Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/14/2004
Publication Date: 5/21/2004
Citation: Breksa III, A.P., Manners, G.D. 2004. Determination of limonin d-ring lactone hydrolase activity by solid phase extraction with indirect fluorescence detection. Journal of Agricultural and Food Chemistry. Vol 52, No. 12, p. 3772-3775. Interpretive Summary: Freeze damage or physical damage to citrus fruit, including juicing, initiates the formation of the bitter dilactone limonoids from non-bitter monolactone precursors. This phenomenon is referred to as delayed bitterness and is a major problem for both fresh fruit and juice producers worldwide. The major limonoid associated with delayed bitterness is limonin. The formation of limonin from limonoate A-ring lactone (LARL) is catalyzed by the acidic pH of citrus juice and the enzyme limonin D-ring lactone hydrolase (LDLH). The identification of commercial cultivars with reduced susceptibility to delayed bitterness is of considerable interest to citrus producers throughout the world. A method for the evaluation of LDLH activity associated with delayed bitterness in citrus is described. The method utilizes solid phase extraction (SPE) for the isolation of limonin A-ring limonoate (LARL), which is subsequently converted to limonin and quantitated by fluorescence. The method we have described is ideally suited for the task of rapidly characterizing both purified and crude preparations of LDLH enzymes. Compared to other reported procedures, this method is more sensitive than thin-layer chromatography methods, does not require radioactive materials and is more efficient than an ion-pairing HPLC method.
Technical Abstract: A method for the evaluation of Limonin D-ring lactone hydrolase (LDLH) LDLH activity associated with delayed bitterness in citrus is described. The method utilizes solid phase extraction (SPE) for the isolation of limonin A-ring limonoate (LARL), which is subsequently converted to limonin and quantitated by fluorescence. The fluorescence method is capable of quantifying the formation LARL in concentrations as low as 75 nanograms and is applicable to both purified and crude enzyme preparations. The coupling of SPE with fluorescence detection allows for the high throughput of samples.