The mature cassava (cultivar TME 419) roots, grown under similar environmental conditions and cultural practices, harvested from the research farm of the Department of Agricultural Engineering, Delta State University of Science and Technology (DSUST), Nigeria, were used for this study. The cassava was planted in April 2024 and harvested in April 2025.

The moringa leaves extract, clove seed extract, and turmeric rhizomes extract, were obtained from the bioprocessing laboratory, of the Department of Agricultural Engineering, DSUST. The extracts were prepared using ethanol, by following standard procedure. The plant materials were air-dried (24–30 °C) for 10 days, sorted to remove foreign bodies, and pulverized using a laboratory milling machine. Each plant material particulate was mixed with food-grade ethanol at a ratio of 1:10 (w/v). The container was hermetically sealed and left to stand for 5 days at room temperature (23 – 34 °C). The mixture was stirred gently for every 12 h, to enhance the effectiveness of extraction procedure. Subsequently, the filtrate was recovered from the mixture through filtration (using a Whatman No. 1 filter paper), and the extract was concentrated by drying it in a water bath at 40 °C (28, 29). This lower evaporating temperature was selected to avoid the thermal degradation, of the volatile phytochemicals (30). Basically, food grade ethanol (80% purity, CAS, 64–17-5, Molecular Weight of 46.069 g/mol) was selected over methanol (methyl alcohol), because of the toxicity associated with methanol, which is harmful to human beings (31).

All the chemicals used in this experimental investigation were of the analytical grade. DPPH (2,2-diphenyl-1-picrylhydrazyl) powder, ethanol, Folin–Ciocalteu reagent, nutrient agar (NA), and methanol were of the analytical grade and manufactured by Fisher Scientific, USA. Additionally, the UV–Vis spectrophotometer was produced by Thermo Scientific, Massachusetts, USA. The electronic scale (model: WT50001KF and accuracy of 0.1 g) was manufactured by WANT Balance Instrument Co., Ltd., China. The digital pH meter (model: GSI-011, resolution of 0.1 pH, and accuracy of +0.01pH) was produced by Globe Scientific Instruments India. The digital TDS meter (model: TDS 652, resolution of 0.1 ppm, and accuracy of ±0.5%) was manufactured by Electronics India company, India; while the BOD meter (Model: BOD-573, accuracy of BOD 5), was manufactured by Wincom Company Ltd., China. The HPLC system (model: Vanquish Flex UHPLC, max pressure of 100 MPa, flow rate range of 0.00 to 8.00 mL/min) was manufactured by Thermo Fisher Scientific, Massachusetts, USA.

The investigational mix design ratios used in this research are presented in Table 1. The concentration of each plant additive was standardized comparative to the cassava product (roots or flour) used for the experiment. Specifically, 2% (w/w) indicates that 2% of the cassava roots/flour weight used for each experimental unit. The soaking solutions were prepared, by dissolving the appropriate amount of the treatment, in tap water at room temperature. Notably, the plant powders were not dissolved in water, but rather they were blended with the dried cassava flour. These lower CF blending ratios (1% plant powder and ≤12% plant extract), were adopted to prevent nutrients antagonism, safeguard heath safety, and promote collaboration with earlier documented research. Basically, the 11 treatments and the two controls will provide comprehensive data, which will aid ANOVA analysis, leading to better reliable results.

Particularly, tap water was used in soaking the cassava roots this study instead of distill water, mainly due to these reasons: cost implications, ease of replication, and the soaking medium commonly used in previous studies. A large volume of water (about 200 L) is required for the soaking operation; therefore, the use of tap water will make the research cost-effective and viable, especially in low- and medium-income countries, where distilled water will not be easily accessible. Therefore, the utilization of the tap water, instead of distill water will enhance consistency and reproducibility of this research as well as factual household application. Additionally, since tap water does not undergo further treatments apart from filtration (unlike distilled water), it will contain some minerals, physicochemical, and biological attributes, that enhance microbial fermentation of the cassava roots. Similarly, borehole water has characteristics similar to tap water in this experimental setup, since the tap water used in this study is underground water (borehole water), which just undergoes filtration as the only treatment.

Moringa leaves were chosen due to their potent nutritional value (Flavonoids, vitamins proteins and minerals), as their essential compounds will enhance the nutritional quality, of the processed cassava flour (32). Clove seeds (Syzygium aromaticum) and turmeric rhizome (Curcuma longa) were chosen due to their high antioxidant activities, as these materials are rich in curcumin, flavonoids, phenolic acids, eugenol, gallic acid, and tannins (33, 34). This will help degrade toxic compounds (such as heavy metals and HCH), potentially reduce the GI level, and enhance the nutrient contents of the cassava flour. According to these scholars (35, 36), clove seeds, turmeric, and Moringa leaves contain large amounts of phytochemicals such as eugenol, curcumin, and moringinine, which are highly effective in inhibiting microbial growth. Apart from their nutritional and antioxidant values, these plant materials are easily accessible across the world, particularly in cassava-producing countries, where they are used for pharmaceutical and dietary applications. This eliminates the fear of food poisoning at lower concentrations, as utilized in this experimental mix design ratio in the practical workflow.

The cassava roots were harvested at peak maturity (12 months after planting), peeled and washed physically with tap water. Then, the washed roots were inspected manually, to discard all rotten or pest-infested roots, as these damaged roots can interfere with the experiment’s results. Approximately 10 kg of the cleaned cassava roots were cut into medium pieces and completely immersed in 10 L of water. The soaking duration was 48 h at room temperature (24^o − 32 °C), without water replacement; and the water was gently agitated every 6 h, to prevent buildup of anaerobic pockets, improve uniform chemical reactions throughout the water. After soaking, the roots were sun-dried (32 ± 4 °C) for 24 h, and then oven dry (65 ± 1 °C) until the moisture content declined to approximately 8%. These drying parameters were chosen to preserve the nutritional values of the cassava roots, ensure the microbial safety of the cassava flour, improve grinding efficiency, and reduce power consumption. The dried roots were ground with a burr mill and screened with a 150 μm sieve to obtain the cassava flour. Then, the flour was blended with the right amount of plant powder, and was packaged in sealed, air-tight containers, since CF has a hygroscopic nature.

The CF fat content was determined using Soxhlet extraction, following AOAC (38) guidelines.

The Kjeldahl nitrogen method was utilized to assess the protein concentration, following the AOAC (38) guidelines as explained by Nilusha et al. (8). 1.0 g of the CF was added to 20 mL of H_eSO₄ in a Kjeldahl flask, and digested in the presence of selenium as a catalyst, until a clear solution was attained. The ammonia content liberated from the solution, by the addition of NaOH was titrated against HCl, and the protein level in each specimen was computed by using Equation 4.

Where N - calculated nitrogen value.

The results gotten from this scientific investigation were evaluated through analysis of variance (ANOVA), to determine if the treatments have significant influence on the cassava flour. Also, the mean values obtained were separated, by employing the Duncan’s Multiple Range Test (DMRT) tool, at the 5% significance level. Each test was performed three times, and the averages and standard deviations were recorded. These results were presented in tables and figures. Additionally, the Principal Component Analysis (PCA) statistical tool, developed with Python software as used, in results visualization, along with further interpretation of the results.

The results of the tap water, and the soaking water recovered after the 48-h soaking are presented in Tables 2, 3. As seen in Tables 2, 3, the microbial load, physicochemical characteristics, and heavy metals (HMs) content of the water varied widely, and these factors were substantially influenced (p ≤ 0.05), by the treatment options (F1–F11). This supports previous reports of Olaniyan et al. (42), which indicated that the leachate from cassava root, substantially affected the microbial load, and the physicochemical properties of water and the environment. The low bacterial counts in the tap water (2.21 log₁₀ cfu/mL), was drastically increased to 2.59 log₁₀ cfu/mL, after soaking the cassava roots in untreated water. This can be linked to organic effluent discharged from the roots into the water, which enhanced microbial activity, through the provision of essential nutrients and suitable environmental conditions (43). It was observed that the TBC recorded in the plant extracts treated water, decreased dramatically, as the treatment concentrations increases. This signifies the antimicrobial effect of the extracts used in the bio-fortification process. Eugenol and curcumin have a strong ability to inhibit bacterial growth, by increasing cell wall absorptivity level, which disrupts nutrient utilization and ultimately leads to cell lysis (44, 45). Also, the phytochemicals such as moringinine and phenolic acids, present in the treatments are very effective, in inhibiting peptidoglycan production. This leads to the formation of bacterial cells with very fragile and porous cell membranes, hence exposing the cells to osmotic shock (46).

Furthermore, it was noted that the water pH becomes more acidic, in the presence of the treatments, and the water acidity was approximately directly proportional to the treatment’s concentration. This scenario can be attributed to the organic acids and other phenolics, discharged by the treatments and cassava roots into the water. The elevated water acidity recorded in treatments F10 and F11, will have antimicrobial effects, because a lower pH environment suppresses most microbial functionality (47). High water acidity helps to increase metal solubility and mobilization (48), and this can be connected to the higher concentrations of HMs and TDS, recorded in the water from the treatment units, particularly in treatments F8 to F11. The non-linear increase in the water BOD level can be linked to the antimicrobial effects, initiated by existence of pharmacologically active constituents in the extracts, leading to a reduction in microbial utilization of organic materials (49). Remarkably, the concentrations of Cd, Pb, Cr, and As in the water, increased as the treatment concentration increases, with the optimal levels recorded in the F10 and F11 samples (Table 3). This major drawback (from the environmental toxicological perspective), can be attributed to the favorable conditions provided by the extracts, in dissolving and leaching metallic compounds from the cassava roots; however, this situation may have a positive impact on the cassava roots, as some of the toxic metals will be degraded, resulting in safer cassava-based diets. This study’s findings have underscored the impact of plant extracts on water used in agricultural processes, particularly cassava root fermentation, and underscore the paramount need for proper remediation before discarding into the environment to protect public health.

The concentrations of the phytates, oxalates, tannins, and HCN in the CF samples, which were subjected to different treatments (F1–F11), along with the two control samples, Control A and Control B, are presented in Table 4. It was noted that the treatments have significant effects on the anti-nutritional parameters (p ≤ 0.05). Conspicuously, this study’s results indicate that, the plain water soaking process minimized anti-nutrient contents in the cassava flour. This aligned with these authors (50, 51) reports, which documented that soaking is a food processing method that helps to reduce the phytates, oxalates, and tannins in plant materials by leaching these compounds into the standing water. The HCN levels recorded in this study were less than those reported by Nebiyu and Getachew (20) for three cassava varieties. The reduction in the concentration of anti-nutrients observed in the Control B sample can be attributed to the activities of naturally occurring fermenting microbes, such as Lactobacillus, Leuconostoc, and Saccharomyces species, which are present in cassava roots. These microorganisms aid in the degradation of harmful compounds, plus enhance the biotransformation process (5, 17).

Furthermore, the findings highlighted that the CF displayed a progressive range of phytates, oxalates, and tannins concentrations, with treatments F1 to F4 showing lower oxalate contents, while treatments F8 to F11 exhibited higher oxalate contents. F3 and F4 exhibited the lowest phytates, oxalates, and tannins concentrations, nearly matching the results obtained in the carrier control sample. Conversely, hybridized treatments F10 and F11 exhibited the maximum phytates, oxalates, and tannins concentrations. This is an indication that phytochemical enrichment plays an essential role in accumulation of the anti-nutrients in the cassava flour. The significant increase in the levels of CF phytates, oxalates, and tannins can be attributed to two major mechanisms: plant extracts leaching into the water, which leads to an increase of these compounds concentration in the soaking water, and the phytochemical infusion process, which helps to increase the anti-nutrient content in the root’s skin through diffusion (52). Additionally, incorporating 1.0% plant powder into the milled flour will help increase the concentrations of phytates, oxalates, and tannins in the CF. Clove, turmeric and moringa are rich in phytochemicals - such as tannins and other phenolic compounds, help increase the concentrations of phytates, oxalates, and tannins in the treated material, by infusing these compounds into the roots (50, 53–55). Also, the decline in the HCN concentration of the soaked-processed CF, may be linked to the hydrolysis of cyanogenic glucoside in the process, which is enhanced through enzymatic and phytochemical actions (20, 21). This reaction was facilitated by the plant extracts in the soaking water, as supported by the treatment results.

Most importantly, the experimental outcomes revealed that, the treatments were able to suppress the flour’s HCN levels within the recommended threshold of 10 mg/kg DW (1 mg/100 g DW) by FAO/WHO, basically for cassava and its derived products, which is considered safe for human consumption (56). This research HCN levels were significantly smaller, than the values documented by Anim et al. (25). The consumption of excessive phytates, oxalates, and tannins impairs the bioavailability of most essential nutrients by producing indigestible substrates that adhere to minerals and proteins in the diet; however, at lower concentrations, they exhibit beneficial antioxidant and antimicrobial properties (53, 55, 57). Phytates tend to inhibit iron absorption, oxalates have the potential to increase kidney stone risk, and tannins can inhibit protein digestibility (58). Hydrogen cyanide toxicity includes respiratory failure, coma, tropical ataxic neuropathy, Konzo, and histotoxic hypoxia (59).

The results of the CF’s vitamins and proximate components are presented in Table 5. The data revealed that the soaking process, and the treatment interventions, has a significant influence on the nutritional quality of the cassava flour (p ≤ 0.05). Notably, the proximate components of the CF significantly increased with increasing treatment concentration across all the extracts used. As seen in Table 5, apart from the fat content, the nutrients levels varied widely in the CF samples, with the lowest values recorded in Control B, and the maximum values documented in the F11 CF specimen. The increment in the enriched CF nutritional quality can be associated, with the phytochemical contributions from the plant extracts. Phytochemicals such as eugenol, flavonoids, saponins, moringinine, and curcuminoids, enhance the nutritional value of the enriched CF through three major pathways. They improve the permeability of cassava root cells, thus encouraging the diffusion of essential nutrients from the soaking water into the root tissues, as the treated soaking water tends to contain higher nutrients content. Additionally, they facilitate the infusion of nutrients into the CF during blending, and also help retard nutrient degradation during the CF processing unit operations (5, 60–63).

This study’s findings corroborated reports published by previous scholars, which stated that soaking cassava roots and other plant materials, in plain water leads to a decline in their nutritional value, as a consequence of leaching and diffusion of nutrients into the immersion solution (64, 65, 103). The carotenoid levels documented in this in vitro investigation were greater than the results, published by Taleon et al. (64) and Odoemelam et al. (16) from similar processing methods. This study vitamin C and protein results corroborate the previous research findings of Lu et al. (9); while they were higher compared to the review results documented by Bayata et al. (66). Otondi et al. (2) reported that integrating 20 to 60% of chia seed (Salvia hispanica L.) flour, into cassava flour helps to increase the nutritional content of the enriched CF, and this is similar to the findings obtained in this research. Also, the protein, carbohydrates, and fat contents achieved in this research, were far less than the outcomes reported by Katunzi-Kilewela for cassava flour blended with 5–25% of chia seed flour. The protein and carbohydrate levels recorded in this current study, align with the findings of Widowati et al. (11), regarding CF produced from microorganisms-assisted fermented cassava roots. Similarly, Nilusha et al. (8) reported similar findings regarding protein and carbohydrate content, for CF processed through soaking treatment.

Also, during the soaking process, microbial actions will enhance the degradation of volatile bioactive compounds, macronutrients, and other nitrogenous compounds, leading to a reduction in the nutritional quality of the root (11, 67). Remarkably, the smaller fat contents achieved in the enriched CF, particularly F10 and F11 enriched flour samples, are advantageous, as lower fat levels facilitate prolonged shelf life, reduce rancidity, and improve the stability of flour-based diets, coupled with preventing the incidences of obesity and cardiovascular hazards. The results indicated that the processing enriched the CF carotenoid levels, thereby enhancing their potential to combat illnesses linked to vitamin A deficiency. Vitamins C and E are antioxidants that immensely help to neutralize free radicals in the body, enhance immune responses, and improve enzymatic performance, thus playing essential roles in preventing oxidative stress in the body (68, 69).

The results of the HMs concentration in the CF are presented in Table 6, and Figure 1. The results revealed that the treatments have a significant influence on the CF heavy metals content (p ≤ 0.05). Generally, Control A exhibited the highest heavy metals concentration, while F11 recorded the lowest HMs levels. This indicates that the bio-fortification treatments significantly curtailed all the levels of HMs. The results revealed that, the treatments were more effective in reducing Pb and Cd concentrations in the bio-processed flour, when compared to As and Cr, which showed a higher stability rate. Interesting, the F11 specimens exhibited about a 50% reduction (cumulatively), in the four HMs analyzed in this research. The reduction in HMs concentration can be attributed to the remediation effects of the treatments. These treatments were able to facilitate the leaching of HMs from the cassava roots into the soaking water, as confirmed by the results presented in Table 2. Some of the metallic salts are fairly soluble in warm water, leading to the degradation of the HMs’ concentration (70). Moreover, bioactive compounds – including polyphenols, alkaloids, and organic acids, have strong chelating effects, which cause them to bind heavy metals (HMs) and produce stable complexes. This inhibits heavy metals absorption into the CF matrix, primarily by reducing their mobility and bioavailability potentials (71). Also, phytochemicals in the treatments enhance the enzymatic hydrolysis of HMs, which provides an effective decontamination effect; through increase precipitation and oxidation rates (36, 45, 60, 72).

The Pb, Cr, and Cd concentrations recorded across the various treatment groups, were less than the values reported by Dibofori-Orji and Edori (73) and Ande et al. (74), for cassava flour sampled from other geographical locations. Also, Anim et al. (25) reported a higher concentration of Pb in cassava sampled from remediated goldmine fields. However, the Pb concentrations achieved in this study, were similar to the results documented by Isaac et al. (75), for flour produced from Dankasa and Shiga banza cassava varieties. Furthermore, though the arsenic and cadmium values recorded in this research were generally lower than the findings of Ogah and Ozioma (76), their chromium concentrations were within the range of values documented in this study. In addition, Asomugha et al. (77) reported higher levels of As and Pb, although their Cd concentrations aligned with the results obtained in this research for the enriched CF samples. The inconsistency in HMs concentrations reported by various scholars, can be linked to geographical locations, field practices, type of treatment adopted, laboratory errors, and other anthropogenic factors (78).

These HMs (Pb, As, Cr, and Cd) are considered toxic metals, because they play insignificant roles in human nutrition and in the medical field. Rather, their trace concentrations in the body have many health complications. Lead poisoning can result in coma, neurological defects, anemia, and convulsions (25). Cadmium toxicity includes renal disorder and carcinogenicity (27); while arsenic toxicity has been linked to cardiovascular diseases and cancer (79). Additionally, higher chromium concentrations have been associated with respiratory and gastrointestinal problems (26). Interestingly, the results obtained in this study never exceeded the maximum permissible limits of 300 μg/kg DW for Pb, and 200 μg/kg DW for Cd and As, as approved by WHO/FAO (56, 80).

The total phenolic content (TPC), and DPPH radical scavenging activity (DRSA), of the cassava flour samples results are presented in Table 7 and Figures 2. It was noted that the applied treatments, had significant influence on the both parameters evaluated (p ≤ 0.05). It was noted that the carrier control unit, had the lowest TPC and DPPH values - 125.33 mg GAE/100 g DW and 34.33%, respectively, which can be linked to the leaching of the active phytochemicals into the plain soaking water. According to Plaskova and Mlcek (81), the soaking of cassava roots leads to their decreased antioxidant capacity, through the diffusion of essential phytochemicals from the plant material into the water. The TPC and DRSA results recorded in this study were greater, than those reported by Nilusha et al. (8), for CF samples produced from several cassava cultivars, which can be correlated with the treatments applied in this research. However, this study’s documented values similar the values stated by Bamidele (5) for microbial-assisted fermented CF, substantiating the antioxidant effects of the treatments.

Soaking with natural additives, alters the free radical scavenging activity of the cassava root flour, leading to the release of bioactive metabolites within the root, facilitating a biotransformation process and resulting in enhanced antioxidant activity (5, 82). Additionally, the experimental findings show that the treatments (F1–F11) demonstrated significant potential in enhancing both TPC and DPPH levels of the cassava flour. The results depicted that F10 and F11 samples exhibited the maximum TPC and DPPH values, signifying their enhanced antioxidant capacity and medicinal properties. Particularly, the higher amounts of antioxidants recorded in Treatments F7 to F11, can be linked to the larger concentrations of protein and vitamins documented in these particular treatments (Table 5).

Besides, there is a stronger tendency that the extracts will facilitate the bio-synthesis of some new phenolics through advanced hydrolysis of certain organic acids, which act as transitional compounds for secondary metabolic routes (83). This action can also be linked to the rapid increase observed in the TPC and antioxidant activity of the treated CF samples. Higher TPC and DPPH values help in preventing oxidative stress-related disorders (84). Muscolo et al. (85) stated that antioxidants play a major role in the prevention of chronic ailments such as cancer, diabetes, neurodegenerative, and cardiovascular disorders, by inhibiting the occurrence of oxidative stress and cells inflammation. Phenolic compounds encourage healthy gut performance, by enhancing appropriate microbial activity and sustaining microbial balance (5, 86).

According to Uguru et al. (69), the effects of organic additives are influenced by the phytochemical metabolisms of the bio-agents, which differ widely among the bio-extracts, leading to their unique antioxidant behaviors. These results highlighted that increasing the treatment’s concentration automatically leads to an increase in the antioxidant level, which has several nutritional and medicinal advantages. However, high concentrations of these extracts can have a negative impact on the flavor and textural profile of the flour, and the accumulation of toxic compounds and heavy metals in the flour, above the public health safe limits.

The results of the glycemic index (GI) of the CF samples are presented in Table 8. It was noted that the soaking and bio-fortification have significant impacts on the CF samples GI values. The untreated sample (Control A) recorded the maximum GI value of 78.00, while the F11 specimen had the minimum GI value of 38.00. Particularly, the Control B specimen had a slightly lower GI value of 69.33, which suggests that soaking in plain water alone plays some critical role in reducing the GI value of the CF. Pronounced GI reduction (from 65.67 to 55.33) was recorded between samples from treatments F1 and F6, though the lowest GI values (50.33 to 38.00) were documented between samples from treatments F7 and F11. This highlighted that some of the treatments were more effective than others, and notably, the single extract-based treatments (F1 to F6), were not potent enough to drastically modify the CF glycemic behavior. Remarkably, the GI values reported in this study were similar to those obtained by Wylis Arief et al. (87) and Adefegha et al. (88) for bioprocessed cassava-based foods. However, Ogbuji and David-Chukwu (89) reported a higher GI value of 84.06 for fermented cassava roots, while Nwaliowe et al. (90) documented GI values that ranged from 86.42 to 89.09 for four cassava cultivars. Also, Eyinla et al. (91) reported a higher Glycemic Index (GI) value of 91 for the processed cassava product.

The results revealed that the treatments were able to reduce the flour GI value to the safer limit range. It has been recommended that low GI foods have glycemic index values that range from 0 to 55, moderate GI foods range from 56 to 69, and high GI foods have values greater than or equal to 70 (92). Glycemic index is a nutritional hazard indicator, and its higher values tend to cause rapid and substantial spikes in blood sugar levels. Diets with lower GI values are highly appropriate, mostly for patients with diabetes and cardiovascular diseases (93). The reduction in the GI levels can be attributed to, the antioxidant properties of the treatments, leaching of soluble carbohydrates during soaking, and microbial actions on the cassava roots (94). Antioxidants obstruct α-amylase and α-glucosidase performances, which critical for glucose production. Additionally, antioxidants inhibit enzyme activity and starch hydrolysis, thereby reducing the glycemic index. Flavonoids, gingerols, phenolic acids, and curcuminoids have strong potential to impede glucose mobility in the bloodstream, by seriously interfering with the basic SGLT1 and GLUT2 actions (19, 95).

Furthermore, Figure 3 shows the conceptual flowchart, illustrating how high phytochemical concentrations in the treatments suppress the glycemic index of the CF. It has been scientifically proven that flavonoids and phenolic acids retard α-amylase and α-glucosidase activity. This leads to a reduction in the rate at which the starch content of the flour is converted into glucose (96). Additionally, polyphenols are effective in improving insulin sensitivity and glucose utilization in the human body. Specifically, the reduction in glucose production will lead to lower glucose absorption into the bloodstream, resulting in the inhibition of rapid spikes in blood sugar. Consequently, this will provide greater health benefits against diabetes and obesity, making the enriched CF more appropriate for dietetic management of protracted diseases.

The results of the Pearson correlation analysis are presented in Table 9. The Pearson correlation revealed high positive correlations (r ≥ 0.90) between oxalates, tannins, carbohydrates, protein, vitamins, TPC, and DPPH levels of the enriched CF. On the contrary, there were negative correlations between the nutritional quality and antioxidant characteristics and the toxic metals. Additionally, there was a good relationship between the anti-nutrients, and the carbohydrate contents of the enriched CF. It can be seen that the anti-nutrients - phytates, oxalates, and tannins, have moderate to strong correlations among themselves, which is an indication that the treatments increase these anti-nutrients concentrations simultaneously. Elevated concentrations of oxalates, phytates, and tannins in diets tend to reduce essential minerals bioavailability, leading to nutrient deficiency diseases (97).

Also, the results revealed that there was a strong relationship among the vitamins (Carotenoids, Vitamins B, C, and E), which indicates that the treatments enhance the vitamins’ content of the CF. The vitamins were also observed to strongly correlate with the antioxidant parameters (TPC and DPPH) and protein content, affirming their synergistic role in enhancing the antioxidant activity of the fortified CF. Additionally, it was noted that the TPC and DPPH had an inverse relationship with HCN, but a moderate negative correlation with GI of the enriched CF samples; in contrast, the TPC and DPPH were found to have a very strong relationship with the CF protein content. This is an indication that the antioxidants performed dual functions: increasing the nutritional quality value of the CF, and also reducing the HCN toxicity and GI risks of the CF. Additionally, the Pearson correlation analysis established that there was a negative relationship (r – 0.4 to −0.8) between the toxic metals (Pb, Cd, As, and Cr) and the vitamins, proteins, and antioxidants (Table 9). This supports the idea that the bioactive extract treatments, help to inhibit the accumulation of toxic metals in the cassava flour. On the contrary, the heat map revealed that there was a strong relationship among the toxic metals, HCN, GI, and the fat content of the CF.

Notably, the protein has a very strong connection with the TPC. This demonstrates that extract treatments help improve the nutritional and antioxidant capacity of the CF, buttressing the previous findings of Moo-Huchin et al. (98). The heatmap emphasizes that there was a strong negative correlation between HCN and the vitamin content of the flour. Remarkably, the results highlighted that HCN and GI have negative relationship with the essential nutrients such as proteins and vitamins. This is one of the major goals of this research, which is reducing the cassava flour HCN and GI levels to safe limit, making it suitable for diabetic and other vulnerable patients. The results also depicted that the CF fat content has a moderate to strong negative relationship with the vitamins, antioxidants, and proteins. This depicts that the treatments help decrease the fat content of the CF and, at the same time, increase the antioxidant-nutrient balance of the flour, which is advantageous to human metabolic health.

This is affirmed by the strong positive correlations between the vitamins and protein, as shown in the Pearson correlation table. For instance, protein and B vitamins relationship (r = 0.954**), and protein and vitamin C relationship (r = 0.842**). Also, the TPC and DPPH were closely aligned, which indicates that these factors share a common antioxidant capacity and contribute to the health-promoting features of cassava flour. According to Kalogerakou and Antoniadou (99), antioxidants play essential roles in human health, including the enhancement of reproductive health, immune function, and healthy vision, together with neutralizing free radicals. On the contrary, the GI and fat content formed a cluster, whose position was almost opposite to that of the vitamins and antioxidants. Notably, this validates this study’s results, which reported that the flour’s GI and fat levels were inversely proportional to the concentrations of vitamins and antioxidants. This aligns with the Ansari et al. (100) report, which stated that large phytochemicals’ concentrations have the potential to lower the GI levels in diets, by inhibiting carbohydrate digestion rate, thereby lowering the blood glucose level.

The results of the PCA biplots are presented in Figures 4–6. Figure 4 affirmed that the treatment greatly affects the microbial performance and HMs content of the soaking water. For instance, the plain tap water was located apart from all the other points, which indicates that it has noticeably lower levels of HMs, TBC, TDS, and BOD, compared to the water samples from Control B and F1 through F11. Also, treatments F10 and F11 show a strong positive relationship with Cd, Pb, and Cr, as their cluster was strongly separated along the PC1 axis. Treatments F1, F2, and F7 show moderate HMs contamination, while treatments F5 and F6 were located around moderate pH and lower HMs contamination. On the contrary, the pH position was located opposite the BOD and TDS orientations, suggesting an inverse correlation - higher water acidity may lead to increase organic matter content, and greater heavy metals solubility. These observations corroborate Zhao et al. (48)‘s report on surface water quality. Conclusively, the PCA biplot has further affirmed that bio-extracts treatments had distinct influence on the water quality, resulting to elevated heavy metals accumulation, and inhibition of microbial activities. Since the water was not spiked with additional metallic or biological compounds, the variations in the concentrations of the parameter (primarily the increases in HMs and TDS), can be linked to the leaching of soluble compounds from the cassava roots into the soaking water.

The PCA biplot in Figure 5, further revealed the multivariate visualization of how the anti-nutritional parameters, varied among the treated and untreated cassava flour samples. It was observed that Control A is located at the extreme ends of both PC1 and PC2 axes, which confirms its extremely high levels of anti-nutrients. The stark separation of Control A from the other points further proved that, the soaking technique and other bio-additive treatments substantially altered the nutritional safety profile of the flour. Particularly, the PCA biplot obviously establishes that, the various treatments substantially modify the balance between HCN and other anti-nutrient compounds, which is essential for food security and public health. According to Chiwona-Karltun et al. (18) and Samtiya et al. (101), prolonging the soaking duration and complementary treatment, greatly helped reduce the HCN level of CF, as well as improving the flour’s nutritional value.

Figure 1 enhance better understanding of the effectiveness of the treatments. It was noted that protein, carotenoids, B vitamins, vitamin C, and vitamin E were located around the PC1 positive side, while fat was located on the PC1 negative side. This suggested that the essential nutrients were highly correlated, as well as their concentrations increased together in the same pattern. Additionally, the PCA results revealed that the carbohydrates behave slightly independently of the vitamins and protein behaviors. The Control A position indicated that this sample’s nutritional value, is significantly lower than that of samples F1 to F11, affirming its baseline composition. Also, treatments F1–F4 are clustered in the moderate nutrient region, which indicates that these enriched CF samples have moderate micronutrient concentrations. The F8–F11 enriched flour samples, particularly the F10 and F11 samples, are situated around the positive axes of PC1 and PC2, which indicates that there is a substantial enrichment of vitamins, proteins, and carotenoids in the flour, along with a lower fat content. The negative relationship between fat and other nutrients indicates that the treatments tend to enhance the dietary quality of the flour and reduce the less desirable nutrients.

Figure 2 further provides a better understanding of the interactions, between the CF specimens and the four toxic metals (Pb, Cd, As, Cr). For instance, the Pb and Cd locations suggested a strong positive correlation; hence, a reduction in one HM concentration, will lead to a decline in the second HM concentration. Notably, the PCA results showed that the HMs concentrations in treatments F1 to F4 remained closely related to that of the Control B. It was observed that the F11 sample was located far from the contamination vectors, indicating that this treatment plan, has the highest efficiency in degrading the toxic metals’ concentrations, especially Pb, Cd, and As.

Additionally, the Figure 6 shows that there was a direct proportional relationship, between TPC and DPPH, affirming the role of phenolic compounds as natural antioxidants. The increase in the antioxidants can be attributed to the improvement, of enzymatic reactions (hydrolysis and oxidation inhibition processes), which were facilitated by the presence of the plant extracts in the soaking water; thereby, promoting the breakdown of complex phytochemical compounds within the root tissues, into simpler bioavailable phenolic compounds (5).

This study was based on only one cassava variety, harvested from one location in one planting season; hence the results obtained cannot be used to generalize cassava flour quality. This is because plant varieties, soil and climatic conditions, can significantly affect the biochemical properties of cassava roots. Though this research will not experience water quality variability as a limitation, since the tap water used was collected at once from the same tap, subsequent reproducibility of this research using tap water, may face water quality variability limitations. This is because ground water quality, such as: heavy metal content, physicochemical properties, and microbial composition - is highly dependent on factors like sampling location, depth, season, method, and storage conditions (102). Therefore, it is recommended that, future comprehensive research be carried out on multiple cassava cultivars, multiple planting seasons, and locations to obtain robust information and to develop broad-based, enriched cassava flour.