain naturally occurring actives in milk, thus representing a significant share in developed areas of the milk industry, especially in developed countries.
At present, the international organization and the countries around the world do not issue identification standards for the sterilization temperature of milk, so that it is necessary to develop a method for identifying the sterilization temperature of pasteurized milk, guide the production of pasteurized milk, ensure the quality of pasteurized milk, ensure the rights and interests of consumers and promote the healthy development of the dairy industry.
Disclosure of Invention
The invention aims to provide a method for identifying the pasteurization temperature of pasteurized milk and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
raw milk (raw milk), also known as raw fresh milk, refers to regular milk expressed from healthy dairy animals with or without filtration and cooling, but without any heating and other sterilization treatments. Pasteurized milk refers to drinking milk from fresh cow's milk by cleaning (filtering and off-line), cooling, standardizing, homogenizing, pasteurizing, cooling and filling.
A method of identifying pasteurized milk sterilization temperature comprising the steps of:
(1) Determining the content of beta-lactoglobulin and furoic acid in fresh milk;
(2) Detecting the content of beta-lactoglobulin and furfuryl amino acid in pasteurized milk and calculating the content difference of the beta-lactoglobulin and furfuryl amino acid from the corresponding substances in raw and fresh milk;
(3) According to the predictive modelCalculating the sterilization temperature of the pasteurized milk;
in the method, in the process of the invention,X 1 : the content of beta-lactoglobulin in pasteurized milk is different from that of beta-lactoglobulin in fresh milk in unit mg/L;X 2 : the content of furfuryl amino acid in pasteurized milk is different from the content of furfuryl amino acid in fresh milk, and the unit is mg/100g protein.
The present invention defines the "pasteurization temperature" specifically as the "pasteurization temperature employed in the pasteurization process of pasteurized milk at high temperature (72.5 ℃ -120 ℃) for a short time (15 s). The pasteurized milk in the method of the invention is not added with nutrition enhancers, or is added with minerals and/or vitamins only.
The method for measuring the content of the beta-lactoglobulin comprises the following steps:
(1) Sampling: taking at least 200 mL liquid milk as an experimental sample, and preserving the sample at 0-4 ℃;
(2) Preparing the upper machine liquid: sucking a 2 mL sample, placing the sample in a 50 mL centrifuge tube, adding 15 mL ultrapure water, and uniformly mixing; adding 35-45 mu L of glacial acetic acid into the centrifuge tube, shaking uniformly, and regulating the pH to 4.6; placing the centrifuge tube in a high-speed centrifuge, and centrifuging at 10000 rpm and 4deg.C for 10 min; taking out the centrifuge tube, discarding fat at the upper part and sediment at the bottom, sucking the middle clear liquid by using a syringe, and passing through a microporous filter membrane of 0.22 mu m for detection by a machine;
(3) Determination of beta-lactoglobulin content
1) Chromatographic reference conditions
Chromatographic column: symmetry C4 chromatographic column, 4.6 mm X250 mm,300 a, 5 μm particle size; or equivalent;
column temperature: 30 ℃;
a detector: a PDA detector;
sample injection volume: 50. mu L;
mobile phase: acetonitrile is mobile phase A;0.1 The% trifluoroacetic acid solution is mobile phase B;
elution gradients are as follows:
time (min) Flow rate mL/min Acetonitrile (%) 0.1% trifluoroacetic acid solution (%)
0 1.0 30 70
5 1.0 55 45
10 1.0 60 40
12 1.0 30 70
16 1.0 30 70
2) Quantification of
Beta-lactoglobulin content is expressed as concentration C, and the numerical value is expressed as mg/L, and is calculated according to formula (1):
……………………(1)
wherein:
C: the content of beta-lactoglobulin in the sample is mg/L;
C i : the content of beta-lactoglobulin in the upper machine liquid is mg/L;
V 1 : the volume of the milk sample is in mL;
V 2 : the volume of ultrapure water is in mL.
The precision control requirement of the method for measuring the content of the beta-lactoglobulin is as follows: the absolute difference of the two independent test results obtained under the repeatability condition is not more than 10% of the arithmetic mean value, and the absolute difference of the two independent test results obtained under the repeatability condition is not more than 20% of the arithmetic mean value.
The method for measuring the content of the furfuryl amino acid comprises the following steps:
(1) Sampling: taking at least 200 mL liquid milk as an experimental sample, and preserving the sample at 0-4 ℃;
(2) Preparation of hydrolysate: sucking a 2 mL sample, placing the sample in a sealed heat-resistant test tube, adding a 6 mL hydrochloric acid solution with the concentration of 10.6 mol/L, uniformly mixing, slowly introducing the high-purity nitrogen into the test tube for 1-2 min, sealing the test tube, placing the test tube in a drying box, heating the test tube at 110 ℃ for 1 h, gently shaking the test tube, and continuing heating until 23 h-24 h is heated; after heating, taking out the test tube from the drying oven, cooling and filtering to obtain hydrolysate;
(3) Determination of protein content in hydrolysate: absorbing 2 mL the hydrolysate prepared in the step (2), and measuring the protein content in the sample solution according to GB/T5009.5;
(4) Purifying the hydrolysate: the C18 column was mounted on a syringe, wetted with 5 mL methanol and 10 mL water, respectively; sucking the hydrolysis liquid in the step (2) of 0.500 and mL into an extraction column, and slowly pushing the hydrolysis liquid into the C18 extraction column by using an injector; absorbing 3 mol/L hydrochloric acid solution to slowly elute the sample in the extraction column to 3 mL;
(5) Determination of the furoic acid content: the HPLC method and/or UPLC method are adopted for measurement;
(6) Calculating the content of furoic acid: the content of the furoic acid is calculated by mass fractionWThe values are expressed in mg/100g protein and are calculated according to the formula (2):
……………………(2)
wherein:
W: the content of furfuryl amino acid in the sample is mg/100g protein;
A t : testing the value of the furfuryl amino acid peak area in the sample;
A std : the value of the furoic acid peak area in the furoic acid standard solution;
C std : concentration of the furoic acid standard solution is in mg/L;
D: dilution fold at the time of measurementThe number of the product is the number,D=6；
M: protein concentration in grams per liter (g/L) in the sample hydrolysate.
The precision control requirement of the method for measuring the content of the furoic acid is as follows: the absolute difference of the two independent test results obtained under the repeatability condition is not more than 10% of the arithmetic mean value, and the absolute difference of the two independent test results obtained under the repeatability condition is not more than 20% of the arithmetic mean value.
Further, chromatographic reference conditions of the HPLC method in the determination method of the furfuryl amino acid content are as follows:
chromatographic column: c18 silica gel column, 250 mm ×4.6mm, 5 μm particle size; or equivalent;
column temperature: 32 ℃;
mobile phase: 0.1% trifluoroacetic acid solution as mobile phase A and methanol as mobile phase B;
elution gradients are as follows:
sequence number Time min Flow rate mL/min Mobile phase A% Mobile phase B%
1 － 1.00 100.0 0.0
2 16.00 1.00 86.8 13.2
3 16.50 1.50 100.0 0.0
4 30.00 1.00 0.0 100.0
The measuring method comprises the following steps:
using the mixed solution of the mobile phase A and the mobile phase B to balance the chromatographic system according to the proportion of 50:50 at the flow rate of 1 mL/min, injecting 20-50 mu L of 3 mol/L hydrochloric acid solution into the balance column to detect the purity of the solvent; and 10 mu L of the solution to be detected is injected to determine the content of furoic acid.
Further, chromatographic reference conditions of a UPLC method in the determination method of the furoic acid content are as follows:
chromatographic column: WATERS ACQUITY UPLC ® HSS T3 column 1.8 μm,100 mm ×2.1 mm; or equivalent;
column temperature: 30. the temperature is lower than the temperature;
mobile phase: 6 g/L KCl solution is a mobile phase A, methanol is a mobile phase B, and pure water is a mobile phase C;
elution conditions: mobile phase A,0.4 mL/min;
the measuring method comprises the following steps:
sequentially balancing the chromatographic system at a flow rate of 0.4 mL/min by using a mobile phase B, C, A respectively, and injecting 2-5 mu L of 3 mol/L hydrochloric acid solution to balance the column so as to detect the purity of the solvent; 2. Mu.L of the solution to be measured was injected to determine the furoic acid content.
The identification method of pasteurized milk sterilization temperature can be used for pasteurized milk production and quality detection.
Compared with the prior art, the invention has the outstanding effects that:
the identification method of the invention is mainly aimed at pasteurized milk without nutrition enhancer or with minerals and vitamins only. Identification of pasteurized milk sterilization temperature is a key point in ensuring the quality of pasteurized milk and directing its production. At present, no similar standard at home and abroad can be referred to, the invention creatively provides an identification method of pasteurized milk sterilization temperature, and the established prediction model can accurately predict the pasteurized milk sterilization temperature within the range of 75-120 ℃ with the accuracy of +/-1 ℃. Not only can guide the production of pasteurized milk and ensure the quality of pasteurized milk, lay a technical foundation for the supervision of pasteurized milk, but also can promote the technical innovation of China in the field to be in the international advanced level.
The identification method further improves the authority of the dairy product detection method, and plays an important role in improving the quality of pasteurized milk, guaranteeing the awareness of consumers, promoting the healthy development of milk industry in China and the like.
The method for identifying the pasteurized milk sterilization temperature and the application according to the invention are further described below with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a plot of the difference in beta-lactoglobulin content in pasteurized and raw fresh milk versus processing temperature for different processing temperatures;
figure 2 is a plot of the difference in the levels of furfuryl amino acids in pasteurized and raw milk versus processing temperature for different processing temperatures.
Detailed Description
Example 1 determination of the content of furoic acid
The milk undergoes Maillard reaction during heating to produce furoic acid (epsilon-N-2-furanmethyl-L-lysine) from protein and sugar. The protein content of the sample is measured after the sample is hydrolyzed by hydrochloric acid, the hydrolysate is extracted by C18, and the furoic acid sample is analyzed by High Performance Liquid Chromatography (HPLC) or ultra high performance liquid chromatography (UPLC) under an ultraviolet (280 nm) detector, and furoic acid is obtained by an external standard method. And finally calculating the content of furfuryl amino acid in every hundred grams of protein. In the method for measuring the content of furoic acid according to the present invention, only the reagent confirmed to be analytically pure and primary water in GB/T6682 are used.
(1) Reagent(s)
Methanol (CH) 3 OH): chromatographic purity.
3 mol/L hydrochloric acid solution: 2.5 mL concentrated hydrochloric acid (12 mol/L) was added to 7.5. 7.5 mL water, and the mixture was mixed well.
10.6 mol/L hydrochloric acid solution: 88 mL concentrated hydrochloric acid (12 mol/L) is added into 12 mL water and mixed evenly.
0.1% trifluoroacetic acid solution: 1 mL trifluoroacetic acid (chromatographic purity) was dissolved in water, the volume was set to 1L, and the ultrasonic degassing was performed for 35 min.
6 g/L KCl solution (m/v): 6 g KCl was accurately weighed and dissolved in a portion of water to a constant volume of 1L, and passed through a 0.45 μm aqueous filter membrane and subjected to ultrasonic deaeration for 35 min.
Standard stock solution of furoic acid (epsilon-N- (2-furanmethyl) -L-lysine): the standard furfuryl amino acid is converted into a standard stock solution of 200 mug/mL by using 3 mol/L hydrochloric acid solution, and can be stored for 24 months at-20 ℃.
Standard working solution of furoic acid: the micropipette draws 250. Mu.L of standard stock solution in a 25 mL volumetric flask and uses 3 mol/L hydrochloric acid solution to determine volume to prepare 2. Mu.g/mL furoic acid standard working solution.
High purity nitrogen: 99.99 Percent of the total weight of the composition.
(2) Instrument for measuring and controlling the intensity of light
High performance liquid chromatograph: equipped with a gradient elution system, and an ultraviolet detector or a diode array detector.
Kjeldahl apparatus.
C18 extraction column: column capacity 500 mg.
Drying box: 110 ℃ +/-2 ℃.
Sealing the heat-resistant test tube: the volume was 20 mL.
A syringe: 10 And (3) mL.
Volumetric flask: the volumes were 25 mL and 1L, respectively.
Ultra-high performance liquid chromatograph: provided with an ultraviolet detector or a diode array detector.
Organic phase filtration membrane: 0.45 μm.
Aqueous phase filter membrane: 0.45 μm.
The determination of the furoic acid content comprises the following steps:
(1) Sampling: taking at least 200 mL liquid milk as an experimental sample, and preserving the sample at 0-4 ℃.
(2) Preparation of hydrolysate: sucking a 2 mL sample, placing the sample in a sealed heat-resistant test tube, adding a 6 mL hydrochloric acid solution with the concentration of 10.6 mol/L, uniformly mixing, slowly introducing the high-purity nitrogen into the test tube for 1-2 min, sealing the test tube, placing the test tube in a drying box, heating the test tube at the temperature of 110 ℃ for about 1 h, gently shaking the test tube, and continuing heating until 23 h-24 h is heated. And after heating, taking the test tube out of the drying box, cooling and filtering to obtain the hydrolysate.
(3) Determination of protein content in hydrolysate: and (3) absorbing 2 mL of the hydrolysate prepared in the step (2), and measuring the protein content in the sample solution according to GB/T5009.5.
(4) Purifying the hydrolysate: mounting the C18 extraction column on a syringe; wetting the extraction column with 5 mL methanol and 10 mL water respectively, and keeping the extraction column in a wet state; sucking the hydrolysis liquid in the step (2) of 0.500 and mL into an extraction column, and slowly pushing the hydrolysis liquid into the C18 extraction column by using an injector; the sample in the extraction column was slowly eluted by pipetting 3 mol/L hydrochloric acid to 3 mL.
(5) Determination of the furoic acid content: the following two methods were used for measurement. (a) HPLC method; (b) UPLC method.
(a) HPLC method:
1) Chromatographic reference conditions
Chromatographic column: c18 silica gel column, 250 mm ×4.6 mm,5 μm particle size, or equivalent.
Column temperature: 32 ℃.
Mobile phase: the 0.1% trifluoroacetic acid solution is mobile phase a and methanol is mobile phase B.
Elution gradient: see table 1.
TABLE 1 elution gradient
Sequence number Time min Flow rate mL/min Mobile phase A% Mobile phase B%
1 － 1.00 100.0 0.0
2 16.00 1.00 86.8 13.2
3 16.50 1.50 100.0 0.0
4 30.00 1.00 0.0 100.0
2) Measurement
Equilibrate the chromatography system with a mixture of mobile phase A and mobile phase B (50:50) at a flow rate of 1 mL/min; injecting 20-50 mu L of 3 mol/L hydrochloric acid solution into the equilibrium column to detect the purity of the solvent; and (3) injecting 10 mu L of the solution to be detected (namely the purified hydrolysate obtained in the step (4)) to determine the content of the furoic acid.
(b) UPLC method
1) Chromatographic reference conditions
Chromatographic column: WATERS ACQUITY UPLC ® HSS T3 column 1.8 μm (100 mm X2.1 mm), or equivalent.
Column temperature: 30. DEG C.
Mobile phase: the 6 g/L KCl solution is mobile phase A, methanol is mobile phase B, and pure water is mobile phase C.
Elution conditions: mobile phase A,0.4 mL/min.
2) Measurement
The chromatography system was equilibrated sequentially with mobile phase B, C, A at a flow rate of 0.4 mL/min, respectively. The column was equilibrated by injecting 2. Mu.L to 5. Mu.L of a 3 mol/L hydrochloric acid solution to examine the purity of the solvent. 2 mu L of the solution to be tested (namely the purified hydrolysate obtained in the step (4)) is injected to measure the content of the furoic acid.
(6) Calculation result of furoic acid content: the content of the furoic acid is calculated by mass fractionWValues are expressed in milligrams per hundred grams of protein (mg/100 g protein) calculated according to formula (1):
……………………(1)
wherein:
W: the content of furfuryl amino acid in the sample is expressed in milligrams per hundred grams of protein (mg/100 g protein);
A t : testing the value of the furfuryl amino acid peak area in the sample;
A std : the value of the furoic acid peak area in the furoic acid standard solution;
C std : concentration of the furoic acid standard solution is in mg/L;
D: dilution factor in measurementD=6)；
m: protein concentration in grams per liter (g/L) in the sample hydrolysate.
The result is retained until the decimal point is later.
The precision of the determination method of the furoic acid content is controlled as follows:
the absolute difference of the two independent test results obtained under the repeatability condition is not more than 10% of the arithmetic mean value, and the absolute difference of the two independent test results obtained under the repeatability condition is not more than 20% of the arithmetic mean value.
The detection limits and quantitative limits of the HPLC and UPLC detection methods are shown in Table 2, respectively.
TABLE 2 detection limits and quantitative limits of HPLC and UPLC methods for determination of furoic acid content
Detection method Sample application amount (mu L) Minimum detected concentration (mg/L) Minimum quantitative concentration (mg/L) Detection limit (mg/100 g protein) Limit of quantification (mg/100 g protein)
HPLC 10 0.05 0.1 1.0 2.0
UPLC 2 0.01 0.1 0.2 0.4
Example 2 method for determining beta-lactoglobulin content
Casein and denatured whey proteins in milk may be isoelectric precipitated at ph=4.6. Centrifugation and filtration can separate acid soluble whey proteins. The beta-lactoglobulin in acid-soluble whey protein can be analyzed by reverse phase high performance liquid chromatography (RP-HPLC) on a uv (280 nm) detector to quantify the beta-lactoglobulin content by an external standard. In the method of measuring β -lactoglobulin according to the present invention, only the reagent confirmed to be analytically pure and primary water in GB/T6682 were used.
(1) Reagent(s)
Acetonitrile (C) 2 H 3 N): chromatographic purity.
Trifluoroacetic acid (CF) 3 CO 2 H) The method comprises the following steps Chromatographic purity.
Acetic acid (C) 2 H 4 O 2 ): analytically pure.
Beta-lactoglobulin standard intermediate working solution: after converting the beta-lactoglobulin standard substance into its purity, 2.5. 2.5 mg/mL of standard working solution was prepared with ultrapure water, and the solution was allowed to stand at 4℃for 1 week.
Beta-lactoglobulin standard working solution: quantitatively transferring the beta-lactoglobulin standard intermediate working solution in a 2 mL volumetric flask, and preparing 5, 10, 20, 50, 100, 200 and 500 mg/L beta-lactoglobulin standard working solution by using ultrapure water to fix the volume.
(2) Instrument for measuring and controlling the intensity of light
High performance liquid chromatograph: equipped with a gradient elution system, and an ultraviolet detector or a diode array detector.
Hitachi high speed centrifuge: is provided with a refrigerating function.
Volumetric flask: the volume was 2 mL.
Microporous filter membrane: the organic and aqueous phases are all used, 0.22 μm.
A syringe: 1 mL.
Centrifuge tube: 50 And (3) mL.
The measurement of the beta-lactoglobulin content comprises the following steps:
(1) Sampling: taking at least 200 mL liquid milk as an experimental sample, and preserving the sample at 0-4 ℃.
(2) Preparing the upper machine liquid: the sample of 2 mL is sucked, placed in a 50 mL centrifuge tube, added with 15 mL ultrapure water and mixed well. Adding about 35-45 mu L of glacial acetic acid into the centrifuge tube, shaking uniformly, and regulating the pH to 4.6. The centrifuge tube was placed in a high speed centrifuge and centrifuged at 10000 rpm at 4℃for 10 min. The centrifuge tube was removed, the upper fat and bottom pellet were discarded, the middle supernatant was aspirated with a syringe, and passed through a 0.22 μm microporous filter membrane for on-machine detection.
(3) Determination of beta-lactoglobulin content:
1) Chromatographic reference conditions
Chromatographic column: symmetry C4 column, 4.6 mm X250 mm,300 a, 5 μm particle size, or equivalent.
Column temperature: 30 ℃.
A detector: PDA detector.
Sample injection volume: 50. mu L.
Mobile phase: acetonitrile is mobile phase A;0.1 The% trifluoroacetic acid solution is mobile phase B.
Elution gradient: see table 3.
Table 3 beta-lactoglobulin content determination gradient elution procedure
Time (min) Flow rate mL/min Acetonitrile (%) 0.1% trifluoroacetic acid solution (%)
0 1.0 30 70
5 1.0 55 45
10 1.0 60 40
12 1.0 30 70
16 1.0 30 70
2) Quantification of
Beta-lactoglobulin content is expressed as concentration C, and values are expressed in milligrams per liter (mg/L), calculated according to formula (2):
……………………(2)
wherein:
C: the content of beta-lactoglobulin in the sample is expressed in milligrams per liter (mg/L);
C i : the content of beta-lactoglobulin in the upper machine liquid is expressed in milligrams per liter (mg/L);
V 1 : the volume of the milk sample in milliliters (mL);
V 2 : volume of ultrapure water in milliliters (mL).
The calculation result is kept to the last two bits of the decimal point.
The precision of the method for measuring the content of the beta-lactoglobulin is controlled as follows:
the absolute difference of the two independent test results obtained under the repeatability condition is not more than 10% of the arithmetic mean value, and the absolute difference of the two independent test results obtained under the repeatability condition is not more than 20% of the arithmetic mean value.
The detection limit of the HPLC method for measuring the beta-lactoglobulin content in the invention is 1.67. 1.67 mg/L.
Example 3 identification method of pasteurized milk temperature
The invention identifies the sterilization temperature of pasteurized milk by detecting two indexes of furfuryl amino acid and beta-lactoglobulin, wherein the quantification of furfuryl amino acid is related to milk-derived proteins. Therefore, substances added with non-milk-derived proteins in the product can interfere with the identification result. The current product standards of liquid milk in China comprise pasteurized milk, sterilized milk, seasoned milk and AD calcium milk, and the products of various raw materials such as pure cow milk, seasoned milk, vitamin-fortified milk, mineral-fortified milk, fruit juice, eggs and the like are more varieties in the market. Meanwhile, GB14880-2012 "national food safety Standard food nutrient supplement use Standard" prescribes that lactoferrin and Casein phosphopeptide can be added to prepared milk, and these two protein-containing components can also produce furoic acid during heating. Based on the above situation, the application scope of the present invention is further defined as: the invention is applicable to pasteurized milk without nutrition enhancers or with minerals and vitamins only.
The sterilization temperature of pasteurized milk without the addition of a nutrition enhancer, or with the addition of minerals and/or vitamins alone, is identified below based on the amounts of furfuryl amino acids and beta-lactoglobulin contained in raw and pasteurized milk.
Raw milk, pasteurized milk with different sterilization temperatures, and content of furoic acid and beta-lactoglobulin
Fresh milk was processed using a pasteurized milk production line (a dairy processing plant) at different pasteurization temperatures and the levels of furfuryl amino acids and beta-lactoglobulin in the pasteurized milk produced under different conditions were measured according to the assay methods of examples 1-2.
The results of the content of furfuryl amino acid and beta-lactoglobulin in fresh milk and pasteurized milk produced under different conditions are shown in table 4, and the results show that the content of furfuryl amino acid gradually increases and the content of beta-lactoglobulin gradually decreases with the increase of the processing temperature; and the higher the processing temperature, the larger the difference between the content of furfuryl amino acid and beta-lactoglobulin in the pasteurized milk and the corresponding value in raw milk is.
TABLE 4 Fungicide and beta-lactoglobulin content in pasteurized milk produced under different pasteurization temperature conditions
(II) pasteurized milk sterilization temperature identification method
Figure 1 is a plot of the fit of the difference in beta-lactoglobulin content to the processing temperature in pasteurized and raw fresh milk at different temperatures. Figure 2 is a graph of fit of the difference in the levels of furfuryl amino acids in pasteurized and raw milk at different temperatures to the processing temperature. Combining fig. 1 and 2 to obtain a fitting curve of the difference value of beta-lactoglobulin and furoic acid in pasteurized milk at different temperatures and the corresponding substances in fresh milk, wherein the fitting curve is as follows(wherein:X 1 represents the content difference of beta-lactoglobulin in mg/L compared with fresh milk;X 2 represents the difference in the content of furoic acid compared with fresh milk, unit mg/100g protein), R 2 =0.994, which can be used for the prediction of pasteurized milk sterilization temperatures in the range 75 ℃ to 120 ℃.
Application example identification of pasteurized milk sterilization temperature produced in a dairy processing plant (model verification)
A batch of pasteurized milk produced in a dairy process is model validated, the validated sample is 14 parts total, including 1 batch of secondary milk, 13 batches total, differing in gradient temperature from 60 ℃ to 120 ℃ by 5 ℃, wherein the normal pasteurized milk is 10 parts.
Referring to table 5, it is the beta-lactoglobulin and furin content in pasteurized milk and the difference in content from the corresponding substances in raw fresh milk. The results in Table 5 show that as the heating temperature increases, the bran amino acid content gradually increases and the beta-lactoglobulin content gradually decreases; and the higher the processing temperature, the larger the difference between the content of furfuryl amino acid and beta-lactoglobulin in the pasteurized milk and the corresponding value in raw milk is. And according to the prediction model established in example 3, the predicted sterilization temperatures are shown in table 5 according to the difference between the corresponding contents of beta-lactoglobulin and furfuryl amino acid in different pasteurized milks and raw milks.
TABLE 5 Fumonic acid and beta-lactoglobulin content in pasteurized milk
The results in Table 5 show that the prediction model established by the invention can accurately predict the sterilization temperature of pasteurized milk within the range of 75-120 ℃ with the accuracy of +/-1 ℃.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.

Claims (7)

1. The method for identifying the pasteurized milk sterilization temperature is characterized in that the sterilization temperature is the sterilization temperature adopted in the pasteurized milk sterilization process at a high temperature for a short time, the high temperature is 75-120 ℃, and the short time is 15s; the pasteurized milk is not added with nutrition enhancer, or is only added with minerals and/or vitamins;
the method comprises the following steps:
(1) Determining the content of beta-lactoglobulin and furoic acid in fresh milk;
(2) Detecting the content of beta-lactoglobulin and furfuryl amino acid in pasteurized milk and calculating the content difference of the beta-lactoglobulin and furfuryl amino acid from the corresponding substances in raw and fresh milk;
(3) According to the predictive modelCalculating the sterilization temperature Y of the pasteurized milk;
in the method, in the process of the invention,X 1 : the content of beta-lactoglobulin in pasteurized milk is different from that of beta-lactoglobulin in fresh milk in unit mg/L;X 2 : the content of furfuryl amino acid in pasteurized milk is different from the content of furfuryl amino acid in raw milk, and the unit is mg/100g protein;
the method for measuring the content of the beta-lactoglobulin comprises the following steps:
1) Sampling: taking at least 200 mL liquid milk as an experimental sample, and preserving the sample at 0-4 ℃;
2) Preparing the upper machine liquid: sucking a 2 mL sample, placing the sample in a 50 mL centrifuge tube, adding 15 mL ultrapure water, and uniformly mixing; adding 35-45 mu L of glacial acetic acid into the centrifuge tube, shaking uniformly, and regulating the pH to 4.6; placing the centrifuge tube in a high-speed centrifuge, and centrifuging at 10000 rpm and 4deg.C for 10 min; taking out the centrifuge tube, discarding fat at the upper part and sediment at the bottom, sucking the middle clear liquid by using a syringe, and passing through a microporous filter membrane of 0.22 mu m for detection by a machine;
3) Determination of beta-lactoglobulin content: measuring by HPLC method, and quantifying by external standard method;
the method for measuring the content of the furfuryl amino acid comprises the following steps:
1) Sampling: taking at least 200 mL liquid milk as an experimental sample, and preserving the sample at 0-4 ℃;
2) Preparation of hydrolysate: sucking a 2 mL sample, placing the sample in a sealed heat-resistant test tube, adding a 6 mL hydrochloric acid solution with the concentration of 10.6 mol/L, uniformly mixing, slowly introducing high-purity nitrogen into the test tube for 1-2 min, sealing the test tube, placing the test tube in a drying oven, heating the test tube at 110 ℃ for 1 h, gently shaking the test tube, and continuing heating until 23 h-24 h is heated; after heating, taking out the test tube from the drying oven, cooling and filtering to obtain hydrolysate;
3) Determination of protein content in hydrolysate: absorbing 2 mL the hydrolysate prepared in the step 2), and measuring the protein content in the sample solution according to GB/T5009.5;
4) Purifying the hydrolysate: the C18 column was mounted on a syringe, wetted with 5 mL methanol and 10 mL water, respectively; sucking the hydrolysis liquid in the step 2) of 0.5 mL into an extraction column, and slowly pushing the hydrolysis liquid into a C18 extraction column by using an injector; absorbing 3 mol/L hydrochloric acid solution to slowly elute the sample in the extraction column to 3 mL;
5) Determination of the furoic acid content: the quantitative analysis was performed by HPLC or UPLC, and external standard method.
2. The method of identifying pasteurized milk sterilization temperature according to claim 1, characterized in that: chromatographic conditions for beta-lactoglobulin content determination:
chromatographic column: symmetry C4 column, 4.6 mm ×250 mm,300 a, 5 μm particle size;
column temperature: 30 ℃;
a detector: PDA detector, detection wavelength: 280nm;
sample injection volume: 50. mu L;
mobile phase: acetonitrile is mobile phase A;0.1 The% trifluoroacetic acid solution is mobile phase B;
elution gradients are as follows:
time (min) Flow rate mL/min Acetonitrile (%) 0.1% trifluoroacetic acid solution (%) 0 1.0 30 70 5 1.0 55 45 10 1.0 60 40 12 1.0 30 70 16 1.0 30 70
。
3. The method of identifying pasteurized milk sterilization temperature according to claim 2, characterized in that: the precision control requirement of the method for measuring the content of the beta-lactoglobulin is as follows: the absolute difference of the two independent test results obtained under the repeatability condition is not more than 10% of the arithmetic mean value, and the absolute difference of the two independent test results obtained under the repeatability condition is not more than 20% of the arithmetic mean value.
4. The method of identifying pasteurized milk sterilization temperature according to claim 1, characterized in that: the calculation of the content of the furoic acid: the content of the furoic acid is calculated by mass fractionWThe values are expressed in mg/100g protein and are calculated according to the formula (1):
……………………(1)
wherein:
W: the content of furfuryl amino acid in the sample is mg/100g protein;
A t : testing the value of the furfuryl amino acid peak area in the sample;
A std : in the standard solution of the furoic acid,a value of the furfuryl amino acid peak area;
C std : concentration of the furoic acid standard solution is in mg/L;
D: the dilution factor is used for the measurement,D=6；
M: protein concentration in grams per liter (g/L) in the sample hydrolysate.
5. The method of identifying pasteurized milk sterilization temperatures according to claim 4, wherein: the chromatographic conditions for measuring the content of the furoic acid by adopting the UPLC method are as follows:
chromatographic column: WATERS ACQUITY UPLC ® HSS T3 column 1.8 μm,100 mm ×2.1 mm;
column temperature: 30. the temperature is lower than the temperature;
mobile phase: 6 g/L KCl solution is a mobile phase A, methanol is a mobile phase B, and pure water is a mobile phase C;
elution conditions: mobile phase A,0.4 mL/min;
a detector: ultraviolet detector, detection wavelength: 280nm;
the measuring method comprises the following steps:
sequentially balancing the chromatographic system at a flow rate of 0.4 mL/min by using a mobile phase B, C, A respectively, and injecting 2-5 mu L of 3 mol/L hydrochloric acid solution to balance the column so as to detect the purity of the solvent; 2. Mu.L of the solution to be measured was injected to determine the furoic acid content.
6. The method of identifying pasteurized milk sterilization temperature according to claim 1, characterized in that: the precision control requirement of the method for measuring the content of the furfuryl amino acid is as follows: the absolute difference of the two independent test results obtained under the repeatability condition is not more than 10% of the arithmetic mean value, and the absolute difference of the two independent test results obtained under the repeatability condition is not more than 20% of the arithmetic mean value.
7. Use of the method for identifying the pasteurization temperature of pasteurized milk according to any one of claims 1 to 6 for pasteurized milk production and testing.
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