Objective : 1) To determine the nutrient content (protein, carbohydrate and lipid) in feather hydrolysate.
2) To analyse the amino acid content in feather hydrolysate meal.
3) To investigate the growth of tilapia after being fed with feather hydrolysate meal.
4) To observe the effect of feather hydrolysate meal in tilapia at the histological level.
Protein is one of the main nutrient components and used efficiently as a source of energy that required by fish. Adequate amount of protein is important for maintenance and voluntary activity besides for growth (Lovell, 1998). Previous studies have been conducted to find out the potential of various sources of protein for fish. For examples were soya protein (Koumi et al., 2009), sweet potato (Ipomeae batatas) (Adewolu, 2008) and moringa (Moringa oleifera Lam) leaves (Richter et al., 2003).
Poultry- feather meal also showed its potential as a source of protein for animals feedstuff (Hasan et al., 1997). However, the general technique used to produce poultry- feather meal which through physical and chemical treatment caused some essential amino acids such as methionine, lysine and tryptophane were destroyed (Fakhfakh- Zouari et al., 2010). Thus, this study will be conducted to provide an alternative source of protein for fish feedstuff from feather keratin by using biological technique. This technique will perform ecologically friendly.
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Feathers are one of the major waste products in poultry processing industry. Accumulation of them in environment could harm the quality of environment. They can be a potential source of pollutant or disease if not well managed. Thus, the management of them is one of the vital aspects in environmental management. Furthermore, under natural condition, feathers take long time to degrade. Thus, an efficient technique is needed to degrade them faster.
Feathers are composed mainly of keratin which is a type of fibrous protein (Fakhfakh - Zouri et al., 2010; Suntornsuk and Suntornsuk, 2003 Sangali and Brandelli, 2000). Extensive cross- linking by disulfide bridges, hydrogen bonds and other hydrophobic bonds contribute to the mechanical strength and resistance to proteases such as trypsin, pepsin and papain (Fakhfakh - Zouri et al., 2010; Corrêa et al., 2010; Park and Son, 2009; Grazziotin et al., 2006).
Generally, feathers were processed to form feather meal through physical and chemical treatments (Park and Son, 2009; Bertsch and Coello, 2005). As a result, some essential amino acids such as methionine, lysine, histidine and tryptophan were destroyed (Park and Son, 2009; Bertsch and Coello, 2005). Consequently, utilization of feather meal is limited due to deficiencies of nutritionally amino acids (Bertsch and Coello, 2005).
Previous studies have been showed that some microorganisms such as fungi, actinomyces and bacteria showed their potential in degradation of feathers (Suntornsuk and Suntornsuk, 2003). Most of the bacteria that showed the potential of feather degradation from genus bacillus. For examples are Bacillus polymyxa, Bacillus cereus (Åaba 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 process to reduce the stability of disulfide bond in keratin. This technique is ecologically friendly and low cost for the degradation of feathers.
Besides that, Bacillus sp. KHAYAT which previously isolated also showed their potential in feather degradation within 7 days. In this study, this strain of bacteria will be used in degradation of feather. Later, the product of degraded feather called as feather hydrolysate will be analyzed and investigated to find out its potential as source of protein for tilapia by feeding trial.
Significance of study
To investigate the potential of feather hydrolysate as a source of protein for animal feedstuff in future.
5.1) Feathers Sampling.
Feathers sampling will be done at local poultry processing industry. Feathers will be washed with extensively tap water and rinsed few times with distilled water. Then, they will be dried in oven at 60°C, 24 H. After that, they will be kept in sealed plastic bag and stored at room temperature before used.
5.2) Bacterial growth.
Bacillus sp. KHAYAT which previously isolated bacteria will be used in this study. Bacteria will be grown on nutrient agar. After 24 H incubation, single colony of bacteria will be inoculated and grown in nutrient broth. Then, bacteria will be used for production of feather hydrolysate.
5.3) Production of Feather Hydrolysate.
Always on Time
Marked to Standard
Bacillus sp. KHAYAT will be cultured in basal salt media containing feather for feather degradation. After 24 H, 1 mL of Bacillus sp. KHAYAT in nutrient broth will be transferred into 250 mL conical flasks that contain 50 mL basal salt media (NaCl 0.5g/L, MgSO4 0.1g/L, KH2PO40.7g/L,K2HPO4 1.4g/L) containing 0.5 g of feathers. Degradation of feathers by the bacteria will be done within 7 days at orbital shaker with 150 rpm/cycle (until reach maximum degradation). After degradation period, degraded feather in the media will be filtered. The supernatant will be used in further analysis and also as the source 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 using Bradford Assay. Amount of protein in feather hydrolysate will be determined from a standard curve of protein while carbohydrate content in feather hydrolysate will be determined by using Nelson-Somogyi Method. The amount of carbohydrate will be determined from a standard curve of glucose. The amount of lipid will be determined by using Soxhlet Method. These analyses will be conducted on commercial fish meal too as comparison to feather hdrolysate meal.
5.5) Preparation of Experimental Diet (Feather Hydrolysate Meal, Fish Meal and Meal without Protein Source).
For preparing feather hydrolysate meal, the supernatant will be mixed together with dextrin as the carbohydrate source, sunflower oil as lipid source, vitamin and mineral premix, α-cellulose as fiber and sodium carboxymethyl cellulose as binder. The ingredients for fish meal and meal without protein source will be prepared as the same way except for protein source which will be replaced with fish meal while nothing added for source of protein in meal without protein source. The concentration of nutrient in three different meals will be adjusted at the same amount. The mixture will be dried in oven at 50°C in the formed of pellet. Then, the pellets will be grinded by hand mortar and sieve to obtained particle sizes ranging between 0.3 and 0.5mm.
5.6) Analysis of Amino Acids Content in Feather Hydrolysate Meal, Fish Meal and Meal without Protein Source.
The amino acids analysis will be conducted by using Reverse Phase High Performance Liquid Chromatography (RPHPLC) Method. The standard amino acids and internal standard of protein will be analyzed together with the sample of meals. The protein samples will be hydrolyzed to its individual amino acids with hydrochloride before the amino acids analysis. The analysis will be performed by using Agilent Liquid Chromatograph. The graph obtained will be indicated the presence of amino 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 covers will be dried in oven with 100°C. Then, they will be transferred into desiccator and cooled. After that, weight of each crucible porcelains will be recorded (W1) till constant weight obtained. Next, 2 g of samples will be put into porcelain crucible. Later, the porcelain crucible will be placed in temperature controlled furnace preheated to 600°C for 2 H. After that, the crucible porcelain will be transferred directly to desiccator, cooled and immediately weighted (W2). The percentage of ash will be reporting to first decimal place based on the below formula:
% Ash (w/w) = [(W2 - W1)/(2g - W1)] x 100
5.7.2) Analysis of Dry Matter (AOAC, 2000).
2 g of samples will be dried at 100°C to constant weight. Then, percentage of dry matter will be calculated as follow:
% DM = 100 - % LOD, with
% LOD = (wt loss on drying (g) - 2 g) x 100
DM : Dry matter
LOD : 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 decimal place (W1). Then, samples will be dried for 5 H at 100°C. Beakers that will be used for fat determination will be dried at least 1 H at 100°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. Next, soxhlet apparatus will be used for extraction and distillation. Diethyl ether will be added for fat extraction. Extraction will be done for minimum of 4 H with condensation rate 5 to 6 per second. Then, distillation of ether will be done. After distillation process done, beakers that used for fat determination will be placed in the hood to remove all traces of ether by evaporation. Next, beakers will be placed in 100°C gravity convection oven no longer than 30 min. Then, beakers will be transferred into the desiccator and weighed will be reported (W3). The percentage of crude fat will be calculated as follow:
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% 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 potassium sulfate, 0.04 g anhydrous copper sulfate, 0.5 to 1.0 g alundum granules will be added. Then, 20 mL sulfuric acid will be added. Next, flask will be placed on preheated burner. Heating will be done until white fumes clear bulb of flask and continue heating for 90 min. Then, flask will be cooled followed with adding of 250 mL distilled water and cool to room temperature (less than 25°C). Next, distillation step will be conducted. Titration flask will be prepared by adding appropriate volume accurately measured acid standard solution to amount of water (VHCl) so that condenser tip is immersed. For reagent blank, 1 mL of acid will be pipetted and approximately 85 mL water will be added. 3 to 4 drops methyl red indicator solution will be added. Then, 2 to 3 drops of tributyl citrate will be added into digestion flask to reduce foaming. Another 0.5 to 1.0 g of alundum granules will be added. After that, flask will be slowly down side, 45% sodium hydroxide solution (approximately 80 mL) will be added sufficiently to make mixture strongly alkali. Then, flask will be connected immediately to distillation apparatus and will be distilled at about 7.5 boil rate (temperature set to bring 250 mL water at 25°C to boil in 7.5 min) until at least 150 mL distillate is collected in titrating flask. Digestion flask and titrating flask will be removed and the condenser tube will be rinsed with distilled water as the flask is being removed. Then, excess acid will be titrated with standard sodium hydroxide solution to orange endpoint (colour change from red to orange to yellow) and volume will be recorded to nearest 0.01 mL (VNaOH). Reagent blank will titrated similarly. The calculation will be done as below:
%N = [(mL of HCl x molarity of HCl) - (mL of NaOH x molarity of NaOH)] x 1.4007 / 1g x 100 Thus,
Crude protein (%) (DM basis) = %N x 6.25
5.6) Preparation of Tilapia and Feeding Trial.
Tilapia juveniles will be obtained and acclimatized in laboratory by giving them fish meal. The acclimatization will be conducted for 14 days. After that, they will be fasting for 24H before the experimental feeding conducted. Then, 30 tilapia juveniles with the average 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 feeding will be conducted three times per day (800, 1200, and 1600). The system will be maintained at a photoperiod of 12H light/ 12H dark. The weight of juveniles will be recorded once a week to adjust the feeding amount required. The feeding trial will be conducted till 4 weeks. At the end 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 growth rate (SGR, %), relative growth rate (RGW, %), feed conversion ratio (FCR, g/g), feed intake (g/day) and protein efficiency ratio (PER) will be calculated.
5.9) Observation of Effects of Feather Hydrolysate Meal on Tilapia at Histological Level.
The kidney and muscle of tilapia juveniles will be dissected for histological examination. The tissue will be cut and soaked in 4% of formalin for about 2H. Then, the tissue will be put into a serial ethanol ranging 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. Next, embedded process will be conducted with paraffin in the cassette. Lastly, microtome will be used to obtain a very thin slice of tissue for microscopic observation.
5.10) Statistical Analysis
Statistical analysis will be done by using ANOVA.
Flow Chart of Methodology
Feathers + Bacteria
(Degradation process: To collect the supernatant)
Determination of Nutrient Content in Feather Hydrolysate
(Protein, carbohydrate and lipid)
Preparation of Experimental Diets
(Feather Hydrolysate Meal, Fish Meal and Meal without Protein Source).
Analysis of Amino Acids Content in Experimental Diets
Determination of Proximate Chemical Analysis in Experimental
(Dry Matter, Ash, Crude Protein and Crude Lipid)
Preparation of Tilapia and Feeding Trial
Observation of Effects of Feather Hydrolysate Meal on Tilapia at Histological Level
Since the main composition of feathers is protein, the feather hydrolysate will be expected to show high protein content. The essential amino acids content in feather hydrolysate will be expected not destroyed since biological method is used for feather degradation instead of physical and chemical treatment. Tilapias also will be expected to show high percentage of weight gain compared to the fish meal and meal without protein source. Lastly, feather hydrolysate meal also will be expected to show no negative effects on the histology level of tilapia.
Contribution of Study toward Country
To provide an alternative source of protein for animal feedstuff with low cost and nutritive in future.