Isolation and after autoclaving. The hydrolyzate was neutralized

Isolation and Hydrolysis of Casein from Milk Angelica Carlos, Ymarie Catacutan, Arriah Celicious*, Russel Dauigoy Department of Psychology, University of Santo Tomas, Manila, Philippines Abstract: Casein, a protein, was isolated from non-fat milk through isoelectric precipitation. A percent yield of 69.89% was obtained from this process. An acid hydrolyzate of casein was acquired from the method of acid hydrolysis. The isolated casein was observed to have undergone a qualitative change before and after autoclaving.

The hydrolyzate was neutralized for the subsequent color reactions. Introduction: During lactation, mammals produce milk through their mammary glands. Milk from cows consists of 87.1% water, 3.4% protein, 3.9% fats, 4.8% lactose, and 0.

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7% minerals.6 It is a source of nutrients for many animals, and it is used in the synthesis of many dairy products in the market today. Milk is a mixture of various types of proteins—most of which are present in small amounts. The basic constituents of living organisms are proteins. This is a macromolecule, having one or more long chains of amino acids bonded together by peptide bond.

A linear chain of bonded amino acids is called a polypeptide.1 Proteins can further be categorized into two types: globular and fibrous proteins. Fibrous proteins are elongated, filamentous proteins that often provide strength and protection to tissues and cells.

Globular proteins, on the other hand, are compact, spherical proteins that are involved in metabolic functions. While fibrous proteins are water-insoluble, globular proteins are soluble in water. The proteins found in milk are classified into three main groups, sorted based on their behaviors and forms: lactalbumins, lactogiobulins, and caseins.

These are all considered to be globular proteins. Furthermore, they are deemed to be complete proteins because they contain the amino acids imperative for building blood and tissue. Casein is considered the main protein. Itis a heterogeneous mixture of proteins in milk that contain phosphorous, and it is exists in milk as calcium salt and calcium caseinate. It forms micelle complexes, and constitutes 80% of mammalian milk.

3 Alpha, beta, and kappa-casein constitute the family of proteins that casein has. Aside from kappa-caseins, they are all naturally hydrophobic. With high amounts of phosphate, kappa-caseins can render water-soluble casein. Proteins are relatively easy to isolate from other biomolecules—carbohydrates, lipids, and nucleic acids. Nevertheless, it is difficult to separate them from each other due to their similar structures and properties. It is useful, therefore, to utilize their miniscule differences to be able to isolate them.

One method of protein isolation is isoelectric precipitation, wherein pH amounts and molecular charges come into play. At a certain pH value, also known as the isoelectric point, an amino acid does not migrate in an electric field and has a neutral charge—a net charge equal to zero.5 This is the pH where amino acids in a mixture can be separated. At 20?C, casein’s isoelectric point is 4.6. A protein can be further broken down to its individual amino acids that make it up.

The breaking down of protein into its component amino acids is a method called hydrolysis. This experiment aims to isolate casein from non-fat milk through isoelectric precipitation, and treat the isolated casein with acid or alkaline hydrolysis. Methodology: A.

Isolation of Casein 5 grams of powdered non-fat dry milk was dissolved in 20 milliliter of warm distilled water inside a 100 milliliter beaker. The solution was warmed to 55?C on a hot plate. A thermometer was used to monitor the temperature of the solution. The beaker was removed from the hot plate. The initial pH of the solution was taken note of. As the solution was being stirred with a stirring rod, 7 drops of 10% CH3COOH or acetic acid were added.

A large amorphous mass was waited to form in the solution. The mass was separated from the solution via decantation. The crude caffeine was dried with the use of filterpapers. The crude casein was weighed, and the percent yield was calculated. The crude casein was equally split into two portions—one portion was for the preparation of acid hydrolysis, and the other portion was encased with aluminum foil and stored for the following experiment’s color reactions test. B. Acid Hydrolysis and Neutralization The crude casein was cut into small pieces.

The casein fragments were transferred into a 50 milliliter Erlenmeyer flask. 4 moles 8 normal sulfuric acid was added in the Erlenmeyer flask. The Erlenmeyer flask was plugged with cotton.

The flask was covered with aluminum foil at the opening, and labeled. The appearance of the casein in the solution was taken note of. The solution was autoclaved at 15 psi for 5 hours. After autoclaving, the appearance of the sample was taken note of. The autoclaved sample was diluted with 15 milliliter of distilled water, and all the contents were transferred to a 250 milliliter beaker. The sample was neutralized by adding half a spatula of solid barium hydroxide. Saturated barium hydroxide was added until the pH read was around 7.

00. The neutralized solution was filtered. 7 milliliter of the filtrate was collected. Results and discussion: Weight of Non-fat Milk 5.0024 grams Initial pH 4.62 Final pH 4.60 Drops of 10% Acetic Acid Used 106 Weight of Crude Casein 3.

4960 grams Percent Yield 69.89% Appearance before hydrolysis Cream-colored, suspended in turbid solution Appearance after hydrolysis Black solution with black precipitate The data measured and observed are shown on the table above. The percent yield was computed as such: crude casein g x 100 = percent yield of non-fat milk g casein 3.

4960 grams x 100 = 69.89% casein 5.0024 grams Table 1. Results on Casein Isolation and HydrolysisThe isolation process of casein was commenced by dissolving the non-fat milk in 15 mL warm distilled water. A non-fat milk was used to prevent casein from binding with the fat molecules when heated.

Fat is present in whole milk, and as the name of the non-fat milk implies, it does not contain any fat molecule. This absence of fat gives way to an easier isolation of casein. The non-fat milk solution was heated to 55?C, but not higher, because this prevented the exposure of proteins. Beyond this temperature, the other proteins present in milk—lactalbumins and lactogiobulins—will become denatured. In this instance, the folds of globular proteins become disorganized and untangled. These uncoiled and disorganized proteins then coalesce and generate a solid mass in the solution, rendering an impure casein isolation.2 The isolation, as well as coagulation, of casein from non-fat milk utilized the alteration of pH. At isoelectric pH, the protein is uncharged.

This means that the charges at the amino side chains are equal to zero. Normally, proteins have charged amino side chains that have polar interactions with water. Due to the neutral charge brought about at isoelectric pH, the intermolecular repulsions are minimized, and the protein molecules coagulate, forming a solid mass that displays minimum water solubility. Since the pH of milk is around 6.

60, and the isoelectric pH of casein is 4.60, a dilute solution of acetic acid was used to lower the pH to the isoelectric point of casein. The isolated casein furthermore underwent acid hydrolysis. The peptide bonds present between amino acids are broken down with the use of a strong acid in the process of acid hydrolysis. In contrast to alkaline hydrolysis, this proceeds without racemization and with less destruction of certain amino acids. In this process, H2SO4 or sulfuric acid was used over other strong acids due to it being easily removed after hydrolysis. Prior to hydrolysis, the isolated casein was cream-colored, suspended in a turbid solution. A black solution with black precipitate was observed after hydrolysis.

This change is of great importance because it signified the destruction of the protein Tryptophan. When tryptophan is destroyed, it is converted to humin, a black pigment that gives the black color to the caseinsolution after hydrolysis.4 The proteins threonine and serine were also denatured in the process. Moreover, acid hydrolysis was accompanied by autoclaving during the experimentation. The process of autoclaving served as a catalyst during hydrolysis; the heat and pressure from autoclaving accelerate the hydrolysis of amino acids. Conclusion: In order to isolate casein from other proteins in powdered non-fat milk, the solution was subjected to a method called isoelectric precipitation. A percent yield casein of 69.

89% was obtained from the experiment. Acid hydrolysis broke down the crude casein into amino acids. It converted and destroyed certain amino acids such as tryptophan to humin, and denatured threonine and serine. References: (1) Berg, J., Tymoczko, J., & Stryer, L. (2002). Biochemistry, 5th edition.

New York: W H Freeman. (2) Denniston, K., Topping, J., & Caret, R.

(2004). General, Organic and Biochemistry. New York: McGraw Hill Companies. (3) Fang, Z., Visker, M., Miranda, G., Delacroix-Buchet, A.

, Bovenhuis, H., & Martin, P. (2016). The relationships among bovine ?S-casein phosphorylation isoforms suggest different phosphorylation pathways. Journal of Dairy Science, 8168-8177. (4) Iqbal, S., & Mido, Y.

(2005). Food Chemistry. New Delhi: Discovery Publishing House. (5) Kirkwood, J.

, Hargreaves, D., O’Keefe, S., ; Wilson, J. (2015).

Using isoelectric point to determine the pH for initial protein crystallization trials. Bioinformatics, 1444–1451. (6) Nokhodchi, A., ; Martin, G. (2015). Pulmonary Drug Delivery: Advances and Challenges.

Chichester: John Wiley ; Sons.


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