毛细管区带电泳测定液态奶及奶粉中的A2 β-酪蛋白及总β-酪蛋白

doi:10.3724/SP.J.1123.2019.11005

摘要/Abstract

摘要：

β-酪蛋白（β-CN）是牛乳中的主要酪蛋白。它包含13种变异体，其中A2是牛β-CN的主要变异体之一。定量测定A2 β-CN对A2乳制品的评价十分重要。该文建立了对液态奶和奶粉中A2 β-CN及总β-CN分离并定量的毛细管区带电泳（CZE）分析方法。使用熔融石英毛细管（50 μm×30/40 cm有效/总长度），以50 mmol/L Na₂HPO₄+140 mmol/L柠檬酸+0.2%（质量分数）HPMC+4 mol/L尿素溶液（pH为2.7）作为分离缓冲溶液，在214 nm下检测。总β-CN和A2 β-CN的校准峰面积与其质量浓度间呈良好的线性关系，相关系数在0.9968~0.9997之间，加标回收率为85.5%~106.4%。对两款巴氏奶和两款奶粉中A2 β-CN和总β-CN进行分析，A2 β-CN和总β-CN的日内精密度分别为2.4%~4.7%和2.6%~4.8%，日间精密度分别为4.0%~6.3%和3.9%~6.7%。所建立的CZE方法可分离液态奶及奶粉中的A2 β-CN，并可对A2 β-CN及总β-CN进行定量。通过计算A2 β-CN在总β-CN中的占比来评价A2乳制品的质量，为保护消费者的利益提供了方法依据。

关键词: 毛细管区带电泳, A2 β-酪蛋白, 总β-酪蛋白, 液态奶, 奶粉

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

冯慿, 丁晓静, 高铁, 杜茹芸, 陈泓序. 毛细管区带电泳测定液态奶及奶粉中的A2 β-酪蛋白及总β-酪蛋白[J]. 色谱, 2020, 38(6): 722-729.

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.

图/表 9

图1 CZE方法分析β-CN标准品的电泳图

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.

图2 CZE方法分析除β-CN外其他主要牛奶蛋白标准品混合物的电泳图

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.

表1 5种主要β-CN变异体中氨基酸序列的差别

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

表2 CE和LC-HRMS方法分析β-CN标准品中B、A1和A2变异体的校准峰面积

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

图3 CZE方法分析(a) A2巴氏奶和(b)普通(A1/A2)巴氏奶的电泳图

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.

图4 CZE方法分析(a) A2全脂奶粉和(b) A2巴氏奶的电泳图

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.

图5 CZE方法分析(a)普通(A1/A2)脱脂奶粉和(b) A2全脂奶粉的电泳图

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.

表3 日内和日间精密度

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

表4 液态奶和奶粉样品中总β-CN、A2 β-CN的含量以及A2 β-CN的占比

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

参考文献

1	Swaisgood H E. Handbook of Milk Composition. San Diego, USA: Academic Press, 1995: 464
2	Givens I , Aikman P , Gibson T , et al. Food Chem, 2013, 139: 549
3	Doan D N , Vicky A S , Francesco B , et al. Food Chem, 2020, 307: 125532
4	Kaminski S , Cieslinska A , Kostyra E . J Appl Genet, 2007, 48 (3): 189
5	Parashar A , Saini R K . Int J Bioassays, 2015, 4 (12): 4611
6	Caroli A M , Savino S , Bulgari O , et al. Molecules, 2016, 21: 141
7	He M , Sun J Q , Jiang Z Q , et al. Nutr J, 2017, 16: 72
8	Kullenberg de Gaudry D , Lohner S , Schmucker C , et al. Nutr Rev, 2019, 77: 278
9	Sheng X Y , Li Z L , Ni J Y , et al. JPGN, 2019, 69 (3): 376
10	Jung T H , Hwang H J , Yun S S , et al. Korean J Food Sci An, 2017, 37 (6): 940
11	Li J B , Liu W J , Wang X G , et al. China Dairy Cattle, 2017, 6: 31
11	李建斌, 刘文娇, 王秀革, et al. 中国奶牛, 2017, 6: 31
12	Duarte-Vazquez M A , Garcia-Ugalde C R , Alvarez B E , et al. J Food Sci Technol, 2018, 55 (5): 1942
13	Bonfatti V , Grigoletto L , Cecchinato A , et al. J Chromatogr A, 2008, 1195 (1/2): 101
14	Gu S Q, Deng X J, Zhao C M, et al. China Patent, 201810316627.6.2018-09-11
14	古淑清, 邓晓军, 赵超敏, 等.中国专利, 201810316627.6.2018-09-11
15	Fuerer C , Jenni R , Cardinaux L , et al. J Dairy Sci, 2019, 103 (2): 2019
16	Recio I , Olieman C . Electrophoresis, 1996, 17 (7): 1228
17	Strickland M , Johnson M E , Broadbent J R . Electrophoresis, 2001, 22 (8): 1510
18	Li Z Y, Liu Y, Chen Y. China Patent, 201810300669.0.2019-10-18
18	李周勇, 刘宇, 陈云.中国专利, 201810300669.0.2019-10-18
19	Liu Y, Chen W, Chen Y. China Patent, 201610784180.6. 2016-12-07
19	刘宇, 陈伟, 陈云.中国专利, 201610784180.6.2016-12-07
20	Feng P , Fuerer C , McMahon A . J AOAC Int, 2017, 100 (2): 510

[1]	邢家溧, 张子庚, 郑睿行, 徐晓蓉, 毛玲燕, 承海, 沈坚. 固相萃取-超高效液相色谱-串联质谱法检测婴幼儿奶粉中的7种链格孢霉毒素[J]. 色谱, 2022, 40(2): 156-164.
[2]	曲栗, 古淑青, 张嘉麒, 赵超敏, 邓晓军. 柱前衍生-超高效液相色谱法测定3种奶粉中18种氨基酸[J]. 色谱, 2021, 39(5): 472-477.
[3]	罗芳, 郭泽华, 曹成喜, 樊柳荫, 张薇. 基于毛细管电泳和离子迁移率经验公式测定洛伐他汀的绝对淌度和解离常数[J]. 色谱, 2021, 39(12): 1362-1367.
[4]	夏张悦怿, 杨丙成, 章飞芳, 梁鑫淼. 替米考星磁性表面分子印迹聚合物的制备及应用[J]. 色谱, 2019, 37(9): 932-938.
[5]	Elijah N. MCCOOL, 孙良亮. 比较纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱用于自顶向下蛋白质组学[J]. 色谱, 2019, 37(8): 878-886.
[6]	励炯, 孙岚, 金朦娜, 王红青, 邱红钰. 分散固相萃取-超高效液相色谱-串联质谱法测定奶粉中7种非选择性环氧化酶抑制药物[J]. 色谱, 2019, 37(2): 177-182.
[7]	周晓晴, 吕小丽, 万建春, 郭平, 郭丹, 席慧婷. 高效液相色谱-串联质谱法测定奶粉中氯酸盐和高氯酸盐[J]. 色谱, 2019, 37(10): 1064-1070.
[8]	聂雪梅, 董旭阳, 许秀丽, 张峰. 液态奶加工过程中反映工艺条件标志物的研究进展[J]. 色谱, 2019, 37(10): 1084-1089.
[9]	许兵兵, 李晓敏, 张庆合, 朱维晃. 气相色谱-串联质谱法测定婴儿奶粉中16种邻苯二甲酸酯类化合物[J]. 色谱, 2018, 36(8): 786-794.
[10]	程建桥, 张峰, 许秀丽, 洪云鹤, 吴寒秋, 王传胜. 超高效液相色谱-高分辨质谱法筛查婴幼儿配方奶粉中17种头孢菌素类药物残留[J]. 色谱, 2018, 36(8): 809-816.
[11]	李洁, 鞠香, 王艳丽, 田其燕, 申中兰, 胡梅, 刘艳明, 祝建华. 3种前处理方法处理婴幼儿配方奶粉中二十二碳六烯酸、花生四烯酸、亚油酸和α-亚麻酸的差异性[J]. 色谱, 2018, 36(8): 827-832.
[12]	陈泓序, 屈锋. 毛细管电泳技术在单克隆抗体药物分析中的应用[J]. 色谱, 2018, 36(3): 195-208.
[13]	韩疏影, 俞慧敏, 宋易霖, 邓海山, 柴川, 池玉梅. 基质固相分散萃取和固相萃取-高效液相色谱-三重四极杆-复合线性离子阱质谱同时测定奶制品中6种雌激素[J]. 色谱, 2018, 36(3): 285-291.
[14]	励炯, 王姣斐, 邱红钰, 李玮. 改进的高效液相色谱法测定婴幼儿配方奶粉中5种核苷酸[J]. 色谱, 2017, 35(8): 832-836.
[15]	严丽娟, 李文斌, 洪煜琛, 吴敏, 徐敦明, 林立毅, 顾春燕. 液相色谱-大气压化学电离串联质谱法测定婴幼儿配方奶粉中的维生素D[J]. 色谱, 2017, 35(4): 427-431.