Search results for: unnatural amino acids

Authors: Navpreet Kaur, Himkusha Thakur, Nirmal Prabhakar

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The most controversial issue in crop production is the use of Organophosphate insecticides. This is evident in many reports that Organophosphate (OP) insecticides, among the broad range of pesticides are mainly involved in acute and chronic poisoning cases. OPs detection is of crucial importance for health protection, food and environmental safety. In our study, a nanocomposite of poly (3,4 ethylenedioxythiophene) (PEDOT) and multi-walled carbon nanotubes (MWCNTs) has been deposited electrochemically onto the surface of fluorine doped tin oxide sheets (FTO) for the analysis of malathion OP. The -COOH functionalization of MWCNTs has been done for the covalent binding with amino groups of AChE enzyme. The use of PEDOT-MWCNT films exhibited an excellent conductivity, enables fast transfer kinetics and provided a favourable biocompatible microenvironment for AChE, for the significant malathion OP detection. The prepared biosensors were characterized by Fourier transform infrared spectrometry (FTIR), Field emission-scanning electron microscopy (FE-SEM) and electrochemical studies. Various optimization studies were done for different parameters including pH (7.5), AChE concentration (50 mU), substrate concentration (0.3 mM) and inhibition time (10 min). Substrate kinetics has been performed and studied for the determination of Michaelis Menten constant. The detection limit for malathion OP was calculated to be 1 fM within the linear range 1 fM to 1 µM. The activity of inhibited AChE enzyme was restored to 98% of its original value by 2-pyridine aldoxime methiodide (2-PAM) (5 mM) treatment for 11 min. The oxime 2-PAM is able to remove malathion from the active site of AChE by means of trans-esterification reaction. The storage stability and reusability of the prepared biosensor is observed to be 30 days and seven times, respectively. The application of the developed biosensor has also been evaluated for spiked lettuce sample. Recoveries of malathion from the spiked lettuce sample ranged between 96-98%. The low detection limit obtained by the developed biosensor made them reliable, sensitive and a low cost process.

Keywords: PEDOT-MWCNT, malathion, organophosphates, acetylcholinesterase, biosensor, oxime (2-PAM)

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Authors: Yasir Arafat, Asma Shah, Hua Shao

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Background and Aims: Legume/cereal intercropping is used worldwide for enhancement in biomass and yield of cereal crops. However, because of intercropping, the belowground biological and chemical interactions and their effect on physiological parameters and yield of crops are limited. Methods: Wheat faba bean (WF) intercropping was designed to understand the underlying changes in the soil's chemical environment, soil microbial communities, and effect on growth and yield parameters. Experimental plots were established as having no root partition (NRP), semi-root partition (SRP), complete root partition (CRP), and their sole cropping (CK). Low molecular weight organic acids (LMWOAs) were determined by GC-MS, and high throughput sequencing of the 16S rRNA gene was carried out to screen microbial structure and composition in different root partitions of the WF intercropping system. Results: We show that intercropping induced a shift in the relative abundance of some genera of plant growth promoting rhizobacteria (PGPR) such as Allorhizobium, Neorhizobium, Pararhizobium, and Rhizobium species and resulted in better growth and yield performance of wheat. Moreover, as the plant's distance of wheat from faba beans decreased, the diversity of microbes increased, and a positive effect was observed on physiological traits and crop yield. Furthermore, an abundance and positive correlations of palmitic acid, arachidic acid, stearic acid, and 9-Octadecenoic with PGPR were recorded in the root zone of WF intercropping, which can play an important role in this facilitative mechanism of enhancing growth and yield of cereals. Conclusion: The two treatments clearly affected soil microbial and chemical composition, which can be reflected in growth and yield enhancement.

Keywords: intercropping, microbial community, LMWOAs, PGPR, soil chemical environment

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Authors: Szu-Chieh Yu, Pei-Chin Chiand, Chih-Yi Lin, Yi-Wen Chien

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UV radiation from sunlight cause numbers of acute and chronic skin damage which can result in inflammation, immune changes, physical changes and DNA damage that facilitates skin aging and the development of skin carcinogenesis. Reactive oxygen species (ROS) are generated by excessive solar UV radiation, resulting in oxidative damage to cellar components, proteins, lipids, and nucleic acids. Thus, antioxidation plays an important role that protects skin against ROS-induced injury. Safflower (Carthamus tinctorius L.) is an important Chinese medicine contained abundance flavones and hydroxysafflor yellow A (HSYA) which is main active ingredient. HSYA is part of quinochalcone and has unique structures of hydroxy groups that provided the antioxidant effect. In this study, the aim was to investigate the protective role of HYSA in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism. The HaCaT cells were UVA-irradiated and the effects of HYSA on cell viability, reactive oxygen species generation, DNA fragmentation and lipid peroxidation were measured. The mRNA expression of matrix metalloproteinase Ι (MMP Ι), cyclooxygenase-2 (COX-2) were determined by RT-PCR. In this study, UVA exposure lead to decrease in cell viability and increase in reactive oxygen species generation in HaCaT cells. HYSA could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, HYSA can reduce inflammation through inhibition the mRNA expression of MMP Ι and COX-2. Our results suggest that HSYA can act as a free radical scavenger while keratinocytes were photodamaged. HYSA could be a useful natural medicine for the protection of epidermal cells from UVA-induced damage and will be developed into products for skin care.

Keywords: HaCaT keratinocytes, hydroxysafflor yellow A (HSYA), MMP Ι, oxidative stress

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Authors: Maira C. M. Fonseca, Maria Aparecida N. Sediyama, Rosana Goncalves R. das Dores, Sanzio Mollica Vidigal, Alberto C. P. Dias

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Among Brazilian plant diversity, many species are used as food and considered minor crops (non-conventional plant foods) (NCPF). Arrowroot (Maranta arundinacea) is a NCPF from which starch is extracted from rhizome do not have gluten. Thus, arrowroot flower starch can be consumed by celiac people. Additional, some medicinal and functional proprieties are assigned to arrowroot leaves which currently are underutilized. In Brazil, it’s cultivated mainly by small scale farmers and there is no specific recommendation for fertilization. This work aimed to determinate the best fertilization for rhizome production and to verify its influence in phenolic composition and antioxidant activity of leaf extracts. Two arrowroot varieties, “Common” and “Seta”, were cultivated in organic system at state of Minas Gerais, Brazil, using cattle manure with three levels of nitrogen (N) (0, 300 and 900 kg N ha-1). The experiment design was in randomized block with four replicates. The highest production of rhizomes in both varieties, “Common” (38198.24 kg ha-1) and “Seta” (43567.71 kg ha-1), were obtained with the use of 300 kg N ha-1. With this fertilization, the total aerial part, petiole and leaf production in the varieties were respectively: “Common” (190.312 kg ha-1; 159.312 kg ha-1; 31.100 kg ha-1) and “Seta” (207.656 kg ha-1; 180.539 kg ha-1; 27.062 kg ha-1). Methanolic leaf extracts were analysed by HPLC-DAD. The major phenolic compounds found were caffeioylquinic acids, p-coumaric derivatives and flavonoids. In general, the production of these compounds significantly decreases with the increase levels of nitrogen (900 kg N ha-1). With 300 kg N ha-1 the phenolic production was similar to control. The antioxidant activity was evaluated using DPPH method and was detected around 60% of radical scavenging when 0.1 mg/mL of plant extracts were used. We concluded that fertilization with 300 kg N ha-1 increased arrowroot rhizome production, maintaining phenolic compounds yield at leaves.

Keywords: antioxidant activity, non-conventional plants, organic fertilization, phenolic compounds

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Authors: Lali Shanshiashvili, Marika Chikviladze, Nino Mamulashvili, Maia Sepashvili, Nana Narmania, David Mikeladze

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Purpose: During demyelinating inflammatory diseases and after the damage of the myelin sheet, myelin-derived proteins, including myelin basic protein (MBP), are secreted into the extracellular space. MBP shows extensive post-translational modifications, including the deimination of arginine residues. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than the unmodified one. It is hypothesized that MBP could change the inflammatory response in astrocytes. Methods: MBP was isolated and purified from bovine brain white matter. Primary astrocyte cultures were prepared from whole brains of 2-day-old Wistar rats. For evaluation of glutamate uptake/release in astrocytes following treatment of cells with MBP charge isomers, Glutamate Assay Kit was used. The expression of EAAT-2 (excitatory amino acid transporters), peroxisome proliferator-activated receptor gamma (PPAR- γ), inhibitor of nuclear factor kappa B (IkB), and high mobility group protein B1 (HMGB1) in astrocytes were assayed by Western Blot analysis. Results: This study investigated the action of deiminated isomer (C8) on the cultured primary astrocytes and compared its effects with the effects of unmodified C1 isomers. The study found that C8 and C1 MBP differently act on the uptake and release of glutamate in astrocytes: nonmodified C1 MBP increases the uptake of glutamate and does not change the release, whereas C8 decreases the release of glutamate but does not alter the uptake. Nevertheless, both isomers increased the expression of PPAR-γ and EAAT2 in the same intensity. However, immunostaining and Western Blots of cell lysates showed a decrease of IkB and increased expression of HMGB1 after the treatment of astrocytes by C8. Moreover, in the presence of C8, astrocytes release more nitric oxide than unmodified C1 isomers. Conclusion: These data suggest that the deiminated isomer of MBP evokes an inflammatory response and enhances the ability of astrocytes to release proinflammatory mediators through activation of NF-kB after the breakdown of myelin sheets. Acknowledgment: This research was supported by the SRNSF Georgia RF17_534 grant.

Keywords: myelin basic protein, glutamate, deimination, astrocytes, inflammation

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Authors: Narayana Murthy Akurathi, Vijaya Lakshmi Marella

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The literature is reviewed with regard to online Super critical fluid extraction (SFE) coupled directly with supercritical fluid chromatography (SFC) -mass spectrometry that have typically more sensitive than conventional LC-MS/MS and GC-MS/MS. It is becoming increasingly interesting to use on-line techniques that combine sample preparation, separation and detection in one analytical set up. This provides less human intervention, uses small amount of sample and organic solvent and yields enhanced analyte enrichment in a shorter time. The sample extraction is performed under light shielding and anaerobic conditions, preventing the degradation of thermo labile analytes. It may be able to analyze compounds over a wide polarity range as SFC generally uses carbon dioxide which was collected as a by-product of other chemical reactions or is collected from the atmosphere as it contributes no new chemicals to the environment. The diffusion of solutes in supercritical fluids is about ten times greater than that in liquids and about three times less than in gases which results in a decrease in resistance to mass transfer in the column and allows for fast high resolution separations. The drawback of SFC when using carbon dioxide as mobile phase is that the direct introduction of water samples poses a series of problems, water must therefore be eliminated before it reaches the analytical column. Hundreds of compounds analysed simultaneously by simple enclosing in an extraction vessel. This is mainly applicable for pharmaceutical industry where it can analyse fatty acids and phospholipids that have many analogues as their UV spectrum is very similar, trace additives in polymers, cleaning validation can be conducted by putting swab sample in an extraction vessel, analysing hundreds of pesticides with good resolution.

Keywords: super critical fluid extraction (SFE), super critical fluid chromatography (SFC), LCMS/MS, GCMS/MS

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Authors: Mustafa Durmuş, Yesim Ozogul, Esra Balıkcı, Saadet Gokdoğan, Fatih Ozogul, Ali Rıza Köşker, İlknur Yuvka

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Food scientists and researchers have paid attention to develop new ways for improving the nutritional value of foods. The application of nanotechnology techniques to the food industry may allow the modification of food texture, taste, sensory attributes, coloring strength, processability, and stability during shelf life of products. In this research, the effects of nanoemulsions based on commercial oils for vacuum-packed sea bass fillets stored at 2±2°C were investigated in terms of the sensory, chemical (total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), peroxide value (PV) and free fatty acids (FFA), pH, water holding capacity (WHC)) and microbiological qualities (total anaerobic bacteria and total lactic acid bacteria). Physical properties of emulsions (viscosity, the particle size of droplet, thermodynamic stability, refractive index, and surface tension) were determined. Nanoemulsion preparation method was based on high energy principle, with ultrasonic homojenizator. Sensory analyses of raw fish showed that the demerit points of the control group were found higher than those of treated groups. The sensory score (odour, taste and texture) of the cooked fillets decreased with storage time, especially in the control. Results obtained from chemical and microbiological analyses also showed that nanoemulsions significantly (p<0.05) decreased the values of biochemical parameters and growth of bacteria during storage period, thus improving quality of vacuum-packed sea bass.

Keywords: quality parameters, nanoemulsion, sea bass, shelf life, vacuum packing

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Authors: Delgado-Meza M., Minor-Pérez H.

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Protease is the generic name of enzymes that hydrolyze proteins. These are classified in the subgroup EC3.4.11-99X of the classification enzymes. In food technology the proteolysis is used to modify functional and nutritional properties of food, and in some cases this proteolysis may cause food spoilage. In general, seafood and rainbow trout have accelerated decomposition process once it has done its capture, due to various factors such as the endogenous enzymatic activity that can result in loss of structure, shape and firmness, besides the release of amino acid precursors of biogenic amines. Some studies suggest the use of protease inhibitors from legume as biological regulators of proteolytic activity. The enzyme inhibitors are any substance that reduces the rate of a reaction catalyzed by an enzyme. The objective of this study was to evaluate the reduction of the proteolytic activity of enzymes in extracts of rainbow trout with protease inhibitors obtained from chickpea flour. Different proportions of rainbow trout enzyme extract (75%, 50% and 25%) and extract chickpea enzyme inhibitors were evaluated. Chickpea inhibitors were obtained by mixing 5 g of flour in 30 mL of pH 7.0 phosphate buffer. The sample was centrifuged at 8000 rpm for 10 min. The supernatant was stored at -15°C. Likewise, 20 g of rainbow trout were ground in 20 mL of phosphate buffer solution at pH 7.0 and the mixture was centrifuged at 5000 rpm for 20 min. The supernatant was used for the study. In each treatment was determined the specific enzymatic activity with the technique of Kunitz, using hemoglobin as substrate for the enzymes acid fraction and casein for basic enzymes. Also biuret protein was quantified for each treatment. The results showed for fraction of basic enzymes in the treatments evaluated, that were inhibition of endogenous enzymatic activity. Inhibition values compared to control were 51.05%, 56.59% and 59.29% when the proportions of endogenous enzymes extract rainbow trout were 75%, 50% and 25% and the remaining volume used was extract with inhibitors. Treatments with acid enzymes showed no reduction in enzyme activity. In conclusion chickpea flour reduced the endogenous enzymatic activity of rainbow trout, which may favor its application to increase the half-life of this food. The authors acknowledge the funding provided by the CONACYT for the project 131998.

Keywords: rainbouw trout, enzyme inhibitors, proteolysis, enzyme activity

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Authors: L. Piñeiro, N. Cobas, L. Gómez-Limia, S. Martínez, I. Franco

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The swordfish (Xiphias gladius) is a large oceanic fish of high commercial value, which is widely distributed in waters of the world’s oceans. They are considered to be an important source of high quality proteins, vitamins and essential fatty acids, although only half of the population follows the recommendation of nutritionists to consume fish at least twice a week. Swordfish is consumed worldwide because of its low fat content and high protein content. It is generally sold as fresh, frozen, and as pieces or slices. The aim of this study was to evaluate the effect of salting and frying on the composition of the water-soluble vitamins (B₂, B₃, B₉ and B₁₂) and fat-soluble vitamins (A, D, and E) of swordfish. Three loins of swordfish from Pacific Ocean were analyzed. All the fishes had a weight between 50 and 70 kg and were transported to the laboratory frozen (-18 ºC). Before the processing, they were defrosted at 4 ºC. Each loin was sliced and salted in brine. After cleaning the slices, they were divided into portions (10×2 cm) and fried in olive oil. The identification and quantification of vitamins were carried out by high-performance liquid chromatography (HPLC), using methanol and 0.010% trifluoroacetic acid as mobile phases at a flow-rate of 0.7 mL min^-1. The UV-Vis detector was used for the detection of the water- and fat-soluble vitamins (A and D), as well as the fluorescence detector for the detection of the vitamin E. During salting, water and fat-soluble vitamin contents remained constant, observing an evident decrease in the values of vitamin B₂. The diffusion of salt into the interior of the pieces and the loss of constitution water that occur during this stage would be related to this significant decrease. In general, after frying water-soluble and fat-soluble vitamins showed a great thermolability with high percentages of retention with values among 50–100%. Vitamin B₃ is the one that exhibited higher percentages of retention with values close to 100%. However, vitamin B₉ presented the highest losses with a percentage of retention of less than 20%.

Keywords: frying, HPLC, salting, swordfish, vitamins

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Authors: Patarasuda Chaisupa, R. Clay Wright

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The selection of appropriate biomolecular targets is a crucial aspect of biopharmaceutical development. The Auxin-Inducible Degron Degradation (AID) technology has demonstrated remarkable potential in efficiently and rapidly degrading target proteins, thereby enabling the identification and acquisition of drug targets. The AID system also offers a viable method to deplete specific proteins, particularly in cases where the degradation pathway has not been exploited or when the adaptation of proteins, including the cell environment, occurs to compensate for the mutation or gene knockout. In this study, we have engineered an improved AID system tailored to deplete proteins of interest. This AID construct combines the auxin-responsive E3 ubiquitin ligase binding domain, AFB2, and the substrate degron, IAA17, fused to the target genes. Essential genes of fungi with the lowest percent amino acid similarity to human and plant orthologs, according to the Basic Local Alignment Search Tool (BLAST), were cloned into the AID construct in S. cerevisiae (AID-tagged strains) using a modular yeast cloning toolkit for multipart assembly and direct genetic modification. Each E3 ubiquitin ligase and IAA17 degron was fused to a fluorescence protein, allowing for real-time monitoring of protein levels in response to different auxin doses via cytometry. Our AID system exhibited high sensitivity, with an EC50 value of 0.040 µM (SE = 0.016) for AFB2, enabling the specific promotion of IAA17::target protein degradation. Furthermore, we demonstrate how this improved AID system enhances quantitative functional studies of various proteins in fungi. The advancements made in auxin-inducible protein degradation in this study offer a powerful approach to investigating critical target protein viability in fungi, screening protein targets for drugs, and regulating intracellular protein abundance, thus revolutionizing the study of protein function underlying a diverse range of biological processes.

Keywords: synthetic biology, bioengineering, molecular biology, biotechnology

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Authors: Paula Buldres, Jorge Toledo

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Objectives: This study aimed to characterize potential probiotic strains isolated from canine breast milk for use in dogs with enteropathies. Methodology: Six canine breast milk strains, one canine colostrum strain, and one control porcine breast milk strain were characterized. According to its functional properties of resistance to acids, different concentrations of bile salts, and pancreatin, its presumptive properties of safety and inhibitory effect on pathogens, non-cytotoxic characteristics, and adhesion to the intestine. The immunomodulatory effect of formulations with better probiotic characterization in vitro and in vivo was also analyzed. Results: Two strains characterized as potential probiotics were obtained, which corresponded to the canine strains (TUCO-16 and TUCO-17), presenting resistance to acidic pH, bile salts, and pancreatin, as well as an inhibitory effect on pathogenic Escherichia coli, Salmonella sp., and Clostridium perfringens. Strains TUCO-16 and TUCO-17 induced a significant increase in the expression of TNF-α and IL-8 in canine macrophages, respectively. Expression analyses of pattern recognition receptors in DH82 cells suggest that TUCO-16 and TUCO-17 might increase the TLR2 expression marker, and porcine strain (TUCO-4) increases the NOD2 expression marker. Based on the count obtained and the encapsulation yield, the best formulations correspond to FOS-Inulin for the TUCO-17 and TUCO-4 strains; Maltodextrin-Inulin for TUCO-16. All the strains are non-cytotoxic. The strain that showed the highest adhesion to intestinal epithelial cells was TUCO-17 with the FOS-Inulin formulation. On the other hand, the probiotics decreased the expression of pro-inflammatory markers in vivo, both in the intestine and in the spleen of mice. Conclusion: The combination of these three strains under study (TUCO-16, TUCO-17, and TUCO-4) would cover the probiotic properties in formulation and immunomodulation of all the markers under study.

Keywords: probiotics, gastrointestinal infec, dog, probiotic formulation, immunomodulatory probiotics

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Authors: Michael Eickermann, Juergen Junk

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The Western honey (Apis mellifera) is threatened by a number of parasites, especially the devastating Varroa mite (Varroa destructor) is responsible for a high level of mortality over winter, e.g., in Europe and USA. While the use of synthetic pesticides or organic acids has been preferred so far to control this parasite, breeding strategies for less susceptible honey bees are in early stages. Hygienic behavior can be an important tool for controlling Varroa destructor. Worker bees with a high level of this behavior are able to detect infested brood in the cells under the wax lid during pupation and remove them out of the hive. The underlying processes of this behavior are only partly investigated, but it is for sure that hygienic behavior is heritable and therefore, can be integrated into commercial breeding lines. In a first step, breeding lines with a high level of phenotypic hygienic behavior have been identified by using a bioassay for accurate assessment of this trait in a long-term national breeding program in Luxembourg since 2015. Based on the artificial infestation of nucleus colonies with 150 phoretic Varroa destructor mites, the level of phenotypic hygienic behavior was detected by counting the number of mites in all stages, twelve days after infestation. A nucleus with a high level of hygienic behavior was overwintered and used for breeding activities in the following years. Artificial insemination was used to combine different breeding lines. Buckfast lines, as well as Carnica lines, were used. While Carnica lines offered only a low increase of hygienic behavior up to maximum 62.5%, Buckfast lines performed much better with mean levels of more than 87.5%. Some mating ends up with a level of 100%. But even with a level of 82.5% Varroa mites are not able to reproduce in the colony anymore. In a final step, a nucleus with a high level of hygienic behavior were build up to full colonies and located at two places in Luxembourg to build up a drone congregation area. Local beekeepers can bring their nucleus to this location for mating the queens with drones offering a high level of hygienic behavior.

Keywords: agiculture, artificial insemination, honey bee, varroa destructor

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Authors: Anusha Atmakuri, R. D. Tyagi, Patrick Drogui

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Landfilling is the most familiar and easy way to dispose solid waste. Landfill is generally received via wastes from municipal near to a landfill. The waste collected is from commercial, industrial, and residential areas and many more. Landfill leachate (LFL) is formed when rainwater passes through the waste placed in landfills and consists of several dissolved organic materials, for instance, aquatic humic substances (AHS), volatile fatty acids (VFAs), heavy metals, inorganic macro components, and xenobiotic organic matters, highly toxic to the environment. These components of LFL put a load on it, hence it necessitates the treatment of LFL prior to its discharge into the environment. Various methods have been used to treat LFL over the years, such as physical, chemical, biological, physicochemical, electrical, and advanced oxidation methods. This study focuses on the combination of biological and electrochemical methods- extracellular polymeric substances and electrocoagulation(EC). The coupling of electro-coagulation process with extracellular polymeric substances (EPS) (as flocculant) as pre and\or post treatment strategy provides efficient and economical process for the decontamination of landfill leachate contaminated with suspended matter, metals (e.g., Fe, Mn) and ammonical nitrogen. Electro-coagulation and EPS mediated coagulation approach could be an economically viable for the treatment of landfill leachate, along with possessing several other advantages over several other methods. This study utilised waste substrates such as activated sludge, crude glycerol and waste cooking oil for the production of EPS using fermentation technology. A comparison of different scenarios for the treatment of landfill leachate is presented- such as using EPS alone as bioflocculant, EPS and EC with EPS being the 1st stage, and EPS and EC with EC being the 1st stage. The work establishes the use of crude EPS as a bioflocculant for the treatment of landfill leachate and wastewater from a site near a landfill, along with EC being successful in removal of some major pollutants such as COD, turbidity, total suspended solids. A combination of these two methods is to be explored more for the complete removal of all pollutants from landfill leachate.

Keywords: landfill leachate, extracellular polymeric substances, electrocoagulation, bioflocculant.

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Authors: Avinash Vellore Sunder, Shikha Shah, Pramod P. Wangikar

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The enzymatic conversion of nitriles to carboxylic acids by nitrilases has gained significance in the green synthesis of several pharmaceutical precursors and fine chemicals. While nitrilases have been characterized from different sources, the industrial application requires the identification of nitrilases that possess higher substrate tolerance, wider specificity and better thermostability, along with the development of an efficient bioprocess for producing large amounts of nitrilase. To produce large amounts of nitrilase, we developed a fed-batch fermentation process on defined media for the high cell density cultivation of E. coli cells expressing the well-studied nitrilase from Alcaligenes fecalis. A DO-stat feeding approach was employed combined with an optimized post-induction strategy to achieve nitrilase titer of 2.5*105 U/l and 78 g/l dry cell weight. We also identified 16 novel nitrilase sequences from genome mining and analysis of substrate binding residues. The nitrilases were expressed in E. coli and their biocatalytic potential was evaluated on a panel of 22 industrially relevant nitrile substrates using high-throughput screening and HPLC analysis. Nine nitrilases were identified to exhibit high activity on structurally diverse nitriles including aliphatic and aromatic dinitriles, heterocyclic, -hydroxy and -keto nitriles. With fed-batch biotransformation, whole-cell Zobelia galactanivorans nitrilase achieved yields of 2.4 M nicotinic acid and 1.8 M isonicotinic acid from 3-cyanopyridine and 4-cyanopyridine respectively within 5 h, while Cupravidus necator nitrilase enantioselectively converted 740 mM mandelonitrile to (R)–mandelic acid. The nitrilase from Achromobacter insolitus could hydrolyze 542 mM iminodiacetonitrile in 1 h. The availability of highly active nitrilases along with bioprocesses for enzyme production expands the toolbox for industrial biocatalysis.

Keywords: biocatalysis, isonicotinic acid, iminodiacetic acid, mandelic acid, nitrilase

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Authors: Attila Kiss, Erzsébet Némedi, Zoltán Naár

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Due to the rapidly growing number of conscious customers in the recent years, more and more people look for products with positive physiological effects which may contribute to the preservation of their health. In response to these demands Food Science Research Institute of Budapest develops and introduces into the market new functional foods of guaranteed positive effect that contain bioactive agents. New, efficient technologies are also elaborated in order to preserve the maximum biological effect of the produced foods. The main objective of our work was the development of new functional biscuits fortified with physiologically beneficial ingredients. Bakery products constitute the base of the food nutrients’ pyramid, thus they might be regarded as foodstuffs of the largest consumed quantity. In addition to the well-known and certified physiological benefits of lysine, as an essential amino acid, a series of antioxidant type compounds is formed as a consequence of the occurring Maillard-reaction. Progress of the evoked Maillard-reaction was studied by applying diverse sugars (glucose, fructose, saccharose, isosugar) and lysine at several temperatures (120-170°C). Interval of thermal treatment was also varied (10-30 min). The composition and production technologies were tailored in order to reach the maximum of the possible biological benefits, so as to the highest antioxidant capacity in the biscuits. Out of the examined sugar components, theextent of the Maillard-reaction-driven transformation of glucose was the most pronounced at both applied temperatures. For the precise assessment of the antioxidant activity of the products FRAP and DPPH methods were adapted and optimised. To acquire an authentic and extensive mechanism of the occurring transformations, Maillard-reaction products were identified, and relevant reaction pathways were revealed. GC-MS and HPLC-MS techniques were applied for the analysis of the 60 generated MRPs and characterisation of actual transformation processes. 3 plausible major transformation routes might have been suggested based on the analytical result and the deductive sequence of possible occurring conversions between lysine and the sugars.

Keywords: Maillard-reaction, lysine, antioxidant activity, GC-MS and HPLC-MS techniques

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Authors: Michael P. Mannino, Gerald W. Hart

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The majority of glucose metabolism proceeds through glycolytic pathways such as glycolysis or pentose phosphate pathway, however, about 5% is shunted through the hexosamine biosynthetic pathway, producing uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). This precursor can then be incorporated into complex oligosaccharides decorating the cell surface or remain as an intracellular post-translational-modification (PTM) of serine/threonine residues (O-GlcNAcylation, OGN), which has been identified on over 4,000 cytosolic or nuclear proteins. Intracellular OGN has major implications on cellularprocesses, typically by modulating protein localization, protein-protein interactions, protein degradation, and gene expression. Additionally, OGN is known to have an extensive cross-talk with phosphorylation, be in a competitive or cooperative manner. Unlike other PTMs there are only two cycling enzymes that are capable of adding or removing the GlcNAc moiety, O-linked N-aceytl glucosamine Transferase (OGT) and O-linked N-acetyl glucoamidase (OGA), respectively. The activity of OGT has been shown to be sensitive to cellular UDP-GlcNAc levels, even changing substrate affinity. Owing to this and that the concentration of UDP-GlcNAc is related to the metabolisms of glucose, amino acid, fatty acid, and nucleotides, O-GlcNAc is often referred to as a nutrient sensing rheostat. Indeed OGN is known to regulate several signaling pathways as a result of nutrient levels, such as insulin signaling. Dysregulation of OGN is associated with several disease states such as cancer, diabetes, and neurodegeneration. Improvements in glycomics over the past 10-15 years has significantly increased the OGT substrate pool, suggesting O-GlcNAc’s involvement in a wide variety of signaling pathways. However, O-GlcNAc’s role at the receptor level has only been identified in a case-by-case basis of known pathways. Examining the OGN of the plasma membrane (PM) may better focus our understanding of O-GlcNAc-effected signaling pathways. In this current study, PM fractions were isolated from several cell types via ultracentrifugation, followed by purification and MS/MS analysis in several cell lines. This process was repeated with or without OGT/OGA inhibitors or with increased/decreased glucose levels in media to ascertain the importance of OGN. Various pathways are followed up on in more detailed studies employing methods to localize OGN at the PM specifically.

Keywords: GlcNAc, nutrient sensitive, post-translational-modification, receptor

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Authors: Abanoub Mikhael, Darryl Hardie, Derek Smith, Helena Petrosova, Robert Ernst, David Goodlett

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Lipopolysaccharide (LPS) is a hallmark virulence factor of Gram-negative bacteria. It is a complex, structurally het- erogeneous mixture due to variations in number, type, and position of its simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of intact R-type lipopolysaccharide complex mixture (lipooligo- saccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and un- equivocal structural assignments. In addition to FAIMS gas phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na-H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.

Keywords: lipopolysaccharide, ion mobility MS, Kendrick mass defect, Tandem mass spectrometry

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Authors: Hamidreza Khodaei, Ali Daryabeigi Zand

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Ovulation is a physiologic process with an inflammatory response that depends on a coordinated activity of gonadotropins and steroid hormones, and inflammatory mediators such as cytokines, prostaglandins, leptin, nitric oxide (NO), etc. Conjugated linoleic acid (CLA) is composed of polyunsaturated fatty acids (PUFA) found in dairy products, beef, and lamb. There is strong evidence that dietary CLA affects mediators involved in ovulation. The objective of this study is to evaluate the impacts of various doses of dietary CLA on systemic and local hormones and parameters involved in ovulation. In this case-control research, 80 (50 ± 2-day old) female mice were randomly divided into 4 groups (C as control treatment and T1, T2 and T3 are considered as the treatment groups). There were four replicates in each group, and there were five mice in every replicate (20 mice, in total). The mice in the control group were fed with no CLA in their diet, but the ones in the treatment group received 0.1, 0.3 and 0.5g/kg of CLA (replacing corn oil in the diet), respectively for four months. After that, blood samples were obtained from the tails of animals that displayed estrus signs and estradiol (E2), progesterone (P4), LH, FSH, NO, leptin and TNFα were measured. In addition, the impacts of CLA on the ovarian production of prostaglandins (PGs) and NO were studied. The data were analyzed by SAS software. CLA considerably decreased serum levels of FSH (p < 0.05), LH, estradiol, NO, leptin and TNFα (p < 0.01). In addition, CLA decreased progesterone levels, but this effect was statistically not significant. The significantly adverse effects of CLA were observed in the ovarian production of PGE2 and PGF2α (p < 0.01). It seems that CLA may play an important role in reducing the ovulation rate in mice as CLA negatively affected female reproduction and it had adverse effects on systemic and local hormones involved in ovulation.

Keywords: conjugated linoleic acid, nitric oxide, ovary, ovulation, prostaglandin, gonadotropin

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Authors: Vikash Yadav, Ashish Arora

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Microorganisms have self-defense mechanisms to protect themselves from toxic environments. Phenolic acid decarboxylase(pad) is responsible for the defense against toxicity caused by phenolic acids, converting them into less toxic vinyl derivatives. The transcription of the pad gene is regulated by a negative transcription factor, phenolic acid decarboxylase regulators (PadR), in a substrate-inducible manner. The PadR family members share the conserved DNA-binding features and interact with the operator DNA using a winged helix-turn-helix (wHTH) motif, which contains a three-helix motif and a β-stranded wing. The members of this family function as transcriptional regulators that are involved in various cellular survival processes, such as toxin production, detoxification, multidrug resistance, antibiotic biosynthesis, and carbon catabolism. Rv1176 of Mycobacterium tuberculosis H37Rv has been assigned to the PadR family protein that remains to be structurally and functionally uncharacterized. To reveal the structural mechanism by which Rv1176 could regulates effector-responsive transcription, several experiments were performed, including Electrophoretic Mobility Shift Assay (EMSA) for DNA protein interaction, differential scanning calorimetry (DSC) and Differential Scanning Fluorimetry (DSF) for temperature and ligand-dependent protein stability, Circular Dichroism (CD) spectroscopy for secondary structure analysis. Further, to evaluate the functional role of Rv1176, the intracellular survival of recombinant M. smegmatis was examined in murine macrophage cell line J774A.1 and different stressed conditions like oxidative, pH, and nutritive stress. All these studies demonstrated that Rv1176 could behave as a transcription regulator and its expression in recombinant M. smegmatis increases intracellular survival.

Keywords: EMSA, Mycobacterium tuberculosis, PadR family protein, transcriptional regulator

Procedia PDF Downloads 53

Authors: Harveen Kaur Tatla, Parisa Niknejad, Rajender Gupta, Bipro Ranjan Dhar, Mohd. Adana Khan

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Sewage sludge management presents a pressing challenge in the realm of wastewater treatment, calling for sustainable and efficient solutions. This study explores the integration of Hydrothermal Liquefaction (HTL) and Anaerobic Digestion (AD) as a promising approach to address the complexities associated with sewage sludge treatment. The integration of these two processes offers a complementary and synergistic framework, allowing for the mitigation of inherent limitations, thereby enhancing overall efficiency, product quality, and the comprehensive utilization of sewage sludge. In this research, we investigate the optimal sequencing of HTL and AD within the treatment framework, aiming to discern which sequence, whether HTL followed by AD or AD followed by HTL, yields superior results. We explore a range of HTL working temperatures, including 250°C, 300°C, and 350°C, coupled with residence times of 30 and 60 minutes. To evaluate the effectiveness of each sequence, a battery of tests is conducted on the resultant products, encompassing Total Ammonia Nitrogen (TAN), Chemical Oxygen Demand (COD), and Volatile Fatty Acids (VFA). Additionally, elemental analysis is employed to determine which sequence maximizes energy recovery. Our findings illuminate the intricate dynamics of HTL and AD integration for sewage sludge management, shedding light on the temperature-residence time interplay and its impact on treatment efficiency. This study not only contributes to the optimization of sewage sludge treatment but also underscores the potential of integrated processes in sustainable waste management strategies. The insights gleaned from this research hold promise for advancing the field of wastewater treatment and resource recovery, addressing critical environmental and energy challenges.

Keywords: Anaerobic Digestion (AD), aqueous phase, energy recovery, Hydrothermal Liquefaction (HTL), sewage sludge management, sustainability.

Procedia PDF Downloads 48

Authors: N. Gopal, K. Jaasminerjiit, L. Z. Xiang

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Quinazoline-4(3H)-one ring system has been consistently regarded as promising privileged structural icon owing to its pharmacodynamic versatility in many of its synthetic derivatives as well as in several naturally occurring alkaloids. The literature reveals that 2nd & 3rd positions of the quinazolin-4(3H)-one pharmacophore are the target for substitution with other moieties. On the other hand, sulphanilamide derivatives and isatin moiety also displayed valuable biological activities. Hence, it was thought worthwhile to study the effects of three pharmacophoric moieties like quinazolinone, sulphanilamide and isatin in a single molecule for the better analgesic activity with lower toxicity. Series of novel 2,3-disubstituted quinazolin-4(3H)-one derivatives have been synthesised from the intermediate Schiff base of 2-(4’-substitutedphenyl)-3-[(N-2-oxoindolin-3-ylidene)-4”-sulphonamidophenyl]-quinazolin-4(3H)-one derivatives, which was prepared from reacting 2-(substituted phenyl)-4H-benzo[d][1,3]-oxazin-4-one with sulphanilamide. The required benzoxazinone derivatives were prepared by reacting anthranilic acid with benzoyl chloride. All the compounds structure was characterised by using H1 NMR, IR and Mass spectroscopy. The intermediate Schiff base and final Mannich base compounds were evaluated for their analgesic activity by acetic acid-induced writhing method at the dose of 25mg/kg, 50 mg/kg, and 100 mg/kg (bw) and Diclofenac (25mg/kg of body weight) will be used as the reference drugs. From the results of the study, it has been observed that final Mannich base showed a better analgesic activity when compared to the parent Schiff bases, it was found that compound substituted with N-methyl piperazine at 1st position of the indole nucleus of the final quinazolinone derivatives (GA4B1) i.e. 2-(4’-methoxy phenyl)-3-[4-(N-(1-N-methyl piperazine amine) methyl-2-oxo indoilin-3-ylidene] benzenesulfonyl quinazolin-4(3H)-one increases the analgesic activity and among the synthesised compounds, GA4B1 exhibited quite superior analgesic activity. The remaining Schiff bases and Mannich base derivatives exhibited moderate analgesic activity. All the compounds showed a dose dependent activity. None of the synthesised compound showed ulcer index whereas the standard drug, diclofenac [25 mg/kg (bw)] showed significantly higher gross ulcer index values.

Keywords: analgesic activity, isatin, mannich base, quinazolin-4(3H)-one

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Authors: M. C. Muya, L. J. Erasmus

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Twenty four new born male Holstein calves were divided into two treatments groups and used to evaluate the effects of M. elsdenii NCIMB 41125. The first groups were dosed with 50 ml containing 108 CFU/mL of M. elsdenii NCIMB 41125 (Me) and the control calves were not dosed. Within each of the two treatments groups, calves were divided into three treatment groups (Not dosed: 7 d, 14 d and 21 d vs dosed Me 7 d, Me14 and Me21 d (treatments), each groups contained 4 calves within which two calves were euthanized at 24 h and two calves at 72 h. Calves entered the trial until euthanize at whether 24 or 72 H after dosing time. After receiving colostrum for 3 consecutive days after birth, calves were fed whole milk and had free access to a commercial calf starter pellet and fresh water. Fecal grab samples were taken from each calf in duplicate +24 h or +72 h relative to dosing. Immediately after euthanizing, the digestive tract was harvested, and duplicate rumen and colon digesta samples collected for VFA’s determination and DNA extraction for bacteria count using 16s RNA PCR probe technique. Independent two t-test was performed to compare mean volatile fatty acids. Mixed-effects linear regressions were performed to establish relationships between: 1) M. elsdenii and Me, and between VFA’s and Me using SAS (2009). M. elsdenii NCIMB 41125 was detected in the faeces, colon and rumen of dosed calves at both +24H and +72H and ranged from 1.6 x 106 to 4.9 x 109 cfu/ml, indicating its potential to colonize in the digestive tract of calves. There was a strong positive relationship (R²=0.96; P < 0.0001) between M. elsdenii NCIMB 41125 and M. elsdenii population (cfu/ml) in the rumen, suggesting that the increase in M. elsdenii was due to increased M. elsdenii NCIMB 41125. An increase in butyrate was observed from +24 h to +72 h when calves were dosed on both d 7 and 14. Results showed that Me presented a positive relationship with butyrate (P < 0.001, R² = 0.43) and a concomitant negative relationship with acetate (P = 0.017, R² = -0.33). These results suggest that dosing pre-weaned dairy calves with M. elsdenii NCIMB 41125 has the potential to alter ruminal VFA production through increasing proportions of butyrate at the expense of propionate.

Keywords: calves, megasphaera elsdenii, rumen fermentation, bacteria

Procedia PDF Downloads 369

Authors: Sara D. Garduno-Diaz, Ramona Milcheva, Chanyu Xu

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Women’s reproductive stages (pre-pregnancy, pregnancy, and lactation) represent a time of higher micronutrient needs. With a healthy food selection as the first path of choice to cover these increased needs, tandem micronutrient supplementation is often required. Because pregnancy and lactation should be treated with care, all supplements consumed should be of quality ingredients and manufactured through controlled processes. This work describes the process followed for the development of plant-based multiple micronutrient supplements aimed at addressing the growing demand for natural ingredients of non-animal origin. A list of key nutrients for inclusion was prioritized, followed by the identification and selection of qualified raw ingredient providers. Nutrient absorption into the food matrix was carried out through natural processes. The outcome is a new line of products meeting the set criteria of being gluten and lactose-free, suitable for vegans/vegetarians, and without artificial conservatives. In addition, each product provides the consumer with 10 vitamins, 6 inorganic nutrients, 1 source of essential fatty acids, and 1 source of phytonutrients each (maca, moringa, and chlorella). Each raw material, as well as the final product, was submitted to microbiological control three-fold (in-house and external). The final micronutrient mix was then tested for human factor contamination, pesticides, total aerobic microbial count, total yeast count, and total mold count. The product was created with the aim of meeting product standards for the European Union, as well as specific requirements for the German market in the food and pharma fields. The results presented here reach the point of introduction of the newly developed product to the market, with acceptability and effectiveness results to be published at a later date.

Keywords: fertility, lactation, organic, pregnancy, vegetarian

Procedia PDF Downloads 126

Authors: L. Obeid, A. Bee, D. Talbot, S. Abramson, M. Welschbillig

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The adsorption process is one of the most efficient methods to remove pollutants from wastewater provided that suitable adsorbents are used. In order to produce environmentally safe adsorbents, natural polymers have received increasing attention in recent years. Thus, alginate and chitosane are extensively used as inexpensive, non-toxic and efficient biosorbents. Alginate is an anionic polysaccharide extracted from brown seaweeds. Chitosan is an amino-polysaccharide; this cationic polymer is obtained by deacetylation of chitin the major constituent of crustaceans. Furthermore, it has been shown that the encapsulation of magnetic materials in alginate and chitosan beads facilitates their recovery from wastewater after the adsorption step, by the use of an external magnetic field gradient, obtained with a magnet or an electromagnet. In the present work, we have studied the adsorption affinity of magnetic alginate beads and magnetic chitosan beads (called magsorbents) for methyl orange (MO) (an anionic dye), methylene blue (MB) (a cationic dye) and p-nitrophenol (PNP) (a hydrophobic pollutant). The effect of different parameters (pH solution, contact time, pollutant initial concentration…) on the adsorption of pollutant on the magnetic beads was investigated. The adsorption of anionic and cationic pollutants is mainly due to electrostatic interactions. Consequently methyl orange is highly adsorbed by chitosan beads in acidic medium and methylene blue by alginate beads in basic medium. In the case of a hydrophobic pollutant, which is weakly adsorbed, we have shown that the adsorption is enhanced by adding a surfactant. Cetylpyridinium chloride (CPC), a cationic surfactant, was used to increase the adsorption of PNP by magnetic alginate beads. Adsorption of CPC by alginate beads occurs through two mechanisms: (i) electrostatic attractions between cationic head groups of CPC and negative carboxylate functions of alginate; (ii) interaction between the hydrocarbon chains of CPC. The hydrophobic pollutant is adsolubilized within the surface aggregated structures of surfactant. Figure c shows that PNP can reach up to 95% of adsorption in presence of CPC. At highest CPC concentrations, desorption occurs due to the formation of micelles in the solution. Our magsorbents appear to efficiently remove ionic and hydrophobic pollutants and we hope that this fundamental research will be helpful for the future development of magnetically assisted processes in water treatment plants.

Keywords: adsorption, alginate, chitosan, magsorbent, magnetic, organic pollutant

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Authors: Jamboor K. Vishwanatha

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Among the potent anti-cancer agents, curcumin has been found to be very efficacious against various cancer cells. Despite multiple medicinal benefits of curcumin, poor water solubility, poor physiochemical properties and low bioavailability continue to pose major challenges in developing a formulation for clinical efficacy. To improve its potential application in the clinical area, we formulated poly lactic-co-glycolic acid (PLGA) nanoparticles. The PLGA nanoparticles were formulated using solid-oil/water emulsion solvent evaporation method and then characterized for percent yield, encapsulation efficiency, surface morphology, particle size, drug distribution within nanoparticles and drug polymer interaction. Our studies showed the successful formation of smooth and spherical curcumin loaded PLGA nanoparticles with a high percent yield of about 92.01±0.13% and an encapsulation efficiency of 90.88±0.14%. The mean particle size of the nanoparticles was found to be 145nm. The in vitro drug release profile showed 55-60% drug release from the nanoparticles over a period of 24 hours with continued sustained release over a period of 8 days. Exposure to curcumin loaded nanoparticles resulted in reduced cell viability of cancer cells compared to normal cells. We used a novel non-covalent insertion of a homo-bifunctional spacer for targeted delivery of curcumin to various cancer cells. Functionalized nanoparticles for antibody/targeting agent conjugation was prepared using a cross-linking ligand, bis(sulfosuccinimidyl) suberate (BS3), which has reactive carboxyl group to conjugate efficiently to the primary amino groups of the targeting agents. In our studies, we demonstrated successful conjugation of antibodies, Annexin A2 or prostate specific membrane antigen (PSMA), to curcumin loaded PLGA nanoparticles for targeting to prostate and breast cancer cells. The percent antibody attachment to PLGA nanoparticles was found to be 92.8%. Efficient intra-cellular uptake of the targeted nanoparticles was observed in the cancer cells. These results have emphasized the potential of our multifunctional curcumin nanoparticles to improve the clinical efficacy of curcumin therapy in patients with cancer.

Keywords: polymeric nanoparticles, cancer therapy, sustained release, curcumin

Procedia PDF Downloads 300

Authors: María del Carmen Recio-Ruiz, Ramiro Ruiz-Rosas, Juana María Rosas, José Rodríguez-Mirasol, Tomás Cordero

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Nowadays, fossil resources are main precursors for fuel production. Due to their contribution to the greenhouse effect and their future depletion, there is a constant search for environmentally friendly feedstock alternatives. Biomass residues constitute an interesting replacement for fossil resources because of their zero net CO₂ emissions. One of the main routes to convert biomass into energy and chemicals is pyrolysis. In this work, conventional pyrolysis of different biomass residues highly available such as almond shells, hemp hurds, olive stones, and Kraft lignin, was studied. In a typical experiment, the biomass was crushed and loaded into a fixed bed reactor under continuous nitrogen flow. The influence of temperature (400-800 ºC) and heating rate (10 and 20 ºC/min) on the pyrolysis yield and composition of the different fractions has been studied. In every case, the mass yields revealed that the solid fraction decreased with temperature, while liquid and gas fractions increased due to depolymerization and cracking reactions at high temperatures. The composition of every pyrolysis fraction was studied in detail. The results showed that the composition of the gas fraction was mainly CO, CO₂ when working at low temperatures, and mostly CH₄ and H₂at high temperatures. The solid fraction developed an incipient microporosity, with narrow micropore volume of 0.21 cm³/g. Regarding the liquid fraction, pyrolysis of almond shell, hemp hurds, and olive stones led mainly to a high content in aliphatic acids and furans, due to the high volatile matter content of these biomass (>74 %wt.), and phenols to a lesser degree, which were formed due to the degradation of lignin at higher temperatures. However, when Kraft lignin was used as bio-oil precursor, the presence of phenols was very prominent, and aliphatic compounds were also detected in a lesser extent.

Keywords: Bio-oil, biomass, conventional pyrolysis, lignocellulosic

Procedia PDF Downloads 113

Authors: Navpreet Kaur, Himkusha Thakur, Nirmal Prabhakar

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One of the most publicized and controversial issue in crop production is the use of agrichemicals- also known as pesticides. This is evident in many reports that Organophosphate (OP) insecticides, among the broad range of pesticides are mainly involved in acute and chronic poisoning cases. Therefore, detection of OPs is very necessary for health protection, food and environmental safety. In our study, a nanocomposite of poly (3,4 ethylenedioxythiophene) (PEDOT) and multi-walled carbon nanotubes (MWCNTs) has been deposited electrochemically onto the surface of fluorine doped tin oxide sheets (FTO) for the analysis of malathion OP. The -COOH functionalization of MWCNTs has been done for the covalent binding with amino groups of AChE enzyme. The use of PEDOT-MWCNT films exhibited an excellent conductivity, enables fast transfer kinetics and provided a favourable biocompatible microenvironment for AChE, for the significant malathion OP detection. The prepared PEDOT-MWCNT/FTO and AChE/PEDOT-MWCNT/FTO nano-biosensors were characterized by Fourier transform infrared spectrometry (FTIR), Field emission-scanning electron microscopy (FE-SEM) and electrochemical studies. Electrochemical studies were done using Cyclic Voltammetry (CV) or Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS). Various optimization studies were done for different parameters including pH (7.5), AChE concentration (50 mU), substrate concentration (0.3 mM) and inhibition time (10 min). The detection limit for malathion OP was calculated to be 1 fM within the linear range 1 fM to 1 µM. The activity of inhibited AChE enzyme was restored to 98% of its original value by 2-pyridine aldoxime methiodide (2-PAM) (5 mM) treatment for 11 min. The oxime 2-PAM is able to remove malathion from the active site of AChE by means of trans-esterification reaction. The storage stability and reusability of the prepared nano-biosensor is observed to be 30 days and seven times, respectively. The application of the developed nano-biosensor has also been evaluated for spiked lettuce sample. Recoveries of malathion from the spiked lettuce sample ranged between 96-98%. The low detection limit obtained by the developed nano-biosensor made them reliable, sensitive and a low cost process.

Keywords: PEDOT-MWCNT, malathion, organophosphates, acetylcholinesterase, nano-biosensor, oxime (2-PAM)

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Authors: Tatjana Jurić, Bojana Blagojević, Denis Uka, Ružica Ždero Pavlović, Boris M. Popović

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Natural deep eutectic solvents (NADES) represent a class of modern systems that have been developed as a green alternative to toxic organic solvents, which are commonly used as extraction media. It has been considered that hydrogen bonding is the main interaction leading to the formation of NADES. The aim of this study was phytochemical characterization and determination of the antioxidant and antibacterial activity of Mentha piperita leaf extracts obtained by six choline chloride-based NADES. NADES were prepared by mixing choline chloride with different hydrogen bond donors in 1:1 molar ratio following the addition of 30% (w/w) water. The mixtures were then heated (60 °C) and stirred (650 rpm) until the clear homogenous liquids were obtained. The Mentha piperita extracts were prepared by mixing 75 mg of peppermint leaves with 1 mL of NADES following by the heating and stirring (60 °C, 650 rpm) within 30 min. The content of six phenolics in extracts was determined using HPLC-PDA. The dominant compounds presented in peppermint leaves - rosmarinic acid and luteolin 7-O-glucoside, were extracted by NADES at a similar level as 70% ethanol. The microdilution method was applied to test the antibacterial activity of extracts. Compared with 70% ethanol, all NADES systems showed higher antibacterial activity towards Pseudomonas aeruginosa (Gram -), Staphylococcus aureus (Gram +), Escherichia coli (Gram -), and Salmonella enterica (Gram -), especially NADES containing organic acids. The majority of NADES extracts showed a better ability to neutralize DPPH radical than conventional solvent and similar ability to reduce Fe3+ to Fe2+ ions in FRAP assay. The obtained results introduce NADES systems as the novel, sustainable, and low-cost solvents with a variety of applications.

Keywords: antibacterial activity, antioxidant activity, green extraction, natural deep eutectic solvents, polyphenols

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Authors: Norhidayu Muhamad Zain

Abstract:

Gelatin, a purified protein derived mostly from porcine and bovine sources, is used widely in food manufacturing, pharmaceutical, and cosmetic industries. However, the presence of any porcine-related products are strictly forbidden for Muslim and Jewish consumption. Therefore, analytical methods offering reliable results to differentiate the sources of gelatin are needed. The aim of this study was to differentiate the sources of gelatin (porcine and tilapia fish) using Fourier transform infrared- attenuated total reflection (FTIR-ATR) combined with two dimensional infrared (2DIR) correlation analysis. Porcine gelatin (PG) and tilapia fish gelatin (FG) samples were diluted in distilled water at concentrations ranged from 4-20% (w/v). The samples were then analysed using FTIR-ATR and 2DIR correlation software. The results showed a significant difference in the pattern map of synchronous spectra at the region of 1000 cm⁻¹ to 1100 cm⁻¹ between PG and FG samples. The auto peak at 1080 cm⁻¹ that attributed to C-O functional group was observed at high intensity in PG samples compared to FG samples. Meanwhile, two auto peaks (1080 cm⁻¹ and 1030 cm⁻¹) at lower intensity were identified in FG samples. In addition, using 2D correlation analysis, the original broad water OH bands in 1D IR spectra can be effectively differentiated into six auto peaks located at 3630, 3340, 3230, 3065, 2950 and 2885 cm⁻¹ for PG samples and five auto peaks at 3630, 3330, 3230, 3060 and 2940 cm⁻¹ for FG samples. Based on the rule proposed by Noda, the sequence of the spectral changes in PG samples is as following: NH₃⁺ amino acid > CH₂ and CH₃ aliphatic > OH stretch > carboxylic acid OH stretch > NH in secondary amide > NH in primary amide. In contrast, the sequence was totally in the opposite direction for FG samples and thus both samples provide different 2D correlation spectra ranged from 2800 cm-1 to 3700 cm⁻¹. This method may provide a rapid determination of gelatin source for application in food, pharmaceutical, and cosmetic products.

Keywords: 2 dimensional infrared (2DIR) correlation analysis, Fourier transform infrared- attenuated total reflection (FTIR-ATR), porcine gelatin, tilapia fish gelatin

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Authors: Daniela Rivera-Tobar Mario Perez-Won, Roberto Lemus-Mondaca, Gipsy Tabilo-Munizaga

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Different dietary trends worldwide seek to consume foods with anti-inflammatory properties, rich in antioxidants, proteins, and unsaturated fatty acids that lead to better metabolic, intestinal, mental, and cardiac health. In this sense, food matrices with high protein content based on macro and microalgae are an excellent alternative to meet the new needs of consumers. An emerging and environmentally friendly technology for producing protein matrices is ultrasonic agglomeration. It consists of the formation of permanent bonds between particles, improving the agglomeration of the matrix compared to conventionally agglomerated products (compression). Among the advantages of this process are the reduction of nutrient loss and the avoidance of binding agents. The objective of this research was to optimize the ultrasonic agglomeration process in matrices composed of Spirulina (Arthrospira platensis) powder and Cochayuyo (Durvillae Antartica) flour, by means of the response variable (Young's modulus) and the independent variables were the process conditions (percentage of ultrasonic amplitude: 70, 80 and 90; ultrasonic agglomeration times and cycles: 20, 25 and 30 seconds, and 3, 4 and 5). It was evaluated using a central composite design and analyzed using response surface methodology. In addition, the effects of agglomeration on thermophysical and microstructural properties were evaluated. It was determined that ultrasonic compression with 80 and 90% amplitude caused conformational changes according to Fourier infrared spectroscopy (FTIR) analysis, the best condition with respect to observed microstructure images (SEM) and differential scanning calorimetry (DSC) analysis, was the condition of 90% amplitude 25 and 30 seconds with 3 and 4 cycles of ultrasound. In conclusion, the agglomerated matrices present good macro and microstructural properties which would allow the design of food systems with better nutritional and functional properties.

Keywords: ultrasonic agglomeration, physical properties of food, protein matrices, macro and microalgae

Procedia PDF Downloads 37