Graduate Thesis Or Dissertation
 

The Role of Ascorbic Acid, β-Cyclodextrins, and Alternative Substrates in Dictating Rates of Polyphenol Oxidase-Catalyzed Browning in Potato-Based Reaction Mixtures

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  • Enzymatic browning is a consequence of a series of reactions initiated by polyphenol oxidase (PPO)-catalyzed quinone formation. Unwanted browning negatively affects food quality and lowers consumer acceptability. Thus, there is general interest in strategies to control enzymatic browning. Cyclodextrins are cyclic glucan oligosaccharides that form inclusion complexes with a number of potential PPO substrates; thus, there is interest in using these compounds as anti-browning agents. Therefore, a study was done to determine the effect of β-cyclodextrins (βCyD) on PPO-catalyzed reactions in a model potato system. Potato enzyme extracts and semi-purified (acetone powder) potato PPO were used as enzyme sources. Substrates included phenolics endogenous to potatoes. Reaction time-courses were followed spectrophotometrically. Extents of βCyD inhibition of PPO-catalyzed reactions were shown to be substrate specific and can be quantitatively accounted for based on degrees of βCyD substrate sequestration. There was no evidence for direct irreversible βCyD inactivation of potato PPO. An apparent “direct PPO inactivation” by βCyD is shown to result from a sequence of sequestration-dependent reactions that occur in commonly employed assay systems for the quantification of PPO in fruits and vegetables. The ability to accurately measure the activity of PPO in complex matrices is essential for a) quantifying the amount of active enzyme in foods, b) following the fractionation of enzyme during processing and handling, and c) doing kinetic analyses to assess the impact of PPO modifiers. The most common activity quantification methods for this enzyme are spectrophotometric. A problem encountered when using such methods is interference due to the presence of ascorbic acid (AA). AA is widely distributed in plant (fruits and vegetables)-based foods and is often used as an enzyme “protecting agent” during PPO extraction; thus, it is commonly associated with PPO activity measurements. A study was done to determine the nature of AA’s effect on spectrophotometric determinations of PPO activity. Potato enzyme extracts and semi-purified potato PPO were used as enzyme sources. Substrates included phenolics endogenous to potatoes. PPO activities were determined based on initial velocity measurements; typically using the first linear increases in absorbance post-lag phase as indicative of initial velocities. AA inactivation of PPO was observed only in the presence of substrate; thus, there was no evidence for direct AA inactivation of PPO. The inactivation of PPO attributed to AA is thus substrate mediated. The extent of AA-dependent inactivation of PPO in model systems varied between substrates. AA-dependent inactivation of PPO was greatest when catechol was the primary substrate, presumably due to catechol being the most effective mechanism-based inhibitor of PPO. AA prevented enzymatic browning in potato PPO extracts, presumably due to its reduction of the quinones generated as a result of the PPO-catalyzed reaction. The problems associated with the presence of AA in PPO assay mixtures could be circumvented by using ascorbate oxidase to remove AA immediately prior to the addition of catechol as the primary assay substrate or by using chlorogenic acid as the primary assay substrate. There is considerable interest in using natural PPO inhibitors to control browning in fruit and vegetable products. p-Coumaric acid, a common secondary metabolite of plants, has been studied as an inhibitor of polyphenol oxidases/tyrosinases from several foods (e.g., mushroom, apple, potato). However, studies on the use of p-coumaric acid for the inhibition of PPO-initiated browning in actual food systems are limited. Therefore, a study was done to ascertain the efficacy of using p-coumaric acid to limit PPO-initiated browning in fresh potato juice. βCyD was included in this study to determine the potential of using it in combination with p-coumaric acid for browning inhibition. Diluted fresh potato juice and semi-purified potato PPO with tyrosine and/or chlorogenic acid were used as representative reaction mixtures. Tyrosine and chlorogenic acid are the primary PPO substrates found in potatoes. Color was characterized spectrophotometrically and/or colorimetrically. Extents of browning inhibition were shown to be reaction system-dependent. Browning in potato juice per se was unexpectedly enhanced by the addition of p-coumaric acid. This was interpreted as p-coumaric acid acting as an alternative substrate with significantly higher browning efficiency. Addition of βCyD to p-coumaric acid spiked potato juice slightly reduced the extent of color enhancement; extents of browning under these conditions were higher than that observed in the native potato juice (i.e., potato juice without the addition of p-coumaric acid). The addition of p-coumaric acid to any of the model reaction mixtures (i.e., those containing semi-purified enzyme and substrates) significantly inhibited browning and the addition of βCyD slightly enhanced the inhibitory effect. The discrepancy in p-coumaric acid effects on browning inhibition in different reaction systems is postulated to be due to non-enzyme and non-substrate components in potato juice that participate in the post-PPO reaction sequences that ultimately lead to brown color formation.
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  • Ongoing Research
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  • 2019-04-15 to 2020-05-16

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