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Lokan in english name is mud clam. It 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 consumption it increasing day by day. In peninsular Malaysia, this species are normally found in Pantai Remis,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.
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 'polimesoda expansa' protein will be perform 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 produced the protein hyydolysate 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 'Caorbiculidae' are reported from indo pacific region (Ingole et al.,2002). There are Polymesoda erosa, P.bengalensis and P. 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 white colour gray, yellowish, blackish stripes depends on the habitat in which they live. Clam size between 4 to 15 cm which is shaped like a seashell shell but has smoother surface and no ribs-clack as shellfish. 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 manmad 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 defined 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.
2.3 The Production of protein hydrolysate
There are two method most widely used for protein hydrolysis which is 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. Proteolytic enzymes from microorganisms and plants are most suitable for preparing fish protein hydrolysates. Alcalase, an alkaline enzyme produced from Bacillus licheniformis, and papain from a plant source Carica papaya have been found to be the best enzymes for the preparation of functional fish protein hydrolysates (FPH) by many researchers (Kristinsson and Rasco,2000).
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).
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 and optimal temperature around 50â°C. Flavourzyme 500 L has a declared activity of 500 L APU/g. (Slizyte et al., 2005).
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 DH 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 DH. 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).
Osmometry is a fast method that can be used for many reactions. Its limitations are that it cannot be used in highly viscous solutions or solutions with a high concentration of solutes, such as salt, used as preservatives during long reactions. The content of nonprotein compounds in the substrate, which are hydrolyzed by other activities (for example, amylase) in the protease preparation, can also make it impossible to correlate osmometer readings with DH values of the protein.
The TNBS method is based on the reaction of primary amino groups with trinitro-benzene-sulfonic acid (TNBS) reagent(Adler-Nissen 1979). However, the method does have its drawbacks. It is laborious, and it is not possible to obtain results quickly enough during hydrolysis to follow the process closely. In addition, 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
To provide a basis for developing a suitable method, a reaction was selected 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 , particularly 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 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 very important in increasing the solubility of these proteins. High solubility of fish protein hydrolysate over a wide range of pH is a substantially useful characteristic for many food applications.
Water Holding capacity
Water holding capacity refers to the abilty 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. Fish protein hydrolysates are highly hygroscopic. The presence of polar groups such as COOH and NH that increase during enzymatic hydrolysis has a substantial effect on the amount of adsorbed water and moisture sorption isothermal for these materials. Some 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). Hydrolysate are also water-soluble and surface active and promote oil-in-water emulsion, due to their hydrophilic and hydrophobic functional groups. Emulsifying properties of hydrolyzed protein can also be improved by controlling the extent of hydrolysis. There is a relationship between %DH and emulsifying properties of fish hydrolysates. Extensive hydrolysis generally result in a drastic loss of 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.Proteins have interfacial properties,which are important for their application as for example emulsifiers in sausages or protein concentrates in dressings
220.127.116.11 Foaming properties
The amphiphilic nature of proteins make a foaming formation possible. A protein may have an excellent foamability but it may not necessarily produce a stable foam. Foaming abilty of protein hydrolysate is governed by the size of peptide. Fish protein hydrolysate from with its reduction in molecular weight presented an improved foamability. 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.
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(Kristinsson and Rasco,2000) and enzyme /substrate specificity. Moreover, hyrolysate powders containing higher amounts of lipids had higher fat absorption ability while a positive relationship between fat absorption and amount of phospholipids was observed in the hydrolysate samples. Lipid residues retained in dried protein hydrolysate after hydrolysis must be lower than 0.5% to reduce development of rancid taste during storage (Slizyte et al 2005).
18.104.22.168 Sensory Properties
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. The mechanism of bitterness is not very that the presence of bile in the raw material may also influence the development of bitterness in fish protein hydrolysate (Dauksas et al.,2004). 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. Hydrolysis of protein results in exposing buried hydrophobic peptides, which are readily able to interact with the taste buds, resulting in detection of 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 funtioanl 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.
2.5.2 Chemical composition and nutritive value
As we know, fish muscle contains the 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. Infrared spectroscopy (FTIR) provides information about the chemical composition and conformational structure of food components. It has also been used to study changes in the secondary structure of fish collagen and gelatin (Muyonga et al., 2004). Its application to the characterization of protein hydrolysates is very limited, nevertheless, FTIR spectra of whey and casein hydrolysates have been found to correlate to various functional properties such as emulsion and foam forming capacities (Van der Ven et al., 2002).
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. An ultrafiltration membrane system could be a useful method for obtaining peptide fractions with a desired molecular size and enhanced biological activity . This system has been successfully applied in the fractionation and functional characterisation of squid skin gelatin hydrolysates (Lin & Li, 2006); and also as a first step in the isolation and further purification of antioxidant peptides from similar sources (Kim et al., 2001; Mendis et al., 2005).
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).
Amino acid composition
The biological activity of a peptide is widely recognized to be based on the amino acid composition. The amino acid composition is important in proteinhydrolysates
because of the nutritional value (essential amino acids) and also has an influence on the functional properties.According to Amino acid profiles of squid hydrolysate (based on 76% moisture containing hydrolysate ) are Asp, Glu, Ser,Gly, His, Arg, Thr, Arg, Ala, Pro, Tyr, Val,Met,Cys,Ile,Leu,Phe,Lys.In previous work, tuna and giant squid skin gelatins have been shown to have very different physicochemical properties, mainly based on differences in amino acid composition (higher Hyp and Hyl content in squid) and molecular weight distribution (absence of cross-linked Î±-chains in squid).Amount of free amino acids was determined by high pressure liquid chromatography (HPLC).
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).In an enzymatic hydrolysis, the capacity of the proteaseto cut peptide bonds is dependent on physical interactionsbetween the substrate (raw material) and the enzyme(protease) in the aqueous environment present during hydrolysis. As a greater portion of the hydrophobic amino acids will reside within hydrophobic regions of the peptide chain in the raw materials, it is likely that the access for the protease to these hydrophobic region might be limited (Chothia 1974).
Emulsifying capacity of Lokan and hydrolysate will be determined by method described in Swift et al. (1961), in Hill (1996). Briefly, Lokan and hyrolysate will be mixed with distilled water to a final protein concentration of 0.3%, to 50 mL of protein suspension, 9 mL sunflower seed oil will be added. The mixture will be homogenized for 30 s at high speed at room temperature with a propeller impeller and, immediately afterwards, will be centrifuged at 2,000 x g at 20C for 5 min. The volume of the emulsion will be measured for each sample.Emulsifying capacity will be calculated as the ratio of the volume from the emulsion
formed and the initial mixture.
Emulsion stability of each Lokan and Hydrolysate will be determined by heating the emulsion to 80Î¿C for 30 min. The emulsion volume will be measured after heating. The emulsion stability is expressed as a percentage of the remaining emulsifying capacity after the heating period.
Foam capacity and foam stability of Lokan and hydrolysates will be tested according to the method of Shahidi, Xiao-Quing, and Synowiecki (1995). Fifty millilitres of protein hydrolysates solution (0.1%, 0.5% and 2%) will be homogenised in a 100 ml cylinder at a
speed of 16,000 rpm with an Ultraturrax T25 (Janke and Kunkel IKA-Labortechnick, Staufen, Germany) to incorporate the air for 1 min. The total volume will be measured at 0, 0.5, 1, 5 10, 20 and 30 min after whipping. Foam capacity will be expressed as foam expansion at 0 min, which will be calculated according the following equation (Shate & Salunkhe, 1981):
Foam expansion ( % ) = [ (A - B) / B ] x 100
Where A is the volume after whipping (ml) and B is the volume before whipping (ml).
Foam stability will be calculated as follows:
Foam stability ( % ) = [ (A - B) / B ] x 100
Where A is the volume at 0.5, 1, 5 10, 20 and 30 min after whipping(ml) and B is the volume before whipping (ml).
Total nitrogen recovery
In order to determine the nitrogen recovery, protein in the soluble fish hydrolysate will be measured by the Biuret method (Layne 1957), using bovine serum albumin as a standard protein. Absorbance was measured at 540 nm in a UV/vis spectrophotometer ( Lambda 35 perkin elmer , USA).Nitrogen recovery was calculated according to Liaset et al. (2002) as follows:Total nitrogen in hydrolysate / total nitrogen in the minced tuna head Ã- 100.
Infrared spectroscopy (FTIR) provides information about the chemical composition and conformational structure of hydroysate will be determined according to method by ( Gomez et al.,2010).Infrared spectra between 4000 and 650 cm-1 will be recorded using a Perkin Elmer Spectrum 400 Infrared Spectrometer (Perkin Elmer Inc, Waltham, MA, USA) equipped with an ATR prism crystal accessory. The spectral resolution was 4 cm-1. Measurements will be performed at room temperature using approximately 25 mg of the freeze-dried hydrolysates and peptide fractions, which will be placed on the surface of the ATR crystal, and pressed with a flat-tip plunger under spectra with suitable peaks will be obtained. All experiments will be performed at triplicate. Background will be subtracted using the Spectrum software version 6.3.2 (Perkin Elmer Inc.). The spectra were baseline corrected
at 1812 cm-1 and normalized (mean normalisation option) for comparison purposes. The amide I band was Fourier self-deconvoluted using a resolution enhancement factor of 1.4. The area of the main FTIR bands will be calculated and the values will be used for the multivariate analysis.