Microbial Community Dynamics In

Microbial community dynamics in traditionally fermented milk PhD thesis Anneloes E Groenenboom Wageningen University and Research 2 Table of contents Chapter 1. Introduction 5 Chapter 2. Microbial communities from spontaneously fermented foods as model system to study eco-evolutionary dynamics 13 Chapter 3. Robust sampling and preservation of DNA for microbial community profiling in field experiments 25 Chapter 4. Microbial population dynamics during traditional production of Mabisi, a spontaneously fermented milk product from Zambia. A field trial. 33 Chapter 5. Does change in bacterial species composition of natural communities reflect adaptation to a new environment? 49 Chapter 6. Bacterial community dynamics in Lait caillé, a traditional product of spontaneous fermentation from Senegal. 65 Chapter 7. General discussion 79 Summary 91 References 95 3 4 1. Introduction This thesis is about Mabisi. In the western world, and maybe anywhere but Zambia, Mabisi is an unknown product. In this country in the middle of Africa, however, Mabisi is a phenomenon; a widely appreciated fermented milk product, which is consumed almost daily by men, women, and children from a very young age. What makes this fermented milk so interesting that I studied it for four years? It is the diversity we find in the bacterial communities that make Mabisi. This PhD thesis is part of a larger project funded by NWO WOTRO on enhancing nutrition security though traditional fermented products (1). In this thesis I would like to show the microbial communities that are responsible for the fermentation and how we can use the constituting bacteria to learn about bacterial community dynamics over time. This was done by combining three disciplines: 1) Food Microbiology, on the conversion of compounds by bacteria during spontaneous fermentation; 2) Evolution, on the changes in the fermenting community over time under selection pressure; 3) Ecology, on the roles and niches bacteria take within the fermenting community (Figure 1.1). My work does not only advance science; results of this project will also be used for improvements in production and legal regulation around Mabisi as well as upscaling production processes to ensure widespread availability of Mabisi thereby improving public health. This final aspect I will discuss in the General Discussion. For now, I would like to start by introducing Mabisi. Figure 1.1 My thesis, linking three disciplines in one PhD project. Combining disciplines has added value compared to isolated disciplinary studies: 1) Using microbial communities from fermented foods is a great way to tackle questions in less trackable microbiota (2), 2) As sequencing costs are decreasing, analysing genetic changes in microbes used for fermentation becomes more accessible (3), 3) The eco-evolutionary dynamics are studied more and more (4), and 4) in this thesis I combine these disciplines for a complete understanding of community dynamics (5,6). Mabisi In many African countries local fermented products with unique properties are produced. Those products offer a great potential to enhance the nutritional status and livelihood of local producers and consumers (7). Zambia also has numerous endogenous fermented products (5), comparable to well known (Western) equivalents such as yoghurt, wine, beer, and sauerkraut. Mabisi is a non-alcoholic, milk-based product, which is consumed mainly in rural areas and is sold at local markets (Figure 1.3). Producers are almost exclusively women, who produce Mabisi according to ancient traditions. Overall, Mabisi has a slightly firm consistency, a mildly sour taste and a perceived shelf-life of a few weeks at ambient temperatures. It is made from raw cow milk that is kept in a calabash of up to 40 litres to allow fermentation over the course of two days at ambient temperatures (Figure 1.2), and variations of this production method are prevalent. For instance, as calabashes are becoming less available, cheaper fermentation vessels are found in plastic buckets and metal milk containers (Figure 1.3). Also, there are variations in the fermentation process itself, such as churning before and during fermentation, draining part of the milk and adding raw milk to 5 the remainders. These production methods result in Mabisi of very diverse organoleptic properties and consistency (8). Independent of production method, Mabisi is a widely consumed product throughout Zambia. It represents a tasty and nutritious product with a long shelf-life, but is also thought to have certain health benefits such as the prevention and cure of diarrhoea. Mabisi contains a mix of lactic acid bacteria in combination with other fermenting bacteria, which might possess probiotic properties (5,9). Producers mostly consume their product with their family members at home and also sell their product on the local market to have an additional source of income. Increased production and consumption of Mabisi has important implications for nutrition security, public health as well as woman entrepreneurship, which are important aspects of the Sustainable Development Goals (SDGs) formulated by the United Nations. In Zambia, half of the produced raw cow milk goes to waste, while food insecurity and malnutrition persist at rural household level due to gender imbalances, differences in household involvement in agriculture and post-harvest losses caused by malfunctioning value chains of local products. Thus, converting raw milk into Mabisi, which is safe and nutritious, contributes to preventing food losses and promotes general health. Increased production is not as straightforward as it sounds. Traditional Mabisi is produced by spontaneous fermentation, meaning that the milk is not pasteurised, and no bacterial starter culture is added to start the fermentation. As a result of this production method, Mabisi cannot be sold at formal markets under the national food safety law. A Mabisi-like product is currently on the market in urban areas, called Lacto-Mabisi. For this Mabisi alternative, milk is pasteurised and a bacterial starter culture of cream cheese is added, resulting in an end product with organoleptic properties that differ from those of traditional Mabisi. Consequently it has a lower consumer preference than traditional Mabisi (10). Also, the health benefits of this products are not proven. To improve national health, traditional Mabisi should be consumed nationwide, also in urban areas. Producing Mabisi with pasteurised milk could yield a safer product than when raw milk is used. However, this would require a starter culture that closely resembles the characteristics of the original microbial community. The formulation of such a starter culture requires the establishment of the relation between product characteristics, quality and the microbial community in Mabisi. Moreover, the spontaneous fermentation of Mabisi might result in a safe and stable product that does not need to be banned from the official markets. To determine this, knowledge about the bacterial communities responsible for the spontaneous fermentation should be extended (5). Figure 1.2 Basic production method of Mabisi. Many variations on this method exist (8). Picture adapted from Schoustra et al. (5). 6 Figure 1.3 Mabisi producers who use different types of containers. Mabisi is produced at home and sold on the local market or along the main (national) roads. Mabisi produced in calabashes is sometimes transferred to plastic buckets for ease of transport and hygiene on the market. The calabash can then be used for another fermentation cycle. In other cases, Mabisi is produced and sold in plastic buckets or bottles and metal buckets. Pictures by Sijmen Schoustra. Spontaneous fermentation Many fermented products are made with a defined starter culture, typically consisting of one or several microbial strains with known characteristics. Well-known examples include yoghurt, bread, and wine. Sometimes starter cultures are undefined and relatively complex, such as the starter culture for Gouda cheese (11). In contrast, products from spontaneous fermentation are not inoculated with a starter culture, but rather are fermented by whichever bacterial community is present in the raw material or residues on the fermentation equipment. This often results in a fermenting community with a high species diversity. Examples of Western products made through spontaneous fermentation are sauerkraut, sourdough, and parmesan cheese. In the latter case, 24 hours fermented whey is added to a new batch of raw cow milk before overnight fermentation (12). This method, in which an amount of finished product provides the starter culture for the fermentation of the new raw ingredients, is called back-slopping (13). In this thesis I distinguish two types of back-slopping: active back-slopping, where a part of finished product is actively added to a new batch of raw material, and passive back-slopping, where the transfer of starter culture to the new raw material is not an active part of the production methods but rather “co-incidental”; via, for example, the surface of the fermentation vessel or the re-use of other equipment. Back-slopping forms the (in many cases only) inoculum for the next fermentation and could be compared to the addition of a starter culture. There is however a difference between back-slopping and the addition of an undefined starter culture. Where in the case of Gouda-type cheeses the starter culture comes from the same frozen source, in back-slopping the source of the starter culture is the

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