1. INTRODUCTION
Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, DNA replication,responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific three-dimensional structure that determines its activity.
Milk is the most nutritionally complete food found in nature.Cow's milk and goat's milk are almost identical in every respect.The only important nutrients lacking in milk are iron and vitamin C. There are 3 main protein in milk: casein,lactalbumin and lactoglobulin.
Eggs are one of the few foods that are used throughout the world regardless of religion and ethnic group (Stadelman and Cotterill, 2001). The chicken egg is one of the perfectly preserved biological items found in nature and is also considered as the best source of protein, lipids, vitamins, and minerals.
Eggs consist of 3 main components: eggshell (9–12%), egg white (60%), and yolk (30–33%). Whole egg is composed of water (75%), proteins (12%), lipids (12%), and carbohydrates and minerals (1%; Kovacs-Nolan et al., 2005). Proteins present in egg are distributed among the egg white and yolk, whereas lipids are mainly concentrated in the yolk. Yolk is covered with the vitelline membrane and mainly consists of water (50%), protein (15–17%), lipids (31–35%), and carbohydrates (1%). Protein present in egg yolk consists of lipovitellins (36%), livetins (38%), phosvitin (8%), and low-density lipoproteins (17%). Also, yolk contains 1% carotinoides, which makes it yellow in color (Stadelman and Cotterill, 2001). In this experiment we will be isolating the casein from milk and egg albumin.
1.1 Theories and Principles
Milk is a mixture of many types of proteins, most of them present in very small amounts. Milk proteins are classified into three main groups of proteins on the basis of their widely different behaviors and forms of existence. They are caseins (80%), whey proteins and minor proteins. Casein is a heterogeneous mixture of phosphorous containing proteins in milk. Casein is present in milk as calcium salt and calcium caseinate. It is a mixture of alpha, beta and kappa caseins to form a cluster called micelle. These micelles are responsible for the white opaque appearance of milk.
Casein, like proteins, are made up of many hundreds of individual amino acids. Each may have a positive or a negative charge, depending on the pH of the [milk] system. At some pH value, all the positive charges and all the negative
charges on the [casein] protein will be in balance, so that the net charge on the
protein will be zero.
That pH value is known as the isoelectric point (IEP) of the protein and is generally the pH at which the protein is least soluble. For casein, the IEP is approximately 4.6 and it is the pH value at which acid casein is precipitated. In milk, which has a pH of about 6.6, the casein micelles have a net negative charge and are quite stable. During the addition of acid to milk, the negative charges on the outer surface of the micelle are neutralized (the phosphate groups are protonated), and the neutral protein precipitates.
The same principle applies when milk is fermented to curd. The lactic acid bacillus produces lactic acid as the major metabolic end-product of carbohydrate [lactose in milk] fermentation. The lactic acid production lowers the the pH of milk to the IEP of casein. At this pH, casein precipitates. (McMaster University Chem Lab Manual)
1.2 Objectives
The objective of this experiment is to isolate casein from milk and to isolate egg albumin
2. METHODOLOGY
The elderly known technique for casein separation is the physicochernical one. It even supports the casein definition (Gordon and Kaplan, 1972). Lowering milk pH to 4.6 leads to casein precipitation. The precipitate is washed several times in order to reach a satisfactory degree of purification. AlI types of acid can be employed as precipitants but the most used ar hydrochloric and sulphuric acids. Because of po or valorization of acid casein wheys, recent techniques of so called ionie acidification (Triballat, 1979; Rialland and Barbier, 1980, were recently developed. They are found on exchange of milk cations (Na t , K+, Ca++) with protons (H+) brought by ion exchange resins. The resulting wheys have a lower mineraI content, especially the ones coming from the Bridel process (Rialland et Barbier, 1980) which do not contain any acid anions. Another advantage of this last process is an increase of
casein yield due to the retention in the curd of the main proteose-peptone consequently of an hysteresis effect of solubility of this component (Pierre et Douin, 1984).
Inorganic salts can be utilized for the precipitation of proteins, with ammonium sulfate being the most common. The concentration of ammonium sulfate required for precipitation varies from protein to protein and should be determined empirically. Typically, ammonium sulfate is used in a series of steps performed at 2 to 8 C. For example, ammonium sulfate is added in increments to a concentration of 20% of saturation while gently stirring and allowed to dissolve and equilibrate between additions. Any precipitate is removed and discarded. This step typically yields macromolecules such as ribosomes, membrane fragments and even denatured proteins. This precipitation is then followed by increasing the ammonium sulfate concentration to 50% of saturation, in which the protein of interest is “salted-out” and collected via centrifugation. The remaining supernatant may contain additional “contaminating” proteins, which are then discarded. The collected precipitate can be resuspended in the minimal volume of buffer suitable for the next step in the purification process, typically via dialysis. Thus concentration, purification and buffer exchange are performed in one process. Sodium chloride can also be utilized in a similar fashion as ammonium sulfate but with lower yield and typically an increase in denaturation of proteins. In addition, magnesium sulfate, potassium or sodium phosphate, potassium or sodium acetate and other sulfate and phosphate salts can be used with varying success. Precipitation and purification in general are very protein specific and require a great deal of optimization. For more information about this subject or other SAFC Biosciences’ products and services, please contact our Technical Services department.
The materials used in this experiment were the following : test tube, beaker, graduated cylinder, fitration paper, thermometer and stirring rod. While the chemicals used were : 1% AgNO3, dilute HNO3, 95% Alcohol, HCL, (NH
₄
)₂
SO₄
, acetone, 1 N CH3COOH.2.2 Procedure
2.2.1 ISOLATION OF CASEIN FROM MILK
Prepare a beaker to evaporize milk then dilute to water and heat for 40C, after diluted, add 10% of hydrochloride solution dropwise and stir vigorously and thoroughly. Wait for another 10 minutes for precipitation. Stir for 5 minutes and pipette. A watch glass was then prepared for washing the curd in 5 mL of distilled water, stir vigorously and decanting to remove the residue. Wash until the chloride that was filtrate was free, if it was turn to yellow, the chloride is still present. Add 1-2 drops of HNO3 in the water to avoid losing of casein due to peptization. And let the curd dry in filter paper and wash using 95% alcohol, stir vigorously for 3 minutes. Then filter and remove the alcohol left by pressing the precipitate between filter paper extract and use acetone. Then weight the watch glass and calculate the casein yield in g/ml.
The egg white was separated fom the yolk from one medium sized chicken egg. The yolk was discarded and the volume of the egg white was measured using a pre-weighed beaker and The weight was determined. 0.1 mL of 1N acetic acid was added .It was stirred gently until no ovoglobulin precipitation was observed. A cheese cloth was used to filter in 250ml beaker and buffered saturated ammonium sulfate was added. After 30 minutes the mixture was transferred to the test tube and it was centrifuge. The clear yellow centrifuge was discard and was transferred to a beaker then buffered ammonium sulfate was added. It was stirred gently until precipitation observed. The mixture then was refrigerated.
3. RESULTS AND DISCUSSION
Proteins may be classified broadly in two general categories: fibrous andglobular. Globular proteins are those that tend to fold back on themselves into compactunits that approach nearly spheroidal shapes. These types of proteins do not formintermolecular interactions between protein units (H bonds, and so on) as fibrousproteins do, and they are more easily solubilized as colloidal suspensions. There arethree kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins. All areglobular. Albumins are globular proteins that are soluble in water and in dilute saltsolutions. They are, however, denatured and coagulated by heat. The second mostabundant protein types in milk are the lactalbumins. Once the caseins have beenremoved, and the solution has been made acidic, the lactalbumins can be isolated byheating the mixture to precipitate them. The typical albumin has a molecular weight of about 41,000.
A third type of protein in milk is the lactoglobulins. They are present in smaller amounts than the albumins and generally denature and precipitate under the sameconditions as the albumins. The lactoglobulins carry the immunological properties of milk. They protect the young mammal until its own immune systems have developed.
The experiment is on Casein isolation from non-fat milk, hydrolysis andneutralization. The first step in the experiment was the isolation of casein from milk (highland pasteurized milk). The casein was precipitated by warming the milk and adding10% acetic acid. It is important that the heating not be excessive or the acid too strong,because these conditions also hydrolyze lactose into its components, glucose andgalactose. It is necessary to avoid an excess of acid, since
the latter dissolves some of the protein. The 10% acetic acid was added continuously until the pH reaches 4.6.Calcium caseinate has its isoelectric (neutrality) point at pH 4.6. Therefore, it isinsoluble in solutions of pH less than 4.6. The pH of milk is about 6.6; therefore casein has a negative charge at this pH and is solubilized as a salt. If acid is added to milk, thenegative charges on the outer surface of the micelle are neutralized (the phosphategroups are protonated) and the neutral protein precipitates:
Ca-caseinate + 2H + ---> casein + Ca2+
Figure 1. Precipitation of Casein
As the pH falls the charge on casein falls and it precipitates. Hence milk curdles as it sours, or the casein precipitates more completely at low pH.
Table 1. Results from the isolation of casein from milk
Mass of watch glass + filter paper (g)
Mass of Residue + watch glass + filter
paper (g) Whey highland pasteurize d milk
28.5 g
29.5 g
10.5 mL
Table 1 show the results from the experiment. However, casein % was not calculated due to the reason that this experiment was not finished.
wt. of egg white ( mL )
wt. of filtrate egg white ( mL )
chicken egg 30 mL 19 mL
Ovalbumin is an almost spherical glycophosphoprotein which is the most common protein in egg white, representing nearly 60 % of the whole. The peptide chain consists of 385 amino acids (MW = 42,700 Da). Hydroxyl groups from the side chains of serine-68 and serine-344 are esterified with phosphoric acid, and an oligosaccharide is attached to the side chain of asparagine-292. In this experiment, egg albumin was isolated from egg white. However due to the shortage of time, this study is not finish. Those we were not able to discuss the results.
4. CONCLUSION
In this experiment, the isolation of casein from milk and the isolation of
egg albumin were unsuccessful. Due to the shortage of time, the study
resulted to a failure those prevent us to discuss the results.
5. REFERENCES
McKee, T. McKee, J.R. 2003. Biochemistry-The Molecular Basis of Life. 3rd
Edition.McGraw-Hill Companies, Inc
Retrieved 15 July 2010 from http://en.wikipedia.org/wiki/Egg_white
P. E. Stein et al., J. Mol. Biol. 1991, 221, 941. DOI: 10.1016/0022-2836(91)80185-W Chick H., Martin C. J.1913. The Precipitation of egg albumin by ammonium sulfate. A contribution to the theory of the “Salting Out” of proteins. Biochem. J.7:380–398. Asenjo, Juan A., Separation Processes in Biotechnology, New York: Marcel Dekker, 1990. 329-58. 2.
Janson, Jan-Christer and Lars Ryden, Protein Purification, New York: VCH Publishers, Inc., 1989. 10-24.
http://webcache.googleusercontent.com/search?
q=cache:http://www.weedemandreap.com/2013/05/milk-showdown-cow-vs-sheep-vs-goat-which-is-best.html
McMaster University, Chem2o6 Lab Manual, 1997,
http://www.chemistry.mcmaster.ca/chem2o6/labmanual/
Minard, B., Chem35 Synthetic Experiments, PennState University, 2002 http://courses.chem.psu.edu/chem35/HTML/Experiments/Exp112.pdf
