L’objectif de cette thèse était d’isoler de nouvelles glycoside-hydrolases à partir d’une souche de Bacillus issue d’un Biotope sud-tunisien. Cette souche a montré des potentialités à produire une amylase et une lichenase à 45°C et à pH 9. La production de ces deux hydrolases a été optimisée en fermentation solide sur millet, une agro-ressource de faible coût. Cette optimisation a été conduite en adoptant la méthodologie des plans d’expériences. Nous avons ainsi obtenu des niveaux de production de l’ordre de 540 Unités d’activités amylase par gramme de substrat solide et 503 U/g d’activité lichenase. Ces deux protéines ont été par la suite purifiées et caractérisées biochimiquement. L’amylase présente un pH et une température d’activité optimaux de 5 et 70°C, respectivement. La lichenase a montré une thermoactivité et une thermostabilité remarquables qui la distinguent des lichenases précédemment décrites. En effet, l’enzyme conserve plus de 20% de son activité à 100°C, et plus de 60% de son activité après une incubation de 30 min à 90°C. Le gène codant pour cette protéine a été isolé par la construction d’une banque fosmidique dans E. coli. La comparaison de sa séquence avec la banque de données NCBI a montré que le gène de la lichenase UEB-S possède une très forte homologie avec celle de Bacillus subtilis 168, avec les positions de deux acides aminés seulement qui divergent. Un modèle de la lichenase construit au cours de cette étude laisse supposer que l’un de ces deux acides aminés (Val 69) pourrait être impliqué dans sa thermostabilité, et ce en modifiant la géométrie du site de fixation au calcium / The aim of this thesis was to isolate new glycoside hydrolases from a Bacillus strain isolated from a Biotope in the south of Tunisia. This strain was able to produce a lichenase and an amylase at 45 ° C and pH 9. The production of these two hydrolases was optimized in solid state fermentaion using millet, a low cost. agro-resource as solid substrate. This optimization was carried out using response surface methodology (RSM) based on Doehlert design. We obtained production levels of around 540 units of amylase activity per gram of solid substrate and 503 U / g of lichenase activity.Both proteins were subsequently purified and characterized biochemically. The amylase has a pH and a temperature optimum of activity of 5 and 70 ° C, respectively. The lichenase showed a remarkable thermostability which distinguish it from described lichenases. Indeed, the enzyme retained more than 20% of its activity at 100 ° C, and more than 60% of its activity after incubation for 30 min at 90 ° C. The gene encoding this protein was isolated by the construction of genomic a library in E. coli. Comparison of its sequence with the NCBI database showed that the gene coding for UEB-S lichenase has a very high homology with that of Bacillus subtilis 168, with a difference in the position of only two amino acids A model for UEB-S lichenase built during this study suggests that one of these two amino acids (Val 69) could be involved in its thermostability probabely by changing the geometry of the calcium binding site

New food innovation is largely based on consumer demand, and currently many consumers demand healthy foods with clean label ingredient statements and plant-based origins. Sprouted grain products meet these qualifications and thus are growing in popularity. Sweetened products have been made from oats by adding exogenous amylase enzymes to hydrolyze starch into sugars. The purpose of this study was to create a clean label oat sweetener using endogenous enzymes. First, amylase activity under various sprouting conditions was determined for 4 hulless and 10 in-hull oat varieties. Paul (hulless variety) and Horsepower (in-hull variety) had the highest amylase activity after sprouting 120 h at 16°C. The amylase activity in these two varieties was then further optimized by determining the highest amylase activity occurred by sprouting for 120 hours at 24°C. Second, amylase activity was determined for these two varieties after oven-drying and freeze-drying of sprouted oats, followed by a 4-week ambient storage period. Paul decreased in alpha-amylase activity for both oven-dried and freeze-dried samples, whereas Horsepower remained constant in its amylase activity for oven-dried and freeze-dried samples. Stored samples were also analyzed for susceptibility to lipid oxidation using SPME-GC-MS. All hexanal levels rose during the 4-week storage study except for the oven-dried Paul samples which began high and decreased over time. Third, a slurry of sprouted Horsepower oats, oat flour, and water was incubated at 45, 55, and 65°C to determine the optimal temperature needed to create a sweetened paste for use in oat-based food products. Incubation at 55°C had the highest initial rate of sugar production as measured by normal phase HPLC. Amount of sugar produced increased over time and plateaued at 6 h.

Nous passons plus des trois quarts de notre vie dans l'état postprandial. Pourtant, une plus grande attention a été accordée à l'étude du métabolisme à jeun qu'à l'impact de l'état postprandial sur la santé.Il est prouvé scientifiquement qu'une alimentation optimale pour la santé passe par la prise en compte de l'impact glycémique des aliments au-delà de leur simple teneur en glucides. Un déterminant important de l'impact glycémique de notre alimentation est l'amidon, qui joue un rôle clé dans la nutrition humaine en fournissant jusqu'à 50% de l'apport énergétique total. S'il est établi que la cinétique de digestion des aliments riches en amidon est un élément essentiel de leur impact glycémique, les contributions de chaque étape digestive à ce processus restent un sujet de débats. Afin de mieux comprendre les facteurs qui peuvent influencer la réponse glycémique aux aliments riches en amidon, et d’identifier de nouvelles stratégies pour atténuer leur impact glycémique, il est essentiel d'élargir notre compréhension du processus digestif de l'amidon. Cette thèse visait à étudier la digestion de repasriches en amidon (pain et pâtes), à réévaluer la contribution l’amylase salivaire à l'aide de digestions semi-dynamiques in vitro, et à mener une étude chez l'Homme pour déterminer l'effet de boissons (jus de citron et thé) sur : la réponse glycémique au pain, l'apport ad libitum, et la digestion gastrique étudiée par imagerie par résonance magnétique (IRM). Nos résultats apportent une base scientifique à l'élaboration d'une stratégie simple et efficace pour réduire la réponse glycémique aux aliments riches en amidon dans des repas de tous les jours. / All of us spend over three quarters of our lives in the postprandial state. Still, more attention has been dedicated to the study of the fasting metabolism than to the impact of the postprandial state on health.Scientific evidence supports that an optimum diet for health requires consideration of the glycaemic impact of foods in preference to consideration of carbohydrate content alone. An important determinant of the glycaemic impactof our diets is starch, which plays a key role in human nutrition, supplying up to 50% of the total energy intake. If it is clear that the digestion rate of starch-rich foods is an important determinant of their glycaemic impact, the contribution of each digestive stage to this process remains controversy. To better understand the factors that can influence the glycaemic response to starch-rich foods, and to identify new strategies to attenuate the glycaemic impact of starch-rich diets, it is essential to expand our understanding of thedigestive process of starch. The aims of this PhD were to study the digestionof starch-rich meals (bread and pasta), to reevaluate the contribution of salivary amylase using semi-dynamic in vitro digestions, and to conduct a human study to determine the effect of drinks (lemon juice and tea) on: the glycaemic response to bread, ad libitum intake, and gastric digestion assessed by magnetic resonance imaging (MRI). Our results provide scientific rationale for the development of a simple and effective strategy to reduce the glycaemicresponse to starch-rich foods in everyday-life meals.

Introduction: The presence of an endotracheal tube (ETT) increases the risk for microaspiration of secretions around the ETT. Biomarkers of pepsin and salivary amylase may be used to identify microaspiration in intubated patients because of their naturally occurring presence in the stomach or oral cavity and non-occurrence in the respiratory tract. Microaspiration may be difficult to detect until pulmonary complications, such as lung injury or infection, occur. This study assessed the presence of pepsin and salivary amylase in oral and tracheal secretions of ventilated adults. Method(s): This is a secondary analysis of data collected from 11 critically ill, adult patients on mechanical ventilation (MV) enrolled in a study to identify cues for ETT suctioning. Two paired samples of oral and tracheal secretions were suctioned when clinically indicated. Tracheal secretions were suctioned with a closed system, and oral secretions were obtained with an oropharyngeal catheter. Specimens were analyzed for total pepsin, pepsin A, pepsin C, and salivary amylase according to established assays. Results: Of 11 subjects, the majority were men (n=8), on enteral feedings (n=9) via a feeding tube placed in the stomach (n=7), and intubated with a continuous subglottic suction ETT (n=8). Median values: age, 62 years; duration of MV, 5.5 days; ETT cuff pressure 24 cm H2O; head of bed, 30degrees]. Pepsin was in measured in both oral (30.5 ng/mL; n=8) and tracheal secretions (11.1 ng/mL; n=7); Similar findings were noted for pepsin A (oral 14.7 ng/mL, n=7; sputum 7.4 ng/mL, n=6) and pepsin C (oral 14.7, n=7; tracheal 7.4, n=6). Salivary amylase (mean micro]mol/min/mL) was present in all oral secretions (359.8) and in the sputum of 6 subjects (1.8). Discussion & Conclusions: The majority of intubated patients on MV had both pepsin and salivary amylase in their sputum, likely due to microaspiration of secretions.; This finding suggests greater efforts are needed to reduce patients' risk. Ongoing strategies to prevent gastric reflux are important such as head of bed elevation and monitoring residuals. Presence of salivary amylase within tracheal secretions may indicate a need for more frequent oropharyngeal suctioning as part of routine care of intubated patients. Analysis shows no variations of the presence of pepsin or salivary amylase in relation to feeding tube placement or type of ETT. Generalizability is limited by the small sample size.

Both animal and human studies suggest that cognitive bias toward negative information, such as that observed in major depression, may arise through the interaction of cortisol (CORT) and norepinephrine (NE) within the amygdala. To date, there is no published account of the relationship between endogenous NE and CORT levels and cognitive bias. The present study examined salivary CORT and salivary alpha-amylase (sAA), an indirect measure of NE, in relation to masked affective priming of words in young female participants. Women with higher salivary CORT showed increased priming to negative word pairs only when sAA was also high; when sAA was low, no effect of CORT on priming was observed. These results are in line with previous research indicating that increased CORT is linked to enhanced processing of negative information. However, our findings extend this literature in providing evidence that CORT predicts enhanced processing of negatively valenced information only in the presence of higher sAA.

In this study, hydrolytic enzymes from sunn pest (Eurygaster integriceps) and cotton bollworm (Helicoverpa armigera) midguts were identified and characterized in terms of their optimum pH, Km and Vmax values. Hydrolytic activities were also tested for inhibition by several protease and alpha-amylase inhibitors which can be used for the development of insect resistant plants through transgenic technologies. For sunn pest midgut, a low proteolytic activity, belonging mostly to trypsin-like and leucine aminopeptidase-like proteases, and a very high alpha-amylase activity was found in sunn pest midgut, reflecting its high carbohydrate diet. Proteolytic activities could not be inhibited by natural protease inhibitors (SBTI and aprotinin) but inhibited significantly by a general serine protease inhibitor PMSF and metalloprotease inhibitors CdCl2 and CuCl2. alpha-Amylase activity of sunn pest midgut is resistant to inhibition by bean alpha-amylase inhibitor, but inhibited by chickpea, wheat and maize alpha-amylase inhibitors by 26 %, 37 % and 40 %, respectively. For cotton bollworm midgut, a very high proteolytic activity, belonging to serine and metalloprotease type, was detected. alpha-Amylase activity was lower compared to sunn pest midgut, but there were higher and diverse type of proteases, might be reflecting its wide range of host preference. Proteolytic activity was significantly inhibited by both natural protease inhibitors (SBTI and aprotinin). It was also inhibited by several synthetic protease inhibitors (PMSF, E-64, TPCK, CdCl2, CuCl2, Chymostatin). alpha-Amylase activity was inhibited by 60 % by wheat alpha-amylase inhibitor, while maize, chickpea and bean alpha-amylase inhibitors had no effect on cotton bollworm midgut alpha-amylase activity.

Master of Science / Department of Grain Science and Industry / Yong Cheng Shi / Maltogenic amylases are widely being used as an antistaling agent in baking industry. However, their action on starch in granular, swelled and dispersed forms, important components formed during bread baking, is largely unknown. Actions of two maltogenic amylases- A and -B on waxy maize starch (WMS) (100% amylopectin) and normal wheat starch (NWS) (~25% amylose) were studied and compared. For any given starch type, starch form, and hydrolysis time, maltogenic amylase-B hydrolyzed both starches more than maltogenic amylase-A as seen through sugar profile analysis indicating its higher degree of multiple attack action (DMA). Their action on non reducing ends blocked compound, p nitrophenol maltoheptaoside, confirmed their endo action. Maltogenic amylase-B showed a higher endo to total enzyme activity ratio than maltogenic amylase-A at any given enzyme weight. Greater MW reduction of dispersed starches by maltogenic amylase-B indicates its higher level of inner chain attack (LICA). Interestingly, MW distributions profiles of swelled starch hydrolysates did not show significant differences irrespective of swelling temperatures. Both enzymes showed differences in oligosaccharides compositions in dispersed and swelled starches’ reaction mixtures with sugars of degree of polymerization (DP) > 2 being degraded to glucose and maltose during later stages. For granular starches, enzymes followed a random pattern of formation and degradation of sugars with DP >2. MW distributions of hydrolyzed granular starches did not show significant shift until at the end of 24h when a low MW peak was observed. Morphological study of granular starches showed that maltogenic amylase-A mainly caused pinholes on WMS while maltogenic amylase-B caused surface corrosion with fewer pinholes. For NWS, both enzymes degraded A granules with deep cavities formation during later stages. A decrease in crystallinity of granular starches means that enzymes were able to hydrolyze both amorphous and crystalline regions. These results indicate that maltogenic amylase-B with a high LICA and high DMA possesses a better starch binding domain which can decrease the starch MW without affecting bread resilience. Strucuture of maltogenic amylase-A modified amylopectin (AP) in relation to its retrogradation was also studied. AP retrogradation was completely inhibited at % DH ≥ 20. MW and chain length distributions of debranched residual AP indicated with increase in % DH, a high proportion of unit chains with DP ≤ 9 and low proportion of unit chains with DP ≥ 17 were formed. Higher proportion of short outer AP chains which cannot participate in double helices formation supports the decrease and eventually complete inhibition of retrogradation. Thus, maltogenic amylase-A can play a very powerful role in inhibiting starch retrogradation even at limited DH (%).

No description available.

Recently, with the development of new technology, researchers have focused on physiological predictors of aggressive behavior, specifically cortisol and alpha amylase. Gordis, Granger, Susman, and Trickett (2006) found the interaction between cortisol and alpha-amylase significantly predicted parent reports of aggression indicating that low levels of physiological reactivity was associated with higher levels of problem behavior. While this research has provided valuable information about aggressive behavior, a major limitation is the majority of research focuses on males, or has not examined gender differences explicitly. This study expanded on work by Gordis et al. (2006) and other researchers on the HPA axis and sympathetic nervous system responses and aggression by using a larger sample, focusing on female adolescents, examining both physical and relational aggression, and utilizing parent and adolescent reports of aggressive behavior. Based on prior literature, I expected that lower levels of salivary cortisol taken at the beginning of the interview and the beginning of the stress task would be associated with higher levels of physical and relational aggression in girls. I also hypothesized that lower levels of cortisol and α-amylase reactivity will be associated with higher levels of physical and relational aggression. Finally, I hypothesized that lower levels of cortisol reactivity coupled with higher levels of α-amylase reactivity will be associated with lower levels of aggressive behavior. Participants in the current study live in moderate- to high-violence areas in Richmond, VA. Participants were 146 adolescent females who were enrolled in a larger longitudinal study on coping with exposure to violence. Most of the adolescents were African-American (91.1%) with a mean age of 13.9 years old (range from 11-17). The changes in physiological responses were monitored during the interview process which included the administration of the Social Competence Interview (SCI). Aggression was measured using the Child Behavior Checklist and Problem Behavior Frequency Scales. In the analyses, I controlled for pubertal status, medication usage, race, and time of day which are all factors that can influence the level of cortisol and alpha-amylase. Results indicated that higher levels of basal cortisol were associated with higher levels of aggressive behavior. In contrast to previous research and prediction, results indicated that symmetry in α-amylase and cortisol predicted lower levels of self-reported physical aggression in girls. Asymmetry in the two systems was associated with higher levels of self-reported physical aggression. These results contribute to the mixed results on female physiological responses and aggression. It also provides support for symmetry in cortisol and α-amylase as a predictor of lower levels of aggressive behavior. Studying a child’s physiological reactions to stress can give insight into behavior regulation, help identify adolescents for prevention/intervention, and serve as markers of treatment progress. These data suggest that physiological associations with aggression may not be the same for males and females, or for youth living in extremely stressful circumstances. Further research is needed to replicate these finding, and specifically to compare these patterns of associations across gender.

No description available.