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Effect of preheating and other process parameters on whey protein reactions during skim milk powder manufacture
ARTICLE IN PRESS
International Dairy Journal 15 (2005) 501–511 www.elsevier.com/locate/idairyj
Effect of preheating and other process parameters on whey protein reactions during skim milk powder manufacture D.J. Oldfielda,c,, M.W. Taylora, H. Singhb a
Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand b Riddet Centre, Massey University, Palmerston North, New Zealand c Whole Protein Technical Centre, Fonterra Cooperative Group Ltd, Edgecumbe, New Zealand Received 23 March 2004; accepted 15 September 2004
Abstract Skim milk powder was manufactured in a milk powder plant using different preheating temperatures, concentrate heating temperatures and spray drying temperatures. Varying the preheating conditions from 70 1C for 52 s to 120 1C for 52 s had a marked effect on the denaturation of b-lactoglobulin A, b-lactoglobulin B, a-lactalbumin, bovine serum albumin (BSA), and immunoglobulin G. In contrast, varying concentrate heating temperature (65–74 1C) and inlet/outlet air dryer temperature (200/ 101 1C–160/89 1C) had a minimal effect on whey protein denaturation. Most of the whey protein denaturation and association with the casein micelle occurred in the preheating section of the powder plant. Aggregation of b-lactoglobulin (b-lg) and BSA predominantly involved disulphide bonds. Although, greater than 90% of the b-lg and BSA was denatured after preheating at 120 1C for 52 s, the extent of association with the casein micelle was lower, 50% for b-lg and 75% for BSA. r 2004 Elsevier Ltd. All rights reserved. Keywords: Skim milk powder; Evaporation; Spray drying; Denaturation; Aggregation; Whey protein
1. Introduction Skim milk powder (SMP) is widely used as an ingredient in many formulated foods. Soups, sauces, recombined evaporated milk, confectionery and bakery products all benefit from the functional properties provided by skim milk powder. The powder can be tailored to a specific end-use by manipulating processing conditions during powder manufacture. The manufacturing process for SMP involves heating the skim milk (known as preheating), concentrating the skim milk solids by evaporation to 45–50% total solids, heating the skim milk concentrate and then spray drying the milk concentrate to produce a powder. Preheating Corresponding author. Whole Protein Technical Centre, Fonterra Cooperative Limited, Edgecumbe, New Zealand. Tel.: +64 7 304 7000; Fax +64 6 304 7063. E-mail address: david.oldfi[email protected] (D.J. Oldfield).
0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.09.004
conditions are used to a large extent to control the functional properties of the powder. A number of changes occur in milk during preheating: whey protein denaturation, association of denatured whey proteins with the casein micelle, transfer of soluble calcium and phosphate to the colloidal phase, destruction of bacteria and inactivation of enzymes (Singh & Newstead, 1992). However, it is the extent of whey protein denaturation that is used to broadly classify SMP for use in different applications. Heat classification is controlled by the degree of preheating and is measured by the whey protein nitrogen index (WPNI). Depending on the severity of the preheating conditions, SMP can be classified as a low-, medium- or high-heat powder. Although there are a large number of studies on heatinduced whey protein denaturation in fresh milk (Hillier, Lyster, & Cheeseman, 1979; Manji & Kakuda, 1986; Dannenberg & Kessler, 1988) they have largely
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been carried out by heating milk in water and oil baths under laboratory scale conditions. Moreover, the rate of heating is important and commercial heating equipment can produce quite different levels of whey protein reactions compared to heating on a laboratory scale (Corredig & Dalgleish, 1996a; Oldfield, Singh, & Taylor, 1998b). The effect of different processing steps in milk powder manufacture, on total whey protein denaturation has been investigated by measuring the WPNI of samples collected at different points in a powder plant (Swanson & Sanderson 1964; O’Connor, McKenna, & O’Sullivan, 1969; de Vilder & Martens, 1976). However, WPNI only provides an overall level of protein denaturation. Individual whey proteins have different levels of thermal stability in milk and there are very few studies reporting how the denaturation of individual whey proteins is affected by milk powder manufacture. Singh and Creamer (1991) investigated the effect of high heat treatments (110–120 1C) and a low heat treatment (72 1C for 15 s) on the denaturation and aggregation reactions of a-lactalbumin (a-la) and blactoglobulin (b-lg) at different steps during milk powder manufacture. High heat treatments denatured most of the whey proteins, evaporation further increased the extent of denaturation and spray drying had only a small effect. However, little is known of the whey proteins most susceptible to heat denaturation, immunoglobulins and bovine serum albumin (BSA), and what effects different processing steps have on their denaturation. In addition to preheat treatment before evaporation, concentrate heating temperature and dryer inlet/outlet temperatures are known to affect important physical properties of milk powder such as moisture, solubility index and bulk density (de Vilder, Martens, & Naudts, 1976, 1979; Baldwin, Baucke, & Sanderson, 1980; Kelly, Kelly & Harrington, 2002). However, the effect of concentrate heating and drying temperatures on the heat-induced reactions of individual whey proteins has not been reported.
Despite the importance of milk powder manufacture and the wide range of uses for milk powders, very little is understood of the chemical changes that occur during processing, especially during evaporation and spray drying. This research work was carried out to investigate the effect of milk powder processing conditions on whey protein reactions.
2. Materials and methods 2.1. Skim milk powder manufacture Whole milk obtained from Fonterra Cooperative Ltd (Longburn, New Zealand) was separated without pasteurisation at 55 1C, at the Fonterra Research Centre (FRC) pilot plant. The skim milk was then processed into skim milk powder. Eight different runs were carried out with a combination of different preheating temperatures, concentrate heating temperatures and dryer inlet/outlet temperatures (Table 1). Subsequently these set of eight runs were repeated at a different time of the dairying season. It was not possible to average the data from the two trials because of differences in milk composition. Hence, the results of one trial are presented here: similar trends were observed for the other trial. An exception was the IgG results, which had the same initial concentration in both trials. Thus the IgG results presented are an average of both trials, and a pooled standard deviation was calculated. Chilled skim milk from the silo tanks were passed through a number of tubular heat exchangers housed in the calandria (steam chests) of the evaporator effects. This heated the milk from 8 to 65 1C. Skim milk was preheated by direct steam injection (DSI) to either 70, 80, 100 or 120 1C, pumped through a 52 s holding tube and then into a flash vessel, which instantaneously reduced the temperature of the heated milk to 70–72 1C. Skim milk was concentrated in a three effect
Table 1 Processing conditions used in the manufacture of skim milk powder and properties of the powders manufactured Run No.
1 2 3 4 5 6 7 8
Processing conditions (average temperatures in 1C)7standard deviationa
Powder properties (average value)7standard deviation
Preheating
Concentrate heating
Dryer inlet air
Dryer outlet air
Moisture (% w w1)
Insolubility index (mL)
WPNI (mg g1)
7070.1 7070.1 7070.1 7070.0 7070.2 12070.5 10070.1 8070.1
6970.9 6571.1 7470.8 6970.7 6971.5 6970.3 6970.2 6970.3
18071.1 18070.3 18070.4 20071.9 16070.5 18070.3 18070.3 18070.8
9071.2 9270.4 9270.6 10171.1 8970.7 8770.4 8770.2 8972.0
4.470.0 4.670.1 4.670.1 4.070.3 4.470.1 5.070.3 4.670.1 4.470.3
0.270.1 0.370.1 0.170.0 0.370.3 0.270.0 0.170.0 0.170.0 0.170.0
8.070.3 7.870.1 7.970.3 7.970.1 7.970.2 1.570.1 2.770.1 7.370.9
WPNI: Whey Protein nitrogen index. a Processing conditions were recorded during each run. These data was averaged and the standard deviation calculated.
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falling film evaporator (Weigand, Karlsruhe, Germany) at a feed rate of 1700 L h1. The temperatures of the 1st, 2nd and 3rd effects were 72, 53 and 41 1C, respectively. Concentrated skim milk (milk concentrate) exiting from the last effect at 47–49% total solids (TS) was then heated in a scraped surface heat exchanger (Model VT422, APV Crepaco Inc., Chicago, Illinois) to either 65, 69 or 74 1C. The heated milk concentrate took 80 s to reach the nozzle atomizer of a pilot scale dryer with an integral fluid bed (IFB dryer, FRC design, FRC, Palmerston North, New Zealand). The feed rate of the milk concentrate to the dryer was 140 L h1. The inlet and outlet temperatures were varied from 160 to 200 1C and from 87 to 100 1C, respectively. The integral fluid bed was supplied with air at 85 1C. The powder was dried to a final target moisture content of o5%. 2.2. Sample collection from the milk powder plant For each run, skim milk from the flash vessel, milk concentrate from the 1st, 2nd and 3rd effects and milk concentrate just before the spray dryer (after concentrate heating) were collected once the plant had reached steady state conditions. All the samples were cooled to approximately room temperature in an ice water bucket. The milk concentrate samples were then diluted, using distilled and deionised water, to the total solids (TS) of the original skim milk (9% TS) and stirred for 2 h. Skim milk powder (SMP) samples, obtained from the powder outlet of the dryer, were reconstituted (9% TS) in distilled and deionised water at 40 1C, and stirred for 2 h at room temperature. 2.3. Protein analysis The original skim milk, samples collected from the milk powder plant, and reconstituted milk powder samples, were ultracentrifuged at 175,000 g for 1 h at 20 1C (Beckman L8-80 M, Beckman, Palo Alto, California). After ultracentrifugation, the supernatant was carefully poured off for protein analysis. The supernatants were analysed for b-lactoglobulin genetic variant A (b-lg A), b-lactoglobulin genetic variant B (b-lg B), a-lactablumin (a-la) and bovine serum albumin (BSA) by polyacrylamide gel electrophoresis (PAGE). Quantitative PAGE analysis was performed on a mini-gel electrophoresis unit (Biorad Laboratories, Richmond, CA, USA), under non-dissociating (nativePAGE), dissociating but non-reducing (SDSNR-PAGE) and dissociating and reducing (SDSR-PAGE) conditions as described by Oldfield, Singh, Taylor, and Pearce (1998a) and Oldfield et al. (1998b). The gels were scanned on a Computing Densitometer (Molecular Dynamics, Sunnyvale, CA) and the integrated intensities
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of the individual protein bands were calculated by a software program, ImageQuant (Molecular Dynamics). Standard curves prepared from purified b-lg A, b-lg B, a-la and BSA (Sigma Corp, St. Louis, MO, USA) were used to determine individual whey protein concentrations (g kg1). Under SDSNR- and SDSR-PAGE running conditions b-lg A and b-lg B bands ran together as a single total b-lg band. The standard error of the PAGE analysis was 6%. The different PAGE methods identified a number of forms of protein, and the following classifications are based on the electrophoretic running conditions used. Quantitative native-PAGE, run under non-dissociating conditions, was used to determine the amount of ‘‘native’’ b-lg A, b-lg B, a-la and BSA present. As the samples were cooled to room temperature before ultracentrifugation and PAGE analysis, any loss from native PAGE should be considered the result of irreversible denaturation leading to aggregate formation. Thus, under the running conditions it was not possible to differentiate between native protein, unfolded monomeric protein and lactosylated whey protein (Kinghorn, Norris, Paterson, & Otter, 1995). Two types of aggregates in the supernatant were quantified by SDSNR- and SDSR-PAGE. SDSNRPAGE conditions (0.2% (w v1) SDS) measured both ‘‘native’’ protein and aggregates in the supernatant that were not linked via disulfide bonds. These aggregates are likely to have formed through non-covalent and hydrophobic interactions and are referred to as ‘‘hydrophobic aggregates’’. Analysis of the supernatant by SDSR-PAGE measured ‘‘total whey protein’’ present. Under the dissociating and reducing conditions (0.2% (w v1) SDS+5% (v v1) 2-mercaptoethanol) disulfide bonds and hydrophobic interactions were broken and all the aggregates, both disulfide-linked and hydrophobic, dissociated into monomers, which could be resolved on the gel along with any native protein present. The amount of whey protein that co-sedimented with the casein micelles was calculated from the difference in concentration between the whey proteins in the original skim milk and the total whey protein in the supernatant (quantified by SDSR-PAGE) of the sample. Whey proteins that co-sedimented with the casein micelles were assumed to be ‘‘associated with the micelle’’ primarily via sulphydryl-disulfide interchange reactions between b-lg and k-casein at the micelle surface. It is possible that b-lg self aggregation formed insoluble aggregates that were large enough to also sediment with the micelles under the centrifugation conditions used. These aggregates would be undistinguishable from associated b-lg/k-casein complexes. Immunoglobulin G (IgG) concentration in the supernatant was determined by an affinity column (HiTrap affinity column, Protein G, Pharmacia, Uppsala,
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Sweden) according to the method of Kinghorn et al. (1995). Standard error of the readings was 70.01 g kg1. 2.4. Concentration and dilution calculations To correct for the changes in protein concentration due to dilution/concentration effects, the concentration of whey protein measured in the supernatant was recalculated back to an equivalent concentration in the original skim milk. This calculation was carried out in two steps. Centrifugation causes an increase in the concentration of whey proteins due to the casein micelles sedimenting, and thus water associated with the sedimented micelles is unavailable as solvent (van Boekel, & Walstra, 1989). Thus the first step was to recalculate the concentrations of individual whey proteins in the supernatant of ultracentrifuged samples to their respective value in the sample before centrifugation, by multiplying by a correction factor (F) as described by van Boekel, & Walstra (1989) (Eq. (1)). In the second step, the concentration of individual whey proteins in the sample prior to centrifugation was converted to a concentration in the original skim milk. This was done by comparing the total solids of the original skim milk with the total solids of the sample that was centrifuged (Eq. (1)). Total solids were measured by drying for 5 h in a 102 1C oven (McDowell, 1972). C milk ¼ C supernatant F
TSmilk ; TSsample
tion using an activity coefficient. The activity coefficients used were 0.3 for milk concentrates (Nieuwenhuijse, Timmermans, & Walstra, 1988) and 0.4 for skim milk at 9% TS (Greets et al., 1983). After milk is heated, the Ca2+ concentration and Ca2+ activity decrease. This decrease is reversible upon cooling milk. The recovery of Ca2+ activity at a constant temperature (20 1C) follows a logarithmic relationship, and extrapolation of the logarithmic relationship back to 1 min allows for a calculation of the Ca2+ activity close to heating conditions (Greets et al., 1983). The logarithmic relationship was used to calculate the Ca2+ activities of skim milk samples from the flash vessel, and milk concentrate samples (undiluted: 48% TS) from the 3rd effect and from just before the spray dryer (concentrate heating samples). Samples were cooled in iced water to 20 1C within 4 min, and then measured with a calcium ion selective electrode. In addition, the logarithmic relationship was used to analyse reconstituted skim milk powders by extrapolating the Ca2+ activity readings back to 1 min, 1 h and 24 h after reconstitution. Milk powder samples were reconstituted in 40 1C water, stirred for 1 h, and cooled to room temperature (20 1C) before they were measured with the calcium ion selective electrode, over a period of 3 h. The same samples were then held at 4 1C for 20 h, and warmed to 20 1C before readings were taken.
(1)
where Cmilk is the concentration in the original skim milk (g kg1), Csupernatant the concentration in the supernatant (g kg1), F the correction factor (=0.917), TSmilk the total solids of the original skim milk (% w w1), and TSsample the total solids of the sample (% w w1). Duplicate samples of concentrate from the 1st effect of the evaporator were analysed to determine the standard error of the individual whey protein concentrations. 2.5. Ca2+ activity measurements The Ca2+ concentrations in the original skim milk, milk concentrates and reconstituted skim milk powder were determined using a calcium ion selective electrode (F2112Ca Radiometer, Copenhagen, Denmark) attached to a digital readout pH meter (Radiometer), using the method of Greets, Bekhof, and Scherjon (1983). Samples were measured at 20 1C in a 100 mL beaker, with moderate stirring. Calibrations were performed using CaCl2/KCl standards (Radiometer) with ionic strengths of 0.08 and 0.2 mol L1 for milk and concentrated milk sample readings, respectively. The Ca2+ activity was calculated from the Ca2+ concentra-
2.6. Proximate analysis Total nitrogen (TN) was determined using the IDF standard method (1993a). Skim milk powder was analysed for whey protein nitrogen index (WPNI) using the method of Sanderson (1970). Insolubility index was determined using the IDF method (1988). Powder moisture (IDF, 1993b) and fat content (IDF, 1987) were measured. Ash content of the milk powder was measured after incineration in a muffled furnace at 550 1C. Reported values are an average of two analyses. The pH of the original skim milk, milk concentrate and reconstituted milk powder samples were measured at 20 1C using an Orion pH meter and probe (Model 9155/56 probe, Model 720 pH/ISE meter; Orion Research Incorporated, Boston, MA). Reported values are an average of two analyses.
3. Results and discussion 3.1. Milk powder manufacture The concentration of individual whey proteins in the original skim milk was: b-lg A 2.2 g kg1, b-lg B 2.3 g kg1, a-la 0.80 g kg1, BSA 0.25 g kg1 and IgG
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0.43 g kg1. Moisture content of the SMP was typically around 4.0–4.6%, except for run 6, which had the highest moisture content of 5.0%, probably due to the low inlet dryer temperature (160 1C). The insolubility index of powders ranged from 0.1 to 0.3 mL. The pH of the original skim milk was 6.67. Different preheating temperatures, concentrate heating temperatures and drying conditions had very little effect on pH, measured at 20 1C. However, as expected, there was a decrease in pH during the evaporation process. The variation in pH for different preheating temperatures was 70.03. Average pH values were: flash vessel 6.66, 1st effect 6.64 (12% TS), 2nd effect 6.47 (20% TS), 3rd effect 6.17 (48% TS), and milk concentrate just before the spray dryer 6.16 (after concentrate heating). After SMP was reconstituted to 9% TS, the pH returned to a value (pH 6.68) similar to that of the original skim milk. 3.2. Ca2+ activity The Ca2+ activities of skim milk, milk concentrates (not diluted; 48% TS) and reconstituted skim milk powder (9% TS) are shown in Table 2. The original skim milk had an average Ca2+ activity of 0.91 mmol L1 with a standard error of 70.02 mmol L1. Greets et al. (1983) reported a Ca2+ activity of 0.82–0.88 mmol L1 and Nieuwenhuijse et al. (1988) a value of 0.87–0.90 mmol L1 for raw milk. Preheating at all the temperatures investigated reduced Ca2+ activity (Table 2). Heating milk causes a transfer of Ca and P from the soluble to the colloidal phase (Nieuwenhuijse et al., 1988; Singh & Newstead, 1992). This would reduce ionic calcium and is the likely cause of the decreased Ca2+ activity during preheating. Evaporating the preheated milk increased Ca2+ activity. This is a consequence of an increase in the level of soluble Ca. However, although there is a five fold increase in milk concentration, the soluble Ca only increases by a factor of 2.2 as there is a shift in soluble calcium to the colloidal phase when milk is concentrated
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(Le Graet & Brule, 1982). Hence, the observed increase in Ca2+ activity caused by evaporation (e.g. from 0.74 to 1.00 mmol L1 for 70 1C preheating) was not as great as the increase in concentration (9% to 48% TS). Heating the milk concentrate (48% TS) at 69 1C for 80 s resulted in a decrease in Ca2+ activity. The difference in pH before (6.17) and after (6.16) heating the milk concentrate was minimal. Thus pH change was unlikely to be the cause of the decrease in Ca2+ activity due to concentrate heating. The observed reduction in Ca2+ activity after concentrate heating may have been caused by a heat-induced shift of Ca2+ into the colloidal phase. Heating milk concentrate (31.3% TS) under sterilisation conditions (120 1C) also causes a decrease in Ca2+ activity (Nieuwenhuijse et al., 1988). Reconstituted SMP (9% TS) had a lower Ca2+ activity 1 min after reconstitution, compared with the original skim milk. Holding the reconstituted SMP for 1 h at 20 1C increased Ca2+ activities, and holding at 4 1C for 20 h resulted in a further increase. However, the Ca2+ activity remained below that of the original skim milk. Thus the changes in Ca2+ activity caused by the powder manufacturing process were only partially reversible over time, when the SMP was reconstituted. The Ca2+ activities of reconstituted SMP made using 70 and 80 1C preheating treatments had slightly higher Ca2+ activities than those made using 100 and 120 1C. The pH of reconstituted samples varied slightly (70.02) over the time they were held for analysis. However, the effect of this pH change on Ca2+ activity would be relatively small; approximately 70.025 mmol L1 based on the data of Augustin and Clarke (1991). 3.3. Irreversible whey protein denaturation during powder manufacture The levels of native b-lg A, b-lg B, a-la and BSA (quantified by native-PAGE analysis) at different stages in the milk powder process are shown in Figs. 1A, 1B, 2A and 2B, respectively. Data shown is from experimental runs 1, and 6 to 8 (Table 1). The level of native
Table 2 Ca2+ activities of original skim milk, milk concentrates and reconstituted skim milk powders (measured at 20 1C) Sampling pointa
Original skim milk Flash vessel 3rd Effect Concentrate heating Powder 1 minb Powder 1 hb Powder 24 hb a
Ca2+ activity (mmol L1) 7 standard error. Skim milk heated at different preheating temperatures 70 1C
80 1C
100 1C
120 1C
0.9170.02 0.7470.01 1.0070.01 0.8770.01 0.6170.01 0.7570.02 0.8470.01
0.9170.02 0.6570.02 0.9570.02 0.6070.04 0.5970.01 0.7070.01 0.7770.01
0.9170.02 0.7270.01 0.8170.01 0.7570.01 0.5070.02 0.6470.02 0.7370.02
0.9170.02 0.5170.01 0.8270.01 0.5770.01 0.5470.01 0.6670.01 0.7270.02
Description of sampling points shown in Fig. 1 legend. Ca2+ activity of reconstituted skim milk powder 1 min, 1 h and 24 h (held for 20 h at 4 1C) after reconstitution.
b
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80
80
Residual native α-la (%)
100
Residual native β-lg A (%)
100
60
40
20
0 Milk
Fl
1E
(A)
2E
3E
CH
60
40
20
0 Milk
Powder
Sampling point
Fl
1E
(A)
2E
3E
CH
Powder
CH
Powder
Sampling point
100
80
60
40
20
0 Milk
(B)
Residual native BSA (%)
Residual native β-lg B (%)
100
Fl
1E
2E
3E
CH
80
60
40
20
0 Milk
Powder
Sampling point
Fl
(B) Fig. 1. Loss of b-lg A (A) and b-lg B (B) from samples collected during milk powder manufacture. Preheating temperatures; 70 (&), 80 (J), 100 (B), 120 1C (W), for 52 s. Sampling point abbreviations; original skim milk (Milk), flash vessel after preheating (Fl), concentrate from 1st, 2nd and 3rd evaporator effects (1E, 2E and 3E, respectively), heated concentrate sampled just before the spray dryer (CH) and reconstituted skim milk powder (Powder). An example of the standard error for duplicate samples is shown for concentrate from the 1st effect.
IgG at different stages is shown in Table 3. All concentrations were calculated back to the total solids of the original skim milk using Eq. (1). The processing step that had the largest effect on native whey protein concentration was preheating. As the preheating temperature increased from 70 to 120 1C, the amount of native whey protein in samples from the flash vessel decreased. It should be noted that preheating at 80, 100 or 120 1C for 52 s was sufficient to denature all the IgG. The concentration of individual whey proteins after preheating by DSI (from the flash vessel) were generally comparable with concentrations calculated from kinetic parameters derived on a DSI heated, UHTplant (Oldfield et al., 1998a). For example, after a preheat of 80 1C for 52 s, calculated concentrations of 2.0, 2.0 and 0.75 g kg1, were comparable with the measured concentrations of 2.0, 2.1 and 0.74 g kg1, for
1E
2E
3E
Sampling point
Fig. 2. Loss of a-la (A) and BSA (B) from samples collected during milk powder manufacture. Preheating temperatures; 70 (&), 80 (J), 100 (B), 120 1C (W), for 52 s. See Fig. 1 legend for a description of the sampling points. An example of the standard error for duplicate samples is shown for concentrate from the 1st effect. Table 3 Native IgG concentration sampled at different points in the powder manufacturing process Processing step
native IgG concentration (g kg1) Average of trial 1 and 2b
Original skim milk Flash vessela 1st effect 2nd effect 3rd effect Concentrate heating Powder
0.43 0.34 0.37 0.37 0.35 0.36 0.35
a
Preheating condition 70 1C for 52 s. Pooled standard deviation 0.027 g kg1.
b
b-lg A, b-lg B and a-la, respectively. In addition, after a preheat treatment of 70 1C for 52 s the calculated concentration of IgG (0.37 g kg1) was comparable to the average concentrations of 0.35 g kg1.
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During evaporation and drying there were only minor changes in the levels of native whey proteins, in comparison with the effect of preheating. A slight decrease occurred in the concentration of a-la during evaporation after preheating at 70, 80 and 120 1C (Fig 2a). This was also observed for BSA preheated at 70 1C. Preheating at 70 1C for 52 s reduced the concentration of native IgG (Po0:05), by 20% (Table 3). However, evaporation and drying had little further effect on the concentration of IgG (Po0:05). In order to determine the effect of concentrate heating temperature and dryer inlet/outlet temperatures, the lowest preheating condition of 70 1C for 52 s was chosen for runs 2–5 (Table 1). Thus any observed changes in the level of individual native whey protein was likely to be caused by the changes in concentrate heating or dryer temperatures. Concentrate from the third effect of the evaporator was heated to either 65, 69 or 74 1C and took 80 s to be pumped to the nozzle. Samples collected just before the dryer (nozzle) showed that varying the concentrate heating temperature from 65 to 74 1C had a negligible effect on the concentration of individual native whey proteins (Table 4). For example, the concentration of IgG did not change significantly when the concentrate heating temperature was varied (Po0:05). The dryer inlet and outlet air temperatures were manipulated (Table 4). Overall, any changes when the inlet/outlet air temperature was increased from 180 1C/ 90 1C to 200 1C/101 1C or decreased to 160 1C/89 1C were relatively minor, in comparison with the effect of preheating. Changing the inlet/outlet air temperatures did not significantly affect the concentration of IgG in the powder (Po0:05). The b-lg and a-la results from this study showed that preheating conditions had the biggest effect on whey protein denaturation and that the subsequent processing steps of evaporation, concentrate heating and drying had a relatively minor effect. Furthermore, the minor whey proteins, BSA and IgG, considered most heat
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sensitive in milk (Singh & Creamer, 1991), also followed similar trends. With the evaporator used in this study, minimal denaturation of all the whey proteins was observed, including the most heat sensitive ones, BSA and IgG. This is probably due to the evaporation temperature decreasing and the total solids levels in the milk increasing with each subsequent evaporator effect. The critical temperature above which irreversible denaturation occurs in milk is 70 1C (de Wit & Swinkels, 1980). Furthermore, the development of thiol groups, important in the formation of disulfide linked aggregates, begins at 72 1C (Kirchmeier, El–Shobery, & Kamal, 1984). Thus denaturation may be expected to occur in the first effect at 70 1C, however it would be unlikely to occur in the last effect at 41 1C, if the effect of temperature was solely considered. However, the residence time in the first effect of the evaporator was 1 min, and based on kinetic data for b-lg and a-la (Dannenberg & Kessler, 1988) the level of denaturation would be expected to be minimal (1%). The increasing total solids concentration, which occurs as evaporation progresses, also has an effect on the extent of whey protein denaturation. McKenna and O’Sullivan (1971) observed that increasing the concentration of skim milk solids from 9% to 44% TS reduces the degree of whey protein denaturation caused by heating at 80 1C for 20 min.This concentration effect is complicated, because individual whey proteins show different responses to total solids concentration. The heat-resistance of b-lg and immunoglobulin increases with increasing total solids in milk and cheese whey (Nielsen, Coulter, Morr, & Rosenau, 1973; Hillier et al., 1979; Ibrahim, Madkor, & Hewedy, 1995; Anema 2000). In contrast, the rate of a-la denaturation is virtually unaffected in the range of 9.6–38.4% TS in skim milk (Anema, 2001). However, a-la has a greater heat resistance than b-lg, BSA and IgG in milk at normal solids (Dannenberg & Kessler, 1988; Singh & Creamer, 1991; Oldfield et al., 1998a) and is thus unlikely to
Table 4 Effect of concentrate heating and dryer inlet and outlet temperatures on the concentration of individual native whey proteins in reconstituted SMP Processing conditions
Runa no.
b-lg A
b-lg B
a-la
BSA
IgGb
2 1 3
1.7 1.9 1.8
2.2 2.1 2.1
0.63 0.64 0.71
0.21 0.21 0.22
0.36 0.34 0.32
5 1 4
1.8 1.9 1.7
2.1 2.1 2.1
0.60 0.64 0.55
0.21 0.21 0.21
0.36 0.34 0.32
Concentrate heating temperature (1C) 65 69 74 Dryer inlet/outlet temperature (1C) 160/89 180/90 200/101 a
Individual native whey protein concentration (g kg1)
Preheating condition for these runs were 70 1C for 52 s (see Table 1). IgG results averaged over trial 1 and 2. Pooled standard deviation 0.027 g kg1.
b
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undergo significant levels of denaturation at evaporation temperatures. The protective effect of increased total solids is due to increased lactose concentration, although lactose does not appear to affect a-la denaturation (Plock, Spiegel, & Kessler, 1998). Moreover, increasing total protein concentration without increasing lactose and milk salt concentration has been shown to result in an increase in b-lg, a-la and BSA denaturation (Law & Leaver, 1997). After exiting the last effect of the evaporator, concentrate at 41 1C was heated to 65–74 1C prior to drying. This was to reduce concentrate viscosity, thereby improving atomisation and powder properties (de Vilder et al., 1979; Baldwin et al., 1980; Oldfield, Teehan, & Kelly, 2000a). Heat sensitive BSA and IgG were not greatly affected by the concentrate heating step (Table 4), even though the temperature was raised from 41 1C to 65–74 1C. The high level of total solids (48%) in the milk concentrate probably retarded the rate of whey protein denaturation (Anema, 2000). In addition, the effective holding time of 80 s from the concentrate heater to the spray dryer nozzle was insufficient to cause extensive denaturation. The range of spray drying conditions used (inlet air 160–200 1C and outlet air 87–101 1C) had little effect on the denaturation of the individual whey proteins, including BSA and IgG. Previous workers also found that spray drying had only a minor effect on the denaturation of b-lg A, b-lg B and a-la (Singh & Creamer, 1991; Guyomarc’h, Warin, Muir, & Leaver, 2000). Although there are a large number of spray dryer designs and configurations, the temperature of a droplet within the drying chamber is typically between the wet bulb temperature and the outlet air temperature, and does not exceed 70 1C until almost all the water has been evaporated (Singh & Newstead, 1992). As the concentrate is atomised it contacts the hot inlet air and water evaporates rapidly from the droplet surface. Evaporation quickly cools the droplet to a temperature above the wet bulb temperature (40–50 1C) and also cools the surrounding hot air. Thus the high inlet air temperature conditions, e.g. 200 1C, are unlikely to cause denaturation. As the droplet dries and falls to the bottom of the dryer its temperature will approach that of the outlet air temperature. Thus outlet air temperature is important and affects properties such as the insolubility index (de Vilder et al., 1976). In this work, high insolubility index values of 0.3 (Table 1) and 0.7 mL were observed when the inlet and outlet air temperature was raised to 200 and 101 1C, respectively. However this is unlikely to be caused by extensive whey protein denaturation, but rather by casein aggregation (Knipschildt, 1969). Other whey protein reactions could have occurred during milk powder manufacture. For example, the early stages of the Maillard reaction (lactosylation) have
been reported in heated milks (Leonil et al., 1997) and milk powders (Guyomarc’h et al., 2000). Spray drying and in particular the combination of low inlet and low outlet air temperature are critical to minimising lactosylation (Guyomarc’h et al., 2000). 3.4. Whey protein aggregation and association with the micelle Samples collected from the milk powder plant were analysed by PAGE techniques to quantify the different types of whey protein aggregates present. The distribution of different b-lg, and BSA aggregates formed during milk powder manufacture (preheating at 100 and 120 1C) are shown in Figs. 3 and 4, respectively. As the samples were cooled, and in the case of concentrates diluted to 9% TS before ultracentrifugation, any changes observed are likely to be the result of irreversible reactions. The extent of heat-induced reactions was not great enough at the preheating temperatures of 70 and 80 1C to observe any clear trends, and
Fig. 3. b-lg aggregates formed during skim milk powder manufacture. Preheating temperatures; 100 1C (A) and 120 1C (B), for 52 s. b-Lg species: native (U), hydrophobic aggregates (///), disulphide-linked aggregates (XXX) and aggregates associated with the micelle (’). See Fig. 1 legend for a description of the sampling points.
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and BSA, which allows them to readily participate in thiol/disulfide interchange reactions. Previous studies have shown that the level of denatured b-lg is highly correlated with the level of disulfide linked b-lg aggregates, in heated skim milk (Oldfield et al., 1998a) and in heated concentrated skim milk (Anema, 2000). Approximately 50% of the b-lg and 75% of the BSA were associated with the micelle after preheating at 120 1C for 52 s. There was a small increase in the amount of b-lg associated with the casein micelle in samples from the 3rd effect and after concentrate heating. However, the levels of b-lg associated with the micelle in reconstituted powder samples were similar to levels obtained in samples from the flash vessel. Singh and Creamer (1991) reported similar observations in milk concentrates and powders. A number of changes occur during evaporation that may promote heat-induced association reactions. The pH decreased during evaporation from 6.66 to 6.17, and this would have reduced protein charge and therefore facilitate association reactions. Greater levels of b-lg and a-la association with the casein micelle have been observed in heated milks adjusted to more acidic pH (Corredig & Dalgleish, 1996b; Oldfield, Singh, Taylor, & Pearce, 2000b; Anema & Li, 2003). In addition, Ca2+ activity increased as a result of evaporation (Table 2) and this may also have promoted association reactions (Smits, & van Brouwershaven, 1980; Visser, Minihan, Smits, Tjan, & Heertje, 1986). Fig. 4. BSA aggregates formed during skim milk powder manufacture. Preheating temperatures; 100 1C (A) and 120 1C (B), for 52 s. BSA species; native (U), hydrophobic aggregates (///), disulphide-linked aggregates (XXX) and aggregates associated with the micelle (’). See Fig. 1 legend for a description of the sampling points.
the results for a-la were variable due to the lower levels of denaturation and are not shown. Most of the b-lg and BSA heat-induced reactions occurred in the preheating section, after which there was relatively little change, with the reconstituted powder approximating the sample obtained from the flash vessel (after preheating). Preheating at 100 1C or 120 1C for 52 s caused extensive denaturation of b-lg and BSA (470–90%). After preheating, denatured b-lg and BSA were present as either aggregates in the supernatant or had co-sedimented with the casein micelles (assumed to be associated with the casein). The denatured b-lg and BSA formed aggregates in the supernatant that were predominantly disulfide-linked, with a small amount of hydrophobic aggregates (i.e. non-convalently linked aggregates). The predominance of disulfide-linked aggregates is probably due to the thiol group and a number of intermolecular disulfide bonds present in b-lg
4. Conclusions Irreversible denaturation of major (b-lg A, b-lg B and a-la) and minor (IgG and BSA) whey proteins in skim milk occurred mainly during preheating. In comparison, the effect of evaporation and spray drying was relatively minor. Varying concentrate heating and spray drying inlet/outlet temperatures over a range typical for milk powder manufacture did not greatly affect denaturation of the whey proteins measured. b-Lg and BSA formed mainly disulphide-linked aggregates. The levels of b-lg and BSA associated with the casein micelle were lower than their respective levels of denaturation. Aggregation and association of b-lg and BSA occurred predominantly during preheating, after which evaporation and spray drying had a negligible effect.
Acknowledgements The authors gratefully acknowledge Fonterra (formerly the New Zealand Dairy Board) for financial support, Mr. J. Grant and Mr. C. Knight for operating the milk powder plant, Dr. K. N. Pearce for useful discussion during the research and Fonterra Research
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Centre (formerly the New Zealand Dairy Research Institute) for the chemical analyses they performed and the use of the milk powder plant.
References Anema, S. G. (2000). Effect of milk concentration on the irreversible thermal denaturation and disulfide aggregation of b-lactoglobulin. Journal of Agricultural and Food Chemistry, 48, 4168–4175. Anema, S. G. (2001). Kinetics of the irreversible thermal denaturation and disulfide aggregation of a-lactalbumin in milk samples of various concentrations. Journal of Food Science, 66(1), 2–9. Anema, S. G., & Li, Y. (2003). Effect of pH on the association of denatured whey proteins with casein micelles in heated reconstituted skim milk. Journal of Agricultural and Food Chemistry, 51, 1640–1646. Augustin, M. A., & Clarke, P. T. (1991). Calcium ion activities of cooled and aged reconstituted and recombined milks. Journal of Dairy Research, 58, 219–229. Baldwin, A. J., Baucke, A. G., & Sanderson, W. B. (1980). The effect of concentrate viscosity on the properties of spray dried skim milk powder. New Zealand Journal of Dairy Science and Technology, 15, 289–297. Corredig, M., & Dalgleish, D. G. (1996a). The binding of alactalbumin and b-lactoglobulin to casein micelles in milk treated by different heating systems. Milchwissenschaft, 51, 123–127. Corredig, M., & Dalgleish, D. G. (1996b). Effect of temperature and pH on the interactions of whey proteins with casein micelles in skim milk. Food Research International, 29, 49–55. Dannenberg, F., & Kessler, H. G. (1988). Reaction kinetics of the denaturation of whey proteins in milk. Journal of Food Science, 53, 258–263. de Vilder, J., & Martens, R. (1976). Heat classification of skim milk powder. II. influence of the heat treatment during manufacture. Revue de l’agriculture,, 29(4), 887–900. de Vilder, J., Martens, R., & Naudts, M. (1976). Influence of process variables on some whole milk powder characteristics. Milchwissenschaft, 31, 396–400. de Vilder, J., Martens, R., & Naudts, M. (1979). The influence of the dry matter content, the homogenization and heating of concentrate on physical characteristics of whole milk powder. Milchwissenschaft, 34, 78–84. de Wit, J. N., & Swinkels, G. A. M. (1980). A differential scanning calorimetric study of the thermal denaturation of bovine blactoglobulin. Biochimica et Biophysica Acta, 624, 40–50. Greets, J. P., Bekhof, J. J., & Scherjon, J. W. (1983). Determination of calcium ion activities in milk with an ion selective electrode. A linear relationship between the logarithm of time and the recovery of calcium ion activity after heat treatment. Netherlands Milk and Dairy Journal, 37, 197–211. Guyomarc’h, F., Warin, F., Muir, D. D., & Leaver, J. (2000). Lactosylation of milk proteins during the manufacture and storage of skim milk powder. International Dairy Journal, 10, 863–872. Hillier, R. M., Lyster, R. L. J., & Cheeseman, G. C. (1979). Thermal denaturation of a-lactalbumin and b-lactoglobulin in cheese whey: effect of total solids concentration and pH. Journal of Dairy Research, 46, 103–111. Ibrahim, F. S., Madkor, S. A., & Hewedy, M. M. (1995). The effect of pH, sugars and calcium ion concentration on the thermal stability of whey proteins. Egyptian Journal of Dairy Science, 23, 177–188. IDF Standard 9C (1987). Dried milk, dried whey, dried buttermilk and dired butter serum determination of fat content (Rose Gottlieb reference method). International Dairy Federation, Brussels, Belgium.
IDF Standard 129A (1988). Dried milk and dried milk products. Determination of insolubility index. International Dairy Federation, Brussels, Belgium. IDF provisional standard 20B (1993a). Milk. Determination of nitrogen content. International Dairy Federation, Brussels. IDF provisional standard 26A (1993b). Dried milk and dried cream. Determination of water content. International Dairy Federation, Brussels. Kelly, J., Kelly, P. M., & Harrington, D. (2002). Influence of processing variables on the physicochemical properties of spray dried fat-based milk powders. Lait, 82, 401–412. Kinghorn, N. M., Norris, C. S., Paterson, G. R., & Otter, D. C. (1995). Comparison of capillary electrophoresis with traditional methods to analyse bovine whey proteins. Journal of Chromatography A, 700, 111–123. Knipschildt, M. E. (1969). Recent developments in drying techniques. Journal of the Society of Dairy Technology, 22(4), 201–216. Kirchmeier, O., El-Shobery, M., & Kamal, N. M. (1984). Heat treatment of milk and development of SH-groups. Milchwissenschaft, 39, 715–717. Law, A. J. R., & Leaver, J. (1997). Effect of protein concentration on rates of thermal denaturation of whey proteins in milk. Journal of Agricultural and Food Chemistry, 45, 4255–4261. Le Graet, Y., & Brule, G. (1982). Effect of concentration and drying on mineral equilibria of skim milk and retentates. Lait, 62, 113–125. Leonil, J., Molle, D., Fauquant, J., Maubois, J. L., Pearce, R. J., & Bouhallab, S. (1997). Characterization by ionisation mass spectrometry of lactosyl b-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose binding site. Journal of Dairy Science, 80(10), 2270–2281. Manji, B., & Kakuda, Y. (1986). Thermal denaturation of whey proteins in skim milk. Canadian Institute of Food Science and Technology Journal, 19, 161–166. McDowell, A. K. R. (1972). A comparison of various methods for the estimations of solids-non-fat in milk and whey. Journal of Dairy Research, 39, 251–259. McKenna, B. M., & O’Sullivan, A. C. (1971). Whey protein denaturation in concentrated skim milks. Journal of Dairy Science, 54, 1075–1077. Nielsen, M. A., Coulter, S. T., Morr, C. V., & Rosenau, J. R. (1973). Four factor response surface experimental design for evaluating the role of processing variables upon protein denaturation in heated whey systems. Journal of Dairy Science, 56(1), 76–83. Nieuwenhuijse, J. A., Timmermans, W., & Walstra, P. (1988). Calcium and phosphate partitions during the manufacture of sterilized concentrated milk and their relations to the heat stability. Netherlands Milk and Dairy Journal, 42, 387–421. O’Connor, F., McKenna, B. M., & O’Sullivan, A. C. (1969). Bacteriological and whey protein denaturation aspects of heating processes used in the manufacture of low-heat skim-milk powder. Irish Journal of Agricultural Research, 8, 417–430. Oldfield, D. J., Singh, H., Taylor, M. W., & Pearce, K. N. (1998a). Kinetics of Denaturation and aggregation of whey proteins in skim milk heated in an ultra-high temperature (UHT) pilot plant. International Dairy Journal, 8, 311–318. Oldfield, D. J., Singh, H., & Taylor, M. W. (1998b). Association of blactoglobulin and a-laclabumin with the casein micelles in skim milk heated in an ultra-high temperature plant. International Dairy Journal, 8, 765–770. Oldfield, D. J., Teehan, C. M., & Kelly, P. M. (2000a). The effect of preheat treatment and other process parameters on the coffee stability of instant whole milk powder. International Dairy Journal, 10, 659–667. Oldfield, D. J., Singh, H., Taylor, M. W., & Pearce, K. N. (2000b). Heat-induced interactions of b-Lactoglobulin and a-Lactalbumin
ARTICLE IN PRESS D.J. Oldfield et al. / International Dairy Journal 15 (2005) 501–511 with the casein micelle in pH adjusted skim milk. International Dairy Journal, 10, 509–518. Plock, J., Spiegel, T., & Kessler, H. G. (1998). Influence of the lactose concentration on the denaturation kinetics of whey proteins in concentrated sweet whey. Milchwissenschaft, 53, 389–393. Sanderson, W. B. (1970). Determination of undenatured whey protein nitrogen in skim milk powder by dye binding. New Zealand Journal of Dairy Science and Techonology, 5, 46–52. Singh, H., & Creamer, L. K. (1991). Denaturation, aggregation and heat stability of milk protein during the manufacture of skim powder. Journal of Dairy Research, 58, 269–283. Singh, H., & Newstead, D. F. (1992). Aspects of proteins in milk powder manufacture. In Fox, P. F. (Ed.), Advanced Dairy
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Chemistry-1: Proteins (pp. 735–765). England: Elsevier Science Publishers Ltd. Smits, P., & van Brouwershaven, J. H. (1980). Heat-induced association of b-lactoglobulin and casein micelles. Journal of Dairy Research, 47, 313–325. Swanson, A. M., & Sanderson, W. B. (1964). The effect of heat treatments given to skim milk and skim milk concentrate before drying. Cereal Science Today, 9, 292–296. van Boekel, M. A. J. S., & Walstra, P. (1989). Steric exclusion of serum proteins with respect to (para)casein micelles. Netherlands Milk and Dairy Journal, 43, 437–446. Visser, J., Minihan, A., Smits, P., Tjan, S. B., & Heertje, I. (1986). Effects of pH and temperature on the milk salt system. Netherlands Milk and Dairy Journal, 40, 351–368.
International Dairy Journal 15 (2005) 501–511 www.elsevier.com/locate/idairyj
Effect of preheating and other process parameters on whey protein reactions during skim milk powder manufacture D.J. Oldfielda,c,, M.W. Taylora, H. Singhb a
Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand b Riddet Centre, Massey University, Palmerston North, New Zealand c Whole Protein Technical Centre, Fonterra Cooperative Group Ltd, Edgecumbe, New Zealand Received 23 March 2004; accepted 15 September 2004
Abstract Skim milk powder was manufactured in a milk powder plant using different preheating temperatures, concentrate heating temperatures and spray drying temperatures. Varying the preheating conditions from 70 1C for 52 s to 120 1C for 52 s had a marked effect on the denaturation of b-lactoglobulin A, b-lactoglobulin B, a-lactalbumin, bovine serum albumin (BSA), and immunoglobulin G. In contrast, varying concentrate heating temperature (65–74 1C) and inlet/outlet air dryer temperature (200/ 101 1C–160/89 1C) had a minimal effect on whey protein denaturation. Most of the whey protein denaturation and association with the casein micelle occurred in the preheating section of the powder plant. Aggregation of b-lactoglobulin (b-lg) and BSA predominantly involved disulphide bonds. Although, greater than 90% of the b-lg and BSA was denatured after preheating at 120 1C for 52 s, the extent of association with the casein micelle was lower, 50% for b-lg and 75% for BSA. r 2004 Elsevier Ltd. All rights reserved. Keywords: Skim milk powder; Evaporation; Spray drying; Denaturation; Aggregation; Whey protein
1. Introduction Skim milk powder (SMP) is widely used as an ingredient in many formulated foods. Soups, sauces, recombined evaporated milk, confectionery and bakery products all benefit from the functional properties provided by skim milk powder. The powder can be tailored to a specific end-use by manipulating processing conditions during powder manufacture. The manufacturing process for SMP involves heating the skim milk (known as preheating), concentrating the skim milk solids by evaporation to 45–50% total solids, heating the skim milk concentrate and then spray drying the milk concentrate to produce a powder. Preheating Corresponding author. Whole Protein Technical Centre, Fonterra Cooperative Limited, Edgecumbe, New Zealand. Tel.: +64 7 304 7000; Fax +64 6 304 7063. E-mail address: david.oldfi[email protected] (D.J. Oldfield).
0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.09.004
conditions are used to a large extent to control the functional properties of the powder. A number of changes occur in milk during preheating: whey protein denaturation, association of denatured whey proteins with the casein micelle, transfer of soluble calcium and phosphate to the colloidal phase, destruction of bacteria and inactivation of enzymes (Singh & Newstead, 1992). However, it is the extent of whey protein denaturation that is used to broadly classify SMP for use in different applications. Heat classification is controlled by the degree of preheating and is measured by the whey protein nitrogen index (WPNI). Depending on the severity of the preheating conditions, SMP can be classified as a low-, medium- or high-heat powder. Although there are a large number of studies on heatinduced whey protein denaturation in fresh milk (Hillier, Lyster, & Cheeseman, 1979; Manji & Kakuda, 1986; Dannenberg & Kessler, 1988) they have largely
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been carried out by heating milk in water and oil baths under laboratory scale conditions. Moreover, the rate of heating is important and commercial heating equipment can produce quite different levels of whey protein reactions compared to heating on a laboratory scale (Corredig & Dalgleish, 1996a; Oldfield, Singh, & Taylor, 1998b). The effect of different processing steps in milk powder manufacture, on total whey protein denaturation has been investigated by measuring the WPNI of samples collected at different points in a powder plant (Swanson & Sanderson 1964; O’Connor, McKenna, & O’Sullivan, 1969; de Vilder & Martens, 1976). However, WPNI only provides an overall level of protein denaturation. Individual whey proteins have different levels of thermal stability in milk and there are very few studies reporting how the denaturation of individual whey proteins is affected by milk powder manufacture. Singh and Creamer (1991) investigated the effect of high heat treatments (110–120 1C) and a low heat treatment (72 1C for 15 s) on the denaturation and aggregation reactions of a-lactalbumin (a-la) and blactoglobulin (b-lg) at different steps during milk powder manufacture. High heat treatments denatured most of the whey proteins, evaporation further increased the extent of denaturation and spray drying had only a small effect. However, little is known of the whey proteins most susceptible to heat denaturation, immunoglobulins and bovine serum albumin (BSA), and what effects different processing steps have on their denaturation. In addition to preheat treatment before evaporation, concentrate heating temperature and dryer inlet/outlet temperatures are known to affect important physical properties of milk powder such as moisture, solubility index and bulk density (de Vilder, Martens, & Naudts, 1976, 1979; Baldwin, Baucke, & Sanderson, 1980; Kelly, Kelly & Harrington, 2002). However, the effect of concentrate heating and drying temperatures on the heat-induced reactions of individual whey proteins has not been reported.
Despite the importance of milk powder manufacture and the wide range of uses for milk powders, very little is understood of the chemical changes that occur during processing, especially during evaporation and spray drying. This research work was carried out to investigate the effect of milk powder processing conditions on whey protein reactions.
2. Materials and methods 2.1. Skim milk powder manufacture Whole milk obtained from Fonterra Cooperative Ltd (Longburn, New Zealand) was separated without pasteurisation at 55 1C, at the Fonterra Research Centre (FRC) pilot plant. The skim milk was then processed into skim milk powder. Eight different runs were carried out with a combination of different preheating temperatures, concentrate heating temperatures and dryer inlet/outlet temperatures (Table 1). Subsequently these set of eight runs were repeated at a different time of the dairying season. It was not possible to average the data from the two trials because of differences in milk composition. Hence, the results of one trial are presented here: similar trends were observed for the other trial. An exception was the IgG results, which had the same initial concentration in both trials. Thus the IgG results presented are an average of both trials, and a pooled standard deviation was calculated. Chilled skim milk from the silo tanks were passed through a number of tubular heat exchangers housed in the calandria (steam chests) of the evaporator effects. This heated the milk from 8 to 65 1C. Skim milk was preheated by direct steam injection (DSI) to either 70, 80, 100 or 120 1C, pumped through a 52 s holding tube and then into a flash vessel, which instantaneously reduced the temperature of the heated milk to 70–72 1C. Skim milk was concentrated in a three effect
Table 1 Processing conditions used in the manufacture of skim milk powder and properties of the powders manufactured Run No.
1 2 3 4 5 6 7 8
Processing conditions (average temperatures in 1C)7standard deviationa
Powder properties (average value)7standard deviation
Preheating
Concentrate heating
Dryer inlet air
Dryer outlet air
Moisture (% w w1)
Insolubility index (mL)
WPNI (mg g1)
7070.1 7070.1 7070.1 7070.0 7070.2 12070.5 10070.1 8070.1
6970.9 6571.1 7470.8 6970.7 6971.5 6970.3 6970.2 6970.3
18071.1 18070.3 18070.4 20071.9 16070.5 18070.3 18070.3 18070.8
9071.2 9270.4 9270.6 10171.1 8970.7 8770.4 8770.2 8972.0
4.470.0 4.670.1 4.670.1 4.070.3 4.470.1 5.070.3 4.670.1 4.470.3
0.270.1 0.370.1 0.170.0 0.370.3 0.270.0 0.170.0 0.170.0 0.170.0
8.070.3 7.870.1 7.970.3 7.970.1 7.970.2 1.570.1 2.770.1 7.370.9
WPNI: Whey Protein nitrogen index. a Processing conditions were recorded during each run. These data was averaged and the standard deviation calculated.
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falling film evaporator (Weigand, Karlsruhe, Germany) at a feed rate of 1700 L h1. The temperatures of the 1st, 2nd and 3rd effects were 72, 53 and 41 1C, respectively. Concentrated skim milk (milk concentrate) exiting from the last effect at 47–49% total solids (TS) was then heated in a scraped surface heat exchanger (Model VT422, APV Crepaco Inc., Chicago, Illinois) to either 65, 69 or 74 1C. The heated milk concentrate took 80 s to reach the nozzle atomizer of a pilot scale dryer with an integral fluid bed (IFB dryer, FRC design, FRC, Palmerston North, New Zealand). The feed rate of the milk concentrate to the dryer was 140 L h1. The inlet and outlet temperatures were varied from 160 to 200 1C and from 87 to 100 1C, respectively. The integral fluid bed was supplied with air at 85 1C. The powder was dried to a final target moisture content of o5%. 2.2. Sample collection from the milk powder plant For each run, skim milk from the flash vessel, milk concentrate from the 1st, 2nd and 3rd effects and milk concentrate just before the spray dryer (after concentrate heating) were collected once the plant had reached steady state conditions. All the samples were cooled to approximately room temperature in an ice water bucket. The milk concentrate samples were then diluted, using distilled and deionised water, to the total solids (TS) of the original skim milk (9% TS) and stirred for 2 h. Skim milk powder (SMP) samples, obtained from the powder outlet of the dryer, were reconstituted (9% TS) in distilled and deionised water at 40 1C, and stirred for 2 h at room temperature. 2.3. Protein analysis The original skim milk, samples collected from the milk powder plant, and reconstituted milk powder samples, were ultracentrifuged at 175,000 g for 1 h at 20 1C (Beckman L8-80 M, Beckman, Palo Alto, California). After ultracentrifugation, the supernatant was carefully poured off for protein analysis. The supernatants were analysed for b-lactoglobulin genetic variant A (b-lg A), b-lactoglobulin genetic variant B (b-lg B), a-lactablumin (a-la) and bovine serum albumin (BSA) by polyacrylamide gel electrophoresis (PAGE). Quantitative PAGE analysis was performed on a mini-gel electrophoresis unit (Biorad Laboratories, Richmond, CA, USA), under non-dissociating (nativePAGE), dissociating but non-reducing (SDSNR-PAGE) and dissociating and reducing (SDSR-PAGE) conditions as described by Oldfield, Singh, Taylor, and Pearce (1998a) and Oldfield et al. (1998b). The gels were scanned on a Computing Densitometer (Molecular Dynamics, Sunnyvale, CA) and the integrated intensities
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of the individual protein bands were calculated by a software program, ImageQuant (Molecular Dynamics). Standard curves prepared from purified b-lg A, b-lg B, a-la and BSA (Sigma Corp, St. Louis, MO, USA) were used to determine individual whey protein concentrations (g kg1). Under SDSNR- and SDSR-PAGE running conditions b-lg A and b-lg B bands ran together as a single total b-lg band. The standard error of the PAGE analysis was 6%. The different PAGE methods identified a number of forms of protein, and the following classifications are based on the electrophoretic running conditions used. Quantitative native-PAGE, run under non-dissociating conditions, was used to determine the amount of ‘‘native’’ b-lg A, b-lg B, a-la and BSA present. As the samples were cooled to room temperature before ultracentrifugation and PAGE analysis, any loss from native PAGE should be considered the result of irreversible denaturation leading to aggregate formation. Thus, under the running conditions it was not possible to differentiate between native protein, unfolded monomeric protein and lactosylated whey protein (Kinghorn, Norris, Paterson, & Otter, 1995). Two types of aggregates in the supernatant were quantified by SDSNR- and SDSR-PAGE. SDSNRPAGE conditions (0.2% (w v1) SDS) measured both ‘‘native’’ protein and aggregates in the supernatant that were not linked via disulfide bonds. These aggregates are likely to have formed through non-covalent and hydrophobic interactions and are referred to as ‘‘hydrophobic aggregates’’. Analysis of the supernatant by SDSR-PAGE measured ‘‘total whey protein’’ present. Under the dissociating and reducing conditions (0.2% (w v1) SDS+5% (v v1) 2-mercaptoethanol) disulfide bonds and hydrophobic interactions were broken and all the aggregates, both disulfide-linked and hydrophobic, dissociated into monomers, which could be resolved on the gel along with any native protein present. The amount of whey protein that co-sedimented with the casein micelles was calculated from the difference in concentration between the whey proteins in the original skim milk and the total whey protein in the supernatant (quantified by SDSR-PAGE) of the sample. Whey proteins that co-sedimented with the casein micelles were assumed to be ‘‘associated with the micelle’’ primarily via sulphydryl-disulfide interchange reactions between b-lg and k-casein at the micelle surface. It is possible that b-lg self aggregation formed insoluble aggregates that were large enough to also sediment with the micelles under the centrifugation conditions used. These aggregates would be undistinguishable from associated b-lg/k-casein complexes. Immunoglobulin G (IgG) concentration in the supernatant was determined by an affinity column (HiTrap affinity column, Protein G, Pharmacia, Uppsala,
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Sweden) according to the method of Kinghorn et al. (1995). Standard error of the readings was 70.01 g kg1. 2.4. Concentration and dilution calculations To correct for the changes in protein concentration due to dilution/concentration effects, the concentration of whey protein measured in the supernatant was recalculated back to an equivalent concentration in the original skim milk. This calculation was carried out in two steps. Centrifugation causes an increase in the concentration of whey proteins due to the casein micelles sedimenting, and thus water associated with the sedimented micelles is unavailable as solvent (van Boekel, & Walstra, 1989). Thus the first step was to recalculate the concentrations of individual whey proteins in the supernatant of ultracentrifuged samples to their respective value in the sample before centrifugation, by multiplying by a correction factor (F) as described by van Boekel, & Walstra (1989) (Eq. (1)). In the second step, the concentration of individual whey proteins in the sample prior to centrifugation was converted to a concentration in the original skim milk. This was done by comparing the total solids of the original skim milk with the total solids of the sample that was centrifuged (Eq. (1)). Total solids were measured by drying for 5 h in a 102 1C oven (McDowell, 1972). C milk ¼ C supernatant F
TSmilk ; TSsample
tion using an activity coefficient. The activity coefficients used were 0.3 for milk concentrates (Nieuwenhuijse, Timmermans, & Walstra, 1988) and 0.4 for skim milk at 9% TS (Greets et al., 1983). After milk is heated, the Ca2+ concentration and Ca2+ activity decrease. This decrease is reversible upon cooling milk. The recovery of Ca2+ activity at a constant temperature (20 1C) follows a logarithmic relationship, and extrapolation of the logarithmic relationship back to 1 min allows for a calculation of the Ca2+ activity close to heating conditions (Greets et al., 1983). The logarithmic relationship was used to calculate the Ca2+ activities of skim milk samples from the flash vessel, and milk concentrate samples (undiluted: 48% TS) from the 3rd effect and from just before the spray dryer (concentrate heating samples). Samples were cooled in iced water to 20 1C within 4 min, and then measured with a calcium ion selective electrode. In addition, the logarithmic relationship was used to analyse reconstituted skim milk powders by extrapolating the Ca2+ activity readings back to 1 min, 1 h and 24 h after reconstitution. Milk powder samples were reconstituted in 40 1C water, stirred for 1 h, and cooled to room temperature (20 1C) before they were measured with the calcium ion selective electrode, over a period of 3 h. The same samples were then held at 4 1C for 20 h, and warmed to 20 1C before readings were taken.
(1)
where Cmilk is the concentration in the original skim milk (g kg1), Csupernatant the concentration in the supernatant (g kg1), F the correction factor (=0.917), TSmilk the total solids of the original skim milk (% w w1), and TSsample the total solids of the sample (% w w1). Duplicate samples of concentrate from the 1st effect of the evaporator were analysed to determine the standard error of the individual whey protein concentrations. 2.5. Ca2+ activity measurements The Ca2+ concentrations in the original skim milk, milk concentrates and reconstituted skim milk powder were determined using a calcium ion selective electrode (F2112Ca Radiometer, Copenhagen, Denmark) attached to a digital readout pH meter (Radiometer), using the method of Greets, Bekhof, and Scherjon (1983). Samples were measured at 20 1C in a 100 mL beaker, with moderate stirring. Calibrations were performed using CaCl2/KCl standards (Radiometer) with ionic strengths of 0.08 and 0.2 mol L1 for milk and concentrated milk sample readings, respectively. The Ca2+ activity was calculated from the Ca2+ concentra-
2.6. Proximate analysis Total nitrogen (TN) was determined using the IDF standard method (1993a). Skim milk powder was analysed for whey protein nitrogen index (WPNI) using the method of Sanderson (1970). Insolubility index was determined using the IDF method (1988). Powder moisture (IDF, 1993b) and fat content (IDF, 1987) were measured. Ash content of the milk powder was measured after incineration in a muffled furnace at 550 1C. Reported values are an average of two analyses. The pH of the original skim milk, milk concentrate and reconstituted milk powder samples were measured at 20 1C using an Orion pH meter and probe (Model 9155/56 probe, Model 720 pH/ISE meter; Orion Research Incorporated, Boston, MA). Reported values are an average of two analyses.
3. Results and discussion 3.1. Milk powder manufacture The concentration of individual whey proteins in the original skim milk was: b-lg A 2.2 g kg1, b-lg B 2.3 g kg1, a-la 0.80 g kg1, BSA 0.25 g kg1 and IgG
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0.43 g kg1. Moisture content of the SMP was typically around 4.0–4.6%, except for run 6, which had the highest moisture content of 5.0%, probably due to the low inlet dryer temperature (160 1C). The insolubility index of powders ranged from 0.1 to 0.3 mL. The pH of the original skim milk was 6.67. Different preheating temperatures, concentrate heating temperatures and drying conditions had very little effect on pH, measured at 20 1C. However, as expected, there was a decrease in pH during the evaporation process. The variation in pH for different preheating temperatures was 70.03. Average pH values were: flash vessel 6.66, 1st effect 6.64 (12% TS), 2nd effect 6.47 (20% TS), 3rd effect 6.17 (48% TS), and milk concentrate just before the spray dryer 6.16 (after concentrate heating). After SMP was reconstituted to 9% TS, the pH returned to a value (pH 6.68) similar to that of the original skim milk. 3.2. Ca2+ activity The Ca2+ activities of skim milk, milk concentrates (not diluted; 48% TS) and reconstituted skim milk powder (9% TS) are shown in Table 2. The original skim milk had an average Ca2+ activity of 0.91 mmol L1 with a standard error of 70.02 mmol L1. Greets et al. (1983) reported a Ca2+ activity of 0.82–0.88 mmol L1 and Nieuwenhuijse et al. (1988) a value of 0.87–0.90 mmol L1 for raw milk. Preheating at all the temperatures investigated reduced Ca2+ activity (Table 2). Heating milk causes a transfer of Ca and P from the soluble to the colloidal phase (Nieuwenhuijse et al., 1988; Singh & Newstead, 1992). This would reduce ionic calcium and is the likely cause of the decreased Ca2+ activity during preheating. Evaporating the preheated milk increased Ca2+ activity. This is a consequence of an increase in the level of soluble Ca. However, although there is a five fold increase in milk concentration, the soluble Ca only increases by a factor of 2.2 as there is a shift in soluble calcium to the colloidal phase when milk is concentrated
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(Le Graet & Brule, 1982). Hence, the observed increase in Ca2+ activity caused by evaporation (e.g. from 0.74 to 1.00 mmol L1 for 70 1C preheating) was not as great as the increase in concentration (9% to 48% TS). Heating the milk concentrate (48% TS) at 69 1C for 80 s resulted in a decrease in Ca2+ activity. The difference in pH before (6.17) and after (6.16) heating the milk concentrate was minimal. Thus pH change was unlikely to be the cause of the decrease in Ca2+ activity due to concentrate heating. The observed reduction in Ca2+ activity after concentrate heating may have been caused by a heat-induced shift of Ca2+ into the colloidal phase. Heating milk concentrate (31.3% TS) under sterilisation conditions (120 1C) also causes a decrease in Ca2+ activity (Nieuwenhuijse et al., 1988). Reconstituted SMP (9% TS) had a lower Ca2+ activity 1 min after reconstitution, compared with the original skim milk. Holding the reconstituted SMP for 1 h at 20 1C increased Ca2+ activities, and holding at 4 1C for 20 h resulted in a further increase. However, the Ca2+ activity remained below that of the original skim milk. Thus the changes in Ca2+ activity caused by the powder manufacturing process were only partially reversible over time, when the SMP was reconstituted. The Ca2+ activities of reconstituted SMP made using 70 and 80 1C preheating treatments had slightly higher Ca2+ activities than those made using 100 and 120 1C. The pH of reconstituted samples varied slightly (70.02) over the time they were held for analysis. However, the effect of this pH change on Ca2+ activity would be relatively small; approximately 70.025 mmol L1 based on the data of Augustin and Clarke (1991). 3.3. Irreversible whey protein denaturation during powder manufacture The levels of native b-lg A, b-lg B, a-la and BSA (quantified by native-PAGE analysis) at different stages in the milk powder process are shown in Figs. 1A, 1B, 2A and 2B, respectively. Data shown is from experimental runs 1, and 6 to 8 (Table 1). The level of native
Table 2 Ca2+ activities of original skim milk, milk concentrates and reconstituted skim milk powders (measured at 20 1C) Sampling pointa
Original skim milk Flash vessel 3rd Effect Concentrate heating Powder 1 minb Powder 1 hb Powder 24 hb a
Ca2+ activity (mmol L1) 7 standard error. Skim milk heated at different preheating temperatures 70 1C
80 1C
100 1C
120 1C
0.9170.02 0.7470.01 1.0070.01 0.8770.01 0.6170.01 0.7570.02 0.8470.01
0.9170.02 0.6570.02 0.9570.02 0.6070.04 0.5970.01 0.7070.01 0.7770.01
0.9170.02 0.7270.01 0.8170.01 0.7570.01 0.5070.02 0.6470.02 0.7370.02
0.9170.02 0.5170.01 0.8270.01 0.5770.01 0.5470.01 0.6670.01 0.7270.02
Description of sampling points shown in Fig. 1 legend. Ca2+ activity of reconstituted skim milk powder 1 min, 1 h and 24 h (held for 20 h at 4 1C) after reconstitution.
b
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506
80
80
Residual native α-la (%)
100
Residual native β-lg A (%)
100
60
40
20
0 Milk
Fl
1E
(A)
2E
3E
CH
60
40
20
0 Milk
Powder
Sampling point
Fl
1E
(A)
2E
3E
CH
Powder
CH
Powder
Sampling point
100
80
60
40
20
0 Milk
(B)
Residual native BSA (%)
Residual native β-lg B (%)
100
Fl
1E
2E
3E
CH
80
60
40
20
0 Milk
Powder
Sampling point
Fl
(B) Fig. 1. Loss of b-lg A (A) and b-lg B (B) from samples collected during milk powder manufacture. Preheating temperatures; 70 (&), 80 (J), 100 (B), 120 1C (W), for 52 s. Sampling point abbreviations; original skim milk (Milk), flash vessel after preheating (Fl), concentrate from 1st, 2nd and 3rd evaporator effects (1E, 2E and 3E, respectively), heated concentrate sampled just before the spray dryer (CH) and reconstituted skim milk powder (Powder). An example of the standard error for duplicate samples is shown for concentrate from the 1st effect.
IgG at different stages is shown in Table 3. All concentrations were calculated back to the total solids of the original skim milk using Eq. (1). The processing step that had the largest effect on native whey protein concentration was preheating. As the preheating temperature increased from 70 to 120 1C, the amount of native whey protein in samples from the flash vessel decreased. It should be noted that preheating at 80, 100 or 120 1C for 52 s was sufficient to denature all the IgG. The concentration of individual whey proteins after preheating by DSI (from the flash vessel) were generally comparable with concentrations calculated from kinetic parameters derived on a DSI heated, UHTplant (Oldfield et al., 1998a). For example, after a preheat of 80 1C for 52 s, calculated concentrations of 2.0, 2.0 and 0.75 g kg1, were comparable with the measured concentrations of 2.0, 2.1 and 0.74 g kg1, for
1E
2E
3E
Sampling point
Fig. 2. Loss of a-la (A) and BSA (B) from samples collected during milk powder manufacture. Preheating temperatures; 70 (&), 80 (J), 100 (B), 120 1C (W), for 52 s. See Fig. 1 legend for a description of the sampling points. An example of the standard error for duplicate samples is shown for concentrate from the 1st effect. Table 3 Native IgG concentration sampled at different points in the powder manufacturing process Processing step
native IgG concentration (g kg1) Average of trial 1 and 2b
Original skim milk Flash vessela 1st effect 2nd effect 3rd effect Concentrate heating Powder
0.43 0.34 0.37 0.37 0.35 0.36 0.35
a
Preheating condition 70 1C for 52 s. Pooled standard deviation 0.027 g kg1.
b
b-lg A, b-lg B and a-la, respectively. In addition, after a preheat treatment of 70 1C for 52 s the calculated concentration of IgG (0.37 g kg1) was comparable to the average concentrations of 0.35 g kg1.
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During evaporation and drying there were only minor changes in the levels of native whey proteins, in comparison with the effect of preheating. A slight decrease occurred in the concentration of a-la during evaporation after preheating at 70, 80 and 120 1C (Fig 2a). This was also observed for BSA preheated at 70 1C. Preheating at 70 1C for 52 s reduced the concentration of native IgG (Po0:05), by 20% (Table 3). However, evaporation and drying had little further effect on the concentration of IgG (Po0:05). In order to determine the effect of concentrate heating temperature and dryer inlet/outlet temperatures, the lowest preheating condition of 70 1C for 52 s was chosen for runs 2–5 (Table 1). Thus any observed changes in the level of individual native whey protein was likely to be caused by the changes in concentrate heating or dryer temperatures. Concentrate from the third effect of the evaporator was heated to either 65, 69 or 74 1C and took 80 s to be pumped to the nozzle. Samples collected just before the dryer (nozzle) showed that varying the concentrate heating temperature from 65 to 74 1C had a negligible effect on the concentration of individual native whey proteins (Table 4). For example, the concentration of IgG did not change significantly when the concentrate heating temperature was varied (Po0:05). The dryer inlet and outlet air temperatures were manipulated (Table 4). Overall, any changes when the inlet/outlet air temperature was increased from 180 1C/ 90 1C to 200 1C/101 1C or decreased to 160 1C/89 1C were relatively minor, in comparison with the effect of preheating. Changing the inlet/outlet air temperatures did not significantly affect the concentration of IgG in the powder (Po0:05). The b-lg and a-la results from this study showed that preheating conditions had the biggest effect on whey protein denaturation and that the subsequent processing steps of evaporation, concentrate heating and drying had a relatively minor effect. Furthermore, the minor whey proteins, BSA and IgG, considered most heat
507
sensitive in milk (Singh & Creamer, 1991), also followed similar trends. With the evaporator used in this study, minimal denaturation of all the whey proteins was observed, including the most heat sensitive ones, BSA and IgG. This is probably due to the evaporation temperature decreasing and the total solids levels in the milk increasing with each subsequent evaporator effect. The critical temperature above which irreversible denaturation occurs in milk is 70 1C (de Wit & Swinkels, 1980). Furthermore, the development of thiol groups, important in the formation of disulfide linked aggregates, begins at 72 1C (Kirchmeier, El–Shobery, & Kamal, 1984). Thus denaturation may be expected to occur in the first effect at 70 1C, however it would be unlikely to occur in the last effect at 41 1C, if the effect of temperature was solely considered. However, the residence time in the first effect of the evaporator was 1 min, and based on kinetic data for b-lg and a-la (Dannenberg & Kessler, 1988) the level of denaturation would be expected to be minimal (1%). The increasing total solids concentration, which occurs as evaporation progresses, also has an effect on the extent of whey protein denaturation. McKenna and O’Sullivan (1971) observed that increasing the concentration of skim milk solids from 9% to 44% TS reduces the degree of whey protein denaturation caused by heating at 80 1C for 20 min.This concentration effect is complicated, because individual whey proteins show different responses to total solids concentration. The heat-resistance of b-lg and immunoglobulin increases with increasing total solids in milk and cheese whey (Nielsen, Coulter, Morr, & Rosenau, 1973; Hillier et al., 1979; Ibrahim, Madkor, & Hewedy, 1995; Anema 2000). In contrast, the rate of a-la denaturation is virtually unaffected in the range of 9.6–38.4% TS in skim milk (Anema, 2001). However, a-la has a greater heat resistance than b-lg, BSA and IgG in milk at normal solids (Dannenberg & Kessler, 1988; Singh & Creamer, 1991; Oldfield et al., 1998a) and is thus unlikely to
Table 4 Effect of concentrate heating and dryer inlet and outlet temperatures on the concentration of individual native whey proteins in reconstituted SMP Processing conditions
Runa no.
b-lg A
b-lg B
a-la
BSA
IgGb
2 1 3
1.7 1.9 1.8
2.2 2.1 2.1
0.63 0.64 0.71
0.21 0.21 0.22
0.36 0.34 0.32
5 1 4
1.8 1.9 1.7
2.1 2.1 2.1
0.60 0.64 0.55
0.21 0.21 0.21
0.36 0.34 0.32
Concentrate heating temperature (1C) 65 69 74 Dryer inlet/outlet temperature (1C) 160/89 180/90 200/101 a
Individual native whey protein concentration (g kg1)
Preheating condition for these runs were 70 1C for 52 s (see Table 1). IgG results averaged over trial 1 and 2. Pooled standard deviation 0.027 g kg1.
b
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undergo significant levels of denaturation at evaporation temperatures. The protective effect of increased total solids is due to increased lactose concentration, although lactose does not appear to affect a-la denaturation (Plock, Spiegel, & Kessler, 1998). Moreover, increasing total protein concentration without increasing lactose and milk salt concentration has been shown to result in an increase in b-lg, a-la and BSA denaturation (Law & Leaver, 1997). After exiting the last effect of the evaporator, concentrate at 41 1C was heated to 65–74 1C prior to drying. This was to reduce concentrate viscosity, thereby improving atomisation and powder properties (de Vilder et al., 1979; Baldwin et al., 1980; Oldfield, Teehan, & Kelly, 2000a). Heat sensitive BSA and IgG were not greatly affected by the concentrate heating step (Table 4), even though the temperature was raised from 41 1C to 65–74 1C. The high level of total solids (48%) in the milk concentrate probably retarded the rate of whey protein denaturation (Anema, 2000). In addition, the effective holding time of 80 s from the concentrate heater to the spray dryer nozzle was insufficient to cause extensive denaturation. The range of spray drying conditions used (inlet air 160–200 1C and outlet air 87–101 1C) had little effect on the denaturation of the individual whey proteins, including BSA and IgG. Previous workers also found that spray drying had only a minor effect on the denaturation of b-lg A, b-lg B and a-la (Singh & Creamer, 1991; Guyomarc’h, Warin, Muir, & Leaver, 2000). Although there are a large number of spray dryer designs and configurations, the temperature of a droplet within the drying chamber is typically between the wet bulb temperature and the outlet air temperature, and does not exceed 70 1C until almost all the water has been evaporated (Singh & Newstead, 1992). As the concentrate is atomised it contacts the hot inlet air and water evaporates rapidly from the droplet surface. Evaporation quickly cools the droplet to a temperature above the wet bulb temperature (40–50 1C) and also cools the surrounding hot air. Thus the high inlet air temperature conditions, e.g. 200 1C, are unlikely to cause denaturation. As the droplet dries and falls to the bottom of the dryer its temperature will approach that of the outlet air temperature. Thus outlet air temperature is important and affects properties such as the insolubility index (de Vilder et al., 1976). In this work, high insolubility index values of 0.3 (Table 1) and 0.7 mL were observed when the inlet and outlet air temperature was raised to 200 and 101 1C, respectively. However this is unlikely to be caused by extensive whey protein denaturation, but rather by casein aggregation (Knipschildt, 1969). Other whey protein reactions could have occurred during milk powder manufacture. For example, the early stages of the Maillard reaction (lactosylation) have
been reported in heated milks (Leonil et al., 1997) and milk powders (Guyomarc’h et al., 2000). Spray drying and in particular the combination of low inlet and low outlet air temperature are critical to minimising lactosylation (Guyomarc’h et al., 2000). 3.4. Whey protein aggregation and association with the micelle Samples collected from the milk powder plant were analysed by PAGE techniques to quantify the different types of whey protein aggregates present. The distribution of different b-lg, and BSA aggregates formed during milk powder manufacture (preheating at 100 and 120 1C) are shown in Figs. 3 and 4, respectively. As the samples were cooled, and in the case of concentrates diluted to 9% TS before ultracentrifugation, any changes observed are likely to be the result of irreversible reactions. The extent of heat-induced reactions was not great enough at the preheating temperatures of 70 and 80 1C to observe any clear trends, and
Fig. 3. b-lg aggregates formed during skim milk powder manufacture. Preheating temperatures; 100 1C (A) and 120 1C (B), for 52 s. b-Lg species: native (U), hydrophobic aggregates (///), disulphide-linked aggregates (XXX) and aggregates associated with the micelle (’). See Fig. 1 legend for a description of the sampling points.
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and BSA, which allows them to readily participate in thiol/disulfide interchange reactions. Previous studies have shown that the level of denatured b-lg is highly correlated with the level of disulfide linked b-lg aggregates, in heated skim milk (Oldfield et al., 1998a) and in heated concentrated skim milk (Anema, 2000). Approximately 50% of the b-lg and 75% of the BSA were associated with the micelle after preheating at 120 1C for 52 s. There was a small increase in the amount of b-lg associated with the casein micelle in samples from the 3rd effect and after concentrate heating. However, the levels of b-lg associated with the micelle in reconstituted powder samples were similar to levels obtained in samples from the flash vessel. Singh and Creamer (1991) reported similar observations in milk concentrates and powders. A number of changes occur during evaporation that may promote heat-induced association reactions. The pH decreased during evaporation from 6.66 to 6.17, and this would have reduced protein charge and therefore facilitate association reactions. Greater levels of b-lg and a-la association with the casein micelle have been observed in heated milks adjusted to more acidic pH (Corredig & Dalgleish, 1996b; Oldfield, Singh, Taylor, & Pearce, 2000b; Anema & Li, 2003). In addition, Ca2+ activity increased as a result of evaporation (Table 2) and this may also have promoted association reactions (Smits, & van Brouwershaven, 1980; Visser, Minihan, Smits, Tjan, & Heertje, 1986). Fig. 4. BSA aggregates formed during skim milk powder manufacture. Preheating temperatures; 100 1C (A) and 120 1C (B), for 52 s. BSA species; native (U), hydrophobic aggregates (///), disulphide-linked aggregates (XXX) and aggregates associated with the micelle (’). See Fig. 1 legend for a description of the sampling points.
the results for a-la were variable due to the lower levels of denaturation and are not shown. Most of the b-lg and BSA heat-induced reactions occurred in the preheating section, after which there was relatively little change, with the reconstituted powder approximating the sample obtained from the flash vessel (after preheating). Preheating at 100 1C or 120 1C for 52 s caused extensive denaturation of b-lg and BSA (470–90%). After preheating, denatured b-lg and BSA were present as either aggregates in the supernatant or had co-sedimented with the casein micelles (assumed to be associated with the casein). The denatured b-lg and BSA formed aggregates in the supernatant that were predominantly disulfide-linked, with a small amount of hydrophobic aggregates (i.e. non-convalently linked aggregates). The predominance of disulfide-linked aggregates is probably due to the thiol group and a number of intermolecular disulfide bonds present in b-lg
4. Conclusions Irreversible denaturation of major (b-lg A, b-lg B and a-la) and minor (IgG and BSA) whey proteins in skim milk occurred mainly during preheating. In comparison, the effect of evaporation and spray drying was relatively minor. Varying concentrate heating and spray drying inlet/outlet temperatures over a range typical for milk powder manufacture did not greatly affect denaturation of the whey proteins measured. b-Lg and BSA formed mainly disulphide-linked aggregates. The levels of b-lg and BSA associated with the casein micelle were lower than their respective levels of denaturation. Aggregation and association of b-lg and BSA occurred predominantly during preheating, after which evaporation and spray drying had a negligible effect.
Acknowledgements The authors gratefully acknowledge Fonterra (formerly the New Zealand Dairy Board) for financial support, Mr. J. Grant and Mr. C. Knight for operating the milk powder plant, Dr. K. N. Pearce for useful discussion during the research and Fonterra Research
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Centre (formerly the New Zealand Dairy Research Institute) for the chemical analyses they performed and the use of the milk powder plant.
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