Leaf Protein Concentrate of Ficus septica as a Plant-Based Aquafeed Ingredient for Nile Tilapia Oreochromis niloticus

2.1 Collection of plant materials

Fresh leaves of F. septica were collected from unmanaged agricultural land in Kediri, East Java, Indonesia, from October 2024 until February 2025. Leaves with mature size, green color and intact surface were selected manually during sampling. Leaves showing signs of disease, insect damage, yellowing, or physical injury were not used. The collected leaves were placed in clean polyethylene bags and transported directly to the laboratory on the same day. All samples were processed immediately after collection to avoid quality deterioration during storage. Before processing, the leaves were washed several times using running tap water to remove attached soil, dust, sand, and other foreign materials. Residual surface water was drained before the next processing step.

2.2 Preparation of leaf protein concentrate

Fresh leaves of F. septica were processed to produce LPC through extraction, coagulation, and drying procedures. Collected leaves were washed several times using running tap water to remove attached soil, dust, sand, and foreign particles from the leaf surface. Clean leaves were drained to remove excess surface water before thermal treatment. Fresh leaves were blanched in hot water at 70–80 ℃ for 2 min to reduce enzymatic activity in the leaf tissue and maintain the stability of the extracted material during processing. After blanching, the leaves were transferred immediately into ice water to stop further heat exposure. Surface moisture was removed before mechanical blending.

Blanched leaves were blended with distilled water at a ratio of 1:5 (w/v) using a laboratory blender until a homogeneous slurry was obtained. The blending process disrupted leaf tissue structure and released intracellular soluble components into the liquid phase. The slurry was filtered through muslin cloth and pressed manually to separate the liquid extract from fibrous residue. The remaining fibrous material was discarded after extraction, while the filtrate was collected as green leaf extract for the next coagulation process.

The collected extract was heated at 100 ℃ for 10 min under continuous observation. Protein precipitation began to appear during heating treatment. After heating, the extract temperature was reduced to approximately 75 ℃ before acidification. Glacial acetic acid was added slowly into the extract while pH was monitored continuously using a digital pH meter. Acid addition continued until pH 4.5–4.6 was reached to initiate protein coagulation. Calcium sulfate (CaSO₄) was added at a concentration of 0.75 g L⁻¹ during coagulation to support curd formation and improve separation of protein aggregates from the liquid fraction.

The coagulated mixture was left undisturbed for 15 min until protein curd formation became clearly visible. Protein curd was separated manually from the remaining liquid fraction by pressing through the filtration cloth. The resulting wet curd was collected and used for drying treatment. Wet curd was dried using a laboratory spray dryer operated at an inlet temperature of 160 ± 5 ℃ and outlet temperature of 80 ± 3 ℃. The liquid suspension entered the spray dryer through a peristaltic pump and two fluid nozzle system at a flow rate of approximately 5 mL min⁻¹. Drying continued until stable powder formation was obtained inside the collection chamber. The LPC production parameters are shown in Table 1.

Table 1. Leaf protein concentrate (LPC) production parameters

The dried product was ground into fine powder using a laboratory grinder to improve sample uniformity before storage and feed preparation. Final LPC powder was stored in airtight containers at room temperature before nutritional analysis and experimental diet formulation. The present study focused on nutritional composition, antioxidant activity, amino acid profile, mineral composition, digestibility, and growth response of LPC in Nile tilapia diets. Protein recovery yield during LPC production was not evaluated in the present work.

2.3 Analysis of proximate composition

Proximate composition analyses of raw leaf powder, LPC, and experimental diets were conducted following published methods [9, 10]. All samples were dried before analysis to reduce moisture variation during measurement. Dried samples were ground using a laboratory grinder until a homogeneous fine powder was obtained. The powder was stored in airtight containers at room temperature before chemical analysis. Each parameter was analyzed in triplicate using separate sample portions to improve analytical consistency and reduce measurement variation between replicates.

Moisture content was determined using the oven drying method. Approximately 2 g of each sample was weighed using an analytical balance and transferred into pre-weighed moisture dishes. Samples were dried in a hot air oven at 105 ℃ until constant weight was obtained. Drying continued for several hours with repeated weighing intervals until no further reduction in sample weight was detected. Moisture percentage was calculated from the difference between the initial sample weight and the final dry weight after oven drying.

Crude protein content was analyzed using the Kjeldahl method based on total nitrogen determination. Approximately 0.5 g of the sample was digested using concentrated sulfuric acid in the presence of catalyst tablets under high temperature conditions until a clear digest solution was obtained. The digestion process converted organic nitrogen compounds into ammonium sulfate. After digestion, the solution was cooled and diluted with distilled water before distillation. Sodium hydroxide solution was added during distillation to release ammonia from the digested sample. Released ammonia was trapped in the boric acid receiving solution and titrated using standardized hydrochloric acid solution. Total nitrogen content was calculated from acid titration values and converted into crude protein using a nitrogen conversion factor of 6.25.

Crude lipid content was determined using Soxhlet extraction with petroleum ether as the extraction solvent. Approximately 2 g of dried sample was wrapped in filter paper and inserted into extraction thimbles. Samples were extracted continuously for several hours until lipid fractions were completely dissolved in the solvent system. Petroleum ether containing dissolved lipid fractions was evaporated after extraction, and the remaining lipid residue was dried before weighing. Crude lipid percentage was calculated based on the weight difference before and after the extraction procedures.

Ash content was measured using dry combustion in a muffle furnace. Approximately 2 g of dried sample was placed into pre-weighed porcelain crucibles and heated gradually before furnace combustion. Samples were combusted at 550 ℃ for several hours until all organic materials were completely removed and only mineral residue remained. Crucibles were cooled in a desiccator before final weighing. Ash content was expressed as a percentage of mineral residue remaining after complete combustion.

Crude fiber analysis was conducted using sequential acid and alkali digestion procedures. Defatted samples obtained after lipid extraction were boiled in sulfuric acid solution under controlled heating conditions. The remaining residue was filtered and washed repeatedly using distilled water to remove soluble materials. The residue was then subjected to sodium hydroxide digestion, followed by filtration and repeated washing procedures. The remaining indigestible material was dried in an oven and combusted in a muffle furnace. Crude fiber content was calculated from the weight difference between dried residue and ash residue after combustion.

Nitrogen-free extract content was calculated by difference using total proximate composition values, including moisture, crude protein, crude lipid, crude fiber, and ash content. Gross energy values were estimated using physiological fuel conversion factors based on protein, lipid, and carbohydrate fractions of each sample. All analytical data were expressed on a dry matter basis.

2.4 Antioxidant properties determination

Total phenolic content (TPC) was determined using the Folin–Ciocalteu method and expressed as mg gallic acid equivalent (GAE) g⁻¹ sample [11]. Sample extract was mixed with Folin–Ciocalteu reagent, followed by incubation before absorbance measurement using a spectrophotometer.

Total flavonoid content (TFC) was analyzed using the aluminum chloride colorimetric method and expressed as mg quercetin equivalent (QE) g⁻¹ sample. Formation of flavonoid complexes during the reaction was measured spectrophotometrically after incubation.

Antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay (DPPH), Ferric Reducing Antioxidant Power assay (FRAP), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization assay (ABTS). DPPH radical scavenging activity was expressed as IC₅₀ values (µg mL⁻¹). Lower IC₅₀ values indicated stronger antioxidant activity. Ferric reducing antioxidant power (FRAP) analysis was expressed as µmol Fe²⁺ g⁻¹ sample based on the reducing capacity of the extract.

ABTS radical scavenging activity was expressed as a percentage inhibition after reaction between the sample extract and ABTS radical solution. All measurements were performed in triplicate.

2.5 Amino acid and mineral analyses

Essential amino acid composition of leaf powder and LPC samples was determined using high-performance liquid chromatography (HPLC) following acid hydrolysis procedures. Amino acid concentrations were expressed as a percentage of total protein. Mineral composition, including calcium (Ca), phosphorus (P), potassium (K), magnesium (Mg), iron (Fe), zinc (Zn), manganese (Mn), and sodium (Na), was analyzed using atomic absorption spectrophotometry (AAS) after wet digestion of samples. Mineral concentrations were expressed as mg 100 g⁻¹ dry matter.

2.6 Experimental diets

Four isonitrogenous experimental diets were formulated to contain different inclusion levels of F. septica LPC: 0% (control), 10% (FS-LPC10), 20% (FS-LPC20), and 30% (FS-LPC30) (Table 2).

Table 2. Formulation of experimental diets containing different inclusion levels of F. septica leaf protein concentrate (LPC) (% dry matter basis)

Diet ingredients were thoroughly mixed with water to obtain homogeneous dough, pelleted using a laboratory pelletizer, and dried at room temperature prior to storage. The proximate composition of the experimental diets was analyzed before feeding trials to ensure comparable nutritional quality among treatments.

Experimental diets were pelleted using a laboratory pelletizer equipped with a 2-mm die. Pellets were dried at room temperature for approximately 24 h until the moisture content fell below 10%. No obvious differences in pellet integrity or water stability were observed among treatments during feeding. Although water stability was not quantitatively determined, all diets maintained acceptable physical integrity during feeding and showed no visible differences in pellet durability or acceptance among treatments. For digestibility determination, chromic oxide (Cr₂O₃) was included at 0.5% of the diet as an inert marker. To maintain the total dietary formulation at 100%, Cr₂O₃ replaced an equivalent proportion of wheat flour in each experimental diet.

2.7 Fish and feeding trial

Nile tilapia (O. niloticus) (weight 5 g; total length of 7-10 cm) was purchased from a local hatchery in Kediri, East Java, Indonesia. Fish were transported to the laboratory using aerated plastic bags and transferred carefully into acclimatization tanks. The acclimatization period lasted for two weeks before the feeding trial started.

During acclimatization, the fish were maintained under laboratory culture conditions and fed a commercial diet three times daily. Fish condition, feeding response, swimming activity, and external body appearance were observed every day. Fish showing abnormal swimming, weak feeding response, body injury, fin damage, scale loss, discoloration, or disease symptoms were not used in the experiment. Only active fish with normal swimming behavior, stable feeding response, intact body surface, and uniform size were selected as experimental animals.

After acclimatization, the fish were distributed randomly into experimental tanks. Each treatment consisted of three replicates. One experimental unit used a 60 L fiberglass tank containing 15 fish. Continuous aeration was supplied in all tanks during the experimental period to maintain dissolved oxygen availability.

The feeding trial was conducted for 60 days. Water temperature was maintained at 27–29 ℃ during the experiment. Dissolved oxygen and pH were monitored regularly using portable water quality instruments. Fish were fed experimental diets three times daily until apparent satiation. Feeding activity was monitored during each feeding period. Uneaten feed, fecal materials, and organic residues were removed from the tanks every day to maintain water quality conditions. Approximately 30% of the water volume was replaced daily throughout the feeding period.

2.8 Apparent digestibility coefficients

Apparent digestibility coefficients (ADC) of dry matter, crude protein, crude lipid gross energy, and crude fiber were determined using the indirect digestibility method with chromic oxide (Cr₂O₃) as an inert marker in the experimental diets. Chromic oxide was incorporated into each diet formulation at 0.5% inclusion level before pellet preparation to allow the determination of nutrient digestibility based on marker concentration differences between diets and fecal samples. Experimental diets containing chromic oxide were mixed thoroughly to ensure uniform marker distribution before pelletizing and drying procedures.

Fecal samples were collected from each treatment group during the feeding trial for digestibility analysis. Feces were collected carefully from the tank bottom by siphoning approximately 1 h after feeding to reduce nutrient leaching into the water and minimize contamination from uneaten feed particles. Collection procedures were conducted slowly to maintain fecal integrity during sampling. Collected feces from each replicate tank were pooled and transferred into clean sample containers for further processing.

Fecal samples were rinsed gently using distilled water to remove attached debris and remaining impurities from the sample surface. Clean fecal samples were dried in a laboratory oven at 60 ℃ until a stable dry weight was obtained. Dried samples were ground into fine powder and stored in airtight containers before chemical analysis. Proximate composition analysis of fecal samples and experimental diets was conducted to determine dry matter, crude protein, crude lipid, crude fiber, and gross energy contents. Chromic oxide concentration in diets and fecal samples was also analyzed for digestibility calculation.

ADC of nutrients was calculated using conventional ADC equations based on the ratio of chromic oxide concentration and nutrient content between diet and feces. Dry matter digestibility was calculated using marker concentration values, while nutrient digestibility coefficients were determined from the relationship between nutrient concentration and marker levels in diets and fecal samples. All digestibility calculations were conducted separately for each replicate tank, and the obtained values were used for statistical analysis.

2.9 Growth performance and feed utilization

Growth performance and feed utilization parameters were evaluated at the end of the 60-day feeding trial using fish growth data and feed consumption records obtained from each experimental tank. Fish were not fed for 24 h before final sampling to reduce variation caused by gut content and digestive materials inside the gastrointestinal tract. At the beginning and end of the experiment, all fish from each tank were counted and weighed collectively to obtain total biomass values for each replicate unit. Individual body weight values were calculated from the average total biomass and total fish number in each tank. Fish handling during weighing procedures was conducted carefully to reduce physical stress and injury during sampling activities.

Water quality parameters were monitored throughout the experimental period. Water temperature ranged from 27-30 ℃, dissolved oxygen ranged from 6.6-7.5 mg L⁻¹, and pH ranged from 6.8-7.0. Water quality remained within acceptable ranges for Nile tilapia culture during the study.

Final weight (g) was determined from the average body weight of fish at the end of the feeding period. Weight gain (WG) was calculated from the difference between the final body weight and the initial body weight of fish during the experimental period using Eq. (1):

$WG(g)= Final \,weight - Initial \,weight$                (1)

Specific growth rate (SGR) was calculated using natural logarithm values of final and initial body weight divided by total culture duration. The obtained value was expressed as percentage growth per day using the Eq. (2):

$\begin{gathered}{SGR}\left(\%{day}^{-1}\right)= \frac{\ln ({Final \,weight })-\ln ({ Initial\, weight})}{{Culture \,period}} \times 100\end{gathered}$              (2)

Feed conversion ratio (FCR) was determined from the relationship between total feed intake and body WG during the feeding period. Feed intake was calculated as the difference between the feed offered and uneaten feed recovered after feeding. Total feed consumption in each tank was recorded throughout the experiment and used for calculation using the following Eq. (3):

$F C R=\frac{{ Feed \,intake }}{{ Weight\, gain }}$                (3)

Protein efficiency ratio (PER) was calculated to evaluate protein utilization efficiency based on body WG and total protein intake from experimental diets. Total protein intake was calculated from feed consumption data and dietary crude protein content using the following Eq. (4):

$P E R=\frac{{Weight \,gain}}{ {Protein \,intake}}$             (4)

Survival rate (SR) was determined from the number of fish remaining at the end of the experiment compared with the initial number of fish stocked in each tank. Dead fish were recorded daily throughout the feeding trial. Survival percentage was calculated using the following Eq. (5):

$S R(\%)=\frac{{Final \,fish \,number}}{{Initial \,fish \,number}} \times 100$            (5)

All calculations were performed for each experimental tank and used as replicate data for statistical analysis.

2.10 Data analysis

All data obtained from proximate composition, antioxidant activity, amino acid composition, digestibility, growth performance, and feed utilization analyses were expressed as mean ± standard deviation (SD). Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) version 25. Data normality and homogeneity of variance were evaluated using Shapiro–Wilk and Levene's tests, respectively, prior to further analyses.

Differences among dietary treatments were analyzed using one-way analysis of variance (ANOVA). When significant differences were detected, Tukey's multiple range test was applied to compare treatment means. Statistical significance was accepted at p < 0.05.

For proximate composition, amino acid composition, mineral composition, and antioxidant activity analyses, comparisons between F. septica leaf powder and LPC were performed using independent-samples t-tests. Statistical significance was accepted at p < 0.05.

For chemical analyses, n = 3 refers to three analytical replicates. For digestibility, growth performance, and feed utilization evaluations, n = 3 refers to three independent replicate tanks, with each tank considered the experimental unit. Values are presented as mean ± SD.

2.11 Ethics

The study followed institutional guidelines for the ethical use of experimental animals.

The present study showed that processing F. septica leaves into LPC increased the nutritional quality of the material and improved its suitability as an aquafeed ingredient for Nile tilapia. The LPC contained higher crude protein, essential amino acids, minerals, phenolic compounds, flavonoids, and antioxidant activity than the original leaf powder. The concentration process also reduced crude fiber content, which improved the nutritional characteristics of the plant biomass. Potassium, calcium, and phosphorus remained dominant minerals in the LPC, while leucine, arginine, lysine, and valine were detected at higher levels after processing. The increase of FRAP and ABTS activity, together with lower DPPH IC50 values, indicated that antioxidant compounds remained stable during extraction, coagulation, and drying processes. However, the physiological effects of these antioxidant compounds in fish were not assessed in the present study and therefore require further investigation.

Dietary inclusion of LPC improved ADC of dry matter protein, lipid, and energy in Nile tilapia. Fish fed the FS-LPC20 diet produced the highest final weight, WG, SGR, and PER, together with lower FCR values. Growth performance at 30% inclusion level decreased compared with the FS-LPC20 treatment, although fish performance still remained higher than the control diet. SR remained high in all dietary treatments during the feeding period. The results showed that moderate inclusion of F. septica LPC improved nutrient utilization and feed efficiency in tilapia culture. The study also showed that terrestrial leaf biomass can be processed into a protein-enriched ingredient through simple extraction and coagulation procedures. F. septica grows naturally in unmanaged terrestrial areas and does not directly compete with conventional food crops. Utilization of this biomass provides an alternative approach for the development of plant-based aquafeed ingredients. Under the present laboratory conditions, dietary inclusion of 20% F. septica LPC produced the most favorable digestibility and growth responses. However, commercial-scale application, long-term safety evaluation, antinutritional-factor characterization, and economic feasibility analyses require further investigation.

Several limitations should be acknowledged. Antioxidant activity was evaluated only through in vitro assays (TPC, TFC, DPPH, FRAP, and ABTS), whereas physiological biomarkers such as superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde, hematological indices, and immune responses were not assessed. Consequently, the present findings demonstrate the antioxidant potential of the ingredient itself but do not confirm biological antioxidant effects in Nile tilapia.