3.1. Proximal chemical analysis of raw materials
The proximate chemical analysis of the flours and gelatin is presented in Table 4.
Table 4
Proximal chemical analysis of raw materials used in the formulation of an extruded food.
Raw materials | Moisture (%) | Lipids (%) | Crude fiber (%) | Protein (%) | Ash (%) | Carbohydrates (%) |
Moringa flour | 7.32 ± 0.06b | 9.89 ± 0.21c | 2.12 ± 0.13c | 20.24 ± 0.43b | 11.53 ± 0.07c | 48.90 ± 0.21b |
Sardine meal | 6.82 ± 0.05a | 9.37 ± 0.37c | 1.18 ± 0.05b | 66.67 ± 0.29c | 15.67 ± 0.06d | 0.43 ± 0.08a |
Corn flour | 10.89 ± 0.09d | 5.10 ± 0.15b | 1.76 ± 0.60bc | 5.22 ± 0.02a | 0.32 ± 0.01a | 76.71 ± 0.36c |
Gelatin | 9.10 ± 0.10c | 1.48 ± 0.57a | 0.00 ± 0.00a | 88.38 ± 0.27d | 1.09 ± 0.03b | 0.17 ± 0.06a |
Equal letters in the same column indicate that there is no statistically significant difference (P ≤ 0.05).
Statistically significant differences (p ≤ 0.05) were observed in the protein content across all raw materials. The highest content was noted in gelatin, which is primarily used as a binder rather than a protein source due to incorporation challenges during the extrusion process if used in excess (Barreto-Curriel et al., 2018). Sardine meal exhibited high protein content, establishing its role as the principal protein source. This is attributed to its complete essential amino acid profile and good nutrient digestibility (Daniel, 2018). Moringa flour also presented significant protein content (20.24%), higher compared to other vegetable proteins such as wheat, barley, quinoa, amaranth, and rice (6.7% − 16.49%). Thus, it can be employed as a protein source to partially substitute sardine meal in aquafeed, considering its unique amino acid profile not commonly found in other vegetable flours (Awadalkareem et al., 2008; Mota et al., 2016; Awadelkarim et al., 2018; Manuel et al., 2019; Abdel-Latif et al., 2022).
Moringa flour and sardine meal demonstrated the highest lipid content relative to the other raw materials analyzed. The lipid values for moringa flour were similar to those reported by Nurcahyani et al. (2019), affirming it as a viable lipid source for aquafeed, fulfilling the requirements for Oreochromis niloticus. In contrast, corn flour displayed higher lipid values compared to the 1.7% reported by Sánchez et al. (2014). The ash content was notably high for sardine meal but lower than the approximately 21% reported for fishmeal by Tarhouni et al. (2019)d rez-Viveros (2017). The highest crude fiber content was observed in moringa flour (2.12%), albeit lower than values reported by Abiodun et al. (2012) and Adewumi (2014), who recorded 7.73% and 7.9% respectively. This result is ideal for aquafeed formulation, where diets with less than 8% fiber are preferred as higher fiber content could affect the expansion of extruded feeds (Mondal et al., 2016; Martin et al., 2019).
Moisture levels varied significantly (p ≤ 0.05) across all raw materials, with the highest being found in corn flour (10.89%), a result lower than the 12.43% reported by Pérez-Viveros (2017). With the exception of corn flour, the moisture content of all ingredients was less than 10%. However, according to Magan and Aldred (2007), the suitable moisture percentage for corn storage is 14%. The ash content also varied among all raw materials, with sardine meal having the highest value (15.67%), akin to the 16.13% reported by Kirimi et al., (2019). This high ash content could be attributed to the mineral content and presence of inorganic compounds in the raw materials, such as head bones, backbone, and viscera (Janbakhsh et al., 2018). Lastly, corn flour exhibited the highest carbohydrate values (76.71%), similar to the 81.6% reported by Sánchez et al. (2014). Moringa also had high carbohydrate content (48.90%), comparable to the values reported by Teixeira et al. (2014) and Idris et al. (2017), who reported values of 44.3% and 44.4%, respectively. Both these sources can potentially be used as an energy source for Oreochromis niloticus, considering reports indicating the successful utilization of up to 30% carbohydrates in the feeding of this species (Boonanuntanasarn et al., 2018).
The use of moringa flour as a partial substitute for sardine meal in aquafeed presents an efficient strategy for producing sustainable and high-quality feed, while addressing the challenges posed by overfishing and limited resource availability.
3.2. Physicochemical characterization of the extruded aquafeed
The impact of sardine meal and moringa flour concentrations, as well as moisture and temperature on the expansion index (EI), bulk density, water solubility index (WSI), water absorption index (WAI), hardness, and buoyancy, are illustrated in Fig. 1 and Fig. 2 respectively.
Based on the physicochemical characterization of the extruded aquafeed, the EI was negatively influenced by the higher concentration of sardine meal (36.56%) and moringa flour (42.91%) (Fig. 1a). This effect may be attributable to the lipid and fiber content of the raw materials (Table 4), especially the moringa flour, which contains a significant amount of lipids. These lipids interfere with the shear force, resulting in less mechanical damage to the starch and fiber chains and thereby lower expansion (Delgado et al., 2021), and consequently, a high bulk density of extruded aquafeeds (Fig. 1b). The extrusion process conditions also affected the EI; a decrease was observed at lower temperatures and higher moisture (Fig. 2a). The water in the mixture acts as a lubricant, increasing the outflow from the extruder, reducing barrel pressure and the mechanical shear of the screw, resulting in lower expansion of the extruded aquafeed (Neder-Suárez et al., 2021).
WSI was greater when the concentrations of sardine meal and moringa flour were lower (Fig. 1c); this suggests increased structural disorganization of the corn flour starch used in aquafeed formulation, generating shorter chains of amylose and amylopectin, which is beneficial for assimilation (Samuelsen et al., 2013). The temperature and moisture used in the extrusion process (Fig. 2c) caused a decrease in WSI at higher extrusion temperatures and higher processing moisture. This decrease is attributable to the extrusion process, wherein higher water content reduces the mechanical shear of the screw inside the barrel, leading to less structural disorganization of the starch and fibers (Singh et al., 2016; Yousf et al., 2017; Ye et al., 2018; Delgado et al., 2021).
WAI increased as the concentrations of sardine meal and moringa flour decreased in the aquafeed, likely due to the reduction of lipids in the mixture, which allows for greater damage to the structure of the starch and thus a higher capacity for water absorption (Delgado et al., 2021). For WAI, it was shown that an increase in the moisture content of the mixture and the processing temperature (16% and 132.5°C respectively) correlates with higher WAI values (4.01 g water/g sample). These conditions permit partial gelatinization of starch, releasing amylose and amylopectin chains, which increase the ability of the extruded feed to retain water (Singh et al., 2007; Neder-Suárez et al., 2020).
Aquafeeds with higher contents of moringa flour and sardine meal (42.91% and 36.56% respectively) showed increased hardness values (Fig. 1e), leading to more compact extruded feeds, which was corroborated by the EI results (Fig. 1a). This was associated with a decrease in water absorption capacity (Fig. 1d), and an increase in bulk density (Fig. 1b) (Samuelsen et al., 2013; Ahmad, 2019). The buoyancy of the extruded aquafeeds was inversely proportional to the hardness results, with lower buoyancy percentages observed when the hardness of the aquafeed increased (Figs. 1e,f). The moisture content during extrusion had a negative impact on the hardness of the extruded aquafeeds. This is attributed to the smaller amount of water in the mixture, which forms fewer pores at the extruder outlet due to minimal evaporation of water, resulting in compact extrudates (Kannadhason et al., 2009; Wang et al., 2021). This condition resulted in a decrease in buoyancy percentages (Figs. 2e,f).
The observed impacts on physicochemical properties of the aquafeed suggest that both the moringa flour and sardine meal concentration, as well as the extrusion conditions, play a pivotal role in defining the quality of the final product. The obtained aquafeeds exhibited enhanced water solubility index (WSI) values, and hardness, suggesting that the replacement of sardine meal with moringa flour does not compromise the feed's physical properties, on the contrary, it can be beneficial in the application.
3.3 Optimization and validation of extruded aquafeed
Table 5 presents the characterization of the optimal diets for generating aquafeed, as well as commercial aquafeeds 1 and 2.
Table 5
Physicochemical characterization of optimal extruded diets and commercial feeds.
Diet and control | EI | Bulk density (g/cm3) | WSI (%) | WAI (g water/g sample) | Hardness (N) | Buoyancy (%) |
Diet 2 | 1.68 ± 0.04b | 0.54 ± 0.02 a | 13.68 ± 0.24c | 3.69 ± 0.06c | 29.63 ± 2.78b | 100 ± 0.00b |
Diet 96 | 1.55 ± 0.03a | 0.68 ± 0.02 c | 12.40 ± 0.60b | 3.32 ± 0.12ab | 32.01 ± 3.05c | 100 ± 0.00b |
Diet 53 | 1.74 ± 0.04c | 0.53 ± 0.03 a | 13.38 ± 0.48bc | 4.04 ± 0.09d | 30.97 ± 3.00bc | 100 ± 0.00b |
Diet 16 | 1.66 ± 0.04b | 0.61 ± 0.03 b | 12.47 ± 0.30b | 3.50 ± 0.08bc | 29.80 ± 2.94b | 100 ± 0.00b |
Control 1 | ND | ND | 8.06 ± 0.55a | 3.16 ± 0.16a | 12.21 ± 1.22a | 86.67 ± 5. 77a |
Control 2 | ND | ND | 8.43 ± 0.31a | 3.53 ± 0.01bc | 48.85 ± 4.86d | 100 ± 0.00b |
Equal letters in the same column indicate that there is no statistically significant difference (P ≤ 0.05).
ND: Not determined, EI: Expansion index, WSI: Water solubility index and WAI: Water absorption index.
Control 1: commercial aquafeed with 2.4 mm of diameter.
Control 2: commercial aquafeed with 3.5 mm of diameter.
The data regarding the optimal diets analyzed (Table 5) illustrate that a lower expansion index (EI) correlated with a higher bulk density. The water solubility index (WSI) values obtained in all optimal diets exceeded those of controls 1 and 2 (8.06% and 8.43%). The WSI values of diets 96 and 16 (12.40% and 13.36%, respectively) resembled those projected in the numerical optimization presented in Table 3 (12.49% and 13.59%, respectively). Meanwhile, the values for diets 2 and 16 (13.67% and 12.47%) diverged from the predictions in Table 3 (11.90% and 14.02%). These variations in solubility could stem from the incorporation of sardine meal and moringa flour as protein sources, which also contribute fiber content to the final composition of the aquafeed (Yousf et al., 2017), and from the processing conditions. As reported by Kallu et al. (2017), the fiber content in an extruded food impacts its solubility. Increased solubility in aquafeed is beneficial because it signifies good digestibility of the starch from corn flour, which correlates to its degree of gelatinization and structural disorganization during the extrusion process (Ye et al., 2018).
The water absorption index (WAI) results for optimal diets, except for diet 96, demonstrated statistically significant differences compared to control 1. The WAI of diets 2, 16, and 53 (3.69, 3.5, and 4.04 g water/g sample, respectively) were statistically similar to control 2 (3.53 g water/g sample). These values are adequate for the application of aquafeed as they correlate to the greater presence of short starch chains post-extrusion, which is promoted by structural disorganization (Lu et al., 2019). Moreover, the fiber content in the diet also affects the absorption capacity of the aquafeed, as reported by Kallu et al. (2017), who found lower WAI values with the inclusion of fiber.
The hardness values of all optimal diets differed significantly from controls 1 and 2, being higher than control 1 and lower than control 2. These results can be attributed to the inclusion of moringa flour in the diet, which contributes a higher fiber content, leading to cracked and therefore more fragile extrudates (Martin et al., 2019). However, the predicted results for diets 2 and 16 (27.93 N and 27.25 N, respectively) were within the ranges observed in this study (26.85 N – 32.41 N and 26.6 N – 32.93 N, respectively). Lastly, the buoyancy results of all optimal diets were like those of control 2, achieving the values predicted in the numerical optimization of 100% for diets 2, 16, and 96, while for diet 53 it was 99.98%, thus fulfilling the buoyancy requirement for Nile tilapia, which feeds on the surface of the pond.
3.3.1. Proximal chemical analysis of optimal diets and controls
The moisture content in the optimal diets for generating aquafeed ranged from 7–9%, exhibiting statistical differences (p ≤ 0.05) compared to control 2 (5.47%) and control 1 (9.59%). However, all diets had moisture contents below 10% (Table 6), which, as reported by Teruel (2002), is ideal for aquafeed storage to prevent mold growth, this demonstrates the practical viability of these feeds for long-term storage and transport, key factors for large-scale aquaculture operations.
Table 6
Proximal chemical analysis of optimal extruded diets and commercial feeds.
Diet and control | Moisture (%) | Lipids (%) | Crude fiber (%) | Protein (%) | Ash (%) | Carbohydrates (%) |
Diet 2 | 8.57 ± 0.10c | 10.71 ± 0.46 c | 1.60 ± 0.01c | 36.33 ± 0.26c | 10.96 ± 0.30b | 31.36 ± 1.18b |
Diet 96 | 8.56 ± 0.04c | 10.76 ± 0.52 c | 1.60 ± 0.01c | 36.16 ± 0.26bc | 11.91 ± 0.09c | 30.75 ± 0.11b |
Diet 53 | 8.40 ± 0.04c | 8.86 ± 0.34 b | 1.15 ± 0.01b | 35.72 ± 0.25b | 11.70 ± 0.14c | 34.53 ± 0.46c |
Diet 16 | 7.86 ± 0.11b | 4.55 ± 0.14 a | 0.87 ± 0.00a | 33.84 ± 0.01a | 11.92 ± 0.2c | 40.56 ± 0.47d |
Control 1 | 9.59 ± 0.03d | 17.23 ± 0.35 d | 0.87 ± 0.00a | 39.49 ± 0.01d | 8.43 ± 0.05a | 24.39 ± 0.36a |
Control 2 | 5.47 ± 0.05a | 9.42 ± 0.24 b | 2.48 ± 0.09d | 33.84 ± 0.01a | 8.41 ± 0.03a | 40.38 ± 0.25d |
Equal letters in the same column indicate that there is no statistically significant difference (P ≤ 0.05).
Control 1: commercial aquafeed with 2.4 mm of diameter.
Control 2: commercial aquafeed with 3.5 mm of diameter.
Regarding the lipid content (Table 6), all the optimal diets had lipid contents of less than 10%. Notably, diet 53 (8.86%) was statistically similar (p ≤ 0.05) to control 2 (9.42%), while all diets had lower contents than control 1 (17.23%). As per Ng and Chong's (2004) findings, which stipulate a minimum requirement of 5% and an optimal range of 10% − 15% for lipid content, diet 16 (4.55%) fell below the minimum value, whereas diets 2 and 96 met the optimal requirement for lipid content.
The crude fiber results of all the diets (Table 6) were under 2%, which is lower than that of control 2 (2.48%). Fiber content should not exceed 10% of the diet as high fiber content negatively affects growth by decreasing gut passage time (Anderson et al. 1984; Liu et al., 2013). The protein content of the optimal diets for generating aquafeed (Table 6) fell between 33.84% and 36.33%. Diet 16 was statistically similar (p ≤ 0.05) to control 2 (33.84%). Compared to control 1 (39.49%), all diets had lower protein content. However, in accordance with the Oreochromis niloticus requirements reported by Liu et al. (2013), diets 2, 96, and 53, with contents greater than 35%, are suitable for a fry stage culture, while diet 16, with a protein content of 33.84%, can be used in a juvenile stage culture.
The ash content in all the optimal diets (Table 6) exceeded 10%, presenting statistically different values (p ≤ 0.05) compared to controls 1 and 2 (8.43% and 8.41%, respectively). This increase can be attributed to the incorporation of a 3% mineral premix into the diet and the amount of minerals and inorganic compounds present in the sardine meal (Janbakhsh et al., 2018). Finally, the carbohydrate content in the diets (Table 6) ranged between 30% and 41%. Diet 16 was statistically similar (p ≤ 0.05) to control 2 (40.38%). Compared to control 1 (24.39%), all diets had higher values. Nonetheless, these carbohydrate contents can serve as an energy source for Oreochromis niloticus (Anderson et al 1984; Boonanuntanasarn et al., 2018).
On the nutritional aspect, the formulated aquafeeds met the recommended nutrient requirements for Oreochromis niloticus, demonstrates that sustainable aquafeed production is feasible with the inclusion of alternative protein sources like moringa flour as a viable alternative protein source in aquafeed formulation. However, the optimization of feed formulation and extrusion parameters is critical to ensure the feed's physicochemical and nutritional quality. Future studies should look into the long-term effects of these aquafeeds on fish health and growth performance to further validate the effectiveness of this approach in aquaculture.
This study focused on extrusion parameters and nutrient requirements for Oreochromis niloticus, however, the analyze an in-depth cost-benefit analysis considering factors such as the availability and price fluctuations of sardine meal and moringa flour, and changes in production and operational costs, will be beneficial for practical applications. Furthermore, as moringa flour is rich in essential amino acids, vitamins, and minerals, future research should also consider its impact on the immunity and disease resistance of farmed fish species. Additionally, the effect of moringa-inclusive diet on the taste and texture of the farmed species, and subsequently, consumer preference, is a valuable avenue to explore.
Finally, this study provides an important foundation for the utilization of alternative protein sources in aquafeed. However, it is essential to mention that the effects of varying ingredient composition in aquafeeds can differ between fish species, owing to differences in their dietary preferences and digestive physiology. Consequently, while the current findings are encouraging, further research is warranted to extend the application of this methodology to other species.