Utilization of Chicken Eggshells as a Source of Calcium to Enhance the Nutritional Value and Quality Characteristics of Crackers

Utilization of Chicken Eggshells as a Source of Calcium to Enhance the Nutritional Value and Quality Characteristics of Crackers | 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 Utilization of Chicken Eggshells as a Source of Calcium to Enhance the Nutritional Value and Quality Characteristics of Crackers Mokhtar M. Salama, Sameh M. Ghanem, Mostafa M. Kadry, Shaoyun Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5398539/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract In recent years, there has been a growing demand for healthy snacks, leading to new products containing health-promoting components. Eggshells have emerged as a novel calcium source for human consumption, with potential for use in food products. Hence, this research aimed to examine the effects of substituting wheat flour with different levels (4%, 8%, and 12%) of chicken eggshell powder (CEsP) on the nutritional value, physiochemical properties of produced crackers, and to measure in vivo calcium absorption. The findings of the research indicated that the boiling process of CEsP effectively eliminated any microbial growth on the surface. CEsP supplementation led to an increase in the ash content of the crackers without altering the fat and protein contents, resulting in a notable decrease in the calorie content. Furthermore, the replacement of WF with CEsP led to an elevation in the mineral content, particularly calcium, which increased from 25.01 to 2446.1 mg/100 g with a higher replacement ratio of 12%. Moreover, the physical properties of the crackers showed no significant changes when using a replacement ratio of up to 8% of the produced crackers. Additionally, the in vivo evaluation of calcium absorption revealed that the highest absorption rate (44.07%) was achieved at the 8% replacement level. Based on these findings, the addition of CEsP to crackers improved their nutritional quality and physicochemical properties up to an 8% replacement level. Thus, the study proposes using CEsP as a dietary calcium supplement to develop novel calcium-enriched food products. Crackers Chicken eggshell Wheat flour Quality characteristics Figures Figure 1 1. Introduction Osteoporosis, a condition resulting from calcium deficiency, has become one of the most significant global health issues in recent years. It is estimated that approximately 200 million people worldwide suffer from calcium deficiency [ 1 ]. Studies have indicated that this deficiency can be attributed to various factors, including the high cost of milk and its products, which prevents some individuals from purchasing them in many countries. Additionally, certain people face health issues, such as lactose intolerance, that limit their consumption of milk and its products [ 2 ]. Therefore, it is imperative to explore the development of functional foods that can fulfill the daily calcium needs of these individuals. The global production of eggs has consistently increased over the past decade, with an observed growth of more than 30% from 2009 to 2019 [ 3 ]. Eggshells are a common household waste generated regularly from households and food processing facilities. Due to the low cost and high nutrient content of eggs, their consumption has been on the rise globally, leading to a 150% increase in egg productivity over the past three decades. However, this increased production has resulted in environmental and health challenges due to the accumulation of chicken eggshells [ 4 ]. In various countries, including Egypt, egg product companies and food manufacturers, such as those producing baked goods and mayonnaise, generate a significant amount of waste from chicken eggshells, contributing to environmental issues. In Egypt specifically, the annual egg production in 2016 reached a record of 510,000 tons [ 5 ]. The egg product industry in Egypt alone produced approximately 56,100 tons of chicken eggshells in the same year [ 5 ]. Almost all of the eggshell is considered waste, and the disposal of these discarded eggshells contributes to food waste, resulting in significant environmental damage by increasing the global carbon footprint (3.3 Gtonnes of CO2 equivalent in 2007) when they are buried [ 6 ]. This contributes to greenhouse gas emissions, one of the major causes of global warming [ 7 ], and leads to microbial proliferation due to the substantial amount of waste generated [ 8 ]. Additionally, the disposal of Eggshell waste poses challenges in terms of cost and utilization of landfill space. Hence, it is of utmost importance to prioritize enhancing the value of eggshells and incorporating them effectively into the food processing sectors. Chicken eggshells represent approximately 9–12% of the weight of a medium-sized egg (54–60 g), and the main component responsible for their formation is calcium carbonate. Humans can utilize eggshells as a source of calcium and other minerals such as magnesium and zinc [ 9 ]. [ 10 ] conducted a study indicating that every 2.7 g of chicken eggshells can provide sufficient calcium to meet the daily needs of adults. Adequate calcium intake, ranging from 200–1200 mg daily, is crucial for maintaining healthy bone status, particularly after menopause, beginning with early infancy [ 11 ]. Chicken eggshells consist of around 95% calcium carbonate (CaCO3) and contain valuable elements like strontium and boron, which play crucial roles in the prevention of osteoporosis [ 12 ]. Numerous studies have demonstrated the potential of chicken eggshells as a source of calcium [ 7 , 12 , 13 ]. Previous research has also indicated the possibility of incorporating eggshells as a calcium source in various food products, including bread [ 14 , 15 ], biscuits [ 16 – 18 ], muffins [ 19 ], chocolate cakes [ 20 ], yogurt [ 21 ], and more, without causing any significant alteration in flavor. Crackers, known for their distinct flavors, long shelf life, and affordable prices, are popular snack foods consumed daily [ 22 ]. Various materials have been used to fortify crackers and enhance their functionality. Examples of such materials include germinated lentil extracts [ 23 ], hibiscus residues [ 24 ], and tuna bone bio-calcium powder [ 25 ]. Therefore, this study aimed to examine the impacts of substituting wheat flour (WF) with varying levels (4%, 8%, and 12%) of CEsP on the chemical composition, mineral content, and physical characteristics of the resulting crackers. Additionally, this work aimed to evaluate the potential biological effects of the produced crackers through in vivo assessments. 2. Materials and methods The wheat flour (72%) used in this study was obtained from the South Cairo Mills Company located in Giza, Egypt. Chicken eggs, other ingredients, and materials required for cracker production were purchased from the local market in Nasr City, Cairo, Egypt. Chemicals used in the analytical procedures were sourced from El-Gamhouria Trading Chemicals and Drugs Company, situated in Cairo, Egypt. 2.1. Preparation of samples 2.1.1. Preparation of chicken eggshell powder CEsP was processed following the method described by [ 20 ]. The eggshells were collected and washed inside and outside using a sponge under running water. They were then placed in boiling water for 30 minutes and subsequently subjected to a drying and sterilization process at a temperature of 135 ± 3ºC for 15 minutes using a hot air oven (Labocon Systems Limited, Hampshire, UK). The dried eggshells were ground using a grinder (Braun AG, No, 4122, Germany), and the resulting powder was sieved to obtain CEsP. The CEsP was packed in polyethylene containers and stored in a refrigerator at 4 ± 1ºC until further use. 2.1.2. Preparation of cracker blends For the preparation of cracker blends, following the method by [ 26 ], WF was substituted with 0%, 4%, 8%, and 12% of CEsP, respectively. Each flour mixture was blended and homogenized separately before being packed in tightly sealed polyethylene bags. These bags were stored at room temperature (20 ± 3ºC) for subsequent analysis and processing. The composition of the prepared blended powders is presented in Table 1 . Table 1 Ingredients recipe for preparation crackers blends (g / 100 g). Ingredients Control Crackers- CEsP samples Substitution levels 4% 8% 12% Wheat flour 60.50 58.08 55.66 53.24 Eggshell powder 2.42 4.84 7.26 Vegetable oil 7.50 7.50 7.50 7.50 Sucrose 1.00 1.00 1.00 1.00 Baking powder 1.50 1.50 1.50 1.50 Salt 1.00 1.00 1.00 1.00 Distilled water 28.50 28.50 28.50 28.50 CEsP : Chicken Eggshell Powder 2.1.3. Produce of cracker samples: To produce the crackers, all the ingredients were weighed based on a batch size of 100 g. The dry components were combined, followed by the addition of oil to the mixture. Water was subsequently added to the dough, which was kneaded for 1 minute using a food processor (Moulinex, France) until a smooth, homogeneous, and cohesive dough was obtained. The dough was then left to rest for 4 minutes at room temperature (20 ± 3ºC). After resting, the dough was rolled out, cut into desired shapes, and left for 10 minutes at room temperature. The crackers were baked in a thermal oven at 200 ± 3°C for 12 minutes and then cooled for 30 minutes at room temperature before being stored in sealed plastic containers until further use. Three separate batches were produced from each blend to replicate [ 26 ]. 2.2. Analytical Methods: 2.2.1. Chicken eggshell powder microbiological examination: The total bacterial count (TBC), Coliform , Salmonella , and Staphylococcus were determined following the methodology outlined by [ 27 ]. 2.2.2. Proximate chemical composition, energy values, and minerals content: The proximate chemical components (moisture, ash, ether extract, crude protein, and fiber) of the tested WF, CEsP, and cracker samples were assessed using the methodologies outlined in the reference [ 28 ]. The difference calculated carbohydrate content: % carbohydrates = 100 - (% crude protein + % ash + % ether extract + % crude fiber). The energy values in kcal/100 g for the cracker samples were calculated using the method described by [ 29 ]. The calculation formula used was as follows: Energy value (kcal/100 g) = 4 (carbohydrate + protein) + 9 (fat). The mineral content (calcium, magnesium, phosphorus, potassium, sodium, and zinc) of WF, CEsP, and cracker samples was determined according to the [ 28 ]. 2.2.3. Physical and textural properties: Physical parameters such as length, width, weight, thickness, and puffiness of the cracker samples were measured based on the procedure outlined by [ 30 ]. Hardness was assessed using a texture analyzer (Stable Micro Systems, Godalming, Surrey, UK), as reported by [ 25 ]. 2.2.4. Biological investigation: In this study, a total of twenty-five young male albino rats weighing approximately 80–90 grams were purchased from the animal house. The biological experiments were conducted at the animal house laboratory at the Agricultural Research Center (ARC) in Giza, Egypt, According to the protocol approved by the Agricultural Research Center (ARC) and the Institutional Animal Care and Use Committee (IACUC) reviewers (IACUC protocol number: ARC-AZU-62-24), and following the 2014 Egyptian Constitution, Article 45, the official decrees of the Ministry of Agriculture No. 27 (1967), the World Organization for Animal Health (WOAH), and the Guide for the Care and Use of Laboratory Animals, 8th Edition (2011). The rats were housed separately in stainless steel cages under controlled environmental conditions, including a constant temperature of 22°C and a 12-hour light-dark cycle. They had unrestricted access to food and water throughout the experiment. Before the commencement of the study, a group of rats were provided with a well-balanced diet (AIN-93) for one week to acclimatize to the experimental conditions. The experimental diets were prepared by mixing the finely ground crackers and a mixture of casein with vitamins at a rate of 60 and 1.5 gm per kg, respectively, to provide the nutritional needs of the rats. After the adaptation period, the rats were divided into five groups: Control (-) (rats fed on crackers free of eggshells), Control (+) (rats fed on crackers free of eggshells mixed with CaCO3 at a ratio of 3.5 g/kg), C4 (rats fed on crackers with a replacement ratio of 4% of WF by CEsP), C8 (rats fed on crackers with a replacement ratio of 8% of WF by CEsP), and C12 (rats fed on crackers with a replacement ratio of 12% of WF by CEsP). Each group consisted of five rats. The rats were fed experimental diets for four weeks. At the end of the experiment, the rats underwent an overnight fasting period, and measurements were taken for total food intake, final body weights of the rats, and the amount of weight gained during the experimental period. The food efficiency ratio was determined using the following equation: Food efficiency ratio = body weight gain/food intake [ 32 ]. 2.2.5.1. In vivo calcium absorption measurement: To estimate calcium absorption in vivo, only 3.5 grams of calcium carbonate were added for each kilogram of crackers free of eggshells in the control group (+), according to [ 18 ]. Calcium absorption determination was also performed in the other groups (control group (-), C4, C8, and C12). The rats were provided with experimental diets for four days. On the fourth day, the amount of feed intake during the previous 24 hours was calculated. The feces were collected, dried in the air, and crushed. The amount of calcium in the consumed food and feces was estimated using an atomic absorption spectrophotometer (Perkin Elmer 4100 ZL., Perkin Elmer Inc., Wellesley, USA) (Varian AA240; Varian Inc., Australia) based on the method described in [ 28 ], and the amount of calcium absorbed was determined using the following equation: 2.2.5.2. Determination of calcium level in blood serum: The calcium level in the collected blood serum samples was calculated using the method provided by [ 33 ]. 2.2.5.3. Parathyroid hormone (PTH) assay: Parathyroid hormone (PTH) in blood serum samples was measured using conventional biochemical analysis methods, as described by [ 34 ]. The Enzyme-Linked Immunosorbent Assay (ELISA) technique was employed using commercially available test kits. 2.2.5. Statistical analysis: The data obtained from our study were expressed as mean values along with their corresponding standard deviations. Statistical analysis was performed using SPSS software (version 20.0, IBM Software, Inc., Chicago, USA, 2018) in a completely randomized design. The statistical analysis included a one-way ANOVA conducted at a significance level of 5%, followed by Duncan's multiple range tests (P < 0.05). 3. Results and discussion 3.1. CEsP microbiological quality Results in Table 1S refer to the microbial content of the CEsP after the boiling process. The results reported in the table indicate that the eggshells were entirely free of microbes, as the total count of bacteria indicated a negative result, and no microbial numbers appeared. The results also indicated that the CEsP was free of coliform, salmonella, and staphylococcus after the boiling process. The obtained results are consistent with those obtained by [17] and [35] , which state that the boiling process was sufficient to get rid of the microbes that may be present on the eggshell, making the shells safe to be added to food without causing problems. 3.2. The proximate chemical composition, caloric value, and mineral content (mg/100 g) of eggshell powder compared to wheat flour. Data in Table 2 reveal the difference in the content of crude protein, fat, ash, carbohydrates, crude fiber, and energy value between the raw materials (WF and CEsP) on a dry basis. The results indicate significant differences (P<0.05) between the studied raw materials in their chemical components. From the data, it can be observed that WF had greater quantities of moisture, crude protein, and carbohydrate (11.31%, 10.64%, and 87.16%, respectively) compared to CEsP, which had values of 1.02%, 3.31%, and 1.38%, respectively. In contrast, the same table shows that the ash content in CEsP was approximately 120 times higher than that found in WF (0.62% vs. 94.82% on a dry weight basis). Therefore, CEsP is considered an excellent source of ash content. These results are consistent with those provided by [18, 20] . Significant differences were observed between WF and CEsP regarding the caloric value. CEsP had the lowest caloric value (18.24 Kcal/100g) compared to WF (400.83 Kcal/100g). Table 2 provides information on the major macro and micro elements such as magnesium (Mg), calcium (Ca), phosphorus (P), sodium (Na), potassium (K), and zinc (Zn) in both WF and CEsP. The table shows a comparison between WF and CEsP samples regarding their mineral content. As shown in the results, calcium constituted the highest proportion among the macro-elements in CEsP, with a content of approximately 35238.00 mg/100g. CEsP also exhibited elevated levels of magnesium, sodium, and zinc (288.78 mg/100g, 84.32 mg/100g, and 47.33 mg/100g, respectively) compared to WF, while WF was superior in potassium (297.08 mg/100g vs. 48.12 mg/100g, respectively). Similarly, the data demonstrate that CEsP and WF contained a similar amount of phosphorus (118.61 mg/100g and 132.11 mg/100g, respectively). Therefore, CEsP was superior in calcium, magnesium, sodium, and zinc, while WF was superior in potassium and relatively superior in phosphorus. These findings align with the findings reported by [36] . As a result, CEsP stands out for its high content of various tested minerals, both macro and micro-elements. Therefore, there is potential for the reutilization of CEsP in food fortification. In general, CEsP can be regarded as a valuable source of essential macro and micro-elements. Table 2: The proximate chemical composition, caloric value, and mineral content of eggshell powder compared with wheat flour. Compounds (%) WF CEsP Moisture 11.31±0.26 a 1.02±0.04 b Crude Protein 10.64±0.31 a 3.31±0.14 b Ether extract 1.07±0.04 a 0.31±0.02 b Ash 0.62±0.03 b 94.82±0.68 a Crude Fiber 0.51±0.03 a 0.18±0.01 b Carbohydrates 87.16±0.42 a 1.38±0.07 b Energy value (kcal/100 g) 400.83±3.28 a 18.24±1.02 b Minerals (mg/100g) Calcium 24.92±0.43 Cb 35238.00±4.89 Aa Magnesium 129.17±0.98 Bb 288.78±1.62 Ba Phosphorus 132.11±1.21 Ba 118.61±0.92 Cb Potassium 297.08±1.54 Aa 48.12±0.71 Eb Sodium 5.91±0.17 Db 84.32±1.05 Da Zinc 0.84±0.04 Eb 47.33±0.53 Ea M±SD: The mean and standard deviation values for different letters in the same row are significantly different ( P <0.05). WF: Wheat Flour. CEsP: Chicken Eggshell Powder 3.3. Effect of replacement of WF by CEsP on quality criteria of cracker samples: Table 3 presents the proximate chemical composition, caloric value, and mineral content of the cracker samples, which were influenced by various replacement levels (4%, 8%, and 12%) of WF with CEsP. The control sample, without any addition of CEsP, is also included in the table. Based on the results, there were no significant differences (P≤0.05) in the protein, fat, and fiber contents of the cracker samples as the substitution level of WF by CEsP increased from 4% to 12%. The protein content ranged from 12.81% to 13.07%, the fat content ranged from 10.73% to 10.89%, and the fiber content ranged from 0.63% to 0.67%. These values were slightly lower compared to the control sample, which had a protein content of 13.18%, fat content of 10.94%, and fiber content of 0.69%. In contrast, the moisture content exhibited a slight and gradual increase as the substitution level of WF by CEsP increased from 4% to 12% compared to the control sample. A similar trend was observed for the ash content, which gradually increased with the replacement percentage from 4% to 12%. However, the increase was more pronounced in the case of ash content, with values rising from 0.84% in the control sample to 10.98% in the cracker samples at a 12% replacement ratio. Additionally, it is evident that as the levels of CEsP increased in the cracker samples, the caloric value decreased. These findings could be attributed to the gradual decrease in carbohydrates resulting from the substitution of CEsP. In general, it can be concluded that cracker samples containing CEsP exhibited favorable nutritional quality in terms of ash, protein, and crude fiber contents. These results align with the findings of [18] , who observed an increase in ash content without affecting fat and protein contents when CEsP was supplemented in biscuits at various levels (5%, 10%, 15%, and 20%). Additionally, a significant reduction in the caloric value of the biscuits was observed. Similar findings were reported by [37] about white bread. The mineral content of both the control sample and the tested cracker samples with replacement ratios of 4%, 8%, and 12% of WF by CEsP were determined and recorded in Table 3. Mineral elements were measured, and significant differences were demonstrated between all formulations. The amount of calcium greatly increased at a 4% replacement ratio compared to the control, which increased from 25.01 mg/100 g to 855.6 mg/100 g. This increase was consistently observed for 8% and 12% CEsP addition, which were found to be 1680.8 mg/100 g and 2449.5 mg/100 g, respectively. Based on the results obtained, the calcium content in crackers was greatly influenced by the high level of calcium in CEsP compared to the calcium content in WF. Besides calcium, the results show that the mineral contents in crackers containing CEsP increased for magnesium, sodium, and zinc. The magnesium content in crackers increased in the range of 135.4 mg/100 g to 154.8 mg/100 g from the control up to 12% supplementation of CEsP. Sodium was high in crackers where the substitution ratio of WF by CEsP reached 12%, recorded as 20.40 mg/100 g compared to 14.73 mg/100 g in the control sample. For zinc content, the control sample showed 1.07 mg/100 g, then increased to 4.54 mg/100 g after WF substitution by 12% of CEsP. For potassium content, the control crackers showed 344.2 mg/100 g, then decreased to 325.9 mg/100 g in a substitution ratio of 12% of WF by CEsP. Also, when the replacement level of WF by CEsP increased from 4% to 12%, the phosphorus content decreased slightly from 190.4 mg/100 g to 187.9 mg/100 g when compared with the control sample, which recorded 191.3 mg/100 g. These findings are consistent with similar results reported by [17, 38] . Table 3: Proximate chemical composition, caloric value, and minerals content of crackers prepared by substituting wheat flour with eggshell powder (on a dry weight basis). Compounds (g/100g) Control Sample Crackers- CEsP Samples Substitution levels 4% 8% 12% Moisture 4.28±0.15 d 4.83±0.21 bc 5.24±0.26 b 5.91±0.18 a Crude Protein 13.18±0.41 a 13.07±0.32 a 12.94±0.35 a 12.81±0.29 a Ether extract 10.94±0.26 a 10.89±0.42 a 10.82±0.33 a 10.73±0.27 a Ash 0.84±0.05 d 4.18±0.18 c 7.31±0.17 b 10.98±0.45 a Crude Fiber 0.69±0.03 a 0.67±0.02 a 0.66±0.04 a 0.63±0.03 a Carbohydrates 74.35±0.84 a 71.19±0.91 b 68.27±1.09 c 64.85±1.02 d Energy value (kcal/100 g) 448.58±7.34 a 435.05±8.70 b 422.22±8.73 c 407.21±7.67 d Minerals (mg/100g) Calcium 25.01±0.61 Dd 855.6±2.25 Ac 1680.8±2.82 Ab 2449.5±3.77 Aa Magnesium 135.4±1.55 Cd 142.1±0.83 Dc 147.7±1.32 Dbc 154.8±1.24 Da Phosphorus 191.3±0.66 Ba 190.4±1.81 Ca 188.7±0.91 Ca 187.9±0.72 Ca Potassium 344.2±2.14 Aa 338.0±1.60 Bab 331.8±1.42 Bbc 325.9±1.20 Bc Sodium 14.73±0.27 Ed 16.51±0.44 Ec 18.66±0.45 Eb 20.40±0.36 Ea Zinc 1.07±0.02 Fd 2.23±0.16 Fc 3.35±0.14 Fb 4.54±0.22 Fa M±SD: The mean and standard deviation values for different letters in the same row are significantly different ( P <0.05). CEsP: Chicken Eggshell Powder M±SD: The means and standard deviation values with different lowercase letters in the same row or different capital letters in the same column are significantly different ( P <0.05). CEsP: Chicken Eggshell Powder 3.4. Effect of replacement WF by CEsP on physical properties of cracker samples The findings regarding the physical properties of cracker samples prepared by partial replacement of WF with ascending levels (4%, 8%, and 12%) of CEsP are shown in Table 4 . Based on the results, it can be observed that all cracker samples containing CEsP did not exhibit significant differences (p>0.05) in terms of length, width, thickness, and weight compared to the control sample. The average weight of the crackers prepared with different levels (4%, 8%, and 12%) of CEsP (2.99 g, 3.09 g, and 3.15 g) was also not significantly higher than that of the control (2.92 g). This could be attributed to the higher moisture retention during the baking process. The puffiness and hardness in cracker samples prepared by partial replacement of WF with ascending levels (4%, 8%, and 12%) of CEsP were lower compared with those made from 100% wheat flour. These findings suggest that the presence of CEsP does not significantly impede gas retention in the cracker samples. Such differences in the physical properties could be attributed to properties in the raw materials such as eggshell flour and wheat flour. These findings align with the results documented by [30] . Table 4 : Physical properties of produced cracker samples Treatments Length (cm) Width (cm) Thickness (T) (cm) Weight (g) Puffiness (%) (T-T 0 )/T 0 × 100 Hardness (N) Control 3.01±0.11 a 2.00±0.10 a 0.30±0.02 a 2.92±0.21 a 42.85±0.65 a 23.73±0.25 a Substitution levels 4% 3.03±0.14 a 2.00±0.13 a 0.30±0.02 a 2.99±0.18 a 42.85±0.41 a 23.02±0.40 ab 8% 3.06±0.12 a 2.01±0.08 a 0.29±0.01 a 3.09±0.14 a 38.09±0.43 b 21.55±0.36 bc 12% 3.10±0.12 a 2.03±0.10 a 0.28±0.03 a 3.15±0.19 a 33.33±0.72 c 19.60±0.21 c M±SD: The mean and standard deviation values for different letters in the same column are significantly different ( P <0.05). T: Thickness of baked cracker, T 0 : Thickness of cracker dough. 3.5. The impact of replacing WF with CEsP on biological parameters. 3.5.1. Body weight, body weight gain, and food efficiency ratio of tested rats: Table 5 shows the effect of cracker sample feeding on male albino rats' body weight, body weight gain, and food efficiency ratio. The statistics in the Table demonstrate that the body weight gain of rat groups feeding on cracker samples with a substitution level of 12% of WF by CEsP was significantly lower (P≤0.05) than those values of control groups and other substitution levels (4% and 8%) groups, which recorded 58.3, 64.5, 65.2, 65.6, and 66.4 g, respectively. The results also showed that the rat groups fed on cracker samples with a substitution level of 12% of WF by CEsP demonstrated a significant decrease (P≤0.05) in feed intake compared to other treated groups. Following an investigation conducted on rats, [40] concluded that diets rich in calcium led to a reduction in weight gain. Similarly, [41] conducted a study involving adults aged 20 to 59 years and found evidence of an inverse relationship between dietary calcium intake and high body mass index. The food efficiency ratio results demonstrated that the rat groups consuming cracker samples with 4% and 8% substitution of wheat flour (WF) with calcium eggshell powder (CEsP) had significantly higher food efficiency ratios compared to the other rat groups (P≤0.05). The variations in food intake corresponded to the growth rate and feed efficiency observed among the different samples. These findings support the hypothesis that the addition of various cracker samples does not inhibit diet digestion in a specific manner. Table 5: Initial and final body weight gain, total food intake, and food efficiency ratio of rats fed on tested cracker samples. Groups Initial body weight (g) Final body weight (g) Body weight gain (g) Total food intake (g/rat/month) Food efficiency ratio (%) Control (-) 85.0±1.50 b 149.5±3.00 b 64.5±2.10 d 289.3±7.30 a 22.29±0.30 c Control (+) 88.1±1.70 a 153.3±4.10 a 65.2±1.30 bc 281.8±4.80 b 23.13±0.20 b C4 82.3±2.40 c 147.9±3.60 c 65.6±1.20 b 276.9±6.50 d 23.69±0.40 a C8 80.8±0.70 d 147.2±2.40 c 66.4±1.80 a 278.4±7.00 c 23.85±0.30 a C12 81.4±1.10 d 139.7±2.60 d 58.3±0.90 e 269.3±5.90 e 21.64±0.20 d The mean and standard deviation values for the same superscript letters in the same column are not significantly different ( P ˃0.05). Control (-): rats fed on crackers free CEsP; Control (+): rats fed on crackers free CEsP with CaCO 3 ; C4 – rats fed on crackers with replacement ratio 4% of WF by CEsP; C 8 – rats fed on crackers with replacement ratio 8% of WF by CEsP; C 12 – rats fed on crackers with replacement ratio 12% of WF by CEsP 3.5.2. In vivo calcium absorption Fig. 2 shows the percentage of calcium absorbed from the cracker samples. The results indicate that the percentage of calcium absorbed from the control (-) and control (+) cracker samples was 28.79% and 33.15%, respectively, while the values of calcium absorbed from the cracker samples containing eggshell powder were 40.29%, 44.07%, and 36.11% for the rats fed on cracker samples with a replacement ratio of 4% of WF by CEsP (C4), crackers with a replacement ratio of 8% of WF by CEsP (C8), and crackers with a replacement ratio of 12% of WF by CEsP (C12), respectively. Based on the results obtained, the group of rats (C8 group) that consumed cracker samples with an 8% replacement ratio of wheat flour (WF) with eggshell powder exhibited the highest percentage of calcium absorption. Fig. 2: In vivo calcium absorption (%) Control (-): rats fed on crackers free CEsP; Control (+): rats fed on crackers free CEsP with CaCO 3 ; C4 : rats fed on crackers with replacement ratio 4% of WF by CEsP; C8 : rats fed on crackers with replacement ratio 8% of WF by CEsP; C12 : rats fed on crackers with replacement ratio 12% of WF by CEsP From the same figure, it is also clear that when the percentage of eggshell powder used in the preparation of crackers was increased from 8% to 12%, a notable reduction was observed in the percentage of absorbed calcium, and this may be because when the percentage of eggshell powder was increased, it caused a high calcium content, which exceeded the rats' dietary calcium requirements. This phenomenon may arise from a potential delay in the breakdown of the mineral during its passage through the intestines [10]. Similarly, [42] demonstrated a positive impact of calcium supplementation through eggshell powder in humans, particularly when the mineral intake is adequate. From the above, eggshell powder is one of the most important sources rich in calcium, and it is characterized by a high level of bioavailability. [43] evaluated the bioavailability of calcium from eggshells and compared it with calcium carbonate using lab rats. The results showed a significant increase in calcium content in the femur compared to calcium carbonate. In a study by [44] , the researchers investigated the potential of rats to absorb and utilize calcium from a fortified diet containing various calcium sources, including eggshell powder. The findings revealed that eggshell powder exhibited a remarkably high capacity for absorption and utilization. Similarly, studies conducted in humans have shown that consuming eggshell powder has positive effects on bone mineral density [45, 46], suggesting that eggshell is a superior source of bioavailable calcium. 3.5.3. Serum calcium and parathyroid hormone (PTH) levels of tested rats: The levels of serum calcium and parathyroid hormone (PTH) in the rats' serum fed on different cracker samples are shown in Table 6. From the obtained results, it can be noticed that significant differences (P<0.05) were observed in the calcium levels between rats fed on different cracker sample diets. Also, the same table indicates that the serum calcium level in rats fed on cracker samples containing eggshell powder was higher than that in other rat groups. This observation may be due to the increase in chicken eggshell powder level in the diet, which affects the bioavailability of calcium. By enhancing the absorption of calcium, it leads to an improvement in bone mineral density and a decrease in the risk of developing osteoporosis. Table 6: Effect of crackers containing WF or CEsP on serum calcium and parathyroid hormone (PTH) in the tested rats. Groups Serum calcium (mg/dl) PTH (µg/ dl) Control (-) 8.40±0.20 e 21.70±0.41 a Control (+) 9.20±0.32 d 15.16±0.20 b C4 9.89±0.26 c 14.56±0.18 c C8 11.22±0.11 b 12.79±0.22 d C12 13.08±0.23 a 11.04±0.16 e The mean and standard deviation values for different letters in the same column are significantly different ( P <0.05). Control (-): rats fed on crackers free CEsP; Control (+): rats fed on crackers free CEsP with CaCO 3 ; C4: rats fed on crackers with replacement ratio 4% of WF by CEsP; C8 : rats fed on crackers with replacement ratio 8% of WF by CEsP; C12 : rats fed on crackers with replacement ratio 12% of WF by CEsP In contrast, parathyroid hormone (PTH) levels in rats fed on cracker samples containing eggshell powder were lower than those in other rat groups. Interestingly, the parathyroid hormone levels were significantly decreased (P<0.05) in all rat groups fed crackers containing chicken eggshell powder compared to other rat groups. This decrease is also more noticeable with increasing substitution levels from 8 to 12% of WF by CEsP. Furthermore, according to the above table, it is evident that rats fed a high-calcium diet exhibited elevated serum calcium levels, resulting in a decrease in blood parathyroid hormone (PTH) levels. Conversely, rats fed a low-calcium diet displayed the opposite effect. Similar findings were reported in a study conducted by [34, 47], where male rats fed low-calcium and very low-calcium diets consistently for several weeks demonstrated a significant increase in plasma PTH levels. 3.5.4. Provides the recommended dietary allowances of calcium by produced crackers: The recommended daily allowance (RDA) for calcium intake varies between 800 and 1300 mg per day for different age groups [48]. However, the specific calcium requirements differ from one country to another. For instance, in India, the recommended intake ranges from 500 to 800 mg/day for children, while in Australia, it varies from 500 to 1000 mg/day for the same age group. Inadequate levels of calcium in the body result in low bone mineral density (BMD), which, if persistent, can lead to osteoporosis and hypocalcemia. Additionally, due to the increasing life expectancy, the population is experiencing a higher proportion of elderly individuals who are highly susceptible to osteoporosis. Without dietary changes, this group remains at risk in the future. Therefore, it is crucial to augment the calcium content in the population's diet to mitigate the risk of osteoporosis among the elderly, both presently and in the future. To meet the daily calcium requirements, various calcium-fortified food products, such as fortified orange juice, flour, and pasta, are available in the market. These products often utilize calcium derived from sources such as cattle bone powder and milk powder [20]. The calcium content in cracker samples with a replacement ratio of 8% of WF by CEsP was 1680.8 mg/100g. Considering that the calcium absorption was 44.07%, the intake of bioavailable calcium in the cracker samples was 740.76 mg/100g. As given in the obtained data in Table 2S , it can be noticed that the number of fortified crackers (8% replacement ratio of WF by CEsP) that can provide the daily recommended dietary allowances of calcium ranged from 135.0 to 175.5 g of cracker products for different age categories. Furthermore, it is advisable to consume calcium in smaller quantities at different intervals throughout the day rather than consuming a large amount all at once. This approach promotes better availability and utilization of calcium in the body. Studies have demonstrated that the bioavailability of calcium increases when it is consumed with food rather than on an empty stomach [49]. The practice of consuming crackers in smaller portions at least twice a day aligns with this recommendation and offers advantages in terms of calcium utilization by the body. 4. Conclusion This study introduces a novel approach to fortifying crackers with chicken eggshell powder (CEsP) to enhance their calcium content and produce functional crackers. The findings demonstrate that incorporating CEsP as a supplement in the preparation of crackers significantly increases the overall ash percentage and specifically boosts the calcium content while simultaneously reducing the caloric value of the crackers. Additionally, the results indicate that the addition of CEsP, up to 8%, does not cause any notable alterations in the physicochemical properties of the crackers. Based on these outcomes, CEsP proves to be a valuable source of highly bioavailable calcium that can be effectively utilized in food products. Moreover, employing CEsP in food production serves as an environmentally friendly solution for managing eggshell waste, thus mitigating potential environmental issues associated with its accumulation. Declarations Conflict of interest The authors declare that they have no conflict of interest. Informed consent Informed consent to participate and consent to publish were obtained from all participants in this study. Consent for publication All authors have reviewed and approved the final version of the manuscript, and we confirm our consent for its publication. We also declare that there are no ethical or legal issues concerning the publication of this work. Funding The authors declare that no funding was received for this research. Author Contribution Taher Abdelnaby: Writing-original draft & Methodology. Mostafa M. Kadry: Discussion, Investigation & Formal Analysis. Sameh M. Ghanem: Conceptualization, Resources, Supervision. Mokhtar M. Salama: Visualization. Shaoyun Wang: Writing-review & editing. Acknowledgements The authors would like to thank everyone who helped with this study. 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Ponce, A.M.G., et al., Asociación entre la ingesta de calcio dietético y el índice de masa corporal elevado en adultos mexicanos de 20 a 59 años de edad: estudio de corte transversal. 2013. Fina, B.L., L.R. Brun, and A. Rigalli, Increase of calcium and reduction of lactose concentration in milk by treatment with kefir grains and eggshell . International Journal of Food Sciences and Nutrition, 2016. 67(2): p. 133–140. Brun, L.R., et al., Chicken eggshell as suitable calcium source at home . International journal of food sciences and nutrition, 2013. 64(6): p. 740–743. Bao, S., W. Windisch, and M. Kirchgessner, Calcium bioavailability of different organic and inorganic dietary Ca sources (citrate, lactate, acetate, oyster-shell, eggshell, β‐tri‐Ca phosphate) . Journal of animal physiology and animal nutrition, 1997. 78(1‐5): p. 154–160. Schaafsma, A., et al., Positive effects of a chicken eggshell powder-enriched vitamin–mineral supplement on femoral neck bone mineral density in healthy late post-menopausal Dutch women . British Journal of Nutrition, 2002. 87(3): p. 267–275. Sakai, S., et al., Effects of eggshell calcium supplementation on bone mass in postmenopausal Vietnamese women . Journal of Nutritional Science and Vitaminology, 2017. 63(2): p. 120–124. He, Y.-H., et al., The calcium-sensing receptor affects fat accumulation via effects on antilipolytic pathways in adipose tissue of rats fed low-calcium diets . The Journal of nutrition, 2011. 141(11): p. 1938–1946. Ross, A.C., et al., The 2011 Dietary Reference Intakes for Calcium and Vitamin D: what dietetics practitioners need to know . Journal of the American Dietetic Association, 2011. 111(4): p. 524–527. Rafferty, K., G. Walters, and R.P. Heaney, Calcium fortificants: overview and strategies for improving calcium nutriture of the US population . Journal of food science, 2007. 72(9): p. R152-R158. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Jan, 2025 Reviews received at journal 17 Jan, 2025 Reviewers agreed at journal 09 Jan, 2025 Reviews received at journal 04 Jan, 2025 Reviewers agreed at journal 30 Dec, 2024 Reviewers invited by journal 20 Dec, 2024 Editor assigned by journal 20 Dec, 2024 Submission checks completed at journal 19 Dec, 2024 First submitted to journal 05 Nov, 2024 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-5398539","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392676971,"identity":"8f555dda-f9eb-42f8-ae76-adca0cc374a0","order_by":0,"name":"Mokhtar M. Salama","email":"","orcid":"","institution":"Al-Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Mokhtar","middleName":"M.","lastName":"Salama","suffix":""},{"id":392676972,"identity":"f25b706a-240e-431d-abc8-05a15545b100","order_by":1,"name":"Sameh M. Ghanem","email":"","orcid":"","institution":"Al-Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Sameh","middleName":"M.","lastName":"Ghanem","suffix":""},{"id":392676973,"identity":"2b847287-f63d-4806-a5a7-cdefd1773539","order_by":2,"name":"Mostafa M. Kadry","email":"","orcid":"","institution":"Al-Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Mostafa","middleName":"M.","lastName":"Kadry","suffix":""},{"id":392676976,"identity":"5f75ce59-5c5e-4e0e-b05e-a8bcfb85c655","order_by":3,"name":"Shaoyun Wang","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Shaoyun","middleName":"","lastName":"Wang","suffix":""},{"id":392676979,"identity":"b91e3e54-f4ac-4916-9789-4ef1199ade0b","order_by":4,"name":"Taher Abdelnaby","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYFACHiCusGFgkADzmInRANJyJo1ULYxth0nQYs9+9uDjijPn5eVnNz/7wFBhndjA3n4Bvy08ecmGZypuGzbOOWY8g+FMemIDz5kCAg7LMZNsOHObsVkiwRjkwsQGiZwE/Fr435j/bGw7Z98mkf6ZgfEfUIv8GwJaJHLMGBvbDiT2SOQAbWkA2cJ+AL+WG2+MgQ5LTp4hkVPMkHAs3biNJwevDgb2/hzDjw0VdrbzZ6RvZvhQYy3bz378AX49KADkCTYGHgMStEBtJsWWUTAKRsEoGAEAAHnpRMC9um52AAAAAElFTkSuQmCC","orcid":"","institution":"Al-Azhar University","correspondingAuthor":true,"prefix":"","firstName":"Taher","middleName":"","lastName":"Abdelnaby","suffix":""}],"badges":[],"createdAt":"2024-11-06 01:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5398539/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5398539/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72149893,"identity":"0b830dbd-0513-47b3-bb94-a1939ec594d6","added_by":"auto","created_at":"2024-12-23 08:06:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12641,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 2: In vivo calcium absorption (%)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5398539/v1/e9b808e5072a856bf620ae0b.png"},{"id":72151489,"identity":"c8e11975-4b10-4661-8af9-aadc81bde21a","added_by":"auto","created_at":"2024-12-23 08:22:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1204719,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5398539/v1/1910ff26-fbe7-46fa-92f6-dab499c96175.pdf"},{"id":72149894,"identity":"6c3d2c27-04cf-4547-909a-e51f28bde350","added_by":"auto","created_at":"2024-12-23 08:06:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16657,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5398539/v1/4fb8609c4f49f8971dd41637.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Utilization of Chicken Eggshells as a Source of Calcium to Enhance the Nutritional Value and Quality Characteristics of Crackers","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOsteoporosis, a condition resulting from calcium deficiency, has become one of the most significant global health issues in recent years. It is estimated that approximately 200\u0026nbsp;million people worldwide suffer from calcium deficiency [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Studies have indicated that this deficiency can be attributed to various factors, including the high cost of milk and its products, which prevents some individuals from purchasing them in many countries. Additionally, certain people face health issues, such as lactose intolerance, that limit their consumption of milk and its products [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, it is imperative to explore the development of functional foods that can fulfill the daily calcium needs of these individuals.\u003c/p\u003e \u003cp\u003eThe global production of eggs has consistently increased over the past decade, with an observed growth of more than 30% from 2009 to 2019 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Eggshells are a common household waste generated regularly from households and food processing facilities. Due to the low cost and high nutrient content of eggs, their consumption has been on the rise globally, leading to a 150% increase in egg productivity over the past three decades. However, this increased production has resulted in environmental and health challenges due to the accumulation of chicken eggshells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn various countries, including Egypt, egg product companies and food manufacturers, such as those producing baked goods and mayonnaise, generate a significant amount of waste from chicken eggshells, contributing to environmental issues. In Egypt specifically, the annual egg production in 2016 reached a record of 510,000 tons [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The egg product industry in Egypt alone produced approximately 56,100 tons of chicken eggshells in the same year [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlmost all of the eggshell is considered waste, and the disposal of these discarded eggshells contributes to food waste, resulting in significant environmental damage by increasing the global carbon footprint (3.3 Gtonnes of CO2 equivalent in 2007) when they are buried [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This contributes to greenhouse gas emissions, one of the major causes of global warming [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and leads to microbial proliferation due to the substantial amount of waste generated [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, the disposal of Eggshell waste poses challenges in terms of cost and utilization of landfill space. Hence, it is of utmost importance to prioritize enhancing the value of eggshells and incorporating them effectively into the food processing sectors.\u003c/p\u003e \u003cp\u003eChicken eggshells represent approximately 9\u0026ndash;12% of the weight of a medium-sized egg (54\u0026ndash;60 g), and the main component responsible for their formation is calcium carbonate. Humans can utilize eggshells as a source of calcium and other minerals such as magnesium and zinc [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] conducted a study indicating that every 2.7 g of chicken eggshells can provide sufficient calcium to meet the daily needs of adults. Adequate calcium intake, ranging from 200\u0026ndash;1200 mg daily, is crucial for maintaining healthy bone status, particularly after menopause, beginning with early infancy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Chicken eggshells consist of around 95% calcium carbonate (CaCO3) and contain valuable elements like strontium and boron, which play crucial roles in the prevention of osteoporosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated the potential of chicken eggshells as a source of calcium [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous research has also indicated the possibility of incorporating eggshells as a calcium source in various food products, including bread [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], biscuits [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], muffins [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], chocolate cakes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], yogurt [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and more, without causing any significant alteration in flavor.\u003c/p\u003e \u003cp\u003eCrackers, known for their distinct flavors, long shelf life, and affordable prices, are popular snack foods consumed daily [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Various materials have been used to fortify crackers and enhance their functionality. Examples of such materials include germinated lentil extracts [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], hibiscus residues [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and tuna bone bio-calcium powder [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to examine the impacts of substituting wheat flour (WF) with varying levels (4%, 8%, and 12%) of CEsP on the chemical composition, mineral content, and physical characteristics of the resulting crackers. Additionally, this work aimed to evaluate the potential biological effects of the produced crackers through in vivo assessments.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe wheat flour (72%) used in this study was obtained from the South Cairo Mills Company located in Giza, Egypt. Chicken eggs, other ingredients, and materials required for cracker production were purchased from the local market in Nasr City, Cairo, Egypt. Chemicals used in the analytical procedures were sourced from El-Gamhouria Trading Chemicals and Drugs Company, situated in Cairo, Egypt.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation of samples\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Preparation of chicken eggshell powder\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCEsP was processed following the method described by [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The eggshells were collected and washed inside and outside using a sponge under running water. They were then placed in boiling water for 30 minutes and subsequently subjected to a drying and sterilization process at a temperature of 135\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026ordm;C for 15 minutes using a hot air oven (Labocon Systems Limited, Hampshire, UK). The dried eggshells were ground using a grinder (Braun AG, No, 4122, Germany), and the resulting powder was sieved to obtain CEsP. The CEsP was packed in polyethylene containers and stored in a refrigerator at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026ordm;C until further use.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Preparation of cracker blends\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the preparation of cracker blends, following the method by [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], WF was substituted with 0%, 4%, 8%, and 12% of CEsP, respectively. Each flour mixture was blended and homogenized separately before being packed in tightly sealed polyethylene bags. These bags were stored at room temperature (20\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026ordm;C) for subsequent analysis and processing. The composition of the prepared blended powders is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIngredients recipe for preparation crackers blends (g / 100 g).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIngredients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eCrackers- CEsP samples\u003c/p\u003e \u003cp\u003eSubstitution levels\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWheat flour\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e58.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEggshell powder\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVegetable oil\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSucrose\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBaking powder\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSalt\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDistilled water\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eCEsP\u003c/b\u003e: Chicken Eggshell Powder\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Produce of cracker samples:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo produce the crackers, all the ingredients were weighed based on a batch size of 100 g. The dry components were combined, followed by the addition of oil to the mixture. Water was subsequently added to the dough, which was kneaded for 1 minute using a food processor (Moulinex, France) until a smooth, homogeneous, and cohesive dough was obtained. The dough was then left to rest for 4 minutes at room temperature (20\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026ordm;C). After resting, the dough was rolled out, cut into desired shapes, and left for 10 minutes at room temperature. The crackers were baked in a thermal oven at 200\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C for 12 minutes and then cooled for 30 minutes at room temperature before being stored in sealed plastic containers until further use. Three separate batches were produced from each blend to replicate [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Analytical Methods:\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Chicken eggshell powder microbiological examination:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe total bacterial count (TBC), \u003cem\u003eColiform\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e were determined following the methodology outlined by [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Proximate chemical composition, energy values, and minerals content:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe proximate chemical components (moisture, ash, ether extract, crude protein, and fiber) of the tested WF, CEsP, and cracker samples were assessed using the methodologies outlined in the reference [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The difference calculated carbohydrate content: % carbohydrates\u0026thinsp;=\u0026thinsp;100 - (% crude protein + % ash + % ether extract + % crude fiber). The energy values in kcal/100 g for the cracker samples were calculated using the method described by [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The calculation formula used was as follows: Energy value (kcal/100 g)\u0026thinsp;=\u0026thinsp;4 (carbohydrate\u0026thinsp;+\u0026thinsp;protein)\u0026thinsp;+\u0026thinsp;9 (fat).\u003c/p\u003e \u003cp\u003eThe mineral content (calcium, magnesium, phosphorus, potassium, sodium, and zinc) of WF, CEsP, and cracker samples was determined according to the [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Physical and textural properties:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePhysical parameters such as length, width, weight, thickness, and puffiness of the cracker samples were measured based on the procedure outlined by [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Hardness was assessed using a texture analyzer (Stable Micro Systems, Godalming, Surrey, UK), as reported by [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Biological investigation:\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn this study, a total of twenty-five young male albino rats weighing approximately 80\u0026ndash;90 grams were purchased from the animal house. The biological experiments were conducted at the animal house laboratory at the Agricultural Research Center (ARC) in Giza, Egypt, According to the protocol approved by the Agricultural Research Center (ARC) and the Institutional Animal Care and Use Committee (IACUC) reviewers (IACUC protocol number: ARC-AZU-62-24), and following the 2014 Egyptian Constitution, Article 45, the official decrees of the Ministry of Agriculture No. 27 (1967), the World Organization for Animal Health (WOAH), and the Guide for the Care and Use of Laboratory Animals, 8th Edition (2011). The rats were housed separately in stainless steel cages under controlled environmental conditions, including a constant temperature of 22\u0026deg;C and a 12-hour light-dark cycle. They had unrestricted access to food and water throughout the experiment. Before the commencement of the study, a group of rats were provided with a well-balanced diet (AIN-93) for one week to acclimatize to the experimental conditions. The experimental diets were prepared by mixing the finely ground crackers and a mixture of casein with vitamins at a rate of 60 and 1.5 gm per kg, respectively, to provide the nutritional needs of the rats. After the adaptation period, the rats were divided into five groups: Control (-) (rats fed on crackers free of eggshells), Control (+) (rats fed on crackers free of eggshells mixed with CaCO3 at a ratio of 3.5 g/kg), C4 (rats fed on crackers with a replacement ratio of 4% of WF by CEsP), C8 (rats fed on crackers with a replacement ratio of 8% of WF by CEsP), and C12 (rats fed on crackers with a replacement ratio of 12% of WF by CEsP). Each group consisted of five rats. The rats were fed experimental diets for four weeks. At the end of the experiment, the rats underwent an overnight fasting period, and measurements were taken for total food intake, final body weights of the rats, and the amount of weight gained during the experimental period.\u003c/p\u003e\u003cp\u003eThe food efficiency ratio was determined using the following equation:\u003c/p\u003e\u003cp\u003eFood efficiency ratio\u0026thinsp;=\u0026thinsp;body weight gain/food intake [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.1. In vivo calcium absorption measurement:\u003c/h2\u003e \u003cp\u003eTo estimate calcium absorption in vivo, only 3.5 grams of calcium carbonate were added for each kilogram of crackers free of eggshells in the control group (+), according to [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Calcium absorption determination was also performed in the other groups (control group (-), C4, C8, and C12). The rats were provided with experimental diets for four days. On the fourth day, the amount of feed intake during the previous 24 hours was calculated. The feces were collected, dried in the air, and crushed. The amount of calcium in the consumed food and feces was estimated using an atomic absorption spectrophotometer (Perkin Elmer 4100 ZL., Perkin Elmer Inc., Wellesley, USA) (Varian AA240; Varian Inc., Australia) based on the method described in [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and the amount of calcium absorbed was determined using the following equation:\u003c/p\u003e\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1734940472.png\" style=\"width: 472px;\"\u003e\u003cbr\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.2. Determination of calcium level in blood serum:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe calcium level in the collected blood serum samples was calculated using the method provided by [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.3. Parathyroid hormone (PTH) assay:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eParathyroid hormone (PTH) in blood serum samples was measured using conventional biochemical analysis methods, as described by [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The Enzyme-Linked Immunosorbent Assay (ELISA) technique was employed using commercially available test kits.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Statistical analysis:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe data obtained from our study were expressed as mean values along with their corresponding standard deviations. Statistical analysis was performed using SPSS software (version 20.0, IBM Software, Inc., Chicago, USA, 2018) in a completely randomized design. The statistical analysis included a one-way ANOVA conducted at a significance level of 5%, followed by Duncan's multiple range tests (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003ch2\u003e3.1.\u0026nbsp;CEsP microbiological quality\u003c/h2\u003e\n\u003cp\u003eResults in Table 1S refer to the microbial content of the CEsP after the boiling process. The results reported in the table indicate that the eggshells were entirely free of microbes, as the total count of bacteria indicated a negative result, and no microbial numbers appeared. The results also indicated that the CEsP was free of coliform, salmonella, and staphylococcus after the boiling process. The obtained results are consistent with those obtained by [17] and [35]\u003cstrong\u003e,\u003c/strong\u003e which state that the boiling process was sufficient to get rid of the microbes that may be present on the eggshell, making the shells safe to be added to food without causing problems.\u003c/p\u003e\n\u003ch2\u003e3.2.\u0026nbsp;The proximate chemical composition, caloric value, and mineral content (mg/100 g) of eggshell powder compared to wheat flour.\u003c/h2\u003e\n\u003cp\u003eData in\u0026nbsp;Table 2\u0026nbsp;reveal the difference in the content of crude protein, fat, ash, carbohydrates, crude fiber, and energy value between the raw materials (WF and CEsP) on a dry basis. The results indicate significant differences (P\u0026lt;0.05) between the studied raw materials in their chemical components. From the data, it can be observed that WF had greater quantities of moisture, crude protein, and carbohydrate (11.31%, 10.64%, and 87.16%, respectively) compared to CEsP, which had values of 1.02%, 3.31%, and 1.38%, respectively. In contrast, the same table shows that the ash content in CEsP was approximately 120 times higher than that found in WF (0.62% vs. 94.82% on a dry weight basis). Therefore, CEsP is considered an excellent source of ash content. These results are consistent with those provided by [18, 20]\u003cstrong\u003e.\u003c/strong\u003e Significant differences were observed between WF and CEsP regarding the caloric value. CEsP had the lowest caloric value (18.24 Kcal/100g) compared to WF (400.83 Kcal/100g).\u003c/p\u003e\n\u003cp\u003eTable 2\u0026nbsp;provides information on the major macro and micro elements such as magnesium (Mg), calcium (Ca), phosphorus (P), sodium (Na), potassium (K), and zinc (Zn) in both WF and CEsP. The table shows a comparison between WF and CEsP samples regarding their mineral content. As shown in the results, calcium constituted the highest proportion among the macro-elements in CEsP, with a content of approximately 35238.00 mg/100g. CEsP also exhibited elevated levels of magnesium, sodium, and zinc (288.78 mg/100g, 84.32 mg/100g, and 47.33 mg/100g, respectively) compared to WF, while WF was superior in potassium (297.08 mg/100g vs. 48.12 mg/100g, respectively).\u003c/p\u003e\n\u003cp\u003eSimilarly, the data demonstrate that CEsP and WF contained a similar amount of phosphorus (118.61 mg/100g and 132.11 mg/100g, respectively). Therefore, CEsP was superior in calcium, magnesium, sodium, and zinc, while WF was superior in potassium and relatively superior in phosphorus. These findings align with the findings reported by [36]\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eAs a result, CEsP stands out for its high content of various tested minerals, both macro and micro-elements. Therefore, there is potential for the reutilization of CEsP in food fortification. In general, CEsP can be regarded as a valuable source of essential macro and micro-elements.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;2: The proximate chemical composition, caloric value, and mineral content of eggshell powder compared with wheat flour.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompounds (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWF\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCEsP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMoisture\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e11.31\u0026plusmn;0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e1.02\u0026plusmn;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrude Protein\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e10.64\u0026plusmn;0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e3.31\u0026plusmn;0.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEther extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e1.07\u0026plusmn;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e0.31\u0026plusmn;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAsh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e0.62\u0026plusmn;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e94.82\u0026plusmn;0.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrude Fiber\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e0.51\u0026plusmn;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e0.18\u0026plusmn;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarbohydrates\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e87.16\u0026plusmn;0.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e1.38\u0026plusmn;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnergy value (kcal/100 g)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e400.83\u0026plusmn;3.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e18.24\u0026plusmn;1.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 596px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinerals (mg/100g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCalcium\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e24.92\u0026plusmn;0.43\u003csup\u003eCb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e35238.00\u0026plusmn;4.89\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMagnesium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e129.17\u0026plusmn;0.98\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e288.78\u0026plusmn;1.62\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhosphorus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e132.11\u0026plusmn;1.21\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e118.61\u0026plusmn;0.92\u003csup\u003eCb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePotassium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e297.08\u0026plusmn;1.54\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e48.12\u0026plusmn;0.71\u003csup\u003eEb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSodium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e5.91\u0026plusmn;0.17\u003csup\u003eDb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e84.32\u0026plusmn;1.05\u003csup\u003eDa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZinc\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 154px;\"\u003e\n \u003cp\u003e0.84\u0026plusmn;0.04\u003csup\u003eEb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 203px;\"\u003e\n \u003cp\u003e47.33\u0026plusmn;0.53\u003csup\u003eEa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eM\u0026plusmn;SD: The mean and standard deviation values for different letters in the same row are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). \u0026nbsp;WF: Wheat Flour. CEsP: Chicken Eggshell Powder\u003c/p\u003e\n\u003ch2\u003e3.3.\u0026nbsp;Effect of replacement of WF by CEsP on quality criteria of cracker samples:\u003c/h2\u003e\n\u003cp\u003eTable \u0026nbsp;3 presents the proximate chemical composition, caloric value, and mineral content of the cracker samples, which were influenced by various replacement levels (4%, 8%, and 12%) of WF with CEsP. The control sample, without any addition of CEsP, is also included in the table.\u003c/p\u003e\n\u003cp\u003eBased on the results, there were no significant differences (P\u0026le;0.05) in the protein, fat, and fiber contents of the cracker samples as the substitution level of WF by CEsP increased from 4% to 12%. The protein content ranged from 12.81% to 13.07%, the fat content ranged from 10.73% to 10.89%, and the fiber content ranged from 0.63% to 0.67%. These values were slightly lower compared to the control sample, which had a protein content of 13.18%, fat content of 10.94%, and fiber content of 0.69%. In contrast, the moisture content exhibited a slight and gradual increase as the substitution level of WF by CEsP increased from 4% to 12% compared to the control sample. A similar trend was observed for the ash content, which gradually increased with the replacement percentage from 4% to 12%. However, the increase was more pronounced in the case of ash content, with values rising from 0.84% in the control sample to 10.98% in the cracker samples at a 12% replacement ratio. Additionally, it is evident that as the levels of CEsP increased in the cracker samples, the caloric value decreased.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings could be attributed to the gradual decrease in carbohydrates resulting from the substitution of CEsP. In general, it can be concluded that cracker samples containing CEsP exhibited favorable nutritional quality in terms of ash, protein, and crude fiber contents. These results align with the findings of\u0026nbsp;[18]\u003cstrong\u003e,\u003c/strong\u003e who observed an increase in ash content without affecting fat and protein contents when CEsP was supplemented in biscuits at various levels (5%, 10%, 15%, and 20%). Additionally, a significant reduction in the caloric value of the biscuits was observed. Similar findings were reported by [37] about white bread.\u003c/p\u003e\n\u003cp\u003eThe mineral content of both the control sample and the tested cracker samples with replacement ratios of 4%, 8%, and 12% of WF by CEsP were determined and recorded in Table \u0026nbsp;3. Mineral elements were measured, and significant differences were demonstrated between all formulations. The amount of calcium greatly increased at a 4% replacement ratio compared to the control, which increased from 25.01 mg/100 g to 855.6 mg/100 g. This increase was consistently observed for 8% and 12% CEsP addition, which were found to be 1680.8 mg/100 g and 2449.5 mg/100 g, respectively. Based on the results obtained, the calcium content in crackers was greatly influenced by the high level of calcium in CEsP compared to the calcium content in WF. Besides calcium, the results show that the mineral contents in crackers containing CEsP increased for magnesium, sodium, and zinc. The magnesium content in crackers increased in the range of 135.4 mg/100 g to 154.8 mg/100 g from the control up to 12% supplementation of CEsP. Sodium was high in crackers where the substitution ratio of WF by CEsP reached 12%, recorded as 20.40 mg/100 g compared to 14.73 mg/100 g in the control sample. For zinc content, the control sample showed 1.07 mg/100 g, then increased to 4.54 mg/100 g after WF substitution by 12% of CEsP. For potassium content, the control crackers showed 344.2 mg/100 g, then decreased to 325.9 mg/100 g in a substitution ratio of 12% of WF by CEsP. Also, when the replacement level of WF by CEsP increased from 4% to 12%, the phosphorus content decreased slightly from 190.4 mg/100 g to 187.9 mg/100 g when compared with the control sample, which recorded 191.3 mg/100 g. These findings are consistent with similar results reported by [17, 38]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable \u0026nbsp;3: Proximate chemical composition, caloric value, and minerals content of crackers prepared by substituting wheat flour with eggshell powder (on a dry weight basis).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompounds (g/100g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 320px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrackers-\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CEsP Samples\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSubstitution levels\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMoisture\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e4.28\u0026plusmn;0.15\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e4.83\u0026plusmn;0.21\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e5.24\u0026plusmn;0.26\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e5.91\u0026plusmn;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrude Protein\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e13.18\u0026plusmn;0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e13.07\u0026plusmn;0.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e12.94\u0026plusmn;0.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e12.81\u0026plusmn;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEther extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e10.94\u0026plusmn;0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e10.89\u0026plusmn;0.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e10.82\u0026plusmn;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e10.73\u0026plusmn;0.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAsh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.84\u0026plusmn;0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e4.18\u0026plusmn;0.18\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e7.31\u0026plusmn;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e10.98\u0026plusmn;0.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrude Fiber\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.69\u0026plusmn;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.67\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.66\u0026plusmn;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.63\u0026plusmn;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarbohydrates\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e74.35\u0026plusmn;0.84\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e71.19\u0026plusmn;0.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e68.27\u0026plusmn;1.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e64.85\u0026plusmn;1.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnergy value (kcal/100 g)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e448.58\u0026plusmn;7.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e435.05\u0026plusmn;8.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e422.22\u0026plusmn;8.73\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e407.21\u0026plusmn;7.67\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 627px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinerals (mg/100g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCalcium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e25.01\u0026plusmn;0.61\u003csup\u003eDd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e855.6\u0026plusmn;2.25\u003csup\u003eAc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1680.8\u0026plusmn;2.82\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2449.5\u0026plusmn;3.77\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMagnesium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e135.4\u0026plusmn;1.55\u003csup\u003eCd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e142.1\u0026plusmn;0.83\u003csup\u003eDc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e147.7\u0026plusmn;1.32\u003csup\u003eDbc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e154.8\u0026plusmn;1.24\u003csup\u003eDa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhosphorus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e191.3\u0026plusmn;0.66\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e190.4\u0026plusmn;1.81\u003csup\u003eCa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e188.7\u0026plusmn;0.91\u003csup\u003eCa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e187.9\u0026plusmn;0.72\u003csup\u003eCa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePotassium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e344.2\u0026plusmn;2.14\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e338.0\u0026plusmn;1.60\u003csup\u003eBab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e331.8\u0026plusmn;1.42\u003csup\u003eBbc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e325.9\u0026plusmn;1.20\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSodium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e14.73\u0026plusmn;0.27\u003csup\u003eEd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e16.51\u0026plusmn;0.44\u003csup\u003eEc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e18.66\u0026plusmn;0.45\u003csup\u003eEb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e20.40\u0026plusmn;0.36\u003csup\u003eEa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZinc\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.07\u0026plusmn;0.02\u003csup\u003eFd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2.23\u0026plusmn;0.16\u003csup\u003eFc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e3.35\u0026plusmn;0.14\u003csup\u003eFb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e4.54\u0026plusmn;0.22\u003csup\u003eFa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eM\u0026plusmn;SD: The mean and standard deviation values for different letters in the same row are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u0026nbsp;CEsP: Chicken Eggshell Powder\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM\u0026plusmn;SD: The means and standard deviation values with different lowercase letters in the same row or different capital letters in the same column are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). \u0026nbsp;CEsP: Chicken Eggshell Powder\u003c/p\u003e\n\u003ch2\u003e3.4.\u0026nbsp;Effect of replacement WF by CEsP on physical properties of cracker samples\u003c/h2\u003e\n\u003cp\u003eThe findings regarding the physical properties of cracker samples prepared by partial replacement of WF with ascending levels (4%, 8%, and 12%) of CEsP are shown in \u003cstrong\u003eTable \u0026nbsp;4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBased on the results, it can be observed that all cracker samples containing CEsP did not exhibit significant differences (p\u0026gt;0.05) in terms of length, width, thickness, and weight compared to the control sample. The average weight of the crackers prepared with different levels (4%, 8%, and 12%) of CEsP (2.99 g, 3.09 g, and 3.15 g) was also not significantly higher than that of the control (2.92 g). This could be attributed to the higher moisture retention during the baking process. The puffiness and hardness in cracker samples prepared by partial replacement of WF with ascending levels (4%, 8%, and 12%) of CEsP were lower compared with those made from 100% wheat flour. These findings suggest that the presence of CEsP does not significantly impede gas retention in the cracker samples. Such differences in the physical properties could be attributed to properties in the raw materials such as eggshell flour and wheat flour. These findings align with the results documented\u0026nbsp;by [30]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e:\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePhysical properties of produced cracker samples\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"681\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWidth\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThickness (T)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeight (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePuffiness (%)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(T-T\u003csub\u003e0\u003c/sub\u003e)/T\u003csub\u003e0\u003c/sub\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e\u0026times;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHardness\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.01\u0026plusmn;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.00\u0026plusmn;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.30\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.92\u0026plusmn;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e42.85\u0026plusmn;0.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e23.73\u0026plusmn;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstitution levels\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.03\u0026plusmn;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.00\u0026plusmn;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.30\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.99\u0026plusmn;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e42.85\u0026plusmn;0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e23.02\u0026plusmn;0.40\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.06\u0026plusmn;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.01\u0026plusmn;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.29\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.09\u0026plusmn;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e38.09\u0026plusmn;0.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e21.55\u0026plusmn;0.36\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.10\u0026plusmn;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.03\u0026plusmn;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.28\u0026plusmn;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.15\u0026plusmn;0.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e33.33\u0026plusmn;0.72\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e19.60\u0026plusmn;0.21\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eM\u0026plusmn;SD: The mean and standard deviation values for different letters in the same column are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u0026nbsp;T: Thickness of baked cracker, T\u003csub\u003e0\u003c/sub\u003e: Thickness of cracker dough.\u003c/p\u003e\n\u003ch2\u003e3.5.\u0026nbsp;The impact of replacing WF with CEsP on biological parameters.\u003c/h2\u003e\n\u003ch3\u003e3.5.1. \u0026nbsp; Body weight, body weight gain, and food efficiency ratio of tested rats:\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eTable \u0026nbsp;5\u003c/strong\u003e shows the effect of cracker sample feeding on male albino rats\u0026apos; body weight, body weight gain, and food efficiency ratio.\u003c/p\u003e\n\u003cp\u003eThe statistics in the Table demonstrate that the body weight gain of rat groups feeding on cracker samples with a substitution level of 12% of WF by CEsP was significantly lower (P\u0026le;0.05) than those values of control groups and other substitution levels (4% and 8%) groups, which recorded 58.3, 64.5, 65.2, 65.6, and 66.4 g, respectively. The results also showed that the rat groups fed on cracker samples with a substitution level of 12% of WF by CEsP demonstrated a significant decrease (P\u0026le;0.05) in feed intake compared to other treated groups. Following an investigation conducted on rats, [40] concluded that diets rich in calcium led to a reduction in weight gain. Similarly, [41] conducted a study involving adults aged 20 to 59 years and found evidence of an inverse relationship between dietary calcium intake and high body mass index.\u003c/p\u003e\n\u003cp\u003eThe food efficiency ratio results demonstrated that the rat groups consuming cracker samples with 4% and 8% substitution of wheat flour (WF) with calcium eggshell powder (CEsP) had significantly higher food efficiency ratios compared to the other rat groups (P\u0026le;0.05). The variations in food intake corresponded to the growth rate and feed efficiency observed among the different samples. These findings support the hypothesis that the addition of various cracker samples does not inhibit diet digestion in a specific manner.\u003c/p\u003e\n\u003cp\u003eTable \u0026nbsp;5: Initial and final body weight gain, total food intake, and food efficiency ratio of rats fed on tested cracker samples.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"673\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInitial body weight (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFinal body weight (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBody weight gain (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal food intake\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(g/rat/month)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFood efficiency ratio (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl (-)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e85.0\u0026plusmn;1.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e149.5\u0026plusmn;3.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e64.5\u0026plusmn;2.10\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e289.3\u0026plusmn;7.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e22.29\u0026plusmn;0.30\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl (+)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e88.1\u0026plusmn;1.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e153.3\u0026plusmn;4.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e65.2\u0026plusmn;1.30\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e281.8\u0026plusmn;4.80\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e23.13\u0026plusmn;0.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e82.3\u0026plusmn;2.40\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e147.9\u0026plusmn;3.60\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e65.6\u0026plusmn;1.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e276.9\u0026plusmn;6.50\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e23.69\u0026plusmn;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e80.8\u0026plusmn;0.70\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e147.2\u0026plusmn;2.40\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e66.4\u0026plusmn;1.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e278.4\u0026plusmn;7.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e23.85\u0026plusmn;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e81.4\u0026plusmn;1.10\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e139.7\u0026plusmn;2.60\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e58.3\u0026plusmn;0.90\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e269.3\u0026plusmn;5.90\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e21.64\u0026plusmn;0.20\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe mean and standard deviation values for the\u0026nbsp;same superscript\u0026nbsp;letters in the same column are not significantly different (\u003cem\u003eP\u003c/em\u003e ˃0.05). \u003cstrong\u003eControl (-):\u0026nbsp;\u003c/strong\u003erats fed on crackers free CEsP; \u003cstrong\u003eControl (+):\u0026nbsp;\u003c/strong\u003erats fed on crackers free CEsP with CaCO\u003csub\u003e3\u003c/sub\u003e;\u0026nbsp;C4\u0026nbsp;\u0026ndash;\u0026nbsp;rats fed on\u0026nbsp;crackers with replacement ratio 4% of WF by CEsP; C\u003csub\u003e8\u003c/sub\u003e \u0026ndash; rats fed on crackers with replacement ratio 8% of WF by CEsP; C\u003csub\u003e12\u003c/sub\u003e \u0026ndash; rats fed on crackers with replacement ratio 12% of WF by CEsP \u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e3.5.2. \u0026nbsp; In vivo calcium absorption\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eFig. 2 shows the percentage of calcium absorbed from the cracker samples. The results indicate that the percentage of calcium absorbed from the control (-) and control (+) cracker samples was 28.79% and 33.15%, respectively, while the values of calcium absorbed from the cracker samples containing eggshell powder were 40.29%, 44.07%, and 36.11% for the rats fed on cracker samples with a replacement ratio of 4% of WF by CEsP (C4), crackers with a replacement ratio of 8% of WF by CEsP (C8), and crackers with a replacement ratio of 12% of WF by CEsP (C12), respectively. Based on the results obtained, the group of rats (C8 group) that consumed cracker samples with an 8% replacement ratio of wheat flour (WF) with eggshell powder exhibited the highest percentage of calcium absorption.\u003c/p\u003e\n\u003cp\u003eFig.\u0026nbsp;2: In vivo calcium absorption (%)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eControl (-):\u0026nbsp;\u003c/strong\u003erats fed on crackers free CEsP; \u003cstrong\u003eControl (+):\u0026nbsp;\u003c/strong\u003erats fed on crackers free CEsP with CaCO\u003csub\u003e3\u003c/sub\u003e; \u0026nbsp; \u003cstrong\u003eC4\u003c/strong\u003e: rats fed on crackers with replacement ratio 4% of WF by CEsP; \u003cstrong\u003eC8\u003c/strong\u003e: rats fed on crackers with replacement ratio 8% of WF by CEsP; \u003cstrong\u003eC12\u003c/strong\u003e: rats fed on crackers with replacement ratio 12% of WF by CEsP \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom the same figure, it is also clear that when the percentage of eggshell powder used in the preparation of crackers was increased from 8% to 12%, a notable reduction was observed in the percentage of absorbed calcium, and this may be because when the percentage of eggshell powder was increased, it caused a high calcium content, which exceeded the rats\u0026apos; dietary calcium requirements. This phenomenon may arise from a potential delay in the breakdown of the mineral during its passage through the intestines [10]. Similarly, [42]\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edemonstrated a positive impact of calcium supplementation through eggshell powder in humans, particularly when the mineral intake is adequate.\u003c/p\u003e\n\u003cp\u003eFrom the above, eggshell powder is one of the most important sources rich in calcium, and it is characterized by a high level of bioavailability. [43] evaluated the bioavailability of calcium from eggshells and compared it with calcium carbonate using lab rats. The results showed a\u0026nbsp;significant increase in calcium content in the femur compared to calcium carbonate. In a study by\u0026nbsp;[44]\u003cstrong\u003e,\u003c/strong\u003e the researchers investigated the potential of rats to absorb and utilize calcium from a fortified diet containing various calcium sources, including eggshell powder. The findings revealed that eggshell powder exhibited a remarkably high capacity for absorption and utilization. Similarly, studies conducted in humans have shown that consuming eggshell powder has positive effects on bone mineral density [45, 46], suggesting that eggshell is a superior source of bioavailable calcium.\u003c/p\u003e\n\u003ch3\u003e3.5.3. \u0026nbsp; Serum calcium and parathyroid hormone (PTH) levels of tested rats:\u003c/h3\u003e\n\u003cp\u003eThe levels of serum calcium and parathyroid hormone (PTH) in the rats\u0026apos; serum fed on different cracker samples are shown in Table \u0026nbsp;6. From the obtained results, it can be noticed that significant differences (P\u0026lt;0.05) were observed in the calcium levels between rats fed on different cracker sample diets. Also, the same table indicates that the serum calcium level in rats fed on cracker samples containing eggshell powder was higher than that in other rat groups. This observation may be due to the increase in chicken eggshell powder level in the diet, which affects the bioavailability of calcium. By enhancing the absorption of calcium, it leads to an improvement in bone mineral density and a decrease in the risk of developing osteoporosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable \u0026nbsp;6: Effect\u003cem\u003e\u0026nbsp;\u003c/em\u003eof crackers containing WF or CEsP on serum calcium and parathyroid hormone (PTH) in the tested rats.\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerum calcium\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mg/dl)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePTH\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g/ dl)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl (-)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e8.40\u0026plusmn;0.20\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e21.70\u0026plusmn;0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl (+)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e9.20\u0026plusmn;0.32\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e15.16\u0026plusmn;0.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e9.89\u0026plusmn;0.26\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e14.56\u0026plusmn;0.18\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e11.22\u0026plusmn;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e12.79\u0026plusmn;0.22\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e13.08\u0026plusmn;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e11.04\u0026plusmn;0.16\u003csup\u003ee\u003c/sup\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\u003eThe mean and standard deviation values for different letters in the same column are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). \u003cstrong\u003eControl (-):\u0026nbsp;\u003c/strong\u003erats fed on crackers free CEsP; \u003cstrong\u003eControl (+):\u0026nbsp;\u003c/strong\u003erats fed on crackers free CEsP with CaCO\u003csub\u003e3\u003c/sub\u003e; \u003cstrong\u003eC4:\u0026nbsp;\u003c/strong\u003erats fed on crackers with replacement ratio 4% of WF by CEsP; \u003cstrong\u003eC8\u003c/strong\u003e: rats fed on crackers with replacement ratio 8% of WF by CEsP; \u003cstrong\u003eC12\u003c/strong\u003e: rats fed on crackers with replacement ratio 12% of WF by CEsP \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast, parathyroid hormone (PTH) levels in rats fed on cracker samples containing eggshell powder were lower than those in other rat groups. Interestingly, the parathyroid hormone levels were significantly decreased (P\u0026lt;0.05) in all rat groups fed crackers containing chicken eggshell powder compared to other rat groups. This decrease is also more noticeable with increasing substitution levels from 8 to 12% of WF by CEsP.\u003c/p\u003e\n\u003cp\u003eFurthermore, according to the above table, it is evident that rats fed a high-calcium diet exhibited elevated serum calcium levels, resulting in a decrease in blood parathyroid hormone (PTH) levels. Conversely, rats fed a low-calcium diet displayed the opposite effect. Similar findings were reported in a study conducted by [34, 47], where male rats fed low-calcium and very low-calcium diets consistently for several weeks demonstrated a significant increase in plasma PTH levels.\u003c/p\u003e\n\u003ch3\u003e3.5.4. \u0026nbsp; Provides the recommended dietary allowances of calcium by produced crackers:\u003c/h3\u003e\n\u003cp\u003eThe recommended daily allowance (RDA) for calcium intake varies between 800 and 1300 mg per day for different age groups [48]. However, the specific calcium requirements differ from one country to another. For instance, in India, the recommended intake ranges from 500 to 800 mg/day for children, while in Australia, it varies from 500 to 1000 mg/day for the same age group. Inadequate levels of calcium in the body result in low bone mineral density (BMD), which, if persistent, can lead to osteoporosis and hypocalcemia. Additionally, due to the increasing life expectancy, the population is experiencing a higher proportion of elderly individuals who are highly susceptible to osteoporosis. Without dietary changes, this group remains at risk in the future. Therefore, it is crucial to augment the calcium content in the population\u0026apos;s diet to mitigate the risk of osteoporosis among the elderly, both presently and in the future. To meet the daily calcium requirements, various calcium-fortified food products, such as fortified orange juice, flour, and pasta, are available in the market. These products often utilize calcium derived from sources such as cattle bone powder and milk powder [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe calcium content in cracker samples with a replacement ratio of 8% of WF by CEsP was 1680.8 mg/100g. Considering that the calcium absorption was 44.07%, the intake of bioavailable calcium in the cracker samples was 740.76 mg/100g. As given in the obtained data in\u003cstrong\u003e\u0026nbsp;Table 2S\u003c/strong\u003e, it can be noticed that the number of fortified crackers (8% replacement ratio of WF by CEsP) that can provide the daily recommended dietary allowances of calcium ranged from 135.0 to 175.5 g of cracker products for different age categories. Furthermore, it is advisable to consume calcium in smaller quantities at different intervals throughout the day rather than consuming a large amount all at once. This approach promotes better availability and utilization of calcium in the body. Studies have demonstrated that the bioavailability of calcium increases when it is consumed with food rather than on an empty stomach [49]. The practice of consuming crackers in smaller portions at least twice a day aligns with this recommendation and offers advantages in terms of calcium utilization by the body.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis study introduces a novel approach to fortifying crackers with chicken eggshell powder (CEsP) to enhance their calcium content and produce functional crackers. The findings demonstrate that incorporating CEsP as a supplement in the preparation of crackers significantly increases the overall ash percentage and specifically boosts the calcium content while simultaneously reducing the caloric value of the crackers. Additionally, the results indicate that the addition of CEsP, up to 8%, does not cause any notable alterations in the physicochemical properties of the crackers. Based on these outcomes, CEsP proves to be a valuable source of highly bioavailable calcium that can be effectively utilized in food products. Moreover, employing CEsP in food production serves as an environmentally friendly solution for managing eggshell waste, thus mitigating potential environmental issues associated with its accumulation.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eInformed consent\u003c/h2\u003e\n\u003cp\u003eInformed consent to participate and consent to publish were obtained from all participants in this study.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors have reviewed and approved the final version of the manuscript, and we confirm our consent for its publication. We also declare that there are no ethical or legal issues concerning the publication of this work.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe authors declare that no funding was received for this research.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eTaher Abdelnaby: Writing-original draft \u0026amp; Methodology. Mostafa M. Kadry: Discussion, Investigation \u0026amp; Formal Analysis. Sameh M. Ghanem: Conceptualization, Resources, Supervision. Mokhtar M. Salama: Visualization. Shaoyun Wang: Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank everyone who helped with this study.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003e\u0026ldquo;Data is provided within the manuscript or supplementary information files\u0026rdquo;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVijayakumar, R. and D. B\u0026uuml;sselberg, \u003cem\u003eOsteoporosis: An under-recognized public health problem: Local and global risk factors and its regional and worldwide prevalence\u003c/em\u003e. Journal of Local and Global Health Science, 2016. 2016(1): p. 2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoh, Y., et al., \u003cem\u003eFactors affecting willingness to get assessed and treated for osteoporosis\u003c/em\u003e. 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R152-R158.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Crackers, Chicken eggshell, Wheat flour, Quality characteristics","lastPublishedDoi":"10.21203/rs.3.rs-5398539/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5398539/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent years, there has been a growing demand for healthy snacks, leading to new products containing health-promoting components. Eggshells have emerged as a novel calcium source for human consumption, with potential for use in food products. Hence, this research aimed to examine the effects of substituting wheat flour with different levels (4%, 8%, and 12%) of chicken eggshell powder (CEsP) on the nutritional value, physiochemical properties of produced crackers, and to measure in vivo calcium absorption. The findings of the research indicated that the boiling process of CEsP effectively eliminated any microbial growth on the surface. CEsP supplementation led to an increase in the ash content of the crackers without altering the fat and protein contents, resulting in a notable decrease in the calorie content. Furthermore, the replacement of WF with CEsP led to an elevation in the mineral content, particularly calcium, which increased from 25.01 to 2446.1 mg/100 g with a higher replacement ratio of 12%. Moreover, the physical properties of the crackers showed no significant changes when using a replacement ratio of up to 8% of the produced crackers. Additionally, the in vivo evaluation of calcium absorption revealed that the highest absorption rate (44.07%) was achieved at the 8% replacement level. Based on these findings, the addition of CEsP to crackers improved their nutritional quality and physicochemical properties up to an 8% replacement level. Thus, the study proposes using CEsP as a dietary calcium supplement to develop novel calcium-enriched food products.\u003c/p\u003e","manuscriptTitle":"Utilization of Chicken Eggshells as a Source of Calcium to Enhance the Nutritional Value and Quality Characteristics of Crackers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 08:06:46","doi":"10.21203/rs.3.rs-5398539/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-23T13:43:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-17T12:41:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178557333385235561658436844131890387093","date":"2025-01-09T12:47:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-04T19:39:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48847668148870682147502501250144925709","date":"2024-12-30T17:20:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-20T06:05:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-20T05:25:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-19T12:05:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Food","date":"2024-11-06T01:10:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3dd197ae-59af-4f97-86c0-a5fb6985f757","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T10:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-23 08:06:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5398539","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5398539","identity":"rs-5398539","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}