Fecal Inoculum Research Articles

Effect of dietary fibre and condensed tannins concentration from various fibrous feedstuffs on <i>in vitro gas</i> production kinetics with rabbit faecal inoculum

1. Alvarez J.L., Marguenda I., García-Rebollar P., Carabaño R., De Blas C., Corujo A., García-Ruiz A.I., 2007. Effects of type and level of fibre on digestive physiology and performance in reproducing and growing rabbits. World Rabbit Sci. 15, 9 –17 CrossRef Google Scholar

Preparation of a standardised faecal slurry for ex-vivo microbiota studies which reduces inter-individual donor bias

BackgroundIn-vitro gut fermentation systems provide suitable models for studying gut microbiota composition and functionality. However, such methods depend on the availability of donors and the assumption of reproducibility between microbial communities before experimental treatments commence. The aim of this study was to develop a frozen standardised inoculum (FSI) which minimizes inter-individual variation and to determine its stability over time using culture-dependent and culture-independent techniques. ResultsA method for the preparation difference of a FSI is described which involves pooling the faecal samples, centrifugation and pelleting of the cell biomass and finally homogenising the cell pellets with phosphate buffer and glycerol. Using this approach, no significant difference in total anaerobe cell viability was observed between the fresh standardised inoculum (before freezing) and the 12days post freezing FSI. Moreover, Quantitative PCR revealed no significant alterations in the estimated bacterial numbers in the FSI preparations for any of the phyla. MiSeq sequencing revealed minute differences in the relative abundance at phylum, family and genus levels between the FSI preparations. Differences in the microbiota denoted as significant were limited between preparations in the majority of cases to changes in percentage relative abundance of ±0.5%. The independently prepared FSIs revealed a high degree of reproducibility in terms of microbial composition between the three preparations. ConclusionsThis study provides a method to produce a standardised human faecal inoculum suitable for freezing. Based on culture-dependent and independent analysis, the method ensures a degree of reproducibility between preparations by lessening the effect of inter-individual variation among the donors, thereby making the system more suitable for the accurate interpretation of the effects of experimental treatments.

Fecal Gas Production of Ten Common Horse Feeds Supplemented With Saccharomyces cerevisiae

The study aimed to assess the nutritive value of 10 feeds (grains and forages) commonly used in horse nutrition in Mexico, on the basis of their chemical composition, in vitro organic matter digestibility (IVOMD) and in vitro gas production measurements with or without the supplementation of Saccharomyces cerevisiae (SC) at 4 mg/g DM. Fecal inoculum was obtained from 4 adult English Thoroughbred horses fed on restricted amount of concentrate and oat hay ad libitum. Substrates tested were: 6 concentrates (corn gluten meal, soybean meal, steam-rolled corn, steam-rolled barley, oat grain, and wheat bran) and 4 roughages (soybean hulls, corn stover, alfalfa hay, and oat hay). Gas production (GP) was recorded at 2, 4, 6, 8, 10, 12, 14, 24, 48, and 70 hours using the pressure transducer technique. Some ingredient × yeast interactions were observed (P ≤ .020) for the asymptotic GP and GP at 48 and 70 hours of incubation. Yeast addition increased (P < .001) the asymptotic GP of concentrates compared to roughages. Concentrate feeds had higher (P < .05) GP and lower (P < .001) rate of GP compared to roughages without yeast. From 24 to 70 hours of incubation, forages with or without yeast had lower (P < .05) GP compared to concentrates supplemented with SC. Forages had higher fermentation pH compared to concentrates but lower (P < .05) metabolizable energy, IVOMD, and microbial protein production compared to concentrates. Supplementation with SC increased (P < .05) the asymptotic GP of oat grain, soybean meal, soybean meal, steam-rolled barley, steam-rolled corn, wheat bran, corn stover, and oat hay, without affecting the rate of GP or lag time of oat grain, soybean meal, wheat bran, corn stover, and oat hay. Moreover, supplementation with SC increased (P < .05) metabolizable energy, IVOMD, and microbial protein production of steam-rolled barley, wheat bran, and corn stover, without affecting (P > .05) the fermentation of other feeds. Supplementation with SC improved fermentation of feeds with higher effects on concentrates compared to roughages. It was concluded that although SC mainly improves concentrate utilization by horses, it also improves fiber digestion when used on high-roughage diets fed to horses.

Effect of Partial Replacement of Steam Rolled Corn With Soybean Hulls or Prickly Pear Cactus in the Horse's Diet in the Presence of Live Saccharomyces cerevisiae on In Vitro Fecal Gas Production

The aim of the study was to evaluate the fecal fermentation of partial replacing steam rolled corn with soybean hulls (SH) or prickly pear cactus (PC) as energy source in horse diets, in the presence of Saccharomyces cerevisiae. Steam rolled corn was replaced with SH at 0% (control), 7.5% (SH75), and 15% (SH150) in the first trial, whereas it was replaced with PC at 0% (control; the same of the first trial), 7.5% (PC75), and 15% (PC150) in the second trial. Yeast of S. cerevisiae was added at 0, 2, and 4 mg/g dry matter (DM) of incubated substrates. Fecal inoculum was obtained from four adult English Thoroughbred horses fed on an amount of commercial concentrate and oat hay ad libitum. Interactions observed between PC rations and yeast doses for the asymptotic gas production (GP), the rate of GP and carbon dioxide (CO2) production during some incubation hours. Moreover, with no effect due to SH rations (P > .05), increased (P < .05) rate of GP was observed with the ration PC75 compared with other rations. Besides, PC75 and PC150 rations with 0 mg yeast/g DM linearly decreased (P < .05) CO2 production at some incubation hours. However, SH75 and SH150 ration had increased (P = .005) DM degradability (DMD). Yeast addition at 2 mg/g DM increased the asymptotic GP (P = .048) with the SH75 and PC150 rations. The level of 4 mg yeast/g DM increased the asymptotic GP (P = .048) from the SH150 ration. Yeast addition at 2 and 4 mg/g DM increased (P < .05) ​the asymptotic GP from PC75 and PC150 rations, respectively, with increasing DMD with the both doses. Yeast addition increased (P < .05) CO2 production from SH75, SH150, PC75, and PC150 rations. It could be concluded that SH and PC can replace steam rolled corn at levels of 7.5% to 15% without negative effect of fermentation kinetics and with better fermentation performance in the presence of yeast at 2 mg/g DM of substrates.

In Vitro Fermentation of Porcine Milk Oligosaccharides and Galacto-oligosaccharides Using Piglet Fecal Inoculum.

In this study, the in vitro fermentation by piglet fecal inoculum of galacto-oligosaccharides (GOS) and porcine milk oligosaccharides (PMOs) was investigated to identify possible preferences for individual oligosaccharide structures by piglet microbiota. First, acidic PMOs and GOS with degrees of polymerization 4-7 were depleted within 12 h of fermentation, whereas fucosylated and phosphorylated PMOs were partially resistant to fermentation. GOS structures containing β1-3 and β1-2 linkages were preferably fermented over GOS containing β1-4 and β1-6 linkages. Upon in vitro fermentation, acetate and butyrate were produced as the main organic acids. GOS fermentation by piglet inoculum showed a unique fermentation pattern with respect to preference of GOS size and organic acids production.

Wheat bran promotes enrichment within the human colonic microbiota of butyrate-producing bacteria that release ferulic acid.

SummaryCereal fibres such as wheat bran are considered to offer human health benefits via their impact on the intestinal microbiota. We show here by 16S rRNA gene‐based community analysis that providing amylase‐pretreated wheat bran as the sole added energy source to human intestinal microbial communities in anaerobic fermentors leads to the selective and progressive enrichment of a small number of bacterial species. In particular, OTUs corresponding to uncultured Lachnospiraceae (Firmicutes) related to E ubacterium xylanophilum and B utyrivibrio spp. were strongly enriched (by five to 160 fold) over 48 h in four independent experiments performed with different faecal inocula, while nine other Firmicutes OTUs showed > 5‐fold enrichment in at least one experiment. Ferulic acid was released from the wheat bran during degradation but was rapidly converted to phenylpropionic acid derivatives via hydrogenation, demethylation and dehydroxylation to give metabolites that are detected in human faecal samples. Pure culture work using bacterial isolates related to the enriched OTUs, including several butyrate‐producers, demonstrated that the strains caused substrate weight loss and released ferulic acid, but with limited further conversion. We conclude that breakdown of wheat bran involves specialist primary degraders while the conversion of released ferulic acid is likely to involve a multi‐species pathway.

In Vitro Gas, Methane, and Carbon Dioxide Productions of High Fibrous Diet Incubated With Fecal Inocula From Horses in Response to the Supplementation With Different Live Yeast Additives

In a randomized block design experiment, the effect of fecal inocula from horses supplemented with live yeast (Saccharomyces cerevisiae) in diets containing 50% oat straw on in vitro total gas (gas production [GP]), methane (CH4), and carbon dioxide (CO2) productions as indicators of hindgut activity was assessed. Three commercial products of S. cerevisiae were tested (1) Biocell F53 (YST53), (2) Procreatin 7 (YST07), and (3) Biosaf SC47 (YST047). For the incubations, each product was added at 0 (control without yeast addition), 2, or 4 mg/g dry matter (DM). Fecal inocula for incubations with each treatment was obtained from Quarter Horse mares fed the same yeast additives for 15 days, resulting in four different fecal inocula (FI53, FI07, FI47, and FI00). The fecal content mixed with the culture media were used to inoculate three identical runs of incubation in bottles containing 1-g DM of substrate (a mixture of concentrate and oat straw [1:1 DM]). The GP, CH4, and CO2 productions were measured at 2, 4, 6, 8, 10, 12, 24, and 48 hours postincubation. Addition of additives YST53 and YST07 at 2 mg/g DM resulted in higher asymptotic GP (linear effect, P = .021) and GP during the first 12 hours of incubation (linear effect, P < .05) compared with control without yeast addition, with the highest value being for the dose 2 mg/g DM with the fecal inoculum FI53. The additive YST47 at all doses with fecal inoculum FI47 had lower GP (linear effect, P < .05) at different incubation hours compared with control. The additive YST53 increased GP, CH4, and fermentation kinetics at the dose 2 mg/g DM with decreasing CH4 production by 78% at 4 mg/g DM at 24 hours of incubation. Addition of YST53 at 2 and 4 mg/g DM with fecal inoculum FI53 enhanced fermentation kinetics (P < .05) compared with control and other additives at different doses. It can be concluded that the yeast additive Biocell F53 was the most effective at doses of 2 and 4 mg/g DM compared with other Saccharomyces strains to attain a more favorable hindgut fermentation to digest fibrous roughages by horses.

Adding mucins to an in vitro batch fermentation model of the large intestine induces changes in microbial population isolated from porcine feces depending on the substrate.

Adding mucus to in vitro fermentation models of the large intestine shows that some genera, namely lactobacilli, are dependent on host-microbiota interactions and that they rely on mucosal layers to increase their activity. This study investigated whether this dependence on mucus is substrate dependent and to what extent other genera are impacted by the presence of mucus. Inulin and cellulose were fermented in vitro by a fecal inoculum from pig in the presence or not of mucin beads in order to compare fermentation patterns and bacterial communities. Mucins increased final gas production with inulin and shifted short-chain fatty acid molar ratios (P<0.001). Quantitative real-time PCR analyses revealed that Lactobacillus spp. and Bifidobacterium spp. decreased with mucins, but Bacteroides spp. increased when inulin was fermented. A more in-depth community analysis indicated that the mucins increased Proteobacteria (0.55 vs 0.25%, P=0.013), Verrucomicrobia (5.25 vs 0.03%, P=0.032), Ruminococcaceae, Bacteroidaceae and Akkermansia spp. Proteobacteria (5.67 vs 0.55%, P<0.001) and Lachnospiraceae (33 vs 10.4%) were promoted in the mucus compared with the broth, while Ruminococcaceae decreased. The introduction of mucins affected many microbial genera and fermentation patterns, but from PCA results, the impact of mucus was independent of the fermentation substrate.

Monitoring the fibrolytic potential of microbial ecosystems from domestic and wild ruminants browsing tanniferous forages.

Although the rumen microbiome has been reported to synthesize a rich source of symbiotic enzymes (exocellulase, endocellulase, hemicellulase and cellobiase), the digestion of tropical C4 grasses and browses by ruminants is still limited. Therefore, this study aimed to unveil potential fibrolytic microbial ecosystems from giraffe, kudu, impala and consortia (A1 [giraffe + kudu], A2 [giraffe + impala], A3 [kudu + impala], and A4 [giraffe + kudu + impala]) browsing tanniferous plants, which can be used to improve forage utilization in domesticated goat. Crude protein enzyme extracts (CPZ) from fresh faecal samples were precipitated by 60% ammonium sulphate and assayed for exocellulase, endocellulase and hemicellulase by incubating with crystalline cellulose, carboxymethyl cellulose and xylan at 38 °C with optimum pH of 5.5 to 6.5 for 1, 2, and 48 h, respectively. Enzyme specific activities were defined as μg of reducing sugar/mg CPZ. In vitro fermentation study was done by transferring 33 mL of fresh faecal inoculum into 67 mL of salivary buffer containing 1 g Acacia sieberiana and incubating for 72 h at 38 °C. Apparent degradability (APDeg), true degradability (TD), neutral detergent fibre degradability (NDFdeg), acid detergent fibre degradability (ADFdeg), microbial yield (MY), metabolizable energy (ME) and total gas emitted (Gas) were measured. Exocellulase activities were higher (P < 0.05) in all wild animals and consortia than those in goat except for A4. Minimal differences in hemicellulase activities (P < 0.05) were observed among goat and wild animals and consortia, while endocellulase activity was generally higher (P < 0.05) in goat than that in the rest of the systems. Apart from A3, TDeg, NDFdeg and ADFdeg were higher (P < 0.05) in all microbial ecosystems from wild animals and consortia than those in goat. Apparent degradability, MY and ME also varied (P < 0.05) among these systems. Giraffe, Kudu and A3 produced lower (P < 0.05) gas than the goat system. This study showed that microbial ecosystems from wild browsers (especially impala) and consortia possess a higher potential to digest tanniferous forage with less enteric gas production compared with domesticated goat, hence those microbiome could be exploited as microbial feed additives for improving digestibility and reducing enteric gas production in domesticated goat. Improvements of goat's digestibility will depend on the survival and establishment of microbial species in the rumen as well as their fibrolytic and symbiotic potential including tannin tolerance.

Comparison of rumen fluid inoculum vs. faecal inoculum on predicted methane production using a fully automated in vitro gas production system

The aim of the present study was to compare in vitro methane (CH4) production and other fermentation parameters from different substrates incubated either in rumen or faecal inoculum. Five different substrates were incubated in two different inocula and gas recordings were made using an automated in vitro gas production system. The substrates were: particulate matter obtained from reticulo-rumen (RR) digesta and faeces (FC) by wet sieving, timothy hay (H), first cut grass silage (S) and a mixture of grass silage and barley (50:50; SB). One gram of each substrate was incubated either in 60ml buffered rumen or faecal inoculum taken from Swedish lactating dairy cows for 48h. The results indicated that in vitro total gas production, predicted in vivo CH4 production and the ratio of CH4 production to total gas production were greater (P<0.01) for substrates incubated in rumen inoculum as compared to faecal inoculum. Mean of predicted in vivo CH4 production was greater for substrates incubated in rumen inoculum (23.5ml/g DM, 29.0ml/g OM) as compared to faecal inoculum (11.2ml/g DM, 14.3ml/g OM). Predicted CH4 production based on volatile fatty acids (VFA) stoichiometry equations (CH4VFA) showed no difference in CH4 per mol VFA (P=0.44) between the two sources of inoculum used for all substrates. Molar proportions of propionate were higher and that of butyrate were lower (P<0.01) for all substrates incubated in faecal inoculum compared to rumen inoculum. No difference (P=0.13) in molar proportions of acetate was observed. Digestibility of neutral detergent fibre (aNDFomD) and true organic matter digestibility (TOMD) were lower (P<0.01) for all substrates incubated in faecal inoculum compared to rumen inoculum. It can be concluded that using faecal inoculum tended to give lower values of predicted in vivo CH4 production as compared to rumen inoculum. The discrepancy between observed and stoichiometric CH4 production suggests an existence of acetogenesis in the hindgut.

Cultivation of stable, reproducible microbial communities from different fecal donors using minibioreactor arrays (MBRAs).

BackgroundContinuous-flow culture models are one tool for studying complex interactions between members of human fecal microbiotas because they allow studies to be completed during an extended period of time under conditions where pH, nutrient availability, and washout of waste products and dead cells can be controlled. Because many of the existing well-validated continuous-flow models are large and complex, we were interested in developing a simpler continuous-flow system that would allow microbial community dynamics to be examined in higher throughput while still maintaining complex microbial communities. To this end, we developed minibioreactor arrays (MBRAs), small volume bioreactors (15 ml) that allow simultaneous cultivation of up to 48 microbial communities in a single anaerobic chamber.ResultsWe used MBRA to characterize the microbial community dynamics of replicate reactors inoculated from three different human fecal donors and reactors seeded with feces pooled from these three donors. We found that MBRA could be used to efficiently cultivate complex microbial communities that were a subset of the initial fecal inoculum (15–25 % of fecal OTUs initially observed). After an initial acclimation period of approximately 1 week, communities in each reactor stabilized and exhibited day-to-day variation similar to that observed in stable mouse fecal communities. Replicate reactors were predominately populated by shared core microbial communities; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units (OTUs). Consistent with differences between fecal donors, MBRA communities present in reactors seeded with different fecal samples had distinct composition and structure.ConclusionsFrom these analyses, we conclude that MBRAs can be used to cultivate communities that recapitulate key features of human fecal communities and are a useful tool to facilitate higher-throughput studies of the dynamics of these communities.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0106-5) contains supplementary material, which is available to authorized users.

Production, Purification, and in Vitro Evaluation of the Prebiotic Potential of Arabinoxylooligosaccharides from Brewer’s Spent Grain

Brewer's spent grain (BSG) samples were subjected to a two-step aqueous processing (starch extraction and autohydrolysis) in order to assess their potential as a raw material for obtaining a mixture of arabinoxylooligosaccharides (AXOS) suitable to be use as prebiotics for elderly. After hydrothermal treatment, the liquors were refined by a sequence of purification and conditioning steps including membrane filtration, enzymatic hydrolysis, and ion exchange. The presence of both substituted (degree of polimerization (DP) = 2-10) and unsubstituted (DP = 2-16) oligosaccharides made up of xylose and arabinose (AXOS) were confirmed in purified mixtures (in which total OS content = 84% w/w) by using chromatographic techniques and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Finally, AXOS were evaluated for their prebiotic activity by in vitro fermentation assays using fecal inocula from elderly people, demonstrating that AXOS were slightly better substrates than FOS, in terms of bacterial population shifts as in the production of SCFA.

The influence of supplementing heifer diets with &lt;i&gt;Saccharomyces cerevisiae&lt;/i&gt; yeast on the activity of polysaccharidases in the rumen

1. Effect of dietary fibre and condensed tannins concentration from various fibrous feedstuffs on in vitro gas production kinetics with rabbit faecal inoculum K. Kara Journal of Animal and Feed Sciences CrossRef

Kiwifruit fibre level influences the predicted production and absorption of SCFA in the hindgut of growing pigs using a combined in vivo-in vitro digestion methodology.

Combined in vivo (ileal cannulated pig) and in vitro (faecal inoculum-based fermentation) digestion methodologies were used to predict the production and absorption of SCFA in the hindgut of growing pigs. Ileal and faecal samples were collected from animals (n 7) fed diets containing either 25 or 50 g/kg DM of kiwifruit fibre from added kiwifruit for 14 d. Ileal and faecal SCFA concentrations normalised for food DM intake (DMI) and nutrient digestibility were determined. Ileal digesta were collected and fermented for 38 h using a fresh pig faecal inoculum to predict SCFA production. The predicted hindgut SCFA production along with the determined ileal and faecal SCFA were then used to predict SCFA absorption in the hindgut and total tract organic matter digestibility. The determined ileal and faecal SCFA concentrations (e.g. 8·5 and 4·4 mmol/kg DMI, respectively, for acetic acid for the low-fibre diet) represented only 0·2-3·2 % of the predicted hindgut SCFA production (e.g. 270 mmol/kg DMI for acetic acid). Predicted production and absorption of acetic, butyric and propionic acids were the highest for the high-fibre diet (P0·05). In conclusion, determined ileal and faecal SCFA concentrations represent only a small fraction of total SCFA production, and may therefore be misleading in relation to the effect of diets on SCFA production and absorption. Considerable quantities of SCFA are produced and absorbed in the hindgut of the pig by the fermentation of kiwifruit.

Evaluation of an optimal preparation of human standardized fecal inocula for in vitro fermentation studies

This study investigated the optimal preservation approach to prepare human feces as inoculum for in vitro fermentations as an alternative to the use of fresh feces. The four treatments studied were: Treatment 1) fresh feces resuspended in dialysate solution+glycerol; Treatment 2) fresh feces resuspended in dialysate solution+glycerol and then stored at −80°C; Treatment 3) fecal sample frozen with 1.5g glycerol; and Treatment 4) fecal sample frozen. All the treatments contained 8.75g of feces, 3.5ml dialysate and 4.9ml glycerol when inoculated in TIM-2 in vitro system. Treatment 1 (fresh fecal preparation) was used as a reference.The effects were evaluated in terms of i) metabolic activity and ii) composition of the microbiota using fermentation experiments in the TIM-2 in vitro system. In all treatments, high levels of acetate were produced followed by n-butyrate and propionate. However, the metabolic activity of the bacteria, in terms of short-chain fatty acid production, was affected by the different treatments. Microbiota composition was analyzed using the IS-pro profiling technique. Diversity in Actinobacteria, Firmicutes, Fusobacteria and Verrucomicrobia and Proteobacteria groups seemed to be preserved in all treatments whereas it was observed to decline in the Bacteroidetes group. Preparing a human fecal inoculum resuspended in dialysate solution with glycerol and then stored at −80°C showed high similarities to the results obtained with fresh feces, and is proposed as the optimal way to freeze fecal material as an alternative to fresh feces for in vitro fermentation studies.

Characterisation and in vitro fermentation of resistant maltodextrins using human faecal inoculum and analysis of bacterial enzymes present

Abstract A commercially available resistant maltodextrin (RMD), which is made out of corn starch by chemically and enzymatically modification, consists of atypical starch linkages and a low average molecular mass of 2 kDa. These characteristics of RMD make it rather complicated to identify with any chromatographic or masspectrometric method at this moment. A batch fermentation with human inocula over 48 h under anaerobic conditions showed that the degradation of the indigestible RMD is slow in comparison to dietary fibres like FOS. The RMD is only fermentable for ca. 60% and no specific oligosaccharide within the RMD population seems to be preferentially utilised by the microbiota present. Short chain fatty acid (SCFA) production, with acetic acid as the main SCFA, started after ca. 5 h of fermentation, while the increase of SCFA at 11 h was concurrent with the major degradation of the oligosaccharides of RMD. The microbiota composition as analysed by HITchip, revealed that RMD slightly stimulated the growth of bifidobacteria in comparison to the blank. The activity of carbohydrate degrading enzymes, produced by the microbiota during the in vitro fermentation, revealed that potential capacity to degrade typical starch linkages was high, but the RMD was only slowly and partly degradable during the incubation of 20 h.

Simultaneous synthesis of mixtures of lactulose and galacto-oligosaccharides and their selective fermentation

Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with β-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product distribution in terms of prebiotic potential. The selectivity of fermentation of each carbohydrate alone as well as mixtures of both was determined using pH-controlled anaerobic batch cultures with faecal inoculum. Within the experimental range considered, lactulose/GOS molar ratio of 4 resulted in the highest selectivity for Bifidobacterium and Lactobacillus/Enterococcus, so this ratio was selected as the target for the synthesis of lactulose from fructose and lactose with Aspergillus oryzae β-galactosidase. Synthesis was optimized using response surface methodology, considering temperature, initial concentrations of acceptor sugars and fructose/lactose molar ratio as key variables, with the aim of maximizing lactulose yield at the optimal product distribution in terms of prebiotic potential (lactulose/GOS molar ratio of 4). Under optimal conditions (50°C, 50%w/w total initial concentrations of sugars and fructose/lactose molar ratio of 6.44), lactulose yield of 0.26g of lactulose produced per g of initial lactose was obtained at the optimal product distribution.

Increased oxidative and nitrosative reactions during digestion could contribute to the association between well-done red meat consumption and colorectal cancer

Uncured and nitrite-cured pork were subjected, raw, cooked (65°C, 15min) or overcooked (90°C, 30min), to an in vitro digestion model, which includes mouth, stomach, duodenum, and colon phases. Heating of uncured meat resulted in a pronounced increase in lipid and protein oxidation products throughout digestion. Nitrite-curing had an antioxidant effect during digestion, but this effect disappeared when the meat was overcooked, resulting in up to ninefold higher 4-hydroxy-2-nonenal concentrations compared with digested nitrite-cured raw and cooked pork. Colonic digesta contained significantly higher concentrations of the NOC-specific DNA adduct O6-carboxy-methylguanine when pork underwent a more intense heating procedure, independent of nitrite-curing, depending strongly on the fecal inoculum used. Since processed meats are usually nitrite-cured, the present study suggests that overcooking processed meat is likely to result in the formation of genotoxic compounds during digestion and should, therefore, be avoided.

Micronized fibres affect in vitro fermentation under normal buffered and osmotic stress conditions using porcine inocula.

In this in vitro study, the modified Hohenheim gas test was used to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum, when fermenting a standard pig diet with varying levels of crude protein (CP; 20, 24 and 28% CP), and supplemented with one of three fibre sources manufactured by micronization treatment. These were wheat envelopes (MWE), pea fibre (MPF) and lupine fibre (MLF). For comparison, inulin was used. As intestinal bacteria have to cope with varying osmotic conditions in their ecosystem, fermentation was performed under normal buffered and osmotic stress conditions. After 24 h of fermentation, total gas production and ammonia production were measured. In addition, the effect of MWE and inulin on short-chain fatty acid (SCFA) production and numbers of total eubacteria, Lactobacillus spp., Bifidobacterium spp., Enterobacteriaceae, Enterococcus spp., Clostridium cluster XIVa and Clostridium cluster IV, were determined using quantitative real-time PCR. Under normal buffered conditions, supplementation of MWE resulted in increased (p < 0.05) SCFA, acetic, propionic and valerianic acid production at CP levels of 20 and 28%. There was an increase (p < 0.05) in ammonia production for the micronized supplements, and for MWE an increased (p < 0.05) branched-chain proportion was observed, possibly due to higher availability of protein for fermentation which was released during the micronization process. Osmotic stress conditions reduced (p < 0.05) total gas as well as total SCFA, acetic and propionic acid production for all treatments, while cell counts were increased (p < 0.05) for Bifidobacterium spp., Enterococcus spp. and Lactobacillus spp. Under normal buffered conditions in combination with 24 and 28% CP levels, lactobacilli were increased for MWE, compared to inulin (p < 0.05). In conclusion, micronized supplements such as MWE may beneficially modulate pigs' intestinal microbiota by increasing SCFA production in addition to a selective proliferation of lactobacilli.

In vitro Fermentation of Structural Carbohydrate-Rich Feeds Using Faecal Inoculum from Pigs

Seven feeds were tested in vitro using faecal inoculum from pigs. Sugar beet pulp, wheat bran, soybean hulls, grapecake, glutamic beet pulp, citrus by-product and fructo-oligosaccharides (FOS) were fermented for 96 h. Cumulative gas production was measured as indicator of the fermentation kinetics. At the end of incubation organic matter disappearance and fermentation end-products (short-chain fatty acids and NH3) were also measured. The gas production profiles were fitted with a multi-phasic model. Significant differences were detected between grapecake and FOS: the very low gas production for the first one was probably due to the high lignin and tannins contents of this by-product, while the higher organic matter cumulative gas volume (OMCV) and organic matter disappearance (OMD) values for FOS were due to the high soluble fibre proportion. Soybean hulls and citrus by-product, showed similar values of degradability and gas production and were characterised by different fermentation profiles. Grapecake showed the lower fermentation, while citrus by-product was characterized high gas and short-chain fatty acids production. These characteristics could be particularly useful to optimize the caecum-colon fermentation in order to obtain a high butyrate acid production.