Activités amylase et lichenase d'une nouvelle souche de Bacillus. Production sur milieu solide et caractérisation.

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 45C 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 70C, 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 100C, et plus de 60% de son activité après une incubation de 30 min à 90C. 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 calciumThe 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 siteTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Water-soluble polysaccharides from Opuntia stricta Haw. fruit peels: Recovery, identification and evaluation of their antioxidant activities

Opuntia stricta Haw. is considered as one of the most common cactus plant growing in Tunisia. Extracting valuable compounds from its fruit peel, considered as by-product, is drawing more and more attention, making it on the verge of commercialization. Water-soluble polysaccharides were extracted from Opuntia stricta Haw. peels, and their chemical composition assessed using thin layer chromatography. The antioxidant activities of the extracted polysaccharides were assessed using 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity, total antioxidant activity and reducing power capacity. The extraction yield of water-soluble polysaccharides was 7.53±0.86%. The chemical composition revealed the presence of rhamnose, arabinose, glucose, mannose, galactose and galacturonic acid. The infra-red spectroscopic analysis showed a similar structure to that of Opuntia ficus-indica polysaccharide peels. Additionally, the extracted polysaccharides exhibited high antioxidant activities. In fact, the free radical scavenging activity (half inhibition concentration = 6.5 mg ml-1 with 94.9% inhibition at 50 mg ml-1), the total antioxidant activity (100 μg ascorbic acid equivalent at 50 mg polysaccharides) and the reducing power capacity (absorbance 700 nm = 0.7 at 50 mg ml-1), appeared to be interesting compared to natural and synthetic antioxidants. Therefore, water-soluble polysaccharides from Opuntia stricta Haw. fruit peels could be a natural alternative to replace synthetic antioxidants

Profiling beneficial phytochemicals in a potato somatic hybrid for tuber peels processing: phenolic acids and anthocyanins composition

The purpose of this study was to characterize the peels of a CN1 somatic hybrid obtained from two dihaploid potato lines (Cardinal H14 and Nicola H1) in terms of the health‐promoting phenolic compounds (phenolic acids and anthocyanins). The CN1 hybrid is defined by a pink tuber skin color making it different from the light‐yellow‐skinned “Spunta,” which is the most commonly grown potato cultivar in Tunisia. Oven‐dried peel samples derived from CN1 hybrid and cv. Spunta were ground, and phenolic compounds were extracted with water or methanol for quantification. Lyophilized peels were used for the phenolic acid and anthocyanin analyses. Higher total quantities of phenolic compounds were recovered in methanol extracts compared with water extracts. A slightly higher concentration of phenolic acids (100 mg/100 g DW) was obtained in the lyophilized peels extract of CN1 hybrid than in the cv. Spunta corresponding sample (83 mg/100 g DW). The profiles of the chlorogenic acid isomers were almost identical in both of CN1 hybrid and cv. Spunta. Caffeic acid (CA) and three caffeoylquinic acids (CQAs): 3‐CQA, 4‐CQA, and 5‐CQA, were identified from both genotypes, 5‐CQA being the dominant form in both potatoes. Since the CN1 hybrid has a pink skin color, its anthocyanin profile was also determined. The anthocyanin quantity in the CN1 peels was 5.07 mg/100 g DW, involving six different anthocyanins that were identified within the extract, namely, Pelargonidin‐3‐rutinoside‐5‐glucoside, peonidin‐3‐rutinoside‐5‐glucoside, coumaroyl ester of pelargonidin‐3‐rutinoside‐5‐glucoside, coumaroyl ester of peonidin‐3‐rutinoside‐5‐glucoside, feruloyl ester of pelargonidin‐3‐rutinoside‐5‐glucoside, and feruloyl ester of peonidin‐3‐rutinoside‐5‐glucoside. These results suggest that the peel waste of CN1 somatic hybrid can be considered as a promising source of high‐value compounds for food industry

Profiling beneficial phytochemicals in a potato somatic hybrid for tuber peels processing: phenolic acids and anthocyanins composition

The purpose of this study was to characterize the peels of a CN1 somatic hybrid obtained from two dihaploid potato lines (Cardinal H14 and Nicola H1) in terms of the health-promoting phenolic compounds (phenolic acids and anthocyanins). The CN1 hybrid is defined by a pink tuber skin color making it different from the light-yellow-skinned "Spunta," which is the most commonly grown potato cultivar in Tunisia. Oven-dried peel samples derived from CN1 hybrid and cv. Spunta were ground, and phenolic compounds were extracted with water or methanol for quantification. Lyophilized peels were used for the phenolic acid and anthocyanin analyses. Higher total quantities of phenolic compounds were recovered in methanol extracts compared with water extracts. A slightly higher concentration of phenolic acids (100 mg/100 g DW) was obtained in the lyophilized peels extract of CN1 hybrid than in the cv. Spunta corresponding sample (83 mg/100 g DW). The profiles of the chlorogenic acid isomers were almost identical in both of CN1 hybrid and cv. Spunta. Caffeic acid (CA) and three caffeoylquinic acids (CQAs): 3-CQA, 4-CQA, and 5-CQA, were identified from both genotypes, 5-CQA being the dominant form in both potatoes. Since the CN1 hybrid has a pink skin color, its anthocyanin profile was also determined. The anthocyanin quantity in the CN1 peels was 5.07 mg/100 g DW, involving six different anthocyanins that were identified within the extract, namely, Pelargonidin-3-rutinoside-5-glucoside, peonidin-3-rutinoside-5-glucoside, coumaroyl ester of pelargonidin-3-rutinoside-5-glucoside, coumaroyl ester of peonidin-3-rutinoside-5-glucoside, feruloyl ester of pelargonidin-3-rutinoside-5-glucoside, and feruloyl ester of peonidin-3-rutinoside-5-glucoside. These results suggest that the peel waste of CN1 somatic hybrid can be considered as a promising source of high-value compounds for food industry

Date Seeds as a Natural Source of Dietary Fibers to Improve Texture and Sensory Properties of Wheat Bread

The aim of this work was to investigate the effect of date seed water-soluble polysaccharides (DSP) and hemicellulose (DSH) as dietary fiber sources in enhancing the wheat bread’s quality. DSP and DSH were extracted from the three date seed varieties Deglet Nour, Ghars Souf, and Allig. The extraction yields ranged from 3.8% to 6.14% and from 13.29% to 18.8%, for DSP and DSH, respectively. DSP and DSH showed interesting functional properties and were incorporated at 0.5% and 0.75% (w/w) in wheat flour with low bread-making quality (FLBM). The results showed that the addition of 0.75% DSH significantly improved the alveograph profile of the dough, and in a more efficient way than that of DSP. Furthermore, bread evaluation revealed that the addition of DSH considerably improved the volume (by 24.22%) and the texture profile of bread (decrease of the hardness and chewiness by 41.54% and 33.81%, respectively), compared to control bread (prepared with FLBM). A sensory analysis showed that the better overall acceptability was found for bread supplemented with DSH. Results in this work demonstrate that hemicellulose fraction extracted from date seeds (DSH) and added with a level of 0.75% to FLBM represents the component that improved bread quality the best

A highly thermostable lichenase from <em>Bacillus</em> sp UEB-S: biochemical and molecular characterization

International audienceA highly thermostable and alkaline lichenase was isolated from the newly isolated strain Bacillus UEB-S. Single step purification was achieved by heating the enzyme extract for 30 min at 90 degrees C. The enzyme was a monomeric protein with a molecular weight of 28 kDa. The optimal temperature and pH for UEB-S lichenases activity were 60 degrees C and 6.0, respectively. More remarkably, the purified lichenase was stable over a broad range of temperature and pH. It retained more than 60% of its activity after incubation at 90 degrees C for 30min. Substrate specificity studies revealed that the enzyme is a true lichenase. A genomic library was screened. It allows the identification of a gene that encodes a putative lichenase showing 98% identity with the lichenase from Bacillus subtilis 168. Sequence comparison revealed that the two enzymes differed by two mutations at positions 69 and 83, where Va169 and Ser83 are replaced by Met and Ala amino acids, respectively. Therefore, a theoretical structural model was built using the lichenase from B. subtilis 168 Pdb code (3 o5sA) structure as template. Comparison of the two 3D structures suggested that Va169 stabilizes a calcium-binding site and could be involved in the higher stability of the enzyme

Potential application of Bacillus subtilis SPB1 lipopeptides in toothpaste formulation

Toothpaste is a gel dentifrice used with a toothbrush as an accessory to clean, keep and promote oral hygiene. The literature review suggests that there are many different formulations of toothpastes and that each of their individual components present specific functions. The concentration of the toothpaste ingredients must be appropriately chosen taking into account the purposes of the toothpaste. Biosurfactants are considered as suitable molecules for application in many formulations such as in toothpaste one. In the present work, two dentifrice formulations were investigated and their efficiencies were tested using chemical surfactant agent and lipopeptide biosurfactant isolated from Bacillus subtilis SPB1. The physicochemical properties were analyzed considering several tests mainly spreading ability, water activity, pH, foaming and cleaning tests. The obtained results indicated that the SPB1 biosurfactant was as efficient as the chemical surfactant confirming its potential utilization in toothpaste formulation compared to the commercial one. The evaluation of the antimicrobial activity of the formulated dentifrice was carried out against eight bacteria. The results demonstrated that the biosurfactant-based product exhibited an important antimicrobial activity, which was very effective against Enterobacter sp and Salmonella typhinirium

Efficiency of potato peels as a low-cost adsorbent for methylene blue dye removal from aqueous solutions

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A laundry detergent compatible lichenase: Statistical optimization for production under solid state fermentation on crude millet

The optimization of lichenase production by Bacillus sp. UEB-S in solid state fermentation (SSF) with millet as solid support, was carried out using response surface methodology (RSM) based on Doehlert design. Four variables (inoculum volume, millet-to-moisture ratio temperature and duration of fermentation) were regarded as factors in the optimization process. The maximum enzyme activity predicted by the model was 684 +/- 90 U/g of dry substrate when using 1.5 x 10(9) CFU/g as inoculation level and 6.16:1 (mL/g) as moisture ratio, for 4.6 days of cultivation at 35 degrees C. Using these conditions, 503 U/g of enzyme activity was experimentally obtained, which is in agreement with the predicted one (525 +/- 76 U/g). The enzyme was stable toward non-ionic surfactants and oxidizing agents. In addition, it showed a good stability and compatibility with a wide range of commercial solid detergents suggesting that lichenase from UEB-S is a potential candidate in detergent formulation. (C) 2012 Elsevier B.V. All rights reserved