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Author: Subject: Homemade Protein Powder - protein assaying needed
rannyfash
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[*] posted on 31-8-2014 at 08:10
Homemade Protein Powder - protein assaying needed


I can understand if a few might not consider this as chemistry and more of a cooking technique.

recently I've been working on my own soylent recipes, soylent being a drink that contains everything needed to survive, Link here to my recipe (do not attempt to make this, it tastes rank, I'm working on getting it to taste nicer) the most expensive ingredient is protein powder and I could not understand why it was so expensive, I could only find limited information on how it was made, it might be obvious to some but I am very impressed it can be made like cheese, in short the soluble proteins were aqueously extracted and precipitated by lowering the pH to its isoelectric point.

Starting with 100g of raw dried split peas, the peas were soaked overnight and macerated, the volume of water used was not recorded but enough is needed to create a roughly viscous slurry, this was then filtered through cheesecloth by gravity to avoid leaks then squeezing was used to drain the rest of the liquid, after leaving the collected liquid undisturbed for an hour it was decanted leaving starch residue at the bottom, the liquid was heated in a pan, when the liquid was just starting to boil, the pan was removed from heat and 2 table spoons of vinegar was added with brief stirring, after leaving for 20 minutes for the protein to flocculate, the mixture was gravity filtered to obtain a low density rubbery chunk, this was dried in an oven and ground to obtain 20.15g of a free flowing powder.

This powder based on 100% extraction should be 24.6g protein and 1.2g of fat according to the nutritional data, as the non/less polar compounds flocculate including the fats, because of this assuming the yield of protein to be 19g and that dried split peas can be bought cheapest in bulk at 90 pence a kilo from this website, on average the cost of protein excluding energy costs would work out as £4.74 per kilo of protein experimentally and £3.66 a kilo of protein if a 100% yield was obtained, compared to the cheapest commercial pea protein at MyProtein for £7.60 (since removing the 2.5kg option this is now £10.99 per kilo), my estimates on yield are only calculations based on nutritional estimates, I understand its flawed, which is why a protein assay is needed, Id also like to add that the commercial protein has a nasty taste that has not been reproduced in my own experiments.

Although what I've done isn't original I've shared what knowledge I have on this subject, if any of you are body-builders or you're on a vegan diet I hope you find it a useful supplement and I hope someone can share with me the knowledge on protein assaying, I've read a little and it seems a little bit costly for a hobbyist such as myself to send off a sample to a company to analyse the overall protein content.

[Edited on 31-8-2014 by rannyfash]

[Edited on 31-8-2014 by rannyfash]
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phlogiston
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[*] posted on 31-8-2014 at 11:56


The biuret test is simple and should give reasonable results. Because you have very large amounts of protein, you don't need a very sensitive method.

Ideally, you would want to use a spectrofotometer to measure the light absorbance at a specific wavelength, but you can probably get a pretty good estimate by eye if you make a series of calibration solutions to compare with.

Other popular protein assays are the Bradford assay and the BCA method.
Both are very simple to perform, but some of the reagents may be harder to acquire.





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rannyfash
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[*] posted on 1-9-2014 at 05:19


I had gone over those techniques but dissolving the powder in liquid just obtains a cloudy solution, my immediate thought was to break the protein into smaller chains with acid or alkali but that would destroy some of the amino acids, just realised that even if the amino acids are converted into other compounds they will most likely still be ligands, in which case it should still be all right, give me a week and ill be back with data, thanks for the help
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[*] posted on 1-9-2014 at 07:23


That is most likely because the proteins are denatured, which occured when you boiled/precipitate them (they unfold, exposing their hydrophobic surfaces on the outside which causes them to aggregate and precipitate).

Two ideas:

1. Try dissolving it in an 8M urea solution. That often works (apply some heat to help the refolding/solubilization) and is compatible with the bradford assay. I have not checked if it is compatible with other methods.

2. Alternatively, measure total nitrogen content. That should closely correlated with the protein+amino acid content.


BTW, most of the free amino acids survive alkaline or acid hydrolysis conditions (this is a common first step in determining the amino acid composition of proteins), but then you can't use a protein assay anymore (they only detect peptides, not free amino acids).






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rannyfash
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[*] posted on 1-9-2014 at 09:55


thanks for the info, exactly the protein is denatured and needs solubilizing, id prefer not to use ammonia, using biuret reagent (my preferred choice for its simplicity) it would give a dark blue tetraamine complex with copper which would make it hard to distinguish without a spectrometer, i think im going to heat a small sample with acid until it looks clear and use biuret reagent, why don't amino acids form copper complexes? and i read this from the university of oxford, are the amino acid degradations negligible?
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rannyfash
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[*] posted on 1-9-2014 at 10:04


scrap that, kjeldahl method looks very enticing, I'll post the data regardless of which I choose
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