3rd International Symposium on Food Rheology and Structure. 511. EFFECT OF ... INTRODUCTION. Whey proteins are widely used as food ingredients.
3rd International Symposium on Food Rheology and Structure
EFFECT OF PROTEOLYSIS ON THE RHEOLOGICAL PROPERTIES OF A WHEY PROTEIN CONCENTRATE D. Torres and M. P. Gonçalves CEQUP/Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. ABSTRACT Whey protein concentrate (WPC) was hydrolysed with pepsin during 5, 10 and 15 minutes. Gelation properties of the intact WPC and digests were measured by small deformation rheology. Reverse phase high performance liquid chromatography was performed with the objective to study the relation between gelation properties of the samples and their protein and peptide composition. A decrease in heat gelling properties of hydrolysates was observed.
INTRODUCTION Whey proteins are widely used as food ingredients because of their excellent nutritional value and functional properties. Enzymatic hydrolysis can affect both nutritional and functional properties of whey protein concentrates (WPC). Hydrolysis induces modification on solubility, viscosity, gelation, and emulsifying and foaming properties [1, 2]. In this work, a previous study on the influence of the degree of hydrolysis of whey proteins in their heat gelling properties is described. MATERIALS AND METHODS Materials. A commercial WPC powder containing approximately 69.6% protein was used for the experiments. All chemicals were analytical grade. Methods. WPC hydrolysis. The commercial WPC was used as substrate for pepsin. The enzymatic degradation was performed at 60 ºC and pH 2.0 during 5, 10 and 15 minutes (H5, H10, H15 respectively). After hydrolysis the three suspensions were lyophilised. The protein and ash content of the powders were determined by standard methods. Protein dispersions preparation. WPC and hydrolysates were then hydrated with distilled water (12% protein) by slow stirring until the dispersion was complete. The mineral content was adjusted with NaCl. Rheological measurements. Rheological measurements were performed with an AR2000 advanced rheometer (TA Instruments). For this purpose, a rough acrylic plate geometry was used (40 mm diameter). Before the experiments, the sample was covered with a thin layer of liquid paraffin to prevent evaporation. The protein dispersion was heated at a rate of 2 ºC/min from 20 to 80 ºC. After an equilibration period of 4 hours at 80 ºC, the mechanical spectrum was
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recorded (0.06283-62.83 rad.s ). The sample was then cooled at a rate of 2 ºC/min from 80 to 20 ºC and the temperature was maintained during 1 hour at 20 ºC. Finally, another mechanical spectrum -1 (0.06283-62.83 rad.s ) was recorded. During the temperature, time and frequency sweep experiments a maximum shear strain of 1% was stated. Temperature and time sweeps were performed at 1 Hz. Analytical reverse-phase HPLC. RP-HPLC was performed with a Chrompack P-300-RP column mounted on a Jasco HPLC system. Twenty microliters of sample were injected and separated at a flow rate of 0.5 mL/min. Buffer A was 99% water1% trifluoroacetic acid and buffer B was 80% acetonitrile-19.1% water-0.1% trifluoroacetic acid. The gradient cycle consisted of 90% A for 5 min, 80 % A in 10 min, 75 % A in 5 min, 70 % A in 3 min, 60 % A in 10 min, 55% A in 4 min; 50% A in 1 min, 30% A in 3 min, 0% A in 2 min, 0% A for 5 min. Absorbance at 215 nm was recorded. RESULTS AND DISCUSSION Figure 1 represents the structure development of gels during the heating and cooling processes. The hydrolysis process markedly affects the gelling properties of the proteins. The gelling temperature, defined as G’/G” crossover, for WPC and H5 is 78.3 and 79.7 ºC respectively. The H10 and H15 start gelling during the holding period B after 5 and 22 minutes respectively. α-lactoalbumin and β-lactoglobulin are selectively affected by this hydrolysis process (figure 2A). After 5 minutes, the remaining α-lactoalbumin is only vestigial whereas β-lactoglobulin is still 85% of the initial amount. After 15 minutes of hydrolysis, the remaining β-lactoglobulin decreased to about 40% (figure 2B). The chromatograms of the hydrolysates show a broadening of the β-lactoglobulin peak, relatively to that of the WPC chromatogram probably due to some degree of denaturation during the first steps of hydrolysis. Various unidentified small peptides are liberated continually during the hydrolysis indicating the specificity of the enzyme. According to some authors, the molecular weight decrease and the hydrophobicity of protein hydrolysates may be the causes for the observed decrease on their functional properties [3].
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The gels strengthen considerably as the temperature is decreased from 80 to 20°C, pointing out the importance of hydrogen bonds in gel structure. As compared to the spectra at 80°C (results not shown), the mechanical spectra at 20°C show larger values for G’ and G”. These spectra frame a section of the viscoelastic plateau, as usual in the case of heat-set globular protein gels, with G’>>G” all over the frequency window (figure 3). Both moduli increase with frequency for all the gels studied. However, whilst G' (ω) increases steadily with frequency, G'' (ω) shows a minimum which is -1 more clearly seen for WPC gel at § 0.2 rad.s . In all cases, tan(δ)=G”/G’ keeps low values (under 0.2).
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Figure 1. Effect of hydrolysis on thermal gelation. Ŷ:WPC, Ƒ: Hydrolysate 5 minutes, Ÿ: Hydrolysate 10 minutes, ż: Hydrolysate 15 minutes. The solid line represents the temperature profile. A: heating period 20-80 ºC (30 min), B: Holding period 80 ºC (210 min), C: cooling period 80-20 ºC (30 min), D: holding period 20 ºC (60 min). B
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Figure 2. RP-HPLC chromatograms of the samples (A). Loss degree of β-lactoblobulin during hydrolysis (B).
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Figure 3. Effect of hydrolysis on frequency dependence of the gel moduli at 20 ºC (B). Ŷ:WPC, Ƒ: Hydrolysate 5 minutes, Ÿ: Hydrolysate 10 minutes, ż: Hydrolysate 15 minutes
From our results, it seems that hydrolysis results in a gel weakening but actually it affects little the shape of the gelation curve or that of the mechanical spectra of the gels, and therefore does not modify intrinsically the mechanism of aggregation and the structure of the gels.
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ACKNOWLEDGEMENTS Financial support from FCT (Project POCTI/36452/ QUI/1999) is gratefully acknowledged. REFERENCES [1] van der Ven C, Gruppen H, de Bont DBA, Voragen AGJ: Emulsion properties of casein and whey protein hydrolysates and the relation with other hydrolysate characteristics, J. Agric. Food Chem. 49 (2001) 5005-5012. [2] Boza JJ, Martínez-Augustin O, Gil A: Nutritional and antigenic characterization of an enzimatic whey protein hydrolysate, J. Agric. Food Chem. 43 (1995) 872-875. [3] Sanchez C, Pouliot M, Gauthier SF, Paquim P: Thermal aggregation of whey protein isolate containing microparticulated or hydrolysed whey proteins, J. Agric. Food Chem. 45 (1997) 23842392.
