A colorimetric method for rapid determination of the binding degree of thermotropic whey protein and casein

Abstract

The present invention belongs to the technical field of analysis and detection, and relates to a method for quickly determining the degree of binding of thermotropic whey protein and casein by colorimetry. The method comprises the following steps: firstly determining the proportion of denatured whey protein in the total protein in the dairy product, then removing the whey protein not bound to the casein, then destroying the disulfide bond and washing the protein precipitate, and then redissolving the protein precipitate. The absorbance of the blank sample and the sample at 280 nm is measured to obtain A. 280,blank and A 280 Then measure the absorbance at 412nm and get A 412，DTNB and A 412，blank Value, OD value = (A 412，DTNB ‑A 412，blank ) / (A 280 ‑A 280 , blank ), draw a standard working curve; the OD value of the milk to be tested was measured after the above steps, and the ratio of denatured whey protein to total protein was checked by the standard working curve, according to the formula C 酪 (%) = C 总 / (100‑C 总 )×100, and calculate the binding degree of whey protein and casein. The present invention shortens the sample preparation process, reduces the measurement cost, improves the measurement efficiency and repeatability, uses a small amount of reagents, and is simple to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, relates to dairy product detection, and in particular to a method for quickly determining the binding degree of thermotropic whey protein and casein by colorimetry. Background Art

[0002] During the heat treatment of milk, whey proteins denature and aggregate. Some of the denatured whey proteins bind to casein micelles through disulfide bonds and hydrophobic interactions, with the amount of binding increasing with increasing heating intensity. Slight fluctuations in heating conditions can affect the degree of whey protein denaturation and aggregation. The distribution of denatured whey protein aggregates within the whey phase and on the casein surface can further affect milk processing characteristics. Unstable heating due to process or equipment issues can lead to unstable denaturation and uneven distribution of whey proteins, which in turn affects the quality and stability of the final product.

[0003] The amount of whey protein bound to casein micelles significantly affects the quality characteristics of various dairy products, such as yogurt and cheese. Therefore, determining the amount of whey protein bound to casein micelles plays a crucial role in controlling the quality and stability of dairy products. However, there are currently no standardized methods for this in China. Conventional protein analysis methods, such as capillary electrophoresis and liquid chromatography-mass spectrometry, are complex and expensive to maintain, making them unsuitable for large-scale determinations.

[0004] It is very necessary to develop a low-cost and easy-to-operate detection method. Summary of the Invention

[0005] In view of the shortcomings of the existing technical means for quantitative analysis of milk protein, such as complex operation and high cost, the present invention aims to disclose a method for rapidly determining the binding degree of thermotropic whey protein and casein by colorimetry.

[0006] Technical Solution

[0007] A method for quickly determining the degree of binding between thermotropic whey protein and casein by colorimetry comprises the following steps:

[0008] A. Preparation of Standard Samples

[0009] Heat the standard milk at 65°C, 75°C, 85°C, and 95°C for 5 min to obtain sample emulsions with different whey protein binding amounts, set aside, and determine the ratio of denatured whey protein to total protein in the sample in g / 100g.

[0010] B. Removal of whey protein not bound to casein

[0011] Take 100 μL of sample emulsion and mix it with sodium acetate buffer in a volume ratio of 1:10, then centrifuge to remove

[0012] Remove the supernatant whey to obtain protein;

[0013] C. Disruption of disulfide bonds and washing of protein precipitates

[0014] After the protein obtained by centrifugation was drained, 800 μL of dithiothreitol (DTT) reagent was added and heated at 45°C and 1600 rpm for 5 minutes to fully dissolve it. Then, 820 μL of trichloroacetic acid solution was added and mixed evenly by vortexing. After standing for 5 minutes, the protein precipitate was centrifuged to obtain a protein precipitate. The protein precipitate was washed with sodium acetate buffer solution, vortexed for 5 minutes, and the supernatant was removed by centrifugation and drained.

[0015] D. Redissolution of protein precipitate

[0016] Add 1 mL of Tris-urea-glycine buffer to the drained protein, heat at 45°C and 1600 rpm for 5 min to fully dissolve, and centrifuge;

[0017] E. Absorbance determination, calculation and drawing of standard working curve

[0018] Pipette 200 μL of sample supernatant into a cuvette, add 800 μL of Tris-urea-glycine buffer and mix evenly. The blank sample is 1 mL of Tris-urea-glycine buffer. Measure the absorbance of the blank sample and sample at 280 nm to obtain A. 280,blank and A 280 value;

[0019] Add 100 μL of DTNB reagent to the cuvettes containing blank and sample respectively, mix well, and measure the absorbance at 412 nm after 5 minutes to obtain A 412，DTNB and A 412，blank The calculation formula is OD value = (A 412，DTNB -A 412，blank ) / (A 280 -A 280,blank ),

[0020] The OD values ​​of the series of samples were plotted into a standard working curve, where the horizontal axis is the ratio of denatured whey protein to total protein C 乳清 :C 总 , unit is g / 100g, the ordinate is OD value;

[0021] F. Measure the OD value of the milk product to be tested according to steps BE, and find the ratio of denatured whey protein to total protein from the standard working curve. 酪 (%) = C 总 / (100-C 总 )×100, calculate the binding degree C of whey protein and casein酪 , unit: g / 100g.

[0022] In a preferred disclosed embodiment of the present invention, the pH value of the standard milk in step A is 6.7-6.8, and the standard milk is liquid skim milk or skim milk powder.

[0023] Furthermore, when the standard milk is skim milk powder, an emulsion with a mass percent concentration of 9% is prepared.

[0024] In a preferred embodiment of the present invention, the total protein content of the standard milk in step A is 3.2-3.5%.

[0025] In a preferred embodiment of the present invention, in step A, the sample emulsion is freeze-dried, and the ratio of denatured whey protein to total protein in the sample is quantitatively determined by capillary electrophoresis, with the unit being g / 100g.

[0026] In a preferred embodiment of the present invention, the centrifugation in step B is performed at a centrifugal force of 15000 g and repeated at least twice to fully remove the protein in the whey phase.

[0027] In a preferred disclosed embodiment of the present invention, the pH value of the sodium acetate buffer in step B is 4.5.

[0028] In a preferred disclosed embodiment of the present invention, the mass fraction of the trichloroacetic acid solution in step C is 24%.

[0029] In a preferred disclosed embodiment of the present invention, the pH value of the sodium acetate buffer in step C is 4.5.

[0030] In a preferred embodiment of the present invention, the centrifugation in step C is performed at a centrifugal force of 15000 g.

[0031] In a preferred embodiment of the present invention, the pH value of the Tris-urea-glycine buffer in step D is 8.3.

[0032] In a preferred embodiment of the present invention, the centrifugation in step D is performed at a centrifugal force of 15000 g.

[0033] In a preferred embodiment of the present invention, the standard deviation of the measurement accuracy of the sample to be tested in step F is less than 5%.

[0034] The degree of binding between whey protein and casein during heating is a key factor affecting the quality of many dairy products, and currently, methods for this purpose are lacking in China. Quantitative protein analysis using methods such as high-performance liquid chromatography, capillary electrophoresis, and liquid chromatography-mass spectrometry (LC-MS / MS) is not only expensive and time-consuming, but also requires complex sample preparation and a long cycle. The present invention utilizes the precipitation of casein at its isoelectric point, pH 4.6, to remove unbound whey protein. The presence of tryptophan groups in both whey protein and casein, when measured at 280 nm for total protein content in their isolates, quantitatively analyzes whey protein content using the principle that whey protein contains cysteine, which reacts with dinitrobenzoic acid to produce a yellow color.

[0035] Beneficial effects

[0036] The colorimetric method employed in the present invention not only shortens the sample preparation process and reduces measurement costs, but also improves measurement efficiency and repeatability. This invention fully considers the principle that cysteine, a characteristic of whey protein, reacts with dinitrobenzoic acid to produce a yellow color, effectively avoiding interference with the quantitative analysis of whey protein and casein. The method uses a small amount of reagents and is simple to operate, avoiding the large amounts of chemical elution reagents required by other chromatographic methods, saving costs while also avoiding environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 . Standard curve of OD values ​​of samples containing different amounts of whey protein complexation. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below with reference to the following examples so that those skilled in the art can better understand the present invention. However, the present invention is not limited to the following examples.

[0039] Example 1

[0040] A method for rapidly determining the degree of binding between thermotropic whey protein and casein by colorimetry, comprising the following steps:

[0041] (1) Use a pipette to draw 100 μL of heated skim milk sample into a 2 mL microcentrifuge tube, add 1 mL of 0.1 mol / L pH 4.5 sodium acetate buffer,

[0042] (2) Shake the mixture at 1600 rpm and 45°C for 5 min using a thermomixer, then centrifuge at 15,000 g for 5 min, remove the supernatant, and repeat twice;

[0043] (3) Add 800 μL of dithiothreitol (DTT) reagent to the centrifuge tube containing the protein precipitate, and use a thermomixer at 1600 rpm and 45°C for 5 min to dissolve the protein precipitate. Add 820 μL of 24% trichloroacetic acid solution, vortex, and let stand for 5 min. Centrifuge at 15,000 g for 5 min, and remove the supernatant.

[0044] (4) Add 1 mL of 0.1 mol / L pH 4.5 sodium acetate buffer to the centrifuge tube, vortex, and centrifuge at 15,000 g for 5 min. Remove the supernatant.

[0045] (5) Add 1 ml of Tris-urea-glycine buffer to the centrifuge tube, shake at 1600 rpm and 45°C for 5 min using a thermomixer until the protein is fully dissolved, and centrifuge at 15,000 g for 5 min to obtain a clear supernatant;

[0046] (6) 200 μL of supernatant was drawn into a cuvette, and 800 μL of tris-urea-glycine buffer was added and mixed evenly. The tris-urea-glycine buffer was used as a blank sample, and the absorbance values ​​were measured at wavelengths of 280 nm and 412 nm, respectively. 100 μL of cysteine ​​dye DTNB reagent was added to the cuvette, mixed evenly, and then allowed to stand for 5 min before measuring the absorbance. The ratio of the absorbance of whey protein to the absorbance of total protein was calculated using the following formula: OD value = (A 412，DTNB -A 412，blank ) / (A 280 -A 280,blank );

[0047] (7) Use the OD value of the standard sample to draw a standard curve;

[0048] (8) The whey protein content of the sample to be tested is determined according to steps (1) to (7). After the OD value is measured, the ratio of whey protein to total protein (C 总 : g / 100g), and the degree of binding between whey protein and casein (C 酪 :g / 100g), C 酪 (%) = C 总 / (100-C 总 )×100.

[0049] Example 2

[0050] A method for rapidly determining the degree of binding between thermotropic whey protein and casein by colorimetry, comprising the following steps:

[0051] (1) Dissolve skim milk powder in deionized water at a mass ratio of 9% and stir at 40°C for 1 h. Pipette 200 μL of the reconstituted skim milk sample into a 2 mL microcentrifuge tube and add 1 mL of 0.1 mol / L pH 4.5 sodium acetate buffer solution.

[0052] (2) Shake the mixture at 1600 rpm and 45°C for 5 min using a thermomixer, then centrifuge at 15,000 g for 5 min, remove the supernatant, and repeat twice;

[0053] (3) Add 800 μL of dithiothreitol (DTT) reagent to the centrifuge tube containing the protein precipitate, and use a thermomixer at 1600 rpm and 45°C for 5 min to dissolve the protein precipitate; add 820 μL of 24% trichloroacetic acid solution, vortex, let stand for 5 min, and then centrifuge at 15,000 g for 5 min, and remove the supernatant;

[0054] (4) Add 1 mL of 0.1 mol / L pH 4.5 sodium acetate buffer to the centrifuge tube, vortex, and centrifuge at 15,000 g for 5 min. Remove the supernatant.

[0055] (5) Add 1 ml of Tris-urea-glycine buffer to the centrifuge tube, shake at 1600 rpm and 45°C for 5 min using a thermomixer until the protein is fully dissolved, and centrifuge at 15,000 g for 5 min to obtain a clear supernatant;

[0056] (6) Pipette 200 μL of supernatant into a cuvette, add 800 μL of tris-urea-glycine buffer and mix evenly, use tris-urea-glycine buffer as a blank sample, measure the absorbance at 280 nm and 412 nm, add 100 μL of cysteine ​​dye DTNB reagent to the cuvette, mix evenly, let it stand for 5 minutes, and then measure the absorbance; the ratio of whey protein absorbance to total protein absorbance is calculated using the following formula:

[0057] OD value = (A 412 ,DTNB- A412，blank ) / (A 280 -A 280,blank );

[0058] (7) Use the OD value of the standard sample to draw a standard curve;

[0059] (8) The whey protein content of the sample to be tested is determined according to steps (1) to (7). After the OD value is measured, the ratio of whey protein to total protein (C 总: g / 100g), and the degree of binding between whey protein and casein (C 酪 : g / 100g).

[0060] C 酪 (%) = C 总 / (100-C 总 )×100.

[0061] The precise determination of the present invention is determined by measuring a standard sample containing 0.1 g / g of whey protein 9 times. The relative standard deviation (RSD) of the sample absorbance is 3.15%, which is within 5%, and has very good precision and repeatability.

[0062] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the description of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for rapidly determining the degree of binding between thermotropic whey protein and casein by colorimetry, characterized in that: The steps include: A. Heat standard milk at 65°C, 75°C, 85°C, and 95°C for 5 min to obtain sample emulsions with different whey protein binding amounts. Set aside and determine the ratio of denatured whey protein to total protein in the samples (g / 100g). B. Taking 100 μL of the sample emulsion and mixing it with sodium acetate buffer at a volume ratio of 1:10, centrifuging and removing the supernatant whey to obtain protein, wherein the pH value of the sodium acetate buffer is 4.5; C. After draining the protein obtained by centrifugation, add 800 μL of dithiothreitol (DTT) reagent and heat at 45°C and 1600 rpm for 5 minutes to fully dissolve it. Then, add 820 μL of trichloroacetic acid solution and vortex to mix evenly. After standing for 5 minutes, centrifuge to obtain a protein precipitate. Wash the protein precipitate with sodium acetate buffer solution, vortex to disperse it for 5 minutes, centrifuge to remove the supernatant, and drain. The mass fraction of the trichloroacetic acid solution is 24%, and the pH value of the sodium acetate buffer is 4.

5. D. Add 1 mL of Tris-urea-glycine buffer to the drained protein, heat at 45°C and 1600 rpm for 5 minutes to fully dissolve, and centrifuge. The pH of the Tris-urea-glycine buffer is 8.

3. E. Pipette 200 μL of sample supernatant into a cuvette, add 800 μL of Tris-urea-glycine buffer and mix well. The blank sample is 1 mL of Tris-urea-glycine buffer. Measure the absorbance of the blank sample and sample at 280 nm to obtain A. 280 , blank and A 280 value, Add 100 μL of DTNB reagent to the cuvettes containing blank and sample respectively, mix well, and measure the absorbance at 412 nm after 5 minutes to obtain A 412，DTNB and A 412，blank The calculation formula is OD value = (A 412，DTNB -A 412，blank ) / (A 280 -A 280 , blank ), The OD values ​​of the series of samples were plotted into a standard working curve, where the horizontal axis is the ratio of denatured whey protein to total protein C 乳清 :C 总 , unit is g / 100g, the ordinate is OD value; F. Measure the OD value of the milk product to be tested according to steps BE, and find the ratio of denatured whey protein to total protein from the standard working curve. 酪 (%) = C 总 / (100- C 总 ) × 100, calculate the binding degree C of whey protein and casein 酪 , unit: g / 100g.

2. The method for rapidly measuring the degree of binding of thermotropic whey protein and casein by colorimetry according to claim 1, wherein: The pH value of the standard milk in step A is 6.7-6.8, and it is liquid skim milk or skim milk powder.

3. The method for rapidly measuring the degree of binding of thermotropic whey protein and casein by colorimetry according to claim 2, wherein: When the standard milk in step A is skim milk powder, an emulsion with a mass percent concentration of 9% is prepared.

4. The method for rapidly measuring the degree of binding of thermotropic whey protein and casein by colorimetry according to claim 1, wherein: The total protein content of the standard milk in step A is 3.2-3.5%.

5. The method for rapidly measuring the degree of binding of thermotropic whey protein and casein by colorimetry according to claim 1, wherein: In step A, the sample emulsion is freeze-dried, and the ratio of denatured whey protein to total protein in the sample is quantitatively determined by capillary electrophoresis, with the unit being g / 100g.

6. The method for rapidly determining the degree of binding of thermotropic whey protein and casein by colorimetry according to claim 1, wherein: The centrifugation in step B is performed at a centrifugal force of 15000 g and repeated at least twice.

7. The method for rapidly determining the degree of binding of thermotropic whey protein and casein by colorimetry according to claim 1, wherein: The centrifugation in step C was performed at a centrifugal force of 15000 g.

8. The method for rapidly determining the degree of binding of thermotropic whey protein and casein by colorimetry according to claim 1, wherein: The centrifugation in step D was performed at a centrifugal force of 15000 g.

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