Yolk Protein Research Articles

Assessing the structural and foaming property changes in egg yolk proteins due to malondialdehyde: Experimental and molecular docking studies

This study evaluated the effects of varying levels of malondialdehyde (MDA) on the structural and foaming properties of the egg yolk proteins (EYPs), and the interaction between them was explored by molecular docking. The results showed that oxidative modification due to MDA increased the carbonyl content of EYPs by 4.49 times. Simultaneously, the total sulfhydryl content was reduced by 21.47%, and the solubility of EYPs was significantly decreased (p < 0.05). Continuous oxidation disorders the previously ordered structure of EYPs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that some proteins underwent crosslinking and aggregation with increased MDA oxidation, aligning with changes in particle size and zeta-potential. Moderate oxidation (<1 mmol/L) enhanced the foaming capacity and foam stability of EYPs. Additionally, molecular docking results uncovered favorable interactions between MDA and specific EYPs, primarily through hydrogen bonding. This research offers valuable insights into managing the functional and quality changes of yolk products during processing.

Comparative analysis on the properties of egg yolk lipids extracted by different extraction methods.

Egg yolk lipids are important components in egg yolk. Compared with proteins, minerals, vitamins and pigments in egg yolk, there is a lack of systematic research on extraction methods and characteristics of yolk lipid. Therefore, in this study, the extraction of egg yolk lipids by supercritical CO2 fluid extraction (SFE), subcritical propane extraction (SPE) and ethanol solvent extraction (SE) was studied, and the differences in composition and physicochemical properties of the three yolk lipids were analyzed. The egg yolk lipids extracted by SFE had the advantages of high unsaturated fatty acid content, low saponification value, acid value, peroxide value, and high content of neutral lipids such as triglyceride and cholesterol, but the polar phospholipids were completely retained in the yolk powder. The efficiency of lipid extraction of SPE was the highest among all the three methods, but the extraction capacity to the polar phospholipid was in the middle of the three methods. The efficiency of lipid extraction of SE was low, but the extraction capacity to the polar phospholipid was the highest. Suitable extraction methods could be selected based on the specific application requirements of egg yolk lipids and low-fat egg yolk powders.

The juvenile hormone analogue, pyriproxifen, alters protein and fat composition of Tenebrio molitor larvae

Abstract Maximising the yield of product from livestock is common practice in the agriculture industry and there is potential to extend this practice to the emerging insect industry, to produce high-quality, sustainable protein. Tenebrio molitor larvae, commonly called yellow mealworms, were fed for 28 days on wheat bran containing the juvenile hormone analogue, pyriproxifen at either 2 mg pyriproxifen/kg wheat bran (JH-PL) or 15 mg pyriproxifen/kg wheat bran (JH-PH). As expected, pupation was inhibited in both pyriproxifen treated groups and significant changes in nutritional composition were observed. Pyriproxifen treated mealworms had a higher protein content per 100 grams of dried material, while fat content was reduced 68% in JH-PH compared to control. These changes were associated with an increase in moisture content and reduction in energy content. The fatty acid profile of extracted fat also displayed significant alterations, with pyriproxifen treated mealworms showing an increase in proportions of saturated fatty acids, reduction in oleic acid but no effect on linoleic acid. The amino acid composition also exhibited a change in composition as a result of pyriproxifen treatment, including an increase in the essential amino acid, lysine, in JH-PH treated mealworms. This change in amino acid profile was associated with a change in the protein composition as observed on SDS-PAGE, with the appearance of a new band identified as the egg-yolk protein, vitellogenin, which has lipid-transporter activity. Hence, pyriproxifen treatment of mealworms has a repartitioning effect, resulting in an increase in the proportion of protein and a decrease in fat on a dry matter basis, demonstrating that mealworm nutrient composition can be manipulated to provide a higher value feed ingredient.

Divalent metal ions under low concentration environment improved the thermal gel properties of egg yolk

To improve the thermal gel properties of egg yolk, the effect of several valence metal ions (K+, Ca2+, Mg2+ and Fe3+) with different concentrations (0-0.72%) on the rheological, gel, and structural properties of egg yolk were investigated. Results showed that monovalent and divalent ions were beneficial to the formation of uniform and dense gel network, especially with the addition of 0.72% magnesium ion, which further improved gel hardness, water holding capacity (WHC) and viscoelastic properties, the properties of egg yolk gel increased with the increase of the concentration of mono-bivalent metal ions. Adding ferric ion remarkably increased the average particle size (d4,3) and apparent viscosity of egg yolk, destroying the disulfide bonds and the hydrophobic interactions in gel. Fourier transform infrared spectroscopy (FT-IR) and fluorescence spectra analysis revealed that metal ions promoted the hydrophobic aggregation among egg yolk proteins and induced the transition of protein secondary structure from ordered to disordered. This work will provide a theoretical reference for the development of low salt and nutrient fortified egg yolk products.

Effect of high-hydrostatic pressure on the digestibility of egg yolk and granule.

The impact of high hydrostatic pressure (HHP) on protein digestibility of egg yolk and egg yolk granule was evaluated by static in vitro digestion using the standardized INFOGEST 2.0 method. The degree of hydrolysis (DH) and the phospholipid content were determined during digestion, and the protein and peptide profiles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase-high pressure liquid chromatography (RP-HPLC). The results showed that HHP induced protein aggregation in egg yolk and granule, mainly by disulfide bridges, which were not disrupted in the oral phase. Proteolysis during the gastric phase improved egg yolk and granule protein solubility, regardless of whether HHP was applied. However, the extent of the samples' digestibility was not affected, with DH values ranging from 15% to 20%. During the intestinal phase, the DH of egg yolk protein (∼40%) was higher than that of the granule (∼25%), probably due to the denser structure of the granule reducing the accessibility of intestinal enzymes. The DH, peptide, and protein profiles of control and HHP-treated egg yolk showed similar protein digestion behaviors for both gastric and intestinal phases. Among the different proteins, only the digestibility of β-phosvitin in HHP-treated granule was enhanced. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin with the potential to generate bioactive phosvitin-derived phosphopeptides. PRACTICAL APPLICATION: High hydrostatic pressure, mainly used as a preservation process, did not impair the nutritional quality of the egg yolk and granule proteins but improved the susceptibility of phosvitin (protein contained in egg yolk) proteolysis to produce bioactive phosphopeptides. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin.

The effects of sucrose/NaCl combined pickling on the textural characteristics, moisture distribution, and protein aggregation behavior of egg yolk.

Salted egg yolks have a tender, loose, gritty, and oily texture and are commonly employed as fillings in baked goods. This study investigated the formation mechanism of egg yolk gels using three different pickling methods: NaCl, sucrose, and mixed groups. The results revealed that of these pickling methods, egg yolks pickled with the mixture had the lowest moisture content (11.59% at 25°C and 10.21% at 45°C), almost no free water content, and the highest hardness (19.11 N at 25°C and 31.01 N at 45°C). Intermolecular force measurements indicated that pickling with the mixture mitigated the surface hardening effect of sucrose and facilitated protein cross-linking. Moreover, confocal laser scanning microscopy of the egg yolk gels pickled with the mixture displayed macromolecular aggregates and oil exudation, suggesting that this method partially disrupted the lipoprotein structure and notably promoted yolk protein aggregation and lipid release. Overall, egg yolks formed a dense gel via the mixed pickling method owing to the ionic concentration and dehydration effects. These findings show the impact of NaCl and sucrose in pickling egg yolks, providing a crucial foundation for developing innovative and desirable egg yolk products. PRACTICAL APPLICATION: This study introduces a novel pickling strategy that combines sucrose and NaCl for egg yolk processing. The egg yolk pickled using this method exhibited improved quality according to the evaluated textural characteristics, moisture distribution, and protein aggregation behavior. The findings may broaden the use of sucrose as a pickling agent for egg yolk processing and provide new ideas for developing and producing pickled eggs and other food products.

Effect of salting and dehydration treatments on the physicochemical and gel properties of hen and duck egg yolks, plasma and granules.

Salted hen egg yolks are less oily and less flavorful than salted duck egg yolks. However, hen eggs have a more adequate market supply and have a broader application prospect than duck eggs. In the present study, egg yolks, plasma, and granules were dehydrated by adding 1% NaCl to simulate traditional curing process of salted egg yolk. The changes in the pickling process of hen egg yolks (HEY) and duck egg yolks (DEY) plasma and granules were compared to reveal the gelation mechanism and the underlying causes of quality differences in salted HEY and DEY. Salted HEY can be compared with the changes in DEY during the pickling process to provide a theoretical basis for the quality improvement of salted HEY to salted DEY. The results showed that both plasma and granules were involved in gel formation, but exhibited different aggregation behaviors. Based on the intermolecular forces, the HEY proteins achieved aggregation mainly through hydrophobic interactions and DEY proteins mainly through covalent binding. According to spin-spin relaxation time, HEY gels immobilized a large amount of lipid and interacted strongly with lipids. DEY gels showed much free lipid and had weak interaction with lipid. The microstructure showed that HEY proteins were easily unfolded to form a homogeneous three-dimensional gel network structure after salting, whereas heterogeneous aggregates were formed to hinder the gel development in DEY. Changes in protein secondary structure content showed that pickling can promote the transformation of the α-helices to β-sheets structure in HEY gels, whereas more α-helices structure was formed in DEY gels. The present study has demonstrated that different gelation behaviors of hen and duck egg yolk proteins (especially in plasma) through salting treatment led to the difference in the quality of salted HEY and DEY. © 2024 Society of Chemical Industry.

Co-enzymatic hydrolysis enhanced thermal stability of mayonnaise

With the accelerated pace of life, frozen bakery products and ready-to-eat hot food are gradually emerging. Mayonnaise, a commonly used flavor sauce, is also applied to these products. However, with the large amount of oil and fat precipitation of mayonnaise in the reheating process of the product, which affects its application in the food industry, the thermal stability of mayonnaise has been gradually paid attention to. In this study, the thermal stability of mayonnaise was effectively improved by the combined enzymatic hydrolysis of liquid egg yolk (LEY) with phospholipase A2 (PLA2) and flavor protease. It was found that the moderate addition of flavor protease could effectively reduce the surface hydrophobicity and interfacial tension of egg yolk protein and improve its emulsification stability. Moreover, the hydrolysis reduced the molecular weight of yolk protein, thus reducing the thermal aggregation effect of yolk protein due to heating, which in turn reduced the loss of protein at the O/W interface and improved the thermal stability of mayonnaise.

Methionine Supplementation Alleviates the Germ Cell Apoptosis Increased by Maternal Caffeine Intake in a C. elegans Model.

Caffeine (1,3,7-trimethylxanthine) is a widely consumed bioactive substance worldwide. Our recent study showed that a reduction in both reproduction and yolk protein production (vitellogenesis) caused by caffeine intake were improved by vitamin B12 supplementation, which is an essential co-factor in methionine metabolism. In the current study, we investigated the role of methionine in the reproduction of caffeine-ingested animals (CIAs). We assessed the effect of methionine metabolism on CIAs and found that caffeine intake decreased both methionine levels and essential enzymes related to the methionine cycle. Furthermore, we found that the caffeine-induced impairment of methionine metabolism decreased vitellogenesis and increased germ cell apoptosis in an LIN-35/RB-dependent manner. Interestingly, the increased germ cell apoptosis was restored to normal levels by methionine supplementation in CIAs. These results indicate that methionine supplementation plays a beneficial role in germ cell health and offspring development by regulating vitellogenesis.

Advances in Understanding the Antioxidant and Antigenic Properties of Egg-Derived Peptides.

Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were characterised in terms of antioxidant and IgE-binding properties, using a combination of in vitro and in silico methods. Based on the degree of hydrolysis (DH) results, the egg yolk proteins are better substrates for all the tested enzymes (DH of 6.2-20.1%) compared to those from egg whites (DH of 2.0-4.4%). The SDS-PAGE analysis indicated that pepsin and proteinase K were more efficient compared to trypsin in breaking the intramolecular peptide bonds of the high molecular weight egg proteins. For all the tested substrates, enzyme-assisted hydrolysis resulted in a significant increase in antioxidant activity, suggesting that many bioactive peptides are encrypted in inactive forms in the parent proteins. The hydrolysates obtained with proteinase K exhibited the highest DPPH radical scavenging activity (124-311 µM Trolox/g protein) and the lowest residual IgE-binding capacity. The bioinformatics tools revealed that proteinase K is able to break the integrity of the main linear IgE-binding epitopes from ovalbumin and ovomucoid. It can be concluded that proteinase K is a promising tool for modulating the intrinsic properties of egg proteins.

Proteomic Analysis of Egg Yolk Proteins During Embryonic Development in Wanxi White Goose.

To investigate the alterations of yolk protein during embryonic development in Wanxi white goose, the egg yolk protein composition at days 0, 4, 7, 14, 18, and 25 of incubation (D0, D4, D7, D14, D18, and D25) was analyzed by two-dimensional gel electrophoresis combined with mass spectrometry. A total of 65 spots representing 11 proteins with significant abundance changes were detected. Apolipoprotein B-100, vitellogenin-1, vitellogenin-2-like, riboflavin-binding protein, and serotransferrin mainly participated in nutrient (lipid, riboflavin, and iron ion) transport, and vitellogenin-2-like showed a lower abundance after D14. Ovomucoid-like were involved in endopeptidase inhibitory activity and immunoglobulin binding and exhibited a higher expression after D18, suggesting a potential role in promoting the absorption of immunoglobulin and providing passive immune protection for goose embryos after D18. Furthermore, myosin-9 and actin (ACTB) were involved in the tight junction pathway, potentially contributing to barrier integrity. Serum albumin mainly participated in cytolysis and toxic substance binding. Therefore, the high expression of serum albumin, myosin-9, and ACTB throughout the incubation might protect the developing embryo. Apolipoprotein B-100, vitellogenin-1, vitellogenin-2-like, riboflavin-binding protein, and serotransferrin might play a crucial role in providing nutrition for embryonic development, and VTG-2-like was preferentially degraded/absorbed.

Foaming and structural properties of egg yolk components microgel particles and their adsorption characteristics at the air-water interface

In this study, microgel particles of egg yolk and its components were constructed by inducing and modifying their structure via the high-pressure homogenization (HPH). In order to compare their structural characterization and reveal their adsorption mechanism at the air-water interface, yolk components were also combined with egg white protein (EWP) to achieve their microgels for investigation. Results suggested that after HPH, foam stabilities of egg yolk protein microgel (EYM) and egg yolk plasma microgel (EPM) were significantly improved, whereas no significance occurred on egg yolk granules microgel (EGM). Interestingly, after HPH, the adsorption capacity of the microgels from combination of yolk components and EWP exhibited a significant increasing trend. However, their interfacial adsorption stability was poor. Fluorescence spectrum and surface tension revealed that more hydrophilic groups and lower surface tension would occur on EY and EP after HPH, whilst no significant effects would happen on EG, which was consistent with the results of foaming characteristics. SDS-PAGE exhibited that there was no obvious change in the protein bands of egg yolk and its components, indicating that egg yolk and component proteins did not undergo covalent crosslinking or degradation with HPH treatment. In summary, our study prepares for the first time of the microgel particles from egg yolk components for stabilizing the air-water interface. In addition, we have revealed the relationship between the physicochemical properties, microstructures and interfacial adsorption properties of the microgel particles from egg yolk components, and have explored their mechanisms in depth.

Molecular Modeling of the Adsorption of an Egg Yolk Protein on a Water-Oil Interface.

Egg yolk contains several molecular species with emulsifying properties, such as proteins and phospholipids. In particular, these molecules have both polar and nonpolar parts and thus can act as surfactants. One of the most surface-active proteins from egg yolk low-density lipoproteins is the so-called Apovitellenin-1. Experimental studies have been hindered by difficulties in isolating individual species from egg yolk lipoproteins. The purpose of this work was to assess the emulsifying properties of Apovitellenin-1 and any potential cooperative or competitive behavior in the presence of phospholipids. To do so, molecular simulations were carried out in a liquid-liquid interfacial system consisting of water and soybean oil, with varying concentrations of phospholipids and for different spatial configurations. To evaluate the conformational stability of the protein at the water-oil interface, the Gibbs free energy was computed from Metadynamics simulations as a function of the distance from the interface and of the radius of gyration. Moreover, a detailed analysis was also performed to determine which peptide residues were responsible for the protein adsorption at the oil-water interface as well as the lowering of the interfacial tension. Lastly, we combined the simulation results with a thermodynamic model to predict the interfacial tension behavior at increasing protein bulk concentration, which cannot be measured experimentally.

Investigation into the impact of enzyme treatment on the structural characteristics of egg yolk protein and its correlation with flavor constituents

The effects of three different enzyme treatments on the structure and volatile compounds of egg yolk protein were investigated. The secondary and tertiary structures, as well as volatiles, were characterized using ultraviolet spectroscopy (UV), fourier transform infrared (FTIR) spectroscopy, free sulfhydryl group analysis, and gas chromatography-mass spectrometry (GC-MS). FTIR analysis revealed a decrease in the contents of intermolecular, intramolecular, and intermolecular antiparallel β-sheets while an increase in random coils was observed. The contents of α-helices and β-turns did not show significant changes, indicating a reduction in protein compactness. Enzymatic treatment led to an increase in hydrophobic interaction, electrostatic interaction, and hydrogen bond formation within the egg yolk hot gel (EYG). Structural characterization demonstrated that EYG formed large uniform air chamber cavities after lipase hydrolysis (LEYG), while EYG hydrolyzed by papain (PEYG) and by flavor protease (FEYG) exhibited more compact structures. In addition, the binding of proteins to flavor substances inhibited the release of volatile flavor compounds. These findings provide insights into enhancing the flavor and texture of egg yolk by modulating its protein structure.

Diets comprising hen egg yolk and milk proteins as potential alternatives to shark egg-based diets for larvae of the Japanese eel Anguilla japonica

Diets comprising hen egg yolk and milk proteins as potential alternatives to shark egg-based diets for larvae of the Japanese eel Anguilla japonica

RNAi-Mediated Knockdown of Acidic Ribosomal Stalk Protein P1 Arrests Egg Development in Adult Female Yellow Fever Mosquitoes, Aedes aegypti.

After taking a blood meal, the fat body of the adult female yellow fever mosquito, Aedes aegypti, switches from a previtellogenic state of arrest to an active state of synthesizing large quantities of yolk protein precursors (YPPs) that are crucial for egg development. The synthesis of YPPs is regulated at both the transcriptional and translational levels. Previously, we identified the cytoplasmic protein general control nonderepressible 1 (GCN1) as a part of the translational regulatory pathway for YPP synthesis. In the current study, we used the C-terminal end of GCN1 to screen for protein-protein interactions and identified 60S acidic ribosomal protein P1 (P1). An expression analysis and RNAi-mediated knockdown of P1 was performed to further investigate the role of P1 in mosquito reproduction. We showed that in unfed (absence of a blood meal) adult A. aegypti mosquitoes, P1 was expressed ubiquitously in the mosquito organs and tissues tested. We also showed that the RNAi-mediated knockdown of P1 in unfed adult female mosquitoes resulted in a strong, transient knockdown with observable phenotypic changes in ovary length and egg deposition. Our results suggest that 60S acidic ribosomal protein P1 is necessary for mosquito reproduction and is a promising target for mosquito population control.

Yolk proteins of the schistosomiasis vector snail Biomphalaria glabrata revealed by multi-omics analysis

Vitellogenesis is the most important process in animal reproduction, in which yolk proteins play a vital role. Among multiple yolk protein precursors, vitellogenin (Vtg) is a well-known major yolk protein (MYP) in most oviparous animals. However, the nature of MYP in the freshwater gastropod snail Biomphalaria glabrata remains elusive. In the current study, we applied bioinformatics, tissue-specific transcriptomics, ovotestis-targeted proteomics, and phylogenetics to investigate the large lipid transfer protein (LLTP) superfamily and ferritin-like family in B. glabrata. Four members of LLTP superfamily (BgVtg1, BgVtg2, BgApo1, and BgApo2), one yolk ferritin (Bg yolk ferritin), and four soma ferritins (Bg ferritin 1, 2, 3, and 4) were identified in B. glabrata genome. The proteomic analysis demonstrated that, among the putative yolk proteins, BgVtg1 was the yolk protein appearing in the highest amount in the ovotestis, followed by Bg yolk ferritin. RNAseq profile showed that the leading synthesis sites of BgVtg1 and Bg yolk ferritin are in the ovotestis (presumably follicle cells) and digestive gland, respectively. Phylogenetic analysis indicated that BgVtg1 is well clustered with Vtgs of other vertebrates and invertebrates. We conclude that, vitellogenin (BgVtg1), not yolk ferritin (Bg yolk ferritin), is the major yolk protein precursor in the schistosomiasis vector snail B. glabrata.

Maternal High-Fat Diet Affects the Contents of Eggs and Causes Abnormal Development in the Medaka Fish.

Maternal nutritional status can affect development and metabolic phenotypes of progeny in animals. The effects of maternal diet are thought to be mediated mainly by changes inside oocytes such as organelles, maternal RNAs, and metabolites. However, to what extent each factor contributes to offspring phenotypes remains uncertain, especially in viviparous mammalian systems, where factors other than oocytes, such as placenta and milk, need to be considered. Here, using the medaka fish as an oviparous vertebrate model, we examined whether maternal high-fat diet (mHFD) feeding affects offspring development and what kind of changes occur in the contents of mature eggs. We found that mHFD caused the high frequency of embryonic deformities of offspring, accompanied by downregulation of transcription- and translation-related genes and zygotic transcripts at the blastula stage. Transcriptomic and metabolomic analyses of mature eggs suggested decreased catabolism of amino acids and glycogen, moderate upregulation of endoplasmic reticulum stress-related genes, and elevated lipid levels in mHFD eggs. Furthermore, high-fat diet females showed a higher incidence of oocyte atresia and downregulation of egg protein genes in the liver. These data suggest that attenuated amino acid catabolism triggered by decreased yolk protein load/processing, as well as elevated lipid levels inside eggs, are the prime candidates that account for the higher incidence of embryonic deformities in mHFD offspring. Our study presents a comprehensive data on the changes inside eggs in a mHFD model of nonmammalian vertebrates and provides insights into the mechanisms of parental nutritional effects on offspring.

Genome-Wide Identification of Vitellogenin Gene Family and Comparative Analysis of Their Involvement in Ovarian Maturation in Exopalaemon carinicauda.

Vitellogenin (Vtg) is a precursor of yolk proteins in egg-laying vertebrates and invertebrates and plays an important role in vitellogenesis and embryonic development. However, the Vtg family remains poorly characterized in Exopalaemon carinicauda, a major commercial mariculture species found along the coasts of the Yellow and Bohai Seas. In this study, 10 Vtg genes from the genomes of E. carinicauda were identified and characterized. Phylogenetic analyses showed that the Vtg genes in crustaceans could be classified into four groups: Astacidea, Brachyra, Penaeidae, and Palaemonidae. EcVtg genes were unevenly distributed on the chromosomes of E. carinicauda, and a molecular evolutionary analysis showed that the EcVtg genes were primarily constrained by purifying selection during evolution. All putative EcVtg proteins were characterized by the presence of three conserved functional domains: a lipoprotein N-terminal domain (LPD_N), a domain of unknown function (DUF1943), and a von Willebrand factor type D domain (vWD). All EcVtg genes exhibited higher expression in the female hepatopancreas than in other tissues, and EcVtg gene expression during ovarian development suggested that the hepatopancreas is the main synthesis site in E. carinicauda. EcVtg1a, EcVtg2, and EcVtg3 play major roles in exogenous vitellogenesis, and EcVtg3 also plays a major role in endogenous vitellogenesis. Bilateral ablation of the eyestalk significantly upregulates EcVtg mRNA expression in the female hepatopancreas, indicating that the X-organ/sinus gland complex plays an important role in ovarian development, mostly by inducing Vtg synthesis. These results could improve our understanding of the function of multiple Vtg genes in crustaceans and aid future studies on the function of EcVtg genes during ovarian development in E. carinicauda.

Vitamin B12 Supplementation Improves Oocyte Development by Modulating Mitochondria and Yolk Protein in a Caffeine-Ingested Caenorhabditis elegans Model.

Vitamin B12 is an essential cofactor involved in the function of two enzymes: cytosolic methionine synthase and mitochondrial methylmalonic-CoA mutase. In our previous studies, caffeine (1,3,7-trimethylxanthine), the most popular bioactivator, was shown to reduce yolk protein (vitellogenin) and fertility in a Caenorhabditis elegans model. Based on the previous finding that methionine supplementation increases vitellogenesis in C. elegans, we investigated the role of vitamin B12 in methionine-mediated vitellogenesis during oogenesis in caffeine-ingested animals (CIA). Vitamin B12 supplementation improved vitellogenesis and reduced oxidative stress by decreasing mitochondrial function in CIA. Furthermore, the decreased number of developing oocytes and high levels of reactive oxygen species in oocytes from CIA were recovered with vitamin B12 supplementation through a reduction in mitochondrial stress, which increased vitellogenesis. Taken together, vitamin B12 supplementation can reverse the negative effects of caffeine intake by enhancing methionine-mediated vitellogenesis and oocyte development by reducing mitochondrial stress.