Analysis Of Short-chain Fatty Acids Research Articles

Metatranscriptome analysis of the microbial fermentation of dietary milk proteins in the murine gut

Undigestible food ingredients are converted by the microbiota into a large range of metabolites, predominated by short chain fatty acids (SCFA). These microbial metabolites are subsequently available for absorption by the host mucosa and can serve as an energy source. Amino acids fermentation by the microbiota expands the spectrum of fermentation end-products beyond acetate, propionate and butyrate, to include in particular branched-SCFA. Here the long-term effects of high protein-diets on microbial community composition and functionality in mice were analyzed. Determinations of the microbiota composition using phylogenetic microarray (MITChip) technology were complemented with metatranscriptome and SCFA analyses to obtain insight in in situ expression of protein fermentation pathways and the phylogenetic groups involved. High protein diets led to increased luminal concentrations of branched-SCFA, in accordance with protein fermentation in the gut. Bacteria dominantly participating in protein catabolism belonged to the Lachnospiraceae, Erysipelotrichaceae and Clostridiaceae families in both normal- and high- protein diet regimes. This study identifies the microbial groups involved in protein catabolism in the intestine and underpins the value of in situ metatranscriptome analyses as an approach to decipher locally active metabolic networks and pathways as a function of the dietary regime, as well as the phylogeny of the microorganisms executing them.

Tributyl phosphate assisted hollow-fiber liquid-phase microextraction of short-chain fatty acids in microbial degradation fluid using capillary electrophoresis-contactless coupled conductivity detection

A tributyl phosphate assisted hollow-fiber liquid-phase microextraction coupled with capillary electrophoresis-contactless coupled conductivity detection (HF-LPME/CE-C4D) method has been developed for trace analysis of common short-chain fatty acids (SCFAs) without derivatization. Under the optimum conditions, ten SCFAs including a pair of isomers were well separated from their homologous FAs and the main coexisting inorganic anions within 40 min. Tributyl phosphate assisted HF-LPME produced excellent purification and enrichment for the model sample with high-salt matrix, microbial degradation fluid, and the limits of detection could reach 0.072–0.67 ng/mL (S/N = 3). Owing to its high sensitivity, good linearity, and acceptable recovery, this proposed method provided a sensitive and environment-friendly alternative for trace analysis of SCFAs in complicated samples.

P.1.020 - Targeting the gut microbiome to reverse microglia activation and stress-induced immune priming in ageing

Ageing is associated with an increased neuroinflammation and a deteriorating brain function. Gastrointestinal microbiota has been implicated as key mediators in the communication between the gut and the brain [1], and regulating microglia function and maturation [2]. A dysregulation of this communication may be critical in mediating age-related decline in brain physiology. Thus, targeting the gut microbiota by prebiotics, non-digestible fibres fermented by colonic bacteria, may alter microglia activation state and brain function in ageing. The aim of this study was to determine if administration of prebiotics affect age-related changes in gut microbiota profile, stress and immune function. Male young adult (8 weeks) and middle-aged C57BL/6 mice (10 months) received chow enriched with 10% oligofructose-enriched inulin (FOS-Inulin: mixture of 92±2% Inulin and 8±2% Fructooligosaccharide, Orafti ® Synergy1; Beneo/Belgium) or control chow (CTR) for 12 weeks. After 3 weeks of diet intervention the effects on stress and immunity were assessed. Microglia activation and peripheral immune cell activation were investigated by flow cytometry. Caecal samples were collected for 16S bacterial rRNA sequencing and short-chain fatty acid analysis. Parametric data was analysed by one-way ANOVA post-hoc Tukey, non-parametric data by Kruskal-Wallis post-hoc Dunn’s. FOS-Inulin profoundly altered the caecal gut microbiota in young adult and middle-aged mice with significant changes of the gut microbiota profile on the family (Verrucomicrobiaceae, Kruskal-Wallis p=0.0002, Prevotellaceae p=0.0015, Rikenallaceae p=0.0002) and genus level (Akkermansia p=0.0006, Alistipes p=0.0004, Bacteroides, p=0.0003). Moreover, beta-diversity analysis suggests that FOS-Inulin drives gut microbiota changes reversing the aging microbiota phenotype towards a young adult phenotype. This was associated with an altered caecal short chain fatty acid profile and an increased caecum size in young adult and middle-aged mice (one-way ANOVA F= F (3, 35) = 23.53, p Targeting the microbiome with prebiotics altered the gut microbiota profile in young adult, but also in middle-aged mice. This was concomitant with effects on stress-induced peripheral immune cell priming and microglia activation. The regulatory effect of prebiotics on monocyte infiltration into the brain and accompanied regulation of age-related microglia activation highlight a potential pathway by which prebiotics can modulate the peripheral immune response and alter neuroinflammation in ageing. Our data highlight a novel strategy for the amelioration of age-related neuroinflammatory pathologies and brain function.

Are carnivore digestive separation mechanisms revealed on structure-rich diets?: Faecal inconsistency in dogs (Canis familiaris) fed day old chicks.

Pronounced variations in faecal consistency have been described anecdotally for some carnivore species fed a structure-rich diet. Typically two faecal consistencies are distinguished, namely hard and firm versus liquid and viscous faeces. It is possible that a separation mechanism is operating in the carnivore digestive tract, as in many herbivore species. Six beagle dogs were fed two experimental diets in a cross-over design of 7 days. Test diets consisted of chunked day old chicks differing only in particle size (fine = 7.8 mm vs coarse = 13 mm) in order to vary dietary structure. Digestive retention time was measured using titanium oxide (TiO2) as marker. The total faecal output was scored for consistency and faecal fermentation profiles were evaluated through faecal short-chain fatty acid (SCFA) and ammonia (NH3) analyses. A total of 181 faecal samples were collected. Dietary particle size did not affect faecal consistency, fermentative end products nor mean retention time (MRT). However, a faecal consistency dichotomy was observed with firm faeces (score 2–2.5) and soft faeces (score 4–4.5) being the most frequently occurring consistencies in an almost alternating pattern in every single dog. Firm and soft faeces differed distinctively in fermentative profiles. Although the structure difference between diets did not affect the faecal dichotomy, feeding whole prey provoked the occurrence of the latter which raises suspicion of a digestive separation mechanism in the canine digestive tract. Further faecal characterisation is however required in order to unravel the underlying mechanism.

GC-MS Analysis of Short-Chain Fatty Acids in Feces, Cecum Content, and Blood Samples.

Short-chain fatty acids, the end products of fermentation of dietary fibers by the gut microbiota, have been shown to exert multiple effects on mammalian metabolism. For the analysis of short-chain fatty acids, gas chromatography-mass spectrometry is a very powerful and reliable method. Here, we describe a fast, reliable, and reproducible method for the separation and quantification of short-chain fatty acids in mouse feces, cecum content, and blood samples (i.e., plasma or serum) using gas chromatography-mass spectrometry. The short-chain fatty acids analyzed include acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and heptanoic acid.

In vitro fermentation of copra meal hydrolysate by chicken microbiota.

The aim of this study was to carry out preliminary investigations on the in vitro fermentation selectivity of copra meal hydrolysate (CMH) by chicken gut microbiota. The ileum and cecum contents from three 35-day-old birds were used as inocula. Yeast mannooligosaccharide (yeast-MOS) or α-mannan was selected as a positive control. Batch culture fermentation with fecal bacteria was performed at 42°C for 24h in an anaerobic chamber. Samples were collected at 0, 6, 12, 18 and 24h of fermentation and evaluated using real-time PCR and short-chain fatty acid (SCFA) analysis. Results showed that the medium containing ileum and both CMH and yeast-MOS substrates led to an increase in the growth of the dominant groups as Lactobacillus, Enterobacteriaceae and Enterococcus spp. compared with 0-h fermentation. Campylobacter spp. and Bifidobacterium spp. were not detected in any samples. A significant decrease in Acinetobacter was observed in all substrates tested after 6h of fermentation (P<0.05). Only the sample from CMH fermentation showed a significantly greater reduction in the population of Pseudomonas after 18-h fermentation with ileum content (P<0.05). Propionate was the main fermentation product found in both ileum and cecum fermentation followed by lactate and acetate. CMH can be utilized by ileum and cecum microbial of chickens, and CMH has a generally desirable effect on the microbiota. CMH has the potential for use as a supplementary diet with similar or improved benefits and lower costs compared to commercial prebiotics. Further experiments in animal trials would seem to be justified.

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OBJECTIVES/SPECIFIC AIMS: Recent data suggest that fecal microbiota and intraluminal organic acids may play an important role in irritable bowel syndrome (IBS) pathogenesis through effects on intestinal secretion and motility. Understanding their contribution will be critical in developing diagnostic and treatment strategies. Objectives and goals of this study will be to: (1) compare fecal microbiota and fecal organic acids in IBS patients and controls and (2) investigate the association between colonic transit and fecal microbiota in IBS patients and controls. METHODS/STUDY POPULATION: We propose a prospective investigation of fecal organic acids, colonic transit and fecal microbiota in 36 IBS patients and 18 healthy controls. The target population will be adults ages 18–65 years meeting Rome IV criteria for IBS (both diarrhea predominant and constipation-predominant, IBS-D, and IBS-C) and asymptomatic controls. Exclusion criteria are: (a) history of microscopic colitis, inflammatory bowel disease, celiac disease, cancer, chronic infectious disease, immunodeficiency, uncontrolled thyroid disease, liver disease, or elevated AST/ALT&gt;2.0x the upper limit of normal, (b) prior radiation therapy of the abdomen or abdominal surgeries with the exception of appendectomy or cholecystectomy&gt;6 months before study initiation, (c) ingestion of prescription, over the counter, or herbal medications affecting gastrointestinal transit or study interpretation within 6 months of study initiation for controls or within 2 days before study initiation for IBS patients, (d) pregnant females, (e) antibiotic usage within 3 months prior to study participation, (f) prebiotic or probiotic usage within the 2 weeks prior to study initiation, (g) tobacco users. Primary outcomes will be fecal bile acid excretion and profile, short-chain fatty acid (SCFA) excretion and profile, colonic transit, and fecal microbiota. Secondary outcomes will be stool characteristics based on responses to validated bowel diaries. Stool samples will be collected from participants during the last 2 days of a 4-day 100-g fat diet and split into 3 samples for fecal microbiota, SCFA, and bile acid analysis and frozen. Frozen aliquots will be shipped to the Metabolite Profiling Facility at Purdue University and the Mayo Clinic Department of Laboratory Medicine and Pathology for SCFA and bile acid measurements, respectively. Analysis of fecal microbiota will be performed in the research laboratory of Dr. David Nelson in collaboration with bioinformatics expertise affiliated with the Nelson lab. Colonic transit time will be measured with the previously validated method using radio-opaque markers. Generalized linear models will be used as the analysis framework for comparing study endpoints among groups. RESULTS/ANTICIPATED RESULTS: This study seeks to examine the innovative concept that specific microbial signatures are associated with increased fecal excretion of organic acids to provide unique insights on a potential mechanistic link between altered intraluminal organic acids and fecal microbiota. DISCUSSION/SIGNIFICANCE OF IMPACT: Results may lead to development of targets for novel therapies and diagnostic biomarkers for IBS, emphasizing the role of the fecal metabolome.

Gut Microbiota Analysis in Rats with Methamphetamine-Induced Conditioned Place Preference.

Methamphetamine abuse is a major public health crisis. Because accumulating evidence supports the hypothesis that the gut microbiota plays an important role in central nervous system (CNS) function, and research on the roles of the microbiome in CNS disorders holds conceivable promise for developing novel therapeutic avenues for treating CNS disorders, we sought to determine whether administration of methamphetamine leads to alterations in the intestinal microbiota. In this study, the gut microbiota profiles of rats with methamphetamine-induced conditioned place preference (CPP) were analyzed through 16S rRNA gene sequencing. The fecal microbial diversity was slightly higher in the METH CPP group. The propionate-producing genus Phascolarctobacterium was attenuated in the METH CPP group, and the family Ruminococcaceae was elevated in the METH CPP group. Short chain fatty acid analysis revealed that the concentrations of propionate were decreased in the fecal matter of METH-administered rats. These findings provide direct evidence that administration of METH causes gut dysbiosis, enable a better understanding of the function of gut microbiota in the process of drug abuse, and provide a new paradigm for addiction treatment.

Identification of weak and gender specific effects in a short 3\xa0weeks intervention study using barley and oat mixed linkage \u03b2-glucan dietary supplements: a human fecal metabolome study by GC-MS

IntroductionMixed-linkage (1→3),(1→4)-β-d-glucans (BG) reduce cholesterol level and insulin response in humans. Despite this, their role in human metabolism and a mode of action remains largely unknown.ObjectivesTo investigate the effects of three structurally different BG on human fecal metabolome in a full cross-over intervention using GC-MS metabolomics.MethodsOver three weeks of intervention, young healthy adults received food supplemented with BG from oat, two different BG from barley or a non-fiber control in a full cross-over design. Untargeted metabolomics and short chain fatty acid analysis was performed on day three fecal samples. ANOVA-simultaneous component analysis was applied to partition the data variation according to the study design, and PLS-DA was used to select most discriminative metabolite markers.ResultsUnivariate and multivariate data analysis revealed a dominating effect of inter-individual variances followed by a gender effect. Weak effects of BG intake were identified including an increased level of gamma-amino-butyrate and palmitoleic acid in males and a decreased level of enterolactone in females. Barley and oat derived BG were found to influence the human fecal metabolome differently. Barley BG increased the relative level of formate in males and isobutyrate, isovalerate, 2-methylbutyrate in females. In total 15, 3 and 11 human fecal metabolites were significantly different between control vs. BG, control vs. oat BG, and barley BG vs. oat BG, respectively.ConclusionsThe study show that human fecal metabolome largely reflects individual (∼28% variation) and gender (∼15% variation) differences, whereas the treatment effect of the BG (∼8% variation) only manifests in a few key metabolites (primarily by the metabolites: d-2-aminobutyric acid, palmitoleic acid, linoleic acid and 11-eicosenoic acid).

Development of a fast and cost-effective gas chromatography-mass spectrometry method for the quantification of short-chain and medium-chain fatty acids in human biofluids.

The quantification of short-chain and medium-chain fatty acids is becoming more and more relevant in fecal and plasma samples due to their biological impact, which has been associated with colon rectal cancer and fiber consumption. For these reasons, a fast, cost-effective, and reproducible analytical method is highly required. In this research, a gas chromatography-mass spectrometry method based on full scan and multiple reaction monitoring (MRM) acquisition modes were optimized and validated for the analysis of short-chain and medium-chain fatty acids in three biological samples: human fecal water, fecal fermentation supernatants, and human plasma. Several extraction solvents (acidified water, diethyl ether, dichloromethane, ethyl acetate, and methyl tert-butyl ether (MTBE) were further evaluated, demonstrating that the latter was clearly the most suitable solvent with recoveries from 75.4 to 124.4% and coefficient of variations lower than 20%. The applicability of the GC-MS method was tested, for instance, acetic acid was quantified by using samples of plasma and feces from healthy donors at mean values of 66.9μM and 24.5mM, respectively. The optimized protocol could successfully find applications within multi-compartment human studies. In parallel, a second pilot experiment on fecal fermentation supernatants indicated that the proposed protocol is suitable to follow the formation of SCFAs during in vitro fermentation by the human gut microbiota. In summary, the present work provided an improved GC-MS method for precise and accurate quantification of SCFAs and MCFAs in human feces and plasma.

Interrelationships between Inflammatory Biomarkers, Fecal and Plasma Short Chain Fatty Acids

Short chain fatty acids (SCFA) have been associated with improved glycemia and to reductions in inflammatory responses in cell and rodent models. Low grade inflammation has been associated with insulin resistance. We measured fecal and plasma SCFA and inflammatory biomarkers in response to a proprietary oligosaccharide intervention. 22 overweight and obese men and women participated in a three‐way crossover study that included 3 levels of oligosaccharide: 0, 15 and 30 g/d. for 3 weeks. During each treatment, a collection of 5 days' feces were combined and analyzed for SCFA. At the end of each treatment, a meal challenge protocol was employed in which participants consumed half their daily dose of oligosaccharide with breakfast followed by a standard lunch. We measured plasma acetate, propionate and butyrate in 12 blood draws over the course of a 9 hour test day and inflammatory cytokines at 3 time points, 0, 195 and 435 min following the breakfast test meal. All SCFA analyses were conducted by GC‐MS. Inflammatory cytokines were measured on an 8‐plex chemiluminescent MSD plate except for adiponectin and CRP which employed an Integra 400 bio‐analyzer. Breath hydrogen and methane were collected to characterize participants as either methane producers (MP) or non‐producers (NM). Adiponectin was positively associated with fecal SCFA (p&lt;0.05). TNF‐α and IL‐10 were negatively associated with various plasma SCFA AUC segments (p&lt;0.05). Neither CRP nor IL‐6 was associated with either fecal or plasma SCFA. Fecal SCFA concentrations did not correlate well with plasma SCFA AUCs. Fecal propionate and butyrate were lower in MP than NM. Breath methane was negatively associated with SCFA production in MP. Plasma and fecal SCFA may predict some inflammatory responses but differences in the time of collections and the span of collections may reflect different temporal relationships to cytokines.Support or Funding InformationFunding provided by USDA CRIS 2032‐51530‐022‐OOD and the West Coast Metabolomics Center (U24 DK097154)

Study of the inhibitory effect of ferulic acid on short-chain fatty acids in milk samples by dispersive liquid–liquid micro-extraction in combination with fluorescence derivatization

ABSTRACTA sensitive high-performance liquid chromatography (HPLC) method was developed to study the influence of ferulic acid on the formation of volatile fatty acids and lactic acid in milk and soybean milk samples. Volatile fatty acids were extracted by liquid–liquid micro-extraction using chloroform and acetonitrile as the extraction and disperser solvents, respectively. The analytes were derivatized with 2-(5-benzoacridine)ethyl-p-toluenesulfonate that showed excellent fluorescence property and made the sensitive HPLC analysis of short-chain fatty acids become possible. The optimized HPLC sensitivity was in the range of 1.1–1.9 µg L−1. Ferulic acid was added in milk and soybean milk samples to study its preservative effect. The results indicated that ferulic acid with concentration of 0.2% (m/v) could effectively reduce the formation of short-chain fatty acids.

A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human stool derivatized with 12C- and 13C-labelled aniline

A novel liquid chromatography tandem mass spectrometry (LCMSMS) method for the quantitative measurement of gut microbial-derived short-chain fatty acids (SCFAs) in human infant stool has been developed and validated. Baseline chromatographic resolution was achieved for 12 SCFAs (acetic, butyric, caproic, 2,2-dimethylbutyric, 2-ethylbutyric, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic, pivalic and valeric acids) within an analysis time of 15min. A novel sequential derivatization of endogenous and spiked SCFAs in stool via 12C- and 13C-aniline respectively, facilitated the accurate quantitation of 12C-aniline derivatized endogenous SCFAs based on calibration of exogenously 13C-derivatized SCFAs. Optimized quenching of derivatization agents prior to LCMSMS analysis further reduced to negligible levels the confounding chromatographic peak due to in-line derivatization of unquenched aniline with residual acetic acid present within the LCMS system. The effect of residual acetic acid, a common LCMS modifier, in analysis of SCFAs has not been addressed in previous SCFA assays. For the first time, a total of 9 SCFAs (acetic, butyric, caproic, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic and valeric acids) were detected and quantitated in 107 healthy infant stool samples. The abundance and diversity of SCFAs in infant stool vary temporally from 3 weeks onwards and stabilize towards the end of 12 months. This in turn reflects the maturation of infant SCFA-producing gut microbiota community. In summary, this novel method is applicable to future studies that investigate the biological roles of SCFAs in paediatric health and diseases.

Development of a sequential injection-liquid microextraction procedure with GC-FID for analysis of short-chain fatty acids in palm oil mill effluent

Short-chain fatty acids, such as acetic, propionic, butyric, iso-valeric and valeric acids, play an important role in methanogenesis activity for biogas production processes. Thus, simple and rapid procedures for monitoring the levels of short-chain fatty acids are requisite for sustaining biogas production. This work presents the development of a sequential injection-liquid microextraction (SI-LME) procedure with GC-FID analysis for determination of short-chain fatty acids. GC-FID was employed for detection of the short-chain fatty acids. Calibration curves were linear with good coefficients of determination (r2>0.999), using methacrylic acid as the internal standard. Limits of quantification (LOQ) were in the range of 0.03–0.19mM. The SI-LME procedure employed tert-butyl methyl ether (TBME) as the extracting solvent. Various SI-LME conditions were investigated and optimized to obtain the highest recovery of extraction. With these optimized conditions, an extraction recovery of the five key short-chain fatty acids of 67–90% was obtained, with less than 2% RSD (n=3). The final SI-LME procedure employed two fluidic zones of TBME with a single aqueous fluidic zone of sample sandwiched between the TBME zones, with 5 cycles of flow reversal at a flow rate of 5µL/s for the extraction process. Intra- and inter-day precision values were 0.5–4.0% RSD and 3.3–4.8% RSD, respectively. Accuracy based on percentage of sample recovery were in the range of 69–96, 102–107, and 82–101% (n=4) for acetic, propionic and butyric acids, respectively. The proposed method was applied for the measurement of short-chain fatty acids in palm oil mill effluents used in biogas production in a factory performing palm oil extraction process. The SI-LME method provides improved extraction performance with high precision, and is both simple and rapid with its economical extraction technique. The SI-LME procedure with GC-FID has strong potential for use as a quality control process for monitoring short-chain fatty acid levels in biogas production.

An in situ extraction and derivatization method for rapid analysis of short-chain fatty acids in rat fecal samples by gas chromatography tandem mass spectrometry

Rapid determination of SCFAs in rat fecal samples by GC-MS/MS combined with in situ extraction and derivatization.

Colonic Transit Time Is a Driven Force of the Gut Microbiota Composition and Metabolism: In Vitro Evidence.

Background/AimsHuman gut microbiota harbors numerous metabolic properties essential for the host’s health. Increased intestinal transit time affects a part of the population and is notably observed with human aging, which also corresponds to modifications of the gut microbiota. Thus we tested the metabolic and compositional changes of a human gut microbiota induced by an increased transit time simulated in vitro.MethodsThe in vitro system, Environmental Control System for Intestinal Microbiota, was used to simulate the environmental conditions of 3 different anatomical parts of the human colon in a continuous process. The retention times of the chemostat conditions were established to correspond to a typical transit time of 48 hours next increased to 96 hours. The bacterial communities, short chain fatty acids and metabolite fingerprints were determined.ResultsIncrease of transit time resulted in a decrease of biomass and of diversity in the more distal compartments. Short chain fatty acid analyses and metabolite fingerprinting revealed increased activity corresponding to carbohydrate fermentation in the proximal compartments while protein fermentations were increased in the lower parts.ConclusionsThis study provides the evidence that the increase of transit time, independently of other factors, affects the composition and metabolism of the gut microbiota. The transit time is one of the factors that explain some of the modifications seen in the gut microbiota of the elderly, as well as patients with slow transit time.

Sensitive and Simplified Detection of Antibiotic Influence on the Dynamic and Versatile Changes of Fecal Short-Chain Fatty Acids.

Short-chain fatty acids (SCFAs), produced by anaerobic fermentation of mainly indigestible dietary carbohydrates by gut microbiota, have a profound influence on intestinal function and host energy metabolism. Antibiotics may seriously disturb the balance of fecal SCFAs. To evaluate the impacts of antibiotics on fecal SCFAs produced by gut microbiota, a simple, reproducible and accurate gas chromatography (GC) method, which can simultaneously analyze seven SCFAs in fecal samples, was developed and validated. The ranges of detection and quantitation of the SCFAs reached 0.0868 ~ 0.393 and 0.261 ~ 1.18 μg·mL-1 respectively, in an optimized protocol for SCFAs extraction and analysis that used 10 mL 75% ethanol aqueous solution containing 1% HCl, without ultrasonication. The technique exhibited excellent intra-day (relative standard deviation (RSD) ≤ 2.54%) and inter-day (RSD ≤ 4.33%) precisions for all the SCFAs. Later, we administered broad-spectrum antibiotics, cefdinir or azithromycin to rats and analyzed the alterations in fecal SCFAs. The total amount, types and distribution of nearly all fecal SCFAs were significantly altered during the administration and even after withdrawal of the antibiotics in rats. The effects of cefdinir on the SCFAs were more pronounced than those of azithromycin. Our findings suggest SCFAs may serve as sensitive indicators to monitor the influences of antibiotics on SCFAs originated by intestinal bacteria. Our improved SCFAs analysis method is a potential platform for a standard clinical test of the effects of new antibiotics on SCFAs.

Bioactive compounds from regular diet and faecal microbial metabolites.

Short-chain fatty acids (SCFAs) formation by intestinal bacteria is regulated by many different factors, among which dietary fibre is currently receiving most attention. However, since fibre-rich foods are usually good dietary sources of phenolic compounds, which are also known to affect the microbiota, authors hypothesize that the regular intake of these bioactive compounds could be associated with a modulation of faecal SCFA production by the intestinal microbiota. In this work, food intake was recorded by means of a validated Food Frequency Questionnaire. Fibres were determined using Marlett food composition tables, and phenolic compounds were obtained from Phenol-Explorer Database. Analysis of SCFA was performed by gas chromatography-flame ionization/mass spectrometry and quantification of microbial populations in faeces by quantitative PCR. Klason lignin and its food contributors, as predictors of faecal butyrate production, were directly associated with Bacteroides and Bifidobacterium levels, as well as lignans with Bacteroides. Also, anthocyanidins, provided by strawberries, were associated with faecal propionate and inversely related to Lactobacillus group. These results support the hypothesis we put forward regarding the association between some vegetable foods (strawberries, pasta, lentils, lettuce and olive oil) and faecal SCFA. More studies are needed in order to elucidate whether these associations have been mediated by the bacterial modulatory effect of the bioactive compounds, anthocyanins, lignans or Klason lignin, present in foodstuffs.

Reduction of Salmonella Typhimurium by Fermentation Metabolites of Diamond V Original XPC in an In Vitro Anaerobic Mixed Chicken Cecal Culture.

Fermentation metabolites of Diamond V Original XPC™ (XPC), a biological product derived from yeast fermentation, were evaluated for their ability to reduce the Salmonella Typhimurium population using an in vitro mixed anaerobic culture system containing cecal microbiota to simulate chicken hindgut conditions. Four different samples were prepared: anaerobic mixed culture containing (1) feed only, (2) cecal only (ceca were harvested from 42 days old broiler chickens), (3) feed and cecal contents, and (4) feed, cecal contents, and 1% XPC. Two experimental conditions were investigated: Group 1, in which the cecal content was added at the same time as a S. Typhimurium marker strain and Group 2, in which the cecal content was preincubated for 24 h prior to the inoculation with the S. Typhimurium marker strain. The mixed cultures were incubated anaerobically at 37°C, and the S. Typhimurium marker strain was enumerated at 0, 24, and 48 h. Analysis of short chain fatty acids was also conducted for 24 h. In the Group 1 experiment, adding XPC did not exhibit significant reduction of S. Typhimurium. However, the presence of XPC resulted in rapid reduction of S. Typhimurium in Group 2. S. Typhimurium was reduced from 6.81 log10 CFU/ml (0 h) to 3.73 log10 CFU/ml and 1.19 log10 CFU/ml after 24 and 48 h, respectively. These levels were also 2.47 log10 and 2.72 log10 lower than the S. Typhimurium level recovered from the control culture with feed and cecal contents, but without XPC. Based on these results, it appears that the ability of XPC to reduce S. Typhimurium requires the presence of the cecal microbiota. Short chain fatty acid analysis indicated that acetate and butyrate concentrations of cultures containing XPC were twofold greater than the control cultures by 24 h of anaerobic growth. Results from the present study suggest that dietary inclusion of XPC may influence cecal microbiota fermentation and has the potential to reduce Salmonella in the cecum. Implications of these findings suggest that XPC may decrease preharvest levels of Salmonella in broilers and layers.

A rapid method for quantifying free and bound acetate based on alkylation and GC-MS analysis.

BackgroundAcetyl-CoA is a key metabolic intermediate with roles in the production of energy and biomass, as well as in metabolic regulation. It was recently found that acetate is crucial for maintaining acetyl-CoA production in hypoxic cancer cells. However, the availability of free acetate in the tumor environment and how much tumor cells consume remains unknown. Similarly, much is still to be learned about changes in the dynamics and distribution of acetylation in response to tumor-relevant conditions. The analysis of acetate is non-trivial, and to help address these topics, we developed a rapid and robust method for the analysis of both free and bound acetate in biological samples.ResultsWe developed a sensitive and high-throughput method for the analysis of acetate based on alkylation to its propyl derivative and gas chromatography-mass spectrometry. The method facilitates simultaneous quantification of both 12C- and 13C-acetate, shows high reproducibility (< 10 % RSD), and has a wide linear range of quantification (2–2000 μM). We demonstrate the method’s utility by measuring free acetate uptake by cultured cancer cells and by quantifying total acetylation (using hydrolysis) in separate cellular compartments. Additionally, we measure free acetate in tissues and bio-fluids and show that there are considerable differences in acetate concentrations between organs in vivo, providing insights into its complex systemic metabolism and availability for various types of tumors.ConclusionsOur approach for the quantification of acetate is straightforward to implement using widely available equipment and reagents, and will aid in in-depth investigation of various aspects of acetate metabolism. It is also readily adaptable to the analysis of formate and short-chain fatty acids, making it highly relevant to the cancer metabolism community.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-016-0157-5) contains supplementary material, which is available to authorized users.