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Frontiers in Food Science and Technology ·

Volume 6 - 2026 | https://doi.org/10.3389/frfst.2026.1850082

Formulation and in vitro prospectus of probiotics, beetroot, and vitamin B12 encapsulated in alginate beads

  • Tanhid Mansuri1
  • Rajesh Hadia2
  • Annu3*corresponding author
  • Samaresh Pal Roy1
  • Aayush Panchal4
  1. 1Department of Pharmaceutics, Sardar Patel College of Pharmacy, Anand, Gujarat, India
  2. 2Department of Pharmacy, Sumandeep Vidyapeeth University, Vadodara, Gujarat, India
  3. 3School of Pharmaceutical Sciences, Shri Guru Ram Rai University, Dehradun, India
  4. 4School of Life and Medical Sciences, University of Hertfordshire, Hatfield, United Kingdom
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Abstract

The current research aims to design and develop encapsulated beads (Ebs) loaded with Lactobacillus reuteri (L. reuteri), beetroot, and Vitamin B12. The Ebs were prepared using the ionic gelation method and optimized via factorial design. The prepared Ebs were optimized for their particle size, drug entrapment efficiency, and structural analysis. Moreover, the Ebs were evaluated for their drug release under gastric and intestinal conditions. The particle size of Ebs ranges from 1.15 to 1.20 mm. The % drug entrapment for Vitamin B12, Beetroot, and L. reuteri were 97.09% ± 3.25%, 87.9% ± 5.85%, and 87.5% ± 4.97%, respectively. The viability of L. reuteri in Ebs was 4.2 × 108 Cfu/g. SEM images revealed round-shaped Ebs. FTIR spectra revealed the successful incorporation of drugs within the Ebs. The cumulative release of Vitamin B12, Beetroot, and L. reuteri from Ebs was 87.02% ± 5.67%, 78.4% ± 7.87%, and 87.5% ± 6.68%, respectively, over 24 h, indicating sustained release profiles. In contrast, the marketed sublingual formulation of Vitamin B12 showed a rapid release of 70.23% ± 5.68%, within 2 h. Results confirm the successful preparation and optimization of L. reuteri - Vitamin B12 and beetroot-loaded Ebs, which may be a promising approach to addressing Vitamin B12 deficiency.

Keywords

1 Introduction

Vitamin B12 is a vital water-soluble micronutrient, predominantly found in foods like meat, fish, eggs, and dairy products (animal-derived products) (Akbari et al., 2022). Despite fortification efforts in plant-based products, the deficiency of Vit B12 remains a significant health issue, contributing to megaloblastic anemia, neurocognitive impairments, and systemic health deterioration (Katouzian and Jafari, 2016; Siddiq and Pascall, 2018). In India, deficiency rates exceed 75%, largely attributed to the fact that over 650 million people are deficient in Vitamin B12 due to vegetarian dietary patterns, cultural practices, and contributing lifestyle factors such as smoking, alcohol consumption, and chronic antacid use (Allen, 2009). The highest prevalence of Vit B12 deficiency is observed among individuals aged 21–40 years (28.5%), with a greater burden in females (29.7%) than in males (19.2%) (Gupta et al., 2024).

According to the European Food Safety Authority (EFSA Panel on Dietetic ProductsNutrition and Allergies, 2015), the recommended dietary intake of vitamin B12 is 4.0 μg per day for adults, while the U.S. National Academy of Medicine advises a slightly lower intake of 2.5 μg per day (Institute of Medicine (US) Panel on Dietary Antioxidants and Related Compounds, 2000). A typical Western diet provides between 4 and 7 μg of Vit B12 daily, which is generally sufficient to maintain normal physiological functions in healthy individuals (Bor et al., 2010; Baart et al., 2021). During pregnancy and lactation, the daily requirement increases by approximately 0.5–1.0 μg to support maternal health and fetal development (EFSA Panel on Dietetic ProductsNutrition and Allergies, 2015). Clinical evidence indicates that both oral supplementation of 1000 μg per day and intramuscular administration involving nine injections over 3 months are equally effective in correcting Vit B12 deficiency in otherwise healthy individuals (Castelli et al., 2011).

Vit B12 deficiency commonly arises from inadequate intake, especially among vegetarians and vegans due to limited dietary sources (Júnior et al., 2022). Malabsorption is another major cause, often linked to pernicious anemia, gastrointestinal disorders, or reduced gastric acid secretion (Green et al., 2017; Allen, 2009; Stabler, 2013). Food-bound cobalamin malabsorption is now recognized as a leading cause in the elderly (Dali-Youcef and Andres, 2009). Long-term use of medications such as metformin and proton pump inhibitors can also impair absorption and contribute to deficiency, which may be reversible after drug discontinuation (Langan and Goodbred, 2017; Lam et al., 2013; Andrès et al., 2004; De Jager et al., 2010).

Probiotics are live microorganisms that confer beneficial effects on health when consumed in sufficient quantities (Guarner and Schaafsma, 1998; Chen et al., 2015). Effective probiotic strains must withstand gastric acidity, adhere to the intestinal epithelium, and colonize the gut (Hawrelak et al., 2013; Dunne et al., 2001). Lactobacillus reuteri, a gram-positive, facultative anaerobe, is commonly used for its probiotic properties and its ability to synthesize essential vitamins such as B12 and folate (Fujisawa et al., 1992; Santos et al., 2008; Thomas et al., 2016). Vitamin B12 is crucial to produce reuterin, an antimicrobial compound generated from glycerol via a B12-dependent enzyme (Taranto et al., 2003; Sriramulu et al., 2008; Gu et al., 2015). Certain strains, including L. reuteri CRL1098 and JCM1112, have shown potential in B12 biosynthesis and mitigating deficiency (Molina et al., 2009; Morita et al., 2008), although some may produce pseudo-B12 with reduced bioavailability in humans.

Beetroot (Beta vulgaris) is a nutrient-rich source containing proteins, dietary fiber, nitrates, and betalains (Sentkowska and Pyrzynska, 2023; Georgiev et al., 2010). Betalains possess strong antioxidant activity, mitigating oxidative stress (Fu et al., 2020). Microencapsulation is an advanced protective delivery system that enhances the stability, bioavailability, and targeted release of labile bioactives (Dias et al., 2015). It is especially effective in probiotic encapsulation, promoting gastrointestinal survival and controlled intestinal release (Kailasapathy, 2014; Phoem et al., 2015).

Common encapsulating biopolymers such as chitosan, alginate, pectin, gelatin, starch, and whey proteins are favored for their natural origin, biocompatibility, and non-toxicity (Loh and Ting, 2015; Nazzaro et al., 2012). Alginate forms ionic cross-linked hydrogels with Ca2+ ions, offering enhanced structural stability and improved probiotic viability (Martin et al., 2015). Microencapsulation enhances the physicochemical stability of sensitive compounds, masks undesirable organoleptic properties, and enables controlled release under targeted physiological conditions (Meyers et al., 1998; Bakry et al., 2016; Piletti et al., 2019).

The current study is designed to encapsulate Lactobacillus reuteri, Vitamin B12, and beetroot extract within sodium alginate beads to protect against gastric acidity and sustain intestinal release over 24 h, thereby improving Vitamin B12 status and absorption.

2 Materials

Vit B12 was procured from Combitic Global Caplet Pvt. Ltd., Sonipat, India. Lactobacillus reuteri culture was obtained from Meteoric Biopharmaceuticals, India. Fresh beetroot (Beta vulgaris) was collected from the local market. Sodium alginate and calcium chloride dihydrate were supplied by Chemdyes Corporation, Rajkot, India. Acetic acid, sodium hydroxide, and hydrochloric acid were purchased from Chem Think Lab, Ankleshwar, India. MRS agar, peptone, LB broth, and potassium dihydrogen phosphate were obtained from S.D. Fine Chemicals Ltd., Mumbai, India. Tetracycline was obtained from Sisca Research Laboratories Pvt. Ltd., Maharashtra, India; sodium chloride from Krishna-Chem Industry, Vadodara, India; and sodium dihydrogen phosphate from Chemdyes Corporation, Rajkot, India.

2.1 Beetroot extract procurement

Beetroot Extract Preparation: Fresh beetroot (Beta vulgaris, locally cultivated variety) was procured from the local market in Anand, Gujarat, India. The root was washed, peeled, and cut into small pieces. An aqueous extract was prepared by blending 100 g of beetroot with 200 mL of distilled water, then filtering through Whatman No. 1 filter paper. The filtrate was concentrated under reduced pressure at 50 °C using a rotary evaporator (Buchi, Switzerland) and stored at 4 °C until use. Total betalain content was estimated spectrophotometrically at 476 nm. Formulation Composition: Each batch of microcapsules was prepared using 2.5% (w/v) sodium alginate containing: Vitamin B12 at 5 mg/10 mL of polymeric solution, Beetroot extract at 1 mL/10 mL (equivalent to ∼50 mg dry extract), and L. reuteri suspension at 1 × 109 CFU/mL incorporated at 1:1 v/v ratio with the polymeric solution.

2.1.1 Formulation development

2.1.1.1 Probiotic bacteria activation

Freeze-dried Lactobacillus reuteri was reactivated using Luria–Bertani (LB) broth. A total of 0.25 g of LB broth powder was dissolved in 10 mL of distilled water and sterilized by autoclaving at 121 °C for 15 min. After cooling, 0.005 g of L. reuteri powder was aseptically inoculated under laminar airflow and incubated aerobically at 30 °C for 24 h. Subsequent serial dilutions and the pour-plate technique were used for activation. Selective cultivation was performed on MRS agar supplemented with tetracycline (50 μg/mL). For preparation, 3.36 g MRS agar was dissolved in 50 mL of distilled water, autoclaved, and cooled. Then, 0.001 g tetracycline was added aseptically. Plates were poured under sterile conditions and incubated at 30 °C for 48 h (Ganje et al., 2024).

2.1.1.2 Formulation and development of vitamin B12, beetroot, and L. Reutri beads

Ebs were formulated using the ionic gelation method. A uniform polymeric solution was prepared by dissolving sodium alginate (2.5% w/v) in distilled water with continuous magnetic stirring. A minimal amount of acetic acid was added to improve solution stability and homogeneity. Lactobacillus reuteri cells were harvested by centrifugation (10 min) and resuspended in sterile 0.1% peptone water to preserve cell viability. The suspension was homogenized, then vitamin B12 and beetroot extract were incorporated, yielding a uniform bioactive core mixture. This mixture was extruded dropwise into a 5% (w/v) calcium chloride dihydrate solution using a syringe needle, allowing gelation and formation of alginate beads. The cross-linking bath was continuously stirred to ensure uniform bead formation. The microcapsules were then hardened, collected, and washed with 0.1% sterile peptone water to eliminate residual CaCl2. The resulting microcapsules exhibited uniform morphology, mechanical integrity, and favorable encapsulation properties, confirming the effectiveness of the optimized formulation. Ebs are demonstrated in Figure 1.

Encapsulating beads of L.reuteri, vitamin B12, and beetroot.
Figure 1. Encapsulating beads of L.reuteri, vitamin B12, and beetroot.

3 Evaluation of prepared Ebs

3.1 Particle size analysis

The Ebs were evaluated for particle size using a calibrated optical microscope. Optical microscopy was performed using a Labomed CxL compound microscope (Labo America Inc., USA) equipped with a calibrated ocular micrometer. Bead diameter was measured at 40× objective magnification. For each batch, a minimum of 10 beads were randomly selected and measured. The stage micrometer was used to calibrate the ocular scale prior to measurements. Mean diameter and standard deviation were calculated from these measurements (Minghua et al., 2004; Ganje et al., 2024).

3.2 Entrapment efficiency of Ebs

In 100 mL of methanol, Ebs equivalent to 6 mg, was dissolved, and the solution was then analyzed using a UV-Vis spectrophotometer at the λmax of Vit B12. The total content of Vitamin B12 and beetroot extract was quantified. Entrapment efficiency (%EE) was calculated using the following equation (Couto et al., 2023):

% EE = Total drug − Free drug / Total drug × 100

3.2.1 UV-visible method validation for analysis of drug entrapment and In vitro drug release

The key validation parameters are: Specificity: Confirmed by the absence of beetroot extract absorption at 361 nm within the tested concentration range (verified by recording the pure beetroot extract spectrum under the same conditions). Linearity: Calibration curve R 2 = 0.999 over the range 1–20 μg/mL of Vitamin B12 in methanol. Accuracy/Recovery: Spiked recovery experiments showed 98.2% ± 1.4% recovery of known Vitamin B12 concentrations added to dissolved microcapsule matrices. Precision: Intraday %RSD <2.0%; Interday %RSD <2.5% (n = 3). LOD/LOQ: 0.28 μg/mL and 0.85 μg/mL respectively (calculated from signal-to-noise ratio). These validation parameters confirm the suitability of the UV-Vis method for entrapment efficiency.

3.3 Encapsulation yield (L. reuteri)

The prepared Ebs (0.5 g) were dissolved in sodium citrate solution and gently agitated at 30–40 rpm for 30–40 min to ensure complete release of the encapsulated cells. The resulting mixture was then subjected to serial dilution using sterile 0.1% peptone water. Appropriate dilutions were spread onto MRS agar plates and incubated at 37 °C for 24 h. After incubation, the number of viable bacteria was counted and expressed as colony-forming units per gram (CFU/g) of microcapsules. The encapsulation yield was subsequently determined using the following formula (Ganje et al., 2024):

% Yield = Number of viabe cells present in microcapsules / Total number of cells in microcapsules × 100

3.4 Enumeration of viable cells

For cell viability assessment, 0.1 g of alginate Ebs was dispersed in 9.9 mL of 10% sodium citrate dihydrate and vortex-mixed for 10 min to facilitate release of the entrapped microorganisms. The resulting solution was serially diluted, and appropriate dilutions were plated in duplicate on MRS agar plates. These plates were then incubated aerobically at 37 °C for 48 h. Following incubation, the number of viable cells was determined using the standard plate count technique and expressed as colony-forming units per gram (CFU/g) (Ganje et al., 2024).

3.5 Surface characterization using electron microscope

The Ebs samples were fixed to aluminum stubs using double-sided carbon adhesive tape and then sputter-coated with a thin gold layer under vacuum to improve electrical conductivity. The prepared samples were examined using a Scanning Electron Microscope (SEM) operated at an accelerating voltage ranging from 5 to 15 kV. High-magnification images were captured to assess the surface features, morphological appearance, and structural integrity of the microcapsules (Popovic et al., 2021).

3.6 Differential scanning colorimetry (DSC)

DSC analysis was performed using a Perkin Elmer Pyris 6 DSC (Massachusetts, USA) to evaluate the thermal behavior of Vitamin B12, beetroot, and sodium alginate-based Ebs. Samples were sealed in aluminum pans and scanned from 40 °C to 400 °C at a heating rate of 5 °C/min to assess thermal transitions and drug-polymer interactions.

3.7 Fourier transform infrared (FTIR)

FTIR analysis was conducted to identify functional groups and assess molecular interactions between core components (Vitamin B12, beetroot, L. reuteri) and the polymeric matrix. FTIR analysis was performed using a Bruker Alpha II ATR-FTIR Spectrometer (Bruker Optik GmbH, Ettlingen, Germany). Freeze-dried Ebs were mixed with KBr in a 1:100 ratio, then compressed into transparent pellets using a hydraulic press. Spectra were recorded from 4000 to 400 cm-1 at a resolution of 4 cm-1. Characteristic peaks were analyzed to evaluate chemical compatibility and structural integrity of the microcapsule formulation (Popovic et al., 2021).

3.8 Survival of free L. reuteri in simulated gastrointestinal fluid

To assess gastric tolerance, a known concentration of L. reuteri suspension was inoculated into 20 mL of Simulated Gastric Fluid (SGF) and incubated at 37 °C, with shaking at 50 rpm to simulate gastric peristalsis. Separate flasks were used for each time point (5, 60, and 120 min) to prevent cross-contamination. At each interval, 10 µL of the mixture was sampled, serially diluted in 0.1% sterile peptone water, and plated on MRS agar. Plates were incubated anaerobically at 37 °C for 48–72 h. Viability was expressed as CFU/mL, indicating L. reuteri survival under acidic gastric conditions (Huang et al., 2015).

Note: Simulated Gastric Fluid (SGF) was prepared according to the United States Pharmacopeia specifications: 0.1 N hydrochloric acid (pH 1.2) containing pepsin at 3.2 mg/mL (activity: 800 U/mg; Sigma-Aldrich). The SGF was freshly prepared on the day of the experiment and pre-warmed to 37 °C before use. Simulated Intestinal Fluid (SIF) was a phosphate buffer (pH 7.4) containing pancreatin (10 mg/mL). Incubation was maintained at 37 °C with orbital shaking at 50 rpm to simulate peristaltic movement.

3.9 In vitro drug release

A two-phase in vitro dissolution study was conducted to simulate gastrointestinal transit and evaluate the release profile and protective efficiency of microcapsules containing Vitamin B12 and Lactobacillus reuteri. In the gastric phase, microcapsules were placed in 900 mL of 0.1 N HCl and tested using a USP Type II (paddle) apparatus at 37 °C, 50 rpm for 2 h. At intervals (15, 30, 60, 90, 120 min), 5 mL aliquots were withdrawn, filtered, and analyzed via UV-Vis spectrophotometry at 361 nm to quantify Vitamin B12 release. Simultaneously, viable L. reuteri counts were determined via CFU enumeration on MRS agar. After 2 h, microcapsules were rinsed and transferred to the intestinal phase using 900 mL of phosphate buffer (pH 7.4) maintained at 37 °C and 50 rpm. Samples were collected at 150, 180, 210, and 240 min, and analyzed similarly for Vitamin B12 release and bacterial viability. This biphasic system assessed controlled release and probiotic survival under simulated gastrointestinal conditions (Mazzocato et al., 2019).

3.10 Statistical analysis

All experiments were performed in triplicate (n = 3) unless otherwise specified. Data are expressed as mean ± standard deviation, and statistical comparisons between groups were performed using one-way ANOVA with p < 0.05 considered statistically significant.

4 Results and discussion

4.1 Particle size

The mean particle size of the Ebs, measured using an optical microscope, ranged from 1.15 to 1.20 m ± 3.47% (n = 10), consistent with the size range reported by Razavi et al. (2021).

4.2 Entrapment efficiency vitamin B12, beetroot, and L. reuteri

The drug entrapment efficiency (%EE) of Vitamin B12 and beetroot in the Ebs was 97.09% ± 4.87% and 87.9% ± 3.76%, (n = 3) respectively, indicating minimal drug loss and effective polymer encapsulation. The enhanced entrapment may be attributed to the optimized drug-to-polymer ratio, supporting improved therapeutic potential and formulation stability, consistent with findings by Mudgil et al. (2022).

4.3 Enumeration of viable cells

The viable counts of Lactobacillus reuteri Ebs were 4.2 × 10^8 CFU/g, indicating successful entrapment of the probiotic cells within the sodium alginate matrix (Mudgile et al., 2024).

4.4 Surface morphology via scanning electron microscopy (SEM)

SEM analysis revealed spherical Ebs ranging from 1.128 mm to 1.228 mm, which were in close agreement with the particle size measurements obtained by optical microscopy. The Ebs showed no signs of crystallization or particle aggregation, indicating structural integrity and formulation uniformity (Mudgil et al., 2024; Qi et al., 2019). The absence of particle agglomeration indicates effective encapsulation and good physicochemical stability of the formulation. Figure 2 represents the SEM images of Ebs.

Scanning Electron Microscopy (SEM) images showing the surface morphology of the developed formulation. **(A)** Images showing the measurement calipers/measurement lines. **(B)** Corresponding image of the Beads without measurement annotations. **(C)** Images with internal structural features of the Beads. (D) Higher-magnification image showing the Beads’ fine internal texture. **(E)** Magnified image showing internal structural features of the Beads. **(F)** Image of the Beads with measurement calipers indicating the measured dimensions.
Figure 2. Scanning Electron Microscopy (SEM) images showing the surface morphology of the developed formulation. (A) Images showing the measurement calipers/measurement lines. (B) Corresponding image of the Beads without measurement annotations. (C) Images with internal structural features of the Beads. (D) Higher-magnification image showing the Beads’ fine internal texture. (E) Magnified image showing internal structural features of the Beads. (F) Image of the Beads with measurement calipers indicating the measured dimensions.

4.5 Differential Scanning Calorimeter (DSC) of encapsulating beads

Figure 3 shows the DSC thermograms of Ebs and Table 1 shows the thermal transitions of Ebs. . The thermogram revealed distinct endothermic transitions, indicating thermal behavior. The shifted thermal transitions observed in the alginate bead thermogram, relative to those of the pure components, confirm successful polymer–drug interactions and physical encapsulation. The high ΔH value at 148.76 °C suggests a significant phase transition related to the alginate-bioactive composite matrix. These findings are consistent with Bajaj et al. (2020), who reported similar DSC peak shifts in co-encapsulated vitamin B12 and D3 spray-dried systems, in which polymer–drug interactions resulted in transition temperature shifts of 20 °C–30 °C relative to pure-drug peaks. The disappearance of individual drug peaks in the bead thermogram further supports successful encapsulation rather than simple physical mixing (Bajaj et al., 2020).

Differential Scanning Calorimeter (DSC) of microencapsulated beads.
Figure 3. Differential Scanning Calorimeter (DSC) of microencapsulated beads.

TABLE 1. Summary of DSC thermal transitions.

SamplePeak temp. (°C)ΔH (J/g)Assignment
Sodium alginate92.26169.313Moisture desorption/glass transition
Sodium alginate242.96106.893Thermal degradation onset
Vitamin B1279.3774.997Moisture loss
Vitamin B12172.0362.111Melting/decomposition
Vitamin B12355.49264.958Complete degradation
Alginate beads148.762102.819Major melting/phase transition
Alginate beads203.6725.877Secondary transition
Alginate beads219.0435.990Early degradation event

4.6 Fourier transform infrared spectroscopy of encapsulated beads

The FTIR spectrum of Vitamin B12, L. reuteri, and beetroot-loaded microcapsules confirmed the presence of key functional groups from beetroot and encapsulating excipients (Figure 4). A broad peak at 3341.29 cm-1 corresponds to O–H stretching, indicating hydroxyl groups from polysaccharides and pigments. The band at 1604.54 cm-1 is attributed to C=C/C=N stretching of betalain chromophores. Peaks at 1429.13 cm-1 (C–H bending), 1080.11 cm-1, and 1028 cm-1 (C–O stretching) confirm the presence of sodium alginate and beetroot components. The absorption at 821.06 cm-1 suggests aromatic = C–H bending, thereby validating the successful incorporation of the phytochemical into the microencapsulated beads. Table 2 represents the FTIR spectral analysis of the EBs showing absorption bands, frequency ranges, functional groups.

Fourier transform infrared spectroscopy microcapsules.
Figure 4. Fourier transform infrared spectroscopy microcapsules.

TABLE 2. FTIR characteristics of alginate beads.

Observed frequency (cm-1)Standard frequency range (cm-1)Functional group
3341.293200–3550O–H stretching
1604.541580–1640C=C/C=N stretching
1429.131400–1450C–H bending
1080.111050–1100C–O stretching
1028.11000–1050C–O stretching
821.06800–900= C–H out-of-plane bending

4.7 Survival of L. reuteri in Simulated Gastric Fluid

Figure 5 shows the viability of free L. reuteri cells. The initial bacterial load at (pH 1.2) over 120 min was 4.8 × 108 CFU/g. The survival was monitored to assess acid tolerance under gastrointestinal conditions.

Survival of L. reuteri in simulated gastric fluid (SGF).
Figure 5. Survival of L. reuteri in simulated gastric fluid (SGF).

4.8 In vitro drug release study

4.8.1 L. reuteri release at pH 7.4

Dissolution studies demonstrated the controlled release of L. reuteri from microcapsules in intestinal fluid at pH 7.4. At 30 min, 1.2 × 108 CFU/g was released, indicating a gradual release profile. By 24 h, the release reached 4.2 × 108 CFU/g, corresponding to ∼87.5% ± 6.68% release efficiency, confirming sustained release (Figure 6).

Release of L. reuteri in simulated intestinal fluid at pH 7.4 (n = 3).
Figure 6. Release of L. reuteri in simulated intestinal fluid at pH 7.4 (n = 3).

4.8.2 In vitro release of vitamin B12 and beetroot from encapsulated beads

In vitro studies were performed at pH 7.4 (simulated intestinal fluid) to elucidate the release rate, release pattern, and the release mechanism of Vitamin B12 and beetroot from encapsulated beads. The encapsulated beads exhibited an initial release of 22.72% for Vitamin B12% and 13.4% for beetroot in phosphate buffer. The cumulative release reached 87.02% ± 5.67% for Vitamin B12% and 70.40% ± 7.87% for beetroot over 24 h, demonstrating a sustained-release profile. In contrast, the marketed Vitamin B12 sublingual tablet released 70.23% ± 5.68% of the drug within 2 h, indicating rapid drug release (Figure 7). Statistical analysis (n = 3) using one-way ANOVA showed a significant difference (p < 0.01) between the encapsulated beads and the marketed formulation, confirming the superior controlled-release performance of the developed formulation. Furthermore, negligible release of both Vitamin B12 and beetroot was observed at pH 1.2 during the initial 2 h, demonstrating the acid resistance of the alginate beads and their ability to protect the encapsulated compounds under simulated gastric conditions, thereby facilitating targeted intestinal release. These findings are consistent with previous reports by Carlan et al. (2017) and Couto et al. (2023).

%Cumulative release of vitamin B12, beetroot and vitamin B12 sublingual tablet from encapsulated beads (n = 3).
Figure 7. %Cumulative release of vitamin B12, beetroot and vitamin B12 sublingual tablet from encapsulated beads (n = 3).

The cumulative release data for Vitamin B12 and beetroot were fitted to various kinetic models, including zero-order, first-order, Higuchi, and Korsmeyer–Peppas models (Figure 8). Among these, the Korsmeyer–Peppas model provided the best fit (R 2 = 0.992) with a release exponent (n = 0.45), indicating that drug release followed a Fickian diffusion-controlled mechanism through the swollen alginate matrix. This release behavior is consistent with diffusion through hydrated polymer networks and agrees with previous studies on alginate-based Vitamin B12 delivery systems reported by Carlan et al. (2017) and Couto et al. (2023). The cumulative release profiles of Vitamin B12, beetroot, and the marketed Vitamin B12 sublingual tablet are presented in Figure 7 (n = 3).

*In vitro* drug release mechanism of Vitamin B12 from encapsulated beads using zero-order, first-order, higuchi and korsmeyer-peppas model.
Figure 8. In vitro drug release mechanism of Vitamin B12 from encapsulated beads using zero-order, first-order, higuchi and korsmeyer-peppas model.

5 Results and discussion

This study successfully developed and evaluated an alginate-based microencapsulation system containing Vitamin B12, Lactobacillus reuteri, and beetroot extract for targeted delivery. Preformulation investigations, including UV spectroscopy, DSC, and FTIR analyses, confirmed the identity, compatibility, and physicochemical stability of the incorporated bioactive components. Microscopic examination and colony-forming unit (CFU) determination verified the viability and purity of L. reuteri prior to encapsulation. Additional characterization techniques, including DSC, PXRD, DLS, zeta potential, CLSM, moisture content, and water activity, would provide further physicochemical insight.

In vitro release studies revealed sustained release of Vitamin B12, Beetroot, and probiotic. The encapsulated bacteria exhibited enhanced resistance under simulated gastric conditions and efficient release in simulated intestinal fluid. These findings demonstrate that the developed multiparticulate delivery platform effectively preserves bioactive integrity, enhances gastrointestinal protection, and enables controlled intestinal availability, highlighting its potential as a multifunctional formulation to address Vitamin B12 insufficiency and support overall health.

6 Conclusion

The current research showed that incorporating a micronutrient, a probiotic microorganism, and a plant-derived bioactive ingredient into a single delivery platform could be a promising multifunctional nutraceutical approach. This formulation has potential applications in managing Vitamin B12 deficiency and promoting gastrointestinal and immune health. Further in vivo studies and long-term stability evaluations are warranted to establish its clinical efficacy.

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Footnotes

Edited by: Soubhagya Tripathy, Indian Institute of Technology Kharagpur, India

Reviewed by: Ashutosh Rai, Indian Institute of Vegetable Research (ICAR), India

Ammar Altemimi, University of Basrah, Iraq

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

The author(s) declared that financial support was not received for this work and/or its publication.

Data availability

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Author contributions

TM: Writing – original draft, Software, Conceptualization, Methodology, Data curation, Investigation. RH: Formal Analysis, Project administration, Writing – review and editing. Annu: Validation, Project administration, Supervision, Writing – review and editing, Visualization, Funding acquisition, Resources. SR: Writing – review and editing, Visualization, Software. AP: Funding acquisition, Formal Analysis, Writing – review and editing.

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Citation

Mansuri, T., et al. (2026). Formulation and in vitro prospectus of probiotics, beetroot, and vitamin B12 encapsulated in alginate beads. Frontiers in Food Science and Technology, Volume 6. https://doi.org/
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6 - 2026

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Copyright © 2026 Mansuri, Hadia, Annu, Roy and Panchal.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Annu

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