Elsevier

Food Chemistry

Volume 199, 15 May 2016, Pages 439-446
Food Chemistry

Different modes of inhibition for organic acids on polyphenoloxidase

https://doi.org/10.1016/j.foodchem.2015.12.034Get rights and content

Highlights

Abstract

It is still unclear whether the inhibitory effect of organic acid on polyphenoloxidase (PPO) is due to the reversible inhibition or decrease of pH. In this study, cinnamic acid, citric acid and malic acid inhibited PPO in different modes. Results showed that the inhibition by cinnamic acid resulted from reversible inhibition, while the decrease of pH was the main cause for citric acid and malic acid. The kinetic results showed that cinnamic acid reversibly inhibited PPO in a mixed-type manner. Fluorescence emission spectra indicated that cinnamic acid might interact with PPO and quench its intrinsic fluorescence, while the decrease of the fluorescence intensity induced by citric acid or malic acid was due to the acid-pH. Cinnamic acid bound to PPO and induced the rearrangement of secondary structure. Molecular docking result revealed cinnamic acid inserted into the hydrophobic cavity of PPO by forming π–π stacking.

Introduction

Polyphenoloxidase (PPO) is a group of copper-containing enzymes, which are widely distributed in animals, plants, fungi and bacteria (Mayer, 2006). PPO is responsible for the browning of damaged fruits and vegetables by catalyzing two different reactions: the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones, which polymerize to brown pigments subsequently (Espín et al., 1998, Virador et al., 2009). Besides the color changes, enzymatic browning deteriorates the nutritional quality of fruits and vegetables and cause a reduction of market value. Therefore, the inactivation of PPO is necessary for controlling browning. Over the years, many methods have been used to adjust the PPO activity. For example, heating (Gouzi, Depagne, & Coradin, 2012), high hydrostatic pressure (Yi et al., 2012), and addition of chemical reagents such as sodium sulfite (Palma-Orozco, Ortiz-Moreno, Dorantes-Álvarez, Sampedro, & Nájera, 2011) and organic acids. Organic acids are widely used to inhibit PPO activity. Many studies have been conducted on the inhibitory effect of various organic acids on PPO in recent years. The commonly used organic acids are citric acid (Liu et al., 2013), ascorbic acid (Landi, Degl’Innocenti, Guglielminetti, & Guidi, 2013), oxalic acid (Yoruk & Marshall, 2003a) and cinnamic acid (Shi, Chen, Wang, Song, & Qiu, 2005).

Cinnamic acid is an organic acid occurring naturally in a number of plants that has low toxicity and a broad spectrum of biological activities (Sova, 2012). Cinnamic acid and its derivatives are widely used in food industry (Hu et al., 2014). Citric acid and malic acid naturally present in some fruits, and they are widely used in food processing to diminish browning and had been shown to inhibit PPO (Yoruk & Marshall, 2003b). Recently, Shi et al. (2005) and Hu et al. (2014) reported that cinnamic acid and its derivatives strongly inhibited PPO activity. Altunkaya and Gökmen (2008) and Queiroz, da Silva, Lopes, Fialho, and Valente-Mesquita (2011) found that citric acid showed effective inhibition of PPO activity. Nevertheless, to our knowledge, only enzymatic activity assay was conducted in these studies. It is still unclear whether the inhibitory effect of organic acid on PPO is due to the reversible inhibition or decrease of pH. Few reports have mentioned the mode of inhibition for organic acids on PPO, especially the difference among these commonly used organic acids. The lack of understanding in inhibitory mode of organic acids on PPO has seriously affected the application of organic acids on PPO.

In our previous study, it was found that as the concentration of citric acid increased, the activity of PPO decreased gradually (Liu et al., 2013). However, the mode of inhibition for citric acid on PPO was not clear. Consequently, in this study, different inhibitory effects of organic acids on PPO were evaluated. The inhibition kinetics and binding constant were determined. Furthermore, conformational changes and the interaction between cinnamic acid and PPO were provided. The objective of the study was to distinguish between the mode of action as reversible inhibition and decrease of pH, and to provide more information for application of organic acids as PPO inhibitors.

Section snippets

Materials

Mushroom (Agaricus bisporus) polyphenoloxidase (T3824-50 Ku, 3130 μ/mg) was purchased from Sigma Chemical Co. (St. Louis, MO). Cinnamic acid, citric acid, malic acid, and reaction substrates (l-DOPA and catechol) were of analytical grade, purchased from Aladdin Chemicals Co. (Shanghai, China). All other chemicals were of analytical grade, solutions were prepared in double-distilled water.

Preparation of cinnamic acid, citric acid and malic acid solution

Preparation of cinnamic acid, citric acid and malic acid solution was carried out according to Liu et al.

Effect of cinnamic acid, malic acid and citric acid on activity of PPO

The effect of malic acid, citric acid and cinnamic acid on PPO activity was studied. As shown in Fig. 1A, the relative activity of PPO decreased with increasing concentrations of organic acids. At the same concentration, cinnamic acid treated PPO showed lower relative activity than citric acid and malic acid treated samples. Same results were found in Fig. 1B with substrate l-DOPA, the inhibitory effect of cinnamic acid was much stronger than that of citric acid or malic acid. When the

Conclusions

In this study, the different inhibitory modes of inhibition for organic acids on PPO were investigated. The extent of inhibition by citric acid and malic acid was mainly dependent on the decrease of pH. Compared with pH, reversible inhibition played a more important part in the inhibition of PPO. Cinnamic acid induced a mixed-type inhibition on PPO during the oxidation of catechol and l-DOPA. The inhibitory effect of cinnamic acid on the l-DOPA oxidation was stronger than catechol oxidation.

Acknowledgment

This study was supported financially by the National Natural Science Foundation of China (No. 31460435).

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