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Evaluation of the effects of alternative physical approach on the metabolism and functional traits of useful microorganisms
Full text linkStato dell’arte: I microrganismi probiotici negli alimenti potrebbero causare cambiamenti significativi nel sapore e nella reologia, a causa del loro metabolismo attivo. È importante, quindi, controllare il metabolismo dei microrganismi probiotici negli alimenti, senza influenzare negativamente la vitalità e le caratteristiche funzionali. Un modo per superare questo problema è l'utilizzo di colture starter attenuate attraverso l’utilizzo di metodi fisici o chimici. Alcuni autori hanno studiato l'omogeneizzazione come metodo per attenuare/modulare il metabolismo delle colture starter nei prodotti caseari (Lanciotti et al., 2004, 2006, 2007); in questo progetto di tesi ho utilizzato una nuova tecnologia emergente, gli ultrasuoni. (i) È stato effettuato uno screening delle combinazioni possibili di ultrasuoni (US) (potenza/durata) su diversi microrganismi probiotici e sono state studiate le caratteristiche probiotiche e tecnologiche dopo l'esposizione agli US. (ii) Sono stati studiati gli effetti degli US sul rilascio di componenti intracellulari. (iii) È stata valutata l'interazione dei ceppi attenuati con il microbiota intestinale utilizzando la fermentazione in vitro (batch culture fermentation). Problematiche: (i) Non si conoscono gli effetti dell’attenuazione mediante US sulle caratteristiche tecnologiche/probiotiche dei microrganismi; (ii) non è chiara la risposta delle cellule microbiche al trattamento con gli US e il danno che ne deriva, in quanto in letteratura sono disponibili pochi dati; (iii) non sono disponibili dati sull'interazione di ceppi attenuati con il microbiota intestinale. Obiettivi: (i) Scelta della migliore combinazione di ultrasuoni (potenza/durata), in grado di evitare la post-acidificazione senza compromettere la vitalità dei ceppi, studio dei cambiamenti delle caratteristiche probiotiche e tecnologiche che i microrganismi potrebbero subire a seguito dell’attenuazione; (ii) valutazione del rilascio di componenti intracellulari (acidi nucleici e proteine) dopo l'applicazione degli ultrasuoni; e (iii) studio dell’effetto di ceppi attenuati sul microbiota intestinale. Pianificazione della ricerca: Nella prima parte sono stati usati tre diversi generi di microrganismi probiotici: Lactobacillus, Bifidobacterium, Propionibacterium. I ceppi sono stati trattati con ultrasuoni e sono state studiate le caratteristiche tecnologiche e probiotiche. Nella seconda parte, sono stati studiati gli effetti degli US sul rilascio di componenti intracellulari. I ceppi sono stati studiati a seguito del trattamento fisico per valutare il rilascio di costituenti intracellulari (acidi nucleici, proteine) ed eventuali lesioni della membrana cellulare. Nell'ultima parte, è stata studiata l'interazione di ceppi attenuati con il microbiota intestinale. Questo studio è stato condotto presso l'Università di Roehampton (Regno Unito). Materiali e metodi: (i) Caratteristiche tecnologiche: Prove di acidificazione in mezzo di laboratorio, crescita a diverse temperature, pH e contenuto di sale; caratteristiche probiotiche: antibiotico resistenza, sopravvivenza a pH 2,5 e in presenza di sali biliari (0,3%), idrofobicità e formazione di biofilm. (ii) La valutazione del danno sulle cellule microbiche è stato determinato attraverso letture spettrofotometriche a 260 e 280 nm per quantificare il rilascio di DNA e proteine. (iii) L'interazione dei ceppi attenuati con il microbiota intestinale è stata valutata utilizzando la fermentazione in vitro (batch culture fermentation). Risultati: (i) Le migliori combinazioni in grado di evitare la post-acidificazione sono state le seguenti: potenza, 60%; tempo, 6 minuti; impulsi, 2 s per Lactobacillus e Bifidobacterium, 40%, 8 min P. jensenii; 60%, 4 min P. freudenreichii subsp. freudenreichii. Gli US non hanno influenzato la vitalità a 45 °C e a pH 9, ma hanno determinato una diminuzione della crescita microbica a pH 4 (lattobacilli e bifidobatteri). Tuttavia, gli US non hanno influenzato il GI dei propionibatteri. L'effetto dell'attenuazione potrebbe essere migliorato con la refrigerazione. Il trattamento con gli US non ha influenzato la maggior parte dei tratti tecnologici, ma ha causato un aumento della suscettibilità ad alcuni antibiotici. Per quanto riguarda i tratti probiotici, gli US hanno causato un aumento di idrofobicità per L. reuteri e P. freudenreichii spp. freudenreichii. Questi risultati sono stati confermati con l'adesione alle cellule Caco-2 per L. reuteri. L. reuteri attenuato ha registrato un significativo aumento di idrofobicità (dal 3 al 25%) e una maggiore adesione alle cellule Caco-2. Inoltre, gli US hanno migliorato la stabilità dei biofilm nel tempo e questo risultato ha confermato i dati ottenuti con l'idrofobicità. (ii) Il rilascio di acidi nucleici e proteine, evidenzia che la membrana cellulare potrebbe essere un altro target del trattamento fisico. (iii) Per quanto riguarda gli effetti degli US sul microbiota intestinale: i ceppi attenuati non hanno influenzato il microbiota intestinale, ma in alcuni casi sono stati evidenziati effetti positivi. Importanza e impatto della ricerca di dottorato: Il metabolismo attivo dei microrganismi probiotici potrebbe costituire un problema quando questi ultimi vengono aggiunti agli alimenti. Infatti alcuni ceppi di batteri lattici continuano a produrre acido lattico e causano post-acidificazione (diminuzione del pH durante la conservazione). Pertanto, è importante controllare il loro metabolismo. Un modo possibile per controllare il metabolismo dei probiotici negli alimenti è l'attenuazione attraverso metodi fisici o chimici. Una delle tecnologie emergenti sono gli ultrasuoni (US). Questo approccio è stato utilizzato per evitare la post-acidificazione in una bevanda commerciale di riso (Bevilacqua et al., 2016). La presente tesi di dottorato ha contribuito a valutare gli effetti dell'attenuazione mediante ultrasuoni, su alcune caratteristiche tecnologiche e probiotiche, testando tre diversi generi di ceppi probiotici. Inoltre, questo progetto di dottorato ha studiato i cambiamenti in termini di caratteristiche tecnologiche e probiotiche che i microrganismi probiotici potrebbero subire a seguito dell'attenuazione; il rilascio di proteine, acidi nucleici. Infine, la novità di questa tesi di dottorato è stata lo studio degli effetti di ceppi attenuati sul microbiota intestinale. Proposte future: una prospettiva futura potrebbe essere focalizzata sull'uso degli US per migliorare o modulare l'adesione dei ceppi probiotici, considerando l'aumento di idrofobicità e la maggiore adesione alle cellule Caco-2. È importante studiare gli effetti di altri ceppi attenuati sul microbiota intestinale modulando le variabili del trattamento.Scientific background: Probiotics in foods could lead to significant changes in food flavor and rheology, due to their active metabolism. A possible way to overcome this problem is the attenuation of probiotics through a physical or chemical method. Some authors studied homogenization as a way to attenuate/modulate the metabolism of starter cultures in dairy products (Lanciotti et al., 2004, 2006, 2007); in this project I have used a new emerging technology, the ultrasounds. (i) A screening of the ultrasound (US) (power/duration) on different probiotic microorganisms was perfomed, and were studied the probiotic and technological characteristics after US-exposure. (ii) The effects of US on the release of intracellular components, was investigate. (iii) The interaction of attenuated strains with gut microbiota was evaluated, using in vitro batch culture fermentation. Open questions: (i) Few data are available on effects of US on probiotics and technological characteristics of probiotic strains; (ii) few data are available on the effect of attenuation with ultrasound on the sub-lethal injury; (iii) no data are available on the interaction of attenuated strains with gut microbiota. Aims: (i) Choice of the best combination of ultrasound to avoid post-acidification without affecting the viability of the strains, and study of the probiotic and technological characteristics to evaluate if attenuation could change them; (ii) study the release of intracellular components (nucleic acids and proteins) after the application of ultrasound; and (iii) evaluation the effects of attenuated strains on gut microbiota. Planning of the research: In the first part three different genera of probiotics were used: Lactobacillus, Bifidobacterium, Propionibacterium. The strains were treated with ultrasound and studied for technological and probiotic characteristics. In the second part, the effects of US on the release of intracellular components, was investigated. The strains were studied after physical treatment to assess the release of intra-cellular constituents (nucleic acids, proteins) and injury of the membrane. In the last part, the interaction of attenuated strains with gut microbiota, was studied. This study was carried out at University of Roehampton (UK). Materials and Methods: (i) Technological traits: acidification in lab medium, growth at different temperatures, pHs and salt content; probiotic traits: antibiotic-resistance, survival at pH 2.5 and in the presence of 0.3% bile salt, hydrophobicity, and biofilm formation. (ii) Injury characterization was evaluated by leakage of UV-absorbing substances. (iii) The interaction of attenuated strains with gut microbiota was evaluated, using in vitro batch culture fermentation. Results: (i) The best combinations to avoid post-acidification were the following: power, 60%; time, 6 min; pulse, 2 s for Lactobacillus and Bifidobacterium, 40%, 8 min P. jensenii; 60%, 4 min P. freudenreichii subsp. freudenreichii. US did not affect viability at 45 °C or at pH 9, but it determined a decrease of microbial growth to pH 4 (lactobacilli and bifidobacteria). However, the US did not affect the GI of propionibacteria. The effect of attenuation could be enhanced by the storage under refrigeration. US-treatment did not affect most of the technological traits, but generally caused an increase of susceptibility to some antibiotics. Concerning probiotic traits, US caused an increase of hydrophobicity for L. reuteri and P. freudenreichii spp. freudenreichii, after US-exposure. These results were confirmed with adhesion to Caco-2 cells for L. reuteri. US-attenuated L. reuteri experienced a significant increase of hydrophobicity (from 3 to 25%) and a higher adhesion to Caco-2 cells. Moreover, US improved the stability of the biofilm over the time, and this result confirmed the data obtained with hydrophobicity. (ii) The release of nucleic acids and proteins was found, highlighting that cell membrane could be another target physical treatments. (iii) Concerning the effects of US on gut microbiota, the ultrasound didn’t affect the gut microbiota, but in some cases, it could have a positive effect. Significance and Impact of PhD research: A main drawback of probiotics in foods can relate to their active metabolism, some strains of lactic acid bacteria continue to produce lactic acid and cause post-acidification (the decrease of pH within the storage). Therefore, it is important to control their metabolism. A possible way to control the metabolism of probiotic in foods is the attenuation through physical or chemical methods. One of the emerging technologies is ultrasound (US). This approach was used to avoid post-acidification in a commercial rice drink (Bevilacqua et al., 2016). The present PhD thesis contributed to evaluate the effects of attenuation with ultrasound, on some technological and probiotic strains, testing three different genera of probiotic strains. Moreover, this PhD project has investigate the changes that may affect probiotic strains after attenuation; the release of proteins, nucleic acids. Finally, the novelty of this PhD thesis was the study of the effects of attenuated strains on gut microbiota. Future trends: A future perspective could be a focus on the use of US to improve or modulate the adhesion of probiotic strains, considering the increase of hydrophobicity and the higher adhesion to Caco-2-cells. It is important to investigate the effects of other attenuated strains on gut microbiota by modulating the variables of the treatment. Key words: Hydrophobicity, acidification, growth, attenuation, gut-microbiota, proteins, nucleic acid, ultrasound, attenuated microorganisms, probiotics, lactic acid bacteria
Use of Central Composite Design in food microbiology: a case study on the effects of secondary phenolis on lactic acid bacteria from olives
No full textThis paper focuses on the use of statistical Design of Experiments (DoE) to investigate the effects of two anti-lactic acid bacteria compounds on growth and metabolism of lactobacilli isolated from Italian table olives.
As phenolic compounds were used p-coumaric and vanillic acids (0.0-0.4%) that were combined with salt (0.0-6.0%) and glucose (0.0-4.0%) through a Central Composite Design. As test organisms were used three strains of Lactobacillus plantarum (5 log cfu/ml); samples were stored at 37° C and cell counts and pH were evaluated periodically.
The growth of lactobacilli was affected in a significant way by salt, p-coumaric and vanillic acids, being the salt the most significant factor after 24 h (short storage time), then replaced by p-coumaric acid. p-coumaric acid played also a significant role on the acidifying ability, expressed as decrease of pH of the medium: microbial metabolism, in fact, appeared as completely inhibited at 0.2% of p-coumaric acid
Comparison of direct microbial count procedures for planktonics and sessiles enumeration.
No full textUltrasound attenuation of lactobacilli and bifidobacteria: Effect on some technological and probiotic properties
No full textAttenuation can be regarded as a tool to modulate the metabolism of probiotic bacteria and, consequently, a strategy to reduce the acidification of the active drinks. Attenuation can be done through chemical and physical approaches and ultrasound (US) is a possibility, previously tested to modulate the metabolism of lactic acid bacteria inoculated in a rice drink, but no data are available on the effect of this treatment of the overall profile of probiotic bacteria. Therefore, the main topic of this paper was to study the effect of US-attenuation on some properties of Lactobacillus reuteri, Lactobacillus plantarum, Bifidobacterium longum, and Bifidobacterium infantis (survival at pH 2, and 2.5, and with 0.3% of bile salt added, hydrophobicity, acidification, and growth at different temperatures, pH or in presence of 7% NaCl). A preliminary screening was done by using 3 power levels (40, 60, and 80%) and 3 different treatment times (2, 4, and 6 min); immediately after sonication, acidification and viable count were tested. The best combination to avoid post-acidification was the following one: power, 60%; time, 6 min; pulse, 2 s. The effect of this combination on the overall profile of the test strains (functional and technological properties) was studied. This combination exerted a positive effect on the hydrophobicity and adhesion to Caco-2 cells of L. reuteri, although the growth at pH 4 was negatively affected. In the other strains, there a was negative effect on acid and bile resistance
Effects of different environmental stresses on cell surface hydrophobicity of lactobacilli, bifidobacteria and propionibacteria
No full textMarinated Sea Bream Fillets Enriched with Lactiplantibacillus plantarum and Bifidobacterium animalis subsp. lactis: Brine Optimization and Product Design
No full textThis study aimed to design marinated sea bream fillets, inoculated with either Lactiplantibacillus plantarum (strains 11, 68, 69) or Bifidobacterium animalis subsp. lactis DSM 10140. In the first step, the optimization of brine composition was performed through a centroid; the factors of the design were citric acid, vinegar, and salt. As a result of optimization, the optimal composition of brine was set to 0.75% citric acid, 55% vinegar, and 3% NaCl. In the second step, sea bream fillets were inoculated with L. plantarum strain 69 and B. animalis subsp. lactis, marinated and then packed in a conditioning solution (oil or diluted brine); the samples were stored at 4 °C for 21 days. The viability of the strains and sensory scores were assessed. The bacteria retained a high viability throughout storage (21 days); however, the sensory scores were at their highest level for 4 days. In particular, sensory assessment suggested a preference for a conditioning solution with oil, rather than with a diluted brine. In addition, a slightly higher preference was found for B. animalis subsp. lactis
Autochthonous lactic acid bacteria with probiotic aptitudes as starter cultures for fish-based products
No full textThis study focused on the selection of lactic starters with probiotic properties for the production of fermented fish-products by the use of a multivariate approach (Cluster Analysis and Principal Component Analysis). Seventy-five isolates were recovered from fish intestinal microbiota and characterized by evaluating phenotypical, technological and probiotic traits; the most promising isolates were molecularly identified and then used into fish fermented sausage production. Namely, data from technological characterization were modelled through Growth Index and used as input to run a preliminary selection. Thus, 15 promising strains were selected and subjected to probiotic characterization; considering the results from probiotic tests, 3 promising strains were finally chosen (11, 68 and 69), identified as members of the genus Lactobacillus and used for the validation at laboratory level through the assessment of their performances for the production of fermented fish sausages. The results were promising as the use of the selected strains reduced the fermentation time (2 days) ensuring a good microbiological quality of the final product
A narrative review on the use of probiotics in several diseases. Evidence and perspectives
Full text linkGut microbiota is a complex ecosystem, strictly linked to health and disease, as a balanced composition (referred as eubiosis) is necessary for several physiological functions, while an unbalanced composition (dysbiosis) is often associated to pathological conditions and/or diseases. An altered microbiota could be positively affected and partially restored through probiotic supplementation, among others. This review addresses the effects of probiotics in several conditions, used as case-studies (colorectal cancer, neuro-psychiatric diseases, intestinal diseases, obesity, diabetes, metabolic syndrome, immune system, and musculoskeletal system disorders) by pointing out the clinical outcomes, the mode of action, mainly related to the production of short chain fatty acids (SCFA), the impact of probiotic dose and mode of supplementation, as well as trying to highlight a hit of the most used genera
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