Biological Values And Estimation Of Proteins, Essential Amino Acids And Lipids

The biological value, along with the estimation of proteins, essential amino acids, and lipids, provides a comprehensive framework for evaluating nutritional quality. These measures are critical for developing dietary guidelines, enhancing food formulations, and addressing nutritional deficiencies in various populations.

1. Biological Values And Estimation Of Proteins, Essential Amino Acids And Lipids.

2. Content 2 Introduction Of Proteins & Amino Acid. Biological Values. Protein Determination. Introduction Of Lipids. Estimation Of Lipids.

3. 3 INTRODUCTION OF PROTEINS & AMINO ACIDS Protiens : Peptide Bond : The bond between the α-nitrogen atom of one amino acid and the carbonyl carbon of a second Amino Acid ; They are building components of proteins. Organic compounds that contain both amino and carboxylic acid functional groups. Proteins are biomolecules comprised of  amino acid  residues joined together by  peptide bonds . The components of proteins include carbon,  hydrogen ,  oxygen , sulfur , nitrogen, and sometimes phosphorus.

4. 4 Types : Essential Amino Acids :- Histidine : ( M etal ion chelation, scavenging of reactive oxygen & nitrogen ) Isoleucine : ( G rowth, immunity, protein metabolis ) Leucine : (To generate ATP ) Lysine : ( H elp the body absorb calcium ) Methionine : (   A n antioxidant ) Phenylalanine : ( P roduction of the neurotransmitters ) Threonine : (A ids proper fat metabolism in the liver ) Tryptophan : ( T o help make melatonin and serotonin ) Valine : ( Promotes muscle growth and tissue repair )

5. 5 Bio-Logical Values : Biological value refers to the proportion of protein retained in the body for growth or maintenance and expressed in percent of nitrogen absorbed. Biological Value =   Biological Value = In : Nitrogen Ingested Fn : Nitrogen In Feces ( On protein diet ) Fc : Nitrogen In Feces ( On protein free diet ) Un : Nitrogen in Urine ( On protein diet ) Uc : Nitrogen in Urine ( On protein free diet )  

6. 6 Biological Value of An Egg with Amino Acid percentage. Example :-

7. 7 Methods of Estimation :- Protein Determination :- Importance of Protein Determination:- Plasma normally contains about 6.5 to 8.5 g/Dl protein and serum about 4% less in our human body and its essential to analyse the activity of protein. Its necessary to to estimate protein concentration in Cell Biology, Biochemistry & Pharma. Methods used Routenely ; Biuret Method Bradford Method Folin -Lowry Method Kjeldahi Method Ultra-violet Method & Mass Spectroscopy Fluorimetric Method Colorimetric Methods.

8. 8 Biuretic Method :- Principle ; The –CO-NH- bond (peptide) in polypeptide chain reacts with copper sulphate in an alkaline medium to give a purple colour . The complex absorbs light in 540 nm .   Preparation ; Take distilled water (500 ml) and dissolve pentavalent copper sulphate (1.5gm) and sodium-potassium tartrate (6gm) (known as Rochelle salt). + 2 molar hydroxide (375 ml) & mix both solutions Procedure ; T ake 3 dry and clean test tubes, A dd 1 or 2 mL of the test solution, albumin and deionised water in the test tubes. Add Biuret reagent (1-2 mL) in each test tube.  Now shake the solution well and let it stand for 5 minutes.  Finally, observe how the colour changes.  Observation Interpretation No Change in Colour ( sol’n reamians blue ) Proteins are unavailable Solution turn Violet from blue Proteins are available Solution turn pink from blue Peptides are available Observation ;

9. Graph ; Visuals ; 9

10. 10 Lowry Method :- 2% Na2CO3 in 0.1 N NaOH 1% NaK Tartrate in H2O 0.5% CuSO4.5 H2O in H2O Reagent I: 48 ml of A, 1 ml of B, 1 ml C Reagent II- 1 part Folin -Phenol [2 N]: 1 part water BSA Standard - 1 mg/ ml Add 0.2 ml of BSA working standard in 5 test tubes and make up to 1ml using distilled water. Add 4.5 ml of Reagent I and incubate for 10 minutes. After incubation add 0.5 ml of reagent II and incubate for 30 minutes. The first reaction is the reduction of copper ions under alkaline conditions, which forms a complex with peptide bonds. The second is the reduction of Folin-Ciocalteu reagent by the copper-peptide bond complex, that gives blue color to the solution. The complex absorbs light in 650 to 750 nm. Reagents ; Principle ; It is based on two chemical reactions. Procedure ;

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12. 12 Bradford Method :- Principle ; Bradford method is based on the ability of proteins to bind to coomassie brilliant blue dye and form a complex whose extinction coefficient is much greater than that of free dye. The protein in solution can be measured quantitatively Reagents ; Dissolve 100mg of Coomassie-Brilliant blue G250 in 50 ml of 95% Ethanol. BSA Standard. Procedure ; Prepare various concentration of standard protein solutions from the stock solution (say 0.2, 0.4, 0.6, 0.8 and 1.0 ml ) into series of test tubes and make up the volume to 1 ml . Add 5.0 ml of coomassie brilliant blue to each tube Wait for 10-30minutes and read each of the standards and each of the samples at 595nm.

13. 13 Graph ; Visuals ;

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15. 15 Intoduction of Lipids : - Classification of Lipids N on-saponifiable lipid : It cannot be disintegrated into smaller molecules. E.G :- cholesterol, triglyceride s, S aponifiable lipid : It can be disintegrated into smaller molecules. E.G :- waxes, phospholipids Polar Lipids : It forms a barrier with an external water environment, are utilized in membrane. E.g :- sphingolipids Non-Polar Lipids : Nonpolar lipids, namely triglycerides, are utilized as fuel and to store energy. Lipids are heterogeneous groups of compound, including fats, wax, Steroids and oily compounds that are soluble in organic solvents and insoluble in polar solvents such as water. Properties ; Lipids are greasy nonpolar stored in the adipose tissue of the body. Lipids are energy-rich organic molecules, which provide energy for different life processes. Lipids are a mechanical barrier dividing a cell from the external environment known as the cell membrane.

16. 16 ESTIMATION OF THE TOTAL AMOUNT OF LIPIDS There are mainly two metods for Lipid Estimation :- Bligh and Dyer Method : It is a method where extraction and partitioning are simultaneous. The lipid extraction of incubation medium, tissue homogenates or cell suspensions can be done. The extraction can be carried out in a single tube . Folch Method : The Folch Method is one of the most commonly used biochemical procedures used for isolating lipids from biological samples.

17. 17 BLIGH & DYER METHOD Principle : It is t he standard method for the determination of total lipids. Methanol, chloroform and water are added to the sample in a two-step extraction and, after phase separation, lipids are quantified in the chloroform phase. By later lipid extracted by Vaccum Distillation. Procedure : S ample containing 1 ml water (1 ml cell suspension, homogenized tissue, plasm) add3.75 ml of a mixture chloroform/methanol (1/2) and vortex during 10-15 min A dd 1.25 ml chloroform with mixing 1 min and 1.25 ml water with mixing another minute before centrifugation. C ollect the lower phase after centrifugation and redissolved in a small volume of chloroform/methanol (2/1) & Collect lower phase. Dry the Lower Phase By Vaccum Distillation.

18. 18 Step : 1 :- Bligh Dyer Chemical Reaction Step : 2 :- Vaccum Distillation Result : Weigh the Lipid Extract for lipid Concentration.

19. 19 FOLCH METHOD Principle : The Folch method is based on the partitioning of lipids in a biphasic mixture of chloroform and methanol. Methanol disrupts hydrogen bonds between lipids and protein following addition of an organic solvent such as chloroform. Procedure : First, a cell homogenate is equilibrated with a 25% volume saline solution and stirred. This mixture is left to stand until biphasic separation. The tissue is homogenized with chloroform/methanol (2/1) to a final volume 20 times the volume of the tissue sample (1 g in 20 ml of solvent mixture). After dispersion,the mixture is centrifuged to separate the two phases. Remove the upper phase by siphoning after that the lower chloroform phase containing lipids is evaporated under vacuum distillation.

20. 20 Step :1 :- Folch Mechanism Step :2:- Vaccum Distillation Result : Weigh the Lipid Extract for lipid Concentration.

21. 21 References :  A Guide to Amino Acid and Protein Nutrition: Dr Robert Wolf. Amino Acid Analysis : Methods & Protocols; Peter Hunziker . Methods for Protein Analysis: A Practical Guide for Laborat0ory Protocols.  Robert A Copeland. https://link.springer.com/book/10.1007/978-1-59745-198-7. https://www.britannica.com/science/protein. Lipid Analysis : William A. W. https://www.sciencedirect.com/book/9780128171059/lipids. https://www.britannica.com/science/lipid.

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