Identification of a new oat ß-amylase by functional proteomics.

Oat (Avena sativa L.) seed extracts exhibited a high degree of catalytic activity including amylase activities. Proteins in the oat seed extracts were optimized for their amylolytic activities. Oat extract with amylolytic activity was separated by SDS-PAGE and a major protein band with an apparent molecular mass of 53 kDa was subjected to tryptic digestion. The generated amino acid sequences were analyzed by liquid chromatography­tandem mass spectrometry (LC/ESI/MS/MS) and database searches. These sequences were used to identify a partial cDNA from expressed sequence tags (ESTs) of A. sativa L. Based upon EST sequences, a predicted full-length gene was identified, with an open reading frame of 1464 bp encoding a protein of 488 amino acid residues (AsBAMY), with a theoretical molecular mass of 55 kDa identified as a ß-amylase belonging to the plant ß-amylase family. Primary structure of oat ß-amylase (AsBAMY) protein indicated high similarity with other ß-amylase from other cereals such as wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale) with two conserved Glu residues (E184 and E378) assigned as the "putative" catalytic residues which would act as an acid and base pair in the catalytic process. In addition, a 3D-model of AsBAMY was built from known X-ray structures and sequence alignments. A similar core (ß/α)8-barrel architecture was found in AsBAMY like the other cereal ß-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (ß/α)8-barrel domain suggesting an accessibility of the non-reducing end of the substrate and thus confirming the results of AsBAMY exo-acting hydrolase.

Citrus lemon essential oil: chemical composition, antioxidant and antimicrobial activities with its preservative effect against Listeria monocytogenes inoculated in minced beef meat.

BACKGROUND: Lemon (Citrus limon) is a flowing plant belonging to the Rutaceae family. Citrus plants constitute one of the main valuable sources of essential oil used in foods and medicinal purposes. METHODS: In this study, we assessed chemical composition, antioxidant and antimicrobial activities of C. limon essential oil (ClEO) with its preservative effect against Listeria monocytogenes inoculated in minced beef meat. Gas chromatography/mass spectrometry (GC-MS) was used to identify the major components of the obtained ClEO. The antioxidant activities of this ClEO were determined according to the ß-carotene bleaching assay, as well as by 2.2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. For antimicrobial activity, agar well diffusion method was used and the minimum inhibitory concentrations (MICs) as well as the minimum fungicidal concentrations (MFCs) were determined. The in situ effect of the ClEO was evaluated through physicochemical parameters (pH and thiobarbituric acid reactive substances (TBARS), as well as against L. monocytogenes in minced beef meat model. RESULTS: Twenty one components were identified in the ClEO and the two dominant compounds were limonene (39.74%) and ß-Pinene (25.44%). This ClEO displayed an excellent DPPH scavenging ability with an extract concentration providing 50% inhibition (IC50) of 15.056 µg/ml and a strong ß-carotene bleaching inhibition after 120 min of incubation with an IC50 of 40.147 µg/ml. The MICs varied from 0.039 to 1.25 mg/ml for Gram positive bacteria and from 0.25 to 2.5 mg/ml for Gram-negative bacteria. The meat preserving potential of ClEO was investigated against L. monocytogenes. ClEO successfully inhibited development of L. monocytogenes in minced beef meat. The application of ClEO at a 0.06 and 0.312 mg/g, may open new promising opportunities for the prevention of contamination from and growth of pathogenic bacteria, particularly L. monocytogenes, during minced beef meat storage at 4 °C. Additionally, during storage period, physicochemical values (pH and TBARS) were higher in control meat than treated meat with ClEO suggesting an efficient antioxidant activity of ClEO. CONCLUSION: It was suggested that the ClEO may be a new potential source as natural antimicrobial and antioxidant agents applied in food systems and pharmaceutical industry.

Oil from pumpkin (Cucurbita pepo L.) seeds: evaluation of its functional properties on wound healing in rats.

BACKGROUND: Increasing natural drug demand for pharmaceutical uses has encouraged scientifics all over the world to explore medicinal plants recognized as efficient remedies. In this context, extracted oil from pumpkin seeds (Cucurbita pepo L.) is an interesting target, as it is composed with prominent pharmacological properties to possible wound healing treatments. METHODS: The composition and content of certain bioactive constituents of the cold pressed oil obtained from pumpkin seeds (Cucurbita pepo L.) were analyzed and studied for their wound healing properties. Uniform wounds were induced on the dorsum of 18 rats, randomly divided into three groups. The wounds were photographed, and topically treated with saline solution (control group), 0.13 mg/mm(2) of a reference drug ("Cicaflora cream®"), and 0.52 µl/mm(2) of pumpkin's oil each 2 days until the first group is completely healing and so far biopsies were histologically assessed. RESULTS: The composition and content of tocopherols, fatty acids, and phytosterols were determined. The results showed an excellent quality of pumpkin oil with high content of polyunsaturated fatty acids (Linoleic acid: 50.88 ± 0.106 g/100 g of total fatty acids), tocopherols (280 ppm) and sterols (2086.5 ± 19.092 ppm). High content of these bioactive components were in agreement with an efficient wound healing by the mean of an in vivo study. In fact, morphometric assessment and histological findings revealed healed biopsies from pumpkin oil treated group of rats, unlike untreated group, and a full re-epithelialization with reappearance of skin appendages and well organized collagen fibers without inflammatory cells. CONCLUSIONS: This study showed the significance of oil from pumpkin seeds (Cucurbita pepo L.) as a promising drug to healing wounds in animal assays. As a whole, pumpkin's oil would be recommended in the nutritional and medicinal purposes.

Effect of treated and untreated domestic wastewater on seed germination, seedling growth and amylase and lipase activities in Avena sativa L.

BACKGROUND: Oats (Avena sativa L.) are a potential economically viable source of lipids and starch for use in foods. The aim of this study was to determine the effect of treated and untreated urban wastewater on seed germination, growth parameters and lipase and amylase activities in A. sativa. RESULTS: Untreated wastewater was highly toxic in nature and had an inhibitory effect on seed germination and seedling growth. However, after bacterial treatment, its toxicity was significantly reduced and it showed improved seed germination. It was observed that treated wastewater had no inhibitory effect on seedling growth parameters. However, A. sativa seeds treated with untreated effluent showed reduced lipase and amylase activities. CONCLUSION: Treated wastewater could be used for irrigation purposes provided that it satisfies other conditions fixed by legislation.

Insight into cellulose-based-nanomaterials - A pursuit of environmental remedies.

Nature has given several unique features to one of the most abundant and inexhaustible biopolymers on the earth, i.e., cellulose. Besides, biodegradability, and cost-effectiveness, cellulose possesses attractive properties such as the ability to undergo chemical and structural modification, plus its light weight and thermal and mechanical stability. Cellulose originates from natural sources, including being significant components of plants (ca. 33%), wood (ca. 50%), and cotton (ca. 90%). It can also be synthesized and modified further into a variety of functionalized nanomaterials for diversified sectors, such as bio-medical, food, customer care, and environmental services. Considering the significant growth in product development and interdisciplinary cellulose-based research, the proposed chapter will let the reader gain knowledge about in-vitro extraction, synthesis of nanomaterials, and applications to resolve ongoing environmental challenges.

Prediction and analysis of GH14 family ß-amylases in oat seedling extract: Structure and function insights using in silico approaches.

Oat (Avena sativa L.) seedling extract exhibited a high degree of catalytic activities. Bioinformatics were used to identify ß-amylases as abundant enzymes in the oat seedling extract. These identified oat enzymes are a member of the GH14 family. Proteins in the Avena sativa seedling extract were separated by SDS-PAGE and 2 major protein bands with an apparent molecular weights of 53 and 42 kDa were the subject of this study. These materials were digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS and database searches. These sequences were used to identify cDNAs from expressed sequence tags (EST) and Transcriptome Shotgun Assembly (TSA) of Avena sativa. Based upon EST and TSA sequences, at least 6 predicted different sequences were identified and assigned as ß-amylases. Insights into structural characterization of the oat predicted ß-amylases were analyzed using in silico approaches. The identified ß-amylases conserved the two Glu residues assigned as the "putative" catalytic residues, which would act as an acid and base pair in the catalytic process. A similar core (ß/α)8-barrel architecture was found in the predicted oat ß-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (ß/α)8-barrel domain. This suggests an accessibility of the non-reducing end of the substrate towards the oat ß-amylases and thus confirming that are exo-acting hydrolases. The results provide a detailed view of the main residues involved in catalysis in this kind of enzyme.

New insights into phospholipases in oat (Avena sativa) from bioinformatic analysis.

Phospholipases from plants in particular from oat (Avena sativa) could not be purified to homogeneity due to their association with other proteins. Interestingly, bioinformatics is a useful tool for the identification of such new sequences of enzymes. The Avena sativa phospholipases could be identified by functional proteomics and bioinformatics analysis with the aid of database searches. Based upon Transcriptome Shotgun Assembly (TSA) sequences, predicted genes were identified for Avena sativa PLD, PLA, and PLC, and assigned as AsPLD1, AsPLA2_1, and AsPiPLC1, respectively. Insights into the structural characterization of the oat predicted enzymes were analyzed using in silico approaches. Our results on sequence analysis of the oat phospholipases provide a detail view of the main residues' characteristics of such biocatalysts.

Structural and functional characterisation of two novel durum wheat annexin genes in response to abiotic stress.

Abiotic stress results in massive loss of crop productivity throughout the world. Understanding the plant gene regulatory mechanisms involved in stress responses is very important. Annexins are a conserved multigene family of Ca-dependent, phospholipid-binding proteins with suggested functions in response to environmental stresses and signalling during plant growth and development. Annexins function to counteract oxidative stress, maintain cell redox homeostasis and enhance drought tolerance. A full-length cDNA of two genes (TdAnn6 and TdAnn12) encoding annexin proteins were isolated and characterised from Tunisian durum wheat varieties (Triticum turgidum L. subsp. durum cv. Mahmoudi). Analyses of the deduced proteins encoded by annexin cDNAs (TdAnn6 and TdAnn12) indicate the presence of the characteristic four repeats of 70-75 amino acids and the motifs proposed to be involved in Ca2+ binding. Gene expression patterns obtained by real-time PCR revealed differential temporal and spatial regulation of the two annexin genes in durum wheat under different abiotic stress conditions such as salt (NaCl 150mM), osmotic (10% polyethylene glycol 8000), ionic (LiCl 10mM), oxidative (H2O2), ABA (100µM), salicylic acid (10mM), cold (4°C) and heat (37°C) stress. The two annexin genes were not regulated by heavy metal stress (CdCl2 150µM). Moreover, heterologous expression of TdAnn6 and TdAnn12 in yeast improves its tolerance to abiotic stresses, suggesting annexin's involvement in theses stress tolerance mechanisms. Taken together, our results show that the two newly isolated wheat annexin might play an active role in modulating plant cell responses to abiotic stress responses.

The LmSAP gene isolated from the halotolerant Lobularia maritima improves salt and ionic tolerance in transgenic tobacco lines.

The A20/AN1 zinc-finger domain-containing proteins of the stress-associated proteins (SAPs) family are fast emerging as potential candidates for biotechnological approaches to improve abiotic stress tolerance in plants. We identified LmSAP, one of the SAPs genes in Lobularia maritima (L.) Desv., a halophyte brassicaceae, through its transcript accumulation in response to salinity and ionic stresses. Sequence homology analysis revealed that LmSAP contains two conserved zinc-finger domains A20 and AN1. Phylogeny analyses showed that LmSAP exhibited high amino acid sequence identity to other plant SAPs. Heterologous expression of LmSAP in yeast increased cell tolerance to salt and osmotic stress. In addition, the overexpression of LmSAP conferred high salt and ionic tolerance to transgenic tobacco plants. Transgenic tobacco seedlings showed higher survival rates and antioxidant activities under salt and ionic stresses. Enhanced antioxidant activities paralleled lower malondialdehyde and superoxide anion O2- levels in the LmSAP transgenic seedlings. Overall, our results suggest that overexpression of LmSAP enhanced salt tolerance by maintaining ionic balance and limiting oxidative and osmotic stresses.

Oat (Avena sativa L.): Oil and Nutriment Compounds Valorization for Potential Use in Industrial Applications.

Oat is a promising plant for the future. It is edible and beneficial thanks to its nutritional, medicinal and pharmaceutical uses and, hence, recognized to be useful for a healthier world. The assessment of the vital functions of oat components is important for industries requiring correct health labelling, valid during the shelf life of any product. Oil, enzymes and other biomolecules of nutraceutic or dietary usage from oats would be valorized for this purpose. Although oats have a unique and versatile composition including antioxidants and biomolecules indispensable for health, they are undervalued in comparison with other staple cereals such as wheat, barley and rice. Furthermore, oats, apart from maize, comprise a high oil content used for a wide range of beneficial purposes. In addition, they contain beta glucan that has proven to be very helpful in reducing blood cholesterol levels and other cardiovascular diseases risks. In fact, there is diversity in the composition and content of the beneficial oat components within their genotypes and the different environmental conditions and, thus, oats are amenable to be enhanced by agronomic practices and genetic approaches.

Optimised amylases extraction from oat seeds and its impact on bread properties.

Statistical approaches were employed for the optimisation of the extraction of amylolytic activity from oat (Avena sativa) seeds. The application of the response surface methodology allows us to determine a set of optimal conditions (ratio seed weight/buffer volume 0.1, germination days 10 days, temperature 20 °C and pH 5.6). Experiments carried out under these conditions led to amylase production yield of 91 U/g. Its maximal activity was in the pH 5.6 and at 55 °C. Study of the incorporation of the optimised oat extract into the bread formulation revealed an improvement of the sensory quality and the textural properties of fresh and stored bread. Three-dimensional elaborations of Confocal Laser Scanning Microscopy (CLSM) images were performed on crumb of the different breads to evaluate the influence of amylase activity on microstructure. The result showed improved baking characteristics as well as overall microscopic and macroscopic appearance.

Protective effects of oat oil on deltamethrin-induced reprotoxicity in male mice.

Oats (Avena sativa L.), which are used in foods, are a potential economically viable source of oil. The purpose of this study was to determine the efficiency of oats oil to alleviate oxidative damage of testis induced by deltamethrin, which is a pyrethroid pesticide that exerts a wide range of effects on non-targeted organisms. The reprotoxicity caused by orally administered deltamethrin (DEL) to mice can be effectively antagonized by the beneficial effects of oats oil (OO) as an antioxidant. Thirty-two male albino mice were divided into four equal groups: a control group, a group of mice given deltamethrin (5 mg per kg b.w.), a group administered deltamethrin after receiving oats oil (6 g per kg b.w.), and a group receiving only OO. Exposure to deltamethrin at a dose of 5 mg per kg b.w. per day caused oxidative stress in testis, proven by a decrease in the epididymal sperm count and motility, an increase in the number of abnormal morphologies in spermatozoa and a significant increase of lipid peroxidation (LP) in the testis when compared to control animals. Co-administration of oats oil to the DEL-treated mice ameliorated the testicular biochemical parameters as well as the histological impairments in testis. We concluded that oats oil ameliorated the toxic effects of deltamethrin in testis explored by reduced LP and improved total sperm density, motility and morphology in mice spermatozoa, suggesting its role as a potential antioxidant.

Essential oil from Artemisia phaeolepis: chemical composition and antimicrobial activities.

Artemisia phaeolepis, a perennial herb with a strong volatile odor, grows on the grasslands of Mediterranean region. Essential oil obtained from Artemisia phaeolepis was analyzed by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. A total of 79 components representing 98.19% of the total oil were identified, and the main compounds in the oil were found to be eucalyptol (11.30%), camphor (8.21%), terpine-4-ol (7.32%), germacrene D (6.39), caryophyllene oxide (6.34%), and caryophyllene (5.37%). The essential oil showed definite inhibitory activity against 10 strains of test microorganisms. Eucalyptol, camphor, terpine-4-ol, caryophyllene, germacrene D and caryophyllene oxide were also examined as the major components of the oil. Camphor showed the strongest antimicrobial activity; terpine-4-ol, eucalyptol, caryophyllene and germacrene D were moderately active and caryophyllene oxide was weakly active. The study revealed that the antimicrobial properties of the essential oil can be attributed to the synergistic effects of its diverse major and minor components.

Characterization of essential oil from Citrus aurantium L. flowers: antimicrobial and antioxidant activities.

Citrus aurantium L. essential oil is commonly used as a flavouring agent. In the present study, the essential oil of fresh Citrus aurantium L. (CaEO) flowers cultivated in North East of Tunisia (Nabeul) was analyzed by GC-FID and GC-MS. 33 compounds were identified, representing 99% of the total oil. Limonene (27.5%) was the main component followed by E-nerolidol (17.5%), α-terpineol (14%), α-terpinyl acetate (11.7%) and E. E-farnesol (8%). The antimicrobial activity of the CaEO was evaluated against a panel of 13 bacteria and 8 fungal strains using agar diffusion and broth microdilution methods. Results have shown that the CaEO exhibited moderate to strong antimicrobial activity against the tested species. The investigation of the mode of action of the CaEO by the time-kill curve showed a drastic bactericidal effect after 5 min using a concentration of 624 µg/ml. The antioxidant activities of the CaEO were assayed by DPPH and beta carotene tests. Results showed that CaEO displayed an excellent DPPH scavenging ability with an IC50 of 1.8 µg/ml and a strong Beta-carotene bleaching inhibition after 120 min of incubation with an IC50 of 15.3 µg/ml. The results suggested that the CaEO possesses antimicrobial and antioxidant properties, and is therefore a potential source of active ingredients for food and pharmaceutical industry.