Hostname: page-component-77c89778f8-cnmwb Total loading time: 0 Render date: 2024-07-19T02:18:53.547Z Has data issue: false hasContentIssue false

Determination of total antioxidant capacity of milk by CUPRAC and ABTS methods with separate characterisation of milk protein fractions

Published online by Cambridge University Press:  03 March 2015

Sema Demirci Çekiç
Affiliation:
Faculty of Engineering, Department of Chemistry, Istanbul University, 34320 Istanbul, Turkey
Aslı Demir
Affiliation:
Faculty of Engineering, Department of Chemistry, Istanbul University, 34320 Istanbul, Turkey
Kevser Sözgen Başkan
Affiliation:
Faculty of Engineering, Department of Chemistry, Istanbul University, 34320 Istanbul, Turkey
Esma Tütem
Affiliation:
Faculty of Engineering, Department of Chemistry, Istanbul University, 34320 Istanbul, Turkey
Reşat Apak*
Affiliation:
Faculty of Engineering, Department of Chemistry, Istanbul University, 34320 Istanbul, Turkey
*
*For correspondence; e-mail: rapak@istanbul.edu.tr

Abstract

Most milk-applied antioxidant assays in literature are based on the isolation and quantification of individual antioxidative compounds, whereas total antioxidant capacity (TAC) gives a more holistic picture due to cooperative action of antioxidants. Recently, the cupric reducing antioxidant capacity (CUPRAC) method has been modified to measure the antioxidant capacities of thiol-containing proteins, where the classical ammonium acetate buffer – that may otherwise precipitate proteins– was replaced with concentrated urea buffer (able to expose embedded thiol groups of proteins to oxidative attack) adjusted to pH 7.0. Thus, antioxidant capacity of milk was investigated with two competing TAC assays, namely CUPRAC and ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid))/persulphate, because only these assays were capable of evaluating protein contribution to the observed TAC value. As milk fat caused turbidity, experiments were carried out with skim milk or defatted milk samples. To determine TAC, modified CUPRAC method was applied to whole milk, separated and redissolved protein fractions, and the remaining liquid phase after necessary operations. Both TAC methods were investigated for their dilution sensitivity and antioxidant power assessment of separate milk fractions such as casein and whey. Proteins like β-lactoglobulin and casein (but not simple thiols) exhibited enhanced CUPRAC reactivity with surfactant (SDS) addition. Addition of milk protein fractions to whole skim milk produced significant ‘negative-biased’ deviations (up to −26% relative standard error) from TAC absorbance additivity in the application of the ABTS method, as opposed to that of the CUPRAC method less affected by chemical deviations from Beer's law thereby producing much smaller deviations from additivity (i.e. the property of additivity is valid when the measured TAC of a mixture is equal to the sum of individual antioxidant capacities of its constituents).

Type
Research Article
Copyright
Copyright © Proprietors of Journal of Dairy Research 2015 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Apak, R, Güçlü, K, Özyürek, M, Karademir, SE & Altun, M 2005 Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method. Free Radical Reearch 39 949961Google Scholar
Bergmann, G, von Oepen, B & Zinn, P 1987 Improvement in the definitions of sensitivity and selectivity. Analytical Chemistry 59 25222526CrossRefGoogle Scholar
Çekiç, SD, Sözgen Başkan, K, Tütem, E & Apak, R 2009 Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols. Talanta 79 344351Google Scholar
Çekiç, SD, Kara, N, Tütem, E, Sözgen Başkan, K & Apak, R 2012 Protein−incorporated serum total antioxidant capacity measurement by a modified CUPRAC (CUPric Reducing Antioxidant Capacity) method. Analytical Letters 45 754763CrossRefGoogle Scholar
Chen, J, Lindmark-Månsson, H, Gorton, L & Åkesson, B 2003 Antioxidant capacity of bovine milk as assayed by spectrophotometric and amperometric methods. International Dairy Journal 13 927935Google Scholar
Claiborne, A, Yeh, JI, Mallett, TC, Luba, J, Crane, EJ III, Charrier, V & Parsonage, D 1999 Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation. Biochemistry 38 1540715416Google Scholar
Creighton, TE 1997 Protein folding coupled to disulfide bond formation. Biological Chemistry 378 731744Google Scholar
Egwin, OC & Gruber, HJ 2001 Spectrophotometric measurement of Mercaptans with 4,4′-dithiodipyridine. Analytical Biochemistry 288 188194Google Scholar
Elias, RJ, Kellerby, SS & Decker, EA 2008 Antioxidant activity of proteins and peptides. Critical Reviews of Food Science and Nutrition 48 430441Google Scholar
Ellman, GL 1959 Tissue sulphydryl groups. Archives of Biochemistry and Biophysics 82 7077Google Scholar
Miller, NJ 1998 Nonvitamin plasma antioxidants. In Methods in Molecular Biology, Vol. 108, pp. 285297 (Ed. Armstrong, D). Totowa, NJ: Humana Press, Inc.Google Scholar
Noyhouzer, T, Kohen, R & Mandler, D 2009 A new approach for measuring the redox state and redox capacity in milk. Analytical Methods 1 9399Google Scholar
Pan, Y, Stocks, BB, Brown, L & Konermann, L 2009 Structural characterization of an integral membrane protein in its natural lipid environment by oxidative methionine labeling and mass spectrometry. Analytical Chemistry 81 2835Google Scholar
Papp, E, Szaras, P, Korcsmaros, T & Csermely, P 2006 Changes of endoplasmic reticulum chaperone complexes, redox state, and impaired protein disulfide reductase activity in misfolding á1-antitrypsin transgenic mice. FASEB Journal 20 E235E244Google Scholar
Re, R, Pellegrini, N, Proteggente, A, Pannala, A, Yang, M & Rice-Evans, C 1999 Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radial Biology & Medicine 26 12311237Google Scholar
Sava, N, Van der Plancken, I, Claeys, W & Hendrickx, M 2005 The kinetics of heat-induced structural changes of beta-lactoglobulin. Journal of Dairy Science 88 16461653Google Scholar
Sözgen Başkan, K, Tütem, E, Özer, N & Apak, R 2013 Spectrophotometric and chromatographic assessment of contributions of carotenoids and chlorophylls to the total antioxidant capacities of plant foods. Journal of Agricultural and Food Chemistry 61 1137111381Google Scholar
Suetsuna, K, Ukeda, H & Ochi, H 2000 Isolation and characterization of free radical scavenging activities peptides derived from casein. Journal of Nutritional Biochemistry 11 128131Google Scholar
Van den Berg, R, Haenen, GRMM, Van den Berg, H & Bast, A 1999 Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures. Food Chemistry 66 511517Google Scholar
Wedemeyer, WJ, Welker, E, Narayan, M & Scheraga, HA 2000 Disulfide bonds and protein folding. Biochemistry 39 42074216Google Scholar
Yagi, M, Sakurai, K, Kalidas, C, Batt, CA & Goto, Y 2003 Reversible unfolding of bovine β-Lactoglobulin mutants without a free thiol group. Journal of Biological Chemistry 278 4700947015Google Scholar
Zulueta, A, Maurizi, A, Frígola, A, Esteve, MJ, Coli, R & Burini, G 2009 Antioxidant capacity of cow milk, whey and deproteinized milk. International Dairy Journal 19 380385CrossRefGoogle Scholar