Harmonizing to ( NASS-USDA, 2001 ) , Catfishs ( order Siluriformes ) is now the 4th most popular fish merchandise consumed in United States. In Malaysia, mudcat is identified as one of the most-preferred Marine fish for day-to-day ingestion that can be found in swamps, paddy Fieldss and watercourses. There are studies on production of good quality fish oils from herring ( Aidos et al. , 2003 ) and catfish ( Sathivel et al. , 2003 ) byproducts.
Much of the oil in mudcat is in the entrails, which contain about 33 per centum fats or lipoids which could be converted into comestible oil or biodiesel merchandise. The oil extracted from fish, are rich in polyunsaturated fatty acids ( PUFAs ) particularly those of the omega-3 household, chiefly eicosapentaenoic acid ( EPA ; C20:5n-3 ) and docosahexaenoic acid ( DHA ; C22:6n-3 ) . Although fish is a dietetic beginning of na?’3 fatty acids, fish do non synthesize them ; they obtain them from the algae ( microalgae in peculiar ) or plankton in their diet ( Falk-Petersen, 1998 ) .
The importance of PUFAs in human wellness and nutrition is good recognised. Many clinical and epidemiologic surveies have shown positive functions for n-3 PUFA in baby development, malignant neoplastic disease, cardiovascular diseases and more late, in assorted mental unwellnesss, including depression, attention-deficit hyperactivity upset and dementedness ( Riediger et al. , 2009 ) . In add-on, fish oil besides helps to forestall encephalon ripening and Alzheimer ‘s disease ( Kyle, 1999 ) . The consequence of long concatenation n-3 PUFA when handling asthma has been late reviewed by Reisman et Al. ( 2006 ) , on intestine diseases ( Razack and Seidner, 2007 ) , psoriasis ( Zulfakar et al. , 2007 ) and in bar some types of malignant neoplastic disease, including colon, chest and prostate ( Marchioli, 2001 ) .
The accent on the importance of omega-3 long-chain PUFA has led to the commercial handiness of purified fish oil addendums that are available in wellness nutrient shops. The International Society for the survey of Fatty Acids and Lipids ( ISSFAL ) recommended Adequate Intakes ( AIs ) of a lower limit of 0.22 g/day for DHA and EPA combined, while the British Nutrition Foundation ( BNF ) has recommended a desirable population consumption of 1.1 g ( females ) and 1.4 g ( males ) of EPA and DHA/day.
There are some extraction methods used to pull out oil from fish in order to give better quality oil extraction and all right quality merchandise at the terminal. The conventional fish processing methods for oil extraction including rendering procedure and hydrodistillation. These methods can lend to the lost, denaturation, or decomposition of the cherished nutritionary fish oil. Hydrodistillation gives some disadvantages due to heat instability of the oils and loss of certain water-soluble constituents ( Damjanovic, 2003 ) . It is besides involves significant energy ingestion to run long hours of extraction. The other conventional method of pull outing oil from fish is by rendering, a procedure in which high heat is used to pull out fat or oil chiefly from carnal tissues. This method used high temperature that can do the constituents of fish oil most susceptible to thermic debasement.
Consequently, there is an increasing demand today for new extraction technique with possibilities for mechanization, shorter extraction clip and lower dissolver ingestion. Pressurised Liquid Extraction ( PLE ) engineering and its applications have been developed and used by assorted research workers ( Bautz, Polzer et al. , 1998 ; Richter and Covino, 2000 ) . It has besides been applied for the analysis of nutrient and biological samples ( Schafer, 1998 ) .
In this survey, an experimental designed was planned and the experiment is performed with PLE which sample in the cell is pressurized with certain force per unit area, heated to want temperature and sample is extracted statically for specific period clip in order to obtain highest output of extracted oil with the highest sum of the marker compound ( EPA and DHA ) in the extraction merchandises. This PLE extraction utilizing H2O and hexane as dissolvers and the obtained consequence will be compared with the extracted output from conventional methods.
One of the major drawbacks of oil incorporating a high sum of omega-3 PUFA, such as fish oils, is their high susceptibleness to oxidization, which involve the formation of toxic merchandises as peroxides or volatile compounds comparative to non-desirable off-flavours. Therefore, optimal processing, storage and packaging of fish oils are indispensable to continue omega-3 PUFA from oxidization. The original of fish oil in liquid signifier make it hold limited shelf-life and trouble in storage because the temperature will impact the formation of peroxide value. This was reported by Pak ( 2005 ) that during storage at 4oC, peroxide values of several fish oils reached the acceptable bound of 8 meq/kg after 60 or 90 yearss and at -18 oC at least 150 yearss. This fact showed that the storage temperature had of import effects on the storage stableness of fish oil.
The usage of antioxidants is one of the most common methods to forestall fish oil oxidization. However, this method still ca n’t alter the physical belongingss of fish oil. One engineering for get the better ofing these jobs and besides cut downing oxidization of omega-3 fatty acids is encapsulation of fish oil. Encapsulation or microencapsulation with a surfacing stuff has besides been proposed as scheme to retard lipid auto-oxidation, better the oil stableness, protract its shelf-life and command off-flavours ( Matsuno and Adachi, 1993 ) .
Cyclodextrin, derived from enzymatically modified amylum molecules is used to encapsulate fish oil. The application of cyclodextrin in pharmaceutical, decorative and nutrient industries is extended because it is cheap and non-toxic. Szente and Szejtli ( 2004 ) reviewed the applications of cyclodextrin as a nutrient ingredient in association with encapsulation engineering.
In this survey, I?-cyclodextrin ( I?-CD ) will be used as encapsulating agent for fish oil ( FO ) and the attendant Field-grade officer: I?-CD inclusion composite will be characterise utilizing Fourier Transform Infrared Spectroscopy ( FTIR ) , Differential Scanning Calorimeter ( DSC ) and Field Emission Scanning Electron Microscopy ( FESEM ) . The stableness of inclusion composite in comparing to command ( original catfish oil ) will besides be investigated in this survey.
Significance of survey
This new engineering to pull out catfish oil utilizing Pressurised Liquid Extraction ( PLE ) will hold several advantages ; more effectual, rapid, selective in extraction and dependable compared to conventional methods. The possibility of altering several extractions variables such as temperature, force per unit area and volume of dissolver is a promising features of this analytical tool to acquire the optimise consequence in term of output and besides the marker compounds ( EPA and DHA ) . This survey besides will be important enterprise in microencapsulation of fish oil since the features and stableness of the attendant inclusion composite will be besides determined. Water soluble powdery signifier of fish oil can be applied in assorted Fieldss such as nutrient, pharmaceutical and medical field. Besides, it can be used as a theoretical account survey for future research by using encapsulation engineering to organize inclusion composite of any fish oil that contain high in PUFAs particularly EPA and DHA.
Aims of survey
The aims of this survey are to
develop Pressurised Liquid Extraction ( PLE ) method for catfish oil extraction in comparing to conventional extraction methods ( rendering and hydrodistillation ) .
encapsulate and characterize I?-cyclodextrin: catfish oil inclusion composites.
find the stableness of the inclusion composite in comparing to command ( catfish oil ) .
Scope and restriction
Application and optimization of new extraction method ( PLE ) for mudcat ( Clarius batrachus ) oil utilizing H2O and solvent n-hexane. The attendant fish oil will be compared with conventional extraction techniques ; rendering and hydrodistillation. The quality will be besides compared with commercially available fish oil ( Menhaden oil, Sigma ) . The extracted fish oil that have the highest quality in term of output, EPA and DHA will be used for encapsulation survey. Phase solubility survey will be carried out to find the molar ratio of encapsulation. The usage of I?-cyclodextrin as encapsulating agent for catfish oil will be besides investigated. Word picture of inclusion composite formed will be besides carried out utilizing instrumental analysis ; FTIR, FESEM and DSC. The stableness of the attendant inclusion composite and fish oil will be determined utilizing Rancimat analysis. The analysis of EPA and DHA can be really disputing due to sensitiveness of fish oil to oxidization. The unsaturation of fatty acids makes the fish oil more vulnerable for spoilage than the other oils. Sample for PLE extraction have to be carefully prepared in term of sample size and wet content which can significantly consequence the quality of attendant fish oil. The analysis of Response Surface Methodology ( RSM ) package can be hard in order to acquire important theoretical account and non important deficiency of fit informations.
2.1 Fish Oil ( FO )
Fish oils are rich in polysaturated fatty acids ( PUFAs ) particularly those of the omega-3 household, Docosahexaenoic Acid ( DHA ) and Eicosapentanaenoic Acid ( EPA ) which are the biological active or active agents in fish oil Chen et Al. ( 2003 ) . Due to its high content of PUFAs, including EPA and DHA, fish oils are extremely susceptible to oxidative spoilage and it produces a rancid odor and spirit in the oil by cut downing its nutritionary quality, doing the formation of other unwanted substances ( Frankell, 1996 ) . However, efforts to integrate fish oil into nutrient preparations has had limited success because of “ fishy ” spirits in finished products-the chief job of nutrient enrichment with omega-3 acids. One engineering for get the better ofing these jobs and besides cut downing oxidization of omega-3 fatty acids is encapsulation of fish oil.
Encapsulation engineering has been used in nutrient industry for more than 60 old ages as a manner to supply liquid and solid ingredients as an effectual barrier for environmental and/ or chemical interactions until release is desired ( Reineccius, 1994 ) . The encapsulation procedure makes it possible to transform the oil into a pulverization in which little droplets of oil are surrounded by a “ shell ” of proteins or saccharide. This will better the oxidative stableness of fish oil and extends its shelf life to 12-24 months when stored in a dry cool environment. In add-on, it has been proved that the usage of fish oil microcapsules offers good consequences in the design of functional nutrients as pick to make full sandwich cookies ( Borneo et al. , 2007 ) and dairy merchandises ( yogurt, fresh cheese, butter ) ( Kolanowski and WeiI?brodt, 2007 ) .
Szente and Szejtli ( 2004 ) reviewed the applications of cyclodextrin ( Cadmium ) as a nutrient ingredient in association with encapsulation engineering. In general, the cyclodextrin inclusion method is the better known technique for encapsulating active substances ( He et al. , 2007 ) . Cadmium can solubilise and brace active compounds on the molecular graduated table by using the molecular inclusion method.
Cyclodextrins are cyclic oligosaccharides dwelling of six I±-cyclodextrin, seven I?-cyclodextrin, eight I?-cyclodextrin or more glucopyranose units linked by I±- ( 1,4 ) bonds. The inside of the molecule is formed by H atoms and glycosidic O span atoms, which give the pit hydrophobic character and interact with assorted organic molecules. Guest molecules, with suited dimensions to suit inside the inside, can be included into the cyclodextrin molecules to organize agent-cyclodextrin inclusion composites. This interaction is through the hydrophobic groups of the guest molecules with the walls of the pit of the cyclodextrins. .
I?-CD as a molecular encapsulant allows the flavour quality and measure to be preserved to a greater extent and longer period compared to other encapsulants besides it can provides length of service to the nutrient point [ 21 ] S. Munoz-Botella, B. del Castillo and M.A. MartyI‹n, Cyclodetrin belongingss and applications of inclusion complex formation, Ars. Pharm. 36 ( 1995 ) , pp. 187-198. Position Record in Scopus | Cited By in Scopus ( 13 ) ( Munoz-Botella et al. , 1995 ) . [ 3 ] J. Szetjli, Introduction and general overview of cyclodextrin chemical science, Chem. Rev. 98 ( 1998 ) , pp. 1743-1753.Klaypradit and Huang, 2007 reported that the encapsulation of fish oil significantly retards oxidization. It can dissemble the obnoxious smells caused by volatile oxidization merchandises and enhances the smell of fish oil-enriched merchandises ( Castro et al. , 2004 ) .
2.4 Inclusion composite techniques
Several techniques are used to organize cyclodextrin composites, co-precipitation, working, freezing drying, spray drying and suspension techniques. For co-precipitate method, this method is the most widely used method in the research lab. Cyclodextrin is dissolved in H2O and the invitee is added while stirring the cyclodextrin solution. The concentration of I?-cyclodextrin can be every bit high as approximately 20 % if the invitee can digest higher temperatures. If a sufficiently high concentration is chosen, the solubility of the cyclodextrin-guest composite will be exceeded as the complexation reaction returns or as chilling is applied. In many instances, the solution of cyclodextrin and invitee must be cooled while stirring before a precipitate is formed. The precipitate can be collected by pouring, centrifugation or filtration ( Waleczek et al. , 2003 ) .
2.4.2 Kneading method
Kneading method is when the sample and cyclodextrin is blending in howitzer and kneads for 45 proceedingss before dried and sieved to organize complex molecules ( Zhang et al. , 2005 ) .
2.4.3 Physical mixture
Physical mixture is done by prepared dry-pestling in a howitzer and kneaded for 5 proceedingss to obtain homogeneous blend ( Zhang et al. , 2005 ) .
2.5 Phase solubility survey
Formation of inclusion composites in solution can be detected by the stage solubility method described by Higuchi and Connors ( 1965 ) . This method is carried out by adding increasing sums of I?-cyclodextrin to an aqueous suspension of an extra sum of invitee ( in this instance is referred as fish oil ) . The suspensions are shaken at changeless temperature until equilibrium is reached. The solid atoms are removed and the solution is analyzed for the entire concentration of invitee dissolved. The solubility of the invitee is now plotted against the I?-cyclodextrin concentration.
2.6 Word picture of inclusion composite
There are techniques used to qualify the inclusion composite and the interaction between them. In this survey, Fourier Transform Infrared Spectroscopy ( FTIR ) , Differential Scanning Calorimeter ( DSC ) and Field Emission Scanning Electron Microscopy ( FESEM ) will be used.
2.6.1 Fourier Transform Infrared Spectroscopy ( FTIR )
A measuring technique for roll uping infrared spectra. FTIR can observe really little changes in bond lengths and angles in supermolecules and therefore has emerged as a powerful tool to look into the structural alterations in the molecules in item ( Garip et al. , 2009 ) . Zaibunnisa et al. , 2009 in her survey of inclusion composite between oleoresin and E¤-cyclodextrin showed that cyclodextrin composite was formed with guest molecules treating carbonyl groups. The displacement to higher wavelength Numberss indicated that the junction in the oleoresin was reduced in the presence of cyclodextrin.
2.6.2 Differential Scanning Calorimeter ( DSC )
This thermo analytical technique is used in analyzing stage passages, such as thaw, glass passages or exorthemic decomposition. The disappearing of thermic events of guest molecules when they are examined as Cadmium composites is by and large taken as cogent evidence of existent inclusion ( Pralhad and Rajendrakumar, 2004 ) . Farcas et al. , 2006 in his survey of word picture of Lasix composite in I?-CD showed DSC secret plan of pure furosemide drug pulverization shows a crisp endorthermic extremum near 210 oC, which attributed to ferosemide runing temperature while two endorthermic extremums were observed in the temperature scope between 100-110 oC and 330 oC in the thermogram of I?-CD. The DSC form of ferosemide: I?-CD inclusion composite prepared by physical commixture demo the presence of extremums of both pure compounds.
Figure 1 DSC thermograms of Lasix: I?-CD inclusion composite by physical mixture.
Beginning: Farcas et Al. ( 2006 )
2.6.3 Field Emission Scanning Electron Microscopy ( FESEM )
It is a type of negatron microscope that contains information about the sample ‘s surface topography, composing and electrical conduction belongingss. It besides gives information on the size and form of samples. Farcas et Al. ( 2006 ) in his survey of word picture of Lasix composite in I?-CD by utilizing FESEM measuring observed that pure Lasix ( guest molecule ) is characterised by the presence of crystalline atom of regular size while pure I?-CD ( host molecule ) appears as crystalline atoms without a definite form. The physical mixture of Lasix: I?-CD showed the crystalline construction of both furosemide and I?-CD.
2.7 Quality and stableness of fish oil
Fish oil is extremely susceptibility to oxidative spoilage ( Huss, 1988 ) because of its high content of polyunsaturated fatty acids. Oxidative stableness is one of the most of import quality indexs in fish oils. Peroxide value ( PV ) and thiobarbituric acid ( TBA ) analysis are normally used to measure of fish oil stableness and monitoring impairment during storage.
Regulatory bureaus have established the bounds for quality and acceptableness of oils for human ingestion which 8 milliequivalent O2 /kg of oil is the bound of acceptableness of PV and 7 -8 milligram malonaldehyde/kg of oil ( Huss, 1988 ) for TBA value. The original of fish oil in liquid signifier make it hold limited shelf-life and trouble in storage because the temperature will impact the formation of peroxide value. This was reported by Pak ( 2005 ) that during storage at 4oC, peroxide values of several fish oils reached the acceptable bound of 8meq/kg after 60 or 90 yearss and at -18 oC at least 150 yearss. This fact showed that the storage temperature had of import effects on the storage stableness of fish oil.
In fish oils, the initiation period ( IP ) is the clip before the highest addition of lipids oxidization. The new official method that allows the coincident and rapid finding of initiation period or Oil Stability Indexes ( OSI ) to be performed is by utilizing Metrohm ‘s 743 Rancimat. The accelerated oxidative trial Rancimat has been used extensively during the last old ages to find oxidative stableness of fats and oils under standardised conditions ( Rossell, 1994 ) . Refering lipidic nutrients, application of Rancimat to intact nutrients would give a more realistic representation of what may happen during storage than using the extracted lipoids, with the extra advantage of avoiding any old, time-consuming handling of samples through the extraction measure.
In the Rancimat, a watercourse of purified air is passed through a sample of oil or fat that is held in a warming block. The outflowing air from the sample is so bubbled through a vas containingA deionised H2O. The conduction of the H2O is continually monitored. Once the oil starts to oxidize, volatileA organic acids, preponderantly formic acid, are swept by the wastewater watercourse, through the deionised H2O, doing an addition in the conduction of the H2O. The OSI is defined as the point at which there is the maximal alteration in the rate of oxidization and is evaluated automatically in the Rancimat.
3.1 Sample readying
Farmed Catfish is obtained from a local provider in Seremban, Negeri Sembilan. The fish will be gutted, washed, minced in a commercial liquidizer and stored at -20 OC until be analyzed.
3.2 Method of extraction
3.2.1 Rendering -Water cookery
Extraction followed the method by Sheu and Chen ( 2002 ) . Flesh sample will be boiled with a sample/water ratio ( 1:2w/w ) in glass beakers for 30, 40, or 50 min, individually. After remotion of the flesh residues, the liquid will be cooled to room temperature and the fat bed will be removed utilizing a separatory funnel.
Extraction followed the method by Zaibunnisa et al. , 2009. Flesh samples ( 300g ) will be hydrodistilled for 8 hour utilizing a Dean Stark- type setup. The distillations will be collected and liquid-liquid extractions will be carried out to divide the oil fraction from H2O.
3.2.3 Pressurised Liquid Extraction ( PLE )
Sample will be land and dried in oven at 40 oC for nightlong for pre-treatment. Samples ( 5g ) will be assorted together with diatomaceous Earth ( 5g ) with ratio before been placed into 22 milliliters cells with cellulose filter at the bottom terminal. Solvent is added. In this experiment, different dissolvers of n-hexane and H2O will be used to look into the influence of solvent mutual opposition on the output of oil. The cell will be pressurized with 1000 pounds per square inch, heated to want temperature ( 70 oC ) and sample is extracted statically for specific period clip ( 5 min ) . Others status will be standardized ; flush volume, 100 % ; purging clip, 60s and inactive rhythm, 1.The infusion will be removed from cell and cell will be flushed with fresh dissolver. When extraction is complete, Nitrogen gas will be compressed to travel all dissolver from cell to phial for analysis. The infusion will be evaporated to dryness ( rotary evaporator ) to cipher output on dry weight footing. Optimization of PLE extraction can be acquiring by utilizing Response Surface Methodology ( RSM ) .
3.3 The physical and chemical analysis of fish oil
3.3.1 Determination of free fatty acid composing
The extracted lipoid will undergo transesterification to bring forth fatty acerb metyl ester ( FAME ) harmonizing to Kinsella et Al. ( 1977 ) . Sodium methoxide and hexane will be used for the readying of the Fatty Acid Methyl Esters ( FAME ) . The fatty acid methyl ester of samples will be analyzed by the gas chromatography ( HEWLET PACKARD, HP 6890 Series, USA ) which is equipped with Flame Ionization Detector ( FID ) . A capillary column ( SGE, 50 m length and 0.22 millimeters diameter ) is used to divide the fatty acerb constituents. The temperature of the injection port and sensor will be set at 260A°C. The oven temperature will be programmed to increase from 50 to 230A°C at a rate of 4A°C per min. One microliter of each sample will be injected manually in extras with the split 40. Fatty acerb extremums in the samples will be identified by comparing the keeping times with that of reliable mention criterions. The consequences will be expressed as comparative per centums.
3.3.2 Peroxide value analysis
The peroxide value will be determined utilizing AOAC method ( AOAC, 1995 ) . Each fat sample ( 5g ) will be assorted with 30 milliliters HOAc-CHCl3 and 0.5 milliliter saturated KI solution. After agitating for 1 min, 30 milliliter of distilled H2O will be added and assorted. A 0.01 N Na2S2O3 will be used for titration. Consequences will be expressed as factory equivalents/kg oil ( meq/kg ) .
3.3.3 Thiobarbituric acid ( TBA ) value analysis
Thiobarbituric acid ( TBA ) value will be followed by distillment method ( Tarladgis et al. , 1960 ) . Optical denseness ( OD ) will be measured against a reagent space at 538nm. The TBA value will be obtained by multiplying the optical density ( OD ) by a invariable of 7.8.
3.3.4 Color analysis
Color of the samples will be measured utilizing a minolta saturation metre ( CR-200 Minolta, Japan ) . The colour readings will be expressed by CIE ( L* a* b* ) system ( RafaelA et al. , 2004 ) . L* , a* and b* indicates whiteness/darkness, redness/greenness and blueness/yellowness, severally. The maximal value for L* is 100, which would be a perfect reflecting diffusor. The lower limit for L* would be zero, which would be black. The a* and b* axes have no specific numerical bounds. Positive a* is ruddy and negative a* is green. Positive b* is xanthous and negative b* is bluish.
3.4 Encapsulation and word picture catfish oil: I?-cyclodextrin
Phase solubility surveies will be carried out following method of Higuchi and Connors ( 1965 ) to find the molar ratio of FO: I?-CD. An extra sum of fish oil ( 20mg ) will be added to screw-capped phials incorporating I?-CD in 5.0 milliliter of ethyl alcohol: H2O ( 25:75 v/v ) solution at assorted concentrations, runing from 0 to 15 Mm for I?-CD. The phials will be shaked at 30 OC for 48 hours in a H2O bath until reached equilibrium. The samples will be centrifuged at 3000rpm for 10 proceedingss. After attainment of equilibrium, the contents of the tubing will be filtered through Whatman filter paper. The infusions will be pooled together and evaporated to 1 milliliter. Inclusion composite of catfish oil: I?-CD will be prepared by utilizing:
3.4.1 Co-precipitation method
Co-precipitation method will be carried out following method of Waleczek et al. , 2003. FO will be added to screw-capped phials incorporating I?-CD in ethyl alcohol: H2O ( 25:75 v/v ) mixture of 5ml. The phials will be agitating at 30 OC until equilibrium reached in H2O bath for 48 hours. The samples will be centrifuged at 3000 revolutions per minute for 10 proceedingss. The supernatant will be decanted to organize the complex as microcrystalline precipitate. The merchandise obtained will be dried in oven at 40 OC for 48 hours. The dried mass will be sieved through 150Aµm mesh.
3.4.2 Kneading method
Kneading method will be carried out following method of Zhang et al. , 2005. A molar ratio of I?-CD and fish oil that will be determined by stage solubility survey will be added in howitzer and kneaded for 45minutes. While working, 40 % of ethyl alcohol: H2O ( 25:75 v/v ) mixture will be added to the mixture to keep proper consistence. The merchandises will be dried in oven at 40 OC for 48hours. The dried mass will be sieved through 150 Aµm mesh.
3.4.3 Physical method
Kneading method will be carried out following method of Zhang et al. , 2005. For control, physical mixture of the same weight ratio of fish oil: I?-CD will be prepared by dry-pestling in a howitzer and kneaded for 5 proceedingss to obtain homogeneous blend.
Word picture of catfish oil: I?-CD inclusion composite will be done by FTIR, FESEM and DSC.
3.5 Stability of fish oil
Oil Stability Index ( OSI ) will be determined at 100 OC and 20 milliliter air/hr utilizing Rancimat setup following AOCS Method ( AOCS, 1994 ) . The Rancimat setup will be used with two rating manners, I ) initiation period ( clip matching to inflection point in the oxidization curve ) and ii ) clip to delta K ( clip needed to accomplish a specific difference in conduction, here will be selected is in 25AµS cm-1.
3.6 Statistical Analysis
Data analysis will be done by utilizing analysis of discrepancy ( ANOVA ) . Comparison of agencies will be carried out by Duncan ‘s multiple scope trials ( P & lt ; 0.05 ) . Statiscally analysis will be performed by utilizing SPSS 10 for Windows.