In-process real-time probiotic phenotypic strain identity tracking: The use of Fourier transform infrared spectroscopy

Abstract

Probiotic bacteria, capable of conferring benefits to the host, can present challenges in design, development, scale-up, manufacturing, commercialization, and life cycle management. Strain identification is one of the main quality parameters; nevertheless, this task can be challenging since established methodologies can lack resolution at the strain level for some microorganisms and\or are labor-intensive and time-consuming. Fourier transform infrared spectroscopy (FTIRS) has been largely used for the investigation of pathogenic species in the clinical field, whereas only recently has been proposed for the identification of probiotic strains. Within the probiotic industrial production, bacterial strains can be subjected to stressful conditions that may affect genomic and phenotypic characteristics; therefore, real-time monitoring of all the sequential growth steps is requested. Considering the fast, low-cost, and high-throughput features, FTIRS is an innovative and functional technology for typing probiotic strains from bench-top experiments to large-scale industrial production, allowing the monitoring of stability and identity of probiotic strains. In this study, the discriminatory power of FTIRS was assessed for four Lactiplantibacillus plantarum probiotic strains grown under different conditions, including temperatures (30 and 37°C) and medium (broth and agar), after consecutive sub-culturing steps. A comparison between the generated spectra with pulsed-field gel electrophoresis (PFGE) profiles was also performed. FTIRS was not only able to distinguish the strains of L. plantarum under different growth conditions but also to prove the phenotypic stability of L. plantarum type strain LP-CT after six growing steps. Regardless of the growth conditions, FTIRS spectra related to LP-CT constituted a unique hierarchical cluster, separated from the other L. plantarum strains. These results were confirmed by a PFGE analysis. In addition, based on FTIRS data, broth cultures demonstrated a higher reproducibility and discriminatory power with respect to agar ones. These results support the introduction of FTIRS in the probiotic industry, allowing for the step-by-step monitoring of massive microbial production while also guaranteeing the stability and purity of the probiotic strain. The proposed novel approach can constitute an impressive improvement in the probiotic manufacturing process.

A Comprehensive Methodology for Microbial Strain Typing Using Fourier-Transform Infrared Spectroscopy

“…The IR Biotyper ® is also applicable for food quality control to verify if the starter culture strains added during production remain consistent throughout the process. It has been employed to accurately differentiate four Lactiplantibacillus plantarum probiotic strains [ 44 ]. In this study, it demonstrated high discriminatory power even when the strains were cultivated under different conditions.…”

“…In this study, it demonstrated high discriminatory power even when the strains were cultivated under different conditions. Interestingly, the growth medium (broth and agar) did not affect its ability to distinguish the four probiotic strains [ 44 ]. In fact, broth cultures exhibited higher reproducibility and discriminatory power compared to agar cultures.…”

Application of Fourier Transform Infrared Spectroscopy to Discriminate Two Closely Related Bacterial Species: Bacillus anthracis and Bacillus cereus Sensu Stricto

“…Notably, the IR Biotyper ® (IRBT) system (Bruker Daltonics, Bremen, Germany) based on FTIRS technology was launched in 2017 as a promising system in the field of microbial strain typing. Multiple successful applications have been reported [13] in the field of food, veterinary and water microbiology [14][15][16], as well as in hospital hygiene [17][18][19][20][21] and probiotic production [22,23].…”