COMPOSITION AND THERMAL BEHAVIOUR OF WHEY PROTEIN PREPARATIONS UNDER ACIDIC CONDITIONS

J. Dairying, Foods & H5. 25 (1) : 8-14, 2006 COMPOSITION AND THERMAL BEHAVIOUR OF WHEY PROTEIN PREPARATIONS UNDER ACIDIC CONDITIONS Ashish Kumar Singh, Nirankar Nath and Sumit Arora! G.B. Pant University of Agriculture and Technology, Pantnagar-263145,India. ABSTRACT Whey protein preparations namely UF-retentate, freeze-dried whey protein concentrate (WPC) and spray dried WPC, were investigated for their composition and thermal behavior at low ph. These preparations differed in their chemical constituents. The protein, lactose, fat and ash content of these preparations were in the range of 58.92-73.00%, 14.48-27.70%,5.12-8.18% and 4.95 5.00%, respectively. Ultrafiltration procedureincreased the proteinand fat contentin WPC significantly and simultaneously reduced levels of other constituents. Whey protein solutions (2% protein) made by dissolving these preparations exhibited maximum thermal stability at ph 3.5 and their stability decreased drastically above ph 4.0. Similarly soluble protein content was maximum at ph 3.5 and minimum at ph 4.5. UF-retentate showed highest thermal stability under acidic conditions fouowed by freeze dried WPC and spray dried WPC INTRODUCTION Whey is a byproduct of cheese and casein manufacture, and contains approximately 20% of the original milk proteins. Volume of the dairy fluid is on the rise concurrent with global increase in the cheese production and to, a lesser extent, casien (McIntosh eta11998). Huge volumes of whey represent a massive and growing protein resource available to food and related industries. Whey is usually considered a waste product and disposed of in the most cost effective manner or processed into low-value commodities like whey powder. Whey proteins are globular and heat denaturable. They are not coagulated by rennet or precipitated by acid. Unique nutritional, therapeutic and functional properties of whey proteins make them an ideal ingredient in formulations ofwide array of nutraceutical products. Whey proteins are one of the few proteins that are soluble at low ionic strength over the entire ph (2-8) range encountered in food applications 1987; Mulvihill 1991). Whey proteins can be used in various forms in protein rich bevrages such as concentrated whey, lactose hydrolyzed dried whey, whey protein concentrates and whey protein isolates. One of the major problems encountered in effective utilization of whey protein preparations in acidic beverages is large variation found in the composition and functional properties of most of the commercially available products. In addition to this, certain processing operations like forewarming, pasteurization, during cheese or casein preparations, freezing or drying during whey protein concentrate (WPC) manufacturing process, cause partial denaturation of whey proteins. Solubility behaviour and thermal stability of these partially denatured whey proteins influences their functional properties and utilization in food products. Therefore, the main objective of the present investigation is to elucidate the compositional and thermal behaviour of whey protein preparations under acidic ph. (Jayaprakasha and Brueckner, 1999). In MATERIAL AND METHODS undenatured form, whey proteins remain Buffalo milk cheddarcheese whey (ph soluble at their isoelectric point. The unique 6.8, T.S.S. 6 Brix and protein content 0.77%) solubility of whey proteins enable them to be was collected from the Experimental Dairy of used in fruit juices, milk-based beverages, soy National Dairy Research Institute; Karnal for drinks, fermented dairy beverages, fruit yams, the preparationofuf-retentate andfreeze-dried jellies, carbonated beverages, etc (Hoogstraten, WPC. Spray dried WPC was gifted by Mahan *Corresponding Author!Dairy Chemistry Division, NDRI, Karnal-132001

Whey protein Moisture Protein preparation (%) (%) UF-Retentate 87.21 58.92 Freeze dried WPC 7.43 68.90 Spray dried WPC 4.01 73.05 S.E.M. ± 0.079 0.126 CD llt 1% 0.415 0.659 Vol. 25, No.1, 2006 Table 1. Chemical constituent of whey protein preparations Proteins Ltd. (Kosikalan) and it was prepared by ultrafiltration of casein whey and spray drying the UF-retentate Preparation of UF- Retentate and Freeze Dried WPC Buffalo milk cheddarcheese whey was passed through a centrifugal separator (Tetra Pak India Pvt. Ltd,. Pune) to remove casein fines and residual fat. Clarified whey was heated at 74 C for 15 seconds and rapidly cooled to 4 C in a plate heat exchanger. The ph of whey was adjusted to ph 7.0 with food grade 1 N NaOH and subjected to ultrafiltration process, using a pilot scale module hollow fibre membrane unit (Ramicon membrane type PM 50). Inlet and outlet pressure in the UF unit was 1.80 bar and 0.5 bar, respectively. Temperature of circulating whey was maintained at 48 ± lac and average flux rate of permeate was 9.80 Uhour. The process was continued till the 80% volume reduction was obtained. Equal amount of distilled water (50 C) was added to UF-whey retentate and first diafilteration was carried out. Process was stopped, when the refractometeric solids in retentate reached to 12.5 Brix. For the preparations of freeze dried WPC, second ultrafiltration ofultrafiltered whey was executed till 85% volume reduction was achieved. UFretentate was further subjected to second and third diafiltration so as to achieve 90% reduction in its volume. UF- retentate obtained was freeze dried at 55 C and4.3 torr in a table top freeze drier (Freeze Mobile II Virtis Gardeners NY Model 10MR-IR). The UFretentate, freeze-dried and spray dried WPC were analysed for moisture, proteins, ash, lactose and calcium content according to AOAC methods (AOAC, 1997). Lactose Fat Ash Calcium (%) (%) (%) (mg/loog) 27.70 7.20 5.11 0.462 18.32 8.18 4.95 0.392 14.48 5.12 5.01 1.14 0.16 0.028 0.035 0.0071 0.83 0.15 0.18 0.037 Thermal denaturation of whey proteins in acidic ph Whey protein samples were dissolved in distilled water to obtain a 2% protein solution. Thesolution was divided into five lots of 50 ml each and their ph was adjusted to 3.5, 3.75, 4.0, 4.25 and 4.5 using 50% lactic acid solution. Samples (10 ml) were poured in screw capped test tubes (15 x 176 mm) and the tubes were placed in temperaturecontrolled water bath set at 95 ± 0.5 C. Tubes were heated to a geometric centre temperature of 92.5 C as determined in preliminary experiments and held for 5 min. Tubes were removed from the water bath, and quickly cooled to room temperature by placing them in an ice bath. The degree of denaturation of WPC, soluble protein content and heatstability profile were estimated. Thesoluble protein was determined by centrifuging the sample at 5000 rpm for 20 min in a Remi centrifuge and the protein content in supernatant was estimated by micro-kjeldhal method using conversion factor of 6.38. Degree ofdenaturation was measured as turbidimetric measurements using a spectrophotometer (Specord 200, Analytikjena, Version 2.1E) at 900 nm and expressedasabsorbanceindex (A.I) (Jelen and Buchheim, 1984). HEAT STABIUTY PROFILE To study the heat stability profile, the protein samples were prepared as in previous experiment (2.2) and after ph adjustment, sampleswere allowed toequilibratefor 10min. Then samples were centrifuged (Remi Centrifuge) at 3000 rpm for 5 min to remove 9

10 J. DAIRYING, FOODS & H.S. E.H 3.50 3.75 4.00 4.25 4.50 Table.2. Thermal denaturation index (A.I.P of three whey protein solutions (2% Protein) at different ph. (ph) (WPC) ph xwpc Spray driedwpc 0.27 1.40 1.76 2.42 2.56- F value ** ** ** Freeze dried WPC 0.14 b 0.47 b O.79 b 1.07 b 1.21 b ±S.E.M. 0.014 0.011 0.025 UF-Retentate 0.09' 0.40' 0.73 b 1.05 b 1.11 ' C.D. at5% 0.04 0.03 0.07 = - :: ll... :E = = 100 90 80 70 60 50 40 30 I 20 10 o 3.5 3.75 4 ph Spray Dried WPC Freeze Dried W PC ---.- UF Retentate... 4.25 4.5 Fig. : Effect of ph on soluble protein content of whey protein preparation upon heating. any turbidity. Two ml of each sample in duplicate was taken in heat stability tubes and were heated in an oil bath maintained at 100 ± 1 C. The time taken by each sample to coagulate, which was judged by the formulation of white flakes, was recorded using stopwatch. STATISTICAL ANALYSIS Data obtained were expressed as mean values with standard errors. In all experiments one-way/two-way analysis of variance (ANOVA) with subsequent least significant difference (LSD) test was applied for multiple sample comparison. This was done to test for any significant differences at 5% level of significance (Snedecor and Cochran, 1994). RESULTS AND DISCUSSIONS Chemical composition of whey protein preparations Protein Chemical make up of freeze-dried WPC, spray dried WPC and UF-Retentate is presented in Table 1. Samples of WPC differ considerably in their composition. The protein content varied from 58.93 to 73.05% (on dry weight basis) in these three WPC samples. The difference in their protein content may be due to the procedure involved in their manufacture and the source of whey. The spray dried and freeze dried WPC were subjected to more than two stage diafilteration, which is reported to increase the protein level of resulting WPC (Jayaprakasha, 1992). UF- retentate, which was

',' ph 3.50 3.75 4.00 4.25 4.50 Vol. 25, No.1, 2006 Table 3. Soluble protein in heat-treated Whey protein solutions (2% Protein) at different ph. Spray driedwpc Freeze dried WPC UF-Retentate 1.37' 1.50 b 1.78 c 1.09' 1.24 b 1.41 c 0.86' 1.03 b 1.22 c 0.40' 0.62 b 0.66 b 0.35' 0.59 b 0.61 b F value ±S.E.M. (ph) ** 0.02 (WPC) ** 0.01 ph x wpc NS 0.03 Values with same superscripts in a. row are not significantly different (P<0.05) from each other diafiltered once, had lowest protein content FAT among the three type samples. Protein content has been reported in the range of 72.0 to 76.6% for eight commercial samples from different manufacturers in Europe (Morr and Foegeding 1990) whereas it was found to vary from 28 80% for WPC samples available in Germany (Jayaprakasha, 1992). All these variations may be because of the differences in manufacturing process, source of raw material and also on the basis of changes made for desired functionality in finished product. LACTOSE Lactose is another important constituent of WPC and the lactose content was 14.48, 18.32 and 27.70 % in spray dried, freeze-dried and UF-retentate, respectively (Table 1). All the three samples differed significantly in their lactose content. As the protein content increased in retentate during ultrafiltration, there was reduction in lactose content because of its removal in permeate stream. Similar results repofted by Trantnik and Kersev (1991), who found that upon diafiltration lactose.a ash content of WPC reduced to a minimum values of 32.8 and 4.7% with corresponding increase in protein level to 52.4% from 52,5.2 and 35% for lactose, ash and protein, respectively. Lactose content in WPCs can be reduced by employing diafilteration or lactose crystallization in whey using very fine lactose monohydrate as seeding material (Jayaprakasha etal., 1995). CD at 5% 0.05 0.04 0.09 Fat content in three WPC samples ranged from 5.12 to 8.18%, being lowest in spray dried and highest in freeze dried on dry matter basis. Lower fat content in spray dried WPC maybe due to raw material Le. skim milk, used for making casein whey, whereas for manufacture of freeze dried and UF-retentate, mixed milk cheddar cheese whey was utilized. Slightly lower fat content Le. 5.2 (Jayaprakasha, 1992) and 5.95 (Vijayakumar and Sangwan, 2000) are reported in WPC samples prepared by ultrafiltration of mixed milk cheddar cheese whey. This difference maybedue todifference in clarification process. The higherlipid content in WPC is due to the fact that the residual lipids in whey are concentrated along with protein during ultrafiltration/diafiltration (';arper, 1984). Significant proportions of these lipids are phospholipids (Morr and Foegedigd, 1990); most probably derived from the rupture of fat globule membranes. Presence of residual lipid has been reported to adversely affect the functiening properties of WPC (Vaghela and Kilara, 1996). ASH AND CALCIUM Ash conb;mt of WPC samples ranged from 4.95-5.12 per cent (Table 1). There was no significant difference among the three samples. As previously reported, ultrafiltration process removes mineral in permeate stream and there was continuous reduction in ash content on subsequent diafilteration. Wide II

I. "...' 12 J. DAIRYING. FOODS & H.S. 10 9 8 7 6.5! 5 I i= 4 3 2! t I, 0 i _Spray dried W PC _Freeze dried W PC _UF Rete.lote 3.5 3.75 ph 4 4.25 Fil.2: Thermal stability profile of whey protein preparations. variations (3.36-15.03%) were reported in ash content of four commercial samples (Jayaprakasha, 1992). There was apparent difference in the calcium content of two WPCS and UF-retentate. Spray dried WPC contained highest amount of calcium (1.14%). Spray dried WPC was manufactured from acid whey, which are known to contain higher amount of calcium as compare to sweet whey. Calcium contenf in freeze dried WPC is almost similar to one prepared by Vijayakumar and Sangwan (2000). The US specifications for WPC are: Protein (min) 25%, fat 0.2-10%, ash 2-15%, lactose (max) 60% and moisture 1-6%. Except moisture all criteria is fulfilled bythe WPC used in our study. Thermal behaviour of whey proteins prepared by different methods DENATURATION INDEX Denaturation index, a measurement of protein denaturation as a result of thermal treatment, is presented in Table 2. Denaturation index of all the three whey protein preparations increased with increase in ph from 3.5 to 4.5. Among the three WPC solutions, spray dried whey protein concentrate showed maximum A.I. value at all ph values followed by freeze dried WPC and UF-retentate. It indicates that spray drying process commonly employed to manufacture commercial WPC has a significant effect on protein denaturation. The denaturation index was significantly affcted by the type of WPC (P<0.05) and ph (P<0.05). The interaction between types of WPC and ph was also significant (P<0.05). However, all WPC solutions heated at ph 3.5, showed no visible aggregation and sedimentation, during the course of int'estigation. But slight sedimentation was observed in spray dried WPC solution, after keeping the tubes overnight. High thermal stability of whey protein solutions at ph below 3.9 is reported by several workers (Patoka et af, 1986; Jelen and Buchheim, 1984). They further reported that Hie stabilizing effect against. heat denaturation was maximum at or below ph 3.4. Both!loLa and P-lg undergo structural changes below ph 4.0 leading to thermal behaviour, which may be different from that at ph above 4.0. The dimer configuration of P 19 fraction dissociates at or below ph 3.5 due to electrostatic repulsion and the resulting monomers are resistant to coagulation (Harwalkar et al, 1985). In addition to these reports Bernal and Jelen (1985) sighted the possible effects ofminorcontaminants in WPC for high thermal stability at ph <3.5. All the..

WPC samples showed coagulation and aggregation at ph above 4.0. In UF-retentate, there was a visible slimy aggregate formation and these aggregates were loosely floating in the liquid. The low denaturation index of UFretentate may be attributed to the less thermal treatment given to mixed milk during cheese processing (batch type pasteurization at 63 C for 30 min.). Spray dried WPC had highest AI values (0.27-2.56) because of the application of higher air temperature during drying. Hall and Iglesias (1998) reported similar results and showed that degree of denaturation increased with increase in air temperature. Freezing and freeze drying process is also reported to denature the proteins if not carefully controlled. Hence, freeze dried WPC exhibited intermediate denaturation index. Another explanation that may be put forward for different thermal behaviour of three WPC' is because of differences in the levels of their constituents (Table1), specially lactose content. Lactose has been shown to be most effective in preventing the thermal aggregation of whey proteins in WPC (Morr and Ha, 1993, Ibrahim et al, 1995). The stabilization of the native protein structure by sugars is explained by preferential hydration of the protein molecules in an aqueous sugar solution (Kulmyrzaev et a12000). Protein Solubility The soluble protein content of heated whey protein solutions ranged from 1.37 to 1.78, 1.09 to 1.41,0.86 to 1.22, 0.40 to 0.66 and 0.35 to 0.61% at ph 3.5, 3.75, 40, 4.25 and 4.5 respectively (Table 3). Soluble protein followed the similar trend as denaturatlon index. At ph 3.5, 68.5-89 percent proteins remain soluble (Fig 1) though the corresponding AI value varies from 0.09 to 0.27. Above ph3.75there was appreciable loss of protein solu,bility in all the samples. Except spray dried whey protein solution, no visible coagulation or aggregation was observed in Vol. 25, No.1, 2006 13 other whey protein preparations. The overall results show that although the whey proteins are quite resistant to precipitation at or below ph 3.5, they are still denatured by heat as applied in this experiment. Protein solubility of WPC solutions was found to differ significantly with ph and methods of preparation involved (P<0.05). The interaction between the ph and type of whey protein preparations was non-significant (Table 3). This indicates that manufacturing process and the composition of whey proteins concentrates has a pronounced effect on theirthermal behaviour. Partially denatured whey ';>roteins are highly soluble Cit ph 6.0, whereas they exhibit markedly lower solubility at their isoelectric point (Jayaprakasha and Brueckner, 1999). Pasteurization ofwhey has no significant effect onsolubility, butheatingofuf-retentate caused a significant reduction in solubility of resulting WPC (Morr, 1987). Ibrahim et al. (1995) reported a recovery of90% for soluble proteins from whey protein samples heated at 900C for 10 min at 4.5 but above this Ph only 50% recovery was obtained. According to Bernal and Jelen (1985) approximately 50% whey proteins get precipitated at ph >3.8. A similar trendwas observedduringpresentinvef.tigation and heavy precipitation was observed at or above ph 4.0 (Figure 1). Above ph,t9, the repulsive action becomes very strong and protein-protein interactions are more likely to occur, resulting precipitation of proteins. Thermal stability It is measured as the time taken by whey-protein solutions to coagulate, varied with ph and WPC samples except at ph 3.5, where there was no coagulation or flake formation even after heating for more than one hour. The three whey proteins exhibited different heat stability at ph 3.75 (Figure 2). UF-retentate showed maximum thermal stability followed by freeze-dried and spray dried WPC solutions. However, at ph 4.0 or above there was no

14 J. DAIRYING, FOODS & H.S. apparent differences in thermal stability between spray dried and freeze dried WPC. Jelen and Buchheim (1984) also observed higher heat stability of whey proteins in acid whey preparations below ph 3.9 and concluded that the effect appeared to be due to higher heat resistance. REFERENCE On the basis of the results, it may be concluded thatstability of whey proteins under acidic conditions depends on the type of whey, methods involve in their preparation and the ph of the solutions. The whey proteins can'be utilized for fortification of fruit juices and other acidic drinks by maintainingthe ph below3.5. AOAC (1997). Official Methods ofanalysis. 16 th Edn. Association of Official Analytical Chemists. Washington D.C., U.SA Bernal, V. and Jelen, P. (1985). J. Dairy Sci. 68: 2847-2852 Hall, S.M. and Iglesias, O. (1998). FoodSci. Technollntem. 3: 381-383. Harper, W. J. (1984). J.Dairy. Sci. 67: 2745-2754 Harwalkar, V. R. etal (1985). Mi/chwissenschaft. 40: 31-34 Harwalkar,V.R. (1980). J. Dairy. Sci. 63: 1043-1051 Hoogstratan, J.J. (1987). IDFBulletin No.17: 212-215 pp. Ibrahim, ES. etal. (1995). Egyptian J. DairySci. 23: 177-188. Jayaprakasha, H. M. and Brueckner, H. (1999). J. Food Sci. Technol36: 199-204. Jayaprakasha, H.M. etal (1995). Japanese J. Dairy FoodSci. 44: 113-121 Jayaprkasha, H. M. (1992). Ph.D. Thesis. National Dairy Research Institute. Deemed University, Kamal, India. Jelen, P. and Buchhheim, W. (1984). Mi/chwissenschalt, 39: 215-218 Kulmyrzaev, A. etal (2000). J. Agric. Food Chern. 48: 1593-1597 Mcintosh, G.H. etal (1998). Int. DairyJ. 8: 425-434 Morr, C.V and Ha, E.Y.W. (1993). Crit. Rev. Food Sci. & Nutri. 33: 431-476. Morr, C.v. (1987). J. Food Sci. 52: 312-319. Morr, C.v. and Foegeding, EA (1990). Food Technol44: 100-112. Mulvihill, D.M. (1991). FoodRes. 51: 65-73 Patocka, Jeta/. (1986). Mi/chwissenschaft. 41: 490-493 Snedecor, GW. and Cochran, w.g. (1994). Statistical Methods- 8 th Ed. Affiliated East-West Press, Iowa State Univ. Press Tratnik, J. and Kersev, J.C. (1991). Mi/chwissenschaft46: 91-97. Vaghela, M. and. Kilara A. (1996). J. Dairy Sci. 79: 1172-1183 Vijyakumar, B. and Sangwan, R.B. (2000). Indian J.Dairy Sci. 53: 248-254._-,,- -- -._---.. -=-