Hi.
I am at the beginning of my research project about the in-vitro protein digestibility of edible fungi.
I am wondering about methods to determine the food protein content.
As from what I have read, there are three different approaches to food protein content. 1. Crude protein (Total N x conversion factor), 2. Spectrophotometric method (Biuret, BCA, Lowry, Bradford, UVvis). They either suffer from overestimation (non-protein nitrogen by Kjeldahl and Dumas) and underestimation (extraction yield). The other is:
3. Amino acid analysis.
This is first done by acid hydrolysis. followed by measurement using GC/LC either derivatized or using MS. The problem with this is the high initial cost.
Derivatization is done so that the amino acid can be measured by fluorescence/UV detector.
And, the function of LC is for separating the amino acids to be measured by the detector as different peaks in the chromatogram.
Correct me if there is something wrong with my understanding of the basic theory.
My question is:
If my objective is to only measure the food protein content (total amino acids) and do not wish to know each of the amino acid profiles, isn't it possible to neutralize then derivatize (ex. using OPA) the acid hydrolysate and measure them using a spectrophotometer (340nm) instead without using any chromatography system? I cannot find any reference that performs this approach.
Maybe is there anything missed and overlooked with this approach to determine the food protein content?
Thanks
Perhaps one reason why the method you suggested is not used is that OPA will react with all the primary amines in the sample, not just the amino acids derived from proteins. Using chromatography allows the separation of the components by retention time. A set of amino acid standards can be used to identify the peaks in the chromatogram that are due to amino acids from proteins, and to quantify them.
If you don't want to know the amino acid profile. Better go for the Kjeldahl method. But careful with the conversion factor. I'm not sure about the conversion factor for edible fungi.
Mr. Wang,
The research task can be carried out accurately, precisely, selectively and sensitively only via mass spectrometry; however, employing our own-authored (my and my co-author's innovation according to the shown authorship, below) innovative stochastic dynamic quantitative method and model equations allowing direct quantification of analytes in condensed phases as well as their exact 3D structural analysis. The method is validated via chromatographic experiment.
Please, consider works [1,2] and reference sections shown, therein.
[1] Analytical Chemistry Letters, 10 (2020) 703-721; Stochastic Dynamic Mass Spectrometric Approach to Quantify Reserpine in Solution; Bojidarka Ivanova, Michael Spiteller.
[2] Steroids, 164 (2020) 108750 Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller.
Consider the content of the following dicussion, as well. Perhaps, it shall be useful.
[https://www.researchgate.net/post/Analysis_of_Mercapturic_acids_in_German_Equas_Proficiency_testing_G-EQUAS_PTs]
Adam B Shapiro Thank you for the answer.
Rohit Thirumdas Bojidarka B. Ivanova Thank you for the suggestion.
Hi Ricky.
If you want to determine protein content of your sample. I would suggest Kjeldahl or Dumas method (Total N* conversion factor). If you use Spectrophotometric assays (Bradford, BCA), you would be determining N content ( soluble protein) in the supernatant. If you want to determine the digestibility i recommend doing OPA and Dumas/Kjeldahl. DH= free amino groups/total protein.
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