I have some samples from microbial fermentation of pea flour. They are aqueous samples and contain solubilized starch (the bacteria use the starch as their carbon source). I want to measure the amount of residual starch in the samples. This starch is resistant to enzymatic hydrolysis, so I need to do acid hydrolysis to measure the starch content. However, acid hydrolysis hydrolyses every other compound in the sample, including mono and oligosaccharides. I am looking for a method to separate the mono and oligosaccharides first, and then measure the polysaccharides or starch in the samples. I was thinking of using 80% ethanol to separate the mono and oligosaccharides, but since the samples are aqueous, ethanol is also soluble in water, so I am not sure how I can extract them from water solution. Could you please help me and let me know how I can separate the mono and oligosaccharides and then measure the starch content?
You have to concentrate or evaporate the sample first at low temperatures and then do extraction with 80% ethanol .
One possible method for separating mono and oligosaccharides from starch in your aqueous samples is to use size-exclusion chromatography (SEC). SEC is a type of chromatography that separates molecules based on their size and shape, with larger molecules eluting first and smaller ones eluting later.
Here is a possible procedure you could use:
Materials:
- SEC column (e.g., Sephadex G-50 or G-75)
- Buffer (e.g., 50 mM sodium acetate)
- Aqueous samples
- Glucose standards for calibration curve
- Glucose assay kit
Procedure:
Note: The SEC method may not completely separate the starch from the mono and oligosaccharides, but it should reduce their concentration in the fraction containing the starch, making it easier to accurately measure the starch content.
4/20/23
Dear Laleh,
There are several things you can try.
1. If you add 2 volumes of ethanol to the starch solution. The ethanol will compete w/ the hydroxyls of the glucose monomers of the starch molecules for the water that holds the starch in solution. This essentially "ties up" the water so it's unavailable to hold the starch in solution, causing it to precipitate. The oligosaccharides will remain in solution and can be removed by pouring off the supernatant after a centrifugation step. You can wash the starch pellet w/ 67-80% EtOH. After recovering the washed starch pellet, you can re-dissolve it w/ water and do acid hydrolysis.
2. You can also remove the mono- & oligosaccharides by dialyzing the starch solution vs. water or a suitable buffer.
Are you sure that the starch is resistant to enzyme hydrolysis? What enzymes have you tried? A combination of alpha-amylase + glucoamylase may do the job for you.
I hope this information helps you. Good luck w/ your research.
Bill Colonna Iowa State University, Ames, IA USA [email protected]
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