HPLC Analysis of α-lactalbumin and β-lactoglobulin in Bovine Milk with C4 and C18 Column

HPLC Analysis of α-lactalbumin and β-lactoglobulin in Bovine Milk with C4 and C18 Column

Abstract: Reversed-phase high-performance liquid chromatography (RP-HPLC) method with C4 column and C18 column for

analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteristic of two columns were

compared. Shiseido Proteonavi C4 column (250 mm×4.6 mm×5 μm) and Shiseido CAPCELL PAK SG 300 C18 column (250 mm×

4.6 mm×5 μm) were used in the experiment. Phase A was composed of 0.1% (V/V) trifluoroacetic acid (TFA) in ultrapure water and

Phase B (organic phase) was composed of 0.1% TFA in acetonitrile. Gradient elution was taken. Flow rate was 1 mL • min-1. The

detection wavelength was 215 nm. The injection volume was 20 μL and the column temperature was 30℃. The results showed that linear

relationship was good and recovery of α-lactalbumin and β-lactoglobulin was 86.12%-104.38%, C18 column had stronger ability to resist

acid and more stable, and the method with C4 column had excellent sensitivities and good separation.

Key words: RP-HPLC, bovine milk, α-lactalbumin, β-lactoglobulin, C4 column, C18 column

CLC number: TS252.7; TS207.3 Document code: A Article ID: 1006-8104(2012)-03-0076-07

Introduction

About 20% of totle proten in bovine milk is whey protein (Eigel et al., 1984) which is only next to casein. Whey protein contains α-lactalbumin, β-lactoglobulin, bovine serum albumin, lactoferrin, immunoglobulin, glycomacropeptide and growth factors (Mercier and Gauthier, 2004). In these proteins α-lactalbumin and β-lactoglobulin are two main typical components and are about 70%-80% of whey protein (Korhonen et al., 1998). Whey protein is widely utilized in the food industry due to its nutritional value and versatile functional properties (Alomirah and Alli, 2004). For example, α-lactalbumin binds metal cations such as Ca2+ and Zn2+ and β-lactoglobulin binds both fatty acids and retinol (Permyakov and Berliner, 2000;

Kontopidis et al., 2004). They provide body with many essential nutrients.　With the development of dairy industry in recent years, a lot of fortified dairy products with whey proteins have appeared in the market; however, some of these products are adulterated with protein hydrolysates of the plant, melamine or other toxic substances for higher protein content and lower cost (Luykx et al., 2007; Sun et al., 2010; Enne et al., 2005). Therefore, it is very important to detect and monitor these two typical whey proteins in milk and dairy products for quality control purpose. 　At present, the main detection methods of whey proteins are capillary electrophoresis (Rehder-silinski and Mcgown, 2003) , HPLC (Visser et al., 1991; Bobe et al., 1998; Dziuba et al., 2001) and surface plasmon resonance (Billakanti et al., 2010). Reversed-phase

Received 25 April 2011Supported by the Project of Science & Technology Plans in Heilongjiang Province in the 11th Five-year Period (GB07B407)Kong Xiao-yu (1985-), male, Master, engaged in the research of quality detection and analysis in dairy. E-mail: [email protected]* Corresponding author. Liu Ning, professor, Ph. D, engaged in the research of nutrition and food safety. E-mail: [email protected]

Kong Xiao-yu1, 2, Wang Jing1, 2, Tang Yan-jun2, Li Dan-dan2, Zhang Nan-nan2, Jiang Jin-dou1, and Liu Ning1, 2*

1 National Dairy Engineering & Research Center, Harbin 150086, China 2 Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, China

September 2012 Vol. 19 No. 3 76-82Journal of Northeast Agricultural University (English Edition) Available online at www.sciencedirect.com

ScienceDirect

E-mail: [email protected]

high-performance liquid chromatography (RP-HPLC) is one effective method in analyzing whey proteins, and it has advantage of superior precision and re-coveries, etc. Some types of packing such as C3 (pro-pyl), C4 (butyl), C8 (octyl), C18 (octadecyl), cyanopropyland phenyl groups are available for determinationof proteins (Wu and Greenblatt, 1995). Generally C18

(octadecyl) is widely used while C4 (butyl) is seldom used for analyzing of α-lactalbumin and β-lactoglobulin in bovine milk. Hence in our experiment, we used RP-HPLC with Shiseido Proteonavi C4 column (250 mm×4.6 mm×5 μm) and Shiseido CAPCELL PAK SG 300 C18 column (250 mm×4.6 mm×5 μm), respectively to develop a method that determined α-lactalbumin and β-lactoglobulin, then compared performance and characteristics of the two columns for the purpose of deciding which column was better.

Materials and Methods

ReagentsAcetonitrile was purchased from Fisher Corporation (HPLC grade, America); Trifluoroacetic acid was purchased from Guangfu Corporation (TFA, HPLC grade, China). Water was purified by Milli-Q system (Millipore, Bedford, MA, USA), α-lactalbumin and β-lactoglobulin were purchased from Sigma Corporation (St Louis, MO, USA).

InstrumentsWaters 2695 HPLC, Waters 2487 UV detector and Empower chromatography workstation software were from Waters Corporation, America. Proteonavi C4 column (250 mm×4.6 mm×5 μm) and CAPCELL PAK SG 300 C18 column (250 mm×4.6 mm×5 μm) were purchased from Shiseido, Japan.

Standard solution and calibration curveA certain amount of α-lactalbumin and β-lactoglobulin was weigheds respectively, then a volume of deionized water was added to appropriate concentration, and diluted to different concentrations progressively. The

series solutions of the two proteins were determinated by HPLC with two columns and the results were used to develop calibration curves.

RecoverySome bovine milk was weighed, and three different weights (50%, 70% and 100% content of the sample) standard proteins of α-lactalbumin and β-lactoglobulin were added to the bovine milk, then the solutions were determined by HPLC with two columns. Recovery was calculated as the followings.　ξ=(m1–m)/m0

　ξ was recovery; m was content of the sample; m1 was content of the sample that add the standard proteins; and m0 was the added content. m1 and m were the average of repeated measurements.

Repeatability and reproducibilityTotal 10 bovine milk samples were determined by HPLC with two columns, and each sample was deter-mined three times. The results were used to calculate the relative standard deviation of peak area. To show the reproducibility, the bovine milk was detected by HPLC by two columns once a day in 7 days.

Sample treatmentTwo liquid milk products were prepared as samples for determination. One was fresh bovine milk purchased from local farm and the other was liquid milk product of some brands purchased from supermarket. They were treated respectively as the followings: adding 1 mol • L-1 hydrochloric acid, standing for 15 min so that casein was precipitated at its isoelectric point. Then the supernatant was skimmed by centrifugation(2 000×g for 15 min at 4℃). All samples were stored at 4℃, and filtered through a 0.45 μm membrane before HPLC analysis.

Results and Discussion

Separation conditionsIn pre-experiment, full spectrum scanning was used

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Kong Xiao-yu et al. HPLC Analysis of α-lactalbumin and β-lactoglobulin in Bovine Milk with C4 and C18 Column ·77·

to select the largest absorption wavelength of whey proteins, and 210, 215, 230 and 280 nm were selected as the detection wavelength. The results showed that 215 nm was the best detection wavelength. In order to determine elution conditions, organic phase rose from 0% to 100% in 60 min. Flow rate was 1 mL • min-1.Calculated the resolution according to retention time. Higher concentrations of TFA in mobile phase caused hydrophobicity of proteins larger, due to protonated basic groups becoming uncharged by forming ion-pair with TFA. And lower concentration of TFA accelerated silanophilic interactions with amino groups of proteins (Hamada et al., 2004). 0.02%, 0.05%, 0.08% and 0.1% of TFA were added to the mobile phase, respectively, and then HPLC analysis began. The results showed that there was a significant fluctuation when the TFA concentration was below 0.05%. When the TFA concentration was closer to 0.1%, the shape of peak was good. 25℃, 30℃, 35℃ and 40℃ were chosen as measured temperature, and the results revealed that temperature had no significant effect on the separation. The flow rate was chosen at 0.5, 0.8 and 1.0 mL • min-1, respectively. The results revealed the best peak was at 1.0 mL • min-1, the peaks were relatively wide under other flow rate conditions. 10, 20 and 40 μL were selected as the sample injection volume. When the injection volume was 10 μL, the repetitiveness of the chromatogram was very poor, because it was too small to inject a kind of volume, and the protein content was uneven. When the injection volume was 40 μL, the proteins content was too large, which reduced the resolution.　Based on the above results, the last separation condition was obtained as the followings. Phase A was composed of 0.1% (V/V) TFA in ultrapure water and Phase B (organic phase) was composed of 0.1% TFA in acetonitrile. Flow rate was 1 mL • min-1. The detection wavelength was 215 nm, the injection volume was 20 μL and the column temperature was 30℃. The elution gradient of C4 column was set as the followings: 0-22 min 20%-44% B, 22-25 min 44%-45% B, 25-35 min 45%-50% B, 35-50 min

50%-20% B. The elution gradient of C18 column was set as the followings: 0-35 min 30%-50% B, 35-40 min 50%-30% B.　The chromatogram detection of bovine milk with C4 column and C18 column are shown in Fig. 1. The efficiency of the monolithic silica columns was increasing with the decreasing of n-alkyl chain length, so separation of C18 column was weaker than that of C4 column (Skudas et al., 2007). As shown in Fig. 1,C4 column was superior to C18 column in separat-ing β-lactoglobulin, but in separating α-lactalbuminit was significantly weaker than C18 column. An unknown peak was appeared in the chromatogram of α-lactalbumin by C18 column, while C4 column was not.

Linear relationship and detection limitThe regression equations were calculated by the concentration and its peak area. The linear equations of two kinds of proteins, linear range, correlation coefficient and detection limit are shown in Table 1, with the signal to noise ratio (S/N)=3 detection limits of confirming every material. Two kinds of proteins in bovine milk must be within the linear range when choosing linear range. Otherwise, regression equations can not be used for the calculation of results. As it is shown, comparing through the linear relation to two kinds of columns, the two correlation coefficients of C18 column were a little greater than those of C4 column, so C18 column was a little more accurate than C4 column. The detection limit expressed the sensitivity of the column. In our study, the detection limit of α-lactalbumin and β-lactoglobulin with C4 column were 0.008 and 0.012 mg • mL-1, respectively, which was a half lower than that of C18 column, so the sensitivity of C4 column was obviously superior to that of C18 column.

RecoveryA certain bovine milk sample was selected, and recovery experiment was carried on. The content ofα-lactalbumin and β-lactoglobulin in samples and


Journal of Northeast Agricultural University (English Edition)·78· Vol. 19　No. 3　2012

results are shown in Table 2. Recovery of α-lactalbu-min and β-lactoglobulin with C4 column was 86.12%-104.38% while with C18 column was 90.21%-102.38%. It is known that long-chain phases like C8 or C18 can be lower recovery of large proteins due to the high hydrophobicity. But according to the results, we

could find out that recovery of C18 column was better than that of C4 column. So stability of C18 column was slightly better than that of C4 column. In this study, the phenomenon was observed that recovery approached 100%, if the added content approached original content, otherwise, the recovery was lower.

Fig.1 Chromatogram detection of bovine milk with C18 (A) and C4 (B) columnChromatogram detection of bovine milk with Shiseido Proteonavi C4 column (250 mm×4.6 mm×5 μm) in A and Shiseido CAPCELL PAK SG 300 C18 column (250 mm×4.6 mm×5 μm) in B. Peak 1 was α-lactalbumin; Peak 2 was β-lactoglobulin B; Peak 3 was β-lactoglobulin A.

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Column Protein Linear range(mg • mL-1) Intercept (a) Slope (b) Correlation

coefficient (r)Detection limit

(mg • mL-1) (S/N=3)

C4

α-lactalbumin 0.1-1.0 5.104×106 1.419×107 0.9992 0.008

β-lactoglobulin 0.1-2.0 1.985×106 8.815×106 0.9984 0.012

C18

α-lactalbumin 0.1-1.0 –1.131×106 2.060×107 0.9995 0.017

β-lactoglobulin 0.1-2.0 –8.876×105 1.164×107 0.9992 0.026

Table 1　Regression equations, linear ranges and correlation coefficients of two whey protein standards



Repeatability and reproducibilityThe repeatability of C4 and C18 columns showed the stability of two kinds of columns well, and peak areaRSD and retention time RSD of columns were app-roaching. The results are shown in Table 3. Retention time RSD and peak area RSD of α-lactalbumin withC4 was 0.1 and 0.7, respectively while 0.3 and 0.6 withC18. Retention time RSD and peak area RSD of β-lacto-globulin were greater than those of α-lactalbumin, due to β-lactoglobulin of bovine milk including two natural variants β-lactoglobulin A and β-lactoglobulin B (Croguennec et al., 2004). Reproducibility RSD was significantly higher than repeatability RSD. 　In the experiment, the interesting phenomenon was observed. The determination results of the first 5 days showed that the protein content was invariable in the bovine milk, and there were not any changes. But from the sixth day on, the protein content obviously reduced, and retention time RSD increased. The retention RSDs of C4 column rose from 0.1 to 0.4 for α-lactalbumin and from 0.3 to 0.6 for β-lactoglobulin, the retention RSDs of C18 column rose from 0.1 to 0.4 for α-lactal-bumin and from 0.3 to 0.4 for β-lactoglobulin, the variation of C4 column was obviously greater than

that of C18 column. Acidity of mixed mobile phase was stronger, because pH of the mobile phase was about 2, the damage to the columns was very serious, even if some companies had already put out the chromatogram column with stronger acidproof performance. Accord-ing to these data, we summarized that the acidproof of C18 column was better than that of C4 column.

Milk product analysisThe milk samples taken from local farm and super-market were determined by RP-HPLC with C4 and C18

column respectively, and each sample was determined for 10 times. The results are shown in Table 4. We could observe that α-lactalbumin and β-lactoglobulin contents of sample 1 were 1.0212 and 2.1342 with C4

and 1.1213 and 2.1296 with C18, respectively, which were obviously higher than those of sample 2. Thecause was the sample coming from local farm andwithout any treatments, keeping well in the transporta-tion way, and the microbes having not decomposed protein, though microbes could break down the protein in the bovine milk (Belotia et al., 2002) . While sample 2 came from the supermarket, sterilized and dealt, whey protein of bovine milk lost in the processing course (Considine et al., 2007).

Table 2　Recoveries of two proteins

Column Proteins Original (mg) Added (mg) Found (mg) Recovery n (%)

C4

α-lactalbumin 0.64010.25 0.8554 86.120.48 1.1097 97.830.88 1.4465 91.64

β-lactoglobulina 1.79040.74 2.4602 90.511.40 3.1625 98.012.21 4.0972 104.38

C18

α-lactalbumin 0.64140.21 0.8308 90.21 0.47 1.1465 97.040.95 1.6036 101.29

β-lactoglobulina 1.78480.73 2.5081 90.411.31 3.0328 95.272.51 4.3545 102.38

n, Every addition level was done for three parallel samples, and every parallel sample was determined for three times repeatedly, the results were averaged; a, β-lactoglobulin included β-lactoglobulin A and β-lactoglobulin B.



Conclusions

We used Shiseido Proteonavi C4 column and Shiseido CAPCELL PAK SG 300 C18 column to determinate α-lactalbumin and β-lactoglobulin in bovine milk. As it is shown, C4 column was better in separating β-lactoglobulin and superior to C18 column, but in separating α-lactalbumin, and it was significantly weaker than C18 column. An unknown peak was appeared in separating α-lactalbumin chromatogram with C18 column, while C4 column was not. The coeffi-cient correlation of C18 column was greater than thatof C4 column, so C18 column was more accurate than C4 column. In our study, the detection limit of C4 column was lower than that of C18 column, so the sensitivity of C4 column was obviously superior to that of C18 column. Recovery of C4 column was 86.12%-104.38%. Recover of C18 column was 90.21%-102.38%. Recovery RSD of C18 column was less than that of C4 column.

So the stability of C18 column was slightly betterthan that of C4 column. We summarized the acidproof of C18 column which was better than that of C4 column. C4 column represented the short chain column. And C18 column represented the long chain column.This study provided the necessary information for the column of short chain packing and the column of long chain packing by the comparison with C4 and C18 column.

Kong Xiao-yu and Wang Jing equal to the contribution of this paper,

and tied for the first author.

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Table 4　Sample variety of two kinds of whey protein content

m, Every sample was determined for three times repeatedly and the results were averaged; n, Every sample was determined for three times repeatedly, and the results were averaged; a, β-lactoglobulin included β-lactoglobulin A and β-lactoglobulin B; b, RSD was the relative standard deviation.

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HPLC Analysis of α-lactalbumin and β-lactoglobulin in Bovine Milk with C4 and C18 Column