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  Section: Plant Lab Protocols
 
 
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Methodology for Anti-nutritional Factor

 
     
 
Trypsin inhibitor
 
The food legumes are rich sources of proteins and their importance has been very well recognized in human nutrition, particularly in the countries where cereals form the staple diet of the people. Though grain legumes are rich in proteins, historically they are also known to possess a wide variety of chemical substances which interfere with the nutritive value when ingested by man or animals. Among such antinutritional factors, enzyme inhibitors, the substances which have the ability to inhibit the activity of the enzymes are present in an appreciable quantity in the grains. Most of the enzyme inhibitors have been found in the seeds of the various plants, but are not necessarily restricted to the seeds alone. The protease inhibitors present in the plants are the inhibitors of the enzymes - trypsin, chymotrypsin, papain, elastase, carboxypeptidase A and B, pronase, pepsin etc. It was observed by Osborne and Mendel as early as 1917 that heat treatment improved the nutritive value of soybean proteins. Read and Haas could give a plausible explanation to this observation in 1938 by demonstrating the presence of trypsin inhibitor (TI) in the raw seeds and its inactivation by heat.
 
Chemically trypsin inhibitors (TI) are proteinaceous molecules. The soybean TI has been crystallized and is called Kunitz protein.
 
 



Principle

The trypsin inhibitor activity is measured indirectly by inhibiting the activity of trypsin. A synthetic substrate (BAPNA) is subjected to hydrolysis by trypsin to produce yellow colored p-nitroanilide. The degree of inhibition by the extract of the yellow color production is measured at 410nm.
 
 
Materials
30% Glacial Acetic Acid (v/v)
 
Trypsin
Dissolve 6.25mg lyophilized trypsin and make up to 25mL with 0.001M HC1. Dilute 2mL of this solution to 25mL for assay.
 
Substrate: Benzoyl-DL-Arginine-paranitroanilide (BAPNA)
Completely dissolve 40mg BAPNA in 0.5mL of dimethyl sulphoxide and then make up to 100mL with Tris-HCl buffer pH 8.2.

 
Tris-HCl Buffer pH 8.2
Weigh 6.05g Tris (hydroxymethyl aminomethane) and 2.94g CaCl2.H2O, dissolve in 900mL water, adjust to pH 8.2 with dil. HCl and make up to 1000mL with distilled water.
 
Source of TI
Extract 0.5g sample in 25mL water by grinding in a prechilled mortar and pestle. Extract the ground sample in a refrigerator for 2-3h with occasional shaking for complete extraction of TI. Centrifuge the homogenate at 12,000rpm for 20 min at 4-6°C. Dilute 1mL of the supernatant to 10mL with distilled water and use as TI source.
 
 


Procedure
1.
Pipette out 0 to 1mL of extract in duplicate sets of test tubes, one to serve as endogenous (E) and the other test (T).
2.
Make up the volume to 2mL with buffer in the endogeneous set.
3.
Make up the volume to 1mL in the test set.
4.
Add 1mL of trypsin solution (20mg) to each tube in the test set. Pipette out into a separate test tube 1mL of buffer and 1mL of trypsin solution for standard(S).
5.
Incubate all the tubes in a water-bath at 37°C.
6.
After a few minutes, add 2.5mL of substrate (1mg BAPNA) to each tube.
7.
Allow the reaction to proceed for 10-60 min at 37°C.
8.
Stop the reaction by adding 0.5mL of 30% glacial acetic acid.
9.
Read the absorbance at 410nm in a spectrophotometer.
10.
Determine the protein content in the extract by the method of Lowry et al.
 
 
Calculation
Find out T-S absorbance.
 
Plot the absorbance against the volume of extract.
 
Determine the aliquot size of the extract required to inhibit 50% of the trypsin activity (S/2). That aliquot size is considered to be one unit of trypsin inhibitor.
 
One unit of activity corresponds to that amount of trypsin inhibitor in fig protein which gives 50% inhibition of enzyme activity under experimental conditions. The trypsin inhibitor activity is expressed as trypsin inhibitor units (TIU) per gram sample or per mg protein. (The dilutions of trypsin inhibitor source were made in such a way that 0.5mL produces 50% inhibition.)
 
 



References

1. Kakade, M L, Rackie, J J, McGhee, J E and Puski, G (1974) Cereal Chem 51 376.
2. Chitra, R and Sadasivam, S (1986) Food Chemistry 21 315.
 
 
     
 
 
     




     
 
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