Fructooligosaccharides Metabolism Research Articles

A γ-glutamylcysteine ligase AcGCL alleviates cadmium-inhibited fructooligosaccharides metabolism by modulating glutathione level in Allium cepa L.

Fructooligosaccharides (FOS) are important carbohydrates in plants. Cadmium (Cd) toxicity limits growth and development in several plant species. Whether FOS metabolism is affected by Cd and the molecular mechanisms of tolerance of the effects of Cd toxicity in plants remain enigmatic. In the present study, FOS metabolism was analyzed under Cd stress in onion (Allium cepa L.). Results showed that Cd stress can inhibit FOS accumulation in onion, followed by the upregulation of a putative onion γ-glutamylcysteine ligase gene AcGCL. Heterologous expression of the AcGCL protein in Escherichia coli revealed that this recombinant enzyme has GCL activity. Furthermore, overexpressing AcGCL significantly increased glutathione (GSH) accumulation in young onion roots under Cd treatment, accompanied by increased phytochelatin (PC) amount, and increased transcript expression of GSH synthetase (GS), and phytochelatin synthase (PCS) genes. Notably, compared with control, overexpressing AcGCL ameliorated Cd phytotoxicity on onion FOS metabolism, which correlated with increased FOS synthesis. Taken together, these results suggest that the function of AcGCL as a γ-glutamylcysteine ligase can alleviate Cd inhibited FOS metabolism by modulating GSH levels in onion.

The local transcriptional regulators SacR1 and SacR2 act as repressors of fructooligosaccharides metabolism in Lactobacillus plantarum

BackgroundIn Lactobacillus plantarum, fructooligosaccharides (FOS) metabolism is controlled by both global and local regulatory mechanisms. Although catabolite control protein A has been identified as a global regulator of FOS metabolism, the functions of local regulators remain unclear. This study aimed to elucidate the roles of two local regulators, SacR1 and SacR2, in the regulation of FOS metabolism in L. plantarum both in vitro and in vivo.ResultsThe inactivation of sacR1 and sacR2 affected the growth and production of metabolites for strains grown on FOS or glucose, respectively. A reverse transcription-quantitative PCR analysis of one wild-type and two mutant strains (ΔsacR1 and ΔsacR2) of L. plantarum identified SacR1 and SacR2 as repressors of genes relevant to FOS metabolism in the absence of FOS, and these genes could be induced or derepressed by the addition of FOS. The analysis predicted four potential transcription factor binding sites (TFBSs) in the putative promoter regions of two FOS-related clusters. The binding of SacR1 and SacR2 to these TFBSs both in vitro and in vivo was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation, respectively. A consensus sequence of WNNNNNAACGNNTTNNNNNW was deduced for the TFBSs of SacR1 and SacR2.ConclusionOur results identified SacR1 and SacR2 as local repressors for FOS metabolism in L. plantarum. The regulation is achieved by the binding of SacR1 and SacR2 to TFBSs in the promoter regions of FOS-related clusters. The results provide new insights into the complex network regulating oligosaccharide metabolism by lactic acid bacteria.

Functional analysis of the role of CcpA in Lactobacillus plantarum grown on fructooligosaccharides or glucose: a transcriptomic perspective

BackgroundThe catabolite control protein A (CcpA) is a master regulator of many important cellular processes in Gram-positive bacteria. In Lactobacillus plantarum, CcpA directly or indirectly controls the transcription of a large number of genes that are involved in carbohydrate metabolism, aerobic and anaerobic growth, stress response and metabolite production, but its role in response to different carbon sources remains unclear.ResultsHere a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth phase of wild-type and ccpA mutant strains of L. plantarum ST-III using fructooligosaccharides (FOS) or glucose as the sole carbon source. The inactivation of ccpA significantly affected the growth and production of metabolites under both carbon sources. About 15% of the total genes were significantly altered between wild-type and ccpA strains grown on glucose and the value is deceased to 12% when these two strains were compared on FOS, while only 7% were obviously changed due to the loss of CcpA when comparing strains grown on glucose and FOS. Although most of the differentially expressed genes mediated by CcpA are glucose dependent, FOS can also induce carbon catabolite repression (CCR) through the CcpA pathway. Moreover, the inactivation of ccpA led to a transformation from homolactic fermentation to mixed fermentation under aerobic conditions. CcpA can control genes directly by binding in the regulatory region of the target genes (mixed fermentation), indirectly through local regulators (fatty acid biosynthesis), or have a double effect via direct and indirect regulation (FOS metabolism).ConclusionOverall, our results show that CcpA plays a central role in response to carbon source and availability of L. plantarum and provide new insights into the complex and extended regulatory network of lactic acid bacteria.

CcpA-Dependent Carbon Catabolite Repression Regulates Fructooligosaccharides Metabolism in Lactobacillus plantarum

Fructooligosaccharides (FOSs) metabolism in Lactobacillus plantarum is controlled by two gene clusters, and the global regulator catabolite control protein A (CcpA) may be involved in the regulation. To understand the mechanism, this study focused on the regulation relationships of CcpA toward target genes and the binding effects on the catabolite responsive element (cre). First, reverse transcription-PCR analysis of the transcriptional organization of the FOS-related gene clusters showed that they were organized in three independent polycistronic units. Diauxic growth, hierarchical utilization of carbohydrates and repression of FOS-related genes were observed in cultures containing FOS and glucose, suggesting carbon catabolite repression (CCR) control in FOS utilization. Knockout of ccpA gene eliminated these phenomena, indicating the principal role of this gene in CCR of FOS metabolism. Furthermore, six potential cre sites for CcpA binding were predicted in the regions of putative promoters of the two clusters. Direct binding was confirmed by electrophoretic mobility shift assays in vitro and chromatin immunoprecipitation in vivo. The results of the above studies suggest that CcpA is a vital regulator of FOS metabolism in L. plantarum and that CcpA-dependent CCR regulates FOS metabolism through the direct binding of CcpA toward the cre sites in the promoter regions of FOS-related clusters.

Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity.

Although fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a β-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome- and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum.

Fructo-oligosaccharide effects on serum cholesterol levels. An overview.

To address the effects of fructooligosaccharides (FOS) intake on serum cholesterol levels. We performed a search for scientific articles in MEDLINE database from 1987 to 2014, using the following English keywords: fructooligosaccharides; fructooligosaccharides and cholesterol. A total of 493 articles were found. After careful selection and exclusion of duplicate articles 34 references were selected. Revised texts were divided into two topics: "FOS Metabolism" and "FOS effects on plasma cholesterol." The use of a FOS diet prevented some lipid disorders and lowered fatty acid synthase activity in the liver in insulin-resistant rats. There was also reduction in weight and total cholesterol in beagle dogs on a calorie-restricted diet enriched with short-chain FOS. Another study found that 2g FOS daily consumption increased significantly serum HDL cholesterol levels but did not ensure a significant reduction in levels of total cholesterol and triglycerides.. Patients with mild hypercholesterolemia receiving short-chain FOS 10.6g daily presented no statistically significant reduction in serum cholesterol levels. However, when FOS was offered to patients that changed their lifestyle, the reduction of LDL cholesterol and steatosis was higher. Fructooligosaccharides intake may have a beneficial effect on lipid metabolism and regulation of serum cholesterol levels in individuals that change their lifestyle. FOS supplementation use in diets may therefore be a strategy for lowering cholesterol.

Fructo-oligosaccharide effects on blood glucose: an overview

To identify the current status of scientific knowledge in fructo-oligosaccharides (FOS), non-conventional sugars that play an important role in glycemia control. We performed a search for scientific articles in MEDLINE and LILACS databases, from January 1962 to December 2011, using English/Portuguese key words: "blood glucose/glicemia", "prebiotics/prebióticos" and "dietary fiber/fibras na dieta". From an initial number of 434 references, some repeated, 43 references published from 1962 to 2011 were included in this study. The selected texts were distributed in three topics: (1) metabolism of FOS, (2) FOS and experimental studies involving glucose and (3) human studies involving glucose and FOS. Five studies have shown that the use of FOS reduces the fecal content and increases intestinal transit time. Experimental studies have shown that dietary supplementation with high doses (60 g/Kg) of propionate, a short-chain fatty acid decreased glycemia. The use of lower doses (3 g/kg) did not produce the same results. Study in subjects with diabetes type II showed that the addition of 8 grams of FOS in the diet for 14 days, caused a reduction in serum glucose. In another study with healthy subjects, there were no changes in glycemic control. This review demonstrates that consumption of FOS has a beneficial influence on glucose metabolism. The controversies appear to be due to inadequate methodological designs and/or the small number of individuals included in some studies.

Fructooligosaccharides metabolism and effect on bacteriocin production in Lactobacillus strains isolated from ensiled corn and molasses

Fructo- (FOS) and galacto-oligosaccharides have been used to promote the growth of probiotics, mainly those from Lactobacillus genus. However, only few reports have evaluated the effect of prebiotics on bacteriocins activity and production. In this work, we characterized the effect of FOS supplementation on the growth, lactic and acetic acids production, and antimicrobial activity of crude extracts obtained from Lactobacillus strains isolated from ensiled corn and molasses. Seven out of 28 isolated Lactobacillus, belonging to Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus brevis, showed antimicrobial activity against Listeria innocua. Among them, the strain L. plantarum LE5 showed antimicrobial activity against Listeria monocytogenes and Enteroccocus faecalis; while the L. plantarum LE27 strain showed antimicrobial effect against L. monocytogenes, E. faecalis, Escherichia coli and Salmonella enteritidis. This antimicrobial activity in most of the cases was obtained only after FOS supplementation. In summary, these results show the feasibility to increase the antimicrobial activity of Lactobacillus bacteriocins by supplementing the growth medium with FOS.

Identification of a Putative Operon Involved in Fructooligosaccharide Utilization by Lactobacillus paracasei

The growth and activity of some Lactobacillus and Bifidobacterium strains are stimulated by the presence of nondigestible fructooligosaccharides (FOS), which are selectively fermented by specific intestinal bacteria. Consumption of FOS, therefore, enriches for those bacteria that possess metabolic pathways necessary for FOS metabolism. In this study, a DNA microarray consisting of 7,680 random genomic library fragments of Lactobacillus paracasei 1195 was used to examine genes involved in the utilization of FOS in this organism. Differential expression profiles between cells grown on FOS and those grown on glucose provided a basis for identifying genes specifically induced by FOS. Several of the FOS-induced genes shared sequence identity with genes encoding beta-fructosidases and components of phosphoenolpyruvate-dependent phosphotransferase systems (PTS). These genes were organized in a putative operon, designated the fos operon, that may play an essential role in FOS utilization. The complete 7,631-bp nucleotide sequence of the putative fos operon was determined and consists of fosABCDXE genes, which encode a putative fructose/mannose PTS (FosABCDX) and a beta-fructosidase precursor (FosE). The latter contains an N-terminal signal peptide sequence and cell wall sorting signals at the C-terminal region, suggesting its localization at the cell wall. Inactivation of the fosE gene led to impaired growth on FOS and other beta-fructose-linked carbohydrates. Transcriptional analysis by reverse transcriptase PCR suggested that fosABCDXE was cotranscribed as a single mRNA during growth on FOS. Expression array analysis revealed that when glucose was added to FOS-grown cells, transcription of the FOS-induced genes was repressed, indicating that FOS metabolism is subject to catabolite regulation.

The Metabolism of the Fructooligosaccharides in Onion Bulbs: A Comprehensive Review

In the past two decades, there has been vast expansion in the research of fructooligosaccharides (FOS), including their chemistry, biochemistry and enzymology in higher plants. However, in spite of these considerable advances in fructan science, many other aspects of the mechanisms of fructan metabolism have not been fully understood. The bulbing and yield of dry onion cultivars vary, but they depend on the contents of the high dry matter and non-structural carbohydrates (fructooligosaccharides) which contribute to keepa- bility. The knowledge of the mechanisms of the synthesis and the degradation of the FOS occurring during growth and storage are of great interest. Important progress has been made in the research area of onion FOS, and in addition to their role as accessible reserve carbohydrates in bulbs during sprouting, FOS partici- pate in many physiological processes of production, protection and preservation of the bulbs. This review aims to contribute to a better understanding of the metabolism of FOS in onion bulbs, based on recent inves- tigations. The activities of the main enzymes involved in the synthesis and the hydrolysis of the FOS during the pre-harvest (growth) and post-harvest (storage) life of the bulbs are reviewed, although present knowledge is too limited to explain clearly the mechanisms triggering the enzymes activities or the mechanisms by which FOS contribute to quality and long keepability of the bulbs.

Metabolism of fructooligosaccharides by Lactobacillus paracasei 1195.

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [(3)H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) alpha-1,2 linked to two, three, or four fructose (F) units (GF(2), GF(3), and GF(4), respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF(2) and GF(3) was rapid, whereas little GF(4) uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.

Metabolism of fructo-oligosaccharides by Bifidobacterium spp.

Bifidobacterium adolescentis ATCC 15703, B. longum ATCC 15707, and B. thermophilum ATCC 25525 were examined for the ability to grow with fructo-oligosaccharides (FOS) as carbohydrate sources. The three species produced cell-associated β-fructosidases (inulinases) capable of hydrolysing FOS. Maximum activity was obtained with short-chain FOS with degrees of polymerization (DP) of between three and five (neosugars). The B. thermophilum inulinase was induced by inulin, a long-chain FOS with DP=35, while the enzymes from the other two strains were constitutive. Production of inulinase by all three strains was regulated by catabolite repression. Inulinase activity of the three Bifidobacterium spp. was similar when grown with 0.5% inulin as the carbohydrate source; however, B. thermophilum grew much more rapidly. All three strains utilized crude Jerusalem artichoke flour (JAF) as a carbohydrate source, suggesting that JAF might have commercial application as a food or feed additive to stimulate bifidobacteria in the gut.