Fluorescent markers for strains in mixed cheese starter cultures

Transcription

1 Fluorescent markers for strains in mixed cheese starter cultures fermentation, starter cultures, fluorescent proteins Svetlana Alexeeva MSc - 6 months Cheese manufacturing relies on the use of starter cultures consisting of single strains or mixtures of different strains and species of mainly lactic acid bacteria. Mixed-dairy starter cultures can be regarded as complex ecosystems containing undefined blends of strains belonging the species Lactococcus lactis and Leuconostoc sp. For the cheese manufacturer, it is relevant to identify which product characteristics of the end-product are determined by the metabolic and physiological properties of the individual strains in the starter culture. For that reason, individual strains need to be tracked and enumerated while being part of a complex microbial community. The objective of this project is to develop fluorescently labeled strains which can be easily traced in complex mixtures of microbes. To monitor the population dynamics we will make use of different fluorescent markers (geneticallyencoded fluorescent proteins) integrated into chromosome of each individual strain. In this project we will exploit an array of microbiological, molecular, genetic and analytical techniques, as well as fluorescence microscopy and flow cytometry.

2 The role of prophages in generation and maintenance of biodiversity fermentation, starter cultures, biodiversity, bacteriophages, fluorescent proteins Svetlana Alexeeva Starter cultures are a combination of various lactic acid bacteria (LAB) used in manufacture of fermented milk products such as cheeses, buttermilk and sour cream. Any bacterial strain can be infected by virulent phages or bear integrated into its chromosome prophages (or temperate phages), bacteriophages which can choose between a lytic and lysogenic pathway of development. Bacteriophages of lactic acid bacteria appear constantly in milk fermentations, they can kill the starter culture and slow or stop the fermentation. It has long been recognized that the complex composition (biodiversity) of mixed starter cultures provides increased robustness to emerging phages as compared to a single strain starters. On the other hand, it has recently been postulated that the presence of bacteriophages in microbial consortia is the driving force for generation and maintenance of the biodiversity. This project aims to investigate the presence and activity of temperate phages in a standard complex starter cheese starter culture (Ur) and its role in generation and maintenance of starter culture biodiversity. Seven characterized lineages of Lactococcus lactis isolated from Ur starter culture will be tested for presence and activity of temperate phages. Phage susceptibility profiles of various Lactococcus lactis strains will be analyzed and the isolated phages will be genetically characterized and classified. Prophage-cured derivatives of the 7 original strains will be made. Next, changes in the distribution of individual strains and population dynamics will be investigated in mixtures of prophage-cured derivatives versus original strains. To follow the population dynamics we will make use of different fluorescent markers (genetically-encoded fluorescent proteins) integrated into chromosome of each individual strain. In this project we will exploit an array of microbiological, molecular, genetic and analytical techniques, fluorescence microscopy and flow cytometry. 1. Heidelberg, J.F., et al., Germ warfare in a microbial mat community: CRISPRs provide insights into the co-evolution of host and viral genomes. PLoS One, (1): p. e Rodriguez-Valera, F., et al., Explaining microbial population genomics through phage predation. Nat Rev Microbiol, (11): p

3 Metabolic pathways involved in aroma production by non-saccharomyces yeast species in novel food fermentation processes aroma formation, yeast, non-conventional yeast Irma van Rijswijck 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 Pichia fabianii and Pichia kudriavzevii) possess interesting metabolic properties which contribute to a successful fermentation process. These properties make them interesting candidates for the development of new types of fermented foods and beverages. Objective/approach: The emphasis of this study is the regulation of aroma formation in two non-conventional yeast species, Pichia fabianii and Pichia kudriavzevii, isolated from fermenting masau fruits. Special focus will be on the physiological role of aroma compounds in the growth and development of the two non-conventional yeast species. 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 DOI: /yea.3050 Aroma formation by the yeast Pichia fabianii.

4 Genome analysis and functionality of aroma pathways in the nonconventional yeast species Pichia fabianii and P. kudriavzevii aroma formation, yeast, non-conventional yeast Irma van Rijswijck 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 Pichia 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. Objective/approach: The complete genome sequences of Pichia 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 DOI: /yea.3050 Scanning EM pictures of Pichia sp. growing on a solid surface.

5 Plasmid profiling of mixed starter culture isolates plasmids, starter cultures, functionalities, population dynamics Maciek Spus Judith Wolkers Mixed starter culture for cheese production called Ur consists of many strains of two species of bacteria: Lactococcus lactis and Leuconostoc mesenteroides (1). These strains possess traits interesting from the technological point of view: citrate metabolism, protease activity, bacteriophage resistance etc. Partially those traits are encoded by chromosomal DNA but often are located on plasmids. Plasmids are mobile, circular genomic elements which can be transferred between cells, lost or acquired in the evolution processes. Ur starter culture is a stable community and the insight on the plasmid content shifts in scope of evolution is one of the most important aspects which might contribute to this stability. Determine the plasmid content before and after experimental evolution and relate it to population dynamics and functionality in complex starter culture. Defined blends of well-defined isolates from different genetic lineages of Ur starter culture will be sequentially propagated in milk for extended number of generations. Plasmid content will be isolated before from individuals and at the end of the experiment from many single colony isolates. Finally, plasmid content will be sequenced. 1. Erkus, O., De Jager, V. C., Spus, M., Van Alen-Boerrigter, I. J., Van Rijswijck, I. M., Hazelwood, L., Smid, E. J. (2013). Multifactorial diversity sustains microbial community stability. The ISME journal. doi: /ismej

6 Characterisation of evolved complex starter cultures fermentation, starter cultures, adaptive evolution Maciek Spus Judith Wolkers-Rooijackers Cheese manufacturing relies on the use of starter cultures consisting of single strains or mixtures of different strains and species of mainly lactic acid bacteria. Mixed-dairy starter cultures can be regarded as complex ecosystems containing (undefined) blends of strains belonging the species Lactococcus lactis and Leuconostoc sp. For the cheese manufacturer, it is relevant to identify which product characteristics of the end-product are determined by the metabolic and physiological properties of the individual strains in the starter culture. By changing the propagation conditions, the strain composition of the complex mixture will change after prolonged cultivation for many generations. This will evoke competition between strains and leads to genetic adaptations of the strains in the culture (adaptive evolution). With the induced shift in relative abundance of particular strains in the starter culture, the overall functionality of the starter culture changes. Our approach opens up new possibilities for the development of specialized starter cultures. The objective is to determine functional and genetic properties of evolved complex dairy starter cultures. Starter cultures which have been subjected for over 1000 generations to a particular propagation regime, will be analyzed for their functional properties. More specifically, the evolved cultures will be compared with the original culture by analysis of: acidification rate in normal milk, biomass yield, Lactococcus:Leuconostoc ratio, relative abundance proteolytic strains, flavor profiles and cell lysis in high and low salt regimes. In addition, strain diversity analysis can be performed for a number of relevant phenotypes using molecular techniques such as qpcr. Smid, E.J., O. Erkus, M. Spus, J. C.M. Wolkers-Rooijackers, S. Alexeeva and M. Kleerebezem Functional implications of the microbial community structure of undefined mesophilic starter cultures. Microbial Cell Factories 13(Suppl 1):S2.

7 Aroma production by extremely slow growing lactic acid bacteria fermentation, near-zero growth, aroma formation, lactic acid bacteria Oscar van Mastrigt Judith Wolkers-Rooijackers Fundamental knowledge of microbial physiology and cellular regulation is obtained mainly from fast growing microorganisms in batch cultures. Natural microbial communities generally live in relative famine conditions with low specific growth rate due to limited supply of nutrients and energy sources. Analogously, under specific industrial fermentation conditions, microorganisms may experience strongly restricted access to nutrients for longer periods of time. For example, lactic acid bacteria (LAB) endure long periods of extremely low nutrient availability during the maturation phase which is part of the production process of for instance dried sausages and cheese. Despite these harsh conditions, several LAB succeed to survive under these conditions for months and continue to contribute to flavor and aroma formation in the product matrix. The goal is to improve and steer aroma formation using high cell density/high viability cultures growing at growth rates approaching zero. In this project, the impact of extremely slow growth rates on physiology and metabolism of Lactococcus lactis subsp. lactis biovar diacetylactis will be studied using so-called retentostat cultivation technique. Ercan, O., E. J. Smid and M. Kleerebezem Quantitative physiology of Lactococcus lactis at extreme low growth rates. Environmental Microbiol. 15: Ercan, O., M. Wels, E. J. Smid and M. Kleerebezem Molecular and metabolic adaptations of Lactococcus lactis at near-zero growth rates. App. Environ. Microbiol. doi: /AEM Principles of chemostat and retentostat cultures (left and center respectively) and the time course of biomass development in a retentostat culture (right).