Quantification of radicals formed during heating of β-lactoglobulin with glucose in aqueous ethanol

Highlights

• Radicals formed during Maillard reactions may be quantified by spin trapping.

• Glucose reaction competes with β-lactoglobulin oxidation.

• Quinones protect β-lactoglobulin against oxidation better than parent phenols.

Abstract

Heating β-lactoglobulin alone, or in the presence of glucose, at moderately elevated temperatures (50, 70 and 80 °C) in 30% aqueous ethanol at pH 6.0, 7.0 and 8.0 led to an iron catalysed formation of highly reactive radicals at concentrations of up to 1.0 mmol/mol protein as trapped by α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN) and quantified by ESR spectroscopy. Oxidation of β-lactoglobulin was favoured by increasing temperature and high pH conditions, whereas presence of glucose decreased oxidation, indicating that oxidation of β-lactoglobulin involves either amine or thiol side chain group blocked as oxidation substrate by reaction with glucose as a reducing sugar. The oxidation of β-lactoglobulin was hampered by the phenolic antioxidant 4-methylcatechol and to an even larger extent by the oxidised quinone form 4-methyl-1,2-benzoquinone, suggesting that it is the quinone forms of phenolic antioxidants which compete with reducing sugars in protecting proteins against oxidation.

Introduction

Maillard reactions (MR) play a key role for food quality, as MR may deplete important nutrients and under some conditions result in potentially toxic reaction products. However, other reaction products of MR are aroma compounds and colouring components characteristic for both heat processed and dried foods. MR occurring in vivo have also drawn attention, as products from MR are known to be involved in metabolic complications from diabetes and cardiovascular diseases. These complications have been related to reactions of glucose with proteins in the body, generating what are known as advanced glycation end products (AGEs). The complex mechanism behind MR in food and in vivo was recently reviewed and related to their physiological effects (Poulsen et al., 2013). Model systems consisting of single proteins in combination with specific reducing sugars were found most useful for a better understanding of the reactions leading to formation of AGEs and especially reactions between β-lactoglobulin or bovine serum albumin with specific reducing sugars have been studied (Chevalier et al., 2001, Chobert et al., 2006; Easa, Hill, Mitchell, & Taylor, 1996). β-Lactoglobulin is a bovine whey protein with 162 amino acid residues and mass of about 18.3 kDa, and important for MR during heat processing of dairy products (Verheul, Pedersen, Roefs, & de Kruif, 1999). β-Lactoglobulin easily denatures during such heat processing and reacts freely with reducing sugars, including glucose, lactose and ribose (Chobert et al., 2006, Chevalier et al., 2001).

Highly reactive radicals are formed as intermediates in MR as the result of oxidative processes related to proteins and lipids and also are occurring during heat processing of food (Yin, Andersen, Thomsen, Skibsted, & Hedegaard, 2013). Most of these radicals are of fleeting existence and have escaped detection even using electron spin resonance (ESR) spectroscopy. However, a recently developed method based on the spin trapping technique have made detection of such highly reactive radicals by ESR possible for food related systems (Yin, Andersen et al., 2013). At more advanced stages of MR, but prior to significant products browning, stable radicals are formed and have been identified by direct measurement using ESR as pyrazinium radical cations (Hofmann, Bors, & Stettmaier, 1999a). Pyrazinium radical cations are suggested to be formed by fragmentation of Schiff bases or by condensation of glycolaldehyde, the reduced form of glyoxal, as a reaction intermediate of MR, with amino acids prior to browning (Hofmann et al., 1999a). Focus so far has, however, been on formation of stable radicals as part of the MR rather than on the highly reactive radicals suggested to be formed during heat processing. Experimental evidence for these highly reactive radicals being formed during heating of proteins with reducing sugars is still lacking, and their significance in protein oxidation competing with MR is somewhat speculative. Since oxidative steps are known to be involved in some of the pathways in glycation of proteins, antioxidants like plant phenols may be active also as anti-glycation agents both in food and after ingestion in vivo by preventing formation of highly reactive radical precursors to AGEs, acrylamide or other toxic compounds. Notably, antioxidants often work well at room temperature, but lose their effect at elevated temperatures. Although it was found that formation of AGEs is inhibited by chlorogenic acid and epicatechin in vivo (Jang et al., 2009, Tsuji-Naito et al., 2009), chlorogenic acid and epicatechin may also have other effects at elevated temperature typical for food processing, during which the plant phenols may get oxidised.

Accordingly, it seems timely to study the formation of highly reactive radicals as reaction intermediates in a high molecular weight food model based on a well-defined protein and a reducing sugar. The formation of radicals was followed by ESR using a spin trapping technique recently used to detect highly reactive radicals in a Maillard reaction model system. The whey protein β-lactoglobulin alone and in mixtures with glucose was heated and the effect of pH at reduced water activity (a_w), presence of oxygen and iron as a pro-oxidative transition metal, on formation of reactive radicals was quantified. In addition, the role of phenolic antioxidants as possible inhibitors for formation of such radicals was explored for the conditions relevant for heat treatment of food.