Lactobacillus is a bacterial genus belonging to LAB (Lactic Acid Bacteria) and they are among the most common probiotics. Recent guidelines on probiotics, issued by the Italian Ministry of Health, state that, on the basis of the available literature, the amount sufficient to obtain a temporary colonization of the intestine by a probiotic strain is at least 10^9 living cells. A microorganism can be defined as a probiotic strain if it is of human origin, if it survive to the gastrointestinal tract, resisting the acidity of the stomach and the action of bile, and it should have immunostimulant activity. In addition, the strain should be able to adhere to the mucosa causing no toxicity, and to produce substances with antibacterial activity against some pathogens. The aim of the work was the isolation and identification of lactobacilli of human origin. It was also deepened the study of their amensalistic properties, with particular attention to the resistance to gastrointestinal transit and their antagonism against pathogenic microorganisms. Three hundred fifty-nine lactobacilli strains were isolated from swabs of healthy people and identified using molecular techniques based on the study of 16S rDNA. The identification of some strains was confirmed by further analysis DHPLC V1 and V3 of 16S rDNA. The strains were subjected to the evaluation of the resistance to bile salts and low pH, to the production of hydrogen peroxide and more particularly, it has been evaluated the ability to produce substances with antibacterial activity. Finally, the attention was focused on the characterization of an active supernatant produced by an oral strain. The isolation of the substance provided chromatographic procedures such as SEC (Size Exclusion Chromatography using Sephadex 50) and SPE (Reverse Phase Chromatography using C18 column). The results were shown that the active fraction has a low molecular weight and for its chemical-physical characteristics is not a common bacteriocin, for this reason are on going further chromatographic studies using columns with increasing polarity (C4, phenyl, cyano, and amino) . Future outlooks are focused on the identification of the molecule in question, by MALDI-TOF and ESI-TOF and then optimizing the whole process to standardize the entire method. In this way, the opportunity to bring to light new molecules will be possible, with the ultimate goal of being able to take advantage from these antibacterial substances.
I lattobacilli appartengono ai LAB (Lactic Acid Bacteria) e sono tra i più comuni microrganismi probiotici. Le recenti linee guida, rilasciate dal Ministero della Salute, affermano che, sulla base della letteratura disponibile, la quantità sufficiente per ottenere una colonizzazione temporanea da parte di un ceppo probiotico è di almeno 10^9 cellule viventi. Un microrganismo per essere definito tale, deve essere di origine umana, avere attività immunostimolante e sopravvivere al tratto gastrointestinale, resistendo all'acidità dello stomaco e all'azione della bile. Inoltre, il ceppo deve essere in grado di aderire alla mucosa non causando tossicità, e di produrre sostanze ad attività antibatterica contro alcuni patogeni. Scopo del lavoro è stato l'isolamento e l’identificazione di lattobacilli di origine umana. Inoltre, è stato approfondito lo studio delle proprietà amensalistiche, con particolare attenzione alla resistenza al transito gastro-intestinale e all’antagonismo nei confronti dei microorganismi patogeni. Trecencinquantanove ceppi di lattobacilli sono stati isolati da tamponi ottenuti da persone sane e identificati tramite tecniche molecolari basate sulla 16S rDNA. Per una selezione di ceppi l identificazione di alcuni ceppi è fatta anche la tecnica DHPLC applicata agli amplificati delle regioni V1 e V3 della 16S rDNA. In tutti ceppi è stata la valutata la resistenza ai sali biliari e al pH acido, la produzione di perossido di idrogeno, ed infine è stata valutata la capacità di produrre sostanze ad attività antibatterica. Particolare attenzione posta alla caratterizzazione delle componenti antibiotiche di un surnatante prodotto da un ceppo di origine orale. L isolamento della sostanza inibitric è stato condotto mediante tecniche cromatografiche quali SEC (Size Exclusion Chromatography mediante Sephadex 50) e SPE (Reverse Phase Chromatography mediante colonna C18). I risultati hanno dimostrato che sostanza contenuta nella frazione attiva possiede un peso molecolare basso e per le sue caratteristiche chimico-fisiche non è, molto probabilmente, batteriocina, per questo motivo sono in avanzamento ulteriori studi cromatografici con colonne a polarità crescente (C4, fenile, ciano e ammino). I progetti futuri sono focalizzati sull’identificazione della molecola in questione, tramite analisi MALDI-TOF e ESI-TOF, e successivamente sull’ottimizzazione del processo di produzione e purificazione. In tal modo si avrà l’opportunità di portare alla luce nuove molecole con l’obiettivo finale di poter sfruttare l’attività antibatterica di tali sostanze.
Amensalistic activity of Lactobacillus spp., isolated from human samples / Fuochi, Virginia. - (2015 Dec 10).
Amensalistic activity of Lactobacillus spp., isolated from human samples
FUOCHI, VIRGINIA
2015-12-10
Abstract
Lactobacillus is a bacterial genus belonging to LAB (Lactic Acid Bacteria) and they are among the most common probiotics. Recent guidelines on probiotics, issued by the Italian Ministry of Health, state that, on the basis of the available literature, the amount sufficient to obtain a temporary colonization of the intestine by a probiotic strain is at least 10^9 living cells. A microorganism can be defined as a probiotic strain if it is of human origin, if it survive to the gastrointestinal tract, resisting the acidity of the stomach and the action of bile, and it should have immunostimulant activity. In addition, the strain should be able to adhere to the mucosa causing no toxicity, and to produce substances with antibacterial activity against some pathogens. The aim of the work was the isolation and identification of lactobacilli of human origin. It was also deepened the study of their amensalistic properties, with particular attention to the resistance to gastrointestinal transit and their antagonism against pathogenic microorganisms. Three hundred fifty-nine lactobacilli strains were isolated from swabs of healthy people and identified using molecular techniques based on the study of 16S rDNA. The identification of some strains was confirmed by further analysis DHPLC V1 and V3 of 16S rDNA. The strains were subjected to the evaluation of the resistance to bile salts and low pH, to the production of hydrogen peroxide and more particularly, it has been evaluated the ability to produce substances with antibacterial activity. Finally, the attention was focused on the characterization of an active supernatant produced by an oral strain. The isolation of the substance provided chromatographic procedures such as SEC (Size Exclusion Chromatography using Sephadex 50) and SPE (Reverse Phase Chromatography using C18 column). The results were shown that the active fraction has a low molecular weight and for its chemical-physical characteristics is not a common bacteriocin, for this reason are on going further chromatographic studies using columns with increasing polarity (C4, phenyl, cyano, and amino) . Future outlooks are focused on the identification of the molecule in question, by MALDI-TOF and ESI-TOF and then optimizing the whole process to standardize the entire method. In this way, the opportunity to bring to light new molecules will be possible, with the ultimate goal of being able to take advantage from these antibacterial substances.File | Dimensione | Formato | |
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