Monitoring the Aroma Compounds of Vicia faba L var. Major and var. Minor

Abstract

The volatile compounds of Vicia faba. L var. minor and var. major seeds were evaluated by headspace-solid phase micro-extraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). The total identification percentages of the extracted volatiles were 95.5% and 98.3%, respectively. The number of aroma compounds detected was 28. Among them, 15 compounds were determined in the emission of whole legume seeds of minor cultivar and 22 from the major one. The volatiles were classified into five chemical classes, i.e., monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, apocarotenes, and non-terpene derivatives. Aldehydes and alkanes were considered as the most abundant constituents in non-terpene derivatives, followed by esters, alcohols, phenols, phenones, and hydrocarbons. A wide difference in term of volatiles was observed between major and minor faba bean cultivars. This study can provide useful information about the specific volatile characteristics for each cultivar and its possible use in the conception of legume-based ingredients and for pertinent breeding programs.

1. Introduction

Vicia faba, also known in the culinary sense as the broad bean, fava bean, or faba bean, horse bean, field bean, bell bean, Windsor or tic bean is a flowering plant in the pea and bean family Fabaceae native to North Africa and south-western Asia, and extensively cultivated elsewhere [1, 2]. It is grown as a winter annual in warm temperate and subtropical areas. Hardier cultivars grown in the Mediterranean region can tolerate winter temperatures of −10°C without serious injury, whereas the hardiest European cultivars can tolerate up to −15°C [3]. Although usually classified in the same genus Vicia as the vetches, some botanists treat it in a separate monotypic genus Faba. Four subspecies including V. faba ssp. minor, V. faba ssp. equina, V. faba ssp. major, and V. faba ssp. Paucijuga were reported by Cubero [4]. However, in [5], Muehlbauer and Tullu classified faba beans based on seed size. Botanically, faba beans were divided into two subspecies: paucijuga and faba. Faba subspecies was, also, subdivised into V. faba var. minor with small, and rounded seeds (1 cm long), V. faba var. equina with medium-sized seeds (1.5 cm) and V. faba var. major with large broad flat seeds (2.5 cm).

Faba bean seeds are very important crops due to their high protein content ranging from 20% to 41%, depending on variety [6]. Using legumes in human diet could potentially prolong life and maintain well-being [7]. They also contain adequate proportions of carbohydrates and oil which increases their food value [8]. Recently, the consumption of legumes was increased over the world and that due to their high nutritional value and beneficial health effects. Neverthelss, the pulses off flavors, in general and in faba bean seeds in special, limit their use [9]. The undesirable aroma of legumes is due to their emission of volatiles developping from the fatty acid oxidation catalyzed by lipoxygense. Aldehydes, alcohols, ketones, acids, and pyrazines categories were responsible for the musty, beany, grassy, earthy, and leafy unwanted odors in faba bean seeds [10].

To enhance the use of pulses worldwide, it is necessary to define methods to ameliorate the flavor of legumes. In fact, an agreeable flavor is an important aspect for any successful product [11]. Technologies and methods such as control of oxidation, germination, dehulling, enzymatic treatment, heat processing, fermentation, milling, and cultivar selection has been described to prevent the off-flavors development and accumulation in pulses [12, 13, 14]. The off-flavors in faba bean seeds were released during harvest, processing, and storage time [15]. To enhance the suitablity of legumes odors, it is relevant to start with raw pulses that emitted low desirable odors to guarantee that the development of off-flavors keeps at the lowest level during processing conditions [10, 16]. Because of the limited information available which dealing with the unwanted odors in faba beans, the purpose of this study was the monitoring of the volatiles emitted by faba bean seeds var. major and var. minor using the headspace solid-phase microextraction coupled to gas chromatography with mass spectrometry HS-SPME-GC-MS (Figure 1).

Figure 1.

Graphical abstract of the presented study.

2. Materiels and methods

2.1 Chemicals and reagents

A pure reference compounds including heptanal, benzaldehyde, octanal, (E)-2-octenal, decanal, dodecanal, all n-hydrocarbons, butyl butyrate, 6-methyl-5-hepten-2-one, naphthalene, acetophenone, phenol, β-pinene, p-cymene, limonene, γ-terpinene, 1,8-cineole, α-terpineol, verbenone, bornyl acetate, longifolene, geranylacetone (E + Z isomers), 1-octanol, 1-decanol, ethyl benzoate, 2-undecanone, α-thujone + β-thujone mixture, and pulegone were purchased from Sigma, Aldrich, Supelco and Merck and used to compare retention times and mass spectra.

2.2 Sample preparation

Major and minor faba bean cultivars were used in this study, named Mamdouh and Najeh, respectively. The bean seeds were offred by the Field Crops Laboratory of the National Institute of Agricultural Research (INRAT). 5 g of similar size seeds, without any physical damages, were choosen. The seeds were stored at 4°C in an opaque aluminum bag until analysis.

2.3 Headspace solid-phase micro extraction (HS-SPME)

The headspace spontaneous volatile emissions of the two vicia faba cultivars were sampled by means of HS-SPME. Three replications were considered for each legume. The sample was kept at room temperature for 30 min. A Solid Phase Micro-Extraction (SPME) device (Supelco, Bellefonte, PA, USA) covered with poly-dimethyl-siloxane (PDMS, 100 μm) was used, adapted according to the fabricator instructions. After that, the fiber was removed and transmitted to the injection port of the GC-MS system. All the desorption conditions were the same for all the seeds. Furtheremore, blanks were conducted before starting the first SPME extraction. Quantitative comparisons of relative peaks areas were assessed among the same chemicals in the different samples.

2.4 Gas chromatography coupled with mass spectrometry (GC-MS)

Gas chromatography-electron impact mass spectrometry (GC-EIMS) analyses were assessed using an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS (Agilent Technologies Inc., Santa Clara, CA, USA) capillary of 12 column (30 m × 0.25 mm; coating thickness 0.25 μm) and an Agilent 5977B single quadruple mass detector (Agilent Technologies Inc., Santa Clara, CA, USA). The conditions of sample analysis were as following: injector and transfer line temperatures 220 and 240°C, respectively; oven temperature programmed from 60 to 240°C at 3°C/min; carrier gas helium at 1 mL/min; split ratio 1:25. The acquisition parameters were as follows: full scan; scan range: 30–300 m/z; scan time: 1.0 s.

2.5 Compounds identification

The determination of the volatile compounds was established by comparability of the retention times with those of the authentic samples, comparing their linear retention indices (LRI) relative to the series of n-hydrocarbons. Computer matching was also used against commercial and laboratory-developed mass spectra library built up from pure substances and components of known mixtures and MS literature data [17].

3. Results and discussion

In total, 28 volatiles were detected by HS-SPME-GC-MS. Among them, 15 were identified in var. minor and 22 in var. major; accounting for 98.3% and 95.4% of total emission, respectively. The flavor constituents were divided into five chemical classes, including monoterpenes hydrocarbons (MH), sesquiterpenes hydrocarbons (STH), apocarotenes (AP), non-terpenes derivatives (NTD), and oxygenated monoterpenes (OM). Faba bean var. major emitted 61.4% of NTD, 19.4% of MH, 9.3% of OM, 4.8% of AP, and 0.6% of STH volatiles. On the other hand, 64.7% of NTD, 27.9% of MH, 3.4% of STH, 1.2% of OM, and 1.1% of AP were detected in the bouquet of the minor cultivar (Table 1). This data is in accordance with the studies of Khrisanapant [18] and Rajhi et al. [10]. Indeed, these authors demonstrated that NTD group was the major chemical class in legumes.

Constituents	Vicia faba major	V. faba minor
Monoterpene hydrocarbons	19.4	27.9
Oxygenated monoterpenes	9.3	1.2
Sesquiterpene hydrocarbons	0.6	3.4
Phenylpropanoids	0	0
Apocarotenes	4.8	1.1
Non-terpene derivatives	61.4	64.7
Total identified (%)	95.5	98.3

Table 1.

Chemical classes percentage of volatiles compounds in major and minor faba bean seeds.

3.1 Non terpenes derivatives constituents (NTD)

The individual volatile profiles of both cultivars are summarized in Figure 2. NTD volatiles identified in this study were: 5 aldehydes (heptanal, nonanal, decanal, undecanal, and dodecanal), 5 alkanes (n-dodecane, n-undecane, n-tridecane, n-tetradecane, and n-pentadecane), esters (butyl butyrate and ethyl octanoate), one phenol (phenol), 1 phenone (acetophenone), alcohol (phenylethyl alcohol), and hydrocarbon (naphthalene). The most abundant chemical group was aldehyde; including nonanal, which has a fat, citrus, and green flavor, decanal which is soap, orange peel and tallow like aroma, and undecanal, which has a waxy type of flavor. These chemicals were emitted by var. minor (8.2%, 2.6%, and 0.6%, respectively) and var. major (7.6%, 5.6%, and 0.9%, respectively). The heptanal aldehyde, which has a strong fruity odor [19], was registered only in the individual profile of var. major (7.1%). However, the dodecanal aldehyde, which has a soapy type of odor [19], was only found in the volatile bouquet of var. minor (0.3%). These results are in good agreement with a previous investigation of volatile compounds of other legumes, in particular soybeans, mung beans, cowpeas, peas, chickpeas, orange lentils and adzuki beans [18, 20, 21].

Figure 2.

NTD volatile compounds identified in faba bean var. minor and var. major.

Alkanes were the second dominant class in the NTD volatiles. Among the detected alkanes, two constituents were emitted by both cultivars: the n-tridecane and n-tetradecane (Figure 2). n-undecane and n-pentadecane were registered in the emission profile of var. major (1.6% and 2.6%, respectively). However, the alkane n-dodecane was characterized in the individual profile of var. minor. The high content in alkanes, was also previously reported by Oomah et al. [20] for different types of P. vulgaris. Their abundance legume seeds may be explained by the occurrence of lipid peroxidation, which causes the formation of the characteristic aroma of dry pulses, since alkanes are mainly obtained from oxidative reaction of lipids [11, 20].

Two esters were identified using the HS-SPME-GC-MS: the butyl butyrate, which has a fruity odor [19], is emitted by major and minor cultivars (6.3% and 12.4%, respectively), and the ethyl octanoate, presented a strong fruity and flowers odor [19], was found only in the bouquet of the latter one (2.5%). The NTD phenol was emitted by var. minor with a percentage identification of 27.4%.

This result agrees with that of Mebazaa et al. [22] and Ramadan et al. [23]. In their study on black, dark red kidney and pinto beans, Oomah et al. [20] demonstrated that the volatile profiles of studied P. vulgaris pulses were rich in aldehydes, alkanes, esters, and ketones.

3.2 Oxygenated monoterpenes

Seven constituents belong to the oxygenated monoterpenes were detected in both cultivars using HS-SPME-GC-MS. Among them five such as camphor, borneol, menthol, carvone, and bornyl acetate were appeared in the emission profile of var. major (2.8%, 0.9%, 1.7%, 1.3%, and 2.6%, respectively). However, the minor cultivar emitted much more α-terpineol (1%) and verbenone (0.2%) (Figure 3).

Figure 3.

OMT volatile compounds identified in faba bean var. minor and var. major.

3.3 Sesquesternes hydrocarbons, apocarotenes, and monoterpenes hydrocarbons constituents

The individual profiles of emitted aroma revealed that two sesquiterpene hydrocarbons were detected; the valencene and longifolene. The major compound emitted by each cultivar was the monoterpene hydrocarbon limonene (19.4 for var. major and 27.9%) (Figure 4).

Figure 4.

STH, AP, and MTH volatile compounds identified in faba bean var. minor and var. major.

3.4 Beany flavors

Hexanal, (E,E)-2,4-decadienal, (E,E)-2,4-nonadienal, nonanal, 2-pentylfuran, and 1-hexanol have been screened and considered as beany flavor markers in pulses [24, 25]. These are generated from the oxidation of unsaturated fatty acids (linoleic and linolenic) by an enzymatic pathway [15, 26, 27]. In this study, only one beany flavor marker was identified: nonanal, which emitted in a comparable amount by both cultivars (8.2 and 7.6% for var. minor and var. major, respectively). These beany flavors are responsible for the unwanted and unpleasant flavor of faba beans.

3.5 Volatiles and faba bean breeding

Unfortunately, pulses including faba bean seeds are not widely used in the world to their highest potential due to their inappreciated flavors, the poor digestibility of their proteins and their long cooking time [9]. One of the most significant sensory elements of food is its aroma, which is the result of the interaction between taste and smell perceptions. Unprocessed legumes emit volatile compounds as a result of the oxidation of fatty acids, which is mediated by lipoxygenase, during the harvesting, processing, and storage processes, giving them the musty, grassy, and bean-like flavors [9, 28]. Aldehydes, alcohols, ketones, acids, and pyrazines are frequently linked to the components of off-flavors in legumes. It is crucial to discuss technology that enhance the flavor of pulses in order to promote the consumption of legumes worldwide. A successful product must, in fact, have a flavor that consumers like. To prevent the development of off odors in legumes, germination, oxidation control, heat processing, enzymatic treatment, and cultivar selection have all been described [9]. To ensure that the formation of unwanted scent stays at the lowest possible level during processing and storage, it is crucial to start with raw materials that exhibit few off flavors. A selection of raw material, in term of volatiles of different cultivars of legumes should be performed. Thus, this type of study is very important to select a legume cultivars which had the lowest emission of off-flavors and then these latter cultivars can be selected as important breeding lines based on their organoleptic proprieties.

4. Conclusions

In this study, we performed the monitoring of the volatiles of faba bean var. minor and var. major using HS-SPME-GC-MS. The data revealed a difference between both profiles; that can be explained by the varietal effect. This study can provide useful information about the specific volatile characteristics for each cultivar and its possible use in the conception of legume-based ingredients and for pertinent breeding programs.