What are the analytical tests that can be done to test the vitality of microencapsulated bacteria?
You can use number of bacteria count before and after microencapsulation
Is there any other tests that we can perform apart from the viability counts ?
Viability of microencapsulated bacteria could also be determined by a dead and alive bacterial staining procedure, which is visualized by either fluorescence or confocal microscopy.
The viability of the microencapsulated bacteria could also be determined by assessing the production of ATP which is determined by flow cytometry. ATP is a direct measure of biological activity. ATP is quantified by measuring the light produced through its reaction with luciferase. The amount of light produced is directly proportional to the amount of living microorganisms present in the sample.
Thank you very much Deon Neveling for your detailed answer. May be I haven't formulated my question in a right way?
Let me formulate my doubt in this way:
Apart from the viability test ( either through plate count/ confocal/ flow cytometry), what are the other parameters that determine the vitality of bacteria ? Of course, viability is the most important factor.
Evaluation of pH over time is one analysis that can be done for testing the fermentation activity of the bacteria ? Is there any other tests/ parameters that can be performed/analysed to determine the fermentation activity of microencapsulated lactobacillus.?
You could measure the substrate (glucose) consumption rate. The consumption can be correlated to the growth.
Hi Divya. What is the aim of the experiment. You are encapsulating the Lactobacillus species, would you like to determine whether these bacteria are viable after encapsulation or would you like to determine whether they are metabolically active after encapsulation?
The measurement of ATP production would definitely be an indication that these cells are metabolically active. I agree with the previous comment that you could look at the consumption of glucose or you could also look at the production of a metabolic end product such as lactic acid. One could then determine the concentration of either of these compounds by HPLC after fermentation.
Thank you very much David Dietz and Deon Neveling. It really helps.
You are absolutely right. I want to know whether they are metabolically active after encapsulation.
Do any one know the standard protocol and sample preparation for HPLC for determining the concentration of lactic acid bacteria or the consumption of glucose by lactobacillus after fermentation?
Hey Divya. So the options you would have to determine whether your Lactobacillus specie is metabolically active after encapsulation is, either quantifying the production of ATP by flow cytometry, quantifying glucose consumption or quantifying the production of metabolic end product such as lactic acid by HPLC.
Lactic acid could either be quantified by a Lactic acid assay kit or HPLC.
D- and L-Lactic acid assay kits are available from Abacam:
L-Lactate Assay Kit (Colorimetric) (ab65331)
D-Lactate Assay Kit (Colorimetric) (ab83429)
For HPLC determination please refer to the following article, if you do not have access let me know and I will forward you a copy.
D.P. Neveling, A. Endo, L.M.T. Dicks (2012). Fructophilic Lactobacillus kunkeei and Lactobacillus brevis Isolated from Fresh Flowers, Bees and Bee-hives. Current Microbiology, (65)507–515, DOI 10.1007/s00284-012-0186-4.
If you go for glucose:
The yield coefficient will be strain specific, I recommend to determine it explicitely for your strain.
Easy way: Measure bio dry matter and glucose concentration for several samples in a batch - in the exponential phase the yield should be the same.
If Glucose is the only metabolite to measure, I would not use HPLC (because of time/sample). I would apply an enzymatic assay or DNS, look here:
https://www.researchgate.net/post/A_quicker_method_to_to_quantify_glucose_in_a_fermentation_sample
I agree with Dr. Dietz. If you do decide to use glucose consumption as an indicator of metabolic activity then an DNS assay to determine the glucose concentration would be much better than using HPLC.
Thank you Deon Nevling. I do not have the access. Could you please send to me?
Thank you David Dietz. It’s a good recommendation.
Thank you Jai Ghosh. Do you have papers on it?
Thank you Ghasem Najafpour for your detailed expainations. Do you have any papers / protocol on measuring the respiration quotient of microencapsulated lactobacillus.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts