Isolation of Casein from Milk and Powdered Milk

Isolation of Casein from draw and Powdered Milk1.1 Introduction(Walsh, 2002) stated that, proteins are biological macromolecules composed of amino social diseases proteins be of one or more polypeptide which are the chain of amino stiflings integrated by peptide bonds.Alberts et al., (2013) detailed that, amino acids of proteins is either hy wastehobic or hydrophilic in nature. Therefore the resulting polypeptide chain shows an amphipathic characteristic. Hydrophilic amino acids exist peripher all(prenominal)y in some biological system and they are highly body of irrigate soluble. Whereas some amino acid does not exist the polar groups to the environment.The well-nigh important factors that influence protein solubility are structure, size, charge and the solvent (Burgess, and Deutscher, 2009). excessively Burgess, and Deutscher (2009) stated that, once the hardihood obtained, the etymon can be scattered by centrifugation or precipitation.Protein falls are aggregates o f protein molecular large enough to be visible and to be collected by centrifugation. The distribution of hydrophilic and hydrophobic residues at the surface of a protein determines its solubility properties. (Rosenburg, 2006). haste is mainly do for concentrate the target protein. And it is attained by adding reagents such as salts (ammonium convert) or organic solvents (acetone or ethanol). (Hatti-Kaul and Mattiasson, 2003)1.1.1 Isolation of casein paintMilk contains three kinds of proteins caseins, lact ovalbumins, and lactoglobulins, all of which are globular proteins. (Spurlock, 2014). Ahluwalia and Dhingra, (2005) stated that, Casein is a combination of phosphoproteins pass oning in milk and cheese.it is existing to the amount of 3% in milk on with 4-5% of lactose and 3-4% of fats and the rest is water system. Caseins exist in micelles which are composed of fill in micelles linked by the characteristic of hydrocolloid which are freely suspended in the aqueous phase of milk. (Tarte, 2009). Casein can be electrophoretically fractioned into four major components alpha-, beta-, gamma-and kappa- casein. Casein develops precipitation from milk at pH 4.6, which has a electronegative charge when compare to the pH of the milk. Therefor it can be accrue as salt by adding acids. (Miller, Jarvis and McBean, 2006).1.2 ObjectivesTo learn the methods of protein precipitation and to relate the solubility of protein with its structure.To learn the methods of closing off of casein from milk and to determine the percentage of casein presented in the (powdered) milk.1.3 Materials rivulet tubesBeakersPipetteClampFiltering paperElectronic sleepWatch glassBunsen burnerAlbumin sampleAmmonium sulphateSodium hydroxideCopper convertEthanolPicric acid course nitratePowdered milkWarm water1.4 Methodology1.4.1 hurry by saltsAlbumin, 3.00ml was interpreted into a leaven tube, ammonium sulfate was added to it and was abstruse until the ascendant gets saturated. The so lution was allowed to stand for nigh 5 minutes and filtered by development filter paper. The biuret strain was done to the filtered solution. 3.00 ml of filtered solution was taken into other test tube and same amount of NaOH was added to it, CuSO4 was added drop by drop.1.4.2 presumption by organic solventsAlbumin, 1.00 ml was taken into a test tube employ a pipette. And 4.00 ml of ethanol was added .the solution was conglomerate well and was allowed to stand.1.4.3 Precipitation by acidic agentsPicric acid solution, 1.00 ml was added into 1.00ml of albumin solution.1.4.4 Precipitation by heavy metal ionsLead nitrate, 8 drops were added into 1.00 ml of albumin.1.4.5 Precipitation by heat and acidAlbumin, 10 ml was taken into a test tube and the upper part of the solution was held all over the Bunsen flame. After the observation few drops of 1% acetic acid were added.1.4.6 Isolation of caseinPowdered milk (non-fat), 17.5 g was weighed by using electronic balance and was diss olved by adding 62.5 ml of warm water in a 200ml beaker. Acetic acid (10%) was added in a drop wise manner with stirring until the liquid changes in to clear solution. the obtained solution was filtered by using clamp, filtering material and beaker. The military issue casein was allowed to dry and was weighed using electronic balance. Biuret test was done for the filtered solution. 3.00 ml of filtered solution was taken into another test tube and same amount of NaOH was added to it, CuSO4 was added drop by drop.1.5 ResultsTestObservationInterferencePrecipitation by metal ionsWhite dissimulation precipitationProteins can be precipitated by metal ions(positive for proteins)Precipitation by heat and acidInitially cloudy white precipitation was find on the upper part of the solution and by adding acetic acid white colourize precipitation was observe.Proteins can be precipitate by heat and acid(positive for proteins)Precipitation by organic solventsWhite color precipitation was dis coveredProteins can be precipitate by organic solvents(positive for proteins)Precipitation by acidic agentWhite color precipitation was observedProteins can be precipitate by acidic agents. (positive for proteins)Precipitation of saltsBiuret testWhite color precipitation was observed.Purple color ring was observedProteins can be precipitate by salts.Positive for proteins.Isolation of caseinBiuret test for filtrationCasein 13.01g was weighedPurple color ring was formed in filtered casein solutionYield %= 100= 100= 74.30%Positive for proteins.1.6 DiscussionsPrecipitation of protein can be obtained by isoelectric precipitation method. isoelectric precipitation is the most wide employ method (Fox and McSweeney, 2003). Proteins can be precipitated by bringing their pH to its isoelectric point in which protein solubility is very low. (Shankara, 2008)Proteins can be precipitate by salts in two ways, half saturation with ammonium sulfate and full saturation with ammonium sulfate. Rashm i, (2002) stated that, distinct proteins show different precipitation reaction towards diverse agents. The full saturation with ammonium sulfate was done in the laboratory. Also the filtrate was tested by biuret reagent, resulted proud color. Compounds with two or more peptide bonds give a violet color with alkaline copper sulfate (Rashmi, 2002)Proteins are strong in solution when they are enclosed by entirely hydrogen-bonded water molecules, as water molecules with additional hydrogen bonding ability have greater stochasticity and are more aggressive. (Chaplin, 2014) hydrated sphere decrease the non - augury. Higher the diameter of the sphere higher the solubility. For an example, it is easy to precipitate globulin from proteins by adding salts, than albumin because globulin has downhearted diameter of hydrated sphere when compare to albumin.The similar concept is used in precipitating proteins by organic solvents and acidic agents. Organic solvents guide the hydrated sphere a nd decrease solubility resulting change magnitude precipitation. Acids neutralize the polarity of the hydrated sphere and decrease solubility in order to increase precipitation.Denaturation occur on heating or adding acidic agents to proteins. Therefor its change the polarity of a protein by changing the arrangements of polar and non-polar groups within the molecule. little polarity decrease the solubility and increases the precipitation.Precipitation by heavy metal ions lead nitrate was used instead of lead acetate or mercuric nitrate. Shankara(2008) stated that, metal ions which are positively charged colligate with negatively charged groups of the protein producing precipitation as metal-proteinate complex.According to the percentage of yield and from the result of biuret test of the filtrate, there can be some proteins present in the filtrate. Because, the milk contains about 3.5% protein by weight and of the total protein, about 80% is casein and 20% is whey protein. (Miller , Jarvis and McBean, 2006)Filtration of casein can be done in two ways. Such as, gravitational filtration and sucktional filtration.1.7. ConclusionProteins were precipitated by using metal ions, heat, organic solvents, acidic agents and salts.The percentage of yield casein of the sample is 74.30%.ReferencesAhluwalia, V. and Dhingra, S. (2005). College Practical Chemistry. Online Google Books. acquirable at http//books.google.lk/books?id=1OgRECl_nwMCpg=PA276dq=isolation+of+caseinhl=ensa=Xei=s0rBU_jZEtOzuATSzICACgved=0CCYQ6AEwAQv=onepageq=isolation of caseinf=false Accessed 12 Jul. 2014.Alberts, B., Bray, D., hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. (2013). Essential Cell Biology, Fourth Edition. 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Online Google Books. Available at http//books.google.lk/books?id=C-wrQaaXxj0Cpg=PA132dq=principle+of+milk+proteinhl=ensa=Xei=lgjCU_DhDZGTuASf_oGoAgved=0CCcQ6AEwAQv=onepageq=principle of milk proteinf=false Accessed 13 Jul. 2014.Walsh, G. (2002). Proteins. Online Google Books. Available at http//books.google.lk/books?id=EXTEjL2wTnYCprintsec=frontcoverdq=proteinhl=ensa=Xei=oEjBU4nXFZeTuAS62YGYDQved=0CD8Q6AEwBgv=onepageq=proteinf=false Accessed 12 Jul. 2014.