Science Serving Society

Development of a Rapid Identification Method for Food Bacteria and Molds:  MALDI-TOF MS Project

Mitsuru Katase, PhD, Fuji Oil Co., Ltd., Osaka, Japan, Expert members of Microbiological Food Safety Task Force in ILSI Japan, NITE Biological Resource Center (NBRC*)

Spoilage of food products due to microorganisms is one of major problems. Information on the safety risk should be obtained as soon as possible when the spoilage occurs. Identification of the spoilage bacteria and molds is extremely essential, but current methods need hours or days, namely they are very time-consuming. We evaluated a novel rapid technique which gives results in minutes for identification of food microorganisms and will verify the standard protocol. The novel technique is based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), which is run at low cost: $ 0.20/sample by protein fingerprinting. Microbial strain library is very important for fingerprinting with molecules composed mainly of microorganism-specific ribosomal protein. But, as this technique started in the clinical field, the library included mainly clinical bacteria. We, Microbiological Food Safety Task Force as one made much effort to convince the equipment manufacturers to expand the library for spoilage bacteria in food industry and provided them with known microbial strains owned by Japan Canners Association. We also held a symposium to share the information with food industry in Japan. The activity finished a feasibility study of application of new methodologies to identification of food microorganisms. However, mold identification by MALDI-TOF MS is still unstable and difficult. Therefore, it has started the collaborative study of the methodologies for molds with NBRC*. As mentioned above, the key to success so far was to design an elaborate framework of the technique and to network various stakeholders. Once the technique is fully established, it will drastically speed up the product inspections to ensure the microbiological food safety of products and to solve microbiological problems. And, it is expected that all stakeholders in the food industry will share the information on the new technique with each other, which will surely strengthen the whole industry and benefit the consumers.

*A national research institute of Japan 

Evaluation of the Acute and Chronic Food Consumption by the Brazilian Population through the Family Budget Survey (POF 2008-2009 – IBGE) to Assess Pesticide Dietary Exposure

Laura B. Valério; Mariana C. N. Pais; Andreia N. O. Jardim; CRISTIANA L. CORRÊA*; Heloísa Kalvan; Márcia Pala; Renata Volpi; Simone Guimarães * Scientific Director at IBTox – Brazilian Institute of Toxicology and Scientific Coordinator at the Agrochemical´s Task Force/ILSI Brazil.

In Brazil, the Family Budget Surveys (POF) are performed periodically by the IBGE (Brazilian Institute of Geography and Statistics), which aims at obtaining information on the consumption habits of the families surveyed, using households as collection units (IBGE, 2017). The information on the individual consumption of food products were obtained through the Food Consumption Unit, also known as POF 7, in the survey performed in 2008/2009, in which subjects from 10 years of age were asked to provide a detailed report of the daily food consumption during two non-consecutive days (IBGE, 2011). The results were listed in the final consumption form, i.e., as raw (in natura), processed or prepared (recipes) food products. Therefore, in order to use these consumption data in risk assessment studies of dietary residues and contaminants, prepared food products were dismembered into their initial ingredients and processed food products were associated to their respective natural foods. At the end of this process, the amounts of each recipe item corresponding to the fresh ingredient could be added to the amounts of these same products directly consumed as raw (in natura) foods. The process described above was performed in the first phase of the project. At the end, the average individual consumption of raw foods by the Brazilian population in the 5 main regions (North, South, Middlewest, Northeast and Southeast) was calculated with the aim of using this data in the chronic exposure evaluation of chemical substances present in food products. However, for the acute exposure calculation, which occurs in a single meal or during 24 hours, the highest consumption values are used, often referring to the 97.5 percentile of the consumption distribution, also known as large portion (LP) of consumption. This evaluation addresses the risk of ingesting a chemical substance present at high concentrations in a food product, which in turn will be consumed in high amounts, in one single day. Given the importance of the acute exposure evaluation both nationally and worldwide, and the current discussions regarding this subject, the second phase was developed using the data obtained in the first phase. For the first time, a public tool was created with national and realistic values of large portion of food consumption (both LP and LPbw), as well as reference of a number of national unit weight (U) of commodities, thus contributing to the country’s scientific development and enabling the performance of acute dietary risk assessment with more current data of consumption by the Brazilian population. The data generated will be further used in the acute and chronic dietary risk assessment, aiming to understand if it is safe to consume food products containing pesticide residues in Brazil.

Validation of Safety Control Measures and Pathogen Reduction Steps for the Safe Production of Traditional Artisanal Dairy Products from the Mesoamerican Region

Jessie Usaga and D. Viquez, University of Costa Rica, Costa Rica; E. Wong, Department of Food Technology, University of Costa Rica (UCR)

Artisanal dairy products have caused numerous foodborne outbreaks in the Mesoamerican region due to deficient application of Good Manufacturing Practices and a lack of safety control measures, such as milk pasteurization. Nevertheless, most regional producers still rely on traditional approaches, including dry salting, mild heat treatments, and mild acidification, as sole pathogen reduction steps to ensure the safety of highly consumed traditional products. However, there is a lack of scientific evidence to confirm the effectiveness of these measures in controlling the pathogens of public health concern. Therefore, this project aims to obtain science-based data to determine the adequacy of the currently applied control measures, and to develop guidelines for the safe processing of these products. Three highly consumed products in the region (a dry salted cheese, a traditional fermented milk, and a stretched-curd cheese) are under investigation. At least 15 local producers have been visited (3 independent visits per producer), to evaluate compliance of GMPs, based on local regulations, and to document the formulation and processing conditions followed by these producers. No formal scientific guidelines regarding these products are yet available. During visits, products were sampled to characterize their physicochemical properties, and to evaluate their microbial quality (presence of generic E. coli and L. monocytogenes has been determined). Overall, basic GMPs were not in compliance and a large proportion of the visited producers do not apply a pasteurization step to the milk, and do not consistently apply any standardized control measures to ensure the safety and microbial stability of their products. Models of three common dairy products were developed at a pilot plant scale, based on producers’ specifications: dry salted cheese, fermented milk, and pulled-curd cheese. For the dry salted cheese, the effects of salting method (dry and moist) and cheese size (two different sizes commonly used by cheese makers) on the pH, water activity, sodium content and probability of pathogen growth are under investigation. For the case of fermented milk, the effect of factors such as fermentation temperature (refrigeration, room temperature, 37ºC) and use of starter cultures, on the growth of lactic acid bacteria and milk acidification kinetics have been evaluated. Finally, for the pulled-curd cheese the heat penetration during the curd stretching step and the potential of pathogen growth in the cheese (after molding) have been analyzed. The most important findings collected so far have been employed to develop standardized guidelines and teaching materials (in Spanish and lay terms), to encourage the safe production of these products. Training activities have also been held to validate the adequacy of these materials. This project will contribute to the training of artisanal producers and hopefully will eventually facilitate the establishment of regional dairy safety regulations.

Latin American Health and Nutrition Study – Part II

Mauro Fisberg, The Instituto Pensi, Fundação Jose Luiz Egydio Setubal, Hospital Infantil Sabara, São Paulo, Brazil; The Departamento de Pediatria, Universidade Federal de São Paulo, São Paulo, Brazil. Georgina Gómez, Department of Biochesmistry, School of Medicine, University of Costa Rica.

The ELANS (Latin American Health and Nutrition Study) is a transversal, multicenter study conducted in adolescents and adults living in urban areas of eight Latin America countries (Argentina, Brazil, Chile, Colombia, Costa Rica, Ecuador, Peru and Venezuela). The aim of the study was to provide up-to-date, reliable and comparable data of dietary intake, physical activity, and nutritional status among representative urban populations stratified by region, cultural background, socioeconomic status, age and gender. This study adds new scientific-based evidence to describe the interplay among datary intake, physical activity, and anthropometric measurements.

A random complex, multistage sampling was performed to select a representative sample of the urban household population, to obtain a final sample of 9,218 individuals, 15–65 y of age, stratified by geographical location, gender, age and socioeconomic status. Two non-consecutive 24-hour dietary recalls collected between 2014 and 2015 were used to evaluated dietary intake and processed by Nutritional Data System for Research (NDS-R). Physical Activity (PA) was estimated with the International Questionnaire of Physical Activity (IPAQ), and an objective measure of the PA for 40 % of the sample was obtained through the use of the accelerometers during 7 valid days.

Overall, mean energy intake (EI) was 1959 kcal, with a balanced distribution of macronutrients (54% carbohydrates, 30% fats and 16% proteins). Main food sources of energy were grains, pasta, and bread (28%), followed by meat and eggs (19%), oils and fats (10%) and non-alcoholic homemade beverages (6%) and ready-to-drink beverages (6%). More than 25% of EI was provided from food sources rich in sugar and fat, sugary drinks, pastries, chips, and candies. Meanwhile, only 18% of EI was from food sources rich in fiber and micronutrients, such as whole grains, roots, fruits, vegetables, beans, fish and nuts. No critical differences were observed by sex or age. A high prevalence (>95%) of inadequate dietary intake of vitamin D, was observed in both genders for all eight countries. Significant differences were observed in dietary intake of Calcium and Vitamin C between countries related to the differences on c food sources and only an inadequate intake of iron was observed among Brazilians specially in fertile age woman. The prevalence of physically active individuals was 52,5%, ranging from 35.4% in Venezuela to 78.3% in Ecuador. Men were more active than women in all eight countries. A high prevalence of excess weight (BMI>25 kg/m2) is present in all countries, accounting for 60% of the participants. This prevalence is higher in women than in men in all countries except for Argentina and Chile. Colombia is the country with the lowest prevalence of obesity (15%), while Chile is the country with the highest (27%).

Since this is a unique study that provide up-dated data on nutrition and physical activity in Latin Americans with a detailed, simultaneous and standardized methodology, the results of this study would be of the greatest actionable value to determine the dietary patterns and energy and nutrient intakes for the developing of dietary recommendations and policies to address the adverse consequences of inappropriate dietary patterns and physical inactivity.

Gene Drive Technology for Malaria Control

Dr. Andrew Roberts, ILSI Research Foundation, USA

Malaria continues to be a devastating disease, especially in sub-Saharan Africa where the deadliest malaria parasite (Plasmodium falciparum) is transmitted by the most efficient vector mosquitoes (Anopheles gambiae). Concerted efforts to reduce the incidence of malaria in the 21st century through a combination of public awareness, medication, pesticide control of vectors and the widespread distribution of insecticide treated bed nets has achieved impressive gains. However, insecticide resistant vectors, drug resistant plasmodium and the unsustainable cost of control efforts threaten to reverse these gains.

Malaria control efforts are exploring new technologies to support existing control methods, and one of these is the use of gene drives targeting the vector mosquito Anopheles gambiae. Designed to introduce a rapidly spreading genetic element into wild mosquito populations to either suppress the vector or reduce its ability to vector the disease, gene drives have the potential to help malaria control efforts reach the critical “last mile” goal of breaking the cycle of parasitism and ending malaria. But before a gene drive can be used in the environment, regulators need to understand how to assess the potential risks.

In cooperation with the Foundation for the National Institutes of Health (FNIH) and the New Partnership for African Development (NEPAD), the ILSI Research Foundation is working with African scientists and regulators to introduce the science behind the gene drive concept and begin the task of scoping future risk assessments. This process is referred to as “problem formulation” and, done well, facilitates a transparent and credible risk assessment to inform future decision making.

Cristiana Corrêa, PhD

Cristiana Leslie Corrêa is the Technical Director at Planitox – The Science-based Toxicology Company and Scientific Director at the Brazilian Institute of Toxicology (IBTox), Campinas, S.P; Brazil. Her research interests focus on Human Health Risk Assessment, working as a toxicologist consultant in registration and reevaluation process of chemicals, food ingredients and agrochemicals. Cristiana has written some books, chapters of books and peer-reviewed publications in the field of Toxicology. She is a Member of the International Life Science Institute (ILSI)/Brazil in the Scientific Committee. Cristiana earned a Masters Degree in Toxicological Analysis and PhD in Toxicology at the University of São Paulo, Brazil and became a specialist in Toxicological Risk Assessment at the Postgraduate Education in Toxicology Program at Wageningen University, The Netherlands.

Mauro Fisberg, PhD, MD

Dr. Fisberg is a pediatrician and nutrologist in Sao Paulo, Brazil. He is the coordinator of the Nutrology and Feeding Difficulties Center- Pensi Institute - Fundação José Luiz Setubal - Sabará Children's Hospital Sao Paulo- Brazil. He is also an Associate Professor of Pediatrics, Escola Paulista de Medicina - Federal University of Sao Paulo- UNIFESP. Dr. Fisberg serves as a Scientific Coordinator of the Healthy Life Style Task Force and is member of the Board ILSI Brazil. He is a past President and General Secretary of the Latin American Society for Pediatric Research-SLAIP as well as an alumnus of the World Hunger Program- United Nations University and the Kellog's Foundation Leadership Program- Partners of the Americas.

Georgina Gómez, ND MSc.

MSc. Gómez is a full professor and researcher at the University of Costa Rica where she has been working and studying for the last 25 years. She is a nutritionist, who graduated from the University of Costa Rica with a Masters Degree in Biochemistry and a Masters Degree in Human Nutrition. As a principal investigator of the Latin American Nutrition and Health Study (ELANS)-Costa Rica, she is leading the dietary assessment of urban Costa Rican population.

Andrew Roberts, PhD

Dr. Andrew F. Roberts is the Deputy Executive Director of the ILSI Research Foundation, where he is responsible for programs addressing environmental risk assessment and food safety assessment for biotechnology. Dr. Roberts joined the ILSI Research Foundation in December 2009 as the Deputy Director of the Center for Environmental Risk Assessment (CERA), where his first tasks included developing tools and materials for use in training and capacity building related to the problem formulation approach to environmental risk assessment published by the Research Foundation (Wolt et al 2009). He has served as the coordinator for CERA’s capacity building projects under the USAID funded South Asia Biosafety Program (SABP) and the World Bank funded Partnership for Biosafety Risk Assessment and Regulation, in Bangladesh, Pakistan, and Vietnam as well as providing technical support for capacity building work in Brazil, India, Japan, Chile, and South Africa. In January of 2015 he became the director of CERA as well as the Center for Safety Assessment of Food and Feed (CSAFF), which works on food and feed safety assessment for foods derived from genetically engineered plants. In January 2017, he became the Deputy Executive Director of the ILSI Research Foundation.

Prior to joining ILSI RF, Dr. Roberts worked at the U.S. Department of Agriculture in several different capacities, all related to the regulation of agricultural biotechnology. He began his career at USDA as an AAAS Risk Policy Fellow in the Office of Science of Biotechnology Regulatory Services (BRS), the group responsible for regulating genetically engineered plants at USDA. After spending a year in the New Technologies office of the Foreign Agricultural Service serving as the lead for USDA’s efforts related to the Cartagena Protocol on Biosafety, he returned to BRS to serve in the International Affairs branch where he remained until joining ILSI RF.

Dr. Roberts received his Ph.D. in Cell and Developmental Biology from Rutgers University where he worked on signal transduction in the model nematode C. elegans.

Jessie Usaga, PhD

Dr. Usaga currently holds a position as Associate Professor in the Department of Food Technology at the University of Costa Rica. She received a B.S. degree in Food Technology from the University of Costa Rica in 2006, and a Ph.D. degree in Food Science, with minors in microbiology and food engineering, at Cornell University in 2014, where she co-authored 8 peer-reviewed publications regarding juice and beverage safety assurance. Between 2016 and 2017, Dr. Usaga was a Visiting Associate Professor at Cornell University, where she managed the laboratory at the High Pressure Processing (HPP) Validation Center, worked on developing protocols for the validation of high pressure processed foods and conducted several microbial challenge studies and research. Her area of expertise includes product development, food microbiology, food quality and safety. Her current primary focus of research emphasizes on processing and microbiology of traditional and artisanal foods, including the application of traditional thermal processing and nonthermal emerging technologies for safety assurance (UV light, membrane filtration, and HPP). Dr. Usaga is a Process Authority for acidified foods, and an active member of ILSI Mesoamerica, IFT, and IAFP. She promotes the involvement of students with these organizations through their participation in international product development and research competitions. Her mentees have been awarded with first place in multiple competitions organized by IFT, IUFoST, and the Chinese Institute of Food Science and Technology. This year, Dr. Usaga is honored to receive the Malaspina International Scholars Travel Award which motivates her to pursue her personal and professional aspirations of promoting food safety through research and extension initiatives and supporting local food companies by providing training and technical advice in food processing and safety.