Yellow, ripened (yellow with green tips) bananas were treated with nitrogen gas for 12, 24 and 48 h at 20 • C to study the effects of anaerobic conditions on the fruit quality, including the production of volatile compounds and activity of alcohol acetyltransferase (AAT) during the post-harvest period. Significantly higher concentrations of acetaldehyde and ethanol were found in the treated fruit than in the untreated bananas after removal from the conditions. The 48-h treatment showed higher levels of ethanol and acetaldehyde after storage. Immediately after releasing the fruit from the anaerobic atmosphere, the amounts of isobutyl acetate and isoamyl acetate decreased. The production of ethyl acetate increased markedly in each treatment unit 1 day after removing the fruit. Regardless of recovery from the production of isobutyl acetate and isoamyl acetate 1 day after treatment, the banana-like aroma was denatured because of the production of high levels of ethyl acetate and ethanol. The increase in AAT was slightly lower in nitrogen-treated fruit than untreated fruit. However, the activity in treated fruit was sufficient for ester production in bananas. The endogenous alcohol levels were increased during nitrogen gas treatment in the fruit due to the inhibition of ester production. Colour development of the bananas was greatly inhibited by the treatments, while the sugar content did not show any differences between the treated and untreated fruit. The results suggest that, even if bananas are removed from the short-term anaerobic conditions, a loss in quality, except sweetness, easily occurs afterwards, especially the characteristic aroma of the fruit during the post-harvest period.
The effect of anaerobic conditions on acetate ester biosynthesis in ripened banana pulp was investigated. Incubation of the pulp in less than 1% O(2) resulted in a significant reduction in the formation of ethyl acetate. Regardless of the presence of a large amount of endogenous ethanol and the remaining exogenous isobutyl alcohol after complete anaerobic incubation with the pulp, the production of acetate ester decreased. The effect of addition of pyruvate, isobutyl alcohol, acetate, and methyl hexanoate on acetate ester formation in 100% N(2) was also investigated. The addition of pyruvate and isobutyl alcohol to the pulp gave lower acetate esters in N(2) than in air, whereas the pulp incubated with acetate and isobutyl alcohol produced more acetate ester in both conditions. Therefore, the lack of acetyl CoA, or more precisely acetate, in the tissue is the main reason for the inhibition of acetate ester formation under anaerobic conditions. The activity of beta-oxidation measured by incubation with methyl hexanoate was detected only in the samples incubated in air. The formation of acetyl CoA, derived from pyruvate through mitochondria and through beta-oxidation, was inhibited by anaerobic conditions, which suggests that mitochondrial activity and/or beta-oxidation are essential for ester biosynthesis.
Butanol vapor feeding to ripe banana pulp slices produced abundant butyl butanoate, indicating that a portion of butanol molecules was converted to butanoate/butanoyl-CoA via butanal, and further biosynthesized to ester. A similar phenomenon was observed when feeding propanol and pentanol, but was less pronounced when feeding hexanol, 2-methylpropanol and 3-methylbutanol. Enzymes which catalyze the cascade reactions, such as alcohol dehydrogenase (ADH), acetyl-CoA synthetase, and alcohol acetyl transferase, have been well documented. Aldehyde dehydrogenase (ALDH), which is presumed to play a key role in the pathway to convert aldehydes to carboxylic acids, has not been reported yet. The conversion is an oxygen-independent metabolic pathway and is enzyme-catalyzed with nicotinamide adenine dinucleotide (NAD+) as the cofactor. Crude ALDH was extracted from ripe banana pulps, and the interference from ADH was removed by two procedures: (1) washing off elutable proteins which contain 95% of ADH, but only about 40% of ALDH activity, with the remaining ALDH extracted from the pellet residues at the crude ALDH extraction stage; (2) adding an ADH inhibitor in the reaction mixture. The optimum pH of the ALDH was 8.8, and optimum phosphate buffer concentration was higher than 100 mM. High affinity of the enzyme was a straight chain of lower aldehydes except ethanal, while poor affinity was branched chain aldehydes.
Acetyl-CoA synthetase (ACS) activity in fully ripened bananas was determined by HPLC. The ACS activity was higher for acetate as a substrate than for butyrate. The higher substrate specificity for acetate than for butyrate evidences that banana produces a great amount of acetate esters and a less amount of butyrate esters. The K. for MgCl2 for butyryl-CoA formation was 5-fold higher than that for acetyl-CoA formation. ACS activity was assayed in mitochondrial, microsomal, and cytosol fractions isolated from ripened banana pulp and the highest total enzyme activity was detected in the cytosol fraction. This is the first report on the presence of this enzyme, ACS, in fruit pulp.
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