Experiment 1: Isolation of Casein, Hydrolysis and Neutralization Rachel Mae Estrada*, Lloyd Jeffrey Gatbonton, Lawrence Go, Kristina

Carla Grant (Group 3)

Department of Biological Sciences, University of Santo Tomas, Manila, Philippines

Abstract
Casein protein is a milk protein extract recognized for its excellent amino acid profile, slow digestion, and interesting peptides (casomorphins, casokinins, casoxins, etc). In this experiment, Casein was isolated, hydrolyzed and neutralized from non-fat powdered milk. Casein was first isolated by adding 10% Acetic acid until if formed an amorphous mass after which, the mass was weighed and a percentage yield of 73.36% was computed. The isolated casein was further hydrolyzed and neutralized for the characterization.

Introduction Proteins, from the Greek proteios, meaning first, are a class of organic compounds which are present in and vital to every living cell. In the form of skin, hair, callus, cartilage, muscles, tendons and ligaments, proteins hold together, protect, and provide structure to the body of a multicelled organism. In the form of enzymes, hormones, antibodies, and globulins, they catalyze, regulate, and protect the body chemistry. In the form of hemoglobin, myoglobin and various lipoproteins, they affect the transport of oxygen and other substances within an organism. The total protein component of milk is composed of numerous specific proteins. The primary group of milk proteins are the caseins. There are 3 or 4 caseins in the milk of most species; the different caseins are distinct molecules but are similar in structure. All other proteins found in milk are grouped together under the name of whey proteins. The major whey proteins in cow milk are beta-lactoglobulin and alpha-lactalbumin. The major milk proteins, including the caseins, -lactoglobulin and a-lactalbumin, are synthesized in the mammary epithelial cells and are only produced by the mammary gland. The immunoglobulin and serum albumin in milk are not synthesized by the epithelial cells. Instead, they are absorbed from the blood (both serum albumin and the

immunoglobulins). An exception to this is that a limited amount of immunoglobulin is synthesized by lymphocytes which reside in the mammary tissue (called plasma cells). These latter cells provide the mammary gland with local immunity. Caseins have an appropriate amino acid composition that is important for growth and development of the nursing young. This high quality protein in cow milk is one of the key reasons why milk is such an important human food. Caseins are highly digestible in the intestine and are a high quality source of amino acids. Most whey proteins are relatively less digestible in the intestine, although all of them are digested to some degree. When substantial whey protein is not digested fully in the intestine, some of the intact protein may stimulate a localized intestinal or a systemic immune response. This is sometimes referred to as milk protein allergy and is most often thought to be caused by -lactoglobulin. Milk protein allergy is only one type of food protein allergy. Caseins are composed of several similar proteins which form a multi-molecular, granular structure called a casein micelle. In addition to casein molecules, the casein micelle contains water and salts (mainly calcium and phosphorous). Some enzymes are associated with casein micelles, too. The micellar structure of casein in milk is an important part of the mode of digestion of milk in the stomach and intestine, the basis for many of the milk products industries (such as the cheese industry), and the basis for our ability to easily separate some proteins and other components from cow milk. Casein is one of the most abundant organic components of milk, in addition to the lactose and milk fat. Individual molecules of casein alone are not very soluble in the aqueous environment of milk. However, the casein micelle granules are maintained as a colloidal suspension in milk. If the micellar structure is disturbed, the micelles may come apart and the casein may come out of solution, forming the gelatinous material of the curd. This is part of the basis for formation of all non-fluid milk products like cheese. Casein protein, like other protein sources, provides a rich amino acid supply to the body. Current data suggest that exercise can increase protein needs and that

increased protein intakes can improve the response to exercise training. Casein protein is slowly digested and this property of casein makes it optimal for consuming during the day as snacks in the form of dairy products or as a protein shake. Since casein slowly enters the blood stream, it has a negligible impact on protein synthesis. However, casein does have a powerful effect in suppressing protein breakdown. This may promote a better protein status over time. Casein protein makes up approximately 80% of the protein in milk. The beneficial properties of casein are partly a result of the amino acid composition and partly a result of the active peptides (the unique amino acid chain configurations that make up casein). In order to prevent the denaturing (or destruction) of the interesting peptides, appropriate processing techniques are required. Since milk protein isolates contain 80% casein, often people will use the terms milk protein isolate as casein interchangeably. The objectives of the experiment were to isolate casein from milk by isoelectric precipitation and to obtain information about the composition of the proteins using hydrolysis and neutralization. Methods and Materials For the isolation of casein from milk, 5 grams of powdered non-fat milk (Nesvita) was dissolved in 20mL warm distilled water. The solution was heated to 55C on a hot plate. The beaker was removed from the hot plate. A solution of 10% acetic acid was added while stirring with a stirring rod. The acid solution was added continuously until the ph reaches 4.6. The precipitated casein was stirred until it formed a large amorphous mass. The precipitate was filtered off by suction filtration or gravity filtration. For the hydrolysis of protein, acid hydrolysis was assigned to the group. To half of the protein isolate, 5mL of 8N H2SO4 was added. The flask was labelled and plugged with a piece of cotton. The flask was autoclaved at 5 psi for 5 hours. The appearance of the sample after autoclaving was noted. The hydrolyzate was diluted with 15 mL of distilled water. The hydrolyzate was then neutralized by adding solid Ba(OH)2 little by little until it reached pH 5. The Ba(OH)2 was dissolved completely before adding more.

Saturated Ba(OH)2 was added until the solution reached pH 7 after adding the solid one, upon reaching pH 5. A pH meter was used to monitor the pH of the solution while neutralizing. The neutralized solution was filtered and the liquid gathered (6.6 mL) was labelled and submitted for the characterization. Results and Discussion Proteins may be classified broadly in two general categories: fibrous and globular. Globular proteins are those that tend to fold back on themselves into compact units that approach nearly spheroidal shapes. These types of proteins do not form intermolecular interactions between protein units (H bonds, and so on) as fibrous proteins do, and they are more easily solubilized as colloidal suspensions. There are three kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins. All are globular. Albumins are globular proteins that are soluble in water and in dilute salt solutions. They are, however, denatured and coagulated by heat. The second most abundant protein types in milk are the lactalbumins. Once the caseins have been removed, and the solution has been made acidic, the lactalbumins can be isolated by heating 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 same conditions 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 and neutralization. The first step in the experiment was the isolation of casein from non-fat milk (Nesvita). The casein was precipitated by warming the powdered milk and adding 10% 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 and galactose. 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 is insoluble 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, the negative charges on the outer surface of the micelle are neutralized (the phosphate groups 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. Weight of sample Initial pH of milk solution Initial volume of 10% acetic acid Total volume used of 10% acetic acid Weight of dried casein Volume of neutralized casein collected 5.1406 g 6.13 6 mL 2.4 mL 3.7714 g 6.6 mL

Table 1. Results of the experiment

The weight of the non-fat powdered milk used in the experiment was 5.1406 g. The dried casein collected was 3.7714 g giving a percentage yield of 73.36 %.    
Figure 2. Calculation of the Percentage Yield of Casein

After the isolation of casein from non-fat milk, protein hydrolysis and neutralization followed. In the experiment, the isolated protein was subjected to acid hydrolysis. An 8N H2SO4 was added to the isolated casein and was autoclaved for five hours at 5 psi. Before autoclaving, the casein was colour beige and it was in its solid form and liquid 8N H2SO4 was added in the Erlenmeyer flask. After autoclaving, the solution was already a dark brown liquid. The hydrozylate was diluted with 15 mL of distilled water and was subjected to neutralization by adding solid Ba(OH)2 until reaching ph 5. While saturated Ba(OH)2

was added until it reached ph 7 after the solution reached ph 5. Its initial pH was 0.87 because the solution contained 8N H2SO4 upon autoclaving. Acid hydrolysis is the most common method for hydrolyzing a protein sample before amino acid analysis. The acid hydrolysis technique can contribute to the variation of the analysis due to complete or partial destruction of several amino acids. Tryptophan is destroyed; serine and threonine are partially destroyed; methionine might undergo oxidation; and cysteine is typically recovered as cystine (but cystine recovery is usually poor because of partial destruction or reduction to cysteine). Acid hydrolysis of proteins before analysis disturbs the original equilibrium between the two compounds so that the composition of the hydrolysate no longer reflects that of the protein. In neutralizing the casein, solid Ba(OH)2 was added to the hydrolyzate followed by saturated Ba(OH)2. Simplification of the procedure for isolating the mixture of free amino acids was the cause of using Ba(OH)2 as neutralizing agent. Conclusion Casein is precipitated by simply adjusting the pH of the milk to be sufficiently acidic that the protein is insoluble, taking care not to acidify too much so that the lactose does not hydrolyze. Lowering the pH leads to dissolution of calcium phosphate until, at the isoelectric point (pH 4.6), all phosphate is dissolved and the caseins precipitate. The other proteins remain water-soluble in acidic solution, but they can also be precipitated and isolated by merely heating the acidic solution and filtering. The isolated casein is insoluble in water, alcohol, and ether, but dissolves in alkaline and some acidic solutions. Proteins are least soluble in water at their isoelectric points and are more soluble at higher or lower pH's. The solubilitiy at pH's different than the isoelectric point appears to be due to the presence of an excess of cationic groups or anionic groups on the surface of the protein. If the protein is, for instance, negatively charged at a pH larger than the isoelectric point, when two proteins bump into each other, the net negative charge repels them and they do not aggregate as one would expect from large molecules. However, at the protein's isoelectric point there is no net charge. When a negative end of one protein bumps into a positive end of another protein, electrostatic attraction causes the two proteins to stick together. Other proteins run into the "dimer"

and join the group. Eventually, enough proteins aggregate together that the protein precipitates. In this experiment, casein( pI = 4.6) will be precipitated from milk (pH = ca. 7) by the addition of glacial acetic acid. Fats, which are present in whole milk, are not isolated in this experiment because powdered non-fat milk is used. References Voet D, Voet JG. (2004). Biochemistry Vol 1 3rd ed. Wiley Van Holde KE, Mathews CK. (1996). Biochemistry. Menlo Park, Calif: Benjamin/Cummings Pub. Co., Inc. Murray RF, Harper HW, Granner DK, Mayes PA, Rodwell VW. (2006). Harper's Illustrated Biochemistry. Jones, Alicia Noelle (2002). "Density of Milk". The Physics Factbook. Greenwood, Norman N.; Earnshaw, A. (1997), Chemistry of the Elements (2nd ed.)