Key Aroma Compounds in Melons - American Chemical Society

Chapter 22

Key Aroma Compounds in Melons Their Development and Cultivar Dependence 1

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S. Grant Wyllie, David N. Leach, Youming Wang, and Robert L. Shewfelt Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0596.ch022

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Centre for Biostructural and Biomolecular Research, University of Western Sydney, Hawkesbury, Richmond, New South Wales 2753, Australia Department of Food Science and Technology, Georgia Station, University of Georgia, Griffin, GA 30323

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Fruits from most C. mello var. reticularis cultivars typically exhibit a strong and characteristic aroma which is often used by the consumer as an indicator of quality. The odor significance of the aroma components from a range of melon cultivars has been assessed by GC sniffing and aroma dilution techniques. The identification of the key compounds considered to be important contributors to the aroma of high quality melons and descriptors of their aroma character is presented. Many of these aroma compounds are formed during the intense biosynthetic process of ripening from free amino acids in the fruit. The changes in the individual free amino acids, sugars, organic acids and volatiles during the development of the fruit have been determined and possible relationships examined.

Sweetness and aroma are known to be among the most important quality indicators of melon fruit for consumers. To further our understanding of the factors that determine these parameters the changes in the concentrations of sugars, aroma volatiles, free amino acids and organic acids during the development and ripening of Cucumis melo cv Makdimon fruit have been determined. The increase in sugar concentration during ripening is due to the rapid accumulation of sucrose, a process which can only proceed if the fruit remains attached to the plant. Some amino acids known to be precursors to the aroma volatiles show significant increases in concentration during ripening. This increase correlates with that of total volatiles production. In contrast to sugar production, aroma production continues after harvest but sensory evaluation by gas chromatography olfaction methods shows that the aroma profile of fruit harvested only three or four days before full maturity is significantly different from that of fruit harvested at full maturity. Thus for this type of melon the time window available to a producer to harvest fruit having adequate quality and shelf life is quite small. 0097-6156/95/0596-0248$12.00/0 © 1995 American Chemical Society

In Fruit Flavors; Rouseff, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0596.ch022

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There is a large body of evidence that suggests that for many fruits, those ripened on the plant have superior flavour and eating qualities than those harvested before the fully ripened state is reached. However in many cases fruit ripened in this way have a reduced shelf life. Growers are faced with the dilemma of when to pick their crop to obtain maximum eating quality and yet obtain a product which will adequately withstand subsequent handling and distribution environments. Different fruits vary greatly in their ability to meet these two competing criteria. Melons particularly those of the type Cucumis melo var reticularis, commonly known as rockmelon, muskmelon or cantaloupe are a fruit in which this conflict is particularly sharply delineated. The factors that determine the consumer acceptance of melons have been the subject of a number of investigations (1-4) but as yet there seems to be no general agreement on the ones that are reliable indicators of overall fruit quality. Final sugar concentration or sweetness is however certainly one of the major determinants of melon quality. Unlike some fruits, melons have no starch reserve to act as a sugar source during ripening. The large increase in the sugar level that occurs over the ripening period is achieved by the rapid importation of carbohydrates into the fruit from the plantf5j. This importation process can take place effectively only if the plant and the fruit remain attached and the leaf structures are both adequate and biosynthetically active; conditions which may not be met if they are diseased or damaged in some way. Thus to obtain desirable sugar levels melons should be picked when very close to the fully ripe condition. However if picked in this condition the resulting fruit may have a shelf life of only a further four to eight days depending on subsequent handling conditions. On the other hand the factors governing aroma production, one of the other important consumer quality parameters, are largely unknown. Therefore as part of our ongoing investigation of the chemical and biochemical changes in melons and their relationship to fruit quality a detailed study of the development of aroma and aroma precursors during fruit development has been undertaken. A t the same time other data on changes in the content and composition of sugars, organic acids and a range of minerals has been obtained. Some limited radioactive tracer work with melonsf 7) and more extensive work with other fruit such as bananas and strawberries^ which have similar aroma constituents to melons, show that amino acids are the precursors of many of the aroma volatile compounds formed during ripening. TTie structural skeletons of many of the melon volatiles can clearly be derived from amino acids by a series of well documented biochemical transformations (Figure 1). For example valine can be converted to a range of esters containing the 2-methylpropyl structure, isoleucine to those containing the 2-methylbutyl structure, leucine to the 3methylbutyl structure and methionine to the thioether group of esters recently identifiedfP-iJj. Alanine is also of considerable interest since this compound, when subjected to the same series of transformations, can conceivably supply both the ethyl group and the acetate group found in many of the aroma volatiles. Thus a very large proportion of the compounds that constitute the total melon aroma profile contain structural elements which could be derived from valine, isoleucine, methionine and alanine. It could therefore be expected that aroma development during ripening may depend on the types and concentrations of amino acids available for biosynthesis in the fruit at this time. These, in turn, may depend on the variety or cultivar of the particular melon or on the husbandry practises or conditions to which the plants were exposed.


250

FRUIT FLAVORS

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Figure 1. Pathways for the conversion of some amino acids to volatile esters.


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Experimental Procedures. Melons used in this study were from authenticated seed, cv Makdimon, obtained from commercial seed producers. They were grown in a weather protected environment using a hydroponic system with a controlled nutrient mix and therefore all the melons were subjected to the same nutrient and growing regimes. Flowers were tagged on the day of anthesis with inspections being carried out every day during the most intense flowering periods. Three fruit from each age group were selected, stored at 4C and extracted within 24 h. Melon samples were taken from the whole fruit by cutting it into longitudinal sections, the edible portion (middle-mesocarp) removed and cut into small pieces (5x5 cm). For G C olfactory work samples prepared as above were subjected to simultaneous distillation-extraction (SDE) for 1.5 h using pentane as the extracting solvent. The extract was concentrated (1 mL) in a Kuderna-Danish flask attached to a Snyder column using a bath temperature of 45C. For the G C effluent sniffing experiments(GCO) the concentrated extracts were chromatographed using a Pye Unicam G C V . The outlet from a nonpolar column (J&W D B 1 , 30 m χ 0.32 mm i.d., 1.0/im film thickness) was divided 1:1 using an outlet splitter (SGE,Australia) with one arm connected to an F I D detector and the other to a sniffing port (SGE,Australia) flushed with humidified air at 500 mL/min. Chromatographic conditions were: initial ten^perature: 60C; initial time: 2 min; program rate: 4C/min; final temperature: 200C; injector temperature: 220C; detector temperature: 220C; carrier gas: N at 10 psi. The sensory response to the column effluent was recorded as outlined by Miranda-Lopez et al. (14) except that the response was recorded in parallel with the F I D using the second channel of a computing integrator ( D A P A Scientific Pty. Ltd., Perth, Australia). The resultant aromagram contains a series of peaks which record the intensity and time of the response. Descriptors of each response were recorded during each run on a tape recorder that was subsequently synchronised with the aromagram. The amino acids after extraction and ion exchange cleanup following the procedure of MacKenzie and Holme(15) were analysed by gas chromatography in the form of their T B D M S derivatives using cycloleucine as an internal standard(76,). Sugars and organic acids were converted to their trimethyl silylethers and determined simultaneously by gas chromatography using the method of Chapman and Senter(77). Volatiles composition and concentration in the samples of melon flesh were determined using a headspace sampler ( H P 19393) coupled to a gas chromatograph. Approximately 5g of flesh was obtained by using a cork borer to remove a number of cores from around the equator of the fruit. This was immediately sealed in a headspace vial, placed in the headspace sampler at a bath temperature of 100C, equilibrated for 15min before analysis on an H P 5890 G C fitted with a 30m χ 0.32mm i.d. O V 1 fused silica column. 2

Results and discussion. Carbohydrates. The changes in the concentration of the total sugars with time in the developing melon fruit are shown in Figure 2. This plot shows the typical sigmoidal increase exhibited by melon fruit during ripening^.



252

FRUIT FLAVORS

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Citric acid Fructose Glucose Sucrose

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35 Days after anthesis

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Figure 3. Changes in glucose, fructose, sucrose and citric acid during fruit development.



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The onset of ripening is indicated by an increase in total sugars beginning at thirty days after anthesis. This is followed by a rapid rise in carbohydrate concentration over the next five to six days until a maximum is reached on day forty four. Also plotted in this figure are the p H changes that occur in the fruit during development. These two curves are strongly correlated and pose some questions about, for example, the p H dependence of the enzymes involved in the biochemical changes associated with ripening, particularly those involved in the switch from sucrose degradation to sucrose synthesis. The changes in the concentrations of the three major saccharides of melon fruit, glucose, fructose and sucrose during development are shown in Figure 3. The increase in total sugars seen during the ripening process can be seen to be entirely due to the accumulation of sucrose, the concentrations of glucose and fructose remain essentially constant over the life of the fruit. This is in agreement with the results reported by McCollum et sl.(18) for a closely related cultivar. The p H changes observed during ripening are not due to changes in the concentration of citric acid, the only significant organic acid present in melons since this showed little variation over the life of the fruit(Fig. 3). Volatiles. Changes in the total volatiles concentration during fruit development are shown in Figure 4. Very few volatiles were detected in the unripe melon. In the ripening process volatiles production appears to lag sugar production by five to six days but shows a very rapid increase thereafter that maximises simultaneously with sugar production. The changes in the concentrations of the predicted precursor amino acids valine, isoleucine, leucine, methionine and alanine are shown in figures 5 and 6. A l l of these amino acids show considerable increases in concentration that coincide with the ripening process. Valine, leucine and isoleucine all show increases commencing about thirty five days after anthesis, the same day that the volatiles also begin to make an appearance. Methionine which is also the precursor of ethylene, the ripening hormone, shows a steady increase between day thirty and the attainment of maturity. The increase in alanine concentration lags behind that of the other amino acids by six or seven days but then undergoes very rapid growth to become one of the most abundant amino acids in the mature fruit. This may be because its production is controlled via a different biochemical pathway or because alanine through its conversion to acetyl coenzyme and/or ethanol provides so many of the ester substrates that it begins to rise only when ester biogenesis is slowing down. In the aroma profile of cv Makdimon about twenty four percent of the volatiles contain the ethyl group, forty five percent the acetate group(both able to be derived from alanine), eighteen percent the 2-methylbutyl group (from isoleucine) and ten percent the 2-methyl propyl group( from valine). The importance of these amino acids as aroma precursors can therefore be appreciated. Aroma Development. The time course of volatiles production lags that of sugar production by several days. On the other hand the generation of volatiles coincides with the increasing concentrations of certain amino acids some of which are clearly aroma precursors. The increases of these amino acids could occur by increased translocation from the plant or by production in some pathway within the fruit itself or by some combination of both. Our evidence, together with that



254

FRUIT FLAVORS

30

35

45

40

50

Days after anthesis

Figure 4. Changes in Total Sugars and Total Volatiles during fruit development.

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Figure 5. Changes in the concentrations of valine, leucine and isoleucine during fruit development.



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of other workersftf, 19) suggests that the aroma development continues after the fruit is separated from the plant and hence at least the latter of these possibilities is operating. Our preliminary work does however indicate that the aroma development of the separated fruit does not proceed in the same manner as that of a vine ripened fruit. The aroma profiles of melons harvested at half slip and stored at room temperature for three days( i.e. till the predicted time of full slip) were compared with those harvested at full slip and analysed immediately. This comparison was carried out utilising both conventional gas chromatography and gas chromatographic olfactory analysis. The gas chromatographic comparison revealed that the half slip material had only about half of the total volatiles concentration of the full slip material. The traces from the aromagrams are shown in Figure 7. The much greater complexity of the full slip material is immediately obvious. This could be due to two factors. Firstly the greater concentration of the full slip extract means that more of the constituents exceed their odour threshold and hence make a contribution to the aroma profile. Secondly, compounds that appear or increase their concentrations later in the ripening process may now change the aroma profile. The quantitative importance of the volatiles derived from branched chain amino acids in the aroma extract of cv Makdimon has been referred to above. The sensoiy importance of these volatiles to the aroma profile has been determined by G C O and aroma extract dilution analysis(20,). These techniques show that ethyl 2-methylbutanoate(from isoleucine) and methyl 2-methylpropanoate (from valine) are among the most significant contributors to this melon's aroma profile. These two compounds were also identified as the most potent odorants in an unspecified muskmelon using aroma extract dilution analysis(27,). Significantly it is the concentrations of compounds such as ethyl 2-methylbutanoate, ethyl 2methylpropanoate, 2-methylbutylacetate, 2-methyl propylacetate and the thioether esters that vaiy markedly from variety to variety and cultivar to cultivarf10,13,22). This suggests that the conversion of amino acids to esters involves enzyme specific pathways which differ between melon varieties and cultivars and hence control their characteristic sensoiy profiles. Comparison of our amino acid analyses of a highly aromatic melon such as cv Makdimon with those of a melon of low volatiles content such as cv Alice shows that the concentrations of free amino acids found in these widely different melons do not vary significantly. The difference in volatiles concentration therefore is not due to different availabilities of amino acid substrate and must lie elsewhere in the biogenetic pathway. Fruit Quality Considerations. Examination of Figure 4 shows that if fruit of adequate consumption quality i.e. having at least nine to ten percent total sugars and high aroma values and a reasonable shelf life is to be obtained, the harvest time window is quite small, no more than two to three days before the fully ripe stage is reached. For this judgement to be reliably made the producer needs a sensitive and field portable indicator of fruit development. The gas sensing device reported by Miles et a\.(23) and the near infrared device described by Du\\(24) appear to show promise in this area. A well controlled and rapid fruit distribution system from grower to consumer will also be required.


256

FRUIT FLAVORS


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Days after anthesis

Figure 6. Changes in the concentration of alanine and methionine during fruit development.

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Acknowledgments. The American Chemical Society for financial assistance. Faculty of Science and Technology, UWS, Hawkesbury. University of Georgia Dr. Β McGlasson, Faculty of Agriculture and Horticulture, U W S , Hawkesbury for advice and encouragement.


M r B. Corliss, Connoisseur's Choice, Sydney for the husbandry of the melon crop. Literature Cited 1 Aulenbach, B.B. and Worthington, J.T. Hort-Science. 1974, 9, 136-137. 2 Bianco, V.V. and Pratt, H. J. Amer. Soc. Hort. Sci.1977,102, 127-133. 3 Yamaguchi, M.; Hughes, D.L.; Yabumoto, K. and Jennings, W.G. Scientia Horticulturae,1977, 6, 59-70. 4 Mutton, L.L.; Cullis, B.R. and Blakeney, A.B. J. Sci. Food Agric. 1981, 32, 385-391. 5 Hubbard, N.L.; Huber, S.C. and Pharr, D.M. Plant Physiol., 1989, 89, 15271534. 6 Yabumoto, K.; Jennings, W.G.; Yamaguchi, M .J.Food Sci. 1977, 42, 32-37. 7 Schreier,P.; Chromatographic Studies of the Biogenesis of Plant Volatiles; Huthig, Heidelberg, 1984; pp 64-76. 8 Homatidou, V.; Karvouni, S. and Dourtoglou, V. In Flavors and Off-Flavors; Charlambous, G., Ed.; Proceedings of the 6th International Flavor Conference, Crete, Greece. Elsevier,Amsterdam, 1989; pp 1011-1023. 9 Homatidou, V.; Karvouni, S.; Dourtoglou, V. and Poulos, C.N. J. Agric. Food Chem. 1992, 40, 1385-1388. 10 Buttery, R.G.; Seifert, R.M.; Ling, L.C.; Soderstom, E.L.; Ogawa, J.M. and Turnbaugh,J.G. J. Agric. Food Chem. 1982, 30, 1208-1211. 11 Horvat, R.J. and Senter, S.D. J. Food Sci. 1987, 52(4), 1097-1098. 12 Wyllie, S.G. and Leach, D.N. J. Agric. Food Chem., 1990, 38, 2042-2044. 13 Wyllie, S.G. and Leach, D.N. J. Agric. Food Chem., 1992, 40, 253-256. 14 Miranda-Lopez, R.; Libbey, L.M.; Watson, B.T. and McDaniel, M.R. J. Food Sci. 1992,57(4), 985-1019. 15 MacKenzie, S.L. and Holme, K.R. J.Chromatogr. 1984, 299, 387-396. 16 MacKenzie, S.L., Tenaschuk, D. and Fortier, G. J. Chromatogr., 1987, 387, 241- 253. 17 Chapman, G.W. and Horvat, R.J. J. Agric. Food Chem. 1989, 37, 947-950. 18 McCollum, T.G.; Huber, D.J. and Cantliffe, D.J. J. Amer. Soc. Hort. Sci. 1988, 113, 399-403. 19 Chachin, Κ and Iwata, T. Bull. Univ. Osaka Pref., 1988, 40, 27-35. 20 Wyllie, S.G., Leach, D.N. Wang, Y. and Shewfelt, R.L. In Sulfur Compounds in Foods, Mussinan, C.J. ;Keelan, M.E. Eds. ACS Symposium Series 564, American Chemical Society, Washington, D.C. 1994. 21 Schieberle, P.; Ofner, S. and Grosch, W. J. Food Sci.,1990, 55, 193-195. 22 Yabumoto, K., Yamaguchi, M. and Jennings, W.G. Food Chem. 1978, 3, 7-16. 23 Benady, M.; Simon, J.E.; Charles,D.J. and Miles G.E., 1992 International Summer Meeting, American Society of Agricultural Engineers, Paper No. 92-6055, 1992. 24 Dull, G.G., Birth, G.S., Smittle, D.A. and Leffler, R.G. J. Food Sci., 1989, 54, 393-395. RECEIVED January 24, 1995


Key Aroma Compounds in Melons - American Chemical Society

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