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Article

Effects of Natural Fermentation Time on Chemical Composition, Antioxidant Activities, and Phenolic Profile of Cassava Root Flour

by
Oluwaseun Peter Bamidele
Department of Food Science and Technology, University of Venda, Thohoyandou 0950, South Africa
Appl. Sci. 2025, 15(15), 8494; https://doi.org/10.3390/app15158494
Submission received: 23 June 2025 / Revised: 23 July 2025 / Accepted: 28 July 2025 / Published: 31 July 2025

Abstract

This study aimed to determine the impact of natural fermentation time on the chemical composition and antioxidant activities of cassava flour. Samples of flour were fermented for intervals of 12, 24, and 48 h and compared with the control (0 h). The results indicated clear differences in the chemical composition of these samples. The pH value was reduced, TTA increased, and TSS decreased. This is due to the action of lactic acid bacteria during fermentation. The TPC value also increased with fermentation time, achieving 2.95 mg GAE/g after 48 h, compared to 1.35 mg GAE/g initially. Antioxidant activities improved significantly; total antioxidant capacity surged from 23.50 µmol TE/g to 69.81 µmol TE/g over the 48 h fermentation period, based on ABTS, DPPH, and FRAP assays. Protein content also improved significantly, increasing from 1.82% to 3.10%, while the hydrogen cyanide content declined from 25.14 mg/100 g to 5.34 mg/100 g, signifying reduced nutritional risk. An increase in minerals was also noted, with calcium showing the highest concentration of 41.35 mg/100 g after 48 h of fermentation. These findings demonstrate the effectiveness of fermenting cassava flour by enhancing its chemical composition and antioxidant properties while lowering antinutrients, which improves its value in functional foods.

1. Introduction

In some parts of Africa, Latin America, and Asia, cassava (Manihot esculenta Crantz) is a critically important starchy root crop [1]. Cassava is capable of fitting into the region’s agricultural infrastructure. However, cassava roots lack proteins, vital micronutrients, and bioactive compounds essential to human health [2]. Cassava also contains several antinutritional factors, the most notable of which are cyanogenic glycosides, which can pose dangers if not appropriately processed [3]. Processing methods such as fermentation (natural or controlled) have helped to mitigate risks associated with hydrogen cyanide and improve the nutritional value of cassava root flour and its functional properties [4].
Fermentation is a metabolic process in which microorganisms like yeast or bacteria convert sugars into acids, alcohol, or gases under anaerobic conditions [5]. There are four types of fermentation methods (lactic acid, alcoholic, acetic acid, and mixed fermentation). Natural fermentation is often categorized under lactic acid fermentation [6].
Natural fermentation is a biological transformation in which wild yeasts and bacteria metabolize sugars, producing alcohol, acid, or gas and in the process, altering a food’s flavor, aroma, and shelf life. This procedure begins spontaneously, drawing on microorganisms already living on the ingredient itself or floating in the surrounding air, and it does not depend on a supplied starter culture [7]. The time frame of fermentation is a significant factor in detoxifying cyanogenic glycosides, starch hydrolysis, and changes in phytochemicals that occur during the fermentation period [8]. Nutrient bioavailability and the functionality and sensory properties of cassava-based products can be significantly improved after fermentation. During fermentation, biochemical changes include modulating radical scavenging entities such as total phenolics, flavonoids, and other bioactive molecules [9].
The fermentation of cassava is supported by a wide range of microorganisms, especially lactic acid bacteria (LAB) and other yeasts and fermenting microbes that occur naturally in cassava [10]. This technique is beneficial from an economic and ecological standpoint and is practiced by many societies in which cassava is a staple food.
The fermentation processes for cassava root flour take place through different stages, consisting of several biochemical changes that impact the chemical profile of the flour [11]. Different types of fermentation can hydrolyze and enzymatically degrade complex carbohydrates, proteins, and fibers into simpler constituents [12]. It has been documented that fermentation processes enhance cassava-based products’ nutritional value and safety by lowering the antinutritional factors such as cyanogenic glycosides, phytates, and tannins [13].
Along with enhancing the digestibility of macronutrients and phytochemicals, natural fermentation also changes the antioxidant properties of cassava root flour. Microbial actions during fermentation may release bound phenolic compounds alongside other bioactive metabolites, thus enhancing the antioxidant activity [14]. These changes support the demand for functional foods containing natural antioxidants, improving fermented cassava products’ health benefits [15]. Neutralizing oxidative stress, which is associated with chronic conditions like cardiovascular disease, diabetes, and neurodegenerative diseases, requires antioxidants [16]. With the emphasized benefits of fermentation processes, which include the bioavailability of nutrients in the body during digestion and the boosting of antibodies, there seems to be a gap in knowledge regarding how different fermentation times and conditions impact the biochemical constituents and the antioxidant properties of cassava root flour. Most of the available literature focuses on controlled fermentation processes, with little or no attention given to the effects of natural fermentation. Therefore, this study aims to determine the effects of natural fermentation with varying time intervals on cassava roots’ flour chemical composition, antinutritional factors, antioxidant activity and phenolic profile.

2. Materials and Methods

Cassava root was purchased from a local farmer in Offa, Kwara State, Nigeria, and transported to the Pilot Plant in the Department of Food Technology, Federal Polytechnic Offa, Kwara State, Nigeria. All the chemicals used for the analyses were standard grade.

2.1. Sample Preparation

The cassava root was washed with clean water before manual peeling. The peeled cassava roots were diced into smaller pieces before natural fermentation (10:00 local time in Nigeria). The methods of Oyeyinka et al. [17] were used to ferment the cassava roots. Unfermented cassava roots served as the control, and the sample fermentation was terminated after 12, 24, and 48 h, respectively. After fermentation, the fermented cassava root was mashed by hand and dried using a hot air oven. The dried cassava root was milled (Lab Hammer mill, LHM-A110/LHM-S110, SciTek, Jinan, China) and sieved using a mesh size of 500 μm. The cassava root flour was packed inside zippered plastic bags and stored at 8 °C for analysis.

2.2. Methods

2.2.1. Determination of pH, Total Soluble Solids (°Brix), and Total Titratable Acidity (TTA)

A pH meter (Hanna Instrument, Poroa de Varzim, Portugal) was used to determine the pH of the samples. Total soluble solids were determined in °Brix using a handheld refractometer previously adjusted to zero with distilled water (Hanna Instruments, Villafranca Padovana, Italy). The titratable acidity of the samples was determined using the AOAC method [18].

2.2.2. Determination of Crude Protein, In Vitro Protein Digestibility (IVPD), and Vitamin C

The crude protein was determined using the AOAC [18] Official Method 991.20. A 2 g sample was added to the digestion flask. A total of 1 g of copper sulfate, along with sodium sulfate (catalyst) in the ratio 1:10, respectively, and 5 mL of concentrated sulfuric acid were also added to the digestion flask. The flask was positioned into the digestion block in the fume cupboard and heated until frothing ceased, giving a clear and light blue-green colouration. The mixture was then allowed to cool and diluted with 30 mL of distilled water, and 30 mL of 40%NaoH was added. The distillation apparatus was connected. The ammonia released by boric acid was then treated with 0.01 mL of hydrochloric acid until the green colour changed to purple. The percentage of nitrogen in the sample was calculated using the following formula:
% Nitrogen ( N )   =   V H C l × N H C l × 14 a m p l e   w e i g h t   ×   100
Note: volume of HCl consumed to the endpoint of titration (V), normality of HCl used is about 0.01 N(N), and molecular weight is that of nitrogen [16]. Conversion of nitrogen percentage into % protein = F%N, where F is the conversion factor (in most cases, 6.25).
The IVPD was determined using the method of Awobusuyi et al. [19]. About 100 mg of the fermented cassava root flour was measured into a 50 mL centrifuge tube. A total of 35 mL of 0.1 M phosphate buffer containing freshly prepared pepsin at a concentration of 1.5 mg/mL was introduced into the tube. The sample was then incubated in a shaking water bath at 37 °C for 2 h to allow enzymatic digestion. Following the incubation, digestion was halted by the addition of a 2 M NaOH solution. The resulting suspension underwent centrifugation at 4800 rpm for 20 min at 4 °C. The supernatant was removed, and the remaining pellet was rinsed with 15 mL of 0.1 M phosphate buffer (pH 7). Another round of centrifugation was performed, after which the pellet was separated by filtration using Whatman No. 3 filter paper, with the filtrate being discarded. The residual undigested protein on the filter paper was then dried at 80 °C for 2 h, and its protein content was quantified using the Dumas combustion method.
The quantification of vitamin C (L-ascorbic acid) was conducted using the dye-titration technique, as outlined in AOAC 967.21. The ascorbic acid content in fermented cassava root flour samples was assessed following the AOAC [18] protocol. Approximately 20 mg of cassava root flour was diluted to a final volume of 50 mL using 0.1 M oxalic acid, which acted as a metal chelator, and the mixture was subsequently filtered. A 5 mL aliquot of the filtrate was transferred into a beaker using a pipette and titrated against a standardized 2,6-dichlorophenol indophenol dye solution. The endpoint of the titration was indicated by a color change from orange to pink. The titration procedure was performed in triplicate, and the obtained titer values were used to calculate the ascorbic acid concentration, expressed as mg per 100 g of fermented cassava root flour.
Determination of antinutritional factors of the cassava root flour. The antinutrients phytate, hydrogen cyanide (915.03), and trypsin inhibitors in the cassava root flour were determined using the AOAC rapid test method [18].

2.2.3. The Total Phenolic Content (TPC), ABTS, DPPH, FRAP

The total phenolic content (TPC) was measured using a modified method from Tezcan et al. [20]. First, 300 µL of a diluted fermented cassava root flour extract (made by mixing 1 mL of the sample with 100 mL of water) was mixed with 1.5 mL of diluted Folin–Ciocalteu reagent (5 times dilution), 1.2 mL of a 7.5% sodium carbonate solution, and a methanol–water mixture (6:4 ratio). This mixture was left at room temperature for 60 min. Subsequently, the absorbance was measured at 760 nm using a Hitachi Model 100-20 spectrophotometer (Hitachi, Tokyo, Japan). The results were reported as milligrams of gallic acid equivalent per milliliter (mg GAE/mL).
To measure antioxidant activity, the ABTS+ radical scavenging test was used, following a modified version of the method by Awika et al. [21], adapted for 96-well plates. The ABTS+ stock solution (7 mM) was prepared by mixing equal parts of ABTS (8 mg/mL in water) and potassium persulfate (2.45 mM in water), and the mixture was left in the dark at room temperature for 12–16 h. This stock was diluted 1:29 with phosphate-buffered saline (PBS, pH 6.9) to obtain a working solution. A standard curve was created using Trolox at different concentrations (0 to 800 µM, in methanol). For the test, 10 µL of the sample (prepared for HPLC) was mixed with 190 µL of ABTS+ working solution in a microplate well and kept in the dark for 30 min. Absorbance was then measured at 750 nm using a Multiscan FC reader. The antioxidant activity was calculated by comparing the absorbance drop with the Trolox standard and expressed as micrograms of Trolox equivalent per milliliter (µg TE/mL).
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was determined following a slightly modified version of the method by Noreen et al. [22]. A 130 µL portion of the sample was mixed with a 0.1 mM DPPH solution and kept in the dark for 30 min. The absorbance was then measured at 510 nm. Control samples were prepared similarly, substituting methanol for the test sample, and methanol alone was used as the blank. The percentage of DPPH radical scavenging was calculated using the appropriate formula.
DPPH   Scavenging   % = ( A c o n t r o l A s a m p l e ) A c o n t r o l × 100
The ferric reducing antioxidant power (FRAP) assay was carried out using a modified version of the method developed by Benzie and Strain [23]. About 2 mg of the sample extract was mixed with 1 mL of FRAP reagent. This reagent was obtained by combining a 300 mM sodium acetate buffer (pH 3.6), a 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) solution, and a 20 mM FeCl3·6H2O solution in a 10:1:1 ratio. The mixture was then diluted with water to reach a total volume of 4 mL. It was incubated in a water bath at 37 °C for 30 min. After incubation, the absorbance was read at 593 nm using a Hitachi Model 100-20 spectrophotometer (Hitachi, Tokyo, Japan).

2.3. Determination of Mineral Content of the Cassava Root Flour

The mineral content of the samples was determined using the AOAC-2011.14. Minerals (calcium, magnesium, iron, and zinc) of the fermented cassava root flour were determined by employing the AOAC [18] method on the digestion of the sample with a mixture of concentrated nitric acid, sulfuric acid, and perchloric acid (10:0:5:2, v/v) using an atomic absorption spectrophotometer (GBC 904AA; Berlin, Germany). The absorbance was read at 880 nm (Spectronic 21 D, Miltonroy, Ivyland, PA, USA), and KH2PO4 (Merck, India Limited, Mumbai, India) served as the standard.

2.4. GC–MS/MS Analysis of Phenolic Acids in Cassava Root Flour

2.4.1. Sample Preparation

To extract phenolic acids, 100 mg of cassava root flour was added to 1 mL of 70% methanol in a test tube. The mixture was vortexed and incubated at 60 °C for 3 h. Following extraction, 130 µL of the supernatant was freeze-dried overnight. The dried residue was then derivatized with 30 µL of N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 µL of acetonitrile at 60 °C for 30 min. One microliter of the derivatized sample was injected into the GC-MS/MS system in splitless mode using an insert placed in a 2 mL GC vial.

2.4.2. Chromatographic Separation

Phenolic acids were separated using a gas chromatograph (Trace 1300, Thermo Scientific, Waltham, MA, USA) coupled to a triple quadrupole mass spectrometer (TSQ 8000, Thermo Scientific, Waltham, MA, USA). The separation was performed on a non-polar Rxi-5Sil MS capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness), with helium as the carrier gas at a constant flow rate of 1 mL/min. The injector temperature was set to 250 °C, and the injection was performed in splitless mode. The oven temperature program was as follows: initial temperature of 100 °C held for 4 min, ramped to 180 °C at 10 °C/min and held for 2 min, then increased to 320 °C at 20 °C/min and held for 5 min. The mass spectrometer operated in selected reaction monitoring (SRM) mode. The ion source and quadrupole temperatures were maintained at 250 °C and 150 °C, respectively, with the transfer line temperature also set to 250 °C. Phenolic compounds are identified by analyzing their unique fragmentation patterns and retention times. The resulting mass spectra are compared with known standards to identify the compounds.

2.5. Scanning Electron Microscopy of Cassava Root Flour

The microstructure of cassava root flour was examined using scanning electron microscopy (JSM 6610-LV, JEOL, Chicago, IL, USA), following the methods of Murungweni et al. [24]. Prior to imaging, the samples were coated with a thin layer of gold–palladium and mounted on stubs. SEM imaging was conducted at a magnification of 1000× with a scale of 50 μm.

2.6. Pasting Properties of Cassava Root Flour

Pasting characteristics were assessed using a Rapid Visco Analyzer (RVA) Series 4 (Newport Scientific, Warriewood, NSW, Australia). Approximately 3 g of cassava flour was combined with 25 mL of distilled water in an RVA canister. The paddle was placed centrally, and the canister was inserted into the RVA. The standard 14 min profile was applied, beginning with a 1 min hold at 50 °C, heating to 91 °C over 4 min, holding at 91 °C for 3 min, cooling to 50 °C over 4 min, and holding again at 50 °C for 2 min. All measurements were conducted in triplicate.

2.7. Statistical Analysis

One-way analysis of variance (ANOVA) was used to analyze the results generated from the study using IBM SPSS version 25. All the analyses were carried out in triplicate.

3. Results and Discussions

3.1. The pH, TTA, TSS, Crude Protein, IVPD, Vitamin C, and Antinutritional Factors

The results in Table 1 demonstrate the progressive biochemical and nutritional transformations occurring in cassava root flour during natural fermentation over a 48 h period. The results of pH, total titratable acidity (TTA), total soluble solids (TSS), crude protein, in vitro protein digestibility (IVPD), vitamin C content, and antinutritional factors (phytates, hydrogen cyanide, and trypsin inhibitor activity) provide key insights into the functional and nutritional implications of fermentation.
There was a significant decrease in pH from 6.50 (control) to 4.20 (48 h), indicating increased microbial activity and acid production during fermentation. This acidification is attributed to the metabolic activity of lactic acid bacteria (LAB) and yeasts, which ferment available carbohydrates into organic acids, primarily lactic acid, during fermentation. The corresponding increase in TTA (from 0.12% to 0.81% lactic acid) further confirms the acidification process. The total soluble solids (TSS), representing simple sugars and soluble compounds, decreased significantly from 5.82 °Brix (control) to 2.12 °Brix (48 h). This reduction is expected, as fermentative microorganisms utilize sugars as an energy source for growth and metabolism, producing organic acids and bioactive compounds as by-products.
There was a notable increase in crude protein content from 1.82% in the control to 3.01% after 48 h of fermentation. This enhancement in crude protein content may be attributed to microbial biomass accumulation [25]. This finding is in line with the findings of Terefe et al. [26], who reported an increase in crude protein of solid-state fermented maize flour.
In vitro protein digestibility (IVPD), an indicator of protein bioavailability, increased significantly from 47.32% in unfermented cassava root flour (control) to 78.51% at 48 h. This improvement can be attributed to proteolytic enzyme activity from fermenting microbes, which break down complex proteins into peptides and free amino acids [27]. Additionally, fermentation reduces protein-bound antinutrients, further enhancing digestibility [28]. This result is similar to that reported by Anyiam et al. [29], who reported an increase in the in vitro protein digestibility of fermented cassava mahewu after 42 h.
The vitamin C content of the samples (unfermented and fermented cassava root flour) increased from 20.52 mg/100 g in the control to 35.23 mg/100 g at 48 h. The increase in the vitamin C content may be due to microbial synthesis of ascorbic acid by certain bacteria and yeasts and increased enzymatic conversion of precursor molecules [30]. The improved vitamin C content indicates fermentation’s potential to enhance cassava flour’s nutritional profile, particularly in regions with prevalent vitamin C deficiencies.
Natural fermentation significantly decreased phytates, hydrogen cyanide (HCN) levels, and trypsin inhibitor activity (TIA), all major antinutritional factors in cassava roots. Phytates, known for chelating essential minerals and reducing bioavailability, decreased from 205.43 mg/100 g (control) to 72.72 mg/100 g (48 h). This reduction is linked to the enzymatic action of microbial phytases, which hydrolyze phytate into lower inositol phosphates and free phosphorus [31]. Hydrogen cyanide, a toxic compound in raw cassava, showed a significant decline from 25.14 mg/100 g (control) to 5.34 mg/100 g (48 h). This detoxification occurs due to microbial linamarase activity, which facilitates the enzymatic hydrolysis and volatilization of cyanogenic glycosides [32]. Trypsin inhibitor activity (TIA), which interferes with protein digestion, decreased from 19.57 TIU/mg in the control to 5.45 TIU/mg after 48 h. The reduction is likely due to microbial proteases degrading trypsin inhibitors, thus improving protein bioavailability and digestibility [33].

3.2. Total Phenolic Contents, Antioxidant Activities, and Mineral Content

The results of total phenolic content (TPC), antioxidant activities (ABTS, DPPH, FRAP), and mineral composition (calcium, magnesium, iron, and zinc) in cassava root flour over different fermentation durations (0 h, 12 h, 24 h, and 48 h) are shown in Table 2. The results highlight significant improvements in TPC, antioxidant properties, and mineral content, suggesting that natural fermentation enhances cassava root flour’s nutritional and functional qualities. It should be noted that the TPC validity is shown in relation to other complementary indirect antioxidant activities such as DPPH, FRAP, and ABST that are determined in this study.
Phenolic compounds are natural antioxidants that contribute to a plant-based foods’ functional and health benefits [34]. The TPC increased from 1.35 mg GAE/g in the control to 2.97 mg GAE/g at 48 h of fermentation. This enhancement can be attributed to microbial enzyme activity (e.g., polyphenol oxidases and glucosidases), which break down bound phenolic compounds into their free, more bioavailable forms [35]. Fermentative breakdown of complex carbohydrates, which release bound phenolic compounds, leads to an increase in TPC [9].
A similar trend was observed for antioxidant activities, measured using ABTS, DPPH, and FRAP assays. ABTS radical scavenging activity increased from 23.50 µmol TE/g (control) to 69.81 µmol TE/g at 48 h, indicating an improvement in the overall antioxidant potential of cassava root flour. The DPPH radical scavenging activity (IC50 values) increased progressively from 40.33 µg/mL (control) to 92.61 µg/mL after 48 h, suggesting a more substantial capacity to neutralize free radicals. The FRAP (ferric reducing antioxidant power) values followed a similar pattern, increasing from 35.23 µmol Fe2+/g in the control to 82.30 µmol Fe2+/g after 48 h, indicating improved reducing power and metal ion chelation ability. The increase in the antioxidant activities of the fermented cassava root flour may be attributed to an increase in the TPC, which involves the action of microorganisms in breaking down bound polyphenols in the samples. These findings suggest that natural fermentation enhances the antioxidant potential of cassava root flour, making it a more valuable ingredient for functional foods and nutraceutical applications.

3.3. Mineral Content

The fermentation process of the cassava root flour improved the bioavailability of essential minerals such as calcium, magnesium, iron, and zinc. Calcium content, essential for bone health and enzymatic functions, increased from 32.50 mg/100 g (control) to 41.35 mg/100 g at 48 h [36]. Magnesium, a crucial cofactor for enzymatic reactions, showed a gradual increase from 21.81 mg/100 g to 26.12 mg/100 g, indicating improved availability. The iron content increased from 5.42 mg/100 g (control) to 6.82 mg/100 g at 24 h, stabilizing afterwards, highlighting fermentation’s role in enhancing iron solubility and reducing inhibitory compounds, and the zinc content, which plays a vital role in immune function and enzyme regulation, increased steadily from 1.74 mg/100 g to 2.62 mg/100 g after 48 h of fermentation. The increase in the mineral content of the naturally fermented cassava root flour may be attributed to phytate reduction, as previously demonstrated in Table 1, which reduces mineral chelation and enhances bioavailability [37]. Also, cassava’s microbial metabolism and structural breakdown during fermentation increase mineral extractability and solubility [12]. This finding is in line with the work of Onyango et al. [38], who reported an increase in zinc content of fermented cassava root.
The findings from this study demonstrate that natural fermentation is an effective technique for improving cassava root flour’s nutritional quality. The progressive acidification during fermentation, enhanced protein digestibility, increased vitamin C levels, and significant reduction in anti-nutrients highlight its potential as a sustainable bio-processing approach. The observed trends suggest that longer fermentation times (48 h) may be optimal for maximizing nutritional benefits. The enhancement of phenolic compounds, antioxidant activity, and mineral content in fermented cassava flour suggests its potential use in functional foods and dietary supplements, especially for populations at risk of micronutrient deficiencies.

3.4. Pasting Properties of Naturally Fermented Cassava Root Flour Using Different Fermentation Times

All the parameters of the pasting properties underwent a uniform decline with the increase in fermentation time for cassava root flour from 0 h (control) to 48 h, as depicted in Table 3. As pointed out previously, peak viscosity, which is the lowest value of rapid Visco units (RVU) recorded for 48 h of fermentation, decreased significantly from 420.3 ± 4.2 RVU (control) to 329.5 ± 3.7 RVU. The increase in viscosity peak significantly diminished commensurate with time, culminating in a 21.6% decrease when compared to the control sample, which is suggestive of starch granule or sequential amylose chain degradation, likely due to enzymatic, i.e., amylolytic activity, during fermentation. This has been extensively documented in the literature as a characteristic of fermentation, particularly in the case of cereals or tuber starches [39,40].
The trough, which is the lowest point of viscosity reflecting the minimum viscosity after peak value during constant heating [41], showed a similar declining trend (from 281.5 ± 3.7 RVU at 0 h to 239.3 ± 3.1 RVU at 48 h). Breakdown, calculated as the difference between peak viscosity and trough, reflects the stability of swollen granules when exposed to shear and heat.
Breakdown reduced from 138.8 ± 2.8 RVU (control) to 90.2 ± 1.9 RVU (48 h), a 35% decrease. This indicates greater resistance to disintegration induced by shear, most likely due to partial hydrolysis of starch molecules resulting in smaller, more stable granules.
Final viscosity, which is the viscosity after cooling and indicates the extent of gel formation, was also reduced from 372.2 ± 3.1 RVU (control) to 318.4 ± 3.2 RVU (48 h). The setback, the difference between the final viscosity and trough, indicates retrogradation and amylose re-association of the sample. Setback decreased from 90.7 ± 2.2 RVU to 79.1 ± 2.1 RVU, indicating that retrogradation was lower in the fermented samples. This is likely due to the reduction in amylose chains during fermentation, where they were converted to shorter disassociating dextrin, which limits molecular re-association upon cooling [42].
The change in pasting temperature (the temperature at which viscosity begins to increase) ranges from 72.1 ± 0.3 °C (control) to 69.8 ± 0.3 °C (48 h). This change indicates that the energy necessary for starch gelatinization is likely lower during fermentation due to a partial hydrolysis of starches, which facilitates water absorption. Peak time, or time to reach peak viscosity, also reduced slightly from 4.3 ± 0.1 min to 4.0 ± 0.1 min, indicating faster gelatinization in fermented samples.
These changes are likely due to the hydrolytic impact of fermentation with microbial or auto-fermentative enzymes, like α-amylases and glucoamyloglucosidases that polymerize old starch polysaccharides into smaller pieces and oligosaccharides [42]. Fermentation increases the starch gelatinization temperature and enthalpy and reduces the setback viscosity of food samples. This is due to an increase in the relative crystallinity of starch and a decrease in the amorphous region of starch granules by microorganisms. On the other hand, improvement in retrogradation means better suitability for porridge or baked goods, especially when those foods need to have smooth textures and long shelf life. However, lower viscosity might be a problem for some applications requiring high gel strength, i.e., some noodle or dumpling recipes.

3.5. Phenolic Profile of Cassava Root Flour Naturally Fermented Using Different Times (mg/g)

The phenolic compounds present in the naturally fermented cassava root flour with various fermentation durations are presented in Table 4. The data from the table suggests that there is a notable increase in the individual and total phenolic compound concentrations with increasing fermentation time, from 0 to 48 h. All six phenolic compounds (gallic acid, caffeic acid, p-coumaric acid, ferulic acid, vanillic acid, and syringic acid) were found to have a cumulative increase in concentration over time. Gallic acid demonstrated the highest increase, from 1.32 ± 0.1 mg/g (control) to 2.74 ± 0.1 mg/g at 48 h (107.6% increase), followed by caffeic acid (0.84 ± 0.1 mg/g to 2.01 ± 0.1 mg/g, 139.3% increase). p-Coumaric acid, ferulic acid, vanillic acid, and syringic acid also significantly increased by 159.6%, 107.4%, 97.9%, and 97.1%, respectively. The trends shown in the data likely assume that fermentation significantly promotes the liberation or biosynthesis of phenolic compounds, which may result from some form of microbial or enzymatic process.
Gallic and caffeic acids underwent a significant increase, which could be due to their higher solubility or preferential release from bound forms, like glycosides or esters, during fermentation. Ferulic and p-coumaric acids, often bound with the arabinoxylans of the cell walls of cereals, likely increased in bioavailability due to hydrolytic cleavage by microbial esterases or β-glucosidases [43]. The smaller increases noticed in the vanillic and syringic acids could suggest slower release kinetics or lower starting concentrations.
The phenolic content increased from 4.17 ± 0.3 mg/g (control) to 9.11 ± 0.1 mg/g at 48 h, a relative increase of 118.5%. This significant increase is consistent with the individual contributions of each phenolic acid and suggests that some unmeasured compounds might be released. The pattern most likely indicates fermentation processes breaking more complex phenolic structures, such as tannins or lignins, into simpler identifiable types [34].
The augmentation of phenolic content is likely to be a consequence of microbial interaction that occurs during the fermentation process. Yeasts and lactic acid bacteria (LAB), involved in fermentation, are known to hydrolyze phenolic compounds through the action of feruloyl esterases, β-glucosidases, decarboxylases, and other glycosidic enzymes, yielding free forms [34]. For instance, the release of ferulic acid is generally associated with LAB-mediated cleavage of arabinoxylan–phenolic complexes, together with hydrolysis of the arabinoxylan backbone [44]. Furthermore, it is possible that some phenolic derivatives are synthesized via microbial metabolism, such as gallic acid resulting from the hydrolysis of decarboxylated hydroxycinnamic acids like caffeic acid to dihydrocaffeic acid [45].
The increased bioavailability of phenolics and the resulting antioxidant activities of the functional compounds present in fermented products are significant from a nutritional perspective. The presence of phenolic compounds, which are known as strong antioxidants, can increase the health benefits of the product, acting as an anti-inflammatory and anti-carcinogenic agent. For example, gallic and caffeic acids are noted to exhibit high radical-scavenging activity [46], and ferulic acid enhances gut health by promoting microbial balance in the gut [47]. These phenolic compounds with “chain-breaking antioxidant” properties work by giving a hydrogen atom from their hydroxyl group to a free radical, particularly peroxyl radicals [48]. Higher phenolic content can also be beneficial from a food processing standpoint because it may decrease the oxidative rancidity and improve the shelf life of a product [49].
These results indicate that a 48 h fermentation period seems to increase the release of phenolic content, which can be used to optimize industrial fermentation protocols. However, further extending fermentation time may impact other measures of quality, such as texture or flavor, as noted in a study regarding the fermentation and the pasting properties of starch, where viscosity was inversely proportional to fermentation time [17].

3.6. Scanning Electron Microscopy Images of Naturally Fermented Cassava Root Flour Using Different Fermentation Times

Figure 1A–D presents a series of scanning electron microscopy (SEM) images illustrating the morphological changes in fermented cassava root flour over a 48 h period. These observations provide insights into the structural stability and degradation of starch granules as affected by microorganisms during fermentation. The control sample starch granules remain intact, since there was no fermentation of the sample. Cassava starch granules are typically spherical to oval, with a size range of 5–35 µm (average ~15 µm) [50], although the diameter was not measured in this study. Their smooth surface and truncated shape (Figure 1A) distinguish them from other starches like maize or potato.
After 12 h (Figure 1B), significant morphological changes are observed. The starch granules lose their smooth, oval appearance and begin to exhibit surface irregularities, with some granules appearing shrunken or collapsed. This suggests the onset of structural degradation, possibly due to enzymatic activity or microorganism actions. There was a gradual disintegration of granules. The naturally fermented cassava root flour showed that the degradation process of the starch granules was intensified (Figure 1C). The starch granule surface becomes more irregular, since the enzymes and the microorganism have more resident time to break down bonds and work on the starch granules.
The disintegration of the starch granules and other components of the sample (naturally fermented cassava root flour) continued at 48 h of fermentation (Figure 1D). The complete loss of structural integrity indicates advanced degradation, likely due to the combined effects of microbial activity and enzymatic actions. At this stage, the starch granules are broken into smaller parts due to the breaking of bonds (α-1,4 and β-1,6 glycosidic bonds). Fermenting the cassava root increases the digestion rate and promotes good health [5]. Therefore, it aids the prebiotic potential for carbohydrates. These findings are in line with the report of Oyeyinka et al. [17], who fermented cassava starch for 72 h.
The SEM images reveal a progressive decline in cassava root flour’s granule integrity over 48 h, from intact starch granules at 0 h to severely degraded structures by 48 h. These findings emphasize the susceptibility of cassava root flour granules under suboptimal conditions and provide a foundation for improving its cultivation in Africa, where food insecurity is an ongoing problem. Further studies are needed to correlate these morphological changes in cassava root flour granules with those of other root crops.

4. Conclusions

The results underscore the importance of time and natural fermentation as a cost-effective, scalable bioprocessing technique that enhances the nutritional value, total phenolic content, phenolic profile, antioxidant capacity, and mineral content of cassava root flour. Differences in fermentation time display positive effects on the quality of cassava root flour. The increase in fermentation time improves cassava root flour’s nutritional qualities and health-promoting properties and decreases peak viscosity and final viscosity during pasting. There is an increase with time in the phenolic profile of the fermented cassava root flour, indicating good health-promoting properties with an increase in fermentation time. These findings are particularly relevant for food industries, small-scale processors, and communities dependent on cassava root flour as a staple food.
One of the limitations of this study is that different varieties of cassava root may exhibit different results. Also, fermentation environments may affect the action of the microorganisms. Therefore, it is recommended that the variety of the cassava roots used must be determined, and the fermentation environment must be specified for future study.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data for this study will be made available on reasonable request to the author.

Acknowledgments

The author would like to acknowledge the laboratory staff and students of the Department of Food Technology, Federal Polytechnic Offa, Kwara State, Nigeria, along with the laboratory staff of the Department of Food Science and Technology, University of Venda, for their support.

Conflicts of Interest

The author declares no conflict of interest.

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Figure 1. Scanning electron microscopy images showing the effects of natural fermentation (with different fermentation times) on cassava root flour. 0 h is the control (A), 12 h is 12 h of fermentation (B), 24 h is 24 h of fermentation (C), and 48 h is 48 h of fermentation (D).
Figure 1. Scanning electron microscopy images showing the effects of natural fermentation (with different fermentation times) on cassava root flour. 0 h is the control (A), 12 h is 12 h of fermentation (B), 24 h is 24 h of fermentation (C), and 48 h is 48 h of fermentation (D).
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Table 1. Effects of natural fermentation time on cassava root flour pH, TTA, TSS, crude protein, IVPD, Vitamin C, and antinutritional factors.
Table 1. Effects of natural fermentation time on cassava root flour pH, TTA, TSS, crude protein, IVPD, Vitamin C, and antinutritional factors.
SamplesControl12 h24 h48 h
pH6.50 d ± 0.56.08 c ± 0.44.90 b ± 0.34.20 a ± 0.5
TTA (% lactic acid)0.12 a ± 0.10.22 b ± 0.20.58 c ± 0.30.81 d ± 0.4
TSS (°Brix)5.82 d ± 0.54.91 c ± 0.33.20 b ± 0.32.12 a ± 0.2
Crude Protein (%)1.82 a ± 0.32.30 b ± 0.42.64 c ± 0.63.01 d ± 0.7
IVPD (%)47.32 a ± 0.257.71 b ± 0.469.20 c ± 0.378.51 d ± 0.5
Vitamin C (mg/100 g)20.52 a ± 0.525.81 b ± 0.729.72 c ± 0.535.23 d ± 0.3
Phytate (mg/100 g)205.43 d ± 2.5162.31 c ± 1.8120.52 b ± 1.272.72 a ± 0.9
Hydrogen Cyanide (mg/100 g)25.14 d ± 0.720.23 c ± 0.911.62 b ± 0.65.34 a ± 0.4
Trypsin Inhibitor Activity (TIU/mg)19.57d ± 0.815.22 c ± 0.712.13 b ± 0.65.45 a ± 0.4
Mean values with different superscripts in a column are significantly (p ≤ 0.05) different from each other.
Table 2. Effects of natural fermentation time on cassava root flour’s total phenolic content, antioxidant activities, and mineral content.
Table 2. Effects of natural fermentation time on cassava root flour’s total phenolic content, antioxidant activities, and mineral content.
SamplesControl12 h24 h48 h
TPC (mg GAE/g)1.35 a ± 0.31.95 b ± 0.52.56 c ± 0.42.97 d ± 0.6
ABTS (µmol TE/g)23.50 a ± 0.437.92 b ± 0.654.40 c ± 0.769.81 d ± 0.8
DPPH (IC50, µg/mL)40.33 a ± 0.860.24 b ± 0.578.40 c ± 0.692.61 d ± 0.7
FRAP (µmol Fe2+/g)35.23 a ± 0.652.73 b ± 0.868.51 c ± 0.782.30 d ± 0.8
Calcium (mg/100 g)32.50 a ± 1.235.61 b ± 1.338.91 c ± 0.441.35 d ± 0.5
Magnesium (mg/100 g)21.81 a ± 0.923.24 b ± 1.024.60 c ± 1.126.12 d ± 0.7
Iron (mg/100 g)5.42 a ± 0.26.11 b ± 0.26.82 c ± 0.36.82 c ± 0.3
Zinc (mg/100 g)1.74 a ± 0.12.10 b ± 0.12.40 c ± 0.32.62 d ± 0.1
Mean values with different superscripts in a column are significantly (p ≤ 0.05) different from each other.
Table 3. Pasting properties of fermented cassava root flour at different fermentation times.
Table 3. Pasting properties of fermented cassava root flour at different fermentation times.
Fermentation Time (h)Peak Viscosity (RVU)Trough (RVU)Breakdown (RVU)Final Viscosity (RVU)Setback (RVU)Pasting Temp (°C)Peak Time (min)
0 (Control)420.3 d ± 5.2281.5 d ± 4.7138.8 d ± 3.6372.2 d ± 4.190.7 d ± 2.772.1 d ± 1.34.3 d ± 0.3
12398.4 c ± 4.9270.2 c ± 5.4128.2 c ± 4.3359.8 c ± 4.489.6 c ± 2.571.4 c ± 1.44.2 c ± 0.4
24362.7 b ± 4.5254.6 b ± 4.9108.1 b ± 1.6336.3 b ± 3.581.7 b ± 2.870.5 b ± 1.24.1 b ± 0.2
48329.5 a ± 5.9239.3 a ± 3.190.2 a ± 1.9318.4 a ± 6.279.1 a ± 2.769.8 a ± 1.34.0 a ± 0.3
Mean values with different superscripts in a column are significantly (p ≤ 0.05) different from each other. RVU = rapid Visco unit.
Table 4. Phenolic profile of cassava root flour naturally fermented using different times.
Table 4. Phenolic profile of cassava root flour naturally fermented using different times.
Phenolic Compound
(mg/g)
0 h (Control)12 h24 h48 h
Gallic acid1.32 a ± 0.11.68 b ± 0.12.15 c ± 0.22.74 d ± 0.1
Caffeic acid0.84 a ± 0.11.21 b ± 0.21.64 c ± 0.12.01 d ± 0.1
p-Coumaric acid0.52 a ± 0.10.73 b ± 0.10.98 c ± 0.11.35 d ± 0.2
Ferulic acid0.68 a ± 0.10.89 b ± 0.11.14 c ± 0.11.41 d ± 0.1
Vanillic acid0.47 a ± 0.10.63 b ± 0.10.78 c ± 0.10.93 d ± 0.1
Syringic acid0.34 a ± 0.10.42 b ± 0.20.54 c ± 0.10.67 d ± 0.1
Total phenolics4.17 a ± 0.35.56 b ± 0.27.23 c ± 0.29.11 d ± 0.1
Values are mean ± standard deviation (n = 3). Different superscript letters in the same row indicate significant differences (p < 0.05).
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Bamidele, O.P. Effects of Natural Fermentation Time on Chemical Composition, Antioxidant Activities, and Phenolic Profile of Cassava Root Flour. Appl. Sci. 2025, 15, 8494. https://doi.org/10.3390/app15158494

AMA Style

Bamidele OP. Effects of Natural Fermentation Time on Chemical Composition, Antioxidant Activities, and Phenolic Profile of Cassava Root Flour. Applied Sciences. 2025; 15(15):8494. https://doi.org/10.3390/app15158494

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Bamidele, Oluwaseun Peter. 2025. "Effects of Natural Fermentation Time on Chemical Composition, Antioxidant Activities, and Phenolic Profile of Cassava Root Flour" Applied Sciences 15, no. 15: 8494. https://doi.org/10.3390/app15158494

APA Style

Bamidele, O. P. (2025). Effects of Natural Fermentation Time on Chemical Composition, Antioxidant Activities, and Phenolic Profile of Cassava Root Flour. Applied Sciences, 15(15), 8494. https://doi.org/10.3390/app15158494

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