Effect of Enzymatic Hydrolysis on the Nutritional and Functional Properties of Nile Tilapia (Oreochromis niloticus) Proteins

MATERIALS AND METHODS

The Tilapia fish (Oreochromis niloticus) was purchased from a local fresh water products market in Wuxi, China, on the 8th January 2010 and were transported within 24 h in ice boxes to the School of Food Science and Technology (SFST) laboratory of Jiangnan University, Wuxi, Jiangsu, People’s Republic of China. The fish (450-580 g fish^-1 with length range of 25-30 cm fish^-1) were prepared using the handling method; disemboweled, beheaded and skin removed before thoroughly washing with clean water to remove contaminants or unwanted particles. Fish muscle retrieved with care, separating the bones from the meat, chopped into pieces about 0.25 cm. Hot Water Dip (HWD) sample was obtained by sinking a portion of the chopped meat in hot water 95±5°C and maintained for 15 min (HWD), hence endogenous enzyme was inactivated and furthers impurities and some oil removed.

Table 1:	Optimum conditions for hydrolysis of Nile Tilapia (Oreochromis niloticus) with different proteases
*AU (Anson units) is the amount of enzyme that under standard conditions digests hemoglobin at an initial rate that produces an amount of trichloroacetic acid-soluble product which gives the same color with the Filon reagent as one milliequivalent of tyrosine released per minute. †LAPU (Leucine aminopeptidase unit) is the amount of enzyme that hydrolyzes 1 μmol of leucine-p-nitroanilide per minute

It was allowed to cool at room temperature, eventually vacuum packed in polyethylene bags. The sample was kept frozen at -20°C for subsequent analysis.

Alcalase 2.4 L is a bacterial endoproteinase from a strain of Bacillus licheniformis was obtained from Novozymes China Inc. and stored at 4°C for subsequent analysis. Prior to the hydrolysis process, the sample was thawed overnight in a refrigerator, 4±1°C. All chemical reagents used in the experiments were of analytical grade. The experiment was carried out in the SFST laboratory from January to April 2010.

Preparation of fish protein hydrolysates and concentrates: HWD sample was hydrolyzed with three different enzymes, under the conditions given in Table 1, based on optimum hydrolysis conditions. One hundred grams of samples were weighed into a vessel immersed in a water bath maintained at appropriate temperature and 700 mL of distilled water was added to make a suspension. The suspension was, for each enzyme, adjusted to appropriate pH and preheated to appropriate temperature; then (1%) enzyme substrate ratio (w/w) was used for all samples with continuous stirring. Hydrolysis was carried out for 5 h. Seventy five millliliter aliquots were taken after 30, 60, 90, 120, 160 and 300 min. After hydrolysis, the enzymes were inactivated by placing in boiling water for 15 min. The hydrolysate was allowed to cool down and centrifuged at 7,500x g for 15 min at 4°C with a D-3756 Osterode am Harz model 4515 centrifuge (Sigma, Hamburg, Germany). The tilapia Fish Protein Hydrolysates (FPH) and the raw samples were lyophilized (fish protein concentrate-FPC) and stored at -20±2°C until used. All experiments were performed in triplicate and the results are the average of the three values.

Amino acid analysis: The dried samples were digested with HCl (6 M) at 110°C for 24 hr under nitrogen atmosphere. Reversed phase high performance liquid chromatography (RP-HPLC) analysis was carried out in an Agilent 1100 (Agilent Technologies, Palo Alto, CA, USA) assembly system after precolumn derivatization with o-phthaldialdehyde (OPA). Each sample (1 μL) was injected on a Zorbax 80 A C18 column (i.d. 4.6x180 mm, Agilent Technologies, Palo Alto, CA, USA) at 40°C with detection at 338 nm. Mobile phase A was 7.35 mM L^-1 sodium acetate/triethylamine/ tetrahydrofuran (500:0.12:2.5, v/v/v), adjusted to pH 7.2 with acetic acid, while mobile phase B (pH 7.2) was 7.35 mM L^-1 sodium acetate/methanol/acetonitrile (1:2:2, v/v/v). The amino acid composition was expressed as g of amino acid per 100 g of protein.

Nutritional parameters: The nutritional parameters of Nile tilapia protein hydrolysates and the concentrates were calculated using their amino acid composition including:

The FAO/WHO reference pattern of essential amino acid requirements (g/100 g of protein) (FAO, 2007) was used as the standard.

(1)

(2)

(3)

Determination of molecular weight: The samples were determined using a Waters™ 600E Advanced Protein Purification System (Waters Corporation, Milford, MA, USA). A TSK gel, 2000SWXL (7.8x300 mm) column was used with 10% acetonitrile + 0.1% TFA in HPLC grade water as the mobile phase. The calibration curve was obtained by running bovine carbonic anhydrase (29,000 Da), horse heart cytochrome C (12,400 Da), bovine insulin (5800 kDa), bacitracin (1450 Da), Gly-Gly-Tyr-Arg (451 kDa) and Gly-Gly-Gly (189 Da). The total surface area of the chromatograms was integrated and separated into eight ranges, expressed as a percentage of the total area.

Determination of surface hydrophobicity: Surface hydrophobicity of HWDH and HWDC were determined by using the fluorescence 1-anilino-8-naphthalene sulfonate (ANS) binding method (Hayakawa and Nakai, 1985). HWDH and HWDC solutions (0.0015, 0.003, 0.006, 0.012, 0.015%, w/v) were prepared in 0.01 M phosphate buffer (pH 7.0) and vortexed homogeneously. Ten microliter of 8 mM ANS in 0.01 M phosphate buffer (pH 7.0) was added into each of 4.0 mL of the protein solutions, then mixed well by vortexing for 10 sec. Fluorescence intensity of these solutions were measured at 390 nm of excitation and 484 nm emmision using a Kontron Spectrofluorometer (model SFM23/B; Kontron Ltd., Zurich, Switzerland). The surface hydrophobicity plotted as the slope of fluorescence intensity against protein concentration and was calculated by linear regression.

Nitrogen Solubility (NS): Nitrogen solubility was determined according to the procedure of Diniz and Martin (1997), with slight modifications. Samples were dispersed in distilled water (10 g L^-1) and pH of the mixture was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 with either 0.5 N HCL or 0.5 N NaOH while continually shaking (Lab-Line Environ-Shaker; Lab-Line Instrument, Inc., Melrose Park, IL, USA) at room temperature for 35 min. A 25 mL aliquot was then centrifuged at 4000x g for 35 min. A 15 mL aliquot of the supernatant was analyzed for nitrogen (N) content by the Kjeldahl method and the NS was calculated according to equation:

(4)

Oil-Holding Capacity (OHC): Oil-Holding Capacity (OHC) of tilapia FPH was determined as the volume of edible oil held by 0.5 g of material according to the method of Shahidi et al. (1995). A 0.5 g sample of each FPH was added to 10 mL soybean oil (Gold Ingots Brand, QS310002012787, Suzhou, People’s Republic of China) in a 50 mL centrifuge tube and vortexed for 30 sec in triplicate. The oil dispersion was centrifuged at 3000x g for 25 min. The free oil was decanted and the OHC was determined by weight difference.

Water-Holding Capacity (WHC): To determine the Water Holding Capacity (WHC) of tilapia FPH, the method outlined by Diniz and Martin (1997), with slight modifications. Triplicate samples (0.5 g) of samples were dissolved with 10 mL of distilled water in centrifuge tubes and vortexed for 30 sec. The dispersions were allowed to stand at room temperature for 30 min, centrifuged at 3000x g for 25 min. The supernatant was filtered with Whatman No.1 filter paper and the volume retrieved was accurately measured. The difference between initial volumes of distilled water added to the protein sample and the volume retrieved. The results were reported as mL of water absorbed per gram of protein sample.

Emulsifying Capacity (EC): Emulsifying capacity was measured using the procedure described by Rakesh and Metz (1973), with modification. A 0.5 g of each freeze-dried sample was transferred into a 250 mL beaker and dissolved in 50 mL of 0.5 N NaCl and then 50 mL of soybean oil (Gold Ingots Brand, QS310002012787, Suzhou, P.R. China) was added. The homogenizer equipped with a motorized stirrer driven by a rheostat Ultra-T18 homogenizer (Shanghai, China) was immersed in the mixture and operated for 120 sec at 10,000 rpm to make an emulsion. The mixture was transferred to centrifuge tubes, maintained in water-bath at 90°C for 10 min and then centrifuged at 3000x g for 20 min. Emulsifying capacity was calculated as in equation:

(5)

where, V_A is the volume of oil added to form an emulsion, V_R is the volume of oil released after centrifugation and W_S is the weight of the sample.

Foaming Capacity (FC) and Foam Stability (FS): Estimation of foaming capacity was done following the method of Bernard-Don et al. (1991) with minor modifications. Thirty milliliter of 30 g L^-1 aqueous dispersion was mixed thoroughly using an Ultra-Turrax 25 homogenizer at 9,500 rpm for 3 min in a 250 mL graduated cylinder. The total volume of the protein dispersion was measured immediately after 30 sec. The difference in volume was expressed as the volume of the foam. Foam stability was determined by measuring the fall in volume of the foam after 60 min.

Bulk Density (BD): Bulk density of freeze-dried tilapia hydrolysates was estimated with approximately 3 g of each sample packed into 25 mL graduated cylinders by gently tapping on the lab bench 10 times. The volume was recorded and bulk density was reported as g mL^-1 of the sample.

In vitro Protein Digestibility (IVPD): In vitro Protein Digestibility (IVPD) was carried out according to the method described by Elkhalil et al. (2001), with slight modifications. Twenty mg of tilapia FPH (HWDPC and HWDPH) samples were digested in triplicate in 10 mL of trypsin (0.2 mg mL^-1 in 100 mM Tris-HCl buffer, pH 7.6). The suspension was incubated at 37°C for 2 h. Hydrolysis was stopped by adding 5 mL 50% trichloroacetic acid (TCA). The mixture was allowed to stand for 30-35 min at 4°C and was then centrifuged at 10,000x g for 25 min using a D-3756 Osterode AM Harz Model 4515 Centrifuge (Sigma, Hamburg, Germany). The resultant precipitate was dissolved in 5 mL of NaOH and protein concentrate was measured using the Kjeldahl method. Digestibility was calculated as follows:

(6)

where, A is total protein content (mg) in the sample and B is total protein content (mg) in the TCA precipitate.

Statistical analysis: Data are result of at list three determinations. One way Analysis of Variance (ANOVA) was used to determine the statistical difference at (p<0.05), using Origin Pro Version 8.0.

RESULTS AND DISCUSSION

Enzymatic hydrolysis with different proteases: The enzymatic hydrolysis of Nile Tilapia (Oreochromis niloticus) with Alcalase 2.4 L showed the highest increase in protein content of HWDH during the first 240 min of hydrolysis 89.86%. The amount of proteins released decreased slightly to 86.64% (Fig. 1). Neutrase solubilized 81.92% of protein during the first 240 min of hydrolysis and the amount solubilized increased to 82.54% with the remaining hydrolyzing period. Flavourzyme showed an increase in protein solubilization during the first 240 min of hydrolysis with 73.12% of the protein being solubilized (Fig. 1). The results of our study exhibited a behavior that is, similar to Adler-Nissen (1986) and Panyam and Kilara (1996). The high efficiency of Alcalase 2.4 L may be a result of a high frequency of potential cleavable sites in HWD which may have contributed to the high degree of solubilization. Alcalase 2.4 L was selected for the current study because of its high yield under optimum conditions, cost effectiveness and readily available.

Amino acid analysis: HWDH contains all the essential amino acids in good proportion as compared to HWDC. The results in Table 2 indicated that the amino acid composition of HWDH and HWDC closely resembles each other. The predominant amino acids amongst the non essential amino acids were aspartic acid, glutamic acid and alanine; those amongst the essential amino acids were lysine, threonine and leucine (Table 2).

Fig. 1:

Amount of protein solubilized by enzymatic hydrolysis of Nile tilapia (Oreochromis niloticus) by different proteases. Value represent the Mean±SD of n = 3 duplicate assays

Table 2:	Comparative amino acid profiles of Nile tilapia (Oreochromis niloticus) protein hydrolysates and its concentrates g/100 g of protein
The data are means and standard deviations of triplicate AFAO/WHO/UNU energy and protein requirements (2007); BRequirements for methionine + cysteine; CRequirements for phenylalanine + tyrosine; DAspartic acid + asparagines; ECysteine + cysteine; FGlutamic acid + glutamine

Both samples have a well-balanced amino acid composition. Moreover, Most of the essential amino acids of their proteins were at a higher level than the Food and Agricultural Organization/World Health Organization reference. These values are generally in accordance with previous publications (Usydus et al., 2009; Vidotti et al., 2003). However, Tryptophan and cystine were much less in HWDC compared with that of HWDH.

Nutritional parameters: Protein is one of the essential nutrients in the human diet. Both the amount and quality of protein provided by a food are important. The protein quality, also known as the nutritional or nutritive value of a food, depends on its amino acid content and on the physiological utilization of specific amino acids after digestion, absorption and minimal obligatory rates of oxidation. Because direct assessment of protein nutritional value in human subjects is impractical for regulatory purposes, methods based on in vitro (chemical) and in vivo bioassays for assessment of protein quality have been developed. Herein, a case is made for the use of amino acid data as a basis for estimation of nutritional quality of fish proteins. The ratio of essential to total amino acids, amino acid score and PER of HWDH and HWDC are shown in Table 3. HWDH and HWDC have a higher ratio of essential to total amino acids than the pattern recommended by FAO/WHOUNU. HWDC had the highest ratio of 49.05% compared to HDWH with a ratio 44.52%.

Table 3:	Nutritional parameters of Nile tilapia (Oreochromis niloticus) protein hydrolysates and its concentrates
The data are means and standard deviations of triplicate; E/T, proportion of essential amino acids (E) to total amino acids (T); PER, predicted protein efficiency ratio

Fig. 2:

Molecular weight distribution of Nile tilapia (Oreochromis niloticus) proteins. (a) HWDC and (b) HWDH

Predicted PER values of HWDH and HWDC all exceeded 2.00, which describes a protein of good and high quality (Friedman, 1996). HWDC have the highest PER values (3.11, 3.55 and 3.35%) for PER I, II and III respectively. The PER values of HWDH and HWDC were rather satisfactory when compared with the standard casein PER of 2.5 (Friedman, 1996). However, total essential amino acid scores for HWDH and HWDC reached the FAO/WHO requirement (2007) for the essential amino acids for children (Table 3).

Molecular weight distribution: The Gel Permeation Chromatography (GPC) using an HPLC system was used to study molecular weight distribution profiles of HWDH and HWDC. Figure 2a, and b show the molecular size distribution profiles of HWDH and HWDC. The chromatographic data indicated that the HWDH composed of lower molecular weight peptides whose peaks ranged from 328-1876 Da (Fig. 2). However, HWDC composed of much higher molecular weight polypeptides whose peaks ranged from 214-19,576 Da. In this study, results revealed that HWDH has lower molecular weight distribution; this is probably associated with higher functional attribute.

Fig. 3:

Fluorescence intensity of Nile tilapia (Oreochromis niloticus) proteins

These findings are in agreement with observations from other studies and support the fact that functional properties are highly influenced by molecular weight distribution (Wang et al., 2006; Kim et al., 2007).

Surface hydrophobicity of proteins: The surface hydrophobicity value is an indicator of the number of hydrophobic groups on the surface of a protein in contact with the polar aqueous environment. The surface hydrophobicity, is an index of the protein’s capacity for intermolecular interaction and hence its functionality. The surface hydrophobicities (So) of HDWH (168.01) and HDWC (200.28), respectively and the linear relationships between protein concentration and fluorescence intensity are shown in Fig. 3. Present result follow similar trend of Achouri and Zhang (2001). The surface hydrophobicity of a protein is an index of the number of hydrophobic groups on its surface in contact with the polar aqueous environment. Changes in surface hydrophobicity as result of proteolysis; influences the functional properties especially the interfacial properties of the hydrolysates. There was a significant difference in surface hydrophobicity between HDWH and HWDC. In the native proteins, the hydrophobic amino acids are buried in the central core of the protein molecule. This feature is lost when protein is denatured or hydrolyzed into shorter peptides (Wang et al., 1999).

Protein solubility: Solubility is one of the most important characteristics of proteins because it is not only important by itself, but also influences other functional properties. Good solubility of proteins is required in many functional applications, especially for emulsions, foams and gel, because soluble proteins provide a homogenous dispersibility of the molecules in colloidal systems and enhanced the interfacial properties (Zayas, 1997).

The solubilities of HDWH and HWDC at pH 2.0 to pH 12.0 are presented in Fig. 4. At pH 4.0 and 5.0, near the isoelectric point at which the net charge of the original protein is minimized (83.27 and 82.23%) and consequently more protein-protein interactions and fewer protein-water interactions occur (Adler-Nissen, 1976; Chobert et al., 1988). Above pH 6.0, the nitrogen solubilities increased rapidly with an increase in pH up to 12.0. The solubility of HDWH reached 95.23%, while solubility for HWDC was 89.25% at pH 12.0 (Fig. 4). These trends in solubilities are in agreement with (Choi et al., 2009; Sathivel et al., 2009). An increase in the extent of enzymatic hydrolysis corresponded to a considerable increase in the nitrogen solubility, over the pH range studied, indicating a positive relationship (Fig. 4). It has been suggested that an increase in the solubility of protein hydrolysates over that of the original protein is due to the reduction of its secondary structure and also to the enzymatic release of smaller polypeptide units from the protein (Adler-Nissen, 1986; Chobert et al., 1988).

Fig. 4:

Effect of pH on nitrogen solubility of Nile tilapia (Oreochromis niloticus) proteins. Value represent the Mean±SD of n = 3 duplicate assays

Table 4:	Functional properties of Nile tilapia (Oreochromis niloticus) protein hydrolysates and concentrates
Values are Means±SD of three determinations. * indicate significant difference and ** insignificant difference at (p<0.05)

Water/oil holding capacity (WHC/OHC): The functional properties of proteins in a food system depend in part on the water-protein interaction. WHC refers to the ability of the protein to imbibe water and retain it against gravitational force within a protein matrix, such as protein gels, beef and fish muscle, it is positively correlated with water-binding capacity (Foh et al., 2010). The WHC of HWDH was 1.77 and 2.43 mL g^-1, respectively, with an insignificant difference (p<0.05) (Table 4). Interactions of water and oil with proteins are very important in the food systems because of their effects on the flavor and texture of foods. Intrinsic factors affecting water binding of food protein include amino acids composition, protein conformation and surface hydrophobicity/polarity (Kamara et al., 2009b; Barbut, 1999).

For oil holding capacity, HWDC was higher (3.30 mL g^-1) while, HWDH was (2.23 mL g^-1), with a significant difference (p<0.05) (Table 4). Present results corroborated to other fish proteins studied (Diniz and Martin, 1997). Further more, high oil absorption is essential in the formulation of food systems like sausages, cake, batters, mayonnaise and salad dressings.

Emulsifying Capacity (EC): The EC is a measure of the effectiveness of proteinaceous emulsifiers (Pearce and Kinsella, 1978). The ability of proteins to form stable emulsions is important owing to the interactions between proteins and lipids in many food systems. An increase in the number of peptide molecules and exposed hydrophobic amino acid residues due to hydrolysis of proteins would contribute to an improvement in the formation of emulsions. From the results, HWDH (21.40 mL 0.5 g ^-1) shows an appreciable EC than HWDC (20.40 mL 0.5 g ^-1) with a significant difference (p<0.05) (Table 4).

Fig. 5:

Foam stability of Nile tilapia (Oreochromis niloticus) proteins. Value represent the Mean±SD of n = 3 duplicate assays

Present results were similar to Wasswa et al. (2008) and Abdul-Hamid et al. (2002).

Foam capacity and stability (FC and FS): Proteins are good foaming agents, since they can rapidly diffuse to the air-water interface and they form a strong cohesive and elastic film by partial unfolding. Foaming properties are correlated with amount of hydrophobic amino acids exposed at the surface of the protein molecule (Wang et al., 1999). Dispersed proteins lower the surface tension at the water-air interface, thus creating foaming capacity (Turgeon et al., 1992).

To have foam stability, protein molecules should form continuous intermolecular polymers enveloping the air bubbles, since intermolecular cohesiveness and elasticity are important to produce stable foams (Kamara et al., 2009a). A significant increase was observed in the foaming capacity of HWDH (124.5 g mL^-1) compared to HWDC (80.3 g mL^-1) with a significant difference (p<0.05) (Table 4). An improvement in foaming capacity for enzymatically modified food proteins is reported by Adler-Nissen (1986). The foam stability of the HWDC was found to be less than that of HWDH. Enzymatic hydrolysis of Nile tilapia proteins caused an increase in the foam volume initially and then a decrease with time. The foam stability values ranged from 124.5 to 37.2 and 80.3 to 32.33 g mL^-1 for HWDH and HWDC respectively (Fig. 5). Present results were similar to (Wasswa et al., 2008; Abdul-Hamid et al., 2002).

Bulk density: Bulk density is a measure of heaviness of the powder. Moreover, bulk density is an important parameter that determines the packaging requirement of a product. Further more; Bulk density signifies the behavior of a product in dry mixes. Also, it varies with the fineness of the particles. HDWH and HWDC had varying bulk densities of 0.53 and 0.35 g mL^-1, respectively with an insignificant difference (p<0.05) (Table 4). Present results obtained for HDWH and HWDC were similar compared to reported values (Wasswa et al., 2007). The low bulk density of HWDH and HWDC was due to its lower particle density and the large particle size. High bulk density is disadvantageous for the formulation of weaning foods, where low density is required (Kamara et al., 2009a).

In vitro Protein Digestibility (IVPD): The in vitro protein digestibility of HWDH and HWDC were significantly different (p<0.05). The in vitro protein digestibilities of both samples were evaluated by the release of TCA-soluble nitrogen, after incubation time of 120 min at 37°C. Table 4 shows that all the protein samples exhibited very good trypsin digestibility. Nonetheless, HWDH had higher digestibility value (93.20%) while HWDC was lower (87.60%). This probably resulted from pre-hydrolysis processing, which led to the existence of fewer attack sites being available to the enzymes in the digestibility assay. Prsent results are within the values reported by Abdul-Hamid et al. (2002).

CONCLUSION

Conclusively, understanding change of Nile tilapia complex during enzymatic hydrolysis can be useful for producing modified proteins with the desired nutritional parameters and functionality. From the results presented here, it is proposed that excellent solubility of the protein hydrolysates could be attributed to reduce size of the polypeptides obtained after proteolysis. The results indicated close relationships between functional properties and molecular size of the modified Nile tilapia. The amino acids of both samples were higher than FAO/WHO requirement for both infants and adults. All the estimated nutritional parameters based on amino acids composition showed that Nile tilapia protein hydrolysates and concentrates have good nutritional quality. Furthermore, the hydrolysates that are obtained also have an effect on improving functionality such as solubility, foaming properties and other important properties of proteins than the concentrate. Not only these hydrolysates can be used as food additives to improve the functionality but also improving the nutritional profile by incorporating them in selected foods. Nile tilapia protein hydrolysates could excellent applications for future product development by virtue of their nutritional and functional properties.

ACKNOWLEDGMENTS

Authors wish to thank the earmarked fund for Modern Agro-industry Technology Research System (NYCYTX-49-22), PCSIRT0627 and 111project-B07029 for providing financial support to carry out this research.