Ex. 14 Lactic Acid Fermentation Introduction Lactic acid fermentation has been used by humankind in food production for thousands of years. Lactobacillus, Leuconostoc, Streptococcus, and Bifidobacterium are common bacterial genera that can ferment simple sugars in food to produce lactic acid and other organic acids, influencing the flavor, nutritional content, and preservation of the foods. Fermentation is a type of metabolism the occurs in the absence of oxygen. An anoxic environment helps prevent growth of some harmful microbes that could contaminate foods, and the acidic byproducts produced during fermentation also have preservative properties. Additional ingredients are often added during fermentation that can provide seasoning and also help ensure that the culturing process is biased towards growth of fermentative microbes, and that the growth of potentially harmful contaminant microbes is prevented. Sauerkraut is a fermented food produced by immersing cabbage in salt water, and then then allowing fermentative growth of bacteria in anoxic conditions. The presence of the salt and the anoxic environment help limit the growth of undesirable bacteria and fungi. The fermentation of sauerkraut occurs through growth of a succession of microorganisms. The first colonizers are only slightly tolerant of low pH and lactic acid, and as they grow, residual oxygen is consumed and acid is produced, leading to a reduction in solution pH. As the pH drops, other bacteria that are more acid tolerant become dominant in the culture (for example, Leuconostoc mesenteroides and Lactobacillus sp.). Leuconostoc cells are described as heterolactic bacteria because they produce approximately equal amounts of lactic acid, ethanol, and CO2. Eventually, increasing acidity will inhibit growth of Leuconostoc as well, and other fermenters such as Lactobacillus sp. will begin to predominate in the sauerkraut culture. Lactobacilli are described as homolactic bacteria because they produce lactic acid as their main fermentation byproduct. Eventually, enough lactic acid from bacterial fermentation accumulates to cause full inhibition of bacterial growth, thereby allowing preservation of the sauerkraut for a long time. Preserved foods like sauerkraut were especially valuable during wintertime, when fresh produce was hard to come by. Learning Objectives Be able to describe how Sauerkraut ferments over time Why is sauerkraut prepared with salt and kept in an anoxic environment? How does pH change, and how does this influence the succession of bacterial species? - - - Be able to describe homolactic vs heterolactic fermentation Lab Period 1 (Set up a sauerkraut culture) Materials and Supplies One deMan-Rogosa-Sharpe (MRS) agar plate: selective for Lactobacillus One Tomato Juice (TJ) agar plate: selective for Leuconostoc Anaerobic jars, pH paper, hot plate, aluminum foil, tablespoon, cutting board, knife. - Green cabbage, table salt (sodium chloride), large plastic container (~2–4 Liters) to contain the cabbage mixture. Plastic bags, to line the cabbage container. Ziplock bags, to contain macerated cabbage, water, and salt. When sealing the bag, the air in the bag should be minimized to help reduce the amount of oxygen present for the fermentation process. Balance, weighing dishes, Bunsen burner, striker, inoculating loop. Procedure 1) The sauerkraut will be prepared by your TA, with some help of some volunteers who are comfortable. Chop the cabbage into even, small pieces and put into zip-lock bags. Make sure that the zip-lock bag is open, on the scale, and tared to get an accurate reading of the weight. The lab scales can only handle up to 400 grams of weight, so don't force them to go over. Make enough bags of cabbage for at least each bench to get one and label the weight on the front of the bag. 2) After making the bags of cabbage, pass them out to the benches and start adding the salt. This style of fermentation requires 3 % (by weight) table salt to be added to the bag of cabbage. Calculate the proper amount of salt for the weight specifically written on your bag of cabbage. 3) Once you have added salt, massage and mash the cabbage until it is wilted and there is a significant amount of cabbage juice in the bag (be careful not to break the bag). This maceration process can take up to 30 minutes. 4) Once you judge that the cabbage is fully macerated, use a transfer pipette to collect about 1mL of the liquid, and add to a microcentrifuge tube. 5) Add your cabbage to the communal cabbage bucket and toss the used ziplock bag into the biohazard waste. 6) Using your centrifuge tube of cabbage juice, and correct streaking technique, streak the MRS and TJ plates. Note: MRS agar is a green/brown color and TJ agar is a dark red color. 7) Measure the pH of the cabbage juice and record in Table 14.1. This is the initial measurement for comparison to see how pH changes as the bacteria colonize the sauerkraut culture. 8) Using a loop, transfer a drop of cabbage juice to a microscope slide, and prepare a wet mount for viewing with the microscope. See Ex. 6 if you need to refresh your memory of what a wet mount is and how it is prepared. See Ex. 5 if you need a refresher on how to correctly use the microscope. 9) Write down your results in Table 14.3. Keep in mind that the sauerkraut hasn't had much time to begin the fermentation process, so consider how this may affect your observations, and predict how these observations are likely to change as the fermentation process progresses over the coming days. 10) Incubate the MRS and TJ plate in your lab section's anaerobic jar until the next lab session. 11) Your TA will place some zip-lock bags of water on top of the sauerkraut to keep as much oxygen out as possible during the fermentation process. Lab Period 2 (observations of sauerkraut fermentation) Materials and Supplies One MRS, one TJ agar plate. pH paper, micropipettes, Bunsen burner, striker, inoculating loop. Reagents for Gram staining procedure. Optical microscope with phase contrast, microscope slides, coverslips. Procedure for observing results of Lab 1 sauerkraut fermentation 1) Observe the growth on the TJ and MRS streak plates from the previous lab. Record observations (growth, colony morphology, density) in Table 14.2. Consider the reasons for growth, or lack thereof. - - 2) If you observe colonies on the plates, make wet mount slides, and also perform a Gram stain, and observe them using the microscope. If no growth occurred, skip to step 7). 3) Making wet mounts or gram stains from colonies is different from liquid cultures, because plate cultures contain a greater density of cells, requiring dilution. Start by using adding a drop of water to the slide (use the DI water from the Gram stain kit). 4) Sterilize the inoculating loop, let it cool, and then pick up a small part of a colony from the most dominant colony type on one of the culture plates. Spread the microbes in the in the water on the slide, and form a smear. Do not use an excessive amount of the colony, or the cells will be too dense to allow good visualization of individual cells under the microscope. 5) For each colony type, use this technique to make one slide for a typical wet mount (adding a coverslip to the smear), and one slide smear that you will allow to air dry for Gram staining. 6) Prepare and the observe the Gram stained slides by microscopy. Note that Leuconostoc species are Gram positive cocci that often form chains, while Lactobacillus species are Gram positive bacilli that often form chains. Make general observations, and keep in mind that it is not possible to definitively identify bacterial species from microscopy alone. 7) Record your observartions in Table 14.2, 14.3, and 14.4. Dispose of the plates from last class into the biohazard bin before moving on. 8) Check on the class sauerkraut culture to see how it is changing. As in the previous lab session, use a transfer pipette to collect about 1 mL of the fermented liquid to place into a centrifuge tube. 9) Streak some of the culture on fresh MRS and TJ plates. Incubate the plates in the anaerobic jar again until next class. 10) Measure the pH of the sauerkraut liquid, and record the results in Table 14.1. Lab Period 3 (observations of sauerkraut fermentation) Materials and Supplies - 1 MRS agar plate and 1 Tomato Juice (TJ) agar plate pH paper, centrifuge tubes, Gram staining supplies Procedure 1) Observe the streak plates from the previous lab and note differences. Again, if you have any viable colonies, prepare wet mounts and Gram stains of representative colonies and record your results in Tables 14.2, 14.3, and 14.4. Dispose of your plates from last class before moving on. 2) Use a transfer pipette to collect a sample of sauerkraut liquid to place into a centrifuge tube. Does the sauerkraut look different this time? Is it smelling different? 3) Remove a sample with your loop and streak on MRS and TJ agars. Incubate the plates in the anaerobic jar until next class. 4) Record the pH of the liquid (Table 14.1). A change in pH towards more acidic indicates that fermentation has occurred and the bacteria are producing acid. Were there any bubbles in the fermented liquid? If so, what are they from? Lab Period 4 (observing sauerkraut) Procedure 1) Examine your plates streaked from the sauerkraut culture in the previous lab session. Record results in Table 14.2. 2) Prepare a wet mount and Gram stain from colonies on each plate. Measure the pH of the sauerkraut fermentation. Record all results in Tables 14.1, 14.3, and 14.4. 3) Dispose of your plates from last class. 4) Make any final observations of how the sauerkraut has changed during the course of the fermentation./n