We are studying different parameters associated with the growth of a yeast in a culture media containing yeast extract, lactose as the carbon source, and peptones (e.g. casein peptone) as the nitrogen source.
We are looking for a technique in order to follow the nitrogen source consumption (either as small peptides or amino acids), in order to complement biomass and lactose removal determinations.
We need a simple, preferably colorimetric method, and we know that proteins quantification assays based in dyes binding as the Bradford method are not useful as we are working in a molecular weight range of peptides too small.
Which method/s can you recommend for this purpose?
The simplest way I can think of is to use a radioactive tracer in the nutrient source. This could work nicely with 14C-lactose. You would just measure how much radioactivity remained in the medium, how much was in the cell pellet, and how much was released as CO2 (you need a way of collecting the radioactive CO2, such as by bubbling the gas released by the culture through a sodium hydroxide solution).
Unfortunately, nitrogen has no radioisotope with sufficiently long half-life for the other part of the experiment.
My next idea is to use 15N as the tracer. It is a rare stable isotope, detectable by NMR, but 15N-labeled peptides might be too expensive to use as a feedstock in your experiment. 15N materials are available here: http://www.isotope.com/products/
Total nitrogen in the medium can be measured by the Kjeldahl method:
http://pubs.acs.org/doi/abs/10.1021/ac60038a038?journalCode=ancham
Maybe you can try the old methods listed here:
http://www.jbc.org/content/62/1/1.full.pdf
or perhaps HPLC may be the best way to differentiate proteins, peptides and free amino acids.
Dear colleagues, thank you very much for your responses.
We need simple and economic methods, as we are interested in following Nitrogen consumption by yeast along time in a culture. We are interested in colorimetric assays. We have used the ninhydrin method, but as far as I know, it only provides a measure of aminoacidic nitrogen, and the reaction has low reproducibility. Methods for quantifyig free ammonium seem not to be appropriate in this case, as only a small fraction of nitrogen consumed by yeasts (peptides and amino acids) is finally released as ammonium, and most of nitrogen compounds are incorporated by yeast in order to grow.
Did you try this?
Abernathy, D. G., Spedding, G. and Starcher, B. (2009), Analysis of Protein and Total Usable Nitrogen in Beer and Wine Using a Microwell Ninhydrin Assay. Journal of the Institute of Brewing, 115: 122–127. doi:10.1002/j.2050-0416.2009.tb00356.x
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