Polyphenol oxidase from bayberry (Myrica rubra Sieb. et Zucc.) and its role in anthocyanin degradation

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Abstract

Polyphenol oxidase (PPO) was extracted from bayberry (Myrica rubra Sieb. et Zucc. cv. Biqi), and its partial biochemical characteristics were studied. Stable and highly active PPO extracts were obtained using insoluble polyvinylpolypyrrolidone (PVPP) in sodium phosphate, pH 7.0, buffer. The highest PPO activity was observed in the ripe fruits. Optimum pH and temperature for bayberry PPO activity were pH 6.0 and T = 30 °C with 0.1 M catechol as substrate. PPO showed activity using the substrates of catechol, gallic acid and protocatechuic acid, but no activity with the substrates (+)-catechin, p-coumaric acid or caffeic acid. Km and Vmax values were 313 mM and 3.26ΔOD/min/g FW, respectively, with catechol as the substrate. Bayberry PPO did not act directly on cyanidin 3-glucoside but the rate of anthocyanin degradation was stimulated by the addition of gallic acid.

Introduction

Bayberry (Myrica rubra Sieb. et Zucc.) is one of six Myrica species native to China, belonging to the genus Myrica in the family Myricaceae (Chen, Xu, Zhang, & Ferguson, 2004). Bayberry is noted for its attractive red colour and exquisite flavours and is praised as a “precious southern Yangtze fruit of early summer” (Chen et al., 2004, Li et al., 1992). Unfortunately, bayberries are very perishable fruits, partly due to ripening in the hot and rainy season of June or July and with high respiration rates. The non-epicarp flesh exposed to the atmosphere is susceptible to bruising and mold rot. Most bayberry cultivars can only be stored with commercial value for 3 days at 20–22 °C and 9–12 days at 0–2 °C (Xi & Zheng, 1993). When stored at 1.0 ± 1.0 °C and chlorine dioxide is released slowly to the packaging bag, bayberries have a marketable life of 21 days (Li & Ma, 2004), which is the longest storage time reported for these fruits. Bayberry juice is an alternative product for longer consumption in which the Biqi cultivar has always been used as the major material because of its large production and excellent quality. However, the colour instability and haze formation during juice processing and storage are major problems facing food technologists (Chen et al., 1994, Li et al., 2002, Zhong, 2002).

Polyphenol oxidase (E.C. 1.14.18.1; PPO) in berry fruits plays an important role in the colour qualities and commercial properties of the fruits and their derived products. PPO and d-catechin together caused the loss of 50–60% of the strawberry anthocyanin pigments after 24 h at room temperature with the formation of a precipitate (Wesche-Ebeling & Montgomery, 1990a). PPO could even act directly on the blueberry anthocyanin degradation in crushed fresh blueberries, and the addition of chlorogenic acid stimulated browning reactions and pigment destruction (Kader, Rovel, Girardin, & Metche, 1997). Skrede, Wrolstad, and Durst (2000) demonstrated that endogenous PPO in blueberry fruits was responsible for anthocyanin degradation during juice processing. Yokotsuka and Singleton (1997) also showed that PPO was involved in anthocyanin destruction during grape juice processing.

The major anthocyanin in bayberry fruits has been identified as cyanidin 3-glucoside, which represents over 95% of the total pigments (Bao et al., 2005, Ye et al., 1994). One of the degradation mechanisms of this o-diphenolic anthocyanin is enzymatic oxidation, including PPO catalyzing o-diphenol to o-quinone and then acting with cyanidin 3-glucoside to generate cyanidin 3-glucoside o-quinone with partial regeneration of the o-diphenolic cosubstrate (Kader et al., 1998, Peng and Markakis, 1963). Phenolic compounds with o-dihydroxy, such as chlorogenic acid (Kader et al., 1997), caffeoyltartaric acid (Sarni, Fulcrand, Souillol, Souquet, & Cheynier, 1995), catechol (Peng & Markakis, 1963), gallic acid (Prabha & Patwardhan, 1986) and catechin (Wesche-Ebeling & Montgomery, 1990a) are good substrates for PPO. In our previous study (Fang, Zhang, & Wang, 2007), gallic acid and protocatechuic acid were detected in the bayberry fruits. However, little is known about the PPO in bayberry fruits and its involvement in anthocyanin degradation. The purpose of the present work was to develop a procedure for the extraction of PPO from bayberry fruits. Its partial characterization, the variation of its activity during bayberry maturity, and its role in anthocyanin degradation were also studied.

Section snippets

Plant material

The bayberry cultivar Biqi was used in the experiment since it is the dominant cultivar in industry. Fruits of unripe, ripe and overripe stages were harvested on June 18, 23, and 28, 2005, respectively. Fruits of different maturity were obtained from the same orchard in Cixi, Zhejiang province, China, and transported to our laboratory in 5 h. On arrival, 1 kg of undamaged fruits was homogenized using a blender and the pulps were used immediately for physicochemical determinations. Another 2 kg

Selection of conditions for enzyme assay

For selection of the most suitable buffer compositions to extract PPO from bayberry tissue, several were employed, as described by Cano et al. (1996). The increase of molar concentration of sodium phosphate buffer increased the extraction of PPO activity nearly twofold. However, the increase of ionic strength by addition of sodium chloride and the addition of detergent Triton X-100 gave only slight increase of PPO activity in extracts of both low and high concentration buffers (Table 1). The t

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