Bioprospecção do potencial tecnológico, probiótico e avaliação da segurança de bactérias láticas endógenas de queijos artesanais brasileiros

Author(s)

Larissa Pereira Margalho

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia de Alimentos
• Defense date: 2020-03-03

Examining board members

Anderson de Souza Sant'Ana; Antonio de Anchieta Câmara Júnior; Carlos Augusto Fernandes de Oliveira; Adriano Gomes da Cruz; Fernanda Bovo Campagnollo

• Advisor: Anderson de Souza Sant'Ana

Abstract

Brazilian artisanal cheeses (BAC) have unique characteristics, which reflect the history, culture, and lifestyle of traditional producing communities. The endogenous microbiota of these cheeses is mainly composed of lactic acid bacteria (LAB). These microorganisms play a crucial role in biochemical reactions that precede different textures, aromas, and flavors. This study aimed to investigate the technological and probiotic properties of LAB isolated from BAC produced in the five regions of Brazil. Chapter one shows results about the technological potential (acidification rate, proteolysis, lipolysis, antimicrobial activity, and growth under different pH conditions, bile salts, and sodium chloride) of 1,002 BAL strains isolated from 578 samples of BAC. For this purpose, high-throughput screening was performed using 96-well microplates, and all data analyzed using software R. The best strains (n=220) were selected due to its potential to be applied in dairy cultures. Chapter two aimed to investigate the occurrence of Enterococcus sp. strains and to access its safety and potential application as dairy cultures, due to the controversial role of Enterococci sp. in food. In chapter three, properties related to diacetyl (related to flavor), exopolysaccharide (EPS) (related to texture), and antimicrobials (with protein origin) formation of selected LAB (n =220) was accessed. Chapter four focused on studying some probiotic properties of the same LAB strains (n=220), as resistance to conditions found in the gastrointestinal tract (GIT), analysis of adhesion, and presence of virulence genes. Besides, a strain of Lactobacillus plantarum (1QB77) was chosen and used as adjunct culture in the manufacturing of microcheeses, in co-culture with cocktails containing Listeria monocytogenes and enterotoxigenic Staphylococcus aureus strains, separately. The fifth chapter aimed to apply a method called evolutionary engineering (EV) (or adaptative engineering) in order to improve the performance of Lb. plantarum (1QB77) in the initial step of the cheese process (acidification of milk). The results showed a great diversity of LAB with technological potential belonging to Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus genus. The Lb. plantarum species was the most recurrent in BAC, showing a great heterogeneity intra-species, according to cheese and region of isolation. There was an association between some properties of LAB and source of isolation, such as the production of antimicrobials with protein origin active against L. monocytogenes and Minas Artesanal cheeses and production of lipolytic enzymes with cheeses from Marajó. The association between high-performance screening methods and multivariate analysis provided a rational selection of a high number of strains, making it possible to group them according to their features in probiotic, biopreservative, and adjunct cultures, which may contribute to safety, quality and enhancement of regional cultures in the dairy process. The Enterococcus genus was considered a natural component of BAC microbiota, mainly in Marajó, Coalho, and Manteiga cheeses, and 63% had the potential for application as a starter or biopreservative cultures, due to the absence of hemolysins and susceptibility to vancomycin. Strain Lb. plantarum 1QB77 met the main in vitro criteria for the evaluation of probiotic potential being able to reduce counts of pathogens tested along microcheeses process by up to 4 logarithmic units from the whole process (22 days of ripening) after submission to the simulated conditions of the GIT. The EV method used to improve the performance of Lb. plantarum 1QB77 increased milk acidification rates of evolved strains in six times, compared to parental (non-evolved) strains, and increased the viability of evolved strains by 90% when exposed to simulated GIT conditions. The microcheese production protocol (~ 190 mg) developed in this study showed results within those presented in large scale production (~ 0.5-1 kg) for medium moisture cheeses, configuring an excellent alternative for studies involving new formulations, new cultures, but with a drastic reduction in costs. In short, BAC has proved to be an excellent source of endogenous lactic acid bacteria with high potential to be applied as an adjunct or biopreservative cultures in the production of dairy products (AU)

• FAPESP's process: 15/25641-4 - Lactic acid bacteria in Brazilian artisanal cheeses: application of Evolutionary Engineering for culture improvement aiming at accelerating cheese ripening
• Grantee: Larissa Pereira Margalho
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)