Batch Culture Fermentation Research Articles

Rapid Screening of Methane-Reducing Compounds for Deployment in Livestock Drinking Water Using In Vitro and FTIR-ATR Analyses

Several additives have been shown to reduce enteric methane emissions from ruminants when supplied in feed. However, utilising this method to deliver such methane-reducing compounds (MRCs) in extensive grazing systems is challenging. Use of livestock drinking water presents a novel method to deliver MRCs to animals in those systems. This work evaluated 13 MRCs for suitability to be deployed in this manner. Compounds were analysed for solubility and stability in aqueous solution using Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy. Furthermore, aqueous solutions of MRCs were subjected to variations in temperature and starting pH of water used to assess solubility and stability of the MRCs in simulated water trough conditions, also using FTIR-ATR spectroscopy. In vitro batch culture fermentations were carried out using a medium-quality tropical grass feed substrate, to simulate pastures consumed by cattle in extensive grazing systems. Measurements were made of total gas and methane production, in vitro dry matter digestibility (IVDMD), and volatile fatty acid (VFA) concentration. Of the MRCs tested, 12 were found to be soluble and stable in water using the FTIR method employed, whilst the other could not be measured. Of the 12 soluble and stable MRCs, one containing synthetic tribromomethane (Rumin8 Investigational Veterinary Product) reduced methane production by 99% (p = 0.001) when delivered aqueously in vitro, without a reduction in IVDMD (p = 0.751), with a shift towards decreased acetate and increased propionate production and decreased total VFA production (p < 0.001). Other compounds investigated also appeared suitable, and the methods developed in this study could be used to guide future research in the area.

Optimizing the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators.

Crop pests and pathogens annually cause over $220 billion in global crop damage, with insects consuming 5%-20%of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes, and agricultural practices. Double-stranded RNA(dsRNA) is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialization of dsRNA-based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimized the design of plasmid DNA constructs to produce dsRNA biocontrols in Escherichia coli, by employing a wide range of alternative synthetic transcriptional terminators before measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstratethat batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight.

Optimizing Biogas Production: Comparative Analysis Of Organic Substrates For Enhanced Gas Yield

Study’s Novelty/Excerpt This study uniquely evaluates the biogas optimization potential of various locally sourced substrates, including cow dung, poultry dung, human, and pig manure, through a comparative analysis over a 15-day period using custom-made biodigesters. The research reveals that poultry dung yields the highest gas production, significantly outperforming other substrates with a gas production ratio of 0.20 on day 15, indicating its superior effectiveness. These findings contribute valuable insights into substrate efficiency for biogas production, suggesting poultry dung as a highly effective substrate and proposing co-digestion strategies to further enhance biogas yields. Full Abstract This research aimed to investigate the utilization of various locally sourced substrates in the biogas optimization process and compare the gas yield to determine the most efficient domestic substrate. The samples tested included cow dung, poultry dung, human, and pig manure. A comparative analysis of gas production over a 15-day period was carried out at 3-day intervals using four custom-made biodigesters with batch culture fermentation. The findings revealed that digester 4, utilizing poultry dung, exhibited the highest gas output. The gas production ratios for human, cow, pig, and poultry dung on day 15 were 0.10:0.11:0.12:0.20, equating to percentages of 18.86%, 20.78%, 22.64%, and 37.73% respectively. The notably higher percentage for poultry dung suggests its superior effectiveness as a substrate for biogas production. Alternatively, the co-digestion of cow and/or poultry dung could be considered as a strategy to enhance biogas production.

Enrichment and characterization of human-associated mucin-degrading microbial consortia by sequential passage.

Mucin is a glycoprotein secreted throughout the mammalian gastrointestinal tract that can support endogenous microorganisms in the absence of complex polysaccharides. While several mucin-degrading bacteria have been identified, the interindividual differences in microbial communities capable of metabolizing this complex polymer are not well described. To determine whether community assembly on mucin is deterministic across individuals or whether taxonomically distinct but functionally similar mucin-degrading communities are selected across fecal inocula, we used a 10-day in vitro sequential batch culture fermentation from three human donors with mucin as the sole carbon source. For each donor, 16S rRNA gene amplicon sequencing was used to characterize microbial community succession, and the short-chain fatty acid profile was determined from the final community. All three communities reached a steady-state by day 7 in which the community composition stabilized. Taxonomic comparisons amongst communities revealed that one of the final communities had Desulfovibrio, another had Akkermansia, and all three shared other members, such as Bacteroides. Metabolic output differences were most notable for one of the donor's communities, with significantly less production of acetate and propionate than the other two communities. These findings demonstrate the feasibility of developing stable mucin-degrading communities with shared and unique taxa. Furthermore, the mechanisms and efficiencies of mucin degradation across individuals are important for understanding how this community-level process impacts human health.

Comparison of prebiotic candidates in ulcerative colitis using an in vitro fermentation model.

This study explored the effect of three different prebiotics, the human milk oligosaccharide 2'-fucosyllactose (2'-FL), an oligofructose-enriched inulin (fructo-oligosaccharide, or FOS), and a galacto-oligosaccaride (GOS) mixture, on the faecal microbiota from patients with ulcerative colitis (UC) using in vitro batch culture fermentation models. Changes in bacterial groups and short-chain fatty acid (SCFA) production were compared. In vitro pH controlled batch culture fermentation was carried out over 48 h on samples from three healthy controls and three patients with active UC. Four vessels were run, one negative control and one for each of the prebiotic substrates. Bacterial enumeration was carried out using fluorescence in situ hybridization with flow cytometry. SCFA quantification was performed using gas chromatography mass spectrometry. All substrates had a positive effect on the gut microbiota and led to significant increases in total SCFA and propionate concentrations at 48 h. 2'-FL was the only substrate to significantly increase acetate and led to the greatest increase in total SCFA concentration at 48 h. 2'-FL best suppressed Desulfovibrio spp., a pathogen associated with UC. 2'FL, FOS, and GOS all significantly improved the gut microbiota in this in vitro study and also led to increased SCFA.

Modulation of gut microbiota by chickpea-derived proteins and peptides with antioxidant capabilities

Chickpea is one of the most widely consumed pulses, and chickpea protein has been reported to have superior protein bioavailability among different pulses. The high protein level in chickpea supplies nutrition and active peptides with potential antioxidant, antihypertensive, and anticancer properties. The objective of this study was to assess the impact of chickpea-derived proteins and peptides on gut microbial modulation and its relationship with antioxidant activities. In vitro fecal batch culture fermentation showed that chickpea albumin peptides had the highest antioxidant ability, drastically promoted Bifidobacterium growth, and enhanced the levels of lactic bacteria (i.e., Pediococcus, Weissella), and an acetate/propionate-producing bacteria Veillonella. The albumin peptides also stimulated the highest generation of short-chain fatty acids (SCFAs), and suppressed the generation of byproducts, including ammonia and indole. These findings indicated that antioxidant activity performs a crucial role in colonic fermentation and that chickpea-derived albumin peptides with outstanding antioxidant ability and prebiotic potential can be applied as functional food ingredients.

Oligofructose alone and in combination with 2'fucosyllactose induces physiologically relevant changes in γ-aminobutyric acid and organic acid production compared to sole 2'fucosyllactose supplementation: an in vitro study.

We explored the potential for the prebiotic oligofructose and prebiotic candidate 2'fucosyllactose, alone and in combination (50:50 blend) to induce physiologically relevant increases in neurotransmitter (γ-aminobutyric acid, serotonin, tryptophan, and dopamine) and organic acid (acetate, propionate, butyrate, lactate, and succinate) production as well as microbiome changes using anaerobic pH-controlled in vitro batch culture fermentations over 48h. Changes in organic acid and neurotransmitter production were assessed by gas chromatography and liquid chromatography and, bacterial enumeration using fluorescence in situ hybridization, respectively. Both oligofructose and oligofructose/2'fucosyllactose combination fermentations induced physiologically relevant concentrations of γ-aminobutyric acid, acetate, propionate, butyrate, and succinate at completion (all P ≤ .05). A high degree of heterogeneity was seen amongst donors in both neurotransmitter and organic acid production in sole 2'FL fermentations suggesting a large responder/nonresponder status exists. Large increases in Bifidobacterium, Lactobacillus, and Bacteroides numbers were detected in oligofructose fermentation, smallest increases being detected in 2'fucosyllactose fermentation. Bacterial numbers in the combined oligofructose/2'fucosyllactose fermentation were closer to that of sole oligofructose. Our results indicate that oligofructose and oligofructose/2'fucosyllactose in combination have the potential to induce physiologically relevant increases in γ-aminobutyric and organic acid production along with offsetting the heterogenicity seen in response to sole 2'fucosyllactose supplementation.

Effect of mixtures of legume species on ruminal fermentation, methane, and microbial nitrogen production in batch and continuous culture (RUSITEC) systems

The effect of cicer milkvetch ( Astragalus cicer L.) and sainfoin ( Onobrychis viciifolia Scop.) on ruminal fermentation, methane production, and microbial nitrogen synthesis was assessed in two experiments. Experiment 1 analyzed two legumes, cicer milkvetch and sainfoin at two stages (vegetative and late flower) incubated with alfalfa ( Medicago sativa) at five inclusion rates 0:100; 25:75, 50:50, 75:25, and 100:0 (as DM) in batch culture. Experiment 2 analyzed vegetative cicer milkvetch and alfalfa incubated in ratios of 25:75, 50:50, 75:25, and 100:0 (as DM) in continuous culture systems (RUSITEC). In batch culture, increased dry matter disappearance (DMD), and propionate percentage (%total), and reduced methane (mg·g−1 DMD) occurred with vegetative cicer milkvetch inclusion. In RUSITEC, DMD linearly increased ( P < 0.01), acetate:propionate ratio quadratically decreased, while ammonia (NH3-N) concentration ( P < 0.01) and butyrate percentage (%total) linearly decreased ( P < 0.05) with increasing inclusion of cicer milkvetch. No differences were observed for methane (CH4) production (mg·g−1 DMD), or short chain fatty acid (SCFA) production (mmol·day−1). Microbial nitrogen synthesis and efficiency of protein synthesis linearly increased ( P < 0.05) with increased inclusion of cicer milkvetch. Results suggest cicer milkvetch may result in synchronicity of energy and nitrogen during rumen fermentation, which could enhance cattle production.

Exploring the feasibility of biological hydrogen production using seed sludge pretreated with agro-industrial wastes

The effect of applying agro-industrial waste (AIW), such as potash extract (PE), cassava-steep wastewater (CSWW), and corn-steep liquor (CSTL), as an alternative material to pretreat digested cattle slurry (DCS) for biological hydrogen production was examined. In this study, the pretreated (PT) DCS was employed for H2 fermentation in batch cultures utilising glucose and sucrose as substrates. The result showed that, at 55 °C and pH 5.5, the pretreated DCS's daily volumetric hydrogen production (VHP) was higher than the untreated DCS. Although heat-shocked DCS produced a higher daily VHP of 135 NmL H2 g−1 VS on the second day using glucose as substrates, it is followed by PE-PT DCS, which gave a peak daily VHP of 115 NmL H2 g−1 VS but at a shorter time. When sucrose was the carbon source, the highest peaks were recorded in all the laboratory reactors on day two, with the highest daily VHP of 211 NmL H2 g−1 VS achieved in PE-PT DCS digesters. After the different DCS PT studies, the dominant phylum Firmicutes, represented by the Clostridium and Ruminococcus, were the most abundant bacteria compared to the untreated DCS, which was more diverse. Further research is required to optimise the conditions for AIW DCS pretreatment.

Mechanistic Modelling of Biomass Growth, Glucose Consumption and Ethanol Production by Kluyveromyces marxianus in Batch Fermentation.

This paper presents results concerning mechanistic modeling to describe the dynamics and interactions between biomass growth, glucose consumption and ethanol production in batch culture fermentation by Kluyveromyces marxianus (K. marxianus). The mathematical model was formulated based on the biological assumptions underlying each variable and is given by a set of three coupled nonlinear first-order Ordinary Differential Equations. The model has ten parameters, and their values were fitted from the experimental data of 17 K. marxianus strains by means of a computational algorithm design in Matlab. The latter allowed us to determine that seven of these parameters share the same value among all the strains, while three parameters concerning biomass maximum growth rate, and ethanol production due to biomass and glucose had specific values for each strain. These values are presented with their corresponding standard error and 95% confidence interval. The goodness of fit of our system was evaluated both qualitatively by in silico experimentation and quantitative by means of the coefficient of determination and the Akaike Information Criterion. Results regarding the fitting capabilities were compared with the classic model given by the logistic, Pirt, and Luedeking-Piret Equations. Further, nonlinear theories were applied to investigate local and global dynamics of the system, the Localization of Compact Invariant Sets Method was applied to determine the so-called localizing domain, i.e., lower and upper bounds for each variable; whilst Lyapunov's stability theories allowed to establish sufficient conditions to ensure asymptotic stability in the nonnegative octant, i.e., R+,03. Finally, the predictive ability of our mechanistic model was explored through several numerical simulations with expected results according to microbiology literature on batch fermentation.

Efficacy of Triphala extracts on the changes of obese fecal microbiome and metabolome in the human gut model.

Triphala is a mixture of tree fruits obtained from Terminalia chebula, Terminalia bellerica, and Phyllanthus emblica. It is one of the Ayurveda medicinal recipes used to treat health diseases such as obesity. The chemical composition analysis of Triphala extracts obtained from an equal portion of three fruits was performed. The contents of total phenolic compounds (62.87±0.21mg gallic acid equivalent/mL), total flavonoids (0.24±0.01mg catechin equivalent/mL), hydrolyzable tannins (177.27±10.09mg gallotannin equivalent/mL), and condensed tannins (0.62±0.11mg catechin equivalent/mL) were observed in Triphala extracts. The 1mg/mL of Triphala extracts was applied to batch culture fermentation which contained the feces from voluntarily obese female adults (body mass index of 35.0-40.0kg/m2) for 24h. The extraction of DNA and metabolites was each conducted on the samples obtained from batch culture fermentation within and without Triphala extracts treatment. The 16S rRNA gene sequencing and untargeted metabolomic analysis were carried out. There was no statistically significant difference between Triphala extracts and control treatments on the changes in microbial profiles (p-value <0.05). While the metabolomic analysis showed statistically significant differences of 305 up-regulated and 23 down-regulated metabolites in the treatment of Triphala extracts when compared with the control (p-value <0.05 and fold-change ≥2) belonging to 60 pathways. The pathway analysis revealed that Triphala extracts play an important role in the activation of phenylalanine, tyrosine and tryptophan biosynthesis. In this study, phenylalanine and tyrosine were identified metabolites which involve in the regulation of energy metabolism. The treatment of Triphala extracts possesses the induction of phenylalanine, tyrosine and tryptophan biosynthesis in fecal batch culture fermentation of obese adults and therefore it can be suggested as a probable herbal medicinal recipe for obesity treatment.

In vitro prebiotic activity of rhLf and galactooligosaccharides on infant intestinal microbiota.

human lactoferrin (Lf) and human milk oligosaccharides possess a wide range of functions. So, the present study focusses on the role of Lf and/or galactooligosaccharides (GOS) in the modulation of gut microbiota composition. recombinant human lactoferrin (rhLf) was added to the first infant formula (0.10, 0.15, 0.20 %) alone or in combination with GOS (1 %) in vessels of a small-scale batch culture fermentation model. Short-chain fatty acids (SCFAs), microbial population groups, and pH were monitored through fermentation for 24 hours. insignificant changes were observed in pH values and acetic acid accumulated during fermentation. Propionic acid content has been insignificantly increased while butyric acid has been insignificantly decreased. Moreover, increments in all bacterial groups except for Bacteroides were observed through the fermentation process. Lactobacillus and Bifidobacterium showed an increase in relation to initial time over the fermentation process, demonstrating the prebiotic effect of lactoferrin and GOS. After 24 hours of fermentation, all tested ingredients showed significant similarities in Enterococcus for controls except for 0.20 % rhLf + 1 % GOS, which provoked a diminution of Enterococci growth. despite the importance of the batch culture fermentation technique in uncovering the prebiotic activity of food ingredients, it is not useful for detecting the prebiotic nature of Lf due to its nature as a protein. Thus, Lf maybe shows its prebiotic activity on the gut microbiota through other mechanisms.

Oligofructose, 2’fucosyllactose and β-glucan in combination induce specific changes in microbial composition and short-chain fatty acid production compared to sole supplementation

In this study, we explored the effects that the prebiotic inulin-type fructans, and prebiotic candidates: 2'fucosyllactose and β-glucan from barley, singular and in combination had on microbial load, microbiome profile, and short-chain fatty acid production. This was carried out as a prescreening tool to determine combinations that could be taken forward for use in a human intervention trial. Effects of inulin-type fructans, 2'fucosyllactose and β-glucan from barley in singular and combination on microbial load and profile and short-chain fatty acid production (SCFA) was conducted using in vitro batch culture fermentation over 48 h. Changes in microbial load and profile were assessed by fluorescence in situ hybridization flow cytometry (FISH-FLOW) and 16S rRNA sequencing, and changes in SCFA via gas chromatography. All substrates generated changes in microbial load and profile, achieving peak microbial load at 8 h fermentation with the largest changes in profile across all substrates in Bifidobacterium (Q<0.05). This coincided with significant increases in acetate observed throughout fermentation (Q<0.05). In comparison to sole supplementation combinations of oligofructose, β-glucan and 2'fuscosyllactose induced significant increases in both propionate and butyrate producing bacteria (Roseburia and Faecalibacterium praunitzii), and concentrations of propionate and butyrate, the latter being maintained until the end of fermentation (all Q<0.05). Combinations of oligofructose, with β-glucan and 2'fucosyllactose induced selective changes in microbial combination and SCFA namely Roseburia, F. praunitzii, propionate and butyrate compared to sole supplementation.

Modulation of gut microbiota and their metabolites by functional mulberry juice non-thermally pasteurized using microfiltration

Background: Mulberry fruit is a source of phenolic compounds and has biological properties. This study aimed to assess the effects of isomaltooligosaccharide (IMO) added to mulberry juice on prebiotic activity and gut fermentation properties.Objectives: The study aimed to produce mulberry juice supplemented with IMO, which might generate short chain fatty acids (SCFA), and to examine the effects of a prebiotic activity after fecal fermentation.Materials and methods: Functional mulberry juices were prepared with three different levels of isomaltooligosaccharide (IMO), namely 0% (MBI0), 2% (MBI2), and 8% (MBI8). The study tested mulberry juices supplemented with IMO, which generated short-chain fatty acids (SCFA), phenolic metabolites, and favored beneficial gut bacteria, and examined the prebiotic activity after fecal fermentation by the colonic microbiota. A crossflow hollow fiber microfiltration system with a 0.22 μm pore size was employed to deliver permeates considered non-thermally pasteurized juices.Results: The results show that short chain fatty acids (SCFA) included large propionic acid and butyric acid concentrations at 48h. The percent of bifidobacteria significantly increased to 5.03% and 17.53% in 24 hours fermentation of MBI2 and MBI8, respectively. After fecal batch culture fermentation, some anthocyanin metabolites such as 3-(2-hydroxyphenyl) propionic acid, 3,4-dihydroxybenzaldehyde, L-phenylalanine, and aminocaproic acid, were detected. Therefore, IMO can serve as a potential prebiotic ingredient added in mulberry juice for promoting the growth of beneficial gut microbiota.Conclusion: The results show that IMO favored beneficial microorganisms in the gut and contributed to biologically active compounds such as metabolites of polyphenols and anthocyanins in the gut.Keywords: Mulberry, isomaltooligosaccharide, prebiotic, gut microbiota, metabolites

Prebiotic Properties of Exopolysaccharides from Lactobacillus helveticus LZ-R-5 and L. pentosus LZ-R-17 Evaluated by In Vitro Simulated Digestion and Fermentation.

The in vitro digestion and fermentation behaviors of Lactobacillus helveticus LZ-R-5- and L. pentosus LZ-R-17-sourced exopolysaccharides (LHEPS and LPEPS) were investigated by stimulated batch-culture fermentation system. The results illustrated that LHEPS was resistant to simulated saliva and gastrointestinal (GSI) digestion, whereas LPEPS generated a few monosaccharides after digestion without significant influence on its main structure. Additionally, LHEPS and LPEPS could be consumed by the human gut microbiota and presented stronger bifidogenic effect comparing to α-glucan and β-glucan, as they promote the proliferation of Lactobacillus and Bifidobacterium in cultures and exhibited high values of selectivity index (13.88 and 11.78, respectively). Furthermore, LPEPS achieved higher contents of lactic acid and acetic acid (35.74 mM and 45.91 mM, respectively) than LHEPS (35.20 mM and 44.65 mM, respectively) during fermentation for 48 h, thus also resulting in a larger amount of total SCFAs (110.86 mM). These results have clearly indicated the potential prebiotic property of EPS fractions from L. helveticus LZ-R-5 and L. pentosus LZ-R-17, which could be further developed as new functional food prebiotics to beneficially improve human gut health.

The microbiome modulating potential of superheated steam (SHS) treatment of dietary fibres

In this study, wheat and oat bran fibres were used to investigate the potential beneficial prebiotic-enhancing effects of superheated steam (SHS) treatment. Following SHS treatment of fibres, in-vitro simulated gastric and intestinal digestion followed by batch culture fermentation was performed. 16S rRNA gene sequencing and short chain fatty acid analysis were conducted, which demonstrated that SHS treatment was associated with significant differences in beta diversity and the relative abundances of several microbial taxa. Principal Coordinate Analysis (PCoA) revealed significant separation between SHS-treated oat and non-treated oat (p = 0.013), and between SHS-treated wheat and non-treated wheat following in vitro batch fermentation (p = 0.042). Abundances of Ruminococcus were found to be significantly higher by 1.09-log in SHS-treated oat when compared with non-treated oat while abundances of Escherichia_Shigella, and Alistipes were significantly lower by 1.47-log and 0.39-log, respectively in the SHS-treated oat samples than the non-treated oat samples (p = 6.85E-06, p = 0.002037, p = 0.002458; respectively). These data demonstrate that SHS treatment of dietary fibre enhanced its effectiveness to modulate the gut microbiome.Industrial Relevance: Superheated steam (SHS) treatment has emerged as an effective method to improve the shelf-life of grains by reducing lipid hydrolysis and oxidation. Our results revealed that SHS treatment of wheat and oat brans led to increased physiological and chemical properties including higher water-binding capacity, water-extractable arabinoxylan, and total phenolics as well as improvement in human gut microbiota. Our results suggest that this method could be implemented in the food industry in order to improve beneficial properties of oat and wheat brans.

The FocA channel functions to maintain intracellular formate homeostasis during Escherichia coli fermentation.

FocA translocates formate/formic acid bi-directionally across the cytoplasmic membrane when Escherichia coli grows by fermentation. It remains unclear, however, what physiological benefit is imparted by FocA, because formic acid (pKa=3.75) can diffuse passively across the membrane, especially at low pH. Here, we monitored changes in intra- and extracellular formate levels during batch-culture fermentation, comparing a parental E. coli K-12 strain with its isogenic focA mutant. Our results show that, regardless of the initial pH in the culture, FocA functions to maintain relatively constant intracellular formate levels during growth. Analysis of a strain synthesizing a FocAT91A variant with an exchange in a conserved threonine residue within the translocation pore revealed the strain accumulated formate intracellularly and imported formate poorly, but in a pH-dependent manner, which was different to uptake by native FocA. We conclude that FocA maintains formate homeostasis, using different mechanisms for efflux and uptake of the anion.

The Effects of Commonly Consumed Dietary Fibres on the Gut Microbiome and Its Fibre Fermentative Capacity in Adults with Inflammatory Bowel Disease in Remission.

Introduction: It has been suggested that the gut microbiome of patients with inflammatory bowel disease (IBD) is unable to ferment dietary fibre. This project explored the in vitro effect of fibre fermentation on production of short-chain fatty acids (SCFA) and on microbiome composition. Methods: Faecal samples were collected from 40 adults (>16 y) with IBD (n = 20 with Crohn’s disease and n = 20 with ulcerative colitis) in clinical remission and 20 healthy controls (HC). In vitro batch culture fermentations were carried out using as substrates maize starch, apple pectin, raftilose, wheat bran, α cellulose and a mixture of these five fibres. SCFA concentration (umol/g) was quantified with gas chromatography and microbiome was profiled with 16S rRNA sequencing. Results: Fibre fermentation did not correct the baseline microbial dysbiosis or lower diversity seen in either patients with CD or UC. For all fibres, up to 51% of baseline ASVs or genera changed in abundance in HC. In patients with IBD, fermentation of fibre substrates had no effect on species or genera abundance. Production of SCFA varied among the different fibre substrates but this was not different between the two IBD groups and compared to HC after either 5 or 24 h fermentation. Conclusions: Despite extensive microbial dysbiosis, patients with IBD have a similar capacity to ferment fibre and release SCFA as HC. Fibre supplementation alone may be unlikely to restore to a healthy status the compositional shifts characteristic of the IBD microbiome.

P705 The effects of commonly consumed dietary fibres on the gut microbiome and its fibre fermentative capacity in adults with inflammatory bowel disease in remission

Abstract Background It has been suggested that the gut microbiome of patients with inflammatory bowel disease (IBD) is unable to ferment dietary fibre. This project explored the in vitro effect of fibre fermentation on production of short chain fatty acids (SCFA) and on microbiome composition. Methods Faecal samples were collected from 40 adults (&amp;gt;16 y) with IBD (n=20 with Crohn’s disease and n=20 with Ulcerative colitis) in clinical remission and 20 healthy controls (HC). In vitro batch culture fermentations were carried out using as substrates maize starch, apple pectin, raftilose, wheat bran, α cellulose and a mixture of these 5 fibres. SCFA concentration (umol/g) was quantified with gas chromatography and microbiome was profiled with 16S rRNA sequencing. Results Fibre fermentation did not correct the baseline microbial dysbiosis or lower diversity seen in either CD or UC. For all fibres, up to 51% of baseline ASVs or genera changed in abundance in HC. In patients with IBD, fermentation of fibre substrates had no effect on species or genera abundance. Production of SCFA varied among the different fibre substrates but this was not different between the two IBD groups and compared to HC after either 5 or 24 hour fermentation. Conclusion Despite extensive microbial dysbiosis, patients with IBD have a similar capacity to ferment fibre and release SCFA as HC. Fibre supplementation alone may be unlikely to restore to a healthy status the compositional shifts characteristic of the IBD microbiome.

Media optimization for SHuffle T7 Escherichia coli expressing SUMO-Lispro proinsulin by response surface methodology

BackgroundSHuffle is a suitable Escherichia coli (E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced in E. coli as inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7 E. coli expressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation.ResultsFactors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2 concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor.ConclusionThe proposed media was suitable for high cell density fermentation of E. coli SHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.