Papaya Seed as a Potential Protein Source: Proposed Extraction Methods and Future Recommendations

3.1. Nutritional and Compositional profile of Papaya Seeds

Papaya pulp is commonly used in processed foods like jams, juices, and jellies because of its high nutritional content. However, the seeds, which make up 20% of the fruit's weight, are frequently discarded as waste, even though they are a valuable source of bioactive compounds [10]. Papaya seeds have also been traditionally used as a food ingredient in some African countries. In Hausa (Nigeria), the seeds are used to prepare daddawa, a fermented food condiment. Different parts of C. papaya, including the fruit, seeds, leaves, bark, and latex, have also been traditionally used for various medicinal purposes [11]. These traditional uses indicate that papaya seeds have applications beyond their treatment as discarded fruit-processing residue and add to the interest in their nutritional and bioactive composition. Papaya seeds have been reported to contain compounds that help treat intestinal worms, ulcers, sickle cell disorder, and poisoning [12]. The proximate composition of papaya seeds varies with ripening stage and processing history (Table 1). Ripe and unripe papaya seeds showed relatively similar protein contents of 26.9% and 26.1%, respectively. However, differences were observed in other components, with ripe seeds showing higher fiber (33.5%) and fat (28.6%), while unripe seeds contained higher carbohydrate (17.8%) and slightly higher ash (8.4%) contents. The relatively similar protein contents between the two ripening stages suggest that protein concentration may be less affected by maturity than some other seed components, although the available data are insufficient to establish a general trend. Greater variation is observed when results from different studies on non-defatted papaya seeds are considered. Reported protein contents range from 24.33 to 27.80%, while carbohydrate, fiber, and fat contents range from 11.67–48.91%, 8.02–22.60%, and 0.65–28.61%, respectively. This variability indicates that proximate composition is not uniform across papaya seed samples and may be influenced by factors such as cultivar, maturity, cultivation conditions, analytical procedures, and processing history. The particularly wide variation in carbohydrate and fat contents is important when comparing reported protein values because changes in the proportions of major components can substantially affect the relative composition of the seed material.

Defatting further demonstrates the effect of processing on the apparent protein concentration. The protein content increased to 44.4% after defatting compared with 24.33–27.80% reported for non-defatted seeds (Table 1). This increase should be interpreted as relative protein enrichment resulting from lipid removal, rather than an increase in the absolute amount of protein. Defatting may nevertheless be an important pretreatment for protein extraction because it reduces the lipid fraction and increases the protein content of the starting material. However, the defatted material still contained a relatively high fiber content (31.8%), indicating that protein remains associated with a substantial non-protein matrix. This matrix may influence protein solubilization, separation, and the functional properties of the recovered protein. Therefore, the composition of the starting material and the use of pretreatments, such as defatting, should be considered when comparing protein extraction studies of papaya seeds [5, 12-15]. Overall, the available compositional data indicate that papaya seeds contain a substantial protein fraction, but their composition varies considerably among samples and processing conditions. This variability is important for protein valorization because the initial lipid, fibre, carbohydrate, and protein contents can influence both the efficiency of extraction and the composition of the final protein product.

3.2. Protein Composition and Amino Acid Profile of Papaya Seed

Papaya seed comprises different protein fractions, with globulins representing the major fraction (53.90%). Marfo et al. [16] also reported differences in protein solubility according to extraction medium, with the highest solubility of seed protein observed in 5% NaCl (23.77%) and a solubility peak around pH 8. Electrophoretic analysis further indicated heterogeneity among the soluble protein fractions, with different molecular-weight bands detected in the water-, NaCl-, and NaOH-soluble fractions. These findings indicate that papaya seeds contain heterogeneous protein fractions with different solubility characteristics, which may influence their behavior during subsequent protein extraction. In a recent study by Thanasegaran et al. [10], the characterization of papaya seeds was conducted. In Fourier-Transform Infrared Spectroscopy (FTIR), prominent protein bands, including amide I and II vibrations typical of seed storage proteins, were observed. Several bands in the fingerprint region were also noted, corresponding to C–O and C–O–C stretching vibrations of polysaccharides, particularly cellulose and pectin, as well as C–H bending of aliphatic chains. Pretreatment processes such as defatting can be used to decrease fat content and increase protein extraction efficiency. Traditionally, conventional solvents such as hexane are used for defatting. However, hexane is derived from non-renewable sources and has a strong environmental impact and poses high risks to operators' safety. It is highly flammable, and when released into the atmosphere, it can react with other pollutants to form ozone and other photochemical oxidants. Hexane is also recognized as neurotoxic due to its solubility in neural lipids. Therefore, alternative solvents are being explored for the defatting process. For instance, ethanol, along with CO₂ and bio-based ethyl acetate and acetone, has shown compatibility with lipid extraction and safe use for food components, ingredients, and raw biomass processing [17].

The amino acid profile of papaya seeds showed considerable variation between the reported studies. Glutamic acid (4.76–12.40 g/100 g protein), leucine (3.84–7.78 g/100 g protein), and arginine (2.90–6.44 g/100 g protein) were among the amino acids reported at relatively higher concentrations. Essential amino acids cannot be synthesized in sufficient quantities by the human body and therefore must be obtained through the diet. Among them, leucine plays important roles in muscle protein synthesis and tissue repair, while isoleucine contributes to energy metabolism and hemoglobin synthesis. Threonine is involved in protein biosynthesis. Among the essential amino acids, isoleucine, lysine, and threonine were present in good amounts, as shown in Table 2. Methionine was consistently among the least abundant essential amino acids (1.30–1.56 g/100 g protein) and is important for normal cellular functions. Glutamate and serine were also present in substantial amounts; glutamate plays roles in nervous system functions, while serine participates in protein biosynthesis and brain development. Cysteine was reported at a relatively low concentration (1.07 g/100 g protein) and contributes to glutathione metabolism. The presence of these essential amino acids in papaya seeds therefore contributes to their nutritional value as a potential plant protein source [12, 18-20]. Although papaya seeds provide essential amino acids, the relatively low methionine content suggests that sulfur-containing amino acids may represent a nutritional limitation. Therefore, amino acid composition alone should not be used to measure protein quality; comparison with reference amino acid patterns, together with digestibility measurements, is required.

3.3. Anti-nutritional Factors and Processing Considerations

Papaya seeds contain several anti-nutritional compounds that should be considered when evaluating their use as a food or protein ingredient. Reported compounds include phytic acid, tannins, oxalates, and trypsin inhibitors. In papaya seed meal, phytic acid, tannins, and oxalate were reported at approximately 23.3, 10.6, and 1.89 mg/100 g dry weight, respectively, while trypsin inhibitor activity was reported as 1.77 TIU/mg protein [18]. These compounds may affect nutritional quality by reducing mineral bioavailability or interfering with protein digestion [20]. However, the reported levels are relatively low, and their nutritional significance may depend on cultivar, processing conditions, and the amount consumed. The in vitro protein digestibility of papaya seed flour was reported to be 80.69%, indicating good protein digestibility. The relatively low levels of antinutritional factors, particularly trypsin inhibitors and tannins, may contribute to the relatively high digestibility. This suggests that papaya seed protein has potential nutritional value, although further studies are needed to confirm its overall protein quality [20].

Processing can therefore be important for reducing these compounds before or during protein recovery. In other plant materials, conventional approaches such as soaking, cooking, fermentation, germination, and other treatments have been investigated for reducing antinutritional factors in plant materials. Conventional treatments such as soaking, boiling, and germination can reduce antinutritional factors and improve protein digestibility, although they may require additional processing time. Emerging non-thermal technologies, including ultrasound and other physical treatments, have also been investigated for modifying plant-protein structure and functionality, with potential effects on digestibility and nutritional quality [21]. Fermentation substantially reduced the antinutritional factors in papaya seeds. Oxalate decreased from 210.1 to 40.2 mg/100 g, phytic acid from 102.0 to 68.0 mg/100 g, tannin from 15.5 to 8.3 mg/100 g, and trypsin inhibitor from 2431.2 to 63.0 mg/100 g. The significant reduction in trypsin inhibitor indicates that fermentation may be an effective processing approach to improve the nutritional quality of papaya seeds, although its effectiveness may vary with fermentation conditions [22]. However, direct evidence demonstrating the removal of these antinutritional factors during papaya seed protein extraction remains limited.

4.1. Conventional Method of Protein Extraction

Protein extraction from papaya seeds has been investigated mainly through alkaline solubilization followed by acid precipitation. The reported studies show that extraction conditions affect both protein recovery and the composition of the final protein product. Wang et al. [23] investigated alkaline extraction from defatted papaya seed flour and optimized the solid-to-liquid ratio, pH, temperature, and extraction time. The highest reported extraction yield was 48.53%, obtained at a solid-to-liquid ratio of 1:40, pH 9.3, 50°C, and 120 min. The extracted protein also showed increased emulsifying and foaming properties as protein concentration increased, indicating its potential for food applications. In comparison, another study used defatting followed by alkaline solubilization and acid precipitation to obtain a protein isolate. The isolate contained 88.08% protein content, compared to 32.40% in the defatted flour and 25.18% in the full-fat flour [24, 25]. The 48.53% reported by Wang et al. [23] represents the extraction rate, whereas the 88.08% reported by Kadiri et al. [24] represents the protein content of the final isolate. Extraction yield generally refers to the amount of total extract obtained relative to the initial raw material. Protein recovery represents the proportion of protein initially present in the raw material that is recovered following extraction. In contrast, protein content refers to the proportion of protein in the final extracted fraction and therefore primarily reflects the degree of protein enrichment or concentration of the extract. Consequently, these parameters should not be used interchangeably when comparing protein extraction studies [23-25].

The results of Kadiri et al. [24] also demonstrate the importance of pH during protein recovery. Protein concentration in the isolate was lower at acidic pH and increased to 88.08% at pH 10. The lower protein concentration around pH 4 is consistent with reduced protein solubility near the isoelectric region, whereas the higher concentration under alkaline conditions was associated with increased electrostatic repulsion and protein unfolding. However, higher pH does not necessarily result in better protein quality. The experiment noted that extreme alkaline conditions may promote protein denaturation and negatively affect the functional properties of the isolate. Thus, alkaline extraction requires a balance between improving protein solubilization and maintaining the structural and functional quality of the recovered protein. Overall, the two studies show that alkaline extraction can effectively recover and concentrate papaya seed proteins, but the optimum conditions differ considerably. Wang et al. [23] achieved the reported extraction rate within 120 min at pH 9.3, whereas Kadiri et al. [24] used pH 10 with a 4h extraction period to produce a highly concentrated protein isolate. This difference indicates that optimization depends not only on maximizing protein solubilization but also on the desired characteristics of the final protein product. Conventional protein extraction commonly relies on aqueous acidic or alkaline media. Although these approaches can facilitate protein solubilization, harsh alkaline conditions may promote protein denaturation and structural alterations, whereas acidic conditions may be less effective at disrupting the plant matrix [26].

4.2. Emerging Extraction Methods for Protein Extraction

The limitations of conventional alkaline extraction have prompted the investigation of alternative approaches to improve protein recovery under milder or less chemically intensive conditions (Fig 2). These approaches include enzyme-assisted extraction, physical-assisted technologies such as Pulsed Electric Field (PEF), High-Voltage Electrical Discharge (HVED), and ultrasound, as well as solvent-based approaches such as deep eutectic solvents (DESs). However, the environmental and processing advantages of these methods depend on their operating conditions, energy requirements, and downstream processing needs. As research to date shows, papaya seeds are a rich source of phytochemicals, including phenolic compounds; however, these compounds may interfere with protein extraction and affect the quality of the recovered protein fraction. Phenolic compounds can interact with proteins and amino acids, potentially reducing the availability of certain amino acids such as methionine and lysine. Under alkaline extraction conditions, phenolic compounds may undergo oxidation to reactive quinones, which can interact with protein functional groups, including amino (-NH₂) and thiol (-SH) groups, through covalent and non-covalent interactions. Such interactions may promote protein–phenolic complex formation and influence protein solubility, recovery, and functionality [27]. Therefore, appropriate pretreatment or extraction strategies may be required to minimize protein–phenolic interactions and improve the efficiency and quality of papaya seed protein extraction.

The potential of aqueous enzymatic extraction was demonstrated by Wang et al. [28], who used papain to extract papaya seed protein. Response surface methodology identified a solvent-to-material ratio of 1:19, a temperature of 49°C, a hydrolysis time of 5 h, an enzyme concentration of 2%, and a pH of 7.0 as the optimum conditions, with a reported extraction rate of 84.96%. This value was higher than the 48.53% extraction rate reported by Wang et al. [23] using alkaline extraction. However, the difference cannot be attributed solely to the use of papain because the two studies differed in extraction mechanism, solid-to-liquid ratio, pH, temperature, and processing time. In addition, the enzymatic process required 5h compared with 120 min for the alkaline process and required an additional enzyme input. Therefore, the results indicate the potential of enzymatic extraction to improve protein recovery. The longer processing time and enzyme requirement should also be considered when evaluating its practical application. Enzyme-assisted extraction also requires controlled pH and temperature conditions suitable for the selected enzyme. Carbohydrases and proteases may therefore require different processing conditions depending on their optimum activity. Although enzymatic treatment can facilitate protein release by disrupting interactions between proteins and other components of the plant matrix, it may require relatively long processing times and can increase energy and operational costs. Enzymatic treatment may also alter protein–carbohydrate interactions, which can influence the composition and properties of the recovered protein fraction [26].

PEF and HVED provide a different approach by promoting physical disruption of the seed matrix. Parniakov et al. [6] compared conventional aqueous extraction with PEF and HVED treatment of papaya seeds. PEF and HVED were applied at approximately 13.3 and 40 kV/cm, respectively, and HVED produced higher protein concentrations in the extract than conventional aqueous extraction and PEF. The experiment proposed that shock waves and cavitation generated during electrical discharge could disrupt the seed matrix and facilitate the release of intracellular compounds. They further suggested that HVED could affect interactions between phenolic compounds and other components of the seed matrix, potentially facilitating the release of proteins and phenolic compounds. Ultrasound-assisted extraction represents another potential physical approach. Acoustic cavitation generated during ultrasonication can produce localized shear forces that disrupt plant tissues and protein aggregates, potentially facilitating the release of intracellular proteins. Ultrasound can also modify protein conformation and intermolecular interactions, thereby affecting solubility, digestibility, and techno-functional properties. In enzyme-assisted processes, ultrasound may additionally improve enzyme accessibility to protein substrates and facilitate hydrolysis. However, these effects are strongly dependent on frequency, intensity, treatment time, temperature, and the characteristics of the protein material. Excessive treatment can produce undesirable structural changes; therefore, ultrasound conditions should be optimized rather than assuming that higher treatment intensity will yield better results. Studies in other plant-protein systems have also investigated the use of ultrasound to reduce antinutritional factors and modify protein allergenicity [21]. However, these effects are dependent on the protein source and processing conditions and cannot currently be assumed for papaya seed protein. Therefore, ultrasound should be considered a potential technology for papaya seed protein extraction rather than an established method for this raw material.

Evidence from other seed proteins further demonstrates the potential of emerging extraction technologies. Anyiam et al. [29] compared Enzyme-Assisted (EAE), Ultrasound-Assisted (UAE), and Microwave-Assisted Extraction (MAE) with conventional alkaline extraction for Moringa oleifera seeds. The emerging methods improved protein yield, protein content, solubility, and techno-functional properties compared with conventional extraction. Protein yield ranged from 14.60–30.08%, while protein content ranged from 80.47–86.61%. Phytate and trypsin inhibitor levels were also reduced, while in vitro protein digestibility increased to 88.11–93.81%. Although UAE and MAE provided high protein yield and purity, EAE provided the best balance of functionality and digestibility. These findings demonstrate that the choice of extraction technology can influence not only protein recovery but also the nutritional and functional quality of the recovered protein.

DESs possess strong solubilizing properties for various non-volatile compounds and have negligible volatility, which can reduce solvent loss during extraction. Protein extraction using DES can be performed through both liquid–liquid and solid–liquid approaches, with solid–liquid extraction commonly involving direct contact between the plant material and the DES. Compared with conventional aqueous or organic solvents, DESs provide a different hydrogen-bonding environment that may influence protein–matrix interactions and protein solubilization. Conventional extraction under strongly acidic or alkaline conditions can promote protein unfolding, expose hydrophobic or hydrophilic groups, and potentially alter protein structure. In contrast, the hydrogen-bond network formed between the hydrogen-bond acceptor and hydrogen-bond donor components of DES may facilitate interactions with protein molecules without necessarily causing chemical modification of the protein structure. However, the extent to which DES preserves protein structure depends on the composition of the DES and extraction conditions and should therefore be experimentally evaluated rather than assumed [26].

DES-assisted extraction has also been investigated in other seed proteins. Wang et al. [30] reported that DES extraction of Moringa oleifera seed protein affected protein recovery, foaming properties, and secondary structure, with relatively less alteration of protein secondary structure under the investigated conditions. Similarly, a comparative study of kenaf seed protein reported that DES-assisted extraction produced proteins with a more ordered structure than conventional alkaline extraction. The DES-extracted proteins also showed a more balanced distribution of hydrophilic and hydrophobic amino acids. Both the choline chloride: urea (ChCl: U)-based DES and conventional alkaline extraction produced proteins with good functional properties, while ChCl: U was identified as a promising alternative for kenaf seed protein extraction [31]. These findings suggest that DES may influence protein structure and functionality differently from conventional alkaline extraction, although its performance depends on the specific DES composition and extraction conditions. DES-assisted extraction also presents challenges, including the relatively high viscosity of many DES systems, which can limit mass transfer, as well as limited selectivity and difficulties in separating the recovered protein from the DES phase. These factors are particularly important when the recovered protein is intended for food applications.

Overall, emerging extraction technologies offer distinct advantages over conventional alkaline extraction. Enzyme-assisted extraction has demonstrated high reported extraction rates but requires longer processing and enzyme addition; HVED can enhance protein release through physical disruption but may yield extracts that require additional purification; ultrasound has the potential to improve protein accessibility and modify protein functionality, although papaya-specific evidence remains limited; and DESs offer extraction medium but require careful consideration of viscosity, selectivity, and downstream protein recovery. Importantly, higher extraction recovery alone should not be used to determine the most suitable technology. Protein purity, structural integrity, digestibility, techno-functional properties, energy requirements, processing time, and downstream purification should also be considered when evaluating these approaches.