Hazards and Control of Risks in Artesanal Meat Products in Portugal Thesis submitted to the Universidade Católica Portuguesa, to attain the degree of PhD in Biotechnology with specialisation in Microbiology by Maria Isabel Pereira da Silva Campelos July 2012 Hazards and Control of Risks in Artesanal Meat Products in Portugal Thesis submitted to the Universidade Católica Portuguesa, to attain the degree of Ph.D. in Biotechnology with specialisation in Microbiology by Maria Isabel Pereira da Silva Campelos Under the supervision of Professor Paula Teixeira Under the co-supervision of Professor Paul Gibbs Under the co-supervision of Professor Tim Hogg July 2012 ―The known is finite, the unknown infinite; intellectually we stand on an islet in the midst of an illimitable ocean of inexplicability. Our business in every generation is to reclaim a little more land.‖ Thomas Henry Huxley. 1884 ―Remember that all models are wrong; the practical question is how wrong do they have to be to not be useful‖. George Edward Pelham Box, 1987 ―The best model of a cat is another cat or, better, the cat itself‖, Norbert Wiener, 1970 To my Father General Introduction GENERAL INTRODUCTION ―Salpicão de Vinhais‖ and ―Chouriça de Vinhais‖ are traditional dry-fermented smoked meat sausages produced in the Vinhais, a small region of Trás-os-Montes. These sausages are registered as Protected Geographical Indication (PGI) and are a significant economic and cultural asset. The production of this kind of product is mainly empirical, developed and perfected through centuries. The scientific knowledge of this sausage variety is limited. Hence, the collection of sound, scientific knowledge regarding its microbiological and chemical characteristics is a necessity and a helpful tool for producers, retailers, official control agencies and consumers, providing for much needed information regarding quality and food safety. Quantitative microbiological risk assessments aim at evaluating the risks, namely the probabilities and severities of adverse health effects for consumers, resulting from the exposure to pathogenic microorganisms present in foods, and implementing appropriate management options. Governments need risk assessment in order to select risk management measures for food trade at national and international levels, avoiding risks to the population. But also, risk assessment can be useful at production, at retailer, and at consumer levels. This work aims to collect scientific data that could, partially, fill the gaps in knowledge regarding these products, analyse the data and develop a risk-based study, according to an internationally accepted framework and finally, to explore the effect of hypothetical risk management measures on the safety of consumers, regarding traditional dry fermented meat smoked sausages. i General Introduction To perform a quantitative microbiological risk assessment, relevant data was either extracted from experimental data and when this was not possible, from relevant scientific literature. To perform the exposure assessment, a probabilistic model was developed. Since the physico-chemical (intrinsic) characteristics of the final product traditional dry fermented smoked sausages, fall near the boundaries that allow, or not, growth of L. monocytogenes, a growth/no growth equation, specific for the conditions of ―Salpicão‖ and ―Chouriça de Vinhais‖ was developed to be included in the model. Though official organisations like the European Food Safety Agency, state the relevance of growth/no growth boundaries, few risk assessments incorporate this in their exposure assessment models. Traditional dry fermented smoked sausages present low to medium risk to the health of consumers. The use of more restrictive Performance Objectives during processing and/or distribution will result in a further reduction of risk. A more rigorous control of final product water activity and the use of a bacteriocinogenic bioprotective culture during production may contribute significantly to reducing the risk of listeriosis in consumers of these Traditional Dry Fermented Smoked Sausages ii Abstract ABSTRACT ―Salpicão de Vinhais‖ and ―Chouriça de Vinhais‖ are traditional dry-fermented smoked meat sausages produced in Vinhais, a small region of Trás-os-Montes. The scientific knowledge of this sausage variety is limited. This work aims to collect scientific data that could, partially, fill the gaps in knowledge regarding these products, analyse the data and develop a risk-based study, according to an internationally accepted framework and finally, to explore the effect of hypothetical risk management measures on the safety of consumers, regarding traditional dry fermented meat smoked sausages. Seventy seven samples of ―Salpicão‖ and ―Chouriça de Vinhais‖ were purchased from producers, local markets and retail stores. Their microbiological and physical chemical characteristics were analyzed. The same analyses were performed on the products during the production processes. The raw materials and ingredients were also analyzed. Regarding the pathogenic flora, Staphylococcus aureus, spores of sulphite-reducing clostridia, Escherichia coli 0157:H7, Yersinia spp. and Salmonella spp., were not detected in any of the samples analyzed; Listeria monocytogenes was detected in 14.3% of the samples. The manufacturing process, namely fermentation, ripening/drying and smoking reduced the numbers of pathogen and hygiene indicator microorganisms. According to the Commission Regulation (EC) 2073/2005, 39% of the contaminated samples of ―Salpicão‖ and ―Chouriça de Vinhais‖ were able to support growth of L. monocytogenes. A quantitative microbiological risk assessment using a probabilistic model was developed. Considering the Portuguese population, with data obtained in this iii Abstract work, the calculated risk of listeriosis, for the intermediate age sub-population was 0.1297 cases per year, for the perinatal sub-population, 1.3695 cases per year, and elderly sub-population 0.1995 cases per year. This means that Traditional Dry Fermented Smoked Sausages constitute a low risk for the Intermediate Age and Elderly population (less than 1 case per annum) and an intermediate risk for the Perinatal populations, considering this last group of consumers as adult pregnant women (1-10 cases per annum). The effect of several putative risk management actions such as the adoption of a Performance Objective of 10 CFU/g, 1 CFU/g and 0.04 CFU/g reduced the risk of listeriosis for all sub-groups, at the end of shelf life, by 25.5%, 58.4% and 58.6%, respectively. The effect of bacteriocinogenic strain of Pediococcus acidilactici HA6111-2, previously isolated from ―alheira‖, was studied on both ―Salpicão‖ and ―Chouriça‖, in a challenge test against a cocktail of Listeria innocua, at pilot scale. A reduction of the numbers of Listeria innocua was achieved in all batches, being the biphasic equation the model that gave the better fit. The use of a bacteriocinogenic strain added to the batter reduced the risk of listeriosis for all sub-groups by 26.7%; obtaining a final product with aw below 0.92, supposedly below the growth limits of L. monocytogenes, reduces the risk by 8.6% in all sub- groups of population; the combination of the bacteriocinogenic strain and the limit of aw 0.92 reduces the risk by 41.0%. Traditional dry fermented smoked sausages present low to medium risk to the health of consumers. The use of more restrictive Performance Objectives during processing and/or distribution will result in a further reduction of risk. iv Abstract A more rigorous control of final product water activity and the use of a bacteriocinogenic bioprotective culture during production may contribute significantly to reducing the risk of listeriosis in consumers of these Traditional Dry Fermented Smoked Sausages (TDFSS). v vi Resumo RESUMO O Salpicão de Vinhais e a Chouriça de Vinhais são enchidos fermentados, secos e fumados, produzidos em Vinhais, uma pequena região de Trás-os-Montes. Este trabalho pretende recolher dados científicos que possam preencher, ainda que parcialmente, os lapsos no conhecimento deste tipo de produtos, de acordo com uma abordagem aceite internacionalmente e, finalmente, explorar o efeito potencial que as medidas de gestão de risco podem ter na segurança dos consumidores, relativamente a enchidos tradicionais, fermentados, secos e fumados. Setenta e sete amostras de ―Salpicão‖ e ―Chouriça de Vinhais‖ foram compradas em produtores, mercados locais e hipermercados. As suas características microbiológicas e físico-químicas foram analizadas. As mesmas análises foram realizadas ao produto em curso, em diferentes fases do processo produtivo. As matérias-primas e ingredientes também foram analisadas. Relativamente à flora patogénica, não foram detectados esporos de Staphylococcus aureus, esporos do género Clostridia sulfito-redutores, Escherichia. coli 0157:H7, Yersinia spp. e Salmonella spp; Listeria monocytogenes foi detectada em 14.3% das amostras. O processo de fabrico, nomeadamente fermentação, maturação/secagem e fumagem reduziram o número de patogénicos e microrganismos indicadores de higiene. De acordo com o Regulamento da Comissão (CE) 2073/2005, 39% das amostras de ―Salpicão‖ e ―Chouriça de Vinhais‖ contaminadas eram capazes de permitir o crescimento de L. monocytogenes. Foi desenvolvida uma avaliação quantitativa de risco microbiológico. Considerando a população portuguesa, com os dados obtidos neste trabalho, o risco calculado de listeriose seria de 0.1297 casos por vii Resumo