Growth and carcass traits were submitted in detail in a companion paper (unpublished data). The results are briefly shown here for contextual reasons. We have demonstrated that the production performance was improved (p < 0.05) when 10% of soybean meal was replaced with C. vulgaris, in comparison to the remaining groups, but not different from the control (p > 0.05). Namely, at the end of experiment (day 40) the body weight, weight gain, and feed intake of the control and CV10% group were 2801 and 2819 g, 70.80 and 63.87 g/day, and 40,425 and 40,075 g/pen, respectively. Compared with the control group, birds supplemented with higher concentrations of C. vulgaris (15% and 20%) had lower body weight (2587 and 2342 g), weight gain (70.80 and 63.87 g/day), and feed intake (37,382 and 35,922 g/pen), while feed conversion ratio did not differ between control and CV groups.
From a nutritional point of view, the quality of meat, pigment concentrations, and antioxidant activity is dependent on the inclusion levels and increase with C. vulgaris inclusion. Furthermore, the addition of C. vulgaris in broilers feed was an efficient way to significantly increase the concentration of DHA+EPA and improve the n-6/n-3 ratio in broilers breast meat without affecting texture and sensory acceptance of the meat. The present study aimed to complement primary nutritional data with the effect of microalgae in broilers feed on mineral, amino acid composition, and digestibility of meat.
3.1. Chemical Composition, Cholesterol Level, and Energy Value
The effect of dietary microalgae on the proximate composition of breast meat is presented in
Table 4. The incorporation of 15 and 20%
C. vulgaris resulted in an increase of protein content in the meat (
p < 0.05). Ash content was increased in all groups where this microalga was included, although no significant differences were found between them. Fat content was significantly lower (
p < 0.05) in the breast muscle of broilers that were fed a diet with
C. vulgaris, in comparison to those fed the control diet. No significant differences were found for moisture, cholesterol, and energy contents between groups.
It is evident from the results that meat from groups of broilers fed
C. vulgaris was richer in protein. The contribution of proteins to the total energetic value of control, CV10%, CV15%, and CV20% breast was 76.11, 85.34, 92.90, and 90.73%, respectively. Such higher protein content could be related to the higher availability of amino acids originated from the
Chlorella proteins, an improved absorption of nutrients, or both. Accordingly, Mirzaie et al. [
22] reported that 10 g/kg dietary supplementation with
Chlorella by-products significantly increased jejunum villus heights and crypt depths, increased absorption area, and consequently feed utilization. Furthermore, Kang et al. [
23] and Janczyk et al. [
24] found that
C. vulgaris addition in feed significantly increased
Lactobacillus spp. in the broiler’s intestines and caeca of laying hens. This is particularly relevant as beneficial microorganisms in the intestinal tract enhance digestion and nutrient absorption [
23]. As such, and in agreement with our results, Kalbe et al. [
25] found protein increase in pork from pigs fed Schizochytrium spp. Furthermore, it has been suggested that DHA supplementation stimulates muscle protein synthesis in growing pigs [
26]. This agrees with findings from the present study, where DHA was significantly higher in experimental groups fed
C. vulgaris (up to 16 times; submitted data). Additionally, Waldroup et al. [
27] showed that greater amount of proteins and single amino acids in broiler feed resulted in elevated protein content of meat. As presented in
Table 3, the diets with
C. vulgaris inclusion resulted in higher crude proteins in broiler feed due to an increase in amino acids, mostly lysine and phenylalanine. Our results corroborate previous studies reporting a slight increase in crude proteins from broiler feed when microalgae were used as a protein source in high concentrations [
9,
28]. This change in feed proteins is due to differences in protein content of soybean meal and microalgae used. While crude protein of soybean meal in the present study was 43%, according to standardized soybean meal protein content on the feed market [
29],
C. vulgaris from the present study contained 46% of proteins (
Table 1). The major determinant of protein deposition is the dietary supply of amino acids [
30]. However, it was suggested that after achieving optimal feed requirements for crude protein and lysine, birds’ growth rates reach their maximum, and further supplementation only results in a plateau effect [
31]. When feed contains equal to or more than 210 g/kg of crude proteins and 1.22% of lysine, further supplementation does not increase muscle protein deposition [
32]. Even though the crude protein level in the present study was high, the lysine concentration was below 1.22%. Furthermore, it is worth mentioning that broiler hybrids have different growth requirements and genetic potential. Thus, we speculate that higher protein content in feed led to higher amino acid content available for absorption and de novo synthesis of muscle proteins within genetically predetermined broiler potential. Apart from the aforementioned, the elevated protein level can be partially attributed to the lower-fat participation in the relative weight of meat samples from broilers fed
C. vulgaris compared to control.
Regarding fat content, the Food Advisory Committee [
33] stated that meat containing less than 5% is considered to be “lean meat”. In the present study, fat content in the breast meat ranged from 0.92 to 3.40%. However, “low fat” health claims can be applied only to breast meat from broilers fed
C. vulgaris groups that had a fat content lower than 3 g/100 g [
19,
34]. Corroborating our study, lower intramuscular fat in meat from pigs fed 2 g/d
Spirulina platensis was reported by Šimkus et al. [
35]. Namely, a lower amount of fat in the muscle is due to fatty acid modification of diets. Diets enriched with n-3 PUFA are associated with reduced fat deposition [
36]. The microalgae, including
C. vulgaris, are a source of n-3 PUFA [
9]. In a companion paper with the same experimental design (data unpublished) the amount of n-3 PUFA in breast meat from broilers fed
C. vulgaris increased between 2.6 and 5.4 times relative to control. In addition, De Tonnac et al. [
37] suggested that the inclusion of DHA in the feed inhibited the expression of sterol regulatory element-binding protein 1, a transcription factor that regulates the expression of genes encoding lipogenic enzymes, and resulted in reduced lipid contents in pig muscle. As mentioned above DHA was significantly higher in meat from broilers fed microalgae, which could influence regulation of the same or similar proteins in birds.
The increase in ash content of the meat as a consequence of Chlorella inclusion is likely due to the greater amount of minerals in meat from the broilers fed
Chlorella (
Table 4) and is related to the mineral composition levels of the different diets. This subject will be addressed later in this Discussion.
Moisture and cholesterol content did not differ among breast meat from different dietary treatments (
p > 0.05). Raw poultry meat has approximately 27 to 90 mg cholesterol/100 g [
38,
39]. In the present study, the cholesterol level from the breast meat was in that range and was not affected by dietary treatment. Such concur with results from previous studies using microalgae inclusion in broilers feed [
9,
11].
3.3. Mineral Composition and Bioaccessibility
The macro- and micromineral contents for raw and cooked breast meat are presented in
Table 6. The major minerals present in raw and cooked meats were macromineral K, followed by P, Na, and Mg. The levels of Ca, Fe, Cu, Zn, and Mn in the groups from different dietary treatments ranged from 4.69 to 6.66, 1.09 to 1.58, 0.053 to 0.065, 1.02 to 1.27, and 0.01 to 0.025 g/100 g in raw and from 4.83 to 7.22, 1.11 to 1.69, 0.13 to 0.15, 1.03 to 1.64, and 0.024 to 0.029 in cooked meat samples, respectively.
The literature data on chicken meat mineral composition are limited. Values found for the most of the measured minerals in the present study were in the ranges given by other authors for raw breast meat [
43,
44]. The K, Ca, Mg, P and Fe content of the breast were significantly increased by 15 and 20% dietary inclusion of
C. vulgaris (
p ˂ 0.05). It has been previously reported that the microalgae
C. vulgaris is rich in minerals, in particular potassium, phosphorus, magnesium, and iron [
45,
46,
47].
One of the most important findings of the present study is that microalgae inclusion in broilers diet enhanced iron content in breast meat. While red meat is the most important food source of iron in the diet, white meat is perceived as healthier than red meat by consumers [
48], despite containing a significantly lower amount of this mineral. Iron deficiency is one of the most widespread nutritional problems resulting in different pathological conditions, including anemia [
49]. The iron content in breast meat, like other minerals, was found to be strongly dependent on the
C. vulgaris inclusion. In the present study addition of 10, 15, and 20% of
C. vulgaris increased iron content by 8.7, 45.1, and 33.2% respectively, by comparison to controls, reaching an iron content similar to those of red meats. The addition of
C. vulgaris also increased the phosphorus amount in breast meat. This increase is partially explained by
C. vulgaris being a good source of phosphorus and in another part due to the chemical form of phosphorus found in microalgae. While the form of phosphorus found in plants is organic phytic acid, the phosphorus is stored in microalgae in the form of polyphosphate granules [
50]. Thus, apart from
C. vulgaris inclusion, other dietary factors in broiler feed should also be considered. In this regard, it is important to mention that soybean contains antinutritional factors including phytic acid and its salt, phytates. Phytate are heat-stable and cannot be eliminated by the heat treatment during the processing of soybean meal [
51]. In addition, the concentration of phytates is lower in the hulls than in the cotyledons, which is why dehulling and oil extraction from whole soybean will lead to an increase in phytate concentration [
52,
53]. The phytate content ranges approximately from 1.4 to 1.6% [
54] in soybean meal, and from 1.00 to 1.47% on a dry matter basis in soybean [
55], respectively. Despite being represented in low amounts, phytic acid and phytate have the capacity to chelate positively charged cations, especially calcium, iron, zinc, and magnesium, and reduce their bioavailability in poultry [
56]. Consequently, lower absorption of these elements could lead to their lower deposition in meat in the control group, where soybean meal content was higher between 10 and 20% compared to experimental groups. However, this explanation cannot apply to zinc, since contrarily to other minerals, zinc was lower in broiler meat by the addition of
C. vulgaris in diet. Although not significant, the decrease in Zn in meat could be a result of its lower absorption due to an antagonism with iron or copper [
57], which increased in meat from all groups fed
C. vulgaris by 22.64%. The decrease of Zn content was also noted by Saeid et al. [
57] in pork from pigs fed diets containing
Spirulina maxima biomass enriched with Cu. In addition to Zn, Na was the only element decreasing in meat with
C. vulgaris addition. Despite being an essential element, it is well established that high sodium intake increases blood pressure and the risk of cardiovascular disease. The WHO recommends reducing the salt intake to <5 g daily [
58]. Although meat products are labeled as “high salt source” and not meat per se, the lower sodium amount in meat contributes to overall salt reduction in the diet. All breasts had a Na content lower than 0.12 g/100 g, hence a “low sodium/salt” claim can be made [
19].
Few reports address the effect of cooking on the mineral content of chicken meat. The cooking process is important for safety and to enhance the sensory characteristics of meat [
59]. However, thermal processing influences nutrient content in meat, including minerals [
60]. In the present study, cooked breast meat showed higher Ca, Mg, P, Fe, Cu, Zn, and Mn contents and lower contents of Na and K than raw samples (
Table 6). Findings from this study agree with previously reported results. Thermal processed meat from different animal species was characterized by a higher content of most minerals, with an exception of Na and K as compared to raw meat [
60,
61,
62,
63]. Tomovic et al. [
64] reported that boiling pork loin increases the mineral content as the consequence of cooking loss, corresponding to a higher concentration of all elements. Purchas et al. [
61] reported that although cooking increased mineral content of most elements, the mineral concentration in the dry matter of raw and cooked lean was similar except for sodium and potassium, which concentrations were lower in dry matter of cooked meat. Namely, divalent minerals bind to proteins which are not likely to decrease during cooking while sodium and potassium, monovalent elements, are released from meat into meat juice. Thus, in our study the relative increase of divalent elements in cooked meat is due to loss of meat juice during cooking and increase level of proteins in relative weight of the cooked sample compared to the raw one.
In the present study, considering the contribution of iron to the diet, an intake of 100 g of cooked breast meat would provide between 1.11 (control) and 1.69 (CV15%) mg/100 g, corresponding to 7.93–12.07% of the daily reference intakes (RDI) for adult women and men. Furthermore, the intake of 100 g of cooked breast would provide between 18.17-(C) and 19.43% (CV20%) K and between 34.28% (CV10%) and 35.98% (CV20%) P of daily reference intakes (RDI) for adults [
19]. For P and K, chicken breast provides at least 15% of the reference daily intake, therefore, it could be labeled as “source of P and K” according to regulations EC No 1924/2006 and EU No 1169/2011. Furthermore, the amount of Cu in cooked broilers breast from CV 20% group is reaching 15% of RDI for this mineral.
Although the determination of mineral content in raw and cooked meat certifies that the matrix is a good source or high in content of particular minerals, the contents of minerals available from food sources to humans is evaluated by their bioaccessibility. Minerals bioaccessibility, expressed as the percentage of mineral remaining in the digesta (fraction obtained after in vitro digestion using INFOGEST protocol), are presented in
Figure 1.
An increasing trend of bioaccessibility was observed for most of the minerals in meat when
C. vulgaris was included in broilers feed, with exception of Cu. There was no significant difference (
p > 0.05) in Fe, Zn, and Cu bioaccessibility for the control breast meat and meat from broilers fed
C. vulgaris. Results presented in
Figure 1 indicate that the minerals are only partially available to be absorbed by the human organism. Higuera et al. [
65] reported that the mineral bioaccessibility in lamb meat decreased by cooking process. Namely, denaturation of proteins can create additional binding sites between protein and metals, leading to metal trapping, thus reducing bioavailability. Additionally, some amino acids such as lysine, methionine, phenylalanine, histidine, and cystine have an affinity for ion [
66]. The meat from this study was rich in those amino acids (
Table 5), in particular lysine. Meat from the control group had higher amount of lysine and cystine compared to CV15% and CV20% groups which can partially be responsible for lower bioaccessibility of aforementioned elements in meat from control.
Mn bioaccessibility significantly increased by
C. vulgaris addition (
p ≤ 0.05). Mn was the mineral with the highest bioaccessibility, reaching up to 99%, with the highest bioaccessible fraction being achieved for breast meat from broilers fed 20%
C. vulgaris. Foods from plant origin are the main source of Mn in human diet [
67]. However, considering the present dietary role of meat and its average daily intake, Mn in meat is important since this essential mineral influences growth, enzymatic defense systems against oxidation and immune system [
68,
69]. Thus, any strategy leading to increase bioaccessibility would provide significant contributions to the amount of Mn ready to be absorbed. After Mn, the most bioaccessible minerals were Mg, P, and Fe. Furthermore, analysis of the effects of
C. vulgaris dietary inclusion on meat mineral bioaccessibility showed that despite the dose-dependent increase observed, only the addition of 20% of this microalga significantly increased (
p ≤ 0.05) K, Ca, Mg, and P bioaccessibility (
p ≤ 0.05) compared to control. Although the inclusion of lower amounts of microalgae in broiler feed did not affect mineral bioaccessibility, it should be highlighted that cooked meat from groups fed
C. vulgaris had higher concentration of minerals; thus, greater concentration of Fe, P, K, Ca, Mg, and Mn was available for absorption in the body, compared with the control meat. In addition, even if the amount of Zn was higher in control meat, the bioaccessible concentration in the digesta was similar to those in
C. vulgaris groups, since the bioaccessibility (%) for this mineral was higher in meat from broilers fed this microalga.
It is difficult to compare values of in vitro digestion between studies due to different digestion protocols and enzymes used, different duration of digestion, sample preparation, and sample chemical compositions and method used to calculate the digestibility. In the case of mineral digestion, bile salts are an additional factor that has to be considered. While Rousseau et al. [
70], found that Zn bioaccessibility was drastically reduced by adding bile salts in comparison to in vitro digestion where only enzymes were used, Muleya et al. [
71] reported that not only bile salt but pancreatine can also interfere in Zn and Fe mineral binding and decrease bioaccessibility. Those authors showed that isotopic labeling of reagent iron and zinc is appropriate to accurately determine their bioaccessibility in grains and legumes based on a modified INFOGEST in vitro digestion method. Muleya et al. [
71] also emphasized that saturated solutions of pancreatin and bile used in the INFOGEST in vitro digestion method, precipitate during centrifugation with the potential to adsorb metals into the residue. They suggest that traditional method for calculation of mineral bioaccessibility can either overestimate or underestimate Zn and Fe bioaccessibility leading to misinterpretation of results. No data on microalgae dietary regime on meat digestibility is available nor was the INFOGEST method used to evaluate mineral bioaccessibility from chicken meat; thus, it is not possible to compare our results with existing literature. Da Silva et al. [
72] reported that Fe bioaccessibility was greater than 80% in baby food samples that contained chicken meat. Those findings are in line with those obtained in present study (84.03–88.33%;
Figure 1). In an opposite trend, Menezes et al. [
73] reported that the bioaccessibility in thermally processed chicken for Ca, Cu, Fe, Mg, and Zn ranged between 8 and 22%, 8 and 12%, 8 and 16%, 10 and 26%, and 8 and 16%, respectively. However, these authors used dialyzed fraction while in the present study soluble fraction was used. Câmara et al. [
74], compared dialysis and solubility methods to determine bioaccessibility of minerals in school meals, including dishes containing chicken (chicken with vegetable stew and chicken in sauce). These authors found that the percentage of mineral solubility was higher for Fe, Zn, and Cu than dialyzed mineral percentage because the mineral may be bound to compounds of molecular sizes in excess of the pore size of the dialysis membrane. This could explain the difference between studies.