Determination of A2 β-casein and total β-casein in cow milk and milk powder by capillary zone electrophoresis

doi:10.3724/SP.J.1123.2019.11005

Abstract

Abstract:

β-casein (β-CN) is one of the major casein proteins in cow milk. There are 13 different variants documented for β-CN in cow milk, among which A1 and A2 are the major variants. The separation and quantitation of A2 β-CN are imperative for A2 dairy products. A new capillary zone electrophoresis (CZE) method with UV detection at 214 nm was established for the separation and quantification of the A2 variant and total β-CN content in cow milk and milk powders. The separation of β-CN variants was achieved on bare fused silica capillaries (50 μm×30/40 cm (effective/total length)). The separation buffer was a mixture of 4 mol/L urea, 0.2% (mass fraction) hydroxypropyl methylcellulose, 140 mmol/L citric acid, and 50 mmol/L disodium hydrogen phosphate buffer (pH 2.7). The corrected peak areas and the concentrations of total β-CN and the A2 variant showed good linearity, with correlation coefficients (r²) ranging from 0.9968 to 0.9997. The intra-day precisions for A2 β-CN and total β-CN determination in four samples (two pasteurized milk samples and two milk powder samples) were in the ranges of 2.4%-4.7% and 2.6%-4.8%, respectively. The inter-day precisions for A2 β-CN and total β-CN determination in four samples were in the ranges of 4.0%-6.3% and 3.9%-6.7%, respectively. The recoveries of A2 and total β-CN ranged from 85.5% to 106.4%. With the established CZE method, the A2 β-CN variant and total β-CN protein in liquid and powder bovine milk products could be separated and accurately quantified. By calculating the A2 β-CN content in the total β-CN, the quality of A2 dairy products can be evaluated, and this in turn would aid in the protection of consumer rights.

Key words: capillary zone electrophoresis (CZE), A2 β-casein, total β-casein, liquid milk, milk powder

FENG Ping, DING Xiaojing, GAO Tie, DU Ruyun, CHEN Hongxu. Determination of A2 β-casein and total β-casein in cow milk and milk powder by capillary zone electrophoresis[J]. Chinese Journal of Chromatography, 2020, 38(6): 722-729.

Figures/Tables 9

Fig. 1 Electropherogram of β-CN standard by capillary zone electrophoresis (CZE) CE conditions: background electrolyte (BGE), citric acid and phosphate buffer (50 mmol/L Na2HPO4+140 mmol/L citric acid+0.2% (mass fraction)HPMC+4 mol/L urea); capillary, fused silica capillary (30/40 (effective/total length), 50 μm i.d.); injection, pressure 3.5 kPa, 10 s; voltage, 20 kV; capillary temperature, 38 ℃; sample storage temperature, 25 ℃; detection, UV at 214 nm. Peaks: 1. B β-CN; 2. A1 β-CN; 3. A2 β-CN.

Fig. 2 Electropherogram of main milk protein standards except of β-CN CE conditions were the same as those in Fig. 1. Peaks: 1. β-lactoglobulin; 2. bovine serum albumin; 3. α-lactalbumin; 4. lactoferrin; 5. IgG; 6. αS1-CN; 7. αS2-CN.

Table 1 Differences in the amino acid sequences among the five main variants of β-CN

Variant	35 site	37 site	67 site	93 site	122 site
Data from https://www.uniprot.org/uniprot/; the different amino acid was highlighted as italic.
A2	serine-P	glutamic acid	proline	methionine	Serine
A1	serine-P	glutamic acid	histidine	methionine	serine
B	serine-P	glutamic acid	histidine	methionine	arginine
C	serine	lysine	histidine	methionine	serine
I	serine-P	glutamic acid	proline	leucine	serine

Table 2 Peak areas of variants in β-CN standard by CE and LC-HRMS

Variant	Corrected peak areas/%
Variant	CE	LC-HRMS
B	7.61	7.94
A1	32.10	32.17
A2	60.28	59.89

Fig. 3 Electropherograms of (a) A2 pasteurized milk and (b) A1/A2 pasteurized milk by CZE CE conditions were the same as those in Fig. 1. Peaks: 1. αS1-CN; 2. αS2-CN; 3. B β-CN; 4. A1 β-CN; 5. A2 β-CN.

Fig. 4 Electropherograms of (a) A2 whole milk powder and (b) A2 pasteurized milk by CZE CE conditions were the same as those in Fig. 1. Peaks: 1. αS1-CN; 2. αS2-CN; 3. A2 β-CN; 4. A2 β-CN with one lactose; 5. A2 β-CN with two lactose.

Fig. 5 Electropherograms of (a) A1/A2 skim milk powder and (b) A2 whole milk powder by CZE CE conditions were the same as those in Fig. 1. Peaks: 1. αS1-CN; 2. αS2-CN; 3. A2 β-CN; 4. A2 β-CN with one lactose; 5. A2 β-CN with two lactose; 6. B β-CN; 7. A1 β-CN.

Table 3 Intra- and inter-day precisions

Sample	Days	Runs	A2 β-CN			Total β-CN
Sample	Days	Runs	Content^*	RSD(r)	RSD(I)	Content^*	RSD(r)	RSD(I)
*g/L for milk; g/100 g for milk powder. RSD(r): intraday RSD; RSD(I): interday.
A2 pasteurized milk	6	12	9.93	2.4	6.3	10.58	2.6	6.7
A1/A2 pasteurized milk	3	18	7.32	2.6	4.0	12.59	2.8	3.9
A2 whole milk powder	7	13	7.93	4.4	5.4	8.27	4.1	5.0
A1/A2 skim milk powder	7	13	9.45	4.7	4.6	13.73	4.8	5.4

Table 4 Contents of total β-CN and A2 β-CN and the percentages of A2 in total β-CN in liquid milk and milk powder samples

Sample	Calculated total β-CN^*	Detected
Sample	Calculated total β-CN^*	Total β-CN^*	A2 β-CN^*	A2/Total β-CN/%
* g/L for milk; g/100 g for milk powder. UHT: ultra high temperature.
A2 pasteurized milk 1	9.2	10.6	9.9	93.9
A2 pasteurized milk 2	9.2	9.9	9.5	95.9
A2 pasteurized milk 3	9.2	9.6	9.2	95.2
A1/A2 pasteurized milk 1	10.4	12.6	7.3	58.1
A1/A2 pasteurized milk 2	8.4	9.6	5.7	58.9
A1/A2 UHT milk 1	9.2	8.9	5.2	58.3
A1/A2 UHT milk 2	8.4	8.2	5.2	63.6
A1/A2 UHT milk 3	9.2	10.2	6.4	62.7
A2 whole milk powder 1	6.9	8.3	7.9	95.9
A2 whole milk powder 2	6.9	7.3	7.1	97.0
A1/A2 skim milk powder 1	11.5	13.7	9.5	68.8
A1/A2 skim milk powder 2	11.5	13.1	8.9	67.6

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