Food Chemistry 139 (2013) 549–552

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Food Chemistry

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Short communication

Proportions of A1, A2, B and C b-casein protein variants in retail milk in the UK

Ian Givens a,⇑, Patricia Aikman a, Trevor Gibson b, Ronald Brown a

a Food Production and Quality Division, School of Agriculture, Policy and Development, University of Reading, Reading RG6 6AR, United Kingdomb The Chemical Analysis Facility, University of Reading, Reading RG6 6AD, United Kingdom

a r t i c l e i n f o

Article history:Received 1 October 2012Received in revised form 20 December 2012Accepted 28 January 2013Available online 10 February 2013

Keywords:MilkA1 b-caseinA2 b-casein

0308-8146/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2013.01.115

⇑ Corresponding author.E-mail address: d.i.givens@reading.ac.uk (I. Givens

a b s t r a c t

The A1 variant protein of the b-casein family has been implicated in various disease states although muchevidence is weak or contradictory. The primary objective was to measure, for the first time, the propor-tions of the key b-casein variant proteins in UK retail milk over the course of one year. In total, 55 samplesof semi-skimmed milk were purchased from five supermarkets over the course of a year and the propor-tions of the A1, A2, B and C casein variant proteins were measured, using high resolution HPLC–MS. Theresults showed that b-casein in UK retail milk comprises approximately 0.58, 0.31, 0.07 and 0.03 A2, A1, Band C protein variants, respectively. The proportion of A2 is higher than some early studies would predictalthough the reasons for this and any implications for health are unclear.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Casein proteins represent about 0.8 of total protein in cows’milk, about 0.36 of which is b-casein (Fox, 2003). Within b-casein,there are a number of protein variants which are genetically deter-mined, with A1, A2, B and C proteins being most common in cattlebreeds of European origin. These variants are generally consideredto be co-dominant, so an A1/A2 heterozygous cow will expressboth genotypic variants in her milk, and thus standard purchasedmilk, which is derived from many individual animals, is likely tocontain a range of different b-casein variants. A1, B and C b-caseinhave a histidine residue at position 67 that allows an enzymaticcleavage to occur during digestion, releasing the seven amino acidpeptide, b casomorphin 7 (bCM-7; De Noni & Cattaneo, 2010),whereas the A2 variant has a proline at position 67 so that bCM-7 is not released. bCM-7 has opioid properties (Brantl, Teschem-acher, Henschen, & Lottspeich, 1979) and some ecological studieshave suggested that bCM-7 and related compounds may be in-volved in the aetiology of a range of chronic diseases, includingType 1 diabetes, ischaemic heart disease, autism and schizophrenia(Elliott, Harris, Hill, Bibby, & Wasmuth, 1999; McLachlan, 2001),although a recent review by the European Food Safety Authority(EFSA, 2009) concluded that ‘a cause-effect relationship betweenthe oral intake of bCM-7 or related peptides and aetiology or causeof any suggested non-communicable diseases cannot be estab-lished’. However, EFSA (2009) also concluded that there was a lackof detailed knowledge on milk protein variant composition across

ll rights reserved.

).

Europe, leading to an inability to assess the exposure of individualsto different b-casein variants.

The aim of this study was to assess the proportions of the key b-casein variant proteins in UK retail milk over the course of oneyear.

2. Materials and methods

2.1. Milk samples

Samples of semi-skimmed pasteurised milk were purchasedfrom five major supermarkets (Asda, Morrisons, Sainsburys, Tescoand Waitrose) within an 8 km range of the laboratory for11 months from November 2008 to October 2009. No sampleswere collected in April. In total, 55 samples were therefore col-lected, i.e. 11 samples of each supermarket brand. Semi-skimmedmilk was chosen as it now represents the major form of liquid milkpurchased (65%; DairyCo, 2012a) and consumed by adults and chil-dren (Bates et al., 2012). Milk samples were taken directly to thelaboratory and after mixing; sub-samples were analysed for totalprotein and total casein concentrations by mid-infrared spectros-copy (FOSS Warrington, UK) and the remainder stored at �20 �C.

2.2. Analysis of milk proteins by high resolution HPLC–MS

Frozen milk samples were thawed, hand-mixed and 1 ml mixedwith 1 ml of an 8 M urea buffer containing 20 mM dithiothreitol.The solution was thoroughly mixed and then left to stand forapproximately 1 h, followed by centrifugation for 5 min at 1200gand 4 �C. The upper fat layer was then removed and 0.5 ml, fromthe middle of the lower aqueous layer, was removed and diluted

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to 2 ml with distilled water. This final solution was then filtered,using a disposable syringe filter (pore size 0.45 lm) prior to injec-tion of a 5 ll aliquot into the HPLC system.

HPLC separation of the proteins involved a modification of themethod of Bonfatti, Grigoletto, Cecchinato, Gallo, and Carnier(2008). The method of Bonfatti et al. (2008) used UV detection at214 nm only, whereas, in our modified method, the concentrationof trifluoroacetic acid (TFA) added to the HPLC buffers was reducedto also enable more successful and specific detection by high reso-lution mass spectrometry (HRMS). HPLC separation of the milkproteins was achieved using an Agilent 1100 binary gradient HPLCsystem (Agilent Technologies UK Ltd., Wokingham, UK) with atemperature-controlled column oven and cooled automatic liquidsampler. A C18 reversed phase column (150 mm long � 2.1 mminternal diameter) with 30 nm pore size and 5 lm particle sizewas used with a guard column, using the same stationary phaseto protect the main analytical column. The column (and guard sys-tem) was supplied by Hichrom Ltd. (Theale, Berkshire, UK) andconsisted of an ACE 5 C18-300 column and used a very shallowmobile phase gradient. Mobile phase A, consisting of a solutionof 0.01% TFA in HPLC grade water (Rathburn Chemicals Ltd., Walk-erburn, UK) and mobile phase B, 0.01% TFA in far UV HPLC gradeacetonitrile (Rathburn Chemicals Ltd., Walkerburn, UK). A constantflow rate of 0.2 ml/min was used and the column was held at atemperature of 45 �C. The mobile phase gradient consisted of astart at 33% B for 5 min, 35% B over 4 min, 37% B over 9 min, 40%B over 5 min, 41% B over 5.5 min and held for 0.5 min. The gradientwas then made 43% B over 15 min, followed by equilibration backto 33% B over 7 min and held for 10 min prior to the next injection.This method allowed the separation of variants A1, A2, B and C ofb-casein sufficiently for identification and quantification purposes.The elution order was B, C, A1 and A2 over a time period of 23–27 min.

Detection of the b-casein variants was achieved, using both UVat 214 nm and HRMS in series with the UV detector. The massspectrometer used was a Bruker Microtof QII high resolution quad-rupole time of flight instrument (Bruker Instruments, Coventry,UK), which accepted the full flow of the column eluant via the stan-dard electrospray ionisation source. The HRMS was calibrated

Fig. 1. For a typical milk the, (a) mass spectrometry (MS) base peak and (b) ultrachromatograms of the (c) A2 and (d) A1 b-casein variant proteins.

using Agilent Tuning Mix (product number G2421A) over the massrange m/z 500–3500. Each protein that eluted from the columnproduced a unique spectral envelope of multiple charged clusters;peak abundance being centred around the 22+ charge state. Brukerdeconvolution software allowed the production of a singly chargedspectrum MH+ or Mr value, M being the monoisotopic molecularweight of the protein determined, using the UniProtKB online data-base (Data Analysis version 4.0 SP1, Build 253; Bruker DaltonikGmbH, Bremen), followed by modifying the sequence as appropri-ate, using Data Analysis & Sequence Editor (Bruker Daltonics BioTools version 3.1, Build 1.36: Bruker Daltonik GmbH, Bremen). Thisinformation confirmed the identity of the variant and relativequantitation was determined by integrating either UV absorptionor mass chromatograms produced from abundant multiplycharged clusters, e.g. 22+ charge state. Fig. 1 shows, for a typicalmilk, the MS base peak and UV chromatograms of the b-casein re-gion, together with extracted ion chromatograms of the A1 and A2b-caseins.

2.3. Statistical analysis

The effects of supermarket and month of year purchased weredetermined by analysis of variance using a general linear model(Mintab 16; Minitab Inc., State College, PA, USA). Tukey’s pairwisemultiple comparison test was used to identify which treatmentswere significantly different from each other when the significancewas P < 0.05.

3. Results

Table 1 shows the protein and total casein contents of the milk,together with the proportions of A1, A2, B and C b-caseins in thetotal b-casein proteins measured. Overall, casein represented about0.77 of total protein and, within the b-casein variant proteins mea-sured, A1, A2, B and C b-caseins represented 0.31, 0.58, 0.07 and0.03 of total b-casein. Month of sampling had a significant(P > 0.05) influence on all determinations except casein expressedas g/100 g of milk protein. Milk protein and total casein tended

violet (UV) chromatograms of the b-casein region together with extracted ion

Table 1Effect of month and supermarket on protein, casein and b-casein variant proteins in retail milk.

Milk proteins Month of sampling SEMA Effect of

Nov Dec Jan Feb Mar May Jun July Aug Sept Oct Month Supermarket

Protein (g/100 g milk) 3.35a 3.32ab 3.29abc 3.26abc 3.22bc 3.22bc 3.20c 3.20bc 3.26abc 3.22abc 3.31abc 0.026 *** **

Casein (g/100 g milk) 2.60a 2.58ab 2.55abc 2.53abc 2.50bc 2.49bc 2.48c 2.48c 2.50bc 2.50abc 2.57abc 0.019 *** **

Casein (g/100 g protein) 77.5 77.6 77.4 77.4 77.6 77.5 77.7 77.3 76.9 77.5 77.5 0.24 NSB NSA1 b-casein (g/100 g total b-

caseins)35.2a 33.5ab 34.5ab 26.5c 29.3bc 29.5abc 29.3bc 31.1abc 32.6abc 32.2abc 32.5abc 1.40 *** NS

A2 b-casein (g/100 g total b-caseins)

56.1 57.5 55.9 62.1 60.7 62.0 59.1 57.2 57.2 56.9 57.7 1.51 * NS

B b-casein (g/100 g total b-caseins)

6.43b 6.83ab 7.02ab 8.74a 7.40ab 6.24b 7.92ab 7.29ab 6.57b 7.35ab 6.20b 0.465 ** **

C b-casein (g/100 g total b-caseins)

2.20d 2.21d 2.49cd 2.69bcd 2.59bcd 2.23d 3.69ab 4.36a 3.66abc 3.56abcd 3.64abc 0.278 *** NS

a,b,c,d Values in the same row with different superscripts differ significantly (P < 0.05); Results are the least square means of eight determinations.A SEM, standard error of the mean.B NS, not significant P > 0.05.

* P < 0.05.** P < 0.01.*** P < 0.001.

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to be highest in autumn/early winter and lowest in summer. Thiswas also the case for A1 b-casein whilst A2 b-casein tended to behigher in late winter/spring. There were fewer significant effectsof supermarket and those observed for protein, casein and B b-casein were all due to one supermarket consistently having signif-icantly lower values than the others.

4. Discussion

The measured concentrations of protein were typical of UK milk(DairyCo, 2012b), although the proportion of it represented bycasein was somewhat lower than earlier reports (Fox, 2003). Toour knowledge, this is the first study to report the proportions ofthe various b-casein variant proteins in UK retail milk which isnow almost entirely produced by Holstein-Friesian cows. Earlystudies in the US indicated that A1 b-casein was the predominantb-casein variant in milk from Holstein-Friesian cows (0.56 of totalb-casein; Ng-Kwai-Hang, Hayes, Moxley, & Monardes, 1985) andBuchberger (1995) reported that the situation in northern Europewas similar (A1 0.46–0.71 of total b-casein). More recently a studywith milk from Holstein-Friesian cows in southwest Germanyshowed that A2 b-casein was the main b-casein variant (A2 0.67,A1 0.30 of total b-casein; Çardak, 2005) and this was also the casein a study in New Zealand (A2 0.55, A1 0.37; Venn, Skeaff, Brown,Mann, & Green, 2006). The protein phenotype seen in the currentstudy showed that, overall, the proportion of A2 b-casein was al-most twice that of A1 b-casein, with proportions similar to thosein the A1/A2 milk reported by Venn et al. (2006) and this con-firmed the low proportions of B and C b-caseins. Whether the high-er proportion of A2 b-casein in the current results than would havebeen predicted by earlier studies reflects changes in dairy cowbreeding in the UK and hence genotype in recent years is notknown.

Whilst significant effects of month were observed for all b-case-in variants, the magnitude of the effect was small and will not sub-stantially alter the mass of each protein consumed in a givenvolume of milk. The reasons for the effects of month are, however,not known. The recent combined data from years 1, 2 and 3 of therolling UK National Diet and Nutrition Survey (Bates, Prentice, &Swan, 2012) confirmed that the mean liquid milk intake of childrenaged 4–10 years was 214 and 185 ml/d for boys and girls, respec-tively, of which 52% was semi-skimmed for both genders. These in-take data include non-consumers and therefore intake byconsumers only will be somewhat higher. Assuming that about

0.36 of casein is b-casein (Fox, 2003), intakes of 200 to 300 ml/dof milk would represent a daily dose of 0.6–0.9 g of A1 b-caseinand 1.1–1.6 g of A2 b-casein. The implication if any, of such a doseof A1 b-casein on child health is not known, although Kost et al.(2009) have provided some recent data that support the hypothe-sis that reduced bCM-7 clearance is a risk factor for delay in psy-chomotor development and other diseases, such as autism.

Further definitive studies are clearly required to confirmwhether or not A1 b-casein and its associated bCM-7 peptide haveany role in neurological disorders such as autism. Such studies willinform whether or not there would be benefits to the UK from pro-ducing milk with different proportions of b-casein variants thanobserved at present.

5. Conclusions

This study has shown that b-casein in UK retail milk comprisesapproximately 0.58, 0.31, 0.07 and 0.03 of A2, A1, B and C proteinvariants, respectively. The proportion of A2 is higher than someearly studies would predict although the reasons for this and anyimplications for health remain unclear.

Acknowledgements

We are grateful to Dr. Kirsty Kliem for the collection and provi-sion of the milk samples. The research was supported by the Uni-versity of Reading.

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