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There has been many researches regarding to aquaculture life such as fish and squid. It is has been used in the production of protein hydrolysate . For example in the recovery of fish waste by enzymatic hydrolysis of protein residues (Guérard et al.,2001; Rajapakse et al., 2005). Protein hydrolysate that come from squid can give many advantages value in aquaculture feed ingredient and organic fertilizer such as a protein source in microdiets for gilthed seabream sparus aurata larvae. Protein hydrolysate from the sea species is also has a great potential in the production of antioxidant hydrolysates and peptides such as Alaska Pollack (Kim et al., 2001), as well as from several squid species, such as Giant squid (Dosidicus gigas) (Mendis et al.,2005 ; Giménez et al.,2009), Jumbo flying squid (Dosidicus eschrichitii Streenstrup) (Lin & Li,2006) or squid (Todarodes pacificus) (Nam et al., 2008).
A number of enzymes have been used for the production of fish protein hydrolysates, including trypsin, chymotrypsin, pepsin, Properase E, Pronase, collagenase, bromelain and papain (Kim et al., 2001; (Mendis et al., 2005a; Lin & Li, 2006; Yang et al., 2008). In this research, the two different enzyme which is Alcalase and flavourzyme will be use to know which enzyme indicate the best product of squid protein hydrolysate.
This research also will be done because there are only little information regarding protein hydrolysate from squid 'Laligo duvaucelly ' and their functional properties and composition. It is because this type of squid has low economic value, large capture volumes and largely sold as an unprocessed product, mainly as raw product to Asian countries.Moreover, human consumption on this species is still limited.
1.2 Significance of study
The hydrolysis of squid 'Laligo duvaucelly ' protein will be perform with two different enzymes Alcalase and Flavourzyme. Enzymatic hydrolysis of protein is preferred method for improving functionality and avoiding destruction of products due to the extremes of chemical and physical treatments . From that, we can know which enzyme will produce less bitter hydrolsate product by the processing techniques for seafood is needed to convert them into more marketable, valuable and acceptable products. It's also the enzymatic protein hydrolysates will generate a certain amount of free amino acids and short chain peptides in the given of the several advantages in functional properties such as improved solubility, heat stability,water binding ability and increased nutritional quality.
1.3 Objective of study
The objective of this research is to evaluate of characterization in protein hydrolsate from squid (Laligo duvaucelly) with two different microbial enzymes ; Alcalase and flavourzyme.
The specific objectives of the research include to:-
Determine the protein content in squid (Laligo duvaucelly) hydrolysate.
Determine the functional properties and composition of squid (Laligo duvaucelly)
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.
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
22.214.171.124 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. 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,DH(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 hydolysate samples.
126.96.36.199 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 (Dauks as 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 thse 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.
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).
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).
Material and Method
3.1 Raw materials
Squid (Loligo duvaucelly) will buy at pasar malam meru and immediately frozen on board at -20 °C. The squid will immediately transfer to the laboratory at UITM. Then, squid will immediately wash with chlorinated water and will be placed in plastic containers and stored frozen at âˆ’18Â°C for pending use.
3.1.1 Microbial Enzymes
The enzymatic process will be perform 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).All of the following analyses will be perform in triplicate.
3.2 Preparation of squid protein hydrolysate
The protein hydrolysate will be prepared according to the method by Adler-Nissen (1986) with a slight of modification. 30 g of minced squid will be suspended in 120 ml of distilled water. The mixture was incubated in a circulated water bath at 60â°C. The pH of the mixture was 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. Hydrolysates will be centrifuged at 14000g for 10 min. Supernatants obtained was freeze-dried at -20oC using the SANYO-Biomedical freeze dryer. This above step will be repeated by using the second enzyme flavourzyme.
3.3 Chemical composition
Moisture content will be determined following AOAC by placing approximately 2 g of sample into a preweighted aluminum dish. Samples 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 raw materials and freeze-dried hydrolysates will be determined using the Kjeldahl method (AOAC 2005). Total lipid in samples will be determined by Soxhlet apparatus (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).
3.4 Nitrogen Recovery
Nitrogen recovery will be calculated according to Liaset et al. (2002) as follows:
Total nitrogen in hydrolysate / total nitrogen in the minced squid Ã- 100.
3.5 Degree of hydrolysis
The hydrolysis was carried out using the pH-stat method (Adler-Nissen, 1986). 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 was 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 % = B x NB x 100
Î± x MP x htot
B = Volume of 1.0M NaOH
NB = 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)
3.6 Amino acid composition
Samples 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 Determination of Functional Properties
The solubility of the hydrolysate compounds from squid will be determine 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 add to 500 mg of dry sample, 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 kept under stirring for 45 min. The dispersion will be transfer to a 50-mL volumetric glass, the volume being completed with the buffer solution. The protein dispersion will 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, through the method described by Lowry et al. (1951). The solubility rate will be determine according to Eq. 2.
S = A x 50 x 100 (2)
W x P/100
where A is the protein concentration in the supernatant (mg mLâˆ’1), W is the weight of the samples (mg), and P is the percentage of protein in the sample.
Water (WHC) and Oil (OHC) Holding Capacity
The capacity to hold water and oil will be determined according to the methodology described by El Khalifa et al. (2005) adjusted to laboratory conditions. For the water holding capacity (WHC), 1 g of each hydrolysate compound 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 was 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.
Emulsification capacity will be measured by mixing 5 ml of rapeseed oil with 5 ml of a 1% FPH solution in water and homogenising (Ultra-Turrax TP 18/10) in 15 ml graded Nunc centrifuge tubes 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 (1 day at room temperature) and centrifugation at 2400 x g for 3 min. Tests will be performed in duplicate.
Foam stability 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.
Determination of rancidity by 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 rancind odor 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. There were asked to open one plastic bag at a time and make three depp sniff of the content and recorded the scale. The sensory evaluation session will be repeated three time.
The colour of powdered hydrolysate was measured by chromameter CR400 (Konica Minalto). L*, a* and b* parameters indicate lightness, redness and yellowness, respectively. Measurement was performed in triplicate.
3.8 Fatty acid analysis
Fats will be extracted from the sample and converted to free fatty acids by saponification. The fatty acids will be converted to their methyl esters and into heptane. Internal standards will be employed for estimation of actual fatty acids present in the fat. Identification/ quantification of fatty acids will be achieved by gas chromatography, the former being resolved by elution times (AOAC, 2005).
3.9 FTIR-ATR spectroscopy
Infrared spectra between 4000 and 650 cm-1 were recorded using a Perkin Elmer Spectrum 400 Infrared Spectrometer equipped with an ATR prism crystal accessory. The spectral resolution was 4 cm-1. Measurements will performed at room temperature using approximately 25 mg of the freeze-dried hydrolysates and peptide fractions, which were placed on the surface of the ATR crystal, and pressed with a flat-tip plunger under spectra with suitable peaks were obtained. All experiments will be performed at least in duplicate. The area of the main FTIR bands was calculated and the values were used for the multivariate analysis.
4.0 Measurement of antioxidant activity
Antioxidant activity of squid Laligo duvaucelly hydrolysate will be determined according to procedure described by Mansour and Khalil(2000). Two milligram Î²-carotene will be dissolved in 20ml chloroform. Three milliliter aliquot of the solution were then added to 40 mg linoleic acid and 400 mg tween 40. Chloroform will be evaporated using the rotary evaporator set at 500C. 100ml oxygenated distilled water will be added to the Î²-carotene emulsion and will be mixed well. 0.12ml antioxidant extract was then mixed with 3ml oxygenated Î²-carotene emulsion as will be prepared earlier and will be incubated at 500C for 120 min. This mixture will be taken out every 15 min interval to record the absorbance at 470nm. For control, distilled water will be used instead of the antioxidant extract. Degradation rate of the extracts will be calculated according to the following equation:
Sample degradation rate = Ln (a/b) x 1/t
Ln= natural log, a = initial absorbance at time 0, b= absorbance at time 0,15 min.,etc,t= time in min
Antioxidant activity (%) will be expressed as percentage inhibition relative to control using the following equation :
Degradation rate control - degradation rate of sample x 100
Degradation rate of control
4.0 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).