Search results for: RT-PCR assay

Authors: Yuhua Wang, Mu Li, Jinggen Liu

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Aim: To investigate the different effects of heroin and milk in activating the corticostriatal system that plays a critical role in reward reinforcement learning. Methods: Male SD rats were trained daily for 15 d to self-administer heroin or milk tablets in a classic runway drug self-administration model. Immunohistochemical assay was used to quantify Arc protein expression in the medial prefrontal cortex (mPFC), the nucleus accumbens (NAc), the dorsomedial striatum (DMS) and the ventrolateral striatum (VLS) in response to chronic self-administration of heroin or milk tablets. NMDA receptor antagonist MK801 (0.1 mg/kg) or dopamine D1 receptor antagonist SCH23390 (0.03 mg/kg) were intravenously injected at the same time as heroin was infused intravenously. Results: Runway training with heroin resulted in robust enhancement of Arc expression in the mPFC, the NAc and the DMS on d 1, 7, and 15, and in the VLS on d 1 and d 7. However, runway training with milk led to increased Arc expression in the mPFC, the NAc and the DMS only on d 7 and/or d 15 but not on d 1. Moreover, runway training with milk failed to induce increased Arc protein in the VLS. Both heroin-seeking behavior and Arc protein expression were blocked by MK801 or SCH23390 administration. Conclusion: The VLS is likely to be critically involved in drug-seeking behavior. The NMDA and D1 receptor-dependent Arc expression is important in drug-seeking behavior.

Keywords: arc, mesocorticolimbic system, drug rewarding behavior, NMDA receptor

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Authors: Riddhi Trivedi, Shrenik Shah

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For Si RNA to be delivered various biodegradable polymers are trialed by many researchers. One of them is Chitosan (CS) nanoparticles which have been extensively studied for siRNA delivery but the stability and efficacy of such particles are highly dependent on the types of cross-linker used. Hence the attempts are made in this study with PGA To address this issue, three common cross-linkers; Ethylene glycol diacrylate (ED) and poly-D-glutamic acid (PGA) were used to prepare siRNA loaded CS-ED/PGA nanoparticles by ionic gelation method. The nanoparticles which were obtained were compared for its characterization in terms of its physicochemical properties i.e. particle size of the resultant particles, zeta potential, its encapsulation capacity in the polymer. Among all the formulations prepared with different crosslinker PGA siRNA had the smallest particle size (ranged from 120 ± 1.7 to 500 ± 10.9 nm) with zeta potential ranged from 22.1 ± 1.5 to +32.4 ± 0.5 mV, and high entrapment ( > 91%) and binding efficiencies. Similarly, CS-ED nanoparticles showed better siRNA protection during storage at 4˚C and as determined by serum protection assay. TEM micrographs revealed the assorted morphology of CS-PGA-siRNA nanoparticles in contrast to irregular morphology displayed by CS-ED-siRNA. All siRNA loaded nanoparticles were found to give initial burst release which after some time followed by a sustained release of siRNA which were loaded inside. All the formulations showed concentration-dependent cytotoxicity with when cytotoxicity performed by HeLa and normal vero cell lines.

Keywords: chitosan, siRNA, cytotoxicity, cell line study

Procedia PDF Downloads 299

Authors: Osama H. Elsayed, Mohsen M. S. Asker, Mahmoud A. Swelim, Ibrahim H. Abbas, Aziza I. Attwa, Mohamed E. El Awady

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Hydroxy marilone C is a bioactive metabolite was produced from the culture broth of Streptomyces badius isolated from Egyptian soil. hydroxy marilone C was purified and fractionated by silica gel column with a gradient mobile phase dicloromethane (DCM) : Methanol then Sephadex LH-20 column using methanol as a mobile phase. It was subjected to many instruments as Infrared (IR), nuclear magnetic resonance (NMR), Mass spectroscopy (MS) and UV spectroscopy to the elucidation of its structure. It was evaluated for antioxidant, cytotoxicity against human alveolar basal epithelial cell line (A-549) and human breast adenocarcinoma cell line (MCF-7) and antiviral activities; showed that the maximum antioxidant activity was 78.8 % at 3000 µg/ml after 90 min. and the IC50 value against DPPH radical found about 1500 µg/ml after 60 min. By Using MTT assay the effect of the pure compound on the proliferation of A-549 cells and MCF-7 cells were 443 µg/ml and 147.9 µg/ml, respectively. While for detection of antiviral activity using Madin-Darby canine kidney (MDCK) cells the maximum cytotoxicity was at 27.9% and IC50 was 128.1µg/ml. The maximum concentration required for protecting 50% of the virus-infected cells against H1N1 viral cytopathogenicity (EC50) was 33.25% for 80 µg/ml. This results indicated that the hydroxy marilone C has a potential antitumor and antiviral activities.

Keywords: hydroxy marilone C, production, bioactive compound, Streptomyces badius

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Authors: Natasha Mandal, Rakesh Singh Moirangthem

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The present work reports a theoretical model to develop a plasmonic biosensor for the detection of glucose concentrations in human blood and urine as the abnormality of glucose label is the major cause of diabetes which becomes a life-threatening disease worldwide. This study is based on the surface plasmon resonance (SPR) sensor applications which is a well-established, highly sensitive, label-free, rapid optical sensing tool. Here we have introduced a sandwich assay of two dielectric spacer layers of MgF2 and BaTiO3which gives better performance compared to commonly used SiO2 and TiO2 dielectric spacers due to their low dielectric loss and higher refractive index. The sensitivity of our proposed sensor was found as 3242 nm/RIU approximately, with an excellent linear response of 0.958, which is higher than the conventional single-layer Au SPR sensor. Further, the sensitivity enhancement is also optimized by coating a few layers of two-dimensional (2D) nanomaterials (e.g., Graphene, h-BN, MXene, MoS2, WS2, etc.) on the sensor chip. Hence, our proposed SPR sensor has the potential for the detection of glucose concentration in blood and urine with enhanced sensitivity and high affinity and could be utilized as a reliable platform for the optical biosensing application in the field of medical diagnosis.

Keywords: biosensor, surface plasmon resonance, dielectric spacer, 2D nanomaterials

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Authors: D. Tohlob, E. Abo Hashem, N. Ghareeb, M. Ghanem, R. Elfarahaty, S. A. Roberts, P. Pemberton, L. Mohiyiddeen, W. G. Newman

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Gonadotropin stimulation is used in females undergoing assisted reproductive treatment for ovulation induction, but ovarian response is variable and unpredictable in these women. More effective protocols and individualization of treatment are needed to increase the success rate of IVF/ICSI cycles. We genotyped seven variants reported in previous studies to be associated with ovarian response (number of ova retrieved and total gonadotropin dose) in women undergoing IVF treatment including FSHR variants Asn 680 Ser (c.2039 A > G), Thr 307 Ala (c. 919 > A), -29 G > A, HRG c.610 C > T gene, BMP15 -9 C > G, AMH Ile 49 Ser (c.146 G > T), and AMHR -489A˃G in 118 Egyptian females attending Mansoura Integrated Fertility Center in Egypt, these females were undergoing their first cycle of controlled ovarian hyper stimulation for IVF/ICSI treatment. They were analyzed by TaqMan allelic discrimination assay in Manchester Center of Genomic Medicine. We found no evidence of any significant difference (p value < 0.05) in the number of eggs retrieved or the gonadotropin dose used between individuals in all genotypes except for HRG c.610 C > T gene polymorphism where regression analysis gives a p value of 0.04 with a fewer eggs number in TT genotyped females. These results indicate that these variants do not provide sufficient clinically relevant data to individualize the treatment protocols.

Keywords: controlled ovarian hyperstimulation, gene variants, ovarian response, assisted reproduction

Procedia PDF Downloads 319

Authors: Fdhila Walid, Bayar Sihem, Khouidi Bochra, Maâtouk Fethi, Ben Amor Feten, Hajer Hentati, Mahdhi Abdelkarim

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The presence of resistant bacteria in the oral cavity can be the major cause of dental antibiotic prophylaxis failure. Multidrug efflux has been described for many organisms, including bacteria and fungi as part of their drugs resistance strategy. The potential use of probiotic bacteria can be considered as a new alternative in the prevention or cure of oral cavity diseases. In this study, different Bacillus strains isolated from the environment were isolated and characterized using biochemical and molecular procedures. The inhibitory activity against different pathogenic bacteria and yeast strains was tested using diffusion agar assay method. Our data revealed that the tested strains have an antimicrobial effect against the pathogenic strains such as Streptococcus mutants. The inhibitory effect was variable depending from the probiotic and pathogenic strains. The obtained result demonstrated that Bacillus can be used as a potential candidates probiotic and help in the prevention and treatment of oral infections, including dental caries, periodontal disease and halitosis. Our data, partly encourage the use of probiotic strains because they do not produce acid which can contribute to faster installation decay and these are spore-forming bacteria that can withstand the stress of the oral cavity (acids, alkalis, and salty foods).

Keywords: probiotic, pathogenic bacteria, yeast, oral cavity

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Authors: Atefeh Esmailnejad, Gholam Reza Nikbakht Brujeni

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The major histocompatibility complex (MHC) is the best-characterized genetic region associated with susceptibility and/or resistance to a wide range of infectious diseases, autoimmune diseases and immune responses to vaccines. It has been demonstrated that there is an association between the MHC and resistance to Marek disease, Newcastle disease, Rous sarcoma tumor, Avian leucosis, Fowl cholera, Salmonellosis and Pasteurellosis in chicken. The present study evaluated the MHC polymorphism and its association with antibody response to Newcastle (ND) vaccine in Iranian native chickens. The MHC polymorphism was investigated using LEI0258 microsatellite locus by PCR-based fragment analysis. LEI0258 microsatellite marker is a genetic indicator for MHC, which is located on microchromosome 16 and strongly associated with serologically defined MHC haplotypes. Antibody titer against ND vaccine was measured by Haemaglutination Inhibition (HI) assay. Statistical analysis was performed using SPSS software (version 21). Total of 13 LEI0258 microsatellite alleles were identified in 72 samples which indicated a high genetic diversity in the population. The association study revealed a significant influence of MHC alleles on immune responses to Newcastle vaccine. 311 and 313 bp alleles were significantly associated with elevated immune responses to Newcastle vaccine (p<0.05). These results would be applicable in designing and improving the populations under selective breeding.

Keywords: chicken, LEI0258, MHC, Newcastle vaccine

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Authors: Ibrahim Khalil Ibrahim, Enas Mohamed Shahine, Abeer Shawky El Hadedy, Emmanuel Kamal Aziz Saba, Ghada Salah Attia Hussein

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Osteoarthritis (OA) is a multifactorial disease characterized by a progressive degradation of articular cartilage and is the leading cause of disability in elderly persons. IL-18 contributes to the destruction of cartilage and bone in the disease process of arthritis. The aim of the study was to investigate the role of IL-18 in primary knee OA patients. Serum level of IL-18 was assessed by enzyme-linked immunosorbent assay in 30 primary knee OA patients and compared to 20 age and gender-matched healthy volunteers as a control group. Radiographic severity of OA was assessed by Kellgren and Lawrence (KL) global scale. Pain, stiffness and functional assessment were done using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). OA patients had significantly higher serum IL-18 level than in control group (420.93 ± 345.4 versus 151.03 ± 144.16 pg/ml, P=0.001). Serum level of IL-18 was positively correlated with KL global scale (P=0.001). There were no statistically significant correlations between serum level of IL-18 and pain, stiffness, function subscales and total WOMAC index scores among the studied patients. In conclusions, IL-18 has a role in the pathogenesis of OA and it is positively correlated with the radiographic damage of OA.

Keywords: Interlukin-18, knee osteoarthritis, primary osteoarthritis, WOMAC scale

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Authors: Alaa F. A. Bakr, Sherein S. Abdelgayed, Osama. S. EL-Tawil, Adel M. Bakeer

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Objective: This study was carried out to evaluate the cytotoxic and genotoxic effect of tartrazine in both male and female albino rats. Methodology: Forty adult male (20) and female (20) Sprague Dawley albino rats (120 - 150g) were obtained and distributed into four experimental groups; Group I; 10 untreated males, Group II; 10 untreated females, Group III; 10 treated males, and Group IV; 10 treated females. Body weight was recorded weekly, reduced glutathione (RGH), lipid peroxidation (SOD), and superoxide dismutase activity (MDA) in liver tissue were carried out, histopathological studies of brain, liver, and kidneys were performed, COMET assay was performed, all values were statistically analyzed. Results: Decrease in the activity of RGH and SOD in the treated groups were reported, but there was a more significant decrease in the female treated group. MDA was increased in treated groups with tartrazine, moreover, it was more significant in the female treated group. Multiple histological lesions were developed in brain, liver, and kidneys. COMET showed positive results. Conclusion: Our study concluded that Tartrazine has a cytotoxic and genotoxic effect on albino rats and it was more significant in females than males.

Keywords: tartrazine, cytotoxicity, genotoxicity, histopathology, albino rats

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Authors: Meriche Hacene, Gadiri Sabiha

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Introduction: Allergy to animal hair is hypersensitivity to animal appendages to look for in front of any rhinoconjunctivitis or asthma. An anamnesis associated with the prick-tests makes it possible to guide the diagnosis, which will be supplemented in case of doubt by specific immunoglobulin E (IgE) assays. The objective of our study is to study the characteristics of patients sensitized to animal hair. Patients and methods: Retrospective study conducted on 105 adult patients and 69 children over a period of 3 years, including patients who received a specific IgE assay (respiratory panel and pediatric panel) by immunodot method. Result: 105 adult patients, including 74 women and 31 men, with an average age of 41 years, of which 8.5% had sensitization to animal hair (5 men and 4 women), namely: cat (5%), horse (4.7%) and dog (3.8%). For the 69 children, a slight female predominance was noted (56%), with an average age of 7.5 years, of which (13%) are sensitized to animal hair (5 girls and 4 boys): cat (10%), while awareness of dog and horse hair was less frequent with an identical prevalence of (4.34%). The dominant symptoms are rhinorrhea and sneezing for both categories, respectively (40% and 26.6% in adults and 23% for both symptoms in children). Cross-sensitization was observed in the 2 series: 1 single cat-dog and cat-horse case and 2 dog-horse cases in adults. In children 100% of patients with sensitization to dog hair had cross-sensitization to cat hair, only 1 case was observed for cat-horse cross-reactivity. Conclusion: This work shows that allergy to animal hair is common. Studies on more representative samples are recommended.

Keywords: children, allegy to animals, specific Ig E, hypersensitivity

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Authors: Zakieh Rostamzadeh , Nariman Sepehrvand-Zahra Shirmohamadi

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Background: Cytomegalovirus (CMV) infection is one of the most common infectious problems following kidney transplantation. In this study, we are aimed to investigate the CMV infection in the setting of renal transplant recipients in Urmia-Iran, using both ELISA and polymerase chain reaction (PCR) methods. Methods: Ninety-six renal transplant recipients were selected randomly and enrolled in a cross-sectional study. Blood sampling was done via venipuncture, and all sera were investigated for anti-CMV IgM, and the seropositive cases in association with 14 randomly selected seronegative cases were investigated with PCR assay. Results: Thirty-three patients (34.3%) were seropositive for anti-CMV IgM, 3 patients (3.1%) were in borderline range, and 60 patients (62.5%) were seronegative. By considering the patients with borderline anti-CMV IgM levels as seropositive, 37.5% were seropositive for anti-CMV IgM. Among 36 seropositive cases, the CMV infection was confirmed in 19 (52.7%) of them using PCR. Age (P = 0.40), educational status (P = 0.77), history of pre-transplantation dialysis (0.52), history of blood transfusion (P = 0.52), and immunosuppressive regimen were not statistically different among recipients with positive versus negative CMV PCR study results. Conclusion: The seroprevalence of CMV infection was demonstrated to be high in renal transplant recipients of Urmia-Iran. The rate was higher compared to several previous reports in the literature. ELISA method has an appropriate sensitivity to screen the recipients for CMV infection but considering its relatively low specificity, the seropositive cases are better to be confirmed by further PCR study.

Keywords: cytomegalovirus, renal transplantation, ELISA, IgM, PCR

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Authors: Mekhaldi Abdelkader, Bouzned Ahcen, Djibaoui Rachid, Hamoum Hakim

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This study presents an attempt to evaluate the antioxidant and antimicrobial activity of methanolic extract and essential oils prepared from the leaves of sage (Salvia officinalis L.). The content of polyphenols in the methanolic extract of the leaves from Salvia officinalis extract was determined by spectrophoto- metrically, calculated as gallic acid and catechin equivalent. Antioxidant activity was evaluated by free radical scavenging activity using 2,2-diphenylpicryl-1-picrylhydrazyl (DPPH) assay. The plant essential oil and methanol extract were also subjected to screenings for the evaluation of their antioxidant activities using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) test. While the plant essential oil showed only weak antioxidant activities, its methanol extract was considerably active in DPPH (IC50= 37.29µg/ml) test. Appreciable total phenolic content (31.25mg/g) was also detected for the plant methanol extract as gallic acid equivalent in the Folin–Ciocalteu test. The plant was also screened for its antimicrobial activity and good to moderate inhibitions were recorded for its essential oil and methanol extract against most of the tested microorganisms. The present investigation revealed that this plant has rich source of antioxidant properties. It is for this reason that sage has found increasing application in food formulations.

Keywords: antibacterial activity, antioxidant activity, flavonoid, polyphenol, salvia officinalis

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Authors: Ayse Demirbas, Bruce A. Welt, Yavuz Yagiz

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Oxidation of polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish causes loss of product quality. Oxidative rancidity causes loss of nutritional value and undesirable color changes. Therefore, powerful antioxidant extracts may provide a relatively low cost and natural means to reduce oxidation, resulting in longer, higher quality and higher value shelf life of foods. In this study, we measured effects of red cabbage antioxidant on lipid oxidation in fresh tilapia filets using thiobarbituric acid reactive substances (TBARS) assay, peroxide value (PV) and color assesment analysis. Extraction of red cabbage was performed using an efficient microwave method. Fresh tilapia filets were dipped in or sprayed with solutions containing different concentrations of extract. Samples were stored for up to 9 days at 4°C and analyzed every other day for color and lipid oxidation. Results showed that treated samples had lower oxidation than controls. Lipid peroxide values on treated samples showed benefits through day-7. Only slight differences were observed between spraying and dipping methods. This work shows that red cabbage antioxidant extracts may represent an inexpensive and all natural method for reducing oxidative spoilage of fresh fish.

Keywords: antioxidant, shelf life, fish, red cabbage, lipid oxidation

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Authors: Kasi Viswanadh Matte, Abhisheh Kumar Mehata, M.S. Muthu

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Brest cancer, up-regulated with human epidermal growth factor receptor type-2 (HER-2) led to the concept of developing HER-2 targeted anticancer therapeutics. Docetaxel-loaded D-α-tocopherol polyethylene glycol succinate 1000 conjugated chitosan (TPGS-g-chitosan) nanoparticles were prepared with or without Trastuzumab decoration. The particle size and entrapment efficiency of conventional, non-targeted and targeted nanoparticles were found to be in the range of 126-186 nm and 74-78% respectively. In-vitro, MDA-MB-231 cells showed that docetaxel-loaded non-targeted and HER-2 receptor targeted TPGS-g-chitosan nanoparticles have enhanced the cellular uptake and cytotoxicity with a promising bioadhesion property, in comparison to conventional nanoparticles. The IC50 values of non-targeted and targeted nanoparticles from cytotoxic assay were found to be 43 and 223 folds higher than DocelTM. The in-vivo pharmacokinetic study showed 2.33, and 2.82-fold enhancement in relative bioavailability of docetaxel for non-targeted and HER-2 receptor targeted nanoparticles, respectively than DocelTM, and after i.v administration, non-targeted and targeted nanoparticle achieved 3.48 and 5.94 times prolonged half-life in comparison to DocelTM. The area under the curve (AUC), relative bioavailability (FR) and mean residence time (MRT) were found to be higher for non-targeted and targeted nanoparticles compared to DocelTM. Further, histopathology results of non-targeted and targeted nanoparticles showed less toxicity on vital organs such as lungs, liver, and kidney compared to DocelTM.

Keywords: breast cancer, HER-2 receptor, targeted nanomedicine, chitosan, TPGS

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Authors: Randa M. Mostafa, Y. Moustafa

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During our daily life, we are continuously exposed to the extremely low frequency electromagnetic fields (ELF-EMFs) generated by electric appliances. The possible relation between exposure to (ELF-MFs) and adverse health effects has attracted and passed through long debate sessions. Extremely low frequency is a term used to describe radiation frequencies below 300 Hertz (Hz).It is very important for public health because of the widespread use of electrical power at 50-60 Hz in most countries. This study set out to investigate the impact of chronic exposure of male rats to 50- Hz, 1 mTesla (ELF-EMF) of over periods of 1, 2, and 4 weeks on concentration of serum FSH, LH, and testosterone hormones. 60 male albino rats were divided into 6 groups and were continuously exposed to 50-Hz, 1 m Tesla (ELF-EMF) generated by magnetic field chamber for periods of 1, 2, and 4 weeks. For each experimental point, sham treated group was used as a control. Assay of serum testosterone LH, and FSHwere performed. Serum testosterone showed no significant changes. FSH showed significant increase than sham exposed group after 1 week of field exposure. LH showed significant increase than sham exposed group only after 4 weeks of field exposure. A future detailed molecular studies must be carried out to figure out and may be able to explain the possible interactions between ELF-EMF and hypothalamic-pituitary gonadal axis.

Keywords: extremely low frequency electromagnetic fields, testosterone, follicular stimulating hormone, LH

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Authors: Nicole Nazario Bayon, Prathima Prabhu Tumkur, Nithin Krisshna Gunasekaran, Krishnan Prabhakaran, Joseph C. Hall, Govindarajan T. Ramesh

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Titanium nitride (TiN) nanoparticles have sparked interest over the past decade due to their characteristics such as thermal stability, extreme hardness, low production cost, and similar optical properties to gold. In this study, TiN nanoparticles were synthesized via a thermal benzene route to obtain a black powder of nanoparticles. The final product was drop cast onto conductive carbon tape and sputter coated with gold/palladium at a thickness of 4 nm for characterization by field emission scanning electron microscopy (FE-SEM) with energy dispersive X-Ray spectroscopy (EDX) that revealed they were spherical. ImageJ software determined the average size of the TiN nanoparticles was 79 nm in diameter. EDX revealed the elements present in the sample and showed no impurities. Further characterization by X-ray diffraction (XRD) revealed characteristic peaks of cubic phase titanium nitride, and crystallite size was calculated to be 14 nm using the Debye-Scherrer method. Dynamic light scattering (DLS) analysis revealed the size and size distribution of the TiN nanoparticles, with average size being 154 nm. Zeta potential concluded the surface of the TiN nanoparticles is negatively charged. Biocompatibility studies using MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay showed TiN nanoparticles are not cytotoxic at low concentrations (2, 5, 10, 25, 50, 75 mcg/well), and cell viability began to decrease at a concentration of 100 mcg/well.

Keywords: biocompatibility, characterization, cytotoxicity, nanoparticles, synthesis, titanium nitride

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Authors: I. Sani, F. Bello, I. M. Fakai, A. Abdulhamid

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Abelmoschus esculentus L. fruit is very common especially in northern part of Nigeria, but people are ignorant of its medicinal and pharmacological benefits. Preliminary phytochemical screening, antioxidant potential and mineral composition of the dried form of this fruit were determined. The Phytochemical screening was conducted using standard methods. Antioxidant potential screening was carried out using Ferric Reducing Antioxidant Power Assay (FRAP) method, while, the mineral compositions were analyzed using an atomic absorption spectrophotometer by wet digest method. The result of the qualitative phytochemical screening revealed that the fruits contain saponins, flavonoids, tannins, steroids, and terpenoids, while, anthraquinone, alkaloids, phenols, glycosides, and phlobatannins were not detected. The quantitative analysis revealed that the fruits contain saponnins (380 ± 0.020 mg/g), flavonoids (240±0.01 mg/g), and tannins (21.71 ± 0.66 mg/ml). The antioxidant potential was determined to be 54.1 ± 0.19%. The mineral composition revealed that 100 g of the fruits contains 97.52 ± 1.04 mg of magnesium (Mg), 94.53 ± 3.21 mg of calcium (Ca), 77.10 ± 0.79 mg of iron (Fe), 47.14 ± 0.41 mg of zinc (Zn), 43.96 ± 1.49 mg of potassium (K), 42.02 ± 1.09 mg of sodium (Na), 0.47 ± 0.08 mg of copper (Cu) and 0.10 ± 0.02 mg of lead (Pb). These results showed that the Abelmoschus esculentus L. fruit is a good source of antioxidants, and contains an appreciable amount of phytochemicals, therefore, it has some pharmacological attributes. On the other side, the fruit can serve as a nutritional supplement for Mg, Ca, Fe, Zn, K, and Na, but a poor source of Cu, and contains no significant amount of Pb.

Keywords: Abelmoschus esculentus Fruits, antioxidant potential, mineral composition, phytochemical screening

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Authors: Dina Fatmawati, Sofia Mubarika, Mae Sri Wahyuningsih

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Chemotherapy for breast cancer is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous study, the combination of Doxorubicin (Dox) and ethanolic extract of Dioscorea esculenta tuber ((EED) was found to have a synergistic effect on T47D human breast cancer cell line. In this study, we investigated the apoptotic effect of the combination on T47D human breast cancer cells and normal fibroblasts cell line and its effects on the expression of Caspase-3 and cleaved poly (ADP-Ribose) Polymerase-1 (cPARP-1) protein. T47D cell lines and fibroblasts cells were treated with the combination of Dox and EED. Apoptotic effect of the combination was determined using flow cytrometry assay. Protein expressions were determined by immunocytochemistry staining. The percentage of apoptotic cells were significantly higher in T47D cell lines (75%) than that of in fibroblast cells (23%). The expression of Caspase 3 (84.53%) and cPARP-1 (83.36%) were significantly higher in the cancer cell lines than those of normal cells. These results indicate that the combination of doxorubicin and Dioscorea esculenta is a promising candidate for the treatment of breast cancer cells.

Keywords: Dioscorea esculenta, Doxorubicin, apoptosis, immunocytochemistry, cancer cells

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Authors: Jae Young Jang, Jong Hoon Ahn, Jae-Woong Lim, So Young Kang, Mi Kyeong Lee

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Rhus verniciflua is commonly known as a lacquer tree in Korea. Stem barks of R. verniciflua have been used as an immunostimulator in traditional medicine. It contains phenolic compounds and is known for diverse biological activities such as antioxidant and antimicrobial activity. However, it also causes allergic dermatitis due to urushiols derivatives. For the development of active natural resources with less toxicity, the content of phenolic compounds and urushiols of different parts of R. verniciflua such as stem barks, lignum and leaves were quantitated by colorimetric assay and HPLC analysis. The urushiols content were the highest in stem barks, and followed by leaves. The lignum contained trace amount of urushiols. The phenolic contents, however, were the most abundant in lignum, and followed by leaves and stem barks. These results clear showed that the content of urushiols and phenolic differs depending on the parts of R. verniciflua. Antimicrobial activity of different parts of R. verniciflua against fish pathogenic bacteria was also investigated using Edwardsiella tarda. Lignum of R. verniciflua was the most effective in antimicrobial activity against E. tarda and phenolic constituents are suggested to be active constituents for activity. Taken together, phenolic compounds are responsible for antimicrobial activity of R. verniciflua. The lignum of R. verniciflua contains high content of phenolic compounds with less urushiols, which suggests efficient antimicrobial activity with less toxicity. Therefore, lignum of R. verniciflua are suggested as good sources for antimicrobial activity against fish bacterial diseases.

Keywords: different parts, phenolic compounds, Rhus verniciflua, urushiols

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Authors: Nina Tsvetkova, Rumen Harizanov, Aleksandra Ivanova, Iskra Rainova, Nina Yancheva-Petrova, Dimitar Strashimirov, Raina Enikova, Mihaela Videnova, Eleonora Kaneva, Iskren Kaftandjiev, Viktoria Levterova, Ivan Simeonovski, Nikolay Yanev, Georgi Hinkov

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The Pneumocystis jirovecii (P. jirovecii) and protozoan of the genera Acanthamoeba, Cryptosporidium, and Toxoplasma gondii are opportunistic pathogens that can cause life-threatening infections in immunocompromised patients. Aim of the study was to evaluate the coinfection rate with opportunistic protozoal agents among Bulgarian patients diagnosed with P. jirovecii pneumonia. Thirty-eight pulmonary samples were collected from 38 patients (28 HIV-infected) with P. jirovecii infection. P. jirovecii DNA was detected by real-time PCR targeting the large mitochondrial subunit ribosomal RNA gene. Acanthamoeba was determined by genus-specific conventional PCR assay. Real-time PCR for the detection of a Toxoplasma gondii and Cryptosporidium DNA fragment was used. Pneumocystis DNA was detected in all 38 specimens; 28 (73.7%) were from HIV-infected patients. Three (10,7%) of them were co-infected with T. gondii and 1 (3.6%) with Cryptosporidium. In the group of non-HIV-infected (n=10), Cryptosporidium DNA was detected in an infant (10%). Acanthamoeba DNA was not found in the tested samples. The current study showed a relatively low rate of coinfections of Cryptosporidium spp./T. gondii and P. jirovecii in the Bulgarian patients studied.

Keywords: coinfection, opportunistic protozoal agents, Pneumocystis jirovecii, pulmonary infections

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Authors: Mona Marzouk, Nesrine Hegazi, Aliaa Ragheb, Mona El Shabrawy, Salwa Kawashty

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The whole plant of Matthiola longipetala (Brassicaceae) was extracted by 70% methanol to give the total aqueous methanol extract (AME), which was defatted by hexane yielded hexane extract (HE) and defatted AME (DAME). HE was analyzed through GC/MS assay and revealed the detection of 28 non-polar compounds. In addition, the chemical investigation of DAME led to the isolation and purification of twelve flavonoids and three chlorogenic acids. Their structures were interpreted through chemical (complete and partial acid hydrolysis) and spectroscopic analysis (MS, UV, 1D and 2D NMR). Among them, nine compounds have been isolated for the first time from M. longipetala. Moreover, LC-ESI-MS analysis of DAME was achieved to detect additional 46 metabolites, including phospholipids, organic acids, phenolic acids and flavonoids. The biological activity of AME, HE and DAME against alloxan inducing oxidative stress and diabetes in male rats was investigated. Diabetes was induced using a single dose of Alloxan (150 mg/kg b.wt.). HE and DAME significantly increased serum GSH content in rats (37.3±0.7 and 35.9±0.6 mmol/l) compared to diabetic rats (21.8±0.3) and vitamin E (36.2±1.1) at P<0.01. Also, HE, DAME and AME revealed a significant acute anti-hyperglycemic effect potentiated after four weeks of treatment with blood glucose levels of 96.2±5.4, 98.7±6.1 and 98.9±8.6 mg/dl, respectively, compared to diabetic rats (263.4±7.8) and metaformin group (81.9±2.4) at P<0.01.

Keywords: Brassicaceae, Flavonoid, LCMS/MS, Matthiola

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Authors: Srisopa Ruangnoo, Arunporn Itharat

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The ethanolic and aqueous extracts of Parkia javanica Merr. germinating seeds and Parkia speciosa Hassk. seeds were evaluated for cytotoxic activity against three different types of human cancer cell lines including colon cancer (LS174T), breast cancer (MCF-7) and prostate cancer (PC3) using sulforhodamine B (SRB) assay. The fresh plant parts were divided into 2 parts. The first part was extracted by maceration with 95% ethanol for 3 days and then filtered, and the filtrates were evaporated by rotary evaporator. The other part was squeezed and filtered. Then the filtrates were dried by freeze dryer. The screening found that the aqueous extract of P. javanica Merr. germinating seeds exhibited more than 70% inhibition (at concentration 50 µg/ml) against all types of human cancer cells. The aqueous extract of P. javanica Merr. germinating seeds showed the highest cytotoxic activity against MCF-7 with the IC50 value as 5.63 µg/ml. The aqueous extract of P. javanica Merr. germinating seeds also showed high cytotoxic activity against PC3 and LS174T with the IC50 values as 10.79 and 11.40 µg/ml, respectively. In conclusion, P. javanica Merr. germinating seed is a natural source of anticancer activity and further research to isolate active compounds from this plant should be undertaken.

Keywords: cytotoxic activity, Parkia javanica Merr., Parkia speciosa Hassk., human cancer cell lines

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Authors: Gizem Buldum, Alexander Bismarck, Athanasios Mantalaris

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Cellulose is the most abundant biopolymer on earth and it is currently being utilised in a multitude of industrial applications. Over the last 30 years, attention has been paid to the bacterial cellulose (BC), since BC exhibits unique physical, chemical and mechanical properties when compared to plant-based cellulose, including high purity and biocompatibility. Although Acetobacter xylinum is the most efficient producer of BC, it’s long doubling time results in insufficient yields of the cellulose production. This limits widespread and continued use of BC. In this study, E. coli BL21 (DE3) or E. coli HMS cells are selected as host organisms for the expression of bacterial cellulose synthase operon (bcs) of A.xylinum. The expression system is created based on pET-Duet1 and pCDF plasmid vectors, which carry bcs operon. The results showed that all bcs genes were successfully transferred and expressed in E.coli strains. The expressions of bcs proteins were shown by SDS and Native page analyses. The functionality of the bcs operon was proved by congo red binding assay. The effect of culturing temperature and the inducer concentration (IPTG) on cell growth and plasmid stability were monitored. The percentage of plasmid harboring cells induced with 0.025 mM IPTG was obtained as 85% at 22˚C in the end of 10-hr culturing period. It was confirmed that the high output cellulose production machinery of A.xylinum can be transferred into other organisms.

Keywords: bacterial cellulose, biopolymer, recombinant expression system, production

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Authors: S. Lekhavat, T. Kajsongkram, S. Sang-han

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Dried ginger was extracted and investigated the effect of ethanol concentration and enzyme pre-treatment on its bioactive compounds in solvent extraction process. Sliced fresh gingers were dried by oven dryer at 70 °C for 24 hours and ground to powder using grinder which their size were controlled by passing through a 20-mesh sieve. In enzyme pre-treatment process, ginger powder was sprayed with 1 % (w/w) cellulase and then was incubated at 45 °C for 2 hours following by extraction process using ethanol at concentration of 0, 20, 40, 60 and 80 % (v/v), respectively. The ratio of ginger powder and ethanol are 1:9 and extracting conditions were controlled at 80 °C for 2 hours. Bioactive compounds extracted from ginger, either enzyme-treated or non enzyme-treated samples, such as total phenolic content (TPC), 6-Gingerol (6 G), 6-Shogaols (6 S) and antioxidant activity (IC50 using DPPH assay), were examined. Regardless of enzyme treatment, the results showed that 60 % ethanol provided the highest TPC (20.36 GAE mg /g. dried ginger), 6G (0.77%), 6S (0.036 %) and the lowest IC50 (625 μg/ml) compared to other ratios of ethanol. Considering the effect of enzyme on bioactive compounds and antioxidant activity, it was found that enzyme-treated sample has more 6G (0.17-0.77 %) and 6S (0.020-0.036 %) than non enzyme-treated samples (0.13-0.77 % 6G, 0.015-0.036 % 6S). However, the results showed that non enzyme-treated extracts provided higher TPC (6.76-20.36 GAE mg /g. dried ginger) and Lowest IC50 (625-1494 μg/ml ) than enzyme-treated extracts (TPC 5.36-17.50 GAE mg /g. dried ginger, IC50 793-2146 μg/ml).

Keywords: antioxidant activity, enzyme, extraction, ginger

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Authors: Yen-Ni Teng, Hsing-Yi Chen, Hsien-An Pan, Yung-Ming Lin, Hany A. Omar, Jui-Hsiang Hung

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Leucine-rich repeats and WD repeat domain containing 1 (LRWD1) is highly expressed in the testes of healthy males. On the other hand, LRWD1 is significantly down-regulated in the testicular tissues of patients with severe spermatogenic defects. In our study, the downregulation of LRWD1 expression by shRNA caused a significant reduction of cell growth and mitosis and a noteworthy increase in the cell microtubule atrophy rate. Here, we used EMBOSS CpG plot analysis to explore the promoter region of LRWD1 gene. We found that CpG islands are located between positions -253 to +5 nucleotides upstream from the LRWD1 transcription start site. Luciferase reporter assay revealed that the hypermethylation of the LRWD1 promoter reduced the transcription activity in cells. In addition, quantitative methylation-specific PCR and immunostaining showed that the methylation inhibitor, 5-Aza-2'-deoxycytidine, increased LRWD1 promoter activity, LRWD1 mRNA, protein expression and cell viability. Whereas, the methylation activator, S-adenosylmethionine, caused opposite effects. The overexpression of p53 and Nrf2 in NT2/D1 cells increased LRWD1 promoter activity while 5-fluorodeoxyuridine decreased it. In conclusion, this study highlights evidence that the methylation status of LRWD1 promoter is associated with LRWD1 expression. Since the expression level of LRWD1 plays an important role in spermatogenesis, the methylation status of LRWD1 may serve as a novel molecular diagnostic or therapeutic approach in male's infertility.

Keywords: LRWD1, DNA methylation, p53, Nrf2

Procedia PDF Downloads 148

Authors: So Youn Park, Yi Sle Lee, Ki Whan Hong, Chi Dae Kim

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The role of metabolism in the pathogenesis of osteoarthritis is an emerging field. Metabolic alterations may be a role in osteoarthritis (OA) pathogenesis, and these changes influence joint destruction via several cytokine. Especially, in OA patients, levels of IL-1β are elevated in the synovial fluid, synovial membrane, subchondral bone, and cartilage. The IL-1β is activated by NLRP3 inflammasomes, and NLRP3 inflammasomes are cytosolic complexes that drive the production of other inflammatory cytokines, including IL-1β. In this study, we examined that SIRT1 suppresses IL-1β through inhibiting NLRP3 inflammasomes and SIRT1 ameliorates osteoarthritis. OA fibroblasts were isolated from synovium of OA patients. IL-1β and NLRP3 were detected in synovium of OA patients by immunohistochemistry. Lipopolysaccharides (LPS) stimulated the expression of active IL-1β mRNA in OA fibroblasts and combination of LPS, and adenosine triphosphate increased more the expression of active IL-1β in OA fibroblasts. The level of IL-1β was measured by western blot and ELISA assay. NLRP3 inflammasomes complex were measured by western blot. SIRT1 did not inhibit expression of NLRP3 inflammasome. So caspase-1, apoptotic speck-like protein containing a caspase recruitment domain (ASC) and NLRP3 protein were expressed in OA fibroblasts. But SIRT1 suppressed activation of IL-1β by inhibiting activity of caspase-1 via NLRP3 inflammasome in OA fibroblasts under LPS plus ATP stimulation. These results suggest that SIRT1 is a modulator of NLRP3 inflammasomes in OA fibroblasts and ameliorate IL-1β, so expression of SIRT1 in OA fibroblast may be a potential strategy for OA inflammation treatment.

Keywords: osteoarthritis, inflammasome, SIRT1, IL-1beta

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Authors: Nematollah Gheibi, Nader Divan Khosroshahi, Mahdi Mohammadi Ghanbarlou

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Introduction: Nowadays, the phenomenon of bacterial resistance is one of the most important challenge of the health community in the world. Propolis is most important production of bee colonies that collected from of various plants. So far, a lot of investigations carried out about its antibacterial effects. Material and methods: Thirty gram of propolis prepared as ethanolic extract and after different process of purification, 7.5 gr of its pure form were obtained. Propolis compounds identification was performed by TLC and VLC methods. The HPLC spectrum obtaining from propolis ethanolic extract was compared with some purified standard phenolic and flavonoid substances. Antibacterial effects of ethanol extract of purified propolis were evaluated on two strains of Staphylococcus aureus and Pseudomonas aeruginosa and their MIC was determined by the microdillution assay. Results: Ethanolic propolis extraction analyzed by TLC were resulted to confirm several phenolic and flavonoid compounds in this extract and some of the confirmed by HPLC technique. Minimum inhibitory concentration (MIC) for standard Staphylococcus aureus (ATCC25923) and Pseudomonas aeruginosa (ATCC27853) strains were obtained 2.5 mg/ml and 50 mg/ml respectively. Conclusion: Bee Propolis is a mix organic compound that has a lot of beneficial effects such as anti-bacterial that emphasized in this investigation. It is proposed as a rich source of natural phenolic and flavonoids compounds in designing of new biological resources for hygienic and medical applications.

Keywords: propolis, Staphylococcus aureus, Pseudomonas aeruginosa, antibacterial

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Authors: Dana A. Thal, Heike Kahlert, Fritz Scholz

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Thiol molecules are known to easily form self-assembled monolayers (SAM) on Au surfaces. Depending on the thiol’s structure, surface modifications via SAM can be used for electrode sensor development. In the presented work, 1-decanethiol coated polycrystalline Au electrodes were applied to indirectly assess the radical scavenging potential of plant compounds and extracts. Different plant compounds with reported antioxidant properties as well as an extract from the plant Gynostemma pentaphyllum were tested for their effectiveness to prevent SAM degradation on the sensor electrodes via photolytically generated radicals in aqueous media. The SAM degradation was monitored over time by differential pulse voltammetry (DPV) measurements. The results were compared to established antioxidant assays. The obtained data showed an exposure time and concentration dependent degradation process of the SAM at the electrode’s surfaces. The tested substances differed in their capacity to prevent SAM degradation. Calculated radical scavenging activities of the tested plant compounds were different for different assays. The presented method poses a simple system for radical scavenging evaluation and, considering the importance of the test system in antioxidant activity evaluation, might be taken as a bridging tool between in-vivo and in-vitro antioxidant assay in order to obtain more biologically relevant results in antioxidant research.

Keywords: alkanethiol SAM, plant antioxidant, polycrystalline Au, radical scavenger

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Authors: Abimbola M. Enitan, John O. Odiyo, Feroz M. Swalaha

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The study of microbial ecology and their function in anaerobic digestion processes are essential to control the biological processes. This is to know the symbiotic relationship between the microorganisms that are involved in the conversion of complex organic matter in the industrial wastewater to simple molecules. In this study, diversity and quantity of bacterial community in the granular sludge taken from the different compartments of a full-scale upflow anaerobic sludge blanket (UASB) reactor treating brewery wastewater was investigated using polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR). The phylogenetic analysis showed three major eubacteria phyla that belong to Proteobacteria, Firmicutes and Chloroflexi in the full-scale UASB reactor, with different groups populating different compartment. The result of qPCR assay showed high amount of eubacteria with increase in concentration along the reactor’s compartment. This study extends our understanding on the diverse, topological distribution and shifts in concentration of microbial communities in the different compartments of a full-scale UASB reactor treating brewery wastewater. The colonization and the trophic interactions among these microbial populations in reducing and transforming complex organic matter within the UASB reactors were established.

Keywords: bacteria, brewery wastewater, real-time quantitative PCR, UASB reactor

Procedia PDF Downloads 259

Authors: Ana M. Guzman, Claudia M. Rodriguez, Pedro F. B. Brandao, Elianna Castillo

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Cadmium (Cd) is a carcinogenic metal to which humans are exposed mainly due to its presence in the food chain. Lactic acid bacteria have the capability to bind cadmium and thus the potential to be used as probiotics to treat this metal toxicity in the human body. The main objective of this study is to evaluate the potential of native lactic acid bacteria, isolated from Colombian fermented cocoa, to remove cadmium from aqueous solutions. An initial screening was made with the Lactobacillus plantarum JCM 1055 type strain, and Cd was quantified by atomic absorption spectroscopy (AAS). Lb. plantarum JCM 1055 was grown in ½ MRS medium to follow growth kinetics during 32 h at 37 °C, by measuring optical density at 600 nm. Washed cells, grown for 18 h, were adjusted to obtain dry biomass concentrations of 1.5 g/L and 0.5 g/L for removal assays in 10 mL of Cd(NO₃)₂ solution with final concentrations of 10 mg/Kg or 1.0 mg/Kg. The assays were performed at two different pH values (2.0 and 5.0), and results showed better adsorption abilities at higher pH. After incubation for 1 h at 37 °C and 150 rpm, the removal percentages for 10 mg/Kg Cd with 1.5 g/L and 0.5 g/L biomass concentration at pH 5.0 were, respectively, 71% and 50%, while the efficiency was 9.15 and 4.52 mg Cd/g dry biomass, respectively. For the assay with 1.0 mg/Kg Cd at pH 5.0, the removal was 100% and 98%, respectively for the same biomass concentrations, and the efficiency was 1.63 and 0.56 mg Cd/g dry biomass, respectively. These results suggest the efficiency of Lactobacillus strains to remove cadmium and their potential to be used as probiotics to treat cadmium toxicity and reduce its accumulation in the human body.

Keywords: cadmium removal, fermented cocoa, lactic acid bacteria, probiotics

Procedia PDF Downloads 171