Phenyllactic Acid Produced by Geotrichum candidum Reduces Fusarium sporotrichioides and F. langsethiae Growth and T-2 Toxin Concentration

Abstract

Fusariumsporotrichioides and F. langsethiae are present in barley crops. Their toxic metabolites, mainly T-2 toxin, affect the quality and safety of raw material and final products such as beer. Therefore, it is crucial to reduce Fusarium spp. proliferation and T-2 toxin contamination during the brewing process. The addition of Geotrichum candidum has been previously demonstrated to reduce the proliferation of Fusarium spp. and the production of toxic metabolites, but the mechanism of action is still not known. Thus, this study focuses on the elucidation of the interaction mechanism between G.candidum and Fusarium spp. in order to improve this bioprocess. First, over a period of 168 h, the co-culture kinetics showed an almost 90% reduction in T-2 toxin concentration, starting at 24 h. Second, sequential cultures lead to a reduction in Fusarium growth and T-2 toxin concentration. Simultaneously, it was demonstrated that G. candidum produces phenyllactic acid (PLA) at the early stages of growth, which could potentially be responsible for the reduction in Fusarium growth and T-2 toxin concentration. To prove the PLA effect, F. sporotrichioides and F.langsethiae were cultivated in PLA supplemented medium. The expected results were achieved with 0.3 g/L of PLA. These promising results contribute to a better understanding of the bioprocess, allowing its optimization at an up-scaled industrial level.

Keywords: F. langsethiae; F. sporotrichioides; G. candidum; T-2 toxin; biocontrol agent; mycotoxin.; phenyllactic acid.

Figure 1

Microbial dry weight analysis in control cultures (G. candidum, F. langsethiae 2297 and F. sporotrichioides 186 alone) and in co-culture experiments (G. candidum with F. langsethiae 2297 and G. candidum with F. sporotrichioides 186).

Figure 2

T-2 concentration (µg/L) and phenyllactic acid (PLA) concentration (g/L) in co-culture experiments. Panel A: Co-culture experiment of G. candidum and F. langsethiae 2297. Panel B: Co-culture experiment of G. candidum and F. sporotrichioides 186 (One-way ANOVA, Tukey’s multiple comparisons post-hoc test, ** p-value < 0.01; *** p-value < 0.001) ND = not detectable.

Figure 3

PLA concentration (g/L) and G. candidum biomass (g/L) in Ym medium (Panel A) and PLA specific production (g PLA/g dry weight) in Ym medium (Panel B).

Figure 4

Sequential culture of F. langsethiae 2297 inoculated in pre-fermented medium by G. candidum and incubated 7 days at 22 °C. Panel A: Dry weight of F. langsethiae 2297 (g/L) in comparison with PLA concentration (g/L). Panel B: T-2 concentration (µg/L) in comparison with PLA concentration (g/L). One-way ANOVA, Dunnett multiple comparisons post-hoc test, * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; ns = not significant).

Figure 5

Sequential culture of F. sporotrichioides 186 inoculated in pre-fermented medium by G. candidum and incubated for 7 days at 22 °C. Panel A: Dry weight of F. sporotrichioides 186 (g/L) in comparison with PLA concentration (g/L). Panel B: T-2 concentration (µg/L) in comparison with PLA concentration (g/L). One-way ANOVA, Dunnett multiple comparisons post-hoc test, * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; ns = not significant).

Figure 6

Effect of PLA on the dry weight of F. langsethiae 2297 (A) and T-2 toxin concentration (B) (One-way ANOVA, Dunnett multiple comparisons post-hoc test, *** p-value < 0.001).

Figure 7

Effect of phenyllactic acid (PLA) on dry weight of F. sporotrichioides 186 (A) and T-2 toxin concentration (B) (One-way ANOVA, Dunnett multiple comparisons post-hoc test, * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001).

Figure 8

Specific production of T-2 toxin by F. langsethiae 2297 (A) and F. sporotrichioides 186 in Ym medium supplemented with pure phenyllactic acid (PLA) (B) and incubated 7 days at 22 °C (One-way ANOVA, Dunnett multiple comparisons post-hoc test, ns = not significant).

Figure 9

Hypothetical phenyllactic acid biosynthesis pathway. Adapted from Chaudhari and Gokhale (2016) [47]. AAT: amino acid transferase; D-LDH: D-lactate dehydrogenase; L-LDH: L-lactate dehydrogenase.