Spray Encapsulation of Iron in Chitosan Biopolymer for Tea Fortification

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Introduction
Micronutrient defciency is the most widespread risk to human health.Iron defciency anemia afects an estimated 2 billion people worldwide.Although the world has progressed in reducing infant mortality, millions of children die before the age of fve because of preventable diseases for which malnutrition is the prominent cause [1].Iron defciency combined with decreased absorption of iodine and vitamin A leads to additional nutritional disorders including mental retardation, brain damage, early childhood blindness, and increased severity of infectious diseases.Nutritional defciencies are preventable by simple modifcations in dietary intake; for example, consumption of iron-rich food, nutritional supplements, and iron-fortifed food can make a signifcant diference.Women of childbearing age (15-44 years) are most vulnerable, due to iron loss in menstruation and pregnancy.Food fortifcation is a process of adding vitamins and minerals to staple foods or beverages.It makes frequently eaten foods more nutritious without relying on consumers to change their habits [2][3][4].Next to water, tea is the most popular beverage in the world irrespective of race, gender, or socioeconomic status.Over the past decade, global per capita tea consumption has risen by 2.5 percent, primarily driven by the signifcant growth in tea-producing nations.Developing and emerging economies, particularly in East Asia, Africa, Latin America, the Caribbean, and the Near East, have been at the forefront of this expansion in demand [5][6][7].Tis hot beverage is particularly common in developing countries including India, Turkey Bangladesh, China, Pakistan, Iran, and Sri Lanka, where on average people consume 120 mL of tea every day [6,8].Tis regular and increasing consumption presents an opportunity to utilize tea as a safe and efective iron fortifcation vehicle.Research data on micronutrient fortifcation, specifcally iron in black and milk tea, are limited.Te food engineering research group at the University of Toronto made a signifcant contribution to exploring micronutrient fortifcation of black tea and provided valuable insights for further investigations and advancements in the feld of fortifcation of tea products [9][10][11].
Tea is rich in polyphenolic compounds that are valuable for their antioxidant activities that can scavenge free radicals and chelate metals [12,13].Tea polyphenols with catechol or gallol substituents form stable-coloured complexes with iron.At the low pH of the stomach, these polyphenols may be converted into quinones which also form stable complexes with iron.Tis makes tea a challenging vehicle for iron fortifcation, as it reduces iron bioavailability and the antioxidant capacity of tea [10,14].Te target populations for the present project are developing nations in South Asia, where tea is most often prepared with milk.Tis adds an additional technical challenge to the overall iron fortifcation of tea because it raises pH, encouraging iron-polyphenol complex formation.Te resulting discolouration becomes a barrier to consumer acceptance.
Ferrous iron (Fe ++ ) is the most bioavailable form of iron.It is converted to the ferric form (Fe +++ ) by oxidation, which can be triggered by alkaline conditions, oxidizing agents such as those present in the air, high humidity, and phenolic compounds [15].Generally, food fortifed with ferrous compounds that undergo oxidation exhibit low iron bioavailability, poor taste, and discolouration reducing consumer acceptability [16].
A study conducted by Dueik et al. found that the chelating agent EDTA can compete with tea polyphenols, and at 1 : 2 molar ratio of iron to EDTA, it prevents ironpolyphenol complex formation at pH 5 [17].Te iron-EDTA complex is highly bioavailable despite iron being present in the ferric form [18]. Te 1 : 2 ratio of iron to EDTA in tea was shown to maintain iron solubility after undergoing a pH adjustment similar to digestion, suggesting that iron bioavailability is preserved [11].Te reaction is dependent on pH, polyphenol concentration, and brewing time.When combined with milk, the infusion of tea exhibits a striking hue of milky orange, which is visually captivating and highly appealing to consumers [19].Unfortunately, in the presence of milk, due to higher pH and possibly increased calcium, iron reacts with polyphenols even in the presence of EDTA to form a dark coloured complex that is unappetizing to some consumers [20][21][22].
Because the formation of stable iron-polyphenol complexes depends on the oxidation of iron, antioxidants may be an alternative to EDTA to prevent iron-polyphenol complex formation [23].Reference [24] conducted a study investigating the manner in which reducing agents interfere with iron-polyphenol reaction in food.Catechol, gallic acid, catechin, cafeic acid, and chlorogenic acid were all tested with ferric sulphate and reducing agents (ascorbic acid, sodium bisulphite, and hydroxylamine).It was found that for all of these phenolic compounds, ascorbic acid and sodium bisulphate eliminated colour formation.Also, the addition of ascorbic acid to commercial products including black tea, green tea, cofee, hot chocolate, and banana baby cereal was shown to reduce colour formation due to added ferrous iron [24].Furthermore, ascorbic acid (vitamin C) is well known to increase iron absorption in general.However, in the presence of milk, sodium ascorbate (selected over ascorbic acid to minimize pH shift) resulted in the same colour issue as EDTA.
Terefore, there are two chemical strategies that may be employed to increase iron bioavailability and reduce, but not eliminate, colour formation in tea prepared with milk: use of a completing chelating agent that forms a bioavailable complex with iron (e.g., EDTA) and optionally a reducing agent/antioxidant added to prevent the oxidation necessary to form stable iron-polyphenol complexes (e.g., ascorbate).
Although additives are needed to increase iron bioavailability in the presence of tea, microencapsulation provides an opportunity to form a physical barrier between iron and polyphenols to prevent discolouration in the beverage.In this way, iron may remain unnoticeable to the consumer while delivering 30-100% of RDI of iron for a healthy adult [25].
Chitosan has gained signifcant attention in recent years mainly because of its novel industrial applications in food, pharmaceutical drug delivery, and medical products [26].It is a pseudonatural, cationic, hydrophilic, nontoxic, biodegradable, polyaminosaccharide, which is structurally similar to cellulose.It is chemically derived from partial deacetylation of chitin, which is a natural animal polysaccharide obtained from the hard outer skeleton of shrimp.Chitosan is biodegradable by enzymes, including chitosanase, papain, cellulase, and acid protease [27].Its molecular weight (MW) and number of deacetylation (DA) units vary in diferent chitosan polymers.It is insoluble in water, organic solvents, and aqueous bases but soluble in acids such as acetic, nitric, hydrochloric, perchloric, and phosphoric acid.Its solubility depends on the amount of protonated amino groups in the polymeric chain [27][28][29].Te positively charged free amino groups of chitosan can form chemical complexes with many negatively charged polyanionic polymers and small molecules.Cross-linking of chitosan has been utilized for producing microcapsules with improved functional properties and controlled release properties.
Sodium tripolyphosphate (TPP) is a nontoxic food additive, used to prepare cross-linked chitosan microparticles.Te positively charged amino group (NH 3 + , protonated in acidic solution) of chitosan reacts with negatively charged phosphate groups (PO 4 −3 ) in TPP [30].Tis leads to the 2 Journal of Food Quality formation of biocompatible cross-linked chitosan with improved functionality [30][31][32].Spray drying based chitosan-iron microcapsules have been previously prepared by our research group [33].
Te present research focuses on the prevention of ofcolour formation while maintaining iron bioavailability in iron-fortifed tea prepared with milk.Optimization of chitosan microcapsule production and the efects of additional secondary coating, hydrophobic surface overcoating, and cross-linking in improving iron-loading capacity, iron release profle, morphology of microparticles, and their suitability in milk tea were investigated.Iron-fortifed tea samples were made by adding iron microcapsules to tea, and the resultant colour of tea was observed.Final formulations combining iron microcapsule absorption enhancers, EDTA, and sodium citrate, as well as chitosan microcapsules, were tested for sensory attributes.® 1000 mg/L Fe in nitric acid) were purchased from Sigma Aldrich, St. Louis, USA.Reagent grade disodium EDTA and sodium ascorbate were purchased from Supelco ® Inc., and dichloromethane was purchased from Caledon Laboratories Ltd., Ontario, Canada.Maltodextrin (DE 7-10) was purchased from Cerestar USA, Inc. JVS Foods India provided soy stearin and black tea samples.Milli Q water was used throughout the experiments.

Spray-Drying Parameters.
Microcapsules were produced using a B-290 mini-spray dryer (Buchi, Switzerland).Spray-drying parameters were optimized with some modifcation of our earlier published method [33].Briefy, an atomizing gas fow rate of 667 std L/h at 618 kPa (90 psi) and an aspirator operating at 5.5 Pa were kept constant throughout the experiments using a standard 0.7 mm diameter nozzle tip.Te inlet feed fow rate was controlled by varying the peristaltic pump speed depending on solution viscosity.Te inlet air temperature (135-160 °C) was controlled in each experiment to keep the outlet temperature below 80 °C.

Microcapsule Preparation.
Te microcapsules were collected at the bottom of the cyclone separator from a sample collection vessel.At the end of each experiment, samples were transferred into clean sample bottles and weighed to determine the yield.Process yield % (equation ( 1)) was calculated by dividing the amount of the resultant powder from the collection vessel by the total solid content in the initial feed solution: weight of premix (g) weight of solids in feed (g) * 100. (1)

Primary Chitosan Microcapsule Production.
Chitosan fakes were dissolved in a 1% w/w aqueous solution of acetic acid.Complete solubility was achieved by keeping the system at room temperature overnight.Chitosan produces a highly viscous solution even at a low concentration; therefore, it is useful to determine the highest possible chitosan concentration for a better solid yield of the powder with spray drying.Chitosan concentrations of 0.1%, 0.2%, 0.5%, 1.0%, 1.5%, and 2.0% w/w were used.Iron salt was added at concentrations of 15, 20, 30, 40, 50, and 60% by weight of total solids in 1% chitosan solution.Ferrous sulphate was mixed with constant stirring at 1500 rpm for about 15 min before spray drying.

Cross-Linked Chitosan Preparation.
Chitosan was crossed-linked using sodium tripolyphosphate.After mixing the chitosan solution with the iron salt (50% and 60% by wt.) for 10 min, sodium triphosphate (10-15 mL per 100 mL chitosan as 1% w/v solution) was added dropwise to the chitosan solution with constant stirring using a laboratory mixer (Silverson Machine Ltd., UK).An opaque suspension of cross-linked chitosan was spray-dried to obtain encapsulated microparticles.

Microcapsules with Added Maltodextrin.
A clear solution of maltodextrin (10% w/v) was prepared and added (1 : 1) to the 1% w/w chitosan solution containing ferrous sulphate (30 and 40% wt.) under mild stirring for 10-15 min.Te fnal solution was spray-dried.While added maltodextrin increased the solid content of the chitosan solution, it also reduced solution viscosity.

Microcapsule with Hydrophobic Surface Coating.
With the intention of stabilizing these microcapsules in an aqueous medium and improving the surface properties and increasing the hydrophobicity of the microcapsules, a hydrophobic surface coating was applied.Approximately, 20 g of chitosan microcapsules was placed on a rotating pan, inclined at 45 °and rotating speed to ∼50 rpm.In a 250 mL spraying fask, 3.5 g of melted soy stearin dissolved in a 1 : 4 mixture of water and dichloromethane was kept warm on a hot plate.Te solution was sprayed onto the free-fowing particles in the pan to ensure uniform coating.

Total Iron Content and Loading
Efciency.Total iron content in microcapsules was analyzed to determine iron loading capacity and iron release from the capsules.Te microcapsules were digested by the addition of 10 mL conc.HNO 3 to 0.1 g of the powder in a microwave-assisted acid digestion system (MARS 6, John Morris Scientifc Pty Ltd.) with the temperature raised to 200-210 °C via microwave irradiation.Te solution obtained was quantitatively transferred to a 25 mL fask and brought to volume with deionized water.As needed, this was further diluted to a known volume (1 : 10) with 5% w/v nitric acid.Te samples were then analyzed by using ICP-AES (Optima 7300 DV ICP AES), calibrated with a 1000 mg/L Fe standard (Merck) solution.Iron concentration was calculated using the following equation: Te success of overall spray-drying process parameters was determined by calculating iron-loading efciency using the following equation:

Surface Analysis by X-Ray Photoelectron Spectroscopy (XPS).
Te spray-dried particles produced using a two-fuid nozzle system result in a matrix structure, with a possibility of uncoated particles on the microcapsule surface.To quantify the elements present on the sample surface at nanometer depth, X-ray photoelectron spectroscopy (XPS) or electron spectroscopy was employed.Te selected chitosan microcapsules were analyzed using the Termo Scientifc ™ K-Alpha ™ XPS system at the Surface Interface Ontario Facility at the University of Toronto, Canada.

Sensory Analysis of Iron-Fortifed Indian-Style Tea.
Tea was brewed in RO water and/or milk to represent common tea preparation methods used in India.Briefy, 250 g of water, skim milk, or whole milk was brought to boil.Ten, tea leaves (1% or 3% w/w Indian black tea) and ironcontaining chitosan microcapsules (delivering at least 4 mg iron per cup) were added together with Na 2 EDTA at a 1 : 2 molar ratio [17].Te tea was boiled for further 4-6 min after the addition of the iron microcapsules and chelating agent.Water loss due to boiling was measured after cooling the tea to 35 °C.Pictures of diferent tea preparations were taken in a white light box.Te Hunter colour (L * a * and b * ) of tea samples at 40 °C was determined, and △E values of selected samples were calculated using an NR series precision colorimeter (3 nH).Delta E was measured as the diference between the colour of samples and unfortifed tea designated as two points in the lab colour space [34].A blind taste test was conducted on the acceptability of regular and iron-fortifed tea.Participants were asked to fll in a survey to rate each sample of tea based on 5 parameters: overall, favour, colour, mouthfeel, and aroma.Tere were 8 participants, of whom 2 were not able to sense aroma.Te participants rated the parameters using a 5-point scale, which was weighted: really bad (x1), bad (x2), neutral (x3), good (x4), and very good (x5).Te responses for each parameter were multiplied by their respective weight and totaled.

Results and Discussion
Spray-drying technology is relatively cheap and is widely used in a single-step large microencapsulation for foods and pharmaceuticals.Depending on the starting feed material and process conditions, a very fne (2-50 μm) to large (2-3 mm) sized particles can be obtained [35,36].In the present study, ferrous sulphate, as a core iron source, and chitosan, as a primary coating agent, were used, with a variety of wall materials.All the formulation variables with their codes discussed in this paper are summarized in Table 1.

Spray-Drying Yield and Iron-Loading Efciency.
Initially, iron-free chitosan solution was spray-dried, and the optimum spray solution concentration was found to be 1% w/v of chitosan with a maximum yield of 62.5% by wt (Figure 1).Te 2% w/w solution became highly viscous and resulted in the lowest yield, and therefore, it was impractical for spray drying.Several formulation and process variables were investigated to enhance productivity, loading efciencies, and improved functional properties of capsules for use in tea fortifcation.Te process yields and loading effciencies of iron microcapsules are presented in Figure 2.
Te yield was between 58-78%, consistent with the 72% yield reported earlier, using chitosan as the primary coating [37].
Te moderate yield of chitosan (CH15%-40% w/w) can be attributed to the fact that it produces a viscous solution at low concentration, which readily adheres to the spray dryer glass chamber during process optimization and leads to the loss of feed mass.Te material sticks to the spray chamber and could not be recovered, thus lowering the overall yield of the dried powder [38].Maltodextrin has diverse applications as a food additive including bulking and flm formation, lowering viscosity, favour and fat binding, and reducing oxygen permeability in encapsulation matrices [39].Te process yield was expected to increase by reducing solution viscosity by adding a secondary polymer.Te incorporation of maltodextrin successfully decreased the viscosity and stickiness of the chitosan solution, leading to fewer instances of nozzle clogging during spraying and minimal deposition on the drying chamber walls.As anticipated, the loading efciency improved (reaching 90.84%) when 10% w/v maltodextrin was added to chitosan with a 30% w/w iron payload.However, this improvement in loading efciency could not be maintained when the iron concentration was increased to 40%.
Chitosan and polyphosphate groups can be linked with either ionic interaction or protonation.Both PO 4 −3 and OH − are present in sodium tripolyphosphate at higher pH and compete to react with the amino (NH 3 + ) group of chitosan [30,31].At higher pH, deprotonation is a dominant mechanism of interaction due to the change in pH of chitosan solution [40].At lower pH, the predominant mechanism is the ionic interaction between the negatively charged phosphate group and the positively charged amino groups.When a cross-linking step was introduced at a higher iron concentration (50% and 60% w/w iron), the iron-loading efciency signifcantly improved to 70.92% and 76.12%, respectively (Figure 2).Intermolecular and intramolecular cross-linking of native chitosan likely leads to a stable threedimensional molecular network, which helped stabilize iron in the microcapsules.Alternately, Fe +2 and SO 4 −2 molecules present in the solution may have interacted with the sites available on the polymer.Further studies should be conducted to understand the mechanism at a molecular level.

Size and Morphology of Microcapsules.
Te morphology of a spray-dried particle can be described by its size, shape, internal structure, and surface properties.Many drying process parameters, including feed solution concentration, the solubility of the excipient, drying temperature, and ratio between the drying time and difusion coefcient, afect the morphology of the particle [41].During spray drying, a droplet of liquid feed undergoes the constant rate of drying until the polymer concentration at the surface becomes very high, resulting in precipitation of the polymer forming a flm around the core [42,43].Te fnal evaporation from microcapsules formed largely depends on the physicochemical properties of the polymer or polymer matrix, e.g., solution viscosity, molecular weight, permeability, and elastic modulus.It was found that the molecular weight of the polymer (which afects viscosity) played a signifcant role in the size and shape of microcapsules [44].Scanning electron microscopy (SEM) of a representative sample was used to take the images of chitosan-based microcapsules formed with diferent formulation variables (Figures 3(a)-3(f )).Spraydried iron-containing particles usually spherical, with low variability, in size and shape, were observed.Slight deformation of particles was observed when iron was added at all concentrations unlike in chitosan particles without iron (Figure 3(a)).Addition of ferrous sulphate slightly altered the viscosity of the 1% chitosan solution at all iron concentrations (Figures 3(b)-3(f )).Incorporating maltodextrin did not reduce the irregularity of particle surfaces; however, a reduced incidence of surface aberrations was observed with a slight increase in particle size (Figures 3(d)-3(e)).Te average size of microcapsules calculated by using image analysis software (ImageJ) is presented in Table 2 along with Journal of Food Quality 6 Journal of Food Quality surface elemental composition.Te size of most of the chitosan-based particles was in the range of 2-4 μm, while addition of maltodextrin increased the overall size of particles (∼10 μm).

Surface Composition and Relative Atomic Abundance.
Relative atomic abundance at the surface of microcapsules (0-10 nm depth) has been examined using XPS.In this method, X-rays are used to irradiate the sample surface.Elemental chemical states C1s, O1s, S2p, and Fe2p were chosen as primary XPS regions to quantify elements.Average particle size was determined by SEM.

Journal of Food Quality
Electrons ejected from the sample surface are counted, and their energy spectrum is recorded.A representative spectrum of elements is generated from the sequences of energies from the bound state of electrons at the surface above the background.Te peak intensity and peak positions were quantifed to determine the elemental and chemical composition of the material at the surface.XPS spectra of chitosan microcapsules with and without loaded iron were recorded for C1s, Fe2p, O1s, and S2p, representing dominant elements in chitosan and ferrous sulphate.Comparing the samples, it was observed that increasing iron with a fxed chitosan coating led to more exposed iron at a depth of 10 nm (Table 2).However, incorporating maltodextrin as a secondary coating material increased the coating to core ratio by 10%, resulting in improved surface protection.Exposed surface iron was reduced from 2.68% and 3.52% to 1.52% and 1.50%, respectively, with the addition of 10% w/w maltodextrin for 30% and 40% w/w.Cross-linking, on the other hand, did not appear to have any major impact on surface iron exposure (Table 2).

Iron Release.
Te investigation of iron release served two primary purposes: to assess the in vitro digestibility profle and to examine the efect of boiling temperature, which mimics teabrewing conditions.Te objective was to determine whether particles would retain iron until the tea was consumed, allowing for subsequent release and absorption in the digestive system, thereby ensuring bioavailability.In other words, the coating was expected to completely dissolve in stomach acids while maintaining its integrity at high temperatures for at least 10-15 minutes.Te iron release profle of selected microparticles is presented in Figures 4-6.It was observed that release increased with an increase in iron loading in the microparticles in constant boiling water Figure 4.An intriguing phenomenon was observed in the samples with the same iron loading but diferent surface treatments.In the case of CHFe40MDST, which featured a hydrophobic soy stearin surface overcoat, lower iron release was observed compared to the CH40MD sample with the same iron loading (Figure 5).Tis diference in iron release was evident between the 10-minute and 25-minute time frame.Chitosan-based microcapsules exhibited signifcant iron release in an acidic environment (Figure 5).Sample CH30Fe, with the lowest iron content, released most of it within 30 minutes.In contrast, sample CH40MDST, which had a hydrophobic coating on the outer surface, exhibited distinct release kinetics compared to CHFeMD with the same iron loading and a burst efect.Te hydrophobic surface coating of CH40MDSTresisted the dissolution and release of iron in the acidic medium for 30-90 minutes.Furthermore, cross-linking at two diferent iron concentrations also resulted in diferent release profles, signifcantly impacting iron release, even at higher iron concentrations.For example, CH60TPP released 60% of iron within two hours, while CH50TPP released 30% total iron.Except for the soy stearin coating, all of the microcapsules demonstrated a desirable burst efect in the acidic medium, attributed to the solubility of chitosan under acidic conditions.Iron release at pH 7 using phosphate bufer (Figure 6) was measured.Samples prepared with native non-crosslinked chitosan with 30-40% iron loading released as little as 5-6% of iron after 1.5 hours, indicating reverse-enteric behaviour of chitosan polymers.However, tripolyphosphateinduced cross-linking of chitosan not only modifed the viscosity of the solution, which was observed during the sample preparation, but also altered the binding properties and afected the solubility of chitosan at neutral pH.Both CHFe50TPP and CHFe60TPP showed drastic release differences at neutral pH during the same time frame.However, both 30% and 40% iron loading led to similar iron release by the end of two hours.

Iron-Fortifed Indian-Style Milk
Tea. Te addition of EDTA prevented iron-polyphenol complex formation in iron-fortifed tea in the absence of milk [11].However, in India, tea is generally prepared by boiling tea granules with water and milk.Te pH of this tea is higher (∼6.0-6.4)(Table 3) than that of tea brewed in water (pH ∼4.3-4.5).At higher pH, iron-polyphenol complex formation is more likely to occur.Also, the evaporation of water as a result of boiling increases polyphenol concentration in tea.Te complex interactions between milk components, iron, tea polyphenols, and iron absorption enhancers (EDTA or ascorbate) contribute to the darker colour of iron-fortifed tea when prepared in milk [20].For visual evaluation, unencapsulated iron with EDTA as a chelating agent or sodium ascorbate (also at a 1 : 2 molar ratio) as a reducing agent was tested.Te colour of the beverage resembles hot chocolate which is expected to be unacceptable to the average consumer (Figure 7).Physical entrapment of iron is required by an inert coating material that can delay ironpolyphenol complex formation until tea is consumed.Unfortunately, most encapsulating agents used in the food and pharmaceutical industry are readily dissolved at high temperatures.It was hoped that the chitosan coating would delay iron release sufciently for acceptable organoleptic quality in milk tea.Iron capsules with various formulation variables were tested by preparing iron-fortifed tea.After analyzing the total iron content of each microcapsule (Table 3), the specifed amount of powder and EDTA (1 : 2 Fe: EDTA molar ratio) were added to the tea.As in all previous formulations, the taste of the tea was acceptable, and the colour of the tea is the foremost indicator of the success of a formulation.To compare the diference between regular tea and tea with unencapsulated iron, positive and negative controls were also prepared.Figure 8 shows the visual comparison of colour after fve min of cooked milk tea with selected formulation variables 2-5 (listed in Table 3) along with regular tea and unencapsulated ferrous sulphate.After a few minutes, tea prepared with CH30Fe and CH40FeMD started to develop a darker colour, while the colour of cross-linked CH50TPP remained stable for more than 15 min of cooking.Lower delta E values, presented in Table 3 (△E � 6.60), and slight visible diference (Figure 8) of colour from regular tea led us to further sensory evaluation.

8
Journal of Food Quality Cross-linked CH50TPP was selected and assessed for sensory profle analysis as presented in (Table 4).Sensory testing was conducted to score CH50TPP with and without the addition of chelating agents.Two selected agents were disodium EDTA and sodium ascorbate.Also, an additional sample (CH50TPP + EDTA, Cap) was prepared where EDTA was added (1 : 2 molar ratio) in the microcapsules before spray drying as part of coating formulation.It was observed that the most acceptable tea overall was tea-containing iron microcapsules and disodium EDTA added separately as a powder.It was also ranked highest for favour and colour.Te mouthfeel was slightly worse than plain or regular tea, and there is room for improvement in the domain of aroma.Tea with added iron microcapsules in the absence of disodium EDTA was tested to determine if disodium EDTA had a negative efect on acceptability.However, acceptability was higher when disodium EDTA was present.It can be inferred that EDTA has the ability to chelate exposed iron from the microcapsules, thus protecting against colour changes.Tea formulations incorporating both iron microcapsules and disodium EDTA could be advantageous particularly in terms of colour acceptability in addition to improving Fe bioavailability.Tea with iron microcapsules and ascorbate powder added separately matched the acceptability of plain tea in every category including the highly ranked mouthfeel and aroma.However, implementing such a strategy would require further testing into the adequate amount of ascorbate to be added to tea to improve iron bioavailability.Terefore, the most promising formulation for iron-fortifed tea contains iron microcapsules and disodium EDTA added as a separate powder.While chitosan was found to be the best coating material, it still has some limitations.Te high viscosity at low concentrations limits its use at low solid loading.Addition of maltodextrin increased the solid concentration and improved the spray process and coat to core ratio, but no additional advantage for its use in sensory or iron release profle was observed.Cross-linking, on the other hand, provides an opportunity to utilize the fexibility of chitosan as a coating material with desired and improved functional properties.Further investigation can be conducted to determine the optimal concentration of the cross-linking agent, such as TPP or other suitable cross-linkers.In

Conclusions
We proposed to fortify black tea with iron in a manner that does not impact the sensory properties of Indian-style milk tea upon brewing while maintaining its iron bioaccessibility.In order to prevent the formation of colour due to the complexation of iron with polyphenols in the presence of milk, iron can be encapsulated to inhibit its release and interaction with tea polyphenols prior to tea consumption.Iron complexes dissociate in the stomach, and absorbable FeEDTA complexes re-form as pH is raised in the intestine.Terefore, it is suggested that iron be separated from the tea until it is ingested by using a polymer coating such as chitosan.However, more work will be required to fnd an encapsulation formulation that can be stabilized in hot tea for the typical duration of consumption, preferably for 30 min or more.Considering that many commercial sources of chitosan are derived from animal-based materials, there may be limitations to its use for populations where animal products are avoided due to vegetarianism or religious reasons.Terefore, it is recommended that plant-based chitosan formulations be explored as an alternative to animal-based chitosan.Tis could improve the applicability and acceptance of chitosan-based products among diverse populations with varying dietary preferences and cultural considerations.3.

Figure 3 :
Figure 3: (a-f ) SEM images of the selected chitosan microcapsules containing various concentrations of iron and secondary coating, taken at 1000-4000x magnifcation.

Figure 6 :
Figure 6: Iron release profle of selected microcapsules at pH 7 in phosphate bufer at 37 °C for 2 hours.

Figure 8 :
Figure 8: Indian-style milk tea brewed with iron microcapsules and EDTA (2-4), visual colour comparison with regular tea as a positive control (1), and unencapsulated iron as a negative control (6) as listed in Table3.

Table 1 :
Description of formulation variables and codes.

Table 2 :
Relative atomic abundance of elements at the surface of microcapsules analyzed by XPS at 10 nm depth.

Table 3 :
Colour and pH of milk tea fortifed with various iron microcapsules and EDTA (value ± SD, where n � 3).
Te iron concentration in tea was adjusted to 4 mg per cup of tea.

Table 4 :
Sensory profle of milk tea fortifed with selected iron microcapsules, disodium EDTA, and sodium ascorbate.