Rapid discrimination of Lentilactobacillus parabuchneri biofilms via in situ infrared spectroscopy

Microbial contamination in food industry is a source of foodborne illnesses and biofilm-related diseases. In particular, biogenic amines (BAs) accumulated in fermented foods via lactic acid bacterial activity exert toxic effects on human health. Among these, biofilms of histamine-producer Lentilactobacillus parabuchneri strains adherent at food processing equipment surfaces can cause food spoilage and poisoning. Understanding the chain of contamination is closely related to elucidating molecular mechanisms of biofilm formation. In the present study, an innovative approach using integrated chemical sensing technologies is demonstrated to fundamentally understand the temporal behavior of biofilms at the molecular level by combining mid-infrared (MIR) spectroscopy and fluorescence sensing strategies. Using these concepts, the biofilm forming capacity of six cheese-isolated L. parabuchneri strains (IPLA 11151, 11150, 11129, 11125, 11122 and 11117) was examined. The cut-off values for the biofilm production ability of each strain were quantified using crystal violet (CV) assays. Real-time infrared attenuated total reflection spectroscopy (IR-ATR) combined with fluorescence quenching oxygen sensing provides insight into distinct molecular mechanisms for each strain. IR spectra showed significant changes in characteristic bands of amides, lactate, nucleic acids, and extracellular polymeric substances (i.e., lipopolysaccharides, phospholipids, phosphodiester, peptidoglycan, etc.), which are major contributors to biofilm maturation involved in the initial adhesion processes. Chemometric methods including principal component analysis and partial least square-discriminant analysis facilitated the rapid determination and classification of cheese isolated L. parabuchneri strains unambiguously differentiating the IR signatures based on their ability to produce biofilm. All biofilms were morphologically characterized by confocal laser scanning microscopy on relevant industrial equipment surfaces. In summary, this innovative approach combining MIR spectroscopy with luminescence sensing enables real-time insight into the molecular composition and formation of L. parabuchneri biofilms.

This work is part of the “Break Biofilms” project that has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska Curie Grant Agreement No. 813439. Special thanks to Dra. Ana Belen Campelo and the Technical Scientific Service of Dairy Research Institute (IPLA-CSIC) for their help with CLSM analysis. The IR lab team at the Institute of Analytical and Bioanalytical Chemistry (IABC) is gratefully acknowledged for their advices and discussions on real-time infrared characterization and chemometric modeling.

Peer reviewed