Lene Jespersen

Occurrence and taxonomic characteristics of strains of Saccharomyces cerevisiae predominant in African indigenous fermented foods and beverages

Indigenous fermented foods and beverages play a major role in the diet of African people. The predominant yeast species seen is Saccharomyces cerevisiae, involved in basically three groups of indigenous fermented products: non-alcoholic starchy foods, alcoholic beverages and fermented milk. These products are to a great extent made by spontaneous fermentation and consequently S. cerevisiae often coexists with other microorganisms even though a microbiological succession usually takes place both between and within species. The functions of S. cerevisiae are mainly related to formation of alcohols and other aroma compounds, but stimulation of e.g. lactic acid bacteria, improvement of nutritional value, probiotic effects, inhibition of undesired microorganisms and production of tissue-degrading enzymes may also be observed. Several different isolates of S. cerevisiae have been shown to be involved in the fermentations and some of the isolates show pheno- and genotypic characteristics that deviate from those normally recognised for S. cerevisiae.

Specific spoilage organisms in breweries and laboratory media for their detection

The Gram positive bacteria are generally regarded as the most hazardous beer spoilage organisms in modern breweries, especially the lactobacilli: L. brevis, L. lindneri, L. curvatus, L. casei, L. buchneri, L. coryneformis, L. plantarum, L. brevisimilis, L. malefermentans and L. parabuchneri and the pediococci: P damnosus, P. inopinatus and P. dextrinicus. Micrococcus kristinae is the only species within the micrococci relevant to brewing. The Gram negative strictly anaerobic bacteria are apparently increasing in importance and include Pectinatus cerevisiiphilus, Pectinatus frisingensis and Selenomonas lacticifex, reported as obligate beer spoilage organisms: Zymophilus raffinosivorans as a potential beer spoilage organism; Megasphaera cerevisiae as an obligate spoilage organism of low alcohol beer and Zymomonas mobilis as capable of spoiling primed beer. With improved process technology the importance of aerobic bacteria has decreased and the same applies for the Gram negative aerobic bacteria Hafnia protea and Enterobacter cloacae which are capable of surviving beer fermentation. Beer spoilage organisms include several so-called wild yeasts, of which Saccharomyces species are generally considered the most important. Even though the detection of beer spoilage organisms by cultivation in laboratory media does not always provide the specificity and the sensitivity required, the use of selective media and incubation conditions still appears to be the method preferred by breweries. The media used depend on the type of sample, the specificity required and, for detection of wild yeasts, to some extent, the characteristics of the culture yeast. Among the media reported so far no single medium can be used to detect all members within a group of specific beer spoilage organisms and further work on the development of improved substrates are required both for bacteria and wild yeasts.

Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis

Samples of Coffea arabica were collected during the different stages of the fermentation from two production sites in Tanzania. The yeasts community was identified by genotyping using ITS–PCR and sequence analysis of the D1/D2 domain of the 26S rRNA gene. For confirmation, denaturating gradient gel electrophoresis (DGGE) of PCR‐amplified 26S rRNA gene was performed to detect yeast directly from coffee samples without cultivation. Yeast counts were in the range 4.0 × 104 − 5.0 × 107 CFU/g with an increase during fermentation. Three yeasts species were dominant. The predominant yeast found during fermentation and drying was Pichia kluyveri. Pichia anomala was found in high numbers during drying of coffee beans. Hanseniaspora uvarum was the predominant yeast during fermentation but decreased during drying. Kluyveromyces marxianus, Candida pseudointermedia, Issatchenkia orientalis, Pichia ohmeri and Torulaspora delbrueckii occurred in concentrations of 103 CFU/g or below in coffee samples. Saccharomyces cerevisiae and Candida xestobii were not isolated by cultivation, but by the DGGE technique. A good agreement was found between the sequence analysis of the D1/D2 domain of the 26S rRNA gene and sequencing of the DGGE bands. Sequences of yeast isolates determined in this study have been deposited in the GenBank database under Accession Nos AY305664–AY305669 and AY305672–AY305683. Sequences of DGGE bands have also been deposited in the GenBank database under Accession Nos AY314789–AY314802. Copyright

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE).

The yeast populations associated with the fermentation of Ghanaian cocoa were investigated using denaturing gradient gel electrophoresis (DGGE). Samples were collected at 12–24 h intervals from heap and tray fermentations, at three different fermentation sites and different periods during the season. Eukaryotic universal primers were used to amplify a fragment of the 26S rRNA gene. The DGGE profiles were relatively complex, underlining that the fermentation of cocoa is a complex microbial process. The identities of selected fragments in the denaturing gels were revealed by sequencing. Hanseniaspora guilliermondii, Candida krusei and Pichia membranifaciens were detected from most fermentations, indicating their possible important role in the fermentation of Ghanaian cocoa. Saccharomyces cerevisiae and Candida zemplinina were almost exclusively detected during tray fermentations. The developed DGGE protocol was compared with traditional culture‐based isolations. The results were comparable but slightly different, as one yeast species (C. zemplinina) was only detected using DGGE. On the other hand, Trichosporon asahii yielded only faint bands in the denaturing gels, despite the fact that it was detected using culture‐based methods. Analysis of pure cultures showed that the targeted region of the 26S rRNA gene was poorly amplified in T. asahii, whereas all other investigated isolates were amplified efficiently using the chosen PCR approach. Cluster analysis revealed that the DGGE profiles clustered according to fermentation method and fermentation site. Furthermore, clustering according to progress in the fermentation was observed. The DGGE technique therefore seems to offer a relatively fast and reliable method for studying yeast population dynamics during cocoa fermentations. The nucleotide sequences determined in this study have been assigned Genbank Accession Nos AY762330–AY762349. Copyright

Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll‐like receptor‐2‐dependent mechanism

Lactobacilli are probiotics that, among other health‐promoting effects, have been ascribed immunostimulating and virus‐preventive properties. Certain Lactobacillus spp. have been shown to possess strong interleukin‐12 (IL‐12) ‐inducing properties. As IL‐12 production depends on the up‐regulation of type I interferons (IFNs), we hypothesized that the strong IL‐12‐inducing capacity of Lactobacillus acidophilus NCFM in murine bone‐marrow‐derived dendritic cells (DCs) is caused by an up‐regulation of IFN‐β, which subsequently induces IL‐12 and the double‐stranded RNA binding Toll‐like receptor‐3 (TLR‐3). The expression of the genes encoding IFN‐β, TLR‐3, IL‐12 and IL‐10 in DCs upon stimulation with L. acidophilus NCFM was determined. Lactobacillus acidophilus NCFM induced a much stronger expression of Ifn‐β, Il‐12 and Il‐10 compared with the synthetic double‐stranded RNA ligand Poly I:C, whereas the levels of expressed Tlr‐3 were similar. Whole genome microarray gene expression analysis revealed that other genes related to viral defence were significantly up‐regulated and among the strongest induced genes in DCs stimulated with L. acidophilus NCFM. The ability to induce IFN‐β was also detected in another L. acidophilus strain (X37), but was not a property of other probiotic strains tested, i.e. Bifidobacterium bifidum Z9 and Escherichia coli Nissle 1917. The IFN‐β expression was markedly reduced in TLR‐2−/− DCs, dependent on endocytosis, and the major cause of the induction of Il‐12 and Tlr‐3 in DCs stimulated with L. acidophilus NCFM. Collectively, our results reveal that certain lactobacilli trigger the expression of viral defence genes in DCs in a TLR‐2 manner dependent on IFN‐β.

Beneficial effects of probiotic and food borne yeasts on human health.

Besides being important in the fermentation of foods and beverages, yeasts have shown numerous beneficial effects on human health. Among these, probiotic effects are the most well known health effects including prevention and treatment of intestinal diseases and immunomodulatory effects. Other beneficial functions of yeasts are improvement of bioavailability of minerals through the hydrolysis of phytate, folate biofortification and detoxification of mycotoxins due to surface binding to the yeast cell wall.

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin

Aims:  To identify the dominant micro‐organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development.

Gene Transcription and Virulence Potential of Listeria monocytogenes Strains After Exposure to Acidic and NaCl Stress

Gene transcription and virulence potential of two strains of Listeria monocytogenes, EGD-e and 4140, were compared by quantitative real-time polymerase chain reaction and in a Caco-2 in vitro model after exposure to acidic (pH 5.5) and NaCl (4.5% w/v) stress. Strain-dependent differences in gene transcription were observed both after exposure to shock (six genes) and after long-term adaptation to stress (18 genes). In the shock experiments, a transient induction of clpC and clpE was seen for both strains, while transient induction of sigB, inlA, and inlB was observed for strain 4140 only; actA was only induced in EGD-e after NaCl shock. The long-term stress experiments were included to imitate the stress conditions encountered by L. monocytogenes when present in food products. Long-term adaptation of EGD-e to acidic stress induced transcription of iap and repressed flaA, while genes related to stress response and invasion (clpC, clpP, inlA, inlB, prfA, and sigB) were induced in 4140. Long-term adaptation of EGD-e to NaCl stress increased transcription of genes important for the intracellular life cycle (actA, hly, iap, inlA, inlB, plcA, plcB, and prfA), while few changes were observed for 4140. Experiments with Caco-2 confirmed that long-term adaptation of EGD-e and 4140 to acidic and NaCl stress is capable of increasing the virulence potential: an improved adhesion to Caco-2 was observed for both EGD-e and 4140 after acidic and NaCl stress, and increased invasion was seen for both strains after long-term NaCl stress. The fact that several virulence genes were up-regulated and that adhesion and invasion properties were increased demonstrate that certain environmental conditions in food products might influence the virulence potential of L. monocytogenes.

Taxonomic and Ecological Diversity of Food and Beverage Yeasts

Fermentation has been used for several thousand years as an effective and low-cost resource to preserve the quality and safety of foods. Apart from this primary role, fermentation adds value and enhances nutritional quality and digestibility through biological enrichment, and provides dietary enrichment through aroma and flavour production and modifying textures of food substrates. All these changes are operated by microorganisms, which, naturally present (spontaneous fermentations) or added (inoculated fermentation) in raw materials, break down complex carbohydrates and proteins into more easily digestible elements. Among the fermentation microorganisms, yeasts are undoubtedly the most important group of microorganisms that are exploited for commercial purposes. Yeasts used in food fermentation processes modify original materials organoleptically, physically and nutritionally and for this they have been used for millennia in bread-making and production of alcoholic beverages. A diversity of fermented foods, which vary according to geographical area and cultural preference, are produced across the globe and the individual and peculiar characteristics which distinguish and typify each food are determined by the expression of the wide biodiversity of the fermenting microorganisms, such as yeasts. In Western countries yeasts are involved in the production of kefir from milk, beer from barley and hops, and wine from grapes, while in developing countries, where the lack of resources limits the use of techniques such as vitamin enrichment of food and the use of capital-intensive processes for food preservation, food fermentation contributes substantially to food security. In these countries there is a wide diffusion of traditional fermented foods, produced from both edible and inedible raw materials, based on local customs and generally consumed as dietary staples. Here we report the ecology and the role of yeasts involved in fermented food and beverages, focusing on the dominant species and their effect on the product quality, summarised in Table 2.1. Chapter 2

Molecular genetic identification of Saccharomyces sensu stricto strains from African sorghum beer

Genetic relationships of 24 phenotypically different strains isolated from sorghum beer in West Africa and the type cultures of the Saccharomyces sensu stricto species were investigated by universally primed polymerase chain reaction (PCR) analysis, microsatellite fingerprinting and PCR-restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers. The results demonstrate that internal transcribed spacer (ITS) PCR-RFLP analysis with the endonucleases HaeIII, HpaII, ScrFI and TaqI is useful for discriminating S. cerevisiae, S. kudriavzevii, S. mikatae from one another and from the S. bayanus/S. pastorianus and S. cariocanus/S. paradoxus pairs. The sorghum beer strains exhibited the same restriction patterns as the type culture of S. cerevisiae CBS 1171. PCR profiles generated with the microsatellite primer (GTG)(5) and the universal primer N21 were almost identical for all isolates and strain CBS 1171. Despite phenotypic peculiarities, the strains involved in sorghum beer production in Ghana and Burkina Faso belong to S. cerevisiae. However, based on sequencing of the rDNA ITS1 region and Southern hybridisation analysis, these strains represent a divergent population of S. cerevisiae.