ISOLATION OF CASEIN FROM MILK AND ACID/BASE HYDROLYSIS AND

NEUTRALIZATION
Alambra Barrion Bravo Chavez Escobar Gonong Yap

INTRODUCTION

PROTEINS
It

is naturally occurring, unbranched polymer, with monomer unit called amino acids. Proteins are chains of amino acid molecules connected by peptide bonds. It is a peptide in which at least 50 amino acid residues are present. It can be categorized in two types: Fibrous Globular

FIBROUS PROTEINS
tend

to form long and strong repeated sets of amino acid residues Unlike globular proteins, it forms intermolecular interactions between the side chains of the residues

GLOBULAR PROTEINS
tend

to fold back on themselves into compact units that approach nearly spheroidal shapes do not form intermolecular interactions between protein units

PROTEIN HYDROLYSIS
Protein

that has been hydrolyzed or broken down into its component amino acids bonds of proteins are hydrolyzed by either strong acid or strong base.

Peptide

MILK
It

is a complex biological fluid with high amount of proteins, lipids, and minerals. It is a good source of calcium and phosphorus but it is deficient in iron, and a poor source of ascorbic acid. Milk proteins contain all 9 essential amino acids required by humans. The function of milk is to supply nutrients such as essential amino acids required for the growth of the newborn.

CASEIN
It

is the main protein in milk It is a phosphoprotein which has phosphate groups attached to the hydroxyl groups of some of the amino acids side-chains. Casein exists in milk as a calcium salt, calcium caseinate. Calcium caseinate has an isoelectric point of pH 4.6

CASEIN
Casein consists of a fairly high number of proline peptides, which do not interact. There are also no disulfide bridges. As a result, it has relatively little tertiary structure. Because of this, it cannot denature It is relatively hydrophobic It is found in milk as a suspension of particles called casein miscelles which show some resemblance with surfactanttype miscellae

OBJECTIVES OF THE EXPERIMENT

To isolate casein from non-fat milk by isoelectric precipitation. hydrolyze casein with an acid or a base

To

ISOLATION OF CASEIN FROM NON-FAT MILK (MILK MAGIC)

PROCEDURE AND RESULTS

5g powdered non-fat dry milk

--dissolve in 20mL distilled H2O --heat the sol n to 55oC on hot plate --take note of initial pH --add 10% CH3COOH dropwise until pH of solution reaches 4.6

casein

PROTEIN HYDROLYSIS SCHEMATIC DIAGRAM

casein
--filter by gravity filtration

casein (residue)

whey (filtrate)

--dry between filter papers and weigh --calculate % yield --divide into two portions
Acid/Base hydrolysis Color reactions

ACID HYDROLYSIS PROCEDURE

protein isolate
-add 4mL 8N H2SO4 -label flask, plug with cotton, and cover with aluminum foil -note appearance before autoclaving -autoclave the flask at 15psi for 5 hrs. -note appearance after autoclaving -dilute hydrolyzate with 15mL d.H2O and transfer to 250mL beaker

-neutralize the hydrolyzate by adding spatula full of Ba(OH)2 -check pH using litmus paper (red purple); confirm using pH paper -if not yet neutralized, add saturated Ba(OH)2 solution dropwise -filter off precipitate formed and wash with 2mL hot H2O twice -If the volume of the filtrate is less than 15mL, make up to 15mL then proceed to color reactions

Acid hydrolyzate

Before autoclaving Group 1 Group 3 Group 5 Group 7 Group 9 Dark brown liquid with thin brown residues White solution Dark brown liquid with thin brown residues White solution

After autoclaving Clear yellow liquid Black solution with black precipitate Clear yellow liquid Black solution

Brownish-black White pieces of casein in solution with brown clear liquid flakes Colorless/clear liquid with suspended casein particles Brown to black solution with light brown particles

Group 11

BASE HYDROLYSIS PROCEDURE

protein isolate
-add 5mL boiling H2O and 2.5g Ba(OH)2 -label flask, plug with cotton, and cover with aluminum foil -note appearance before autoclaving -autoclave the flask at 15psi for 5 hrs. -note appearance after autoclaving -dilute hydrolyzate with 15mL d.H2O and transfer to 250mL beaker

-neutralize the hydrolyzate by adding 1.0mL 16N H2SO4 -check pH using litmus paper (red purple); confirm using pH paper -if not yet neutralized, add 8N H2SO4 solution dropwise -filter off precipitate formed and wash with 2mL hot H2O twice -If the volume of the filtrate is less than 15mL, make up to 15mL then proceed to color reactions

Base hydrolyzate

Before autoclaving Group 2 Group 4 Group 6 Group 8 Group 10 Cloudy white solution Cloudy solution Yellowish form, smooth texture Solid light yellow product

After autoclaving Yellow cloudy solution Turbid, curd forms, yellow color Yellowish liquid with small amounts of white precipitate

Yellowish soulition with lumps of undissolved casein Yellowish liquid with small amounts of white precipitate

Group 12

Solid light yellow product

DATA AND RESULTS

Percent Yield Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 65.7% 78.1% 72.5% 65.9% 73.4% 54.6%

Percent Yield Group 7 Group 8 Group 9 Group 10 Group 11 Group 12 48% 66.3% 69.9% 69%

PERCENT YIELD:

DISCUSSION

PRINCIPLES INVOLVED IN ISOLATION OF CASEIN

Isoelectric Precipitation Acid Hydrolysis Alkaline Hydrolysis Neutralization

ISOELECTRIC PRECIPITATION
Precipitation- formation of a solid in a solution or inside another solid during a chemical reaction. When the reaction occurs in a liquid, the solid formed is called the precipitate. The precipitation from suspension of a protein when the pH is at the isoelectric point

ISOELECTRIC PH
Protein is uncharged Positive charge = Negative charge Minimized intermolecular repulsions Displays minimum water solubility

ISOELECTRIC PH
pH of milk = 6.6 Isoelectric pH of casein= 4.6 10% Acetic Acid- phosphate groups present in casein is protonated and the neutral protein precipitates. causes the casein micelles to destabilize/aggregate by decreasing the pH. aggregation occurs as a result of entropically driven hydrophobic interactions precipitates casein by coagulation Increases solubility of organic calcium and phosphorous in the micelle

ISOELECTRIC PRECIPITATION

pH< 4.6
soluble

pH= 4.6
insoluble

pH= 4.6

pH> 4.6
soluble

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.

CASEIN MICELLE

A: a submicelle; B: protruding chain; C: Calcium phosphate; D: -casein; E: phosphate groups

ACID HYDROLYSIS
Acid

hydrolysis proceeds without racemization and with less destruction of certain amino acids (S, T, C, R) than alkaline treatment. It is most likely the method of choice in the analysis of proteins and polypeptides. Tryptophan is destroyed by acid hydrolysis. In acid hydrolysis, the acid itself also acts as a catalyst Yields burry-brown solution.

WHY AUTOCLAVE THE HYDROLYZATE

Heating with strong H2SO4 There is complete destruction of tryptophan, a severe loss of cysteine and minor losses of serine and threonine.

BASE HYDROLYSIS
Serine, Threonine, Cystein, and Arginine

are destroyed in base hydrolysis. It is not used much because it destroys more amino acids compared with acid hydrolysis. Ba (OH)2 The base itself also acts as a catalyst.

NEUTRALIZATION
Ba(OH)2 -neutralizing

agent Procedure for isolating mixture of free amino acids

CONCLUSION
Casein

was isolated through isoelectric precipitation with the use of a weak acid, acetic acid. The isoelectric pH of casein is 4.6 Casein isolated was hydrolyzed using a stron acid (H2SO4) and a strong base (Ba(OH)2). W is destroyed in acid hydrolysis S,T,R,C are destroyed in base hydrolysis.