International Journal of Biotechnology and Recent Advances

ISSN: 2639-4529

International Biotechnology and Research Conference

April 25-27, 2018, Rome, Italy
Poster Session Abstracts
DOI: 10.18689/2639-4529.a1.003

Native Cell Membrane Nanoparticles System for Membrane Proteins

Weihua Qiu1,2*, Ziao Fu3, Guoyan G. Xu1, Robert A. Grassucci4, Yan Zhang1, Joachim Frank4,5, Wayne A. Hendrickson4,6,7 and Youzhong Guo1,2

1Department of Medicinal Chemistry, Virginia Commonwealth University, USA
2Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, USA
3Integrated Program in Cellular, Molecular, and Biomedical Studies, Columbia University, USA
4Department of Biochemistry and Molecular Biophysics, Columbia University, USA
5Department of Biological Sciences, Columbia University, USA
6Department of Physiology and Cellular Biophysics, Columbia University, USA
7New York Structural Biology Center, USA

We devised a native cell membrane nanoparticles system, which we applied in a single-particle cryo-EM study of the multidrug exporter AcrB. Lipid-AcrB nanoparticles were prepared directly from membranes without any use of detergents. A 3D reconstruction in C1 symmetry achieved a final density map at 3.2 Å resolution, an atomic model of quasi-C3-symmetric AcrB was fitted to this map, and the residual density revealed many ordered lipid molecules. Most remarkably, a central cavity between the three transmembrane domains contains a 24-lipid patch of well-ordered bilayer structure. Inner leaflet lipid chains pack in a hexagonal array like that in phosphatidylethanolamine crystal structures, whereas the outer leaflet has highly irregular packing. Protein side chains interact with both leaflets and participate in the hexagonal pattern. The AcrB export mechanism requires reorganization of the lipid bilayer structure. This system should be broadly applicable for membrane protein structural biology and structure-based drug discovery and development.

Biography:
Weihua Qiu received B.S. degree in biotechnology from Henan Normal University, China in 1999 and Ph.D in biological sciences from the University of Texas at Austin, USA in 2010. She then joined in Rutgers University and Columbia University for postdoctoral training with Drs. Aaron Shatkin and Susan Steinberg. Since 2016, she worked with Dr. YouzhongGuo in Virginia Commonwealth University. Her current research focus on membrane protein structural biology as a lab manager and postdoctoral scientist.

Microbial Production of Natural 2-Phenylethanol

Chreptowicz Karolina* and Mierzejewska Jolanta

Warsaw University of Technology, Faculty of Chemistry, Poland

Thanks to its pleasant rose flavor as well as antibacterial and antifungal properties, 2-phenylethanol (2-PE) has huge market demand. After vanillin, it is the second-most-used additive in perfumery, while also being a component in the food and pharmaceutical industries. Although nowadays most 2-PE originates from chemical synthesis, biotechnological production with yeast is becoming increasingly more attractive since it gives a final product classified as natural.

The presented work is focused on microbial production of natural 2-PE. The first stage of the study was the search for yeast strains, isolated from the natural environment, that will efficiently produce 2-PE. With the best producer, we have developed a complete technology of 2-PE production - starting from the biotransformation stage and ending with pure product. In a batch culture conducted in a 5-l bioreactor, we obtained 3.6 g/l 2-PE after 72 h. As 2-PE titer of 2-4 g/l in broth is toxic for yeast, we achieved maximum concentration in a simple batch-culture. Therefore, to enhance productivity we tested extractive fermentation as one of several in situ product removal (ISPR) techniques. We showed for the first time that rapeseed oil can be successfully applied for this purpose. In addition, it is also an excellent biomaterial with promising use in the food or cosmetic industries. Recently, our work has focused on reducing the production costs and, to this end, we tested organic waste from agriculture and food processing as cheap feedstock. This approach has two advantages: lowering the price of the culture medium and better management of harmful wastes. Currently, we are working on downstream processing to combine all stages in one complete technology.

This work was financially supported by the National Science Centre, Poland (2016/21/D/NZ9/01605).

Biography:
Karolina Chreptowicz is currently a Ph.D. candidate at the Warsaw University of Technology. She earned her Bachelor and then Master degree in Industrial Biotechnology at the Warsaw University of Technology, Faculty of Chemistry, Poland. Since 2013, she has been working with Dr. Jolanta Mierzejewska in the field of yeast biotechnology. At present, in her Ph.D. thesis, she is involved in the development of a laboratory-scale technology for the production of natural 2-phenylethanol - starting from the biotransformation stage by separating and purifying the final product.

Determination and Quantification of Heavy Metals using Infrared Spectroscopy and Chemometric Techniques

R.J. Delgado Macuil*, A.C. Benítez Rojas, M. Rojas Lopez and O. Zaca Moran

Centro de Investigación en Biotecnología Aplicada, Instituto Politécnico Nacional, México

Actually the determination and quantification of heavy metals in food has a high priority for public health, quality systems and food safety, these elements can be added to the food production system at different stages of the agri-food chain and it is essential to monitor his presence or absence through it. Until now, the methodology to perform this task is based on atomic absorption spectrophotometry that requires at least a couple of days to obtain a reliable result. The aimed of this work is develop and validate an alternative analytical method for the determination of heavy metals in several matrices, based on numerical methods and the correlation between spectrometric techniques; Atomic Force and Infrared by Fourier transform.

The presence of Hg and Pb at different concentrations seem to modify the milk spectrum in the region of proteins, lipids and OH; at 1636, 1337, 550, 3300 and 3350 cm-1. The presence of As, Cd, Cr Hg and Pb at concentrations even of 0.1 ppb generate characteristic IR spectra that can help to identify their presence in water. By Principal Components Analysis (PCA), was possible have a better discrimination of the samples in the same matrix (milk). Where the cloud of points in the different regions of interest, show a good discrimination for the seven metals used in this work.

Biography:
Raul Delgado Macuil born in Puebla Mexico. He received B.S. degree in electronics from BUAP by Physical-Mathematics School in 1994; and he received the PhD in Optics from Astrophysical Optics y Electronics National Institute in 2005; both institutions in Mexico. Head research in the nanobiotechnology and biosensors fields in the Applied Biotechnology Research Center, a National Politechnique Institute center in Mexico. As author or co-author, about 70 scientific and professional papers have been published. He is a head of 21 national projects and more than 25 graduate and postgraduate thesis has been directed, have more than three thousand nationals and international congress participations.

Solid-phase Enzyme Catalysis of DNA end Repair Avoids Heat Treatment and Reduces GC-bias in Next-Generation Sequencing of Human Genomic DNA

Aihua Zhang*, Shaohua Li, Lynne Apone, Xiaoli Sun, Lixin Chen, Laurence M. Ettwiller, Bradley W. Langhorst, ChristopherJ. Noren and Ming-QunXu

New England Biolabs, USA

Next-generation sequencing (NGS) has caused a revolution in both research and diagnosis. NGS analysis relies on preparation of a representative, non-biased library evenly distributed across the entire genome under investigation. This critical task has become increasingly challenging since biases found in the current methods of the NGS library preparation produce uneven coverage and compromisethe quality of NGS analysis. In this report we have identified a systematic sequence bias during construction of amplification-free human DNA libraries for the Illumina sequencing platform. Our study indicates that inefficient processing of AT-rich DNA in the major steps that comprise library construction results in under-representation of the extremely high AT-content fraction in human genomic libraries. We have demonstrated a new strategy by employing DNA modifying enzymes conjugated to magnetic beads in construction of amplification-free human DNA libraries. We show that this method significantly lowers the sequence coverage bias of the libraries on Illumina sequencing platform.

Biography:
Dr. Aihua Zhang graduated from Hunan medical college, Hunan, China in 1984 and did her postdoctoral training at MGH, Harvard U.S.A. She has been working as a research scientist at New England Biolabs, Inc. since 1995. Her current research interest is validating immobilized enzymes for NGS library prep.

The Mechanism of Action of Zingerone in the Pacemaker Potentials of Interstitial Cells of Cajal in Murine Small Intestine

Byung Joo Kim1,2*, Iksung Kim1,2, Hyun Jung Kim1,2 and Jung Nam Kim1,2

1Division of Longevity and Biofunctional Medicine, Pusan National University School of Korean Medicine, Republic of Korea
2Healthy Aging Korean Medical Research Center, Pusan National University School of Korean Medicine, Republic of Korea

Background: Zingerone, a major component found in ginger root, is clinically effective for the treatment of various diseases. Interstitial cells of Cajal (ICCs) are the pacemaker cells responsible for slow waves in the gastrointestinal (GI) tract. We investigated the effects of zingerone on the pacemaker potentials of ICCs to assess its mechanisms of action and its potential as a treatment for GI tract motility disorder.

Methods: We isolated ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs.

Results: Under the current clamping mode, zingerone inhibited pacemaker potentials of ICCs concentration-dependently. These effects were blocked not by capsazepine, a transient receptor potential vanilloid 1 (TRPV1) channel blocker, but by glibenclamide, a specific ATP-sensitive K+channel blocker. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor), LY294002 (a phosphoinositide 3-kinase inhibitor), and calphostin C (a protein kinase C (PKC) inhibitor) did not block the effects of zingerone on the pacemaker potentials relative to treatment with zingerone alone. However, zingerone-induced pacemaker potential inhibition was blocked by 1H[1,2,4] oxadiazolo [4,3a] quinoxalin1one (ODQ; a guanylate cyclase inhibitor), KT5823 (a protein kinase G (PKG) inhibitor), and L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor). In addition, zingerone stimulated cyclic guanosine monophosphate (cGMP) production in ICCs. Finally, pretreatment with PD98059 (a p42/44 mitogen-activated protein kinase (MAPK) inhibitor), SB203580 (a p38 MAPK inhibitor), and SP600125 (cJun Nterminal kinases (JNK)specific inhibitor) blocked the zingerone-induced pacemaker potential inhibition.

Conclusion: These results suggest that zingerone concentration-dependently inhibits pacemaker potentials of ICCs via NO/cGMP-dependent ATP-sensitive K+channels through MAPK-dependent pathways. Taken together, this study shows that zingerone may have the potential for development as a GI regulation agent.

Biography:
Byung Joo Kim, has completed his PhD from Seoul National University and also postdoctoral studies from Seoul National University. He is the Professor at the Division of Longevity and Biofunctional Medicine, Pusan National University School of Korean Medicine, Yangsan, Republic of Korea. His research interest is in the action mechanisms of traditional medicine and interstitial cells of Cajal in gastrointestinal tract.

Determination of Optimal Sterilization Types for In Vitro Propagation of Walnuts Cultivars in Georgia

Iveta Megrelishvili*, Ekaterine Bulauri, Maia Kukhaleishvili and Tamar Chipashvili

Georgian Technical University, Biotechnology Center, Georgia

Microbial diseases have been reported in the walnut orchards due to different reasons in recent years in Georgia. For the purpose of walnuts tissue cultures reproduction, it is necessary to determine the optimal type of sterilization of initial material.

The aims of this study were determined optimal sterilization type to propagate walnuts cultivars ‘Pedro’, and ‘Chandler’ using tissue culture technique.

The initial explants (unimodal micro cuttings with a length between 1 and 1.2 cm) of three cultivars were collected from the young 2-3 years old walnuts orchards, Dzevera, ShidaKartli, Georgia. Two sterilized types were used for in vitro propagation of walnuts: I.1-2% hypochlorite 10-15 min, followed 70 % alcohol -30 min and 3 times sterile distillate water II. 0.1% mercuric chloride 5 min and 3 times sterile distillate water.

It was revealed that microbial contamination were 58.97% using 0.1% mercury chloride and relatively high by sodium hipocloride-85.12%.

DKW medium was used for in vitro cultivation of walnuts cultivars supplemented with 0.1 mg/L IBA, 1 mg/L BAP and sucrose 3%. The pH of culture medium was adjusted at 5.5 before adding the gelling agent and autoclaving. Micro cuttings were kept at 25±2°C under a 16 h photoperiod.

Finally, optimal sterilization types of initial explants (Combination II) was determined. Walnuts cultivars: ‘Pedro’ and ‘Chandler’ were propagated using tissue culture methods first time in Georgia.

This study led to develop an effective method for micro propagation of Juglansregia, which enable us to establish new walnuts orchards in Georgia.

Biography:
Iveta Megrelishvili has completed his PhD at the age of 28 years from Ivane Javakhishvili Tbilisi State University. She is the main research scientist of Georgian Technical Univeristy, Biotechnology Center and Head of Virology Lab, Scientific-Research Center of Agriculture. He has published more than 8 papers in reputed journals and has a great experience in the field of plant biotechnology, plant virology and molecular biology.

Definition of Optimal In Vitro Conditions for Different Maturity Potato Cultivars

Maia Kukhaleishvili*, Ekaterine Bulauri, Tamar Shamatava, Tamar Chipashvili and Iveta Megrelishvili

Georgian Technical University, Biotechnology Center, Georgia

The research aim was to determine optimal in vitro condition for propagation different maturity potato cultivars. Potato varieties were collected from in vitro potato collection of Georgian Technical University, Biotechnology Center according to their maturity: Early cultivars: Viviana, Red Sonia, Bellarosa, Vineta, Anushka; Medium early: Donata, Bernina, Madeira, Sante, Laura. Medium late: Brodie, Shepody, Jelly, Carola, Desiree.

The influence of three types of combinations with temperature, humidity, light and photoperiod was studied on all three maturity potato cultivarʼs in vitro development:I. T-23-25° C, H-80%, Lux-5-5500, 16h; II. T-25-27° C, H-75%, Lux-5-5500,16h: III. T-27-29° C, H-70%, Lux-5-5500, 16h;The results were evaluated after 17 days of reproduction. Best in vitro condition for all researched potato varieties was selected for their leave colors, rooting, and shoot formation. All potato cultivars morphological characterization was variable depending on the type of in vitro condition. It was revealed early maturity cultivars had maximum potential for in vitro propagation (5-6 nodes, average rooting 92% and shoot formation 94%) on combination: T-23-25° C, H-80%, Lux-5-5500, 16h. Combination: T-25-27° C, H-75%, Lux-5-5500,16h was best for medium early cultivars (5-6 nods, rooting 89% and shoot formation 90%). The perfect in vitro reproduction of medium late varieties (5-6 nodes, average rooting 87% and shoot formation 82%) was observed on the combination: T-27-29° C, H-70%, Lux-5-5500, 16h.

As it is known, for in vitro developments of early medium and late medium potato cultivars are necessary 22-26 days, but our results were obtained in 17 days. Finally, optimal in vitro condition for propagation different maturity potato cultivars was defined.

Biography:
Maia Kukhaleishvili has completed his PhD at the age of 58 years from ST. Andrew the First Called Georgian University of the Patriarchate of Georgia. She is the director of Georgian Technical University, Biotechnology Center- Scientific-Research Center. She has published more than 10 papers in reputed journals and has a great experience in the field of Agriculture and Biotechnology.

Affinity Protein Purification Resulting in Protein Sequence without Remaining Amino Acid Residues

Heba A Hosiny1*, Balint Hajdu1, Zita Fabian1, Eniko Hermann1 Wojtek Bal2 and Bela Gyurcsik1

1University of Szeged, Department of Inorganic and Analytical Chemistry, Hungary
2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland Purification of proteins is vital for the characterization of the function, structure and interactions of the protein of interest and efficiently carried out by affinity chromatographic methods. The specific interaction with the appropriate resins requires fusion affinity tags, such as e.g. the oligo-His, the maltose binding protein or glutathione-S-transferase tag. These sequences are encoded by the plasmids used for protein expression. The affinity tags have to be cleaved off after the target protein is selectively bound to the solid support. This is performed by specific proteases. These enzmes are expensive and mostly they leave few extra amino acids at the terminus, which may interfere with the structure and function of the purified protein.

Recent studies in our research group focused on the protein constituents of new artificial nucleases. Artificial DNA nucleases have provided scientists with the unprecedented ability to probe, regulate, and manipulate the human genome. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat-Cas9 system (CRISPR/Cas9) represent a powerful array of tools that can bind to and cleave a specified DNA sequence. Our ressearch focused on new type of ZFNs with intramolecular alloteric activation. The computer design of such enzymes requires the precise protein sequence to be obtained. Using the Ni(II)-affinity chromatography for e.g. zinc finger protein purification requires the complete removal of the oligo-His affinity tag. However, this was not possible to achieve by the traditional methods. We redesigned the cloning region of pET21a plasmid. The new approach is to cleave the His-tag by Ni(II) instead of proteases. Therefore, the new plasmid encodes the affinity tag with a Ni(II)-sensitive cleavage site at its N terminus. The precise gene of the protein is inserted into the cloning region by the help of BsmBI restriction endonuclease, so that the recognition site is deleted in the cleavage/ligation procedure. We performed the cloning, expression and purification procedures and will report the results on the poster.

Biography:
Heba Alaa Eldeen Hosiny Abd Elhameed is a PhD student in the University of Szeged. She is from Egypt, where she has been working as Assistant lecturer at Zagazig University. Presently she is examining new types of artificial nucleases by means of various biophysical methods within the frame of a Stipendium Hungaricum PhD scholarship and under supervision of Dr. Bela Gyurcsik.

Development of the Method of Isolation, Culturing and Assessment of Metabolic Ability of Bacteria from Species Desulfovibrio Desulfuricans

Katarzyna Ciemniak*, Aleksandra Wańczyk, Paulina Maciejewska, Piotr Łysakowski, Joanna Kobus-Cisowska, Piotr Kubiak and Daria Szymanowska

Faculty of Food Science and Nutrition, Poznan University of Life Sciences, Poland

Bacteria of the genus Desulfovibrio are gram-negative absolute an aerobic microorganisms, usually rod-shaped. They have the ability to carry out the processes of dissimilatory reduction of sulphates to hydrogensulphide taking part in the sulfur cycle in nature. A typical species of this genus is Desulfovibrio desulfuricans, occurring mainly in deeper layers of soil, sediments of water, industrial sewage, oilfields as well asanimal and human stool, sometimes causing infections of the digestive system.

The aim of the work was to develop a procedure for the isolation of Desulfovibrio desulfuricans from environmental samples using various selection factors. In addition, the metabolism of isolates have been tested, including their ability to grow in substrates with various carbon sources and the utilization of diesel fuel as the only carbon source as well as the ability to reduce sulphates. The tests were carried out under relatively anaerobic conditions using soil samples contaminated with petroleum derivatives. Tests showing metabolic abilities were carried out in bioreactors maintaining sterility regimes and appropriate gas conditions. The increase in biomass concentration, substrate loss and sulphate reductions were analyzed.

As a result of the work, Desulfovibrio desulfuricans monocultures were obtained using the antibiotic selection factor. The conducted culturing tests allowed for the selection of parameters allowing for an efficient biomass multiplication as well as the assessment of the influence of parameters such as pH, temperature, agitator speed and gas conditions on the metabolicconditions of Desulfovibrio desulfuricans. In addition, the ability of bacteria to utilize diesel oil and to reduce sulphate was analyzed.

Biography:
Katarzyna Ciemniak is currently a Ph.D. candidateat the Poznan University of Life Sciences. She completed her Engineerʼs and then Masterʼsdegree in Industrial Biotechnology at the Department of Biotechnology and Food Microbiology at Poznan University of Life Sciences, Poland. Since 2017 she has been working with Dr. hab. Daria Szymanowska – Powałowska in the field including pharmaceutical chemistry and microbiology, carrying out tests for bacterial resistance on complexes of antibiotics with cyclodextrins. Since the beginning of her doctoralstudies she has been participating in a project concerning research on the activity of Desulfovibrio desulfuricans bacteria in the process of decomposition of petroleum-derived waste.

Violacein as a Bioactive Compound of Microbiological Origin - Bioproduction and Anti-Melanoma Activity

Patrycja Kowalska*, Beata Gad, Anna Sobiepanek, Tomasz Kobiela and Małgorzata Milner- Krawczyk

Faculty of Chemistry, Warsaw University of Technology, Poland

Violacein is an organic chemical compound that is derived from indole. Microorganisms capable of producing violacein are characterized by a dark purple coloration of the colonies. As shown in recent years, this compound has antibacterial, antifungal and anticancer properties. Studies on violaceinʼs influence on various cell lines, conducted by many independent teams, indicate that it acts on tumor lines in small concentrations, and the mechanism of its action is depended on the cell type. In some cases, violacein induces apoptosis in cancer cells without affecting normal cells.

We isolated new strain, capable to violacein bioproduction. On the basis of comparative analysis of the 16S rRNA coding sequence the strain was classified as Janthinobacterium lividum (KP16). We established optimal culture conditions, as well as extraction and purification methods of this intracellular secondary metabolite: 2-fold diluted lysogenic broth (LB) as a growth medium and 5 days culture at 15 – 20 °C with shaking at 110-120 rpm. The obtained methanolic extracts were purified in the system of acetone : chloroform : ammonia (1 : 2 : 0.01) on a silica beads (Silica gel 60 with grains 0.015 - 0.040 mm). After optimizing the extraction and purification methods, the violacein was obtained with a purity of over 95%. Subsequently, studies concerning proliferative and metabolic activity of violacin-treated melanoma cell lines were performed. The results showed a clear selective effect of violacein on metastatic cells.

Biography:
Patrycja Kowalska is currently a MSc.Eng. student at the Warsaw University of Technology. She is going to specialized in Medicine and Cosmetics at Faculty of Chemistry WUT. She earned her Bachelorʼs Engineering degree in Biotechnology at the Warsaw University of Technology, Faculty of Chemistry, Poland. During her BSc studies she started working with Ph.D. Małgorzata Milner-Krawczyk in the field of bacteria biotechnology and violacein bioproduction. Nowadays studies ordinance on cell lines studies and potentially used of violacein.

Separation of Proteins on Ion-exchange Resins

Aleksandra Zawodnia* and Wojciech Białas

Poznan University of Life Sciences, Poland

Lactoferrin (Lf) is a biologically active glycoprotein from the transferrin family, mainly found in milk. Lf exhibits antimicrobial, immunomodulating and anti-cancer activity. Lab-scale experiments were carried out to generate input data for simulations of an industrial plant for Lf production. Lf was purified using an ion exchange chromatography. Three ion exchange resins have been tested: Amberlite XAD761, Dowex 50WX4 and SP-Sepharose. Two different elution methods were employed: linear gradient and step gradient. The bicinchoninic acid protein assay method with BSA protein standard was used for further analysis of protein fractions. The composition of the different fractions was investigated by using SDS-PAGE electrophoresisto assess their purity. The conceptual project and economic analysis of a plant for production of LF were done using the software SuperPro Designer version 10.

Best results were obtained when SP-Sepharose was used as an stationary phase leading to a total recovery of Lf from column. The resin capacity determined using pure Lf was equal to 60 mg Lf/ml.The linear gradient resulted in the separation of lactoferrin into Lf a and Lf b, while in the step gradient, only one peak of lactoferrin was obtained. However, the most favorable was separation in step gradient, becausein this method, higher concentrations of lactoferrin were obtained. Simulation results performed in SuperPro Designer indicate that the step method was more economical than the linear gradient method; therefore changing the Lf production method to step could reduce the cost of Lf production from milk whey.

Biography:
Aleksandra Zawodnia is a graduate student of the Poznan University of Life Sciences (Poland). In 2017 she earned M.Sc. degree in Biotechnology, on the Faculty of Agriculture and Bioengineering. At present, she works as a microbiologist at Poznan University of Life Sciences, Department of Biotechnology and Food Microbiology. She is interested in industrial biotechnology and environmental microbiology.

Development of Eubiotic Preparation for Domestic Livestock

Aleksandra Wanczyk*, Katarzyna Ciemniak, Paulina Maciejewska, Wojciech Białas, Piotr Kubiak, Joanna Kobus-Cisowska and Daria Szymanowska

Poznan University of Live Sciences, Poland

Animal feeding is a key element in cost-effective production. beneficial effects can be achieved with wholesome feeds based on high quality roughage and nutritious fodder, vitamin and mineral additives. Yeast are industrially interesting microorganisms. Their dry weight is composed of lysine- and threonine–rich protein in 35-65%. Yeast are a rich source of B-complex vitamins and ergosterol. They contain fat and carbohydrates which give them high energy content. They are also easily digested.

Herbs are also functionally interesting. Their preparations contain beneficial substances, such as flavonoids, anthocyanines, glycosides, tannin and tannines, secoiridoids, mucilage, essential oils, alkaloids, polyphenols and mineral salts. These compounds have antistress, antibacterial, antiviral and antifungal properties stimulate the appetite of livestock and help maintain physiological balance. Oils of thyme, basil and cumin enhance appetite; stimulate secretion of bile and digestive enzymes, and intestinal motility, which improves assimilation of nutrients. Thyme, basil, cumin and camomile oils show activity against pathogenic bacteria, including Clostridium perfringens and E.coli.

The aim of this work was to develop a multifunctional eubiotic preparation composed of two species of yeast and a herbal complex. Fed-batch cultures were conducted in 6.6 L bioreactors. Glucose, molasses, sucrose, whey and corn steep liquor were investigated as substrates. Growth of biomass, substrate utilization and concentration of metabolites were monitored. Kinetic indicators were calculated in order to allow comparison of substrates. From various tested herbal additives, garlic, oregano and thyme extracts were selected based on the results of antioxidant potential and antibacterial activity. Addition of these extracts can improve digestion, nutrient assimilation and immunity of farm animals.

This work is the result of cooperation with BAF Inc. for the purpose of the project entitled “Development of a novel recipe and production process of a eubiotic preparation containing two symbiotic yeast species”. Research was financed by POIR.02.03.02-30-0047/17.

Biography:
Aleksandra Wanczyk is a graduate with a Masterʼs Degree in Biotechnology at Poznan University of Life Sciences in Poland. She is currently working as a microbiologist at the Department of Biotechnology and Food Microbiology of Poznan University of Life Sciences. Her research interests are in the field of yeast biotechnology.

Development of Multifunctional Diet Supplements Containing Alpha-Ketoglutaric Acid

Paulina Maciejewska1*, Aleksandra Wańczyk1, Katarzyna Ciemniak1, Judyta Cielecka-Piontek2, Joanna Kobus-Cisowska1, Piotr Kubiak1 and Daria Szymanowska1

1Poznan University of Life Sciences, Poland
2Poznan University of Medical Sciences, Poland

α-Ketoglutaric acid (KGA) is an organic compound containing both a carboxyl and a ketone group. KGA, also referred to as 2-oxoglutamate, 2-ketoglutaric acid, 2-oxoglutaric acid, is a rate-determining intermediate in the tricarboxylic acid cycle and amino acid metabolism and has a crucial role in cellular energy metabolism. The aim of this project was to develop a new line of probiotics aimed at 5 groups of recipients - athletes, people after antibiotic therapy and chemotherapy, future mothers and children with gut and psychology syndrome (GAPS).

The developed products are multi-component liquids and gels, which makes them a novelty on the market, as most probiotic supplements are dry. Bacteria and bacterial metabolites (including KGA), vitamins and minerals and plant extracts with proven health-promoting properties are the functional ingredients of the products. Combination of probiotic bacteria and KGA is another reason that makes the developed product assortment unique on domestic market of functional food.

Four research tasks related to the development of the microbiological process of KGA synthesis and the production technology of new probiotic products containing KGA were undertaken in order to achieve the assumed goal.

This work is a result of cooperation with Living Food sp.z.o.o., which implements the project entitled “The development of new functional products produced on the basis of a consortium of probiotic strains, alpha-ketoglutaric acid and a vitamin-mineral complex.” The work is financed by the National Center for Research and Development. No. Application for co-financing: POIR.01.01.01-00-0685/17.

Biography:
Paulina Maciejewska graduated from Poznan University of Life Sciences in Poland, where she received M.S. degree in Biotechnology on the Faculty of Agriculture and Bioengineering in 2017. She works at University of Life Sciences in Poland, Faculty of Food Science and Nutrition, Department of Biotechnology and Food Microbiology as a microbiologist. She is involved in the development of microbiological synthesis of alpha-ketoglutaric acid. She is interested in industrial biotechnology and food microbiology.

Direct Screening and Isolation of Microorganisms for a Biofertilizer Formulation

Laura Jeannette García Barrera1*, Karim Hassam Montalvo Aguilar1 and Francisco Roberto Quiroz Figueroa2

1Instituto Politécnico Nacional, Centro de Investigación en Biotecnología Aplicada, México
2Instituto Politécnico Nacional, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad Sinaloa, México

Among genomic tools, metagenomics has the potential for characterizing large-scale population of bacterial genomes from environmental samples, without the need of isolation and growth. In this work, a metagenomic survey for bacteria present in three different substrates, namely soil, compost and digestate, were carried out. Bacteria belonging to genus with agricultural importance, such as plant growth promotion (PGP), were selected for further analysis. Briefly, bacteria with putative PGP activities were isolated and cultivated toward the goal of formulating a biofertilizer. Samples from soil, compost and digestate were culture in selective media (NBRIP, Pikovskaya Agar, Ashby Agar, NFB, R2A, FMA and Chitin Agar for the isolation of Lysobacter, Varioborax, Azospirillum, and Paenibacillus). A total of 152 strains were isolated, corresponding to 47, 42 and 63 from soil, compost and digestate; respectively. Then, pure bacterial strainswere assessed as PGP agents by evaluating the production of indol aceticacid, gibberellic acid, and siderophore, as well as seed germination. Forty bacterial strains that showed PGP activity were tested for antagonistic behaviors. At last, 17strains were selected for PGP effects on Dactylisglomerata L. Strains with the higher PGP activities were identified as Microbacterium sp., Enterobacter ludwigii and Rahnella aquatilis. Taken together, the combination of metagenomic information and classical lab procedures allowed to formulate a novel biofertilizer for plant growth promotion, which is the first step to improve crop productivity.

Biography:
M. C. Laura Jeannette García Barrera is a Researcher of CIBA-IPN, México. She studied Environmental Engineering at UPAEP, México. She has a Masterʼs degree in Sciences in the specialty of Biotechnology from CINVESTAV, México. Has experience evaluating the antifungal activity of plant extracts and works with molecular biology of viruses, bacteria and fungi. Has participated in projects in collaboration with the industry and is currently studying the use of biofertilizers and their impact on microbial communities in soils.

Digestate Concentration and Harvest Intervals Effects on Yield of Forage Grasses and Legumes

Laura Jeannette García Barrera*, Job Jonathan Castro Ramos, Hilzamara Guadalupe Larios Peña, Ismael Eulogio Sarmiento, Stefani Aletse Meza Zamora and Rigoberto Castro Aguilar

Instituto Politécnico Nacional/Centro de Investigación en Biotecnología Aplicada, Mexico

Knowing the effect of digestate that itʼs the anaerobic digestion subproduct of solid residues of cattle, on the yield of forage grasses and legumes, is a justification to reduce the use of agrochemicals. The aim was to evaluate the effect of concentration, harvest frequency and irrigation frequency on the components of the yield of Ryegrass and White clover. To do this, evaluate digestate concentrations (20, 40 and 60%), harvest frequencies (4, 5 and 6 weeks) and irrigation frequencies (15 and 30 days) were applied. The variables were: Dry matter, weight of leaves, stems, petiole, plant height, growth rate of the crop. The digestate was acquired in Chapingo University. A factorial design with arrangement 3*3*2 was used, and mean comparisons were made by Tukey (P<0.05), by the GLM procedure of the SAS software. The results showed that the highest yield, weight of leaves, stems, growth rate of the crop and height of plant was with the treatment of 60% of digestate, harvest frequency every four week and the irrigation every 15 days in both species (P<0.01). It is concluded that the concentration, harvest frequencies and digestate irrigation frequencies affect the yield and can be used as substitute of agrochemicals fertilizer.

Biography:
M. C. Laura Jeannette García Barrera is a Researcher of CIBA-IPN, México. She studied Environmental Engineering at UPAEP, México. She has a Masterʼs degree in Sciences in the specialty of Biotechnology from CINVESTAV, México. Has experience evaluating the antifungal activity of plant extracts and works with molecular biology of viruses, bacteria and fungi. Has participated in projects in collaboration with the industry and is currently studying the use of biofertilizers and their impact on microbial communities in soils.

Natural Herbs for the Treatment of Skin Ulcers in Diabetic Animal Models

Diana Sami1*, Hana Heiba2 and Ahmed Abdellatif1,2

1Department of Biotechnology Program, The American University in Cairo, Egypt
2Department of Biology, The American University in Cairo, Egypt

Chronic skin ulcers resulting from pressure ulcers or diabetes, affect nearly 2 million people each year and account for an annual healthcare costs of about $50 billion. Skin ulcers are commonly associated with elderly, bedridden, and debilitated patients, spinal cord injury and patients undergoing major orthopedic surgery. Diabetes increases the risk of skin ulcers because of its association with nerve damage (neuropathy), poor circulation and infection. Current treatment of skin ulcers includes traditional wound dressings, antibiotics, and debridement to remove necrotic tissues. One major disadvantage of using excessive antibiotics in skin ulcer treatment is antibiotics resistance. Herbal antimicrobials are expected to be non-cytotoxic, antibacterial, anti-inflammatory effect and will promote skin ulcer healing. Our aim in this study is to investigate various formulas of natural herbs e.g. Curcumin, and Ginsengs to enhance skin ulcer healing in animal models of skin ulcers. Our preliminary data show promising results in skin healing improvement both in Diabetic and Non diabetic pressure ulcer models. Ongoing work is verifying the healing and antibacterial potential of such formulas.

Biography:
Diana Sami is currently a graduate student in the Biotechnology program at the American University in Cairo (AUC). She earned her Bachelor degree in pharmaceutical Science from faculty of pharmacy, Helwan University, Egypt in 2011. She then obtained a Diploma in healthcare and hospital management from the American University in Cairo (AUC) in 2014. At present, Diana is interested in the development of cost effective treatment for skin ulcers using natural herbs.

Novel Antibacterial and Anti-Fungal Agent from Eicchornia Crassipes

Bharat Kwartra

St. Markʼs Sec Public school, India

Introduction

Bacterial and fungal infection in humans and plants is an undiscriminating concern to countries worldwide. Large number of it occurs in developing countries with poor economic condition. Eichhornia crassipes, also known as water hyacinth in English, is a floating hydrophytic plant that is notorious for its invasive ability. Due to its prolificness, the plant constantly and quickly causes eutrophication and retardation of the flow of the water it lives in. This results in the need of constant removal of this plant by the people. This research has led to the discovery of using the abundantly found Eichhornia crassipes, as a cheap yet effective source of antibacterial and antifungal agent. Method and Experiment Design

The experiment was conducted in two sets, each set is divided into two parts and each part was subdivided into three elements of different microbes.

SET 1, Part 1

1. The ethanol extract of Eichhornia crassipes is first purified through centrifugation and pasteurisation, and then diluted into 3 different concentrations; 75%, 50% and 25%. In three trials, each of the three Eichhornia crassipes extract concentrations is tested against the three bacterias: Staphylococcus aureus, Streptococcus sobrinus and Escherichia coli, in vitro, by the cylinder plate assay method.

2. Water is used as negative control and ampicillin is used as positive control and comparison, being a representative of conventional antibacterial agents.

3. The size of zone of inhibition produced after incubation will be the measure of the antibacterial effect of the extract.

SET 1, Part 2

1. The ethanol extract of Eichhornia crassipes is first purified through centrifugation and pasteurisation, and then diluted into 3 different concentrations; 75%, 50% and 25%. In three trials, each of the three Eichhornia crassipes extract concentrations is tested against the two fungi: Magnaporthe grisea, Fusarium oxysporum and Aspergillus niger, in vitro, by the cylinder plate assay method.

2. Water is used as negative control and fluconazole is used as positive control and comparison, being a representative of conventional Anti fungal agents.

3. The size of zone of inhibition produced after incubation will be the measure of the Anti fungal effect of the extract.

SET 2, Part 1

A second set of experiment is conducted to test whether the extract is bacteriostatic or bactericidal. The second experiment involves:

1. The transfer of the inhibition zone liquid into a new nutrient media.

2. Presence of bacteria on the new media after 24 hours incubation will determine the type of the antibacterial activity.

SET 2, Part 2

A second set of experiment is conducted to test whether the extract is fungistatic or fungicidal. The second experiment involves: 1. The transfer of the inhibition zone liquid into a new nutrient media. 2. Presence of fungus on the new media after 24 hours incubation will determine the type of the antibacterial activity.

Result and Analysis

SET 1, Part 1

The negative control shows no inhibition effect, indicated by the absence of inhibition zone. The highest inhibition effect is produced by the 75% concentration,. The smallest inhibition effect is produced by the 25% concentration. The Ampicillin produces higher inhibition effect than the extracts.

SET 1, Part 2

The negative control shows no inhibition effect, indicated by the absence of inhibition zone. The highest inhibition effect is produced by the 75% concentration,. The smallest inhibition effect is produced by the 25% concentration.

SET 2, Part 1

Presence of bacteria in the new nutrient media of the experiment indicates that the extract is bacteriostatic.

SET 2, Part 2

Absence of fungi in the new nutrient media of the experiment indicates that the extract is Fungicidal.

Three new molecules were derived from the extracts using Beta lactamase test, lanosterol tests and various other tests.

Conclusion: The antibacterial property of Eichhornia crassipes is due to the presence of two bioactive chemicals (derived); flavonoid and alkaloid.

The Anti fungal property of Eichhornia crassipes is due to the presence of bioactive chemicals (derived) similar to fluconazole.

Eichhornia crassipes extract possesses antibacterial and Anti fungal properties, and is bacteriostatic and fungicidal respectively. Therefore, Eichhornia crassipes is a good and cheaper substitute of antibacterial due to its easy accessibility and abundance.

Biography:
Bharat Kwatra is a Student of High school in India, but very keen and enthusiastic about microbes and research related to biochemistry, which make him different from others. He is an active Member of American Microbial society, Indian microbial society, European Research trade and development society and World Association for Scientific Research and Technical Innovation at very young age. He is very passionate about his research which encourages him to look forward for gaining knowledge. From the age of 15 years, he is involved in doing research on various health issues. He received first prize in international conference, 2017 and Won Youth research forum at Beneficial microbes, USA. He is also being awarded as Youngest Researcher in his state. He has published Three international journals and have seven patents. He is being recently working on Single cell protein with Department of microbiology at his school.

The Effect of Stress on Behavior and Immunity in Wistar Rats

Sansri Soraya*, Bairi Abed El Madjid and Haloui Meriem

University Badji Mokhtar Annaba, Algeria

Stress is a major current problem both in humans and in animals and implement strategies to limit sometimes adverse effects. In addition, exposure to stress causes behavioral and immune disorder in rodents. Experimentally, this modification is based on the intensity and type of exerted stress.

The objective of this work is to study the effect of three types of stresses, acute restraint, and predation by separation to assess immune and behavioral changes in the Wistar rat. Comparison between the three types of stresses, Behavioral and adaptive changes in the rat are an attempt to identify the behavioral parameters evaluated from the open fields andmaze.

Our results on stressed rats showed the following:

  • Increased anxiety with onset of depression evaluated in tests in an elevated cross maze and open field.
  • Impairment of spatial memory.
  • Disturbance of the immune system cells and humoral response (monocytes and lymphocytes)
  • .

Evaluation of Phenomenological Variables by Applying Compost and Digestate to Different Concentrations in Lettuce Cultivation (Lectuca Sativa L.)

Jeisel Delgado Flores1*, Job Jhonathan Castro Ramos1, Rigoberto Castro Rivera1 and Patricia Ibarra Torres2

1Instituto Politécnico Nacional, Centro de investigación en Biotecnología Aplicada, México, 2Universidad Politécnica de Guanajuato, Ingeniería Agroindustrial, México.

Increase in world population has caused an increasing demand for food, especially those of vegetable origin. Consequently, to meet this demand, farmers have used agrochemicals to improve crop yields. The objective of this work was to evaluate the growth of lettuce (Lactuca sativa L.) in response to the application of two types of organic fertilizers: compost and digestate, each of them obtained from organicwaste and cowdung; respectively. Plants with untreated soil were grown as a negative control, while plants grown in the presence of a chemicalfertilizer (NPK 17-17-17) were established as positive controls. In the case of plants treated with compost, concentrations of 20%, 40%, 50%, 60% and 100% were used; while for digestate they were 20%, 40%, 60% and 80% with frequencies of 15 and 30 days. The height of the plant, fresh weight, leaf area and root were measured. The experiments showed that the best compost treatment was the 100% application, in which the height of the plants was 52% higher and the fresh weights even times higher than the negative control. The Best digestate treatment was at a concentration of 80% and a watering frequency of 15 days. In the latter case, the height of the plants and fresh weightwas 65% and 89% greater tan the negative control. In summary, it is posible to improve the growth of lettuce by applying compost or digestate; however, more experiments are needed to find the right combination.

Biography:
Jeisel Delgado Flores is a masterʼs student in the area of Applied Biotechnology at CIBA-IPN, Mexico. She is an engineer in food industries graduated from the InstitutoTecnológico del Altiplano (ITAT), Tlaxcala, México. She has experience in training people from rural communities, to process fruits and vegetables, dairy products and meat as an alternative to food preservation. She also has work as a researcher in conjunction with ITAT to find option to water treatment contaminated with heavy metals. Currently, her research work focuses on the development of a biofertilizer applicable to crops for human consumption.

Screening, Characterization, and Biocatalytic Capacity of Lipase Producing Wild Yeasts from Makiling Forest Reserve (MFR) Philippines

Julianne Vilela1*, Charisse Leanne Legaspi3, Flora Querubin3, Jarel Elgin Tolentino3, Joseph Martin Paet3, AngelitoPaguio Jr3 and Francisco Elegado2

1Philippine Genome Center Agriculture, University of the Philippines Los Baños, Philippines
2National Institite of Molecular Biology and Biotechnology (BIOTECH) University of the Philippines Los Baños, Philippines
3Graduate School, University of the Philippines Los Baños, Philippines

Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are enzymes generally used in several industrial applications. However, even with the numerous industrial applications of lipases, there are limited studies aiming to characterize and optimize lipase activity especially that of yeast lipase-producing microorganisms. This work aims to select and identify lipase-producing yeasts isolated from Makiling Forest Reserve (MFR), Philippines and to optimize conditions to maximize lipase production. A total of 144 wild yeasts were tested for their lipase producing potential and strain B1-7 showed the highest lipase activity in both solid and liquid selection media (7.6 EAI and 0.082 U/mL-min activity, respectively). Strain B1-7 was molecularly identified by sequencing the ITS1-5.8S-ITS2 region of the fungal rRNA operon DNA as Cryptococcus flavescens. The optimum conditions for maximum lipase activity (0.66 U/mL-min) of the putative C. flavescens strain obtained using Response Surface Method (Box-Behnken Design) were 5.0 C:N value, pH 6.0 and 0.5% inducer. Lipase activity was significantly affected by the C:N to percent inducer interaction (p= 0.010) and % inducer (p = 0.040). After a 72h fed-batch fermentation experiment, lipase activity was 0.115 U/mL-min. A negative correlation (R2= -0.426) was observed between lipase activity and biomass suggesting that lipase production did not depend on biomass. Moreover, no change in lactose concentration was observed suggesting that it was not used as the primary source of carbon. Hence, we can exploit the potential of producing a new strain for industrial application.

Biography:
Ms. Julianne Vilela is a University Researcher and Bioinformatician at the Philippine Genome Center, University of the Philippines. Ms. Vilela extensive training includes: Genome-wide Association Studies (GWAS), Quantitative Genetics and Genomics at Iowa State University, Iowa, USA, and Plant Genome and Bioinformatics Training at Boyce Thompson Institute for Plant Sciences, Cornell University, USA.

Evaluation of a Panel of CirRNAs Expression as a Novel Potential Biomarker in Hepatocellular Carcinoma

Kerolos Atalla Shehata3*, Marwa Matboli1, Ayman El-Sayed Shafei2, Ahmed M Ashry3, Kamal M Kamal3, Mohammed Ali Agag3, Ibrahim Reda3, Hana M Abdelzaher3 and Mahmoud A Ali2

1Medical Biochemistry and Molecular biology, Department, Faculty of Medicine, Ain Shams University Medical Research Institute, Cairo, Egypt
2Biomedical Research Department, Armed Forces College of Medicine, Cairo, Egypt
3Undergraduate Student, Armed Forces College of Medicine, Cairo, Egypt

Background: Circular RNAs are a newly validated type of non-coding RNAs recently found to be deregulated in several human cancers. Accurate and specific non-invasive biomarkers are urgently needed for the diagnosis and prognosis of hepatocellular carcinoma (HCC).

Patients and Methods: We performed bioinformatic analysis to retrieve a novel panel of circRNAs potentially relevant in HCC. We examined their expression in the sera of sixty-eight HCC patients, sixty chronic hepatitis C(CHC) patients and 36 healthy controls using RT-qPCR. We examined the performance characteristics of the selected circRNA biomarker panel in comparison to alpha fetoprotein. In addition, we performed cox regression analysis to correlate between their expression levels and patient survival.

Results: The circRNA-based biomarker panel (hsa_circ_00156, hsa_circ -000224 and hsa_circ -00520) showed strong biomarker potential with relatively high sensitivities and specificities. The combined panel showed superior performance characteristics to those of AFP.

Conclusion: Through our analysis of this preliminary data, we believe that this novel circRNA-based biomarker panel could potentially be used in the diagnosis and prognosis of HCC.

A Novel Alpha(1-2)-Fucosylation and Actin Dependent Uptake Vehiculates Doxorubicine-Loaded Liposomes into Highly Proliferative Cells

Domenico Nolfi1*, Cinzia Della Giovampaola1, Antonietta Capone1, Claudia Bonechi2, Agnese Magnani2 and Floriana Rosati1

1University of Siena, Department of Life Sciences, Italy
2University of Siena, Department of Biotechnology, Italy

Finding new strategies to direct drug carrying nanovesicles towards specific cellular targets is one of the major goals of Biomedical Research today. Recently, we found a fucosylated structure exclusive of high proliferative cells, which is responsible for a peculiar uptake mechanism alternative to the classic ones and dependent on alpha(1-2)fucosylation. This structure, detectable by using the fucose-binding lectin from Lotus tetragonolobus (LTL), was first observed in CVEC and then in A431 and DU145. It appears as a network of tubules extending between the perinuclear region and the cellular periphery. LTL binding sites were also found to be exposed at the cell surface in a very restricted region. The uptake capacity of the LTL-positive tubular structure was then demonstrated by testing the lectin with living cells. Other lectins tested in the same way were found to enter the cells through the classical endocytotic mechanism.

In order to exploit this new uptake mechanism as drug delivery system, we constructed and tested in DU145 doxorubicin-loaded liposomes functionalized with LTL (LTL-Dox-L). We confirmed that the LTL-Dox-L enters the cells as the corresponding lectin and that by this vector the intracellular delivery of the drug was much more than that entered via unmodified doxorubicin-loaded liposomes. By electron microscopy we also demonstrated that liposomes enter the cells one by one in tiny tubules that never fuse with lysosomes. Therefore, liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity.

More recent results have indicated the actin cytoskeleton as responsible of the integrity of the tubular structure: destruction of actin filaments with Cytochalasin D gives rise to the gradual disappearance of the tubular LTL positive structure, until it remains as vesicles in the perinuclear region. The treatment with Cytochalasin D also strongly decreases the entrance in the cells of LTL-Dox-L.

Biography:
Domenico Nolfi was born in Sicily on the 12th of December, 1991. He is currently undertaking a PhD at the department of Life Sciences of the University of Siena, where he earned a masterʼs degree in Molecular and Cellular Biology in 2016 after completing a bachelorʼs degree in Biological Sciences in 2014. His fields of interest are Glycobiology and Cellular Biology and his research focuses on a alpha-2 fucosylation-dependent uptake machanism and its role in targeting of drug delivery systems. In his spare time, he loves cooking and dancing.

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