526 Journal ofFood Protection Vol. 42, No.6, Pages 526-534 (June, 1979) Copyright© 1979, International Association of Milk, Food, and Environmental Sanitarians Naturally-Occurring and Ethylene-Induced Phenolic Compounds in the Carrot Root S. K. SARKARI* and C. T. PHAN2 Department ofFood Science, University ojAlberta, Edmonton, Alberta T6G 2N2, Canada and Department des Sciences Biologiqus, Universite de Montreal, Cas Postale 6128, Montreal, Quebec H3C 317, Canada (Received for publication August 14, 1978) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 ABSTRACT periods of storage (3). The carrots processed soon after Carrots accumulate phenols and often develop off-flavor and color harvest were free from this bitter flavor. In an attempt to after long periods of storage. To investigate probable causes for such find a solution to this bitterness problem, investigations physiological disorder, the effect of ethylene on various aspects of metabolism of carrot roots was studied. Ethylene, when applied at into various aspects of carrot production, including moderate level (100 ppm), caused an increase in total phenol content of cultural and post-harvest handling practices, were the roots. It caused an increased accumulation of the phenols normally initiated by various workers at the Agricultural present in the tissue, especially isochlorogenic acid. Moreover, Experiment Station in Geneva, New York. Atkin (3) relatively longer exposure to a moderate level (100 ppm) and short reported the results of a survey of various cultural exposure to high levels (2000 and 50,000 ppm) of ethylene induced formation of new compounds, viz, isocoumarin, eugenin, and two practices of carrot growers. Application of oils (to control others yet unidentified. Studies with [l-14C]acetate, [2- 14C]malonate and weeds), insecticides, fertilizers, and crop rotation were [3-14C]acetoacetate indicated that the newly synthesized compounds the major management procedures. The results of his are probably synthesized via the acetate pathway. Ethylene stimulated survey failed to provide any correlation between above the rate of 0 2 uptake and C02 evolution by carrot slices, indicating cultural practices and the development of bitterness in probable relationship of glucose metabolism with de novo synthesis of "stress-metabolites". Studies with specifically labelled glucose showed carrots. However, Atkin always observed that whenever that both the Embden-Meyerhof-Parnas (EMP) and the Pentose bitterness occurred, it did so in refrigerated storage. Phosphate (PP) pathways operate in carrots, and that ethylene The nature of the compound(s) responsible for preferentially stimulated the EMP pathway. Like ethylene, dinitro­ bitterness in carrots was investigated by Sondheimer (24). phenol (DNP) induced isocoumarin synthesis in carrots. Methylene blue, an electron acceptor often used for stimulating glucose He suggested that bitter flavor in carrots was caused by catabolism via the PP pathway, also induced isocoumarin synthesis in the presence of several compounds and that 8-hydroxy-3- carrots. The effect of cycloheximide. an inhibitor of protein synthesis, methyl-6-methoxy-3,4-dihydroisoioumarin was one of suggested that the de novo synthesis of enzyme protein(s) might be them. He named this compound as 'isocoumarin', and required for ethylene-induced isocoumarin synthesis in carrots. In con­ this name has been used ever since. clusion, it appears that ethylene triggers changes in the metabolism of carrots during storage, which result in, among other things, synthesis What causes induction of isocoumarin synthesis in of so-called "stress-metabolites," namely isocoumarin and eugenin and carrots was the subject of further investigation, and related compounds. various workers suggested that ethylene might be a "triggering factor" (5,6, 7). Carrot root is an important component of the vegetable portion of our diet. It is a good source of Although ethylene was suggested as a causative agent vitamins, minerals and fiber. And it adds rich color and for synthesis of isocoumarin in carrots on the basis of aroma to our food. On the basis of the United States isocoumarin's ability to fluoresce under U .V .light (5,6, 7), Department of Agriculture statistics for 1970 (1), carrot no attempt was made by these workers to isolate and root has been ranked tenth in terms of nutritional value characterize isocoumarin from ethylene-treated carrots among 38 other fruits and vegetables, and seventh for its to ascertain whether the observed fluorescence was contribution to nutrition. caused only by isocoumarin and not any other Current advancement in agriculture has enabled us to compound(s). Further, the question whether isocoumarin produce more carrots than can be marketed as fresh is the only compound synthesized in carrots in the produce, which leaves a large amount of produce to be presence of ethylene was unanswered until we started our processed in some form for later use. The time lag investigation some 12 years later. Our results show that between harvest and processing, or future marketing as not only isocoumarin but three additional related fresh produce, appears to be crucial in relation to compounds are synthesized de novo in carrots after undesirable color and flavor development in carrots (8). exposure to ethylene. Further, the levels of existing During the early 1950s, processing industries in the phenols, including hydroxy-cinnamic acid derivatives, eastern U.S. and Canada encountered a problem of increased considerably in ethylene-treated carrots. bitterness in some carrots that were processed after some The cinnamic acid derivatives, namely various 'University ofAlberta. phenolic compounds encompassing flavonoids, lignins, 'Universite de Montreal. and tannins, although classified by plant physiologists as PHENOLIC COMPOUNDS IN CARROTS 527 secondary metabolites, occupy an important place in ether to obtain two fractions: (a) ether-soluble and (b) food color and flavor. Therefore, it must be recognized ether-insoluble. The presence of phenolic compounds in by people dealing with foods of plant origin that unless the ether-soluble fraction was not detectable in carrots adequate care in handling and storage of fresh produce is stored at 3 ± 1 C and ca. 90% relative humidity, and in taken, undesirable color and/or flavor development may the absence of ethylene. No isocoumarin or any other result from various phenolic compounds that are present related compounds were present in the extract. in these tissues. The ether-insoluble portion revealed the presence of a We, in this paper, will describe the results of our number of phenolic compounds on the paper chromato­ studies on the effects of ethylene on various phenolic gram (Table 2). Three of these spots were identified by compounds in carrot roots. co-chromatography with known compounds; caffeic acid (I), isochlorogenic acid (II) and chlorogenic acid (III). EXPERIMENTAL The rest of the spots were not identified as such but All the carrots used for experiments were grown at the Horticultural Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 Research Station, Brooks, Alberta. These were stored at 3 ± 1 C and 98% relative humidity, in the absence of light. The methods of extraction, separation and identification of various phenolic compounds have been published previously (13.17-21), and a detailed description will not be given here. However, it should be noted that we have used intact carrots as well as slices for our studies. The slices were used either for feeding experiments with labelled compounds or to augment the effects of ethylene on carrots over a short time, resulting from an increased surface for exposure to ethylene. The entire phenol extract of carrots was classified, according to (I) solubility, into ether-soluble and methanol-soluble (or ether-insoluble) fractions. The composition of extracts was analyzed by paper, thin-layer and gas chromatography. (II) RESULTS AND DISCUSSION E]Ject ofstoroge on phenol content ofcarrots The total phenol content of carrots stored at 3 ± 1 C HOO~COH ~ increased steadily with time. Figure 1 shows that the HO o-t-CH=CH-o amount of phenols in 100 g of carrots increased from 35 ::,...1 to 210 mg during a period of 9 months. Chubey and I ' OH Nylund (8) observed a similar increase in phenol content OH of carrot roots during storage at various temperatures. They found that the amount of phenol accumulated was (III) (IV) the highest at 10 C. Our laboratory is currently looking at the effect of storage on phenol content of potatoes. Probable physiological basis for phenol accumulation ncH=CH-COOH in carrots during storage will be described later, but from Ho-V a practical standpoint increased phenol content poses I OCH additional problems in post-harvest handling. Carrots 3 richer in phenols are more susceptible to surface ( V) browning and other concomitant problems (8). The concern over increased phenol content in carrots in relation to surface browning is heightened because of p-coumaric acid (IV), ferulic acid (V) and caffeic acid the existence of a concentration gradient from core to the (I) were identified by thin-layer and gas-liquid­ surface of the carrot root (fable 1). About 85o/oof phenol chromatography after acid and alkaline hydrolysis of the is present in the 1-mm thick surface layer of the root. original extract (19). We found that under the above This would easily explain why carrots with bruises on the storage conditions the levels of hydroxycinnamic acid surface tend to develop brown color (8). Although an derivatives increased with storage time. increase in phenols in carrots augments the potential for Effect of ethylene on phenol content ofcarrots surface browning, no bitterness, as described by The situation is quite different, however, if carrots are Sondheimer (24), was detected. stored even for a short time in a refrigerated storage in Composition ofphenolic compounds in carrots the presence of ethylene.