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bovine milk mixtures
International Dairy Journal 21 (2011) 831e838
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The distributions of major whey proteins in acid wheys obtained from caprine/bovine and ovine/bovine milk mixtures Mirjana B. Pesic a, *, Miroljub B. Barac a, Miroslav M. Vrvic b, c, Nikola M. Ristic a, Ognjen D. Macej a, Sladjana P. Stanojevic a, Aleksandar Z. Kostic a a b c
Institute of Food Technology and Biochemistry, Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11081 Belgrade, Serbia Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia Institute of Chemistry, Technology and Metallurgy, Department of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 August 2010 Received in revised form 8 April 2011 Accepted 10 April 2011
The distributions of major whey proteins in acid wheys from different caprine/bovine and ovine/bovine milk mixtures were investigated using native-polyacrylamide gel electrophoresis (PAGE). Significantly different distributions of major whey proteins as individual proteins or as the sum of the same protein from different species were established. The caprine major whey proteins were dominant in mixtures with 10%, 20% and 30% bovine milk. The total b-lactoglobulin to a-lactalbumin (b-LGs/a-LAs) ratios ranged from 1.37 to 2.12 for caprine/bovine acid wheys. The corresponding ratios for ovine/bovine acid wheys were in the range 2.59e2.12. Linear relationships were estimated among the amounts of added bovine milk and the percentages of individual major whey proteins in all milk analysed, with square correlation coefficients from 0.990 to 0.997. These correlations enabled the use of native-PAGE as simple, reliable and low cost analytical method for determination the distributions of major whey proteins in caprine/bovine acid wheys. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Whey, mainly resulting from bovine cheese production, is normally transformed into valuable whey protein products such as concentrates and isolates, due to the high nutritional and functional properties of its component proteins (Pereira, Díaz, & Cobos, 2002). The major whey proteins, a-lactalbumin (a-LA) and b-lactoglobulin (b-LG) are strongly correlated with these properties. a-LA has good emulsifying and foaming properties, but shows poor gelation characteristics (Rodiles-López et al., 2008). However, native b-LG has excellent gelling and foaming properties (Foegeding, Davis, Doucet, & McGuffey, 2002). In addition, these proteins possess important biological properties such as immune system modulation, anticarcinogenic activity and various metabolic features (Madureira, Pereira, Gomes, Pintado, & Xavier Malcata, 2007). In Mediterranean countries, traditional cheeses are mainly manufactured from ovine and caprine milk or from a mixture of milk from different species (Hernández-Ledesma, Recio, Ramos, & Amigo, 2002). The milk of these species is almost totally processed into cheese, resulting in concomitant increases in the
* Corresponding author. Tel./fax: þ381 11 2199711. E-mail address: [email protected] (M.B. Pesic). 0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.04.002
volume of whey. In the past, part of this whey was used for animal feed or processed into whey cheeses but the rest was treated as a waste. Nowadays, the production of whey protein concentrate from ovineecaprine sources exist, but all cheese whey cannot be transformed and is still often discarded, especially caprine whey (Pandya & Ghodke, 2007). One of the reasons for less extended exploitation of small ruminants’ milk whey is the insufficient number of studies focused on functional and biological properties of its proteins, in contrast to numerous studies that have been carried out on bovine whey. It was reported that ovine and caprine wheys have a unique whey protein composition compared with that of bovine whey (Moatsou, Hatzinaki, Samolada, & Anifantakis, 2005). Ovine whey is characterised by higher protein content in comparison with caprine and bovine whey and also by a higher proportion of b-LG, whereas caprine whey had comparatively high levels of a-LA and higher total amounts of sum of b-LG and a-LA than bovine whey (Casper, Wendorff, & Thomas, 1998; Moatsou et al., 2005; Slacanac et al., 2010). Whey protein concentrate (WPC) from ovine sources demonstrated significantly better foaming and gelation properties than bovine WPC due to a higher b-LG content and lower ash content (Casper, Wendorff, & Thomas, 1999). The use of membrane technology in the production of ovine WPC and hydrolysis of ovine
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whey proteins were also reported as a means to achieve enhanced functional properties of ovine whey (Díaz, Pereira, & Cobos, 2004, 2006; Misún, Curda, & Jelen, 2008; Pereira et al., 2002). The information concerning functional properties of caprine whey is more limited (Casper et al., 1999; Pintado, da Silva, & Malcata, 1999). The study of Casper et al. (1999) revealed the superiority of caprine WPC in foaming and gelling properties compared with bovine WPC. These authors also observed significantly better emulsifying properties for caprine WPC at low pH, produced from rennet whey, than for bovine and ovine WPC, due to higher levels of a-LA. In addition, caprine and ovine milk proteins, including those from wheys, are also important sources of bioactive angiotensin converting enzyme inhibitory peptides and antihypertensive peptides. Some major reviews exist concerning their nutritional and therapeutic values (Park, Juárez, Ramos, & Haenlein, 2007; Sla canac et al., 2010). Insufficient information is available for wheys obtained from milk mixtures, despite the fact that cheeses made from the mixtures of milk from different species are produced in significant quantity. In Spain, these type of cheeses account for about 40% of the cheese production (Picon et al., 2010a, 2010b). In Greece, 72% of annually produced whey come from the Feta cheese production (Philippopoulos & Papadakis, 2001). The majority of literature data concerning the milk mixtures refer to cheese properties (Bonczar, Regu1a-Sardat, Pustkowiak, & Zebrowska, 2009; Boumba, Voutsinas, & Philippopoulos, 2001; Calvo & Balcones, 1998; Dmytrów, Mituniewicz-Ma1ek, & Dmytrów, 2010; Picon et al., 2010a, 2010b) and detection of milk adulterations (Ferreira & Caçote, 2003; Rodríguez, Ortiz, Sarabia, & Gredilla, 2010). To our knowledge, studies about distributions of major whey proteins in wheys obtained from milk mixtures are not available. Information on the relative proportions of these proteins is important for prediction of functional and nutritional attributes of whey products. For these reasons, a study that examines protein compositions of caprine/bovine and ovine/bovine wheys is necessary for evaluation of the potential for food-grade products from these sources of whey. A list of methods has been reported in the literature for the determination of whey protein fractions: (1) electrophoretic techniques such as isoelectric focussing (Mayer, 2005), capillary electrophoresis (Cartoni, Coccioli, Jasionowska, & Masci, 1999; Miralles, Ramos, & Amigo, 2005) and two dimensional techniques (Lindmark-Månsson, Timgren, Aldén, & Paulsson, 2005); (2) various high performance liquid chromatography methods (Ferreira & Caçote, 2003; Rodríguez et al., 2010); (3) immunological methods (Costa, Ravasco, Miranda, Duthoit, & Roseiro, 2008) and (4) mass spectrometry methods (Chen, Chang, Chung, Lee, & Ling, 2004; } ller et al., 2008). Kaminarides and Koukiassa (2002) reported Mu that most accurate and sensitive methods are based on immunological and electrophoretic techniques. Recently, Lin, Sun, Cao, Cao, and Jiang (2010) proposed a convenient and lower cost method, native-polyacrylamide gel electrophoresis (PAGE), as an useful analytical tool for simultaneous qualitative and quantitative analysis of bovine whey proteins in raw, heat-treated and commercial bovine milk. Also, we recently reported that this technique is suitable for the detection and quantification of adulteration in caprine/bovine and ovine/bovine milk mixtures (Pesic et al., 2011). However, little attention has been paid to the use of native-PAGE in determination of each whey protein in milk mixtures. The objective of this study was therefore to evaluate the distributions of major whey proteins in acid wheys obtained from different caprine/bovine and ovine/bovine milk mixtures using native-PAGE. Results of this study will help to identify trends and relative proportions of major whey proteins from each milk species in mixtures that may be unique and useful for the production of different whey-based food ingredients. Furthermore, estimation of
distributions of major whey proteins can be carried out using a convenient and lower cost method that needs no more training than other modern techniques. 2. Materials and methods 2.1. Chemicals and reagents Bovine whey protein standards: bovine serum albumin (BSA),
a-lactalbumin (ba-LA) and b-lactoglobulins (bb-LGs) in genetic variants A (bb-LGA) and B (bb-LGB) were obtained from Sigma (St. Louis, MO, USA). Ovine and caprine whey proteins standards were prepared in our laboratory (Pesic et al., 2011). All reagents used in this study were electrophoresis grade and dissolved in Ultrapure water (Ultra-pure water system, SG ver.1.11, Waters, USA, LLC). 2.2. Sample preparation Fresh milk from goats, cows and ewes was collected from local farms. Ovine milk was obtained from a single flock of Virtemberg ewes, caprine milk from a single flock of Saanen goats and bovine milk from a single herd of Holstein-Friesian cows. The ovine and caprine milk samples were collected during the complete morning milking of four animals of each flock. The bovine sample was obtained after complete morning milking of 82 animals. All samples were collected in July and August. After addition of 0.02% of sodium azide, milk samples were skimmed as has been recently described (Pesic et al., 2011) and were stored at 4 C until used. Protein compositions of pure milk were determined using official AOAC methods (AOAC, 1995) for total nitrogen and nonprotein nitrogen (NPN). Non-casein nitrogen (NCN) was determined as described for total nitrogen after removal of casein by precipitation at pH 4.6 for bovine milk, or pH 4.2 for caprine and ovine milk. The total protein (TP), casein (CN) and whey protein (WP) were calculated as described by Anema and Stanley (1998). Average protein compositions (g kg1) were as follows: TP 31.3, CN 25.2, WP 6.1 for bovine milk; TP 27.6, CN 21.0, WP 6.6 for caprine milk; TP 55.1, CN 42.8, WP 11.7 for ovine milk. Caprine/bovine and ovine/bovine milk mixtures were prepared by mixing 3%, 5%, 10%, 30%, 50%, 70% and 90% (v/v) of bovine milk with “non-bovine” milk. Acid wheys from all milk mixtures were obtained by isoelectric precipitation at pH 4.2 with 4 M HCl (Pesic et al., 2011). 2.3. Native-polyacrylamide gel electrophoresis Native-PAGE was performed according to the procedure previously described (Pesic et al., 2011) using electrophoresis unit LKB2001-100, with power supply LKB-Macrodrive 5 and cooling unit LKB-Multitemp (LKB, Sweden). Briefly, samples of acid wheys from caprine, bovine and caprine/bovine milk mixtures were diluted 1:2, whereas those from ovine and ovine/bovine milk mixtures were diluted 1:3 with the sample buffer. The separating gels were 7% (w/v) and stacking gels were 5% (w/v). Aliquots of 5 mL of the diluted samples were loaded on to the gels. The gels were run for 3 h to completion and were stained for 1 h followed by two destaining steps with continuous agitation. Gels were scanned and analysed using SigmaGel software version 1.1 (Jandal Scientific, San Rafael, CA, USA). 2.4. Statistical analysis All experiments were repeated three times. The experimental data were analysed using Statistica software ver 6.0 (StatSoft Co., Tulsa, OK). Means comparisons were conducted using t-test for independent samples. Regression analysis was also conducted.
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Fig. 1. Electrophoretic (A) and densitometric (B) patterns of pure caprine (a), bovine (j) and caprine/bovine acid wheys from milk mixtures with 3 (b), 5 (c), 10 (d), 20 (e), 30 (f), 50 (g), 70 (h) and 90% (i) of bovine milk. Peak identification: 1, BSA from caprine and bovine milk; 2, cb-LG; 3, ca-LA; 4, ba-LA; 5, bb-LGB; 6, bb-LGA.
3. Results and discussion Figs. 1 and 3 show electrophoretic and densitometric patterns of acid whey proteins from pure bovine, caprine and ovine
milk as well as from caprine/bovine and ovine/bovine milk mixtures. The upper part of the gels containing immunoglobulins was omitted from gels and densitograms. The identification of proteins was done by using the standards of bovine
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Table 1 Mean percentages of three independent determinations (standard deviation) of major whey proteins of acid wheys obtained from different caprine/bovine milk mixtures.a Acid wheys
ca-LA
cb-LG
cMWP
ba-LA
bb-LGB
0% Bovine 3% Bovine 5% Bovine 10% Bovine 20% Bovine 30% Bovine 50% Bovine 70% Bovine 90% Bovine 100% Bovine
33.5 0.16 32.4A 0.56 31.9A 0.10 28.3 0.37 25.3 0.59 21.6 0.31 16.4 0.10 10.6 0.11 5.5 0.24 e
45.9 0.17 44.5 0.35 43.8 0.11 38.4 0.50 34.2 0.77 29.6 0.50 22.5 0.10 14.5 0.41 7.3 0.24 e
79.4 0.33 76.9 0.91 75.7 0.10 66.7 0.87 59.5 1.35 51.2 0.81 38.9 0.20 25.1 0.53 12.8 0.49 e
e 3.6a 0.04 4.4a 0.20 6.6a 0.10 9.4a 0.23 12.0 0.30 16.1 0.10 20.1a 0.10 24.5a 0.06 28.6 0.10
e 3.4a 0.13 4.2a 0.18 6.7a 0.12 9.0a 0.26 11.0 0.32 15.6 0.09 20.1a 0.10 24.5a 0.23 27.9 0.10
bb-LGA e 4.1 0.10 5.1 0.23 7.6 0.20 11.1 0.32 13.5 0.43 19.0 0.19 22.9 0.10 27.9 0.22 32.7 0.36
bb-LGs
b-LGs/a-LAs
bMWP
e 7.5 0.13 9.3 0.42 14.3 0.10 20.1 0.58 24.5 0.75 34.6 0.10 43.0 0.11 52.4 0.45 60.6 0.35
e 11.1 0.16 13.7 0.61 20.9 0.10 29.5 0.81 36.5 0.44 50.7 0.11 63.1 0.10 76.9 0.51 89.2 0.30
1.37 0.01 1.44A 0.01 1.46A 0.01 1.51 0.01 1.56 0.02 1.61 0.04 1.76 0.01 1.88 0.02 1.99 0.02 2.12 0.02
a Abbreviations: ca-LA, caprine a-lactalbumin; cb-LG, caprine b-lactoglobulin; cMWP, the sum of caprine major whey proteins (ca-LA þ cb-LG); ba-LA, bovine a-lactalbumin; bb-LGB, bovine b-lactoglobulinB; bb-LGBA, bovine b-lactoglobulinA; bb-LGs, the sum of bovine b-lactoglobulinB and bovine b-lactoglobulinA; bMWP, the sum of bovine major whey proteins (ba-LA þ bb-LGs); b-LGs/a-Las, the ratio of total b-lactoglobulins (bovine þ caprine) and total a-lactalbumins (bovine þ caprine) present in milk mixtures. Means with the same superscript lower case letters in the same row and the same superscript upper case letters in the same column were not significantly different at p < 0.05. Means without letters in the same row or column were significantly different at p < 0.05.
whey proteins and whey proteins of caprine and ovine milk prepared in our laboratory. Also, detection was done by comparing electrophoregrams from our previous study (Pesic et al., 2011).
3.1. Caprine/bovine acid wheys The order of individual whey protein bands are based on overall protein charge and molecular size and are in Fig. 1 as follows: blood serum albumin (BSA) from caprine and bovine acid wheys, caprine b-lactoglobulin (cb-LG), caprine a-lactalbumin (ca-LA), ba-LA, bbLGB and bb-LGA. The relative proportions of major whey proteins in each acid whey analysed are shown in Table 1. Caprine whey proteins from pure caprine acid whey had a higher percentage of ca-LA compared with that obtained by Moatsou et al. (2005), but lower than as reported by Pintado and Malcata (1996). The latter study reported that the lactation period is a very important factor in distribution of major whey proteins in caprine whey. The ca-LA reached a maximum in May, then decreased until October, whereas cb-LG reached a minimum in April and July and increased significantly until June and October. In July, the ratio of cb-LG to ca-LA had the lowest value. Therefore, the higher value of ca-LA in this study was expected since the milk was collected in July. The percentage of cb-LG is similar to that reported by others (Moatsou et al., 2005; Pintado & Malcata, 1996).
The distribution of major bovine whey proteins in pure bovine acid whey completely corresponds to data reported by Lin et al. (2010) who also used native-PAGE for protein quantification. With increased addition of bovine milk in caprine milk, stepwise from 3 to 90%, frontal bands of ba-LA, bb-LGB and bb-LGA became increasingly apparent, whereas bands of ca-LA and cb-LG decreased proportionally (Table 1). There were great differences among the distributions of individual whey proteins among the analysed mixtures. Addition of 10% of bovine milk in caprine milk resulted in participation of bovine major whey proteins with about 20% of total detected whey proteins. The mixture with equal percentages of caprine and bovine milk showed dominant major whey proteins profile with dominant percentages of major bovine whey proteins (51% against 39%). The caprine major whey proteins were dominant in mixtures of 10%, 20% and 30% bovine milk. Functional properties and nutritional values of whey proteins mainly depend on a-LA and b-LGs, amounts and ratios, the distributions of the sum of caprine and bovine a-lactalbumins (a-LAs) and caprine and bovine b-lactoglobulins (b-LGs) among analysed mixtures are given in Fig. 2. The highest percentages of a-LAs were in the mixtures with 3e20% of bovine milk. Further addition of bovine milk into mixtures increased the percentages of b-LGs and decreased the percentages of a-LAs to 60.6% and 28.6%, respectively, which are the values for pure bovine major whey proteins. Also, the mixture with 30% of added bovine milk had the same percentage of a-LAs (mean value 33.6 0.21%) as percentage of ca-
Table 2 Linear regressions among percentages of major caprine and bovine acid whey proteins and volume percentages of bovine milk in acid wheys of caprine/bovine milk mixtures.a
Fig. 2. Distributions of the sum of b-lactoglobulins ( ) and a-lactalbumins (-) from caprine and bovine milk among the analysed caprine/bovine acid wheys. Within a parameter, bars without common letter differ (p < 0.05). Means were of triplicate determinations.
Whey protein
Regression equation
n
r
r2
p
ca-LA cb-LG ba-LA bb-LGB bb-LGA bb-LGs b-LGs a-LAs
y ¼ 32.57 0.318x y ¼ 44.53 0.435x y ¼ 3.80 þ 0.241x y ¼ 3.56 þ 0.239x y ¼ 4.47 þ 0.274x y ¼ 8.04 þ 0.514x y ¼ 50.88 þ 0.100x y ¼ 35.56 0.065x
10 10 9 9 9 9 10 10
0.996 0.996 0.996 0.998 0.996 0.997 0.890 0.946
0.993 0.992 0.993 0.995 0.991 0.994 0.792 0.895
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
a Abbreviations: n, number of points considered for the regression (each point represents the average of three replications); r, correlation coefficient; r2, square correlation coefficient; p, probability; ca-LA, caprine a-lactalbumin; cb-LG, caprine b-lactoglobulin; ba-LA, bovine a-lactalbumin; bb-LGB, bovine b-lactoglobulinB; bbLGBA, bovine b-lactoglobulinA; bb-LGs, the sum of bovine b-lactoglobulinB and bovine b-lactoglobulinA; b-LGs, total b-lactoglobulins (bovine þ caprine) present in milk mixtures; a-Las, total a-lactalbumins (bovine þ caprine) present in milk mixtures.
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Fig. 3. Electrophoretic (A) and densitometric (B) patterns of pure ovine (a), bovine (j) and ovine/bovine acid wheys from milk mixtures with 3 (b), 5 (c), 10 (d), 20 (e), 30 (f), 50 (g), 70 (h) and 90% (i) of bovine milk. Peak identification: 1, BSA from ovine and bovine milk; 2, oa-LA; 3, ob-LG þ ba-LA; 4, bb-LGB; 5, bb-LGA.
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LA in pure caprine acid wheys. The b-LGs/a-LAs ratios ranged from 1.37 to 2.12 (Table 1). The great variability in acid whey proteins profiles among the different caprine/bovine milk mixtures offer the potential for the manufacture of a wide range of whey-based food ingredients. For example, acid wheys with high proportions of a-LAs have the potential for enhanced emulsification at lower pH, whereas acid wheys with higher proportions of b-LGs could be utilised for whey products with enhanced foaming and gelation properties. From nutritional and biological standpoints, acid wheys enriched in a-LAs could be useful for infant formula application or production of whey ingredients with anti-cancer effects. The significance of bLGs as binding and carrier proteins is well known (Madureira et al., 2007), so acid wheys enriched in these proteins could be utilised for manufacture of food ingredients with enhanced carrier function. Although the caprine major whey proteins are closely homologous to bovine counterparts, some differences still exist. It was established that cb-LG differs in six and ca-LA in twelve amino acids from bovine bb-LGB and ba-LA which induce structural and electrophoretic differences (Jenness, 1980). Caprine major whey proteins are more heat-sensitive than bovine ones which could influence thermally-induced gelling properties (Pintado et al., 1999; Raynal-Ljutovac, Park, Gaucheron, & Boulhallab, 2007). Therefore, knowledge of the origin of detected individual major whey proteins as well as their quantity in bovine/caprine milk mixtures is very important in predicting applications of these acid wheys. Regression analysis of the data revealed a close correlation among percentages of added bovine milk in caprine milk and the relative proportions of individual major whey proteins (Table 2). High values of correlation coefficients indicate that native-PAGE could be good tool for determining the distributions of major whey proteins in caprine/bovine milk mixtures. Taking into consideration that the production of caprine milk and speciality cheeses is mainly concentrated in small plants, a low cost, accurate and simple method for rapid profiling of major whey proteins is particularly beneficial. It should be mentioned that protein profiles of major whey proteins in different milk mixtures could varies significantly due to the fact that caprine whey proteins are influenced by breed, stage of lactation, feeding climate, season and udder health status (Park et al., 2007), which further emphasises the necessity for a simple and reliable method. 3.2. Ovine/bovine acid wheys The order of individual whey proteins with increasing electrophoretic mobility in Fig. 3 was as follows: BSA from both ovine and
bovine acid wheys, ovine a-lactalbumin (oa-LA), ovine b-lactoglobulins (ob-LGs) and ba-LA, bb-LGB and bb-LGA. The separation of genetic variants of ob-LGs could not be achieved. Also, the difference among ob-LGs and ba-LA could not be distinguished. The lack of better separation was also observed in our recent study (Pesic et al., 2011) as well as by others (Amigo, Ramos, Calhau, & Barbosa, 1992; Mayer, 2005). Furthermore, it was observed that the detection of bovine major whey proteins in the mixture with 3% of bovine milk was not possible. The distributions of detected major acid whey proteins were presented in Table 3. The pure ovine acid whey had lower percentages of both major whey proteins than the pure bovine. These values agree closely with those reported by others (Pintado & Malcata, 1996; Pintado et al., 1999), but disagree with data reported by Moatsou et al. (2005) who found higher values for ob-LGs and smaller values for oa-LA. It is known that the distribution of major ovine whey proteins varies throughout the season, in early lactation the proportion of ob-LGs are lower whereas the proportion of oa-LA are higher than in middle lactation (Casper et al., 1998). The samples of ovine milk used in this study were collected in August, in early lactation, so the protein profile of major whey proteins in acid whey obtained was reasonable. Although the percentage of ob-LGs was lower than that of bbLGs, the ob-LGs/oa-LA ratio was higher than the same ratio for bovine acid whey and was 2.59 0.01. The percentages of ob-LGs and ba-LA were estimated as the sum of these proteins, but their individual proportions could be established from the initial ratio of bb-LGs to ba-LA in pure bovine milk. This ratio did not substantially change in the mixtures, so it was possible to calculate the percentages of bovine a-LA and hence, the percentages of oblactoglobulins in all mixtures (Table 3). Also, it is possible to make these calculations from the initial ratio of ob-lactoglobulins to oaLA in pure ovine milk. According to the data obtained, great differences in major whey proteins profiles among the analysed mixtures existed. The ratios of the sum of b-LGs to a-LAs were gradually changed from 2.59 to 2.12 with stepwise additions of bovine milk to ovine milk. The decrease of b-LGs/a-LAs ratios were due to an increase of a-LAs in the mixtures. This conclusion resulted from regression analysis of data showed in Table 3. Regression equations presented in Table 4 revealed close relationships among the amount of added bovine milk and percentages of major whey proteins, as was also shown in caprine/bovine milk mixtures. The slopes of regression lines obtained for ob-LGs and bb-LGs had the same values, which indicated that the sum of these proteins did not significantly change among analysed mixtures. On the other hand, the slopes of regression lines obtained for ba-LA and oa-LA revealed that the
Table 3 Mean percentages (standard deviation) of three independent determinations of major whey proteins of acid wheys obtained from different ovine/bovine milk mixtures.a Acid wheys 0% Bovine 5% Bovine 10% Bovine 20% Bovine 30% Bovine 50% Bovine 70% Bovine 90% Bovine 100% Bovine
oa-LA 21.8 0.10 20.4 0.13 19.6 0.19 18.4 0.19 16.0 0.10 11.5 0.27 8.4 0.10 4.2 0.05 e
ob-LGs þ ba-LA a
56.5 0.17 55.3A 0.28 55.6A 0.27 54.6 0.55 51.1 0.10 44.6 0.11 41.0 0.11 34.1 0.10 28.0a 0.12
ob-LGsb a
56.5 0.17 52.7 0.30 51.0 0.35 47.2 0.48 41.5 0.16 29.8 0.13 22.2a 0.17 10.7 0.10 e
ba-LAc
bb-LGB
bb-LGA
bb-LGs
b-LGs/a-LAs
e
e 2.3 0.02 4.2 0.13 7.2a 0.16 9.3a 0.10 14.3 0.10 17.7 0.10 22.5 0.10 27.5 0.19
e
e 5.5 0.04 9.8 0.15 15.7 0.15 20.4 0.51 31.4 0.10 39.9 0.12 49.7 0.10 59.3 0.10
2.59 0.01 2.53A 0.03 2.51A 0.01 2.44C 0.01 2.42C 0.01 2.33 0.02 2.28 0.02 2.19 0.01 2.12 0.01
2.6 0.02 4.6 0.10 7.4a 0.10 9.6a 0.24 14.8 0.10 18.8 0.10 23.4 0.10 28.0a 0.11
3.2 0.02 5.6 0.26 8.5 0.10 11.1 0.43 17.1 0.10 22.2a 0.21 27.2 0.10 31.8 0.16
a Abbreviations: oa-LA ¼ ovine a-lactalbumin, ob-LGs ¼ ovine b-lactoglobulins, ba-LA ¼ bovine a-lactalbumin, bb-LGB ¼ bovine b-lactoglobulinB, bb-LGBA ¼ bovine blactoglobulinA, bb-LGs ¼ the sum of bovine b-lactoglobulinB and bovine b-lactoglobulinA, b-LGs/a-LAs ¼ the ratio of total b-lactoglobulins (bovine þ ovine) and total alactalbumins (bovine þ ovine) present in milk mixtures. Means with the same superscript lower case letters in the same row and with the same superscript upper case letters in the same column were not significantly different at p < 0.05. Means without letters in the same row or column were significantly different at p < 0.05. b These values are calculated from the sum of ob-LGs þ ba-LA and data obtained for ba-LA. c These values are calculated from ratio of bb-LGs to ba-LA estimated in pure bovine milk. Values obtained for bb-LGs were divided with coefficient 2.12.
M.B. Pesic et al. / International Dairy Journal 21 (2011) 831e838 Table 4 Linear regressions among percentages of major ovine and bovine acid whey proteins and volume percentages of bovine milk in acid wheys of ovine/bovine milk mixtures.a Whey protein
Regression equation
n
r
r2
p
oa-LA ob-LGs þ ba-LA ob-LGs ba-LA bb-LGB bb-LGA bb-LGs
y ¼ 21.67 0.193x y ¼ 58.22 0.274x y ¼ 56.81 0.531x y ¼ 1.42 þ 0.257x y ¼ 1.62 þ 0.245x y ¼ 2.43 þ 0.286x y ¼ 4.09 þ 0.531x
8 9 9 9 8 8 8
0.998 0.986 0.995 0.996 0.995 0.998 0.997
0.997 0.972 0.991 0.992 0.990 0.997 0.994
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
a Abbreviations: n, number of points considered for the regression (each point represents the average of three replications); r, correlation coefficient; r2, square correlation coefficient; p, probability; oa-LA, ovine a-lactalbumin; ob-LGs, ovine blactoglobulins; ba-LA, bovine a-lactalbumin; bb-LGB, bovine b-lactoglobulinB; bbLGBA, bovine b-lactoglobulinA; bb-LGs, the sum of bovine b-lactoglobulinB and bovine b-lactoglobulinA.
percentages of ba-LA increased faster than the decrease of oa-LA percentages. So, the sum of oa-LA and ba-LA percentages in ovine/ bovine acid wheys had the higher values than in pure ovine acid whey. This resulted in lower values of b-LGs/a-LAs ratio in ovine/ bovine acid wheys than in pure ovine one. Thus, acid wheys from ovine/bovine milk mixtures were enriched in a-LAs. The significance of a-LAs was commented on above, so further research is needed to investigate possible applications of ovine/ bovine acid wheys as functional food ingredients. It is of note that ovine whey proteins, as caprine whey proteins, exhibit different degree of thermal denaturation, gelling properties and other functional properties compared with bovine counterparts, so knowledge of protein profiles in acid wheys obtained from ovine/ bovine milk mixtures deserve attention. 4. Conclusions Results of this study highlighted that caprine/bovine and ovine/ bovine acid wheys showed great differences in the relative proportions of the major whey proteins in both as individual whey proteins and as the sum of a-lactalbumins and b-lactoglobulins from different species. The values obtained indicated unique distributions of major whey proteins. The impacts of these differences on the technological behaviours and functional properties of the acid wheys from these milk mixtures need to be studied. Native-PAGE could be very useful as simple, reliable and low cost analytical method for determination the distributions of major whey proteins in caprine/bovine and ovine/bovine acid wheys, especially knowing that whey proteins composition could vary throughout the seasons. The need for better utilisation of wheys makes our research results interesting for further scientific work. Acknowledgements This investigation was supported by the grant of the Ministry of Science and Technological Development of Serbia (Project No. III46009). References Amigo, L., Ramos, M., Calhau, L., & Barbosa, M. (1992). Comparison of electrophoresis, isoelectric focusing, and immunodiffusion in determinations of cows’ and goats’ milk in Serra da Estrela cheeses. Lait, 72, 95e101. Anema, S. G., & Stanley, D. J. (1998). Heat-induced, pH-dependent behaviour of protein in caprine milk. International Dairy Journal, 8, 917e923. AOAC. (1995). Methods of analysis (16th ed.). Washington, DC: Assoc. Official Analytical Chemists.
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Bonczar, G., Regu1a-Sardat, A., Pustkowiak, H., & Zebrowska, A. (2009). Effect of mixing of ewe’s and cow’s milk on bundz cheese properties. Food Science Technology Quality, 16, 96e106. Boumba, V. A., Voutsinas, L. P., & Philippopoulos, C. D. (2001). Composition and nutritional value of commercial dried whey products from feta cheese manufacture. International Journal of Dairy Technology, 54, 141e145. Calvo, M. M., & Balcones, E. (1998). Influence of heat treatment on rennet clotting properties of mixtures of cow’s, ewe’s, and goat’s milk and on cheese yield. Journal of Agricultural and Food Chemistry, 46, 2957e2962. Cartoni, G., Coccioli, F., Jasionowska, R., & Masci, M. (1999). Determination of cows’ milk in goats’ milk and cheese by capillary electrophoresis of the whey protein fractions. Journal of Chromatography A, 846, 135e141. Casper, J. L., Wendorff, W. L., & Thomas, D. L. (1998). Seasonal changes in protein composition of whey from commercial manufacture of caprine and ovine specialty cheeses. Journal of Dairy Science, 81, 3117e3122. Casper, J. L., Wendorff, W. L., & Thomas, D. L. (1999). Functional properties of whey protein concentrates from caprine and ovine specialty cheese wheys. Journal of Dairy Science, 82, 265e271. Chen, R.-K., Chang, L.-W., Chung, Y.-Y., Lee, M.-H., & Ling, Y.-C. (2004). Quantification of cow milk adulteration in goat milk using high-performance liquid chromatography with electrospray ionization mass spectrometry. Rapid Communications in Mass Spectrometry, 18, 1167e1171. Costa, N., Ravasco, F., Miranda, R., Duthoit, M., & Roseiro, L. B. (2008). Evaluation of a commercial ELISA method for the quantitative detection of goat and cow milk in ewe milk and cheese. Small Ruminant Research, 79, 73e79. Díaz, O., Pereira, C. D., & Cobos, A. (2004). 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Hispánico cheese manufactured using frozen ovine milk curds scalded at different temperatures. International Dairy Journal, 20, 344e351. Picon, A., Gaya, P., Fernández-García, E., Rivas-Cañedo, A., Ávila, M., & Nuñez, M. (2010b). Proteolysis, lipolysis, volatile compounds, texture, and flavor of Hispánico cheese made using frozen ewe milk curds pressed for different times. Journal of Dairy Science, 93, 2896e2905. Pintado, M. E., & Malcata, F. X. (1996). Effect of thermal treatment on the protein profile of whey from ovine and caprine milk throughout lactation. International Dairy Journal, 6, 497e518. Pintado, M. E., da Silva, J. A. L., & Malcata, F. X. (1999). Comparative characterization of whey protein concentrates from ovine, caprine and bovine breeds. Food Science and Technology e Lebensmittel-Wissenschaft & Technologie, 32, 231e237.
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The distributions of major whey proteins in acid wheys obtained from caprine/bovine and ovine/bovine milk mixtures Mirjana B. Pesic a, *, Miroljub B. Barac a, Miroslav M. Vrvic b, c, Nikola M. Ristic a, Ognjen D. Macej a, Sladjana P. Stanojevic a, Aleksandar Z. Kostic a a b c
Institute of Food Technology and Biochemistry, Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11081 Belgrade, Serbia Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia Institute of Chemistry, Technology and Metallurgy, Department of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 August 2010 Received in revised form 8 April 2011 Accepted 10 April 2011
The distributions of major whey proteins in acid wheys from different caprine/bovine and ovine/bovine milk mixtures were investigated using native-polyacrylamide gel electrophoresis (PAGE). Significantly different distributions of major whey proteins as individual proteins or as the sum of the same protein from different species were established. The caprine major whey proteins were dominant in mixtures with 10%, 20% and 30% bovine milk. The total b-lactoglobulin to a-lactalbumin (b-LGs/a-LAs) ratios ranged from 1.37 to 2.12 for caprine/bovine acid wheys. The corresponding ratios for ovine/bovine acid wheys were in the range 2.59e2.12. Linear relationships were estimated among the amounts of added bovine milk and the percentages of individual major whey proteins in all milk analysed, with square correlation coefficients from 0.990 to 0.997. These correlations enabled the use of native-PAGE as simple, reliable and low cost analytical method for determination the distributions of major whey proteins in caprine/bovine acid wheys. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Whey, mainly resulting from bovine cheese production, is normally transformed into valuable whey protein products such as concentrates and isolates, due to the high nutritional and functional properties of its component proteins (Pereira, Díaz, & Cobos, 2002). The major whey proteins, a-lactalbumin (a-LA) and b-lactoglobulin (b-LG) are strongly correlated with these properties. a-LA has good emulsifying and foaming properties, but shows poor gelation characteristics (Rodiles-López et al., 2008). However, native b-LG has excellent gelling and foaming properties (Foegeding, Davis, Doucet, & McGuffey, 2002). In addition, these proteins possess important biological properties such as immune system modulation, anticarcinogenic activity and various metabolic features (Madureira, Pereira, Gomes, Pintado, & Xavier Malcata, 2007). In Mediterranean countries, traditional cheeses are mainly manufactured from ovine and caprine milk or from a mixture of milk from different species (Hernández-Ledesma, Recio, Ramos, & Amigo, 2002). The milk of these species is almost totally processed into cheese, resulting in concomitant increases in the
* Corresponding author. Tel./fax: þ381 11 2199711. E-mail address: [email protected] (M.B. Pesic). 0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.04.002
volume of whey. In the past, part of this whey was used for animal feed or processed into whey cheeses but the rest was treated as a waste. Nowadays, the production of whey protein concentrate from ovineecaprine sources exist, but all cheese whey cannot be transformed and is still often discarded, especially caprine whey (Pandya & Ghodke, 2007). One of the reasons for less extended exploitation of small ruminants’ milk whey is the insufficient number of studies focused on functional and biological properties of its proteins, in contrast to numerous studies that have been carried out on bovine whey. It was reported that ovine and caprine wheys have a unique whey protein composition compared with that of bovine whey (Moatsou, Hatzinaki, Samolada, & Anifantakis, 2005). Ovine whey is characterised by higher protein content in comparison with caprine and bovine whey and also by a higher proportion of b-LG, whereas caprine whey had comparatively high levels of a-LA and higher total amounts of sum of b-LG and a-LA than bovine whey (Casper, Wendorff, & Thomas, 1998; Moatsou et al., 2005; Slacanac et al., 2010). Whey protein concentrate (WPC) from ovine sources demonstrated significantly better foaming and gelation properties than bovine WPC due to a higher b-LG content and lower ash content (Casper, Wendorff, & Thomas, 1999). The use of membrane technology in the production of ovine WPC and hydrolysis of ovine
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whey proteins were also reported as a means to achieve enhanced functional properties of ovine whey (Díaz, Pereira, & Cobos, 2004, 2006; Misún, Curda, & Jelen, 2008; Pereira et al., 2002). The information concerning functional properties of caprine whey is more limited (Casper et al., 1999; Pintado, da Silva, & Malcata, 1999). The study of Casper et al. (1999) revealed the superiority of caprine WPC in foaming and gelling properties compared with bovine WPC. These authors also observed significantly better emulsifying properties for caprine WPC at low pH, produced from rennet whey, than for bovine and ovine WPC, due to higher levels of a-LA. In addition, caprine and ovine milk proteins, including those from wheys, are also important sources of bioactive angiotensin converting enzyme inhibitory peptides and antihypertensive peptides. Some major reviews exist concerning their nutritional and therapeutic values (Park, Juárez, Ramos, & Haenlein, 2007; Sla canac et al., 2010). Insufficient information is available for wheys obtained from milk mixtures, despite the fact that cheeses made from the mixtures of milk from different species are produced in significant quantity. In Spain, these type of cheeses account for about 40% of the cheese production (Picon et al., 2010a, 2010b). In Greece, 72% of annually produced whey come from the Feta cheese production (Philippopoulos & Papadakis, 2001). The majority of literature data concerning the milk mixtures refer to cheese properties (Bonczar, Regu1a-Sardat, Pustkowiak, & Zebrowska, 2009; Boumba, Voutsinas, & Philippopoulos, 2001; Calvo & Balcones, 1998; Dmytrów, Mituniewicz-Ma1ek, & Dmytrów, 2010; Picon et al., 2010a, 2010b) and detection of milk adulterations (Ferreira & Caçote, 2003; Rodríguez, Ortiz, Sarabia, & Gredilla, 2010). To our knowledge, studies about distributions of major whey proteins in wheys obtained from milk mixtures are not available. Information on the relative proportions of these proteins is important for prediction of functional and nutritional attributes of whey products. For these reasons, a study that examines protein compositions of caprine/bovine and ovine/bovine wheys is necessary for evaluation of the potential for food-grade products from these sources of whey. A list of methods has been reported in the literature for the determination of whey protein fractions: (1) electrophoretic techniques such as isoelectric focussing (Mayer, 2005), capillary electrophoresis (Cartoni, Coccioli, Jasionowska, & Masci, 1999; Miralles, Ramos, & Amigo, 2005) and two dimensional techniques (Lindmark-Månsson, Timgren, Aldén, & Paulsson, 2005); (2) various high performance liquid chromatography methods (Ferreira & Caçote, 2003; Rodríguez et al., 2010); (3) immunological methods (Costa, Ravasco, Miranda, Duthoit, & Roseiro, 2008) and (4) mass spectrometry methods (Chen, Chang, Chung, Lee, & Ling, 2004; } ller et al., 2008). Kaminarides and Koukiassa (2002) reported Mu that most accurate and sensitive methods are based on immunological and electrophoretic techniques. Recently, Lin, Sun, Cao, Cao, and Jiang (2010) proposed a convenient and lower cost method, native-polyacrylamide gel electrophoresis (PAGE), as an useful analytical tool for simultaneous qualitative and quantitative analysis of bovine whey proteins in raw, heat-treated and commercial bovine milk. Also, we recently reported that this technique is suitable for the detection and quantification of adulteration in caprine/bovine and ovine/bovine milk mixtures (Pesic et al., 2011). However, little attention has been paid to the use of native-PAGE in determination of each whey protein in milk mixtures. The objective of this study was therefore to evaluate the distributions of major whey proteins in acid wheys obtained from different caprine/bovine and ovine/bovine milk mixtures using native-PAGE. Results of this study will help to identify trends and relative proportions of major whey proteins from each milk species in mixtures that may be unique and useful for the production of different whey-based food ingredients. Furthermore, estimation of
distributions of major whey proteins can be carried out using a convenient and lower cost method that needs no more training than other modern techniques. 2. Materials and methods 2.1. Chemicals and reagents Bovine whey protein standards: bovine serum albumin (BSA),
a-lactalbumin (ba-LA) and b-lactoglobulins (bb-LGs) in genetic variants A (bb-LGA) and B (bb-LGB) were obtained from Sigma (St. Louis, MO, USA). Ovine and caprine whey proteins standards were prepared in our laboratory (Pesic et al., 2011). All reagents used in this study were electrophoresis grade and dissolved in Ultrapure water (Ultra-pure water system, SG ver.1.11, Waters, USA, LLC). 2.2. Sample preparation Fresh milk from goats, cows and ewes was collected from local farms. Ovine milk was obtained from a single flock of Virtemberg ewes, caprine milk from a single flock of Saanen goats and bovine milk from a single herd of Holstein-Friesian cows. The ovine and caprine milk samples were collected during the complete morning milking of four animals of each flock. The bovine sample was obtained after complete morning milking of 82 animals. All samples were collected in July and August. After addition of 0.02% of sodium azide, milk samples were skimmed as has been recently described (Pesic et al., 2011) and were stored at 4 C until used. Protein compositions of pure milk were determined using official AOAC methods (AOAC, 1995) for total nitrogen and nonprotein nitrogen (NPN). Non-casein nitrogen (NCN) was determined as described for total nitrogen after removal of casein by precipitation at pH 4.6 for bovine milk, or pH 4.2 for caprine and ovine milk. The total protein (TP), casein (CN) and whey protein (WP) were calculated as described by Anema and Stanley (1998). Average protein compositions (g kg1) were as follows: TP 31.3, CN 25.2, WP 6.1 for bovine milk; TP 27.6, CN 21.0, WP 6.6 for caprine milk; TP 55.1, CN 42.8, WP 11.7 for ovine milk. Caprine/bovine and ovine/bovine milk mixtures were prepared by mixing 3%, 5%, 10%, 30%, 50%, 70% and 90% (v/v) of bovine milk with “non-bovine” milk. Acid wheys from all milk mixtures were obtained by isoelectric precipitation at pH 4.2 with 4 M HCl (Pesic et al., 2011). 2.3. Native-polyacrylamide gel electrophoresis Native-PAGE was performed according to the procedure previously described (Pesic et al., 2011) using electrophoresis unit LKB2001-100, with power supply LKB-Macrodrive 5 and cooling unit LKB-Multitemp (LKB, Sweden). Briefly, samples of acid wheys from caprine, bovine and caprine/bovine milk mixtures were diluted 1:2, whereas those from ovine and ovine/bovine milk mixtures were diluted 1:3 with the sample buffer. The separating gels were 7% (w/v) and stacking gels were 5% (w/v). Aliquots of 5 mL of the diluted samples were loaded on to the gels. The gels were run for 3 h to completion and were stained for 1 h followed by two destaining steps with continuous agitation. Gels were scanned and analysed using SigmaGel software version 1.1 (Jandal Scientific, San Rafael, CA, USA). 2.4. Statistical analysis All experiments were repeated three times. The experimental data were analysed using Statistica software ver 6.0 (StatSoft Co., Tulsa, OK). Means comparisons were conducted using t-test for independent samples. Regression analysis was also conducted.
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Fig. 1. Electrophoretic (A) and densitometric (B) patterns of pure caprine (a), bovine (j) and caprine/bovine acid wheys from milk mixtures with 3 (b), 5 (c), 10 (d), 20 (e), 30 (f), 50 (g), 70 (h) and 90% (i) of bovine milk. Peak identification: 1, BSA from caprine and bovine milk; 2, cb-LG; 3, ca-LA; 4, ba-LA; 5, bb-LGB; 6, bb-LGA.
3. Results and discussion Figs. 1 and 3 show electrophoretic and densitometric patterns of acid whey proteins from pure bovine, caprine and ovine
milk as well as from caprine/bovine and ovine/bovine milk mixtures. The upper part of the gels containing immunoglobulins was omitted from gels and densitograms. The identification of proteins was done by using the standards of bovine
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Table 1 Mean percentages of three independent determinations (standard deviation) of major whey proteins of acid wheys obtained from different caprine/bovine milk mixtures.a Acid wheys
ca-LA
cb-LG
cMWP
ba-LA
bb-LGB
0% Bovine 3% Bovine 5% Bovine 10% Bovine 20% Bovine 30% Bovine 50% Bovine 70% Bovine 90% Bovine 100% Bovine
33.5 0.16 32.4A 0.56 31.9A 0.10 28.3 0.37 25.3 0.59 21.6 0.31 16.4 0.10 10.6 0.11 5.5 0.24 e
45.9 0.17 44.5 0.35 43.8 0.11 38.4 0.50 34.2 0.77 29.6 0.50 22.5 0.10 14.5 0.41 7.3 0.24 e
79.4 0.33 76.9 0.91 75.7 0.10 66.7 0.87 59.5 1.35 51.2 0.81 38.9 0.20 25.1 0.53 12.8 0.49 e
e 3.6a 0.04 4.4a 0.20 6.6a 0.10 9.4a 0.23 12.0 0.30 16.1 0.10 20.1a 0.10 24.5a 0.06 28.6 0.10
e 3.4a 0.13 4.2a 0.18 6.7a 0.12 9.0a 0.26 11.0 0.32 15.6 0.09 20.1a 0.10 24.5a 0.23 27.9 0.10
bb-LGA e 4.1 0.10 5.1 0.23 7.6 0.20 11.1 0.32 13.5 0.43 19.0 0.19 22.9 0.10 27.9 0.22 32.7 0.36
bb-LGs
b-LGs/a-LAs
bMWP
e 7.5 0.13 9.3 0.42 14.3 0.10 20.1 0.58 24.5 0.75 34.6 0.10 43.0 0.11 52.4 0.45 60.6 0.35
e 11.1 0.16 13.7 0.61 20.9 0.10 29.5 0.81 36.5 0.44 50.7 0.11 63.1 0.10 76.9 0.51 89.2 0.30
1.37 0.01 1.44A 0.01 1.46A 0.01 1.51 0.01 1.56 0.02 1.61 0.04 1.76 0.01 1.88 0.02 1.99 0.02 2.12 0.02
a Abbreviations: ca-LA, caprine a-lactalbumin; cb-LG, caprine b-lactoglobulin; cMWP, the sum of caprine major whey proteins (ca-LA þ cb-LG); ba-LA, bovine a-lactalbumin; bb-LGB, bovine b-lactoglobulinB; bb-LGBA, bovine b-lactoglobulinA; bb-LGs, the sum of bovine b-lactoglobulinB and bovine b-lactoglobulinA; bMWP, the sum of bovine major whey proteins (ba-LA þ bb-LGs); b-LGs/a-Las, the ratio of total b-lactoglobulins (bovine þ caprine) and total a-lactalbumins (bovine þ caprine) present in milk mixtures. Means with the same superscript lower case letters in the same row and the same superscript upper case letters in the same column were not significantly different at p < 0.05. Means without letters in the same row or column were significantly different at p < 0.05.
whey proteins and whey proteins of caprine and ovine milk prepared in our laboratory. Also, detection was done by comparing electrophoregrams from our previous study (Pesic et al., 2011).
3.1. Caprine/bovine acid wheys The order of individual whey protein bands are based on overall protein charge and molecular size and are in Fig. 1 as follows: blood serum albumin (BSA) from caprine and bovine acid wheys, caprine b-lactoglobulin (cb-LG), caprine a-lactalbumin (ca-LA), ba-LA, bbLGB and bb-LGA. The relative proportions of major whey proteins in each acid whey analysed are shown in Table 1. Caprine whey proteins from pure caprine acid whey had a higher percentage of ca-LA compared with that obtained by Moatsou et al. (2005), but lower than as reported by Pintado and Malcata (1996). The latter study reported that the lactation period is a very important factor in distribution of major whey proteins in caprine whey. The ca-LA reached a maximum in May, then decreased until October, whereas cb-LG reached a minimum in April and July and increased significantly until June and October. In July, the ratio of cb-LG to ca-LA had the lowest value. Therefore, the higher value of ca-LA in this study was expected since the milk was collected in July. The percentage of cb-LG is similar to that reported by others (Moatsou et al., 2005; Pintado & Malcata, 1996).
The distribution of major bovine whey proteins in pure bovine acid whey completely corresponds to data reported by Lin et al. (2010) who also used native-PAGE for protein quantification. With increased addition of bovine milk in caprine milk, stepwise from 3 to 90%, frontal bands of ba-LA, bb-LGB and bb-LGA became increasingly apparent, whereas bands of ca-LA and cb-LG decreased proportionally (Table 1). There were great differences among the distributions of individual whey proteins among the analysed mixtures. Addition of 10% of bovine milk in caprine milk resulted in participation of bovine major whey proteins with about 20% of total detected whey proteins. The mixture with equal percentages of caprine and bovine milk showed dominant major whey proteins profile with dominant percentages of major bovine whey proteins (51% against 39%). The caprine major whey proteins were dominant in mixtures of 10%, 20% and 30% bovine milk. Functional properties and nutritional values of whey proteins mainly depend on a-LA and b-LGs, amounts and ratios, the distributions of the sum of caprine and bovine a-lactalbumins (a-LAs) and caprine and bovine b-lactoglobulins (b-LGs) among analysed mixtures are given in Fig. 2. The highest percentages of a-LAs were in the mixtures with 3e20% of bovine milk. Further addition of bovine milk into mixtures increased the percentages of b-LGs and decreased the percentages of a-LAs to 60.6% and 28.6%, respectively, which are the values for pure bovine major whey proteins. Also, the mixture with 30% of added bovine milk had the same percentage of a-LAs (mean value 33.6 0.21%) as percentage of ca-
Table 2 Linear regressions among percentages of major caprine and bovine acid whey proteins and volume percentages of bovine milk in acid wheys of caprine/bovine milk mixtures.a
Fig. 2. Distributions of the sum of b-lactoglobulins ( ) and a-lactalbumins (-) from caprine and bovine milk among the analysed caprine/bovine acid wheys. Within a parameter, bars without common letter differ (p < 0.05). Means were of triplicate determinations.
Whey protein
Regression equation
n
r
r2
p
ca-LA cb-LG ba-LA bb-LGB bb-LGA bb-LGs b-LGs a-LAs
y ¼ 32.57 0.318x y ¼ 44.53 0.435x y ¼ 3.80 þ 0.241x y ¼ 3.56 þ 0.239x y ¼ 4.47 þ 0.274x y ¼ 8.04 þ 0.514x y ¼ 50.88 þ 0.100x y ¼ 35.56 0.065x
10 10 9 9 9 9 10 10
0.996 0.996 0.996 0.998 0.996 0.997 0.890 0.946
0.993 0.992 0.993 0.995 0.991 0.994 0.792 0.895
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
a Abbreviations: n, number of points considered for the regression (each point represents the average of three replications); r, correlation coefficient; r2, square correlation coefficient; p, probability; ca-LA, caprine a-lactalbumin; cb-LG, caprine b-lactoglobulin; ba-LA, bovine a-lactalbumin; bb-LGB, bovine b-lactoglobulinB; bbLGBA, bovine b-lactoglobulinA; bb-LGs, the sum of bovine b-lactoglobulinB and bovine b-lactoglobulinA; b-LGs, total b-lactoglobulins (bovine þ caprine) present in milk mixtures; a-Las, total a-lactalbumins (bovine þ caprine) present in milk mixtures.
M.B. Pesic et al. / International Dairy Journal 21 (2011) 831e838
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Fig. 3. Electrophoretic (A) and densitometric (B) patterns of pure ovine (a), bovine (j) and ovine/bovine acid wheys from milk mixtures with 3 (b), 5 (c), 10 (d), 20 (e), 30 (f), 50 (g), 70 (h) and 90% (i) of bovine milk. Peak identification: 1, BSA from ovine and bovine milk; 2, oa-LA; 3, ob-LG þ ba-LA; 4, bb-LGB; 5, bb-LGA.
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M.B. Pesic et al. / International Dairy Journal 21 (2011) 831e838
LA in pure caprine acid wheys. The b-LGs/a-LAs ratios ranged from 1.37 to 2.12 (Table 1). The great variability in acid whey proteins profiles among the different caprine/bovine milk mixtures offer the potential for the manufacture of a wide range of whey-based food ingredients. For example, acid wheys with high proportions of a-LAs have the potential for enhanced emulsification at lower pH, whereas acid wheys with higher proportions of b-LGs could be utilised for whey products with enhanced foaming and gelation properties. From nutritional and biological standpoints, acid wheys enriched in a-LAs could be useful for infant formula application or production of whey ingredients with anti-cancer effects. The significance of bLGs as binding and carrier proteins is well known (Madureira et al., 2007), so acid wheys enriched in these proteins could be utilised for manufacture of food ingredients with enhanced carrier function. Although the caprine major whey proteins are closely homologous to bovine counterparts, some differences still exist. It was established that cb-LG differs in six and ca-LA in twelve amino acids from bovine bb-LGB and ba-LA which induce structural and electrophoretic differences (Jenness, 1980). Caprine major whey proteins are more heat-sensitive than bovine ones which could influence thermally-induced gelling properties (Pintado et al., 1999; Raynal-Ljutovac, Park, Gaucheron, & Boulhallab, 2007). Therefore, knowledge of the origin of detected individual major whey proteins as well as their quantity in bovine/caprine milk mixtures is very important in predicting applications of these acid wheys. Regression analysis of the data revealed a close correlation among percentages of added bovine milk in caprine milk and the relative proportions of individual major whey proteins (Table 2). High values of correlation coefficients indicate that native-PAGE could be good tool for determining the distributions of major whey proteins in caprine/bovine milk mixtures. Taking into consideration that the production of caprine milk and speciality cheeses is mainly concentrated in small plants, a low cost, accurate and simple method for rapid profiling of major whey proteins is particularly beneficial. It should be mentioned that protein profiles of major whey proteins in different milk mixtures could varies significantly due to the fact that caprine whey proteins are influenced by breed, stage of lactation, feeding climate, season and udder health status (Park et al., 2007), which further emphasises the necessity for a simple and reliable method. 3.2. Ovine/bovine acid wheys The order of individual whey proteins with increasing electrophoretic mobility in Fig. 3 was as follows: BSA from both ovine and
bovine acid wheys, ovine a-lactalbumin (oa-LA), ovine b-lactoglobulins (ob-LGs) and ba-LA, bb-LGB and bb-LGA. The separation of genetic variants of ob-LGs could not be achieved. Also, the difference among ob-LGs and ba-LA could not be distinguished. The lack of better separation was also observed in our recent study (Pesic et al., 2011) as well as by others (Amigo, Ramos, Calhau, & Barbosa, 1992; Mayer, 2005). Furthermore, it was observed that the detection of bovine major whey proteins in the mixture with 3% of bovine milk was not possible. The distributions of detected major acid whey proteins were presented in Table 3. The pure ovine acid whey had lower percentages of both major whey proteins than the pure bovine. These values agree closely with those reported by others (Pintado & Malcata, 1996; Pintado et al., 1999), but disagree with data reported by Moatsou et al. (2005) who found higher values for ob-LGs and smaller values for oa-LA. It is known that the distribution of major ovine whey proteins varies throughout the season, in early lactation the proportion of ob-LGs are lower whereas the proportion of oa-LA are higher than in middle lactation (Casper et al., 1998). The samples of ovine milk used in this study were collected in August, in early lactation, so the protein profile of major whey proteins in acid whey obtained was reasonable. Although the percentage of ob-LGs was lower than that of bbLGs, the ob-LGs/oa-LA ratio was higher than the same ratio for bovine acid whey and was 2.59 0.01. The percentages of ob-LGs and ba-LA were estimated as the sum of these proteins, but their individual proportions could be established from the initial ratio of bb-LGs to ba-LA in pure bovine milk. This ratio did not substantially change in the mixtures, so it was possible to calculate the percentages of bovine a-LA and hence, the percentages of oblactoglobulins in all mixtures (Table 3). Also, it is possible to make these calculations from the initial ratio of ob-lactoglobulins to oaLA in pure ovine milk. According to the data obtained, great differences in major whey proteins profiles among the analysed mixtures existed. The ratios of the sum of b-LGs to a-LAs were gradually changed from 2.59 to 2.12 with stepwise additions of bovine milk to ovine milk. The decrease of b-LGs/a-LAs ratios were due to an increase of a-LAs in the mixtures. This conclusion resulted from regression analysis of data showed in Table 3. Regression equations presented in Table 4 revealed close relationships among the amount of added bovine milk and percentages of major whey proteins, as was also shown in caprine/bovine milk mixtures. The slopes of regression lines obtained for ob-LGs and bb-LGs had the same values, which indicated that the sum of these proteins did not significantly change among analysed mixtures. On the other hand, the slopes of regression lines obtained for ba-LA and oa-LA revealed that the
Table 3 Mean percentages (standard deviation) of three independent determinations of major whey proteins of acid wheys obtained from different ovine/bovine milk mixtures.a Acid wheys 0% Bovine 5% Bovine 10% Bovine 20% Bovine 30% Bovine 50% Bovine 70% Bovine 90% Bovine 100% Bovine
oa-LA 21.8 0.10 20.4 0.13 19.6 0.19 18.4 0.19 16.0 0.10 11.5 0.27 8.4 0.10 4.2 0.05 e
ob-LGs þ ba-LA a
56.5 0.17 55.3A 0.28 55.6A 0.27 54.6 0.55 51.1 0.10 44.6 0.11 41.0 0.11 34.1 0.10 28.0a 0.12
ob-LGsb a
56.5 0.17 52.7 0.30 51.0 0.35 47.2 0.48 41.5 0.16 29.8 0.13 22.2a 0.17 10.7 0.10 e
ba-LAc
bb-LGB
bb-LGA
bb-LGs
b-LGs/a-LAs
e
e 2.3 0.02 4.2 0.13 7.2a 0.16 9.3a 0.10 14.3 0.10 17.7 0.10 22.5 0.10 27.5 0.19
e
e 5.5 0.04 9.8 0.15 15.7 0.15 20.4 0.51 31.4 0.10 39.9 0.12 49.7 0.10 59.3 0.10
2.59 0.01 2.53A 0.03 2.51A 0.01 2.44C 0.01 2.42C 0.01 2.33 0.02 2.28 0.02 2.19 0.01 2.12 0.01
2.6 0.02 4.6 0.10 7.4a 0.10 9.6a 0.24 14.8 0.10 18.8 0.10 23.4 0.10 28.0a 0.11
3.2 0.02 5.6 0.26 8.5 0.10 11.1 0.43 17.1 0.10 22.2a 0.21 27.2 0.10 31.8 0.16
a Abbreviations: oa-LA ¼ ovine a-lactalbumin, ob-LGs ¼ ovine b-lactoglobulins, ba-LA ¼ bovine a-lactalbumin, bb-LGB ¼ bovine b-lactoglobulinB, bb-LGBA ¼ bovine blactoglobulinA, bb-LGs ¼ the sum of bovine b-lactoglobulinB and bovine b-lactoglobulinA, b-LGs/a-LAs ¼ the ratio of total b-lactoglobulins (bovine þ ovine) and total alactalbumins (bovine þ ovine) present in milk mixtures. Means with the same superscript lower case letters in the same row and with the same superscript upper case letters in the same column were not significantly different at p < 0.05. Means without letters in the same row or column were significantly different at p < 0.05. b These values are calculated from the sum of ob-LGs þ ba-LA and data obtained for ba-LA. c These values are calculated from ratio of bb-LGs to ba-LA estimated in pure bovine milk. Values obtained for bb-LGs were divided with coefficient 2.12.
M.B. Pesic et al. / International Dairy Journal 21 (2011) 831e838 Table 4 Linear regressions among percentages of major ovine and bovine acid whey proteins and volume percentages of bovine milk in acid wheys of ovine/bovine milk mixtures.a Whey protein
Regression equation
n
r
r2
p
oa-LA ob-LGs þ ba-LA ob-LGs ba-LA bb-LGB bb-LGA bb-LGs
y ¼ 21.67 0.193x y ¼ 58.22 0.274x y ¼ 56.81 0.531x y ¼ 1.42 þ 0.257x y ¼ 1.62 þ 0.245x y ¼ 2.43 þ 0.286x y ¼ 4.09 þ 0.531x
8 9 9 9 8 8 8
0.998 0.986 0.995 0.996 0.995 0.998 0.997
0.997 0.972 0.991 0.992 0.990 0.997 0.994
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
a Abbreviations: n, number of points considered for the regression (each point represents the average of three replications); r, correlation coefficient; r2, square correlation coefficient; p, probability; oa-LA, ovine a-lactalbumin; ob-LGs, ovine blactoglobulins; ba-LA, bovine a-lactalbumin; bb-LGB, bovine b-lactoglobulinB; bbLGBA, bovine b-lactoglobulinA; bb-LGs, the sum of bovine b-lactoglobulinB and bovine b-lactoglobulinA.
percentages of ba-LA increased faster than the decrease of oa-LA percentages. So, the sum of oa-LA and ba-LA percentages in ovine/ bovine acid wheys had the higher values than in pure ovine acid whey. This resulted in lower values of b-LGs/a-LAs ratio in ovine/ bovine acid wheys than in pure ovine one. Thus, acid wheys from ovine/bovine milk mixtures were enriched in a-LAs. The significance of a-LAs was commented on above, so further research is needed to investigate possible applications of ovine/ bovine acid wheys as functional food ingredients. It is of note that ovine whey proteins, as caprine whey proteins, exhibit different degree of thermal denaturation, gelling properties and other functional properties compared with bovine counterparts, so knowledge of protein profiles in acid wheys obtained from ovine/ bovine milk mixtures deserve attention. 4. Conclusions Results of this study highlighted that caprine/bovine and ovine/ bovine acid wheys showed great differences in the relative proportions of the major whey proteins in both as individual whey proteins and as the sum of a-lactalbumins and b-lactoglobulins from different species. The values obtained indicated unique distributions of major whey proteins. The impacts of these differences on the technological behaviours and functional properties of the acid wheys from these milk mixtures need to be studied. Native-PAGE could be very useful as simple, reliable and low cost analytical method for determination the distributions of major whey proteins in caprine/bovine and ovine/bovine acid wheys, especially knowing that whey proteins composition could vary throughout the seasons. The need for better utilisation of wheys makes our research results interesting for further scientific work. Acknowledgements This investigation was supported by the grant of the Ministry of Science and Technological Development of Serbia (Project No. III46009). References Amigo, L., Ramos, M., Calhau, L., & Barbosa, M. (1992). Comparison of electrophoresis, isoelectric focusing, and immunodiffusion in determinations of cows’ and goats’ milk in Serra da Estrela cheeses. Lait, 72, 95e101. Anema, S. G., & Stanley, D. J. (1998). Heat-induced, pH-dependent behaviour of protein in caprine milk. International Dairy Journal, 8, 917e923. AOAC. (1995). Methods of analysis (16th ed.). Washington, DC: Assoc. Official Analytical Chemists.
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