Odour-active compounds in baby banana Fruit (Musa acuminata AA Simmonds cv. Bocadillo)

ABSTRACT

The contribution of individual compounds to the typical aroma of baby banana cv. Bocadillo fruit would be useful to assess aroma changes caused in the course of fruit processing. Potent odourants in baby banana fruit were determined by combined headspace solid-phase microextraction GC–Olfactometry (HS-SPME-GC–O), solvent-assisted flavour evaporation, gas chromatography−olfactometry (GC–O), gas chromatography-mass spectrometry (GC-MS), aroma extract dilution analysis (AEDA) and calculation of odour activity values. Twenty-three constituents were considered as potentially odour-active compounds contributing to the typical fruit aroma, from which 2-pentyl acetate, 3-methylbutyl acetate, 3-methylbutyl butanoate, 3-methylbutyl 3-methylbutanoate, hexanal, (E)-2-hexenal and 2-pentyl 3-methylbutanoate were the most important contributors to the aroma. HS-SPME-GC-O has a great potential as fast and simple tool to control aroma quality of baby banana fruit.

KEYWORDS:

• Aroma extract dilution analysis
• Baby banana
• Gas chromatography–olfactometry
• Odour activity values
• Solid-phase microextraction

Introduction

Banana (Musa spp., family Musaceae) is a tropical fruit with a pleasant flavour, widely consumed throughout the world. According to the consumption, it is divided in two main groups: the proper banana or dessert banana, which is eaten raw, and plantain being processed by cooking before consumption. Most cultivars are derived from two diploid wild species, M. acuminata (A genome) and M. balbisiana (B genome). Edible clones are considered as to the relative contribution of M. acuminata and M. balbisiana. Most are triploids, although some diploids and tetraploids are also edible.^[1]

Numerous studies have been performed on the aroma constituents of banana.^[2-6] The volatile profile of banana has been established using different extraction and analytical methods on different cultivars from Philippines and Taiwan,^[7,8] Ivory Coast,^[9] Costa Rica and Canary Islands,^[10] Honduras,^[11] Madeira,^[12,13] Cuba,^[14] Guadeloupe,^[5] Thailand^[15] and Brazil.^[16] As a result of these studies, nearly 250 volatile compounds have been identified for several fresh and processed banana products.^[17] Regardless of the isolation method used (e.g., static or dynamic headspace, solvent extraction, simultaneous distillation-extraction [SDE], or solid-phase microextraction [SPME]), the major volatile compounds are generally esters (3-methylbutyl, 2-methylpropyl, and other uncommon esters), alcohols and ketones; however, only some of them have been recognised as banana flavour contributors.

At present, it has become clear that in order to understand a fruit aroma, the analysis of volatile composition alone is not sufficient. It is also necessary to examine closely the odour-active compounds isolated by adequate techniques as well as understand their roles in the overall aroma profile of the fruit. Some studies have determined the sensorial contribution of each volatile compound to the aroma of Cavendish banana. The ester fraction contributes to fruity note.^[5,7] The 3-methylbutyl esters derived from acetic, butanoic and 3-methylbutanoic acids were found to be key components of the fruity odour.^[7,9,11] Carbonyl compounds and alcohols contribute to the green-woody and herbal note.^[2,7] Using SDE, 139 volatile compounds were identified in the volatile compounds in cv. Bocadillo grown in Colombia, and by calculating odour activity values (OAVs), 34 of them could be considered odour-active compounds in this banana cultivar.^[18] Recently, 31 volatiles were found as odour-active compounds and contribute to the typical aroma of banana cv. Giant Cavendish by applying headspace SPME and SDE, combined with GC-FID, GC-MS, aroma extract dilution analysis (AEDA), and OAVs.^[19] The contribution of individual compounds to the typical aroma would be to assess aroma changes caused in the course of fruit processing, that is, from the post-harvest storage until thermal procedures by industrial processing.

The baby banana fruit (Musa acuminata AA Simmonds cv. Bocadillo), also known as Bocadillo, Finger Banana, Ladyfinger Banana, Nino Banana, Murapo and Orito, is one of the sweetest and smallest banana cultivars (10–12 cm in length). It is cultivated mainly in Colombia, Ecuador, Costa Rica, Venezuela and Kenya. This cultivar represents $244 million global exportations of Colombia and currently ranks third in the exportation in comparison with coffee and flowers.^[20] However, detailed information about the aroma-active compounds in baby banana fruit is still lacking.

The aim of the present study was to identify and rank, according to their potential aromatic importance, the odourants responsible for the flavour characteristics of baby banana fruit. In order to do that, two different ranking strategies have been followed: a first semi-quantitative strategy based on the direct GC–Olfactometry of the volatiles extracted by headspace SPME, and a second one combining solvent-assisted flavour evapouration (SAFE) and quantitative GC in order to calculate OAVs. This is the first report on the aroma-active compounds in fresh banana fruit using SAFE technique.

Materials and methods

Fruits

Banana fruits (Musa acuminata AA Simmonds cv. Bocadillo), selected with a similar ripening degree, were harvested in a commercial plantation located in Antioquia, Colombia from the 2016 crop season and immediately transported to the laboratory by Nativa Produce SAS Co. Three batches of four ripe fruits in each were cut and rapidly homogenised in a commercial homogeniser. These batches were used for subsequent analyses.

Chemicals and reagents

Pure reference standards for GC analyses were purchased from Sigma–Aldrich (St. Louis, MO), except 2-pentyl acetate, 2-pentyl 3-methylbutanoate and 2-heptyl butanoate, which were synthesised by esterification in the laboratory. Hexyl (E)-3-hexenoate was generously supplied by Dallant S.A. (Barcelona, Spain). An n-alkane solution (C₈–C₃₂) was acquired from Sigma–Aldrich (St. Louis, MO). To this solution, a mixture of C₅–C₇ n-alkanes was added. Anhydrous sodium sulphate, sodium chloride, diethyl ether and methyl nonanoate were purchased from Merck (Darmstadt, Germany).

Standard chemical analysis

Soluble solids, total acidity (as malic acid) and pH value were performed on the fruit pulp according to standard methods.^[21]

Headspace solid-phase microextraction analysis

Volatile compounds from the headspace of fruit homogenates were isolated using 50/30 μm DVB/CAR/PDMS fibres (Supelco Park, Bellefonte, PA, USA). This fibre was selected for the analysis because it trapped the volatile compounds from banana.^[19] Fibres were conditioned prior to each sampling step and immediately used after conditioning. SPME extractions conditions were: 3 g of stirred homogenate, 3 mL of Milli-Q water and 1 g NaCl contained in a 15 mL-vial sealed with a PTFE-lined screw cap, and 40°C for 30 min. Extraction was made by triplicate.

Isolation of volatile compounds by solvent-assisted flavour evapouration

Homogenised pulp (200 g) was mixed with 300 ml of Milli-Q water (previously saturated with NaCl) and 100 μL of a standard solution of methyl nonanoate (2 mg/mL). The mixture was extracted with recently redistilled diethyl ether (2 × 100 mL) on an autoshaker at room temperature for 10 min. After centrifugation (6000 min⁻¹; 5 min), the supernatants were combined, and the residue was discarded. The organic layer was isolated in a separatory funnel, and the aqueous layer was discarded. The organic phase was subjected to SAFE^[22] at 40°C, and the organic phase was dried over anhydrous Na₂SO₄ and concentrated to 0.6 mL using a Vigreux column (15 cm × 1 cm i.d.) and then to 0.2 mL with a gentle nitrogen stream. The volatile compounds isolated by SAFE were evaluated by two experts by smelling a drop of the extract onto a cardboard smelling strip as done by perfumers. After evapouration of the solvent, both experts agreed that the isolate evoked the characteristic odour of the fruit, thereby indicating that the method used was appropriate.

GC-FID and GC-MS analysis

A Konik 4000A gas chromatograph (Konik, Barcelona) with a flame ionisation detector (FID) was used. The capillary columns were DB-5 ms and DB-Wax (each, 30 m × 0.25 mm × 0.25 μm; J & W Scientific, Folsom, CA, USA). The oven temperature was held at 50°C for 2 min and then raised to 250°C at 4°C/min and held for 10 min. Hydrogen was used as a carrier gas at a flow rate was 1 mL/min. Injector and detector temperatures were 250°C. For the SAFE extracts, 1 μL was injected in 1:10 split mode, and for SPME extracts, splitless mode (2 min) was applied. The retention times of a series of n-alkanes (C₅–C₃₂) was used to calculate the retention indices for all identified compounds and for reference standards. Quantitative analyses of the odour-active compounds exhibiting dilution factor higher than 8 were performed by internal standard method. Methyl nonanoate was added as internal standard to the homogenised pulp before extraction by SAFE distillation. To determine the response factor for each compound, calibration lines were made using a series of solutions of varying nominal concentrations containing each analyte (IS/analyte, 1:4 until 4:1), where the slope was assumed as the response factor. An identical amount of the internal standard was added to each solution, and the corresponding chromatograms were obtained.^[23] All data were obtained by triplicate.

GC-MS analyses were performed on a Hewlett–Packard 6890N series II (Agilent, Palo Alto, CA) gas chromatograph with the same fused capillary columns, temperature programme, and helium carrier gas flow rate as in GC-FID. EIMS, electron energy, 70 eV; ion source and connecting parts temperature, 250°C. The acquisition was performed in scanning mode (mass range m/z 35–400 u). Compounds were preliminarily identified by use of NIST 05, Wiley 6, NBS 75k, Adams 2001, and in-house Flavorlib libraries, and then the identities of most were confirmed by comparison of their linear retention indices with those of reference standards or with published data.^[24]

Direct SPME-GC–O

The procedure was performed according to an earlier study.^[19] GC–O analyses were achieved with a gas chromatograph Konik 4000A instrument (Konik, Barcelona). The sniffing port was a cylindrically shaped aluminium device with a bevelled top and a central drill hole connected to the capillary. Oven temperature was held at 250°C, carrier gas (hydrogen) flow rate was 1 mL/min and nitrogen (30 mL/min) was used as makeup gas. The SPME fibre was introduced into the GC port (splitless mode for 2 min, injector temperature at 250°C). Because no chromatographic separation was carried out by the short capillary, volatile compounds arrived simultaneously at the sniffing port. For each SPME analysis, a trained panel of three assessors perceived and evaluated the overall odour. Then they evaluated with the direct GC–O device the SPME extract to check its similarity to the reference using a 10 cm scale ranging from 0 (close to the reference) to 10 (far from the reference). Assessors had to smell the reference before each sample evaluation.

Gas chromatography–olfactometry analysis of SPME extract

The odour-active compounds captured on SPME fibre were analysed by GC–O on a Hewlett–Packard 6890 N equipped with a DB-Wax column (30 m × 0.25 mm × 0.25 μm; J & W Scientific, USA), connected via two fused silica capillaries (25 cm × 0.25 mm i.d.) to a FID and an ODO II sniffing port. The SPME fibre was desorbed for 5 min into a 0.75 mm i.d. liner in the injection port at 250°C. Operating conditions were the same as described before for GC-FID. The GC effluent was split 1:1 between the FID and the sniffing port (both at 250°C). A non-humidified airflow (30 mL/min) was used as the makeup gas. Three assessors evaluated the effluents separately. For each odour stimulus, the assessors recorded the detection time and gave an odour description. GC–O frequency analysis was performed following the methodology described earlier.^[25] Odour quality and detection times were marked in the chromatogram. Analyses were in triplicate for each assessor. Compounds which were detected with the same descriptors, at least three times, were considered as odour-active compounds.

Gas chromatography–olfactometry analysis of SAFE extract

GC–O analyses were performed with a Hewlett–Packard 6890 N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) and a DB-Wax column (30 m × 0.25 mm × 0.25 μm; J & W Scientific, USA), connected with two fused silica capillaries (25 cm × 0.25 mm i.d.) to an FID and an ODO II sniffing port (SGE International Pty. Ltd., Ringwood, Australia). The temperature programme and carrier gas flow rate were identical to the GC-FID method. Detected odours (quality and detection times) were marked in the chromatogram. The GC–O analyses were performed by two assessors.

Aroma extract dilution analysis

The extract obtained by SAFE was stepwise diluted with diethyl ether (1 + 1) until no odourant was detectable by sniffing of the high dilution. GC–O was performed with 1 μL aliquots, and the FD factors obtained by two assessors were averaged. The odour activity of each compound, which is expressed as flavour dilution (FD) factor, was determined as the greatest dilution at which that compound could still be detected.

Odour detection threshold determinations

Odour detection thresholds for some compounds were determined by a previously described multiple paired comparison test.^[26] The statistical analyses for determining the odour detection thresholds involved calculating the concentration corresponding to 50% positive responses from the total judgements. The calculation was made from the linear regression of percentage detection against log concentration. The 95% confidence limit calculated for the threshold values was used as a measure of error.

Results and discussion

The fruit pulp had the following general characteristics: soluble solids 24.5% ± 0.3%, total acidity 0.25% ± 0.01% (as malic acid), and pH 5.11 ± 0.02, which are typical of a mature baby banana cv. Bocadillo fruit. Taking into account that DVB/CAR/PDMS fibre provided the most representative odour for banana according to an earlier study,^[19] the overall aroma extraction efficiency was assessed by direct SPME-GC–O. The similarity on a scale 1–10 determined for the SPME extract the overall odour with respect to the reference sample was 8.9 ± 0.5. Therefore, this fibre was considered adequate to capture the odour to that of the fresh fruit.

GC–O was applied to DVB/CAR/PDMS extract to find odourant zones in the olfactogram and then detect the aroma compounds potentially responsible for these odours. The odours detected by the assessors, together with the compounds tentatively identified as being responsible for those odour impressions, are given in Table 1. Eighteen compounds were elucidated as contributing to the overall baby banana aroma. Among them, 2-pentyl acetate (banana), 3-methylbutyl acetate (ripe banana), 3-methylbutyl butanoate (fruity, banana-like) and 3-methylbutyl 3-methylbutanoate (fruity, banana peel) were the compounds with higher detection frequencies in the SPME extract. These last three esters were the most abundant in the gas chromatogram and also had high detection frequencies.

Table 1. Baby banana odour-active compounds identified by SPME-GC–O.

Compound	RIa	RIp	Odour description	Detection frequency
Ethyl acetate	615	884	Ethereal-fruity, sweet	3
2-Pentanone	688	983	Banana	6
2-Methylpropyl acetate	770	1018	Ethereal, rum-like	4
Hexanal	803	1080	Green greasy	7
2-Pentyl acetate	854	1075	Banana	9
(E)-2-Hexenal	858	1220	Intense green-fruity	8
3-Methylbutyl acetate	881	1125	Ripe banana	9
2-Methylpropyl butanoate	958	1161	Fruity	4
3-Methylbutyl butanoate	974	1256	Fruity, banana-like	9
2-Pentyl 3-methylbutanoate	1088	1274	Banana	8
3-Methylbutyl 3-methylbutanote	1106	1294	Fruity, banana peel	9
2-Heptyl butanoate	1207	1401	Fruity green	6
Hexyl (E)-3-hexenoate	1218	1414	Fruity green	7
Hexyl 3-methylbutanoate	1244	1425	Sweet banana	6
3-Methylbutyl hexanoate	1254	1465	Fruity	5
(Z)-4-Hepten-2-yl 3-methylbutanoate^a	1274	–	Banana-like	6
Eugenol	1366	2146	Spicy	7
Elemicin	1560	2239	Spicy	8

RI_a and RI_p = retention index in DB-5 ms and DB-Wax columns.

^bTentative identification (only by matching LRI and mass spectra from libraries).

The use of SPME combined with GC–O does not provide immediate information about the contribution of all odourants to the overall aroma. The major reason is that only the high and medium volatile compounds are isolated by the headspace SPME. Consequently, to investigate the contribution of each volatile compound to baby banana aroma, the compounds had to be quantified in the fruit homogenate by means of SAFE. To the best of our knowledge, there are not reports that have employed SAFE technique until this date.

Addition of salt to break enzymatic reactions and the use of non-elevated temperatures during the SAFE workup was selected to avoid artefact formation and compound degradation. As a result, the smell of the SAFE extract still clearly represented the typical aroma characteristics of the fresh baby banana fruits. Aroma isolate was submitted to AEDA, which resulted in 26 odour-active compounds with FD ≥ 8, which have been arranged in retention index order from DB-5 ms column (Table 2). Positive identifications were based on matching retention indices and mass spectra with those of standard reference compounds analysed under identical experimental conditions. The volatiles with the highest FD values were 2-pentyl acetate, 3-methylbutyl acetate and 3-methylbutyl butanoate. These esters have been reported as important odourants in different banana cultivars^{[5,7,9,11,19]} Pino et al.^[18] reported 3-methylbutyl acetate and 3-methylbutyl butanoate as odour-active compounds in this banana cultivar by calculating only OAVs. Other odourants with FD factor = 512 were two aldehydes (hexanal and (E)-2-hexenal), 2-pentyl 3-methylbutanoate and 3-methylbutyl 3-methylbutanoate. Only the aldehydes were previously found as odour-active compounds in this banana cultivar.^[18]

Table 2. Odour-active (FD ≥ 8) volatile compounds detected in SAFE extract from baby banana cv. Bocadillo.

Compound	Odour threshold (μg kg^−1)	Conc ± SD (μg kg^−1)	Odour description	FD^a	OAV^b
Ethyl acetate	5000^c	2800 ± 140	Ethereal-fruity, sweety	8	<1
2-Pentanone	50^d	4000 ± 176	Banana, sweety	128	80
3-Methylbutan-1-ol	220^e	5940 ± 292	Fruity-winey	32	27
2-Methylpropyl acetate	66^f	396 ± 16	Ethereal	16	6
Hexanal	2.4^e	698 ± 35	Green grassy	512	291
2-Pentyl acetate	4^g	3000 ± 179	Banana	1024	750
(E)-2-Hexenal	17^f	2990 ± 161	Green-intense fruity	512	176
(Z)-3-Hexen-1-ol	70^f	210 ± 11	Fresh grass	8	3
3-Methylbutyl acetate	2^c	2000 ± 74	Ripe banana	1024	1000
2-Heptanone	140^c	978 ± 32	Fruity-spicy	16	7
2-Heptanol	400^h	235 ± 9	Fruity, pungent	8	<1
2-Methylpropyl 2-methylpropanoate	30^f	58 ± 2	Sweet-fruity	8	2
2-Methylpropyl butanoate	30^f	689 ± 27	Fruity	64	23
Butyl butanoate	100^f	300 ± 13	Sweet-fruity	32	3
Butyl 3-methylbutanoate	17^f	103 ± 5	Ethereal-fruity	32	6
3-Methylbutyl butanoate	0.1^c	894 ± 36	Banana	1024	9000
2-Pentyl 3-methylbutanoate	3^g	695 ± 24	Banana	512	233
3-Methylbutyl 3-methylbutanoate	65^h	974 ± 41	Banana peel	512	15
2-Heptyl butanoate	23^g	295 ± 14	Fruity green	256	13
Hexyl (E)-3-hexenoate	60^g	776 ± 27	Fruity green	256	13
Hexyl 2-methylbutanoate	22^f	197 ± 9	Fruity, pungent	32	9
Hexyl 3-methylbutanoate	20^g	1000 ± 441	Sweet banana	128	50
3-Methylbutyl hexanoate	51^g	964 ± 32	Fruity	64	19
(Z)-4-Hepten-2-yl 3-methylbutanoate^j	nd^i	1400 ± 602	Banana-like	64	nd
Eugenol	6^c	194 ± 9	Spicy	128	33
Elemicin	100^h	213 ± 14	Spicy	32	2

^aFlavour dilution factor. ^bOdour activity values were calculated by dividing the concentrations by the respective odour threshold. ^cReference 26. ^dReference 27. ^eReference 28. ^fReference 29. ^gDetermined in the present work. ^hReference 30. ⁱno datum. ^jtentative identification (only by matching LRI and mass spectra from libraries).

The OAVs were calculated for the odourants with higher FD values (see Table 2). Two of the odourants identified as potentially relevant by AEDA were found with OAVs<1, and therefore, they should not contribute to baby banana flavour. The potentially important odourants obtained with the odour activity approach is a refinement of that provided by the AEDA and corrects some of the limitations of the AEDA technique.

Regarding correspondences between SPME-GC–O and OAV approaches, SPME-GC–O has been very effective, since with a very small effort, it was possible to identify six out of the seven most important odourants, attending to the OAV criteria. Only hexanal ranked high attending to OAV did not have a high SPME-GC–O score. On the other hand, SPME-GC–O seems to overscore only one odourant (elemicin) in comparison with OAV criteria. The former had a higher GC–O detection frequency and an OAV of only two. AEDA ranking was definitively very different to that provided by OAV or by SPME-GC–O. The definitive role played by the odourants will have to be finally judged using different reconstitution techniques and sensory evaluation. Only then it will be possible to make an exact judgement of the discrepancies observed between the two ranking procedures.

Conclusion

This study revealed potent odourants that are responsible for the overall aroma of baby banana cv. Bocadillo fruit by application of aroma extract dilution analysis and odour activity values. A total of 23 odourants were considered as potentially odour-active compounds, from which 2-pentyl acetate, 3-methylbutyl acetate, 3-methylbutyl butanoate, 3-methylbutyl 3-methylbutanoate, hexanal, (E)-2-hexenal, and 2-pentyl 3-methylbutanoate were the most odour-active compounds. The high similarity of results between the OAV and HS-SPME-GC–O approaches suggests that the latter has a great potential as a fast and simple tool to control aroma quality of baby banana fruit.