Effect of Selected Browning Inhibitors on Phenolic Metabolism in Stem Tissue of Harvested Lettuce Francisco A. Toma ´ s-Barbera ´ n,* ,† Marı ´a I. Gil, † Marisol Castan ˜ er, † Francisco Arte ´s, † and Mikal E. Saltveit ‡ Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 4195, Murcia 30080, Spain, and Mann Laboratory, Department of Vegetable Crops, University of California, Davis, California 95616 Wound-induced changes in phenolic metabolism causes stem browning (butt discoloration) in harvested lettuce. Stem tissue near the harvesting cut exhibited increased phenylalanine ammonia- lyase (PAL) activity and accumulation of caffeic acid derivatives. These o-diphenols can be oxidized by the enzyme polyphenol oxidase (PPO) to produce brown pigments. This browning reaction can readily be followed by measuring a* values. Browning was reduced by washing stem disks with solutions of 0.3 M calcium chloride, 1.0 mM 2,4-dichlorophenoxyacetic acid (2,4-D), or 0.5 M acetic acid. These browning inhibitors appear to act in different ways. Calcium chloride decreased PAL activity to 60% of the control, but did not substantially affect the accumulation of phenolic compounds. The mechanism of calcium action could be to decrease PPO activity or to preserve membrane structure. PAL activity was inhibited 60% by 2,4-D, and the biosynthesis of phenolic compounds was strongly inhibited but not suppressed. Acetic acid completely inhibited PAL activity and the production of wound-induced phenolics. PAL was irreversibly inhibited by acetic acid, and this may explain its role as a browning inhibitor. Keywords: Lettuce; Lactuca sativa; postharvest storage; butt discoloration; phenolic metabolism; PAL; browning inhibition INTRODUCTION The shelf life of minimally processed fruits and vegetables is often limited by enzymatic browning (Vamos-Vigyazo, 1981). The organoleptic and biochemi- cal characteristics of fruits and vegetables are also strongly modified by the appearance of brown pigments. Oxidative browning is mainly due to the enzyme polyphe- nol oxidase (EC 1.14.18.1) (PPO), a mixed function oxidase which catalyzes the hydroxylation of monophe- nols to o-diphenols and, in a second step, the oxidation of colorless o-diphenols to highly colored o-quinones (Sapers and Hicks, 1989; Janovitz-Klapp et al., 1990). The o-quinones condense spontaneously with other o-quinones and with many constituents of foods such as proteins, reducing sugars, etc. to form high molecular weight polymers which precipitate yielding the brown, red, or dark pigments characteristic of browned fruit and vegetable tissues (McEvily et al., 1992). PPO has a broad specificity toward different phenolic substrates, and the brown pigments resulting from these phenolics differ widely in color intensity (Nicolas et al., 1993). Browning is one of the main causes of quality loss during the postharvest storage of head lettuce and minimally processed lettuce (Ilker et al., 1977; Mateos et al., 1993). Extensive investigations have been con- ducted to understand the biochemical basis of lettuce browning and to find physical or chemical treatments to prevent these discolorations (Hyodo et al., 1978; Ke and Saltveit, 1986; Siriphanich and Kader, 1985; Fujita et al., 1991; Heimdal et al., 1994; Chazarra et al., 1996). In a recent study we described the beneficial effect of washing the cut stem end (i.e., the butt) of lettuce heads with different organic acid solutions to prevent butt discoloration (Castan ˜ er et al., 1996). Prevention of ethylene-induced browning (russet spotting) by calcium and synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4- D) has also been reported (Ke and Saltveit, 1986). Wounding generally induces increased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity and increased phenolic metabolism in many plant tissues (Dixon and Paiva, 1995). In addition, wounding induces cellular decompartmentalization which allows mixing of phe- nolic substrates and PPO, leading to the development of browning (Mayer, 1987). Browning can be delayed by storage at low temperatures, but it nevertheless occurs after a lag period. Lettuce butt discoloration is a type of browning induced by wounding. Harvesting lettuce entails wound- ing the stem butt end, thus inducing all the above mentioned changes leading to the formation of brown pigments and butt discoloration. Similar changes also occur in iceberg lettuce midribs during the storage of minimally processed cut lettuce (Mateos et al., 1993). The objective of this work was to study changes in phenolic metabolism during the development of wound- induced lettuce stem browning and to understand how washing treatments with calcium chloride, 2,4-D, and acetic acid solutions can prevent browning through their effects on phenolic metabolism. MATERIALS AND METHODS Lettuce. Commercially grown and harvested crisphead (Iceberg) lettuce were obtained from a local wholesale market and transported to the laboratory where they were stored at 0 °C until used. Preparation of Stem Disks. Stem cylinders were excised from lettuce heads with a 2.5 cm diameter stainless steel cork- borer, by pushing the cork-borer up the stem from the butt * Address correspondence to this author (fax +34-68- 266 613; e-mail fatomas@natura.cebas.csic.es). † CEBAS (CSIC). ‡ University of California, Davis. 583 J. Agric. Food Chem. 1997, 45, 583-589 S0021-8561(96)00478-5 CCC: $14.00 © 1997 American Chemical Society