Similar In Vitro Ileal Fermentation Outcomes Validate the Use of a Pig Ileal Inoculum in an In Vitro Fermentation Assay for the Adult Human.

BACKGROUND: An in vivo/in vitro ileal fermentation assay using growing pigs has shown important fermentability and organic acid production. This assay could be used to study human foods but needs validation. OBJECTIVES: A pig inoculum for studying human ileal fermentation was used to compare the in vitro fermentation of fiber substrates using ileal inocula prepared from growing pigs or human ileostomates. METHODS: Ten pigs (19 ± 4.5 kg bodyweight, mean ± standard deviation) received a diet containing human foods. After 2 wk, ileal digesta were collected 5 h postmeal. Five recruited human ileostomates incorporated the same human foods into their diet for a week before consuming 2 meals similar to the pigs' diet. Ileal effluents were then collected from 2 to 6 h postmeal. The porcine ileal digesta and human ileal effluents were used for microbial analysis and in vitro fermentation of arabinogalactan, fructooligosaccharides, and pectin. RESULTS: The in vitro organic matter fermentability of arabinogalactan, fructooligosaccharides, and pectin was similar (P > 0.05) between the pig and human ileal inocula (34 ± 2.13% on average). Regardless of substrates, the propionic and lactic acid production was similar between humans and pigs (P > 0.05). Ninety percent of the ileal bacterial genera were found in similar (P > 0.05) numbers in pigs and human ileostomates, which accords with the similar (P > 0.05) Shannon diversity index and predicted metabolic activity. However, some of the most abundant genera were different between species, such as Granulicatella which had 83-fold greater (P ≤ 0.05) numbers in human ileostomates, and Lactobacillus had 272-fold greater (P ≤ 0.05) numbers in pigs. CONCLUSIONS: The in vitro ileal fermentation patterns were similar across species despite some ileal microbial compositional differences, suggesting that the growing pig could be used as a model to provide an ileal inoculum for studying ileal fermentation in adult humans. This trial was registered at the Australian New Zealand Clinical Trials registry as ACTRN12622000813785.

Gut microbiota-mediated metabolism of green tea catechins and the biological consequences: An updated review.

Multiple beneficial effects have been attributed to green tea catechins (GTCs). However, the bioavailability of GTCs is generally low, with only a small portion directly absorbed in the small intestine. The majority of ingested GTCs reaches the large intestinal lumen, and are extensively degraded via biotransformation by gut microbiota, forming many low-molecular-weight metabolites such as phenyl-γ-valerolactones, phenolic acids, butyrate, and acetate. This process not only improves the overall bioavailability of GTC-derived metabolites but also enriches the biological activities of GTCs. Therefore, the intra- and inter-individual differences in human gut microbiota as well as the resulting biological contribution of microbial metabolites are crucial for the ultimate health benefits. In this review, the microbial degradation of major GTCs was characterized and an overview of the in vitro models used for GTC metabolism was summarized. The intra- and inter-individual differences of human gut microbiota composition and the resulting divergence in the metabolic patterns of GTCs were highlighted. Moreover, the potential beneficial effects of GTCs and their gut microbial metabolites were also discussed. Overall, the microbial metabolites of GTCs with higher bioavailability and bioactive potency are key factors for the observed beneficial effects of GTCs and green tea consumption.

In vitro and in vivo fermentation models to study the function of dietary fiber in pig nutrition.

The importance of dietary fiber (DF) in animal diets is increasing with the advancement of nutritional research. DF is fermented by gut microbiota to produce metabolites, which are important in improving intestinal health. This review is a systematic review of DF in pig nutrition using in vitro and in vivo models. The fermentation characteristics of DF and the metabolic mechanisms of its metabolites were summarized in an in vitro model, and it was pointed out that SCFAs and gases are the important metabolites connecting DF, gut microbiota, and intestinal health, and they play a key role in intestinal health. At the same time, some information about host-microbe interactions could have been improved through traditional animal in vivo models, and the most direct feedback on nutrients was generated, confirming the beneficial effects of DF on sow reproductive performance, piglet intestinal health, and growing pork quality. Finally, the advantages and disadvantages of different fermentation models were compared. In future studies, it is necessary to flexibly combine in vivo and in vitro fermentation models to profoundly investigate the mechanism of DF on the organism in order to promote the development of precision nutrition tools and to provide a scientific basis for the in-depth and rational utilization of DF in animal husbandry. KEY POINTS: • The fermentation characteristics of dietary fiber in vitro models were reviewed. • Metabolic pathways of metabolites and their roles in the intestine were reviewed. • The role of dietary fiber in pigs at different stages was reviewed.

In vitro conversion of ellagic acid to urolithin A by different gut microbiota of urolithin metabotype A.

The metabolite urolithin A, a metabolite of the dietary polyphenol ellagic acid (EA), has significant health benefits for humans. However, studies on the gut microbiota involved in ellagic acid metabolism are limited. In this study, we conducted in vitro fermentation of EA using human intestinal microbiome combined with antibiotics (vancomycin, polymyxin B sulfate, and amphotericin B). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis demonstrated that the production capacity of urolithin A by gut microbiota co-treated with polymyxin B sulfate and amphotericin B (22.39 µM) was similar to that of untreated gut microbiota (24.26 µM). Macrogenomics (high-throughput sequencing) was used to analyze the composition and structure of the gut microbiota. The results showed that the abundance of Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum in the gut microbiota without antibiotic treatment or co-treated with polymyxin B sulfate and amphotericin B during EA fermentation was higher than that in other antibiotic treatment gut microbiota. Therefore, B. longum, B. adolescentis, and B. bifidum may be new genera involved in the conversion of EA to urolithin A. In conclusion, the study revealed unique interactions between polyphenols and gut microbiota, deepening our understanding of the relationship between phenolic compounds like EA and the gut microbiota. These findings may contribute to the development of gut bacteria as potential probiotics for further development. KEY POINTS: • Intestinal microbiome involved in ellagic acid metabolism. • Gram-positive bacteria in the intestinal microbiome are crucial for ellagic acid metabolism. • Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum participate in ellagic acid metabolism.

Turmeric rhizomes reduced in vitro methane production and improved gas production and nutrient degradability.

The present study aimed to evaluate the effect of dry turmeric rhizomes on in vitro biogas production and diet fermentability. Turmeric rhizomes were included at gradually increased levels: 0, 0.5, 1, 1.5 and 2% of a diet containing per kg dr matter (DM): 500 g concentrate feed mixture, 400 g berseem hay and 100 g rice straw, and incubated for 48 h. Gas chromatography-mass spectrometry analysis showed that ar-turmerone, α-turmerone and ß-turmerone were the major bioactive compounds in the rhizomes. Turmeric rhizomes increased (p < 0.01) asymptotic gas production (GP) and rate and lag of CH4 production and decreased (p < 0.01) rate of GP, lag of GP, asymptotic CH4 production and proportion of CH4 production. Turmeric rhizome administration linearly increased (p < 0.01) DM and fiber degradability and concentrations of total short-chain fatty acids, acetic and propionic acids and ammonia-N and quadratically (p < 0.05) decreased fermentation pH. It is concluded that including up to 2% turmeric rhizomes improved in vitro ruminal fermentation and decreased CH4 production.

Effects of 2'-fucosyllactose on the composition and metabolic activity of intestinal microbiota from piglets after in vitro fermentation.

BACKGROUND: As indigestible carbohydrates, milk oligosaccharides possess various benefits for newborns, mainly through intestinal microbiota, among which 2'-fucosyllactose (2'-FL) is the most predominant milk oligosaccharide. However, knowledge about the fermentative characteristics of 2'-FL in the gut remains limited, especially in the small intestine. The aim of this study is to explore the differential fermentability of 2'-FL by the small and large intestinal microbiota of piglets using fructo-oligosaccharide (FOS) and lactose as controls in an in vitro batch fermentation experiment. During fermentation, microbial composition was characterized along with gas production and short-chain fatty acid production. RESULTS: 2'-Fucosyllactose showed differential fermentability in jejunal and colonic fermentation. Compared with the colon, 2'-FL produced less gas in the jejunum than in the FOS and lactose groups (P < 0.05). Meanwhile, 2'-FL exhibited a different influence on the microbial composition and metabolism in the jejunum and colon compared with FOS and lactose. In the jejunum, compared with the FOS and lactose groups, the 2'-FL group showed a higher abundance of Bacteroides, Prevotella, and Blautia, but a lower abundance of Streptococcus and Lactobacillus (P < 0.05), with a higher level of propionate and a lower level of lactate during fermentation (P < 0.05). In the colon, compared with the FOS and lactose groups, 2'-FL increased the abundance of Blautia, Faecalibacterium, and Lachnospiraceae FCS020, but decreased the abundance of Prevotella_9, Succinivibrio, and Megasphaera (P < 0.05) with an increase in acetate production (P < 0.05). CONCLUSION: Overall, the results suggested that the small intestinal microbiota had the potential to ferment milk oligosaccharides. Meanwhile, in comparison with FOS and lactose, 2'-FL selectively stimulated the growth of propionate-producing bacteria in the jejunum and acetate-producing bacteria in the colon. These results demonstrated the differences in fermentation properties of 2'-FL by small and large intestinal microbiota and provided new evidence for the application of 2'-FL in optimizing gut microbiota. © 2023 Society of Chemical Industry.

In vitro fermentation characteristics of different types of fiber-rich ingredients by pig fecal inoculum.

BACKGROUND: Dietary fibers with varying physicochemical properties have different fermentation characteristics, which may differently impact host health. The present study aimed to determine the fermentation characteristics including gas production kinetics, short-chain fatty acids (SCFAs) production and microbial composition of different fibrous ingredients using in vitro fermentation by fecal microbiota. RESULTS: Sugar beet pule (SBP), wheat bran (WB), dried corn distillers grains with solubles (DDGS), rice bran (RB) and alfalfa meal (AM) were selected to fermentation in vitro for 36 h. The results showed that SBP had the greatest gas production. SBP had the highest in vitro dry matter fermentability (IVDMF) and production of acetate, propionate and total SCFAs, followed by WB, which were all greater than DDGS, AM and RB. The alpha-diversity was higher in the DDGS, AM and RB groups than in the WB and SBP groups. Differences in microbial community composition were observed among groups. The relative abundance of Treponema was highest in WB group. RB group showed lower Prevotella abundance than other groups but had higher Succinivibrio abundance. Interestingly, the Lactobacillus reached the highest abundances in the DDGS group. Correlation analysis indicated that the relative abundance of Treponema and Prevotella was positively associated with the gas production, IVDMF and SCFAs, whereas norank_f_Muribaculaceae, Rikenellaceae_RC9_gut_group, Lysinibacillus and Succinivibrio were the opposite. CONCLUSION: Collectively, WB and SBP were fermented rapidly by fecal microbiota compared to DDGS, AM and RB. Different fiber sources have different fiber compositions and fermentation properties that affect the microbial compositins and SCFAs production. © 2024 Society of Chemical Industry.

Novel techniques for the mass production of nutritionally improved, fungus-treated lignocellulosic biomass for ruminant nutrition.

BACKGROUND: Laboratory-scale experiments have shown that treatment with selective lignin-degrading white-rot fungi improves the nutritional value and ruminal degradability of lignocellulosic biomass (LCB). However, the lack of effective field-applicable pasteurization methods has long been recognized as a major obstacle for scaling up the technique for fungal treatment of large quantities of LCB for animal feeding. In this study, wheat straw (an LCB substrate) was subjected to four field-applicable pasteurization methods - hot-water, formaldehyde fumigation, steam, and hydrated lime - and cultured with Pleurotus ostreatus grain spawn for 10, 20, and 30 days under solid-state fermentation. Samples of untreated, pasteurized but non-inoculated and fungus-treated straws were analyzed for chemical composition, aflatoxin B1 (AFB1 ), and in vitro dry matter digestibility (IVDMD), in vitro total gas (IVGP), methane (CH4 ), and volatile fatty acid (VFA) production. RESULTS: During the 30-day fungal treatment, steam and lime pasteurized straws had the greatest loss of lignin, resulting in marked improvements in crude protein (CP), IVDMD, IVGP, and total VFAs. Irrespective of the pasteurization method, the increase in IVDMD during fungal treatment was linearly (R2 = 0.77-0.92) related to lignin-loss in the substrate during fungal treatment. The CH4 production of the fungus-treated straw was not affected by the pasteurization methods. Aflatoxin B1 was within the safe level (<5 µg kg-1 ) in all pasteurized, fungus treated straws. CONCLUSION: Steam and lime were promising field-applicable pasteurization techniques to produce nutritionally improved fungus-treated wheat straw to feed ruminants. Lime pasteurization was more economical and did not require expensive energy inputs. © 2023 Society of Chemical Industry.

Catabolism of phenolics from grape peel and its effects on gut microbiota during in vitro colonic fermentation.

BACKGROUND: Grape peels, the main by-products of wine processing, are rich in bioactive ingredients of phenolics, including proanthocyanidins, flavonoids and anthocyanins. Phenolics have the function of regulating intestinal microbiota and promoting intestinal health. From the perspective of the dietary nutrition of grape peel phenolics (GPP), the present study aimed to investigate the influence of GPP on the composition and metabolism of human gut microbiota during in vitro fermentation. RESULTS: The results indicated that GPP could decrease pH and promote the production of short-chain fatty acids. ACE and Chao1 indices in GPP group were lower than that of the Blank group. GPP enhanced the levels of Lachnospiraceae UCG-004, Bacteroidetes and Roseburia, but reduced the Firmicutes/Bacteroidetes ratio. Kyoto Encyclopedia of Proteins and Genome enrichment pathways related to phenolic acid metabolism mainly included flavonoid, anthocyanin, flavone and flavonol biosynthesis. Gut microbiota could accelerate the release and breakdown of phenolic compounds, resulting in a decrease in the content of hesperetin-7-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-rutinoside etc. In vitro antibacterial test found that GPP increased the diameters of the inhibition zones of Escherichia coli and Staphylococcus aureus in a dose-dependent manner. CONCLUSION: The results of the present study revealed that GPP might be a potential prebiotic-like to prevent diseases by improving gut health. The findings could provide a theoretical basis for the potential to exploit GPP as dietary nutrition to maintain intestinal function. © 2024 Society of Chemical Industry.

Effects of Saccharomyces cerevisiae and Bacillus subtilis on in vitro fermentation in the rumen of Hu sheep.

BACKGROUND: The demand for animal products is increasing in developing countries due to population growth. However, livestock production contributes significantly to global warming, accounting for 25%. Probiotics can help improve livestock efficiency by enhancing gut microbes and fat metabolism. They can modify rumen populations, enhance fermentation, reduce methane emissions and improve feed digestion. In this study, the goal was to determine the most effective method of reducing methane emissions in the rumen of sheep in vitro by adding different concentrations of Saccharomyces cerevisiae and Bacillus subtilis. RESULTS: Adding 8 × 106 CFU g-1 S. cerevisiae during fermentation reduced pH levels after 48 h. This also increased the concentrations of NH3-N, microbial protein and total gas production. At the same time, it decreased methane emissions. Furthermore, adding 20 × 106 CFU g-1 B. subtilis to the mixture increased total gas production (TGP) and methane production, with the highest production observed after 48 h. However, it did not affect pH levels after 48 h. CONCLUSION: It can be concluded that S. cerevisiae had significantly increased microbial protein and NH3-N concentrations after fermentation without altering pH. Additionally, the addition of S. cerevisiae enhanced TGP and reduced methane emissions. It is worth noting that TGP increased because B. subtilis was added at a concentration of 20 × 106 CFU g-1, with no significant differences between concentrations. Therefore, we recommend adding S. cerevisiae and B. subtilis to the diet at doses of 8 and 20 × 106 CFU g-1, as it resulted in higher TGP and reduced methane emissions. © 2024 Society of Chemical Industry.

Anti-inflammatory activity of ß-glucans from different sources before and after fermentation by fecal bacteria in vitro.

BACKGROUND: ß-Glucans are widely sourced and have various physiological effects, including anti-inflammatory effects. However, the strength of the anti-inflammatory activity of ß-glucans from different sources remains unknown due to the lack of rapid and effective biomarkers. This study therefore aimed to screen out the ß-glucans with strong anti-inflammatory activity from five different sources and to further screen out possible biomarkers in metabolites after fermenting the ß-glucans with gut microorganisms. RESULTS: The results showed that all five ß-glucans inhibited the production of lipopolysaccharide (LPS)-induced pro-inflammatory mediators and suppressed the mRNA expression level of TLR4/MyD88. Their anti-inflammatory mechanisms involved the inhibition of intracellular reactive oxygen species (ROS) production and suppression of mRNA expression of the NF-κB pathway and JNK pathway. Among them, barley ß-glucan exhibited the strongest anti-inflammatory effect, followed by Ganoderma lucidum ß-glucan. Enhanced anti-inflammatory activity of ß-glucan was found after fermentation and may be related to the increased abundance of metabolites such as vanillin, dihydroxyphenylacetic acid, caffeic acid, acetic acid, butyric acid, and lactic acid. They were strongly positively correlated to the abundance of beneficial bacteria such as Blautia, suggesting that the production of those metabolites may be responsible for the flourishing of the beneficial bacteria. CONCLUSION: In conclusion, barley was a preferred raw material for the preparation of ß-glucans with strong anti-inflammatory activity. Vanillin, dihydroxyphenylacetic acid, caffeic acid, acetic acid, butyric acid, and lactic acid were the possible biomarkers that could be utilized to evaluate the anti-inflammatory effect of ß-glucans. © 2023 Society of Chemical Industry.

In vitro gut microbiome response to carbohydrate supplementation is acutely affected by a sudden change in diet.

BACKGROUND: Interactions between diet, stress and the gut microbiome are of interest as a means to modulate health and performance. Here, in vitro fermentation was used to explore the effects of a sudden change in diet, 21 days sole sustenance on the Meal, Ready-to-Eat (MRE) U.S. military combat ration, on inter-species competition and functional potential of the human gut microbiota. Human fecal samples collected before and after MRE intervention or consuming a habitual diet (HAB) were introduced to nutrient-rich media supplemented with starch for in vitro fermentation under ascending colon conditions. 16S rRNA amplicon and Whole-metagenome sequencing (WMS) were used to measure community composition and functional potential. Specific statistical analyses were implemented to detect changes in relative abundance from taxa, genes and pathways. RESULTS: Differential changes in relative abundance of 11 taxa, Dorea, Lachnospira, Bacteroides fragilis, Akkermansia muciniphila, Bifidobacterium adolescentis, Betaproteobacteria, Enterobacteriaceae, Bacteroides egerthii, Ruminococcus bromii, Prevotella, and Slackia, and nine Carbohydrate-Active Enzymes, specifically GH13_14, over the 24 h fermentation were observed as a function of the diet intervention and correlated to specific taxa of interest. CONCLUSIONS: These findings suggest that consuming MRE for 21 days acutely effects changes in gut microbiota structure in response to carbohydrate but may induce alterations in metabolic capacity. Additionally, these findings demonstrate the potential of starch as a candidate supplemental strategy to functionally modulate specific gut commensals during stress-induced states.

Interaction between ß-glucans and gut microbiota: a comprehensive review.

Gut microbiota (GMB) in humans plays a crucial role in health and diseases. Diet can regulate the composition and function of GMB which are associated with different human diseases. Dietary fibers can induce different health benefits through stimulation of beneficial GMB. ß-glucans (BGs) as dietary fibers have gained much interest due to their various functional properties. They can have therapeutic roles on gut health based on modulation of GMB, intestinal fermentation, production of different metabolites, and so on. There is an increasing interest in food industries in commercial application of BG as a bioactive substance into food formulations. The aim of this review is considering the metabolizing of BGs by GMB, effects of BGs on the variation of GMB population, influence of BGs on the gut infections, prebiotic effects of BGs in the gut, in vivo and in vitro fermentation of BGs and effects of processing on BG fermentability.

Regulation of gut microbiota and alleviation of DSS-induced colitis by vitexin.

PURPOSE: Vitexin is one of the flavonoids in millet and has a variety of biological activities. However, the function of vitexin on colitis is not clear. This research studied the regulation of vitexin on colitis and investigated the possible mechanisms. METHODS: An in vitro fermentation model was used to evaluate the regulation of vitexin on gut microbiota of patients with inflammatory bowel disease (IBD). At the same time, an acute colitis mice model induced by dextran sodium sulfate (DSS) was used to evaluate the effects of vitexin on intestinal inflammation, barrier and gut microbiota. RESULTS: In this study, it was found that vitexin altered the structure of gut microbiota by decreasing harmful bacteria, such as Veillonella, Terrisporobacter, Klebsiella, Paeniclostridium, and increasing beneficial bacteria, such as Parabacteroides, Flavonifractor, Blautia after in vitro fermentation with the feces of colitis patients. Further, DSS-induced colitis mice models revealed that vitexin treatment significantly improved colitis symptoms, maintained intestinal barrier and down-regulated the expression of inflammatory factors, such as IL-1ß and TNF-α. In addition, vitexin also improved the diversity of gut microbiota of colitis mice by decreasing the abundance of harmful bacteria. CONCLUSION: This research suggested that vitexin could alleviate colitis by regulating gut microbiota and attenuated gut inflammation.

Buckwheat (Fagopyrum esculentum) Hulls Are a Rich Source of Fermentable Dietary Fibre and Bioactive Phytochemicals.

Pseudo-cereals such as buckwheat (Fagopyrum esculentum) are valid candidates to promote diet biodiversity and nutrition security in an era of global climate change. Buckwheat hulls (BHs) are currently an unexplored source of dietary fibre and bioactive phytochemicals. This study assessed the effects of several bioprocessing treatments (using enzymes, yeast, and combinations of both) on BHs' nutrient and phytochemical content, their digestion and metabolism in vitro (using a gastrointestinal digestion model and mixed microbiota from human faeces). The metabolites were measured using targeted LC-MS/MS and GC analysis and 16S rRNA gene sequencing was used to detect the impact on microbiota composition. BHs are rich in insoluble fibre (31.09 ± 0.22% as non-starch polysaccharides), protocatechuic acid (390.71 ± 31.72 mg/kg), and syringaresinol (125.60 ± 6.76 mg/kg). The bioprocessing treatments significantly increased the extractability of gallic acid, vanillic acid, p-hydroxybenzoic acid, syringic acid, vanillin, syringaldehyde, p-coumaric acid, ferulic acid, caffeic acid, and syringaresinol in the alkaline-labile bound form, suggesting the bioaccessibility of these phytochemicals to the colon. Furthermore, one of the treatments, EC_2 treatment, increased significantly the in vitro upper gastrointestinal release of bioactive phytochemicals, especially for protocatechuic acid (p < 0.01). The BH fibre was fermentable, promoting the formation mainly of propionate and, to a lesser extent, butyrate formation. The EM_1 and EC_2 treatments effectively increased the content of insoluble fibre but had no effect on dietary fibre fermentation (p > 0.05). These findings promote the use of buckwheat hulls as a source of dietary fibre and phytochemicals to help meet dietary recommendations and needs.

Effects of Four Extraction Methods on Structure and In Vitro Fermentation Characteristics of Soluble Dietary Fiber from Rape Bee Pollen.

In this study, soluble dietary fibers (SDFs) were extracted from rape bee pollen using four methods including acid extraction (AC), alkali extraction (AL), cellulase extraction (CL) and complex enzyme extraction (CE). The effects of different extraction methods on the structure of SDFs and in vitro fermentation characteristics were further investigated. The results showed that the four extraction methods significantly affected the monosaccharide composition molar ratio, molecular weight, surface microstructure and phenolic compounds content, but showed little effect on the typical functional groups and crystal structure. In addition, all SDFs decreased the Firmicutes/Bacteroidota ratio, promoted the growth of beneficial bacteria such as Bacteroides, Parabacteroides and Phascolarctobacterium, inhibited the growth of pathogenic bacteria such as Escherichia-Shigella, and increased the total short-chain fatty acids (SCFAs) concentrations by 1.63-2.45 times, suggesting that the bee pollen SDFs had a positive regulation on gut microbiota. Notably, the SDF obtained by CE exhibited the largest molecular weight, a relatively loose structure, higher extraction yield and phenolic compounds content and the highest SCFA concentration. Overall, our results indicated that CE was an appropriate extraction method of high-quality bee pollen SDF.

A new equipment for continuous measurement of methane production in a batch in vitro rumen system.

A new rumen batch fermentation system that allows continuous measures of total gas (GP) and methane production (MP) was tested. The fermentation system is composed of glass bottles connected to gas counters (Ritter Apparatebau GmbH & Co. KG) and an infrared gas analyser that measures the methane concentration. The system allows direct and continuous measurement of GP and MP for accurate kinetic studies. The aim of the work was to test the rumen fermentation system and compare the GP and MP kinetics obtained. Barley meal (BM), alfalfa hay (AH), corn silage (CS), and soya bean hulls (SH) were used as substrates in four consecutive fermentation runs. Cumulative volumes of GP and MP and the percentage of methane on total GP were recorded continuously until 48 h and average values at 1 h intervals were fitted with an exponential model with a lag phase reaching a good fit (R2 > 0.992). GP and MP reached the highest plateau levels for SH (1836 and 370 ml, respectively; p < 0.01) and the lowest for AH (1000 and 233 ml, respectively). The remaining substrates showed intermediate values. MP kinetics showed a discrete lag phase (from 0.09 to 1.12 h), whereas it was equal to zero for the total GP (except for SH). The methane concentration in gas flowing increased rapidly at the beginning of fermentation (from 0.35 to 0.95 h-1 ) and reached a plateau after approximately 8-12 h. In conclusion, the rumen fermentation system evaluated generates methane data comparable to those reported in the literature and allows simple continuous measurement of methane release throughout fermentation.

Dynamics of in vitro rumen methane production after nitrate addition.

The present study aimed to assess the dynamics of rumen methane (CH4) production following the addition of NaNO3. This was done using an in vitro rumen fermentation system that ensures continuous gas and methane assessments. Four different levels of NaNO3 were used to get the final nitrate concentrations of 0.5, 1.0, 1.5, and 2.0 mg/ml of rumen fluid. For each dose, corresponding controls contained sodium chloride and urea were realised to ensure comparable levels of sodium and nitrogen. The addition of nitrates had slight effect on the intensity of fermentation because the total gas produced minus CH4 (total methane-free gas) only went down at the highest dose (2.0 mg/ml), and the final concentrations of SCFA were the same at all doses. The most evident effect was a modification of the SCFA profile (low concentrations of propionate and valerate, progressive increments of acetate, and decreases of butyrate) and a reduction in overall CH4 production. The CH4 yield for the 0.5 mg/ml dose was not different from control in the entire fermentation. Yield of the 1.0 mg/ml dose was significantly lower than the control group (p < 0.05) only within the initial 24-h period, and higher dosages (1.5 and 2.0 mg/ml) were lower during the entire fermentation (p < 0.01). Methane yields were well fitted with the Gompertz model, but only the highest level of nitrate inclusion had a significant impact on the majority of model parameters (p < 0.01). The linear regressions between CH4 yields (y) and the amounts of nitrates (x) at progressive fermentation durations (e.g. 6, 12, 24, and 48 h) produced equations with increasing absolute slopes (from -0.069 to -0.517 ml/mg of nitrate). Therefore, nitrate reduced rumen CH4 yield in a dose-dependent manner: the impact of low doses was primarily observed at the initial stages of fermentation, whereas high doses exhibited effectiveness throughout the entire fermentation process. In conclusion, in batch fermentation systems, the dose effect of nitrates on methane yield was time dependent.

Construction of recombinant Pichia pastoris strains for ammonia reduction by the gdhA and glnA regulatory genes in laying hens.

Ammonia emissions have become an important environmental challenge for the livestock industry. Probiotics are often used as additives to reduce ammonia, and the ammonia reduction efficiency of common probiotics is approximately 20-40%. In this study, we constructed a gdhA recombinant Pichia pastoris strain, glnA recombinant Pichia pastoris strain and gdhA-glnA Pichia pastoris recombinant strain using the gdhA and glnA genes, which have the potential function of reducing ammonia emissions. The results of in vitro fermentation showed that compared with the control, wild-type Pichia pastoris and pPICZA strains, the gdhA, glnA and gdhA-glnA recombinant strains significantly reduced ammonia emissions in laying hens (P < 0.05), with emission reduction efficiencies of 63.95%, 65.68% and 74.04%, respectively. The reason may be that the recombinant Pichia pastoris strains can convert ammonium nitrogen into amino acids for self-growth through ammonia assimilation, and reduce the pH, uric acid and urea content in the intestinal tract of livestock and poultry, and urease activity. Therefore, the construction of recombinant strains can provide technical support for reducing ammonia pollution in the livestock industry.

Sodium butyrate reduces ammonia emissions through glutamate metabolic pathways in cecal microorganisms of laying hens.

Ammonia emission is an important problem that needs to be solved in laying hen industries. Sodium butyrate (SB) is considered to have potential for reducing ammonia production because of its ability to improve nitrogen metabolism. In this in vitro fermentation study, we presented a correlation analysis of the metatranscriptome and metaproteome of lay hen cecal microorganisms, in order to identify important proteins and pathways involved in ammonia production reduction due to sodium butyrate supplementation. The results showed that sodium butyrate supplement decreased the production of ammonia by 26.22% as compared with the non-sodium butyrate supplementation (CK) group. The SB group exhibited a lower concentration of ammonium nitrogen (NH4+-N) and a decreased pH. Sodium butyrate promoted the uric acid concentration and lowered the uricase activity in the fermentation broth of laying hens cecal content. Notably, the 'alanine, aspartate and glutamate metabolism' category was more abundant in the SB group. The addition of sodium butyrate increased the expression of glutamate dehydrogenase (GDH) gene in cecal microbiota (e.g., Ruminococcus sp. and Bacteroides sp.) in vitro. The metaproteome analysis results showed that the expression of GDH with NADPH as coenzyme (NADPH-GDH) was up-regulated in cecal microbiota by sodium butyrate supplement. Our results indicate that sodium butyrate can affect glutamate metabolism through regulating the expression of glutamate dehydrogenase in cecal microorganisms, thereby reducing ammonia production. This study reveals that glutamate dehydrogenase-mediated glutamate metabolism play a key role in ammonia emission reduction in laying hen and provide theoretical basis for further developing ammonia production reduction approach.