
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00780-6
10.1016/j.psj.2024.104201
104201
METABOLISM AND NUTRITION
Lipidomics combined with random forest machine learning algorithms to reveal freshness markers for duck eggs during storage in different rearing systems
Chen Mengying *†1
Gong Lan *1
Zhu Lei *
Fang Xiaomin *
Zhang Can †
You Zhaorong ‡
Chen Huimin §
Wei Ruicheng rcwei79@163.com
*2
Wang Ran *3
⁎ Institute of Quality Safety and Nutrition of Agricultural Products, Jiangsu Academy of Agricultural Sciences, Jiangsu Provincial Key Laboratory of Food Quality and Safety-Province and Ministry jointly built the cultivation base of the State Key Laboratory, Nanjing 210014, China
† College of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
‡ Gaoyou Duck Egg Association, Yangzhou 225600, China
§ SCIEX, China
2 Corresponding author: rcwei79@163.com
3 Co-Corresponding author.
1 The authors contributed equally to this work (co-first author).

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https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The differences in lipids in duck eggs between the 2 rearing systems during storage have not been fully studied. Herein, we propose untargeted lipidomics combined with a random forest (RF) algorithm to identify potential marker lipids based on ultra-performance liquid chromatography‒mass spectrometry (UPLPC-MS/MS). A total of 106 and 16 differential lipids (DL) were screened in egg yolk and white, respectively. In yolk, metabolic pathway analysis of DLs revealed that glycerophospholipid metabolism and sphingolipid metabolism were the key metabolic pathways in the traditional free-range system (TFS) during storage, glycosylphosphatidylinositol-anchored biosynthesis and glyceride metabolism were the key pathways in the floor-rearing system (FRS). In egg white, the key pathway in both systems is the biosynthesis of unsaturated fatty acids. Combined with RF algorithm, 12 marker lipids were screened during storage. Therefore, this study elucidates the changes in lipids in duck eggs during storage in 2 rearing systems and provides new ideas for screening marker lipids during storage. This approach is highly important for evaluating the quality of egg and egg products and provides guidance for duck egg production.

Key words

duck egg
2 rearing system
untargeted lipidomic
random forest algorithm
marker compound
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pmcINTRODUCTION

Duck eggs are the second largest poultry egg population in the world (Lin et al., 2016). Duck eggs are a healthy food item that are both economically and nutritionally accessible, and they contain essential macronutrients such as readily digested proteins and fats, as well as micronutrients such as vitamins and minerals (Miranda et al., 2015). Most duck eggs need to undergo different storage durations from production to sale. In the USA and Canada, eggs are kept at low temperatures during storage, transportation, and sales. However, full cold-chain infrastructure is not accessible for the storage and delivery of duck eggs in developing or impoverished nations (Meng et al., 2022). In addition, to make items such as salted duck eggs or pidan, which are easily prepared and stored products, some producers deceive consumers by using expired or spoiled duck eggs. Sometimes, this lack of freshness occasionally does not manifest as an observable alteration in color or odor. But freshness has a great influence on the processing of duck eggs (Chang et al., 2021; Sun et al., 2023).

The functional properties of eggs can be impacted by external conditions, such as breed, rearing system, temperature, humidity, storage time and handling conditions (Liu et al., 2023). Freshness is a crucial parameter for ensuring the production of safe and high-quality commodities. However, the evaluation of freshness is often based on sensory quality, and changes in functional nutrients are not clearly defined. The assessment of egg freshness is still challenging due to the lack of robust chemical markers and screening technique (Cavanna et al., 2018). Random forest (RF) algorithms are among the most accurate prediction machine learning methods and have been successfully applied in various fields, including biomedical exploration and food quality evaluation (Cutler et al., 2007; Cardinal et al., 2020; Breda et al., 2024). Gu et al. revealed key marker compounds of pork freshness during storage based on an RF model (Gu et al., 2023). Furthermore, the rearing system is a significant nongenetic factor that affects production benefits and egg quality in duck husbandry (Guo et al., 2021). As potential substitutes for traditional free-range rearing systems (TFSs), floor-rearing systems (FRSs) are the current predominant dryland duck rearing systems. The lipid deposition may be affected by the rearing systems (Samman et al.,2009). However, the different influence of these 2 systems on storage quality and lipid characteristics of duck eggs is poorly understood.

Lipidomics has been widely applied to analysis of food composition, quality identification and traceability in food research by identifying hundreds of lipids in an unbiased way at the molecular level (Han, 2016; Liu et al., 2020; Wu et al., 2020; Wang et al., 2023). During long-term storage, eggs undergo lipid deterioration, such as oxidation and hydrolysis (Lv et al., 2023). Changes in lipid profiles negatively influence flavor, color, nutritive value, safety and processing and can affect the purchasing behavior of customers (Yamamoto et al., 2021). Studies have shown that the content of polyunsaturated fatty acids (PUFA) decreases and that of monounsaturated fatty acids (MUFAs) and saturated fatty acids (SFAs) increases during egg storage, thereby markedly reducing the nutritional and edible value of eggs (Drabik et al., 2021). Moreover, lipid oxidation products change the structure and function of other components and affect the quality and functional characteristics of stored eggs (Bao et al., 2019). Zhang et al. reported that lipid oxidation in egg white can influence the protein foaming properties during egg storage and processing periods (Zhang et al., 2022). Moreover, identifying key lipids involved in metabolic regulation can be accomplished by comparing changes in the lipid metabolic network under different conditions and ultimately revealing the mechanism of lipid action during various biological activities (Zhang et al., 2023). Thus, as a key component of eggs, the mechanism of how the lipid composition affects the freshness of eggs during storage is not fully understood.

Gaoyou duck eggs as agro-product geographical indications of China have excellent egg quality. Freshness is one of the main factors influencing the quality of salted duck egg. As a result, research into how rearing systems and storage time could impact duck egg quality is required. To our knowledge, although there are some reports of changes in the lipid groups of egg yolk during storage, the relationship between lipid transformation and sensory quality is still unclear, which largely limits the progress of research on the relationship between storage time and the mechanism of spoilage. Meanwhile, few studies have used lipidomics to identify biomarkers during duck egg storage. To bridge this research gap, this study proposed a method in which untargeted lipidomics is coupled with an RF machine model algorithm to identify potential marker compounds and analyse their effects on freshness change during egg storage by UHPLC–MS/MS. Therefore, the aim of this study was to provide a comprehensive profile of lipids in duck eggs in different rearing systems, to provide additional data to support the determination of duck eggs freshness, and to propose a new approach for identifying marker compounds. These results are helpful for evaluating the quality of egg and egg products and provides guidance for duck egg production.

MATERIALS AND METHODS

Sample Preparation

Fresh duck eggs from 2 different rearing systems (lake region free-range rearing system, TFS; floor-rearing system, FRS) laid for 24 h were obtained from the core area of Gao You (Jiangsu, China) and used in this study. A total of 150 eggs in each group were divided into 5 groups. The plants were kept at 22°C±2°C and 60% relative humidity. Sampling was carried out at 0, 7, 14, 21 and 28 d, and 6 samples were randomly selected each time. The quality of the duck eggs was first determined, after which the raw egg yolk was separated from the egg white. The yolk membrane was pierced to collect the egg yolk. Then, the yolk and egg white obtained from each sample were combined and mixed evenly. The samples were quickly frozen in liquid nitrogen and stored at 80°C for further study.

Determination of Egg Qualities During Storage

Six eggs were randomly selected from thirty duck eggs collected from each rearing system at 5 storage time points to determine egg quality, including egg weight (EW), Haugh unit (HU), egg white height (EWH), yolk color (YC), yolk index (YI), eggshell thickness (EST) and pH. The EW, HU, and YC of the duck eggs were analyzed by an egg multitester (Robotmation EMT-7300; Tokyo, Japan). The quality of the duck eggs was determined as described previously (Dang et al., 2023). The yolk, albumen, and eggshell from each egg were separated and weighed to calculate the composition ratio of the eggs (Shi et al., 2022; Tan et al., 2022).

Lipid Extraction

The samples were thawed on ice. Then, egg yolk (70 mg) and egg white (140 mg) were homogenized with 200 µL of water, and each sample was homogenized at 6.1 M/S for 20 s with a steel ball. After the addition of 1 mL of the extraction solvent (MTBE: MeOH = 3:1, v/v), the mixture was ultrasonicated for 20 min at 4°C, followed by standing for 30 min at room temperature. The solution was centrifuged at 15,000 × g for 15 min at 10°C, after which the upper organic solvent layer was obtained and dried under nitrogen. The lipid extracts of egg yolk and egg white were redissolved in 400 μL and 200 μL 90% isopropanol/acetonitrile/water (v/v/v= 30:65:5), respectively, for LC–MS/MS analysis.

LC‒MS/MS Method for Lipid Analysis

Reverse-phase chromatography was selected for LC separation using a CSH C18 column (1.7 µm, 2.1 mm × 100 mm, Waters). Finally, 1 µL of sample was injected. Solvent A was acetonitrile-water (6:4, v/v) with 10 mM ammonium formate, and solvent B was acetonitrile-isopropanol (1:9, v/v) with 10 mM ammonium formate. The initial mobile phase was 20% solvent B, and the flow rate was 300 µL/min. The temperature was held for 1 min, increased to 60% solvent from 1 min to 4 min, linearly increased to 98% solvent B in 25 min and held for 1 min, and then equilibrated at 20% solvent B for 4 min.

A ZenoTOF 7600 system (SCIEX, Framingham, MA) equipped with an external calibrant delivery system (CDS) was used for mass spectrometry analysis. Information-dependent acquisition (IDA) was performed for MS/MS analysis in both positive electrospray ionization (ESI+) and negative electrospray ionization (ESI−) modes. In ESI+ mode, the MS parameters were as follows: declustering potential, 80 V; collision energy, 30 V; ion spray voltage, +5500 V; mass range, m/z 50 to 1200; gas 1 and gas 2 pressure, 50 psi; curtain gas pressure, 35 psi; and interface heater temperature, 600°C. The parameters for analyses conducted in ESI− mode were as follows: declustering potential, −80 V; collision energy, −30 V; ion spray voltage, −4,500 V; and mass range, m/z 50 to 1,200.

Data Processing and Statistics

The raw LC-MS data files were converted into mzXML format by MSConvert. For lipid identification, the MS/MS spectrum of each feature was searched by MS-DIAL software version 24 with an integrated LipidBlast12 database (containing 180,378 lipids). Intensity of each peak was recorded. We used relative quantifications in this study based on the peak integration of the MS/MS spectra. The statistical analyses, including missing-data imputation with KNN method and data normalization with probabilistic quotient normalization method, were performed using metaX (Liu et al., 2020). Additionally, lipid species were distinguished by using strict criteria consisting of high mass accuracy of precursor ion (<0.01 Da), fragment ion (<0.05 Da), and ion intensity score related to the consistency of the MS/MS fragmentation, and the parameters RT tolerance, MS1 (primary MS profile) accurate mass tolerance, MS2 (secondary MS profile) accurate mass tolerance, and identification score cut-off value were set at 5 min, 0.01 Da, 0.05 Da, and 80%, respectively. The screening of differentially abundant lipids (DLs) fulfilled the criteria of a cut-off value of log2 fold change (FC) (relative intensity) > 1.5 and < 0.67, P < 0.01, and variable importance in the projection (VIP) > 1. MetaboAnalyst 5.0 software (http://www.metaboanalyst.ca) was used to perform hierarchical clustering (HCA), principal component analysis (PCA), partial least squares discrimination analysis (PLS-DA), random forest (RF), and subject operating characteristic (ROC) curve analysis and visualization of metabolic lipids. The metabolic pathways associated with DLs were elucidated through the Kyoto Encyclopedia of Genes and Genomes (KEGGwww.genome.jp/kegg) pathway database. Data were presented as mean ± standard deviation (SD). Additionally, Pearson correlation analysis was performed using SPSS 21.0 (IBM Corp., New York, NY) to identify potentially key marker compounds associated with duck egg freshness.

RESULTS AND DISCUSSION

Egg Quality After Different Storage Durations in 2 Rearing Systems

The freshness of eggs is crucial to their quality. Table S1 displays the variations in EW, HU, EWH, YC, YI, EST and pH, which serve as significant markers of egg freshness throughout storage. In this study, EW, EWH, HU, YI, EST and moisture content gradually decreased during the storage period, resulting in a significant change (P < 0.05). The decrease in EW during storage might be related to the loss of moisture from the eggs and eggshell thickness (Wardy et al., 2014). Specifically, EWH and HU exhibited the same trend: their values decreased notably after 14 d (d 14, P < 0.05), reaching minimum values of 4.12 mm and 49.13 mm, respectively, at d 28. This demonstrates a decrease in freshness. The pH of duck eggs during the early storage period increases due to the decomposition of carbonic acid solution in egg white; then, the pH decreases slightly and remains stable after d 21 (Wardy et al., 2013). The gradual liquefaction of the yolk and weakening of the yolk membrane, which are primarily caused by water diffusing into the egg white, are indicated by a decrease in the YI (Torrico et al., 2014).

EW, EWH, HU and YC values were affected by the rearing system during storage (P < 0.05). Compared with those in the TFS, the EWH and HU in the FRS were greater at d 28, and the YC was lower at d 28. The YC values increased from 13.38 ± 0.66 to 14.32 ± 0.32 in the FRS (P < 0.05). Notably, in the TFS, the EW and YC values were greater than those in the FRS, which may be related to differences in breeding patterns. YC is mainly deposited into the yolk by carotenoids and lutein in the diet. In the TFS, laying ducks feed on natural grass, fish and shrimp, which contain large amounts of protein, lipids, and vitamins. This process is conducive to the deposition of egg yolk color. The rearing system had no effect on the other parameters of egg quality. As shown in Table. In S1, there was a significant difference in nutrient composition between d 1 and 21 or 28 (P < 0.05). By comparison, the protein content of the 2 groups on d 28 was greater than that on d 1, whereas the moisture content was lower. With increasing storage duration, the fat content of the TFS group significantly changed on d 28 (P < 0.05), while that of the FRS group was significantly greater than that on d 1 only on d 21 (P < 0.05). The increase in protein and fat content may be caused by the decrease in moisture content.

Generation of Lipid Profiles in Duck Eggs

We identified lipid profiles with a UPLC-ESI-Zeno TOF 7600 based on wide-scale ion scanning spectra. Total ion current (TIC) diagrams were acquired from QC samples in ESI+ and ESI− modes to identify lipid compounds. The chromatographic peak intensity and retention time of the QC samples basically overlapped, indicating that the stability of the instrument and reliability of the data were good (Figure 1). False-positive ions were filtered by a coefficient of variation (CV) > 30% at the stage of data quality control. The ESI+ mode signals were mainly those of acylcarnitine (CAR), cholesteryl ester (CE), ceramide (Cer), diglyceride (DG), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), EtherDG, EtherLPC, EtherLPE, EtherPC, EtherPE, monoglyceride (MG), N-acyl ethanolamine (NAE), triglyceride (TG), sterol lipid (ST), and sphingomyelin (SM) components, while the ESI− mode signals were mainly those of cardiolipin (CL), free fatty acid (FFA), LPE, lysophosphatidylinositol (LPI), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and oxidized phosphatidylethanolamine (OxPE) and oxidized phosphatidylinositol (OxPI) components.Figure 1 Overlapping TIC plots of 6 QC samples obtained from egg yolk ESI+ (A), egg yolk ESI− (B), egg white ESI+ (C), and egg yolk ESI− (D) samples.

Figure 1

Overall lipidomic analysis of egg yolk and egg white samples. A total of 831 lipid species and 6 lipid classes were detected and tentatively identified in 2 rearing systems of egg yolk. The lipids were mainly composed of 6 categories: glycerophospholipids (GPs, 50.66%, 421/831), glycerolipids (GLs, 35.74%, 297/831), fatty acyls (FAs, 5.78%, 48/831), sphingolipids (SPs, 4.69%, 39/831), STs (2.17%, 18/831), and prenol lipids (PRs, 0.24%, 2/831) (Fig. 2A, B). Specifically, 16 subclasses of GPs were identified. PC was the most abundant GP in egg yolk, accounting for 53.2% of the GPs. This result is consistent with previous findings that yolk may be one of the richest natural sources of phospholipids (Ali et al., 2017). There were 108 kinds of PCs containing unsaturated fatty acid chains, accounting for 92.31% of the total PCs. The amount of lipids in the GL group was second only to that in GP, with a relative content of 32.1%. TG, which belongs to the GL group, was the most abundant lipid species and accounted for 28.2% of the total GLs, followed by DG (4.0%). We found that selected lipid species, namely, PC (16:0/22:6), PC (16:0/20:4), LPC (16:0), TG (16:0/18:1/18:2), PC (16:0/18:2), PC (16:0/18:1), TG (16:1/18:1/18:1), and TG (16:0/18:1/18:1), were consistently present in high abundance in all the duck egg yolk tested. Most of them contained one or more unsaturated fatty acid chains. These findings were expected and were similar to those of other studies reporting that chicken egg yolk is rich in various lipid species (Chin et al., 2023).Figure 2 The percentages of lipid classes are shown, and the percentages (%) in the pie chart indicate the number of each lipid relative to the total amount of lipids detected (A). Numbers of lipid species identified by using LC/MS-based lipidomic analysis in egg yolks (B). Percentages of lipid classes in egg white; the percentages (%) in the pie chart indicate the number of each lipid relative to the total amount of lipids detected (C). Numbers of lipid species identified by using LC/MS-based lipidomic analysis in egg whites (D).

Figure 2

A total of 176 lipids were detected in egg white in 2 rearing systems divided into 5 classes: GL (56.82%, 100/176), GP (23.86%, 42/176), FA (11.36%, 20/176), SP (6.25%, 11/176), and ST (1.14%, 2/176). Furthermore, the lipid classes could be divided into 20 subclasses, including 8 GL species, 6 GP species, 2 FA species, 3 SP species, and 1 ST species (Figures 2C and 2D). PR was not detected in the egg yolk compared with the egg white. Previous studies have shown that pregnenolone belongs to PRs and its metabolic derivatives have anti-inflammatory, anticancer, and nerve-protective functions (Morrow et al., 2024). TG was the most abundant subclass in egg white, accounting for 51 kinds of lipids. Among them, 22 kinds of polyunsaturated fatty acids were found, accounting for 43.14% of the total TG content. The number of DGs was second only to the number of TGs, with 33 DGs present. There were 25 PUFAs, accounting for 75.76% of the total DGs. In addition, the number of UFAs in PCs and EtherPEs is also advantageous. The more unsaturated groups there are, the faster the lipid oxidation rate is (Shimizu and Iwamoto, 2022); therefore, it is speculated that TG, DG, PC, and EtherPE are relatively susceptible to oxidation.

Lipid Composition of Duck Eggs in the 2 Rearing Systems

Under the 2 rearing systems, the distributions of lipid classes in yolk were similar. However, there was a difference in lipid content. The relative content of total lipids in the TFS was significantly greater than that in the FRS, mainly in the GP, GL and SP groups (Figure 3D). Among these subclasses, TFS eggs had notably greater relative expression levels of the DG, TG, EtherPC, PC, PE and LPC subclasses than did FRS eggs (Figure 3C). A further comparison of the lipidomic profiles between the TFS and FRS and the identification of DLs were carried out using PCA (Figure 3A). PCA values of 47.4% (PC1) and 16.5% (PC2) indicated that there were many differences between the rearing systems. Compared with those in the FRS, the number of upregulated species (98) in the TFS was greater than that of downregulated species (14). The most upregulated lipids belonged to the GL and GP classes, and the subclasses were mainly PC (44), EtherPC (12), TG (9), DG (8) and PE (8). The TFS ducks forage for algae and crustaceans, which are rich in a variety of phospholipids and natural pigments, resulting in duck eggs with a high phospholipid content, red yolk and deep color. Compared with those in TFS eggs, 11 GP and 3 GL classes were downregulated in FRS eggs. TG (18:0/20:2/18:3), TG (16:0/18:1/22:5) and TG (18:1/20:1/18:2) were unique species in FRS eggs.Figure 3 PCA score plot of lipids in egg yolks from the 2 systems (A). PCA score plot of lipids in egg whites under 2 systems (B). The relative expression levels of lipids in egg yolk were greater in the 2 systems (C). Relative content of total lipids in egg yolk in the 2 systems (D). Relative contents of PC O (18:1/20:4) and SM (34:1;2O) in egg whites from the 2 systems (E).

Figure 3

In the 2 rearing systems, the subclass of lipids in egg white was the same, although there were certain discrepancies in the relative total amount of each subclass. The TFS was 3.03% greater than the relative total amount of FRS, which was due to the higher relative content of GP and SP, specifically the higher content of DG, EtherPC, PE, and SM. PCA was used to distinguish the egg white of the 2 groups; PC1 accounted for 38.8% of the data variance, and PC2 accounted for 16.6% of the data variance (Figure 3B); these 2 groups could be clearly distinguished. There were obvious differences in the egg white lipid composition among the different rearing systems. In the TFS eggs, the relative contents of Cer and SM were 2-10 times those in the FRS eggs. In addition, the relative contents of PC O (18:1/20:4) and SM (34:1;2O) were significantly greater than those in the FRS eggs (Figure 3E). Notably, only 2 kinds of AHexSIS were detected in the whole egg white experiment. The relative content of the 2 AHexSISs in the FRS eggs was much greater than that in the TFS eggs, which was 13.91 times greater. AHexSIS, which is often detected in plants, is an ST and is closely related to hormone regulation in the human body.

Analysis of Duck Egg Lipids and Screening of DLs During Storage

To further study the lipidomic changes between the 2 rearing systems during storage, multivariate statistical analysis was carried out on the data. First, the duck eggs from the different rearing systems were analyzed via PCA. The first 2 PCs (PC1 and PC2) were extracted to explain the data variance. In egg yolk, PC1 and PC2 explained 40.1% and 12.3% of the data variance in the TFS group, respectively, and 24.2% and 12.3% of the data variance in the FRS group, respectively. In egg white, PC1 and PC2 explained 35.2% and 18.2% of the data variance in the TFS group, respectively, and 19.2% and 16.5% of the data variance in the FRS group, respectively (Figure 4). The PCA score plot showed that the yolk in the different rearing systems partially overlapped but still showed a trend towards separation. This showed that the reproducibility of the samples was good and that the lipidomic data of the egg yolk changed significantly after different storage durations. To eliminate random errors irrelevant to the research purpose, a supervised PLS-DA model was constructed by partial least squares analysis. The model was verified to ensure that it was reliable, and VIP values were obtained. Consequently, we used the P value, FC and VIP value to screen for DLs (S2).Figure 4 PCA score plot of TFS (A) and FRS (B) egg yolk samples; PCA score plot of TFS (C) and FRS (D) egg white samples.

Figure 4

Egg yolk DL analysis. Heatmaps (Figures 5A and 5B) were generated to visualize the dynamic changes in different lipid classes in egg yolk from eggs of ducks in the 2 rearing systems during different storage periods. The storage time had no significant effect on the type of lipid species but had an effect on the lipid content. In the TFS treatment, the total relative expression of lipids continued to decrease with storage time and decreased 46.0% during the 28 d of storage. GP and GL decreased from d 1 to d 7, increased slightly from d 7 to d 14, and then decreased gradually to d 28. This decrease was attributed mainly to DGs, EtherPCs, TGs and PCs. After removing the same lipid molecules, 106 DLs were identified in the 2 rearing systems. In the TFS group, 74, 24, 5 and 13 lipids were significantly changed in duck eggs at d 7, 14, 21 and 28, respectively. On d 7 of storage, 30 PCs and 11 TGs were significantly reduced, and the number of PCs and DGs subsequently decreased on d 14, 21 and 28. There were changes in 5 SM compounds on d 28, and the relative content decreased by 1.7 to 2.2 times. The DLs were then loosely grouped into 4 different categories (FA, GP, GL, and SP groups). There were more than 50% of the different lipids under different rearing systems changed significantly during storage. In addition, more than 50% of the different lipids decreased over time. This is consistent with the decreasing trend of the total fat content. GP is susceptible to oxidation due to its greater number of unsaturated bonds and emulsification. Phospholipase and lipase have significant effects on lipid hydrolysis during egg storage. TG is hydrolyzed by lipase to produce FFAs, glycerol, etc. FFAs increase in volatility because of the hydrolysis of GP and GL (Zhang et al., 2021). Among them, the most highly upregulated lipids were FA18:2 and FA20:4. The antioxidant activity of egg yolk phospholipids is opposite to that of SFA chains, and most of the PUFA chains in PCs are at the sn-2 position. Under the action of phospholipases, these chains are hydrolysed into FAs, lysophospholipids, choline phosphate and other substances.Figure 5 Changes in lipid classes in TFS (A) and FRS (B) egg yolks. Changes in lipid subclasses in the TFS (C) and FSR (D) egg whites.

Figure 5

In the FRS eggs, the majority of the molecular species (GP, SP, and ST) changed slightly from d 1 to 14. From d 21 to 28, the changes were significant, and the relative content decreased. This slight change possibly occurred because antioxidant components, such as carotenoids and superoxide dismutase, play important roles in inhibiting lipid oxidation (Benedé and Molina, 2020). With the degradation of antioxidants and the accumulation of lipid oxidation products, the rate of lipid oxidation intensifies (Buszewski et al., 2017). Three DLs were found from d 1 to 7: FA 22:6, TG (18:1/20:1/18:2), and SM (36:2;2O). Four DLs, three of which were PEs, changed between d 7 and d 14. There were 9 DLs from d 14 to 21, and these lipids were concentrated in the TG group (7/9). Only 2 DLs were observed in the final stage, both of which were EtherPCs, namely, PC O (18:1/22:4), and PC O (18:0/22:4). In total, in contrast to those in the TRS eggs, the number of changes in DLs in the FRS eggs was significantly less.

Egg white DL analysis. Considering that the number of lipids detected in egg white was relatively small compared to that in egg yolk, a heatmap of lipid subclasses was generated to show the dynamic changes in the relative content of egg white during storage in the 2 rearing systems (Figures 5C and 5D). Among the 2, the relative contents of TG and DG were most notable. After removing the same lipid molecules, a total of 16 DLs were identified between 2 systems; these DLs were in 5 lipid subclasses (EtherPEs, FAs, LPCs, PEs, TGs). In TFS eggs, 4 lipids, namely, FFAs (18:1, 18:2, 20:4, and 22:6), were significantly upregulated from d 1 to d 7. From d 7 to d 14, there were 10 DLs, all of which were significantly upregulated, including 9 TGs. No DLs were identified from d 14 to 21. During d 21 to 28, only the LPC (16:0) concentration significantly increased. Chang et al. reported that the extracted ion chromatograph (EIC) of LPC (16:0) showed obvious variation in signal abundance between fresh and stored eggs (Chang et al., 2021). In contrast, in our study, the reported concentration of LPC (16:0) after storage was significantly lower than that in fresh eggs. This may be attributed to the fact that Chang et al. analyzed whole eggs, while we only analyzed egg white. In addition, the differences may also be related to the site of the sample, the species of egg, the feeding method, and the duration of storage. Additionally, FA 18:2, FA 20:4, and FA 22:6 exhibited an increasing trend during the storage period, with increases of 1.47, 1.99, and 4.10, respectively, while FA 22:6 exhibited the greatest increase. Once again, it was confirmed that the more unsaturated groups there were, the faster the rate of oxidation was.

In the FRS eggs, the total content of lipids increased by 1.69%, and the EtherPC, FA and PE subclasses played important roles, increasing in content by 92.33%, 77.09% and 32.35%, respectively. With the extension of storage time, the relative content of AHexSIS decreased in the FRS eggs but increased in the TFS eggs. From d 1 to d 7, 5 DLs—FA 18:1, FA 18:2, FA 20:4 and FA 22:6—were significantly upregulated, and PE 38:4 was significantly downregulated. There were no DLs between d 7 to 14 and d 14 to 21. From d 21 to d 28, PE O to 40:6 was significantly upregulated. Moreover, the FA 18:1, FA 18:2, and FA 20:4 contents consistently increased during storage. A comparison of the rearing systems revealed that the TG concentration change was mainly in the TFS eggs, and the FA change was observed more in the FRS eggs. The FFA content showed an upwards trend, and the LPC content showed a downwards trend. The increase in FFA content was mainly attributed to phospholipid degradation; however, the products of phospholipid degradation was shown to mainly include FFAs and lysophospholipids (Li et al., 2022), and the LPC content first increased and then decreased in both systems. It is speculated that LPC is hydrolyzed after production to generate glycerol, base, phosphoric acid, etc.

In particular, the relative contents of TG (14:0/14:0/16:1), TG (14:0/15:1/15:1), TG (14:0/15:0/16:0), and TG (14:0/16:0/16:1) in TFS eggs were significantly lower than those in FRS eggs on d 1; however, these lipids were markedly upregulated with prolonged storage until the contents matched those in FRS eggs. All these TGs contained C14:0 saturated fatty acids.

Lipid Metabolism Pathway Analysis

In egg yolk. Pathway analysis is necessary for further elucidating changes in lipid metabolism during storage (Table S2). In TFS, the key metabolic pathways are involved in glycerophospholipid metabolism and sphingolipid metabolism. GPs are among the main components of cell membranes. They play an important role in preventing the rupture and lysis of cell membranes and maintaining the stability of cells. Glycerophospholipid metabolism mainly involves the hydrolysis of PE by phospholipase to produce LPE, which is consistent with the downregulation of the relative content of PE and the upregulation of LPE. However, the relative levels of both PC, LPC (20:0), LPC (22:0), LPC (18:0) and LPC (18:3) were significantly decreased after egg storage, and such discoveries were found in other study (Chang et al., 2021). One possible reason is the vitelline membrane weakens and lose its strength as egg quality deteriorates over time. The phospholipid cleavage process in the yolk may be catalyzed by the endogenous enzyme lysophospholipase D, which is found in the albumen. The endogenous enzyme originating in the albumen, lysophospholipase D, for instance, may enter the yolk and catalyze the cleavage of phospholipids. Hence, this phospholipid catabolism can affect YI value decline. The products of GP metabolism are easily oxidized and decomposed into aldehydes and ketones, which result in bad odors in egg yolk. SPs are mainly distributed in cell membranes, plasma and lipoproteins. They are closely related to the signal transduction regulation of growth, differentiation, ageing, apoptosis and death in mammalian cells (Espinoza and Snider, 2024). Combined with the decrease in the relative levels of SM and Cer, SP metabolism is caused mainly by the catalytic decrease in SM, which results in the production of Cer and the phospholipid choline. Then, Cer can be glycosylated, phosphorylated, and deacylated to produce a series of metabolites. In the FRS eggs, the key pathways enriched were involved in glycosylphosphatidylinositol (GPI)-anchored biosynthesis and glyceride metabolism. GPI biosynthesis is the only way for proteins to bind to cell membranes. In this study, the process chiefly involved the synthesis of phosphoethanolamine and the eventual generation of PE. Glyceride metabolism primarily relates to the hydrolysis of triglycerides by lipase to produce free fatty acids, which is consistent with the notable upregulation of the relative content of FFAs in the later stage. FFAs can further oxidize and deteriorate, causing the undesirable bitter taste of egg yolk (Rannou et al., 2013). The study showed TRS eggs possess high phospholipids and carotenoids, and YC did not change significantly during storage time. But the diversity and amount of GP differed considerably when compared to FRS. The freshness of stored eggs decreases significantly due to oxidation processes caused by reactive oxygen species (ROS). The antioxidants of GP in eggs exert antioxidative effects in response to ROS. We hypothesized that GP may engage in antioxidant processes, postponing the egg color shift in the yolk (Yuan et al., 2021).

In egg white. According to the analysis of the metabolic pathways of egg white, the biosynthesis of unsaturated fatty acids, which includes the synthesis of oleic acid, linoleic acid, arachidonic acid (AA), and docosahexaenoic acid (DHA), which are produced by fatty acyl-coenzymes through a series of enzymatic reactions, was the most significant and influential pathway among the 2 rearing systems. The biosynthesis pathway of unsaturated fatty acids is related to fat deposition (Wang et al., 2020), and the relative contents of FA 18:1, FA 18:2, FA 20:4, and FA 22:6 significantly increased after storage. During egg white storage and processing periods, lipid oxidation often occurs and affects the protein structural changes (Zhang et al., 2022). In the process of accelerated oxidation, all significantly TG were down-regulated, of which TG (46:3| 14:1_16:1_16:1) showed the highest fold-change value at 14 d in TRS. This process may influence the HU and EWH.

Screening of Marker Lipids During Storage

The purpose of this study was to further elucidate the effect of storage time on the quality of eggs from ducks in different rearing systems and to screen for marker lipids. Lipids in different culture modes were visually classified and identified by a random forest (RF) model. RF models consist of classification and regression tree structure classifiers (Moradi et al., 2024), with relatively high prediction accuracy and a combination of the required set of features (Svetnik et al., 2003). During decision tree building, 1/3 of the samples were excluded from the collection sample of the decision tree, which were called out-of-bag (OOB) data. These OOB data can be used to test samples to obtain an unbiased estimate of classification error (OOB error) (Gu et al., 2023).

In egg yolk. According to the sample variability shown in the PCA plot and multiple group attempts, the samples were divided into d 1∼14 and d 21∼28 to construct an RF model for discriminant analysis. After the RF was established, the OOB error rate was 0.167 in TFS and 0.133 in FRS. Based on the average decreasing accuracy, the 40 key lipids (Table S3) were selected, and the area under the receiver-operator (ROC) curve (AUC) was calculated to evaluate the performance of the key lipids. The closer the AUC was to 1, the stronger the labelling performance was. The AUC for TFS was 0.997, whereas that for FRS was 0.959 (Figures 6A and 6B), suggesting that these lipids could be potential markers of lipids for duck egg storage.Figure 6 ROC curve analysis of TFS (A) and FRS (B) in egg yolk; ROC curve analysis of TFS (C) and FRS (D) in egg white.

Figure 6

To determine the marker lipids of duck eggs during storage, correlation analysis between egg quality and DLs was first performed based on the P value, FC and VIP and subsequently combined with the potential marker lipids obtained by the RF model (Figure 7A). DG (18:0/18:1), DG (16:0/18:0), DG (17:1/18:1), DG (18:0/18:0), and Cer (18:1;2O/24:1) were screened out in the TFS samples. These parameters were positively correlated with the significance of the high HU and EWH values (P < 0.01). DG and Cer are intermediate products of glycerophospholipid metabolism and sphingolipid metabolism. The relative content of the 5 marker lipids increased with increasing storage time, and the freshness of the duck eggs decreased. Liu et al. reported that DG and GPL are key lipids and metabolic pathways involved in egg storage (Liu et al., 2023). TG (16:0/18:1/16:3) was obtained as a marker lipid in the FRS and was positively correlated with EWH (P < 0.05) (Table S5).Figure 7 ROC curve analysis of individual marker lipids.

Figure 7

In egg white. The samples were divided into d 1 to 7 and d 14 to 28 to construct the RF model with the best classification result. In this case, the OOB error rate obtained for TFS data was 0.033, whereas that obtained for FRS data was 0.067. Considering the number of overall lipids in egg white, the first 20 key lipids (Table S4) were selected for construction of the ROC curve. The AUC for TFS was 1, whereas that for FRS was 0.964 (Figures 6C and 6D).

Consistent with the methods used to identify the marker lipids in egg yolk (Figure 7B), 3 marker lipids were obtained from the TFS: FA 22:6, TG (14:0/14:0/16:1) and TG (15:0/16:1/18:1). These parameters were negatively correlated with HU and EWH. Among them, FA22:6 was positively correlated with YC (P < 0.05). In the FRS, 4 marker lipids were obtained: FA18:1, FA18:2, FA20:4, and FA22:6. All 4 lipids were negatively correlated with HU and EWH. FFAs are mainly products of lipid hydrolysis and are easily oxidized to hydroperoxide compounds, increasing lipid oxidation. This leads to the oxidation of protein in egg white, affects the structure and function of protein, and subsequently reduces the freshness and nutritional value of eggs (Table S6).

CONCLUSIONS

In this study, UHPLC‒MS/MS lipidomic technology was successfully used to analyze the changes in the lipid composition of duck eggs during storage in 2 rearing systems. As a consequence, among the quality indices of the duck eggs, EW, EWH, HU and YC were significantly affected by the rearing system. After 28 d of storage, EW and YC in TFS eggs were higher than in FRS eggs, and EWH and HU were lower than in FRS eggs. According to the lipidomic data, 831 lipids and 6 lipid classes were identified in egg yolk, with the highest relative content being associated with GPs. A total of 176 lipids and 5 lipid classes were identified in egg white, with the highest relative content observed for GLs. The total relative content of lipids in the TFS eggs was greater than that in the FRS eggs. Multivariate statistical analysis was used to screen for DLs, after which pathway analysis was performed. In the yolk, after removal of the same lipid molecules, 106 DLs were identified under the 2 rearing systems. GP metabolism and sphingolipid metabolism were the key metabolic pathways in the TFS eggs, and GPI-anchored biosynthesis and glyceride metabolism were the key pathways in the FRS eggs. In egg white, 16 DLs were identified, and the biosynthesis of UFAs was the key metabolic pathway in both rearing systems. RF machine algorithms were combined to identify marker lipids during storage. In egg yolk, DG (18:0/18:1), DG (16:0/18:0), DG (17:1/18:1), DG (18:0/18:0), and Cer (18:1;2O/24:1) were identified as marker lipids in TFS. TG (16:0/18:1/16:3) was identified in the FRS eggs. In egg white, FA 22:6, TG (14:0/14:0/16:1), and TG (15:0/16:1/18:1) were identified in the FRS eggs. FA 18:1, FA 18:2, FA 20:4, and FA 22:6 were identified in the FRS eggs. These marker lipids were significantly correlated with the quality of the eggs stored in duck eggs. In conclusion, this study described the comprehensive lipid profile of duck eggs during storage in different rearing systems, provided a new idea for screening marker lipids in eggs, and provided a theoretical basis for evaluating the changes in duck egg quality during storage, which was ultimately conducive to evaluating the quality of duck eggs and duck egg products.

DISCLOSURES

The authors declare no conflicts of interest.

Appendix Supplementary materials

Image, application 1

Image, application 2

ACKNOWLEDGMENTS

This work was supported by the Jiangsu Provincial Key Laboratory of Food Quality and Safety (2022-SBGJZZ-16 ); “Gao You Duck Egg” Geographical Indication Agricultural Products Protection Project.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104201.
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