I am preparing gastrointestinal fluids to test yeast survivability in-vitro.
The protocol requires me to prepare two solutions, gastric juice and intestinal fluid, requiring me to add pepsin and pancreatin respectively after the autoclaving.
There are MCE, PTFE and PVDF filters available in the lab, and my concern is possible loss of enzymatic activity due to using unsuitable filter. Which one, or another, do you suggest for preparing such solutions?
10/20/22
Dear Emre,
I can't tell you which filter is the best one to use. However, this is something that you can easily test for yourself. You can try running small batches of the pepsin & pancreatin separately through each type of filter and measure the enzymatic activity of filtered vs. unfiltered enzymes. One thing to keep in mind is that membrane filters are not always "benign". Sometimes they bind some of the material (in your case, protein) that you are trying to filter. If you look in the catalogs of companies that sell membrane filters, they often describe a particular membrane as exhibiting "Low protein binding". If you follow the above suggestion and find less enzyme activity in the filtrate of one or more of the membranes, it could be that some of the enzyme protein was bound by the filter. To check this, it would be a good idea to measure not only the enzyme activity in the filtered- and unfiltered preparation, but also the protein concentration in the filtered- and unfiltered sample.
Something you mentioned in the first part of your question: Exactly what are you going to autoclave? Autoclaving pepsin, pancreatin, gastric juice and intestinal fluid will denature enzymes and other proteins that they contain.
I hope this information helps you.
Bill Colonna Iowa State University, Ames, Iowa, USA [email protected]
Polysulphone would be suitable, as it shows little protein binding.
What protocol are you following? Is the pepsin and pancreas used sterile?
Dear W.J. Colonna, Engelbert Buxbaum , Kübra Cinar Topcu thank you for your answers.
I am following the protocol of (Pennacchia et al., 2008):
- For gastric juice: 6.2g/l NaCl, 2.2g/l KCl, 0.22g/l CaCl2, 1.2g/l NaHCO3, 3g/l Pepsin (added through membrane filtration after autoclaving the above solution through membrane filtration), at adjusted pH 2.5 (pre-autoclave)
- For intestinal juice: 1.29g/l NaCl, 0.23g/l KCl, 6.4g/l NaHC3, 0.3% (w/v) bile salts, 0.1% (w/v) pancreatin (added through membrane filtration after autoclaving the above solution) at adjusted pH 7.5 with HCl (pre-autoclave)
Me and my colleague ran two separate experiments, one with plate counting method, and the other with flow cytometry. Our results were different regarding the survivability after exposing the yeast cells to the intestinal juice. The difference was she used PTFE membrane filter to add pancreatin to the solution, and I tried PVDF membrane for that, but had some issues with solubility of the enzyme and its filtration, therefore continued the experiment without pancreatin.
I am sharing the article with you. I hope that will be useful
"Probiotic properties of lactic acid bacteria strains isolated from pastırma"
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