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  Section: Plant Lab Protocols
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Methodology for Amino Acids and Proteins

Protein digestibility in vitro
Protein quality determination is important in the production of nutritionally improved varieties of cereals and pulses. The nutritive value of a protein depends primarily on its capacity to supply needs of nitrogen and essential amino acids. Although the chemically determined amino acid composition is used to measure the quality of a protein, the biological availability of these amino acids is the real measure of the quality of the proteins. The availability of amino acids depends on the extent of digestibility of proteins by the proteolytic enzymes of the alimentary tract.
Digestibility of a protein can be assessed using rats which is termed in vivo digestibility. It can also be measured using proteolytic enzymes and called in vitro protein digestibility. The results obtained with the later procedure agreed well with in vivo protein digestibility as measured in the rats.
The procedure described here is that of Hsu et al1 which was later modified by Satterlee et al2.

Four proteolytic enzymes are used to digest the protein and the pH changed due to the release of amino acids at a fixed time interval is measured. By using the formula - % digestibility = 234.84 – 22.56 X, where X is the pH after 20min incubation, the in vitro digestibility is arrived at.
Powdered sample which passes through 80-mesh screen
Glass distilled water
Three enzyme solution: 1.6mg trypsin, 3.1mg chymotrypsin and 1.3mg peptidase per mL in glass distilled water.

rgin-left:.25in;text-indent:-9.0pt;line-height:normal;"> Bacterial protease solution: 7.95mg protease (type IV from Streptomyces griseus) per mL in glass distilled water.
Add 10mL of glass distilled water to the powdered sample (amount of sample is adjusted so as to contain 6.25mg protein/mL).
Allow the sample to hydrate for at least 1h (not longer than 25h) at 5°C.
a) Equilibrate the sample to pH 8.0 at 37°C.
b) Equilibrate the three enzyme solution to pH 8.0 at 37°C.
Add 1mL of three enzyme solution to the sample suspension and stir while being held at 37°C.
After exactly 10min from the time of addition of three enzyme solution (still stirring) add 1mL of the bacterial protease solution.
Immediately transfer the solution to 55°C water bath.
Nine minutes after adding the bacterial enzyme, transfer the solution back to 37°C water bath (in total 19min after the addition of the three enzyme solution).
Measure the pH of the hydrolysate exactly 10min after the addition of bacterial enzyme. This is called the 20min pH.

In vitro protein digestibility is calculated using the following equation:-
% digestibility = 234.84 – 22.56 X
where X is the pH after 20min incubation
First run a control (HNRC sodium caseinate) before each set of samples and this must have a 20min pH of 6.42 ±0.05. this control is needed to ensure the presence of proper enzyme activity prior to any running samples.

1. Hsu, H W, Sutton, N E, Banjo, M O, Satterlee, L D and Kendrick, J G (1978) Fd Technol 32 69.
2. Satterlee, L D, Marshall, H F and Tennyson, J M (1979) J Am Oil Chem Soc 56 103.


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