Nutraceutical approach to the modulation of cholesterol metabolism: in vitro and in vivo studies

Negli ultimi anni, si sono diffusi nuove strategie per il trattamento delle malattie cardiovascolari, che possano supportare una terapia medica, o in alcuni casi, sostituirla. Infatti, l’abbandono delle terapie è il più importante problema di salute pubblica del mondo occidentale, soprattutto per le malattie croniche. Ciò è dovuto alla complessità delle terapie farmacologiche e ai numerosi e in alcuni casi gravi effetti collaterali dei farmaci somministrati. Di conseguenza, una riduzione di questi effetti migliorerebbe le condizioni di vita del paziente e quindi diminuirebbe il rischio di abbandono della terapia. Per ottenere ciò, è possibile affiancare al trattamento farmacologico una terapia nutraceutica, consistente nella somministrazione di complessi molecolari o microorganismi, provenienti da piante, latte o cibi funzionali. Lo scopo generale di questo studio è indagare le attività ipolipidemizzanti di un composto nutraceutico e di un ceppo batterio specifico nel modello animale che presenta elevati alti livelli plasmatici di colesterolo. Inoltre, sono stati analizzati gli effetti del trattamento nutraceutico sui meccanismi fisiologici che contrastano la creazione della placca aterosclerotica come l’efflusso di colesterolo dalle “foam cells” presenti nell’ateroma, o la riduzione dell’assorbimento intestinale di colesterolo. La presente tesi è divisa in due parti. Nella prima parte, abbiamo analizzato la capacità dei Bifidobacteria di ridurre i livelli di colesterolo nel medium di crescita. Dall’analisi, si è osservato che vari ceppi del genere Bifidobacteria presentano un’ampia capacità di assimilazione del colesterolo all’interno della cellula batterica, in particolare il Bifidobacterium bifidum PRL2010. Le analisi di trascrittomica del Bb PRL2010 incubato in presenza di colesterolo, hanno rivelato un significativo aumento dei livelli di trascrizione di geni codificanti trasportatori e riduttasi, responsabili del meccanismo di accumulo all’interno della cellula batterica e della conversione del colesterolo in coprostanolo. L’attività ipolipidemizzante del Bb PRL2010 è stata poi valutata nel modello murino, mostrando la modificazione del microbiota dei topi trattati dopo somministrazione del batterio in questione. Nella seconda parte del progetto di ricerca, abbiamo indagato sugli effetti di un composto coperto da brevetto, chiamato “Ola”, sull’efflusso di colesterolo di criceti trattati con questo composto nutraceutico. L’efflusso di colesterolo è il primo step del meccanismo fisiologico noto come Trasporto Inverso del Colesterolo, che consente l’eliminazione del colesterolo dalle placche aterosclerotiche, attraverso l’interazione fra le HDL, presenti nella circolazione sanguigna, e specifici trasportatori delle foam cells, come ABCA1/G1 e SR-BI. In seguito, le lipoproteine rilasciano il colesterolo alle cellule epatiche, dove è metabolizzato ed escreto attraverso le feci. Per valutare l’effetto dell’Ola sul profilo lipidico dei criceti, sono state condotte analisi in vitro. I risultati mostrano un aumento dell’efflusso di colesterolo in cellule che esprimono il trasportatore ABCA1, comparato con il gruppo controllo. Questi due studi mostrano come l’approccio nutraceutico può essere un importante modo per contrastare l’aterosclerosi. Come mostrato in letteratura, gli effetti dei composti nutraceutici sull’aterosclerosi e su altre malattie croniche, hanno portato a un ampio uso come supporto alle terapie farmacologiche, ed in alcuni casi hanno rimpiazzato la terapia farmacologica stessa.

Characterization of virus-like particles associated with the human faecal and caecal microbiota

This work represents an investigation into the presence, abundance and diversity of virus-like particles (VLPs) associated with human faecal and caecal samples. Various methodologies for the recovery of VLPs from faeces were tested and optimized, including successful down-stream processing of such samples for the purpose of an in-depth electron microscopic analysis, pulsed-field gel electrophoresis and efficient DNA recovery. The applicability of the developed VLP characterization method beyond the use of faecal samples was then verified using samples obtained from human caecal fluid.

Avaliação do efeito de um probiótico na performance zootécnica e microbiota entérica em coelhos (Oryctolagus cuniculus) de produção

A cunicultura é uma atividade pecuária em crescente desenvolvimento e isso traduz-se em novos desafios. Durante muitos anos a criação de coelhos recorreu em demasia ao uso de antimicrobianos com o objetivo de tratar e prevenir o aparecimento de diversas doenças. Paralelamente, estes compostos foram também usados como “promotores de crescimento”, visando essencialmente uma melhoria da eficiência digestiva. Porém, o uso indiscriminado destas substâncias levantou questões de saúde pública, como a emergência de estirpes bacterianas multirresistentes e a inerente disseminação de genes de resistência, possivelmente transferíveis ao Homem através da cadeia alimentar. No presente, existe uma enorme pressão para a adoção de estratégias que possibilitem uma redução massiva na quantidade de antimicrobianos administrados a espécies pecuárias. Este trabalho visou contribuir para o estudo de uma alternativa ao uso de antimicrobianos - os probióticos – enquanto suplementos alimentares constituídos por microrganismos vivos capazes de equilibrar a microbiota intestinal do hospedeiro. Para tal, foram constituídos dois grupos de coelhos com base na alimentação: i) grupo antibiótico, com acesso a um alimento composto suplementado com antibióticos e ii) o grupo probiótico alimentado com a mesma dieta, mas sem antibióticos e inoculado com um probiótico constituído por Escherichia coli e Enterococcus spp.. Ao longo de 22 dias de estudo foram monitorizados alguns indicadores produtivos e efetuadas recolhas periódicas de fezes para estudo microbiológico. A análise dos resultados zootécnicos permitiram verificar que o uso de probióticos em detrimento de antibióticos parece promover o crescimento de coelhos, tornando-se um método mais rentável na produção cunícula. Através de genotipagem por ERIC-PCR e PFGE, pretendeu-se verificar se as estirpes estranhas ao trato gastrointestinal dos coelhos seriam capazes de coloniza-lo, permanecendo ao longo do tempo de estudo. O facto de as estirpes inoculadas no probiótico terem sido encontradas ao longo dos dias de estudo nos coelhos aos quais foram administradas, sugere que os efeitos observados na performance zootécnica estejam relacionados com as estirpes administradas no probiótico, pelo que este poderá ser um sistema viável na substituição de antibióticos na alimentação de coelhos de produção.

Infection by Cryptosporidium parvum in renal patients submitted to renal transplant or hemodialysis

The frequency of infection by Cryptosporidium parvum was determined in two groups of renal patients submitted to immunosuppression. One group consisted of 23 renal transplanted individuals, and the other consisted of 32 patients with chronic renal insufficiency, periodically submitted to hemodialysis. A third group of 27 patients with systemic arterial hypertension, not immunosuppressed, was used as control. During a period of 18 months all the patients were submitted to faecal examination to detect C. parvum oocysts, for a total of 1 to 6 tests per patient. The results showed frequencies of C. parvum infection of 34.8%, 25% and 17.4%, respectively, for the renal transplanted group, the patients submitted to hemodialysis and the control group. Statistical analysis showed no significant differences among the three groups even though the frequency of C. parvum infection was higher in the transplanted group. However, when the number of fecal samples containing C. parvum oocysts was taken in account, a significantly higher frequency was found in the renal transplanted group.

In vitro fermentation of carbohydrates by porcine faecal inocula and their influence on Salmonella Typhimurium growth in batch culture systems

The aim of this study was to evaluate in vitro the influence of fermentable carbohydrates on the activity of porcine microbiota and survival of Salmonella Typhimurium in a batch culture system simulating the porcine hindgut. The carbohydrates tested were xylooligosaccharides, a mixture of fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). These ingredients stimulated the growth of selected Bifidobacterium and Lactobacillus species in pure cultures. In batch cultures, the carbohydrates influenced some fermentation parameters. For example, GEO and FIN significantly increased lactic acids compared with the control (no added carbohydrate). With the exception of LAC, the test carbohydrates increased the production of short-chain fatty acid (SCFA) and modified SCFA profiles. Quantitative analysis of bacterial populations by FISH revealed increased counts of the Bifidobacterium group compared with control and, with exception of FOS, increased Lactobacillus, Leuconostoc and Weissella spp. counts. Salmonella numbers were the lowest during the fermentation of LAC. This work has looked at carbohydrate metabolism by porcine microbiota in a pH-controlled batch fermentation system. It provides an initial model to analyse interactions with pathogens.

What do we mean when we refer to Bacteroidetes populations in the human gastrointestinal microbiota?

Recent large-scale cloning studies have shown that the ratio of Bacteroidetes to Firmicutes may be important in the obesity-associated gut microbiota, but the species these phyla represent in this ecosystem has not been examined. The Bacteroidetes data from the recent Turnbaugh study were examined to determine those members of the phylum detected in human faecal samples. In addition, FISH analysis was performed on faecal samples from 17 healthy, nonobese donors using probe Bac303, routinely used by gut microbiologists to enumerate BacteroidesPrevotella populations in faecal samples, and another probe (CFB286) whose target range has some overlap with that of Bac303. Sequence analysis of the Turnbaugh data showed that 23/519 clones were chimeras or erroneous sequences; all good sequences were related to species of the order Bacteroidales, but no one species was present in all donors. FISH analysis demonstrated that approximately one-quarter of the healthy, nonobese donors harboured high numbers of Bacteroidales not detected by probe Bac303. It is clear that Bacteroidales populations in human faecal samples have been underestimated in FISH-based studies. New probes and complementary primer sets should be designed to examine numerical and compositional changes in the Bacteroidales during dietary interventions and in studies of the obesity-associated microbiota in humans and animal model systems.

Whole-grain wheat breakfast cereal has a prebiotic effect on the human gut microbiota: a double-blind, placebo-controlled, crossover study

Epidemiological studies have shown an inverse association between dietary intake of whole grains and the risk of chronic disease. This may be related to the ability to mediate a prebiotic modulation of gut microbiota. However, no studies have been conducted on the microbiota modulatory capability of whole-grain (WG) cereals. In the present study, the impact of WG wheat on the human intestinal microbiota compared to wheat bran (WB) was determined. A double-blind, randomised, crossover study was carried out in thirty-one volunteers who were randomised into two groups and consumed daily 48g breakfast cereals, either WG or WB, in two 3-week study periods, separated by a 2-week washout period. Numbers of faecal bifidobacteria and lactobacilli (the target genera for prebiotic intake), were significantly higher upon WG ingestion compared with WB. Ingestion of both breakfast cereals resulted in a significant increase in ferulic acid concentrations in blood but no discernible difference in faeces or urine. No significant differences in faecal SCFA, fasting blood glucose, insulin, total cholesterol (TC), TAG or HDL-cholesterol were observed upon ingestion of WG compared with WB. However, a significant reduction in TC was observed in volunteers in the top quartile of TC concentrations upon ingestion of either cereal. No adverse intestinal symptoms were reported and WB ingestion increased stool frequency. Daily consumption of WG wheat exerted a pronounced prebiotic effect on the human gut microbiota composition. This prebiotic activity may contribute towards the beneficial physiological effects of WG wheat.

The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota.

We have examined the gut bacterial metabolism of pomegranate by-product (POMx) and major pomegranate polyphenols, punicalagins, using pH-controlled, stirred, batch culture fermentation systems reflective of the distal region of the human large intestine. Incubation of POMx or punicalagins with faecal bacteria resulted in formation of the dibenzopyranone-type urolithins. The time course profile confirmed the tetrahydroxylated urolithin D as the first product of microbial transformation, followed by compounds with decreasing number of phenolic hydroxy groups: the trihydroxy analogue urolithin C and dihydroxylated urolithin A. POMx exposure enhanced the growth of total bacteria, Bifidobacterium spp. and Lactobacillus spp., without influencing the Clostridium coccoides–Eubacterium rectale group and the C. histolyticum group. In addition, POMx increased concentrations of short chain fatty acids (SCFA) viz. acetate, propionate and butyrate in the fermentation medium. Punicalagins did not affect the growth of bacteria or production of SCFA. The results suggest that POMx oligomers, composed of gallic acid, ellagic acid and glucose units, may account for the enhanced growth of probiotic bacteria.

A human volunteer study to assess the impact of confectionery sweeteners on the gut microbiota composition.

Sweeteners are being sourced to lower the energetic value of confectionery including chocolates. Some, especially non-digestible carbohydrates, may possess other benefits for human health upon their fermentation by the colonic microbiota. The present study assessed non-digestible carbohydrate sweeteners, selected for use in low-energy chocolates, for their ability to beneficially modulate faecal bacterial profiles in human volunteers. Forty volunteers consumed a test chocolate (low-energy or experimental chocolate) containing 22·8 g of maltitol (MTL), MTL and polydextrose (PDX), or MTL and resistant starch for fourteen consecutive days. The dose of the test chocolates was doubled every 2 weeks over a 6-week period. Numbers of faecal bifidobacteria significantly increased with all the three test treatments. Chocolate containing the PDX blend also significantly increased faecal lactobacilli (P = 0·00 001) after the 6 weeks. The PDX blend also showed significant increases in faecal propionate and butyrate (P = 0·002 and 0·006, respectively). All the test chocolates were well tolerated with no significant change in bowel habit or intestinal symptoms even at a daily dose of 45·6 g of non-digestible carbohydrate sweetener. This is of importance not only for giving manufacturers a sugar replacement that can reduce energetic content, but also for providing a well-tolerated means of delivering high levels of non-digestible carbohydrates into the colon, bringing about improvements in the biomarkers of gut health.

The type and quantity of dietary fat and carbohydrate alter faecal microbiome and short-chain fatty acid excretion in a metabolic syndrome ‘at-risk’ population syndrome.

An obese-type human microbiota with an increased Firmicutes:Bacteroidetes ratio has been described that may link the gut microbiome with obesity and metabolic syndrome (MetS) development. Dietary fat and carbohydrate are modifiable risk factors that may impact on MetS by altering the human microbiome composition. We determined the effect of the amount and type of dietary fat and carbohydrate on faecal bacteria and short chain fatty acid (SCFA) concentrations in people ‘at risk’ of MetS.

In vitro batch cultures of gut microbiota from healthy and ulcerative colitis (UC) subjects suggest that sulphate-reducing bacteria levels are raised in UC and by a protein-rich diet.

Imbalances in gut microbiota composition during ulcerative colitis (UC) indicate a role for the microbiota in propagating the disorder. Such effects were investigated using in vitro batch cultures (with/without mucin, peptone or starch) inoculated with faecal slurries from healthy or UC patients; the growth of five bacterial groups was monitored along with short-chain fatty acid (SCFA) production. Healthy cultures gave two-fold higher growth and SCFA levels with up to ten-fold higher butyrate production. Starch gave the highest growth and SCFA production (particularly butyrate), indicating starch-enhanced saccharolytic activity. Sulphate-reducing bacteria (SRB) were the predominant bacterial group (of five examined) for UC inocula whereas they were the minority group for the healthy inocula. Furthermore, SRB growth was stimulated by peptone presumably due to the presence of sulphur-rich amino acids. The results suggest raised SRB levels in UC, which could contribute to the condition through release of toxic sulphide.

Influence of galacto-oligosaccharide mixture (B-GOS) on gut microbiota, immune parameters and metabonomics in elderly persons

It is recognised that ageing induces various changes to the human colonic microbiota. Most relevant is a reduction in bifidobacteria, which is a health-positive genus. Prebiotics, such as galacto-oligosaccharides (GOS), are dietary ingredients that selectively fortify beneficial gut microbial groups. Therefore, they have the potential to reverse the age-related decline in bifidobacteria and modulate associated health parameters. We assessed the effect of GOS mixture (Bimuno (B-GOS)) on gut microbiota, markers of immune function and metabolites in forty elderly (age 65-80 years) volunteers in a randomised, double-blind, placebo (maltodextrin)-controlled, cross-over study. The intervention periods consisted of 10 weeks with daily doses of 5·5 g/d with a 4-week washout period in between. Blood and faecal samples were collected for the analyses of faecal bacterial populations and immune and metabolic biomarkers. B-GOS consumption led to significant increases in bacteroides and bifidobacteria, the latter correlating with increased lactic acid in faecal waters. Higher IL-10, IL-8, natural killer cell activity and C-reactive protein and lower IL-1β were also observed. Administration of B-GOS to elderly volunteers may be useful in positively affecting the microbiota and some markers of immune function associated with ageing.

Impact of palm date consumption on microbiota growth and large intestinal health: a randomised, controlled, cross-over, human intervention study

The reported inverse association between the intake of plant-based foods and a reduction in the prevalence of colorectal cancer may be partly mediated by interactions between insoluble fibre and (poly)phenols and the intestinal microbiota. In the present study, we assessed the impact of palm date consumption, rich in both polyphenols and fibre, on the growth of colonic microbiota and markers of colon cancer risk in a randomised, controlled, cross-over human intervention study. A total of twenty-two healthy human volunteers were randomly assigned to either a control group (maltodextrin-dextrose, 37·1 g) or an intervention group (seven dates, approximately 50 g). Each arm was of 21 d duration and was separated by a 14-d washout period in a cross-over manner. Changes in the growth of microbiota were assessed by fluorescence in situ hybridisation analysis, whereas SCFA levels were assessed using HPLC. Further, ammonia concentrations, faecal water genotoxicity and anti-proliferation ability were also assessed using different assays, which included cell work and the Comet assay. Accordingly, dietary intakes, anthropometric measurements and bowel movement assessment were also carried out. Although the consumption of dates did not induce significant changes in the growth of select bacterial groups or SCFA, there were significant increases in bowel movements and stool frequency (P<0·01; n 21) and significant reductions in stool ammonia concentration (P<0·05; n 21) after consumption of dates, relative to baseline. Furthermore, date fruit intake significantly reduced genotoxicity in human faecal water relative to control (P<0·01; n 21). Our data indicate that consumption of date fruit may reduce colon cancer risk without inducing changes in the microbiota.

Impact of increasing fruit and vegetables and flavonoid intake on the human gut microbiota

Epidemiological studies have shown protective effects of fruits and vegetables (F&V) in lowering the risk of developing cardiovascular diseases (CVD) and cancers. Plant-derived dietary fibre (non-digestible polysaccharides) and/or flavonoids may mediate the observed protective effects particularly through their interaction with the gut microbiota. The aim of this study was to assess the impact of fruit and vegetable (F&V) intake on gut microbiota, with an emphasis on the role of flavonoids, and further to explore relationships between microbiota and factors associated with CVD risk. In the study, a parallel design with 3 study groups, participants in the two intervention groups representing high-flavonoid (HF) and low flavonoid (LF) intakes were asked to increase their daily F&V intake by 2, 4 and 6 portions for a duration of 6 weeks each, while a third (control) group continued with their habitual diet. Faecal samples were collected at baseline and after each dose from 122 subjects. Faecal bacteria enumeration was performed by fluorescence in situ hybridisation (FISH). Correlations of dietary components, flavonoid intake and markers of CVD with bacterial numbers were also performed. A significant dose X treatment interaction was only found for Clostidium leptum-Ruminococcus bromii/flavefaciens with a significant increase after intake of 6 additional portions in the LF group. Correlation analysis of the data from all 122 subjects independent from dietary intervention indicated an inhibitory role of F&V intake, flavonoid content and sugars against the growth of potentially pathogenic clostridia. Additionally, we observed associations between certain bacterial populations and CVD risk factors including plasma TNF-α, plasma lipids and BMI/waist circumference.

The gut microbiota: a major player in the toxicity of environmental pollutants?

Exposure to environmental chemicals has been linked to various health disorders, including obesity, type 2 diabetes, cancer and dysregulation of the immune and reproductive systems, whereas the gastrointestinal microbiota critically contributes to a variety of host metabolic and immune functions. We aimed to evaluate the bidirectional relationship between gut bacteria and environmental pollutants and to assess the toxicological relevance of the bacteria–xenobiotic interplay for the host. We examined studies using isolated bacteria, faecal or caecal suspensions—germ-free or antibiotic-treated animals—as well as animals reassociated with a microbiota exposed to environmental chemicals. The literature indicates that gut microbes have an extensive capacity to metabolise environmental chemicals that can be classified in five core enzymatic families (azoreductases, nitroreductases, β-glucuronidases, sulfatases and β-lyases) unequivocally involved in the metabolism of >30 environmental contaminants. There is clear evidence that bacteria-dependent metabolism of pollutants modulates the toxicity for the host. Conversely, environmental contaminants from various chemical families have been shown to alter the composition and/or the metabolic activity of the gastrointestinal bacteria, which may be an important factor contributing to shape an individual’s microbiotype. The physiological consequences of these alterations have not been studied in details but pollutant-induced alterations of the gut bacteria are likely to contribute to their toxicity. In conclusion, there is a body of evidence suggesting that gut microbiota are a major, yet underestimated element that must be considered to fully evaluate the toxicity of environmental contaminants.