Selective fermentation of gentiobiose-derived oligosaccharides by human gut bacteria and influence of molecular weight

Gentiooligosaccharides and alternansucrase gentiobiose acceptor products were fractionated by their degree of polymerization (DP) on a Bio-Gel P2 column. Fractions were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, and incubated with human faecal bacteria under anaerobic conditions at 37 degrees C. The growth of predominant gut bacteria on the oligosaccharides was evaluated by fluorescence in situ hybridization and a prebiotic index (PI) was calculated. Lower DP gentiooligosaccharides (DP2-3) showed the highest selectivity (PI of 4.89 and 3.40, respectively), whereas DP4-5 alternansucrase gentiobiose acceptor products generated the greatest values (PI of 5.87). The production of short-chain fatty acids was also determined during the time course of the reactions. The mixture of DP6-10 alternansucrase gentiobiose acceptor products generated the highest levels of butyric acid but the lowest levels of lactic acid. Generally, for similar molecular weights, alternansucrase gentiobiose acceptor products gave higher PI values than gentiooligosaccharides.

High temperature reduction of metmyoglobin in aqueous muscle extracts

Oxymyoglobin in aqueous extracts of fresh beef longissimus dorsi muscles was initially oxidised to metmyoglobin during heat treatments at temperatures in the range 50-70 degreesC. The metmyoglobin then underwent reduction to a red pigment that was shown spectrally to be identical to oxymyoglobin. The formation of oxymyoglobin involved a heat induced precipitate that when removed from the solution, allowed oxidation to metmyoglobin to occur. However, on re-addition of the precipitate further reduction to oxymyoglobin took place. Dialysis of the muscle extract prior to heating markedly inhibited the reduction but addition of NADH to the dialysate permitted further reduction. The precipitate plus NADH caused oxymyoglobin formation in the presence of metmyoglobin but neither the precipitate nor NADH alone induced this formation. It is concluded that the initial conversion of oxymyoglobin to metmyoglobin on heating fresh beef muscle extracts was reversible and that the reverse reaction depended on the presence of both NADH and a muscle protein.

Measurement of ionic calcium in milk

There are many reports in the literature regarding the effects of ionic calcium on reactions related to casein micelle stability, such as heat stability, ethanol stability and susceptibility to gelation, sediment formation and fouling. However, experimental evidence supporting these assertions is much less readily available. This paper evaluates three selective ion electrode systems for measuring ionic calcium directly in milk as well as looking at the effects on pH reduction and addition of calcium chloride. The best electrode system was the Ciba Corning 634 system, which was designed for blood but has been modified for milk. This was found to be reproducible and stable when calibrated daily and allowed direct measurements to be taken on milk in 70 s. This has been found to perform well now for 3 years. The other systems were not so useful, as they took longer to stabilize, but may be useful for higher ionic calcium concentrations, which are found in acidified milk products. Reducing the pH increased ionic calcium and reduced ethanol stability. Calcium chloride addition reduced pH, increased ionic calcium and reduced the ethanol stability. Readjusting the pH to its value before calcium addition reduced the ionic calcium, but not back to its original value. Milks from individual cows showed wide variations in their ionic calcium concentrations. This establishes the methodology for a more detailed investigation on measurement of ionic calcium in milks from individual cows and from bulk milks, to allow a better understanding of its role in casein micelle stability.

Selective separation of beta-lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB

The selective separation of whey proteins was studied using colloidal gas aphrons generated from the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). From the titration curves obtained by zeta potential measurements of individual whey proteins, it was expected to selectively adsorb the major whey proteins, i.e., bovine serum albumin, alpha-lactalbumin, and beta-lactoglobulin to the aphrons and elute the remaining proteins (lactoferrin and lactoperoxidase) in the liquid phase. A number of process parameters including pH, ionic strength, and mass ratio of surfactant to protein (M-CTAB/M-TP) were varied in order to evaluate their effect on protein separation. Under optimum conditions (2 mmol/l CTAB, M-CTAB/M-TP = 0.26-0.35, pH 8, and ionic strength = 0.018 mol/l), 80-90% beta-lactoglobulin was removed from the liquid phase as a precipitate, while about 75% lactoferrin and lactoperoxidase, 80% bovine serum albumin, 95% immunoglobulin, and 65% alpha-lactalbumin were recovered in the liquid fraction. Mechanistic studies using zeta potential measurements and fluorescence spectroscopy proved that electrostatic interactions modulate only partially the selectivity of protein separation, as proteins with similar surface charges do not separate to the same extent between the two phases. The selectivity of recovery of beta-lactoglobulin probably occurs in two steps: the first being the selective interaction of the protein with opposite-charged surfactant molecules by means of electrostatic interactions, which leads to denaturation of the protein and subsequent formation and precipitation of the CTAB-beta-lactoglobulin complex. This is followed by the separation of CTAB-beta-lactoglobulin aggregates from the bulk liquid by flotation in the aphron phase. In this way, CGAs act as carriers which facilitate the removal of protein precipitate. (c) 2005 Wiley Periodicals, Inc.

Selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia

Aims/hypothesis Recent evidence suggests that a particular gut microbial community may favour occurrence of the metabolic diseases. Recently, we reported that high-fat (HF) feeding was associated with higher endotoxaemia and lower Bifidobacterium species (spp.) caecal content in mice. We therefore tested whether restoration of the quantity of caecal Bifidobacterium spp. could modulate metabolic endotoxaemia, the inflammatory tone and the development of diabetes. Methods Since bifidobacteria have been reported to reduce intestinal endotoxin levels and improve mucosal barrier function, we specifically increased the gut bifidobacterial content of HF-diet-fed mice through the use of a prebiotic (oligofructose [OFS]). Results Compared with normal chow-fed control mice, HF feeding significantly reduced intestinal Gram-negative and Gram-positive bacteria including levels of bifidobacteria, a dominant member of the intestinal microbiota, which is seen as physiologically positive. As expected, HF-OFS-fed mice had totally restored quantities of bifidobacteria. HF-feeding significantly increased endotoxaemia, which was normalised to control levels in HF-OFS-treated mice. Multiple-correlation analyses showed that endotoxaemia significantly and negatively correlated with Bifidobacterium spp., but no relationship was seen between endotoxaemia and any other bacterial group. Finally, in HF-OFS-treated-mice, Bifidobacterium spp. significantly and positively correlated with improved glucose tolerance, glucose-induced insulin secretion and normalised inflammatory tone (decreased endotoxaemia, plasma and adipose tissue proinflammatory cytokines). Conclusions/interpretation Together, these findings suggest that the gut microbiota contribute towards the pathophysiological regulation of endotoxaemia and set the tone of inflammation for occurrence of diabetes and/or obesity. Thus, it would be useful to develop specific strategies for modifying gut microbiota in favour of bifidobacteria to prevent the deleterious effect of HF-diet-induced metabolic diseases.

Increased n-6 polyunsaturated fatty acids do not attenuate the effects of long-chain n-3 polyunsaturated fatty acids on insulin sensitivity or triacylglycerol reduction in Indian Asians

Background: Indian Asians in Western countries have a higher rate of coronary artery disease than do the indigenous white populations, and this higher rate may be influenced by a dietary imbalance of n-6 and n-3 polyunsaturated fatty acids (PUFAs). Objective: The objective of the study was to test the hypothesis that a high background dietary intake of n-6 PUFA attenuates the effects of fish-oil supplementation on insulin sensitivity and associated blood lipids of the metabolic syndrome. Design: Twenty-nine Indian Asian men were recruited to participate in a 12-wk dietary intervention trial. Volunteers were randomly assigned to receive either a moderate or a high n-6 PUFA diet featuring modified oils and spreads over a 6-wk period. After this 6-wk period, both groups were supplemented with 4.0 g fish oil/d (2.5 g eicosapentaenoic acid + docosahexaenoic acid) for an additional 6 wk in combination with the dietary treatment. Volunteers participated in a postprandial study and an insulin sensitivity test after the 6-wk dietary intervention and again after the fish-oil supplementation period. Results: There was no significant time X treatment interaction for blood lipids or insulin action after dietary intervention with the moderate or high n-6 PUFA diets in combination with fish oil. After the 6-wk period of fish oil supplementation, fasting and postprandial plasma triacylglycerol concentrations decreased significantly. Conclusion: The background dietary n-6 PUFA concentration did not modulate the effect of fish-oil supplementation on blood lipids or measures of insulin sensitivity in this ethnic group.

Effect of chymosin reduction and salt substitution on the properties of white salted cheese

A whey salts mixture was used as a partial substitute for sodium chloride to provide a modified Na:K ratio (1:3.4) in the manufacture of white salted cheese using ultrafiltration. Reduction of chymosin addition from 20 to 8 mu L kg(-1) of cheese was also investigated. Variation of salt and chymosin levels did not result in any significant differences in composition and physicochemical properties. The rates of proteolysis in terms of water-soluble nitrogen (WSN) and nitrogen soluble in 12% trichloroacetic acid (TCA-SN) were affected by chymosin levels but not by salt treatment. Urea-PAGE electrophoretic analysis of caseins from the cheeses manufactured using three levels of chymosin and two salt types showed that the hydrolysis of alpha(s1)-casein was higher than for beta-caseins but the differences between the cheeses were not significant (P > 0.05). The chymosin level did not have a significant effect (P > 0.05) on hardness and fracturability, suggesting that any variation in hardness due to the initial hydrolysis was being confounded by other variables. Cheeses including the whey salts product were harder and more fracturable (P < 0.01) than the cheese treated with NaCl only. Both hardness and fracturability values decreased (P < 0.05) over the maturation period. The scores for bitterness were low; neither the effects of salt nor chymosin levels were significant (P > 0.05). (c) 2005 Elsevier Ltd. All rights reserved.

Effect of chymosin and salt reduction on the quality of ultrafiltrated white-salted cheese

This study demonstrated that both chymosin and salt-in-moisture (SM) were important factors for proteolysis in the manufacture of ultrafiltrated white-salted cheese, with significant effects on water-soluble nitrogen and nitrogen soluble in trichloroacetic acid. In contrast, the levels of free amino acids were not significantly affected by chymosin and salt treatments. The cheeses made using high levels of chymosin with low SM had lower levels of residual α(s1)- and β-casein at the end of ripening. On texture profile analysis, the hardness and fracturability of the cheeses significantly increased with SM and decreased during ripening. Increases in chymosin significantly contributed to the overall weakening of the structure throughout ripening. Bitter flavour was detected after 12 weeks in the cheese made with the higher chymosin level and lower SM, which could be the result of accumulation of γ-casein fractions. The sensory data indicated that the hedonic responses for low chymosin with low SM cheeses were good and acceptable in flavour, which may be due to the moderate levels of proteolysis products.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Protocol for the PINCER trial: a cluster randomised trial comparing the effectiveness of a pharmacist-led IT-based intervention with simple feedback in reducing rates of clinically important errors in medicines management in general practices

Background: Medication errors are an important cause of morbidity and mortality in primary care. The aims of this study are to determine the effectiveness, cost effectiveness and acceptability of a pharmacist-led information-technology-based complex intervention compared with simple feedback in reducing proportions of patients at risk from potentially hazardous prescribing and medicines management in general (family) practice. Methods: Research subject group: "At-risk" patients registered with computerised general practices in two geographical regions in England. Design: Parallel group pragmatic cluster randomised trial. Interventions: Practices will be randomised to either: (i) Computer-generated feedback; or (ii) Pharmacist-led intervention comprising of computer-generated feedback, educational outreach and dedicated support. Primary outcome measures: The proportion of patients in each practice at six and 12 months post intervention: - with a computer-recorded history of peptic ulcer being prescribed non-selective non-steroidal anti-inflammatory drugs - with a computer-recorded diagnosis of asthma being prescribed beta-blockers - aged 75 years and older receiving long-term prescriptions for angiotensin converting enzyme inhibitors or loop diuretics without a recorded assessment of renal function and electrolytes in the preceding 15 months. Secondary outcome measures; These relate to a number of other examples of potentially hazardous prescribing and medicines management. Economic analysis: An economic evaluation will be done of the cost per error avoided, from the perspective of the UK National Health Service (NHS), comparing the pharmacist-led intervention with simple feedback. Qualitative analysis: A qualitative study will be conducted to explore the views and experiences of health care professionals and NHS managers concerning the interventions, and investigate possible reasons why the interventions prove effective, or conversely prove ineffective. Sample size: 34 practices in each of the two treatment arms would provide at least 80% power (two-tailed alpha of 0.05) to demonstrate a 50% reduction in error rates for each of the three primary outcome measures in the pharmacist-led intervention arm compared with a 11% reduction in the simple feedback arm. Discussion: At the time of submission of this article, 72 general practices have been recruited (36 in each arm of the trial) and the interventions have been delivered. Analysis has not yet been undertaken.