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Development
of Nano-Encapsulated Arthrospira platensis (Spirulina)-Based Fish Feed
Pellets: Formulation, Characterization, and Biological Evaluation
Abstract
The application of nanotechnology in aquafeed
formulation offers a promising strategy to improve nutrient bioavailability,
feed efficiency, and fish health. Arthrospira platensis (commonly known
as spirulina) is a protein-rich microalga containing bioactive compounds such
as phycocyanin, carotenoids, vitamins, and essential fatty acids. This study
aimed to develop and evaluate nano-encapsulated spirulina-based fish feed
pellets. Spirulina extract was encapsulated using chitosan–tripolyphosphate
ionic gelation to produce nanoparticles (50–180 nm). The nanoparticles were
incorporated into extruded fish feed pellets and evaluated for physicochemical
characteristics, proximate composition, water stability, and biological
performance in Nile tilapia (Oreochromis niloticus). Results indicated
improved feed conversion ratio (FCR), specific growth rate (SGR), and enhanced
immune parameters in fish fed nano-spirulina diets compared with conventional
spirulina diets. The findings demonstrate that nano-encapsulation enhances
spirulina stability and bioavailability, supporting its application in
sustainable aquaculture.
Keywords: nanotechnology,
spirulina, nanoencapsulation, aquafeed, bioavailability, tilapia.
1. Introduction
Aquaculture is one of the fastest-growing
food production sectors globally and plays a critical role in food security
(Food and Agriculture Organization of the United Nations [FAO], 2022). Feed
represents approximately 60–70% of operational costs in intensive aquaculture
systems, necessitating innovations to improve feed efficiency and
sustainability.
Arthrospira platensis
(spirulina) is widely recognized as a high-value microalga with protein content
ranging from 60–70%, containing essential amino acids, polyunsaturated fatty
acids, vitamins, minerals, and bioactive pigments such as phycocyanin and
β-carotene (Becker, 2013). Numerous studies have reported its
immunostimulatory, antioxidant, and growth-promoting effects in fish (Belay,
2002; Abdel-Tawwab & Ahmad, 2009).
Nanotechnology has emerged as a
transformative approach in feed science. Nano-encapsulation improves nutrient
stability, protects sensitive bioactive compounds from degradation, enhances
intestinal absorption, and enables controlled release mechanisms (Handy et al.,
2012). Chitosan-based nanoparticles are particularly attractive due to their
biocompatibility, biodegradability, and mucoadhesive properties (Calvo et al.,
1997).
This study aimed to (1) synthesize and
characterize nano-encapsulated spirulina, (2) formulate extruded fish feed
pellets incorporating nano-spirulina, and (3) evaluate growth performance and
immune responses in Nile tilapia (Oreochromis niloticus).
2. Materials and Methods
2.1 Preparation of Spirulina Extract
Dried spirulina powder (Arthrospira
platensis) was suspended in distilled water (1:10 w/v) and subjected to
magnetic stirring for 24 h at 4°C. The suspension was centrifuged at 10,000 rpm
for 20 min, and the supernatant was collected as crude extract.
2.2 Synthesis of Nano-Encapsulated Spirulina
Nano-encapsulation was performed using ionic
gelation. Chitosan (0.2% w/v) was dissolved in 1% acetic acid solution.
Spirulina extract was added dropwise into the chitosan solution under
continuous stirring. Sodium tripolyphosphate (TPP, 0.1%) was then added to
induce crosslinking. The mixture was homogenized using ultrasonication (20 kHz,
10 min).
2.3 Nanoparticle Characterization
- Particle size and polydispersity index (PDI):
Dynamic Light Scattering (DLS)
- Surface charge: Zeta potential analysis
- Morphology: Scanning Electron Microscopy (SEM)
- Encapsulation efficiency (EE%): Spectrophotometric analysis at 620 nm (phycocyanin absorption peak)
2.4 Feed Formulation and Pellet Production
Four experimental diets were prepared:
- Control (without spirulina)
- Conventional spirulina (10%)
- Nano-spirulina (5%)
- Nano-spirulina (10%)
All diets were iso-nitrogenous (30% crude
protein) and iso-energetic. Pellets were produced using low-temperature
extrusion (<60°C), dried at 45°C until moisture <10%, and stored in
airtight containers.
2.5 Experimental Design
60-day feeding trial was conducted using Nile
tilapia (Oreochromis niloticus) juveniles (initial weight: 10 ± 0.5 g).
Fish were randomly distributed into 12 tanks (triplicate per treatment) under a
completely randomized design.
2.6 Growth and Immune Parameters
- Weight gain (WG)
- Specific growth rate (SGR)
- Feed conversion ratio (FCR)
- Survival rate (SR)
- Lysozyme activity
- Respiratory burst activity
2.7 Statistical Analysis
Data were analyzed using one-way ANOVA
followed by Tukey’s post hoc test (p < 0.05).
3. Results
3.1 Physicochemical Characterization of
Nano-Encapsulated Spirulina
The physicochemical properties of
nanoparticles play a crucial role in determining their stability,
bioavailability, and functional performance in aquafeed applications. Particle
size influences surface area, dissolution rate, and interaction with the intestinal
epithelium, while the polydispersity index (PDI) reflects the uniformity of
particle distribution. A low PDI value (<0.3) generally indicates a narrow
size distribution and homogeneous nanoparticle system, which is desirable for
consistent biological activity.
Zeta potential is a key indicator of
colloidal stability, representing the surface charge of nanoparticles and their
electrostatic repulsion capacity. Absolute zeta potential values greater than
±30 mV are typically associated with stable colloidal dispersions due to
reduced particle aggregation. Encapsulation efficiency (EE%) and loading
capacity (LC%) are critical parameters that determine the proportion of
spirulina bioactive compounds successfully entrapped within the chitosan matrix
and their potential delivery efficiency. The physicochemical characteristics of
the synthesized nano-encapsulated spirulina are presented in Table 1.
Table 1. Physicochemical characteristics of
nano-encapsulated spirulina (mean ± SD, n = 3).
|
Parameter |
Value |
|
Particle size (nm) |
118.4
± 21.6 |
|
Polydispersity Index (PDI) |
0.21
± 0.03 |
|
Zeta potential (mV) |
+31.8
± 2.4 |
|
Encapsulation efficiency (%) |
82.3
± 3.1 |
|
Loading capacity (%) |
18.7
± 1.9 |
Nanoparticles showed spherical morphology
with uniform distribution under SEM analysis. The positive zeta potential
indicated colloidal stability and mucoadhesive potential.
3.2 Proximate Composition of Experimental
Diets
The proximate composition of experimental
diets was analyzed to ensure that all treatments were nutritionally comparable
and met the dietary requirements of Nile tilapia. Maintaining iso-nitrogenous
and iso-energetic conditions is essential in feeding trials to ensure that
observed differences in growth performance and physiological responses are
attributable to the tested ingredient (nano-encapsulated spirulina), rather
than variations in basic nutrient levels.
All diets were formulated to contain
approximately 30% crude protein and comparable gross energy levels, consistent
with recommended requirements for juvenile Oreochromis niloticus.
Standard analytical procedures (AOAC methods) were applied to determine crude
protein, lipid, fiber, ash, moisture, and energy content. The inclusion of
conventional spirulina and nano-spirulina was carefully adjusted by balancing
other protein and energy sources to avoid confounding nutritional effects. The
proximate composition results are presented in Table 2.
Table 2. Proximate composition of
experimental diets (% dry matter basis, mean ± SD, n = 3).
|
Parameter |
Control |
Spirulina
10% |
Nano-Spirulina
5% |
Nano-Spirulina
10% |
|
Crude protein (%) |
30.2
± 0.4 |
30.5
± 0.3 |
30.4
± 0.5 |
30.6
± 0.4 |
|
Crude lipid (%) |
7.8
± 0.2 |
8.1
± 0.3 |
8.0
± 0.2 |
8.2
± 0.3 |
|
Crude fiber (%) |
4.5
± 0.2 |
4.8
± 0.2 |
4.6
± 0.3 |
4.9
± 0.2 |
|
Ash (%) |
9.1
± 0.3 |
9.4
± 0.4 |
9.2
± 0.3 |
9.6
± 0.4 |
|
Moisture (%) |
8.6
± 0.4 |
8.4
± 0.5 |
8.5
± 0.3 |
8.3
± 0.4 |
|
Gross energy (kcal/kg) |
4,210
± 35 |
4,235
± 41 |
4,228
± 37 |
4,240
± 39 |
No significant differences (p > 0.05) were
observed in protein or energy levels, confirming iso-nitrogenous and
iso-energetic diets.
3.3 Physical Quality of Pellets
Physical quality parameters are critical
indicators of feed performance in aquaculture, as they directly influence feed
intake, nutrient retention, and environmental impact. Water stability
determines the resistance of pellets to disintegration in water, thereby
reducing nutrient leaching and maintaining feed integrity during feeding.
Floatability is particularly important in tilapia culture systems, where
floating pellets allow better feed monitoring and minimize wastage. Pellet
hardness reflects structural strength, affecting handling durability and
resistance to mechanical breakage during storage and transport.
In the present study, incorporation of
nano-encapsulated spirulina significantly improved all measured physical
characteristics compared to control and conventional spirulina diets. The
enhancement in water stability and hardness is likely attributable to the
interaction between chitosan-based nanoparticles and feed matrix components,
forming a stronger cross-linked network structure. Moreover, the nano-sized
particles may have improved matrix homogeneity, contributing to increased
structural cohesion and buoyancy. These improvements suggest that
nano-spirulina not only enhances nutritional functionality but also positively
modifies the technological properties of feed pellets.
Table 3. Physical characteristics of pellets
(mean ± SD, n = 3).
|
Parameter |
Control |
Spirulina
10% |
Nano-Spirulina
5% |
Nano-Spirulina
10% |
|
Water stability (%) |
83.7
± 2.1ᵃ |
84.5
± 2.4ᵃ |
89.8
± 1.7ᵇ |
92.3
± 1.5ᶜ |
|
Floatability (%) |
76.2
± 3.4ᵃ |
78.5
± 3.1ᵃ |
85.6
± 2.8ᵇ |
88.9
± 2.6ᶜ |
|
Pellet hardness (N) |
21.5
± 1.2ᵃ |
22.1
± 1.3ᵃ |
24.8
± 1.1ᵇ |
26.4
± 1.4ᶜ |
Different superscript letters within rows
indicate significant differences (p < 0.05).
3.4 Growth Performance
Growth performance parameters were evaluated
to determine the biological efficacy of nano-encapsulated spirulina in
improving nutrient utilization and overall production efficiency in Nile
tilapia (Oreochromis niloticus). To ensure experimental validity, fish
across all treatments had statistically similar initial body weights,
confirming uniform stocking conditions prior to the feeding trial. Growth
responses were assessed using standard aquaculture performance indicators,
including final weight, weight gain, specific growth rate (SGR), feed
conversion ratio (FCR), and survival rate.
Specific growth rate reflects daily biomass
increment relative to body weight, while FCR represents feed utilization
efficiency and is a key economic indicator in aquaculture production systems.
Improvements in these parameters are typically associated with enhanced
digestibility, nutrient absorption, and metabolic efficiency. The
nano-encapsulation approach was hypothesized to increase the bioavailability of
spirulina bioactive compounds—such as phycocyanin, essential amino acids,
vitamins, and antioxidants—by improving intestinal interaction and reducing
nutrient degradation. The growth performance results after 60 days of feeding
are presented in Table 4.
Table 4. Growth performance of Nile
tilapia after 60 days (mean ± SD, n = 3).
|
Parameter |
Control |
Spirulina 10% |
Nano-Spirulina 5% |
Nano-Spirulina 10% |
|
Initial weight (g) |
10.1 ± 0.5 |
10.2 ± 0.4 |
10.1 ± 0.6 |
10.0 ± 0.5 |
|
Final weight (g) |
38.6
± 2.4ᵃ |
46.8
± 2.7ᵇ |
51.2
± 2.9ᶜ |
55.9
± 3.1ᵈ |
|
Weight gain (g) |
28.5
± 2.2ᵃ |
36.6
± 2.3ᵇ |
41.1
± 2.6ᶜ |
45.9
± 2.8ᵈ |
|
SGR (%/day) |
2.15
± 0.07ᵃ |
2.48
± 0.09ᵇ |
2.67
± 0.08ᶜ |
2.82
± 0.10ᵈ |
|
FCR |
1.62
± 0.05ᶜ |
1.41
± 0.04ᵇ |
1.28
± 0.03ᵃᵇ |
1.19
± 0.03ᵃ |
|
Survival rate (%) |
91.3
± 2.1ᵃ |
93.5
± 1.9ᵃᵇ |
95.7
± 1.6ᵇ |
97.2
± 1.3ᵇ |
Different superscripts indicate significant
differences (p < 0.05).
3.5 Immune and Hematological Parameters
Evaluation of innate immune parameters was
conducted to assess the immunomodulatory potential of nano-encapsulated
spirulina in Nile tilapia (Oreochromis niloticus). In teleost fish, the
innate immune system serves as the primary defense mechanism against pathogens,
particularly in intensive aquaculture systems where stress and disease
outbreaks are common. Therefore, enhancement of non-specific immunity is a critical
indicator of functional feed efficacy.
Lysozyme activity represents an important
humoral defense component capable of lysing bacterial cell walls, while
respiratory burst activity reflects the production of reactive oxygen species
(ROS) by phagocytic cells during pathogen elimination. Total leukocyte count
indicates cellular immune status, and hemoglobin concentration serves as a
physiological indicator of overall health and oxygen transport capacity.
Improvements in these parameters suggest enhanced immune competence and better
physiological condition.
The nano-encapsulation strategy was
hypothesized to increase the bioavailability and stability of spirulina-derived
bioactive compounds, such as phycocyanin, polysaccharides, and antioxidant
molecules, thereby stimulating immune cell activity more effectively than
conventional spirulina supplementation. The results of innate immune response
parameters are presented in Table 5.
Table 5. Innate immune response parameters
(mean ± SD, n = 3).
|
Parameter |
Control |
Spirulina
10% |
Nano-Spirulina
5% |
Nano-Spirulina
10% |
|
Lysozyme activity (U/mL) |
18.4
± 1.6ᵃ |
24.7
± 1.8ᵇ |
29.3
± 2.1ᶜ |
33.8
± 2.4ᵈ |
|
Respiratory burst (OD 540 nm) |
0.21
± 0.02ᵃ |
0.28
± 0.03ᵇ |
0.33
± 0.02ᶜ |
0.37
± 0.03ᵈ |
|
Total leukocyte (×10³/mm³) |
22.6
± 1.9ᵃ |
26.4
± 2.2ᵇ |
29.7
± 2.1ᶜ |
32.1
± 2.3ᵈ |
|
Hemoglobin (g/dL) |
7.8
± 0.4ᵃ |
8.6
± 0.5ᵇ |
9.1
± 0.4ᶜ |
9.5
± 0.5ᶜ |
Nano-spirulina significantly enhanced innate
immune responses compared to conventional spirulina.
3.6 Intestinal Histomorphology
Intestinal histomorphological evaluation was
performed to determine whether dietary nano-encapsulated spirulina influenced
gut structural integrity and absorptive capacity in Nile tilapia (Oreochromis
niloticus). The morphology of the intestinal mucosa is a key determinant of
nutrient digestion and absorption efficiency, and structural modifications are
often associated with improved growth performance and feed utilization.
Villus height is widely recognized as an
indicator of absorptive surface area; increased villus length enhances contact
between digesta and epithelial cells, thereby facilitating nutrient uptake.
Meanwhile, goblet cells are responsible for mucus secretion, which plays an
essential role in mucosal protection, lubrication, and barrier function against
pathogenic microorganisms. An increase in goblet cell density may reflect
enhanced mucosal defense and improved gut health status.
The incorporation of nano-spirulina was
hypothesized to promote intestinal development through improved bioavailability
of bioactive compounds such as phycocyanin, essential amino acids, and
antioxidant molecules. These compounds may stimulate epithelial cell
proliferation and modulate gut-associated immune responses, leading to
structural improvements in the intestinal mucosa. The histomorphological
parameters of the intestine are presented in Table 6.
Table 6. Intestinal histomorphology (mean ±
SD, n = 3).
|
Parameter |
Control |
Spirulina
10% |
Nano-Spirulina
5% |
Nano-Spirulina
10% |
|
Villus height (µm) |
412
± 28ᵃ |
478
± 31ᵇ |
526
± 34ᶜ |
571
± 37ᵈ |
|
Goblet cells (cells/field) |
14.2
± 1.3ᵃ |
17.8
± 1.5ᵇ |
20.4
± 1.7ᶜ |
23.1
± 1.8ᵈ |
Statistical Statement
All data were expressed as mean ± standard
deviation (SD). Statistical analysis was performed using one-way ANOVA followed
by Tukey’s HSD test at p < 0.05. Assumptions of normality and homogeneity of
variance were verified prior to analysis.
4. Discussion
Nano-encapsulation improved spirulina
stability and bioavailability, as evidenced by enhanced growth and immune
responses. The positive zeta potential likely improved adhesion to intestinal
mucosa, enhancing nutrient absorption. Comparable findings were reported by
Abdel-Tawwab & Ahmad (2009), who observed improved tilapia growth with
spirulina supplementation.
Chitosan nanoparticles have previously
demonstrated enhanced delivery of bioactive compounds due to their mucoadhesive
properties (Calvo et al., 1997). Furthermore, nano-sized particles increase
surface area, facilitating improved enzymatic interaction and absorption (Handy
et al., 2012).
Improved FCR suggests enhanced nutrient
utilization efficiency, potentially reducing nitrogenous waste in aquaculture
systems and supporting environmental sustainability goals promoted by FAO
(2022).
5. Conclusion
Nano-encapsulated spirulina significantly
enhances growth performance, feed efficiency, and immune responses in Nile
tilapia. The integration of nanotechnology in aquafeed production offers a
promising strategy for sustainable aquaculture. Further research is recommended
to evaluate long-term safety, economic feasibility, and regulatory compliance
for commercial-scale application.
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