Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. Nowadays, researchers and producers are looking for probiotic strains with proven efficacy towards specific applications in order to choose the most suitable strain for the required use. Coree s.r.l. is a company dealing with the screening of potential new probiotic strains to be developed in food and food supplements specifically designed for the first phase of the life, from delivery to weaning. Food is strictly correlated to probiotic efficacy and it can be used as a deliver system to colonize the human intestine. To date, the delivery of probiotics through novel functional food represents a very interesting opportunity for manufacturers and customers. The aim of my PhD project was to study if new isolated strains of Lactobacillus spp. and Bifidobacterium spp. were able to take advantage of the interaction with their substrate and express this synergism with improved beneficial features to the host by considering an important stage of life, the weaning phase. In light of this evidence, the first part of the Ph.D. investigated whether probiotic bacteria, when grown in weaning food matrices, changed their properties, such as the adhesion to the gut or the regulation of various genes expression by human intestinal cells. We used some conventional complementary foods, such as carrot, apple puree, rice cream, and oat-based beverage, to evaluate the possible variations in terms of post-fermentation adhesiveness. Results showed that Lactobacillus spp. and Bifidobacterium spp. strains, when cultured in complementary foods, were able to improve their adhesiveness to the gut mucosa, showing a synergic effect between food matrix and probiotic microorganism. Moreover, it has been demonstrated that the ability to ferment weaning foods and to adhere to the human intestinal cell lines was strain-specific and not correlated to the genus of microorganisms. Furthermore, data have evidenced that bacterial strains isolated from plants possessed good ability to adhere to gut, and, thus, the adhesion properties is not influenced by the origins. These results were published an original article titled “The Synergistic Interaction between Probiotics and Food Affects Their Beneficial Features” on the journal “Advances in Nutrition and Food” (January 2020). In parallel, genome analysis was carried out to new isolated bacterial strains of COREE in order to better characterize their profiles. Next-Generation Sequencing (NGS) approach was used to find the hypothetical presence of plasmids and antibiotic-resistance genes and determine the bacterial fermentation type. During fermentation, bacterial strains produce a wide range of metabolites, some of which are bioavailable and bioactive with health properties that can be absorbed by the gastrointestinal cells and exert their effect at the systemic level. Therefore, metabolomic approach was applied to provide information about the different functional and nutritional properties of oat-based beverage fermented by Lactobacillus spp. and Bifidobacterium spp. strains. Oat is widely appreciated for its beneficial properties for adult and infant health, and currently, more food products are available for infant phase. Firstly, we evaluated the metabolites profile of oat beverage following in vitro gastrointestinal digestion by UHPLC-Q-TOF and HPAEC-PAD analysis. The untargeted metabolomics allowed to investigate the profile of metabolites released by oat-based beverage at the end of digestion. The results showed that flavonoids, phenolic acids, amino acids, and steroids were the major class of compounds identified in oat before any treatment. After the in vitro digestion, amino acids, peptides, and phenolic acids were the most released compounds. Afterward, the impact of co-fermentation by Lactobacillus spp. and Bifidobacterium spp. strains on the oat metabolites profile was evaluated. The fermentation process decreased the levels of antinutrients (lignans and phytic acid), a class of compounds that reduces the correct absorption of important micronutrients. Additionally, the digested profile of fermented oat beverage showed a major release of amino acids, vitamins, polyphenols, and phytochemical substances respect to the unfermented condition. Moreover, oat fiber component represents the most bioactive metabolite, consisting predominantly of β-glucans. For this reason, ion chromatography technique (HPAEC-PAD) was used to analyze the impact of digestion and fermentation on the oligosaccharidic component. Data indicated that the bacterial strains did not modify the oligosaccharides profile preserving its prebiotic capacity. These data have been submitted as original article entitled " The combined effect of fermentation of lactic acid bacteria and in vitro digestion on metabolomic and oligosaccharide profile of oat beverage” on the journal “Food Research International”. Successively, a simulated digestion/absorption model by using the enterocytes-type cells (Caco-2) allowed to examine the differences between the absorbed metabolites of fermented and non-fermented food matrix. The assimilated profile of non-fermented oat-based beverage was found rich in amino acids and myo-inositol. The linoleic acid, one of the most abundant fatty acid in Avena S., was found totally unabsorbed revealing as substrate for the intestinal microflora. Regarding the effect of fermentation on the absorbed profiles, results revealed that the bacterial strains did not alter drastically the metabolites profiles but induced few and strain-specificity effects that increased the absorption of specific compounds. This work suggests the relevance of culture starter in the development of fermented functional food. The fermentation by selected bacterial strains, even belonging to the same species, produced two food matrices with similar chemical profiles, but with a rather different effect on host metabolism. These findings are submitting as original article titled " In-vitro assessment of the strain-specific impact of fermented oat on the absorption of different metabolites” on the journal “Food Research International”.
I probiotici sono definiti come "microrganismi vivi che, se somministrati in quantità adeguate, conferiscono un beneficio alla salute dell'ospite". Al giorno d'oggi, ricercatori e produttori sono alla ricerca di nuovi ceppi probiotici con provata efficacia per specifiche applicazioni al fine di scegliere il ceppo più adatto all'uso richiesto. Coree s.r.l. è un'azienda che si occupa dello screening di nuovi ceppi probiotici per lo sviluppi di alimenti ed integratori alimentari specificamente pensati per la prima fase della vita, dalla nascita allo svezzamento. La matrice alimentare è strettamente correlata all'efficacia probiotica e può essere utilizzata come sistema di somministrazione al fine di colonizzare l'intestino umano. Ad oggi, la somministrazione di probiotici attraverso nuovi alimenti funzionali rappresenta un'opportunità molto interessante per i produttori ed i consumatori. Lo scopo di questo progetto di dottorato è stato quello di studiare se nuovi ceppi probiotici di Lactobacillus spp. e Bifidobacterium spp. fossero in grado di trarre vantaggio dall'interazione con il loro substrato ed esprimere questo sinergismo per migliorarne le caratteristiche benefiche nei confronti dell'ospite in una importante fase della vita, quella dello svezzamento. Alla luce di queste evidenze, la prima parte del dottorato ha indagato se i batteri probiotici, coltivati in matrici alimentari per lo svezzamento, cambiassero le loro proprietà, come l'adesione alla parete intestinale oppure la regolazione di espressione di vari geni da parte delle cellule intestinali umane. Sono stati utilizzati alcuni alimenti convenzionali della fase dello svezzamento, come carota, purea di mela, crema di riso e bevanda a base di avena, per valutare le possibili variazioni in termini di adesività post-fermentazione. I risultati hanno mostrato che i ceppi di Lactobacillus spp. e Bifidobacterium spp., se coltivati nelle matrici sopracitate, erano in grado di migliorare la loro adesività alla mucosa intestinale dimostrando un effetto sinergico tra matrice alimentare e microrganismo probiotico. È stato inoltre dimostrato che la capacità di fermentare determinati alimenti per lo svezzamento e di aderire alle linee cellulari intestinali umane era ceppo-specifica e non correlata al genere dei microrganismi. I dati hanno evidenziato che i ceppi batterici isolati dalle piante possedevano una buona capacità di aderire all'intestino e, quindi, le proprietà di adesione non sono influenzate dall’origine del ceppo. I risultati ottenuti sono stati pubblicati in un articolo scientifico sulla rivista "Advances in Nutrition and Food"(Gennaio 2020). In parallelo, l'approccio Next-Generation Sequencing (NGS) è stato impiegato per studiare i genomi dei nuovi ceppi batterici di proprietà di COREE s.r.l. e valutare l’eventuale presenza di plasmidi, geni di antibiotico-resistenza ed analizzare il profilo metabolico. Durante la fermentazione, i ceppi batterici producono una vasta gamma di metaboliti, alcuni dei quali sono composti biodisponibili e bioattivi con effetti positivi. Pertanto, un approccio metabolomico è stato applicato per fornire ulteriori informazioni sulle proprietà funzionali e nutrizionali della bevanda a base di avena fermentata da ceppi di Lactobacillus spp. e Bifidobacterium spp. L'avena è ampiamente apprezzata per i suoi effetti salutistici nell’adulto e nel bambino, ed attualmente sono disponibili sempre più prodotti alimentari per la fase infantile. Tramite gli strumenti UHPLC-Q-TOF e HPAEC-PAD, è stato determinato l’impatto della fermentazione e della digestione sul profilo dei metaboliti della bevanda vegetale. I risultati hanno mostrato che, nel latte di avena tal quale, le classi di composti più abbondanti erano flavonoidi, acidi fenolici, aminoacidi e steroidi. Dopo la digestione in vitro, i profili metabolici hanno indicato che aminoacidi, peptidi ed acidi fenolici erano le molecole più rilasciate a livello intestinale della bevanda di avena. Successivamente, è stato valutato l'impatto della co-fermentazione di ceppi di Lactobacillus spp. e Bifidobacterium spp. Il processo di fermentazione ha diminuito i livelli di antinutrienti, come lignani ed acido fitico, una classe di composti correlata al malassorbimento di importanti micronutrienti. Il profilo digerito della bevanda di avena fermentata ha presentato un maggiore rilascio di aminoacidi, vitamine, polifenoli e sostanze fitochimiche rispetto alla condizione non fermentata. Secondo quanto riportato dalla letteratura scientifica, la fibra rappresenta la parte più bioattiva dell’avena, formata prevalentemente da β-glucani. La tecnica analitica di cromatografia ionica (HPAEC-PAD) ha permesso di analizzare l'impatto della digestione e della fermentazione sulla sua componente oligosaccaridica. I risultati hanno indicato che i ceppi batterici non hanno modificato il profilo degli oligosaccaridi e che quindi la sua capacità prebiotica è stata preservata. Questi dati sono stati pubblicati sulla rivista "Food Research International" (Febbraio 2021). Successivamente, un modello di assorbimento in vitro con le cellule enterocyte-type Caco-2 ha permesso di esaminare le differenze tra i metaboliti assorbiti della bevanda di avena fermentata e non fermentata. In particolare, la bevanda di avena è stata fermentata da due diversi blend di ceppi probiotici, appartenenti alla stessa specie. Il profilo assorbito della bevanda non fermentata è risultato ricco di aminoacidi e di myo-inositolo. L'acido linoleico, uno degli acidi grassi più abbondanti in Avena S., è stato trovato totalmente non assorbito rivelandosi un potenziale substrato per il microbiota intestinale. Dopo il processo di fermentazione, i risultati hanno mostrato che il profilo dei metaboliti assorbiti non è stato alterato drasticamente ma si è riscontrato un aumento di assorbimento di composti specifici e diversi tra i due blend utilizzati, indicando un effetto della ceppo-specificità. Questo lavoro ha suggerito l’importanza dello starter di coltura nello sviluppo di alimenti fermentati. La fermentazione da specifici ceppi batterici selezionati, anche appartenenti alla stessa specie, ha prodotto due matrici alimentari con simili profili chimici, ma con un effetto diverso sul metabolismo cellulare delle Caco-2. Questi risultati saranno presto sottomessi sulla rivista "Food Research International".
BOCCHI, SERENA, Study of the metabolites involved in the symbiotic relationship between probiotic and host, MORELLI, LORENZO, LUCINI, LUIGI, ELLI, MARINA, Università Cattolica del Sacro Cuore Piacenza:Ciclo XXXIII [https://hdl.handle.net/10807/284996]
Study of the metabolites involved in the symbiotic relationship between probiotic and host
Bocchi, Serena
2021
Abstract
Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. Nowadays, researchers and producers are looking for probiotic strains with proven efficacy towards specific applications in order to choose the most suitable strain for the required use. Coree s.r.l. is a company dealing with the screening of potential new probiotic strains to be developed in food and food supplements specifically designed for the first phase of the life, from delivery to weaning. Food is strictly correlated to probiotic efficacy and it can be used as a deliver system to colonize the human intestine. To date, the delivery of probiotics through novel functional food represents a very interesting opportunity for manufacturers and customers. The aim of my PhD project was to study if new isolated strains of Lactobacillus spp. and Bifidobacterium spp. were able to take advantage of the interaction with their substrate and express this synergism with improved beneficial features to the host by considering an important stage of life, the weaning phase. In light of this evidence, the first part of the Ph.D. investigated whether probiotic bacteria, when grown in weaning food matrices, changed their properties, such as the adhesion to the gut or the regulation of various genes expression by human intestinal cells. We used some conventional complementary foods, such as carrot, apple puree, rice cream, and oat-based beverage, to evaluate the possible variations in terms of post-fermentation adhesiveness. Results showed that Lactobacillus spp. and Bifidobacterium spp. strains, when cultured in complementary foods, were able to improve their adhesiveness to the gut mucosa, showing a synergic effect between food matrix and probiotic microorganism. Moreover, it has been demonstrated that the ability to ferment weaning foods and to adhere to the human intestinal cell lines was strain-specific and not correlated to the genus of microorganisms. Furthermore, data have evidenced that bacterial strains isolated from plants possessed good ability to adhere to gut, and, thus, the adhesion properties is not influenced by the origins. These results were published an original article titled “The Synergistic Interaction between Probiotics and Food Affects Their Beneficial Features” on the journal “Advances in Nutrition and Food” (January 2020). In parallel, genome analysis was carried out to new isolated bacterial strains of COREE in order to better characterize their profiles. Next-Generation Sequencing (NGS) approach was used to find the hypothetical presence of plasmids and antibiotic-resistance genes and determine the bacterial fermentation type. During fermentation, bacterial strains produce a wide range of metabolites, some of which are bioavailable and bioactive with health properties that can be absorbed by the gastrointestinal cells and exert their effect at the systemic level. Therefore, metabolomic approach was applied to provide information about the different functional and nutritional properties of oat-based beverage fermented by Lactobacillus spp. and Bifidobacterium spp. strains. Oat is widely appreciated for its beneficial properties for adult and infant health, and currently, more food products are available for infant phase. Firstly, we evaluated the metabolites profile of oat beverage following in vitro gastrointestinal digestion by UHPLC-Q-TOF and HPAEC-PAD analysis. The untargeted metabolomics allowed to investigate the profile of metabolites released by oat-based beverage at the end of digestion. The results showed that flavonoids, phenolic acids, amino acids, and steroids were the major class of compounds identified in oat before any treatment. After the in vitro digestion, amino acids, peptides, and phenolic acids were the most released compounds. Afterward, the impact of co-fermentation by Lactobacillus spp. and Bifidobacterium spp. strains on the oat metabolites profile was evaluated. The fermentation process decreased the levels of antinutrients (lignans and phytic acid), a class of compounds that reduces the correct absorption of important micronutrients. Additionally, the digested profile of fermented oat beverage showed a major release of amino acids, vitamins, polyphenols, and phytochemical substances respect to the unfermented condition. Moreover, oat fiber component represents the most bioactive metabolite, consisting predominantly of β-glucans. For this reason, ion chromatography technique (HPAEC-PAD) was used to analyze the impact of digestion and fermentation on the oligosaccharidic component. Data indicated that the bacterial strains did not modify the oligosaccharides profile preserving its prebiotic capacity. These data have been submitted as original article entitled " The combined effect of fermentation of lactic acid bacteria and in vitro digestion on metabolomic and oligosaccharide profile of oat beverage” on the journal “Food Research International”. Successively, a simulated digestion/absorption model by using the enterocytes-type cells (Caco-2) allowed to examine the differences between the absorbed metabolites of fermented and non-fermented food matrix. The assimilated profile of non-fermented oat-based beverage was found rich in amino acids and myo-inositol. The linoleic acid, one of the most abundant fatty acid in Avena S., was found totally unabsorbed revealing as substrate for the intestinal microflora. Regarding the effect of fermentation on the absorbed profiles, results revealed that the bacterial strains did not alter drastically the metabolites profiles but induced few and strain-specificity effects that increased the absorption of specific compounds. This work suggests the relevance of culture starter in the development of fermented functional food. The fermentation by selected bacterial strains, even belonging to the same species, produced two food matrices with similar chemical profiles, but with a rather different effect on host metabolism. These findings are submitting as original article titled " In-vitro assessment of the strain-specific impact of fermented oat on the absorption of different metabolites” on the journal “Food Research International”.File | Dimensione | Formato | |
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