Eggshell Membrane Research Articles

One‐Step Synthesis of Hydrogen‐Bonded Microcapsules for pH‐Triggered Protein Release

One‐step microfluidic approach in which interfacial complexation of hydrogen bonding polymers is utilized to prepare pH‐responsive microcapsules is presented. Using water‐in‐oil‐in‐oil‐in‐water (W1/O1/O2/W2) triple‐emulsion droplets as templates, it is shown that polymeric microcapsules with an aqueous core and a shell that consists of two complementary hydrogen bonding polymers, poly(propylene oxide)(PPO) and poly(methacrylic acid)(PMAA), can be prepared in a robust manner. The presence of a buffer layer enables controlled interfacial complexation of PMAA and PPO at the water/oil interface, followed by spontaneous dewetting of the oil droplet to result in hydrogen‐bonded microcapsules dispersed in aqueous media. In addition, it is demonstrated that the permeability of the PPO/PMAA membrane can be readily tuned by varying the molecular weight of PMAA. Furthermore, it is shown that the resulting PPO/PMAA microcapsules are stable at a high salt concentration (>1 m NaCl) unlike analogous capsules prepared through electrostatic complexation while they release the encapsulated protein above the critical pH of which the PMAA ionizes and results in disassembly of the shell membrane.

A drug-incorporated-microparticle-eggshell-membrane-scaffold (DIMES) dressing: A novel biomaterial for localised wound regeneration

Chronic wounds affect millions of people annually and have emotional and financial implications in addition to health issues. The current treatment for chronic wounds involves the repeated use of bandages and drugs such as antibiotics over an extended period. A cost-effective and convenient solution for wound healing is the development of drug-incorporated bandages. This study aimed to develop a biocompatible bandage made of drug-incorporated poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs) and eggshell membrane (ESM) for cornea wound healing. ESM has desirable properties for wound healing and can be isolated from eggshells using acetic acid or ethylenediaminetetraacetic acid (EDTA) protocols. Fluorescein isothiocyanate-labelled Bovine Serum Albumin (FITC-BSA) was used as a model drug, and the PLGA MPs were fabricated using a solvent extraction method. The MPs were successfully attached to the fibrous layer of the ESM using NaOH. The surface features of the ESM samples containing MPs were studied using a field emission scanning electron microscope (FESEM) and compared with blank ESM images. The findings indicated that the MPs were attached to the ESM fibres and had similar shapes and sizes as the control MPs. The fibre diameters of the MPs samples were assessed using Fiji-ImageJ software, and no significant changes were observed compared to the blank ESM. The surface roughness, Ra values, of the MPs incorporated ESM samples were evaluated and compared to the blank ESM, and no significant changes were found. Fourier transform infrared (FTIR) spectroscopy was used to analyse the chemical Composition of the bandage, and the spectra showed that the FBM were effectively incorporated into the ESM. The FTIR spectra identified the major peaks of the natural ESM and the PLGA polymer in the bandage. The bandage was transparent but had a reduced visibility in the waterproof test card method. The bandage achieved sustained drug release up to 10 days and was found to be biocompatible and non-toxic in a chorioallantoic membrane (CAM) assay. Overall, the drug-incorporated PLGA MPs-ESM bandage has great potential for treating chronic wounds.

Preparation and characterization of propolis reinforced eggshell membrane/ GelMA composite hydrogel for biomedical applications

Gelatin methacrylate-based hydrogels (GelMA) were widely used in tissue engineering and regenerative medicine. However, to manipulate their various chemical and physical properties and create high-efficiency hydrogels, different materials have been used in their structure. Eggshell membrane (ESM) and propolis are two nature-derived materials that could be used to improve the various characteristics of hydrogels, especially structural and biological properties. Hence, the main purpose of this study is the development of a new type of GelMA hydrogel containing ESM and propolis, for use in regenerative medicine. In this regard, in this study, after synthesizing GelMA, the fragmented ESM fibers were added to it and the GM/EMF hydrogel was made using a photoinitiator and visible light irradiation. Finally, GM/EMF/P hydrogels were prepared by incubating GM/EMF hydrogels in the propolis solution for 24 h. After various structural, chemical, and biological characterizations, it was found that the hydrogels obtained in this study offer improved morphological, hydrophilic, thermal, mechanical, and biological properties. The developed GM/EMF/P hydrogel presented more porosity with smaller and interconnected pores compared to the other hydrogels. GM/EMF hydrogels due to possessing EMF showed compressive strength up to 25.95 ± 1.69 KPa, which is more than the compressive strength provided by GM hydrogels (24.550 ± 4.3 KPa). Also, GM/EMF/P hydrogel offered the best compressive strength (44.65 ± 3.48) due to the presence of both EMF and propolis. GM scaffold with a contact angle of about 65.41 ± 2.199 θ showed more hydrophobicity compared to GM/EMF (28.67 ± 1.58 θ), and GM/EMF/P (26.24 ± 0.73 θ) hydrogels. Also, the higher swelling percentage of GM/EMF/P hydrogels (343.197 ± 42.79) indicated the high capacity of this hydrogel to retain more water than other scaffolds. Regarding the biocompatibility of the fabricated structures, MTT assay results showed that GM/EMF/P hydrogel significantly (p-value < 0.05) supported cell viability. Based on the results, it seems that GM/EMF/P hydrogel could be a promising biomaterial candidate for use in various fields of regenerative medicine.

Stabilization of zirconia nanoparticles by collagen protein and calcium carbonate extracted from eggshell and its biodegradation, radical scavenging and mineralization activity

In this current study,collagen protein extracted from eggshell membrane and calcium carbonate (a main component of eggshell) are used as additive to enhance the stability and hardness of the zirconia crystals. Five different samples are prepared by adding membrane containing eggshell content as, 1 g, 2 g, 3 g, 4 g and 5 g in aqueous zirconium oxychloride sol. Phase purity is confirmed by XRD and FTIR. Phase pure and dense particles divulges the high hardness (∼1389 HV) and fracture toughness (12.89 MPa m1/2).FESEManalysis illustrates the formation of dense, well separated, non-agglomerated and spherical nanoparticles at 5 g of eggshell content. Eggshell works as a surfactant and stabilizer for formation of phase pure tetragonal spherical nanoparticles. Biodegradation study of optimized tetragonal zirconia (t-ZrO2)in phosphate buffered saline (PBS) presents that minor change in weight and hardness after 72 h of immersion. Antioxidant study shows the 96% of radical scavenging activity (RSA). In vitro bio-mineralization study shows the formation of new bone after 5 weeks. After 5 weeks all pores were filled and minerals were deposited on the surface of the scaffolds. SEM images confirms that eggshell-zirconia composite form new bone. So eggshell addition results in formation of phase pure t-ZrO2 nanoparticles with well-defined boundaries which exhibit higher hardness, fracture toughness, stability and enhanced antioxidant properties.

Biocompatibility and histological responses of eggshell membrane for dental implant-guided bone regeneration.

Guided bone regeneration (GBR) utilizing eggshell membrane (ESM) as a potential biomaterial for dental implant therapy augmentation was explored in this study. ESM, an environmentally friendly waste product, possesses collagen-rich characteristics. The biocompatibility and histological responses of ESM were investigated in a rat model. Twelve young adult Wistar rats were used in this study. ESM samples were implanted in subcutaneous and intramuscular pockets, and samples were collected at 48 hours, 4 weeks, and 8 weeks post-implantation. Histological analysis revealed the changes in ESM over time. Results showed that ESM maintained its structural integrity, induced a moderate cellular response, and exhibited slow degradation, indicating potential biocompatibility. However, the lack of organized collagen arrangement in ESM led to the formation of irregular and polymorphic spaces, allowing cell migration. Encapsulation of ESM by newly proliferating collagen fibers and multinucleated giant cells was observed at later time points, indicating a foreign body reaction. Crosslinking might improve its performance as a separation membrane, as it has the potential to resist enzymatic degradation and enhance biomechanical properties. In conclusion, ESM demonstrated biocompatibility, slow degradation, and lack of foreign body reaction. While not suitable as a complete separation membrane due to irregular collagen arrangement, further research involving crosslinking could enhance its properties, making it a viable option for guided bone regeneration applications in dental implant therapy. This study highlights the potential of repurposing waste materials for medical purposes and underscores the importance of controlled collagen structure in biomaterial development.

Congo red dye removal from aqueous solution by encapsulated eggshell membrane using central composite design

Eggshell is solid wastes produced from the food industry. Commonly, eggshell and its membrane used to treat water pollution such as heavy metal removal and also dye removal. Thus, on the treatment process, it’s was having the material handling problem, which was needed filtration method to remove out the adsorbent from the solution, and it was not efficient when commercializing in the wastewater treatment plant. Therefore, this study was using alcohol-sodium alginate (PVA/SA) to encapsulate the eggshell membrane, and this encapsulation method also would be able to enhance the performance of raw material on the dye adsorption. In this study, the eggshell membrane (ESM) are prepared in bead form, and the optimization of the bead are performed by the removal of congo red dye at several parameters such as pH, initial dye concentration, contact time, and dosage of ESM. The central composite design was selected to optimize the adsorption process by generating 30 runs of the experiment using software Design Expert® version 11. The Fourier Transforms Infrared Spectroscopy (FTIR) analysis and Brunauer-Emmett-Teller (BET) analysis are used to characterize the ESM bead. There was found out that main functional group on ESM bead contribute on adsorption process were carbonyl group, hydroxyl group and ESM bead also having the 42.74 nm of pore size. The optimize point for each parameter was 12.63mg/L of initial dye concentration, 6.12 g of ESM dosage, pH 2.19 and 12.83 min of contact time and the optimum congo red dye removal efficiency was 98.86 %.

Haemato-biochemical alterations associated with the use of eggshell membrane as a dressing material for full-thickness wounds in a rabbit model

Haemato-biochemical alterations associated with the use of eggshell membrane as a dressing material for full-thickness wounds in a rabbit model

Eggshell-Waste-Derived Calcium Acetate, Calcium Hydrogen Phosphate and Corresponding Eggshell Membranes

The development of innovative transformation techniques for various wastes generated by the agri-food industry is one of the goals of sustainable waste management oriented toward “zero-waste” approach. This includes the production of bulk and fine chemicals, bioactive compounds, enzymes and functional materials from various waste. The present research shows the possibility of the production of value-added products from eggshell waste using chemical transformation by acetic and o-phosphoric acid at a laboratory scale level. Eggshell-derived calcium acetate monohydrate and calcium hydrogen phosphate, both of high purity, as well as corresponding eggshell membranes as valuable by-products, were produced. Chemical transformation of 100 g of eggshell waste with 10% (w/v) acetic acid resulted in 111.41 ± 2.13 g of calcium acetate monohydrate and 2.90 ± 0.14 g of eggshell membranes, with a chemical yield in calcium acetate monohydrate of 79.16 ± 1.98%. On the other hand, the transformation of 100 g of eggshell waste in 15% (w/v) o-phosphoric acid yielded 77.06 ± 6.21 g of calcium hydrogen phosphate and 2.94 ± 0.08 g of eggshell membranes, with a chemical yield in calcium hydrogen phosphate of 71.36 ± 5.58%. Eggshell-derived calcium hydrogen phosphate met all prescribed criteria for food-grade additive, as determined by physicochemical analysis. On the other hand, calcium acetate monohydrate met the majority, except water insoluble and formic acid and oxidizable impurities content. Based on the obtained results, it can be concluded that the proposed eggshell waste transformation process by two different acids might be useful for the production of calcium acetate and calcium hydrogen phosphate as food-grade additives, as well as eggshell membranes as valuable by-products.

Eggshell Membrane Mediated Synthesis of CdO–NiO Nanocomposite for Multifunctional Applications

Eggshell Membrane Mediated Synthesis of CdO–NiO Nanocomposite for Multifunctional Applications

Synthesis and Assessment of Antimicrobial Composites of Ag Nanoparticles or AgNO3 and Egg Shell Membranes.

Engineering research has been expanded by the advent of material fusion, which has led to the development of composites that are more reliable and cost-effective. This investigation aims to utilise this concept to promote a circular economy by maximizing the adsorption of silver nanoparticles and silver nitrate onto recycled chicken eggshell membranes, resulting in optimized antimicrobial silver/eggshell membrane composites. The pH, time, concentration, and adsorption temperatures were optimized. It was confirmed that these composites were excellent candidates for use in antimicrobial applications. The silver nanoparticles were produced through chemical synthesis using sodium borohydride as a reducing agent and through adsorption/surface reduction of silver nitrate on eggshell membranes. The composites were thoroughly characterized by various techniques, including spectrophotometry, atomic absorption spectrometry, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy, as well as agar well diffusion and MTT assay. The results indicate that silver/eggshell membrane composites with excellent antimicrobial properties were produced using both silver nanoparticles and silver nitrate at a pH of 6, 25 °C, and after 48 h of agitation. These materials exhibited remarkable antimicrobial activity against Pseudomonas aeruginosa and Bacillus subtilis, resulting in 27.77% and 15.34% cell death, respectively.

Methods of In Ovo and Ex Ovo Ostrich Embryo Culture with Observations on the Development and Maturation of the Chorioallantoic Membrane.

Culture of shell-free and windowed eggs for drug testing and other experiments has been perfected for smaller eggs such as those of chickens, where the developing blood vessels of the chorioallantoic membrane (CAM) become accessible for manipulative studies. However, due to the thickness and hardness of the ostrich egg shell, such techniques are not applicable. Using a tork craft mini rotary and a drill bit, we established windowed egg, in-shell-membrane windowed egg, and in-shell-membrane shell-free methods in the ostrich egg, depending on whether the shell membranes were retained or not. Concomitant study of the developing CAM revealed that at embryonic day 16 (E16), the three layers of the CAM were clearly delineated and at E25, the chorionic capillaries had fused with the epithelium while the CAM at E37 had reached maturity and the chorion and the allantois were both 3-4 times thicker and villous cavity (VC) and capillary-covering cells were well delineated. Both intussusceptive and sprouting angiogenesis were found to be the predominant modes of vascular growth in the ostrich CAM. Development and maturation of the ostrich CAM are similar to those of the well-studied chicken egg, albeit its incubation time being twice in duration.

Multicomponent SERS imprinted bio-membrane based on eggshell membrane for selective detection of spiramycin in water

A novel imprinting membrane (Ag/CuO/CNTs/Eggshell membrane -Molecularly Imprinted polymeric Membrances, ACCE-MIMs) based on surface enhanced Raman scattering (SERS) is successfully synthesized for the specific detection of spiramycin (SP). Firstly, CNTs and CuO are successively loaded on eggshell membrane (ESM) to increase the uniformity and sensitivity by virtue of the unique reticular structure of CNTs and excellent chemical enhancement of CuO. Through the abundant amino and carboxyl groups on ESM, Ag nanoparticles (Ag NPs) are anchored and growth on the surface of membrane by two in-situ reduction process and form a three-dimensional (3D) architecture. Eventually, the ACCE-MIMs are synthetized by silicon-based sol-gel method. With the help of imprinted polymers, the ACCE-MIMs present anti-interference performance during selective detection. The sensor presents a great liner relationship (R2 = 0.9965) between SP concentration and the Raman peak intensity when the concentration of SP ranges between 1 × 10−6 M ∼1 × 10−12 M. The limit of detection concentration is 1 × 10−12 M and the enhancement factor (EF) is 0.2956 × 107. The ACCE-MIMs exhibits sensitive detection property and this procedure provides a promising future for detection process.

Eggshell translucency in late-phase laying hens and its effect on egg quality and physiological indicators.

Eggshell translucency severely affects external egg quality, and variations in the eggshell or eggshell membrane are considered the structural basis of the trait. Research has shown that 1.85% additional mixed fatty acids in the diet would greatly decrease the occurrence of eggshell translucency. Only a few studies have examined the phenotypic regularity of eggshell translucency with the increasing age of hens. Therefore, two strains, 1139 Rhode Island Red-White (RIR-White) and 836 Dwarf Layer-White (DWL-White), were used, and from each strain, 30 hens each that consecutively laid translucent or opaque eggs at 67 wks of age were selected. Subsequently, eggshell translucency, internal quality and external quality of eggs, and total cholesterol, albumin, calcium binding protein and other physiological indicators related to lipid, lipoprotein, and calcium metabolisms at the 75th, 79th, and 83rd wks of age in the late phase of the laying cycle were determined. Results: (1) In terms of flocks, for both strains, the translucency scores of the translucent groups were significantly higher than those of the opaque groups (P < 0.05); in terms of individuals, 81.1% RIR-White and 82.8% DWL-White hens consecutively laid eggs of the same or similar translucency, indicating the stability of the trait with increasing hen age; (2) In RIR-White, the eggshell strength of the translucent group at 75 weeks was significantly higher than that of the opaque group (P < 0.05); in DWL-White, the eggshell membrane thickness of the translucent group at the 75th and 83rd weeks was significantly lower than that of the opaque group (P < 0.05); (3) Compared to the opaque groups, the translucent groups had lower total cholesterol content in both RIR-White and DWL-White, lower albumin content in DWL-White at the 79th weeks (P < 0.05), and higher calcium-binding protein (CALB1) in RIR-White at the 83rd weeks (P < 0.05). In summary, this study illustrates the stability of eggshell translucency in late-phase laying hens and provides a reference of physiological indicators for exploring the formation of translucent eggs.

Milk lactose removal by β-galactosidase immobilized on eggshell membrane

Milk lactose removal by β-galactosidase immobilized on eggshell membrane

Transformation of eggshell waste to egg white protein solution, calcium chloride dihydrate, and eggshell membrane powder

Abstract The present study investigated the possibility of complete utilization of eggshell waste (ESW) transforming it to adherent egg white protein solution, calcium chloride dihydrate, and eggshell membranes (ESM). Adherent egg white protein solution was obtained by washing ESW three times with distilled water at 25°C, followed by filtration, and analyzed for the protein content and lysozyme activity. ESM and calcium chloride were obtained simultaneously by the exposure of washed eggshells to 5% hydrochloric acid treatment at 25°C for 3 h, followed by separation by filtration. The separated ESM were washed, dried, and milled to powder and analyzed for protein and lipid content. The calcium chloride solution was exposed to the neutralization of excess hydrochloric acid by calcium hydroxide, followed by evaporation to one-tenth of volume. Calcium chloride crystals were precipitated from the concentrated solution with acetone, separated by filtration, dried at 110°C, and analyzed for chemical composition and purity. The obtained results revealed that 100 g of ESW can be transformed to 1.61 ± 0.34 g of adherent white proteins containing 485,821 U of lysozyme activity, 2.84 ± 0.16 g of ESM powder, and 108.74 ± 3.62 g of calcium chloride dihydrate of high purity.

Imaging and positioning through scattering media with double-helix point spread function engineering.

Double-helix point spread function (DH-PSF) microscopy has been developed for three-dimensional (3D) localization and imaging at super-resolution but usually in environments with no or weak scattering. To date, super-resolution imaging through turbid media has not been reported. We aim to explore the potential of DH-PSF microscopy in the imaging and localization of targets in scattering environments for improved 3D localization accuracy and imaging quality. The conventional DH-PSF method was modified to accommodate the scanning strategy combined with a deconvolution algorithm. The localization of a fluorescent microsphere is determined by the center of the corresponding double spot, and the image is reconstructed from the scanned data by deconvoluting the DH-PSF. The resolution, i.e., the localization accuracy, was calibrated to 13nm in the transverse plane and 51nm in the axial direction. Penetration thickness could reach an optical thickness (OT) of 5. Proof-of-concept imaging and the 3D localization of fluorescent microspheres through an eggshell membrane and an inner epidermal membrane of an onion are presented to demonstrate the super-resolution and optical sectioning capabilities. Modified DH-PSF microscopy can image and localize targets buried in scattering media using super-resolution. Combining fluorescent dyes, nanoparticles, and quantum dots, among other fluorescent probes, the proposed method may provide a simple solution for visualizing deeper and clearer in/through scattering media, making in situ super-resolution microscopy possible for various demanding applications.

Bilayered skin substitute incorporating rutin nanoparticles for antioxidant, anti-inflammatory, and anti-fibrotic effect

Hypertrophic scarring in large burns and delayed healing in chronic wounds are consequences of prolonged and aggravated inflammation, sustained infiltration of immune cells, free radical generation, and abundance of inflammatory mediators. Therefore, it is imperative to curb hyperinflammation to expedite wound healing. In this study, rutin nanoparticles (RNPs) were synthesized without an encapsulant and incorporated into eggshell membrane powder-crosslinked gelatin-chitosan cryogels to impart antioxidant and anti-inflammatory properties for treating hyperinflammation. The resultant nanoparticles were found to be 17.53 ± 4.03 nm in size and were stable at room temperature for a month with no visible sedimentation. RNPs were found to be non-cytotoxic and exhibited anti-inflammatory (by increasing IL-10 levels) and antioxidant properties (by controlling the generation of reactive oxygen species and enhancing catalase production in human macrophages). Additionally, RNPs were found to reduce α-SMA expression in fibroblasts, thereby demonstrating their anti-scarring effect. In vivo studies with a bilayered skin substitute constituting an RNP-incorporated cryogel proved that it is biocompatible, does not induce renal toxicity, aids wound healing, and induces better re-epithelialization than the control groups at the initial stages. Thus, RNP-incorporated cryogels containing bilayered skin substitutes are an advanced and novel alternative to commercial dermo-epidermal substitutes that lack anti-inflammatory or anti-scarring properties.

Aging-associated increased nitric oxide production is a potential cause of inferior eggshell quality produced by aged laying hens

It is important to prolong the productive life of laying hens without compromising their welfare. Therefore, in this study, we aimed to identify the cause for inferior quality egg production of aged hens by investigating the aging-associated molecular changes related to eggshell formation in the isthmic and uterine mucosae and determining whether nitric oxide plays a role in decreasing the quality of eggs produced by aged hens. Young (35 weeks old) and aged (130 weeks old) White Leghorn laying hens were used in this study to determine the effects of age on the expression of proteins related to eggshell membranes formation in the isthmus and eggshell biomineralization and nitric oxide production in the uterus. Nitric oxide synthesis during the ovulatory cycle was examined in twenty-five laying hens (46–52 weeks old) euthanized at 0, 4, 7, 16, and 24 h after oviposition. S-Nitroso-N-acetylpenicillamine (a nitric oxide donor) was added to the cultured isthmic and uterine mucosal cells to examine the effects of nitric oxide on the expression of genes related to eggshell membranes formation and eggshell biomineralization, respectively. The results showed that the protein abundance of collagen I and V in the isthmic mucosa and collagen V in the eggshell membranes were lower in aged hens than in young hens. The mRNA expression levels of calbindin, osteopontin, and ovocalyxin-36 and the protein abundance of calbindin and carbonic anhydrase-2 were lower in the uterine mucosa of aged hens than in that of young hens. Nitric oxide synthesis was higher in the uterine mucosa of aged hens than in that of young hens. Nitric oxide downregulated the mRNA expression levels of osteopontin and ovocalyxin-36 in cultured uterine mucosal cells. Our results indicated that the eggshell quality decreases with aging due to molecular changes in the uterine mucosa affecting the eggshell membrane formation and eggshell biomineralization. Moreover, nitric oxide overproduction may play a role in this dysfunction.

The influence of eggshell powder and limestone on the rheological properties of cement pastes

The paper presents the effect of the powders obtained from egg-shells and limestone on the rheological properties of cement pastes. Both powders were added to Portland cement CEM I 42.5 R, 30% by mass. The higher yield stress and plastic viscosity of the cement pastes prepared with the addition of eggshell powder, compared to the cement pastes made with limestone powder, were explained by the influence of the eggshell membrane and its chemical structure. The membrane, which absorbs water and expands in the cement paste, is causing an increase in rheological parameters. The membrane, which absorbs water and swells in the cement paste, causes an increase in viscosity and thixotropy compared to the neat cement slurry and slurry with limestone meal added as a result of particle bonding.

Could eggshell membrane be an adjuvant for recombinant Hepatitis B vaccine?: A preliminary investigation

BackgroundDespite the invasiveness of the Hepatitis B infection, its vaccines are only formulated with FDA-approved alum-based adjuvants, which poorly elicit a lasting immune response, hence the need for a more effective adjuvant system. This study evaluated the immunogenicity and toxicity of eggshell membranes (ESM) when administered as an adjuvant for the recombinant HBV vaccine (rHBsAg) in albino mice. Differential white blood cell analysis, as well as the titer measurement of Immunoglobulin G, subclass G1 and G2a on indirect ELISA, was performed to measure the immune-modulatory potentials of ESM. Moreover, analysis of the liver marker enzyme (AST and ALT) and body/liver weights was performed to ascertain the toxicity level of ESM. Finally, Immuno-informatic analysis was used to investigate the immune-modulatory potential of individual member proteins of ESM.ResultsOur results showed a significant improvement in the experimental group's lymphocyte count after boost-dose administration compared to the controls, whereas there was no significant change in the granulocyte population. Furthermore, the formulations (ESM-rHBsAg) significantly improved IgG and IgG1 titers after each successive immunization. Body/liver weight and liver function showed ESM non-toxic to mice. The immunoinformatic analysis discovered ovalbumin, lysozyme-C, and UFM-1 as the member proteins of ESM with immune-modulatory activities of activating antigen-presenting cells (APC).ConclusionThis study has provided a clue into the potential valorization of eggshell membranes and their peptides as an adjuvant for vaccines such as HBV. We recommend more in-depth molecular analysis to support our findings as well as foster real-life application.