1. Introduction
Macroalgae have been increasingly cultivated for numerous industrial applications, including biotechnological and nutritional purposes. Indeed, seaweeds are valuable sources of bioactive and prebiotic compounds (e.g., polysaccharides), minerals, vitamins (i.e., complex B, C, and E), pigments, essential amino acids, and bioactive peptides, with some species being good sources of polyunsaturated fatty acids [
1].
Brown algae (
Phaeophyceae), such as
Laminaria sp., represent a large proportion of cultured seaweed biomass that can be used for feed and food [
2].
Laminaria sp. is composed of bioactive polysaccharides (e.g., laminarin and fucoidan) with potential health benefits [
3], as well as iodine and antioxidant carotenoids, chlorophylls, and vitamin E [
1]. Although
Laminaria sp. has low lipid content (up to 1.3% dry matter, DM), its fatty-acid profile may be rich in some polyunsaturated fatty acids (PUFA), such as arachidonic (20:4n-6, ARA) and eicosapentaenoic (EPA, 20:5n-3) acids [
4,
5], which are beneficial for human health [
6]. In addition, dietary supplements of algal polysaccharide extracts were reported to modulate pigs’ gut microbiota, which can have an impact on lipid metabolism [
7]. The nutritional and bioactive properties of
Laminaria sp. were shown to enhance poultry meat quality [
8,
9,
10], when these algae were used as feed supplements.
Several reports evaluated the potential of
Laminaria sp. extracts as feed supplements for monogastric animals, mostly in the form of laminarin and fucoidan extracts [
11,
12]. However, to the best of our knowledge and despite the potential of using
Laminaria sp. as a feed ingredient, such application was scarcely reported [
13,
14,
15]. Indeed, high dietary levels of macroalga can compromise nutrient digestibility due to the presence of an intricate cell wall that is resistant to degradation by digestive enzymes, thus trapping other valuable nutrients and preventing their intestinal absorption [
16]. In particular, brown seaweeds have a specific cell wall structure mainly composed of gel-forming alginate crosslinked with phenolic compounds and fucose-containing sulfated polysaccharides tightly linked with minor contents of cellulose [
17]. Therefore, the use of exogenous carbohydrate-active enzymes (CAZymes) to degrade the brown macroalga cell wall is a promising strategy to increase the bioavailability of nutrients in poultry diets added with algae. Although there are some challenges related to large-scale and cost-effective algae production, the use of feed enzymes might allow seaweeds to be used as partial replacement sources of conventional and unsustainable feed ingredients (e.g., corn), enhancing the nutritional value of brown seaweeds by degrading algal non-starch polysaccharides [
1]. This could be a solution to profit from the high biomass of macroalgae that can be produced per surface area and hinder the current food–feed–biofuel competition for conventional sources [
1]. Commercially available CAZyme mixtures containing xylanases and β-glucanases have been widely incorporated in cereal-based diets for poultry to increase their nutritional value [
18]. However, to date, there are no reports about the inclusion of exogenous enzymes in seaweed-added poultry diets. However, recent studies tested the benefits on growth and meat quality of using commercial (Rovabio
® Excel AP) and recombinant CAZymes as supplements in microalga-containing diets for broiler chickens [
19,
20]. In addition, alginate lyases and cellulases were shown to degrade
Laminaria digitata biomass for biotechnological applications [
21,
22]. Moreover, in a recent in vitro study, an individual alginate lyase from a family 7 polysaccharide lyase (PL7) partially disrupted the
L. digitata cell wall and released monounsaturated fatty acids, such as 18:1c9, and monosaccharides from algal biomass [
23]. However, no in vivo assay was conducted in order to analyze the effect on broiler chicken growth and meat quality of supplementing recombinant alginate lyase in a diet incorporated with
L. digitata. Therefore, the present study aimed to test if dietary supplementation with alginate lyase or a commercial carbohydrase would counteract the potential deleterious effects of adding high levels of
L. digitata to the diet. Thus, feed enzymes are expected to improve the nutritional value of poultry meat by releasing algae bioactive compounds with benefits for human health. This would increase the importance and utilization of brown algae as a feed ingredient to partially replace corn.
4. Discussion
The dietary inclusion of 15%
L. digitata as a partial substitute of corn impaired animal growth performance. Indeed, reductions of 11% in final BW and 14% in ADG, along with a consequent increase of 10% in feed conversion ratio, were found in birds fed the macroalga treatment without the addition of enzymes. Although there was a numerical difference between the initial BW (day 21) of broilers from the control and LA treatment due to a considerable variability of values, it was not significant and, thus, this parameter cannot be considered a conditioning factor of final BW (day 35). In addition, the difference in BW at day 35 (192 g) between control and LA treatment was almost four times higher than that found at day 21 (49.9 g), which was attributed to the dietary incorporation of algae. The commercial CAZyme mixture with major enzymatic activities of xylanase and β-glucanase slightly counterbalanced the negative effects on BW and ADG caused by LA treatment, which was also found with the recombinant alginate lyase for ADG and feed conversion ratio. The negative effects on chicken growth caused by dietary
L. digitata were probably due to the high inclusion rate of alga, since low doses of seaweed extract were described to enhance growth performance of broilers [
11,
12] and pigs [
35,
36,
37,
38,
39,
40]. For instance, final BW was increased in broiler chickens fed
Laminaria spp. extract supplemented at 20 mg/mL in water [
10], and feed efficiency was enhanced in pigs fed with up to 0.03% of
Laminaria sp. extracted polysaccharides (e.g., laminarin and fucoidan) [
35,
37,
39]. Conversely, Ventura et al. [
15] found that the incorporation of
U. rigida, at more than 10% in a broiler starter diet, increased feed conversion ratio, which was suggested to be due to the presence of high amount of indigestible algal polysaccharides. In addition, Zahid et al. [
41] showed that a brown alga mixture led to a numerical decrease in final BW when fed at 20% to 40% to chicks. Recently, Stokvis et al. [
14] described an increase in feed conversion ratio in broilers fed 10% of the brown macroalga,
Saccharina latissima. The latter results were previously described by Bikker et al. [
42] as being caused by the high mineral and non-starch polysaccharide contents of brown macroalgae. In the present study, there were considerable high levels of minerals in the seaweed-containing diets, but broilers’ mortality was less than 3%, with few animals presenting diarrhea that compromised their growth and health, conversely to what was suggested by Bikker et al. [
42]. Therefore, the presence of algal indigestible polysaccharide in the diets was probably the cause of animal growth impairment. Despite the fact that nutrient digestibility was not evaluated, the increase by more than 40% in ileal viscosity in chickens fed LA and LAR diets in comparison with the control and LAE might have reduced feed passage and consequently nutrient digestion and absorption by trapping valuable nutrients [
43]. The increment in ileal viscosity caused by the dietary incorporation of
L. digitata was probably due to the presence of hydrocolloidal and anionic polysaccharides in the macroalgal cell wall (e.g., alginates) [
44] that are largely indigestible by monogastric animals and can increase medium viscosity [
45]. Similar effects were previously reported when 15% or 10% of the microalgae
Arthrospira platensis [
19] and
Chlorella vulgaris [
20], respectively, were fed to broiler chicks. However, contrary to what was described for microalga, the recombinant enzyme reversed the effect on ileal viscosity caused by the macroalga treatment. Therefore, it is possible that alginate lyase partially degraded the
L. digitata cell wall, as previously shown in vitro [
23], and, to some extent, disrupted gelling and hydroscopic polymers formed by crosslinking between cell-wall phenolic compounds and alginate [
17]. In addition, the xylanases and β-glucanases in LAR treatment could not specifically hydrolyze algal polysaccharides, and, as expected, an increase in intestinal viscosity was also observed when the LA diet was supplemented with the commercial carbohydrase mixture.
Furthermore, in the present study, the increase in jejunum and ileum lengths with all macroalga-containing treatments and of cecum length with LA and LAE treatments could indicate a morphological intestinal modification to compensate growth impairment and, thus, increase nutrient absorption. However, it is possible that this effect was not just a compensatory mechanism but also the result of algal polysaccharide bioactivity. In fact, laminarin and fucoidan extracted from
Laminaria sp. were previously shown to increase duodenal villous height in piglets [
46], even though their effect on intestinal length was not reported.
Considering meat quality, the pH of thigh meat was significantly, although slightly, increased by the LA treatment relative to control. The pH was shown to be a determinant factor of myofibrillar protein denaturation in red muscles [
47] with a potential effect on meat sensory attributes and carcass traits [
48]. However, pH values in the thigh, similarly to those found in the breast, were within the range previously described for poultry meat [
10,
49] and, thus, this parameter was not be associated with any modification of meat quality. Moreover, the color of breast meat was influenced by dietary macroalga, with a decrease in a* value promoted by the LA treatment. A similar result was reported by Tavaniello et al. [
10] in chicken breast muscle with in-water
Laminaria spp. supplementation, and by Rajauria et al. [
50] in pork with a low dietary level (0.53% feed) of
Laminaria spp. extract. In the present study, the analytical difference in meat color was not distinguishable by the naked eye in the raw meat or even by the trained sensory panel exposed to cooked meat. Therefore, this modification would not have a negative impact on the appearance of meat for consumers. The effect of macroalga treatments on meat redness might be explained by the 1.4-fold increase in total carotenoids in breast and thigh muscles. In addition, meat color was probably determined by myoglobin concentration and oxidation status in the muscle, since oxidation of myoglobin into oxymyoglobin is responsible for the bright cherry-red color sought by consumers [
51]. In fact, an interaction between positively charged proteins in meat, such as oxymyoglobin, and algal anionic polysaccharides may have occurred with a consequent effect on meat redness. This aspect was suggested by Moroney et al. [
52] to justify a decrease in a* value, in a concentration-dependent manner, in pig meat pulverized with
L. digitata extract containing fucoidan and laminarin. The fact that the use of alginate lyase could counterbalance the effect of macroalga on meat redness corroborates the phenomenon described by Moroney et al. [
52]. Interestingly, the dietary supplementation with commercial CAZyme mixture led to a similar result to that found with the LAE treatment, although minor degradation activities toward
L. digitata polysaccharides were previously reported for xylanases and β-glucanases [
23].
The increase in total carotenoids and chlorophylls in chicken breast and thigh meats provides benefits for consumers and enhances the nutritional value of meat. For instance, fucoxanthin, which is the major carotenoid present in brown seaweeds, was shown to have antioxidant, antitumor, and anti-inflammatory properties [
53]. Although chlorophyll metabolism and function have been scarcely studied, Viera et al. [
54] reported that, in mice, chlorophylls are converted into pheophorbides or pheophytins, absorbed in the intestine, and eventually transported to tissues. This process of conversion of chlorophylls into their derivatives and uptake by cells was also demonstrated in vitro with seaweeds, such as
Laminaria ochroleuca [
55,
56]. Chlorophylls and their derivatives were shown to have important functional functions, such as the ability to trap mutagens and antioxidant activities. The latter activities include free-radical-scavenging properties and metabolic activation of detoxification pathways [
57]. As a matter of fact, chlorophylls, such as chlorophyll
a, were identified as antioxidants having a synergistic activity with vitamin E because of their ability to scavenge peroxyl radicals [
58]. Regarding vitamin E homologs, the treatments with macroalga had no effect on the amount of the major homolog α-tocopherol, which was within the range described for broiler chicks [
19,
59]. However, γ-tocopherol was decreased with macroalga-containing treatments in the breast and with LAE treatment in the thigh. The fact that a considerably low amount of γ-tocopherol (up to 0.075 µg/g) was obtained in both meats indicates the reduced biological impact of these results. In addition, the concentration of γ-tocopherol was about 10 times lower than that found in previous studies [
19,
20,
59], which was mostly due to the decrease in γ-tocopherol amount, between 38.5% and 72.3%, in the present control diet compared with previous diets. Moreover, the sensory panel detected a decrease in juiciness and flavor of breast meat with LAE treatment compared with the control. The mechanisms responsible for the changes in the panel perception of meat juiciness and flavor remain to be explained. However, although these parameters were significantly discriminated, they changed by less than 1.0 and, thus, no major impacts on consumer acceptability of meat are expected. Indeed, meats from all treatments were positively (>4.0 points) scored for overall acceptability without differences for this parameter.
The dietary treatments influenced the total lipid amount and the fatty-acid profile of breast and thigh. In fact, treatments with
L. digitata led to a reduction in total lipids in both meats and, thus, to an increase in meat leanness, which was described as one of the major attributes determining consumers’ decisions toward meat [
60].
Nevertheless, meats from all treatments were considered lean (total fat < 5%) [
61], with an average of total lipids of 1.8% for the breast and 2.6% for the thigh. In addition, treatments with macroalgae promoted the accumulation of total PUFA with increases in n-6 and n-3 PUFAs in the breast and in n-3 PUFAs in the thigh, although the latter was not significant with the commercial CAZyme supplementation. The higher accumulation of n-3 PUFA in meat with alga-added diets was mainly due to a relative increment in specific n-3 long-chain (LC) PUFAs, including 20:5n-3, 22:5n-3, and 22:6n-3. Similar findings were previously reported when
Laminaria spp. were used as a supplement for broiler chickens, except for the effect on 20:5n-3 [
10]. Furthermore, Islam et al. [
9] described that dietary supplementation with a mixture (1:1) of
Laminaria japonica and charcoal at up to 1% led to an increase in 22:6n-3 proportion in duck meat. Considering the low lipid content (1.31% DM) of
L. digitata, it is possible that the beneficial contribution of this macroalga for n-3 LC PUFA proportion in meat was not just a result of the high percentage of these fatty acids in seaweed biomass, but also a consequence of the bioactivity of algal polysaccharides. In fact, previous reports in pigs showed that polysaccharides extracted from
Laminaria spp. (i.e., fucoidan and laminarin) can modify gut microbiota and, consequently, the production of short-chain fatty acids [
7]. The latter were shown to be involved in lipid metabolism and de novo synthesis of fatty acids in the liver [
62]. Although the effect of algal polysaccharides on the gut microbiota and lipid metabolism was not assessed in the present study, the high levels of
Laminaria spp. incorporated in the broiler diet could have potentiated the action of polysaccharides toward fatty-acid metabolism. The relative increase in n-6 and n-3 PUFAs is particularly relevant, considering the low efficiency of conversion pathways of 18:3n-3 and 18:2n-6 into n-3 LC-PUFA and n-6 LC-PUFA, respectively, and the reduced human intake of PUFA, mostly n-3 PUFA [
63]. The importance of enriching chicken meat with n-3 LC-PUFA is linked to the benefits of these fatty acids for human health, since n-3 LC-PUFAs are associated with enhanced cognitive abilities and suppression of chronic diseases, such as rheumatoid arthritis, atherosclerosis, and coronary heart disease [
6,
63].
Herein, the effect of treatments on individual fatty-acid proportions led to an increase in PUFA/SFA ratio in the breast and a decrease in n-6/n-3 PUFA ratio in breast and thigh with macroalga-containing treatments. Therefore, the dietary incorporation of
L. digitata improved meat nutritional value, since a higher PUFA/SFA ratio and lower n-6/n-3 PUFA ratio have been used as indicators of healthier meat [
64]. However, the n-6/n-3 PUFA ratio was considerably above the maximum recommended value of 4.0 [
64], which was essentially due to a predominance of 18:2n-6 (more than 30%) among all fatty acids present in meat.
Moreover, the treatments with macroalga led to a significant increase in iodine and bromine in the breast muscle of chickens and, consequently, to an increment in total microminerals in meat. A similar result for iodine was found in the adipose tissue and muscle of piglets fed a diet supplemented with 0.116% or 0.186%
L. digitata [
65]. In the present study, the accumulation of iodine and bromine in meat was explained by a considerable increase in each one of these compounds in alga-added diets compared with the control diet. In fact,
L. digitata presented high levels of iodine (4399 mg/kg DM) and bromine (474 mg/kg DM), which are within the range of values already reported, due to the ability of seaweed to concentrate mineral compounds from seawater [
1]. The increase in iodine in poultry meat can be favorable for human health, particularly considering the low iodine intake of nearly one-third of the global population, mainly children and pregnant women, with a consequent impairment of thyroid hormone synthesis [
66,
67]. The lack of thyroid hormones compromises cellular metabolism and development of organs, especially the brain, and eventually leads to iodine deficiency disorders [
67]. Although a higher accumulation of iodine in meat can improve its nutritional value, an excessive intake of this mineral should be monitored to avoid pathological problems. In the present study, meat from treatments with macroalgae contained an average of 0.35 mg of iodine/100 g, which is above the recommended daily intake of 0.15 mg/day for an adult person [
67] ingesting 100 g of meat per day. Therefore, a meat with an accumulation of iodine higher than that with the control (0.01 mg/100 g) but lower than with macroalgae would be more favorable. In addition, bromine has no proven nutritional benefits, and it is considered a food contaminant and a potentially toxic element [
68]. However, the amount of bromine present in the meat from treatments with macroalgae (average of 790 µg/100 g; 11.3 µg/kg BW/d for 70 kg of BW) is far below the acceptable daily intake of 1000 µg/kg BW/day [
68].