Identification of the aroma compounds in Vitex doniana sweet: free and bound odorants

Abstract

Background

Most often, the glycosidically-bound aroma compounds are released during industrial processing or pre-treatment of fruits. This usually introduces modification to the aroma notes of such fruits. Therefore, there is the need to understand the contribution of these bound aroma compounds to the overall aroma of a given fruit. In recent years research studies have reported on the free- and bound volatile compounds of several fruits. However, there is no report yet on Vitex doniana sweet.

Results

Results of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) of free and glycosidically-bound aroma-active compounds from Vitex doniana sweet revealed a total of 35 compounds in the free fraction, and 28 compounds were in the bound fraction respectively. Whilst the major group of compounds in the free fraction were terpenes, alcohols, and esters, the bound fraction consisted of ketones, alcohols, terpenes and norisoprenoids.

Conclusion

A comparative analysis of the aroma potencies of the free and bound volatile fractions revealed that; free fraction exhibited strong potency for the fruity and floral notes, and the bound fraction produced more of the flowery, caramel-like and cherry-like notes. In addition results of odour activity values showed that ethylbutanoate, [beta]-damascenone, ethyl-2-methyl propionate, linalool, hexyl acetate and (Z)-rose oxide contributed highly to the sweet prune-like aroma of the fruit.

Lasekan Chemistry Central Journal (2017) 11:19 DOI 10.1186/s13065-017-0247-7

Identication ofthe aroma compounds inVitex doniana sweet: free andbound odorants

Ola Lasekan*

Background

Vitex doniana sweet (Vds) is the edible fruit that belongs to the family Lamiaceae. There are about 250 species in this family [1]. V. doniana sweet is the most abundant and widespread of this genus in the Savannah regions. The fruit is commonly called ucha koro, oori-nla and mfudu or mfulu in Swahili. V. doniana sweet is oblong, about 3cm long. It is green when immature, and purplish-black on ripening with a starchy black pulp. Each fruit contains one hard conical seed which is about 1.52.0 cm long and 11.2cm wide. The fruit which tastes like prunes is rich in nutrients including vitamins A (0.27mg1001g

DB), B1 (18.33mg1001g DB), B2 (4.80mg1001g DB), B6 (20.45mg1001g DB) and C (35.58mg1001g DB)

respectively [2]. The fruit which is consumed fresh can

be made into jam and wine [3]. V. doniana sweet has a unique sweet prune-like aroma when ripened. Although, a number of sugars [4], amino acids and minerals [5] have been reported in Vds, however, there is no study yet on the components responsible for the unique sweet prune-like aroma of the Vds. Studies have shown that fruits aromatic components are either in the free form, or bound to sugar in the form of glycosides [68].

Most often, the glycosidically-bound aroma compounds are released during industrial processing or pre-treatment of fruits. This usually introduces modication to the aroma notes of such fruits [9]. Whilst several studies have reported on the free and glycosidically-bound volatiles in fruits such as strawberry [8], mango [10], raspberry [11], lychee [12], blackberry [6], acerola [7] and a host of other fruits, there has been no study on the volatile constituents of Vitex doniana sweet.

*Correspondence: [email protected] of Food Technology, University Putra Malaysia, 43400 Serdang, Malaysia

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This study aimed at providing an insight into the free and glycosidically-bound aroma compounds of Vitex doniana sweet.

Results anddiscussion

The volatile fractions of both free and glycosidically bound V. doniana sweet, separated on two columns (DBFFAP and SE-54) of dierent polarity are shown in Table1 and Fig.1. A total of 35 compounds were identied in the free fraction while only 28 compounds were detected in the bound fraction. In general, the aroma compounds identied in both fractions were made up of alcohols (7), aldehydes (2), acids (2), esters (11), terpenes (9), ketones (3), norisoprenoids (7), and a phenol. The most important ones in terms of concentration and the numbers identied in the free fraction were the terpenes (43%), alcohols (29%), and esters (25%). On the other hand, in the bound fraction, the ketones, were the most abundant (29%) followed by the alcohols (26%), terpenes (20%) and the norisoprenoids (13%).

In the free fraction of the sweet black plum, the major aroma-active compounds (>300 g kg1) were linalool, 2-phenylethanol, 3-methyl-but-3-en-1-ol, ethyl cinna-mate, ethylbutanoate, hexyl acetate, methyl octanoate, methyl hexanoate, ethyl-2-methylpropionate, geraniol, and (Z)-3-hexen-1-ol. These compounds accounted for 88.8% of the aroma in the free fraction. In addition, most of these compounds were previously reported in several fruits such as lychee, strawberry, cherry and oranges [8, 1214] either in the free or bound form. The identication of signicant numbers of fatty acid esters such as methylbutanoate, ethylbutanoate and methyl hexanoate is an indication of the possible contribution of lipid metabolism in the biogenesis of Vds aroma. Volatile esters are produced by virtually all fruit species during ripening. Most volatile esters have avour characteristics described as fruity [15]. Worthy of note was the high concentration of linalool (5121gkg1) in the Vds. This oral-like terpene alcohol which is produced from isopentenyl pyrophosphate via the universal isoprenoid intermediate geranyl pyrophosphate, and membrane-bound enzymes such as linalool synthase [16] has been reported in lychee [17], Coastal Rican guava [18], mangaba fruit [19] and black velvet tamarind [20]. Another compound of interest is the honey-like 2-phenyl ethanol which produced a signicant concentration in the free fraction. The odorant is an important avour compound in the food and cosmetic industries.

The major volatile compounds in the bound fraction of the Vds were; 4-hydroxy--ionol, guaiacol, y-jasmolac-tone, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, acetophe-none, linalool and 3-methyl-but-3-en-1-ol (Table 1). In

comparison to the free volatile compounds, which were mainly alcohols, esters and terpenes, the bound volatiles proles included alcohols, ketones, and norisoprenoids. While most of the alcohols detected in the free fraction, were found in the bound form, there were fewer esters identied in the bound form. Only methyl octanoate was detected in both fractions. The reason for this observation is not farfetched because glycosidically bound volatiles are organic compounds in which the aglycone is volatile. This aglycone must be bounded to the sugar via glycosidic bond, for which these compounds have to have an OH, SH, or NH. Thus aldehydes, esters and terpenes are not able to form glycosidical bonds. Although, similar alcohol proles were obtained from both free and bound fractions, the concentrations of the alcohols in the bound fraction were signicantly (P<0.05) lower to that of the free fraction. Of interest is the high abundance of 3-methyl-but-3-en-1-ol in both fractions. The presence of this compound in the bound form attested to the fact that it is an important intermediate in various biosynthetic pathways. In addition, signicant numbers of odorous norisoprenoids were detected in the bound fraction. Among them were the oral 4-hydroxy--ionol, the spicy 3-oxo--ionol, 4-oxo--ionol and the owery -damascenone. Most of these compounds have been detected in several fruits such as grape [21], apple [22], raspberry [11] and passion fruit [23]. Also, identied in trace amounts (<10gkg1) in the bound fraction were the two isomers (I & II) of theaspirane.

However, to gain an insight into the contribution of the aroma compounds to the aroma notes of the free and bound fractions, the 36 odorants detected through aroma extract dilution analysis (AEDA) as the key odorants were quantied. The avour dilution (FD) factors obtained for the key odorants ranged from 2 to 512 (Table2). Results revealed an array of aroma notes as shown in Table 2. The seventeen odorants with FD factors 16 were further investigated. The results of the quantitation showed that linalool was the predominant compound in both the free (5121gkg1) and the bound (506gkg1) fractions respectively (Table 3). This was followed by 2-phenyl ethanol (2457 g kg1) in the free fraction and acetophenone in the bound fraction. However, a comparative analysis of the aroma potencies revealed that the free volatile fraction of the Vds exhibited more potency for the ethyl-2-methylpropionate, -damascenone and ethylbutanoate as exemplied by their high odour activity values (OAVs) (Table3). On the other hand, the bound fraction recorded higher OAVs for -damascenone and linalool respectively. Also, the OAVs indicated that hexyl acetate, ethyl-2-methylpropionate, ethylbutanoate, linalool, -damacenone and (Z)-rose oxide contributed to the sweet prune-like aroma of the Vds. Interestingly,

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Table 1 The concentration ofvolatile compounds (free andbound) identied inVitex doniana sweet (gkg1 ofpulp)

Compounds1 LR1 LR2 Free Bound

Alcohols

3-Methyl-but-3-en-1-ol 1209 720 1046 33.0a 570 23.6b

2/3-Methyl-butanol 1213 738 153 11.4a 102 10.6b

(Z)-3-Hexen-1-ol 1389 858 312 17.2a 23 2.0b

Hexan-1-ol 1079 872 60 3.5a 33 1.5b

2,6-Dimethylcyclohexanol 1112 979 tr tr 1-Octen-3-ol 1451 979 tr tr 2-Phenylethanol 1911 1117 2457 151.0a 97 5.9b

Aldehydes

2-Phenylethanal 1037 tr 21 2.1a

Benzaldehyde 1524 1517 tr 35 3.2a

Acids

2-Ethyl hexanoic acid 1129 tr Nd Acetic acid 1428 600 18 2.7a 19 0.8a

Esters

Ethyl-2-methylpropionate 961 758 315 26.0 Nd

Methylbutanoate 981 723 205 16.0a tr Ethylbutanoate 1028 803 604 112.0 Nd

1-Pentyl acetate 1170 919 37 4.3 Nd

Methyl hexanoate 1000 433 45.1 Nd

Butyl butanoate 1218 995 65 5.6 Nd

2-Heptyl acetate 1259 1040 tr tr Hexyl acetate 1270 1014 522 101.6 Nd

(Z)-3-Hexenyl acetate 1325 1007 125 2.5a tr Methyl octanoate 1137 475 96.0a 35 1.5b

Ethyl cinnamate 2167 1469 715 117.0 Nd

Terpenes

Limonene 1185 1030 127 9.3 Nd

(E)--Ocimene 1250 1156 tr Nd Borneol 1253 885 tr tr (Z)-Rose oxide 1337 40 5.0 Nd

(E)--Bergamotene 1415 tr Nd Linalool 1540 1103 5121 107.0a 506 19.4b

-Terpineol 1582 1195 216 5.0a 57 6.7b

Geranial 1715 1277 114 4.5 Nd

Geraniol 1840 341 13.4a 79 8.6b

Ketones

Acetophenone 1067 42 6.0b 437 15.6a

4-Hydroxy-2,5-dimethyl-3(2H)-furanone 2038 1070 50 2.6b 326 15.0a

-Jasmolactone 2176 Nd 186 11.7

Phenol

Guaiacol 1842 1089 Nd 231 14.3

Norisoprenoids

Theaspirane isomer I 1280 Nd tr

Theaspirane isomer II 1308 Nd tr

-Damascenone 1801 1389 tr 21 1.7a

4-Hydroxy--ionol 1601 Nd 162 10

-Ionone 1933 1491 260 12.0a trb

3-Oxo--ionol 1938 Nd 100 12.5

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Table 1 continued

Compounds1 LR1 LR2 Free Bound

4-Oxo--ionol 1943 Nd 141 7.9

Total 13,900 g kg1 3236 g kg1 Alcohols 29.1% 26.1%

Esters 25.2% 1.36% Terpenes 43% 20.1%Ketones 0.66% 29.3%

Nop. 1.91% 13.3%

MeanSD (n=3) with dierent superscript along the same row are signicantly dierent (P<0.05)

LR1, DB-FFAP; LR2, SE-54; tr trace amount (<10gkg1), Nd not detected, Nop norisoprenoids LRI linear retention index on column 1, LR2 linear retention index on column 2

1 Compounds were identied by comparing their retention indices on DB-FFAP and SE-54 columns, their mass spectra, and odour notes were compared with their respective reference odorants data

compounds with high concentration such as 2-phenyl ethanol (2457 g kg1), geraniol and methyl butanoate gave low OAVs. Therefore, their contribution to the aroma note of the Vds can be assumed to be low.

Sensory evaluation of both bound and free odorants of V. doniana sweet revealed distinct aroma characteristics. For instance, while the free fraction was characterised by the owery and fruity notes, the bound fraction exhibited cherry-like, owery, and caramel notes (Fig. 2). However to determine which compounds are responsible for

the perceived aroma notes, a more detailed analysis on aroma models and omission test will be required.

Conclusion

The study has revealed for the rst time the aroma proles of the free and glycosidically bound fractions of V. doniana sweet. In the free fraction, the predominant compounds were the terpenes, alcohols and esters. The glycosidically bound fraction was composed of ketones, alcohols, terpenes and norisoprenoids. Results of the OAVs revealed

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Table 2 Key odorants (free and bound) detected in Vitex doniana sweet

No Compound Odour impression DB-FFAP FD

1 Ethyl-2-methylpropionatea Fruity 961 322 Methylbutanoatea Fruity 981 1283 Ethylbutanoatea Banana-like 1028 16 4 2-Phenylethanalb Honey-like 1037 45 Acetophenonea Cherry-like 1067 5126 Hexan-1-ola Green, blooming 1079 27 2,6-Dimethylcyclohexanolc 112 Nd 8 2-Ethyl hexanoic acida 1129 Nd 9 1-Pentyl acetatea Herbal-like 1170 210 Limonenea Orange-like 1185 16 11 3-Methylbut-3-en-1-ola Slightly apple-like 1209 8 12 2/3-Methylbutanola Solvent 1213 413 Butyl butanoatea Fruity, pineapple 1218 32 14 (E)--Ocimeneb Flowery, blooming 1250 6415 Borneolb Camphor-like 1253 2 16 2-Heptyl acetatea Woody, rum-like 1259 217 Hexyl acetatea Fruity 1270 16 18 (Z)-3-Hexenyl acetatea Fresh, pear-like 1337 8 19 (Z)-Rose oxidea Rose-like 1337 16 20 (Z)-3-Hexen-1-ola Green 1389 8 21 (E)--Bergamoteneb oral 1415 822 Acetic acida Sweaty 1428 4 23 1-Octen-3-ola Mushroom-like 1451 224 Benzaldehydea Almond-like 1521 1625 Linaloola Flowery 1540 16 26 -Terpineola Floral 1582 8 27 4-Hydroxy--ionola Floral 1601 1628 Geraniala Rose-like 1715 8 29 -Damascenonea Flowery 1801 1630 Geraniola Rose-like 1840 1631 Guaiacola Smoky 1842 4 32 2-Phenylethanola Honey-like 1911 16 33 -Iononea Floral, violet-like 1933 4 34 3-Oxo--ionolc Spicy 1938 2 35 4-Hydroxy-2,5-dimethyl-

3(2H)-furanonea

Caramel-like 2038 16

36 Ethyl cinnamatea Flowery, sweet 2167 32

Nd not determined, FD avour dilution

a GC retention and MS data in agreement with that of the reference odorants

b GC retention and MS data in agreement with spectra found in the library

c Tentatively identied by MS matching with library spectra

that while the free volatile fraction of the V. doniana sweet exhibited strong potency for the fruity and oral notes; the bound volatile fraction produced more of owery, caramel and cherry-like notes. In addition, results have shown that ethylbutanoate, -damascenone, ethyl-2-methyl propionate, linalool, hexyl acetate and (Z)-rose oxide contributed highly to the sweet prune-like aroma of V. doniana sweet.

Materials andmethods

Fruit material

Freshly harvested ripe Vitex doniana sweet (purple black in colour) (Fig.3) (300 fruits) grown in Owo, southwest Nigeria, were purchased from a local producer and stored (20 C, 85% RH). The fruits were 2.83.2 cm in length, 1.21.4cm in width and contained one hard conical seed each which is about 1.52.0 cm long and 1.01.2cm wide. Quartering method [24] was used to select fruits for aroma analysis. At harvest, fruit had 10.5o brix and a titratable acidity of 0.86% malic acid equivalent.

Reagents andstandards

Ethanol, methanol and dichloromethane were purchased from Merck (Darmstadt, Germany), while sodium dihydrogen phosphate-1-hydrate,l- (+) -ascorbic acid, and citric acid were obtained from Panreac (Barcelona,

Spain). Sodium uoride and ethyl acetate were purchased from Fluka (Buchs, Switzerland). Almond -glucosidase was obtained from Sigma Chemical (St. Louis, MO). Amberlite XAD-2 resins were purchased from Sigma-Aldrich (Poole, Dorset, UK) and pure water was from a Milli-Q purication system (Millipore, Bedford, MA, USA). An alkane solution (C8C24; 20 mgL1 dichloromethane) was used to calculate the linear retention index (LRI) for each analyte. Other reagents were of analytical grade.

The following reference chemicals: Acetic acid, methyl butanoate, ethyl-2-methyl propionate, ethyl butanoate, 2-ethylhexanoic acid, 3-methylbutanol, (Z)-3-hexen-1-ol, hexanol, octen-3-ol, benzaldehyde, 3-methyl-but-3-en-1-ol, 2-phenylethanol, 1-pentyl acetate, limonene, 3-methylbut-3-en-1ol, acetophenone, butylbutanoate, (E)--ocimene, 2-heptyl acetate, hexyl acetate, (Z)-3-hexenyl acetate, (Z)-rose oxide, (Z)-3-hexenol, (E)--bergamotene, 1-octen-3-ol, linalool, -terpineol, 4-hydroxy--ionol, geranial, geraniol, guaiacol, -damascenone, -ionone, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, ethylcinnamate were from Sigma-Aldrich (St. Louis, MO). Stock standard solutions of 103 or 104 gmL1 of each compound was prepared as described earlier [25].

Fractionation offree aroma compounds ofsweet black plum

Fruit pulp (500g) was blended with 700mL of distilled water. After 30s, the mixture was centrifuged at 3000g

and 4 C for 15 min. The supernatant was ltered through a bed of Celite. The clear Vds juice (300 mL) was applied onto an Amberlite XAD-2 adsorbent in a (302cm) glass column. The column was washed with 250 mL of deionised water and 200 mL of n-pentane/

diethyl ether mixture (1/1 v/v). The eluted extract was

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Table 3 A comparative analysis ofthe aroma potency ofcompounds withavour dilution (FD) values16 inVitex doni

ana sweet

No Compounds Conc.(gkg1fresh fruit) offractions

Threshold (gkg1 ofH2O) [ref.] OAVs

Free Bound Free Bound

1 Ethyl-2-methylpropionate 315 Nd 0.1 [4] 3150 Nd2 Methylbutanoate 205 <10 28 [4] 7 <13 Ethylbutanoate 604 Nd 5 x 102 [4] 120,800 Nd4 Acetophenone 42 437 65 [5] <1 75 Limonene 127 Nd 210 [1] <1 Nd6 Butylbutanoate 65 Nd 100 [2] <1 Nd7 (E)--Ocimene <10 Nd Nd Nd8 Hexyl acetate 522 Nd 2 [4] 261 Nd9 (Z)-Rose oxide 40 Nd 0.5 [1] 80 Nd10 Benzaldehyde <10 35 350 [5] <1 <111 Linalool 5121 506 15 [3] 341 3412 4-Hydroxy--ionol Nd 162 Nd Nd13 Geraniol 79 341 40 [4] 2 914 -Damascenone <10 26 2 x 103 [4] 5000 10,500 15 2-Phenylethanol 2457 97 1000 [4] 3 <116 4-Hydroxy-2,5-dimethyl-3(2H)-furanone 50 326 40 [4] 1 817 Ethyl cinnamate 715 Nd Nd Nd

Nd not detected, OAVs odour activity values[1] Maarse [29], [2] Takeoka etal. [30], [3] Lasekan & Ng [20], [4] Rychlik etal. [31], [5] Buttery etal. [32] OAVs, calculated by dividing concentration with threshold value in water

dried over anhydrous sodium sulphate and concentrated to 1 mL [26]. The concentrated extract (i.e. free fraction of the sweet black plum) was used for the GCMS and GCO analyses. The experiment was carried out in triplicate.

Bound aroma compounds ofthe V. doniana sweet

After the free fraction was obtained from the Amberlite XAD-2 glass column, the glycosidic extract adsorbed on the column was collected by washing it with 250mL of methanol. The obtained extract was dried over

anhydrous sodium sulphate and similarly concentrated as the free fraction. The concentrated bound fraction was re-dissolved in 100 mL of phosphate-citrate buer (0.2M, pH 5.0) and washed (2) with 45mL of n-pentane/diethyl ether (1/1, v/v) to remove any free fraction. One mililiter of an almond -glucosidase solution (5 unit mg1 solid, concentration of 1 unit mL1 buer) was added to the glycosidic extract and incubated overnight at 37 C [27]. The liberated aglycones were extracted with 30 mL of n-pentane/diethyl ether

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Aroma prole determination

Fresh Vds (40 g) were placed inside glass containers (7 cm 3.5 cm) and were orthonasally analysed as described earlier [20]. Reference odorants used were:

Acetophenone (cherry-like), linalool (Flowery), (Z)-rose oxide (rose-like), 4-hydroxy-2,5-dimethyl-3(2H)-fura-none (caramel-like) and hexyl acetate (fruity). Panellists rated the intensities of each descriptor on an unstructured scale from 0 to 10, where 0 = not detectable, 5=weak, and 10=strong. Final results were presented in a web plot.

Statistical analysis

Statistical analyses were carried out with SPSS version 16.0 Windows (SPSS Inc., Chicago, IL). Signicance of dierences between means was tested by one-way analysis of variance (ANOVA). Results were expressed as meanSD (standard deviation) of triplicate analyses.

Acknowledgements

The author is grateful for the extensive nancial support of the Fundamental Research Scheme (No. 5524558) at the University Putra Malaysia.

Competing interests

The author declares that he has no competing interests.

Received: 18 July 2016 Accepted: 14 February 2017

(1/1, v/v) (2). The combined extracts were dried over anhydrous sodium sulphate, ltered and concentrated as described earlier [26]. The concentrated extract was used for the GCMS analysis and the experiment was carried out in triplicate.

GCMS andGCFID analyses

A Shimadzu (Kyoto, Japan) QP-5050A GCMS equipped with a GC-17 A Ver.3, a ame ionization detector (FID) and tted dierently with columns DB-FFAP and SE-54 (each, 30 m 0.32 mm i.d., lm thickness 0.25 m;

Scientic Instrument Services, Inc., Ringoes, NJ) was employed. The gas chromatographic and mass spectro-metric conditions were the same as described previously by Lasekan & Ng, [20]. The HP Chemstation Software was employed for the data acquisition and mass spectra were identied using the NIST/NB575K database.

Gas chromatographyolfactometry

A Trace Ultra 1300 gas chromatograph (Thermo Scientic, Waltham, MA, USA) tted with a DB-FFAP column (30m0.32mm i.d., lm thickness, 0.25m, Scientic

Instrument Services, Inc., Ringoes, NJ) and an ODP 3 olfactory Detector Port (Gerstel, Mulheim, Germany), with additional supply of humidied purge air, was operated as earlier reported by Lasekan etal. [25]. The split ratio between the sniffing port and the FID detector was 1:1. Two replicate samples were snied by three trained panel-lists who presented normalised responses, reproducibility and agreement with one another. The GCO analysis was divided into three parts of 20min and each panellist participated in the sniffing. An aroma note is valid only when the three panellists were able to detect the odour note.

Identication andquantication

The linear retention indices were calculated according to Kovats method using a mixture of normal parafn C6C28 as external references. The identication of volatiles was carried out by comparing their retention indices, mass spectra data and odour notes with those of the reference odorants, literature data or with the data bank (NIST/NB575K). Quantitative data were obtained by relating the peak area of each odorant to that of the corresponding external standard and were expressed as gkg1.

Aroma extracts dilution analysis (AEDA)

The extracts of the free and bound fractions were diluted step wise twofold with dichloromethane by volume to obtain dilutions of 1:2, 1:4, 1:8, and 1:16 and so on. Each obtained dilution was injected into the GCO. The highest dilution in which an aroma compound was observed is referred to as the FD factor of that compound [28].

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