Effect of Processing Methods on Mineral Bioaccessibility in Common Beans (Phaseolus Vulgaris L.)

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Wabi Bajo Nagessa, Borges Chambal, Custodia Macuamule This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4873186/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Mineral bioavailability is a concern in legume based foods due to presence of Antinutritional factors. Zinc and iron deficiency is widespread in low-income countries because of the low consumption of animal products due to their unaffordable costs. The objective of this study was to evaluate the effects of processing conditions on the in vitro mineral bioaccessibility of small black common beans cultivated in Mozambique. The samples were collected from ‘Instituto de Investigação Agrária de Moçambique’ (IIAM), Maputo, Mozambique. Soaking (in water and sodium bicarbonate), germination (for 24 and 48 hours at 25oC), and cooking (ordinary and pressure) were applied. The oven-dried and finely ground samples of processed beans were in vitro digested and analyzed for mineral bioaccessibility. The in vitro gastrointestinal simulation assay was performed and the dialyzable part of the samples was used for iron, zinc, and copper bioaccessibility determination. Though there is a slight difference, all the processing methods investigated in this study; soaking, germination, and cooking treatments affected the mineral bioaccessibility of beans. The in vitro iron, zinc, and copper bioaccessibility was enhanced by the respective processing methods as compared to the control sample. The iron bioaccessibility was observed to be 2.22% for water soaking and 2.59% for sodium carbonate soaking, 2.75% for 24-hour germination 4.27% for 48-hours germination, and 3.56% for ordinary cooking and 7.79% for pressure cooking. The in vitro iron bioaccessibility is relatively low as compared to that of zinc. In vitro, Zinc bioaccessibility was found to be the same for water and sodium bicarbonate soaking which was 6.94%. Germination for 24 and 48 hours resulted in zinc bioaccessibility of 7.58% and 10.08% respectively while zinc bioaccessibility of 6.52% for ordinary cooking and 8.41% for pressure cooking was achieved. The respective in vitro copper bioaccessibility obtained for soaking in water and sodium bicarbonate was found to be 6.04% and 6.78% which are almost similar. Similarly, the germination for 24 and 48 hours showed copper bioaccessibility of 7.01% and 7.63% in respective order whereas it was observed that copper bioaccessibility of 5.79% for ordinary cooking and 8.50% for pressure cooking was achieved in this study. This shows that it is expected that the solubility of pressure-cooked beans in the intestine and the release of minerals from its matrix is high. The result illustrates that the pressure cooking of presoaked beans had the greater value for all mineral's bioaccessibility except for zinc, and it can be concluded that the processing techniques could help improve minerals' bioaccessibility in beans which in turn helps to combat malnutrition and ensure food security in developing countries. Health sciences/Health care/Disease prevention/Nutritional supplements Health sciences/Health care/Nutrition Health sciences/Health care/Public health Beans in vitro digestion mineral bioaccessibility bioavalability malnutrition Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Micronutrient deficiencies in societies rely on the legumes as staple food, particularly beans-based diet, is a problem highly related with effect of these antinutritional factors. Zinc and iron deficiency is widespread in low-income countries because of a low consumption of animal products with a high content of bioavailable dietary iron and zinc due to their unaffordable costs. The consumption of cereals and legumes which are high in antinutritional factors is common in developing regions [1], [2]. Among legumes, which provide reasonable amount of both macro-and-micronutrients, common beans ( Phaseolus vulgaris L .), are widely produced and consumed in Africa including Mozambique and Ethiopia. Common beans are the most commonly consumed legume worldwide, and produced for direct human consumption [3], [4]. They are a staple food and the major source of iron for populations in Eastern Africa and Latin America. The dried seeds of common beans are an important source of dietary protein for millions of people throughout the tropics, complementing those amino acids lacking in diets based on cereals :lysine and tryptophan as well as good source of minerals iron, copper and zinc [5], [6]. Besides, common bean is a nutritious food low in fat and high in protein. They are also part of many traditional diets, playing a major role in vegetarian diets being consumed in different dishes together with other food products in different countries [7], [8]. Consumption in medium to high amounts of beans is being associated with a decrease in some types of cancer, lower incidence of degenerative diseases and reduction of diseases such as diabetes, cardiovascular diseases and even neoplasms [9], [10]. However, beans are known to be rich in anti-nutritional factors such as phytates, tannin and , oligosaccharides which reduce the digestion and bioavailability of nutrients in beans [11]. Phytate is one of the main factors limiting the bioavailability of mineral cations in common beans, and polyphenols might hinder the absorption of nutrients [12] The low iron absorption has been reported to be attributed to inhibitory compounds [13]. The consumption of common beans at wider level is limited by the presence of these anti-nutritional factors. They affects nutritional quality especially protein digestion and mineral absorption [14]. The poor bioavailability of essential minerals leads to public health problem such as leading to iron deficiency anemia and Zinc Deficiency. As a consequence, low utilization of the common beans results in food insecure, malnourished and unproductive society which indirectly affects the sustainable development of the country. The role of traditional or improved processing techniques in reducing ANFs and improving sensory quality of beans have been reviewed well [15]. The beans, before their consumption, are cooked, and though most of the consumers prepare food at household level. It was also reported in previous studies that different processing techniques including soaking, cooking, germination, fermentation and enzymatic treatments can contribute for reducing ANFS and improved bioaccessibility/bioavailability of nutrients especially minerals and protein, improve sensory properties [16]. According to Matella et al ., [17], soaking and germination can increase the utilization of kidney beans. Bioaccessability is the amount of substance that is solubilized in the gastrointestinal environment. It is the fraction that is released from food matrix into the gastrointestinal tract during the digestion process, and thus, available for intestinal absorption and can enter the bloodstream [18]. Therefore, to be absorbed, nutrients must first be released from the food matrix and modified in the gastrointestinal tract. As a consequence, in vitro digestion models that mimic the complex physicochemical and physiological conditions of the human GI tract have been developed and used for the purpose of studying nutrients bioaccessibility (Hur et al ., 2011). This research was conducted on small black common beans variety released and culivated by Instituto de Investigacao Agraria de Mozambique , to evaluate effects of different processing techniques (hydrothermal and others) on in vitro minerals bioaccessibility bean through reduction of ANFs. This study achieved the enhanced of mineral bioaccessibility as the phytate and tannin content of beans were reduced as a functions of different processing techniques. The improved bioaccessibility of minerals contributes to combat micronutrient deficiency. MATERIALS AND METHODS Description of Study Area The laboratory experiment was conducted at three faculties of Eduardo Mondlane University including Chemical Engineering (food technology) laboratory, Microbiology Laboratory and Department of Chemistry (central laboratory) faculty of Engineering, Veterinary, and Sciences, respectively at Eduardo Mondlane University. The site is located at Mozambique, Maputo. The area is located in the Southeast coast of Africa at 18.6677 o S, 35.5296 o E. Materials Chemicals and reagents The reagents used for the experiment were pancreatin from porcine pancreas (P3292-SIGMA ALDRICH, USA), Pepsin from porcine gastric mucosa (P7000-SIGMA ALDRICH, USA), Glycine hydrochloride (G2879-SIGMA ALDRICH, USA), molecular porous membrane tubing (MWCO-8000Da, Rancho Dominguez CA 90220-6425 US, & Canada) and Sodium Bicarbonate. Collection and Preparation of Raw Materials The matured, unbroken and free of defects black small common bean samples (A 222 -bean variety) were collected from ‘ Instituto de Investigação Agraria de Moçambique’ (IIAM), Maputo, Mozambique. The beans were cultivated in the year 2020 cropping season and harvested in December, 2020. About 5 kg of the collected bean samples were sorted for sands, husks, soils and any foreign materials and kept at room temperature prior to processing by respective processing methods; soaking, cooking and germination. The control sample (raw bean) was milled by laboratory mill (ZM200-Retsch Rheinische Straße 3642781 Haan, Germany) and stored at room temperature prior to mineral analysis and in vitro digestion assay. Treatment’s setup The collected beans were subjected to three different processing methods with two levels. The processing methods studied in the experiment were soaking (water and NaHCO 3 ), cooking of previously soaked beans in water (ordinary and Pressure), and germination (1 and 2 days). The sample size was 7 including the control sample which was not subjected to any processing techniques. Processing of Methods of Beans The raw beans samples were sorted, cleaned, and ground using miller (ZM200-Retsch Rheinische Straße 3642781 Haan, Germany) to use as a control sample for anti-nutritional, minerals analysis, and in vitro digestion. The remaining beans samples were processed using three processing methods: soaking, germination, and cooking. Soaking germination and cooking were performed following method of Emire & Rakshit [20] & Kaur & Kapoor [21], and [22] for pressure cooking. The pH of water and sodium bicarbonate (NaHCO 3 ) solution before and after processing was measured by PH meter during the conduction of an experiment. Soaking The cleaned common beans seed free of broken seeds and other foreign matter were soaked for 12h in distilled water and (0.05%) NaHCO 3 solution at 1:3 ratios (seed-to-solution) at ambient temperature. The pH of the water and sodium bicarbonate solutions were 7.2 and 8.84 respectively. The soaked seeds were washed twice with water followed by rinsing with distilled water and was then dried in an oven at 60 ○ C. The dried samples were ground, stored in vacuum plastic for further analysis. Germination The common beans were weighed (120g) into plastic bowl and soaked in distilled water (bean-to-water ratio of 1:5w/v) for 12 h at room temperature. The seeds were then incubated to germinate in an incubator at 25 o C for 24 and 48 hours in aluminium foil and muslin clothes. The distilled water was sprinkled on the seeds (twice a day) during germination period. The germinated beans were dried in a hot air oven at 60°C, ground using sample mill and stored in vacuum plastic bags for further analysis. Cooking The previously soaked beans in tap water for 12 hours were cooked in conventional cooking pan with a seed to water ratio 1:6 at 100 o C for 1.47 hours (ordinary cooking) and 100 o C for 38 minutes (pressure cooker). The cooked samples were mashed manually with mortar and pistle, and dried in a hot air oven maintained at 60 ○ C and then, ground to a fine powder and stored in vacuum plastic bags for further analysis. The figures 1 (A & B), 2 (A & B) and figure 3 shows the pictures taken during laboratory experiment. Determination of Moisture Content The empty petridishes were oven dried for 1h at 105 o C, transferred to the desiccators cooled for 30 min, and weighed. The prepared beans flours were mixed thoroughly. About 2.00g of fresh samples were put into the previously dried and weighed petridishes. The dishes and their contents were placed in the drying oven and allowed to dry for 1h at 105 o C. Then, after drying, the petridishes and their contents were taken out of the oven and cooled in desiccators and reweighed. The amount of water present in a sample is considered to be equal to the loss of weight after drying the sample to constant weight at a temperature near the boiling point of water (AOAC 925.10). Determination of total ash content Ash was determined by incineration of known weights of the samples in a muffle furnace at 550 o C until a white ash was obtained. Organic matter was burned off and the inorganic material remaining is cooled and weighed. Heating was carried out in stages, first to derive the water, then to char the product thoroughly and finally to ash at 550 o C in a muffle furnace. The ashing dish was placed into a muffle furnace for 30 min at 550 o C. The dishes were removed and cooled in desiccators (with granular silica gel) for about 30 min at room temperature; each dish was weighed to the nearest g. About 4 g of flour sample was added into each dish. The dishes were placed on a hot plate under a fume-hood and the temperature was slowly increased until smoking ceases and the samples become thoroughly charred. The dishes were placed inside the muffle furnace at 550 o C for 18 h, and removed from the muffle and then placed in desiccators for 1h to cool. The ash was clean and which in appearance. When cooled to room temperature, each dish + ash was reweighed. Weight of total ash was calculated by difference and expressed as percentage of the fresh sample. In vitro Mineral Bioaccessibility Assay The in vitro gastrointestinal digestion assay was conducted to determine the bioaccessible portions of minerals in beans treated with different processing methods. The determination of mineral bioaccessibility was performed based on the in vitro digestion method described by [23] . The gastric (phase) digestion simulation was performed by weighing 4 grams of ground bean was suspended in previously prepared 24mL of 20mM glycine-HCl buffer solution. Then, the pH was adjusted to 2.0 by adding 2M HCl, and 1mL pepsin enzyme solution prepared by dissolving 1.6 grams of pepsin in 10mL 20mM glycine-HCl buffer was added in to the suspended samples. The suspension was then incubated in hybridization shaker (Sl-30H, BIBBY SCIENTIFIC LIMITED STONE, STAFFORDSHIRE, ST15 OSA, UK) for duration of 2 hours at 37.4 o C. Pepsin enzyme was inactivated by putting sample solutions into ice box prior to the commencement of intestinal phase. The pH of the gastric digested suspension was adjusted to 7.0 with 1 Molar NaHCO 3 to simulate intestinal (phase) condition. To the gastric digested samples, 10mL of a pancreatin enzyme solution previously prepared from 0.4 gram of Pancreatin in 100mL of distilled water was added, and a previously rinsed dialysis membrane (with distilled water) containing 2mL of dionized water was placed in the digestion system. Then, it was again incubated under the same condition for 2 hours. Finally, the dialysis membrane was removed from the system and the contents of minerals in the dialysate were quantified by ICP-AES. The bioaccessibility of minerals in terms of percent was calculated by the following formula. Determination of mineral concentration in beans The minerals content of the beans were determined according to Official methods of analysis [24], and Inductively Coupled plasma Atomic Emission Spectroscopy (ICP-AES-9820 Plasma Atomic SHIMADZU South Africa) was used for determination minerals. Two grams of the sample flours were weighed, and 10 ml of the mixture of HNO 3 (65%v/v) and HCl (37%v/v) was added. To digest the samples, the mixture was placed on the heating mantle, and digestion was stopped when about 2ml of the solution was left in the digestion cup. The sample was then diluted with ultrapure water, centrifuged and filtered using filter paper (Whatman No.40). The clear sample solution of about 14ml was used for each mineral analysis by ICP-AES. The mineral content of the sample was calculated by the following formula and expressed as mg/kg dry weight basis. Where: Volume-volume of sample solution analyzed Mass-mass of sample used for acid digestion Concentration-Concentration of minerals in the analyzed solution Preparation of standard The ULTRASPEC Certified aqueous Reference Material of 100 µg/mL was used to prepare standard solutions for mineral analysis. The series of standard solutions containing 100, 200, 400, 600, 800, 1200 and 1600 µg/L were prepared from mother solutions (100 µg/mL). The standard solutions were used to prepare the calibration curve where a graph of concentration vs intensity of standard solutions was plotted. Experimental Design and Data analysis Completely Randomized Design was used, and the experiments were conducted in triplicate and data was subjected to Analysis of variance (ANOVA), and least significant difference (LSD test) was employed to separate the means. RESULTS AND DISCUSSIONS The effect of processing methods on moisture and Ash content of beans The moisture and ash content of processed beans along with control analyzed in the experiment is presented in Table 1. The statistical analysis showed that the moisture and ash contents are significantly different among treatments. Table 1: Some compositions of processed beans Treatments Parameters Moisture (%) Ash (%) Dry matter (% Untreated (control) beans 7.13 ±1.16 C 6.72±0.31 A 92.87±1.16 A Soaked in water overnight 7.81 ±0.52 BC 6.09±0.57 A 92.19±0.52 AB Soaked in NaHCO 3 overnight 7.32±0.19 A 6.32±0.036 A 92.67±0.193 C Germinated for 24 hours at 25 o C 8.69±0.88 BC 5.29±0.29 AB 91.31±0.88 AB Germinated for 48 hours at 25 o C 8.79±1.76 BC 4.43±0.81 B 91.21±1.76 AB Soaked overnight in water+ Ordinary cooking 8.73±0.94 BC 5.62±0.18 AB 91.27±0.94 AB Soaked overnight in water+ pressure cooking 9.48±0.44 B 5.91±1.31 AB 90.52±0.44 B CV 10.97 11.15 1.10 LSD 1.70 1.52 1.07 [1] All the values are the mean of triplicate results. The means assigned with different letters in the column are significantly different. The moisture, ash, and dry matter content of processed beans is presented in Table 1. The statistical analysis showed that the moisture, ash and dry matter content of beans sample were affected by processing methods. The moisture content was a bit higher after soaking in water and sodium bicarbonate as compared to the control. Germination for 12–48 hours as soaking followed by ordinary cooking was found to have almost similar moisture contents. However, soaking followed by pressure cooking resulted in the highest moisture content. This could be due to the absorption of water by the seed before cooking. This could be due to the typical functional properties of the bean matrix because soaking allows the water to disperse in the protein fraction and starch granules, which helps soften the texture of beans. Ash, or the total mineral contents of beans, is a very important parameter and can be affected by the processing methods. The total mineral content of the beans decreased due to the processing methods applied as compared to the control, with germination for 24 hours resulting in the lowest 5.29% and soaking in sodium bicarbonate resulting in the highest 6.32% total mineral contents following the control sample. Since almost all of the processing methods applied include the steeping of the beans in water except the soaking in sodium bicarbonate treatment, the loss of ash content due to the leaching of mineral elements occurred. This result agrees with the previous study, where the ash content of baobab seeds decreased from 8.93% (raw) to 5.84% and 8.93% to 6.12% by soaking in water and sprouting, respectively [25]. According to Sarmento et al. [26], the dark black beans were found to have a 3.80% ash content, which is lower than the current result. The previous study reported that the ash content of common beans was in the range of 3.90%–5.70% [26]. The similar results on the effect of processing techniques on the ash content of field beans reported a reduction from 3.00% to 2.66%, 3.00% to 2.83%, and 3.00% to 2.05% soaking for 18 hours, germination for 40 hours, and cooking of presoaked beans, respectively [27]. The dry matter content of the beans was also studied, and it was observed that the dry matter content was inversely correlated with the moisture content, which is also scientifically valid. Effect of processing Methods on Minerals bioaccessibility In this study, the effect of processing methods on the bioaccessibilities of iron, zinc, and copper was studied. The statistical analysis showed that the mineral bioaccessibility (BA) of common beans was affected by processing methods. The in vitro mineral bioaccessibility was determined in terms of dialyzable portions of minerals after in vitro digestion. The summarized results of mineral bioaccessibility are presented in Table 2. Effects of soaking, germination and cooking on bioaccessibility of Iron Soaking, germination and cooking treatments greatly affected the in vitro iron bioaccessibility. The iron BA was significantly different among treatments at P<0.05, and the values lie in the range of 1.41%-7.79% with the values representing control samples and pressure cooking of presoaked beans respectively. The respective iron BA values for beans soaked in water and soaked in sodium bicarbonate for 12 hours (overnight) was 2.22% and 2.59%. According to Afify et al ., (2011), in vitro iron bioavailability of white sorghum varieties found to be increased from 8.02−13.16% to 14.62−20.75% after application of soaking treatment which is higher than the result of this study. Table 2: The Iron, Zinc and Copper bio-accessibilities of processed beans Treatments Mineral bioaccessibility (%) Iron (Fe) Zinc (Zn) Copper (Cu) Untreated (control) beans 1.41±0.30 B 5.46±2.88 A 5.88±3.07 A Soaked in water overnight 2.22±0.37 B 6.94±2.01 A 6.04±0.40 A Soaked in NaHCO 3 overnight 2.59±0.84 B 6.94±0.19 A 6.78±0.86 A Germinated for 24-hours 2.72±1.01 B 7.58±0.47 A 7.09±1.75 A Germinated for 48-hours 4.27±2.20 B 10.08±4.32 A 7.63±0.21 A Soaked overnight in water+ Ordinary cooking 3.56±1.91 B 6.52±1.53 A 5.79±1.16 A Soaked overnight in water+ pressure cooking 7.79±0.19 A 8.41±4.12 A 8.50±0.20 A Grand Mean 3.51 7.49 6.81 [2] All the values are the mean of triplicate results. The means assigned with different letters along the columns are significantly different. This variation in bioavailability/bioaccessibility could be due to the difference in crop type and genetic background. The soaking process (Water and sodium bicarbonate) was resulted in the enhanced iron BA as compared to untreated beans. In principle, this result correlates with Ruel and Levin [29] who reported that soaking flour for 24 hours resulted in the increment of the amount of soluble iron by tenfold, though there is difference in level of increment in bioaccessibility. However, Sabrina et al. Sabrina et al ., [30] reported the lower bioaccessibility of iron (0.2%) for water-soaked black beans followed by ordinary cooking, which is lower as compared to this study. The in vitro solubility of iron was enhanced by 2–23% due to the application of soaking [31]. The soaking process can soften the seeds and enhance the degradation of complex matrices, including phytate salt, which in turn results in enhanced iron bioaccessibility. Germination is also another processing method that results in enhanced iron bioaccessibility. In this study, in vitro iron bioaccessibility of 2.72% (2 folds) and 4.27% (4 folds) was achieved by germinating for 24 hours and 48 hours, respectively, which is a 2 fold and 4 fold increases as compared to untreated beans. The role of germination and spreading in increasing mineral bioavailability by reducing tannin and phytic acid was previously reported [32]. It was also stated by [33] that germination improves the bioavailability of minerals like iron, calcium, copper, manganese, and zinc. The current investigation is also in agreement with the previous study, which showed a significant increase in the in vitro iron bioavailability of lentil, cowpea, chickpea, and green gram following germination for 24 hours [32]. Heat treatment or cooking was also found to be the other processing method that resulted in enhanced iron BA in beans. It was observed that cooking presoaked beans resulted in an iron BA result of 3.56% (ordinary cooking) and 7.93% (pressure cooking), which is higher than the unprocessed or control beans in this study. This shows that cooking greatly enhanced the mineral bioavailability as compared with the control. This could be due to the effect of thermal energy on the food matrix (complex salt degradation), especially by the steam that circulates in the pressure cooker. This result is almost correlated with the results reported by other authors in previous studies. Previous research conducted on different food products showed that the bioavailability of iron in kidney beans was found to be 9.40% [8] . The previous study showed that presoaked, regular pan-cooked cowpeas were reported to have an iron biao accessibility of 8.92%, and pressure-cooked beans had 44.44% iron BA, which is by far greater than that of this study [37]. This variation may be due to the crop type, crop composition, and agroecological differences. The amount of soluble and bioaccessible iron was increased by the application of a pressure cooker. A similar effect was reported by previous studies, which found that cooking improved the absorption and digestibility of iron[34]. According to [28], the iron solubility, another way of determining mineral BA, was reported to be 8.02–13.60% for the raw sorghum grains, 14.62–20.75% for the soaked ones, and 16.67–20.63% for the germinated grains. It has also been reported that soaking and germination are processes that activate the endogenous phytase present in the plant material. Soaking may also lead to a phytate reduction through water solubilization and the subsequent leaching of some phytic acid salts [35]. The less or lack of mineral bioaccessibility improvement after application of appropriate processing techniques could be because PA as IP6 is not degraded well to lower forms of inositol phosphate. According to [36] , 90% IP6 degradation is required to result in a two-fold increase in the bioavailability of iron. Generally, the result of the current study showed that the highest iron BA was achieved by pressure cooking of presoaked beans, followed by germination for 48 hours. Previous studies stated that the iron from plant sources that is non-haem has a lower rate of absorption (2–20%) as compared to animal sources (haem) iron (15-35%) [29], [38]. Non-haem iron is less bioavailable due to the presence of phytate, complex, and insoluble salts. Individual iron status and requirements, sources, contents, and meal compositions are among the factors that contribute to the lower bioaccessibility and availability of non-haem iron [39]. For iron from plant foods (nonheme iron), the most important thing is to enhance its bioavailability by lowering phytic acid and tannins or increasing the use of promoters such as ascorbic acid. The plant source of iron is less bioavailable, and the raw agricultural commodities need to be treated with different processing methods that help enhance the absorption and bioavailability of nutrients. As a consequence, it is advisable to apply various processing methods that can enhance the mineral bioavailability of plant-based foods. This is very important from the point of view of nutrition because most low-income and rural consumers rely on legume-based foods. Iron is a mineral of importance from a public health point of view, especially for children and pregnant and lactating women. To help protect these groups from iron deficiency anemia, It is also an essential micronutrient that causes anemia when consumed insufficiently and exists in every cell of the human body. Iron contributes to several metabolic functions, including hemoglobin transport and storage, electron transport, and energy production [40], [41]. Iron is a micronutrient of public health importance and an essential part of hemoglobin for oxygen transport and myoglobin for transporting and storing oxygen in the muscle and releasing it when needed during muscle contraction. It also takes part in facilitating the electron transfer system, ATP synthesis, and the formation of red blood cells and their function [42]. Iron is crucial for the differentiation and growth of epithelial tissue. It forms highly-toxic hydroxyl radicals, thus involved in the killing of bacteria by neutrophils; a component of enzymes critical for the functioning of immune cells (e.g., ribonucleotide reductase involved in DNA synthesis); involved in the regulation of cytokine production and action [43]; iron-rich status promotes the M2-like macrophage phenotype and negatively regulates the M1 pro-inflammatory response [44]. Involved in the regulation of cytokine production and action, it is required for the generation of pathogen-killing (reactive oxygen species) ROS by neutrophils during an oxidative burst[45]. In industrialized countries, well-nourished adults can have 3-5 grams of iron, of which about 65% is in the form of hemoglobin. The rest of the iron in the body exists in the form of myoglobin, other heme compounds that promote intracellular oxidation, or is stored as ferritin in the reticuloendothelial system and cells of liver hepatocytes, bone marrow, and spleen [46]. The beans are excellent sources of iron, which provides reasonable amounts in our daily diet. The recommended daily intake (RDI) for iron falls between 8 and 11 mg for men and women, respectively. Approximately, beans provide 0.75-0.95 mg of zinc per ½ cup serving, or approximately 10–11% of the RDI for women and 6–8% of the RDI for men [47]. The current study also showed that to obtain a reasonable amount of bioaccessible iron, it is important to apply pressure cooking. Since iron is crucial from nutrition as well as a health point of view, it is required to consider the appropriate processing techniques of legumes and cereals, which can help improve its bioavailability, especially in developing countries. Effects of soaking, germination and cooking on Bioaccessibility of Zinc The BA of zinc was found to be in the range of 5.46%–10.08%, with the respective values representing control and germination for 48 hours, respectively (Table 2). Soaking in water and sodium bicarbonate contributed to the improved zinc in vitro bioaccessibility of 5.46%–6.74% and 5.46%–6.743%, respectively. This improvement in the bioaccessibility of zinc for both the soaking types (water and sodium bicarbonate) is relatively low as compared to other treatments. This could be due to the short soaking time or the genetic background of the beans investigated. The Suliburska & Krejpcio (2014) study reported a lower result of zinc bioaccessibility than this study for hazelnuts (1.9%) and a nearly similar result for white sorghum varieties (7.35%−9.73% to 9.07−10.72% due to the application of soaking). Application of germination also contributed to the improved bioaccessible zinc in beans. The respective mean values of in vitro zinc bioaccessibility is presented in Table 2. In this study, germination treatment showed the increased zinc bioaccessibility as compared to the untreated beans which resulted in lower in vitro zinc bioaccessibility of 5.46%. The germination time of 24-hours and 48-hours resulted in 1.4-and 2-folds enhancement in zinc bioaccessibility. Similar results with regard to the role of germination in improving the bioavailability and absorption of minerals have been reported in previous studies. For instance, enhancement of zinc bioavailability in sorghum varieties was achieved due to the application of germination [28]. An ordinary and pressure cooking of pre-soaked (overnight) beans affected the in vitro bioaccessibility of zinc at P<0.05, as shown in Table 2. In vitro zinc bioaccessibility achieved was 6.52% and 8.41% for ordinary and pressure cooking, respectively. The cooking type had a significant effect on zinc bioaccessibility, and the best result was achieved by the application of pressure cooking as compared to ordinary cooking and control. This could be due to thermal energy or steam and the pressure applied to the seed matrix. However, this result is lower than the zinc bioaccessibility value reported by [37] for Brazilian cowpeas bred, which was in the range of 38.81%–52.78% after application of ordinary cooking. This kind of variation could be made as a result of different factors, like differences in cooking conditions, analytical procedures followed, and personal errors during analysis. Generally, germination for 48 hours resulted in the highest zinc BA in this study. This may be due to the activation of the phytase enzyme by presoaking and germination for 2 days, which in turn allowed further degradation of phytate and made the zinc ion free of complex formation. However, contradictory results were documented in previous studies in which the highest bioaccessibility of minerals was achieved by pressure cooking presoaked beans [30]. The current study showed that in vitro zinc bioaccessibility is higher than that of other minerals. The highest in vitro zinc bioaccessibility obtained in this study agrees with previous results reported for common beans [48]. Zinc plays an important role in nutrition and health. It exerts its ubiquitous effects on immune function, disease resistance, and general health. Analysis of animal and human studies examining the in vivo effects of nutritional zinc deficiency and supplementation on immune cells and their function underscores the essential role of zinc in the normal development and function of many key tissues, cells, and effectors of immunity [49]. Zinc also helps maintain the integrity of the skin and mucosal membrane (e.g., cofactor for metalloenzymes required for cell membrane repair [50]. It plays a central role in the cellular growth and differentiation of immune cells [51] and nd activates the phagocytic activity of peritoneal macrophages for E. coli and S. aureus [52]. Effect of soaking, germination and cooking on bioaccessibility of copper The effects of soaking, germination, and cooking treatments were studied and resulted in a significant enhancement of copper bioaccessibility. The in vitro copper BA results for soaking, germination, and cooking were found to be 6.04% (soaking in water), 6.78% (soaking in NaHCO3), 7.06% (germination of 24 hours), 7.63% (germination of 48 hours), 5.79% (ordinary cooking), and 8.50% (pressure cooking). A slight increment in the bioaccessibility of copper was observed for soaking treatment. Germination played a vital role in increasing the bioaccessibility of copper in common beans. Though there was no significant difference between the two-germination times in improving bioaccessibility, germinating for 24- and 48-hours resulted in 7.06% and 7.63% bioaccessible copper, respectively, and this shows the germination treatment enhanced the amount of bioaccessible copper. Cooking, on the other hand, contributed to enhancing the amount of bioaccessible minerals in beans. The in vitro copper bioaccessibility of 5.79% and 8.50% was obtained by ordinary and pressure cooking, respectively. The enhancement of mineral bioaccessibility could be due to the degradation of phytate during thermal treatment by pressure cooking. A similar study reported that 70% BA of copper was achieved by applying pressure cooking to black beans [30]. This difference might occur due to the nature of the bean sample, the analytical methods applied, and the procedures applied during processing. Processing methods like fermentation, drying, hydration, cooking, and germination could affect the BA of minerals due to the interference with the food matrix. According to [53], the soaking of foods or raw grain seeds in water increases the amount of soluble minerals through passive diffusion or leaching of water-soluble anti-nutritional factors, including sodium, potassium, or magnesium phytate. The bioavailability of minerals in foods can be affected by the presence of phytates, tannins, and polyphenols. ANFs are shown to cause the complexing, inhibition, and binding of minerals to form complex salts, in turn decreasing their bioaccessibility [54]. Thermal treatment (cooking) can enhance the bioaccessibility of minerals due to the softening of the food matrix and consequently the release of protein-bound minerals [55], and by reducing solubility inhibitors such as phytates, tannins, and phenolic compounds [56]. The current study reveals that Cu and Zn bioaccessibility is better than that of iron. This could be due to the nature of the minerals, in that their capacity to be solubilized when they are in a complex form like phytate is different. The study on the solubility and hydrolysability of metal phytate complexes and metal species has shown that Cu, Zn, and Fe are more soluble. The author also illustrated that the acidic PH (5) resulted in greater solubility and hydrolyzability of phytates than the nearly neutral PH (7.5) [57]. Copper is an important cofactor of cytochrome C-oxidase, a component of the mitochondrial respiratory chain, and is involved in the metabolism of iron. The role of copper in neutrophils and monocytes enhances NK cell activity [58]. It maintains intracellular antioxidant balance, suggesting an important role in the inflammatory response [59]. The beans, as a good source of copper, must undergo appropriate processing to make the copper available for intestinal absorption. It has been reported that pressure cooking is an appropriate processing method to reduce antinutritional factors and, in turn, enhance the bioaccessibility of minerals [60]. CONCLUSIONS AND RECOMMENDATIONS The common bean is one of the most consumed legumes in Africa, especially in the southern and eastern parts of the continent. Being a good source of plant-based proteins such as Fe, Ca, Zn, and copper, it is consumed alone or mixed with other foods. The effect of processing methods on in vitro mineral bioaccessibility was studied in this experiment. This study reveals that different processing techniques, including soaking, germination, and cooking, affected the in vitro mineral bioaccessibility of common beans. In vitro iron, zinc, and copper bioaccessibility was found to be enhanced by soaking, germination, and cooking. Though all of the processing methods resulted in enhanced in vitro mineral bioaccessibility, pressure cooking was found to be the best processing method for increasing the in vitro bioaccessibility of iron and copper. This indicates that thermal cooking with a pressure cooker significantly degrades the bean matrix and reduces its anti-nutritional content. It is therefore recommended to be used in societies where there is a pressure cooker facility. The ordinary cooking of presoaked beans is also used in this study. Ordinary cooking, which is traditionally used in rural areas of bean-producing countries, also resulted in a better result in increased in vitro mineral bioaccessibility following pressure cooking. Germination for 48 hours showed the highest in vitro zinc bias (10.08%) as compared to others in this study. The beans are widely consumed alone or mixed with other dishes in African countries. Considering this reality, the appropriate processing techniques, especially soaking in water followed by ordinary cooking, should be applied to enhance the bioaccessibility of essential minerals. Generally, the in vitro bioaccessibility of essential minerals in the human body (Fe) and the essential micronutrients widely known for their optimal growth and reproduction roles (Cu and Zn) could be enhanced by the application of different processing methods. The Agricultural Research Centers should also train the rural communities that are relying on legumes and beans in particular as protein and essential mineral sources and closely follow them to practice appropriate and easily applicable processing techniques. Agricultural research and academic institutions should mainly focus on rural communities and provide training on how to process legume crops to improve nutritional value in terms of bioavailability. Declarations Acknowledgement I would also like to thank staffs of University of Eduardo Mondlane Eng. Asimina Esmail Sulemane, Dr. Adolfo Firmino Condo, and Eng. Virginia Ivone Gongole for their technical support during the laboratory works. I am also thankful to European Union (EU) for financial support and Intra-Africa Academic Mobility Scheme (MOUNAF) project coordinators for their sincere supports. Conflict of interest All authors declare no conflict of interest in this manuscript. Data Availability All data generated or analysed during this study are included in this published article. References R. B. Canani et al. , “Zinc inhibits cholera toxin-induced, but not Escherichia coli heat-stable enterotoxin-induced, ion secretion in human enterocytes,” J. Infect. Dis. , vol. 191, no. 7, pp. 1072–1077, 2005, doi: 10.1086/428504. H. A. & J. A. R. 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1","display":"","copyAsset":false,"role":"figure","size":48741,"visible":true,"origin":"","legend":"\u003cp\u003eCommon bean seeds for the experiment (A) and bean soaking (B)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4873186/v1/f03dbf3b4e2b7351f7c473d4.jpg"},{"id":64439696,"identity":"a0d73681-f758-45e0-9044-460421f78bfe","added_by":"auto","created_at":"2024-09-13 07:58:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79195,"visible":true,"origin":"","legend":"\u003cp\u003eGerminated common bean seeds for 24hrs (A) and 48hrs (B)\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4873186/v1/ec0094e04be997e22ba7c988.jpg"},{"id":64439693,"identity":"5fc064ec-7ef9-46bd-86ab-2ff3cc32f28e","added_by":"auto","created_at":"2024-09-13 07:58:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30984,"visible":true,"origin":"","legend":"\u003cp\u003eOrdinary (Right-hand) and pressure (Left-hand) cooking of common bean\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4873186/v1/02608eb6dadf06b08e2c8b00.jpg"},{"id":64440552,"identity":"cb534ed4-4033-4f95-964f-73e2500ce6c4","added_by":"auto","created_at":"2024-09-13 08:06:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67155,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of in-vitro digestion pr\u003cem\u003eocess Source: \u003c/em\u003e[23]\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4873186/v1/14b1d22f4a62a8a04dae2594.jpg"},{"id":100069264,"identity":"4deb715b-1b95-47ed-8b2a-616e48a7c30c","added_by":"auto","created_at":"2026-01-12 16:12:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1107596,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4873186/v1/7f81d5cb-30ab-48aa-bf22-0db869cf3869.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Processing Methods on Mineral Bioaccessibility in Common Beans (Phaseolus Vulgaris L.)","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMicronutrient deficiencies in societies rely on the legumes as staple food, particularly beans-based diet, is a problem highly related with effect of these antinutritional factors. Zinc and iron deficiency is widespread in low-income countries because of a low consumption of animal products with a high content of bioavailable dietary iron and zinc due to their unaffordable costs. The consumption of cereals and legumes which are high in antinutritional factors is common in developing regions\u0026nbsp;[1], [2].\u0026nbsp;Among legumes, which provide reasonable amount of both macro-and-micronutrients, common beans (\u003cem\u003ePhaseolus vulgaris L\u003c/em\u003e.), are widely produced and consumed in Africa including Mozambique and Ethiopia. Common beans are the most commonly consumed legume worldwide, and produced for direct human consumption\u0026nbsp;[3], [4].\u0026nbsp;They are a staple food and the major source of iron for populations in Eastern Africa and Latin America.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dried seeds of common beans are an important source of dietary protein for millions of people throughout the tropics, complementing those amino acids lacking in diets based on cereals :lysine and tryptophan as well as good source of \u0026nbsp; minerals iron, copper and zinc\u0026nbsp;[5], [6]. Besides, common bean is a nutritious food low in fat and high in protein. They\u0026nbsp;are also part of many traditional diets, playing a major role in vegetarian diets being consumed in different dishes together with other food products in different countries\u0026nbsp;[7], [8].\u0026nbsp;Consumption in medium to high amounts of beans is being associated with a decrease in some types of cancer, lower incidence of degenerative diseases and reduction of diseases such as diabetes, cardiovascular diseases and even neoplasms\u0026nbsp;[9], [10].\u003c/p\u003e\n\u003cp\u003eHowever, beans are known to be rich in anti-nutritional factors such as phytates, tannin and , oligosaccharides which reduce the digestion and bioavailability of nutrients in beans\u0026nbsp;[11]. \u0026nbsp;Phytate is one of the main factors limiting the bioavailability of mineral cations in common beans, and polyphenols might hinder the absorption of nutrients\u0026nbsp;[12]\u0026nbsp;The low iron absorption has been reported to be attributed to inhibitory compounds\u0026nbsp;[13]. \u0026nbsp;The consumption of common beans at wider level is limited by the presence of these anti-nutritional factors.\u0026nbsp;They affects nutritional quality especially protein digestion and mineral absorption\u0026nbsp;[14].\u0026nbsp;The poor bioavailability of essential minerals leads to public health problem such as\u0026nbsp;leading to iron deficiency anemia and Zinc Deficiency. As a consequence, low utilization of the common beans results in food insecure, malnourished and unproductive society which indirectly affects the sustainable development of the country. The role of traditional or improved processing techniques in reducing ANFs and improving sensory quality of beans have been reviewed well\u0026nbsp;[15]. The beans, before their consumption, are cooked, and though most of the consumers prepare food at household level. It was also reported in previous studies that different processing techniques including\u0026nbsp;soaking, cooking, germination, fermentation and enzymatic treatments\u0026nbsp;can contribute for reducing ANFS and improved bioaccessibility/bioavailability of nutrients especially minerals and protein, improve sensory properties\u0026nbsp;[16].\u0026nbsp;According to\u0026nbsp;Matella \u003cem\u003eet al\u003c/em\u003e.,\u0026nbsp;[17], soaking and germination can increase the utilization of kidney beans.\u0026nbsp;Bioaccessability is the amount of substance that is solubilized in the gastrointestinal environment. It is the fraction that is released from food matrix into the gastrointestinal tract during the digestion process, \u0026nbsp;and thus, available for intestinal absorption and can enter the bloodstream\u0026nbsp;[18].\u0026nbsp;\u0026nbsp;Therefore,\u0026nbsp;to be absorbed, nutrients must first be released from the food matrix and modified in the gastrointestinal tract. As a consequence, in vitro digestion models that mimic the complex physicochemical and physiological conditions of the human GI tract have been developed and used for the purpose of studying nutrients bioaccessibility\u0026nbsp;(Hur \u003cem\u003eet al\u003c/em\u003e., 2011).\u0026nbsp;This research was conducted on small black common beans variety released and culivated by \u003cem\u003eInstituto de Investigacao Agraria de Mozambique\u003c/em\u003e, to evaluate effects of different processing techniques (hydrothermal and others) on in vitro minerals bioaccessibility bean through reduction of ANFs. This study achieved the enhanced of mineral bioaccessibility as the phytate and tannin content of beans were reduced as a functions of different processing techniques. The improved bioaccessibility of minerals contributes to combat micronutrient deficiency.\u0026nbsp;\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003ch2\u003eDescription of Study Area\u003c/h2\u003e\n\u003cp\u003eThe laboratory experiment was conducted at three faculties of Eduardo Mondlane University including Chemical Engineering (food technology) laboratory, Microbiology Laboratory and Department of Chemistry (central laboratory) faculty of Engineering, Veterinary, and Sciences, respectively at Eduardo Mondlane University. The site is located at Mozambique, Maputo. The area is located in the Southeast coast of Africa at 18.6677\u003csup\u003eo\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003eS, 35.5296\u003csup\u003eo\u0026nbsp;\u003c/sup\u003eE.\u003c/p\u003e\n\u003ch2\u003eMaterials\u0026nbsp;\u003c/h2\u003e\n\u003ch3\u003eChemicals and reagents\u003c/h3\u003e\n\u003cp\u003eThe reagents used for the experiment were pancreatin from porcine pancreas (P3292-SIGMA ALDRICH, USA), Pepsin from porcine gastric mucosa (P7000-SIGMA ALDRICH, USA), Glycine hydrochloride (G2879-SIGMA ALDRICH, USA), molecular porous membrane tubing (MWCO-8000Da, Rancho Dominguez CA 90220-6425 US, \u0026amp; Canada) and Sodium Bicarbonate.\u003c/p\u003e\n\u003ch3\u003eCollection and Preparation of Raw Materials\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eThe matured, unbroken and free of defects black small common bean samples (A\u003csub\u003e222\u003c/sub\u003e-bean variety) were collected from \u0026lsquo;\u003cem\u003eInstituto de Investiga\u0026ccedil;\u0026atilde;o Agraria de Mo\u0026ccedil;ambique\u0026rsquo;\u003c/em\u003e (IIAM), Maputo, Mozambique. The beans were cultivated in the year 2020 cropping season and harvested in December, 2020. \u0026nbsp;About 5 kg of the collected bean samples were sorted for sands, husks, soils and any foreign materials and kept at room temperature prior to processing by respective processing methods; soaking, cooking and germination. The control sample (raw bean) was milled by laboratory mill (ZM200-Retsch Rheinische Stra\u0026szlig;e 3642781 Haan, Germany) and stored at room temperature prior to mineral analysis and in vitro digestion assay.\u003c/p\u003e\n\u003ch3\u003eTreatment\u0026rsquo;s setup\u003c/h3\u003e\n\u003cp\u003eThe collected beans were subjected to three different processing methods with two levels. The processing methods studied in the experiment were soaking (water and NaHCO\u003csub\u003e3\u003c/sub\u003e), cooking of previously soaked beans in water (ordinary and Pressure), and germination (1 and 2 days). The sample size was 7 including the control sample which was not subjected to any processing techniques.\u003c/p\u003e\n\u003ch2\u003eProcessing of Methods of Beans\u003c/h2\u003e\n\u003cp\u003eThe raw beans samples were sorted, cleaned, and ground using miller (ZM200-Retsch Rheinische Stra\u0026szlig;e 3642781 Haan, Germany) to use as a control sample for anti-nutritional, minerals analysis, and in vitro digestion. The remaining beans samples were processed using three processing methods: soaking, germination, and cooking. Soaking \u0026nbsp; germination and cooking were performed following method of Emire \u0026amp; Rakshit [20] \u0026amp; Kaur \u0026amp; Kapoor [21], and [22] for pressure cooking. The pH of water and sodium bicarbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e) solution before and after processing was measured by PH meter during the conduction of an experiment.\u003c/p\u003e\n\u003ch3\u003eSoaking\u003c/h3\u003e\n\u003cp\u003eThe cleaned common beans seed free of broken seeds and other foreign matter were soaked for 12h in distilled water and (0.05%) NaHCO\u003csub\u003e3\u003c/sub\u003e solution at 1:3 ratios (seed-to-solution) at ambient temperature. The pH\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eof the water and sodium bicarbonate solutions were 7.2 and 8.84 respectively. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe soaked seeds were washed twice with water followed by rinsing with distilled water and was then dried in an oven at 60\u003csup\u003e○\u003c/sup\u003eC. The dried samples were ground, stored in vacuum plastic for further analysis. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eGermination\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003e\u0026nbsp;The common beans were weighed (120g) into plastic bowl and soaked in distilled water (bean-to-water ratio of 1:5w/v) for 12 h at room temperature. The seeds were then incubated to germinate in an incubator at 25\u003csup\u003eo\u003c/sup\u003eC for 24 and 48 hours in aluminium foil and muslin clothes. The distilled water was sprinkled on the seeds (twice a day) during germination period. The germinated beans were dried in a hot air oven at 60\u0026deg;C, ground using sample mill and stored in vacuum plastic bags for further analysis.\u003c/p\u003e\n\u003ch3\u003e\u0026nbsp;Cooking\u003c/h3\u003e\n\u003cp\u003eThe previously soaked beans in tap water for 12 hours were cooked in conventional cooking pan with a seed to water ratio 1:6 at 100\u003csup\u003eo\u003c/sup\u003eC for 1.47 hours (ordinary cooking) and 100\u003csup\u003eo\u003c/sup\u003eC for 38 minutes (pressure cooker). The cooked samples were mashed manually with mortar and pistle, and dried in a hot air oven maintained at 60\u003csup\u003e○\u003c/sup\u003eC and then, ground to a fine powder and stored in vacuum plastic bags for further analysis. The figures 1 (A \u0026amp; B), 2 (A \u0026amp; B) and figure 3 shows the pictures taken during laboratory experiment.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDetermination of Moisture Content\u003c/h2\u003e\n\u003cp\u003eThe empty petridishes were oven dried for 1h at 105\u003csup\u003eo\u003c/sup\u003eC, transferred to the desiccators cooled for 30 min, and weighed. The prepared beans flours were mixed thoroughly. About 2.00g of fresh samples were put into the previously dried and weighed petridishes. The dishes and their contents were placed in the drying oven and allowed to dry for 1h at 105\u003csup\u003eo\u003c/sup\u003eC. Then, after drying, the petridishes and their contents were taken out of the oven and cooled in desiccators and reweighed. The amount of water present in a sample is considered to be equal to the loss of weight after drying the sample to constant weight at a temperature near the boiling point of water (AOAC 925.10).\u003c/p\u003e\n\u003cp\u003e\u003cimg 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width=\"479\" height=\"40\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of total ash content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAsh was determined by incineration of known weights of the samples in a muffle furnace at 550\u003csup\u003eo\u003c/sup\u003eC until a white ash was obtained. Organic matter was burned off and the inorganic material remaining is cooled and weighed. Heating was carried out in stages, first to derive the water, then to char the product thoroughly and finally to ash at 550\u003csup\u003eo\u003c/sup\u003eC in a muffle furnace. The ashing dish was placed into a muffle furnace for 30 min at 550\u003csup\u003eo\u003c/sup\u003eC. The dishes were removed and cooled in desiccators (with granular silica gel) for about 30 min at room temperature; each dish was weighed to the nearest g. About 4 g of flour sample was added into each dish. The dishes were placed on a hot plate under a fume-hood and the temperature was slowly increased until smoking ceases and the samples become thoroughly charred. The dishes were placed inside the muffle furnace at 550\u003csup\u003eo\u003c/sup\u003eC for 18 h, and removed from the muffle and then placed in desiccators for 1h to cool. The ash was clean and which in appearance. When cooled to room temperature, each dish + ash was reweighed. Weight of total ash was calculated by difference and expressed as percentage of the fresh sample.\u003c/p\u003e\n\u003ch2\u003eIn vitro Mineral Bioaccessibility Assay\u003c/h2\u003e\n\u003cp\u003eThe in vitro gastrointestinal digestion assay was conducted to determine the\u0026nbsp;bioaccessible portions of minerals in beans treated with different processing methods.\u0026nbsp;The determination of mineral bioaccessibility was performed based on the in vitro digestion method described by\u0026nbsp;[23]\u003cstrong\u003e. \u0026nbsp;\u003c/strong\u003eThe gastric (phase) digestion simulation was performed by weighing 4 grams of ground bean was suspended in previously prepared 24mL of 20mM glycine-HCl buffer solution. Then, the pH was adjusted to 2.0 by adding 2M HCl, and 1mL pepsin enzyme solution prepared by dissolving 1.6 grams of pepsin in 10mL 20mM glycine-HCl buffer was added in to the suspended samples. The suspension was then incubated in hybridization shaker (Sl-30H, BIBBY SCIENTIFIC LIMITED STONE, STAFFORDSHIRE, ST15 OSA, UK) for duration of 2 hours at 37.4\u003csup\u003eo\u003c/sup\u003eC. \u0026nbsp; Pepsin enzyme was inactivated by putting sample solutions into ice box prior to the commencement of intestinal phase.\u003c/p\u003e\n\u003cp\u003eThe pH of the gastric digested suspension was adjusted to 7.0 with 1 Molar NaHCO\u003csub\u003e3\u003c/sub\u003e to simulate intestinal (phase) condition. To the gastric digested samples, 10mL of a pancreatin enzyme solution previously prepared from 0.4 gram of Pancreatin in 100mL of distilled water was added, and a previously rinsed dialysis membrane (with distilled water) containing 2mL of dionized water was placed in the digestion system. Then, it was again incubated under the same condition for 2 hours. Finally, the dialysis membrane was removed from the system and the contents of minerals in the dialysate were quantified by ICP-AES. The bioaccessibility of minerals in terms of percent was calculated by the following formula. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"625\" height=\"51\"\u003e\u003c/p\u003e\n\u003ch2\u003eDetermination of mineral\u0026nbsp;concentration in beans\u003c/h2\u003e\n\u003cp\u003eThe minerals content of the beans were determined according to Official methods of analysis\u0026nbsp;[24], and Inductively\u0026nbsp;Coupled\u0026nbsp;plasma Atomic Emission Spectroscopy\u0026nbsp;(ICP-AES-9820 Plasma Atomic SHIMADZU South Africa)\u0026nbsp;was used for determination minerals. Two grams of the sample flours were weighed, and 10 ml of the mixture of HNO\u003csub\u003e3\u003c/sub\u003e (65%v/v) and HCl (37%v/v) was added. To digest the samples, the mixture was placed on the heating mantle, and digestion was stopped when about 2ml of the solution was left in the digestion cup. The sample was then diluted with ultrapure water, centrifuged and filtered using filter paper (Whatman No.40). The clear sample solution of about 14ml was used for each mineral analysis by ICP-AES. The mineral content of the sample was calculated by the following formula and expressed as mg/kg dry weight basis.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAh0AAAA0CAYAAAAuXIowAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAA9dSURBVHhe7Z3Lix1FG4cr397LqKsgQYxZiIggo0IwiwheUBHB6BjjQlCiUQloRCUqLmLiBZ2AaIzBgIgSQwJujPECuogEjBdGNCjESAjiarz/AfnOU9O/4U2nz2XO9GnnHH8P1Jyu6qrq7qqerrfeeqtq0YkWyRhjjDFmwPyv+DXGGGOMGSgWOowxxhjTCBY6jDHGGNMIFjqMMcYY0wgWOowxxhjTCBY6jDHGGNMIFjqMMcYY0wgWOowZQX777bfiqD/mm94YY6qw0GHMiPHtt9+mycnJwtcfP/zwQ3rhhRcKnzHG1IOFDmNGiJ9//jk9++yzafPmzVloWLRo0ezv/fffn5544ol8zK/44IMP0llnnZXDcdddd1268sor05IlSyx4GGNqxcugGzNCIDBs27YtnX/++dl/2WWXpQ0bNqSrr746nXPOOWlqaiqddtpp6Zprrkk//fRTjkOau+66azZO/CSU8zPGmPlgTYcxIwJaDmwxooBw9tlnp3PPPTf/wiWXXJLPHz16NPvhqquuSqtXr84Cx8TERBE6A8LIzp07C58xxswPCx3GjAiffPJJuueeewrfDGWDUPzlsGPHjqXp6ems4Xj33XeL0BnQfuzevbvwjQ4IaAw3Yf/SCcrj9ddfL3zGmPliocOYEeG9995LF110UeFL6fPPP09fffVVeuaZZ2ZtM9BarFmzJh8r7Pfff0/Lli2btenAxkOgIRkbG8uNdN3IfqRpELoQODZt2pQ1P524/fbbc5lGGxhjTP9Y6DDmXwCBgEa3Tg4dOpQuvPDCwpeyMSjaiw8//DA9+uij+Zhf/DqGjz/+OB05ciSHbd++PX3//fc5XCB4/Prrr4WvPvbt21ccNQuC1/r162eHnFQXOI7LUI4MRw1C8DLmv4aFjhECNXA3dXGv0NuNPd7/OhhkVjVIvVJOL4GgTv7444/ZhnQurFu3Lttz0Oi+8cYb6dprry3OzIANyOHDhwtffZx++unFUbPs2LEjXXHFFYVvpi42btyYn5vjKlauXJn27t1b+Iwx/WKhY0RAXfzXX391VRf3yvXXX5/ef//9nG9TIDDRONP4MYVTAhTj6hdccEEO57zCUc0Txnni42LDHtPxC6jWlY4w5aVppQhuSqOxfMqAYYoVK1bkcPWMuRdNQdVQBWk0/bRbepyIz871ZVvR6b7qgum1CEC4L7/88pR36LzzzsvvViQOi6g8FcYxTnVBGalMGK4o25SAnlNpYn46x6/KgPIpXxfa1W8ErUVZODvjjDOKo2risJUxZh60PjRmyGn10k60equFr15avb8T27dvL3yDZenSpbPX4roHDhw40WogUAfkY+A5x8fH8/H09HQ+pzQTExMnnn/++XysdFNTUzne2NhYDic95QWkI40gX/k5xz0I3Y/gWPlzTRxhvabft29fTi94dt37rl278jnuGzrdVyTmB/j7dRE9H3Df3AN+ypJyxlG+lAXE+DwLz0YcnodnUflTHvFZOKcyIn35nN5x0sc84/12ql9Rfj4oX68M96VnMsb0jzUdQw49w9deey0bxQ0C1mi47777GhnPbjUi6d57783H2B2g6pYtgdTed9xxR9YagHqr6oVeeuml+RdI12pEcq+deBhLAs+xZcuW3BPmueLMDOIpj157tuSPbQRu8eLFuUdPD5u8O1EeWuDZly9fno/RBgCrgkI/9wWt/+++XeSbb77JC4UBGjC0HgxR3HjjjXnoBcc9o4FB07Bnz55Ze5Hjx4/n8iAOz3Hrrbemr7/+Op8ro/qsgnNoXACtRMwz0ql+RUtgqdS2RNCqSOsCBw8eLI6MMfPBQseQw8yExx9//KSPr9TLUvXjsM+QapoPquBYqm+5+LHlw97qPaYXX3yxCBkczJLQ0AH3SwPCQlage9q/f/8pNgeCxlDDAKT76KOPZtXryvfMM89MrR5rblhp6Fu94Rxe5u+//z6lYVJYuwar1cPOvyy6Rb5zSc+zq2Hj2fFHo1BRdV9ifHy8q3CodyPWcTf+/PPPvNYHyM5n7dq1efhN13vkkUey8PvAAw+krVu35jBAQKA8iMd9f/rpp3ldEKh6ll9++SWHIej0gtLrt5f65d6ZXhwpDx8hOEW4n1WrVhU+Y0zftP45zZCCypcqlBpeSOWMahuknicc9XhUI6MWV7w4vBFBbU5+qLMHCffB/XAtqcgBtbbCufeoxleYygInFT154I/PSFrKg3CeV3nF9KBjqdTjtShHfuN50JAJ+era3dLr+qQlndLjh273FaGOla4dnOf55wLlV37HuP8yDG2UhzNIR7jum2PlVX4WlRHPrzQ8f7syIFzlqPtpV78Rlb/uI+YfneB8fB+NMf1joWOI4cNc9fEHPpp8LIE4+rDrQy0I10ea36qPNPABrxJIzMIBwapb41iu/24gaM5VSBkGeC7Kqt37LihTv/fG1IeHV4YYLPilqu6Vskr7ueeeyzMWWu9C5cwFwV4dLD5lFi6sHsqaG3XCMAR7t4waDBsya6fbbC9sVWRnZMyooKF2XBxub4I5CR00Vkz/kw0Av/hjI2ZmGHRlMkbOugwyPozouth7MHaPbcNjjz2Ww2644YZsiKk4l19++ezLh4vTDyN8nMnHLFxkqFnX+ir8XyNoIswYY0YDvvF0TuhoHjhwILcNc7Hxmi89Cx18gFhQh94wjRY3zBbaGI/Jyn4YoLGmca0LrRfQND/++GP+ZcZEGWYOUD+aAcIxDnRMHIwsMfJT2NTUVFvBQrMmqtY9MAuHu+++O7311lttOwJouoQ6Ee146qmn0pNPPtlxVokxZriIu0bTPixdujQbcDdFz0IHHyCmHdKQ6YZRO46Pj+fjYUGNdV189913xdHJqGEfFFqqWnXRD8zwiGq2W265Je3atas4ezKaRfLPP//kX7MwQUB49dVX0+TkZBFyMi+//HIWLKlvViFtN9sFbQlTYtut0GmMGU6q2gxN02+EVuPYExgSVi20E5GlumYakEazBkAW6Rgv4ogXZxYIjN1k3IiLhmyyvq/KHz/hxNG1CJOxGHF1b3LcB+EcY2BHXOUjQ0ziKCzmpxkK0ZEmPqeoq2wEcYjfJFyvk1Ed5UecTm4uRozGGGMGB+1Q0/TcatFgxEa0ChpOBAJNraSBIp0ab8DPgyoMQYbGVZAWvxo34pEGOMexGmKEC/y6nuLSsBFGQ8+1orAU84uQhrgIEuTLPRCXtDjyqsqPMqlqSLlGLK86yibybwkd3d4BY4wxCxu1g3KxHRo0tQkdNMjEocGO0NDS4IpyPhIcBI1+1GxEqs7RSJcFlFiApIlCgeKUIU6760aIF/PjWboJHXWVTYR47c4RPl9XBeHx/uqkfH07u4XojBklopa/KeY0e+XYsWPF0anImLS8vDNjzO3GjaEcnyWS2xmucQ4jVtkg4I4ePXrKaoKRbhs5Raqui7EdG1ZhcMeGXP3M4KirbHqlVa/zdv2g1S47uXZGt1X3YGe30BxEOygclv98H2IYs8MIj2GkgxiG4xuwENNjJB/D9L8bw3ALIX3520PahZieuDEMB02lL4MtR6sjWfgaovWP1BPtVP301tEeaOijqjePtkEQJ/aWy5oHev7tpK5O56BK08G1YppOmo6qvKMmBcrxyvmL+Jx1lU2EeO3ODQquF8vCGGPMcIO2I2rcB03Pmo4HH3wwrwuBRKrpeOxnoc23sIhtNb7p6aefztIY6Pxtt92W/VVoCp/yZEMvtAmaN0xesqzVOe2jwTnuR9erAi1I1fRB8mf6J1qMTqBJEaRhimmZQ4cO5WtIuixTV9n8m6g+vMW3McaMDqtXr857JzVGIXz0BD11euckw8nwEpsF4BeNiM4TN/bukaZ0jhkgOLQnykvQi1c4WgTNFgHOEVdpYv4Kl0ZG41W4KMmRp/LmntvFA56PcPKkly8Ng3r8aDF0XWktys8JdZWNkBYkls0g0fV4XmOMMcOHtO7RNc0i/rQubIYMNCYtYSS1BJe83XgVjOGhGaqjihljZcl0bRFvhhfeHXYNZp0d3g9WJGwJlT2vyYE2Dw2ilwc3xsyVORmSmoUDQzYIHVokrIp33nmnOJo/bO3N/iumHqLBV0RGigh5g4ChOoYkN23alJe2ZwG78Tku8MdwJ8Ns3YYmjTGmjIWOIWZiYiLt2bOn8J1Ku1lA/cBa/TfffHPhM/MFDRVC40svvVSEzNjNIHQgBAxqNdudO3em9evXn/Ru9POeoBXB3qmTPZUxxpSx0DHEsM8Gxqi9fPjpOat3DSxzrV61XLvetTYQ88Zf9cF06LVr12bjbO1n88orr5wiEKCZQKNA/bDBogx6ScMUbtWdNCbEJR5hmiIZ2bFjR95DqQryVn4cd3tHVq5cmfbu3Vv4jDGmOxY6hhjNiunlw79kyZI8H1uzcdgUjOES7D3WrVuXtSbtetfEJU6dmhMzA4IHM5kQItB8xA3ZAM0EdUc9UV8HDx7M4dhiEJ9w6vX48eM5fMuWLemzzz5L09PTlfY31H+nety4cWPe+A9NRrd3xDOZjDFzxULHkEODg4FnJ20HPVR2C0VA0WY/TIFmx2DOdRo6ocfL+YcffrgIMXWyatWqvFMzOzajuZLwIJYvX56FCLQXu3fvLkJntFyE4Vi0Dz8wbMPGfRgRv/322zmsF1asWJFuuummdPHFF2dbD+j1HTHGmF6x0DHk0EDQYGEcWEbre9C7HRsby+pwqedR5W/dujX3Yll7pGqXQdI/9NBDufGylqNe0GgwAwQhEC0CwoMEwrguC4IAjT1aCzQNWn13zZo1eRfYI0eOZMEAbRd1yy6yX3zxRdqwYUO68847c9wI2pGYP+BHWOEdYM6+1sHp9o5I62KMMT3T+qCYEYA1QljrQ2umQHk9klaDk/2suaH1RuQ4xxxuQXzWEtHmeqZeVO7UBeWuelM49QNaJ4Y61DovpFE4jnoiD9ZsUR2zxovyiBAW6zS+B6BrEB7P4crvCHGj3xhjuuF1OkYIerqLFy+e7TF3gp41RqjEpaeLceGbb745u1aDjBulajejAXWNloTp1N20V53eEd61/fv3p82bNxexjTGmOx5eGSFoDHoROABVOap2xuuXLVuWVfcSOABhwwLH6IGgsW3btjQ5OTkrWLaj3TvCTJnDhw9b4DDGzBlrOowxxhjTCNZ0GGOMMaYRLHQYY4wxphEsdBhjjDGmESx0GGOMMaYRLHQYY4wxphEsdBhjjDGmESx0GGOMMaYBUvo/IRvfaH+qEaIAAAAASUVORK5CYII=\" height=\"52\" width=\"541\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere: Volume-volume of sample solution analyzed\u003c/p\u003e\n\u003cp\u003eMass-mass of sample used for acid digestion\u003c/p\u003e\n\u003cp\u003eConcentration-Concentration of minerals in the analyzed solution\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003ePreparation of standard\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe ULTRASPEC Certified aqueous Reference Material of 100 \u0026micro;g/mL was used to prepare standard solutions for mineral analysis. The series of standard solutions containing 100, 200, 400, 600, 800, 1200 and 1600 \u0026micro;g/L were prepared from mother solutions (100 \u0026micro;g/mL). The standard solutions were used to prepare the calibration curve where a graph of concentration vs intensity of standard solutions was plotted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompletely Randomized Design was used, and the experiments were conducted in triplicate and data was subjected to Analysis of variance (ANOVA), and least significant difference (LSD test) was employed to separate the means.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSIONS","content":"\u003ch2\u003eThe effect of processing methods on moisture and Ash content of beans\u003c/h2\u003e\n\u003cp\u003eThe moisture and ash content of processed beans along with control analyzed in the experiment is presented in Table 1. The statistical analysis showed that the moisture and ash contents are significantly different among treatments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1: Some compositions of processed beans\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMoisture (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAsh (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDry matter (%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUntreated (control) beans\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.13\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u0026plusmn;1.16\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.72\u0026plusmn;0.31\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e92.87\u0026plusmn;1.16\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSoaked in water overnight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.81\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u0026plusmn;0.52\u003csup\u003e\u0026nbsp;BC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.09\u0026plusmn;0.57\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e92.19\u0026plusmn;0.52\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSoaked in NaHCO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eovernight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.32\u0026plusmn;0.19\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.32\u0026plusmn;0.036\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e92.67\u0026plusmn;0.193\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGerminated for 24 hours at 25\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8.69\u0026plusmn;0.88\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.29\u0026plusmn;0.29\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e91.31\u0026plusmn;0.88\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGerminated for 48 hours at 25\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8.79\u0026plusmn;1.76\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.43\u0026plusmn;0.81\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e91.21\u0026plusmn;1.76\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSoaked overnight in water+ Ordinary cooking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8.73\u0026plusmn;0.94\u003csup\u003e\u0026nbsp;BC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.62\u0026plusmn;0.18\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e91.27\u0026plusmn;0.94\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSoaked overnight in water+ pressure cooking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.48\u0026plusmn;0.44\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.91\u0026plusmn;1.31\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e90.52\u0026plusmn;0.44\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eCV\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e10.97\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e11.15\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e1.10\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eLSD\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e1.70\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e1.52\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e1.07\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e[1]\u003cem\u003e\u0026nbsp;All the values are the mean of triplicate results. The means assigned with different letters in the column are significantly different.\u003c/em\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe moisture, ash, and dry matter content of processed beans is presented in Table 1. The statistical analysis showed that the moisture, ash and dry matter content of beans sample were affected by processing methods. The moisture content was a bit higher after soaking in water and sodium bicarbonate as compared to the control. Germination for 12\u0026ndash;48 hours as soaking followed by ordinary cooking was found to have almost similar moisture contents. However, soaking followed by pressure cooking resulted in the highest moisture content. This could be due to the absorption of water by the seed before cooking. This could be due to the typical functional properties of the bean matrix because soaking allows the water to disperse in the protein fraction and starch granules, which helps soften the texture of beans.\u003c/p\u003e\n\u003cp\u003eAsh, or the total mineral contents of beans, is a very important parameter and can be affected by the processing methods. The total mineral content of the beans decreased due to the processing methods applied as compared to the control, with germination for 24 hours resulting in the lowest 5.29% and soaking in sodium bicarbonate resulting in the highest 6.32% total mineral contents following the control sample. Since almost all of the processing methods applied include the steeping of the beans in water except the soaking in sodium bicarbonate treatment, the loss of ash content due to the leaching of mineral elements occurred. This result agrees with the previous study, where the ash content of baobab seeds decreased from 8.93% (raw) to 5.84% and 8.93% to 6.12% by soaking in water and sprouting, respectively [25]. According to Sarmento et al. [26], the dark black beans were found to have a 3.80% ash content, which is lower than the current result. The previous study reported that the ash content of common beans was in the range of 3.90%\u0026ndash;5.70% [26]. The similar results on the effect of processing techniques on the ash content of field beans reported a reduction from 3.00% to 2.66%, 3.00% to 2.83%, and 3.00% to 2.05% soaking for 18 hours, germination for 40 hours, and cooking of presoaked beans, respectively [27]. The dry matter content of the beans was also studied, and it was observed that the dry matter content was inversely correlated with the moisture content, which is also scientifically valid.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEffect of processing Methods on Minerals bioaccessibility\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eIn this study, the effect of processing methods on the bioaccessibilities of iron, zinc, and copper was studied. The statistical analysis showed that the mineral bioaccessibility (BA) of common beans was affected by processing methods. The in vitro mineral bioaccessibility was determined in terms of dialyzable portions of minerals after in vitro digestion. The summarized results of mineral bioaccessibility are presented in Table 2.\u003c/p\u003e\n\u003ch3\u003eEffects of soaking, germination and cooking on bioaccessibility of Iron\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eSoaking, germination and cooking treatments greatly affected the in vitro iron bioaccessibility. The iron BA was significantly different among treatments at P\u0026lt;0.05, and the values lie in the range of 1.41%-7.79% with the values representing control samples and pressure cooking of presoaked beans respectively. The respective iron BA values for beans soaked in water and soaked in sodium bicarbonate for 12 hours (overnight) was 2.22% and 2.59%. According to Afify \u003cem\u003eet al\u003c/em\u003e., (2011), \u0026nbsp;in vitro iron bioavailability of white sorghum varieties found to be increased \u0026nbsp;from 8.02\u0026minus;13.16% to 14.62\u0026minus;20.75% after application of soaking treatment which is higher than the result of this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2: The Iron, Zinc and Copper bio-accessibilities of processed beans\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.44444444444444%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"55.55555555555556%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eMineral bioaccessibility (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.18518518518518%\" valign=\"top\"\u003e\n \u003cp\u003eIron (Fe)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.48148148148148%\" valign=\"top\"\u003e\n \u003cp\u003eZinc (Zn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eCopper (Cu)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003eUntreated (control) beans\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e1.41\u0026plusmn;0.30\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\" valign=\"top\"\u003e\n \u003cp\u003e5.46\u0026plusmn;2.88\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e5.88\u0026plusmn;3.07\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003eSoaked in water overnight\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e2.22\u0026plusmn;0.37\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\" valign=\"top\"\u003e\n \u003cp\u003e6.94\u0026plusmn;2.01\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e6.04\u0026plusmn;0.40\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003eSoaked in NaHCO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eovernight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e2.59\u0026plusmn;0.84\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\" valign=\"top\"\u003e\n \u003cp\u003e6.94\u0026plusmn;0.19\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e6.78\u0026plusmn;0.86\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;Germinated for 24-hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e2.72\u0026plusmn;1.01\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\" valign=\"top\"\u003e\n \u003cp\u003e7.58\u0026plusmn;0.47\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e7.09\u0026plusmn;1.75\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003eGerminated for 48-hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e4.27\u0026plusmn;2.20\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\" valign=\"top\"\u003e\n \u003cp\u003e10.08\u0026plusmn;4.32\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e7.63\u0026plusmn;0.21\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003eSoaked overnight in water+ Ordinary cooking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e3.56\u0026plusmn;1.91\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\" valign=\"top\"\u003e\n \u003cp\u003e6.52\u0026plusmn;1.53\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e5.79\u0026plusmn;1.16\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003eSoaked overnight in water+ pressure cooking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e7.79\u0026plusmn;0.19\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\" valign=\"top\"\u003e\n \u003cp\u003e8.41\u0026plusmn;4.12\u003csup\u003eA\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e8.50\u0026plusmn;0.20\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003eGrand Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e3.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\" valign=\"top\"\u003e\n \u003cp\u003e7.49 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e6.81 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e[2]\u003cem\u003e\u0026nbsp;All the values are the mean of triplicate results. The means assigned with different letters along the columns are significantly different.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis variation in bioavailability/bioaccessibility could be due to the difference in crop type and genetic background. The soaking process (Water and sodium bicarbonate) was resulted in the enhanced iron BA as compared to untreated beans. In principle, this result correlates with Ruel and Levin [29] who reported that soaking flour for 24 hours resulted in the increment of the amount of soluble iron by tenfold, though there is difference in level of increment in bioaccessibility. However, Sabrina et al. \u0026nbsp;Sabrina \u003cem\u003eet al\u003c/em\u003e., \u0026nbsp;[30] reported the lower bioaccessibility of iron (0.2%) for water-soaked black beans followed by ordinary cooking, which is lower as compared to this study. The in vitro solubility of iron was enhanced by 2\u0026ndash;23% due to the application of soaking [31]. The soaking process can soften the seeds and enhance the degradation of complex matrices, including phytate salt, which in turn results in enhanced iron bioaccessibility.\u003c/p\u003e\n\u003cp\u003eGermination is also another processing method that results in enhanced iron bioaccessibility. In this study, in vitro iron bioaccessibility of 2.72% (2 folds) and 4.27% (4 folds) was achieved by germinating for 24 hours and 48 hours, respectively, which is a 2 fold and 4 fold increases as compared to untreated beans. The role of germination and spreading in increasing mineral bioavailability by reducing tannin and phytic acid was previously reported [32]. It was also stated by [33] that germination improves the bioavailability of minerals like iron, calcium, copper, manganese, and zinc. The current investigation is also in agreement with the previous study, which showed a significant increase in the in vitro iron bioavailability of lentil, cowpea, chickpea, and green gram following germination for 24 hours [32].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHeat treatment or cooking was also found to be the other processing method that resulted in enhanced iron BA in beans. It was observed that cooking presoaked beans resulted in an iron BA result of 3.56% (ordinary cooking) and 7.93% (pressure cooking), which is higher than the unprocessed or control beans in this study. This shows that cooking greatly enhanced the mineral bioavailability as compared with the control. This could be due to the effect of thermal energy on the food matrix (complex salt degradation), especially by the steam that circulates in the pressure cooker. This result is almost correlated with the results reported by other authors in previous studies. Previous research conducted on different food products showed that the bioavailability of iron in kidney beans was found to be 9.40%\u0026nbsp;[8]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe previous study showed that presoaked, regular pan-cooked cowpeas were reported to have an iron biao accessibility of 8.92%, and pressure-cooked beans had 44.44% iron BA, which is by far greater than that of this study\u0026nbsp;[37]. This variation may be due to the crop type, crop composition, and agroecological differences. The amount of soluble and bioaccessible iron was increased by the application of a pressure cooker. A similar effect was reported by previous studies, which found that cooking improved the absorption and digestibility of iron[34]. According to\u0026nbsp;[28], the iron solubility, another way of determining mineral BA, was reported to be 8.02\u0026ndash;13.60% for the raw sorghum grains, 14.62\u0026ndash;20.75% for the soaked ones, and 16.67\u0026ndash;20.63% for the germinated grains. It has also been reported that soaking and germination are processes that activate the endogenous phytase present in the plant material. Soaking may also lead to a phytate reduction through water solubilization and the subsequent leaching of some phytic acid salts\u0026nbsp;[35]. The less or lack of mineral bioaccessibility improvement after application of appropriate processing techniques could be because PA as IP6 is not degraded well to lower forms of inositol phosphate. According to\u0026nbsp;[36]\u0026nbsp;, 90% IP6 degradation is required to result in a two-fold increase in the bioavailability of iron.\u003c/p\u003e\n\u003cp\u003eGenerally, the result of the current study showed that the highest iron BA was achieved by pressure cooking of presoaked beans, followed by germination for 48 hours. Previous studies stated that the iron from plant sources that is non-haem has a lower rate of absorption (2\u0026ndash;20%) as compared to animal sources (haem) iron (15-35%)\u0026nbsp;[29], [38]. Non-haem iron is less bioavailable due to the presence of phytate, complex, and insoluble salts. Individual iron status and requirements, sources, contents, and meal compositions are among the factors that contribute to the lower bioaccessibility and availability of non-haem iron\u0026nbsp;[39]. For iron from plant foods (nonheme iron), the most important thing is to enhance its bioavailability by lowering phytic acid and tannins or increasing the use of promoters such as ascorbic acid. The plant source of iron is less bioavailable, and the raw agricultural commodities need to be treated with different processing methods that help enhance the absorption and bioavailability of nutrients. As a consequence, it is advisable to apply various processing methods that can enhance the mineral bioavailability of plant-based foods. This is very important from the point of view of nutrition because most low-income and rural consumers rely on legume-based foods.\u003c/p\u003e\n\u003cp\u003eIron is a mineral of importance from a public health point of view, especially for children and pregnant and lactating women. To help protect these groups from iron deficiency anemia, It is also an essential micronutrient that causes anemia when consumed insufficiently and exists in every cell of the human body. Iron contributes to several metabolic functions, including hemoglobin transport and storage, electron transport, and energy production [40], [41]. Iron is a micronutrient of public health importance and an essential part of hemoglobin for oxygen transport and myoglobin for transporting and storing oxygen in the muscle and releasing it when needed during muscle contraction. It also takes part in facilitating the electron transfer system, ATP synthesis, and the formation of red blood cells and their function [42]. \u0026nbsp; Iron is crucial for the differentiation and growth of epithelial tissue. It forms highly-toxic hydroxyl radicals, thus involved in the killing of bacteria by neutrophils; a component of enzymes critical for the functioning of immune cells (e.g., ribonucleotide reductase involved in DNA synthesis); involved in the regulation of cytokine production and action [43]; iron-rich status promotes the M2-like macrophage phenotype and negatively regulates the M1 pro-inflammatory response [44]. Involved in the regulation of cytokine production and action, it is required for the generation of pathogen-killing (reactive oxygen species) ROS by neutrophils during an oxidative burst[45].\u003c/p\u003e\n\u003cp\u003eIn industrialized countries, well-nourished adults can have 3-5 grams of iron, of which about 65% is in the form of hemoglobin. The rest of the iron in the body exists in the form of myoglobin, other heme compounds that promote intracellular oxidation, or is stored as ferritin in the reticuloendothelial system and cells of liver hepatocytes, bone marrow, and spleen [46]. \u0026nbsp;The beans are excellent sources of iron, which provides reasonable amounts in our daily diet. The recommended daily intake (RDI) for iron falls between 8 and 11 mg for men and women, respectively. Approximately, beans provide 0.75-0.95 mg of zinc per \u0026frac12; cup serving, or approximately 10\u0026ndash;11% of the RDI for women and 6\u0026ndash;8% of the RDI for men [47]. The current study also showed that to obtain a reasonable amount of bioaccessible iron, it is important to apply pressure cooking. Since iron is crucial from nutrition as well as a health point of view, it is required to consider the appropriate processing techniques of legumes and cereals, which can help improve its bioavailability, especially in developing countries.\u003c/p\u003e\n\u003ch3\u003eEffects of soaking, germination and cooking on Bioaccessibility of Zinc\u003c/h3\u003e\n\u003cp\u003eThe BA of zinc was found to be in the range of 5.46%\u0026ndash;10.08%, with the respective values representing control and germination for 48 hours, respectively (Table 2). Soaking in water and sodium bicarbonate contributed to the improved zinc in vitro bioaccessibility of 5.46%\u0026ndash;6.74% and 5.46%\u0026ndash;6.743%, respectively. This improvement in the bioaccessibility of zinc for both the soaking types (water and sodium bicarbonate) is relatively low as compared to other treatments. This could be due to the short soaking time or the genetic background of the beans investigated. The Suliburska \u0026amp; Krejpcio (2014) study reported a lower result of zinc bioaccessibility than this study for hazelnuts (1.9%) and a nearly similar result for white sorghum varieties (7.35%\u0026minus;9.73% to 9.07\u0026minus;10.72% due to the application of soaking). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eApplication of germination also contributed to the improved bioaccessible zinc in beans. The respective mean values of in vitro zinc bioaccessibility is presented in Table 2. In this study, germination treatment showed the increased zinc bioaccessibility as compared to the untreated beans which resulted in lower in vitro zinc bioaccessibility of 5.46%. The germination time of 24-hours and 48-hours resulted in 1.4-and 2-folds enhancement in zinc bioaccessibility. \u0026nbsp;Similar results with regard to the role of germination in improving the bioavailability and absorption of minerals have been reported in previous studies. For instance, enhancement of zinc bioavailability in sorghum varieties was achieved due to the application of germination [28]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn ordinary and pressure cooking of pre-soaked (overnight) beans affected the in vitro bioaccessibility of zinc at P\u0026lt;0.05, as shown in Table 2. In vitro zinc bioaccessibility achieved was 6.52% and 8.41% for ordinary and pressure cooking, respectively. The cooking type had a significant effect on zinc bioaccessibility, and the best result was achieved by the application of pressure cooking as compared to ordinary cooking and control. This could be due to thermal energy or steam and the pressure applied to the seed matrix. However, this result is lower than the zinc bioaccessibility value reported by [37] for Brazilian cowpeas bred, which was in the range of 38.81%\u0026ndash;52.78% after application of ordinary cooking. This kind of variation could be made as a result of different factors, like differences in cooking conditions, analytical procedures followed, and personal errors during analysis. Generally, germination for 48 hours resulted in the highest zinc BA in this study. This may be due to the activation of the phytase enzyme by presoaking and germination for 2 days, which in turn allowed further degradation of phytate and made the zinc ion free of complex formation. However, contradictory results were documented in previous studies in which the highest bioaccessibility of minerals was achieved by pressure cooking presoaked beans [30]. The current study showed that in vitro zinc bioaccessibility is higher than that of other minerals. The highest in vitro zinc bioaccessibility obtained in this study agrees with previous results reported for common beans [48]. Zinc plays an important role in nutrition and health. It exerts its ubiquitous effects on immune function, disease resistance, and general health. Analysis of animal and human studies examining the in vivo effects of nutritional zinc deficiency and supplementation on immune cells and their function underscores the essential role of zinc in the normal development and function of many key tissues, cells, and effectors of immunity [49]. Zinc also helps maintain the integrity of the skin and mucosal membrane (e.g., cofactor for metalloenzymes required for cell membrane repair [50]. It plays a central role in the cellular growth and differentiation of immune cells [51] and nd activates the phagocytic activity of peritoneal macrophages for E. coli and S. aureus [52].\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eEffect of soaking, germination and cooking on bioaccessibility of copper\u003c/h3\u003e\n\u003cp\u003eThe effects of soaking, germination, and cooking treatments were studied and resulted in a significant enhancement of copper bioaccessibility. The in vitro copper BA results for soaking, germination, and cooking were found to be 6.04% (soaking in water), 6.78% (soaking in NaHCO3), 7.06% (germination of 24 hours), 7.63% (germination of 48 hours), 5.79% (ordinary cooking), and 8.50% (pressure cooking). A slight increment in the bioaccessibility of copper was observed for soaking treatment. Germination played a vital role in increasing the bioaccessibility of copper in common beans. Though there was no significant difference between the two-germination times in improving bioaccessibility, germinating for 24- and 48-hours resulted in 7.06% and 7.63% bioaccessible copper, respectively, and this shows the germination treatment enhanced the amount of bioaccessible copper.\u003c/p\u003e\n\u003cp\u003eCooking, on the other hand, contributed to enhancing the amount of bioaccessible minerals in beans. The in vitro copper bioaccessibility of 5.79% and 8.50% was obtained by ordinary and pressure cooking, respectively. The enhancement of mineral bioaccessibility could be due to the degradation of phytate during thermal treatment by pressure cooking. A similar study reported that 70% BA of copper was achieved by applying pressure cooking to black beans [30]. This difference might occur due to the nature of the bean sample, the analytical methods applied, and the procedures applied during processing. Processing methods like fermentation, drying, hydration, cooking, and germination could affect the BA of minerals due to the interference with the food matrix. According to [53], the soaking of foods or raw grain seeds in water increases the amount of soluble minerals through passive diffusion or leaching of water-soluble anti-nutritional factors, including sodium, potassium, or magnesium phytate. The bioavailability of minerals in foods can be affected by the presence of phytates, tannins, and polyphenols. ANFs are shown to cause the complexing, inhibition, and binding of minerals to form complex salts, in turn decreasing their bioaccessibility [54].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThermal treatment (cooking) can enhance the bioaccessibility of minerals due to the softening of the food matrix and consequently the release of protein-bound minerals [55], \u0026nbsp;and by reducing solubility inhibitors such as phytates, tannins, and phenolic compounds [56]. The current study reveals that Cu and Zn bioaccessibility is better than that of iron. This could be due to the nature of the minerals, in that their capacity to be solubilized when they are in a complex form like phytate is different. The study on the solubility and hydrolysability of metal phytate complexes and metal species has shown that Cu, Zn, and Fe are more soluble. The author also illustrated that the acidic PH (5) resulted in greater solubility and hydrolyzability of phytates than the nearly neutral PH (7.5) [57]. Copper is an important cofactor of cytochrome C-oxidase, a component of the mitochondrial respiratory chain, and is involved in the metabolism of iron. The role of copper in neutrophils and monocytes enhances NK cell activity [58]. It maintains intracellular antioxidant balance, suggesting an important role in the inflammatory response [59]. The beans, as a good source of copper, must undergo appropriate processing to make the copper available for intestinal absorption. It has been reported that pressure cooking is an appropriate processing method to reduce antinutritional factors and, in turn, enhance the bioaccessibility of minerals [60].\u003c/p\u003e\n\u003cdiv id=\"ftn2\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"CONCLUSIONS AND RECOMMENDATIONS","content":"\u003cp\u003eThe common bean is one of the most consumed legumes in Africa, especially in the southern and eastern parts of the continent. Being a good source of plant-based proteins such as Fe, Ca, Zn, and copper, it is consumed alone or mixed with other foods. The effect of processing methods on in vitro mineral bioaccessibility was studied in this experiment. This study reveals that different processing techniques, including soaking, germination, and cooking, affected the in vitro mineral bioaccessibility of common beans. In vitro iron, zinc, and copper bioaccessibility was found to be enhanced by soaking, germination, and cooking. Though all of the processing methods resulted in enhanced in vitro mineral bioaccessibility, pressure cooking was found to be the best processing method for increasing the in vitro bioaccessibility of iron and copper. This indicates that thermal cooking with a pressure cooker significantly degrades the bean matrix and reduces its anti-nutritional content. It is therefore recommended to be used in societies where there is a pressure cooker facility. The ordinary cooking of presoaked beans is also used in this study. Ordinary cooking, which is traditionally used in rural areas of bean-producing countries, also resulted in a better result in increased in vitro mineral bioaccessibility following pressure cooking.\u003c/p\u003e\n\u003cp\u003eGermination for 48 hours showed the highest in vitro zinc bias (10.08%) as compared to others in this study. The beans are widely consumed alone or mixed with other dishes in African countries. Considering this reality, the appropriate processing techniques, especially soaking in water followed by ordinary cooking, should be applied to enhance the bioaccessibility of essential minerals. Generally, the in vitro bioaccessibility of essential minerals in the human body (Fe) and the essential micronutrients widely known for their optimal growth and reproduction roles (Cu and Zn) could be enhanced by the application of different processing methods. The Agricultural Research Centers should also train the rural communities that are relying on legumes and beans in particular as protein and essential mineral sources and closely follow them to practice appropriate and easily applicable processing techniques. Agricultural research and academic institutions should mainly focus on rural communities and provide training on how to process legume crops to improve nutritional value in terms of bioavailability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI would also like to thank staffs of University of Eduardo Mondlane Eng. Asimina Esmail Sulemane, Dr. Adolfo Firmino Condo, and Eng. Virginia Ivone Gongole for their technical support during the laboratory works. \u0026nbsp;I am also thankful to European Union (EU) for financial support and Intra-Africa Academic Mobility Scheme (MOUNAF) project coordinators for their sincere supports.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflict of interest in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eR. B. Canani \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Zinc inhibits cholera toxin-induced, but not Escherichia coli heat-stable enterotoxin-induced, ion secretion in human enterocytes,\u0026rdquo; \u003cem\u003eJ. Infect. 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Macuamule, \u0026ldquo;Effects of processing methods on phytate and tannin content of black small common beans ( Phaseolus vulgaris L .) cultivated in Mozambique Effects of processing methods on phytate and tannin content of black small common beans ( Phaseolus vulgaris L .) cu,\u0026rdquo; \u003cem\u003eCogent Food Agric.\u003c/em\u003e, vol. 9, no. 2, 2023, doi: 10.1080/23311932.2023.2289713.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Beans, in vitro digestion, mineral bioaccessibility, bioavalability, malnutrition","lastPublishedDoi":"10.21203/rs.3.rs-4873186/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4873186/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMineral bioavailability is a concern in legume based foods due to presence of Antinutritional factors. Zinc and iron deficiency is widespread in low-income countries because of the low consumption of animal products due to their unaffordable costs. The objective of this study was to evaluate the effects of processing conditions on the in vitro mineral bioaccessibility of small black common beans cultivated in Mozambique. The samples were collected from ‘Instituto de Investigação Agrária de Moçambique’ (IIAM), Maputo, Mozambique. Soaking (in water and sodium bicarbonate), germination (for 24 and 48 hours at 25oC), and cooking (ordinary and pressure) were applied. The oven-dried and finely ground samples of processed beans were in vitro digested and analyzed for mineral bioaccessibility.\u003c/p\u003e\n\u003cp\u003eThe in vitro gastrointestinal simulation assay was performed and the dialyzable part of the samples was used for iron, zinc, and copper bioaccessibility determination. Though there is a slight difference, all the processing methods investigated in this study; soaking, germination, and cooking treatments affected the mineral bioaccessibility of beans. The in vitro iron, zinc, and copper bioaccessibility was enhanced by the respective processing methods as compared to the control sample. The iron bioaccessibility was observed to be 2.22% for water soaking and 2.59% for sodium carbonate soaking, 2.75% for 24-hour germination 4.27% for 48-hours germination, and 3.56% for ordinary cooking and 7.79% for pressure cooking. The in vitro iron bioaccessibility is relatively low as compared to that of zinc. In vitro, Zinc bioaccessibility was found to be the same for water and sodium bicarbonate soaking which was 6.94%.\u003c/p\u003e\n\u003cp\u003eGermination for 24 and 48 hours resulted in zinc bioaccessibility of 7.58% and 10.08% respectively while zinc bioaccessibility of 6.52% for ordinary cooking and 8.41% for pressure cooking was achieved. The respective in vitro copper bioaccessibility obtained for soaking in water and sodium bicarbonate was found to be 6.04% and 6.78% which are almost similar. Similarly, the germination for 24 and 48 hours showed copper bioaccessibility of 7.01% and 7.63% in respective order whereas it was observed that copper bioaccessibility of 5.79% for ordinary cooking and 8.50% for pressure cooking was achieved in this study. This shows that it is expected that the solubility of pressure-cooked beans in the intestine and the release of minerals from its matrix is high. The result illustrates that the pressure cooking of presoaked beans had the greater value for all mineral's bioaccessibility except for zinc, and it can be concluded that the processing techniques could help improve minerals' bioaccessibility in beans which in turn helps to combat malnutrition and ensure food security in developing countries.\u003c/p\u003e","manuscriptTitle":"Effect of Processing Methods on Mineral Bioaccessibility in Common Beans (Phaseolus Vulgaris L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-13 07:58:27","doi":"10.21203/rs.3.rs-4873186/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-01T06:55:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-29T08:13:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-23T14:48:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1074898665055399715388882090718334726","date":"2024-09-08T16:17:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93047536278600326349416137148006829739","date":"2024-09-05T03:42:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-05T03:21:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-27T11:30:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-08-21T02:36:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-17T07:21:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-07T08:37:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa7065af-55bf-4013-b987-e5a0432178af","owner":[],"postedDate":"September 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":37282505,"name":"Health sciences/Health care/Disease prevention/Nutritional supplements"},{"id":37282506,"name":"Health sciences/Health care/Nutrition"},{"id":37282507,"name":"Health sciences/Health care/Public health"}],"tags":[],"updatedAt":"2026-01-12T16:03:13+00:00","versionOfRecord":{"articleIdentity":"rs-4873186","link":"https://doi.org/10.1038/s41598-025-34639-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-06 15:57:20","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2024-09-13 07:58:27","video":"","vorDoi":"10.1038/s41598-025-34639-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34639-3","workflowStages":[]},"version":"v1","identity":"rs-4873186","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4873186","identity":"rs-4873186","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}