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Article

Evaluation of In Vitro Cytoprotective Activity, Antioxidant Activity and Proteomic Profiles of Novel Sorghum-Based Fermented Beverages

by
David R. Katerere
1,2,
Abel Navarré Dopazo
3,
Raffaele Sessa
4,
Silvia Trombetti
4,
Michela Grosso
4,* and
Luana Izzo
3
1
Department of Pharmaceutical Science, Tshwane University of Technology, Pretoria 0001, South Africa
2
Department of Pharmacy, Manica University, Lusaka P.O. Box 32379, Zambia
3
Department of Pharmacy, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy
4
Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy
*
Author to whom correspondence should be addressed.
Beverages 2026, 12(1), 9; https://doi.org/10.3390/beverages12010009
Submission received: 17 November 2025 / Revised: 11 December 2025 / Accepted: 22 December 2025 / Published: 8 January 2026
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Abstract

Fermentation, one of the oldest food processing techniques, is known to play a pivotal role in improving the nutritional and functional characteristics of cereals, with positive implications for gut health and overall well-being. The present study aims to examine the phenolic acids, peptides, and potential bioactive properties of 2 novel sorghum-based fermented beverages, Niselo and Delishe. A total of 48 phenolic compounds were identified through targeted and untargeted Ultra-High Performance Liquid Chromatography coupled with a Quadrupole Orbitrap High-Resolution Mass Spectrometer (UHPLC–Q-Orbitrap HRMS) analyses, revealing a higher content of phenolic acids in Niselo and a prevalence of flavonoids in Delishe. Niselo exhibited enhanced in vitro cytoprotective and reactive oxygen species (ROS)-scavenging activity and displayed a clear cytoprotective effect against hydrogen peroxide-induced oxidative stress in Caco-2 cells. Proteomic profiling using tryptic digestion revealed that Niselo has a substantial abundance of fragments of peptides matching several stress-related and antioxidant proteins, indicating a superior stress-response and/or defense capability. Overall, these findings highlight the functional potential of sorghum-based fermented beverages, supporting their role as health-promoting products.

1. Introduction

Sorghum bicolor is a staple cereal widely cultivated and consumed in Africa and South East Asia [1,2,3], where it is prepared as boiled kernels, porridges, and traditional beverages. Sorghum is classified into red, black, white, and brown varieties and each has distinct phytochemical and nutritional attributes [4,5]. White sorghum has recently been identified as a promising source of antioxidant polyphenols [6,7,8] while red sorghum contains high tannin and resistant starch [8,9,10,11].
Eleusine coracana, commonly known as finger millet, is one of the minor cereals that is increasingly becoming popular in Southern and Eastern Africa and used as a staple in low-income groups in India [12,13,14,15]. Though its hectarage is low, it is known for its restorative effects in convalescent patients. The health benefits are attributed to its polyphenol and dietary fiber, as well as high calcium and potassium content [16,17,18].
In Southern Africa, sorghum, and to some extent, finger millet, is fermented into alcoholic opaque beer (commonly known as umqombothi or muchaiwa/doro) or non-alcoholic drinks commonly called maheu/mageu/motoho [19,20]. The subjects of this paper are Niselo, a probiotic-rich non-dairy yogurt developed in South Africa under the SANBio BioFISA II program, and Delishe, a millet–sorghum probiotic beverage created for East African markets with support from Innovate UK/Unilever. These products have been recently developed and commercialized by NutriGO-SA, a spin-out from Tshwane University of Technology.
Fermentation of grains with lactic acid bacteria (LAB) has been shown to enhance functional food properties [21,22]. Indeed, LAB fermentation increases crude fiber, polyphenol availability, and antioxidant capacity, while also introducing probiotic organisms that improve gut colonization and immune modulation [23,24].
Additionally, LAB degrade antinutritional compounds such as oxalates, phytates, and complex carbohydrates that would otherwise impede nutrient absorption, thereby enhancing the digestibility of foods and increasing the bioavailability of nutrients such as vitamins and minerals [25,26]. Besides producing beneficial metabolites, fermentation may improve sensory qualities and shelf stability [27,28].
Beyond probiotic enrichment, sorghum fermentation contributes to microbiota modulation by generating prebiotic substrates and short-chain fatty acids (SCFAs), which play a role in satiety, fat metabolism, and intestinal health [29,30]. Recent studies further highlight that fermented sorghum products can reduce oxidative stress, improve anti-inflammatory markers, and support metabolic health in both animal and human models [9,31,32].
The aim of this study was to comprehensively evaluate the chemical profile and biological effects of two novel sorghum-based fermented beverages, Niselo and Delishe, through a multidimensional analytical framework. The present study may enhance our understanding of the effects associated with the consumption of sorghum-based beverages and may provide new insights into the diverse biological properties arising from specific sorghum formulations.

2. Materials and Methods

2.1. Reagents and Standards

Analytical (LC-MS grade) water, methanol, and formic acid (FA) were provided by Merck (Milan, Italy). Polyphenol standards, including quinic acid, protocatechuic acid, catechin, chlorogenic acid, epicatechin, caffeic acid, p-coumaric acid, ferulic acid, naringin, luteolin-7-glucoside, rutin hydrate, quercetin-3b-glucoside, diosmin, apigenin-7O-glucoside, kaempferol-3-O-glucoside, ellagic acid, daidzein, quercetin, naringenin, genistein, luteolin, and apigenin, were purchased from Merck (Milan, Italy). Niselo and Delishe were obtained from the manufacturer Nutrigo Pty Ltd. in Pretoria, South Africa.

2.2. Polyphenols Extraction

One gram of lyophilized sample was weighed into a 50 mL tube, to which a mixture (v/v) of methanol and water 80:20, both containing 0.1% FA, was added. The tubes were vortexed (ZX3, VEPL Scientifica Srl, Usmate (MB), Italy for 3 min and sonicated (LBS1, Zetalab Srl, Padova, Italy) for 15 min, after which they were centrifuged (X3R, Heraeus Multifuge, Thermo Fisher Scientific, Milan, Italy; 5000 rpm, 4 °C, 5 min) and the supernatant was recovered. The resulting pellet was re-extracted under the same conditions, and the supernatant generated was combined with the previous one. The supernatant was filtered (0.22 µm) and diluted 1:2 with 0.1% FA methanol prior to analysis.

2.3. Proteomics Sample Preparation

The EasyPep Mini MS kit (ThermoFisher Scientific, Rockford, IL, USA) was used. Briefly, 10 mg of the lyophilized sample was weighed into an Eppendorf tube, to which 100 µL of lysate solution containing 1 µL of universal nuclease was added. Content was homogenized and left for 30 min at room temperature without agitation to release protein fraction, after which tubes were centrifuged (16,000× g, 10 min). A volume of 70 µL of supernatant was extracted, and the final volume was brought to 100 µL with lysate solution. Next, 50 µL of reduction solution and 50 µL of alkylation solution were added to each tube, which were incubated at 95 °C for 10 min. Afterwards, the samples were allowed to cool to room temperature, and 50 µL of trypsin solution was added for sample digestion to each tube. The tubes were incubated at 37 °C for 24 h, after which 50 µL of the digestion stop solution was added per tube. Finally, digested samples were purified using Solid Phase Extraction (SPE) C18 columns (Phenomenex, Castel Maggiore, Italy). The final eluted volume (300 µL) was dried using a nitrogen flow and resuspended in 100 µL of 0.1% FA water for analysis.

2.4. UHPLC-Q-Orbitrap HRMS Analysis

2.4.1. Polyphenols

Polyphenols were determined using a Vanquish DAD-Flex UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) utilizing a micro-degasser (GPL-3400RS), a refrigerated autosampler (WPS-3000), a solvent delivery pump (HPG-3400RS), and a thermostatic column oven (TCC-3100). Chromatographic separation was performed on a Kinetex F5 column (100 × 2.1 mm, 2.6 µm) thermostated at 30 °C (Phenomenex). Phases used consisted of 0.1% FA water (A) and 0.1% FA methanol (B), and the following gradient (phase A) was followed, with a flow of 0.4 mL/min for 13 min: 0 min, 100%; 0.5 min, 100%; 1.5 min 30%; 8 min, 15%; 11 min, 100%; 13 min, 100%, with an injection volume of 5 µL.
Mass spectrometry analysis was performed on a Q Exactive Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) using negative ionization (electrospray ionization interface, ESI−) with a scan range of 80 to 1200 m/z and a maximum injection time of 200 ms, in full MS and all ion fragment (AIF) mode according to a previous reported method [33]. The mass resolving power in full MS was set at 70,000 FWHM and the automatic gain control (AGC) at 1 × 106, while in AIF, they were 17,500 FWHM and 1 × 105 for the AGC, as well as a collision energy of 15, 30, and 45 eV. Quan/Qual Browser Xcalibur software, version number 4.7.69.37 was used for data processing (Thermo Fisher, Waltham, MA, USA). For the identification and quantification of polyphenols in the targeted analysis, standards were injected at a concentration of 5 ppm to 0.0024 ppm using 1/2 dilutions in 0.1% FA methanol, while identification in untargeted analysis was performed using the theoretical mass of the compounds.

2.4.2. Proteomics

Peptide chromatographic separation was performed on an Aeris Peptide XB-C18 (Phenomenex, Torrance, CA, USA) column (100 × 2.1 mm, 1.7 µm) thermostated at 45 °C. A 28 min gradient method was used, with water (phase A) and ACN (phase B), both containing 0.1% FA, with 0.25 mL/min flow and the following parameters (A): 0 min, 94%; 3 min, 94%; 14 min, 70%; 15 min, 65%; 16 min, 20%; 18 min, 20%; 18.1 min, 94%; 28 min, 94%. Injection volume was 20 µL.
Mass spectrometry analysis was conducted using positive ionization mode (ESI+) in full MS and data-dependent mode (dd-MS2). The mass resolving power in full MS was set at 70,000 FWHM, the automatic gain control (AGC) at 3 × 106, and a maximum injection time of 200 ms, with a scan range of 375–1500 m/z, while in dd-MS2 mode the mass resolving power was set at 17,500 FWHM, the AGC at 1 × 105, and a maximum injection time of 300 ms. An isolation window of 2 m/z was set, and the collision energy was 28 eV. The charge exclusions were set at 1, 7, 8, or >8, with a dynamic exclusion of 5 s. Proteome Discoverer software, version number3.1.1.93, was used for data processing. The UniProt database was used to compare data obtained by mass spectrometry, using the repositories corresponding to the organisms Lactobacillus rhamnosus, Sorghum bicolor, Eleusine coracana, and Zea mays (Gene Ontology). Searches were performed considering two failed cleavages with complete proteolytic specificity (trypsin), with a peptide length between 6 and 50 residues, ±10 ppm for the precursor mass tolerance and ±0.02 Da for the fragment masses, and oxidized methionine as a possible residue modification, establishing a maximum precursor mass shift range of ±15.9 Da to accommodate methionine oxidation. In turn, a mass shift range of ±42.01 Da was considered for the acetylation of the terminal residue, as well as ±57.02 Da for the carbamidomethylation of the initial residue. For proteomics final analysis, only proteins with a number of ≥2 unique peptides were included.

2.5. Cell Culture

Caco-2 cells, originally obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), were routinely subcultured at ~50% confluence and maintained under standard conditions at 37 °C in a humidified atmosphere containing 5% CO2. Cells were grown in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich), 4 mM stable L-glutamine (GlutaMax; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and 1× penicillin–streptomycin solution (Pen-Strep; Sigma Aldrich). To prevent mycoplasma contamination, cultures were routinely screened using the PCR Mycoplasma Test Kit (AppliChem A3744, Darmstadt, Germany). Once cells reached the logarithmic growth phase, they were trypsinized, subcultured for 48 h, and subsequently starved in a fresh serum-free medium prior to downstream in vitro experiments.

2.6. Cytotoxicity Assays

To determine potential cytotoxicity, Delishe and Niselo fermented beverage samples were vacuum freeze-dried and subsequently diluted in complete DMEM to final concentrations of 0.05, 0.1, 0.25, 0.5, 0.75 and 1 mg/mL. Cytotoxicity towards Caco-2 cells was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, as previously described [34,35]. Briefly, Caco-2 cells at logarithmic growth phase were seeded in a 96-well plate at a density of 1 × 104 cells/well in 100 μL of complete medium. After 48 h, cells were synchronized by 2 h serum starvation in serum-free DMEM, then treated with the fermented beverages for 24 h. Following treatment, the supernatant was carefully removed and replaced with 90 μL of fresh DMEM per well. Subsequently, 10 μL of MTT labeling reagent (Cell Proliferation Kit I; Roche, Mannheim, Germany) was added to each well and the plates were incubated for 4 h at 37 °C to allow formation of formazan crystals by metabolically active cells. After incubation, 100 µL of solubilization buffer (10% SDS in 0.01 M HCl) was added to each well, according to the manufacturer’s instructions. Absorbance was measured at 570 nm with a reference wavelength of 690 nm using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Cell viability was calculated as follows: (absorbance of the treated cells/absorbance of the untreated control) × 100.

2.7. H2O2-Induced Cytotoxicity Assays

To evaluate the cytoprotective effects of Niselo and Delishe beverages on the cell damage under pro-oxidative conditions, Caco-2 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 1 × 104 cells/well and in 100 μL of complete medium and incubated for 48 h prior to the MTT assay. Cells were then synchronized by incubation in serum-free DMEM for 2 h prior to treatment. Cells were divided into three experimental groups: untreated control, H2O2-stressed control, and test groups. The untreated control group was maintained in serum-free DMEM at 37 °C for 26 h without treatment. For the test groups, synchronized cells were incubated in serum-free DMEM supplemented with freeze-dried fermented beverage powders at final concentrations of 0.25 mg/mL (low dose), 0.5 mg/mL (medium dose), and 1 mg/mL (high dose), and incubated for 24 h. After treatment, the cells were washed with DPBS and incubated for an additional 2 h in serum-free medium. In parallel, the H2O2-stressed group was first incubated with the indicated concentrations of fermented beverages in serum-free DMEM for 24 h, followed by a 2 h incubation in fresh DMEM containing 1 mM H2O2 as the oxidative stimulus. Cells treated with hydrogen peroxide alone were used as a positive control. After treatment, the culture supernatants were removed and replaced with 90 μL of DMEM per well. MTT labeling reagent (Cell Proliferation Kit I; Roche) was added and plates were incubated for 4 h at 37 °C in a humidified atmosphere containing 5% CO2 to allow formazan crystal formation. Subsequently, 100 µL of solubilization buffer (10% SDS in 0.01 M HCl) was added to each well, according to the manufacturer’s instructions. Cell viability, based on the mitochondrial NAD(P)H-dependent activity, was quantified by measuring the absorbance using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) as detailed above.

2.8. Intracellular Reactive Oxygen Species (ROS) Accumulation Assays

To compare the effects of Niselo and Delishe beverages on intracellular ROS neutralization under H2O2-induced oxidative stress, intracellular ROS levels were detected using the fluorescent probe H2DCF-DA (2′7′-dichlorodihydrofluorescein diacetate), through a spectrofluorometric assay, as previously described [36]. Synchronized Caco-2 cells were treated following the procedure detailed in Section 2.7 with 0.25, 0.5 and 1 mg/mL of each fermented beverage for 48 h. After treatment, the cells were washed twice with Dulbecco’s Phosphate-Buffered Saline (DPBS) and incubated with 5 µM H2DCF-DA, diluted in Hank’s Balanced Salt Solution (HBSS), for 20 min at 37 °C in the dark. After staining, the excess dye was removed and the cells were washed twice with 1× DPBS. Subsequently, cells were incubated in fresh HBSS containing 1 mM H2O2, as described above, for the H2O2-stressed control group. Fluorescence was measured using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek) with excitation and emission wavelengths set at 485 and 538 nm, respectively. Data were expressed as fluorescence percentage relative to the untreated control group.

2.9. Statistical Analysis

Statistical analysis was carried out using one-way or two-way analysis of variance (ANOVA), followed, where appropriate, by Dunnett’s multiple comparisons test or multiple Student’s t-test. Differences were considered statistically significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (* p ≤ 0.05 and ** p ≤ 0.001, respectively, vs. untreated control or a p ≤ 0.05 and aa p ≤ 0.001 for Niselo vs. Delishe treatments).

3. Results

3.1. Analysis of Polyphenol Profiles in Niselo and Delishe Fermented Beverages

Chromatographic and MS/MS parameters for targeted and untargeted polyphenols are shown in Table 1 and Table 2. In total, 48 phenolic compounds were identified in the Niselo and Delishe samples, 22 through targeted analysis and 26 through untargeted analysis. The data revealed distinct compositional patterns between the samples, with some compounds showing clear predominance in one matrix over the other. Table 3 and Figure 1 show the concentration (mg/100 g) of targeted polyphenols. Niselo shows particularly high levels of caffeic acid (1.821 mg/100 g), ferulic acid (1.696 mg/100 g), and p-coumaric acid (0.780 mg/100 g), indicating a strong presence of hydroxycinnamic acids. Delishe displays much lower amounts of these acids but is richer in flavan-3-ols such as catechin (6.204 mg/100 g) and epicatechin (1.638 mg/100 g), as well as in protocatechuic acid (1.768 mg/100 g). Flavonoid glycosides, including luteolin-7-glucoside, kaempferol-3-O-glucoside, and rutin hydrate, are present in both samples, although their concentrations are generally slightly higher in Delishe. Naringenin and luteolin, key flavonoids, occur in comparable amounts between the two extracts. Overall, Niselo is characterized by a profile dominated by phenolic acids, while Delishe shows a flavan-3-ol–rich composition with elevated levels of catechins and protocatechuic acid. These differences reflect variations in raw material composition, potentially influencing the antioxidant and sensory properties of the two samples.
A retrospective UHPLC-Q-Orbitrap HRMS analysis was performed to explore the polyphenolic profile beyond the targeted compounds. As authentic standards were not available, absolute quantification was not feasible; therefore, the data are reported as (%) relative area of untargeted polyphenols identified, expressed as both average area and (%) average relative area (Table 4). This semi-quantitative approach provides an overview of the relative distribution and abundance of untargeted polyphenols among samples.
Delishe exhibits higher levels of certain oxidized fatty acids (e.g., trihydroxy-octadecanoic acid), whereas Niselo shows a greater abundance of typical phenolic compounds (e.g., caffeoylglycerol, naringenin hexoside). Trihydroxy-octadecanoic acid clearly dominates in Delishe (52.0%), while in Niselo it accounts for only 20.8%, suggesting a higher presence of lipid oxidation-derived metabolites in Delishe. Caffeoylglycerol and its derivatives (1-O-, 2-O-, and 1,3-di-O-caffeoylglycerol) are more abundant in Niselo, indicating a potentially higher content of caffeic acid-based phenolic compounds. Naringenin hexoside I is markedly more abundant in Niselo (10.3%) than in Delishe (4.1%), suggesting a more pronounced flavonoid composition in Niselo. Other compounds, such as caffeic acid hexoside and saccharide, also exhibit moderate yet consistent differences, with higher relative abundances detected in Niselo. The major difference in composition is that Niselo is made with 100% sorghum, while Delishe is 50% sorghum and 50% finger millet.

3.2. Effects of Niselo and Delishe Fermented Beverages on Cell Viability

MTT assay was performed to evaluate the potential cytotoxicity exerted by Niselo and Delishe fermented beverages on Caco-2 cells at different concentrations defined on the basis of literature data [37]. The Caco-2 cell model was chosen because these cells are widely used as a standardized in vitro model of the human intestinal epithelium to assess absorption, bioavailability, and cytoprotective effects of food, beverage, and nutraceutical compounds. Recognized by the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for predicting intestinal bioavailability, this model is also frequently applied to compare fermented and non-fermented plant products, particularly to evaluate fermentation-enhanced antioxidant and anti-inflammatory properties in vitro [38,39,40,41]. As shown in Figure 2, within the concentration range of 0.01 mg/mL to 1 mg/mL, both fermented beverages showed comparable effects on cell viability (above 91% as compared to untreated cells) with no significant differences in cytotoxicity between treated and untreated cells after 24 h.

3.3. Intracellular ROS Levels in Caco-2 Cells Treated with Delishe and Niselo Beverages

The effect of Delishe and Niselo on intracellular ROS generation in Caco-2 cells was evaluated after 24 h treatment using a fluorometric assay with the specific probe H2DCF-DA. Cells treated with 1 mM H2O2 alone were used as a positive control. As shown in Figure 3, the measurement of intracellular ROS levels in cultures pre-incubated with low, medium and high concentrations of Delishe or Niselo beverages revealed no significant differences compared to untreated cells under basal conditions after 24 h. Subsequently, Caco-2 cells pretreated with Delishe and Niselo beverages and then subjected to oxidative stress showed that ROS accumulation significantly decreased in cells challenged with 1 mM H2O2 compared to cells treated with H2O2 alone (positive control) (Figure 4). This reduction was observed for both beverages even at the lowest concentration of 0.25 mg/mL. However, cells pretreated with Niselo and subsequently exposed to oxidative stress exhibited lower ROS levels than those pretreated with Delishe at the medium concentration of 0.5 mg/mL (114% vs. 125.5%, respectively). In accordance with the cell viability data, these results indicate that the antioxidant metabolites present in the bioaccessible fraction of the Niselo fermented beverage were sufficient to neutralize a greater proportion of excess ROS in cells, suggesting that the enhanced protective effect observed is, at least in part, mediated by ROS-scavenging antioxidant mechanisms [42,43].

3.4. Cell Viability and Cytoprotective Effects of Delishe or Niselo Beverages in Caco-2 Cells Under Oxidative Stress

It has been reported that compounds such as polyphenols, flavonoids, and extracellular products by lactic acid bacteria fermentation play a protective role in mitigating H2O2-triggered oxidative stress in Caco-2 cells [44]. Therefore, based on the different polyphenol profiles of these two beverages, to better understand the potential protective effects of these components, we investigated the antioxidant activity of Niselo and Delishe beverages in a Caco-2 cell model. MTT assays were conducted to assess mitochondrial metabolic activity as an indirect indicator of cell viability and the cytoprotective effect of the two fermented beverages against oxidative stress. For this purpose, Caco-2 cells were incubated in serum-free DMEM supplemented with freeze-dried fermented beverage powders at three different final concentrations: 0.25 mg/mL (low dose), 0.5 mg/mL (medium dose), and 1 mg/mL (high dose), for 24 h at 37 °C. Caco-2 cells maintained in serum-free DMEM for 24 h were used as the untreated control. After pre-treatment, cells were exposed to fresh DMEM containing 1 mM H2O2 as an oxidative stimulus for 2 h as an oxidative stimulus that was chosen according to previous studies showing significant Caco-2 cell death under these conditions [37,45]. In cells treated with hydrogen peroxide alone, used as a cell death positive control, cell viability significantly decreased to 72.5% compared to untreated controls (Figure 2). Caco-2 cells pretreated with the Delishe beverage at different concentrations showed a gradual and significant reduction in cell viability under oxidative stress compared to untreated cells. Specifically, cell viability values were 79.7%, 72.4 and 68.2% at 0.25, 0.5 and 1 mg/mL, respectively, which were similar to those observed in the positive control group (treated with 1 mM H2O2 alone). In contrast, cells pretreated with the Niselo fermented beverage exhibited a cytoprotective effect against hydrogen peroxide-induced oxidative stress even at the lowest dose, with a more pronounced effect observed at the 0.5 and 1 mg/mL concentrations, where cell viability was significantly higher (91.75% vs. 72.47% and 89.3% vs. 68.2%, respectively) compared to Delishe pretreatment (Figure 5). These findings are consistent with the different profiles of polyphenols and flavonoids observed in the Niselo beverage by UHPLC-MS analysis compared to the Delishe fermented beverage.

3.5. Proteomic Profiles in Niselo and Delishe Fermented Beverages

The proteomic analysis reveals notable quantitative and functional differences between the two samples, Niselo and Delishe (Table 5). Niselo shows a substantial abundance of several stress-related and antioxidant proteins, including subtilisin-chymotrypsin inhibitors (A0A921Q906, A0A921QAA6), glutaredoxin domain-containing protein (C5YBX1), and superoxide dismutase (A0A317YKC7), indicating a potentially stronger stress-response or defense capability. Proteins involved in cell organization and biogenesis, such as the uncharacterized protein A0A1B6PL34, also display higher relative abundance in Niselo. Delishe exhibits a higher abundance of metabolic enzymes related to energy production and carbohydrate metabolism, such as phosphoglycerate kinase (A0A3L6ELN6), glyceraldehyde-3-phosphate dehydrogenase (A0A1D6HCF4), and elongation factor Tu (Q6UBQ8). This pattern suggests a more active primary metabolism in Delishe. Most of the detected proteins originate from Sorghum bicolor, which represents the main source of the proteomic profile in both samples. The abundance ratio (Niselo/Delishe) is generally greater than 1, often ranging between 1.4 and 3.0, indicating higher expression or accumulation of these proteins in Niselo. This suggests that Niselo possesses a richer complement of defense- and stress-response proteins, which could be associated with enhanced oxidative stability or physiological adaptation. Conversely, proteins originating from Zea mays and Lactobacillus rhamnosus exhibit abundance ratios below 1, meaning that they are more abundant in Delishe. Overall, Niselo is characterized by a proteomic profile enriched in defense- and stress-related proteins, while Delishe displays higher levels of enzymes involved in primary metabolic processes. The heat map highlights differences in protein abundance between Niselo and Delishe as shown in Figure 6.

4. Discussion

Fermentation is recognized not only for its ability to enhance digestibility and improve the functional and sensory properties of foods, but also for its significant contribution to nutritional enrichment, particularly in cereals [46,47]. Numerous studies have demonstrated that fermentation can reduce antinutritional factors, increase bioactive compound availability, and promote beneficial microbial profiles in cereal matrices [48,49].
In many African regions, traditional fermented beverages are predominantly cereal-derived, representing an essential component of the diet and local food culture [50,51,52]. These products are of considerable importance to human health owing to their nutritional, nutraceutical, and biofunctional properties [53]. Evidence indicates that the consumption of cereal-based fermented beverages enhances the bioavailability of macro- and micro-nutrients, particularly through the enzymatic breakdown of complex carbohydrates, proteins, and phytates during fermentation [54,55].
Moreover, cereal fermentates are rich sources of vitamins, dietary fiber, flavonoids, phenolic compounds, antioxidants, amino acids, and bioactive peptides, many of which exhibit documented antioxidant, anti-inflammatory, and immunomodulatory activities [56,57,58]. The combined nutritional and functional properties of these beverages highlight their potential role in supporting gut health, modulating metabolic pathways, and contributing to overall well-being [54,59,60].
This study aimed to explore the bioactive health potential of two novel sorghum-based fermented beverages: Niselo (100% Sorghum bicolor) and Delishe (50% Eleusine coracana, finger Millet/50% Sorghum).
The targeted polyphenol analysis (Table 3 and Figure 1) revealed clear differences between Niselo and Delishe in phenolic composition. Both products show the presence of phenolic acids and flavonoids that explain the antioxidant and functional food properties [12,61]. Niselo exhibited higher concentrations of caffeic acid (1.821 mg/100 g), ferulic acid (1.696 mg/100 g), and p-coumaric acid (0.780 mg/100 g). The hydroxycinnamic acids are known for their strong radical-scavenging and metal-chelating properties. Delishe, on the other hand, was richer in protocatechuic acid (1.768 mg/100 g), catechin (7.278 mg/100 g), and epicatechin (1.626 mg/100 g). These flavonoids are potent hydrogen-donating antioxidants and modulate enzyme and signaling pathways [62]. These findings align with previous reports [62,63]. In this study, the protective effects of the fermented beverages against oxidative stress were evaluated using the Caco-2 cell line, an intestinal epithelium model widely recognized for its strong physiological relevance [41,64,65]. MTT analyses confirmed that neither Niselo nor Delishe fermented beverages exerted cytotoxic effects across the tested concentrations, with cell viability remaining comparable to untreated controls.
Under oxidative stress, both fermentates significantly reduced intracellular ROS accumulation; however, Niselo consistently showed a more pronounced cytoprotective effect. Even at the lowest tested dose, its bioactive metabolites more effectively counteracted H2O2-induced oxidative damage, suggesting direct ROS neutralization. This result aligns with the predominance of phenolic acids in Niselo, revealed by UHPLC–MS analysis, compounds that are readily absorbed by intestinal epithelial cells and are recognized as potent direct scavengers of ROS [66,67]. In contrast, Delishe contained higher levels of flavanols, which possess well-documented antioxidant properties, but operate mainly through indirect or more complex mechanisms. Previous studies report that flavanols can modulate ROS generation in Caco-2 cells by influencing endogenous cellular defense pathways [68]. This mechanistic distinction could explain, at least in part, the moderate but significant antioxidant activity observed for Delishe, which appears less immediate and less intense than that of Niselo.
Given the diversity of their phenolic profiles, combining sorghum and finger millet could offer synergistic antioxidant benefits. The phenolic acids in sorghum confer oxidative stability during processing, while the flavonoids from finger millet enhance overall radical scavenging potential and may contribute to cardioprotective and anti-inflammatory effects [62,69]. Such complementary interactions have been proposed as a rationale for developing multigrain functional foods with enhanced health value [70]. Proteomic profiling reinforced these in vitro findings, revealing that Niselo contains higher levels of stress- and defense-related proteins, including superoxide dismutase, glutaredoxin-domain proteins, and subtilisin-chymotrypsin inhibitors. Stress-related and antioxidant proteins have been widely reported in proteomics studies as key factors for plant defense and stress response, contributing to enhanced oxidative stability and stress tolerance [71,72,73]. The proteomic data confirm the strong influence of raw material composition. As reported by Ali et al., 2025, the drought and heat stress significantly limit crop growth and productivity, thus showing that proteomic profiles can help identify the molecular mechanisms underlying Sorghum’s adaptation to these combined stresses [71,74]. It is important to note that this study lacks a direct comparison with unfermented Sorghum. Therefore, the observed effects cannot be exclusively attributed to fermentation. Furthermore, although the approach used in the present study contributes to a better understanding of the effects derived from the consumption of fermented sorghum beverages, it is to be noted that in vitro studies may not accurately predict the biological efficacy of these products, as they do not reflect the full complexity of living organisms and cannot reproduce essential physiological processes. To overcome these limitations, in vivo studies should be conducted to evaluate bioavailability, metabolic modifications and systemic interactions, thereby offering a more robust and comprehensive evaluation of the biological activity highlighted by in vitro studies.

5. Conclusions

Fermentation of sorghum and millets enhances their nutritional and functional food properties. In this study, we generated evidence to validate the biological effects and potential health attributes of multigrain beverages formulated from African cereals. The two fermented products, Niselo and Delishe, showed distinct phenolic and proteomic profiles, which translated into different antioxidant activities at the cellular level. Specifically, we characterized their phenolic composition using both targeted and untargeted UHPLC–Q-Orbitrap HRMS approaches. In parallel, we evaluated the biological activity by assessing cytotoxicity, intracellular ROS modulation, and the cytoprotective capacity of the beverages in Caco-2 cells exposed to H2O2-induced oxidative stress. Finally, a tryptic digestion followed by LC–MS/MS analysis was performed to identify and quantify peptide fragments and their corresponding proteins, thereby elucidating the proteomic signatures of the two beverages.
The integration of chemical, in vitro biological and proteomic data highlights the importance of cereal biodiversity and grain combinations in determining the biofunctional properties of fermented beverages. These findings suggest that African cereal fermentates represent promising candidates for the development of health-promoting fermented beverages with potential benefits on oxidative balance/stress, gut health, and overall well-being. Further in vivo studies and clinical evaluations will be essential to validate these effects and support their application in the functional beverage and nutraceutical sectors.

Author Contributions

Conceptualization, D.R.K., M.G. and L.I.; methodology, D.R.K., A.N.D., R.S. and S.T.; software, R.S. and S.T.; validation, M.G., and L.I.; investigation, D.R.K., A.N.D., R.S. and S.T.; resources, D.R.K.; data curation, M.G. and L.I.; writing—original draft preparation, M.G. and L.I.; writing—review and editing, M.G. and L.I.; visualization, M.G. and L.I.; supervision, M.G. and L.I.; project administration, M.G. and L.I.; funding acquisition, D.R.K. All authors have read and agreed to the published version of the manuscript.

Funding

Through its Division of Research Capacity Development under the Research Capacity Development Initiative—Mid-Career Scientist Programme from funding received from the South African National Treasury. The content and findings reported/illustrated are the sole deduction, view and responsibility of the researcher and do not reflect the official position and sentiments of the SAMRC.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The UniProt database was used to compare data obtained by mass spectrometry, using the repositories corresponding to the organisms Lactobacillus rhamnosus, Sorghum bicolor, Eleusine coracana, and Zea mays (Gene Ontology). The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

During the preparation of this manuscript/study, the author(s) used Perplexity.ai for the purposes of literature search. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

D.R.K. was involved in the formulation of both products. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. The authors declare no conflict of interest.

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Figure 1. Graphical representation of targeted phenolic compounds in the analyzed samples. Compounds are classified according to their chemical groups (phenolic acids, flavanols, flavanones, flavones, flavonols, and isoflavones). Results are expressed as mean values (mg/100 g) ± standard deviation (SD). Statistical analysis was performed using multiple Student’s t-test. Differences were considered significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (aa p ≤ 0.001 for Niselo vs. Delishe targeted compounds).
Figure 1. Graphical representation of targeted phenolic compounds in the analyzed samples. Compounds are classified according to their chemical groups (phenolic acids, flavanols, flavanones, flavones, flavonols, and isoflavones). Results are expressed as mean values (mg/100 g) ± standard deviation (SD). Statistical analysis was performed using multiple Student’s t-test. Differences were considered significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (aa p ≤ 0.001 for Niselo vs. Delishe targeted compounds).
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Figure 2. Cytotoxicity evaluation of Delishe and Niselo beverages in Caco-2 cells. Cell viability was assessed by the MTT assay after 24 h of treatment with increasing concentrations (0.05, 0.1, 0.25, 0.5, 0.75 and 1 mg/mL) of each fermented beverage. Results as expressed as mean ± SD of three independent experiments and reported as fold change relative to untreated control cells (set as 100%). No significant differences were observed between treated and control cells, nor between Delishe and Niselo treatments.
Figure 2. Cytotoxicity evaluation of Delishe and Niselo beverages in Caco-2 cells. Cell viability was assessed by the MTT assay after 24 h of treatment with increasing concentrations (0.05, 0.1, 0.25, 0.5, 0.75 and 1 mg/mL) of each fermented beverage. Results as expressed as mean ± SD of three independent experiments and reported as fold change relative to untreated control cells (set as 100%). No significant differences were observed between treated and control cells, nor between Delishe and Niselo treatments.
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Figure 3. Evaluation of intracellular ROS level in Caco-2 cells incubated with Delishe or Niselo beverages at different concentrations (0.25, 0.5 and 1 mg/mL) for 24 h under basal conditions. ROS were assessed by the H2DCF-DA assay. Cells treated with 1 mM H2O2 alone served as a positive control. Results expressed as mean ± SD of three independent experiments and reported as fold change relative to untreated control cells (set as 100%). No significant differences were observed between treated and control cells, nor between Delishe and Niselo treatments. Differences were considered significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (** p ≤ 0.001 versus untreated control).
Figure 3. Evaluation of intracellular ROS level in Caco-2 cells incubated with Delishe or Niselo beverages at different concentrations (0.25, 0.5 and 1 mg/mL) for 24 h under basal conditions. ROS were assessed by the H2DCF-DA assay. Cells treated with 1 mM H2O2 alone served as a positive control. Results expressed as mean ± SD of three independent experiments and reported as fold change relative to untreated control cells (set as 100%). No significant differences were observed between treated and control cells, nor between Delishe and Niselo treatments. Differences were considered significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (** p ≤ 0.001 versus untreated control).
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Figure 4. Evaluation of intracellular ROS levels in Caco-2 cells pre-incubated with Delishe or Niselo beverages and exposed to pro-oxidant conditions. Cells were treated at different concentrations of Delishe and Niselo (0.25, 0.5 and 1 mg/mL) for 24 h and subsequently exposed to oxidative stress induced by 1 mM H2O2, as assessed by H2DCF-DA assay. Cells treated with 1 mM H2O2 alone served as a positive control. Pretreatment with both beverages significantly reduced ROS accumulation compared to the positive control, even at the lowest concentration. A more pronounced decrease was observed in Niselo-pretreated cells (0.5 mg/mL) as compared to Delishe-pretreated cells at the same concentration. Results are expressed as mean ± SD of three independent experiments and reported as fold change relative to untreated control cells (set as 100%). Differences were considered significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (** p ≤ 0.001 vs. untreated control; # p ≤ 0.05; ## p ≤ 0.001 vs. H2O2-positive control; a p ≤ 0.05; for Niselo vs. Delishe pretreatment).
Figure 4. Evaluation of intracellular ROS levels in Caco-2 cells pre-incubated with Delishe or Niselo beverages and exposed to pro-oxidant conditions. Cells were treated at different concentrations of Delishe and Niselo (0.25, 0.5 and 1 mg/mL) for 24 h and subsequently exposed to oxidative stress induced by 1 mM H2O2, as assessed by H2DCF-DA assay. Cells treated with 1 mM H2O2 alone served as a positive control. Pretreatment with both beverages significantly reduced ROS accumulation compared to the positive control, even at the lowest concentration. A more pronounced decrease was observed in Niselo-pretreated cells (0.5 mg/mL) as compared to Delishe-pretreated cells at the same concentration. Results are expressed as mean ± SD of three independent experiments and reported as fold change relative to untreated control cells (set as 100%). Differences were considered significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (** p ≤ 0.001 vs. untreated control; # p ≤ 0.05; ## p ≤ 0.001 vs. H2O2-positive control; a p ≤ 0.05; for Niselo vs. Delishe pretreatment).
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Figure 5. Evaluation of cell viability and cytoprotective effects of Delishe or Niselo beverages in Caco-2 cells under oxidative stress. After 24 h pretreatment with freeze-dried beverage powders (0.25, 0.5 and 1 mg/mL), cells were exposed to 1 mM H2O2 for 2 h. Cells treated with 1 mM H2O2 alone served as a positive control for cytotoxicity. Delishe pretreatment resulted in a significant dose-dependent decrease in cell viability, whereas Niselo pretreated cells showed a marked cytoprotective effect against H2O2-induced oxidative stress, particularly at 0.5 and 1 mg/mL, where viability remained significantly higher compared to Delishe-treated cells. Results are expressed as mean ± SD of three independent experiments and reported as fold change relative to untreated control cells (set as 100%). Differences were considered significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (* p ≤ 0.05; ** p ≤ 0.001 vs. untreated control; # p ≤ 0.05 vs. H2O2-positive control; a p ≤ 0.05 for Niselo vs. Delishe pretreatment).
Figure 5. Evaluation of cell viability and cytoprotective effects of Delishe or Niselo beverages in Caco-2 cells under oxidative stress. After 24 h pretreatment with freeze-dried beverage powders (0.25, 0.5 and 1 mg/mL), cells were exposed to 1 mM H2O2 for 2 h. Cells treated with 1 mM H2O2 alone served as a positive control for cytotoxicity. Delishe pretreatment resulted in a significant dose-dependent decrease in cell viability, whereas Niselo pretreated cells showed a marked cytoprotective effect against H2O2-induced oxidative stress, particularly at 0.5 and 1 mg/mL, where viability remained significantly higher compared to Delishe-treated cells. Results are expressed as mean ± SD of three independent experiments and reported as fold change relative to untreated control cells (set as 100%). Differences were considered significant at p-value ≤ 0.05 and highly significant at p-value ≤ 0.001 (* p ≤ 0.05; ** p ≤ 0.001 vs. untreated control; # p ≤ 0.05 vs. H2O2-positive control; a p ≤ 0.05 for Niselo vs. Delishe pretreatment).
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Figure 6. Heat map illustrating the results of the proteomic analysis of three biological replicates (A, B, and C) from Niselo and Delishe samples. The colour gradient represents variations in protein abundance across samples.
Figure 6. Heat map illustrating the results of the proteomic analysis of three biological replicates (A, B, and C) from Niselo and Delishe samples. The colour gradient represents variations in protein abundance across samples.
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Table 1. Chromatographic retention time and MS/MS parameters for 22 targeted polyphenols in samples.
Table 1. Chromatographic retention time and MS/MS parameters for 22 targeted polyphenols in samples.
CompoundsLOQ (ppm)Retention Time (min)Measured Mass (m/z)Theorical Mass (m/z)Accuracy (∆ ppm)Chemical Formula
Quinic acid0.00490.72191.05506191.05528−1.466C7H12O6
Protocatechiuc acid0.01953.93153.01811153.01857−3.006C7H6O4
Catechin0.00494.14289.07220289.072050.519C15H14O6
Chlorogenic acid0.00494.18353.08847353.087801.898C16H18O9
Epicatechin0.00984.28289.07248289.071961.799C7H6O4
Caffeic acid0.00494.32179.03442179.03455−0.726C9H8O4
p-coumaric acid0.01954.53163.03994163.039373.496C9H8O3
Ferulic acid0.07804.58193.05014193.05016−0.104C10H10O4
Naringin0.00494.60579.17279579.172121.157C27H32O14
Luteolin-7-glucoside0.00494.66447.09418447.093631.230C21H20O11
Rutin hydrate0.01954.68609.14648609.14673−0.410C27H32O17
Quercetin-3b-glucoside0.00494.71463.08862463.08884−0.475C21H20O12
Diosmin0.00494.75607.16736607.167180.296C28H32O15
Apigenin-7O-glucoside0.00494.80431.09906431.098601.067C21H20O10
Kaempferol-3O-glucoside0.00494.86447.09421447.093631.297C21H20O11
Ellagic acid0.00494.87300.99911300.99924−0.432C14H6O8
Daidzein0.00495.04253.05609253.05699−3.556C15H10O4
Quercetin0.00495.31301.03595301.035082.890C15H10O7
Naringenin0.01955.38271.06143271.06186−1.586C15H12O5
Genistein0.01955.48269.04614269.045552.193C15H10O5
Luteolin0.01955.47285.04086285.040620.842C15H10O6
Apigenin0.00495.78269.04572269.045550.632C15H10O5
Table 2. Chromatographic retention time and MS/MS parameters for 26 untargeted polyphenols in samples.
Table 2. Chromatographic retention time and MS/MS parameters for 26 untargeted polyphenols in samples.
Untargeted PolyphenolsRetention Time (min)Chemical FormulaTheorical Mass (m/z)Measured Mass (m/z)Accuracy
(∆ ppm)
1-O-coumaroylglycerol4.42C12H14O5237.07685237.076950.422
1-0-caffeylglycerol4.16C12H14O6253.07176253.072041.106
2-O-caffeoylglycerol4.28C12H14O6253.07176253.072041.106
7,3′,4′-trihydroxyflavone5.79C15H10O5269.04555269.046142.193
7,3′,4′,5′-tetrahydroxy flavanone4.77C15H12O6287.05611287.056852.578
Eriodictyol5.08C15H12O6287.05611287.05672.055
Chrysoeriol5.88C16H12O6299.05611299.056641.772
Dihydroxy-octadecadienoic acid6.3C18H32O6311.22278311.223482.249
Dihydroxy-octadecenoic acid6.51C18H34O6313.23843313.239072.043
Trihydroxy-octadecenoic acid5.38C18H34O6329.23335329.233951.822
Caffeic acid hexoside4.16C15H18O6341.08781341.088321.495
Saccharide4.32C16H20O10371.09837371.099061.859
1,3-O-coumaroyl-caffeoyl-glycerol5.16C21H20O8399.10854399.10801−1.328
1,3-O-coumaroyl-feruloyl-glycerol5.45C22H22O8413.12419413.124881.670
1,3-O-dicaffeoylglycerol4.96C21H20O9415.10346415.10411.542
1-O-caffeoyl-2-O-glucosylglycerol I4.07C18H24O11415.12458415.125121.301
1-O-caffeoyl-2-O-glucosylglycerol II4.14C18H24O11415.12458415.125311.759
Naringenin hexoside I4.52C21H22O10433.11402433.114410.900
Naringenin hexoside II4.75C21H22O10433.11402433.11461.339
Naringenin hexoside III4.88C21H22O10433.11402433.114621.385
1,3-O-diferuloylglicerol5.51C23H24O9443.13476443.135471.602
Luteolin hexoside I4.57C21H20O11447.09328447.094061.745
Luteolin hexoside II4.66C21H20O11447.09328447.094332.349
Eriodictyol-O-hexoside4.32C21H22O11449.10893449.109561.403
N1−N4-dicaffeoyl-spermidine4.25C25H31N3O6468.21401468.214511.068
N1−N8-caffeoyl-feruloyl-spermidine4.37C26H33N3O6482.22966482.230561.866
Table 3. Quantification of targeted phenolic compounds in the analyzed samples Niselo e Delishe, determined by UHPLC-Q-Orbitrap high-resolution mass spectrometry (HRMS). Results are expressed as mean values (mg/100 g) ± standard deviation (SD).
Table 3. Quantification of targeted phenolic compounds in the analyzed samples Niselo e Delishe, determined by UHPLC-Q-Orbitrap high-resolution mass spectrometry (HRMS). Results are expressed as mean values (mg/100 g) ± standard deviation (SD).
Targeted PolyphenolsNiseloDelishe
mg/100 gSDmg/100 gSD
Phenolic acids
Quinic acid0.0550.0040.0140.002
Protocatechiuc acid0.1240.0071.7680.041
Chlorogenic acid0.0910.0040.0680.002
Caffeic acid1.8210.0110.0230.001
Ellagic acid<LOQ-<LOQ-
Ferulic acid1.6960.0030.9230.005
p-coumaric acid0.780.009nf-
Flavanols
Catechin<LOQ-7.2780.052
Epicatechinnf-1.6260.009
Flavanones
Naringin0.0470.0010.0250.001
Naringenin0.5660.0010.5050.008
Flavones
Luteolin-7-glucoside0.0630.0010.040.002
Luteolin0.1010.0010.150.002
Apigenin-7O-glucoside0.0590.0040.0150.001
Apigenin0.0370.0010.0480.001
Flavonols
Rutin hydrate0.230.0070.0880.001
Quercetin-3b-glucoside<LOQ-0.0220.002
Diosmin0.0540.0090.0240.001
Kaempferol-3O-glucoside0.1130.0030.0350.001
Quercetinnf-0.0190.001
Isoflavones
Genistein0.0880.0020.1140.005
Daidzein<LOQ-<LOQ-
Total phenols5.925 3.881
Table 4. Relative area (%) of untargeted polyphenols identified in the analyzed samples. The results are reported as average peak area and average relative area (%).
Table 4. Relative area (%) of untargeted polyphenols identified in the analyzed samples. The results are reported as average peak area and average relative area (%).
Untargeted PolyphenolsAverage AreaAverage Relative Area (%)
NiseloDelisheNiseloDelishe
1-O-coumaroylglycerol21,486,46110,187,7175.32.0
1-O-caffeylglycerol4,795,1842,084,4331.20.4
2-O-caffeoylglycerol52,248,57019,312,52812.93.8
7,3′,4′-trihydroxyflavone8,351,78011,941,9172.12.4
7,3′,4′,5′-tetrahydroxy flavanone69,2231,420,2050.00.3
eriodictyol17,480,52213,144,4424.32.6
chrysoeriol3,610,6673,530,9210.90.7
dihydroxy-octadecadienoic acid11,826,43227,447,2412.95.4
dihydroxy-octadecenoic acid36,843,09386,826,8899.117.1
trihydroxy-octadecenoic acid84,007,160264,078,43220.852.0
Caffeic acid hexoside5,772,4585,237,4931.41.0
saccharide23,069,16710,844,0735.72.1
1,3-O-coumaroyl-caffeoyl-glycerol8,928,7931,800,4122.20.4
1,3-O-coumaroyl-feruloyl-glycerol8,080,5413,253,1652.00.6
1,3-O-dicaffeoylglycerol18,495,4613,149,4854.60.6
1-O-caffeoyl-2-O-glucosylglycerol I6,076,5811,499,8771.50.3
1-O-caffeoyl-2-O-glucosylglycerol II119,434114,5680.00.0
naringenin hexoside I41,455,32620,711,02010.34.1
naringenin hexoside IINF1,237,2240.00.2
naringenin hexoside III1,947,173877,9970.50.2
1,3-O-diferuloylglicerol1,207,265407,7200.30.1
luteolin hexoside I5,720,9913,037,2261.40.6
luteolin hexoside II9,129,5173,457,0702.30.7
eriodictyol-O-hexoside17,111,8966,613,6714.21.3
N1−N4-dicaffeoyl-spermidine14,462,0085,082,6843.61.0
N1−N8-caffeoyl-feruloyl-spermidine1,352,112349,6130.30.1
Table 5. Results of the proteomic analysis performed on the Niselo and Delishe samples include the identified peptide sequences, which are mapped to their corresponding proteins, providing a comprehensive overview of the protein composition in both samples.
Table 5. Results of the proteomic analysis performed on the Niselo and Delishe samples include the identified peptide sequences, which are mapped to their corresponding proteins, providing a comprehensive overview of the protein composition in both samples.
ProteinPeptideAverage Scaled Abundance (%)Abundance Ratio (Niselo/Delishe)
Accession NumberDescriptionOrganismMolecular Weight (kDa)Biological FunctionMatching Peptides SequenceProtein Coverage (%)Unique PeptidesNiseloDelishe
A0A921Q906Subtilisin-chymotrypsin inhibitor-2ASorghum bicolor8Stress response[K].DKPDADIFVLPVGSPVTR.[D]603147.752.32.901
[R].IFVDTVAETPR.[V]
[K].QSWPEVVGLSVEEAK.[K]
A0A921QAA6Subtilisin-chymotrypsin inhibitor CI-1BSorghum bicolor8Stress response[K].DMPNAYIQVLPVGSPVTLDIRPDR.[V]603134.165.92.479
[K].DMPNAYIQVLPVGSPVTLDIRPDR.[V]
[K].TSWPEVLGMSIK.[E]
[K].EATEIILK.[D]
A0A1Z5R5E6Non-specific lipid-transfer proteinSorghum bicolor11.5Transport[R].GISGLNAGNAASIPSK.[C]403120.179.91.524
[R].GQGSAPSAGCCSGVR.[S]
[K].CGVSVPYTISTSTDCSR.[V]
C5YBX1Glutaredoxin domain-containing proteinSorghum bicolor13.4Stress response[K].AIELDVESDGPELQNALK.[E]383156.843.22.588
[K].LVPLLTEAGAIAGSTSK.[T]
[K].EIVASAPLVVFSK.[T]
C5XQX6Bowman-Birk serine protease inhibitors family domain-containing proteinSorghum bicolor9-[K].ECTSWSGVYTCDDLLTK.[C]322132.167.91.818
[R].DFLPEGCPCK.[T]
Q6UBQ8Elongation factor TuLacticaseibacillus rhamnosus25.9Protein metabolism[K].TLDLGEAGDNVGVLLR.[G]30466.7133.30.491
[R].DLLTEYDYPGDDIPVVR.[G]
[K].VGDEVEIVGLVDK.[V]
[K].SVVTGLEMFHK.[T]
[R].QVGVNYIVVFLNK.[C]
A0A1B6PL34Uncharacterized proteinSorghum bicolor23.5Cell organization and biogenesis; protein metabolism; other metabolic processes; stress response[R].LPENADLDSVAASLDNGVLTVR.[F]273152.647.53.013
[R].DEAAAVSPLSDVGLLADPFR.[I]
[R].ETPDAHEIVVDVPGMR.[R]
A0A921TZF3Starch synthase, chloroplastic/amyloplasticSorghum bicolor66Other metabolic processes[R].FAFSDFPELNLPER.[F]2613108.491.61.367
[K].EALQAEVGLPVDR.[NK]
[R].LSVDCNVVEPADVK.[K]
[R].FSLLCQAALEAPR.[I]
[K].DAWDTSVVSEIK.[MX]
[R].VLTVSPYYAEELISGIAR.[G]
[R].GCELDNIMR.[L]
[RK].NCMIQDLSWK.[G-V]
[K].YDVSTAVEAK.[A]
[K].VVGTPAYEEMVK.[N]
[K].IYGPDAGTDYK.[D]
[K].IPLVAFIGR.[L]
[R].FEPCGLIQLQGMR.[YV]
[K].VVGTPAYEEMVK.[N]
C5YDE5OleosinSorghum bicolor16.2Developmental processes;other biological processes[R].GGTGGGAGGYGDYNR.[G]253112.787.31.258
[R].GGGAGMYGESQQQQQK.[Q]
[K].QGAMMTAIK.[A]
A0A1Z5SBV8Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain-containing proteinSorghum bicolor13.1Transport[K].TVASCGVALPR.[C]172115.884.31.258
[K].AQQGCLCQFAK.[N]
A0A1D6K7T5ChitinaseZea mays22.6Other metabolic processes; stress response[R].ELAAFFGQTSHETTGGTR.[G]15201000.01
[R].GAADQFQWGYCFK.[E]
A0A921V3I9SHSP domain-containing proteinSorghum bicolor26.9Stress response[K].VMVEDDTLVIR.[G]143118.981.11.443
[R].LFDDAVGFPMATR.[R]
[R].LPWDIVEDDK.[E]
A0A317YKC7Superoxide dismutaseZea mays18.7Other metabolic processes;other biological processes[K].AVAVLGSSEGVK.[G]142154.245.81.817
[R].AVVVHADPDDLGK.[GD]
A0A3L6DUH5Ubiquitin-40S ribosomal protein S27aZea mays20.9Protein metabolism; other metabolic processes[K].TITLEVESSDTIDNVK.[AS]132115.684.41.339
[K].ESTLHLVLR.[L]
A0A1W0VZJ3Phytocyanin domain-containing proteinSorghum bicolor26.6-[R].SGPFFFISSDEDR.[C]112126.473.51.757
[R].LQAAAVGSSSGSSVLR.[L]
C5X3B9PeroxiredoxinSorghum bicolor24.2Other biological processes[R].AVIAPSVSDEEAR.[K]112136.263.82.049
[K].VTYPILADPGR.[D]
A0A3L6EJI2Heat shock protein, mitochondrialZea mays30.4Stress response[R].VAVEDGVLVIEGEK.[R]92134.865.22.125
[K].DGVLYVTVPR.[T]
A0A7S7FP27Glyceraldehyde-3-phosphate dehydrogenaseLacticaseibacillus rhamnosus36.7Other metabolic processes[R].VYAEPQAQNIPWVK.[N]8253.2146.80.363
[K].AIGLVIPELNGK.[L]
A0A3L6ELN6Phosphoglycerate kinaseZea mays42.4Other metabolic processes[K].ELDYLVGAVANPK.[K]7217.8182.20.102
[K].GVTTIIGGGDSVAAVEK.[VA]
C5X0T3Cupin type-1 domain-containing proteinSorghum bicolor57.8-[R].TLLGPEIAAAFGAR.[E]73127.472.61.840
[R].GGPFEFFGFTTSAR.[R]
[R].NSYGWTVSVDK.[H]
A0A1D6PKZ8Histone H2BZea mays25.7-[K].QVHPDIGISSK.[A]7268.7131.30.520
[K].SVETYK.[I]
A0A921RTP7EF-hand domain-containing proteinSorghum bicolor46.2-[R].EADVDGDGQINYEEFVK.[VM]7265.1134.90.480
[K].DQNGFISAAELR.[H]
A0A1D6HCF4Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating)Zea mays55.7Other metabolic processes[R].VPTVDVSVVDLTVR.[IL]6229.8170.20.158
[RK].AASFNIIPSSTGAAK.[AVLG]
A0A1D6IAS7UTP--glucose-1-phosphate uridylyltransferaseZea mays59.5Other metabolic processes[K].VLQLETAAGAAIR.[FSV]5201000.010
[K].SIPSIVELDSLK.[VS]
A0A921UHX0Plant antimicrobial peptide domain-containing proteinSorghum bicolor52.2Stress response[K].ACEWQYGEDTPR.[K]42129.970.11.564
[R].YEDQPWR.[T]
A0A3L6DXC0Histone H4Zea mays106.8-[R].ISGLIYEETR.[GR]22127.172.92.096
[K].TVTAMDVVYALK.[R]
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Katerere, D.R.; Navarré Dopazo, A.; Sessa, R.; Trombetti, S.; Grosso, M.; Izzo, L. Evaluation of In Vitro Cytoprotective Activity, Antioxidant Activity and Proteomic Profiles of Novel Sorghum-Based Fermented Beverages. Beverages 2026, 12, 9. https://doi.org/10.3390/beverages12010009

AMA Style

Katerere DR, Navarré Dopazo A, Sessa R, Trombetti S, Grosso M, Izzo L. Evaluation of In Vitro Cytoprotective Activity, Antioxidant Activity and Proteomic Profiles of Novel Sorghum-Based Fermented Beverages. Beverages. 2026; 12(1):9. https://doi.org/10.3390/beverages12010009

Chicago/Turabian Style

Katerere, David R., Abel Navarré Dopazo, Raffaele Sessa, Silvia Trombetti, Michela Grosso, and Luana Izzo. 2026. "Evaluation of In Vitro Cytoprotective Activity, Antioxidant Activity and Proteomic Profiles of Novel Sorghum-Based Fermented Beverages" Beverages 12, no. 1: 9. https://doi.org/10.3390/beverages12010009

APA Style

Katerere, D. R., Navarré Dopazo, A., Sessa, R., Trombetti, S., Grosso, M., & Izzo, L. (2026). Evaluation of In Vitro Cytoprotective Activity, Antioxidant Activity and Proteomic Profiles of Novel Sorghum-Based Fermented Beverages. Beverages, 12(1), 9. https://doi.org/10.3390/beverages12010009

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