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There is growing interest in Sub-Saharan Africa for substituting maize with climate-smart crops like sorghum and millets in local food processing. We conducted a survey to explore current variations in processing and consumption practices for Mahewu, a traditionally fermented cereal beverage from Zimbabwe. Processing involved cooking a cereal porridge, adding cereal flour or malt as a starter ingredient, and fermenting for 12–48 h. Ingredient availability was the main driver for porridge ingredient choice (42% of respondents) with the most preferred being maize (55% of respondents), pearl millet (22%) and sorghum (9%). Final product taste had the most influence on starter ingredient choice, with most respondents preferring pearl millet flour (23%), finger millet malt (22%), wheat flour (17%), and sorghum malt (13%). Our study proves that maize can be replaced with sorghum and millet in Mahewu processing, thus increasing the climate-resilience of future food systems, and demonstrates that traditional practices harbour clues for adapting current practices.

Bifidobacterium breve is a common habitant of the human gut and is used as probiotic in functional foods. B. breve has to cope with multiple stress conditions encountered during processing and passage through the human gut, including high temperature, low pH and exposure to oxygen. Additionally, during industrial processing and in the gut, B. breve could encounter nutrient limitation resulting in reduced growth rates that can trigger adaptive stress responses. For this reason, it is important to develop culture methods that elicit resistance to multiple stresses (robustness) encountered by the bacteria. To investigate the impact of caloric restriction on robustness of the probiotic B. breve NRBB57, this strain was grown in lactose-limited chemostat cultures and in retentostat for 21 days, at growth rates ranging from 0.4 h-1 to 0.00081 h-1. Proteomes of cells harvested at different growth rates were correlated to acid, hydrogen peroxide and heat stress survival capacity. Comparative proteome analysis showed that retentostat-grown cells had significantly increased abundance of a variety of stress proteins involved in protein quality maintenance and DNA repair (DnaJ, Hsp90, FtsH, ClpB, ClpP1, ClpC, GroES, RuvB, RecA), as well as proteins involved in oxidative stress defence (peroxiredoxin, ferredoxin, thioredoxin peroxidase, glutaredoxin and thioredoxin reductase). Exposure to three different stress conditions, 45 °C, pH 3, and 10 mM H2O2, showed highest stress resistance of retentostat cells sampled at week 2 and week 3 grown at 0.0018 and 0.00081 h-1. Our findings show that cultivation at near-zero growth rates induces higher abundance of stress defence proteins contributing to the robustness of B. breve NRBB57, thereby offering an approach that may support its production and functionality.

Background: Lytic bacteriophages infect and lyse bacteria and, as a by-product, may affect diversity in microbial communities through selective predation on abundant bacterial strains. We used a complex dairy starter named Ur to investigate population dynamics of Lactococcus lactis, Lactococcus cremoris and Leuconostoc mesenteroides strains in terms of constant-diversity and periodic selection models.Methods: To mimic the starter Ur, we designed blends of 24 strains representing all eight previously identified genetic lineages in the starter culture. The blends were propagated by daily transfers in milk for over 500 generations in the presence or absence of a cocktail of lytic bacteriophages. The relative abundance of genetic lineages of L. lactis, L. cremoris and Lc. mesenteroides strains present in the complex blend, as well as phage presence, were monitored.Results: Control blends without phage predation showed decreased strain diversity, leading to a stable state due to the domination of the fittest strain(s) of a particular lineage according to periodic selection dynamics. However, in phage-challenged blends, predation caused a large shift in the microbial composition by killing the fittest and sensitive strains.Conclusion: It was demonstrated that phage-challenged blends maintained their diversity at the level of genetic lineages, thus providing experimental support for the constant-diversity dynamics model in a complex microbial community.

Vitamin K2 is essential for maintaining human health. It is required for blood coagulation and contributes to cardiovascular and bone health. Therefore, vitamin K2 enrichment in the diet is of high interest for human health. Bacteria are the natural producers of vitamin K2, and known producers such as Lactococcus lactis are key players in the production of fermented foods. This fact offers opportunities to enhance vitamin K2 levels in food. Knowledge on vitamin K2 production in L. lactis is important for vitamin K2 enriched in fermented foods (a background introduction is provided in chapter 1).In this thesis study, initially, L. lactis strains were screened for vitamin K2 content, and the impact of various cultivation conditions was examined (chapter 3). It was observed that significant strain diversity existed in terms of specific concentrations and titers of vitamin K2. In L. lactis ssp. cremoris MG1363, aerated cultivation conditions and carbon sources like fructose or trehalose, were found to increase the vitamin K2 content as compared to static cultivation and glucose as carbon source. In quark fermentation, it was consistently observed that altered carbon source (fructose) and aerobic cultivation of the L. lactis MG1363 pre-culture resulted in elevated vitamin K2 concentrations in the quark product.Next, an adaptive laboratory evolution (ALE) strategy was applied to obtain natural vitamin K2 overproducing L. lactis strains (chapter 4). By propagating strain MG1363 in aerated conditions, Three evolved strains were selected that showed improved stationary phase survival in oxygenated conditions. In comparison to the original strain MG1363, the evolved strains showed increased vitamin K2 content and exhibited high resistance against hydrogen peroxide-induced oxidative stress. Genome sequencing and proteomic analysis provided explanations for the enhanced oxidative stress resistance, but the mechanisms underlying elevated vitamin K2 content in the evolved strains

Listeria monocytogenes is a facultative anaerobe responsible for a severe infection called listeriosis, which primarily affects immunocompromised individuals. This foodborne pathogen can activate adaptive stress responses supporting its survival in a range of stress conditions encountered in food production and host environments. The capacity to utilize alternative substrates for growth is crucial for the survival of L. monocytogenes when other efficient nutrients such as glucose are unavailable. In recent years more and more evidence has been presented that so-called Bacterial Microcompartments (BMCs) play an essential role in the utilization of specific host-derived substrates by enteric pathogens that result from degradation of phospholipids and metabolism of mucus-derived saccharides, including ethanolamine and 1,2-propanediol. Notably, these compounds are also encountered in a wide range of food products, and could therefore also contribute to transmission of pathogens to the host. It has been suggested that BMCs also play a role in L. monocytogenes ecophysiology and pathogenicity, but experimental evidence supporting these claims are very scarce. In this thesis, we studied to understand the role of bacterial microcompartments in Listeria monocytogenes growth, stress adaptation and virulence.

Folate is a B-vitamin with an important role in health and disease. The optimal folate status with regard to human health remains controversial. A low intake of natural folate as well as excessive intake of synthetic folic acid, were previously linked to an increased risk of colorectal cancer or with aberrant molecular pathways related to carcinogenesis in some studies. Importantly, most studies conducted so far, solely focused on dietary intake or circulating levels of folate in relation to cancer risk. Notably, diet or dietary supplements are not the only sources of folate. Several bacteria in the gastrointestinal tract can synthesize B-vitamins, including folate, in quantities that resemble dietary intake. The impact of bacterial folate biosynthesis concerning human health and disease remains unexplored. This review highlights current insights into folate biosynthesis by intestinal bacteria and its implications for processes relevant to cancer development, such as epigenetic DNA modifications and DNA synthesis. Moreover, we will reflect on the emerging question whether food-grade or intestinal bacteria can be considered a potential target to ensure sufficient levels of folate in the gastrointestinal tract and, hence the relevance of bacterial folate biosynthesis for disease prevention or treatment.Keywords: Folate, biosynthesis, colon, intestinal bacteria, colorectal cancer, DNA methylation, one-carbon metabolism

Traditional fermented products are an important part of the diet in many African countries. These products can provide much of the needed proteins, vitamins and other micro-nutrients to its consumers, which include young children. Also, traditional fermented products are rooted in the local context and have high social and cultural value. Increasing our understanding of these spontaneously fermented products can be used to increase the value chain of these products in order to increase economic stability of producers as well as nutritional intake in rural and urban areas. Most traditional fermented products are produced by spontaneous fermentation, meaning that there is no starter culture added to initiate fermentation. As a consequence these products contain a diverse microbial community, which are responsible for the characteristics of the product. Often a back-slopping method is used, where a portion of previously fermented product inoculates fresh raw material. Unknowingly, producers of these products have domesticated these bacterial communities and allowed the species to co-evolve within their community. In this way, these bacterial communities form a model system for ecological and evolutionary research. Historically, experimental evolution was mainly performed with single organisms. In recent years the interest in how individuals evolve in the context of ecosystems is growing. This eco-evolutionary research needs model systems that can represent natural communities in their interactions and complexities, while still being a tangible model system. In this thesis I investigated the microbial communities that are responsible for the fermentation and used 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 pres

Fermented dairy products are an important part of the human diet because they provide nutrients and extend the shelf life of raw milk, which is perishable. In Africa, there are several traditionally fermented milk products (TFMPs) mostly, produced at household level for domestic consumption such as mabisi from Zambia. Mabisi is made by spontaneous fermentation of raw milk at ambient temperature in a calabash, plastic or metal container for two days. The main aim of this study was to characterise the microbial community composition of mabisi and optimise its production process. Thus, a survey was carried out to determine the different production practices/methods of mabisi, key production parameters, uses and microbial community composition. Then, a laboratory experiment was carried out to determine the effect of fermentation temperature on the quality of mabisi and its microbial community composition. Furthermore, a field experiment was also conducted to determine the microbial dynamics of three types of mabisi. We found seven different production methods of mabisi, which include tonga, barotse, backslopping, illa, creamy, cooked and thick-tonga mabisi. Tonga mabisi was found to be the most popular and widely produced mabisi variant throughout the country by all ethnic groups. The key production parameters identified were: type of fermentation container, fermentation temperature, backslopping, alternate removal of whey in combination with addition of raw milk, heating and cooling, fermentation time and finally, agitation (churning). We further found that mabisi is a versatile product with various uses. The microbial community composition of mabisi from across the country was unravelled and showed that the top 10 most abundant genera were Lactococcus, Lactobacillus, Streptococcus, Enterobacter, Klebsiella, Kluyvera, Aeromonas, Acinetobacter, Buttiauxella and Citrobacter, which belong to two phyla, Firmicutes and Proteobacteria. The microbial community composition was

Fermentation is the oldest technology used in the production of many traditional cereal-based beverages across Africa. Munkoyo, a spontaneously fermented cereal-based beverage from Zambia and Democratic Republic of Congo is one of those beverages and the focal product studied in this thesis. It is consumed by large parts of the population, as an energy drink during travel or work on the field and is also consumed at social gatherings like wedding festivals and funerals. The processing of Munkoyo has to date not been standardized and greatly varies between different regions and processors. This may affect the quality and the safety of the beverage. Optimization of the processing methods and microbial composition of Munkoyo to attain good quality and a safe product can be a milestone leading to increased consumption of the beverage. Like all fermented foods, processing of Munkoyo relies on microbial activity transforming raw materials (cereals such as maize, millet and sorghum) into a processed product. The thesis focused on understanding the art of making Munkoyo by surveying different processors and linked this to the microbial community composition of fermenting microbes and their functionality in producing aroma compounds. Further, we investigated whether the source of the fermenting bacteria could lie in the Rhynchosia roots that are added during processing. Chapter 2 describes a survey using a semi structured questionnaire and focus group discussions in Lusaka, Chongwe, Chibombo and Mumbwa. The survey revealed that Munkoyo is consumed by the entire population, as an energy drink, during long hours of manual work, at social gatherings and is mainly processed at household level. Characterization of the bacterial communities of over 90 samples with 16S amplicon sequencing on DNA extracted from the entire bacterial community revealed six families of mainly lactic acid bacteria to dominate the bacterial communities. Chapter 3 reports that there are principally three

Cashew nut is a cash crop in tropical regions of Africa including Benin, providing substantial revenues to small-scale farmers and contributing significantly to the national gross domestic product. In Beninese context, about 95% of raw cashew nuts are exported. Therefore, the production of raw cashew nuts of good quality is mandatory to enter the international market. Moreover, health issues related to the consumption of nuts contaminated with mycotoxins are of high concern for human being. The objective of this research was to investigate the occurrence of mycotoxins and other secondary metabolites of Aspergillus sp. on raw cashew nuts and to evaluate farmers’ practices that affect the quality of raw nuts.In surface disinfected samples 90.2% of raw kernels were contaminated by strains of A. section Nigri in the northern guinea (NG) zone compared to 87.2% in the southern guinea (SS) zone. The level of contamination of raw cashew kernels by strains of A. section Flavi was 6.7% in the NG zone whereas it was 4.6% in the SS zone. When non disinfected kernels were plated, A. section Nigri was predominant in both NG and SS zones, with percentages of 89.7% and 93.4%, respectively. Ten and 15.7% of raw cashew kernels were contaminated by strains of A. section Flavi in NG and SS zones, respectively. A. flavus, A. tamari, A. costaricaensis, A. minisclerotigenes and A. nomius were identified as strains in A. section Flavi. The average water content and the cashew nut count were respectively 8.6% and 172 nuts/kg in the NG zone and 8.7% and 174 nuts/kg in the SS zone.The occurrence of aflatoxins, analysed by ultra-high performance liquid chromatography tandem mass spectrometry, showed that none of the analysed samples was positive for natural occurrence of aflatoxins with a detection limit of 0.05 - 0.2 µg/kg. All the above results showed that, at present, cashew nuts from Benin were in the range of good quality for export.Seven species of black aspergilli were isolated based on

Lactic acid bacteria (LAB) are extensively used for the production of fermented foods from both animal and plant origin, such as cheese, yoghurt, kimchi and sauerkraut. Their predominant function in these processes is to produce organic acids, mainly lactic acid, thereby lowering the pH, which contributes to the safety and shelf life of the product. Nowadays, many fermented foods are made using starter cultures for a consistently safe product of constant quality. In Dutch-type cheeses, the starter cultures consist of various strains of mainly two species: Lactococcus lactis and Leuconostoc mesenteroides. L. lactis subsp. cremoris and L. lactis subsp. lactis are considered to be the main acid producers that dominate during the early stages of cheese manufacturing when nutrients are abundant. During cheese ripening, nutrients are scarce and LAB encounter long periods of nutrient limitation leading to slow growth. In particular, L. lactis biovar diacetylactis and Lc. mesenteroides survive these periods of extreme nutrient limitation and still contribute to aroma formation. In this thesis, the dairy-isolated strains Lactococcus lactis biovar diacetylactis FM03P and Leuconostoc mesenteroides FM06 were studied focussing on the effect of near-zero growth rates on their quantitative physiology and aroma formation capacity. Moreover, possible strategies to enhance aroma formation at near-zero growth rates are described. The genomes of both lactic acid bacteria strains were sequenced, including the plasmids they harboured, (Chapter 2) revealing that their genomes carried clear signatures of adaptation to the dairy environment. The plasmids of L. lactis FM03P and the genes they carried were further characterised in Chapter 3. Plasmid-encoded functions that were identified include lactose utilisation, citrate uptake, oligopeptide uptake and peptide degradation, bacteriophage resistance, uptake of cations (magnesium and manganese), exopolysaccharide production, and stress resist

Esters are essential for the flavor and aroma of fermented products, and are mainly produced by alcohol acyl transferases (AATs). A recently discovered AAT family named Eat (Ethanol acetyltransferase) contributes to ethyl acetate synthesis in yeast. However, its effect on the synthesis of other esters is unknown. In this study, the role of the Eat family in ester synthesis was compared to that of other Saccharomyces cerevisiae AATs (Atf1p, Atf2p, Eht1p, and Eeb1p) in silico and in vivo. A genomic study in a collection of industrial S. cerevisiae strains showed that variation of the primary sequence of the AATs did not correlate with ester production. Fifteen members of the EAT family from nine yeast species were overexpressed in S. cerevisiae CEN.PK2-1D and were able to increase the production of acetate and propanoate esters. The role of Eat1p was then studied in more detail in S. cerevisiae CEN.PK2-1D by deleting EAT1 in various combinations with other known S. cerevisiae AATs. Between 6 and 11 esters were produced under three cultivation conditions. Contrary to our expectations, a strain where all known AATs were disrupted could still produce, e.g., ethyl acetate and isoamyl acetate. This study has expanded our understanding of ester synthesis in yeast but also showed that some unknown ester-producing mechanisms still exist.

Yeast are used as workhorses to convert hopped wort into beer. Conventionally, such yeasts belong to the genus Saccharomyces and most research on fermentation of wort for the production of beer has focussed on the species Saccharomyces cerevisiae and Saccharomyces pastorianus. Recently, there is an increasing interest in unravelling features of non-conventional yeast species for beer innovation. In this thesis, features of yeast isolates belonging to the species: Cyberlindnera fabianii, Pichia kudriavzevii and S. cerevisiae (all isolated from fermented masau (Ziziphus mauritiana) fruits in Zimbabwe), were studied with focus on aroma production. Additionally, a novel approach was used to apply these yeasts in co-cultivation with Brewers’ yeast (S. cerevisiae) for beer innovation. The characteristics and quality of the beer are mainly determined by aroma compounds in the final product such as esters, alcohols, aldehydes and acids. Yeast use various metabolic pathways such as glycolysis, the fermentative pathway, the tricarboxylic acid (TCA) cycle and the Ehrlich pathway to produce aroma compounds or the precursors for the synthesis thereof (Chapter 1). Among the aroma compounds, esters are of major importance, especially since they are perceived by the human olfactory system at very low concentrations. In general, esters are desirable compounds in beers due to their fruity flavour. Examples are isoamyl acetate (banana), isobutyl acetate (fruity, sweet), phenylethyl acetate (rose, apple, honey), ethyl acetate (sweet pear), ethyl hexanoate (apple, aniseed) and ethyl octanoate (sour apple). Together with an extensive range of other volatile organic compounds (VOCs) these compounds were previously profiled using headspace solid-phase-micro-extraction gas-chromatography mass-spectrometry (GCMS). Interestingly, comparative profiling of aromas showed that C. fabianii produces significantly higher amounts of isoamyl acetate and ethyl acetate compared to S. cerevisiae. It has

Item does not contain fulltext, DNA based microbial community profiling of food samples is confounded by the presence of DNA derived from membrane compromised (dead or injured) cells. Selective amplification of DNA from viable (intact) fraction of the community by propidium monoazide (PMA) treatment could circumvent this problem. Gouda cheese manufacturing is a proper model to evaluate the use of PMA for selective detection of intact cells since large fraction of membrane compromised cells emerges as a background in the cheese matrix during ripening. In this study, the effect of PMA on cheese community profiles was evaluated throughout manufacturing and ripening using quantitative PCR (qPCR). PMA effectively inhibited the amplification of DNA derived from membrane compromised cells and enhanced the analysis of the intact fraction residing in the cheese samples. Furthermore, a two-step protocol, which involves whole genome amplification (WGA) to enrich the DNA not modified with PMA and subsequent sequencing, was developed for the selective metagenome sequencing of viable fraction in the Gouda cheese microbial community. The metagenome profile of PMA treated cheese sample reflected the viable community profile at that time point in the cheese manufacturing.

Biofilms consist of microorganisms attached to a surface and embedded in a protective matrix of extracellular polymeric substances. Within a biofilm, micro-organisms are protected from harsh environmental conditions including those resulting from cleaning and disinfecting agents leading to food safety and quality issues after dispersal of life biofilm cells and subsequent contamination of foods. In this thesis, single and multi-strain biofilm formation by Lactobacillus plantarum isolates was characterised including the model strain L. plantarum WCFS1 and food spoilage isolates. Analysis of the L. plantarum single strain biofilm formation showed a role for proteins and/or proteinaceous material in surface colonization and extracellular DNA as components of the biofilm matrix. The relevance of lysis for the build-up of the biofilm matrix with eDNA was demonstrated using L. plantarum WCFS1 mutants affected in the production of cell wall polysaccharides resulting in altered cell surface composition and mutants lacking cell wall lytic enzymes that showed decreased cell lysis. Dual and multi-strain biofilms were studied using quantitative PCR and next generation sequencing based on detection of strain specific alleles in competitive planktonic and surface-attached biofilm growth models. In multi-strain cultures, the performance of individual strains generally correlated with their performance in pure culture, and relative strain abundance in multi-strain static biofilms positively correlated with the relative strain abundance in suspended (planktonic) cultures. Performance of individual strains in dual-strain biofilms was highly influenced by the presence of the secondary strain, and in most cases no correlation between the relative contributions of viable planktonic cells and viable cells in the biofilm was noted. The next generation sequencing approach provided additional insights in the performance of twelve individual L. plantarum strains in static and dynamic flow co

Undefined mixed complex starter cultures are broadly used in Gouda-type cheese production due to their robustness to phage predation, resilience for changes in environmental conditions and aroma compounds production ability during ripening. These microbial communities of lactic acid bacteria prior their isolation and deposition in starter culture collections were continuously used at the farm-level production facilities. Thus, one can consider undefined mixed complex starters as domesticated microbial communities. The process of domestication was facilitated by humans who have been continuously repeating successful fermentations using part of previous batch as inoculum (i.e. back-slopping). Therefore, a term ‘community breeding’ can describe this human-driven domestication of microbial communities. Community breeding of a model complex starter Ur led to establishment of a simple two-species composition of Lactococcus lactis and Leuconostoc mesenteroides represented by, in total, 8 genetic lineages. At the same time, this simple microbial community displays a high degree of intraspecies diversity, presumably caused by evolutionary processes of horizontal gene transfer, genome decay and mutations. Such diversity at strain level is particularly interesting in the context of continuous bacteriophage predation pressure present in this microbial community. It is thought that constant-diversity (CD) dynamics, based on the ‘kill-the-winner’ principles, is operational in Ur starter at the strain level. According to CD model, the fittest strain(s), which feed on the most abundant substrate, will be selected against due to density-dependent phage predation. The control of the fittest strain abundance by bacteriophages opens space for differentiation of strains via eco-evolutionary feedbacks. In particular, strains of complex starter culture not only adapted to quickly acidify milk (via efficient consumption of lactose and protein to peptides degradation), but concurrently, to

Mutandabota is a dairy product consumed as a major source of proteins and micronutrients in Southern Africa. In this study the microbial safety of traditional and a variant of mutandabota fermented with the probiotic Lactobacillus rhamnosus yoba (yoba mutandabota) was investigated by challenging the products with five important food pathogens: Listeria monocytogenes, Salmonella spp., Campylobacter jejuni, Escherichia coli O157:H7 and Bacillus cereus. Pasteurized full-fat cow's milk was used for producing traditional and yoba mutandabota, and was inoculated with a cocktail of strains of the pathogens at an inoculum level of 5.5 log cfu/mL. Survival of the pathogens was monitored over a potential consumption time of 24 h for traditional mutandabota, and over 24 h of fermentation followed by 24 h of potential consumption time for yoba mutandabota. In traditional mutandabota (pH 3.4 ± 0.1) no viable cells of B. cereus and C. jejuni were detected 3 h after inoculation, while L. monocytogenes, E. coli O157:H7 and Salmonella spp. significantly declined (P <0.05), but could still be detected (<3.5 log inactivation) at the end of the potential consumption time. This indicated that consumption of traditional mutandabota exposes consumers to the risk of food-borne microbial infections. In yoba mutandabota, L. rhamnosus yoba grew from 5.5 ± 0.1 log cfu/mL to 9.1 ± 0.4 log cfu/mL in the presence of pathogens. The pH of yoba mutandabota dropped from 4.2 ± 0.1 to 3.3 ± 0.1 after 24 h of fermentation, mainly due to organic acids produced during fermentation. Only Salmonella spp. was able to grow in yoba mutandabota during the first 9 h of fermentation, but then decreased in viable plate count. None of the tested pathogens were detected (> 3.5 log inactivation) after 3 h into potential consumption time of yoba mutandabota. Inactivation of pathogens in mutandabota is of public health significance because food-borne pathogens endanger public health upon consumption of contaminated foo

The objective of this study was to evaluate the presence of Aspergillus section Flavi and A. section Nigri in cashew nuts harvested in the Northern Guinea (NG) and Southern Sudanian (SS) agro-ecological zones of Benin. Also, the presence of aflatoxins was investigated. For detection of fungal contamination, a total of100 kernels/sample (with disinfection) and 40 kernels/sample (without disinfection) were plated. Seventy samples from fourteen villages were used. Aflatoxins occurrence was analysed on 84 samples by ultraehigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The average water content and the cashew nuts count were respectively 8.6% and 172 nuts/kg in NG and 8.7% and 174 nuts/kg in SS. Significant differences between villages in both zones were found for both water content and nuts count. In disinfected samples, strains of Aspergillus section Nigri were predominant, inNG and SS zones (90.2% and 87.2%) respectively. When non disinfected kernels were plated, A. section Nigri was predominant in both NG and SS zones, with percentages of 89.7% and 93.4%, respectively. None of the 84 nuts samples were positive for natural occurrence of aflatoxins with a detection limit of 0.05e0.2 mg/kg.

Contains fulltext : 153413.pdf (publisher's version ) (Open Access), This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in L. lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation of L. lactis to conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zero-growth adaptation.

Contains fulltext : 155358.pdf (publisher's version ) (Open Access), This paper describes the transcriptional adaptations of nongrowing, retentostat cultures of Lactococcus lactis to starvation. Near-zero-growth cultures (mu = 0.0001 h(-1)) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a recovery period of another 24 h by reinitiating the medium supply to the retentostat culture. During starvation, the viability of the culture was largely retained, and the expression of genes involved in transcription and translational machineries, cell division, and cell membrane energy metabolism was strongly repressed. Expression of these genes was largely recovered following the reinitiation of the medium supply. Starvation triggered the elevated expression of genes associated with synthesis of branched-chain amino acids, histidine, purine, and riboflavin. The expression of these biosynthesis genes was found to remain at an elevated level after reinitiation of the medium supply. In addition, starvation induced the complete gene set predicted to be involved in natural competence in L. lactis KF147, and the elevated expression of these genes was sustained during the subsequent recovery period, but our attempts to experimentally demonstrate natural transformation in these cells failed. Mining the starvation response gene set identified a conserved cis-acting element that resembles the lactococcal CodY motif in the upstream regions of genes associated with transcription and translational machineries, purine biosynthesis, and natural transformation in L. lactis, suggesting a role for CodY in the observed transcriptome adaptations to starvation in nongrowing cells.

This paper describes the transcriptional adaptations of nongrowing, retentostat cultures of Lactococcus lactis to starvation. Near-zero-growth cultures (? = 0.0001 h?1) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a recovery period of another 24 h by reinitiating the medium supply to the retentostat culture. During starvation, the viability of the culture was largely retained, and the expression of genes involved in transcription and translational machineries, cell division, and cell membrane energy metabolism was strongly repressed. Expression of these genes was largely recovered following the reinitiation of the medium supply. Starvation triggered the elevated expression of genes associated with synthesis of branched-chain amino acids, histidine, purine, and riboflavin. The expression of these biosynthesis genes was found to remain at an elevated level after reinitiation of the medium supply. In addition, starvation induced the complete gene set predicted to be involved in natural competence in L. lactis KF147, and the elevated expression of these genes was sustained during the subsequent recovery period, but our attempts to experimentally demonstrate natural transformation in these cells failed. Mining the starvation response gene set identified a conserved cis-acting element that resembles the lactococcal CodY motif in the upstream regions of genes associated with transcription and translational machineries, purine biosynthesis, and natural transformation in L. lactis, suggesting a role for CodY in the observed transcriptome adaptations to starvation in nongrowing cells., Biotechnology, Applied Sciences

The current knowledge of the physiology and gene expression of industrially relevant microorganisms is largely based on laboratory studies under conditions of rapid growth and high metabolic activity. However, in natural ecosystems and industrial processes, microbes frequently encounter severe calorie restriction. As a consequence, microbial growth rates in such settings can be extremely slow and even approach zero. Furthermore, uncoupling microbial growth from product formation, while cellular integrity and activity are maintained, offers perspectives that are economically highly interesting. Retentostat cultures have been employed to investigate microbial physiology at (near-)zero growth rates. This minireview compares information from recent physiological and gene expression studies on retentostat cultures of the industrially relevant microorganisms Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger. Shared responses of these organisms to (near-)zero growth rates include increased stress tolerance and a downregulation of genes involved in protein synthesis. Other adaptations, such as changes in morphology and (secondary) metabolite production, were species specific. This comparison underlines the industrial and scientific significance of further research on microbial (near-)zero growth physiology., Biotechnology, Applied Sciences

This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in L. lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation of L. lactis to conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zero-growth adaptation., Biotechnology, Applied Sciences

Fermented products have high nutritional value and constitute an important part of the Chinese dietary profile; they are also gaining popularity throughout the world. Daqu is a traditional natural fermentation starter culture that has a significant impact on the quality and flavour of Chinese liquor and vinegar. A review of the literature was conducted focusing on the classification, composition, and manufacture of Daqu. The review provided a preliminary understanding of the link between the fermentation process and the characteristics of the final Daqu product. Then the occurrence, levels, and diversity of microorganisms were studied in different types of Daqu produced by various fermentation processes. The results showed that Bacillus licheniformis and Saccharomycopsis fibuligera were present in all the tested samples of Daqu. Regional comparisons showed Staphylococcus gallinarum and Staphylococcus saprophyticus in southern Daqu. The fungi Sm. fibuligera and Lichtheimia ramosa were found in low/medium-temperature Daqu and Thermomyces lanuginosus occurred in high-temperature Daqu. In order to study the functionality of Daqu and the contribution of the predominant microorganisms to alcoholic fermentation, the mesophilic and thermophilic bacteria and spores, Enterobacteriaceae, lactic acid bacteria, yeasts, and moulds present in the core and outer portions of Fen-Daqu were isolated. The isolates were identified by culture-dependent sequencing of rRNA genes (16S rRNA for bacteria; 18S rRNA, 26S rRNA, and ITS rRNA for fungi). A succession of fungi, lactic acid, and Bacillus spp. was associated with prevailing acidity, moisture content, and temperature during Daqu fermentation. The predominant species in fermentation were B. licheniformis, Pediococcus pentosaceus, Lactobacillus plantarum, Pichia kudriavzevii, Wickerhamomyces anomalus, Sacchromyces cerevisiae, and Sm. fibuligera. One strain of the each of the above-mentioned predominant species, with the highest

Inaugural lecture upon taking up the post of Personal Professor of Food Microbiology at Wageningen University on 11 June 2015. 'Microbial cultures deliver the typical characteristics of fermented food products through their metabolic activity, by secretion of enzymes in the food matrix and by their particular growth and survival behaviour. Knowledge of the structural complexity and population dynamics provides explanations for compositional stability and overall performance of such cultures. Especially, insight into microbial interactions delivers new design rules for robust composite starter cultures with predictable and desirable industrial performance.'

Daqu is a fermentative saccharification agent that is used to initiate fermentation in the production of Chinese liquor and vinegar. Different types of Daqu can be distinguished based on the maximum fermentation temperature, location of production, and raw materials used. We aimed to characterize and distinguish the different types of Daqu using a culture-independent cloning method. The lowest microbial diversity was found in Daqu produced at high-temperature. Principal component analysis (PCA) was used to compare the bacterial composition of Daqu from different regions (i.e., northern Daqu and southern Daqu). Staphylococcus gallinarum and Staphylococcus saprophyticus were found in southern Daqu, and were absent in northern Daqu. The fungi Saccharomycopsis fibuligera and Lichtheimia ramosa dominated in low/medium-temperature Daqu, whereas Thermomyces lanuginosus occurred in high-temperature Daqu. Our study identified potential biomarkers for the different types of Daqu, which can be useful for quality control and technology development of liquor or vinegar production.

The objective of this study was to investigate the effect of preculturing of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 under sublethal stress conditions on their survival and metabolite formation in set-yoghurt. Prior to co-cultivation with yoghurt starters in milk, the two probiotic strains were precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor. The activity of sublethally precultured probiotics was evaluated during fermentation and refrigerated storage by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated adaptive stress responses of the two probiotic strains resulting in their viability improvement without adverse influence on milk acidification. A complementary metabolomic approach using SPME-GC/MS and 1H-NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Principal component analysis revealed substantial impact of the activity of sublethally precultured probiotics on metabolite formation demonstrated by distinctive volatile and non-volatile metabolite profiles of set-yoghurt. Changes in relative abundance of various aroma compounds suggest that incorporation of stress-adapted probiotics considerably influences the organoleptic quality of product. This study provides new information on the application of stress-adapted probiotics in an actual food-carrier environment

The current knowledge of the physiology and gene expression of industrially relevant microorganisms is largely based on laboratory studies under conditions of rapid growth and high metabolic activity. However, in natural ecosystems and industrial processes, microbes frequently encounter severe calorie restriction. As a consequence, microbial growth rates in such settings can be extremely slow and even approach zero. Furthermore, uncoupling microbial growth from product formation, while cellular integrity and activity are maintained, offers perspectives that are economically highly interesting. Retentostat cultures have been employed to investigate microbial physiology at (near-)zero growth rates. This minireview compares information from recent physiological and gene expression studies on retentostat cultures of the industrially relevant microorganisms Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger. Shared responses of these organisms to (near-)zero growth rates include increased stress tolerance and a downregulation of genes involved in protein synthesis. Other adaptations, such as changes in morphology and (secondary) metabolite production, were species specific. This comparison underlines the industrial and scientific significance of further research on microbial (near-)zero growth physiology.