Isolation of Casein From Milk and Acid
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Transcript of Isolation of Casein From Milk and Acid
ISOLATION OF CASEIN FROM MILK AND ACID/BASE HYDROLYSIS AND NEUTRALIZATION
Alambra
Barrion
Bravo
Chavez
Escobar
Gonong
Yap
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
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
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
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
-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
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
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.
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.