Bioprospecting Opuntia ficus-indica (L.) Mill cladode fractions for improved functionality in livestock feed

Opuntia ficus-indica offers significant health benefits for livestock in arid regions, yet the potential of its cladode components for specialised feed formulation has not been fully explored. The present study investigated the potential of freeze-dried cladode components from O. ficus-indica (var. Morado) for use in livestock feed. The cladodes were separated into peel, mucilage and insoluble fibre, and then freeze-dried and pulverised into powder. Analyses included powder yield, moisture content (MC), hydration parameters [water solubility (WS), water holding capacity (WHC), swelling capacity (SC) and hygroscopicity], pigments, bioactive compounds, anti-nutritional factors [total tannin content (TTC) and total alkaloid content (TAC)] and antioxidant activities including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric reducing antioxidant power (FRAP). The peel powder had the highest powder yield (7.6%) and lower MC (5.57%), while displaying significantly higher total chlorophyll (188 µg/g) and β-carotene (5.9 mg/g) and the strongest antioxidant activity, with the lowest IC 50 values for both DPPH and ABTS (0.005 and 0.03 mg/mL, respectively), compared to mucilage and insoluble fibre powders. The mucilage powder exhibited the highest water solubility (48.3%), while the insoluble fibre powder showed the highest WHC (16.95 g dH 2 O/g DW) and SC (16.55 g) and the lowest hygroscopicity (37.91 g/100 g). TTC and TAC were within ranges of 0.4–0.97 mg TAE/g DW and 0.78–1.74 g/100 g DW, respectively, across all samples. FTIR analysis confirmed the abundance of functional groups in the peel powder, with the strongest absorption bands corresponding to C–O stretching vibrations. Principal component analysis (PCA) validated these findings, associating the highest pigment content, bioactive content and antioxidant properties with the peel powder. Therefore, these results suggest O. ficus-indica cladode peels as valuable sources of bioactive compounds that may be studied further for antioxidant functions in livestock, while mucilage and insoluble fibre may be further investigated for enhanced hydration properties of livestock feeds.

Livestock production, especially in arid and semi-arid regions, is frequently constrained by the scarcity and low quality of conventional feed resources, particularly during prolonged dry seasons caused by intense droughts (Baris, 2023). This persistent challenge necessitates exploring alternative, climate-resilient feed options that provide essential functional properties and improve animal performance, health, and nutrition. Cactus pear (Opuntia ficus-indica L. Mill), a plant highly adapted to drought conditions due to its unique crassulacean acid metabolism and efficient water retention capacity, has emerged as a promising sustainable forage (Pastorelli et al., 2022; Dutra et al., 2024). The Opuntia ficus-indica components, including the fruit and cladodes, are rich sources of bioactive and nutritious compounds suitable for use in various value-added products. The cladodes, which are the focus of this study, are reported as valuable sources of highly digestible carbohydrates and various bioactive constituents, including antioxidants, pigments, vitamins, and minerals (García et al., 2015; Andreu et al., 2018; Blando et al., 2019; de Wit and Fouché, 2021). Despite the O. ficus-indica cladodes’ low crude protein compared to some traditional forages like soybean, their high digestibility and energy content make them an excellent supplementary feed, particularly when combined with other protein-rich sources to form hybrid feed formulations (Tonisi et al., 2026).

Due to their highlighted functional benefits, the cladodes of O. ficus-indica have found valuable applications as cheap alternative fodder for livestock (Mahrose, 2021), such as ruminants, in both fresh and sun-dried forms (Tonisi et al., 2026). However, drying methods such as sun-drying have been reported to deplete nutrients such as vitamin C, calcium, and water-soluble carbohydrates, as well as bioactive compounds, in agricultural products, including animal feed (Ndawula et al., 2004; Çoklar and Akbulut, 2017; Mahlogonolo, 2024; Arcanjo et al., 2025). As such, dehydration methods, specifically freeze-drying, the method of choice in this study, have emerged as a viable technique for preserving nutrients and bioactive compounds in livestock feed. Among various dehydration techniques, including open sun, shade, hybrid, microwave, and air convective, freeze-drying has shown superior results for preserving the biochemical composition of alfalfa hay (Kaplan et al., 2024). Similarly, freeze-drying has been shown to retain and enhance total phenolic and flavonoid content, as well as antioxidant capacity, in seaweed (Ullah et al., 2025). The main disadvantages of freeze-drying are its prohibitive cost, high energy consumption, and lengthy processing times. Given these demerits relative to its merits, freeze-drying is primarily used in this study as a standardisation and benchmarking tool to assess maximum preservation and accurate profiling of bioactive compounds, nutrients, and functional properties, rather than as the most suitable method for the commercial drying of animal feed. As a foundational study, prioritising a processing technique that minimally degrades valuable compounds is essential for revealing the true biochemical and biological characteristics of the plant material.

Processing of O. ficus-indica cladodes can yield different fractions, such as peel, mucilage and insoluble fibre, each with potentially distinct nutritional, biological and functional properties. The mucilage, a complex polysaccharide-rich hydrocolloid, is known for its functional properties, such as efficient water solubility (Mukaila et al., 2025), which may be attributed to soluble polysaccharides and could increase the energy content of livestock feed. The peel and fibrous residues, often considered by-products in mucilage processing, are rich in phenolics and dietary fibre, respectively. The presence of phenolic compounds may potentially increase biologically protective properties of livestock feed, such as anti-inflammatory and antioxidant activities (Zeghbib et al., 2022). Fibrous compounds may possess varying hydration functions that serve as crucial indicators of potential gastrointestinal benefits in livestock. Therefore, exploring the specific potential of these components as tailored animal feed supplements is essential. Furthermore, valorising these fractions aligns with FAO (2013) climate-smart strategies by addressing feed shortages and supporting circular economy principles through the repurposing of agricultural by-products into value-added products.

Despite extensive research validating the use of whole cactus cladodes as forage (Mayer and Cushman, 2019; Mabotja, 2020; Nyambali et al., 2022; Pastorelli et al., 2022; Moshidi et al., 2025); utilising the whole cladode prevents more diversified feed applications. A notable research gap exists regarding comparisons of the functional properties and biological activities of O. ficus-indica cladode fractions. Addressing this gap may unlock the full potential of O. ficus-indica, cladodes, allowing the design of specialised feed formulations for diverse livestock categories. In addition, South Africa is one of the countries that harbour various cultivars of O. ficus-indica, predominantly utilised for fruit production, while cladode production is an overlooked feed source that may mitigate livestock feed shortages during droughts. Thus, appraising the components of O. ficus-indica cladodes provides valuable information for future studies investigating their diverse applications as nutrient-rich, drought-resistant feed ingredients or fortifiers. This approach is highly relevant for arid regions, including South Africa, where continuous droughts foster research that will help increase the adoption of drought-resilient feed sources, such as O. ficus-indica cladodes, as a reliable livestock feed.

Therefore, the study described herein aims to evaluate and compare the physical characteristics, hydration properties, phytochemical and antioxidant activities, as well as the presence of functional groups of freeze-dried peel, mucilage and insoluble fibre fractions derived from O. ficus-indica cladodes to stimulate future research that may further study their potential as sustainable animal feed supplements. The study hypothesised that the freeze-dried peel, mucilage, and insoluble fibre fractions would exhibit distinct, and fraction-specific characteristics. This research will also provide a valuable foundation for in vitro and in vivo investigations aimed at maximising the use of this drought-tolerant plant to enhance livestock nutrition and performance, particularly in drought-prone regions.

Plant material collection

O. ficus-indica (L.) Mill (var. Morado) cladodes were harvested from a 10-year-old cactus pear orchard at Ubali farm, located in Kamelfontein, Pretoria, South Africa (25°38′31.9″S, 28°27′33.0″E). The plant material was transported to the Postharvest and Agro-processing Research Centre (PARC) at the University of Johannesburg, Gauteng province, South Africa.

Preparation of peel, mucilage and insoluble fibre

The cladodes were thoroughly washed with running tap water for approximately 2 min and cut into uniform pieces (20 × 20 mm) using a sterilised kitchen knife. Peels were then removed from the cut pieces and set aside for further processing. The intact pulp obtained after peel removal was then microwaved at 800 W for 4 min before being homogenised in an electric blender (Nutri1200, Milex, Japan) to obtain a slurry (Shinga and Fawole, 2023). Mucilage (supernatant) was then extracted from the slurry by centrifugation (Thermo Fisher Scientific, Biofuge Stratos, United Kingdom) for 10 min at 8000 × g and 4 °C. Sediments obtained after centrifugation, referred to as the insoluble fibre, were also collected. The different components (peel, mucilage, and insoluble fibre) were then packed in different airtight plastic bags and frozen at -80 °C before freeze-drying after 24 h.

Preparation of freeze-dried peel, mucilage and insoluble fibre powders

The frozen samples (peel, mucilage and insoluble fibre) were freeze-dried (HRFDL, Harvest Right, Salt Lake City, USA) for 72 h at a pressure and temperature of 0.05 mbar and –50 °C, respectively. The freeze-dried samples were then pulverised (Knife Mill Pulverisette 11, Fritsch, Germany) into fine powders, packed in airtight containers and stored in a dark place at room temperature until further analysis.

Analysis of the freeze-dried powders

Physical properties

The powder yield was measured on a dry matter (DM) basis relative to the fresh weight. Moisture content was determined using a Kern DBS moisture analyzer (DBS60-3, Kern and Sohn, Ziegelel, Balingen, Germany). Colour attributes, including the L* (lightness/darkness), a* (redness/greenness), and b* (yellowness/blueness), were measured using a calibrated chromometer (CR-10 plus, Konica Minolta, Osaka, Japan), while chroma and hue angle were calculated from the a* and b* values. Detailed analytical procedures are provided in Supplementary Section S1 of the Supplementary Material.

Hydration properties

Water solubility (%) and water holding capacity (g dH 2 O/g dry sample weight) of the freeze-dried powders were determined following the methods described by Cegledi et al. (2022) and Zain et al. (2021), respectively. The procedures reported by Lokeswari et al. (2021) and Cegledi et al. (2022) were used to determine the powder swelling capacity (g/g) and hygroscopicity (g/100 g), respectively. Detailed analytical procedures are provided in the Supplementary File S2.

Chlorophyll a, b and total chlorophyll content

Chlorophyll pigments (a and b) and total chlorophyll content were determined according to the method described by Olarewaju et al. (2017). Briefly, a powder sample (1 g) was mixed with 5 mL of 80% (v/v) acetone solution in the dark and centrifuged (Thermo Fisher Scientific, Biofuge Stratos, United Kingdom) at 15 000 × g for 30 min at 5 °C. The absorbance of the supernatants was measured at 470, 646, and 663 nm, using a microplate reader (AMR-100, Hangzhou Allsheng Instruments, Hangzhou, China). Thereafter, the detection of total chlorophyll (TChl) was calculated using Equations 1–3.

Where Chl a and Chl b denote chlorophyll a and b content, subsequently used to calculate TChl. All measurements were conducted in triplicate.

β-carotene and vitamin C content

The determination of β-carotene was performed under dim lights. Briefly, the powder sample (1 g) was mixed with 30 mL of ethanol (96%) and hexane solution (1:1). The mixture was vortexed (Vortex mixer-300, Gemmy Industrial Corp., Taipei, Taiwan), after which the mixture was sonicated (Labotec 705, Johannesburg, Gauteng, South Africa) for 10 min at a temperature below 30 °C, maintained using ice. The sonicated samples were then centrifuged (Thermo Fischer Scientific, Biofuge, Stratos, United Kingdom) for 5 min at 4 °C and 8 400 × g, and the absorbances of the resultant supernatants were measured at a wavelength of 470 nm using a microplate reader (AMR-100, Hangzhou Allsheng Instruments, Hangzhou, China). A β-carotene standard curve (R2 = 0.9987, concentration: 0.0125-0.2 mg/mL) was prepared, and the results were expressed as mg β-carotene/100 g dry weight (mg β-carotene/100 g DW). The experiment was conducted in triplicate.

Vitamin C was measured according to the procedure reported by Shinga et al. (2025). Powdered samples (0.125 g) were mixed with 5 mL of metaphosphoric acid and vortexed (30s) (Vortex mixer-300, Gemmy Industrial Corp., Taipei, Taiwan) prior to sonication for 10 min using an ultrasonic bath (Labotec 705, Johannesburg, Gauteng, South Africa). The mixture was then centrifuged at 4 °C and 10 000 × g for 10 min. Aliquots (50 µL) of the supernatants were mixed with 100 µL of 2, 6-dichlorophenolindophenol in a 96-well plate and incubated at room temperature in the dark for 30 min. After incubation, absorbance was measured at 515 nm using a microplate reader (AMR-100, Hangzhou Allsheng Instruments, Hangzhou, China). A vitamin C standard curve (R = 0.9993, concentration: 0.05-0.25 mg/mL) was prepared, and the results were expressed as mg ascorbic acid equivalent per gram of dry weight (mg AAE/g DW).

Total phenolic content, total tannin content, total flavonoid content and total alkaloid content

Total phenolic content (TPC), total flavonoid content (TFC), total tannin content (TTC), and total alkaloid content (TAC) were determined using the extracts prepared following the procedure described in the Supplementary Material (Supplementary Section S2). The measurement of TPC was determined according to a previously described method (Notardonato et al., 2023). Briefly, each sample (500 µL) was mixed with 500 µL of Folin-Ciocalteu reagent (2N), incubated for 2 min at room temperature, and then sodium carbonate (2 500 µL of 2%) was added. Thereafter, the samples were vortexed (Vortex mixer-300, Gemmy Industrial Corp., Taipei, Taiwan) and incubated in the dark at room temperature for 40 min. The samples’ absorbances were measured at 725 nm against a blank (50% methanol) using a microplate reader (AMR-100, Hangzhou Allsheng Instruments, Hangzhou, China). A gallic acid standard curve (R2 = 0.9969, concentration: 0.02-0.06 mg/mL) was prepared and the results were expressed as mg gallic acid equivalent per gram of dry weight (mg GAE/g DW).

The method described by Notardonato et al. (2023) was followed to determine TTC. In brief, polyvinylpolypyrrolidone (PVPP, 100 mg) was added to a mixture containing 1 mL of 50% methanol and 1 mL of an extract. The mixture was then vortexed (Vortex mixer-300, Gemmy Industrial Corp., Taipei, Taiwan) for 30s and incubated at 4 °C for 15 min in the dark. After incubation, the samples were vortexed again for 30s and then centrifuged (Thermo Fischer Scientific, Biofuge, Stratos, United Kingdom) at 3000 × g for 10 min. The obtained supernatant (500 µL) was used to measure total tannins using the total phenolic method described above. The TTC was calculated as the difference between the total phenolic content before and after treatment with PVPP, and the results were expressed as tannic acid equivalents per gram of the sample’s dry weight (mg TAE mg TAE/g DW).

The TFC was determined following the method described by Yang et al. (2009). In brief, 120 µL of distilled water and 75 µL of 5% sodium nitrite were added to a sample extract of 250 µL. After incubation for 5 min, 150 µL of freshly prepared 10% aluminium chloride was added to the reaction mixture. After additional incubation of 5 min at room temperature, a volume of 500 µL (1M sodium hydroxide) was added, followed by the addition of 775 µL of distilled water to bring the mixture to a final volume of 3 mL. The absorbance was immediately measured using a wavelength of 510 nm using a microplate reader (AMR-100, Hangzhou Allsheng Instruments, Hangzhou, China) against a blank (50% methanol). A standard curve (R2 = 0.9913, concentration: 0.005-0.08 mg/mL) was prepared using catechin and the TFC was expressed as mg of catechin equivalent per gram of dry weight (mg CE/g DW).

The TAC of the samples was quantified according to the method described by Adeniyi et al. (2009). Briefly, the sample (2 g) was mixed with 30 mL of 10% acetic acid (prepared in absolute ethanol) and incubated for 4h at room temperature. The resultant mixture was then filtered (Filter discs-90 MM, Ahlstrom Munktell, Helsinki, Finland) and evaporated to a quarter of its original volume using a water-bath (55-70 °C). Thereafter, concentrated ammonia solution was added dropwise until the precipitation was complete. The precipitate was allowed to settle, and a pre-weighed filter disc was used to collect it. The collected residue was washed three times with 1% ammonia solution, and the filter paper was dried in an oven at 60 °C to a constant weight. The difference between the weights of the pre-weighed and dried filter disk was used to calculate the alkaloid content, which was expressed as g/100 g. All analyses were conducted in triplicate.

Antioxidant activity

Radical scavenging activity against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical was determined using the extracts prepared following the procedure described in the Supplementary Material (Supplementary Section S2). To measure the scavenging activity of the samples against DPPH, the method described by Karioti et al. (2004) was followed. Various concentrations (0.001, 0.002, 0.005, 0.01, 0.02, 0.03 mg/mL) of the extracts were used, from which 15 µL of each of the prepared extracts was transferred into Eppendorf tubes, followed by the addition of 735 µL of aqueous methanol. Methanolic DPPH (0.1 mM, 750 µL) was added to the mixture, which was incubated for 30 min at room temperature in the dark. Ascorbic acid was used as a positive control. The absorbance was measured at 517 nm using a spectrophotometer under dim light. The experiment was conducted in triplicate. DPPH radical scavenging activity was calculated using the following formula shown in Equation 4.

Where A E is the absorbance of the tested sample while A B is the absorbance of the DPPH solution only. The analysis was conducted in triplicate. The IC 50 values were calculated to obtain the concentration of samples required to inhibit 50% of the DPPH radical.

ABTS radical scavenging activity of the samples was measured according to a previously reported method (Jurić et al., 2023). A working solution was prepared by mixing equal volumes of 7 mM ABTS and 2.4 mM potassium persulfate and incubating the mixture at room temperature for 12h in the dark. The obtained ABTS working solution (1 mL) was mixed with 60 mL of methanol to obtain an absorbance of 0.706 ± 0.001 units measured at 734 nm using a microplate reader (AMR-100, Hangzhou Allsheng Instruments, Hangzhou, China). Various concentrations (0.02, 0.04, 0.06, 0.08, 0.1, 0.12 mg/mL) of the sample or standard were prepared from which 75 µL was transferred into a clean centrifuge tube, followed by the addition of 1425 µL of ABTS working solution and incubation for 7 min in the dark at room temperature. The absorbance was measured at 734 nm against a blank (50% methanol). The final ABTS radical scavenging activity of the extracts and that of Trolox (positive control) was calculated using the following formula shown in Equation 5.

Where Abs control is the absorbance of ABTS radical 50% methanol, A b s s a m p l e is the absorbance of the ABTS radical sample extract/standard. The analysis was conducted in triplicate. The IC 50 values were calculated to obtain the concentration of samples required to inhibit 50% of the ABTS radical.

The ferric reducing antioxidant power (FRAP) of the samples was determined according to the method reported by Thaipong et al. (2006). Each powder sample (2 g) was added to 30 mL of 50% methanol and the extraction was done as reported in the Supplementary Material (Supplementary Section S2). Thereafter, a 150 µL aliquot was mixed with 2850 µL of the FRAP reagent solution, prepared by adding 25 mL of acetate buffer, 2.5 mL of 2, 4, 6-Tris (2-pyridyl)-s-triazine, and 2.5 mL of ferric chloride. The resulting reaction mixture was incubated for 30 min in the dark, and absorbance was measured at 593 nm using a microplate reader (AMR-100, Hangzhou Allsheng Instruments, Hangzhou, China). A standard curve (R2 = 0.9946, concentration: 0.0-0.25 mg/mL) was prepared using Trolox, and the FRAP was expressed as µM of Trolox equivalent per gram of dry weight (µM TE/g DW).

ATR-FTIR analysis

Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy (ATR-FTIR) (Cary 630 FTIR, Agilent Technologies, Santa Clara, California) was used to analyze the presence of functional groups in the powder samples. The resultant spectra were interpreted using the Microlab software. Each spectrum was measured over the spectral range of 4000 to 400 cm -1, and the instrument resolution was set to 4 cm -1. The obtained ATR-FTIR spectra were plotted using Origin software (Version 8.5.1, OriginLab, Northampton, USA).

Statistical analysis

The study used a completely randomised design (n = 3), and data generated were analyzed using one-way analysis of variance (ANOVA) in STATISTICA 14.0 (TIBCO, Tulsa, OK, United States). Duncan’s Multiple Range Test was used to separate the means at the 5% significance level. The results were expressed as means ± standard error (SE). Graphical data illustrations were created using Microsoft Excel (Version 2502, Microsoft, Redmond, WA, USA). Principal component analysis (PCA) was performed using RStudio (Version 2026.01.0, Posit Software, PBC, Boston, MA, United States).

Powder yield, colour and moisture content

As demonstrated in Table 1, the peel exhibited a powder yield that was approximately 10 times higher compared to that of mucilage and insoluble fibre. This high recovery makes peel the most feasible fraction for bulk production and supports its valorisation as a functional feed ingredient for livestock production. Meanwhile, the powder yield of mucilage and insoluble fibre showed no significant difference from each other, regardless of their compositional variations. The lower powder yield is attributed to the mucilage’s high water content, which reduces the solid yield. For insoluble fibre, its lower yield can be related to the low quantities obtained after separation from the mucilage. Furthermore, although insoluble fibre occupied a large volumetric space, it produced a lower recovery weight than mucilage, likely due to differences in physical and structural properties such as porosity, particle size, and bulk density. Generally, the mucilage powder yield (0.9%) in this study was significantly lower than reported in the literature. For instance, Luna-Zapién et al. (2023) reported mucilage powder yield ranging from 13.25 to 17.1% from four varieties of cactus pear, while Harrak (2021) reported 5.1 to 6.9% across different maturities of O. ficus-indica cladodes. These discrepancies likely reflect differences in cultivar, geographical location, climate, agronomic practices, cladode age, and mainly extraction methods. Notably, this study involved peeling the cladodes before extraction, whereas previous studies did not; this procedural difference likely accounts for the lower yield observed, especially given the significant yield differences between the peel and mucilage (Table 1).

Given that moisture content (MC) directly influences the final powder mass and production yield (Tontul and Topuz, 2017), measuring MC was critical. Interestingly, across the tested samples, the peel powder, despite displaying the highest overall yield, exhibited the lowest MC, together with mucilage. Conversely, the insoluble fibre, which showed a lower yield, recorded the highest moisture content (Table 1). These findings indicate that there is no direct, proportional link between the amount of residual MC and the final powder yield. The higher MC observed in insoluble fibre powder can be attributed to its porous, rigid, and cell-wall matrix structure, which physically traps free water that makes it difficult to remove during the drying process. In addition, MC is a key indicator of storage stability and the susceptibility of powders to microbial growth, particularly mold development. The low moisture content (<10%) in all samples indicates good powder stability, potential resistance to microbial proliferation, and a prolonged shelf-life for storage and future use, especially in areas that experience droughts and animal feed shortages. While the present MC results are similar to previously reported values for oven-dried mucilage powder (4.5–5.9%), Sepúlveda et al. (2007), they differ significantly from those of freeze-dried mucilage powders reported for different cultivars (15.39–16.23%) (Du Toit et al., 2019a). These discrepancies may be attributed to various factors such as cultivar selection, extraction methods, drying conditions and the source of mucilage.

Colour parameters of plant-derived powders act as indicators for the preservation or degradation of functional pigments, which are key to the plant’s biological and health-promoting properties. Thus, to comprehensively assess the colour characteristics of the O. ficus-indica cladode components, colour parameters (L, a, b, C, and h°) were analyzed. Regarding L* values, the mucilage (80.6) and insoluble fibre (74.4) powders had higher mean values and were significantly lighter than the peel powder (55.7), and these differences are further supported by the visual images shown in Table 1. The L* results corroborate the a* values, which reflected the intense greener colour (-5.5) exhibited by the peel powder, suggesting a higher retention of chlorophyll pigment in the peel compared to other fractions. As shown in Table 1, a decrease in greenness corresponds with an increase in C* values, reflecting a shift towards yellowness, especially in mucilage and insoluble fibre powder samples. Peel and insoluble fibre powders exhibited higher h° (approximately -1.4 to -1.6 radians, or -80° to -92°) compared to the mucilage powder, which showed a significantly lower value (approximately 0.5 radians or 28°). This disparity, driven by the lower content of green pigments in the mucilage powder, further corroborates the previously observed colour results. Although the L* values of mucilage powder colour results from the present study were similar to those reported by Du Toit et al. (2019a) (77 to 83), other colour parameters like C* (23 to 24), h° (approximately 100°), a* (-4.5 to -6.1), and b* (23 to 24), differed significantly, reflecting differences in the mucilage extraction processes. Furthermore, as literature regarding the powder processing of cactus cladode into peel and insoluble fibre components is scarce, this study represents a foundational analysis of colour attributes of these distinct O. ficus-indica components.

Water solubility, water holding capacity, swelling capacity and hygroscopicity

Hydration properties are integral parameters that strongly inform the final quality of livestock feed and how they may potentially influence the gastrointestinal status of livestock (Huang et al., 2022). To assess the potential of the cladode powders for reconstitution into other feed components, their potential gut functionality and storage stability, the hydration properties, including water solubility (WS), water holding capacity (WHC), swelling capacity (SC), and hygroscopicity, were measured (Figures 1A–D). As illustrated in Figure 1A, the cladode component significantly influenced the WS of the produced powder. Mucilage exhibited the highest solubility, demonstrating a value that is 3-6-fold greater than that of the peel and insoluble fibre powder (Figure 1A), which can be attributed to the presence of water-soluble polysaccharides (Oh and Kim, 2022; Adhikary et al., 2023). Despite using an improved extraction technique that removed insoluble fibre, the present study observed lower mucilage powder solubility compared to literature values of 76–80% from other Opuntia spp. cultivars (Du Toit et al., 2019b). Conversely, the low WS expressed by the insoluble fibre and peel powders indicates varying proportions of rigid, lignocellulosic components, which do not dissolve in aqueous systems. Further, the lowest solubility of peel powder may have been driven by the additional presence of waxy hydrophobic cuticles. Nutritionally, these results suggest that mucilage can potentially offer more water-soluble polysaccharides when tested in feed diets than peel and insoluble fibre.

The WHC of the powder samples aligns with their solubility profiles (Figures 1A, B). Mucilage powder, which demonstrated the highest WS, exhibited a significantly lower WHC of 2.3 g dH 2 O/g DW compared to the peel and insoluble fibre powders that expressed a range of 15-16.95 g dH 2 O/g DW. The lower WHC of the mucilage powder is similar that of the previously dried mucilage samples extracted from certain Opuntia spp (de Andrade Vieira et al., 2021). The high WHC exhibited by the peel and insoluble fibre suggests the possible varying presence of structural non-starch polysaccharides such as pectin in these samples (Cui et al., 2013). Furthermore, the grinding process likely played a crucial role in the observed WHC results by disrupting the cell walls of the plant material, increasing surface area, and exposing water-binding polysaccharides. In comparison with traditional feeds, the insoluble fibre and peel powders exhibited significantly higher WHC than maize stover, which had a value of 8.3 g dH 2 O/g DW (Ndou et al., 2013), indicating their potential as superior sources of dietary fibre.

Along with WHC, the SC is a critical parameter for evaluating the presence and type of dietary fibre in food and feed materials (Knudsen, 2001). Figure 1C indicates that insoluble fibre exhibited a significantly higher SC (>75%) compared to peel and mucilage. This observation can be explained by the presence of hydrophilic compounds and dietary fibrous components that form expansive, water-absorbing hydrocolloid networks in aqueous environments (Cheng et al., 2025). These results corroborate the WHC findings (Figure 1B) and reinforce the fact that the insoluble fibre exhibits good water retention capacity. In addition, high SC achieved by the insoluble fibre may potentially support efficient gut functionality in livestock, as it is noted as a function of dietary fibre with substantial SC for livestock gut health (Celi et al., 2017). As fibrous plant material with significant SC may offer prebiotic benefits in gut microbiota, in vitro and in vivo fermentation studies are required to confirm their prebiotic effects in animal digestive systems. Compared to commercial fibres commonly used in feed formulations, the SC of the insoluble fibre powder in this study exceeds that of inulin (Rosell et al., 2009). Therefore, the insoluble fibre may be a better source of dietary fibre components than the peel and mucilage, suggesting potential functional and nutritional benefits as a feed ingredient, pending further studies to identify specific fibre types.

Similar to MC, the hygroscopic behavior of a powder material is a crucial factor in assessing its storage stability and chemical composition (Juarez-Enriquez et al., 2017). As shown in Figure 1D, hygroscopicity varied significantly across the powder samples, with the mucilage exhibiting the highest moisture absorption at 83%, while insoluble fibre and peel ranged between 38–41%. Notably, despite having high hygroscopicity, the mucilage displayed the lowest residual MC compared to other samples (Table 1), whereas the opposite trend was observed for insoluble fibre. These contrasting findings suggest that, in addition to MC, powder product stability requires the assessment of hygroscopicity. The high hygroscopicity of the mucilage powder indicates an abundance of amorphous sugars, rendering it highly reactive with water molecules in its surrounding environment (Mikhailov et al., 2009). Previous studies have shown that a high hygroscopicity (>70% relative humidity) of this magnitude leads to biodeterioration, significant ochratoxin A accumulation in poultry compound feed, and nutrient loss in stored corn grain (Rao et al., 2016; Coradi et al., 2025). The high hygroscopicity of mucilage may limit its direct use in dry feed premixes, making encapsulation necessary for stabilisation. Nevertheless, the low hygroscopicity of insoluble fibre and peel powders facilitates stable storage and potential resistance to microbial growth. Consequently, under controlled conditions, these materials may offer a viable solution for stocking against future feedstock shortages.

Chlorophyll and β-carotene content

According to Figures 2A, B, the peel powder contained the highest concentrations of chlorophyll a and b (chl a and chl b) than the mucilage and insoluble fibre (>5–20 times higher), indicating that these photosynthetic pigments are highly embedded in the peel. Total chlorophyll (TChl) content exhibited the same pattern of results observed for chl a and chl b (Figure 2C). These findings are supported by the colour images and the higher greenness values (Table 1). Conversely, the mucilage displayed the lowest chl a and chl b concentrations, consistent with its lower greenness value (Table 1). The peel powder analyzed in this study showed higher chlorophyll content than prickly pear pulp and whole fruit reported in previous literature (Cano et al., 2017). This discrepancy is likely due to the processing technique employed in this study, which concentrated chlorophyll pigments in the peel, making it a potential valuable by-product for animal feed. Given that chlorophyll pigments are linked to antioxidant benefits in livestock nutrition (Gadzama et al., 2024; Martins et al., 2022; Sadok et al., 2009), the higher chl a and chl b contents identified in this study indicate that the peel powder may be an acceptable feed ingredient worthy of future exploration.

Among plant pigments, β-carotene, a carotenoid, is an essential precursor of vitamin A associated with positive implications for animal nutrition. As illustrated in Figure 2D, β-carotene concentration was significantly higher in the peel powder than in the insoluble fibre and mucilage powders. In this study, the β-carotene content in the peel surpassed the 0.0005 mg/g reported for other O. ficus-indica cladodes (Heba et al., 2020). Additionally, these levels exceeded those found in conventional feedstuffs like whole soybean (0.11–0.15 mg/g) and soybean hull meals (0.04–0.13 mg/g) (Pickworth et al., 2012). However, the nutritional benefits of β-carotene must be validated at practical inclusion levels following feed processing and storage. Overall, these results suggest that the peel powder may be a source of substantial plant pigments that can enhance both health and colour when tested in feed formulations.

Vitamin C, total phenolics, total flavonoids, total tannins and total alkaloids contents

Assessing total phenolic content (TPC), total flavonoid content (TFC), total tannin content (TTC), and total alkaloid content (TAC) was crucial, as these compounds are also known for a variety of health-promoting benefits in livestock feed. Cladode fractionation did not reveal significant variations in vitamin C content, which remained uniform at approximately 12 mg AAE/g DW across the different components studied (Figure 3A). This suggests that, as a water-soluble compound, vitamin C is evenly distributed across the cladode components rather than bound to structural tissues. The present vitamin C content results are superior to the 0.029 mg/g reported for O. stricta cladode powder by Izuegbuna et al. (2019). The higher vitamin C content could be attributed to the freeze-drying method employed, which is known to preserve and concentrate heat-labile compounds while evaporating water, unlike the oven-drying technique that degrades heat-sensitive compounds, as reported in the literature (Okoli et al., 2025; Alibas and Ipek, 2026). While citrus by-products are commonly used as sources of vitamin C for livestock such as poultry, they contain significantly less of this nutrient compared to the studied O. ficus-indica cladode fractions (Figure 3A; Khitma et al., 2018). Therefore, the powders from peel, mucilage and insoluble fibres of O. ficus-indica cladode could be evaluated as supplementary sources of vitamin C, especially for heat-stressed chickens requiring antioxidants.

While differences were not statistically significant in vitamin C content, the mucilage and peel powders exhibited higher TPC (2.00-2.14 mg GAE/g DW) compared to the insoluble fibre (1.07 mg GAE/g DW), effectively doubling the concentration (Figure 3B). A similar trend was observed for TFC, which varied between 0.02 and 0.06 mg CE/g DW (Figure 3C), suggesting a distinct spatial localisation of these compounds within the cladode. The significant disparity in TPC and TFC between the mucilage powder and insoluble fibre suggests that these compounds are largely loosely bound, leading to their migration into the mucilage during centrifugation. Compared to literature, the TPC of the peel and mucilage powders was higher than that of freeze-dried commercial cladodes and corn husks used for animal feed, but lower than that of wild O. ficus-indica cladodes (Martínez-Soto et al., 2015; Cai et al., 2024). Meanwhile, the TFC was substantially lower than most reported values; for instance, dehydrated cladodes in literature showed a TFC range of 0.19-0.35 mg QE/g DW (Martínez et al., 2020). Overall, peel and mucilage are superior sources of polyphenols compared to insoluble fibre, providing bioactive compounds that may potentially enhance antioxidative status, combat oxidative stress, and improve livestock product quality (Serra et al., 2021). Moreover, as livestock feed research shifts toward using natural bioactive sources, the peel and mucilage represent viable alternatives to synthetic antioxidants.

As illustrated in Figure 3D, the TTC of the investigated cladode powders ranged from 0.4 to 0.9 mg TAE/g DW. The mucilage powder contained significantly higher tannin levels compared to the insoluble fibre powder but showed similar levels to the peel powder. These results suggest that the tannins are freely bound and also serve a defensive function against environmental stressors; hence, their higher concentrations in the mucilage and peel. The tannin levels observed in this study were lower than the maximum value (6.45 mg GAE/g DW) reported for Nigerian O. ficus-indica cladodes (Dib et al., 2013). According to Ahmed et al. (2025), these tannin levels are below the dietary threshold of approximately 30 mg TE/g DW recommended for ruminants. Depending on the tannin type, dietary inclusion level and targeted livestock, low tannin concentrations may contribute beneficial antioxidant, antimicrobial, and anti-parasitic effects, rather than as limiting anti-nutritional factors (Ahmed et al., 2025). Furthermore, the reduced presence of tannins may indicate higher bioavailability of key nutrients, such as crude protein, carbohydrates, and dietary fibres, thereby enhancing the overall nutritional value of the cladode fractions (da Silva Aguiar et al., 2023). As with tannins, low alkaloid levels are also crucial in plant materials used as animal feed to avoid antinutritional effects. According to Figure 3E, O. ficus-indica cladode fractionation reveals component-specific alkaloid distribution, with the highest concentration (1.7 g/100 g) in the peel powder and the lowest (0.7 g/100 g) in the mucilage powder, supporting the findings of Ginestra et al. (2009), despite their use of different extraction solvents. These low levels, particularly in the mucilage, are considered tolerable for animal nutrition. The low levels of alkaloids and tannins in this study suggest that cladode fractionation and freeze-drying may have effectively reduced these antinutritional components by partitioning them among fractions based on their chemical properties. Due to the limited research on the alkaloid and tannin content of O. ficus-indica cladodes, these findings provide critical insights into the anti-nutritional status of various cladode fractions.

Antioxidant activity

As shown in Figures 4A, B, all powder samples demonstrated dose-dependent DPPH and ABTS radical scavenging activities. Compared with selected plant-derived feed materials reported in the literature, the peel powder in this study demonstrated superior antioxidant potential, achieving 90.6% DPPH inhibition at a concentration of 0.02 mg/mL, compared with 95.51% inhibition at 0.2 mg/mL reported for alfalfa extracts (Chen et al., 2020). The superior radical scavenging activities of the peel powder are supported by IC 50 values, which were approximately 4 and 3 times lower than those of the mucilage and insoluble fibre powders, respectively. Further supporting these results, the FRAP assay confirmed superior ferric reducing power in the peel compared to the other powders (Supplementary Figure S5). This enhanced activity in the peel correlates with its higher total phenolic, flavonoid, alkaloid, and tannin contents (Figures 3B–D). These bioactive compounds possess multiple phenolic hydroxyl groups, enabling them to donate hydrogen atoms or electrons to neutralise free radicals. While the peel powder exhibited an IC 50 value higher than ascorbic acid (positive control), it demonstrated an IC 50 value lower than that of Trolox (positive control) (along with the mucilage), indicating strong antioxidant potency (Figures 4A, B). Plant materials with strong antioxidant activity have been reported to elevate feed efficiency and strengthen immunity against oxidative stress in livestock (Nefzi et al., 2026). As such, based on these results, future work should investigate the antioxidant potency of the peel powder in feed formulations and in vivo trials. In addition, it is essential to profile the secondary metabolites within the tested O. ficus-indica cladode powders to identify key biomarkers, understand their mechanisms of action, and establish methods to maximise their concentrations.

ATR-FTIR spectra

FTIR analysis was used to identify functional groups in the tested samples, providing insights into the chemical classes present and their influence on the observed biological properties. As shown in Figure 5, the O-H stretching band appeared in all freeze-dried powders, with higher intensities in the peel, supporting a higher concentration of phenolic compounds. This observation coincides with the results reported by Mohlamonyane et al. (2025), which demonstrated that the peel contains more flavonoid compounds than the insoluble fibre. Furthermore, the higher O-H functional group intensity in the peel likely supports the efficient antioxidant activity observed in Figure 5. The C-H stretching bands were detected exclusively in the peel and mucilage powders, with the peel exhibiting the highest intensity (78.29%), suggesting a significant presence of lipids. These lipids likely contributed to the previously observed hydrophobic nature of the peel (Figure 1A). These results align with previous spectroscopic studies, which strongly associate higher intensities at 2924 cm -1 with a significant presence of lipids, such as triacylglycerols (Guillén and Cabo, 2002; Zielińska et al., 2022). Regarding the C=C stretching frequency, all extracts showed peaks, with transmittance values of 64.08% (peel), 87.69% (mucilage), and 96.14% (insoluble fibre) (Supplementary Figures S2–S4). These C=C bonds indicate the potential presence of alkenes, such as y-terpinene, carotenes, ethylene, and limonene.

The peaks at 1239.3 cm - ¹ (peel) and 1246 cm - ¹ (mucilage) are likely due to C-N stretching vibrations, suggesting the presence of proteinaceous components within these samples. Supporting these results, the insoluble fibre exhibited weaker C-N stretches, indicating lower protein concentration, as this component consists primarily of structural polysaccharides. Furthermore, all extracts displayed C-O stretching bonds at 1028.7 cm - ¹ (peel) and 1026.9 cm - ¹ (mucilage and insoluble fibre), with transmittance values of 35.82%, 74.44%, and 89.34%, respectively. These frequencies indicate the presence of carbonyl groups, with the highest concentration observed in the peel. These results are further corroborated by Bano and Irfan (2019); Ishaya et al. (2024), and Yahaya (2024), who attributed this band to the presence of glycosidic bonds (C–O–C) and C–O stretching vibration of cellulose molecules. Overall, the functional group analysis indicates that the peel contains the highest concentration of these compounds, consistent with the measured physicochemical properties.

Principal component analysis

Principal component analysis (PCA) was performed to understand how the fractionation of the O. ficus-indica cladode into mucilage, peel, and insoluble fibre affected the quality and functionality of the developed powders. The analysis revealed a clear effect of the cladode fractionation on the measured physicochemical variables (Figure 5). Together, PC1(60.9%) and PC2(29.6%), explained 90.5% of the accumulative variance, as stipulated in Figure 6. The values of variable contributions and loadings constituting PC1 and PC2 are detailed in the Supplementary File (Supplementary Table S1) to support these observations. The distinct separation of the powder samples in the biplot supports the variations observed in the investigated parameters. Notably, PC1 was dominated by powder yield, colour parameters (L, a, b, C), chlorophyll pigments, β-carotene, and antioxidant capacity. These variables clustered near the peel powder, indicating that the peel fraction performed better for these quality attributes than the other samples. The negative correlation between chlorophyll pigments (chl a, chl b, and TChl) and the colour attribute a* supports the findings shown in Table 1, reinforcing that higher chlorophyll concentrations result in more negative a* values, and lower values of L, b, and C*. In addition, a higher concentration of chlorophyll pigments indicates a more intense green colour in the tested product. The positioning of bioactive compounds such as β-carotene and antioxidant variables (DPPH IC 50, ABTS IC 50 and FRAP) near the peel powder can be used to support the results presented in Figure 2D and Figures 4D respectively. These strong relationships of the peel fraction suggest the β-carotene as an additional bioactive compound that might have contributed to the observed anti-radical and reducing ability of the peel powder compared to mucilage and insoluble fibre.

For PC2, the major contributors, including SC, WHC, hygroscopicity, WS, MC, h° and vitamin C demonstrated strong correlation with the mucilage and insoluble fibre. The bioactive compounds (TPC, TFC and TTC) showed an intermediatory positioning between the mucilage and the peel fraction, which was negatively correlated to the insoluble fibre. The strong correlation of SC and MC, and their positioning closer to the insoluble fibre sample, supports the earlier observations (Table 1; Figure 1C) where the insoluble fibre fraction expressed higher contents of both variables. Conversely, SC and MC showed a negative association with antioxidant and bioactive compounds, aligning with the lower concentrations of phenolics, flavonoids, and tannins and lower DPPH and ABTS radical scavenging activities observed in the insoluble fibre. Meanwhile, within PC2, hygroscopicity and WS showed a strong association with the mucilage powder, with a positive correlation confirming the powder’s hydrophilic nature. Conversely, hygroscopicity displayed a direct negative relationship with WHC; as shown in Figures 1B, D, the mucilage exhibited a high hygroscopic nature and a lower overall water adsorption capacity. In addition, it is worth noting that, although vitamin C was observed evenly distributed across the samples (Figure 3A), the PCA revealed its strongest association with the mucilage and insoluble fibre than the with peel.

In summary, PCA confirmed functional differentiation among the fractions as the peel was associated mainly with pigments, bioactive and antioxidant indices, whereas mucilage and insoluble fibre were associated mainly with hydration-related properties relevant to feed texture and gut functionality.

This study provides valuable insights into the biological variations among O. ficus-indica cladode components and the need to further study their potential implications for livestock feed applications. The results validate the hypothesis that freeze-dried peel, mucilage, and insoluble fibre have distinct, component-specific properties, suggesting that each can be investigated for specialised needs in livestock feed. Specifically, the peel shows high concentrations of pigments and bioactive compounds, including chlorophyll, β-carotene, flavonoids, and phenolics, which could be responsible for the greater antioxidant activity observed in the peel, indicating potential for future evaluation as a feed supplement with substantial antioxidant properties. Concurrently, mucilage and insoluble fibre powders exhibited excellent hydration properties, which may be further evaluated for feed texture modification and gut functionality in livestock. Processing cladodes into these separate components introduces a potentially scalable processing technique for various feed industries and livestock farmers, whose adoption can be further evaluated by techno-economic and sustainability assessments. Cladode fractionation may also enhance the feed value of the cladodes by creating tailored cladode fractions that may be investigated for feed specific purposes. However, addressing some of the observed potential challenges, such as the high hygroscopicity of the mucilage powder, also requires further studies, including encapsulation, to enhance its stability during storage. Further research is also required to profile nutritional components, including proximate composition, minerals, and heavy metals, for safety evaluation. Given the peel’s superior bioactive profile and high radical scavenging activity, investigations into its antimicrobial, anti-inflammatory, and parasiticidal properties are recommended. Finally, assessing the influence of O. ficus-indica cultivars and cladode maturity stages is essential to identify the optimal cultivar and harvest time for desired properties.