Research note: Changes in chicken egg yolk metabolome during its spray drying and storage

Abstract

The differences in metabolites between fresh egg yolk (FEY), spray-dried egg yolk powder (SEY), and stored egg yolk powder (S-SEY) were quantitatively compared through metabolomic analysis. Total of 1004 metabolites were identified in the three groups of egg yolk samples. In pairwise group analysis, 242 differential metabolites were identified in FEY and SEY, 311 differential metabolites were identified in FEY and S-SEY, and 157 differential metabolites were identified in SEY and S-SEY. The analysis of differential metabolites with the highest abundance showed that amino acids, carbohydrates and lipids in FEY would undergo oxidation reactions after spray drying and storage and thus led to significant changes in the type and abundance of metabolites. The representative differential metabolites were then screened out for judging the freshness of egg yolk powder. Therefore, the results are highly important for evaluating the quality of egg yolk powder and provide important information for understanding the nutritional changes of egg yolk after spray drying and storage.

Introduction

The demand for egg powder has gradually increased accompanied by the development of food industry and importance of food safety. Compared to fresh egg and liquid egg, egg powder has significant advantages in shelf life and transportation costs. However, egg powder may undergo more changes during drying and long-term storage. Especially the nutritional changes in egg yolk powder may be more significant, as it contains abundant lipids and minerals in addition to protein. The impact of drying treatment on the physicochemical properties of yolk powder was investigated, and results showed that the spray drying temperature and storage conditions would modify the water content, water activity and the particle size distribution of yolk powder (Rannou et al., 2015). The previous work of our research group had studied in detail the impact of spray drying treatment on the functional characteristics and physical properties of egg yolks. Compared to fresh egg yolks, after spray drying treatment, the gelation and emulsification properties of egg yolk powder were significantly reduced, as well as the surface hydrophobicity was decreased, and the zeta potential was enhanced (Hu et al., 2023). In addition to these changes, lipidomics analysis also showed that lipid oxidation and hydrolysis of phospholipids were occurred after spray drying treatment and storage (Luo et al., 2023).

Therefore, this study focuses on the changes in the perspective of water-soluble metabolites in egg yolk to further supplement the changes during spray drying and storage. We employed quantitative metabolomic analysis based on LC-MS/MS to comprehensively compare the metabolites between fresh egg yolk (FEY), spray-dried yolk powder (SEY) and stored spray-dried yolk powder (S-SEY). By analyzing the changes in metabolite abundance, we aim to provide new insights into the effects of spray drying and storage on egg yolk.

Materials and methods

Preparation of FEY, SEY and S-SEY

Fresh chicken eggs (within 72 h of laying, 56.0 ± 2.0 g) from Roman hens (40-50 weeks, caged) were purchased from Sichuan Sundaily Village Ecological Food Co. Ltd. (Mianyang, Sichuan). Egg yolks were separated from the white and were collected. Fresh egg yolk (FEY, 500mL) sample was obtained by magnetic stirring at 4 °C for 1 h. The FEY was kept at 65 °C for 6 min, and then spray-dried (Bilon-6000Y, Bilang Instrument Manufacturing Co., Ltd., Shanghai, China) to prepared spray-dried egg yolk powder (SEY) with the final moisture content less than 4%. The stored SEY samples (S-SEY) was prepared by storing the SEY sample at 60 °C for 3 days (Zang et al., 2023). The preparation of each set of samples (FEY, SEY, S-SEY) was repeated three times. All samples were stored at -80 °C until analyzed.

Metabolites extraction

All samples were freeze-dried before metabolite extraction to ensure that their water content was 4 %±0.5. The metabolites extraction was performed by adding 20 mg samples (FEY, SEY and S-SEY) into 400 μL of 70% methanol aqueous solution (with the internal standard mixture) and then vortexed for 3 min. After that, ultrasound treatment (KQ5200E, Kunshan Shumei Ultrasonic Instruments Co., Ltd., Kunshan, China) was performed in the ice bath for 10 min and then vortexed for another 1 min. The samples were then left for 30 min at -20 °C and centrifuged at 12000 ×g (5424R, Eppendorf, Germany) for 10 min at 4 °C. The supernatant was aspirated and centrifuged at the same condition to obtain the extracted metabolites solution for future LC-MS/MS analysis. Metabolite extraction was performed independently for each repeated sample.

LC-MS/MS analysis of metabolites

The extracted samples were analyzed using an ultra-performance liquid chromatography (UPLC) device (ExionLC AD, SCIEX, Darmstadt, Hessian, Germany) in conjunction with a tandem mass spectrometry device (MS/MS, QTRAP 5500, SCIEX). The liquid chromatography separation was carried out on a Waters Acquity UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm) with a flow rate of 0.4 mL/min at 40 °C. The mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. The sample injection volume was 2 μL. The gradient program was mobile phase B 5% at 0 min, 90% at 11.0-12.0 min, 5% at 12.1-14.0 min. The temperature of electron-spray ionization was 550 °C, ion spray voltage was 5500 V (positive) and -4500 V (negative). Ion source gas I, gas II, curtain gas was set at 55, 60, and 25.0 psi, respectively. The collision-activated dissociation was set as “high”. Scan and detect each ion pair in the QTRAP based on optimized clustering potential and collision energy. Metabolome analysis was conducted three replicates for each group.

Data processing and analysis

The metabolites were qualitatively analyzed (Analyst 1.6.3, AB Sciex, Foster City, CA, USA) using the self-established metabolic standard database (Metware database, Wuhan Metware Biotechnology Co., Ltd.) based on the retention time, ion pair information, and secondary spectrum data. Metabolites were quantified through the multiple reaction monitoring (MRM) mode of QTRAP (Wang et al., 2022). Metabolite abundance was expressed as mean ± standard deviation. One-way analysis of variance was performed using GraphPad Prism 8.0 software (p < 0.05) (Wang et al., 2023). Discrimination of significant differences in metabolites between groups were screened with variable importance in projection (VIP) of > 1 and fold change (FC) in abundance of > 2.00 (or < 0.50).

Results and discussion

Differentially abundant metabolites (DAMs) in FEY, SEY and S-SEY

A total of 1004 metabolites were detected in FEY, SEY, and S-SEY. Among them, 373 kinds of “amino acid and its metabolite” were identified, accounting for 37.2% of the metabolite species. Other major metabolites included 114 kinds of “organic acid and its derivatives”, 98 kinds of “fatty acyls”, 79 kinds of “nucleotide and its metabolites”, 64 kinds of “heterocyclic compounds”, 62 kinds of “benzene and substituted derivatives”, 61 kinds of “glycerolphospholipids (GPs)”, 46 kinds of “carbohydrate and its metabolites”, 45 kinds of “alcohols and amines”, 20 kinds of “coenzymes and vitamins”, and 42 kinds of other metabolites.

Abundance-based calculations revealed that the categories with the highest metabolite abundance were “amino acid and its metabolites” and “GPs”. In FEY, SEY and S-SEY, “amino acid and its metabolites” accounted for 40.74%, 37.96%, and 35.67%, respectively (Fig. 1A). Following closely behind it, “GPs” accounted for 31.52% (FEY), 32.25% (SEY), and 34.41% (S-SEY), respectively. In addition, the abundance of “fatty acyls” in FEY, SEY and S-SEY were 5.73%, 5.87% and 6.71%, respectively. “Coenzymes and vitamin metabolite” in FEY, SEY and S-SEY were accounted for 3.90%, 4.22% and 4.23% respectively.

Fig. 1.

Quantitative metabolomic analysis of egg yolk during spray drying and storage. A, Comparison of the abundance content of metabolite species in FEY, SEY and S-SEY. B, PCA plots of the metabolomes; C, venn diagram of DAMs caused by spray drying (FEY vs SEY) and storage (SEY vs S-SEY). D-F, the top five DAMs with the largest variable importance in projection (VIP) value in the pairwise comparisons. FEY, fresh egg yolk; SEY, spray-dried egg yolk; S-SEY, stored SEY.

The results of PCA analysis showed significant differences among metabolites of FEY, SEY and S-SEY (Fig. 1B). The three repeated samples in the same group were relatively clustered in the PCA diagram, showing a good repeatability and high stability. The three groups (FEY, SEY and S-SEY) were separated from each other. PC1 and PC2 were 47.46% and 17.08%, respectively, which contributed a cumulative difference of 64.54%, indicating significant inter-group differences in the metabolic profiles of the three groups of samples. The DAMs between groups were screened with VIP > 1 and FC > 2.00 (or < 0.50). In the comparative analysis of FEY and SEY, 242 DAMs were identified and of these, 110 DAMs were shared by both groups, 30 DAMs were unique to FEY, and 102 were unique to SEY. The top 5 DAMs with the largest VIP value were N-acetyl-L-leucine, indole, 13(R)-hydroxyoctadecadienoic acid (13(R)-HODE), N-phenylacetylglycine, and 11,12-epoxy-eicosotrienic acid (11,12-EET) (Fig. 1D). During the subsequent storage process (60 °C for 3 days), 157 DAMs were identified between SEY and S-SEY, with 39 metabolites disappeared and 41 metabolites were newly generated after storage. The representative DAMs were 2-aminoethanesulfonic acid, 13(R)-HODE, 2-phenylacetamide, 15-hydroxy-eicosatetraenoic acid ((±)15-HETE), and 9-hydroxy-eicosatetraenoic acid ((±)9-HETE) (Fig. 1E). Accumulation changes were observed during the whole spray drying and storage process: a total of 311 DAMs were screened between the comparison between S-SEY and FEY. Including 114 newly generated metabolites in S-SEY, 39 metabolites present in FEY but disappearing in S-SEY, and 158 DAMs identified in both FEY and S-SEY. The representative DAMs were 13(R)-HODE, 2-(4-hydroxyphenyl)ethanol, 12-hydroxy-eicosatetraenoic acid ((±)12-HETE), D-glucoronic acid, and (±)high-proline (Fig. 1F). In pair-to-pair comparison, although the top 5 DAMs were different, most of them belonged to amino acid metabolites or lipid oxides, indicating that amino acids and lipids were oxidized reactions during spray drying and storage. Interestingly, 13(R)-HODE was the shared representative DAMs, which abundance was significantly increased with spray drying and storage treatment.

It was worth noting that the most representative DAMs in pairwise comparisons were different. Compared with FEY, SEY exhibited 242 DAMs, among them 90 DAMs were continuously existed after the subsequent storage process. Meanwhile, 67 DAMs were newly exhibited in S-SEY during storage in comparison to SEY (Fig. 1C). These results suggested that both spray drying and storage processes produced new DAMs, and some kinds of the metabolites produced by spray drying could undergo continuous changes in subsequent storage processes. Therefore, the analysis of these representative DAMs helped us to understand the details of changes in egg yolk powder during spray drying and storage.

Oxidation of amino acids and carbohydrates in egg yolk during spray drying and storage

Given that the total abundance of amino acid metabolites in egg yolk is more than 20 times that of carbohydrate metabolites, the metabolome data of this study did not detect the occurrence of widespread Maillard reactions. For example, the abundance of alkaline amino acids (L-lysine, L-arginine, L-histidine) and reducing sugars (D-fructose, D-glucose) in FEY did not change significantly after spray drying or during subsequent storage. However, the oxidation of amino acids and carbohydrates was observed (Fig. 2A). The abundance of oxidized amino acid derivatives such as (±)-high-proline, pyroglutamic acid, methionine sulfoxide, N(6),N(6)-dimethyl-L-lysine, and O-phospho-l-tyrosine were substantially increased in SEY and S-SEY. The primary oxidation products of carbohydrates such as D-galacturonic acid, 2-keto-D-gluconic acid, glucuronic acid, 1,6-dehydro-β-D-glucose, and D-glucosamine were abundant in FEY, but their abundance decreased significantly after spray drying and storage, suggesting these metabolites were further oxidized during spray drying and storage. In addition, the abundance of D-glucose 1,6-bisphosphate, L-gulonolactone, and sucrose 6′-monophosphate increased significantly after spray drying, indicating that phosphorylation and lactonization reaction occurred widely under heat treatment.

Fig. 2.

Changes in abundance of representative amino acids (A), lipids (B), vitamins (C), and PCA plots of the small peptide metabolites (D) in egg yolk during spray drying and storage. FEY, fresh egg yolk; SEY, spray-dried egg yolk; S-SEY, stored SEY. Significant differences (p < 0.05) between groups are indicated by different letters (a-c).

Oxidation and hydrolysis of lipids during egg yolk spray drying and storage

Compared with FEY, the abundance of lipid in SEY was same, or increased, but rarely decreased (Fig. 2B). For example, the abundance of the most representative lipids with the highest abundance in egg yolk (LPE(0:0/16:0), LPC(0:0/18:1), LPE(18:1/0:0), FFA(18:2), FFA(16:0), and carnitine C2:0 showed no significant change during the spray drying and storage. However, the oxidation of lipids in egg yolk were observed after spray drying and storage. There were 17, 34, and 38 kinds of “oxidized lipids” were identified in FEY, SEY, and S-SEY, respectively. The total abundance of these identified “oxidized lipids” in SEY and S-SEY increased by 37.99% and 74.52%, respectively, compared to FEY. Especially, the abundance of oxidation products of long-chain polyunsaturated fatty acids, such as hydroxylipids and epoxides, increased rapidly in SEY and S-SEY. The abundance of 5-hydroxy-eicosatetraenoic acid ((±)5-HETE), (±)15-HETE, 13(R)HODE, and 11,12-EET, in S-SEY was 8.48, 38.65, 42.86, and 80.24 times higher than their abundance in FEY, respectively. Similarly, our previous work also found that oxidation and hydrolysis of lipids occurred in egg yolk not only at heat treatment of spray drying but also even at lower heat treatment intensities (68.5°C for 22 min) (Luo et al., 2023; Wang et al., 2023). Besides, lipids oxidation also occurred in fresh egg yolks during storage, especially glycerophospholipid and glycerolipid (Liu et al., 2023). The phenomenon was confirmed in the present metabolomics analysis: the total abundance of 58 kinds of lysophospholipids and 27 kinds of FFAs in S-SEY increased by 8.75% and 23.28% compared with FEY, respectively.

Changes in vitamin during egg yolk spray drying and storage

Eggs are rich in vitamins, which are involved in the regulation of chicken embryo development. Among the 20 coenzymes and vitamins identified, B vitamins were the most abundant, such as pantothenate (B₅), biotin (B₇), and thiamine (B₁). In general, vitamins in food are easily lost during processing. B vitamins, as an important vitamin in eggs yolk, did not decrease or disappear significantly in SEY and S-SEY after spray drying treatment and storage treatment compared to FEY, indicating that the instantaneous high temperature treatment did not cause damage (Fig. 2C). The similar phenomenon was found in the other research. For example, Wang et al (2023) found that the abundance of vitamin A in egg was not reduced significantly after boiling because it might bind the fat-soluble protein into a stable complex (Wang et al., 2023). Besides, newly metabolite N-methylnicotinamide with high anti-inflammatory effect was produced in S-SEY during storage due to the oxidation by nicotinamide (B3). Therefore, the study on the change of vitamin content in the processing of egg yolk powder has important guiding significance for the development of high value-added egg products.

Potential biomarker metabolites in egg yolk during spray drying and storage

In this study, a total of 230 kinds of peptides were identified. Compared with FEY, 11 peptides in SEY disappeared and 13 emerged after spray drying. After storage, 4 peptides were added and 13 were lost in S-SEY compared with SEY. The lost peptides during spray drying and storage may be degraded or involved in chemical reactions to produce other derivatives, while the new peptides may arise from the degradation of proteins. As the species and abundance of egg yolk peptides vary greatly during spray drying and storage, they can be used as potential markers of the degree of egg yolk processing. PCA analysis of 230 kinds of peptides showed that PC1 and PC2 were 51.74% and 12.0%, respectively (Fig. 2D). The variation between the peptides was higher than that of the whole metabolome in PCA analysis. Therefore, the species and abundance information of peptides in egg yolk is very meaningful for determining the degree of egg yolk processing and storage time.

The key metabolites identified and screened could be applied in practical contexts. First, they could predict the shelf life of egg yolk powder during storage. Second, it was found that lipid oxidation was continuous, which suggested that the process of spray drying and packaging should try to avoid the entry of oxygen. For example, the egg powder packaging could be filled with nitrogen. Third, due to the heat treatment of spray drying would cause the degradation of macro-molecules, the temperature could be appropriately reduced in the actual processing to reduce the degradation of nutrients.

Conclusion

To study the effects of spray drying and storage on egg yolk powder, the metabolites of FEY, SEY and S-SEY were quantitatively analyzed by LC-MS/MS. Amino acids and its metabolites, organic acid and its derivative and fatty acyl were the three major metabolite categories that were identified. The change of the representative metabolites obtained in pairwise comparisons inferred that spray drying and storage would cause the oxidation of amino acids and lipids as well as the phosphorylation of carbohydrates. Representative DAMs were screened as potential biomarkers to determine the degree of processing and the shelf life of egg yolk powder. These changes in FEY, SEY and S-SEY metabolites obtained through high-throughput metabolome analysis offer new insights into the safety, trophism and storability of SEY.

Funding

This research was supported by the National Natural Science Foundation of China (32072236), and Sichuan Science and Technology Program (2024NSFSC1266). Sichuan Innovation Team Project of National Modern Agro-Industry Industry Technology System (SCCXTD-2024-24), and Modern Agro-Industry Technology Research System (CARS-40-K25).

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in the present study.

CRediT authorship contribution statement

Beibei Wang: Investigation, Data curation, Writing – original draft. Xialei Liu: Data curation. Shugang Li: Resources, Writing – review & editing. Shijian Dong: Data curation. Putri Widyanti Harlina: Methodology, Writing – review & editing. Jinqiu Wang: Writing – original draft. Fang Geng: Conceptualization, Writing – original draft, Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.