INTRODUCTION

The rapid development of agriculture and industry has increased agricultural product consumption, resulting in an increase in agricultural waste and by-products. If allowed to build up, it pollutes the environment and poses a safety risk. These by-products offer intrinsic safety benefits as well as valuable elements that can be completely utilized or turned into high-value goods. Eggs are made up of four parts: eggshells, eggshell membranes, egg white and egg yolk, all of which have excellent functional properties and a wide range of biological activities (Chen et al., 2019). China is the world’s biggest egg producer, followed by the United States and India, with annual egg production exceeding 33 million tonnes in 2020, (Li et al., 2020). Egg by-products such as eggshells, eggshell membranes (ESM), salted egg whites (SEW), defatted yolk proteins (DYP), which are mostly derived from food industries and household garbage referring to baked foods, egg powder, sauce, yolk phospholipids and other foods have become abundant, as egg consumption has increased. Fortunately, egg wastes have been repurposed, as their economic and nutritional significance has become increasingly recognized. ESM can be used in the synthesis of peptides, food-borne compounds and food supplements, for example. DYP has been utilized to make peptides, dietary supplements and feeds, whereas SEW has been used to make hydrolysates and food additives. These findings show that egg leftovers hold a lot of promise. Waheed et al. (2020) have evaluated eggshell applications such as eggshell calcium, animal feed and plant fertilizer, however other egg by-products are sparse.

Some researchers have recently indicated that various egg by-products can be used in peptide manufacturing or food applications, however a new and systematic evaluation of egg by-product utilization is needed. The primary arguments are focused on egg by-product preparation, functional qualities, biological activities and food uses. Some researchers have recently indicated that various egg by-products can be used in peptide manufacturing or food applications, however a new and systematic evaluation of egg by-product utilization is needed. Considering the nutritious nature of ESM, many studies have researched its recycling and effective usage. ESM is now employed in biomaterials, agriculture, medicine and bodily health promotion.

Eggshell membrane protein and hydrolysates preparation

Collagen is a structured protein that helps to keep the extracellular matrix’s structure and functioning intact. It is mostly derived from animal skin and bones, albeit these animal tissues are fraught with controversy. Although, ESM collagen included less than 10% organic matrix, it had a mild allergic reaction and a high bio-safety. As a result, ESM can be utilized to extract collagen as a preferred option. Because ESM contains non-collagen proteins, alkali treatment is required before acid-extracted collagen (Kheirabadi, hadi Razavi et al., 2018). The recovery rate of collagen produced by acid extraction, on the other hand, was poor (8.35 %). By using response surface technology, Mohammadi et al. were able to extract collagen at a 30% extraction rate (Mohammadi et al., 2016). It’s possible that enzymes aided collagen disintegrate more effectively. Despite the fact that ESM can be utilized directly, their applications are limited, due to their potent cross-linking and base tolerance. Protein hydrolysis is an effective process for preparing hydrolysates or peptides. Zhao et al. demonstrated an enzymatic hydrolysis assistance with sodium sulfite technique for preparing ESM-derived peptides that was rapid, green and efficient and the product had good antioxidant properties. It’s possible that sodium sulfite breaks the disulfide link in ESM protein, loosening the structure and making it more receptive to enzymatic reactions (Zhao et al., 2019). Microbial fermentation could yield bioactive peptides in addition to protease hydrolysis.

Eggshell membrane and hydrolysate functional characteristics

egg byproductsSolubility, foaming, gelling and emulsifying capabilities are all functional features of protein. Due to their insolubility and many disulphide linkages, ESM is challenging to use successfully. Enzymatic hydrolysis and microbial fermentation are two approaches now being used to increase its value. When compared to the hydrolysate obtained without ultrasonic pre-treatment, the ESM hydrolysates obtained under optimal ultrasonic conditions of 95.74 percent amplitude ( 28.06 min, and solid-liquid ratio of 1:30 had better solubility, foaming and emulsifying properties. It’s possible that ultrasonic treatment triggered protein conformational change resulting in more proteins and peptides adsorbed on the interface, lowering interfacial tension and improving functional characteristics (Jain & Anal, 2016). The emulsion stabilized by ultrasound-prepared ESM hydrolysate, on the other hand, displayed poor emulsion stability. The addition of resistant starch improved emulsion stability and effectively delayed oil oxidation, but the emulsion flocculated and released fatty acids, as it went through the small intestine. Understanding how to manage the digestive behaviour of these emulsions could lead to new developments in ESM nutrition delivery systems. Lactobacillus fermentation is an environmentally friendly method of producing bioactive peptides. Protein hydrolysis into oligopeptides was catalyzed by Lactobacillus envelope protease and these hydrolysates were further hydrolysed into smaller peptides and amino acids by intracellular peptidases. Jain et al. fermented ESMs with Lactobacillus and discovered that the hydrolysates showed good foaming (36.7%), emulsifying (94.6 m2/g), solubility (90.7%) and biological activity (Jain & Anal, 2017).

Eggshell Membrane and Hydrolysate Biological Activities

Antibacterial: ESM effectively protects eggs from spoilage and deterioration caused by external dust and microorganisms, thanks to its compositions containing defensins, ovotransferrin and keratin peptides. These compounds’ antimicrobial properties have already been established (Moreno-Fernandez, et al., 2020). Furthermore, ESM’s antibacterial effectiveness against Staphylococcus aureus and Pseudomonas aeruginosa was mostly determined by its size and concentration (Kulshreshtha, et al, 2020). All of the hydrolysates displayed antibacterial activity, however the inhibitory effect on gram-positive bacteria was more pronounced than on gram-negative bacteria, which could be due to differences in bacterial cell membrane sensitivity. Because it was significantly similar with lipopolysaccharide binding protein, Li et al. recently discovered that ESM nanocomposite material has outstanding antibacterial action against both gram-positive and gram-negative bacteria (Li, et al., 2019). These innovative designs not only improved the antibacterial properties of ESM and hydrolysates, but also provided useful information for the creation of antibacterial materials.

Promoting wound healing:

The skin’s role as the body’s biggest organ is to shield it from external stimuli. The patient does not tolerate the protracted pathogenic periods and problems. As a result, developing cost-effective materials to promote wound healing, particularly for large-area wounds, is critical. ESM is a high-collagen industrial waste that has the potential to be used as a wound-healing material. In the early stages of wound healing, ESM also promoted keratinocyte proliferation by activating the activities of metalloproteinases-2 (MMP2) and MMP9 in the early stages (Vuong et al., 2018). Liu et al. discovered that nano-silver modified ESM expedited wound healing by increasing epithelial regeneration and granulation formation, which improved wound healing and antibacterial activity. The synergistic effects of composite materials on wound healing have recently been discovered (Liu et al., 2020). Chitosan, for example, dramatically improved ESM’s antibacterial activity, boosted wound exudate absorption, and prevented additional wound worsening (Li, Ma, Ahn, & Huang, 2019). In summary, the effects of ESM and hydrolysates on wound healing have primarily been studied in vitro and further data is needed, despite the fact that ESM has been shown to preserve the gut barrier (Xiao, N., et al., 2021).

Anti-inflammatory:

Inflammation is a natural part of the human body. Chronic inflammatory disorders like inflammatory bowel disease and arthritis, on the other hand, are caused by excessive inflammation. Fresh egg protein, pickled egg protein, phospholipids, peptides, and amino acids have all been widely researched for their anti-inflammatory properties (Moreno-Fernandez et al., 2020). Because of its components, ESM has the ability to reduce inflammation. Shi et al. confirmed that ESM hydrolysates lowered proinflammatory cytokine production in vitro and relieved excessive proinflammatory cytokine secretion and expression in colitis mice. (Shi, et al., 2014). ESM’s ability to modulate inflammation via the nuclear factor kappa-b signalling pathway was also verified in vitro .However, because ESM has a poor digestibility (46%) and more than 80% of ESM protein was absorbed and utilized in the bodies of rats, this anti-inflammatory effect in vitro was difficult to fully reflect the digestion and absorption of ESM in vivo. It was used by the intestinal microbiota and played a vital role in the remission of colitis. ESM powder, on the other hand, reduced colon inflammation by healing intestinal epithelium damage and controlling intestinal microbiota imbalances. Furthermore, through controlling the intestinal flora and improving lipid metabolism, ESM powder lowered serum triglycerides and liver total cholesterol in high-fat fed rats. These findings suggested that ESM acted biologically through hydrolysates or the gut flora. Arthritis is an autoimmune illness that causes discomfort and damage to joints. In rats, ESM was found to reduce joint inflammation, cartilage degradation and periosteum development. Clinical trials also revealed that ESM alleviated joint pain and damage, with the effect being synergistic when nutritional supplements were included (Xiao, N., et al., 2021).

Antioxidant:

BiologicalChanges in the body’s redox equilibrium are referred to as oxidative stress. Excessive oxidative stress hastens the onset of a variety of illnesses, including colitis and liver fibrosis. The potential of ESM protein hydrolysate extracted with 3-mercaptopropionic acid to scavenge radicals was validated by Huang et al. In Caco-2 cells, the ESM hydrolysates generated by multi-enzyme hydrolysis had a safe effect and reduced oxidative stress (Shi, Kovacs-Nolan, Jiang, Tsao, & Mine, 2014a). The antioxidant activity of small molecular weight hydrolysates was higher than that of large molecular weight hydrolysates, owing to the fact that tiny molecular peptides increased the activities and expressions of antioxidant enzymes. Small peptides had more aromatic and hydrophobic amino acids in terms of amino acid makeup. The antioxidant activity of peptides was found to be directly connected to its composition and molecular weight. However, its antioxidant activity was influenced by secondary structure, hydrophobicity and charge. These structural variations were especially important for peptide synthesis and activity determination when single peptides exhibited stronger antioxidant activity than hydrolysates. New insights for studying the structure-activity relationship of peptides came from computer modelling and molecular docking technology. Excessive oxidative stress exacerbated fibrosis in the liver. ESM has been demonstrated to reduce liver fibrosis in vivo by partially downregulating the c-jun and c-fos signalling pathways (Xiao et al., 2020).

Food applications of Eggshell Membrane and Hydrolysates

Although, it is typically employed in wastewater recycle, heavy metal filtering and biological materials, ESM as a food by-product has the potential to be exploited as food-borne materials. Supplementing with ESM (500 mg/day) alleviated arthritic pain and lowered the expression of the cartilage degradation marker CTX-II in a clinical trial (Ruff et al., 2020). By replacing typical non-degradable plastic film, edible film protects meals against oxygen, germs and odour invasion. It should have good functional features like barrier, film-forming, safety and degradability. Natural biological macromolecules have broad development prospects in food packaging materials, due to their outstanding safety and degradability. Mohammadi et al. found that the interaction of ESM gelatin and chitosan changed the functional properties of composite film, which gave composite film a uniform and dense microstructure at an appropriate ratio, thereby showing the potential as food packaging materials (Mohammadi et al., 2018). The ESM-soy protein isolate composite film loaded with eugenol had better structural and functional properties (Li et al., 2020). These evidences showed that ESM had the great potential applied into new food materials. In summary, although the biological activities of ESM and their hydrolysates have been extensively studied, their functional properties and food applications are relatively lacking. Additionally, the current protein extraction of ESM is mainly focused on collagen, but collagen only accounts for 10% of the ESM quality. In conclusion, whereas ESM and its hydrolysates have been widely researched for their biological activity, their functional characteristics and food uses have received less attention. Furthermore, present ESM protein extraction focuses mostly on collagen, whereas collagen only accounts for 10% of ESM quality. The 70% residual is still wasteful, which goes against the maximization of resource utilization idea. Importantly, biological activities of leftover organic matrix, such as hyaluronic acid have been demonstrated, implying that ESM still have significant development potential.

Conclusion and Future Prospects

Although, egg leftovers are frequently discarded, they are not trash. Although, the necessity of recycling wastes has been recognized based on current research, in-depth studies are absent. In fact, several procedures need to be addressed further, such as the worth of faulty and broken eggs. For large-scale processing of egg by-products and combination extraction of numerous bioactive components, efficient and cost-effective separation equipment and procedures are required. Adding egg by-products increased the functional characteristics of foods, but stability and feasibility still need to be addressed. After adding egg by-products, consider the flavour, taste and acceptability of the cuisine. In addition, in vivo biological activity, bioavailability, long-term safety and clinical consequences are required. Computer technology and Artificial Intelligence can help with the target research on a technical level. Future product development will focus on collaborative assembly with additional bioactive substances.

References:

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About the Authors:
Hamna Wahab, Dr. Faslu Rahman & Anand T.S.

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