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. 2021 Apr 3;7(4):e06666. doi: 10.1016/j.heliyon.2021.e06666

Metabolite profile of Bambara groundnut (Vigna subterranea) and dawadawa (an African fermented condiment) investigation using gas chromatography high resolution time-of-flight mass spectrometry (GC-HRTOF-MS)

Janet Adeyinka Adebiyi a,∗, Patrick Berka Njobeh a, Oluwafemi Ayodeji Adebo a, Eugenie Kayitesi b,∗∗
PMCID: PMC8050003  PMID: 33889778

Abstract

Metabolite profile provides an overview and avenue for the detection of a vast number of metabolites in food sample at a particular time. Gas chromatography high resolution time-of-flight mass spectrometry (GC-HRTOF-MS) is one of such techniques that can be utilized for profiling known and unknown compounds in a food sample. In this study, the metabolite profiles of Bambara groundnut and dawadawa (unhulled and dehulled) were investigated using GC-HRTOF-MS. The presence of varying groups of metabolites, including aldehydes, sterols, ketones, alcohols, nitrogen-containing compounds, furans, pyridines, acids, vitamins, fatty acids, sulphur-related compounds, esters, terpenes and terpenoids were reported. Bambara groundnut fermented into derived dawadawa products induced either an increase or decrease as well as the formation of some metabolites. The major compounds (with their peak area percentages) identified in Bambara groundnut were furfuryl ether (9.31%), bis (2-(dimethylamino)ethyl) ether (7.95%), 2-monopalmitin (7.88%), hexadecanoic acid, methyl ester (6.98%), 9,12-octadecadienoic acid (Z,Z) and 2-hydroxy-1-(hydroxymethyl)ethyl ester (5.82%). For dehulled dawadawa, the significant compounds were palmitic acid, ethyl ester (17.7%), lauric acid, ethyl ester (10.2%), carbonic acid, 2-dimethylaminoethyl 2-methoxyethyl ester (7.3%), 9,12-octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester (5.13%) and maltol (4%), while for undehulled dawadawa, it was indoline, 2-(hydroxydiphenylmethyl) (26.1%), benzoic acid, 4-amino-4-hydroximino-2,2,6,6-tetramethyl-1-piperidinyl ester (8.2%), 2-undecen-4-ol (4.7%), 2-methylbutyl propanoate (4.7%) and ë-tocopherol (4.3%). These observed metabolites reported herein provides an overview of the metabolites in these investigated foods, some of which could be related to nutrition, bioactivity as well as sensory properties. It is important to emphasize that based on some of the metabolites detected, it could be suggested that Bambara groundnut and derived dawadawa might serve as functional foods that are beneficial to health.

Keywords: Fermented condiment, GC-HRTOF-MS, Legume, Metabolites, Profiling

Fermented condiment; GC-HRTOF-MS; Legume; Metabolites; Profiling.

1. Introduction

Fermentation is a biochemical process that results in modifications (increase/decrease) as well as formation (synthesis) of metabolites. These metabolites contribute to the nutritional qualities, taste, shelf life, safety, aroma, health promoting properties and overall composition of fermented foods. Traditionally, these fermented foods are produced from legumes or cereals and undergo various forms of fermentation, such as alkali fermentation, lactic acid fermentation, acetic acid fermentation and alcoholic fermentation (Oliveira et al., 2014). The fermentation process whereby the pH of a legume increases to alkaline levels and possibly pH values of 9 and above is known as alkaline fermentation (Omafuvbe et al., 2002) and such is due to breakdown of proteins to ammonia, peptides and amino acids (Wang and Fung, 1996; Kiers et al., 2000).

In African and Asian countries, several alkaline fermented food condiments, such as dawadawa, thua-nao, iru, natto and soumbala, are mostly produced from legumes and from Bambara groundnut (Fadahunsi and Olubunmi, 2009; Parkouda et al., 2009; Ademiluyi and Oboh, 2011; Akanni et al., 2018a; Muhammad et al., 2018). These fermented condiments are processed mostly through natural fermentation; however, this is not always the case, as some undergo controlled fermentation. During the production of dawadawa, raw legume seeds are soaked, manually dehulled and boiled to soften the seeds. The boiled softened raw seeds are wrapped with leaves (such as banana leaves), kept in sacks or bags and incubated in a plastic bowl/calabash/earthen pot for three to five days (the fermentation period is usually based on human discretion) (Onawola et al., 2012). The most important and major processing step for this product is fermentation and has been proven to enhance the organoleptic and beneficial health properties of fermented legumes (Oboh et al., 2009; Ademiluyi and Oboh, 2011; Chinma et al., 2020).

Monitoring of these metabolic, biochemical, physicochemical and structural changes occurring during the fermentation process may be somewhat difficult, necessitating the utilization of techniques and robust equipment, which can provide a better overview of these metabolites. Gas chromatography–mass spectrometry is a non-biased, comprehensive and sensitive technological system used for the detection of diverse volatile and semi-volatile metabolites (Adebo et al., 2021). It has advantages of better resolution, high sensitivity, good reproducibility and, with the necessary databases, makes identification of compounds relatively easier (Hu et al., 2018). Particularly for gas chromatography coupled with high-resolution time of flight mass spectrometry, it is a powerful and highly effective analytical tool with excellent capabilities including a better chromatographic separating capability over a wide mass range with an accurate mass measurement (Brits et al., 2018; Kewuyemi et al., 2020). The exact mass information and mass resolution provided by high-resolution time-of-flight mass spectrometry (HR-TOFMS) can enhance target identification of compound and also assist in the identification of unknown compounds (Ubukata et al., 2015).

While few authors have studied the composition of dawadawa (Akanni et al., 2018a; Onyenekwe et al., 2012; Adebiyi et al., 2019), there is still no study providing a comparison of the metabolite profile of two types of dawadawa (dehulled and unhulled) from Bambara groundnut (BGN) obtained through natural fermentation. Thus, this study was aimed to profile metabolites in BGN and dawadawa (dehulled and unhulled) using GC-HRTOF-MS, envisaging that the metabolites would be beneficial to consumers of these products.

2. Materials and methods

2.1. Raw materials and sample preparation

Bambara groundnuts (mixed varieties) (i.e. brown, cream and red) used in this study were procured from a local farmer in Limpopo Province, South Africa. These were subsequently sorted to remove extraneous material or debris and cleaned with water.

2.2. Fermentation of Bambara groundnut into dawadawa

The production of both dehulled and unhulled dawadawa has been previously described in our earlier study Adebiyi et al. (2019). Briefly, the BGN was soaked for 24 h in water and dehulled manually, the dehulled raw seeds were rinsed with water and boiled for 1 h. Later, the boiled and cooled BGN seeds were spread and covered with a sterile banana leaves, wrapped in jute bags and incubated at 35 °C for 84 h. For the unhulled dawadawa (UHD), the hulls or seed coats were not removed after soaking and were incubated at 35 °C for 120 h. The fermentation time and temperature conditions (35 °C for 84 h and 35 °C for 120 h, for DD and UHD, respectively) that were used for producing the dawadawa samples was guided by the optimized results earlier achieved and reported in Adebiyi et al. (2020). The samples were freeze-dried at −55 °C for 24 h (LyoQuest Telstar Technologies, Spain) and stored at 4 °C prior to analysis.

2.3. Sample preparation for metabolite profiling

At the initial stage of sample preparation, different extraction solvents (all analytical grade) [100% acetonitrile (ACN), 100% methanol (MeOH), 100% water (H2O), 80% ACN in H2O, 80% MeOH in H2O, 50% ACN:MeOH (v/v), 1% HCl in MeOH, ACN:MeOH:H2O (4,4,2, v/v/v) and isopropanol:ACN:H2O (4,4,2, v/v/v)] were used to investigate for a possible range of available metabolites in the samples.

An informed compromise was reached and extraction using 80% MeOH in H2O was finally adopted based on a wider range of relevant metabolites detected on the GC-HRTOF-MS system. Briefly, 10 mL of 80% MeOH was added to 1 g of freeze-dried sample, agitated and the mixture sonicated in an ultrasonic bath (Integral Systems Ultrasonic Bath UMC 5, Labotec, South Africa) at 4 °C for 1 h. The mixture was then centrifuged at 3 500 rpm for 5 min at 4 °C (Eppendorf 5702R, Merck South Africa), transferred into a 250 mL round bottom flask and concentrated at 30 °C, under pressure using a rotary evaporator (Buchi, Switzerland). The extract was reconstituted with 1 mL chromatographic grade MeOH (Sigma Aldrich, Germany) and filtered into a dark amber vial for analysis. The extraction was done in triplicates for each sample.

2.4. GC-HRTOF-MS analysis

A mass calibration of the instrument was performed prior to analysis on the LECO Pegasus GC-HRTOF-MS system (LECO Corporation, St Joseph, MI, USA) and subsequent sample analyses done using the method of Adebo et al. (2019). The samples were then analyzed on the GC-HRTOF-MS system equipped with an Agilent 7890A (Agilent Technologies, Inc., Wilmington, DE, USA) gas chromatograph running in a high-resolution. This was coupled to a Gerstel MPS multipurpose autosampler (Gerstel Inc. Germany) and analytical column was a Rxi®-5ms (30 m × 0.25 mm ID × 0.25 μm) (Restek, Bellefonte, USA). One microlitre (μL) of each sample was injected (in a splitless mode) with helium as the carrier gas at a constant flow rate (1 mL/min). The transfer line and inlet temperatures were 225 °C and 250 °C respectively. The oven temperature was initially set at 70 °C, held for 0.5 min, ramped at 10 °C/min to 150 °C and held for 2 min. The oven temperature was later ramped at 10 °C/min to 330 °C and held for 3 min Triplicate extraction for each sample were respectively injected once into the GC-HR-TOF-MS equipment as well as solvent blanks to observe impurities and possible contamination.

2.5. Data analysis

From the data obtained, peak picking, retention time alignment, peak matching and detection were done on the ChromaTOF-HRT® software (LECO Corporation, St Joseph, MI, USA). Other data processing parameters adopted included a signal to noise ratio of 100 and a minimum match similarity of >70% prior to when compound name is assigned, using the Mainlib, Feihn and NIST metabolomics database by comparing the molecular formula, retention time and mass spectra data. Percentage peak areas were subsequently calculated, and the respective observed m/z fragments obtained from the ChromaTOF-HRT® data station were recorded after which the metabolite class was annotated with corresponding m/z fragments and molecular formula.

3. Results and discussion

3.1. Metabolites of Bambara groundnut and dawadawa profiled using GC-HRTOF-MS

The metabolic compounds of BGN and the two dawadawa produced (unhulled and dehulled) were analyzed using GC-HRTOF-MS. This is the first report to the best of our knowledge to profile and investigate the metabolites of BGN, unhulled (UHD) and dehulled (DD) dawadawa using GC-HRTOF-MS. In total, 134 metabolites were identified, and their identities presented in Table 1. Figure 1 represents the GC-HRTOF-MS chromatogram of BGN, DD and UHD samples. The group of compounds detected were terpenes and terpenoids (2%), amines (2%), sulphur related compounds (4%), ketones (7%), pyridines (3%), vitamins (2%), esters including fatty acid methyl and ethyl esters (37%), alcohols including sterols (7%), phenols (6%) and other miscellaneous compounds (20%). Eight compounds were identified in both dawadawa products, 29 in BGN, 42 in only DD, 17 in only UHD and 12 in all the samples analyzed (Figure 2A). Generally, more metabolites were detected in DD samples as compared to UHD, which might be attributed to increased microbial activity enhancing metabolic activities and better breakdown and/or formation of compounds. This was also the observation in an earlier study (Adebiyi et al., 2019; Adebiyi, 2020) and can be related to higher antioxidant activities and antinutritional factors (ANFs) in UHD as compared to DD samples, which might influence microbial activities. Some of the compounds identified in Table 1, were not detected in the raw BGN, but observed in DD and UHD samples. It can thus be speculated that these compounds were presumably produced during fermentation. The major metabolites that were only found in the fermented condiments include 9,12-octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester (5.13%), carbonic acid, 2-dimethylaminoethyl 2-methoxyethyl ester (7.3%), lauric acid, ethyl ester (10.2%), maltol (4%) and palmitic acid, ethyl ester (17.7%) for dehulled dawadawa and 2-methylbutyl propanoate (4.7%), 2-undecen-4-ol (4.7%), benzoic acid,4-amino-4-hydroximino-2,2,6,6-tetramethyl-1-piperidinyl ester (8.2%), ë-tocopherol (4.3%) and indoline, 2-(hydroxydiphenylmethyl)- (26.1%) for unhulled dawadawa samples (Table 1).

Table 1.

Metabolites identified in Bambara groundnut and dawadawa (dehulled and unhulled) samples.

tR (min) Compound name and metabolite class Observed m/z m/z fragments MF Percentage peak areas
BGN DD UHD
Acids
04:47 6-Methylbicyclo[2.2.1]hept-2-ene-5-carboxylic acid 122.4808 65.9583, 105.1135 C9H12O2 ND ND 4.69
06:27
 1-Hydroxycyclohexanecarboxylic acid
 131.6358
 68.0502, 98.9424
 C7H12O3
 ND
 ND
 3.46
Alcohols
05:03 2-undecen-4-ol 192.9805 71.0490, 131.0703 C11H22O ND 1.98 4.70
06:02 Maltol 126.0312 55.0180, 71.0128 C6H6O3 4.85 4.00 ND
06:03 Phenylethyl alcohol 122.0728 91.0544, 122.0728 C8H10O ND 1.36 ND
17:00
 1-Hexadecanol
 196.2187
 55.0544, 83.0856
 C16H34O
 0.10
 ND
 ND
Aldehyde
08:11
 à-Ethylidenebenzeneacetaldehyde
 146.0728
 115.0544, 138.0913
 C10H10O
 ND
 0.06
 ND
Amines
14:13 1-Naphthalenamine, N-ethyl- 171.1045 129.0702, 156.0810 C12H13N ND 0.12 ND
12:12 N-acetylphenethylamine 163.0994 30.0342, 104.0623 C10H13NO ND 0.62 ND
13:20
 p-Aminobiphenyl
 169.0888
 141.0700, 167.0733
 C12H11N
 ND
 0.12
 ND
Benzenes
07:53 Benzene, 1,3-bis(1,1-dimethylethyl)- 190.1711 124.0756, 175.1482 C14H22 ND 0.04 ND
10:13 Benzeneethanol, à-(phenylmethyl)- 208.2062 92.0622, 103.0544 C15H16O ND 0.49 ND
24:55
 Benzeneethanamine, 2-fluoro-á,3,4-trihydroxy-N-isopropyl-
 226.2167
 59.0367, 72.0445
 C11H16FNO3
 0.66
 0.21
 ND
Esters
03:55 Benzoic acid, 4-amino-, 4-hydroximino-2,2,6,6-tetramethyl-1-piperidinyl ester 120.4566 80.4620, 83.5630 C16H23N3O3 ND ND 8.23
06:56 Benzofenac methyl ester 174.1069 61.0106, 91.0211 C16H15ClO3 0.05 ND ND
07:08 Benzoic acid, 4-amino-, 4-acetoxy-2,2,6,6-tetramethyl-1-piperidinyl ester 153.0501 107.1252, 120.4566 C18H26N2O4 0.17 ND 1.23
08:27 Cyclobutanecarboxylic acid, 2-dimethylaminoethyl ester 151.1098 58.0653, 71.0730 C9H17NO2 ND 0.38 ND
09:18 Propanoic acid, 2-methyl-, 2,2-dimethyl-1-(1-methylethyl)-1,3-propanediyl ester 329.0325 43.0543, 71.0492 C16H30O4 0.34 0.25 ND
09:37 Propanoic acid, 2-methyl-, 3-hydroxy-2,2,4-trimethylpentyl ester 174.1206 71.0492, 89.0598 C12H24O3 0.33 ND ND
10:19 Fumaric acid, ethyl 2,3,5-trichlorophenyl ester 167.1065 99.0442, 127.0390 C12H9Cl3O4 0.07 ND 0.57
10:34 Fumaric acid, monoamide, N,N-dimethyl-, 3-chlorophenyl ester 185.0676 98.0602, 126.0552 C12H12ClNO3 ND 0.16 ND
11:04 Phthalic acid, 3,4-dichlorophenyl methyl ester 194.0571 77.0386, 163.0392 C15H10Cl2O4 0.22 ND ND
11:04 Phthalic acid, methyl 4-(2-phenylprop-2-yl)phenyl ester 283.0486 103.0139, 163.0307 C24H22O4 ND 0.10 0.33
11:08 4-Butylbenzoic acid, 2-dimethylaminoethyl ester 161.1200 58.0653, 71.0731 C15H23NO2 ND 0.19 ND
12:02 3,4-Dimethyl-2-(3-methyl-butyryl)-benzoic acid, methyl ester 208.5497 54.5083, 191.4042 C15H20O3 ND ND 3.42
12:33 Ethyl 2-cyano-3-methylbutanoate 153.9684 68.0387, 82.5285 C8H13NO2 0.10 ND 0.61
12:57 6-Methoxythymyl 2-methylbutyrate 180.2455 121.4904, 165.0691 C16H24O3 ND ND 0.42
13:23 Phthalic acid, monoamide, N-ethyl-N-(3-methylphenyl)-, ethyl ester 194.0570 149.0235, 177.0545 C19H21NO3 0.04 ND ND
13:25 Butyric acid, thio-, S-hexyl ester 194.1546 73.0543, 71.0492 C10H20OS ND 0.23 ND
15:42 Fumaric acid, butyl 2-phenylethyl ester 267.9997 104.0623, 203.0943 C16H20O4 ND 0.33 ND
16:43 Ethyl 13-methyl-tetradecanoate 270.2554 88.0520, 101.0599 C17H34O2 ND 0.17 ND
16:56 Phthalic acid, heptyl tridec-2-yn-1-yl ester 460.9532 57.0701, 149.0236 C28H42O4 0.42 ND ND
17:45 Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, methyl ester 292.2035 147.0808, 277.1799 C18H28O3 0.02 ND ND
17:55 DL-Alanine, N-methyl-N-(byt-3-yn-1-yloxycarbonyl)-, tridecyl ester 224.1825 86.0966, 154.0738 C22H39NO4 ND 1.67 ND
17:57 Phthalic acid, 8-chlorooctyl nonyl ester 236.2140 148.8379, 205.4445 C25H39ClO4 0.56 0.94 0.30
17:57 Phthalic acid, 2-chloropropyl heptyl ester 224.0991 149.0235, 205.0860 C18H25ClO4 0.53 ND 0.28
17:58 Phthalic acid, 8-chlorooctyl decyl ester 224.1005 103.0392, 149.0235 C26H41ClO4 0.57 0.33 0.60
18:20 Fumaric acid, 2,6-dimethoxyphenyl dodec-2-en-1-yl ester 213.1026 68.0386, 153.9559 C24H34O6 ND 1.10 2.60
18:30 L-Proline, N-valeryl-, decyl ester 219.0079 55.1733, 84.0285 C20H37NO3 ND 0.86 ND
20:10 2-Methylbutyl propanoate 142.8595 56.5643, 70.0906 C8H16O2 ND ND 4.68
21:00 Octanoic acid, 2-dimethylaminoethyl ester 218.0598 58.0652, 72.0808 C12H25NO2 2.48 1.98 ND
22:28 Carbonic acid, 2-dimethylaminoethyl 2-methoxyethyl ester 194.1912 58.0652, 71.0729 C8H17NO4 4.65 7.33 ND
23:11 Phthalic acid, dicyclohexyl ester 300.2082 149.0236, 167.0342 C20H26O4 0.47 0.11 0.82
24:06 Isophthalic acid, phenylethyl undecyl ester 267.0185 104.0825, 131.6355 C27H36O4 ND 0.02 0.46
24:19 9,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester 348.0901 67.0543, 262.2299 C21H38O4 5.82 5.14 ND
24:29 Octadecanoic acid, 2,3-dihydroxypropyl ester 359.3167 74.0362, 98.0728 C21H42O4 2.32 ND ND
25:30 Butylphosphonic acid, decyl 4-(2-phenylprop-2-yl)phenyl ester 472.3099 221.1319, 457.2876 C29H45O3P 0.06 ND ND
25:30 Succinic acid, 2-chloro-6-fluorophenyl phenethyl ester 400.9841 105.0699, 279.2308 C18H16ClFO4 ND 0.32 ND
25:37 Succinic acid, 3,4-dimethylphenyl 2-(dimethylamino)ethyl ester 312.3026 58.0135, 71.7611 C16H23NO4 0.37 0.16 ND
25:37 Carbonic acid, 2-dimethylaminoethyl isobutyl ester 186.1471 58.0652, 71.0729 C9H19NO3 0.21 0.27 ND
29:25 Urs-12-en-24-oic acid, 3-oxo-, methyl ester, (+)- 427.3893 189.1643, 218.2032 C31H48O3 0.14 ND ND
30:39
 Olean-12-en-28-oic acid, 3-oxo-, methyl ester
 452.3664
 203.1796, 262.1931
 C31H48O3
 0.74
 ND
 ND
Fatty acid ethyl esters
17:08 Pentadecanoic acid, ethyl ester 270.2552 88.0520, 101.0599 C17H34O2 ND 0.25 ND
18:00 9-hexadecenoic acid, ethyl ester 282.2556 69.0699, 88.0521 C18H34O2 ND 0.26 ND
18:14 Lauric acid, ethyl ester 228.2055 88.0521, 101.0600 C14H28O2 ND 10.15 ND
18:19 Palmitic acid, ethyl ester 285.2786 88.0522, 101.0601 C18H36O2 ND 17.74 ND
23:29
 Stearic acid, ethyl ester
 312.2990
 88.0520, 101.0599
 C20H40O2
 ND
 1.41
 ND
Fatty acid methyl esters
15:59 Myristic acid, methyl ester 256.2399 88.0521, 101.0600 C16H32O2 ND 0.75 ND
17:30 Hexadecanoic acid, methyl ester 270.2556 74.0363, 87.0442 C17H34O2 6.98 ND ND
19:15 9,12-Octadecadienoic acid, methyl ester 294.2561 81.0699, 95.0858 C19H34O2 2.55 ND ND
19:19 trans-13-Octadecenoic acid, methyl ester 296.2714 55.0543, 74.0363 C19H36O2 0.73 ND ND
19:31 Octadecanoic acid methyl ester 298.2871 74.0363, 143.1070 C19H38O2 1.61 ND ND
23:00
 Cerotic acid, methyl ester
 356.3559
 74.0363, 87.0442
 C27H54O2
 ND
 1.41
 ND
Fatty acid
18:05
 Palmitic acid
 256.2404
 60.0207, 73.0284
 C16H32O2
 4.03
 ND
 ND
Fatty acid derivatives
20:29 Myristic acid amide 227.2204 59.0367, 72.0445 C14H29NO ND 1.04 ND
22:55
 2-monopalmitin
 331.2852
 104.0738, 128.5062
 C19H38O4
 7.88
 3.65
 ND
Furans
03:35 Furanoeudesma-1,4-diene 108.0683 47.0327, 64.0181 C15H18O ND 1.50 ND
07:46 3-Butene-1,2-diol, 1-(2-furanyl)- 128.0357 49.0073, 97.0286 C8H10O3 2.39 1.90 0.32
19:45
 Furfuryl ether
 176.0922
 81.0335, 143.0342
 C10H10O3
 9.31
 ND
 ND
Ketones
04:09 Hex-4-yn-3-one 95.8902 67.0060, 68.0471 C6H8O ND ND 1.02
06:02 3-Acetoxy-2-methyl-pyran-4-one 129.0913 71.0128, 126.0312 C8H8O4 3.57 3.67 ND
07:41 2-Coumaranone 134.0364 78.0464, 106.0414 C8H6O2 ND 0.12 ND
08:44 Ethanone, 1-(2-hydroxy-5-methylphenyl)- 150.0677 107.0493, 135.0442 C9H10O2 0.44 0.30 ND
09:47 7-Chloro-1,3,4,10-tetrahydro-10-hydroxy-1-[[2-[1-pyrrolidinyl]ethyl]imino]-3-[3-(trifluoromethyl)phenyl]-9(2H)-acridinone 268.9973 84.0809, 132.0548 C26H25ClF3N3O2 0.91 0.15 3.75
11:04 2-(6-Chloro-3-nitro-4-phenyl-quinolin-2-ylsulfanyl)-1-(2,3-dihydro-benzo[1,4]dioxin-6-yl)- ethanone 194.0574 132.5996, 163.0307 C25H17ClN2O5S ND 0.12 0.36
17:32 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione 276.1718 175.119, 205.0861 C17H24O3 0.11 ND ND
23:20 2-methoxy-,2-octen-4-one, 152.0474 99.0443, 114.0677 C9H16O2 0.19 0.08 ND
28:04
 3,6,13,16-tetraoxatricyclo[16.2.2.2(8,11)]tetracosa-8,10,18,20,21,23-hexaene-2,7,12,17-tetrone
 380.0489
 208.0519, 341.0657
 C20H16O8
 0.08
 ND
 ND
Nitrogenous compounds
04:48 Indoline, 2-(hydroxydiphenylmethyl)- 314.5135 103.0505, 118.4013 C21H19NO ND ND 26.11
07:34
 Indole, 3-(2-(diethylamino)ethyl)-
 130.6049
 85.5811, 129.5840
 C14H20N2
 ND
 ND
 0.16
Others (Miscellaneous compounds)
03:45 N-[3,3′-dimethoxy-4'-(2-piperidin-1-yl-acetylamino)-biphenyl-4-yl]-2-piperidin-1- yl-acetamide 128.0471 93.0701, 98.0364 C28H38N4O4 0.55 5.14 ND
05:50 Succinic anhydride 102.0283 36.5607, 55.5498 C4H4O3 ND ND 6.71
06:22 Decamethylcyclopentasiloxane 358.0680 73.0469, 266.9992 C10H30O5Si5 0.12 0.05 ND
06:27 N,N-Dimethylglycine 103.0631 42.0338, 58.0653 C4H9NO2 ND 0.62 ND
06:39 1H-Imidazole-4-methanol 98.0364 69.0335, 97.0286 C4H6N2O ND 2.31 ND
06:52 Thiourea, N-(3-methyl-2-pyridinyl)-N'-[(tetrahydro-2-furanyl)methyl]- 332.0663 44.0733, 150.0677 C12H17N3OS ND 0.12 ND
07:32 Catecholborane 120.0570 100.0759, 148.0994 C6H5BO2 ND 0.71 ND
07:42 2-Benzoxazolamine, N-(1,1-dimethylethyl)- 153.9813 105.0764, 133.6238 C11H14N2O ND ND 0.32
08:43 Dodecamethylcyclohexasiloxane 434.0840 73.0468, 341.0179 C12H36O6Si6 0.76 0.91 1.69
08:44 Phenyl-1,2-diamine, N,4,5-trimethyl- 149.8967 106.1084, 134.6487 C9H14N2 ND ND 0.36
09:53 Benzaldehyde, 3-methoxy-4-[(2-methylphenyl)methoxy]- 195.1248 105.0701, 132.0810 C16H16O3 ND 1.00 ND
11:15 4,4′-Dichlorodibutyl ether 158.0202 91.0312, 93.0280 C8H16Cl2O 0.03 ND ND
11:40 Tetradecamethylcycloheptasiloxane 508.1064 73.0467, 281.0513 C14H42O7Si7 2.04 1.72 ND
11:50 Tetradonium Bromide 165.0703 58.0653 C17H38BrN 0.13 ND ND
12:58 2,3,5,6-Tetrafluoroanisole 180.0782 137.0569, 165.0548 C7H4F4O ND 0.23 ND
13:14 3-Methyl-4-phenyl-1H-pyrrole 157.0886 127.5468, 155.9884 C11H11N ND 1.19 1.12
13:24 2-propynenitrile, 3-fluoro- 69.0591 53.4495, 81.5012 C3FN ND ND 1.05
14:20 Hexadecamethyl-cyclooctasiloxane 580.1260 73.0468, 355.0702 C16H48O8Si8 1.71 1.41 1.53
17:09 2,7-Dimethylcarbazole 195.1039 140.0702, 167.0726 C14H13N ND 0.03 ND
17:50 1-Methyl-2,5-dipropyldecahydroquinoline 195.4010 86.6185, 166.1360 C16H31N ND ND 0.15
19:41 2-(1-Pyrrolidinyl)ethyl 4-propoxysalicylate 238.7279 83.5285, 96.8999 C16H23NO4 ND ND 0.43
19:42 Monoethanolamine stearic acid amide 282.2785 85.0523, 98.0602 C20H41NO2 3.21 ND ND
20:47 3-Cyclopentylpropionamide, N,N-dimethyl- 170.1547 45.0574, 87.0680 C10H19NO 0.72 0.12 ND
22:07 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)- 244.1207 125.0709, 153.0660 C14H16N2O2 ND 0.22 ND
22:25 Bis(2-(Dimethylamino)ethyl) ether 156.1014 58.0652, 71.0729 C8H20N2O 7.95 0.25 ND
24:55 Acetaldehyde, diethylhydrazone 114.0678 71.0492, 99.0443 C6H14N2 ND 0.09 ND
26:59
 S-[2-[N,N-Dimethylamino]ethyl]N,N-dimethylcarbamoyl thiocarbohydroximate
 218.0808
 58.0652, 71.0729
 C8H17N3O2S
 0.20
 ND
 ND
Phenols
09:14 2-methyl-6-tert-butylphenol 164.1196 121.0649, 149.0963 C11H16O 0.02 ND ND
12:02 2,4-Di-tert-butylphenol 206.1665 57.0700, 191.1431 C14H22O 2.00 ND ND
12:04 Phenol, 2,4,6-tris(1-methylethyl)- 220.1824 177.1274, 205.1588 C15H24O 0.13 0.13 0.56
12:05 Butylated Hydroxytoluene 220.1824 43.0179, 205.1589 C15H24O 0.19 0.12 0.65
12:57 2-tert-Butyl-4-methoxyphenol 180.0781 137.0596, 165.0548 C11H16O2 0.35 ND ND
16:48 Resorcinol/3-Hydroxyphenol 110.0602 82.0289, 201.1147 C6H6O2 ND 0.15 ND
16:52 Taxicatigenin 153.9559 68.0387, 124.5019 C8H10O3 ND ND 0.58
22:14
 Phenol, 2,2′-methylenebis[6-(1,1-dimethylethyl)-4-methyl-
 340.2401
 161.0964, 177.1277
 C23H32O2
 3.52
 0.76
 5.21
Pyridines
11:18 o-phenylpyridine 154.9679 126.5207, 155.9841 C11H9N ND ND 0.29
11:19 m-Phenylpyridine 155.0730 127.0544, 156.0764 C11H9N ND 0.46 0.23
15:05 4-pyridinamine, N-[(4-methoxyphenyl)methylene]- 212.1306 91.0544, 197.1073 C13H12N2O ND 0.14 ND
20:56
 2,6-diphenyl-pyridine,
 231.1044
 58.0653, 202.0777
 C17H13N
 ND
 0.16
 ND
Sterols
28:08 Ergosta-5,24-dien-3-ol, (3á)- 384.3342 281.2269, 314.2608 C28H46O 0.04 ND ND
28:11 Campesterol 400.3701 145.1014, 213.1642 C28H48O 0.49 0.12 ND
28:24 Stigmasterol 412.3710 83.0856, 159.1172 C29H48O 1.39 0.41 ND
28:53 Stigmasta-5,24(28)-dien-3-ol, (3á,24Z)- 412.3703 314.2609, 281.2269 C29H48O 0.51 0.15 ND
29:02 Cycloeucalenol 412.3684 95.0857, 107.0858 C30H50O 0.17 ND ND
29:06
 Olean-12-en-3-ol
 426.3862
 203.1797, 218.2032
 C30H50O
 0.29
 ND
 ND
Sulphur related compounds
04:13 Dimethyl trisulfide 125.9627 78.9671, 127.9585 C2H6S3 ND 1.88 ND
07:16 2,5-dihydrothiopene 86.0364 45.0336, 57.0336 C4H6S ND 1.52 ND
08:18 Hemineurine 143.0401 85.0108, 112.0217 C6H9NOS ND 0.10 ND
20:45 O-Ethyl S-2-diethylaminoethyl ethylphosphonothiolate 257.7809 85.6057, 98.9677 C10H24NO2PS ND ND 1.21
21:12
 1H-Indole-3-carbonitrile, 2-(4-chlorobenzenesulfonylmethyl)-1-methyl-
 225.1118
 169.1224, 201.1485
 C17H13ClN2O2S
 ND
 0.08
 ND
Terpenes and Terpenoid
04:56 Eucalyptol 154.1354 81.0700, 93.0701 C10H18O 0.26 ND ND
07:02 Naphthalene 128.0622 76.0307, 99.0442 C10H8 0.34 0.27 ND
28:47
 Clionasterol
 414.3864
 145.1015, 213.1643
 C29H50O
 0.61
 0.15
 ND
Vitamins
26:08 ë-Tocopherol 402.3499 137.0599, 177.0914 C27H46O2 3.43 1.05 4.30
26:51 ç-Tocopherol 416.3654 151.0755, 191.1069 C28H48O2 1.77 0.41 3.28
23:58 dl-7-azatryptophan 204.0760 88.0336, 131.0524 C10H11N3O2 ND 0.28 1.22
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tR – Retention time; m/z – mass-to-charge ratio; MF – Molecular formula; ND- Not detected; BGN – Bambara groundnut, DD – Dehulled dawadawa; UHD – Unhulled dawadawa

Figure 1.

Figure 1

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GC-HRTOF-MS chromatogram of the BGN (Bambara groundnut), DD (Dehulled dawadawa) and UHD (Unhulled dawadawa) samples.

Figure 2.

Figure 2

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(A) Venn diagram showing the relationship between the metabolites in BGN (Bambara groundnut), DD (Dehulled dawadawa) and UHD (Unhulled dawadawa) samples, (B) Pie chart showing percentage distribution of the compounds.

Esters were the principal compounds reported in this study. Other similar studies on fermented condiments contradict this observation, with pyrazines being the major constituent in sonru, afitin, iru (Azokpota et al., 2008), acids the dominant group in castor oil bean fermented condiment (Ojinnaka and Ojimelukwe, 2013), aldehydes in locust bean daddawa, soybean and melon seed ogiri (Onyenekwe et al., 2012), while aldehydes, acids and ketones were reported to dominate dawadawa from BGN using Bacillus species (Akanni et al., 2018a). Nevertheless, esters are major metabolite groups common to several fermented condiments in Africa and are mostly formed during fermentation by esterification of alcohols with fatty acids (Fan and Qian, 2005). Chemical reactions between alcoholic metabolites as well as microbial acidic metabolites could also lead to the formation of esters during fermentation. Their contributions to food aroma/odour are important, combined with the fact that esters at ambient temperatures are highly volatile and their perception thresholds are much lower compared to their alcohol precursors (Nogueira et al., 2005). Compounds belonging to the esters group constitutes 29% (Figure 2B) of the total metabolites recorded and were more prominent in the DD as compared to UHD. Phthalic acid 8-chlorooctyl decyl ester, phthalic acid dicyclohexyl ester and phthalic acid 8-chlorooctyl nonyl ester were the major esters detected in BGN, DD and UHD (Table 1).

The identified compounds in this study could be as a result of the breakdown and constituents in BGN such as proteins, lipids and other bioavailable compounds through the activities of the microbial enzymes. As reflected in the GC-HRTOF-MS data presented in Table 1, fermentation of BGN into derived dawadawa led to the formation, increase, as well as decrease of some compounds. Formation of these constituents could be attributed to the presence of microorganisms involved in the fermentation process and other processing factors as well as operations involved during dawadawa preparation (Azokpota et al., 2010). Compounds belonging to an acid group were only present in UHD samples, which can be attributed to the relatively longer fermentation period for the UHD samples. Acids are sometimes considered as undesirable compounds that confer unpleasant characteristics such as rancid, sweaty and pungent flavors (Frauendorfer and Schieberle, 2008), although they have been reported to confer some acidic, fruity and sour notes in fermented foods (Park et al., 2013).

Compounds belonging to the sulphur related group were mostly present in the DD with none detected in the BGN, in agreement with the study of Akanni et al. (2018b), in which sulphur-related compounds were equally not detected in the raw BGN. Dimethyl trisulfide (sulphur related compound) is known to confer meaty, sulfureous, eggy, alliaceous, cooked, savory, and onion note (Liu et al., 2012). It can also be identified as a possible product of amino acid metabolism (Tamman et al., 2000). Speculated possible amino acid degradation and significantly (p ≤ 0.05) different values in the amino acid of BGN and derived dawadawa (Adebiyi et al., 2019) could also explain the detected amine-related and nitrogenous compounds (Table 1).

Both ketones and aldehydes are formed by fatty acids beta-oxidation as well as oxidation catalyzed by lipoxygenase and hyper-oxidase enzymes, yielding important flavor compounds (Nzigamasabo, 2012). Aldehydes are not only flavor components, but also known as vital reactants associated with heterocyclic compounds formation (Ziegleder, 2009). Ketones are generally derived from amino acid and lipid degradation with the presence of these compounds having an impact on food flavor (Adebo et al., 2018). Nine ketones were detected, i.e. six from DD and three from UHD. The ketones in UHD decreased, as compared to the raw BGN, whereas there was a slight increase of the ketone group in DD (relative to the percentage peak areas). Dehulling of the seedcoats exposed fats related components to more oxygen coupled with removal of available antioxidants in the hull. This thus suggests that fat oxidation would likely be higher in the dehulled samples as compared to the unhulled samples (Akkad et al., 2019). Therefore, an increased level of ketones in the dehulled samples might be due to partial oxidation of the alcohols as well as synthesis through several metabolic pathways, particularly reduction of methyl-ketone (Curioni et al., 2002; Akkad et al., 2019). This may be associated with the disappearance and/or reduction of some ketones in BGN and dawadawa.

Alcohols constituted 3% of metabolites in Figure 2B and are generated by reduction reaction of corresponding aldehydes and oxidation of acids (Pham et al., 2008). According to Estrella et al. (2004), aldehydes and ketones are relatively unstable intermediate compounds and can easily be reduced to alcohols. In total, four alcohols were detected in this study, i.e. 2-undecen-4-ol at high levels in two fermented samples, maltol in BGN and DD, phenylethyl alcohol in DD, with 1-hexadecanol in BGN. The phenylethyl alcohol compound has a rose-like odor and is known as one of the major Korean fermented soy sauce odor-active compounds (Lee et al., 2006), suggesting that these alcohol-related compounds might contribute to the flavor of these dawadawa samples. A decrease in the number of alcohols in the fermented samples might be due to the heat treatment (i.e. cooking) applied during processing (Cho et al., 2017; Wang et al., 2019).

The pyridines group was not detected in the BGN except in the dawadawa samples, indicative of a formation of these compounds. Pyridines are usually formed during cooking of food (Gupta et al., 2019) or meat, probably due to the reaction of amino acids with alkanals (Hui, 2012). They are classified as the flavor component of beer and as important organoleptic compounds of foods from cocoa, peanuts, cheeses, beans and barley (Maga, 1981). Due to the physical properties of BGN (hard to cook phenomenon), the seeds are usually cooked briefly then dehulled (depending on the product) prior to fermentation, which might explain the occurrence of pyridines in this study.

Three vitamin-related compounds (Table 1) were detected in dawadawa samples except for dl-7-azatryptophan, which was completely absent in BGN. Other notable vitamins observed were ç-Tocopherol and ë-Tocopherol, which are forms of vitamin E. Not only is vitamin E of nutritional and dietary importance, but it also functions as an antioxidant by preventing the propagation of lipid peroxidation (Frei, 2004). It was observed that in dl-7-azatryptophan, the peak area of UHD (1.22%) was higher than that of DD (0.28%), while UHD has the highest percentage peak area in all the vitamins reported. Furans are heterocyclic compounds, known to possess sweet, roasted, burnt, caramel and sugar notes as previously reported in dawadawa (Akanni et al., 2018a; Azokpota et al., 2008).

Phenols are a major group of antioxidants and of great significance due to their biological and free radical scavenging activities (Koleva et al., 2018). Compounds belonging to the phenol group were also identified in this study. There was formation of taxicatigenin, which is also known as 3,5-dimethoxyphenol in UHD sample. 3,5-dimethoxyphenol belongs to methoxyphenols (a class of compounds comprising of a methoxy group) and connected to the benzene ring of a phenol moiety. The occurrence of this compound and its presence in only UHD could further explain its higher antioxidant activity in a previous study (Adebiyi, 2020), as taxicatigenin is a bioactive compound with potential antioxidant activity (Nithya et al., 2018). Bioactive compounds are also known to inhibit microbial growth that might have contributed to lesser microbial activity in UHD samples, resulting in reduced pH values (Adebiyi et al., 2020). Compounds belonging to the sterols group were common in raw BGN but none of these sterols were detected in UHD. There are claims that naturally occurring plant cholesterols may promote the health of animals and humans once consumed regularly either as food supplements or naturally in foods for a reasonable amount of time (Ogbe et al., 2015).

Fatty acid methyl esters were common in raw BGN, with the formation of methyl esters (myristic acid and cerotic acid methyl esters) in DD, while none of the fatty acid methyl esters were detected in UHD. This difference might have been due to the cooking process adopted (i.e., boiling), as heat treatment is known to affect fatty acid constituents of foods (Ouazib et al., 2015). Hexadecanoic acid methyl ester and octadecanoic acid methyl ester were detected only in raw BGN and are both known as the most abundant saturated fatty acids in nature, reported in plants, animals, lower organisms and functions in cells as specific proteolipids (i.e. connected to internal cysteine residues through thioester bonds) (Anonymous, 2013).

4. Conclusion

A total of 134 metabolites were detected in Bambara groundnuts and derived dawadawa samples using GC-HRTOF-MS. From the two fermented samples, dehulled samples had the highest number of metabolites as compared to their unhulled counterparts. Compounds identified included esters, ketones, phenols, flavor related compounds and constituents that could confer organoleptic properties, nutritional and functional benefits of BGN and derived dawadawa. The BGN seeds and dehulled dawadawa possess beneficial components that can potentially be incorporated into human diets for health benefits. Further investigations into the quantification of the metabolites in this study are still needed, particularly for those significant metabolites obtained in all three samples. These would not only provide a better understanding of legume fermentation, but also assist in providing an insight into these significant metabolites that could potentially be biomarkers of dawadawa.

Declarations

Author contribution statement

Janet Adeyinka Adebiyi: Conceived and designed the experiments; Performed the experiments; Wrote the paper.

Patrick Berka Njobeh, Eugenie Kayitesi: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.

Oluwafemi Ayodeji Adebo: Performed the experiments; Analyzed and interpreted the data.

Funding statement

This work was supported by the National Research Foundation (NRF) of South Africa (120751) and the NRF of South Africa National Equipment Programme (99047).

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Contributor Information

Janet Adeyinka Adebiyi, Email: janetaadex@gmail.com.

Eugenie Kayitesi, Email: eugenie.kayitesi@up.ac.za.

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