Development of an in vitro system simulating bucco-gastric digestion to assess the physical and chemical changes of food.

The release of nutrients from solid food depends on the physical and chemical characteristics of substrates, and on dynamic physiological events including pH, gastric emptying and enzymatic secretion. Our laboratory has developed an in vitro digestive system mimicking mouth and stomach processes to determine physical and chemical changes of bread during digestion. To simulate oral-phase digestion, bread was minced and subjected to in vitro amylase digestion, releasing 219 +/- 11 g oligosaccharides/kg total carbohydrate. During the gastric phase, bread proteins, which are converted into insoluble aggregated proteins during breadmaking, were emptied in various states of peptic digestion: undigested aggregated proteins and degraded proteins of intermediate and low molecular weight. The mean particle size of ground bread decreased progressively to the end of the gastric digestion (from 292 to 109 microm). The in vitro digestive system proved to be a useful tool for understanding the dynamic digestion of various food components held within the structure of a food matrix.

Genes induced by growth hormone in a model of adipogenic differentiation.

A substantial number of GH regulated genes have been reported in mature hepatocytes, but genes involved in GH-initiated cell differentiation have not yet been identified. Here we have studied a well-characterised model of GH-dependent differentiation, adipogenesis of 3T3-F442A preadipocytes, to identify genes rapidly induced by GH. Using the suppression subtractive hybridisation technique, we have identified eight genes induced within 60 min of GH treatment, and verified these by northern analysis. Six were identifiable as Stat 2, Stat 3, thrombospondin-1, oncostatin M receptor beta chain, a DEAD box RNA helicase, and muscleblind, a developmental transcription factor. Bioinformatic approaches assigned one of the two remaining unknown genes as a novel 436 residue serine/threonine kinase. As each of the identified genes have important developmental roles, they may be important in initiating GH-induced adipogenesis.

Particle size of solid food after human mastication and in vitro simulation of oral breakdown.

Mastication, the first step in food digestion, results in the breakdown of solid food and its lubrication with saliva. Although the rate and extent of starch digestion are closely dependent on the way food is chewed, this factor has not been adequately considered in the preparation of food for in vitro digestion experiments. The purpose of this study was to determine the size distribution of starchy food particles before swallowing and to use an in vitro mincing procedure to simulate how food is divided up during chewing. Foods differing in texture and size (bread, spaghetti and tortiglioni) were chewed by 12 healthy subjects and spat out before swallowing. Chewing time and saliva impregnation were measured for each mouthful. The particle sizes resulting from experiments with chewed and minced bread and pasta were analysed respectively by light laser diffraction and image analysis. Chewing time was longer for bread than pasta, resulting in higher saliva impregnation. Chewed bread showed a bimodal distribution of particle size (30 microns, 500 microns), whereas both kinds of pasta produced particles of similar size (0.5 to 30 mm2) after mastication. Mincing reproduced the division of bread and pasta as achieved by chewing in an acceptable way. From our results it seems that the size of particles resulting from mastication depends on food texture. We succeeded by wetting and mincing food to prepare food in a similar bolus-like form before swallowing. Mincing provides a simple means of simulating the reduction of food into particles for in vitro digestion studies.

Relationships between transit time in man and in vitro fermentation of dietary fiber by fecal bacteria.

OBJECTIVE: To assess the effects of drug-induced changes in mean transit time (MTT) on the activity of human fecal flora in vitro. METHODS: The activity of fecal flora was estimated by the ability of a fecal inoculum to ferment a substrate (beet fiber) in vitro in a batch system for 24 h. The inoculum was collected from 8 healthy volunteers studied during three 3-week randomized periods, who received a controlled diet alone (control period) or the same diet with either cisapride or loperamide. Cisapride and loperamide were adjusted in order to halve and double MTT measured during the control period. At the end of each period, the percentage disappearance of the initial added substrate and the concentration and the profile of short-chain fatty acids (SCFAs), were determined. RESULTS: In the control period, the pH of the inoculum and SCFA concentration were inversely related to MTT (P=0.0001). Individual SCFA production was also significantly related to MTT (P<0.01). Cisapride-reduced transit time was associated with a significant rise in the concentrations of total SCFAs (P<0.05), propionic and butyric acids (P<0.05) and the percentage substrate disappearance (P<0.05). Inverse relations were observed during the loperamide period. Moreover, MTT was inversely related to the percentage substrate disappearance (P<0.001), SCFA production (P<0.001) and butyrate production (P<0.0005). CONCLUSION: Changes in MTT alter bacterial activity and modify the bacterial pathways affecting the proportion of individual SCFAs. European Journal of Clinical Nutrition (2000) 54, 603-609

Bioavailability of starch in bread rich in amylose: metabolic responses in healthy subjects and starch structure.

OBJECTIVE: This study investigated whether postprandial metabolic responses to bread could be lowered by substituting high amylose maize starch for a part of the flour. DESIGN AND SUBJECTS: Eight healthy subjects consumed test meals of equivalent nutritional composition based on white wheat bread, bread rich in amylose (HAWB) and spaghetti as a breakfast meal. Blood samples were collected to measure insulin and glucose concentration during two hours after consumption. The degree of starch crystallinity was investigated by X-ray diffraction and DSC analysis. RESULTS: HAWB produced low glycaemic (60 +/- 18) and insulinaemic (57 +/- 20) indexes similar to those of spaghetti (83 +/- 46, 61 +/- 16). In vitro amylase hydrolysis of the three foods showed that high amylose content in HAWB significantly lowered starch degradation in bread without affecting hydrolysis kinetics. Addition of amylose in dough increased the resistant starch content of HAWB (14% of dry matter). The resistant starch fraction was mainly composed of crystalline amylose (B-type X-ray diffraction pattern, melting temperature 105 degrees C) attributable to native high amylose maize starch incompletely gelatinised during bread-cooking. CONCLUSIONS: Bread produced by the substitution of high amylose maize starch for a part of wheat flour showed a low glycaemic index. Resistant starch in HAWB corresponded to native crystalline amylose not gelatinised during normal bread-processing conditions.

Physical and chemical transformations of cereal food during oral digestion in human subjects.

Chemical and physical transformations of solid food begin in the mouth, but the oral phase of digestion has rarely been studied. In the present study, twelve healthy volunteers masticated mouthfuls of either bread or spaghetti for a physiologically-determined time, and the levels of particle degradation and starch digestion before swallowing were compared for each food. The amounts of saliva moistening bread and spaghetti before swallowing were, respectively, 220 (SEM 12) v. 39 (SEM 6) g/kg fresh matter. Particle size reduction also differed since bread particles were highly degraded, showing a loss of structure, whereas spaghetti retained its physical structure, with rough and incomplete reduction of particle size. Starch hydrolysis was twice as high for bread as for spaghetti, mainly because of the release of high-molecular-mass alpha-glucans. The production of oligosaccharides was similar after mastication of the two foods, respectively 125 (SEM 8) and 92 (SEM 7) g/kg total starch. Starch hydrolysis, which clearly began in the mouth, depended on the initial structure of the food, as in the breakdown of solid food. These significant physical and chemical degradations of solid foods during oral digestion may influence the entire digestive process.

Digestive and metabolic effects of potato and maize fibres in human subjects.

The physiological effects of dietary fibres in humans are due to their physico-chemical properties. However, it is difficult to predict these effects simply by measuring certain characteristics in vitro. Studies in human subjects are still required to assess the effectiveness of new substrates. The aim of the present study in healthy human subjects was to evaluate the effects of two novel fibres, potato (PF) and maize (MF), on fasting and postprandial blood concentrations of carbohydrate and lipid metabolites as well as on stool output and transit time. The chemical composition, water-binding capacity (WBC) and fermentative properties of the fibres were also characterized in order to determine their possible involvement in digestive and metabolic effects. Stools, as well as breath and blood samples, were collected after consumption for 1 month of either a basal diet (control) or a basal diet supplemented with fibre (15 g/d). MF resisted fermentation better than PF and had lower digestibility. However, both fibres increased faecal output of dry matter, neutral sugars and water. There was an inverse relationship between stool weight and orofaecal transit time, although only MF significantly reduced transit time. Orocaecal transit was lengthened by PF, probably because of its high WBC. PF ingestion also decreased postprandial plasma levels of total and esterified cholesterol but had no effect on fasting concentrations. In contrast, MF lowered fasting cholesterolaemia and increased free:esterified cholesterol. These particular physiological and fermentative properties suggest that PF and MF would be suitable ingredients in a healthy diet.

Relations between transit time, fermentation products, and hydrogen consuming flora in healthy humans.

BACKGROUND/AIMS: To investigate whether transit time could influence H2 consuming flora and certain indices of colonic bacterial fermentation. METHODS: Eight healthy volunteers (four methane excretors and four non-methane excretors) were studied for three, three week periods during which they received a controlled diet alone (control period), and then the same diet with cisapride or loperamide. At the end of each period, mean transit time (MTT) was estimated, an H2 lactulose breath test was performed, and stools were analysed. RESULTS: In the control period, transit time was inversely related to faecal weight, sulphate reducing bacteria counts, concentrations of total short chain fatty acids (SCFAs), propionic and butyric acids, and H2 excreted in breath after lactulose ingestion. Conversely, transit time was positively related to faecal pH and tended to be related to methanogen counts. Methanogenic bacteria counts were inversely related to those of sulphate reducing bacteria and methane excretors had slower MTT and lower sulphate reducing bacteria counts than non-methane excretors. Compared with the control period, MTT was significantly shortened (p < 0.05) by cisapride and prolonged (p < 0.05) by loperamide (73 (11) hours, 47 (5) hours and 147 (12) hours for control, cisapride, and loperamide, respectively, mean (SD)). Cisapride reduced transit time was associated with (a) a significant rise in faecal weight, sulphate reducing bacteria, concentrations of total SCFAs, and propionic and butyric acids and breath H2 as well as (b) a significant fall in faecal pH and breath CH4 excretion, and (c) a non-significant decrease in the counts of methanogenic bacteria. Reverse relations were roughly the same during the loperamide period including a significant rise in the counts of methanogenic bacteria and a significant fall in those of sulphate reducing bacteria. CONCLUSIONS: Transit time differences between healthy volunteers are associated with differences in H2 consuming flora and certain indices of colonic fermentation. Considering the effects of some fermentation products on intestinal morphology and function, these variations may be relevant to the pathogenesis of colorectal diseases.

Chronic consumption of short-chain fructooligosaccharides by healthy subjects decreased basal hepatic glucose production but had no effect on insulin-stimulated glucose metabolism.

We aimed to study the effects of chronic ingestion of short-chain fructooligosaccharides (FOS), an indigestible carbohydrate, on hepatic glucose production, insulin-mediated glucose metabolism, erythrocyte insulin binding, and blood lipids in healthy subjects. Twelve healthy volunteers received either 20 g FOS/d or sucrose for 4 wk in a double-blind crossover design. FOS did not modify fasting plasma glucose and insulin concentrations. Mean (+/- SEM) basal hepatic glucose production was lower after FOS than after sucrose consumption (2.18 +/- 0.10 compared with 2.32 +/- 0.09 mg.kg-1, min-1, respectively; P < 0.02, paired Student's t test). However, neither insulin suppression of hepatic glucose production nor insulin stimulation of glucose uptake measured by hyperinsulinemic clamp was significantly different between the two dietary periods. Erythrocyte insulin binding was also comparable. Serum triacylglycerols, total and high-density- lipoprotein cholesterol, apolipoproteins A-I and B, and lipoprotein(a) were not modified by FOS. To try to understand why FOS did not increase serum lipids, the in vitro production of short-chain fatty acids from FOS was evaluated by using human fecal inoculum and compared with that from lactulose, which was found to increase serum lipids. FOS produced an acetate-propionate ratio two times lower than that of lactulose. We conclude that 4 wk of 20 g FOS/d decreased basal hepatic glucose production but had no detectable effect on insulin-stimulated glucose metabolism in healthy subjects. The colonic fermentation pattern of undigestible carbohydrates may be relevant to predicting their metabolic effects.

In vitro fermentation by human faecal bacteria of total and purified dietary fibres from brown seaweeds.

The in vitro degradation of dietary fibre from three brown seaweeds (Himanthalia elongata, Laminaria digitata and Undaria pinnatifida) was studied, using human faecal flora. Two sets of fibre were tested: (1) total algal fibres extracted from the whole algae, mainly composed of alginates, and (2) purified fibres (sulphated fucans, Na-alginates and laminarans) representative of those contained in the whole brown algae. Mannuronate, one algal component, was also investigated. Substrate disappearance and short-chain fatty acid (SCFA) production were monitored after 6, 12 and 24 h fermentation. Gas production was followed hourly during the first 9 h and then at 12 and 24 h. Sugarbeet fibre was used as a fermentation reference substrate. According to the fermentative indices used, most of each of the total algal fibres disappeared after 24 h (range 60-76%) but, unlike the reference substrate, they were not completely metabolized to SCFA (range 47-62%). Among the purified algal fibres, disappearance of laminarans was approximately 90% and metabolism to SCFA was approximately 85% in close agreement with the fermentation pattern of reference fibres. Sulphated fucans were not degraded. Na-alginates exhibited a fermentation pattern quite similar to those of the whole algal fibres with a more pronounced discrepancy between disappearance and production of SCFA: disappearance was approximately 83% but metabolism was only approximately 57%. Mannuronate was slowly fermented but its metabolism corresponded to its disappearance from the fermentative medium. Thus, the characteristic fermentation pattern of the total fibres from the three brown algae investigated was attributed to the peculiar fermentation of alginates, and mannuronate was shown not to be directly involved.

Estimation of the fermentability of dietary fibre in vitro: a European interlaboratory study.

Five European laboratories tested a simple in vitro batch system for dietary fibre fermentation studies. The inoculum was composed of fresh human faeces mixed with a carbonate-phosphate buffer complex supplemented with trace elements and urea. Five dietary fibre sources (cellulose, sugarbeet fibre, soyabean fibre, maize bran and pectin) were used by each laboratory on three occasions to determine pH, residual non-starch polysaccharides (NSP) and short-chain fatty acid production during fermentation. Cellulose and maize bran degradabilities were very low (7.2(SE 10.8) and 6.2 (SE 9.1)% respectively after 24 h), whereas pectin and soyabean fibre were highly degraded (97.4 (SE 4.4) and 91.1 (SE 3.4)% respectively after 24 h). Sugarbeet fibre exhibited an intermediate level of degradability (59.5 (SE 14.9)%). Short-chain fatty acid production was closely related to NSP degradation (r 0.99). Although each variable was ranked similarly by all laboratories, some differences occurred with respect to absolute values. However, the adaptation of donors to the experimental substrates was not an influential factor. Interlaboratory differences could be reduced either by adding less substrate during incubations or using less-diluted inocula. In vitro fermentations with inocula made from human faeces and from rat caecal contents gave similar results. There was a close correspondence between the data obtained in the present experiment and those previously published in in vivo studies in the rat using the same fibres. The in vitro batch system tested during the present study provides a rapid means of obtaining quantitative estimates of the fermentation and the estimation of the energy content of new sources of dietary fibre.

Sugar composition of dietary fibre and short-chain fatty acid production during in vitro fermentation by human bacteria.

The aim of the present study was to assess the relationship between the disappearance of dietary fibre sugars and the production of individual short-chain fatty acids (SCFA). The bacterial degradation of five dietary fibres whose sugars were quantified was investigated in vitro using a human faecal inoculum. Involvement of the main fibre sugars in SCFA production was evaluated by a stepwise multiple linear regression. The results show first that the nature and chiefly the associations between the fibre sugars were key variables in the fermentability. Second, the nature and the amounts of SCFA produced were closely related to the in vitro fermentation of the main sugars available: uronic acids seemed to be principally involved in the production of acetic acid whereas the production of propionic acid could be promoted by the fermentation of glucose and, to a lesser extent, by that of xylose and arabinose. Xylose tended to have a greater impact than uronic acids and glucose on the production of butyric acid. Thus, it would be possible to predict which SCFA could be specifically produced during the fermentation of a fibre, as far as the chemical composition and structure of this fibre are known.

Short-chain fatty acids do not alter jejunal motility in man.

Short-chain fatty acids (SCFAs) alter ileal and colonic motility, but their effects on duodenojejunal motility are unknown. Simultaneous jejunal manometric recordings and hydrogen breath tests after lactulose were performed in eight healthy subjects during continuous duodenal infusion of either saline or SCFAs. These experiments were conducted in the fasting state and postprandially. The effects of various boluses of SCFAs on duodenojejunal motility were also determined in six subjects. During the fasting period, the number and characteristics of migrating motor complex, prolonged propagated contractions, discrete clustered contractions, motility index, and orocecal transit time were similar during saline and SCFAs. Similarly, the motility index and the duration of the postprandial period were not different between SCFAs and saline after the meal. The motility index was significantly increased after each of the 100-ml boluses (saline or SCFAs), but was not altered after the 12.5-ml boluses, suggesting a volume-related effect. Thus, SCFAs do not seem to affect proximal small bowel motility in healthy humans.

Formaldehyde content of milk in goats fed formaldehyde-treated soybean oil-meal.

Formaldehyde is used in ruminant feeding for different purposes including the protection of dietary proteins from ruminal degradation. The formaldehyde content of milk of goats fed various levels of formaldehyde-treated soybean oil-meal has been determined by using a sensitive HPLC method. Results showed a significant linear correlation between ingested formaldehyde and formaldehyde concentration in milk. About 0.02% of ingested formaldehyde was excreted in milk, as free formaldehyde.

Breath hydrogen response to lactulose in healthy subjects: relationship to methane producing status.

In order to assess the relationship between methane (CH4) producing status and the breath excretion of hydrogen (H2) in healthy subjects, breath CH4 and H2 were simultaneously measured for 14 hours after oral ingestion of 10 g lactulose in 65 young volunteers. Forty were breath CH4 producers and 25 were not. Statistically significant differences were observed between both groups, with lower values for CH4 producers recorded for the following parameters: fasting basal value of breath H2 (8.1 (4.9) v 5.2 (3.7) ppm, p less than 0.05), mouth-to-caecum transit time (68 (24) v 111 (52) min, p less than 0.005), and breath H2 production measured as area under the curve 13.1 (6.9) v 8.8 (3.8) 10(3) ppm/min, p less than 0.02). There was no significant correlation between individual production of breath H2 and CH4. These results indicate that the response to lactulose depends on breath CH4 producing status. In clinical practice, defining normal values of mouth-to-caecum transit time without knowledge of breath CH4 producing status may lead to misinterpretation of the H2 breath test.

Pasta cooking time: influence on starch digestion and plasma glucose and insulin responses in healthy subjects.

The influence of pasta cooking time on starch digestion and plasma glucose and insulin responses was studied in 12 healthy subjects. During 3 consecutive days, one of three pasta test meals (50 g starch) cooked for 11, 16.5, and 22 min was served to each volunteer in a random order. Hydrogen and methane breath excretion was measured after pasta ingestion; plasma responses were compared with those of an equivalent oral glucose-tolerance test. No significant differences were found between cooking times and plasma indices, orocecal transit time, or incremental hydrogen excretion (delta peak hydrogen). With one exception, pasta meals that were completely absorbed were ingested by methane producers. Postprandial delta peak hydrogen was significantly lower in methane than in nonmethane producers (p less than 0.02). These results point to a lack of influence of cooking time on nutritional characteristics of pasta and suggest that starch malabsorption determined by breath-hydrogen-test criteria may be underestimated in methane producers.