CHINESE JOURNAL OF ANALYTICAL CHEMISTRYVolume 37, Issue 11, November 2009 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2009, 37(11), 1667–1670.

Received 4 April 2009; accepted 23 June 2009 * Corresponding author. E-mail: zhangqh@nim.ac.cn This work was supported by the National Natural Science Foundation of China (No. 20775075) and the National Key Technologies RD Program (No. 2006BAF07B03). Copyright © 2009, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(08)60146-2

RESEARCH PAPER

Determination of Major Bovine Milk Proteins by Reversed Phase High Performance Liquid Chromatography WANG Juan1,2, ZHANG Qing-He2,*, WANG Zhi-Hua1, LI Hong-Mei21 State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China 2 National Institute of Metrology, Beijing 100013, China

Abstract: A reversed-phase high-performance liquid chromatography (RP-HPLC) for the quantification of six major bovine milk proteins ( -casein ( -CN), s2-casein ( s2-CN), s1-casein ( s1-CN), -casein ( -CN), immunoglobulins (IgG) and whey) was developed. The separation and quantification of these major proteomic components were achieved with an Agilent Zorbax 300SB-C8 analytical column (250 mm × 4.6 mm I.D., 5 m) at 45 °C with detection wavelength at 214 nm. The gradient elution was carried out with solvents of 0.1% TFA in water and 0.1% TFA in acetonitrile at a flow rate of 0.8 ml min–1. The method validation consisted of testing the linearity, repeatability, reproducibility and accuracy. The recoveries of six target proteins spiked in milk were in the range from 74.8% to 132.5%. Nine different commercial milk brands were used, the difference of the concentration and relative ration of -CN, s2-CN, s1-CN, -CN and whey were found.

Key Words: Reversed-phase high-performance liquid chromatography; Bovine milk; Protein

1 Introduction

Bovine milk contains 3.0%–3.5% (w/V) of proteins. 80% of the proteins consist of caseins, and the remaining 20% are whey or serum proteins. The casein (CN) group can be subdivided into -CN, s2-CN, s1-CN and -CN, with the approximate proportions of 1:4:1:4. Whey proteins include

-lactalbumin ( -La) and -lactoglobulin ( -Lg) with bovine serum albumin (BSA) and immunoglobulins (Ig) as minor constituents. These proteins contain eight amino acids considered essential amino acids with balanced amino acid composition, high biological potency and high rate of utilization. However, these essential amino acids cannot be synthesized in the human body. Indeed, dairy products constitute some of the major sources of proteins to human beings and therefore represent a considerable market. To control the quality of milk and avoid adding adulterants, these major milk proteins should be detected and monitored. At present, methods employed to analyze the fractions of milk

proteins are liquid chromatography[1–5], gel electrophoresis[6,7], isoelectric focusing [8], and so on.

In this study, a reversed-phase high-performance liquid chromatography (RP-HPLC) for detecting six major proteins in milk was optimized and validated. Nine different commercial milk brands were used; the difference of the concentration and relative ration of -CN, s2-CN, s1-CN, -CN and whey were found.

2 Experimental

2.1 Instruments and reagents

Shimadzu binary pump (LC-20AT) coupled to a diode array detector (SPD-M20A) and LC Systems software were used. CEM protein analyzer (Sprint, USA) was used to analyze the total protein content.

Guanidine hydrochloride (GdnHCl) and Bis-Tris (> 99%) were purchased from Beijing Biotechnology and

WANG-Juan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(11): 1667–1670

dl-Dithiothreitol (Lot R1/83/030, > 99%) was purchased from Inalco (USA). Purified major proteins -CN, S1-CN, S2-CN, -CN, whey and IgG were purchased from Sigma-Aldrich

Company. The purity of the proteins was tested with HPLC in our laboratory. Trifluoroacetic acid (TFA) and HPLC-grade acetonitrile were purchased from Tedia (USA). The nine kinds of milk samples were purchased from a total supermarket. 2.2 Sample preparation 2.2.1 Purified protein

A certain amount of purified protein was weighed, and a

volume of a sample buffer consisting of 6 M GdnHCl, 19.5 mM dithiothreitol and 5.37 mM tri-sodium citrate (pH 7) was added. After one hour of incubation at room temperature, the sample was dissolved using a vortex mixer (IKA MS 3D S25, Germany) until there is no noticeable foaming production. Dilution of the solution containing 4.5 M GdnHCl and the chromatographic eluent A in the ratio of 1:3 was then carried out. Standard concentrations were -CN, 5.354 g l–1; S1-CN, 2.637 g l–1; S2-CN, 0.423 g l–1; -CN, 7.52 g l–1; IgG, 2.160 g l–1; and whey, 3.088 g l–1, respectively. 2.2.2 Liquid milk

Five milliliter of liquid milk was poured directly from the

commercial package to an ultracentrifugation tube made of polypropylene. A certain mass of liquid milk was weighed and the sample buffer was added in a 1:1 ratio (V/V). The sample was centrifuged at 14800 rpm at 4 °C for 20 min using a Sigma 3k30 (Sartorius Instruments, Germany) ultracentrifuge. The same method was carried out for the purified proteins after the fat layer was removed. 2.3 Chromatographic conditions

Separations were performed on a reversed-phase analytical

column C8 (Zorbax 300SB-C8 RP, Agilent Technologies) with 5 m particle size and 30 nm pore diameter (150 mm × 4.6 mm I.D.), and the column temperature was kept at 45 °C. Gradient elution was carried out with two solvents. Solvent A consisted of a mixture of 0.1% trifluoroacetic acid (TFA) and water, and solvent B consisted of a mixture of 0.1% TFA and acetonitrile. The flow rate was 0.8 ml min–1. Detection wavelength was set at 214 nm.

The optimized elution gradient conditions were selected as follows: linear gradient from 33% to 35% B in 5 min, from 35% to 37% in 4 min, from 37% to 40% B in 9 min, then from 40% to 41% in 4 min, followed by an isocratic elution at 41% B in 5 min, and then linear gradient from 41% to 43% in 1 min, from 43% to 45% in 8 min, a final increase to 90% in 4 min,

and then to the starting condition in 0.1 min.

3 Results and discussion 3.1 Optimization of pretreatment conditions

The pretreatment processes of the milk samples include

protein denaturation, incubation, and fat removal. Protein precipitation is avoided in the pretreatment processes. Several pretreatment methods and reagents for analysis of whey protein in milk have been studied by Bobe et al[9]. A solution containing sodium citrate, DTT and GdnHCl (pH 7) was added directly into the milk, and then the milk was incubated at room temperature and analyzed by HPLC after centrifuging and skimming.

In a study by Bordin et al[10], a solution containing 6 mM sodium citrate, 20 mM DTT and 5 M GdnHCl (pH 7) was added to the milk in 1:1 ratio (v/v). The sample was shaken for 10 s, incubated for 1 h at room temperature, and the skim is centrifuged, then HPLC was performed. However, according to method followed by Bordin, clear solution could not be obtained, and flocculation occurred after half an hour. Only four casein proteins can be separated and detected, and the peaks of whey proteins could not be found. In this study, we found that by adding 0.1 M Bis-Tris to the above-mentioned solution, clear solution and a good shape of chromatographic peak of whey proteins can be obtained. 3.2 Optimization of chromatographic conditions 3.2.1 Choice of column and detection wavelength

The main factors influencing the protein separation by

RP-HPLC are pore diameter of the packings and the length of bonded alkyl chain. Compared with 30 nm pore size C18 column, proteins’ peak shape was poor in column with 10 nm silica-bonded phase. There were no significant difference in the peak shapes for the separation of the casein proteins on 30 nm pore size C4, C8 and C18 columns; however, the best separation could be achieved for s-CN and -CN on the C8. Therefore, Agilent Zorbax 300SB-C8 column was used in this study.

Detection wavelength was set at 214 nm, because it was the relatively low baseline noise and strong signal for the proteins, compared with 200 and 280 nm. 3.2.2 Optimization of mobile phase conditions

In this study, the mobile phase conditions were optimized

based on the mobile phase composition, gradient, and flow rate. The retention times of the major eluted peaks coincided with retention times of standard protein fractions. The identification of peaks of milk proteins were confirmed by comparison with

WANG-Juan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(11): 1667–1670

commercial standards and with chromatographic profiles of individual milk samples in literatures[1–5]. The proteins were eluted in the following order: -CN, S2-CN, S1-CN, -CN, whey and IgG.

Under the optimum conditions, on the basis of comparisons with the retention time of standard proteins, BSA coeluted with -CN (Fig.1). However, BSA was not detectable in milk

samples in the study by Bonfatti et al[10]. Chromatograms relative to milk samples and soy milk were

obtained using the optimized elution (Fig.1). It could be seen that the separation can be achieved between plant proteins and the main bovine milk proteins; thus, this procedure was suitable for analysis of bovine milk, which contains illegally added plant proteins. 3.3 Linearity, accuracy and precision 3.3.1 Linearity and Accuracy

The detector’s response was studied by varying the injection

volume of the protein solutions from 0.5 l to 20 l. For all the proteins, the regression coefficients (R2) of the linear relationship between injection volume of the proteins and peak areas were exceeded 0.999. Parameters of calibration curves are listed in Table 1.The recovery tests were carried out for the two replicates at two spike level. The results are listed in Table 2. The average recoveries ranged from 74.8% to 132.5% (n = 2) for the spiked 6 proteins. 3.3.2 Repeatability and reproducibility

Relative standard deviations (RSDs) of retention times and

peak areas obtained in the analysis of repeatability and reproducibility are presented in Table 3, which indicated that the precision (repeatability and reproducibility) of the method was acceptable. The RSDs of retention times were below 1.1% in one instrument (repeatability) and below 2.0% on the other instrument in the same laboratory (reproducibility) .The RSDs of peak areas were below 4.7% in one instrument (repeatability) and below 5.7% in the other instrument in the same laboratory (reproducibility).

3.4 Quantification of proteins in commercially available

milks The concentrations of various proteins in nine different milk

brands were determined by the developed method. The results are shown in Table 4. The concentration of total major protein in six domestic milk samples ranged from 3.05% to 3.56%, and for three abroad milk samples, it ranged from 3.26% to 3.38%.It was close to the result obtained by the protein analyzer.

Fig.1 Chromatogram of commercially available milk and soy milk

Table 1 Linear ranges, regression equations and correlation coefficient of six proteins standards

Proteins Injected amount ( g) Regression equations Correlation coefficient (R2) -CN 0.88–35.04 y = 1.111 × 109x + 9.903 × 104 0.9999 S2-CN 0.21–8.25 y = 5.324 ×108x + 1.823 × 105 0.9997 S1-CN 1.32–51.39 y = 9.654 ×108x + 4.562 × 105 0.9999 -CN 2.68–107.09 y = 9.402 × 108x + 4.130 × 104 0.9995

Igg 1.08–43.20 y = 1.488 × 108x – 2.815 × 104 0.9999 Whey 10.29–102.93 y = 4.953 × 108x – 9.077 × 104 0.9997

Table 2 Recoveries of six target proteins spiked in milk

Proteins Original (g l–1) Added level (g l–1) Found (g l–1) Recovery (%)

12.88 20.03 94.3 S1-CN 7.88

2.84 10.43 89.6 S2-CN 3.51 1.56 1.30 83.3

6.84 7.59 80.7 -CN 2.10

1.36 3.23 83.1 10.64 20.35 91.9

-CN 10.57 1.72 11.86 74.8 5.76 7.88 124.7

Igg 0.70 0.80 1.76 132.5 19.82 25.99 107.8

Whey 4.63 2.91 7.30 91.8

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The percentages of whey were in the range of 14.5%–20.0%

in all the nine milk samples, which varies indifferent brands (6 kinds of Domestic, 3 kinds of Foreign). This result was similar to that in the literature[11]. The average concentration percentages of -CN, s2-CN, s1-CN and -CN were 8%, 11%, 27% and 35%, respectively, with the relative proportion of 1.0:1.3:3.2:4.2. These results were in agreement with the results reported in the literature[11]. However, distinctness has found in Foreign 3, the concentration of caseins was the highest, whereas the concentration of whey was the lowest.

Table 3 Repeatability and reproducibility of analysis method Repeatability Reproducibility

Proteins Retention time RSD (%)

Peak area RSD (%)

Retention time RSD (%)

Peak area RSD (%)

S1-CN 0.3 0.7 1.3 2.1

S2-CN 0.5 4.7 1.9 4.0 -CN 1.1 2.0 2.0 1.7 -CN 0.3 2.0 0.5 1.5

Igg 0.8 2.2 0.2 5.7 Whey 0.1 3.9 0.7 0.2

Table 4 Concentration of proteins in 9 commercially available milks

CTMPC (%)a C (%)b P (%)c

Milk CHPLC CCEM TE (%)d -CN s2-CN s1-CN -CN Whey -CN S2-CN S1-CN -CN Whey

Domestic 1 3.05 3.14 –2.9 0.23 0.35 0.81 1.05 0.61 7.55 11.4 26.6 34.5 20.0 2 3.05 3.07 –0.7 0.25 0.34 0.84 1.10 0.52 8.20 11.1 27.6 36.1 17.1 3 3.23 3.12 3.6 0.27 0.36 0.90 1.13 0.57 8.36 11.2 27.9 35.0 17.6 4 3.24 3.15 2.8 0.26 0.36 0.88 1.18 0.56 8.03 11.0 27.2 36.5 17.3 5 3.39 3.25 4.3 0.28 0.37 0.95 1.20 0.59 8.26 10.9 28.0 35.4 17.4 6 3.56 3.26 9.2 0.28 0.42 0.99 1.22 0.65 7.86 11.8 27.8 32.3 18.3

Foreign 1 3.27 3.51 –7.0 0.29 0.36 0.86 1.19 0.57 8.88 10.9 26.3 36.5 17.5 2 3.26 3.49 –6.7 0.29 0.36 0.86 1.15 0.60 8.91 10.9 26.4 35.3 18.4 3 3.38 3.45 –2.0 0.36 0.38 0.84 1.31 0.49 10.65 11.3 24.8 38.7 14.5

a: Total proteins concentration (TMPC); b: Concentration of individual protein in milk; c: Proportion of individual protein in milk; d: Relative error.

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