Dear researchers,
We are currently trying to make plant extracts using ultrasound in water. It includes many filtrations though different size filters. The smallest one used is 7-8 um pore size. Knowing the fact bacteria and their spores are smaller than that, it is clear the extract needs to be additionaly sterilized. We tried to use filters usually used for small amount of liquids (syringe + membrane filters 0.22 um) but the filtration speed was about one drop per minute which would make not possible to get decent amounts of extracts.
I recently ready about ultrasound + temperature sterilization methods and thought maybe during the extraction process many microorganisms might have been killed? Perhaps it is an option to add heat (up to 60 C) and ultrasound during extraction process to get final product free of microorganisms? Or perhaps to treat the final product with heat + additional ultrasound? Maybe you have some other suggestions we can try?
I need to add that we want to test the antimicrobial activity of the extracts. Therefore we need as pure extracts as possible, without added preservatives and without damaging sterilization methods on biological active compounds. We are considering simple freezing in -20 C for longer storage time. We don't have access to liquid nitrogen for freeze drying.
Hey,
I think it would be helpful if you would explain your extraction method a little bit more in detail (do you use other solvents than water?) and on which bioactive compounds you are focussed. Are these compounds proteins or secondary metabolites? If you focus on proteins you have to be really careful and I would not even heat it up to 60°C or use freeze-thaw cycles. But if you're interested in secondary metabolites you can use more harsh methods.
If you have access to an evaporator you could add a solvent like MeOH for sterilization, then evaporate and resolve it in water for bioactivity analysis.
Dried or frozen parts of the plants are made into powder and mixed with solvent - in our case water. Left for 3 - 8 hours (depending on plant characteristics) for water to be fully absorbed. Then with ultrasound plant cell walls are disrupted and active substances are released. 150 W / g, 10 - 30 min, 30 C. Then it is left alone 6 - 24 hours for all substances to transfer to solvent - water. Then we have many step filtration with the smallest filter being F1002 grade which according to specification filters particles in size 7-8 um.
Our aim is to study biological active compounds from plant extracts with antimicrobial activity. I am not sure proteins are contributing to this ability. However, I just have started to research this field and might be wrong.
We plan to test extracts with agar diffusion (with agar wells) method against some microorganisms and measuring the inhibitory zone.
Honestly, I don't know if you really need to perform an additional sterilization step. Freezing, drying, and pulverization of the plant parts should strongly diminish microbial contaminations. If you store your final extract properly (as example at -20°C) and just use it for the antimicrobial assays, which should not incubate to long (maybe 3-4 days), I don't believe that you will have microbial contaminations.
Otherwise you could make an additional autoclaving step. This would be fast and simple and regarding your sample preparation before I don't think that you could damage any interesting compound.
In my opinion a biological active compound should also get through a heat treatment without being damaged. Otherwise this compound is not that interesting.
You could consider irradiation (either X-Ray or Gamma-Ray) if you find that heat damages the activity of the compounds you are interested in.
Neither methanol (or ethanol), freezing nor freeze drying will sterilize.
Sonication step will reduce contamination but, unless you've appropriately validated sterility, think you're effectively testing a ~24 hour microbial culture of your plant extract. The zoi revaluates that culture - not the plant extract per se. Doubt you can establish and maintain sterility so at least determine level of contamination after extraction then filter sterilize. Autoclaving will clearly denature many biochemicals and volatilize essential oil voc's. Gamma may also denature some compounds but doubt seriously if you will access that technology.
Suggest "extraction " step should be better controlled (9-32 hours is a pretty wide range) but you might start with testing the immediate supernatant after sonication.
If you're planning to publish - please be aware that many journals no longer accept reports of antimicrobial efficacy of crude extracts from previously untested plants. They demand novelty in the extract itself - esp. a novel compound responsible for efficacy.
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