Inclusion of limited amounts of extruded legumes plus cereal mixes in normocaloric or obesogenic diets for rats: effects on intestinal microbiota composition.

BACKGROUND: Differences in the composition of the intestinal microbiota and energetic metabolism between lean and obese populations have been described. Legume consumption has been reported to modulate intestinal microbiota composition. However, to the best of our knowledge, no information can be found in the literature on the effects of consumption of diets containing extruded legume plus cereal mixes on the intestinal microbiota composition of rats. Our purpose was to evaluate the effects on lipids profile (see the accompanying paper) and intestinal microbiota composition (current paper) of incorporating this new food ingredient in normocaloric and obesogenic diets. RESULTS: Intestinal and fecal qPCR-based microbial composition of rats fed the extruded legumes plus cereal mixes differed (P < 0.05) from controls. Obesogenic diets did not affect bacterial counts. However, the inclusion of the extruded mixes reduced (P < 0.05) log10 counts in some bacterial groups and increased (P < 0.05) counts of Lactobacilli, while others remained unaffected. PCoA at the genus level grouped together Lactobacillus reuteri, Akkermansia miciniphila and species from Parabacteroides, Prevotella, Rikenellaceae, and Lactobacillus with extruded legume plus cereal diets. Feeding on extruded legumes plus cereal mixes was associated with increased mRNA expression of the cytokines IL6 and TNF-α and decreased expression of TLR4. CONCLUSIONS: Our results show that the inclusion in the feed of limited amounts of extruded legumes plus cereal mix, providing a diet that is closer to a normal human one, did modulate the intestinal microbiota composition. Taken together, these results point to the protective, health-promoting properties of extruded legume plus cereal mixes.

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.

Inclusion of Limited Amounts of Extruded Legumes Plus Cereal Mixes in Normocaloric or Obesogenic Diets for Rats: Effects on Lipid Profile.

Overweight and obesity are regarded as world epidemics and are major risk factors for a number of chronic diseases, including diabetes, cardiovascular diseases, and cancer. Two new highly palatable extruded mixes based on rice and pea (Pisum sativum) or kidney bean (Phaseolus vulgaris) meals were incorporated into normocaloric or obesogenic diets for rats at a low inclusion level (25%). Our purpose was to evaluate the effects of dietary incorporation of this new food ingredient on lipid profile. Organs (heart, liver, kidneys, spleen, stomach, small intestine, colon, cecum) and visceral fat relative weights were different (p < 0.01) from controls for animals fed the obesogenic diets and in rats fed extruded diets with respect to controls. Faecal excretion of bile acids was higher (p < 0.01) for rats fed extruded mixes compared with controls. The inclusion of extruded mixes replacing part of the casein in the control diet lowered liver cholesterol and triglycerides (p < 0.001) and plasma low-density lipoprotein (LDL; p < 0.01) values, although plasma high-density lipoprotein (HDL) was unaltered. Both the inclusion of extruded mixes and the use of obesogenic diets resulted in significantly (p < 0.001) different long chain fatty acid (LCFA) profiles in liver and visceral fat. Incorporating extruded legume plus cereal mixes beneficially influenced lipid metabolism, and would therefore deserve closer attention in human intervention studies, particularly with adolescents. To our knowledge, this is the first report on the nutritional and physiological effects of extruded legume plus cereal mixes.

Controlling bacterial physiology for optimal expression of gene reporter constructs.

Bacterial biosensors for the detection of pollutants are based on the regulatory elements that control the corresponding degradation pathways. An increasing number of catabolic pathways under the control of specific regulators are now known to be influenced by the presence of alternative carbon sources, which to different extents repress expression of the pathway despite the presence of the inducer. The molecular basis underlying the control of each catabolic pathway is different, although all sense a high energy state of the cell resulting from the presence of more favourable carbon sources. Biosensor tests mimicking field conditions point to global regulation being relevant for biosensor performance; thus, this global regulation must be taken into account when designing whole-cell biosensors.

Species-specific repetitive extragenic palindromic (REP) sequences in Pseudomonas putida.

Pseudomonas putida KT2440 is a soil bacterium that effectively colonises the roots of many plants and degrades a variety of toxic aromatic compounds. Its genome has recently been sequenced. We describe that a 35 bp sequence with the structure of an imperfect palindrome, originally found repeated three times downstream of the rpoH gene terminator, is detected more than 800 times in the chromosome of this strain. The structure of this DNA segment is analogous to that of the so-called enterobacteriaceae repetitive extragenic palindromic (REP) sequences, although its sequence is different. Computer-assisted analysis of the presence and distribution of this repeated sequence in the P.putida chromosome revealed that in at least 80% of the cases the sequence is extragenic, and in 82% of the cases the distance of this extragenic element to the end of one of the neighbouring genes was <100 bp. This 35 bp element can be found either as a single element, as pairs of elements, or sometimes forming clusters of up to five elements in which they alternate orientation. PCR scanning of chromosomes from different isolates of Pseudomonas sp. strains using oligonucleotides complementary to the most conserved region of this sequence shows that it is only present in isolates of the species P.putida. For this reason we suggest that the P.putida 35 bp element is a distinctive REP sequence in P.putida. This is the first time that REP sequences have been described and characterised in a group of non-enterobacteriaceae.

Role of the ptsN gene product in catabolite repression of the Pseudomonas putida TOL toluene degradation pathway in chemostat cultures.

The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. We show that the ptsN gene product IIA(Ntr) participates in this repression. Crc, involved in yeast extract-dependent repression in batch cultures, did not influence expression when cells were growing in a chemostat with succinate at maximum rate.

Integration of signals through Crc and PtsN in catabolite repression of Pseudomonas putida TOL plasmid pWW0.

Toluene degradation in Pseudomonas putida KT2440 pWW0 plasmid is subjected to catabolite repression. Pu and P(S1) promoters of the pWW0 TOL plasmid are down-regulated in vivo during exponential growth in rich medium. In cells growing on minimal medium, yeast extract (YE) addition mimics exponential-phase rich medium repression of these promoters. We have constructed and tested mutants in a series of global regulators described in Pseudomonas. We describe that a mutant in crc (catabolite repression control) partially relieves YE repression. Macroarray experiments show that crc transcription is strongly increased in the presence of YE, inversely correlated with TOL pathway expression. On the other hand, we have found that induced levels of expression from Pu and P(S) in the presence of YE are partially derepressed in a ptsN mutant of P. putida. PtsN but not Crc seems to directly interfere with XylR activation at target promoters. The effect of the double mutation in ptsN and crc is not the sum of the effects of each independent mutation and suggests that both regulators are elements of a common regulatory pathway. Basal expression levels from these promoters in the absence of inducer are still XylR dependent and are also repressed in the presence of yeast extract. Neither crc nor ptsN could relieve this repression.

Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals: evidence from continuous cultures.

Expression of the genes of the alkane degradation pathway encoded in the Pseudomonas putida OCT plasmid are subject to negative and dominant global control depending on the carbon source used and on the physiological status of the cell. We investigated the signals responsible for this control in chemostat cultures under conditions of nutrient or oxygen limitation. Our results show that this global control is not related to the growth rate and responds to two different signals. One signal is the concentration of the carbon source that generates the repressing effect (true catabolite repression control). The second signal is influenced by the level of expression of the cytochome o ubiquinol oxidase, which in turn depends on factors such as oxygen availability or the carbon source used. Since under carbon limitation conditions the first signal is relieved but the second signal is not, we propose that modulation mediated by the cytochrome o ubiquinol oxidase is not classical catabolite repression control but rather a more general physiological control mechanism. The two signals have an additive, but independent, effect, inhibiting induction of the alkane degradation pathway.

Molecular characterization of Pseudomonas putida KT2440 rpoH gene regulation.

The rpoH gene of Pseudomonas putida KT2440 encoding the heat-shock sigma factor sigma(32) was cloned and sequenced, and the translated gene product was predicted to be a protein of 32.5 kDa. The unambiguous role of the gene as a sigma factor was confirmed because the cloned P. putida gene complemented the growth defect, at 37 and 42 degrees C, of an Escherichia coli rpoH mutant strain. Primer extension analysis showed that in P. putida the rpoH gene is expressed from three promoters in cells growing at 30 degrees C. Two of them, P1 and P3, share homology with the sigma(70)-dependent promoters, while the third one, P2, shows a typical sigma(24)-consensus sequence. The pattern of transcription initiation of the rpoH gene did not change in response to different stresses, i.e. a sudden heat shock or the addition of aromatic compounds. However, the predicted secondary structure of the 5' region of the mRNA derived from the three different promoters suggests regulation at the level of translation efficiency and/or mRNA half-life. An inverted repeat sequence located 20 bp downstream of the rpoH stop codon was shown to function as a terminator in vivo in P. putida growing at temperatures from 18 to 42 degrees C.