Dietary Modulation on sn-Position Distribution and Its Impact on Emulsifying and Nutritional Properties in Egg Yolk Lipids

Dietary Modulation on sn-Position Distribution and Its Impact on Emulsifying and Nutritional Properties in Egg Yolk Lipids | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dietary Modulation on sn-Position Distribution and Its Impact on Emulsifying and Nutritional Properties in Egg Yolk Lipids Xiaodan Zhang, Xuan Ji, Wenqian Guan, Ying Gao, Sharina Qi, Shijian Dong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7844056/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Eggs are recognized as nutrient-dense foods, and precise modulation of yolk lipid structures is critical for enhancing their functional properties. In this study, laying hens fed with three different diets of corn-dried distiller grains with solubles (DDGS), whole grains and flaxseed, respectively, were chosen to investigate how dietary composition remodeled and modulate structures as well as functional properties of yolks. A multi-scale approach integrating GC-MS, MALDI-TOF-MS, and 1 H NMR was applied. The results showed that yolks from hens fed with the DDGS diet contained trans-oleic acid (C18:1n9t) and palmitic acid (C16:0). The high level of monounsaturated fatty acids (MUFA) was found to improve the flexibility of the lipoprotein interface, thereby enhancing emulsifying activity. The cholesterol content in egg yolks was determined to be the lowest (0.84 mg/g) in the whole-grain diet, but an accumulation of phosphatidylserine was observed, which may disrupt the ω-6/ω-3 balance and increase the risk of oxidation. In yolks from the flaxseed diet, the docosahexaenoic acid (DHA) was preferentially deposited at the sn-2 position of triacylglycerols, promoting higher bioavailability of long-chain polyunsaturated fatty acids. The causal mechanisms underlying the differential regulation of yolk functionality by dietary components were elucidated. This was achieved through the specific remodeling of lipid molecular structures, which included the sn-2 positional distribution of fatty acids and variations in phospholipid subclasses. It aims to provide a novel integrated approach for the design of “phenomenon-substance-mechanism” on eggs via precision dietary formulation, further promoting a transformation of the poultry egg industry from compositional modification to structural empowerment. Diet Egg yolk Lipid structure Functional properties Phospholipid subclasses Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Eggs are considered one of the most complete natural nutrient carriers and high-quality protein sources in human diet [1] . As the second major component in eggs, yolk lipids account for 30 to 35 percent, and are not merely an energy source, instead, their unique composition and structure, including fatty acid profiles, distributions of phospholipid classes, and molecular organization of lipids, are recognized as the core determinants of the nutritional quality, processing functionality, and bioavailability of eggs [2] . These unique functions have placed eggs in a key position within the food industry, demonstrated by their applications in fields of baking, sauces, and infant complementary foods [3–5] . However, the relatively high levels of saturated fat (approximately 3 g per 100 g) and cholesterol (approximately 200 to 300 mg per 100 g) in the lipid composition of conventional eggs are considered insufficient to meet the precise nutritional requirements of special populations such as patients with cardiovascular diseases and infants [6] . Hence, it is urgent to upgrade the functional egg industry with the growing demand of consumers for the nutritional and health attributes of foods. The quality and functional properties of eggs are significantly influenced by multiple factors, including the age and breed of hens, dietary additives, and storage conditions. In particular, the functional quality and nutritional composition of yolk are primarily enhanced from a natural perspective through modifications in poultry feed composition. Meanwhile, the development of functional eggs has been largely focused on the enrichment of target components such as omega-3 fatty acids, low cholesterol, and high levels of vitamins. Accordingly, the contents of specific nutrients in eggs can be increased by altering the feed composition of laying hens, whereby nutritionally fortified eggs are produced and referred to as “designer eggs.” [7] . Extensive evidence has been provided demonstrating that the yolk fatty acid profile is significantly influenced by dietary fatty acid composition. For instance, supplementing laying hen diets with flaxseed-rich in α-linolenic acid enhances the deposition of long-chain n-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA), in egg yolks [8] . Plant-based antioxidants in feed also improve yolk oxidative stability [9, 10] . These changes modify not only the nutritional value but also the processing functionality of yolk lipids, as well as their emulsifications. The emulsifying property of eggs is significantly improved when flaxseed oil is supplemented in the diet [11] . Although numerous studies have been conducted on dietary regulation of yolk lipids, the focus has mainly been placed on the overall composition of fatty acids rather than on the remodeling of fine molecular structures. Information regarding phospholipid subclasses, sn-positions of triacylglycerols, and the structural organization of lipid molecules remains limited. At present, diverse techniques have been applied to the identification of food lipids. Traditional lipid analysis methods, such as GC-MS, are used to quantify fatty acid species with precision, but the positional information of acyl chains has not been resolved [12] . Although UPLC-TOF-MS based lipidomics has been recognized to offer advantages in throughput and sensitivity, clear limitations have also been noted in structural elucidation of lipids, discrimination of isomers, cost efficiency, and dynamic range, with particular difficulties in distinguishing structural isomers. The limitations of existing single techniques have been recognized to drive the integration of multi-scale approaches, by which new opportunities have been provided for in-depth elucidation of lipid structure-function relationships. MALDI-TOF-MS has been applied for the characterization of nearly all lipid classes, including nonpolar lipids such as triacylglycerols (TAG) [13] . The regional isomeric distribution can be rapidly localized by 1 H NMR through the splitting patterns of glycerol backbone protons, and quantitative analysis can be performed with simple sample preparation without the need for derivatization, standards, or calibration curves [14] . The combined application of these two techniques is expected to reveal the essential characteristics of yolk lipid structures that can be regulated by dietary interventions across molecular, mesoscopic, and macroscopic dimensions. Based on this rationale, eggs produced from hens fed with DDGS, whole grains, and flaxseed were selected as the experimental objects, and a multi-scale strategy combining GC-MS, 1 H NMR, and MALDI-TOF-MS was employed. Lipidomics, structural elucidation, and functional characterization techniques were integrated to investigate the differential mechanisms by which three representative functional diets influenced yolk lipid molecular structures and functional properties. The study was designed to elucidate the specific remodeling effects of diversified diets on yolk lipid molecular structures and to clarify how such structural changes were cascaded to drive the differentiation of key functions, including emulsification, oxidative stability, and nutritional digestibility. The experimental evidence was provided for the precise design of functional-egg diets, nutritional targeting, and product development. While support was also offered for the transformation and upgrading of the poultry egg industry from “compositional modification” to “structural empowerment” and “functional customization.” 2 Materials and Methods 2.1 Sample Preparation The experiment employed a single-factor completely randomized design. A total of 1,200 healthy laying hens (28 weeks old) (purchased from Sichuan Shengdile Village Ecological Food Co., Ltd., Mianyang, China) were randomly divided into three groups (n = 400 per group) and fed the following diets: Table 1 The composition and chemical composition of the basic diet of egg chickens Name of raw material (%) T1 T2 T3 Corn 61.43 63.03 58.19 Soybean Meal 18.60 21.40 22.90 Corn Gluten Meal 4.34 4.01 0.91 Corn DDGS 4.00 -- -- Flaxseed Meal -- -- 7.00 Soybean Oil 0.30 0.34 -- Premix(Vitamin & Mineral Premix) 11.22 11.22 11.22 Note: T1-T3 represented the corn DDGS group, corn-soybean meal group, and flaxseed group respectively. The composition of the basal diets was presented in Table 1 . The experimental period was 8 weeks with ad libitum access to feed and water. Eggs (50 eggs/group) were collected from each group on day 56 of the trial for subsequent use. 2.2 Emulsifying Properties The emulsifying activity index (EAI) and emulsion stability index (ESI) were determined according to the method described by Sun et al. [15] with modifications. A 1% yolk powder solution (prepared in pH 7.0 PBS buffer) was homogenized with soybean oil (oil-to-water ratio of 1:4) at 10,000 rpm for 1 min. EAI was expressed as the percentage of emulsion height over total dispersion height. For ESI measurement, the samples were heated to 80°C for 30 min, cooled, and then centrifuged prior to analysis. 2.3 Particle Size and Zeta Potential The yolk solutions (1%) were diluted 1:200 with distilled water and filtered through a 0.45 µm membrane. The particle size distribution (Z-average), polydispersity index (PDI), and zeta potential were measured by dynamic light scattering (Malvern Zetasizer Nano ZS) at 25°C. Each sample was tested in triplicate. 2.4 Malondialdehyde (MDA) Content MDA content was measured using a commercial assay kit to evaluate lipid oxidation. 2.5 Cholesterol Content Following the method of Laila & Putra et al. [16] with minor modifications. A 3 g portion of yolk homogenate was weighed and placed into a 25 mL colorimetric tube, dissolved with distilled water, and diluted to the mark before being mixed thoroughly to obtain a yolk dilution. An aliquot of 1 mL of the yolk dilution was accurately transferred into a 50 mL beaker, followed by the addition of 3 mL of 10% potassium hydroxide solution and 10 mL of anhydrous ethanol solution, and the mixture was stirred. Direct saponification was carried out in a 60°C water bath for 1 h (stirred once every 15 min), after which the mixture was removed. After cooling, 50 mL of petroleum ether was added and mixed with a glass rod for extraction, and the supernatant was transferred into a separatory funnel. The supernatant was repeatedly washed three times with distilled water, and the total volume was measured. A 0.5 mL portion was then taken into a small beaker, evaporated to dryness in a 60°C water bath, and the residue was dissolved in 2 mL of methanol solution and 2 mL of ferric chloride–phosphoric acid color reagent. After cooling to room temperature, the yolk dilution was replaced by solvent as the blank, and absorbance was measured at 550 nm using a microplate reader. Cholesterol content was calculated according to the following formula (1). $$\:\text{Cholesterol\:content\:(mg/100g)=}\frac{\text{C×4×2×V×25×100}}{\text{W×1000}}$$ 1 where C represented the cholesterol concentration in the extract (µg/mL), V represented the volume of the extract (mL), and W represented the mass of the homogenized egg yolk (g). 2.6 In Vitro Digestibility of Fatty Acids Gastrointestinal digestive fluids were prepared according to the method of Martos et al. [17] . 1 g portion of yolk sample intestinal digest was weighed, and an appropriate amount of anhydrous sodium sulfate was added. Subsequently, 50 mL of petroleum ether (30–60°C) was introduced, mixed thoroughly, and left to stand overnight. The mixture was then filtered, and 25 mL of the filtrate was collected. To the filtrate, 50 mL of anhydrous ethanol was added, followed by the addition of 2–3 drops of phenolphthalein. Titration was carried out with 0.01 mol/L NaOH solution. The degree of fat digestion was characterized by the amount of free fatty acids, and the free fatty acid content was calculated according to the following formula (2). $$\:\text{Free\:fatty\:acid\:(NaOH/g)=4×}\frac{\text{A×F}}{\text{m}}$$ 2 where A represented the titrant volume, mL; F represented the titrant concentration, 0.01 mol/L; m represented the egg yolk sample mass, g. 2.7 Fatty Acid Composition An appropriate amount of egg-yolk powder was mixed with 90 mL of chloroform-methanol (v:v = 2:1), and ultrasonic extraction was carried out for 20 min. The mixture was then filtered, and the filtrate was evaporated to dryness using rotary evaporation. Subsequently, the petroleum ether and anhydrous sodium sulfate were added for a second extraction. The extract was centrifuged at 3000 r/min for 5 min, the ether phase was collected, and the petroleum ether was removed by evaporation. Fatty acid methyl esters (FAMEs) was prepared following Watkins et al. [18] . 300 mg of yolk oil was saponified with NaOH-methanol at 60°C for 30 min, and this was followed by methylation with boron trifluoride-methanol at 60°C for 30 min. After cooling, n-hexane and saturated NaCl were added, and the upper ether phase was collected for GC-MS analysis. GC-MS was performed on an Agilent 7890B-5977B system with an HP-88 column (60 m × 0.25 mm × 0.2 µm). Helium was used as the carrier gas (0.8 mL/min), injection volume was 1 µL (split 10:1), and the oven program ranged from 40°C to 250°C with stepwise increments. Fatty acids were quantified by peak area normalization. 2.8 Lipid Molecular Species Lipid analysis was performed using MALDI-TOF-MS (Bruker UltrafleXtreme) [19] . The matrix solution was prepared with 2,5-dihydroxybenzoic acid (DHB, 10 mg/mL dissolved in 70% acetonitrile–0.1% TFA). 1 mg portion of yolk powder was mixed with the matrix solution (1:1, v/v), spotted onto the target, and dried. Data were acquired in the positive ion reflectron mode within the m/z range of 500–1000, with a laser energy of 25% and an accumulation of 500 shots. Lipid molecular species were identified by matching m/z values to the LIPID MAPS database ( http://www.lipidmaps.org ) with a mass error of ± 0.02 Da. The triacylglycerols (TG) and phospholipids (PC/SM) were verified through characteristic fragment ions. Relative abundances within the m/z range of 400–1000 were normalized to the total ion current intensity. 2.9 sn-Position of Fatty Acids The acyl positions of lipids were analyzed by 1 H NMR (Bruker Avance III HD 600 MHz) [20] . A 50 mg portion of yolk powder was dissolved in 0.6 mL of CDCl₃, centrifuged, and the supernatant was transferred into a 5 mm NMR tube. The sampling parameters were set at 298 K with 64 scans and a spectral width of 12 ppm. All spectra were processed in the same manner using MestReNova software. 2.10 Statistical Analysis The result data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed using SPSS 26.0 (p < 0.05). Principal component analysis (PCA) was carried out in MetaboAnalyst, where lipid composition (GC-MS data) and functional properties were dimensionally reduced after UV standardization, and the principal component loading matrix was extracted. A value of p < 0.05 was considered statistically significant. 3. Results and Discussion 3.1 Effects of Different Diets on Yolk Emulsifying Activity and Emulsion Stability Emulsifying activity (EAI) and emulsion stability (ESI) are the core functional attributes of egg yolk as natural emulsifiers and directly determine its performance in food applications. These interfacial properties are largely determined by lipid molecules, particularly by the behavior of phospholipids and lipoproteins at the oil–water interface. In addition, these properties are influenced by lipid composition (degree of saturation and phospholipid classes) and molecular conformation (cis/trans isomerism) [21] . As shown in Fig. 1 , the EAI of corn DDGS eggs (EY1, 0.28) was found significantly higher than that of other groups (p 0.05). It was noted that the emulsifying stability of all three dietary groups remained at a relatively high level, but the differences among groups were not significant (p > 0.05). This phenomenon may be explained by the convergence of interfacial membrane stability, which was maintained primarily through the combined effects of phospholipids and apolipoproteins on the mechanical strength of the interface. Particle size served as a key parameter describing the size of lipoprotein aggregates or emulsion droplets in yolk solutions, while zeta potential was a reliable indicator of surface charge density and interactions between proteins and emulsifiers [22] . Greater uniformity (lower polydispersity index, PDI) and higher surface charge magnitude (larger absolute zeta potential) generally contributed to improved physical stability of emulsions [23] . As shown in Fig. 1 D, the zeta potential values of the three yolk solutions were all negative, suggesting that negative charges were adsorbed onto the particle surfaces in the emulsions. Among them, EY1 exhibited the narrowest particle size distribution, the smallest particle size (3500 nm), and the lowest PDI (0.34), with a zeta potential absolute value of 12.5 mV (Fig. 1 C). In contrast, EY2 showed the widest particle size distribution, a relatively larger average particle size (3550 nm), and the highest PDI (0.38). The peak of EY3 was located in the middle with a moderate distribution range; although its average particle size was the largest (3600 nm), the absolute value of its zeta potential was the highest at 12.8 mV. Taken together, EY1 yolk particles were evaluated to exhibit better monodispersity and higher solution stability. EY2 particles were found to be more dispersed and prone to flocculation or stratification, while EY3 demonstrated the best solution stability. The observed differences in stability might be attributed to the intrinsic properties of fatty acids, such as headgroup polarity, charge, and their accumulation within the interfacial protein layer. In addition, surface charge could also be influenced by the contents of proteins adsorbed at the interface, implying that yolks richer in long-chain unsaturated fatty acids might alter protein adsorption behavior and consequently shift zeta potential values [24] . Based on the particle size and stability results of yolk solutions, the smallest emulsion particles with the highest emulsifying activity were formed by EY1. In contrast, the particles of EY2 were found to be heterogeneous in size, and the emulsions were less uniformly dispersed. The emulsion particles formed by EY3 yolk exhibited the highest surface charge density and were evaluated to possess the greatest stability. 3.2 Effects of Different Diets on Yolk Oxidative Stability and Fatty Acid Digestibility Lipid oxidation is a key factor leading to quality deterioration of yolks and yolk-based products, resulting in undesirable flavors, discoloration, and loss of nutritional value. Malondialdehyde (MDA), a secondary product of lipid peroxidation, was measured as an indicator of oxidative stress and lipid oxidation levels [25] . As shown in Fig. 2 A, significant differences in MDA concentrations were observed among the dietary groups. Specifically, EY1 exhibited the highest value (3.3981 nmol/g), followed by EY2 (3.03718 nmol/g), while EY3 had the lowest concentration (2.43629 nmol/g), indicating superior oxidative stability in flaxseed-fed yolks. Previous studies have shown that yolks are highly susceptible to oxidation due to their elevated fat content. A high proportion of PUFA, particularly long-chain PUFA containing multiple double bonds, was especially prone to oxidative reactions [26] . However, flaxseed was naturally rich in lignans and other antioxidants that could scavenge free radicals and interrupt chain reactions, thereby protecting yolk lipids from oxidation [27] . Therefore, differences in oxidative stability were attributed not only to PUFA levels and species but also to their positional distribution (sn-position) and local molecular environment. Fatty acid digestibility served as a core indicator of yolk nutritional value, as it directly determined lipid bioavailability [28] . The composition of lipid molecules, including glyceride configuration, phospholipid distribution, and fatty acid chain characteristics, was profoundly influenced by the digestive process through the regulation of emulsion stability, enzymatic hydrolysis efficiency, and the absorption interface [29] . Significant differences in the simulated gastrointestinal digestibility of fatty acids among the three yolks were observed (Fig. 2 B). The highest digestibility was measured in EY3 (93.15%), followed by EY2 (90.52%), whereas the lowest value was detected in EY1 (84.79%) (p < 0.05). Since fatty acids in foods are primarily present in triacylglycerols (TG), medium-chain fatty acids were hydrolyzed more rapidly than long-chain fatty acids. Under the action of intestinal lipase, hydrolysis of lipids is mainly carried out at the sn-1,3 positions of TG, whereas sn-2 fatty acids were usually absorbed directly in the form of sn-2 monoacylglycerols, with potentially higher efficiency of digestion and absorption [30] . In addition, the accessibility of digestive enzymes was influenced by droplet size and interfacial composition, such as phospholipids and proteins [31] . In summary, the oxidative stability and the release rate of fatty acids during the in vitro simulated digestion of yolk lipids were significantly influenced by dietary composition. Given that lipid digestive efficiency was highly dependent on sn-2 positional fatty acids, it was hypothesized that the high digestibility of EY3 might be associated with the specific enrichment of PUFA at the sn-2 position. 3.3 Effects of Different Diets on Yolk Fatty Acid Composition and Cholesterol Content Fatty acid composition and cholesterol content represented core components of yolk quality, as they determined both nutritional value and functional properties. Dietary composition served as a major external factor shaping the yolk lipid profile, since fatty acids from feed can be directly deposited in yolk or modified during metabolism before deposition [32] . As shown in Table 1 , total saturated fatty acids (SFA) were highest in EY3 (59.5%), significantly greater than in EY2 (35.4%) and EY1 (18.6%) (p < 0.05). The SFA content in the three yolk samples was dominated by palmitic acid (PA, C16:0) and stearic acid (SA, C18:0), with the highest levels observed in EY3, where PA and SA were found to be 31.539 ± 0.28 mg/g and 9.273 ± 0.002 mg/g, respectively. For monounsaturated fatty acids (MUFA), the total content was found to be highest in EY1 (61%), significantly higher than in EY2 (41.7%) and EY3 (1.7%) (p < 0.05). Additionally, the content of trans oleic acid (C18:1n-9t) in EY1 was notably high (12.665 ± 0.108 mg/g), which may be associated with the metabolic characteristics of Corn DDGS. Corn DDGS was recognized as being rich in protein, fat, and fiber, and its fat composition was characterized by a high content of unsaturated fatty acids. However, partial isomerization may be induced during processing under high-temperature conditions, leading to the formation of trans fatty acids [33] . Regarding PUFA content, the highest total content was determined in EY3 (38.8%), significantly higher than in EY2 (22.9%) and EY1 (20.4%) (p < 0.05). In particular, docosahexaenoic acid (DHA) (2.422 ± 0.114 mg/g) was found to be significantly higher in EY3 than in the other two egg groups (p < 0.01). Additionally, EY3 exhibited the lowest n-6/n-3 PUFA ratio, which was associated with improved antioxidant status and reduced lipid peroxidation [34] . This result was consistent with studies reported in the literature, which suggested that the addition of flaxseed to the diet improved the egg health lipid index [35] . It was noted that the value of directly supplementing human diets with whole flaxseed or flaxseed oil was considered limited [36] . In contrast, eggs enriched in long-chain n-3 PUFA could be obtained by supplementing laying hens with flaxseed, thereby providing a source of nutritional supplementation. Significant differences were also observed in yolk cholesterol levels. The order of cholesterol content was EY1 (1220 µg/g) > EY3 (1090 µg/g) > EY2 (840 µg/g). The diet of EY2 was mainly composed of corn (63.03%) and soybean meal (21.4%), without the addition of specific ingredients such as flaxseed meal or corn DDGS. As a result, the fatty acid composition was relatively simple and dominated by cis fatty acids, with a relatively lower energy density, and the metabolic products of the feed were less likely to promote cholesterol synthesis. In addition, soybean meal was known to be rich in phytosterols such as β-sitosterol, whose structure was similar to that of cholesterol [37] . By occupying the NPC1L1 intestinal transporter, the absorption and conversion rate of cholesterol was thereby reduced, which was considered one of the reasons for the lowest cholesterol content observed in EY2. The above nutritional results confirmed that yolk core components, including the fatty acid profile and cholesterol, were remodeled by dietary composition. A high content of MUFA (particularly oleic acid) was detected in EY1, which was associated with enhanced flexibility of the lipoprotein interface (with the highest emulsifying activity observed in Fig. 1 A). In EY2, the lowest cholesterol level was achieved through its lower energy density and the inhibitory effect on cholesterol conversion. In EY3, a high content of beneficial n-3 PUFA (especially DHA) together with the lowest n-6/n-3 ratio was measured, conferring improved oxidative stability (Fig. 2 ). Table 2 Component content of egg yolk in three kinds of diet Type of fatty acid EY1 (mg/g) EY2 (mg/g) EY3 (mg/g) C15:0 0.023 ± 0.001 b 0.02 ± 0.08 b 0.061 ± 0.001a C16:0 6.237 ± 0.012 c 13.853 ± 0.032b 31.539 ± 0.283a C17:0 0.003 ± 0.06 c 0.057 ± 0.003b 0.23 ± 0.014a C18:0 0.052 ± 0.001b 0.007 ± 0.03c 9.273 ± 0.002a C20:0 1.142 ± 0.012b 3.766 ± 0.031a ND SFA 7.958 ± 0.499C 18.703 ± 1.066b 42.603 ± 1.796a C16:1 0.226 ± 0.019b 0.177 ± 0.003b 0.734 ± 0.002a C17:1 0.068 ± 0.01b 0.063 ± 0.01b 0.212 ± 0.002a C18:1 0.118 ± 0.006c 21.808 ± 0.005a 0.265 ± 0.001b C18:1n-9t 12.665 ± 0.103a ND 0.131 ± 0⁶ C18:1n-9c 13.075 ± 0.06 ND ND MUFA 26.152 ± 0.138a 22.048 ± 0.007b 1.343 ± 0.003c C18:2 n-6c (LA) 7.124 ± 0.112c 10.486 ± 0.221b 23.465 ± 0.013a C20:2 0.082 ± 0.002a 0.062 ± 0.001b 0.077 ± 0.01a C20:3 n-6 (DGLA) 0.068 ± 0.001c 0.075 ± 0.002b 0.162 ± 0.01a C20:4 n-6 (ARA) (Arachidonic acid) 1.179 ± 0.016b 1.166 ± 0.03c 1.193 ± 0.002a C22:4 n-6 0.031 ± 0.001c 0.051 ± 0.01b 0.226 ± 0.002a C22:5 n-6 0.015 ± 0.001c 0.053 ± 0.002b 0.269 ± 0.004a C22:6n-3 (DHA) 0.263 ± 0.0136b 0.212 ± 0.003b 2.422 ± 0.114a PUFA 8.763 ± 0.114c 12.106 ± 0.247b 27.814 ± 0.094a cholesterol 1.22 ± 1.32a 0.84 ± 0.857c 1.09 ± 0.774b 3.4 MALDI-TOF-MS Identification of Yolk Lipids from Different Diets The diversity of lipid molecules in egg yolk directly influenced interfacial behavior, oxidative sensitivity, and digestive dynamics [38] . Lipidomic profiling of yolks from the three dietary groups was performed using MALDI-TOF-MS. The spectral data were annotated and normalized through the LIPID MAPS database (v2.3). In positive-ion mode, lipid spectra were recorded with a detection threshold set at relative intensity (Rel. Intens.) > 0.01. A total of 189 lipid species belonging to 12 major classes were identified, forming a framework that encompassed core categories such as phospholipids (PLs) and glycerolipids (GLs). The overall relative composition of lipids across the three yolk types was shown in the heatmap (Fig. 3 A), with radar plots illustrating relative abundance patterns (Fig. 3 B). The relative abundance of lipids was reflected by red and blue colors, with large differences observed between PLs and GLs. In EY1, relatively higher levels of medium-chain PC and PE were detected, which were highly consistent with its optimal ESI (Fig. 1 A). These lipid molecules were considered to play a central role in constructing stable emulsifying films with low interfacial tension. In EY2, the highest relative levels of phosphatidylserine (PS) and phosphatidic acid (PA) were measured. The negative charge of PS was suggested to cause electrostatic repulsion and membrane instability, which provided a molecular-level explanation for its poorer particle homogeneity (high PDI) and potential oxidative risk (higher MDA). By contrast, long-chain TG molecules were found to be enriched in EY3, leading to interfacial molecular conformations favorable for uniform emulsion distribution. The result was consistent with the earlier inference regarding fatty acids and yolk emulsion stability. At the same time, TG, as the main storage form of neutral lipids, was emphasized to have its sn-positional distribution as a critical factor for digestive and absorptive efficiency. In conclusion, diet was identified as the key factor driving lipid profile differentiation. By MALDI-TOF-MS analysis, characteristic “fingerprint” lipids of yolks derived from different diets were revealed, and significant differences in yolk lipid composition were confirmed to be induced by dietary factors. 3.5 Identification of Phospholipid (PL) Molecular Species in Yolks from Different Diets In terms of the differentiated remodeling of PLs, the relative contents of PE and PC subclasses followed the order EY1 > EY2 > EY3, whereas the relative content of PS subclasses followed the order EY2 > EY1 > EY3 (Fig. 4 A). The high levels of PC and PE in EY1 were attributed to the inhibition of LPA generation and the reduction of PC degradation by trans fatty acids derived from Corn DDGS (4%). Meanwhile, bile acids were adsorbed by soybean meal fiber (adsorption rate ≥ 50%), resulting in the inhibition of lipase activity and the subsequent accumulation of PS as a metabolic intermediate. Consequently, the highest PS content was detected in EY2. The common phospholipid subclasses identified in yolks from different diets included PC(35:8), PC(35:9), PS(36:5), and PE(40:11) (Fig. 4 B). The relative abundance of medium-chain PC was found to be the highest in EY1, which was attributed to the synergistic metabolic effects of the high-corn formulation and Corn DDGS. A significant accumulation of PS(36:5) in EY2 directly reflected the metabolic imprint of ω-6 linoleic acid from the corn–soybean diet, consistent with the conservative deposition pattern of fatty acids in grain-based diets [39] . In EY3, the phospholipid distribution was characterized by a “dispersed low-energy” pattern, with lower relative intensities compared to the other two yolk groups. This phenomenon was suggested to result from the activation of the phospholipase A2 (PLA2) pathway by flaxseed supplementation, which promoted phospholipid hydrolysis and the release of free DHA. However, due to the inhibited expression of endogenous phospholipid synthases in the yolk, DHA was more likely to be stored in the form of neutral lipids [40] . The three most abundant phospholipids across yolks were shown in Fig. 4 C. Notably, phosphatidic acid (PA 51:10) was detected only in EY3. As the simplest glycerophospholipid, PA is an essential signaling molecule in both intracellular and extracellular pathways, which further indicated that lipid metabolic activity was elevated in EY3 and may be related to flaxseed-mediated regulation of lipase activity. Overall, high levels of medium-chain PC and PE were formed in EY1 yolk, leading to enhanced emulsion stability. In EY2, the accumulation of PS was found to exacerbate oxidative stress, whereas in EY3, DHA was driven toward storage in TG, resulting in dual benefits of efficient neutral lipid incorporation of DHA and improved membrane oxidative stability. 3.6 Identification of Glycerolipid (GL) Molecular Species in Yolks from Different Diets In terms of glycerolipids, the relative abundance of triacylglycerols (TGs) followed the order EY3 > EY1 > EY2, and diacylglycerols (DGs) were most abundant in EY1, followed by EY2 and then EY3. The total TG content in EY3 reached 71.5%, in which strong signals of TG(51:8; O3) (C16:0-C22:6-C22:6) and TG(51:9) were identified, confirming the efficient deposition of DHA in TG (Fig. 5 ). This result was consistent with the preferential incorporation of flaxseed-derived fatty acids into TG, corresponding to the characteristic profile of EY3 with high TG/DG and low PC/PE. It was also indicated that hens utilized available precursor ALA for the biosynthesis of EPA and DHA through the activity of hepatic FA desaturases and elongases [41] . Notably, DHA contains six double bonds and was theoretically more susceptible to oxidation than common PUFA. However, the oxidized DG(55:13) in EY3 showed a relative content of only 0.08, one quarter of that in grain eggs (0.34), consistent with the results of oxidative stability. In contrast, the accumulation of oxidized glycerides in EY2 suggested that the corn–soybean diet lacked exogenous antioxidants, resulting in the upregulation of lipoxygenase (LOX) activity and triggering a lipolysis–oxidation cascade [342 Furthermore, the preferential deposition of DHA in TG rather than in DHA-PC/PE in EY3 revealed the limitation of yolk phospholipidization capacity. In EY1, the proportions of TG(51:9) (0.79) and TG(51:8) (0.69) were relatively high, which was likely attributed to corn DDGS in the diet (rich in ω-6 linoleic acid) and fatty acid deposition. From the analysis of GLs, DHA enrichment in yolk TG was driven by flaxseed. TG(51:8) was suggested to contain three DHA molecules (C22:6), indicating that EY3 may contain approximately 150–200 mg of DHA, reaching the high-end standard of the industry. In the corn-soybean diet, the absence of exogenous antioxidants resulted in the activation of adipose triglyceride lipase (ATGL) and lipoxygenase (LOX), leading to the accumulation of oxidized TG in EY2. In EY1, the high level of n-6 linoleic acid (C18:2n6) was preferentially incorporated into TG, with significant expression of TG(51:9) and TG(51:8), thereby forming a conservative deposition pattern of n-6 PUFA. 3.7 Differential Lipid Analysis of Egg Yolks from Different Diets In the principal component analysis (PCA) plot (Fig. 6 A), the samples of the three types of eggs were displayed along PC1 (explaining 68% of the variance) and PC2 (explaining 17.6% of the variance). The lipid samples of the three egg yolks were clearly separated in the plot, and the intergroup separation was further emphasized, suggesting that the lipid composition of egg yolks was affected by the diet, thereby allowing different types of eggs to be distinguished at the lipid level. The partial least squares discriminant analysis (PLS-DA) of differential lipids was employed as the central approach through which the logic chain of dietary intervention-lipid metabolism remodeling-functional differentiation was elucidated. As shown in the PLS-DA plot of differential lipids in egg yolks (Fig. 6 B), lipids with VIP values greater than 1, including LPS, PC, and PE classes, were identified as the key biomarkers distinguishing the three egg sources. It was indicated that the differences among the sample groups were mainly driven by phospholipids, which might be related to the regulation of oxidative levels, emulsion stability, and membrane fluidity, whereas the contribution of ether lipids was relatively low. Pairwise comparisons of lipid abundances across groups were further visualized by volcano plots (Fig. 6 C). Each point was used to represent a lipid molecule, with purple points being used to indicate downregulated lipids, orange points being used to indicate upregulated lipids, and gray points being used to indicate lipids without significant differences. The horizontal axis was designated to represent the log 2 FC value, while the vertical axis was designated to represent the p-value. The greater the values along both axes were, the more significant the changes in the selected lipid species were considered to be. For the EY3 and EY2 groups, lipid biosynthesis and ether-linked modifications were detected to be enhanced in EY3, which implied a potential elevation of antioxidant capacity. When EY3 was compared with EY1, an increase in lipid synthesis-particularly of glycerolipids and ether phospholipids was detected, accompanied by an improvement in membrane stability. In the comparison of EY1 and EY2, basal metabolic lipids (short-chain glycerolipids) were found to be predominant in EY1, with a relatively higher proportion of high-quality phospholipids, thereby confirming its favorable emulsifying activity. Interestingly, in the differential analysis between EY3 and the other two types of eggs, TG(51:8) was consistently identified as being significantly upregulated, indicating that EY3 might possess unique characteristics in fat storage or energy metabolism. The egg yolk lipid architecture of the three egg types was directly remodeled by dietary intervention, resulting in markedly distinct lipid fingerprints. The intergroup variations were predominantly driven by polyunsaturated phospholipids. In addition, TG(51:8) was identified as a characteristic biomarker among the groups, providing a key target for the lipid engineering design of flaxseed eggs. 3.8 Determination of Fatty Acid sn-Position in Yolk Lipids from Different Diets The complementarity of ¹H NMR and MALDI-TOF-MS was evidenced, showing that dietary components differentially affected egg yolk lipid structures by regulating fatty acid composition, phospholipid distribution, and oxidative stability. Through chemical shifts (ppm) and coupling constants, information on the chemical environment of hydrogen atoms in molecules was provided, allowing direct elucidation of functional groups, stereoisomers, and dynamic structures [43] . From Fig. 7 , it was observed that, apart from the solvent peaks of DMSO (δ 2.51 ppm) and HDO (δ 3.13 ppm), the major lipid classes and specific lipid species present in the three egg yolks were profiled. For the characteristic peaks of TG, the signal at 5.21 ppm was assigned to the CH-O-CO protons of the glycerol backbone, while the signals at δ 1.24 ppm and δ 1.17 ppm were attributed to the -(CH₂)n- methylene chains of long-chain fatty acids. The signal at 0.95 ppm represented the terminal -CH₃ methyl group of fatty acid chains. Since prominent broad peaks were consistently observed at δ 1.25–1.30 ppm in all three egg yolks, it was demonstrated that TG in the yolks was connected through a glycerol methylene backbone with long-chain fatty acid -(CH₂)n- chains. Specifically, cis FA (δ 2.00-2.10 ppm) and trans FA (δ 1.98–2.04 ppm) were detected in EY1, while trans FA was absent in EY2, which was consistent with the results of fatty acid composition analysis. In addition, the sn-2 glycerol lipids (δ 5.25–5.30 ppm) of EY3 exhibited the most intense signal among the three egg types, suggesting the highest relative abundance. These lipids, being bound to long-chain fatty acids, were more readily utilized by the human body, in agreement with the digestibility results. Regarding specific lipids, the characteristic signals of PC were assigned to the choline headgroup [-N(CH₃)₃] (δ 3.13 ppm) and the CH₂-O-PO₄⁻ protons of the phospholipid glycerol backbone (δ 4.28 ppm). Consequently, a higher phospholipid content was indicated in EY1, which may be associated with lipoprotein remodeling regulated by distillers’ grains, thereby enhancing interfacial charge density and improving emulsifying properties. In EY2, the cholesterol content (δ 0.66 ppm) was the lowest, corroborating the results of lipidomics and compositional analyses. In contrast, the lowest phosphatidylcholine content was observed in EY3, consistent with the GLs lipid analysis. Moreover, for specific fatty acids (DHA/ALA), the DHA signals (δ 0.8–1.0 ppm) and ALA signals (δ 1.24 ppm) in EY3 were more prominent. In summary, direct evidence for key structural features such as the sn-2 position, trans configuration, and phospholipid headgroups was revealed by ¹H NMR, forming a complete chain of mutual validation with lipidomic and functional data. In EY1, the highest phosphatidylcholine content (δ 3.13, 4.28 ppm) was identified, which was suggested to enhance emulsifying properties by increasing interfacial charge density. In EY2, no trans fatty acids were detected, while cholesterol (δ 0.66 ppm) was found to be the lowest. In EY3, the strongest lipid signals at the sn-2 position (δ 5.25–5.30 ppm) were detected, pointing to abundance of long-chain fatty acids such as DHA at this position, closely associated with its higher digestibility. 3.9 Correlation Analysis of Diet Composition, Lipid Structure, and Functional Properties The directional design of dietary components was implemented to precisely regulate the molecular structures of yolk lipids, thereby cascade-driving the coordinated changes in functional properties such as nutritional value, emulsifying stability, and oxidative stability. The DDGS diet was evidenced to enhance the enrichment of trans-oleic acid (C18:1n9t) in PC, and the high MUFA content was identified to markedly improve the emulsifying activity of EY1 by reducing interfacial tension. The flaxseed diet was demonstrated to direct DHA to be specifically located at the sn-2 position of triglycerides (as confirmed by ¹H NMR), where selective hydrolysis of the sn-1,3 positions by pancreatic lipase released sn-2 monoglycerides, thereby improving the digestibility of EY3. The soybean meal diet was found to inhibit cholesterol absorption due to plant sterols, while abnormal accumulation of phosphatidylserine (PS) was identified in EY2, whose high charge density induced electrostatic repulsion, ultimately decreasing emulsion stability. Correlation analysis was employed as a key approach to elucidate the intrinsic associations among multidimensional yolk indicators under diversified dietary regulation. The results demonstrated the core finding of this study that the remodeling of lipid molecular structures drove functional differentiation. In Fig. 8 , significant correlations among variables were clearly presented. A very strong positive correlation was observed between emulsifying activity and MUFA (r = 0.88), while a strong positive correlation was also observed between emulsifying activity and PUFA (r = 0.84), which was in accordance with the role of long-chain PUFAs (e.g., DHA) in flaxseed eggs in improving interfacial fluidity. However, a strong negative correlation was observed between emulsifying activity and SFA (r = -0.88), highlighting the inhibitory effect of droplet crystallization tendency induced by high SFA on interfacial behavior, which offset part of the positive effect of PUFA. Emulsifying activity was driven by MUFA through the optimization of lipoprotein interfacial properties, corroborating the mechanism by which MUFA significantly enhanced emulsification capacity via improved flexibility and spreadability of LDL interfacial membranes. In contrast, phospholipid charge repulsion (high PS) and structural disorder in grain-based eggs resulted in larger droplet size and higher PDI (0.38), markedly weakening emulsification performance. Moreover, the specific enrichment of PUFA at the sn-2 position was identified as the key factor simultaneously enabling efficient digestion and absorption (positive correlation) and ultra-low oxidation (negative correlation). The phospholipid profiles revealed by lipidomics and the structural resolution were demonstrated to serve as a mechanistic bridge linking the intrinsic biochemical relationships among variables. Overall, dietary components were evidenced to remodel yolk lipid molecular structures, thereby driving the directional reconstruction of lipid metabolic networks and differentially regulating three major functional properties. Nutritional characteristics were dominated by the positional distribution of fatty acids at the sn-2 site, which determined bioavailability. Emulsifying properties were regulated by the charge of PC/PE headgroups and molecular order, which in turn affected interfacial stability. Oxidative stability was determined by the storage form of PUFAs in long-chain triglycerides as well as the cooperation of endogenous antioxidant systems. 4. Conclusion In this study, mechanisms diversifying diets (DDGS, cereals, flaxseed) specifically remodeled yolk lipid molecular structures to drive functional differentiation were elucidated using multi-scale integrated techniques. In EY1, the enrichment of MUFA (61%) and trans-oleic acid (C18:1n9t, 12.665 µg/g) was identified, leading to the formation of a layered structure of medium-chain phosphatidylcholine (PC 35:8). Although this structure was associated with peak emulsifying activity, a significant increase in malondialdehyde content (MDA 3.40 nmol/g) was also observed, indicating that the system became markedly more sensitive to oxidative reactions. In EY2, abnormal accumulation of PS (62.3%) was detected, leading to reduced interfacial stability, while the competitive inhibition by dietary phytosterols resulted in the lowest cholesterol content (0.84 mg/g). In EY3, DHA was shown to be preferentially located at the sn-2 position of triglycerides, producing synergistic effects that enabled high digestibility (93.15%) and low oxidation, although SFA accumulation (59.5%) was found to weaken emulsifying activity. Furthermore, by dissecting the mechanisms through which lipid structures influenced functional properties, the enrichment of PUFAs at the sn-2 position was identified as the core pathway for improving digestion and absorption. In addition, phospholipid profiles were shown to govern emulsifying stability by regulating interfacial charge and membrane strength, whereas trans configurations and lipid densification were identified as the key inducers of oxidative susceptibility and digestive inhibition. Finally, correlation analysis confirmed that lipid molecular structures were the fundamental driving force of functional differentiation. This study therefore provided new targets for the design of functional eggs based on “structure-enabled” strategies, and suggested that future optimization could be achieved by adjusting dietary composition to enhance sn-2 PUFA positioning and eliminate trans fats, thereby overcoming the bottleneck in the coordinated improvement of nutritional utilization and oxidative stability. Declarations CRediT authorship contribution statement Xiaodan Zhang : Writing-original draft, Investigation, Data curation; Xuan Ji : Methodology, Investigation; Wenqian Guan : Methodology, Formal analysis; Sharina Qi : Validation, Data curation; Shijian Dong : Methodology, Data curation; Jijun Wu : Resources, Validation; Ying Gao : Formal analysis, Writing-review & editing; Shugang Li : Supervision, Resources, Project administration, Funding acquisition, Conceptualization, Writing-review & editing. Declaration of competing interest These 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. Funding The work is supported by Project of National Key Research and Development Program of China (No.2022YFD2101001), the Earmarked Fund for China Agriculture Research System (CARS-40-K25, CARS-40-S11), the Project of National Natural Science Foundation of China (No. 32172226), the Special Fund for Anhui Agriculture Research System (AHCYJSTX-NCPJG-15), the Cooperative Projects of Hefei University of Technology-Anhui Rongda Food Co. Ltd. (No. W2020JSKF0489). Author Contribution **Xiaodan Zhang:** Writing-original draft, Investigation, Data curation; **Xuan Ji:** Methodology, Investigation; **Wenqian Guan:** Methodology, Formal analysis; **Sharina Qi:** Validation, Data curation; **Shijian Dong:** Methodology, Data curation; **Jijun Wu:** Resources, Validation; **Ying Gao:** Formal analysis, Writing-review & editing; **Shugang Li** : Supervision, Resources, Project administration, Funding acquisition, Conceptualization, Writing-review & editing. References M. Salahuddin, A. A. A. Wareth, K. A. Rashwan, et al. The role of egg-derived nutrients in Alzheimer’s disease: Exploring potential benefits and biological insights. Food Bioscience 2024, 62, 105096. https://doi.org/10.1016/j.fbio.2024.105096 M. I. Sultana, B. T. Molla, S. Bae, et al. Isolation, characterization, proteome, miRNAome, and the embryotrophic effects of chicken egg yolk nanovesicles (vitellovesicles). Scientific Reports 2023, 13, 4204. https://doi.org/10.1038/s41598-023-31012-0 J. L. Deleu, S. Melis, E. Wilderjans, et al. Protein network formation during pound cake baking: The role of egg yolk and its fractions. Food Hydrocolloids 2017, 63, 226–232. https://doi.org/10.1016/j.foodhyd.2016.07.036 C. P. Mezquita, J. Morales, J. Palma, et al. Stability of lutein obtained from Muriellopsis sp. biomass and used as a natural colorant and antioxidant in a mayonnaise-like dressing sauce. CyTA - Journal of Food 2019, 17, 517–526. https://doi.org/10.1080/19476337.2019.1609091 R. Suhag. Egg yolk, a multifunctional emulsifier: New insights on factors influencing and mechanistic pathways in egg yolk emulsification. Applied Sciences 2024, 14, 9692. https://doi.org/10.3390/app14219692 J. M. Miranda, X. Antón, C. Redondo-Valbuena, et al. Egg and egg-derived foods: Effects on human health and use as functional foods. Nutrients 2015, 7, 706–729. https://doi.org/10.3390/nu7010706 K. Zaheer. An updated review on chicken eggs: Production, consumption, management aspects and nutritional benefits to human health. Food and Nutrition Sciences 2015, 6, 1208–1220. https://doi.org/10.4236/fns.2015.613126 O. S. Dudek, M. Pietras, M. Puchała, et al. Camelina sativa oil and camelina cake as sources of polyunsaturated fatty acids in the diets of laying hens: Effect on hen performance, fatty acid profile of yolk lipids, and egg sensory quality. Annals of Animal Science 2020, 20 (4), 1365–1377. https://doi.org/10.2478/aoas-2020-0035 D. Mierliță, S. Daraban, A. C. Teușdea, et al. Effect of dietary cold-pressed hempseed cake supplemented with tomato waste on laying hen performance and egg yolk lipid profile and antioxidant status before and after storage. Animals 2024, 14, 3444. https://doi.org/10.3390/ani14233444 D. Tadesse, N. Retta, W. Esatu, et al. Fatty acid content and oxidative stability in eggs and breast muscle of Sasso chickens fed different levels of Dodonaea angustifolia polyphenol in flaxseed-enriched diets. Agriculture 2024, 14, 993. https://doi.org/10.3390/agriculture14070993 H. Aro, T. Rokka, J. Valaja, et al. Functional and sensory properties of hen eggs with modified fatty acid compositions. Food Function 2011, 2 (11), 671–677. https://doi.org/10.1039/c1fo10132c E. N. S. Doomun, S. Loke, S. O’Callaghan, et al. A simple method for measuring carbon-13 fatty acid enrichment in the major lipid classes of microalgae using GC–MS. Metabolites 2016, 6 (4), 42. https://doi.org/10.3390/metabo6040042 A. Najla, C. Elon, K. D. T. Tastet, et al. Fractional factorial design of MALDI-TOF-MS sample preparations for the optimized detection of phospholipids and acylglycerols. Analytical Chemistry 2016, 88 (12), 6301–6308. https://doi.org/10.1021/acs.analchem.6b00512 M. C. Francesca, M. Nadia, R. O. Cesare, et al. Triacylglycerol composition and chemical–physical properties of cocoa butter and its derivatives: NMR, DSC, X-ray, rheological investigation. International Journal of Molecular Sciences 2023, 24 (4), 2090. https://doi.org/10.3390/ijms24032090 Y. Sun, Y. Yang, H. Jin, et al. Ultrasound-assisted pH-shifting treatment: Effects on egg yolk protein interfacial adsorption and emulsification properties. Food Chemistry 2025, 482, 144172. https://doi.org/10.1016/j.foodchem.2025.144172 U. Laila, T. J. Putra. The influence of inter-day colorimetric method in quantification of chicken meat total cholesterol with lipid extraction–saponification pretreatment. IOP Conference Series: Materials Science and Engineering 2019, 633, 012048. https://doi.org/10.1088/1757-899x/633/1/012048 G. Martos, R. López-Fandiño, E. Molina. Immunoreactivity of hen egg allergens: Influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk. Food Chemistry 2013, 136 (2), 775–781. https://doi.org/10.1016/j.foodchem.2012.07.106 B. A. Watkins, S. Feng, A. K. Strom, et al. Conjugated linoleic acids alter the fatty acid composition and physical properties of egg yolk and albumen. Journal of Agricultural and Food Chemistry 2003, 51 (23), 6870–6876. https://doi.org/10.1021/jf026156v E. Alves, T. Melo, F. Rey, et al. Polar lipid profiling of olive oils as a useful tool in helping to decipher their unique fingerprint. LWT – Food Science and Technology 2016, 74, 371–377. https://doi.org/10.1016/j.lwt.2016.07.071 P. Abraham, S. Soledad, P. Trinidad, et al. ¹H NMR to analyse the lipid profile in the glyceride fraction of different categories of Iberian dry-cured hams. Food Chemistry 2022, 383, 132371. https://doi.org/10.1016/j.foodchem.2022.132371 M. Anton. Egg yolk: Structures, functionalities and processes. Journal of the Science of Food and Agriculture 2013, 93 (12), 2871–2880. https://doi.org/10.1002/jsfa.6247 J. Jin, D. Cheng, D. Du, E. Olayemi, D. Xiao, T. Li, et al. Effect of emulsifier–fat interactions and interfacial competitive adsorption of emulsifiers with proteins on fat crystallization and stability of whipped-frozen emulsions. Food Hydrocolloids 2020, 101, 105491. https://doi.org/10.1016/j.foodhyd.2019.105491 D. J. McClements. Nanoscale nutrient delivery systems for food applications: Improving bioactive dispersibility, stability, and bioavailability. Journal of Food Science 2015, 80 (7), N1602–N1611. https://doi.org/10.1111/1750-3841.12919 P. Garcia-Moreno, C. Jacobsen, P. Marcatili, S. Gregersen, M. T. Overgaard, et al. Emulsifying peptides from potato protein predicted by bioinformatics: Stabilization of fish oil-in-water emulsions. Food Hydrocolloids 2020, 101, 105529. https://doi.org/10.1016/j.foodhyd.2019.105529 M. Fasiangova, G. Borilova. Impact of selenium supplementation on the oxidation stability of eggs. World’s Poultry Science Journal 2017, 73 (1), 175–184. https://doi.org/10.1017/s0043933916000854 K. Liang, H. Zu, X. Wang. Effect of storage on n-3 PUFA-enriched eggs. CyTA – Journal of Food 2020, 18 (1), 102–107. https://doi.org/10.1080/19476337.2020.1713896 N. Sun, J. Chen, Z. Bao, et al. Egg yolk phosphatidylethanolamine: Extraction optimization, antioxidative activity, and molecular structure profiling. Journal of Food Science 2019, 84 (5), 1002–1011. https://doi.org/10.1111/1750-3841.14512 G. Santos Sánchez, B. Miralles, A. Brodkorb, et al. Current advances for in vitro protein digestibility. Frontiers in Nutrition 2024, 11, 1404538. https://doi.org/10.3389/fnut.2024.1404538 Y. Guo, Z. Cai, Y. Xie, et al. Synthesis, physicochemical properties, and health aspects of structured lipids: A review. Comprehensive Reviews in Food Science and Food Safety 2020, 19 (2), 759–800. https://doi.org/10.1111/1541-4337.12537 A. Serra, G. Conte, F. Ciucci, et al. Dietary linseed supplementation affects the fatty acid composition of the sn-2 position of triglycerides in sheep milk. Journal of Dairy Science 2018, 101 (8), 6742–6751. https://doi.org/10.3168/jds.2017-14188 T. Zhang, J. Xu, Y. Wang, et al. Health benefits of dietary marine DHA/EPA-enriched glycerophospholipids. Progress in Lipid Research 2019, 75, 100997. https://doi.org/10.1016/j.plipres.2019.100997 A. Irawan, N. Ningsih, Hafizuddin, et al. Supplementary n-3 fatty acid sources on performance and formation of omega-3 in eggs of laying hens: A meta-analysis. Poultry Science 2022, 101 (12), 101566. https://doi.org/10.1016/j.psj.2022.101566 P. E. Kiełb, J. Tumanowicz, A. Zachwieja, et al. Changes in fatty acid levels during in vitro ruminal fluid incubation with different proportions of maize distillers dried grains (DDGS). Agriculture 2023, 13 (4), 763. https://doi.org/10.3390/agriculture13040763 A. T. Ebeid. The impact of incorporation of n-3 fatty acids into eggs on ovarian follicular development, immune response, antioxidative status and tibial bone characteristics in aged laying hens. Animal 2011, 5 (10), 1554–1562. https://doi.org/10.1017/s1751731111000619 B. Omri, R. Chalghoumi, L. Izzo, et al. Effect of dietary incorporation of linseed alone or together with tomato–red pepper mix on laying hens’ egg yolk fatty acids profile and health lipid indexes. Nutrients 2019, 11, 813. https://doi.org/10.3390/nu11040813 M. D. Baucells, N. Crespo, A. C. Barroeta, et al. Incorporation of different polyunsaturated fatty acids into eggs. Poultry Science 2000, 79 (1), 51–59. https://doi.org/10.1093/ps/79.1.51 L. Xiang, X. Yan, M. Yuqian, et al. The bioavailability and biological activities of phytosterols as modulators of cholesterol metabolism. Molecules 2022, 27, 523. https://doi.org/10.3390/molecules27020523 S. Lioliopoulou, A. G. Papadopoulos, T. F. Mantzouridou, et al. Pomegranate peel dietary supplementation in laying hens affects egg yolk color and eggshell quality, decreases yolk lipid oxidation and influences yolk lipidomic profile. Journal of Applied Poultry Research 2024, 33, 100495. https://doi.org/10.1016/j.japr.2024.100495 T. O. Toomer, P. Maharjan, L. K. Harding, et al. Effects of full-fat high-oleic soybean meal in layer diets on performance, egg quality and chemical composition. Poultry Science 2024, 103 (3), 104074. https://doi.org/10.1016/j.psj.2023.104074 K. Nafiseh, Z. Ali, F. Ata, et al. Effects of flaxseed consumption on plasma lipids, lipoprotein-associated phospholipase A2 activity and gut microbiota composition in patients with coronary artery disease. Nutrition and Health 2022, 28 (5), 535–546. https://doi.org/10.1177/02601060221091016 H. M. M. Balnave, E. A. Akter, C. Geier, et al. Choline and methionine supplementation in layer hens fed flaxseed: Effects on hen production performance, egg fatty acid composition, tocopherol content, and oxidative stability. Poultry Science 2021, 100, 101299. https://doi.org/10.1016/j.psj.2021.101299 Y. Tang, C. Zhou, Z. Yu, et al. Formation of lipid-derived volatile products through lipoxygenase (LOX)- and hydroperoxide lyase (HPL)-mediated pathway in oat, barley and soybean. Food Chemistry: X 2024, 22, 101514. https://doi.org/10.1016/j.fochx.2023.101514 K. Cho, E. P. Hansen. Structure identification of novel compounds using simple IR, ¹H, and ¹³C NMR spectroscopy and computational tools. Bulletin of the Korean Chemical Society 2020, 41 (1), 78–83. https://doi.org/10.1002/bkcs.11924 Additional Declarations No competing interests reported. Supplementary Files Abstractgraphic.tif Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Oct, 2025 Reviews received at journal 27 Oct, 2025 Reviews received at journal 22 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers invited by journal 13 Oct, 2025 Editor assigned by journal 13 Oct, 2025 Submission checks completed at journal 13 Oct, 2025 First submitted to journal 12 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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18:47:30","extension":"png","order_by":47,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59204,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/ab793872947f675347e9281a.png"},{"id":94597238,"identity":"2f4590c2-eab2-4a1a-bae4-fd0186035646","added_by":"auto","created_at":"2025-10-28 18:46:18","extension":"png","order_by":48,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7594,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/1d34b3c1f9e2c4617509ade2.png"},{"id":94597439,"identity":"21813cb0-c460-42c4-8f07-e7248000005e","added_by":"auto","created_at":"2025-10-28 18:47:36","extension":"png","order_by":49,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107197,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/e1bdcdb702c6c21833199636.png"},{"id":94597953,"identity":"523688a8-044c-4cc4-a828-1a3b6925fd7e","added_by":"auto","created_at":"2025-10-28 18:50:27","extension":"xml","order_by":50,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122596,"visible":true,"origin":"","legend":"","description":"","filename":"5189aebda2034d36b9942856f677a3271structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/7c44d013bdd2aeed0c54b1da.xml"},{"id":94598047,"identity":"9e60d42a-56ba-40be-ba89-e5bd760d4734","added_by":"auto","created_at":"2025-10-28 18:50:59","extension":"html","order_by":51,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":129488,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/05d49bd647ac547d11182cc7.html"},{"id":94597792,"identity":"3c9bb7e8-2ba3-4191-b416-c7530d13e446","added_by":"auto","created_at":"2025-10-28 18:49:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":775708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmulsification activity (A), emulsification stability (B), particle size (C) and Zeta potential (D) of eggs on different diets\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/6b2b521689637c7ab94dad80.png"},{"id":94597650,"identity":"62b978b6-f940-4776-a0f9-6b7986442a6b","added_by":"auto","created_at":"2025-10-28 18:48:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":596810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMalondialdehyde content (A), fatty acid digestibility (B) of three diet eggs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/7641307b550b0072fd79093a.png"},{"id":94597634,"identity":"09f4410c-9925-4fe6-a87e-1d4fc57b63df","added_by":"auto","created_at":"2025-10-28 18:48:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2233650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipid caloric profile of egg yolk in three diets (A); Radar chart of main lipids in egg yolk of three diets (B)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/54b1a849483749a8ade5fc53.png"},{"id":94597390,"identity":"52adb07b-46a8-4cb1-b803-994dbf8b7c03","added_by":"auto","created_at":"2025-10-28 18:47:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11453584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal relative content of phospholipid subtypes in egg yolks from different diets (A); Relative content of identical phospholipid subtypes in egg yolks from different diets (B); Phospholipid lipid with the highest relative intensity in egg yolks from different diets (C)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/43c0c1f5fdaf1921f98c0034.png"},{"id":94597625,"identity":"a6f9feb9-d71c-46d0-9293-6ca4119fe859","added_by":"auto","created_at":"2025-10-28 18:48:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10868149,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal relative content of different dietary egg yolk lipid subtypes (A); Relative content of the same lipid subtypes in egg yolk from different diets (B); Lipid subtypes with the highest relative intensity in egg yolk from different diets (C)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/7f21fec6639259a15bd83ca1.png"},{"id":94597948,"identity":"a3660eeb-3841-4915-a0d3-ee2aadf5d3e2","added_by":"auto","created_at":"2025-10-28 18:50:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1327001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCA of egg yolk lipids from different diets (A); PLS-DA of differences in egg yolk lipids from different diets (B); Lipid volcano diagram of different diet egg yolks (C)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/afb06336e84ea3b83a0b0d4c.png"},{"id":94597720,"identity":"3d51a8d1-fbab-4775-b9c4-87ce48a61a19","added_by":"auto","created_at":"2025-10-28 18:48:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":149687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR of egg yolk lipids in three diets\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/e6bb19ad759fccf6dc6d7c5f.png"},{"id":94597649,"identity":"e8d0ae26-6444-4af5-9139-06d3ce65b83a","added_by":"auto","created_at":"2025-10-28 18:48:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2458069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between lipid molecules and functional characteristics of egg yolk in three diets Figure\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/94b249e4153506f5e2c6e30b.png"},{"id":94600344,"identity":"b1c372ef-fc53-415b-8ffb-95e9870d1e94","added_by":"auto","created_at":"2025-10-28 19:16:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21427926,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/8c4c15d0-4065-447c-a3ab-09b582a8279a.pdf"},{"id":94597317,"identity":"442818fe-6bd7-4727-b184-22513fabd689","added_by":"auto","created_at":"2025-10-28 18:46:53","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":504493,"visible":true,"origin":"","legend":"","description":"","filename":"Abstractgraphic.tif","url":"https://assets-eu.researchsquare.com/files/rs-7844056/v1/4cd20a768ba44a46e1b53e20.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dietary Modulation on sn-Position Distribution and Its Impact on Emulsifying and Nutritional Properties in Egg Yolk Lipids","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eEggs are considered one of the most complete natural nutrient carriers and high-quality protein sources in human diet \u003csup\u003e[1]\u003c/sup\u003e. As the second major component in eggs, yolk lipids account for 30 to 35 percent, and are not merely an energy source, instead, their unique composition and structure, including fatty acid profiles, distributions of phospholipid classes, and molecular organization of lipids, are recognized as the core determinants of the nutritional quality, processing functionality, and bioavailability of eggs \u003csup\u003e[2]\u003c/sup\u003e. These unique functions have placed eggs in a key position within the food industry, demonstrated by their applications in fields of baking, sauces, and infant complementary foods \u003csup\u003e[3\u0026ndash;5]\u003c/sup\u003e. However, the relatively high levels of saturated fat (approximately 3 g per 100 g) and cholesterol (approximately 200 to 300 mg per 100 g) in the lipid composition of conventional eggs are considered insufficient to meet the precise nutritional requirements of special populations such as patients with cardiovascular diseases and infants \u003csup\u003e[6]\u003c/sup\u003e. Hence, it is urgent to upgrade the functional egg industry with the growing demand of consumers for the nutritional and health attributes of foods.\u003c/p\u003e\u003cp\u003eThe quality and functional properties of eggs are significantly influenced by multiple factors, including the age and breed of hens, dietary additives, and storage conditions. In particular, the functional quality and nutritional composition of yolk are primarily enhanced from a natural perspective through modifications in poultry feed composition. Meanwhile, the development of functional eggs has been largely focused on the enrichment of target components such as omega-3 fatty acids, low cholesterol, and high levels of vitamins. Accordingly, the contents of specific nutrients in eggs can be increased by altering the feed composition of laying hens, whereby nutritionally fortified eggs are produced and referred to as \u0026ldquo;designer eggs.\u0026rdquo; \u003csup\u003e[7]\u003c/sup\u003e. Extensive evidence has been provided demonstrating that the yolk fatty acid profile is significantly influenced by dietary fatty acid composition. For instance, supplementing laying hen diets with flaxseed-rich in α-linolenic acid enhances the deposition of long-chain n-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA), in egg yolks \u003csup\u003e[8]\u003c/sup\u003e. Plant-based antioxidants in feed also improve yolk oxidative stability \u003csup\u003e[9, 10]\u003c/sup\u003e. These changes modify not only the nutritional value but also the processing functionality of yolk lipids, as well as their emulsifications. The emulsifying property of eggs is significantly improved when flaxseed oil is supplemented in the diet \u003csup\u003e[11]\u003c/sup\u003e. Although numerous studies have been conducted on dietary regulation of yolk lipids, the focus has mainly been placed on the overall composition of fatty acids rather than on the remodeling of fine molecular structures. Information regarding phospholipid subclasses, sn-positions of triacylglycerols, and the structural organization of lipid molecules remains limited.\u003c/p\u003e\u003cp\u003eAt present, diverse techniques have been applied to the identification of food lipids. Traditional lipid analysis methods, such as GC-MS, are used to quantify fatty acid species with precision, but the positional information of acyl chains has not been resolved \u003csup\u003e[12]\u003c/sup\u003e. Although UPLC-TOF-MS based lipidomics has been recognized to offer advantages in throughput and sensitivity, clear limitations have also been noted in structural elucidation of lipids, discrimination of isomers, cost efficiency, and dynamic range, with particular difficulties in distinguishing structural isomers. The limitations of existing single techniques have been recognized to drive the integration of multi-scale approaches, by which new opportunities have been provided for in-depth elucidation of lipid structure-function relationships. MALDI-TOF-MS has been applied for the characterization of nearly all lipid classes, including nonpolar lipids such as triacylglycerols (TAG) \u003csup\u003e[13]\u003c/sup\u003e. The regional isomeric distribution can be rapidly localized by \u003csup\u003e1\u003c/sup\u003eH NMR through the splitting patterns of glycerol backbone protons, and quantitative analysis can be performed with simple sample preparation without the need for derivatization, standards, or calibration curves \u003csup\u003e[14]\u003c/sup\u003e. The combined application of these two techniques is expected to reveal the essential characteristics of yolk lipid structures that can be regulated by dietary interventions across molecular, mesoscopic, and macroscopic dimensions.\u003c/p\u003e\u003cp\u003eBased on this rationale, eggs produced from hens fed with DDGS, whole grains, and flaxseed were selected as the experimental objects, and a multi-scale strategy combining GC-MS, \u003csup\u003e1\u003c/sup\u003eH NMR, and MALDI-TOF-MS was employed. Lipidomics, structural elucidation, and functional characterization techniques were integrated to investigate the differential mechanisms by which three representative functional diets influenced yolk lipid molecular structures and functional properties. The study was designed to elucidate the specific remodeling effects of diversified diets on yolk lipid molecular structures and to clarify how such structural changes were cascaded to drive the differentiation of key functions, including emulsification, oxidative stability, and nutritional digestibility. The experimental evidence was provided for the precise design of functional-egg diets, nutritional targeting, and product development. While support was also offered for the transformation and upgrading of the poultry egg industry from \u0026ldquo;compositional modification\u0026rdquo; to \u0026ldquo;structural empowerment\u0026rdquo; and \u0026ldquo;functional customization.\u0026rdquo;\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Sample Preparation\u003c/h2\u003e\u003cp\u003eThe experiment employed a single-factor completely randomized design. A total of 1,200 healthy laying hens (28 weeks old) (purchased from Sichuan Shengdile Village Ecological Food Co., Ltd., Mianyang, China) were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;400 per group) and fed the following diets:\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe composition and chemical composition of the basic diet of egg chickens\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName of raw material (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean Meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn Gluten Meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn DDGS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlaxseed Meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean Oil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePremix(Vitamin \u0026amp; Mineral Premix)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: T1-T3 represented the corn DDGS group, corn-soybean meal group, and flaxseed group respectively.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe composition of the basal diets was presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The experimental period was 8 weeks with ad libitum access to feed and water. Eggs (50 eggs/group) were collected from each group on day 56 of the trial for subsequent use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Emulsifying Properties\u003c/h2\u003e\u003cp\u003eThe emulsifying activity index (EAI) and emulsion stability index (ESI) were determined according to the method described by Sun et al. \u003csup\u003e[15]\u003c/sup\u003e with modifications. A 1% yolk powder solution (prepared in pH 7.0 PBS buffer) was homogenized with soybean oil (oil-to-water ratio of 1:4) at 10,000 rpm for 1 min. EAI was expressed as the percentage of emulsion height over total dispersion height. For ESI measurement, the samples were heated to 80\u0026deg;C for 30 min, cooled, and then centrifuged prior to analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Particle Size and Zeta Potential\u003c/h2\u003e\u003cp\u003eThe yolk solutions (1%) were diluted 1:200 with distilled water and filtered through a 0.45 \u0026micro;m membrane. The particle size distribution (Z-average), polydispersity index (PDI), and zeta potential were measured by dynamic light scattering (Malvern Zetasizer Nano ZS) at 25\u0026deg;C. Each sample was tested in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Malondialdehyde (MDA) Content\u003c/h2\u003e\u003cp\u003eMDA content was measured using a commercial assay kit to evaluate lipid oxidation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cholesterol Content\u003c/h2\u003e\u003cp\u003eFollowing the method of Laila \u0026amp; Putra et al. \u003csup\u003e[16]\u003c/sup\u003e with minor modifications. A 3 g portion of yolk homogenate was weighed and placed into a 25 mL colorimetric tube, dissolved with distilled water, and diluted to the mark before being mixed thoroughly to obtain a yolk dilution. An aliquot of 1 mL of the yolk dilution was accurately transferred into a 50 mL beaker, followed by the addition of 3 mL of 10% potassium hydroxide solution and 10 mL of anhydrous ethanol solution, and the mixture was stirred. Direct saponification was carried out in a 60\u0026deg;C water bath for 1 h (stirred once every 15 min), after which the mixture was removed. After cooling, 50 mL of petroleum ether was added and mixed with a glass rod for extraction, and the supernatant was transferred into a separatory funnel. The supernatant was repeatedly washed three times with distilled water, and the total volume was measured. A 0.5 mL portion was then taken into a small beaker, evaporated to dryness in a 60\u0026deg;C water bath, and the residue was dissolved in 2 mL of methanol solution and 2 mL of ferric chloride\u0026ndash;phosphoric acid color reagent. After cooling to room temperature, the yolk dilution was replaced by solvent as the blank, and absorbance was measured at 550 nm using a microplate reader. Cholesterol content was calculated according to the following formula (1).\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{Cholesterol\\:content\\:(mg/100g)=}\\frac{\\text{C\u0026times;4\u0026times;2\u0026times;V\u0026times;25\u0026times;100}}{\\text{W\u0026times;1000}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere C represented the cholesterol concentration in the extract (\u0026micro;g/mL), V represented the volume of the extract (mL), and W represented the mass of the homogenized egg yolk (g).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 In Vitro Digestibility of Fatty Acids\u003c/h2\u003e\u003cp\u003eGastrointestinal digestive fluids were prepared according to the method of Martos et al. \u003csup\u003e[17]\u003c/sup\u003e. 1 g portion of yolk sample intestinal digest was weighed, and an appropriate amount of anhydrous sodium sulfate was added. Subsequently, 50 mL of petroleum ether (30\u0026ndash;60\u0026deg;C) was introduced, mixed thoroughly, and left to stand overnight. The mixture was then filtered, and 25 mL of the filtrate was collected. To the filtrate, 50 mL of anhydrous ethanol was added, followed by the addition of 2\u0026ndash;3 drops of phenolphthalein. Titration was carried out with 0.01 mol/L NaOH solution. The degree of fat digestion was characterized by the amount of free fatty acids, and the free fatty acid content was calculated according to the following formula (2).\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\text{Free\\:fatty\\:acid\\:(NaOH/g)=4\u0026times;}\\frac{\\text{A\u0026times;F}}{\\text{m}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere A represented the titrant volume, mL; F represented the titrant concentration, 0.01 mol/L; m represented the egg yolk sample mass, g.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Fatty Acid Composition\u003c/h2\u003e\u003cp\u003eAn appropriate amount of egg-yolk powder was mixed with 90 mL of chloroform-methanol (v:v\u0026thinsp;=\u0026thinsp;2:1), and ultrasonic extraction was carried out for 20 min. The mixture was then filtered, and the filtrate was evaporated to dryness using rotary evaporation. Subsequently, the petroleum ether and anhydrous sodium sulfate were added for a second extraction. The extract was centrifuged at 3000 r/min for 5 min, the ether phase was collected, and the petroleum ether was removed by evaporation. Fatty acid methyl esters (FAMEs) was prepared following Watkins et al. \u003csup\u003e[18]\u003c/sup\u003e. 300 mg of yolk oil was saponified with NaOH-methanol at 60\u0026deg;C for 30 min, and this was followed by methylation with boron trifluoride-methanol at 60\u0026deg;C for 30 min. After cooling, n-hexane and saturated NaCl were added, and the upper ether phase was collected for GC-MS analysis. GC-MS was performed on an Agilent 7890B-5977B system with an HP-88 column (60 m \u0026times; 0.25 mm \u0026times; 0.2 \u0026micro;m). Helium was used as the carrier gas (0.8 mL/min), injection volume was 1 \u0026micro;L (split 10:1), and the oven program ranged from 40\u0026deg;C to 250\u0026deg;C with stepwise increments. Fatty acids were quantified by peak area normalization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Lipid Molecular Species\u003c/h2\u003e\u003cp\u003eLipid analysis was performed using MALDI-TOF-MS (Bruker UltrafleXtreme) \u003csup\u003e[19]\u003c/sup\u003e. The matrix solution was prepared with 2,5-dihydroxybenzoic acid (DHB, 10 mg/mL dissolved in 70% acetonitrile\u0026ndash;0.1% TFA). 1 mg portion of yolk powder was mixed with the matrix solution (1:1, v/v), spotted onto the target, and dried. Data were acquired in the positive ion reflectron mode within the m/z range of 500\u0026ndash;1000, with a laser energy of 25% and an accumulation of 500 shots. Lipid molecular species were identified by matching m/z values to the LIPID MAPS database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.lipidmaps.org\u003c/span\u003e\u003cspan address=\"http://www.lipidmaps.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with a mass error of \u0026plusmn;\u0026thinsp;0.02 Da. The triacylglycerols (TG) and phospholipids (PC/SM) were verified through characteristic fragment ions. Relative abundances within the m/z range of 400\u0026ndash;1000 were normalized to the total ion current intensity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 sn-Position of Fatty Acids\u003c/h2\u003e\u003cp\u003eThe acyl positions of lipids were analyzed by \u003csup\u003e1\u003c/sup\u003eH NMR (Bruker Avance III HD 600 MHz) \u003csup\u003e[20]\u003c/sup\u003e. A 50 mg portion of yolk powder was dissolved in 0.6 mL of CDCl₃, centrifuged, and the supernatant was transferred into a 5 mm NMR tube. The sampling parameters were set at 298 K with 64 scans and a spectral width of 12 ppm. All spectra were processed in the same manner using MestReNova software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Statistical Analysis\u003c/h2\u003e\u003cp\u003eThe result data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way analysis of variance (ANOVA) was performed using SPSS 26.0 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Principal component analysis (PCA) was carried out in MetaboAnalyst, where lipid composition (GC-MS data) and functional properties were dimensionally reduced after UV standardization, and the principal component loading matrix was extracted. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Effects of Different Diets on Yolk Emulsifying Activity and Emulsion Stability\u003c/h2\u003e\u003cp\u003eEmulsifying activity (EAI) and emulsion stability (ESI) are the core functional attributes of egg yolk as natural emulsifiers and directly determine its performance in food applications. These interfacial properties are largely determined by lipid molecules, particularly by the behavior of phospholipids and lipoproteins at the oil\u0026ndash;water interface. In addition, these properties are influenced by lipid composition (degree of saturation and phospholipid classes) and molecular conformation (cis/trans isomerism) \u003csup\u003e[21]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the EAI of corn DDGS eggs (EY1, 0.28) was found significantly higher than that of other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas no significant difference was observed between whole-grain eggs (EY2) and flaxseed eggs (EY3) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). It was noted that the emulsifying stability of all three dietary groups remained at a relatively high level, but the differences among groups were not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This phenomenon may be explained by the convergence of interfacial membrane stability, which was maintained primarily through the combined effects of phospholipids and apolipoproteins on the mechanical strength of the interface.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eParticle size served as a key parameter describing the size of lipoprotein aggregates or emulsion droplets in yolk solutions, while zeta potential was a reliable indicator of surface charge density and interactions between proteins and emulsifiers \u003csup\u003e[22]\u003c/sup\u003e. Greater uniformity (lower polydispersity index, PDI) and higher surface charge magnitude (larger absolute zeta potential) generally contributed to improved physical stability of emulsions \u003csup\u003e[23]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, the zeta potential values of the three yolk solutions were all negative, suggesting that negative charges were adsorbed onto the particle surfaces in the emulsions. Among them, EY1 exhibited the narrowest particle size distribution, the smallest particle size (3500 nm), and the lowest PDI (0.34), with a zeta potential absolute value of 12.5 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In contrast, EY2 showed the widest particle size distribution, a relatively larger average particle size (3550 nm), and the highest PDI (0.38). The peak of EY3 was located in the middle with a moderate distribution range; although its average particle size was the largest (3600 nm), the absolute value of its zeta potential was the highest at 12.8 mV. Taken together, EY1 yolk particles were evaluated to exhibit better monodispersity and higher solution stability. EY2 particles were found to be more dispersed and prone to flocculation or stratification, while EY3 demonstrated the best solution stability. The observed differences in stability might be attributed to the intrinsic properties of fatty acids, such as headgroup polarity, charge, and their accumulation within the interfacial protein layer. In addition, surface charge could also be influenced by the contents of proteins adsorbed at the interface, implying that yolks richer in long-chain unsaturated fatty acids might alter protein adsorption behavior and consequently shift zeta potential values \u003csup\u003e[24]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBased on the particle size and stability results of yolk solutions, the smallest emulsion particles with the highest emulsifying activity were formed by EY1. In contrast, the particles of EY2 were found to be heterogeneous in size, and the emulsions were less uniformly dispersed. The emulsion particles formed by EY3 yolk exhibited the highest surface charge density and were evaluated to possess the greatest stability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effects of Different Diets on Yolk Oxidative Stability and Fatty Acid Digestibility\u003c/h2\u003e\u003cp\u003eLipid oxidation is a key factor leading to quality deterioration of yolks and yolk-based products, resulting in undesirable flavors, discoloration, and loss of nutritional value. Malondialdehyde (MDA), a secondary product of lipid peroxidation, was measured as an indicator of oxidative stress and lipid oxidation levels \u003csup\u003e[25]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, significant differences in MDA concentrations were observed among the dietary groups. Specifically, EY1 exhibited the highest value (3.3981 nmol/g), followed by EY2 (3.03718 nmol/g), while EY3 had the lowest concentration (2.43629 nmol/g), indicating superior oxidative stability in flaxseed-fed yolks. Previous studies have shown that yolks are highly susceptible to oxidation due to their elevated fat content. A high proportion of PUFA, particularly long-chain PUFA containing multiple double bonds, was especially prone to oxidative reactions \u003csup\u003e[26]\u003c/sup\u003e. However, flaxseed was naturally rich in lignans and other antioxidants that could scavenge free radicals and interrupt chain reactions, thereby protecting yolk lipids from oxidation \u003csup\u003e[27]\u003c/sup\u003e. Therefore, differences in oxidative stability were attributed not only to PUFA levels and species but also to their positional distribution (sn-position) and local molecular environment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFatty acid digestibility served as a core indicator of yolk nutritional value, as it directly determined lipid bioavailability \u003csup\u003e[28]\u003c/sup\u003e. The composition of lipid molecules, including glyceride configuration, phospholipid distribution, and fatty acid chain characteristics, was profoundly influenced by the digestive process through the regulation of emulsion stability, enzymatic hydrolysis efficiency, and the absorption interface \u003csup\u003e[29]\u003c/sup\u003e. Significant differences in the simulated gastrointestinal digestibility of fatty acids among the three yolks were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The highest digestibility was measured in EY3 (93.15%), followed by EY2 (90.52%), whereas the lowest value was detected in EY1 (84.79%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Since fatty acids in foods are primarily present in triacylglycerols (TG), medium-chain fatty acids were hydrolyzed more rapidly than long-chain fatty acids. Under the action of intestinal lipase, hydrolysis of lipids is mainly carried out at the sn-1,3 positions of TG, whereas sn-2 fatty acids were usually absorbed directly in the form of sn-2 monoacylglycerols, with potentially higher efficiency of digestion and absorption \u003csup\u003e[30]\u003c/sup\u003e. In addition, the accessibility of digestive enzymes was influenced by droplet size and interfacial composition, such as phospholipids and proteins \u003csup\u003e[31]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn summary, the oxidative stability and the release rate of fatty acids during the in vitro simulated digestion of yolk lipids were significantly influenced by dietary composition. Given that lipid digestive efficiency was highly dependent on sn-2 positional fatty acids, it was hypothesized that the high digestibility of EY3 might be associated with the specific enrichment of PUFA at the sn-2 position.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effects of Different Diets on Yolk Fatty Acid Composition and Cholesterol Content\u003c/h2\u003e\u003cp\u003eFatty acid composition and cholesterol content represented core components of yolk quality, as they determined both nutritional value and functional properties. Dietary composition served as a major external factor shaping the yolk lipid profile, since fatty acids from feed can be directly deposited in yolk or modified during metabolism before deposition \u003csup\u003e[32]\u003c/sup\u003e. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, total saturated fatty acids (SFA) were highest in EY3 (59.5%), significantly greater than in EY2 (35.4%) and EY1 (18.6%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The SFA content in the three yolk samples was dominated by palmitic acid (PA, C16:0) and stearic acid (SA, C18:0), with the highest levels observed in EY3, where PA and SA were found to be 31.539\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 mg/g and 9.273\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 mg/g, respectively. For monounsaturated fatty acids (MUFA), the total content was found to be highest in EY1 (61%), significantly higher than in EY2 (41.7%) and EY3 (1.7%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the content of trans oleic acid (C18:1n-9t) in EY1 was notably high (12.665\u0026thinsp;\u0026plusmn;\u0026thinsp;0.108 mg/g), which may be associated with the metabolic characteristics of Corn DDGS. Corn DDGS was recognized as being rich in protein, fat, and fiber, and its fat composition was characterized by a high content of unsaturated fatty acids. However, partial isomerization may be induced during processing under high-temperature conditions, leading to the formation of trans fatty acids \u003csup\u003e[33]\u003c/sup\u003e. Regarding PUFA content, the highest total content was determined in EY3 (38.8%), significantly higher than in EY2 (22.9%) and EY1 (20.4%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In particular, docosahexaenoic acid (DHA) (2.422\u0026thinsp;\u0026plusmn;\u0026thinsp;0.114 mg/g) was found to be significantly higher in EY3 than in the other two egg groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, EY3 exhibited the lowest n-6/n-3 PUFA ratio, which was associated with improved antioxidant status and reduced lipid peroxidation \u003csup\u003e[34]\u003c/sup\u003e. This result was consistent with studies reported in the literature, which suggested that the addition of flaxseed to the diet improved the egg health lipid index \u003csup\u003e[35]\u003c/sup\u003e. It was noted that the value of directly supplementing human diets with whole flaxseed or flaxseed oil was considered limited \u003csup\u003e[36]\u003c/sup\u003e. In contrast, eggs enriched in long-chain n-3 PUFA could be obtained by supplementing laying hens with flaxseed, thereby providing a source of nutritional supplementation.\u003c/p\u003e\u003cp\u003eSignificant differences were also observed in yolk cholesterol levels. The order of cholesterol content was EY1 (1220 \u0026micro;g/g)\u0026thinsp;\u0026gt;\u0026thinsp;EY3 (1090 \u0026micro;g/g)\u0026thinsp;\u0026gt;\u0026thinsp;EY2 (840 \u0026micro;g/g). The diet of EY2 was mainly composed of corn (63.03%) and soybean meal (21.4%), without the addition of specific ingredients such as flaxseed meal or corn DDGS. As a result, the fatty acid composition was relatively simple and dominated by cis fatty acids, with a relatively lower energy density, and the metabolic products of the feed were less likely to promote cholesterol synthesis. In addition, soybean meal was known to be rich in phytosterols such as β-sitosterol, whose structure was similar to that of cholesterol \u003csup\u003e[37]\u003c/sup\u003e. By occupying the NPC1L1 intestinal transporter, the absorption and conversion rate of cholesterol was thereby reduced, which was considered one of the reasons for the lowest cholesterol content observed in EY2.\u003c/p\u003e\u003cp\u003eThe above nutritional results confirmed that yolk core components, including the fatty acid profile and cholesterol, were remodeled by dietary composition. A high content of MUFA (particularly oleic acid) was detected in EY1, which was associated with enhanced flexibility of the lipoprotein interface (with the highest emulsifying activity observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In EY2, the lowest cholesterol level was achieved through its lower energy density and the inhibitory effect on cholesterol conversion. In EY3, a high content of beneficial n-3 PUFA (especially DHA) together with the lowest n-6/n-3 ratio was measured, conferring improved oxidative stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComponent content of egg yolk in three kinds of diet\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType of fatty acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEY1 (mg/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEY2 (mg/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEY3 (mg/g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC15:0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.023\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.061\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC16:0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.237\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.853\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.539\u0026thinsp;\u0026plusmn;\u0026thinsp;0.283a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC17:0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.057\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC18:0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.052\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.273\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC20:0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.142\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.766\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSFA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.958\u0026thinsp;\u0026plusmn;\u0026thinsp;0.499C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.703\u0026thinsp;\u0026plusmn;\u0026thinsp;1.066b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42.603\u0026thinsp;\u0026plusmn;\u0026thinsp;1.796a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC16:1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.226\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.177\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.734\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC17:1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.068\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.063\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.212\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC18:1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.118\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.808\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.265\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC18:1n-9t\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.665\u0026thinsp;\u0026plusmn;\u0026thinsp;0.103a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.131\u0026thinsp;\u0026plusmn;\u0026thinsp;0⁶\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC18:1n-9c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.075\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMUFA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26.152\u0026thinsp;\u0026plusmn;\u0026thinsp;0.138a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.048\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.343\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC18:2 n-6c (LA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.124\u0026thinsp;\u0026plusmn;\u0026thinsp;0.112c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.486\u0026thinsp;\u0026plusmn;\u0026thinsp;0.221b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.465\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC20:2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.082\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.062\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC20:3 n-6 (DGLA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.068\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.075\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.162\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC20:4 n-6 (ARA)\u003c/p\u003e\u003cp\u003e(Arachidonic acid)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.179\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.166\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.193\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC22:4 n-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.031\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.051\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.226\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC22:5 n-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.015\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.053\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.269\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC22:6n-3 (DHA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.263\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0136b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.212\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.422\u0026thinsp;\u0026plusmn;\u0026thinsp;0.114a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePUFA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.763\u0026thinsp;\u0026plusmn;\u0026thinsp;0.114c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.106\u0026thinsp;\u0026plusmn;\u0026thinsp;0.247b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.814\u0026thinsp;\u0026plusmn;\u0026thinsp;0.094a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003echolesterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.857c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.774b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 MALDI-TOF-MS Identification of Yolk Lipids from Different Diets\u003c/h2\u003e\u003cp\u003eThe diversity of lipid molecules in egg yolk directly influenced interfacial behavior, oxidative sensitivity, and digestive dynamics \u003csup\u003e[38]\u003c/sup\u003e. Lipidomic profiling of yolks from the three dietary groups was performed using MALDI-TOF-MS. The spectral data were annotated and normalized through the LIPID MAPS database (v2.3). In positive-ion mode, lipid spectra were recorded with a detection threshold set at relative intensity (Rel. Intens.)\u0026thinsp;\u0026gt;\u0026thinsp;0.01. A total of 189 lipid species belonging to 12 major classes were identified, forming a framework that encompassed core categories such as phospholipids (PLs) and glycerolipids (GLs).\u003c/p\u003e\u003cp\u003eThe overall relative composition of lipids across the three yolk types was shown in the heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with radar plots illustrating relative abundance patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The relative abundance of lipids was reflected by red and blue colors, with large differences observed between PLs and GLs. In EY1, relatively higher levels of medium-chain PC and PE were detected, which were highly consistent with its optimal ESI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These lipid molecules were considered to play a central role in constructing stable emulsifying films with low interfacial tension. In EY2, the highest relative levels of phosphatidylserine (PS) and phosphatidic acid (PA) were measured. The negative charge of PS was suggested to cause electrostatic repulsion and membrane instability, which provided a molecular-level explanation for its poorer particle homogeneity (high PDI) and potential oxidative risk (higher MDA). By contrast, long-chain TG molecules were found to be enriched in EY3, leading to interfacial molecular conformations favorable for uniform emulsion distribution. The result was consistent with the earlier inference regarding fatty acids and yolk emulsion stability. At the same time, TG, as the main storage form of neutral lipids, was emphasized to have its sn-positional distribution as a critical factor for digestive and absorptive efficiency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn conclusion, diet was identified as the key factor driving lipid profile differentiation. By MALDI-TOF-MS analysis, characteristic \u0026ldquo;fingerprint\u0026rdquo; lipids of yolks derived from different diets were revealed, and significant differences in yolk lipid composition were confirmed to be induced by dietary factors.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Identification of Phospholipid (PL) Molecular Species in Yolks from Different Diets\u003c/h2\u003e\u003cp\u003eIn terms of the differentiated remodeling of PLs, the relative contents of PE and PC subclasses followed the order EY1\u0026thinsp;\u0026gt;\u0026thinsp;EY2\u0026thinsp;\u0026gt;\u0026thinsp;EY3, whereas the relative content of PS subclasses followed the order EY2\u0026thinsp;\u0026gt;\u0026thinsp;EY1\u0026thinsp;\u0026gt;\u0026thinsp;EY3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The high levels of PC and PE in EY1 were attributed to the inhibition of LPA generation and the reduction of PC degradation by trans fatty acids derived from Corn DDGS (4%). Meanwhile, bile acids were adsorbed by soybean meal fiber (adsorption rate\u0026thinsp;\u0026ge;\u0026thinsp;50%), resulting in the inhibition of lipase activity and the subsequent accumulation of PS as a metabolic intermediate. Consequently, the highest PS content was detected in EY2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe common phospholipid subclasses identified in yolks from different diets included PC(35:8), PC(35:9), PS(36:5), and PE(40:11) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The relative abundance of medium-chain PC was found to be the highest in EY1, which was attributed to the synergistic metabolic effects of the high-corn formulation and Corn DDGS. A significant accumulation of PS(36:5) in EY2 directly reflected the metabolic imprint of ω-6 linoleic acid from the corn\u0026ndash;soybean diet, consistent with the conservative deposition pattern of fatty acids in grain-based diets \u003csup\u003e[39]\u003c/sup\u003e. In EY3, the phospholipid distribution was characterized by a \u0026ldquo;dispersed low-energy\u0026rdquo; pattern, with lower relative intensities compared to the other two yolk groups. This phenomenon was suggested to result from the activation of the phospholipase A2 (PLA2) pathway by flaxseed supplementation, which promoted phospholipid hydrolysis and the release of free DHA. However, due to the inhibited expression of endogenous phospholipid synthases in the yolk, DHA was more likely to be stored in the form of neutral lipids \u003csup\u003e[40]\u003c/sup\u003e. The three most abundant phospholipids across yolks were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. Notably, phosphatidic acid (PA 51:10) was detected only in EY3. As the simplest glycerophospholipid, PA is an essential signaling molecule in both intracellular and extracellular pathways, which further indicated that lipid metabolic activity was elevated in EY3 and may be related to flaxseed-mediated regulation of lipase activity.\u003c/p\u003e\u003cp\u003eOverall, high levels of medium-chain PC and PE were formed in EY1 yolk, leading to enhanced emulsion stability. In EY2, the accumulation of PS was found to exacerbate oxidative stress, whereas in EY3, DHA was driven toward storage in TG, resulting in dual benefits of efficient neutral lipid incorporation of DHA and improved membrane oxidative stability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Identification of Glycerolipid (GL) Molecular Species in Yolks from Different Diets\u003c/h2\u003e\u003cp\u003eIn terms of glycerolipids, the relative abundance of triacylglycerols (TGs) followed the order EY3\u0026thinsp;\u0026gt;\u0026thinsp;EY1\u0026thinsp;\u0026gt;\u0026thinsp;EY2, and diacylglycerols (DGs) were most abundant in EY1, followed by EY2 and then EY3. The total TG content in EY3 reached 71.5%, in which strong signals of TG(51:8; O3) (C16:0-C22:6-C22:6) and TG(51:9) were identified, confirming the efficient deposition of DHA in TG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This result was consistent with the preferential incorporation of flaxseed-derived fatty acids into TG, corresponding to the characteristic profile of EY3 with high TG/DG and low PC/PE. It was also indicated that hens utilized available precursor ALA for the biosynthesis of EPA and DHA through the activity of hepatic FA desaturases and elongases \u003csup\u003e[41]\u003c/sup\u003e. Notably, DHA contains six double bonds and was theoretically more susceptible to oxidation than common PUFA. However, the oxidized DG(55:13) in EY3 showed a relative content of only 0.08, one quarter of that in grain eggs (0.34), consistent with the results of oxidative stability. In contrast, the accumulation of oxidized glycerides in EY2 suggested that the corn\u0026ndash;soybean diet lacked exogenous antioxidants, resulting in the upregulation of lipoxygenase (LOX) activity and triggering a lipolysis\u0026ndash;oxidation cascade \u003csup\u003e[342\u003c/sup\u003e Furthermore, the preferential deposition of DHA in TG rather than in DHA-PC/PE in EY3 revealed the limitation of yolk phospholipidization capacity. In EY1, the proportions of TG(51:9) (0.79) and TG(51:8) (0.69) were relatively high, which was likely attributed to corn DDGS in the diet (rich in ω-6 linoleic acid) and fatty acid deposition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFrom the analysis of GLs, DHA enrichment in yolk TG was driven by flaxseed. TG(51:8) was suggested to contain three DHA molecules (C22:6), indicating that EY3 may contain approximately 150\u0026ndash;200 mg of DHA, reaching the high-end standard of the industry. In the corn-soybean diet, the absence of exogenous antioxidants resulted in the activation of adipose triglyceride lipase (ATGL) and lipoxygenase (LOX), leading to the accumulation of oxidized TG in EY2. In EY1, the high level of n-6 linoleic acid (C18:2n6) was preferentially incorporated into TG, with significant expression of TG(51:9) and TG(51:8), thereby forming a conservative deposition pattern of n-6 PUFA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Differential Lipid Analysis of Egg Yolks from Different Diets\u003c/h2\u003e\u003cp\u003eIn the principal component analysis (PCA) plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), the samples of the three types of eggs were displayed along PC1 (explaining 68% of the variance) and PC2 (explaining 17.6% of the variance). The lipid samples of the three egg yolks were clearly separated in the plot, and the intergroup separation was further emphasized, suggesting that the lipid composition of egg yolks was affected by the diet, thereby allowing different types of eggs to be distinguished at the lipid level. The partial least squares discriminant analysis (PLS-DA) of differential lipids was employed as the central approach through which the logic chain of dietary intervention-lipid metabolism remodeling-functional differentiation was elucidated. As shown in the PLS-DA plot of differential lipids in egg yolks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), lipids with VIP values greater than 1, including LPS, PC, and PE classes, were identified as the key biomarkers distinguishing the three egg sources. It was indicated that the differences among the sample groups were mainly driven by phospholipids, which might be related to the regulation of oxidative levels, emulsion stability, and membrane fluidity, whereas the contribution of ether lipids was relatively low.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePairwise comparisons of lipid abundances across groups were further visualized by volcano plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Each point was used to represent a lipid molecule, with purple points being used to indicate downregulated lipids, orange points being used to indicate upregulated lipids, and gray points being used to indicate lipids without significant differences. The horizontal axis was designated to represent the log\u003csub\u003e2\u003c/sub\u003eFC value, while the vertical axis was designated to represent the p-value. The greater the values along both axes were, the more significant the changes in the selected lipid species were considered to be.\u003c/p\u003e\u003cp\u003eFor the EY3 and EY2 groups, lipid biosynthesis and ether-linked modifications were detected to be enhanced in EY3, which implied a potential elevation of antioxidant capacity. When EY3 was compared with EY1, an increase in lipid synthesis-particularly of glycerolipids and ether phospholipids was detected, accompanied by an improvement in membrane stability. In the comparison of EY1 and EY2, basal metabolic lipids (short-chain glycerolipids) were found to be predominant in EY1, with a relatively higher proportion of high-quality phospholipids, thereby confirming its favorable emulsifying activity. Interestingly, in the differential analysis between EY3 and the other two types of eggs, TG(51:8) was consistently identified as being significantly upregulated, indicating that EY3 might possess unique characteristics in fat storage or energy metabolism.\u003c/p\u003e\u003cp\u003eThe egg yolk lipid architecture of the three egg types was directly remodeled by dietary intervention, resulting in markedly distinct lipid fingerprints. The intergroup variations were predominantly driven by polyunsaturated phospholipids. In addition, TG(51:8) was identified as a characteristic biomarker among the groups, providing a key target for the lipid engineering design of flaxseed eggs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Determination of Fatty Acid sn-Position in Yolk Lipids from Different Diets\u003c/h2\u003e\u003cp\u003eThe complementarity of \u0026sup1;H NMR and MALDI-TOF-MS was evidenced, showing that dietary components differentially affected egg yolk lipid structures by regulating fatty acid composition, phospholipid distribution, and oxidative stability. Through chemical shifts (ppm) and coupling constants, information on the chemical environment of hydrogen atoms in molecules was provided, allowing direct elucidation of functional groups, stereoisomers, and dynamic structures \u003csup\u003e[43]\u003c/sup\u003e. From Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, it was observed that, apart from the solvent peaks of DMSO (δ 2.51 ppm) and HDO (δ 3.13 ppm), the major lipid classes and specific lipid species present in the three egg yolks were profiled. For the characteristic peaks of TG, the signal at 5.21 ppm was assigned to the CH-O-CO protons of the glycerol backbone, while the signals at δ 1.24 ppm and δ 1.17 ppm were attributed to the -(CH₂)n- methylene chains of long-chain fatty acids. The signal at 0.95 ppm represented the terminal -CH₃ methyl group of fatty acid chains. Since prominent broad peaks were consistently observed at δ 1.25\u0026ndash;1.30 ppm in all three egg yolks, it was demonstrated that TG in the yolks was connected through a glycerol methylene backbone with long-chain fatty acid -(CH₂)n- chains. Specifically, cis FA (δ 2.00-2.10 ppm) and trans FA (δ 1.98\u0026ndash;2.04 ppm) were detected in EY1, while trans FA was absent in EY2, which was consistent with the results of fatty acid composition analysis. In addition, the sn-2 glycerol lipids (δ 5.25\u0026ndash;5.30 ppm) of EY3 exhibited the most intense signal among the three egg types, suggesting the highest relative abundance. These lipids, being bound to long-chain fatty acids, were more readily utilized by the human body, in agreement with the digestibility results. Regarding specific lipids, the characteristic signals of PC were assigned to the choline headgroup [-N(CH₃)₃] (δ 3.13 ppm) and the CH₂-O-PO₄⁻ protons of the phospholipid glycerol backbone (δ 4.28 ppm). Consequently, a higher phospholipid content was indicated in EY1, which may be associated with lipoprotein remodeling regulated by distillers\u0026rsquo; grains, thereby enhancing interfacial charge density and improving emulsifying properties. In EY2, the cholesterol content (δ 0.66 ppm) was the lowest, corroborating the results of lipidomics and compositional analyses. In contrast, the lowest phosphatidylcholine content was observed in EY3, consistent with the GLs lipid analysis. Moreover, for specific fatty acids (DHA/ALA), the DHA signals (δ 0.8\u0026ndash;1.0 ppm) and ALA signals (δ 1.24 ppm) in EY3 were more prominent.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn summary, direct evidence for key structural features such as the sn-2 position, trans configuration, and phospholipid headgroups was revealed by \u0026sup1;H NMR, forming a complete chain of mutual validation with lipidomic and functional data. In EY1, the highest phosphatidylcholine content (δ 3.13, 4.28 ppm) was identified, which was suggested to enhance emulsifying properties by increasing interfacial charge density. In EY2, no trans fatty acids were detected, while cholesterol (δ 0.66 ppm) was found to be the lowest. In EY3, the strongest lipid signals at the sn-2 position (δ 5.25\u0026ndash;5.30 ppm) were detected, pointing to abundance of long-chain fatty acids such as DHA at this position, closely associated with its higher digestibility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Correlation Analysis of Diet Composition, Lipid Structure, and Functional Properties\u003c/h2\u003e\u003cp\u003eThe directional design of dietary components was implemented to precisely regulate the molecular structures of yolk lipids, thereby cascade-driving the coordinated changes in functional properties such as nutritional value, emulsifying stability, and oxidative stability. The DDGS diet was evidenced to enhance the enrichment of trans-oleic acid (C18:1n9t) in PC, and the high MUFA content was identified to markedly improve the emulsifying activity of EY1 by reducing interfacial tension. The flaxseed diet was demonstrated to direct DHA to be specifically located at the sn-2 position of triglycerides (as confirmed by \u0026sup1;H NMR), where selective hydrolysis of the sn-1,3 positions by pancreatic lipase released sn-2 monoglycerides, thereby improving the digestibility of EY3. The soybean meal diet was found to inhibit cholesterol absorption due to plant sterols, while abnormal accumulation of phosphatidylserine (PS) was identified in EY2, whose high charge density induced electrostatic repulsion, ultimately decreasing emulsion stability. Correlation analysis was employed as a key approach to elucidate the intrinsic associations among multidimensional yolk indicators under diversified dietary regulation. The results demonstrated the core finding of this study that the remodeling of lipid molecular structures drove functional differentiation. In Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, significant correlations among variables were clearly presented. A very strong positive correlation was observed between emulsifying activity and MUFA (r\u0026thinsp;=\u0026thinsp;0.88), while a strong positive correlation was also observed between emulsifying activity and PUFA (r\u0026thinsp;=\u0026thinsp;0.84), which was in accordance with the role of long-chain PUFAs (e.g., DHA) in flaxseed eggs in improving interfacial fluidity. However, a strong negative correlation was observed between emulsifying activity and SFA (r = -0.88), highlighting the inhibitory effect of droplet crystallization tendency induced by high SFA on interfacial behavior, which offset part of the positive effect of PUFA. Emulsifying activity was driven by MUFA through the optimization of lipoprotein interfacial properties, corroborating the mechanism by which MUFA significantly enhanced emulsification capacity via improved flexibility and spreadability of LDL interfacial membranes. In contrast, phospholipid charge repulsion (high PS) and structural disorder in grain-based eggs resulted in larger droplet size and higher PDI (0.38), markedly weakening emulsification performance. Moreover, the specific enrichment of PUFA at the sn-2 position was identified as the key factor simultaneously enabling efficient digestion and absorption (positive correlation) and ultra-low oxidation (negative correlation). The phospholipid profiles revealed by lipidomics and the structural resolution were demonstrated to serve as a mechanistic bridge linking the intrinsic biochemical relationships among variables.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOverall, dietary components were evidenced to remodel yolk lipid molecular structures, thereby driving the directional reconstruction of lipid metabolic networks and differentially regulating three major functional properties. Nutritional characteristics were dominated by the positional distribution of fatty acids at the sn-2 site, which determined bioavailability. Emulsifying properties were regulated by the charge of PC/PE headgroups and molecular order, which in turn affected interfacial stability. Oxidative stability was determined by the storage form of PUFAs in long-chain triglycerides as well as the cooperation of endogenous antioxidant systems.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, mechanisms diversifying diets (DDGS, cereals, flaxseed) specifically remodeled yolk lipid molecular structures to drive functional differentiation were elucidated using multi-scale integrated techniques. In EY1, the enrichment of MUFA (61%) and trans-oleic acid (C18:1n9t, 12.665 \u0026micro;g/g) was identified, leading to the formation of a layered structure of medium-chain phosphatidylcholine (PC 35:8). Although this structure was associated with peak emulsifying activity, a significant increase in malondialdehyde content (MDA 3.40 nmol/g) was also observed, indicating that the system became markedly more sensitive to oxidative reactions. In EY2, abnormal accumulation of PS (62.3%) was detected, leading to reduced interfacial stability, while the competitive inhibition by dietary phytosterols resulted in the lowest cholesterol content (0.84 mg/g). In EY3, DHA was shown to be preferentially located at the sn-2 position of triglycerides, producing synergistic effects that enabled high digestibility (93.15%) and low oxidation, although SFA accumulation (59.5%) was found to weaken emulsifying activity. Furthermore, by dissecting the mechanisms through which lipid structures influenced functional properties, the enrichment of PUFAs at the sn-2 position was identified as the core pathway for improving digestion and absorption. In addition, phospholipid profiles were shown to govern emulsifying stability by regulating interfacial charge and membrane strength, whereas trans configurations and lipid densification were identified as the key inducers of oxidative susceptibility and digestive inhibition. Finally, correlation analysis confirmed that lipid molecular structures were the fundamental driving force of functional differentiation. This study therefore provided new targets for the design of functional eggs based on \u0026ldquo;structure-enabled\u0026rdquo; strategies, and suggested that future optimization could be achieved by adjusting dietary composition to enhance sn-2 PUFA positioning and eliminate trans fats, thereby overcoming the bottleneck in the coordinated improvement of nutritional utilization and oxidative stability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cb\u003eCRediT authorship contribution statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eXiaodan Zhang\u003c/b\u003e: Writing-original draft, Investigation, Data curation; \u003cb\u003eXuan Ji\u003c/b\u003e: Methodology, Investigation; \u003cb\u003eWenqian Guan\u003c/b\u003e: Methodology, Formal analysis; \u003cb\u003eSharina Qi\u003c/b\u003e: Validation, Data curation; \u003cb\u003eShijian Dong\u003c/b\u003e: Methodology, Data curation; \u003cb\u003eJijun Wu\u003c/b\u003e: Resources, Validation; \u003cb\u003eYing Gao\u003c/b\u003e: Formal analysis, Writing-review \u0026amp; editing; \u003cb\u003eShugang Li\u003c/b\u003e: Supervision, Resources, Project administration, Funding acquisition, Conceptualization, Writing-review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\u003cp\u003eThese 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.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe work is supported by Project of National Key Research and Development Program of China (No.2022YFD2101001), the Earmarked Fund for China Agriculture Research System (CARS-40-K25, CARS-40-S11), the Project of National Natural Science Foundation of China (No. 32172226), the Special Fund for Anhui Agriculture Research System (AHCYJSTX-NCPJG-15), the Cooperative Projects of Hefei University of Technology-Anhui Rongda Food Co. Ltd. (No. W2020JSKF0489).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**Xiaodan Zhang:** Writing-original draft, Investigation, Data curation; **Xuan Ji:** Methodology, Investigation; **Wenqian Guan:** Methodology, Formal analysis; **Sharina Qi:** Validation, Data curation; **Shijian Dong:** Methodology, Data curation; **Jijun Wu:** Resources, Validation; **Ying Gao:** Formal analysis, Writing-review \u0026amp; editing; **Shugang Li** : Supervision, Resources, Project administration, Funding acquisition, Conceptualization, Writing-review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eM. Salahuddin, A. A. A. Wareth, K. A. Rashwan, et al. The role of egg-derived nutrients in Alzheimer’s disease: Exploring potential benefits and biological insights. Food Bioscience 2024, 62, 105096. https://doi.org/10.1016/j.fbio.2024.105096\u003c/li\u003e\n\u003cli\u003eM. I. Sultana, B. T. Molla, S. Bae, et al. Isolation, characterization, proteome, miRNAome, and the embryotrophic effects of chicken egg yolk nanovesicles (vitellovesicles). Scientific Reports 2023, 13, 4204. https://doi.org/10.1038/s41598-023-31012-0\u003c/li\u003e\n\u003cli\u003eJ. L. Deleu, S. Melis, E. Wilderjans, et al. Protein network formation during pound cake baking: The role of egg yolk and its fractions. Food Hydrocolloids 2017, 63, 226–232. https://doi.org/10.1016/j.foodhyd.2016.07.036\u003c/li\u003e\n\u003cli\u003eC. P. Mezquita, J. Morales, J. Palma, et al. Stability of lutein obtained from Muriellopsis sp. biomass and used as a natural colorant and antioxidant in a mayonnaise-like dressing sauce. CyTA - Journal of Food 2019, 17, 517–526. https://doi.org/10.1080/19476337.2019.1609091\u003c/li\u003e\n\u003cli\u003eR. Suhag. Egg yolk, a multifunctional emulsifier: New insights on factors influencing and mechanistic pathways in egg yolk emulsification. Applied Sciences 2024, 14, 9692. https://doi.org/10.3390/app14219692\u003c/li\u003e\n\u003cli\u003eJ. M. Miranda, X. Antón, C. Redondo-Valbuena, et al. Egg and egg-derived foods: Effects on human health and use as functional foods. Nutrients 2015, 7, 706–729. https://doi.org/10.3390/nu7010706\u003c/li\u003e\n\u003cli\u003eK. Zaheer. An updated review on chicken eggs: Production, consumption, management aspects and nutritional benefits to human health. Food and Nutrition Sciences 2015, 6, 1208–1220. https://doi.org/10.4236/fns.2015.613126\u003c/li\u003e\n\u003cli\u003eO. S. Dudek, M. Pietras, M. Puchała, et al. Camelina sativa oil and camelina cake as sources of polyunsaturated fatty acids in the diets of laying hens: Effect on hen performance, fatty acid profile of yolk lipids, and egg sensory quality. Annals of Animal Science 2020, 20 (4), 1365–1377. https://doi.org/10.2478/aoas-2020-0035\u003c/li\u003e\n\u003cli\u003eD. Mierliță, S. Daraban, A. C. Teușdea, et al. Effect of dietary cold-pressed hempseed cake supplemented with tomato waste on laying hen performance and egg yolk lipid profile and antioxidant status before and after storage. Animals 2024, 14, 3444. https://doi.org/10.3390/ani14233444\u003c/li\u003e\n\u003cli\u003eD. Tadesse, N. Retta, W. Esatu, et al. Fatty acid content and oxidative stability in eggs and breast muscle of Sasso chickens fed different levels of Dodonaea angustifolia polyphenol in flaxseed-enriched diets. Agriculture 2024, 14, 993. https://doi.org/10.3390/agriculture14070993\u003c/li\u003e\n\u003cli\u003eH. Aro, T. Rokka, J. Valaja, et al. Functional and sensory properties of hen eggs with modified fatty acid compositions. Food Function 2011, 2 (11), 671–677. https://doi.org/10.1039/c1fo10132c\u003c/li\u003e\n\u003cli\u003eE. N. S. Doomun, S. Loke, S. O’Callaghan, et al. A simple method for measuring carbon-13 fatty acid enrichment in the major lipid classes of microalgae using GC–MS. Metabolites 2016, 6 (4), 42. https://doi.org/10.3390/metabo6040042\u003c/li\u003e\n\u003cli\u003eA. Najla, C. Elon, K. D. T. Tastet, et al. Fractional factorial design of MALDI-TOF-MS sample preparations for the optimized detection of phospholipids and acylglycerols. Analytical Chemistry 2016, 88 (12), 6301–6308. https://doi.org/10.1021/acs.analchem.6b00512\u003c/li\u003e\n\u003cli\u003eM. C. Francesca, M. Nadia, R. O. Cesare, et al. Triacylglycerol composition and chemical–physical properties of cocoa butter and its derivatives: NMR, DSC, X-ray, rheological investigation. International Journal of Molecular Sciences 2023, 24 (4), 2090. https://doi.org/10.3390/ijms24032090\u003c/li\u003e\n\u003cli\u003eY. Sun, Y. Yang, H. Jin, et al. Ultrasound-assisted pH-shifting treatment: Effects on egg yolk protein interfacial adsorption and emulsification properties. Food Chemistry 2025, 482, 144172. https://doi.org/10.1016/j.foodchem.2025.144172\u003c/li\u003e\n\u003cli\u003eU. Laila, T. J. Putra. The influence of inter-day colorimetric method in quantification of chicken meat total cholesterol with lipid extraction–saponification pretreatment. IOP Conference Series: Materials Science and Engineering 2019, 633, 012048. https://doi.org/10.1088/1757-899x/633/1/012048\u003c/li\u003e\n\u003cli\u003eG. Martos, R. López-Fandiño, E. Molina. Immunoreactivity of hen egg allergens: Influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk. Food Chemistry 2013, 136 (2), 775–781. https://doi.org/10.1016/j.foodchem.2012.07.106\u003c/li\u003e\n\u003cli\u003eB. A. Watkins, S. Feng, A. K. Strom, et al. Conjugated linoleic acids alter the fatty acid composition and physical properties of egg yolk and albumen. Journal of Agricultural and Food Chemistry 2003, 51 (23), 6870–6876. https://doi.org/10.1021/jf026156v\u003c/li\u003e\n\u003cli\u003eE. Alves, T. Melo, F. Rey, et al. Polar lipid profiling of olive oils as a useful tool in helping to decipher their unique fingerprint. LWT – Food Science and Technology 2016, 74, 371–377. https://doi.org/10.1016/j.lwt.2016.07.071\u003c/li\u003e\n\u003cli\u003eP. Abraham, S. Soledad, P. Trinidad, et al. ¹H NMR to analyse the lipid profile in the glyceride fraction of different categories of Iberian dry-cured hams. Food Chemistry 2022, 383, 132371. https://doi.org/10.1016/j.foodchem.2022.132371\u003c/li\u003e\n\u003cli\u003eM. Anton. Egg yolk: Structures, functionalities and processes. Journal of the Science of Food and Agriculture 2013, 93 (12), 2871–2880. https://doi.org/10.1002/jsfa.6247\u003c/li\u003e\n\u003cli\u003eJ. Jin, D. Cheng, D. Du, E. Olayemi, D. Xiao, T. Li, et al. Effect of emulsifier–fat interactions and interfacial competitive adsorption of emulsifiers with proteins on fat crystallization and stability of whipped-frozen emulsions. Food Hydrocolloids 2020, 101, 105491. https://doi.org/10.1016/j.foodhyd.2019.105491\u003c/li\u003e\n\u003cli\u003eD. J. McClements. Nanoscale nutrient delivery systems for food applications: Improving bioactive dispersibility, stability, and bioavailability. Journal of Food Science 2015, 80 (7), N1602–N1611. https://doi.org/10.1111/1750-3841.12919\u003c/li\u003e\n\u003cli\u003eP. Garcia-Moreno, C. Jacobsen, P. Marcatili, S. Gregersen, M. T. Overgaard, et al. Emulsifying peptides from potato protein predicted by bioinformatics: Stabilization of fish oil-in-water emulsions. Food Hydrocolloids 2020, 101, 105529. https://doi.org/10.1016/j.foodhyd.2019.105529\u003c/li\u003e\n\u003cli\u003eM. Fasiangova, G. Borilova. Impact of selenium supplementation on the oxidation stability of eggs. World’s Poultry Science Journal 2017, 73 (1), 175–184. https://doi.org/10.1017/s0043933916000854\u003c/li\u003e\n\u003cli\u003eK. Liang, H. Zu, X. Wang. Effect of storage on n-3 PUFA-enriched eggs. CyTA – Journal of Food 2020, 18 (1), 102–107. https://doi.org/10.1080/19476337.2020.1713896\u003c/li\u003e\n\u003cli\u003eN. Sun, J. Chen, Z. Bao, et al. Egg yolk phosphatidylethanolamine: Extraction optimization, antioxidative activity, and molecular structure profiling. Journal of Food Science 2019, 84 (5), 1002–1011. https://doi.org/10.1111/1750-3841.14512\u003c/li\u003e\n\u003cli\u003eG. Santos Sánchez, B. Miralles, A. Brodkorb, et al. Current advances for in vitro protein digestibility. Frontiers in Nutrition 2024, 11, 1404538. https://doi.org/10.3389/fnut.2024.1404538\u003c/li\u003e\n\u003cli\u003eY. Guo, Z. Cai, Y. Xie, et al. Synthesis, physicochemical properties, and health aspects of structured lipids: A review. Comprehensive Reviews in Food Science and Food Safety 2020, 19 (2), 759–800. https://doi.org/10.1111/1541-4337.12537\u003c/li\u003e\n\u003cli\u003eA. Serra, G. Conte, F. Ciucci, et al. Dietary linseed supplementation affects the fatty acid composition of the sn-2 position of triglycerides in sheep milk. Journal of Dairy Science 2018, 101 (8), 6742–6751. https://doi.org/10.3168/jds.2017-14188\u003c/li\u003e\n\u003cli\u003eT. Zhang, J. Xu, Y. Wang, et al. Health benefits of dietary marine DHA/EPA-enriched glycerophospholipids. Progress in Lipid Research 2019, 75, 100997. https://doi.org/10.1016/j.plipres.2019.100997\u003c/li\u003e\n\u003cli\u003eA. Irawan, N. Ningsih, Hafizuddin, et al. Supplementary n-3 fatty acid sources on performance and formation of omega-3 in eggs of laying hens: A meta-analysis. Poultry Science 2022, 101 (12), 101566. https://doi.org/10.1016/j.psj.2022.101566\u003c/li\u003e\n\u003cli\u003eP. E. Kiełb, J. Tumanowicz, A. Zachwieja, et al. Changes in fatty acid levels during in vitro ruminal fluid incubation with different proportions of maize distillers dried grains (DDGS). Agriculture 2023, 13 (4), 763. https://doi.org/10.3390/agriculture13040763\u003c/li\u003e\n\u003cli\u003eA. T. Ebeid. The impact of incorporation of n-3 fatty acids into eggs on ovarian follicular development, immune response, antioxidative status and tibial bone characteristics in aged laying hens. Animal 2011, 5 (10), 1554–1562. https://doi.org/10.1017/s1751731111000619\u003c/li\u003e\n\u003cli\u003eB. Omri, R. Chalghoumi, L. Izzo, et al. Effect of dietary incorporation of linseed alone or together with tomato–red pepper mix on laying hens’ egg yolk fatty acids profile and health lipid indexes. Nutrients 2019, 11, 813. https://doi.org/10.3390/nu11040813\u003c/li\u003e\n\u003cli\u003eM. D. Baucells, N. Crespo, A. C. Barroeta, et al. Incorporation of different polyunsaturated fatty acids into eggs. Poultry Science 2000, 79 (1), 51–59. https://doi.org/10.1093/ps/79.1.51\u003c/li\u003e\n\u003cli\u003eL. Xiang, X. Yan, M. Yuqian, et al. The bioavailability and biological activities of phytosterols as modulators of cholesterol metabolism. Molecules 2022, 27, 523. https://doi.org/10.3390/molecules27020523\u003c/li\u003e\n\u003cli\u003eS. Lioliopoulou, A. G. Papadopoulos, T. F. Mantzouridou, et al. Pomegranate peel dietary supplementation in laying hens affects egg yolk color and eggshell quality, decreases yolk lipid oxidation and influences yolk lipidomic profile. Journal of Applied Poultry Research 2024, 33, 100495. https://doi.org/10.1016/j.japr.2024.100495\u003c/li\u003e\n\u003cli\u003eT. O. Toomer, P. Maharjan, L. K. Harding, et al. Effects of full-fat high-oleic soybean meal in layer diets on performance, egg quality and chemical composition. Poultry Science 2024, 103 (3), 104074. https://doi.org/10.1016/j.psj.2023.104074\u003c/li\u003e\n\u003cli\u003eK. Nafiseh, Z. Ali, F. Ata, et al. Effects of flaxseed consumption on plasma lipids, lipoprotein-associated phospholipase A2 activity and gut microbiota composition in patients with coronary artery disease. Nutrition and Health 2022, 28 (5), 535–546. https://doi.org/10.1177/02601060221091016\u003c/li\u003e\n\u003cli\u003eH. M. M. Balnave, E. A. Akter, C. Geier, et al. Choline and methionine supplementation in layer hens fed flaxseed: Effects on hen production performance, egg fatty acid composition, tocopherol content, and oxidative stability. Poultry Science 2021, 100, 101299. https://doi.org/10.1016/j.psj.2021.101299\u003c/li\u003e\n\u003cli\u003eY. Tang, C. Zhou, Z. Yu, et al. Formation of lipid-derived volatile products through lipoxygenase (LOX)- and hydroperoxide lyase (HPL)-mediated pathway in oat, barley and soybean. Food Chemistry: X 2024, 22, 101514. https://doi.org/10.1016/j.fochx.2023.101514\u003c/li\u003e\n\u003cli\u003eK. Cho, E. P. Hansen. Structure identification of novel compounds using simple IR, ¹H, and ¹³C NMR spectroscopy and computational tools. Bulletin of the Korean Chemical Society 2020, 41 (1), 78–83. https://doi.org/10.1002/bkcs.11924\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"agricultural-products-processing-and-storage","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Agricultural Products Processing and Storage](https://link.springer.com/journal/44462)","snPcode":"44462","submissionUrl":"https://submission.springernature.com/new-submission/44462/3","title":"Agricultural Products Processing and Storage","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Diet, Egg yolk, Lipid structure, Functional properties, Phospholipid subclasses","lastPublishedDoi":"10.21203/rs.3.rs-7844056/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7844056/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEggs are recognized as nutrient-dense foods, and precise modulation of yolk lipid structures is critical for enhancing their functional properties. In this study, laying hens fed with three different diets of corn-dried distiller grains with solubles (DDGS), whole grains and flaxseed, respectively, were chosen to investigate how dietary composition remodeled and modulate structures as well as functional properties of yolks. A multi-scale approach integrating GC-MS, MALDI-TOF-MS, and \u003csup\u003e1\u003c/sup\u003eH NMR was applied. The results showed that yolks from hens fed with the DDGS diet contained trans-oleic acid (C18:1n9t) and palmitic acid (C16:0). The high level of monounsaturated fatty acids (MUFA) was found to improve the flexibility of the lipoprotein interface, thereby enhancing emulsifying activity. The cholesterol content in egg yolks was determined to be the lowest (0.84 mg/g) in the whole-grain diet, but an accumulation of phosphatidylserine was observed, which may disrupt the ω-6/ω-3 balance and increase the risk of oxidation. In yolks from the flaxseed diet, the docosahexaenoic acid (DHA) was preferentially deposited at the sn-2 position of triacylglycerols, promoting higher bioavailability of long-chain polyunsaturated fatty acids. The causal mechanisms underlying the differential regulation of yolk functionality by dietary components were elucidated. This was achieved through the specific remodeling of lipid molecular structures, which included the sn-2 positional distribution of fatty acids and variations in phospholipid subclasses. It aims to provide a novel integrated approach for the design of \u0026ldquo;phenomenon-substance-mechanism\u0026rdquo; on eggs via precision dietary formulation, further promoting a transformation of the poultry egg industry from compositional modification to structural empowerment.\u003c/p\u003e","manuscriptTitle":"Dietary Modulation on sn-Position Distribution and Its Impact on Emulsifying and Nutritional Properties in Egg Yolk Lipids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-28 18:04:47","doi":"10.21203/rs.3.rs-7844056/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T09:43:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T07:09:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T08:17:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323607095118569575313371836418092707245","date":"2025-10-16T03:28:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78994394782012881345036693892242907755","date":"2025-10-14T04:35:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-14T01:31:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T00:41:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-13T23:53:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Agricultural Products Processing and Storage","date":"2025-10-13T03:12:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"agricultural-products-processing-and-storage","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Agricultural Products Processing and Storage](https://link.springer.com/journal/44462)","snPcode":"44462","submissionUrl":"https://submission.springernature.com/new-submission/44462/3","title":"Agricultural Products Processing and Storage","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"80b62ce9-5e50-42b3-b102-cf3382dd0fc6","owner":[],"postedDate":"October 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T08:39:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-28 18:04:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7844056","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7844056","identity":"rs-7844056","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}