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Comparison of selective hydrolysis of α-lactalbumin by acid Protease A and Protease M as alternative to pepsin: potential for β-lactoglobulin purification in whey proteins

Published online by Cambridge University Press:  07 February 2019

Katarina Lisak Jakopović*
Laboratory for Technology of Milk and Milk products, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000, Zagreb, Croatia
Seronei Chelulei Cheison
Global Applied Science & Technology (GAST), Mars GmbH, Eitzerstr 215, D-27283 Verden (Aller), Germany
Ulrich Kulozik
Chair for Food and Bioprocess Engineering, ZIEL Institute for Food & Health, Technical University of Munich, Weihenstephaner Berg 1, D-85354 Freising, Germany
Rajka Božanić
Laboratory for Technology of Milk and Milk products, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000, Zagreb, Croatia
Author for correspondence: Katarina Lisak Jakopović, Email:


The experiments reported in this research paper examine the potential of digestion using acidic enzymes Protease A and Protease M to selectively hydrolyse α-lactalbumin (α-La) whilst leaving β-lactoglobulin (β-Lg) relatively intact. Both enzymes were compared with pepsin hydrolysis since its selectivity to different whey proteins is known. Analysis of the hydrolysis environment showed that the pH and temperature play a significant role in determining the best conditions for achievement of hydrolysis, irrespective of which enzyme was used. Whey protein isolate (WPI) was hydrolysed using pepsin, Acid Protease A and Protease M by randomized hydrolysis conditions. Reversed-phase high performance liquid chromatography was used to analyse residual proteins. Regarding enzyme selectivity under various milieu conditions, all three enzymes showed similarities in the reaction progress and their potential for β-Lg isolation.

Research Article
Copyright © Hannah Dairy Research Foundation 2019 

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Alomirah, HF and Alli, I (2004) Separation and characterization of β-lactoglobulin and α-lactalbumin from whey and whey protein preparations. International Dairy Journal 14, 411419.CrossRefGoogle Scholar
Bramaud, C, Aimar, P and Daufin, G (1997) Optimisation of a whey protein fractionation process based on the selective precipitation of α-lactalbumin. Le Lait 77, 411423.CrossRefGoogle Scholar
Chatterton, DEW, Smithers, G, Roupas, P and Brodkorb, A (2006) Bioactivity of β-lactoglobulin and α-lactalbumin-Technological implications for processing. International Dairy Journal 16, 12291240.CrossRefGoogle Scholar
Cheison, SC, Schmitt, M, Leeb, E, Letzel, T and Kulozik, U (2010) Influence of the temperature and the degree of hydrolysis on the peptide composition of trypsin hydrolysates of β-lactoglobulin: analysis by LC-ESI-TOF/MS. Food Chemistry 121, 457467.CrossRefGoogle Scholar
Cheison, SC, Leeb, E, Toro-Sierra, J and Kulozik, U (2011) Influence of hydrolysis temperature and pH on the selective trypsinolysis of whey proteins and potential recovery of native alpha-lactalbumin. International Dairy Journal 21, 166171.CrossRefGoogle Scholar
Cheison, SC, Bor, EK, Faraj, AK and Kulozik, U (2012) Selective hydrolysis of α lactalbumin by Acid Protease A offers potential for β-lactoglobulin purification in whey proteins. LWT – Food Science and Technology 49, 117122.CrossRefGoogle Scholar
Creamer, LK, Nilsson, HC, Paulsson, MA, Coker, CJ, Hill, JP and Jimenez-Flores, R (2004) Effect of genetic variation on the tryptic hydrolysis of bovine b-lactoglobulin A, B, and C. Journal of Dairy Science 87, 40234032.CrossRefGoogle Scholar
Custodio, MF, Goulart, AJ, Marques, DP, Giordano, RC, Giordano, RLC and Monti, R (2005) Hydrolysis of cheese whey proteins with trypsin, chymotrypsin and carboxypeptidase. Alimentação e Nutrição 16, 105109.Google Scholar
Diermayr, P and Dehne, L (1990) Kontrollierte enzymatische proteinhydrolyse im bereich niedriger pH-werte. Zeitschrift für Lebensmitteluntersuchung und –Forschung A 190, 516520.CrossRefGoogle Scholar
Foegeding, EA, Davis, JP, Doucet, D and McGuffey, MK (2002) Advances in modifying and understanding whey protein functionality. Trends in Food Science & Technology 13, 151159.CrossRefGoogle Scholar
Galvão, CM, Silva, AF, Custódio, MF, Monti, R and Giordano, RL (2001) Controlled hydrolysis of cheese whey proteins using trypsin and α-chymotrypsin. Applied Biochemistry and Biotechnology 91–93, 761776.CrossRefGoogle ScholarPubMed
Kinekawa, YI and Kitabatake, N (1996) Purification of β-lactoglobulin from whey protein concentrate by pepsin treatment. Journal of Dairy Research 79, 350356.CrossRefGoogle ScholarPubMed
Konrad, G and Kleinschmidt, T (2008) A new method for isolation of native α-lactalbumin from sweet whey. International Dairy Journal 18, 4754.CrossRefGoogle Scholar
Konrad, G, Lieske, B and Faber, W (2000) A large-scale isolation of native β-lactoglobulin: characterization of physicochemical properties and comparison with other methods. International Dairy Journal 10 713721.CrossRefGoogle Scholar
Lisak, K, Toro-Sierra, J, Božanić, R, Kulozik, U and Cheison, SC (2013) Chymotrypsin selectively digests β-lactoglobulin in whey protein isolate away from enzyme optimal conditions: potential for native α-lactalbumin purification. Journal of Dairy Research 80, 1420.CrossRefGoogle ScholarPubMed
Lisak Jakopović, K, Barukčić, I and Božanić, R (2016) Physiological significance, structure and isolation of α-lactalbumin. Mljekarstvo 66, 113.Google Scholar
Mailliart, P and Ribadeau-Dumas, B (1988) Preparation of β-lactoglobulin and p-lactoglobulin-free proteins from whey retentate by NaCl salting out at low pH. Journal of Food Science 53, 743745.CrossRefGoogle Scholar
Maletić, M, Aleksić, N, Vejnović, B, Nikšić, D, Kulić, M, Đukić, B and Ćirković, D (2016) Polymorphism of κ-casein and β-lactoglobulin genes in Busha and Holstein Friesian dairy cows in Serbia. Mljekarstvo 66, 198205.CrossRefGoogle Scholar
Maté, HJ and Krochta, JM (1994) Beta-lactoglobulin separation from whey proteine isolate on a large-scale. Journal of Food Science 59, 11111114.CrossRefGoogle Scholar
Otte, J, Zakora, M, Qvist, KB, Olsen, C and Barkholt, V (1998) Hydrolysis of bovine β-lactoglobulin by Various proteases and identification of selected peptides. International Dairy Journal 7, 835848.CrossRefGoogle Scholar
Permyakov, EA and Berliner, LJ (2000) α-Lactalbumin: structure and function. FEBS Letters 473, 269274.CrossRefGoogle ScholarPubMed
Schmidt, DG and Poll, JK (1991) Enzymatic hydrolysis of whey proteins. Hydrolysis of α-lactalbumin and β-lactoglobulin in buffer solutions by proteolytic enzymes. Netherlands Milk and Dairy Journal 45, 225240.Google Scholar
Smithers, GW (2008) Whey and whey proteins–from ‘gutter-to-gold’. International Dairy Journal 18, 695704.CrossRefGoogle Scholar
Toro-Sierra, J, Tolkach, A and Kulozik, U (2013) Fractionation of α-lactalbumin and β-lactoglobulin from whey protein isolate using selective thermal aggregation, an optimized membrane separation procedure and resolubilization techniques at pilot plant scale. Food and Bioprocess Technology 6, 10311043.CrossRefGoogle Scholar