S. M. Martín-Orúe

Assessing the survival of transgenic plant DNA in the human gastrointestinal tract

The inclusion of genetically modified (GM) plants in the human diet has raised concerns about the possible transfer of transgenes from GM plants to intestinal microflora and enterocytes. The persistence in the human gut of DNA from dietary GM plants is unknown. Here we study the survival of the transgene epsps from GM soya in the small intestine of human ileostomists (i.e., individuals in which the terminal ileum is resected and digesta are diverted from the body via a stoma to a colostomy bag). The amount of transgene that survived passage through the small bowel varied among individuals, with a maximum of 3.7% recovered at the stoma of one individual. The transgene did not survive passage through the intact gastrointestinal tract of human subjects fed GM soya. Three of seven ileostomists showed evidence of low-frequency gene transfer from GM soya to the microflora of the small bowel before their involvement in these experiments. As this low level of epsps in the intestinal microflora did not increase after consumption of the meal containing GM soya, we conclude that gene transfer did not occur during the feeding experiment.

The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation.

beta-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

In vitro fermentation of carbohydrates by porcine faecal inocula and their influence on Salmonella Typhimurium growth in batch culture systems

The aim of this study was to evaluate in vitro the influence of fermentable carbohydrates on the activity of porcine microbiota and survival of Salmonella Typhimurium in a batch culture system simulating the porcine hindgut. The carbohydrates tested were xylooligosaccharides, a mixture of fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). These ingredients stimulated the growth of selected Bifidobacterium and Lactobacillus species in pure cultures. In batch cultures, the carbohydrates influenced some fermentation parameters. For example, GEO and FIN significantly increased lactic acids compared with the control (no added carbohydrate). With the exception of LAC, the test carbohydrates increased the production of short-chain fatty acid (SCFA) and modified SCFA profiles. Quantitative analysis of bacterial populations by FISH revealed increased counts of the Bifidobacterium group compared with control and, with exception of FOS, increased Lactobacillus, Leuconostoc and Weissella spp. counts. Salmonella numbers were the lowest during the fermentation of LAC. This work has looked at carbohydrate metabolism by porcine microbiota in a pH-controlled batch fermentation system. It provides an initial model to analyse interactions with pathogens.

Excretion of purine derivatives in cows: endogenous contribution and recovery of exogenous purine bases

Two dry cows fitted with simple cannula in the rumen and duodenum, and fed with a mixed diet (straw:barley, 50:50), were used to determine endogenous losses and response of urinary purine derivatives (PDs) to duodenally infused yeast-RNA. Duodenal flow of purine bases (PBs) was determined by a dual marker system, and 15N was infused continuously into the rumen to label microbial PBs. The isotope dilution of urinary PDs in relation to duodenal PBs confirmed the presence of an endogenous fraction (236 μmol/kg LW0.75) bigger than in sheep and lower than values estimated in cows with impaired rumen fermentation. Excretion of PDs increased linearly in response to incremental supply of PBs with an equimolar recovery of 0.84 mol/mol. However, net recovery of duodenal PBs was 0.67 for the basal diet and 0.65, 0.90, 0.79 and 0.82 for the four levels of PB infusion. It is suggested that differences in digestibility between yeast-RNA and duodenal PBs might explain differences in recovery estimations.

Quantification and chemical composition of mixed bacteria harvested from solid fractions of rumen digesta: effect of detachment procedure

Four ewes were given two diets made up with two ratios of rolled barley grain and ammonia treated straw (80/20, diet C and 20/80 diet R) twice a day. Two microbial markers, purine bases (PB) and 15N, were used as internal and external markers, respectively. Ruminal bacteria from the liquid (LAB) and solid (SAB) fractions of digesta were harvested from samples obtained at 1 and 6 h after feeding. Bacteria were separated from the particulate material by cooling plus homogenisation (B) and also by applying tertiary butanol (TB), methylcellulose (M) or changes of temperature (CHT). The most effective procedures to remove bacteria from the solid phase were M and CHT although CHT showed the highest level of losses and the lowest total recovery of the bacterial pellet. There were no differences between B and TB in both detaching efficiency and total recovery ratio of adherent bacteria. Ratio of recovery of detached material as a bacterial pellet was 32.0, 32.2, 33.3 and 27.8% for B, TB, M and CHT, respectively. Diet did not interact with detaching efficiency of the experimental treatment although concentration of total N (g/100 g OM) and PB (μmol/g OM) content concentration were higher in pellets obtained with diet C (9.11 and 125) than with R (8.20 and 107, respectively, P<0.05). Postprandial differences were not significant. Bacterial samples extracted from the liquid phase contained significantly (P<0.001) more total N (9.21 vs. 8.51) PB (160.5 vs. 104.3) and PB/N (1.73 vs. 1.23 μmol/mg) than those samples extracted from the solid phase. There were no differences in the chemical composition of the microbial sample after detachment by B, BT and M but bacterial extract obtained after CHT treatment showed significant changes in PB, N content and PB/N ratio (P<0.01).

Degradation of transgenic DNA from genetically modified soya and maize in human intestinal simulations.

The inclusion of genetically modified (GM) foods in the human diet has caused considerable debate. There is concern that the transfer of plant-derived transgenes to the resident intestinal microflora could have safety implications. For these gene transfer events to occur, the nucleic acid would need to survive passage through the gastrointestinal tract. The aim of the present study was to evaluate the rate at which transgenes, contained within GM soya and maize, are degraded in gastric and small bowel simulations. The data showed that 80 % of the transgene in naked GM soya DNA was degraded in the gastric simulations, while no degradation of the transgenes contained within GM soya and maize were observed in these acidic conditions. In the small intestinal simulations, transgenes in naked soya DNA were degraded at a similar rate to the material in the soya protein. After incubation for 30 min, the transgenes remaining in soya protein and naked DNA were 52 (sem 13.1) % and 34 (sem 17.5) %, respectively, and at the completion of the experiment (3 h) these values were 5 % and 3 %, respectively. In contrast to the soya transgene, the maize nucleic acid was hydrolysed in the small intestinal simulations in a biphasic process in which approximately 85 % was rapidly degraded, while the rest of the DNA was cleaved at a rate similar to that in the soya material. Guar gum and tannic acid, molecules that are known to inhibit digestive enzymes, did not influence the rate of transgene degradation in soya protein. In contrast guar gum reduced the rate of transgene degradation in naked soya DNA in the initial stages, but the polysaccharide did not influence the amount of nucleic acid remaining at the end of the experiment. Tannic acid reduced the rate of DNA degradation throughout the small bowel simulations, with 21 (sem 5.4) % and 2 (sem 1.8) % of the naked soya DNA remaining in the presence and absence of the phenolic acid, respectively. These data indicate that some transgenes in GM foods may survive passage through the small intestine.

Large bowel fermentation of maize or sorghum-acorn diets fed as a different source of carbohydrates to Landrace and Iberian pigs

Twenty-four finishing pigs (twelve Iberian and twelve Landrace) were used in a growing and slaughtering experiment. Animals were fed two diets differing in their ingredients, maize (diet C) or sorghum-acorn (diet A). At an average weight of 107.0 kg pigs were slaughtered and hindgut digesta sampled to study the effect of breed and diet on large bowel fermentation. Flows of digesta to the hindgut compartment were estimated based on an indigestible flow marker (Cr2O3) and were higher in Iberian than in Landrace pigs (P<0.001), and higher in animals fed diet A than diet C (P=0.07). The higher flows in Iberian pigs were mainly associated with a higher voluntary feed intake (3.50 v. 2.70 kg/d, P<0.01) and lower ileal digestibility of NSP (-12.8 v. 47.8, P<0.01). Differences between diets were mainly associated with a lower ileal digestibility of starch from diet A (89.2 v. 96.9 %, P=0.06), although no differences in the resistant starch content were observed in vitro. Fermentation of different carbohydrates through the large bowel showed that NSP-glucose had lower digestibility in Iberian than in Landrace pigs (62.5 v. 94.2 %, P<0.001), but no differences were observed in starch, or other NSP-fibre fractions (arabinose, xylose and galactose). The type and amount of carbohydrates reaching the large bowel were related to the diet but also to breed, and promoted differences in the fermentative activity associated with different volatile fatty acid patterns and changes in microbial enzymic activity.

Effect and interaction between wheat bran and zinc oxide on productive performance and intestinal health in post-weaning piglets.

The inclusion of fibre has been studied as an alternative to antimicrobials in weaning pig diets, while ZnO is used as an effective method to prevent post-weaning diarrhoea. However, it has not been investigated to what extent these two strategies interact with each other. The present study was designed to evaluate the effects of including wheat bran (WB) and ZnO alone or combined in the diet of early-weaning pigs on productive performance and microbial activity in the gastrointestinal tract (trial 1). A total of sixty-four piglets were distributed in a 2 × 2 factorial combination of two levels of WB (0 v. 40 g/kg) and ZnO (0 v. 3 g/kg) in the diet. The inclusion of ZnO in the diet improved the feed intake and growth of the animals and reduced the incidence of diarrhoea. The inclusion of WB increased SCFA concentrations and decreased Escherichia coli counts. However, simultaneous incorporation of WB and ZnO increased E. coli counts. Two in vitro trials were also designed to clarify hypotheses derived from the in vivo test: (1) the ability of WB and other fibre sources to bind E. coli in vitro (trial 2) and (2) the in vitro interactions between WB and ZnO with respect to E. coli growth (trial 3). We can conclude that incorporation of WB in the diet improved gut health by modulating the activity and composition of the microbial population. The negative interaction between WB and ZnO raises the interest of considering the inclusion of phytase enzymes to reduce the therapeutic levels of ZnO in post-weaning diets.

Evaluation of the inclusion of a mixture of organic acids or lactulose into the feed of pigs experimentally challenged with Salmonella Typhimurium.

A mixture of organic acids and lactulose for preventing or reducing colonization of the gut by Salmonella Typhimurium was evaluated in pigs. A total of 63 4-week-old commercial piglets were randomly distributed into three different experimental dietary groups: a plain diet without additives (PD) and the same diet supplemented with either 0.4% (w/v) formic acid and 0.4% lactic acid (w/v) (AC) or 1% (w/v) lactulose (LC). After 7 days of adaptation, two-thirds of the pigs (14 from each diet) were challenged with a 2-mL oral dose of 10(8)CFU/mL of Salmonella Typhimurium, leaving the remaining animals unchallenged (UC). After 4 and 10 days post-challenge, pigs were euthanized and the ileum and caecum content were aseptically sampled to (a) quantify lactic, formic, and short-chain fatty acids (SCFA), (b) quantify bacterial populations and Salmonella by fluorescence in situ hybridization and (c) qualitatively analyse bacterial populations through denaturing gradient gel electrophoresis (DGGE). Modification of fermentation products and counts of some of the bacterial groups analysed in the challenged pigs receiving the treatments AC and LC were minimal. Treatments only influenced the bacterial diversity after 10 days post-challenge, with AC generating a lower number of DGGE bands than UC (P<0.05). Neither the inclusion of a mixture of 0.4% (w/v) formic and 0.4% (w/v) lactic acids nor of 1% (w/v) lactulose in the feed influenced numbers of Salmonella in the ileum and caecum of experimentally challenged pigs.

Lactulose and Lactobacillus plantarum, a Potential Complementary Synbiotic To Control Postweaning Colibacillosis in Piglets

ABSTRACT The potential of a prebiotic oligosaccharide lactulose, a probiotic strain of Lactobacillus plantarum, or their synbiotic combination to control postweaning colibacillosis in pigs was evaluated using an enterotoxigenic Escherichia coli (ETEC) K88 oral challenge. Seventy-two weanlings were fed four diets: a control diet (CTR), that diet supplemented with L. plantarum (2 × 1010 CFU · day−1) (LPN), that diet supplemented with 10 g · kg−1 lactulose (LAC), or a combination of the two treatments (SYN). After 7 days, the pigs were orally challenged. Six pigs per treatment were euthanized on days 6 and 10 postchallenge (PC). Inclusion of lactulose improved the average daily gain (ADG) (P < 0.05) and increased lactobacilli (P < 0.05) and the percentage of butyric acid (P < 0.02) in the colon. An increase in the ileum villous height (P < 0.05) and a reduction of the pig major acute-phase protein (Pig-MAP) in serum (P < 0.01) were observed also. The inclusion of the probiotic increased numbers of L. plantarum bacteria in the ileum and colon (P < 0.05) and in the total lactobacilli in the colon and showed a trend to reduce diarrhea (P = 0.09). The concentrations of ammonia in ileal and colonic digesta were decreased (P < 0.05), and the villous height (P < 0.01) and number of ileal goblet cells (P < 0.05) increased, at day 10 PC. A decrease in plasmatic tumor necrosis factor alpha (TNF-α) (P < 0.01) was also seen. The positive effects of the two additives were combined in the SYN treatment, resulting in a complementary synbiotic with potential to be used to control postweaning colibacillosis.