Mechanisms non-enzymatic browning in orange juice during storage

Highlights

• Browning and Reactive Carbonyl Species were monitored in orange juice for 8 weeks.

• Changes in RCS concentrations were positively correlated with color formation.

• 3-Deoxyglucosone and acetol significantly increased color formation.

• The Maillard reaction was reported as the main pathway of orange juice browning.

• Tryptophan and glutamine significantly impacted RCS and color formation.

Abstract

The role of Reactive Carbonyl Species (RCS) derived from the Maillard reaction and ascorbic acid degradation on brown color formation was investigated in orange juice during storage. Eight RCS were monitored in aseptic juice over an 8-week period under refrigerated (4 °C) and accelerated conditions (35 °C). Significant changes in RCS concentrations were reported and positively correlated with color formation. Recombination experiments demonstrated the significant role of 3-deoxyglucosone and acetol on color formation as well as their interactions with glyoxal and methylglyoxal that lead to an increase in browning. Isotopic enrichment techniques further identified fructose as the main precursor of RCS, indicating the important role of Maillard reaction as a mechanism of non-enzymatic browning during orange juice storage. Finally, among the amino acids, tryptophan and glutamine showed the largest percentage losses in orange juice during storage and were reported to significantly impact the RCS composition and color formation.

Introduction

Orange juice is a popular beverage worldwide and significantly contributes to the daily fruit consumption in most countries. Fruit intake is highly impacted by factors such as cost, convenience, health benefit and sensory appeal (Pollard, Kirk, & Cade, 2002). Orange juice meets many of these requirements with valuable nutritional content and highly appreciated sensory properties. However, during storage under uncontrolled environmental conditions juice browning is occurring and gives rise to negative color and flavor changes that ultimately affects the consumer experience.

Juice browning can occur through enzymatic and non-enzymatic pathways. Enzymatic browning results from the oxidation of phenolic compounds into quinones by the polyphenol oxidase enzyme (PPO) (Martinez & Whitaker, 1995). The use of additives and/or heat treatment can inhibit enzymatic browning during juice processing (Campos et al., 1996, Sanchez-Vega et al., 2009). However, the mechanisms of the non-enzymatic browning reaction are complex and have not been adequately characterized, challenging the development of effective mitigation strategies.

Ascorbic acid degradation and the Maillard reaction have been identified as the main non-enzymatic reaction pathways responsible for juice browning (Bharate & Bharate, 2014). Historically, ascorbic acid has been defined as the main contributor of browning in citrus juice as its degradation follows a linear trend, significantly correlating to brown color formation. Multiple pathways can occur simultaneously during non-enzymatic browning reactions in food systems, and focusing on simple correlations between main precursors and browning can overlook contributing reaction mechanisms. For example, reducing sugars such as glucose and fructose do not always exhibit significant changes in concentration during storage as they can be simultaneously generated from sucrose hydrolysis. In general, the role (and mechanisms) of sugar reactions on juice browning has not been as well defined as the mechanisms of ascorbic acid degradation.

Based on existing knowledge, juice manufacturers have developed strategies for mitigation, primarily utilizing additives or supplemental processes (Bharate & Bharate, 2014). However, recent consumer trends are oriented towards the use of natural ingredients and processes that are in line with sustainability and preservation of the environment. For these reasons, a more comprehensive understanding of browning reactions in juice is needed and will allow for the development of mitigation strategies that utilize natural ingredients and support simple label practices.

Reactive Carbonyl Species (RCS) are generated from reducing sugars during the Maillard reaction. RCS are potent intermediates in the Maillard reaction and play a central role in flavor and color formation (Cämmerer et al., 2002, Kroh et al., 2008). In apple juice, RCS have been identified as key precursors of color formation during storage has been recently demonstrated (Paravisini & Peterson, 2018). The causative effect of specific RCS on browning in apple juice was demonstrated for glyoxal and methylglyoxal. Based on these findings, the use of endogenous apple phenolic compounds was proved to significantly reduce these RCS levels and thus suppress browning formation during storage. In lemon juice, the accumulation of RCS is thought to be associated with browning formation through the breakdown of ascorbic acid but was not able to adequately explain color formation (Clegg & Morton, 1965).

Additionally, the catalytic role of amino acids on citrus juice browning has been previously reported (Clegg, 1966, Handwerk and Coleman, 1988). In fruits, multiple factors are known to impact the composition of amino acids. In apple, for example, the concentration of γ-amino butyric acid is significantly impacted by the storage conditions of the fruit (Deewatthanawong & Watkins, 2010). The variety of the fruit also impact the intrinsic amino acid composition. Gaining more knowledge regarding the role of amino acids in orange juice browning beyond their catalytic effect is needed.

The overall goals of this study were (1) to define the contribution of RCS from ascorbic acid and Maillard reaction pathways on orange juice browning and (2) to characterize the impact of amino acids on RCS and color formation.