1. Introduction
Shell eggs [
1] are among the few foods that are consumed by people of all ages, regardless of religion or ethnicity worldwide [
2]. The chicken egg is one of nature’s flawlessly preserved biological materials [
1], as well as a delectable and easy-to-digest source of excellent nutrients for humans [
3,
4]. Egg yolk contains nutritive and non-nutritive compounds that are beneficial to human health, according to a study by Attia et al. [
5]. However, Elkin [
6] recommends against consuming shell eggs because they have been shown to elevate cholesterol levels and are related to hypercholesterolemia and risk factors for coronary heart disease, so they should be consumed in moderation [
5]. Weggemans et al. [
7] reported an increase in blood cholesterol concentration, which was believed to be the result of high egg intake and which may contribute to various cardiovascular risks. However, since the scientific evidence linking dietary cholesterol (from eggs) to the risk of cardiovascular diseases is more or less insubstantial, Qureshi et al. [
8] found no correlation between egg intake and increase in blood cholesterol concentrations, implying that the cardiovascular risk is largely dependent on increase in serum total and LDL-cholesterol [
8]. It was also reported that since eggs increase satiety, they are now being used as a weight-loss treatment. Qureshi et al. [
8] found that eating one egg per day does not increase the risk of stroke or ischemic stroke, and that the belief that higher egg intake among diabetics is linked to an increased risk of coronary artery disease warrants further research. The chicken egg is considered as a functional food with an edible portion made up of water (74%), high-quality proteins (12%), lipids (12% of polyunsaturated fatty acids (PUFA) and phospholipids), carbohydrate (<1%), as well as vitamins and minerals [
9,
10]. Antioxidant and nutraceuticals properties were found in egg proteins (such as ovalbumin, ovotransferrin, phosvitin), egg lipids (phospholipids), micronutrients such as vitamin A and E, selenium, and carotenoids [
11,
12]. Furthermore, the chicken egg can be also enriched with antioxidants (natural or synthetic) via poultry feed manipulations [
13].
Selenium is an important micro-mineral that acts primarily as an antioxidant through selenoproteins such as glutathione peroxidase (GPX), thioredoxin reductases (TrxR), and selenoprotein P (Sepp1) [
14]. By catalyzing the reduction of hydrogen peroxide and hydroperoxide activities, they protect cells and tissues from oxidative damage [
15,
16]. Surai et al. [
17] highlighted that selenium-enriched eggs provided up to 50% of the daily Se requirements and were high in nutrients like omega 3, vitamin E, and D, selenium, and antioxidants like lutein. Pham-huy et al. [
18] highlighted the potential role of antioxidants in disease prevention and health maintenance. Egg storage time can cause chemical changes in the eggs, deteriorating their quality [
19]. Egg yolk is an excellent source of nutrients for humans [
20], and abundant in polyunsaturated fatty acids that are prone to oxidation, with a coefficient of digestion comparable to milk and a superior biological value of proteins [
13]. Progressive storage time changes the physicochemical properties of eggs [
21]. The concentration and ratio of saturated and unsaturated fatty acids changes due to oxidation, particularly during storage.
Lipid peroxidation is a process that deteriorates egg quality, specifically yolk lipid stability during storage. It degrades the nutritional quality of an egg causing undesirable changes in taste, flavor, odor, color depreciation, and toxic substances [
21]. The prevention of these effects can be achieved by supplementing hen feed with selenium as an antioxidants source. Antioxidants including selenium and vitamin E play a crucial role in anti-lipid oxidation protective processes [
22]. Human Se intake is often lower than the recommended daily allowance [
23], necessitating its consumption via other food sources. Poultry eggs can be enriched or fortified by adding selenium (inorganic or organic) compounds to the hen’s diets [
24]. Organic Se, such as Se-enriched yeast, Se-proteinate produced enzymatically by hydrolyzed soy protein, nano-Se, and Se-amino acids [
25], has recently become available.
Saccharomyces cerevisiae has been established as the main strain used for aerobic fermentation in Se-enriched media with sodium selenite as Se source, which accumulates and incorporates into organic Se-containing compounds to produce Se-yeast [
26]. Organic Se supplements have several advantages over inorganic Se supplements, including increased egg Se concentration, improved oxidative stability, and a better fatty acid profile of the stored egg content [
13,
25].
There is also a new organic Se-enriched dietary source derived from bacteria (
Stenotrophomonas maltophilia (ADS18)) [
27], which may provide health-related benefits to humans through animal products. To our knowledge, no research has been done on its (Se-enriched bacterial proteins of ADS18) use in layers. Therefore, this study aims to examine the effects of different dietary Se sources (sodium selenite, selenium yeast,
Stenotrophomonas maltophilia enriched bacterial protein (ADS18) on egg yolk color, egg yolk, and breast meat antioxidant profile, total antioxidant capacity (TAC), and oxidative stability for fresh and stored egg yolk at 4 ± 2 °C for 14 days.
4. Discussion
Consumers’ preference for egg selection has now shifted from yolk cholesterol content or fatty acid profile to its color [
46]. The dietary supplementation of carotene plays a vital role in egg yolk color intensification [
36]; pigment (carotene) synthesis in hen eggs is feasible with its supplementation via diet ingredients [
47]. Microorganisms like algae, fungi, some bacteria, and plants were reported to synthesize carotene pigments [
48]. In the present study, the L* (lightness), a* (red), b* (yellow), chroma (saturation or color intensity), and hue (color tone) of egg yolks were calculated using the RYCF and CIELAB photo-calorimetric determination systems. The findings showed that dietary supplementation with organic selenium increased egg yolk yellowness and decreased yolk brightness while not affect egg yolk redness in all treatment groups and for both the fresh and post-stored eggs. Similarly, hens provided organic Se supplementation had higher yolk color (fresh and stored) values, as calculated by the Yolk Color Fan
® (Roche) scale; however, this scale only determines the sequence number of a stripe range. Since there is a lack of research on the effects of Se on egg yolk color traits measured by a Chroma Meter, some literature findings containing antioxidants are used to support or refute these findings. Gouveia et al. [
49] found that xanthophylls are ingested by chickens via the intestinal tract, assimilated into triglyceride-rich lipoproteins (chylomicrons), released into the blood (circulatory system), and transported to the yolk [
50]. Since the hens were fed the same basal diet except for Se supplementation (inorganic or organic), the increased yolk color in the treated group may be attributed to the accumulation of xanthophylls in the yolk.
The current findings are consistent with previous results [
51], who observed a linear increase in egg yolk color score (RYCF) with dietary incorporation of marine algae (
Spirulina platensis) at 0.1 to 0.2% (6.3–7.6) and 1.5 to 2.5% (10.55–11.66), respectively, compared to the negative control. Similarly, Park et al. [
52] observed similar results when hens were fed with dietary marine microalgae (
Schizochytrium) powder. Studies on Se supplementation (inorganic or organic) on the egg yolk color are lacking. However, a study by Omri et al. [
36] found that supplementing 2% tomato and red pepper mixture reduced (
p < 0.05) the lightness of yolk color. The egg yolk color index was stated to increase from 8.5 to 14.6 when 130 g of dried tomato peel per kg was added to the diet [
53]. Arpasova et al. [
54] observed that hens fed lower amounts of Se had lighter egg yolks than those fed higher Se. This resulted in a deeper egg yolk color in the organic (ADS18 or Se-Yeast) treated group. On the contrary, Omri et al. [
48] reported an increase in egg yolk redness and decreased yellowness with colorimetric determination when evaluating the effects of
Arthrospira platensis (spirulina) supplementation on laying hens. However, the latter author [
55] found that eggs stored at 4 °C for 30 days corresponding to linseeds-fed hens had increased yellowness and decreased redness. Nonetheless, there are few studies on the impact of storage on egg yolk color. The yolk pigmentation stability of omega 3 (ω-3) enriched eggs stored at room temperature (26.5 °C) and refrigeration (7.9 °C) for 35 d was found to be decreased [
56]. Fresh and stored egg yolks saturation (C*) displayed a similar trend of significant differences, but there were no dietary effects on hue angle values to either of the treatment groups. The use of Se supplementation (organic) as an antioxidant will help to reduce the use of synthetic pigments as feed additives in diets of laying hens. Therefore, its stability over a defined period of storage needs to be of primary concern to researchers. It is important to fix undesirable changes (be it chemical, enzymatic, or physical) in appearance and color as well as the quality of the nutrients contained in layers diet, as some can damage and lead to pigment losses during storage. In hens fed different Se sources, the contrast analysis showed significant (
p < 0.05) differences in the brightness and yellowness of the egg yolk (fresh or stored) and pronounce in the basal diet, inorganic and organic (ADS18 or Se-yeast) diets. For the Chroma (C*) index, there were significant differences between the treatments over the trial period, but no difference was observed for the Hue (H*) index.
Carotenoids are lipid-soluble pigments in plants, insects, birds, and aquatic animals that result from the pigment carotene or carotenoids [
57]. They function as a color pigmentation of orange, yellow, or sometimes red. Carotenoids in egg yolk are solely dependent on nutrients available in the feed and thus vary by egg types [
58]. The antioxidant Se can help improve the color of egg yolks. Organic selenium or vitamin E supplementation, for example, increased egg yolk carotene concentration [
59]. The color of the egg yolk is influenced by oxycarotenoids (xanthophyll pigments) resulting from the hen’s diet, which are lost when oxidized [
60]. They are connected to lipoproteins, which are transported to the egg yolk of an egg [
61]. The yolk color response to antioxidants influences the stability of lipid-soluble carotenoids available in a hen’s diet or body [
62]. Furthermore, carotenoid is an antioxidant that acts as feather dye, vitamin A precursor, and other related endocrine and immune-related functions in poultry [
63]. Total carotenoids were measured in this study. As a result, in addition to age-related macular degeneration, the two major egg carotenoids (lutein and zeaxanthin) play an important role in eye macular disease by protecting against light-induced oxidative damage as their mechanism of action [
64]. They can attract blue light until it has a passive antioxidant effect on photoreceptor cells [
64]. Owing to their possession of double bonds, they have ability to produce a highly resonance-stabilized C-centered radical that help to scavenge hydroxyl and superoxide radicals [
65]. Many of these effects are linked to its biological antioxidant function [
66]. In this study, the organic bacterial protein (ADS18) had a higher total carotenoid content than the inorganic and basal diet groups. This is consistent with previous findings by Karadas et al. [
67], who found a 22-fold increase in carotenoid content in hens supplemented with carotenoids in eggs during pre-and post-hatch studies compare to controls. In vivo studies using dried tomato peel revealed a 2.7-fold increase in β-carotene compared to 1.7 µg/gDM in comparison [
68]. There is a scarcity of information on the impact of storage in hens supplemented with similar treatment to that used in the current research. Similar to the current results, eggs stored at room temperature (26.5 °C) or under refrigeration (7.9 °C) for 35 d had lower total carotenoids egg yolk concentrations (28.55 against 22.09 µg/g) and (28.55 against 23.57 µg/g) [
56]. In contrast, eggs stored at 2 °C for 56 d [
69] and 4 °C for 28 d had no decrease in total carotenoid contents in the egg yolk [
36].
Total egg yolk and breast meat cholesterol were significantly decreased by dietary Se supplementation. Se supplementation was reported by Poirier et al. [
70] to reduce plasma lipids concentrations of total cholesterol, LDL-cholesterol, and VLDL-cholesterol in male Syrian hamsters. Selenium plays a vital role in the hormonal (thyroid) balance of fat metabolism [
71], and its deficiency has been linked to increased 3-hydroxy 3-methylgluatryl CoA (HMG-CoA) reductase activity in liver microsomes [
72]. As an antioxidant form of the active center of GPX, Se may help lower cholesterol levels [
73]. In their review, Brown and Jessup [
74] observed that as the antioxidant level increases in the diet, the cholesterol concentration decreases, and vice versa. Organic (ADS18 > Se-Yeast) Se supplementation, relative to inorganic (Na
2SeO
3) and basal diet fed hens, substantially decreased total egg yolk and breast meat cholesterol. A linear reduction in egg yolk and serum cholesterol levels was recorded with an increase of 0, 5, 10, and 15g MPM/Kg in
Moringa oleifera pod (Lam.) meals [
75]. Supplementing with organic Se and vitamin E has also been shown to lower cholesterol levels in serum and egg yolks [
24]. Radwan et al. [
73] found that nano-Se supplementation reduces total plasma and yolk cholesterol levels (153 mg/dl and 14.0 mg/g) at 0.25 ppm, respectively. Similarly, Attia et al. [
76] and Łukaszewicz et al. [
77] found a significant reduction in plasma cholesterol at 0.25 ppm and 0.3 mg/kg, of dual-purpose breeding hens of Gimmizah and Japanese quails yolk fed-organic selenium. The lower cholesterol observed in fresh, stored egg yolk and refrigerated breast meat may be attributable to differences in cholesterol synthesis control enzymes in chickens [
78]. The reason for cholesterol decrease may be due to the inhibition of sterol biosynthesis by oxysterols. Se supplementation has been shown to increase 15d-PGJ
2 (15-deoxy-Δ-12, 14 prostaglandin J2) production in response to oxidative stress-induced cell protection [
79], a known peroxisome proliferator-activated receptor-γ ligand (PPAR γ) [
80]. The activation of the latter by troglitazone regulates the concentration of sterol regulatory element-binding protein (SREBP)-2, resulting in a decreased cholesterol synthesis [
81].
The egg white and yolk of a chicken egg contain a lot of antioxidant-rich compounds [
9]. Antioxidants are abundant in egg proteins (ovalbumin, ovotransferrin, phosvitin), egg lipids (phospholipids), and micronutrients (vitamin E and A, selenium, and carotenoids) [
9]. The type of flavonoids and phenolic acid that play a role in good antioxidant activity was the bioactive compounds in the egg, particularly the albumen [
82]. Free radicals can be effectively counteracted by the existence and activity of phenolic acids and flavonoids in the system [
82]. Dietary supplementation with Se increased the bioactive (phenolic) content of fresh, stored egg yolk and breast meat in the current research. Phenolics are a major phytochemical class that includes chemical compounds of one or more phenolic groups [
83]. The resulting concentration of antioxidants depends on the phenol group and the double bond, i.e., the lower the concentration of antioxidants, the higher its activity [
84]. In contrast, Siger et al. [
85] found the binding ability to scavenge peroxyl radicals was unconnected with the flavonoid concentration because of the chances of the formation of the phenoxyl radicals. Simple phenolic acids are not easily deposited into chicken egg yolk due to their hydrophilic nature under natural conditions [
86]. Since work is scarce in this regard, comparing the current results to other literature studies is difficult. Untea et al. [
87] recently reported a significant increase in the total polyphenol content of egg yolk with a 0.5% and 1.0% dietary inclusion of bilberry and walnut leaves, respectively. From hens-fed grounded mixtures of 4.5 and 2% of linseeds and fenugreek seeds, a significant increase in total phenol concentration in yolk was observed compared to 4.5% ground linseeds and 4.5% ground linseeds and 1% each of dried tomato and sweet pepper powder, respectively [
53]. A phyto-additives (dried tomato peel) trial on the yolk carotenoids and phenols of laying hens revealed an increase in total phenol content, which was linked to cholesterol reduction [
35]. However, the inclusion of varying levels of dietary fennel seed did not have a significant effect on the total phenol content of egg yolk from
Coturnix coturnix japonica [
40]. Gasecka et al. [
88] found that Se supplementation at concentrations ranging from 0.5 to 5.0 mmol/L increased the biosynthesis of phenolics and flavonoids in mushrooms. Se treatment also increases the phenolic content of green tea [
89], and purple potatoes [
90] with the mechanism still unknown. One potential mechanism might be that Se promotes the accumulation of some sugars, such as glucose, which is a crucial substrate in many metabolic pathways [
90]. Sae-Lee et al. [
89] suggest that Se and Al influenced the content of secondary metabolites such as phenolic substances, but the biosynthesis of these substances is uncertain. Another explanation may be that phenolics and flavonoids have been confirmed to be responsible for antioxidant activity [
9,
91,
92,
93], which could be connected to the improved antioxidant enzyme status and higher selenoproteins gene expression [
94]. To our knowledge, there is no evidence or literature on total phenol content in response to selenium supplementation in egg yolks and breast meat.
Flavonoids are forms of antioxidants that are water-soluble and have glucose groups in the side chain [
95]. The subsequent concentration of antioxidants depends on the phenol group and the double bond presence [
84]. However, owing to the possibility of phenoxyl radicals formation, Siger et al. [
85] found that the capacity to scavenge peroxyl radicals was independent of flavonoid concentration. The flavonoid content of breast meat and fresh and stored egg yolk showed that selenium supplementation did not affect this parameter. In this respect, literature is scarce, making it difficult to compare our findings. Omri et al. [
36] found no differences in flavonoids content of chicken egg yolk supplemented with linseed alone or combined with dried tomato-red pepper mixture before and after storage. Omri and Abdouli [
53], however, found that hens supplemented with sweet pepper and dried tomato and fenugreek seeds had higher flavonoid concentrations in egg yolk (1.53 to 2.96 mg CAE/g) and (1.53 to 3.02 mg CAE/g). There are few studies on the impact of selenium supplementation on the total flavonoid content of fresh and stored egg yolks and breast meat.
Eggs are considered an excellent source of dietary antioxidants [
96]. In this study, dietary Se supplementation influenced the antioxidant activity of fresh and stored eggs as measured by the reduction of Mo (VI) to Mo(V). Whereas only stored eggs were affected by dietary treatments for ferric reducing power activity. These results suggest that dietary addition of Se to layers diet increased chicken eggs antioxidant capacity. There is no research on egg antioxidant activity expressed as AAE or GAE per g in laying hens in the selenium supplementation literature. Wang et al. [
97] recorded higher total antioxidant capacity in eggs from epigallocatechin-3-gallate (EGCG)-fed layers, which is consistent with the present findings. Omri, et al. [
36] also found an improvement in antioxidant activity measured by phosphomolybdenum reduction of a mixture of a diet supplemented with ground linseed (4.5%), dried tomato paste (1%), and sweet pepper powder (1%), stored and slightly in the fresh egg for hens-fed (1%). No significant antioxidant activity of egg yolk or meat was found when measured in the above study with a ferric reduction antioxidant power assay. Similarly, egg yolk total antioxidant activity was positively affected in golden pheasants (
Chrysolophus pictus) fed diets containing various levels of green vegetables [
98]. Organic Se was found to increase the carotenoid content while decreasing cholesterol content in egg yolks in the current study, which may have led to the higher egg antioxidant capacity. It is, therefore, logical to speculate that organic Se supplementation (ADS18 or Se-Yeast) would enhance egg yolk antioxidant capacity, because organic Se prevents carotene oxidation, potentially increasing its deposition. McGraw et al. [
99] stated an increase in egg antioxidant status during hatching and fleeing in goldfinches may be beneficial. However, further research is needed to explain and understand the underlying mechanisms of this response.
The freshness of eggs is one of the consistency parameters affected by storage time, temperature, and relative humidity [
73]. In cells, free radicals can produce reactive substances, which in turn damages cells and tissues. Antioxidants may prevent the damage caused by oxidation. Oxidation intensity of lipids is one of the parameters used as an indicator to assess the freshness of poultry products. MDA is one of the lipid peroxide metabolic products and negatively correlates with the activity of GPX [
100]. The degree of peroxidation of fatty acids (animal products) can be monitored by malondialdehyde (MDA) concentrations, i.e., the higher the MDA concentration and thus the degree of lipid peroxidation. A decrease of the content of MDA observed in egg yolk, breast muscle, thigh, and serum may be due to the increase in the activity of GPX resulting from supplemental dietary form (organic vs inorganic). The advantageous effects of organic Se in layers are connected to its efficacy of being transferred to the egg [
101]. Organic Se was found to improve the oxidative stability of eggs [
24] by reducing the eggshell or fluid’s cellular damage. Generally, due to its antioxidant properties, Se provides fat and protein oxidation stability in the eggs of laying hens fed a dietary Se diet [
22]. A study to investigate the interaction between different Se sources and trace elements about the antioxidant system of laying hens is consistent with the present results [
102]. Egg-laying hens receiving selenomethionine in stored eggs showed decreased lipid peroxidation, probably increasing the shell life of the eggs [
103]. Selenium supplementation at 0.25 ppm showed a significant decrease in MDA content in fresh and stored egg yolk compared with 0.10 ppm supplemented egg yolk [
73]. Wang et al. [
104] reported a significant increase in GPX activity and decrease yolk MDA content when Se to Langshan layer hens was supplemented with 0.3 mg/kg. More egg freshness was observed by the latter author and Gajčević et al. [
105] after a month of storage at 4 °C with 0.4 mg/kg of organic Se supplementation.
The noted increase in MDA content in stored eggs could be attributed to the storage temperature (4 ± 2 °C). Organic Se-supplemented egg yolks, on the other hand, had lower MDA values than inorganic Se and unsupplemented egg yolks. Cimrin et al. [
13] recently recorded lower yolk TBARS values in vitamin E-fed hens eggs at room temperature, although refrigerated eggs showed no dietary effects. In Hy-Line W-36 hens trials, Susceptibility to lipid peroxidation and egg yolk decreased with a combination of increased Se and vitamin E concentrations [
19]. As a result, the authors predicted that advanced storage would increase MDA concentrations [
106]. Skřivan et al. [
17] and Asadi et al. [
25] found significantly increased yolk lipid peroxidation and MDA content when eggs were stored at 20 °C and refrigerated for 7–14 days. On the contrary, the lipid oxidation stability of fresh egg yolks was not improved by dietary inclusion of linseed mixture, dried tomato paste, and sweet red pepper [
36]. The malondialdehyde content of refrigerated egg yolks did not improve after six weeks of storage, according to Nimalaratne et al. [
96]. Nadia et al. [
107] found that there was a significant difference between the treatments with dietary natural antioxidants but not with storage time.
Though hens (layers) are bred to produce eggs, the quality of their meat is important to ensure oxidative stability after supplementing antioxidants to their diets. As a result, measuring lipid peroxidation using MDA content in breast and thigh muscle can be helpful. Poultry meat, due to its high polyunsaturated fatty acid content, is typically susceptible to rapid deterioration. Ahmad et al. [
100] observed significantly reduced lipid peroxidation in fed-selenium yeast chicken breast meat, which is consistent with the current findings. A significant decrease in malondialdehyde (MDA) concentration of 0.15 mg organic Se/kg each in broiler muscle (L-Se-Met or D-Se-Met) compared to inorganic sodium selenite group [
108]. In contrast, dietary supplementation with Se did not affect the concentration of MDA (expressed as TBARs) in lamb muscle over 9 days of storage [
109]. The activity of GPX in serum and liver, as well as free radical inhibition, is found to have a significant effect to reduce the content of MDA in broiler blood-fed 0.30 mg/kg of nano-Se [
110]. However, dietary supplementation with Se did not affect serum MDA concentration [
108].
Lipid peroxidation is a complex pathway in which fatty acyl hydroperoxides form as a free radical chain in the reaction process between unsaturated fatty acids and reactive oxygen species [
100]. Lipid degradation and oxidative rancidity are caused by a sequence of secondary reactions that follow primary autoxidation and result in changes in flavor, nutrient quality loss, and environmental pollution, among others [
111]. Organic selenium (ADS18 > Se-Yeast) supplementation reduced MDA concentration in egg yolks, breast and thigh tissue, and blood throughout the study. The difference in responses between different sources of Se may be attributable to differences in metabolic pathways (inorganic or organic), as organic sources preserved the integrity of muscle cells linked to lipid oxidation and oxidative stability [
112].