Hi, I have carried out milk fermentations with bacterial species and want to check the milk fermentates for antioxidant activities. I centrifuged the milk ferementates after the incubation period and sterile filtered the supernatents into collection tubes for freezing. I have been using the DPPH assay to measure antioxidant activity of the fermentates. Several researchers have suggested that samples be incubated for 30 mins. However, while I do get slight reduction in absorbance values in this period, the samples still remain a purple/violet colour, reflecting that of the colour of the DPPH solution. It is my understanding that when DPPH is reduced it decolourises and turns yellow, thereby indicating the presence of strong antioxidants in a sample. This was noted by using different concentrations of Trolox and observing the colour change and subsequent decreasing absorbance values with a stronger concentration of Trolox. However, while my samples remain purple after 30 mins and several hours thereafter, they do turn yellow after 24 hours, thereby indicating the presence of potential antixoidants. With the trolox standards, this decolourisation happens instantly. Does anyone know why it takes 24 hours for DPPH to change from a purple/violet to yellow colour in the presence of my samples? To the best of my knowledge, I have not found a researcher that incubates the samples for longer than a few hours at most. Any help/explanation would be greatly appreciated.
Regards,
Ken
Dia duit Ken. I tested the antioxidant activity of plain milk and yogurt, and seaweed enriched milk and yogurt using DPPH assay. Have a look at my thesis. Here is the method I used:
Yogurt or milk samples were diluted in methanol (1:10) and 500 µl was added to 3.5
ml DPPH and mixed vigorously using a vortex mixer. Samples were stored in the dark
for 60 min and subsequently centrifuged at 3000g for 10 min at room temperature. The
absorbance of the supernatant was measured at 515 nm using a spectrophotometer . Trolox (0.04 µM) was used as a
standard for comparative purposes. A control was prepared containing DPPH and
methanol only. Data was expressed as % DPPH radical scavenging activity and
calculated as follows:
DPPH activity, % = (1 - (Abs515nm sample / Abs515nm Control)) × 100.
I found that all samples exhibited DPPH scavenging activity apart from most the yogurt samples following in vitro digestion.
Hi,
one of the possible reasons for the late discoloration of DPPH in presence of yours samples is the the nature of the polyphenols and media in which you tested the antioxidant activity. Maybe you should try some other assay such as FRAP or ABTS.
Here is some nice research on the nature of the mechanism of DPPH assay.
Electron-Transfer Reaction of Cinnamic Acids and Their Methyl Esters with the DPPH• Radical in Alcoholic Solutions
Mario C. Foti,*, Carmelo Daquino, and, and Corrada Geraci
The Journal of Organic Chemistry 2004 69 (7), 2309-2314
DOI: 10.1021/jo035758q
Best regards.
Hi A. M. Naim,
Thank you for the rapid response. Your method is very helpful and I will try it to see how it impacts the results I have already obtained using an alternative method. Did you ever find that the absorbance of your sample (milk/yoghurt) was higher than that of your negative control (DPPH and methanol)? This would subsequently mean that there was potentially no antioxidants in your sample although the colour may have changed from purple to yellow. This, from what I understand, is due to the absorbance of the your sample (milk/yoghurt) and as such I have included a colour blank (sample and methanol) in my assay to account for such interference. This was subsequently subtracted from the absorbance of my sample with DPPH. Have you tried this or had any problems with misinterpretation of results due to interference with absorbance from the actual milk/yoghurt sample?
Best Regards,
Ken
Hi Ante,
Thank you for your answer. I will try another one of the assays you have mentioned. Also, thank you for the paper, should be an interesting read.
Best regards,
Ken
Hi Ken. I have never had that experience with the DPPH but I have with other assays. I think the dilution and centrifugal steps may help. Also I concur with Ante that you should try using other antioxidant assault s. I suggest the Total Phenol Content, FRAP and Ferrous Ion Chelating Assay which are quite easy to set up. You could also consider more difficult assays like ORAC.
DPPH should not be incubated overnight. incubation period should be not exceeds 45min.
Hi Ken, the 30 min of incubation you used is what is mostly reported in the literature. During this incubation period (preferably in the dark), it is expected that the antioxidant present in the sample would donate hydrogen to the lone electron in the nitrogen atom of the DPPH, to reduce it to its corresponding hydrazine, which is the principle of the assay. Ordinarily, the stability of DPPH in solution is not indefinite; it gets reduced with time in solution, more so, when it is exposed to the atmosphere, even without adding antioxidant extract. So, the change in colour you observed after 24 hours may suggest that your samples are very low in antioxidants; in that case, you may need to increase the concentration of the extract you are using.
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