Food Microbiology: Lactic Acid Fermentation of Sauerkraut
People have used microorganisms to produce fermented foods for centuries. Common examples include alcoholic beverages, produced from fermented fruits and grains, cheeses, produced from fermented milk, and breads, produced from fermented grain flours. Many vegetables are also fermented to produce foods that have very different flavors and are much less prone to spoilage than the original material. Examples of fermented vegetable products include soy sauce and miso, olives, chocolate, coffee, and pickles.
Cabbage has also been fermented in various fashions by different cultures throughout history. Modern fermented cabbage products range in style from German sauerkraut to Korean kimchi. Despite the very different flavor of these foods, they are all produced in the same way. The ethnic variation occurs with the flavorings that are added (or not) to the cabbage as it ferments. The simplest version, in which no additional seasonings are added, is generally referred to as sauerkraut.
Essentially all fermented cabbage foods utilize the natural microbial community already present on the plant to perform the fermentation. Sauerkraut is thus made by shredding cabbage and mixing it with a final concentration of 2.2% to 2.8% salt. Adding salt to the cabbage achieves two purposes. First, it draws sugar-containing juices out of the shredded leaves so that they can be fermented more easily, and second, the high salt concentration inhibits the growth of most of the microorganisms present on the cabbage leaves. The microbes best suited to grow under these high salt conditions are lactic acid bacteria of the genera Leuconostoc and Lactobacillus. These bacteria become the dominant microorganisms within the community and ferment the plant sugars to produce lactic acid as a waste product.
Growth of the microbial community within a sauerkraut fermentation follows a predictable pattern of community succession. Leuconostocspp. (usually L. mesenteroides) will predominate early during the fermentation. As the concentration of lactic acids increases to about 1%, further growth of Leuconostoc is inhibited by the increasing acidity. At this point, Lactobacillus sp. (typically L. plantarum and L. brevis) become the predominant microorganisms. They can continue to ferment the cabbage to produce lactic acid levels as high as 2.4%, although a more palatable product usually has only about1.3% to 1.7% lactic acid. (The fermentation can be stopped at the appropriate time by canning or refrigerating the product.) The total time required for a sauerkraut fermentation to occur is 2-3 weeks, with the pH dropping from initial levels of about 6.5 to below 3.0. This final combination of high salt and high acidity is what preserves the food product by inhibiting the growth of potential spoilage microorganisms.
In this exercise, we will set up a sauerkraut fermentation as an enrichment for lactic acid bacteria. The protocol for isolating and characterizing the lactic acid bacteria is described in the next exercise.
Materials (per lab bench)
Cabbage
Salt
Glass beaker and Ehrlenmeyer flask
Balance (shared)
Knife, mixing bowl, wooden spoon (shared)
Saran wrap
Procedure
- Each lab bench will set up its own sauerkraut fermentation. First, shred the cabbage into very thin pieces. You should prepare enough to fill the beaker loosely.
- Place the shredded cabbage in a plastic bag and weigh it. Add salt to a final concentration of about 2.5% (i.e., about 2.5g of NaCl for every 100g of cabbage).
- Mix the salt and cabbage together thoroughly by shaking the bag (it should start to look “wet”), then transfer it to your beaker. Press the cabbage down firmly with the base of the Ehrlenmeyer flask to force out any trapped air bubbles. The beaker should be about ½ full at this point, and a thin film of juice should accumulate at the top.
- Fill the Ehrlenmeyer flask with water and place it on top of the cabbage mixture. This will serve as a weight to keep the reaction surfaces in close proximity, to force out gas bubbles as they develop, and to keep air out of the solution. There should be no air bubbles trapped beneath the flask, and a thin layer of fluid should continue to cover the top of the mixture around the edge of the flask. (A small amount of water can be added, if necessary.)
- Leaving the flask in place, wrap a piece of saran wrap around the top of the beaker to retard evaporation. Incubate at room temperature.
- Check the fermentation each lab period to be sure that the top layer of fluid is still present and that no gas bubbles have become trapped. Force out any bubbles by pressing down on the beaker, and replenish the fluid by adding a small amount of tap water, as needed.
Important: It is imperative that the system be maintained in an anaerobic state! (The alternative is full-scale fungal rot instead of bacterial fermentation.) You must therefore be careful to remove any bubbles or air pockets initially present within the cabbage solution, and you must prevent the top of the mixture from drying out during the fermentation period by adding water as necessary.
Isolation & characterization of lactic acid bacteria
The lactic acid bacteria consist of several genera of bacteria that produce lactic acid as a major fermentation product. They are either Gram positive rods or Gram positive cocci and all are catalase negative. Although they are able to grow in the presence of oxygen, they are indifferent to it. They neither use it in their metabolism nor are they harmed by its presence. They are aerotolerant anaerobes, whose only means of extracting energy from the sugars they metabolize is by fermentation, i.e. substrate-level phosphorylation. Their natural habitats are the surfaces of plants, and the cavities and intestinal tracts of animals where sugars are present. Also in these habitats their many nutritional requirements for amino acids, vitamins, purines and pyrimidines can be met by the plant or animal hosts. In general, they form small colonies even on rich media, and have complex nutritional requirements, making them difficult to work with. Some form capsules or slime on media containing sucrose.
Gram positive cocci of the lactic acid bacteria include the genera, Streptococcus (flora of mucous membranes), Leuconostoc (plants, dairy products), Enterococcus (fecal flora), and Lactococcus (dairy and plant products). The Gram positive rods are in the genus Lactobacillus (animal and vegetable food products, gastrointestinal tracts, mammalian vagina). Lactic acid has a pleasant taste in small amounts and for centuries these organisms have been used to produce special foods.
Because the lactics have such a long list of requirements for growth, designing an enrichment for them to the exclusion of other bacteria is difficult if not impossible. Several media have been formulated to support the growth of lactic acid bacteria, but they are not selective. You will use fermenting cabbage (sauerkraut) as sources for these organisms. You will try to isolate one each of Leuconostoc and Lactobacillus.
PERIOD 1
- One student at each bench should test the pH of the 3-6 day old sauerkraut fermentation and share the information with the other five students.
- Each student should streak samples of the sauerkraut onto Elliker agar and TSY + 5% sucrose agar. Incubate at 30°C. Gram stain some sauerkraut juice and note the Gram reaction, size, shape and relative numbers of the varieties of bacteria present.
PERIOD 2 (2 to 4 days after Per.1)
Sample several colonies to identify probable lactic acid bacteria. Many colonies will be small and difficult to work with. Be sure to observe with care! They cannot be ignored! Small inocula are all that is needed (use needle if necessary); Look for colonies of Gram positive cocci that are catalase negative. You want to isolate if possible a Leuconostoc.
Leuconostoc is easily identifiable by its production of polysaccharide slime when grown on a sucrose-containing medium. If you have an isolated slimy colony on TSY + sucrose which is catalase negative and Gram positive cocci, streak it onto an Elliker agar plate. If there are no isolated colonies on the TSY + sucrose plate, choose instead an appropriate colony from the Elliker plate to streak.
If you have no isolated colonies on your plates, resample the original materials with more careful plate streaking.
PERIOD 3 (2 to 4 days after Per.2)
Check isolated colonies from your plate (if it has a single colony type per plate) for Gram stain, shape and catalase reaction. Stock culture onto a TSY + 2% glucose slant. After they have grown move them to the room temperature incubator until Period 7.
Restreak for purity if your plate still shows multiple colony types or no isolation.
PERIOD 4 (Three weeks after Per.1)
- One student at each bench should test the pH of the 3-4 week old sauerkraut and share the information with the other five students.
- Each student should streak a sample of the sauerkraut onto MRS agar. Incubate at 30°C. Gram stain some sauerkraut juice and note the Gram reaction, size, shape and relative numbers of the varieties of bacteria present. How does this sample differ in appearance from the 3-day sample?
PERIOD 5 (2-4 days after Per.4)
Sample several colonies to identify a probable Lactobacillus. Again colonies will be small and difficult to work with. Look for a colony of Gram positive rods that is catalase negative and restreak for purity.
PERIOD 6 (2-4 days after Per.5)
Check an isolated colony from plate (that should have a single colony type per plate) for Gram stain, shape and catalase reaction. Stock culture onto TSY + 2% glucose slants. After they have grown move them to room temperature incubator until Period 7.
PERIOD 7 (2 or more days after Per.6) Characterizing the lactic acid bacteria
Inoculate each isolate into TSY + 2% glucose deeps containing bromcresol purple by stabbing with a sterile loop. Seal with a layber of 4% agar and incubate at 30°C.
These deeps will show fermentation of glucose with a change to yellow color and perhaps the presence of gas, which will tell you if your isolate is homofermentative or heterofermentative.
PERIOD 8 (2 or more days after Per.7)
Read all results and decide as best as possible the partial identification of the isolates.
If their only fermentation product is lactic acid, they are called homofermentative. If there is also CO2and ethanol produced, they are heterofermentive. These two can be distinguished in the inoculated sealed deeps where a pH change will show acid production and bubble will show gas production when CO2(and therefore ethanol) is produced. All of these “lactic acid bacteria” grow both aerobically and anaerobically (by fermentation only), and are Gram positive and catalase negative.
The accompanying flow chart outlines the degree of identification which we will expect with these organisms.
COCCI RODS
Pairs or chains Lactobacillus
No gummy colony gummy colony
on sucrose on sucrose
homofermentative heterofermentative
homofermentative homofermentative heterofermentative
Streptococcus Report as homo- or hetero-
Report as report as Fermentative Lactobacillus
Growth no growth Streptococcus Leuconostoc
At 42 C At 42 C salivarius mesenteroides
See Bergey’s Manual for tests to speciate.
Report as report as
Strep. Strep.
Thermophilus lactis
See Bergey’s Manual for tests that verify species.