Physiological study of Lactobacillus delbrueckii subsp. bulgaricus strains in a novel chemically defined medium

Abstract

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h(-1). MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.

FIG. 1

Growth characteristics of various lactic acid bacteria in MPL. Fresh colonies obtained on MRS plates were inoculated in MPL containing a 2% concentration of either lactose or glucose and the 12 vitamins indicated in Table 1, column 2. OD₆₀₀ was measured after at least 48 h of incubation at either 42 or 30°C. The standard deviation is indicated when two independent experiments were performed.

FIG. 2

Effect of various concentrations of galactose on CNRZ397 growth. Media were inoculated by diluting a preculture (1/100) into MPL-lactose (1%) (open circles) supplemented with galactose at 1% (triangles), 2% (squares), 3% (diamonds), or 4% (filled circles). Growth was followed using a microplate reader as described in Materials and Methods.

FIG. 3

Protein labeling in MPL. CNRZ397 was grown in MPL containing 40 μM methionine. At an OD₆₀₀ of 0.7, [³⁵S]Met was added to the cultures, and samples were withdrawn at different time intervals. Samples equivalent to 0.2 OD₆₀₀ units were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Lanes 1 to 7 correspond to samples incubated with [³⁵S]Met at 10 μCi/ml for 1, 2, 5, 10, 15, 20, or 25 min, respectively. Lanes 8 and 9 correspond to samples labeled for 2 min with 25 or 50 μCi of [³⁵S]Met/ml, respectively.