Show the Fermentation of Food

Fermented foods receive their tangy flavors, shelf stability, and possibly even health benefits from the active presence of “good” bacteria and/or yeast. Use this assay to demonstrate how fermented foods are teaming with bustling communities of microbes.

Be sure to use aseptic technique, described in more detail below, while conducting all aspects of this preparation. Pre-made agar plates can be purchased from a variety of vendors, or made on your own (see recipe below).

1. Collect the number of agar plates you will need, and label each with a sharpie marker to include your initials, date, ferment being plated, and the phrase “DO NOT OPEN”.
2. Set up 2 tubes per ferment you plan on testing, and label each tube accordingly.
   • Tube A will be prepared at room temperature.
   • Tube B will be boiled before plating.
3. For each ferment being tested, transfer approximately 1ml of fermentation liquid to Tube A, and 1ml of ferment liquid to Tube B.
   • Each time you go to transfer materials from the source to a tube, or from tube to tube, be sure that the transferring instrument is sanitized each time, or use a new sterile transfer pipette.
4. Keep set A tubes on the countertop. Boil sample set B for 10 minutes by placing the tubes in boiling water or a heat block set to 100 °C.
   • by boiling the ferment, you are presumably killing all of the microbes present.
5. Pour the liquid in each tube onto the correspondingly labeled agar plate.
6. Spread the liquid culture evenly around the agar plate using a sterile swab or cotton swab.
7. Return the lid to the agar plate, and allow the liquid to dry.
8. Turn the plate upside down (so that the agar side is up) and leave on the countertop to grow for 1-4 days.
9. Record observations, room temperature, and other factors that may be relevant.
10. At the end of the growth period, compare the difference between Tube Set A (unboiled) and Tube Set B (boiled).

Do you notice a difference between the unboiled and boiled ferments after plating?

1. Turn your sterilized jar on its side. Start loading vegetable spears into the jar, being sure that the length of vegetable pieces is shorter than the height of your jar
2. Add approximately 20g of celtic sea salt (salt volumes vary, but if you cannot measure by weight: 5 tsp celtic sea salt, 5 tsp table salt, 6 1/4 tsp kosher salt) per pint of pickling volume (scale this if your jar volume is significantly different)
3. Fill the jar to the very top with water.
4. Cap it with the sanitized lid, and swish the liquid around a bit to mix the salt in.
5. Remove the lid, and take a small drop of liquid out (using sterile dropper) and transfer to a pH strip. Record pH — this is time zero. Return lid.
6. Continue measuring and recording pH using sterile dropper on a daily basis to understand how pH changes during the lacto-fermentation process.
7. At the end of 2 weeks, the vegetables should be nice and crispy and ready to eat!

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?