Spatiotemporal digestion and intestinal microbial fermentation properties of Ganoderma lucidum based on in vitro digestive system.

The study of short (SCFAs) and branched chain fatty acids (BCFAs) in human stool related to gastrointestinal diseases, gut microbiota, metabolism and diet has dramatically increased. As a result, a fast, reliable method with minimal pretreatment is needed for quantification of these metabolites (acetic, propionic, isobutyric, butyric, isovaleric, valeric and caproic acid; μg/g stool) in stool. Therefore, a GC-MS method meeting this criterion was developed. Stool samples were homogenized, diluted with 80:20 water:methanol (v/v) and adjusted to a pH of 1.5 - 2.5. Samples were vortexed, centrifuged and directly injected into the GC-MS using pulsed splitless injection offering two-to-three-fold signal enhancement over a 10:1 split injection. DB-FATWAX Ultra Inert Polyethylene Glycol (PEG) Column showed no peak tailing, reduced responses or retention time shifts after 1476 stool injections while other columns failed before 361 injections. A case-control study was conducted using 53 remnant raw stool samples with a positive diagnosis of either ulcerative colitis (UC) or Crohn’s Disease (CD) which comprised the Inflammatory Bowel Disease (IBD) group and were compared to a control group of 21 samples for SCFA and BCFA concentrations. Strong statistical differences were observed between groups whereas the IBD group contained less propionic, butyric and valeric acid (p lessthan 0.05). A receiver operator curve was plotted using the sum of significantly different SCFAs normalized against acetic acid resulting in 96% AUC (95% CI: 0.89 – 0.98) demonstrating potential diagnostical application.

Investigate feasibility of short (SCFA) and branched chain fatty acids (BCFA) in human stool as biomarkers for inflammatory bowel disease (IBD). Interest in fecal SCFAs and BCFAs has increased. Evidence shows strong connections between these metabolites and IBD pathogenesis and pathophysiology. A case-control study of 74 stool samples (21 healthy; 24 ulcerative colitis, UC; 29 Crohn's disease, CD) was quantified using a validated, gas chromatography-mass spectrometry (GC-MS) method for acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acid (μg/g stool). Bristol Stool Form Scale (BSFS) and pH were recorded. Receiver operator curve (ROC) analysis resulted in area under the curve (AUC) value of 0.96 (95% CI: 0.89-0.98, P <0.001), with 92% sensitivity and 81% specificity using the acetic/(propionic+butyric+isovaleric+valeric acid) transformation between healthy and IBD groups. ROC analysis resulted in AUC of 0.83 (95% CI: 0.66-0.92, P <0.001), with 75% sensitivity and 86% specificity using the acetic/(isobutyric/propionic acid) transformation between UC and CD subgroups. Acetic acid was the most abundant SCFA (72/74 samples) and nonsignificantly different between groups (healthy vs. IBD; P ≥0.05, 0.161 and UC vs. CD; P ≥0.025, 0.623). Fecal SCFAs and BCFAs demonstrated feasibility as biomarkers for IBD. Increased sensitivity and specificity were achieved over fecal calprotectin (FCP) tests between healthy and IBD patients. No useable data was found in the literature to use a validation cohort.

In modern societies obesity has become a serious issue which must be urgently addressed. The health implications of neglected obesity are substantial, as not only does it affect individuals' everyday lives, but it also leads to significantly increased mortality due to the development of several disorders such as type-2 diabetes, cardiovascular diseases, cancers, and depression. The objective of this research was to investigate the alterations in selected health markers caused by overweight and obesity in children. The measured parameters were the activity of the fecal enzymes, the concentration of short-chain fatty acids (SCFAs), and the concentration of branched-chain fatty acids (BCFAs). The activity of the fecal enzymes, specifically α-glucosidase, α-galactosidase, β-glucosidase, β-galactosidase, and β-glucuronidase, was determined using spectrophotometry at a wavelength of 400 nm. Furthermore, concentrations of lactic acid, SCFAs (formic, acetic, propionic, butyric, and valeric acids), and BCFAs (isobutyric and isovaleric acids) were determined using the HPLC method. The obtained results reveal that obese children have different fecal enzyme activity and a different profile of fatty acids from children of normal weight. The group of obese children, when compared to children of normal weight, had increased concentrations of BCFAs (p < 0.05) and higher activity of potentially harmful enzymes such as β-glucosidase and β-glucuronidase (p < 0.05). In comparison, children of normal weight exhibited significantly increased concentrations of lactic acid and SCFAs (especially formic and butyric acids) (p < 0.05). Furthermore, their α-glucosidase and α-galactosidase activity were higher when compared to the group of obese children (p < 0.05). These results suggest that the prevalence of obesity has a significant impact on metabolites produced in the gastrointestinal tract, which might result in a higher chance of developing serious diseases.

Short-chain fatty acids (SCFAs) are produced by the fermentation of undigested polysaccharides; they are a group of metabolites resulting from the activity of intestinal bacteria. The main SCFAs are acetic, butyric, propionic, valeric, and caproic acid, and their levels and proportions depend on various factors. The aim of this study was to investigate the relationship between the concentration of SCFAs and the occurrence of specific gastrointestinal symptoms in infants. This study was conducted using faecal samples obtained at 1, 3, 6, and 12 months of age. The SCFA content was measured using gas chromatography. At 1 month, an association was found between butyric acid and flatulence. At 3 months, an association was found between butyric acid and flatulence/gas and between 3,4-methylovaleric acid and mucus in the stool. At 6 months, an association was found between butyric and valeric acids and flatulence. By 12 months, the gastrointestinal symptoms had decreased significantly. This study confirms that there is an association between SCFA levels and the presence of bloating, gas, mucus in the stool, and constipation in the gastrointestinal tract. Higher levels of butyric and valeric acids may lead to an increase in troublesome symptoms, such as flatulence and gas, in the first few months of life but are not associated with the occurrence of intestinal colic. The level of 3,4-methylovaleric acid is associated with the presence of allergies, whereas a decrease in acetic acid and an increase in isovaleric acid may exacerbate defecation problems in infants.

Background: Microbial fermentation of non-digestible carbohydrates and/or protein produces short-chain fatty acids (SCFA), whereas branched-chain fatty acids (BCFA) are produced from protein fermentation. The effects of individual SCFA and BCFA of comparable carbon chain length on adipocyte inflammation have not been investigated. Objective: To compare the effects of SCFA and BCFA on inflammatory mediator secretion in an adipocyte cell culture model designed to recapitulate obesity-associated adipocyte inflammation under normoxic and hypoxic conditions. Methods: The 3T3-L1 adipocytes were cultured (24 h) without (Control, Con) and with 1 mmol/L of SCFA (butyric acid (But) or valeric acid (Val)) or 1 mmol/L of BCFA (isobutyric acid (IsoBut) or isovaleric acid (IsoVal)) and were unstimulated (cells alone, n = 6/treatment), or stimulated with 10 ng/mL lipopolysaccharide (LPS, inflammatory stimulus, n = 8/treatment) or 10 ng/mL LPS + 100 µmol/L of the hypoxia memetic cobalt chloride (LPS/CC, inflammatory/hypoxic stimulus, n = 8/treatment). Results: Compared to Con + LPS, But + LPS reduced secreted protein levels of interleukin (IL)-1β, IL-6, macrophage chemoattractant protein (MCP)-1/chemokine ligand (CCL)2, MCP3/CCL7, macrophage inflammatory protein (MIP)-1α/CCL3 and regulated upon activation, normal T cell expressed, and secreted (RANTES)/CCL5 and decreased intracellular protein expression of the ratio of phosphorylated to total signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NFκB) p65 (p < 0.05). Val + LPS reduced IL-6 secretion and increased MCP-1/CCL2 secretion compared to Con + LPS and exhibited a different inflammatory mediator secretory profile from But + LPS (p < 0.05), indicating that individual SCFA exert individual effects. There were no differences in the secretory profile of the BCFA IsoBut + LPS and IsoVal + LPS (p > 0.05). Alternatively, under inflammatory hypoxic conditions (LPS/CC) Val, IsoVal, and IsoBut all increased secretion of IL-6, MCP-1/CCL2 and MIP-1α/CCL3 compared to Con (p < 0.05), whereas mediator secretion did not differ between But and Con (p > 0.05), indicating that the proinflammatory effects of SCFA and BCFA was attenuated by But. Interestingly, But + LPS/CC decreased STAT3 activation versus Con + LPS/CC (p < 0.05). Conclusions: The decreased secretion of inflammatory mediators that is attributable to But highlights the fact that individual SCFA and BCFA exert differential effects on adipocyte inflammation under normoxic and hypoxic conditions.

Proteins, peptides and amino acids (AAs) that bypass upper gastrointestinal (GI) digestion can be fermented in the colonic regions. This could lead to microbial production of health promoting short-chain fatty acids (SCFAs). Nitrogenous compounds can also be fermented to generate potentially harmful branched chain fatty acids (BCFAs). As collagen hydrolysate (CH) supplements contain a high peptide content, we evaluated whether peptides that undergo intestinal CH digestion and microbial fermentation can generate SCFAs and BCFAs. Two bovine-sourced CH formulations (CH-GL and CH-OPT) underwent digestive processes and microbial fermentation for 24 h in a dynamic GI digestion model containing human fecal matter. After 24 h, CH-OPT showed a significant (p &lt; 0.05) increase in SCFAs (propionic, butyric and valeric acids) in the ascending colonic vessel with no changes observed with CH-GL. Only CH-OPT showed a significant (p &lt; 0.05) increase in BCFAs, also noted in the ascending colon. No significant (p &lt; 0.05) changes to SCFAs and BCFAs were observed in the transverse and descending colons for both CHs. These findings demonstrate that CHs can induce microbial production of SCFAs and BCFAs although this appears to depend on the CH tested. More studies are needed to determine the physiological significance of these microbial metabolites from intake of CH supplements.

The study of short (SCFAs) and branched chain fatty acids (BCFAs) in human stool related to gastrointestinal diseases, gut microbiota, metabolism, and diet has dramatically increased. As a result, a fast, reliable method with minimal pretreatment is needed for quantification of these metabolites (acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acid) in stool. Therefore, a GC-MS method meeting this criterion was developed. A bias sampling study showed no statistical difference (p>0.05) in analyte means when comparing 100 mg subsamples of homogenized to non-homogenized samples (n=6, p values 0.153-0.910). Stool samples were homogenized, diluted with 80:20 water:methanol (v/v), and adjusted to a pH of 1.5-2.5. Samples were vortexed, centrifuged, and directly injected into the GC-MS using pulsed splitless injection offering twofold-to-threefold signal enhancement over a 10:1 split injection. DB-FATWAX Ultra Inert Polyethylene Glycol (PEG) Column showed no peak tailing, reduced responses, or retention time shifts after 1,476 stool injections, while other columns failed before 361 injections. Intra- and inter-day accuracy for stool supernatant samples ranged from -10.21% to 8.88% and -13.25% to 9.91%, while intra- and inter-day precision ranged from 0.21% to 1.21% and 0.89% to 2.84% coefficient of variation (CV), respectively. This method demonstrates excellent linearity (0.9999-1.0000) and low limits of quantification (1.50-8.01 μM). Stool samples proved stable stored at -20°C up to 28 days, and recoveries ranged from 85.04% to 106.59%. Matrix effects in stool are non-significant determined by comparing standard and stool supernatant calibration curve slopes (p>0.05).

Changes in gut microbiota in predigested Hibiscus sabdariffa L calyces and Agave (Agave tequilana weber) fructans assessed in a dynamic in vitro model (TIM-2) of the human colon

Based on the fact that the clostridia isolated from paddy field soil were mostlyproteolytic and that they produced some branched-chain volatile fatty acids on peptone media, the occurrence and the substrates for the production of such acids in paddy soil were investigated. The results obtained are as follows: 1) It was revealed that two branched-chain volatile fatty acids, iso-valeric and iso-butyric acids, were detected In fresh paddy field soil. 2) The paddy soil supplemented with peptone solution produced significant amounts of branched-chain volatile fatty acids-iso-butyric and iso-valeric acidsin addition to straight-chain volatile fatty acids-acetic, propionic, and butyric acids. When glucose was added instead of peptone, not branched-chain fatty and propionic acids were detected but only acetic and butyric acids. These facts indicate that the branched-chain fatty acids may originate from amino acids. 3) When a pair of amino acids, such as proline and leucine or proline-and valine, were sup...

Beach-cast seaweed has recently garnered attention for its nutrient-rich composition, including proteins, carbohydrates, vitamins, minerals, and phytochemicals. This study focuses on the phenolic content and antioxidant potential of five Australian beach-cast seaweed species during in vitro digestion and colonic fermentation. The bioaccessibility of the selected phenolic compounds was estimated and short chain fatty acids (SCFAs) production was determined. Cystophora sp., showed a notable increase in phenolic content (23.1 mg GAE/g) and antioxidant capacity (0.42 mg CE/g) during the intestinal and gastric phases of in vitro digestion. Durvillaea sp. demonstrated a significant release of flavonoids (0.35 mg QE/g), while Phyllosphora comosa released high levels of tannins (0.72 mg CE/g) during the intestinal phase. During colonic fermentation, P. comosa released the highest levels of phenolic compounds (4.3 mg GAE/g) after 2 h, followed by an increase in flavonoids (0.15 mg QE/g), tannins (0.07 mg CE/g), and antioxidant activity (DPPH: 0.12 mg TE/g; FRAP: 0.61 mg TE/g) after 4 h. Moreover, P. comosa released a considerable amount of phenolic compounds during both in vitro digestion and colonic fermentation. All species consistently released phenolic compounds throughout the study. Phloroglucinol, gallic acid, and protocatechuic acid were identified as the most bioaccessible phenolic compounds in all five Australian beach-cast seaweeds in the in vitro digestion. Nevertheless, compound levels declined during the colonic fermentation phase due to decomposition and fermentation by gut microbiota. With regard to SCFAs, P. comosa displayed elevated levels of acetic (0.51 mmol/L) and propionic acid (0.36 mmol/L) at 2 h, while Durvillaea sp. showed increased butyric (0.42 mmol/L) and valeric (0.26 mmol/L) production acid after 8 h. These findings suggest that seaweed such as Cystophora sp., Durvillaea sp., and P. comosa are promising candidates for food fortification or nutraceutical applications, given their rich phenolic content and antioxidant properties that potentially offer gut health benefits.

The connection between gut microbiota and factors like diet is crucial for maintaining intestinal balance, which in turn impacts the host’s overall health. Nannochloropsis gaditana microalgae is a sustainable source of bioactive compounds, mainly known for its used in aquaculture and extraction of bioactive lipids, with potential health benefits whose effects on human gut microbiota are still unknown. Therefore, the goal of this work was to assess the impact of N. gaditana on human gut microbiota composition and derived metabolites by combining the INFOGEST protocol and in vitro colonic fermentation process to evaluate potential effects on human gut microbiota conformation through 16S rRNA gene sequencing and its metabolic functionality. The results have demonstrated the ability of the digests from N. gaditana to significantly modify gut microbiota composition, promoting an increase in beneficial bacterial genera such as Akkermansia, Butyricicoccus, Eisenbergiella, Lachnoclostridium, and Marvinbryantia, in contrast to inulin, after 48 h of colonic fermentation. Additionally, the digests increased the levels of both major and minor short-chain fatty acids (SCFAs), particularly butyric and valeric acids, considered as intestinal biomarkers, and increased ammonium production. This research has demonstrated, for the first time, the potential of N. gaditana microalgae as a sustainable agent for influencing the composition and functionality of human gut microbiota.

Offensive odors which evolve during the decomposition of swine waste have resulted in complaints from neighbors in agricultural areas. This study chronologically monitored the emissions of volatile fatty acids (VFAs) during composting to quantify the release of malodorous compounds. Swine waste/sawdust mixtures and sawdust controls were placed in 208 liter reactor vessels and constantly aerated over a 21-day period. Acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids were quantified in total collections of condensed water vapor and volatiles released from the vessels. Exit gasses were passed through water-cooled condensers, and condensate was collected every 12 hours. Composting conditions were shown to be adequate by measuring carbon to nitrogen ratios, pH, temperature, and moisture contents. The 21-day aerobic treatment decreased all VFAs in the swine waste mixtures by 61 to 100 percent, with a mean of 87 percent. Prior to peak gas volatilization, when the vessel contents attained peak composting temperatures, acetic acid was emitted in the greatest amounts, followed by butyric, propionic, valeric, isovaleric, and isobutyric acids in order of decreasing emissions. Following peak gas volatilization, butyric acid was released in the greatest amounts, followed by acetic, propionic, valeric, isovaleric, and isobutyric acids. The described condensation procedure was shown to be an effective method to quantify malodorous compounds emitted during the decomposition of livestock waste. In addition, aeration was an effective treatment to decrease VFAs and, therefore, odor potential resulting from their emissions.

A study has been made of the formation of some volatile esters in anaerobic fermentation, by Saccharomyces cerevisiae, of a synthetic medium with glucose as the carbon source. The relationship between the rate of formation of ethyl butyrate and the concentration of butyric acid can be expressed by a modification of the Briggs-Haldane equation for monomolecular enzymic reactions; the same is true with ethyl valerate and valeric acid. Butyric acid and valeric acid compete in the ester synthesis, presumably for coenzyme A. The kinetics of such a system of competing substrates is discussed, and the formation of ethyl butyrate and ethyl valerate from butyric acid and valeric acid is shown to satisfy the theory. The constants in the modified Briggs-Haldane equation are calculated for ester formation from butyric acid and valeric acid. The maximum rate is about the same, but the Michaelis constant for butyric acid is about twice that for valeric acid. The formation of ethyl acetate shows some discrepancies. These are discussed.

Sanhua decoction (SHD), a traditional prescription, has long been used in treating ischemic stroke (IS). However, the therapeutic effect of SHD and the associated changes in gut microbiota and short-chain fatty acids (SCFAs) are uncertain. In this study, a rat model of IS was established by the middle cerebral artery occlusion (MCAO). By evaluating the cerebral infarct area and brain tissue pathology, it was found that SHD ameliorated IS-related symptoms in MCAO rats. Using 16S rRNA gene sequencing, we found that SHD reduced abnormally elevated Lactobacillus and opportunistic pathogens such as Desulfovibrio, but increased some beneficial bacteria that produce SCFAs, including Clostridia, Lachnospiraceae, Ruminococcaceae, and Coprococcus. KEGG analysis revealed that SHD regulates several pathways, including D-arginine and D-ornithine metabolism, polyketide sugar unit biosynthesis, and cyanoamino acid metabolism, which are significantly altered in MCAO rats. By gas chromatography-mass spectrometry detection of SCFAs, we found that fecal acetic acid, valeric acid, and caproic acid were significantly increased in MCAO rats, whereas propionic acid and isobutyric acid were decreased. SHD reversed the changes in acetic acid and propionic acid in the model rats and significantly increased fecal butyric acid. In addition, MCAO rats had significantly higher serum levels of acetic acid, butyric acid, isovaleric acid, and valeric acid, and lower levels of caproic acid. Altered serum levels of butyric acid, isovaleric acid, valeric acid, and caproic acid were restored, and the level of isobutyric acid was reduced after SHD administration. Spearman analysis revealed that cerebral infarct area had a strong correlation with Bifidobacterium, Desulfovibrio, Lachnospiraceae, Lactobacillus, acetic acid, valeric acid, and caproic acid. Overall, this study demonstrates for the first time that the effect of SHD on IS may be related to gut microbiota and SCFAs, providing a potential scientific explanation for the ameliorative effect of SHD on IS.

HIV progression is characterized by immune activation and microbial translocation from the gut. Short-chain fatty acids (SCFAs) are essential for gut homeostasis. Decreased intestinal SCFAs play a role in rapid HIV progression. To compare the SCFA profile, intestinal microbiome, and intestinal mucosal injury between patients with HIV (but not AIDS) and healthy controls. This was a prospective study of 15 patients without AIDS and 10 controls conducted between July 2016 and January 2017 at the Institute of Dermatology and Venereology (Sichuan Academy of Medical Sciences). Stool specimens were collected to analyze the microbiome and SCFAs. Blood I-FABP and D-lactate (gut injury markers) were measured as well as T cells in HIV-positive patients. Intestinal mucosa was observed by colonoscopy. Rikenellaceae, Microbacteriaceae, Roseburia, Lachnospiraceae, Alistipes, and Ruminococcaceae were decreased, while Moraxellaceae and Psychrobacter were increased in HIV-positive patients. Butyric acid (p = 0.04) and valeric acid (p = 0.03) were reduced in HIV-positive patients. Colonoscopy revealed no visible damage in all subjects. There were no differences in I-FABP and D-lactate between groups. Butyric and valeric acids mainly positively correlated with Rikenellaceae, Ruminococcaceae, Alistipes, Roseburia, and Lachnospiraceae. CD8+ cells were positively correlated with Proteobacteria. CD4+ cells, and CD4/CD8 were negatively correlated with acetic acid. CD8+ cells were positively correlated with valeric acid. The differences in the distribution of intestinal flora between HIV-infected and healthy individuals, especially some SCFAs, suggest that there is already a predisposition to intestinal mucosa damage in HIV-infected individuals.