Characterization of the Biofilms Formed by Histamine-Producing Lentilactobacillus parabuchneri Strains in the Dairy Environment

Abstract

Lentilactobacillus parabuchneri, a lactic acid bacterium, is largely responsible for the production and accumulation of histamine, a toxic biogenic amine, in cheese. L. parabuchneri strains can form biofilms on the surface of industry equipment. Since they are resistant to cleaning and disinfection, they may act as reservoirs of histamine-producing contaminants in cheese. The aim of this study was to investigate the biofilm-producing capacity of L. parabuchneri strains. Using the crystal violet technique, the strains were first categorized as weak, moderate or strong biofilm producers. Analysis of their biofilm matrices revealed them to be mainly composed of proteins. Two strains of each category were then selected to analyze the influence on the biofilm-forming capacity of temperature, pH, carbon source, NaCl concentration and surface material (i.e., focusing on those used in the dairy industry). In general, low temperature (8 °C), high NaCl concentrations (2-3% w/v) and neutral pH (pH 6) prevented biofilm formation. All strains were found to adhere easily to beech wood. These findings increase knowledge of the biofilm-forming capacity of histamine-producing L. parabuchneri strains and how their formation may be prevented for improving food safety.

Keywords: Lentilactobacillus parabuchneri; biofilms; biogenic amines; histamine.

Figure 1

Biofilm-producing capacity on polystyrene of the histamine-producing Lentilactobacillus parabuchneri strains. The strains were incubated at 37 °C for 48 h. Data represent means ± SD (error bars) of at least three independent experiments. Shading of the same color indicates no significant difference. The producer/nonproducer cut-off (weak producer) is signaled by the dashed line (ODc), the solid line is 2 × ODc (moderate producer) and the dotted line 4 × ODc (strong producer).

Figure 2

Effect of proteinase K, trypsin, DNAse I, RNAse and NaIO₄ on biofilms produced by the strong biofilm producers L. parabuchneri IPLA11150 and IPLA11151. The strains were incubated for 48 h at 37 °C and subjected to treatments with the stated agents for 24 h. Data represent means ± SD (error bars) of three experiments. Values marked with * differ significantly.

Figure 3

Biofilm-producing capacity on polystyrene at different times. The strains were incubated at 37 °C for 12, 24, 48, 72 and 96 h. Data represent means ± SD (error bars) of three experiments. For each strain, values marked with the same letter are not significantly different (p > 0.05 according to the Bonferroni post-hoc test). The producer/nonproducer cut-off (weak producer) is signaled by the dashed line (ODc), the solid line is 2 × ODc (moderate producer) and the dotted line 4 × ODc (strong producer).

Figure 4

Biofilm-producing capacity on polystyrene of the histamine-producing L. parabuchneri strains incubated at 8, 12 and 24 °C. The strains were incubated for 2, 7, 14 and 30 days. Data represent means ± SD (error bars) of three experiments. Values marked with the same letter do not differ significantly (p > 0.05 according to the Bonferroni post-hoc test). The producer/nonproducer cut-off (weak producer) is signaled by the dashed line (ODc), the solid line is 2 × ODc (moderate producer) and the dotted line 4 × ODc (strong producer).

Figure 5

Biofilm-producing capacity on polystyrene of the biogenic amine-producing Lentilactobacillus strains incubated with MRS at pH 4.7 and 6. The strains were incubated for 48 h at 37 °C. Data represent means ± SD (error bars) of three experiments. Values marked with * differ significantly. The producer/nonproducer cut-off (weak producer) is signaled by the dashed line (ODc), the solid line is 2 × ODc (moderate producer) and the dotted line 4 × ODc (strong producer).

Figure 6

Biofilm-producing capacity on polystyrene of the histamine-producing L. parabuchneri strains incubated with MRS supplemented with different carbon sources. The strains were incubated for 48 h at 37 °C. Data represent means ± SD (error bars) of three experiments. Values marked with * differ significantly, considering glucose as the control. The producer/nonproducer cut-off (weak producer) is signaled by the dashed line (ODc), the solid line is 2 × ODc (moderate producer) and the dotted line 4 × ODc (strong producer). Lact: Lactose; Galac: Galactose; Gluc: Glucose.

Figure 7

Effect of NaCl concentration (in % p/v) on biofilm-producing capacity on polystyrene of the histamine-producing L. parabuchneri strains. The strains were incubated for 48 h at 37 °C. Data represent means ± SD (error bars) of three experiments. Values marked with * differ significantly. The producer/nonproducer cut-off (weak producer) is signaled by the dashed line (ODc), the solid line is 2 × ODc (moderate producer) and the dotted line 4 × ODc (strong producer).

Figure 8

Scanning electron microscopy images of L. parabuchneri strain biofilms grown at 37 °C for 48 h on different surfaces: (a) stainless steel; (b) food-grade plastic; (c) beech wood; and (d) rubber. Scale bars (10 μm) are shown on the photomicrographs. Red arrows indicate extracellular polymeric substances in the matrix.