Isolation and Hydrolysis of Casein From Non Fat

ISOLATION AND HYDROLYSIS OF CASEIN FROM NON FAT MILK Acao, Praiseus Awat, Ann Christine Corpuz, Ma. Cristina Escalona, Ropelio Gianan, Amirah 3MB

Proteins One

of the four biomolecules Polymeric; the monomer is an amino acid Amino acids are linked via Peptide bonds Two types of proteins: Globular and Fibrous Some functions of protein include: Regulatory, Catalytic, Structural, Defensive, Storage, Transport, Receptor

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Milk    

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Common food which has high nutritional value 9 essential amino acids found in milk protein Different kinds of milk: Non-fat, Skim Milk, Low fat and pure fat Three proteins found in milk: Casein, lactalbumin and lactaglobulins. All are globular proteins. NON-FAT MILK was used to prevent FAT from 'contaminating' CASEIN. Fats go along with the protein isolation/hydrolysis

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Casein   

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One of the major proteins in milk Most abundant protein in milk (along with whey) Phosphoprotein- contains one or more phosphate groups attached in side chains (usu. serine or threonine) Slow-digesting protein (provides a sustained release of amino acids) A mixture of alpha, beta and kappa caseins form a cluster called micelle which is responsible for the white opaque appearance of milk. Micelle -  any water-soluble aggregate, spontaneously and reversibly, formed from amphiphile (possesses both hydrophobic and hydrophilic elements) molecules. pI is at pH 4.6

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OBJECTIVES   

To isolate casein by Isoelectric precipitation from non-fat milk To subject the isolated casein to acid and base hydrolysis NEUTRALIZE!!

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MATERIALS(NO NEED)     

5g Milk Magic 10% Acetic acid pH meter 8N H2SO4 (for acid hydrolysis) Ba(OH)2 (for base hydrolysis)

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METHODOLOGY 5g milk powder • Add 20mL distilled water • Stir warm to 55°C • Remove from the hot plate

Warm milk solution • Note initial pH • Add dropwise of 10% Acetic acid(HAc) while stirring until pH 4.6 (do not destroy the lumps formed) • Note the volume of HAc used • Let it stand

Casein

• Decant • Dry between filter and tissue papers • Compute for percent yield 7

% Yield casein

=

Actual Yield (g Casein) Theoretical Yield (g Milk) 100

Divide equally into two

Intact casein • Wrap with Aluminum foil and label • Refrigirate

Intact protein for next meeting

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Casein

• Cut in small pieces • Put in 50mL Erlenmeyer flask

Acid Base Hydrolysis Hydrolysis

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• Add 4ml of 8N H2SO4 • Plug with cotton and cover with aluminum foil then label • Note appearance • Autoclave at 15psi for 5 hours

Acid Hydrolyzate

• Check pH using the pH meter • If not yet neutralized, add dropwise of saturated Ba(OH)2 • Check pH again using the pH meter • Filter

Residue (discard)

• Dilute with 15ml distilled H2O • Transfer into 250mL beaker • Neutralize by adding 1 porcelain spatula of Ba(OH)2 then dissolve all of Ba(OH)2 by heating the solution while stirring, do not let it boil

Filtrat e Transfer to

graduated cylinder, make to 7mL

7mL Neutralized Hydrolyzate 9

Methodology 

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Non-fat milk was used so that it would be easier to isolate casein as whole milk contains more proteins. The milk was only warmed so as not to denature the milk. Denature – to cause the tertiary structure of (a protein) to unfold, as with heat, alkali, or acid 10

Methodology   

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Autoclave This was done for the heat-catalysed reaction. This was to free the amino acids in high pressure as the acid/base catalyses the reaction. HIGH PRESSURE = FASTER HYDROLYSIS

Group (Acid hydrolysis)

Before Autoclaving

After Autoclaving

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Casein did not dissolve; Casein pieces with clear liquid

Black Solid

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Chunky, Does not dissolve easily

Black solution with thin residues on the edges of the flask and some are floating

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Clear liquid with white solids

Black Solid

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Clear liquid with white clumps of white casein

Dark brown with white paper-thin brown casein

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Clear solution with white solids

Clear solution with white solids

• Check pH using the pH meter • If not yet neutralized, add dropwise of 8N H2SO4 until ph 7 • Check pH again using the pH meter • Filter

Base Hydrolysis • Add 5mL of boiling distilled H2O • Add 2.0g of Ba(OH)2 • Plug with cotton and cover with aluminum foil then label • Warm gently to dissolve all Ba(OH)2 • Note appearance • Autoclave at 15psi for 5 hours

Residue (discard)

Base Hydrolyzate • Dilute with 15ml distilled H2O • Transfer into 250mL beaker • Neutralize by adding 1mL of 16N H2SO4 dropwise

Filtrat e Transfer to

graduated cylinder, make to 7mL

7mL Neutralized Hydrolyzate 13

Group (Base hydrolysis)

Before Autoclaving

After Autoclaving

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Clay creamy white solid

Yellow orange with white precipitate

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Cloudy, light yellow solution with lumps

Cloudy, light yellow with small lumps

Darkish Yellow

Milk tea color brown

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Results Percent Yield Group 1

69%

Group 2

83.57%

Group 3

62.10%

Group 4

71%

Group 5

68.10%

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Results Percent Yield Group 6 Group 7

84.73%

Group 8

68.26%

Group 9

82.54

Group 10

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Discussion Isoelectric Point (pI) and Protein Solubility The pI of a protein is the pH at which protein is least soluble and as a result it can be easily 'caught' in a mixture or solution. The pH where a molecule has not net charge.

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Discussion Isoelectric Precipitation A

precipitate is a deposit of solid particles settled out of a solution. The milk containing the protein casein has an initial basic pH ranging from 6 to 7 (6.6). Acetic acid was added for it to reach the protein's isoelectric point, which is at pH 4.6. During the addition of the acid (donates H +), the negative charges on the outer surface of the micelle are neutralized (the phosphate groups are protonated), and the neutral protein precipitates.   The casein peptides and micelle structures become disturbed or denatured to form simpler structures which become gelatinous. Casein was obtained by reaching its pI. 18

Discussion Acid/Base Hydrolysis of Protein (Casein) Hydrolysis is the splitting or breaking of a substance into smaller pieces with the use of water. Protein hydrolysis may involve enzymes or chemical reagents and in this case, we used either an acid H2SO4 or a base - Ba(OH)2 for splitting proteins into smaller, amino acids. 19

Acid Hydrolysis   

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Utilizes sulfuric acid (H2SO4) Less amino acids are destroyed Hydrolysis that is frequently used over base hydrolysis IMPORTANT A.A. DESTROYED IS W Tryptophan to HUMIN (Black pigment after autoclave)

Base Hydrolysis 

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Destroys more amino acids than acid hydrolysis therefore it is less frequently used. (Targets S, T, C and R) Utilizes Ba(OH)2 (barium hydroxide) IMPT A.A. destroyed: R Arginine to UREA and ORNITHINE (No physical changes) 21

Neutralization   

Acid hydrolysis: Ba(OH)2 Base hydrolysis: 8N H2SO4 Sulfuric Acid + Barium Hydroxide = Barium Sulfate (Salt) formed in the hydrolyzate for further characterization of A.A. in EXPT 2

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References  

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Amphiphile. (n.d.) Collins English Dictionary – Complete and Unabridged. (1991, 1994, 1998, 2000, 2003). casein. (n.d.) Miller-Keane Encyclopedia and Dictionary of Medicine, Nursing, and Allied Health, Seventh Edition. (2003). Retrieved September 13 2015 from http://medical-dictionary.thefreedictionary.com/casein Clark, J. (2004). The hydrolysis of proteins. Retrieved from http://www.chemguide.co.uk/: http://www.chemguide.co.uk/organicprops/aminoacids/proteinhydrolysis.html denature. (n.d.) American Heritage® Dictionary of the English Language, Fifth Edition. (2011). Jacobs, J. (2015, June 12). What is calcium caseinate? Retrieved from Livestrong.com: http://www.livestrong.com/article/509796-what-is-calcium-caseinate/ micelle. (n.d.) Farlex Partner Medical Dictionary. (2012). Retrieved September 14 2015 from http://medical-dictionary.thefreedictionary.com/micelle Volek, J. Ph.D., R.D. (n.d.). Whey vs. casein protein. Retrieved from Nutrition Express: http://www.nutritionexpress.com/showarticle.aspx?articleid=787 vlab.amrita.edu,. (2011). Isoelectric Precipitation of Proteins: Casein from Milk. Retrieved 13 September 2015, from vlab.amrita.edu/? sub=3&brch=63&sim=158&cnt=1 Brooker, et. al. (2011). Biology. New York, NY: McGraw-Hill. Campbell, M. and Farell, S. (2015). Biochemistry. Canada. Cengage Learning.

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