Lokan or mud clam in English is edible marine bivalves and widely distributed throughout the west indo-pacific region included peninsular Malaysia and Sarawak.This large, heavy bivalve formerly known as Polymesoda erosa is found buried in the stiff mud of the landward fringe of mangroves.It is being able to tolerate long periods of low tide, and has the ability to resume filter-feeding rapidly when inundated. This species is currently used in cooking as delicious seafood and human consume it increasing day by day. In peninsular Malaysia, this species are normally found in Selangor and Perak.
Hydrolysate is defined as any compound produced by hydrolysis process meanwhile protein hydrolysate is a mixture of amino acids prepared by splitting a protein with acid, alkali or enzyme. Protein hydrolysates are used widely in the food industry for various purposes, such as milk replacers, protein supplement, surimi production, beverage stabilizers, and flavor enhancers. In this study, Lokan (polimesoda erosa) protein hydrolysate using two different enzymes alcalase and flavourzyme will be prepared and the characteristics of the protein hydrolysate produced will be evaluated to give a more valuable and marketable product.
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1.2 Problem statement
Polymesoda erosa is the type of clam that are found buried shallowly in mangrove mud and are often harvested food in southern Asia but not on a commercial scale (Peter et al.,2011). There only few information regarding this species. Moreover human consumption on this species is still limited. So , the hydrolysis of Lokan 'Polymesoda erosa' protein will be performed due to the processing techniques for seafood is needed to convert them into more marketable, valuable and acceptable products. Enzymatic hydrolysis of protein is a chosen option because it avoids the extremes of chemical and physical treatments and thus minimizes undesirable reactions. Enzymes such as Alcalase and Flavourzyme will be used because it have been reported to hydrolyze fish protein efficiently (Kristinsson and Rasco 2000; Normah et al. 2005; Dumay et al. 2006; Safari et al. 2009; Martins et al. 2009).
1.3 Significance of study
Sea species is mostly used to produce protein hydrolysate because it is easily to found, edible and halal. A bivalvia species such as Lokan has limited uses and human just consume it as dish for meal.
This research is to produce protein hydrolysate from Lokan ( Polymesoda erosa).
The objective of this work is
To determine the characteristic of Lokan 'polimesoda expansa' protein hydrolysate using two different enzymes Alcalase and Flavourzyme
To produce the protein hydrolysate in order to direct their use towards the best applications in products for direct human consumption.
Polymesoda erosa 'Lokan'
Three species of the mangrove clams belonging to sub genus 'Polymesoda' and family of 'Coarbiculidae' are reported from indo pacific region (Ingole et al.,2002). There are Polymesoda erosa, Polymesoda bengalensis and Polymesoda expansa. Mud clam of the polymesoda erosa will be used because it normally found in our country,Malaysia. This species has been collected from landward side of intertidal mud and mangrove and eat by filtering water and filter out the iron in the water. Lokan is a life that has two hard shells and has colour white gray, yellowish, blackish stripes depends on the habitat in which they live. Clam size between 4 to 15 cm which is shaped like a 'kerang' but has smoother surface and no ribs-clack . It has been investigated that this species is less tolerant of colder waters of the northern and southern extremities of its distribution. This species is also reported found in manmade prawn pond (Bayen et al., 2005). This species cannot be found in everyday market, it can only be found abundant during their season.
Fish Protein Hydrolysate
Protein hydrolysates can be define as protein that chemically or enzymatically broken down to peptides of varying sizes (Adler-Nissen, 1986). The chain length of peptides formed during the hydrolysis process is one of the parameters determining both the functional and the organoleptic properties of the hydrolysate. Normally, fish protein hydrolysate includes 85-90% protein,2-4% lipid and 6-7% ash ( Mackie,1982). Fish protein hydrolysates can be obtained using acid, base, endogenous enzymes and added bacterial or digestive proteases and also through in vitro digestion. Digestion parameters such as time, temperature and pH are tightly controlled to produce protein hydrolysate with the desired properties. Therefore, proper production of concentrated protein products can be used as food ingredients owing to the capability of their functional properties.
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2.3 The Production of protein hydrolysate
Fish protein hydrolysates will be produced by mincing and mixing the raw material with water to a 8â€12% protein concentration before adjusting pH, adding enzymes (0,5â€2,0% of protein weight) and setting the hydrolysis temperature (Thorkelsson and Kristinsson 2009).There are two methods most widely used for protein hydrolysis which are the chemical and biological method. Chemical hydrolysis has been used in industrial practise. Biological process using added enzymes are employed more frequently and enzymes hydrolysis holds the most efficient for the future because it renders the products of high functionality and nutritive value (Pacheco-Aguilar et al.,2008). A variety of commercial enzymes have been tested successfully for hydrolyzing fish and other food proteins. The commercial blends of enzymes are most commonly used. They are usually a combination of endoâ€ end exopeptidases (Kristinsson and Rasco 2002). The endoâ€proteases is Alcalase and the exopeptidase is Flavourzyme. Endoproteases work by cleaving peptide bonds in the interior of polypeptide chains,whereas exopeptidases cleave off amino acids one at a time from the end of polypeptide chains(Thorkelsson and Kristinsson 2009).In order to control the bioactive properties of the hydrolysate it is important to stop the enzyme reaction at a closely defined %DH (degree of hydrolysis) value. All the proteases can be irreversibly inactivated by heat treatment. Cost and the properties of the end product influence the type of enzymes used.
Alcalase has great ability to solublize fish protein and is nonspecific, with an optimum temperature that ranged from 50 to 70Â°C. It has optimal pH range at the value of 8 to 10 that could reduce the risk of microbial contaminations (Chabeaud et al., 2009). According to Adler-Nissen (1986), hydrolysates prepared by using Alcalase had the highest protein recovery and the lowest lipid content than those made using Papain and Neutrase. Moreover, it has been reported that Alcalase treated fish protein hydrolysates had less bitter principles compared to those prepared with papain (Hoyle and Merritt,1994). Furthermore Alcalase has been documented to be a better candidate for hydrolyzing fish proteins based on enzyme cost per activity (Kristinsson and Rasco, 2000b).Generally, Alcalase 2.4 L-assisted reactions have been repeatedly favoured for fish hydrolysis, due to the high degree of hydrolysis that can be achieved in a relatively short time under moderate pH conditions, compared to neutral or acidic enzymes (Aspmo et al., 2005; Bhaskar et al., 2008; Kristinsson and Rasco, 2000; Kristinsson and Rasco, 2000b). The proteases Alcalase are inactive by heating at 50Â°C for 30 minutes or at 85Â°C for 10 minutes after the hydrolysis has been completed.
Flavourzyme is a fungal protease/peptidase complex produced by submerged fermentation of a selected strain of Aspergillus oryzae which has not been genetically modified and are used for the hydrolysis of proteins under neutral or slightly acidic conditions. The optimal working conditions for Flavourzyme 500 L are reported to be at pH 5.0 to 7.0 with an optimal temperature around 50â°C. Flavourzyme 500 L has a declared activity of 500 L APU/g. (Slizyte et al., 2005).
Flavourzyme also can be used to degrade a variety of food proteins extensively, i.e. to degrees of hydrolysis of up to 70% . Extensive hydrolysis of proteins with Flavourzyme produces protein hydrolysates, without the bitterness that often occurs with moderate enzymatic hydrolysis. The Flavourzyme complex can be inactivated by heating at 90Â°C for 10 minutes or at 120Â°C for 5 seconds after the hydrolysis has been completed.
Degree of hydrolysis
Degree of hydrolysis (DH), which indicates the percentage of peptide bonds cleaved (Adler-Nissen,1986), is one of the basic parameters that describes the properties of protein hydrolysate and needs to be controlled during protein hydrolysis. DH demonstrates the four processing variable including substrate, enzyme-substrate ratio, temperature and time (kristinsson and Rasco, 2000). These are essential because several properties of protein hydrolysate are closely related to DH. Hydrolysis of peptides bonds causes several changes such as reduced molecular weight, an increase of amino and carboxyl groups, which increase solubility and destruction of tertiary structure (Nielsen,1997). There are several ways on how DH is measured which is pH-stat, osmometry and the trinitro- benzene-sulfonic acid (TNBS) method.
The pH-stat technique monitors the degree of hydrolysis by adding a base or acid depending on the pH of hydrolysis to keep the pH constant during hydrolysis. The amount of base used is proportional to the degree of hydrolysis. In practical protein hydrolysis experiments, the pH-stat technique is limited to pH conditions higher than around 7 (Adler-Nissen 1986). When hydrolyzing to obtain a high DH above 30%, it is not economically feasible to carry out hydrolysis when using pH-stat (constant pH . 7) as a single enzyme system working efficiently at pH . 7 is not readily available. To obtain a very high DH, a combination of different enzymes is needed. This will include enzymes with highest activity at pH lower than 7, which is out of the range of pH-stat control. Furthermore, the addition of a base during hydrolysis may be undesirable depending on the use of the end product. During a hydrolysis reaction, the alteration of the mixture's freezing point depression can be measured by an osmometer (cryoscope). This can be correlated to DH (Adler- Nissen 1984).
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The TNBS method is the reaction of primary amino groups with trinitro-benzene-sulfonic acid (TNBS) reagent(Adler-Nissen 1979). This method does have its drawbacks because it is laborious, and it is not possible to obtain results quickly enough during hydrolysis to follow the process closely. Other than that, the TNBS reagent is unstable, toxic, and has to be handled carefully due to the risk of explosion. So there is a need for an alternative method without these drawbacks.
2.4.4 OPA Method
The OPA method is a reaction between amino groups and o-phthaldialdehyde (OPA) in the presence of beta-mercaptoethanol forming a colored compound detectable at 340 nm in a spectrophotometer or fluorometrically at 455 nm (Nielsen et al., 2001).
Functional properties and composition of fish protein hydrolysate
Enzymatic hydrolysis of fish protein generates a mixture of free amino acids and varying size of peptides (Santos et al.,2011). This process increases the number of polar group with the coincidental increase in solubility of the hydrolysate (Adler-Nissen, 1986; Kristinsson & Rasco, 2000).Therefore, functional characteristic of protein can be modified. The functional properties of fish protein hydrolysate are important, especially if there are used as ingredients in a food products. The main functional properties of the fish protein hydrolysate are include solubility, water holding capacity, emulsifying, foaming and sensory properties ( Kristinsson and Rasco,2000).
Solubility is the most important characteristic for protein hydrolysate functional properties. Hydrolysate has an excellent solubility at high degree of hydrolysis (Gbogouri et al., 2004). Many of other functional properties, such as emulsification and foaming, are affected by solubility (Gbogouri et al.,2004; Kristinsson & Rasco, 2000). Enzymatic hydrolysis is important in increasing the solubility of these proteins. Both protein hydrolysate produced from enzyme alcalase and flavourzyme with high degree of hydrolysis , had higher solubilities. This lends further support to the findings of Gbogouri et al. (2004) who reported that hydrolysates had an excellent solubility at high degrees of hydrolysis.High solubility of fish protein hydrolysate over a wide range of pH is a substantially useful characteristic for many food applications. In general, the degradation of proteins to smaller peptides leads to more soluble products (Gbogouri et al., 2004 ). The balance of hydrophilic and hydrophobic forces of peptides is another crucial influence on solubility increments (Gbogouri et al., 2004). Enzymatic hydrolysis potentially affects the molecular size and hydrophobicity, as well as polar and ionizable groups of protein hydrolysates. The smaller peptides from myofibrillar proteins are expected to have proportionally more polar residues, with the ability to form hydrogen bonds with water and increase the solubility (Gbogouri et al., 2004). As a summary, hydrolysates with smaller peptides and higher degree of hydrolysis were more soluble.
Water Holding capacity
Fish protein hydrolysates are highly hygroscopic. Hygroscopic refers to the ability of the protein to absorb water and retain it against gravitational force within a protein matrix, such as protein gels or beef and fish muscle.The presence of polar groups such as carboxylic (COOH) and ammonia (NH) that increase during enzymatic hydrolysis has a substantial effect on the amount of adsorbed water and moisture sorption isothermal for these materials. Recent studies have shown that FPH can contribute to increased water holding capacity in food formulations (Wasswa et al., 2008); and addition of FPH from salmon reduced water loss after freezing (Kristinson and Rasco 2000).
Protein are often used as surfactants in emulsion-type processed foods Slizyte et al.,2005). It have interfacial properties,which are important for their application as for example emulsifiers in sausages or protein concentrates in dressings. Hydrolysates are also water-soluble and surface active and promote oil-in-water emulsion, due to their hydrophilic and hydrophobic functional groups with their associated charges (Gbogouri et al., 2004;Rahali, Chobert, Haertle, & Gueguen, 2000). Emulsifying properties of hydrolyzed protein can also be improved by controlling the extent of hydrolysis. There is a relationship between percent degree of hydolysis and emulsifying properties of fish hydrolysates. Extensive hydrolysis generally results in a drastic loss of emulsifying properties (Gbogouri et al.,2004; Kristinsson & Rasco, 2000). With a limited degree of hydrolysis, the hydrolysates have exceptional emulsifying activity and stability (Kristinsson & Rasco, 2000). According to Klompong et al.,2005 Emulsifying properties for both HA and HF decreased with increasing DH in which at low DH hydrolysates exhibited strong emulsifying properties. The molecular weights of the hydrolysate are also influence on emulsifying properties. Suggested that peptide should have a minimum chain length of >20 residues to function as good emulsifiers. Higher contents of larger molecular weight peptides or more hydrophobic peptides contribute to the stability of the emulsion. Thus, hydrolysates with a higher DH had poorer emulsifier properties due to their small peptide size. Small peptides migrate rapidly and adsorb at the interface, but show less efficiency in decreasing the interface tension since they cannot unfold and reorient at the interface like large peptides to stabilize emulsions (Gbogouri et al., 2004; Rahali et al., 2000). Apart from peptide size, amphiphilicity of peptides is important for interfacial and emulsifying properties. Rahali et al. (2000) analyzed amino acid sequence at an oil/water interface and concluded that amphiphilic character was more important than peptide length for emulsion properties. The higher emulsion properties of hydrolysates accompanied their higher solubility (Klompong et al.,2005). Hydrolysates with high solubility can rapidly diffuse and adsorb at the interface. At the same will give better emulsifier properties. Emulsifying properties were influenced by specificity of enzyme (Gauthier, Paquin, Pouliot, &Turgeon, 1993).
184.108.40.206 Foaming properties
The amphiphilic natures of proteins make a foaming formation possible. A protein may have an excellent foamability but it may not necessarily produce stable foam. Foam formation is influenced by three factors, including transportation, penetration and reorganization of molecules at the air-water interface (Klompong et al.,2005). To exhibit good foaming, a protein must be capable of migrating rapidly to the air-water interface; unfolding and rearranging at the interface suggested that the foaming capacity of protein was improved by making it more flexible, by exposing more hydrophobic residues and by increasing capacity to decrease surface tension. The digestion of the protein produces a range of peptides which possess the altered hydrophobicity, charge balance conformation, compared to the native molecule. Protein hydrolysate with reduced molecular weight is flexible in forming a stable interfacial layer and increasing the rate of diffusion to the interface, leading to the improved foamability properties. High molecular weight peptides are generally positively related to foam stability of protein hydrolysates (Van et al., 2002). For the adsorption at the air- water interface, molecules should contain hydrophobic regions. Protein solubility makes an important contribution to the foaming behaviour of protein. The ability and the stability foam of FPH were increased when hydrolysate concentrations increased from 0.1% to 3% (Muzaifa et al.2011). From these finding it revealed that protein hydrolysates from fish waste have a good foam ability and stability. This result is in agreement with Thiansilakul et al (2007) who studied the foam ability protein hydrolysates from round scad (Decapterus maruadsi).
Absorb and hold ability is also important function properties of fish protein hydrolysate(Slizyte et al., 2005). The mechanism of fat absorption is attributed mostly to physical entrapment of the oil. The higher bulk density of the protein, the higher fat absorption is obtained. Fat binding capacity of protein also link with surface hydrophobicity, degree hydrolysis and enzyme or substrate specificity (Kristinsson and Rasco,2000).
Taste and odour are the major sensory properties of fish protein hydrolysates. It must be acceptable to the targeted consumers if the products with the bioactive peptides are to be successful on the market. The sensory properties depend both on the raw material, the kind of protease applied and the hydrolytic conditions. High quality fish sauce has a delicious flavour (Thongthai and Gildberg,2005), but bitter taste is a problem with many fish protein hydrolysates (Kristinsson and Rasco,2002). The problem may be solved either by mild hydrolysis to reduce production of medium size peptides or by extensive hydrolysis to digest the troublesome peptides to free amino acids. But mild hydrolysis normally reduces the yield significantly. Extensive digestion is probably more practical. Although enzymatic hydrolysis of protein develops desirable functional properties, it results in the formation of short chain peptides, thus causing the development of bitter taste in the product. The bitterness strongly restricts the practical uses of these hydrolysate as a food ingredient. It is widely accepted that hydrophobic amino acids of peptides are major factor. Peptides with a molecular weight ranging from 1000 to 6000 Da and with hydrophobic characteristics have been shown most likely to be bitter taste. An extensive hydrolysis to free amino acids is able to decrease the bitterness of these peptides because hydrophobic are far bitter compared with a mixture of free amino acids (Kristinsson and Rasco, 2000). However, free amino acids are undesirable from a functional standpoint. Strict control of hydrolysis and termination at low %DH values therefore is desirable to prevent the development of a bitter taste and to maintain the functional properties. The presence of bile in the raw material may also influence the development of bitterness in fish protein hydrolysate (Dauksas et al.,2004).
2.5.2 Chemical composition of fish protein hydolysate
According to Muzaifa et al (2011), there were no big differences in moisture and fat content of fish protein hydrolysate prepared using Alcalase and Flavourzyme ,while there were differences on protein and ash contain. They were also found that FPH using Alcalase enzyme had higher protein content than FPH using Flavourzyme. Protein content of this FPH is still high and could be an essential source of proteins.The lipid content in FPH was greatly reduced when compared to the raw material, because lipids are usually removed along with the insoluble protein fraction by centrifugal separation (Kristinsson and Rasco 2000b; Nilsang et al. 2005; Ovissipour et al. 2009). Decreasing of lipids content in the protein hydrolysate contributes significantly to the stability of the material towards lipid oxidation (Diniz and Martin 1997; Kristinsson and Rasco 2000b; Nilsang et al. 2005; Shahidi et al. 1995). The lipid fraction is used separately as a feed ingredient or in other commercial applications. The protein recovery ranged from 45.2 to 73.7%. Kristinsson and Rasco (2000a) reported 40.6 to 79.9% nitrogen recovery, corresponding to a hydrolysate with 5 and 10% DH, respectively. Many researchers found that soluble protein recovery would increase by using a longer hydrolysis time and an elevated reaction temperature (Kristinsson and Rasco 2000a, b; Ovissipour et al. 2009).
2.5.3 Molecular Weight Distribution
The average molecular weight of protein hydrolysates is one of the most important factors, which determines their functional properties (Ranathunga et al 2005). An ultrafiltration membrane system could be a useful method for obtaining peptide fractions with a desired molecular size and enhanced biological activity (Gómez-Guillén et al., 2010). This system has been successfully applied in the fractionation and functional characterisation of squid skin gelatin hydrolysates (Lin & Li, 2006).
Amino acid composition
Fish muscle contains nutritive and easily digestible protein with an excellent amino acid composition. Fish protein hydrolysates produce high protein content ranging from 62 to 90% (Slizyte et al.,2005), which depends on the substrate and the preparation.
The biological activity of a peptide is widely recognized to be based on the amino acid composition. The amino acid composition is important in protein hydrolysates because of the nutritional value known as essential amino acids and also has an influence on the functional properties. Hydrolysates that have an intermediate chain length and limited amounts of free amino acids would be valuable as ingredients in formulated and nutritionally balanced fish diets (Pigott and Tucker 2002).
The searobin enzymatically hydrolysate digest obtained with the enzyme Alcalase had a higher amino acid content than the one obtained from Flavourzyme (Sarita et al.,2011). However, both showed essential amino acid amounts consistent with those found in the literature (Abdul-Hamid et al. 2002).The present of amino acid is also creating bitterness taste due to the exposure of interior hydrophobic amino acid side chains. The amino acids that create a bitter taste are valine, isoleucine, phenylalanine, tryptophan, leucine, and tyrosine (Pedersen, 1994). The endo and exopeptidase enzymes mixtures such as KojizymeTM and FlavourzymeTM can minimize the bitterness in the hydrolysed product (Liaset et al., 2000).
Mineral content in shellfish
Shellfish as well as mud clam are rich in essential mineral contents such as Iron, Zinc and Copper. These minerals are needed in the human body. Iron is an essential mineral in the heme molecule of hemoglobin, the component of the red blood cell that carries oxygen in the bloodstream. Therefore, people who do not eat enough iron can suffer from iron-deficiency anemia. Worldwide, about 1 billion people have iron-deficiency anemia, and about 2 billion people are deficient in zinc (Muller et al., 2005). Clams in particular have enough iron in 100 grams to meet about 78% of the Dietary Reference Intake for 19-50-year-old, non-pregnant females and exceed that for adult men starting at age 19 and for women who are 51 years old and older (Food and Nutrition Board, 2004). Zinc is also necessary for a healthy diet. From Wardlaw and Smith,2009 research ,this mineral helps with immune function and is essential for healing of wounds, development of sexual organs and bones, immune function, and cell membrane structure and function. Copper is also an essential mineral in the diet, because it helps to form hemoglobin and collagen (a ubiquitous protein in the body). It is also a part of several enzyme systems, including those that prevent oxidative damage to cell membranes (Dong,2009)
Cholesterol and Caloric content
Shellfish in general are low in caloric and cholesterol values. Cholesterol is essential in the body because it is used to make important compounds such as bile acids (which help to digest fat in the intestine), sex hormones and vitamin D. Cells in the body make about two-thirds of the cholesterol in the body, while the other third comes from foods eaten (Byrd-Bredbenner et al., 2009). However, non-cholesterol sterols (also known as plant sterols or plant stanols) are found in herbivorous mollusks, such as clams and scallops . These non-cholesterol compounds are absorbed from the intestine and can actually decrease the absorption of cholesterol (Byrd-Bredbenner et al., 2009) and therefore have a positive effect on health. According to Dong,2001 shellfish have cholesterol concentrations of less than 80 milligrams per 100 grams (edible portion) and therefore can be consumed by people trying to limit their dietary cholesterol intake.
Antioxidant activities of fish protein hydrolysate
Hydrolysis of protein contains free amino acids and peptides, which have been found to exhibit antioxidative activity. Fish protein hydrolysate have been also been recognized to act as natural antioxidant against lipid oxidation in food model system.Generally, the quenching of free radicals by natural antioxidants has been reported as taking place through hydrogen donation. Certain peptides are electron donors and can react with free radicals to terminate the radical chain reaction (Park et al., 2001). Even though the exact mechanism by which peptides act as antioxidants is not clearly known, some aromatic amino acids and His are reported to play a vital role in this. Mendis et al., (2005) reported on the substantial presence of hydrophobic amino acids in gelatin peptide sequences for observed antioxidant activities. Besides amino acid composition and specific peptide sequences, functional properties of antioxidant peptides are also highly influenced by molecular structure and. Moreover, peptide conformation can lead to both synergistic and antagonistic effects in comparison with the antioxidant activity of amino acids alone (Hernández et al., 2005).
Sensory evaluation is defined as scientific discipline used to evoke, measure, analyze and interpret reactions to those characteristics of foods and materials as they are perceived by the sense of sight, smell, taste, touch and hearing (Stone and Sidel 1993). Due to the essential role of sensory activities in product development, product cost reduction, quality control and quality assurance, more and more foods are using scientific sensory techniques to evaluate food quality. Descriptive sensory tests are the most sophisticated tools in the arsenal of sensory scientists (Lawless & Heymann 1998) and involve the detection (discrimination) and the description of both the qualitative and quantitative sensory aspects using trained panels of from 5 to 100 judges (Meilgaard, Civille & Carr 1999). Panelists must be able to describe the perceived sensory attributes of a sample which include the appearance, aroma, flavor, texture and sound properties. In addition, panelist must learn to differentiate samples quantitatively. There are several different methods that can be used in the descriptive sensory analysis, including the 12 Flavor Profile Method, Texture Profile Method, Quantitative Descriptive AnalysisTM , the SpectrumTM method, Quantitative Flavor Profiling, Free-Choice Profiling and generic descriptive analysis (Murray, Delahunty & Baxter 2001). The panelists are screened from the potential candidates based on their ability to describe the characteristics using verbal descriptors and to detect differences in sensory characters and intensities. All descriptive sensory tests require panel training. For some descriptive sensory procedures, an evaluation test is required to evaluate the success of the training. Descriptive analysis is using as the most comprehensive, flexible and useful sensory method and provides detailed information on all of a products' sensory properties. In the next millennium, it is expected that it will be used increasingly for a wider range of uses (Murray, Delahunty & Baxter 2001)
Material and Method
3.1 Raw materials
Lokan (Poymesoda erosa) will be bought from local market and immediately placed in ice and transported to the laboratory. Upon arrival, the flesh of Polmesoda erosa will be separated manually, washed and then minced using a blender. The minced of squid will be stored in -20 â°C until will be used.
The enzymatic process will be performed with Alcalase and Flavourzyme. Alcalase is a bacterial endopeptidase produced by Bacillus licheniformis. Alcalase 2.4 L with a declared activity of 2.4 Anson Units (AU) gâˆ’1 has optimum enzymatic activity between 50 and 70Â°C, and at pH values between 6 and 10 ( Guerard et al.,2007). Flavourzyme is a fungal protease/peptidase complex produced by submerged fermentation of a selected strain of Aspergillus oryzae. Optimum working conditions reported for Flavourzyme 500 L (with declared activity of 500 LAminopeptidase Units (APU) gâˆ’1) include pH 5 to 7, with an optimum temperature around 50Â°C (Slizyte et al., 2005).
3.2 Preparation of Lokan (Polymesoda erosa) Protein Hydrolysate
Protein hydrolysate will be prepared according to the method by Adler-Nissen (1986). The flesh of Lokan will be cut into small pieces and will be suspended in 120 ml of distilled water and then will be blended. The mixture will be incubated in a circulated water bath at 60â°C. The pH of the mixture will be adjusted to pH 8.5 and maintained constant during hydrolysis using 1.0 M NaOH. Once the pH and temperature has stabilized, Alcalase at enzyme-substrate ratio at 3% will be added and the reactions continue for 2 hours. The enzymatic reaction will be terminated by placing the samples in a water bath at 90 â°C for 15 min with occasional agitation. This is will be followed by centrifugation at 14000g for 10 min. Supernatants obtained will be freeze-dried at -20oC using the SANYO-Biomedical freeze dryer (ALPHA 1-4 (MARTIN CHRIST)). This procedure will be repeated using flavourzyme with condition at 55Â°C and pH 7.
3.3 Degree of Hydrolysis
Degree of hydrolysis (DH) is defined as the percentage ratio between the number of peptide bonds cleaved (h) and the total number of peptide bonds in the substrate studied (htot). The degree of hydrolysis will be determined based on the consumption of base necessary for controlling the mixtureâ€Ÿs pH during the batch assay as in the equation below (Adler-Nissen, 1986).
DH % = Î² x NÎ² x 100
Î± x MP x htot
Î² = Volume of 1.0M NaOH
NÎ² = Molarity of NaOH
Î± = Average degree of dissociation of the NH3 groups
MP = Mass of protein in g
htot = Total number of peptide bonds in the protein substrate (mmol/g protein)
The yield will be determined by the ratio of the mass of hydrolysate and the total weight of the raw Lokan (Polymesoda erosa) meat. The yield obtained will be calculated as follow:
Yield (%) : Weight of powdered hydrolysate x 100
Wet weight of fresh squid
3.5 Chemical Composition
Chemical composition of the Lokan (Polymesoda erosa) and the hydrolysate will be determined following to (AOAC, 2005). The moisture content will be determined by placing approximately 2 g of sample into a preweighted aluminum dish. Lokan (Polymesoda erosa) and hydrolysate will be dried in an oven at 105 Â°C until constant weight will be obtained. The total crude protein (N Ã- 6.25) in Lokan (Polymesoda erosa) and freeze-dried hydrolysates will be determined using the Kjeldahl method (AOAC, 2005). Total lipid in Lokan (Polymesoda erosa) squid and hydrolysate will be determined by Soxhlet extraction (AOAC, 2005 ). Ash content will be determined by charring a pre-dried sample in a crucible at 600 Â°C until constant weight of white ash will be obtained (AOAC, 2005).
3.6 Amino acid composition
Lokan (Polymesoda erosa) and hydrolysate will be hydrolyze with 6N HCl at 110 Â°C for 24 h. Derivatisation has been conducted using ophthaldialdehyde (OPA) prior to HPLC analysis. Breeze 2 HPLC System software will be used to measure amino acid composition while the instrument will be used to analyse is Water 1525 Binary HPLC Pump.
3.7 Fatty acid composition
Fatty acid composition of Lokan (Polymesoda erosa) and hydrolysate will be determined according to ( Lian et al.,2003).Lipids will be extracted for the proximate analysis. The chloroform layer with 1 mg of lipid will be accurately transferred into a test tube, dried under a nitrogen stream in a 40 Â°C water bath, and then mixed with 1 mL of methylene chloride (MeCl2) with 50 Âµg of C21 as internal standard. Transmethylation will be conducted by adding 3 mL of 5% HCl / MeOH, sealing under nitrogen, and placing in aa 70 Â°C oven for 120 min. After cooling, 4 mL of 6% K2CO3 was added and vortexstirred. The MeCl2 layer will be dried under N2 in a 40 Â°C water bath, redissolved in 1 mL of MeCl2, and filtered through a 2.5 Âµm filter into an amber GC vial. The vial was capped under N2 for the analysis of FAMEs, which was performed with a Perkin-Elmer GC equipped with a flame ionization detector. The methyl esters will be separated in a DB-Wax column (30 m x 0.25 mm i.d. x 0.25 ím film thickness) (J&W Scientific, Folsom, CA) under the following operation conditions: injection, 2.0 íL; injector temperature, 250 Â°C; detector temperature, 300 Â°C; flow rate of carrier gas He, 20 mL/min; oven temperature, 50 Â°C, held for 2 min following injection; ramp, 40 Â°C/ min to 200 Â°C, held for 16 min, 210 Â°C, held for 11 min, and 220 Â°C,held for 10 min. The relative content of each fatty acid methyl ester will be reported as percent peak area of total fatty acid methyl esters using the FAME quantitative standard mix and C21 internal standard (Accu- Standard, New Haven, CT).
3.8 Mineral Contents
The minerals will be analyzed according to (Chompoonuch et al.,2006). Mineral contents, namely calcium (Ca), phosphorus (P), iron (Fe), sodium (Na), magnesium(Mg), and potassium (K), of Lokan (Polymesoda erosa) and hydrolysate will be determined using inductively coupled plasma emission spectrometry (ICP-OES, Model Optima 4300 DV, Perkin-Elmer, USA). Lokan (Polymesoda erosa) and hydrolysate (0.5 g) will digested in 9 ml of concentrated nitric acid and 3 ml of hydrofluoric acid using microwave heating at 180 Â± 5 Î¿C for 15 min. Wavelengths for the determination of Ca, Mg, Na, P, K and Fe were 317.9, 285.2, 588.9, 213.6, 766.4 and 248.3 nm,respectively.
3.9 Molecular weight distribution
The molecular weight of Lokan (Polymesoda erosa) and hydrolysate will be analysed by Trycine-SDS-PAGE according to (Giménez et al.,2009), using a 5% stacking gel, a 10% separating gel and a 16.5% resolving gel.The dry hydrolysates will be dissolved (20 mg/ml) in the loading buffer (50 mM tris-HCl, 4% SDS, 12% glycerol, 2% mercaptoethanol and 0.01% bromophenol blue), heat-denatured at 40Î¿C for 30 min and will be run in a Mini Protean II unit at 25 mA/gel. The loading volume is 15 ll in all lanes. Protein bands were stained with Coomassie brilliant Blue R250. The approximate molecular weight of the hydrolysates will be determined using a low molecular weight-SDS market kit consisted of triosephosphate isomerase (26.6 kDa), myoglobin (16.9 kDa), a-lactalbumin (14.4 kDa), aprotinin (6.5 kDa), insulin b chain oxidised (3.5 kDa) and bacitracin (1.4 kDa).
4.0 Cholesterol analysis
Cholesterol of Lokan (Polymesoda erosa) and hydrolysate will be determined according (Magdalena et al.,2008) using gas chromatography. Cholesterol will be extracted from sample of 0.25g of freeze-dried with 15ml of chloroform. After filtration, the solution will be supplemented with chloroform in the measurement container to a volume of 25ml. One ml of acetic anhydride and 0.25ml of sulphuric acid (vi) will be added to 2ml of the filtered obtained. After 5 min the absorption value will be measured in a blind test at a wavelength of 620nm. The result will be presented as mg 100 g-1 of wet weight.
4.1 Caloric Content
Caloric content of Lokan (Polymesoda erosa) and hydrolysate will be determined according to (David et al.,2010). Dried Lokan (Polymesoda erosa) and hydolysate will be blended to a homogeneous mixture using a standard coffee grinder and then will be redried to a constant mass (60.01 g for two consecutive days) to remove any moisture that had accumulated during homogenization. At least two 0.1-0.2-g pellets were formed and ignited in a semi-micro bomb calorimeter ( C5000,IKA WERKE,GB/USA) to measure caloric content. Tests will be performed in triplicate.
4.2 Determination of Functional Properties
4.2.1 Solubility (S)
The solubility of the hydrolysate will be determined according to Morr et al.(1985) with a pH variation in the range of 3 to 11. Two milliliter of a 0.1-M NaCl solution will be added to 500 mg of hydrolysate, forming a homogenous paste. A buffer solution will be added to the solution with the corresponding pH up to a volume of 40 mL. The protein dispersion will be kept under stirring for 45 min. The dispersion will be transferred to a 50-mL volumetric glass, the volume being completed with the buffer solution. The protein dispersion will be centrifuged at 6,000Ã-g for 30 min by using Kubota 5420. Aliquots will be taken from the supernatant in order to identify the soluble protein content, by Kjedaht (AOAC,2005). The solubility rate will be determined according to following equation
S = A x 50 x 100
W x P/100
where A is the protein concentration in the supernatant (mg / mL), W is the weight of the samples (mg), and P is the percentage of protein in the sample.
4.2.2 Water and Oil Holding Capacity
The capacity of hydrolysate to hold water and oil will be determined according to the methodology described by El Khalifa et al. (2005). To determine the water holding capacity (WHC), 1 g hydrolysate will be placed in previously weighted centrifuge tubes, and 14 mL of water will be added. For the oil holding capacity (OHC), 14 mL of corn oil will be added. Both samples will be stirred in a tube stirrer and kept at rest for 30 min at room temperature before being centrifuged at 5,000Ã-g for 25 min. The excess of water or oil will be removed by tube inversion over tissue paper. The difference between the sample's weight before and after water or oil absorption will be taken as the amount of water or oil absorbed. WHC or OHC were expressed as the percentage of water or oil absorbed by gram of sample.
Amount of water or oil absorbed = sample weight before -sample weight after absorption
WHC or OHC (%) = amount of water or oil absorbed x 100
Weight of sample
4.2.3 Emulsifying properties
Emulsification capacity of hydrolysate will be measured by mixing 5 ml of rapeseed oil with 5 ml of a 1% hydrolysate solution in water and homogenising (Ultra-Turrax T 25 D) at 20,000 rpm for 90 s. The emulsion will be centrifuged at 2400 x g for 3 min . The volume of each fraction (oil, emulsion and water) was determined and emulsification capacity will be expressed as millilitres of emulsified oil per 1 g of FPH. Emulsion stability will be expressed as percentage of initial emulsion remaining after a certain time it will be determined at 0, 10, 20, 30, 60, 90 minutes and centrifugation at 2400 x g for 3 min. Tests will be performed in triplicate.
4.2.4 Foam stability
Foam stability of hydrolysate will be measured using the Rudin method (Wilde and Clark 1996) with a slight modification. Briefly, a 40-mL mixture of distilled water and squid batter, with a final protein content of 3%, will be homogenized with a flat impeller (almost parallel to the bottom) at high speed for 1 min. The resulting foamy liquid will be poured into a graduated cylinder and the foam and liquid volumes were recorded.. Foaming stability will be expressed as the volume of foam remaining after allowing the sample to rest at room temperature for 60 min. Tests will be performed in triplicate.
4.3 Determination of sensory evaluation
Twelve panellist will be trained prior to the actual evaluation session (Normah and anida 2004). Freshly prepared sample will be used as a reference odor. Training involved the familiarization of five point descriptive fishy odor, fishy flavour and bitter flavour scales ( 1=absent, 2= very slight, 3= moderate,4=strong ,5 = very strong ). During the sensory evaluation session, panellist will be served with the samples that are kept in scaled plastic bag. For the fishy odor, they will be asked to open one plastic bag at a time and make three depp sniff of the content and record the scale. For the fishy and bitter flavour, they will be asked to taste of the content and record the scale. The sensory evaluation session will be repeated three time.
4.4 Colour measurement
The colour of powdered and liquid hydrolysate will be measured by chromameter CR400 (Konica Minalto). L*, a* and b* parameters indicate lightness, redness and yellowness, respectively. Measurement will be performed in triplicate.
4..5 Determination of antioxidative activities of Lokan (Polymesoda erosa) protein hydrolysate
1) DPPH radical scavenging activities
The scavenging effect of the hydrolysates on a,adiphenyl- b-picrylhydrazyl (DPPH) free radical will be measured as described by Wu et al.( 2003). Diluted hydrolysate (1.5 ml) will be added to 1.5 ml of 0.1 mM DPPH in 95% ethanol. The mixture will be shaken and left for 30 min at room temperature, and the absorbance of the resulting solution will be measured at 517 nm. A lower absorbance represented a higher DPPH scavenging activity. The scavenging effect is expressed as [(Blank absorbance -Sample absorbance)/Blank absorbance] x 100%. Tests will be performed in triplicate.
2) Reducing Power
The reducing power of the hydrolysates will be measured according to the method of Wu et al.( 2003). Diluted hydrolysate (2 ml) will be added to 2 ml of 0.2 M phosphate buffer (pH 6.6) and 2 ml of 1% potassium ferricyanide. The mixture will be incubated at 50 Î¿C for 20 min. Then 2 ml of 10% TCA will be added to the reaction mixture. A volume of 2 ml from each incubated mixture will be mixed with 2 ml of distilled water and 0.4 ml of 0.1% ferric chloride in test tube. After a 10 min reaction, the absorbance of the resulting solution will be measured at 700 nm.Increased absorbance of the reaction mixture indicated increased reducing power. Tests will be performed in triplicate.
3) Oxidative stability during storage
Freeze-dried hyrolysate prepared (2.5g) will be kept in the amber vial and will be closed tightly with screw-cap. After storage at 4 and 25 c for 0,1,2,4 and 6 weeks, the sample will be taken for analyses. Tests will be performed in triplicate.
4.6 Statistical analysis
The data obtained will be analyze using the Analysis of variance ( ANOVA) to determine significance at 5% level. Duncan Multiple Range Test ( DMRT) will be used to identify differences between means. The statistical program wili be use was Statistical Analysis System (SAS,1985).