Biochemical Properties of Polyphenol Oxidases from Ready-to-Eat Lentil (Lens culinaris Medik.) Sprouts and Factors Affecting Their Activities: A Search for Potent Tools Limiting Enzymatic Browning.

Małgorzata Sikora,Anna Jakubczyk,Justyna Bochnak,Michał Świeca,Monika Franczyk,Urszula Złotek

doi:10.3390/foods8050154

Małgorzata Sikora, Anna Jakubczyk + Show 4 more

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https://doi.org/10.3390/foods8050154

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Abstract

Enzymatic browning of sprouts during storage is a serious problem negatively influencing their consumer quality. Identifying and understanding the mechanism of inhibition of polyphenol oxidases (PPOs) in lentil sprouts may offer inexpensive alternatives to prevent browning. This study focused on the biochemical characteristics of PPOs from stored lentil sprouts, providing data that may be directly implemented in improving the consumer quality of sprouts. The purification resulted in approximately 25-fold enrichment of two PPO isoenzymes (PPO I and PPO II). The optimum pH for total PPOs, as well as for PPO I and PPO II isoenzymes, was 4.5–5.5, 4.5–5.0, and 5.5, respectively. The optimal temperature for PPOs was 35 °C. Total PPOs and the PPO I and PPO II isoenzymes had the greatest affinity for catechol (Km = 1.32, 1.76, and 0.94 mM, respectively). Ascorbic acid was the most effective in the inhibition of dark color formation by total PPOs, and showed ca. 62%, 43%, and 24% inhibition at 20-, 2-, and 0.2-mM concentrations. Ascorbic acid, l-cysteine, and sodium metabisulfite (20 mM) significantly inhibited color development in the reactions catalyzed by both isoenzymes of PPO. Ba2+, Fe3+, and Mn2+ (10 mM) completely inhibited PPO activity. This study of the effect of antibrowning compounds and cations on PPO activity provides data that can be used to protect lentil sprouts against enzymatic browning during storage and processing.

Highlights

Polyphenol oxidases (PPOs) (EC 1.14.18.1, EC 1.10.3.1, or EC 1.10.3.2) are widely distributed in the plant kingdom, and their level and activity are dependent on the age, species, variety, maturity, and stress status of plants [1,2]
We report the isolation, partial purification, and biochemical properties of two isoenzymes and total polyphenol oxidases (PPOs) activity in lentil sprouts (Lens culinaris Medik.)
Diethylaminoethyl–Sepharose (DEAE–S), tris(hydroxymethyl)aminomethane (TRIS), ethylenediaminetetraacetic acid sodium salt (EDTA), 4-methylcatechol, gallic acid, caffeic acid, l-cysteine, ascorbic acid, and dl-dithiothreitol were obtained from Sigma-Aldrich (Poznań, Poland)

Summary

Introduction

Polyphenol oxidases (PPOs) (EC 1.14.18.1, EC 1.10.3.1, or EC 1.10.3.2) are widely distributed in the plant kingdom, and their level and activity are dependent on the age, species, variety, maturity, and stress status of plants [1,2]. Three main types of phenol oxidases are known: (I) Monophenol monooxygenase ( called tyrosinase, monophenol oxidase, or cresolase) catalyzes the hydroxylation of monophenol to ortho-diphenol and the oxidation of diphenol to ortho-quinone; (II) diphenol oxidase ( called catechol oxidase, polyphenol oxidase, or o-diphenolase) catalyzes the oxidation of ortho-phenol, but cannot catalyze the oxidation or monooxygenation of metaphenol and para-phenol; and (III) laccase catalyzes the oxidation of ortho-phenol and para-phenol, but cannot catalyze the oxidation of monophenol and metaphenol [3]. As one of the antibrowning agents, ascorbic acid inactivates PPOs irreversibly in the absence of PPO substrates, probably through binding to its active site, preferentially in its oxy form It can reduce reaction products, limiting the formation of a dark color. Citric acid and ethylenediaminetetraacetic acid sodium salt (EDTA) chelate copper at an enzyme-active site [12,13]

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