Biochemistry Laboratory Formal Report CHEMISTRY
Isolation of Casein from Milk
600L EXPT 01 PAGE 01-02
Alexandra Veronica T. Rebosa*, Carlos Diego A. Rozul, Jose Ricardo P. Samson, Murielle F. Santiago Department of Psychology, College of Science
* e-mail:
[email protected]
Abstract The protein casein was isolated from nonfat milk by means of isoelectric precipitation. gradually adding acetic acid to the warmed milk in order to reach the isoelectric pH of milk, which is 4.6, and by filtering the resulting amorphous mass (casein). The obtained casein weighed 2.3382g and its percent yield was 46%.
Keywords: milk, protein, casein, isoelectric pH, isoelectric precipitation Introduction Milk is a very vital substance necessary for the nourishment of animals. It is also a rich source of nutrients such as B vitamins, calcium, fat, and protein. It is especially rich in protein, providing ten essential amino acids necessary for growth and development. A protein is a biomolecule made up of smaller monomers known as amino acids, which are joined together by peptide bonds (McGuire & Beerman, 2012). A peptide bond is a type of covalent bond which is formed when there is a loss of a water molecule between an amino group and a carboxylic acid (Petsko & Ringe, 2004). Amino 1
Biochemistry Laboratory Formal Report acids, also known as the building block of proteins, have three very important parts: (1) a central carbon (C) atom connected to a hydrogen (H) atom, (2) an amino group (---NH2), and (3) a carboxylic group (---COOH or ---COO-). Amino acids also contain a side chain connected to the amino group known as the R group (McGuire & Beerman, 2012). There are three different kinds of proteins found in milk. These proteins are known as caseins, lactalbumins, and lactoglobulins, all of which are globular (Menguito, et. al, 2014). Casein was the only milk protein isolated in the experiment via isoelectric precipitation. Casein is a type of protein found in milk when its pH reaches its isoelectric point at 4.6. It is obtained via the process of isoelectric precipitation, which involves the gradual acidification of the milk using acids such as acetic acid (HAc), in order for the milk to reach its isoelectric pH, the point in which the compound has no net charge (Fox & McSweeney, 2003). The basis for this is because at the milk’s isoelectric point, the protein loses its solubility to interact with its solvent. 5g of powdered nonfat milk and 10% acetic acid were used in isolating the casein in this experiment. This experiment aims to isolate casein from nonfat milk by isoelectric precipitation. Results and discussion For this experiment, the weight of the nonfat milk powder and casein, the initial pH of the milk, the volume of the acetic acid used, and the percent yield of the extracted casein were recorded. The obtained measurements can be found in Table 1 (see below): 2
Biochemistry Laboratory Formal Report Mass of nonfat milk powder Initial pH of milk Volume of acetic acid used Weight of casein % yield of extracted casein
5.0410g 6.55 0.18 mL 2.3382g 46.38%
Table 1. Obtained data from the isolation of casein Based on the data above, the nonfat milk used in the experiment is slightly acidic, though almost neutral given its pH. As the milk was heated and maintained to about 55C, acetic acid was gradually added in order for the milk to reach its isoelectric pH, which is 4.6. Once the milk reached its isoelectric point, the milk started to coagulate into chunks of an amorphous solid due to its diminished solubility. The obtained percent yield of the extracted casein in the experiment was 46.38%, which is to say that the nonfat milk used in the experiment consisted 46% protein. Cow’s milk is known to have around 80% casein protein while human milk is known to have about 40% casein (Breedon, 2012). Although human milk is known to have more or less the same amount of casein as the milk used in the experiment, milk can also have a lower casein content when its fat content is lower. Experimental methodology For the experiment, the protein casein was isolated from nonfat milk. 5g of powdered non-fat milk was dissolved in 20mL of warm distilled water in a 100mL beaker. Afterwards, the solution was heated to 55°C on a hot plate, after which the initial pH of the milk was recorded. 10% acetic acid solution was added to the dissolved mix in drops gradually while being stirred with a stirring rod. The acid solution was 3
Biochemistry Laboratory Formal Report continuously dropped into the nonfat milk mixture until the pH of the nonfat milk reached 4.6. The volume of the acid used was recorded. The mixture was left to stand until an amorphous mass was formed. The amorphous mass, casein, was decanted, pressed, and dried between two pieces of filter paper. Afterwards, the casein was weighed, and its percent yield was computed. Lastly, the casein was split into two portions; the first portion was saved for hydrolysis (the next experiment), and the second portion was stored in the refrigerator. References Fox, P.F. & McSweeney, P.L.H. Advanced Dairy Chemistry: Volume 1: Proteins, Parts A&B; Springer Science & Business Media: New York, USA, 2003; pp 2-6 McGuire, M. & Beerman, K. Nutritional Sciences: From Fundamentals to Food; Cengage Learning: USA, 2012; p 163 Menguito, C.A, Albano, P.B.S., Macabeo, A.P.G., Manansala, T.L., & Santiago, K.S. Basic Laboratory Experiments in Biochemistry; University of Santo Tomas College of Science: Manila, 2014; p 1 Petsko, G.A. & Ringe, D. Protein Structure and Function; New Science Press: London, UK, 2004; p 8 http://www.rpi.edu/dept/chem-eng/Biotech-Environ/PRECIP/precpph.html (retreived on August 6, 2014) http://ons.wvdhhr.org/Portals/20/PDFs/Aunt%20C%20Choosing%20Infant%20Milks%20 andFormulas%20Part2%20commercial%20formulas%204-12%20THIS.pdf` (retrieved on August 6, 2014)
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