I did the experiment a number of times but am still not able to get the expected results. I would like to seek help from everyone. The protocol is as follows:
1. Dissolve maltodextrin and fructo-oligosaccharide in sterile distilled water. (ratio for probiotic : Maltodextrin is 1: 0.6)
2. Add the probiotic culture into the mixture (maltodextrin + FOS) and incubate for 20mins
3. Homogenize the sample at 18,000rpm for 10 min
4. Dry the sample at 45C for 30 min in the drying cabinet
5. Store the sample at room temperature and conduct the viability test periodically.
May I know any mistake in between? The result I obtained was a strange, non-capsulated culture with a higher viability count than the encapsulated culture.
Dear Shi Yiing Tang,
Based on my experience there is several important issue which has a key role in order to increase your work efficiency:
First: use pbs (phosphate buffer solution) pH=7 for making the primary mixture (maltodextrin and fructo-oligosaccharide).
Second: the process of mixing cell culture to primary solution must be done very slowly and in appropriate temperature based on optimum temp. of you bacterium.
Last: I believe that homogenization could be eliminated, due to destructive effect of shear stress on LABs. you can use slight mixing process instead of homogenization.
Regards,
Maybe you should try to dry them for less than 30 min. Drying can kill bacteria.
I agree, perhaps your temperature is to high (or too much time) for your probiotics.
Dear Shi Yiing Tang,
There are several questions/dilemma abut your experimental set-up.
1. Is this protocol from literature? If it is, send message to authors to clarifies any problematic step in experiment.
2. 18000rpm for 10min-this can be very dangerous for bacterial cells. Why so high rotation speed. Try to mix by magnetic stirrer.
3. I do not know is it drying procedure reliable for your experiment. How much water remain in mixture after drying? I belie that freeze drying is better for drying of cells.
Best regards
Steva
Dear shiii
You have several mistakes which can be corrected easily.
1.High homogenization speed makes shear forces that directly damages viable bacteria. So you must use lower speed in long time.
2. you would harvest probiotic capsules or beads by centrifuge methods which you did not mention.
3. mesophil cutures like Lb.casei can hardly survive in that condition, thermophils maybe. Try to keep your counts by freezing methods or lyophilization.
other wise you can use 15% glyserol+pepton to protect your microcapsules
best wishes
Dear Shi Yiing Tang,
Based on my experience there is several important issue which has a key role in order to increase your work efficiency:
First: use pbs (phosphate buffer solution) pH=7 for making the primary mixture (maltodextrin and fructo-oligosaccharide).
Second: the process of mixing cell culture to primary solution must be done very slowly and in appropriate temperature based on optimum temp. of you bacterium.
Last: I believe that homogenization could be eliminated, due to destructive effect of shear stress on LABs. you can use slight mixing process instead of homogenization.
Regards,
Does anyone know what is the best ratio use for probiotic:maltodextrin? Do you think 1:0.6 is too high?Thanks. Regards,
Shi
hey dude... go through a lot of research papers... ur asking like that as if u joined preschool =).... btw the ratio depends on what substrate ur using... it may be added in Substrate:maltodextrin the 4:1 ratio
Dear Shi Yiing Tang,
There are several questions abut your experimental set-up.
1. 18000rpm for 10min-this can be very dangerous for bacterial cells. So you must use lower speed in long time.
2. Use phosphate buffer solution pH=7.0 for making the primary mixture (maltodextrin and fructo-oligosaccharide) and second the process of mixing cell culture to primary solution must be done very slowly and maintained in appropriate temperature.
3. Send message to authors to clarify problematic step in your experiment.
Best wishes
Vinay Verma
Hi, Shi Yiing Tang,
Firstly, you should standardize the protocol of microencapsulation by trial and error basis. It is because if you are following the method of some other researcher or reference method, the type of strain, their temp. optima, resistance to various physical conditions, etc. may vary from your strain.
So starting with ratio of probiotic : Maltodextrin, you should standardize the conditions for setting your protocol. I also believe the homogenization step does not suit well. you may do simply stirring or can use low speed.
Good luck. Hope this will help you!
Hi Ms. Tang,
Based on your experimental protocol, seems like you are following some journals. But you might followed wrong journal that suit your experiments.
Previous answers should be able to help you.
The result you obtained have more non-capsulated might be because of weak encapsulation layer. Try to search several encapsulation ratio and do trial and error experiments, which suit your bacterial strain well. You might also can consider using electron microscope to observe your bacterial strains in the the encapsulated bead.
Hope this will help. Good luck.
One thing check that the homogenisation stage is not locally rapidly increasing the temperature and causing the polymers to uncontrollably phase separate.
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