Development of Sustainable Vegan Pea Protein-Zinc Complex: Characterization, In Vitro Cellular Mineral Uptake, and Application in Functional Biscuit Production

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Introduction
In the current era, there is a growing global concern about the widespread occurrence of micronutrient defciencies, which have signifcant negative impacts on various age groups and result in adverse health consequences.In particular, pregnant women and children under the age of 5 are highly vulnerable to defciencies in essential minerals such as iron, zinc, calcium, magnesium, and copper [1].Tese vital nutrients are regulated by factors including dietary habits, food intake, mineral absorption, and recycling processes within the human body.Inadequate levels of minerals can lead to the development of severe diseases and disorders, directly or indirectly afecting physiological processes, such as compromised tissue oxygen delivery, weakened physical strength, cognitive impairment, reduced productivity, and increased susceptibility to infections [2].Zinc defciency, for instance, can impair cellular signaling, mitochondrial respiration, and intermediary metabolism and may contribute to congenital anomalies, prenatal growth retardation, compromised immune function, and cognitive impairment [3].Te recognition of the importance of addressing micronutrient defciencies has signifcantly increased among governmental, nongovernmental, and industrial entities in recent years.However, diversifying diets to alleviate mineral defciencies is often hindered by factors such as afordability, availability, and religious constraints [4].Moreover, certain foods contain antinutritional properties that afect the absorption of minerals, thereby contributing to defciencies in the human body.Consequently, food fortifcation with external sources of minerals has been identifed as an efective strategy by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO), utilizing existing distribution systems within the food industry [5].
Traditionally, several inorganic salts have been added to fortify food products with minerals, playing a crucial role in factors such as bioavailability, chemical stability, appearance, and uniformity of the fortifed products.Tese inorganic salts have served as a sustainable and economic alternative in combating mineral defciencies and ensuring the nutritional security of large populations for many years [6].However, recent studies have revealed various drawbacks associated with inorganic minerals, such as zinc sulfate due to their toxic efects, raising concerns about their severe impact on human health.Long-term use of inorganic minerals has been observed to lead to toxicity as they accumulate in the human body [7].Te resulting tissue-level toxicity can range from mild symptoms such as nausea and abdominal pain to severe health problems such as liver abnormalities and increased intracranial pressure [4].Additionally, the presence of inorganic minerals in their free form can induce extreme toxicity by generating reactive oxygen species, while excessive long-term use can result in metabolic acidosis [8].Te accumulation of free zinc ions in the human brain has been linked to neurotoxicity.As a result, recent studies have explored alternative approaches involving the interaction of minerals with proteins to form complexes, thereby mitigating the use of inorganic minerals [9].Te study by Shilpashree et al. [10] showed that complexing iron or zinc with whey protein (WP) reduces iron's pro-oxidant activity and increases the bioavailability of both minerals.WP-mineral complexes protected against oxidative stress and provided favorable conditions for mineral absorption compared to free minerals.Moreover, Caco-2 cells exhibited signifcantly higher bioavailability of iron and zinc when delivered as WPmineral complexes.Te research conducted by Shilpashree et al. [11] demonstrated that Fe-and Zn-bound sodium caseinate (S.NaCas) have higher binding capacities than uncomplexed minerals.Tis binding resulted in altered physicochemical properties of the complexes, including changes in solubility, surface charge, and molecular size.Notably, the Fe-bound S.NaCas complex showed signifcantly higher mineral uptake in Caco-2 cells compared with free Fe.Additionally, it promoted greater intracellular iron storage as measured by ferritin formation.Zn-bound S.NaCN also exhibited improved mineral uptake compared with free Zn.
Te incorporation of zinc into protein-based food products has the potential to positively impact their shelf life and oxidative stability.Zinc, an essential trace element, exhibits antioxidant properties and can serve as a cofactor for enzymes involved in scavenging free radicals [12].Tis antioxidant activity contributes to the prevention of oxidation in fats and other susceptible components.Additionally, zinc's interaction with proteins can enhance their overall stability, preventing denaturation and degradation over time [13].Te antimicrobial properties of zinc further inhibit the growth of spoilage microorganisms and pathogens, extending the product's shelf life.However, challenges such as metalinduced oxidation and maintaining optimal concentrations must be carefully addressed [14].Strategies to overcome these challenges include the use of chelating agents, packaging with barrier properties, pH control, and the incorporation of synergistic antioxidants [15].By navigating these considerations, the addition of zinc to proteins can be optimized to harness its benefcial efects on shelf life and oxidative stability without compromising product quality [16].
Te development of a sustainable vegan pea protein concentrate-zinc complex ofers a promising approach to address the growing demand for protein while minimizing environmental impact and promoting human health [17].Pea protein production, compared with its animal-based counterparts, boasts a signifcantly reduced environmental footprint, requiring less land, water, and fertilizer, while simultaneously generating lower greenhouse gas emissions [18].Moreover, the complexation of zinc with pea protein efectively enhances zinc bioavailability, addressing potential nutritional defciencies prevalent in plant-based dietary patterns.Te incorporation of this complex into functional food products, such as biscuits, not only provides enhanced nutritional value but also maintains sensory attributes, ensuring consumer acceptability [19].By promoting the substitution of animal-based protein sources with vegan pea protein concentrate-zinc complexes, we can efectively mitigate the environmental impact of animal agriculture, reduce our reliance on animal resources, and contribute to the establishment of a more sustainable and healthier food system [20].In addition to the abovementioned benefts, the utilization of vegan pea protein concentrate-zinc complexes aligns with the principles of circular economy, promoting resource efciency and waste reduction.Pea protein, a byproduct 2 Journal of Food Biochemistry of pea starch production, would otherwise be discarded, representing a valuable resource left untapped [21].By transforming this byproduct into a functional ingredient, we not only minimize waste generation but also add value to the pea processing chain, fostering sustainable practices within the food industry [22].Furthermore, the incorporation of vegan pea protein concentrate-zinc complexes into food products can contribute to addressing global protein malnutrition, particularly in developing regions [23].Pea protein, as a complete protein source, ofers a viable alternative to animal-based proteins, providing essential amino acids for optimal growth and development.By increasing the availability of plant-based protein sources, we can enhance food security and promote nutritional well-being, particularly among vulnerable populations [24].
In the current study, we investigated the potential of pea protein concentrate (PPC) in forming protein-mineral composites, with a specifc focus on zinc.Trough this investigation, valuable insights into the physical, functional, and binding properties of PPC are expected to be gained, opening up possibilities for its utilization in the development of mineralenriched food products.Previous studies have indicated that proteins from legumes, including those present in PPC, frequently bind with metal ions through functional groups such as carboxyl groups in Glu and Asp, imidazole groups in His, and sulfhydryl groups in Cys.Te abundance of these binding sites in PPC along with its complex structure resulting from interactions such as hydrogen bonding and sulfde bridges, make it an excellent ligand for ferrous and zinc ions through multidentate coordination.Furthermore, the addition of inorganic salts to various food products has posed challenges, causing changes in sensory attributes and rendering them less acceptable for human consumption.For example, sensory analysis of several inorganic minerals, including gluconate, zinc sulfate, and chloride, revealed metallic taste, astringency, glutamate sensation, and bitterness, compromising their acceptability [25].Terefore, this study will also assess the sensory attributes of biscuits fortifed with PPC-Zn composites, aiming to overcome these sensory issues.To the best of our knowledge, despite extensive research on proteins derived from legumes in recent years, the zinc-binding abilities of proteins in a pea protein concentrate and their characterization, as well as the further development of composite biscuits based on PPC-Zn, have not yet been investigated.

Materials.
Vegan protein source such as PPC was procured from Roquette India Pvt. Ltd., located in Ahmedabad, Gujarat, India.Te food-grade minerals such as zinc sulfate monohydrate (ZnSO 4 •H 2 O) were procured from Sigma-Aldrich, St. Louis, MO, USA.Te other reagents used were Folin and Ciocalteu's reagent, sodium carbonate, copper sulfate, sodium potassium tartrate, bovine serum albumin (BSA), sodium hydroxide, and phosphate bufer, and other general chemicals used in this study were procured from Laboratory Reagents and Fine Chemicals, Loba Chemie Pvt. Ltd., Mumbai, India.Ingredients used for biscuit preparation involved refned wheat four, powdered sugar, butter, vanilla essence, baking soda, and salt and were purchased from the local market in Phagwara, Punjab.Te study used acidwashed class "A" certifed glassware throughout the experimentation, and all the reagents and standard solutions were prepared using triple-distilled water.

Preparation of Protein-Zinc Complex (PPC-Zn) and
Fortifed Biscuits.( 1) Preparation of PPC-Zn.Te zincbinding capacity of PPC was assessed through ultrafltration (UF), following the methodology described by Patil et al. [26].Solutions of PPC were prepared at a concentration of 0.01 g/mL.Zinc was incrementally introduced to each protein solution at concentrations spanning from 1 to 10 mmol/L.Tis addition was executed under constant agitation using a magnetic stirrer.Te pH of the protein-zinc complex was set at 6.5, and the complex was subsequently incubated for a duration of 2 h at a temperature of 24 °C, to optimize the interaction between zinc and protein.After centrifugation at 14000 × g for 30 min at 24 °C, the supernatant containing soluble protein and zinc was diligently separated and subsequently underwent further processing via the utilization of Amicon ultrafltration (UF) membrane tubes featuring a molecular weight cut-of of 10 kDa.Te collected fltrate underwent analysis to quantify its zinc content using the inductively coupled plasma-optical emission spectrometry (ICP-OES) technique (Toshvin Analytical Pvt. Ltd., Mumbai, Maharashtra, India).Subsequent to this, a pilot-scale UF membrane system was deployed to generate PPC-Zn.A diafltration procedure was applied to ascertain the presence of free zinc in the gathered fltrate.In particular, 2 mL of 4 M NaOH was added to 20 mL of collected fltrate, followed by centrifugation to acquire a solution devoid of pellets.Lastly, the concentrate was reduced to a quarter of its initial volume and subsequently subjected to spray drying.Te zinc retention and yield in protein-zinc complexes were calculated according to the following equations:

Zinc retention (%) �
The concentration of zinc in the complex Amount of zinc concentration added Yield (%) � Weight of obtained complex (g) Weight of added protein source (g)   × 100  . ( Journal of Food Biochemistry Te ultrafltration (UF) procedure was executed utilizing a Sartorius ultrafltration unit, furnished with a Masterfex Easy-Load pump-7518.Te fltration process employed a Hydrosart membrane featuring a molecular weight cut-of of 10 KDa and a fltration surface area measuring 0.1 m 2 .Te membrane's pressure setting was calibrated to 15 psi, while the cross-fow velocity was sustained at 300 mL/min.To oversee the progress of the fltration process, fow meters and pressure gauges were strategically interconnected to the inlet and outlet points of both the permeate and retentate streams of the membrane.
(2) Preparation of Biscuits.Te procedure for biscuit preparation, encompassing both the control and fortifed variants, adhered to a consistent set of operational steps with specifc diferentiations attributed to the introduction of fortifcation materials.Te control biscuit formulation, following the method outlined by Santosh et al. [27] with minor adaptations, involved a meticulous integration of refned wheat four, powdered sugar, butter, vanilla essence, baking soda, and salt.Te mixture of dry ingredients, namely, refned wheat four, powdered sugar, baking soda, and salt, transpired within a designated mixing vessel.Te infusion of softened butter and vanilla essence followed, prompting manual blending to yield a coherent dough matrix.Subsequent to the dough's preparation, a resting period coincided with the oven preheating to an established temperature of 100 °C, for 10 min.After this preparatory phase, the dough underwent controlled rolling to achieve an approximate thickness of 0.635 cm, facilitated by a lightly foured surface.Te implementation of a biscuit cutter yielded distinct biscuit units, which were subsequently transferred onto a baking sheet and subjected to a controlled baking process at 140 °C for a time span ranging between 15 and 20 min.Verifcation of proper internal baking was ascertained through the use of a toothpick.After baking, the biscuits were cooled, stored in vacuum-sealed pouches, and labeled "B1."In alignment with the methodological framework established, the fortifcation phase was introduced, implicating the incorporation of specifc composite materials.Te integration of zinc fortifcation was facilitated through the use of zinc sulfate monohydrate, individually denoted PPC-Zn composites.Te compositebased biscuit formulation followed an analogous procedure to the control variant, with distinct adjustments attributed to the introduction of 1.0 g of the respective composite powders within each dough.Following this incorporation, the subsequent kneading process homogenized the components.Te doughs were subject to a comparable resting period concurrent with the oven preheating at 100 °C over a 10-min interval.Consistent with the prior methodology, the doughs were uniformly rolled out and biscuit units were delineated through a biscuit cutter.Baking of the fortifed biscuits occurred at 140 °C for 15-20 min, culminating in an externally observed light golden-brown hue.Verifcation of internal completeness of baking was again conducted through a toothpick assessment.Te post-baking cooling duration was consistently observed, prior to the fnalized storage within the vacuum-sealed pouches, denoted as B2.

Analysis of the Pea Protein concentrate-Zinc Complex and Fortifed Biscuits
(1) Protein Content.Te Folin-Lowry method was employed to determine protein content in a sample following the Lowry et al. [28] procedure.A sample preparation involved dissolving 1.0 g of pea protein concentrate powder in 100 mL of distilled water to obtain a 1% protein solution.After adding reagents and vortexing, test tubes were incubated for 30 min at room temperature in the dark.Color intensity was measured at 660 nm, and a standard curve relating concentrations to absorbance was generated using bovine serum albumin.Te sample protein content was determined by interpolation.Te protein content was estimated using the following equation: Protein Content (µg/mL) � Concentration of protein from standard curve × dilution factor. ( (2) Estimation of Zinc Content.Te zinc content within PPC was evaluated according to the methodology outlined in the investigation conducted by Absulijabbar et al. [29].
For quantitative zinc analysis using ICP-OES (inductively coupled plasma-optical emission spectrometry), glass laboratory dishes were immersed in 10% nitric acid (HNO 3 ) for 24 h, followed by cleaning with deionized water and air-drying.(3) Mineral Binding Efciency.Te zinc-protein mineral binding efciency was determined according to the following equation given by Shilpashree et al. [30]: Mineral binding efficiency (%) � The total amount of zinc present in the sample The free form of zinc present in the sample   × 100  .
(4) Functional Properties (1) Protein Solubility (PS) Solubility is one of the most commonly measured functional properties of food proteins.Te analysis of the solubility of the PPC sample was conducted using the following method by Li et al. [31].Sample (1 g) was dissolved in 100 mL of phosphate bufer (0.05 M, pH 7), and the pH of the solution was adjusted to 7 using 0.1 N NaOH.Te solution in a beaker was mixed for 1 hr with the help of a magnetic stirrer at 30 °C followed by centrifugation at 18000 × g for 20 min at 4 °C.Te supernatant was fltered through Whatman No. 1 flter paper, and protein content was determined using Lowry's method.Te percentage of solubility was calculated as follows: Protein solubility (%) � Protein concentration in the supernatant Total protein concentration in the sample (2) Water-Holding Capacity and Oil-Holding Capacity (WHC and OHC) Te water-holding and oil-holding capacities of the PPC were determined using the method outlined by Li et al. [32].Firstly, the weight of the centrifuge tubes was measured and a sample (1 g) was added to it, and again, the weight of the centrifuge tube was measured.Ten, the sample was mixed with 10 mL of distilled water for WHC or 10 mL of canola oil for OHC in a preweight centrifuge tube.Te mixture was allowed to stand for 30 min to reach equilibrium and then centrifuged at 2000 × g for 15 min.Te excess water/oil was carefully decanted, and the remaining wet protein mass was weighed.Te waterholding and oil-holding capacities were calculated as the ratio of the mass of water retained to the initial mass of the sample, expressed as a percentage.(3) Foaming Capacity and Stability Te foaming capacity and stability of pea protein concentrate powder were examined according to the method of Li et al. [32].A sample (1 g) was incorporated into 50 mL of triple-distilled water.Employing a magnetic stirrer at 500 rpm, stirring persisted for 30 min until a uniform solution was formed.Te soluble fraction was gathered to evaluate foaming attributes.After centrifugation at 10000 rpm for 15 min, the sample was vigorously blended at maximum speed using an electric blender for 10 min.Te resulting foam was promptly transferred to a 100 mL measuring cylinder, and initial and fnal volumes were recorded.Foam capacity, indicating volume percentage increase, and foaming stability, denoting volume change after 30 min at room temperature, were calculated.Te percentage change in volume was computed to measure the foaming capacity.Te foaming capacity was calculated using the following equations: Te change in foam volume after 30 min (30 °C) was measured as foam stability and was determined using the following equation: Foaming stability (%) � Volume after resting (mL) − volume before resting (mL) Volume before resting (mL)   × 100  .
Journal of Food Biochemistry (

5) Characterization of PPC-Zn Composites
(1) Scanning Electron Microscope (SEM) Te SEM of the PPC-Zn was measured according to the method described by Relucenti et al. [33].Morphological characteristics of powdered pea protein were evaluated using a feld emission scanning electron microscope (FE-SEM: JEOL JSM-7610F Plus, Tokyo, Japan) equipped with an energy dispersive detector (EDS: MRB EDS, SDD 150 LN2 Free, UK) and a Gold Sputter Coater (ACG World Smart Coater).An amount of 5 mg of pea protein powder was afxed to carbon-coated copper tape.Gold coating, achieved via a sputtering process at 30 mA for 2 min, was executed to improve conductivity.Micrographs were acquired at magnifcations of 500X, 2000X, 25000X, and 5000X, utilizing accelerating voltages of 15 and 20.0 kV.Consistent working distance at 8.0−7.9 mm was upheld across all magnifcations.(2) Termogravimetric Analysis (TGA) Termal stability and mass variation of pea protein powder were assessed employing TGA (TGA 340 Em Prepash, Scinco Co. Ltd., Korea) using the method described by Saadsatkhah et al. [34].Test specimens (15 mg) underwent controlled heating from 10 to 950 °C at a steady pace (10 °C/min) under a nitrogen environment (50 ml/min), while monitoring weight loss for thermal characterization.(3) Fourier Transform Infrared Spectroscopy (FTIR) Te FTIR of the PPC-Zn was evaluated according to the method described by Tiernan et al. [35].FTIR was employed to investigate functional groups in pea protein powder using ATR and pellet accessories (PerkinElmer X400).A sample mixture was prepared by combining 10 mg pea protein powder with 100 mg KBr, and the mixture was deposited onto a pristine machine mirror surface.Data collection spanned 4000 to 400 cm −1 , utilizing air as the background.Transmittance data were acquired and managed using Spectrum 10 software.(4) Particle Size and Zeta Potential Te particle size and zeta potential of the PPC-Zn were examined using the methodology described by Li et al. [36].Te zeta potential and size of pea protein were determined using a particle size and zeta potential analyzer (Microtrac MRB NANO-TRAC Wave II, Osaka, Japan) at 24 °C.A 1% solution of each protein type was prepared and subjected to ultrasonication in an ultrasonic water bath for 10 min.
(6) Mineral Bioavailability (1) In Vitro Mineral Bioavailability Pea protein concentrate-zinc bioavailability was assessed using the procedure of He et al. [37].Te method simulated digestion and used Caco-2 cells to gauge absorption, providing insights into its nutritional impact.(2) Stimulated Gastrointestinal Digestion Pea protein concentrate-zinc complex bioavailability was assessed using the procedure of He et al. [37].
Te experiment mimicked gastrointestinal digestion, aiming to understand zinc absorption and its nutritional implications.In the simulation, 5 mL of protein mixed with 1.90 mL of saliva was agitated at 35 °C and 95 rpm for 10 min.Gastric digestion was followed by 3.1 mL of gastric juice (pH adjusted to 2) incubated at 35 °C for 2 h.Subsequently, a mixture of 1.97 mL of bile and 5.30 mL of duodenal juice was added, simulating intestinal digestion for 3 h.After digestion, ultrafltration separated soluble and insoluble components.Zinc bioavailability was calculated from the soluble fraction of zinc concentration using the following equation, estimating the available zinc for absorption in PPC: (  Te sensory evaluation of biscuit samples was conducted using a nine-point hedonic scale, assessing attributes such as appearance, color, taste, texture, aroma, and overall acceptability.Te panel comprised 30 members, evenly divided between 15 males and 15 females, with ages ranging from 23 to 45 years old.Te sensory analysis was reviewed and approved by the institutional ethical committee with reference number LPU/CA/023/25/03/12026. Te nine-point hedonic scale with 9-like extremely to 5-neither like nor dislike and 1-dislike extremely was used, and samples were coded with specifc code numbers to avoid biasness.Te selected formulations of biscuits were designated as B-1 (control) and B-2 (pea protein concentrate-zinc).Te panelist evaluated biscuits for their appearance, color, taste, texture, aroma, and overall acceptability.All sensory assessments took place in the Food Technology and Nutrition Department of Lovely Professional University, Phagwara, Punjab, India.
(2) Microbial Analysis Microbial assessment of the biscuits encompassed quantifcation of total plate count and yeast and mold count as per the methodology of Pokharel et al. [39].Aseptic protocols were rigorously upheld to obviate extraneous contamination.Biscuit samples were meticulously prepared through sterile techniques and aseptically transferred to sterile containers.Serial dilutions employing sterile diluents were executed.For the total plate count, agar media of relevance were utilized, with subsequent spread plating of diluted samples.Incubation parameters, as stipulated, were adhered to.Following incubation, the discerning enumeration of colonies transpired, with their enumeration culminating in colonyforming units per gram (cfu/g) of biscuit.Analogously, yeast and mold quantifcation employed selective agar media, customized to these microorganisms' needs.Diluted samples were subject to spread plating, succeeded by incubation under specifed conditions.Subsequent colony enumeration allowed for a distinction between yeast and mold colonies.

Statistical Analysis.
Data were presented as the mean-± standard deviation (SD) of the triplicate samples.Statistical analysis was performed using SPSS software version 25.0 (IBM Inc., Chicago, IL, USA), and an independentsamples t-test was applied to compare the means between the biscuit samples at the 0.05 signifcance level.

Results and Discussion
3.1.Zinc-Binding Efciency of PPC.As shown in Figure 1, the mineral binding efciency of the PPC increases signifcantly (p ≤ 0.05) from 89.65 ± 1.26 to 95.35 ± 1.86% up to the addition of 1-5 mM concentration of zinc sulfate in the protein and reaches its maximum at 5 mM concentration of zinc (95.35 ± 1.86%).Tis increase can be attributed to the presence of specifc amino acids and charged side chains within PPC.Since zinc ions carry a positive charge in their ionic form, the negatively charged key amino acids (such as Glu, Asp, Arg, and Gly) present in PPC tend to form ionic bonds, contributing to the formation of complexes with high binding efciency [40].Tis phenomenon is supported by previous studies indicating that zinc binds to pea protein through various amino groups, including amide and carboxyl groups on the protein's side chain and specifc functional groups such as imidazole and thiol.Furthermore, the morphological characteristics of the pea protein concentrate might have contributed to its maximum binding capacity and strong afnity for zinc ions [41].In particular, the protein was found to possess a signifcant number of coordination sites on its surface, leading to a multidentate coordination pattern.Te binding process was observed to be rapid and endothermic, indicating the spontaneous energy-absorbing nature of the interaction [42].As the zinc concentration increases in the range of 5-12 mM, the mineral binding efciency of PPC increases nonsignifcantly (p ≥ 0.05) (95.35 ± 1.86 to 96.04 ± 0.20%).Tis could be attributed due to the saturation of binding sites, altered protein conformation, competitive binding with other ions, pH and ionic changes, solubility limits, and potential protein Journal of Food Biochemistry aggregation.Additionally, elevated zinc sulfate concentrations could facilitate protein-protein interactions, impacting site accessibility and binding efciency [43].Our study aligns with Shilpashree et al. [44], who found maximum zincbinding capacity at 2 mM Zn concentration.Protein samples with the highest zinc-binding capacities, including pea protein concentrate bound with zinc at 5 mM (PPC-Zn), were selected for further investigation.Tis concentration was chosen due to a statistically signifcant increase in zinc-binding capacity observed up to this concentration.Beyond this concentration (5 to 12 mM), a nonsignifcant increase in the mineral binding efciency was observed.A comprehensive analysis will assess their functional properties and characteristic features.In vitro mineral bioavailability studies will determine the availability of bound zinc for human body absorption, and cellular mineral uptake of zinc will be examined to evaluate the proteins' efciency in delivering zinc to cells.

Protein Content of PPC and PPC-Zn. As shown in
Figure 2, the protein content of PPC-Zn (79.02 ± 1.33%) shows a nonsignifcant diference (p ≥ 0.05) as compared to PPC (78.86 ± 1.16%).Te nonsignifcant increase is due to the nature of the zinc-binding interactions, which could involve weak afnities that do not signifcantly alter the overall protein structure.Additionally, the proportion of protein molecules engaged in the zinc-binding process might be relatively small, resulting in an overall limited efect on the protein content [45].

Scanning Electron Microscope (SEM).
As shown in Figure 3(a), the SEM analysis conducted on the pea protein concentrate-zinc complex revealed important structural insights into the interaction between pea protein and zinc ions.In the SEM images, distinct morphological changes were observed in the pea protein samples after zinc complexation compared with the native pea protein.Te native pea protein exhibited a relatively smooth and uniform surface under SEM, consistent with its native structure.However, upon complexation with zinc ions, the surface morphology exhibited variations.Irregularities, including small aggregates or clusters, were noticeable on the surface of the pea protein concentrate-zinc complex.Tis suggests that zinc binding might induce some degree of protein aggregation or conformational changes.Te observed structural alterations could be attributed to the binding of zinc ions to specifc amino acid residues on the pea protein, leading to changes in intermolecular interactions.Tese changes might afect the protein's overall conformation and surface characteristics, ultimately infuencing its aggregation behavior [46].Te surface irregularities and aggregates suggest that zinc binding induces localized structural rearrangements within the protein matrix [42].Tese fndings align with previous studies indicating that metal ion binding can infuence protein structure and interactions [30].

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Particle Size and Zeta Potential. As represented in
Figure 3(b), the zeta potential value of the PPC-Zn (−13.86 ± 0.23 mV) shows a signifcant diference (p ≤ 0.05) as compared to the PPC (−10.89 ± 0.31 mV).Tis observation suggests that the introduction of zinc ions induces a signifcant change in the overall surface charge of the protein.Tis could imply that the binding of zinc makes a signifcant impact on the electrostatic interactions at the protein's surface [47].Similarly, as shown in Figure 3(c), the particle size of PPC-Zn (226.76 ± 0.45 nm) shows a nonsignifcant diference (p ≥ 0.05) compared with PPC (221.76 ± 0.45 nm) (Figure 2(c)).Tis result suggests that zinc coordination at the studied concentrations does not markedly alter the protein's colloidal stability or induce noticeable aggregation efects.

Fourier Transform Infrared (FTIR).
Te FTIR spectra provide valuable insights into the structural changes, as shown in Figure 3(d), which occur in PPC-Zn and PPC.In the spectral region associated with O-H stretching vibrations, the wavenumbers of 3294.711cm −1 and 3302.131cm −1 , indicative of hydroxyl groups, exhibit a slight diference.Tis discrepancy might be attributed to the interaction between zinc ions and hydroxyl groups within the protein, suggesting potential modifcations in the protein's hydrogen bonding network.Te C-H stretching vibrations, represented by wavenumbers of 2926.346cm −1 and 2927.087cm −1 , display minimal variance between PPC and PPC-Zn.Tis suggests that the fundamental carbon-hydrogen bonds, prevalent in aliphatic hydrocarbons, remain relatively unafected by the binding of zinc ions.Te wavenumbers of 1537.793cm −1 and 1536.382cm −1 , corresponding to amide II vibrations arising from N-H bending and C-N stretching interactions, exhibit a slight diference.Tis could imply subtle conformational changes in the protein structure upon zinc binding, potentially afecting its overall stability and functional properties.Observing the wavenumbers of 1435.945cm −1 and 1450.399cm −1 associated with CH 2 bending vibrations reveals the potential for alterations in the secondary structure of the protein due to zinc binding.Similarly, the variations in wavenumbers of 1397.363cm −1 and 1401.339cm −1 , refecting CH 3 bending vibrations, further suggest structural adjustments induced by the binding process.Te region featuring wavenumbers of 1237.845cm −1 and 1234.701cm −1 , possibly linked to C-N stretching vibrations within the peptide backbone, might indicate broader modifcations to the protein's overall structure as a consequence of zinc binding.In the spectral range involving C-O stretching vibrations, the wavenumbers of 1139.247cm −1 and 1109.474cm −1 , corresponding to carbonyl groups, show diferences that hint at changes in the secondary structure of the protein upon zinc interaction.Finally, the fngerprint regions with wavenumbers of 621.072 cm −1 and 616.335 cm −1 represent a diverse set of functional group contributions.Alterations in these regions may provide insights into shifts within the overall molecular environment of the pea protein due to zinc binding.
Comparing the overall intensity of the peaks, it is noticeable that some peaks, such as the one at 1109.474 cm −1 , exhibit signifcant changes in intensity upon zinc binding.
Tis could indicate alterations in the protein's secondary structure and bonding environment caused by the coordination of zinc ions [48].Additionally, zinc ions are known to play crucial roles in stabilizing protein structures and infuencing their functions.Te variations in peak positions and intensities suggest local conformational adjustments and alterations in hydrogen bonding patterns in the protein upon zinc coordination [49].

Termogravimetric Analysis (TGA).
As depicted in Figure 3(e), the thermogravimetric analysis of both PPC and PPC-Zn revealed distinct thermal degradation characteristics across diferent temperature ranges.In the low-temperature range (150 to 250 °C), both PPC and PPC-Zn exhibited comparable weight losses, with delta Y values of approximately 8.75% and 2.65%, respectively.Tis similarity suggests that the initial weight loss is primarily due to the evaporation of moisture and volatile compounds present within the samples.Moving into the intermediate temperature range (400 to 450 °C), both samples experienced signifcant weight loss, as indicated by substantial delta Y values of 42.38% for PPC and 38.35% for PPC-Zn.Tis stage corresponds to the decomposition of protein structures and the release of volatile organic fragments.Even at higher temperatures in the range of 500 to 550 °C, both PPC and PPC-Zn continued to exhibit weight loss, with delta Y values of 14.30% and 39.51%, respectively.Tis weight loss can be attributed to the degradation of residual organic matter and the potential formation of ash content.
Te subtle variations in weight loss observed between PPC and PPC-Zn across the studied temperature intervals suggest that zinc binding has a minimal impact on the overall thermal stability of pea protein.Te consistent degradation patterns imply that the coordination of zinc ions does not induce signifcant alterations in the molecular structure of the protein, thereby maintaining its thermal behavior [50].

Protein Solubility (PS).
As represented in Figure 4(a), the protein solubility of PPC-Zn signifcantly increases (p ≤ 0.05) from 79.83 ± 1.23 to 81.92 ± 1.36%, which is enhanced by 2.55% as compared to PPC.Te signifcant increase in protein solubility due to the structural changes induced by zinc coordination likely favors interactions with the solvent molecules.Zinc binding could disrupt certain internal protein interactions, potentially unfolding compact regions and making them more accessible to water molecules [51].Additionally, the coordination of zinc ions might alter the distribution of charges across the protein's surface.Tis charge rearrangement can impact the protein's interactions with surrounding water molecules.If the exposed hydrophilic regions of the protein increase due to zinc coordination, more water molecules can surround and solvate the protein, further contributing to its improved solubility [52].Journal of Food Biochemistry 13.09% (3.45 ± 0.57 g/g to 3.97 ± 0.63 g/g) and 11.17% (3.26 ± 0.11 g/g to 3.67 ± 0.38 g/g) as compared to PPC.Te observed improvement in WHC and OHC of PPC-Zn compared with PPC can be attributed to the interactions between zinc ions and the protein's structure.Tis interaction potentially leads to enhanced hydration properties as a result of zinc's coordination.Te binding of zinc ions might induce conformational changes within the protein, creating more favorable binding sites for water molecules.Tis conformational adaptation is believed to contribute to the heightened water-holding capacity of the protein [53].Similarly, the increased OHC observed in PPC-Zn can be attributed to the infuence of zinc ions on the protein's interactions with oil molecules.Te presence of zinc ions seems to enhance the protein's afnity for oil components.Tis could be facilitated by modifcations in the protein's surface properties caused by the binding of zinc ions.Tese modifcations likely result in more efective interactions between the protein and oil, leading to improved oil retention [54].

Foaming Capacity and Foaming Stability (FC and FS).
As shown in Figure 4(c), the FC and FS of the PPC-Zn signifcantly increased (p ≤ 0.05) by 2.08% (65.89 ± 1.54 to 67.29 ± 1.23%) and 6.07% (51.03 ± 1.12 to 54.33 ± 1.06%) as compared to PPC.Tis enhancement is likely attributed to altered intermolecular interactions facilitated by zinc binding, potentially leading to enhanced protein unfolding and increased surface activity.Consequently, greater protein availability at the air-water interface promotes the creation of stable foams [55].Furthermore, the improved stability observed in foams generated from PPC highlights the contribution of zinc ions in reinforcing the protein network at the air-water interface.Te coordination of zinc ions potentially fosters cross-linking or electrostatic interactions that strengthen cohesive forces within the protein matrix, Journal of Food Biochemistry resulting in enhanced resistance to foam collapse over time [56].

Zinc Content.
As illustrated in Figure 5(a), the zinc content of the PPC-Zn signifcantly increased (p ≤ 0.05) by 9.86% as compared to zinc sulfate.Te increased zinc content observed in PPC-Zn can be attributed to a combination of factors arising from the binding process between zinc ions and the protein structure.Chelation, a critical mechanism in this process, involves the bonding of zinc ions with specifc amino acid residues within the pea protein structure.Tis chelation not only enhances the stability of the resulting complex but also prevents the undesirable precipitation or aggregation of zinc ions [57].As a result, the chelated zinc ions remain tightly associated with the pea protein, leading to elevated zinc retention and concentration.Furthermore, the interaction between zinc ions and the pea protein molecule induces structural changes that hold potential signifcance [58].Tese structural modifcations may uncover previously concealed amino acid residues, thus creating new interaction sites for zinc ions.Te consequential adjustments in the protein's folding pattern and secondary structure contribute to the formation of additional binding sites for zinc [59].Our investigation concurs with the fndings of Shilpashree et al. [10], where the zinc bioavailability of whey protein-zinc complex increased by 9.26% as compared to whey protein.
3.6.Zinc Bioavailability.As shown in Figure 5(b), the zinc bioavailability of PPC-Zn is 74.22 ± 0.37%, which signifcantly increases (p ≤ 0.05) by 2.26% as compared to zinc sulfate.Tis can be attributed to several factors that interact to enhance the absorption and utilization of zinc by the human body.Firstly, pea protein contains naturally occurring compounds such as phytic acid, which acts as an antinutrient by binding to minerals such as zinc, limiting their absorption in the gut [60].However, during the processing of pea protein, techniques such as enzymatic hydrolysis or fermentation are often employed.Tese processes can break down antinutritional factors such as phytic acid, reducing their inhibitory efects on mineral absorption.Moreover, the protein matrix of pea protein may create a favorable environment for zinc absorption.Te amino acids present in the protein can potentially form complexes with zinc, aiding in its solubilization and subsequent uptake in the small intestine [61].Tis interaction could protect the zinc from binding with other compounds that might inhibit absorption.However, zinc sulfate, although a common dietary zinc source, can present absorption challenges.It dissociates into ionic zinc in the digestive tract, which can compete with other minerals for absorption sites.Additionally, the ionic nature of zinc sulfate might expose it to interactions with dietary components that can reduce its absorption or bioavailability [62].Our study demonstrates concurrence with the investigation conducted by Shilpashree et al. [10].Teir work revealed that the zinc bioavailability of whey protein increased by 13.15%.Te signifcant increase in cellular transport, retention, and uptake of PPC-Zn compared with zinc sulfate can be attributed to several interrelated factors that collectively create a more favorable environment for zinc absorption and utilization within the body's cells.Firstly, the protein matrix of pea protein plays a pivotal role [63].
Te amino acids present in the protein can form chelation complexes with zinc ions.Tis chelation enhances the solubility of zinc, preventing its precipitation and aggregation in the gastrointestinal tract, and facilitates its transport through the intestinal mucosa into the bloodstream.Te chelated form of zinc is more stable and less likely to interact with other dietary components that might inhibit absorption or reduce its bioavailability [64].Furthermore, the protein-bound zinc can be recognized by specifc transporters on the surface of intestinal cells.Tese transporters are designed to recognize and transport amino acid complexes, which allow the zinc to be more efciently taken up by the enterocytes.Tis recognition mechanism can lead to a higher rate of absorption for protein-bound zinc compared with the ionic form found in zinc sulfate [65].Once absorbed, the zinc bound to pea protein might also have advantages in terms of cellular retention.Te chelated form of zinc is less likely to be excreted directly through the kidneys, as it is shielded from the competitive ion transport systems that regulate renal excretion.Tis enhanced retention within the body can contribute to sustained zinc availability for cellular processes [66].Additionally, the potential reduction in antinutritional factors within pea protein through processing can further contribute to increased cellular uptake.Phytic acid, which is present in various plant-based foods, including peas, can hinder mineral absorption.Processing methods that break down phytic acid can alleviate this inhibition, making zinc more accessible for uptake into cells [67].Our study correlates with the research of Shilpashree et al. [10], where they found that cellular transport, retention, and uptake of whey protein increase by 5.86%, 9.09%, and 7.70% as compared to zinc sulfate.Tis experiment investigated the potential of proteinbound zinc for enhancing cellular absorption compared with free zinc.Using Caco-2 cells as a model for human intestinal cells, they evaluated the impact of pea protein concentrate (PPC-Zn) on zinc transport, retention, and uptake.Te study revealed a signifcant increase in all three parameters for PPC-Zn compared with zinc sulfate, suggesting that the protein matrix in PPC-Zn facilitates chelation, improves transport mechanisms, reduces antinutrients, and enhances retention, ultimately leading to improved zinc bioavailability.Tese fndings suggest that PPC-Zn may be a more efective form of zinc supplementation than conventional zinc sulfate, with potential benefts for human health.6(a) and 6(b), the comparative sensory analysis results between the fortifed biscuit sample (B2) and the control (B1).Te sensory attributes of the B2 sample exhibited a notable resemblance to those of the control sample.Sensory scores for both samples exhibited a decline with increasing storage duration.Over the 30-day storage investigation, discernible sensory alterations between B2 and control were absent from day 0 to day 15.Notably, color, favor and taste, aroma, mouthfeel, and overall acceptability garnered panelists' approval for B2 over the control.Troughout the sensory analysis phase, both B2 and B1 samples were stored within aluminum zip lock pouches.B2's sensory traits remained consistent during the storage period of up to 15 days in comparison with the control.Beyond the 15-day mark, a marginal decrease in B2's sensory attributes became apparent in contrast to the control.

Zinc Uptake in Biscuits.
As shown in Figure 7, the cellular transport, retention, and uptake of the PPC-Zn powder-incorporated biscuits signifcantly increase  .77± 0.48%) as compared to zinc sulfate.Tis increase is due to its superior bioavailability, solubility, and particle characteristics that facilitate efcient cellular interaction.PPC-Zn powder forms stable complexes, aiding absorption, and controlled release kinetics ensure sustained zinc supply, enhancing uptake.Synergistic efects with biscuit components further boost transport efciency [68].PPC-Zn powder's unique composition potentially improves digestibility, triggers specifc cellular mechanisms, and promotes metabolic interactions, leading to efective zinc utilization.Its stability in the gastrointestinal environment supports absorption [49].
3.9.Limitations and Drawbacks.Te study primarily focuses on the interaction between PPC and PPC-Zn, leaving unexplored potential interactions with other minerals, which could impact the broader utility of PPC.Additionally, the microbial counts in fortifed biscuits were consistently higher than those in control biscuits throughout storage, highlighting the need for a more comprehensive examination of the longterm microbial stability and safety of the fortifed product.Furthermore, the study does not extensively explore the impact of variations in processing conditions on the formation of PPC-Zn composites, and the economic feasibility of incorporating PPC-Zn into food products remains unaddressed.Clinical validation is essential to confrm the enhanced mineral bioavailability observed in PPC-Zn powder-incorporated biscuits in a human physiological context.

Conclusion
In conclusion, this study successfully developed a sustainable vegan pea protein concentrate-zinc (PPC-Zn) complex for the formulation of functional biscuits.Te PPC-Zn complex exhibited improved functional properties, including solubility, water-holding capacity, oil-holding capacity, foaming capacity, and foaming stability.Te PPC-Zn powderincorporated biscuits exhibited acceptable sensory attributes and improved zinc bioavailability.Te in vitro cellular mineral uptake studies demonstrated that the PPC-Zn complex was readily absorbed and retained by the cells.Te fndings of this study have signifcant implications for the development of functional food products to address the global challenge of zinc defciency, particularly in vegan populations.Te PPC-Zn complex is a promising functional ingredient that can be used to formulate a variety of food products, including biscuits, breads, pasta, and snacks.Future research should focus on evaluating the long-term stability of the PPC-Zn complex in food matrices and under diferent storage conditions.Additionally, clinical trials are needed to assess the efcacy of PPC-Zn powder-incorporated food products in improving zinc intake and status in humans.Overall, this study demonstrates the feasibility of developing sustainable vegan PPC-Zn complexes for the formulation of functional food products with improved zinc bioavailability.

Figure 1 :Figure 2 :
Figure1: Mineral binding efciency of the PPC-Zn (pea protein concentrate-zinc complex).Te results were expressed as the mean ± standard deviation of ≥3 independent replicates, and error bars represent the standard deviation from the mean values, while diferent uppercase letters above each bar represent signifcantly diferent values within samples based on the analysis of the t-test.
According to Figure 4(b), the WHC and OHC of the PPC-Zn signifcantly increased (p ≤ 0.05) by 10

Figure 4 :
Figure 4: (a) Protein solubility, (b) water-and oil-holding capacities, and (c) foaming capacity and foaming stability of the PPC and PPC-Zn.Te results were expressed as the mean ± standard deviation of ≥3 independent replicates, and error bars represent the standard deviation from the mean values, while diferent lowercase letters above each bar represent signifcantly diferent values within samples based on t-tests.PPC-Zn, pea protein concentrate-zinc complex; PPC, pea protein concentrate.

Figure 5 :
Figure5: (a) Zinc content; (b) zinc bioavailability; and (c) cellular transport, retention, and uptake of the PPC-Zn.PPC-Zn, pea protein concentrate-zinc complex.Te results were expressed as the mean ± standard deviation of ≥3 independent replicates, and error bars represent the standard deviation from the mean values, while diferent lowercase letters above each bar represent signifcantly diferent values within samples based on the analysis of the t-test.

Table 1 :
Microbiological analysis of pea protein protein-zinc powder-incorporated biscuits during the storage period.Data are presented as the mean ± standard deviation of the triplicate samples.Means with diferent lowercase letters (a-e) within the column over the storage time and uppercase letters (A and B) within rows between the samples denote signifcant diferences (p ≤ 0.05) based on the t-test.B1 and B2: control biscuit and PPC-Zn powder-incorporated biscuit; PPC-Zn, pea protein concentrate-zinc complex; ND, not detected.±0.19 cfu/g.On days 15, 20, 25, and 30, signifcant distinctions (p ≤ 0.05) surfaced in the yeast and mold count between B1 and B2.Microbial counts rose during the storage duration, with B2 consistently manifesting greater counts than B1.In summary, the results underscore the impact of incorporating PPC-Zn powder on microbial growth.Troughout the storage timeframe, B2 consistently manifested elevated microbial counts compared with B1, albeit remaining within acceptable safety parameters.3.8.2.Sensory Analysis of Biscuits.As represented in Figures 14 Journal of Food Biochemistry (p ≤ 0.05) by 8.41% (23.39 ± 0.63 to 25.54 ± 0.56%), 9.82% (30.65 ± 0.45 to 33.99 ± 0.39%), and 5.83% (55.34 ± 0.34 to 58