Abstract
Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.
Highlights
Lactic acid bacteria (LAB) comprise a group of several genera of microorganisms, which display lactic acid as their major end product of fermentation [1]
The aim of this study was to evaluate the survival of the probiotic strains L. acidophilus La-5 and B. animalis subsp. lactis Bb12 and of the bacteriocinogenic and potentially probiotic strain L. sakei subsp. sakei 2a in synbiotic and probiotic petit-suisse cheeses throughout their shelf-lives challenged by an in vitro assay simulating gastrointestinal conditions
Dead or cellular components of probiotic bacteria are thought to mediate beneficial effects on the host [37], the current consensus is that probiotics should be alive to exert their beneficial effect(s)
Summary
Lactic acid bacteria (LAB) comprise a group of several genera of microorganisms, which display lactic acid as their major end product of fermentation [1]. LAB are indigenous members of the intestinal microbiota of animals and are associated with plants, and fermented food, including meat and dairy products [2]. These microorganisms produce different antimicrobial peptides, known as bacteriocins, which are useful to improve the safety and biopreservation of foods [3]. Bacteriocin-producing Lactobacillus species might play a dual role by acting as agents for food preservation and safety as well as presenting a potential to confer health benefits [9]. Prebiotics are fermentable ingredients that allow specific changes in the composition and/or activity of the gastrointestinal microbiota leading to benefits in host health and well-being [11]
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