A rapid identification method of whey protein based on differential scanning fluorescence and its application

Abstract

The present invention belongs to the field of food detection, and discloses a method and application of rapid identification of whey protein based on differential scanning fluorescence. The method uses differential scanning fluorescence detection technology (DSF) to obtain a thermal stability difference curve with the endogenous fluorescence signal of whey protein itself, and uses the feature extraction and statistical analysis method of the thermal stability difference curve of each whey protein to establish an identification method of five kinds of bovine whey protein. The present invention only requires a simple acetic acid extraction pretreatment, and is suitable for the determination of the denaturation rate of whey protein in milk and dairy products, the distinction of different thermal processing processes, and the rapid identification and detection of different milk sources. The method overcomes the shortcomings of existing whey protein identification and detection methods, such as electrophoresis, chromatography, and chromatography-mass spectrometry, which are limited by cost, operation, and instrumentation, and can be used for rapid, simple, high-throughput, and low-cost dairy product quality control in production practice.

Description

Technical Field

[0001] The invention belongs to the field of food detection, relates to a whey protein identification method, and particularly to a whey protein rapid identification method based on differential scanning fluorescence and application. Background Art

[0002] The type and content of whey protein is one of the important evaluation indicators of the nutritional and commercial value of milk and dairy products. Therefore, its detection method is crucial for the authenticity identification of milk sources, the optimization of dairy processing technology, and the evaluation of the nutritional quality of dairy products. Whey protein contains a variety of active proteins such as α-Lac, β-Lg, IgG, LF, etc., and has high functional characteristics. However, in the commercialization process, heat treatment is a necessary condition to ensure the stability of quality and compliance with hygiene standards during the circulation of dairy products. It can effectively control microbial indicators and stabilize the shelf life, but at the same time it causes changes in protein structure and active functions, and the extent of these changes depends on the temperature and time of heat treatment. Different heat-processed dairy products have different nutritional values ​​and market prices. Therefore, the detection of milk protein content can be used to distinguish dairy products treated with different heat-processing processes, protect the legitimate rights and interests of consumers, and control the quality of dairy production processes. The denaturation rate of whey protein will be different with different degrees of heat processing. The denaturation rate of whey protein has a very important influence on the sensory, texture, and nutrition of dairy products. It is also an important factor affecting the successful processing of yogurt and cheese, and an important evaluation indicator of their quality. Therefore, the method for determining the denaturation rate of whey protein is extremely important and necessary, and is of great significance to improving the quality of dairy products in my country. In addition, goat milk, camel milk, yak milk and other specialty milks have low production and high prices. Driven by economic interests, the phenomenon of adulteration of cow milk is more obvious. Whey protein detection can develop different milk source identification and common milk source authenticity detection methods from the perspective of protein, which plays an important role in the development of specialty dairy industry and protecting consumer safety and rights. Existing whey protein identification and detection methods, such as electrophoresis, chromatography, and chromatography-mass spectrometry, are limited by cost, operation and instruments and can only be carried out in the laboratory. There is still a lack of rapid and simple whey protein detection technology that can be used in production practice.

[0003] Differential scanning fluorimetry (DSF) is a technique that uses program-controlled temperature to detect changes in sample or dye fluorescence signals during a slow temperature rise to detect the thermal denaturation of proteins in a sample. Compared to differential scanning fluorimetry (DSC), the gold standard for thermal stability analysis, DSF has the following advantages:

[104] : (a) Larger applicable concentration range: DSC requires a high concentration of protein to obtain a clear denaturation peak, generally 0.2 to 2 mg / mL, while DSF allows the analysis of samples in the range of 0.005 to 200 mg / mL; (b) Less sample consumption: DSC usually requires a sample volume of 400 μL, while DSF only requires 10 μL; (c) Higher throughput: DSC can only analyze one sample at a time, with a low throughput, while DSF can scan and detect up to 96 samples at a time. Summary of the invention

[0004] The present invention aims at providing a rapid, simple and low-cost whey protein identification detection method and kit which can be used in production practice, aiming at existing methods for detecting whey protein identification, such as electrophoresis, chromatography and chromatography-mass spectrometry, which are limited by cost, operation and instruments and can only be performed in laboratories.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for rapid identification of whey protein based on differential scanning fluorescence comprises the following steps:

[0007] (1) Sample preparation: The target proteins: β-lactoglobulin, α-lactalbumin, bovine serum albumin, bovine immunoglobulin, and bovine lactoferrin were dissolved in 1×PBS to 10 mg / mL as standard protein stock solution, and diluted to different concentrations as needed for later use. Whey protein in dairy samples was extracted using acetate buffer;

[0008] (2) Melting analysis: The sample prepared in step (1) is analyzed by melting to obtain a DSF curve at 350 nm, a DSF curve of a 350 nm / 330 nm ratio channel, and melting peak curves of the two melting curves;

[0009] (3) Data processing: PCA analysis was performed on the melting curve information to extract the characteristic values ​​of each whey protein melting curve or melting peak curve, and a characteristic value melting information library was formed for LDA analysis to distinguish the proteins based on their melting information differences.

[0010] Preferably, the whey protein concentration in step (1) is >0.2 mg / mL, and the pH range is 4.0-7.4.

[0011] Preferably, the dairy product in step (1) is selected from raw milk, milk powder or liquid dairy products.

[0012] Preferably, the dairy product sample in step (1) comprises cow's milk, goat's milk, camel's milk, yak's milk, buffalo's milk, donkey's milk, and horse's milk.

[0013] Preferably, the dairy product sample in step (1) is treated with an acetate buffer at pH 4.0 to 5.0 to extract whey protein.

[0014] Preferably, the DSF detection parameters in step (2) are: scanning temperature range: 35-95°C, heating rate: 6°C / min-30°C / min.

[0015] Based on the application of any of the aforementioned methods for rapid identification of whey proteins based on differential scanning fluorescence, it is applied to the rapid identification of whey proteins from different sources. The specific method is: select a concentration >0.1 mg / mL and a pH of 4.0-7.4 that can obtain stable melting curves of three different sources of IgG, different sources of SA, and different sources of LF, and perform a duplicate DSF melting experiment. Repeat each protein five times to obtain characteristic melting curves and melting peak curves of IgG, SA and LF from different sources, input the entire melting curve information into MetaboAnalyst5.0 for PCA principal component analysis, and identify the protein origins of IgG, SA and LF.

[0016] The application of any of the aforementioned methods for rapid identification of whey protein based on differential scanning fluorescence method: applied to the rapid detection of whey protein denaturation rate, the specific method is: first, the melting peak area of ​​unheat-treated whey protein is used as a control, and the residual active whey protein content under different heat treatment conditions is calculated according to the melting peak area; then, the melting peak area of ​​whey protein extracted from raw milk is used as a control, and the residual active whey protein content in samples with different thermal processing processes is calculated according to the melting peak area.

[0017] Application of any of the aforementioned methods for rapid identification of whey protein based on differential scanning fluorescence: applied to rapid identification and detection of milk with different thermal processing processes, the specific method is: extract whey protein in milk with different thermal processing processes with acetate buffer, take 10 μL of whey protein samples from milk with different thermal processing processes extracted with acetate buffer for melting, repeat each sample 5 times, perform melting analysis, after obtaining the melting curves of milk with different thermal processing processes, input the entire melting curve information into MetaboAnalyst5.0 for PCA principal component analysis for identification.

[0018] Application of any of the aforementioned methods for rapid identification of whey protein based on differential scanning fluorescence: applied to rapid adulteration identification detection of milk from different sources, the specific method is: weigh 0.04g of liquid fresh cow's milk, fresh goat's milk, and fresh camel's milk, respectively, add acetic acid buffer solution of specific pH to make the volume to 1mL, then oscillate and mix, centrifuge at 12000rpm, 4℃, 10min, remove the upper fat and lower precipitate, transfer the middle layer to a clean centrifuge tube, store at -20℃, take 10μL of the sample obtained by the above treatment for melting detection, and obtain the characteristic melting curve of cow, goat, and camel milk whey protein; take 10μL of the prepared mixed solution for melting detection to determine its adulteration detection limit; take 10μL of the treated sample for melting detection to achieve quantitative analysis of cow's milk adulterated in goat milk and cow's milk adulterated in camel milk.

[0019] The beneficial effects of the present invention are embodied in:

[0020] The present invention can obtain the melting curve of protein through DSF, and obtain stable melting curves and melting peak curves of different types of whey proteins and whey proteins from different sources, respectively, and then use the difference of melting curves, combined with difference curve feature extraction and statistical analysis methods, to establish an identification and detection method for five types of bovine whey proteins. The present invention only requires simple acetic acid extraction pretreatment, is suitable for the determination of whey protein denaturation rate in milk and dairy products, the distinction of different thermal processing processes, and the rapid identification and detection of different milk sources, and has strong practical application potential. It can provide guidance for the precise control of the processing technology of dairy enterprises, provide a new solution for the authenticity identification of milk sources, evaluate the nutritional value of milk and dairy products, and promote the healthy development of the dairy industry. The method is simple and fast to operate, has high throughput, does not require professional technicians to operate, and has low detection costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : DSF identification and detection of whey proteins at pH 5.0. Original melting curves, melting peak curves, normalized melting curves, normalized melting peak curves of 5 whey proteins in F350 / 330 channel A) and corresponding PCA analysis graph B); corresponding characteristic value histogram C) and corresponding LDA analysis graph D); original melting curves, melting peak curves, normalized melting curves, normalized melting peak curves of 5 whey proteins in F350 channel E) and corresponding PCA analysis graph F); corresponding characteristic value histogram G) and corresponding LDA analysis graph H).

[0022] In the figure: β-Lg represents β-lactoglobulin; α-Lac represents α-lactalbumin; BIgG represents bovine immunoglobulin; BSA represents bovine serum albumin; BLF represents bovine lactoferrin; in Figure C, V1, V2, V3, V4, and V5 represent five characteristic values ​​extracted from the melting curve, respectively.

[0023] Figure 2 : DSF identification detection of whey proteins from different sources. Normalized melting curves of IgG from different sources in F350 / 330 channel A) and corresponding PCA analysis graph B); Normalized melting curves of LF from different sources C) and corresponding PCA analysis graph D); Normalized melting curves of SA from different sources E) and corresponding PCA analysis graph F);

[0024] In the figure: HIgG represents human immunoglobulin; BIgG represents bovine immunoglobulin; GIgG represents goat immunoglobulin; HLF represents human lactoferrin; BLF represents bovine lactoferrin; GLF represents goat lactoferrin; BSA represents bovine serum albumin; HSA represents human serum albumin.

[0025] Figure 3 : Detection of whey protein denaturation rate based on DSF. Melting peak curve of simulated whey protein after heat treatment under different conditions A) and corresponding melting peak area and proportion of remaining active whey protein content B); Normalized melting curve of bovine milk whey protein with different thermal processing processes C) and corresponding melting peak area and proportion of remaining active whey protein content D).

[0026] In the figure: Control represents a whey protein sample that has not been subjected to any heat treatment as a control; Pasteurized milk represents pasteurized milk; UHT milk represents ultra-high temperature sterilized milk; INF milk represents steam immersion sterilized milk.

[0027] Figure 4 :Identification and detection of milk with different thermal processing processes based on DSF. Normalized melting curves of whey proteins in milk with different thermal processing processes (A) and corresponding PCA analysis graph (B); Normalized melting peak curves (C) and corresponding PCA analysis graph (D).

[0028] In the figure: pasteurized milk means pasteurized milk; UHT milk means ultra-high temperature sterilized milk; INF milk means steam immersion sterilized milk.

[0029] Figure 5 :Identification and detection of milk from different sources based on DSF. Melting curve of whey protein from bovine and goat milk (A) and melting peak curve (B); Melting curve of samples of whey protein from bovine and goat milk with different mixing ratios (C) and its corresponding melting peak curve (D); PCA analysis result diagram (E); Relationship between factor1 and the adulteration ratio of bovine milk whey protein in the PCA result diagram (F); Melting curve of whey protein from bovine and camel milk (G) and melting peak curve (H).

[0030] In the figure, Cow milk-whey represents cow whey protein; Goat milk-whey represents sheep whey protein; Camelmilk-whey represents camel whey protein; the linear equation is y=0.000772833x-0.02273, R 2 =0.99877. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] This method utilizes DSF technology, based on the difference in melting information caused by the different thermal stabilities of the proteins themselves, and uses the fluorescence signal of the protein itself as the signal output mechanism, overcoming the problems of high sample consumption, low throughput, and time consumption of differential scanning calorimetry (DSC) technology. First, a rapid, simple, high-throughput, and low-cost whey protein identification and detection method is established, which is applied to the detection of whey protein denaturation rate in milk and dairy products, the distinction between different thermal processing processes, and the rapid identification and detection of different milk sources.

[0033] Example 1: Differential scanning fluorescence method for identification and detection of bovine whey protein

[0034] 1.1 Preparation of solution

[0035] 1.1.1 PBS buffer (20×): Dissolve the contents of one bag in 50 mL of distilled or deionized water to generate a 20× buffer solution with a pH of 7.4 at 25°C and store at -20°C. Dilute to 1× as needed when using.

[0036] 1.1.2 Preparation of acetate buffer solutions of different pH values: Weigh a certain mass of sodium acetate powder using an analytical balance, dissolve it to 50 mM using ultrapure water, and then adjust the pH of the acetic acid solution (50 mM) using the prepared sodium acetate solution.

[0037] 1.1.3 Dissolution of protein: Weigh 1 mg of each standard protein powder (β-lactoglobulin, α-lactalbumin, bovine immunoglobulin, serum albumin, bovine lactoferrin), add 1× PBS buffer to make up to 0.1 mL, and mix thoroughly. At this time, the concentration of the protein standard stock solution is 10 mg / mL. Dilute with 1× PBS buffer when using.

[0038] 1.2 Sample preparation

[0039] Protein solution dilution: dilute β-lactoglobulin, α-lactalbumin, bovine immunoglobulin, serum albumin, and bovine lactoferrin with 1× PBS buffer to 0.2 mg / mL respectively for later use.

[0040] 1.3 Melting analysis

[0041] 1.3.1 Melting test: Take a clean quartz capillary, absorb 10 μL of protein sample by siphoning, and place the capillary loaded with sample on the detection table of the protein thermal stability analyzer for analysis. Melting parameters: temperature rise range 35-95℃, temperature rise rate 30℃ / min.

[0042] 1.3.2 Data acquisition: Fluorescence measurements were performed at an excitation wavelength of 280 nm. The emission fluorescence signal intensities at 330 nm (F330) and 350 nm (F350) were collected, the F350 / F330-Nano DSF signals were calculated, and the original melting curve at 350 nm, the original melting curve of the 350 nm / 330 nm ratio channel, and the melting peak curves of the two melting curves were obtained.

[0043] 1.3.3 Identification test: A multiple-well melting experiment was performed within the concentration and pH range where the whey proteins could stably show characteristic melting signals (>0.2 mg / mL, pH 4.0-7.4). Each protein was repeated 5 times to obtain the melting curves and melting peaks of the five whey proteins.

[0044] 1.3.4 Blind sample detection: DSF detection was performed on 30 unknown samples using the above reaction system and melting conditions (0.2 mg / mL, pH 5.0). These samples were identified using the established standard library of melting curves, and the unknown samples were predicted based on the classification results of multivariate statistics.

[0045] 1.4 Data Analysis

[0046] The entire melting curve information was input for PCA (principal component analysis, MetaboAnalyst5.0), and the fluorescence signal corresponding to the Tm value of each bovine whey protein melting curve was taken as the eigenvalue to form an eigenvalue melting information library for LDA (linear discriminant analysis, SYSTAT). The signals of the F350 / F330 channel and the F350 channel were processed separately for identification detection.

[0047] Results Figure 1 , Table 1, Table 2.

[0048] The fluorescent signal of the protein comes from the inherent aromatic amino acids such as Trp and Tyr. When the protein molecule is thermally unfolded, the position of the aromatic amino acids changes, causing the change of the fluorescent signal. Due to the different structures of each whey protein, it has different thermal stability, which makes each whey protein have a unique melting curve. Figure 1As shown in A and E, in the 350nm / 330nm ratio channel and the 350nm channel, the five whey proteins β-Lg, α-Lac, BIgG, BSA, and BLF can all stably present characteristic melting curves, and the corresponding melting peak positions are more different, with different Tm values. Normalizing the melting curves can amplify the differences in melting information between the five whey proteins, and the differences between the proteins can be observed more conveniently and intuitively. The five whey proteins were identified by combining the statistical methods PCA and LDA. The identification results are shown in Figure 1 B, D, F, and H all clearly show 5 clusters, corresponding to the five whey proteins tested, and all five proteins show good separation. The PCA results of the protein melting curve, melting peak curve, and corresponding normalized curve, as well as the LDA results of selecting the signal value corresponding to the Tm value of each protein in the normalized melting curve as the characteristic information, can effectively distinguish the five whey proteins, and the accuracy of the LDA results is 100%. The results of the blind sample test are summarized in Table 2. The results show that all 30 samples can be successfully identified, and the detection accuracy rate reaches 100%, indicating that the DSF-based bovine whey protein identification detection strategy is feasible.

[0049] Table 1: Whey protein identification results in different modes of pH 5.0 system

[0050]

[0051] Table 2: Identification of 30 unknown samples using the normalized melting curve LDA method

[0052]

[0053] Example 2: Differential scanning fluorescence method for identification and detection of whey proteins from different sources

[0054] 2.1 Solution preparation

[0055] 2.1.1 Dissolution of protein: Weigh 1 mg of protein powder from different sources (bovine immunoglobulin, sheep immunoglobulin, human immunoglobulin, bovine serum albumin, human serum albumin, bovine lactoferrin, human lactoferrin, sheep lactoferrin), add 1× PBS buffer to make up to 0.1 mL, mix thoroughly, and the concentration of the protein standard stock solution is 10 mg / mL. Dilute with 1× PBS buffer when using.

[0056] The other solutions are prepared in the same manner as in 1.1.

[0057] 2.2 Sample preparation

[0058] Protein solution dilution: dilute bovine immunoglobulin, human immunoglobulin, human immunoglobulin, bovine serum albumin, human serum albumin, bovine lactoferrin, human lactoferrin, and goat lactoferrin with 1× PBS buffer to 0.2 mg / mL respectively for use.

[0059] 2.3 Melting analysis

[0060] The concentration and pH (>0.1 mg / mL, pH 4.0-7.4) that can obtain stable melting curves of three different sources of IgG, different sources of SA, and different sources of LF were selected for a duplicate DSF melting experiment. Each protein was repeated five times to obtain the characteristic melting curves and melting peak curves of different sources of IgG, SA and LF.

[0061] Melting detection steps and data collection are the same as 1.3

[0062] 2.4 Data Analysis

[0063] The entire melting curve information was input for PCA (Principal Component Analysis, MetaboAnalyst 5.0) to identify the protein origins of IgG, SA and LF.

[0064] Results Figure 2 .

[0065] The normalized melting curves of three different IgG sources (BIgG, GIgG, HIgG) are similar, with similar melting characteristics and similar melting peak positions. However, the melting peak shapes and Tm values ​​of proteins from different species are different (78.3℃, 79.2℃, 75.3℃). The melting peak of GIgG is slightly narrower than that of BIgG. There are still differences in the melting information between them. PCA is used to analyze and process the melting information of IgG from different sources. Figure 2 B, it can be accurately clustered into three categories, corresponding to three different species. The amino acid sequence homology between BLF and GLF and HLF is 92.23% and 69.52%, respectively. The normalized melting peak curve obtained by multiple well melting is shown in Figure 2 As shown in Figure C, the melting curves of BLF and GLF are closer in shape, and both are more different from the melting curve of HLF. For example, the melting curve and melting peak of HLF are more biased to the right, the Tm value is larger, and there is no second melting peak. After PCA analysis of these melting information, the three different sources of LF can be well distinguished ( Figure 2 D), and the clustering results show that BLF is closer to the center of GLF and farther from the center of HLF, which is consistent with the amino acid sequence homology between these three proteins. Similarly, BSA and HSA were tested by multiple well melting. The results are as follows Figure 3-5As shown. The melting peaks of BSA and HSA are both inverted peaks, and the melting curves also have similar directions and trends, indicating that the same type of protein from different species has similar melting characteristics, and the difference in melting information between SA from different species is reflected in the melting peak position and Tm value (60.4℃, 62.3℃). We further performed PCA analysis on these melting information data and obtained the following results Figure 2 F, which intuitively shows that BSA and HSA can also be clearly distinguished. The above results show that the melting information obtained by DSF can be used to distinguish and identify the species of whey protein.

[0066] Example 3: Differential scanning fluorescence method for detecting whey protein denaturation rate

[0067] 3.1 Solution preparation

[0068] Same as 1.1.

[0069] 3.2 Sample preparation

[0070] 3.2.1 Simulated whey protein: The standard proteins β-Lg, α-Lac, BIgG, BSA, and BLF prepared in step 1.1 were mixed according to their proportions in raw milk to prepare simulated whey protein samples. The samples were heated according to the commonly used thermal processing conditions as the research object. 20 μL of simulated whey protein was placed in a centrifuge tube and placed in a thermal cycler. The heating conditions were set to 65°C, 30 min, 85°C, 30 min, 75°C, 15 s, and 85°C, 5 s for heat treatment.

[0071] 3.2.2 Whey protein extraction from actual samples of different thermal processes: Weigh 0.4 g of raw milk, pasteurized milk, UHT milk, and INF milk samples, dilute to 1 mL with acetate buffer solution at pH 4.6, and oscillate to mix. Centrifuge at 12000 rpm, 4°C, 10 min to remove the upper fat layer and the lower precipitate (including casein), and transfer the whey protein in the middle layer to a clean centrifuge tube and store at -20°C.

[0072] 3.3 Melting analysis

[0073] 3.3.1 Simulated whey protein melting: 10 μL of the simulated whey protein sample treated in 3.2.1 was respectively aspirated into a capillary tube for melting detection. The simulated whey protein without any heat treatment was used as a control. Each sample was repeated 5 times for melting analysis.

[0074] 3.3.2 Melting of whey protein from actual samples with different thermal processes: Take 10 μL of the whey protein samples with different thermal processes treated in 3.2.2 for melting analysis.

[0075] Melting operation and data collection are the same as 1.3.

[0076] 3.4 Data Analysis

[0077] 3.4.1 Simulated whey protein: The melting peak area of ​​unheat-treated whey protein was used as a control, and the residual active whey protein content under different heat treatment conditions was calculated based on the melting peak area.

[0078] 3.4.2 Whey protein in actual samples of different thermal processing: The melting peak area of ​​whey protein extracted from raw bovine milk was used as a control, and the residual active whey protein content of milk processed by other thermal processes was calculated based on the melting peak area.

[0079] Results Figure 3 .

[0080] When milk is heat treated, whey protein denatures as the protein unfolds and the hydrophobic groups are exposed. Different heat treatment methods have different denaturation degrees for proteins. The melting curves of simulated whey protein samples after heat treatment under different conditions are shown in Figure 2. Figure 3 As shown in Figure A, compared with the melting curve of whey protein without heat treatment, the melting curve of whey protein after heat treatment has changed significantly, which is the result of the effect of heat treatment on the interaction between proteins and the thermal denaturation of some proteins. Figure 3 As shown in Figure C, after two short-term high-temperature sterilizations (75℃ / 15s; 85℃ / 5s), 81-87% of active whey protein remained, while after 65℃ / 30min treatment, only 51% of active whey protein remained. After 85℃ / 30min treatment, the melting peak signal almost disappeared, and the remaining active whey protein was less than 10%. For milk samples with different thermal processing technologies, the melting results and the remaining active whey protein content are shown in Figure C. Figure 3 B, D, the residual active whey protein of pasteurized milk and INF milk is very close, both are 78%, while UHT milk only has about 55% active whey protein. The above results show that DSF can be used to detect the denaturation rate of whey protein.

[0081] Example 4: Differential scanning fluorescence method for identification and detection of milk with different thermal processing processes

[0082] 4.1 Solution preparation

[0083] Same as 1.1.

[0084] 4.2 Sample preparation

[0085] Whey protein from milks processed by different heat treatment processes was extracted using acetate buffer, following the same process as in 3.2.

[0086] 4.3 Melting analysis

[0087] Take 10 μL of the different heat-processed whey protein samples extracted in 4.2 for melting, and repeat each sample 5 times for melting analysis. The melting operation and data collection are the same as 1.3.

[0088] 4.4 Data Analysis

[0089] After obtaining the melting curves of different thermal processing emulsions, the entire melting curve information was input for PCA (principal component analysis, MetaboAnalyst5.0) for identification.

[0090] Results Figure 4 .

[0091] The melting curves and melting peak curves of three commercially available liquid milks with different thermal processing technologies and raw milk without any processing technology are as follows: Figure 4 A and C show that the melting signals of the three samples with different thermal processing techniques have changed significantly compared to raw milk. Each sample has its own unique melting curve, which is related to the loss of active whey protein content and the interaction between them. According to the differences in the melting signals of whey proteins extracted from milk with different thermal processing techniques, they can be distinguished from each other. Samples belonging to the same thermal processing technique will be grouped into the same cluster ( Figure 4 B, D). The clustering results of pasteurized milk and INF milk are relatively close. From the melting information, it can be observed that the melting curves and melting peak curves of the two milks are relatively similar, indicating that the two thermal processing technologies have similar effects on the production of whey protein. The above results show that the method established by the present invention can be used for the rapid identification and detection of milk with different thermal processing technologies.

[0092] Example 5: Differential scanning fluorescence method for detecting adulteration of milk from different sources

[0093] 5.1 Solution preparation

[0094] Same as 1.1.

[0095] 5.2 Sample preparation

[0096] 5.2.1 Extraction of whey protein from milk of different origins: Weigh 0.04 g each of liquid fresh cow milk, fresh goat milk and fresh camel milk, add acetic acid buffer solution of specific pH to make the volume to 1 mL, then shake and mix, centrifuge at 12000 rpm, 4°C, 10 min, remove the upper fat and lower precipitate, transfer the middle layer to a clean centrifuge tube, and store at -20°C.

[0097] 5.2.2 Samples of goat milk whey protein with different adulteration ratios: Take goat milk whey protein (5.0 mg / mL) extracted with acetic acid as the stock solution, and add the extracted bovine milk whey protein (5.0 mg / mL) into the goat milk whey protein in different ratios to prepare a mixed solution, namely: 100%, 80%, 50%, 30%, 10%, 5%, 1%, and 0.1%.

[0098] 5.2.3 Actual sample adulteration detection: Use acetate buffer to extract whey protein in the actual adulterated sample, centrifuge at 12000rpm, 4℃, 10min, remove the upper fat layer and the lower precipitate, transfer the middle layer to a clean centrifuge tube, and store at -20℃.

[0099] 5.3 Melting analysis

[0100] 5.3.1 Melting of whey protein in milk from different sources: Take 10 μL of the sample treated in 5.2.1 for melting detection to obtain the characteristic melting curves of whey protein from bovine, sheep and camel milk.

[0101] 5.3.2 Melting of sheep whey protein samples with different adulteration ratios: Take 10 μL of the mixed solution prepared in 5.2.2 for melting test to determine its adulteration detection limit.

[0102] 5.3.3 Actual sample melting: Take 10 μL of the sample processed in 5.2.3 for melting detection to achieve quantitative analysis of cow milk mixed into goat milk.

[0103] 5.4 Data Analysis

[0104] The information of melting curves of mixed whey proteins of cow and sheep milk with different adulteration ratios was input for PCA (Principal Component Analysis, MetaboAnalyst5.0) for identification detection.

[0105] Results Figure 5 , Table 3.

[0106] Table 3: Identification and test results of actual samples of adulterated goat milk

[0107]

[0108] Melting curve of whey protein in bovine and sheep liquid milk, such as Figure 5 A and B, bovine milk whey protein has two main melting peaks, which are provided by α-Lac and β-Lg, which are ranked first in bovine whey protein content. The melting phenomenon is obviously different from that of raw cow milk. Raw goat milk whey protein has only one melting peak, α-Lac, which rises around 90°C, but no complete melting peak appears. This difference is caused by β-Lg from cow and goat sources. This shows that based on DSF, bovine and goat milk can be distinguished by the difference in whey protein melting information. Figure 5C, D, 8 mixed samples with different proportions produced 8 different melting curves. As the content of bovine milk whey protein increased, the melting curve shape changed from the goat milk whey protein melting curve to the bovine milk whey protein melting curve. The melting information of mixed samples with different proportions was significantly different. The results were analyzed by PCA, and the minimum detection limit was 5%. The results are shown in Figure 5 As shown in Figure E, 7 distinct categories can be obtained. The three samples with the same whey protein composition ratio are divided into a category with the same main component. The mixed samples with different adulteration ratios have different distribution ranges. As the proportion of bovine milk whey protein mixed with goat milk whey protein increases, it is closer to the range where the main component is bovine milk whey protein. The proportion of bovine milk whey protein mixed with the position of its distribution shows a certain linear relationship. Therefore, we use the proportion of bovine milk whey protein as the horizontal axis and factor1 in the PCA diagram as the vertical axis to draw a graph. The results are shown in Figure 1. Figure 5 As shown in F, the results show that there is an obvious linear relationship between different mixing ratios and the distribution position of PCA results. The linear correlation coefficient is 0.99877, and the linear relationship is good. The test results of actual adulterated samples are shown in Table 3. The milk components can be detected in samples with an adulteration ratio greater than 5%, and the calculated adulteration ratio is close to the actual adulteration ratio. The above results show that obtaining the melting information of whey protein through DSF can realize the adulteration detection of bovine and sheep milk, and the adulteration ratio can be judged according to the distribution position of PCA results. The melting curve and melting peak curve of fresh camel milk are shown in Figure 5 As shown in G and H, camel milk whey protein has only one melting peak and lacks the melting peak of β-Lg, which is significantly different from bovine whey protein. This shows that this method can also be used to distinguish between bovine milk and camel milk, and can provide a very potential detection method for the detection of adulteration of specialty milk.

Claims

1. A method for rapid identification of whey protein based on differential scanning fluorimetry, characterized in that: The steps include: (1) Sample preparation: The target proteins: β-lactoglobulin, α-lactalbumin, bovine serum albumin, bovine immunoglobulin, and bovine lactoferrin were dissolved in 1×PBS to 10 mg / mL as standard protein stock solution, and diluted to different concentrations as needed for later use. Whey protein in dairy samples was extracted using acetate buffer; (2) Melting analysis: The sample prepared in step (1) is analyzed by melting to obtain a DSF curve at 350 nm, a DSF curve of a 350 nm / 330 nm ratio channel, and melting peak curves of two melting curves; (3) Data processing: PCA analysis was performed on the melting curve information to extract the characteristic values ​​of each whey protein melting curve or melting peak curve, and a characteristic value melting information library was formed for LDA analysis to distinguish the proteins based on their melting information differences.

2. A method for rapid identification of whey protein based on differential scanning fluorimetry according to claim 1, characterized in that: The whey protein concentration in step (1) is greater than 0.2 mg / mL, and the pH range is 4.0 to 7.

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3. A method for rapid identification of whey protein based on differential scanning fluorimetry according to claim 1, characterized in that: The dairy product in step (1) is selected from raw milk, milk powder or liquid dairy products.

4. The method for rapid identification of whey protein based on differential scanning fluorimetry according to claim 1, characterized in that: The dairy product sample in step (1) includes cow's milk, goat's milk, camel's milk, yak's milk, buffalo's milk, donkey's milk and horse's milk.

5. The method for rapid identification of whey protein based on differential scanning fluorimetry according to claim 1, characterized in that: The dairy product sample in step (1) needs to be treated with an acetate buffer at pH 4.0 to 5.0 to extract whey protein.

6. The method for rapid identification of whey protein based on differential scanning fluorescence according to claim 1, characterized in that: The DSF detection parameters in step (2) are: scanning temperature range: 35 to 95°C, heating rate: 6°C / min to 30°C / min.

7. The use of a method for rapid identification of whey protein based on differential scanning fluorescence according to any one of claims 1 to 5, characterized in that: It is used for rapid identification of whey proteins from different sources. The specific method is as follows: select a concentration of >0.1 mg / mL and a pH of 4.0-7.4 that can obtain stable melting curves of three different sources of IgG, SA, and LF, and perform a duplicate DSF melting experiment. Repeat each protein five times to obtain characteristic melting curves and melting peak curves of IgG, SA, and LF from different sources. Input the entire melting curve information into MetaboAnalyst5.0 for PCA principal component analysis to identify the protein origins of IgG, SA, and LF.

8. The use of a method for rapid identification of whey protein based on differential scanning fluorescence according to any one of claims 1 to 5, characterized in that: It is used for rapid detection of whey protein denaturation rate. The specific method is: first, the melting peak area of ​​unheat-treated whey protein is used as a control, and the residual active whey protein content under different heat treatment conditions is calculated according to the melting peak area; then, the melting peak area of ​​whey protein extracted from raw milk is used as a control, and the residual active whey protein content in samples with different heat processing processes is calculated according to the melting peak area.

9. The use of a method for rapid identification of whey protein based on differential scanning fluorescence according to any one of claims 1 to 5, characterized in that: It is used for rapid identification and detection of milk with different thermal processing processes. The specific method is: use acetate buffer to extract whey protein in milk with different thermal processing processes, take 10 μL of whey protein samples extracted with acetate buffer from milk with different thermal processing processes for melting, repeat each sample 5 times, perform melting analysis, after obtaining the melting curves of milk with different thermal processing processes, input the entire melting curve information into MetaboAnalyst5.0 for PCA principal component analysis for identification.

10. The use of a method for rapid identification of whey protein based on differential scanning fluorescence according to any one of claims 1 to 5, characterized in that: It is applied to the rapid adulteration identification and detection of milk from different sources. The specific method is as follows: weigh 0.04g of liquid fresh cow milk, fresh goat milk and fresh camel milk, respectively, add acetic acid buffer solution with a pH of 4.0-5.0 to make the volume to 1mL, and then oscillate and mix, centrifuge at 12000rpm, 4℃, 10min, remove the upper fat and lower precipitate, transfer the middle layer to a clean centrifuge tube, store at -20℃, take 10μL of the sample obtained by the above treatment for melting detection, and obtain the characteristic melting curve of cow, goat and camel milk whey protein; take 10μL of the prepared mixed solution for melting detection to determine its adulteration detection limit; Take 10 μL of the treated sample for melting detection to achieve quantitative analysis of cow's milk mixed with goat's milk and cow's milk mixed with camel's milk.

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