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    Activity 7 - Quantitative Analysis of Protein - Post Lab.

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    ogheeluv
    This document describes a procedure to quantify the amount of casein protein in different milk samples. Casein was precipitated from cow, carabao, goat and human milk samples by heating the milk to 40°C and adding acetic acid to reach the casein isoelectric point of pH 4.6. The casein curd was then purified by removing fat using ethanol. Weighing of the isolated casein found that cow milk contained the highest amount at 5.01 grams per 20mL, followed by carabao milk at 3.14 grams, while human milk contained the lowest amount at 0.8 grams. The casein content provides information about milk composition and quality for applications in infant nutrition, food production

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    Activity 7 - Quantitative Analysis of Protein - Post Lab.

    Uploaded by

    ogheeluv
    109 views28 pages
    This document describes a procedure to quantify the amount of casein protein in different milk samples. Casein was precipitated from cow, carabao, goat and human milk samples by heating the milk to 40°C and adding acetic acid to reach the casein isoelectric point of pH 4.6. The casein curd was then purified by removing fat using ethanol. Weighing of the isolated casein found that cow milk contained the highest amount at 5.01 grams per 20mL, followed by carabao milk at 3.14 grams, while human milk contained the lowest amount at 0.8 grams. The casein content provides information about milk composition and quality for applications in infant nutrition, food production

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    Quantitative Analysis of Protein in Milk.

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    Activity 7 - Quantitative Analysis of Protein - Post lab.

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    This document describes a procedure to quantify the amount of casein protein in different milk samples. Casein was precipitated from cow, carabao, goat and human milk samples by heating the milk to 40°C and adding acetic acid to reach the casein isoelectric point of pH 4.6. The casein curd was then purified by removing fat using ethanol. Weighing of the isolated casein found that cow milk contained the highest amount at 5.01 grams per 20mL, followed by carabao milk at 3.14 grams, while human milk contained the lowest amount at 0.8 grams. The casein content provides information about milk composition and quality for applications in infant nutrition, food production

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    109 views28 pages

    Activity 7 - Quantitative Analysis of Protein - Post Lab.

    Uploaded by

    ogheeluv
    This document describes a procedure to quantify the amount of casein protein in different milk samples. Casein was precipitated from cow, carabao, goat and human milk samples by heating the milk to 40°C and adding acetic acid to reach the casein isoelectric point of pH 4.6. The casein curd was then purified by removing fat using ethanol. Weighing of the isolated casein found that cow milk contained the highest amount at 5.01 grams per 20mL, followed by carabao milk at 3.14 grams, while human milk contained the lowest amount at 0.8 grams. The casein content provides information about milk composition and quality for applications in infant nutrition, food production

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    Act.

    7
    Quantitative Analysis of Protein
    Estimation of Amount of Casein in Milk
    Proteins are chains of amino acid
    molecules connected by peptide bonds
     Amino acid chains are 20 different amino acids exist
    called polypeptides

    The amino acids within protein chains can bond


    across the chain and fold to form 3-dimensional
    structures.
    This Photo by Unknown Author is licensed under CC BY-SA
    Proteins can be relatively straight or form tightly
    compacted globules or be somewhere in between.

     The term “denatured” is used when proteins unfold from


    their native chain or globular shape.

     Denaturing proteins is beneficial in some instances.


     To allow easy access to the protein chain by enzymes
    for digestion,
     To increase the ability of the whey proteins to bind water and
    provide a desirable texture in yogurt production.
    Milk Protein Chemistry
    2 major categories of milk protein:
    Casein family contains phosphorus and will
    coagulate or precipitate at pH 4.6.
    Serum (Whey) proteins do not contain
    phosphorus, and these proteins remain in
    solution in milk at pH 4.6.
    Casein family of protein in milk
     consists of several types of
    caseins (α-s1, α-s2 , ß, and 6)
    and each has its own amino
    acid composition, genetic
    variations, and functional
    properties. 
      The caseins are suspended in
    milk in a complex called a
    micelle.
    The high phosphate
    content of the casein
    family allows it to
    associate with calcium
    and form calcium
    phosphate salts.

    This Photo by Unknown Author is licensed under CC BY-SA


    Serum (Whey) Protein family
     consists of approximately 50% ß-lactoglobulin, 20% α-lactalbumin,
    blood serum albumin, immunoglobulins, lactoferrin, transferrin,
    and many minor proteins and enzymes.

     Like the other major milk components, each whey protein has its
    own characteristic composition and variations.

     The functions of many whey proteins are not clearly defined, and
    they may not have a specific function in milk but may be an
    artifact of milk synthesis. 
     ß-lactoglobulin is thought to be a carrier of vitamin A.
    Amount of Casein in milk

     Casein is a complete protein source. That means it


    provides all the essential amino acids your body needs for
    growth and repair.

    Casein is a slow digesting protein


    constitutes approximately 80% (29.5 g/L) of the total
    protein in milk
    Learning Objectives:

     Toprecipitate and isolate the casein curd from the


    milk samples by acidification to reach casein’s
    isoelectric point.
     Tocompare the mass of casein curd extracted from
    the different samples.
     To find the milk with the highest casein content
    Materials/Chemicals and Reagents

     20 mL milk sample (cow, carabao, goat) Water bath


     Acetic Acid Stirring rod
     Ethanol Graduated cylinder (100
    mL)
     Watch Glass Filter Paper (Whatman
    No.1)
     100 mL Beaker Buchner funnel with
    suction (vacuum)
    http://www.biosciencenotes.com/wp-content/uploads/2018/07/casein5-300x199.jpg
    Procedure

    Preparation of Milk Samples


    1. Using the graduated cylinder, a 20 ml sample of
    fresh milk was poured in a dry clean beaker.
    2. Each sample was labeled properly.
    Isolation of Casein
    1.the beaker with milk sample was placed into the water
    bath at 40⁰C for 5 minutes.
    2.After 5 minutes, 10 drops of 5% acetic acid was slowly
    added to the beaker with constant stirring.
    3.The samples were kept undisturbed for 10 minutes.
    4.Casein was seen precipitating and forming curd along with
    fat.
    5.The solution was filtered, and the precipitate was
    transferred into another beaker.
    PRINCIPLES:

    Milk samples heated at 40 - 60 deg C.


    The casein component of milk is relatively
    heat-stable, capable of surviving pasteurization
    at 62-71°C.
    Conversely the whey protein component is
    denatured at these temperatures.
    Adding acetic acid dropwise to the milk
    sample.
     Acidifyingthe milk – allows the casein to
    precipitate.
     Toprecipitate the casein, acetic acid is added drop
    by drop until the isoelectric point is reached.
     Theisoelectric point of casein is at pH 4.6 where
    the protein shows the least solubility and gets
    precipitated.
    The isoelectric point of a protein (I.E.P.) is the pH at which
    the molecule is electrically neutral (net charge is zero).

     When a protein is charged e.g. with a negative


    charge, the negative molecules repel each other
    and thus remain in the solution and do not
    precipitate.
     But when these molecules are neutral, they tend
    to precipitate due to the absence of the repelling
    forces and, the high molecular weight of protein.
    Also, at a low pH, casein protein denatures
    (unfold).
    The unfolded proteins are then free to
    interact with each other and clump
    together in a way they could not do when
    they were properly folded.
    The milk takes on a curdled appearance
    from the lumps of proteins that are
    binding one another.
    Purification of Casein

    1. To separate casein from fat in the curd, 10 ml of ethanol


    was added with stirring.
    2. The liquid of dissolved fat was poured out and the solid
    casein was set aside.
    3. The solid casein was washed with cold water and filtered
    using Whatman No.1.
    4. The excess water gently removed from the filter paper.
    5. The precipitate was dried for five minutes.
    Addition of ethanol to the precipitates

     The precipitate doesn’t dissolve in H2O, alcohol


    or ether but dissolves in alkaline solutions (e.g.
    KOH forming potassium caseinate).

     Caseinis insoluble in ethanol and this property


    is used to remove fat from the preparation.
    Weighing of Casein

    1. The solid casein was transferred from filter paper to


    the watch glass.
    2. The weight of casein was measured using a top
    loading balance.
    RESULTS

    Table 1. Casein in milk samples


    Yield of Casein
    Milk Sample
    (Grams)/20ml
    Cow 5.01

    Carabao 3.14

    Human 0.8
    Kulkarni CP. 2017. Analysis of casein precipitation from the various milk
    samples available in market. International Journal of Food Science and
    Nutrition. 2(6): 215-216
    The casein precipitated from the cow milk
    contains more amount of casein protein than
    the goat and buffalo milk samples.
    Incow's milk, approximately 82% of milk protein is
    casein and the remaining 18% is serum, or whey
    protein (milkfacts.info).
    The lower amount of casein in the buffalo milk could
    be due to the more fat content in it (Kulkarni 2017).
    Human milk contains approximately 40% casein,
    and 60% whey.
    Applications:
     For infant nutrition
     Knowing the protein ratio of human milk is considered
    a guideline when manufacturing artificial milk, also
    called formula
     Ifinfant formulas have greater percentage of casein, it
    will be more difficult for the baby to digest.
     Approximately 60-80% of all protein in human milk is
    whey protein.  These proteins have great infection-
    protection properties.
    Applications:

      Forfood technology applications and dairy food


    production.
     The quantitative analysis of casein precipitated from the
    various milk samples provide the ample scope to the
    cottage cheese manufacture.
     Commercially available milk may have variations in casein
    content which may indicate adulteration of milk by adding
    water or other substances.
    Applications:

    • Non-food
    applications of
    casein
    REFERENCES
     Milk Facts. http://milkfacts.info/Milk%20Composition/Protein.htm
     General Properties of Casein.
    https://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/enzyme-r
    eagents/casein.html
     Applied Biochemistry Lab.
    https://walghazzawi.kau.edu.sa/Files/0007119/Files/119837_applied_biochemistry.pdf
     What’s in Breast Milk? American Pregnancy Association.
    https://americanpregnancy.org/first-year-of-life/whats-in-breastmilk/
     Kulkarni CP. 2017. Analysis of casein precipitation from the various milk samples
    available in market. International Journal of Food Science and Nutrition. 2(6): 215-216
     Lonnerdai B, Forsum E. 1985. Casein content of human milk. Am.J. Clin Nutr. 41(1):113-
    120
     Jean-Luc Audic, Bernard Chaufer, Georges Daufin. Non-food applications of milk
    components and dairy co-products: A review. Le Lait, INRA Editions, 2003, 83 (6),
    pp.417-438. 10.1051/lait:2003027

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