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Food Hydrocolloids 15 (2001) 915

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Functional improvement of whey protein concentrate on interaction with pectin


S. Mishra, B. Mann*, V.K. Joshi
Dairy Chemistry Division, National Dairy Research Institute, Karnal 132001, India Received 29 February 2000; received in revised form 20 June 2000; accepted 6 July 2000

Abstract The formation of a proteinpolysaccharide soluble complex is a valuable method for determining the physico-chemical properties of proteins. The present work was undertaken with the objectives of preparation of whey protein concentrate (WPC)pectin complexes and to evaluate the functional properties at different conditions, viz., pH, salt and temperature. WPCpectin complexes were prepared by dry heat treatment and the complex formation was ascertained by gel ltration (using G-100) and centrifugation techniques. Studies on the functional properties of this proteinpolysaccharide complex showed increased solubility at pH 4.6 compared to the control ultraltered whey protein concentrate (UF WPC). Emulsifying properties were also observed to be higher, viz., 98.56% emulsion activity at 0.10% concentration and 99.65% emulsion stability at 0.25% concentration. Better gelation was observed in UF WPCpectin complexes at 8% concentration compared to UF WPC alone. Foaming properties were also observed to be improved in these complexes at pH values (4.6 and 7) and temperature (10 and 308C), whereas, increased foam stability was observed at 1.0 M ionic strength. q 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Whey protein concentrate; Pectin; Proteinpolysaccharide complexes; Functional properties

1. Introduction Whey represents a potentially signicant source of functional protein ingredients for many traditional and novel food products. However, the various treatments employed during the manufacture of whey protein concentrates (WPCs) such as concentration of proteins, rening, purication and extent of drying have a marked inuence on the functional behaviour of proteins. Since whey proteins are very sensitive to heat, the extent of heat treatment given during the manufacture of WPC is very critical because it may cause the denaturation of the proteins which alternatively affects the solubility and other functional properties of the proteins and due to the alteration of these functional properties, there is limited use of these WPCs in various food products. Therefore, in order to increase the applicability of WPCs, functional properties need to be manipulated and the formation of complexes of WPC with polysaccharide appears to be one method to achieve this (Dalev & Simenova, 1995; Dickinson & Galazka, 1991; Hattori, Aiba, Nagasawa, & Takahashi, 1996; Xie & Hettiarachchy, 1997). Since in the food industry, proteins are used as emulsifying agents and polysaccharides as stabi* Corresponding author. Tel.: 191-184-259152; fax: 191-184-250042.

lising agents due to a thickening effect, by combining the two biopolymers one can think of coupling these properties together (Dickinson & Euston, 1991). Moreover, such proteinpolysaccharide complexes can be safely incorporated into the food system thus avoiding many undesirable chemicals. However, to bring the two biopolymers together, covalent coupling and electrostatic interaction can be used. The main advantage of a covalent proteinpolysaccharide hybrid over a non-covalent complex is the retention of molecular integrity and solubility over a wide range of solution conditions (Dickinson & Euston, 1991). Hence, in the present study, covalent complexes of proteinpolysaccharides were prepared by mixing the two biopolymers above the isoelectric point. The functional properties were then evaluated at different conditions, viz., pH, salt concentration and temperature.

2. Materials and methods 2.1. UF WPC The ultraltered whey protein concentrate (UF WPC) used in the experiment contained 71.25% protein by weight, 3.77% moisture by weight, traces of fat, lactose and ash.

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S. Mishra et al. / Food Hydrocolloids 15 (2001) 915

Fig. 1. (a) Gel ltration prole of UF-WPC (0 day at 60 ^ 18C) using Sephadex G-100 with bed size of 0.8 85 cm, ow rate 40 ml/h. (b) Gel ltration prole of UF-WPC (15 day at 60 ^ 18C) using Sephadex G-100 with bed size of 0.8 85 cm, ow rate 40 ml/h. (c) Gel ltration prole of UF-WPCpectin (0 day at 60 ^ 18C) using Sephadex G-100 with bed size of 0.8 85 cm, ow rate 40 ml/h. (d) Gel ltration prole of UF-WPCpectin (15 day at 60 ^ 18C) using Sephadex G-100 with bed size of 0.8 85 cm, ow rate 40 ml/h.

2.2. Pectin Pure pectin of CDH Laboratory Reagent with methoxyl content of about 6% on dry material basis, was used. It was a yellow white odourless powder which solubilises in water forming a viscous opalescent colloidal solution. The maximum content of sulphated ash was 3%. 2.3. Preparation of proteinpolysaccharide complexes Proteinpolysaccharide complexes were prepared by mixing the two biopolymers at equal ratios (weight ratios). For the preparation of these complexes, proteins (WPC) and polysaccharide (Pectin) were taken in equal amounts (by weight) and dissolved in a minimum quantity of water. Equal amounts of biopolymers were taken, as maximum complex formation was observed at this ratio. After adjusting the pH to 7.0, proteinpolysaccharide solutions were mixed, freeze dried and then kept in an oven at 60 ^ 18C for a period of 5, 10 and 15 days as indicated by Dickinson and Galazka (1991). The two biopolymers were mixed at pH 7.0 since it avoids the formation of ionic complexes that might form at lower pH. The formation of the complex

was conrmed by gel ltration and centrifugation techniques. The former method used was the same as described by Morr (1985) with slight modication. For the latter, 1% solution (w/v) was prepared by dissolving proteinpolysaccharide (WPCpectin) mixtures in distilled water and then it was centrifuged at 16,000g for 30 min. The protein content of the supernatant was subsequently estimated by the Kjeldahl method. 2.4. Evaluation of functional properties Functional properties were studied by using UF-WPC and UF-WPCpectin mixtures (kept at 60 ^ 18C for 15 days). 2.4.1. Solubility Solubility was determined according to the method described in the Indian Standards Institution Handbook of Food Analysis, Part XI (ISI, 1981). 2.4.2. Emulsifying properties Emulsions were prepared with an ultrasonic instrument (Branson Sonier Cell Disruptor Model B-12) using different concentrations (0.025, 0.05, 0.1, 0.25, 0.5, 0.75

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Fig. 1. (continued)

and 1% by weight) of samples containing 10% sunower oil. The emulsifying properties were measured by the method of Dalev and Simenova (1995) with slight modication. For emulsion activity (EA), the emulsions were centrifuged in graduated tubes for 5 min at 2600g and the whole volume (wv) of the system and the volume of the emulsion phase (EPV) were measured. Emulsifying activity was expressed as: EA % EPV=wv 100 For emulsion stability (ES), the emulsions were kept at 808C for 30 min, then in an ice bath for 15 min and nally centrifuged at 1300g for 5 min. The ES was calculated by: ES % EPV=wv 100 2.4.3. Gelling behaviour The gelling behaviour was studied by preparing gels at different concentrations (4, 8, 12, 16 and 20% protein by weight) and at different pH values (4.5, 7.5 and 8.5). The gels were prepared by keeping the solutions at 908C for 10 min in a water bath and then transferred to refrigeration

temperature (688C for 24 h). The stability of the gels was further checked by heating the gels at 908C for 10 min. 2.4.4. Foaming properties Foaming capacity and foam stability were determined by the method of Dewit, Hontelez-Backx and Adamse (1988). For this, the proteinpolysaccharide mixture were maintained at two different pH (4.6 and 7.0), temperatures (30 and 108C) and ionic strengths (0 and 1.0). Overrun and foam stability were calculated as follows: Overrun B 2 A=A 100 Stability C=A 100 where A is the volume of liquid prior to whipping, B the total foam volume obtained immediately after whipping, and C the volume of liquid retained in the foam after 1 h at room temperature (i.e. the weight of the foam divided by the density of the liquid).

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Table 1 Percent protein in supernatant of UF WPC and UF WPCpolysaccharide complexes after centrifugation at 16,000g/30 min S.No. 1. 2. 3. 4. Samples UF WPC (0 day) UF WPC (15 day) UF WPCpectin (0 day) UF WPCpectin (15 day) % Protein in supernatant 98.24 78.59 90.34 55.01

Table 2 Percent solubility of UF-WPC and UF-WPCpolysaccharide mixtures at different pH S.No. Samples Solubility a (%) pH 4.6 1. 2. 3. 4.
a

this temperature. The gel ltration of UF WPCpectin (0 day) resulted in the formation of three peaks. Peak I had decreased but broadened in comparison with the peak I of UF WPC (0 and 15 days). Peaks II and III appeared to be slightly merged. This could be due to slight complex formation between UF WPC and pectin on day 0, i.e. by mixing the two biopolymers. The elution prole of UF WPCpectin (15 days at 60 ^ 18C) showed a broader and increased peak I, whereas, peaks II and III were not distinguishable. As the void volume was 55 ml, the appearance of a broader peak in this volume indicated the presence of high molecular weight WPCpectin complexes. Moreover, the disappearance of peaks II and III also supported this view as the protein of these peaks could be involved in complex formation. 3.1.2. Centrifugation technique Here, the complex formation was ascertained by measuring the protein solubility that represents the amount of protein that is not involved in complex formation. In case of UF WPC (0 day), the mean protein solubility was observed to be 98.24% (Table 1) that decreased to 78.59% in UF WPC (15 days at 60 ^ 18C) which may be due to slight denaturation of the proteins during dry heat treatment. Whereas, the mean protein solubility of UF WPCpectin (0 day) was estimated to be 90.34%, which is slightly less than that of UF WPC (0 day), and it could be due to the slight formation of a complex of UF WPC with pectin by just mixing the two. The mean protein solubility of UF WPCpectin (0 day) decreased further from 90.34 to 55.01% of UF WPCpectin (15 days at 60 ^ 18C) which might be due to the formation of the complex by left over protein. 3.2. Functional properties 3.2.1. Solubility The solubility was measured at pH values 4.6 and 7.0 (Table 2). High solubility was observed in both UF WPC

pH 7.0 99.0 ^ 0.6 91.1 ^ 1.1 89.1 ^ 1.6 88.9 ^ 0.7

UF-WPC (0 day) UF-WPC (15 days) UF-WPCpectin (0 day) UF-WPCpectin (15 days) Mean ^ S.E. value.

74.2 ^ 0.8 37.0 ^ 1.4 78.9 ^ 0.6 89.3 ^ 1.3

3. Results and discussion 3.1. Conrmation of complex formation 3.1.1. Gel ltration The elution proles of UF WPC (0 and 15 days at 60 ^ 18C) and UF WPCpectin (0 and 15 days at 60 ^ 18C) are shown in Fig. 1(ad). On gel ltration, the UF WPC (0 day) was observed to resolve into three peaks, i.e. peak I, II and III. Peak I remained almost constant in UF WPC (15 days at 60 ^ 18C), whereas, peak II had slightly merged with peak I and peak III had slightly decreased. This might be attributed to the slight denaturation of UF WPC at

Fig. 2. Emulsion activity (%) of various WPC and WPCpolysaccharide mixtures.

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Fig. 3. ES (%) of various WPC and WPCpolysaccharide mixtures.

and UF WPCpectin at pH 7.0. However, at pH 4.6, the protein solubility in UF WPC (0 day) was found to be less, i.e. 74.25 ^ 0.877 which can be ascribed as the denaturation of UF WPC during processing and this value decreased further to 36.09 ^ 1.375 in UF WPC (15 days at 60 ^ 18C). In the case of UF WPCpectin, solubility increased from 78.91 ^ 0.629 (UF WPCpectin 0 day) to 89.30 ^ 1.259 UF WPCpectin (15 days at 60 ^ 18C). 3.2.2. Emulsifying properties 3.2.2.1. Emulsion activity (EA). UF WPCpectin (15 days) exhibited high EA (94.4%) at 0.025% concentration by weight (0.0125% of UF WPC) and 98.56% at 0.10% concentration by weight (0.05% of UF WPC) in comparison with that of UF WPCpectin (0 day) at respective concen-

trations (Fig. 2). These results were in accordance with the observations of Hattori et al. (1996), who reported increased emulsifying ability in case of b-lactoglobulin (b-lg)alginic acid (ALG) conjugates as compared to the emulsions of b-lg alone. Xie and Hettiarachchy (1997) have described that in emulsion systems containing both protein and polysaccharide, proteins typically form an adsorbed primary layer at the oil water interface, whereas, hydrophilic polysaccharides possibly form a thick secondary layer which enhances the steric stabilising properties on the outside of protein coated droplets (Dickinson, 1994). 3.2.2.2. Emulsion stability (ES). ES was observed to be more in UF WPCpectin (0 day) at 0.025 and 0.05% concentrations by weight (0.0125 and 0.025% of UF WPC by weight) as compared with that of UF WPC (0 day) at

Table 3 Gelling behaviour of UF WPC and UF WPCpolysaccharide mixtures as a function of concentration and pH S.No. Samples (conc.) 4% pH 4.6 1. 2. 3. 4. UF WPC (0 day) UF WPC (15 days) UF WPCpec (0 day) UF WPCpec (15 days) Ppt Ppt Sol Sol 6.5 Sol Sol Sol Sol 8.5 Sol Sol Sol Sol 8% 4.6 Ppt Ppt Sol Sol 6.5 Sol Sol Sol Sol 8.5 Sol Sol Sol Gel 12% 4.6 Ppt Ppt Sol Gel 6.5 Gel Sol Gel Gel 8.5 Gel Sol Gel Gel 16% 4.6 Ppt Ppt Sol Gel 6.5 Gel Sol Gel Gel 8.5 Gel Sol Gel Gel 20% 4.6 Ppt Ppt Gel Gel 6.5 Gel Gel Gel Gel 8.5 Gel Gel Gel Gel

Table 4 Thermal stability of UF WPC and UF WPCpolysaccharide mixture as a function of concentration and pH S.No. Samples (conc.) 4% pH 4.6 1. 2. 3. 4. UF WPC (0 day) UF WPC (15 days) UF WPCpec (0 day) UF WPCpec (15 days) Ppt Ppt Sol Sol 6.5 Sol Sol Sol Sol 8.5 Sol Sol Sol Sol 8% 4.6 Ppt Ppt Sol Sol 6.5 Sol Sol Sol Sol 8.5 Sol Sol Sol Gel 12% 4.6 Ppt Ppt Sol Sol 6.5 Gel Sol Gel Gel 8.5 Gel Sol Gel Gel 16% 4.6 Ppt Ppt Sol Gel 6.5 Gel Sol Gel Gel 8.5 Gel Sol Gel Gel 20% 4.6 Ppt Ppt Gel Gel 6.5 Gel Gel Gel Gel 8.5 Gel Gel Gel Gel

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Table 5 Foaming properties of UF-WPC and UF-WPCpolysaccharide mixtures S.No. Samples Percent overrun a 308C pH 4.6 1. 2. 3. 4.
a

108C pH 7.0 23.5 ^ 0.9 29.3 ^ 1.2 30.6 ^ 0.3 37.3 ^ 1.0 pH 4.6 24.3 ^ 0.4 25.0 ^ 0.9 30.6 ^ 0.8 35.1 ^ 0.9 pH 7.0 23.2 ^ 1.2 24.1 ^ 0.3 34.3 ^ 1.0 33.1 ^ 0.2

UF WPC (0 day) UF WPC (15 day) UF WPCpec (0 day) UF WPCpec (15 days) Mean ^ SE values.

24.7 ^ 0.6 24.8 ^ 1.8 34.2 ^ 0.3 47.8 ^ 2.1

respective concentrations. However, at higher concentrations, i.e. 0.25 and 0.5% concentrations by weight (0.125 and 0.25% of UF WPC by weight), lower ES of UF WPCpectin (0 day) was observed. This decrease in ES can be explained by `depletion occulation' which resulted from the addition of incompatible biopolymers at such higher concentrations (Dickinson & Galazka, 1991). Also UF WPCpectin (15 days at 60 ^ 18C) revealed high (90.97%) ES at 0.025% concentration (0.0125% of UF WPC) and (99.65%) at 0.25% concentration (0.125% of UF WPC) (Fig. 3). Burova, Grinberg, Grinberg, Leontiev, and Tolstoguzov (1992) have also reported the increased ES in legumin pectin complexes due to increased viscosity of the continuous phase. Increased ES in case of b-lgALG conjugates compared to the emulsion of b-lg alone was also reported by some workers (Hattori et al., 1996). It was thought that blgALG conjugates showed good ES because both conjugates were high in net charge, which displayed a strong protective effect of shielding in the emulsier. 3.2.3. Gelling behaviour Precipitates were obtained in UF WPC (0 and 15 days) at pH 4.6. This is in accordance with the ndings of Sternberg, Chiang and Eberts (1976), who claimed that around pH 5.0, a precipitate rather than a gel was produced (Table 3). However, in case of UF WPCpectin (0 and 15 days) at pH 4.6 and low concentration (4 and 8% protein), solutions remained as such (Table 3) due to increased solubility of these complexes (Payen, 1972; Ledward, 1979; Xie &

Hettiarachchy, 1997). But gels were obtained in UF WPCpectin (15 days at 60 ^ 18C) at pH 4.6 and 12% protein concentration which might be due to hydrogen bonding between the OH group of the polysaccharide with NH2 or COOH groups of whey proteins (Bimlesh, 1990). Similarly, at pH 6.5, UF WPC (15 days at 60 ^ 18C) formed gel at concentration (20% protein by weight), whereas UF WPCpectin (15 days at 60 ^ 18C) formed gel at lower concentration of 12% protein by weight which indicated the better gelling ability of UF WPC pectin (15 days at 60 ^ 18C). The observations were similar to the results of Burova et al. (1992) which showed increased gelation in leguminpectin and legumindextran sulphate complexes. Also at pH 8.5, gels were obtained in UF WPCpectin (15 days at 60 ^ 18C) at a lower concentration (8% protein by weight) compared to UF WPCpectin (0 day) and UF WPC (0 day) which formed gel at 12% protein. The thermal stability of the gels was checked by heating the complexes at 908C/10 min (Table 4). The gels of UF WPCpectin (15 days at 60 ^ 18C) remained stable at lower concentration (8% protein). However, not much difference was observed in UF WPC. 3.2.4. Foaming properties 3.2.4.1. Foaming capacity. Increased foaming capacity was observed in UF WPCpectin (0 and 15 days) at

Table 6 Foam stability of different UF WPC and UF WPCpolysaccharide mixtures (foam stability of UF-WPC and UF-WPCpolysccharide complexes at different pH and temperatures) S.No. Samples Foam stability a 308C pH 4.6 1. 2. 3. 4.
a

108C pH 7.0 24.1 ^ 0.01 25.3 ^ 0.2 35.3 ^ 1.2 39.3 ^ 1.2 pH4.6 30.9 ^ 1.8 30.9 ^ 0.1 43.1 ^ 0.6 47.5 ^ 1.3 pH 7.0 25.1 ^ 1.0 27.2 ^ 1.2 39.7 ^ 0.2 42.6 ^ 1.6

UF WPC (0 day) UF WPC (15 days) UF WPCpec (0 day) UF WPCpec (15 days) Mean ^ SE values.

27.9 ^ 0.7 28.5 ^ 0.8 40.1 ^ 0.9 48.4 ^ 1.7

S. Mishra et al. / Food Hydrocolloids 15 (2001) 915 Table 7 Foaming Properties of UF WPC and UF WPCpolysaccharide at ionic strength (1.0 M), 308C S.No. 1. 2. 3. 4.
a

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ered as an alternative way to enhance the applicability of WPC in the food industry. References
Bimlesh, K. (1990). Physico-chemical properties of whey protein concentrates. PhD thesis, National Dairy Research Institute (Deemed University), Karnal, India. Burova, T. V., Grinberg, N. V., Gringberg, V. Y., Leontiev, A. L., & Tolstoguzov, V. B. (1992). Effects of polysaccharides upon the functional properties of 11S globulin of broad beans. Carbohydrate Polymers, 18, 101108. Dalev, P. G., & Simenova, L. S. (1995). Emulsifying properties of protein pectin complexes and their use in oil containing foodstuffs. Journal of Science and Food Agriculture, 68, 203206. DeWit, J. N., Hontelez-Backx, E., & Adamse, M. (1988). Evaluation of functional properties of whey protein concentrates and whey protein isolates. Netherlands Milk and Dairy Journal, 42, 155. Dickinson, E. (1994). Protein-stabilized emulsions. Journal of Food Engineering, 22, 5974 (cited: Journal of Food Science (1997), 62: 1103). Dickinson, E., & Euston, S. R. (1991). Stability of emulsions containing both protein and polysaccharide. In E. Dickson, Food polymers, gels and colloids, Cambridge: The Royal Society of Chemistry. Dickinson, E., & Galazka, V. B. (1991). Emulsion stabilization by ionic and covalent complexes of beta-lactoglobulin with polysaccharides. Food Hydrocolloids, 5, 281296. Hattori, M., Aiba, Y., Nagasawa, K., & Takahashi, K. (1996). Functional improvement of alginic acid by conjugating with beta-lactoglobulin. Journal of Food Science, 61, 11711176. ISI (1981). Handbook of food analysis, part XI dairy products, New Delhi: Indian Standards Institution. Ledward, D. A. (1979). In J. M. V. Blanshard & J. R. Mitchell, Polysaccharides in foods (pp. 205217). London: Butterworths. Morr, C. V. (1985). Composition, physico-chemical and functional properties of reference whey protein concentrates. Journal of Food Science, 50, 1406. Payen, T. A. J. (1972). Light scattering of protein reactivity of polysaccharides, especially of carageenan. Journal of Dairy Science, 56, 629. Sternberg, M., Chiang, J. P., & Eberts, N. J. (1976). Cheese whey proteins with polyacrylic acid. Journal of Dairy Science, 59, 10421050. Xie, Y. R., & Hettiarachchy, N. S. (1997). Xanthan gum effects on solubility and emulsication properties of soy protein isolate. Journal of Food Science, 6, 11011104.

Samples UF-WPC (0 day) UF-WPC (15 days) UF-WPCpec (0 day) UF-WPCpec (15 days)

% Overrun a 23.9 ^ 0.7 22.1 ^ 1.6 34.1 ^ 0.8 34.1 ^ 0.8

Foam stability a % 25.6 ^ 0.6 27.8 ^ 0.8 65.8 ^ 0.8 65.8 ^ 0.8

Mean ^ S.E. values.

temperatures of 30 and 108C compared to UF WPC (0 and 15 days; Table 5). This increase in foaming could be due to increased solubility and molecular exibility of proteins as a result of which protein oriented, interacted and spread at the interface to form thicker and more viscous lms. 3.2.4.2. Foam stability. At pH values of 4.6 and 7.0, temperatures of 30 and 108C and 1.0 M ionic strength, foam stability was observed to be higher in UF WPC pectin (0 and 15 days at 60 ^ 18C) in comparison with the respective pH values, temperatures and ionic strength of UF WPC (0 and 15 days at 60 ^ 18C) (Tables 6 and 7) which might be due to the increase of viscosity of the continuous phase. 4. Conclusion The formation of proteinpolysaccharide complexes is a valuable method for controlling the physico-chemical properties of proteins. In the present study, functional properties like solubility, emulsication, gelation and foaming behaviour of WPC appeared to improve when combined with pectin. Hence, proteinpolysaccharide complexes can be consid-