Raquel Ruiz

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.

Complexity in efflux pump control: cross-regulation by the paralogues TtgV and TtgT.

Pseudomonas putida DOT‐T1E, known for its high tolerance to solvents, possesses three Resistance–Nodulation–Cell Division‐type (RND) efflux pumps, namely TtgABC, TtgDEF and TtgGHI, which are involved in the active extrusion of solvents. Expression of the ttgABC and ttgGHI operons was previously shown to be regulated by the adjacently encoded repressors, TtgR and TtgV, respectively. Upstream of the third RND operon, ttgDEF, is located a putative regulator gene, ttgT. In this study, TtgT is shown to bind to the promoter region of the ttgDEF operon, and to be released from DNA in the presence of organic solvents. In vitro studies revealed that TtgV and TtgT bind the same operator sites in both the ttgDEF and the ttgGHI promoters. However, the affinity of TtgV for the ttgDEF operator was higher than that of TtgT, which, together with the fact that the ttgV promoter seems to be almost twice stronger than the ttgT promoter, explains why TtgV takes over in the regulation of the two efflux pump operons. The functional replacement of the cognate, chromosomally encoded TtgT by the plasmid‐encoded paralogue TtgV illustrates a new mode of efflux pump regulation of which the physiological relevance is discussed.

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.

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.

Transcriptional Regulation of the Toluene Catabolic Pathways

Although in the past century large amounts of aromatic hydrocarbons have been released as a consequence of industrial activity, most of these compounds in the environment are the product of the natural pyrolysis of organic material28. As a consequence, toluene, one of the simplest substituted aromatic compounds of natural origin, is widely distributed in natural environments. Therefore, because living organisms have been in contact with toluene and related compounds through evolutionary periods of time, it is not surprising that bacteria have developed the capability to degrade aromatic compounds such as toluene. In fact, aerobic bacteria able to use toluene as the sole source of carbon and energy are easily isolated from natural samples. Many of these organisms belong to the Pseudomonadaceae.

Stability of retinol in milk during frozen and other storage conditions

ZusammenfassungEs wurde der Einfluß von Lagertemperatur und Lagerzeit auf den Retinolgehalt von vier UHT-Vollmilchproben aus dem Handel ohne hinzugefügte Vitamine mittels HPLC studiert. Bereits nach einem Monat wiesen die bei 30 °C gelagerten Proben signifikante Verluste von Retinol auf. Im allgemeinen stiegen die Verluste von Retinol mit der Länge der Lagerungszeit an. Die Größe des Anstiegs bei einer Lagertemperatur zwischen 30 °C und 40 °C hing von der untersuchten Milch ab. Nach kurzer Tiefkähllagerung (bis zu 60 Tagen) wurde keine Veränderung des Retinolgehaltes beobachtet, aber signifikante Verluste (P≦0.05) waren nach einer Tiefkühllagerung von 4 bis 8 Monaten festzustellen. In der vorliegenden Arbeit wurde auch die Wirkung der Wasseraktivität und der Temperaturbedingungen auf die Stabilität von Retinol während der Lagerung von Vollmilchpulver ohne hinzugefügte Vitamine untersucht. Es wurde beobachtet, daß die Zunahme der Wasseraktivität und Lagertemperatur eine signifikante Abnahme des Retinolgehaltes von Milchpulver verursacht.SummaryThe effect of storage temperature and storage time on the retinol content of four commercial unfortified whole UHT milk samples was studied by HPLC. Significant losses of retinol in these milks were observed after 1 month of storage at 30° C. Losses generally increased with storage time. Increasing the storage temperature from 30° C to 40° C had a variable effect depending on the particular milk. Short periods of frozen storage (up to 60 days) had no effect on the retinol content. However, frozen storage time from 4 to 8 months led to significant (P<0.05) losses in retinol content. The effect of water activity and temperature conditions during storage of unfortified whole milk powder on the stability of retinol was also studied. Increasing the activity of water and temperature of storage significantly lowered the retinol content in milk powder.

Morphology and enzymatic activity of the small intestinal mucosa of Iberian pigs as compared with a lean pig strain

Castrated male Iberian (n = 12) and Landrace × Large White (n = 12) pigs were used to study histological structure and enzymatic activity in the small intestine at 3 points of the productive cycle (BW = 15, 50, and 115 kg). Both strains were fed the same cereal-based diets (DE = 2,799 kcal·kg(-1), and CP = 15%) throughout the entire experimental period. Differences (P < 0.05) in histometrical variables (villus height, width and surface, crypt depth, villus height/crypt depth relationship, mucosal thickness, muscular layer thickness, and number of goblet cells) were found among samples of small intestinal sections (duodenum, jejunum, and ileum) at the 3 productive stages studied. Also, differences (P < 0.05) in histometrical variables of small intestinal samples were found between Iberian and lean pigs at all productive stages, although these differences tended to disappear with age. Differences (P < 0.05) in enzymatic activities (lactase, sucrase, maltase, isomaltase, aminopeptidase, and alkaline phosphatase) of small intestinal samples were found between the different intestinal sections at all productive stages studied. Although differences (P < 0.05) in enzymatic activities of small intestinal samples were found between Iberian and lean pigs at all productive stages, values tended to equalize with pig age. We concluded that differences previously found in dietary nutritional utilization between Iberian and lean strains are likely not due to differences in intestinal absorption or hydrolytic capacity.

Correlations between changes in intestinal microbiota composition and performance parameters in broiler chickens

Growing male Cobb broiler chickens were fed on diets supplemented with additives reported as able to influence intestinal microbiota composition. The diets used were a balanced commercial diet (no additive), inulin (20 g/kg), fructose caramel (FC, 20 g/kg) and the garlic derivative PTS-O (propyl propane thiosulfonate, 45 and 90 mg/kg diet). The composition of the intestinal microbiota was analysed by qPCR at different points of the intestinal tract, and a number of nutritional parameters were also determined. The relative amounts of bacteroides (bacteroides/total bacteria) in the ileal contents correlated (p < 0.05) positively with faecal NDF, ADF, hemicellulose and cellulose digestibility. The relative amounts of Escherichia-Shigella (Escherichia-Shigella/total bacteria) in the crop contents correlated (p = 0.05) negatively with weight gain of broilers. Faecal N digestibility correlated (p < 0.05) negatively with total bacteria in the ileal contents of chickens. The relative amounts of Escherichia-Shigella (Escherichia-Shigella/total bacteria) in the caecal contents correlated (p = 0.05) negatively with faecal fat digestibility of broilers. Total bacteria in ileal or caecal contents of growing chickens correlated (p < 0.05) negatively with ileal N digestibility. The results here reported suggest that positive or negative correlations can be found between performance parameters and changes in intestinal microbiota composition of growing broiler chickens.

Effects of inulin and di-D-fructose dianhydride-enriched caramels on intestinal microbiota composition and performance of broiler chickens.

In vitro and in vivo experiments were designed to evaluate the effectiveness of laboratory-made di-d-fructose dianhydride (DFA)-enriched caramels. The DFA-enriched caramels were obtained from d-fructose (FC), d-fructose and sucrose (FSC), or d-fructose and β-cyclodextrin (FCDC). In the in vitro experiment, raftilose and all caramels increased (P<0.05) l-lactate concentration and decreased (P<0.05) pH. Total short-chain fatty acid concentration was higher (P<0.05) than controls in tubes containing raftilose, FSC, FCDC and commercial sucrose caramel (CSC). Raftilose, and all caramels tested except FSC and FC (1%), increased (P<0.01) lactobacilli log10 number of copies compared with the non-additive control. FSC, FCDC and CSC increased (P<0.01) the bifidobacteria number of copies as compared with controls. All additives, except FCDC, decreased (P<0.01) Clostridium coccoides/Eubacterium rectale log number of copies. Compared with controls, raftilose, FC and CSC led to lower (P<0.01) Escherichia-Shigella and enterobacteria. For the in vivo experiment, a total of 144 male 1-day-old broiler chickens of the Cobb strain were randomly assigned to one of the three dietary treatments for 21 days. Dietary treatments were control (commercial diet with no additive), inulin (20 g inulin/kg diet) and FC (20 g FC/kg diet). Final BW of birds fed FC diet was higher (P<0.01) than controls or inulin-fed birds, although feed: gain values were not different. Feed intake of chickens fed FC was higher (P<0.01) than that of inulin-fed birds but not statistically different from controls. Crop pH values were lower (P<0.01) in birds fed FC diet as compared with control diet, with inulin-fed chickens showing values not different from control- or FC-fed birds. Lower (P<0.05) lactobacilli number of copies was determined in the crop, ileum and caeca of birds fed the inulin diet compared with the control diet. Inulin supplementation also resulted in lower (P<0.05) C. coccoides/E. rectale, bacteroides and total bacteria in caecal contents. Addition of FC to broiler diets gave place to lower (P<0.05) enterobacteria and Escherichia-Shigella in crop and caecal contents compared with controls. The bacteroides number of copies increased (P<0.05) as compared with controls in the ileum, but decreased (P<0.05) in the caeca of chickens fed the FC diet. Energy, ADF, NDF and non-starch polysaccharides faecal digestibilities were greater (P<0.05) than controls in chickens fed diets containing inulin or FC. Fat digestibility was higher (P<0.05) in FC-fed birds compared with controls or inulin-fed chickens. In conclusion, DFA-enriched caramels tested here, particularly FC, may represent a type of new additives useful in poultry production.