Chapter 12 - Lactic Acid Bacteria

12 - 1 An introduction to lactic acid bacteria

Lactic acid bacteria are Gram-positive usually non-motile, non-spore-forming rods and cocci. They lack the ability to synthesize cytochromes and porphyrins (components of respiratory chains) and therefore cannot generate ATP by creation of a proton gradient. The lactics can only obtain ATP by fermentation, usually of sugars. Since they do not use oxygen in their energy production, lactic acid bacteria happily grow under anaerobic conditions, but they can also grow in oxygen's presence. They are protected from oxygen byproducts (e.g. H₂O₂) because they have peroxidases. These organisms, as defined in experiment 3, are aerotolerant anaerobes. They are differentiated from other organisms by their ability to ferment hexoses to lactic acid, hence the name. Lactic acid bacteria can be divided into two groups based upon the products produced from the fermentation of glucose. Homofermentative organisms ferment glucose to two moles of lactic acid, generating a net of 2 ATP per mole of glucose metabolized. Lactic acid is the major product of this fermentation. Heterofermentative lactic acid bacteria ferment 1 mole of glucose to 1 mole of lactic acid, 1 mole of ethanol, and 1 mole of CO₂. One mole of ATP is generated per mole of glucose, resulting in less growth per mole of glucose metabolized. Because of the low energy yields, lactic acid bacteria often grow more slowly than microbes capable of respiration, and produce smaller colonies of 2-3 mm.

It is easy to determine whether a lactic acid bacteria has a homo- or heterofermentative metabolism by the hot-loop test. A major end-product of heterofermentation is CO₂. In a medium containing glucose this gas is highly soluble at high pH and will stay in solution. If, however, the temperature of the solution is increased, CO₂ will become insoluble and will be released in the gaseous form. The hot-loop test consists of growing a test isolate to saturation in a medium containing glucose. After incubation, a wire loop (inoculating loop) is heated to redness and plunged into the broth culture. This causes the liquid around the loop to heat up. If a test organism is heterofermentative, CO₂ bubbles will evolve close to the loop.

The lactic acid bacteria have limited biosynthetic ability, requiring preformed amino acids, B vitamins, purines, pyrimidines and typically a sugar as carbon and energy source. A rich medium is usually employed when cultivating lactics. These multiple requirements restrict their habitats to areas where the required compounds are abundant (animals, plants, and other multicellular organisms). Lactic acid bacteria can grow at temperatures from 5-45°C and not surprisingly are tolerant to acidic conditions, with most strains able to grow at pH 4.4.

The genera of lactic acid bacteria

Lactics are classified by the fermentation pathway used to ferment glucose and by their cell morphology. Lactobacillus are rod shaped organisms that can be either hetero- or homofermentative. They are widespread and can be isolated from many plant and animal sources. Lactobacilli are more tolerant to acid than the other genera of lactic acid bacteria and this property makes them important in the final phases of many food fermentations when other organisms are inhibited by the low pH.

Leuconostoc are ovoid cocci, often in chains. All bacteria of this genus have a heterofermentative mode of metabolism. When grown in media containing sucrose, copious amounts of a slimy polysaccharide (dextran) are produced. Dextran has found uses in medicine as a plasma extender and in biotechnology.

Pediococcus are cocci often found in pairs and tetrads that are strictly homofermentative. Their habitat is restricted mainly to plants. P. cerevisiae has been used as a starter culture for the fermentation of some sausages with great success. Streptococcus are cocci in chains that are distinguished from the Leuconostoc by their strictly homofermentative metabolism. These organisms can be isolated from oral cavities of animals, the intestinal tract, skin, and any foods that come in contact with these environments. While the other genera of lactic acid bacteria rarely cause disease, Streptococcus pyogenes is a common, troublesome pathogen, causing strep throat and rheumatic fever.

Enterococcus and Lactococcus are two recent taxonomic divisions of lactic acid bacteria. These were created to reorganize the large and divergent Streptococcus genus into smaller, more related groups of bacteria. Enterococcus are gram-positive cocci that form pairs or chains. They are distributed widely in the environment, particularly in feces of vertebrates. Strains can grow in the presence of 6.5% NaCl and with 40% bile present.

Lactococcus includes strains that are gram-positive, spherical cells occurring in pairs or chains. They have a strictly homofermentative metabolism and are found in dairy and plant products. For centuries lactic acid bacteria have been used to produce fermented food products including pickles, sauerkraut, sausage, yogurt, cheese, buttermilk, soy sauce, and more. Some examples include Streptococcus thermophilus along with Lactobacillus bulgaricus that are used in the production of yogurt. Also, Lactococcus lactis and S. thermophilus are two strains often used as starter cultures in the production of cheese. Finally, Lactobacillus and Leuconostoc are useful in the fermentation of cabbage to sauerkraut.

12 - 2 Lactic acid bacteria experiment

Below is the protocol that we will follow for investigation of the Lactic Acid Bacteria.

Period 1

Materials

Cultures of the following grown in APT Broth1 or Milk2:

Samples of juice (diluted 1/10) from 4 or 5-day-old and 10 to 12-day-old sauerkraut fermentations (Each pair uses either the younger or the older sample.)

2 tubes of APT Broth (approx. 8-10 ml)

2 tubes of pasteurized or sterilized milk (exactly 5 ml)

5 plates HIAG (HIA plus 5% glucose)

5 plates HIAS (HIA plus 5% sucrose, 0.5% glucose and 0.02% sodium azide)

4 dilution blanks (9 ml)

Pipettors and sterile tips

1 small, clean Erlenmeyer flask

Titration apparatus (with 0.1M NaOH and phenolphthalein)

1. Gram reaction and morphology. Prepare heat-fixed smears from the cultures. After performing the inoculations below, gram-stain the smears and determine the gram reaction, shape and arrangement of the cells for each culture. Reocrd your observations. A stained background will be seen for the organisms which had been grown in milk.
2. Differentiation between homo- and heterofermentative lactic acid bacteria. This simple test which detects CO₂ production by the heterofermentative lactics is helpful in the identifications of the genera. We will differentiate between the almost morphologically-identical genera Lactococcus and Leuconostoc.
3. Inoculate one tube of APT Broth with Lactococcus lactis and another tube with Leuconostoc mesenteroides.
4. Incubate the tubes in your desk drawer for 1-2 days. If the next lab period is more than 2 days away, place the tubes in the rack on the stage. (The tubes will be refrigerated until the day before the next lab period, then incubated at 30°C.)
5. Fermentation of milk. Some (not all!) lactic acid bacteria ferment lactose, the milk sugar.
6. The production of yogurt is accomplished by two organisms, Lactobacillus bulgaricus and Streptococcus thermophilus. The resulting low pH from lactose fermentation causes the coagula-tion of the milk protein (casein), forming a curd. The two organisms produce some flavoring compounds and also growth factors which assist their mutual growth. The following production of pseudo-yogurt will demonstrate the effect of these organisms on milk:
7. To each tube of milk (A and B), add 0.1 ml of L. bulgaricus and 0.1 ml of S. thermophilus. Incubate tube B at 37°C until the next period.
8. Add the contents of tube A to a clean Erlenmeyer flask. Obtain some distilled water in a clean glass from the special tap in the sink, and pipette 5 ml of the water into the flask. At the titration station, add 4 drops of phenolphthalein solution, mix well and titrate with 0.1M NaOH until the indicator shows a trace of pink color persisting for at least 15-20 seconds. Record the volume of NaOH used. Dispose the flask into the tray on the discard cart.
9. Slime production from sucrose. This procedure will demonstrate how some lactic acid bacteria produce slime from sucrose but not from most other sugars. In the case of Leuconostoc mesenteroides and certain other lactics, the enzyme dextran sucrase splits sucrose into glucose and fructose and also polymerizes the glucose into dextran.
10. Streak the culture of L. mesenteroides for isolated colonies on one plate each of glucose and sucrose-containing media, i.e., HIAG and HIAS. (See Materials. The sodium azide in the latter medium will be of no significance in this test.)
11. Incubate at 30°C until the next period.
12. Lactic acid bacteria in the sauerkraut fermentation. Sauerkraut is essentially shredded cabbage which has been altered by the growth and activities of bacteria indigenous to the cabbage (see the introduction). Important in the activities of these organisms and the reduction of spoilage organisms are anaerobic conditions and the initial addition of NaCl (2.5% of the total weight). Proceed as follows with one of the sauerkraut samples provided:
13. Prepare dilutions and platings of the sample (already a 1/10 dilution) onto HIAG and HIAS such that one plate of each medium is made for each of these plated dilutions: 10^-3, 10^-4, 10^-5 and 10^-6.
14. Optionally, make a streak plate of the sauerkraut sample, plating onto HIAS and HIAG.
15. Note that we are using Heart Infusion Agar plus glucose (HIAG) - an all-purpose medium - for the total count.
16. The other medium (HIAS) contains sodium azide and will support the growth of only lactic acid bacteria when incubated aerobically.
17. As HIAS contains sucrose, it will help to differentiate between the two major lactic acid genera in sauerkraut - the slime-forming Leuconostoc and the non-slime-forming Lactobacillus.
18. Incubate at 30°C until the next period.

Period 2

Materials

1 pipette (5 ml)

1 small, clean Erlenmeyer flask

titration apparatus (with 0.1M NaOH and phenolphthalein)

1. Differentiation between homo- and heterofermentative lactic acid bacteria. For each tube, plunge a red-hot inoculating loop fully into the culture. The evolution of gas indicates soluble CO₂ in the medium, a result of heterofermentative metabolism. This test will differentiate effectively between Leuconostoc and the other chain-forming genera of cocci in the lactic acid bacteria group. Observe Figure 12-2 to see a movie of the hot loop test. Record your results (Additional note: Pediococcus and Aerococcus are both homofermentative; Lactobacillus species are either homo- or heterofermentative.)
2. Analysis of the milk fermentation. Note the odor and texture of the pseudo-yogurt in tube B incubated since last period. Titrate as you did for tube A; the 5 ml of distilled water can be added to the tube to help loosen the solid curd.
3. Compare the results of both tubes. Has acid been produced? What effect has this had on the milk?
4. From the amount of NaOH used, the percent lactic acid in the milk can be calculated according to the following formula: (Recall that the sample volume was 5 ml.)

Lactic Acid Formula:

5. Slime production from sucrose. Describe the colonial morphology of Leuconostoc mesenteroides on each medium (glucose vs. sucrose). Poke at the growth on each plate with a sterile loop and note the relative consistencies of the growth. Record and explain your observations.
6. Lactic acid bacteria in the sauerkraut fermentation. If the colonies are difficult to see, reincubate the plates at room temperature and make your observations next period.
7. From the colony counts on HIAG and HIAS, determine the CFUs per ml in the original, undiluted sample for the total count and the count of lactic acid bacteria.
8. Optionally, observe your streak plates of the sauerkraut on the streak plates of HIAS or HIAG.
9. Are any relatively slimy or mucoid colonies apparent on the medium containing sucrose (HIAS)? If so, determine the CFUs per ml of Leuconostoc in the sample.
10. Compare your results with those who used the other sauerkraut sample. What may the relative differences in the various counts mean?

An example of a hot loop test