Effects of cultivation conditions on folate production by lactic acid bacteria

Abstract

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.

FIG. 1.

Chromatograms of folate calibrators (A and C), cell extracts of S. thermophilus before (B and D) and after deconjugation (E) monitored by UV absorption at 280 nm (A and B) and 360 nm (C to E). Peaks of the following folate compounds are shown: 5-formyl tetrahydrofolate-Glu₁ (S and R diastereoisomer) (peak 1), 5-formyl tetrahydrofolate-Glu₂ (peak 2), 5-formyl tetrahydrofolate-Glu₃ (peak 3), 5-formyl tetrahydrofolate-Glu₄ (peak 4), 5-formyl tetrahydrofolate-Glu₅ (peak 5), 5,10-methenyl tetrahydrofolate-Glu₁ (peak 6), 5,10-methenyl tetrahydrofolate-Glu₂ (peak 7), and 5,10-methenyl tetrahydrofolate-Glu₃ (peak 8). Elution time is shown on the x axis, and UV absorption (in milli absorbance units [mAU]) is shown on the y axis.

FIG. 2.

Folate production and distribution in different L. lactis strains grown aerobically and anaerobically with and without hemin in M17 medium. Total folate production per OD₆₀₀ unit (right y axis) (grey bars) and extracellular folate (white bars), intracellular folate (hatched bars), and total folate production (black bars) (left y axis) are shown. Bars: 1, wild-type strain grown anaerobically; 2, wild-type strain grown aerobically; 3, ldh mutant strain grown aerobically; 4, wild-type strain grown anaerobically with hemin; 5, ldh mutant strain grown aerobically with hemin.

FIG. 3.

Influence of PABA and tyrosine on folate production of L. lactis grown in CDM. Extracellular folate (white bars), intracellular folate (hatched bars), and total folate production (black bars) are shown. Strains were grown under the following conditions: without PABA and without tyrosine (bars 1), with 100 μM PABA and 1.2 mM tyrosine (bars 2), with 100 μM PABA but without tyrosine (bars 3), and with 1.2 mM tyrosine but without PABA (bars 4).

FIG. 4.

Total folate production by S. thermophilus NIZO strain B119 grown at different dilution rates (= growth rates) in continuous culture in modified M17 medium (see Materials and Methods).