Evaluation of In vitro bio accessibility and In vivo bioavailability of Iron Biofortified and Fortified wheat samples through Static digestion using Caco-2 cell line and animal model studies: Implications for Nutritional Enhancement

Evaluation of In vitro bio accessibility and In vivo bioavailability of Iron Biofortified and Fortified wheat samples through Static digestion using Caco-2 cell line and animal model studies: Implications for Nutritional Enhancement | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of In vitro bio accessibility and In vivo bioavailability of Iron Biofortified and Fortified wheat samples through Static digestion using Caco-2 cell line and animal model studies: Implications for Nutritional Enhancement Harshitha Arun Pardhe, Krishnaveni Nagappan, S Naveen, Mohit Ananda, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6358588/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Iron deficiency remains a significant nutritional concern globally, particularly in developing nations, attributed to insufficient nutritional status, specifically inadequate iron intake in the diet. This study utilized In vitro static digestion and a Caco-2 cell model to assess iron bioaccessibility in biofortified and fortified wheat flour, along with its cooked form (chapatti), a commonly consumed wheat-based food product. In this study wheat flour and chapatti samples underwent standardized INFOGEST protocol, involving oral, gastric, and intestinal phases, followed by iron bioaccessibility assessment using the Caco-2 cell monolayer model. Compared to conventional wheat types, biofortified and fortified flours exhibited a significant increase in iron content (67.5 ppm and 53.3 ppm, respectively) and iron bioaccessibility 80.7% and 78.3%, respectively. In the cooked form, biofortified wheat demonstrated higher iron bioaccessibility 91.1% compared to regular wheat varieties and the uncooked form. The fortified form showed a bioaccessibility of 90.5%, suggesting the influence of food matrix and processing techniques. Wheat flour serves as an affordable nutrient source, addressing deficiencies for a majority of individuals. The findings contribute to nutritional planning for wheat product consumption and enhance understanding of iron bio accessibility’s role in increasing iron intake and preventing insufficiency in the general population. This research may contribute to better nutrition acceptance and more effective wheat selection. Biofortification Fortification Bio-accessibility Iron Caco-2 cell Bioavailability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The World Health Organization (WHO) defines “Micronutrients” as compounds required in extremely minor quantities i.e., less than 100 mg/day (WHO). They are essential for the synthesis of hormones, enzymes, and other compounds involved in growth and development. (WHO) The untoward outcomes of “Micronutrient Malnutrition” or “Hidden hunger” has serious consequences including but not limited to lethargy, poor health, eyesight complications, stunted growth, mental illness, and early death. (US). The diseases resulting from inadequacies are regarded as serious issues affecting the human health directly and indirectly the development of nations, as well as underappreciated problem of nutrient shortages. Improving nutritional health is one of the major socioeconomic challenges of the era, particularly in light of the ageing and expanding global population [ 19 ]. One such malnutrition is Iron deficiency anaemia affecting approximately 1.62 billion people worldwide. Globally, in low-income countries, iron is considered vital micronutrients for health as their deficiency affects children and pregnant women, i.e., WHO reports it as a contributing factor in 45% of fatalities among children under the age of five and 40% of pregnant women are anaemic [ 1 ] Anaemia is a medical condition characterised by reduced red blood cells (RBCs) and a lack of sufficient oxygen-carrying capacity to meet physiological needs. Inadequate nutritional status, including low intake of iron, contributes to substantial health loss [ 2 – 4 ]. Poor iron status alone can have negative effects on pregnancy outcomes, developmental delays, cognitive impairment, physical and occupational performance [ 5 , 6 ]. This scenario is concerning and seeks for the adoption of suitable and effective nutritional practices and policies for alleviation of this illness. Although numerous methods have been in practice for the prevention of anaemia, they are often inaccessible to remote and underprivileged populations where iron deficiency is most prevalent, as a result there is a need for an effective and strategical approach. In this perspective, iron biofortification and food fortification remains a promising and economical strategy for addressing a specific population with deficiency disorders. Biofortification involves the breeding or genetic alteration of crops to enhance their nutrient content, whereas fortification is the process of adding nutrients to foods during processing [ 7 – 9 ]. Evaluating the bio accessibility and bioavailability of iron in both biofortified and fortified products provides insights into their comparative nutritional benefits. With the primary aim of combating iron deficiency malnutrition, wheat flours and their cooked form (chapatti) are chosen in present research as it is the staple food crop consumed on daily basis in the form of chapatti, roti, bakery products etc., [ 10 – 12 ] In general, diet related factors have a greater impact on the bioavailability of the minerals in food, especially Fe. The balance between variables that either inhibit or increase nutrient absorption in the entire diet determines the overall influence on the nutrient bioavailability [ 13 , 14 ]. This current strategy also aims to analyse the antinutrients such as phytate, in food crops as these compounds can chelate iron to form insoluble complexes in the gastrointestinal tract that, consequently, reduce their bioavailability. However, they also possess benefits that preclude breeders from aiming to remove them entirely, but in connection with the micronutrient deficiency lesser the content of phytate better the absorption of minerals and better the bio accessibility [ 15 – 18 ]. Bio accessibility is the amount of nutrient released from the food matrix and accessible for absorption [ 19 ]. It is usually measured by in vitro simulated digestion in combination with Caco-2 cell model, to assess in vitro bioavailability [ 20 ]. Human intestinal Caco-2 cells are colonic carcinoma cells that display morphological and functional characteristics similar to those of differentiated epithelial cells from small intestinal mucosa [ 21 , 22 ]. As the cells continue to mature upon culture, their morphological and biochemical characteristics more closely resemble those of enterocytes and show highly polarized columnar cells with tight junctions that result in the apical membrane separating from the basolateral membrane. The in vitro method mainly involves digestive enzymes having a substantial impact on the absorption of iron from wheat or its processed form. Digestive enzymes are proteins produced by the body to aid the breakdown of complex nutrients into simpler forms that can be absorbed and utilized by cells [ 21 , 22 ]. In the context of iron absorption from wheat/chapatti two principle digestive enzymes are involved. This process aids in releasing iron from wheat or its products protein complexes, making it more accessible for absorption in the small intestine. Pepsin an enzyme produced in the stomach plays a crucial role in breaking down proteins. It works in an acidic environment, to cleave proteins into smaller peptides. Although it doesn’t directly affect iron absorption, pepsin’s role in protein digestion is essential as wheat and its processed forms contain proteins, and iron in these foods is bound to these proteins which are to be cleaved for further iron absorption in intestine [ 23 ]. The pancreas secretes pancreatic enzymes into the small intestine, such as proteases, amylase and lipases. These enzymes further break down proteins into peptides and amino acids, carbohydrate into simpler sugar and fats in to fatty acids and glycerol, to release iron for absorption. This phase is essential because it prepares food particles for further absorption in the small intestine. These enzymes facilitate the release of iron from food, reduce it to a form suitable (Ferric Fe 3+ to ferrous Fe 2+ ) with the help of enzyme called duodenal cytochrome B (Dcytb) for uptake and transport across the intestinal lining into the blood with the help of divalent metal transporter 1 (DMT 1) transport. Understanding this process highlights the need of a healthy digestive system in ensuring that the body’s iron requirement is satisfied, especially for individuals who rely on non- heme iron sources in their diets [ 24 – 26 ]. Along with in vitro bio accessibility the study on kinetics of the iron gives the better understanding on the concentration/amount of iron entering into the system. Determining bioavailability- (the rate and extent to which the active substance or active moiety is absorbed and becomes available at the site of action) is one of the main goals of kinetics analyses. Bioavailability is usually evaluated by measuring the serum concentration of the particular compounds. Concerning iron, the erythrocyte is the principal site of action, with iron storage sites being of minor importance. Iron bioavailability has been defined in a number of ways [ 27 ] but most definitions agree that it should be a measurable amount of total iron that is taken in by the body and metabolised, or integrated into haemoglobin Therefore, serum concentration is considered relevant in assessing the kinetic parameters. Thus, approaches to kinetics assessment of iron are clearly required [ 28 , 29 ] Thus, in order to properly estimate the minimum micronutrient concentrations that breeders must reach, as well as predict the ability of these interventions to be successful, the amount of the micronutrients present in the ready-to-eat portion of the food and available for absorption must be investigated and also, analysing the anti-nutrient (phytate) effect on iron absorption from wheat flour and cooked form of wheat. The investigation employed a simulated gastrointestinal digestion model to simulate human digestion process. The samples which include wheat flour and chapatti were put through a series of digestive process that included oral, gastric and intestinal phases, followed by assessment of iron bio accessibility using Caco-2 cell monolayer model. In vivo animal model for predicting kinetics parameters includes the study of the absorption, digestion, metabolism Viz Peak Plasma Concentration (Cmax), Time of Peak Concentration (Tmax), Half-Life (T1/2), Clearance (CI) were observed for administered wheat samples rich in iron, in in vivo animal models [ 29 , 30 ]. Materials and Methods The methodology involves food sample preparation, Antinutrient analysis, in vitro digestion, Iron uptake by Caco-2 cell lines and in vivo kinetic parameters assessment. Unless otherwise stated, all the chemicals used in this work are of analytical grade, procured from Sigma-Aldrich, India. Deionized and double distilled water was used in the entire study. Samples collection and preparation of raw material Biofortified (BW) HI-8663, HI-1605 and normal whole wheat varieties (NW), HI-803, GW-H57, H-8753, LOK-303, and LOK-807 were acquired from cultivars across India. Commercially available fortified (F) and normal wheat flours (NF) were acquired from a local market in Tamil Nadu. The collected samples were eventually cleaned and were allowed to dry for 48 hours before being milled in a lab pulveriser Machine (Sieve size − 1). Each flour sample was placed in airtight containers and stored until further use. All the glassware used in the work was cleaned with detergent water, rinsed with 5% nitric acid, repeated rinses with deionized water, and then dried in an oven to make them free from iron contamination. Processing of in-house Food-samples Chapatti, a flat bread of Indian origin is popular in majority of the households. Basically, the chapatti dough was prepared by mixing wheat flour with optimum amount of deionised water. The dough was covered and left to rest at room temperature for 5 minutes. The dough was divided into equal portions and rolled into a round sheet. The chapattis were baked on an earthen pan at low to medium flame 150ºC to 170ºC for about 2-3min on each side. Once the chapatti was evenly baked and had puffed up, it was removed from the heat and cooled at room temperature and crushed to powder and stored for further analysis [ 31 ]. Simulated digestion fluids preparation Simulated salivary Fluids (SSF), simulated gastric fluids (SGF) and simulated intestinal fluid (SIF) were made with corresponding electrolyte stock solution, enzymes, CaCl 2 and water as represented in Table 1 . Table 1 Volumes of electrolyte stock solutions of digestion fluids for a volume of 400 mL diluted with deionised water (1.25× concentrations) Sl no. Stock conc. SSF (pH-7) SGF (pH-3) SIF (pH-7) Salt solutions added g/L M ml mM ml mM ml mM KCl 37.3 0.5 15.1 15.1 6.9 6.9 6.8 6.8 KH 2 PO 4 68 0.5 3.7 3.7 0.9 0.9 0.8 0.8 NaHCO 3 84 1 6.8 13.6 12.5 25 42.5 85 NaCl 117 2 - - 11.8 47.2 9.6 38.4 MgCl 2 (H 2 O) 6 30.5 0.15 0.5 0.15 0.4 0.12 1.1 0.33 (NH 4 ) CO 3 48 0.6 0.06 0.06 0.5 0.5 - - HCl - 6 0.09 1.1 1.3 15.6 0.7 8.4 CaCl 2 (H 2 O) 2 44.1 0.3 0.025 1.5 0.005 0.15 0.04 0.6 In vitro simulated gastrointestinal digestion studies (INFOGEST) [ 32 ] The digestibility of wheat samples was evaluated using the simulated INFOGEST In vitro digestion method. Initially for the oral phase of digestion 5gm of the samples were mixed with α-amylase simulated salivary fluid (SSF) in a 1:2 (w/v) ratio. The mixture (bolus form) was gently stirred and incubated at 37ºC for 5 minutes to begin enzymatic digestion by salivary amylase. The oral phase samples were then stored at -20ºC for future analysis. Following the oral phase, the mixture was given an acidic treatment to simulate the conditions as in the stomach with simulated gastric fluids. The pH was raised to 2.0 by adding hydrochloric acid (HCl) and Pepsin was introduced to facilitate protein digestion. The mixture was incubated at 37°C for 2 hours and the samples from the gastrointestinal phase were kept at -20º Celsius for future analysis. To simulate the conditions in the small intestine, Bile salts and pancreatic enzymes (lipase, amylase and proteases) were added to the gastric digesta. This step allowed for the further digestion of macronutrients and release of nutrients. The mixture was incubated at 37°C for an additional 2 hours and the digested samples were stored at -20ºC. At the end of each phase the samples were collected to analyse the amount of iron content present during the process of digestion. This digesta reflected the changes in nutritional composition that occurred during the in vitro digestion process and represented the partially digested wheat sample. Caco-2 cell culture Caco-2 cells were obtained from American type culture collection and used in experiments at passages 30–40; the cells were cultured in 25 or 75cm 2 culture flasks and seeded at densities of 50000 cells/cm 2 in 6-well plates. The culture medium used was Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and supplemented with 10% (v/v) Fetal bovine serum, 1% (v/v) penicillin/ streptomycin solution, and 1% (v/v) non-essential amino acid. The cells were maintained at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. Caco-2 cells were cultured until they formed a confluent monolayer the medium was changed every 48 hrs. This monolayer represented the intestinal epitheliums which were further seeded in 6 well plates and used 12–14 days after seeding [ 21 , 22 ]. Iron uptake by Caco-2 cell lines Following the INFOGEST digestion, the resulting intestinal digesta was transferred on to the Caco-2 cell monolayer. The purpose was to assess how the digested nutrients/ iron interacted with the cells and absorbed by the cells. The Caco-2 cell studies consist of 6-well culture plates and the samples were split across different plates. The aliquot of the food digest (intestinal phase) was transferred to the well, allowing soluble iron to pass through and was incubated for 22-24hrs. At the end of each experiments, medium was removed from the wells and cells were rinsed twice with ice cold phosphate buffered saline (PBS) and cell monolayer were removed with a cell scarper and placed in Ependroff tubes and stored at -80ºC until further analysis. Cells were analysed for Iron content using ICP-MS, which quantified the extent of iron absorption by the intestinal cells [ 33 , 34 ]. Bioavailability study/In vivo Kinetic studies The in vivo kinetic/bioavailability study was performed at JSS College of Pharmacy, Ooty with the approval from the Institutional Animal Ethics Committee (IAEC), Approval no. JSSCPO/OT/IAEC/06/2023-24. The processed wheat samples were chosen to evaluate the kinetic profile of iron upon oral administration to animal models. Male Wister rats of about 200-250gm were selected and they were grouped into 3 groups of 6 animal each where; Group-I animals were administered with Biofortified samples; Group-II with fortified samples and Group-III with normal wheat flour samples and the comparative studies were carried out for the following groups. Sample assignment Bioavailability of iron from the cooked wheat samples (Biofortified, Fortified and Normal flour samples) were determined by the serum iron concentration curve (SIC) method. This method was based on plotting the serum iron concentrations after oral administration of samples to rats. Prior to the start of the experiment, the male rats were fed with a standard diet and distilled water ad libitum for five days. Then all the rats were kept fasted for 18 hours and randomly distributed into 3 groups having 6 rats each. Freshly prepared samples were orally administrated to the rats to provide 15mg Fe/kg body weight. For the control rats, deionised water alone was orally administered [ 35 ]. Blood collection After 0, 1, 2, 4, 6 8, 12, 24 and 48 hours of oral administration, blood samples were withdrawn (staggered manner) through retro-orbital puncture and serum was collected by centrifugation at 2000g for 10 minutes. Further, serum from all the samples was used for the measurement of serum iron concentration using ICP-MS [ 7 ]. Measurement of Iron content The Iron content in the wheat flour and the in vitro digested samples and serum samples were determined by inductively coupled plasma Mass spectroscopy. Briefly, about 0.3 to 0.5g of samples were weighed and placed into Teflon vessels to which a 6 mL of ICP-MS grade (AD Trace pure) nitric acid and 4–6 mL of ICP-MS grade water was added and placed in microwave digester (Perkin Elmer Titan MPS) with the below mentioned program for cereal matrix (Table 2 ). After digestion, the volume was adjusted to 50 mL using deionised water and introduced into the inductively coupled plasma mass spectrometer. The iron content was measured at specific wavelengths, and concentrations were quantified from standard solutions [ 7 , 36 ]. Table 2 Microwave programme for digestion Temperature Pressure Ramp Hold (min) 120º C 35 5 5 180º C 35 5 20 70º C 35 5 5 Analysis of Phytate (Antinutrient) Phytic acid content of the raw and cooked wheat samples were determined according to the method described by Davies and Reid. Individually one gram of raw sample, cooked samples as well as in vitro digested samples were extracted by taking 40 mL of 0.5M nitric acid in a conical flask and shaken for 1hour on a shaker at 30ᴼC and 80 revolutions per minute. The samples were filtered and 5 mL of 0.08M Ferric chloride was added and boiled for 20 minutes and filtered. The free iron (Fe 3−) remaining in the solution was determined calorimetrically by adding 2 ml of 0.005M ammonium thiocyanate and the iron-binding capacities of the extract was determined by difference [ 37 ]. Statistical Analysis All measurements were carried out in triplicate for each of the samples and results are expressed as mean ± values of standard deviations using Microsoft Excel, 2016 and ANOVA for statistical significance and PK solver software for kinetic parameters. Results and Discussion Iron bio accessibility in wheat and its processed form were analyzed using the Caco-2 cell line method following standard INFOGEST protocol. Kinetic parameters assessed by Pk solver software, and AOAC standards for iron quantification data are presented in below tables. Iron content in Biofortified (B), Fortified (F), Non-biofortified (NB) and Non-fortified (NF) samples were evaluated in both raw and processed/cooked states and are presented in Table 3 and Fig. 1 . According to IFCT standards, Non-biofortified and Non-fortified wheat flours typically contain approximately 41.0 mg/1000g and 39.7 mg/1000g of iron, respectively [ 38 ]. In raw form, biofortified samples displayed iron concentrations ranging from 67.5 ppm to 62.5 ppm, Fortified samples showed values ranging from 32ppm to 53ppm surpassing both non-biofortified and non-fortified samples. In the processed/cooked state, biofortified wheat products exhibited iron concentrations of 60ppm to 61ppm, fortified samples in the processed/cooked state showed 30ppm to 52ppm again surpassing both non-biofortified and non-fortified samples. The results suggest that biofortified wheat products consistently outperformed both NB and NF counterparts in terms of iron. The reduction in the iron content in the processed form can be attributed to several factors such as temperature, chemical reactions/degradation, denaturation of iron that occurs during the processing leading to conversion of iron forms into less soluble forms. Cooking process can also lead to physical changes in structure of the food matrix, which may impact the accessibility and release of iron during analysis. Some amount of iron may leach out into the cooking medium or may be lost due to adherence to cooking surfaces. These losses could contribute to the observed reduction in iron content in the cooked samples [ 15 ]. The increased iron concentrations in BF samples, both in raw and processed states, emphasize the potential of biofortification as an effective strategy to address iron deficiency. Fortified samples also demonstrated improved iron concentration compared to NF samples, indicating the effectiveness of fortification in enhancing iron content. However, biofortified samples consistently exhibited higher iron concentrations, emphasizing the superiority of biofortification over traditional fortification methods. Furthermore, all employed methods in this study to measure iron content exhibited a positive correlation, which was statistically significant at p < 0.05. These findings suggest that the bioavailability of iron in wheat products can be significantly enhanced through fortification and biofortification, contributing to improved nutritional outcomes in populations heavily reliant on wheat-based diets and to assess the potential health benefits of incorporating functional wheat products into regular diets [ 15 ]. Table 3 Iron content in wheat flour and cooked from samples in ppm Sample (lab code) Iron content (wheat flour) Iron content (chapatti) % loss % present NB1 22 ± 0.05 20 ± 0.05 9.0 90.9 NB2 17 ± 0.1 15 ± 0.03 11.7 88.2 B1 67.5 ± 0.02 60 ± 0.04 11.1 88.8 B2 62.5 ± 0.05 61 ± 0.04 2.4 97.6 NF1 22 ± 0.1 17 ± 0.05 22.7 77.2 NF2 20 ± 0.05 16 ± 0.04 20 80 F1 53 ± 0.05 52 ± 0.04 1.8 98.1 F2 32 ± 0.01 30 ± 0.05 6.2 93.7 *NB: Non biofortified, B: Biofortified, NF: Non-fortified, F: Fortified Values are expressed in mean ± standard deviation*n = 3 determinations per sample significant at p < 0.05 In vitro Iron Bio accessibility Non-fortified/non-biofortified wheat samples: In the uncooked form, the Bioaccesible iron content of NB and NF wheat flour ranged from 9.2 ppm to 13.1 ppm, with corresponding bio accessibility percentages between 69.1% and 67.1%. Following the cooking process, the Bio accesible iron content ranged from 12.5 ppm to 11.5ppm, showing a slight decrease, while the bio accessibility percentages increases to a range of 75.7–76.6%. The observed decrease in bio accessible iron content after cooking NB/NF wheat samples may be attributed to various factors, such as heat-induced alterations in the physical and chemical structure of iron compounds [ 10 ]. However, the concurrent increase in bio accessibility percentages post cooking suggests that the cooking process may enhance the bioavailability of iron despite a nominal decrease in total content [ 17 , 18 ]. Fortified/Biofortified wheat samples: Unprocessed samples of F and B wheat flour exhibited a substantial increase in bio accessible iron content, ranging from 40.5 ppm to 50.5ppm. The corresponding bio accessibility percentages were notably higher, ranging from 84.1–86.7%. Moreover, processed samples of F and B wheat flour demonstrated a further enhancement, with bio accessible iron content ranging from 43.8 ppm to 51.6 ppm, and bio accessibility percentages reaching 90.8–92.4%. represented in Table 4 & 5 and Fig. 2 & 3 It was found that the iron was found to be more accessible in the gastric phase compared to the intestinal phase this could be influenced by several factors i.e., solubility of iron where iron tends to be more soluble in acidic nature making it more accessible for absorption once the partially digested food enters into the small intestine, where the pH levels are higher and neutral, the solubility of iron may decrease, affecting its bio accessibility. The composition of the food matrix can significantly influence iron bio accessibility as certain foods such as phytates, and calcium can bind to iron and reduce its absorption. Theses interactions could be more pronounced in the intestinal phase when compared to gastric phase [ 39 ]. In the stomach, iron may form complexes with certain components of the food matrix, which could enhance its solubility and availability. However, in the intestinal phase, these complexes might break down affecting iron release and absorption. Enzymes present in the small intestine further break down food particles, potentially altering the availability of iron. The shift from an acidic to a more neutral environment in the intestine can also impact the chemical state of iron [ 40 ]. The results of the in vitro bio accessibility studies using the Caco-2 cell line reveal interesting insights into the iron accessibility of both NF/NB and F/B wheat flour. The substantial increase in both bio accessible iron content and bio accessibility percentages in F and B wheat flour, especially after processing, highlights the effectiveness of F and B strategies. The higher bio accessibility percentage post-processing may be indicative of improved iron solubility or alterations in the matrix that facilitate better iron release during digestion [ 18 ]. These findings underscore the potential of fortification and biofortification in enhancing the bioavailability of iron in wheat flour. Further investigations into the mechanisms underlying the observed changes in bio accessibility could provide valuable insights for improving the nutritional quality of staple foods. Moreover, the study emphasizes the importance of considering processing methods in biofortification/fortification strategies to optimize the bioaccessibity of essential nutrients. Table 4 Iron content assessment in wheat flour samples at different digestive phases (ppm) Wheat flour Sample lab code Oral Phase Gastric phase Intestinal phase Bioaccesible iron Caco-2 cell absorption NB 22 ± 0.01 19.6 ± 0.01 13.3 ± 0.04 9.2 ± 0.05 % degraded/loss 0 11 16.9 30.8 % available 100 89 83.1 69.1 B 67.1 ± 0.01 60.7 ± 0.005 58.2 ± 0.08 50.5 ± 0.01 % degraded/loss 0.6 9.6 4.1 13.2 % available 99.40 90.4 95.8 86.7 NF 22 ± 0.01 21.4 ± 0.01 19.5 ± 0.07 13.1 ± 0.02 % degraded/loss 0 2.8 8.8 32.8 % available 100 97.2 91.1 67.1 F 53 ± 0.05 50.2 ± 0.01 48.1 ± 0.01 40.5 ± 0.03 % degraded/loss 0 5.3 4.1 15.8 % available 100 94.7 95.8 84.1 *NB: Non biofortified, B: Biofortified, NF: Non-fortified, F: Fortified Values are expressed in mean ± standard deviation; *n = 3 determinations per sample significant at p < 0.05 Table 5 Iron content assessment in cooked form (chapatti) samples at different digestive phases (ppm) Chapatti Sample lab code Oral Phase Gastric phase Intestinal phase Bioaccesible iron Caco-2 cell absorption NB 20 ± 0.01 18.5 ± 0.02 16.5 ± 0.05 12.5 ± 0.05 % degraded/loss 0 7.5 10.8 24.2 % available 100 92.5 89.1 75.7 B 59.2 ± 0.01 57.9 ± 0.01 55.8 ± 0.01 51.6 ± 0.01 % degraded/loss 1.3 2.1 3.6 7.5 % available 98.6 97.8 96.3 92.4 NF 17 ± 0.01 16.9 ± 0.02 15 ± 0.07 11.5 ± 0.05 % degraded/loss 0 0.5 11.2 23.3 % available 100 99.4 88.7 76.6 F 50.6 ± 0.05 49.9 ± 0.06 48.2 ± 0.08 43.8 ± 0.08 % degraded/loss 2.6 1.3 3.4 9.1 % available 97.3 98.6 96.5 90.8 *NB: Non biofortified, B: Biofortified, NF: Non-fortified, F: Fortified Values are expressed in mean ± standard deviation*n = 3 determinations per sample significant at p < 0.05 Anti-nutrients (phytate) IFCT data by Longvah T and Ananthan R revealed that wheat Atta’s phytate level ranges from 632 to 638 mg/100g [ 38 ] Higher phytic acid levels were observed in wheat flour, ranging from 504 mg/100g to 625.8mg/100g, compared to processed samples, where phytate levels ranged from 225.1 mg/100g to 330.2 mg/100g. Notably, after absorption phytate content was found to be higher in digested wheat flour ie, 455.1 to 545.4 mg/100g than in cooked form biofortified wheat with 212.4 mg/100g and fortified wheat with 185.2mg/100g exhibiting significantly lower phytate content than normal wheat, suggesting a potential enhancement in iron absorption. In vitro digestion demonstrated a reduction in phytate content, with bioavailability dropping to 50% in cooked forms. Represented in Table 6 and Fig. 4 & 5 . The percentage of reduction in phytate content during cooking would depend on various factors such as cooking method, duration, temperature, and initial phytate concentration in the raw material. i.e., on heating chemical reaction occurs that break down the phytate structure, in the food matrix exposing the phytate molecules to degradation reducing its concentration, and heating can also lead to inactivation of enzymes responsible for the synthesis of phytate. The observed variations in phytate levels between raw wheat flour and cooked samples indicate the impact of processing on phytate degradation [ 40 ]. The observed variations in phytate levels between raw wheat flour and cooked samples indicate the impact of processing on phytate degradation. The higher phytate content in digested wheat flour suggests a potential limitation in iron absorption, as phytate can form insoluble complexes with iron. The significantly lower phytate content in biofortified and fortified cooked samples is promising, as it implies enhanced iron bioavailability [ 13 , 14 ]. This finding aligns with the increased iron concentrations observed in biofortified wheat product, emphasizing the potential of biofortification to mitigate the inhibitory effects of phytate on iron absorption. The reduction in phytate content during in vitro digestion further supports the perception that processing methods can influence the bioavailability of iron. The drop-in bioavailability suggests the potential for improved iron absorption in the gastrointestinal tract [ 15 – 18 ]. The finding underscores the importance of considering both strategies for addressing iron deficiency and providing valuable insights for future interventions aimed at improving iron status in populations reliant on wheat-based diets. Table 6 Influence of phytic acid on absorption by Caco-2 cell lines in wheat flour and its cooked form Sample (la code) mg/100g Phytate (wheat flour) Phytate after absorption by Caco-2 Phytate (cooked) Phytate after absorption by Caco-2 NB 625.8 ± 0.05 545.4 ± 0.02 330.2 ± 0.05 311.1 ± 0.01 B 575.4 ± 0.06 465.3 ± 0.01 291.5 ± 0.05 212.4 ± 0.02 NF 521.5 ± 0.05 501.8 ± 0.02 243.3 ± 0.03 216.2 ± 0.02 F 504.1 ± 0.05 455.1 ± 0.01 225.1 ± 0.04 185.2 ± 0.01 *NB: Non biofortified, B: Biofortified, NF: Non-fortified, F: Fortified Values are expressed in mean ± standard deviation*n = 3 determinations per sample significant at p < 0.05 In vivo bioavailability studies Once the bio accessibility of the iron content was evaluated for the selected biofortified and fortified wheat samples the bioavailability of iron were analysed through in vivo studies. Pharmacokinetics involves determining the absorption, distribution, metabolism, and excretion (ADME). The plasma concentrations of the analyte were used to evaluate the pharmacokinetic parameters. Measurable iron-blood levels were noticed in all serum samples up to 48 hrs., after oral administration of wheat samples in the form of chapatti i.e., group 1 with biofortified, group 2- fortified and group 3- non-fortified chapatti samples. At particular time intervals of 0, 1, 2, 4, 6, 8, 12, 24, 48 blood was withdrawn, and centrifuged at 2000g for 15min to separate serum for quantification of iron using ICP-MS. A non-compartmental model analysis in PK SOLVER software was utilized to calculate the pharmacokinetic parameters with linear Trapezoidal method. The peak plasma concentration i.e., Cmax of iron in biofortified wheat samples was observed to be 13.557 mg/kg at 2.0 hrs of Tmax. Cmax of iron in fortified samples was found to be 12.724g/kg at 2.0 hrs. Tmax; and Cmax of 8.184mg/kg at 1hrs for normal wheat flour samples. The pharmacokinetic parameters evaluated for the administered wheat samples are summarized in table.7&8 and area under curve was plotted for time (h) vs. concentration (mg/kg) in Figure.6–8. It was clearly evident that processing and cooking clearly improved the bio accessibility as well as the bioavailability of iron. Bio accessibility measures the fraction of a nutrient that is released from the food matrix during digestion and made available for absorption. Bioavailability, on the other hand, refers to the proportion of the absorbed nutrient that is utilized by the body. In co-relation the iron content observed between in vitro bio accessibility and in vivo plasma iron bioavailability can be attributed to several aspects i.e. the invitro bio accessibility studies with Caco-2 cell line, simulate the digestion process but it does not completely replicate the complexities of the human digestive system whereas, the in vivo studies involve the actual digestion and absorption process in the rat animal model, which can be influenced by the factors such as gastric acidity, enzymatic activity and interactions with other food components [ 41 ]. In vivo studies also account for the metabolism and excretion of nutrients, which may affect the measured plasma concentrations over time. Also, the metabolism and the presence of binding proteins in the bloodstream can influence the pharmacokinetic profile of iron. The rate of iron absorption can vary between in vitro and in vivo systems, leading to differences in the timing and magnitude of peak plasma concentrations (Cmax) observed in the two types of studies. In vitro studies may not fully capture the effects of food matrix components on iron absorption, whereas in vivo studies consider the complex interactions between nutrients and other food components present in the diet which influences the maximum plasma iron concentration [ 41 ]. Table 7 Serum Iron content in biofortified, fortified and normal flour processed wheat samples (ppm) Sl no Time Biofortified samples Serum iron Conc. Fortified samples Serum iron Conc. Normal wheat flour serum iron conc. 1 0 0 0 0 2 1 12.375 11.056 8.184 3 2 13.557 12.724 6.847 4 4 13.485 11.226 6.896 5 6 12.687 9.684 6.023 6 8 10.274 9.46 5.829 7 12 10.203 8.938 5.533 8 24 8.898 7.61 5.365 9 48 7.834 6.976 4.337 Table 8 Representation of kinetic parameters in biofortified, fortified and normal wheat chapatti samples (ppm) Sl no. Parameters Unit Values (Biofortified samples) Fortified samples Normal flour samples 1 Lambda_z 1/h 0.007 ± 0.01 0.007 ± 0.005 0.007 ± 0.004 2 T 1/2 h 98.41 ± 0.005 87.83 ± 0.001 94.6 ± 0.001 3 Tmax h 2 ± 0.000 2 ± 0.000 1 ± 0.000 4 Cmax mg/kg 13.5 ± 0.004 12.7 ± 0.009 8.1 ± 0.004 5 Tlag h 0 0 0 6 AUC 0-t mg/kg*h 451.6 ± 0.008 392.5 ± 0.002 254.6 ± 0.005 7 AUC 0-inf_obs mg/kg*h 1563.9 ± 0.003 1276.5 ± 0.004 846.9 ± 0.004 8 AUMC 0-inf_obs mg/kg*h^2 221201.93 ± 0.006 163099.0 ± 0.008 114946.2 ± 0.005 9 MRT 0-inf_obs min 141.43 ± 0.004 127.76 ± 0.004 135.72 ± 0.004 10 Vz/F_obs (mg)/(mg/kg) 1.36 ± 0.005 1.48 ± 0.003 2.41 ± 0.008 11 Cl/F_obs (mg)/(mg/kg)/h 0.009 ± 0.005 0.01 ± 0.001 0.017 ± 0.007 To comprehend the iron content in processed samples, a preliminary assessment of iron levels in raw wheat flour was essential. The iron content in wheat flour varies due to factors like wheat variety, soil conditions, and milling processes. On average, whole wheat contains approximately 4 mg/100g of non-heme iron, less readily absorbed than heme iron found in animal products. Cooking can result in a loss of small traces of iron, influenced by variables such as temperature and duration, affecting bio accessibility and bioavailability. Our study yielded novel findings, particularly in the quantification of iron in various wheat flour varieties, including enriched and Bio accesible studies not reported previously. For instance, Saied et al. investigated nutritional parameters in commercially available wheat flours in Bangladesh, while Niedzielski et al. determined iron levels using UV and AAS techniques. Limited research exists on the comparative mineral content in cereal crops, and various studies, such as those by Akinyele and Shokunbi; Arif et al.; Hailu Kassegn focus on specific aspects but lack comprehensive quantification of iron in different wheat samples. Our comparative assessment of iron estimation in functional wheat varieties underscores the potential of biofortification and fortification in enhancing nutritional content. The significantly higher iron concentration in biofortified and fortified wheat flour, observed in both raw and cooked samples, supports the effectiveness of these strategies in addressing iron deficiency. Iron bio accessibility in chapati samples prepared from these flours demonstrated increased iron content, particularly in biofortified chapati, showcasing the nutritional quality enhancement through biofortification. Several factors contribute to enhanced iron bio accessibility and bioavailability, including fortification, breeding programs for high-iron varieties, and the use of Caco-2 cell lines as an in vitro model and in vivo animal model. While processing marginally deteriorates metal content, our analysis of phytate content revealed lower levels in biofortified wheat samples compared to normal varieties, suggesting the potential for selecting wheat varieties with lower phytate content to improve iron bio accessibility. Overall, this study highlights the multifaceted approaches to enhance iron bioavailability in wheat-based food products and their potential impact on addressing iron deficiency malnutrition. Conclusion In conclusion, this study highlights the potential of biofortified and fortified wheat flour and processed samples as significant dietary sources of bioavailable iron. The importance of considering both iron concentration and factors influencing iron absorption is emphasized through evaluation of iron content as its bio accessibility, bioavailability and phytate content. Future research endeavours should explore the practical implications of these findings on human populations and assess the benefits of selecting wheat cultivars with reduced phytate levels to enhance overall iron bioavailability. Based on the comprehensive analysis of the bio accessibility and bioavailability of iron from different wheat samples (biofortified, fortified and normal wheat samples) conducted it was observed that processing and cooking of the samples, significantly improved the bioavailability of iron from wheat samples. Both the functional wheat forms i.e., biofortified and fortified wheat samples exhibited higher peak plasma concentration Cmax when compared to normal samples, indicating better absorption and utilization of iron. The Tmax i.e., the time to reach the maximum plasma concentration was relatively consistent across all wheat samples analysed, stating a similar rate of absorption regardless of the functional strategies. Implementing these strategies could be highly significant in combating the deficiencies. Our research illuminates the valuable contributions of both biofortified and fortified wheat products to dietary iron intake, with a slight advantage in iron bio accessibility observed in biofortified products. While chapattis offer diverse nutritional benefits, including dietary fiber and a balanced diet that supports increased iron consumption, maintaining a varied and balanced diet remains vital to meeting daily iron needs and preventing iron deficiency. However, it is crucial to recognize the need for further exploration through studies and clinical trials to validate the real-world impact of increased iron bioavailability on human health. Abbreviations WHO- World Health Organization RBCs- Red blood cells Caco-2- Colonic Carcinoma cells Cmax-Peak Plasma concentration Tmax- Time of Peak Concentration T1/2- Half-Life CI- Clearance BW- Biofortified wheat NW - Normal whole wheat F- Fortified NF- Normal wheat flours SSF-Simulated salivary Fluids SGF- Simulated gastric fluids SIF- Simulated intestinal fluid DMEM- Dulbecco’s modified Eagle’s medium IAEC PBS- Phosphate buffered saline IAEC- Institutional Animal Ethics Committee SIC-Serum iron concentration curve Declarations Acknowledgment The authors are grateful to JSS College of Pharmacy, Ooty, and JSS Academy of Higher Education and Research, Mysuru, India for the continuous support and providing the facilities for this study. The authors especially would like to thank ICMR ADHOC [No. F.N.5/9/1323/2020-Nut] for the financial support, The authors are thankful to the Director, DFRL, Mysore, for providing the necessary facilities to conduct the study. Author Contributions All authors made substantial contributions to this manuscript. Harshitha Arun Pardhe conceptualized and drafted the manuscript. Krishnaveni Nagappan supervised the study design. Naveen S provided technical and material support for sample analysis. Rashmi V offered technical assistance for spectrometric analysis. Aditya Kumar Singh conducted in vitro studies analysis and interpretation. Mohit Ananda contributed to data analysis and manuscript drafting. Gullapalli Kowmudi assisted with reviewing and practical implications. Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical Issues The in vivo kinetic/bioavailability study was performed at JSS College of Pharmacy, Ooty with the approval from the Institutional Animal Ethics Committee (IAEC), Approval no. JSSCPO/OT/IAEC/06/2023-24. Clinical trial number Not applicable. Funding This work was supported by the [Indian Council of Medical Research (ICMR) under Grant No. F.N.5/9/1323/2020-Nut ORCID Harshitha Arun Pardhe- http://orcid.org/0000-0003-3826-9219 *Krishnaveni Nagappan- http://orcid.org/0000-0003-0596-9489 Naveen S- http://orcid.org/0000-0003-2981-422X Rashmi V- http://orcid.org/0000-0001-5692-7938 Aditya Kumar Singh- http://orcid.org/0009-0008-0202-8011 Gullapalli Kowmudi- http://orcid.org/0000-0001-9564-0542 Mohit Ananda- https://orcid.org/0000-0002-1142-543X References World Health Organization, (2021). Malnutrition. https://www.who.int/news-room/factsheets/detail/malnutrition (accessed February 2021) G.A. Stevens, J.E. Bennett, Q. Hennocq, Y. Lu, L.M. De-Regil, L. Rogers, G. Danaei, G. Li, R.A. White, S.R. Flaxman et al., Trends and mortality effects of vitamin A deficiency in children in 138 low-income and middle-income countries between 1991 and 2013: a pooled analysis of population-based surveys. 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Whittaker, Comparison of in vitro, animal, and clinical determinations of iron bioavailability: International Nutritional Anemia Consultative Group Task Force report on iron bioavailability. Am. J. Clin. Nutr. 9 (2), 225 (1989). https://doi.org/10.1093/ajcn/49.2.225 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6358588","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451094744,"identity":"394e83c2-a972-4a9b-ac1a-eb3308d7b026","order_by":0,"name":"Harshitha Arun Pardhe","email":"","orcid":"","institution":"Department of Pharmaceutical Analysis,JSS College of Pharmacy, JSS Academy of Higher Education and Research,Ooty,Nilgiris,Tamil 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representation of Iron content assessment in cooked form (chapatti) samples at different digestive phases (ppm)\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6358588/v1/d267b7ca44c507215613c02d.jpg"},{"id":82000255,"identity":"2a9ff106-b28e-4937-8ab4-c75b96798c0e","added_by":"auto","created_at":"2025-05-05 19:24:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79974,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of Phytic acid content in wheat flour and its absorption\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6358588/v1/76f32308be4200d61a98eba1.jpg"},{"id":81999826,"identity":"5e23698f-513b-4a3d-956d-a435d3f364bc","added_by":"auto","created_at":"2025-05-05 19:16:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86639,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of Phytic acid in cooked samples and its absorption\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6358588/v1/8943284fb962074b01435444.jpg"},{"id":81999823,"identity":"6f943d1d-eec9-4fd8-adf5-e07404062afe","added_by":"auto","created_at":"2025-05-05 19:16:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":108133,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Serum time concentration vs. Time curve for biofortified wheat samples.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6358588/v1/2a64ee99d7fe4ea480cab1d5.jpg"},{"id":81999821,"identity":"dcd90b06-eac6-4338-aded-e01fb0e80f22","added_by":"auto","created_at":"2025-05-05 19:16:26","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":107318,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Serum time concentration vs. Time curve for fortified wheat samples.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6358588/v1/0c4aacddb54c8198f8a51cc4.jpg"},{"id":81999830,"identity":"3f9d6d72-8ce9-4753-8faa-7695851a5870","added_by":"auto","created_at":"2025-05-05 19:16:26","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":107617,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Serum time concentration vs. Time curve for normal wheat samples.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6358588/v1/74b2164bd94baabdc89b603e.jpg"},{"id":86582730,"identity":"ddbfc48d-ff06-4f03-860a-4ecc43625197","added_by":"auto","created_at":"2025-07-13 00:01:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2195941,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6358588/v1/eb9de8b1-bdc4-46ec-a68f-f16d4fa26795.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of In vitro bio accessibility and In vivo bioavailability of Iron Biofortified and Fortified wheat samples through Static digestion using Caco-2 cell line and animal model studies: Implications for Nutritional Enhancement","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe World Health Organization (WHO) defines \u0026ldquo;Micronutrients\u0026rdquo; as compounds required in extremely minor quantities i.e., less than 100 mg/day (WHO). They are essential for the synthesis of hormones, enzymes, and other compounds involved in growth and development. (WHO) The untoward outcomes of \u0026ldquo;Micronutrient Malnutrition\u0026rdquo; or \u0026ldquo;Hidden hunger\u0026rdquo; has serious consequences including but not limited to lethargy, poor health, eyesight complications, stunted growth, mental illness, and early death. (US). The diseases resulting from inadequacies are regarded as serious issues affecting the human health directly and indirectly the development of nations, as well as underappreciated problem of nutrient shortages. Improving nutritional health is one of the major socioeconomic challenges of the era, particularly in light of the ageing and expanding global population [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. One such malnutrition is Iron deficiency anaemia affecting approximately 1.62\u0026nbsp;billion people worldwide. Globally, in low-income countries, iron is considered vital micronutrients for health as their deficiency affects children and pregnant women, i.e., WHO reports it as a contributing factor in 45% of fatalities among children under the age of five and 40% of pregnant women are anaemic [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAnaemia is a medical condition characterised by reduced red blood cells (RBCs) and a lack of sufficient oxygen-carrying capacity to meet physiological needs. Inadequate nutritional status, including low intake of iron, contributes to substantial health loss [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Poor iron status alone can have negative effects on pregnancy outcomes, developmental delays, cognitive impairment, physical and occupational performance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This scenario is concerning and seeks for the adoption of suitable and effective nutritional practices and policies for alleviation of this illness. Although numerous methods have been in practice for the prevention of anaemia, they are often inaccessible to remote and underprivileged populations where iron deficiency is most prevalent, as a result there is a need for an effective and strategical approach. In this perspective, iron biofortification and food fortification remains a promising and economical strategy for addressing a specific population with deficiency disorders. Biofortification involves the breeding or genetic alteration of crops to enhance their nutrient content, whereas fortification is the process of adding nutrients to foods during processing [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Evaluating the bio accessibility and bioavailability of iron in both biofortified and fortified products provides insights into their comparative nutritional benefits.\u003c/p\u003e \u003cp\u003eWith the primary aim of combating iron deficiency malnutrition, wheat flours and their cooked form (chapatti) are chosen in present research as it is the staple food crop consumed on daily basis in the form of chapatti, roti, bakery products etc., [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] In general, diet related factors have a greater impact on the bioavailability of the minerals in food, especially Fe. The balance between variables that either inhibit or increase nutrient absorption in the entire diet determines the overall influence on the nutrient bioavailability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This current strategy also aims to analyse the antinutrients such as phytate, in food crops as these compounds can chelate iron to form insoluble complexes in the gastrointestinal tract that, consequently, reduce their bioavailability. However, they also possess benefits that preclude breeders from aiming to remove them entirely, but in connection with the micronutrient deficiency lesser the content of phytate better the absorption of minerals and better the bio accessibility [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Bio accessibility is the amount of nutrient released from the food matrix and accessible for absorption [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It is usually measured by \u003cem\u003ein vitro\u003c/em\u003e simulated digestion in combination with Caco-2 cell model, to assess \u003cem\u003ein vitro\u003c/em\u003e bioavailability [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Human intestinal Caco-2 cells are colonic carcinoma cells that display morphological and functional characteristics similar to those of differentiated epithelial cells from small intestinal mucosa [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As the cells continue to mature upon culture, their morphological and biochemical characteristics more closely resemble those of enterocytes and show highly polarized columnar cells with tight junctions that result in the apical membrane separating from the basolateral membrane. The \u003cem\u003ein vitro\u003c/em\u003e method mainly involves digestive enzymes having a substantial impact on the absorption of iron from wheat or its processed form. Digestive enzymes are proteins produced by the body to aid the breakdown of complex nutrients into simpler forms that can be absorbed and utilized by cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the context of iron absorption from wheat/chapatti two principle digestive enzymes are involved. This process aids in releasing iron from wheat or its products protein complexes, making it more accessible for absorption in the small intestine. Pepsin an enzyme produced in the stomach plays a crucial role in breaking down proteins. It works in an acidic environment, to cleave proteins into smaller peptides. Although it doesn\u0026rsquo;t directly affect iron absorption, pepsin\u0026rsquo;s role in protein digestion is essential as wheat and its processed forms contain proteins, and iron in these foods is bound to these proteins which are to be cleaved for further iron absorption in intestine [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The pancreas secretes pancreatic enzymes into the small intestine, such as proteases, amylase and lipases. These enzymes further break down proteins into peptides and amino acids, carbohydrate into simpler sugar and fats in to fatty acids and glycerol, to release iron for absorption. This phase is essential because it prepares food particles for further absorption in the small intestine. These enzymes facilitate the release of iron from food, reduce it to a form suitable (Ferric Fe\u003csup\u003e3+\u003c/sup\u003e to ferrous Fe\u003csup\u003e2+\u003c/sup\u003e) with the help of enzyme called duodenal cytochrome B (Dcytb) for uptake and transport across the intestinal lining into the blood with the help of divalent metal transporter 1 (DMT 1) transport. Understanding this process highlights the need of a healthy digestive system in ensuring that the body\u0026rsquo;s iron requirement is satisfied, especially for individuals who rely on non- heme iron sources in their diets [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Along with \u003cem\u003ein vitro\u003c/em\u003e bio accessibility the study on kinetics of the iron gives the better understanding on the concentration/amount of iron entering into the system.\u003c/p\u003e \u003cp\u003eDetermining bioavailability- (the rate and extent to which the active substance or active moiety is absorbed and becomes available at the site of action) is one of the main goals of kinetics analyses. Bioavailability is usually evaluated by measuring the serum concentration of the particular compounds. Concerning iron, the erythrocyte is the principal site of action, with iron storage sites being of minor importance. Iron bioavailability has been defined in a number of ways [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] but most definitions agree that it should be a measurable amount of total iron that is taken in by the body and metabolised, or integrated into haemoglobin Therefore, serum concentration is considered relevant in assessing the kinetic parameters. Thus, approaches to kinetics assessment of iron are clearly required [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThus, in order to properly estimate the minimum micronutrient concentrations that breeders must reach, as well as predict the ability of these interventions to be successful, the amount of the micronutrients present in the ready-to-eat portion of the food and available for absorption must be investigated and also, analysing the anti-nutrient (phytate) effect on iron absorption from wheat flour and cooked form of wheat. The investigation employed a simulated gastrointestinal digestion model to simulate human digestion process. The samples which include wheat flour and chapatti were put through a series of digestive process that included oral, gastric and intestinal phases, followed by assessment of iron bio accessibility using Caco-2 cell monolayer model. \u003cem\u003eIn vivo\u003c/em\u003e animal model for predicting kinetics parameters includes the study of the absorption, digestion, metabolism Viz Peak Plasma Concentration (Cmax), Time of Peak Concentration (Tmax), Half-Life (T1/2), Clearance (CI) were observed for administered wheat samples rich in iron, in \u003cem\u003ein vivo\u003c/em\u003e animal models [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe methodology involves food sample preparation, Antinutrient analysis, \u003cem\u003ein vitro\u003c/em\u003e digestion, Iron uptake by Caco-2 cell lines and \u003cem\u003ein vivo\u003c/em\u003e kinetic parameters assessment. Unless otherwise stated, all the chemicals used in this work are of analytical grade, procured from Sigma-Aldrich, India. Deionized and double distilled water was used in the entire study.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSamples collection and preparation of raw material\u003c/h2\u003e \u003cp\u003eBiofortified (BW) HI-8663, HI-1605 and normal whole wheat varieties (NW), HI-803, GW-H57, H-8753, LOK-303, and LOK-807 were acquired from cultivars across India. Commercially available fortified (F) and normal wheat flours (NF) were acquired from a local market in Tamil Nadu. The collected samples were eventually cleaned and were allowed to dry for 48 hours before being milled in a lab pulveriser Machine (Sieve size \u0026minus;\u0026thinsp;1). Each flour sample was placed in airtight containers and stored until further use. All the glassware used in the work was cleaned with detergent water, rinsed with 5% nitric acid, repeated rinses with deionized water, and then dried in an oven to make them free from iron contamination.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProcessing of in-house Food-samples\u003c/h3\u003e\n\u003cp\u003eChapatti, a flat bread of Indian origin is popular in majority of the households. Basically, the chapatti dough was prepared by mixing wheat flour with optimum amount of deionised water. The dough was covered and left to rest at room temperature for 5 minutes. The dough was divided into equal portions and rolled into a round sheet. The chapattis were baked on an earthen pan at low to medium flame 150\u0026ordm;C to 170\u0026ordm;C for about 2-3min on each side. Once the chapatti was evenly baked and had puffed up, it was removed from the heat and cooled at room temperature and crushed to powder and stored for further analysis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eSimulated digestion fluids preparation\u003c/h3\u003e\n\u003cp\u003eSimulated salivary Fluids (SSF), simulated gastric fluids (SGF) and simulated intestinal fluid (SIF) were made with corresponding electrolyte stock solution, enzymes, CaCl\u003csub\u003e2\u003c/sub\u003e and water as represented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVolumes of electrolyte stock solutions of digestion fluids for a volume of 400 mL diluted with deionised water (1.25\u0026times; concentrations)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSl no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eStock conc.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSSF (pH-7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSGF (pH-3)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eSIF (pH-7)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalt solutions added\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e 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align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaHCO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e42.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e47.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e38.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMgCl\u003csub\u003e2\u003c/sub\u003e (H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(NH\u003csub\u003e4\u003c/sub\u003e) CO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro simulated gastrointestinal digestion studies (INFOGEST)\u003c/b\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe digestibility of wheat samples was evaluated using the simulated INFOGEST \u003cem\u003eIn vitro\u003c/em\u003e digestion method. Initially for the oral phase of digestion 5gm of the samples were mixed with α-amylase simulated salivary fluid (SSF) in a 1:2 (w/v) ratio. The mixture (bolus form) was gently stirred and incubated at 37\u0026ordm;C for 5 minutes to begin enzymatic digestion by salivary amylase. The oral phase samples were then stored at -20\u0026ordm;C for future analysis. Following the oral phase, the mixture was given an acidic treatment to simulate the conditions as in the stomach with simulated gastric fluids. The pH was raised to 2.0 by adding hydrochloric acid (HCl) and Pepsin was introduced to facilitate protein digestion. The mixture was incubated at 37\u0026deg;C for 2 hours and the samples from the gastrointestinal phase were kept at -20\u0026ordm; Celsius for future analysis. To simulate the conditions in the small intestine, Bile salts and pancreatic enzymes (lipase, amylase and proteases) were added to the gastric digesta. This step allowed for the further digestion of macronutrients and release of nutrients. The mixture was incubated at 37\u0026deg;C for an additional 2 hours and the digested samples were stored at -20\u0026ordm;C. At the end of each phase the samples were collected to analyse the amount of iron content present during the process of digestion. This digesta reflected the changes in nutritional composition that occurred during the \u003cem\u003ein vitro\u003c/em\u003e digestion process and represented the partially digested wheat sample.\u003c/p\u003e\n\u003ch3\u003eCaco-2 cell culture\u003c/h3\u003e\n\u003cp\u003eCaco-2 cells were obtained from American type culture collection and used in experiments at passages 30\u0026ndash;40; the cells were cultured in 25 or 75cm\u003csup\u003e2\u003c/sup\u003e culture flasks and seeded at densities of 50000 cells/cm\u003csup\u003e2\u003c/sup\u003e in 6-well plates. The culture medium used was Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) with high glucose and supplemented with 10% (v/v) Fetal bovine serum, 1% (v/v) penicillin/ streptomycin solution, and 1% (v/v) non-essential amino acid. The cells were maintained at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air. Caco-2 cells were cultured until they formed a confluent monolayer the medium was changed every 48 hrs. This monolayer represented the intestinal epitheliums which were further seeded in 6 well plates and used 12\u0026ndash;14 days after seeding [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eIron uptake by Caco-2 cell lines\u003c/h3\u003e\n\u003cp\u003eFollowing the INFOGEST digestion, the resulting intestinal digesta was transferred on to the Caco-2 cell monolayer. The purpose was to assess how the digested nutrients/ iron interacted with the cells and absorbed by the cells. The Caco-2 cell studies consist of 6-well culture plates and the samples were split across different plates. The aliquot of the food digest (intestinal phase) was transferred to the well, allowing soluble iron to pass through and was incubated for 22-24hrs. At the end of each experiments, medium was removed from the wells and cells were rinsed twice with ice cold phosphate buffered saline (PBS) and cell monolayer were removed with a cell scarper and placed in Ependroff tubes and stored at -80\u0026ordm;C until further analysis. Cells were analysed for Iron content using ICP-MS, which quantified the extent of iron absorption by the intestinal cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBioavailability study/In vivo Kinetic studies\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e kinetic/bioavailability study was performed at JSS College of Pharmacy, Ooty with the approval from the Institutional Animal Ethics Committee (IAEC), Approval no. JSSCPO/OT/IAEC/06/2023-24. The processed wheat samples were chosen to evaluate the kinetic profile of iron upon oral administration to animal models. Male Wister rats of about 200-250gm were selected and they were grouped into 3 groups of 6 animal each where; Group-I animals were administered with Biofortified samples; Group-II with fortified samples and Group-III with normal wheat flour samples and the comparative studies were carried out for the following groups.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample assignment\u003c/h3\u003e\n\u003cp\u003eBioavailability of iron from the cooked wheat samples (Biofortified, Fortified and Normal flour samples) were determined by the serum iron concentration curve (SIC) method. This method was based on plotting the serum iron concentrations after oral administration of samples to rats. Prior to the start of the experiment, the male rats were fed with a standard diet and distilled water ad libitum for five days. Then all the rats were kept fasted for 18 hours and randomly distributed into 3 groups having 6 rats each. Freshly prepared samples were orally administrated to the rats to provide 15mg Fe/kg body weight. For the control rats, deionised water alone was orally administered [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBlood collection\u003c/strong\u003e \u003cp\u003eAfter 0, 1, 2, 4, 6 8, 12, 24 and 48 hours of oral administration, blood samples were withdrawn (staggered manner) through retro-orbital puncture and serum was collected by centrifugation at 2000g for 10 minutes. Further, serum from all the samples was used for the measurement of serum iron concentration using ICP-MS [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Iron content\u003c/h2\u003e \u003cp\u003eThe Iron content in the wheat flour and the \u003cem\u003ein vitro\u003c/em\u003e digested samples and serum samples were determined by inductively coupled plasma Mass spectroscopy. Briefly, about 0.3 to 0.5g of samples were weighed and placed into Teflon vessels to which a 6 mL of ICP-MS grade (AD Trace pure) nitric acid and 4\u0026ndash;6 mL of ICP-MS grade water was added and placed in microwave digester (Perkin Elmer Titan MPS) with the below mentioned program for cereal matrix (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After digestion, the volume was adjusted to 50 mL using deionised water and introduced into the inductively coupled plasma mass spectrometer. The iron content was measured at specific wavelengths, and concentrations were quantified from standard solutions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMicrowave programme for digestion\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePressure\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRamp\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHold (min)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e120\u0026ordm; C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e180\u0026ordm; C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e70\u0026ordm; C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Phytate (Antinutrient)\u003c/h2\u003e \u003cp\u003e Phytic acid content of the raw and cooked wheat samples were determined according to the method described by Davies and Reid. Individually one gram of raw sample, cooked samples as well as \u003cem\u003ein vitro\u003c/em\u003e digested samples were extracted by taking 40 mL of 0.5M nitric acid in a conical flask and shaken for 1hour on a shaker at 30ᴼC and 80 revolutions per minute. The samples were filtered and 5 mL of 0.08M Ferric chloride was added and boiled for 20 minutes and filtered. The free iron (Fe\u003csup\u003e3\u0026minus;)\u003c/sup\u003e remaining in the solution was determined calorimetrically by adding 2 ml of 0.005M ammonium thiocyanate and the iron-binding capacities of the extract was determined by difference [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll measurements were carried out in triplicate for each of the samples and results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;values of standard deviations using Microsoft Excel, 2016 and ANOVA for statistical significance and PK solver software for kinetic parameters.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003cp\u003eIron bio accessibility in wheat and its processed form were analyzed using the Caco-2 cell line method following standard INFOGEST protocol. Kinetic parameters assessed by Pk solver software, and AOAC standards for iron quantification data are presented in below tables.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIron content\u003c/strong\u003e in Biofortified (B), Fortified (F), Non-biofortified (NB) and Non-fortified (NF) samples were evaluated in both raw and processed/cooked states and are presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. According to IFCT standards, Non-biofortified and Non-fortified wheat flours typically contain approximately 41.0 mg/1000g and 39.7 mg/1000g of iron, respectively [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. In raw form, biofortified samples displayed iron concentrations ranging from 67.5 ppm to 62.5 ppm, Fortified samples showed values ranging from 32ppm to 53ppm surpassing both non-biofortified and non-fortified samples. In the processed/cooked state, biofortified wheat products exhibited iron concentrations of 60ppm to 61ppm, fortified samples in the processed/cooked state showed 30ppm to 52ppm again surpassing both non-biofortified and non-fortified samples. The results suggest that biofortified wheat products consistently outperformed both NB and NF counterparts in terms of iron. The reduction in the iron content in the processed form can be attributed to several factors such as temperature, chemical reactions/degradation, denaturation of iron that occurs during the processing leading to conversion of iron forms into less soluble forms. Cooking process can also lead to physical changes in structure of the food matrix, which may impact the accessibility and release of iron during analysis. Some amount of iron may leach out into the cooking medium or may be lost due to adherence to cooking surfaces. These losses could contribute to the observed reduction in iron content in the cooked samples [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. The increased iron concentrations in BF samples, both in raw and processed states, emphasize the potential of biofortification as an effective strategy to address iron deficiency. Fortified samples also demonstrated improved iron concentration compared to NF samples, indicating the effectiveness of fortification in enhancing iron content. However, biofortified samples consistently exhibited higher iron concentrations, emphasizing the superiority of biofortification over traditional fortification methods. Furthermore, all employed methods in this study to measure iron content exhibited a positive correlation, which was statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. These findings suggest that the bioavailability of iron in wheat products can be significantly enhanced through fortification and biofortification, contributing to improved nutritional outcomes in populations heavily reliant on wheat-based diets and to assess the potential health benefits of incorporating functional wheat products into regular diets [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIron content in wheat flour and cooked from samples in ppm\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample (lab code)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIron content (wheat flour)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIron content (chapatti)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% loss\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% present\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e*NB: Non biofortified, B: Biofortified, NF: Non-fortified, F: Fortified\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eValues are expressed in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation*n\u0026thinsp;=\u0026thinsp;3 determinations per sample significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eIn vitro Iron Bio accessibility\u003c/h2\u003e\n \u003cp\u003eNon-fortified/non-biofortified wheat samples: In the uncooked form, the Bioaccesible iron content of NB and NF wheat flour ranged from 9.2 ppm to 13.1 ppm, with corresponding bio accessibility percentages between 69.1% and 67.1%. Following the cooking process, the Bio accesible iron content ranged from 12.5 ppm to 11.5ppm, showing a slight decrease, while the bio accessibility percentages increases to a range of 75.7\u0026ndash;76.6%. The observed decrease in bio accessible iron content after cooking NB/NF wheat samples may be attributed to various factors, such as heat-induced alterations in the physical and chemical structure of iron compounds [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the concurrent increase in bio accessibility percentages post cooking suggests that the cooking process may enhance the bioavailability of iron despite a nominal decrease in total content [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eFortified/Biofortified wheat samples: Unprocessed samples of F and B wheat flour exhibited a substantial increase in bio accessible iron content, ranging from 40.5 ppm to 50.5ppm. The corresponding bio accessibility percentages were notably higher, ranging from 84.1\u0026ndash;86.7%. Moreover, processed samples of F and B wheat flour demonstrated a further enhancement, with bio accessible iron content ranging from 43.8 ppm to 51.6 ppm, and bio accessibility percentages reaching 90.8\u0026ndash;92.4%. represented in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026amp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026amp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eIt was found that the iron was found to be more accessible in the gastric phase compared to the intestinal phase this could be influenced by several factors i.e., solubility of iron where iron tends to be more soluble in acidic nature making it more accessible for absorption once the partially digested food enters into the small intestine, where the pH levels are higher and neutral, the solubility of iron may decrease, affecting its bio accessibility. The composition of the food matrix can significantly influence iron bio accessibility as certain foods such as phytates, and calcium can bind to iron and reduce its absorption. Theses interactions could be more pronounced in the intestinal phase when compared to gastric phase [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the stomach, iron may form complexes with certain components of the food matrix, which could enhance its solubility and availability. However, in the intestinal phase, these complexes might break down affecting iron release and absorption. Enzymes present in the small intestine further break down food particles, potentially altering the availability of iron. The shift from an acidic to a more neutral environment in the intestine can also impact the chemical state of iron [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. The results of the \u003cem\u003ein vitro\u003c/em\u003e bio accessibility studies using the Caco-2 cell line reveal interesting insights into the iron accessibility of both NF/NB and F/B wheat flour. The substantial increase in both bio accessible iron content and bio accessibility percentages in F and B wheat flour, especially after processing, highlights the effectiveness of F and B strategies. The higher bio accessibility percentage post-processing may be indicative of improved iron solubility or alterations in the matrix that facilitate better iron release during digestion [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. These findings underscore the potential of fortification and biofortification in enhancing the bioavailability of iron in wheat flour. Further investigations into the mechanisms underlying the observed changes in bio accessibility could provide valuable insights for improving the nutritional quality of staple foods. Moreover, the study emphasizes the importance of considering processing methods in biofortification/fortification strategies to optimize the bioaccessibity of essential nutrients.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIron content assessment in wheat flour samples at different digestive phases (ppm)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWheat flour Sample lab code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOral Phase\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGastric phase\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIntestinal phase\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBioaccesible iron Caco-2 cell absorption\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% degraded/loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% available\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% degraded/loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% available\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e86.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% degraded/loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% available\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% degraded/loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% available\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e84.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e*NB: Non biofortified, B: Biofortified, NF: Non-fortified, F: Fortified\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eValues are expressed in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation; *n\u0026thinsp;=\u0026thinsp;3 determinations per sample significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIron content assessment in cooked form (chapatti) samples at different digestive phases (ppm)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChapatti Sample lab code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOral Phase\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGastric phase\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIntestinal phase\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBioaccesible iron Caco-2 cell absorption\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% degraded/loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% available\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% degraded/loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% available\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e92.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% degraded/loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% available\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% degraded/loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% available\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e90.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e*NB: Non biofortified, B: Biofortified, NF: Non-fortified, F: Fortified\u003c/p\u003e\n \u003cp\u003eValues are expressed in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation*n\u0026thinsp;=\u0026thinsp;3 determinations per sample significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-nutrients (phytate)\u003c/strong\u003e IFCT data by Longvah T and Ananthan R revealed that wheat Atta\u0026rsquo;s phytate level ranges from 632 to 638 mg/100g [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e] Higher phytic acid levels were observed in wheat flour, ranging from 504 mg/100g to 625.8mg/100g, compared to processed samples, where phytate levels ranged from 225.1 mg/100g to 330.2 mg/100g. Notably, after absorption phytate content was found to be higher in digested wheat flour ie, 455.1 to 545.4 mg/100g than in cooked form biofortified wheat with 212.4 mg/100g and fortified wheat with 185.2mg/100g exhibiting significantly lower phytate content than normal wheat, suggesting a potential enhancement in iron absorption. \u003cem\u003eIn vitro digestion\u003c/em\u003e demonstrated a reduction in phytate content, with bioavailability dropping to 50% in cooked forms. Represented in Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The percentage of reduction in phytate content during cooking would depend on various factors such as cooking method, duration, temperature, and initial phytate concentration in the raw material. i.e., on heating chemical reaction occurs that break down the phytate structure, in the food matrix exposing the phytate molecules to degradation reducing its concentration, and heating can also lead to inactivation of enzymes responsible for the synthesis of phytate. The observed variations in phytate levels between raw wheat flour and cooked samples indicate the impact of processing on phytate degradation [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. The observed variations in phytate levels between raw wheat flour and cooked samples indicate the impact of processing on phytate degradation. The higher phytate content in digested wheat flour suggests a potential limitation in iron absorption, as phytate can form insoluble complexes with iron. The significantly lower phytate content in biofortified and fortified cooked samples is promising, as it implies enhanced iron bioavailability [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. This finding aligns with the increased iron concentrations observed in biofortified wheat product, emphasizing the potential of biofortification to mitigate the inhibitory effects of phytate on iron absorption. The reduction in phytate content during \u003cem\u003ein vitro\u003c/em\u003e digestion further supports the perception that processing methods can influence the bioavailability of iron. The drop-in bioavailability suggests the potential for improved iron absorption in the gastrointestinal tract [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The finding underscores the importance of considering both strategies for addressing iron deficiency and providing valuable insights for future interventions aimed at improving iron status in populations reliant on wheat-based diets.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eInfluence of phytic acid on absorption by Caco-2 cell lines in wheat flour and its cooked form\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample (la code) mg/100g\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhytate (wheat flour)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhytate after absorption by Caco-2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhytate (cooked)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhytate after absorption by Caco-2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e625.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e545.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e330.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e311.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e575.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e465.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e291.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e212.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e521.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e501.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e243.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e216.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e504.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e455.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e225.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e185.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e*NB: Non biofortified, B: Biofortified, NF: Non-fortified, F: Fortified\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eValues are expressed in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation*n\u0026thinsp;=\u0026thinsp;3 determinations per sample significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eIn vivo bioavailability studies\u003c/h2\u003e\n \u003cp\u003eOnce the bio accessibility of the iron content was evaluated for the selected biofortified and fortified wheat samples the bioavailability of iron were analysed through \u003cem\u003ein vivo\u003c/em\u003e studies. Pharmacokinetics involves determining the absorption, distribution, metabolism, and excretion (ADME). The plasma concentrations of the analyte were used to evaluate the pharmacokinetic parameters. Measurable iron-blood levels were noticed in all serum samples up to 48 hrs., after oral administration of wheat samples in the form of chapatti i.e., group 1 with biofortified, group 2- fortified and group 3- non-fortified chapatti samples. At particular time intervals of 0, 1, 2, 4, 6, 8, 12, 24, 48 blood was withdrawn, and centrifuged at 2000g for 15min to separate serum for quantification of iron using ICP-MS. A non-compartmental model analysis in PK SOLVER software was utilized to calculate the pharmacokinetic parameters with linear Trapezoidal method. The peak plasma concentration i.e., Cmax of iron in biofortified wheat samples was observed to be 13.557 mg/kg at 2.0 hrs of Tmax. Cmax of iron in fortified samples was found to be 12.724g/kg at 2.0 hrs. Tmax; and Cmax of 8.184mg/kg at 1hrs for normal wheat flour samples. The pharmacokinetic parameters evaluated for the administered wheat samples are summarized in table.7\u0026amp;8 and area under curve was plotted for time (h) vs. concentration (mg/kg) in Figure.6\u0026ndash;8. It was clearly evident that processing and cooking clearly improved the bio accessibility as well as the bioavailability of iron.\u003c/p\u003e\n \u003cp\u003eBio accessibility measures the fraction of a nutrient that is released from the food matrix during digestion and made available for absorption. Bioavailability, on the other hand, refers to the proportion of the absorbed nutrient that is utilized by the body. In co-relation the iron content observed between \u003cem\u003ein vitro\u003c/em\u003e bio accessibility and \u003cem\u003ein vivo\u003c/em\u003e plasma iron bioavailability can be attributed to several aspects i.e. the \u003cem\u003einvitro\u003c/em\u003e bio accessibility studies with Caco-2 cell line, simulate the digestion process but it does not completely replicate the complexities of the human digestive system whereas, the \u003cem\u003ein vivo\u003c/em\u003e studies involve the actual digestion and absorption process in the rat animal model, which can be influenced by the factors such as gastric acidity, enzymatic activity and interactions with other food components [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003eIn vivo\u003c/em\u003e studies also account for the metabolism and excretion of nutrients, which may affect the measured plasma concentrations over time. Also, the metabolism and the presence of binding proteins in the bloodstream can influence the pharmacokinetic profile of iron. The rate of iron absorption can vary between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e systems, leading to differences in the timing and magnitude of peak plasma concentrations (Cmax) observed in the two types of studies. \u003cem\u003eIn vitro\u003c/em\u003e studies may not fully capture the effects of food matrix components on iron absorption, whereas \u003cem\u003ein vivo\u003c/em\u003e studies consider the complex interactions between nutrients and other food components present in the diet which influences the maximum plasma iron concentration [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSerum Iron content in biofortified, fortified and normal flour processed wheat samples (ppm)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSl no\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBiofortified samples\u003c/p\u003e\n \u003cp\u003eSerum iron Conc.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFortified samples Serum iron Conc.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal wheat flour serum iron conc.\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.184\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.847\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.485\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.896\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.684\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.829\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.938\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.533\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.898\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.365\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.834\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.337\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRepresentation of kinetic parameters in biofortified, fortified and normal wheat chapatti samples (ppm)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSl no.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUnit\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eValues (Biofortified samples)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFortified samples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal flour samples\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLambda_z\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1/h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT \u003csub\u003e1/2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTmax\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCmax\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTlag\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUC 0-t\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg/kg*h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e451.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e392.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e254.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUC 0-inf_obs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg/kg*h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1563.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1276.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e846.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUMC 0-inf_obs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg/kg*h^2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e221201.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e163099.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e114946.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMRT 0-inf_obs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e141.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e127.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e135.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVz/F_obs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(mg)/(mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCl/F_obs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(mg)/(mg/kg)/h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.009\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.017\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eTo comprehend the iron content in processed samples, a preliminary assessment of iron levels in raw wheat flour was essential. The iron content in wheat flour varies due to factors like wheat variety, soil conditions, and milling processes. On average, whole wheat contains approximately 4 mg/100g of non-heme iron, less readily absorbed than heme iron found in animal products. Cooking can result in a loss of small traces of iron, influenced by variables such as temperature and duration, affecting bio accessibility and bioavailability. Our study yielded novel findings, particularly in the quantification of iron in various wheat flour varieties, including enriched and Bio accesible studies not reported previously. For instance, Saied et al. investigated nutritional parameters in commercially available wheat flours in Bangladesh, while Niedzielski et al. determined iron levels using UV and AAS techniques. Limited research exists on the comparative mineral content in cereal crops, and various studies, such as those by Akinyele and Shokunbi; Arif et al.; Hailu Kassegn focus on specific aspects but lack comprehensive quantification of iron in different wheat samples. Our comparative assessment of iron estimation in functional wheat varieties underscores the potential of biofortification and fortification in enhancing nutritional content. The significantly higher iron concentration in biofortified and fortified wheat flour, observed in both raw and cooked samples, supports the effectiveness of these strategies in addressing iron deficiency. Iron bio accessibility in chapati samples prepared from these flours demonstrated increased iron content, particularly in biofortified chapati, showcasing the nutritional quality enhancement through biofortification. Several factors contribute to enhanced iron bio accessibility and bioavailability, including fortification, breeding programs for high-iron varieties, and the use of Caco-2 cell lines as an \u003cem\u003ein vitro\u003c/em\u003e model and \u003cem\u003ein vivo\u003c/em\u003e animal model. While processing marginally deteriorates metal content, our analysis of phytate content revealed lower levels in biofortified wheat samples compared to normal varieties, suggesting the potential for selecting wheat varieties with lower phytate content to improve iron bio accessibility. Overall, this study highlights the multifaceted approaches to enhance iron bioavailability in wheat-based food products and their potential impact on addressing iron deficiency malnutrition.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study highlights the potential of biofortified and fortified wheat flour and processed samples as significant dietary sources of bioavailable iron. The importance of considering both iron concentration and factors influencing iron absorption is emphasized through evaluation of iron content as its bio accessibility, bioavailability and phytate content. Future research endeavours should explore the practical implications of these findings on human populations and assess the benefits of selecting wheat cultivars with reduced phytate levels to enhance overall iron bioavailability. Based on the comprehensive analysis of the bio accessibility and bioavailability of iron from different wheat samples (biofortified, fortified and normal wheat samples) conducted it was observed that processing and cooking of the samples, significantly improved the bioavailability of iron from wheat samples. Both the functional wheat forms i.e., biofortified and fortified wheat samples exhibited higher peak plasma concentration Cmax when compared to normal samples, indicating better absorption and utilization of iron. The Tmax i.e., the time to reach the maximum plasma concentration was relatively consistent across all wheat samples analysed, stating a similar rate of absorption regardless of the functional strategies. Implementing these strategies could be highly significant in combating the deficiencies. Our research illuminates the valuable contributions of both biofortified and fortified wheat products to dietary iron intake, with a slight advantage in iron bio accessibility observed in biofortified products. While chapattis offer diverse nutritional benefits, including dietary fiber and a balanced diet that supports increased iron consumption, maintaining a varied and balanced diet remains vital to meeting daily iron needs and preventing iron deficiency. However, it is crucial to recognize the need for further exploration through studies and clinical trials to validate the real-world impact of increased iron bioavailability on human health.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eWHO- World Health Organization\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRBCs- Red blood cells\u003c/p\u003e\n\u003cp\u003eCaco-2- Colonic Carcinoma cells\u003c/p\u003e\n\u003cp\u003eCmax-Peak Plasma concentration\u003c/p\u003e\n\u003cp\u003eTmax- Time of Peak Concentration\u003c/p\u003e\n\u003cp\u003eT1/2- Half-Life\u003c/p\u003e\n\u003cp\u003eCI- Clearance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBW- Biofortified wheat\u003c/p\u003e\n\u003cp\u003eNW - Normal whole wheat\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eF- Fortified\u003c/p\u003e\n\u003cp\u003eNF- Normal wheat flours\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSSF-Simulated salivary Fluids\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSGF- Simulated gastric fluids\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSIF- Simulated intestinal fluid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDMEM- Dulbecco’s modified Eagle’s medium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIAEC PBS- Phosphate buffered saline\u003c/p\u003e\n\u003cp\u003eIAEC- Institutional Animal Ethics Committee\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;SIC-Serum iron concentration curve\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to JSS College of Pharmacy, Ooty, and JSS Academy of Higher Education and Research, Mysuru, India for the continuous support and providing the facilities for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors especially would like to thank ICMR ADHOC [No. F.N.5/9/1323/2020-Nut] for the financial support,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to the Director, DFRL, Mysore, for providing the necessary facilities to conduct the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors made substantial contributions to this manuscript. Harshitha Arun Pardhe conceptualized and drafted the manuscript. Krishnaveni Nagappan supervised the study design. Naveen S provided technical and material support for sample analysis. Rashmi V offered technical assistance for spectrometric analysis. Aditya Kumar Singh conducted \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003estudies analysis and interpretation. Mohit Ananda contributed to data analysis and manuscript drafting. Gullapalli Kowmudi assisted with reviewing and practical implications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Issues\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003ekinetic/bioavailability study was performed at JSS College of Pharmacy, Ooty with the approval from the Institutional Animal Ethics Committee (IAEC), Approval no. JSSCPO/OT/IAEC/06/2023-24.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the [Indian Council of Medical Research (ICMR) under Grant No. F.N.5/9/1323/2020-Nut\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHarshitha Arun Pardhe- http://orcid.org/0000-0003-3826-9219\u003c/p\u003e\n\u003cp\u003e*Krishnaveni Nagappan- http://orcid.org/0000-0003-0596-9489\u003c/p\u003e\n\u003cp\u003eNaveen S- http://orcid.org/0000-0003-2981-422X\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Rashmi V- http://orcid.org/0000-0001-5692-7938\u003c/p\u003e\n\u003cp\u003eAditya Kumar Singh- http://orcid.org/0009-0008-0202-8011\u003c/p\u003e\n\u003cp\u003eGullapalli Kowmudi- http://orcid.org/0000-0001-9564-0542\u003c/p\u003e\n\u003cp\u003eMohit Ananda- https://orcid.org/0000-0002-1142-543X\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization, (2021). 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Nutr. \u003cb\u003e9\u003c/b\u003e(2), 225 (1989). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ajcn/49.2.225\u003c/span\u003e\u003cspan address=\"10.1093/ajcn/49.2.225\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biofortification, Fortification, Bio-accessibility, Iron, Caco-2 cell, Bioavailability","lastPublishedDoi":"10.21203/rs.3.rs-6358588/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6358588/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIron deficiency remains a significant nutritional concern globally, particularly in developing nations, attributed to insufficient nutritional status, specifically inadequate iron intake in the diet. This study utilized In vitro static digestion and a Caco-2 cell model to assess iron bioaccessibility in biofortified and fortified wheat flour, along with its cooked form (chapatti), a commonly consumed wheat-based food product. In this study wheat flour and chapatti samples underwent standardized INFOGEST protocol, involving oral, gastric, and intestinal phases, followed by iron bioaccessibility assessment using the Caco-2 cell monolayer model. Compared to conventional wheat types, biofortified and fortified flours exhibited a significant increase in iron content (67.5 ppm and 53.3 ppm, respectively) and iron bioaccessibility 80.7% and 78.3%, respectively. In the cooked form, biofortified wheat demonstrated higher iron bioaccessibility 91.1% compared to regular wheat varieties and the uncooked form. The fortified form showed a bioaccessibility of 90.5%, suggesting the influence of food matrix and processing techniques. Wheat flour serves as an affordable nutrient source, addressing deficiencies for a majority of individuals. The findings contribute to nutritional planning for wheat product consumption and enhance understanding of iron bio accessibility\u0026rsquo;s role in increasing iron intake and preventing insufficiency in the general population. This research may contribute to better nutrition acceptance and more effective wheat selection.\u003c/p\u003e","manuscriptTitle":"Evaluation of In vitro bio accessibility and In vivo bioavailability of Iron Biofortified and Fortified wheat samples through Static digestion using Caco-2 cell line and animal model studies: Implications for Nutritional Enhancement","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 19:16:21","doi":"10.21203/rs.3.rs-6358588/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"58623306-be19-438d-9052-b28777b3b1cb","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-12T23:53:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 19:16:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6358588","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6358588","identity":"rs-6358588","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}