Discovering probiotic microorganisms: invitro, invivo, genetic and omics approaches

Over the past decades the food industry has been revolutionized toward the production of functional foods due to an increasing awareness of the consumers on the positive role of food in wellbeing and health. By definition probiotic foods must contain live microorganisms in adequate amounts so as to be beneficial for the consumer’s health. There are numerous probiotic foods marketed today and many probiotic strains are commercially available. However, the question that arises is how to determine the real probiotic potential of microorganisms. This is becoming increasingly important, as even a superficial search of the relevant literature reveals that the number of proclaimed probiotics is growing fast. While the vast majority of probiotic microorganisms are food-related or commensal bacteria that are often regarded as safe, probiotics from other sources are increasingly being reported raising possible regulatory and safety issues. Potential probiotics are selected after in vitro or in vivo assays by evaluating simple traits such as resistance to the acidic conditions of the stomach or bile resistance, or by assessing their impact on complicated host functions such as immune development, metabolic function or gut–brain interaction. While final human clinical trials are considered mandatory for communicating health benefits, rather few strains with positive studies have been able to convince legal authorities with these health claims. Consequently, concern has been raised about the validity of the workflows currently used to characterize probiotics. In this review we will present an overview of the most common assays employed in screening for probiotics, highlighting the potential strengths and limitations of these approaches. Furthermore, we will focus on how the advent of omics technologies has reshaped our understanding of the biology of probiotics, allowing the exploration of novel routes for screening and studying such microorganisms

A Key Role of Dendritic Cells in Probiotic Functionality

BACKGROUND: Disruption of the intestinal homeostasis and tolerance towards the resident microbiota is a major mechanism involved in the development of inflammatory bowel disease. While some bacteria are inducers of disease, others, known as probiotics, are able to reduce inflammation. Because dendritic cells (DCs) play a central role in regulating immune responses and in inducing tolerance, we investigated their role in the anti-inflammatory potential of probiotic lactic acid bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Selected LAB strains, while efficiently taken up by DCs in vitro, induced a partial maturation of the cells. Transfer of probiotic-treated DCs conferred protection against 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Protection was associated with a reduction of inflammatory scores and colonic expression of pro-inflammatory genes, while a high local expression of the immunoregulatory enzyme indolamine 2, 3 dioxgenase (IDO) was observed. The preventive effect of probiotic-pulsed DCs required not only MyD88-, TLR2- and NOD2-dependent signaling but also the induction of CD4+ CD25+ regulatory cells in an IL-10-independent pathway. CONCLUSIONS/SIGNIFICANCE: Altogether, these results suggest that selected probiotics can stimulate DC regulatory functions by targeting specific pattern-recognition receptors and pathways. The results not only emphasize the role of DCs in probiotic immune interactions, but indicate a possible role in immune-intervention therapy for IBD

Probiotic properties of lactic acid bacteria: in vitro and in vivo study of their antimicrobial and immunomodulatory activity

Aim: The aim of this study was to select potentially probiotic strains and to eludidate their mechanisms of action. Methodology: In this study 116 lactic and bacteria strains were tested for antimicrobial activity against a large number of stains. In all the cases where antimicrobial activity was detected, an attempt was made to elucidate the responsible compounds and to further examine this antimicrobial activity with FT-IR spectroscopy. All selected strains were subjected to a series of in vitro trials for their probiotic potential and their immunomodulation capacity. Finally, two in vivo trials were conducted. Results: Three Lactobacillus strains stand out because of their antimicrobial activity against one Salmonella strain, and S. macedonicus ACA-DC 198 for its activity against the majority of Gramt bacteria. Anti-salmonella activity revealed changes in the structural components of Salmonella cells and the antimicrobial activity of the streptococcus strain was due to its bacterium. The four selected strains were found to have good probiotic properties and differences in their immunodulatory action. The L. fermentum ACA-DC 179 strain was successfully applied to the two in vivo mouse models.Σκοπός: Η επιλογή προβιοτικών στελεχών οξυγαλακτικών βακτηρίων μέσα από μια σειρά in vitro και in vivo πειραμάτων και η κατανόηση του μηχανισμού δράσης των στελεχών αυτών. Μεθοδολογία: Στη διατριβή αυτή μελετήθηκαν 116 στελέχη βακτηρίων για αντιμικροβιακή δράση έναντι ενός μεγάλου αριθμού μικροοργανισμών “στόχων”. Σε όλες τις περιπτώσεις εύρεσης αντιμικροβιακής δράσης έγινε προσπάθεια απομόνωσης και χαρακτηρισμού των υπεύθυνων ενώσεων και μελέτη της δράσης με φασματοσκοπία FT-IR. Παράλληλα, επιλεγμένα στελέχη εξετάστηκαν in vitro για το προβιοτικό τους δυναμικό και για την ανοσορυθμιστική τους ικανότητα. Τέλος, ακολούθησαν δύο in vivo μελέτες σε ζώα. Αποτελέσματα: Τρία στελέχη του γένους Lactobacillus ξεχώρισαν για την αντιμικροβιακή τους δράση έναντι ενός στελέχους Salmonella και το στέλεχος Streptococcus macedonicus ACA-DC 198 για τη δράση του έναντι ενός μεγάλου αριθμού Gramt βακτηρίων. Η αντι-Salmonella δράση συνδέθηκε με μεταβολές στα λιπίδια της κυτταρικής μεμβράνης και στους πολυσακχαρίτες του κυτταρικού τοιχώματος των κυττάρων Salmonella και η δράση του στρεπτόκοκκου με την παραγωγή βακτηριοσίνης. Τα επιλεγμένα στελέχη παρουσίασαν ικανοποιητικό προβιοτικό δυναμικό και διαφοροποιήσεις στην ανοσορυθμιστική τους ικανότητα. Το στέλεχος L. fermentum ACA-DC 179 είχε προστατευτική δράση και στις δύο in vivo μελέτες σε ζώα

Omics Approaches to Assess Flavor Development in Cheese

Cheese is characterized by a rich and complex microbiota that plays a vital role during both production and ripening, contributing significantly to the safety, quality, and sensory characteristics of the final product. In this context, it is vital to explore the microbiota composition and understand its dynamics and evolution during cheese manufacturing and ripening. Application of high-throughput DNA sequencing technologies have facilitated the more accurate identification of the cheese microbiome, detailed study of its potential functionality, and its contribution to the development of specific organoleptic properties. These technologies include amplicon sequencing, whole-metagenome shotgun sequencing, metatranscriptomics, and, most recently, metabolomics. In recent years, however, the application of multiple meta-omics approaches along with data integration analysis, which was enabled by advanced computational and bioinformatics tools, paved the way to better comprehension of the cheese ripening process, revealing significant associations between the cheese microbiota and metabolites, as well as their impact on cheese flavor and quality

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters

Kinetic analysis of the antibacterial activity of probiotic lactobacilli towards Salmonella enterica serovar Typhimurium reveals a role for lactic acid and other inhibitory compounds.

Six Lactobacillus strains including commercial probiotic ones (L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota, L. johnsonii La1, L. plantarum ACA-DC 287 and L. rhamnosus GG) were investigated, through batch fermentations under controlled conditions, for their capacity to inhibit Salmonella enterica serovar Typhimurium SL1344. All lactobacilli displayed strong antibacterial activity toward this Gram-negative pathogen and significantly inhibited invasion of the pathogen into cultured human enterocyte-like Caco-2/TC7 cells. By studying the production kinetics of antibacterial activity and applying the appropriate acid and pH control samples during a killing assay, we were able to distinguish between the effect of lactic acid and other inhibitory compounds produced. The antibacterial activity of L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota and L. rhamnosus GG was solely due to the production of lactic acid. The antibacterial activity of L. johnsonii La1 and L. plantarum ACA-DC 287 was due to the production of lactic acid and (an) unknown inhibitory substance(s). The latter was (were) only active in the presence of lactic acid. In addition, the lactic acid produced was responsible for significant inhibitory activity upon invasion of Salmonella into Caco-2/TC7 cells

Goat Milk with Different Alpha-s1 Casein Genotype (CSN1S1) Fermented by Selected Lactobacillus paracasei as Potential Functional Food

International audienceThe characteristics of fermented milk are affected by the type of milk used and the microorganisms involved in the fermentation process. Goat milk has been widely suggested as a possible alternative to cow milk in allergic subjects, because of the high genetic variability in alpha-s1 casein (CSN1S1) content, which is associated with different technological and nutritional properties of milk. The aim of the study was to evaluate the suitability of goat milk with low and high CSN1S1 to produce fermented milk. In addition, the performance as starter of selected Lactobacillus paracasei FS109 strain compared to no-selected L. paracasei strains was investigated. Initially, the selected L. paracasei FS109 strain was tested for adhesion ability to HT-29 and Caco-2 cells and immunomodulation effect. Then, the strain was used to produce fermented milk from goat milk with a low and high casein CSN1S1 genotype. The results indicated that greater acidifying activity was obtained for L. paracasei FS109 after 24 h of fermentation than the other two strains tested independently by the CSN1S1 genotype. L. paracasei FS109 grew well during fermentation, reaching a higher value (>8.5 log CFU/mL). Interestingly, the same strain maintained a high viable population (about 9 log CFU/mL) during the 30-day cold storage of the product. The present study shows for the first time the suitability of the goat milk with low CSN1S1 genotypes to produce fermented milk and highlight the importance of strain selection in determination of technological and beneficial traits. Combining goat milk with low CSN1S1 and selected strains could be a strategy of improving traditional and functional fermented milk market

RNA Arbitrarily Primed PCR and Fourier Transform Infrared Spectroscopy Reveal Plasticity in the Acid Tolerance Response of Streptococcus macedonicus▿

We have previously reported that an acid tolerance response (ATR) can be induced in Streptococcus macedonicus cells at mid-log phase after autoacidification, transient exposure to acidic pH, or acid habituation, as well as at stationary phase. Here, we compared the transcriptional profiles of these epigenetic phenotypes, by RNA arbitrarily primed PCR (RAP-PCR), and their whole-cell chemical compositions, by Fourier transform infrared spectroscopy (FT-IR). RAP-PCR fingerprints revealed significant differences among the phenotypes, indicating that gene expression during the ATR is influenced not only by the growth phase but also by the treatments employed to induce the response. The genes coding for the mannose-specific IID component, the 1,2-diacylglycerol 3-glucosyltransferase, the 3-oxoacyl-acyl carrier protein, the large subunit of carbamoyl-phosphate synthase, and a hypothetical protein were found to be induced at least under some of the acid-adapting conditions. Furthermore, principal component analysis of the second-derivative-transformed FT-IR spectra segregated S. macedonicus phenotypes individually in all spectral regions that are characteristic for major cellular constituents like the polysaccharides of the cell wall, fatty acids of the cell membrane, proteins, and other compounds that absorb in these regions. These findings provide evidence for major changes in cellular composition due to acid adaptation that were clearly different to some extent among the phenotypes. Overall, our data demonstrate the plasticity in the ATR of S. macedonicus, which reflects the inherent ability of the bacterium to adjust the response to the distinctiveness of the imposed stress condition, probably to maximize its adaptability