Production of antifungals by food-grade microorganisms

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1 Production of antifungals by food-grade microorganisms antifungal compounds, lactic acid bacteria Eddy Smid Prof. Dr J. Hugenholtz (FBR) MSc - 6 months Most food and beverages are continuously subject to contamination by various microorganisms, during primary production, during processing, during storage, during transport and during use/handling by the consumer. The most difficult challenge is to control/limit the growth of fungi since they (their spores) survive high temperatures and low ph s and, as eukaryotic cells, tend to be sensitive to treatments that are also toxic/detrimental to human cells. In this project, we focus on documented/available/well-known cultures of lactic acid bacteria that produce a number of different antifungal components such as phenyllactic acid, cyclic peptides and hydroxy fatty acids. We will use various fermentation approaches to produce high titres of these antifungals and we will test the efficacy of this antifungal fermentation fluid against relevant fungal food spoilers. Ultimately, we would like to provide the food industry with natural/biobased solutions against fungal spoilage instead of the highly unpopular chemical solutions that are currently in use. Schnürer and Magnusson Antifungal lactic acid bacteria as biopreservatives. Trends in Food Science & Technology 16:70 78 Summary current knowledge of antifungals produced by LAB (from: Schnürer and Magnusson, 2006)

2 Novel concepts for fermented vegetables fermented vegetables; lactic acid bacteria; novel foods Eddy Smid MSc 6 months Consumption of fruits and vegetables is essential for a healthy human diet. However, in many countries of the Western world, people do not meet the recommended daily intake of vegetables and fruits. Fermentation of plant raw materials offers an attractive way to boost the daily consumption of fresh-like vegetables and fruits. This project will focus on the exploitation of lactic acid bacteria for the development of novel fermented (combinations of) vegetables. Various fermentable raw materials will be considered: cabbage, horseradish, leek, ginger, seaweed, carrots, cucumber, red beets, garlic and dill. In addition, new formulations of salts with lower sodium content will be investigated. The succession of lactic acid bacteria will be monitored using various techniques and subsequently linked to product characteristics (ph, texture, nutritional value and microbial safety). A commercial producer of fermented vegetables and a professional food designer will participate in the project. Di Cagno, Coda, De Angelis and Gobbetti Exploitation of vegetables and fruits through lactic acid fermentation. Food Microbiology 33: 1-10 Succession of lactic acid bacteria and change of ph during spontaneous fermentation of kimchi. (from: Di Cagno et al, 2013)

3 Genome analysis and functionality of aroma pathways in the nonconventional yeast species Cyberlindnera fabianii and Pichia kudriavzevii aroma formation, yeast, non-conventional yeast Irma van Rijswijck Eddy Smid MSc - 6 months Several different yeast species have been identified as major players in complex microbial communities associated with fermented foods and beverages. Most of our knowledge of yeast is based on Saccharomyces cerevisiae. However, many poorly characterised non-saccharomyces yeast species (such as Cyberlindera fabianii and Pichia kudriavzevii) possess interesting metabolic properties which contribute to a successful fermentation process. For instance, Pichia species were found to be involved in the production of an array of aroma compounds which make these yeast species interesting candidates for the development of new types of fermented foods and beverages. The complete genome sequences of Cyberlindnera fabianii and Pichia kudriavzevii respectively, were recently determined. In this project, the presence and activity of Pichia sp. genes involved in aroma formation pathways will be analysed. The results will be compared with gene presence and activity in a Saccharomyces cerevisiae strain isolated from the same environmental niche (ie fermented Masau fruit). van Rijswijck, I.M.H., J. Dijksterhuis, J. C.M. Wolkers-Rooijackers, T. Abee and E.J. Smid Nitrogen limitation leads to penetrative growth into agar and affects aroma formation in Pichia fabianii, P. kudriavzevii and Saccharomyces cerevisiae. Yeast 31: Scanning EM pictures of Pichia sp. growing on a solid surface

4 Innovation of tempeh using lupine tempeh, fermentation, lupine, soy, novel product Eddy Smid MSc - 6 months Tempeh is a traditional soy product where soybeans are dehulled, soaked and fermented with the fungus Rhizopus oligosporus. Because of its nutritional value (rich in proteins, dietary fibers and vitamins) it is a popular ingredient in the vegetarian and vegan kitchen. There is little variation in traditional tempeh products because soybeans are the main fermentable substrate. To meet consumers demands, new tempeh formulas must be developed. One of the ways is using a sustainable alternative for soybeans. Lupine, a Mediterranean crop that has a high nutritional value, potentially is such an alternative. According to Priatni et al. (2013) lupine tempe showed good overall acceptability (based on quality and sensory tests). This project will focus on developing new tempeh formulas using lupine seeds as substrate. Lupine tempeh will be compared with soy tempeh with respect to different product characteristics, such as texture, colour, microbial safety. A commercial company (de Vegetarische Slager) will participate in the project. Sri Priatni, Anastasia F. Devi, Leonardus B.S. Kardono and Vijay Jayasena Quality and sensory evaluations of tempe prepared from various particle sizes of lupine beans. Jurnal Teknologi Dan Industri Pangan 24.2: 209.

5 Effect of growth rate on plasmid copy number fermentation, growth rate, lactic acid bacteria, plasmid Oscar van Mastrigt Eddy Smid BSc/MSc 4-6 months Fundamental knowledge of microbial physiology and cellular regulation is obtained mainly from fast growing microorganisms in batch cultures. In contrast, natural microbial communities generally live in relative famine conditions with low specific growth rate due to limited supply of nutrients and energy sources. Plasmids are extrachromosomal genetic elements which replicate independent from the chromosome. Sequencing of strains of the lactic acid bacteria Lactococcus lactis and Leuconostoc mesenteroides revealed 7 and 3 different plasmids respectively with plasmid copy numbers (number of plasmids per chromosome) ranging from 1 to 20 when grown in batch cultures. Objective: The goal is to determine the effect of growth rate on the plasmid copy number of the different plasmids. Other relevant parameters which could affect the plasmid copy number might be tested as well. Approach: Different growth rates will be obtained by growing the bacteria in batch, chemostat and retentostat (see graphic below) cultivation. These will lead to high (doubling time of 1 our), intermediate and near-zero growth rates (doubling time of more than 3 months), respectively. qpcr will be used to determine the plasmid copy numbers. Ercan, O., E. J. Smid and M. Kleerebezem Quantitative physiology of Lactococcus lactis at extreme low growth rates. Environmental Microbiol. 15: Principles of chemostat and retentostat cultures (left and center respectively) and the time course of biomass development in a retentostat culture (right).

6 Effect of plasmid copy number on activity of citrate utilization fermentation, plasmid, lactic acid bacteria, citrate, activity, expression Oscar van Mastrigt Eddy Smid MSc 6 months Cheese manufacturing relies on the use of starter cultures consisting of different strains and species of mainly lactic acid bacteria. One of these species is Lactococcus lactis subsp lactis bv diacetylactis. This variant discriminates itself from the other biovars in this species by its ability to utilize citrate and convert it into the buttery aroma compounds acetoin and diacetyl. The gene for citrate transport (CitP) is located on a plasmid. Plasmid copy numbers (number of plasmids per chromosome, PCN) can change dependent on the cultivation conditions. These changes might affect the expression of the genes encoded by the plasmid and their activity. Objective: To determine the effect of plasmid copy number on the activity of citrate utilization and the gene expression of CitP. Approach: L. lactis subsp lactis bv diacetylactis will be grown at conditions resulting in high and low copy numbers of the plasmid encoding CitP. Citrate uptake rates will be determined and gene expression of CitP will be measured. Finally, the resting cultures will be screened for the production of acetoin and diacetyl, which might results from citrate conversion. Figure 1. Plasmid pcit in L. lactis subsp lactis bv diacetylactis FM03