68 CHAPTER 6 FUNCTIONAL PROPERTIES OF PROTEIN HYDROLYSATES 6.1 INTRODUCTION Functional properties can be defined as the overall physicochemical properties of proteins in food systems during processing, storage and consumption. Fish protein hydrolysates (FPH) have good solubility for a wide range of ionic strength and and also tolerate strong heat without precipitating [22]. Fish protein hydrolysate has better foaming and emulsifying properties, thus it may be used as emulsifying and emulsion stabilizing ingredients in a different range of products as well as used in the formation and stabilization of foam-based products. As the size of the peptides is very important for interfacial/surface activity of FPH, the degree of hydrolysis is important [221]. Protein hydrolysates are used predominantly in the food industry for different purposes, such as milk replacers, protein supplement, surimi production, beverage stabilizers, and flavor enhancers [22]. Fish protein hydrolysates, produced by controlled enzymatic hydrolysis, have better nutritional properties such as balanced amino acid composition and high digestibility but are mainly used for animal nutrition [222]. An advantage noticed by subjecting fish meat to enzymatic hydrolysis is the ability to modify and enhance the functional properties of fish proteins. Those hydrolyzed proteins are important, particularly in their use as food ingredients [223]. Enzymatic hydrolysis of fish protein produces a pool of heterogeneous free amino acids, dipeptide, tripeptide, and oligopeptides, which increases the number of polar groups and the solubility of hydrolysate. Therefore, it changed the functional characteristics of proteins, improving their functional quality and availability [11]. Functional properties are related to structure of proteins, such as the sequence and composition of amino acids, molecular weights, conformation, and charge distributed on the molecule [224]. The charge s nature and density facilitate interactions with other components, such as water, ions, lipids, carbohydrates,
69 vitamins, color, and flavor constituents, which depend on factors such as, temperature, ionic strength, which are in turn involved during food preparation, processing, and storage [2]. Byproducts of fish-processing plants, such as downgraded whole fish, heads, skin, and frame bones, are currently used to produce food proteins at low cost [29]. Protein hydrolysate can be used to improve or modify the physicochemical, functional properties such as solubility, fat absorption, and water holding capacity, foaming properties, emulsifying properties and or sensory properties of proteins without losing its nutritional value [21]. The choice of substrate, protease enzyme employed and degree of hydrolysis can greatly affect the physicochemical properties of hydrolysate. Commercial enzyme, Alcalase has been strongly recommended for fish hydrolysis [197]. Produced hydrolysates by enzymatic treatment are containing well defined peptide profiles and there is an extensive review on the application of enzymatic protein hydrolysates in human nutrition [226]. The enzymatic treatment of proteins generates peptides and amino acids, which can modify the biological and functional characteristics of the proteins and improve their quality and offers interesting opportunities for food applications. Hydrophobic and ionic interactions are the prime factors that promote the solubility characteristics of proteins. Hydrophobic interactions influence the proteinprotein interactions and result in poor solubility, whereas ionic interactions influence protein-water interactions and results in improved solubility. Ionic residues on the surface of peptides and proteins introduce electrostatic repulsion between protein molecules and repulsion between hydration shells around ionic groups and these both important factors contribute to high solubility of proteins [227]. The emulsifying properties of fish protein hydrolysates are directly linked to their surface properties or a way which the hydrolysate effectively lowers the interfacial tension between the hydrophobic and hydrophilic components in food. Proteins adsorb to the surface of freshly formed oil droplets during homogenization and form a protective membrane that prevents droplets from coalescing [228]. The amphiphilic nature of proteins makes the foaming possible; the hydrophobic portion of the protein extends into the air and the hydrophilic portion into the aqueous phase. It has been reported by Townsend and Nakai [229] that total hydrophobicity of proteins or
7 the hydrophobicity of exposed or unfolded protein has a significant correlation to foaming formation. 6.2 MATERIALS AND METHODS 6.2.1 Solubility The solubility of fish protein hydrolysates at values from 2-1 was determined by the method of Dong et al., [217]. Reagents 1. HCl 2. NaOH 3. Fish protein hydrolysates 4. Biuret reagent Procedure Briefly, 2 mg of protein hydrolysate sample were dispersed in 2 ml of deionized water and of the mixture was adjusted 1 or 6 N HCl and 1 or 6 N NaOH. The mixture was stirred at room temperature for 3 min and centrifuged at 7g for min. Protein contents in the supernatant were determined using the Biuret method. Total protein content in the sample was determined after solubilization of the sample in. N NaOH. Protein solubility was calculated as follows: Solubility (%) = (Protein content in supernatant/total protein content in sample) 1
71 6.2.2 Emulsifying Properties The emulsifying properties of the fish protein hydrolysates were determined by the methods of Pearce and Kinsella [23]. Reagents 1. Fish protein hydrolysates 2. Vegetable oil 3. Homogenizer 4. Sodium dodecyl sulphate Procedure Briefly, about 6 ml of 1% protein solution of northern whiting fish muscle and visceral mass hydrolysates was mixed separately with 2 ml of vegetable oil in beakers. The was adjusted to 2., 4., 6., 8. and 1. in each beaker respectively. The mix was homogenized using a homogenizer at a speed of 2, rpm for 1 minute. Then l of the emulsion was pipetted out from the bottom of the container at min and 1 minutes and then was mixed with ml of SDS solution. The absorbance was measured at nm using a spectrophotometer. The absorbances measured immediately (A ) and 1 min (A 1 ) after emulsion formations were used to calculate the emulsifying activity index (EAI) and the emulsion stability index (ESI) as follows: EAI (m 2 /g) = 2 2.33 A /. Protein weight (g) ESI (min) = A t / A where as, A = A - A 1 and t = 1 min A = Absorbance, t = time taken to check the emulsion stability
72 6.2.3 Foaming Properties Foaming capacity and stability of protein hydrolysate were determined according to the method of Sathe and Salunkhe [231]. Reagents 1. Fish protein hydrolysates 2. NaOH 3. HCl Procedure Briefly, 2 ml of.% active protein hydrolysates from northern whiting fish muscle and visceral mass were adjusted to 2, 4, 6, 8 and 1 followed by homogenization at a speed of 16, rpm to incorporate the air for 2 min at room temperature. The whipped sample was immediately transferred into a ml measuring cylinder and the total volume was read after 3 s. The foaming capacity was calculated as: Foaming capacity (%) = (A-B/B) 16 Where, A is the volume after whipping (ml), B is the volume before whipping (ml). The whipped sample was allowed to stand at 2 o C for 3 min and the volume of whipped sample was then recorded. Foam stability was calculated as follows: Foaming stability (%) = (A-B/B) 1 Where, A = volume after standing (ml), B = volume before whipping (ml).
73 6.3 RESULTS 6.3.1 Solubility The solubility capacity of active protein hydrolysates from northern whiting fish muscle and visceral mass was tested in different (2, 4, 6, 8 & 1) and the results were showed in the figure (6.1a,b). The solubility capacity of tryptic muscle hydrolysates was above 7% and peptic visceral mass protein hydrolysate was above 2% in all the range of ; however the solubility was poor at 4. Among the two hydrolysates, tryptic muscle protein hydrolysate showed better solubility than peptic visceral mass protein hydrolysates, this may be due to hydrolysis pattern of trypsin could have produced low molecular weight peptides, which can easily solubilize. 6.3.2 Emulsifying Properties The emulsifying ability (EAI) and emulsifying stability (ESI) of active protein hydrolysates generated from northern whiting fish muscle and visceral mass by trypsin and pepsin separately in different (2, 4, 6, 8 & 1) was showed in figure (6.2a,b & 6.3a,b). The emulsifying properties of the hydrolysates were initially increased and then decreased suddenly at 4 and increased gradually with increase in. EAI and ESI were lowest at 4, with coincidental decrease due to poor solubility. The EAI and ESI of both active protein hydrolysates generally increased as moved away from 4, this effect was more pronounced with trypsin hydrolysate of northern whiting muscle. Among the two active hydrolysates, trypsin hydrolysates from muscle showed better emulsifying properties compared with peptic visceral mass hydrolysate, this could be due to the influence of specific enzymes. 6.3.3 Foaming Properties The Foaming properties of active protein hydrolysates of northern whiting fish muscle and visceral mass generated by trypsin and pepsin separately are showed in figure (6.4a,b & 6.a,b). The foaming properties of both the hydrolysates
74 were investigated at different (2, 4, 6, 8, & 1) and were greatly influenced by change in the. Foaming capacity and foaming stability was higher in tryptic muscle hydrolysate of northern whiting fish compared to peptic visceral mass hydrolysates. The foaming capacity and foaming stability of both hydrolysates reached maximum at 6 and gradually decreased upon increasing the. The Foaming properties of active hydrolysates were poor at 4 due to low solubility of hydrolysates in the isoelectric point. Solubility (%) 9 8 7 6 4 3 2 1 C (a) Solubility (%) 8 7 6 4 3 2 1 C (b) Figure 6.1 Solubility of (a) muscle (b) visceral mass protein hydrolysates from northern whiting fish at different values. Data represent standard deviation from triplicate determinations
7 EAI (%) 4 4 3 3 2 1 C (a) EAI (%) 4 4 3 3 2 1 C (b) Figure 6.2 Emulsifying ability of (a) muscle (b) visceral mass protein hydrolysates from northern whiting fish at different values. Data represent standard deviation from triplicate determinations
76 ESI (%) 4 4 3 3 2 1 C (a) ESI (%) 4 4 3 3 2 1 C (b) Figure 6.3 Emulsifying stability of (a) muscle (b) visceral mass protein hydrolysates from northern whiting fish at different values. Data represent standard deviation from triplicate determinations
77 Foaming capacity (%) 4 3 3 2 1 C (a) Foaming capacity (%) 4 4 3 3 2 1 C (b) Figure 6.4 Foaming capacity of (a) muscle (b) visceral mass protein hydrolysates from northern whiting fish at different values. Data represent standard deviation from triplicate determinations
78 Foaming stability 3 Foaming stability (%) 2 1 C (a) Foaming stability (%) 2 1 C (b) Figure 6. Foaming stability of (a) muscle (b) visceral mass protein hydrolysates from northern whiting fish at different values. Data represent standard deviation from triplicate determinations
79 6.4 DISCUSSION 6.4.1 Solubility Both the hydrolysates from northern whiting fish were soluble over a wide range (2-1), in which more than 7% solubility was obtained for trptic muscle protein hydrolysates and more than 2% solubility was obtained for peptic visceral mass protein hydrolysate. The hydrolysates were easily soluble in alkaline to a better extent with compared with acidic. Simillar type of results is reported in many articles. The Protein hydrolysates from yellow stripe trevally (Selaroides leptolepis) fish hydrolysed by alcalase and flavourzyme with different degree of hydrolysis ( %), also showed high solubility (>8%) in the range of 2 12 [189]. The same results were reported in salmon by-products [28] and silver carp [217] which showed decrease in solubility at the 4- and drastically increased with increase in. The result suggested that near 4 proteins hydrolysates with high molecular weight (MW) remaining after hydrolysis were precipitated at this, which was close to the isoelectric point (pi). Generally, the degradation of proteins to smaller peptides leads to more soluble products [189]. The smaller peptides from proteins hydrolysates are expected to have considerably more polar residues, which have the ability to form hydrogen bonds with water and augment solubility [28]. Enzymatic hydrolysis potentially influences the molecular size and hydrophobicity, as well as polar and ionisable groups of protein hydrolysates [232]. The balance of hydrophilic and hydrophobic forces of peptides is another crucial factor on the solubility of protein hydrolysate [28]. Therefore, the differences in solubility of hydrolysates might be determined by the size of peptides, the hydrophobic hydrophilic balance, as well as the charge group of the peptides produced during the hydrolysis process. 6.4.2 Emulsifying Properties Emulsifying activity index (EAI) and emulsion stability index (ESI) of both trptic muscle protein hydrolysate and peptic visceral mass protein hydrolysate of northern whiting fish was investigated after treating with various (2, 4, 6, 8 &
8 1) are shown in Fig. x. EAI and ESI of both active hydrolysates was higher at 2 and decreased suddenly at 4 and increased gradually by increasing the. When considering the effect of on EAI and ESI, the lowest EAI and ESI were found at 4, with coincidental decrease in solubility. Since the lowest solubility occurred at 4, peptides could not move rapidly to the interface. Additionally, the net charge of peptide could be minimized at 4. The trend was similar to that reported in emulsifying properties of in yellow stripe trevally [189] and in round scad, [122] which showed the EAI and ESI were affected by the. The higher EAI of hydrolysates accompanied their higher solubility [233]. Hydrolysates with high solubility can rapidly diffuse and adsorb at the interface. EAI and ESI generally increased as moved away from 4. This effect was more pronounced with both the hydrolysates, thus suggesting that the sequence and composition of amino acids in the hydrolysates might be different, leading to varying charge of the resulting peptides at a particular. Emulsifying properties were influenced by specificity of enzyme [234].The mechanism to generate the emulsion system is attributed to the adsorption of peptides on the surface of freshly formed oil droplets during homogenization and the formation of a protective membrane that inhibits coalescence of the oil droplet [23]. Hydrolysates are surface-active materials and promote oil-in-water emulsion because of their hydrophilic and hydrophobic groups with their associated charges [28]. Apart from peptide size, amphiphilicity of peptides is important for interfacial and emulsifying properties. Rahali et al., [236] analyzed amino acid sequence at an oil/water interface and concluded that amphiphilic character was more important than was peptide length for emulsion properties. 6.4.3 Foaming Properties Foam formation is regulated by three factors, including transportation, penetration and reorganization of molecules at the air water interface. To generate good foaming, a protein must be capable of migrating rapidly to the air water interface, unfolding and rearranging at the interface [237]. The foaming properties of both tryptic muscle protein hydrolysate and peptic visceral mass protein hydrolysate of northern whiting fish was investigated after treating with various (2, 4, 6, 8 &
81 1) are shown in figure (6.4 & 6.). The foaming capacity and foaming stability of protein hydrolysates were greatly affected by. After the exposure of hydrolysates with different, the foaming capacity and foaming stability tended to decrease at 4. The foaming capacity of both protein hydrolysates reached a maximum at 6 with a slight decrease at alkaline (8 & 1). Therefore, net charge could influence the adsorption of the proteins at the air water interface. When the net charge was increased, the foaming property was also increased [1]. Similar type of results are reported by Souissi et al., [238] indicated that foam capacity in sardinella (Sardinella aurita) decreased with increase in, reached maximum at 6 and gradually showed decrease at alkaline. Furthermore, similar results were reported in yellow stripe travelly, where showed major factor for foaming properties [189]. The lowest foaming properties of proteins also coincided with the lowest solubility at their isoelectric [239]. Similarly, for foam stability, the lowest value was found at 4 for both the hydrolysates. The low foam stability was concomitant with the low solubility at 4. Protein solubility makes an important contribution to the foaming property of protein hydrolysates. The of the dispersing medium dramatically influences foaming properties, especially foam stability [229]. Foam stability depends principally on the nature of the film and reflects the extent of protein protein interaction within the matrix [233]. The decreased foam stability at very acidic or alkaline might be due to the repulsion of peptides via ionic repulsion.