The Biosafety and Ethical Review Committee (BERC) of the University of Sargodha, Sargodha, Pakistan, with a clearance number of UOS/IFSN/2023/07, provided ethical approval for this work. The informed consent was obtained from the participants who took part in sensory evaluation and were informed in detail about the purpose of this study.

Fresh eggshells, CLO, eggs, butter, sugar, wheat grains, for the development of commercial straight grade flour (CSGF), and baking powder were purchased locally from native markets of Rawalpindi and Islamabad, Pakistan. It was assured that same trade/brand ingredients were used in each trial to avoid variation in results. All the chemicals and reagents used in this study were of analytical grade and procured from Aladdin Chemicals, Shanghai, China.

By following the procedure adopted by Hassan (2015), the eggshells were collected and membrane removed, followed by washing under running water; then scrubbing was done with a sponge. After scrubbing, eggshells were immersed in a solution of sodium hypochlorite for disinfection, followed by rinsing and drying. In a post-drying procedure, eggshells were crunched to a fine powder using pestle and mortar. Sieving of powdered eggshells was done through 0.18-mm sieve. Obtained powder was then dried in an oven (UNB 100, Memmert, Germany) at 80°C for 15 min. In order to remove all microorganisms from ESP, its sterilization was performed at 136°C for 18 min in an autoclave (MLS-3750, Labo Autoclave, Sanyo, Japan) using the protocols adopted by Kadhim et al. (2022). Packaging of ESP was done in low-grade polyethylene bags for safe storage. A flowchart for the pretreatment of ESP is presented in Figure 1.

For the preparation of flour, tempering of procured wheat grains was done at a moisture level of 15.5% after thorough cleaning. Tempered grains were stored in plastic bins at room temperature for 24 h in order to balance the water content of grains. The post-tempering moisture content was determined by method No. 26-95 of American Association of Cereal Chemists (AACC, 2000). The milling of tempered wheat grains was performed in Brabender Quadrumat Senior mill. By following the AACC (2000) technique No. 26-21A, the obtained milled fractions, such as break flour, reduction flour, bran, and shorts, were weighed and their proportions were determined using the total samples recovered. CSGF was yielded by blending of break roll flour and reduction roll flour ingredients. The fat, fiber, ash, protein, and nitrogen-free extract (NFE) of CSGF were determined using their respective methods provided in AACC (2000).

The mineral contents, such as iron, magnesium, phosphorus, calcium, potassium, and sodium, present in ESP were determined by the wet digestion method using atomic absorption spectrophotometer (5100PC, Norwalk, CT, USA) as described in Association of Official Analytical Chemists (AOAC, 2012), and further elaborated by Hussain et al. (2021). This method is commonly adopted for determination of wide range of macro and micro minerals because of its accuracy, cost effectiveness, and ease of use. A 0.5-g sample of ESP was taken and digestion was performed in a 100-mL conical flask on hot plate with 10-mL nitric acid solution. The sample was then heated for 20 min at 70°C. The same sample was then digested in 5 mL of 60% chloric acid at 190°C. The digestion of the sample was performed until contents in the flask turned clear. The sample volume of 100 mL was achieved by adding deionized water. In order to get standard curves, the samples were run with known strength of standards to determine the contents of iron, magnesium, calcium, and phosphorus. The sodium and potassium contents present in ESP were determined through flame photometer (Sherwood Scientific, UK) by following the method described in AOAC (2012).

Moisture content in ESP was determined by following the method No. 44-15 of AACC (2000). Briefly, 2-g sample was collected in a petri dish that was previously weighed (W₁) and was heated to 105°C for 24 h. After that, the sample was placed for some time in a desiccator. The dried material was weighed again after cooling (W₂). Then the moisture percentage in ESP was estimated by using the following formula:

where W₁ = pre-weighed weight, W₂ = weight after cooling of sample.

The protein content in ESP was determined using Kjeltech apparatus following the Kjeldahl’s method No. 46-30 of AOAC (2000). In the presence of digestion mixture, concentrated H₂SO₄ was used to break down 5 g of ESP. The mixture after digestion was run till a bright green hue was achieved. Then 250 mL of distilled water was used to dilute digestion mixture. Thereafter, 10 mL of diluted sample and 10 mL of 40% NaOH solution were combined for distillation in a distillation equipment. Then 4% boric acid solution was combined with ammonia, and until methyl red indication was attained, the sample was forced-running. The material was then titrated against 0.1-N H₂SO₄ solution. Finally, the following formula was used to estimate nitrogen:

The percentage of protein was calculated by the following expression:

Protein (%) = Nitrogen (%) × 6.25.

High-performance liquid chromatography (HPLC) as reported by Feng et al. (2020) was used to analyze vitamins D and A contents, PUFAs, monounsaturated fatty acids (MUFAs), and other fatty acid-related parameters in CLO. This method was preferred due to high selectivity, rapid separation, simple sample preparation, and accurate quantification. First, 0.5-mL sample of fresh CLO was taken. Then a solution was prepared by mixing 25 mL of methanol with 700 mg of extremely pure potassium hydroxide and potassium methoxide. After that, ascorbic acid solution of 1% weight was prepared by mixing 165 mg of ascorbic acid in 10 mL of methanol. Then 500 mg of CLO was mixed with 2-mL ascorbic acid, followed by 5 mL of MeOK mixture in a glass flask. After that, the mixture was vortexed for 30 s. The flask was then placed in a silicone bath for 30 min at 80°C with reflux. The sample was cooled in ice water before being combined with 5 mL of n-hexane. The solid saponifiable sample was removed from hexane layer and dried under N₂ flux. The dried material was dissolved in 600 mL of MeOH. Standard vitamin D₃ fractions were produced in methanol and kept at 4°C in glass bottles to avoid exposure to sunlight.

The amount of vitamin D₃ was calculated by the following formula:

where

a₁ = peak area of vitamin D₃,

a₂ = peak area of vitamin D₂,

CF = correction factor

(The quotient of the peak areas of vitamins D₃ and D₂, was measured at identical concentrations of vitamin forms),

i = amount of internal standard added (g), and W = weight of sample (g).

Control and supplemented cookies were prepared by following the guidelines provided by Hussain et al. (2023b), using the recipe given in Table 1. Further, the treatment plan is provided in Table 2, where T₀ was control cookies, which had no source of calcium and vitamin D whereas treatments (T₁, T₂, T₃, and T₄) were prepared keeping in mind the recommended daily allowance (RDA) for calcium and vitamin D. Explaining in detail, first the raw materials were weighed with accuracy. After weighing, eggs, butter, and CLO were mixed, followed by beating. Dry ingredients, such as ESP, CSGF, baking powder, and sugar, were added during the beating process. The dough was kneaded with soft hands, then flattened and cut with a cookie cutter. The cookies were placed on greased baking trays and baked for 25 min at 175°C in a preheated oven. After cooling to room temperature, the prepared cookies were packed in bio-oriented polypropylene (BOPP) bags and kept at room temperature in laboratory for further investigations. Flow chart for preparing cookies is presented in Figure 2.

Vitamin D content in ESP-supplemented cookies was estimated by HPLC method using the protocols provided in Section 2.9.

In the current study, the control and supplemented cookies were exposed to sensory evaluation, which includes texture, color, flavor, mouth feel, and overall acceptability. The sensory evaluation was carried out in a laboratory by a panel of 40 semi-trained judges of both genders aged between 25 and 35 years, using a 9-point hedonic scale as guided by Rafique et al. (2023). The panel was briefed about the procedure and coded samples were provided along with evaluation proforma, and their informed consent was obtained. Proforma used for sensory evaluation of cookies is presented in Annexure 1.

This study considered possible variables that were classified in Statistical Package for Social Sciences (SPSS version 16) and Minitab (version 19). The effects of treatments of indicative parameters were examined using both one-way and two-way analyses of variance (ANOVA) with post hoc Tukey test, and between-group comparisons were carried out using the general linear model (GLM). For all statistical outcomes, a p ≤ 0.05 was deemed statistically significant (Hipsey et al., 2019).

Proximate analysis of CSGF showed the following: calcium = 18.5±0.51 mg/100 g, phosphorus = 107.25±1.70 mg/100 g, moisture content = 12.63±0.01%, and protein content = 10.51±0.021% (Table 3). The ash content recorded in CSGF was 0.94±0.01% and crude fiber was recorded as 0.37±0.021 whereas the iron content was 2.1±0.129 mg/100 g. Previous research findings by Ficco et al. (2009) and Mepba et al. (2007) were also in agreement with the results of the present study, as the authors also reported CSGF to be a poor source of calcium, iron, ash, and fiber. Some other research findings reported that the moisture content in CSGF was 13.33±0.60%, protein 11.01±0.7%, crude fat 0.84±0.25%, crude fiber 0.84±0.27%, and the ash content was 1.51±0.51% (Hussain et al., 2023b). Mineral contents in CSGF, such as magnesium 5.51±0.37 mg/100 g, potassium 4.76±0.28 mg/100 g, calcium 18.61±0.66 mg/100 g, and iron 0.700±0.52 mg/100 g were reported by Khan et al. (2017). These findings confirmed the present results with strong agreements.

The proximate and mineral analysis of CSGF demonstrated that while preparing cookies, the incorporation of ESP might be useful in increasing ash, fiber, and mineral contents of the product, as CSGF has comparatively lower contents of these factors.

Results after statistical analysis of proximate and mineral parameters of ESP are shown in Table 4. The presented results revealed that ESP contains calcium 38.5±0.17 mg/100 g, potassium 41.64±0.02 mg/100 g, phosphorus 99.34±0.03 mg/100 g, magnesium 375.52±0.98 mg/100 g, and sodium 87.06±0.025 mg/100 g. On the other hand, ESP contained protein 2.12±0.02%, ash 96.6±0.23%, and moisture 0.53±0.017%. These results were strongly supported by the findings provided by Waheed et al. (2019), as they reported that ESP contained calcium 38 mg/100 g, moisture 0.52%, and protein 2.13%, while phosphorus in ESP was reported as 99.36 mg/100 g. Similar findings were also observed in a study conducted by Murakami et al. (2007), who analyzed ESP and proposed that it is composed of 94% calcium carbonate, 1% of calcium phosphate, and 1% magnesium carbonate. ESP consists of 39% elemental calcium; therefore, it is a promising and effective source of dietary calcium.

The present results of ESP were also in accordance with the results achieved by Bartter et al. (2018), who analyzed calcium content of eggshell in three different studies and discovered it ranging from 360 mg/g to 400 mg/g of eggshell. The present study was also in accordance with the findings of Brun et al. (2013), who reported that eggshell mainly comprises calcium as 38 mg/g, potassium as 1.4 mg/g, sodium as 5.1 mg/g, and phosphorus as 4.4 mg/g. The present results of ESP were also in accordance with some previous findings, which estimated that ESP contained moisture as 0.46%, ash 94.6%, and protein as 3.92%. A high amount of calcium of around 40%, as calcium carbonate 98.43% and calcium phosphate 0.75%, along with magnesium 0.5%, and phosphorus 0.1%, have also been reported in ESP (Afzal et al., 2020; Ali and Badawy, 2017; Ray et al. 2017). The present results and their relevant literature showed ESP to be a good source of ash, fiber, and important minerals, such as calcium and phosphorus.

Analysis of CLO was conducted by HPLC, and as presented in Table 5, the following components were discovered in CLO: omega 3 fatty acids, 1.32±0.22 mg; eicosapentaenoic acid (EPA), 741.25±2.98 mg; dexosahexaenoic acid (DHA), 461.25±2.98 mg; other omega 3 fatty acids, 199±2.44 mg; vitamin D₃, 250.25±1.70 µg; vitamin A, 30,002±1.27 µg; total MUFA, 46.6±0.25 g; total PUFA, 22.475±0.25 g; total saturated fats, 22.57±0.17 g; and cholesterol, 570.25±1.70 mg. These findings showed CLO to be a good source of vitamins A and D, PUFAs, and MUFAs. The chemical composition of CLO presented by Higashi et al. (2014) demonstrated that CLO contained omega 3 fatty acids, 1.4 g; EPA, 740 mg; DHA, 460 mg; vitamin D₃, 250 µg; and other omega 3 fatty acids, 1.4 g per 5 mL. Similar findings were also demonstrated by Hubicka et al. (2020), who estimated that CLO contained high quantity of highly unsaturated fatty acids (HUFAs): DHA, 22:6, and EPA, 20:5. Similar composition was also reported by Almarri et al. (2017), who evaluated that CLO is a rich source of omega 3 fatty acids, HUFAs, and vitamin D₃ (252 µg).

In chemical analysis, FTIR is used to assess a compound’s functional group. Infrared spectroscopy is frequently employed in the food industry to check the quality of products, and food samples are identified and verified using it (Indrianingsih et al., 2024). The FTIR spectrum of control cookies is shown in Figure 5A and that of ESP- and CLO-supplemented cookies is shown in Figure 5B. The absorbance bands of control cookies show at 2,920.4 cm^-1, 1,742.5 cm^-1, and 989.6 cm^-1, whereas the sharp peaks of supplemented cookies show at 2916.6 cm^-1, 1742.5 cm^-1, and 987.7 cm^-1. The absorption of ^–OH hydroxyl group, which occurred from carbohydrates or other substances such carboxylic aldehydes and ketones, was linked to vibration at roughly 2,900–3,000 cm^-1. This is attributed to vitamin D molecules, potential hydration in calcium carbonate, or content of moisture. In contrast, the control sample exhibits a weaker or less prominent peak, suggesting a lower concentration of hydroxyl group, possibly due to the absence of vitamin D supplementation. A strong vibration at a wave number of around 1,742.5 cm^-1 came from the stretching vibration of C=O (carbonyl groups) discovered in vitamin D molecules. This peak is either absent or has lower intensity in the control sample, confirming that vitamin D ester groups are introduced only in the supplemented sample. Similarly, vibrations at 987.7 cm^-1 showed the presence of fat and calcium carbonate in cookies, as CLO and ESP were introduced as replacements of butter and CSGF, and the fat from CLO and calcium carbonate from ESP had high contents of vitamin D and calcium, respectively. These peak intensities suggest increased calcium and vitamin D contents. In contrast, these peaks are weaker in the control sample, indicating a lower concentration of calcium and vitamin D or possibly differences in crystallinity. This FTIR comparison validates the incorporation of calcium and vitamin D, making the supplemented sample nutritionally enhanced compared to the control. The presence of Vitamin D functional groups and stronger calcium carbonate peaks suggests improved bioavailability of essential nutrients.

XRD plots of the control and supplemented cookies are shown in Figures 6A,B. The peaks are sharp and highly resolved that depicted the crystalline structure of ESP-fortified cookies. The ESP’s crystallographic lattice of calcium carbonate was confirmed by diffractogram, which shows a single crystalline phase (calcite) with a peak at 29.4° in accordance with X-ray diffraction pattern. These bands also showed that the crystallinity of calcium in supplemented cookies was not affected by baking (Therdthai et al., 2023). When Kalaycı et al. (2025) stated that the calcium carbonate peak in the XRD results of ESP was 2ϴ = 29.58° for various eggshells, it clearly confirmed the current findings about the existence of high calcium contents in supplemented cookies. The lack of calcium carbonate could be the cause of this peak’s non-observance in the control cookies of this study. The present results are in strong agreement with the findings of that of Hamidi et al. (2017), who studied the crystalline structure of ESP and concluded that ESP could be used as an excellent source of dietary calcium. Cree and Rutter (2015) asserted that biowaste chicken eggshells had a high calcite or calcium carbonate content. The FTIR spectra and XRD patterns of milled ESP were determined to be comparable with the current findings when ESP was used in cookies. Waste eggshells produced by processing industries could be utilized as a calcium fortification agent in various bakery products. The FTIR analysis of raw and boiled chicken ESP showed a similar absorption profile, with peaks at 1,394 cm^–1, 873 cm^–1, and 712 cm^–1, which supported the current findings. In a study conducted by Awogbemi et al. (2020), the XRD characterization of ESP showed that the proportion of calcium carbonate in raw and boiled samples was 79.3% and 99.2%, respectively, while these findings also supported the current results.