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Article

Oat Okara Fermentation: New Insights into the Microbiological and Metabolomic Characterization

by
Federica Meanti
1,
Chiara Mussio
1,
Gabriele Rocchetti
2,
Annalisa Rebecchi
1,*,
Luigi Lucini
3 and
Lorenzo Morelli
1
1
Department for Sustainable Food Process (DiSTAS), Università Cattolica del Sacro Cuore, Via Stefano Leonida Bissolati 74, 26100 Cremona, Italy
2
Department of Animal, Nutrition and Food Sciences (DIANA), Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy
3
Department for Sustainable Food Process (DiSTAS), Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(11), 545; https://doi.org/10.3390/fermentation10110545
Submission received: 18 September 2024 / Revised: 21 October 2024 / Accepted: 23 October 2024 / Published: 24 October 2024
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Versions Notes

Abstract

The importance of the valorization of industrial by-products has led to increasing research into their reuse. In this research, the innovative by-product okara oat flour, derived from the vegetable beverage industry, was studied. Oat okara sourdough was also produced and evaluated. The microbiological identification and typing involved bacterial and yeast isolates from both flour and sourdough. Untargeted metabolomics allowed the identification of biomarkers of fermented flour, such as phenolic classes, post-fermentation metabolites, fatty acids, and amino acids. The microorganisms most found were Weissella confusa, Enterococcus faecium, Pediococcus pentosaceus, and Pichia kudriavzevii, while Saccharomyces cerevisiae appeared only at the end of the sourdough’s back-slopping. Untargeted metabolomics identified a total of 539 metabolites, including phenolic compounds, lipids, amino acids, and organic acids. An increase in polyphenols released from the food matrix was detected, likely because of the higher bio-accessibility of phenolic metabolites promoted by microbial fermentation. Fermentation led to an increase in isoferulic acid, p-coumaric acid, sinapic acid, and a decrease in amino acids, which can be attributed to the metabolism of lactic acid bacteria. Some key markers of the fermentation process of both lactic acid bacteria and yeast were also measured, including organic acids (lactate, succinate, and propionate derivatives) and flavor compounds (e.g., diacetyl). Two bioactive compounds, such as gamma-aminobutyric acid and 3-phenyl-lactic acid had accumulated at the end of fermentation. Taken together, our findings showed that oat okara flour can be considered an excellent raw material for formulating more sustainable and functional foods due to fermentation promoted by autochthonous microbiota.

1. Introduction

The issue of reusing and valorizing industrial by-products has gained increasing importance in recent years. According to the Food and Agriculture Organization [1], the disposal of food processing by-products leads not only to economic losses but also to socio-environmental problems. Accordingly, the focus on finding alternative uses has grown globally in recent years [2].
In this scenario, okara and derived products could have a pivotal role. Okara is a residue obtained from the production of vegetable beverages; the most well-known comes from soybeans. Okara still contains all the insoluble residues of the starting ingredient, so principally the proteinic, fibrous, and lipidic parts [3]. Many studies [4] have been conducted on soybean okara and have led to relevant results. The key components of this product include high contents of proteins, isoflavones, soluble and insoluble fiber, mineral elements, and saponins. These are all chemical classes that could lead to beneficial health feedback.
In fact, supplementing the diet with okara is reported to be related to weight loss because of the high dietary fiber content, improved lipid metabolism, prebiotic effect, and enhanced antioxidant status of the gut [5].
A downside of this product is that it is not easily usable in its raw form: it has a high moisture content that makes it extremely perishable and prone to rapid rancidity [6]. Gupta et al. [7] suggested the need for pre-treatments before its utilization as an ingredient for both food and feed preparations. In this regard, fermentation can be an excellent cost-effective way to boost the by-product’s nutritional properties, making the nutrients present more bioavailable while also increasing its digestibility compared with raw okara. Interestingly, a second method that facilitates storage and handling is to dry the okara pulp. This can also overcome other issues, such as by inactivating anti-nutritional factors, such as trypsin inhibitors, that are deactivated by heat treatment [8]. Soy okara is mainly used to produce functional ingredients; accordingly, Castellanos Fuentes et al. [9] previously used the probiotic Lactobacillus casei for solid-state fermentation, demonstrating that the by-product okara can serve as a valid substrate for probiotics. In addition, Colletti et al. [10] suggested okara flour can be integrated with wheat flour in food products, improving protein and fiber intake. Furthermore, he pointed out another important application of the soy okara by-product in extending the shelf-life of certain foods, such as chocolate biscuits and cheese ravioli; in fact, due to its blandness, it can be used at high concentrations without adversely affecting texture and flavor.
Therefore, several research findings demonstrate soy okara’s great potential as a valuable ingredient, but this by-product can be obtained from the production of other vegetable beverages, such as oat beverages. However, there is very little information about the exploitation of oat okara in food science, and in the case of other grains or legumes, it is completely non-existent. It could be extremely interesting to evaluate oat okara for its beneficial characteristics; for instance, it can be considered gluten-free, and the presence of beta-glucans can represent a natural alternative to commercial hydrocolloids [11]. Furthermore, Holopainen-Mantila et al. [12] evaluated many of the characteristics of oat-based foods, paying particular attention to the qualitative and nutritional aspects of oat proteins, concluding that oats are an excellent source of protein, which, combined with the high presence of dietary fiber and phytochemicals, allows for more balanced nutrition than animal products. In fact, oat protein is considered an emerging protein source: it could replace soy protein in food preparations, as it resembles hulled soy flour in terms of protein quality [13]. It is also important to mention that most prolamin proteins in cereals can generate an allergenic effect because of their high proline and glutamine contents; in contrast, oats have globulin as their main storage protein and, therefore, do not have allergenic potential [14].
Okara is obtained through a liquid–solid separation that allows the fibrous residue to be separated from the vegetable drink [15]. Therefore, it is plausible to assert that the fibrous part retains many of the insoluble, but also soluble, components of the starting cereal, which can then be found in the final okara products. In the last few years, metabolomics has emerged as a valuable technique for tracking metabolite alteration during food processing [16]. Metabolomics allows all the metabolites to be identified simultaneously, thus delineating the sample’s fingerprint to predict and monitor the final product’s nutritional, functional, safety, and quality characteristics [17]. Furthermore, this approach has been used to understand the synergistic interactions between different chemicals better when moving from flour to the fermentation process [18].
Therefore, starting from this background level of information, the following study’s objective was to consider another type of okara, i.e., oat, in the form of flour and sourdough, thus evaluating and fully characterizing its chemical–physical, microbiological, and metabolomic profiles, to understand better its potential to be exploited as a functional ingredient, especially to support muscular systems.

2. Materials and Methods

2.1. Oat Okara Flour and Oat Okara Sourdough Preparation

The oat okara flour (OOF) used in this study was sponsored by the Packtin company (Reggio Emilia, Italy). Flour is produced from the okara of the cereal, from which the moisture is removed; it is then freeze-dried and finally ground. The chemical composition of oat okara flour was protein 41.5%, ash 6.13%, carbohydrate 7.31%, fat 13.6%, and dietary fiber 23.8% (on a dry matter basis) (Table S1).
The OOF sample was used to produce the oat okara sourdough (OOS) with only tap water added. The dough samples were prepared in triplicate. The mixture of water and flour was prepared in 2:1 proportion. All ingredients were mixed with a TK20 kneading machine (Tekno Stamap, Vicenza, Italy) for 12 min and kept at room temperature (25 ± 2 °C) for 24 h, allowing for spontaneous fermentation. Dough was refreshed daily over a period of 30 days and sampled at times 0 (t0), 1 (t1), and 30 days (t2) after fermentation. The dough samples were named OOSt0, OOSt1, and OOSt2. In total, 12 samples were collected.

2.2. Microbiological Analysis and pH Measurements

The OOF was subjected to microbiological analysis to characterize the microbial composition. A total of 10 g of flour was homogenized in 90 mL of salt water (NaCl, 9 g/L) using a Stomacher machine (400 Circulator; International PBI, Milan, Italy) at 260 rpm for 2 min; the homogenization step was repeated twice. The decimal dilutions were carried out and then plated on the following agar plates: Plate Count Agar (PCA, Oxoid, Milan, Italy) incubated at 30 °C for 72 h for the aerobic total microbial count analysis (TMC), Violet Red Bile Glucose Agar (VRBGA, Oxoid) incubated at 37 °C for 24 h for the search of Enterobacteriaceae, Violet Red Bile Agar (VRBA, Oxoid) supplemented with 4 methyl-umbelliferyl-D-glucuronide (MUG) (100 μg/mL Oxoid) incubated at 44 °C for 24 h for the presence of Escherichia coli, MRS Agar (Oxoid, Italy) for lactic acid bacteria (LAB) for 72 h at 30 °C under restricted oxygen conditions achieved using Anaerocult A (Merck, Darmstadt, Germany) (According to ISO 15214/1998) [19], and MRS supplemented with Vancomycin 0.01% (w/v) for heterofermentative LAB. The decimal counts were also performed on Rosa Bengala (RB, Oxoid) with the addition of chloramphenicol 0.01% (w/v) (Boehringer Ingelheim, Germany) for 5 days at 25 °C for yeast and molds and on Slanetz & Bartley (SB, Oxoid) for 48 h at 37 °C in aerobic conditions to assess the presence of enterococci. A total mesophilic anaerobic count was performed on Reinforced Clostridial Medium (RCM, Oxoid) in anaerobic condition at 37 °C for 48 h. In addition, the presence of Bacillus cereus was evaluated on Bacillus Cereus Selective Agar (PEMBA, Oxoid) with added egg yolk emulsion (Oxoid) and Polymixin B supplement (Oxoid) incubated at 30 °C for 48 h. To assess the presence of total aerobic, anaerobic, and Bacillus cereus spores, the flour samples were pasteurized at 80 °C for 10 min and plated using the corresponding medium.
Analyses were carried out in duplicate.
For the OOS samples, microbiological analyses were carried out to evaluate the spontaneous fermentative microflora, total lactic acid bacteria, heterofermentative LAB, yeast, and enterococci counts were performed. Sample preparation for analysis was performed as described above.
Thirty percent of colonies from OOF and OOS samples at different times (t0, t1, t30) were randomly picked up from MRS and MRS and added with Vancomycin, RB, and SB plates to assess the colonies’ morphological characteristics. Isolates were purified via the streaking technique three times on the same medium of isolation. The isolates were frozen in duplicate in a growth medium supplemented with 30% glycerol at −20 °C and −40 °C.
In addition, the pH values of OOF and OOSs were measured in triplicate: 10 g of sample was homogenized with 90 mL of distilled water, and the pH was measured using a digital pH meter (EdgeBlu, Hanna Instruments, Villafranca Padovana, Italy).

2.3. Molecular Characterization of Lactic Acid Bacteria at Strain Level

Genomic DNA from pure cultures of LAB was extracted using the microlysis method (Microzone, Stourbridge, UK) according to the manufacturer’s instructions. The strain level discrimination of LAB isolates was performed by RAPD-PCR (random amplified polymorphic DNA-PCR) method, using the primer RAPD5 (5′ AACGCGCAAC 3′) [20]. RAPD-PCR products were observed on 1% agarose gel and the 100 bp DNA ladder (Promega, Madison, WI, USA) as molecular weight markers in 1X Tris-acetate-EDTA (TAE) buffer solution at 90 V for 120 min and then were visualized with the software Image Lab (Version 3.0.0.07, Bio-Rad, Hercules, CA, USA). The isolates were divided into clusters.

2.4. Genotypic Identifications of Lactic Acid Bacteria Isolates

A representative of each cluster was used for taxonomic identification by performing 16S rRNA amplification using P1 (5′-GCGGCGTGCCTAATACATGC-3′) and P6 (5′-CTACGGCTACCTTGTTACGA-3′) primers [21]. PCR products were run on 1% agarose gel and a 100 bp marker in 1X Tris-acetate-EDTA (TAE) buffer solution at 100 V for 60 min, and the profiles were visualized with the software Image Lab (Bio-Rad). The amplified DNA was then purified with NucleoSpin® Gel and PCR clean-up (MACHEREY-NAGEL, Duren, Germany). The purified PCR products were sequenced by Sanger analysis at the BMR Genomics of Padova, Italy. The 16S ribosomal DNA sequences were then analyzed on the NCBI-BLAST server.

2.5. Genotypic Identifications of Yeasts Isolates

The isolated yeasts were identified by amplification and sequencing of the ribosomal Internal Transcribed Spacer (ITS) region, using ITS1 (5′-GTTTCCGTAGGTGAACTTGC-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers, as previously described [22]. Yeast DNA extraction was performed with a KAPA Biosystem kit (Merck, Darmstadt, Germany) following the manufacturer’s protocol. PCR amplification was carried out in a 25 μL reaction mixture constituted of 4 μL of DNA, 5 μL buffer 5X, 0.5 μL dNTPs 10 mM, 2 μL MgCl2 25 mM, 1 μL of each primer 10 mM, 0.14 μL Taq polymerase (Invitrogen, Carlsbad, CA, USA) and 12.35 μL nuclease-free water. The PCR reaction started with an initial denaturation at 94 °C for 5 min, followed by 29 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, an extension phase at 72 °C for 1 min, and a final extension at 72 °C for 4 min. The PCR products were visualized on a 1.5% gel in 1X Tris-acetate-EDTA (TAE) buffer solution at 100 V for 1 h; as a molecular weight marker, the 100 bp DNA ladder (Promega,) was used. The gel was displayed with the software Image Lab (Bio-Rad). To determine the complete sequences of 5.8S rDNA and the adjacent ITS1 and ITS2 regions, the purified PCR products were sequenced by Sanger analysis at the BMR Genomics of Padova, Italy. These sequences were analyzed to identify the fungi through the BLASTn program (www.ncbi.nlm.nih.gov/BLAST, (accessed on 3 July 2024)).
Strain characterization was assessed by Restriction Fragment Length Polymorphism (RFLP) after digestion of the amplified ITS1-4 region using the restriction enzymes as reported [23]. 4 µL of PCR products were digested with the restriction endonucleases CfoI, HaeIII, and Hinf1 (Promega). Digestion was carried out in a 20 μL reaction mixture consisting of 4 μL of PCR product, 1 μL of restriction endonuclease, 2 μL of buffer, and 13 μL of nuclease-free water. The reaction mixture was prepared for each individual restriction enzyme, with each corresponding to a different buffer (Buffer L, Buffer C, Buffer R). The restriction fragments were observed on a 2% gel in 1X Tris-acetate-EDTA (TAE) buffer solution at 80 V for 1.5 h; as a molecular weight marker, the 100 bp DNA ladder (Promega) was used. The gel was viewed with the software Image Lab (Bio-Rad).

2.6. Extraction of Metabolites and Untargeted Metabolomics Characterization

The analysis was performed in triplicate for both the oat okara flour and the oat okara dough. Briefly, 1 g of each sample was extracted in 10 mL of an 80% methanol solution acidified with 0.1% formic acid. Then, for the oat okara flour, the extraction was promoted by a homogenizer-assisted extractor for 2 min (PT1200E, Polytron, Duluth, GA, USA). Meanwhile, for the oat okara dough, ultrasounds were applied for 10 min at ambient temperature with a maximum power of 120 W. Subsequently, the samples were centrifuged at 5500 rpm for 10 min at 4 °C and stored overnight in the freezer at −18 °C. Finally, the supernatants were filtered through a 0.2 μm syringe cellulose filter and collected in amber vials until further analysis.
The fingerprint of the oat okara flour and dough was conducted using high-resolution mass spectrometry (HRMS) performed on a Q-Exactive™ Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, MA, USA) coupled to a Vanquish ultra-high-pressure liquid chromatography (UHPLC) pump and equipped with heated electrospray ionization (HESI)-II probe (Thermo Scientific, Waltham, MA, USA). For the chromatography step, LC-MS grade water and acetonitrile (Sigma-Aldrich, Milan, Italy) were employed as a mobile phase, using gradient elution (6–94% acetonitrile in 35 min) and 0.1% formic acid as a phase modifier on a BEH C18 column (2.1 mm × 100 mm, 1.7 μm) (Milford, MA, USA) maintained at 35 °C. The injection volume was 6 µL with a constant flow rate of 200 µL/min. Afterward, raw data were processed using the MS-DIAL software (version 4.90), considering the automatic peak finding, LOWESS normalization, and annotation via spectral matching using the FooDB database. In addition, an in-house database containing the main metabolic products and by-products of LAB and yeasts was used to search for potential correlations with the aromatic profile of the product. The features were searched in the mass range of 80–1200 m/z with a minimum peak height of 10,000 cps. For MS and MS/MS analysis, accurate mass tolerance for peak centroiding was set to 0.05 Da and 0.1 Da, respectively. The information on retention time was not considered for the calculation of the total score. The accurate mass tolerance for identification was 0.05 Da for MS and 0.1 Da for MS/MS. Overall, the identification of compounds was based on mass accuracy, isotopic profile, and spectral matching. The total identification score cut-off was 50%, taking into consideration the most common HESI + adducts. Finally, in this experimental work, a confidence level of 2 (i.e., putatively annotated compounds) was achieved in annotation.

2.7. Statistical Analysis

The multivariate statistical analysis of metabolomics data was performed using two different software applications, namely MetaboAnalyst 6.0 and SIMCA 18.0 (Umetrics, Malmo, Sweden). Briefly, raw data were normalized according to the median, transformed into Log2 values, and Pareto-scaled. Afterward, the hierarchical cluster analysis (HCA) was performed unsupervised, while orthogonal projections to latent structures discriminant analysis (OPLS-DA) was used as supervised multivariate statistics. The OPLS-DA model was built considering the influence of storage time (i.e., 0, 1, and 30 days) and also considering the model validation parameters, in particular goodness-of-fit R2Y and goodness-of-prediction Q2Y. Additionally, the variable importance in the projection (VIP) approach was used to discriminate the most relevant metabolites in both the oat okara flour and dough, selecting the minimum threshold values higher than one. Finally, the fold-change variation was evaluated to check both the direction and the intensity of variation of the marker compounds identified by the VIP selection method.

3. Results and Discussion

3.1. Microbiological Evaluation of Oat Okara and pH Measurements

3.1.1. Oat Okara Flour

The results regarding the microorganisms analyzed in oat okara flour are shown in Figure 1. A high aerobic TMC (6.65 log cfu/g) was detected, while anaerobic bacteria were lower (5.37 log cfu/g). Similar values to the TMC were found for total lactobacilli count (6.64 log cfu/g) and heterofermentative lactobacilli (6.61 log cfu/g). Minervini et al. [24], studying 19 different types of Italian sourdough produced with durum wheat flour, associated the high presence of sugars such as maltose, glucose, and fructose and high levels of free amino acids in the flour with bacterial flora in the sourdough composed mainly of obliged heterofermentative LAB. Other microorganisms present in high quantities were enterococci (5.42 log cfu/g) and yeasts (5.36 log cfu/g). Enterobacteriaceae and coliforms were not detected (value < 1 log cfu/g). Bacillus cereus was not found in the flour samples (<2 log cfu/g); however, other authors have detected Bacillus spp. in low-moisture foods such as flour and various starchy foods [25,26].
Similar counts were found for aerobic and anaerobic bacterial spores (about 4 log spores/g). These results showed a high number of spores compared with wholemeal flour (3.1 log spores/g) and white bread flour (about 2 log spores/g) [27].

3.1.2. Microbiological Count of Oat Okara Sourdoughs

The results regarding the spontaneous fermentation of oat okara flour to produce oat okara sourdough are shown in Figure 2. Spontaneous sourdough oats were evaluated at three different refreshment times: t0, t1 (1 day), and t2 (30 days). As in okara flour, the bacterial population of the total and heterofermentative lactobacilli showed the same values and increased by two logs after 1 day of fermentation (from 6.49 log cfu/g for OOSt0 to 8.67 log cfu/g for OOSt1).
After 30 days of back-slopping (t2), the LAB microbiota of the sourdoughs remained stable (around 9 log cfu/g for both total and heterofermentative lactobacilli). A high content of LAB can be associated with the great amount of protein, ash, and micronutrients in oat okara, compared with oat and wheat flour, resulting in an increase in the buffer capacity of flour [28]. Similar values were found by Boreczek et al. [29] in different sourdoughs obtained with wheat, spelt, and rye flour. However, Hüttner et al. [30] found slightly lower values in the spontaneous fermentation of oat flour: after 2 days of refreshments, the LAB population reached a value of 108 CFU/g and remained stable throughout the refreshment process. As in okara flour, the enterococci count was high initially (6.1 and 6.6 log at t0 and t1, respectively) and decreased by about 1 log after 30 days.
LAB from wheat and some unconventional flours originating from Italy revealed that enterococci were the most frequently isolated LAB [31]. Studies [32] on sourdough microbiota evolution have shown that species not specific to Enterococcus, Lactococcus, and Leuconostoc were predominant in the first phase of fermentation. The same results were obtained in different traditional sourdough from the Abruzzo region (central Italy), where the LAB cocci count, including enterococci, ranged between 104 and 106 CFU/g [33].
Regarding the yeast population, the highest value was found after 30 days of refreshments (6.95 log cfu/g), an increase of almost 2 logs over the initial value at T0 (5.12 log cfu/g). It is quite common to record an increase in yeast population at the start of fermentation because of the accelerated carbohydrate metabolism of LAB, which makes monosaccharides available to support yeast growth and development [34]. The LAB and yeast count and their ratio in the range of 100:1 over time highlighted that a performing sourdough had been obtained [35].
The pH trend followed the development of the microbial community: it decreased from 5.13 (OOSt0) to 4.63 after 24 h and stabilized around 4.17 after 30 days of back-slopping, values within the most common pH range of 3.4 to 4.9 [35].

3.2. Molecular Identification and Characterization of the Isolates

In this study, 160 bacterial colonies and 32 yeast colonies were isolated and identified by the molecular technique. The differentiation at the strain level for bacteria was carried out by RAPD-PCR, identifying 21 different biotypes. The RFLP analysis of yeast made it possible to group three different profiles. For each representative RAPD and RFLP profile, one strain was selected and subject to sequencing.
Nine different species of lactic acid bacteria (Weissella confusa, Enterococcus faecium, Pediococcus pentosaceus, Enterococcus lactis, Lactiplantibacillus plantarum, Lactobacillus coryniformis, Leuconostoc pseudomesenteroides, Companilactobacillus paralimentarius, and Levilactobacillus brevis) and three yeast species (Pichia kudriavzevii, Saccharomyces cerevisiae, and Hanseniospora opuntiae) were found (Figure 3). During the fermentation period, variations in the bacterial community have been assessed. In flour and OOSt0, the main bacteria were W. confusa and E. lactis, while in OOSt1, they decreased in favor of an increase in P. pentosaceus and L. mesentorides. Finally, E. faecium, Lb. plantarum, L. coryniformis, C. paralimentarius and L. brevis became dominant in OOSt2. The largest group of species at the beginning of the fermentation was W. confusa, and the typing, carried out by RAPD-PCR analyses, identified four different RAPD profiles. W. confusa is normally present in sourdoughs of different origins; it has been detected in the doughs of many types of cereals as well as legumes [36,37]. In particular, Woo et al. [38] isolated W. confusa from gluten-free cereal-based sourdoughs and stated that it had excellent dough-forming capabilities.
E. faecium was always present in all samples and was most notable in OOSt2. E. faecium is widely present in the sourdoughs formed by various products, even among those defined as gluten-free; for example, Dentice Maidana et al. [39] detected E. faecium during the spontaneous fermentation period of chia flour.
E. lactis was also one of the predominant species, both in the flour and in the sourdough at t0; in the sourdoughs at t1 and t2, however, it was no longer present. E. lactis is not typically found in cereal products but is not entirely uncommon. Divisekera et al. [40] isolated a strain of E. lactis from a variety of fermented finger millet flour typical of Sri Lanka. Indeed, in oat okara, it was detected in the flour and then only in the dough at t0, i.e., the flour with added water.
Overall, four different strains of P. pentosaceus and L. pseudomesenteroides were present after 24 h fermentation. Conversely, Corsetti and Settanni et al. [41] identified only P. pentosaecous in the early phases and not in the stable sourdough. Manini et al. [42] instead indicated P. pentosaceus and L. mesenteroides as part of the predominant components of endogenous LAB development in all refreshment phases of the sourdough fermentation of wheat bran.
Interestingly, during the sourdough’s refreshment period of 30 days, other bacterial species increased, which enriched the microbial pool acting as agents of spontaneous fermentation: Lb. plantarum, L. coryniformis, C. paralimentarius, and, to a lesser extent, L. brevis. Considering the oat sourdough, both P. pentosaceus and L. coryniformis were frequently found by Hüttner et al. [30] and Hanjinia et al. [43]; it is not unusual for P. pentosaceus and Lb. plantarum to be found together in sourdough, as demonstrated by Abedfar et al. [44], and in gluten-free cereal sources [45]. In addition, Alfonzo et al. [46] detected L. pseudomesenteroides in association with P. pentosaceus and Lb. plantarum in sourdough produced with durum wheat semolina in southern Italy. Many studies [47,48,49] then associate the presence of E. faecium in wheat sourdough with microorganisms such as Lb. plantarum, P. pentosaceus, and L. coryniformis.
C. paralimentarius and L. brevis are other LABs frequently found in sourdough: Syrokou et al. [50] and Boujamaai et al. [51] identified it as dominant in two traditional Greek and Moroccan sourdoughs, respectively. Moreover, Cardinali et al. [52] identified C. paralimentarius and L. brevis as the main lactic acid bacteria of type I sourdough produced with Triticum aestivum, and De Vuyst et al. [53] reported that these two species represent an example of a stable LAB combination for type I sourdough.
Additionally, the two yeast species characterizing the flour, OOSt0 and OOSt1, were P. kudriavzevii and H. opuntiae, while after 30 days (OOSt2), S. cerevisiae was the predominant species. The restriction patterns obtained by RFLP evidenced only one profile RFLP for each yeast species. P. kudriavzevii is not an unusual yeast to find in fermented products; in fact, Kahve et al. [54] detected this yeast in several fermented matrices, such as cereals, milk, and vegetables, and he reported that this species had promising capabilities to improve the nutritional quality of food through natural fermentation and, further, showed beneficial probiotic effects. Xu D. et al. and Xu Y. et al. [55,56] also demonstrated that fermentation by P. kudriavzevii in sourdoughs led to better flavor development because of its preference for acid utilization. The presence of H. opuntiae in fermented products is often linked to aroma production, such as in beer [57] and cocoa [58].
S. cerevisiae is the yeast that commonly acts during the formation of doughs and has also been found in association with P. kudriavzevii in traditional and homemade sourdough production in Turkey [59].

3.3. Untargeted Profiling by High-Resolution Mass Spectrometry (HRMS)

Oat okara flour and dough were analyzed using a comprehensive UHPLC-HRMS untargeted approach to determine their chemical profiles. Initially, a hierarchical cluster analysis (HCA) was conducted to identify similarities and differences between the flour and the fermented dough. The results, as shown in Figure 4A, indicated a clear separation of the oat okara flour (OOF) and unfermented dough at time 0 (OOSt0) from the fermented samples (OOSt1 and OOSt2). This observation suggests that the spontaneous fermentative microflora significantly affects the chemical profile of oat okara dough, thus determining a modification of specific classes of metabolites. Interestingly, the HCA findings were corroborated by a supervised multivariate analysis of the fermented doughs (OOSt1 and OOSt2). Particularly, the OPLS-DA model (Figure 4B) demonstrated high-quality parameters in terms of discrimination, with more than acceptable goodness-of-fit (R2 = 0.999) and goodness-of-prediction (Q2 = 0.919) values.
The chemical profile of the flour was then assessed, revealing a total of 539 detected compounds, of which 326 (>62%) were phenolic compounds, followed by lipids (15%), amino acids (>8%), and other molecules (>14%). A semi-quantification of bioactive phenolic compounds was then performed, and the results for anthocyanins, flavan-3-ols, flavonols, other flavonoids, other polyphenols (including lower molecular weight phenolics and remaining classes), phenolic acids, and stilbenes in oat okara flour are shown in Table 1. As a general consideration, phenolic acids were the most abundant subclass of phenolics, followed by the other phenolics group (Oleuropein Eq.) This finding is consistent with other studies [60] that consider oat flour as a promising source of health-promoting bioactive phenolics. Oats are known to contain avenanthramides (i.e., 2c, 2f, and 2p), which uniquely characterize this food matrix, exhibiting in vitro antioxidant properties and health benefits, including protection against chronic diseases such as cardiovascular diseases, cancer, and diabetes [61]. These phenolic compounds were successfully identified by following our untargeted metabolomics-based approach (Table S1). Notably, the avenanthramides 2c, 2f, and 2p, present in the oat okara flour, were not discriminant in the fermented dough. This absence could be attributed to the microorganisms metabolizing these phenolic compounds during fermentation, using them as a substrate [62].
Table 2 highlights some of the most discriminant VIP markers of the OPLS-DA prediction model reported in Figure 4B, together with some not discriminant metabolites (involved in flavor and metabolic activity of the microbial consortia) showing a significant log2 Fold-Change (FC) variation. The remaining VIP discriminant metabolites can be found in Table S1. As a general consideration, the fermentation process resulted in a significant increase in the accumulation of five main phenolic acids in oats (i.e., isoferulic acid, p-coumaric acid, p-coumaroyl malic acid, trans-ferulic acid, and sinapic acid) compared with the non-fermented dough (OOS-T0), especially after 30 days of fermentation. Conversely, three phenolic acids showed a marked and discriminant down-accumulation following the fermentation process, namely 8-4′-dehydrodiferulic acid, vanillic acid, and 24-metyhlcholestanol ferulate (Table 2). Samples fermented 30 days showed significantly lower levels for three amino acids, namely alanine (−1.43), tryptophan (−1.11), and pyroglutamic acid (−0.95). This reduction could be associated with the metabolism of LAB, which decomposes proteins during fermentation to produce small-molecule peptides and free amino acids that are further modified [63].
As far as the discriminant ability of phenolic acids is concerned, Boudaoud et al. [64] previously reported a pivotal role of sourdough yeast–bacteria interactions in changing the metabolism of ferulic acid and its metabolites during fermentation; these authors reported an increase in some specific metabolites, namely 4-vinylguaiacol and dihydroferulic acid. Under our experimental conditions, we measured the discriminant potential of several forms of ferulic acid, recording a marked discriminant ability (as affected by fermentation) for 8-4′-dehydrodiferulic acid (VIP score: 2.43) and trans-ferulic acid (VIP score: 1.50). In addition to the discriminant ability as a function of the fermentation vs. the OOS-T0 sample, we found a marked and increasing accumulation of isoferulic acid (Log2 FC = 2.84) in OOS-t2 samples, thus confirming the ability of the microbial consortia to process these phenolic metabolites during fermentation time.
Finally, to better investigate the potential correlations between microorganisms (LAB and yeast) and the potential flavor of the products (according to the annotated metabolites), we studied the evolution of organic acids and other key metabolites of both LAB and yeasts during dough fermentation. The most important metabolites [65,66] dealing with the metabolic activity of LAB and yeasts in dough fermentation are reported in Table 2.
It is known that LAB can synthesize various organic acids according to their homo-lactic and hetero-lactic fermentation abilities, and depending on the final product produced, these compounds can prevent the spoilage of the formulated food product together with improving its final taste and texture. Although LABs mainly produce lactic acid, they can also produce propionate, acetate, and succinate derivatives [66]. Under our experimental conditions, we found significant accumulation of several organic acids over fermentation time (i.e., after 30 days), including (among the others) lactic acid (Log2FC = 1.39), 2,3-diaminopropionic acid (Log2FC = 1.63), 3-phenyl-lactic acid (Log2FC = 1.62), and succinic acid (Log2FC = 0.31) (Table 2). Interestingly, our findings suggested a role of fermentation in promoting the accumulation of a key metabolite, namely gamma-aminobutyric acid (GABA), recording a higher up-accumulation in OOS-t1 samples (Table 2). GABA is a neurotransmitter obtained by the enzymatic activity of both glutamate decarboxylase and pyridoxal-5′-phosphate. This substance is regarded as one of the bioactive compounds created by LAB that may be beneficial to the consumer’s health [66]. Another very important metabolite promoted by the fermentation of LAB and yeasts was 3-phenyllactic acid (PLA); PLA is known for its strong antimicrobial properties, which can inhibit the growth of pathogenic bacteria and spoilage microorganisms, thus contributing to the safety and shelf life of fermented food products [67]. This is crucial in dough fermentation as it helps maintain the microbial balance. PLA can also contribute to the overall sensory profile and quality of fermented dough [68]. During fermentation, LAB and yeasts metabolize phenylalanine into PLA, influencing the aroma and taste of the final product, thus providing a mildly acidic note and contributing to the complexity of the dough’s taste. Therefore, the production of PLA during fermentation can be considered a key marker of the dynamic interactions between LAB and yeast in the final dough. Another important marker compound recorded is the reduced glutathione (GSH), showing a significant down-accumulation after 30 days of fermentation. The reduction of GSH during prolonged fermentation likely results from its role in counteracting oxidative stress, detoxification of fermentation by-products, and enzymatic degradation. Particularly, GSH plays a crucial role in detoxifying some compounds, such as aldehydes, alcohols, and organic acids that accumulate during fermentation. As these compounds increase over time, GSH may be depleted as it participates in neutralizing them. Therefore, its reduction reflects the dynamic metabolic environment of the fermented dough, together with the ability of some LAB to synthesize it as a defense mechanism to cope with stress conditions [69]. Additionally, we recorded also two key compounds involved in the production of polyamines, such as ornithine and citrulline. Ornithine was up-accumulated in OOS-T2 samples, while an opposite trend was found for citrulline (Table 2). Citrulline is often a precursor to ornithine in metabolic pathways, such as the urea cycle and polyamine synthesis pathways [70]. During fermentation, microorganisms can utilize citrulline as a nitrogen source or for the synthesis of amino acids and polyamines, leading to its depletion over time. On the other hand, the rise in ornithine levels suggests that citrulline is being converted into ornithine. This is likely because of the activity of enzymes such as argininosuccinate lyase, which converts citrulline into ornithine and fumarate in the urea cycle. Additionally, microorganisms can decarboxylate ornithine to produce putrescine, a polyamine important in cellular growth, which might play a role in the fermentation dynamics.
Regarding the metabolic activity of yeast, besides ethanol and carbon dioxide, they can produce metabolites that specifically affect flavor, such as organic acids, diacetyl, higher alcohols from branched-chain amino acids, and esters derived thereof [65]. Under our experimental conditions, we measured both a high discriminant ability and an accumulation trend for diacetyl. This latter, also known as 2,3-butanedione, can be produced by yeasts through both pyruvate catabolism or the Ehrlich pathway; it is reported to contribute to the crumb aroma, although it is also formed by Strecker degradation during baking. After 30 days of fermentation, we measured a marked accumulation of diacetyl in OOS-t2 samples (Log2FC = 4.87), and this trend is rather coherent with the accumulation of S. cerevisiae noticed in Figure 3.

4. Conclusions

This study’s objective was to characterize an innovative ingredient derived from the circular economy to assess its potential as a new functional ingredient. Specifically, okara oat flour and the sourdough derived from it were evaluated. At the microbiological level, a high number of LAB normally present in sourdoughs was found, and interesting species of flavor-building yeasts were present in both the flour and at the beginning of backslopping. This indicates that this industrial by-product has a lot of potential for producing new ingredients. Additionally, a high-resolution UHPLC-Orbitrap-MS approach was applied to assess the untargeted chemical profile of the oat okara flour and the fermented doughs. The provided results suggest that oat by-products may be efficiently exploited as rich sources of phenolic compounds, in particular phenolic acids and low-molecular-weight phenolics. Furthermore, the fermentation process showed an increase in three main phenolic acids, especially after 30 days of fermentation. Organic acids (lactate, succinate, and propionate derivatives) and flavor compounds (e.g., diacetyl) were the best markers of LAB and yeast metabolic activity. This approach provides valuable insights into the valorization of oat by-products. Future studies could be designed to exploit the sourdough of oat okara to produce more sustainable and healthy bakery products, also thanks to a standardization of the fermentation process based on the selection of optimal microbial starters.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation10110545/s1, Table S1: this table contains the following sheets: (A) chemical analysis on oat okara flour. (B) Raw annotation resulting from the untargeted metabolomics analysis by UHPLC-HRMS. (C) Phenolic profile of oat okara flour evaluated through untargeted metabolomics analysis. (D) additional VIP marker compounds resulting from the OPLS-DA supervised discriminant analysis for the comparison of OOS-t1 and OOS-t2 vs. OOSt0.

Author Contributions

Conceptualization and methodology, A.R., G.R. and L.L.; formal analysis, F.M. and C.M.; investigation and data curation, F.M., C.M., A.R. and G.R.; writing—original draft preparation, F.M. and C.M.; writing—review and editing, A.R. and G.R.; supervision, L.L. and L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the project called ‘AURA—Anziani: Una Risorsa da Alimentare/Elderly: A Resource to Feed’, identified under the number F/310136/01-05/X56, is financed under the Ministerial Decree of 31 December 2021 and the Agreement for Innovation of 7 February 2023, between the Ministry of Enterprise and Made in Italy, Panificio Cremona Italia S.r.l. together with Healthy Aging Research Group Società Benefit S.r.l. in acronym H.A.R.G. S.B. S.r.l., Teracell S.r.l., Rivoltini Alimentare Dolciaria S.r.l., Università Cattolica Del Sacro Cuore, aimed at supporting research and development activities, approved by decree dated 1 March 2023 no. 531 of the Director General for Incentives to Enterprises of the Ministry of Enterprise and Made in Italy and registered at the Court of Auditors on 7 April 2023 no. 375.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The dataset from metabolomics is available as Supplementary Material.

Acknowledgments

This study was supported by the Doctoral School on the Agro-Food System (Agrisystem) of the Università Cattolica del Sacro Cuore (Italy).

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Pasarín, V.; Viinikainen, T. Enabling a Legal Environment for the Prevention and Reduction of Food Loss and Waste; Legal Brief 9; Food and Agriculture Organization of the United Nations: Rome, Italy, 2022; pp. 1–10. [Google Scholar]
  2. Scialabba, N.E. Food Wastage Footprint Full-Cost Accounting: Final Report; Food and Agriculture Organization of the United Na-tions: Rome, Italy, 2014; pp. 1–98, e-ISBN 978-92-5-108513-4. [Google Scholar]
  3. Vong, W.C.; Yang, K.L.C.A.; Liu, S.-Q. Okara (soybean residue) biotransformation by yeast Yarrowia lipolytica. Int. J. Food Microbiol. 2016, 235, 1–9. [Google Scholar] [CrossRef] [PubMed]
  4. Čechp, M.; Hercp, P.; Ivanišováp, E.; Kolesárováp, A.; Urminskáp, D.; Grygorievap, O. Okara-by-product from soy processing: Characteristic, properties, benefits, and potential perspectives for industry. Int. J. Exp. Res. Rev. 2022, 28, 66–83. [Google Scholar] [CrossRef]
  5. Swallah, M.S.; Fan, H.; Wang, S.; Yu, H.; Piao, C. Prebiotic impacts of soybean residue (Okara) on eubiosis/dysbiosis condition of the gut and the possible effects on liver and kidney functions. Molecules 2021, 26, 326. [Google Scholar] [CrossRef] [PubMed]
  6. Mok, W.K.; Tan, Y.X.; Chen, W.N. Evaluating the potential of Bacillus subtilis fermented okara as a functional food ingredient through in vitro digestion and fermentation. Food Biotechnol. 2021, 35, 136–157. [Google Scholar] [CrossRef]
  7. Gupta, S.; Lee, J.J.L.; Chen, W.N. Analysis of Improved Nutritional Composition of Potential Functional Food (Okara) after Probiotic Solid-State Fermentation. J. Agric. Food Chem. 2018, 66, 5373–5381. [Google Scholar] [CrossRef]
  8. Li, B.; Qiao, M.; Lu, F. Composition, Nutrition, and Utilization of Okara (Soybean Residue). Food Rev. Int. 2012, 28, 231–252. [Google Scholar] [CrossRef]
  9. Fuentes, A.P.C.; Bengoa, A.; Gagliarini, N.; Abraham, A.; Pla, M.F.d.E.; Flores, S.K. Physicochemical and functional characterisation of a food ingredient based on okara containing probiotics. Food Bioprod. Process. 2022, 135, 74–86. [Google Scholar] [CrossRef]
  10. Colletti, A.; Attrovio, A.; Boffa, L.; Mantegna, S.; Cravotto, G. Valorisation of by-products from soybean (Glycine max (L.) Merr.) processing. Molecules 2020, 25, 2129. [Google Scholar] [CrossRef]
  11. Paz, G.M.; King, J.M.; Prinyawiwatkul, W. High Protein Rice Flour in the Development of Gluten-Free Bread. J. Culin. Sci. Technol. 2020, 19, 315–330. [Google Scholar] [CrossRef]
  12. Holopainen-Mantila, U.; Vanhatalo, S.; Lehtinen, P.; Sozer, N. Oats as a source of nutritious alternative protein. J. Cereal Sci. 2024, 116, 103862. [Google Scholar] [CrossRef]
  13. Mel, R.; Malalgoda, M. Oat protein as a novel protein ingredient: Structure, functionality, and factors impacting utilization. Cereal Chem. 2021, 99, 21–36. [Google Scholar] [CrossRef]
  14. Shewry, P.R.; Beaudoin, F.; Jenkins, J.; Griffiths-Jones, S.; Mills, E.N.C. Plant protein families and their relationships to food allergy. Biochem. Soc. Trans. 2002, 30, 906–910. [Google Scholar] [CrossRef] [PubMed]
  15. Helstad, A.; Marefati, A.; Ahlström, C.; Rayner, M.; Purhagen, J.; Östbring, K. High-Pressure Pasteurization of Oat Okara. Foods 2023, 12, 4070. [Google Scholar] [CrossRef] [PubMed]
  16. Wu, W.; Zhang, L.; Zheng, X.; Huang, Q.; Farag, M.A.; Zhu, R.; Zhao, C. Emerging applications of metabolomics in food science and future trends. Food Chem. X 2022, 16, 100500. [Google Scholar] [CrossRef]
  17. Ferri, M.; Serrazanetti, D.I.; Tassoni, A.; Baldissarri, M.; Gianotti, A. Improving the functional and sensorial profile of cereal-based fermented foods by selecting Lactobacillus plantarum strains via a metabolomics approach. Food Res. Int. 2016, 89, 1095–1105. [Google Scholar] [CrossRef]
  18. Balestra, F.; Laghi, L.; Saa, D.T.; Gianotti, A.; Rocculi, P.; Pinnavaia, G. Physico-chemical and metabolomic characterization of KAMUT® Khorasan and durum wheat fermented dough. Food Chem. 2015, 187, 451–459. [Google Scholar] [CrossRef]
  19. ISO 15214:1998 (En); Microbiology of Food Animal Feeding Stuffs Horizontal Method for the Enumeration of Mesophilic Lactic Acid Bacteria Colony-Count Technique at 30 Degrees, C. International Organisation for Standardization (ISO): Geneva, Switzerland, 1998. Available online: https://www.iso.org/standard/26853.html (accessed on 12 July 2024).
  20. Johansson, M.L.; Quednau, M.; Molin, G.; Ahrné, S. Randomly amplified polymorphic DNA (RAPD) for rapid typing of Lactobacillus plantarum strains. Lett. Appl. Microbiol. 1995, 21, 155–159. [Google Scholar] [CrossRef]
  21. Di Cello, F.; Fani, R. A molecular strategy for the study of natural bacterial communities by PCR-based techniques. Minerva Biotecnol. 1996, 8, 126–134. [Google Scholar]
  22. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR—Protocols and Applications—A Laboratory Manual; Academic Press: Cambridge, MA, USA, 1990; Chapter 38; pp. 315–322. [Google Scholar]
  23. Esteve-Zarzoso, B.; Belloch, C.; Uruburu, F.; Querol, A. Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int. J. Syst. Evol. Microbiol. 1999, 49, 329–337. [Google Scholar] [CrossRef]
  24. Minervini, F.; Di Cagno, R.; Lattanzi, A.; De Angelis, M.; Antonielli, L.; Cardinali, G.; Cappelle, S.; Gobbetti, M. Lactic acid bacterium and yeast microbiotas of 19 sourdoughs used for traditional/typical Italian breads: Interactions between ingredients and microbial species diversity. Appl. Environ. Microbiol. 2012, 78, 1251–1264. [Google Scholar] [CrossRef]
  25. Iurlina, M.O.; Saiz, A.I.; Fuselli, S.R.; Fritz, R. Prevalence of Bacillus spp. in different food products collected in Argentina. LWT 2006, 39, 105–110. [Google Scholar] [CrossRef]
  26. Fangio, M.F.; Roura, S.I.; Fritz, R. Isolation and identification of Bacillus spp. and related genera from different starchy foods. J. Food Sci. 2010, 75, M218–M221. [Google Scholar] [CrossRef] [PubMed]
  27. Pereira, A.P.M.; Stradiotto, G.C.; Freire, L.; Alvarenga, V.O.; Crucello, A.; Morassi, L.L.; Silva, F.P.; Sant’ana, A.S. Occurrence and enumeration of rope-producing spore forming bacteria in flour and their spoilage potential in different bread formulations. LWT 2020, 133, 110108. [Google Scholar] [CrossRef]
  28. Lau, S.W.; Chong, A.Q.; Chin, N.L.; Talib, R.A.; Basha, R.K. Sourdough Microbiome Comparison and Benefits. Microorganisms 2021, 9, 1355. [Google Scholar] [CrossRef]
  29. Boreczek, J.; Litwinek, D.; Żylińska-Urban, J.; Izak, D.; Buksa, K.; Gawor, J.; Gromadka, R.; Bardowski, J.K.; Kowalczyk, M. Bacterial community dynamics in spontaneous sourdoughs made from wheat, spelt, and rye wholemeal flour. Microbiologyopen 2020, 9, e1009. [Google Scholar] [CrossRef]
  30. Hüttner, E.K.; Bello, F.D.; Arendt, E.K. Identification of lactic acid bacteria isolated from oat sourdoughs and investigation into their potential for the improvement of oat bread quality. Eur. Food Res. Technol. 2010, 230, 849–857. [Google Scholar] [CrossRef]
  31. Corsetti, A.; Settanni, L.; López, C.C.; Felis, G.E.; Mastrangelo, M.; Suzzi, G. A taxonomic survey of lactic acid bacteria isolated from wheat (Triticum durum) kernels and non-conventional flours. Syst. Appl. Microbiol. 2007, 30, 561–571. [Google Scholar] [CrossRef]
  32. Alfonzo, A.; Miceli, C.; Nasca, A.; Franciosi, E.; Ventimiglia, G.; Di Gerlando, R.; Tuohy, K.; Francesca, N.; Moschetti, G.; Settanni, L. Monitoring of wheat lactic acid bacteria from the field until the first step of dough fermentation. Food Microbiol. 2017, 62, 256–269. [Google Scholar] [CrossRef]
  33. Corsetti, A.; Settanni, L.; Valmorri, S.; Mastrangelo, M.; Suzzi, G. Identification of subdominant sourdough lactic acid bacteria and their evolution during laboratory-scale fermentations. Food Microbiol. 2007, 24, 592–600. [Google Scholar] [CrossRef]
  34. García-Béjar, B.; Fernández-Pacheco, P.; Carreño-Domínguez, J.; Briones, A.; Arévalo-Villena, M. Identification and biotechnological characterisation of yeast microbiota involved in spontaneous fermented wholegrain sourdoughs. J. Sci. Food Agric. 2023, 103, 7683–7693. [Google Scholar] [CrossRef]
  35. Arora, K.; Ameur, H.; Polo, A.; Di Cagno, R.; Rizzello, C.G.; Gobbetti, M. Thirty years of knowledge on sourdough fermentation: A systematic review. Trends Food Sci. Technol. 2020, 108, 71–83. [Google Scholar] [CrossRef]
  36. Galli, V.; Venturi, M.; Coda, R.; Maina, N.H.; Granchi, L. Isolation and characterization of indigenous Weissella confusa for in situ bacterial exopolysaccharides (EPS) production in chickpea sourdough. Food Res. Int. 2020, 138, 109785. [Google Scholar] [CrossRef] [PubMed]
  37. Mohammadi-Kouchesfahani, M.; Hamidi-Esfahani, Z.; Azizi, M.H. Isolation and identification of lactic acid bacteria with phytase activity from sourdough. Food Sci. Nutr. 2019, 7, 3700–3708. [Google Scholar] [CrossRef] [PubMed]
  38. Woo, S.-H.; Park, J.; Sung, J.M.; Choi, E.-J.; Choi, Y.-S.; Park, J.-D. Characterization of Lactic Acid Bacteria and Yeast from Grains as Starter Cultures for Gluten-Free Sourdough. Foods 2023, 12, 4367. [Google Scholar] [CrossRef]
  39. Maidana, S.D.; Ficoseco, C.A.; Bassi, D.; Cocconcelli, P.S.; Puglisi, E.; Savoy, G.; Vignolo, G.; Fontana, C. Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented chia sourdough. Int. J. Food Microbiol. 2020, 316, 108425. [Google Scholar] [CrossRef]
  40. Divisekera, D.M.W.D.; Samarasekera, J.K.R.R.; Hettiarachchi, C.; Gooneratne, J.; Choudhary, M.I.; Gopalakrishnan, S.; Wahab, A.-T. Lactic acid bacteria isolated from fermented flour of finger millet, its probiotic attributes and bioactive properties. Ann. Microbiol. 2018, 69, 79–92. [Google Scholar] [CrossRef]
  41. Corsetti, A.; Settanni, L. Lactobacilli in sourdough fermentation. Food Res. Int. 2007, 40, 539–558. [Google Scholar] [CrossRef]
  42. Manini, F.; Brasca, M.; Plumed-Ferrer, C.; Morandi, S.; Erba, D.; Casiraghi, M.C. Study of the chemical changes and evolution of microbiota during sourdough like fermentation of wheat bran. Cereal Chem. 2014, 91, 342–349. [Google Scholar] [CrossRef]
  43. Hajinia, F.; Sadeghi, A.; Mahoonak, A.S. The use of antifungal oat-sourdough lactic acid bacteria to improve safety and technological functionalities of the supplemented wheat bread. J. Food Saf. 2020, 41, e12873. [Google Scholar] [CrossRef]
  44. Abedfar, A.; Hosseininezhad, M.; Sadeghi, A.; Raeisi, M.; Feizy, J. Investigation on “spontaneous fermentation” and the productivity of microbial exopolysaccharides by Lactobacillus plantarum and Pediococcus pentosaceus isolated from wheat bran sourdough. LWT 2018, 96, 686–693. [Google Scholar] [CrossRef]
  45. Iosca, G.; De Vero, L.; Di Rocco, G.; Perrone, G.; Gullo, M.; Pulvirenti, A. Anti-Spoilage Activity and Exopolysaccharides Production by Selected Lactic Acid Bacteria. Foods 2022, 11, 1914. [Google Scholar] [CrossRef] [PubMed]
  46. Alfonzo, A.; Ventimiglia, G.; Corona, O.; Di Gerlando, R.; Gaglio, R.; Francesca, N.; Moschetti, G.; Settanni, L. Diversity and technological potential of lactic acid bacteria of wheat flours. Food Microbiol. 2013, 36, 343–354. [Google Scholar] [CrossRef] [PubMed]
  47. Lancetti, R.; Sciarini, L.; Pérez, G.T.; Salvucci, E. Technological Performance and Selection of Lactic Acid Bacteria Isolated from Argentinian Grains as Starters for Wheat Sourdough. Curr. Microbiol. 2020, 78, 255–264. [Google Scholar] [CrossRef]
  48. Salvucci, E.; LeBlanc, J.G.; Pérez, G. Technological properties of Lactic acid bacteria isolated from raw cereal material. LWT 2016, 70, 185–191. [Google Scholar] [CrossRef]
  49. Paramithiotis, S.; Gioulatos, S.; Tsakalidou, E.; Kalantzopoulos, G. Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough. Process. Biochem. 2006, 41, 2429–2433. [Google Scholar] [CrossRef]
  50. Syrokou, M.K.; Themeli, C.; Paramithiotis, S.; Mataragas, M.; Bosnea, L.; Argyri, A.A.; Chorianopoulos, N.G.; Skandamis, P.N.; Drosinos, E.H. Microbial ecology of greek wheat sourdoughs, identified by a culture-dependent and a culture-independent approach. Foods 2020, 9, 1603. [Google Scholar] [CrossRef]
  51. EL Boujamaai, M.; Mannani, N.; Aloui, A.; Errachidi, F.; Ben Salah-Abbès, J.; Riba, A.; Abbès, S.; Rocha, J.M.; Bartkiene, E.; Brabet, C.; et al. Biodiversity and biotechnological properties of lactic acid bacteria isolated from traditional Moroccan sourdoughs. World J. Microbiol. Biotechnol. 2023, 39, 1–11. [Google Scholar] [CrossRef]
  52. Cardinali, F.; Garofalo, C.; Reale, A.; Boscaino, F.; Osimani, A.; Milanović, V.; Taccari, M.; Aquilanti, L. Liquid sourdough from stone-ground soft wheat (Triticum aestivum) flour: Development and exploitation in the breadmaking process. Food Res. Int. 2022, 161, 111796. [Google Scholar] [CrossRef]
  53. De Vuyst, L.; Van Kerrebroeck, S.; Harth, H.; Huys, G.; Daniel, H.-M.; Weckx, S. Microbial ecology of sourdough fermentations: Diverse or uniform? Food Microbiol. 2014, 37, 11–29. [Google Scholar] [CrossRef]
  54. Kahve, H.I. In Vitro Evaluation of the Technological and Probiotic Potential of Pichia kudriavzevii Strains Isolated from Traditional Fermented Foods. Curr. Microbiol. 2023, 80, 1–9. [Google Scholar] [CrossRef]
  55. Xu, D.; Zhang, H.; Xi, J.; Jin, Y.; Chen, Y.; Guo, L.; Jin, Z.; Xu, X. Improving bread aroma using low-temperature sourdough fermentation. Food Biosci. 2020, 37, 100704. [Google Scholar] [CrossRef]
  56. Xu, Y.; Xu, Y.; Zhao, Q.; Xu, X.; Xu, D. The contributions of Pichia kudriavzevii EP1 to sourdough: Acid utilization, oligosaccharides and peptides production and aroma enhancement. Food Biosci. 2024, 59, 104225. [Google Scholar] [CrossRef]
  57. Bourbon-Melo, N.; Palma, M.; Rocha, M.P.; Ferreira, A.; Bronze, M.R.; Elias, H.; Sá-Correia, I. Use of Hanseniaspora guilliermondii and Hanseniaspora opuntiae to enhance the aromatic profile of beer in mixed-culture fermentation with Saccharomyces cerevisiae. Food Microbiol. 2020, 95, 103678. [Google Scholar] [CrossRef] [PubMed]
  58. Papalexandratou, Z.; Lefeber, T.; Bahrim, B.; Lee, O.S.; Daniel, H.-M.; De Vuyst, L. Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations, underlining the importance of these microbial species for a successful cocoa bean fermentation process. Food Microbiol. 2013, 35, 73–85. [Google Scholar] [CrossRef] [PubMed]
  59. Sevgili, A.; Can, C.; Ceyhan, D.I.; Erkmen, O. Molecular identification of LAB and yeasts from traditional sourdoughs and their impacts on the sourdough bread quality characteristics. Curr. Res. Food Sci. 2023, 6, 100479. [Google Scholar] [CrossRef] [PubMed]
  60. Hitayezu, R.; Baakdah, M.M.; Kinnin, J.; Henderson, K.; Tsopmo, A. Antioxidant activity, avenanthramide and phenolic acid contents of oat milling fractions. J. Cereal Sci. 2015, 63, 35–40. [Google Scholar] [CrossRef]
  61. Wang, Y.; Wu, J.; Lv, M.; Shao, Z.; Hungwe, M.; Wang, J.; Bai, X.; Xie, J.; Wang, Y.; Geng, W. Metabolism Characteristics of Lactic Acid Bacteria and the Expanding Applications in Food Industry. Front. Bioeng. Biotechnol. 2021, 9, 612285. [Google Scholar] [CrossRef]
  62. Djorgbenoo, R.; Djorgbenoo, R.; Hu, J.; Hu, J.; Hu, C.; Hu, C.; Sang, S.; Sang, S. Fermented Oats as a Novel Functional Food. Nutrients 2023, 15, 3521. [Google Scholar] [CrossRef]
  63. Huang, W.-Y.; Cai, Y.-Z.; Zhang, Y. Natural phenolic compounds from medicinal herbs and dietary plants: Potential use for cancer prevention. Nutr. Cancer 2009, 62, 1–20. [Google Scholar] [CrossRef]
  64. Boudaoud, S.; Aouf, C.; Devillers, H.; Sicard, D.; Segond, D. Sourdough yeast-bacteria interactions can change ferulic acid metabolism during fermentation. Food Microbiol. 2021, 98, 103790. [Google Scholar] [CrossRef]
  65. De Vuyst, L.; Harth, H.; Van Kerrebroeck, S.; Leroy, F. Yeast diversity of sourdoughs and associated metabolic properties and functionalities. Int. J. Food Microbiol. 2016, 239, 26–34. [Google Scholar] [CrossRef] [PubMed]
  66. Hakim, B.N.A.; Xuan, N.J.; Oslan, S.N.H. A Comprehensive Review of Bioactive Compounds from Lactic Acid Bacteria: Potential Functions as Functional Food in Dietetics and the Food Industry. Foods 2023, 12, 2850. [Google Scholar] [CrossRef] [PubMed]
  67. Yu, S.; Zhou, C.; Zhang, T.; Jiang, B.; Mu, W. Short communication: 3-Phenyllactic acid production in milk by Pediococcus pentosaceus SK25 during laboratory fermentation process. J. Dairy Sci. 2015, 98, 813–817. [Google Scholar] [CrossRef] [PubMed]
  68. Valerio, F.; Lavermicocca, P.; Pascale, M.; Visconti, A. Production of phenyllactic acid by lactic acid bacteria: An approach to the selection of strains contributing to food quality and preservation. FEMS Microbiol. Lett. 2004, 233, 289–295. [Google Scholar] [CrossRef]
  69. Singh, R.; Pophaly, S.D.; Kaushik, J.K.; Tomar, S.K. Current status and emerging role of glutathione in food grade lactic acid bacteria. Microb. Cell Factories 2012, 11, 114. [Google Scholar] [CrossRef]
  70. Premi, L.; Rocchetti, G.; Rossetti, C.; Dallolio, M.; Lucini, L.; Rebecchi, A. Coagulase-negative staphylococci enhance the colour of fermented meat through a complex cross-talk between the arginase and nitric oxide synthase activities. LWT 2024, 202, 116333. [Google Scholar] [CrossRef]
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Figure 1. Microbiological counts in oat okara flour.
Figure 1. Microbiological counts in oat okara flour.
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Figure 2. Results of spontaneous fermentation of oat okara sourdoughs (t0, t1, t2): microbial counts and pH trend during the fermentation process.
Figure 2. Results of spontaneous fermentation of oat okara sourdoughs (t0, t1, t2): microbial counts and pH trend during the fermentation process.
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Figure 3. Oat okara flour and sourdough (t0, t1, t2) isolate identification.
Figure 3. Oat okara flour and sourdough (t0, t1, t2) isolate identification.
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Figure 4. Heat map resulting from the hierarchical clustering analysis (A) and OPLS-DA score plot (B) were both built considering the chemical profile of the different samples (OOF, OOS-t0, OOS-t1, and OOS-t2).
Figure 4. Heat map resulting from the hierarchical clustering analysis (A) and OPLS-DA score plot (B) were both built considering the chemical profile of the different samples (OOF, OOS-t0, OOS-t1, and OOS-t2).
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Table 1. Semi-quantification of phenolic compounds annotated in oat okara flour, expressed as the mean ± standard deviation (n = 3). Data are reported as mg phenolic equivalents (Eq) per 100 g of dry matter (DM).
Table 1. Semi-quantification of phenolic compounds annotated in oat okara flour, expressed as the mean ± standard deviation (n = 3). Data are reported as mg phenolic equivalents (Eq) per 100 g of dry matter (DM).
Phenolic ClassOOF (mg/100 g DM)
Anthocyanins (Cyanidin Eq.)4.95 ± 0.23
Flavan-3-ols (Catechin Eq.)3.22 ± 0.42
Flavonols (Quercetin Eq.)2.70 ± 0.08
Other flavonoids (Luteolin Eq.)6.00 ± 0.12
Other phenolics (Oleuropein Eq.)16.56 ± 1.74
Phenolic acids (Ferulic acid Eq.)29.05 ± 0.21
Stilbenes (Resveratrol Eq.)1.28 ± 0.14
Total phenolics63.76 ± 10.14
Table 2. Discriminant metabolites associated with LAB and yeast metabolic activities. Each metabolite is provided with its Log2 fold change (FC) and VIP score for the comparison against OOS-t0.
Table 2. Discriminant metabolites associated with LAB and yeast metabolic activities. Each metabolite is provided with its Log2 fold change (FC) and VIP score for the comparison against OOS-t0.
MetabolitesLog2FC
OOS-t1 vs. OOS-t0		Log2FC
OOS-t2 vs. OOS-t0		VIP Score
6-Phosphogluconic acid3.625.941.83
Reduced Glutathione−2.79−4.961.73
Diacetyl1.824.871.70
5-Hydroxyindole-3-acetic acid−1.18−3.541.42
Citrulline1.18−1.821.68
2,3-Diaminopropionic acid0.891.63<1
3-phenyllactic acid1.141.62<1
Lactoylglutathione0.451.54<1
Indole-3-acetic acid1.381.42<1
Lactic acid0.591.39<1
NADH1.761.37<1
trans-2-Hexenal1.261.30<1
2-Hydroxy-3-methylbutyric acid1.271.20<1
3-Indolepropionic acid2.351.141.00
4-Aminobutyrate1.871.021.03
Ornithine0.920.77<1
Butyric acid0.440.73<1
Linolenic acid0.630.61<1
2-Hydroxyisobutyric acid0.620.56<1
NAD+−0.380.50<1
Oxidized Glutathione1.37−0.411.43
3-hydroxy-3-methylbutanoic acid0.630.36<1
Succinic acid0.170.31<1
9-hydroxy-10,12-octadecadienoic acid−2.590.251.50
Citric acid−2.540.221.64
Propionic acid−0.42−0.19<1
p-Hydroxyphenyllactic acid0.140.19<1
8-4′-Dehydrodiferulic acid−3.69−8.792.43
p-Coumaroyl malic acid3.565.411.67
1,5-Diferuloylquinic acid2.474.561.65
Sinapic acid1.694.541.65
trans-Ferulic acid1.11−1.591.50
p-Coumaric acid1.833.911.41
Isoferulic Acid1.322.841.18
Vanillic acid−0.49−1.641.04
24-Methylcholestanol ferulate−7.74−7.342.20
Alanine−0.54−1.431.01
Tryptophan0.45−1.111.21
Pyroglutamic acid0.61−0.951.24
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MDPI and ACS Style

Meanti, F.; Mussio, C.; Rocchetti, G.; Rebecchi, A.; Lucini, L.; Morelli, L. Oat Okara Fermentation: New Insights into the Microbiological and Metabolomic Characterization. Fermentation 2024, 10, 545. https://doi.org/10.3390/fermentation10110545

AMA Style

Meanti F, Mussio C, Rocchetti G, Rebecchi A, Lucini L, Morelli L. Oat Okara Fermentation: New Insights into the Microbiological and Metabolomic Characterization. Fermentation. 2024; 10(11):545. https://doi.org/10.3390/fermentation10110545

Chicago/Turabian Style

Meanti, Federica, Chiara Mussio, Gabriele Rocchetti, Annalisa Rebecchi, Luigi Lucini, and Lorenzo Morelli. 2024. "Oat Okara Fermentation: New Insights into the Microbiological and Metabolomic Characterization" Fermentation 10, no. 11: 545. https://doi.org/10.3390/fermentation10110545

APA Style

Meanti, F., Mussio, C., Rocchetti, G., Rebecchi, A., Lucini, L., & Morelli, L. (2024). Oat Okara Fermentation: New Insights into the Microbiological and Metabolomic Characterization. Fermentation, 10(11), 545. https://doi.org/10.3390/fermentation10110545

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