Lactic acid-fermented Moringa oleifera seed–cassava beverage: chemical composition, in vitro antioxidant activity, and dietary contribution

1 Introduction

Dietary diversification is a key strategy for improving nutrition and strengthening food security. This is particularly important in developing regions where people rely heavily on a limited number of staple crops such as cassava and maize (Anyiam et al., 2022a; Hasanah et al., 2024). Such dietary patterns often lead to imbalanced nutrient intake and micronutrient deficiencies, as they are typically high in carbohydrates but limited in essential nutrients. Enhancing food diversity through the development of novel functional foods from underutilized plant resources represents a sustainable approach to improving nutrition and supporting global food security (Varzakas and Smaoui, 2024).

Functional beverages are among the most rapidly expanding segments within the healthy food market (Intrasook et al., 2024; Gull et al., 2025). For instance, the global market for functional foods and beverages was valued at USD 60.5 billion in 2024 and is expected to reach USD 120.3 billion by 2034, reflecting a compound annual growth rate of 7.20% during the forecast period (Emergen, 2024).

This trend is largely driven by increasing consumer awareness of health and well-being, along with increasing recognition of the benefits associated with functional foods.

The development of functional beverages increasingly relies on bioprocessing techniques that enhance both nutritional value and bioactivity. Among these approaches, fermentation is widely recognized as a sustainable technology for producing functional foods and beverages with improved health-promoting properties (Gadhoumi et al., 2024; Seol and Yeo, 2026). Fermentation is a biochemical process in which microorganisms transform food substrates into more stable and functionally enhanced products. This process is often accompanied by the synthesis of beneficial metabolites, including bioactive peptides, and phenolic derivatives (Kumar et al., 2025; Künili et al., 2025), which contribute to improved functional properties. Recent studies have demonstrated that fermented foods possess therapeutic potential across various disease conditions (Mihai et al., 2024; Künili et al., 2025). For example, they have been associated with improved metabolic health, including reduced risk of obesity, hypertension, and type 2 diabetes (Anyiam et al., 2024; Pyo et al., 2024). Fermentation also supports gut health, enhances immune function, and reduces inflammation (Maftei et al., 2024; Mukherjee et al., 2024). In addition, fermentation has been reported to improve nutrient bioavailability by reducing antinutritional factors (ANFs) and enhances sensory properties of food, thereby increasing consumer acceptability (Seol and Yeo, 2026). These benefits highlight fermentation as a sustainable approach for developing functional foods from locally available raw materials. Plant-based ingredients further contribute to this potential because of their abundant nutritional value and health-enhancing properties (Sharma et al., 2025).

Among plant resources, Moringa oleifera has gained significant attention recently, due to its high nutritional value and health-promoting properties, including anti-diabetic, antioxidant, and immunomodulatory effects (Divya et al., 2024; Gul et al., 2025; Mukhtiar et al., 2026). It is widely cultivated in Africa and Asia and has become globally popular because of its multiple uses and adaptability to diverse environmental conditions (Liang et al., 2025; Anyiam et al., 2025). The plant is commonly known as the “miracle tree”. It is drought-tolerant, adaptable to diverse soil conditions, and easy to cultivate. Traditionally, various parts of the plant, such as the leaves, seeds, and stems, have been used in folk medicine to treat a range of ailments across African and Asian communities. Moringa seeds contain high levels of protein (28–51%) with a balanced profile of essential amino acids (Gautier et al., 2022; Anyiam et al., 2025). The ratio of essential to total amino acids (31.17%) meets dietary needs for both children and adults, and their aromatic amino acids are comparable to those in animal proteins like beef and eggs (Anyiam et al., 2025). Moringa seeds are also rich in micronutrients such as iron and vitamins (Gul et al., 2025; Vinodkumar et al., 2025). Their fats are mainly unsaturated, especially oleic acid (65–75%) and α-linolenic acid (44–57%), which support heart health (Divya et al., 2024; Gul et al., 2025). In addition, they contain bioactive compounds like gallic acid, ellagic acid, and kaempferol, although levels vary by region (Wang et al., 2024; Adetuyi et al., 2025). These compounds enhance the nutritional quality and health benefits of moringa-based foods. However, despite these benefits, the use of moringa seeds in food processing is still limited, with only a few value-added products available. This is mainly due to the presence of antinutritional factors, which can negatively impact their sensory qualities.

Cassava (Manihot esculenta), on the other hand, is one of the most widely consumed staple crops in tropical and subtropical regions. Its adaptability to diverse environmental conditions and its role in food security make it an important agricultural resource (Sakadzo et al., 2025; Baci and Jarso, 2026). It provides a major source of dietary energy for millions of people due to its high carbohydrate content. However, cassava is inherently low in protein and certain micronutrients, and it contains ANFs such as cyanogenic glycosides (Sakadzo et al., 2025), which can pose health risks if not properly processed. In Nigeria for instance, cassava is processed into various fermented traditional foods such as garri, fufu, and abacha, which are frequently consumed in many households. In many regions where cassava serves as a staple food, children frequently fail to meet their daily nutrient requirements (Anyiam et al., 2022a). This inadequacy contributes to persistent malnutrition, stunted growth, and developmental challenges among school-aged children.

Several studies have reported the potential of moringa leaf in various beverage applications, such as soursop-based ice cream (Ademosun, 2021), sorghum-based mahewu (Mafukata et al., 2024), and yoghurt-like beverages (Gautier et al., 2022; Adepoju and Selezneva, 2024). However, studies on the application potential of moringa seeds in food processing is limited. A recent study by Aderinola et al. (2024) reported the antidiabetic and anti-inflammatory properties of biscuits fortified with moringa seed powder (MSP). To the best of our knowledge, the combined use of MSP and cassava in the production of fermented beverages has not yet been explored. Therefore, this study aims to develop and investigate the chemical composition, in vitro antioxidant, and potential dietary contribution of a fermented Moringa oleifera seed–cassava beverage (MOSCAB). The inclusion of MSP and cassava in a fermented beverage system may result in synergistic effects that enhance the nutritional quality and functional properties of the final product. Understanding these parameters is essential for supporting its development at both small-scale and industrial levels. Such a novel product could serve as an accessible functional beverage, particularly in regions where both raw materials are readily available.

2 Materials and methods

2.1 Sample collection and preparation

Fresh and fully matured Moringa oleifera seeds were obtained from smallholder farms in Ariam community, Ikwuano Local Government Area, Abia State, South-eastern Nigeria. The seeds were removed from their pods, rinsed with tap water to eliminate surface impurities, and sorted manually to remove damaged seeds. Clean seeds were soaked in water at a ratio of 1:3 (w/v) for 8 hours, after which the seed coats were manually removed. The dehulled seeds were dried in a hot air oven (Memmert-A55/33, Schwabach, Germany) at 50 °C for 36 h (Anyiam et al., 2025). The dried material was milled using an electric blender (RT-04A, Rong Tsong Tech. Co., Taiwan) and sieved through a 0.2 mm mesh to obtain a fine flour. The resulting powder was stored in airtight containers at 4 °C until further use. Fresh cassava tubers were sourced from the National Root Crops Research Institute, Umudike, and transported to the laboratory on the same day. The roots were peeled, washed thoroughly to remove dirt and debris, and cut into smaller pieces. These were dried in an oven at 60 °C for 36 h until a constant weight was achieved, then milled into flour. The flour was sieved to obtain a fine, uniform texture and stored in airtight containers prior to use. All chemicals and reagents used in the study were of analytical grade.

2.2 Product formulation

A binary mixture design was employed to develop blends of moringa seed powder (MSP) and cassava flour. The formulations included 100% cassava flour (control) and cassava–moringa blends at ratios of 80:15 (T15), 70:25 (T25), and 60:35 (T35) (Table 1). The product was designed for potential inclusion in school feeding programs for children aged 3–14 years. The proportion of moringa seed was selected to provide the recommended one-third (33.33%) of the daily nutrient requirements per serving (Uche et al., 2025), based on the reported nutritional composition of MSP (Gul et al., 2025). Moreover, previous studies have reported no signs of toxicity associated with both fermented and non-fermented Moringa oleifera seed supplementation across low, moderate, and high inclusion levels, indicating its safety in animal models (Anyiam et al., 2024; Adetuyi et al., 2025).

Table 1. Product formulation and experimental design.

2.3 Production of MOSCAB through fermentation process

Figure 1 presents the process flow diagram for producing the moringa seed–cassava beverage (MOSCAB). Each blend from Table 1 (100 g) were thoroughly mixed and distilled water (200 mL) was added to form a paste. Subsequently, boiled water (500 mL) was added to achieve a final solid-to-water ratio of 1:7 (w/v). Table sugar (3 g) was added to enhance fermentation process, and the mixture was homogenized (IKA ULTRA-TURRAX T18, Germany) at 7500 × g for 5 min. The slurry was boiled for 15 min with occasional stirring to prevent lump formation, then cooled to room temperature, and inoculated with 2 g of commercial starter culture (YoFlex^®, Chr. Hansen, Hørsholm, Denmark) containing lactobacillus plantarum and Streptococcus thermophilus. The inoculated mixture was incubated at 30 °C to ferment until pH reached approximately 4.5 after 24 h. Fermented final products were refrigerated at 4 °C until analysis (Supplementary Figure 1).

2.4 Evaluations

2.4.1 Proximate analysis

Proximate composition was determined using standard procedures recommended by AOAC International (2019). Moisture content was measured by oven-drying samples at 105 °C to constant weight (AOAC 925.10). Ash content was obtained by incinerating the samples in a muffle furnace at 550 °C (AOAC 923.03). Crude protein was analyzed using the Kjeldahl method, applying a nitrogen conversion factor of 6.25 (AOAC 920.152). Lipid content was extracted with petroleum ether using a Soxtec system (2055, Foss Analytical, Höganäs, Sweden) following AOAC 922.06. Total dietary fiber was determined using sequential acid and alkaline digestion with 1.25% sulfuric acid and 1.25% sodium hydroxide, followed by gravimetric measurement (AOAC 991.43). Carbohydrate content was determined by difference, which involves subtracting the measured components from 100. The energy value was calculated using Equation 1. Before analysis, samples were freeze-dried, and all results are reported on a dry weight basis.

(1) Energy value (Kcal/100g)=(P×4)+(F×9)+(C×4)

Where; P is protein content (g/100g), F is fat content (g/100g) and C is carbohydrate (g/100g).

2.4.2 Analysis for minerals

Mineral content was analyzed using atomic absorption spectrophotometry based on the procedure of Uche et al. (2025), with minor modifications. Briefly, 2 g of each freeze-dried sample was incinerated in a muffle furnace at 550 °C for 3 h. The resulting ash was cooled, dissolved in 10 mL of a mixed acid solution (HCl: HNO₃, 3:1 v/v), and left to stand for 30 min to ensure complete digestion. The solution was then filtered and diluted to a final volume of 50 mL with deionized water. The prepared digest was analyzed using an atomic absorption spectrophotometer (Spectro Analytical Tech. AA-7000, Kleve, Germany), with operating parameters set according to Uche et al. (2025). Calibration was carried out using standard solutions of known concentrations, and all measurements were performed in triplicate. The minerals selected for analysis were based on commonly reported deficiencies among school-aged children in the study area (Anyiam et al., 2022a; Yusuf et al., 2025).

2.4.3 Analysis of vitamins

Vitamins B1 (thiamine) and B2 (riboflavin) were quantified according to AOAC (2019) methods 942.23 and 970.65, respectively, using reversed-phase HPLC (Agilent 1200, Waldbronn, Germany) equipped with a fluorescence detector as described in Uche et al. (2025). Chromatographic separation was carried out using an Eclipse XDB-C18 column (4.6 × 150 mm, 5 µm). The mobile phase comprised 0.01 M sodium phosphate buffer (90%, v/v) and methanol (10%, v/v), delivered at a flow rate of 0.65 mL/min. Niacin (vitamin B3) was quantified using a colorimetric method in accordance with AOAC 944.13, with measurements taken on a UV–Vis spectrophotometer (Biochrom Libra, England). Ascorbic acid (vitamin C) was determined by titration with 2,6-dichlorophenolindophenol following AOAC 967.21. All measurements were performed in triplicate, and calibration curves were established using certified reference standards.

2.5 Analysis of antioxidant activity

2.5.1 Determination of DPPH radical scavenging activity

The DPPH assay was used to assess the free radical scavenging capacity of the samples, following the method of Liang et al. (2023) with slight adjustments. In brief, 1 g of sample was extracted in 20 mL of 80% methanol with continuous shaking for 1 h at room temperature, then centrifuged at 4000 rpm for 10 min. The clear supernatant obtained was used for further analysis. For the assay, 100 µL of the extract was combined with 2.5 mL of 0.3 mM DPPH solution prepared in methanol. The mixture was vortex-mixed for 30 seconds and kept in the dark at room temperature for 30 min. Absorbance was measured at 517 nm using a UV–Vis spectrophotometer (Biochrom Libra, England). Trolox (Sigma Aldrich, USA) was used as the calibration standard (R² = 0.9982), and results were expressed as percentage scavenging activity. The percentage inhibition was calculated using Equation 2, and all analyses were performed in triplicate.

(2) DPPH scavenging activity(%)=A0−A1A0 ×100

A₀ represents the absorbance of the control, while A₁ denotes the absorbance of the sample.

2.5.2 Reducing power assay

Sample reducing ability was evaluated using the FRAP (ferric reducing antioxidant power) assay following the method described by Jafari et al. (2026), with slight modifications. The working reagent was freshly prepared by blending sodium acetate buffer (300 mM, pH 3.6), TPTZ solution (10 mM), and ferric chloride hexahydrate (20 mM) in a 50:5:5 (v/v/v) ratio. An aliquot of 100 µL of the extract was mixed with 3 mL of the FRAP reagent and kept at room temperature in the dark for 15 min. Absorbance was then measured at 593 nm. All determinations were conducted in triplicate. Results were calculated from a Trolox standard curve and expressed as mg Trolox equivalents per 100 g dry weight (mg TE/100 g dwb.).

2.5.3 Total antioxidant capacity assay

Total antioxidant capacity (TAC) was evaluated using the phosphomolybdenum assay following the method described by Liu et al. (2024) with slight modifications. The reaction mixture was prepared using sulfuric acid (0.6 M), sodium phosphate (28 mM), and ammonium molybdate (4 mM). For determination, 300 µL of the sample extract was mixed with 3 mL of the reagent in a sealed tube and incubated at 95 °C for 90 min. After cooling, the absorbance was measured at 695 nm using a blank for reference. The TAC values were obtained from a Trolox calibration curve and expressed as mg Trolox equivalents per 100 g dry weight (mg TE/100 g dwb.).

2.6 Antinutritional factors assay

2.6.1 Determination of tannic acid

Tannic acid content in the MOSCAB samples was measured using the Folin–Denis spectrophotometric method (Arise et al., 2022). A tannic acid stock solution (1 mg/mL) was diluted stepwise to obtain standard concentrations ranging from 0 to 100 µg/mL for calibration. Each sample (1 g) was extracted with 40 mL of 10% (v/v) methanol under continuous shaking for 2 h. The mixture was filtered, and the volume of the filtrate was adjusted to 50 mL. For the assay, 0.5 mL of the extract was mixed with 2.5 mL of Folin–Denis reagent (10%, v/v) and 2 mL of 7.5% sodium carbonate solution. The mixture was incubated at room temperature for 30 min to allow color formation. Absorbance was measured at 765 nm using a spectrophotometer, and tannic acid concentration was determined from the standard curve (R² = 0.9997).

2.6.2 Determination of phytic acid content

Phytic acid in the samples was quantified according to the method of Anyiam et al. (2025). A 2 g portion of each sample was immersed in 100 mL of 2% HCl and left to extract for 5 h at room temperature. The mixture was then filtered using Whatman No. 1 filter paper. From the filtrate, 25 mL was measured into a conical flask, and 5 mL of 0.3% potassium thiocyanate solution was added as an indicator. This was titrated with a freshly prepared 1.05% (w/v) ferric chloride solution until a persistent brownish-yellow color appeared and remained stable for at least 5 min. The phytate content was then determined using Equation 3.

(3) Concentration of phytate(mg/100g)=Titre value×0.1081 Sample weight(g)

2.6.3 Determination of oxalic acid

Oxalate content was measured using a titration method described by Anyiam et al. (2025). Each formulation weighing 1 g was transferred into a 100 mL conical flask and mixed with 75 mL of 3.0 M HCl. The mixture was stirred intermittently for 1 h using a magnetic stirrer, then filtered through Whatman No. 1 filter paper. A 25 mL aliquot of the filtrate was taken and titrated with 0.1 N KMnO₄ solution maintained at 90 °C. The endpoint was reached when a faint pink coloration appeared and remained stable for at least 30 seconds. The oxalic acid concentration was then calculated using Equation 4.

(4) Oxalate(mg/100g)=Vol. KMnO4×630.3sample weight (g)

2.7 In vitro protein digestibility assay

In vitro protein digestibility (IVPD) of the MOSCAB formulations and the control was evaluated using a two-stage enzymatic digestion method adapted from Anyiam et al. (2026a). Protein suspensions (1 g/100 mL) were first adjusted to pH 2 and digested with pepsin at an enzyme-to-substrate ratio of 1:100 (w/w) at 37 °C for 120 min with gentle shaking. The pH was then raised to pH 8, and pancreatin was added at the same enzyme-to-protein ratio. The mixture was further incubated at 37 °C for up to 240 min. Enzymatic activity was terminated by placing the samples in boiling water for 10 min. Undigested proteins were precipitated by adding 10 mL of 10% (w/v) trichloroacetic acid, followed by centrifugation at 8,000 × g for 10 min. The precipitate obtained was neutralized, freeze-dried, and its protein content determined using the Kjeldahl method. The IVPD (%) was calculated as the proportion of digested protein relative to the initial protein content, as shown in Equation 5.

(5) IVPD(%)=Protein before digestion−protein after digestionProtein before digestion ×100

2.8 Sensory evaluation

Sensory evaluation of each MOSCAB formulation was conducted according to the method described by Uche et al. (2025) with slight adjustments. A semi-trained panel of 40 members participated in the assessment. Before the sensory evaluation, panelists were instructed on how to use the 9-point hedonic scale and recruited based on their willingness to participate. The samples were labeled with random three-digit codes and presented in glass cups at 5 °C in a randomized sequence under white lighting in a well-ventilated room. Participants assessed the samples for color/appearance, taste, flavor, mouthfeel, and overall acceptability using the hedonic scale. Drinking water was provided between samples to minimize carryover effects. Ethical clearance for the study was granted by the Human Research Ethics Committee of the Federal Medical Center, Umuahia (FMC/QEH/G.596/Vol.10/662). The purpose of the study was explained to all participants beforehand, and written informed consent was obtained. All responses were recorded anonymously.

2.9 Comparative analysis

A radar plot was generated to visualize the comparative functional and nutritional performance of all formulations and the control. The aim was to identify the formulation with the most favorable overall characteristics for further evaluation of contribution to daily nutrient requirements. To enable comparison across attributes with different units, all variables were normalized. The values of ANFs (phytate, oxalate, and tannin) were inverted to ensure correct interpretation, whereby lower values indicated better performance. The selection criteria for the best formulation were based on overall nutritional quality (particularly protein content, micronutrient levels), IVPD, desirable pH, reduced ANFs, enhanced antioxidant properties, as well as overall sensory acceptability.

2.10 Potential contribution to recommended dietary allowance

From the comparative analysis, sample T25 was selected for evaluation of the potential contribution of nutrients in MOSCAB per serving to the recommended dietary allowance (RDA). The serving size (250 mL) was selected based on reported average daily beverage intake ranges in children (Uche et al., 2025; Hamaimbo et al., 2023). This volume corresponded to a yield of 44.81 g (dry weight) after freeze-drying, which was used as the standard portion for estimating daily nutrient intake using Equation 6. The contribution of MOSCAB to the WHO-recommended dietary allowance for school-aged children (3–14 years) was calculated using Equation 7. The WHO-recommended dietary allowances (RDA) (WHO/FAO, 2007) used to estimate the contribution of nutrients from MOSCAB to school children aged 4–13 years were as follows: protein, 19 and 34 g/day; iron, 10 and 8 mg/day; zinc, 5 and 8 mg/day; calcium, 700 and 1300 mg/day; and vitamin C, 25 and 45 mg/day for the 4–8 and 9–13year age groups, respectively.

(6) Nutrient intake(g/serving)=Nutrient content (g)×Portion size(g)100

(7) RDA contribution (%)=Nutrient intake (g)WHO Recommeded dietary allowance (g/day)×100

The contribution for protein was estimated using IVPD-adjusted protein values. The estimated RDA contributions for other selected micronutrients were based on the amounts obtained from MOSCAB (T25) per serving size, without accounting for actual nutrient bioavailability during digestion.

2.11 Statistical analysis

All analyses were performed in triplicate, and results are expressed as mean ± standard deviation. The data were analyzed using analysis of variance (ANOVA) with SPSS for Windows (version 22.0, SPSS Inc.). Mean comparisons were conducted using the least significant difference (LSD) post hoc test, and statistical significance was set at p< 0.05.

3 Result and discussion

3.1 Effect of moringa seed powder inclusion on pH

The fermentation dynamics of MOSCAB were evaluated by monitoring pH changes during processing. As shown in Table 2, the pH of the formulations after fermentation ranged from 5.22 to 3.48. Increased MSP inclusion at higher concentration resulted in a greater reduction in pH, with sample T35 recording the lowest value (3.48) after 24 h of fermentation. A pH range of 4.0–4.5 is generally recommended for fermented beverages to ensure acceptable sensory and physicochemical quality (Mafukata et al., 2024). The pH values of T15 and T25 (4.58–4.47) fall within or close to this range and may enhance product stability and microbial safety, particularly under refrigeration. Most foodborne pathogens grow optimally at near-neutral pH (6.0–7.5) but are inhibited under acidic conditions, especially below pH 4.5 (Zhang et al., 2026). However, this inhibition depends on factors such as acid tolerance, type of acid, temperature, and water activity. For example, Escherichia coli O157:H7 and Listeria monocytogenes can grow at pH 4.5–4.7, while toxin production by Clostridium botulinum is generally prevented below pH 4.6 (El-Garhi et al., 2025; Anyiam et al., 2026a). The decrease in pH is attributed to organic acid production during fermentation (Akanni et al., 2024; Salvador et al., 2021). These organic acids (such as lactic acid, acetic acid and butyric acid) may also contribute to antimicrobial activity by disrupting microbial cell function. The marked pH reduction in T35 may be due to enhanced microbial activity supported by the high protein and mineral content of MSP which provides growth substrates for fermenting microorganisms. Moreover, MSP may also contribute directly to acidity through inherent organic acids and phenolic compounds such as gallic acid, tannic acid, and kaempferol (Adetuyi et al., 2025; Gul et al., 2025). Similar trends of reducing pH have been reported in various fermented plant-based beverages (Mafukata et al., 2024; Anyiam et al., 2026a). While the lower pH of T35 may improve microbial safety, the excessive acidification could negatively affect sensory quality, particularly by increasing sourness, which may explain its lower acceptability scores recorded in section 3.8.

Table 2. Effect of MSP inclusion on proximate, energy and pH of MOSCAB (g/100g dwb).

Mean with different superscript (a-b-c) are significantly different at (P<0.05) down the columns. T15: 15%; T25: 25%; T35: 35% of moringa seed powder. CHO, Carbohydrate.

3.2 Effect of MSP inclusion on proximate composition

MSP inclusion significantly (p< 0.05) affected proximate composition and energy value of MOSCAB (Table 2). Moisture content of MOSCAB ranged from 8.14–9.17%, with no significant difference (p > 0.05) among samples. A slight increase in moisture was observed in T35, possibly due to the higher moringa seed powder (MSP) content, which contains proteins and dietary fibers that can bind water. Fermentation may also enhance moisture retention by producing smaller hydrophilic compounds that enhance water holding capacity. Although moisture was measured after freeze-drying, fresh beverages typically contain 50–85% moisture (Mafukata et al., 2024; Uche et al., 2025). Such high moisture levels require proper storage, such as refrigeration, to prevent microbial spoilage. Protein content increased progressively from 2.43 to 28.73 g/100 g dwb, with increasing MSP concentration. This is attributed to the high protein content of moringa seeds (30–52%), which contain globulins and albumins as major storage proteins (Anyiam et al., 2025). Fermentation may also increase the relative protein proportion through carbohydrate reduction. In addition, microbial biomass and enzyme synthesis during fermentation may also contribute to improvement in protein content (Arise et al., 2022; Anyiam et al., 2026b). This finding is consistent with previous reports of increased protein content in bread and biscuits fortified with moringa seed as a partial substitute (Bolarinwa et al., 2019; Aderinola et al., 2024). Such improvement in protein content is important because cassava is naturally low in protein, and the addition of MSP can help address protein deficiency, particularly in regions where cassava is a frequently consumed staple crop.

Fat content increased significantly (1.79–15.05 g/100 g) with inclusion of MSP. This is attributed to the high oil content of moringa seeds (23–31.99%) (Gul et al., 2025; Wilcaso et al., 2025). However, a slight decrease in fat was observed in T35 compared to other formulations. This slight reduction in fat could possibly be due to increased microbial activity which may stimulate the increased production of lipolytic enzymes leading to lipid breakdown (Arise et al., 2022; Anyiam et al., 2023). Moderate fat composition of food beverages may improve mouthfeel and sensory quality. Lipid hydrolysis may also release beneficial fatty acids such as oleic and linoleic acids. Previous studies have shown that MSP lipids are rich in unsaturated fatty acids, including oleic (65–75%), linoleic, and α-linolenic acids (Abdalla et al., 2022; Srivastava et al., 2023; Gharsallah et al., 2023), which are considered beneficial for health. These fatty acids are associated with improved lipid metabolism and reduced cardiovascular risk (Srivastava et al., 2023). Hence, the increase in fat content of MOSCAB with inclusion of MSP may not necessarily pose health concerns to consumers.

A similar trend was observed for crude fiber content, which increased at moderate MSP inclusion levels (T15 and T25) but decreased slightly at T35. The initial increase in fiber may be attributed to the presence of structural polysaccharides in moringa seeds. Previous studies have shown that moringa seeds contain 6.0–9.33% crude fiber (Anyiam et al., 2024; Wilcaso et al., 2025), which consists of arabinose and xylose-rich heteropolysaccharides with notable bioactive properties (Hamid et al., 2025). The decrease in fiber at higher inclusion level may be due to intensified microbial activity from availability of more substrate, which may result in rapid degradation of fiber by microbial enzymes such as cellulases and hemicellulases (Anyiam et al., 2026b). Dietary fiber plays an important role in supporting gastrointestinal health, digestion, and beneficial gut microbiota (Anudeep et al., 2016; Hamid et al., 2025). At moderate levels, the increased fiber in samples may enhance these benefits in children. However, excessive fiber may reduce nutrient absorption and negatively affect acceptability. Ash content increased significantly (1.28–3.93 g/100 g) across all formulation with increasing MSP inclusion. This may be attributed to the mineral richness of moringa seeds, including calcium, potassium, magnesium, and iron (Vinodkumar et al., 2025). A slight decrease in ash observed at higher MSP levels (T35) may be due to possible microbial utilization of minerals during fermentation. For instance, a recent study on cereal-based fermented food reported that during fermentation of Medida using Bifidobacterium longum, the iron, magnesium, and zinc contents were lowered, showing that microbial activity can influence mineral levels in the final food matrix (Irakoze et al., 2023; Kitessa, 2024). Despite this slight reduction, ash content remained higher than the control, indicating improved mineral content. Fermentation may also improve mineral bio accessibility through phytate complex degradation by microbial phytase thereby improving the bioavailability of the minerals (Anyiam et al., 2026b; Seol and Yeo, 2026).

Carbohydrate content decreased from 83.41% to 46.34% with increasing MSP. This may be due to substitution of cassava with protein-rich moringa seed flour and microbial utilization of carbohydrates during fermentation, as source of energy (Arise et al., 2022; Anyiam et al., 2026b). This shift in macronutrient distribution may lower glycemic potential and modify metabolic responses of the beverage. The changes in macronutrient composition significantly influenced the energy value of MOSCAB. Energy increased with MSP inclusion, from 368.19 kcal/100 g in the control to 432.25 kcal/100 g in T25, but decreased in T35 (364.09 kcal/100 g). The initial increase is attributed to increase in fat content with MSP inclusion, which provides more energy (9 kcal/g) than protein and carbohydrates (4 kcal/g). The reduction in energy value observed in T35 may be related to the reduction in fat and carbohydrate balance during fermentation by LAB.

3.3 Effect of MSP inclusion on mineral composition of MOSCAB

The mineral composition of different formulations of MOSCAB is presented in Table 3. Significant differences (p< 0.05) were observed among the samples. Inclusion of MSP resulted in substantial increase (P<0.05) in the concentrations of calcium, zinc, copper, and iron compared with the control sample. Particularly, calcium content increased progressively (21.37-112.67 mg/100 g) with increase MSP ratio, representing more than a five-fold increase. Similarly, iron concentration increased from 1.55 mg/100 g in the control to 9.41 mg/100 g in T35 sample. This increase can be attributed to the naturally rich mineral composition of Moringa oleifera seeds. Recent studies have reported that moringa seeds are rich in essential minerals, including potassium, calcium, magnesium, phosphorus, and iron (Gautier et al., 2022; Vinodkumar et al., 2025). For instance, a study by Vinodkumar et al. (2025) reported that moringa seeds contain high levels of calcium (286.2 mg/100 g) and about 9.97-14.2 mg/100g of iron has been reported in the raw seed (Gautier et al., 2022). These minerals are widely distributed within the seed matrix and contribute to its nutritional value. In addition, fermentation has been reported to increase the mineral content of foods. For instance, Anyiam et al. (2023) reported increases in calcium and iron by 45.03% and 22.34%, respectively, in edible termite-enhanced fermented cassava after 24 h of fermentation. The authors attributed this increase to the reduction of ANFs due to microbial enzymatic activities, thereby releasing minerals previously bound within the food matrix.

Table 3. Mineral composition of Moringa seed-enhanced cassava beverage (mg/100g).

Mean with different superscript (a-b-c) are significantly different at (P<0.05) along the columns. Control: cassava; T15:15% moringa seed powder, T25: 25% moringa seed powder; T35: 35% moringa seed powder. Fermented for 24 h at 30°C.

The calcium content of MOSCAB, particularly in T35, is comparable to that reported for cow’s milk (119–127 mg/100 g) (Rincon et al., 2020; Rizzoli, 2022). It is higher than values reported for almond-coconut-based beverage (24.6–31.12 mg/100 g) (Uche et al., 2025), but lower than those found in yoghurt and cheese products (169–183 mg/100 g) (Rizzoli, 2022). The significant increases in calcium, iron, and zinc is important due to their essential physiological roles. Calcium supports bone development and cellular functions in children, iron is required for hemoglobin synthesis and anemia prevention, while zinc is vital for immune function, enzyme activity, and growth (Uche et al., 2025; Yusuf et al., 2025). This is especially relevant for populations that rely heavily on cassava-based diets, which are typically low in essential minerals (Boyiako et al., 2020; Sakadzo et al., 2025). While most minerals increased with MSP substitution, manganese and copper showed a different trend, peaking at T25 and declining slightly at higher inclusion levels (T35). This reduction may be linked to intensified fermentation in T35, as indicated by the decrease in pH. Increased metabolic activity of lactic acid bacteria likely raised the demand for trace minerals that act as enzymatic cofactors. For example, manganese serves as a cofactor for enzymes such as lactate dehydrogenase and manganese-dependent superoxide dismutase, involved in energy metabolism and oxidative stress protection. Similarly, copper functions in redox enzymes like Cu/Zn-superoxide dismutase. The possible utilization of these minerals by active LAB cells may explain their reduced levels at higher MSP inclusion. Despite the reduction, the levels of manganese and copper in T35 remained significantly higher than in the control. The observed increases in mineral concentrations are consistent with the higher ash content recorded in the proximate analysis (Table 2). It is important to note that in this study, only total iron content was quantified, and the oxidation state of the iron (Fe²⁺ or Fe³⁺) was not determined. However, fermentation-induced acidification may enhance mineral solubility and potentially improve bioavailability. Therefore, the increased mineral content of MOSCAB may contribute meaningful amounts of calcium, iron, zinc, manganese, and copper to the diets of school-aged children, although bioavailability was not directly assessed in this study.

3.4 Effect of MSP inclusion on vitamin composition of MOSCAB

The vitamin composition of MOSCAB is presented in Table 4. MSP inclusion led to an increase in most vitamins with increasing concentration across formulations, except for vitamin C, which declined. Riboflavin (0.15–0.64 mg/100 g) and niacin (0.16–0.49 mg/100 g) increased significantly (p< 0.05) with increasing MSP inclusion (T15–T35). This may be attributed to the intrinsic vitamin content of MSP. For example, Coello et al. (2020) reported that moringa seeds contain 1113.5 µg/100g of thiamine and 179.11 µg/100 g of riboflavin. The increase may also be linked to microbial biosynthesis during lactic acid fermentation. Previous reports have shown that several lactic acid bacteria are capable of producing B-group vitamins during food fermentation (Levit et al., 2021). However, thiamine (B1) increased at moderate MSP levels (T15–T25) but declined at the highest inclusion level (T35). This reduction may be associated with intensified fermentation activity, leading to increased microbial utilization of thiamine as a metabolic cofactor. For example, thiamine in its active form, (thiamine pyrophosphate), is essential for the decarboxylation of pyruvate to acetyl-CoA during carbohydrate metabolism for energy generation in fermenting microorganisms.

Table 4. Effect of MSP inclusion on vitamin compositions of MOSCAB (mg/100g dwb).

Mean with different superscript (a-b-c) are significantly different at (P<0.05) along the columns. T15, T25 and T35 are 15, 25 and 35% of moringa seed inclusion.

The significant reduction in vitamin C following MSP inclusion is unexpected. This is because previous studies have reported that moringa seed contain appreciable amounts of vitamin C (45.42–220 mg/100 g) compared to other parts of the plant (Srivastava et al., 2023; Oyewusi and Apata, 2025). This reduction may be attributed to oxidation during processing, as well as the utilization of vitamin C by fermenting microorganisms. Similar decreases in vitamin C content have been documented in fermented foods. For instance, Sangija et al. (2022) observed a reduction (by 88.33–95%) in vitamin C during fermentation of African nightshade, while Anyiam et al. (2023) reported decreased vitamin C composition in fermented cassava after 48 h of fermentation. The authors attributed the losses to oxidation of ascorbic acid to diketogulonic acid during thermal processing. Moreover, during fermentation, microbial activity can also create oxidative conditions by synthesizing ascorbate oxidase that promote the oxidation of vitamin C to dehydroascorbic acid, which can subsequently degrade into inactive compounds (Sangija et al., 2022). Despite this reduction, the vitamin C content of MOSCAB remains higher than that reported for almond–coconut-based beverages (0.88–1.27 mg/100 g) (Uche et al., 2025) and falls within the range reported for yoghurt (1.10–17.44 mg/100 g) (Sobczak et al., 2022). The presence of these vitamins is nutritionally important. Riboflavin and niacin serve as precursors of redox cofactors (i.e., FAD/FMN and NAD⁺/NADP⁺), which are essential for energy metabolism, while vitamin C contributes to antioxidant defense by scavenging reactive oxygen species and regenerating other antioxidants such as α-tocopherol.

3.5 Effect of MSP addition on antinutritional factors

The composition of ANFs in fermented MOSCAB samples varied significantly (P< 0.05) across formulations (Figure 2). The control sample exhibited the highest concentrations of all ANFs evaluated. A significant reduction (P< 0.05) in phytate (84.13–42.93 mg/100 g) and oxalate (114.48–37.63 mg/100 g) was observed in all formulated samples (T15–T35) following the inclusion of MSP and fermentation, compared to the control. In contrast, tannin content showed a different pattern, increasing by 56.31% at moderate MSP inclusion (T15) before declining significantly to 18.02 mg/100 g at T35. Overall, the lowest concentrations of phytate, oxalate, and tannin were recorded in T35, indicating a progressive reduction across the formulations. Phytate, tannin, and oxalate are considered antinutritional factors because they bind to minerals and proteins, leading to decreased protein digestibility and impaired mineral bioavailability (Gautier et al., 2022; Liang et al., 2025; Seol and Yeo, 2026. Previous studies have reported that moringa seeds contain phytic acid (19.29–70 mg/100 g), oxalate (9.71–51.24 mg/100 g), and tannins (6.64–62.6 mg/100 g), with variations influenced by seed maturity and cultivation conditions (León-López et al., 2020; Liang et al., 2025; Anyiam et al., 2025). The presence of these ANFs has been attributed to the plant adaptation mechanisms to harsh environmental conditions, as moringa is known for its resilience to climate variability (Anyiam et al., 2025). The observed reduction in phytate and oxalate contents in the MOSCAB may be attributed to the combined effects of soaking, cooking, and fermentation during processing of the beverage. These methods have been widely reported to be effective in reducing ANFs in plant-based foods (Arise et al., 2022; Liang et al., 2025). Soaking of moringa seeds for 8 h prior to processing likely facilitated the leaching of soluble antinutrients, while thermal processing for starch gelatinization prior to fermentation, may have contributed to the degradation of ANFs. Furthermore, fermentation with a LAB may have enhanced the breakdown of phytate through microbial phytase, which hydrolyze phytic acid into lower inositol phosphates and free phosphate. In addition, fermentation-induced acidification may enhance phytate solubility and thereby facilitating enzymatic degradation (Liang et al., 2025). The results are consistent with previous studies reporting reductions in ANFs during the fermentation of plant-based foods and beverages (Arise et al., 2022; Anyiam et al., 2026b).

The initial increase in tannin content at moderate MSP inclusion (i.e., T15 and T25) may be attributed to the contribution of tannins from moringa seed powder, which has been reported to contain high levels (62.6–196 mg/100g) of tannins (León-López et al., 2020). However, the subsequent decline in tannin content at higher inclusion levels (T35) may be associated with microbial degradation of tannins during fermentation. This process may be mediated by tannase enzymes produced by lactic acid bacteria, which hydrolyze tannins into simpler phenolic compounds (Arise et al., 2022; Anyiam et al., 2023). The overall reduction in ANFs at higher MSP levels indicates that the combined processing techniques used in MOSCAB beverage development (i.e., soaking, cooking, and fermentation) were effective in enhancing the nutritional quality of the product. Although no internationally accepted thresholds for these ANFs are available, the observed significant reduction may account for the improvement in protein digestibility reported in section 3.6. It is also expected to enhance mineral absorption, thereby contributing to meeting the daily nutrient requirements of children. In addition, partial degradation of antinutrients can liberate bound polyphenols that were previously associated with plant cell walls or storage compounds, thereby enhancing the measurable antioxidant activity of the beverage (as discussed in section 3.4). Although phytate, and tannin, are considered ANFs, their presence at moderate levels can provide important health benefits (Srivastava et al., 2023; Liang et al., 2025). Small amount of phytate has been reported to possess antioxidant, anticancer, and hypocholesterolemic properties and may help regulate blood glucose by slowing starch digestion (Pires et al., 2023). similarly, tannins also exhibit strong antioxidant and antimicrobial activities due to their polyphenolic structure, which enables them to scavenge free radicals and inhibit lipid oxidation (Kumar et al., 2025). Therefore, the residual levels of these compounds in the fermented beverage may not only remain within safe dietary limits but could also contribute to the overall functional and health-promoting properties of the product.

3.6 Effect of MSP inclusion on in vitro protein digestibility

The addition of MSP significantly improved the IVPD of fermented MOSCAB (61.58–89.44%) compared to the control (Table 5). The highest IVPD was observed in formulation T35, which had the greatest MSP inclusion. This improvement may be attributed to enhanced fermentation process following MSP incorporation and the reduction of ANFs during fermentation. The incorporation of MSP likely increased the proportion of readily digestible protein while diluting cassava-derived enzyme inhibitors, thereby improving enzyme–substrate interactions (Mafukata et al., 2024). Furthermore, the progressive reduction in pH and increase in acidity following MSP inclusion may have promoted the activity of proteolytic enzymes produced by LAB, thereby facilitating the breakdown of complex proteins (Irakoze et al., 2023; Arise et al., 2022). These findings are consistent with previous studies on protein-enriched fermented foods and beverages. For instance, Mafukata et al. (2024) reported progressive improvements in IVPD (50.60–70.38%) of fermented sorghum-based mahewu fortified with moringa leaf flour at varying substitution levels. Similarly, Anyiam et al. (2026a) observed increased IVPD (67.37–92.37%) in fermented cassava-based beverage improved with enzyme-extracted bambara groundnut protein isolate. A comparable trend was also reported by Uyanwa et al. (2024), who found increased IVPD (76.05–81.06%) in cassava-based cookies supplemented with plant protein flour. These authors attributed the improvements to improved protein hydrolysis during fermentation and the reduction of ANFs such as phytates and tannins. According to the authors, increased microbial activity, due to the availability of nitrogen sources, enhances the synthesis of enzymes such as proteases (Mafukata et al., 2024; Anyiam et al., 2026b). These enzymes hydrolyze complex storage proteins into smaller peptides and free amino acids, thereby improving protein digestibility. The highest IVPD recorded in T35 (89.64%) may therefore be linked to intensified microbial proteolysis. Increased protein availability from MSP likely enhanced organic acid production leading to pH reduction (Table 2), which in turn favors protease activity (Anyiam et al., 2026b). In addition, the greater reduction of ANFs in T35 may have further contributed to improved digestibility. The observed increase in IVPD across formulations is an indication of enhanced protein bioavailability, which may contribute to improved dietary protein intake, particularly among school-aged children.

Table 5. Effect of MSP inclusion on IVPD and antioxidant properties of fermented MOSCAB.

Mean with different superscript (a-b-c) are significantly different at (P<0.05) along the columns. Control: 100% cassava flour, T15: 15% MSP, T25:25% MSP, T35: 35% MSP. TAC, Total antioxidant capacity; FRAP, ferric reducing antioxidant power. IVPD, In vitro protein digestibility.

3.7 Effect of MSP inclusion on the antioxidant properties of fermented MOSCAB

The antioxidant properties of the developed product were evaluated in vitro using DPPH, FRAP, and total antioxidant capacity (TAC), and compared with the control (devoid of MSP). As shown in Table 5, MSP inclusion significantly enhanced antioxidant properties, as evidenced by increased DPPH radical scavenging activity (26.86–86.0%), TAC (8.01–51.02 mg TE/100 g), and FRAP (31.86–122.67 mg TE/100 g), with T35 exhibiting the highest values across all parameters. DPPH evaluates radical scavenging via hydrogen or electron donation, FRAP reflects reducing power through Fe³⁺ to Fe²⁺ conversion, while TAC provides an overall estimate of antioxidant capacity (Liang et al., 2023). The enhanced antioxidant activity observed across the formulations may be attributed to the combined effects of MSP-derived bioactive compounds and fermentation-induced metabolites. According to Seol and Yeo (2026), fermentation can promote antioxidant activity through two mechanisms; (i) microbial biotransformation of polyphenols into lower-molecular-weight compounds with higher bioactivity, (ii) the generation of bioactive peptides via proteolysis. Moringa seeds have been previously reported to be rich in bioactive components, including polyphenols, flavonoids, anthocyanidins, and bioactive polysaccharides (Srivastava et al., 2023; Wilcaso et al., 2025; Hamid et al., 2025), which contribute to free radical scavenging. For example, niazirin, a phenolic glycoside widely distributed in moringa seed, has been reported to mitigate hyperglycemic-induced oxidative stress in experimental models (Wang et al., 2019; Gharsallah et al., 2023). Similarly, arabinogalactan-rich polysaccharides isolated from M. oleifera demonstrated dose-dependent DPPH radical scavenging activity (Hamid et al., 2025). Fermentation-mediated hydrolytic and oxidative enzymes from LAB facilitate the release of bioactive phenolic metabolites from plant cell matrices, including ferulic, p-coumaric, gallic, and caffeic acids, which have been suggested to activate the Nrf2 pathway and enhance antioxidant enzyme expression in fermented foods (Kumar et al., 2025).

Beyond phenolic compounds, LAB fermentation has been shown to improve antioxidant activity through the production of low molecular-weight bioactive peptides with free radical–scavenging properties (Sanjukta and Rai, 2016; Seol and Yeo, 2026). These peptides are originally inactive within their parent proteins but become functional after enzymatic breakdown or microbial fermentation. Although the exact mechanisms are not fully clear, it is proposed that aromatic amino acid residues in their side chains can donate protons to stabilize electron-deficient radicals (Sanjukta and Rai, 2016; Anyiam et al., 2024). In addition, these peptides may contribute to antioxidant activity by directly neutralizing free radicals and by binding pro-oxidant metal ions such as Fe²⁺ and Cu²⁺. This phenomenon has been widely reported in fermented cereal-based beverages and other lactic acid–fermented foods, where microbial proteolysis releases peptides with significant antioxidant and cholesterol lowering effects (Sobczak et al., 2022; Tang et al., 2023; Ikechi et al., 2025). For example, fermented herbal beverages, including Moringa oleifera, exhibited improved DPPH radical scavenging activity (37.15–92.37%) (Don et al., 2024). Similarly, Liu et al. (2026) observed increased antioxidant activity in moringa tea following fermentation with different LAB strains. Metabolomic analysis revealed elevated production of aromatic amino acids, quinic acid derivatives, and flavonoid glycosides (Liu et al., 2026). Likewise, Wang et al. (2024) observed that fermentation of moringa leaf with Monascus purpureus enhanced antioxidant properties by converting tryptophan into metabolites such as melatonin, kynurenine, and 3-hydroxyanthranilic acid, which act as potent antioxidants in vivo (Ding et al., 2023). The higher antioxidant activity observed in T35, despite its lower ascorbate content, may be attributed to the combined effects of phenolic compounds, bioactive peptides, and other fermentation-derived antioxidant metabolites. It may also be associated with increased acidity during fermentation, which could promote enzymatic hydrolysis and the release of antioxidant amino acids such as cysteine, tyrosine, and methionine. For example, cysteine exerts antioxidant activity through its highly reactive thiol (–SH) group, which directly scavenges reactive oxygen species (ROS) (Obike et al., 2026). More importantly, cysteine also serves as the rate-limiting precursor for glutathione synthesis, a key intracellular antioxidant, while also enhancing cellular defense mechanisms through the activation of NRF2 signaling and subsequent upregulation of antioxidant enzymes (Ding et al., 2023; Egbujor et al., 2024). This observation is consistent with the in vivo findings from our previous study on MOSCAB in pellet form using rats (Anyiam et al., 2024). It was observed that administration of the moringa seed-incorporated feed to streptozotocin-induced diabetic rats for 28 days significantly increased catalase and superoxide dismutase activities, elevated glutathione levels, and reduced malondialdehyde concentrations, indicating enhanced antioxidant status. This highlights the potential of moringa seed and cassava in developing functional fermented beverages with health-promoting properties.

3.8 Evaluation of sensory attributes and acceptability of MOSCAB

The sensory attributes and general acceptability of the fermented MOSCAB are presented in Table 6. The results demonstrate that increasing inclusion of MSP in the fermented MOSCAB had a significant effect on all sensory attributes evaluated. The control sample recorded the highest scores for mouthfeel. A progressive decline in this attribute was observed with increasing MSP levels (T15–T35), with T35 showing significantly lower scores across all parameters. However, flavor and color/appearance showed a slightly different trend, with T25 recording the highest score for flavor (6.63), while T15 recorded the highest scores for appearance (6.28), taste (6.88) and overall acceptability (6.45). The progressive decline in color/appearance with increasing MSP (T25–T35) is likely due to the darker pigmentation and possible browning reactions associated with moringa seed components, which can reduce visual appeal. For instance, the higher carotenoid content in moringa seeds which contributes to their strong antioxidant activity (Gautier et al., 2022), may also impart the slight yellow tones observed in MOSCAB (Supplementary Figure 1). The relatively higher appearance and overall acceptability scores observed for T15 indicate moderate acceptability of the product at low MSP inclusion levels. This may be attributed to the contribution of amino acids present in MSP. Previous studies have shown that moringa seed is rich in glutamic acid (20.66 g/100 g), which is known to impart an umami taste to foods in free form (Anyiam et al., 2025). These flavor-enhancing amino acids may partially offset the negative sensory effects associated with tannins and other bitter compounds, thereby maintaining the comparatively favorable acceptability observed in T15.

Table 6. Sensory acceptability of MOSCAB.

The reduction in taste and acceptability at higher MSP inclusion (T25-T35) may be linked to the inherent bitterness of moringa seeds, which is often associated with increased levels of phenolic compounds, saponins, and other bioactive components. For example, glucosinolates, a compound extracted from moringa seed, have been reported to impact bitterness in food (Gautier et al., 2022; Liang et al., 2025). The pre-treatment of MSP by soaking prior to fermentation may have been insufficient to fully degrade these bitter compounds at higher inclusion levels. The decline in mouthfeel following MSP inclusion could be explained by the increased solid content and fiber, from MSP, which may result in a thicker, coarser, or slightly gritty texture, thereby reducing smoothness. Interestingly, the higher flavor score observed in T15 (6.63) suggests that moderate MSP inclusion may contribute desirable aromatic compounds through fermentation-derived metabolites. According to Akanni et al. (2024), LAB fermentation of beverages revealed that alcohols, aldehydes, ketones, esters, and carboxylic acids predominate among the flavor and aroma components. Similar patterns in sensory attributes and general acceptability (6.6–5.3) were reported by Akanni et al. (2024) in a fermented sorghum-based beverage with bambara groundnut flour at different inclusion ratios. Similarly, Mafukata et al. (2024) reported progressively lower sensory attributes, particularly in acceptance ratings (7.62–3.92) and taste (7.33–4.08), in a moringa leaf–sorghum-based beverage. At higher inclusion levels (T35), the beverage received the lowest ratings for all evaluated parameters, with overall acceptability of lower than 5. The lower pH observed in T35 (3.45) may have intensified sourness beyond the optimal range in this formulation, further contributing to the lowest taste and overall acceptability scores. Another possible reason is the possible increased release of free amino acids during fermentation. In particular, branched-chain and aromatic amino acids may contribute to bitterness and undesirable taste changes, as they are associated with bitter perception and the formation of flavor-active compounds (Zhao et al., 2016). The findings suggest that while MSP can enhance certain functional properties and nutritional values, its inclusion level must be carefully optimized to balance nutritional benefits with sensory acceptability.

3.9 Comparative analysis of different formulations

The radar plot provides a visual comparison of the overall nutritional, antioxidant, and antinutritional profiles of the four MOSCAB formulations (Figure 3). The aim was to highlight the formulation with the most favorable overall results for estimation of contribution to daily nutrient requirements. The chart shows that the T35 sample exhibited superior nutritional and bioactive properties compared to the control and other treatments. Specifically, T35 demonstrated the highest values in protein content, IVPD, antioxidant activities, essential minerals (Fe and Ca), and B-complex vitamins. It also showed marked reductions in ANFs, indicating an improved nutritional profile and enhanced functional properties. These results suggest that T35 most effectively enhanced the beverage’s nutritional quality and functional potential. However, sensory evaluation revealed that T35 received the lowest scores for all attributes, including overall acceptability. This was likely due to the excessive sourness from increased acidity beyond the optimal range (pH 4.0-4.5) for fermented beverages (Boyiako et al., 2020; Anyiam et al., 2026a). On the other hand, T15 recorded the highest ratings for overall acceptability and appearance but showed the lowest nutritional values, IVPD, functional activities, and increased phytate and oxalate compared to other formulations. The T25 formulation offered a substantially improved nutritional profile, including higher protein, vitamin, and mineral contents, while maintaining sensory scores within the acceptable range on the 9-point hedonic scale, indicating moderate consumer liking. Therefore, T25 formulation was considered the best formulation, providing a balanced compromise between enhanced nutritional quality, functional properties, and consumer acceptability. Hence, it was selected for further evaluation of its potential contribution to meeting the daily nutrient requirements of school children.

3.10 Nutrient intake and potential contribution to daily recommended intake

The contribution of the fermented MOSCAB to the daily nutrient requirements of school-aged children is presented in Table 7. The values varied substantially with MSP inclusion. For the control sample, all nutrients (except vitamin C) provided lowest contributions to recommended dietary allowance (RDA), with protein, calcium, iron, and zinc supplying only between 2–21% of the RDA. These amounts are far below the recommended one-third threshold (33.3%) for school meals within the study location (Uche et al., 2025; Anyiam et al., 2022b). Vitamin C in the control, however, reached 39.5% of the RDA for children aged 4–8, exceeding the minimum threshold. Previous studies have shown that cassava tuber is a good source of vitamin C, with raw roots containing between approximately 14.9 and 50 mg of ascorbic acid per 100 g (Mohidin et al., 2023). The T25 formulation, containing 25% MSP inclusion, significantly improved the nutritional contribution of several key nutrients. Protein contribution increased to 45.6% for children aged 4–8 and 35.5% for those aged 9–13, slightly exceeding the one-third threshold for the younger age group. Iron and zinc contributions were substantially enhanced, supplying 30.7–38.4% (iron) and 20.6–33.0% (zinc) of the RDA. These improvements can be attributed to the high protein and mineral content of moringa seed flour, as well as the fermentation process as previously discussed, which can reduce ANFs, thereby increasing mineral bioavailability. Calcium contribution, however, remained low (6.38–3.43%), reflecting the relatively low calcium content of both cassava and moringa seed flour. The increase in calcium content following MSP inclusion, (although not sufficient to meet daily requirements), is still beneficial when compared with other plant-based beverages (Rincon et al., 2020; Uche et al., 2025). This suggests the need for additional calcium sources or for children to consume larger portions if the beverage is to serve as a significant nutritional contributor to their daily requirements. Vitamin C contribution in T25 declined to 23.9% (4–8 years) and 13.3% (9–13 years), possibly due to partial degradation of ascorbic acid during fermentation and storage. These results indicate that moderate MSP inclusion can significantly improve protein, iron, and zinc intake, making fermented MOSCAB a potentially valuable functional food for addressing protein-energy and micronutrient deficiencies in school-aged children. However, the low calcium and vitamin C contributions, particularly in older children, suggest that the beverage should be complemented with other calcium- and vitamin C-rich foods (e.g., dairy, leafy vegetables, or fruits) to meet overall nutrient requirements. Moreover, optimization of MSP inclusion levels is critical as excessive levels negatively impact sensory acceptability, as observed in section 3.5 of this study. Therefore, MOSCAB (T25) represents a nutritionally improved formulation that can contribute substantially to school feeding programs, particularly for protein and iron, but should be part of a diversified diet to ensure balanced micronutrient intake. Fermentation-mediated enhancement of mineral bioavailability and protein digestibility further supports the functional potential of MOSCAB as a school meal component.

Table 7. Daily nutrient Intake per serving and potential contribution to RDA.

Values are based on a serving size of 44.81 g. *Protein values are IVPD-adjusted. RDA values based on WHO recommendations: protein (19–34 g), iron (10–8 mg), zinc (5–8 mg), calcium (700–1300 mg), and vitamin C (25–45 mg) for ages 4–8 and 9–13 years, respectively (WHO/FAO, 2007).

3.11 Limitations of the study

Some limitations of this study should be considered when interpreting the findings. First, the estimated potential contribution of the beverage to the RDA was based solely on IVPD-adjusted protein values and the compositional analysis of micronutrients. The limited amino acid profile of MOSCAB and the bioavailability of micronutrients were not included in the estimation, as amino acid profiling was not performed in the present study. However, both the limiting amino acid and the bioavailability of minerals are crucial for accurately determining the actual contribution of nutrients per serving to the RDA. Secondly, IVPD was determined using in vitro assays, which may not fully capture the complexity of protein digestion, absorption, and metabolism in the human system. Therefore, further in vivo studies are recommended to evaluate nutrient absorption and assess the actual nutritional effectiveness of the beverage. Third, the sensory evaluation was conducted with a limited panel of 40 participants, including students and staff, rather than the target population of school children, due to ethical and logistical constraints. As a result, the findings may not fully represent the preferences of the intended consumers. Finally, the study did not assess the shelf-life stability of MOSCAB during storage, which are important for the product’s practical application and long-term consumption. Future research should address these limitations to provide a more comprehensive understanding of the beverage’s nutritional, functional, and sensory properties.

4 Conclusion

The study explored the development of a fermented beverage by blending cassava with nutrient-rich Moringa oleifera seed. The inclusion of Moringa oleifera seed and lactic acid fermentation significantly improved the nutritional composition, antioxidant activity, and in vitro protein digestibility of the cassava-based beverage while reducing antinutritional factors. Although the T35 formulation exhibited the highest nutritional and antioxidant properties, its sensory acceptability was reduced due to increased sourness and slight off-flavors. In contrast, T15 showed higher sensory acceptability but lower nutritional and functional benefits. The T25 formulation provided the best balance between nutritional enhancement, bioactivity, and consumer acceptability, while contributing meaningfully to the recommended daily nutrient intake of school-aged children (4–13 years). These findings highlight the potential of fermented moringa seed–cassava beverage as a sustainable functional food product with enhanced nutritional and health-promoting properties for improving nutrient intake in vulnerable populations, particularly in resource-limited settings.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/finmi.2026.1846828/full#supplementary-material

Supplementary material Supplementary material at the source

Footnotes

Edited by: Marta Laranjo, Universidade de Évora, Portugal

Reviewed by: Shantonu Roy, Indian Institute of Engineering Science and Technology, Shibpur, India

Kinza Fatima, Graduate School of China Academy of Engineering Physics, China