Chromatographic elution profiles, electrophoretic properties and free amino and sulphydryl group contents of commercial sodium caseinates

PII:

ELSEVIER

Inr. Dairy Journal 7 ( 1997) 2 13-220 C:I 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 09%6946/97/$17.00 + 0.00

SO958-6946(97)00010-l

Chromatographic Elution P ‘rofil.es, Electrophoretic Properties and Frlee Amino and Sulphydryl Group Contents of Commercial Sodium Caseinates A. G. Lynchab, D. M. Mulvihill”, A. J. R. Law b, J. Leaverb and D. S. Horneb “Department

of Food Chemistry, University College Cork, Cork, Republic of Ireland hHannah Research Institute, Ayr, KA6 SHL, UK

(Received 20 August 1996; accepted 12 February

1997)

ABSTRACT

The proteins in commercial sodium caseinates and laboratory-prepared, unheated sodium caseinate were studied using anion and cation exchange FPLC, gel permeation FPLC and alkaline urea-PAGE, and free amino and sulphydryl groups were analysed. Anion and cation exchange FPLC profiles showed that the charged residues of constituent proteins in the caseinates were modified to different extents. Commercial caseinates showed an extra peak (pre-cc,,-casein) on cation exchange FPLC, which eluted at a lower salt concentration than that required to elute cc,,-casein; there was little pre-cc,,-casein in the laboratory-prepared caseinate. Gel permeation FPLC showed that the caseinates contained different levels of high molecular weight proteins which were present at very low levels in the laboratory-prepared caseinate. Alkaline urea-PAGE gave good resolution of all proteins in the laboratoryprepared caseinate while in the commercial caseinate samples, protein bands were smeared and a,z-casein was less pronounced. The laboratorv-oreoared caseinate had a higher content of free amino and sulphydryl groups than the commercial caseinates. 0 1997 Elsevier &i&k Ltd

INTRODUCTION

thiol and thiol-thiol reactions and interactions (Walstra and Jenness, 1984; Burton, 1984). Dephosphorylation, covalent bond formation, loss of lysine and fragmentation of the caseins occurred on heating sodium caseinate solutions (2%, w/v, pH 7.0) at 140°C for 60min (Guo et al., 1989). Law et al. (1994) found that caseins in normal milk, pH 6.7, were stable on heating to 110°C for up to 5min, but at or above this temperature the caseins were modified to different extents in comparison with caseins from unheated milk. Differences in functional attributes between caseinates indicate of the that some aspects manufacturing process are poorly controlled. The aim of this study was to assess the chromatographic elution profiles, electrophoretic properties and free amino and sulphydryl contents of a number of commercial sodium caseinates and to compare them with those of a sodium caseinate prepared in the laboratory from unheated milk.

Sodium caseinate is a widely used food ingredient because of its good functional and sensory properties (Fox and Mulvihill, 1983; Mulvihill, 1989, 1992). In cream liqueurs, sodium caseinate stabilizes the fat globules formed during homogenization and also confers a smooth creamy texture on the liqueur (Banks et al., 1983). Differences in the chemical composition and protein complement have been found between batches of sodium caseinate from the same and different manufacturers (Muir and Dalgleish, 1987; Dalgleish and Law, 1988). These differences could adversely affect the characteristic behaviour of products in which the sodium caseinates are used. Some sodium caseinates increased the viscosity of cream liqueurs stored at 45°C to unacceptable levels (Lynch, 1995), even in the presence of a calcium chelating agent. During the conversion of acid casein to sodium caseinate, the protein may be exposed to high temperatures at alkaline pH values. These conditions are known to cause hydrolysis of phosphoseryl residues (Belec and Jenness, 1962a, b; Manson, 1973; Creamer 1977) and and Matheson, the formation of dehydroalanine and lysinoalanine (Annan and Manson, 1981; Creamer and Matheson, 1977). Caseins are random-coil proteins with little tertiary structure (Swaisgood, 1992) and their side chains are readily available for reaction. Other reactions which can occur in caseins at high temperatures and alkaline pH values are hydrolysis of peptide bonds, deamination, cleavage of N-acetylneuraminic acid from rc-casein and various

MATERIALS Caseinates

AND METHODS

and acid casein

Nine commercial sodium caseinates, numbered 1-9, were used in this study. Unheated acid casein was manufactured from bulk milk from the Hannah Research Institute herd (HRI, Ayr KA6 5HL, Scotland, UK) using the method of Hollar et al. (1991). The casein was lyophilized using an Edwards Super Modulyo freeze-drier (Crawley, West Sussex, UK) and stored at -20°C. 213

214

FAST PROTEIN (FPLC)

A. G. Lynch et al. LIQUID

CHROMATOGRAPHY

Anion exchange FPLC

Anion exchange FPLC was carried out on a Mono Q HR5/5 column (Pharmacia AB, Uppsala, Sweden) using the method of Davies and Law (1987) with the following modification: protein (20 mg) was dissolved in 5 mL of a 6 M urea/5 mM bis-tris-propane/7 mM HCl buffer, pH 7.0. The sulphydryl groups were reduced and alkylated by addition of 1OOpL of a cysteamine hydrochloride solution (32mgmL-‘) and 1OOpL of a cystamine dihydrochloride solution (318 mg mL_‘). After 1.5 h at room temperature, the alkylated protein solution (5 mL) was transferred to a dialysis sac (1 cm diameter) and dialysed against 3x 150mL volumes of 3.3 M urea/5 mM bis-tris-propane/7 mM HCI buffer, pH 7.0, (buffer A) for 24 h, with buffer changes after 4 and 16h. The protein solution was then filtered through a, 0.22pm Acrodisc LC PVDF syringe filter (Gelman Sciences, Northampton, UK) and the percentage filterable protein was determined as follows: absorbances of both filtered and unfiltered protein solutions, diluted l/25 with 6M urea, were determined at 280 and 320nm using 1Omm path-length cells in a Cary spectrophotometer [Varian (UK) Ltd, Walton-onThames, Surrey, UK] and converted to protein concentration by reference to a standard curve. The standard curve was obtained by plotiing the relationship between A2s0- 1.7Ajz0 and protein concentration (0 to 6x 10e4g protein (caseinate 6)mL-’ of 6M urea solution, pH 7.0); the % filterable protein was then calculated as follows: % filterable protein =

protein in filtered solution x 100 protein in unfiltered solution

Filtered protein solution (5OOpL) was washed onto the column using 1 mL of buffer A and the caseins eluted at a flow rate of 1 mLmin_’ with a NaCl gradient formed by mixing buffer A with buffer A containing 1 mol L-’ NaCl so that the concentration of NaCl in the column reached 100, 110, 220, 280, 295 and 430mM after elution- of 2, 9, 14, 21.5, 29 and 35mL, respectively. The absorbance of the eluate at 280nm was monitored continuously and the amounts of protein present were calculated using A2801%lcm values (absorption values of 1% protein solutions in a 1 cm light path at 280nm) of 10.0 for a,‘-casein, 4.6 for B-casein, 9.6 for K-casein, 10.1 for. as2-casein, 8.0 for y2plus y3-caseins and 8.2 for the unidentified components, A, B and C (average value for whole casein), as used by Davies and Law (1987); the elution regions designated as y-casein, A; B, Kc-casein, C, /?-, aa-and a,‘-caseins were1 the same as those designated for anion exchange FPLC by Davies and Law (1987). This procedure was repeated for each caseinate. Cation exchange FPLC

solution was adjusted to 7.0 with 1 M NaOH and 2mercaptoethanol (1 pLmL_‘) added to reduce the disulphide bonds of IC- and a,z-caseins. After 1 h, the reduced protein solution was readjusted to pH 5.0 with 1 M HCl. The protein solution was then filtered through a 0.22pm Acrodisc LC PVDF syringe filter and the percentage filterable protein determined as described above, except that an 8 M urea, pH 5.0 buffer was used as the diluent for determining absorbances at 280 and 320 nm. Filtered protein solution (5OOpL) was washed onto the column using 1 mL of a 6 M urea/2OmM acetate buffer, pH 5.0 (buffer B) and the proteins were eluted at a flow rate of 1 mLmin_’ by a salt gradient formed by mixing buffer B with buffer B containing 1 mol L-’ NaCl so that the NaCl concentration in the eluting buffer remained at 0 M for 2 mL and reached 25, 75, 150 and 260 mM after elution of 2.1, 16.5, 26.5 and 42.5 mL, respectively. The absorbance of the eluate at 280 nm was monitored continuously and the concentratis; of protein present calculated using the same Azso ‘cm values as used for anion exchange FPLC. The elution regions designated as unidentified D, /?-casein, unidentified E, IC-, pre-asI-, asI- and as2caseins were similar to those designated for cation exchange FPLC by Hollar ef al. (1991) and Law et al. (1993). This procedure was repeated for each caseinate. Gel permeation FPLC Gel permeation FPLC was performed using a Superose 12 HR10/30 column (Pharmacia AB, Uppsala, Sweden). Protein was dissolved in the eluting buffer (2.5%, w/v, SDS, 7.63 mM Tris and 0.76mM EDTA, pH 8.2) at a concentration of 6mgmL-‘. Samples (IOOpL containing 0.6mg of protein) were washed onto the column with 1 mL of eluting buffer, and eluted at a flow rate of 0.5 mLmin_‘; the absorbance of the eluate was monitored continuously at 280nm. As the identity of the protein eluting in the peaks was unknown, the area under each peak (high or low molecular weight fraction) was expressed as a % of the total area under both peaks, as determined by absorbance at 280 nm. Electrophoresis

Alkaline urea polyacrylamide gel electrophoresis (PAGE) was performed as described by Davies and Law (1977). Electrophoresis was carried out in 4.5%, w/v, polyacrylamide gels (96% polyacrylamide, 4% N,Nmethylenebisacrylamide) containing 4.5 mol L-’ urea and prepared in tris-EDTA-barbitone buffer (25 mM tris/3 mM EDTA.Na2.2H20/27 mM diethylbarbituric acid, pH 7.9fO.l) for 6 h at 200V. Gels were pre-run for 30 min at 300 V to remove impurities before application of 20 PL of a 20mgmL-’ solution of protein in sample buffer to the gel slots. Gels were stained with l%, w/v, Coomassie brilliant blue G-250 (Merck, Darmstadt, Germany) in methanol:water:acetic acid (5: 10:1, v/v/v) and destained with methanol:water:acetic acid (5: 10: 1, v/v/v). Free amino group content

Cation exchange FPLC was performed using a Mono S HR5/5 column (Pharmacia AB, Uppsala, Sweden), using the method of Hollar er al. (1991) except that the protein (20 mg) was dissolved in 5 mL of an 8 M urea/ 20mM acetate buffer, pH 5.0. The pH of the protein

The free amino group content of each caseinate was determined by reaction with ninhydrin (0.2%, v/v, in ethanol; Sigma Chemical Co., St Louis, MO, USA). Protein (0.6mg) was dissolved in 1 mL of distilled water,

Proteins in commercial sodium caseinates

1”

pH 7.0, and 0.5 mL of ninhydrin reagent was added. The solutions were vortexed and boiled in glass Pyrex tubes (covered with glass marbles to prevent evaporation) for 20min. The tubes were cooled and 2.5mL of 50% (v/v) propan-2-01 added to each, and mixed thoroughly by vortexing. The absorbance at 570nm was determined within 1 h after propan-2-01 addition using 1Omm path length cells in a Cary spectrophotometer. A standard curve for free amino groups was prepared by the same procedure using leucine at concentrations in the range 8 x lo-’ to 1.6x lop6 moles L-‘. All assays were performed in duplicate and the results expressed as moles of free amino groups g-’ of protein.

215

AB

I,

1

K-

t

c

1

p-

CQ-

, %1-

Free sulphydryl group content The free sulphydryl (SH) content of each caseinate was determined using Ellman’s reagent (Ellman, 1959) which was prepared by addition of 4mg of 5,5’-dithiobis-(Znitrobenzoic acid), (DTNB) (Pierce Chemicals, 3260 BA Oud-Beijerland, Holland) to 1 mL of reaction buffer (8 M urea/O.1 M NaHzP04.2H20, pH 8.0). Protein was dissolved in 1 mL of 8 M urea solution at a concentration of 30mg mL_r, pH 8.0. The reaction buffer (2.5mL) and the Ellman’s reagent (50 pL) were added to 25OpL of this solution. A blank was prepared by using 25OpL of 8 M urea solution in place of the protein solution. After thorough mixing and incubation at room temperature for 15 min, the absorbance was determined at 412 nm. The SH content of the proteins was determined using a molar extinction coefficient for reduced Ellman’s reagent in 8 M urea of 14,290 at 412 nm (Gething and Davidson, 1972). All assays were performed in duplicate and the results expressed as pmoles SH groups g-’ protein. RESULTS AND DISCUSSION Anion exchange FPLC The anion exchange FPLC profiles of prepared, unheated sodium caseinate sodium caseinates 1 and 7 are shown in the quantitative compositional data for

laboratory(LSC) and Fig. 1 and the protein

20

10

0

30

40

ELUTION VOLUME (ml) Fig. 1. Elution profiles of LSC (--), sodium caseinate 1 (. .) and sodium caseinate 7 (- --) on a Pharmacia Mono Q anion

exchange

FPLC

column,

type HR5/5.

complement, as determined by anion exchange FPLC analysis, of LSC and sodium caseinates 1 to 9 are shown in Table 1. All caseinates gave very reproducible chromatograms; the range of values obtained for proportions of caseins eluting in each region were < 5% of the mean values. The elution profile of LSC showed good separation of all the major caseins, with sharp peaks for K-, B-, q2- and a,,-caseins and with little protein eluting in the ycasein and unidentified regions. In contrast, caseinate 1 had a diffuse elution profile (similar elution profile for caseinate 4), and caseinate 7 had a very diffuse elution profile (similar elution profiles for caseinates 2, 3, 5, 6, 8 and 9). For caseinate 7, the proportion of total filterable protein eluted in the y-casein, A, B and C regions were 8.4, 2.0, 1.7 and 4.8%, respectively, compared to 3.5, 0.8, 1.0 and 1.5% protein, respectively, for LSC. For LSC, 38.6 and 35.7% of total filterable protein eluted in the asI- and fl-casein regions, respectively, compared to 31.3 and 28.4%, respectively, for caseinate 7. Anion exchange FPLC

Table 1. Proportions of Different Casein Fractions Present in the Filterable Laboratory-prepared

Unheated

Sodium

Caseinate

(LSC), as Measured the Total Filterable

Protein of Each Commercial Sodium Caseinate and by Anion Exchange FPLC and Expressed as Percentages of Protein0

Casein fractions Caseinate

I 2 3 4 5 6 7 8 9 LSC

Y-

A

B

K-

6.6 8.5 10.3 5.2 8.0 7.8 8.4 7.7 7.5 3.5

1.6 2.4 2.2 1.4 2.0 2.0 2.0 1.7 1.7 0.8

1.6 2.0 1.7 1.5 1.7 1.6 1.7 1.6 1.4 1.0

10.0 10.8 11.4 10.0 11.7

“Mean values calculated

from the quantitative

11.0 10.8 9.7 10.6 9.5 data obtained

C

2.4 4.3 5.0 2.1 5.2 5.0 4.8 5.4 4.7 1.5 from two anion

B-

42-

31.6 27.0 25.8 32.7 26.1 28.5 28.4 29.0 28.6 35.7 exchange

11.9 12.3 13.9 11.3 15.8 12.9 13.1 13.1 12.5 9.5 FPLC

analyses

asI-

Filterable protein as % of total protein

34.5 33.0 30.0 35.9 29.7 32.0 31.3 31.8 33.1 38.6 for each caseinate.

91.1 94.3 95.4 95.7 95.7 95.0 93.5 95.3 91.1 100.0

216

A. G. Lynch et al.

chromatograms and quantitative data for LSC and caseinates 1 to 9 (Fig. 1 and Table 1) were comparable with the results obtained by anion exchange FPLC analysis of sodium caseinates by Muir and Dalgleish (1987) and Dalgleish and Law (1988). Dalgleish and Law (1988) proposed that dephosphorylation of proteins in the caseinates may have been responsible for the increased amount of protein eluting from the anion exchange FPLC column in the a,z-casein and C regions, compared to casein isolated from unheated milk. Similarly, in the present study, the higher level of protein eluting in the y-casein, A, B, C and a,z-casein regions of the chromatograms of caseinates 1 and 7, compared to LSC, indicated reduction of the net negative charge on the caseins. This may have been caused by loss of phosphoseryl residues during the manufacture of commercial caseinate, leading to earlier elution of the modified proteins from the positively charged anion exchange Creamer and Matheson (1977) found column. increased loss of serine and release of phosphate as the pH of sodium caseinate (5%, w/v, heated at 60°C for 60 min) was raised from 8.0 to 11.5, while Guo et al. in the rate of an increase (1989) observed dephosphorylation of sodium caseinate (2%, w/v, pH 7.0) as temperature was raised from 100 to 140°C for 60min. These results suggest that the commercial caseinates in the present study may have been heated at values. during temperatures and pH different manufacture as the elution profiles and quantitative compositional data for the protein complement of caseinates 1 and 4 indicated that the extent of modification to the net negative charge on the proteins in these caseinates was not as high as for caseinates 2, 3, 5, 6, 7, 8 and 9. Tail edges were present in the cr,r-casein peak of caseinates 1 and 7 which were absent in the a,r-casein peak of LSC (Fig. 1); these tail edges in the commercial caseinates indicated that some caseins may have a high net negative charge, possibly due to the formation of lysinoalanine during caseinate manufacture, as the modified proteins in the tail edges needed a higher salt concentration for elution from the column than the corresponding proteins in LSC. Similarly, Law et al. (1994) found tail edges in the Q- and /?-casein peaks in the anion exchange chromatograms of the pH 4.6insoluble protein isolated from skim milk, pH 6.7, heated at> 120°C for 5 min, but not in chromatograms of the pH 4.6-insoluble protein isolated from unheated milk; they proposed that protein in the tail edges of the asl- and /?-casein peaks had lost positive charge, possibly due to Maillard reaction of s-amino groups of lysine with carbonyl compounds on heating milk at high temperatures. Thus, the processing conditions to which the caseins are exposed (e.g. pH, temperature and duration of heating) during the conversion of acid casein to sodium caseinate may modify the net negative charge on the proteins to different extents.

caseinates l-9 are summarized in Table 2. All caseinates gave very reproducible chromatograms; the range of values obtained for proportions of caseins eluting in each region were < 5% of the mean values. The elution profile of LSC showed good resolution of the major casein peaks with little protein eluting in the pre-orslcasein (referred to as “P” by Law et al., 1994) and unidentified D and E regions compared to caseinates 1 and 7. The elution profile of caseinate 1 (similar to the elution profile for caseinate 4) showed slight modifications compared to LSC, e.g. it had a smaller CX,~casein peak and a greater amount of protein eluting in the pre-a,,-casein region than LSC. The elution profile of caseinate 7 (similar to elution profiles for caseinates 2, 3, 5, 6, 8 and 9) showed substantial modification of all the major casein peaks compared to LSC and caseinate 1; the CQ-, K- and /?-casein peaks of caseinate 7 eluted at lower salt concentrations than similar peaks in caseinate 1 and LSC and more protein eluted in the pre-a,,-casein region with less in the a,,-casein region compared to caseinate 1 and LSC. There appeared to be an inverse relationship between the amount of protein eluting in the asI- and pre-cr,lcasein regions for all samples. LSC had the highest amount, 29.0%, of protein eluting in the a,,-casein region and the lowest amount, 1.4%, of protein in the pre-a,,-casein region; this contrasts with the commercial caseinates values which ranged from 17.2 and 9.0% protein in the a,t- and pre-a,,-casein regions, respectively, for caseinate 8, to 26.3% a,r-casein for caseinate 1 and 2.3% pre-a,,-casein for caseinate 4 (Table 2). Sodium caseinate is susceptible to losses of positively charged amino acid residues on heating at high temperatures and at alkaline pH values (Creamer and Matheson, 1977; Guo et al., 1989). Law et al. (1994) analysed the pH 4.6-insoluble protein recovered from unheated milk and from milk heated at >12O”C for 5min by cation exchange FPLC and found that the level of pre-a,,-casein was higher and that of a,,-casein lower in the protein recovered from the heated sample. They proposed that a,,-casein lost positive charge on

D

,

B-

,E

,

K-

Pa,l-

a,l-

asp

0.3

0 0

Cation exchange FPLC

Cation exchange FPLC chromatograms of LSC and sodium caseinates 1 and 7 are shown in Fig. 2, and the quantitative cation exchange FPLC results for LSC and

10

20

30

40

50

ELUTION VOLUME [ml) Fig. 2. Elution profiles of LSC (), sodium caseinate 1 (. . .) and sodium caseinate 7 (---) on a Pharmacia Mono S cation exchange FPLC column, type HR5/5.

Proteins in commercial sodium caseinates

217

Proportions of Different Casein Fractions Present in the Filterable Protein of Each Commercial Sodium Caseinate and Laboratory-prepared Unheated Sodium Caseinate (LSC), as Measured by Cation Exchange FPLC and Expressed as Percentages of the Total Filterable Protein”

Table 2.

Casein fractions Caseinate

Filterable protein as % of total protein

D

B

E

K-

Pre-a, I-

a,t-

as2-

5.4 9.7 7.5

42.0 40.0 41.8

2.9 4.4 4.4

10.5 11.0 10.8

2.6 5.3 7.5

26.3 21.8 19.0

10.4 8.0 9.1

91.2 88.9 100.0

5 6 7 8 9

4.3 8.6 6.9 6.3 6.3 6.3

41.1 41.3 45.6 43.1 44.3 44.9

3.0 3.6 3.6 3.6 3.9 4.2

10.8 10.9 11.5 11.1 11.1 11.2

2.3 6.0 6.8 7.5 9.0 7.4

25.9 21.4 19.6 20.0 17.2 18.1

12.6 9.0 9.1 9.1 8.4 8.1

89.1 90.2 97.6 94.4 80.3 91.5

LSC

4.8

41.8

1.7

10.2

1.4

29.0

11.2

97.8

I 2 3 4

“Mean values calculated from the quantitative

data obtained from two cation exchange FPLC analyses for each caseinate.

heating milk at high temperatures, possibly through reaction of s-amino groups of lysine. The modified proteins eluted from the negatively charged column at a lower salt concentration than that needed to elute the unmodified proteins in the unheated sample. Similarly, in the present study, the high levels of protein eluting in the pre-cr,i-casein region and the low levels of protein eluting in the cc,,-casein region of the commercial caseinates compared with LSC, may be due to a decrease in net positive charge on cr,i-casein during manufacture, possibly through reaction of s-amino groups of lysine. This could explain why the modified proteins were eluted from the column at lower salt concentrations than that required to elute the unmodified proteins in LSC. However, the elution profiles (Fig. 2) and quantitative compositional data (Table 2) for the protein complement of caseinates 1 and 4 indicated that the extent of modification to the net positive charge of the proteins in these caseinates was not as great as for caseinates 2, 3, 5, 6, 7, 8 and 9. Thus, the conditions to which the caseins are exposed (e.g. pH, temperature and duration of heating) during the manufacture of sodium caseinate may modify the net positive charge on the proteins to different extents. The differences in pH and composition between the anion and cation exchange buffers may explain the variances in the level of filterable protein (expressed as % of total protein) for each caseinate. In the present study, no difficulty was found in filtering the caseinates through a 0.22~pm filter. In contrast, some of the caseinates used by Dalgleish and Law (1988) required a larger pore size filter before application of the samples to the Mono Q FPLC column. Gel electrophoresis Alkaline urea-PAGE gave good resolution of LSC, with distinct bands for individual proteins; however, the commercial caseinates showed variable levels of smearing and less distinct protein bands [Fig. 3(top and bottom)]. The a,*-casein bands were less visible for caseinates 2, 3, 5, 6,7,8, and 9 than for caseinates 1 and 4 and LSC. Fox (1981) and Singh and Fox (1985) showed that a,*-casein was the casein most susceptible to thermal degradation,

and Guo et al. (1989) found that CI,~-and rc-caseins were more susceptible to high temperatures than asl- and Bcaseins. Guo et al. (1989) and Law et al. (1994) showed that as the temperature and duration of heating of sodium caseinate solution or milk increased, protein separation on PAGE became less distinct and smearing of protein bands increased. These results suggest that caseinates 1 and 4 were not heat-treated as severely as the other caseinates during manufacture. Gel permeation FPLC Gel permeation chromatography using conditions which dissociated the caseins but which did not break covalent bonds, resolved the caseins into two major peaks representing high and low molecular weight (MW) fractions (Fig. 4). Caseinates 1, 2, 4 and 9 had the greatest amounts of high MW protein and LSC and caseinate 8 the lowest (Table 3). Gel permeation FPLC indicated modification to the structure and aggregation state of the caseinates during manufacture, which may influence their functional properties. Aggregated caseinates were much less surface active than dispersed caseinates (Mulvihill and Murphy, 1991). Guo (1990) found that the viscosity of heated sodium caseinate solutions was lower than those of unheated caseinates; heat-induced hydrolysis of caseins to lower MW peptides was suggested as being responsible for the lower viscosity of the heated caseinate solutions. In the present study, caseinates 1, 2 and 4 had higher amounts of high MW protein than caseinates 3, 5 and 6. Cream liqueurs made with caseinates 1, 2 or 4 had higher apparent viscosities than liqueurs made with caseinates 3, 5 or 6 after storage for 77days at 45°C (Lynch, 1995), suggesting that a high content of high MW protein may contribute to viscosity increases in cream liqueurs. Free amino group content The free amino group content of LSC was greater than that of any of the commercial caseinates; the free amino group content of these caseinates ranged from 2.2 x lO-4 moles g-’ protein for caseinate 2 to

A. G. Lynch et al.

218

as2-

K-

Y-I 2,3

ABCDEFGH

as1

_-

Qs2-

-II

a-La B- B-L!3 K-

r-y,3

-r -

A

BCDE

Fig. 3 . (Top) Alkaline urea-PAGE of LSC (lane A), whey protein standard (lane B) and sodium caseinates 1, 2, 3. 4, 5 and 7 C-H, respectively). (Bottom) Alkaline urea-PAGE of whey protein standard (lane A), LSC (lane B) and sodium caseinates 6, 8 and 9 (lanes C-E, respectively).

3.6x 10-4molesg-’ protein for caseinate 5 (Table 3). This shows that the caseinates had lost positively charged amino groups during manufacture or that the positively charged groups were unable to react with ninhydrin due to the aggregated state of the proteins. During the manufacture of sodium caseinate, the proteins may be exposed to high temperatures and alkaline pH values which promote reactions involving positively charged amino residues (Creamer and Matheson, 1977). Guo et al. (1989) found reduced lysine content in sodium caseinate heated for long temperatures. Formation of periods at high lysinoalanine and transamidation reactions reduce the free amino group content of caseinate and may also alter structure if intermolecular interactions are

involved. The isopeptides, &-N(j?-aspartyl)lysine and EN(y-glutamyl)lysine, have been detected in dry casein (Walstra and Jenness, 1984) and reactions leading to the formation of these and similar peptides may also occur during the manufacture of sodium caseinate. Free SH group content The free SH content of LSC and caseinate 8 was much higher than that of any of the other caseinates (Table 3). LSC and caseinate 8 also had lower proportions of protein eluting in the high molecular weight fraction on gel permeation FPLC than the other caseinates (Table 3). a,l- and @-Caseins do not contain cysteine residues but K-and a,z-caseins each contains

Proteins in commercial

15

10

sodium caseinates

ELUTION VOLUME Fig. 4. Elution profiles of LSC (-----), a Pharmacia Superose 12 gel permeation

25

20

219

30

(mL)

sodium caseinate 1 (. ,), sodium caseinate 7 (- - -) and sodium caseinate 2 (----) FPLC column, type HR5/5.

on

Table 3. Proportion of High Molecular Weight Proteins”, as Measured by Gel Permeation FPLC (Expressed as Percentages of Total Protein), and the Concentration of Free Amino and Sulphydryl Groups (Determined by Reaction with Ninhydrin and DTNB, Respectivelyh) in Commercial Sodium Caseinates and Laboratory-prepared Unheated-Sodium Caseinate) Free NHz(molesx 10p4g-’ protein)

% High MW

Caseinate

26 24 24 22 21 21 21 18 17 17

2 9 1 4 3 5 6 7 8 LSC

2.2 2.8 2.2 2.9 2.7 3.6 3.3 2.9 3.0 5.5

Free SH (pmolesg-’

protein)

0.8 1.9 0.7 0.8 1.4 1.2 1.8 1.9 4.1 5.6

“High molecular weight proteins were those proteins which eluted in the first 15 mL from the gel permeation FPLC column. Proteins eluted at 15 mL were assumed to have a MW of approximately 65 000; this figure was determined from the data of Law ef at. (1993). ‘Means of duplicates.

two half cystine residues which usually exist as disulphides (Swaisgood, 1992). The differences in free SH content of the caseinates may be due to the different conditions to which the caseinates were manufacture; high heating exposed during temperatures at alkaline pH values promote /?elimination of cysteine to form dehydroalanine which in turn can react with lysine to form lysinoalanine or with cysteine to form lanthionine (Creamer and Matheson, 1977; Annan and Manson, 198 1; Walstra and Jenness, 1984). Other reactions which cysteine undergoes at high temperatures and alkaline pH values include (Walstra and Jenness, 1984): R’ -SH

+ R*-SH

R’-SH

+ R*-S-S-R3

= R’ -S-S-R* = R’-S-S-R*

(1)

the proteins and affect their functional properties. Lee et al. (1992) showed that the active SH content of commercial sodium caseinate was lower when assayed at pH 8.0 than at pH 7.0, probably due to the oxidation of SH groups to -S-S- at alkaline pH values [Eq. (l)]. Snoeren and van der Spek (1977) found complex formation involving thiol reactions between blactoglobulin and CL,~-and K-caseins on heating skim milk at 140°C for 4sec. Thus, intermolecular disulphide-linked proteins may contribute to the high levels of high MW protein in caseinates 1, 2, 4 and 9 as determined by gel permeation FPLC; these caseinates may have been exposed to alkaline pH values, high temperatures or aeration which may accelerate the rate of reactions (1) and (2) above.

+ R3-SH (2)

The above reactions can be intra- or intermolecular, with intermolecular reactions leading to polymerization via covalent linkages which could alter the structure of

CONCLUSIONS The results obtained in this study showed differences in the level of modification to the negative and positive charge, % high MW protein and the free amino and

220

A. G. Lynch et al.

SH group contents of commercial sodium caseinates. The caseinates can be divided into four groups as follows: (1) slight modification of negative and positive charges with low contents of high MW protein, e.g. LSC; (2) slight modifications of negative and positive charges with high contents of high MW protein, e.g. caseinates 1 and 4; (3) appreciable modification of negative and positive charges with low contents of high MW protein, e.g. caseinates 3, 5, 6, 7 and 8 and (4) appreciable modification of negative and positive of high MW protein, e.g. charges with high contents caseinates 2 and 9.

Gething, M. J. H. and Davidson, B. E. (1972) The molar absorption coefficient of reduced Ellman’s reagent: 3carboxylate-4-nitro-thiophenolate. European Journal of Biochemistry 30, 352-353. Guo, M. (1990) Heat-induced

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Hollar, C. M., Law, A. J. R., Dalgleish, D. G. and Brown, R. J. (1991) Separation of the major casein fractions using cation-exchange fast protein liquid chromatography. Journal of Dairy Science 74, 2403-2409.

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