Luis A. Rubio

Monomer and Linkage Type of Galacto-Oligosaccharides Affect Their Resistance to Ileal Digestion and Prebiotic Properties in Rats

A detailed study was performed to compare the in vivo ileal digestibility and modulatory effects in fecal microbiota of novel galacto-oligosaccharides (GOS) derived from lactulose [GOS-Lu; degree of polymerization (DP) ≥2, 14.0% trisaccharides] and commercial GOS derived from lactose (GOS-La; DP ≥3, 35.1% trisaccharides) in growing rats (5 wk old). Rats were fed either a control diet or diets containing 1% (wt:wt) of GOS-Lu or GOS-La for 14 d. Quantitative analysis of carbohydrates from dietary and ileal samples demonstrated that the trisaccharide fraction of GOS-Lu was significantly more resistant to gut digestion than that from GOS-La, as indicated by their ileal digestibility rates of 12.5 ± 2.6% and 52.9 ± 2.7%, respectively, whereas the disaccharide fraction of GOS-Lu was fully resistant to the extreme environment of the upper digestive tract. The low ileal digestibility of GOS-Lu was due to the great resistance of galactosyl-fructoses to mammalian digestive enzymes, highlighting the key role played by the monomer type and linkage involved in the oligosaccharide chain. The partial digestion of GOS-La trisaccharides showed that glycosidic linkages (1→6) and (1→2) between galactose and glucose monomers were significantly more resistant to in vivo gastrointestinal digestion than the linkage (1→4) between galactose units. The absence of GOS-La and GOS-Lu digestion-resistant oligosaccharides in fecal samples indicated that they were readily fermented within the large intestine, enabling both types of GOS to have a potential prebiotic function. Indeed, compared with controls, the GOS-Lu group had significantly more bifidobacteria in fecal samples after 14 d of treatment. The number of Eubacterium rectale also was greater in the GOS-Lu and GOS-La groups than in controls. These novel data support a direct relationship between patterns of resistance to digestion and prebiotic properties of GOS.

Maillard reaction products modulate gut microbiota composition in adolescents

SCOPE Scarce data are available concerning effects of certain bioactive substances such as Maillard reaction products (MRP) on the gut microbiota composition, and the question of how a diet rich in MRP affects gut microbiota in humans is still open. METHODS AND RESULTS Two experiments were conducted. In expt. 1, adolescents consumed diets either high or low in MRP in a two-period crossover trial; in expt. 2, rats were fed diets supplemented or not with MRP model-systems. Intestinal microbiota composition in fecal (adolescents) or cecal (rat) samples was assessed by qPCR analysis. Negative correlations were found in the human assay between lactobacilli numbers and dietary advanced MRP (r = -0.418 and -0.387, for hydroxymethylfurfural and carboxymethyl-lysine respectively, p < 0.05), whereas bifidobacteria counts were negatively correlated with Amadori compounds intake. In the rat assay, total bacteria and lactobacilli were negatively correlated with MRP intake (r = -0.674,-0.675 and -0.676, for Amadori compounds, hydroxymethylfurfural and carboxymethyl-lysine respectively, p < 0.05), but no correlations were found with bifidobacteria. CONCLUSIONS Dietary MRP are able to modulate in vivo the intestinal microbiota composition both in humans and in rats, and the specific effects are likely to be linked to the chemical structure and dietary amounts of the different browning compounds.

Galacto-oligosaccharides derived from lactulose exert a selective stimulation on the growth of Bifidobacterium animalis in the large intestine of growing rats

There is increasing interest in identifying novel dietary nondigestible carbohydrates capable of modulating the composition and/or metabolic activities of the gut microbiota. This work assessed the differential modulatory influence of novel galacto-oligosaccharides derived from lactulose (GOS-Lu) in comparison with commercial galacto-oligosaccharides derived from lactose (GOS-La) in gut microbiota of growing rats (5 weeks old). Rats were fed either a control diet or diets containing 1% (w/w) of GOS-Lu or GOS-La, and cecal and colonic contents were collected after 14 days of treatment. Compared to controls, GOS-Lu had significantly more bifidobacteria within the large intestine, showing a significant and selective increase of Bifidobacterium animalis in the cecum and colon; however, no significant differences in the number of bifidobacteria among GOS-Lu and GOS-La groups were observed. Both types of GOS significantly increased the number of the Eubacterium rectale / Clostridium coccoides group. These findings support a prebiotic role of galactosyl-fructoses in functional foods.

Garlic derivatives (PTS and PTS-O) differently affect the ecology of swine faecal microbiota in vitro

A number of in vitro experiments were designed to evaluate the effects of two different industrial products, namely PROALLIUM-S-DMC and PROALLIUM-SO-DMC (DMC Research Center, Granada, Spain), obtained from garlic (Allium sativum) on the faecal microbiota of pigs. The effects of three different concentrations (50, 200 and 400 ppm) of the active compounds (PTS and PTS-O, respectively) from both industrial products on the gastrointestinal microbiota of pigs were tested. Growth medium without any additive (0 ppm) was used as control. Predominant bacterial groups (total aerobes, total anaerobes, lactobacilli, bifidobacteria, coliforms, enterobacteria, bacteroides and clostridia) were studied. Results showed that both PTS and PTS-O have significant (P<0.01) antimicrobial activity against every group studied, although enterobacteria and coliforms were the most affected populations (P<0.01). Time kill curves for Escherichia coli and Salmonella typhimurium, two common pathogens of pigs, showed that both compounds had a bactericidal effect against these strains. For the bacterial groups here studied, the antimicrobial effect of PTS-O was significantly (P<0.001) stronger than that of PTS. Trials in vivo are in course to study the potential use of these products as alternatives to antibiotics in pig feeds.

Garlic derivative propyl propane thiosulfonate is effective against broiler enteropathogens in vivo

Two experiments were carried out to study the effects of dietary supplementation with the garlic (Allium sativum)-derived product propyl propane thiosulfonate (PTS-O) on the intestinal log(10) number of copies of enteropathogens in broiler chickens, together with their intestinal morphology and growth performance. The additive had no significant effect on feed intake at any dose assayed. In experiment 1 (1 to 21 d of age), the BW of chickens fed on 45 mg of PTS-O/kg of diet was higher (P < 0.01) than that of controls. Birds fed on diets containing 45 and 90 mg of PTS-O/kg of diet had improved (P < 0.01) feed:gain ratios compared with the controls at 21 d of age. Ileal villus height, width and surface area, mucosal thickness, and muscular layer thickness were considerably greater (P < 0.01) than control values in chickens fed 90 mg of PTS-O/kg of diet. The Clostridium perfringens log(10) number of counts was not significantly affected at any dose assayed. The inclusion of PTS-O at both concentrations (45 and 90 mg/kg of diet) resulted in lower (P < 0.01) log(10) number of copies of ileal Salmonella spp. and crop enterobacteria and Escherichia coli. The inclusion of 90 mg of PTS-O/kg of diet also resulted in lower (P < 0.01) enterobacteria and E. coli log(10) numbers of copies in the ileal and cecal contents, respectively. The number of copies of Campylobacter jejuni was not significantly affected. In experiment 2 (15 to 28 d of age), lower (P < 0.01) log(10) number of copies of Salmonella spp. and C. jejuni were determined in the ileal contents of chickens fed on diets containing 135 mg of PTS-O/kg of diet. The addition of 90 mg of PTS-O/kg of diet lowered (P < 0.01) only the number of copies of ileal Salmonella spp. This investigation confirmed previous in vitro data and showed that PTS-O lowered the intestinal numbers of enteropathogens and improved the ileal histological structure and productive parameters of broilers.

The utilization of raw and autoclaved faba beans (Vicia faba L., var. minor) and faba bean fractions in diets for growing broiler chickens

The effects of the inclusion of raw and autoclaved whole faba beans (Vicia faba; RFB and AFB respectively) or faba bean fractions (cotyledons and hulls) in diets for growing broiler chickens (0-4 weeks of age) on performance, intestinal physiology and jejunal histological structure have been studied in three experiments. Significant decreases in body-weight as well as lower food consumption and higher food intake:weight gain ratio were observed in those animals fed on diets containing 250, 350 and 500 RFB/kg in the diet. Birds fed on AFB diets (500 g/kg) had significantly greater body-weights than chicks fed on RFB or raw faba bean cotyledons (RC). Significant increases in the relative lengths of duodenum, jejunum, ileum and caeca, pancreas relative weight, and intestinal transit time of birds fed on diets containing 250, 350 and 500 g RFB/kg compared with control birds were observed. Including AFB (500 g/kg) in the diet significantly increased body-weight and significantly decreased pancreas weight compared with RFB (500 g/kg)-fed birds. The inclusion of RFB hulls had no effect on these variables. Dehulling or autoclaving of faba beans, or both, proved to have no significant effect on relative lengths of duodenum, jejunum, ileum and caeca, nor on caecal volatile fatty acid concentration in birds fed on 500 g faba beans/kg diet. Electron microscopy of the jejunal mucosa revealed discrete hyperplasia of polysomes and mitochondrial swelling in those animals fed on AFB (500 g/kg) or AC (426.4 g/kg). Pronounced strangulations were also observed along the microvilli, whose length was similar to that of control birds. The inclusion of RFB hulls, either autoclaved or raw, led to no ultrastructural changes in the enterocytes, as detected by electron microscopy. Birds fed on diets containing the cotyledons of RFB (RC, 426.4 g/kg) rather than whole RFB showed the same ultrastructural disorders as RFB (500 g/kg)-fed birds. The present study shows that factors other than those usually claimed, i.e. protease inhibitors, phytates, tannins and lectins, may be contributing to the low nutritional value of V. faba seeds for growing chickens.

Pea (Pisum sativum L.) seed albumin extracts show anti-inflammatory effect in the DSS model of mouse colitis

SCOPE This study investigates the preventive effects of two pea (Pisum sativum) seed albumin extracts, either in the presence (pea seed extract [PSE]) or absence (albumin fraction from PSE [AF-PSE]) of soluble polysaccharides, in the dextran sodium sulfate (DSS) induced colitis in mice. METHODS AND RESULTS Male C57BL/6J mice were assigned to five groups: one noncolitic and four colitic. Colitis was induced by incorporating DSS (3.5%) in the drinking water for 4 days, after which DSS was removed. Treated groups received orally PSE (15 g/kg⋅day), or AF-PSE (1.5 g/kg⋅day), or pure soy Bowman-Birk inhibitor (BBI; 50 mg/kg⋅day), starting 2 wk before colitis induction, and maintained for 9 days after. All treated groups showed intestinal anti-inflammatory effect, evidenced by reduced microscopic histological damage in comparison with untreated colitic mice. The treatments ameliorated the colonic mRNA expression of different proinflammatory markers: cytokines, inducible enzymes, metalloproteinases, adhesion molecules, and toll-like receptors, as well as proteins involved in maintaining the epithelial barrier function. Furthermore, the administration of PSE, AF-PSE, or soy BBI restored bacterial counts, partially or totally, to values in healthy mice. CONCLUSION PSE and AF-PSE ameliorated DSS-induced damage to mice, their effects being due, at least partially, to the presence of active BBI.

Organs relative weight and plasma amino acid concentrations in rats fed diets based on whole legume (faba bean, lupin, chickpea, defatted soybean) seed meals or their fractions

The effects on organ relative weights (g per 100 g bw) and plasma amino acid concentrations of diets based in legume (faba bean (Vicia faba), lupin (Lupinus angustifolius), chickpea (Cicer arietinum) and soybean (Glycine max)) seed meals as the only source of protein were studied with growing rats using lactalbumin or casein as controls. Also, legume seed meal extracted globulins were included in control diets replacing lactalbumin to produce legume globulin-based diets, and legume residue fractions, containing most of the starch and/or insoluble fibre (NSP+lignin) from the seed meals, were included in control diets to reach the same amounts present in whole legume seed meal diets. All experimental diets were formulated to contain the same amounts of protein (100 g kg−1) and energy (15.5 kJ g−1), and were supplemented with essential amino acids. Compared with lactalbumin, higher relative weights of gastrointestinal sections were determined in rats fed legume seed meals or their corresponding residue fractions. On the contrary, spleen relative weight was lower in rats fed diets containing lupin, chickpea or soybean meals or extracted globulins, while residue fractions had no effect on it. Thymus relative weight was also lower in rats fed whole chickpea seed meal or any of the extracted legume globulins. Except for chickpea meal, animals fed legume- or legume protein-based diets had lower liver relative weights than controls. Lower proportions (mg g−1 tissue) of glycogen, and lower total protein and RNA, were also determined in the livers of rats fed lupin seed meal. Free plasma concentrations (mM litre −1) of glycine, histidine and arginine were higher, and threonine, leucine and lysine were lower, in rats fed diets based in all legume seed meals of their respective globulin proteins. The possible reasons and implications of these results are discussed. © 1999 Society of Chemical Industry

Characterization of pea (Pisum sativum) seed protein fractions

BACKGROUND Legume seed proteins have to be chemically characterized in order to properly link their nutritional effects with their chemical structure. RESULTS Vicilin and albumin fractions devoid of cross-contamination, as assessed by mass peptide fingerprinting analysis, were obtained from defatted pea (Pisum sativum cv. Bilbo) meal. The extracted protein fractions contained 56.7-67.7 g non-starch polysaccharides kg⁻¹. The vicilin fraction was higher than legumins in arginine, isoleucine, leucine, phenylalanine and lysine. The most abundant amino acids in the albumin fraction were aspartic acid, glutamic acid, lysine and arginine, and the amounts of methionine were more than double than those in legumins and vicilins. The pea albumin fraction showed a clear enrichment of protease inhibitory activity when compared with the seed meal. In vitro digestibility values for pea proteins were 0.63 ±  0.04, 0.88 ±  0.04 and 0.41 ±  0.23 for legumins, vicilins and albumins respectively. CONCLUSION Vicilin and albumin fractions devoid of cross-contamination with other proteins were obtained from pea seed meal. The vicilin fraction also contained low amounts of soluble non-starch polysaccharides and was enriched in isoleucine, leucine, phenylalanine and lysine. In vitro digestibility values for pea proteins were similar or even numerically higher than those for control proteins.

Anti-carcinogenic soyabean Bowman-Birk inhibitors survive faecal fermentation in their active form and do not affect the microbiota composition in vitro

Bowman-Birk inhibitor (BBI) from soyabeans is a naturally occurring protease inhibitor with potential anti-inflammatory and chemopreventive properties within the gastrointestinal tract (GIT). In a previous paper, we reported that significant amounts of BBI-related proteins reach the terminal ileum functionally and biologically active. We have now investigated: (a) if soyabean BBI is biotransformed by faecal microbiota which would reduce its potential colorectal chemopreventive properties and (b) the potential influence of this protease inhibitor on the modulation of faecal microbiota. In vitro incubation studies of native soyabean BBI at a physiological level (93 microM) with mixed faecal samples of pigs for 24 h at 37 degrees C demonstrated that BBI remains active and its intrinsic trypsin and chymotrypsin inhibitory activities were not significantly influenced by the enzymic or metabolic activity of faecal microbiota. Soyabean BBI did not affect the growth of the different bacterial groups studied (lactobacilli, bifidobacteria, bacteroides, coliforms, enterobacteria, clostridia and total anaerobes). It was concluded that protease inhibitory activities, intrinsically linked to the chemopreventive properties of soyabean BBI, were largely unaffected by faecal microbiota in vitro. BBI retains significance, therefore, as a bioactive compound in the human GIT.