The Importance and Analysis of Aflatoxin and Gluten in Corn

Corn Corn or maize (zea mays) is a domesticated plant of the Americas. Like other indigenous plants like beans, squash, melons, and tobacco, European colonists quickly adopted maize agriculture Maize quickly spread to other parts of the world as well.

Corn & Native Americans Corn was grown by Native Americans as staple of their diet Mastered the agronomic method of the “three sisters’ concept: corn, beans and squash Many of these same principles are used today to grow corn.

Corn Today Today corn is the number one crop grown in the United States It is used in a variety of different food, feed and nonfood

Keeping Corn Safe Because corn is so important to our lives and economy we need to keep the corn supply safe to eat. Corn like other foods must be grown, harvested, stored, processed and handled properly to prevent attack by insects and microorganisms that can make corn unsafe to eat.

: Molds One group of organisms that we are very concerned about are molds. Molds are microscopic fungi that can grow on foods and feeds. We are particularly concerned with those that produce toxins or poisons called mycotoxins.

: Alfatoxin One type of mycotoxin that we are concerned about is aflatoxin – which is produced by the mold Aspergillus flavus and other species of molds. This toxin can cause a variety of ailments ranging from vomiting and abdominal pain to liver disease and liver cancers

: Alfatoxin Because mold is in the environment and that it is difficult to keep foods totally free from the molds that produce the toxin, there is a limit of the amount of aflatoxin that can be present in foods. 20 parts/billion (ppb) except milk 0.5 (ppb)

Alfatoxin Determination : Alfatoxin Determination Scientists have methods to determine if aflatoxin is present in corn and other feedstuffs. Today we will learn how to detect aflatoxin in corn. Virtual Laboratory Experiment “Hands-On” Experiment Summary of what we learned

Gluten In contrast to aflatoxin, gluten is not harmful to all people. : Gluten In contrast to aflatoxin, gluten is not harmful to all people. Gluten is associated with disease called Celiac disease. Iron deficiency anemia (low blood count) Musculoskeletal problems (muscle cramps, joint and bone pain) Growth problems and failure to thrive (in children)

: Gluten However, not all people sensitive to gluten get Celiac disease. Instead, some have: dermatitis herpetiformis neurologic conditions such as gluten-sensitive ataxia Seizures Tingling sensation in the legs Loss of balance

: Gluten Gluten is associated with cereal grains. However, the myth that all cereal contain gluten exists. Gluten is actually the protein complex formed through the interaction between gliadin and glutenin. Celiac disease relates to small protein fragments (i.e. peptides) that promote an immune response leading to symptoms of Celiac disease. The peptides are form during digestion of protein or protein complexes such as gluten.

Gluten What triggers the immune response : Gluten What triggers the immune response Prolamin is a class of protein in cereals that gives rise to the specific peptides during digestion Only certain Prolamins break down into the specific peptide that causes an immune response Proteins responsible: Wheat or other Triticum species (gliadin) Barley (hordein) Rye (secalin) Sorghum (kafirin) Oats (avenin) (a minor protein – little impact on Celiac disease)

Activities Activity I – Virtual Laboratory Exercise : Activities Activity I – Virtual Laboratory Exercise Please get into groups of 2 persons (if necessary, 3-4 persons depending on the availability of computers) and enter into the Corn Mold Virtual Laboratory. Please follow the instructions as directed by the virtual laboratory instructor: (http://virtuallab.nmsu.edu/)

Activities Activity II – Hands-On Exercise: Aflatoxin analysis Part A. Black Light Test  

Activities Activity II – Hands-On Exercise: Aflatoxin analysis Part B. Test Kit to Measure Aflatoxin

: Activities Activity III – Hands-On Exercise: Gluten Analysis

Activity II – Hands-On Exercise: Aflatoxin Analysis Remove the ground corn from the test kit. Record the code number on the data sheet (provided by instructor). Find the corn sample located by the black light box that matches your code. This sample will be the one that you will test in under black light. Caution: Do not look directly at the black light. Place the corn sample into the light box. View the corn sample for fluorescence. Note if fluorescence exists (bright yellow or green-yellow color).  

Activity II – Hands-On Exercise: Aflatoxin Analysis Add 10 g ground grain sample to suitable screw cap container. Add 20 ml of 70% Methanol Extraction Solution and shake vigorously for 1-3 minutes to extract aflatoxin from grain sample. Allow sediment to settle for 5 minutes. It is important not to transfer sediment to the test strip since it can interfere with the flow of liquid and this may affect test results. Add 200 µL of diluent (please obtain bottle from instructors) to the sample cup (small plastic cup) using a clean calibrated transfer pipette.

Activity II – Hands-On Exercise: Aflatoxin Analysis Transfer 200 µL of grain extract to a sample cup. Mix the sample using the pipette by drawing and expelling the liquid in and out of the pipette 3 times (be careful not to push liquid from the sample cup during expelling of the liquid). Insert test strip (arrow pointing down) into liquid and allow test to develop. Read results after 5 minutes and interpret according to the Interpreting the Lateral Flow Strip section.

Activity III – Hands-On Exercise: Gluten Analysis Add 5 mL of distilled water to sample provided. Shake the sample for 2 minutes. Allow the sample to sit 3 minutes for the large particles to settle. The liquid portion (i.e. sample) obtained will be used in step 2 below. Follow the test as described below for the extraction of gluten.

Activity III – Hands-On Exercise: Gluten Analysis Follow the test as described below to determine the presence of gluten. Interpret the results

Circle the response for test outcome SUNDAY ACADEMY DATA SHEET Activity Circle the response for test outcome Black Light What was the code number for your sample?   Did sample give positive result? Yes No Aflatoxin Test Was your sample positive or negative for aflatoxin? Positive Negative Did the black light and aflatoxin test results agree? Gluten Test Did your result match the expected result (see instructor)?