ISOLATION OF CASEIN FROM MILK AND ACID/BASE HYDROLYSIS AND NEUTRALIZATION

Alambra

Barrion

Bravo

Chavez

Escobar

Gonong

Yap

INTRODUCTION

PROTEINSIt 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:FibrousGlobular

FIBROUS PROTEINStend 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 PROTEINStend 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

Peptide bonds of proteins are hydrolyzed by either strong acid or strong base.

MILKIt 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.

CASEINIt is the main protein in milkIt 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 surfactant-type miscellae

OBJECTIVES OF THE EXPERIMENT To isolate casein from non-fat milk by

isoelectric precipitation.

To hydrolyze casein with an acid or a base

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% CH3COOHdropwise until pH of solution reaches 4.6

casein

PROTEIN HYDROLYSIS SCHEMATIC DIAGRAM

casein

--filter by gravity filtration

whey(filtrate)

casein(residue)

--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 After autoclaving

Group 1 Dark brown liquid with thin brown residues

Clear yellow liquid

Group 3 White solutionBlack solution with black precipitate

Group 5Dark brown liquid with thin brown residues Clear yellow liquid

Group 7 White solution Black solution

Group 9 White pieces of casein in clear liquid

Brownish-black solution with brown flakes

Group 11Colorless/clear liquid with suspended casein particles

Brown to black solution with light brown particles

BASE HYDROLYSIS PROCEDURE

-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

protein isolate

-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 After autoclaving

Group 2 Cloudy solution Yellow cloudy solution

Group 4 Yellowish form, smooth texture

Turbid, curd forms, yellow color

Group 6 Solid light yellow product

Yellowish liquid with small amounts of white precipitate

Group 8

Group 10 Cloudy white solutionYellowish soulition with lumps of undissolved casein

Group 12 Solid light yellow product

Yellowish liquid with small amounts of white precipitate

Percent Yield

Group 1 65.7%

Group 2 78.1%

Group 3 72.5%

Group 4 65.9%

Group 5 73.4%

Group 6 54.6%

DATA AND RESULTS

Percent Yield

Group 7 69%

Group 8

Group 9 69.9%

Group 10

Group 11 48%

Group 12 66.3%

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

pH= 4.6

pH< 4.6soluble insolubl

e

ISOELECTRIC PRECIPITATION

pH= 4.6

pH> 4.6soluble

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: κphosphate groups

ACID HYDROLYSISAcid 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

catalystYields 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

CONCLUSIONCasein 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 hydrolysisS,T,R,C are destroyed in base hydrolysis.