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Isolation from Chicken Liver and Enzymatic Hydrolysis of Glycogen and Characterization of Carbohydrates through Qualitative Testing

Group 7 2D-Pharmacy, Biochemistry Laboratory, Faculty of Pharmacy, University of Santo Tomas Hannah Co, John Kevin S. Magadia, Jem Marie Magno, Luisa Magsino, Irene Medina ABSTRACT The objectives of the experiment were to isolate polysaccharide from an animal source and characterize carbohydrates according to the qualitative test results. With chicken liver as the animal sample, grinding of sample, heating, filtration and various tests were conducted to extract glycogen from the said sample. Using Molischs and I2 Reaction tests, the turbid, dark yellow Glycogen isolate resulted with the appearance of a violet ring in Molischs test and gave up a clear light yellow solution in I2 Reaction test. After which were the Qualitative testing for glucose, fructose, xylose, lactose, sucrose and galactose. Each carbohydrate was subjected to Benedicts, Barfoeds, Seliwanoffs and Bials-Orcinol tests and gave positive and negative results further detailed in results and discussion. INTRODUCTION Carbohydrates are large group of organic compounds occurring in foods and living tissues and including sugars, starch, and cellulose. They contain hydrogen and oxygen in the same ratio as water (2:1) and typically can be broken down to release energy in the animal body. It can be polyhydroxyaldehydes (aldoses) or polyhydroxyketones (ketoses). Carbohydrates are divided into three general classes depending on the number of carbohydrate molecules they contain. These three classes are Monosaccharides, Oligosaccharides and Polysaccharides. METHODOLOGY I. Isolation and Enzymatic Hydrolysis Using mortar and pestle the Carbohydrate source, Chicken Liver, was ground thoroughly until no lumps were visible. Three milliliters of distilled water was added and sequentially transferred into a beaker for a 30-minute water bath. Occasionally, water has been added to prevent drying. To improve the precipitation of proteins, 1 mL of 0.1% acetic acid was added. Glycogen precipitation was done by adding 5-10 drops of ethanol to 1mL glycogen solution. To conduct Molischs Test, a few drops of Molischs Reagent were added into 1 mL glycogen solution. Two milliliters of concentrated H2SO4 were poured down the side of the tube to form a colored layer. On the other hand, I2 Reaction testing was done by adding a few drops of 0.01 M I2 into 1 mL sample solution. The enzymatic hydrolysis was conducted by placing 10mL of isolated carbohydrate in a beaker and adding 2.3mL of saliva to it. The solution was then allowed to stand for 30 minutes. After standing, the solution was placed into a dialyzing bag and suspended overnight into a small flask filled with 50mL distilled water. On the next meeting, the dialyzing bag was removed and discarded. The solution inside the flask was concentrated by heating over the hot plate until the volume reduced to about 2mL. Afterwards, the concentrated sample was tested for the presence of reducing sugar using the Benedicts test. II. Qualitative Test In separate test tubes, 5 drops of each carbohydrate sample (glucose, fructose, xylose, lactose, sucrose and galactose) was mixed with 1mL of the required reagent for the specific tests. The reagents used for the Benedicts, Barfoeds, Seliwanoffs, and BialsOrcinol tests were Benedicts reagent, Barfoeds solution, Seliwanoffs reagent and Orcinol in concentrated HCl respectively. RESULTS AND DISCUSSION I. Isolation and Enzymatic Hydrolysis Glycogen was successfully extracted from chicken liver. Precipitation of proteins, which was enhanced by the addition of 0.1% acetic acid, was brought about by boiling of the chicken liver with water. During heating, glycogen was left soluble in the solution. The impurities or precipitate was separated from the solution by filtration. Successful extraction was indicated by the acquisition of positive results from glycogen precipitation by ethanol, Molischs test and iodine reaction. Carbohydrates are detected in Molischs test using concentrated H2SO4. This permitted monosaccharides that will be dehydrated with the conc. H2SO4 to form furfural from pentoses, which reacts with 5% a-naphthol in 95% ethanol to form violet ring. The violet ring indicates the presence of carbohydrates. Positive iodine test indicates the presence of glycogen in a solution or a polysaccharide. Polysaccharides react with iodine to form reddish brown precipitate. The actual test resulted with a clear, light yellow

solution that indicates absence of glycogen. The error could have been on the mixing of sample and reagents or on the procedure of heating. The enzymatic hydrolysis was done to test for reducing sugar using the Benedicts test. The alkaline condition of this test causes isometric transformation of ketoses to aldoses resulting in the reduction of blue cupric ion to cuprous oxide, a brick-red precipitate. Actual result shows clear, green solution indicating absence of reducing sugar. II. Qualitative Test Results Benedicts test is used to distinguish non-reducing and reducing ones in carbohydrates. CuSO4, NaOH and tartaric acid are the components present in Benedicts reagent. All monosaccharides can reduce the blue cupric ion Cu+2 ions to brick red Cu2O. The brick red color indicates positive result. Table 1.0 shows the visible result for each corresponding sugar tested. Sugar Visible Result Glucose Brick red ppt (+) Fructose Brick red ppt (+) Xylose Brick red ppt (+) Lactose Brick red ppt (+) Sucrose Brick red ppt (+) Galactose Brick red ppt (+)
Table 1.0 Benedicts Test

Fructose Xylose Lactose Sucrose Galactose

Table 3.0 Seliwanoffs Test

Cherry red coloration (+)

Brown soln (-)

Red orange clear soln (-) Cherry red coloration (+) Red orange clear soln (-)

Bials-Orcinol test shows positive result for pentoses and is used to differentiate pentoses from hexoses. Bials reagent consists of orcinol, concentrated HCl, and ferric chloride (FeCl3). Pentoses are allowed to dehydrate resulting to furfural that condenses orcinol and ferric ion forming blue-green solution. Table 4.0 shows experimentation results. Sugar Visible Result Glucose Light green soln (-) Fructose Green soln (-) Xylose Blue green soln (+) Lactose Green soln (-) Sucrose Green soln (-) Galactose Green soln (-)
Table 4.0 Bials-Orcinol Test

Barfoeds test is more specific than Benedicts test because it shows visible results. Barfoeds test is used to distinguish whether a carbohydrate is monosaccharide or a disaccharide. Its reagent consists of cupric acetate and acetic acid. All monosaccharides are reducing sugars, they reduce Cu+2 ions to a brick red Cu2O when heated. Brick red precipitate indicates positive result. Table 2.0 shows experimentation results. Sugar Visible Result Glucose Brick red ppt (+) Fructose Brick red ppt (+) Xylose Brick red ppt (+) Lactose Clear green soln (-) Sucrose Brick red ppt (+) Galactose Brick red ppt (+)
Table 2.0 Barfoeds Test

REFERENCES 1. Crisostomo, A. C.; Daya, M. L.; et al (2010). Laboratory Manual in Biochemistry. C & E Publishing Inc. 2. 3. Johnson, C.R., Miller, M.J., Pasto D.J. (1998). Experiments and Techniques in Biochemistry. United Stated of America. Prentice Hall Inc. 4. Bettelheim, F.A. and March, J. (1998). Organic and Biochemistry. 3rd ed. New York: Harcout Brace College Publishers

Seliwanoffs test is used for ketoses. It consists of resorcinol in 6M HCl. The concentrated HCl allows ketoses to undergo dehydration than aldoses that further condenses forming a cherry red positive result. Ketoses containing compounds can also yield positive results. Fructose and sucrose are ketoses while lactose glucose, xylose and galactose are aldoses. Table 3.0 shows experimentation results.

Sugar Glucose

Visible Result Clear orange soln (-)