# Lab 5 Bacterial Fermentation and Lactic Acid Bacteria
## "The Sauerkraut Lab"

## BACKGROUND

Ethanol fermentation and lactic acid fermentation are two important steps in food production. While the former is used for making wine and beer, the latter is widely involved in milk-based cheese manufacturing and vegetable-based pickle making (e.g., sauerkraut). Sauerkraut means sour cabbage. By mixing sliced cabbage with about 2.5% salt and packing it tightly in a jar to create an anaerobic condition, the naturally present bacteria will grow, especially lactic acid bacteria (LAB). Under the anaerobic conditions, LAB gradually take over and become the dominant bacterial group. The fermentation can take place at room temperature (~23 ˚C), with the fermentation process taking about three to four weeks. At lower temperatures, the fermentation will take longer or may not occur at all. If the temperature is too high, spoilage will occur before the fermentation is complete. The acidity is one way to check the fermentation completeness; acidity of 1.2% should be achieved in about few weeks, and the end pH should be around 2%.


Figure 5.1 During the fermentation process, the numbers of LAB increased as the fermentation time increased. The pH decreased as the percentage of lactic acid increased (from 1.0% to 1.8%) Figure is adapted from Dr. Matt J. Stasiewicz from UIUC. For more information, please refer to Hutkins (2006). Fermented Vegetables Microbiology and Technology of Fermented Foods. Blackwell Publishing. Page 233-261. Created with BioRender.com.

## OBJECTIVES

1. Practice making sauerkraut.
2. Get familiar with the culturing of LAB.

## MATERIALS

### Food
- Whole cabbage and un-iodized salt

### Media
- MRS agar (check Lactobacilli MRS agar in the media manual) x5
- PCA agar x 5
- VRBA agar x 5
- 0.1% peptone water x 4
- 2% boiled salt water (keep it at room temperature before the lab)

### Supplies
- Cutting board and knife
- Balances
- Spatulas
- Weigh boats
- Airlock or mason jars
- Small ziplock bags
- Whirl-pak bags
- pH meter
- Mixing bowl
- Anaerobic jar with gas packs

## PROCEDURES
### Procedures [VP for lab 5](https://drive.google.com/file/d/1-EZ7v7PwbdyucsHvhUUYnuDSeQq21hFt/view?usp=drive_link)
### STEP 1: Chop the cabbage and mix it with salt

**Figure 5.2** Mixing chopped cabbage with 2% salt and sauerkraut in mason jars. Chop the cabbage into thin strips (<1/8 inch if you can) and get rid of the core (Please trim cabbage heads by removing bruised, damaged, or soiled outer leaves). Weigh about 500 grams of chopped cabbage and mix it with 2.5% salt (12.5 gram of salt) and 1.0% dextrose (5 gram of dextrose). Mix them well in the bowl and let it sit for about 10 min. Press and squeeze tightly to release the juice. Place glass weight directly on cabbage mixture to cover and to compress the cabbage mixture. The preparation of salted cabbage and the fermentation process as well as the fermentation will take place in the sensory building kitchen.

### STEP 2: Plate the salted cabbage for total PCA, VRBA, and MRS counts.


Figure 5.3 MRS plates incubated in an anaerobic jar with gas packs. Weigh **25 grams** of salted cabbage and mix with 225 ml of buffered peptone water. Stomach the sample in 2 min and make dilutions from 1:10 to 1:10,000. Plate 100 µl of each dilution onto PCA and MRS agar and plate 1 ml of each dilution with VRBA using the pour plating-with-overlay method. Also, measure the pH of the salted cabbage (make sure the probe is dipped in the liquid).

### STEP 3: Tightly pack the salted cabbage into the bottom of the jar.

Pack tightly in the jar, which needs to be about 80% full, and add 2% salt water to “seal” the cabbage and fill up the jar. The addition of brine is to create anaerobic conditions. Incubate the jars at 23 ˚C for three weeks. Each team needs to prepare three jars since we need to monitor the fermentation process for three weeks. If 500 grams of cabbage is not enough, chop more and add salt accordingly.

**Starting from next Monday (or Tuesday for the Tuesday groups), in addition to the new labs, one jar of fermented cabbage needs to be taken out for plating (PCA, VRBA, and MRS) and pH checking. The plates need to be counted on Wednesdays or Thursdays.**

## Questions for Discussion

1. How do the MRS counts change from Day 0 to Days 7, 14,