Nano-armoured Vitamin C: A novel approach to micronutrient delivery and natural food preservation in integrated food systems

Nano-armoured Vitamin C: A novel approach to micronutrient delivery and natural food preservation in integrated food systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nano-armoured Vitamin C: A novel approach to micronutrient delivery and natural food preservation in integrated food systems Catherine Antony, Krishnaveni Narayanaswamy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8801688/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The present study highlights the development of nano-encapsulated ascorbic acid delivery systems to address challenges in food preservation and nutritional fortification. Ascorbic acid-producing Acetobacter strains isolated from natural sources were characterised and optimised for fermentation at pH 4–6 and 30°C, achieving 99% substrate conversion within 96 hours. Iron oxide nanoparticles synthesised via coprecipitation were functionalized with biosynthesised ascorbic acid through pH-controlled coating (pH 12.5, 80 0 C, 1 hour). Scanning electron microscopy confirmed uniform coating architecture. Preservation efficacy was tested in fruit juices (apple, grape, orange, and banana) and dairy products (milk and curd), demonstrating that nano-encapsulated formulations achieved approximately 75% antimicrobial activity compared to commercial ascorbic acid. This reduction in microbial loads, from previously uncountable levels to 7–20 CFU at 10^ (-8) dilutions, was notable. The dual-functional vitamin C and iron fortification, and clean label compliance, are due to their biosynthetic origin. This integrated biotechnology-nanotechnology platform establishes proof of concept for next-generation multifunctional food additives that extend shelf life while enhancing nutritional value, providing viable solutions for food industries pursuing natural preservation strategies. Nano-encapsulation Biosynthesised Ascorbic acid Iron oxide nanoparticles Natural food preservative dual fortification Acetobacter fermentation Clean label technology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION The global food industry faces significant challenges in food safety, nutritional security, and adapting to evolving consumer preferences (Barrett, 2021 ). Microbial spoilage and lipid oxidation of food result in annual losses in billions of dollars and pose significant public health risks (Vesković, 2025 ). India’s food processing sector, ranking sixth globally, exemplifies the transition from traditional preservation methods to advanced systems that incorporate sophisticated techniques and functional additives (Lisboa et al., 2024 ). Consumer demand has shifted dramatically toward natural, clean-label products, emphasising transparency and minimal processing over synthetic additives (Antony & Narayanaswamy, 2026 ). Current preservation strategies rely predominantly on antimicrobial agents ( sulphuric acid, nitrates, nitrites) and synthetic antioxidants (BHA, BHT, TBHQ), which effectively extend shelf life but face increasing rejection due to health concerns, potential antimicrobial resistance, regulatory restrictions, and incompatibility with clean-label demands (Ahmed et al., 2022 ). Ascorbic acid (vitamin C) is a multifunctional solution that meets both technical and consumer demands in food preservation (Tissera et al., 2025 ). As a potent antioxidant, it prevents lipid oxidation, pigment discolouration, and nutrient degradation while supporting collagen synthesis, immune function, and iron absorption (Alberts et al., 2025 ). Despite substantial health benefits, including protection against oxidative stress and chronic diseases, ascorbic acid faces critical limitations: instability under processing conditions ( oxygen, elevated temperatures, pH variations, light, metal catalysts), compromising efficacy and nutritional value, plus averse effects from direct supplementation(gastrointestinal distress, nausea), limiting consumer acceptance (Malik et al., 2022 ). Nanotechnology offers transformative solutions through nanoencapsulation, enabling enhanced stability, controlled release, improved bioavailability, and targeted delivery (Mehta et al., 2025 ). Iron oxide nanoparticles exhibit advantageous properties, including biocompatibility, superparamagnetic properties that enable controlled delivery and magnetic recovery, tunable surface chemistry for functionalization, and nutritional value as bioavailable iron supplements, thereby addressing micronutrient deficiencies (Waseem et al., 2025 ). This combination creates synergistic functionality in which ascorbic acid enhances iron absorption while iron oxide nanoparticles protect it from degradation, simultaneously addressing two prevalent global nutritional deficits (Kumari & Chauhan, 2022a ). Biological production through microbial fermentation using Acetobacter species, Gluconobacter oxydans , and Aspergillus niger , offers sustainable alternatives to chemical synthesis, aligning with clean-label requirements while minimising environmental impacts (See et al., 2024 ). Despite advances in nanoencapsulation and biological production methods, significant gaps remain in their combined use for food preservation (Anushree et al., 2025). Most studies have examined these technologies separately rather than together, testing nanoencapsulation in laboratory settings without verifying effectiveness in actual food products (Mehta et al., 2025 ). While some research has improved nutrient delivery or explored production methods, these studies have not demonstrated antimicrobial activity or addressed practical concerns, such as production costs, scaling up to industrial levels (Waseem et al., 2025 ). The food industry requires solutions that simultaneously enhance nutrition, prevent microbial spoilage, meet consumer demand for natural ingredients, and remain economically feasible; yet, few studies have connected laboratory-scale production with nanoparticle technology and testing in real food systems (Acharekar et al., 2025). Therefore, an integrated approach is needed that combines optimised microbial production, nano-encapsulation methods, and characterisation and testing across various food products to develop a practical, multipurpose preservation technology for commercial use (Lavanya et al., 2024). This research addresses the integration gap by developing nano-encapsulated ascorbic acid delivery systems using iron oxide nanocarriers, combining microbial fermentation with Acetobacter strains, controlled synthesis and functionalization of iron oxide nanoparticles, and synthetic validation across diverse food matrices, including fruit juices and dairy products. The iron oxide nanoparticles serve dual functions: as protective carriers that shield ascorbic acid from degradation while enhancing its bioavailability, and as a source of iron supplementation to address micronutrient deficiency. This integrated methodology bridges the gap between fundamental science and industrial applications, offering solutions for natural food preservation while providing scalable platforms for micronutrient fortification. The specific objectives were to isolate and characterise ascorbic acid-producing microorganisms from environmental sources; to optimise fermentation parameters for maximum production; to synthesise iron oxide nanoparticles and encapsulate biosynthesised ascorbic acid within these matrices; to characterize the physicochemical and functional properties of nano encapsulated formulations; to evaluate antimicrobial efficacy and preservation performance; and to compare shelf life extension between nano encapsulated ascorbate, commercial ascorbic acid, and untreated controls across multiple food products. METHODOLOGY Isolation of ascorbic acid-producing bacteria: sample collection and enrichment Acetic acid bacteria capable of direct ascorbic acid biosynthesis were isolated from naturally fermented substrates, including deteriorated fruits (apples and grapes), coconut toddy, and honey. These sources are selected for their high carbohydrate content and their established association with Acetobacter species. Samples were aseptically processed and subjected to selective enrichment using source-specific media formulations. For fruit-derived isolates, the enrichment medium contained glucose (1% w/v), ethanol (0.5% v/v), acetic acid (0.3% v/v), peptone (1.5%w/v), and yeast extract (0.8% w/v). Toddy samples were enriched in medium containing glucose (5% w/v), yeast extract (1% w/v), and cycloheximide (100 ppm) to suppress fungal contamination. All enrichment cultures were incubated at 30 ° C with continuous agitation (180 rpm) for 5 days to promote the proliferation of acetic acid bacteria (Rahman et al., 2024). Selective isolation and culture characterisation Following enrichment, cultures were transferred to differential media for isolation and preliminary identification. Fruit-derived isolates were plated on glucose yeast calcium carbonate (GYC) medium containing (10% w/v), yeast extract (1% w/v), calcium carbonate (2% w/v), and agar (1.5% w/v), adjusted to pH 6.8. The medium was supplemented with 100 mg/L of cycloheximide to inhibit non-target organisms. Plates were incubated aerobically at 30°C for 3-4 days (Lee et al., 2024). Ethanol tolerance and acid production characteristics were verified using Carr medium containing yeast extract (3% w/v), bromocresol green (0.2% w/v), ethanol (5-9% v/v), and agar (2% w/v) for toddy-derived. Isolates, Glucose Yeast Peptone (GYP) medium was employed, consisting of glucose (2% w/v), sodium acetate trihydrate (0.5% w/v), tryptone (0.5% w/v), yeast extract (0.5% w/v), potassium phosphate (0.1% w/v), Tween 80 (0.5% v/v), and agar (1.7% w/v) at pH 6.8. Plates were incubated at 37°C for 3-5 days (Sahoo et al., 2020). Characterisation and identification of isolates Isolates were characterised through Gram staining and comprehensive biochemical profiling, including enzyme assays (catalase, oxidase, and urease), IMVIC tests, triple sugar iron agar tests, and carbohydrate fermentation patterns, to establish morphological features and metabolic capabilities for taxonomic identification (L’Haridon et al., 2020). Optimisation of growth conditions Selected isolates were cultivated in brain heart infusion (BHI) broth to determine the optimal conditions for biomass accumulation and ascorbic acid production. Culture aliquots (200 μL) were inoculated into 15 mL BHI broth in triplicate. Temperature optimisation was conducted by incubating cultures at 4°C, 25°C, 30°C, 37°C, and 45°C for 7 days, while pH optimisation involved adjusting the fermentation medium to pH 4.0, 5.0, 6.0, and 7.0 using 0.1 N NaOH or 0.1 N HCl, with cultures incubated at the optimal temperature for 5 days. Growth was monitored by measuring optical density at 600nm with a UV-visible spectrophotometer to identify conditions that yielded the maximum biomass and ascorbic acid production (Aswini et al., 2020). Ascorbic acid production by fed-batch fermentation Fed-batch fermentation was conducted using optimised Acetobacter strains with sorbitol as the primary carbon source. The production medium contained sorbitol (8% w/v), yeast extract (5% w/v), glycerol (0.05% v/v), magnesium sulfate (0.25% w/v), and calcium carbonate (1.5% w/v). Culture flasks were incubated at 30 °C with continuous agitation at 180 rpm for 96 hours. Following fermentation, the culture broth was centrifuged at 10,000 rpm for 15 minutes, and the cell-free supernatant was collected for ascorbic acid purification (Tucaliuc et al., 2022). Identification and quantification of ascorbic acid Thin-layer chromatography Qualitative identification was performed using silica gel TLC plates with a mobile phase consisting of chloroform, ethanol, acetone, and ammonium hydroxide (2:2:2:1 v/v/v/v). The elution chamber was pre-saturated with solvent vapour for 10-15 minutes. Samples were spotted 6-8 mm from the edge of the plate using a micropipette. Elution continued until the solvent front advanced to 5-10 mm from the top edge. Plates were dried at 60 °C for 10 minutes and visualised under UV light (254 nm), where ascorbic acid exhibited characteristic blue fluorescence. Retention factor (Rf) values were calculated as : Where hx is the distance from the origin to the spot centre, and h 0 is the distance from the origin to the solvent front. Blue fluorescent bands corresponding to ascorbic acid were scraped from the plate, dissolved in distilled water, and centrifuged at 10,000 rpm for 5 minutes to remove silica particles. The supernatant was filtered and used for quantitative analysis (Akasaka, 2013). Iodometric titration Ascorbic acid content was quantified by redox titration. TLC-purified samples (5 mL) were transferred to conical flasks containing 1 mL of starch indicator solution. The mixture was titrated against a standardised iodine solution (0.005 M) until the first persistent blue-black colouration appeared, indicating the formation of the starch-iodine complex. Titrations were performed in triplicate, and the results were recorded as concordant values (Belete et al., 2023). 2,6 – Dichlorophenolindophenol (DCPIP) assay An alternative quantification method employed DCPIP dye reduction. Sample aliquots (5mL) were mixed with equal volumes of distilled water and titrated against standardised DCPIP solution. The endpoint was marked by a persistent colour change from blue to pink, indicating complete oxidation of ascorbic acid. Titrations were performed in triplicate to ensure reproducibility (Nakamura et al., 2013). Synthesis of iron oxide nanoparticles Iron oxide nanoparticles were synthesised via the reverse co-precipitation method. Ferric chloride (FeCl₃, 0.8 g) and ferrous sulfate (FeSO₄, 2.75 g) were dissolved in 50 mL of deionised water under vigorous stirring. Sodium hydroxide (2.75 g) was added dropwise until the solution turned black, indicating the formation of magnetite (Fe₃O₄). The precipitate was collected by centrifugation at 10,000 rpm for 15 minutes and washed repeatedly with distilled water using a magnetic separation technique. The purified nanoparticles were dried in a hot-air oven at 80 °C (Mahmud et al., 2020). Encapsulation of ascorbic acid onto iron oxide nanoparticles The magnetic nanoparticle dispersion was adjusted to pH 12.5 with 10 M ammonium hydroxide to optimise the surface charge for ascorbic acid adsorption. Biosynthesised ascorbic acid (0.5 g) was added to the alkaline dispersion under continuous stirring. The mixture was maintained at 80 °C for 1 hour to facilitate stable coating formation through interaction between ascorbic acid functional groups and the hydroxylated iron oxide surface. The ascorbic acid-functionalised nanoparticles were recovered magnetically, washed with distilled water, and dried at 80 °C (Kumari & Chauhan, 2022b). Characterisation of ascorbic acid-coated nanoparticles Scanning electron microscopy (SEM) Morphological characterisation was performed using scanning electron microscopy to visualise nanoparticle size, shape, and surface coating architecture. SEM analysis provides information on particle size distribution, surface topography, and the uniformity of ascorbic acid encapsulation at nanometre resolution (Mahmud et al., 2020). Food preservation efficacy testing The antimicrobial and preservative potential of ascorbic acid-coated nanoparticles was evaluated across multiple food matrices, including fruit juices (apple, grape, orange and banana) and dairy products (milk and curd) (Teshome et al., 2022). Three experimental groups were established for each food type: Control group : Food products without preservatives. Commercial ascorbic acid group : Products supplemented with commercially available ascorbic acid at standard concentrations. Nanoparticle group : Products fortified with biosynthesised ascorbic acid coated iron oxide nanoparticles at equivalent ascorbic acid concentrations. Samples were stored under standardised conditions, and microbial load was monitored using the viable plate count method. Serial dilutions (10 -6 to 10 -8 ) were prepared, plated on nutrient agar, and incubated at 37 °C for 24 to 48 hours. Colony-forming units (CFU) were enumerated to assess antimicrobial efficacy and shelf-life extension across treatment groups. RESULTS AND DISCUSSION Isolation and characterisation of ascorbic acid-producing bacteria Ascorbic acid-producing Acetobacter strains were isolated from deteriorated fruits and naturally fermented beverages. Samples were aseptically processed and enriched in a selective medium that promotes the growth of acetic acid bacteria, then transferred to GYC and CARR differential media for isolation and preliminary identification. Cultural characterisation on GYC medium : Following 3-4 days of incubation at 37 °C, isolates exhibited characteristic colonial morphology on GYC medium. Colonies were large, mucoid, and slimy in texture, with milky-white to pale yellow colouration (Fig. 1). Clear halos formed around individual colonies due to the solubilization of calcium carbonate by metabolically produced organic acids. This zone of clearance indicated acid production, a characteristic metabolic feature of Acetobacter species, with observed morphology consistent with established profiles of acetic acid bacteria. Cultural characterisation on CARR Medium: Complementary screening on CARR medium containing bromocresol green revealed that colonies initially appeared blue, transitioning to yellow as ethanol oxidation produced acetic acid (Fig.2). This colour change confirmed acid production and ethanol utilisation, characteristic metabolic features of Acetobacter species. Isolates tolerated ethanol concentrations of 5-9 % consistent with acetic acid bacteria. Morphological and biochemical characteristics Morphological characterisation through Gram staining and comprehensive biochemical profiling was performed to establish the taxonomic identity of the isolates. The results, including cell morphology, enzyme activities, metabolic patterns, and carbohydrate fermentation profiles, are presented in Table 1. The biochemical profile confirmed the isolates as Acetobacter species, particularly Acetobacter aceti , based on their characteristic aerobic respiratory metabolism and metabolic versatility, which is typical of acetic acid bacteria within the Acetobacteraceae family. Table 1: Morphological and biochemical characteristics of the isolated strain CHARACTERISTIC RESULT Colony morphology Large, mucoid, milky-white to pale yellow Gram reaction Gram-negative Cell shape Coccoid Catalase Positive Oxidase Positive Indole production Negative Methyl Red Negative Voges-Proskauer Positive Citrate utilization Positive Glucose fermentation Acid + Gas Lactose fermentation Acid + Gas Sucrose fermentation Acid + Gas Mannitol fermentation Acid + Gas Triple Sugar Iron Acid slant/Acid butt (A/A) Urease Negative Optimisation of culture conditions for ascorbic acid production pH optimisation The influence of pH on ascorbic acid biosynthesis was evaluated over a pH range of 4.0-7.0 (Fig. 3). Maximum production occurred at pH 4.0 and 6.0, representing optimal conditions for bacterial growth and enzymatic activity. Production declined substantially at pH values below 4.0 and above 7.0, reflecting the acidophilic nature of Acetobacter species and the optimal activity range of key biosynthetic enzymes. Temperature optimisation Temperature optimisation revealed maximum ascorbic acid production at 30 °C, with significantly lower yields at 4 °C and 40 °C (Fig. 4). This optimal temperature represents a balance between metabolic rate and enzyme stability characteristic of mesophilic Acetobacter species. Suboptimal temperatures (4 °C) reduced enzymatic activity, whereas 40 °C led to enzyme denaturation and cellular stress, limiting ascorbic acid biosynthesis The isolation, characterisation, and optimisation studies established optimal conditions (pH 4.0-6.0, 30 °C) for ascorbic acid production by Acetobacter strains, providing parameters for subsequent fed-batch fermentation and nanoparticle encapsulation applications. Biosynthesis and analytical confirmation of ascorbic acid Fed -batch fermentation production Fed-batch fermentation using Acetobacter species with sorbitol as the carbon source achieved approximately 99 % substrate conversion after 96 hours. Ascorbic acid presence and concentration in the fermentation broth were confirmed through thin-layer chromatography, iodometric titration, and DCPIP dye reduction assay. Qualitative identification by thin-layer chromatography Thin-layer chromatography on silica gel using a chloroform-ethanol-acetone-ammonium hydroxide mobile phase confirmed the presence of ascorbic acid in the fermentation medium. The retention factor (Rf) was calculated using the standard formula: Where hx represents the distance travelled by the substance from the origin (1.7 mm), and h 0 represents the distance travelled by the solvent front from the origin(2.5 mm) Rf = 1.7 / 2.5 = 0.68 The calculated retention factor (Rf = 1.7/2.5 = 0.68) corresponded to ascorbic acid reference standards (Table 2). Under UV light at 254 nm, the separated compound exhibited characteristic blue fluorescence (Fig. 5), confirming the identification of ascorbic acid. Table 2: Reference Retention Factors and UV Fluorescence of Vitamins Vitamin Common Name Rf Value Colour Under UV B₁ Thiamine 0.40 Violet B₂ Riboflavin 0.30 Yellow B₃ Niacin (Nicotinic acid) 0.36 Violet B₆ Pyridoxine 0.49 Blue C Ascorbic acid <0.1* Blue *Reference value from standard vitamin chromatography protocols Quantitative analysis by iodometric titration Ascorbic acid content was determined by iodometric titration based on its oxidation to dehydroascorbic acid. The blue extract from TLC analysis (1g) was dissolved in 5mL of distilled water and titrated with standardised iodine solution in duplicate, showing reproducible results (Table 3). Table 3: Iodometric Titration Results S. No Volume of the Sample Burette reading (ml) Volume of Iodine(mL) Initial Final 1 2 5 ml 5 ml 0.0 0.0 1.25 2.5 1.25 2.5 Iodometric titration of 5mL samples consumed an average of 1.9mL iodine solution (11.4 mL for 30 mL total volume). The endpoint was identified by the formation of a blue-black starch-iodine complex, indicating complete oxidation of ascorbic acid and confirming its presence and reducing capacity in the fermentation medium. Validation by 2,6-Dichlorophenolindophenol (DCPIP) assay Ascorbic acid quantification employed DCPIP dye reduction, based on the conversion of blue DCPIP to its colourless form. Following standardisation with ascorbic acid in the fermentation media, the endpoint was determined by a stable pink colour (≥15 seconds). Titration of 5 mL samples consumed 2.8 mL DCPIP, corresponding to 16.8 mL for the complete 30 mL volume. Three methods, TLC, iodometric titration, and DCPIP reduction, collectively confirmed ascorbic acid biosynthesis. TLC established identity via Rf values and UV response, while titrimetric approaches quantified concentration. Consistent findings across techniques validate the Acetobacter- mediated biotransformation of sorbitol to ascorbic acid as a sustainable production strategy, supporting future scale-up for nanoparticle encapsulation and food preservation applications. Synthesis and characterisation of ascorbic acid-coated iron oxide nanoparticles Iron oxide nanoparticle synthesis Iron oxide nanoparticles were synthesised via coprecipitation using FeCl₃ and FeSO₄ as iron sources with ammonium hydroxide as the precipitating agent. This method produced black magnetite precipitates characteristic of Fe₃O₄ (Fig. 6), yielding magnetically responsive nanoparticles under ambient conditions for subsequent functionalization. Ascorbic acid encapsulation Iron oxide nanoparticles were surface-modified with ascorbic acid through pH and temperature-controlled encapsulation. The magnetic dispersion was adjusted to pH 12.5 with 10 N ammonia to optimise surface charge for ascorbic acid adsorption. Ascorbic acid (0.5 g) was added to the alkaline dispersion, and the mixture was heated at 80 °C with stirring for 1 hour to promote interaction between the ascorbic acid functional groups and the hydroxylated iron oxide surface. The coated nanoparticles were recovered by oven drying; the colour change indicated successful functionalization, reflecting the combined optical properties of the iron oxide core and the organic coating. Morphological characterisation by SEM SEM analysis revealed the nanoparticle morphology and coating structure at nanoscale resolution (Fig.8). The micrographs showed densely packed, spherical nanoparticles with a uniform size distribution. Ascorbic acid formed a conformal coating with complete surface coverage, demonstrating successful encapsulation without significant aggregation. The uniform distribution and spherical geometry confirmed the practicality of synthesis and encapsulation. This coating morphology provides structural protection for ascorbic acid while maintaining accessibility for functional applications. Functional validation: Antioxidant activity retention Ascorbic acid bioactivity post-encapsulation was verified using the methylene blue decolourisation assay. The addition of coated nanoparticles to the methylene blue solution resulted in rapid, complete decolourisation within minutes, as the dye reduced to its colourless leuco form. This transition confirmed that the encapsulated ascorbic acid retains its reducing capacity and antioxidant functionality, thereby maintaining both its nutritional value and chemical reactivity, which are essential for antimicrobial activity. The iron oxide core retained its magnetic properties, enabling efficient magnetic separation and recovery, a key advantage for industrial-scale applications. Characterisation results demonstrate successful synthesis of ascorbic acid-coated iron oxide nanoparticles with retained functionality, uniform morphology, and structural properties suitable for food preservation and nutritional fortification. Antimicrobial efficacy and food preservation performance Antimicrobial efficacy of biosynthesised ascorbic acid-coated iron oxide nanoparticles was evaluated across six food matrices: apple, orange, grape and banana juices, milk and curd. Viable plate count analysis compared untreated controls, nanoparticle-treated, and commercial ascorbic acid-treated samples (Fig.9, Table 4). Control samples exhibited uncontrolled microbial growth (TNTC) at 10⁻⁶ dilutions, confirming rapid spoilage. Commercial ascorbic acid reduced microbial loads to 2-12 CFU at 10⁻⁸ dilutions, while biosynthesised nanoparticles demonstrated substantial efficacy with 7-20 CFU at 10⁻⁸ dilutions across all tested matrices. Table 4: Microbial Colony Counts in Treated and Untreated Food Samples Sl. No Food Sample Dilution Control (CFU) Nano-encapsulated Ascorbic Acid (CFU) Commercial Ascorbic Acid (CFU) Remark 1 Apple juice 10⁻⁶ TNTC 15 12 Highly effective 10⁻⁷ TNTC 13 7 Highly effective 10⁻⁸ TNTC 9 3 Highly effective 2 Grape juice 10⁻⁶ TNTC 14 8 Highly effective 10⁻⁷ TNTC 11 5 Highly effective 10⁻⁸ TNTC 19 2 Highly effective 3 Orange juice 10⁻⁶ TNTC 15 9 Highly effective 10⁻⁷ TNTC 13 7 Highly effective 10⁻⁸ TNTC 11 5 Highly effective 4 Banana juice 10⁻⁶ TNTC 13 8 Highly effective 10⁻⁷ TNTC 12 6 Highly effective 10⁻⁸ TNTC 7 5 Highly effective 5 Milk 10⁻⁶ TNTC 12 7 Highly effective 10⁻⁷ TNTC 10 6 Highly effective 10⁻⁸ TNTC 8 4 Highly effective 6 Curd 10⁻⁶ TNTC 20 10 Highly effective 10⁻⁷ TNTC 15 7 Highly effective 10⁻⁸ TNTC 12 5 Highly effective TNTC = Too Numerous To count. Nano-encapsulated ascorbic acid consistently reduced bacterial counts from TNTC to countable levels across all samples, demonstrating effective antimicrobial activity compared to the control (7-20 CFU). Quantitative assessment of preservation efficacy Comparative analysis revealed that biosynthesised ascorbic acid-coated nanoparticles achieved approximately 75% of the antimicrobial activity of commercial ascorbic acid across all tested food matrices, evidenced by consistently higher colony counts at equivalent dilutions. Despite this moderate reduction in potency, the nanoparticle formulation effectively suppressed microbial growth by several orders of magnitude compared with untreated controls, demonstrating its viability as a functional preservative. Antimicrobial performance varied across food matrices: banana juice exhibited the lowest colony count (7 CFU at 10⁻⁸ ), while curd showed the highest (20 CFU at 10⁻⁶; 12 CFU at 10⁻⁸). These matrix-dependent differences reflect the influence of intrinsic food properties, including pH, nutrient composition, initial microbial load, and physicochemical characteristics, on preservation efficacy. Dual functionality: Preservation and nutritional fortification Beyond antimicrobial efficacy, the nanoparticle formulation demonstrated dual functional potential by simultaneously providing nutritional fortification. The iron oxide nanoparticle core serves as a bioavailable source of iron to address micronutrient deficiency, while the ascorbic acid coating provides vitamin C supplementation. This integrated approach delivers both preservation and nutritional enhancement within a single additive system, representing a significant advancement over conventional single-purpose preservatives. The controlled release characteristics imparted by nanoencapsulation offer additional functional advantages. The gradual liberation of ascorbic acid from the nanoparticle matrix potentially extends the effective preservation duration compared to free ascorbic acid, which may be rapidly consumed through oxidation or degradation reactions in food systems. Furthermore, the biosynthetic origin of the formulation aligns with consumer preferences for natural, clean-label ingredients while maintaining compatibility with existing food processing infrastructure. Food matrix-specific performance Analysis of preservation efficacy across different food categories revealed product-specific performance patterns. In fruit juices, acidic pH naturally complements the antimicrobial action of ascorbic acid, with all juice types showing effective microbial suppression. Orange and apple juices demonstrated intermediate colony counts (9-15 CFU at 10⁻⁶), while banana juice exhibited the most effective suppression at higher dilutions. In dairy products, milk showed moderate colony counts (8-12 CFU across dilutions), while curd exhibited slightly elevated counts (12-20 CFU), potentially attributable to its inherent fermented nature and higher initial microbial populations. These results establish proof of concept for biosynthesised ascorbic acid-coated iron oxide nanoparticles as multifunctional food additives that extend shelf life while simultaneously enhancing nutritional value. The formulation demonstrates practical applicability across diverse food matrices, with performance levels suitable for commercial food preservation applications. FUTURE SCOPE The development of biosynthesised ascorbic acid-coated oxide nanoparticles provides a foundation for future commercial translation. Priority research areas include optimising encapsulation through advanced characterisation (TEM, XRD, FTIR), scaling production via continuous fermentation and automated reactors, and establishing stability profiles under storage and processing conditions. Food matrix-specific formulations require investigation across beverages, dairy, high-fat products, and bakery applications to maximise functionality while minimising sensory impacts. Rigorous toxicological evaluation, following guidelines from the FDA, EFSA, and FSSAI, including acute, chronic, genotoxicity, and reproductive toxicity studies, is essential for regulatory approval, alongside sensory evaluation and consumer acceptance testing. The nano-encapsulation platform offers opportunities to extend the shelf life of other unstable bioactives, such as B-complex vitamins, vitamin E, polyphenols, omega-3 fatty acids, and natural antimicrobials. Stimuli–responsive coatings triggered by pH, temperature, or microbial presence could enable intelligent preservation systems. Human bioavailability studies would validate the nutritional benefits, particularly the synergistic effects of combined vitamin C and iron supplementation in populations prone to deficiency. Life cycle analysis, waste-stream behaviour assessment, and the development of biodegradable alternatives would address environmental stability. Integration with active packaging technologies could create comprehensive preservation systems, transforming this proof of concept into a commercially viable, regulatory-compliant solution for the industry. CONCLUSION This study developed an integrated bioprocess for producing nano-encapsulated ascorbic acid, a dual-functional food additive for preservation and nutritional fortification. Ascorbic acid-producing Acetobacter strains isolated from natural sources were characterised through morphological and biochemical analyses. Fermentation optimisation identified pH 4.0–6.0 and 30°C as optimal conditions, achieving 99% substrate conversion within 96 hours. Biosynthesis was validated via thin-layer chromatography (Rf 0.68), iodometric titration, and DCPIP assay. Iron oxide nanoparticles synthesised by co-precipitation were functionalised with biosynthesised ascorbic acid, producing a uniform coating architecture, as confirmed by SEM. Preservation testing across fruit juices and dairy products showed nano-encapsulated formulations achieved approximately 75% antimicrobial activity relative to commercial ascorbic acid, reducing microbial loads from uncountable levels to 7–20 CFU at 10 − 8 dilutions while providing vitamin C and iron fortification. The approach offers advantages in sustainable biological production, enhanced stability through nanoencapsulation, dual micronutrient fortification, and multimodal antimicrobial action. Successful translation from the laboratory to functional food applications demonstrates commercial viability through scalable fermentation and straightforward nanoparticle synthesis, applicable across diverse food matrices. Future work should optimise encapsulation efficacy, assess long-term stability and safety, evaluate sensory properties, and develop industrial-scale protocols. This proof of fortification agent aligns with clean-label trends and sustainability requirements. Abbreviations % Percentage A/A Acid slant and Acid butt BHA Butylated Hydroxyanisole BHT Butylated Hydroxytoluene CFU Colony Forming Unit DCPIP 2,6 – Dichlorophenolindophenol DNA Deoxyribonucleic Acid EFSA European Food Safety Authority et al. et alii (and others) FAO Food and Agriculture Organisation FDA Food and Drug Administration FSSAI Food Safety and Standards Authority of India FTIR Fourier Transform Infrared spectroscopy GYC Glucose Yeast Calcium carbonate GYP Glucose Yeast Peptone IMViC I ndole, M ethyl Red, V oges-Proskauer, and C itrate nm Nanometres pH Potential of Hydrogen Rf Retention factor rpm Rotation per Minute SEM Scanning Electron Microscopy TBHQ Tert-butylhydroquinone TEM Transmission Electron Microscopy TLC Thin-Layer Chromatography TNTC Too Numerous To Count U.S. United States UV Ultraviolet w/v weight/volume w/w weight/weight XRD X-ray diffraction Declarations ACKNOWLEDGEMENT The authors would like to extend their heartfelt appreciation to the DST-FIST scheme, the Management, and the Department of Microbiology at PSG College of Arts and Science, Coimbatore, for their support and valuable assistance throughout the experiment. CONTRIBUTION OF AUTHORS Catherine Antony: Conceptualisation, Methodology, Experimental design, Data handling, Writing - original draft and editing Dr Krishnaveni N: Validation, supervision and critical review CONFLICT OF INTEREST The authors declare no competing interests. FUNDING Not Applicable DATA AVAILABILITY The data generated and analysed during the study are included in the manuscript. ETHICS STATEMENT Not Applicable CONCENT TO PARTICIPATE Not Applicable CONCENT TO PUBLISH Not Applicable References Ahmed, T. M. K., Bakr, M. M., & Ahmed, Q. A. (2022). Side Effects of Preservatives on Human Life . 2 . Akasaka, K. (2013). Simple Determination of l-Ascorbic Acid on TLC by Visual Detection Using Autocatalytic Reaction. Analytical Sciences , 29 (5), 505–509. https://doi.org/10.2116/analsci.29.505 Alberts, A., Moldoveanu, E.-T., Niculescu, A.-G., & Grumezescu, A. M. (2025). Vitamin C: A Comprehensive Review of Its Role in Health, Disease Prevention, and Therapeutic Potential. Molecules , 30 (3), 748. https://doi.org/10.3390/molecules30030748 Antony, C., & Narayanaswamy, K. (2026). Food preservatives: Natural or synthetic? Archives of Microbiology , 208 (3), 134. https://doi.org/10.1007/s00203-025-04674-9 Aswini, K., Gopal, N. O., & Uthandi, S. (2020). Optimized culture conditions for bacterial cellulose production by Acetobacter senegalensis MA1. BMC Biotechnology , 20 (1), 46. https://doi.org/10.1186/s12896-020-00639-6 Barrett, C. B. (2021). Overcoming Global Food Security Challenges through Science and Solidarity. American Journal of Agricultural Economics , 103 (2), 422–447. https://doi.org/10.1111/ajae.12160 Belete, A., Yisak, H., Chandravanshi, B. S., & Yaya, E. E. (2023). Ascorbic acid content and the antioxidant activity of common fruits commercially available in Addis Ababa, Ethiopia. Bulletin of the Chemical Society of Ethiopia , 37 (2), 277–288. https://doi.org/10.4314/bcse.v37i2.3 Kumari, A., & Chauhan, A. K. (2022a). Iron nanoparticles as a promising compound for food fortification in iron deficiency anemia: A review. Journal of Food Science and Technology , 59 (9), 3319–3335. https://doi.org/10.1007/s13197-021-05184-4 Kumari, A., & Chauhan, A. K. (2022b). Iron nanoparticles as a promising compound for food fortification in iron deficiency anemia: A review. Journal of Food Science and Technology , 59 (9), 3319–3335. https://doi.org/10.1007/s13197-021-05184-4 Lee, D.-H., Kim, S.-H., Lee, C.-Y., Jo, H.-W., Lee, W.-H., Kim, E.-H., Choi, B.-K., & Huh, C.-K. (2024). Screening of Acetic Acid Bacteria Isolated from Various Sources for Use in Kombucha Production. Fermentation , 10 (1), 18. https://doi.org/10.3390/fermentation10010018 L’Haridon, S., Toffin, L., & Roussel, E. (2020). Methanococcoides. In M. E. Trujillo, S. Dedysh, P. DeVos, B. Hedlund, P. Kämpfer, F. A. Rainey, & W. B. Whitman (Eds.), Bergey’s Manual of Systematics of Archaea and Bacteria (1st ed., pp. 1–9). Wiley. https://doi.org/10.1002/9781118960608.gbm00514.pub2 Lisboa, H. M., Pasquali, M. B., dos Anjos, A. I., Sarinho, A. M., de Melo, E. D., Andrade, R., Batista, L., Lima, J., Diniz, Y., & Barros, A. (2024). Innovative and Sustainable Food Preservation Techniques: Enhancing Food Quality, Safety, and Environmental Sustainability. Sustainability , 16 (18), Article 18. https://doi.org/10.3390/su16188223 Mahmud, N., Nasser, M. S., El-Naas, M. H., Ba-Abbad, M. M., Wahab Mohammad, A., Mansour, S., & Benamor, A. (2020). Synthesis and Characterization of Fe3O4 Nanoparticles Using Different Experimental Methods. IOP Conference Series: Materials Science and Engineering , 778 (1), 012028. https://doi.org/10.1088/1757-899X/778/1/012028 Malik, M., Narwal, V., & Pundir, C. S. (2022). Ascorbic acid biosensing methods: A review. Process Biochemistry , 118 , 11–23. https://doi.org/10.1016/j.procbio.2022.03.028 Mehta, J., Pathania, K., & Pawar, S. V. (2025). Recent overview of nanotechnology based approaches for targeted delivery of nutraceuticals. Sustainable Food Technology , 3 (4), 947–978. https://doi.org/10.1039/D5FB00122F Nakamura, H., Hattori, D., Tokunaga, D., & Suzuki, Y. (2013). An isothermal absorptiometric assay for viable microbes using the redox color indicator 2,6-dichlorophenolindophenol. Analytical Biochemistry , 441 (2), 140–146. https://doi.org/10.1016/j.ab.2013.07.010 Rahman, M., Uddin, M. B., Aziz, M. G., Abunaser, M., Haque, M. R., & Siddiki, M. S. R. (2024). Isolation and Characterization of Acetic Acid Bacteria from Pineapple, Sugarcane, Apple, Grape, Pomegranate, and Papaya Fruit. European Journal of Agriculture and Food Sciences , 6 (2), 14–18. https://doi.org/10.24018/ejfood.2024.6.2.772 Sahoo, B. K., Mishra, R. R., & Behera, B. C. (2020). Isolation and Identification of Thermotolerent Acetic Acid Bacteria from Waste Fruits. Asian Journal of Biological and Life Sciences , 9 (2), 209–213. https://doi.org/10.5530/ajbls.2020.9.32 See, X. Z., Yeo, W. S., & Saptoro, A. (2024). A comprehensive review and recent advances of vitamin C: Overview, functions, sources, applications, market survey and processes. Chemical Engineering Research and Design , 206 , 108–129. https://doi.org/10.1016/j.cherd.2024.04.048 Teshome, E., Forsido, S. F., Rupasinghe, H. P. V., & Olika Keyata, E. (2022). Potentials of Natural Preservatives to Enhance Food Safety and Shelf Life: A Review. The Scientific World Journal , 2022 (1), 9901018. https://doi.org/10.1155/2022/9901018 Tissera, C. E., Barnetche, M. E., Silva, O. F., & Fernández, M. A. (2025). Increasing the stability of ascorbic acid through encapsulation in food-grade vesicles: An approach for nutritional improvement. International Journal of Food Science and Technology , 60 (1), vvae027. https://doi.org/10.1093/ijfood/vvae027 Tucaliuc, A., Cîșlaru, A., Kloetzer, L., & Blaga, A. C. (2022). Strain Development, Substrate Utilization, and Downstream Purification of Vitamin C. Processes , 10 (8), 1595. https://doi.org/10.3390/pr10081595 Vesković, S. (2025). In the Global Food System: Addressing Food Losses, Waste, and Safety. In S. Veskovic (Ed.), Natural Food Preservation: Controlling Loss, Advancing Safety (pp. 5–58). Springer Nature Switzerland. https://doi.org/10.1007/978-3-031-85089-9_2 Waseem, S. Mohd., Gaikwad, S. V., Pandit, V. A., & Kapse, N. N. (2025). Iron Oxide Nanoparticles for Catalytic Applications: Synthesis, Characterization, and Environmental Performance. Topics in Catalysis . https://doi.org/10.1007/s11244-025-02204-x Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 05 May, 2026 Reviews received at journal 08 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviewers invited by journal 09 Mar, 2026 Editor invited by journal 01 Mar, 2026 Editor assigned by journal 15 Feb, 2026 Submission checks completed at journal 15 Feb, 2026 First submitted to journal 05 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8801688","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":603288893,"identity":"a2004c2f-2350-43af-b350-a3c25b4475bc","order_by":0,"name":"Catherine Antony","email":"","orcid":"","institution":"PSG College of Arts \u0026 Science","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"","lastName":"Antony","suffix":""},{"id":603288894,"identity":"f8208a8b-623c-49e3-b5a6-ad036cd97e00","order_by":1,"name":"Krishnaveni 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15:03:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1170908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolated Acetobacter colonies appeared blue initially and turned yellow\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/d233444d679d727c00ced137.png"},{"id":104506699,"identity":"3905f540-966e-428b-8444-360f066d54ef","added_by":"auto","created_at":"2026-03-12 15:03:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epH Optimisation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/2b1fab94f06872bf0352d40c.png"},{"id":104506705,"identity":"838c0e36-07f5-4bc4-92c2-db971cac844c","added_by":"auto","created_at":"2026-03-12 15:03:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":31529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemperature Optimisation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/ac3095adcab1b21827417046.png"},{"id":104506702,"identity":"75374656-4ff7-4a2f-8a6e-fe529406fdef","added_by":"auto","created_at":"2026-03-12 15:03:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":617991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThin-layer chromatography\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/49800b38a441de622c1d5dc2.png"},{"id":104506707,"identity":"98a1109b-2a54-4d39-a13c-cb52bfde6fef","added_by":"auto","created_at":"2026-03-12 15:03:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":921353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesis of iron oxide nanoparticle\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/8a6ebe091e473657e1c8fc27.png"},{"id":104506706,"identity":"515144e9-de62-4f96-a486-b7f097e18c2d","added_by":"auto","created_at":"2026-03-12 15:03:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":138718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIron oxide nanoparticle encapsulated with Ascorbic acid\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/d745053a358a6712117ddb4e.png"},{"id":104506703,"identity":"1ba0c416-d5a1-43a2-9220-a3f9d491fd3f","added_by":"auto","created_at":"2026-03-12 15:03:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":554108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScanning Electron Microscopic view of iron oxide encapsulated ascorbic acid\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/e42e09290940dbc71b1e5b35.png"},{"id":104506704,"identity":"c4f11a6e-f671-4c13-b159-6f297753f8b7","added_by":"auto","created_at":"2026-03-12 15:03:15","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1367621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative Preservation Activity Across Food Matrix\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/79716a33f47181c771dc0936.png"},{"id":105032972,"identity":"cbb4ae34-43e6-45c6-81e2-314c354e07f8","added_by":"auto","created_at":"2026-03-20 07:08:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10059625,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8801688/v1/5c4092bd-5e81-490f-a7b3-fae695ae9b9e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nano-armoured Vitamin C: A novel approach to micronutrient delivery and natural food preservation in integrated food systems","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe global food industry faces significant challenges in food safety, nutritional security, and adapting to evolving consumer preferences (Barrett, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Microbial spoilage and lipid oxidation of food result in annual losses in billions of dollars and pose significant public health risks (Vesković, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). India\u0026rsquo;s food processing sector, ranking sixth globally, exemplifies the transition from traditional preservation methods to advanced systems that incorporate sophisticated techniques and functional additives (Lisboa et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consumer demand has shifted dramatically toward natural, clean-label products, emphasising transparency and minimal processing over synthetic additives (Antony \u0026amp; Narayanaswamy, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Current preservation strategies rely predominantly on antimicrobial agents ( sulphuric acid, nitrates, nitrites) and synthetic antioxidants (BHA, BHT, TBHQ), which effectively extend shelf life but face increasing rejection due to health concerns, potential antimicrobial resistance, regulatory restrictions, and incompatibility with clean-label demands (Ahmed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAscorbic acid (vitamin C) is a multifunctional solution that meets both technical and consumer demands in food preservation (Tissera et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). As a potent antioxidant, it prevents lipid oxidation, pigment discolouration, and nutrient degradation while supporting collagen synthesis, immune function, and iron absorption (Alberts et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Despite substantial health benefits, including protection against oxidative stress and chronic diseases, ascorbic acid faces critical limitations: instability under processing conditions ( oxygen, elevated temperatures, pH variations, light, metal catalysts), compromising efficacy and nutritional value, plus averse effects from direct supplementation(gastrointestinal distress, nausea), limiting consumer acceptance (Malik et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nanotechnology offers transformative solutions through nanoencapsulation, enabling enhanced stability, controlled release, improved bioavailability, and targeted delivery (Mehta et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Iron oxide nanoparticles exhibit advantageous properties, including biocompatibility, superparamagnetic properties that enable controlled delivery and magnetic recovery, tunable surface chemistry for functionalization, and nutritional value as bioavailable iron supplements, thereby addressing micronutrient deficiencies (Waseem et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This combination creates synergistic functionality in which ascorbic acid enhances iron absorption while iron oxide nanoparticles protect it from degradation, simultaneously addressing two prevalent global nutritional deficits (Kumari \u0026amp; Chauhan, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Biological production through microbial fermentation using \u003cem\u003eAcetobacter species, Gluconobacter oxydans\u003c/em\u003e, and \u003cem\u003eAspergillus niger\u003c/em\u003e, offers sustainable alternatives to chemical synthesis, aligning with clean-label requirements while minimising environmental impacts (See et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite advances in nanoencapsulation and biological production methods, significant gaps remain in their combined use for food preservation (Anushree et al., 2025). Most studies have examined these technologies separately rather than together, testing nanoencapsulation in laboratory settings without verifying effectiveness in actual food products (Mehta et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While some research has improved nutrient delivery or explored production methods, these studies have not demonstrated antimicrobial activity or addressed practical concerns, such as production costs, scaling up to industrial levels (Waseem et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The food industry requires solutions that simultaneously enhance nutrition, prevent microbial spoilage, meet consumer demand for natural ingredients, and remain economically feasible; yet, few studies have connected laboratory-scale production with nanoparticle technology and testing in real food systems (Acharekar et al., 2025). Therefore, an integrated approach is needed that combines optimised microbial production, nano-encapsulation methods, and characterisation and testing across various food products to develop a practical, multipurpose preservation technology for commercial use (Lavanya et al., 2024).\u003c/p\u003e \u003cp\u003eThis research addresses the integration gap by developing nano-encapsulated ascorbic acid delivery systems using iron oxide nanocarriers, combining microbial fermentation with \u003cem\u003eAcetobacter\u003c/em\u003e strains, controlled synthesis and functionalization of iron oxide nanoparticles, and synthetic validation across diverse food matrices, including fruit juices and dairy products. The iron oxide nanoparticles serve dual functions: as protective carriers that shield ascorbic acid from degradation while enhancing its bioavailability, and as a source of iron supplementation to address micronutrient deficiency. This integrated methodology bridges the gap between fundamental science and industrial applications, offering solutions for natural food preservation while providing scalable platforms for micronutrient fortification. The specific objectives were to isolate and characterise ascorbic acid-producing microorganisms from environmental sources; to optimise fermentation parameters for maximum production; to synthesise iron oxide nanoparticles and encapsulate biosynthesised ascorbic acid within these matrices; to characterize the physicochemical and functional properties of nano encapsulated formulations; to evaluate antimicrobial efficacy and preservation performance; and to compare shelf life extension between nano encapsulated ascorbate, commercial ascorbic acid, and untreated controls across multiple food products.\u003c/p\u003e"},{"header":"METHODOLOGY","content":"\u003cp\u003e\u003cstrong\u003eIsolation of ascorbic acid-producing bacteria: sample collection and enrichment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcetic acid bacteria capable of direct ascorbic acid biosynthesis were isolated from naturally fermented substrates, including deteriorated fruits (apples and grapes), coconut toddy, and honey. These sources are selected for their high carbohydrate content and their established association with \u003cem\u003eAcetobacter\u003c/em\u003e species. Samples were aseptically processed and subjected to selective enrichment using source-specific media formulations. For fruit-derived isolates, the enrichment medium contained glucose (1% w/v), ethanol (0.5% v/v), acetic acid (0.3% v/v), peptone (1.5%w/v), and yeast extract (0.8% w/v). Toddy samples were enriched in medium containing glucose (5% w/v), yeast extract (1% w/v), and cycloheximide (100 ppm) to suppress fungal contamination. All enrichment cultures were incubated at 30\u003csup\u003e \u0026deg;\u003c/sup\u003eC with continuous agitation (180 rpm) for 5 days to promote the proliferation of acetic acid bacteria (Rahman et al., 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelective isolation and culture characterisation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing enrichment, cultures were transferred to differential media for isolation and preliminary identification. Fruit-derived isolates were plated on glucose yeast calcium carbonate (GYC) medium containing (10% w/v), yeast extract (1% w/v), calcium carbonate (2% w/v), and agar (1.5% w/v), adjusted to pH 6.8. The medium was supplemented with 100 mg/L of cycloheximide to inhibit non-target organisms. Plates were incubated aerobically at 30\u0026deg;C for 3-4 days (Lee et al., 2024). \u003c/p\u003e\n\u003cp\u003eEthanol tolerance and acid production characteristics were verified using Carr medium containing yeast extract (3% w/v), bromocresol green (0.2% w/v), ethanol (5-9% v/v), and agar (2% w/v) for toddy-derived. Isolates, Glucose Yeast Peptone (GYP) medium was employed, consisting of glucose (2% w/v), sodium acetate trihydrate (0.5% w/v), tryptone (0.5% w/v), yeast extract (0.5% w/v), potassium phosphate (0.1% w/v), Tween 80 (0.5% v/v), and agar (1.7% w/v) at pH 6.8. Plates were incubated at 37\u0026deg;C for 3-5 days (Sahoo et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterisation and identification of isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolates were characterised through Gram staining and comprehensive biochemical profiling, including enzyme assays (catalase, oxidase, and urease), IMVIC tests, triple sugar iron agar tests, and carbohydrate fermentation patterns, to establish morphological features and metabolic capabilities for taxonomic identification (L\u0026rsquo;Haridon et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimisation of growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSelected isolates were cultivated in brain heart infusion (BHI) broth to determine the optimal conditions for biomass accumulation and ascorbic acid production. Culture aliquots (200 \u0026mu;L) were inoculated into 15 mL BHI broth in triplicate. Temperature optimisation was conducted by incubating cultures at 4\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C, 37\u0026deg;C, and 45\u0026deg;C for 7 days, while pH optimisation involved adjusting the fermentation medium to pH 4.0, 5.0, 6.0, and 7.0 using 0.1 N NaOH or 0.1 N HCl, with cultures incubated at the optimal temperature for 5 days. Growth was monitored by measuring optical density at 600nm with a UV-visible spectrophotometer to identify conditions that yielded the maximum biomass and ascorbic acid production (Aswini et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAscorbic acid production by fed-batch fermentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFed-batch fermentation was conducted using optimised \u003cem\u003eAcetobacter\u003c/em\u003e strains with sorbitol as the primary carbon source. The production medium contained sorbitol (8% w/v), yeast extract (5% w/v), glycerol (0.05% v/v), magnesium sulfate (0.25% w/v), and calcium carbonate (1.5% w/v). Culture flasks were incubated at 30 \u0026deg;C with continuous agitation at 180 rpm for 96 hours. Following fermentation, the culture broth was centrifuged at 10,000 rpm for 15 minutes, and the cell-free supernatant was collected for ascorbic acid purification (Tucaliuc et al., 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification and quantification of ascorbic acid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThin-layer chromatography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQualitative identification was performed using silica gel TLC plates with a mobile phase consisting of chloroform, ethanol, acetone, and ammonium hydroxide (2:2:2:1 v/v/v/v). The elution chamber was pre-saturated with solvent vapour for 10-15 minutes. Samples were spotted 6-8 mm from the edge of the plate using a micropipette. Elution continued until the solvent front advanced to 5-10 mm from the top edge. Plates were dried at 60 \u0026deg;C for 10 minutes and visualised under UV light (254 nm), where ascorbic acid exhibited characteristic blue fluorescence. Retention factor (Rf) values were calculated as :\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"79\" height=\"18\" src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1773327068.png\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere hx is the distance from the origin to the spot centre, and h\u003csub\u003e0 \u003c/sub\u003eis the distance from the origin to the solvent front. Blue fluorescent bands corresponding to ascorbic acid were scraped from the plate, dissolved in distilled water, and centrifuged at 10,000 rpm for 5 minutes to remove silica particles. The supernatant was filtered and used for quantitative analysis (Akasaka, 2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIodometric titration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAscorbic acid content was quantified by redox titration. TLC-purified samples (5 mL) were transferred to conical flasks containing 1 mL of starch indicator solution. The mixture was titrated against a standardised iodine solution (0.005 M) until the first persistent blue-black colouration appeared, indicating the formation of the starch-iodine complex. Titrations were performed in triplicate, and the results were recorded as concordant values (Belete et al., 2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2,6 \u0026ndash; Dichlorophenolindophenol (DCPIP) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn alternative quantification method employed DCPIP dye reduction. Sample aliquots (5mL) were mixed with equal volumes of distilled water and titrated against standardised DCPIP solution. The endpoint was marked by a persistent colour change from blue to pink, indicating complete oxidation of ascorbic acid. Titrations were performed in triplicate to ensure reproducibility (Nakamura et al., 2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of iron oxide nanoparticles \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIron oxide nanoparticles were synthesised via the reverse co-precipitation method. Ferric chloride (FeCl₃, 0.8 g) and ferrous sulfate (FeSO₄, 2.75 g) were dissolved in 50 mL of deionised water under vigorous stirring. Sodium hydroxide (2.75 g) was added dropwise until the solution turned black, indicating the formation of magnetite (Fe₃O₄). The precipitate was collected by centrifugation at 10,000 rpm for 15 minutes and washed repeatedly with distilled water using a magnetic separation technique. The purified nanoparticles were dried in a hot-air oven at 80 \u0026deg;C (Mahmud et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEncapsulation of ascorbic acid onto iron oxide nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe magnetic nanoparticle dispersion was adjusted to pH 12.5 with 10 M ammonium hydroxide to optimise the surface charge for ascorbic acid adsorption. Biosynthesised ascorbic acid (0.5 g) was added to the alkaline dispersion under continuous stirring. The mixture was maintained at 80 \u0026deg;C for 1 hour to facilitate stable coating formation through interaction between ascorbic acid functional groups and the hydroxylated iron oxide surface. The ascorbic acid-functionalised nanoparticles were recovered magnetically, washed with distilled water, and dried at 80 \u0026deg;C (Kumari \u0026amp; Chauhan, 2022b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterisation of ascorbic acid-coated nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphological characterisation was performed using scanning electron microscopy to visualise nanoparticle size, shape, and surface coating architecture. SEM analysis provides information on particle size distribution, surface topography, and the uniformity of ascorbic acid encapsulation at nanometre resolution (Mahmud et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFood preservation efficacy testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial and preservative potential of ascorbic acid-coated nanoparticles was evaluated across multiple food matrices, including fruit juices (apple, grape, orange and banana) and dairy products (milk and curd) (Teshome et al., 2022). Three experimental groups were established for each food type:\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eControl group\u003c/strong\u003e: Food products without preservatives.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eCommercial ascorbic acid group\u003c/strong\u003e: Products supplemented with commercially available ascorbic acid at standard concentrations.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eNanoparticle group\u003c/strong\u003e: Products fortified with biosynthesised ascorbic acid coated iron oxide nanoparticles at equivalent ascorbic acid concentrations.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eSamples were stored under standardised conditions, and microbial load was monitored using the viable plate count method. Serial dilutions (10\u003csup\u003e-6\u003c/sup\u003e to 10\u003csup\u003e-8\u003c/sup\u003e) were prepared, plated on nutrient agar, and incubated at 37 \u0026deg;C for 24 to 48 hours. Colony-forming units (CFU) were enumerated to assess antimicrobial efficacy and shelf-life extension across treatment groups.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eIsolation and characterisation of ascorbic acid-producing bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAscorbic acid-producing \u003cem\u003eAcetobacter\u003c/em\u003e strains were isolated from deteriorated fruits and naturally fermented beverages. Samples were aseptically processed and enriched in a selective medium that promotes the growth of acetic acid bacteria, then transferred to GYC and CARR differential media for isolation and preliminary identification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCultural characterisation on GYC medium\u003c/strong\u003e: Following 3-4 days of incubation at 37 \u0026deg;C, isolates exhibited characteristic colonial morphology on GYC medium. Colonies were large, mucoid, and slimy in texture, with milky-white to pale yellow colouration (Fig. 1). Clear halos formed around individual colonies due to the solubilization of calcium carbonate by metabolically produced organic acids. This zone of clearance indicated acid production, a characteristic metabolic feature of Acetobacter species, with observed morphology consistent with established profiles of acetic acid bacteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCultural characterisation on CARR Medium:\u0026nbsp;\u003c/strong\u003eComplementary screening on CARR medium containing bromocresol green revealed that colonies initially appeared blue, transitioning to yellow as ethanol oxidation produced acetic acid (Fig.2). This colour change confirmed acid production and ethanol utilisation, characteristic metabolic features of \u003cem\u003eAcetobacter\u0026nbsp;\u003c/em\u003especies. Isolates tolerated ethanol concentrations of 5-9 % consistent with acetic acid bacteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological and biochemical characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphological characterisation through Gram staining and comprehensive biochemical profiling was performed to establish the taxonomic identity of the isolates. The results, including cell morphology, enzyme activities, metabolic patterns, and carbohydrate fermentation profiles, are presented in Table 1. The biochemical profile confirmed the isolates as \u003cem\u003eAcetobacter\u003c/em\u003e species, particularly \u003cem\u003eAcetobacter aceti\u003c/em\u003e, based on their characteristic aerobic respiratory metabolism and metabolic versatility, which is typical of acetic acid bacteria within the \u003cem\u003eAcetobacteraceae\u003c/em\u003e family.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Morphological and biochemical characteristics of the isolated strain\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"371\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCHARACTERISTIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRESULT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eColony morphology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLarge, mucoid, milky-white to pale yellow\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGram reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGram-negative\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell shape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCoccoid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIndole production\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNegative\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMethyl Red\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNegative\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVoges-Proskauer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCitrate utilization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGlucose fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcid + Gas\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLactose fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcid + Gas\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSucrose fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcid + Gas\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMannitol fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcid + Gas\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTriple Sugar Iron\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcid slant/Acid butt (A/A)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUrease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNegative\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eOptimisation of culture conditions for ascorbic acid production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003epH optimisation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe influence of pH on ascorbic acid biosynthesis was evaluated over a pH range of 4.0-7.0 (Fig. 3). Maximum production occurred at pH 4.0 and 6.0, representing optimal conditions for bacterial growth and enzymatic activity. Production declined substantially at pH values below 4.0 and above 7.0, reflecting the acidophilic nature of \u003cem\u003eAcetobacter\u003c/em\u003e species and the optimal activity range of key biosynthetic enzymes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature optimisation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTemperature optimisation revealed maximum ascorbic acid production at 30 \u0026deg;C, with significantly lower yields at 4 \u0026deg;C and 40 \u0026deg;C (Fig. 4). This optimal temperature represents a balance between metabolic rate and enzyme stability characteristic of mesophilic \u003cem\u003eAcetobacter\u003c/em\u003e species. Suboptimal temperatures (4 \u0026deg;C) reduced enzymatic activity, whereas 40 \u0026deg;C led to enzyme denaturation and cellular stress, limiting ascorbic acid biosynthesis\u003c/p\u003e\n\u003cp\u003eThe isolation, characterisation, and optimisation studies established optimal conditions (pH 4.0-6.0, 30 \u0026deg;C) for ascorbic acid production by \u003cem\u003eAcetobacter\u0026nbsp;\u003c/em\u003estrains, providing parameters for subsequent fed-batch fermentation and nanoparticle encapsulation applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiosynthesis and analytical confirmation of ascorbic acid\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFed -batch fermentation production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFed-batch fermentation using Acetobacter species with sorbitol as the carbon source achieved approximately 99 % substrate conversion after 96 hours. Ascorbic acid presence and concentration in the fermentation broth were confirmed through thin-layer chromatography, iodometric titration, and DCPIP dye reduction assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQualitative identification by thin-layer chromatography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThin-layer chromatography on silica gel using a chloroform-ethanol-acetone-ammonium hydroxide mobile phase confirmed the presence of ascorbic acid in the fermentation medium. The retention factor (Rf) was calculated using the standard formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"65\" height=\"18\" src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1773327287.png\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere hx represents the distance travelled by the substance from the origin (1.7 mm), and h\u003csub\u003e0\u0026nbsp;\u003c/sub\u003erepresents the distance travelled by the solvent front from the origin(2.5 mm)\u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRf = 1.7 / 2.5 = 0.68\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe calculated retention factor\u0026nbsp;(Rf = 1.7/2.5 = 0.68) corresponded to ascorbic acid reference standards (Table 2). Under UV light at 254 nm, the separated compound exhibited characteristic blue fluorescence (Fig. 5), confirming the identification of ascorbic acid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Reference Retention Factors and UV Fluorescence of Vitamins\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"425\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVitamin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCommon Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRf Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eColour Under UV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB₁\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThiamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eViolet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB₂\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eRiboflavin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eYellow\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB₃\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNiacin (Nicotinic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eViolet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB₆\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePyridoxine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eBlue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAscorbic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026lt;0.1*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eBlue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e*Reference value from standard vitamin chromatography protocols\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative analysis by iodometric titration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAscorbic acid content was determined by iodometric titration based on its oxidation to dehydroascorbic acid. The blue extract from TLC analysis (1g) was dissolved in 5mL of distilled water and titrated with standardised iodine solution in duplicate, showing reproducible results (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Iodometric Titration Results\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"430\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eS. No\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVolume of the\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Burette reading (ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVolume of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIodine(mL)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Initial\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Final\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 5 ml\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 5 ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.0\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 1.25\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1.25\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;2.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIodometric titration of 5mL samples consumed an average of 1.9mL iodine solution (11.4 mL for 30 mL total volume). The endpoint was identified by the formation of a blue-black starch-iodine complex, indicating complete oxidation of ascorbic acid and confirming its presence and reducing capacity in the fermentation medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation by 2,6-Dichlorophenolindophenol (DCPIP) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAscorbic acid quantification employed DCPIP dye reduction, based on the conversion of blue DCPIP to its colourless form. Following standardisation with ascorbic acid in the fermentation media, the endpoint was determined by a stable pink colour (\u0026ge;15 seconds). Titration of 5 mL samples consumed 2.8 mL DCPIP, corresponding to 16.8 mL for the complete 30 mL volume.\u003c/p\u003e\n\u003cp\u003eThree methods, TLC, iodometric titration, and DCPIP reduction, collectively confirmed ascorbic acid biosynthesis. TLC established identity via Rf values and UV response, while titrimetric approaches quantified concentration. Consistent findings across techniques validate the \u003cem\u003eAcetobacter-\u003c/em\u003emediated biotransformation of sorbitol to ascorbic acid as a sustainable production strategy, supporting future scale-up for nanoparticle encapsulation and food preservation applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis and characterisation of ascorbic acid-coated iron oxide nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIron oxide nanoparticle synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIron oxide nanoparticles were synthesised via coprecipitation using FeCl₃ and FeSO₄ as iron sources with ammonium hydroxide as the precipitating agent. This method produced black magnetite precipitates characteristic of Fe₃O₄ (Fig. 6), yielding magnetically responsive nanoparticles under ambient conditions for subsequent functionalization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAscorbic acid encapsulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIron oxide nanoparticles were surface-modified with ascorbic acid through pH and temperature-controlled encapsulation. The magnetic dispersion was adjusted to pH 12.5 with 10 N ammonia to optimise surface charge for ascorbic acid adsorption. Ascorbic acid (0.5 g) was added to the alkaline dispersion, and the mixture was heated at 80 \u0026deg;C with stirring for 1 hour to promote interaction between the ascorbic acid functional groups and the hydroxylated iron oxide surface. The coated nanoparticles were recovered by oven drying; the colour change indicated successful functionalization, reflecting the combined optical properties of the iron oxide core and the organic coating.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological characterisation by SEM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSEM analysis revealed the nanoparticle morphology and coating structure at nanoscale resolution (Fig.8). The micrographs showed densely packed, spherical nanoparticles with a uniform size distribution. Ascorbic acid formed a conformal coating with complete surface coverage, demonstrating successful encapsulation without significant aggregation. The uniform distribution and spherical geometry confirmed the practicality of synthesis and encapsulation. This coating morphology provides structural protection for ascorbic acid while maintaining accessibility for functional applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional validation: Antioxidant activity retention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAscorbic acid bioactivity post-encapsulation was verified using the methylene blue decolourisation assay. The addition of coated nanoparticles to the methylene blue solution resulted in rapid, complete decolourisation within minutes, as the dye reduced to its colourless leuco form. This transition confirmed that the encapsulated ascorbic acid retains its reducing capacity and antioxidant functionality, thereby maintaining both its nutritional value and chemical reactivity, which are essential for antimicrobial activity. The iron oxide core retained its magnetic properties, enabling efficient magnetic separation and recovery, a key advantage for industrial-scale applications. Characterisation results demonstrate successful synthesis of ascorbic acid-coated iron oxide nanoparticles with retained functionality, uniform morphology, and structural properties suitable for food preservation and nutritional fortification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial efficacy and food preservation performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntimicrobial efficacy of biosynthesised ascorbic acid-coated iron oxide nanoparticles was evaluated across six food matrices: apple, orange, grape and banana juices, milk and curd. Viable plate count analysis compared untreated controls, nanoparticle-treated, and commercial ascorbic acid-treated samples \u0026nbsp; (Fig.9, Table 4). Control samples exhibited uncontrolled microbial growth (TNTC) at 10⁻⁶ dilutions, confirming rapid spoilage. Commercial ascorbic acid reduced microbial loads to 2-12 CFU at 10⁻⁸ dilutions, while biosynthesised nanoparticles demonstrated substantial efficacy with 7-20 CFU at 10⁻⁸ dilutions across all tested matrices.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Microbial Colony Counts in Treated and Untreated Food Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSl. No\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFood Sample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDilution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl (CFU)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNano-encapsulated Ascorbic Acid (CFU)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCommercial Ascorbic Acid\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(CFU)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRemark\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 35px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eApple juice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁷\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 35px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eGrape juice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁷\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 35px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eOrange juice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁷\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 35px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eBanana juice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁷\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 35px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eMilk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁷\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 35px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eCurd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁷\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10⁻⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTNTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eHighly effective\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTNTC = Too Numerous To count. Nano-encapsulated ascorbic acid consistently reduced bacterial counts from TNTC to countable levels across all samples, demonstrating effective antimicrobial activity compared to the control (7-20 CFU).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative assessment of preservation efficacy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparative analysis revealed that biosynthesised ascorbic acid-coated nanoparticles achieved approximately 75% of the antimicrobial activity of commercial ascorbic acid across all tested food matrices, evidenced by consistently higher colony counts at equivalent dilutions. Despite this moderate reduction in potency, the nanoparticle formulation effectively suppressed microbial growth by several orders of magnitude compared with untreated controls, demonstrating its viability as a functional preservative. Antimicrobial performance varied across food matrices: banana juice exhibited the lowest colony count (7 CFU at 10⁻⁸ ), while curd showed the highest (20 CFU at 10⁻⁶; 12 CFU at 10⁻⁸). These matrix-dependent differences reflect the influence of intrinsic food properties, including pH, nutrient composition, initial microbial load, and physicochemical characteristics, on preservation efficacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual functionality: Preservation and nutritional fortification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBeyond antimicrobial efficacy, the nanoparticle formulation demonstrated dual functional potential by simultaneously providing nutritional fortification. The iron oxide nanoparticle core serves as a bioavailable source of iron to address micronutrient deficiency, while the ascorbic acid coating provides vitamin C supplementation. This integrated approach delivers both preservation and nutritional enhancement within a single additive system, representing a significant advancement over conventional single-purpose preservatives. The controlled release characteristics imparted by nanoencapsulation offer additional functional advantages. The gradual liberation of ascorbic acid from the nanoparticle matrix potentially extends the effective preservation duration compared to free ascorbic acid, which may be rapidly consumed through oxidation or degradation reactions in food systems. Furthermore, the biosynthetic origin of the formulation aligns with consumer preferences for natural, clean-label ingredients while maintaining compatibility with existing food processing infrastructure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFood matrix-specific performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of preservation efficacy across different food categories revealed product-specific performance patterns. In fruit juices, acidic pH naturally complements the antimicrobial action of ascorbic acid, with all juice types showing effective microbial suppression. Orange and apple juices demonstrated intermediate colony counts (9-15 CFU at 10⁻⁶), while banana juice exhibited the most effective suppression at higher dilutions. In dairy products, milk showed moderate colony counts (8-12 CFU across dilutions), while curd exhibited slightly elevated counts (12-20 CFU), potentially attributable to its inherent fermented nature and higher initial microbial populations. These results establish proof of concept for biosynthesised ascorbic acid-coated iron oxide nanoparticles as multifunctional food additives that extend shelf life while simultaneously enhancing nutritional value. The formulation demonstrates practical applicability across diverse food matrices, with performance levels suitable for commercial food preservation applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUTURE SCOPE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe development of biosynthesised ascorbic acid-coated oxide nanoparticles provides a foundation for future commercial translation. Priority research areas include optimising encapsulation through advanced characterisation (TEM, XRD, FTIR), scaling production via continuous fermentation and automated reactors, and establishing stability profiles under storage and processing conditions. Food matrix-specific formulations require investigation across beverages, dairy, high-fat products, and bakery applications to maximise functionality while minimising sensory impacts. Rigorous toxicological evaluation, following guidelines from the FDA, EFSA, and FSSAI, including acute, chronic, genotoxicity, and reproductive toxicity studies, is essential for regulatory approval, alongside sensory evaluation and consumer acceptance testing. The nano-encapsulation platform offers opportunities to extend the shelf life of other unstable bioactives, such as B-complex vitamins, vitamin E, polyphenols, omega-3 fatty acids, and natural antimicrobials. Stimuli\u0026ndash;responsive coatings triggered by pH, temperature, or microbial presence could enable intelligent preservation systems. Human bioavailability studies would validate the nutritional benefits, particularly the synergistic effects of combined vitamin C and iron supplementation in populations prone to deficiency. Life cycle analysis, waste-stream behaviour assessment, and the development of biodegradable alternatives would address environmental stability. Integration with active packaging technologies could create comprehensive preservation systems, transforming this proof of concept into a commercially viable, regulatory-compliant solution for the industry.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study developed an integrated bioprocess for producing nano-encapsulated ascorbic acid, a dual-functional food additive for preservation and nutritional fortification. Ascorbic acid-producing \u003cem\u003eAcetobacter\u003c/em\u003e strains isolated from natural sources were characterised through morphological and biochemical analyses. Fermentation optimisation identified pH 4.0\u0026ndash;6.0 and 30\u0026deg;C as optimal conditions, achieving 99% substrate conversion within 96 hours. Biosynthesis was validated via thin-layer chromatography (Rf 0.68), iodometric titration, and DCPIP assay. Iron oxide nanoparticles synthesised by co-precipitation were functionalised with biosynthesised ascorbic acid, producing a uniform coating architecture, as confirmed by SEM. Preservation testing across fruit juices and dairy products showed nano-encapsulated formulations achieved approximately 75% antimicrobial activity relative to commercial ascorbic acid, reducing microbial loads from uncountable levels to 7\u0026ndash;20 CFU at 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e dilutions while providing vitamin C and iron fortification. The approach offers advantages in sustainable biological production, enhanced stability through nanoencapsulation, dual micronutrient fortification, and multimodal antimicrobial action. Successful translation from the laboratory to functional food applications demonstrates commercial viability through scalable fermentation and straightforward nanoparticle synthesis, applicable across diverse food matrices. Future work should optimise encapsulation efficacy, assess long-term stability and safety, evaluate sensory properties, and develop industrial-scale protocols. This proof of fortification agent aligns with clean-label trends and sustainability requirements.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e% Percentage\u003c/p\u003e\n\u003cp\u003eA/A Acid slant and Acid butt\u003c/p\u003e\n\u003cp\u003eBHA Butylated Hydroxyanisole\u003c/p\u003e\n\u003cp\u003eBHT Butylated Hydroxytoluene\u003c/p\u003e\n\u003cp\u003eCFU Colony Forming Unit\u003c/p\u003e\n\u003cp\u003eDCPIP 2,6 \u0026ndash; Dichlorophenolindophenol\u003c/p\u003e\n\u003cp\u003eDNA Deoxyribonucleic Acid\u003c/p\u003e\n\u003cp\u003eEFSA European Food Safety Authority\u003c/p\u003e\n\u003cp\u003eet al. et alii (and others)\u003c/p\u003e\n\u003cp\u003eFAO Food and Agriculture Organisation\u003c/p\u003e\n\u003cp\u003eFDA Food and Drug Administration\u003c/p\u003e\n\u003cp\u003eFSSAI Food Safety and Standards Authority of India\u003c/p\u003e\n\u003cp\u003eFTIR Fourier Transform Infrared spectroscopy\u003c/p\u003e\n\u003cp\u003eGYC Glucose Yeast Calcium carbonate\u003c/p\u003e\n\u003cp\u003eGYP Glucose Yeast Peptone\u003c/p\u003e\n\u003cp\u003eIMViC \u003cstrong\u003eI\u003c/strong\u003endole, \u003cstrong\u003eM\u003c/strong\u003eethyl Red, \u003cstrong\u003eV\u003c/strong\u003eoges-Proskauer, and \u003cstrong\u003eC\u003c/strong\u003eitrate\u003c/p\u003e\n\u003cp\u003enm Nanometres\u003c/p\u003e\n\u003cp\u003epH Potential of Hydrogen\u003c/p\u003e\n\u003cp\u003eRf Retention factor\u003c/p\u003e\n\u003cp\u003erpm Rotation per Minute\u003c/p\u003e\n\u003cp\u003eSEM Scanning Electron Microscopy\u003c/p\u003e\n\u003cp\u003eTBHQ Tert-butylhydroquinone\u003c/p\u003e\n\u003cp\u003eTEM Transmission Electron Microscopy\u003c/p\u003e\n\u003cp\u003eTLC Thin-Layer Chromatography\u003c/p\u003e\n\u003cp\u003eTNTC Too Numerous To Count\u003c/p\u003e\n\u003cp\u003eU.S. United States\u003c/p\u003e\n\u003cp\u003eUV Ultraviolet\u003c/p\u003e\n\u003cp\u003ew/v weight/volume\u003c/p\u003e\n\u003cp\u003ew/w weight/weight\u003c/p\u003e\n\u003cp\u003eXRD X-ray diffraction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to extend their heartfelt appreciation to the \u003cstrong\u003eDST-FIST\u003c/strong\u003e scheme, the Management, and the Department of Microbiology at PSG College of Arts and Science, Coimbatore, for their support and valuable assistance throughout the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONTRIBUTION OF AUTHORS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCatherine Antony: Conceptualisation, Methodology, Experimental design, Data handling, Writing - original draft and editing\u003c/p\u003e\n\u003cp\u003eDr Krishnaveni N: Validation, supervision and critical review\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated and analysed during the study are included in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCENT TO PUBLISH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhmed, T. M. K., Bakr, M. M., \u0026amp; Ahmed, Q. A. (2022). \u003cem\u003eSide Effects of Preservatives on Human Life\u003c/em\u003e. \u003cem\u003e2\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eAkasaka, K. (2013). Simple Determination of l-Ascorbic Acid on TLC by Visual Detection Using Autocatalytic Reaction. \u003cem\u003eAnalytical Sciences\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e(5), 505\u0026ndash;509. https://doi.org/10.2116/analsci.29.505\u003c/li\u003e\n \u003cli\u003eAlberts, A., Moldoveanu, E.-T., Niculescu, A.-G., \u0026amp; Grumezescu, A. M. (2025). Vitamin C: A Comprehensive Review of Its Role in Health, Disease Prevention, and Therapeutic Potential. \u003cem\u003eMolecules\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e(3), 748. https://doi.org/10.3390/molecules30030748\u003c/li\u003e\n \u003cli\u003eAntony, C., \u0026amp; Narayanaswamy, K. (2026). Food preservatives: Natural or synthetic? \u003cem\u003eArchives of Microbiology\u003c/em\u003e, \u003cem\u003e208\u003c/em\u003e(3), 134. https://doi.org/10.1007/s00203-025-04674-9\u003c/li\u003e\n \u003cli\u003eAswini, K., Gopal, N. O., \u0026amp; Uthandi, S. (2020). Optimized culture conditions for bacterial cellulose production by Acetobacter senegalensis MA1. \u003cem\u003eBMC Biotechnology\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(1), 46. https://doi.org/10.1186/s12896-020-00639-6\u003c/li\u003e\n \u003cli\u003eBarrett, C. B. (2021). Overcoming Global Food Security Challenges through Science and Solidarity. \u003cem\u003eAmerican Journal of Agricultural Economics\u003c/em\u003e, \u003cem\u003e103\u003c/em\u003e(2), 422\u0026ndash;447. https://doi.org/10.1111/ajae.12160\u003c/li\u003e\n \u003cli\u003eBelete, A., Yisak, H., Chandravanshi, B. S., \u0026amp; Yaya, E. E. (2023). Ascorbic acid content and the antioxidant activity of common fruits commercially available in Addis Ababa, Ethiopia. \u003cem\u003eBulletin of the Chemical Society of Ethiopia\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(2), 277\u0026ndash;288. https://doi.org/10.4314/bcse.v37i2.3\u003c/li\u003e\n \u003cli\u003eKumari, A., \u0026amp; Chauhan, A. K. (2022a). Iron nanoparticles as a promising compound for food fortification in iron deficiency anemia: A review. \u003cem\u003eJournal of Food Science and Technology\u003c/em\u003e, \u003cem\u003e59\u003c/em\u003e(9), 3319\u0026ndash;3335. https://doi.org/10.1007/s13197-021-05184-4\u003c/li\u003e\n \u003cli\u003eKumari, A., \u0026amp; Chauhan, A. K. (2022b). Iron nanoparticles as a promising compound for food fortification in iron deficiency anemia: A review. \u003cem\u003eJournal of Food Science and Technology\u003c/em\u003e, \u003cem\u003e59\u003c/em\u003e(9), 3319\u0026ndash;3335. https://doi.org/10.1007/s13197-021-05184-4\u003c/li\u003e\n \u003cli\u003eLee, D.-H., Kim, S.-H., Lee, C.-Y., Jo, H.-W., Lee, W.-H., Kim, E.-H., Choi, B.-K., \u0026amp; Huh, C.-K. (2024). Screening of Acetic Acid Bacteria Isolated from Various Sources for Use in Kombucha Production. \u003cem\u003eFermentation\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(1), 18. https://doi.org/10.3390/fermentation10010018\u003c/li\u003e\n \u003cli\u003eL\u0026rsquo;Haridon, S., Toffin, L., \u0026amp; Roussel, E. (2020). Methanococcoides. In M. E. Trujillo, S. Dedysh, P. DeVos, B. Hedlund, P. K\u0026auml;mpfer, F. A. Rainey, \u0026amp; W. B. Whitman (Eds.), \u003cem\u003eBergey\u0026rsquo;s Manual of Systematics of Archaea and Bacteria\u003c/em\u003e (1st ed., pp. 1\u0026ndash;9). Wiley. https://doi.org/10.1002/9781118960608.gbm00514.pub2\u003c/li\u003e\n \u003cli\u003eLisboa, H. M., Pasquali, M. B., dos Anjos, A. I., Sarinho, A. M., de Melo, E. D., Andrade, R., Batista, L., Lima, J., Diniz, Y., \u0026amp; Barros, A. (2024). Innovative and Sustainable Food Preservation Techniques: Enhancing Food Quality, Safety, and Environmental Sustainability. \u003cem\u003eSustainability\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(18), Article 18. https://doi.org/10.3390/su16188223\u003c/li\u003e\n \u003cli\u003eMahmud, N., Nasser, M. S., El-Naas, M. H., Ba-Abbad, M. M., Wahab Mohammad, A., Mansour, S., \u0026amp; Benamor, A. (2020). Synthesis and Characterization of Fe3O4 Nanoparticles Using Different Experimental Methods. \u003cem\u003eIOP Conference Series: Materials Science and Engineering\u003c/em\u003e, \u003cem\u003e778\u003c/em\u003e(1), 012028. https://doi.org/10.1088/1757-899X/778/1/012028\u003c/li\u003e\n \u003cli\u003eMalik, M., Narwal, V., \u0026amp; Pundir, C. S. (2022). Ascorbic acid biosensing methods: A review. \u003cem\u003eProcess Biochemistry\u003c/em\u003e, \u003cem\u003e118\u003c/em\u003e, 11\u0026ndash;23. https://doi.org/10.1016/j.procbio.2022.03.028\u003c/li\u003e\n \u003cli\u003eMehta, J., Pathania, K., \u0026amp; Pawar, S. V. (2025). Recent overview of nanotechnology based approaches for targeted delivery of nutraceuticals. \u003cem\u003eSustainable Food Technology\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(4), 947\u0026ndash;978. https://doi.org/10.1039/D5FB00122F\u003c/li\u003e\n \u003cli\u003eNakamura, H., Hattori, D., Tokunaga, D., \u0026amp; Suzuki, Y. (2013). An isothermal absorptiometric assay for viable microbes using the redox color indicator 2,6-dichlorophenolindophenol. \u003cem\u003eAnalytical Biochemistry\u003c/em\u003e, \u003cem\u003e441\u003c/em\u003e(2), 140\u0026ndash;146. https://doi.org/10.1016/j.ab.2013.07.010\u003c/li\u003e\n \u003cli\u003eRahman, M., Uddin, M. B., Aziz, M. G., Abunaser, M., Haque, M. R., \u0026amp; Siddiki, M. S. R. (2024). Isolation and Characterization of Acetic Acid Bacteria from Pineapple, Sugarcane, Apple, Grape, Pomegranate, and Papaya Fruit. \u003cem\u003eEuropean Journal of Agriculture and Food Sciences\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(2), 14\u0026ndash;18. https://doi.org/10.24018/ejfood.2024.6.2.772\u003c/li\u003e\n \u003cli\u003eSahoo, B. K., Mishra, R. R., \u0026amp; Behera, B. C. (2020). Isolation and Identification of Thermotolerent Acetic Acid Bacteria from Waste Fruits. \u003cem\u003eAsian Journal of Biological and Life Sciences\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(2), 209\u0026ndash;213. https://doi.org/10.5530/ajbls.2020.9.32\u003c/li\u003e\n \u003cli\u003eSee, X. Z., Yeo, W. S., \u0026amp; Saptoro, A. (2024). A comprehensive review and recent advances of vitamin C: Overview, functions, sources, applications, market survey and processes. \u003cem\u003eChemical Engineering Research and Design\u003c/em\u003e, \u003cem\u003e206\u003c/em\u003e, 108\u0026ndash;129. https://doi.org/10.1016/j.cherd.2024.04.048\u003c/li\u003e\n \u003cli\u003eTeshome, E., Forsido, S. F., Rupasinghe, H. P. V., \u0026amp; Olika Keyata, E. (2022). Potentials of Natural Preservatives to Enhance Food Safety and Shelf Life: A Review. \u003cem\u003eThe Scientific World Journal\u003c/em\u003e, \u003cem\u003e2022\u003c/em\u003e(1), 9901018. https://doi.org/10.1155/2022/9901018\u003c/li\u003e\n \u003cli\u003eTissera, C. E., Barnetche, M. E., Silva, O. F., \u0026amp; Fern\u0026aacute;ndez, M. A. (2025). Increasing the stability of ascorbic acid through encapsulation in food-grade vesicles: An approach for nutritional improvement. \u003cem\u003eInternational Journal of Food Science and Technology\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e(1), vvae027. https://doi.org/10.1093/ijfood/vvae027\u003c/li\u003e\n \u003cli\u003eTucaliuc, A., C\u0026icirc;șlaru, A., Kloetzer, L., \u0026amp; Blaga, A. C. (2022). Strain Development, Substrate Utilization, and Downstream Purification of Vitamin C. \u003cem\u003eProcesses\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(8), 1595. https://doi.org/10.3390/pr10081595\u003c/li\u003e\n \u003cli\u003eVesković, S. (2025). In the Global Food System: Addressing Food Losses, Waste, and Safety. In S. Veskovic (Ed.), \u003cem\u003eNatural Food Preservation: Controlling Loss, Advancing Safety\u003c/em\u003e (pp. 5\u0026ndash;58). Springer Nature Switzerland. https://doi.org/10.1007/978-3-031-85089-9_2\u003c/li\u003e\n \u003cli\u003eWaseem, S. Mohd., Gaikwad, S. V., Pandit, V. A., \u0026amp; Kapse, N. N. (2025). Iron Oxide Nanoparticles for Catalytic Applications: Synthesis, Characterization, and Environmental Performance. \u003cem\u003eTopics in Catalysis\u003c/em\u003e. https://doi.org/10.1007/s11244-025-02204-x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nano-encapsulation, Biosynthesised Ascorbic acid, Iron oxide nanoparticles, Natural food preservative, dual fortification, Acetobacter fermentation, Clean label technology","lastPublishedDoi":"10.21203/rs.3.rs-8801688/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8801688/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study highlights the development of nano-encapsulated ascorbic acid delivery systems to address challenges in food preservation and nutritional fortification. Ascorbic acid-producing \u003cem\u003eAcetobacter\u003c/em\u003e strains isolated from natural sources were characterised and optimised for fermentation at pH 4\u0026ndash;6 and 30\u0026deg;C, achieving 99% substrate conversion within 96 hours. Iron oxide nanoparticles synthesised via coprecipitation were functionalized with biosynthesised ascorbic acid through pH-controlled coating (pH 12.5, 80\u003csup\u003e0\u003c/sup\u003eC, 1 hour). Scanning electron microscopy confirmed uniform coating architecture. Preservation efficacy was tested in fruit juices (apple, grape, orange, and banana) and dairy products (milk and curd), demonstrating that nano-encapsulated formulations achieved approximately 75% antimicrobial activity compared to commercial ascorbic acid. This reduction in microbial loads, from previously uncountable levels to 7\u0026ndash;20 CFU at 10^ (-8) dilutions, was notable. The dual-functional vitamin C and iron fortification, and clean label compliance, are due to their biosynthetic origin. This integrated biotechnology-nanotechnology platform establishes proof of concept for next-generation multifunctional food additives that extend shelf life while enhancing nutritional value, providing viable solutions for food industries pursuing natural preservation strategies.\u003c/p\u003e","manuscriptTitle":"Nano-armoured Vitamin C: A novel approach to micronutrient delivery and natural food preservation in integrated food systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 15:03:01","doi":"10.21203/rs.3.rs-8801688/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"11542366286763815109207468400259036790","date":"2026-05-05T04:27:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-08T23:46:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191297454053685271749422715969558505831","date":"2026-03-31T16:07:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-09T15:40:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-01T12:55:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T00:41:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-16T00:41:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Nano","date":"2026-02-06T01:51:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"02f48fad-39f3-4063-b771-96e1a6d8722a","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"11542366286763815109207468400259036790","date":"2026-05-05T04:27:05+00:00","index":34,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-12T15:03:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 15:03:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8801688","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8801688","identity":"rs-8801688","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}