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Glycosidically-bound aroma volatile compounds in the skin and pulp of ‘Kensington Pride’ mango fruit at different stages of maturity
Postharvest Biology and Technology 29 (2003) 205 /218 www.elsevier.com/locate/postharvbio
Glycosidically-bound aroma volatile compounds in the skin and pulp of ‘Kensington Pride’ mango fruit at different stages of maturity Herianus J.D. Lalel a, Zora Singh a,*, Soon Chye Tan b a
Horticulture/Viticulture, Muresk Institute of Agriculture, Division of Resources and Environment, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australia b Department of Agriculture, 3 Baron Hay Court, South Perth, Perth, WA 6151, Australia Received 18 July 2002; accepted 6 December 2002
Abstract ‘Kensington Pride’ mangoes (Mangifera indica L.) were harvested at the mature green, half ripe and ripe stages to investigate glycosidically-bound aroma volatiles in their skin and pulp. The aglycones extract was obtained by Amberlite XAD-2 adsorption and methanol elution followed by hydrolysis with b-glucosidase and hemicellulase. Analysis of aglycones was achieved by using gas chromatography coupled with a mass selective detector. The composition and concentrations of glycosidically-bound aroma compounds of the ‘Kensington Pride’ mango were strongly influenced by fruit part and maturity stages. Ninety-two aglycones were detected in the pulp and 85 aglycones were present in the skin. Glycosidically-bound aroma volatile compounds produced via aromatic amino acid metabolites were the most abundant class of compounds found in the skin, whilst in the pulp, terpenes were found to be the most abundant compounds, accounting for 41.18 and 38.04% of the total number of compounds in skin and pulp, respectively. Glycosidically-bound aroma volatile compounds produced via carbohydrate metabolism and terpenes were higher in the skin than in the pulp of mangoes at all maturity stages. Some other compounds, including glycosidically-bound aroma volatile compounds produced via lipid, aromatic amino acid and carotenoid metabolism were higher in the skin than in the pulp only at the mature green and half ripe stage, whilst glycosidically-bound acids were higher in the skin than in the pulp at the half ripe and ripe stage. Most of the glycosidically-bound aroma compounds increased in the pulp as maturity progressed. Amongst all glycosidically-bound aroma compounds only terpenes may contribute to the aroma of fresh ripe ‘Kensington Pride’ mangoes. # 2003 Elsevier B.V. All rights reserved. Keywords: Mangifera indica ; Flavour; Glycosidically-bound volatile compounds; Maturity stages
1. Introduction * Corresponding author. Tel.: /61-8-89266-3138; fax: /618-9266-3063. E-mail address: [email protected] (Z. Singh).
Mango (Mangifera indica L.) is a highly prized fruit due to its attractive flavour, delicious taste and high nutritional value. Aroma, a component
0925-5214/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-5214(02)00250-8
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Table 1 Colour, firmness and TSS of the fruit at harvest Maturity stage
Fruit part Pulp
MG Half ripe Ripe
Firmness (N)
TSS (%)
114.79/10.8 22.69/1.0 17.69/2.9
7.89/0.2 12.49/0.3 15.19/0.3
Skin
Chroma
Hueo
Chroma
Hueo
32.599/2.4 35.629/1.8 29.759/2.5
26.29/2.9 67.99/1.2 60.49/2.1
22.339/1.7 22.819/1.3 26.869/2.1
249.29/2.0 82.29/4.6 66.39/1.2
n/10 fruit; 9/S.D.; MG, mature green.
of flavour, is specific to each cultivar of mango and is an important quality aspect of the ripe mango fruit. More than 270 aroma volatile compounds in different mango varieties have been identified in free form (Sakho et al., 1985; Shibamoto and Tang, 1990). Aroma volatile compounds have also been reported to be present in the mango fruit in the glycosidically-bound form (Adedeji et al., 1992; Sakho et al., 1997; Olle et al., 1998). The sugar moieties of glycosidicallybound aroma volatiles, which have been reported in the mango are a-terpeneyl-b-D-glucopyranosides, a-terpeneyl-6-O -rutinosides and a-terpeneyl-6-O -(a-L-arabinofuranosyl)-b-D-glucopyranosides (Sakho et al., 1997). Aroma compounds (aglycones) can be released from glycosidicallybound compounds by enzymatic or chemical reactions during maturation, storage, industrial pretreatment or processing (Boulanger and Crouzet, 2000). Apart from mangoes, glycosidically-bound aroma volatile compounds have been reported in different fruit including passion fruit (Engel and Tressl, 1983), grape (Gunata et al., 1985; Wirth et al., 2001), papaya (Schwab et al., 1989), apricot, peach and yellow plum (Krammer et al., 1991). The importance of glycosidically-bound aroma volatile compounds and their contribution to fruit aroma has received increasing attention from many researchers in the past decade (Adedeji et al., 1992), and more recently because of the increase in processed fruit products such as juice,
dried fruit, jam, pickles and alcoholic beverages (Crouzet et al., 1997; Mateo and Jimenez, 2000). Thirty-three aroma compounds have been identified in the crude heterosidic extract of the pulp of an unknown African mango cultivar after hydrolysis using b-glucosidase (Adedeji et al., 1992). Moreover, Sakho et al. (1997) identified 27 volatile compounds released from a glycosidically-bound fraction of an ungrafted African mango after incubation with pectinase. Sixty-nine aroma compounds have been found in the mango pulp after enzymatic hydrolysis of their glycosidically-bound fractions, including aliphatic alcohols, acids, aldehydes, ketones, esters, aromatic compounds, terpenes and norisoprenoids (Adedeji et al., 1992; Koulibaly et al., 1992; Sakho et al., 1997; Olle et al., 1998). The majority of research work on glycosidically-bound aroma volatile compounds has been reported from the pulp of fully ripe mangoes. Suarez et al. (1991) reported that glycosidically-bound aroma compounds were distributed in the pulp as well as in the skin of the lulo fruit (Solanum vestissimum D.). Apparently, no research work has been reported on qualitative and quantitative differences in glycosidically-bound aroma compounds in the skin of the mango fruit. Moreover, no research work has been reported either on glycosidicallybound aroma compounds in skin and pulp or their occurrence in different maturity stages of the ‘Kensington Pride’ mango fruit. These observations prompted the current investigation into
H.J.D. Lalel et al. / Postharvest Biology and Technology 29 (2003) 205 /218
glycosidically-bound aroma volatile compounds in the skin as well as in the pulp of the ‘Kensington Pride’ mango fruit at different stages of maturity.
2. Materials and methods
2.1. Plant materials ‘Kensington Pride’ mangoes (M. indica L.) were picked from a commercial orchard located in Chittering (11685?E, 31825?S), Western Australia, during the third week of March 2001 at three maturity stages: mature green (hard with 100% green skin), half-ripe (slightly soft with 50/60% yellow skin), and ripe (eating soft with more than 75% yellow skin). The detailed characteristics of the fruit at these maturity stages at harvest, including colour, firmness and total soluble solids (TSS) are presented in Table 1. Thirty uniform fruit were randomly chosen at each maturity stage for this experiment. Ten fruit were used as a treatment unit and replicated three times. The skin and pulp of the fruit were taken and stored at /18 8C (1 month) prior to analysis for glycosidically-bound aroma compounds.
2.2. Reagents Solvents were purchased from various locations. Distilled n -pentane (99.5% purity with total C5 isomer 99.99%) was purchased from EM Science (Gibbstown, NJ, Canada), dichloromethane (99.5% purity) from Ajax Chemicals (Auburn, NSW, Australia) and methanol (99.9% purity) from Riedel-de Haen (Seelze, Niedersachsen, Germany). n -Paraffin C7 /C22, with a purity of more than 95.5%, and (9/)-2-octanol (98% purity) were obtained from Sigma-Aldrich (Steinheim, Germany). Amberlite XAD-2 (20 /60 mesh), obtained from Supelco (Bellefonte, PA, USA), was treated according to the procedure of Gunata et al. (1985). Almond b-glucosidase and Aspergillus niger hemicellulase from Sigma-Aldrich were also used.
207
2.3. Fruit colour All fruit from each treatment were assessed for their skin colour. Skin colour (L , a* and b* values) was recorded using a Hunter Lab colorFlex 45/0 Spectrophotometer (Hunter Associates Laboratory, Inc., Reston, VA, USA). Chroma was calculated using the formula [(a*2/b *2)1/2], which represents colour saturation from dull (low value) to vivid colour (high value). Hue angle was calculated using the formula tan 1(b*/a *), which is defined as a colour wheel, with red /purple at an angle of 08, yellow at 908, bluish /green at 1808, and blue at 2708 (McGuire, 1992). 2.4. Fruit firmness The firmness of each fruit was measured on two peeled surfaces opposite each other in the equatorial region of the fruit using an electric pressure tester (model EPT-1, Lake City Technical Product Inc., Kelowna, BC, Canada) fitted with a plunger of 11 mm diameter. Fruit firmness was expressed as Newtons. 2.5. Total soluble solids analysis Total soluble solids (TSS) were determined using freshly extracted juice from ten fruit. TSS (%) were determined using an infrared digital refractometer (model PR-101, Atago Co., Ltd., Tokyo, Japan). 2.6. Glycoside extraction Frozen pulp (100 g) and skin (50 g) of the fruit were thawed in 200 or 100 ml of cold (4 8C) 0.1 M phosphate buffer pH 7.0, respectively. Following thorough homogenisation, phase separation was achieved by centrifugation at 5000 rpm for 30 min (4 8C). The supernatant was percolated at 59/ 1 8C at the rate of 90 ml/h through a column (50 /1 cm i.d.) packed with 20 ml of Amberlite XAD-2. The column was then rinsed with 200 ml of Milli-Q water to eliminate water-soluble compounds. Free volatile compounds were removed first by elution with 100 ml of azeotropic pentane/ dichloromethane mixture (2:1, v/v). Glycosidi-
208
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Table 2 Bound aroma volatile compounds produced via lipid metabolism released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg1)
RI Pulp
Skin
MG
HR
R
MG
HR
R
777 854 867 970
n.d. 0.055 n.d. n.d.
0.006 0.059 0.021 tr
0.036 0.088 0.038 0.005
0.020 0.762 0.039 n.d.
0.042 0.235 0.028 n.d.
0.022 0.130 0.018 n.d.
Acids 2-Methylbutanoic acid Hexanoic acid Octanoic acid Dodecanoic acid Tetradecanoic acid Hexadecanoic acid Methyl dihydromalvalate (Z,Z )-9,12-Octadecadienoic acid Heptadecene-(8)-carbonic acid-(1)
846 977 1170 1559 1759 1962 2045 2130 2140
n.d. n.d. n.d. n.d. n.d. 0.060 n.d. n.d. n.d.
tr tr tr n.d. n.d. 0.030 tr n.d. n.d.
tr 0.004 0.014 n.d. n.d. 0.038 0.242 n.d. n.d.
n.d. n.d. n.d. 0.048 n.d. 0.039 n.d. n.d. n.d.
n.d. n.d. 0.052 0.063 0.064 0.330 n.d. 0.018 0.083
n.d. n.d. 0.061 0.028 0.100 0.357 n.d. 0.020 0.072
Esters Isopropyl tetradecanoate Ethyl 9-hexadecanoate Ethyl hexadecanoat Ethyl 9,12,15-octadecatrienoat Ethyl (Z )-9-octadecenoat
1831 1977 1997 2170 2171
n.d. n.d. n.d. n.d. n.d.
tr 0.043 0.025 tr 0.026
0.098 0.937 0.305 0.013 0.554
n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d.
Aldehydes Furfural Tetradecanal Longifolenaldehyde
832 1818 1876
n.d. n.d. n.d.
n.d. n.d. n.d.
0.006 n.d. n.d.
n.d. 0.028 0.032
n.d. 0.074 n.d.
n.d. 0.042 n.d.
Lactones g-Lactone g-Undecalactone
1262 1986
n.d. n.d.
0.007 0.048
0.030 0.119
0.036 n.d.
0.140 n.d.
0.062 n.d.
Aliphatic alcohols 3-Methyl 2-buten-1-ol cis -3-Hexenol 1-Hexanol 1-Heptanol
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
cally-bound volatile components were obtained by subsequent elution with 100 ml of methanol (Boulanger et al., 1999). The methanol fraction containing bound volatile compounds was initially dried over anhydrous sodium sulphate and concentrated to dryness under vacuum at 45 8C to get a crude glycosidically-bound extract. 2.7. Enzymatic hydrolysis Crude glycosidically-bound extract was dissolved in 0.5 ml of 0.1 M phosphate /citrate buffer
(pH 5) and washed with 5/0.5 ml of azeotropic pentane/dichloromethane mixture (2:1, v/v). Following washing, a 0.5 ml mixture containing 5 mg/ ml of almond b-glucosidase and 500 mg/ml of hemicellulase in 0.1 M phosphate /citrate buffer (pH 5) was added to the washed buffer mixture. Enzymatic hydrolysis of glycosides was performed for 16 h at 409/1 8C. After cooling to room temperature, 10 ml 2-octanol (3.08 mg/ml) was added as an internal standard and the released aglycons were extracted with azeotropic pentane/ dichloromethane mixture (5 /0.5 ml). The agly-
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209
Table 3 Bound aroma volatile compounds produced via carbohydrate metabolism released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg1)
RI
Pulp
2,4-Pentanedione 2,5-Dimethyl furan Furaneol 7,7-Dimethylbicyclo[3.3.0]octan-2-one (E )-4-(2?,6?,6?-Trimethyl-1?,2?-epoxy-cyclohexyl)-3-penten-2-one (isomer 1) 9-Decen-2-one (E )-4-(2?,6?,6?-Trimethyl-1?,2?-epoxy-cyclohexyl)-3-penten-2-one (isomer 2) 2-Furanmethanol 7,9-Di-tert -butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione
831 1055 1376 1397 1470 1486 1597 1929
Skin
MG
HR
R
MG
HR
R
n.d. n.d. 0.016 n.d. n.d. n.d. n.d. n.d. 0.131
n.d. n.d. 0.017 n.d. n.d. n.d. n.d. 0.018 0.011
0.011 n.d. 0.081 n.d. n.d. n.d. n.d. 0.117 0.017
0.020 0.007 n.d. 0.305 0.122 0.057 0.063 n.d. n.d.
0.036 n.d. 0.038 0.241 0.061 n.d. 0.785 n.d. n.d.
0.070 n.d. 0.025 0.177 0.035 n.d. 0.292 n.d. n.d.
RI, retention index on HP5-MS column; n.d., not detected; n/3 replications; MG, mature green; HR, half ripe; R, ripe.
con extract was concentrated to a final volume of 0.5 ml in a nitrogen stream at room temperature (209/1 8C). Each maturity stage or part of the fruit (skin and pulp) was analysed in triplicate. A blank experiment was also conducted to check for the possibility of contamination. 2.8. Aglycones analysis A Hewlett-Packard 6890 gas chromatograph (GC) coupled to a Hewlett-Packard 5973 Mass selective (MS) detector with an electron impact mode (EI) generated at 70 eV was used. The fused capillary column HP-5MS (60 m/0.25 mm; film thickness 0.25 mm) was used for separation. Aglycones extract (1 ml) was injected with a split/ splitless mode. The injection temperature was maintained at 260 8C and the column temperature was held at 40 8C for 1 min and increased at the rate of 3 8C/min to 310 8C and then maintained for 20 min. Helium was the carrier gas at the rate of 1.0 ml/min. Identification of the compounds was based on comparison of their mass spectra with those of authentic standards in the Wiley275.L library and their linear retention indices (RI). RI were calculated using n -paraffin standards (Van den Dool and Kratz, 1963). The aglycones were quantified using internal standardisation with consideration that the detector response factor to 2-octanol for all compounds
was equal to one. 2-Octanol was used as the internal standard because of an intermediate behaviour of 2-octanol on monoterpenes and sesquiterpenes (Olle et al., 1998). The compounds, which were present in notable amounts (/0.001 mg/kg) both in the skin and pulp of the fruit at all maturity stages, were statistically analysed. 2.9. Statistical analysis The data were subjected to analysis of variance (ANOVA) using GENSTAT 5 release 4.1 (Lawes Agricultural Trust, Rothamsted Experimental Station, UK). The effects of maturity stage and part of fruit including skin and pulp on various parameters were assessed. Least significant differences (Fisher’s protected LSD) were calculated at P /0.05 following a significant F -test. All the assumptions of analysis were checked to ensure validity.
3. Results 3.1. Glycosidically-bound volatile compounds One hundred and forty four compounds were identified from a heterosidic fraction obtained from the skin and pulp of the ‘Kensington Pride’ mango after enzymatic hydrolysis with b-glucosi-
210
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Table 4 Terpenes released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg 1)
RI
Pulp
Isoprene Northujane Methylene cyclohexane p -Cymene trans -b-Ocimene cis -Linalool furanoxide p -Cymenyl trans -Linalool furanoxide Linalool (2-Methylprop-1-enyl)-cyclohexa-1,3-diene Mentha-1,4,8-triene p -Mentha-1,5-dien-8-ol Epoxy linalool 1,8-Menthadien-4-ol p -Cymen-8-ol a-Terpineol a-Phellandrene epoxide b-Cyclogeraniol trans -(/)-Carveol exo -2-Hydroxycineole Nerol Neral 1-Methyladamantane Geraniol m -Cymene 3-Carene-10-ol Geranial 1(7),3,8-ortho -Menthatriene Tricyclo(3.2.1.0)-octane p -Cymen-a-ol Menth-1-ene-4,8-diol p -Mentha-1,4-dien-7-ol p -Mentha-1,5,8-triene cis -1,2,3,6,7,7a -Hexahydo-7a -methyl-3aH -inden-3a -ol Geranic acid Isocyclocitral 7,7-Dimethylbicyclo[3.3.0]-octane-2-one trans -Sobrerol Z -Citral 2-(2-Hydroxyethyl)-3-methoxy-tricyclo[2.2.21.0*2,6]heptane 4,5-Epoxy-1-isopropyl-4-methyl-1-cyclohexene trans -Chrysanthemal 2-Methyl-6-methylene-1,7-octadien-3-one 2-endo -Hydroxy-2-exo -methylprotoadamantane 8-Hydroxy geraniol 2-Pinen-10-hydroxyperoxide Ledenoxide (I) Capnellane-8-one Caryophylla-3,8(13)-dien-5-b-ol
859 961 1026 1049 1075 1091 1091 1101 1140 1144 1165 1177 1182 1189 1196 1211 1213 1224 1228 1231 1245 1250 1257 1259 1270 1274 1286 1288 1295 1317 1333 1341 1343 1355 1365 1378 1384 1392 1398 1405 1451 1465 1467 1521 1629 1890 1942 1946
Skin
MG
HR
R
MG
HR
R
n.d. n.d. n.d. n.d. n.d. 0.025 n.d. 0.034 0.006 n.d. n.d. n.d. n.d. n.d. 0.014 0.013 n.d. n.d. n.d. n.d. tr tr n.d. n.d. tr 0.005 tr n.d. n.d. 0.029 0.021 n.d. n.d. 0.069 0.020 n.d. 0.037 0.019 n.d. n.d. 0.018 n.d. tr tr 0.065 n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d. 0.027 n.d. 0.040 0.053 n.d. 0.004 0.01 0.008 0.010 0.045 0.036 0.004 0.005 0.008 0.008 0.009 0.052 0.006 0.018 0.042 0.014 0.036 n.d. 0.015 0.095 0.045 0.006 n.d. 0.243 0.019 0.159 0.123 0.044 n.d. 0.021 0.038 0.018 0.181 tr 0.089 n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d. 0.032 n.d. 0.051 0.022 n.d. 0.007 0.015 0.009 0.011 0.429 0.034 0.008 0.009 0.027 0.017 0.026 0.109 0.015 0.325 0.284 0.025 0.135 n.d. 0.015 0.100 0.033 0.008 n.d. 0.381 0.048 0.196 0.139 0.043 n.d. 0.037 0.030 0.023 0.177 tr 0.183 n.d. n.d. n.d. n.d.
0.022 0.022 0.064 0.020 0.009 n.d. 0.099 n.d. 0.008 0.008 0.036 n.d. n.d. 0.043 0.133 0.234 n.d. n.d. 0.023 n.d. n.d. 0.020 0.060 0.113 n.d. 0.136 0.008 0.111 n.d. 0.618 n.d. n.d. 2.195 n.d. n.d. n.d. n.d. n.d. 0.116 n.d. n.d. 0.733 n.d. n.d. n.d. 0.358 0.051 0.048 0.045
0.039 0.011 0.034 0.015 0.011 n.d. 0.067 n.d. 0.024 0.010 0.029 n.d. n.d. 0.029 0.165 0.082 n.d. n.d. 0.018 n.d. 0.007 0.040 0.052 3.528 n.d. 0.059 0.073 0.062 n.d. 0.527 n.d. n.d. 1.751 n.d. n.d. n.d. n.d. n.d. 0.124 n.d. n.d. 0.754 n.d. n.d. n.d. 0.251 0.062 0.093 0.031
0.031 0.011 0.015 0.012 0.012 n.d. 0.047 n.d. 0.013 0.006 0.019 n.d. n.d. 0.022 0.334 0.041 n.d. n.d. 0.013 n.d. n.d. 0.013 0.034 1.279 n.d. 0.032 0.024 0.047 n.d. 0.246 n.d. n.d. 1.083 n.d. n.d. n.d. n.d. n.d. 0.132 n.d. n.d. 0.311 n.d. n.d. n.d. 0.082 0.019 n.d. 0.015
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
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211
Table 5 Bound aroma volatile compounds produced via aromatic amino acid metabolism released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg1)
RI
Pulp
Benzaldehyde Phenol Benzenemethanol Phenylacetaldehyde Benzeneethanol 4-Ketoisophorone Benzoic acid 2-Hydroxy benzoic acid methyl ester Myrtenol 2-(4?-Methylphenyl)-propanal 4-(1-Methylethyl)-benzaldehyde 4-(2-Propenyl)-phenol 2-Xylyl ethanol p-sec -Butylphenol Phenolic compound 4,4a ,5,6,7,8-Hexahydro 2(3H )-naphthalenone Perilla alcohol 2-Methyl-5-(1-methylethyl)-phenol Thymol methyl ether Eugenol 2-Allyl-4-methylphenol trans -Sobrerol 5-Methyl-2-(1-methylethyl)-phenol a,a-Dimethoxy-m -xylene 2-Hydroxy-benzeethanol 1-(2-Hydroxyphenyl)-ethanone 4-Hydroxy benzoic acid methyl ester Acetovanillone 4-(1,1-Dimethylethyl)-1,2-benzenediol trans -Crysanthenyl acetate 2,4,5-Trimethyl benzenemethanol 2,4,5-Trimethyl-benzenemethanol 2,6-bis(1,1-Dimethylethyl)-4-methyl phenol Ethyl vanillylether 4-(4-Hydroxyphenyl)-2-butanone 2-Allyl-p -cresol 4-Hydroxy-benzoic acid propyl ester cis -Cinerolone Zingerone 1,4-Dimethylindanyl acetate 1-(2,3,4,5-Tetramethylphenyl)-1-butanone Trimethyl-(1-methylethyl)-benzene Coniferyl alcohol Phenolic compound Vomifoliol 1,2,4-Triethyl benzene 2,5-Dimethoxy-4-propoxy-benzaldehyde 1,2-Benzenedicarboxylic acid butyl phenylmethyl ester
963 981 1035 1046 1116 1147 1159 1198 1202 1210 1246 1254 1277 1279 1284 1299 1302 1304 1328 1363 1376 1382 1407 1409 1410 1442 1459 1491 1493 1504 1513 1516 1518 1553 1554 1599 1626 1641 1653 1658 1660 1678 1745 1786 1796 1813 1895
Skin
MG
HR
R
MG
HR
R
tr n.d. 0.059 0.005 0.068 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.039 n.d. n.d. n.d. n.d. 0.025 0.051 0.047 n.d. n.d. n.d. 0.020 n.d. n.d. 0.589 n.d. 0.015 n.d. n.d. 0.018 n.d. n.d. n.d. n.d. 0.179 n.d. n.d. n.d. 0.015 0.018 0.023 n.d. 0.186 n.d. n.d. n.d.
0.006 0.015 0.117 0.004 0.126 0.005 n.d. n.d. 0.005 n.d. 0.016 n.d. 0.103 n.d. n.d. n.d. n.d. 0.019 0.009 0.109 0.017 n.d. n.d. 0.042 0.034 n.d. 1.094 n.d. 0.023 n.d. n.d. 0.030 n.d. 0.013 n.d. n.d. 0.304 n.d. n.d. n.d. 0.037 0.076 0.011 n.d. 0.343 n.d. n.d. n.d.
0.007 tr 0.158 0.006 0.216 tr n.d. n.d. 0.007 n.d. 0.011 n.d. 0.113 n.d. n.d. n.d. n.d. 0.060 0.020 0.134 0.062 n.d. n.d. 0.070 0.084 n.d. 0.983 n.d. 0.029 n.d. n.d. 0.040 n.d. 0.033 n.d. n.d. 0.337 n.d. n.d. n.d. tr 0.100 0.057 n.d. 0.572 n.d. n.d. n.d.
n.d. n.d. 1.695 0.010 0.296 0.006 n.d. 0.036 0.036 0.047 0.041 0.039 0.865 0.233 0.064 0.051 0.120 n.d. n.d. n.d. n.d. 0.144 0.221 n.d. n.d. 0.056 n.d. 0.096 n.d. 0.080 0.146 n.d. 0.263 n.d. 0.086 0.125 n.d. 0.099 0.639 0.562 n.d. 0.401 0.028 0.046 0.459 0.031 0.080 n.d.
n.d. n.d. 1.494 0.011 0.204 0.006 0.012 0.042 0.027 0.029 0.026 n.d. 0.714 0.084 0.058 0.063 0.099 n.d. n.d. n.d. n.d. 0.073 0.171 n.d. n.d. 0.157 n.d. 0.056 n.d. 0.078 0.116 n.d. 0.411 n.d. 0.080 0.100 n.d. 0.106 0.607 0.270 n.d. 0.683 0.042 0.052 0.628 0.052 0.059 0.058
n.d. n.d. 0.427 0.012 0.093 0.004 n.d. 0.013 0.012 0.022 0.016 n.d. 0.307 0.048 0.047 0.030 0.063 n.d. n.d. n.d. n.d. 0.025 0.076 n.d. n.d. 0.013 n.d. 0.028 n.d. 0.029 0.056 n.d. 0.101 n.d. 0.030 0.032 n.d. 0.055 0.244 0.084 n.d. 0.232 0.024 0.019 0.185 n.d. 0.013 0.057
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Table 5 (Continued ) Compound
Concentration (mg kg1)
RI
Pulp
3-Benzyl-1,4-diaza-2,5-dioxo-bicyclo[4.3.0]nonane 1,2-benzenedicarboxylic acid diisooctyl ester n -Nornuciferine
Skin
MG
HR
R
MG
HR
R
0.024 n.d. n.d.
0.015 0.013 tr
0.019 0.131 0.127
0.011 0.324 n.d.
0.022 0.208 n.d.
0.011 0.301 n.d.
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
dase and hemicellulase (Tables 2 /7). Among these compounds, 92 compounds were detected in the pulp and 85 compounds were present in the skin. Aromatic amino acid metabolites were the most abundant class of compounds found in the skin, whilst in the pulp terpenes were found to be the most abundant compounds, which accounted for 41.18 and 38.04% of the total number of compounds in skin and pulp, respectively. Total glycosidically-bound volatiles in the pulp significantly (P 5/0.05) increased during fruit ripening, whilst in the skin the compounds increased and peaked at the half ripe stage (Fig. 1). 3.1.1. Glycosidically-bound volatile compounds produced via lipid metabolism Glycosidically-bound volatile compounds produced via lipid metabolism identified in the pulp
and skin accounted for 23 compounds including aliphatic alcohols, acids, esters, aldehydes and lactones (Table 2). Total glycosidically-bound volatile compounds produced via lipid metabolism were significantly (P 5/0.05) higher in the skin than in the pulp of mature green and half ripe fruit as compared with the ripe fruit (Fig. 2). Total glycosidically-bound volatile compounds produced via lipid metabolism in the pulp increased as ripening progressed, whilst in the skin they remained unchanged. At the ripe stage, total glycosidically-bound volatile compounds produced via lipid metabolism in the pulp were significantly (P 5/0.05) higher than those of the skin. Among glycosidically-bound volatile compounds produced via lipid metabolism, only cis 3-hexenol and hexadecanoic acid were present in
Table 6 Bound aroma volatile compounds produced via carotenoid metabolism released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg 1)
RI
Pulp
2-endo-Hydroxy-2-exo-methylprotoadamantane (9R)-9-Hydroxy-4-7E -megastigmadien-3-one 3-Oxo-7,8-dihydro-a-ionol 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol (isomer 1) 3-Oxo-7,8-dihydro-b-ionol 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol (isomer 2) (9/)-15-Hexadecanolide
1464 1655 1713 1721 1731 1782 1943
Skin
MG
HR
R
MG
HR
R
n.d. 0.051 0.082 0.043 n.d. 0.055 0.011
n.d. 0.194 0.326 0.123 tr 0.167 0.014
n.d. 0.217 0.454 0.155 0.027 0.233 0.023
0.490 n.d. 0.541 0.067 n.d. 0.123 n.d.
0.596 n.d. 1.022 0.143 n.d. 0.222 n.d.
0.279 n.d. 0.359 0.051 n.d. 0.062 n.d.
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
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213
Table 7 Miscellaneous compounds released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg1)
RI Pulp
5-Ethyl-4,6-dimethyl-2-methoxy pyridine Cyclohexadecane Epoxy cyclohexane 5,7-Dihydroxy-dihydroflavone Hexadecanemide 9-Octadecenamide
1498 1881 1883 2182
Skin
MG
HR
R
MG
HR
R
n.d. n.d. n.d. n.d. n.d. n.d.
0.017 n.d. n.d. n.d. 0.014 0.067
tr n.d. n.d. n.d. tr 0.069
n.d. 0.078 0.110 0.060 n.d. n.d.
n.d. 0.560 n.d. 0.116 n.d. n.d.
n.d. 0.896 n.d. 0.033 n.d. n.d.
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
Fig. 1. Effects of maturity stage and fruit part on total glycosidically-bound compounds. Different lower cases on the top of similar shaded bars (fruit parts) or different upper cases on the top of bars of each maturity stage show significant differences (LSD, P 5/0.05). Mt, mature; n/3 replications.
the skin and pulp in notable amounts and were statistically analysed (Table 8). Skin had significantly (P 5/0.05) higher concentrations of cis -3hexenol than the pulp at all maturity stages of the fruit. The concentrations of cis -hexenol in the skin declined as the maturity progressed to the ripe stage, whilst in the pulp it increased. On the other hand, the concentration of hexadecanoic acid in mature green fruit was significantly (P 5/0.05) higher in the pulp than in the skin. However, in half ripe and ripe fruit, its concentration was higher in the skin than in the pulp.
3.1.2. Glycosidically-bound volatile compounds produced via carbohydrate metabolism The concentration of total glycosidically-bound volatile compounds produced via carbohydrate metabolism released by enzymatic hydrolysis was significantly (P 5/0.05) higher in the skin than in the pulp, regardless of maturity stage (Fig. 2). The concentration of total glycosidically-bound volatile compounds produced via carbohydrate metabolism in the skin was significantly (P 5/0.05) higher at the half ripe stage as compared with the mature green and ripe stage. In the pulp, they increased as ripening progressed. Six ketones were found in the skin, whilst four compounds were found in the pulp (Table 3). 3.1.3. Terpenes The trend of changes in total terpenes released after incubation with b-glucosidase and hemicellulase as maturity progressed was similar to the trend of total ketones (Fig. 2). The concentration of terpenes in the skin was significantly (P 5/0.05) higher at the half ripe stage than in all other maturity stages. In the pulp, the concentration of total terpenes increased significantly (P 5/0.05) as ripening progressed to the ripe stage. In number, terpenes found in the pulp were more than in the skin (Table 4), whilst in quantity terpenes found in the skin were significantly higher than in the pulp irrespective of the fruit maturity stage (Fig. 2). Among total numbers of identified terpenes released by enzymatic hydrolysis, oxygenated ter-
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Fig. 2. Effects of maturity stage and fruit part on glycosidically-bound aroma volatile compounds produced via lipid metabolism (A), carbohydrate metabolism (B), terpenes (C), aromatic amino acid metabolism (D), carotenoid metabolism (E), and unknown pathways (F). Different lower cases on the top of similar shaded bars (fruit parts) or different upper cases on the top of bars of each maturity stage show significant differences (LSD, P 5/0.05). Mt, mature; n/3 replications.
penes accounted for 75% (Table 4). The most abundant terpene found in the skin was geraniol followed by p -mentha-1,5,8-triene and trans -chrysanthemal. In the pulp, the most abundant terpene was p-cymen-8-ol followed by cis -1,2,3,6,7,7a hexahydro-7a-methyl-3a H-inden-3a -ol and geraniol. In mature green and half ripe mangoes, pcymen-8-ol, a-terpineol and 3-carene-10-ol were found to be significantly (P 5/0.05) higher in the skin than the pulp, whilst in ripe fruit the trend was reversed (Table 8). Linalool was significantly (P 5/0.05) higher in the skin of mature green mangoes than in the pulp, but the trend was reversed in half ripe and ripe fruit. 3.1.4. Glycosidically-bound volatile compounds produced via aromatic amino acid metabolism Total glycosidically-bound volatile compounds produced via aromatic amino acids metabolism
released by enzymatic hydrolysis in the skin reduced as the maturity progressed to the ripe stage, whilst in the pulp its concentration exhibited the reverse trend (Fig. 2). In mature green and half ripe mangoes, glycosidically-bound volatile compounds produced via aromatic amino acid metabolism were significantly (P 5/0.05) higher in the skin than that in the pulp. At the ripe stage, glycosidically-bound volatile compounds produced via aromatic amino acid metabolism were significantly higher in the pulp than in the skin. Benzenemethanol was the most abundant glycosidically-bound volatile compound produced via aromatic amino acid metabolism in the skin, while 4-hydroxy benzoic acid methyl ester was the most abundant in the pulp (Table 5). The concentrations of benzenemethanol, 2-xylyl ethanol, phenylacetaldehyde and trimethyl-(1-methylethyl)benzene were significantly (P 5/0.05) higher in
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215
Table 8 Effects of maturity stage and fruit part on bound aroma volatile compounds Compound
Concentration (mg kg1)
Fruit part MG
cis -3-Hexenol Hexadecanoic acid Benzyl alcohol (benzenemethanol) Phenylacetaldehyde Benzeneethanol 2-Xylyl ethanol p -Cymen-a-ol Trimethyl-(1-methylethyl)-Benzene Coniferyl alcohol 3-Benzyl-1,4-diaza-2,5Dioxobicyclo[4.3.0]nonane Vomifoliol Linalool p -Cymen-8-ol a-Terpineol 3-Carene-10-ol 3-Oxo-7,8-dihydro-a-ionol 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol (isomer 1) 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol (isomer 2)
Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin
0.055 0.762 0.060 0.039 0.059 1.695 0.005 0.010 0.068 0.296 0.039 0.865 0.029 0.618 0.018 0.401 0.023 0.028 0.024 0.011 0.186 0.459 0.006 0.008 0.014 0.133 0.013 0.234 0.005 0.136 0.082 0.541 0.043 0.067 0.055 0.123
HR aA cB bB aA aA cB bA aB aA cB aA cB aA cB aA bB bA bB cB aA aA bB aA aB aA aB aA cB aA cB aA bB aA bB aA bB
0.059 0.235 0.030 0.330 0.117 1.494 0.004 0.011 0.126 0.204 0.103 0.714 0.095 0.527 0.076 0.683 0.011 0.042 0.015 0.022 0.343 0.628 0.053 0.024 0.045 0.165 0.036 0.082 0.014 0.059 0.326 1.022 0.123 0.143 0.167 0.222
LSD (P/0.05)
R abA bB aA bB abA bB aA bB bA bB bA bB bA bB bA cB aA cB aA bB bA cB cB cA bA bB bA bB bA bB bA cB bA cB bA cB
0.088 0.130 0.038 0.357 0.158 0.427 0.006 0.012 0.216 0.093 0.113 0.307 0.100 0.246 0.100 0.232 0.057 0.024 0.019 0.011 0.572 0.185 0.022 0.013 0.429 0.334 0.034 0.041 0.025 0.032 0.454 0.359 0.155 0.051 0.233 0.062
bA aB aA cB bA aB cA cB cB aA bA aB bA aB bA aB cB aA bB aA cB aA bB bA cB cA bA aA cB aA cB aA cB aA cB aA
0.030 0.018 0.084 0.001 0.016 0.043 0.031 0.030 0.003 0.002 0.038 0.002 0.021 0.009 0.003 0.049 0.010 0.014
Means within the same row (fruit part) followed by the same lower cases are not significantly different (P /0.05); means within the same column (maturity stage) followed by the same upper cases are not significantly different (P /0.05), n/3 replications; MG, mature green; HR, half ripe; R, ripe.
the skin than in the pulp at all maturity stages (Table 8). As maturity progressed to the ripe stage, the concentrations of benzenemethanol and 2xylyl ethanol declined. In mature green and half ripe fruit, benzeneethanol and coniferyl alcohol were significantly higher in the skin than in the pulp. However in the ripe fruit, the concentrations were higher in the pulp than in the skin.
3.2. Glycosidically-bound volatile compounds produced via carotenoid metabolism The concentration of glycosidically-bound carotenoids metabolites (norisoprenoids) in the pulp increased as the maturity progressed to the ripe stage (Fig. 2). The concentration of norisoprenoids was significantly (P 5/0.05) higher in the skin of
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half ripe fruit than in mature green and ripe fruit. 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol isomer 1 (RI /1721), isomer 2 (RI/1782) and 3-oxo-7,8dihydro-a-ionol were significantly (P 5/0.05) higher in the skin of green mature and half ripe mangoes than in the pulp, while in ripe mangoes the trend was reversed (Table 8). 3.2.1. Glycosidically-bound aroma volatile compounds produced via unknown pathways Two cyclic alkanes and three nitrogen-containing compounds were detected only in the pulp, whilst one flavone was found only in the skin (Table 7). Cyclohexadecane increased during fruit ripening. All the glycosidically-bound amides were only detected in the pulp of the half ripe and ripe mangoes. 5,7-Dihydroxy-dihydroflavone peaked at half ripe stage.
4. Discussion Hydrolysis of glycosidically-bound aroma volatile compounds in the skin and pulp of the ‘Kensington Pride’ mango, using b-glucosidase and hemicellulase, showed qualitative and quantitative differences in the skin and pulp at various stages of maturity. Most of the compounds were higher in the skin than in the pulp, especially in mature green and half ripe mangoes, as compared with the ripe ones (Figs. 1 and 2). This may due to the higher concentrations of these compounds as free compounds (unbound volatile) in the skin than in the pulp. We found that free volatile aldehydes, fatty acids and terpenes were higher in the skin than in the pulp (data not shown). The high concentration of lipophilic compounds can destroy the cell membranes, and therefore, glycosidation usually takes place as one of the protective mechanisms in plant tissue (Stahl-Biskup et al., 1993). The content of glycosidically-bound volatile compounds produced via lipid metabolism, carbohydrate metabolism, aromatic amino acid metabolism, terpene and carotenoid metabolism generally declined in the skin at the ripe stage, but the trend was reversed in the pulp (Fig. 2). Probably during ripening some glycosidically-
bound aroma compounds were transported from the skin to the pulp of the fruit, since during ripening the integrity of cell walls declines as fruit softens (Gomez-Lim, 1997). It may be argued that some of the aroma compounds were being synthesised (glycosidised) in the pulp during ripening as some of the glycosidically-bound aroma compounds were not present in the skin. Amongst glycosidically-bound volatile compounds produced via lipid metabolism released by enzymatic hydrolysis, cis -3-hexenol, 1-hexanol, 1-heptanol and g-octalactone have been found in the free form (unpublished data). The presence of glycosidically-bound cis -3-hexenol, hexanoic acid, tetradecanoic acid and hexadecanoic acid, on the other hand, have been reported in other mango cultivars (Adedeji et al., 1992; Sakho et al., 1997; Olle et al., 1998). Other glycosidically-bound aroma volatile compounds produced via lipid metabolism including 1-hexanol and 1-heptanol have also been reported to be present in papayas (Schwab et al., 1989) and grapes (Wirth et al., 2001), whilst 2-methylbutanoic acid, octanoic acid and dodecanoic acid have been reported in apricots, peaches and yellow plums (Krammer et al., 1991). Except for cis -3-hexenol, lactones and aldehydes, the contribution of other glycosidically-bound volatile compounds produced via lipid metabolism to the overall aroma is probably low because they generally have a high odour threshold (Buttery et al., 1988; Baek and Cadwallader, 1999). Terpenes, including linalool, p -mentha-1,5,8triene, p-cymen-8-ol, 4-terpineol and a-terpineol, were found in bound and free forms (Lalel et al., 2003). Most of these compounds have low odour thresholds (Masanetz and Grosch, 1998) and, therefore, they may contribute to the overall aroma of the fruit. The presence of glycosidically-bound 3-carene-10-ol may be related to 3carene, the second most abundant free terpene (Lalel et al., 2003). Linalool and its epoxy or furanoxide forms have been reported to be found in other mangoes in glycosidically-bound forms (Adedeji et al., 1992; Sakho et al., 1997). Similar to most glycosidic compounds, glycosidically-bound volatile compounds produced via aromatic amino acid and carotenoid metabolism
H.J.D. Lalel et al. / Postharvest Biology and Technology 29 (2003) 205 /218
were higher in the skin than in the pulp of the fruit (Fig. 2). Glycosidation of these compounds may also be higher in the skin than in the pulp. During ripening they may also be transported from the skin to the pulp. Although benzyl alcohol (benzenemethanol) was the most abundant glycosidically-bound volatile compound produced via aromatic amino acid metabolites in the skin, it may not contribute to the overall aroma of the fruit because of its high odour threshold (10 000 ppb) (Buttery et al., 1988). Benzyl alcohol (benzenemethanol), having a balsamic earthy smell (Parada et al., 2000) has also been found in other mango cultivars bound with b-D-glucopyranoside or 6-O -(a-L-rhamnophyranosyl)/b-D-glucopyranoside (Adedeji et al., 1992; Sakho et al., 1997). Phenylacetaldehyde, which is only present at the level of 4 /6 ppb in pulp or 10/12 ppb in skin, may contribute to the overall aroma of the fruit if it is released during ripening or processing, as its odour threshold is 4 ppb (Buttery et al., 1988). While the presence of this compound in a glycosidicallybound form has not been reported in mangoes, its presence has been reported in papaya (Schwab et al., 1989) and apricot (Krammer et al., 1991). As a free aroma compound, it has been found in the ‘Alphonso’ mango, giving a pungent green, floral and sweet smell (MacLeod and de Troconis, 1982). The content of most of glycosidically-bound volatile compounds produced via carotenoid metabolism (norisoprenoids) present in the ‘Kensington Pride’ mango (Table 6) were more than their reported threshold (B/1 ppb) (Buttery et al., 1988). Amongst the glycosidically-bound norisoprenoids found, 3-oxo-7,8-dihydro-a-ionol, 9-hydroxy-4,7megastigmadien-3-one and vomifoliol have also been found in other mango cultivars (Adedeji et al., 1992; Sakho et al., 1997; Olle et al., 1998). Although none of these norisoprenoids were found in the free form, these compounds may relate to bionone, which was found in the ‘Kensington Pride’ mango in a free form (Lalel et al., 2003). These norisoprenoids may be important for the aroma of processed mango products, as some of them are found in canned mango puree (Hunter et al., 1974). In conclusion, the composition and concentration of glycosidically-bound aroma compounds of
217
the ‘Kensington Pride’ mango were strongly influenced by fruit part and maturity stage. Glycosidically-bound volatile compounds produced via aromatic amino acid metabolism and terpenes were higher in the skin than in the pulp of mangoes at all maturity stages. Some other glycosidically-bound aroma volatile compounds including glycosidically-bound volatile compounds produced via lipid, aromatic amino acid and carotenoid metabolism were higher in the skin than in the pulp only in mature green and half ripe fruit, whilst glycosidically-bound acids were higher in the skin than in the pulp in half ripe and ripe fruit. Most of glycosidically-bound aroma compounds increased in the pulp as maturity progressed. Amongst all glycosidically-bound aroma compounds, probably only terpenes may contribute to the aroma of fresh ripe ‘Kensington Pride’ mangoes.
Acknowledgements The authors thank Mr S. Bambarderi and Dr Peter Sheppard for technical assistance during analysis and Professor B. Kagi, School of Applied Chemistry for providing GC and GC /MS facilities. We are thankful to Robyn Blake for critically reading this manuscript. H.J.D. Lalel also thankfully acknowledges the financial support from University of Nusa Cendana, Kupang, Indonesia under auspicious of DUE-project funded by World Bank.
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Glycosidically-bound aroma volatile compounds in the skin and pulp of ‘Kensington Pride’ mango fruit at different stages of maturity Herianus J.D. Lalel a, Zora Singh a,*, Soon Chye Tan b a
Horticulture/Viticulture, Muresk Institute of Agriculture, Division of Resources and Environment, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australia b Department of Agriculture, 3 Baron Hay Court, South Perth, Perth, WA 6151, Australia Received 18 July 2002; accepted 6 December 2002
Abstract ‘Kensington Pride’ mangoes (Mangifera indica L.) were harvested at the mature green, half ripe and ripe stages to investigate glycosidically-bound aroma volatiles in their skin and pulp. The aglycones extract was obtained by Amberlite XAD-2 adsorption and methanol elution followed by hydrolysis with b-glucosidase and hemicellulase. Analysis of aglycones was achieved by using gas chromatography coupled with a mass selective detector. The composition and concentrations of glycosidically-bound aroma compounds of the ‘Kensington Pride’ mango were strongly influenced by fruit part and maturity stages. Ninety-two aglycones were detected in the pulp and 85 aglycones were present in the skin. Glycosidically-bound aroma volatile compounds produced via aromatic amino acid metabolites were the most abundant class of compounds found in the skin, whilst in the pulp, terpenes were found to be the most abundant compounds, accounting for 41.18 and 38.04% of the total number of compounds in skin and pulp, respectively. Glycosidically-bound aroma volatile compounds produced via carbohydrate metabolism and terpenes were higher in the skin than in the pulp of mangoes at all maturity stages. Some other compounds, including glycosidically-bound aroma volatile compounds produced via lipid, aromatic amino acid and carotenoid metabolism were higher in the skin than in the pulp only at the mature green and half ripe stage, whilst glycosidically-bound acids were higher in the skin than in the pulp at the half ripe and ripe stage. Most of the glycosidically-bound aroma compounds increased in the pulp as maturity progressed. Amongst all glycosidically-bound aroma compounds only terpenes may contribute to the aroma of fresh ripe ‘Kensington Pride’ mangoes. # 2003 Elsevier B.V. All rights reserved. Keywords: Mangifera indica ; Flavour; Glycosidically-bound volatile compounds; Maturity stages
1. Introduction * Corresponding author. Tel.: /61-8-89266-3138; fax: /618-9266-3063. E-mail address: [email protected] (Z. Singh).
Mango (Mangifera indica L.) is a highly prized fruit due to its attractive flavour, delicious taste and high nutritional value. Aroma, a component
0925-5214/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-5214(02)00250-8
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Table 1 Colour, firmness and TSS of the fruit at harvest Maturity stage
Fruit part Pulp
MG Half ripe Ripe
Firmness (N)
TSS (%)
114.79/10.8 22.69/1.0 17.69/2.9
7.89/0.2 12.49/0.3 15.19/0.3
Skin
Chroma
Hueo
Chroma
Hueo
32.599/2.4 35.629/1.8 29.759/2.5
26.29/2.9 67.99/1.2 60.49/2.1
22.339/1.7 22.819/1.3 26.869/2.1
249.29/2.0 82.29/4.6 66.39/1.2
n/10 fruit; 9/S.D.; MG, mature green.
of flavour, is specific to each cultivar of mango and is an important quality aspect of the ripe mango fruit. More than 270 aroma volatile compounds in different mango varieties have been identified in free form (Sakho et al., 1985; Shibamoto and Tang, 1990). Aroma volatile compounds have also been reported to be present in the mango fruit in the glycosidically-bound form (Adedeji et al., 1992; Sakho et al., 1997; Olle et al., 1998). The sugar moieties of glycosidicallybound aroma volatiles, which have been reported in the mango are a-terpeneyl-b-D-glucopyranosides, a-terpeneyl-6-O -rutinosides and a-terpeneyl-6-O -(a-L-arabinofuranosyl)-b-D-glucopyranosides (Sakho et al., 1997). Aroma compounds (aglycones) can be released from glycosidicallybound compounds by enzymatic or chemical reactions during maturation, storage, industrial pretreatment or processing (Boulanger and Crouzet, 2000). Apart from mangoes, glycosidically-bound aroma volatile compounds have been reported in different fruit including passion fruit (Engel and Tressl, 1983), grape (Gunata et al., 1985; Wirth et al., 2001), papaya (Schwab et al., 1989), apricot, peach and yellow plum (Krammer et al., 1991). The importance of glycosidically-bound aroma volatile compounds and their contribution to fruit aroma has received increasing attention from many researchers in the past decade (Adedeji et al., 1992), and more recently because of the increase in processed fruit products such as juice,
dried fruit, jam, pickles and alcoholic beverages (Crouzet et al., 1997; Mateo and Jimenez, 2000). Thirty-three aroma compounds have been identified in the crude heterosidic extract of the pulp of an unknown African mango cultivar after hydrolysis using b-glucosidase (Adedeji et al., 1992). Moreover, Sakho et al. (1997) identified 27 volatile compounds released from a glycosidically-bound fraction of an ungrafted African mango after incubation with pectinase. Sixty-nine aroma compounds have been found in the mango pulp after enzymatic hydrolysis of their glycosidically-bound fractions, including aliphatic alcohols, acids, aldehydes, ketones, esters, aromatic compounds, terpenes and norisoprenoids (Adedeji et al., 1992; Koulibaly et al., 1992; Sakho et al., 1997; Olle et al., 1998). The majority of research work on glycosidically-bound aroma volatile compounds has been reported from the pulp of fully ripe mangoes. Suarez et al. (1991) reported that glycosidically-bound aroma compounds were distributed in the pulp as well as in the skin of the lulo fruit (Solanum vestissimum D.). Apparently, no research work has been reported on qualitative and quantitative differences in glycosidically-bound aroma compounds in the skin of the mango fruit. Moreover, no research work has been reported either on glycosidicallybound aroma compounds in skin and pulp or their occurrence in different maturity stages of the ‘Kensington Pride’ mango fruit. These observations prompted the current investigation into
H.J.D. Lalel et al. / Postharvest Biology and Technology 29 (2003) 205 /218
glycosidically-bound aroma volatile compounds in the skin as well as in the pulp of the ‘Kensington Pride’ mango fruit at different stages of maturity.
2. Materials and methods
2.1. Plant materials ‘Kensington Pride’ mangoes (M. indica L.) were picked from a commercial orchard located in Chittering (11685?E, 31825?S), Western Australia, during the third week of March 2001 at three maturity stages: mature green (hard with 100% green skin), half-ripe (slightly soft with 50/60% yellow skin), and ripe (eating soft with more than 75% yellow skin). The detailed characteristics of the fruit at these maturity stages at harvest, including colour, firmness and total soluble solids (TSS) are presented in Table 1. Thirty uniform fruit were randomly chosen at each maturity stage for this experiment. Ten fruit were used as a treatment unit and replicated three times. The skin and pulp of the fruit were taken and stored at /18 8C (1 month) prior to analysis for glycosidically-bound aroma compounds.
2.2. Reagents Solvents were purchased from various locations. Distilled n -pentane (99.5% purity with total C5 isomer 99.99%) was purchased from EM Science (Gibbstown, NJ, Canada), dichloromethane (99.5% purity) from Ajax Chemicals (Auburn, NSW, Australia) and methanol (99.9% purity) from Riedel-de Haen (Seelze, Niedersachsen, Germany). n -Paraffin C7 /C22, with a purity of more than 95.5%, and (9/)-2-octanol (98% purity) were obtained from Sigma-Aldrich (Steinheim, Germany). Amberlite XAD-2 (20 /60 mesh), obtained from Supelco (Bellefonte, PA, USA), was treated according to the procedure of Gunata et al. (1985). Almond b-glucosidase and Aspergillus niger hemicellulase from Sigma-Aldrich were also used.
207
2.3. Fruit colour All fruit from each treatment were assessed for their skin colour. Skin colour (L , a* and b* values) was recorded using a Hunter Lab colorFlex 45/0 Spectrophotometer (Hunter Associates Laboratory, Inc., Reston, VA, USA). Chroma was calculated using the formula [(a*2/b *2)1/2], which represents colour saturation from dull (low value) to vivid colour (high value). Hue angle was calculated using the formula tan 1(b*/a *), which is defined as a colour wheel, with red /purple at an angle of 08, yellow at 908, bluish /green at 1808, and blue at 2708 (McGuire, 1992). 2.4. Fruit firmness The firmness of each fruit was measured on two peeled surfaces opposite each other in the equatorial region of the fruit using an electric pressure tester (model EPT-1, Lake City Technical Product Inc., Kelowna, BC, Canada) fitted with a plunger of 11 mm diameter. Fruit firmness was expressed as Newtons. 2.5. Total soluble solids analysis Total soluble solids (TSS) were determined using freshly extracted juice from ten fruit. TSS (%) were determined using an infrared digital refractometer (model PR-101, Atago Co., Ltd., Tokyo, Japan). 2.6. Glycoside extraction Frozen pulp (100 g) and skin (50 g) of the fruit were thawed in 200 or 100 ml of cold (4 8C) 0.1 M phosphate buffer pH 7.0, respectively. Following thorough homogenisation, phase separation was achieved by centrifugation at 5000 rpm for 30 min (4 8C). The supernatant was percolated at 59/ 1 8C at the rate of 90 ml/h through a column (50 /1 cm i.d.) packed with 20 ml of Amberlite XAD-2. The column was then rinsed with 200 ml of Milli-Q water to eliminate water-soluble compounds. Free volatile compounds were removed first by elution with 100 ml of azeotropic pentane/ dichloromethane mixture (2:1, v/v). Glycosidi-
208
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Table 2 Bound aroma volatile compounds produced via lipid metabolism released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg1)
RI Pulp
Skin
MG
HR
R
MG
HR
R
777 854 867 970
n.d. 0.055 n.d. n.d.
0.006 0.059 0.021 tr
0.036 0.088 0.038 0.005
0.020 0.762 0.039 n.d.
0.042 0.235 0.028 n.d.
0.022 0.130 0.018 n.d.
Acids 2-Methylbutanoic acid Hexanoic acid Octanoic acid Dodecanoic acid Tetradecanoic acid Hexadecanoic acid Methyl dihydromalvalate (Z,Z )-9,12-Octadecadienoic acid Heptadecene-(8)-carbonic acid-(1)
846 977 1170 1559 1759 1962 2045 2130 2140
n.d. n.d. n.d. n.d. n.d. 0.060 n.d. n.d. n.d.
tr tr tr n.d. n.d. 0.030 tr n.d. n.d.
tr 0.004 0.014 n.d. n.d. 0.038 0.242 n.d. n.d.
n.d. n.d. n.d. 0.048 n.d. 0.039 n.d. n.d. n.d.
n.d. n.d. 0.052 0.063 0.064 0.330 n.d. 0.018 0.083
n.d. n.d. 0.061 0.028 0.100 0.357 n.d. 0.020 0.072
Esters Isopropyl tetradecanoate Ethyl 9-hexadecanoate Ethyl hexadecanoat Ethyl 9,12,15-octadecatrienoat Ethyl (Z )-9-octadecenoat
1831 1977 1997 2170 2171
n.d. n.d. n.d. n.d. n.d.
tr 0.043 0.025 tr 0.026
0.098 0.937 0.305 0.013 0.554
n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d.
Aldehydes Furfural Tetradecanal Longifolenaldehyde
832 1818 1876
n.d. n.d. n.d.
n.d. n.d. n.d.
0.006 n.d. n.d.
n.d. 0.028 0.032
n.d. 0.074 n.d.
n.d. 0.042 n.d.
Lactones g-Lactone g-Undecalactone
1262 1986
n.d. n.d.
0.007 0.048
0.030 0.119
0.036 n.d.
0.140 n.d.
0.062 n.d.
Aliphatic alcohols 3-Methyl 2-buten-1-ol cis -3-Hexenol 1-Hexanol 1-Heptanol
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
cally-bound volatile components were obtained by subsequent elution with 100 ml of methanol (Boulanger et al., 1999). The methanol fraction containing bound volatile compounds was initially dried over anhydrous sodium sulphate and concentrated to dryness under vacuum at 45 8C to get a crude glycosidically-bound extract. 2.7. Enzymatic hydrolysis Crude glycosidically-bound extract was dissolved in 0.5 ml of 0.1 M phosphate /citrate buffer
(pH 5) and washed with 5/0.5 ml of azeotropic pentane/dichloromethane mixture (2:1, v/v). Following washing, a 0.5 ml mixture containing 5 mg/ ml of almond b-glucosidase and 500 mg/ml of hemicellulase in 0.1 M phosphate /citrate buffer (pH 5) was added to the washed buffer mixture. Enzymatic hydrolysis of glycosides was performed for 16 h at 409/1 8C. After cooling to room temperature, 10 ml 2-octanol (3.08 mg/ml) was added as an internal standard and the released aglycons were extracted with azeotropic pentane/ dichloromethane mixture (5 /0.5 ml). The agly-
H.J.D. Lalel et al. / Postharvest Biology and Technology 29 (2003) 205 /218
209
Table 3 Bound aroma volatile compounds produced via carbohydrate metabolism released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg1)
RI
Pulp
2,4-Pentanedione 2,5-Dimethyl furan Furaneol 7,7-Dimethylbicyclo[3.3.0]octan-2-one (E )-4-(2?,6?,6?-Trimethyl-1?,2?-epoxy-cyclohexyl)-3-penten-2-one (isomer 1) 9-Decen-2-one (E )-4-(2?,6?,6?-Trimethyl-1?,2?-epoxy-cyclohexyl)-3-penten-2-one (isomer 2) 2-Furanmethanol 7,9-Di-tert -butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione
831 1055 1376 1397 1470 1486 1597 1929
Skin
MG
HR
R
MG
HR
R
n.d. n.d. 0.016 n.d. n.d. n.d. n.d. n.d. 0.131
n.d. n.d. 0.017 n.d. n.d. n.d. n.d. 0.018 0.011
0.011 n.d. 0.081 n.d. n.d. n.d. n.d. 0.117 0.017
0.020 0.007 n.d. 0.305 0.122 0.057 0.063 n.d. n.d.
0.036 n.d. 0.038 0.241 0.061 n.d. 0.785 n.d. n.d.
0.070 n.d. 0.025 0.177 0.035 n.d. 0.292 n.d. n.d.
RI, retention index on HP5-MS column; n.d., not detected; n/3 replications; MG, mature green; HR, half ripe; R, ripe.
con extract was concentrated to a final volume of 0.5 ml in a nitrogen stream at room temperature (209/1 8C). Each maturity stage or part of the fruit (skin and pulp) was analysed in triplicate. A blank experiment was also conducted to check for the possibility of contamination. 2.8. Aglycones analysis A Hewlett-Packard 6890 gas chromatograph (GC) coupled to a Hewlett-Packard 5973 Mass selective (MS) detector with an electron impact mode (EI) generated at 70 eV was used. The fused capillary column HP-5MS (60 m/0.25 mm; film thickness 0.25 mm) was used for separation. Aglycones extract (1 ml) was injected with a split/ splitless mode. The injection temperature was maintained at 260 8C and the column temperature was held at 40 8C for 1 min and increased at the rate of 3 8C/min to 310 8C and then maintained for 20 min. Helium was the carrier gas at the rate of 1.0 ml/min. Identification of the compounds was based on comparison of their mass spectra with those of authentic standards in the Wiley275.L library and their linear retention indices (RI). RI were calculated using n -paraffin standards (Van den Dool and Kratz, 1963). The aglycones were quantified using internal standardisation with consideration that the detector response factor to 2-octanol for all compounds
was equal to one. 2-Octanol was used as the internal standard because of an intermediate behaviour of 2-octanol on monoterpenes and sesquiterpenes (Olle et al., 1998). The compounds, which were present in notable amounts (/0.001 mg/kg) both in the skin and pulp of the fruit at all maturity stages, were statistically analysed. 2.9. Statistical analysis The data were subjected to analysis of variance (ANOVA) using GENSTAT 5 release 4.1 (Lawes Agricultural Trust, Rothamsted Experimental Station, UK). The effects of maturity stage and part of fruit including skin and pulp on various parameters were assessed. Least significant differences (Fisher’s protected LSD) were calculated at P /0.05 following a significant F -test. All the assumptions of analysis were checked to ensure validity.
3. Results 3.1. Glycosidically-bound volatile compounds One hundred and forty four compounds were identified from a heterosidic fraction obtained from the skin and pulp of the ‘Kensington Pride’ mango after enzymatic hydrolysis with b-glucosi-
210
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Table 4 Terpenes released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg 1)
RI
Pulp
Isoprene Northujane Methylene cyclohexane p -Cymene trans -b-Ocimene cis -Linalool furanoxide p -Cymenyl trans -Linalool furanoxide Linalool (2-Methylprop-1-enyl)-cyclohexa-1,3-diene Mentha-1,4,8-triene p -Mentha-1,5-dien-8-ol Epoxy linalool 1,8-Menthadien-4-ol p -Cymen-8-ol a-Terpineol a-Phellandrene epoxide b-Cyclogeraniol trans -(/)-Carveol exo -2-Hydroxycineole Nerol Neral 1-Methyladamantane Geraniol m -Cymene 3-Carene-10-ol Geranial 1(7),3,8-ortho -Menthatriene Tricyclo(3.2.1.0)-octane p -Cymen-a-ol Menth-1-ene-4,8-diol p -Mentha-1,4-dien-7-ol p -Mentha-1,5,8-triene cis -1,2,3,6,7,7a -Hexahydo-7a -methyl-3aH -inden-3a -ol Geranic acid Isocyclocitral 7,7-Dimethylbicyclo[3.3.0]-octane-2-one trans -Sobrerol Z -Citral 2-(2-Hydroxyethyl)-3-methoxy-tricyclo[2.2.21.0*2,6]heptane 4,5-Epoxy-1-isopropyl-4-methyl-1-cyclohexene trans -Chrysanthemal 2-Methyl-6-methylene-1,7-octadien-3-one 2-endo -Hydroxy-2-exo -methylprotoadamantane 8-Hydroxy geraniol 2-Pinen-10-hydroxyperoxide Ledenoxide (I) Capnellane-8-one Caryophylla-3,8(13)-dien-5-b-ol
859 961 1026 1049 1075 1091 1091 1101 1140 1144 1165 1177 1182 1189 1196 1211 1213 1224 1228 1231 1245 1250 1257 1259 1270 1274 1286 1288 1295 1317 1333 1341 1343 1355 1365 1378 1384 1392 1398 1405 1451 1465 1467 1521 1629 1890 1942 1946
Skin
MG
HR
R
MG
HR
R
n.d. n.d. n.d. n.d. n.d. 0.025 n.d. 0.034 0.006 n.d. n.d. n.d. n.d. n.d. 0.014 0.013 n.d. n.d. n.d. n.d. tr tr n.d. n.d. tr 0.005 tr n.d. n.d. 0.029 0.021 n.d. n.d. 0.069 0.020 n.d. 0.037 0.019 n.d. n.d. 0.018 n.d. tr tr 0.065 n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d. 0.027 n.d. 0.040 0.053 n.d. 0.004 0.01 0.008 0.010 0.045 0.036 0.004 0.005 0.008 0.008 0.009 0.052 0.006 0.018 0.042 0.014 0.036 n.d. 0.015 0.095 0.045 0.006 n.d. 0.243 0.019 0.159 0.123 0.044 n.d. 0.021 0.038 0.018 0.181 tr 0.089 n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d. 0.032 n.d. 0.051 0.022 n.d. 0.007 0.015 0.009 0.011 0.429 0.034 0.008 0.009 0.027 0.017 0.026 0.109 0.015 0.325 0.284 0.025 0.135 n.d. 0.015 0.100 0.033 0.008 n.d. 0.381 0.048 0.196 0.139 0.043 n.d. 0.037 0.030 0.023 0.177 tr 0.183 n.d. n.d. n.d. n.d.
0.022 0.022 0.064 0.020 0.009 n.d. 0.099 n.d. 0.008 0.008 0.036 n.d. n.d. 0.043 0.133 0.234 n.d. n.d. 0.023 n.d. n.d. 0.020 0.060 0.113 n.d. 0.136 0.008 0.111 n.d. 0.618 n.d. n.d. 2.195 n.d. n.d. n.d. n.d. n.d. 0.116 n.d. n.d. 0.733 n.d. n.d. n.d. 0.358 0.051 0.048 0.045
0.039 0.011 0.034 0.015 0.011 n.d. 0.067 n.d. 0.024 0.010 0.029 n.d. n.d. 0.029 0.165 0.082 n.d. n.d. 0.018 n.d. 0.007 0.040 0.052 3.528 n.d. 0.059 0.073 0.062 n.d. 0.527 n.d. n.d. 1.751 n.d. n.d. n.d. n.d. n.d. 0.124 n.d. n.d. 0.754 n.d. n.d. n.d. 0.251 0.062 0.093 0.031
0.031 0.011 0.015 0.012 0.012 n.d. 0.047 n.d. 0.013 0.006 0.019 n.d. n.d. 0.022 0.334 0.041 n.d. n.d. 0.013 n.d. n.d. 0.013 0.034 1.279 n.d. 0.032 0.024 0.047 n.d. 0.246 n.d. n.d. 1.083 n.d. n.d. n.d. n.d. n.d. 0.132 n.d. n.d. 0.311 n.d. n.d. n.d. 0.082 0.019 n.d. 0.015
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
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211
Table 5 Bound aroma volatile compounds produced via aromatic amino acid metabolism released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg1)
RI
Pulp
Benzaldehyde Phenol Benzenemethanol Phenylacetaldehyde Benzeneethanol 4-Ketoisophorone Benzoic acid 2-Hydroxy benzoic acid methyl ester Myrtenol 2-(4?-Methylphenyl)-propanal 4-(1-Methylethyl)-benzaldehyde 4-(2-Propenyl)-phenol 2-Xylyl ethanol p-sec -Butylphenol Phenolic compound 4,4a ,5,6,7,8-Hexahydro 2(3H )-naphthalenone Perilla alcohol 2-Methyl-5-(1-methylethyl)-phenol Thymol methyl ether Eugenol 2-Allyl-4-methylphenol trans -Sobrerol 5-Methyl-2-(1-methylethyl)-phenol a,a-Dimethoxy-m -xylene 2-Hydroxy-benzeethanol 1-(2-Hydroxyphenyl)-ethanone 4-Hydroxy benzoic acid methyl ester Acetovanillone 4-(1,1-Dimethylethyl)-1,2-benzenediol trans -Crysanthenyl acetate 2,4,5-Trimethyl benzenemethanol 2,4,5-Trimethyl-benzenemethanol 2,6-bis(1,1-Dimethylethyl)-4-methyl phenol Ethyl vanillylether 4-(4-Hydroxyphenyl)-2-butanone 2-Allyl-p -cresol 4-Hydroxy-benzoic acid propyl ester cis -Cinerolone Zingerone 1,4-Dimethylindanyl acetate 1-(2,3,4,5-Tetramethylphenyl)-1-butanone Trimethyl-(1-methylethyl)-benzene Coniferyl alcohol Phenolic compound Vomifoliol 1,2,4-Triethyl benzene 2,5-Dimethoxy-4-propoxy-benzaldehyde 1,2-Benzenedicarboxylic acid butyl phenylmethyl ester
963 981 1035 1046 1116 1147 1159 1198 1202 1210 1246 1254 1277 1279 1284 1299 1302 1304 1328 1363 1376 1382 1407 1409 1410 1442 1459 1491 1493 1504 1513 1516 1518 1553 1554 1599 1626 1641 1653 1658 1660 1678 1745 1786 1796 1813 1895
Skin
MG
HR
R
MG
HR
R
tr n.d. 0.059 0.005 0.068 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.039 n.d. n.d. n.d. n.d. 0.025 0.051 0.047 n.d. n.d. n.d. 0.020 n.d. n.d. 0.589 n.d. 0.015 n.d. n.d. 0.018 n.d. n.d. n.d. n.d. 0.179 n.d. n.d. n.d. 0.015 0.018 0.023 n.d. 0.186 n.d. n.d. n.d.
0.006 0.015 0.117 0.004 0.126 0.005 n.d. n.d. 0.005 n.d. 0.016 n.d. 0.103 n.d. n.d. n.d. n.d. 0.019 0.009 0.109 0.017 n.d. n.d. 0.042 0.034 n.d. 1.094 n.d. 0.023 n.d. n.d. 0.030 n.d. 0.013 n.d. n.d. 0.304 n.d. n.d. n.d. 0.037 0.076 0.011 n.d. 0.343 n.d. n.d. n.d.
0.007 tr 0.158 0.006 0.216 tr n.d. n.d. 0.007 n.d. 0.011 n.d. 0.113 n.d. n.d. n.d. n.d. 0.060 0.020 0.134 0.062 n.d. n.d. 0.070 0.084 n.d. 0.983 n.d. 0.029 n.d. n.d. 0.040 n.d. 0.033 n.d. n.d. 0.337 n.d. n.d. n.d. tr 0.100 0.057 n.d. 0.572 n.d. n.d. n.d.
n.d. n.d. 1.695 0.010 0.296 0.006 n.d. 0.036 0.036 0.047 0.041 0.039 0.865 0.233 0.064 0.051 0.120 n.d. n.d. n.d. n.d. 0.144 0.221 n.d. n.d. 0.056 n.d. 0.096 n.d. 0.080 0.146 n.d. 0.263 n.d. 0.086 0.125 n.d. 0.099 0.639 0.562 n.d. 0.401 0.028 0.046 0.459 0.031 0.080 n.d.
n.d. n.d. 1.494 0.011 0.204 0.006 0.012 0.042 0.027 0.029 0.026 n.d. 0.714 0.084 0.058 0.063 0.099 n.d. n.d. n.d. n.d. 0.073 0.171 n.d. n.d. 0.157 n.d. 0.056 n.d. 0.078 0.116 n.d. 0.411 n.d. 0.080 0.100 n.d. 0.106 0.607 0.270 n.d. 0.683 0.042 0.052 0.628 0.052 0.059 0.058
n.d. n.d. 0.427 0.012 0.093 0.004 n.d. 0.013 0.012 0.022 0.016 n.d. 0.307 0.048 0.047 0.030 0.063 n.d. n.d. n.d. n.d. 0.025 0.076 n.d. n.d. 0.013 n.d. 0.028 n.d. 0.029 0.056 n.d. 0.101 n.d. 0.030 0.032 n.d. 0.055 0.244 0.084 n.d. 0.232 0.024 0.019 0.185 n.d. 0.013 0.057
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Table 5 (Continued ) Compound
Concentration (mg kg1)
RI
Pulp
3-Benzyl-1,4-diaza-2,5-dioxo-bicyclo[4.3.0]nonane 1,2-benzenedicarboxylic acid diisooctyl ester n -Nornuciferine
Skin
MG
HR
R
MG
HR
R
0.024 n.d. n.d.
0.015 0.013 tr
0.019 0.131 0.127
0.011 0.324 n.d.
0.022 0.208 n.d.
0.011 0.301 n.d.
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
dase and hemicellulase (Tables 2 /7). Among these compounds, 92 compounds were detected in the pulp and 85 compounds were present in the skin. Aromatic amino acid metabolites were the most abundant class of compounds found in the skin, whilst in the pulp terpenes were found to be the most abundant compounds, which accounted for 41.18 and 38.04% of the total number of compounds in skin and pulp, respectively. Total glycosidically-bound volatiles in the pulp significantly (P 5/0.05) increased during fruit ripening, whilst in the skin the compounds increased and peaked at the half ripe stage (Fig. 1). 3.1.1. Glycosidically-bound volatile compounds produced via lipid metabolism Glycosidically-bound volatile compounds produced via lipid metabolism identified in the pulp
and skin accounted for 23 compounds including aliphatic alcohols, acids, esters, aldehydes and lactones (Table 2). Total glycosidically-bound volatile compounds produced via lipid metabolism were significantly (P 5/0.05) higher in the skin than in the pulp of mature green and half ripe fruit as compared with the ripe fruit (Fig. 2). Total glycosidically-bound volatile compounds produced via lipid metabolism in the pulp increased as ripening progressed, whilst in the skin they remained unchanged. At the ripe stage, total glycosidically-bound volatile compounds produced via lipid metabolism in the pulp were significantly (P 5/0.05) higher than those of the skin. Among glycosidically-bound volatile compounds produced via lipid metabolism, only cis 3-hexenol and hexadecanoic acid were present in
Table 6 Bound aroma volatile compounds produced via carotenoid metabolism released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg 1)
RI
Pulp
2-endo-Hydroxy-2-exo-methylprotoadamantane (9R)-9-Hydroxy-4-7E -megastigmadien-3-one 3-Oxo-7,8-dihydro-a-ionol 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol (isomer 1) 3-Oxo-7,8-dihydro-b-ionol 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol (isomer 2) (9/)-15-Hexadecanolide
1464 1655 1713 1721 1731 1782 1943
Skin
MG
HR
R
MG
HR
R
n.d. 0.051 0.082 0.043 n.d. 0.055 0.011
n.d. 0.194 0.326 0.123 tr 0.167 0.014
n.d. 0.217 0.454 0.155 0.027 0.233 0.023
0.490 n.d. 0.541 0.067 n.d. 0.123 n.d.
0.596 n.d. 1.022 0.143 n.d. 0.222 n.d.
0.279 n.d. 0.359 0.051 n.d. 0.062 n.d.
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
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Table 7 Miscellaneous compounds released after enzymatic hydrolysis of a heterosidic fraction Compound
Concentration (mg kg1)
RI Pulp
5-Ethyl-4,6-dimethyl-2-methoxy pyridine Cyclohexadecane Epoxy cyclohexane 5,7-Dihydroxy-dihydroflavone Hexadecanemide 9-Octadecenamide
1498 1881 1883 2182
Skin
MG
HR
R
MG
HR
R
n.d. n.d. n.d. n.d. n.d. n.d.
0.017 n.d. n.d. n.d. 0.014 0.067
tr n.d. n.d. n.d. tr 0.069
n.d. 0.078 0.110 0.060 n.d. n.d.
n.d. 0.560 n.d. 0.116 n.d. n.d.
n.d. 0.896 n.d. 0.033 n.d. n.d.
RI, retention index on HP5-MS column; n.d., not detected; tr, trace (B/0.001 mg/kg); n/3 replications; MG, mature green; HR, half ripe; R, ripe.
Fig. 1. Effects of maturity stage and fruit part on total glycosidically-bound compounds. Different lower cases on the top of similar shaded bars (fruit parts) or different upper cases on the top of bars of each maturity stage show significant differences (LSD, P 5/0.05). Mt, mature; n/3 replications.
the skin and pulp in notable amounts and were statistically analysed (Table 8). Skin had significantly (P 5/0.05) higher concentrations of cis -3hexenol than the pulp at all maturity stages of the fruit. The concentrations of cis -hexenol in the skin declined as the maturity progressed to the ripe stage, whilst in the pulp it increased. On the other hand, the concentration of hexadecanoic acid in mature green fruit was significantly (P 5/0.05) higher in the pulp than in the skin. However, in half ripe and ripe fruit, its concentration was higher in the skin than in the pulp.
3.1.2. Glycosidically-bound volatile compounds produced via carbohydrate metabolism The concentration of total glycosidically-bound volatile compounds produced via carbohydrate metabolism released by enzymatic hydrolysis was significantly (P 5/0.05) higher in the skin than in the pulp, regardless of maturity stage (Fig. 2). The concentration of total glycosidically-bound volatile compounds produced via carbohydrate metabolism in the skin was significantly (P 5/0.05) higher at the half ripe stage as compared with the mature green and ripe stage. In the pulp, they increased as ripening progressed. Six ketones were found in the skin, whilst four compounds were found in the pulp (Table 3). 3.1.3. Terpenes The trend of changes in total terpenes released after incubation with b-glucosidase and hemicellulase as maturity progressed was similar to the trend of total ketones (Fig. 2). The concentration of terpenes in the skin was significantly (P 5/0.05) higher at the half ripe stage than in all other maturity stages. In the pulp, the concentration of total terpenes increased significantly (P 5/0.05) as ripening progressed to the ripe stage. In number, terpenes found in the pulp were more than in the skin (Table 4), whilst in quantity terpenes found in the skin were significantly higher than in the pulp irrespective of the fruit maturity stage (Fig. 2). Among total numbers of identified terpenes released by enzymatic hydrolysis, oxygenated ter-
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Fig. 2. Effects of maturity stage and fruit part on glycosidically-bound aroma volatile compounds produced via lipid metabolism (A), carbohydrate metabolism (B), terpenes (C), aromatic amino acid metabolism (D), carotenoid metabolism (E), and unknown pathways (F). Different lower cases on the top of similar shaded bars (fruit parts) or different upper cases on the top of bars of each maturity stage show significant differences (LSD, P 5/0.05). Mt, mature; n/3 replications.
penes accounted for 75% (Table 4). The most abundant terpene found in the skin was geraniol followed by p -mentha-1,5,8-triene and trans -chrysanthemal. In the pulp, the most abundant terpene was p-cymen-8-ol followed by cis -1,2,3,6,7,7a hexahydro-7a-methyl-3a H-inden-3a -ol and geraniol. In mature green and half ripe mangoes, pcymen-8-ol, a-terpineol and 3-carene-10-ol were found to be significantly (P 5/0.05) higher in the skin than the pulp, whilst in ripe fruit the trend was reversed (Table 8). Linalool was significantly (P 5/0.05) higher in the skin of mature green mangoes than in the pulp, but the trend was reversed in half ripe and ripe fruit. 3.1.4. Glycosidically-bound volatile compounds produced via aromatic amino acid metabolism Total glycosidically-bound volatile compounds produced via aromatic amino acids metabolism
released by enzymatic hydrolysis in the skin reduced as the maturity progressed to the ripe stage, whilst in the pulp its concentration exhibited the reverse trend (Fig. 2). In mature green and half ripe mangoes, glycosidically-bound volatile compounds produced via aromatic amino acid metabolism were significantly (P 5/0.05) higher in the skin than that in the pulp. At the ripe stage, glycosidically-bound volatile compounds produced via aromatic amino acid metabolism were significantly higher in the pulp than in the skin. Benzenemethanol was the most abundant glycosidically-bound volatile compound produced via aromatic amino acid metabolism in the skin, while 4-hydroxy benzoic acid methyl ester was the most abundant in the pulp (Table 5). The concentrations of benzenemethanol, 2-xylyl ethanol, phenylacetaldehyde and trimethyl-(1-methylethyl)benzene were significantly (P 5/0.05) higher in
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215
Table 8 Effects of maturity stage and fruit part on bound aroma volatile compounds Compound
Concentration (mg kg1)
Fruit part MG
cis -3-Hexenol Hexadecanoic acid Benzyl alcohol (benzenemethanol) Phenylacetaldehyde Benzeneethanol 2-Xylyl ethanol p -Cymen-a-ol Trimethyl-(1-methylethyl)-Benzene Coniferyl alcohol 3-Benzyl-1,4-diaza-2,5Dioxobicyclo[4.3.0]nonane Vomifoliol Linalool p -Cymen-8-ol a-Terpineol 3-Carene-10-ol 3-Oxo-7,8-dihydro-a-ionol 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol (isomer 1) 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol (isomer 2)
Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin Pulp Skin
0.055 0.762 0.060 0.039 0.059 1.695 0.005 0.010 0.068 0.296 0.039 0.865 0.029 0.618 0.018 0.401 0.023 0.028 0.024 0.011 0.186 0.459 0.006 0.008 0.014 0.133 0.013 0.234 0.005 0.136 0.082 0.541 0.043 0.067 0.055 0.123
HR aA cB bB aA aA cB bA aB aA cB aA cB aA cB aA bB bA bB cB aA aA bB aA aB aA aB aA cB aA cB aA bB aA bB aA bB
0.059 0.235 0.030 0.330 0.117 1.494 0.004 0.011 0.126 0.204 0.103 0.714 0.095 0.527 0.076 0.683 0.011 0.042 0.015 0.022 0.343 0.628 0.053 0.024 0.045 0.165 0.036 0.082 0.014 0.059 0.326 1.022 0.123 0.143 0.167 0.222
LSD (P/0.05)
R abA bB aA bB abA bB aA bB bA bB bA bB bA bB bA cB aA cB aA bB bA cB cB cA bA bB bA bB bA bB bA cB bA cB bA cB
0.088 0.130 0.038 0.357 0.158 0.427 0.006 0.012 0.216 0.093 0.113 0.307 0.100 0.246 0.100 0.232 0.057 0.024 0.019 0.011 0.572 0.185 0.022 0.013 0.429 0.334 0.034 0.041 0.025 0.032 0.454 0.359 0.155 0.051 0.233 0.062
bA aB aA cB bA aB cA cB cB aA bA aB bA aB bA aB cB aA bB aA cB aA bB bA cB cA bA aA cB aA cB aA cB aA cB aA
0.030 0.018 0.084 0.001 0.016 0.043 0.031 0.030 0.003 0.002 0.038 0.002 0.021 0.009 0.003 0.049 0.010 0.014
Means within the same row (fruit part) followed by the same lower cases are not significantly different (P /0.05); means within the same column (maturity stage) followed by the same upper cases are not significantly different (P /0.05), n/3 replications; MG, mature green; HR, half ripe; R, ripe.
the skin than in the pulp at all maturity stages (Table 8). As maturity progressed to the ripe stage, the concentrations of benzenemethanol and 2xylyl ethanol declined. In mature green and half ripe fruit, benzeneethanol and coniferyl alcohol were significantly higher in the skin than in the pulp. However in the ripe fruit, the concentrations were higher in the pulp than in the skin.
3.2. Glycosidically-bound volatile compounds produced via carotenoid metabolism The concentration of glycosidically-bound carotenoids metabolites (norisoprenoids) in the pulp increased as the maturity progressed to the ripe stage (Fig. 2). The concentration of norisoprenoids was significantly (P 5/0.05) higher in the skin of
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half ripe fruit than in mature green and ripe fruit. 6,7-Dehydro-7,8-dihydro-3-oxo-a-ionol isomer 1 (RI /1721), isomer 2 (RI/1782) and 3-oxo-7,8dihydro-a-ionol were significantly (P 5/0.05) higher in the skin of green mature and half ripe mangoes than in the pulp, while in ripe mangoes the trend was reversed (Table 8). 3.2.1. Glycosidically-bound aroma volatile compounds produced via unknown pathways Two cyclic alkanes and three nitrogen-containing compounds were detected only in the pulp, whilst one flavone was found only in the skin (Table 7). Cyclohexadecane increased during fruit ripening. All the glycosidically-bound amides were only detected in the pulp of the half ripe and ripe mangoes. 5,7-Dihydroxy-dihydroflavone peaked at half ripe stage.
4. Discussion Hydrolysis of glycosidically-bound aroma volatile compounds in the skin and pulp of the ‘Kensington Pride’ mango, using b-glucosidase and hemicellulase, showed qualitative and quantitative differences in the skin and pulp at various stages of maturity. Most of the compounds were higher in the skin than in the pulp, especially in mature green and half ripe mangoes, as compared with the ripe ones (Figs. 1 and 2). This may due to the higher concentrations of these compounds as free compounds (unbound volatile) in the skin than in the pulp. We found that free volatile aldehydes, fatty acids and terpenes were higher in the skin than in the pulp (data not shown). The high concentration of lipophilic compounds can destroy the cell membranes, and therefore, glycosidation usually takes place as one of the protective mechanisms in plant tissue (Stahl-Biskup et al., 1993). The content of glycosidically-bound volatile compounds produced via lipid metabolism, carbohydrate metabolism, aromatic amino acid metabolism, terpene and carotenoid metabolism generally declined in the skin at the ripe stage, but the trend was reversed in the pulp (Fig. 2). Probably during ripening some glycosidically-
bound aroma compounds were transported from the skin to the pulp of the fruit, since during ripening the integrity of cell walls declines as fruit softens (Gomez-Lim, 1997). It may be argued that some of the aroma compounds were being synthesised (glycosidised) in the pulp during ripening as some of the glycosidically-bound aroma compounds were not present in the skin. Amongst glycosidically-bound volatile compounds produced via lipid metabolism released by enzymatic hydrolysis, cis -3-hexenol, 1-hexanol, 1-heptanol and g-octalactone have been found in the free form (unpublished data). The presence of glycosidically-bound cis -3-hexenol, hexanoic acid, tetradecanoic acid and hexadecanoic acid, on the other hand, have been reported in other mango cultivars (Adedeji et al., 1992; Sakho et al., 1997; Olle et al., 1998). Other glycosidically-bound aroma volatile compounds produced via lipid metabolism including 1-hexanol and 1-heptanol have also been reported to be present in papayas (Schwab et al., 1989) and grapes (Wirth et al., 2001), whilst 2-methylbutanoic acid, octanoic acid and dodecanoic acid have been reported in apricots, peaches and yellow plums (Krammer et al., 1991). Except for cis -3-hexenol, lactones and aldehydes, the contribution of other glycosidically-bound volatile compounds produced via lipid metabolism to the overall aroma is probably low because they generally have a high odour threshold (Buttery et al., 1988; Baek and Cadwallader, 1999). Terpenes, including linalool, p -mentha-1,5,8triene, p-cymen-8-ol, 4-terpineol and a-terpineol, were found in bound and free forms (Lalel et al., 2003). Most of these compounds have low odour thresholds (Masanetz and Grosch, 1998) and, therefore, they may contribute to the overall aroma of the fruit. The presence of glycosidically-bound 3-carene-10-ol may be related to 3carene, the second most abundant free terpene (Lalel et al., 2003). Linalool and its epoxy or furanoxide forms have been reported to be found in other mangoes in glycosidically-bound forms (Adedeji et al., 1992; Sakho et al., 1997). Similar to most glycosidic compounds, glycosidically-bound volatile compounds produced via aromatic amino acid and carotenoid metabolism
H.J.D. Lalel et al. / Postharvest Biology and Technology 29 (2003) 205 /218
were higher in the skin than in the pulp of the fruit (Fig. 2). Glycosidation of these compounds may also be higher in the skin than in the pulp. During ripening they may also be transported from the skin to the pulp. Although benzyl alcohol (benzenemethanol) was the most abundant glycosidically-bound volatile compound produced via aromatic amino acid metabolites in the skin, it may not contribute to the overall aroma of the fruit because of its high odour threshold (10 000 ppb) (Buttery et al., 1988). Benzyl alcohol (benzenemethanol), having a balsamic earthy smell (Parada et al., 2000) has also been found in other mango cultivars bound with b-D-glucopyranoside or 6-O -(a-L-rhamnophyranosyl)/b-D-glucopyranoside (Adedeji et al., 1992; Sakho et al., 1997). Phenylacetaldehyde, which is only present at the level of 4 /6 ppb in pulp or 10/12 ppb in skin, may contribute to the overall aroma of the fruit if it is released during ripening or processing, as its odour threshold is 4 ppb (Buttery et al., 1988). While the presence of this compound in a glycosidicallybound form has not been reported in mangoes, its presence has been reported in papaya (Schwab et al., 1989) and apricot (Krammer et al., 1991). As a free aroma compound, it has been found in the ‘Alphonso’ mango, giving a pungent green, floral and sweet smell (MacLeod and de Troconis, 1982). The content of most of glycosidically-bound volatile compounds produced via carotenoid metabolism (norisoprenoids) present in the ‘Kensington Pride’ mango (Table 6) were more than their reported threshold (B/1 ppb) (Buttery et al., 1988). Amongst the glycosidically-bound norisoprenoids found, 3-oxo-7,8-dihydro-a-ionol, 9-hydroxy-4,7megastigmadien-3-one and vomifoliol have also been found in other mango cultivars (Adedeji et al., 1992; Sakho et al., 1997; Olle et al., 1998). Although none of these norisoprenoids were found in the free form, these compounds may relate to bionone, which was found in the ‘Kensington Pride’ mango in a free form (Lalel et al., 2003). These norisoprenoids may be important for the aroma of processed mango products, as some of them are found in canned mango puree (Hunter et al., 1974). In conclusion, the composition and concentration of glycosidically-bound aroma compounds of
217
the ‘Kensington Pride’ mango were strongly influenced by fruit part and maturity stage. Glycosidically-bound volatile compounds produced via aromatic amino acid metabolism and terpenes were higher in the skin than in the pulp of mangoes at all maturity stages. Some other glycosidically-bound aroma volatile compounds including glycosidically-bound volatile compounds produced via lipid, aromatic amino acid and carotenoid metabolism were higher in the skin than in the pulp only in mature green and half ripe fruit, whilst glycosidically-bound acids were higher in the skin than in the pulp in half ripe and ripe fruit. Most of glycosidically-bound aroma compounds increased in the pulp as maturity progressed. Amongst all glycosidically-bound aroma compounds, probably only terpenes may contribute to the aroma of fresh ripe ‘Kensington Pride’ mangoes.
Acknowledgements The authors thank Mr S. Bambarderi and Dr Peter Sheppard for technical assistance during analysis and Professor B. Kagi, School of Applied Chemistry for providing GC and GC /MS facilities. We are thankful to Robyn Blake for critically reading this manuscript. H.J.D. Lalel also thankfully acknowledges the financial support from University of Nusa Cendana, Kupang, Indonesia under auspicious of DUE-project funded by World Bank.
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