Foodborne pathogenic microorganisms form biofilms at abiotic surfaces, which is a particular challenge in food processing industries. The complexity of biofilm formation requires a fundamental understanding on the involved molecular mechanisms, which may then lead to efficient prevention strategies. In the present study, biogenic amine producing bacteria, i.e., Lentilactobacillus parabuchneri DSM 5987 strain isolated from cheese were studied in respect with biofilm formation, which is of substantial relevance given their contribution to the presence of histamine in dairy products. While scanning electron microscopy was used to investigate biofilm adhesion at stainless steel surfaces, in situ infrared attenuated total reflection spectroscopy (IR-ATR) using a custom flow-through assembly was used for real-time and non-destructive observations of biofilm formation during a period of several days. The spectral window of 1700–600 cm−1 provides access to vibrational signatures characteristic for identifying and tracking L. parabuchneri biofilm formation and maturation. Especially, the amide I and II bands, lactic acid produced as the biofilm matures, and a pronounced increase of bands characteristic for extracellular polymeric substances (EPS) provide molecular insight into biofilm formation, maturation, and changes in biofilm architecture. Finally, multivariate data evaluation strategies were applied facilitating the unambiguous classification of the observed biofilm changes via IR spectroscopic data.
Antimicrobial materials are considered potential alternatives to prevent the development of biofilm-associated contaminations. Concerns regarding synthetic preservatives necessitate the development of innovative and safe natural antimicrobials. In the present study, we discuss the in situ infrared attenuated total reflection spectroscopy (IR-ATR) investigations of the selective antimicrobial efficiency of chitosan in controlling the growth of Lentilactobacillus parabuchneri biofilms. The protonated charges of chitosan were additionally amplified by structural modification via methylation, yielding quaternized derivative TMC (i.e., N, N, N-trimethyl chitosan). To evaluate antimicrobial effectiveness against L. parab. biofilms, IR-ATR spectroscopy provided information on molecular mechanisms and insights into chemical changes during real-time biofilm inhibition studies. The integrated fiberoptic oxygen microsensors enabled monitoring oxygen (O2) concentration gradients within biofilms, thereby confirming the metabolic oxygen depletion dropping from 4.5 to 0.7 mg L−1. IR studies revealed strong electrostatic interactions between chitosan/its water-soluble derivative and bacteria, indicating that a few hours were sufficient to affect biofilm disruption. The significant decrease in the IR bands is related to the characteristic spectral information of amide I, II, III, nucleic acid, and extracellular polymeric matrix (EPS) produced by L. parabuchneri biofilms. Cell clusters of biofilms, microcolonies, and destabilization of the EPS matrix after the addition of biopolymers were visualized using optical microscopy. In addition, scanning electron microscopy (SEM) of biofilms grown on polystyrene and stainless-steel surfaces was used to examine morphological changes, indicating the disintegration of the biofilm matrix into individual cells. Quantification of the total biofilm formation correlated with the CV assay results, indicating cell death and lysis. The electrostatic interactions between chitosan and the bacterial cell wall typically occur between protonated amino groups and negatively charged phospholipids, which promote permeabilization. Biofilm growth inhibition was assessed by a viability assay for a period of 72 h and in the range of low MIC values (varying 0.01–2%). These results support the potential of chitosan and TMC for bacterial growth prevention of the foodborne contaminant L. parabuchneri in the dairy industry and for further implementation in food packaging.
Unlike other antimicrobial agents, Ag-based composites are stable and currently widely used as broad spectral additives, fighting microbial biofilms and other biological threats. The goal of the present study is to develop a green, multifunctional, and robust antibiofilm water-insoluble coating, inhibiting histamine-producing Lentilactobacillus parabuchneri biofilms. Herein, laser-ablated Ag NPs (L-Ag NPs) were incorporated into and onto a montmorillonite (MMT) surface layer with a simple wet chemical method, provided that the electrostatic interaction between L-Ag NPs and MMT clay led to the formation of L-Ag/MMT nanoantimicrobials (NAMs). The use of MMT support can facilitate handling Ag NPs in industrial applications. The Ag/MMT composite was characterized with transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which confirmed the entrapment of L-Ag NPs into MMT clay. The surface chemical composition was assessed with X-ray photoelectron spectroscopy, proving that Ag NPs were in contact with and deposited onto the surface of MMT. The characteristic L-Ag/MMT band was investigated with UV–vis spectroscopy. Following that, the L-Ag/MMT composite was embedded into a biosafe water-insoluble beeswax agent with a spin coating technique. The antimicrobial ion release kinetic profile of the L-Ag/MMT/beeswax coating through an electrothermal atomic absorption spectroscopy (ETAAS) study supported the controlled release of Ag ions, reaching a plateau at 420 ±80 nM, which is safe from the point of view of Ag toxicity. Microbial biofilm growth inhibition was assessed with real-time in situ Fourier transform infrared attenuated total reflection spectroscopy (FTIR-ATR) in a flow cell assembly over 32 h. The study was further supported by optical density (OD) measurements and SEM on bacteria incubated in the presence of the L-Ag/MMT/beeswax coating.
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.
<p>Microbial biofilms are a crucial problem in many areas including the food processing industry, biotechnology, water quality and medical scenarios. The complexity of biofilm formation and subsequent prevention strategies - requires a fundamental understanding of the involved molecular mechanisms and the possibility of long-term monitoring biofilm formation. Infrared attenuated total reflection (IR-ATR) spectroscopy is a versatile analytical technique for monitoring biofilm formation of bacteria isolates in situ, non-destructively, and close to real time as an innovative approach providing molecular insight into biofilm formation . The utility of IR-ATR to investigate microorganism behavior within biofilms derives from the evanescent field penetrating few micrometers into the biofilm formed directly at the interface of a multi-reflection ATR waveguide and the sample. In the present study, isolates from food biogenic amine (BA)-producing bacteria, <em>Lactobacillus parabuchneri</em> DSM 5987 strains formed in cheese are analyzed for developing a deeper understanding on the formation of biofilms, which are significant contributors to the presence of histamine in dairy food products . Infrared spectra were recorded using a custom flow-through ATR assembly for revealing the metabolism of microorganisms within such biofilms along with the effects of the substrate functionality and culture conditions on the extracellular biopolymeric matrices [3,4]. The appearance of key IR bands in the region of 1600-1200 cm<sup>-1</sup> indicates the production of lactic acid or lactate and the presence of amide groups, while most pronounced intensities in 1140-950 cm<sup>-1</sup> correspond to phospholipids, polysaccharides and nucleic acids. In this study, the spectral region between 1700 and 600 cm<sup>-1</sup> was determined to be the representative region for the identification of <em>Lactobacillus parabuchneri</em> biofilms enabling to study bioadhesion mechanisms and physico-chemical property changes during extended periods of biofilm growth. Real time monitoring has led to concrete steps for inhibition and disintegration via suitable antimicrobials by deposition on the IR inactive region of ATR waveguide. Multivariate data evaluation and classification strategies were applied to enable efficient multiparametric analysis for providing molecular information facilitating a better understanding of biofilm formation, maturation and changes in biofilm architecture via IR spectroscopic data.&#160;<br />&#160;<br /><strong>Keywords:</strong> IR-ATR spectroscopy, <em>in situ</em> monitoring, <em>Lactobacillus parabuchneri</em>, biofilm, ATR waveguide, flow-through ATR, lactic acid, multivariate data analysis.&#160;</p> <p><br /><strong>References:</strong>  Stenclova P, Freisinger S, et al. <em>Appl. Spectro.</em>, <strong>2019</strong>; Vol.73 (4) 424-432  Yunda E, Quil&#232;s F, et al. <em>Biofouling</em>, <strong>2019</strong>; Vol.35 (5) 494-507  Diaz M, del Rio B, et al. <em>Food Microbiol.,</em> <strong>2016</strong>; Vol.7 (591) 85-91  Lorite G, de Souza A, et al. <em>Colloids Surfaces B. Biointerfaces</em>, <strong>2013</strong>; Vol. 102 519-525&#160;</p>
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