Temporal changes in the fermentation characteristics, bacterial community structure and the functionality of the predicted metagenome of a batch fermenter medium containing the upper gastrointestinal enzyme resistant fraction of white sorghum (Sorghum bicolor L. Moench).

Sorghum is a potential prebiotic ascribed to the high native resistant starch (RS) content. Our previous studies on raw sorghum have revealed prominent amino acid fermentation despite the high RS content. Interestingly, autoclaved-freeze-dried sorghum fed rats exhibited beneficial microbial and biochemical profiles. Having a keen interest to reciprocally scrutinize the underlying mechanisms behind these contrasting outcomes, we used an in vitro porcine batch fermentation model. The fermentable substrates in raw and autoclaved-freeze-dried (three cycles) sorghum (AC) after in vitro gastrointestinal digestion fostered similar bacterial community structures, yet with significant differences in the characteristic amylolytic microbial taxa abundance and their temporal variation. Further, significant differences in the concentration of organic acids in raw and AC manifested the differences in the predicted abundance of the underlying pathways of carbohydrate and organic acid metabolism. Thus, this study highlights the propensity of the heat-moisture treatment of sorghum in modifying the fermentability of its RS.

Effects of concentrate levels on intestinal fermentation and the microbial profile in Japanese draft horses.

In racehorses, feeding a high-concentrate diet could cause abnormal fermentation in the hindgut. This feeding management regime is not suitable for the nutritional physiology of horses. However, studies on the hindgut environment have yet to be reported in Japanese draft horses, so feeding management needs to be investigated in these horses. Therefore, the objective of this study was to investigate the effects of a high-concentrate diet on hindgut fermentation in Japanese draft horses. Feces were collected from 20 male Japanese draft horses managed by two stables with different feeding designs (65% weight ratio of concentrate feed, HC; 50% weight ratio of concentrate, MC), and fecal metabolic characteristics and the microbiome were analyzed. Higher lactate concentrations and lower fecal pH levels were observed in the HC group (P=0.0011, P=0.0192, respectively). Fecal microbiome analysis revealed a decrease in microbial diversity (P=0.0360) and an increase in the relative abundance of Streptococcus lutetiensis/equinus/infantarius (P=0.0011) in the HC group. On the other hand, fibrolytic bacteria in the MC group had similarities with Clostridium sacchalolyticum and Ruminococcus albus. This study revealed that overfeeding of concentrates induced abnormal fermentation in the hindgut of Japanese draft horses. This suggests that the establishment of a feeding design based on not only the chemical compositions of feeds but also microbial dynamics is needed.

In vitro colonic fermentation characteristics of barley-koji differ from those of barley.

Barley-koji is prepared by inoculating barley, a beneficial prebiotic source, with the fungi Aspergillus luchuensis mut. kawachii. In this study, the prebiotic effects of barley-koji on human colonic microbiota were evaluated in vitro compared with barley, using pig feces. The enzyme-resistant fraction of the following sample groups each was added to respective fermenters: cellulose, barley (Commander and ß104), and barley-koji (Commander-koji and ß104-koji). Short-chain fatty acid and ammonia-nitrogen production increased and decreased, respectively, in barley-koji and barley groups. Furthermore, the propionate concentration increased in the barley group, showing a positive correlation with the abundance of the genus Dialister. In the barley-koji group, however, acetate and n-butyrate concentrations increased during the early stages of incubation, and the relative abundance of the genus Megasphaera was higher than those of the other genera. Therefore, this study demonstrated that barley-koji might possess beneficial physiological properties for colonic fermentation, which differ from those of barley.

Combined effects of BARLEYmax and cocoa polyphenols on colonic microbiota and bacterial metabolites in vitro.

BARLEYmax, a barley variety, and cocoa polyphenols (CPPs) have been reported to affect bacterial metabolites in the colon. This study aimed to evaluate the combined effects of BARLEYmax and CPPs supplementation on fecal microbiota in vitro using pig feces for 48 h. The relative abundances of the family Clostridiaceae and the genus Clostridium and ammonia-nitrogen production were decreased by both BARLEYmax and CPP supplementation, and there was a positive correlation between their abundances and the ammonia-nitrogen concentration. Although acetate and n-butyrate production was decreased by CPP supplementation, their concentrations were maintained at a higher level in the BARLEYmax + CPP group than in the cellulose (control) and cellulose + CPP groups. Therefore, this study demonstrated that a combination of BARLEYmax and CPPs may be beneficial in maintaining higher short-chain fatty acid production and the elimination of potentially harmful factors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00959-z.

Insects as Novel Ruminant Feed and a Potential Mitigation Strategy for Methane Emissions.

This study is the first to evaluate the chemical composition and impacts of four different edible insects, Acheta domesticus (A.d), Brachytrupes portentosus (B.p), Gryllus bimaculatus (G.b), and Bombyx mori (B.m), on the digestibility, rumen fermentation, and methane production when used as a substitute for 25% of the soybean meal (SBM) in a ruminant diet through in vitro incubation. The dietary treatments were 100% grass hay, 60% grass hay + 40% SBM, 60% grass hay + 30% SBM + 10% A.d, 60% grass hay + 30% SBM + 10% B.p, 60% grass hay + 30% SBM + 10% G.b, and 60% grass hay + 30% SBM + 10% B.m. The experiment was conducted as a short-term batch culture for 24 h at 39 °C, and the incubation was repeated in 3 consecutive runs. Chemical analysis of the insects showed that they were rich in fat (14-26%) with a high proportion of unsaturated fatty acids (60-70%). Additionally, the insects were rich in protein (48-61%) containing all essential amino acids and the amino acid profiles of the insects were almost the same as that of SBM. The inclusion of insects did not affect nutrient digestibility or the production of volatile fatty acids but did increase the production of ammonia-nitrogen. The addition of G.b and B.m led to decrease in methane production by up to 18% and 16%, respectively. These results reveal that substitution of 25% SBM in the diet with the tested insects had no negative impacts, and their potential to reduce methane production is an environmental benefit.

Deciphering the colonic fermentation characteristics of agavin and digestion-resistant maltodextrin in a simulated batch fermentation system.

Gut microbial fermentation of soluble dietary fibers promotes general and substrate-specific health benefits. In this study, the fermentation characteristics of two soluble branched-dietary fibers, namely, agavin (a type of agave fructans) and digestion-resistant maltodextrin (RD) were investigated against cellulose, using a simulated colonic fermenter apparatus employing a mixed culture of swine fecal bacteria. After 48 h of complete fermentation period, the microbial composition was different among all groups, where Bifidobacterium spp. and Lactobacillus spp. dominated the agavin treatment, while the members of the families Lachnospiraceae and Prevotellaceae dominated the RD treatment. Agavin treatment exhibited a clearly segregated two-phased prolonged fermentation trend compared to RD treatment as manifested by the fermentation rates. Further, the highest short-chain fatty acids production even at the end of the fermentation cycle, acidic pH, and the negligible concentration of ammonia accumulation demonstrated favorable fermentation attributes of agavin compared to RD. Therefore, agavin might be an effective and desirable substrate for the colonic microbiota than RD with reference to the expressed microbial taxa and fermentation attributes. This study revealed a notable significance of the structural differences of fermentable fibers on the subsequent fermentation characteristics.

The Efficacy of Plant-Based Bioactives Supplementation to Different Proportion of Concentrate Diets on Methane Production and Rumen Fermentation Characteristics In Vitro.

This In Vitro study was conducted to investigate the impact of plant-bioactives extract (PE), a combination of garlic powder and bitter orange extract, on methane production, rumen fermentation, and digestibility in different feeding models. The dietary treatments were 1000 g grass/kg ration + 0 g concentrate/kg ration (100:0), 80:20, 60:40, 40:60, and 20:80. The PE was supplemented at 200 g/kg of the feed. Each group consisted of 6 replicates. The experiment was performed as an In Vitro batch culture for 24 h at 39 °C. This procedure was repeated in three consecutive runs. The results of this experiment showed that supplementation with PE strongly reduced methane production in all kinds of feeding models (p < 0.001). Its efficacy in reducing methane/digestible dry matter was 44% in the 100:0 diet, and this reduction power increased up to a 69.2% with the inclusion of concentrate in the 20:80 diet. The PE application significantly increased gas and carbon dioxide production and the concentration of ammonia-nitrogen, but decreased the pH (p < 0.001). In contrast, it did not interfere with organic matter and fiber digestibility. Supplementation with PE was effective in altering rumen fermentation toward less acetate and more propionate and butyrate (p < 0.001). Additionally, it improved the production of total volatile fatty acids in all feeding models (p < 0.001). In conclusion, the PE combination showed effective methane reduction by improving rumen fermentation characteristics without exhibiting adverse effects on fiber digestibility. Thus, PE could be used with all kinds of feeding models to effectively mitigate methane emissions from ruminants.

Frozen Autoclaved Sorghum Enhanced Colonic Fermentation and Lower Visceral Fat Accumulation in Rats.

As raw sorghum is not able to influence considerable colonic fermentation despite its higher resistant starch (RS) content, our study aimed to investigate the effects of frozen autoclaved sorghum on colonic fermentation. Fischer 344 rats were fed frozen cooked refined (S-Rf) and whole (S-Wh) sorghum diets and were compared against α-corn starch (CON) and high amylose starch (HAS) fed rats for zoometric parameters, cecal biochemical and microbiological parameters. Sorghum fed rats exhibited significantly lower feed intake and visceral adipose tissue mass compared to CON. Bacterial alpha diversity was significantly higher in the sorghum fed rats compared to HAS and the two sorghum fed groups clustered together, separately from HAS and CON in the beta diversity plot. Serum non-High Density Lipoprotein cholesterol and total cholesterol in S-Rf group were significantly lower compared to CON, while total fecal bile excretion was also significantly higher in the two sorghum fed groups. Lower visceral adiposity was correlated with lower feed intake, RS content ingested and cecal short chain fatty acid (SCFA) contents. Thus, higher RS inflow to the colon via frozen autoclaved sorghum might have influenced colonic fermentation of RS and the resultant SCFA might have influenced lower adiposity as manifested by the lower body weight gain.

In vitro fermentation potential of the residue of Korean red ginseng root in a mixed culture of swine faecal bacteria.

Agricultural by-products such as the ginseng residue contain dietary fibre. This study was aimed at investigating the intestinal fermentation potential of the residue of Korean red ginseng root using an in vitro simulator of the colon using swine fecal bacteria. pH-Controlled glass fermentors were used to conduct a small scale in vitro batch fermentation under anaerobic conditions for 48 h. One of the following substrates was included in each fermentor: commercial cellulose (CEL), xylooligosaccharide (XOS), and crude ginseng-insoluble fibre (CGF). The pH was lower (p < 0.05) and the production of total short chain fatty acid was increased (p < 0.05) in the XOS and CGF groups compared with the CEL group after 6 h of incubation. The α-diversity analysis of the microbial community at 48 h showed that the number of bacterial species was (p < 0.05) reduced in the XOS and CGF groups compared with that in the CEL group. ß-Diversity of the microbial population at 48 h showed that all groups were clustered differently. The relative abundance of Bifidobacterium and Prevotella in the CGF group were significantly (p < 0.05) higher than those in the CEL and XOS groups. Ammonia nitrogen production in the XOS and CGF groups was (p < 0.05) lower after 6 h of incubation, and skatole production in the CGF group was (p < 0.05) lower at 48 h than that in the CEL group. These results suggested that the ginseng residue might be fermentable in the large intestine and thus would promote the maintenance of a healthy colonic environment in the host.

Dietary adzuki bean paste dose-dependently reduces visceral fat accumulation in rats fed a normal diet.

The aim of this study was to evaluate the dose-dependent effect of adzuki bean (Vigna angularis) paste (ABP) on visceral fat accumulation in rats. ABP is a rich source of indigestible carbohydrates (18.5%) with fiber and resistant starch (RS) contents of 14.5% and 4.0%, respectively. Animals were fed one of the following diets, control (CON), 30% ABP or 58.9% ABP for 28 days. The daily dietary energy intake was lowered (p < 0.05) and reduced visceral fat accumulation and lower serum lipid levels were observed in ABP fed groups. ABP consumption dose-dependently increased (p < 0.05) the daily fecal lipid and fecal acidic sterol excretions. On the other hand, cecal content and fecal moisture content in the 58.9% ABP group were greater (p < 0.05) than the CON group, while there was no significant difference between the two ABP fed groups. Both 30% and 58.9% ABP diets had significantly (p < 0.05) higher contents of cecal acetic, propionic and n-butyric acids, and lowered cecal pH, independently of the ABP dose. Microbial community data of rats fed ABP diets exhibited higher alpha-diversities than the rats fed CON diet, based on the Shannon Index and the number of observed species index, where the two ABP groups exhibited a similar alpha diversity. The weighted UniFrac-based principal coordinate analysis plot of cecal microbial community data showed that the ABP had a substantial effect on the cecal microbial composition. Furthermore, cecal bacterial 16S rRNA gene sequencing revealed that the ABP supplemented diets decreased the ratio of Firmicutes to Bacteroidetes. These findings suggested that the cecal fermentation of fiber and RS in ABP, might have decreased the energy intake, altered the gut microbiota composition, increased fecal lipid output, and thereby reduced fat accumulation in rats.

Impacts of Mootral on Methane Production, Rumen Fermentation, and Microbial Community in an in vitro Study.

Methane mitigation strategies have a two-sided benefit for both environment and efficient livestock production. This preliminary short-term in vitro trial using Mootral (garlic and citrus extracts), a novel natural feed supplement, was conducted to evaluate its efficacy on rumen fermentation characteristics, methane production, and the bacterial and archaeal community. The experiment was performed as a batch culture using rumen fluid collected from sheep, and Mootral was supplemented in three concentrations: 0% (Control), 10%, and 20% of the substrate (50% Grass:50% Concentrate). The rumen fermentation data and alpha diversity of microbial community were analyzed by ordinary one-way analysis of variance. The relative abundance and statistical significance of families and operational taxonomic units (OTUs) among the groups were compared by Kruskal-Wallis H test using Calypso software. After 24-h incubation at 39°C, Mootral in a dose-dependent manner improved the production of total volatile fatty acids and propionate while it reduced the acetate proportion and acetate/propionate ratio. The total produced gas was two times higher in the Mootral-supplemented groups than control (P < 0.01), while the proportion of methane in the produced gas was reduced by 22% (P < 0.05) and 54% (P < 0.01) for 10 and 20% Mootral, respectively. Mootral did not change pH, digestibility, and ammonia-nitrogen. Microbial community analyses showed that Mootral effectively changed the ruminal microbiome. The bacterial community showed an increase of the relative abundance of the propionate-producing family such as Prevotellaceae (P = 0.014) and Veillonellaceae (P = 0.030), while there was a decrease in the relative abundance of some hydrogen-producing bacteria by Mootral supplementation. In the archaeal community, Methanobacteriaceae was decreased by Mootral supplementation compared with control (P = 0.032), while the Methanomassiliicoccaceae family increased in a dose-dependent effect (P = 0.038). The results of the study showed the efficacy of the new mixture to alter the ruminal microbial community, produce more propionate, and reduce microbial groups associated with methane production, thus suggesting that Mootral is a promising natural mixture for methane reduction from ruminants.

In Vivo Colonic Fermentation of Sorghum (Sorghum bicolor L.): Important Correlations Observed among the Physiological Parameters of Cecum, Liver, Adipose Tissue and Fasting Serum Lipid Profile.

High amylose corn starch (HAS), whole grain sorghum (S-Wh), refined sorghum (S-Rf) and α-corn starch (CON) diets were fed to animals for 1 mo aiming to examine the physiological effects of resistant starch inclusion in the diet from grains. HAS exhibited significantly lower feed intake, final body weight, serum lipid profile with significantly higher cecal parameters and short chain fatty acid (SCFA) contents. S-Wh group exhibited significantly higher body weight, feed intake and serum lipid parameters compared to other 3 groups. Cecal fermentation was not seemed to be prominent in the CON, S-Wh and S-Rf groups with respect to lower cecal parameters and SCFA contents. The cecal microbial compositions in HAS, S-Wh and CON/S-Rf exhibited 3 distinct clusters suggesting a significant effect of the cecal microbial composition on cecal parameters, SCFA contents and physiological parameters.

Purple Sweet Potato Polyphenols Differentially Influence the Microbial Composition Depending on the Fermentability of Dietary Fiber in a Mixed Culture of Swine Fecal Bacteria.

The prevalence of many chronic diseases which have been associated with poor nutrition may be reduced by the positive modulation of colonic microbiota. In this study, we assess the effects of purple sweet potato polyphenols (PSP) in a mixed culture of swine fecal bacteria during in vitro colonic fermentation using pig colonic digest. Jar fermenters were used to conduct a small scale in vitro colonic fermentation experiments under the anaerobic condition for 48 h. Jar fermenters were assigned to one of the following groups: Cellulose, cellulose + PSP, inulin, and inulin + PSP. The present study revealed that the polyphenolic content of purple sweet potato could modulate the colonic microbiota by differentially increasing the population of beneficial bacteria and decreasing the pathogenic bacteria depending on cellulose and inulin. Accordingly, PSP might be a material conducive for improving the conditions for the fermentation of partly-fermentable dietary fiber. Besides, PSP was also responsible for the drastic reduction of putrefactive products, especially p-cresol to a significant level. Our results suggest that PSP could alter the microbial composition depending upon the fermentability of dietary fiber and has the potential to maintain a stable and healthy colonic environment that will ultimately alleviate chronic diseases development and confer health benefits to the host.

Colonic fermentation of water soluble fiber fraction extracted from sugarcane (Sacchurum officinarum L.) bagasse in murine models.

Biochemical effects of the water soluble fiber fraction of sugarcane bagasse (BSF) fermented in the colon was examined to evaluate its potential health promoting effects. A feeding experiment involving Fischer 344 rats, was conducted with 3 experimental diets containing, cellulose (CON), a commercial xylo-oligosaccharide (XYO) and BSF (BGS). Cumulative feed intake was significantly lower in XYO group while cecal weight was significantly higher. Acetic and propionic acid contents in the cecal content were significantly higher in the BGS and XYO, respectively. Total short chain fatty acid content was significantly higher in BGS and XYO resulting significantly lower cecal pH. Beneficial bacteria such as Bifidobacterium, Blautia, Akkermansia and Roseburia abundance was significantly higher in the XYO and BGS groups. Further, mucin and immunoglobulin-A contents were significantly higher in BGS group compared to CON group. Thus, BSF exhibited its ability to enhance the intestinal and systemic health upon fermentation in the colon.

The Performance of an Oral Microbiome Biomarker Panel in Predicting Oral Cavity and Oropharyngeal Cancers.

The oral microbiome can play a role in the instigation and progression of oral diseases that can manifest into other systemic conditions. These associations encourage the exploration of oral dysbiosis leading to the pathogenesis of cancers. In this study, oral rinse was used to characterize the oral microbiome fluctuation associated with oral cavity cancer (OCC) and oropharyngeal cancers (OPC). The study cohort consists of normal healthy controls (n = 10, between 20 and 30 years of age; n = 10, above 50 years of age), high-risk individuals (n = 11, above 50 years of age with bad oral hygiene and/or oral diseases) and OCC and OPC patients (n = 31, HPV-positive; n = 21, HPV-negative). Oral rinse samples were analyzed using 16S rRNA gene amplicon sequencing on the MiSeq platform. Kruskal-Wallis rank test was used to identify genera associated with OCC and OPC. A logistic regression analysis was carried out to determine the performance of these genera as a biomarker panel to predict OCC and OPC. In addition, a two-fold cross-validation with a bootstrap procedure was carried out in R to investigate how well the panel would perform in an emulated clinical scenario. Our data indicate that the oral microbiome is able to predict the presence of OCC and OPC with sensitivity and specificity of 100 and 90%, respectively. With further validation, the panel could potentially be implemented into clinical diagnostic and prognostic workflows for OCC and OPC.

Food Starch Structure Impacts Gut Microbiome Composition.

Starch is a major source of energy in the human diet and is consumed in diverse forms. Resistant starch (RS) escapes small intestinal digestion and is fermented in the colon by the resident microbiota, with beneficial impacts on colonic function and host health, but the impacts of the micro- and nanoscale structure of different physical forms of food starch on the broader microbial community have not been described previously. Here, we use a porcine in vitro fermentation model to establish that starch structure dramatically impacts microbiome composition, including the key amylolytic species, and markedly alters both digestion kinetics and fermentation outcomes. We show that three characteristic food forms of starch that survive digestion in the small intestine each give rise to substantial and distinct changes in the microbiome and in fermentation products. Our results highlight the complexity of starch fermentation processes and indicate that not all forms of RS in foods are degraded or fermented in the same way. This work points the way for the design of RS with tailored degradation by defined microbial communities, informed by an understanding of how substrate structure influences the gut microbiome, to improve nutritive value and/or health benefits.IMPORTANCE Dietary starch is a major component in the human diet. A proportion of the starch in our diet escapes digestion in the small intestine and is fermented in the colon. In this study, we use a model of the colon, seeded with porcine feces, in which we investigate the fermentation of a variety of starches with structures typical of those found in foods. We show that the microbial community changes over time in our model colon are highly dependent on the structure of the substrate and how accessible the starch is to colonic microbes. These findings have important implications for how we classify starches reaching the colon and for the design of foods with improved nutritional properties.

Monitoring of gene expression in Fibrobacter succinogenes S85 under the co-culture with non-fibrolytic ruminal bacteria.

Fibrobacter succinogenes is one of the most pivotal fibrolytic bacterial species in the rumen. In a previous study, we confirmed enhancement of fiber digestion in a co-culture of F. succinogenes S85 with non-fibrolytic ruminal strains R-25 and/or Selenomonas ruminantium S137. In the present study, mRNA expression level of selected functional genes in the genome of F. succinogenes S85 was monitored by real-time RT-PCR. Growth profile of F. succinogenes S85 was similar in both the monoculture and co-cultures with non-fibrolytics. However, expression of 16S rRNA gene of F. succinogenes S85 in the co-culture was higher (P < 0.01) than that of the monoculture. This finding suggests that metabolic activity of F. succinogenes S85 was enhanced by coexistence with strains R-25 and/or S. ruminantium S137. The mRNA expression of fumarate reductase and glycoside hydrolase genes was up-regulated (P < 0.01) when F. succinogenes S85 was co-cultured with non-fibrolytics. These results indicate the enhancement of succinate production and fiber hydrolysis by F. succinogenes S85 in co-cultures of S. ruminantium and R-25 strains.

Involvement of recently cultured group U2 bacterium in ruminal fiber digestion revealed by coculture with Fibrobacter succinogenes S85.

In a previous study, we reported the ecological significance of uncultured bacterial group U2 in the rumen. In this study, the involvement of a recently cultured group U2 bacterium, strain R-25, in fiber digestion was tested in coculture with the fibrolytic bacterium Fibrobacter succinogenes S85. Dry matter (DM) digestion, growth and metabolites were examined in culture using rice straw as the carbon source. Although strain R-25 did not digest rice straw in monoculture, coculture of strain R-25 and F. succinogenes S85 showed enhanced DM digestion compared with that for F. succinogenes S85 monoculture (36.9 ± 0.6% vs. 32.8 ± 1.3%, P < 0.05). Growth of strain R-25 and production of the main metabolites, d-lactate (strain R-25) and succinate (F. succinogenes S85), were enhanced in the coculture. Enzyme assay showed increased activities of carboxymethylcellulase and xylanase in coculture of strain R-25 and F. succinogenes S85. Triculture including strain R-25, F. succinogenes S85 and Selenomonas ruminantium S137 showed a further increase in DM digestion (41.8 ± 0.8%, P < 0.05) with a concomitant increase in propionate, produced from the conversion of d-lactate and succinate. These results suggest that the positive interaction between strains R-25 and F. succinogenes S85 causes increased rice straw digestion.