There is a need to search into alternative source of gelatin due to religious and health issues. In This study gelatin was extracted from chicken legs by modified acid extraction process and later lyophilized. Various tests were carried out such as identification, proximate analysis, pH, gel strength, clarity, viscosity and thermal analysis to characterize the gelatin. The result shows that the gelatin average yield obtained was 5.18%, pH 3.8, moisture content 7.17%, total protein content 93.77%, total fat content 0.93% and total ash 1.57%. The bloom strength was found high compared with commercially available bovine gelatin. All test results for the gelatin comply with pharmaceutical standard as stated in standard pharmacopeias.
Keywords: amino acid, characterization, chicken, extraction, gelatin.
Pratical Application: This benefits the poultry industry whereby waste material such as chicken leg can be processed to high value commercial gelatin. The chicken gelatin as an alternative source is acceptable for human consumption both for health or religious beliefs and adhering to the standard required in pharmaceutical and food industry.
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Gelatin is a series of protein products derived by partial hydrolysis of animal collagen (Gomez-Guillen, 2001). Due to its unique properties, it is widely used as biopolymers in food, pharmaceutical, cosmetics and photographic applications. In the pharmaceutical industry, it is well known to be used in the manufacturing of hard or soft capsules and wound care products, as a matrix for implants, injectables, drug delivery microspheres, and intravenous infusions (Saddler & Horsey, 1987; Pollack, 1990; Rao, 1995). In the food industry, gelatin is utilized in confections (mainly for providing chewiness, texture and foam stabilization), low-fat spreads (to provide creaminess, fat reduction and mouthfeel), dairy (to provide stabilization and texturization), baked goods (to provide emulsification, gelling and stabilization), and meat products (to provide water-binding) (Johnston-Banks, 1990; Schrieber & Gareis, 2007). As it has low calories and high protein, is recommended for use in foodstuff to enhance protein levels especially in body-building foods. Furthermore, it is used as a stabilizer in live attenuated viral vaccines for immunization against measles, mumps, rubella, Japanese encephalitis, rabies, diphtheria and tetanus toxin (Burke, Hsu & Volkin, 1999).
The need for alternative sources of gelatin arises from religious and health issues surrounding the current main sources of gelatins, namely porcine and bovine. Islam and Judaism prohibit consumption of porcine-based materials and bovine-based materials are not acceptable by Hindus and Buddhists (Sattar et al., 2004; Easterbrook et al., 2008). Outbreak of Bovine Spongiform Encephalopathy (BSE or Mad Cow Disease) caused concern on the safety of bovine tissue-derived collagens and gelatins, thus leading for the studies into new alternative sources mainly from fish and poultry. There are several studies being carried out for gelatins from the skin of various fish species (Jamilah & Harvinder, 2002; Gudmundsson, 2002; Irwandi & Che Man, 2009; Jamilah et al., 2011), but reported allergic reactions to bovine, porcine and/or fish gelatin particularly used in injectables has lead investigation to be carried out on poultry source (Kelso et al., 1993; Sakaguchi et al., 1995; Sakaguchi et al., 2000; Bogdanovic et al., 2009; Roger & Stokes, 2005).
Increase in chicken breeding in Malaysia from 2004 to 2009 shows the poultry meat production increased from 191,655.95 tonnes to 208,332.52 tonnes, indicating a tremendous increase in the demand of chicken production and consumption (data from Ministry of Agriculture, Malaysia, 2009). This has caused an increase in the production of waste material such as the chicken legs, which consists of high amount of proteins. Conversion of these high protein waste materials into value-added products could yield additional income which has both economic and waste management benefits, as its composition is not much different from those of bovine or porcine source (Eastoe et al, 1958). The low antigenicity of avian collagen and the growing demand for non-bovine collagen are two valuable reasons for further investigation of the functional properties of avian collagen in numerous applications (Cliche, 2003).
The aim of this study is to optimize the extraction technique of gelatin from poultry waste by-products in order to obtain a high yield, food and pharmaceutical-grade gelatin. Characterize the extracted gelatin based on existing pharmaceutical and food standards.
Materials and Methods
Chicken legs were purchased from a wholesaler in Kajang, Selangor where the shank to toes was obtained from chicken which were slaughtered for consumption in the open market, and processed immediately. All reagents used were of analytical grade and the experiments were carried out in triplicates. The bovine gelatin was obtained from Sigma Life Science USA, batch no: 126K00531 for comparative studies.
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Preparation of Chicken Leg Gelatin
The preparation of gelatin from chicken legs was performed using a modified procedure described by Nichlos-Simonnot, 1997; Muyonga, 2004; Gomez-Guillen et al., 2005 and Gomez-Guillen et al., 2009. About 600 g of chicken legs, were weighed and washed with water to remove the scales. They were then chopped into small pieces and later washed with about 10 litres of distilled water to remove blood from the bone marrow and other residues. After crushing further using a laboratory blender for about 3 - 5 minutes, they were washed again with 10 litres of distilled water to remove the blood and other debris.
The crushed chicken legs were then soaked in 2 litres of 0.5 N HCl and stirred at room temperature using a magnetic stirrer. During this treatment the wet ossein formed was filtered. The ossein obtained was washed with 20 litres of distilled water to remove the remaining acid and other impurities. Then to the wet ossein, 600 ml of distilled water was added and heated with continuous stirring at a constant temperature of 45Â°C Â± 2Â°C for 1 hour. The mixture was then filtered and the liquid was collected using muslin and the pH was adjusted using NaOH.
The clear liquid obtained was checked for its pH and pre-freeze at -80Â°C for about 4 days. Then the frozen clear supernatant was lyophilized in a Labconco Freeze Dry System (Model 77530-11, England) in a vacuum environment of 400 Ã- 10Â¬Â³Mbars at -50 to -41Â°C for 100 hours.
Identification test was carried out as outlined in British Pharmacopoeia 2010 for gelatin where the biuret method for determination of protein presence.
Yield was calculated based on the percentage of dried gelatin obtained over wet weight of chicken legs.
a. Determination of Moisture Content
The moisture content was determined using HR 73 Halogen Moisture Analyzer, utilizing Switch-Off Criterion number 3, as recommended in the operational manual (Mettler Toledo, Switzerland).
b. Ash Content
The test was carried out as outlined by Gelatin Manufacturers Institute of America (GMIA) by igniting the crucibles in a muffle furnace at 500Â°C Â± 25Â°C for an hour. After an hour the furnace door was left open and the crucible was removed from the furnace and cooled to room temperature in a dessicator. The empty crucible was weighed and recorded (W1). Approximately 3 g of gelatin was weighed into the crucible and the weight was recorded (W2). The gelatin was distributed evenly in the crucible and was charred on heating mantle until no smoke evolves. Then it was transferred into a muffle furnace at 500 Â± 25Â°C until the ash was grey in colour (about 6 hours). The crucible was weighed and recorded (W3). The content of ash was calculated as:
% Of Ash = (W3 - W1) X 100
c. Total Protein Content
The total protein measurement was carried out by using Foss Tecator Digestion System (Model 2020/2300) based on the Kjeldahl method for the analysis of total nitrogen content as a marker, using conversion factor of 5.4 to estimate protein content. The method was outlined in AOAC 17th edition and Handbook for Kjeldahl Digestion by FOSS DK-3400 Hilleroed, Denmark.
d. Total Fat Content
The total fat content determination was carried out using Soxtec 1047 System Hydrolyzing Unit and FOSS Soxtec Avanti 2055 Manual Extraction Unit. 1 g of celite was added to glass thimbles. Then 1.5 g Â± 0.001 g of control sample was weighed into a glass thimble and 1.5 g Â± 0.001 g of the gelatin was weighed into another glass thimble. The sample weight (W1) was recorded, empty aluminium extraction cups was weighed (W2) and extraction cup weight after fat extraction and cooling in dessicator (W3). The total fat content was calculated as:
% Fat = (W3 - W2) X 100
The pH value was determined as outlined in the British Pharmacopoeia 2010 for gelatin, using SevenEasy pH Meter S20 (Mettler Toledo, Switzerland). Standardization of the pH meter was carried out with pH 4.01, 7.00 and 9.21 buffers.
The clarity test was carried out as outlined in Gelatin Manufacturers Institute of America by using UV/Vis Spectrometer (Perkin Elmer Lambda 25). 7.50 g Â± 0.001 g of gelatin into a Bloom jar and 105 ml Â± 0.2 ml of deionized water was added. The gelatin particles was stirred and allowed to stand for 1 - 3 hours at room temperature. The gelatin was dissolved in a 65oC water bath for 10 - 15 minutes with continuous stirring using a glass rod. The sample was removed into a 45oC water bath and held until the temperature is 45 Â± 1oC. Transmittance at 640 nm was recorded, using deionized water as blank.
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Determination of Gel Strength
The gel strength of 6.67% gelatin gel was performed as indicated in British Pharmacopoeia 2010, using QTS Texture Analyser (CNS Farnell, England) with a radius cylinder probe of 11.8 mm.
The viscosity test was carried out by using Rheocalc V3.2 specification which was manufactured by Brookfield Engineering Labs. Viscosity of 6.67% gelatin solution (in cP) was determined using Brookfield DV III Ultra Rheometer (Model RV, USA), equipped with a CPE 41Z spindle and sample cup CPE-44PSYZ, measuring at 60Â°C Â± 2Â°C temperature within the range of 240 - 250 rpm.
The thermal analysis was carried out using the differential scanning calorimetric (DSC 6000 Perkin Elmer, U.S). It is connected to a chillier (C6, Perkin Elmer, U.S) and a thermal gas station (Perkin Elmer, U.S) to control the flow of the purge gas. The DSC was set with nitrogen as the purging gas at a flow rate of 20 ml/minute. Hermetically-sealed aluminium 20ml pans were used. Indium and zinc (Perkin Elmer U.S) were use to calibrate the DSC. Each samples weighing between 1.0 - 1.7 mg were scanned from -20Â°C to 370Â°C at heat flow rate of 10Â°C/min. The heat of fusion (DH), glass transition (tg) and melting point were determined from the resulting thermogram.
Results and Discussion
Identification test of chicken leg gelatin as in figure 1 showed, development of violet colour when added with blue copper (II) sulphate solution in an alkaline pH, thus confirming the presence of protein in the extracted chicken leg gelatin ( Zhou et al., 2006,British Pharmacopoeia 2010; Chang, 2010). The gelatine obtain is white spongy type which is due to lyophilisation. There is no odour in dry state but slight aroma when dissolved.
The yield and properties of gelatin depend on the kind of raw materials, pre-treatment and process method in the preparation of collagen. The average yield of chicken leg gelatin as summarized in table 1 was about 5.18%. In cases of fish gelatin the extraction yield from skin ranged about 5.5% - 21% of the weight of raw material (Gimenez et al, 2005a, 2005b; Grossman & Bergman 1992; Jamilah & Harvinder, 2002; Muyonga 2004; Karim & Bhat, 2009; Tavakolipour H, 2011). This yield was due to wet weight of crushed bones along with it bone marrows and other impurities before crushing. It was also found fish skin at temperature of 45oC and at extraction time between 15-60 minutes was optimal condition for gelatin preparation compared to the backbone and head of fish (Kolodziejsha, 2007).
The proximate analysis of chicken leg gelatin is summarized as in table 2. The moisture content varied not only with the extent of drying but also introduction of humidity during handling and storage (Ockerman and Hansen, 1988). The high protein content shows that the gelatin obtained is highly pure. The fat content could be reduced further by degreasing process being done before extraction (See et al., 2010). Low ash content shows that pre-treatment process was efficient in demineralising the bone. It also shows that the washing steps were sufficient in removing the acid and alkali residues while achieving the desired pH range (Ockerman and Hansen, 1998).
Variation in pH could be contributed by the different type and concentration of chemical used during the pre-treatment. The difference between the chicken leg gelatin and bovine gelatin is shown in table 1.
The clarity test as shown in figure 2 indicates that the chicken leg gelatin was lower compared to the bovine gelatin which is commercially available. This hazy or colloidal solution largely depends on efficiency of filtration process during extraction. However the clarity can be improved by treating the gelatin solution with ion exchange resin (Arnesen, 2007).
Bloom or gel strength is a measure of hardness, stiffness, strength, firmness and compressibility of the gel at a particular temperature and is influenced by concentration and molecular weight (Ockerman and Hansen, 1998). Result of bloom strength of chicken leg gelatin and bovine gelatin is shown as in table 1. The high bloom strength of chicken legs gelatin could be due to high molecular weight resulted from low extraction temperature as compared to extraction being carried out at high temperature. It was well established that hydrogen bonds between water molecules and free hydroxyl groups of amino acid in gelatin are essential for the gelatin's gel strength (Babel, 1998; Arnesen et al., 2002) and higher hydroxyproline content, the higher the gel strength of the gelatin (Sarabia et al., 2000).
Shear stress-shear rate data of gelatin solution were tested for various rheological models using the software provided along with rheometer. Some researchers have reported a clearly Newtonian behaviour for gelatine (Marcotte, 2001). The viscosity for the chicken gelatin at 60Â°C was in the range of 4.69 - 4.76 cP, compared to bovine gelatin was in the range of 4.51 - 4.57 cP as shown in figure 3. Thus showing that both the chicken gelatin and bovine gelatin has the similar flow characteristics which are Newtonian as shown in figure 4.
The DSC study as shown in figure 5 shows, that the onset and peak melting point of chicken leg gelatin was comparable with bovine gelatin. The chicken gelatin due to its origin from mammalian source has the same properties as that of other mammalian sources. It also indicating that the gelatins from different sources exhibit different degrees of plasticization (Rahman et al., 2008)
The present results shows the chicken leg gelatin obtained complies with the standard pharmacopeia requirements, indicating its characteristics and properties are similar to other mammalian and fish gelatin. This gives benefit to the poultry industry whereby waste material such as chicken leg can be processed to high value commercial gelatin, which can be used in pharmaceutical applications or as excipients and in food industry. It also indicates that the chicken gelatin could be acceptable for human consumption both for health or religious beliefs and as an alternative source that meets the standard required. It is estimated from the above study that about 5.342kg of dry gelatin can be produced from 100kg of chicken leg or at least from 1,250 slaughtered chickens.