Water-Soluble Polysaccharides from Ephedra alata Stems: Structural Characterization, Functional Properties, and Antioxidant Activity.

In this study, the physicochemical characterization, functional properties, and antioxidant activity of polysaccharides extracted from Ephedra alata (EAP) were investigated. EAP were extracted in water during 3 h with a liquid/solid ratio of 5 in a water bath at 90 °C. The structure of the extracted EAP was examined by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and gas chromatography-mass spectrometry (GC-MS). The functional properties and biochemical activities of EAP were determined. The chemical analysis revealed that the contents of carbohydrates, uronic acid, and proteins were 73.24% ± 1.24%, 6.82% ± 0.57%, and 6.56% ± 0.36%, respectively. The results showed that the extracted EAP essentially contain three functional groups: C=O, C-H, and O-H. SEM images showed that EAP present numerous high porosity particles. The monosaccharide composition revealed a polymer composed of glucose (43.1%), galactose (36.4%), mannose (14.9%), arabinose (3.7%), and gluconic acid (1.7%). EAP showed interesting functional properties (solubility, oil holding capacity, foaming and emulsion properties). Finally, the results revealed that EAP displayed excellent antihypertensive and antioxidant activities. Overall, EAP present a promising natural source of food additives, antioxidants, and antihypertensive agents.

Fungal ß-1,3-1,4-glucanases: production, proprieties and biotechnological applications.

ß-1,3-1,4-glucanases (or lichenases; EC 3.2.1.73) comprise one of the main enzymes used in industry during recent decades. These enzymes hydrolyze ß-glucans containing ß-1,3 and ß-1,4 linkages, such as cereal ß-glucans and lichenan. The ß-1,3-1,4-glucanases are produced by a variety of bacteria, fungi, plants and animals. A large number of microbial ß-1,3-1,4-glucanases have potential application in industrial processes, such as feed, food and detergent industries. The present review summarizes the available studies with respect to ß-1,3-1,4-glucanases production conditions, enzyme biochemical properties and potential industrial application. © 2018 Society of Chemical Industry.

Seed oil extraction from red prickly pear using hexane and supercritical CO2 : assessment of phenolic compound composition, antioxidant and antibacterial activities.

BACKGROUND: Investigating Opuntia species for their seed oil content is of much importance owing to their potential use for food and in cosmetic applications. These oils have an important content in unsaturated fatty acids as well as antioxidant compounds (e.g. polyphenols, vitamin E), which have been associated with the prevention of some chronic diseases. Moreover, Opuntia stricta oils possess important antimicrobial activities. For instance, the main focus of this study was to compare the effectiveness of conventional (hexane extraction) and novel (supercritical (SC)-CO2 ) extraction methods for the recovery of oil and phenolic compounds from O. stricta seeds. The oil yield of both extracts was then compared and the polyphenol content and composition of both extracts were determined by liquid chromatography-high-resolution mass spectrometry. Additionally, antioxidant (DPPH assay) and antimicrobial activities (disc diffusion method) of O. stricta seed oils were determined. RESULTS: The oil yield (based on Soxhlet's method) of O. stricta seeds was determined using SC-CO2 (49.9 ± 2.2%), and hexane (49.0 ± 1.5%). Although obtaining similar oil extraction yields using the two methods, the extracted oil using SC-CO2 was more enriched in polyphenols (172.2 ± 11.9 µg gallic acid equivalents (GAE) g-1 oil) than that extracted using hexane (76.0 ± 6.9 µg GAE g-1 of oil). Polyphenol profiles showed that the SC-CO2 process led to the yield of more compounds (45) than that using hexane extraction (11). Moreover, the antioxidant and antimicrobial activities of SC-CO2 extract showed a high percentage of inhibition. CONCLUSION: SC-CO2 extraction of O. stricta seed oil led to extraction of oil with a similar yield to that with hexane extraction, but with higher polyphenol content. The extract containing polyphenols exhibited high antioxidant and antibacterial properties, demonstrating their great potential as feedstock for high-oil quality. © 2016 Society of Chemical Industry.

Effect of xylan oligosaccharides generated from corncobs on food acceptability, growth performance, haematology and immunological parameters of Dicentrarchus labrax fingerlings.

The objective of this study was to determine the effect of two levels of inclusion of xylan oligosaccharides (XOS) extracted from corncob on growth, feed utilization, immune status and disease resistance of Mediterranean sea bass (Dicentrarchus labrax) fingerlings. Specimens of 4.75 ± 0.69 g at initial density of 2.7 ± 0.13 kg/m(3) were fed during 12 weeks at 0 g kg(-1) diet, 5 g kg(-1) diet and 10 g kg(-1) diet, dietary XOS level of inclusion in a commercial sea bass diet. Feeding the fish at both XOS dietary inclusion levels significantly increased weight gain, protein efficiency ratio and feed conversion ratio. Feeding of supplemented diets to fish led to reducing mortalities after challenging with A. hydrophila. The haematological and immunological parameters were assayed in both pre-challenged and post-challenged groups. There was an increased trend in red blood corpuscles, white blood corpuscles, pack cell volume, haemoglobin (Hb %) and serum protein content in treated groups over the control as time elapsed with the feeding trials. The serum immunoglobulin level and lysozyme activity showed an increased trend in the fed groups. Histological features of the liver showed lower lipid vacuolization and regular-shaped morphology of hepatocytes around the sinusoidal spaces denoting a better utilization of dietary nutrients supported with the morphometric data. In conclusion, XOS added at a designated dose (5 g kg(-1) diet) in the diet improves growth and stimulates the immunity and makes D. labrax fingerlings more resistant to infection by A. hydrophila.

Functional characterization of Penicillium occitanis Pol6 and Penicillium funiculosum GH11 xylanases.

Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of heteroxylans constituting the lignocellulosic plant cell wall. Xylanases from the GH11 family are considered as true xylanases because of their high substrate specificity. In order to study in depth a crucial difference in the thumb region between two closely related xylanases from Penicillium in terms of kinetic parameters and inhibition sensitivity, the GH11 xylanases from Penicillium occitanis Pol6 (PoXyn3) and from Penicillium funiculosum (PfXynC) were heterologously expressed in Pichia pastoris. The PoXyn3 and PfXynC cDNAs encoding mature xylanases were cloned into pGAPZαA vectors and integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase constitutive promoter. PfXynC was expressed as a His-tagged recombinant protein and purified from the supernatant homogeneity by a one-step purification protocol using immobilized metal affinity chromatography. The recombinant PoXyn3 was purified using a single anion-exchange chromatography. The purified recombinant enzymes were optimally active at 45°C and pH 4.0 for PoXyn3 and 40°C and pH 3.0 for PfXynC. The measured kinetic parameters (k(cat) and Vmax) showed that PfXynC was five times more active than PoXyn3 irrespective of the substrate whereas the apparent affinity (K(m)) was similar. The recombinant enzymes showed distinct sensitivity to the Triticum aestivum xylanase inhibitor TAXI-I.

Cloning and constitutive expression of His-tagged xylanase GH 11 from Penicillium occitanis Pol6 in Pichia pastoris X33: purification and characterization.

High-level constitutive expression of xylanase GH11 from Penicillium occitanis Pol6 termed PoXyn2 was achieved using the methylotrophic yeast Pichia pastoris. The PoXyn2 cDNA encoding for a mature xylanase of 320 amino acids was subcloned into the pGAPZαA vector, to construct recombinant xylanse with six histidine residues at the N-terminal and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. Activity assay and SDS-PAGE demonstrate that the His-tagged xylanase was extracellularly expressed in P. pastoris and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). The purified PoXyn2 showed a single band on SDS-PAGE with an apparent molecular weight of 30 kDa. The xylanase activity was optimal at pH 3.0 and 50°C. The specific activity measured for Oat Spelt Xylan was 8549.85 U mg(-1). The apparent The K(M) and V(max) values were 8.33±0.7 mg ml(-1)and 58.82±0.9 µmol min(-1) ml(-1), respectively, as measured on Oat Spelt Xylan. This is the first report demonstrating the possibility of mass production of P. occitanis xylanase using P. pastoris.

Purification and biochemical characterization of extracellular beta-glucosidases from the hypercellulolytic Pol6 mutant of Penicillium occitanis.

The Pol6 mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases and beta-glucosidase production with high cellulose degradation efficiency (Jain et al., Enzyme Microb Technol, 12:691-696, 1990; Hadj-Taieb et al., Appl Microbiol Biotechnol, 37:197-201, 1992; Ellouz Chaabouni et al., Enzyme Microb Technol, 16:538-542, 1994; Ellouz Chaabouni et al., Appl Microbiol Biotechnol, 43:267-269, 1995). In this work, two forms of beta-glucosidase (beta-glu 1 and beta-glu 2) were purified from the culture supernatant of the Pol6 strain by gel filtration, ion exchange chromatography, and preparative anionic native electrophoresis. These enzymes were eluted as two distinct species from the diethylamino ethanol Sepharose CL6B and anionic native electrophoresis. However, both behaved identically on sodium dodecyl sulfate polyacrylamide gel electrophoresis (MW, 98 kDa), shared the same amino acid composition, carbohydrate content (8%), and kinetic properties. Moreover, they strongly cross-reacted immunologically. They were active on cellobiose and pNPG with Km values of 1.43 and 0.37 mM, respectively. beta-glu 1 and beta-glu 2 were competitively inhibited by 1 mM of glucose and 0.03 mM of delta-gluconolactone. They were also significantly inhibited by Hg(2+) and Cu(2) at 2 mM. The addition of purified enzymes to the poor beta-glucosidase crude extract of Trichoderma reesei increased its hydrolytic efficiency on H(3)P0(4) swollen cellulose but had no effect with P. occitanis crude extract. Besides their hydrolytic activities, beta-glu 1 and beta-glu 2 were endowed with trans-glycosidase activity at high concentration of glucose.

Adsorptive removal of malachite green from aqueous solutions by almond gum: Kinetic study and equilibrium isotherms.

This work aimed at investigating the potential of almond gum as low cost adsorbent for the removal of the cationic dye; malachite green from aqueous solutions. Almond gum was first analyzed by scanning electron microscopy (SEM) and Fourier transforms infrared spectroscopy (FTIR), and then the adsorption behavior was studied in batch system. The effects of the adsorption parameters (adsorbent dose, pH, contact time, particle size, initial dye concentration, temperature and agitation) on the dye removal have been studied. Adsorption equilibrium and isotherms were evaluated depending on temperature using the isotherms of Freundlich, Langmuir, and Tempkin. The obtained result showed that both Langmuir and Freundlich models were adapted to study the dye sorption. The maximum adsorption capacities were equal to 172.41mg/g, 181.81mg/g, and 196.07mg/g at 303.16K, 313.16K, and 323.16K, respectively. The kinetics of sorption were following the pseudo-second order model. The thermodynamic changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG) indicated that the adsorption of malachite green at the surface of almond gum is endothermic and occurs spontaneously. Desorption experiments were conducted to regenerate almond gum, showing great desorption capacity when using HCl at pH 2.

Antioxidant Properties of Water-Soluble Gum from Flaxseed Hulls.

Soluble flaxseed gum (SFG) was extracted from flax (Linum usitatissimum) hulls using hot water, and its functional groups and antioxidant properties were investigated using infrared spectroscopy and different antioxidant assays (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), reducing power capacity, and ß-carotene bleaching inhibition assay), respectively. The antioxidant capacity of SFG showed interesting DPPH radical-scavenging capacity (IC50 SFG = 2.5 mg·mL(-1)), strong ABTS radical scavenging activity (% inhibition ABTS = 75.6% ± 2.6% at 40 mg·mL(-1)), high reducing power capacity (RPSFG = 5 mg·mL(-1)), and potent ß-carotene bleaching inhibition activity (IC50 SFG = 10 mg·mL(-1)). All of the obtained results demonstrate the promising potential use of SFG in numerous industrial applications, and a way to valorize flaxseed hulls.

Purification and characterization of two low molecular weight endoglucanases produced by Penicillium occitanis mutant Pol 6.

Two endoglucanases (EGs), EG A and EG B, were purified to homogeneity from Penicillium occitanis mutant Pol 6 culture medium. The molecular weights of EG A and EG B were 31,000 and 28,000 kDa, respectively. The pI was about 3 for EG A and 7.5 for EG B. Optimal activity was obtained at pH 3.5 for both endoglucanases. Optimal temperature for enzyme activity was 60 degrees C for EG A and 50 degrees C for EG B. EG A was thermostable at 60 degrees C and remained active after 1 h at 70 degrees C. EGs hydrolyzed carboxymethylcellulose, phosphoric acid swollen cellulose, and beta-glucan efficiently, whereas microcrystalline cellulose (Avicel) and laminarin were poorly hydrolyzed. Only EG B showed xylanase activity. Furthermore, these EGs were insensitive to the action of glucose and cellobiose but were inhibited by the divalent cations Hg2+, Co2+, and Mn2+.

Biological Activities of xylooligosaccharides generated from garlic straw xylan by purified xylanase from Bacillus mojavensis UEB-FK.

A newly isolated Bacterium strain named UEB-FK was selected from Tunisian Sahara, exhibiting the highest clear zone on agar plates containing oat spelt xylan by staining with Congo red. On the basis of 16S rDNA sequence analysis, this strain was identified as Bacillus mojavensis. This strain produced extracellular xylanase. Xylanase from the strain was purified to homogeneity and had an apparent molecular weight of 14 kDa. The K m and V max values of the purified xylanase on oat spelt xylan were 3.85 mg/mL and 250.02 U/mg, respectively. The optimum pH and temperature for the enzyme were found to be 4.0 and 50 °C, respectively, and the enzyme exhibited significant heat stability. In addition, the enzyme was found to be stable in a wide range of pH (3-9). The main hydrolysis products yielded from garlic straw-extracted xylan were xylobiose and xylotriose. The antioxidant and antibacterial activities of xylan oligosaccharide (XOS) were investigated. As regards to the in vitro antioxidant activities, the XOS showed a important DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity (IC50 = 0.45 mg/mL) and a high ß-carotene bleaching (IC50 = 2.2 mg/mL). Furthermore, XOS had a high antimicrobial activity against Klebsiella pneumoniae, Enterococcus faecalis, Bacillus thuringiensis, and Pseudomonas aeruginosa.

Structural data and biological properties of almond gum oligosaccharide: application to beef meat preservation.

Enzymatic hydrolysis of almond gum generates low molecular weight oligosaccharides (OAG) with a yield of 33.5%. The generated oligosaccharides were purified and identified. OAG analyses show that the most prominent residues were galactose and arabinose with traces of xylose, rhamnose, glucose and mannose. The glycosyl linkage positions were analyzed using gas chromatography-mass spectrometry showing a main chain composed of galactose units [ → 3)-Gal-(1 → ] branched mainly with arabinose residues [Ara-(1 → ]. The antioxidant and antimicrobial activities of OAG were investigated. As regards the in vitro antioxidant activities, the OAG showed a high total antioxidant activity (347 µg ascorbic acid equivalent/mL), an important DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity (IC50 = 0.64 mg/mL) and a high reducing capacity (RP0.5AU = 3.6 mg/mL). Furthermore, OAG had a high antimicrobial activity against Salmonella thyphimirium, Bacillus cereus, Actinomycetes sp, Klebsiella pneumoniae, Escherichia coli, Alternaria alternate and Candidat albicans. Finally, OAG efficiency was tested using 0.5%; 0.75% and 1% concentrations in beef meat preservation. Microbial growth and lipid oxidation were monitored during 9 days at 4 °C. The results showed significant inhibitions (p < 0.05) of lipid oxidation and microbial growth in ground beef meat containing OAG.

Production of xylooligosaccharides by immobilized His-tagged recombinant xylanase from Penicillium occitanis on nickel-chelate Eupergit C.

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0-4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.

Immobilization of His-tagged recombinant xylanase from Penicillium occitanis on nickel-chelate Eupergit C for increasing digestibility of poultry feed.

Recombinant xylanase 2 from Penicillium occitanis expressed with an His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49%. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0-4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Thermodynamically, increase in enthalpy and free energy change after covalent immobilization could be credited to the enhanced stability. Immobilized xylanase could be reused for 10 consecutive cycles retaining 60% of its initial activity. It was found to be effective in releasing reducing sugar from poultry feed. Immobilization on Eupergit C is important due to its mechanical resistance at high pH and temperature. Hence, considerable stability and reusability of bound enzyme may be advantageous for its industrial application.

A new raw-starch-digesting α-amylase: production under solid-state fermentation on crude millet and biochemical characterization.

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using 1.38 × 10(9) CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at 37 degrees C, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and 70 degrees C, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

Purification and properties of a thermostable xylanase GH 11 from Penicillium occitanis Pol6.

An extracellular, endo-ß-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS-PAGE with an apparent molecular weight of 30 kDa. The xylanase activity was optimal at pH 3.0 and 65 °C. The specific activity measured for oat spelt xylan was 2,368 U mg(-1). The apparent K(m) and V(max) values were 8.33 mg ml(-1) and 58.82 µmol min(-1) ml(-1), respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962 bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88 kDa.

Purification and characterization of a low molecular weight of beta-mannanase from Penicillium occitanis Pol6.

The highest beta-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified beta-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 degrees C and pH 4.0. It was stable for 30 min at 70 degrees C and has a broad pH stability (2.0-12.0). ManIII showed K (m), V (max), and K (cat) values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s(-1) with locust bean gum as substrate, respectively. It was inhibited by mannose with a K (I) of 0.610(-3) mg/ml. ManIII was activated by CuSO4 and CaCl2 (2.5 mM). However, in presence of 2.5 mM Co2+, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.

New thermostable amylase from Bacillus cohnii US147 with a broad pH applicability.

A new thermophilic bacterial strain identified as Bacillus cohnii US147 was isolated from the southern Tunisian soil. The identification was based on physiological tests and molecular techniques related to the 16S ribosomal ribonucleic acid. The isolated strain produced amylase, which was purified. This amylase had an apparent molecular mass of 30 kDa as estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Amylase US147 showed K (m) and V (max) values of 0.7 mg/ml and 2.2 U/ml, respectively, with starch as the substrate. The enzyme was active in acid and basic pH and had a maximal activity on starch at pH 9 and 70 degrees C. The enzyme was stable at pH 9 for 72 h and retained half of its activity after incubation at 70 degrees C for 150 min. A partially inhibition (15%, 25%, 23%, 20%, and 22%) was obtained with 1 mM SDS, 1 mM NaBO(3), 1 mM H(2)O(2,) 1 mM Zn(+2), and 5 mM ethylenediamine tetraacetic acid (EDTA), respectively. The amylase recovered its original activity by the addition of 10 mM Ca (2+) to the 5 mM EDTA. These properties indicated a possible use of this amylase in starch saccharification, in detergent, and in other industrial applications.