Purpose
Aflatoxin is a mycotoxin commonly found in nut and corn products. The Aflatoxin B1 variant of these mycotoxins is produced by the Aspergillus Flavus and Aspergillus Parasiticus, which are prevalent in some agricultural settings. The structure of this toxic molecule is found at the top-right of this page. The image below illustrates the growth of the Aspergillus mold on pistachios.
The metabolism of this molecule is localized to the liver, which is why it is often implicated in liver cancer. The effects of this family of molecules on mammalian livers have been well documented for some time. This links to an article from the 1960's that reviews these changes to the livers of a variety of different mammals.
The tolerable limit of Aflatoxin in the United States is set at 20 parts per billion (ppb) per 25 grams of sample. Thus, in this experiment we sought to determine the concentration of Aflatoxin in a 25 gram sample of organic peanut butter from the Maryland Food Co-op.
Aflatoxin is a mycotoxin commonly found in nut and corn products. The Aflatoxin B1 variant of these mycotoxins is produced by the Aspergillus Flavus and Aspergillus Parasiticus, which are prevalent in some agricultural settings. The structure of this toxic molecule is found at the top-right of this page. The image below illustrates the growth of the Aspergillus mold on pistachios.
The metabolism of this molecule is localized to the liver, which is why it is often implicated in liver cancer. The effects of this family of molecules on mammalian livers have been well documented for some time. This links to an article from the 1960's that reviews these changes to the livers of a variety of different mammals.
The tolerable limit of Aflatoxin in the United States is set at 20 parts per billion (ppb) per 25 grams of sample. Thus, in this experiment we sought to determine the concentration of Aflatoxin in a 25 gram sample of organic peanut butter from the Maryland Food Co-op.
Procedure
A VICAM AflaTest immunoaffinity column (see image to the left) was used to isolate pure Aflatoxin from the peanut butter solution. Sample preparation was accomplished by blending 25 g of peanut butter with 5 g NaCl and 125 mL of 70:30, methanol:water. The sample was then filtered using fluted filter paper. 15 mL of the filtrate was diluted with 30 mL of water and subsequently filtered using a a microfiber glass filter. 15 mL of this filtrate was mixed and diluted with 30 mL of deionized water and filtered through a microfiber glass filter. Half of this filtrate (15 mL) was then subjected to the AflaTest column where the flow-thru was allowed to drip at a rate of 1-2 drops per second. At this point, the column retained the molecules of Aflatoxin so the next step was to elute the column. A wash with 10 mL of deionized water preceded stripping of the column with HPLC grade methanol. Prior to loading our sample, the fluorometer was calibrated with a series of standards. Subsequently, 1 mL of eluate was collected and placed in the calibrated fluorometer which then generated a reading of the Aflatoxin concentration in ppb.
VICAM provides an instructional video for its AflaTest. Please consider watching this video, which we have provided for your convenience. Below the video you will find images of the equipment used in this experiment from the lab session itself.
A VICAM AflaTest immunoaffinity column (see image to the left) was used to isolate pure Aflatoxin from the peanut butter solution. Sample preparation was accomplished by blending 25 g of peanut butter with 5 g NaCl and 125 mL of 70:30, methanol:water. The sample was then filtered using fluted filter paper. 15 mL of the filtrate was diluted with 30 mL of water and subsequently filtered using a a microfiber glass filter. 15 mL of this filtrate was mixed and diluted with 30 mL of deionized water and filtered through a microfiber glass filter. Half of this filtrate (15 mL) was then subjected to the AflaTest column where the flow-thru was allowed to drip at a rate of 1-2 drops per second. At this point, the column retained the molecules of Aflatoxin so the next step was to elute the column. A wash with 10 mL of deionized water preceded stripping of the column with HPLC grade methanol. Prior to loading our sample, the fluorometer was calibrated with a series of standards. Subsequently, 1 mL of eluate was collected and placed in the calibrated fluorometer which then generated a reading of the Aflatoxin concentration in ppb.
VICAM provides an instructional video for its AflaTest. Please consider watching this video, which we have provided for your convenience. Below the video you will find images of the equipment used in this experiment from the lab session itself.
Results and Conclusion
The sample mass that we used was 25.3329 grams and we found that there was 0 ppb (parts per billion) in our sample. Thus, there was effectively 0 ppb of Aflatoxin per gram of peanut butter. This was surprising to us given that organic peanuts are grown in the absence of pesticides and other chemicals that kill certain microorganisms and molds. Had time allowed, it would have been interesting to repeat the experiment and seen if the fluorometer would have still yielded 0 ppb on the repeat trial.
The sample mass that we used was 25.3329 grams and we found that there was 0 ppb (parts per billion) in our sample. Thus, there was effectively 0 ppb of Aflatoxin per gram of peanut butter. This was surprising to us given that organic peanuts are grown in the absence of pesticides and other chemicals that kill certain microorganisms and molds. Had time allowed, it would have been interesting to repeat the experiment and seen if the fluorometer would have still yielded 0 ppb on the repeat trial.