Explore the Fermentation of Food

1. Fill 15ml conical tube with 8ml of a sugar solution
2. Mix the 7% yeast solution to be a uniform suspension. Fill the remainder of the tube (~7ml) with yeast solution such that the meniscus rises above the lip of the 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 by emptying the tube, filling with water to the mark, and pouring the water into a graduated cylinder or onto a balance for accurate measurement

What conclusions can you draw about the metabolism efficiency of different substrates by S. cerevisiae?

1. Clean and ideally sanitize equipment (e.g. dishwasher or chemical sanitizer—bleach, StarSan, etc.)
   • Brewing Container: 1 L container e.g. pyrex beaker, wide mouth ball jar, or measuring cup
   • Spoons (2; one for sugar, one for stirring tea)
   • Thermometer
   • Storage Container (size depends on how much broth will be left over e.g. the difference between the volume of your storage jars and 1 L; container should seal with a lid)
2. Heat 1 L water to boiling (e.g. use an electric kettle, pot on the stove, or beaker on a hot plate)
3. Pour ~850 ml of water into the Brewing Container. Let cool to 170F.
4. Add 3 bags of green (or black) tea (~6g tea) and let steep for 10 minutes.
5. Tug on bags 10 times to disperse the brewed tea. Discard tea bags.
6. Add 150 g sucrose (table sugar; ¾ C) and stir until dissolved.
7. Bring the water up to 1 L with the excess boiled water.
8. Cool to room temperature (in a fridge (~30 min), freezer (keep a close eye on it), or on the counter overnight)
9. Label the brewed tea/sugar mixture, and use to start your kombucha culture.
   • Note: this is a good stopping point as the sugar-tea can be stored for several weeks in a sealed container if good sanitation was followed.

What experimental question do you have in terms of how something might affect the production and composition of kombucha?

How will you test this?

Set up your tubes and water bath in such a way that the gas produced in each fermentation will rise to the tops of the tubes and volumes of gas can be marked with a permanent marker or waxed pencil.

In the example at right, plastic tubes were used with a few holes poked into each plastic cap. The tubes were filled with yeast solution and food source (e.g. glucose), covered, and inverted quickly into the water bath. The samples should be held inside the tubes and displaced into the water bath as gas is produced.

A similar setup can be used with test tubes and stoppers with holes or using a glass slide to invert each tube into the bath before sliding off the slide and allowing the fermentation gas produced to displace the yeast solution. You may also be able to use a typical gas collection apparatus from a chemistry laboratory at your school.

Finally, volumes of gas can be determined by emptying a marked tube and filling it with water exactly to the mark. Then that water can be poured into a graduated cylinder of appropriate size to determine the volume of gas produced.

Guiding the Inquiry

Think about how you can design an experiment to test this question, keeping in mind the inclusion of an appropriate control. Remember, a simple design makes for elegant results.

Write out your procedure, step by step to test your experimental question. Create a proper data table to easily record and share your results. Was your hypothesis supported by the data?  Why or why not?