Analysis Of Feather Hydrolysate And Its Potential Biology Essay
Aim: 1 ) To find the alimentary content ( protein, saccharide and lipoid ) in plume hydrolysate.
2 ) To analyze the amino acid content in feather hydrolysate repast.3 ) To look into the growing of Tilapia after being fed with plume hydrolysate repast.4 ) To detect the consequence of plume hydrolysate repast in Tilapia at the histological degree.
Protein is one of the chief alimentary constituents and used expeditiously as a beginning of energy that required by fish. Adequate sum of protein is of import for care and voluntary activity besides for growing ( Lovell, 1998 ) . Previous surveies have been conducted to happen out the potency of assorted beginnings of protein for fish. For illustrations were soya protein ( Koumi et al. , 2009 ) , sweet murphy ( Ipomeae batatas ) ( Adewolu, 2008 ) and moringa ( Moringa oleifera Lam ) leaves ( Richter et al.
, 2003 ) .Poultry- plume repast besides showed its possible as a beginning of protein for animate beings feedstuff ( Hasan et al. , 1997 ) . However, the general technique used to bring forth poultry- plume repast which through physical and chemical intervention caused some indispensable aminic acids such as methionine, lysine and tryptophane were destroyed ( Fakhfakh- Zouari et al. , 2010 ) . Therefore, this survey will be conducted to supply an alternate beginning of protein for fish feedstuff from plume ceratin by utilizing biological technique.
This technique will execute ecologically friendly.Feathers are one of the major waste merchandises in domestic fowl processing industry. Accumulation of them in environment could harm the quality of environment. They can be a possible beginning of pollutant or disease if non good managed. Therefore, the direction of them is one of the critical facets in environmental direction. Furthermore, under natural status, plumes take long clip to degrade. Thus, an efficient technique is needed to degrade them faster.Feathers are composed chiefly of ceratin which is a type of hempen protein ( Fakhfakh – Zouri et al.
, 2010 ; Suntornsuk and Suntornsuk, 2003 Sangali and Brandelli, 2000 ) . Extensive cross- linking by disulfide Bridgess, H bonds and other hydrophobic bonds contribute to the mechanical strength and opposition to peptidases such as trypsin, pepsin and papain ( Fakhfakh – Zouri et al. , 2010 ; Corr & A ; ecirc ; a et al. , 2010 ; Park and Son, 2009 ; Grazziotin et al. , 2006 ) .
By and large, plumes were processed to organize feather repast through physical and chemical interventions ( Park and Son, 2009 ; Bertsch and Coello, 2005 ) . As a consequence, some indispensable aminic acids such as methionine, lysine, histidine and tryptophan were destroyed ( Park and Son, 2009 ; Bertsch and Coello, 2005 ) . Consequently, use of feather repast is limited due to lacks of nutritionally aminic acids ( Bertsch and Coello, 2005 ) .Previous surveies have been showed that some micro-organisms such as Fungis, actinomyces and bacteriums showed their possible in debasement of plumes ( Suntornsuk and Suntornsuk, 2003 ) . Most of the bacteriums that showed the potency of feather debasement from genus B. For illustrations are Bacillus polymyxa, Bacillus Cereus ( A?aba and Rodziewicz, 2010 ) , Bacillus pumilus A1 ( Fakhfakh- Zouari et al.
, 2010 ) and Bacillus megaterium F7- 1 ( Park and Son, 2009 ) . The keratinolytic enzymes of those microoragnisms performed the keratinolysis which is a procedure to cut down the stableness of disulfide bond in ceratin. This technique is ecologically friendly and low cost for the debasement of plumes.Besides that, Bacillus sp. KHAYAT which antecedently isolated besides showed their possible in feather debasement within 7 yearss. In this survey, this strain of bacteriums will be used in debasement of plume.
Subsequently, the merchandise of debauched plume called as plume hydrolysate will be analyzed and investigated to happen out its possible as beginning of protein for Tilapia by feeding test.
Significance of survey
To look into the potency of feather hydrolysate as a beginning of protein for carnal feedstuff in future.
5.1 ) Feathers Sampling.
Feathers trying will be done at local domestic fowl processing industry. Feathers will be washed with extensively tap H2O and rinsed few times with distilled H2O. Then, they will be dried in oven at 60 & A ; deg ; C, 24 H.
After that, they will be kept in certain plastic bag and stored at room temperature before used.
5.2 ) Bacterial growing.
Bacillus sp. KHAYAT which antecedently isolated bacteriums will be used in this survey. Bacteria will be grown on alimentary agar.
After 24 H incubation, individual settlement of bacteriums will be inoculated and grown in alimentary stock. Then, bacteriums will be used for production of feather hydrolysate.
5.3 ) Production of Feather Hydrolysate.
Bacillus sp. KHAYAT will be cultured in basal salt media incorporating plume for feather debasement. After 24 H, 1 milliliter of Bacillus sp.
KHAYAT in alimentary stock will be transferred into 250 mL conelike flasks that contain 50 milliliter basal salt media ( NaCl 0.5g/L, MgSO4 0.1g/L, KH2PO40.7g/L, K2HPO4 1.
4g/L ) incorporating 0.5 g of plumes. Degradation of plumes by the bacteriums will be done within 7 yearss at orbital shaker with 150 rpm/cycle ( until reach maximal debasement ) . After debasement period, degraded plume in the media will be filtered. The supernatant will be used in farther analysis and besides as the beginning of protein for Tilapia.
5.4 ) Determination of Nutrient Content ( Protein, Carbohydrate and Lipid ) in Feather Hydrolysate
Protein content in feather hydrolysate will be determined by utilizing Bradford Assay.
Amount of protein in feather hydrolysate will be determined from a standard curve of protein while saccharide content in feather hydrolysate will be determined by utilizing Nelson-Somogyi Method. The sum of saccharide will be determined from a standard curve of glucose. The sum of lipoid will be determined by utilizing Soxhlet Method. These analyses will be conducted on commercial fish repast excessively every bit comparing to feather hdrolysate repast.
5 ) Preparation of Experimental Diet ( Feather Hydrolysate Meal, Fish Meal and Meal without Protein Source ) .
For fixing feather hydrolysate repast, the supernatant will be assorted together with dextrin as the saccharide beginning, sunflower oil as lipid beginning, vitamin and mineral premix, ?-cellulose as fibre and Na carboxymethyl cellulose as binder. The ingredients for fish repast and repast without protein beginning will be prepared as the same manner except for protein beginning which will be replaced with fish repast while nil added for beginning of protein in repast without protein beginning. The concentration of food in three different repasts will be adjusted at the same sum. The mixture will be dried in oven at 50 & A ; deg ; C in the formed of pellet.
Then, the pellets will be grinded by manus howitzer and screen to obtained atom sizes runing between 0.3 and 0.5mm.
6 ) Analysis of Amino Acids Content in Feather Hydrolysate Meal, Fish Meal and Meal without Protein Source.
The amino acids analysis will be conducted by utilizing Reverse Phase High Performance Liquid Chromatography ( RPHPLC ) Method. The standard amino acids and internal criterion of protein will be analyzed together with the sample of repasts. The protein samples will be hydrolyzed to its single amino acids with hydrochloride before the amino acids analysis. The analysis will be performed by utilizing Agilent Liquid Chromatograph. The graph obtained will be indicated the presence of aminic acids in the samples.
5.7 ) Determination of Proximate Chemical Analysis in Feather Hydrolysate Meal, Fish Meal and Meal without Protein Source.
5.7.1 ) Analysis of Ash ( AOAC, 2000 ) .
Porcelains crucible with their screens will be dried in oven with 100 & A ; deg ; C. Then, they will be transferred into desiccator and cooled. After that, weight of each crucible porcelains will be recorded ( W1 ) boulder clay changeless weight obtained. Following, 2 g of samples will be put into porcelain crucible. Subsequently, the porcelain melting pot will be placed in temperature controlled furnace preheated to 600 & A ; deg ; C for 2 H. After that, the crucible porcelain will be transferred straight to desiccator, cooled and instantly weighted ( W2 ) . The per centum of ash will be describing to first denary topographic point based on the below expression:% Ash ( w/w ) = [ ( W2 – W1 ) / ( 2g – W1 ) ] x 100
7.2 ) Analysis of Dry Matter ( AOAC, 2000 ) .
2 g of samples will be dried at 100 & A ; deg ; C to constant weight.
Then, per centum of dry affair will be calculated as follow:% DM = 100 – % LOD, with% LOD = ( wt loss on drying ( g ) – 2 g ) x 100Diabetes mellitus: Dry affairLOD: Loss of Drying
5.7.3 ) Analysis of Crude Fat ( AOAC, 2000 ) .
2 g of samples into a thimble will be weighed and recorded to the first denary topographic point ( W1 ) .
Then, samples will be dried for 5 H at 100 & A ; deg ; C. Beakers that will be used for fat finding will be dried at least 1 H at 100 & A ; deg ; C and cooled in the desiccator. The beakers will be weighed and recorded ( W2 ) . When the drying period is over, samples will be transferred into the desiccator. Following, soxhlet setup will be used for extraction and distillment. Diethyl ether will be added for fat extraction. Extraction will be done for lower limit of 4 H with condensation rate 5 to 6 per second. Then, distillment of quintessence will be done.
After distillment procedure done, beakers that used for fat finding will be placed in the goon to take all hints of quintessence by vaporization. Following, beakers will be placed in 100 & A ; deg ; C gravitation convection oven no longer than 30 min. Then, beakers will be transferred into the desiccator and weighed will be reported ( W3 ) . The per centum of petroleum fat will be calculated as follow:% Crude fat: ( W3 – W2 ) x 100 / W1
5.7.4 ) Analysis of Crude Protein ( AOAC, 2000 ) .
1g of samples will be weighed and put into digestion flask. 15 g of K sulphate, 0.04 g anhydrous Cu sulphate, 0.5 to 1.0 g alundum granules will be added. Then, 20 milliliter sulphuric acid will be added. Following, flask will be placed on preheated burner. Heating will be done until white exhausts clear bulb of flask and go on heating for 90 min.
Then, flask will be cooled followed with adding of 250 milliliters distilled H2O and cool to room temperature ( less than 25 & A ; deg ; C ) . Following, distillation measure will be conducted. Titration flask will be prepared by adding appropriate volume accurately measured acerb standard solution to amount of H2O ( VHCl ) so that capacitor tip is immersed. For reagent space, 1 milliliter of acid will be pipetted and about 85 mL H2O will be added.
3 to 4 beads methyl ruddy index solution will be added. Then, 2 to 3 beads of tributyl citrate will be added into digestion flask to cut down frothing. Another 0.
5 to 1.0 g of alundum granules will be added. After that, flask will be easy down side, 45 % Na hydroxide solution ( about 80 milliliter ) will be added sufficiently to do mixture strongly alkali.
Then, flask will be connected instantly to distillment setup and will be distilled at approximately 7.5 furuncle rate ( temperature set to convey 250 mL H2O at 25 & A ; deg ; C to boil in 7.5 min ) until at least 150 mL distillation is collected in titrating flask. Digestion flask and titrating flask will be removed and the capacitor tubing will be rinsed with distilled H2O as the flask is being removed. Then, extra acid will be titrated with standard Na hydrated oxide solution to orange end point ( color alteration from ruddy to orange to yellow ) and volume will be recorded to nearest 0.
01 milliliter ( VNaOH ) . Reagent space will titrated likewise. The computation will be done as below:% N = [ ( milliliter of HCl x molar concentration of HCl ) – ( milliliter of NaOH x molar concentration of NaOH ) ] x 1.4007 / 1g ten 100 Therefore,Crude protein ( % ) ( DM footing ) = % N x 6.25
5.6 ) Preparation of Tilapia and Feeding Trial.
Tilapia juveniles will be obtained and acclimatized in research lab by giving them fish repast. The acclimatisation will be conducted for 14 yearss.
After that, they will be fasting for 24H before the experimental eating conducted. Then, 30 Tilapia juveniles with the norm of weight will be distributed to the three different experimental diets with three replicates for each experimental diet. The initial weight of each juvenile will be recorded. The eating will be conducted three times per twenty-four hours ( 800, 1200, and 1600 ) . The system will be maintained at a photoperiod of 12H light/ 12H dark. The weight of juveniles will be recorded one time a hebdomad to set the eating sum required.
The feeding test will be conducted till 4 hebdomads. At the terminal of experiment, the weight of Tilapia juveniles will be recorded and three replicates of Tilapia juveniles for each experimental diet will be sacrificed for histology experiment. The specific growing rate ( SGR, % ) , comparative growing rate ( RGW, % ) , feed transition ratio ( FCR, g/g ) , feed consumption ( g/day ) and protein efficiency ratio ( PER ) will be calculated.
5.9 ) Observation of Effectss of Feather Hydrolysate Meal on Tilapia at Histological Level.
The kidney and musculus of Tilapia juveniles will be dissected for histological scrutiny. The tissue will be cut and soaked in 4 % of formol for approximately 2H. Then, the tissue will be put into a consecutive ethyl alcohol runing from 70 % , 80 % , 90 % and 100 % with 1H for each concentration.
After that, tissue will be wash with xylene several times until clear tissue is obtained. Following, embedded procedure will be conducted with paraffin in the cassette. Last, microtome will be used to obtain a really thin piece of tissue for microscopic observation.
5.10 ) Statistical Analysis
Statistical analysis will be done by utilizing ANOVA.
Flow Chart of Methodology
Feathers + Bacteria
( Degradation procedure: To roll up the supernatant )
Determination of Nutrient Content in Feather Hydrolysate
( Protein, saccharide and lipoid )
Preparation of Experimental Diets
( Feather Hydrolysate Meal, Fish Meal and Meal without Protein Source ) .
Analysis of Amino Acids Content in Experimental Diets
( RPHPLC )
Determination of Proximate Chemical Analysis in Experimental
( Dry Matter, Ash, Crude Protein and Crude Lipid )
Preparation of Tilapia and Feeding Trial
Observation of Effectss of Feather Hydrolysate Meal on Tilapia at Histological Level
Since the chief composing of plumes is protein, the plume hydrolysate will be expected to demo high protein content. The indispensable amino acids content in feather hydrolysate will be expected non destroyed since biological method is used for feather debasement alternatively of physical and chemical intervention.
Tilapias besides will be expected to demo high per centum of weight addition compared to the fish repast and repast without protein beginning. Last, feather hydrolysate repast besides will be expected to demo no negative effects on the histology degree of Tilapia.
Contribution of Study toward Country
To supply an alternate beginning of protein for carnal feedstuff with low cost and alimentary in future.