Exploring the Impact of Solid-State Fermentation on Fava Bean Flour: A Comparative Study of Aspergillus oryzae and Rhizopus oligosporus

Abstract

Fava bean (Vicia faba L.) is a protein-rich pulse with high nutritional value, but its functional and sensory characteristics limit its application in foods. Solid-state fermentation (SSF) can modify the composition of plant proteins, modulate its functionality, and enhance the sensory aspects. In this study, fava bean flour (FB) was fermented with Aspergillus oryzae and Rhizopus oligosporus to produce FBA and FBR, respectively, ingredients with distinct nutritional, functional, and aroma characteristics. The protein content increased by 20% in FBA and 8% in FBR, while fat levels rose more significantly in FBR (+40%). The overall content of fermentable oligo-, di-, mono-saccharides, and polyols (FODMAPs) decreased by 47% (FBA) and 57% (FBR), although polyol production by A. oryzae was observed. SSF improved the nutritional profile of FBA and FBR, with a notable increase in the concentration of essential amino acids observed, and a reduction in most antinutrients, with the exception of trypsin inhibitors. SSF resulted in the formation of aggregates, which increased the particle size and reduced protein solubility. Emulsions prepared with the fermented ingredients separated faster, and the foaming capacity of both FBA and FBR was decreased, but an increase in water-holding capacity was observed. SSF resulted in the production of predominantly savoury-associated aroma compounds, with compounds characteristic of metallic and mouldy aromas reduced. These results indicate the potential of SSF to transform FB with enhanced nutritional value and improved sensory and functional properties.

Full text

Citation: Gautheron, O.; Nyhan, L.;
Torreiro, M.G.; Tlais, A.Z.A.; Cappello,
C.; Gobbetti, M.; Hammer, A.K.;
Zannini, E.; Arendt, E.K.; Sahin, A.W.
Exploring the Impact of Solid-State
Fermentation on Fava Bean Flour: A
Comparative Study of Aspergillus
oryzae and Rhizopus oligosporus.Foods
2024,13, 2922. https://doi.org/
10.3390/foods13182922
Academic Editors: Maria Lucia
Guerra Monteiro, Denes do Rosario
and Yhan Da Silva Mutz
Received: 12 August 2024
Revised: 10 September 2024
Accepted: 13 September 2024
Published: 15 September 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
foods
Article
Exploring the Impact of Solid-State Fermentation on Fava
Bean Flour: A Comparative Study of Aspergillus oryzae and
Rhizopus oligosporus
Ophélie Gautheron 1, Laura Nyhan 1, Maria Garcia Torreiro 2, Ali Zein Alabiden Tlais 3, Claudia Cappello 3,
Marco Gobbetti
3
, Andreas Klaus Hammer
4
, Emanuele Zannini
1,5
, Elke K. Arendt
1, 6,
* and Aylin W. Sahin
1
1School of Food and Nutritional Sciences, University College Cork, T12 YN60 Cork, Ireland;
o.gautheron@umail.ucc.ie (O.G.); lnyhan@ucc.ie (L.N.); e.zannini@ucc.ie (E.Z.); aylin.sahin@ucc.ie (A.W.S.)
2Mogu srl, Via S. Francesco, 62, 21020 Inarzo, VA, Italy; mgt@mogu.bio
3Faculty of Agricultural, Environmental and Food Sciences, Free University of Bozen-Bolzano, Piazza
Università, 1, 39100 Bolzano, BZ, Italy; alizeinalabiden.tlais@unibz.it (A.Z.A.T.);
claudia.cappello@unibz.it (C.C.); marco.gobbetti@unibz.it (M.G.)
4
Fraunhofer Institute for Molecular Biology and Applied Ecology, Ohlebergsweg 12, 35392 Giessen, Germany;
andreas.hammer@ime.fraunhofer.de
5Dipartimento di Biologia Ambientale, Sapienza Universitàdi Roma, 00185 Rome, RM, Italy
6APC Microbiome Ireland, University College Cork, T12 YT20 Cork, Ireland
*Correspondence: e.arendt@ucc.ie
Abstract: Fava bean (Vicia faba L.) is a protein-rich pulse with high nutritional value, but its functional
and sensory characteristics limit its application in foods. Solid-state fermentation (SSF) can modify
the composition of plant proteins, modulate its functionality, and enhance the sensory aspects. In
this study, fava bean flour (FB) was fermented with Aspergillus oryzae and Rhizopus oligosporus to
produce FBA and FBR, respectively, ingredients with distinct nutritional, functional, and aroma
characteristics. The protein content increased by 20% in FBA and 8% in FBR, while fat levels rose
more significantly in FBR (+40%). The overall content of fermentable oligo-, di-, mono-saccharides,
and polyols (FODMAPs) decreased by 47% (FBA) and 57% (FBR), although polyol production by A.
oryzae was observed. SSF improved the nutritional profile of FBA and FBR, with a notable increase
in the concentration of essential amino acids observed, and a reduction in most antinutrients, with
the exception of trypsin inhibitors. SSF resulted in the formation of aggregates, which increased
the particle size and reduced protein solubility. Emulsions prepared with the fermented ingredients
separated faster, and the foaming capacity of both FBA and FBR was decreased, but an increase in
water-holding capacity was observed. SSF resulted in the production of predominantly savoury-
associated aroma compounds, with compounds characteristic of metallic and mouldy aromas reduced.
These results indicate the potential of SSF to transform FB with enhanced nutritional value and
improved sensory and functional properties.
Keywords: aroma; metabolites; nutrition; techno-functionalities; antinutritional factors; fava bean
flour; solid-state fermentation
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
Foods 2024,13, 2922. https://doi.org/10.3390/foods13182922 https://www.mdpi.com/journal/foods

Foods 2024,13, 2922 2 of 27
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
–
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, mi-
croorganisms 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.
2. Materials and Methods
2.1. Raw Materials and Starter Cultures
Fava bean flour (FB) (Müller’s Mühle, Gelsenkirchen, Germany) was fermented (Mogu,
Inarzo, Italy) with Aspergillus oryzae (from a commercial koji starter from Starter Cultures,
Amsterdam, The Netherlands) or Rhizopus oligosporus (from a commercial tempeh starter
from Top Cultures, Zoersel, Belgium). The starters were prepared according to Chutrtong
and Bussabun (2014) [
20
], with modifications. Briefly, clear sporulation on MYA medium
was lyophilised (alpha 1-2 LDplus lyophiliser, 230 V, CHRIST, Germany) and mixed with
sterilised rice flour (Oryza sativa, Riseria d’Italia S.r.l.), in a 9:1 weight ratio, using a labo-
ratory mill. The starter was then stored in Mogu’s (Inarzo, Italy) growing room, which is
equipped with a temperature-controlled AC system, for up to 60 days at 25 ◦C.
Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise
stated.
2.2. Solid-State Fermentation
The fava beans were fermented according to Gautheron et al. (2024) [
21
]. Briefly, the
fava bean substrate was washed to remove any unwanted contaminants or residues that
may have negatively impacted fermentation [
22
], and rehydrated by immersion in tap

Foods 2024,13, 2922 3 of 27
water (in a 1:2 ratio of substrate to tap water by volume) for 15 min. After draining, the
substrate was autoclaved (121 ◦C for 15 min) and subsequently cooled to 30 ◦C.
The R. oligosporus tempeh-like fermentation was performed according to Erkan et al.
(2020) with slight changes [
23
]. For pH adjustment to 4.5–5.5, 25 mL of wine vinegar was
added to 500 g of the dry substrate during the cooling phase. For inoculation, 1.5 g of the
tempeh starter was added to 500 g of the dry substrate and mixed thoroughly to ensure the
homogenous distribution of spores. The substrate was tightly packed onto stainless steel
trays (3.8 L volume) with a polycarbonate lid. Inoculation was performed manually under
a laminar flow hood, keeping clean conditions but not completely sterile. The substrate
and inoculum were mixed manually.
The A. oryzae koji-like fermentation was performed according to the method described
by Kim et al. (2012) [
24
]. For this, 1.5 g of the koji starter was added to 500 g of the
substrate, mixed thoroughly, and compacted onto stainless steel trays (3.8 L volume) with
a polycarbonate lid. Inoculation was performed manually under a laminar flow hood,
keeping clean conditions but not completely sterile. The substrate and inoculum were
mixed manually.
The inoculated substrates were incubated on heating mats (24
×
52 cm, 220 V, Lerway)
at 28
◦
C (tempeh) or 30
◦
C (koji), controlled by a thermostat (ITC-308, 220 V, Inkbird,
Shenzen, China) which was placed into the middle of the substrate bed. For A. oryzae, the
substrate was mixed 24 h after inoculation to ensure complete colonisation, dissipate heat,
and promote aeration. After 48 h (for R. oligosporus) and 72 h (for A. oryzae), the substrate’s
surface was covered with mycelium, forming a compact cake. The fermented substrate was
freeze-dried, then ground into flour (particle size ≤100–200 µm).
2.3. Compositional Analysis
Compositional analysis was performed as described by Gautheron et al. (2024) [21].
Carbohydrates
Starch (resistant, digestible, and total) was determined using the Megazyme kit K-
RAPRS (Megazyme, Bray, Ireland). Dietary fibre was determined using the K-RINTDF
method (Megazyme, Bray, Ireland). Sugars (glucose, fructose, sucrose, maltose, and galac-
tose), and FODMAPs (polyols: arabitol, sorbitol, and mannitol; oligosaccharides: raffi-
nose/stachyose, verbascose, kestose, and nystose)were extracted as described by Ispiryan
et al. (2019), and separated and quantified via high-performance anion-exchange chro-
matography coupled with pulsed amperometric detection (HPAEC-PAD) on a Dionex
TM
ICS-5000+ system (Thermo Scientific, Sunnyvale, CA, USA) [
25
]. All carbohydrate mea-
surements were performed using authentic reference standards.
2.4. Protein Characteristics
2.4.1. Total Amino Acids and Free Amino Acids
Total amino acids were assessed following hydrolysis under acidic, oxidative-acidic,
or alkaline conditions using a Sykam S433 amino acid analyser (Fürstenfeldbruck, Ger-
many). External calibration was applied, following the method outlined by Ahlborn et al.
(2019) [
26
]. The true protein content was calculated by summing up the amino acid residues,
factoring in the added water amount for peptide bond hydrolysis.
Free amino acid concentrations were determined by MS-Omics Aps (Vedbæk, Den-
mark). The ingredients underwent derivatisation with methyl chloroformate, following a
slightly modified version of the protocol outlined by Smart et al. (2010) [
27
], as reported by
Gautheron et al. (2024) [21].
2.4.2. Sulfhydryl (SH) Groups
Exposed, free, and total SH groups were quantified using Ellman’s method, as de-
scribed by Gautheron et al. (2024) [21].

Foods 2024,13, 2922 4 of 27
2.4.3. Protein Solubility
Protein solubility was measured at the native pH and pH 7 using the Kjeldahl method
(AACC Method 46-12.01 [28]), as described by Jaeger et al. (2023) [29].
2.4.4. SDS-PAGE
The protein profile of the ingredients was determined by SDS-PAGE, as described by
Gautheron et al. (2024) [21].
2.5. Techno-Functional Properties of the Ingredients
2.5.1. pH and Total Titratable Acidity (TTA)
The pH and TTA of the ingredients were assessed following the method outlined by
Jaeger et al. (2023) [29].
2.5.2. Water- and Oil-Holding Capacity
Water-holding capacity (WHC) and oil-holding capacity (OHC) were measured in
accordance with the method of Boye et al. (2010), with slight modifications [
1
], as described
by Gautheron et al. (2024) [21].
2.5.3. Foaming Properties
For this, 2% (w/w) dispersions of the ingredients were prepared in distilled water, and
foaming capacity and foam stability were assessed according to Gautheron et al. (2024) [
21
].
2.5.4. Minimum Gelling Concentration
The minimum gelling concentration was determined following the method of Vogel-
sang et al. (2020) [30].
2.5.5. Emulsion Characteristics
Emulsion stability was evaluated using the method described by Jaeger et al. (2023) [
29
].
2.5.6. Particle Size
The particle size distribution of the protein ingredients was measured by laser diffrac-
tion using the Mastersizer 3000 (Malvern Instruments Ltd., Worcestershire, UK) equipped
with the AERO-S attachment. The refractive index of the particles was set to 1.45, and the
absorption index was set to 0.001.
2.5.7. Colour
The ingredients’ colour was measured using a ChromaMeter CR-400 (Konica Minolta,
Osaka, Japan), based on the CIE L*a*b* colour space system, as described by Gautheron
et al. (2024) [
21
]. The differential colour index value was calculated using the following
equation
∆E=q(∆L∗)2+(∆a∗)2+(∆b∗)2(1)
where ∆L∗=L∗
FB −L∗
FB A/FBR,∆a∗=a∗
FB −a∗
FB A/FBR, and ∆b∗=b∗
FB −b∗
FB A/FBR
2.6. Antinutrients
Antinutritional factors of the ingredients were determined as follows. All results are
expressed as dry matter and were analysed in duplicate.
2.6.1. Phytic Acid
Phytic acid concentrations were measured using a phytic acid (phytate)/total phos-
phorus kit (Megazyme International, Ireland), which quantified the phosphorus released
by the enzymatic action of phytases. Briefly, 1 g of each ingredient was mixed with 20 mL
of
0.66 M
HCl and incubated overnight at 25–28
◦
C. The ingredients were then centrifuged
for 10 min at 18,516 rcf, and the pH of the supernatant was adjusted to 7 using 0.75 M

Foods 2024,13, 2922 5 of 27
NaOH. The extracts were processed to determine the concentrations of free and total phos-
phorus using a phosphorus calibration curve, as per the manufacturer’s instructions. The
phosphorus content was used to estimate the phytic acid concentrations via a mathematical
formula provided in the instruction manual.
2.6.2. Total Saponin
The total saponin content was quantified using a modified version of the method
by Lai et al. (2013), as detailed by Krause et al. (2023) [
31
,
32
]. Briefly, 0.5 g of each
ingredient was defatted using 10 mL of petroleum ether by continuous shaking for 4 h.
After evaporation of the solvent, 20 mg of the residues were extracted by mixing with 5 mL
of 80% (v/v) methanol for 4 h. The mixture was centrifuged at 7916 rcf for 10 min at 4
◦
C,
and the supernatants were stored in the dark at 4
◦
C until use. To prepare the assay, 0.1 mL
of the ingredient’s extract, 0.4 mL of 80% (v/v) methanol, 0.5 mL of a freshly prepared 8%
ethanolic vanillin solution, and 5 mL of 72% sulfuric acid were mixed in an ice-water bath.
The mixture was then heated at 60
◦
C for 10 min and cooled in ice water. Absorbance was
measured at 544 nm against a reagent blank, and the results were expressed as mg saponin
per gram of extract based on a standard curve of saponin in 80% aqueous methanol.
2.6.3. Condensed Tannins
Condensed tannins were quantified using the vanillin assay, as described by Krause
et al. (2023) [
32
]. The extracts were prepared by mixing 200 mg of the ingredient with
10 mL of absolute methanol for 20 min in rotating screw-cap culture tubes. The supernatant
was collected by centrifugation (SL16R centrifuge, Thermo Fischer Scientific, Waltham,
MA, USA) at 2740 rcf for 10 min, and 1 mL of the ingredients and catechin standards were
transferred in duplicate to glass tubes and heated to 30
◦
C in a water bath. Simultaneously,
the vanillin reagent was prepared by mixing equal parts of 1% (w/v) vanillin in absolute
methanol and 8% (v/v) concentrated HCl in absolute methanol. Next, 5 mL of the preheated
vanillin reagent was added to one set of ingredients and standard tubes, while 5 mL of a
preheated 4% aqueous HCl solution was added to the second set of tubes, with a 1 min
interval between additions. The ingredients were incubated at 30
◦
C for 20 min, and the
absorbance values were measured by a UV-1800 spectrophotometer (Shimadzu, Kyoto,
Japan) at 500 nm at 1 min intervals. Condensed tannins were expressed as catechin equiva-
lents (CE) mg/g, calculated from the standard curve obtained by plotting the absorbance
at 500 nm against the reagent blanks.
2.6.4. Trypsin Inhibitor Activity (TIA)
Trypsin inhibitor activity (TIA) was assessed using a spectrophotometric assay follow-
ing the AOCS Method Ba 12a-2020 33]. One gram of the sample was extracted with 10 mL
of a 0.15 M phosphate buffer pH 8.1 at 4
◦
C overnight. Extracts (200
µ
L) were incubated
with 250
µ
L of a trypsin solution (0.004% trypsin in 0.025 M glycine HCl buffer) and diluted
to 1 mL with at phosphate buffer of pH 8.1. Then 2.5 mL of a 0.001 M BAPNA solution
(dissolved in a minimum volume of DMSO with a phosphate buffer, pH 8.1), previously
warmed to 37
◦
C, was added. After incubating the mixture at 37
◦
C for 15 min in a shaking
water bath, the reaction was stopped by adding 300
µ
L of 30% (v/v) glacial acetic acid. In
this method, one trypsin unit was defined as a 0.02 unit increase in absorbance measured
by the UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) at 410 nm under the 5 mL
assay conditions outlined in the protocol [
33
]. TIA is expressed as trypsin inhibitor units
per mg of dry matter.
2.6.5. Chymotrypsin Inhibitor Activity (CIA)
Chymotrypsin inhibitor activity (CIA) was evaluated by following the spectropho-
tometric method outlined by Alonso et al. (2000) [
34
]. Ingredients were extracted by
mixing the ingredients with 0.05 M Tris-HCl buffer (pH 7.6) overnight at a ratio of 1:10
(ingredient–buffer) (w/v). Next, 50
µ
L of the extracts were combined with a 0.005% chy-

Foods 2024,13, 2922 6 of 27
motrypsin solution prepared in 0.05 M Tris-HCl buffer (pH 7.6) (100
µ
L), followed by
dilution to 1 mL. Subsequently, 2.5 mL of 0.001 M benzoyl-L-tyrosine ethyl ester (BTEE),
heated to 30
◦
C, was added to the ingredients, and the absorbance values were immediately
recorded at 256 nm by the UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). In this
assay, one chymotrypsin unit corresponded to a 0.01 unit increase in absorbance of the
reaction mixture.
2.7. Microscopy
Microscopy was performed using a scanning electron microscope (SEM), according to
the method reported by Atzler et al. (2021) [35].
2.8. Olfactometry
Olfactometry analyses were conducted externally by AromaLAB GmbH (Martinsried,
Germany), as described by Gautheron et al. (2024) [21].
2.9. Fungal Metabolites
Organic acids were determined by MS-Omics Aps (Vedbæk, Denmark). The ingredi-
ents underwent derivatisation with methyl chloroformate, following a slightly modified
version of the protocol outlined by Smart et al. (2010) [27], as reported by Gautheron et al.
(2024) [
21
]. Ergosterol concentrations were determined according to the method described
by Bickel-Haase et al. (2024) [36].
2.10. Statistical Analysis
All analyses were performed in triplicate unless otherwise specified. The obtained
results were assessed for normality, followed by a one-way ANOVA with a post hoc Tukey
test (p< 0.05) was conducted using IBM SPSS Statistics software, version 28 (Armonk, NY,
USA). In cases where equal variances were not assumed, correction using the Welch test
and Games Howell post hoc test (p< 0.05) was applied. Non-normally distributed data
were analysed using Kruskal–Wallis tests (p< 0.05).
Microsoft Excel Version 2407 (Microsoft Corporation, Redmond, WA, USA) was used
to perform correlation analysis and regressions.
3. Results
3.1. Composition of Ingredients
The composition of FB, FBA, and FBR are displayed in Table 1. FB had a moisture
content of 12.06 g/100 g, while a decrease was observed in FBA and FBR due to the
drying operation during the SSF process. FB had a protein content of 24.58%, which
increased by 20.3% and 8.1% in FBA and FBR, respectively. The fat content in FB was 2.27%
in total, derived mainly from linoleic acid (39.2%), oleic acid (23.3%), and palmitic acid
(12.8%). Stearic acid and linolenic acid also contributed to the total fat, with 3.1% and 2.2%,
respectively. The complete fatty acid profile can be found in the supporting information (cf.
Appendix A, Table A1). With the application of fermentation, the fat content was higher
in FBR compared with FBA. The fatty acid profile showed an increase in palmitic acid,
oleic acid, and linoleic acid for both genera. However, fermentation with R. oligosporus
led to a further increase in stearic acid (+200%) and linolenic acid (+140%) compared
with FB. Regarding carbohydrates, FB displayed a mono-/disaccharide concentration of
2.21%, derived solely from sucrose, and a total oligosaccharide concentration comprising
mainly verbascose (3/4) and raffinose/stachyose (1/4). Total starch showed a balance
between digestible starches (51.4%) and resistant starches (48.6%). Finally, total dietary
fibre represented 27.88%, mainly from the insoluble fraction (92.5%). After SSF, a reduction
in total mono-/disaccharides was observed in FBA, while it increased in FBR. Sucrose was
entirely consumed by A. oryzae, whereas it was reduced only by a quarter by R. oligosporus.
An increase in glucose, maltose, and galactose was observed in both fermented ingredients,
while fructose production was also noted in the FBR. Oligosaccharides decreased more

Foods 2024,13, 2922 7 of 27
significantly in FBA, with the values of raffinose/stachyose and verbascose reduced by
94.8% and 95.3%, respectively. In comparison, raffinose/stachyose remained constant in
FBR, and verbascose decreased by 77.0%. Small quantities of kestose and nystose were
detected in both ingredients. The SSF process led to a reduction in resistant starch by
two-thirds and three-quarters for FBA and FBR, respectively. Digestible starch showed an
increase of 18.0% in FBA. Finally, fermentation resulted in a decrease in the total dietary
fibre content, with a significant reduction in the insoluble fraction (
−
38% in FBA and
−
36%
in FBR). Both soluble dietary fibre fractions increased in the fermented ingredients.
Table 1. Composition of fava bean flour (FB), fava bean flour fermented by Aspergillus oryzae (FBA),
and fava bean flour fermented by Rhizopus oligosporus (FBR). The results are expressed on a dry matter
basis, in g/100 g ±standard deviation. n.d. indicates not detected.
g/100 g
FB FBA FBR
Moisture 12.06 ±0.09 8.37 ±0.13 7.54 ±0.03
g/100 g DM
Protein 24.58 ±0.18 29.56 ±0.06 26.57 ±0.18
Total Nitrogen 4.95 ±0.22 5.78 ±0.20 5.87 ±0.19
Total fats 2.27 ±0.14 2.65 ±0.31 3.18 ±0.37
Fatty acid profile
Palmitic acid 0.29 ±0.05 0.36 ±0.06 0.43 ±0.06
Stearic acid 0.07 ±0.01 0.06 ±0.01 0.21 ±0.05
Oleic acid 0.53 ±0.07 0.60 ±0.08 0.79 ±0.10
Linoleic acid 0.89 ±0.11 1.05 ±0.13 0.96 ±0.12
Linolenic acid 0.05 ±0.01 0.05 ±0.01 0.12 ±0.02
Carbohydrates
Mono-/disaccharides 2.21 ±0.08 a1.67 ±0.27 b3.67 ±0.15 c
Glucose n.d. a0.93 ±0.16 b1.07 ±0.03 b
Fructose n.d. an.d. a0.13 ±0.01 b
Sucrose 2.21 ±0.08 an.d. b1.67 ±0.03 c
Maltose n.d. a0.71 ±0.11 b0.75 ±0.07 b
Galactose n.d. a0.03 ±0.00 b0.06 ±0.00 c
Oligosaccharides 3.74 ±0.24 a0.21 ±0.01 b1.64 ±0.05 c
Raffinose/stachyose 0.96 ±0.05 a0.05 ±0.00 b0.98 ±0.04 a
Verbascose 2.78 ±0.19 a0.13 ±0.00 b0.64 ±0.01 c
Kestose n.d. a0.01 ±0.00 b0.01 ±0.00 c
Nystose n.d. a0.03 ±0.00 b0.01 ±0.00 c
Starch
Digestible starch 25.69 ±0.55 a34.62 ±1.69 b28.43 ±2.12 a,b
Resistant starch 24.31 ±0.46 a9.43 ±0.96 b6.39 ±0.37 c
Total starch 50.00 ±0.87 a44.04 ±2.28 b34.83 ±2.15 c
Dietary fibre
Soluble low molecular weight (SDFS) 1.17 ±0.06 a2.45 ±0.06 b3.16 ±0.13 c
Soluble high molecular weight (SDFP) 0.92 ±0.35 a2.18 ±0.29 b2.46 ±0.28 b
Insoluble (IDF) 25.79 ±0.68 a15.93 ±0.22 b16.45 ±1.17 b
Total dietary fibre (TDF) 27.88 ±0.59 a20.59 ±0.44 b22.07 ±0.34 b
Ash 3.57 ±0.22 3.90 ±0.26 3.92 ±0.26
Results within the same row that share the same letter do not differ significantly (p< 0.05).
The nitrogen-to-protein conversion factors for FB, FBA, and FBR are shown in Table 2.
FB displayed a conversion factor of 4.97, while fermentation with A. oryzae increased it and
fermentation with R. oligosporus decreased it.

Foods 2024,13, 2922 8 of 27
Table 2. Nitrogen-to-protein conversion factor for fava bean flour (FB), fava bean flour fermented
by Aspergillus oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR), used to
calculate protein solubility.
Kjeldahl Conversion Factor
Fava bean flour (FB) 4.97
Fava bean flour fermented with Aspergillus oryzae (FBA) 5.11
Fava bean flour fermented with Rhizopus oligosporus (FBR) 4.52
3.2. FODMAP Analysis
The FODMAP contents of the ingredients are given in Table 3. Fermentation with R.
oligosporus resulted in no change in polyols, while an increase of 1.82 g/100 g (arabitol and
mannitol) was detected in FBA. Compared with FB, the oligosaccharide content of FBA
decreased by almost 18-fold, while the oligosaccharide content in FBR decreased to a lesser
extent, by approximately 40%. Overall, the two fermented ingredients showed a reduction
in total FODMAPs, with a drop of 45.6% (FBA) and 56.1% (FBR).
Table 3. FODMAP content in fava bean flour (FB), fava bean flour fermented by Aspergillus oryzae
(FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The results are expressed on
a dry matter basis, in g/100 g
±
standard deviation. * EF denotes excess fructose, calculated as
glucose–fructose. n.d. indicates not detected.
FODMAP g/100 g DM
FB FBA FBR
Excess fructose (EF) * - - -
Glucose n.d. a0.93 ±0.16 b1.07 ±0.03 b
Fructose n.d. an.d.a0.13 ±0.01 b
∑Polyols 0.01 ±0.00 a1.83 ±0.21 b0.01 ±0.00 a
Arabitol n.d. a1.23 ±0.14 bn.d. a
Sorbitol 0.01 ±0.00 an.d.b0.01 ±0.00 c
Mannitol n.d. a0.60 ±0.07 bn.d. a
∑Oligosaccharides 3.74 ±0.24 a0.22 ±0.01 b1.64 ±0.05 c
Raffinose/stachyose 0.96 ±0.05 a0.05 ±0.00 b0.98 ±0.04 a
Verbascose 2.78 ±0.19 a0.13 ±0.00 b0.64 ±0.01 c
Kestose n.d. a0.01 ±0.00 b0.01 ±0.00 c
Nystose n.d. a0.03 ±0.00 b0.01 ±0.00 c
Total fructans - - -
∑FODMAPs 3.75 ±0.24 a2.04 ±0.22 b1.65 ±0.05 b
Results within the same row that share the same letter do not differ significantly (p< 0.05).
3.3. Amino Acid Profile
Table 4displays the total amino acid profile, and their percentages in relation to
the 2007 WHO/FAO recommendation for adults and children aged >3 years old [
37
].
Fermentation with A. oryzae or R. oligosporus resulted in an increase in most essential amino
acids, except for isoleucine, leucine, and threonine. The largest increases observed in FBR
were for methionine (+147.6%), cysteine (+70.0%), and histidine (+69.5%). In FBA, the valine
concentration increased by 31.8%. FB provided >100% of all essential amino acids except the
sulfur amino acids (SAA) (83% of requirements), but fermentation resulted in FBA and FBR
providing 132% and 164% of the SAA requirements, respectively. Regarding nonessential
amino acids, larger reductions were observed, mainly for arginine, which decreased by
21.5% in FBA and 31.4% in FBR. Although the free amino acids (Table 5) showed higher
concentrations after fermentation with A. oryzae, most of them increased after fermentation
with both fungi, with the exception of asparagine (FBA and FBR), aspartic acid (FBR), and
glutamic acid (FBR).

Foods 2024,13, 2922 9 of 27
Table 4. Amino acid profiles of fava bean flour (FB), fava bean flour fermented by Aspergillus oryzae
(FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The total amino acids were
quantified and expressed as g/100 g protein
±
standard deviation, and the daily requirements for
each essential amino acid were determined according to the WHO (2007).
FB FBA FBR
Level (g/100 g
protein) * % Requirement ** Level (g/100 g
protein) % Requirement Level (g/100 g
protein) % Requirement
Essential AA
Histidine 2.98 ±0.42 198.92 3.52 ±0.15 234.65 5.05 ±0.42 336.81
Isoleucine 5.26 ±0.74 175.48 5.23 ±0.18 174.35 5.05 ±0.66 168.29
Leucine 9.74 ±1.36 165.05 9.40 ±0.36 159.40 8.93 ±1.16 151.34
Lysine 8.07 ±1.12 179.39 8.72 ±0.33 193.80 8.31 ±1.13 184.72
Methionine 0.63 ±0.08 1.22 ±0.05 1.56 ±0.01
Cysteine 1.20 ±0.14 1.68 ±0.09 2.04 ±0.03
Methionine + cysteine 1.83 ±0.22 83.27 2.90 ±0.13 131.78 3.61 ±0.04 163.89
Phenylalanine 5.18 ±0.72 5.48 ±0.23 5.50 ±0.71
Tyrosine *** 3.25 ±0.45 3.39 ±0.19 3.73 ±0.50
Phenylalanine + tyrosine 8.42 ±1.18 221.68 8.87 ±0.42 233.29 9.23 ±1.22 242.92
Threonine 5.00 ±0.70 217.42 4.99 ±0.20 217.07 4.89 ±0.62 212.42
Tryptophan 0.60 ±0.06 100.23 0.93 ±0.02 154.59 0.95 ±0.05 158.76
Valine 4.56 ±0.64 116.96 6.01 ±0.22 154.22 6.06 ±0.81 155.33
Nonessential AA
Alanine 5.09 ±0.71 5.65 ±0.20 7.49 ±0.97
Arginine 11.50 ±1.60 9.03 ±0.41 7.89 ±1.09
Aspartic acid + asparagine 14.21 ±1.98 13.55 ±0.49 13.17 ±1.71
Glutamic acid + glutamine 21.67 ±3.02 20.59 ±0.76 19.51 ±2.44
Proline 4.56 ±0.64 5.05 ±0.19 5.07 ±0.61
Serine 6.84 ±0.95 6.51 ±0.26 5.97 ±0.78
Glycine 5.53 ±0.77 5.23 0.18 5.12 ±0.65
* Calculated by dividing the amino acid content (g/100 g DM) by the true protein content (sum of amino
acid residues). ** Calculated by determining the ratio of each essential amino acid per 100 g of protein to the
requirement specified by the WHO (2007). *** Tyrosine, although not an essential amino acid, is included because
its requirement is combined with that of phenylalanine.
Table 5. Free amino acid content of fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The results are expressed
on a dry matter basis in g/100 g ingredient ±standard deviation.
g/100 g Ingredient DM
Free AA FB FBA FBR
Alanine 0.044 ±0.002 0.180 ±0.002 0.186 ±0.008
Asparagine 0.041 ±0.017 0.026 ±0.002 0.028 ±0.002
Aspartic acid 0.126 ±0.078 0.188 ±0.003 0.066 ±0.004
Cysteine 0.000 ±0.001 0.011 ±0.001 0.006 ±0.000
Glutamic acid 0.239 ±0.108 0.335 ±0.020 0.206 ±0.011
Glutamine 0.002 ±0.002 0.024 ±0.002 0.026 ±0.001
Glycine 0.002 ±0.001 0.008 ±0.000 0.007 ±0.000
Histidine 0.004 ±0.001 0.103 ±0.009 0.090 ±0.003
Isoleucine 0.011 ±0.007 0.135 ±0.002 0.095 ±0.007
Leucine 0.009 ±0.006 0.148 ±0.003 0.093 ±0.006
Lysine 0.033 ±0.016 0.229 ±0.023 0.133 ±0.005
Methionine 0.001 ±0.001 0.021 ±0.001 0.003 ±0.000
Ornithine 0.003 ±0.002 0.018 ±0.001 0.032 ±0.001
Phenylalanine 0.046 ±0.028 0.151 ±0.003 0.113 ±0.006
Proline 0.003 ±0.002 0.018 ±0.000 0.012 ±0.001
Serine 0.007 ±0.003 0.097 ±0.005 0.053 ±0.003
Threonine 0.006 ±0.004 0.089 ±0.003 0.055 ±0.004
Tryptophan 0.010 ±0.007 0.043 ±0.002 0.022 ±0.001
Tyrosine 0.015 ±0.009 0.099 ±0.004 0.059 ±0.003
Valine 0.014 ±0.009 0.106 ±0.001 0.079 ±0.006
3.4. Techno-Functional Properties
The techno-functional properties of FB, FBA, and FBR are displayed in Table 6.

Foods 2024,13, 2922 10 of 27
Table 6. Techno-functional properties of fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The results are presented
as mean ±standard deviation.
FB FBA FBR
pH 6.65 ±0.01 a6.26 ±0.03 b6.22 ±0.01 b
TTA (mL 0.1 M NaOH/10g ingredient) 10.98 ±0.03 a24.37 ±0.51 b26.60 ±0.36 c
SH groups (µmol SH/g protein)
Exposed 4.78 ±0.30 a3.67 ±0.33 b0.68 ±0.03 c
Free 5.97 ±0.25 a5.55 ±0.91 a6.10 ±0.17 a
Total 101.13 ±8.43 a108.17 ±9.09 a70.61 ±4.70 b
Water-holding capacity (%) 64.77 ±0.17 a77.05 ±1.33 b75.64 ±0.71 c
Oil-holding capacity (%) 78.42 ±0.54 a,b 75.64 ±0.71 a79.42 ±0.74 b
Foaming capacity (%) 19.73 ±3.12 a11.27 ±1.30 b9.81 ±2.56 b
Foam stability (%) 93.64 ±5.53 a100.00 ±0.00 a13.89 ±2.41 b
Minimum gelling concentration (%) 24.00 ±0.00 a18.00 ±0.00 b17.00 ±0.00 c
Protein solubility (%)
Native pH 100.13 ±2.06 a50.68 ±1.77 b25.55 ±0.99 c
pH 7 92.63 ±4.59 a85.06 ±1.55 b32.48 ±0.50 c
Separation rate (%/s) 0.016 ±0.002 a0.028 ±0.001 b0.030 ±0.002 b
Particle size distribution (µm)
D [4,3] 67.67 ±0.81 a516.00 ±10.54 b439.33 ±2.08 c
Dv (10) 12.23 ±0.06 a32.63 ±1.70 b37.03 ±0.61 b
Dv (50) 36.90 ±0.26 a414.00 ±31.43 b335.33 ±16.17 b
Dv (90) 174.00 ±2.65 a1196.67 ±20.82 b1020.00 ±10.00 c
Colour
L* value 90.92 ±0.50 a74.82 ±0.15 b66.72 ±0.67 c
a* value −1.44 ±0.02 a4.32 ±0.07 b4.29 ±0.07 b
b* value 16.59 ±0.47 a21.50 ±0.00 b19.98 ±0.33 c
Differential colour index ∆E- 17.80 ±0.59 25.11 ±0.23
Results within the same row that share the same letter do not differ significantly (p< 0.05).
3.4.1. pH and TTA
The pH and TTA are presented in Table 6. FB exhibited a pH value of 6.65 and a TTA
of 10.98 mL/10 g. Fermentation resulted in a decrease in pH of 0.39 (FBA) and 0.43% (FBR).
The TTA increased significantly by 121.9% (FBA) and 142.3% (FBR).
3.4.2. Sulfhydryl (SH) Groups
Exposed, free, and total SH groups are presented in Table 6. FB showed values
of 4.78
µ
mol SH/g protein (exposed), 5.97
µ
mol SH/g protein (free), and 101.13
µ
mol
SH/g protein (total). A significant change in the concentration of exposed SH groups was
observed after fermentation, decreasing by 23% and 86% in FBA and FBR, respectively.
In contrast, fermentation resulted in no significant changes in the concentration of free
SH groups, with values of 5.55–6.10
µ
mol SH/g protein determined in FB, FBA, and FBR.
Compared with FB, the total SH groups were significantly lower in FBR (
−
30.2%), whereas
no significant difference was observed after fermentation with A. oryzae.
3.4.3. Water- and Oil -Holding Capacity
The water and oil-holding capacity values are shown in Table 6. FB had a water-
holding capacity (WHC) of 64.77% and an oil-holding capacity (OHC) of 78.42%. Fermenta-
tion increased the WHC of FBA and FBR by 19.0% and 16.8%, respectively. The OHC values
determined for FBA and FBR were not significantly different from those of FB; however,
FBR had a significantly higher OHC (79.42%) than FBA (75.64%).

Foods 2024,13, 2922 11 of 27
3.4.4. Foaming Properties
The foaming properties of the ingredients are given in Table 6. FB showed a foaming
capacity of 19.73% and a foam stability of 93.64%. The foaming capacity was reduced
by 8.46% (FBA) and 9.92% (FBR) by the SSF process. The foam stability increased to its
maximum with A. oryzae, while it decreased significantly by 85.2% with R. oligosporus.
3.4.5. Minimum Gelling Concentration
The minimum gelling concentration for the three ingredients is presented in Table 6.
FB required 24 g/100 g to produce a gel, while fermentation reduced this value to 18 g/100
g and 17 g/100 g for FBA and FBR, respectively.
3.4.6. Protein Solubility
The protein solubility of the ingredients is presented in Table 6. FB showed a protein
solubility of 92–100% at its native pH and pH 7. Fermentation significantly reduced the
protein solubility at all pH values. At native pH, the protein solubility of FBA (85.06%)
was slightly lower than that of FB, with this further decreasing to 50.67% at pH 7. Even
more significant reductions in protein solubility were observed after fermentation with R.
oligosporus, with solubilities of 32.48% and 25.55% determined for FBR at the native pH and
pH 7, respectively.
3.4.7. Emulsifying Characteristics
The separation rates of the ingredients are given in Table 6. The transmission profiles
are shown in Figure 1. The FB emulsion showed the slowest phase separation (0.016%), with
a gradual increase in transmission. For the FBA and FBR emulsions, the almost immediate
increase in the transmission of light along the ingredients’ cuvettes and the formation of a
cream layer (visible on the left side of the profile), and a sediment layer (visible on the right
side of the profile) show evidence of quicker separation (0.028–0.030%/s).
Foods 2024, 13, x FOR PEER REVIEW 12 of 30
more significant reductions in protein solubility were observed after fermentation with R.
oligosporus, with solubilities of 32.48% and 25.55% determined for FBR at the native pH
and pH 7, respectively.
3.4.7. Emulsifying Characteristics
The separation rates of the ingredients are given in Table 6. The transmission profiles
are shown in Figure 1. The FB emulsion showed the slowest phase separation (0.016%),
with a gradual increase in transmission. For the FBA and FBR emulsions, the almost im-
mediate increase in the transmission of light along the ingredients’ cuvees and the for-
mation of a cream layer (visible on the left side of the profile), and a sediment layer (visible
on the right side of the profile) show evidence of quicker separation (0.028–0.030%/s).
Figure 1. Light transmission profiles of emulsions of fava bean flour (A), fava bean flour fermented
with Aspergillus oryzae (B), and fava bean flour fermented with Rhizopus oligosporus (C) as a function
of the position. The left side of each graph represents the top of the ingredient’s cuvee. Red and
green lines indicate the initial and latest transmission profiles, respectively.
3.4.8. Particle Size
The particle size characteristics are provided in Table 6. FB had the smallest particle
size, with an average D [4,3] value of 67.67 μm, while the fermented ingredients had larger
particle sizes, with 516.00 μm (FBA) and 439.33 μm (FBR). The three particle size percen-
tiles of FB also showed significantly lower values after the SSF process. The highest Dv
(10) value was determined for FBR, while FBA presented the highest Dv (50) and Dv (90)
values. Furthermore, the particle size distributions of the three ingredients exhibited a
bimodal behaviour (see Appendix B, Figure A1).
3.4.9. Colour
The L*, a*, and b* colour values are listed in Table 6. FB showed an L* value of 90.92,
an a* value of −1.44, and a b* value of 16.59. SSF reduced the lightness of the ingredients
by 17.7% (FBA) and 26.6% (FBR), while slight increases in the a* values and in b* values
were observed, indicating more intense red and yellow tones. Overall, FBR showed a
higher differential colour index than FBA, indicating a more significant colour difference
compared with the unfermented fava bean flour.
3.4.10. Protein Profile
The protein profiles of FB, FBA, and FBR are presented in Figure 2. FB displayed
peptides with molecular weights ranging from ~5 kDa to ~100 kDa, with bands predomi-
nantly visible around ~20 kDa, ~37 kDa, ~50 kDa, and just below 75 kDa. After fermenta-
tion, most of the bands decreased in intensity, and were visible between ~5 kDa and ~37
kDa, with a more pronounced band at ~20 kDa.
Figure 1. Light transmission profiles of emulsions of fava bean flour (A), fava bean flour fermented
with Aspergillus oryzae (B), and fava bean flour fermented with Rhizopus oligosporus (C) as a function
of the position. The left side of each graph represents the top of the ingredient’s cuvette. Red and
green lines indicate the initial and latest transmission profiles, respectively.
3.4.8. Particle Size
The particle size characteristics are provided in Table 6. FB had the smallest particle
size, with an average D [4,3] value of 67.67
µ
m, while the fermented ingredients had larger
particle sizes, with 516.00
µ
m (FBA) and 439.33
µ
m (FBR). The three particle size percentiles
of FB also showed significantly lower values after the SSF process. The highest Dv (10)
value was determined for FBR, while FBA presented the highest Dv (50) and Dv (90) values.
Furthermore, the particle size distributions of the three ingredients exhibited a bimodal
behaviour (see Appendix B, Figure A1).
3.4.9. Colour
The L*,a*, and b* colour values are listed in Table 6. FB showed an L* value of 90.92,
an a* value of
−
1.44, and a b* value of 16.59. SSF reduced the lightness of the ingredients

Foods 2024,13, 2922 12 of 27
by 17.7% (FBA) and 26.6% (FBR), while slight increases in the a* values and in b* values
were observed, indicating more intense red and yellow tones. Overall, FBR showed a
higher differential colour index than FBA, indicating a more significant colour difference
compared with the unfermented fava bean flour.
3.4.10. Protein Profile
The protein profiles of FB, FBA, and FBR are presented in Figure 2. FB displayed pep-
tides with molecular weights ranging from ~5 kDa to ~100 kDa, with bands predominantly
visible around ~20 kDa, ~37 kDa, ~50 kDa, and just below 75 kDa. After fermentation,
most of the bands decreased in intensity, and were visible between ~5 kDa and ~37 kDa,
with a more pronounced band at ~20 kDa.
Foods 2024, 13, x FOR PEER REVIEW 13 of 30
Figure 2. Protein profiles of the fava bean ingredients, with the reference ladder in the first position
(L), followed by fava bean flour FB (A), fava bean flour fermented with Aspergillus oryzae (B), and
fava bean flour fermented with Rhizopus oligosporus (C).
3.5. Microscopy
Figure 3 illustrates the SEM micrographs of the ingredients. The differences in mor-
phology between FB and the fermented ingredients (FBA and FBR) are notable. FB con-
tained greater numbers of round and “free” particles. The network of mycelia was observ-
able in the FBA and FBR, resulting in a more compact structure and a rougher surface,
which also reflects the larger particle size of the ingredients.
Figure 3. Representative scanning electron micrographs of fava bean flour (A), fava bean flour fer-
mented with Aspergillus oryzae (B), and fava bean flour fermented with Rhizopus oligosporus (C). The
magnification shown is 1000×.
3.6. Antinutrients
Figure 2. Protein profiles of the fava bean ingredients, with the reference ladder in the first position
(L), followed by fava bean flour FB (A), fava bean flour fermented with Aspergillus oryzae (B), and
fava bean flour fermented with Rhizopus oligosporus (C).
3.5. Microscopy
Figure 3illustrates the SEM micrographs of the ingredients. The differences in mor-
phology between FB and the fermented ingredients (FBA and FBR) are notable. FB con-
tained greater numbers of round and “free” particles. The network of mycelia was observ-
able in the FBA and FBR, resulting in a more compact structure and a rougher surface,
which also reflects the larger particle size of the ingredients.

Foods 2024,13, 2922 13 of 27
Foods 2024, 13, x FOR PEER REVIEW 13 of 30
Figure 2. Protein profiles of the fava bean ingredients, with the reference ladder in the first position
(L), followed by fava bean flour FB (A), fava bean flour fermented with Aspergillus oryzae (B), and
fava bean flour fermented with Rhizopus oligosporus (C).
3.5. Microscopy
Figure 3 illustrates the SEM micrographs of the ingredients. The differences in mor-
phology between FB and the fermented ingredients (FBA and FBR) are notable. FB con-
tained greater numbers of round and “free” particles. The network of mycelia was observ-
able in the FBA and FBR, resulting in a more compact structure and a rougher surface,
which also reflects the larger particle size of the ingredients.
Figure 3. Representative scanning electron micrographs of fava bean flour (A), fava bean flour fer-
mented with Aspergillus oryzae (B), and fava bean flour fermented with Rhizopus oligosporus (C). The
magnification shown is 1000×.
3.6. Antinutrients
Figure 3. Representative scanning electron micrographs of fava bean flour (A), fava bean flour
fermented with Aspergillus oryzae (B), and fava bean flour fermented with Rhizopus oligosporus (C).
The magnification shown is 1000×.
3.6. Antinutrients
The antinutrient composition of the ingredients is shown in Table 7. The phytic acid
levels in FB (0.397 g/100 g) decreased to a higher extent in FBR (0.214 g/100 g) than in FBA
(0.329 g/100 g), with a similar trend observed for chymotrypsin inhibitor concentrations.
Condensed tannins in FB were eliminated during fermentation by the two genera. The
saponin level in FB was eliminated with A. oryzae, while fermentation with R. oligosporus
reduced saponins by only 19.4%.
Table 7. Content of the antinutritional factors in fava bean flour (FB), fava bean flour fermented
by Aspergillus oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). Values are
presented on a dry matter basis as the mean ±standard deviation.
FB FBA FBR
Phytic acid (g/100 g) 0.397 ±0.001 0.329 ±0.001 0.214 ±0.002
Saponins (GAE mg/g) 0.31 ±0.09 0.00 ±0.00 0.25 ±0.03
Trypsin inhibitors (TIU/mg) 0.00 ±0.00 0.82 ±0.04 4.22 ±0.14
Chymotrypsin inhibitors (CIU/mg) 36.00 ±0.28 25.70 ±0.42 12.20 ±0.28
Condensed tannins (catechin equivalent mg/g)
1.56 ±0.98 0.02 ±0.02 0.00 ±0.00
3.7. Olfactometry
Olfactometric analysis identified a total of 95 odour-active compounds, including acids,
alcohols, aldehydes, ketones, and esters/lactones, as well as one unknown compound (cf.
Appendix C, Table A2). Only compounds with significant differences in intensity between
FB and FBA/FBR were considered (Figure 4). In addition, Word clouds (cf. Appendix C,
Figure A2) were created for each ingredient, based on the aromas associated with the
compounds detected (which may be identical).
Among the compounds selected, FB mainly contained trans-4,5-epoxy-(E)-2-decenal
(metallic), methional (boiled potato), and acetic acid (vinegar). An unpleasant chemical
compound characterised by a mouldy aroma, 2,4,6-trichloroanisole, was also detected.
In addition, other aroma-active compounds more characteristic of “savoury” aromas
were also present, but with slightly less intensity, such as 2-methoxyphenol (smoky),
2-acetyl-1-pyrroline (roasty), and butanoic acid (cheese). On the other hand, “sweeter”
aroma-associated compounds were also detected, such as 2-phenylethanol (honey), ethyl-3-
methylbutanoate (fruity), phenylacetaldehyde (honey), and maltol (caramel). The intensity
of the abovementioned savoury and sweet aroma compounds were enhanced by fermen-
tation, while the compounds associated with the metallic and mouldy aromas decreased.
SSF also led to the formation of new compounds in FBA and FBR, which, with the excep-
tion of
γ
-dodecalactone (peach aroma), relatively strengthened the savoury profile of the
raw material. These compounds included 2/3-methylbutanal (malty), 2,3-butanedione
(butter), 2-methylpropanoic acid (cheese), 2-methyl-3-(methyldithio)furan (meaty), and 3-
methylpentanoic acid (cheese). Sufur compounds such as dimethyltrisulfide and dimethyl-
tetrasulfide (only in FBR) also introduced a cabbage-like aroma to the aromatic profile of

Foods 2024,13, 2922 14 of 27
the fermented products. An earthy characteristic also appeared due to the presence of
certain pyrazine compounds. Overall, in terms of intensity, the biggest changes seemed to
occurr with R. oligosporus.
Foods 2024, 13, x FOR PEER REVIEW 16 of 30
Figure 4. Changes in aromas’ intensity during fermentation relative to fava bean flour, based on the
GC-FID peak area (ΔFBA = FBA-FB and ΔFBR = FBR-FB) for compounds with a significant differ-
ence (of at least 1 for one of the fermented ingredients).
3.8. Fungal Metabolites
Changes in the concentration of organic acids during fermentation are shown in Fig-
ure 5. Fermentation resulted in significant decreases in the concentrations of citric acid,
cis-aconitic acid, and isocitric acid, with the most notable reduction observed after fer-
mentation with A. oryzae. In contrast, lactic acid, malic acid, and succinic acid predomi-
nantly increased in FBA and FBR after SSF, with small increases in fumaric acid and 2-
oxoglutaric acid levels also being observed. Higher concentrations were observed in FBR.
-1 -0.5 0 0.5 1 1.5 2 2.5 3
2/3-methylbutanal
2,3-butanedione
ethyl-3-methylbutanoate
2-/3-methyl-1-butanol
1-octen-3-one
2-acetyl-1-pyrroline
dimethyltrisulfide
2,3,5-trimethylpyrazine
acetic acid
methional
2-vinyl-3,5-dimethylpyrazine
2-methylpropanoic acid
1-octanol
butanoic acid
phenylacetaldehyde
2-methyl-3-(methyldithio)furan
dimethyltetrasulfide
(E)-2-undecenal
2-acetyl-2-thiazoline
3-methylpentanoic acid
2,4,6-trichloroanisole
(E)-β-damascenone, (E)-β-damascone
2-hydroxy-3,4-dimethyl-2-cyclopenten-1-one
2-methoxyphenol
2-phenylethanol
trans-2,3-epoxydodecanal
maltol
trans-4,5-epoxy-(E)-2-decenal
isoeugenol
γ-dodecalactone
Difference in intensity [-]
ΔFBR
ΔFBA
Figure 4. Changes in aromas’ intensity during fermentation relative to fava bean flour, based on the
GC-FID peak area (
∆
FBA = FBA-FB and
∆
FBR = FBR-FB) for compounds with a significant difference
(of at least 1 for one of the fermented ingredients).
3.8. Fungal Metabolites
Changes in the concentration of organic acids during fermentation are shown in
Figure 5
. Fermentation resulted in significant decreases in the concentrations of citric acid,
cis-aconitic acid, and isocitric acid, with the most notable reduction observed after fermen-
tation with A. oryzae. In contrast, lactic acid, malic acid, and succinic acid predominantly
increased in FBA and FBR after SSF, with small increases in fumaric acid and 2-oxoglutaric
acid levels also being observed. Higher concentrations were observed in FBR.
The ergosterol concentrations of the fermented ingredients were measured after SSF, as
an indicator of fungal biomass, and are presented in Table 8. The results showed a slightly
higher value with A. oryzae compared with R. oligosporus.

Foods 2024,13, 2922 15 of 27
Foods 2024, 13, x FOR PEER REVIEW 17 of 30
Figure 5. Changes in the organic acid concentrations of fava bean flour fermented with Aspergillus
oryzae (FBA) or Rhizopus oligosporus (FBR) compared with fava bean flour (FB) (ΔFBA = FBA-FB and
ΔFBR = FBR-FB) detected by MS-Omics and expressed as g/100 g on a dry maer basis.
The ergosterol concentrations of the fermented ingredients were measured after SSF,
as an indicator of fungal biomass, and are presented in Table 8. The results showed a
slightly higher value with A. oryzae compared with R. oligosporus.
Table 8. Ergosterol concentrations of fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR).
Ingredient Ergosterol (mg/100 g)
Fava bean flour (FB) 0.0 ± 0.0
Fava bean flour + Aspergillus oryzae (FBA) 195.1 ± 2.8
Fava bean flour + Rhizopus oligosporus (FBR) 187.6 ± 0.6
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 fer-
mentation (SSF) has been shown to enhance nutritional properties by reducing antinutri-
tional 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 poly-
ols, 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 lev-
els, 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, carote-
noids, 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
-0.300 -0.250 -0.200 -0.150 -0.100 -0.050 0.000 0.050 0.100 0.150
Succinic acid
Lactic acid
2-Oxoglutaric acid
Malic acid
cis-Aconitic acid
Citric acid
Isocitric acid
Fumaric acid
Concentration [g/100 g DM]
ΔFBR
ΔFBA
Figure 5. Changes in the organic acid concentrations of fava bean flour fermented with Aspergillus
oryzae (FBA) or Rhizopus oligosporus (FBR) compared with fava bean flour (FB) (
∆
FBA = FBA-FB and
∆FBR = FBR-FB) detected by MS-Omics and expressed as g/100 g on a dry matter basis.
Table 8. Ergosterol concentrations of fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR).
Ingredient Ergosterol (mg/100 g)
Fava bean flour (FB) 0.0 ±0.0
Fava bean flour + Aspergillus oryzae (FBA) 195.1 ±2.8
Fava bean flour + Rhizopus oligosporus (FBR) 187.6 ±0.6
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

Foods 2024,13, 2922 16 of 27
(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 cardio-
vascular 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

Foods 2024,13, 2922 17 of 27
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 hydropho-
bic 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

Foods 2024,13, 2922 18 of 27
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 (
∆
E) 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 vine-
gar 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 decar-
boxylated 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, methion-
ine, 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, pheny-
lalanine, and methionine, respectively [
85
–
87
]. Methionine also formed the sulfur com-
pounds 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.

Foods 2024,13, 2922 19 of 27
5. Conclusions
SSF with the filamentous fungi A. oryzae and R. oligosporus resulted in changes in the
nutritional, functional, and aromatic profile of fava bean flour. An increase in the protein
and fat content were observed in the fermented ingredients, while levels of starch, fibre, and
oligosaccharides generally decreased. The nutritional quality of FB was improved, fulfilling
the WHO’s recommended daily amino acid requirements for all essential amino acids, with
a reduction in most antinutrients observed. In addition, the fungi produced acids during
their metabolism, with a sharp increase in concentrations of malic, lactic, and succinic acids,
while a significant decrease in citric acid was observed. In terms of the techno-functional
properties, the WHC of FBA and FBR increased, as did the foam stability for FBA. On the
other hand, the OHC, minimum gelling concentrations, and foaming properties decreased.
After the SSF process, the emulsions separated more rapidly, and an increase in particle size
was observed. SSF modulated the aroma profile, mainly intensifying compounds associated
with savoury aromas such as cheese, malty, cabbage, vinegar, roasty, and butter-like aromas,
although some sweeter aromas, such as peach and caramel, were also identified. Overall,
the aroma changes were more intense in the FBR, with SSF aiding in the reduction of the
flour’s metallic and mouldy aromas. In a comparison of the two fermented products, FBA
may offer a superior nutritional profile, with a higher protein content, lower fat and sugar
contents, higher protein solubility, higher foam stability, and less significant colour changes.
However, fermentation with R. oligosporus could be more effective for the production of
desirable aroma-associated compounds and organic acids, while also increasing dietary
fibre and reducing FODMAP contents. In summary, both ingredients produced by SSF of
fava bean flour with A. oryzae and R. oligosporus present interesting nutritional, functional,
and aroma characteristics. Future research should focus on the investigation of their sensory
properties, food applications, and consumer acceptance.
Author Contributions: Conceptualisation, A.W.S., E.K.A., and E.Z.; data curation, O.G.; methodology,
O.G., A.Z.A.T., C.C., A.K.H., and M.G.T.; visualisation O.G.; writing—original draft preparation, O.G.;
review and editing, A.W.S., L.N., E.K.A., M.G.T., A.Z.A.T., C.C., M.G., A.K.H., and E.Z.; supervision,
A.W.S. and E.K.A.; project administration, L.N.; resources M.G.; funding acquisition, E.Z. and E.K.A.
All authors have read and agreed to the published version of the manuscript.
Funding: This project was funded by the SMART PROTEIN Project of the European Union’s Horizon
2020 Research and Innovation Program (No. 862957).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article, further inquiries can be directed to the corresponding author.
Acknowledgments: The authors wish to thank Celia Segura Godoy for her assistance with the
analysis of dietary fibre.
Conflicts of Interest: Maria Garcia Torreiro was employed by the company Mogu srl. The remaining
authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.

Foods 2024,13, 2922 20 of 27
Appendix A. Total Fatty Acid Profile
Table A1. Total fatty acid profile of fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The results are expressed
on a dry matter basis in g/100 g ±standard deviation. n.d. = not detected.
Fatty Acid Profile g/100 g DM
FB FBA FBR
Lauric acid 12:0 0.01 ±0.00 n.d. n.d.
Myristic acid 14:0 0.00 ±0.00 0.01 ±0.00 0.01 ±0.00
Pentadecanoic acid 15:0 0.00 ±0.00 0.01 ±0.00 0.01 ±0.00
Palmitic acid 16:0 0.29 ±0.05 0.36 ±0.06 0.43 ±0.06
Hexadecenoic acid 16:1 0.00 ±0.00 0.02 ±0.00 0.02 ±0.00
Isoeptadecanoic acid 17:0 n.d. n.d. n.d.
14-Methyl hexadecanoic acid 17:0 n.d. 0.00 ±0.00 n.d.
Margaric acid 17:0 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00
Heptadecenoic acid 17:1 n.d. n.d. 0.02 ±0.00
Stearic acid 18:0 0.07 ±0.01 0.06 ±0.01 0.21 ±0.05
Oleic acid 18:1 0.53 ±0.07 0.60 ±0.08 0.79 ±0.10
Linoleic acid 18:2 0.89 ±0.11 1.05 ±0.13 0.96 ±0.12
Conjugated linoleic acid 18:2 n.d. n.d. n.d.
Linolenic acid 18:3 0.05 ±0.01 0.05 ±0.01 0.12 ±0.02
Arachidic acid 20:0 0.02 ±0.00 0.01 ±0.00 0.02 ±0.00
Eicosenoic acid 20:1 0.01 ±0.00 0.01 ±0.00 0.01 ±0.00
Heneicosylic acid 21:0 n.d. n.d. n.d.
Behenic acid 22:0 0.00 ±0.00 n.d. 0.01 ±0.00
Docosanoic acid 22:1 n.d. n.d. n.d.
Lignoceric acid 24:0 n.d. n.d. 0.01 ±0.00
Appendix B. Particle Size Distribution
Foods 2024, 13, x FOR PEER REVIEW 22 of 30
Appendix A. Total Fay Acid Profile
Table A1. Total fay acid profile of fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The results are expressed
on a dry maer basis in g/100 g ± standard deviation. n.d. = not detected.
Fay acid profile g/100 g DM
FB FBA FBR
Lauric acid 12:0 0.01 ± 0.00 n.d. n.d.
Myristic acid 14:0 0.00 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
Pentadecanoic acid 15:0 0.00 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
Palmitic acid 16:0 0.29 ± 0.05 0.36 ± 0.06 0.43 ± 0.06
Hexadecenoic acid 16:1 0.00 ± 0.00 0.02 ± 0.00 0.02 ± 0.00
Isoeptadecanoic acid i17:0 n.d. n.d. n.d.
14-Methyl hexadecanoic acid
a
17:0 n.d. 0.00 ± 0.00 n.d.
Margaric acid 17:0 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Heptadecenoic acid 17:1 n.d. n.d. 0.02 ± 0.00
Stearic acid 18:0 0.07 ± 0.01 0.06 ± 0.01 0.21 ± 0.05
Oleic acid 18:1 0.53 ± 0.07 0.60 ± 0.08 0.79 ± 0.10
Linoleic acid 18:2 0.89 ± 0.11 1.05 ± 0.13 0.96 ± 0.12
Conjugated linoleic acid 18:2 n.d. n.d. n.d.
Linolenic acid 18:3 0.05 ± 0.01 0.05 ± 0.01 0.12 ± 0.02
Arachidic acid 20:0 0.02 ± 0.00 0.01 ± 0.00 0.02 ± 0.00
Eicosenoic acid 20:1 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
Heneicosylic acid 21:0 n.d. n.d. n.d.
Behenic acid 22:0 0.00 ± 0.00 n.d. 0.01 ± 0.00
Docosanoic acid 22:1 n.d. n.d. n.d.
Lignoceric acid 24:0 n.d. n.d. 0.01 ± 0.00
Appendix B. Particle Size Distribution
Figure A1. Particle size distribution for fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The results are expressed
as the volume density (%) as a function of size (µm).
Figure A1. Particle size distribution for fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The results are expressed
as the volume density (%) as a function of size (µm).

Foods 2024,13, 2922 21 of 27
Appendix C. Aroma Profile
Table A2. Full aroma profile of fava bean flour (FB), fava bean flour fermented by Aspergillus
oryzae (FBA), and fava bean flour fermented by Rhizopus oligosporus (FBR). The results include the
compounds’ names, odour quality, and intensities.
Compound 1Odour Quality 2RI2(FFAP) Intensity 3,4
FB FBA FBR
Methylpropanal Malty <900 0 0.75 0.5
2/3-Methylbutanal Malty 920 0 2.5 2.5
Methylbutanoate Fruity 973 0 0.5 0.75
2,3-Butanedione Butter 987 0 1.5 1.75
Unknown Onion, garlic 1019 0 0 0.5
Unknown Gas 1042 0 0 0.5
Ethyl-2-methylbutanoate Fruity 1050 0.5 1.25 1
Ethyl-3-methylbutanoate Fruity 1069 0.75 1.25 1.75
Hexanal Grassy 1088 1 1.5 1.75
3-Methyl-2-buten-1-thiol Beer 1108 0.5 0.5 1
2-Heptanone Fruity 1178 0 0 0.5
2-/3-Methyl-1-butanol Malty 1207 0 0.5 1.75
(Z)-4-heptenal Fishy 1241 0 0.5 0.5
3-Hydroxy-2-butanone Butter 1280 0 0.75 0.5
Octanal Citrus 1285 0 0 0.5
1-Octen-3-one Mushroom 1295 0.75 1.5 1.75
2-Acetyl-1-pyrroline Roasty 1329 0.5 2.25 2.25
1-Mercapto-2-propanone Catty 1363 0.75 0.5 0.5
Dimethyltrisulfide Cabbage 1366 0 1.5 2
(E)/(Z)-1,5-octadien-3-one Geranium 1368 0 0 0.75
2,3,5-Trimethylpyrazine Earthy 1408 0 1 1.25
2-Isopropyl-3-
methoxypyrazine Bell pepper 1425 0.5 0 0
2-Furfurylthiol Coffee 1428 0.5 0 0
Acetic acid Vinegar 1433 1.25 3 3
Methional Boiled potato 1450 1.5 2.5 2.5
2-Ethyl-3,5-
dimethylpyrazine Earthy 1455 1 1.5 1
(Z)-2-nonenal Fatty 1495 0.5 0 1
2-Isobutyl-3-
methoxypyrazine Bell pepper 1518 0.5 0 0.5
(E)-2-nonenal Cardboard 1523 0.75 0.5 1
Linalool Floral 1544 1 0.5 0.5
2-Vinyl-3,5-
dimethylpyrazine Earthy 1551 0 0.5 1.5
2-Methylpropanoic acid Cheese 1554 0 3 3
1-Octanol Citrus 1559 0 1 0
2-Acetyl-1,4,5,6-
tetrahydropyridine Roasty 1564 0.5 0 0
(E,Z)-2,6-nonadienal Cucumber 1574 1 0 0.5
(Z)-2-decenal Fatty 1603 0.5 0 0
Benzyl mercaptane Cress 1611 0.5 1 0.75

Foods 2024,13, 2922 22 of 27
Table A2. Cont.
Compound 1Odour Quality 2RI2(FFAP) Intensity 3,4
FB FBA FBR
2-Acetylpyrazine Roasty 1618 0 0 0.5
Butanoic acid Cheese 1621 0.5 1.75 1.75
Phenylacetaldehyde Honey 1632 0.5 1.25 1.5
2-Methyl-3-
(methyldithio)furan Meaty 1658 0 1 1.25
2-/3-Methylbutanoic acid Cheese 1663 3 3 2.75
(E,E)-2,4-nonadienal Fatty 1692 1.25 1.25 1
3-Methyl-2,4-nonandione Floral 1711 0.5 0.5 0
Methionol Boiled potato 1717 0 0.5 0
Dimethyltetrasulfide Cabbage 1720 0 0 1.5
Unknown Onion, gravy 1726 0.5 0.75 0.75
Pentanoic acid Cheese 1731 0 0.75 0.5
(E)-2-undecenal Metallic 1740 0 1 1
2-Acetyl-2-thiazoline Roasty 1749 0 1 1
Ethyl-2-phenylacetate Honey, floral 1757 0.5 0.5 0
Unknown Mouldy, musty 1769 0 0 2
3-Methylpentanoic acid Cheese 1771 0 0.5 1
2,4,6-Trichloroanisole Mouldy 1794 1 0 0
2-Phenylethylacetate Honey 1797 0 0.5 0
(E,E)-2,4-decadienal Fatty 1803 0.75 1.25 1.5
(E)-β-damascenone,
(E)-β-damascone Apple 1809 0 0.5 1
Geosmin Earthy 1812 0.5 0 1
2-Propionyl-2-thiazoline Roasty 1821 0.5 0 0
Calamenene Clove 1826 0 0.5 0
3-Mercapto-1-hexanol Rhubarb 1832 1.5 1.25 1.25
Hexanoic acid Goat 1835 0.5 1 0.5
Geraniol Rose 1844 1 0.5 0.5
2-Hydroxy-3,4-dimethyl-2-
cyclopenten-1-one Caramel 1850 0 1 1
2-Methoxyphenol Smoky 1853 1 1.5 2
(E,E,Z)-2,4,6-nonatrienal Oatflakes 1868 1.5 0.5 1.25
2-Phenylethanol Honey 1900 1 0 2
2-Ethyl-3-mercapto-1-
hexanol Meaty 1952 0 0 0.5
Trans-2,3-epoxydodecanal Citrus, soapy 1958 0 0 1
Maltol Caramel 1961 0.5 1.5 1.5
Trans-4,5-epoxy-(E)-2-
decenal Metallic 1997 2 1.5 1
4-Ethyl-2-methoxyphenol Smoky 2016 0 0 0.5
p-anisaldehyde Aniseed 2019 0 0 0.5
γ-Nonalactone Peach 2023 1 1 0.5
Furaneol Caramel 2026 2 2.25 1.75

Foods 2024,13, 2922 23 of 27
Table A2. Cont.
Compound 1Odour Quality 2RI2(FFAP) Intensity 3,4
FB FBA FBR
Octanoic acid Goat 2039 0 0 0.5
γ-(Z)-2-nonenolactone Coconut 2065 0 0 0.5
3-/4-Methylphenol Phenolic 2074 0.5 0.5 0.5
2,6-Dichlorophenol Phenolic,
medical 2100 0 0 0.75
γ-Decalactone Peach 2133 1 1 1
Unknown Perfume 2137 1 0.5 0.5
Eugenol Clove 2157 1.25 1 1
3-/4-Ethylphenol Phenolic 2173 0.5 0.5 0.5
2-Methoxy-4-vinylphenol Clove 2187 0.75 1 1
Sotolon Seasoning 2197 1.75 2.25 2.25
2-Aminoacetophenone Foxy 2210 1 1.5 1.75
Decanoic acid Soapy 2247 0 0 0.5
3-/4-Propylphenol Phenolic 2257 0 0.5 0
Isoeugenol Ham 2337 0 1 1
γ-Dodecalactone Peach 2378 0 1.5 1.25
Coumarine Woodruff 2448 0.5 0 0.5
3-Methylindole Fecal 2485 0.5 0.5 0
Phenylacetic acid Honey 2546 1.75 2.25 2
Vanillin Vanilla 2562 1.5 2 1.5
Phenylpropanoic acid Goat >2600 0.5 1 0.5
1
Compounds were identified on the basis of their linear retention indices, odour qualities, and odour thresholds
as perceived at the sniffing port.
2
Linear retention index.
3
Intensity: 0 = not detectable; 1 = weakly detectable;
2 = unequivocally detectable; 3 = intensely detectable; steps of 0.5 possible. 4n.d. = not determined.
Foods 2024, 13, x FOR PEER REVIEW 26 of 30
Isoeugenol Ham 2337 0 1 1
γ-Dodecalactone Peach 2378 0 1.5 1.25
Coumarine Woodruff 2448 0.5 0 0.5
3-Methylindole Fecal 2485 0.5 0.5 0
Phenylacetic acid Honey 2546 1.75 2.25 2
Vanillin Vanilla 2562 1.5 2 1.5
Phenylpropanoic acid Goat >2600 0.5 1 0.5
1
Compounds were identified on the basis of their linear retention indices, odour qualities, and
odour thresholds as perceived at the sniffing port.
2
Linear retention index.
3
Intensity: 0 = not detect-
able; 1 = weakly detectable; 2 = unequivocally detectable; 3 = intensely detectable; steps of 0.5 possi-
ble.
4
n.d. = not determined.
Figure A2. Word clouds for FB (A), FBA (B), and FBR (C) generated from the aroma profiles detected
by olfactometry. Larger font sizes reflect the predominance of aromas associated with the com-
pounds.
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