Isolation of Casein From Milk and Acid

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

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

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that has been hydrolyzed or broken down into its component amino acids

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wo isolate casein from non-fat milk by isoelectric precipitation.

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




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 --filter by gravity filtration


    

   
 

--dry between filter papers and weigh --calculate % yield --divide into two portions   
   



  


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 -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

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Before autoclaving

After autoclaving

Group 1

Dark brown liquid with thin brown residues

Clear yellow liquid

Group 3

White solution

Group 5

Dark brown liquid with thin brown residues

Clear yellow liquid

Group 7

White solution

Black solution

Group 9

Brownish-black White pieces of casein in solution with brown clear liquid flakes

Group 11

Colorless/clear liquid with suspended casein particles

Black solution with black precipitate

Brown to black solution with light brown particles

  
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 -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


  


Before autoclaving

After autoclaving

Group 2

Cloudy solution

Yellow cloudy solution

Group 4

Yellowish form, smooth texture

wurbid, curd forms, yellow color

Group 6

Solid light yellow product

Yellowish liquid with small amounts of white precipitate

Group 10

Cloudy white solution

Yellowish soulition with lumps of undissolved casein

Group 12

Solid light yellow product

Yellowish liquid with small amounts of white precipitate

Group 8

*'''* %& ÷ercent 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%

÷ercent Yield Group 7

69%

Group 8 Group 9

69.9%

Group 10 Group 11

48%

Group 12

66.3%

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" Ú' Isoelectric ÷recipitation ë Acid Hydrolysis ë Alkaline Hydrolysis ë Neutralization ë


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 ÷recipitation- 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. ë whe precipitation from suspension of a protein when the pH is at the isoelectric point ë


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  ë ÷ositive charge = Negative charge ë Minimized intermolecular repulsions ë Displays minimum water solubility ë


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 !%#" ë &'(ë ë- 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 ·               ë


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agent ë ÷rocedure for isolating mixture of free amino acids

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