Rates of production of acetate, propionate, and butyrate in the rumen of lactating dairy cows given normal and low-roughage diets

Five lactating dairy cows with a permanent cannula in the rumen were given ( kg DM/d) a normal diet (7.8 concentrates, 5.1 hay) or a low-roughage (LR) diet (11.5 concentrates, 1.2 hay) in two meals daily in a two-period crossover design. Milk fat (g/kg) was severely reduced on diet LR. To measure rates of production of individual volatile fatty acids (VFA) in the rumen, 0.5 mCi 1-C-14-acetic acid, 2-C-14-propionic acid, or 1-C-14-n-butyric acid were infused into the rumen for 22 h at intervals of 2 to 6 d; rumen samples were taken over the last 12 h. To measure rumen volume, we infused Cr-EDTA into the rumen continuously, and polyethylene glycol was injected 2 h before the morning feed. Results were very variable, so volumes measured by rumen emptying were used instead. Net production of propionic acid more than doubled on LR, but acetate and butyrate production was only numerically lower. Net production rates pooled across both diets were significantly related to concentrations for each VFA. Molar proportions of net production were only slightly higher than molar proportions of concentrations for acetate and propionate but were lower for butyrate. The net energy value (MJ/d) of production of the three VFA increased from 89.5 on normal to 109.1 on LR, equivalent to 55 and 64% of digestible energy, respectively. Fully interchanging, three-pool models of VFA C fluxes are presented. It is concluded that net production rates of VFA can be measured in non-steady states without the need to measure rumen volumes.

The F test for comparing two normal variances: correct and incorrect calculation of the two-sided p-value?

This article illustrates that not all statistical software packages are correctly calculating a p-value for the classical F test comparison of two independent Normal variances. This is illustrated with a simple example, and the reasons why are discussed. Eight different software packages are considered.

Dietary glycated protein modulates the colonic microbiota towards a more detrimental composition in ulcerative colitis patients and non-ulcerative colitis subjects

AIM: To investigate the effect of native, heated and glycated bovine serum albumin (BSA) on the ulcerative colitis (UC) and non-UC colonic microbiota in vitro. METHODS AND RESULTS: Continuous flow culture (CFC) models of the human colonic microbiota inoculated with faeces from UC and non-UC volunteers were maintained on BSA as growth substrate. Changes in bacterial populations and short-chain fatty acids were determined. UC and non-UC microbiota differed significantly in microbial populations, with elevated numbers of sulfate-reducing bacteria (SRB) and clostridia in the microbiota from UC patients. Compared with native BSA, glycated BSA modulated the gut microbiota of UC patients in vitro towards a more detrimental community structure with significant increases in putatively harmful bacteria (clostridia, bacteroides and SRB; P < 0.009) and decreases in dominant and putatively beneficial bacterial groups (eubacteria and bifidobacteria; P < 0.0004). The levels of beneficial short-chain fatty acids were significantly decreased by heated or glycated BSA, but were increased significantly by native BSA. CONCLUSION: The UC colonic microbiota maintained in CFC was significantly modified by glycated BSA. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that dietary glycated protein may impact upon the composition and activity of the colonic microbiota, an important environmental variable in UC.

Estimation following selection of the largest of two normal means

This paper considers the problem of estimation when one of a number of populations, assumed normal with known common variance, is selected on the basis of it having the largest observed mean. Conditional on selection of the population, the observed mean is a biased estimate of the true mean. This problem arises in the analysis of clinical trials in which selection is made between a number of experimental treatments that are compared with each other either with or without an additional control treatment. Attempts to obtain approximately unbiased estimates in this setting have been proposed by Shen [2001. An improved method of evaluating drug effect in a multiple dose clinical trial. Statist. Medicine 20, 1913–1929] and Stallard and Todd [2005. Point estimates and confidence regions for sequential trials involving selection. J. Statist. Plann. Inference 135, 402–419]. This paper explores the problem in the simple setting in which two experimental treatments are compared in a single analysis. It is shown that in this case the estimate of Stallard and Todd is the maximum-likelihood estimate (m.l.e.), and this is compared with the estimate proposed by Shen. In particular, it is shown that the m.l.e. has infinite expectation whatever the true value of the mean being estimated. We show that there is no conditionally unbiased estimator, and propose a new family of approximately conditionally unbiased estimators, comparing these with the estimators suggested by Shen.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INFalpha and INFgamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INF alpha and INF gamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

In vitro three-stage continuous fermentation of gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from maltodextrin as the source, were evaluated for their fermentability by the human colonic microflora. The selectivity of growth of desirable bacteria in the human colon was studied in a three-stage continuous model of the human large intestine. Populations of bacteria, and their fluctuations as a response to the fermentation, were enumerated using fluorescent in situ hybridization (FISH). The gluco-oligosaccharides resulted in increases in numbers of bifidobacteria and the Lactobacillus/Enterococcus group in all 3 vessels of the system, representing the proximal, transverse and distal colonic areas. The prebiotic indices of the glucooligosaccharides were 2.29, 4.23 and 2.74 in V1, V2 and V3 respectively.

In vitro fermentation of mixed linkage glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

The aim of this study was to develop selectively fermented (prebiotic) carbohydrate molecules which would also result in the generation of butyric acid. Glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from various types of maltodextrins were evaluated for their fermentation by mixed cultures of human colonic microflora. The selectivity of growth of desirable bacteria (bifidobacteria, lactobacilli) was studied in stirred pH-controlled (6.8) batch cultures. Bacterial populations were enumerated using fluorescent in situ hybridization (FISH). Gluco-oligosaccharides resulted in significantly (P<0.05) increased numbers of bifidobacteria and lactobacilli within 24 hours. Bacteroides, clostridial and eubacterial populations were slightly decreased at 48 h. There was very little difference in selectivity between the maltodextrin substrates and the products, although maltodextrin displayed a slightly less selective fermentation than the gluco-oligosaccharide products, also stimulating the growth of bacteroides, clostridia and eubacteria. Gluco-oligosaccharides, produced from G19 maltodextrin, resulted in the best prebiotic effect with the highest prebiotic index (PI) of 5.90 at 48 hours. Acetate, propionate and butyrate were all produced from glucooligosaccharides, derived from G19 maltodextrin, at 48 hours but no lactate or formate were detected.

A novel galactooligosaccharide mixture increases the bifidobacterial population numbers in a continuous in vitro fermentation system and in the proximal colonic contents of pigs in vivo

Prebiotics are nondigestible food ingredients that encourage proliferation of selected groups of the colonic microflora, thereby altering the composition toward a more beneficial community. In the present study, the prebiotic potential of a novel galactooligosaccharide (GOS) mixture, produced by the activity of galactosyltransferases from Bifidobacterium bifidum 41171 on lactose, was assessed in vitro and in a parallel continuous randomized pig trial. In situ fluorescent hybridization with 16S rRNA-targeted probes was used to investigate changes in total bacteria, bifidobacteria, lactobacilli, bacteroides, and Clostridium histolyticum group in response to supplementing the novel GOS mixture. In a 3-stage continuous culture system, the bifidobacterial numbers for the first 2 vessels, which represented the proximal and traverse colon, increased (P < 0.05) after the addition of the oligosaccharide mixture. In addition, the oligosaccharide mixture strongly inhibited the attachment of enterohepatic Escherichia coli (P < 0.01) and Salmonella enterica serotype Typhimurium (P < 0.01) to HT29 cells. Addition of the novel mixture at 4% (wt:wt) to a commercial diet increased the density of bificlobacteria (P < 0.001) and the acetate concentration (P < 0.001), and decreased the pH (P < 0.001) compared with the control diet and the control diet supplemented with inulin, suggesting a great prebiotic potential for the novel oligosaccharide mixture. J. Nutr. 135: 1726-1731, 2005.

Potential roles and clinical utility of prebiotics in newborns, infants, and children: proceedings from a global prebiotic summit meeting, New York City, June 27-28, 2008

Initial bacterial colonization, including colonization with health-positive bacteria, such as bifidobacteria and lactobacilli, is necessary for the normal development of intestinal innate and adaptive immune defenses. The predominance of beneficial bacteria in the gut microflora of breast-fed infants is thought to be, at least in part, supported by the metabolism of the complex mixture of oligosaccharides present in human breast milk, and a more adult-type intestinal microbiota is found in formula-fed infants. Inadequate gut colonization, dysbiosis, may lead to an increased risk of infectious, allergic, and autoimmune disorders later in life. The addition of appropriate amounts of selected prebiotics to infant formulas can enhance the growth of bifidobacteria or lactobacilli in the colonic microbiota and, thereby, might produce beneficial effects. Among the substrates considered as prebiotics are the oligosaccharides inulin, fructo-oligosaccharides, galacto-oligosaccharides, and lactulose. There are some reports that such prebiotics have beneficial effects on various markers of health. For example, primary prevention trials in infants have provided promising data on prevention of infections and atopic dermatitis. Additional well-designed prospective clinical trials and mechanistic studies are needed to advance knowledge further in this promising field. (J Pediatr 2009;155:S61-70).

Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products

The metabolism of chlorogenic acid., naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-D-hydroxyphenyl)propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols. (C) 2003 Elsevier Inc. All rights reserved.

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.

Pectins and pectic-oligosaccharides inhibit Escherichia coli O157 : H7 Shiga toxin as directed towards the human colonic cell line HT29

Pectins and pectic-oligosaccharides, as derived by controlled enzymatic hydrolysis, were evaluated for their ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7. Both types of material resulted in some degree of protection but this was significantly higher (P > 0.01) with the oligosaccharide fractions (giving 90-100% cell survival, compared to 70-80% with the polymer). An effect of methylation on the protective effect was detected with lower degrees being more active. The pectic-oligosaccharides and galabiose, the minimum toxin receptor analogue, were shown to inhibit toxicity and were both protective at 10 mg ml(-1), but not at lower concentrations. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.