Isolation of Casein From Milk

In this experiment, casein was isolated from milk by means of isoelectric precipitation. A percent yield of 5% was obtained by the group.

Introduction

Milk is an opaque white or bluish-white liquid secreted by the mammary glands of female mammals, serving for the nourishment of their young. This liquid, as secreted by cows, goats or certain other animals are used by humans as food and as a source of dairy products such as cheese and butter. Milk composition differs widely among species. Factors causing these variances include: the type of protein; the proportion of protein, fat, and sugar; the levels of various vitamins and minerals; and the size of the butterfat globules, and the strength of the curd. On average, cow milk contains 3.4% protein, 3.6% fat, and 4.6% lactose, 0.7% minerals and supplies 66 kcal of energy per 100 grams. Bovine milk normally contains 30-35 grams of protein per liter. Of which, 80% is arranged in casein micelles.

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Figure 1. Model of Casein Supramolecule

Containing a fairly high number of proline residues, which do not interact and no disulfide bridges, casein has, as a result, relatively minimal tertiary structure. It is comparatively hydrophobic, resulting to its poor solubility in water. Showing only limited resemblance with surfactant-type micellae in a sense that the hydrophilic parts reside at the surface and are spherical, casein is found in milk as a suspension of particles called "casein micelles." On the other hand, the interior of a casein micelle is highly hydrated. The caseins in the micelles are held together by calcium ions and hydrophobic interactions.

Casein’s isolelectric point is 4.6. It has a negative. The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein becomes zero. At a solution pH that is above the pI the surface of the protein is predominantly negatively charged and therefore like-charged molecules will exhibit repulsive forces. Likewise, at a solution pH that is below the pI, the surface of the protein is predominantly positively charged and repulsion between proteins occurs. However, at the pI the negative and positive charges cancel, repulsive electrostatic forces are reduced and the attraction forces predominate. The attraction forces will cause aggregation and precipitation. The pI of most proteins is in the pH range of 4-6.

Mineral acids, such as hydrochloric and sulfuric acid are used as precipitants. The greatest disadvantage to isoelectric point precipitation is the irreversible denaturation caused by the mineral acids. For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins, rather than the target protein. The precipitation of casein during cheesemaking, or during production of sodium caseinate, is an isoelectric precipitation.tive charge in milk since milk’s pH is 6.6.1 Results and Discssion

Casein was isolated from milk by means of isoelectric precipitation. A percent yield of % was obtained by the group. Table 1 presents the data and results obtained from the experiment which includes: (1) the weight of powdered milk, (2) the initial pH, (3) the final pH, (4) the volume of the acetic acid used, (5) the weight of casein and (6) the percent yield. In order to compute for the percent yield, the weight of the isolated casein was divided by the weight of the powdered milk, and then multiplied by 100%.

Table 1. Data and results of the experiment: Isolation of Casein from Milk. Before autoclaving, the isolate was a white-yellowish solid with smooth texture. After autoclaving, it turned into a brown solution with black precipitate. The filtrate was a yellowish solution. The general principle behind this experiment is that when casein is at its isoelectric point, it is generally at the pH where it is least soluble. As a result, casein precipitates at this pH. To explain further, casein is present in milk as calcium salt, calcium caseinate. It is a mixture of alpha, beta and kappa caseins to form a cluster called micelle. These micelles were responsible for the white opaque appearance of milk.

The casein, as proteins, is made up of many hundreds of individual amino acids, each of which 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 pH of milk to the IEP of casein. At this pH, casein precipitates.

2. Experimental

5g of powdered non-fat dry milk was dissolved in 20 mL warm distilled water in a 100-mL beaker. The solution was heated on a hot plate to 55°C. The beaker was then removed from the hot plate. The initial pH of the milk solution was noted. A solution of 10% acetic acid was then added dropwise whole while being stirred by a stirring rod. The acid solution was continuously added until the pH reached 4.6. The volume of the acetic acid used was noted. The solution was left standing until a large amorphous mass was formed.

The isolated casein was dried between filter papers. The casein was weighed and the percent yield was determined. The isolated casein was then divided into two portions. One portion was used for acid/base hydrolysis. The other portion was stored in the refrigerator (to be characterized later using various chemical tests).

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