Fingerprinting of volatile profiles of sprouted and unsprouted seeds and flours of Phaseolus vulgaris using HS-SPME/GC-MS

Fingerprinting of volatile profiles of sprouted and unsprouted seeds and flours of Phaseolus vulgaris using HS-SPME/GC-MS | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fingerprinting of volatile profiles of sprouted and unsprouted seeds and flours of Phaseolus vulgaris using HS-SPME/GC-MS Imene Rajhi, Rim Nefissi Ouertani, Fatma Rajhi, Haythem Mhadhbi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5024199/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The impact of germination and milling on volatile compounds (VOCs) of chickpea cultivar was evaluated using HS-SPME/GC-MS technique. In total, 35 VOCs were identified. In this study, 28 active odors were characterized in seeds and 23 in flours: 17 in raw seeds, 24 in sprouted grains, 21 in raw flours, and 17 in germinated flours; they accounted for 98.9, 96.6, 97.9, and 98.8% of total emissions, respectively. The VOCs were classified into six chemical classes i.e., monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), apocarotenes (AP), phenylpropanoids (PP), nitrogen/sulfur derivatives (NSD), and non-terpene derivatives (NTD). Germination and milling processes decreased clearly the emission of NTD VOCs and increased the MH, OM, PP, AC, and NSD VOCs. Aldehydes, the most interest constituents giving the undesirable odors of legumes, was diminished in sprouted seed and flour versions. This study can provide useful information on the conception of legume–based ingredients combined with specific volatile characteristics in order to reduce unwanted odors and definitely for pertinent breeding programs. HS-SPME-GC-MS dry beans germination seeds flours VOCs Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Fabaceae , also known as legumes, are the second largest plant family (Kouris-Blazos et al., 2016). The Fabaceae are a large family of approximately 18,000 plant species, including herbs, trees, climbers, and shrubs. However, humans consume only a limited number of species (Rajhi et al., 2021). Legumes are divided into two categories namely pulses and oilseeds. The term pulses refer to the dried seeds, including peas ( Pisum sativum ), lentils ( Lens culinaris ), lupins ( Lupinus spp .), chickpeas ( Cicer arietinum ), common beans ( Phaseolus vulgaris ), faba beans ( Vicia faba L.) and fenugreek ( Trigonella foenum-graecum L.) and the second group is formed by soybeans ( Glycine max ) and peanuts ( Arachis hypogaea ) (Roland et al., 2017). Additionally, legumes are considered as the most important source of nutrients after cereals. In fact, they are esteemed as a low-priced and sustainable meat alternative in developing countries (Kouris-Blazos et al., 2016). Pulses are a valuable food, providing 20-45% of protein, including essential amino acids, 60% of carbohydrates and 5-37% of fiber. In addition, legumes have an important nutraceutical advantage due essentially to their content of bioactive compounds protecting against cancer, cardiovascular and degenerative diseases (Messina, 2016). Dry beans ( Phaseolus vulgaris ) are well known as a nutrient-dense plant food which furnish exceptional nutrient content. At approximately 22%, dry beans have nearly double the amount of protein compared with common cereal grains, with a lower content of carbohydrates and fat (Uebersax et al., 2022). Nevertheless, the use of pulses in the culinary arts is restricted due to the existence of a specific off-flavors (Rolland et al, 2017). The undesirable odors of unprocessed legume seeds, such as, beany, grassy, earthy, and leafy are due to the generation of volatiles organic compounds (COVs) from the oxidation of fatty acid catalyzed by lipoxygenase during harvest, processing, and storage conditions (Rajhi et al., 2022a). In general, the pulse undesirable odors constituents are associated to aldehydes, alcohols, pyrazines, terpenes, ketones, and acids. A satisfactory flavor is a critical feature of any merchandise. To enhance the pulse consumption worldwide, it might be essential to establish technologies to improve their odors including heat processing, dehulling, fermentation, milling, and germination (Rajhi et al., 2022b). Germination ameliorates the nutritional properties of seed pulses by decreasing anti-nutritional factors, such as phytases, trypsin inhibitors, and unwanted beany flavors produced by the oxidation of unsaturated fatty acids by lipoxygenase (Simons, 2011). In general, the information in the literature related to the off-flavors in legumes is limited. Thus, the aims of this work were (1) to investigate the impact of germination and milling on the volatile compounds (VOCs) of three dry beans cultivars (navy, kidney, and black) using headspace solid phase microextraction coupled to gas chromatography with mass spectrometry (HS-SPME/GC-MS) and (2) to determinate the discriminating compounds in raw and germinated seeds and flours using a multivariate analysis (PCA). Materials And Methods 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. Sample preparation Three cultivars of dry bean seeds (Navy, Kidney, and Black beans) were used in this study. Similar size seeds, without any physical damages, were selected. They were stored at 4°C in an opaque aluminum bag until analysis. The seeds were stored at 4°C in an opaque aluminum bag until analysis. Germination and milling processes For the germination process, raw seeds were surface disinfected in HgCl 2 (0.1%) for 1 min and then rinsed thoroughly using sterilized distilled water. The moisturized seeds were sown in petri dishes containing laboratory paper moistened with water. The petri dishes were then incubated into the germinator at 20°C, 99 % humidity, and in dark. Five days later, the sprouted seeds were dried in a vacuum oven at 45°C for 6 h. The raw seeds were named Navy beans-S, Black beans-S, and Kidney beans-S. The germinated seeds were named G-Navy beans-S, G-Black beans-S, and G-Kidney beans-S. To obtain the flour, 30 g of each type of legume seeds were crushed using a domestic blender at room temperature. Then, the flour was passed through a 100-mesh sieve. All the analyses were performed in triplicate. The crushed raw and germinated flours were named, respectively, Navy beans-F, Black beans-F, Kidney beans-F, G-Navy beans-F, G-Black beans-F, and G-Kidney beans-F. Headspace solid-phase micro extraction (HS-SPME) The headspace spontaneous volatile emissions of the whole and crushed seeds were sampled by means of HS‐SPME. Triplicates were assessed for each sample. Each replicate was let to equilibrate for 30 min at room temperature before sampling. A Solid Phase Micro‐Extraction (SPME) device (Supelco, Bellefonte, PA, USA) coated with poly-dimethyl-siloxane (PDMS, 100 μm) was utilized, preconditioned according to the manufacturer instructions. Then, the fiber was withdrawn into the needle and transferred to the injection port of the GC‐MS system. The desorption conditions were identical for all the samples. Additionally, blanks were carried out before each first SPME extraction, and randomly repeated during each series. Quantitative comparisons of relative peaks areas were performed between the same chemicals in the different samples. 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 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 (Ascrizzi et al. 2017). Compounds identification The identification of the volatile compounds was based on a comparison 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 (Davies 1990). Statistical analysis Principal Component Analysis ( XLSTAT software version 2014), and clustering were used to analyze data. Principal Component Analysis (PCA) was implemented using the two sets of data from whole seeds and flours of legumes. Every incorporated matrix involved 7 chemical classes in columns and different types of cultivars in rows. The PCA analysis was carried out to (1) detect differences and similarities between the two different legume cultivars as well as to evidence possible correlation between chemical classes and legumes, (2) determine grouping and separation of cultivars and, finally, (3) identification of discriminant compound related to each cultivar for both forms. Results Overall, 79 VOCs were identified by HS-SPME-GC-MS in the emission profiles of raw and sprouted seeds of different legume cultivars. The detected and identified VOCs belonged to seven chemical classes, including monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), sesquiterpene hydrocarbons (STH), phenylpropanoids (PP), apocarotenes (AC), nitrogen/sulfur derivatives (NSD), and non-terpene derivatives (NTD). The emitted VOCs were identified using HS-SPME/GC-MS. 3.1. Impact of germination on volatiles compounds of dry bean seeds To evaluate the impact of germination of dry bean cultivars on the emission of volatile compounds, raw seeds (Navy beans-S, Black beans-S, and Kidney beans-S) and germinated seeds (G-Navy beans-S, G-Black beans-S, and G-Kidney beans-S), were used. In total, 40 VOCs were identified in raw seeds: 18 for Navy beans-S, 28 for Kidney beans-S, and 30 for Black beans-S; they accounted for 98.1, 99.0, and 97.3% of total emissions, respectively. In sprouted seeds, 31 VOCs were characterized as follows: 25 for G-Navy beans-S and G-Black beans-S, and 14 for G-Kidney beans-S, accounted for 99.8, 95.6, and 97.1%, of the total emissions, respectively. As shown in Figure 1a, the most abundant chemical classes were NTD for Navy beans-S (61.4%), PP for Kidney beans-S (43.0%), and STH for Black beans-S (41.9%) following by MH (11.8%), OM (22.6%), and NTD (19.1%), respectively. The third most representative chemical classes for these samples were PP (10.2%), NTD (15.6%), and PP (16.9%), respectively. In the case of VOCs emitted by the germinated seeds, the number was as follow: 25 for G-Navy beans-S, 14 for G-Kidney beans-S, and 25 for G-Black beans-S, accounting for 99.8, 97.1, and 95.6% of the total emissions, respectively. The individual volatile profiles of raw and germinated seeds were quite different. In sprouted seeds, the composition of VOCs was changed. The germination process was accompanied by an increase of NTD constituents for all dry bean samples. Figure 1b shows that the most abundant chemical class for G-Navy beans-S, G-Kidney beans-S, and G-Black beans-S was NTD (79.8, 83.6, 68.5%, respectively), following by AP (6.4%), OM (10.6, and 12.2%), respectively. As regards to the VOCs belong to STH class, they were totally absent in G-Navy beans-S and G-Kidney beans-S and were present only in small amount in G-Black beans-S (2.5%). In the case of PP constituents, these were identified in small amounts in all germinated cultivars compared to raw samples (Figure 1a and b). Among the identified VOCs, 39 NTD constituents were emitted by all samples: 10 for unsprouted Navy beans-S, 8 for Kidney beans-S,13 for Black beans-S, 19 for G-Navy beans-S, 9 for G-Kidney beans-S, and 12 for G-Black beans-S (Table 1). Nevertheless, the respective emissions were quite different. Figure 2 shows the different percentages identification of NTD components in sprouted legumes compared to their corresponding unsprouted versions. It can be seen from Fig.2 a and b that seed germination was accompanied by an increase of aldehydes for the three cultivars. An augmentation of the emission of alkanes, ketones, alcohols and esters was recorded especially in the profile of G-Black beans-S (Fig. 2a and b). A different profile was monitored in Navy and Black beans. In the aroma profiles of raw and germinated beans seeds only 3 VOCs were shared the six versions of beans, such as nonanal (NTD), decanal (NTD), and limonene (MH) (Table 1). Naphthalene (NTD), ( E )-Anethole, and β-caryophyllene presented the highest percentages in Navy beans-S, Kidney beans-S and Black beans-S, respectively. However, they dropped dramatically as the seeds germinated. A general increase in the percentages of nonanal, decanal and n -tetradecane was observed in sprouted seeds. Indeed, nonanal becomes the dominant emitted volatiles in all germinated cultivars. As the seeds germinated, new constituents were emitted, including heptanal (NTD), benzaldehyde (NTD), octanal (NTD), 2-ethyl-1-hexanol (NTD), 6-methylheptyl 2-propenoate (NTD), 1-undecene (NTD), ( Z )-3-pentadecene (NTD), 1,8-cineole (OM), dihydrocitronellol (OM), carvacrol (OM), p -cymene (MH). On the other hand, because of sprouting, many VOCs disappeared in the emission bouquet such as ( E )-2-nonenal (NTD), p -anisaldehyde (NTD), n -undecane (NTD), naphthalene (NTD), linalool (OM), carvone (OM), α-humulene (STH), ar -curcumene (STH), and ( E )-anethole (PP). 3.2. Impact of germination on volatiles compounds of dry bean flours To evaluate the impact of germination and milling of dry beans cultivars on the emission of VOCs, raw (Navy beans-F, Black beans-F, and Kidney beans-F), and germinated (G-Navy beans-F, G-Black beans-F, and G-Kidney beans-F) flours were used. For these samples 56 VOCs were identified and classified into seven chemical classes: NTD (18), OM (13), STH (10), MH (8), PP (4), NSD (1), and AC (1). The impact of germination and milling was summarized in Table 1, which revealed that each flour sample had a different volatile emission compared to its corresponding seed. The total percentage identification for Navy beans-F, Kidney beans-F, and Black beans-F were 95.2, 98.3, and 98.9%, respectively. However, for the germinated samples were 99.8, 98.9. and 99.7%, respectively. Fig. 1c and d clearly shows that the most representative classes in un-sprouted Navy beans-F and Black beans-F was MH (53.5, and 43.5%, respectively), following by NTD for the former one (38.6%) and PP for the latter sample (33.5%). A different profile was recorded in Kidney beans-F, which emit much more PP VOCs (30.9%), followed by MH (29.2%) and OM (24.5%) (Fig.1c). One constituent belong to NSD class was identified only in the profile of unsprouted black beans flour (1,2-benzisothiazole). The germination process was substantially accompanied by an increase of NTD constituents in all sprouted flour samples, which is the dominant chemical class emitted by G-Navy beans-F and G-Kidney beans-F (45.3, and 31.2%, respectively) (Fig.1c). The second most representative class in these samples was OM for the first et MH for the second version. However, G-Black beans-F showed a reverse behavior compared to other germinated legume flours. In fact, 40.2% of the total emitted VOCs belonged to MH, 23.4% to PP, and 18.8% to NTD VOCs. Among the 2 NTD VOCs; 12 were identified in raw and 13 in sprouted flours (Table 1). NTD VOCs in flours from raw samples consisted of alkanes (5), aldehydes (3), alcohols (1), esters (1), and divers (2). However, the emission bouquet was changed when the germinated seeds were minced: alkanes (4), aldehydes (3), ketones (1), esters (1), and divers (4). The aldehydes VOCs in flours obtained from raw Navy beans-F, Kidney beans-F and Black beans-F constituted 3.4, 1.5, 0.2%, respectively (Fig.1d). Nevertheless, the emission of these VOCs was increased when the sprouted seeds were minced (20.8, 6.8, and 4.9% for G-Navy beans-F, G-Kidney beans-F and G-Black beans-F, respectively). Regarding the alkanes content, there were significant differences between raw and germinated dry legumes. These VOCs diminished in G-Navy beans-F and increased in other cultivars, when compared to their corresponding raw flours (Fig.1d). Alcohols and esters were totally absent in germinated samples. In the aroma profiles of raw and germinated flours only one constituent was shared by all samples, namely limonene with its lemon like odor (MH) (Table 1). The identification percentages of this volatile were increased after the milling of raw seeds, reaching 53.5, 19.6, 29.5 % for Navy beans-F, Kidney beans-F, and Black beans-F, respectively. However, as a result of germination, the emission of limonene was decreased reaching 16.1, 15.9, and 19%, respectively in sprouted flours. Limonene was the most dominant constituent for all flours except for Kidney beans-F, which emit much more ( E )-anethole (25.9%). The NTD VOCs nonanal and 6-methyl-5-hepten-2-one, with their fruity odor, were detected in the bouquet profile of unsprouted seeds, but when these samples were crushed, these components disappeared. When the sprouted seeds were minced many constituents including benzaldehyde, 6-methyl-5-hepten-2-one, methyl 2-ethylhexanoate, 3.5-octadien-2-one, 1-undecene, 1,8-cineole, and carvacrol newly appeared compared to raw pulse flours. On the contrary, some others disappeared in germinated flours such as γ-nonalactone, p -anisaldehyde, phenylethyl alcohol, naphthalene, and carvone (Table 1). 3.3. Comparison of the volatile constituents among the raw and germinated legume seeds and flours and identification of the discriminating compounds The PCA plot setup for raw and germinated dry bean seeds and flours is shown in Fig.3. The PC1 and PC2 axes explained 61.11% of the total variance (37.60 % and 23.51%, respectively) and were correlated to NTD and AC, respectively. By analyzing the scores-plot in the area defined by both axes, the samples were divided inti four groups. Group 1 is situated in the upper right of the scores-plot and correlated positively to PC1 and PC2 is constituted by Kidney beans-S and Black beans-S. Such a group is characterized by the highest content of PP. Group 2 is placed in the bottom right of the scores-plot, and correlates positively to PC1 and negatively to PC2; it is formed by Navy beans-S, G-Kidney beans-S, G-Kidney beans-S, and G-Navy beans-S. this group is distinguish especially by the emission of NTD volatiles. Group 3 is situated in the left bottom of the scores-plot and correlates negatively to both axes and it formed by Navy beans-F, G-Kidney beans-F, and G-Navy beans-F. These legume versions are characterized by the presence of MH and NSD in their volatile profiles. Group 4 is located in the upper left side of the scores plot and correlated negatively to PC1 and positively to PC2 and it is constituted by Black beans-F, Kidney beans-F, and G-Black beans-F. This group is characterized by the highest content of OM, AP, and PP. 3.4. Correlations among the chemical classes Correlations among the different volatile chemical classes were analyzed to study the relations among them in raw and germinated dry bean seeds and flours. Table 2 shows the coefficients of the Pearson’s correlation among all samples (Table 2). Data demonstrated a significant positive correlation among MH and NSD, MH and OM, and OM and AC (r=0.794; 0.370; 0.392, respectively). A poor positive correlation was also detected among MH and PP, and MH and AP (r=0.111; and 0.155, respectively). A positive correlation means that when one variable moves higher or lower, the other variable moves in the same direction with the same magnitude. However, a negative correlation was observed among STH and OM, and NSD and PP (r= -0.104; and -0.170, respectively). A negative correlation indicates that both variables move in the opposite direction. 3.5. Hierarchical cluster analysis All collected data were submitted to hierarchical cluster analysis to detect the effect of sprouting and crushing on the seeds of dry beans (Fig. 4). The resulting heatmap indicates that samples are distributed in three major clusters: C1 is formed by Navy beans-S, G-Navy beans-S, G-Black beans-S, and G-Kidney beans-S. Cluster 2 is composed by Black beans-S and Kidney beans-S. Finally, cluster 3 is constituted by Black beans-F, Kidney beans-F, Navy beans-F, G-Navy beans-F, G-Black beans-F, and G-Kidney beans-F. The heat map is a colored representation of the data. The red stands indicate the low values of the studied parameters, the black presented the intermediate values, and the green the high values. Discussion Germination is cost-effective process that furnish an important content of bioactive constituents (Duenas et al., 2016, Rajhi et al., 2022c). In addition, germinated beans believed to be a powerful approach to boost antioxidant activities without increasing the legume off-flavors (Xu et al., 2019). Germinated seeds have been widely consumed as food ingredients due to the popular belief that sprouting provides significant nutritional savor benefits through unsprouted grains (Rajhi et al., 2024). Germination is the process by which plant hormones and digestive enzymes such as amylases, proteases, and lipases are produced, causing starch, protein, and lipid destruction, respectively. These alterations can significantly impact the function and quality of sprouted pulses. Indeed, an augmentation of lipase content can stimulate the autoxidation of lipids and then provoke the generation of undesirables’ odors in germinated seeds (Finnie et al., 2019). The identification of VOCs by HS-SPME/GC-MS has shown to be an efficient method to evaluate the effect of sprouting and milling on the VOCs of foods (Oomah et al., 2007; Khrisanapant et al., 2019; Akkad et al., 2019; Rajhi et al., 2023). Xu et al. (2019) found that dietary changes during sprouting are influenced by the food components and the germination conditions. In the present study, the germination process resulted in a noticeable increase of NTD VOCs, due essentially to the clear increase of aldehyde content in all seed and flour samples; with a spectacular augmentation in G-Kidney beans-S (Table 1, Fig. 1, and Fig. 2). This result was also confirmed by Akkad et al. (2021) and Rajhi et al. (2022d and e) who reported that longer sprouting times on faba bean and lentil seeds, have a negative effect since the beany off-flavors increased. Aldehydes are the major VOCs of interest, since these have a great impact on the volatile profile of legumes, and these determine their distinctive flavor. Aldehydes, the carbonyl derivatives, including heptanal (which has a fatty, citrusy, and rancid odor), benzaldehyde (with an almond and burnt sugar aroma), and octanal (with its distinctive fat, soapy, lemon and green aroma), were identified only in sprouted beans compared to unsprouted versions (Qiao et al., 2008). Additionally, VOCs such as nonanal (fatty and citrus-like odors) and decanal (sweet and floral aroma) were detected in the emission bouquet of sprouted dry bean flours compared to their corresponding unsprouted ones (Acree and Arn, 2004). Food flavor can be substantially influenced by lipid oxidation, which provoke the generation of potent odorants such as saturated and unsaturated aldehydes (Belitz et al., 2009). Conversely to aldehydes, alkanes make only a small contribution to the general food aroma (Fahlbusch et al., 2003). Alkanes were identified as the second most abundant VOCs emitted by raw and germinated seeds. The germination did not affect the emission of this volatile. These results are in good agreement with a previous investigation of VOCs of other legume in particular faba bean cultivars (Akkad et al., 2021; Rajhi et al., 2021). An exception was recorded for G-Black beans-S; which emit much more alkanes compared to its corresponding raw version. This high content in alkanes, especially in dry beans, was also previously reported by Oomah et al. (2007) for different types of P. vulgaris . Their abundance may be explained by the occurrence of lipid peroxidation, which causes the formation of the characteristic aroma of dry legumes, since alkanes are mainly obtained from oxidative reaction of lipids (Shahidi et al., 1986). Additionally, comparing the volatile profiles of two different chickpeas (desi and kabuli), alkanes were considered as one of their main aroma constituents (Ghosh et al., 2020). Four alcohols were newly emitted by sprouted seeds compared to raw ones (Table 1). Alcohols in legumes are the result of the oxidation of lipids due to the presence of dehydrogenases. In dry beans, some alcohols were associated to their particular aroma, i.e. grassy and green ones (Shahidi et al., 1986). In general, alcohols are undesirable ones, being considered causes of off-flavors (Khrisanapant et al., 2019). The milling process was accompanied by a decrease of aldehydes and alkanes. A comparable trend was registered in milled brown rice; the lipid-derived compounds such aldehydes, alkanes, and alcohols decreased as the degree milling increased (Sun and Siebenmorgen, 1994). The products of the oxidative degradation of unsaturated fatty acids and amino acids including octanal which has a chemical, metallic and burnt odor, nonanal which has a fat, citrusy, and green smell, and decanal with its soapy, orange peel and tallow-like odor, and green flavor, were more abundant in germinated seeds and flours than in raw ones (Akkad et al., 2021). Different classes of terpenes were also characterized in this study, including OM, MH and STH (Table 1). The profiles of raw and sprouted seeds and flours were quite different. As a result of germination, the identification percentages of all terpenes were decreased in sprouted seeds compared to raw versions. Nevertheless, the milling was accompanied with an important augmentation of MH VOCs. A different result was presented by Rajhi et al. (2022d), when they studied the effect of germination on the VOCs of faba beans. Indeed, they demonstrate that flour samples showed a similar trend compared to the seed versions, especially as regards the appearance of terpenes volatiles. Unlike NTD VOCs, which are formed after lipid peroxidation, terpenes are obviously biosynthesized in legumes (Wink, 2013). The most abundant terpene in the investigated legumes was limonene, which is present in all samples. This volatile compound has a pleasant citrus and fresh odor (Mosciano, 2000). This result in good agreement with the volatile profile that was previously characterized in P. vulgaris (Oomah et al., 2007). Conclusion The effect of sprouting and milling on the aroma attributes of three dry bean cultivars using headspace SPME sampling coupled to GC-MS system was assigned. The individual volatile profiles of studied legume seeds and flours varied not only by the type of treatment (germination and milling) but also between the varieties. Germination is a biological process in which the bioavailability of nutrients like protein will be increased, the anti-nutrients like tannins that are known as contributors to the bitterness of certain legumes, will be decreased. In this study, we observed that NTD volatiles appeared in all cultivars, and they were considered as the abundant chemical class for Navy beans-S and all sprouted seeds and flours except for G-Black beans-F. At the same time, MH constituents, which was the principal chemical class emitted by unsprouted Navy and Black beans; significantly increased during the germination process. Additionally, the PCA analysis was performed to discriminate among dry bean cultivars and their flours. The finding from this study is the identification of the discriminated constituents for each cultivar and the corresponding flour, under sprouting conditions. This data could be used in legume-based receipt to impart desirable aroma properties based on the presence of certain volatile compounds. Declarations Funding: This research received no external funding. References Acree, T., Arn, H., Flavornet and Human Odor Space. http://www.flavornet.org/ f lavornet.html. Akkad, R., Kharraza, E., Hanb, J., Housec, J.D., Curtisa, J.M., 2019. Characterization of the volatile flavor compounds in low and high tannin faba beans (Vicia faba var. minor) grown in Alberta, Canada. Food Res. Int. 120, 285–294. Akkad, R., Buchko, A., Johnston, S.P., Han, H., House, J.D., Curtis, J.M., 2021. Sprouting improves the flavour quality of faba bean flours. Food Chem. 364. Ascrizzi, R., Gonz´alez-Rivera, J., Pomelli, C.S., Chiappe, C., Margari, P., Costagli, F., 2017. Ionic liquids, ultra-sounds and microwaves: An effective combination for a sustainable extraction with higher yields. The cumin essential oil case. React. Chem. Eng. 2, 577–589. Belitz, H. D., Grosch, W., Schieberle, P. (2009). Lipids. In Food chemistry (4th ed., pp. 158–247). Berlin: Springer-Verlag. Davies, N.W., 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on Methyl Silicon and Carbowax 20M phases. J. Chromatogr. A. 503, 1–24. Duenas, M., Sarmento, T., Aguilera, Y., Benitez, V., Moll, E., Esteban, R.M., Martín- Cabrejas, M.A., 2016. Impact of cooking and germination on phenolic composition and dietary fibre fractions in dark beans (Phaseolus vulgaris L.) and lentils (Lens culinaris L.). LWT - Food Sci. TECHNOL 66, 72–78. Fahlbusch, K.G., Hammerschmidt, F.J., Panten, J., Pickenhagen, W., Schatkowski, D., Bauer, K., Surburg, H., 2003. Flavours and fragrances. Ullmann’s Encyclopedia of industrial chemistry (6). 15. Ullmann’s Encyclopedia of industrial chemistry (73–198). Wiley-VCH Verlag, Weinheim. Ghosh, A., Dadhich, A., Bhardwaj, P. J., Babu, N., & Kumar, V. (2020). Comparative analysis of metabolites in contrasting chickpea cultivars. Journal of Plant Biochemistry and Biotechnology, 29(2), 253–265. Finnie, S., Brovelli, V., Nelson, D., 2019. Sprouted grains as a food ingredient. In H. Feng, B. Nemzer, & J. W. DeVries (Eds.), Sprouted grains: Nutritional value, production, and applications. AACC International, pp. 113–142. Kouris-Blazos, A., and Belski, R. (2016). Health benefits of legumes and pulses with a focus on Australian sweet lupins. Asia Pacific Journal of Clinical Nutrition, 21(1), 1–17. Khrisanapant, P., Kebede, B., Ying Leong, S., Indrawati, O., 2019. A comprehensive characterisation of volatile and fatty acid profiles of legume seeds. Foods 8, 651. Messina, M. J. (2016). Legumes and soybeans: Overview of their nutritional profiles and health effects. Asia Pacific Journal of Clinical Nutrition, 25(1), 1–17. Oomah, B.D., Liang, L.S.Y., Balasubramanian, P., 2007. Volatile Compounds of dry beans (Phaseolus vulgaris L.). Plant Foods Hum. Nutr. 62, 177. Qiao, Y., Xie, B.J., Zhang, Y., Zhang, Y., Fan, G., Yao, X.L., Pan, S.Y., 2008. Characterization of aroma active compounds in fruit juice and peel oil of Jinchen sweet orange fruit (Citrus sinensis (L.) Osbeck) by GC-MS and GC-O. Molecules 13 (6), 1333–1344. Rajhi I., Ben Mansour R., Mhadhbi H.: Germination Effect on Phenolic Composition and Antioxidants Activities of Faba Bean Cultivar. JMRHS 7 (4), 3130-3136 (2024) Rajhi I., Baccouri B., Rajhi F., Guido F., Mhadhbi H.: Monitoring the Aroma Compounds of Vicia faba L var. Major and var. Minor. Case Studies of Breeding Strategies in Major Plant Species. IntechOpen, 2023. Rajhi I., Baccouri B., Rajhi F., Mhadhbi H., Flamini G.: HS-SPME-GC-MS characterization of volatile chemicals released from microwaving and conventional processing methods of fenugreek seeds and flours. Ind. Crops Prod. 182 : 114824, 2022a. https://doi.org/10.1016/j.indcrop.2022.114824 Rajhi I., Boulaaba M., Baccouri B., Rajhi F., Mhadhbi H., Flamini G.: Assessment of dehulling effect on volatiles, phenolic compounds, and antioxidant activities of faba bean seeds and flours. S. Afr. J. Bot. 147 : 741-753, 2022b. Rajhi I., Ben Mansour R., Baccouri B., Amri M., Mhadhbi H.: Sprouting characteristics and associated changes in antioxidant activities and phenolic composition of faba bean cultivars. Agrochimica. 60 (4), 2022c. Rajhi I., Baccouri B., Rajhi F., Hammami J., Souibgui M., Amri M. Mhadhbi H., Flamino G.: Evaluation of germination effect on volatile compounds of different faba bean cultivars using HS-SPME/GC-MS. J. Food Compos. Anal. 112 : 104692, 2022d. https://doi.org/10.1016/j.jfca.2022.104692 Rajhi I., Baccouri B., Rajhi F., Mhadhbi H., Flamini G.: Monitoring the volatile compounds status of whole seeds and flours of legume cultivars. Food Biosci. 41 : 101-105, 2021. https://doi.org/10.1016/j.fbio.2021.101105 Rajhi I., Baccouri B., Rajhi F., Hammami J., Abbes Z., Mhadhbi H., Flamino G.: HS-SPME-GC-MS combined with chemometrics to assess the impact of germination, dehulling, and milling on flavor attributes of brown and green lentils (Lens culinaris subsp. culinaris). S. Afr. J. Bot. 150 : 1102-1110, 2022e. Roland, W.S.U., Pouvreau, L., Curran, J., van de Velde, F., de Kok, P.M.T., 2017. Flavor aspects of pulse ingredients. Cereal Chem. 94, 58–65. Shahidi, F., Rubin, L. J., D’Souza, L. A. (1986). Meat flavour volatiles: A review of the composition, techniques of analysis, and sensory evaluations. Critical Reviews in Food Science and Nutrition, 24, 141–243. Sun, H., and Siebenmorgen, T. J. 1993. Rice milling quality affected by kernel thickness. Cereal Chem. 70:727-733. Simons, R., 2011. Prenylated Isoflavonoids from Soya and Licorice: Analysis, Induction and In Vitro Estrogenicity. Ph.D. thesis. Wageningen University, Wageningen, the Netherlands. Uebersax, M.A.; Cichy, K.A.; Gomez, F.E.; Porch, T.G.; Heitholt, J.; Osorno, J.M.; Kamfwa, K.; Snapp, S.S.; Bales, S. Dry beans (Phaseolus vulgaris L.) as a vital component of sustainable agriculture and food security—A review. Legume Sci. 2023, 5, e155. Xu, M., Jin, Z., Lan, Y., Rao, J., Chen, B., 2019. HS-SPME/GC-MS/Olfactometry Combined with Chemometrics to Assess the Impact of Germination on Flavor Attributes of Chickpea, Lentil, and Yellow Pea Flours. Food Chem. 280, 83–95. Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5024199","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":353792031,"identity":"524364be-d37a-4321-a3a4-a973687ddd77","order_by":0,"name":"Imene Rajhi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACNiiVwMbewMAMYgJpYrXwHIBoAdLEgQQGiQQitfCJHT72uKKGL49P8o3h54IKGwYeaQJ62KTT0g3PHGMrZpPOMZaecSaNgYcvgZCWHDPJBja2xDbpHANp3rbDDPY8BBzGJp3/TbLhH1CL5Bnj3yAtPIS15LBJNrYBtUjwmEkTqSXNTLKxD6iFJ63MmudMGg9BLfKzk59JNnw7lji//fDm2zwVNnIEtUDBMSDmMACxiNTAwFADxOwPiFU9CkbBKBgFIwwAANcSNX2ZNGahAAAAAElFTkSuQmCC","orcid":"","institution":"Center of Biotechnogy of Borj Cédria","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Imene","middleName":"","lastName":"Rajhi","suffix":""},{"id":353792032,"identity":"b0027be2-4f65-4f57-b67b-bc39e51ce5c9","order_by":1,"name":"Rim Nefissi Ouertani","email":"","orcid":"","institution":"Center of Biotechnogy of Borj Cédria","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rim","middleName":"Nefissi","lastName":"Ouertani","suffix":""},{"id":353792033,"identity":"6eb512b6-e9ac-4e27-829a-2c241a3e0648","order_by":2,"name":"Fatma Rajhi","email":"","orcid":"","institution":"University of Gabès","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fatma","middleName":"","lastName":"Rajhi","suffix":""},{"id":353792034,"identity":"b59ed0ff-62ff-4762-a8e4-092fb4c370ee","order_by":3,"name":"Haythem Mhadhbi","email":"","orcid":"","institution":"Center of Biotechnogy of Borj Cédria","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haythem","middleName":"","lastName":"Mhadhbi","suffix":""},{"id":353792035,"identity":"5ec6daa1-76d0-4604-939f-feee1c40b370","order_by":4,"name":"Guido Flamini","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guido","middleName":"","lastName":"Flamini","suffix":""}],"badges":[],"createdAt":"2024-09-03 10:30:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5024199/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5024199/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64656064,"identity":"da5cb8b6-50d3-4c72-be7f-48a728d0f0b2","added_by":"auto","created_at":"2024-09-17 06:51:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24981,"visible":true,"origin":"","legend":"\u003cp\u003ePercentages of different chemical classes emitted by un-sprouted seeds (a), sprouted seeds (b), unsprouted flours (c), and sprouted flours (d) using SPME-GC-MS. With MH: monoterpene hydrocarbons, OM: oxygenated monoterpenes, STH: sesquiterpene hydrocarbons, AP: apocarotenes, NSD: nitrogen/sulfur derivatives, and NTD: non-terpene derivatives.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5024199/v1/583db8571fb1254833784db8.png"},{"id":64656066,"identity":"edc5119b-e1e2-4dda-876a-bf85101ba7fc","added_by":"auto","created_at":"2024-09-17 06:51:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18029,"visible":true,"origin":"","legend":"\u003cp\u003ePercentages of different groups of NTD VOCs emitted by un-sprouted seeds (a), sprouted seeds (b), unsprouted flours (c), and sprouted flours (d) using SPME-GC-MS.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5024199/v1/d2e52e6eb0c4b84447045f08.png"},{"id":64656605,"identity":"3d79f172-f019-48b0-8818-0ffcc4df1a43","added_by":"auto","created_at":"2024-09-17 06:59:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32035,"visible":true,"origin":"","legend":"\u003cp\u003ePlots from the PCA demonstrating the contribution of the volatile chemical classes and the grouping of raw (Navy beans-S, Kidney beans-S, Black beans-S, Navy beans-F, Kidney beans-F, and Black beans-F) and germinated (G-Navy beans-S, G-Kidney beans-S, G-Black beans-S, G-Navy beans-F, G-Kidney beans-F, and G-Black beans-F) samples. With: G: Germinated, S: Seeds, and F: Flours.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5024199/v1/d95101e7ebd3cb190a10caf5.png"},{"id":64656067,"identity":"0f7dee22-fe9f-4858-9c53-1801e154114d","added_by":"auto","created_at":"2024-09-17 06:51:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26419,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap based on Euclidean distances using different chemical classes of of raw (Navy beans-S, Kidney beans-S, Black beans-S, Navy beans-F, Kidney beans-F, and Black beans-F) and germinated (G-Navy beans-S, G-Kidney beans-S, G-Black beans-S, G-Navy beans-F, G-Kidney beans-F, and G-Black beans-F) samples. The different clusters C1, C2, and C3 correspond to the individualized groups. With: G: Germinated, S: Seeds, and F: Flours\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5024199/v1/5fb488cf08301d6a5ac2729e.png"},{"id":64657415,"identity":"80ea59b1-2681-47ed-89f8-ab34cdd6d051","added_by":"auto","created_at":"2024-09-17 07:07:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":521313,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5024199/v1/7296f8c9-d34b-42a5-bb4f-13604ea5f6e7.pdf"},{"id":64656065,"identity":"74d504cb-6648-4fe5-bf21-2c8a28929ab9","added_by":"auto","created_at":"2024-09-17 06:51:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":50610,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5024199/v1/a42996256a1149e19f689ee2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFingerprinting of volatile profiles of sprouted and unsprouted seeds and flours of \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e using HS-SPME/GC-MS\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e, also known as legumes, are the second largest plant family (Kouris-Blazos et al., 2016). The Fabaceae are a large family of approximately 18,000 plant species, including herbs, trees, climbers, and shrubs. However, humans consume only a limited number of species (Rajhi et al., 2021). Legumes are divided into two categories namely pulses and oilseeds. The term pulses refer to the dried seeds, including\u0026nbsp;peas (\u003cem\u003ePisum sativum\u003c/em\u003e), lentils (\u003cem\u003eLens culinaris\u003c/em\u003e), lupins (\u003cem\u003eLupinus spp\u003c/em\u003e.), chickpeas (\u003cem\u003eCicer arietinum\u003c/em\u003e), common beans (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e), faba beans (\u003cem\u003eVicia faba\u003c/em\u003e L.) and fenugreek (\u003cem\u003eTrigonella foenum-graecum\u003c/em\u003e L.)\u0026nbsp;and the second group is formed by soybeans\u0026nbsp;(\u003cem\u003eGlycine max\u003c/em\u003e) and peanuts (\u003cem\u003eArachis hypogaea\u003c/em\u003e) (Roland et al., 2017). Additionally, legumes are considered as the most important source of nutrients after cereals. In fact, they are esteemed as a low-priced and sustainable meat alternative in developing countries (Kouris-Blazos et al., 2016).\u0026nbsp;Pulses are a valuable food, providing 20-45% of protein, including essential amino acids, 60% of carbohydrates and 5-37% of fiber. In addition, legumes have an important nutraceutical advantage due essentially to their content of bioactive compounds protecting against cancer, cardiovascular and degenerative diseases (Messina, 2016). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDry beans (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e) are well known as a nutrient-dense plant food which furnish exceptional nutrient content. At approximately 22%, dry beans have nearly double the amount of protein compared with common cereal grains, with a lower content of carbohydrates and fat (Uebersax et al., 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNevertheless, the use of pulses in the culinary arts is restricted due to the existence of a specific off-flavors (Rolland et al, 2017). The undesirable odors of unprocessed legume seeds, such as, beany, grassy, earthy, and leafy are due to the generation of volatiles organic compounds (COVs) from the oxidation of fatty acid catalyzed by lipoxygenase during harvest, processing, and storage conditions (Rajhi et al., 2022a). In general, the pulse undesirable odors constituents are associated to aldehydes, alcohols, pyrazines, terpenes, ketones, and acids. A satisfactory flavor is a critical feature of any merchandise. To enhance the pulse consumption worldwide, it might be essential to establish technologies to improve their odors including heat processing, dehulling, fermentation, milling, and germination (Rajhi et al., 2022b). Germination ameliorates the nutritional properties of seed pulses by decreasing anti-nutritional factors, such as phytases, trypsin inhibitors, and unwanted beany flavors produced by the oxidation of unsaturated fatty acids by lipoxygenase (Simons, 2011).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn general, the information in the literature related to the off-flavors in legumes is limited. Thus, the aims of this work were (1) to investigate the impact of germination and milling on the volatile compounds (VOCs) of \u0026nbsp;three dry beans cultivars (navy, kidney, and black) using headspace solid phase microextraction coupled to gas chromatography with mass spectrometry (HS-SPME/GC-MS) and (2) to determinate the discriminating compounds in raw and germinated seeds and flours using a multivariate analysis (PCA).\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eChemicals and reagents\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 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,\u0026nbsp;\u0026beta;-pinene, p-cymene, limonene,\u0026nbsp;\u0026gamma;-terpinene, 1,8-cineole,\u0026nbsp;\u0026alpha;-terpineol, verbenone, bornyl acetate, longifolene, geranylacetone (E + Z isomers), 1-octanol, 1-decanol, ethyl benzoate, 2-undecanone,\u0026nbsp;\u0026alpha;-thujone +\u0026nbsp;\u0026beta;-thujone mixture, and pulegone were purchased from Sigma, Aldrich, Supelco and Merck and used to compare retention times and mass spectra.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree cultivars of dry bean seeds (Navy, Kidney, and Black beans) were used in this study. Similar size seeds, without any physical damages, were selected. They were stored at 4\u0026deg;C in an opaque aluminum bag until analysis. The seeds were stored at 4\u0026deg;C in an opaque aluminum bag until analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGermination and milling processes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the germination process, raw seeds\u0026nbsp;were surface disinfected in HgCl\u003csub\u003e2\u003c/sub\u003e (0.1%) for 1 min and then rinsed thoroughly using sterilized distilled water. The moisturized seeds were sown in petri dishes containing laboratory paper moistened with water. The petri dishes were then incubated into the germinator at 20\u0026deg;C, 99 % humidity, and in dark. Five days later, the sprouted seeds were dried in a vacuum oven at 45\u0026deg;C for 6 h.\u0026nbsp;The raw seeds were named Navy beans-S, Black beans-S, and Kidney beans-S.\u0026nbsp;The germinated seeds were named G-Navy beans-S, G-Black beans-S, and G-Kidney beans-S.\u003c/p\u003e\n\u003cp\u003eTo obtain the flour,\u0026nbsp;30 g\u0026nbsp;of each type of\u0026nbsp;legume seeds were crushed using a domestic blender at room temperature. Then, the flour was passed through a 100-mesh sieve. All the analyses were performed in triplicate. The crushed raw and germinated flours were named, respectively, Navy beans-F, Black beans-F, Kidney beans-F, G-Navy beans-F, G-Black beans-F, and G-Kidney beans-F.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHeadspace solid-phase micro extraction (HS-SPME)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe headspace spontaneous volatile emissions of the whole and crushed seeds were sampled by means of HS‐SPME. Triplicates were assessed for each sample. Each replicate was let to equilibrate for 30 min at room temperature before sampling. A Solid Phase Micro‐Extraction (SPME) device (Supelco, Bellefonte, PA, USA) coated with poly-dimethyl-siloxane (PDMS, 100\u0026nbsp;\u0026mu;m) was utilized, preconditioned according to the manufacturer instructions. Then, the fiber was withdrawn into the needle and transferred to the injection port of the GC‐MS system. The desorption conditions were identical for all the samples. Additionally, blanks were carried out before each first SPME extraction, and randomly repeated during each series. Quantitative comparisons of relative peaks areas were performed between the same chemicals in the different samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGas Chromatography Coupled with Mass Spectrometry (GC\u003c/strong\u003e\u003cstrong\u003e‐\u003c/strong\u003e\u003cstrong\u003eMS)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGas 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 column (30 m \u0026times; 0.25 mm: coating thickness 0.25\u0026nbsp;\u0026mu;m) 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 \u0026deg;C, respectively; oven temperature programmed from 60 to 240 \u0026deg;C at 3 \u0026deg;C/min; carrier gas helium at 1 mL/min; split ratio 1:25. The acquisition parameters were as follows: full scan; scan range: 30\u0026ndash;300 m/z; scan time: 1.0 s (Ascrizzi et al. 2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompounds identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe identification of the volatile compounds was based on a comparison of the retention times with those of the authentic samples, comparing their linear retention indices (LRI) relative to the series of \u003cem\u003en\u003c/em\u003e‐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 (Davies 1990).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrincipal Component Analysis\u0026nbsp;(\u003cem\u003eXLSTAT\u0026nbsp;\u003c/em\u003esoftware version 2014), and clustering were used to analyze data. Principal Component Analysis (PCA) was implemented using the two sets of data from whole seeds and flours of legumes. Every incorporated matrix involved 7 chemical classes in columns and different types of cultivars in rows. The PCA analysis was carried out to (1) detect differences and similarities between the two different legume cultivars as well as to evidence possible correlation between chemical classes and legumes, (2) determine grouping and separation of cultivars and, finally, (3) identification of discriminant compound related to each cultivar for both forms.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOverall, 79 VOCs were identified by HS-SPME-GC-MS in the emission profiles of raw and sprouted seeds of different legume cultivars. The detected and identified VOCs belonged to seven chemical classes, including monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), sesquiterpene hydrocarbons (STH), phenylpropanoids (PP), apocarotenes (AC), nitrogen/sulfur derivatives (NSD), and non-terpene derivatives (NTD).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe emitted VOCs were identified using HS-SPME/GC-MS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1. Impact of germination on volatiles compounds of dry bean seeds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the impact of germination of dry bean cultivars on the emission of volatile compounds, raw seeds (Navy beans-S, Black beans-S, and Kidney beans-S) and germinated seeds (G-Navy beans-S, G-Black beans-S, and G-Kidney beans-S), were used.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn total, 40 VOCs were identified in raw seeds: 18 for Navy beans-S, 28 for Kidney beans-S, and 30 for Black beans-S; they accounted for 98.1, 99.0, and 97.3% of total emissions, respectively. In sprouted seeds, 31 VOCs were characterized as follows: 25 for G-Navy beans-S and G-Black beans-S, and 14 for G-Kidney beans-S, accounted for 99.8, 95.6, and 97.1%, of the total emissions, respectively. As shown in Figure 1a, the most abundant chemical classes were NTD for Navy beans-S (61.4%), PP for Kidney beans-S (43.0%), and STH for Black beans-S (41.9%) following by MH (11.8%), OM (22.6%), and NTD (19.1%), respectively. The third most representative chemical classes for these samples were PP (10.2%), NTD (15.6%), and PP (16.9%), respectively. In the case of VOCs emitted by the germinated seeds, the number was as follow: 25 for G-Navy beans-S, 14 for G-Kidney beans-S, and 25 for G-Black beans-S, accounting for 99.8, 97.1, and 95.6% of the total emissions, respectively. The individual volatile profiles of raw and germinated seeds were quite different. In sprouted seeds, the composition of VOCs was changed. The germination process was accompanied by an increase of NTD constituents for all dry bean samples. Figure 1b shows that the most abundant chemical class for G-Navy beans-S, G-Kidney beans-S, and G-Black beans-S was NTD (79.8, 83.6, 68.5%, respectively), following by AP (6.4%), OM (10.6, and 12.2%), respectively. As regards to the VOCs belong to STH class, they were totally absent in G-Navy beans-S and G-Kidney beans-S and were present only in small amount in G-Black beans-S (2.5%). In the case of PP constituents, these were identified in small amounts in all germinated cultivars compared to raw samples (Figure 1a and b). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the identified VOCs, 39 NTD constituents were emitted by all samples: 10 for unsprouted Navy beans-S, 8 for Kidney beans-S,13 for Black beans-S, 19 for G-Navy beans-S, 9 for G-Kidney beans-S, and 12 for G-Black beans-S (Table 1). Nevertheless, the respective emissions were quite different. Figure 2 shows the different percentages identification of NTD components in sprouted legumes compared to their corresponding unsprouted versions. It can be seen from Fig.2 a and b that seed germination was accompanied by an increase of aldehydes for the three cultivars. An augmentation of the emission of alkanes, ketones, alcohols and esters was recorded especially in the profile of G-Black beans-S (Fig. 2a and b). A different profile was monitored in Navy and Black beans.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the aroma profiles of raw and germinated beans seeds only 3 VOCs were shared the six versions of beans, such as nonanal (NTD), decanal (NTD), and limonene (MH) (Table 1). Naphthalene (NTD), (\u003cem\u003eE\u003c/em\u003e)-Anethole, and \u0026beta;-caryophyllene presented the highest percentages in Navy beans-S, Kidney beans-S and Black beans-S, respectively. However, they dropped dramatically as the seeds germinated. A general increase in the percentages of nonanal, decanal and \u003cem\u003en\u003c/em\u003e-tetradecane was observed in sprouted seeds. Indeed, nonanal becomes the dominant emitted volatiles in all germinated cultivars. As the seeds germinated, new constituents were emitted, including heptanal (NTD), benzaldehyde (NTD), octanal (NTD), 2-ethyl-1-hexanol (NTD), 6-methylheptyl 2-propenoate (NTD), 1-undecene (NTD), (\u003cem\u003eZ\u003c/em\u003e)-3-pentadecene (NTD), 1,8-cineole (OM), dihydrocitronellol (OM), carvacrol (OM), \u003cem\u003ep\u003c/em\u003e-cymene (MH). On the other hand, because of sprouting, many VOCs disappeared in the emission bouquet such as (\u003cem\u003eE\u003c/em\u003e)-2-nonenal (NTD),\u0026nbsp;\u003cem\u003ep\u003c/em\u003e-anisaldehyde (NTD), \u003cem\u003en\u003c/em\u003e-undecane (NTD), naphthalene (NTD), linalool (OM), carvone (OM),\u0026nbsp;\u0026alpha;-humulene (STH),\u0026nbsp;\u003cem\u003ear\u003c/em\u003e-curcumene (STH), and (\u003cem\u003eE\u003c/em\u003e)-anethole (PP). \u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Impact of germination on volatiles compounds of dry bean flours\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the impact of germination and milling of dry beans cultivars on the emission of VOCs, raw (Navy beans-F, Black beans-F, and Kidney beans-F), and germinated (G-Navy beans-F, G-Black beans-F, and G-Kidney beans-F) flours were used. For these samples 56 VOCs were identified and classified into seven chemical classes: NTD (18), OM (13), STH (10), MH (8), PP (4), NSD (1), and AC (1). The impact of germination and milling was summarized in Table 1, which revealed that each flour sample had a different volatile emission compared to its corresponding seed. The total percentage identification for Navy beans-F, Kidney beans-F, and Black beans-F were 95.2, 98.3, and 98.9%, respectively. However, for the germinated samples were 99.8, 98.9. and 99.7%, respectively. Fig. 1c and d clearly shows that the most representative classes in un-sprouted Navy beans-F and Black beans-F was MH (53.5, and 43.5%, respectively), following by NTD for the former one (38.6%) and PP for the latter sample (33.5%). A different profile was recorded in Kidney beans-F, which emit much more PP VOCs (30.9%), followed by MH (29.2%) and OM (24.5%) (Fig.1c). One constituent belong to NSD class was identified only in the profile of unsprouted black beans flour (1,2-benzisothiazole). \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe germination process was substantially accompanied by an increase of NTD constituents in all sprouted flour samples, which is the dominant chemical class emitted by G-Navy beans-F and G-Kidney beans-F (45.3, and 31.2%, respectively) (Fig.1c). The second most representative class in these samples was OM for the first et MH for the second version. However, G-Black beans-F showed a reverse behavior compared to other germinated legume flours. In fact, 40.2% of the total emitted VOCs belonged to MH, 23.4% to PP, and 18.8% to NTD VOCs. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the 2 NTD VOCs; 12 were identified in raw and 13 in sprouted flours (Table 1). NTD VOCs in flours from raw samples consisted of alkanes (5), aldehydes (3), alcohols (1), esters (1), and divers (2). However, the emission bouquet was changed when the germinated seeds were minced: alkanes (4), aldehydes (3), ketones (1), esters (1), and divers (4). The aldehydes VOCs in flours obtained from raw Navy beans-F, Kidney beans-F and Black beans-F constituted 3.4, 1.5, 0.2%, respectively (Fig.1d). Nevertheless, the emission of these VOCs was increased when the sprouted seeds were minced (20.8, 6.8, and 4.9% for G-Navy beans-F, G-Kidney beans-F and G-Black beans-F, respectively). Regarding the alkanes content, there were significant differences between raw and germinated dry legumes. These VOCs diminished in G-Navy beans-F and increased in other cultivars, when compared to their corresponding raw flours (Fig.1d). Alcohols and esters were totally absent in germinated samples. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the aroma profiles of raw and germinated flours only one constituent was shared by all samples, namely limonene with its lemon like odor (MH) (Table 1). The identification percentages of this volatile were increased after the milling of raw seeds, reaching 53.5, 19.6, 29.5 % for Navy beans-F, Kidney beans-F, and Black beans-F, respectively. However, as a result of germination, the emission of limonene was decreased reaching 16.1, 15.9, and 19%, respectively in sprouted flours. Limonene was the most dominant constituent for all flours except for Kidney beans-F, which emit much more (\u003cem\u003eE\u003c/em\u003e)-anethole (25.9%). The NTD VOCs nonanal and 6-methyl-5-hepten-2-one, with their fruity odor, were detected in the bouquet profile of unsprouted seeds, but when these samples were crushed, these components disappeared. When the sprouted seeds were minced many constituents including benzaldehyde, 6-methyl-5-hepten-2-one, methyl 2-ethylhexanoate, 3.5-octadien-2-one, 1-undecene, 1,8-cineole, and carvacrol newly appeared compared to raw pulse flours. On the contrary, some others disappeared in germinated flours such as\u0026nbsp;\u0026gamma;-nonalactone, \u003cem\u003ep\u003c/em\u003e-anisaldehyde, phenylethyl alcohol, naphthalene, and carvone (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Comparison of the volatile constituents among the raw and germinated legume seeds and flours and identification of the discriminating compounds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PCA plot setup for raw and germinated dry bean seeds and flours is shown in Fig.3. The PC1 and PC2 axes explained 61.11% of the total variance (37.60 % and 23.51%, respectively) and were correlated to NTD and AC, respectively. By analyzing the scores-plot in the area defined by both axes, the samples were divided inti four groups. Group 1 is situated in the upper right of the scores-plot and correlated positively to PC1 and PC2 is constituted by Kidney beans-S and Black beans-S. Such a group is characterized by the highest content of PP. Group 2 is placed in the bottom right of the scores-plot, and correlates positively to PC1 and negatively to PC2; it is formed by Navy beans-S, G-Kidney beans-S, G-Kidney beans-S, and G-Navy beans-S. this group is distinguish especially by the emission of NTD volatiles. Group 3 is situated in the left bottom of the scores-plot and correlates negatively to both axes and it formed by Navy beans-F, G-Kidney beans-F, and G-Navy beans-F. These legume versions are characterized by the presence of MH and NSD in their volatile profiles. Group 4 is located in the upper left side of the scores plot and correlated negatively to PC1 and positively to PC2 and it is constituted by Black beans-F, Kidney beans-F, and G-Black beans-F. This group is characterized by the highest content of OM, AP, and PP. \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Correlations among the chemical classes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrelations among the different volatile chemical classes were analyzed to study the relations among them in raw and germinated dry bean seeds and flours. Table 2 shows the coefficients of the Pearson\u0026rsquo;s correlation among all samples (Table 2). Data demonstrated a significant positive correlation among MH and NSD, MH and OM, and OM and AC (r=0.794; 0.370; 0.392, respectively). A poor positive correlation was also detected among MH and PP, and MH and AP (r=0.111; and 0.155, respectively). A positive correlation means that when one variable moves higher or lower, the other variable moves in the same direction with the same magnitude. However, a negative correlation was observed among STH and OM, and NSD and PP (r= -0.104; and -0.170, respectively). A negative correlation indicates that both variables move in the opposite direction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Hierarchical cluster analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll collected data were submitted to hierarchical cluster analysis to detect the effect of sprouting and crushing on the seeds of dry beans (Fig. 4). The resulting heatmap indicates that samples are distributed in three major clusters: C1 is formed by Navy beans-S, G-Navy beans-S, G-Black beans-S, and G-Kidney beans-S. Cluster 2 is composed by Black beans-S and Kidney beans-S. Finally, cluster 3 is constituted by Black beans-F, Kidney beans-F, Navy beans-F, G-Navy beans-F, G-Black beans-F, and G-Kidney beans-F. The heat map is a colored representation of the data. The red stands indicate the low values of the studied parameters, the black presented the intermediate values, and the green the high values.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGermination is cost-effective process that furnish an important content of bioactive constituents (Duenas et al., 2016, Rajhi et al., 2022c). In addition, germinated beans believed to be a powerful approach to boost antioxidant activities without increasing the legume off-flavors (Xu et al., 2019). Germinated seeds have been widely consumed as food ingredients due to the popular belief that sprouting provides significant nutritional savor benefits through unsprouted grains (Rajhi et al., 2024).\u0026nbsp;Germination is the process by which plant hormones and digestive enzymes such as amylases, proteases, and lipases are produced, causing starch, protein, and lipid destruction, respectively. These alterations can significantly impact the function and quality of sprouted pulses. Indeed, an augmentation of lipase content can stimulate the autoxidation of lipids and then provoke the generation of undesirables\u0026rsquo; odors in germinated seeds\u0026nbsp;(Finnie et al., 2019).\u003c/p\u003e\n\u003cp\u003eThe identification of VOCs by HS-SPME/GC-MS has shown to be an efficient method to evaluate the effect of sprouting and milling on the VOCs of foods (Oomah et al., 2007; Khrisanapant et al., 2019; Akkad et al., 2019; Rajhi et al., 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXu et al. (2019) found that dietary changes during sprouting are influenced by the food components and the germination conditions. In the present study, the germination process resulted in a noticeable increase of NTD VOCs, due essentially to the clear increase of aldehyde content in all seed and flour samples; with a spectacular augmentation in G-Kidney beans-S (Table 1, Fig. 1, and Fig. 2).\u0026nbsp;This result was also confirmed by Akkad et al. (2021) and Rajhi et al. (2022d and e) who reported that longer sprouting times on faba bean and lentil seeds, have a negative effect since the beany off-flavors increased.\u0026nbsp;Aldehydes are the major VOCs of interest, since these have a great impact on the volatile profile of legumes, and these determine their distinctive flavor. Aldehydes, the carbonyl derivatives, including heptanal (which has a fatty, citrusy, and rancid odor), benzaldehyde (with an almond and burnt sugar aroma), and octanal (with its distinctive fat, soapy, lemon and green aroma), were identified only in sprouted beans compared to unsprouted versions (Qiao et al., 2008). Additionally, VOCs such as nonanal (fatty and citrus-like odors) and decanal (sweet and floral aroma) were detected in the emission bouquet of sprouted dry bean flours compared to their corresponding unsprouted ones (Acree and Arn, 2004). Food flavor can be substantially influenced by lipid oxidation, which provoke the generation of potent odorants such as\u0026nbsp;saturated and unsaturated aldehydes\u0026nbsp;(Belitz et al., 2009). Conversely to aldehydes, alkanes make only a small contribution to the general food aroma (Fahlbusch et al., 2003). Alkanes were identified as the second most abundant VOCs emitted by raw and germinated seeds. The germination did not affect the emission of this volatile. These results are in good agreement with a previous investigation of VOCs of other legume in particular faba bean cultivars (Akkad et al., 2021; Rajhi et al., 2021). An exception was recorded for G-Black beans-S; which emit much more alkanes compared to its corresponding raw version. This high content in alkanes, especially in dry beans, was also previously reported by Oomah et al. (2007) for different types of \u003cem\u003eP. vulgaris\u003c/em\u003e. Their abundance may be explained by the occurrence of lipid peroxidation, which causes the formation of the characteristic aroma of dry legumes, since alkanes are mainly obtained from oxidative reaction of lipids (Shahidi et al., 1986). Additionally, comparing the volatile profiles of two different chickpeas (desi and kabuli), alkanes were considered as one of their main aroma constituents (Ghosh et al., 2020). Four alcohols were newly emitted by sprouted seeds compared to raw ones (Table 1). Alcohols in legumes are the result of the oxidation of lipids due to the presence of dehydrogenases. In dry beans, some alcohols were associated to their particular aroma, i.e. grassy and green ones (Shahidi et al., 1986). In general, alcohols are undesirable ones, being considered causes of off-flavors (Khrisanapant et al., 2019).\u003c/p\u003e\n\u003cp\u003eThe milling process was accompanied by a decrease of aldehydes and alkanes. A comparable trend was registered in milled brown rice; the lipid-derived compounds such aldehydes, alkanes, and alcohols decreased as the degree milling increased (Sun and Siebenmorgen, 1994). The products of the oxidative degradation of unsaturated fatty acids and amino acids including octanal which has a chemical, metallic and burnt odor, nonanal which has a fat, citrusy, and green smell, and decanal with its soapy, orange peel and tallow-like odor, and green flavor, were more abundant in germinated seeds and flours than in raw ones (Akkad et al., 2021). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDifferent classes of terpenes were also characterized in this study, including OM, MH and STH (Table 1). The profiles of raw and sprouted seeds and flours were quite different. As a result of germination, the identification percentages of all terpenes were decreased in sprouted seeds compared to raw versions. Nevertheless, the milling was accompanied with an important augmentation of MH VOCs. A different result was presented by Rajhi et al. (2022d), when they studied the effect of germination on the VOCs of faba beans. Indeed, they demonstrate that flour samples showed a similar trend compared to the seed versions, especially as regards the appearance of terpenes volatiles. Unlike NTD VOCs, which are formed after lipid peroxidation, terpenes are obviously biosynthesized in legumes (Wink, 2013). The most abundant terpene in the investigated legumes was limonene, which is present in all samples. This volatile compound has a pleasant citrus and fresh odor (Mosciano, 2000). This result in good agreement with the volatile profile that was previously characterized in \u003cem\u003eP. vulgaris\u003c/em\u003e (Oomah et al., 2007). \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe effect of sprouting and milling on the aroma attributes of three dry bean cultivars using headspace SPME sampling coupled to GC-MS system was assigned. The individual volatile profiles of studied legume seeds and flours varied not only by the type of treatment (germination and milling) but also between the varieties. Germination is a biological process in which the bioavailability of nutrients like protein will be increased, the anti-nutrients like tannins that are known as contributors to the bitterness of certain legumes, will be decreased. In this study, we observed that NTD volatiles appeared in all cultivars, and they were considered as the abundant chemical class for Navy beans-S and all sprouted seeds and flours except for G-Black beans-F. \u0026nbsp;At the same time, MH constituents, which was the principal chemical class emitted by unsprouted Navy and Black beans; significantly increased during the germination process. Additionally, the PCA analysis was performed to discriminate among dry bean cultivars and their flours. The finding from this study is the identification of the discriminated constituents for each cultivar and the corresponding flour, under sprouting conditions. This data could be used in legume-based receipt to impart desirable aroma properties based on the presence of certain volatile compounds.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research received no external funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcree, T., Arn, H., Flavornet and Human Odor Space. http://www.flavornet.org/ f lavornet.html.\u003c/li\u003e\n\u003cli\u003eAkkad, R., Kharraza, E., Hanb, J., Housec, J.D., Curtisa, J.M., 2019. Characterization of the volatile flavor compounds in low and high tannin faba beans (Vicia faba var. minor) grown in Alberta, Canada. Food Res. Int. 120, 285\u0026ndash;294. \u003c/li\u003e\n\u003cli\u003eAkkad, R., Buchko, A., Johnston, S.P., Han, H., House, J.D., Curtis, J.M., 2021. Sprouting improves the flavour quality of faba bean flours. Food Chem. 364.\u003c/li\u003e\n\u003cli\u003eAscrizzi, R., Gonz\u0026acute;alez-Rivera, J., Pomelli, C.S., Chiappe, C., Margari, P., Costagli, F., 2017. Ionic liquids, ultra-sounds and microwaves: An effective combination for a sustainable extraction with higher yields. The cumin essential oil case. React. Chem. Eng. 2, 577\u0026ndash;589.\u003c/li\u003e\n\u003cli\u003eBelitz, H. D., Grosch, W., Schieberle, P. (2009). Lipids. In Food chemistry (4th ed., pp. 158\u0026ndash;247). Berlin: Springer-Verlag.\u003c/li\u003e\n\u003cli\u003eDavies, N.W., 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on Methyl Silicon and Carbowax 20M phases. J. Chromatogr. A. 503, 1\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eDuenas, M., Sarmento, T., Aguilera, Y., Benitez, V., Moll, E., Esteban, R.M., Mart\u0026iacute;n- Cabrejas, M.A., 2016. Impact of cooking and germination on phenolic composition and dietary fibre fractions in dark beans (Phaseolus vulgaris L.) and lentils (Lens culinaris L.). LWT - Food Sci. TECHNOL 66, 72\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eFahlbusch, K.G., Hammerschmidt, F.J., Panten, J., Pickenhagen, W., Schatkowski, D., Bauer, K., Surburg, H., 2003. Flavours and fragrances. Ullmann\u0026rsquo;s Encyclopedia of industrial chemistry (6). 15. Ullmann\u0026rsquo;s Encyclopedia of industrial chemistry (73\u0026ndash;198). Wiley-VCH Verlag, Weinheim.\u003c/li\u003e\n\u003cli\u003eGhosh, A., Dadhich, A., Bhardwaj, P. J., Babu, N., \u0026amp; Kumar, V. (2020). Comparative analysis of metabolites in contrasting chickpea cultivars. Journal of Plant Biochemistry and Biotechnology, 29(2), 253\u0026ndash;265.\u003c/li\u003e\n\u003cli\u003eFinnie, S., Brovelli, V., Nelson, D., 2019. Sprouted grains as a food ingredient. In H. Feng, B. Nemzer, \u0026amp; J. W. DeVries (Eds.), Sprouted grains: Nutritional value, production, and applications. AACC International, pp. 113\u0026ndash;142.\u003c/li\u003e\n\u003cli\u003eKouris-Blazos, A., and Belski, R. (2016). Health benefits of legumes and pulses with a focus on Australian sweet lupins. Asia Pacific Journal of Clinical Nutrition, 21(1), 1\u0026ndash;17. \u003c/li\u003e\n\u003cli\u003eKhrisanapant, P., Kebede, B., Ying Leong, S., Indrawati, O., 2019. A comprehensive characterisation of volatile and fatty acid profiles of legume seeds. Foods 8, 651.\u003c/li\u003e\n\u003cli\u003eMessina, M. J. (2016). Legumes and soybeans: Overview of their nutritional profiles and health effects. Asia Pacific Journal of Clinical Nutrition, 25(1), 1\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eOomah, B.D., Liang, L.S.Y., Balasubramanian, P., 2007. Volatile Compounds of dry beans (Phaseolus vulgaris L.). Plant Foods Hum. Nutr. 62, 177.\u003c/li\u003e\n\u003cli\u003eQiao, Y., Xie, B.J., Zhang, Y., Zhang, Y., Fan, G., Yao, X.L., Pan, S.Y., 2008. Characterization of aroma active compounds in fruit juice and peel oil of Jinchen sweet orange fruit (Citrus sinensis (L.) Osbeck) by GC-MS and GC-O. Molecules 13 (6), 1333\u0026ndash;1344.\u003c/li\u003e\n\u003cli\u003eRajhi I., Ben Mansour R., Mhadhbi H.: Germination Effect on Phenolic Composition and Antioxidants Activities of Faba Bean Cultivar. JMRHS 7 (4), 3130-3136 (2024)\u003c/li\u003e\n\u003cli\u003eRajhi I., Baccouri B., Rajhi F., Guido F., Mhadhbi H.: Monitoring the Aroma Compounds of Vicia faba L var. Major and var. Minor. Case Studies of Breeding Strategies in Major Plant Species. IntechOpen, 2023. \u003c/li\u003e\n\u003cli\u003eRajhi I., Baccouri B., Rajhi F., Mhadhbi H., Flamini G.: HS-SPME-GC-MS characterization of volatile chemicals released from microwaving and conventional processing methods of fenugreek seeds and flours. Ind. Crops Prod. \u003cstrong\u003e182\u003c/strong\u003e: 114824, 2022a.\u003c/li\u003e\n\u003cli\u003ehttps://doi.org/10.1016/j.indcrop.2022.114824\u003c/li\u003e\n\u003cli\u003eRajhi I., Boulaaba M., Baccouri B., Rajhi F., Mhadhbi H., Flamini G.: Assessment of dehulling effect on volatiles, phenolic compounds, and antioxidant activities of faba bean seeds and flours. S. Afr. J. Bot. \u003cstrong\u003e147\u003c/strong\u003e: 741-753, 2022b.\u003c/li\u003e\n\u003cli\u003eRajhi I., Ben Mansour R., Baccouri B., Amri M., Mhadhbi H.: Sprouting characteristics and associated changes in antioxidant activities and phenolic composition of faba bean cultivars. Agrochimica. \u003cstrong\u003e60\u003c/strong\u003e (4), 2022c. \u003c/li\u003e\n\u003cli\u003eRajhi I., Baccouri B., Rajhi F., Hammami J., Souibgui M., Amri M. Mhadhbi H., Flamino G.: Evaluation of germination effect on volatile compounds of different faba bean cultivars using HS-SPME/GC-MS. J. Food Compos. Anal. \u003cstrong\u003e112\u003c/strong\u003e: 104692, 2022d. \u003c/li\u003e\n\u003cli\u003ehttps://doi.org/10.1016/j.jfca.2022.104692\u003c/li\u003e\n\u003cli\u003eRajhi I., Baccouri B., Rajhi F., Mhadhbi H., Flamini G.: Monitoring the volatile compounds status of whole seeds and flours of legume cultivars. Food Biosci. \u003cstrong\u003e41\u003c/strong\u003e: 101-105, 2021.\u003c/li\u003e\n\u003cli\u003ehttps://doi.org/10.1016/j.fbio.2021.101105\u003c/li\u003e\n\u003cli\u003eRajhi I., Baccouri B., Rajhi F., Hammami J., Abbes Z., Mhadhbi H., Flamino G.: HS-SPME-GC-MS combined with chemometrics to assess the impact of germination, dehulling, and milling on flavor attributes of brown and green lentils (Lens culinaris subsp. culinaris). S. Afr. J. Bot. \u003cstrong\u003e150\u003c/strong\u003e: 1102-1110, 2022e.\u003c/li\u003e\n\u003cli\u003eRoland, W.S.U., Pouvreau, L., Curran, J., van de Velde, F., de Kok, P.M.T., 2017. Flavor aspects of pulse ingredients. Cereal Chem. 94, 58\u0026ndash;65.\u003c/li\u003e\n\u003cli\u003eShahidi, F., Rubin, L. J., D\u0026rsquo;Souza, L. A. (1986). Meat flavour volatiles: A review of the composition, techniques of analysis, and sensory evaluations. Critical Reviews in Food Science and Nutrition, 24, 141\u0026ndash;243.\u003c/li\u003e\n\u003cli\u003eSun, H., and Siebenmorgen, T. J. 1993. Rice milling quality affected by kernel thickness. Cereal Chem. 70:727-733.\u003c/li\u003e\n\u003cli\u003eSimons, R., 2011. Prenylated Isoflavonoids from Soya and Licorice: Analysis, Induction and In Vitro Estrogenicity. Ph.D. thesis. Wageningen University, Wageningen, the Netherlands.\u003c/li\u003e\n\u003cli\u003eUebersax, M.A.; Cichy, K.A.; Gomez, F.E.; Porch, T.G.; Heitholt, J.; Osorno, J.M.; Kamfwa, K.; Snapp, S.S.; Bales, S. Dry beans (Phaseolus vulgaris L.) as a vital component of sustainable agriculture and food security\u0026mdash;A review. Legume Sci. 2023, 5, e155. \u003c/li\u003e\n\u003cli\u003eXu, M., Jin, Z., Lan, Y., Rao, J., Chen, B., 2019. HS-SPME/GC-MS/Olfactometry Combined with Chemometrics to Assess the Impact of Germination on Flavor Attributes of Chickpea, Lentil, and Yellow Pea Flours. Food Chem. 280, 83\u0026ndash;95.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HS-SPME-GC-MS, dry beans, germination, seeds, flours, VOCs","lastPublishedDoi":"10.21203/rs.3.rs-5024199/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5024199/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The impact of germination and milling on volatile compounds (VOCs) of chickpea cultivar was evaluated using HS-SPME/GC-MS technique. In total, 35 VOCs were identified. In this study, 28 active odors were characterized in seeds and 23 in flours: 17 in raw seeds, 24 in sprouted grains, 21 in raw flours, and 17 in germinated flours; they accounted for 98.9, 96.6, 97.9, and 98.8% of total emissions, respectively. The VOCs were classified into six chemical classes i.e., monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), apocarotenes (AP), phenylpropanoids (PP), nitrogen/sulfur derivatives (NSD), and non-terpene derivatives (NTD). Germination and milling processes decreased clearly the emission of NTD VOCs and increased the MH, OM, PP, AC, and NSD VOCs. Aldehydes, the most interest constituents giving the undesirable odors of legumes, was diminished in sprouted seed and flour versions. This study can provide useful information on the conception of legume–based ingredients combined with specific volatile characteristics in order to reduce unwanted odors and definitely for pertinent breeding programs. ","manuscriptTitle":"Fingerprinting of volatile profiles of sprouted and unsprouted seeds and flours of Phaseolus vulgaris using HS-SPME/GC-MS","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-17 06:51:25","doi":"10.21203/rs.3.rs-5024199/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d709fdc3-4021-4023-b7b8-1b68194984ee","owner":[],"postedDate":"September 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-17T06:51:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-17 06:51:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5024199","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5024199","identity":"rs-5024199","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}