Food Chemistry 145 (2014) 496–504

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Characterisation of metabolic profile of banana genotypes, aiming at biofortified Musa spp. cultivars Cristine Vanz Borges a,1,2, Vanusia Batista de Oliveira Amorim b,3, Fernanda Ramlov c,4, Carlos Alberto da Silva Ledo b,3, Marcela Donato c,4, Marcelo Maraschin c,4, Edson Perito Amorim b,⇑,1 a b c

Federal University of Recôncavo da Bahia, P.O. Box 44380-000, Cruz das Almas, Bahia, Brazil Embrapa Cassava & Fruits, P.O. Box 44380-000, Cruz das Almas, Brazil Federal University of Santa Catarina, Plant Morphogenesis and Biochemistry Laboratory, P.O. Box 476 88049-900, Florianopolis, Brazil

a r t i c l e

i n f o

Article history: Received 15 February 2013 Received in revised form 19 June 2013 Accepted 12 August 2013 Available online 22 August 2013 Keywords: Musa spp. Bioactive compounds Biofortification Functional foods

a b s t r a c t The banana is an important, widely consumed fruit, especially in areas of rampant undernutrition. Twenty-nine samples were analysed, including 9 diploids, 13 triploids and 7 tetraploids, in the Active Germplasm Bank, at Embrapa Cassava & Fruits, to evaluate the bioactive compounds. The results of this study reveal the presence of a diversity of bioactive compounds, e.g., catechins; they are phenolic compounds with high antioxidant potential and antitumour activity. In addition, accessions with appreciable amounts of pVACs were identified, especially compared with the main cultivars that are currently marketed. The ATR-FTIR, combined with principal components analysis, identified accessions with distinct metabolic profiles in the fingerprint regions of compounds important for human health. Likewise, starch fraction characterisation allowed discrimination of accessions according to their physical, chemical, and functional properties. The results of this study demonstrate that the banana has functional characteristics endowing it with the potential to promote human health. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Brazil is the fifth largest producer of bananas in the world. In 2012, it produced approximately 6.83  106 ton an area of 520,000 hectares (IBGE, 2013). However, there are few cultivars, adapted to commercial exploitation, with agronomic potential and tolerance to pests and diseases that bear organoleptic fruits with good post-harvest characteristics and nutritional and functional value. The Banana Active Germplasm Bank of Embrapa Cassava & Fruits holds approximately 400 accessions and is recognised as a source of genetic variability of interest for the selection of genotypes of Musa spp. that are rich in functional compounds; this germ bank seeks to introduce an important goal to banana breeding programmes, i.e., the biofortification of the banana (Amorim et al., 2011). ⇑ Corresponding author. Tel.: +55 75 13312 8058. E-mail addresses: [email protected] (C.V. Borges), [email protected] (Vanusia Batista de Oliveira Amorim), [email protected] (F. Ramlov), [email protected] (Carlos Alberto da Silva Ledo), [email protected] (M. Donato), [email protected] (M. Maraschin), [email protected] (E.P. Amorim). 1 C.V. Borges and E.P. Amorim contributed equally to this work. 2 Tel./fax: +55 75 3621 2002. 3 Tel.: +55 75 13312 8058. 4 Tel.: +55 48 3721 4812; fax: +55 48 3721 5335. 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.041

Recent research indicates that deficiency in micronutrients, especially in vitamin A, affects millions of people in developing countries (WHO, 2012). Vitamin A is obtained from foods through the consumption of carotenoids known as pro-vitamin A. There are several types of carotenoids in fruits and vegetables, and approximately 50 carotenoids have been shown to have pro-vitamin A activity. However, b-carotene is the most important of these carotenoids and the most abundant in food, followed by a-carotene and b-cryptoxanthin, which have half of the vitamin A activity of b-carotene (Rodriguez-Amaya, 2001). More recently, studies on the provitamin A carotenoid contents of the banana have found that commercial cultivars, especially those of the Cavendish subgroup, do not contain significant amounts of these compounds (Amorim et al., 2011; Davey, Bergh, Markham, Swennen, & Keulemans, 2009; Englberger et al., 2010; Mattos, Amorim, Cohen, Amorim, & Silva, 2010). However, genotypes with high levels of these pigments have been detected, e.g., ‘Bantol Red’, ‘Pusit’, ‘Iholena Lele’, ‘Henderneyargh’, ‘Katimor’, ‘Chek Porng Mean’ (Davey et al., 2009), Micronesian Fe’i cultivars ‘Utin lap’ and ‘Utinwas’ (Englberger et al., 2006), ‘Jaran’, ‘Malbut’ (Mattos et al., 2010), ‘Jari Buaya’, ‘Khai’ and ‘NBA-14’ (Amorim et al., 2011), which demonstrates that these traits can be successfully improved in breeding programmes. Phenolic compounds, e.g., flavonoids and phenolic acids, belong to an important class of secondary metabolites, due to their recognised antioxidant activity; this property confers food quality on the

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fruit and provides potential benefits to human health (Amorim et al., 2011). This class of compounds is also relevant to the biofortification of genotypes of Musa spp. Antioxidants neutralise free radicals and inhibit the initiation chain or interrupt the chain of propagation of oxidative reactions, converting free radicals into less harmful molecules and repairing oxidative damage in human cells. As is the case for carotenoids, phenolic compounds are not found in abundance in the Cavendish commercial cultivars (Vijayakumar, Presannakumar, & Vijayalakshmi, 2008). However, several studies have identified banana genotypes in germplasm banks with outstanding levels of these secondary metabolites and great diversity of content among accessions (Amorim et al., 2011; Sulaiman et al., 2011), which demonstrates the potential of bananas to be biofortified through breeding programmes. The main constituent of banana pulp is starch, a polysaccharide that is a major component of the human diet. There appears to be a consensus in the literature that increased proportions of plant polysaccharides in the diet, including starch, have positive effects on human health. The structural arrangement and relative contents of starch constituents in the granules, namely amylose and amylopectin, define the functionality of starch and affect its qualitative attributes and the half-life of the fruit after harvesting. The present work assumes, as a working hypothesis, a relationship between genetic variability and the profile of the carotenoidic composition and phenolic compounds of the accessions of the banana from Active Germplasm Bank of Embrapa Cassava & Fruits. This study aimed to determine the profile of carotenoids and phenolic acids in pulp fruits of banana genotypes, in connection with the programme for the selection of superior genotypes for breeding, aiming at generating biofortified cultivars of Musa spp. In addition, descriptive and classificatory models were built, based on unsupervised chemometric methods applied to the set of spectral data of mid-infrared vibrational spectroscopy (FTIR). High-performance liquid chromatography was used as an analytical strategy for the discrimination of accessions of interest. Finally, the ratio of crystalline to amorphous phases of the starch granules was determined, based on the FTIR spectral data.

2. Materials and methods 2.1. Collection and preparation of samples Bunches from 29 accessions of banana from different genomic groups maintained in the Active Germplasm Bank of Embrapa Cassava & Fruits (Table 1) were used to determine the phenolic contents, carotenoid profiles, and structural characteristics of the starchy fraction by Fourier transform mid-infrared vibrational spectroscopy (FTIR). These accessions were selected from the results of Amorim et al. (2011). When the fruits reached the sixth stage of maturation (completely yellow), they were cut into slices (5 cm) and dried in an oven (45 °C, constant weight). The dried biomass was ground manually in the presence of liquid N2 into banana pulp flour. The compounds of interest were then extracted. Three replicates were performed for each banana accession.

2.2. Extraction and analysis of phenolic compounds and total flavonoids Samples (1 g dry weight) of banana pulp flour were added to 10 ml of distilled-deionised water and incubated (25 °C, 30 min) with agitation to extract the compounds of interest. The aqueous extracts were recovered by filtration on cellulose membranes under vacuum.

Table 1 Accessions of banana belonging to the Active Germplasm Bank of Embrapa Cassava & Fruits. Accessions

Ploidy level

Subgroup/subspecies

Origin

028003-01 Jari Buaya Lidi Jaran Pipit Malbut Tuu Gia F3P4 M48 Nanica Wasolay Caipira Nam Highgate Gros Michel Orotawa Williams Torp Thap Maeo Saba Champa Madras Figo Cinza Bucaneiro Calypso Ambrosia Tropical

AA(C) AA(C) AA(C) AA(C) AA(C) AA(C) AA(C) AA(H) AA(H) AA(C) AAA AAA AAA AAA AAA AAA AAA AAA AAB ABB ABB ABB AAAA AAAA AAAA AAAB

(Tuu Gia  Calcutta 4)

Brazil Malaysia Indonesia Indonesia Indonesia Papua New Guinea Hawaí Ecuador Ecuador Brazil Papua New Guinea Brazil Tailand Unknown Brazil France Belgium Papua New Guinea Brazil Costa Rica France Brazill Santa Lucia Santa Lucia Santa Lucia Brazil

Porp Maravilha Teparod

AAAB AAAB ABBB

ssp. burmannica

Hybrid Hybrid Cavendish Ibota

Gros michel Cavendish

Saba Bluggoe (Highgate  2n) (Highgate  2n) (Highgate  2n) (Yangambi n.2  M53)

Papua New Guinea Brazil Tailand

C: cultivated; H: hybrid.

Aliquots (1 ml/sample) of the aqueous extract were used to determine the total phenol content, using the Folin–Ciocalteau reagent, as described by Randhir, Preethi, and Kalidas (2002). The concentrations were calculated with the help of an external gallic acid (Sigma) standard curve (y = 1.7219x, r2 = 0.99) and expressed as mg of gallic acid equivalents per gramme of flour. The samples of banana pulp flours (1 g dry weight) were added to 10 ml of methanol and incubated at 25 °C/30 min in the absence of light for the extraction of flavonoid compounds. The extracts were filtered on cellulose supports under vacuum, concentrated under reduced pressure, and resolubilised in 5 ml of methanol. A sample volume of 0.5 ml was added to 2.5 ml of ethanol and 0.5 ml of aluminium chloride (2% w/v in methanol), followed by incubation (1 h) and reading of the absorbance of the extracts (420 nm). The quantification of total flavonoids was carried out using the quercetin standard curve (y = 0.009x, r2 = 0.99), and the values were expressed as the means ± standard deviation of three consecutive readings of the samples. Aliquots (10 ll) of the aqueous extract of 9 accessions of banana plants, selected from among the 29 accessions for their higher content of total phenolic compounds and flavonoids (UV–visible spectrophotometry), were injected into a liquid chromatography (LC10A Shimadzu) equipped with a reversed-phase column (C18 Shim-Pack CLC-ODS, 250 mm  4.6 mm, 5 lm Ø–40 °C) coupled with a pre-column (C18 Shim-Pack CLC-ODS, 30 mm  4.6 mm, 5 lm Ø) and a UV–visible spectrophotometric detector (k = 280 nm). Elution used H2O:AcOH:n-BuOH (350:1:10, v/v/v) as a mobile phase at a flow rate of 0.8 ml/min. The identification of compounds of interest was performed by comparing the retention times of samples with those of standard compounds (gallic acid, epicatechin, gallocatechin, and 3,4 dihydroxybenzoic acid) and by co-chromatography under the same experimental conditions. The quantification of phenolic acids was performed using

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external standard curves of gallic acid (y = 19,466x, r2 = 0.99), gallocatechin (y = 79,509x, r2 = 0.99) and protocatechuic acid (y = 14412x, r2 = 0.99). The results were expressed in lg per 100 g of pulp (dry weight) and refer to the average of three consecutive injections for each sample (n = 3). 2.3. Extraction and analysis of carotenoid compounds The samples were prepared according to Aman, Carle, Conrad, Beifuss, and Schieber (2005), with modifications relevant to the extraction of carotenoids. The samples (1 g) were added to 10 ml of hexane:acetone solution (1:1, v/v) containing 1 ml of butylated hydroxytoluene (1 mg BHT/100 ml of organosolvent), incubated (1 h, in the absence of light) and filtered on a cellulose support under vacuum; the supernatant was recovered and concentrated in a rota-evaporator. The organosolvent extract was resolubilised in 3 ml of hexane and centrifuged (9920.06g). The supernatant was recovered and concentrated under nitrogen gas streaming. Aliquots (10 ll) of the organosolvent extracts of 12 of the 29 accessions of banana plants selected for their higher content of total carotenoid compounds (>0.25 lg/g dry weight, UV–visible spectrophotometry – data not shown) were injected into the liquid chromatography (LC-10A Shimadzu) system equipped with a thermostatted (35 °C) C18 reversed-phase column (Vydac 201TP54, 250 mm  4.6 mm, 5 lm Ø) coupled to a pre-column (C18 Vydac 201TP54, 30 mm  4.6 mm, 5 lm Ø) and a spectrophotometric detector (450 nm). Methanol:acetonitrile (90:10, v/v) was used for elution at a rate of 1 ml/min. The identification of compounds of interest was carried out via co-chromatography and comparison of retention times of samples with those of standard compounds (lutein, zeaxanthin, a-carotene, and trans/cis-b-carotene, Sigma–Aldrich, USA) run under the same experimental conditions. The quantification of carotenoids was carried out using external lutein (y = 7044x, r2 = 0.99) and b-carotene (y = 1019x, r2 = 0.99) standard curves, employing the values of the peak areas of interest for the calculations of the analyte concentrations. The results were expressed in lg per gramme of pulp (dry weight) and refer to the average of three consecutive injections per sample (n = 3). 2.4. Determination of the retinol activity equivalent and vitamin A Pro-vitamin A carotenoids have distinct types of vitamin A activity, due to differences in their chemical structures. The concept of retinol activity equivalent considers that 1 lg of retinol, 12 lg of t-BC, or 24 lg of other pro-vitamin A carotenoids corresponds to one retinol activity equivalent (Yeum & Russel, 2002). The conversion factors for other possible isomers of t-BC and tAC are not known; therefore, for all these compounds (if present), we utilised a conversion factor of 24:1, per lg of ingested carotenoid (Davey et al., 2009). On the basis of these conversion factors, it is possible to calculate RAEs and thus the net vitamin A nutritional value of the fruit from each accession. 2.5. Chemical structural characterisation of the flour samples by Fourier transform mid-infrared vibrational spectroscopy (FTIR) Samples of banana pulp flour (10 mg/sample) and standards of amylose and amylopectin (Sigma–Aldrich) were submitted to FTIR vibrational spectroscopy in Bruker IFS 55 equipment with a glycerin-sulphate detector (DTGS) and attenuated total reflectance accessories (ATR, Golden Gate). One hundred and twenty-eight scans per sample were collected in spectral windows of 4000– 500 waves cm 1 at a resolution of 4 waves cm 1. Three spectra were collected for each sample. Spectral data were analysed with the OPUS software system (Bruker BioSpin, version 5.0), taking into

account the delimitation of the spectral window of interest (3000–600 waves cm 1), the correction of the baseline, the normalisation of the signal/noise ratio, and smoothing. The values of the absorbances of the infrared bands at 1047 and 1022 waves cm 1 were collected after the processing of the spectra; the heights of the absorption bands of interest (referenced at baseline) were used to calculate the ratio between the crystalline and amorphous phases (i.e., R 1047/1022) of the starch granules of the accessions of Musa spp. (Kuhnen et al., 2010; Van Soest, Tournois, Wit de, & Vliegenthart, 1995). A control spectrum (background) was obtained, in the absence of a sample, on the crystal of the ATR accessory. 2.6. Statistical and chemometric analysis The contents of the compounds of interest were expressed as means ± standard error of the means (n = 3) and subjected to analysis of variance. The averages were grouped by the Scott & Knott test at 5% probability. Subsequently, the results of phenolic compounds obtained by HPLC were also subjected to principal component analysis (PCA). The ATR-FTIR dataset was used to construct a descriptive model, based on the calculation of principal components with the aid of the statistical packages Pirouette (see 4.5., Infometrics, Inc., USA) and Past (v. 2:16, University of Oslo, Norway). 3. Results and discussion 3.1. Contents of total phenols and flavonoids The contents of total phenols and flavonoids varied widely among the banana accessions (Table 2). The average content of total phenols was 24.2 mg GAE/100 g and ranged from 2 mg GAE/ 100 g (Caipira) to 95 mg GAE/100 g (Highgate). For flavonoids, the average concentration was 2.41 mg QE/100 g and ranged from 0.40 (Caipira) to 7.45 mg QE/100 g (Wasolay). These results agree with those found in studies carried out with other genotypes of Musa spp. that revealed a sufficiently high degree of genetic variability in the content of these metabolites within the germplasm. This finding is a positive factor for breeding programmes aimed at increasing the levels of these compounds in commercial genotypes of bananas (Mattos et al., 2010; Bennett, Shiga, Hassimotto, Rosa, Lajolo & Cardenunsi, 2010; Sulaiman et al., 2011). Furthermore, the accession Highgate had average concentrations of total phenolics that were four times higher than those of the Williams cultivar and five times higher than those of the Nanica cultivar, two representatives of the Cavendish subgroup and the most commonly cultivated by farmers in several countries. 3.2. Chromatographic analysis (HPLC) of phenolic compounds Nine accessions with high levels of total phenols and total flavonoids (UV–visible spectrophotometry – Table 2) were selected to identify and quantify phenolic compounds in the samples of banana fruit pulp under study via RP-HPLC–UV–Vis. Four phenolic compounds were identified: epicatechin, gallocatechin, gallic acid, and 3,4 dihydroxybenzoic acid (protocatechuic acid) (Table 3). Other phenolic compounds have also been identified in banana pulp with this technique, including naringenin (Aurore, Parfait, & Fahrasmane, 2009). Gallocatechin was the major compound in the aqueous extract of the pulp of the banana accessions under study (average of 360 lg/100 g). The triploid ‘Highgate’ had the highest content (591 lg/100 g) of this secondary metabolite (Table 3). The triploid ‘Nam’ had the highest average content of epicatechin (114 lg/

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Table 2 Average and standard deviation of the content of total phenols and total flavonoids in fruits of 29 banana accessions of the Active Germplasm Bank of Embrapa Cassava & Fruits. Accessions

Ploidy level

Total phenols (mg GAE/100 gdw1)

Total flavonoids (mg QE/100 gdw1)

028003-01 Jari Buaya Lidi Jaran Pipit Malbut Tuu Gia F3P4 M48 Nanica Wasolay Caipira Nam Highgate Gros Michel Orotawa Williams Torp Thap Maeo Saba Champa Madras Figo Cinza Bucaneiro Calipso Ambrosia Tropical Porp Maravilha Teparod

AA(C) AA(C) AA(C) AA(C) AA(C) AA(C) AA(C) AA(H) AA(H) AAA AAA AAA AAA AAA AAA AAA AAA AAA AAB ABB ABB ABB AAAA AAAA AAAA AAAB AAAB AAAB ABBB

21.0 ± 1.20i 20.9 ± 1.21i 9.00 ± 0.13p 28.0 ± 1.28f 6.00 ± 0.89r 13.0 ± 1.10m 12.0 ± 0.12n 15.0 ± 0.15l 4.02 ± 0.06s 18.7 ± 1.18j 56.7 ± 1.56c 2.01 ± 0.14t 38.3 ± 1.10d 95.3 ± 1.35a 76.0 ± 1.07b 17.0 ± 1.20k 22.3 ± 1.17h 18.3 ± 1.02j 21.0 ± 1.03i 29.3 ± 1.29e 26.0 ± 1.20g 17.3 ± 1.16k 76.3 ± 1.76b 11.3 ± 1.11o 13.0 ± 1.13m 13.0 ± 0.13m 11.0 ± 0.11o 7.01 ± 0.13q 4.02 ± 0.04s

1.52 ± 0.10n 2.58 ± 0.21h 0.79 ± 0.035r 0.82 ± 0.015r 0.40 ± 0.021t 0.67 ± 0.015s 1.87 ± 0.26l 0.77 ± 0.021r 0.44 ± 0.012t 4.97 ± 0.16f 7.45 ± 0.13a 0.40 ± 0.010t 1.89 ± 0.06l 5.56 ± 0.10c 6.75 ± 1.32b 1.72 ± 0.15m 1.31 ± 0.26p 2.35 ± 0.10i 1.49 ± 0.26n 5.25 ± 0.25e 2.07 ± 0.64j 2.33 ± 0.25i 5.46 ± 0.61d 1.43 ± 0.10o 1.43 ± 0.15q 2.80 ± 0.05g 2.20 ± 0.26k 1.97 ± 0.21k 1.24 ± 0.15p

Mean Minimum Maximum CV (%)

– – – –

24.2 2.01 95.3 1.83

2.41 0.40 7.45 1.79

C, cultivated; H, hybrid; CV, coefficient of variation; GAE, gallic acid equivalents; QE, quercetin equivalents. Averages followed by the same letter in the columns belong to the same group by the Scott and Knott (1974) test at 5% probability. 1 dw: dry weight.

Table 3 Average concentration of phenolic compounds (lg/100 g ± standard deviation) determined by HPLC of banana pulp flours in 9 accessions of the Active Germplasm Bank of Embrapa Cassava & Fruits. Accession

Ploidy level

Epicatechin

Gallocatechin *

Gallic acid

Protocatechuic acid

Saba Jaran Bucaneiro 028003-01 Gros-Michel Wasolay Champa Madras Nam Highgate

ABB AA AAAA AA AAAA AAA ABB AAA AAA

nd 33.6 ± 1.71c 101 ± 6.01b 105 ± 8.29b nd nd nd 114 ± 6.80a nd

324 ± 6.74e 149 ± 5.01g 410 ± 15.5d 152 ± 6.21g 510 ± 23.9b 418 ± 18.2d 482 ± 13.3c 204 ± 10.9f 591 ± 26.4a

4.32 ± 0.8d 10.2 ± 0.40a 7.73 ± 0.21b 0.61 ± 0.06i 1.82 ± 0.20g 2.31 ± 0.31f 2.62 ± 0.62e 1.21 ± 0.21h 5.05 ± 0.42c

1.58 ± 0.60c 0.67 ± 0.03e 1.39 ± 0.81d 0.66 ± 0.08e 2.07 ± 0.62b 1.93 ± 0.21b 1.82 ± 0.61b 1.70 ± 0.90c 5.91 ± 0.41a

Mean Minimum Maximum CV (%)

– – – –

39.3 nd 114 8.25

360 149 591 2.26

3.97 1.21 10.2 2.77

1.97 0.66 5.91 5.71

CV: coefficient of variation; nd (not detected). Averages followed by the same letter in the columns belong to the same group by the Scott and Knott (1974) test at 5% de probability.

⁄

100 g). Bennett et al. (2010) analysed triploids of Musa spp. and identified epicatechin and gallocatechin at levels similar to those found in this study (33.9 –460 lg/100 g and 37.3–542 lg/100 g, respectively) (Fig. S2). Other studies of the genotypes of Musa spp. also identified higher proportions of catechins than of other classes of phenolic compounds (Bennett et al. (2010); Del Mar Verde-Mendez, Forster, Rodriguez-Delgado, Rodriguez-Rodriguez, & Romero, 2003). Catechins are phenolic compounds with high antioxidant potential

and antitumor activity (Dreosti, Wargovich, & Yang, 1997). Studies carried out by Vijayakumar et al. (2008) found that flavonoids from banana pulp are effective in vivo antioxidants that protect the body against various oxidative processes. Lower rates of phenolic acids were found in all accessions studied in this work, with gallic acid in particular at average levels of 10.2 lg/100 g in the diploid ‘Jaran’. The average content of protocatechuic acid was 1.97 lg/100 g, with a maximum value of 5.91 lg/100 g for the triploid ‘Highgate’ (Table 3). It is noteworthy that the occurrence of protocatechuic acid has never been reported

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in samples of banana pulp, although it has been detected in other fruits, such as blueberry and guava (Gutièrrez, Mitchel, & Solis, 2008). In fact, studies indicate that when present, hydroxybenzoic acids are usually found in only small amounts in edible plants (e.g., fruits), with the exception of some red fruits, radishes, and onions (Manach, Scalbert, Morand, Rémézy, & Jiménez, 2004), thus corroborating the results obtained in this work. Phenolic acids, especially gallic acid, also reduce free radicals present in the human body and are therefore important for the prevention of cardiovascular diseases and some types of cancer (Yeh & Yen, 2006). With the aim of establishing a descriptive model of the grouping of varieties according to their phenolic compounds, the quantitative data resulting from HPLC analysis were used in a principal components analysis (PCA). The dispersion of varieties according to the PC1 and PC2 axes is shown in Fig. 1, clearly revealing the existence of three groups of accessions. Together, PC1 (54.77%) and PC2 (52.47%) explained 80.24% of the variance of the chromatographic data set, and the variables gallocatechin and gallic acid showed the highest factorial contribution to PC1 and PC2, respectively. The samples with the highest contents of gallocatechin were grouped in PC1 ( ), while PC2 ( ) contained those with higher levels of gallic acid. On the other hand, accessions with lower contents of these compounds and higher contents of epicatechin were found in PC1 (+) and PC2 (+). These results reveal that accessions clearly group according to their concentration of phenolic compounds, which indicates that PCA is a promising tool to identify and select genotypes to be used in hybridisation schemes focussing on banana biofortification.

The total content of pro-vitamin A carotenoid (pVACs) was calculated as the sum of the concentrations of trans-a-carotene (tAC), trans-b-carotene (t-BC), and cis-b-carotene (c-BC). The results revealed that the carotenoid content of banana fruit consists mostly of pro-vitamin A compounds (84.6%). This observation is also demonstrated in other research works on Musa spp. (Table 4) (Davey et al., 2009; Englberger et al., 2010). The ratio of carotenoids detected in banana pulps is quite different from that found in cultures considered rich in carotenoids, such as maize, in which the major compounds are lutein and zeaxanthin and only 10–20% are pVACs (Ortiz-Monasterio et al., 2007, Kuhnen et al., 2010). This finding highlights the importance of the banana as a potentially useful food for programmes aiming to increase the consumption of vitamin A by populations with great need for this nutrient. The average pVAC content was 231 lg/g (97.9 lg/g of t-BC). The minimum value was found for the Williams cultivar (Cavendish subgroup), where trace amounts of these compounds were detected (t-AC and t-BC); the maximal value of 1164 lg/g (525 lg/g of t-BC) was found in the wild diploid, Jari Buaya. Overall, the carotenoid contents varied significantly in the analysed banana accessions. It was not possible to identify this compound in the widely commercially exploited Williams cultivar. Other studies on Musa spp. (Mello, Lima, & Maciel, 2006) presented similar results. Cultivars of the Cavendish subgroup are characterised by a very light pulp colour (i.e., beige). Importantly, pulp colour is a phenotypic trait positively correlated with the presence of pVAC carotenoids in the banana (Amorim et al., 2011; Davey et al., 2009; Englberger et al., 2010).

3.3. Changes in the content of pro-vitamin A carotenoid (pVACs) within the Musa germoplasm

3.4. Profiles of pro-vitamin A carotenoid (pVACs) in samples of Musa spp.

Previous experiments, performed by lyophilising and oven drying samples of some banana accessions (e.g., Highgate, Wasolay, Jari Buaya, Malbut, Jaran, Williams, Saba, Caipira, Nam, and 02803-01), did not reveal meaningful effects on the carotenoid profile according to the drying technique used (Table 4), so that the oven-dried samples were chosen for further analysis.

In addition to determining total pVACs, it is paramount to discriminate pro-vitamin A compounds because vitamin activity varies among them. trans-b-Carotene is the compound with the highest activity (12 lg of t-BC is equivalent to 1 lg of retinol activity equivalent), while t-AC and c-BC have only half of the activity of t-BC vitamin A. The analyses demonstrated that 44.9% of the total

2.0 1.5 Nam 1.0

028003-01 Gros-Michel

0.5

CP 2: 25.47%

Champa-Madras 0.0

Wasolay Saba

Highgate

-0.5 Bucaneiro -1.0 -1.5 Jaran

-2.0 -2.5 -3.0 -5

-4

-3

-2

-1

0

1

2

3

4

CP 1: 54.77% Fig. 1. Factorial distribution of PC1 and PC2 for the data of average content of phenolic compounds of pulp flour of 9 banana accessions of the Active Germplasm Bank of Embrapa Cassava & Fruits.

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Table 4 Concentration of pVACs (t-BC, t-AC, and c-BC), percentage of pVACS of the total carotenoids, and RAE of oven-dried pulp flour according to the studied accessions of Musa spp. Data shown in parentheses refer to freeze-dried samples of banana pulp flour. Lutein (lg/g dry weight)

Zeaxanthin (lg/g)

Average total of carotenoid (HPLC)

pVACs % of total carotenoid

t-BC % of total pVACs

RAE (lg/g)

20.1e (20.0) 27.9d (28.0) 224a (222) 33.7c (32.9) T (T) nd 12.9f (13.1) nd (nd) nd (nd) nd (nd) 36.4b (37.1)

2.22c (2.19) 2.63c (2.58) 2.28c (2.21) 4.61a (4.01) 3.52b (3.50) 4.04b 2.05c (2.00) 0.57d (0.49) nd (nd) 0.87d (0.72) 0.23e (0.26)

T (T) nd (nd) nd (nd) T (nd) T

99.2

20.9

13.9

99.0

27.0

14.5

99.8

45.1

70.4

98.4

36.4

16

98.8

56.0

18.8

T 2.68a (2.72) 3.72a (3.55) 3.02a (2.19) 3.27a (3.19) 2.05a (2.11)

279 (279) 276 (278) 1167 (1162) 286 (285) 292 (291) 4.04 88.0 (88.9) 16.2 (16.3) 44.7 (44.3) 16.1 (16.2) 321 (322)

– 94.6

– 73.8

– 6.03

73.5

80.4

0.89

93.2

53.6

2.67

74.3

64.7

0.81

99.3

46.3

19.4

29.6 0 224 3.46

1.99 0 4.61 14.3

1.46 0 3.72 20.5

235 4.04 1167 –

84.6 0 99.8 –

43.9 0 80.4 –

13.7 0 70.4 –

Average content of pVACs (lg/g dry weight)

Accessions

Genomic group

Average total content of pVACs (HPLC) (lg/g dry weight)

t-AC

t-BC

c-BC

Highgate

AAA

Wasolay

AAA

Jari Buaya

AA(C)

Malbut

AA(C)

Jaran

AA(C)

Williams Saba

AAA ABB

Caipira

AAA

Nam

AAA

02803-01

AA

Thap Maeo

AAB

277 (276) 273 (275) 1164 (1160) 282 (281) 289 (288) T 83.3 (84.1) 11.9 (12.3) 41.7 (42.1) 12.0 (12.3) 319 (320)

199b (199) 172b (173) 415a (415) 146c (146) 127c (128) T 8.90d (9.05) 2.33d (2.40) 19.4d (20.0) 4.22d (4.34) 135c (136)

57.9d (57.0) 73.7d (74.7) 525a (523) 102c (102) 162b (160) T 61.5d (62.0) 9.56e (9.88) 22.4e (22.1) 7.75e (7.98) 148b (147)

Total average Minimum Maximum CV (%)

– – – –

231 0 (T) 1164 –

104 0 (T) 415 17.1

97.9 0 (T) 525 8.01

Averages followed by the same letter in the columns belong to the same group by the Scott and Knott (1974) test at 5% de probability; pVACs, pro-vitamin A carotenoid; t-AC, trans-a-carotene; t-BC, trans-b-carotene; c-BC, cis-b-carotene; RAE, retinol activity equivalents; T, Trace of carotenoid – outside the measured range of you calibration curve; CV, coefficient of variation; nd (not detected).

pulp of pVAC is composed of t-AC, 42.4% of t-BC, and only 12.8% of c-BC (Table 4). Davey et al. (2009) confirmed the wide variation in vitamin A activity within the germplasm of Musa spp. and concluded that not all of the cultivars with the highest amount of pVACs have high percentages of t-BC. The Jari Buaya accession, for example, which was shown to contain the highest amount of pVACs (1164 lg/g), had only 45.1% t-BC, while the Caipira accession, with only 11.89 lg/g of pVACs, had 80.40% t-BC. In other studies on banana carotenoids, only 2% of the samples had more than 80% t-BC (Davey et al., 2009). In contrast to these results, Englberger et al. (2010) analysed certain cultivars of the Australimusa section with high pVACs contents and found that genotypes with high contents of pVACs consistently had high proportions of t-BC (75– 100%). These differences in the accessions under analysis most likely occur because these accessions belong to distinct sections, with different growth and development patterns than those originating from the Eumusa section. 3.5. Cultivars with high pro-vitamin A carotenoid (pVACs) (high t-BC) According to Davey et al. (2009), the cultivars that had the highest amounts of pVACs (t-BC) were identified in the sections Eumusa and Australimusa, most likely originating from Papua New Guinea, with the exception of the rare Fe’i banana from Central Micronesia. These varieties have different traits and high values of t-BC, i.e., 8.51 lg/100 g of fresh pulp (Englberger et al., 2010). However, our study found accessions from Honduras and Indonesia with higher amounts of t-BC than accessions from Papua New Guinea. Accession Jari Buaya (AA) from Malaysia, had the highest contents of pVAC and t-BC (525 lg/g of dry weight), followed by accessions Thap Maeo (AAB) (148 lg/g) and Jaran (AA) (162 lg/ g), which originate from Honduras and Indonesia, respectively (Table 4). The accessions from Papua New Guinea had the fourth and

sixth highest contents (Malbut and Wasolay), respectively. These results suggest that there is no association between place of origin and pVAC content and that this trait is dispersed in the germplasm of Musa spp. Therefore, it can be concluded that carotenoids and pVACs are quantitative traits determined by multiple alleles that are affected by the environment.

3.6. Characterisation of the metabolic profile of the samples of banana pulp flours by ATR-FTIR spectroscopy Fifteen to twenty bands were identified in the FTIR spectra (3000–600 cm 1 – Supplementary Fig. S1) of the banana pulp flours. Visual analysis of spectral profiles allowed the detection of discrepancies in the presence or absence of certain bands, typically revealing different chemical compositions and metabolic profiles among the accessions. The ATR-FTIR spectra revealed bands at 2927–2885 cm 1 (Supplementary Fig. S1), corresponding to typical signals of aliphatic hydrocarbons. These signals are possibly related to fatty acids or lipids in the samples and derived from the axial and angular deformation of the methyl (–CH3) and/or methylene (CH2) group(s) of the alkyl portion of these compounds (Lambert, Shurvell, Lightner, & Cooks, 2001). The presence of carboxylic groups, associated with the axial deformation of the functional carbonyl group (C@O) typically found in fatty acids, was attributed to the band at 1740 cm 1 (Silverstein, 1994). The presence of protein in the samples can be inferred by the signals between 1650 and 1550 cm 1 related to amines I and II, respectively (Lambert et al., 2001), and between 1543 and 1480 cm 1, associated with amine II (Schulz & Baranska, 2007). In addition, several bands associated with carbohydrates (1200– 850 cm 1) and with compounds with aromatic rings in their structure (such as (poly)phenolics (900–631 cm 1)), were found in all of

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Fig. 2. Scores of PC1 (63.4%). PC2 (22.0%) and PC3 (6.5%) determined, based on the FTIR data for the fingerprint region of carbohydrates (1200–850 cm

the studied accessions (Supplementary Fig. S1 – Lambert et al., 2001). Visual analysis of FTIR spectral profiles has usually not been very efficient, given the complexity of the data generated. An FTIR spectral profile often contains 4000 data points, and such a dimensionality complicates the extraction of information relevant to the objectives of the present study. However, this scenario is typically found in metabolomics studies in which the number of variables is much greater than the number of samples, as observed in this study (Lay, Borgmann, Liyanage, & Wilkins, 2006). Therefore, the analysis of mega-datasets requires the use of powerful computer tools and a data processing approach for the extraction of additional, relevant and accurate information regarding the visual analysis of the ATR-FTIR spectra. For this reason, the ATR-FTIR spectra data were used for principal components analysis (PCA) as a strategy to reduce data dimension without losing relevant information, i.e., data mining. Initially, the entire ATR-FTIR spectral data set, i.e., 3000–600 cm 1 of the accessions under study, was submitted to PC determination to identify distinctive metabolic patterns important to genotype discrimination. However, metabolic patterns of the studied samples could not be detected using such a strategy, prompting us to further apply PCA to the dataset of fingerprint regions of carbohydrates, proteins, and lipids. The PCA was performed for the carbohydrate fingerprint spectral region (1200–850 cm 1) after the removal of sample outliers, e.g., ‘028003-01’, ‘M48’, and ‘Caipira’ (data not presented). The data in Fig. 2 allowed the discrimination of diploids ‘Jari Buaya’ and ‘Lidi’, reflecting differences in the chemical composition of carbohydrates. In this descriptive model, PC1, PC2, and PC3 contributed to explain 91.9% of the variance present in the dataset. The major

1

).

factorial contributions resulted from the bands at 1013, 1024, 1042, 1049 cm 1, associated with starch components, e.g., amylopectin (1013, 1024 cm 1) and amylose (1042, 1049 waves cm 1 – Kuhnen et al., 2010; Millan-Testa, Mendez-Montealvo, Ottenhof, Farhat, & Bello-Pérez, 2005). The PC calculation for the ATR-FTIR dataset of the protein fingerprint region (1650–1500 cm 1), after exclusion of the outlier samples (‘028003-01’, ‘M48’, and ‘Caipira’), identified the accessions Saba (PC1+, PC2+, and PC3 ) and Champa Madras (PC1 , PC2+, and PC3+) as quite divergent. Accessions F3P4, Thap Maeo, and Orotawa grouped into PC1+, PC2+, and PC3 and presented spectral profiles similar to protein compounds. The principal components PC1, PC2, and PC3 contributed to explain 92.0% of the sample variance and the analysis of the factorial contributions indicated that the bands at 1595 cm 1, 1554 cm 1 (PC1+), and 1631 cm 1 (PC2+) were the main variables responsible for the discrimination of accessions. The band at 631 cm 1 corresponds to the compounds with amine I group (1600–1680 cm 1), while the signals at 1554 cm 1 and 1595 cm 1 correspond to amine II groups (Schulz & Baranska, 2007). When considering the fingerprint region associated with the typical lipid signals (2997–2800 cm 1 and 1700–1760 cm 1), the accessions M48 and 028003-01 were considered outliers (data not shown) and were removed from the spectral data set for PC calculation. PC1 and PC2 contributed to explain 85.9% of the variance in the data and the calculation of PC3 (7.2%) added information relevant to the interpretation of the variance of the spectral data. According to this descriptive model, the accessions Lidi, Tuu Gia, and F3P4 (AA genomic group) proved to be discrepant, as they were grouped into PC1+/PC2 /PC3 and showed peculiar charac-

C.V. Borges et al. / Food Chemistry 145 (2014) 496–504

teristics related to lipid composition. The bands at 2882, 2925 cm 1 (triglycerides), and 1727 cm 1 (axial deformation of the C@O group of fatty acids – Schulz & Baranska, 2007) were shown to be factorial contributions to the descriptive model. Finally, previous studies have shown that some bands in the medium infrared spectrum reflect the shape of the macromolecular structures of amylose and amylopectin in starch granules (Wilson & Belton, 1988). The bands at 1047 cm 1 and 1022 cm 1 were sensitive to the proportions of the crystalline and amorphous phases of starch, respectively (Sevenou, Hill, Farhat, & Mitchell, 2002; Van Soest, Tournois, Wit, & Vliegenthart, 1995), and were relevant to the prediction of the structural characteristics of starch-derived products in storage or after processing (Millan-Testa, Mendez-Montealvo, Ottenhof, Farhat, & Bello-Perez, 2005). These bands were detected in all of the ATR-FTIR spectral profiles of the studied samples, and the ratios of the values of their absorbances were used to estimate the degree of crystallinity of the starch granules (R 1047/1022 – Supplementary Table S1). The values of the absorbance ratios of the bands at 1022 cm 1 (amorphous component) and 1047 cm 1 (crystalline component) indicate that the crystalline structure is more dominant than the amorphous phase of the starch granules; the only exception to this observation occurred in the Lidi accession (R 1047/1022 = 0.97). This finding suggests that accessions have different physical–chemical characteristics and digestibility properties and corroborates the findings of the principal component analysis, based on spectral data of the fingerprint region of carbohydrates that clearly distinguished the Lidi accession. The R 1047/1022 values observed (>1.0) are consistent with those found by Millan-Testa et al. (2005) in banana genotypes (R 1047/1022 = 1.12, Musa paradisiaca var. Macho). These findings illustrate the level of ordered structure present in the superficial layers of starch granules, an important trait in the behavioural response of starch post-harvest (i.e., half-life). Finally, the collection of spectra by the ATR-FTIR technique involves the acquisition of information relating to the supramolecular structure of the surface (2 lm in depth) of the starch granule samples (Sevenou et al., 2002). Moreover, considering that the experiments did not use starch samples isolated from banana pulp flour, it is possible to infer that the differences observed among the samples are not related to the organisation of ring structures of starch expansion. Analysed together, the results demonstrated that the construction of a descriptive model based on the ATR-FTIR integral data, i.e., (3000–600 cm 1) of the banana flours under study could not effectively identify discrepant and relevant metabolic profiles. Therefore, this approach is inefficient for the selection and breeding of the species. In a second series of experiments, discriminant descriptive models were built from the segmentation of the spectral profile, using the dataset typically associated with important compounds for human health, e.g., carbohydrates, proteins, and lipids. This analytical strategy allowed the identification of accessions with distinct metabolic profiles for the three classes of primary metabolites under study. The Lidi accession proved to be the most discrepant in the sample population, particularly with respect to its lipid and carbohydrate composition. It is widely known that environmental and genetic variations can qualitatively and quantitatively affect the profile of primary metabolism compounds (Baye, Pearson, & Settles, 2006). However, variation in the metabolic profiles of the accessions of Musa ssp. is assumed to be mostly due to genetic factors because the accessions were grown in the same environment (soil, geographic area, and climate) and received similar culture treatments.

503

4. Conclusion The results of this study reveal great diversity in the content of bioactive compounds (e.g., phenolic compounds and carotenoids) within the germplasm of Musa spp. Accessions with appreciable amounts of these compounds were identified, especially compared with the main cultivars that are currently marketed. In addition, the use of ATR-FTIR, associated with PCA, proved to be a tool with potential for and applicability to banana breeding programmes because it allowed the identification of accessions with distinct metabolic profiles, especially in the fingerprint regions of compounds important to human health (carbohydrates, proteins, and lipids). ATR-FTIR spectroscopy also revealed the structural divergence of starchy components of banana pulp flours, based on their ordered structure, i.e., crystallinity, allowing the discrimination of accessions according to this relevant trait. The results demonstrate the potential use of certain banana accessions in health promotion due to their functional characteristics. Accessions can be distinguished for their levels of bioactive compounds and used in breeding programmes to obtain biofortified cultivars with specific nutritional values that might contribute to increasing the intake of nutrients in disadvantaged populations. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013.08.041.

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