*3.2. Characterization of Vitamin D Nanoemulsion*

The average particle size of the fabricated nanoemulsion was evaluated using Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) via dynamic light scattering (DLS). An ambient temperature of 25 ◦C and scatter angle of 173◦ were set to carry out the measurements. To eliminate the overlapping effects of scattering, a dilution of 500 times was performed with buffer solution of equivalent pH before measurements. A disposable capillary cell of 1 mL was employed for zeta potential measurement.

To analyze morphology, a Morgagni 268D transmission electron microscope (TEM, Fei Electron Optics, Eindhoven, The Netherlands) functioning at 80 kV was employed. On a 400-mesh copper panel coated with carbon foil, one drop of each sample previously diluted 100 times was applied and allowed to dry for two minutes. A negative stain of 2% phosphotungstic acid was then applied to the sample slide and allowed to dry before examination. Observations were conducted at room temperature, using a smaller objective aperture, a lower accelerating voltage of 80 kV, and at increasing magnifications [44].

### *3.3. Stability Study of Vitamin D Nanoemulsion*

The high-performance liquid chromatography (HPLC) analysis was employed for quantification of vitamin D. Waters e2695 separations Module HPLC system (Waters Co., Milford, MA, USA) equipped with autosampler, Waters 2489 dual wavelength UV detector and a column heater was used. The symmetry C8 column by Waters, having 100 Å pore size with 4.6 mm internal diameter, 5 µm particle size, and 250 mm length, was used for the stationary phase and Empower™ software (Version 3, feature release 4) from Waters was used to process and analyze the results.

A primary stock solution of 1 µg/mL was prepared by dissolving vitamin D in HPLC grade methanol. Hence, 50 mg vitamin D was serially diluted to obtain standard solution of desired concentration. The following process parameters were utilized for separation: temperature was maintained at 25 ± 2 ◦C, methanol and HPLC grade water in a ratio of 90:10 (*v*/*v*) were employed as mobile phase, 1.0 mL/min flow rate was set and maintained, sample cooler was set to 15 ◦C, and detection was carried out at 265 nm. The standard solution was analyzed 5 times (*n* = 5) for regression.

Vitamin D nanoemulsion was freshly prepared and stored in light resistant container. Since vitamin D degrades rapidly in the aqueous solution, a comparison was made with the oil solution of vitamin D prepared by mixing a weighed quantity of vitamin D in corn oil. One mL of both samples was diluted to 100 mL using HPLC grade methanol and were then analyzed on day 0, day 15 and day 30.

### *3.4. Soft Confectionary (Nano Gummy) Preparation*

The nanoemulsion based gelled matrix system was developed using gelatin (hydrocolloids), as shown in Figure 1. The matrices were made from a blend of type A and type B gelatin with a bloom strength of 160 g. Different combinations of sweeteners (sorbitol, xylitol, sucralose), citric acid, and flavors (coffee, lime, vanilla, orange, candy) were tested. *Gels* **2022**, *8*, x FOR PEER REVIEW 6 of 16

**Figure 1.** Formulation development process from nano emulsion optimization to gummy formation. **Figure 1.** Formulation development process from nano emulsion optimization to gummy formation.

Briefly, gelatin types A and B in equal quantity were dissolved in a small quantity of water heated to 60 C by stirring at 100 rpm. To this, the previously developed o/w nanoemulsion was added, and the mixture was stirred for 2 min. Sweeteners were then added followed by citric acid, flavoring agent, and corn oil (plasticizer). The influence of corn oil on the matrix properties and product acceptability was studied. The formula of the developed Nano gummy using 10% corn oil is shown in Table 2. Briefly, gelatin types A and B in equal quantity were dissolved in a small quantity of water heated to 60 ◦C by stirring at 100 rpm. To this, the previously developed o/w nanoemulsion was added, and the mixture was stirred for 2 min. Sweeteners were then added followed by citric acid, flavoring agent, and corn oil (plasticizer). The influence of corn oil on the matrix properties and product acceptability was studied. The formula of the developed Nano gummy using 10% corn oil is shown in Table 2.

> Gelatin 10.29 Sorbitol 13.20 Xylitol 30.79 Sucralose 0.38 Citric acid 0.38 Lime flavor 0.66 Corn Oil 10.00

The textural properties of gummy confections were evaluated using two-bite compression tests [45]. Gummies were formulated with different corn oil concentrations (0%, 10%, 20% and 30%) and textural attributes, including hardness and stickiness, were measured. A TA.XT2i Texture Analyzer (Stable Micro Systems, Godalming, UK) was used for the evaluation. All measurements were made using a 5 kg load cell. A cylinder aluminum plate probe with a diameter of 7.5 cm, lubricated using the corn oil was lowered at a speed of 1 mm/s to compress 50% of confectionary jelly cube measuring 10 mm<sup>3</sup> placed on the fixed bottom surface. The equipment was adjusted to zero, trigger force was kept at 0.05 N, and the plate was automatically lowered until the bottom surface of the plate just contacted the table. All the measurements were made in triplicate. Data were obtained and analyzed using Stable Micro System's Exponent software, which evaluates the force–time

Sensory assessment is accomplished by documenting the outcome of the assessor's replies based on the parameters of taste, texture/appearance, aroma, and overall liking after a comprehensive study. A questionnaire was prepared, and sensory assessments were performed using a qualitative 9-point hedonic scale, with 1 being least likely and 9 being most. Participant were given three different gummies, viz. Placebo gummy (nano gummy without vitamin D), Nano gummy, and Marketed gummy (Azveston Healthcare Pvt. Ltd., Bengaluru, India) on Day 1 and again on Day 45 after storage. Volunteers were

curve formed during the compression of the sample [46,47].

**Table 2.** Nano gummy formula with 10 wt. % corn oil.

*3.5. Texture Profile Analysis of Nano Gummy*

*3.6. Sensory Evaluation of Nano Gummy*


**Table 2.** Nano gummy formula with 10 wt. % corn oil.

### *3.5. Texture Profile Analysis of Nano Gummy*

The textural properties of gummy confections were evaluated using two-bite compression tests [45]. Gummies were formulated with different corn oil concentrations (0%, 10%, 20% and 30%) and textural attributes, including hardness and stickiness, were measured. A TA.XT2i Texture Analyzer (Stable Micro Systems, Godalming, UK) was used for the evaluation. All measurements were made using a 5 kg load cell. A cylinder aluminum plate probe with a diameter of 7.5 cm, lubricated using the corn oil was lowered at a speed of 1 mm/s to compress 50% of confectionary jelly cube measuring 10 mm<sup>3</sup> placed on the fixed bottom surface. The equipment was adjusted to zero, trigger force was kept at 0.05 N, and the plate was automatically lowered until the bottom surface of the plate just contacted the table. All the measurements were made in triplicate. Data were obtained and analyzed using Stable Micro System's Exponent software, which evaluates the force–time curve formed during the compression of the sample [46,47].

### *3.6. Sensory Evaluation of Nano Gummy*

Sensory assessment is accomplished by documenting the outcome of the assessor's replies based on the parameters of taste, texture/appearance, aroma, and overall liking after a comprehensive study. A questionnaire was prepared, and sensory assessments were performed using a qualitative 9-point hedonic scale, with 1 being least likely and 9 being most. Participant were given three different gummies, viz. Placebo gummy (nano gummy without vitamin D), Nano gummy, and Marketed gummy (Azveston Healthcare Pvt. Ltd., Bengaluru, India) on Day 1 and again on Day 45 after storage. Volunteers were screened based on inclusion and exclusion criteria and were informed about the study objectives and recruited after providing informed consent. Gender-neutral healthy volunteers who spoke and understood English, aged between 18 and 45 years, and had no medical history or known sensitivities to the materials used in gummy fabrication were eligible to participate. Participants younger than 18 years old, smokers, those having flu or similar manifestation, those who did not speak or comprehend the language of choice, and those who had known sensitivities to the materials used in gummy production were all excluded. Oral hygiene product, food and aerated drinks were restricted 2-h before and throughout the study period. Responses were collected based on the questionnaire, including: How do you like the taste? How do you like the aroma? How do you like the appearance and texture and overall product? One-way ANOVA (regression analysis) and *t*-test with Welch correction, with *p* < 0.05 as significantly different, were used for the evaluation of differences between the gummy and changes over 45 days of storage.

### **4. Results**

### *4.1. Preparation and Optimization of Vitamin D Nanoemulsion*

Based on the previously reported solubility of vitamin D, corn oil was selected as the oily phase. Results of the emulsification study (Figure 2) showed that tween 20 has maximum potential to emulsify corn oil followed by lecithin. The decreasing order of emulsification potential among was as follows: tween 20 (96%) > lecithin (87%) > tween 80 (84%) > tween 60 (84%) > transcutol (72%) > poloxamer 407 (61%) > poloxamer 188 (53%) > span 20 (49%). **4. Results**

uation of differences between the gummy and changes over 45 days of storage.

*Gels* **2022**, *8*, x FOR PEER REVIEW 7 of 16

*4.1. Preparation and Optimization of Vitamin D Nanoemulsion*

From the results obtained, tween 20 and lecithin were selected as surfactant and co-surfactant, respectively, for the preparation of the vitamin D nanoemulsion. > tween 60 (84%) > transcutol (72%) > poloxamer 407 (61%) > poloxamer 188 (53%) > span 20 (49%). From the results obtained, tween 20 and lecithin were selected as surfactant and co-surfactant, respectively, for the preparation of the vitamin D nanoemulsion.

Based on the previously reported solubility of vitamin D, corn oil was selected as the oily phase. Results of the emulsification study (Figure 2) showed that tween 20 has maximum potential to emulsify corn oil followed by lecithin. The decreasing order of emulsification potential among was as follows: tween 20 (96%) > lecithin (87%) > tween 80 (84%)

screened based on inclusion and exclusion criteria and were informed about the study objectives and recruited after providing informed consent. Gender-neutral healthy volunteers who spoke and understood English, aged between 18 and 45 years, and had no medical history or known sensitivities to the materials used in gummy fabrication were eligible to participate. Participants younger than 18 years old, smokers, those having flu or similar manifestation, those who did not speak or comprehend the language of choice, and those who had known sensitivities to the materials used in gummy production were all excluded. Oral hygiene product, food and aerated drinks were restricted 2-h before and throughout the study period. Responses were collected based on the questionnaire, including: How do you like the taste? How do you like the aroma? How do you like the appearance and texture and overall product? One-way ANOVA (regression analysis) and t-test with Welch correction, with *p* < 0.05 as significantly different, were used for the eval-

**Figure 2.** Comparative corn oil emulsification potential of various screened surfactants. **Figure 2.** Comparative corn oil emulsification potential of various screened surfactants.

Data obtained from a grading system based on the visual screening, appearance, and emulsification capacity of formulations prepared using different ratios of Smix and Oil were obtained and are tabulated in Table 1. Data revealed that Smix ratio 2:1 resulted in a clear and stable nanoemulsion. Thus, Smix 2:1 was selected for pseudo ternary analysis to obtain the optimized formulation. The nanoemulsion region is shown by a colored region in the pseudo-ternary phase diagram (Figure 3). From the results, it was evident that 3% corn oil, 3% tween 20 and lecithin in a ratio of 2:1 and 94% water resulted in the optimized vitamin D nanoemulsion utilized for further evaluation. Data obtained from a grading system based on the visual screening, appearance, and emulsification capacity of formulations prepared using different ratios of Smix and Oil were obtained and are tabulated in Table 1. Data revealed that Smix ratio 2:1 resulted in a clear and stable nanoemulsion. Thus, Smix 2:1 was selected for pseudo ternary analysis to obtain the optimized formulation. The nanoemulsion region is shown by a colored region in the pseudo-ternary phase diagram (Figure 3). From the results, it was evident that 3% corn oil, 3% tween 20 and lecithin in a ratio of 2:1 and 94% water resulted in the optimized vitamin D nanoemulsion utilized for further evaluation. *Gels* **2022**, *8*, x FOR PEER REVIEW 8 of 16

**Figure 3.** Pseudo-ternary Phase diagram of optimized nano-emulsion with corn oil, Smix (tween 20 + lecithin; 2:1) and water. **Figure 3.** Pseudo-ternary Phase diagram of optimized nano-emulsion with corn oil, Smix (tween 20 + lecithin; 2:1) and water.

### *4.2. Characterization of Vitamin D Nanoemulsion 4.2. Characterization of Vitamin D Nanoemulsion*

As illustrated in Figure 4B, the average particle size and particle size distribution of the optimized vitamin D nanoemulsion were found to be 118.6 ± 4.3 nm and 0.11 ± 0.30, respectively. The zeta-potential was found to be −27 ± 0.82 mV, indicating that the formed nanoemulsion was stable. As illustrated in Figure 4B, the average particle size and particle size distribution of the optimized vitamin D nanoemulsion were found to be 118.6 ± 4.3 nm and 0.11 ± 0.30, respectively. The zeta-potential was found to be −27 ± 0.82 mV, indicating that the formed nanoemulsion was stable.

the globule, which was not measured using DLS due to difference in principle.

**Figure 4.** (**A**) HI-RES TEM micrograph of NE. (**B**) Particle size distribution of optimized NE.

TEM revealed that the vitamin D nanoemulsion was spherical (Figure 4A). The droplet size measured by TEM (127 ± 7.2 nm) was slightly larger, but not significantly different from that measured by dynamic light scattering (118.6 ± 4.3 nm). The slightly larger size observed in TEM could be due to the measurement of the hydration layer surrounding

+ lecithin; 2:1) and water.

nanoemulsion was stable.

*4.2. Characterization of Vitamin D Nanoemulsion*

**Figure 4.** (**A**) HI-RES TEM micrograph of NE. (**B**) Particle size distribution of optimized NE. **Figure 4.** (**A**) HI-RES TEM micrograph of NE. (**B**) Particle size distribution of optimized NE.

TEM revealed that the vitamin D nanoemulsion was spherical (Figure 4A). The droplet size measured by TEM (127 ± 7.2 nm) was slightly larger, but not significantly different from that measured by dynamic light scattering (118.6 ± 4.3 nm). The slightly larger size observed in TEM could be due to the measurement of the hydration layer surrounding the globule, which was not measured using DLS due to difference in principle.

**Figure 3.** Pseudo-ternary Phase diagram of optimized nano-emulsion with corn oil, Smix (tween 20

As illustrated in Figure 4B, the average particle size and particle size distribution of the optimized vitamin D nanoemulsion were found to be 118.6 ± 4.3 nm and 0.11 ± 0.30, respectively. The zeta-potential was found to be −27 ± 0.82 mV, indicating that the formed

TEM revealed that the vitamin D nanoemulsion was spherical (Figure 4A). The droplet size measured by TEM (127 ± 7.2 nm) was slightly larger, but not significantly different from that measured by dynamic light scattering (118.6 ± 4.3 nm). The slightly larger size observed in TEM could be due to the measurement of the hydration layer surrounding

the globule, which was not measured using DLS due to difference in principle.

### *4.3. Stability Study of Vitamin D Nanoemulsion*

A representative chromatogram of vitamin D is shown in Figure 5A,B. The standard solution of a known concentration of vitamin D (1 µg/mL) was analyzed five times for robustness. The data obtained for the regression plot of vitamin D by HPLC are shown in Table 3.

The linear equation for the calibration plot was used to quantify the concentration of vitamin D on days 0, 15, and 30. Vitamin D in corn oil solution degraded by 5.49% in 15 days and 8.97% in 30 days, whereas the nanoemulsion formulation degraded by 2.94% and 7.63% on day 15 and day 30, respectively (Table 4).


**Table 3.** Average retention area, retention time and peak height of stock solution.

A representative chromatogram of vitamin D is shown in Figure 5A,B. The standard solution of a known concentration of vitamin D (1 μg/mL) was analyzed five times for robustness. The data obtained for the regression plot of vitamin D by HPLC are shown in

**Figure 5.** HPLC Chromatograph of (**A**) Standard solution (**B**) Test sample B. **Figure 5.** HPLC Chromatograph of (**A**) Standard solution (**B**) Test sample B.

**Table 3.** Average retention area, retention time and peak height of stock solution. **Table 4.** Concentration reduction and percent reduction of vitamin D on day 15 and 30.


*4.3. Stability Study of Vitamin D Nanoemulsion*

Table 3.

### 1 4,140,783 7.677 285,363 100 *4.4. Texture Analysis*

Mean 4,136,208.4 7.652 285,315.8 100 Std. Dev 5329.52 0.022 930.21 NA The linear equation for the calibration plot was used to quantify the concentration of vitamin D on days 0, 15, and 30. Vitamin D in corn oil solution degraded by 5.49% in 15 days and 8.97% in 30 days, whereas the nanoemulsion formulation degraded by 2.94% and 7.63% on day 15 and day 30, respectively (Table 4). **Table 4.** Concentration reduction and percent reduction of vitamin D on day 15 and 30. **Test Day Sample A (IU/mL) % Reduction Sample B (IU/mL) % Reduction** Day 0 289,302.01 0 38,282.13 0 Day 15 273,420.01 5.49 37,155.68 2.94 The gummies prepared using different concentrations of corn oil as plasticizer were subjected to texture analysis. The maximum force during the initial compression was used to determine hardness (g) and the negative force with which the probe disconnects from the sample over its way up after compressing was termed as stickiness (g). The mean hardness values and mean stickiness values of each gummy type on day 1, 15, 30, and 45 are shown in Table 5. Gummies without corn oil were softest on day 1 (1540 ± 125 g) but they were extremely soft and difficult to handle and not in the range of an acceptable marketed gummy. Although gummies with 40% corn oil had the lowest stickiness value of the four (76 ± 8 gm on day 45) due the excess of oil present, they were the hardest. As shown in Figure 6, gummies with 10% corn oil had the optimum hardness value (3100 ± 200 g on day 1 and 3600 ± 210 g on day 45), and stickiness was also minimal. It was noted that the gummies prepared with 10% corn oil had excellent hardness and texture up to 30 days and this was retained up to 45 days on further storage.

1 4,130,736 7.663 284,132 100


**Table 5.** (**A**) Mean hardness value (Peak force (g) mean ± STDV) and (**B**) mean stickiness value (negative force (g) mean ± STDV) of each gummy on day 1, 15, 30 and 45.

**Figure 6.** Graphical representation of (**A**) peak hardness cum (**B**) peak stickiness of each gummy on day 1, 15, 30 and 45 and (**C**) force-time curve of 40% corn oil gummy on day 1.

### *4.5. Sensory Analysis*

The Placebo, Nano, and Marketed gummies were compared and the data obtained via sensory evaluation conducted at Day 1 and at Day 45 following storage are presented in Table 6. The highest score for taste was observed for the Marketed gummy. However, the difference between the score compared to placebo and nano gummy was insignificant (*p* > 0.05). Further, storage for a period of 45 days had no detrimental effect on the taste and the scores for all the gummies remained same. The aroma of Placebo and Nano gummies was significantly better than the Marketed gummy (*p* = 0.01; *p* < 0.05). Following 45 days storage, the loss of aroma was observed for all the gummies, but the loss was maximum for the Marketed gummy. Although the appearance and texture of the Marketed gummy was better than Nano gummy (*p* = 0.029; *p* < 0.05), the nano gummy retained both texture and appearance without any significant change (*p* = 0.45) over 45 days of storage. Overall liking of the Nano gummy was comparable to Marketed gummy and was similar on day 1 and day 45 without any significant difference (*p* = 0.4).


**Table 6.** Average scores from the sensory evaluation on Day 1 and day 45.

### **5. Discussion**

The present study explores the potential of a novel nano matrix-based gummy (Nano gummy) to deliver vitamin D. The novel matrix not only prevented the degradation of vitamin D, but also enhances the compliance. Our results suggested that vitamin D was well preserved in O/W nanoemulsion and remained stable over storage. Further, sensory analysis studies suggested that palatability and consumer acceptability were well achieved.

The Nano gummy described in the present work was prepared by a two-step procedure. Firstly, vitamin D loaded nanoemulsions were formed using spontaneous emulsification and aqueous titration procedure. Based on the solubility studies and published literature, corn oil was selected as oil phase. A previous study supported that the in vitro digestibility and bioaccessibility of vitamin D were maximum in long chain triglycerides such as corn oil compared to medium chain triglycerides [34,35]. Studies conducted by Yang et al. [36], Qian et al. [37], and Rao et al. [38] showed similar results where long chain triacylglycerols, viz. corn oil, displayed better bioavailability characteristics of fat-soluble vitamins in contrast to indigestible oils due to the improved solubilization ability of mixed micelles produced by long chain fatty acids [39]. From the results of the emulsification study, tween 20 and lecithin with maximum potential to emulsify corn oil (96% and 87%, respectively) were selected as surfactant and co-surfactant, respectively.

Pseudo-ternary phase diagrams were drawn to locate the nanoemulsion zones, where oil, surfactant, co-surfactant, and water exist as clear and homogeneous dispersions. Ternary phase diagrams provide information on the best combinations to use for the preparation of nanoemulsion. The droplet size of the nanoemulsion is critical as it influences the solubility as well as bioavailability and it is well established that smaller size droplets result in enhanced solubility and bioavailability owing to the higher surface area [34,35].

The phase analysis demonstrated that when the surfactant: co-surfactant ratio was 2:1, the largest proportion of oil was integrated in the nanoemulsion system and smaller size nanoemulsions. The 3% corn oil, 2% tween 20, 1% lecithin, and 94% water resulted in an optimized vitamin D nanoemulsion with globule size 118.6 ± 4.3 nm and particle size distribution of 0.11 ± 0.30. The zeta potential of nanoemulsion is important because its value can be linked to colloidal dispersion stability. In a dispersion, the zeta potential demonstrates the extent of repulsion between contiguous, identically charged particles. A higher zeta potential indicates stability for particles and molecules of sufficiently small size. In cases of lower zeta potential, repulsive forces are weaker compared to attractive forces, leading to the breaking and flocculation of dispersions. Colloids having a large zeta potential (either negative or positive) are deemed electrically stable [48,49]. The optimized formulation displayed zeta-potential of −27 ± 0.82 mV, indicating that the developed nanoemulsions are stable. Using TEM, the structural and dimensional characterization of the optimized vitamin D nanoemulsion was accomplished. The nanoemulsion droplet structure was confirmed to be spherical, and each droplet had a discrete region surrounding it, which suggested vitamin D encapsulation. The size of the nanoemulsion droplets obtained by TEM was in agreement with that obtained by DLS. Blackness in the core of

particles as observed in TEM image could be attributed to the solubilization of vitamin D in the oil.

During the stability study, the results showed that vitamin D nanoemulsion retained the vitamin D concentration quite well, although it was exposed to water in the emulsion. Jiang et al. [23] showcased similar behavior when encapsulating vitamin D in carrier oils and storing away from light, which prevents its otherwise easy degradation. This study confirmed the stability of vitamin D in the nanoemulsion upon storage for extended time periods. The vitamin D degradation was only 7.63% in nanoemulsion compared to 8.97% in oil following 30-day storage. The stability results do not appear to be distinctly different but are significant because in the nanoemulsion, the vitamin D, prone to hydrolysis, was in contact with water.

In the second step, the optimized nanoemulsion was mixed with hydrocolloid matrix to form the Nano gummy. It was hypothesized that the Nano gummy might improve the bioavailability of vitamin D, although this was not tested in the present research. The improved bioavailability of vitamin D from Nano gummy could be explained by two mechanisms. Firstly, gummies are meant for chewing in the mouth where they are mixed with saliva and begin to dissolve. Most likely, the released nanoemulsion from the Nano gummy in the buccal cavity will allow enhanced permeation of vitamin D and thus improved bioavailability. Such enhancement of buccal permeation by nanoemulsions has been previously reported [39,50,51]. Secondly, the released nanoemulsion from the Nano gummy will disperse in the gastric fluid. The nanoemulsion results in the enhanced solubilization of vitamin D and its subsequent absorption from GIT, resulting in improved bioavailability. Thus, the Nano gummy could significantly enhance the bioavailability of vitamin D encapsulated in nanosized emulsion droplets in hydrocolloid matrix [50].

While designing gummies, oil incorporation played an important in the texture properties of gummies. Gummies with corn oil 10% exhibit optimum hardness value throughout the study. The addition of oil led to a steep increase in hardness value, but for gummies the optimum hardness value ranges around 3100 g force. After 3100 g force, the gummies become harder and firmer. Texture profile analysis may further be used to track changes in texture during the drying process of gummy confections. The stickiness results showed that the gummies with the lowest content of oil, i.e., 10% oil, exhibited more adhesiveness throughout the study, as can be observed in Table 5. The stickiness value increases with a decrease in oil content. Stickiness for gummies with 40% oil content was even lower, but they became hard and deteriorated the chewy feel. The texture of the gummies is also affected by their age, because the water content changes over time and water plays an essential element in maintaining texture [52]. Concentrations of other additives were kept constant throughout the experiment. Sucralose is an artificial sweetener, while sorbitol and xylitol are sugar alcohols. These act as sweeteners and impart that likeliness among children to willingly consume the product. Various flavors improve the aroma and overall taste of the product since these factors play an important role in overall liking of the product. Homogenization speeds were limited to 1000 rpm as higher homogenization speeds may lead to instability or breaking of the incorporated emulsion.

Although texture can be measured using both instrumental and sensory techniques, it has been demonstrated that sensory analysis when coupled with instrumental analysis yields better attributes [53] for a consumer-oriented product. The sensory assessment technique is widely used in food product development, its quality control, monitoring of shelf life, consumer acceptance, and some experts suggest it to be one of the most sensitive and reliable approach in certain situations [54]. In the current study, the formulated gummy retained its texture and overall acceptance over 45 days of storage at room temperature. This signifies a stable product in terms of consumer perception. Unvarying taste over storage indicated that excipients were inter compatible and incorporated nano-emulsion jacketed vitamin D quite well. Although the formulated confectionary lost significant aroma on storage based on the sensory scores, this could be improved by modifying flavoring agents.

### **6. Conclusions**

The nanoemulsion-based matrix system using edible oil, emulsifier and hydrocolloid proved to be an effective approach to develop stable, nano enhanced vitamin D gummies that are readily consumer acceptable. The developed Nano gummy could enhance the vitamin D bioavailability. However, prolonged stability studies and gummy evaluation are required before scale up.

**Author Contributions:** Conceptualization, G.K.J. and M.H.W.; methodology, A.G.; software, A.M.A.A.; validation, A.G. and A.Z.; formal analysis, M.Z.A.; investigation, M.Z.A.; resources, F.J.A.; data curation, N.H.; writing—original draft preparation, M.Z.A.; writing—review and editing, M.H.W. and G.K.J.; visualization, M.H.W. and A.M.A.A.; supervision, G.K.J. and F.J.A.; project administration, F.J.A.; funding acquisition, F.J.A. and A.M.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors of this research would like to acknowledge the financial support offered by Taif University Researchers Supporting Project number (TURSP-2020/50), Taif University, Taif, Saudi Arabia. The author: Mohammad Zubair Ahmed is indebted to the Jamia Hamdard Silver Jubilee Research Fellowship, Jamia Hamdard, New Delhi for providing the fellowship.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study as its not required for supplemental food products made with standard established process. No biospecimen or sensitive information like demographics was collected from panelists in any form during the sensory evaluation.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** This study did not report any data (All the data is included in the current manuscript).

**Acknowledgments:** The authors of this research would like to acknowledge the financial support offered by Taif University Researchers Supporting Project number (TURSP-2020/50), Taif University, Taif, Saudi Arabia. The author: Mohammad Zubair Ahmed is indebted to the Jamia Hamdard Silver Jubilee Research Fellowship, Jamia Hamdard, New Delhi for providing the fellowship. The authors also want to extend their gratitude to Department of Scientific and Industrial Research, Government of India for providing grant under CRTDH scheme.

**Conflicts of Interest:** The authors declare no conflict of interests.
