1. Introduction
Pulses are widely consumed as staple foods and serve as a crucial source of dietary protein for a large proportion of the world’s population, especially in regions where the consumption of animal protein is restricted due to limited availability or avoided due to religious or cultural practices [
1].
Fava bean (
Vicia faba L.) is one of the most widely grown pulses, following soybean and pea in terms of area and production [
2]. It is recognised as a source of protein, accounting for 27–40%, with a low fat content (1–3%), composed mainly of oleic acid (monounsaturated fatty acid) and linoleic acid (polyunsaturated fatty acid) [
3,
4]. Fava bean also contains around 13% dietary fibre and 40% starch, as well as essential vitamins and minerals (primarily zinc, potassium, and iron) [
3,
4]. Including pulses in the diet may have potential health benefits, such as reducing the risk of cardiovascular disease by preventing hypertension and hypercholesterolemia [
5]. However, their use in food is still limited, as they contain several bioactive compounds traditionally classified as antinutrients. These antinutritional factors include phytic acid, saponins, tannins, trypsin inhibitors, and flatulence-causing oligosaccharides [
6]. They can have negative effects by reducing the digestibility of proteins and carbohydrates, and interfering with minerals’ bioavailability [
7]. However, processing methods such as dehulling, soaking, cooking, fermentation, and germination can enhance the nutritive value of food legumes by reducing these effects [
8].
Solid-state fermentation (SSF) has been cited in numerous studies for its ability to degrade antinutritional compounds, and in enhancing the sensory, compositional, and functional properties of legumes [
9,
10,
11,
12,
13]. During SSF, the fermenting microorganism grows on a damp, solid substrate with a significantly low water content, allowing close contact between the microorganism and the gaseous oxygen from the air [
14]. During SSF, microorganisms produce enzymes such as amylases, proteases, and lipases, resulting in the breakdown of macronutrients into more digestible compounds, which can enhance the aroma, flavour, and texture [
13].
Filamentous fungi such as
Aspergillus oryzae and
Rhizopus oligosporus are used in industry to manufacture antibiotics, organic acids, and commercial enzymes [
15].
A. oryzae has been used for centuries in Asia to make traditional fermented soy products, while
R. oligosporus was traditionally used in Indonesia to produce tempeh by fermenting soybeans [
16,
17]. Both genera are considered as generally recognised as safe (GRAS) and have been applied in several studies on SSF of pulses [
18,
19]. Chawla et al. (2017) used SSF on black-eyed pea with
A. oryzae and observed positive changes in functional properties such as water- and oil-holding capacities, emulsion and foaming properties, and the enhanced bioavailability and digestibility of iron and zinc [
18]. Another study conducted by Toor et al. (2022), investigating the fermentation of different legumes (chickpea, pigeon pea and soybean) by
R. oligosporus, revealed an increase in protein, ash, and amino acid contents. In addition, the SSF process changed the colour and some functional properties [
19].
The aim of this study was to examine the effects of SSF with A. oryzae and R. oligosporus on the nutritional composition of fava bean flour. Furthermore, the research explored changes in the techno-functional properties and aroma characteristics of the ingredients.
4. Discussion
Aspergillus oryzae and
Rhizopus oligosporus are general recognised as safe (GRAS) fungi that have been used in the food industry for decades [
18,
19]. Fungal solid-state fermentation (SSF) has been shown to enhance nutritional properties by reducing antinutritional factors, and impacting the techno-functional properties, composition, and sensory characteristics of legumes. In this study, fava bean flour was fermented with
A. oryzae and
R. oligosporus, and the effect on the techno-functionality, nutritional profile, and aroma characteristics of the resulting ingredients was investigated.
FODMAPs, an abbreviation for fermentable oligo-, di-, mono-saccharides, and polyols, are a broad category of small nondigestible carbohydrates made up of 1–10 sugar molecules that the small intestine has difficulty absorbing [
38]. The decreased sucrose levels, especially in FBA, indicated its possible utilisation during glycolysis. In carbohydrate metabolism, sucrose is first converted to fructose and glucose, which is transformed into D-glucose-6-phosphate. This molecule can then either be converted into D-fructose-6-phosphate further undergoing glycolysis or enter the pentose phosphate pathway (PPP) [
39,
40]. The PPP is the main source of NADPH, playing a crucial role in fungi by aiding in the production of various important compounds, including polyols, biofuels, carotenoids, and antibiotics [
41]. Zaveri et al. (2022) stated that some
Rhizopus species and strains metabolise sucrose less efficiently than glucose, which would explain the differences in total mono-/disaccharides after SSF between the two genera [
42]. Polyols can also have effects such as bloating, pain, changes in bowel habits, and a laxative effect, particularly in people suffering from irritable bowel syndrome (IBS). These effects are due to the malabsorption of these sugar-alcohols and their rapid fermentation by bacteria in the colon, resulting in the production of gas [
43]. The complete degradation of sucrose by
A. oryzae was likely used for polyol production, with a significant negative correlation observed between sucrose and the polyol content (
p-value: 0.15, r-value: 0.97). According to Kordowska-Wiater (2015), glucose is one of the most efficient precursors for the production of arabitol [
44]. As well as the naturally occurring mannitol present in
A. oryzae cells, an increase in polyols may also be due to the low water activity (a
w) during SSF, with a low a
w resulting in osmotic stress and an accumulation of solutes such as ions, polyols, or amino acids to prevent cellular water loss [
45]. Despite the increase in arabitol and mannitol in FBA, the concentration of galacto-oligosaccharides (GOS) was almost completely reduced, resulting in a decrease in total FODMAPs. FBR had a similar FODMAP content to FBA, but
R. oligosporus reduced the GOS to a lesser extent, with raffinose/stachyose levels remaining unchanged. In accordance with the literature, pulses are considered high in FODMAPs because of their high content of GOS [
46].
Fungal amylases are responsible for breaking down starch into the simple sugars glucose and maltose [
15,
47]. Total starch showed a significant reduction in both fermented ingredients, and this could also be observed in the micrographs. Indeed, the smooth, round, and irregular molecules observed in FB were characteristic of starch molecules [
48,
49]. These molecules were less numerous in the fermented ingredients, particularly in the FBR, which was also reflected in the lower value of total starch. However, the significant reduction in resistant starch (RS), a type of starch which is resistant or less susceptible to enzymatic hydrolysis, must have occurred in another way, with studies showing that mechanical and physical processes such as grinding and autoclaving could make RS less resistant and more accessible for hydrolysis [
50,
51]. Pulses’ amyloses can also form part of a complexation with lipids and thus contribute to the RS content [
50,
52]. These complexes may have been hydrolysed by fungal enzymes, which would explain the increase in digestible starch in the fermented ingredients [
53]. Total dietary fibre (TDF) in the FB showed higher values than in previous studies. Millar et al. (2019) reported a total dietary fibre content of 13.8%, of which the insoluble fraction accounted for two-thirds of this value [
54]. Resistant starch is considered a form of dietary fibre, which may explain why insoluble dietary fibre accounted for such a large proportion (92.5%) of the TDF in FB [
55]. Additionally, Jeraci et al. (1990) stated that the AOAC method for measuring total fibre can be influenced by the presence of certain components such as ash, proteins, tannins, and resistant starches [
56].
The production of fatty acids is part of the general metabolic pathway of fungi, through the release of lipases to hydrolyse lipids [
57,
58]. The increased content of medium-chain fatty acids could also be due to the breakdown of the aforementioned amylose–lipid complexes. FB contained mostly linoleic acid, an essential fatty acid [
59], which is the predominant fatty acid in pulses [
60]. During SSF, both fermented ingredients showed metabolisation of linoleic acid and oleic acid (a monounsaturated fatty acid with health benefits [
61]), as well as palmitic acid (a saturated fatty acid). Additionally,
R. oligosporus produced stearic acid (a saturated fatty acid) and linolenic acid (an essential fatty acid). Saturated fatty acids are generally reduced in food due to their negative effects on cardiovascular diseases [
62]. These changes in the fatty acid composition might be attributed to the increased fat content in the substrate, which acted as an inducing agent for fungal metabolism, explaining the higher total fat content in FBR [
19].
Proteases produced by filamentous fungi are responsible for hydrolysing complex proteins into shorter peptides or their constituent amino acids [
63]. This was demonstrated in the protein profiles of FBA and FBR determined by SDS-PAGE, which showed a decrease in the molecular weight of protein, reflecting changes in the amino acid composition. An important decrease in arginine, an essential precursor for synthesising compounds such as urea, nitric oxide, and glutamate, as well as other amino acids such as proline [
64,
65], was also observed. In addition, arginine, which is a basic amino acid, may have decreased due to its destabilisation by the acidic fermentation conditions [
66]. Regarding essential amino acids, an increase in the levels of sulfur amino acids was observed after SSF, resulting in complete fulfilment of the daily requirements of adults outlined by the WHO [
37]. Filamentous fungi are capable of synthesising cysteine and methionine from serine, with serine being reduced during fermentation in the current study and having a significant negative correlation with cysteine and methionine (cysteine:
p-value: 0.14, r-value: 0.98; methionine:
p-value: 0.19, r-value: 0.96) [
67,
68]. Additionally, some amino acids may have increased due to the breakdown of condensed tannins and insoluble protein complexes by microbial tannase enzymes during fermentation [
19]. While condensed tannins were fully eliminated in both fermented ingredients,
A. oryzae and
R. oligosporus showed different trends with regards to the degradation of antinutrients in the fava bean substrate. Although
A. oryzae is known to secrete a large amount of phytase enzymes which are responsible for the degradation of phytic acid, the reduction in phytic acid was low, while
R. oligosporus showed a higher degree of degradation. A similar trend was previously observed after SSF of a quinoa substrate with
A. orzyzae and
R. oligosporus [
21]. This could also explain the decreased level of starch degradation observed in FBA, as phytic acid can bind starch [
6]. As also observed in a separate study [
21],
A. oryzae reduced chymotrypsin inhibitors to a lower level than
R. oligosporus. However, saponins were fully eliminated in FBA, whereas only a slight reduction was observed in FBR. Saponins are commonly present in pulses and contribute a bitter taste that may limit consumer acceptability [
69]. Furthermore, the formation of trypsin inhibitors may hinder the absorption of dietary proteins, with these compounds capable of binding to the active sites of pancreatic trypsin, resulting in a reduction in the enzyme’s proteolytic activity [
70].
The protein solubility of both ingredients decreased after SSF. Since a lower protein solubility was observed in FBR, as well as a higher concentration of trypsin inhibitors, this may be a reason. Indeed, a negative correlation occurred between trypsin inhibitors and protein solubility (protein solubility at pH 7:
p-value: 0.04, r-value: 0.998; protein solubility at native pH:
p-value: 0.33, r-value: 0.87). However, protein solubility may also have been affected by other factors (intrinsic and extrinsic), such as the observed increase in the protein content of the fermented ingredients. Changes in the amino acid composition and the conformation of proteins have an impact on proteins’ solubility [
71,
72]. Because of the ability of filamentous fungi to assimilate complex substrates, they produce a protein-rich fungal biomass called mycoproteins [
73]. This network of mycelia and porous microstructures was clearly observed in the microscopic images. The aggregated surfaces also resulted in larger particles, which is also an important factor for the reduction in protein solubility [
72].
The hydrolysis of proteins by the proteolytic activity of fungi exposes their hydrophobic and/or hydrophobic sites by unfolding the proteins’ structure [
74,
75], potentially enhancing techno-functional properties such as the gelation, foaming characteristics and emulsifying properties [
76]. In this study, a decrease in the foaming capacity of FBA and FBR and the foam stability of FBR was observed. This could potentially be due to the exposure of hydrophobic sites and an increased likelihood of absorption at the air–water interface, thereby reducing the interfacial tension [
77]. Moreover, it may also be due to extensive protein denaturation, as well as increased particle size [
76,
78]. A significant negative correlation between the mean particle size and foaming capacity (
p-value: 0.17, r-value: 0.96) was observed. Indeed, hydrophobic exposure was also responsible for the formation of aggregates [
74], with aggregates in the powder resulting in significant changes in the techno-functional properties. The increase in the emulsions’ separation rates was also positively correlated with the mean particle size (
p-value: 0.17, r-value: 0.97) [
79]. FBR also showed a higher level of sedimentation in an emulsion, which may be a reflection of its lower protein solubility [
79]. The formation of aggregates can also result in to the development of structures called microcapillaries, which have internal spaces that can physically trap oil, thus increasing the oil-holding capacity (OHC) [
74]. However, in this study, no significant difference in the OHC of fava bean flour was observed after SSF. It is possible that enzymatic hydrolysis may have exposed a more significant amount of hydrophilic binding sites, contributing to the increased water-holding capacity (WHC) of both fermented ingredients [
75]. WHC was also found to have a significant negative correlation with the minimum gelling concentration (
p-value: 0.15, r-value: 0.97). Many studies have linked these two properties, as a high WHC can aid in the binding of water, resulting in a stronger gel structure [
80,
81].
Colour changes (ΔE) were observed in both FB and FBR after fermentation, with both ingredients showing lower L* values as well as higher a* and b* values [
82]. Colour changes may occur during SSF due to fungal growth through the production of mycelia and/or spores [
83]. This could also explain the higher differential colour index of FBR, as different fungal species can produce different mycelia, which vary in colour [
84]. However, this could also be due to the autoclaving process carried out on the ingredients prior to fermentation [
82].
Olfactometric analysis revealed that fermenting fava bean flour with
A. oryzae and
R. oligosporus produced 3-methylpentanoic acid, butanoic acid, and 2-methylpropanoic acid, which are associated with cheese aromas, along with acetic acid, which gives a vinegar aroma. Acetic and butanoic acids have pyruvate as a precursor, a product of sugar metabolism, while 2-methylpropanoic and 3-methylpentanoic acids were derived from their respective aldehydes [
85]. The main pathway for synthesising aromatic compounds begins with the oxidative deamination of amino acids, producing an α-keto acid, which is decarboxylated to form an aldehyde. The aldehyde can then be oxidised to an acid or reduced to an alcohol [
85]. Key amino acids in this process include valine, leucine, isoleucine, methionine, cysteine, phenylalanine, proline, and lysine. Valine, leucine, isoleucine, phenylalanine, and methionine produce aldehydes through the Strecker degradation pathway [
86]. In this study, the Strecker reaction produced 2-methylbutanal (malty), 3-methylbutanal (malty), phenylacetaldehyde (honey), and methional (boiled potato) from isoleucine, leucine, phenylalanine, and methionine, respectively [
85,
86,
87]. Methionine also formed the sulfur compounds dimethyltrisulfide, with a higher content in FBR, and dimethyltetrasulfide, found only in FBR, resulting in a cabbage-like aroma [
86,
88]. Earthy aromas from fermented products are due to pyrazine compounds, which again were slightly more developed in FBR, likely derived from lysine [
86]. 2-Acetyl-1-pyrroline produced a roasty aroma from proline [
86], and cysteine may be the source of the meaty aroma of 2-methyl-3-(methyldithio)furan [
89,
90]. Additionally, sweet aromas such as γ-dodecalactone (peach) and maltol (caramel) were also developed in the fermented products. γ-Dodecalactone, this time predominant in FBA, may have been produced through a different pathway where fungi transform certain fatty acids [
91], with oleic acid being the likely source [
92]. Maltol resulted from the Maillard reaction [
93]. SSF also appeared to enhance the aroma profile by reducing 2,4,6-trichloroanisole and trans-4,5-epoxy-(E)-2-decenal, which gave fava bean flour a mouldy and metallic-like aroma.
Some filamentous fungi are of great interest for their high production of organic acids. Lactic acid was the organic acid produced most by both fungi. Glucose undergoes glycolysis to produce pyruvate and ATP for cellular energy. Pyruvate is then converted to pyruvic acid and further into lactic acid by fungi [
58,
94]. Furthermore, citric acid is typically produced in high amounts during fermentation from two pyruvic acid molecules via the TCA cycle [
95]. However, in this study, citric acid levels were significantly lower, possibly due to the duration of fermentation. Normally, one pyruvic acid molecule becomes acetyl-CoA, and the other becomes oxaloacetic acid, which enters the mitochondria and converts to malic acid, then citric acid with acetyl-CoA [
96]. The significant production of malic acid suggests incomplete fermentation. Fumaric and succinic acids are intermediates of the TCA cycle, and fungi are also capable of using a reducing TCA cycle in the cytosol, converting pyruvic acid to malic acid, oxaloacetic acid, fumaric acid, and finally succinic acid [
96]. This would explain the increase in succinic and fumaric acids. All these acids showed a strong positive correlation with glucose (lactic acid:
p-value 0.09, r-value 0.989; malic acid:
p-value 0.01, r-value 1.00; fumaric acid:
p-value 0.06, r-value 0.996; succinic acid:
p-value 0.13, r-value 0.979). The drop in pH and rise in TTA values during SSF were linked to the fungi’s acid production.