Bioaccessibility of iron in developed pectin iron complex using Citrus

limon Burm F. peels subjected to in-vitro gastro-pancreatic digestion

Singhal, S., Swami Hulle, N. R., & Koidis, A. (2024). Bioaccessibility of iron in developed pectin iron complex
using Citrus limon Burm F. peels subjected to in-vitro gastro-pancreatic digestion. Food Chemistry, 457, Article
140457. [Link]

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Download date:16. thg 4. 2025

 1 Bioaccessibility of iron in developed pectin iron complex using Citrus limon
2 Burm F. peels subjected to in-vitro gastro-pancreatic digestion

3 Somya Singhala,b, Nishant Rachayya Swami Hullea*, Anastasios Koidisb

a
4 Department of Food Engineering and Technology, Tezpur University, Tezpur, Assam, India-
5 784028

b
6 Institute for Global Food Security, School of Biological Sciences, Queen’s University
7 Belfast, Belfast, United Kingdom-BT7 1NN

10 Abstract

11 Pectin from the citrus peel waste has novel applications in food and biomedical industries. The
12 present work focused on addressing iron deficiency, which is a global health concern, by
13 developing a functional ingredient using pectin extracted from Assam lemon (Citrus limon
14 Burm. F) and supplementing iron via the pectin-iron complex (PIC). Extracted pectin was
15 incubated with iron chloride hexahydrate (0.90-1.80 mM) for 180 h to optimize the
16 complexation conditions, with the optimal concentration being 1.36 mM. The iron
17 bioavailability and its absorption in the PIC was assessed using in-vitro simulation digestion
18 and Caco-2 cell monolayers. The bioaccessible form of iron in the developed PIC during the
19 intestinal phase was 5.34±0.16%, which was negligible in pectin. The absorption of
20 bioaccessible iron in the PIC was found to be 2.93±0.03%. The results demonstrated that PIC
21 could reduce iron deficiency and increase fibre intake, leading to several health benefits.

22

23 Keywords: pectin; bioavailability, in-vitro assay, Caco-2 cell model, pectin-iron complex

24 *corresponding author: Dr Nishant R. Swami Hulle

25 E-mail id: nishant@[Link]
26

27

28

29
30 1. INTRODUCTION

31 One of the major nutritional problems present globally is micronutrient malnutrition especially
32 iron (Fe) deficiency affecting millions of people, especially in infants, children, and pregnant
33 women. Iron deficiency is mostly prevalent in school-aged children in developing countries.
34 The deficiency of the same induces anaemia, increased susceptibility to infection, and
35 decreased cognitive abilities and motor activity among individuals (Kinyoki et al., 2021). The
36 World Health Organization (WHO) has proposed three approaches to combat micronutrient
37 malnutrition such as food diversification and education, fortification, and supplementation
38 (Ritchie and Roser, 2024). Fe supplements, however, if taken orally could result in multiple
39 gastrointestinal side effects due to the release of free Fe ions (Ma et al., 2021). Hence,
40 alternative Fe supplements with limited or no side effects are highly desirable.

41 A study reported the effectiveness of a complex formed between a polysaccharide and Fe(III)
42 as an oral Fe supplement. This complex demonstrated favourable characteristics such as water
43 stability, chemical solubility, and minimal side effects (Saini et al., 2014). It was also found to
44 be non-toxic even at high concentrations (Torino et al., 2014). However, there are certain issues
45 linked with the utilization of polysaccharide-Fe(III) complexes such as the acidic pH in gastric
46 fluid during the process of Fe digestion can lead to the degradation of the composition of a few
47 polysaccharide-Fe(III) complexes, causing the release and dissolution of Fe ions (Cheng et al.,
48 2019). Additionally, insoluble ferric compounds may develop in the intestinal juice, which may
49 not be absorbed by the small intestine (Wang et al., 2015). The main challenge for
50 polysaccharide-based carriers, therefore, lies in retaining Fe ions in the presence of gastric
51 juice. Pectin, a substance with excellent gelling properties against gastric acid, is considered a
52 promising solution. Importantly, pectin does not degrade when exposed to digestive enzymes
53 present in the small intestine (Ma et al., 2021). Consequently, employing pectin to transport Fe
54 ions seems to be a logical approach. The development of iron-pectin beads from the ionic
55 gelation method has resulted in a novel system for the delivery of iron to intestinal cells
56 (Ghibaudo et al., 2018). An innovative system for stabilizing and delivering lactic acid bacteria
57 using pectin-iron complex beads showed that beads were stable in simulated digestive
58 conditions and successfully released cultivable bacteria, iron, and pectin in the gut (Ghibaudo
59 et al., 2017).

60 By definition, pectin is a type of anionic polysaccharide that, primarily contains of D-

61 galacturonic acid residues linked together through α-(1→4)-glycosidic bonds along with
62 different neutral sugar residues. Pectin has gained much significance globally due to its diverse
63 range of bioactivities and effectiveness. Pectin is one of the most widely used additives in food
64 and beverage formulations for its ability to gel, thicken, replace fats, and stabilize colloids.
65 Additionally, it has shown promise in biomedical research for drug delivery, antioxidative
66 effects, and promoting wound healing. Pectin has also a significant role in lowering blood
67 cholesterol among patients (Singhal et al., 2022). A patient must consume at least 6 g of pectin
68 each day for their cholesterol to be significantly reduced (Abdulhakeem et al., 2023). Pectin
69 has several advantages when added to food and drug formulations. One of them is the presence
70 of divalent cations in pectin results in the formation of stable gels, making them suitable carriers
71 for delivering bioactive substances (Tyagi et al., 2015) and actively managing obesity and
72 weight loss issues (Abdulhakeem et al., 2023). Pectin from varying sources has been reported
73 to be effective in health management, like lowering cholesterol, which lowers cardiovascular
74 related risks (Naqash et al., 2017). Another reason is pectin has been regarded as harmless,
75 easily accessible, and having low production costs (Abdulhakeem et al., 2023).

76 The present study focuses on extracting and applying pectin obtained from Assam lemon. This
77 variety of Assam lemon is native to the state with a GI Tag and has a unique flavour and
78 morphology, and the peel generally is discarded or pickled. There is no available literature that
79 cites the extraction of pectin from Assam lemon. The present study is cantered on the
80 valorisation of this citrus by-product, which involves extracting pectin from the peel. This
81 approach promotes sustainability and adds value to agricultural waste. In previously cited
82 research works the commercial citrus pectin was used, while the present study is an extended
83 work on the optimization of the valorisation process of Assam lemon using novel techniques.
84 These differences could lead to novel applications or improvements in existing processes.
85 There is limited scientific information available on the formation of pectin-iron complex (PIC)
86 and their characterization for possible applications in food systems. The objective of this
87 investigation is to create a citrus based pectin-iron complex (PIC) to develop a novel iron
88 supplement. This involves complexing ferric ions with citrus pectin. The resultant complex was
89 characterized using Spectrometric techniques, Scanning Electron Microscopy (SEM), Degree
90 of esterification (DE), and galacturonic acid analysis. Moreover, the absorption of Fe from
91 synthesized PIC was studied through in vitro assays and the Caco-2 cell model.

92

93 2. MATERIALS AND METHODS

 94 2.1. Materials and Reagents

95 The freshly harvested lemon samples were collected from the Citrus Garden, Tezpur
96 University, Assam, India, during the period of June-July, 2021. The lemons were washed,
97 peeled and were subjected to tray drying at 45°C/10 h and finely powdered. The lemon peel
98 powder was then sifted via a sieve (40-mesh) and stored at ambient temperature. Caco-2 cells
99 (P-35) were supplied by Prof. Lisa Connolly, School of Biological Sciences, Queen’s
100 University Belfast, UK. Pepsin sourced from porcine gastric mucosa ≥2500 units/mg protein
101 (P7012), pancreatin sourced from porcine pancreas (P7545, 8X USP), and bile extract porcine
102 (B8631) were purchased from Sigma-Aldrich (Dorset, UK). The rest of the reagents were
103 analytical grade purchased from Sigma-Aldrich (Dorset, UK).

104 2.2. Pectin extraction and its purification

105 Citrus pectin from Assam lemon peels was separated using the conventional method described
106 in Singhal et al. (2024). Briefly, 5 g dried peel powder was suspended in 150 mL of citric acid
107 solution (pH 2). The solution was heated at 90°C using a water bath shaker for 60 min and
108 cooled down to room temperature (RT). It was filtered using a cheesecloth to separate any solid
109 particles. The resulting filtrate, containing crude pectin, was collected and then subjected to
110 precipitation with ethanol (99%) in 1:2 (v/v) ratio undisturbed at RT/3 h. Following this, the
111 precipitated pectin underwent centrifugation (6500 rpm/15 min/room temperature), leading to
112 the separation of the wet pectin fraction. Following that, the wet pectin fraction underwent a
113 washing process twice with 150 mL of ethanol to eliminate any remaining monosaccharides
114 and disaccharides. The acquired moist pectin was dried at 40°C in a hot air oven until constant
115 weight was achieved. The obtained dried pectin was finely powdered using a mortar pestle and
116 preserved in a sealed container at RT for further use.

117 2.3. Formulation and experimental design of the PIC

118 The pectin extracted from Assam lemon peels (refer to section 2.2.) was complexed with Fe by
119 modifying the method mentioned in Chirug et al. (2018). Based on the preliminary results, the
120 pectin (0.5% w/v) was suspended in the iron III chloride hexahydrate (FeCl3.6H2O), in a
121 concentration ranging from 0.90 mM to 1.80 mM for 180 h at RT. After incubation, the solution
122 underwent centrifugation at 6000 rpm for 10 min to eliminate unbound Fe from PIC. The PIC
123 was subjected to dialysis against de-ionized water for 48 h/RT, freeze-dried (6 h) and then
124 transferred to a sealed container at RT for further analysis.
125 The formulation of PIC was modelled and optimized using a three level of one-factor response
126 surface methodology design (7 experimental runs). The details of the experimental design have
127 been mentioned in Supplementary Table 1. The parameters of the process and their
128 corresponding range were selected according to initial observations aimed at examining their
129 influence on the response variable, Fe content (ppm) within the PIC. The general linear
130 equation involving process variables was presented in the equation Eq. (1).

131 Y = 𝛽𝛽𝑜𝑜 + 𝛽𝛽1 𝑥𝑥1 + 𝛽𝛽2 𝑥𝑥2 … . . +𝛽𝛽𝑘𝑘 𝑥𝑥𝑘𝑘 …(1)

132 Where, Y is the response variable, 𝛽𝛽𝑜𝑜 is the intercept term, 𝛽𝛽1 , 𝛽𝛽2 , and 𝛽𝛽𝑘𝑘 represent the
133 coefficient associated with the independent variables, and 𝑥𝑥1 , 𝑥𝑥2 , and 𝑥𝑥𝑘𝑘 represent levels of the
134 independent variables considered in the study. All experimental runs were done in triplicates.
135 The effect of independent variables was analysed by employing Design Expert (Version 12) to
136 do regression analysis and ANOVA test. The level of significance was analysed on the basis of
137 F-value at 95% confidence interval.

138 2.4. Determination of Fe content in PIC

139 The Fe content in PIC was measured by ICP-OES (Inductively Coupled Plasma Optical
140 Emission Spectrophotometry) (model: 5100 ICP-OES, Make: Agilent Technologies, USA).
141 The PIC powder (0.100 g) was transferred in labelled 50 mL centrifuge tubes and 2 mL nitric
142 acid (69%) was added to all tubes. After incubating the tubes overnight, 2 mL of hydrogen
143 peroxide was added to the respective tubes and were allowed to release gas for 15 min in a
144 Class II safety cabinet. The tubes were then placed in a microwave digestor’s carousel, and a
145 digestion program consisting of three stages was initiated, which could take a total of 65 min,
146 including than that of cooling process. The chosen program gradually heated the samples for
147 35 min, reaching a temperature of 95°C followed by 30 min digestion. The heating stages were
148 as follows: a 5-min ramp from RT to 55°C, a 10-min hold at 55°C, a 5-min ramp to 75°C, a 10-
149 min hold at 75°C, a final 5-min ramp to 95°C, and a 30-min hold at 95°C. Subsequently, the
150 volumes were adjusted to the final weight of approximately 30 g using deionized water, and
151 the precise masses were recorded. The iron (Fe) content in PIC was then run into the ICP-OES
152 instrument. The determination was conducted in triplicates (Wahengbam et al., 2019).

153 2.5. Physicochemical properties of PIC

154 2.5.1. Proximate analysis
155 Proximate analysis of moisture content, ash content, crude lipid content and crude protein
156 content of pectin and PIC were determined according to AOAC method (2010).

157 2.5.2. Degree of Esterification (DE)

158 An FTIR analysis method was used to determine DE. The term DE denotes the proportion of
159 carboxyl groups (COOH) that have undergone methylation compared with the total number of
160 COOH in a compound. The DE (%) was calculated according to Eq. (2) (Liew et al., 2016):

𝐴𝐴1745
161 𝐷𝐷𝐷𝐷(%) = 𝐴𝐴 × 100 …(2)
1745 +𝐴𝐴1630

162 where, A1745 and A1630 represent the absorbance intensity at wavelengths 1745 cm-1 and 1630
163 cm-1, respectively.

164 2.5.3. Methoxyl group content

165 The methoxyl group content of pectin and PIC was determined using the method of Ranganna
166 (1995) by equation 3.

𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑜𝑜𝑜𝑜 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (𝑚𝑚𝑚𝑚)×𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎×3.1

167 𝑀𝑀𝑀𝑀𝑀𝑀ℎ𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 (%) = 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊ℎ𝑡𝑡 𝑜𝑜𝑜𝑜 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 (𝑔𝑔)
….(3)

168 2.5.4. Galacturonic acid

169 The content of galacturonic acid in the PIC was analysed using the 3,5-dimethyl phenol method
170 described by Kumar and Kumar (2017). In a test tube, 0.25 mL PIC solution (1 mg/mL in
171 distilled water) was mixed with 0.25 mL of sodium chloride solution (2% w/v) and 4 mL of
172 concentrated sulfuric acid (37% w/v). The solution was then heated at 70°C for 10 min followed
173 by cooling to RT. Then, 0.1 mL of a 3,5-dimethyl phenol solution (10 mg of 3,5-dimethyl
174 phenol in 10 mL glacial acetic acid) was added and mixed. Immediately afterwards, the
175 absorbance difference at 450 nm and 400 nm was measured using a UV-Vis spectrophotometer.
176 D-galacturonic acid solutions (0.2-2.0 mg/mL) were used as a reference standard for the
177 calibration curve. Using this calibration curve, the galacturonic acid content of the PIC was
178 calculated. The absorbance difference of the standard was multiplied by a correction factor of
179 1.1.

180 2.6. Structural characterization of PIC

181 2.6.1. Fourier transform infrared spectroscopy (FTIR)
182 About 1 mg of PIC was mixed with 10 mg of potassium bromate and subjected to hydraulic
183 pressing to form a pellet. Subsequently, the scanning of pellet was done using an FTIR
184 spectrophotometer (Model: Nicolet Instruments 410 FTIR, Make: Thermo Scientific, USA)
185 across the frequency (400-4000 cm-1) with a resolution of 4 cm-1 (Ma et al., 2021).

186 2.6.2. X-Ray diffraction (XRD)

187 The PIC’s crystal structures and atomic spacing were examined using XRD. The diffraction
188 patterns of crystals of the PIC were recorded using an XRD instrument (AXS SMART APEX-
189 I, Bruker, Germany). The radiation settings were maintained at 40 kV and 40 mA. The scanning
190 range extended from 2θ of 5° to 80°, and the scanning rate was set at 4 degrees per minute (4
191 deg min-1) (Singhal et al., 2024).

192 2.6.3. Scanning electron microscopy (SEM)

193 Images of the PIC were taken following the application of gold sputter coating to evaluate the
194 surface morphology of the PIC, particularly focusing on single crystals. This investigation was
195 conducted utilizing a dual-beam Focused Ion Beam (FIB) TESCAN LYRA3 instrument (Wang
196 et al., 2020). SEM image of pectin and PIC was captured at a view field from 1.38-3.85 mm
197 with a scale bar indicating a length of 200 µm-1mm and in 75x to 200x magnification range.

198 2.7. Rheological and thermal study of pectin and PIC

199 Pectin and PIC powder were dissolved in distilled water to prepare pectin and PIC solutions
200 with a 1%, 2%, and 3% (w/v) concentration. The rheometer (MCR72, Anton Paar, Austria)
201 equipped with parallel plate geometry (60 mm diameter) and a 1 mm gap between plates was
202 used to conduct dynamic frequency sweeps. The strain (1%) was kept within the linear
203 viscoelastic zone during tests, which were performed at a constant temperature of 25°C over
204 an angular frequency range of 0.1 to 100 rad/s producing storage modulus (Gʹ) and loss
205 modulus (Gʹʹ)

206 The thermal characteristics of the pectin and PIC in a nitrogen environment were measured
207 using a differential scanning calorimeter (DSC 214 Polyma, Netzsch, Germany). A sample of
208 around 10 mg was weighed and sealed with the lid inverted in an aluminium concave. A blank
209 sample was used as a control. The samples were heated from 30-300°C at a rate of 10°C/min.
210 The enthalpy (H), conclusion temperature (Tc), peak temperature (Tp), and onset temperature
211 (To) were obtained from the thermogram.
212 The degrading parameters of pectin and PIC were examined using thermogravimetric (TGA)
213 processing. To evaluate each sample's thermal stability, a thermogravimetric analyzer (TG 209
214 F1 Libra, Netzsch, Germany) was employed. Samples were heated at a rate of 10°C/min to
215 temperatures between 25°C and 600°C in a nitrogen environment.

216 2.8. In vitro Fe release from PIC for assessment of bioaccessible Fe

217 Bioaccessibility of Fe in the pectin and PIC was carried out by the INFOGEST in vitro digestion
218 protocol (Brodkorb et al., 2019). In order to replicate the human digestive system, in-vitro
219 digestion was performed comprising three main steps: oral, gastric, and intestinal digestion. In
220 the oral phase, 1 g of PIC powder was combined with a 30 mL solution containing 140 mM
221 NaCl and 5 mM KCl in a 1:1 (v:v) ratio. Then, added 25 μL CaCl2 (0.3M) and 500 μL of 100
222 mM ascorbic acid solution to the oral bolus. The oral bolus was placed in a water bath shaker
223 at a rate of 200 rpm maintained at 37°C for 2 min. For the gastric phase, 0.5 mL of pepsin
224 solution with an 11000 U/mL concentration was introduced to the previously orally digested
225 sample. The pH of 3 was adjusted using 1 M HCl. The oral bolus was thereafter placed in a
226 shaking water bath for 2 h at 200 rpm and 37°C. For the intestinal phase, the gastric chime after
227 incubation was adjusted to pH 5 using 1 M NaHCO3. Following pH adjustment, a 2.5 mL
228 solution of the pancreatin-bile mixture (0.45 g bile salts and 0.075 g pancreatin in 37.5 mL of
229 0.1 M NaHCO3) was introduced to the gastric chime. After adding 40 μL CaCl2 (0.3 M), the
230 pH was adjusted to 7.0 using 1 M NaOH. The mixture was then incubated for 2 h in a shaking
231 water bath set at 37°C/200 rpm. Following incubation, the samples that had undergone
232 digestion were cooled in ice for 10 min and centrifuged at 3500 g-1 for 1 h at the temperature
233 of 4°C. The resulting supernatants (soluble mineral fraction) were carefully isolated and stored
234 at −80°C for subsequent analysis.

235 2.9. Quantification of bioaccessible Fe

236 The supernatants from the preceding step (Section 2.7.) were analysed for bioaccessible Fe by
237 ICP-OES (Section 2.4.). The % bioaccessibility of Fe in PIC was calculated by using an
238 equation.

𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑚𝑚.𝑓𝑓.
239 %𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 = 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑚𝑚.𝑐𝑐.
× 100 …(4)

240 where: the bioaccessible mineral fraction (bioaccessible m.f.) and the total micronutrient
241 content (m.c.) represents the soluble Fe fraction obtained post simulated digestion and the
242 overall Fe content found in dried PIC (ppm), respectively (Wahengbam et al., 2019).
243 2.10. Fe release study from PIC using Caco-2 cells

244 As mentioned in section 2.7, the soluble m.f. from the in-vitro simulated digestion was used to
245 examine the bioavailability of Fe transfer across Caco-2 cells. The cells were cultured in flasks
246 (75 cm2) and nurtured in a nutrient medium comprising Dulbecco’s minimum essential media
247 (MEM). Fetal bovine serum (10% v/v), sodium pyruvate (1% v/v), 4 mM L-glutamine, and 1%
248 (v/v) penicillin-streptomycin were added to the solution. The cells were incubated (5% CO2
249 and 95% relative humidity) at 37°C under standard conditions (Wahengbam et al., 2019).

250 For testing the bioavailability, 0.1 mL of the cell solution, having 2×105 cells/well for each 6.5
251 mm diameter filter, was seeded in the inserts (apical compartment) of 24 well plates, while 0.6
252 mL of nutrient medium was added to the basolateral compartment. The plate was then
253 incubated with the filter supports at 37°C in an incubator with 5% CO2, and 95% relative
254 humidity for 6 h. After incubation, the apical medium was removed without disturbing the
255 inserts and replaced with 0.1 mL of nutrient medium to remove non-adherent cells thus,
256 reducing the risk of multilayer formation. The nutrient medium was refreshed every 48 hours
257 during the initial 21-29 days after seeding. After this time frame, the growing media was
258 removed. Then, 0.6 mL and 0.1 mL of Ca2+ and Mg2+ free Hank's balance salt solution (HBSS)
259 were poured into the basolateral and apical compartments, respectively, to rinse the cell
260 monolayer. Iron can compete with calcium and magnesium ions for binding sites on transport
261 proteins and chelators. The competition for these binding sites is reduced by utilizing a Ca2+
262 and Mg2+ free solution, which results in more precise studies of iron binding and absorption.
263 After 20 min of incubation at 37°C and 100 rpm, the plates were removed and the HBSS was
264 poured out of both compartments. For the transport experiment, 0.6 mL of HBSS and 0.1 mL
265 of soluble mineral fraction (section 2.4.) were included in the basolateral and apical
266 compartments, respectively. The plates were thereafter subjected to incubation at 37°C for 2 h
267 at 500 rpm in an incubator shaker. The sample was obtained from the basolateral compartment
268 and preserved at -80°C to assess the absorption of Fe and the transportation of bioavailable Fe
269 across the cell monolayer using ICP-OES (section 2.4.). The viabilities of the cell were
270 evaluated using trypan blue exclusion, with typical rates ranging between 92-97% following a
271 2-h exposure period.

𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑥𝑥𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏
272 % 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑜𝑜𝑜𝑜 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 = × 100 …(5)
𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 𝑥𝑥𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎

273 where "transport xbasolateral" represents the quantity of Fe (ppm) that has been carried out via the
274 cell monolayer to reach the basolateral compartment. On the other hand, "bioaccessible xapical"
275 in the apical compartment signifies the quantity of bioaccessible Fe (ppm) (Wahengbam et al.,
276 2019).

277 2.11. Statistical analysis

278 The analysis of data was performed utilizing the one-way analysis of variance (ANOVA)
279 method, and Duncan's mean comparison test was conducted at a significance level of p = 0.05
280 to identify any variations among treatments. The statistical analysis was carried out utilizing
281 IBM SPSS Statistics 20. The experiment is conducted in triplicates and data is represented in
282 mean±standard deviation.

283 3. RESULTS AND DISCUSSION

284 3.1. Formulation and optimization of PIC

285 Based on preliminary results, a one-factor design was implemented keeping 180 h as the
286 constant incubation time and FeCl3.6H2O concentration varying from 0.90 mM to 1.80 mM as
287 an independent variable for optimizing PIC.

288 The Fe concentration (dependent variable) in PIC at different concentrations of FeCl3.6H2O

289 (independent variable) varied between 1238.41 to 3776.74 ppm. According to the employed
290 design, a linear model was evaluated. ANOVA depicted the significance of the model variations
291 compared to the variation of the experimental results. The results of the ANOVA are mentioned
292 in Supplementary Table 2. The F-value of 70.34 indicates that the linear model was statistically
293 significant.

294 The R2 and adjusted R2 values for the obtained Fe concentration were found to be 0.93 and
295 0.92, respectively. These values indicate a strong correlation between the predicted and actual
296 Fe concentrations, with no significant difference noted between them. The coefficient of
297 variation was found to be 10.95%. This lower coefficient indicated minimal deviation between
298 experimental and predicted values, underscoring the accuracy of the model. Furthermore, the
299 adequate precision, which gauges the signal-to-noise ratio, was calculated to be 14.793 for the
300 Fe concentration in PIC. This observation suggests that the model can predict data with more
301 precision. The linear equation, presented in coded form as Eq. (6), includes significant
302 parameters that contribute to the model's predictive capability.

303 I = +2517.89+ 1089.61×A …(6)

304 In Equation (5), the coefficient of parameter ‘A’ was determined to be 1089.61. This value
305 indicates a positive effect of parameter A on the response variable. In other words, increasing
306 the value of parameter ‘A’ contributes positively to the response variable as described by the
307 model. The positive effect of the independent variable on a dependent variable indicates that
308 with an increase in the independent variable value (FeCl3.6H2O) the value of a dependent
309 variable increases. So, the increase of FeCl3.6H2O concentration increases the Fe content in
310 PIC. This might be due to a combination of factors related to the availability of Fe ions and the
311 binding capacity of pectin molecules. As the concentration of FeCl3.6H2O increases, more Fe
312 ions are available in the solution (Persson, 2018). Maksudova et al. (2010) stated that this
313 higher concentration provides more opportunities for Fe ions to bind to the pectin molecules,
314 leading to an increase in Fe content in the PIC. Pectin molecules possess binding sites that can
315 be complex with metal ions such as Fe. However, Maksudova et al. (2010) did not report the
316 limiting concentration of Fe. With a higher concentration of FeCl3.6H2O, more Fe ions are
317 available to bind to these sites, resulting in an increased Fe content in the PIC.

318 The linear equation mentioned in Equation (5) was used to study the optimal conditions of
319 process parameters aiming to achieve a target Fe content of 3000 ppm in PIC. According to the
320 model, an Fe content of 2542 ppm was predicted at 1.36 mM of FeCl3.6H2O with a desirability
321 of 1.000. For validation purposes, experiments were conducted at the optimal conditions,
322 resulting in an observed Fe content in PIC of 2657 ± 23.61 ppm. Under optimum conditions,
323 there was a relative deviation between the experimental and predicted response values under
324 optimal conditions was 1.06%, between the predicted and observed response values. This
325 concludes that the developed linear model accurately predicts the Fe content in PIC under the
326 specified conditions.

327 3.2. Physicochemical properties of PIC

328 3.2.1. Proximate composition

329 This section covers the PIC's physicochemical characteristics. It was found that the moisture
330 content of PIC (19.10 ± 1.12% w/w) and pectin (21.10 ± 1.43% w/w) was non-significant.
331 Likewise, there was no statistical significance found in the protein content of pectin (2.62 ±
332 0.21%) and PIC (2.34 ± 0.17%). On the other hand, Koh et al. (2014) reported that the protein
333 and moisture contents of the pectin derived from jackfruit rinds were 1.53–1.74% and 5.19–
334 9.98% w/w, respectively. Additionally, both PIC and pectin have no fat content. The ash content
335 of PIC (2.785 ± 0.10%) was found to be lower than that of pectin (3.873 ± 0.10%).
336 3.2.2. Degree of Esterification (DE)

337 The term DE stands for the esterified COOH groups inside galacturonic acid chains with
338 methyl or acetyl groups and is essential for determining the quality of pectin. On the basis of
339 DE value, pectin is categorized as either high methoxyl (HM) pectin (DE ≥ 50%), or low
340 methoxyl (LM) pectin (DE ≤ 50%) (Liew et al., 2016). The DE values for the pectin (51.87%)
341 and PIC (50.35%) exceed 50%, indicating that it falls into the category of HM pectin. This type
342 of pectin is commonly utilized in the production of jams and jellies.

343 3.2.3. Galacturonic acid

344 The concentration of galacturonic acid was higher than 65% for pectin and PIC however, pectin
345 had higher galacturonic acid content (84.33 ± 1.44%) compared to PIC (67.39 ± 4.92%), which
346 might be because pectin, after forming PIC, is not solely composed of galacturonic acid
347 molecules. Due to the presence of negatively charged carboxylic groups, galacturonic acid
348 exhibits a strong propensity to bind with Fe ions, leading to the formation of a highly stable
349 complex (Cheng et al., 2019).

350 3.2.4 Methoxyl group content

351 Methoxyl group content has a key role in regulating pectin's gel-forming ability and setting
352 time. The methoxyl group content of pectin and PIC was found to be 2.02 ± 0.01% and 13.52
353 ± 0.04%, respectively. These results, however, were less than those reported for banana (7.03%)
354 and mango (7.33%) peels (Apsara & Pushpalatha, 2002), but they were comparable to dragon
355 fruit pectin (Zaidel et al., 2017), which had 2.98 to 4.34%. This could be due to the partial
356 degradation of pectin. According to Aina et al. (2012), depending on the source and extraction
357 conditions, pectin's methoxyl group concentration can range from 0.20 to 12%. The amount of
358 methoxyl groups in fruits is dependent on their maturity because, as they mature, their sugar
359 content rises while their methoxyl group content falls. A high amount of methoxyl groups
360 improves pectin's capacity to bind sugar and its dispersion quality (Apsara & Pushpalatha,
361 2002).

362 3.3 Structural characterization of PIC

363 3.3.1 FTIR Spectroscopy

364 The FTIR spectra of PIC and extracted pectin are represented in Fig. 1a. The O-H stretching
365 band (3100-3600 cm-1), the C-H stretching bands (2800-3000 cm-1), and the fingerprint region
366 (below 2000 cm-1) were among the distinct sections that were the focus of the spectral
367 investigation. Specifically, bands that contribute to the resonant absorption energy of the
368 pyranose cycle vibrations (950-1200 cm-1) and the area between 1200 and 1800 cm-1 indicating
369 the condition of carboxylic groups, were highlighted (Wang et al., 2014). Table 1 represents
370 the FTIR frequency range and functional groups identified in pectin and PIC.

371 Hydrogen bonding and complexation inside the molecule determine the position of the C=O
372 stretching wave number within these ranges. Delocalization of the C=O group following
373 complexation with a C=C band causes the absorption to shift to a lower wave number (Stuart,
374 2004). Pectin and the PIC lacked lignin and proteins, as indicated by the absence of the bands
375 at about 1600-1500 cm-1 that are attributed to aromatic rings and protein amide (Liu et al.,
376 2010).

377 The variations observed in the bands within specific regions, such as the (C–H) bands at 2929
378 cm-1, 1340 cm-1, and 1240 cm-1, as well as the complex band (C–H) spanning 1000-700 cm-1,
379 were attributed to alterations in the interactions between pectin and Fe (III). Furthermore, in
380 the 1153-1019 cm-1 region, both the spectra of pectin and the PIC exhibited several highly
381 fused bands. These changes likely reflect the complex interplay and molecular interactions
382 between pectin and Fe (III) in the synthesized PIC. The loss of these bands may suggest that
383 following Fe (III) chelation, changes occurred to the pectin's organized structures and
384 glycosidic bridge (Wang et al., 2015).

385 3.3.2 X-Ray diffraction (XRD)

386 To discern the amorphous or crystalline nature of pectin, XRD is a valuable technique utilized
387 to study its physical and functional characteristics. In Fig. 2b, the XRD graphs depict the
388 extracted pectin and the developed PIC. The diffraction patterns of the pectin exhibited a single
389 peak around 2θ of 9.4°. However, upon complexing pectin with ferric ions, freshly diffraction
390 patterns emerged, revealing two peaks approximately 2θ of 6.15° and 10.65° (as shown in Fig.
391 2b). These alterations in the diffraction patterns suggest changes in the crystalline or amorphous
392 properties of pectin induced by the complexation process with ferric ions. The diffraction peak
393 of PIC was sharper and more intense than that of pectin, resulting in a larger crystallite grain
394 size. The broad peaks observed in pectin disappeared following the coordination reaction,
395 suggesting a modification in the crystal morphology. Similar results were observed in the
396 soybean polysaccharide-Fe (III) complex by Gao et al. (2018). The observed higher intensity
397 of pectin compared to the PIC suggests a higher degree of crystallinity in pectin. The esterified
398 C=O related peaks’ reduced intensity in the FT-IR spectrum of pectin implies a correlation
399 between the crystallinity of pectin and its degree of esterification. Jiang et al. (2019) also noted
400 that pectin with a higher DE tends to exhibit a higher degree of crystallinity. The crystallization
401 process is attributed to hydrophobic interactions among methyl ester groups during pectin
402 dehydration.

403 The evolution of the diffraction pattern indicates that the PIC is relatively more amorphous
404 compared to pectin, despite having a larger crystallite grain size. This suggests that the
405 complexation with ferric ions influences the crystalline structure of pectin, potentially altering
406 its physical properties and functionality. Therefore, PIC can be used as a functional ingredient
407 in food products such as jams, jellies, fruit preserves, and confectionery.

408 3.3.3 Scanning Electron Microscopy (SEM)

409 The surface morphologies of extracted pectin and developed PIC are illustrated in Fig. 3. The
410 overall texture of PIC appears relatively smooth and flat compared to that of pectin. This could
411 be due to the coordination bonding present among pectin and Fe. The size of PIC grains is
412 around 1.08-1.59 mm and is relatively well defined than compared to that of pectin. The PIC
413 grains seem to be more cylindrical in shape, while the pectin grains are more rock like shape.
414 Overall, SEM micrographs conclude that PIC exhibited more disordered structures or less well-
415 defined crystal lattices along with a large grain size, which is in agreement with the diffraction
416 patterns of PIC in XRD.

417 3.4 Rheological and thermal study of pectin and PIC

418 The dynamic frequency test was done to study the viscoelastic properties of pectin and PIC
419 solutions at different concentrations (1-3% w/v). The storage modulus (Gʹ) and loss modulus
420 (Gʹʹ) were measured as a function of angular frequency. In all concentrations of pectin and PIC,
421 the Gʹʹ was higher than Gʹ at low frequencies, signifying dominant viscous behaviour. With the
422 increase in angular frequency, Gʹ increased compared to Gʹʹ causing crossover points. In both
423 pectin and PIC solutions, the crossover point was shifted to a lower angular frequency with an
424 increase in pectin and PIC concentration, as seen in Fig. 2. This shifting of crossover points
425 suggests that the solution behaves more elastic at lower frequencies. The stronger elastic
426 characteristics of the solution are caused by increased network formation and molecular
427 interactions like ionic and hydrogen bonding. As a result, Gʹ becomes more prominent over a
428 wider frequency range, indicating a better organized viscoelastic network and stronger gel-like
429 activity in the solution. This behaviour of pectin and PIC solutions is necessary for food
430 industry such as jam and jellies processing and pharmaceutical industry for controlled-release
431 drug delivery systems.

432 The TGA analysis also showed three main regions at 50-200, 200-400 and 400-600°C (Fig.
433 1c). A little weight loss of around 10% to 15% was seen in the first area, most likely as a result
434 of free or absorbed water evaporating in the space between chains. Due to the pyrolytic
435 breakdown of the PIC and pectin chains, there was a significant mass loss (roughly 50%) during
436 the second stage (200–400°C) (Ezzati et al., 2020). First, the galacturonic acid chains
437 underwent extensive thermal degradation. Consequently, the acid side group and carbon in the
438 ring underwent decarboxylation, resulting in the development of various gaseous products and
439 the production of solid char (Wang et al., 2016). According to Ezzati et al. (2020), the third
440 stage result revealed an additional little mass loss associated with the thermal breakdown of
441 char. The solid char containing polyaromatic structures bonded by aliphatic and ketonic groups
442 would partially collapse and stack compactly when the pyrolytic temperature rose (Wang et al.,
443 2016). PIC was shown to have a lower mass residue (13.50%) than pectin (16.59%), most likely
444 as a result of PIC exhibiting a lower DE value (Ezzati et al., 2020). According to Einhorn-Stoll
445 et al. (2007), pectin with a lower DE value has more free carboxylic groups accessible, which
446 can create more hydrogen bonds and accelerate the breakdown of pectin chains.

447 The Fig. 1d shows the DSC thermogram of pectin and PIC. The thermal curve of pectin shows
448 two broad endothermic peaks and one exothermic peak. One of the endothermic peaks was
449 found to be at 79.2°C having an onset at 35.8°C and ending at 152.7°C with the enthalpy of
450 fusion of 0.9047 J/g, and another at 197.9°C, having an onset at 183.7°C and ending at 216.6
451 °C with the enthalpy of fusion of 0.7932 J/g. The thermal curve of pectin also exhibits one
452 sharp exothermic peak at 249.2°C with the onset at 236°C and end at 263.1°C with the enthalpy
453 of fusion of 0.4782 J/g. Whereas, the thermal curve of PIC exhibits one endothermic peak at
454 66.2°C with an onset at 38°C and end at 107.3°C and exothermic peak at 256.0°C having an
455 onset at 236.4°C and an end at 275.7°C with the enthalpy of fusion of 1.527 J/g and 0.2635 J/g,
456 respectively.

457 The endothermic peaks could be caused by hydrogen bonding between galacturonic acid units,
458 the presence of water, or a conformational shift that resulted in the galacturonan ring changing
459 from its more stable 4C1 chair conformation to its inverse 1C4 chair conformation (Einhorn-
460 Stoll & Kunzek, 2009). The peak of PIC shifted somewhat toward a lower temperature, most
461 likely as a result of its altered structure and increased water content. According to Wang et al.
462 (2016), the exothermic peak, on the other hand, represents the thermal degradation of the PIC
463 and pectin samples, which is connected to their chemical ingredient profiles. The exothermic
464 peak of PIC (256.0°C) was higher than that of pectin (249.2°C) indicating that PIC pectin has
465 better thermal stability (Ezzati et al., 2020). Typically, pectin is a food ingredient that may be
466 added to baked goods like cakes, bread, and pastries that are exposed to high temperatures
467 (Combo et al., 2013). Furthermore, the thermal analysis in the present study revealed that PIC
468 had greater thermal stability than pectin, suggesting that PIC can be preferable to pectin when
469 thermal food processing is involved.

470 3.5 In-vitro Fe release from PIC

471 The proportion of Fe remaining in the supernatant of extracted pectin and PIC after in vitro
472 simulated digestion indicates the bioavailable condition of soluble Fe. This bioaccessible Fe
473 has the potential to be absorbed by intestinal cells, increasing its bioavailability and
474 physiological usage inside the body. It can be deduced from Fig. 4b that the PIC's %
475 bioaccessibility was much greater than that of extracted pectin. The total Fe content of the
476 pectin and PIC is 23 ppm and 2657 ppm, respectively. Fe concentration in pectin is negligible
477 after exposure to in vitro conditions. While, after exposure to simulated oral, gastric, and
478 intestinal conditions, the Fe concentration in PIC is 143.978 ± 4.20, 144.235 ± 0.85, and
479 141.913 ± 4.20 ppm, respectively and is statistically insignificant (p<0.05) (Fig. 4a). The
480 concentration of Fe remained unchanged under simulated in vitro conditions, indicating that
481 the structure of the PIC stayed intact even in the acidic environment of the gastric fluid. This
482 stability might be attributed to the existence of a Fe core encased by pectin ligands (Ma et al.,
483 2021). The bioavailability of Fe released from the selected PIC was determined to be 5.34 ±
484 0.16%, indicating effective absorption potential. Similar findings were seen in Gupta et al.
485 (2006). The iron bioavailability in green leafy vegetables such as Digera arvensis Forsk. and
486 Trianthema portulacastrum L. were 7 and 6%, respectively. While in other green leafy
487 vegetables in the study, iron bioavailability was found to be in the range of 16-43%, which is
488 3 to 8-fold of the result found in the present study (Gupta et al., 2006). Additionally, the
489 structure of the PIC shielded the Fe ions during passage through the stomach phase, thereby
490 mitigating potential stomach discomfort induced by the Fe ions and preserving their activity.
491 Consequently, a higher concentration of Fe was transported to and accumulated in the intestinal
492 fluid. These results highlight the possibility of PIC as a novel Fe supplement for addressing Fe
493 deficiency in anaemic patients (Ma et al., 2021). Utilizing Fe-deficient fruits and vegetables
494 such as cucumbers and pineapple to develop functional foods holds tremendous promise for
495 PIC towards better nutrition.

496 3.6 Fe release study from PIC using Caco-2 cells

497 Caco-2 cells may differentiate to become tiny intestinal enterocytes with all the necessary Fe
498 absorption and transport proteins therefore, they are a well-established model for assessing
499 non-heme Fe intake through ferritin synthesis (Eagling et al., 2014). In this study, specialized
500 Caco-2 cells were exposed to pectin and PIC supernatants. The conversion of Fe(III) to Fe(II)
501 has the potential to enhance Fe absorption, as Fe(II) can be efficiently conveyed through
502 enterocytes via the divalent metal-ion transporter 1. This process facilitates the uptake of Fe by
503 the cells, thereby contributing to its bioavailability and physiological utilization (Wang et al.,
504 2019). The average Fe absorption was 77.807 ± 0.697 ppm, representing 2.93% (Fig. 4c) of the
505 total Fe present in PIC while 54.82% of the overall Fe was introduced to the differentiated cells.
506 This result is comparable with Bryszewska et al. (2019), conducting an in-vitro study of iron
507 transport using Caco-2 cells from breads fortified with microencapsulated iron. In their case,
508 the iron transport of four breads with the addition of microencapsulated iron by sourdough
509 fermentation was in the range of 3.23-5.25% (Bryszewska et al., 2019). However, a negligible
510 amount of Fe was present in pectin in the intestinal fluid therefore, there was no chance of Fe
511 uptake by the differentiated Caco-2 cells. The Fe absorption values observed after 2 h of
512 incubation remained consistent throughout the study, with no significant differences observed
513 over time. It is well-established that under saturation conditions, Fe accumulation occurs
514 rapidly, typically showing a sharp increase in Fe uptake within the initial 40-60 min of
515 exposure. This phenomenon indicates the efficient uptake and accumulation of Fe by the cells
516 under the experimental conditions. This causes the Fe concentration within the cells to plateau
517 (Ghibaudo et al., 2018). In the brush border of enterocytes, soluble free inorganic Fe is
518 absorbed at the gut level. While Fe(III) is absorbed through divalent metal transporter 1
519 (DMT1), Fe(II) is absorbed directly. During the first reduction of Fe(III) to Fe(II), DMT1 can
520 then transport the latter over the brush border membrane (Ghibaudo et al., 2018).

521 4 Conclusion

522 This study aimed to utilize pectin extracted from Citrus limon Burm f. peels for the formation
523 of a pectin iron complex. The optimal concentration of FeCl3.6H2O was determined and the
524 bioavailability and absorption of iron were evaluated. During the intestinal phase, an increase
525 in the bioaccessible iron was identified. The findings suggest that PIC could serve as a
526 promising source to address iron deficiency while augmenting dietary fibre intake, potentially
527 offering numerous health advantages to human health. Future work could be increasing the
528 bioavailability of Fe in PIC high enough to meet its recommended daily allowance in the human
529 population and study the form of iron (Fe(III) or Fe(II)) in the PIC reduced by Caco-2 cells.
530 Moreover, the experimental results from this investigation establish the foundation for the
531 potential utilization of PIC along with the in vivo evaluation of its efficacy in addressing Fe
532 deficiency.

533

534 Declaration of Competing Interest

535 The authors assert that they have no conflicting interest.

536 CRediT authorship contribution statement

537 Somya Singhal: Investigation, methodology, writing original draft. Nishant Rachayya Swami
538 Hulle: Conceptualization, review and editing, supervision. Anastasios Koidis: methodology
539 review, editing, supervision.

540

541 Data availability

542 Data will be made available on request.

543

544 Acknowledgements

545 The authors are thankful to the Commonwealth Scholarship Commission (CSC) for funding
546 the study and Queen’s University, Belfast (QUB), UK for providing all necessary facilities for
547 the successful completion of the study. Moreover, we are thankful to Prof Lisa Connolly for
548 supplying Caco-2 cells and cell culture facilities. The first author is thankful to Dr Shagufta
549 Rizwana for her constant support.

550

551

552

553
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