Radhouane Kammoun

Encapsulation in alginate and alginate coated-chitosan improved the survival of newly probiotic in oxgall and gastric juice

This study was undertaken to develop an optimum composition model for the microencapsulation of a newly probiotic on sodium alginate using response surface methodology. The individual and interactive effects of three independent variables, namely sodium alginate concentration, biomass concentration, and hardening time, were investigated using Box-Behnken design experiments. A second ordered polynomial model was fitted and optimum conditions were estimated. The optimal conditions identified were 2% for sodium alginate, 10(10)UFC/ml for biomass, and 30 min for hardening time. The experimental value obtained for immobilized cells under these conditions was about 80.98%, which was in close agreement with the predicted value of 82.6%. Viability of microspheres (96%) was enhanced with chitosan as coating materials. The survival rates of free and microencapsulated Lactobacillus plantarum TN8 during exposure to artificial gastrointestinal conditions were compared. The results revealed that the encapsulated cells exhibited significantly higher resistances to artificial intestinal juice (AIJ) and artificial gastric juice (AGJ). Microencapsulation was also noted to effectively protect the strain from heating at 65 °C and refrigerating at 4 °C. Taken together, the findings indicated that microencapsulation conferred important protective effects to L. plantarum against the gastrointestinal conditions encountered during the transit of food.

Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168

To attempt cost-effective production of US417 phytase in Bacillus subtilis, we developed an efficient system for its large-scale production in the generally recognized as safe microorganism B. subtilis 168. Hence, the phy US417 corresponding gene was cloned in the pMSP3535 vector, and for the first time for a plasmid carrying the pAMβ1 replication origin, multimeric forms of the resulting plasmid were used to transform naturally competent B. subtilis 168 cells. Subsequently, a sequential optimization strategy based on Plackett-Burman and Box-Behnken experimental designs was applied to enhance phytase production by the recombinant Bacillus. The maximum phytase activity of 47 U ml-1 was reached in the presence of 12.5 g l-1 of yeast extract and 15 g l-1 of ammonium sulphate with shaking at 300 rpm. This is 73 fold higher than the activity produced by the native US417 strain before optimization. Characterization of the produced recombinant phytase has revealed that the enzyme exhibited improved thermostability compared to the wild type PHY US417 phytase strengthening its potential for application as feed supplement. Together, our findings strongly suggest that the strategy herein developed combining heterologous expression using a cloning vector carrying the pAMβ1 replication origin and experimental designs optimization can be generalized for recombinant proteins production in Bacillus.

Extraction and characterization of three polysaccharides extracted from Opuntia ficus indica cladodes

The chemical extraction and the characterization of polysaccharides from mucilage (MC), pectin (PC) and total pectic mucilage fraction (TFC) of Opuntia ficus indica cladodes as well as the evaluation of their antioxidant activities was investigated. The FTIR spectroscopic analysis revealed the presence of carboxyl and hydroxyl groups corresponding to polysaccharides. Uronic acid and the total sugar contents of PC were higher than those of TFC and MC whereas ash content of MC was considerably more important. In addition, the findings showed that all the samples had little protein content and low average molecular weight compared to the results mentioned in literature. Furthermore, MC reached not only the highest water (WHC) and oil holding (OHC) capacities (7.81g/g and 1.34g/g, respectively) but also the highest antioxidant properties (DPPH and ABTS scavenging activities, β-carotene bleaching inhibition activity and reducing power). However, PC had the strongest emulsifying and foaming properties. As for TFC, it had low WHC, OHC and emulsifying properties whereas it had higher foaming properties than MC and greater antioxidant properties compared to PC. These outcomes can encourage the use of PC as a surfactant and MC and TFC as natural antioxidants in food and pharmaceutical industries.

Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: Optimization of culture conditions

Aspergillus oryzae S2 was assayed for alpha-amylase production under solid state fermentation (SSF). In addition to AmyA and AmyB already produced in monitored submerged culture, the strain was noted to produce new AmyB oligomeric forms, in particular a dominant tetrameric form named AmyC. The latter was purified to homogeneity through fractional acetone precipitation and size exclusion chromatography. SDS-PAGE and native PAGE analyses revealed that, purified AmyC was an approximately 172 kDa tetramer of four 42 kDa subunits. AmyC was also noted to display the same NH2-terminal amino acid sequence residues and approximately the same physico-chemical properties of AmyA and AmyB, to exhibit maximum activity at pH 5.6 and 60 °C, and to produce maltose and maltotriose as major starch hydrolysis end-products. Soyabean meal was the best substitute to yeast extract compared to fish powder waste and wheat gluten waste. AmyC production was optimized under SSF using statistical design methodology. Moisture content of 76.25%, C/N substrate ratio of 0.62, and inoculum size of 10(6.87) spores allowed maximum activity of 22118.34 U/g of dried substrate, which was 33 times higher than the one obtained before the application of the central composite design (CCD).

Phytase production by Bacillus subtilis US417 in submerged and solid state fermentations

When the variables (inoculum size, methanol and yeast extract) identified to affect phytase production by Bacillus subtilis US417 using Plackett-Burman design were optimized by RSM, a high enzyme production of 112 U/g of wheat bran was attained. Overall, a 5-fold improvement in phytase production was achieved. In SSF, on the other hand, a 4-fold enhancement in enzyme titer was attained (85 U/g of wheat bran). Based on these findings, phytase productivity was higher in SF [2.3 U/(gu2009×u2009h)] than in SSF [1.2 U/(gu2009×u2009h)].

Purification and biochemical characterization of a novel thermostable lichenase from Aspergillus niger US368.

New β-1,3;1,4-glucanase was purified from Aspergillus niger US368. The pure glucanase has a molecular mass of about 32 kDa. The N-terminal sequence of the purified enzyme (A-G-T-N-P-P-I-G-V) was determined. The optimum pH and temperature recorded for enzyme activity were 5 and 60 °C, respectively. It also displayed marked thermostability with a half-life of 30 min at 70 °C. At 37 °C, the enzyme showed 100% stability from pH 3 to 10. The Km and Vmax values exhibited by the enzyme on barley β-glucan were 0.62 mg ml(-1) and 34.46 U ml(-1), respectively. The enzyme is a retaining-one and was only active toward glucan containing β-1,3;1,4-linkages. The production of β-glucanase with barley flour as the sole carbon source was optimized. This is the first report on the purification and characterization of a β-1,3;1,4-glucanase from A. niger. This lichenase could be considered as a candidate for future application particularly in the animal feed industry.

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24°C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).

Ultrasonic extraction of pectin from Opuntia ficus indica cladodes after mucilage removal: Optimization of experimental conditions and evaluation of chemical and functional properties.

Ultrasonic assisted extraction (UAE) of pectin from Opuntia ficus indica (OFI) cladodes after mucilage removal was attempted using the response surface methodology. The process variables were optimized by the isovariant central composite design in order to improve the pectin extraction yield. The optimum condition obtained was: sonication time 70min, temperature 70°C, pH 1.5 and the water-material ratio 30ml/g. This condition was validated and the performance of experimental extraction was 18.14%±1.41%, which was closely linked to the predicted value (19.06%). Thus, UAE present a promising alternative to conventional extraction process thanks to its high efficiency which was achieved in less time and at lower temperatures. The pectin extracted by UAE from OFI cladodes (UAEPC) has a low degree of esterification, high uronic acid content, important functional properties and good anti-radical activity. These results are in favor of the use of UAEPC as potential additive in food industry.

Biocontrol of tomato plant diseases caused by Fusarium solani using a new isolated Aspergillus tubingensis CTM 507 glucose oxidase.

The present study focuses on the potential of glucose oxidase (GOD) as a promising biocontrol agent for fungal plant pathogens. In fact, a new GOD producing fungus was isolated and identified as an Aspergillus tubingensis. GOD (125 AU) has been found to inhibit Fusarium solani growth and spore production. Indeed, GOD caused the reduction of spores, the formation of chlamydospores, the induction of mycelial cords and the vacuolization of mycelium. In vivo assays, GOD acted as a curative treatment capable of protecting the tomato plants against F.xa0solani diseases. In fact, the incidence was null in the curative treatment with GOD and it is around 45% for the preventive treatment. The optimization of media composition and culture conditions led to a 2.6-fold enhancement in enzyme activity, reaching 81.48U/mL. This study has demonstrated that GOD is a potent antifungal agent that could be used as a new biofungicide to protect plants from diseases.

Effect of Aspergillus oryzae CBS 819.72 α-amylase on rheological dough properties and bread quality

An optimum Aspergillus oryzae CBS 819.72 α-amylase production in a laboratory-scale fermentor using a wheat grinding by-product as a sole carbon source (340 U/mL) was obtained after 48 h of batch fermentation under an agitation rate of 900 rpm and a pH maintained constant for 24 h. The application of this α-amylase preparation at an adequate concentration showed positive effects on dough properties and bread quality. Extensographic analysis revealed that while this addition induced a significant increase in maximal dough resistance to extension and area below the curve (energy), it brought a substantial decrease in extensibility. Farinographic results revealed small decreases in terms of time development, water absorption, and dough stability. Bread volume was also observed to undergo a significant increase.