Significance of applied microbiology is determined by the vital role of microorganisms in global natural cycles, technologies, medicine, and ecology. Insitute of Microbiology, Belarus National Academy of Sciences, is the leading research institution of Belarus in the area of applied microbiology, specializing in the evaluation of microbial biodiversity, physiology, and biochemistry; genetic engineering of microorganisms producing bioactive substances; development of biotechnologies for an industry, agriculture, medicine, environmental protection. A crucial aspect of microbiological processes is the provision of superactive strains synthesizing bioactive substances. Both conventional selection methods and genetic engineering are used to attain this goal. The Center for Analytical and Genetic-Engineering Studies set up at the Institute is equipped with up-to-date instrumentation allowing to derive strains with defined properties engaged in an elaboration of biotechnologies lying within the mainstream of global high-tech trends. Molecular-genetic identification of microbial strains is carried out at Belarussian collection of non-pathogenic microorganisms integrated into Institute structure and registered as a member of World Federation of Culture Collections. Apart from main collection stock, special DNA banks were founded for industrial strains, plant pathogens, xenobiotics destructors and bioterioration agents, to ensure the preservation of genomes of promising biotechnological objects; early diagnostics of pathologies affecting forest and agricultural cultures; bioremediation of natural and technogenic environments; evaluation of diverse materials bioresistance. Technologies and production generated at the Institute find the outlet in numeric branches of national economy, including agriculture – biopesticides, microbial fertilizers, probiotics, biodisinfectants, biopreservatives, enzyme preparations; medicine – pharmaceutical technologies, diagnostic kits; industry – food additives, organic acids, alcohols, and makes a solid contribution to its progress.

Linda K Medlin obtained her PhD from Texas A&M University in 1983 and is currently a Research Fellow at the Marine Biological Association of the UK where she co-ordinated the FP7 EU project MIDTAL. During her employement at University of Paris and at Microbia Environnement, she directed two additional FP7 projects Aqua and SMS. All three of these projects involved the making of species specific probes for early warning systems for toxic algae and/or freshwater pathogens and applying them in a microarray or biosensor format and for making phylochips for the analysis of marine biodiversities using microarrays. She has published over 250 research papers including 35 chapters in books. She has participated in over 18 EU grants and many other national funding proposals from the UK and Germany.

Microarrays are oligonucleotides applied to the surface of a glass slide in an ordered array. When rRNA sequences are used, these are called phylochips, which can identify organisms and is a relatively new, innovative microarray application. Phylochips can facilitate monitoring for any microorganism in any environment and visualize its changes in abundance over time for longterm records. We developed in three EU projects: a phylochip for the detection of toxic algae in marine waters and for freshwater pathogens in freshwater and tested them with environmental samples in five countries for the toxic algae and six countries for the freshwater pathogens. Water samples were filtered until they clogged or concentrated into one litre using a kidney dialysis filter, of which free filters are being distributed upon request. Total RNA was extracted using TriReagent, fluorescently labelled and hybridised to the phylochip. The pattern captured via fluorescent excitation in the microarray scanner is exported as an excel file and analyzed based on presence/absence of probe signals in a hierarchical fashion. For a species to be present, probes for higher taxa, viz., genus to kingdom must also be present. Where calibration curves have been made, then the microarray signal can be converted into cell numbers. In a fourth EU project microarray probes were transferred to a SHA coupled to an electrochemical and a colorimeter detection. The electrochemical detection was 16 fold higher than that obtained in earlier applications and the colorimetric detection was automated for a real time in-situ assay in a buoy.

J F Alderete received his PhD from The University of Kansas in 1978. He did Postdoctoral research at The University of North Carolina at Chapel Hill. He has published 140 scientific articles and has 63 book chapters, invited articles, and press releases. His work has been presented at 157 scientific conferences, and he has given seminars at 90 colleges and universities worldwide. He has served in NIH Study Sections, Boards of Scientific Counselors, and National Advisory Councils. He has been a member of several National Academy of Medicine panels.

There is a need for a rapid, inexpensive and accurate serum antibody-based diagnostic with targets of high specificity for screening of large cohorts of women and men at risk of infection by Trichomonas vaginalis. This protist causes the number one nonviral sexually transmitted infection worldwide. The antigen-detection OSOM™ Trichomonas Rapid Test (Seskui Diagnostics) is a lateral flow, immunochromatographic Point-of-Care test that works only for women. During our investigations of the T. vaginalishost interactions, highly immunogenic proteins detected by sera of patients with trichomonosis, but not uninfected controls were identified. Some of these immunogenic proteins include the metabolic enzymes fructose-1,6-bisphosphate aldolase (A), α-enolase (E), and glyceraldehyde-3-phosphate dehydrogenase (G). The epitopes of these proteins were characterized and found to have little or no sequence identity to other eukaryotes, yeasts, and microbial pathogens, including organisms that cause other STIs. We have constructed a new version of an earlier chimeric recombinant String-Of-Epitopes (SOE) protein consisting of 15-mer peptides of epitopes of A, E, and G. This composite protein called AEG:SOE2 was detected by ELISA with highly reactive sera of women and men but not control, negative serum lacking antibody to T. vaginalis. I believe that this approach lends itself to the creation of highly specific immunogenic targets for both detection of serum antibody in patients as well as for future subunit vaccines.

Mariateresa Volpicella has completed her PhD and Postdoctoral studies at the A Moro University. From 2004 she is Researcher in Molecular Biology at the Department of Biosciences, Biotechnology and Biopharmaceutics at the University of Bari. She has published 25 papers in reputed journal. Her recent research activity involves studies on bacteria adaptation to environmental stresses by genomics, transcriptomics and proteomics approaches, and also metagenomics of extreme environments.

Background: Marine salterns are excellent sites for studying the dynamics of the prokaryotic biodiversity at increasing salt concentrations. Metagenomics offers the most direct approach for reliably assessing the microbial diversity including uncultivable prokaryotes. Salterns of Margherita di Savoia (MdS) are located on the East-cost of South Italy. They are the largest salterns in Europe, with a yet unexplored microbiota composition.

 

Objective: Defining the microbiota composition of the salterns of MdS in ponds with increasing salt concentrations. Methodology: eDNA purified from nine ponds with salt concentration in the 4.9-36% range was used for PCR amplification of the V5-V6 hypervariable region of the 16S rRNA gene. NGS of amplicon libraries was carried out by the Illumina MiSeq platform. Obtained reads were analyzed using the BioMas software for taxonomic classification.

 

Results & Conclusions: The microbiota composition of the MdS salterns resulted in a peculiar composition of prokaryotes, quantitatively different from that of other salterns of the Mediterranean area. For example, Archaea are absent at low salt concentrations (4.9-8.4%) and reach their highest concentrations (30-35%) in the high-salinity ponds. In similar ponds of the salterns of Santa Pola (Spain), their presence has been estimated around 90%. Conversely, in the high-salinity ponds of the MdS salterns, the Eubacteria Salinibacter genus is the most represented genus. This study is of particular interest, not only to define the microbiota composition in different salt concentrations, but also for better addressing future functional metagenomics analysis aimed at the identification of biotechnological useful extremozymes.

Mihai Nita-Lazar has a wide expertise in the fields of Microbiology, Molecular Biology and Biochemistry mainly in prestigious laboratories such as the Institute of Microbiology, ETH, Zurich, Switzerland, Department of Biochemistry and Cellular Biology of Stony Brook University, New York, SUA, Department of Cellular and Molecular Biology of Medical University Center from Boston, Massachusetts, SUA and the Department of Microbiology, University of Medicine, Baltimore, Maryland, SUA. He is currently working as Principal Investigator of Bioassay-Biological Analysis Laboratory of the National R&D Institute for Industrial Ecology-ECOIND. He has published more than 40 ISI papers, four books/chapters, h-index 10.

The waterborne pathogenic bacteria, especially the enteric bacteria of human fecal origin have become currently a global public health issue. The detection and quantification of drinking water microorganisms are an essential part of any quality control or water safety management plan interconnected to enteric bacterial pathogens such as Salmonella spp., Shigella spp., Vibrio cholerae or to nonfecal bacterial pathogens such as Legionella spp. The standard methods of detecting waterborne pathogenic bacteria are time-consuming due to the growing step in a specific culture media, followed by isolation, microbiological and/or serological identification and in some cases followed by subspecific characterization. This study aimed to develop a faster, powerful, more sensitive and reproducible diagnostic tool to monitor a specific pathogen contamination in drinking water by specific antibodyantigen interactions. Three pathogenic bacteria such as Pseudomonas aeruginosa, Escherichia coli and Legionella spp. were detectedby immunofluorescence technique with fluorochome tagged antibodies. Our results showed a good specificity of the antibodies in a very complex bacterial mixt as well as a starting detection level from 1 bacteria/ml. Overall, these techniques proved to be a reliable one, time-effective and sensitive for diagnosis and prevention of drinking water quality and waterborne bacterial disease.

Catalina Stoica has completed her PhD in 2016 from University of Bucharest, Ecological Systems and Sustainability Department, Faculty of Biology in Romania. She is currently working as a Research Scientist in Laboratory of Bioassay-Biological Analysis of the National R&D Institute for Industrial Ecology-ECOIND, the only institute in Romania that displays a global approach on industrial ecology and environmental issues. She has published more than 18 papers both as first author and co-author in international journals as well as three co-authored book chapters.

The human population increase led to agricultural and farming development, which is limited by its land availability. Nowadays, a major direction is based on the production efficiency and on the economy of size. The economy of size aims to lower the average cost per unit of production as the production increased. In our study, we used a very abundant and economically efficient nutrient to reduce the feeding costs, but to keep at least the same level of eggs and meat poultry production. The new nutrient formula was based on cellulose fibres, combined to a cellulosic enzymatic food supplement. Our in vitro results showed that by adding a cellulase based compound to the new feeding nutrient the cellulose content decreased, so the poultry could digest the new and economically efficient diet without any duodenal negative impact. In vitro enzyme – substrate assays were performed on 14 celluloses based nutrients (substrates) and 2 cellulase food supplements (enzymes). The results showed a 50% efficiency of cellulose degradation rate during 1h for a 1:1 enzyme-substrate ratio, especially for the BioZyme M6000 food supplement. Moreover, the nutrients and the food supplements were microbiologically tested for the presence of Escherichia coli and Salmonella spp. The results showed the absence of Salmonella spp., but the presence of Escherichia coli in a density ranged between 33 and 1609 CFU/ml. Overall, the results showed an economical viable solution for poultry farms based on a rich cellulose based nutrient supplemented with cellulase food supplement.

Marie Filteau has her expertise in Microbial Ecology, Systems Biology and Evolution. She is a Professor at the Department of Food Science at Laval University in Quebec City, Canada. She has fundamental interests in microbial ecosystems, in microbial interactions, their role in microbiome functions, and their impact on food quality. Her applied research is focused towards maple and meat products.

Maple syrup is a natural sweetener produced from the concentration of maple sap during the North-American spring season. Over the last decades, maple syrup production transitioned from artisanal to industrial, leading to annual productions of nearly ten million gallons in Canada. The increased volumes now justify the need to address quality variation in the product. A range of color grades, but also flavors and defects are encountered in maple syrup. We previously showed that maple sap microbial contamination and predominant populations are correlated with syrup quality. However, the causality underlying these correlations remains to be explored. Are microorganisms directly responsible for changes in quality, or are they only reflecting changes in maple sap chemical composition driven by the tree metabolism? To improve our understanding of maple sap composition variation, we applied a systems biology approach using a collection of barcoded Saccharomyces cerevisiae deletion strains as a biological reporter. By competing these genetically modified yeasts in various maple sap samples, we identified metabolic pathways linked to nutrient utilization that vary over the collecting season, one of which is associated with a major flavor defect. We also identified allantoic acid as the principal nitrogen source in map sap, which may play a major role in modulating microbial contamination. The next step is to identify how these molecules directly contribute to maple syrup quality and how maple sap microorganisms respond to variations in these limiting nutrient sources. Such knowledge may ultimately help devise microbial-based strategies to improve maple syrup quality.

Shaukat Iqbal Hashmi is working as head of Central Analytical Services at National Aquaculture Group, Saudi Arabia. He has completed his M.Sc. from University of Agriculture Faisalabad, Pakistan in the year of 2002. He has more than 13 years of experience in ISO 9001,ISO 14001, ISO 22000, ISO 17025 & HACCP, Gloab GAP, BAP certified organizations.

Aflatoxins are toxic substances and are fungal metabolites (Mycotoxins) produced by certain molds of the genus Aspergillus growing on several raw food commodities. Aflatoxins are highly toxic compounds and can cause both acute and chronic toxicity in humans and many other animals. Aflatoxins consist of about 20 similar compounds but only four are naturally found in foods. These are aflatoxins B1, B2, G1 and G2. Aflatoxin B1 is the most commonly found in food and the most toxic e.g. when lactating cattle and other animals ingest aflatoxins in contaminated feed, toxic metabolites can be formed and may be present in milk. These metabolites, aflatoxin M1 and M2, are potentially important contaminants in dairy products. US food safety regulations include a limit of 20 μg/kg for total aflatoxins (B1, B2, G1 and G2) in all foods except milk and a limit of 0.5 μg /kg for M1 in milk. Higher limits apply in animal feeds. If fish or shrimp are fed by contaminated feed, ingestion of such feed can cause toxic metabolites contamination and can be found in liver of the fish and hepatopancreaz of the shrimp. It is very important to assess the quality of these products by monitoring the aflatoxins residue in fish liver and shrimp hepatopancreaz. There are a variety of methods available for aflatoxins determination e.g. by ELISA or by HPLC but the real challenge is to extract the aflatoxins from liver or hepatopancreaz of fish & shrimp. For extraction of aflatoxins from meat matrix, liver, kidneys etc., 70 % methanol is recommended. Helica Total Aflatoxins protocol for ELISA was followed for analysis and validation. Total aflatoxin Assay is a solid phase direct competitive enzyme immunoassay. An aflatoxin specific antibody optimized to cross react with all four subtypes of aflatoxin was coated to a polystyrene micro well of the ELISA plate. Aflatoxins were extracted from sample with 70% methanol. The extracted sample and HRP-conjugated aflatoxin B1 were mixed and added to the antibody-coated micro well. Aflatoxin from the extracted sample and HRP-conjugated aflatoxin B1 compete to bind with the antibody coated to the micro well. Micro well contents were decanted, and non-specific reactants were removed by washing. An enzyme substrate (TMB) was added and color (blue) was developed. The intensity of the color was directly proportional to the amount of bound conjugate and inversely proportional to the concentration of aflatoxin in the sample or standard. Therefore, as the concentration of aflatoxin in the sample or standard increased, the intensity of the blue color decreased. An acidic stop solution was added which changed the chromogen color from blue to yellow. The micro wells were measured optically by a microplate reader (ELISA reader) with an absorbance filter of 450nm (OD450). The optical densities of the samples were compared to the OD's of the kit standards and an interpretative result was determined.

Alejandro Garrido Maestu is a Research Fellow working at International Iberian Nanotechnology Laboratory, with the expertise in Microbiology and Molecular Biology. At present he is working in a project based on a novel DNA amplification technique (loop-mediated isothermal amplification) for foodborne pathogens detection, combining with microfluidics and gold nanoparticles. This project is expected to have a great impact in the food industry as it will allow fast and accurate detection of the main foodborne pathogens. He has participated in several research projects involving bacterial agents detection, back in Spain and in the University of Florida where he started his Postdoctoral training. He has authored 10 peer-review papers with an h-index of 8, along with 1 book, all focused on food microbiology and bacterial pathogens.

Statement of the Problem: The genus Salmonella continues to be a major health issue. In Europe, more than 90000 of salmonellosis cases were confirmed in 2015 (1.9% increase respect to 2014), being serovar Enteritidis and Typhimurium the most prevalent ones, representing 45.7% and 15.8%. Conventional culture methods are lengthy and time-consuming, for this reason, the development of new methods that allow early detection of these pathogens is of high interest. Molecular methods have the ability of overcoming the previous limitations, being DNA amplification techniques the most attractive approach. In this sense, in recent years isothermal DNA amplification techniques have gained a lot of interest over other more stablished techniques, such as PCR/ qPCR, as providing several added-on advantages (no need of expensive equipment, many alternatives for product detection, etc.) One of these techniques with increase interest is loop-mediated isothermal amplification (LAMP).

 

Methodology: A selection of the most appropriate genetic targets was done, based on previously published studies, being selected invA for Salmonella spp. detection, STM4497 and safA for Typhimurium and Enteritidis serotyping respectively. Specificity and sensitivity assays were accomplished to ensure correct performance, and finally were implemented as the detection strategy after a simple pre-enrichment and DNA extraction. These new methods were compared against qPCR.

 

Findings: The newly designed assays performed excellent in terms of specificity, but presented lower sensitivity than qPCR. This problem was overcome with a pre-enrichment step. The evaluation of the performance of the assays in spiked food samples (turkey, chicken and egg) indicated that they may be implemented in routine food-testing. Conclusion & Significance: The developed methodology is suitable for routine food analysis, and may be used, either in a sequential mode or for directly assessing the presence of a specific serovar. LAMP represents a valid alternative to conventional DNA amplification techniques, and will allow costs reduction.

Rui-feng Mao, was born in 1963, Guilin. In 1983, he has graduated from the Department of biology, Sichuan University, and received a bachelor of science degree in microbiology. In 2011, he has completed his doctoral degree from the school of chemistry and chemical engineering of Guangxi University and received his Ph.D. in engineering. He is currently an associate professor of food science at the school of light industry and food engineering of Guangxi University. He has long  been engaged in the teaching and research work of microbiology and biochemistry. The main research areas include microbial isolation, screening, utilization of renewable resources.

In Guangxi of China, GMP (Good Manufacturing Practice) have been introduced in sugar mills since 2005 to comply with the requirements of the government administration, the clean zone was defined, and physical isolation was enforced as a procedure in sugar production. However, the rate of microbial detection in sugars did not reduce as expected. In this paper, the microbiological investigation and microbiological harm analysis of sugar clean zone in three sugar mills in Guangxi were reported. The methodology of investigating microbial species isolated from sugar cane clean area and its surrounding environment in sugarcane mill, as well as the distribution pattern of environmental microorganisms combined with air flow analysis and microbiological analysis were introduced. The research results provide the basis for control theory and control technology in the analysis of microbial harm in sugarcane mill.

Maria Turtoi is a staff member of Dunarea de Jos University of Galati, Romania. With a solid background training in food science and engineering, she teaches courses such as food unit operations, food packaging, nonconventional technologies and food safety in minimal processing. Her research activity closely follows the teaching activity. However, she has expertise in emerging technologies such as pulsed light and ultraviolet light treatments used to decontaminate the surface of food and food contact materials with the purpose of extending the shelf life of treated food, and enhancing the safety of minimally processed food.

Statement of the problem: Wheat grains are naturally contaminated with bacteria, yeasts and moulds while in the field and during storage, which represent the major cause of grain spoilage. In particular, fungi produce losses related to heating, biochemical changes, production of mycotoxins, decrease of germination capacity, and loss of dry matter. Growth of microorganisms can be prevented by drying of grains to reduce the moisture content and using of chemical preservatives. However, drying methods are expensive and chemical preservatives affect the germination capacity of wheat grains. Pulsed light (PL) is an emerging non-thermal technology able to reduce the microbial population on the surface of food and food contact materials.

 

Methodology: Wheat grains have been exposed to PL at different fluencies (2.23–30.05 J/cm2) and different number of pulses (10–60) in an experimental set-up able to rotate the wheat grains and expose the entire surface to PL. Survival populations of fungi and bacteria have been counted after each PL treatment and compared with fungi and bacteria load of control sample. Germination tests have been performed for PL treated samples and control. Subsequently, wheat grains used to determine the germination capacity have been dried, ground and subjected to analyse antioxidant capacity and enzymatic activity.

 

Findings: PL exposure of wheat grains resulted in reductions of up to 2.3 log CFU/g and 1.2 log CFU/g for fungi and bacteria respectively. Germination capacity of wheat grains has not been affected by PL treatment, but germination rate has been improved due to reduction of microbial load and enhanced of enzymatic activity.

 

Conclusion & Significance: Results confirm the positive effect of PL treatment on wheat grain decontamination and enhancement of germination rate which extend the shelf life of grains and use in food industry.

Amparo Gamero holds a PhD in Food Science and Technology. She works at the Institute of Food Science and Technology of the Spanish Research Council. Her research topic deals with the study of the role of Saccharomyces and non-conventional yeasts in aroma production during food fermentations. She has several SCI publications and has active participation in international conferences.

Saccharomyces cerevisiae is the most common species used in bakery due to its rapid sugar consumption and CO2 production, the most important attributes required to leaven the dough. These attributes have been shown not to be unique to Saccharomyces cerevisiae, but also found in several non-conventional yeast species. Only a reduced number of these non-conventional yeast species presenting potential to be used in bakery have been described. The rest of them remain poorly studied, being a vast untapped potential for the use as leavening agents and flavor producers. In this research work, the potential of several non-conventional yeasts to be used in bakery was assessed employing dough-like conditions in the microbread platform. This platform allows fast screenings of different recipes. In this case, the capabilities of bread leavening and aroma formation of different non-conventional yeast species were tested. The results showed that bread leavened with the non-conventional yeasts Kazachstania gamospora and Wickerhamomyces subpelliculosus had better overall results compared to control baker’s yeast, especially regarding aroma profiles. In this way, this study points out Kazachstania gamospora and Wickerhamomyces subpelliculosus as interesting alternative baker’s yeasts.