Explore the Biochemical Deconstruction of an Egg Sandwich

1. Fill 15ml conical tube with 8ml of a sugar solution (recommended starting concentration is 0.5%, v/v)
   
2. Fill remainder of tube (~7ml) with 7% yeast solution such that the meniscus rises above the lip of the tube. 
   • NOTE: stock yeast solution should be agitated before adding to tube
3. Replace cap onto tube — because of holes, there will be a small squirt of solution to come out. 
   • NOTE: make sure that there are no sizeable air bubbles in the tube
4. Invert the tube, and place in a large beaker filled with water (preheated to 40 °C). Place this beaker into a water bath or onto a hot plate to maintain temperature 
   • NOTE: you can also test the effect of temperature on fermentation by adjusting temperature of water bath or hot plate
5. Immediately mark the bottom of the CO₂ bubble (if there is one). Mark this point at 5 minute intervals for 30 minutes.
6. At the end of the experiment, record the level of CO₂ produced at each time interval

Which substrate(s) did the yeast ferment fastest? Which substrate(s) allowed for the greatest total fermentation?

Based on your results, what does this tell you about the important bonds in sugar and starch molecules?

Which of these food molecules are in your egg sandwich? How do you think your body compares to the yeast when trying to digest these sugars and starches?

PROCEDURE FOR MILK TEST

1. Fill 6 test tubes each with 5 mL of milk.
2. To each test tube containing milk, add one of the following substances:
   • 1 mL water
   • 1 mL vinegar
   • 2 g meat tenderizer
   • 1 mL meat tenderizer solution
   • 1 mL boiled meat tenderizer solution
   • 1 mL boiled vinegar
     
3. Make observations immediately and over the next 30-60 minutes.

What conclusions can you draw about how acid, heat, and enzymes affect milk protein structure?

Which conditions might be starting to make “cheese?”

1. To each tube of foodstuffs, add 1 mL of chloroform:methanol (2:1, v/v) solution.
2. Secure the cap on the tube and vigorously shake the samples for 30 seconds.
3. Place the tubes back into a rack, and remove the caps.
   • At this point, you have created a monophasic system whereby components are dissolving in the solution.
4. Add 267 μL of the 0.73% NaCl solution to each tube to make a chloroform: methanol: water mixture (2:1:0.8, v/v/v).
5. Secure the caps back onto the tubes and again shake vigorously for 30 seconds.
   • The addition of the aqueous salt solution transforms the mixture into a biphasic system, and helps to separate the hydrophobic lipid components from water-soluble (hydrophilic) components.
   • Because chloroform is more dense than methanol and water, the chloroform-lipid phase will move toward the bottom of the tube, and on top of this will sit the less dense methanol-salt phase (thereby creating a biphasic system).
6. Spin conical tubes in a benchtop centrifuge at 2,500 rpm for 2 minutes.
7. Carefully remove the tubes from the centrifuge, being sure not to disturb the phase separation.
   • If you do not have a benchtop centrifuge, you can allow the tubes to sit, undisturbed, for 30-60 minutes, which will allow the phases to completely separate.
8. Transfer the top, methanol-water phase, to a waste container labeled for methanol disposal.
   • If you have interest and ability, this phase can be used to assess non-lipid components of the food materials.
9. Label a fresh glass tube with the appropriate food label.
10. Using a glass pipette, transfer the bottom, chloroform-lipid phase to this freshly labeled glass tube.
    • There will still be a small amount of biomass in the tube, above the chloroform-lipid phase — try to get to the chloroform-lipid phase without disturbing the biomass too much. you can just quickly “push” your glass pipette tip through the biomass to get to the bottom.

What observations can you make about the lipid samples?

10. The lipid is now ready to be analyzed using TLC.

1. Using 2 small pieces of tape, attach the TLC plate to the hotplate such that the bottom of the plate is touching the hotplate.
2. Mark the origin (bottom) of your plate with a pencil and label each lane.
3. Slowly spot the standard in the first lane in increments of 5 μL, allowing the spot to dry.
4. Repeat 3 times for a total of 15 μL standard.
5. Next, slowly spot the lipid extract in the designated lanes in increments of 5 μL, allowing the spots to dry.
6. Repeat 5 times for a total of 25 μL sample.
   • If you do not have micropipettes, you can use a glass Pasteur pipette to add your lipid extract to the plate in a dropwise fashion, being sure that you allow the “spot” to dry in between each drop.
7. Place the plate into the charged TLC chamber, in front of the wick.
8. Secure the lid to the chamber and allow the plate to develop.
9. Once the solvent front nears the top of the plate, remove the plate from the TLC chamber, quickly mark the solvent front with a pencil, and allow it to dry.
   • It typically takes 30-60 minutes for the solvent front to reach near the top of the plate.
10. Place the plate into the iodine chamber for visualization and secure the lid.
11. Once adequately stained (about 3-5 minutes), remove and lightly outline the spots and/or take a picture.

What conclusions can you draw about the polarity of the substances?

Discuss one purpose of this technique in research or industry.