Development of novel procedures to obtain innovative supplements based on phytocompounds and microorganisms.

L\u2019attivit\ue0 di ricerca \ue8 stata svolta al fine di studiare l\u2019effetto di microrganismi fermentanti sui componenti metabolici di matrici vegetali, con l\u2019obiettivo finale di ottenere un integratore alimentare con migliorate propriet\ue0 metaboliche di una selezionata variet\ue0 di agrumi. E' stata creata una collezione di microrganismi (lieviti e batteri) che potranno essere ulteriormente studiati per possibili applicazioni future. Alcuni di questi microrganismi sono stati impiegati nelle prove di fermentazione per valutare l'effetto sul profilo metabolico complessivo. E' stata valutata anche la possibilit\ue0 di aggiungere dei microrganismi probiotici con propriet\ue0 benefiche documentate. Le prove di fermentazione hanno individuato diversi aspetti innovati da approfondire a livello aziendale con prove di fattibilit\ue0 e stabilit\ue0 dei prossibili integratore alimentare realizzabili.The research activity aimed at studying the effect of microrganisms on the metabolic profile of vegetable matrices. A collection of yeasts and bacteria were created. Some of them were chosen to ferment a selected variety of citrus fruits. Two selected probiotics strains with documented properties were added to the product. The analyses performed arose several innovative aspects to be evaluate in pre-indestrial setting with feasibility and stability tests in order to define which food supplement can be obtained

Theoretical outcomes and applications of taxonomic studies on the genera Enterococcus and Lactobacillus

Questo progetto di dottorato, dal titolo \u201cTheoretical outcomes and applications of taxonomic studies on the genera Enterococcus and Lactobacillus\u201d, si colloca nel contesto della tassonomia batterica. Tale disciplina scientifica si propone di descrivere la diversit\ue0 degli organismi viventi con lo scopo di caratterizzarli e di raggrupparli in un sistema gerarchico ordinato. \uc8 convinzione diffusa che il sistema di classificazione pi\uf9 stabile e affidabile sia quello che si basa sulle reali relazioni genealogiche degli organismi. Tuttavia, data la loro semplicit\ue0 strutturale, lo studio dei batteri \ue8 sempre stato fortemente influenzato dallo sviluppo scientifico e dalle tecnologie di indagine disponibili. I microrganismi ricoprono notevole interesse in diverse aree scientifiche, quali la medicina, la genetica, l\u2019ecologia e trovano ampia applicazione nell\u2019industria alimentare, farmaceutica e nel settore ambientale. Lo studio della biodiversit\ue0 esistente \ue8 indispensabile per impiegare al meglio i microrganismi, per scoprire nuove applicazioni biotecnologiche nonch\ue9 per approfondire i rischi derivanti dalla presenza di batteri indesiderati o pericolosi. La tassonomia, inoltre, ha il compito di assegnare un nome univoco ad ogni organismo fino ad ora caratterizzato, in modo tale da consentire lo scambio delle informazioni disponibili tramite l\u2019utilizzo di una terminologia comune e di fornire strumenti utili alla corretta identificazione di nuovi isolati. Nel corso di questo progetto sono state approfondite alcune problematiche tassonomiche legate a specie appartenenti ai generi Lactobacillus ed Enterococcus, che includono batteri di notevole interesse ecologico e applicativo, in particolare, nella produzione di alimenti fermentati dove vengono impiegati come commensali o starter. Alcune specie, inoltre, sono comuni abitanti del tratto gastro-intestinale dell\u2019uomo e degli animali e possono influire sulla composizione della microflora intestinale, sull\u2019assorbimento dei nutrienti e sullo stato di salute dell\u2019ospite. Data la loro diffusione in ambienti cos\uec differenti e il ruolo che ricoprono in diversi settori, l\u2019approfondimento tassonomico di questi generi risulta particolarmente rilevante. Le ricerche svolte nell\u2019ambito di questo progetto sono state affrontate con l\u2019ausilio di approcci differenti a seconda dell\u2019organismo preso in esame, della problematica da risolvere e del livello tassonomico investigato (tra specie distinte, strettamente correlate o fra individui di una stessa specie). Nella prima parte della tesi (capitolo 2) sono state approfondite le relazioni filogenetiche tra enterococchi tramite l\u2019analisi di sequenza di sei diversi marcatori molecolari (16S rDNA, recA, hsp60, pheS, atpA, rpoA) e il confronto dell\u2019organizzazione genica delle regioni adiacenti al gene recA. Questo approccio ha permesso di evidenziare la presenza di gruppi di specie maggiormente correlate tra loro e di valutare un probabile ordine di speciazione. I dati di sequenza ottenuti sono stati analizzati per individuare dei polimorfismi specie-specifici sui geni 16S rRNA, recA e hsp60, in corrispondenza dei quali sono state disegnate delle sonde da impiegare a fini diagnostici. Queste sonde, utilizzate per lo sviluppo di un prototipo di DNA-microarray tramite deposizione meccanica, hanno consentito di differenziare, sulla base dei profili di ibridazione ottenuti, le specie di enterococchi che si ritrovano pi\uf9 frequentemente in campioni clinici e in matrici alimentari, alcune delle quali, come E. durans ed E. hirae, sono in genere difficilmente distinguibili con altri metodi molecolari e fenotipici. Nella seconda parte (capitoli 3 e 4) sono state affrontate due problematiche di classificazione tra specie strettamente correlate (L. ingluviei e L. thermotolerans, L. vaccinostercus e L. durianis), che mostrano una elevata conservazione dei marcatori molecolari e propriet\ue0 fenotipiche molto simili. Pertanto, il loro grado di similarit\ue0 \ue8 stato determinato tramite ibridazione del DNA totale, l\u2019unico metodo attualmente valido per la delimitazione di una specie batterica. Dai risultati ottenuti \ue8 stato possibile accertare che le unit\ue0 tassonomiche non differenziabili rappresentano in realt\ue0 una singola specie, erroneamente annotata nel sistema di classificazione con nomi diversi. Come conseguenza di queste evidenze lo schema di classificazione del genere Lactobacillus e il sistema di nomenclatura di questi organismi \ue8 stato corretto, in modo tale da consentire in futuro una pi\uf9 chiara e non ambigua identificazione di isolati ambientali. Nella terza parte (capitoli 5 e 6) \ue8 stata approfondita la biodiversit\ue0 microbica a livello intraspecifico di due specie che ricoprono un ruolo rilevante in diverse produzioni alimentari, L. plantarum e L. delbrueckii. Per la specie L. plantarum \ue8 stato confrontato il contenuto genico di ceppi di diversa origine tramite l\u2019utilizzo di DNA-microarray commerciali su cui \ue8 rappresentato il genoma completo del ceppo WCFS1, interamente sequenziato nel 2003. Gli esperimenti di ibridazione hanno evidenziato una notevole eterogeneit\ue0 genetica che correla con i profili fenotipici dei ceppi considerati. Un ulteriore approfondimento di queste osservazioni consentir\ue0, tramite l\u2019utilizzo di supporti bioinformatici dedicati, di valutare pi\uf9 chiaramente il legame tra specifici determinanti genici e propriet\ue0 fisiologiche in un\u2019ottica di genomica funzionale. Nel capitolo 6 \ue8 riportata per la prima volta la presenza di due distinte vie metaboliche, una mediata da permeasi e l\u2019altra da un trasportatore fosfoenol-piruvato dipendente, responsabili del trasporto e della degradazione del lattosio nella specie L. delbrueckii. La presenza/assenza e l\u2019espressione di una o dell\u2019altra via metabolica sono legate all\u2019ecologia, alle propriet\ue0 tecnologiche e all\u2019evoluzione delle quattro subspecie attualmente descritte, L. delbrueckii subsp. lactis, bulgaricus, delbrueckii e indicus. I risultati ottenuti costituiscono un originale contributo per valutare con pi\uf9 rigore la flessibilit\ue0 di queste specie batteriche e la funzionalit\ue0 dei singoli isolati che li rendono differentemente adatti a sopravvivere in determinati ambienti (latte, vegetali) e a trovare impiego in diversi processi dell\u2019industria alimentare (produzione di yogurt o formaggi). In conclusione, gli studi tassonomici riportati hanno contribuito a migliorare lo schema di classificazione dei batteri investigati, chiarendo le eventuali ambiguit\ue0 del sistema di nomenclatura. Inoltre, \ue8 evidente come la ricerca di base possa avere notevoli risvolti applicativi quali l\u2019approfondimento di tratti funzionali, utili per la selezione di organismi di rilevanza tecnologica. Non da ultimo, l\u2019eterogeneit\ue0 osservata consente di comprendere pi\uf9 chiaramente la flessibilit\ue0 genetica che spinge questi organismi ad evolvere rapidamente e ad adattarsi all\u2019ambiente che li circonda.The present thesis, titled \u201cTheoretical outcomes and applications of taxonomic studies on the genera Enterococcus and Lactobacillus\u201d, concerns taxonomy of bacteria. This scientific discipline aims at studying all living organisms with the final goal of characterizing and arranging them in an orderly manner on the basis of their similarity and relationships. The classification scheme based on the real genealogical relationships of living organisms is considered the most stable. However, as a consequence of their simple structure, bacteria investigation has been strongly influenced by scientific and technical advances. Microbes certainly play a crucial role in several scientific area, such as medicine, genetics, ecology and they are largely employed in food production industry, pharmacology and environmental science. Thus, it is necessary to study existing biodiversity in order to use microorganisms for human activities, to discover new biotechnological applications and to deep the risks linked to the presence of unwanted or even dangerous bacteria. Moreover, taxonomy assigns a unique name to each described organism, in order to provide information exchange by using a common terminology and to provide tools or methods to improve the correct identification of new isolates. During this project different taxonomic aspects have been faced concerning some species belonging to the genera Lactobacillus ed Enterococcus, which include relevant bacteria for ecological and applicative purpose, in particular, for fermented food production where they are largely employed as natural component or as starter. Some species are marked as common inhabitant of the gastro-intestinal tract of human and animals and they can modulate the microflora composition, nutrient assimilation as well as the general health status of the host. Due to their presence in so different environments and their application in several human area, their study appear particularly interesting and necessary. The present project has been carried out by using different approaches based on the aspect to be evaluated, the organism and the taxonomic rank under investigation (i.e. the comparison of distinct or strictly correlated species and the analysis of strains belonging to the same taxonomic unit). In the first part of this dissertation (chapter 2) is presented an evaluation of the phylogenetic relationships among enterococci by using a comparative sequence analysis of six molecular markers (16S rDNA, recA, hsp60, pheS, atpA, rpoA) and synteny of the region flanking recA gene. These approaches allowed to underline the presence of distinct groups of closely related species and to hypothesize their speciation order. The obtained gene sequence data of three target genes, 16S rRNA, recA e hsp60, were analysed in order to design diagnostic probes in correspondence of species-specific polymorphisms. The designed probes, employed to develop a prototype of spotted DNA-microarray, permitted to distinguish, on the basis of the obtained hybridization profiles, the Enterococcus species which are more frequently detected in clinical samples and in several food production chains, e.g. E. durans and E. hirae, usually hardly differentiated by the use of other molecular and phenotypic methods. In the second part (chapters 3 and 4) other two aspects, involving closely related species (L. ingluviei e L. thermotolerans, L. vaccinostercus e L. durianis) have been faced. These species showed highly conserved molecular markers and similar phenotypic properties. Thus, their degree of similarity has been determined by DNA-DNA hybridization, that is the unique valid method to circumscribe a bacterial species. Obtained results permitted to evidence that these closely related taxa belong to a single species, and they were wrongly classified with different names. As a consequence of these evidences, the classification scheme of Lactobacillus genus and the nomenclature of these organisms were corrected, in order to allow in the future a more clear and unambiguous identification of natural isolates. In the third part of the thesis (chapters 5 and 6) it is reported an exploration of microbial biodiversity at inter-specific level, in particular of two relevant species largely employed in several food production processes, i.e. L. plantarum and L. delbrueckii. Concerning the L. plantarum species, the gene-content of strains of different isolation origin was evaluated by using commercial DNA-microarray representing the complete genome of WCFS1 strain, sequenced in the 2003. Hybridization experiments highlighted an consistent genetic heterogeneity which seems correlatine with the phenotypic profiles of the considered strains. A further evaluation of these observations, by the application of powerful bioinformatic tools, will permit to match specific genetic determinants and physiological properties in a functional genomic perspective. In chapter 6 the presence of two distinct metabolic pathways, responsible for lactose transport and degradation in L. delbrueckii species, is reported for the first time: one involves a permease and the other a phosphoenol-pyruvate dependent system. The presence/absence and transcription regulation of the first or of the second metabolic pathway seemed linked to ecology, technological properties and evolution of the four subspecies, L. delbrueckii subsp. lactis, bulgaricus, delbrueckii and indicus. The presented observations represent an original contribution to appraise the flexibility of this bacterial species and functionality of single isolates made them able to survive in different habits (milk, vegetable) and to be employed in several food industry processes (e.g. yogurt or cheese production). In conclusion, the reported taxonomic studies have contributed to improve the classification scheme of the investigated bacteria and removing some ambiguous aspects of the nomenclature system. Moreover, it is highlight the applications of basic research studies such as a deep evaluation of functional traits and criteria, helpful to the selection of organism of technological relevance. Finally, the observed heterogeneity furnish a more clear view of genetic flexibility which force these organisms to rapidly evolve and to easily adapt to the several environments

Lactobacillus delbrueckii subsp. indicus subsp. nov., isolated from Indian dairy products

Four strains isolated from Indian dairy products and initially identified as Lactobacillus delbrueckii could not be assigned to a definite subspecies because molecular identification and phenotypic traits did not agree with those of recognized subspecies of L. delbrueckii. Hybridization of total DNA (78-86% against type strains of the other three subspecies), AFLP and RAPD-PCR fingerprints, phylogenetic analysis based on 16S rRNA gene sequences and sequence analysis of two coding genes (recA and hsp60), together with phenotypic profiles, indicated that the four strains form a coherent cluster and represent a novel subspecies, for which the name Lactobacillus delbrueckii subsp. indicus subsp. nov. is proposed. The type strain is NCC725T (=LMG 22083T=DSM 15996T)

Lactobacillus casei group effective identification

Lactobacillus casei, L. paracasei and L. rhamnosus form a closely related taxonomic group (Lactobacillus casei-group) within the facultatively heterofermentative lactobacilli. Some strains belonging to these species have been used for a long time as probiotics in a wide range of different products, and they commonly represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavor development. The close genetic relationship among L. casei-group species, and the related starter cultures, hinders the development of an adequate selective identification method useful to understand the role played by each microbial population in the matrix and the routine biochemical tests often fail to discriminate among closely related species occurring in the same ecological environments, like inside ripened cheeses. The aim of this study was a comprehensive analysis of 75 cheese isolates previously identified as L. casei group, combining the results of multiple approaches. Alternative proposed culture media for Lb. casei- group strains selective growth were tested, giving poor discrimination among strains. Futhermore, a species-specific PCR based on existing primers designed on the 16s rDNA was adapted to laboratory conditions and tested on the isolated strains. This approach allowed the identification of the majority of the tested strains, but still leaving some strains with an uncertain identity. These isolates had to be checked with more than one primer pair in order to avoid misidentification due to possible cross-reactions. Finally, a new species-specific multiplex PCR assay was developed using primers designed on a universal protein encoding gene. The amplicon size was able to rapidly distinguish L. casei, L. paracasei and L. rhamnosus strains

Genetic diversity of enterococci from Iranian home-made artisanal dairy products

Enterococci represent a significant part of microbiota in home-made dairy products due to their remarkable intrinsic resistance to critical growth conditions and technological properties. In this study, the predominant coccal lactic acid bacteria (LAB) were isolated from traditional dairy products made and largely consumed in villages of Chahar-Mahalo Bakhtiyari province of Iran. Thirty-six samples of homemadedairy products were collected from scattered households, and a total of 61 grampositive and catalase-negative cocci were analyzed. Genetic fingerprinting (repetitive extragenic palindromic PCR (REP-PCR) and 16S\u201323S intergenic spacer (ITS) amplification) and gene sequencing (16S rRNA gene and pheS gene) were carried out toobtain an unambiguous identification of the isolates at the species level and to highlight genetic biodiversity among wild enterococci. It was shown that 42 out of 61 isolates belonged to Enterococcus spp., and Enterococcus faecium was the most frequentlyisolated species. Intraspecific diversity of the E. faecium species was further investigated, targeting the genes hyl, esp, and msrC, related to virulence and antibiotic resistance, and the results obtained suggest that two strains could be further characterized for possible use as starter cultures. In conclusion, the present study confirms that application of diverse independent molecular techniques is required to clearly identify Enterococcus spp. isolates and to unveil wild strain biodiversity. Moreover, a preliminary molecular screening of selected genetic features represents a fast and feasible approach to select, among a wide collection of isolates, the most promising candidates for further exhaustive characterization of safety and technological traits

Effective identification of Lactobacillus casei group species: Genome-based selection of the gene mutL as the target of a novel multiplex PCR assay

Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity

Lactobacillus durianis Leisner et al. 2002 is a later heterotypic synonym of Lactobacillus vaccinostercus Kozaki and Okada 1983.

The taxonomic status of the species Lactobacillus durianis and Lactobacillus vaccinostercus is briefly summarized and experimental evidence concerning their similarity is presented. Highly similar 16S rRNA gene sequences (99.8% similarity over 1523 bp), partial recA gene sequences (99.5% similarity over 600 bp) and partial hsp60 gene sequences (99.1% similarity over 924 bp) suggest that the two species are closely related. Moreover, a high DNA-DNA binding level (87%) and similar genomic DNA G+C contents (41-44 mol% for both species) as well as similar biochemical characteristics support the evidence that they constitute a single species. Consequently, according to Rules 38 and 42 of the Bacteriological Code, the name Lactobacillus vaccinostercus, the oldest legitimate name, must be maintained and the name Lactobacillus durianis should be considered a later heterotypic synony

Microbiota of high-pressure-processed Serrano ham investigated by culture-dependent and culture-independent methods

The microbiota of Serrano dry-cured ham of different chemical composition, subjected or not to high-pressure processing (HPP), was investigated using culture-dependent and culture-independent methods. Microbial counts were submitted to analysis of variance with physicochemical parameters (aw, NaCl concentration, salt-in-lean ratio and intramuscular fat content) or HPP as main effects. In untreated hams, physicochemical parameters significantly affected counts of aerobic mesophiles, psychrotrophs, and moulds and yeasts. NaCl concentration and fat content influenced the levels of four and three of the five studied microbial groups, respectively, whereas no influence of aw was stated. The HPP treatment had a significant effect on counts of all investigated microbial groups. Culture-independent methods showed the presence of bacteria such as Staphylococcus equorum, Staphylococcus succinus, Bacillus subtilis and Cellulosimicrobium sp., moulds like Penicillium commune, Aspergillus fumigatus, Sclerotinia sclerotiorum, Eurotium athecium and Moniliella mellis, and yeasts like Debaryomyces hansenii and Candida glucosophila. Absence of B. subtilis bands and weaker bands of E. athecium were recorded for HPP-treated hams. The higher microbial levels found in lean ham might result in a quicker deterioration. HPP treatment confirmed its suitability as a procedure to control spoilage microorganisms. DGGE did not seem to be sensitive enough to highlight changes caused by HPP treatment in the microbiota of ham, but contributed to the detection of microbial species not previously found in ham