PREPARATION AND CHARACTERIZATION OF FISH PROTEIN HYDROLYSATES:
PREPARATION OF FPHs:
According to Halim and Sarbon (2016), the yield of fish protein hydrolysates (FPH) prepared accounts for around 8.96 percent of the total weight of the fish used. The inclusion of papain and proteinase k during the enzymatic hydrolysis process catalyzed the breakdown of the Fish's complex amino acid chains into multiple units of smaller and shorter amino acid chains, with the number of peptide bonds varying depending on the enzyme concentration, hydrolysis time, mixture pH, and temperature used for hydrolysis (Srichanun et al., 2014; Jamil et al., 2016). During the entire hydrolysis process, the yield of hydrolysate formed is dependent on the number of broken peptide bonds, which is determined by the number of peptide bonds in protein mass (degree of hydrolysis DH) (Hamid et al., 2015; Halim and Sarbon, 2016). This means that a higher DH contributes to a higher yield of protein hydrolysate.
PROXIMATE COMPOSITION:
The proximate composition of hydrolysates prepared with papain (FPH1) and proteinase k (FPH2) enzymes is shown in Table 1. The hydrolysate made from papain has a high protein content, which is beneficial for hydrolysis because it releases amino acids that improve the functional properties of the fish protein hydrolysate, allowing it to be used as an important source of protein (Baharuddin et al., 2015). When comparing papain hydrolysate to proteinase k hydrolysate, papain hydrolysate produces more ash. Both enzymes generated hydrolysates with significantly low fat and moisture content, suggesting that these hydrolysates have the potential to be used in the food industry. The lipid content of both FPHs has dropped. Fish protein hydrolysates obtained by enzymatic hydrolysis were found to have a lower lipid content (Jemil et al., 2014, Taheri et al., 2012). The membranes of muscle cells begin to round up and form insoluble vesicles during the hydrolysis process, resulting in the removal of membrane structured lipids (Kristinsson and Rasco, 2000).
FUNCTIONAL PROPERTIES:
SOLUBILITY:
Figure 2 depicted the solubility of fish protein hydrolysates made with papain and proteinase K at various pH levels. In comparison to the solubility of the selected sample (unprocessed muscle protein), a notable improvement in the solubility of the FPHs can be seen in the graph. For pH 2, the sample demonstrated solubility of 6.2%, whereas the hydrolysate from FPH1 (papain) reached 75.7%, whereas FPH2 (proteinase K) showed solubility of 73.4%. At pH 10 the solubility of the hydrolysate produced from papain has a solubility of 93.23%. The protein hydrolysate produced by proteinase K has 89.98%. The charge on the weak acidic and basic side chain groups is affected by pH, so the hydrolysates have low solubility at their isoelectric points. (Nazeer et al., 2013). The protein molecules are broken down into smaller peptide units during the hydrolysis process, which explains the FPHs' high solubility at pH 10. (Yin et al., 2010.). The degree of hydrolysis (DH) of the protein hydrolysate is related to its solubility. The peptide bonds are broken during the hydrolysis process, exposing the protein's hydrophilic site (Jamil et al., 2016.). Hydrolysates with exposed hydrophilic sites have a higher solubility since they can form hydrogen bonds with water (Milewski, 2001).
FPH extracted with papain has a higher solubility at pH 10 than hydrolysate from pink perch muscle (Nemipterus japonicas) (Naqash and Nazeer, 2013). As a result, both of these FPHs have a lot of potential as a key component in human and animal food production.
FOAMING PROPERTIES:
Figures 3a and 3b show the foaming capacity and stability of protein hydrolysates made from papain and proteinase K compared to untreated muscle protein (sample). Both hydrolysates have a higher foaming capacity than the sample. The protein hydrolysate made from papain has a slightly higher foaming capacity than the protein hydrolysate made from proteinase K. Foaming capacity was found to be higher at pH 2 and decreases as pH increases. An effective foaming agent demonstrates a high ability to rapidly migrate protein to the air-water interface. The ionic repulsions of peptides at the air-water interface may be responsible for the reduced foaming capacity in high alkaline environments (Klompong et al., 2007). The transportation, penetration, and rearrangement of molecules at the air-water interface are all linked to the foaming properties of protein hydrolysates (Elavarasan et al., 2014). When protein is dispersed in low water tension at the air-water interface, foam is formed (Tanuja et al., 2012). Furthermore, the low molecular weight (MW) of the hydrolysate affects foaming properties, as low MW hydrolysates are unable to sustain the well-ordered, interface orientation of the molecule (Nalinanon et al., 2011). Figure 3b shows that FPH's foaming stability was lower than its foaming capability at all pH levels. The results of this study are close to those of Naqash and Nazeer (2013), who noticed that the foaming properties of pink perch muscle hydrolysate decreased at pH 4 and increased as the pH increased. The results, on the other hand, were higher than Chi et al (2014) recorded foaming ability of Spanish mackerel hydrolysate as 65%.
As shown in fig.3b, both hydrolysates have good foaming stability over a pH range. FPH1 (papain) foams up more consistently than FPH2 (Proteinase kinase). Protein molecules should form continuous intermolecular polymers that envelope the air bubbles because intermolecular cohesiveness and elasticity are needed for producing stable foams. The low surfactant activity of short peptide chains may explain the decrease in foam stability in hydrolysates (Mutilangi et al., 1996). The results of this study are close to those of Naqash and Nazeer (2013), who found that the foaming properties of pink perch muscle hydrolysate decreased at pH 4 and increased as the pH increased.
WATER BINDING CAPACITY:
The water holding capacity of Fish protein hydrolysates FPH 1(papain) and FPH 2(proteinase k) significantly decreased (p<0.05) with increasing hydrolysate concentration from 0.2% to 1.0% (figure 4). The result showed that at 1.0% of both the FPHs had almost the highest surface area to mass ratio, resulting in the highest water holding capacity. In contrast, FPHs with a concentration of 1.0% consist of the least exposed surface that can be imbibed with water, therefore decreasing their water holding capacity. During the hydrolysis process, the complex protein is broken down into shorter amino acid chains, typically exposing the N-terminal (polar groups) which is ready to preferentially bind with the H-bond of water.
The ability of a protein to imbibe water and maintain it against gravity within a protein matrix is referred to as its water-holding ability (Foh et al., 2010). A higher water holding capacity in hydrolysate indicates a higher surface area to the mass ratio in hydrolysate that can interact with water's H-bond (Slizyte et al., 2009). The water-holding ability of hydrolysate was improved due to the low molecular weight of the hydrolysate and the increased concentrations of polar groups exposed during the breakdown of amino acid chains (Taheri et al., 2013.). FPHs can contain hydrophilic polar group amino acids such as serine, threonine, asparagine, and glutamine, which may have a positive impact on the hydrolysates' water-holding capability, which is critical for increasing cooking yield (Halim and Sarbon, 2016). FPH1 has a higher water holding capacity than those of hydrolysates produced from bluewing searobin (3.75 mL/g), tilapia (1.77 – 2.10 mL/g), and zebra blenny (6.10 mL/g) muscles (dos Santos et al., 2011; Foh et al., 2011; Jemil et al., 2014).
OIL BINDING CAPACITY OF FISH PROTEIN HYDROLYSATE:
Figure 5 shows the oil binding capacity of fish protein hydrolysates FPH1 (Papain) and FPH2 (Proteinase K) at different concentrations (0.2%, 0.4%, 0.6%, 0.8% and 1.0%). The FPH 1(papain) at 1.0% showed the highest oil binding capacity, followed by FPHs at 0.6%, 0.8%, 0.2%, and 0.4%. However, the oil binding capacity of FPHs displayed no significant differences (p>0.05) at different hydrolysate concentrations.
Both FPHs have a lower oil binding ability than water-binding capacity. The higher amount of polar than non-polar groups at the N-terminal of amino acid chains can explain this. As a result, FPHs showed lower oil binding than water binding. Oil binding potential is normally correlated to the hydrophobicity of the protein surface, according to dos Santos et al. (2011). Based on these main findings, FPHs at various concentrations usually exhibited similar surface hydrophobic properties, allowing them to naturally bond with oil. The oil binding potential of protein hydrolysates is also influenced by the bulk density of peptides and enzyme/substrate specificity (Cho et al., 2008). The ability of hydrolysate to absorb oil is essential for influencing the taste and functional characteristics of products such as meat, salad dressings, bakery products, and confectionaries (Cho et al., 2008; dos Santos et al., 2011). As a result, both FPHs have been shown to have the potential to bind oil in food products.
FPHs, on the other hand, had an oil binding potential of less than 1.00 mL/g at all concentrations, which was lower than that of striped catfish (1.35 mL/g) and dagaa (3.50 mL/g) muscle hydrolysates (Tanuja et al., 2012; Betty et al., 2014).
EMULSIFYING ACTIVITY:
The emulsifying activity index (EAI) and emulsifying stability index (ESI) showed no significant differences (p>0.05) of both FPHs but FPH1 (papain) shows higher EAI and ESI at all pH levels than FPH2 except for pH 4 because of low solubility at pH 4. the highest EAI of FPH1 at pH 10 (83.89 m2/g) followed by EAI of FPH1 at pH 2 (81.9- m2/g), EAI at pH 8 (78.85 m2/g) and pH 6 (65.02 m2/g), while the highest ESI was at pH 10 (81.11min) followed by ESI at pH 2 (72.49 min), pH 8 (71.87 min) as presented in figure 6a and figure 6b.
Since they are less effective at reducing interfacial stress than larger peptides due to the lack of unfolding and reorientation at the interface, small peptides spread to and absorb quickly at the interface (Gbogouri et al., 2004). Polypeptides undergo structural unfolding as a result of the negative charges formed at high pH (pH 10). This peptide unfolding can induce repulsion while also improving interface orientation. This could lead to better exposure of hydrophilic and hydrophobic residues in peptides, which would promote interaction at the oil-in-water interface (Finger & Mangino, 1991). The effectiveness of the hydrolysate compound in reducing the interfacial stress between the hydrophobic and hydrolytic components in food products is directly related to the emulsifying properties of hydrolyzed compounds (Cho et al., 2008). By altering the surface hydrophobicity and charge of the protective layer surrounding the lipid globules, the pH of the environment may have a major impact on the emulsifying properties of hydrolysate (Taheri et al., 2013). The amino acid composition of the eel protein hydrolysate (EPH) also contributes to its emulsifying properties, in addition to the pH level. The presence of hydrophobic amino acids in hydrolysates may affect their emulsifying properties (Cho et al., 2008). According to studies, the EAI of fish protein hydrolysates was highest at pH 6–10, and lowest at pH 4 (Pacheco-Aguilar et al., 2008; Naqash and Nazeer, 2013), which was consistent with the findings of this research.