Inayara Cristina Alves Lacerda

Enteroxigenic Staphylococcus spp. and other microbial contaminants during production of Canastra cheese, Brazil

Canastra cheese is produced from raw cows milk, and it is made at the farmhouse level using artisanal procedures and natural starters. The aim of this work was to determine the main hygienic-sanitary indicators and enterotoxigenic staphylococcal strains present during the manufacturing of traditional cheese of Serra da Canastra region, Minas Gerais state, Brazil. Samples from 10 farms were studied, and they included: water employed in the process, raw milk, natural starters, cheese curd before salting and cheese after five days of ripening. All water samples exhibited faecal coliform contamination above the maximum acceptable value recommended by Brazilian standards. Pseudomonas aeruginosa and sulfite-reducing clostridia were also isolated from the water samples. In five samples of raw milk faecal coliform were above the limits allowed by the Brazilian legislation. The counts of Staphylococcus spp. in milk were between <2.0 to 4.9 log.cfu.ml-1. The counts of microbiological indicators were higher in natural starters and curd. High levels of faecal and total coliform, as well as molds, were found in the cheese samples. In all cheeses analyzed Staphylococcus spp. were found in levels above 5.0 log.cfu.g-1. The enterotoxins (SE) most frequently produced by Staphylococcus spp. strains were SEB and SEC. A high number of coagulase negative Staphylococcus strains were also enterotoxin producers. None of the samples contained Salmonella spp. or Listeria spp. These results point out a need for improvements in the production process of the artisanal cheese produced at Serra da Canastra in Brazil.

Identification of lactic acid bacteria associated with traditional cachaça fermentations

During the production of traditional cachaca (alembic´s cachaca), contamination of the fermented must is one of the factors leading to economic losses in the beverage manufacturing industry. The diversity of bacterial populations and the role of these microorganisms during the cachaca production process are still poorly understood in Brazil. In our work, the fermentation process was followed in two distilleries located in the state of Minas Gerais. The objective of this work was to identify the populations of lactic acid bacteria present during cachaca fermentation using physiological and molecular methods. Lactic acid bacteria were isolated in high frequencies during all of the fermentative processes, and Lactobacillus plantarum and L. casei were the most prevalent species. Other lactic acid bacteria were found in minor frequencies, such as L. ferintoshensis, L. fermentum, L. jensenii, L. murinus, Lactococcus lactis, Enterococcus sp. and Weissella confusa. These bacteria could contribute to the increase of volatile acidity levels or to the production of compounds that could influence the taste and aroma of the beverage.

Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaça and minas cheese production using culture-independent 16s rRNA gene sequence analysis

We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaca and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6% of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8% of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaca samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9% of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75% of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9% of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent.

Identification of Staphylococcus spp. isolated during the ripening process of a traditional minas cheese

Estudou-se a dinâmica das populacoes de Staphylococcus spp. durante a maturacao do queijo Canastra, em tres fazendas localizadas no estado de Minas Gerais. A presenca dos genes que codificam para a producao das enzimas coagulase (coa), termonuclease (nuc) e producao de enterotoxinas (sea, seb, sec e sed), em linhagens de Staphylococcus isoladas durante os 60 dias de maturacao do queijo foi analisada. Tambem foi investigada a presenca de enterotoxina estafilococica A, C e D nas amostras de queijo. As amostras de queijo com 0, 7, 15, 30 e 45 dias de maturacao apresentaram contagens de Staphylococcus spp. entre 103 e 108ufc / g. Todos os isolados coagulase positivo nos testes fisiologicos apresentaram o gene coa. Nao foi observada associacao entre os resultados obtidos com os testes bioquimicos e aqueles obtidos com a PCR usando iniciadores gene-especificos para linhagens coagulase negativa. Os genes da coagulase e termonuclease ocorreram simultaneamente em 41,3% dos Staphylococcus spp. testados. Nenhum dos isolados de Staphylococcus apresentou os genes que codificam para a producao das enterotoxinas SEA, SEB, SEC ou SED. As enterotoxinas A, C ou D nao foram detectadas em nenhuma das amostras de queijo analisadas.

Selection of starter cultures for the production of sour cassava starch in a pilot-scale fermentation process

Sour cassava starch (Polvilho azedo) is obtained from a spontaneous fermentation conducted by microorganisms from raw materials and fermentation tanks. This product is traditionally used in the baking industry for the manufacture of biscuits and Brazilian cheese breads. However, the end of fermentation is evaluated empirically, and the process occurs without standardization, which results in products of inconsistent quality. Predominant microbiota from a cassava flour manufacturer was isolated in order to select starter cultures for the production of sour cassava starch in a pilot-scale fermentation process. Lactic acid bacteria and yeasts were isolated, enumerated and grouped by Restriction Fragment Length Polymorphism, and PCR fingerprinting, respectively. One isolate of each molecular profile was identified by sequencing of the rRNA gene. LAB were prevalent throughout the entire process. Lactobacillus brevis (21.5%), which produced the highest values of acidity, and Lactobacillus plantarum (13.9%) were among the most frequent species. Pichia scutulata (52.2%) was the prevalent yeast and showed amylolytic activity. The aforementioned species were tested as single and mixed starter cultures in a pilot-scale fermentation process for 28 days. L. plantarum exhibited better performance as a starter culture, which suggests its potential for the production of sour cassava starch.

POTENCIAL RISCO DE INTOXICAÇÃO ALIMENTAR POR Staphylococcus spp. ENTEROTOXIGÊNICOS ISOLADOS DE BOLOS COM COBERTURA E RECHEIO

As Doencas Transmitidas por Alimentos (DTA) sao causadas pelo consumo de alimentos contendo contaminantes fisicos, quimicos ou biologicos e constituem uma preocupacao mundial. Dentre as DTA, uma das principais causas de gastroenterites e a intoxicacao originada pela ingestao de enterotoxinas estafilococicas pre-formadas nos alimentos. O presente estudo teve como objetivo caracterizar as linhagens enterotoxigenicas de Staphylococcus spp. e as enterotoxinas mais frequentes presentes em amostras de bolo confeitado envolvidas em surtos de intoxicacao alimentar. Para atender ao objetivo proposto, realizou-se um levantamento de dados secundarios (2010-2013) e analises microbiologicas de amostras de bolo confeitado (2014-2015), envolvidas sem surtos de toxinfeccao alimentares pelo microrganismo, ocorridos em Minas Gerais. Neste periodo (2010-2015) foram analisadas 32 amostras de bolos confeitados e, de acordo com a analise da ficha de inquerito, os surtos envolveram 683 pessoas, dentre estas, 613 desenvolveram sintomas de intoxicacao alimentar. Os mais frequentes foram vomito, diarreia, nausea, dor abdominal e cefaleia com periodo de incubacao medio de 6h. Dentre as amostras analisadas, em 29 (90,6%) a contagem de Staphylococcus coagulase positiva (SCP) foi superior a 1,0x10 5 UFC/g, sendo que S. aureus foi o agente isolado em 75% dos episodios e Staphylococcus coagulase negativa (SCN) enterotoxigenico em apenas tres. A maioria dos surtos investigados ocorreu em residencias, correspondendo a 59,4%. O perfil enterotoxigenico das cepas de isoladas demonstrou a prevalencia da Enterotoxina Estafilococica A (SEA). Nas amostras do alimento, a enterotoxina detectada com maior frequencia foi a Enterotoxina Estafilococica C (SEC). Palavras-chave: Doencas transmitidas por alimentos, Enterotoxinas estafilococicas, Analise de perigos e pontos criticos de controle, Bolos confeitados, ABSTRACT: The Foodborne Diseases (FBD) are a global concern, originated from the consumption of food with physical, chemical or biological contamination. Among the FBD, a major cause of gastroenteritis is the intoxication caused by ingestion of preformed staphylococcal enterotoxins in foods. This study aimed to describe the enterotoxigenic species of Staphylococcus spp. and its enterotoxins frequently found in filled cake samples involved in food poisoning outbreaks. The data collection and the microbiological analysis were performed with filled cake samples (2014-2015), related with food outbreaks by Staphylococcus spp., which occurred in Minas Gerais state. In this period (2010-2015), 32 samples of filled cakes from outbreaks by enterotoxigenic strains of Staphylococcus spp. were evaluated. These outbreaks involved 683 people and, among these, 613 developed symptoms of food poisoning. The most common symptoms were vomiting, diarrhea, nausea, abdominal pain and headache with an average incubation period of 6 hours. Among the filled cake samples, in 29 of them (90.6%), Staphylococcus coagulase positive count was higher than 1,0x105 CFU/g. S. aureus was isolated agent in 75% of episodes and strains of enterotoxigenic coagulase-negative Staphylococcus (CNS) in only three episodes. The majority of the investigated outbreaks occurred in houses (59.4%). The profile of isolated enterotoxigenic Staphylococcus strains showed the prevalence of Staphylococcal enterotoxin A (SEA). In food samples, the most frequently detected enterotoxin was Staphylococcal enterotoxin C (SEC). Keywords: Foodborne diseases, Staphylococcal enterotoxins, HACCP, Outbreak Food Poisoning, Filled cake,