1. Introduction
Microalgae are a diverse group of photosynthetic organisms that are known as a source of valuable bioactive compounds with numerous benefits for applications in the pharmaceuticals, food, cosmetics, and bioenergy sectors [
1].
Chlorella vulgaris is a unicellular green microalga in the division Chlorophyta and a rich source of high-quality protein due to the important concentrations of essential amino acids. The protein content can range between 42 and 65.5% [
2,
3], with a lysine level of up to 13.2% [
4].
C. vulgaris also contains important amounts of lipids, with a high content of polyunsaturated fatty acids and carbohydrates, minerals, vitamins, phenolic compounds, and pigments, such as chlorophylls and carotenoids [
5,
6]. During the final stage of their metabolism, microalgae undergo a transition to the carotenoid production stage, accumulating elevated quantities of pigments [
7]. Carotenoids frequently present in
C. vulgaris biomass include β-carotene, a precursor of vitamin A, along with lutein, astaxanthin, canthaxanthin, and violaxanthin. Chlorophylls are the predominant pigments within
C. vulgaris cells, surpassing synthetic dyes and being a preferred alternative for food and cosmetic applications [
8]. Additionally, chlorophyll exhibits therapeutic properties that are beneficial for ulcer treatment and liver recovery, promoting enhanced cell growth and repair [
9]. Carotenoids and chlorophylls possess notable antioxidant and radical scavenging activity and have transitioned from being regarded solely as bioactive compounds to emerging as biomarkers signaling the onset of diseases associated with oxidative stress [
10]. Moreover,
C. vulgaris cells contain phenolic compounds recognized for their antioxidant, antifungal, and antibacterial activity. Additionally, it has been reported that the presence of phenolics in
C. vulgaris formulations contributes to their antidiabetic effects [
11].
Microalgae cultivation is recognized as an environmentally friendly process [
12] because it requires simple conditions (CO
2 and sunlight) and can be carried out in non-agricultural lands. Furthermore, microalgae cultivation does not necessitate the use of pesticides, unlike conventional crops, and it yields higher production outputs [
13]. As photosynthetic organisms, microalgae efficiently convert atmospheric carbon dioxide into high-value compounds, playing a role in reducing CO
2 levels in the atmosphere [
14].
Due to its rich composition of essential nutrients, including high levels of natural antioxidants,
C. vulgaris has been used in the nutraceutical industries as a replacement for synthetic antioxidants [
15], and also in animal feeding as an alternative sustainable high-quality protein feed resource [
16]. The growth of the poultry industry increases the demand for agricultural output for feed production. Proteins are the most expensive and most critical components in feed formulation. To address these issues, substantial efforts have been directed towards identifying alternative sustainable high-quality protein feed resources.
C. vulgaris emerges as a valuable source of protein and other nutrients for animal feeding [
17]. Additionally, its abundance of biologically active compounds makes these organisms highly appealing as feed ingredients, offering benefits that extend beyond mere nutrient supply [
18]. Moreover,
C. vulgaris shows potential to mitigate the environmental footprint of poultry production. Studies have shown that incorporating
C. vulgaris into 10-day-old broilers’ diets can result in enhancing growth performance, gut functional characteristics, and bacterial communities [
19]. Moreover, [
20] explored the potential of
C. vulgaris as a candidate alternative to antibiotics in broiler chickens, revealing its influence on affecting the humoral immune response in broiler chicks. The impact of
C. vulgaris in broilers’ feed on the digestibility and mineral bioaccessibility in meat has been studied in [
21], showing an improved nutritional composition of meat in terms of minerals and essential amino acid content.
This study aimed to assess the impact of C. vulgaris supplementation in broilers’ diet, alone or in combination with vitamin E, on meat quality parameters, nutritional value, and oxidative stability during storage time.
2. Materials and Methods
The experimental trial was carried out with the approval (no. 2761/03.03.2021) of the Ethics Committee of the National Research and Development Institute of Animal Biology and Nutrition in Romania. The experimental procedures followed the guidelines specified in Romanian Law 43/2014 regarding the handling and protection of animals used for experimental purposes and complied with Directive 2010/63/EU on the protection of animals used for scientific purposes.
2.1. Experimental Design
An experiment was conducted on 180 COBB 500 broiler chickens (14 days old), assigned into six treatments, following a 2 × 3 factorial arrangement. The initial average weight of birds at 14 days of age was 421.48 g. A corn–soybean meal diet was supplemented with 3 levels of
C. vulgaris (0% (C1), 1% (E1), 2% (E2)), 2 levels of vitamin E (0% (C1), 250 ppm (C2)), and a combination of the two supplements (1%
C. vulgaris + 250 ppm vitamin (E3), 2%
C. vulgaris + 250 ppm vitamin (E4)), as presented in
Table 1. Each group included five replicates with six birds per replicate and thirty chicks per treatment. The plant material used in this study consisted of a powder of microalga
Chlorella vulgaris, supplied by Vivio (Brzozów, Poland).
The chicks were housed in digestibility pens in a poultry experimental hall with controlled environmental conditions (average temperature/total period: 25.08 ± 0.58 °C; humidity: 61.27 ± 4.79%; ventilation/broiler: 0.61 ± 0.16%). The light regimen was 23 h light/1 h darkness. The chicks had free access to feed and water. At the end of the feeding trial, 6 broilers (42 days old) were randomly selected from each treatment group and slaughtered by cervical dislocation. The thigh meat samples without skin were collected to assess the impact of the dietary supplements on the meat quality in terms of proximate composition, vitamin E, fatty acid profile, and oxidative stability parameters. Samples were stored in plastic bags at −80 °C until further analysis. Instrumental color measurements and pH assessment were performed on fresh meat samples.
2.2. Determination of Meat Quality Traits
The pH was measured on thigh muscles from the right side of the bird using a glass penetration pH electrode (HI9025, Hanna Instruments, Woonsocket, RI, USA) at 45 min postmortem. The pH value was determined as the average of three replicate measurements taken on the same muscle. The standard color parameters, lightness (L*), redness (a*), and yellowness (b*), were determined for thigh meats with a Minolta CR-400 Chroma Meter (Minolta Camera Co. Ltd., Osaka, Japan). The final CIELAB color parameters were the average of 3 readings, taken from 3 spots at the surface of thigh meats.
The chroma value (C*) was calculated as explained in [
22], using the following equation:
where a* = redness and b* = yellowness.
The hue angle (h*) was assessed with the following formula:
where a* = redness and b* = yellowness.
The total color difference (ΔE*) in the thigh meat samples between the control diet (C1) and the experimental diets was calculated according to the following equation:
where L* = lightness, a* = redness, and b* = yellowness.
Cooking loss was assessed as described in [
23]. Individual samples of thigh meat with similar weight and size were packed in plastic bags and cooked in a water bath (Memmert, Schwabach, Germany) for 30 min at 85 °C. After cooling at room temperature, the samples were dried with a paper towel and reweighed. The cooking loss was calculated by the following equation:
where W1 = initial sample weight before cooking and W2 = sample weight after cooking.
To evaluate drip loss, samples of fresh thigh muscle were weighed (W1), placed into a plastic bag, and hung in a refrigerator at 4 °C for 24 h [
24]. Subsequently, samples were dried and weighed again (W2), using the following equation:
where W1 = initial sample weight and W2 = final sample weight.
2.3. Chemical Analysis
2.3.1. Proximate Composition
The proximate composition of
C. vulgaris and meat samples was determined using the reference method as described below: crude protein (ISO 5983-2/2009 [
25]) with the Kjeldahl method and a semiautomatic Kjeltec auto 1030 Tecator Instruments (Höganäs, Sweden), crude fat (SR ISO 6492/2001 [
26]) with the method of continuous solvent extraction and the equipment Soxtec 2055 Foss Tecator (Höganäs, Sweden), crude fiber using the intermediary filtration method and the equipment Fibertec 2010 System Foss Tecator (Höganäs, Sweden), and dry matter (ISO 6496/2001 [
27]) and ash (ISO 2171/2010 [
28]) using the gravimetric method and a Nabertherm calcination furnace (Nabertherm GmbH, Lilienthal, Germany).
2.3.2. Fatty Acid Determination
The fatty acid profiling of
C. vulgaris and thigh meat was assessed as outlined in [
29]. Fatty acids (FAs) were converted into FA methyl ester (FAME). Afterward, derivatives of FAs were analyzed using a Perkin Elmer Clarus 500 gas chromatograph (Waltham, MA, USA) equipped with a capillary column with a highly polar stationary phase (Thermo Electron, Waltham, MA, USA), with dimensions of 60 m × 0.25 mm × 0.25 µm film. Detection was conducted using a flame ionization detector (FID), and identification and quantification were achieved by referencing analytical standards.
Lipid quality indexes in thigh meat were determined based on the data obtained from the fatty acid composition of thigh samples, and their relevance to human nutrition was assessed. They were calculated using the following equations [
30,
31,
32,
33,
34,
35]:
2.3.3. Liposoluble Antioxidants
The liposoluble antioxidants (vitamin E and xanthophylls) were analyzed as previously described in [
36]. The extraction procedure involved saponification with ethanolic KOH and extraction with petroleum ether. The final extract was washed with distilled water to remove any alkaline traces and evaporated under vacuum until dry. The concentrated extracts were dissolved in ethanol.
Xanthophylls were analyzed using an HPLC (Finningan Surveyor Plus, Thermo-Electron Corporation, Waltham, MA, USA) with a PDA-UV detector at wavelength 445 nm, and a C18 reversed-phase column with a stationary phase of 5 µm (250 × 4.60 mm i.d.) (Nucleodur, Macherey-Nagel, Duren, Germany). The chromatographic analysis was performed in isocratic conditions, involving a mobile phase which consists of 87% acetone and 13% water, at a flow rate of 1 mL/min.
Vitamin E was analyzed using an HPLC (Vanquish Thermo-Electron Corporation, Waltham, MA, USA), a PDA-UV detector at wavelength 292 nm, and a HyperSil BDS C18 column, with silica gel, dimensions 250 × 4.6 mm, and particle size 5 µm (Thermo-Electron Corporation, Waltham, MA, USA). The mobile phase used was 96% methanol and 4% water, with a flow rate of 1.5 mL/min, in isocratic conditions.
2.3.4. Water-Soluble Antioxidants
The total polyphenol content (TPC) was measured using the Folin–Ciocalteu spectrophotometric method [
30]. A calibration curve of gallic acid was employed to quantify the total phenol content, and the results were expressed as mg of gallic acid equivalents per g of dried sample (mg GAE/g).
The total flavonoid content was determined using the aluminum chloride colorimetric method described in [
30]. The absorbance was measured at 410 nm against a blank using a UV-VIS spectrophotometer (Jasco V-530, Japan Servo Co. Ltd., Tokyo, Japan). A calibration curve with quercetin as the standard was used and the flavonoid content was expressed as mg of quercetin equivalent (QE) per g.
2.3.5. Antioxidant Capacity Analysis
The antioxidant capacity of
C. vulgaris was evaluated using two different spectrophotometric methods for the determination of DPPH and iron chelating ability. A DPPH solution prepared in methanol (0.2 mM) was mixed with the sample extract and distilled water in a ratio of 2:0.4:1.6 (
v/
v/
v). The absorbance was then measured at 517 nm using a spectrophotometer (Jasco V-530, Japan Servo Co., Ltd., Tokyo, Japan). Trolox was used as a reference for standard calibration curves to analyze the DPPH concentration [
37]. The results were expressed as mmol of Trolox equivalents per kg of sample (mmol eq Trolox/kg sample).
To assess the chelating effect on ferrous ions, a method described by [
38] was used. The method involves measuring the absorbance of the purple complex formed when an extract competes with ferrozine for ferrous ions. This method is based on measuring the absorbance of the purple complex formed when an extract competes with ferrozine for ferrous ions. The absorbance was measured using a UV-VIS spectrophotometer (JASCO V-560, Japan Servo Co. Ltd., Tokyo, Japan) at 562 nm, compared to a blank.
2.3.6. Oxidative Stability of Meat
The oxidative stability of the thigh meat samples was assessed by evaluating primary lipid degradation parameters, including peroxide values (PVs), conjugated dienes (CDs), and conjugated trienes (CTs), as well as secondary parameters such as p-anisidine values and thiobarbituric acid-reactive substances (TBARSs). These measurements were conducted using previously described methods [
39].
Additionally, markers of lipid peroxidation included myoglobin derivatives (metmyoglobin, deoxymyoglobin, and oxymyoglobin). All the aforementioned parameters were measured spectrophotometrically using a V-530 Jasco spectrophotometer (Tokyo, Japan), following the methods described in [
39].
2.4. Relative Prevention of Lipid Oxidation—Efficiency Factor
The relative inhibition of lipid oxidation was quantified as described in [
40], as an efficacy ratio on day 7 of storage, calculated by dividing the PV or TBARS value of the control group by that of the experimental groups. A higher efficiency factor indicates greater effectiveness of the dietary treatments in decreasing lipid oxidation (PV or TBARS) in the thigh meat of broilers.
2.5. Statistical Analysis
The analytical data regarding the nutritional composition, lipid quality indexes, and markers of lipid peroxidation assessed in thigh meat samples were analyzed using a 2-way ANOVA, followed by Tukey’s HSD test, with XLStat (Addinsoft, New York, NY, USA). The statistical model included the fixed effects of vitamin E (0 and 250 ppm) and C. vulgaris (0%, 1%, and 2%) as well as the interaction between them. p-values less than 0.05 were considered significant. Each broiler that was chosen for collecting samples of thigh meat was treated as an experimental unit.
3. Results
3.1. Nutritional Composition of C. vulgaris
The results regarding the nutritional composition of
Chlorella vulgaris are presented in
Table 2.
C. vulgaris was found to be a rich source of essential nutrients, including proteins, fatty acids, vitamins, and antioxidants. The nutritional composition analysis highlighted the high protein content of the analyzed microalga, making it a valuable source of high-protein nutritional supplements. The antioxidant analysis of C. vulgaris showed elevated concentrations of vitamin E and xanthophylls, which increase the antioxidant capacity and enhance the nutraceutical potential of C. vulgaris.
The FA profile of
Chlorella vulgaris allowed for the identification of 14 FAs (
Table 2), including saturated (C12:0, C14:0, C15:0, C16:0, C17:0, C18:0 and C20:0), monounsaturated (C14:1, C15:1, C16:1, C17:1 and C18:1), and polyunsaturated (C18:2, C18:3) FAs. The primary fatty acids of
Chlorella were linoleic, palmitic, alpha-linolenic, oleic, heptadecenoic, and heptadecanoic acids.
3.2. Meat Quality Traits
The effect of dietary treatments on the meat quality traits of broilers is shown in
Table 3. Thighs from birds fed with diets supplemented with
C. vulgaris and vitamin E (E3 and E4 groups) had significantly higher values of pH than birds from the control group (C1). Thigh lightness (L*) registered a slight increase in the E3 and E4 groups compared to the control, but the values were not statistically different.
Dietary incorporation of C. vulgaris, including those supplemented with vitamin E, resulted in a minor but statistically significant (p < 0.05) increase in b*, C*, and h*, while a* registered lowered values (p < 0.05) when compared to the control group. Nevertheless, the calculated color differences (ΔE*) between the samples of the studied groups showed a minor variation, which led to the conclusion that no differences in color could be observed. Neither cooking loss nor drip loss was affected by dietary treatments (p > 0.05) in thigh meat.
3.3. Proximate Composition of Meat
The effect of dietary microalgae on the proximate composition of thigh meat is presented in
Figure 1. The incorporation of 1 and 2%
C. vulgaris, alone or with vitamin E, resulted in an increase in protein content in the meat (
p < 0.0001). Ash content was increased in all the groups, although no significant differences were found between them, except for the 2%
C. vulgaris and vitamin E group (E4). Fat content was significantly lower (
p < 0.0001) in the thigh muscle of broilers that were fed a diet with
C. vulgaris or vitamin E or a combination of them, in comparison to those fed the control diet.
3.4. Fatty Acid Profiling of Meat
The effect of
C. vulgaris incorporation, alone or combined with vitamin E, on the fatty acid composition of thigh meats is presented in
Table 4. Dietary treatments induced changes in the fatty acid profile of thigh meat. The percentage of palmitic acid was lower (
p < 0.0001) in the thighs of broilers fed
C. vulgaris and
C. vulgaris + vitamin E diets. Chlorella-fed broilers had a higher concentration of α-linolenic, erucic, and eicosatrienoic acids (
p < 0.0001) in their thigh meat compared to the control group. Linolenic acid was detected in a small amount only in groups supplemented with
C. vulgaris and vitamin E (E3 and E4 groups). The proportions of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) were enhanced in the thighs of broilers fed with
C. vulgaris, with significant increases being observed in the group supplemented with 2%
C. vulgaris and vitamin E (E4). In fact, both DPA and DHA levels increased by 2.01-fold and by 1.60-fold in the 2%
C. vulgaris + vitamin E group (E4). Moreover, the total amount of omega-3 FAs was higher in the thighs of broilers fed with
C. vulgaris.
Concerning the fatty acid ratios, a significantly decreased n-6/n-3 ratio (p < 0.05) in the thigh muscle was observed in groups supplemented with 2% C. vulgaris, alone or with vitamin E (E2 and E4 groups), and also in the 1% C. vulgaris with vitamin E group (E3), when compared with the control. The PUFA/SFA ratio was increased across all dietary treatments (p < 0.0001). Both DFAs and OFAs were influenced by the C. vulgaris supplementation. The highest percentage of hypocholesterolemic fatty acids (DFAs) and the lowest of hypercholesterolemic fatty acids (OFAs) were shown for thighs from the C. vulgaris supplementation groups. The DFA/OFA index registered the highest increase in the 2% C. vulgaris with vitamin E group (E4).
3.5. Lipid Quality Indexes
To estimate the lipid nutritional and health benefits of
C. vulgaris, alone or combined with vitamin E, the lipid quality indexes were calculated (
Table 5). The values of oxidative susceptibility (OS), double-bond index (DBI), iodine value (IV), and peroxidisability index (PI) from meat samples of experimental groups presented a significant (
p < 0.05) increase compared to the control group. A decreasing tendency was observed in the AI of the thighs from broilers fed with
C. vulgaris, a significant reduction (
p < 0.05) being observed in the groups with 2%
C. vulgaris supplementation (E2 and E4). In the same manner, the TI registered the highest significant (
p < 0.05) decrease as an effect of the 2%
C. vulgaris supplementation in broilers’ diet (E2 and E4). The h/H values were significantly higher in
C. vulgaris-supplemented groups when compared to the two control groups. The calculated oxidizability value (COX) was also measured for all meat samples. The results showed a significant increase (
p < 0.05) in the COX value in all the groups, except the E3 group where the differences were not significantly higher. Therefore, the thigh meat from the experimental groups (including C1 and excluding E3) exhibits better oxidative stability and thus has a longer shelf life.
The health-promoting index (HPI) enables the assessment of the impact of fatty acids on cardiovascular diseases. It represents the inverse of the atherogenicity index, indicating inverse values compared to the AI, and, in the present study, registered a favorable increase in the groups with supplemented C. vulgaris. The significant variation in values obtained from the different indexes highlights their distinct meanings and the necessity of using each index under specific conditions.
3.6. Vitamin E Concentration in Thigh Meat
The effect of feeding treatments on the thigh meat content of vitamin E is presented in
Table 6. The concentration of vitamin E was influenced by feeding treatments (
p < 0.05), showing a significant increase in all the groups compared to the control group (C1); the increase ranged between 21.7 and 44.69%. Supplementation of broiler diet with 2%
C. vulgaris and 250 mg/kg vitamin E exhibited a 1.45-fold increase in vitamin E concentration in thigh meat compared to the control group, being the highest level registered in thigh meat in this experiment.
3.7. Oxidative Stability of Meat
Supplementation of broilers’ diets with
C. vulgaris, alone or combined with vitamin E, had no significant effect on the primary oxidation products in thigh meat stored for 7 days in refrigerated conditions (
Table 7), although a decreasing tendency was observed for conjugated dienes, conjugated trienes, and peroxide value in the experimental groups compared to the control group (C1). Regarding the secondary oxidation products, TBARS levels were significantly reduced (
p < 0.05) in the experimental groups relative to the reference group (C1) after 7 days of refrigeration. For p-anisidine, lower concentrations were also observed in the experimental groups, but without statistical significance.
Incorporation of
C. vulgaris, alone or combined with vitamin E, in broilers’ diet led to changes in the hem pigments in thigh meat stored for 7 days in refrigerated conditions (
Figure 2). The results showed a significant increase (
p > 0.05) in the deoxymyoglobin percentage in all the experimental groups compared to the control group, with no significant differences observed between the experimental groups. Another positive effect was the decrease in metmyoglobin concentrations in the thigh meat of the experimental groups, with significant differences noted (
p < 0.05) when compared to the control group. The oxymyoglobin levels did not register any significant differences between the studied groups.
Figure 3 shows the efficiency factor of the dietary treatments in relation to PV and TBARS measurements on day 7 of refrigerated storage of thigh meat. A higher efficiency factor indicates a greater ability of the antioxidants to reduce lipid oxidation (as measured by PV or TBARS) in the meat samples.
After 7 days of storage in refrigerated conditions, thigh meat from the group receiving a diet supplemented with 2% C. vulgaris and vitamin E (E4) showed the highest efficiency factor (lowest degree of lipid oxidation compared to the control group C1), followed by the group fed a diet with 1% C. vulgaris and vitamin E (E3), for both PV and TBARS measurements. The inclusion of C. vulgaris (1% and 2%) in combination with vitamin E (250 mg/kg) in broiler diets exhibited the best prevention of lipid oxidation after 7 days of refrigerated storage, defined by the highest efficiency factors assessed in terms of secondary oxidation products.