Experiment 1: Isolation and Hydrolysis of Casein from Milk Agamata | Briones | Chan | Espineda | Juniosa
Introduction ● Protein (Bollag, Rozycki & Edelstein, 1996) α-Amino acids connected by peptide bonds Primary, secondary, tertiary, and quaternary structures ● Milk (Bollag, Rozycki & Edelstein, 1996) ○ Composed of vitamins, minerals, proteins, carbohydrates, and lipids. ○ Proteins: casein, alpha-lactalbumin and beta-lactoglobulins ○ pH= 6.6 ● Casein (Ferrier, 2014) ○ Alpha (α-s1 and α-s2), beta, and kappa casein ○ Isoelectric pH= 4.6 ○ ○
Objectives ● To isolate casein from non-fat milk by isoelectric precipitation ● To subject isolated casein to acid or alkaline hydrolysis
Methodology
5 g powdered nonfat milk
-- dissolved in 20 mL distilled water -- heated the solution to 55°C -- took note of the initial pH -- added 10 % CH3COOH dropwise until pH of solution reaches 4.6
--decant
Casein (residue)
Whey (filtrate)
-- dried between filter papers then weighed -- calculated the percentage of casein -- divided into 2 portions Half intact casein
Half intact casein
Half intact casein
-- added 4 mL of 8N H2SO4 to the casein in a 50 mL erlenmeyer flask -- autoclaved at 15 psi, 121°C for 5 hours -- diluted with 15 mL distilled water -- neutralized by adding solid Ba(OH)2 until pH is 6 -- added saturated Ba(OH)2 (dropwise) until pH is 7 -- confirmed pH 7 using pH meter -- filtered Filtrate (for color reactions)
Residue (discarded)
Half intact casein
-- added 5 mL boiling water and 2 g of Ba(OH)2 to the casein in a 50 mL erlenmeyer flask -- warmed the flask -- autoclaved at 15 psi, 121°C for 5 hours -- diluted with 15 mL distilled water -- neutralized by adding 1.0 mL of 16N H2SO4 -- added 8N H2SO4 (dropwise) until pH is 7 -- confirmed pH using pH meter -- filtered Filtrate (for color reactions)
Residue (discarded)
Results for Casein Isolation Group
Percentage of isolated casein
Group
Percentage of isolated casein
Group 1
70.46 %
Group 6
66.37 %
Group 2
64.10 %
Group 7
68.07 %
Group 3
67.96 %
Group 8
67.62 %
Group 4
67.23 %
Group 9
68.98 %
Group 5
66.34 %
Results for Acid Hydrolysis Group Number
Appearance of the hydrolyzate before autoclaving
Appearance of the hydrolyzate after autoclaving
Group 1
White soft solids, colorless liquid
Brown fragments, dark brown liquid
Group 3
White clumps in gray turbid liquid
Black precipitate in black solution
Group 5
White solid precipitate with colorless liquid
Black solution with black precipitate
Results for Acid Hydrolysis Group number
Appearance of the hydrolyzate before autoclaving
Appearance of the hydrolyzate after autoclaving
Group 7
Turbid solution with white precipitate
Brownish black solution, black precipitate
Group 9
Clear liquid with protein chunks
Black liquid coloration with brown flakes and precipitate
Results for Alkaline Hydrolysis Group Number
Appearance of the hydrolyzate before autoclaving
Appearance of the hydrolyzate after autoclaving
Group 2
Yellow liquid with white clumps
Turbid, yellow orange liquid, with precipitate
Group 4
Light yellow liquid with small clumps
Light yellow turbid liquid with yellow precipitate
Group 6
Dark yellow solution w/ undissolved casein
Creamy yellow solution with ppt
Group 8
White clumps with yellow liquid
Cream colored precipitate w/ solution
Discussion ● Isolation of Casein ● Acid Hydrolysis ● Alkaline Hydrolysis ● Neutralization
Isolation of Casein by Isoelectric Precipitation Isoelectric Point (pI) - the pH at which protein is least soluble and has a neutral or 0 net charge The pI of casein is approximately 4.6 and it is the pH value at which acid casein is precipitated. Milk has a pH of about 6.6 so when the pH drops to 4.6 the casein then be insoluble and will precipitate. http://www.teachnlearnchem.com/Matter_and_Energy/PDF/casein%20glue.pdf.
Calcium phosphate
Alpha and beta casein
Kappa casein http://www.livestrong.com/article/543320-isolation -of-casein-lactose-from-milk/
Calcium Caseinate
+
H+
Calcium + Casein
Isolation of Casein by Isoelectric Precipitation ● Skim Milk vs. Whole Milk (Bollag, Rozycki & Edelstein, 1996) Skim milk was used because of its 0 fat content unlike whole milk which contains fat that can remain with the casein once it precipitates. ● Heating at 55°C (Boyer, 2009) Other proteins might also hydrolyze
●
Dropwise addition of 10% Acetic Acid (Campbell, 2015) -- during the addition of acid to milk, the negative charges on the outer surface of the micelle are neutralized. The phosphate groups are protonated & the neutral protein precipitation -- pH should reach 4.6
●
Drying the isolated casein between filter papers and tissue papers to remove excess water in the casein
●
Storing in the refrigerator (Garett & Grisham, 2010) -- slow down enzyme activity and prevent growth of microorganism
Protein Hydrolysis ➢
Breakdown of protein into smaller peptides and free amino acids called protein hydrolysates
➢
During Hydrolysis, the peptide bonds in protein molecules break to yield diand tripeptides, and free-amino acids 2 types:
➢
●
Acid Hydrolysis
●
Alkaline Hydrolysis
(Copeland, 1994)
Autoclave Cut into small pieces → Autoclave at 15 psi ● Speeds up hydrolysis ● It is a acid catalyzed reaction ● Breaks the peptide bonds
http://oomyceteworld.net/protocols/autoclave%20operation.pdf.
Acid Hydrolysis ●
Proceeds without racemization wherein all amino acids retain their structural conformation & with less destruction of certain amino acids than alkaline hydrolysis
●
Acid itself acts as the catalyst
●
Addition of 8N H2SO4
●
Tryptophan is usually totally lost in an acid hydrolysis. Cysteine, serine and threonine are partially broken down and asparagine and glutamine are converted to their acidic forms, aspartic acid and glutamic acid.
●
Yields a dark brown or black solution
(Copeland, 1994)
Alkaline Hydrolysis ●
Amino Acids such as Serine, Threonine, Cysteine, and Arginine are destroyed in base hydrolysis
●
With racemization wherein amino acids have their structures altered
●
Not used much because it destroys more amino acids
●
Base itself acts as catalyst
●
Addition of Ba(OH)2
● Arginine converted to urea and ornithine which gives the yellow orange color. (Copeland, 1994)
Neutralization ● A chemical reaction in which a strong acid and a strong base react with each other and reach the neutral pH, pH 7.
● Ba(OH)2 beneficial in complete removal of sulfate ions because the BaSO4 formed is insoluble in water and precipitates out. (Ferrier, 2014)
Using a pH meter ● For Acid Hydrolysis, Crystallized Ba(OH)2 was added until the pH is 6 then saturated Ba(OH)2 solution was added dropwise until the pH is 7. ● For Alkaline Hydrolysis, 16N or the more concentrated H2SO4 was added dropwise until the pH is 8 then 8N H2SO4 was also added dropwise until pH is 7. (Garett & Grisham, 2010)
Possible Errors ●
Spillage
Conclusion ● Casein was isolated from non-fat milk by adding acetic acid which decreased the pH of the casein to 4.6. The percentage of casein is about 64-70%. ● Casein isolated was hydrolyzed using a strong acid, H2SO4 in acid hydrolysis and W was destroyed.
Book References Bollag, D. M., Rozycki, M. D. & Edelstein, S. J. (1996). Protein methods (2nd ed.). USA: Wiley-Liss, Inc. Boyer, R. (2009). Biochemistry Laboratory modern theory and techniques. USA: Pearson Education, Inc. Campbell, M. K. (2015). Biochemistry (8th ed.). USA: Brooks/Cole, Cengage Learning. Copeland, R. A. (1994). Methods for protein analysis: A practical guide to laboratory protocols. USA: Chapman & Hall. Ferrier, E. R. (2014). Biochemistry (6th ed.). USA: Lippincott Williams & Wilkins Garett, R. H. & Grisham, C. M. (2010). Biochemistry (4th ed.). USA: Brooks/Cole, Cengage Learning.
Internet Sources Brennan, J. (2016). Livestrong. Isolation casein & lactose in milk. Retrieved September 15, 2016 from http://www.livestrong.com/article/543320isolation -of-casein-lactose-from-milk/. Chemistry. (n.d.). Chemistry. Casein glue - activity. Retrieved September 16, 2016 from http://www.teachnlearnchem.com/Matter_and_Energy/PDF/ casein%20glue.pdf. Judelson, H. (2004). Operation of the autoclaves. Retrieved September 17, 2016 from http://oomyceteworld.net/protocols/autoclave%20operation .pdf.
