Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

Abomasal infusion of casein, starch and soybean oil differentially affect plasma concentrations of gut peptides and feed intake in lactating dairy cows

The effects of specific nutrients on secretion and plasma concentrations of gut peptides (glucagon-like peptide-1((7-36)) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin-8 (CCK)) differ across species, but are not reported for cattle. Our objective was to determine acute (hours) and chronic (1 week) effects of increased abomasal supply of protein, carbohydrate, or fat to the small intestine on dry matter intake (DMI) and plasma concentrations of GLP-1, GIP, CCK, and insulin. Four mid-lactation Holstein cows were used in a 4 x 4 Latin square design experiment. Treatments were 7-day abomasal infusions of water, soybean oil (500 g/d), corn starch (1100 g/d), or casein (800 g/d). Jugular vein plasma was obtained over 7 h at the end of the first and last day of infusions. Oil infusion decreased DMI on day 7, but total metabolizable energy (ME) supply (diet plus infusate) did not differ from water infusion. Casein and starch infusion had no effect on feed DMI; thus, ME supply increased. Decreased DMI on day 7 of oil infusion was accompanied by increased plasma GLP-1 concentration, but decreased plasma CCK concentration. Increased plasma GIP concentration was associated with increased ME supply on day 7 of casein and starch infusion. Casein infusion tended to increase plasma CCK concentration on both days of sampling, and increased plasma GLP-1 and insulin concentration on day 1 of infusion. The present data indicate a sustained elevation of plasma concentration of GLP-1, but not CCK, may contribute to the reduced DMI observed in dairy cows provided supplemental fat. (C) 2008 Elsevier Inc. All rights reserved.

Production and metabolic effects of site of starch digestion in lactating dairy cattle

Milk solids yield in modern dairy cows has increased linearly over the last 50 years, stressing the need for maximal dietary energy intake to allow genetic potential for milk energy yield to be realized with minimal negative effects on health and reproduction. Feeding supplemental starch is a common approach for increasing the energy density of the ration and supplying carbon for meeting the substantial glucose requirement of the higher yielding cow. In this regard, it is a long held belief that feeding starch in forms that increase digestion in the small intestine and glucose absorption will benefit the cow in terms of energetic efficiency and production response, but data supporting this dogma are equivocal. This review will consider the impact of supplemental starch and site of starch digestion on metabolic and production responses of lactating dairy cows, including effects on feed intake, milk yield and composition, nutrient partitioning, the capacity of the small intestine for starch digestion, and nutrient absorption and metabolism by the splanchnic tissues (the portal-drained viscera and liver). Whilst there appears to be considerable capacity for starch digestion and glucose absorption in the lactating dairy cow, numerous strategic studies implementing postruminal starch or glucose infusions have observed increases in milk yield, but decreased milk fat concentration such that there is little effect on milk energy yield, even in early lactation. Measurements of energy balance confirm that the majority of the supplemental energy arising from postruminal starch digestion is used with high efficiency to support body adipose and protein retention, even in early lactation. These responses may be mediated by changes in insulin status, and be beneficial to the cow in terms of reproductive success and well-being. However, shifting starch digestion from the rumen impacts the nitrogen economy of the cow as well by shifting the microbial protein gained from starch digestion from potentially absorbable protein to endogenous faecal loss.

Visceral tissue mass and rumen volume in dairy cows during the transition from late gestation to early lactation

The objectives were to measure the effects of transition and supplemental barley or rumen-protected protein on visceral tissue mass in dairy cows and the effects of transition and barley on rumen volume and liquid turnover. Cows were individually fed a grass silage-based gestation ration to meet energy and protein requirements for body weight stasis beginning 6 wk before expected calving. A corn silage-based lactation ration was individually fed ad libitum after calving. In the visceral mass study, 36 cows were randomly assigned to one of 3 dietary treatments: basal ration or basal ration plus either 800 g dry matter (DM) of barley meal per day or 750 g DM of rumen-protected soybean protein per day. Cows were slaughtered at 21 and 7 d before expected calving date or at 10 and 22 d postpartum. Visceral mass and rumen papillae characteristics were measured. Diets had little effect on visceral mass. The mass of the reticulo-rumen, small intestine, large intestine, and liver was, or tended to be, greater at 22 d postpartum but not at 10 d postpartum before DM intake had increased. Rumen papillae mass increased at 10 d postpartum, perhaps in response to increased concentrates. Mesenteric fat decreased after calving, reflecting body fat mobilization. Ten rumen-cannulated cows were fed the basal gestation ration alone or supplemented with 880 g of barley meal DM. Rumen volumes and liquid dilution rates were measured at 17 and 8 d before calving and at 10, 20, and 31 d postpartum. Feeding barley had no effects. After calving, rumen DM volume and liquid dilution rate increased, but liquid volume did not increase. Changes in gastrointestinal and liver mass during transition were apparently a consequence of changes in DM intake and nutrient supply and not initiation of lactation per se.

Nitrogen recycling through the gut and the nitrogen economy of ruminants: An asynchronous symbiosis

The extensive development of the ruminant forestomach sets apart their N economy from that of nonruminants in a number of respects. Extensive pregastric fermentation alters the profile of protein reaching the small intestine, largely through the transformation of nitrogenous compounds into microbial protein. This process is fueled primarily by carbohydrate fermentation and includes extensive recycling of N between the body and gut lumen pools. Nitrogen recycling occurs via blood and gut lumen exchanges of urea and NH3, as well as endogenous gut and secretory N entry into the gut lumen, and the subsequent digestion and absorption of microbial and endogenous protein. Factors controlling urea transfer to the gut from blood, including the contributions of urea transporters, remain equivocal. Ammonia produced by microbial degradation of urea and dietary and endogenous AA is utilized by microbial fermentation or absorbed and primarily converted to urea. Therefore, microbial growth and carbohydrate fermentation affect the extent of NH3 absorption and urea N recycling and excretion. The extensive recycling of N to the rumen represents an evolutionary advantage of the ruminant in terms of absorbable protein supply during periods of dietary protein deficiency, or asynchronous carbohydrate and protein supply, but incurs a cost of greater N intakes, especially in terms of excess N excretion. Efforts to improve the efficiency of N utilization in ruminants by synchronizing fermentable energy and N availability have generally met with limited success with regards to production responses. In contrast, imposing asynchrony through oscillating dietary protein concentration, or infrequent supplementation, surprisingly has not negatively affected production responses unless the frequency of supplementation is less than once every 3 d. In some cases, oscillation of dietary protein concentration has improved N retention compared with animals fed an equal amount of dietary protein on a daily basis. This may reflect benefits of Orn cycle adaptations and sustained recycling of urea to the gut. The microbial symbiosis of the ruminant is inherently adaptable to asynchronous N and energy supply. Recycling of urea to the gut buffers the effect of irregular dietary N supply such that intuitive benefits of rumen synchrony in terms of the efficiency of N utilization are typically not observed in practice.

Metabolism of Maillard reaction products by the human gut microbiota - implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.

Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.

Metabolism of Maillard reaction products by the human gut microbiota: implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

Sialyloligosaccharides inhibit cholera toxin binding to the GM1 receptor

It is recognised that cholera toxin (Ctx) is a significant cause of gastrointestinal disease globally, particularly in developing countries where access to uncontaminated drinking water is at a premium. Ctx vaccines are prohibitively expensive and only give short-term protection. Consequently, there is scope for the development of alternative control strategies or prophylactics. This may include the use of oligosaccharides as functional mimics for the cell-surface toxin receptor (GM I). Furthermore, the sialic acid component of epithelial receptors has already been shown to contribute significantly to the adhesion and pathogenesis of Ctx. Here, we demonstrate the total inhibition of Ctx using GM1-competitive ELISA with 25 mg mL(-1) of a commercial preparation of sialyloligosaccharides (SOS). The IC50 value was calculated as 5.21 mg mL(-1). One-hundred percent inhibition was also observed at all concentrations of Ctx-HRP tested with 500 ng mL(-1) GM1-OS. Whilst SOS has much lower affinity for Ctx than GM1-OS, the commercial preparation is impure containing only 33.6% carbohydrate; however, the biantennary nature of SOS appears to give a significant increase in potency over constituent monosaccahride residues. It is proposed that SOS could be used as a conventional food additive, such as in emulsifiers, stabilisers or sweeteners, and are classified as nondigestible oligosaccharides that pass into the small intestine, which is the site of Ctx pathogenesis. (C) 2008 Elsevier Ltd. All rights reserved.

The reaction of flavanols with nitrous acid protects against N-nitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth

Studies have suggested that diets rich in polyphenols Such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D I levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract. (c) 2005 Elsevier Inc. All rights reserved.

Adverse gastrointestinal effects of arginine and related amino acids

Oral supplements of arginine and citrulline increase local nitric oxide (NO production in the small intestine and this may be harmful under certain circumstances. Gastrointestinal toxicity was therefore reviewed with respect to the intestinal physiology of arginine, citrulline, ornithine, and cystine (which shares the same transporter) and the many clinical trials of supplements of the dibasic amino acids or N-acetylcysteine (NAC. The human intestinal dibasic amino acid transport system has high affinity and low capacity. L-Arginine (but not lysine, ornithine, or D-arginine) induces water and electrolyte secretion that is mediated by NO, which acts as an absorbagogue at low levels and as a secretagogue at high levels. The action of many laxatives is NO mediated and there are reports of diarrhea following oral administration of arginine or ornithine ihine. The clinical data cover a wide span of arginine intakes f rom 3 g/d to > 100 g/d, but the standard of reporting adverse effects (e.g. nausea, vomiting, and diarrhea) was variable. Single doses of 3-6 g rarely provoked side effects and healthy athletes appeared to be more susceptible than diabetic patients to gastrointestinal symptoms at individual doses >9 g. This may relate to an effect of disease on gastrointestinal motility and pharmacokinetics. Most side effects of arginine and NAC occurred at single doses of >9 g in adults >140 mg/kg) often when part of a daily regime of similar to>30 g/d (>174 mmol/d). In the case of arginine, this compares with the laxative threshold of the nonabsorbed disaccharide alcohol, lactitol (74 g or 194 mmol). Adverse effects seemed dependent on the dosage regime and disappeared if divided doses were ingested (unlike lactitol). Large single doses of poorly absorbed amino acids seem to provoke diarrhea. More research is needed to refine dosage strategies that reduce this phenomenon. It is suggested that dipeptide forms of arginine may meet this criterion.

Effect of polydextrose on intestinal microbes and immune functions in pigs

Dietary fibre has been proposed to decrease risk for colon cancer by altering the composition of intestinal microbes or their activity. In the present study, the changes in intestinal microbiota and its activity, and immunological characteristics, such as cyclo-oxygenase (COX)-2 gene expression in mucosa, in pigs fed with a high-energy-density diet, with and without supplementation of a soluble fibre (polydextrose; PDX) (30 g/d) were assessed in different intestinal compartments. PDX was gradually fermented throughout the intestine, and was still present in the distal colon. Irrespective of the diet throughout the intestine, of the four microbial groups determined by fluorescent in situ hybridisation, lactobacilli were found to be dominating, followed by clostridia and Bacteroides. Bifidobacteria represented a minority of the total intestinal microbiota. The numbers of bacteria increased approximately ten-fold from the distal small intestine to the distal colon. Concomitantly, also concentrations of SCFA and biogenic amines increased in the large intestine. In contrast, concentrations of luminal IgA decreased distally but the expression of mucosal COX-2 had a tendency to increase in the mucosa towards the distal colon. Addition of PDX to the diet significantly changed the fermentation endproducts, especially in the distal colon, whereas effects on bacteria] composition were rather minor. There was a reduction in concentrations of SCFA and tryptamine, and an increase in concentrations of spermidine in the colon upon PDX supplementation. Furthermore, PDX tended to decrease the expression of mucosal COX-2, therefore possibly reducing the risk of developing colon cancer-promoting conditions in the distal intestine.

The fate of olive oil polyphenols in the gastrointestinal tract: implications of gastric and colonic microflora-dependent biotransformation

We have conducted a detailed investigation into the absorption, metabolism and microflora-dependent transformation of hydroxytyrosol ( HT), tyrosol (TYR) and their conjugated forms, such as oleuropein (OL). Conjugated forms underwent rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of free HT and TYR entering the small intestine. Both HT and TYR transferred across human Caco-2 cell monolayers and rat segments of jejunum and ileum and were subject to classic phase I/II biotransformation. The major metabolites identified were an O-methylated derivative of HT, glucuronides of HT and TYR and a novel glutathionylated conjugate of HT. In contrast, there was no absorption of OL in either model. However, OL was rapidly degraded by the colonic microflora resulting in the formation of HT. Our study provides additional information regarding the breakdown of complex olive oil polyphenols in the GI tract, in particular the stomach and the large intestine.

Prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a randomized, controlled, double-blind, crossover intervention study

BACKGROUND: The absorption of cocoa flavanols in the small intestine is limited, and the majority of the flavanols reach the large intestine where they may be metabolized by resident microbiota. OBJECTIVE: We assessed the prebiotic potential of cocoa flavanols in a randomized, double-blind, crossover, controlled intervention study. DESIGN: Twenty-two healthy human volunteers were randomly assigned to either a high-cocoa flavanol (HCF) group (494 mg cocoa flavanols/d) or a low-cocoa flavanol (LCF) group (23 mg cocoa flavanols/d) for 4 wk. This was followed by a 4-wk washout period before volunteers crossed to the alternant arm. Fecal samples were recovered before and after each intervention, and bacterial numbers were measured by fluorescence in situ hybridization. A number of other biochemical and physiologic markers were measured. RESULTS: Compared with the consumption of the LCF drink, the daily consumption of the HCF drink for 4 wk significantly increased the bifidobacterial (P < 0.01) and lactobacilli (P < 0.001) populations but significantly decreased clostridia counts (P < 0.001). These microbial changes were paralleled by significant reductions in plasma triacylglycerol (P < 0.05) and C-reactive protein (P < 0.05) concentrations. Furthermore, changes in C-reactive protein concentrations were linked to changes in lactobacilli counts (P < 0.05, R(2) = -0.33 for the model). These in vivo changes were closely paralleled by cocoa flavanol-induced bacterial changes in mixed-batch culture experiments. CONCLUSION: This study shows, for the first time to our knowledge, that consumption of cocoa flavanols can significantly affect the growth of select gut microflora in humans, which suggests the potential prebiotic benefits associated with the dietary inclusion of flavanol-rich foods. This trial was registered at clinicaltrials.gov as NCT01091922.