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Full Research Article

Effects of Processed Plant Ingredients (Hydrolyzed Purslane and Bioconverted Mesquite) on Digestibility and Antinutritional Factors in Nile Tilapia (Oreochromis niloticus)

J
Jessica M. Rodríguez-Leyva1
F
Francisco J. Valdez-González1,2
I
Iván E. Díaz-Vázquez4
E
Edith O. Cuevas-Rodríguez3
O
Oscar I. Zavala-Leal1,2
L
Liliana León-López3
L
L. Cuevas-Rodríguez Breidy1,2,*
1Programa de Maestría en Ciencias Biológico Agropecuarias, Universidad Autónoma de Nayarit. Carretera Tepic-Compostela, km 9 C.P. 63780, Xalisco, Nayarit.
2Escuela Nacional de Ingeniería Pesquera, Universidad Autónoma de Nayarit. Carretera a Los Cocos km. 12, Bahía de Matanchén San Blas, Nayarit, México.
3Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Programa Regional de Posgrado en Biotecnología, Culiacán, Sinaloa, México.
4Universidad Tecnológica de la Sierra. Carretera Federal Ruíz-Zacatecas Km 100.9 C.P. 63546, Mesa del Nayar, Nayarit, México.

ABSTRACT

Background: The incorporation of plant-based ingredients in aquafeed represents a sustainable alternative to reduce dependence on animal-derived protein sources such as fishmeal. However, the presence of antinutritional compounds limits nutrient utilization and growth performance in cultured species. This study evaluated the effect of enzymatic hydrolysis and solid-state fermentation on improving the nutritional quality of plant-derived flours.

Methods: Purslane (Portulaca oleracea) flour was enzymatically hydrolyzed, while mesquite pod (Prosopis laevigata) flour underwent solid-state fermentation with Rhizopus oligosporus. Proximate composition, antinutritional factors (tannins and phytates) and apparent digestibility coefficients were determined.

Result: Significant differences (p≤0.05) were observed in chemical composition after treatments. Protein content increased to 22.41±2.28% in purslane flour and 14.18±0.80% in mesquite flour, while lipid content decreased to 1.10±0.24% and 3.30±0.75%, respectively. Both treatments substantially reduced tannin and phytate concentrations, indicating effective degradation of antinutritional compounds. Additionally, the apparent digestibility coefficients of dry matter improved, ranging from 65.79 to 85.73%. These improvements are attributed to enzymatic degradation of fibrous structures and complex polysaccharides, promoting the release of low-molecular-weight peptides and more bioavailable amino acids.

INTRODUCTION

Aquaculture is one of the fastest-growing animal food production sectors worldwide, with Nile tilapia (Oreochromis niloticus) ranking among the most important cultured fish species due to its rapid growth, high survival and adaptability to diverse farming conditions (Arumugam et al., 2023; Cuevas-Rodríguez et al., 2024). However, feed costs remain a major constraint to profitable production, accounting for up to 70% of operational expenses, largely because of the inclusion of fishmeal as the primary protein source (Esmaeili et al., 2017; Rodríguez-Hernández et al., 2025).
       
Fishmeal is widely used in aquaculture feeds because of its high protein content, balanced amino acid profile, palatability and digestibility. Nevertheless, increasing costs and limited availability have intensified the search for alternative protein sources that can partially or completely replace fishmeal without compromising fish performance (Hussain et al., 2024; Nandi et al., 2025). Plant-based ingredients have emerged as promising alternatives, although their utilization is often constrained by the presence of antinutritional factors that reduce nutrient availability and digestibility (Bhatt et al., 2011; Nikmaram et al., 2017; Priyatharshni et al., 2024).
       
Among potential alternative ingredients, purslane (Portulaca oleracea) and mesquite pods (Prosopis laevigata) are of particular interest due to their availability and nutritional value. Purslane is rich in a-linolenic acid and can be produced year-round in tropical and subtropical regions (Masoodi et al., 2011; Zhou et al., 2015). Mesquite pods are abundant in arid and semi-arid regions of Mexico and contain appreciable levels of protein and bioactive compounds (González-Cortazar et al., 2025; Barragán-Longoria et al., 2025). However, both ingredients contain antinutritional compounds that may limit their efficient use in aquafeeds.

Processing techniques such as enzymatic hydrolysis and solid-state bioconversion have demonstrated potential to improve nutrient availability and reduce antinutritional factors in plant-derived feed ingredients. Despite their promise, information regarding the application of enzymatic hydrolysis to purslane and solid-state bioconversion to mesquite pods remains limited, particularly in relation to nutrient digestibility and antinutritional factor reduction in Nile tilapia diets.
       
Therefore, the objective of this study was to evaluate whether enzymatic hydrolysis of purslane and solid-state bioconversion of mesquite pods improve nutrient digestibility and reduce antinutritional factors when used as alternative plant ingredients in diets for Nile tilapia (Oreochromis niloticus).

MATERIALS AND METHODS

The experiment was conducted in 2025 at the Laboratory of Food Biotechnology and Aquaculture Nutrition, Escuela Nacional de Ingeniería Pesquera, Universidad Autónoma de Nayarit. Purslane (Portulaca oleracea) and mesquite pods (Prosopis laevigata) were collected in San Blas, Nayarit, Mexico, dried, milled, sieved (80 μm) and stored at 4°C until use.
       
Four ingredients were evaluated: Natural Purslane Flour (NPF), Hydrolyzed Purslane Flour (HPF), Raw Mesquite Pod Flour (RMPF) and Bioconverted Mesquite Pod Flour (BMPF). NPF and RMPF were used without treatment, whereas HPF was obtained through enzymatic hydrolysis and BMPF through solid-state bioconversion.
 
Enzymatic hydrolysis
 
Hydrolysis of purslane flour was performed according to Li et al. (2006). Flour suspensions were adjusted to pH 8.0 and incubated with Alcalase (4% w/v) at 55°C for 2 h. Enzyme activity was terminated by heating at 100°C for 10 min. The hydrolysates were dried at 55°C for 24 h and stored at 4°C. Hydrolysis efficiency was verified by SDS-PAGE following Laemmli (1970).
 
Solid-state bioconversion
 
Mesquite pod flour was bioconverted following Cuevas-Rodríguez et al. (2004) with modifications. Mesquite pods were treated with 8% acetic acid, cooked, inoculated with Rhizopus oligosporus (1 ×106  spores mL-1) and incubated at 30°C for 48 h. Bioconverted samples were subsequently dehydrated, milled and sieved.
 
Proximate composition and antinutritional factors
 
Proximate composition of ingredients and diets (crude protein, ether extract, ash, crude fiber and nitrogen-free extract) was determined according to AOAC (2012) methods. Phytic acid, tannins, trypsin inhibitor activity and saponins were quantified following Latta and Eskin (1980), Price et al., (1979), Stauffer (1990) and Hiai et al., (1976), respectively.
 
Experimental diets and digestibility assay
 
A reference diet containing 30% protein and 10% lipid was formulated. Apparent digestibility coefficients were determined using the indirect substitution method of Cho and Slinger (1979), replacing 30% of the reference diet with each test ingredient. Experimental diets contained 1% chromic oxide as an inert marker (Table 1). Diets were mixed, pelleted (2 mm), dried at 45°C for 12 h and stored at 4°C.

Table 1: Formulation of the reference and experimental diets for the digestibility bioassay based on Purslane (Portulaca oleracea) Hydrolysis and Mesquite (Prosopis laevigata) for tilapia (Oreochromis niloticus).


 
Fish and rearing conditions
 
Nile tilapia (15±3 g) were stocked at 10 fish per tank in fifteen 250-L conical fiberglass tanks. Continuous aeration maintained dissolved oxygen between 6.21 and 7.15 mg L-1 and water temperature at 27±4°C. Fish were acclimated and fasted for two days before the trial. During the 25-day digestibility assay, fish were fed ad libitum twice daily. Feces were collected by siphoning two hours after feeding, rinsed with distilled water, dried and stored for analysis.
 
Chromic oxide determination and digestibility calculations
 
Chromic oxide concentrations in diets and feces were determined following Furukawa and Tsukahara (1966). Apparent digestibility coefficients for dry matter and protein were calculated according to Maynard et al., (1981) using chromic oxide as an external marker.
 
Statistical analysis
 
Data were tested for normality and homogeneity of variance and analyzed by one-way ANOVA (α = 0.05). Significant differences among treatments were identified using Tukey’s multiple comparison test. Analyses were performed using STATISTICA 7.0 software.

RESULTS AND DISCUTION

Biotechnological processing significantly improved the nutritional quality of both purslane (Portulaca oleracea) and mesquite pod (Prosopis laevigata) flours. Enzymatic hydrolysis increased the protein content of purslane flour by approximately 8%, while solid-state bioconversion increased the protein content of mesquite pod flour and reduced lipid levels in both ingredients (Table 2). Similar effects have been reported for fermented and hydrolyzed plant ingredients, where the utilization of carbohydrates and lipids during processing concentrates the protein fraction and improves nutrient availability (Toor et al., 2022; Yang et al., 2022).

Table 2: Proximate chemical composition (g/kg) of the ingredients used in the diets.


       
The treatments also significantly reduced the principal antinutritional factors, including phytic acid, trypsin inhibitors and saponins (Table 3). The reduction of these compounds is nutritionally relevant because they interfere with nutrient absorption, digestive enzyme activity and protein utilization in fish. Previous studies have shown that enzymatic hydrolysis and microbial fermentation promote the degradation of phytates and protease inhibitors, resulting in improved nutrient availability and digestive efficiency in aquaculture species (Pontes et al., 2021; Rodrigues et al., 2023; Rachmawati et al., 2024). In contrast, tannin concentrations were negligible in purslane flour and remained low in mesquite pod flour after processing, suggesting that tannins are unlikely to limit the use of these ingredients in tilapia diets.

Table 3: Antinutritional content in ingredients used in fish diets.


       
The improvements in chemical composition and antinutritional profile were reflected in the digestibility results. Hydrolyzed purslane flour and bioconverted mesquite pod flour showed significantly higher apparent digestibility coefficients for dry matter and protein than their untreated counterparts. Protein digestibility exceeded 90% in both processed ingredients, indicating that hydrolysis and bioconversion enhanced nutrient accessibility and utilization (Table 4). Similar improvements have been reported in fermented and hydrolyzed plant ingredients used in Nile tilapia diets, where reductions in antinutritional factors and structural carbohydrates increased the availability of amino acids and digestible nutrients (Meyllianawaty and Maulida, 2022; Neves et al., 2024).

Table 4: Mean (±SD) apparent digestibility of dry matter (ADM) and apparent protein digestibility (ADP) of the analyzed ingredients.


       
Overall, the results demonstrate that enzymatic hydrolysis and solid-state bioconversion effectively improve the nutritional value of purslane and mesquite pod flours by increasing protein availability, reducing antinutritional compounds and enhancing nutrient digestibility. These findings support the potential use of both processed ingredients as alternative plant protein sources in Nile tilapia feeds.

CONCLUSION

Enzymatic hydrolysis and solid-state bioconversion significantly improved the nutritional quality of purslane (Portulaca oleracea) and mesquite pod (Prosopis laevigata) flours by increasing protein content, reducing antinutritional factors and enhancing nutrient digestibility in Nile tilapia diets. These results support the potential use of both processed ingredients as sustainable alternative protein sources for Oreochromis niloticus, although further growth trials are required to validate their application under commercial farming conditions.

ACKNOWLEDGEMENT

The present study was supported by Jessica María Rodríguez Leyva received support from SECIHTI as part of her Master of Science program. The authors also wish to thank all the staff and colleagues who contributed to the experimental work and laboratory analyses.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Effects of Processed Plant Ingredients (Hydrolyzed Purslane and Bioconverted Mesquite) on Digestibility and Antinutritional Factors in Nile Tilapia (Oreochromis niloticus)

    J
    Jessica M. Rodríguez-Leyva1
    F
    Francisco J. Valdez-González1,2
    I
    Iván E. Díaz-Vázquez4
    E
    Edith O. Cuevas-Rodríguez3
    O
    Oscar I. Zavala-Leal1,2
    L
    Liliana León-López3
    L
    L. Cuevas-Rodríguez Breidy1,2,*
    1Programa de Maestría en Ciencias Biológico Agropecuarias, Universidad Autónoma de Nayarit. Carretera Tepic-Compostela, km 9 C.P. 63780, Xalisco, Nayarit.
    2Escuela Nacional de Ingeniería Pesquera, Universidad Autónoma de Nayarit. Carretera a Los Cocos km. 12, Bahía de Matanchén San Blas, Nayarit, México.
    3Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Programa Regional de Posgrado en Biotecnología, Culiacán, Sinaloa, México.
    4Universidad Tecnológica de la Sierra. Carretera Federal Ruíz-Zacatecas Km 100.9 C.P. 63546, Mesa del Nayar, Nayarit, México.

    ABSTRACT

    Background: The incorporation of plant-based ingredients in aquafeed represents a sustainable alternative to reduce dependence on animal-derived protein sources such as fishmeal. However, the presence of antinutritional compounds limits nutrient utilization and growth performance in cultured species. This study evaluated the effect of enzymatic hydrolysis and solid-state fermentation on improving the nutritional quality of plant-derived flours.

    Methods: Purslane (Portulaca oleracea) flour was enzymatically hydrolyzed, while mesquite pod (Prosopis laevigata) flour underwent solid-state fermentation with Rhizopus oligosporus. Proximate composition, antinutritional factors (tannins and phytates) and apparent digestibility coefficients were determined.

    Result: Significant differences (p≤0.05) were observed in chemical composition after treatments. Protein content increased to 22.41±2.28% in purslane flour and 14.18±0.80% in mesquite flour, while lipid content decreased to 1.10±0.24% and 3.30±0.75%, respectively. Both treatments substantially reduced tannin and phytate concentrations, indicating effective degradation of antinutritional compounds. Additionally, the apparent digestibility coefficients of dry matter improved, ranging from 65.79 to 85.73%. These improvements are attributed to enzymatic degradation of fibrous structures and complex polysaccharides, promoting the release of low-molecular-weight peptides and more bioavailable amino acids.

    INTRODUCTION

    Aquaculture is one of the fastest-growing animal food production sectors worldwide, with Nile tilapia (Oreochromis niloticus) ranking among the most important cultured fish species due to its rapid growth, high survival and adaptability to diverse farming conditions (Arumugam et al., 2023; Cuevas-Rodríguez et al., 2024). However, feed costs remain a major constraint to profitable production, accounting for up to 70% of operational expenses, largely because of the inclusion of fishmeal as the primary protein source (Esmaeili et al., 2017; Rodríguez-Hernández et al., 2025).
           
    Fishmeal is widely used in aquaculture feeds because of its high protein content, balanced amino acid profile, palatability and digestibility. Nevertheless, increasing costs and limited availability have intensified the search for alternative protein sources that can partially or completely replace fishmeal without compromising fish performance (Hussain et al., 2024; Nandi et al., 2025). Plant-based ingredients have emerged as promising alternatives, although their utilization is often constrained by the presence of antinutritional factors that reduce nutrient availability and digestibility (Bhatt et al., 2011; Nikmaram et al., 2017; Priyatharshni et al., 2024).
           
    Among potential alternative ingredients, purslane (Portulaca oleracea) and mesquite pods (Prosopis laevigata) are of particular interest due to their availability and nutritional value. Purslane is rich in a-linolenic acid and can be produced year-round in tropical and subtropical regions (Masoodi et al., 2011; Zhou et al., 2015). Mesquite pods are abundant in arid and semi-arid regions of Mexico and contain appreciable levels of protein and bioactive compounds (González-Cortazar et al., 2025; Barragán-Longoria et al., 2025). However, both ingredients contain antinutritional compounds that may limit their efficient use in aquafeeds.

    Processing techniques such as enzymatic hydrolysis and solid-state bioconversion have demonstrated potential to improve nutrient availability and reduce antinutritional factors in plant-derived feed ingredients. Despite their promise, information regarding the application of enzymatic hydrolysis to purslane and solid-state bioconversion to mesquite pods remains limited, particularly in relation to nutrient digestibility and antinutritional factor reduction in Nile tilapia diets.
           
    Therefore, the objective of this study was to evaluate whether enzymatic hydrolysis of purslane and solid-state bioconversion of mesquite pods improve nutrient digestibility and reduce antinutritional factors when used as alternative plant ingredients in diets for Nile tilapia (Oreochromis niloticus).

    MATERIALS AND METHODS

    The experiment was conducted in 2025 at the Laboratory of Food Biotechnology and Aquaculture Nutrition, Escuela Nacional de Ingeniería Pesquera, Universidad Autónoma de Nayarit. Purslane (Portulaca oleracea) and mesquite pods (Prosopis laevigata) were collected in San Blas, Nayarit, Mexico, dried, milled, sieved (80 μm) and stored at 4°C until use.
           
    Four ingredients were evaluated: Natural Purslane Flour (NPF), Hydrolyzed Purslane Flour (HPF), Raw Mesquite Pod Flour (RMPF) and Bioconverted Mesquite Pod Flour (BMPF). NPF and RMPF were used without treatment, whereas HPF was obtained through enzymatic hydrolysis and BMPF through solid-state bioconversion.
     
    Enzymatic hydrolysis
     
    Hydrolysis of purslane flour was performed according to Li et al. (2006). Flour suspensions were adjusted to pH 8.0 and incubated with Alcalase (4% w/v) at 55°C for 2 h. Enzyme activity was terminated by heating at 100°C for 10 min. The hydrolysates were dried at 55°C for 24 h and stored at 4°C. Hydrolysis efficiency was verified by SDS-PAGE following Laemmli (1970).
     
    Solid-state bioconversion
     
    Mesquite pod flour was bioconverted following Cuevas-Rodríguez et al. (2004) with modifications. Mesquite pods were treated with 8% acetic acid, cooked, inoculated with Rhizopus oligosporus (1 ×106  spores mL-1) and incubated at 30°C for 48 h. Bioconverted samples were subsequently dehydrated, milled and sieved.
     
    Proximate composition and antinutritional factors
     
    Proximate composition of ingredients and diets (crude protein, ether extract, ash, crude fiber and nitrogen-free extract) was determined according to AOAC (2012) methods. Phytic acid, tannins, trypsin inhibitor activity and saponins were quantified following Latta and Eskin (1980), Price et al., (1979), Stauffer (1990) and Hiai et al., (1976), respectively.
     
    Experimental diets and digestibility assay
     
    A reference diet containing 30% protein and 10% lipid was formulated. Apparent digestibility coefficients were determined using the indirect substitution method of Cho and Slinger (1979), replacing 30% of the reference diet with each test ingredient. Experimental diets contained 1% chromic oxide as an inert marker (Table 1). Diets were mixed, pelleted (2 mm), dried at 45°C for 12 h and stored at 4°C.

    Table 1: Formulation of the reference and experimental diets for the digestibility bioassay based on Purslane (Portulaca oleracea) Hydrolysis and Mesquite (Prosopis laevigata) for tilapia (Oreochromis niloticus).


     
    Fish and rearing conditions
     
    Nile tilapia (15±3 g) were stocked at 10 fish per tank in fifteen 250-L conical fiberglass tanks. Continuous aeration maintained dissolved oxygen between 6.21 and 7.15 mg L-1 and water temperature at 27±4°C. Fish were acclimated and fasted for two days before the trial. During the 25-day digestibility assay, fish were fed ad libitum twice daily. Feces were collected by siphoning two hours after feeding, rinsed with distilled water, dried and stored for analysis.
     
    Chromic oxide determination and digestibility calculations
     
    Chromic oxide concentrations in diets and feces were determined following Furukawa and Tsukahara (1966). Apparent digestibility coefficients for dry matter and protein were calculated according to Maynard et al., (1981) using chromic oxide as an external marker.
     
    Statistical analysis
     
    Data were tested for normality and homogeneity of variance and analyzed by one-way ANOVA (α = 0.05). Significant differences among treatments were identified using Tukey’s multiple comparison test. Analyses were performed using STATISTICA 7.0 software.

    RESULTS AND DISCUTION

    Biotechnological processing significantly improved the nutritional quality of both purslane (Portulaca oleracea) and mesquite pod (Prosopis laevigata) flours. Enzymatic hydrolysis increased the protein content of purslane flour by approximately 8%, while solid-state bioconversion increased the protein content of mesquite pod flour and reduced lipid levels in both ingredients (Table 2). Similar effects have been reported for fermented and hydrolyzed plant ingredients, where the utilization of carbohydrates and lipids during processing concentrates the protein fraction and improves nutrient availability (Toor et al., 2022; Yang et al., 2022).

    Table 2: Proximate chemical composition (g/kg) of the ingredients used in the diets.


           
    The treatments also significantly reduced the principal antinutritional factors, including phytic acid, trypsin inhibitors and saponins (Table 3). The reduction of these compounds is nutritionally relevant because they interfere with nutrient absorption, digestive enzyme activity and protein utilization in fish. Previous studies have shown that enzymatic hydrolysis and microbial fermentation promote the degradation of phytates and protease inhibitors, resulting in improved nutrient availability and digestive efficiency in aquaculture species (Pontes et al., 2021; Rodrigues et al., 2023; Rachmawati et al., 2024). In contrast, tannin concentrations were negligible in purslane flour and remained low in mesquite pod flour after processing, suggesting that tannins are unlikely to limit the use of these ingredients in tilapia diets.

    Table 3: Antinutritional content in ingredients used in fish diets.


           
    The improvements in chemical composition and antinutritional profile were reflected in the digestibility results. Hydrolyzed purslane flour and bioconverted mesquite pod flour showed significantly higher apparent digestibility coefficients for dry matter and protein than their untreated counterparts. Protein digestibility exceeded 90% in both processed ingredients, indicating that hydrolysis and bioconversion enhanced nutrient accessibility and utilization (Table 4). Similar improvements have been reported in fermented and hydrolyzed plant ingredients used in Nile tilapia diets, where reductions in antinutritional factors and structural carbohydrates increased the availability of amino acids and digestible nutrients (Meyllianawaty and Maulida, 2022; Neves et al., 2024).

    Table 4: Mean (±SD) apparent digestibility of dry matter (ADM) and apparent protein digestibility (ADP) of the analyzed ingredients.


           
    Overall, the results demonstrate that enzymatic hydrolysis and solid-state bioconversion effectively improve the nutritional value of purslane and mesquite pod flours by increasing protein availability, reducing antinutritional compounds and enhancing nutrient digestibility. These findings support the potential use of both processed ingredients as alternative plant protein sources in Nile tilapia feeds.

    CONCLUSION

    Enzymatic hydrolysis and solid-state bioconversion significantly improved the nutritional quality of purslane (Portulaca oleracea) and mesquite pod (Prosopis laevigata) flours by increasing protein content, reducing antinutritional factors and enhancing nutrient digestibility in Nile tilapia diets. These results support the potential use of both processed ingredients as sustainable alternative protein sources for Oreochromis niloticus, although further growth trials are required to validate their application under commercial farming conditions.

    ACKNOWLEDGEMENT

    The present study was supported by Jessica María Rodríguez Leyva received support from SECIHTI as part of her Master of Science program. The authors also wish to thank all the staff and colleagues who contributed to the experimental work and laboratory analyses.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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