Evaluation of Dietary Supplements Containing Viable Bacteria by Cultivation/MALDI-TOF Mass Spectrometry and PCR Identification

Abstract

The insufficient quality of products containing beneficial live bacteria in terms of content and viability of labelled microorganisms is an often-reported problem. The aim of this work was to evaluate the quality of dietary supplements containing viable bacteria available in Slovenian pharmacies using plate counting, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and species- or subspecies-specific PCR with DNA isolated from consortia of viable bacteria, from individual isolates, or directly from the products. Twelve percent of the products (3 of 26) contained insufficient numbers of viable bacteria. Eighty-three of the labelled species (111 in total) were confirmed by PCR with DNA from the product; 74% of these were confirmed by PCR with DNA from viable consortium, and 65% of these were confirmed by MALDI-TOF MS analysis of colonies. Certain species in multi-strain products were confirmed by PCR with DNA from viable consortia but not by MALDI-TOF MS, suggesting that the number of isolates examined (three per labelled strain) was too low. With the exception of Lacticaseibacillus casei and closely related species (Lacticaseibacillus rhamnosus and Lacticaseibacillus zeae), PCR and MALDI-TOF identification results agreed for 99% of the isolates examined, although several MALDI-TOF results had lower score values (1.700–1.999), indicating that the species identification was not reliable. The species L. zeae, which appeared in 20 matches of the Biotyper analysis, was identified as L. rhamnosus by PCR. The MALDI-TOF MS analysis was also unsuccessful in detecting Lactobacillus acidophilus La-5 and Bacillus coagulans due to missing peaks and unreliable identification, respectively. Mislabelling was detected by both methods for two putative L. casei strains that turned out to belong to the species Lacticaseibacillus paracasei. PCR remains more successful in subspecies-level identification as long as the database of MALDI-TOF MS spectra is not expanded by building in-house databases. The lack of positive PCR results with viable consortia or colonies, but positive PCR results with DNA isolated directly from the products observed in 10% (11/112) of the labelled strains, suggests the presence of non-culturable bacteria in the products. MALDI-TOF MS is a faster and simpler alternative to PCR identification, provided that a sufficient number of colonies are examined. Generation of in-house library may further improve the identification accuracy at the species and sub-species level.

Microbiological Quality and Resistance to an Artificial Gut Environment of Two Probiotic Formulations

“…These results correlate with the evidence that MALDI-TOF MS has been shown to fail in identifying some species when the number of species-and strain-specific spectra included in the MALDI database is low [45]. A previous study suggested that the development of a dedicated in-house library containing MALDI TOF MS spectra of various reference strains could improve the identification accuracy [46]. Metagenomic sequencing misidentified L. helveticus in Microbiosys.…”

“…In fact, in a previous study analyzing the quality of dietary supplements sold in Slovenia, MALDI-TOF MS alone was not accurate in distinguishing some closely related Lactobacillus species. In these cases, the correct identification was obtained by pairing MALDI-TOF MS with species-specific PCR [46].…”

Lipidomics Characterization of the Microbiome in People with Diabetic Foot Infection Using MALDI-TOF MS

“…M. Hajjar et al 70 utilized MALDI TOF in negative-ion MALDI-TOF for the analysis of LPS or lipid A preparations to identify Francisella tularensis strains. In the food industry, the technique has been employed for evaluating dietary supplements containing live bacteria, as shown by Lorbeg et al 71 In plant research, Wang et al 72 used it for studying lipid distribution in peanut seeds. Importantly, in the realm of microbial identification, particularly for closely related or drug-resistant strains, MALDI’s role is being increasingly recognized.…”

Przeżywalność Środowiskowych Bifidobacterium Spp. Oraz Bakterii Fermentacji Mlekowej W Serach Twarogowych Wyprodukowanych Z Użyciem Kultury Środowiskowej

“…W dostępnych na polskim rynku preparatach zawierających bakterie probiotyczne często spotykana jest zbyt mała liczba drobnoustrojów lub mniejsza liczba gatunków, niż producent deklarował na opakowaniu. Bardzo często obserwuje się niską przeżywalność lub jej brak, zwłaszcza w przypadku bakterii z rodzaju Bifidobacterium spp., w komercyjnych suplementach diety i preparatach medycznych [1,3,19,22,34]. Drobnoustroje te są beztlenowymi, nieruchliwymi, Gram-dodatnimi i nietworzącymi przetrwalników pałeczkami [5].…”