I am trying to check the in protein quality of a chickpea cultivar and for that we need to calculate the in vitro digestibility of the chickpea protein.I am following the protocol of Hsu.et al. but m bit confused how to calculate the digestibility percentage using casein as standard.
Here is How I do it for Sorghum (Sorghum bicolor) but I used in vitro Pepsin assay. This is based on the method on Mertz et al., 1984 as modified by Aboubacar et al., 2001. You digest about 200 mg sample powder in enzyme solution (35 mL) for 2 hours at a pH=2 and temperature =37oC. After 2 hours you stop the digestion by changing the pH (add 2 mL of 2 N sodium buffer) and centrifuge at 4900 g, for 20 minutes while keeping the temperature at 4oC. Discard the supernatant carefully (not to disturb the residue/gell) and wash the residue twice using 20 mL of phosphate buffer (pH=7) centrifuging at the same condition above. Then transfer the residue into aluminum tin and dry at 40oC for 24 hours using a forced air oven. Collect the dried residue, package properly and keep refrigerated.
Do nitrogen (protein) analysis for the digested residue (PCR) and flower (PCF) using LECO combustion method or kjeldahl method. The In vitro Protein Digestibility (PD) in percent, is given by the following formula:
PD (%) = [(PCR-PCF)/PCF]*100
where PCR = protein content of the residue and
PCF = protein content of flour
Links to the above articles are included. I hope this gives you some idea and Good Luck!
http://www.pnas.org/content/81/1/1.full.pdf
http://aaccipublications.aaccnet.org/doi/pdf/10.1094/CCHEM.2001.78.2.160
Thanks a lot Tadesse for the suggestion...I thought of following the same protocol u mentioned about bt unfortunately the kjeldhal machine is nt wrking and so I had to follow the pH drop method of digestibility analysis..I came across many papers where they used caesin as standard bt they hv nt mentioned xactly how they calculate
I am glad you found it useful. I think the pH drop method also gives you something comparable. Good luck!
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