1. Introduction
The increased public consciousness of the health benefits of long-chain n-3 polyunsaturated fatty acids (n-3 LC-PUFA), especially docosahexaenoic acid (DHA), has elevated consumer preference for the products fortified with these desirable nutrients [
1,
2]. DHA-enriched eggs are considered to be the most successful and efficient vehicles to incorporate DHA into the human diet [
3]. Microalgae (MA), the primary producers of DHA, have been proven to be one of the most promising ingredients for DHA-enriched egg production due to their higher enrichment efficiency, production sustainability, and better sensory qualities than the commonly-used fish oil [
4]. In addition to the incorporation of DHA into eggs, dietary MA to hens also produces dose-dependent incorporation of DHA into muscles [
3]. With millions of laying hens going unused as food every year, increasing hen meat with a “healthy appeal” into the supply chain might be a promising way to meet increasing global needs. The use of DHA-enriched meat from hens after DHA-egg production could increase the value and desirability of hen meat or its processed products, given that consumers are already willing to pay more for n-3 LC-PUFA-enriched products [
2]. Hen meat has been historically popular and highly desired in many Asian cultures in the preparation of aromatic and savory broths, owing to its specific flavor and aroma [
5]. In France, boiled older chickens are regarded as delicacies [
6]. Additionally, various methods of processing DHA-enriched hen meat (e.g., sausages) have been suggested to improve consumer acceptability.
However, published MA-based innovation studies have primarily focused on broiler meat and pork [
7,
8]. Little is known about the potential impacts of MA on hen meat. To establish MA-fed hen meat as a nutritional and palatable meat protein source, it is important to evaluate the lipid composition, muscle quality characteristics, and oxidative stability of meat from hens. Therefore, the present study aims to investigate the impacts of dietary DHA-rich MA (
Aurantiochytrium sp.) on fatty acid (FA) profiles and quality attributes of meat from laying hens. In addition, the oxidative stability of the raw and cooked DHA-enriched meat, with or without storage, is determined.
4. Discussion
The experimental results demonstrated dose-dependent accumulations of n-3 LC-PUFA, predominantly DHA, in both breast and thigh muscle of hens receiving 0–2.0% MA. Consistent with our findings, broilers fed DHA-rich microalgae, such as
Aurantiochytrium sp.,
N. oceanica, and
Schizochytrium limacinum, showed increased DHA contents in the breast or thigh muscle compared to those with a control ration [
7,
19,
20]. The species of microalgae used in the current study (
Aurantiochytrium sp.) produces DHA predominantly (
Table 1). However, despite the fact that little EPA was detected in MA-supplemented diets, dose-dependent EPA enrichments in hen meat were found relative to control. The result is in accordance with the findings reported in broilers [
20], which is likely attributed to the retro-conversion of DHA to EPA [
21]. Concomitant with the increases of total n-3 PUFA in breast and thigh, the total n-6 PUFA contents (especially LNA and ARA) linearly decreased with MA supplementation. Our results are in accordance with several recent reports on pigs and broilers with MA supplementation [
22,
23]. The reduction in n-6 PUFA contents probably resulted from the competition of substrates and biosynthesis enzymes between the n-3 and n-6 PUFAs [
24].
Food products that include at least 40 or 80 mg EPA + DHA per 100 g can be marketed as being a “source of n-3 PUFA” and “high in n-3 PUFA”, respectively [
25]. In our present study, the supplementation of 0.5% or more
Aurantiochytrium sp. to hen diets was responsible for the n-3 LC-PUFA (EPA + DHA) concentrations rising to 44.4–137.2 mg per 100 g hen meat, meeting the required standard to be considered a “source of n-3 PUFA” or to qualify as being “high in n-3 PUFA”. In this way, consumption of 100 g meat from hens fed 2.0% MA would provide a mean intake of more than 125 mg of n-3 LC PUFA, supplying about 50% of the daily recommended intake (250 mg per day) for n-3 LC-PUFA [
25]. In comparison, the control hen meat provided only 5.0% of the recommended daily intake. The data reported in broilers also produced similar estimates of enrichment [
26].
The nutritional properties of meat are largely attributed to its fat and FA content, and balanced FA intakes are crucial to decrease the risk of atherosclerosis, cardiovascular, and other related diseases [
1]. Hence, health lipid indices based on the functional impacts of FAs were used in this study for nutritional evaluation of the n-3 LC-PUFA enriched meat [
27]. It is well documented that a dietary PUFA/SFA ratio of above 0.45, coupled with an n-6:n-3 ratio below 4.0, is desirable for the protection of the human cardiovascular system [
28]. In the present study, the increase in DHA contents subsequently resulted in a linear and quadratic reduction in the n-6: n-3 ratio, and a reverse trend was found in PUFA/SFA ratio in breast and thigh muscle. As the MA levels increased up to 2.0%, the n-6:n-3 ratio declined from 12.77 to 1.87 in breast meat and from 12.28 to 2.28 in thigh meat. Our results indicate that the meat from hens fed MA of 1.0% or more complies with the recommendations (below 4.0), while those fed basal or diets containing less than 1.0% MA in the current study do not.
AI and TI are vital parameters indicating the potential for stimulating platelet aggregation, and the h/H ratio is associated with cholesterol metabolism [
29]. From the perspectives of human health, healthy animal products can be characterized by low AI and TI and a high h/H ratio. Generally, the desired AI and TI recommended for human consumption are less than 0.5 and 1.0, respectively [
29]. In this study, the breast meat showed AI and TI of 0.42–0.43 and 0.60–0.82, and the thigh meat showed AI and TI of 0.42–0.44 and 0.62–0.80 with MA supplementation, which were within the recommended ranges. The lowest AI and TI were observed in meat from hens fed 2.0% MA. The better health lipid indices, with lower n-6:n-3 PUFA ratio, as well as higher PUFA:SFA and h/H ratios caused by dietary MA inclusion, were in good agreement with the findings of an earlier study in broilers [
27]. Hence, meat from MA-fed hens could be categorized as “beneficial to human health consumption”, the consumption of which might help reduce the risk of atherosclerosis, cardiovascular, and other related diseases.
Chicken meat, in particular with n-3 PUFA enrichment, is highly prone to oxidative processes [
30]. It has been shown that n-3 PUFA enrichment by the inclusion of fish or flaxseed oils in poultry diets reduced the oxidative stability of chicken meat [
26,
31]. However, the present study demonstrated that MA supplementations at less than 2.0% of the diets did not increase the TBARS values in fresh breast and thigh muscle, which is in good agreement with previous findings with
Schizochytrium sp. [
7] and defatted
N. oceanica [
32] to broilers. The discrepancies from fish or flaxseed oil-supplemented meat may be accredited to the antioxidant properties of microalgae. In addition to DHA enrichment, the
Aurantiochytrium sp. are rich in betacarotene (
Table 1), which is simultaneously transferred into the muscle with the microalgal DHA incorporation. The increased antioxidant components enhance the oxidative stability of muscle via the nonenzyme antioxidant system reflected by the increased DPPH and ABTS radical-scavenging activities. Similar beneficial effects of vitamins in MA on antioxidant status were found in broilers [
7]. On the contrary, a previous study indicated that broilers receiving MA increased the susceptibility to oxidation of meat [
26]. This can likely be explained by the insufficient radical-scavenging activities of the intrinsic antioxidant components to mitigate the lipid oxidation induced by the increased n-3 PUFA enrichment.
The processes of cooking and storage usually aggravate lipid peroxidation in poultry meat [
33]. In comparison with the fresh raw breast and thigh meat, regardless of MA supplemental levels, increased TBARS values were observed in refrigerated and heat-processed meat. After 6 d of refrigerated storage, meat from MA-supplemented hens displayed higher instability compared to those from nonsupplemented birds. Moreover, the dietary n-3 PUFA had a stronger effect on heated meat than on fresh or frozen meat, which is in accordance with the findings of Eder et al. [
33]. The supplementation with 2.0% MA to hen diets resulted in increases by 47.3% and 44.4% of the TBARS content in cooked breast and thigh meat relative to the control. In addition, thigh meat showed more susceptibility to peroxidation than breast meat, which is probably associated with the absolute higher intramuscular fat and PUFA content (
Table S1) in thigh than breast muscle [
34]. Overall, meat enriched with n-3 PUFA with MA supplementation showed reduced oxidative stability during processing or refrigerated storage, indicating that the intrinsic antioxidant components are not sufficient in mitigating lipid oxidation. Additional antioxidant supplementation appears to be a notable strategy for diminishing lipid oxidation of meat [
35]. Recent studies have demonstrated that dietary inclusion of antioxidants such as vitamin E and plant/herb extracts could inhibit lipid peroxidation in fresh, cooked, or frozen stored chicken meat [
36,
37,
38]. As such, additional antioxidants can be included in diets to prevent oxidative deterioration of DHA-enriched meat, especially meat subjected to the processes of cooking or storage.
The present study also demonstrated higher breast muscle yield in MA-supplemented hens than those of the control hens, which is possibly associated with the increased muscle protein synthesis stimulated by MA [
39]. Similar beneficial effects of MA on muscle yield were also found in broilers [
7]. In this sense, the MA-induced raise in the edible meat yield may encourage the interest of abattoirs slaughtering hens for meat production purposes. The increased inclusion levels of MA in hen diets linearly decreased the shearing force of both breast and thigh meat. Although not statistically different, there was a numerically lower shear force in the meat of birds fed MA rather than the control. Generally, the shear force of MA-treated hen meat ranged from 32 to 37 N, which is still regarded as tender (less than 45 N) by consumers [
40]. There were no significant effects on water holding capacity (WHC, determined by the drip loss) of the breast and thigh muscle at 24 h postmortem, following the MA treatment. However, the drip loss of hen meat at 6 d postmortem was significantly decreased in a linear or quadratic response. The significant reduction in WHC during refrigerated storage might be due to the oxidative degradation of membrane phospholipids, which damages the structure and function of membranes and increases the loss of sarcoplasmic fluid [
41]. On the contrary, previous studies indicated that MA supplementation decreased the drip loss of meat in broilers [
7] and pigs [
23]. This phenomenon was explained by low levels of n-3 PUFA enrichment that enabled the muscular cells to build a flexible lipid bilayer membrane, and thus resulting in an increased WHC. These results indicate that the effects of MA on meat quality properties are highly dependent on the included levels, microalgae species, and their chemical composition.