Formation mechanism and stability of egg white fluid gels under ultrahigh-pressure homogenization pretreatment and synergistic heating effect.

The thermal sterilization process of protein beverages inevitably leads to the formation of insoluble thermal aggregates, greatly reducing the texture and taste of protein beverages. In this study, homogenized egg white (HEW) was obtained by ultrahigh-high-pressure (UHP) homogenization pretreatment of egg white (EW), and then a special egg white fluid gel (EWFG) was prepared by water bath heating. The results showed that the optimal conditions for preparing EWFG were three cycles at 20 MPa homogenizing pressure and heating in a water bath at 72℃ for 10 min. Under these conditions, the EWFG was a milky-white homogeneous liquid with an average particle size of about 560 nm. Measurements of the physicochemical properties of HEW and EWFG showed that the UHP homogenization treatment reduced the viscosity of HEW, decreased the particle size of protein aggregates, and increased the zeta potential, which altered the interactions of proteins during the subsequent heating process and facilitated the formation of homogeneous and dispersed EWFG. EWFG showed good stability at pH 6-10 and in low-concentration saline and medium-concentration sucrose solutions. The EWFG obtained by the present treatment is more suitable for factory-scale production and has great potential for protein beverage applications.

Research Note: Comprehensive proteomic, phosphoproteomic, and N-glycoproteomic analysis of chicken egg yolk plasma.

Chicken egg yolk plasma (EYP), the supernatant fraction of egg yolk obtained by water dilution and centrifugation, is a rich source of various bioactive substances and a significant bearer of yolk-emulsifying properties. This study utilized proteomics to conduct a comprehensive and in-depth analysis of both common and modified EYP proteins (phosphorylated proteins and N-glycosylated proteins). Total of 208 proteins were identified in EYP, including 42 phosphorylated proteins with 137 phosphorylation sites and 150 N-glycoproteins with 332 N-glycosylation sites. Among the phosphorylation sites, tyrosine accounted for 80.6%, while the N-glycosylation sites predominantly featured "N-X-T" motifs, accounting for 58.7%. Functional enrichment analysis revealed that most proteins were involved in regulating enzyme activity and inhibition with a particular focus on modulating peptidase activity. Notably, vitellogenins-2 (30 phosphorylation sites, 9 N-glycosylation sites) and apolipoprotein B (10 phosphorylation sites, 56 N-glycosylation sites) were the 2 proteins with the most modification sites. Additionally, EYP was found to contain the highly N-glycosylated complement proteins C3 and C4. These findings provide new insights into the protein composition of EYP and its roles in chicken embryo development and immune defense, offering a theoretical foundation for the application of EYP in various fields.

Effects of Photoperiod and Light Quality on Germination and Growth of Camellia sinensis 'HuangKui'.

Light, as a critical environmental factor, plays a pivotal role in photosynthesis, ultimately influencing the timing of bud flush in tea plants. However, the synergistic effects of different photoperiods and light qualities on the timing of bud flush in the albino tea cultivar 'HuangKui' (later germination variety) remain unknown. Thus, the objective of this study was to investigate the effects of different photoperiods (12L/12D, 14L/10D, 16L/8D, and 18L/6D, where L = the number of daylight hours and D = the number of hours of darkness) and ratios of red (R) to blue (B) light (R/B 1:1, R/B 1:2, R/B 1:3, and R/B 2:1) on the germination and growth of the albino tea variety 'HuangKui'. In our study, we examined how different photoperiods and red light and blue light affected tea germination and growth by investigating the timing of bud flush, photosynthesis, chlorophyll content, and growth indicators. First, our study showed that 'HuangKui' germinated 4 days, 2 days, and 1 day earlier under the 16L/8D photoperiod at the one bud and one leaf period compared with plants cultivated under the 12L/12D, 14L/10D, and 18L/6D photoperiods under light simulating the solar spectrum. Also, the growth of 'HuangKui' was maximumly promoted under the 16L/8D photoperiod treatment. Additionally, the earliest germination of 'HuangKui' was observed for the 16L/8D photoperiod under the R/B 2:1 (red/blue) treatment compared with the other treatments. Moreover, the greatest plant height, length of the new shoots, and new leaf areas were detected in the albino tea variety 'HuangKui' under R/B 2:1. Moreover, the contents of auxin (indole acetic acid, IAA) and trans-zeatin (tZ) under R/B 2:1 were significantly higher than those under the R/B 1:1 and control treatments with the 16L/8D photoperiod. Additionally, the auxin-related expression levels of CsIAA13, CsGH3.1, CsAUX1, and CsARF2 under the R/B 2:1 treatment were significantly higher than those in the control. The expression of CsARR-B, a positive regulator of cytokinin-related genes, was significantly higher under the R/B 2:1 treatment than under the control treatment, while the opposite result was found for the expression of the negative regulator CsARR-A. Therefore, the R/B 2:1 treatment with the 16L/8D photoperiod was an appropriate means of timing the bud flush for the albino tea variety 'HuangKui', which may be related to IAA or tZ signal transduction. In conclusion, our research offers a novel lighting strategy that promotes the germination and growth of albino tea cultivars.

Using salted egg white in steamed bread: Impact on functional and structural characteristics.

Steamed bread has long been an important part of Chinese cuisine. This study investigated the effects of salted egg white (SEW) (5, 10, 15, and 20% w/w) on the quality of steamed breads. Findings revealed that SEW notably enhanced the bread's volume and texture, with a 20% inclusion significantly boosting water retention and rheological properties, albeit reducing bread's lightness. In addition, the H-bond absorption band intensity in the Fourier transform infrared spectroscopy (FTIR) analysis showed increased peak intensities with higher SEW levels, indicative of protein structure alterations. X-ray diffraction confirmed the presence of an amylose-lipid complex. Scanning electron microscope (SEM) and Confocal laser scanning microscope (CLSM) imaging depicted a smooth, consistent protein network with SEW addition. Consumer sensory evaluation responded favourably to the SEW15 steamed bread, suggesting its potential for food industry application. Overall, the study considers SEW an effective ingredient for improving steamed bread quality.

Research note: Preparation and stability of egg white fluidic gel induced by ultrasonic pretreatment.

Ultrasound-induced egg white fluidic gels (UEFG) were prepared through ultrasonic pretreatment followed by subsequent heating. The optimal preparation parameters for UEFG were: ultrasonic power density between 0.2 to 0.8 W/mL, ultrasonic treatment time surpassing 150 s, heating temperature within 70 to 76°C, and heating time under 16 min. The prepared UEFG is a milky white solution with a viscosity lower than that of fresh egg white, and displayed a particle size distribution primarily between 100 and 1,200 nm. Stability assessments conducted over 28 d at 4°C revealed that UEFG remained stable at pH 6-10, with exceptional stability from pH 8 to 10, while it is less stable in highly acidic (pH 2-4) or basic environments (pH 12). The UEFG also showed commendable stability in the presence of salt and sucrose solutions. We report a simple and novel method for preparing UEFG with good flowability after heating treatment. The UEFG has broad applications in the food industry, such as precooked egg white powder, high protein beverages, composite dairy products, etc.

A Novel Food Bore Protein Hydrogel with Silver Ions for Promoting Burn Wound Healing.

Hydrogels have emerged as a promising option for treating local scald wounds due to their unique physical and chemical properties. This study aims to evaluate the efficacy of ovalbumin/gelatin composite hydrogels in repairing deep II-degree scald wounds using a mouse dorsal skin model. Trauma tissues collected at various time points are analyzed for total protein content, hydroxyproline content, histological features, and expression of relevant markers. The results reveal that the hydrogel accelerates the healing process of scalded wounds, which is 17.27% higher than the control group. The hydrogel treatment also effectively prevents wound enlargement and redness of the edges caused by infection during the initial stage of scalding. The total protein and hydroxyproline content of the treated wounds are significantly elevated. Additionally, the hydrogel up-regulates the expression of VEGF (a crucial angiogenic factor) and down-regulates CD68 (a macrophage marker). In summary, this study provides valuable insights into the potential of multifunctional protein-based hydrogels in wound healing.

Inhibition of water-diluted precipitate formation from egg whites by ultrasonic pretreatment: Insights from quantitative proteomics analysis.

The formation of the egg white precipitate (EWP) during dilution poses challenges in food processing. In this paper, the effects of 90 W and 360 W ultrasonic intensities on the inhibition of EWP formation were investigated. The findings revealed that 360 W sonication effectively disrupted protein aggregates, decreasing the dry matter of EWP by 5.24 %, particle size by 57.86 %, and viscosity by 82.28 %. Furthermore, the ultrasonic pretreatment unfolded protein structures and increased the content of ß-sheet structures. Combined with quantitative proteomics and intermolecular forces analysis, the mechanism by which ultrasonic pretreatment inhibited water-diluted EWP formation by altering protein interactions was proposed: ultrasonic pretreatment disrupted electrostatic interactions centered on lysozyme, as well as hydrogen-bonding interactions between ovomucin and water. In conclusion, our research provides valuable insights into the application of ultrasonic pretreatment as a means to control and improve the quality of egg white-based products.

Chicken egg white precipitates induced by water dilution.

Chicken egg whites (whole egg white, EW; thick egg white, TKEW; thin egg white, TNEW) become turbid and are accompanied by the formation of precipitates after being diluted with an equal mass of deionized water. The precipitates of TKEW induced by water dilution (Thick Egg White Precipitates, TKEWP) account for 14.47 % of TKEW total dry matter, much higher than that of thin egg white precipitates (TNEWP) (1.51 %) and whole egg white precipitates (EWP) (5.53 %). Quantitative proteomic analysis identified 27 differentially abundant proteins (p < 0.05) among EW, EWP, TNEWP, and TKEWP. Lysozyme was found to be a key protein in the formation of EW precipitates induced by water dilution, as its abundance was significantly higher in TNEWP and TKEWP. Mucin-5B (α-ovomucin) had the highest abundance in TKEWP, suggesting that its insolubility is one of the important factors contributing to the large formation of TKEWP. This paper systematically studies the formation, characteristics, and composition of egg white precipitation caused by water dilution, and puts forward a new understanding of the processing characteristics of egg white liquid, thus laying a theoretical foundation for further research methods to reduce egg white precipitation by water dilution.

Quantitative N-glycoproteomic analysis of egg yolk powder during thermal processing.

The production of egg yolk powder often involves critical processes such as pasteurization and spray drying, however, these thermal processes will inevitably affect the functional properties of egg yolk (especially gelation and emulsification). The aim of this study was to elucidate the mechanism of the effect of pasteurized egg yolk (P-EY) and spray-dried egg yolk (SD-EY) on the functional properties through quantitative N-glycoproteomic. The results showed, compared with fresh egg yolk (F-EY), emulsifying property of mild heat-treated P-EY was slightly reduced while the gelation property did not undergo significant changes, whereas emulsifying activity (EAI) and gelation strength of vigorously heat-treated SD-EY were significantly reduced by 48.72 % and 35.73 %, respectively. During thermal processing in SD-EY, larger aggregate particles (particle size ∼10 um) were formed, and the surface hydrophobicity was reduced (93.0 %) and the zeta potential was enhanced (62.8 %). The results of quantitative N-glycoproteomic showed that 13 N-glycosylated proteins (APOB, vitellogenin, etc.) were down-regulated while only 2 N-glycosylated proteins were up-regulated; 21 N-glycosylation sites were down-regulated and 2 N-glycosylation sites were up-regulated in SD-EY, suggesting that covalent cross-linking of protein N-glycoproteins may have occurred in the process of spray-drying, which altered molecular physicochemical characteristics of the yolk solution that further affecting the processing properties of egg yolk.

Quantitative proteomics provides new insights into the mechanism of improving rehydration of egg white powder by ultrasonic pretreatment.

Poor rehydration is one of the key factors affecting the functional properties of egg white powder (EWP). Reducing rehydrated precipitates is important for the processing and application of EWP. In this study, effects of ultrasonic pretreatment on the physicochemical and functional properties of EWP rehydration solutions were studied with the aim of revealing the mechanism of ultrasonic pretreatment to improve rehydration. Compared with freeze-dried EWP (FD) and spray-dried EWP (SD), the percentage of ultrasonic pretreated FD (UFD) and ultrasonic pretreated SD (USD) rehydrated precipitates decreased by 13.0 % and 5.6 %, respectively, after ultrasonic pretreatment (0.25 W/mL for 10 min); and the average particle sizes of UFD and USD solutions decreased by 22.5 % and 15.5 %, respectively. Fourier transform infrared spectroscopy showed that ultrasonic pretreatment caused higher ß-sheet content in the protein secondary structure of UFD rehydrated precipitates (49.2 %). Quantitative proteomic analysis revealed a decrease in the abundance of major egg white proteins (ovalbumin, ovotransferrin, ovomucoid and ovomucin) in the rehydrated precipitates of UFD, except for lysozyme. It was also shown that lysozyme-centered aggregates were disrupted in the rehydrated precipitates of UFD. Our research suggests that ultrasonic pretreatment improves EWP rehydration by reducing the interactions between high abundance proteins as well as improving the solubility of high abundance proteins.

Effect of preserved eggs on the health of SD rats, and anti-tumor action of HT-29 cells.

Preserved eggs are traditional alkali-pickled food in China and have been enjoyed by consumers and extensively studied by researchers for their nutritional tastes and their anti-tumor, anti-inflammatory, antioxidant, lipid-lowering, and blood pressure-lowering properties. To study the anti-tumor effects of preserved eggs, this project observed the health on rats, and anti-tumor effects and separated anti-tumor active components on HT-29 cells. SD rats fed for 80 days showed that preserved eggs had no significant effect on weight, food intake, blood pH, liver tissues, or organ indices. Preserved eggs significantly increased blood levels of oxidative stress markers SOD and CAT, decreased MDA levels by 0.46, 0.23, and 0.25 times. Moreover, they also increased the level of IL-2 from 1233 to 1340 pg/mL. Two water-soluble bioactive peptide fractions, B1 and B2, with molecular weights ≥10 kDa were further obtained from preserved eggs by ultrafiltration and Superdex Peptide 10/300 GL. The potential mechanism of B1 and B2 is to activate the internal mitochondrial apoptotic pathway and induce apoptosis by up-regulating the expression of the pro-apoptotic factors cytochrome C, caspase-3, and caspase-9 mRNA in HT-29 cells.

A novel antibacterial hydrogel based on thiolated ovalbumin/gelatin with silver ions to promote wound healing in mice.

Hydrogels could be used as wound dressings, but most protein-based hydrogels lack anti-bacterial effects. Here, we successfully prepared a silver ion cross-linked thiolated protein hydrogel (thiolated Ovalbumin and Gelatin, O3G7). The wound photographs showed that the healing rate (96.23 %) of hydrogel-treated mice was higher than the control group. Meanwhile, the hydrogel increased the granulation tissue's total protein content. Furthermore, it significantly increased the collagen content, consistent with the results of Masson's trichrome (MT) staining and immunohistochemical analysis of type I collagen (ColI). The results of hematoxylin and eosin (H&E) staining showed the growth and proliferation of inflammatory cells, granulation tissue, fibroblasts, blood vessels and hair follicles in acute wounds. O3G7 hydrogel had fewer inflammatory cells and more neovascularization, and hair follicle tissue and intact epidermis could be observed. The results of immunofluorescence and immunohistochemistry showed that the O3G7 group reduced the expression of tumor necrosis factor (TNF)-α (56.87 % of the control group) and upregulated the expression of transforming growth factor (TGF)-ß (1.29 times of the control group). These results suggest that O3G7 hydrogel significantly affects the healing of acute wounds. This study demonstrates that hydrogels prepared from food-derived proteins will be promising and bio-safe candidates in bioengineering.

Translucency mechanism of heat-induced pigeon egg white gel.

In this study, the properties of pigeon egg white (PEW) and chicken egg white (CEW) thermal gels were compared, with the aim of revealing the mechanisms involved in the high transparency of PEW thermal gels. Results demonstrated that PEW gels exhibited higher transparency than CEW gels. Scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis revealed that PEW gels formed a fine chain gel network structure with an average diameter of thermal aggregates (89.84 ± 7.13 nm). The molecular properties of PEW proteins, such as higher content of ß-sheet structures (32.73 %), reactive groups (free sulfhydryl groups, hydrophobic groups), and absolute zeta potential (-3.563 mV), were found to contribute to the formation of smaller thermal aggregates during thermal denaturation. The microrheology measurements showed that these features allowed PEW proteins to interact less with each other and form smaller thermal aggregates during thermal denaturation, which facilitated the formation of fine chain gel networks and thus improved the transparency of the gels. The present study initially reveals the molecular basis of the high transparency of PEW thermal gels and provides a theoretical reference for the development of new highly transparent protein materials.

Elastic and transparent ovalbumin hydrogels formed via succinylation combined with pH-shifting treatment.

Ovalbumin (OVA) is a model protein with extensive research on structure and function, however, the application of OVA in food processing is limited due to its low gelation properties. In this study, thermally-induced highly transparent and elastic hydrogels from OVA pretreated by succinylation combined with pH-shifting method were reported. Transmission electron microscope (TEM) and free sulfhydryl groups determination revealed that the pretreatment induced the stretching of the protein structure and promoted the formation of preliminary aggregates. Further heating the pretreated OVA suspension resulted in a homogeneous and macroporous gel network with thin connecting walls. Such homogeneous gel network structures may be related to the effective modulation of the thermal aggregation efficiency of proteins by succinylation and the high level of protein unfolding by pH-shifting treatments, which synergistically allowed for more active sites to be created during heating to facilitate intermolecular interactions, including hydrogen bonding and hydrophobic interactions. Notably, the method resulted in a 507.14% increase in elasticity, a 60.74% increase in water holding capacity of the OVA hydrogels compared to the native OVA hydrogels without pretreatment. Also, the hydrogels were transparent with 73.11% light transmittance. In conclusion, succinylation and pH-shifting combined treatment could be an effective method for the preparation of OVA hydrogels with superior gelation properties.

Characterization of the Dynamic Gastrointestinal Digests of the Preserved Eggs and Their Effect and Mechanism on HepG2 Cells.

Preserved eggs, an alkaline-fermented food, have been widely searched for their anti-inflammatory activity. Their digestive characteristics in the human gastrointestinal tract and anti-cancer mechanism have not been well explained. In this study, we investigated the digestive characteristics and anti-tumor mechanisms of preserved eggs using an in vitro dynamic human gastrointestinal-IV (DHGI-IV) model. During digestion, the sample pH dynamically changed from 7.01 to 8.39. The samples were largely emptied in the stomach with a lag time of 45 min after 2 h. Protein and fat were significantly hydrolyzed with 90% and 87% digestibility, respectively. Moreover, preserved eggs digests (PED) significantly increased the free radical scavenging activity of ABTS, DPPH, FRAP and hydroxyl groups by 15, 14, 10 and 8 times more than the control group, respectively. PED significantly inhibited the growth, cloning and migration of HepG2 cells at concentrations of 250-1000 µg/mL. Meanwhile, it induced apoptosis by up/down-regulating the expression of the pro-apoptotic factor Bak and the anti-apoptotic gene Bcl-2 in the mitochondrial pathway. PED (1000 µg/mL) treatment resulted in 55% higher ROS production than the control, which also led to apoptosis. Furthermore, PED down-regulated the expression of the pro-angiogenic genes HIF-1α and VEGF. These findings provided a reliable scientific reference for the study of the anti-tumor activity of preserved eggs.

Effect of salt penetration and water migration on cooked salted egg yolk gel during storage: Physicochemical properties, structural characteristics and flavor changes.

There is an urgent need to explore the salt penetration and water migration in the cooked salted egg yolk (CSEY) gel. The aim of this study was to investigate the effect of salt and water on the physicochemical, structural and flavor characteristics of CSEY gel stored at 25 °C. The hardness of the yolk increased significantly (608.0 g â†’ 2730.7 g) during storage. The results of LF-NMR showed that the free water content in the yolk gel was enhanced. The microstructure of CSEY could be observed by SEM, which showed that during storage, polyhedral particles were dispersed due to the heating process. In addition, ethyl acetate (71.9 %) was completely undetectable in the CSEY-21 d. In summary, the sample at and before 14 d of storage had good processing value. Therefore, this work would provide theoretical guidance for the low-sodium pickling of SEY (salted egg yolk) and its suitable storage methods.

Use of peptide-modified nanoparticles as a bacterial cell targeting agent for enhanced antibacterial activity and other biomedical applications.

Bacterial pathogens have posed a serious threat to human health because they are difficult to be eliminated inside cells. Here, an effective design of poly(lactic-co-glycolic) (PLGA) nanoparticles (NPs) modified with antimicrobial peptides and loaded with gentamicin (Gen) was reported with enhanced antibacterial activity and cellular internalization ability. The results showed that the drug loading capacity and encapsulation efficiency of OVTp12-modified NPs were 7.55 % and 81.3 %, respectively. We observed that OVTp12 and OVTp12-modified NPs significantly increased the interaction with Staphylococcus aureus cells. Moreover, OVTp12-modified NPs showed an effective inhibitory effect on S. aureus with low cytotoxicity. The results of cell internalization indicated that OVTp12-modified NPs were markedly higher than that of unmodified nanoparticles when incubated with MC3T3-E1 cells. In conclusion, the bacterial cell-targeting ability of this antimicrobial peptide provides advantages for the treatment of intracellular bacterial infections.

Succinylation Modified Ovalbumin: Structural, Interfacial, and Functional Properties.

In this study, ovalbumin (OVA) was succinylated with the addition of different levels of succinic anhydride, and the structural and functional properties of succinylated OVA (SOVA) were investigated. SDS-PAGE and FTIR spectrum confirmed the covalent attachment of the succinyl group to OVA. Thermal stability and the absolute value of zeta potential (pH 6.0) of SOVA were enhanced by 14.90% and 76.77% higher than that of the native OVA (NOVA), respectively. Circular dichroism (CD) spectra demonstrated that the succinylation decreased the α-helix and increased ß-sheet content to 21.31% and 43.28%, respectively. The content of free sulfhydryl groups increased and intrinsic fluorescence spectra suggested the SOVA became more unfolded and flexible as the degree of succinylation enhanced. Furthermore, succinylation effectively enhanced the solubility and decreased the interface tension (oil-water and air-water interface) of OVA. Compared to NOVA, the emulsifying activity and stability of SOVA were increased by 1.6 times and 1.2 times, respectively, and foaming capacity and stability were enhanced by 2.7 times and 1.5 times, respectively.

Identification and molecular mechanisms of novel antioxidant peptides from two sources of eggshell membrane hydrolysates showing cytoprotection against oxidative stress: A combined in silico and in vitro study.

Eggshell membranes (ESM) from fresh and hatched chicken eggs are important agricultural byproducts. In this study, we investigated the antioxidant and cytoprotective activity of hydrolysates from fresh and hatched ESM, identified the antioxidant peptides and explored their potential molecular mechanism using a combined in silico and in vitro approach. The results showed that the hydrolysates fractions (MW < 3 kDa) of both ESM exhibited excellent antioxidant effects and could protect H2O2-induced RAW264.7 cells by reducing ROS and MDA levels involving the modulation of the Keap1-Nrf2 pathway. Six novel peptides identified by integrated approaches of peptidomics and in silico bioinformatic analysis were synthesized, exhibiting significantly higher ORAC values (629.41-1823.77 µmol TE/mmol) than GSH (397.21 µmol TE/mmol). Among these, KPLCPP, MDGWPR, and LWNPR possessed stronger ABTS scavenging and cytoprotective activities than GSH. All the six peptides could dock onto the Keap1-Kelch domain. Moreover, KPLCPP and LWNPR could regulate the Keap1-Nrf2 pathway and induced the overexpression of antioxidant enzymes including HO-1, SOD and GSH-Px. With the molecular docking and western blot analysis, the underlying molecular mechanism of the ESM antioxidant peptides might be related to the activation of Keap1-Nrf2 pathway by occupying the Nrf2-binding site on Keap1. This study provides a theoretical basis for the application of fresh and hatched ESM antioxidant peptides in functional foods, as well as insights for the identification and the mechanisms research of more food-derived antioxidant peptides.

Nondestructive characterization gender of chicken eggs by odor using SPME/GC-MS coupled with chemometrics.

It's a difficult task for researchers to identify the gender of chicken eggs by nondestructive approach in the early of incubation, which not only could reduce the cost of incubation, but also could improve the welfare of chicks. Therefore, SPME/GC-MS has been applied to investigate its potential as a nondestructive tool for characterizing the differences of odor between male and female chicken eggs during early of incubation and even before hatch. The results showed that more volatiles were found in female White leghorn eggs during early of incubation and 6,10-dimethyl-5,9-undecadien-2-one, 6-methyl-5-hepten-2-one, nonanal, decanal, octanal, 2-nonen-1-ol, etc. were important for the distinction of male and female White leghorn eggs during E1-E9 of incubation. 2-ethyl-1-hexanol; octanal, nonanal, 2,2,4-trimethyl-3-carboxyisopropyl pentanoic acid isobutyl ester; 2-nonen-1-ol, cyclopropanecarboxamide, heptadecane were correlated with gender of unhatched White leghorn, Hy-line brown and Jing fen eggs, respectively. Moreover, sex-related volatiles have been strongly influenced by incubation process and egg breed, and to be related to steroid hormone biosynthesis. What's more, this study enables us to develop a new visual for ovo sexing of chicken eggs and advances our understanding of the biological significance behind volatiles emitted from chicken eggs.