Pollination necessary for the agricultural crop production affects the functions of the ecosystems on earth. In landscapes where wild pollinators are decreasing, honey bees promote the maintenance of plant species, therefore honey bee losses are of great concern. Current honey bee colony losses (Apis mellifera) worldwide are caused by Colony collapse disorder, the mite Varroa destructor and pesticides. This results in the honey bees weakened immune defenses making them susceptible to different diseases. Studies show that long-term natural selection for coexistence, or resistance to Varroa mites by honey bees is possible, but further developments are needed for this application in managed beekeeping. Furthermore, lactic acid bacteria found in honey bees can play a crucial role by improving its immune response. At places where apicultural practices have led to decreased amounts of lactic acid bacteria in the bees, supplementary feeding is a possible treatment solution. The beekeepers' observations of mite reproduction dynamics and the overwintering of strong and healthy honey bees are needed to decrease Varroa treatment with synthetic chemicals. Based on this knowledge, a small survey of beekeepers in Örebro County, Sweden, was conducted in an attempt to determine the status of their bees during the last 5 years. The largest colony loss over the past 5 winters was predominantly in 2012-2013. Varroa mite infestations with disease symptoms were primarily found in the central region. Since the survey was small and time was limited it was only possible to make general conclusions. Deeper understanding of lactic acid bacteria in honey bee societies and their inhibition of different diseases are important for future research.

As the demand for farmed shrimp continues to grow worldwide, the use of probiotics to address the sustainability of aquaculture fisheries has gained much attention. Emerging diseases in shrimp aquaculture, such as acute hepatopancreatic necrosis disease (AHPND), have devastating economic impacts in countries that largely depend on this activity. The relevance of this research lies on the fact that it explores the potential of using probiotics to mitigate the negative effects of AHPND in shrimp aquaculture. The scope of these studies includes survival of probiotic microbes in typical aquaculture water conditions, the effectiveness of probiotics in vitro and in vivo against the pathogenic strain of Vibrio parahaemolyticus that causes AHPND, and the effects of probiotics on the bacterial community composition in aquaculture water and gastrointestinal tract of shrimp after an induced AHPND infection. The microorganisms chosen as probiotics for this research include a lactic acid bacterium, a yeast and a photosynthetic bacterium. Informal feedback from shrimp farmers in Thailand and Vietnam revealed positive results against AHPND when using a commercially available probiotic containing multiple species of microorganisms from these probiotic groups. This research was divided into four studies. The first study (Chapter 2) evaluated the growth of the three different probiotic microbes in two different salinity conditions commonly found in intensive shrimp production systems to determine whether they could be further considered as potential candidates. The hypothesis was that the NaCl concentrations of the media may not have an effect on acid production, growth and cell morphology of the microorganisms being evaluated due to their metabolic mechanisms of adaptation to differences in osmotic pressure. The probiotic microbes were cultured in nutrient media enriched with 1 and 2% NaCl. Microbial survival, acidity and cell morphology between treatments were compared using enumeration by serial dilutions and plating, pH measurements and scanning electron microscopy imaging, respectively. The results showed that salinity levels up to 2% NaCl did not affect the growth of lactic acid bacteria and yeast. Photosynthetic bacteria grown in media with 1% NaCl showed a 24-hour delay in comparison to the control and a prolonged lag phase that lasted 48 hours when the media contained 2% NaCl. Therefore, the hypothesis was partially supported. Based on these results, all three probiotic microbes demonstrated to be suitable for application in aquaculture ponds with up to 2% salinity. The second study (chapter 3) aimed at determining the inhibitory effects of eight different formulations of probiotic solutions against the pathogenic strain of V. parahaemolyticus in vitro. The hypothesis of this study was that probiotic solutions containing whole microbial cultures of multiple microbial types including lactic acid bacteria may have a greater inactivation of the pathogen. The probiotic formulations consisted of individual cultures, combinations of the three probiotic microbes, and a commercially available probiotic formulation. The inhibitory effects were evaluated following a disk diffusion test on solid media by comparing diameters of zones of inhibition, and a challenge test in liquid media by comparing pathogen survival after exposure to probiotic solutions. Findings revealed inhibition zones with greater diameters in disks treated with whole microbial cultures (min: 7.83 mm, max: 11.33 mm) versus disks treated with only supernatants (min: 7.00 mm, max: 8.50 mm). Results from the challenge in liquid media tests showed greater inactivation of the pathogen after 48 h (6.56±0.07 to 5.43±0.03 log10 reduction) when treated with lactic acid bacteria alone and in combination with other microbial types. From these results, the hypothesis was supported and it was concluded that probiotic solutions including a lactic acid bacterium, the combination of lactic acid bacterium and photosynthetic bacterium and the combination of lactic acid bacterium, yeast and photosynthetic bacterium may be used to effectively inhibit AHPND in shrimp aquaculture. The third study (chapter 4) explored the effects of probiotic solutions on live shrimp (Litopenaeus vannamei) pretreated with probiotics for 7 days prior to challenging them with the pathogenic V. parahaemolyticus strain causing AHPND. The hypothesis of this study was that higher shrimp survival and weight gains would be observed when shrimps are exposed to probiotics solutions with multiple microbial types in the water and feed. Water quality parameters (dissolved oxygen, temperature, acidity, salinity and total ammonia nitrogen), difference in shrimp weight increase and shrimp survival were compared between probiotic treatments and controls. Treatments included: (1) a lactic acid bacterium alone (Pro.Sol1), (2) a lactic acid bacterium and a photosynthetic bacterium (Pro.Sol2), (3) the combination of a lactic acid bacterium, a yeast, and a photosynthetic bacterium (Pro.Sol3), and (4) a commercial probiotic (Com.Pro) and the results showed shrimp survival of 11.7, 26.7, 36.7 and 73.3%, respectively. Also, treatments Pro.Sol3 and Com.Pro resulted in higher weight gains (19.7 and 31.2%, respectively) versus the negative control (11.2%). Moreover, onset of the disease was delayed in all treatments as follows: 12 h with Pro.Sol1, 20 h with Pro.Sol2, 22 h with Pro.Sol3, and 26 h with Com.Pro. From these results, the hypothesis was supported and it was concluded that probiotics have the potential to effectively mitigate the effects of AHPND in the shrimp aquaculture. Finally, the fourth study (chapter 5) evaluated the effects of probiotics on the bacterial diversity of the gastrointestinal tract of shrimp as well as variation of bacterial and fungal diversity in the water before and after challenging shrimp with the pathogenic V. parahaemolyticus strain causing AHPND. The hypothesis of this study was that probiotic solutions with multiple microbial types may be able to maintain the microbial composition of the shrimp GI tract and aquaculture water preventing an increase in relative abundance of the family Vibrionaceae. Next generation sequencing was conducted using an Illumina MiSeq™ and primers specific for bacterial V4 hypervariable region of the 16S rRNA gene. The results obtained from the GI tract of shrimp revealed that the relative abundance of the family Vibrionaceae significantly increased in treatments with high mortalities, whereas treatments with higher survivals showed no significant difference in relative abundance of Vibrionaceae family members (P>0.05) in comparison to the negative control. The Shannon diversity index values (abundance and evenness) of the bacterial communities revealed that the treatment with the highest survival had the highest Shannon index value (4.69±0.133) whereas the treatment with lowest survival had the lowest Shannon index value (0.17±0.004). The results obtained from water samples did not show a higher abundance of the family Vibrionaceae, and diversity was maintained after infection (Shannon index 4.64±0.58). Regarding fungal diversity in water samples, Shannon index values revealed no significant changes before (3.627±0.37) and after infection (3.664±0.18) except for Pro.Sol3 (2.859±0.56) and Com.Pro (1.795±0.50), which included yeast in their formulation. Thus, the hypothesis of this study was partially supported since the results revealed that while all probiotics maintained the diversity of microbial composition in the water, only those probiotic solutions with various microbial types in the formulation maintained the diversity of the microbial composition in the GI tract of shrimp providing protection against AHPND.

Les exopolysaccharides (EPS) produits par les bactéries lactiques ont longtemps été considérés comme des composés indésirables dans le vin. En effet, le β-glucane produit par certaines souches bactériennes est responsable d’une altération qui rend le vin impropre à la consommation. Cependant, les polysaccharides produits par Oenococcus oeni, principale espèce de la fermentation malolactique (FML) qui est une étape essentielle participant à la stabilisation et la qualité du vin, n’ont jamais été associés à un quelconque défaut. Notre premier objectif était d'identifier le matériel génétique impliqué dans la production de polysaccharides, en termes d'évolution phylogénétique et de diversité, parmi une collection de 50 souches d’O. oeni. L'analyse bioinformatique des 50 séquences génomiques a révélé la présence de deux loci organisés en opéron et potentiellement impliqués dans la production d'hétéropolysaccharides, mais aussi la présence de gènes isolés de glycosyltransférases et glycosides hydrolases impliqués dans la synthèse d’homopolysaccharides. L’ensemble des souches analysées présentent au moins une des voies de biosynthèse des EPS, suggérant un rôle essentiel chez O. oeni. Par la suite, une approche biochimique des polysaccharides produits a été mise en place et a permis de confirmer les données génomiques. Une glycosyltransférase membranaire, jouant un rôle clé dans la production d'EPS, a été caractérisée afin de mieux comprendre le mécanisme de la synthèse EPS. Par ailleurs, l’étude physiologique des polysaccharides produits a révélé l’existence de deux phénotypes (forme libérée et capsulaire), chacun jouant un rôle dans la survie et l’adaptation de la bactérie au vin et au procédé de lyophilisation industriel, forme sous laquelle les souches commerciales sont proposées pour induire la FML. / Exopolysaccharides (EPS) of lactic acid bacteria have long ago been undesirable in wine. Indeed, β-glucan produced by certain strains of bacteria provokes wine spoilage and makes the wine commercially defective. However, the polysaccharides produced by Oenococcus oeni which is the main species to drive malolactic fermentation (MLF), an essential step for wine stabilization and quality improvement, are not to be blamed for any spoilage effect. Our first aim was to identify the genetic material implicated in polysaccharides production, in terms of phylogenetic evolution and diversity among the strains of our collection. The bioinformatic analysis of 50 O. oeni genomic sequences revealed the presence of two organized loci potentially implicated in the production of heteropolysaccharides and also the presence of isolated genes of glycosyltransferases and glycoside-hydrolases implicated in the production of homopolysaccharides. The presence of at least one biosynthetic pathways in all the strains tested shows the importance of the polysaccharides genes for Oenococcus oeni. Thereafter, we reached-up a biochemical approach of the produced polysaccharides, confirming the results of the bioinformatics research. Initially we characterized a membrane glycosyltranferase, playing a key role in the EPS production. The results allow us to better understand the mechanism of the EPS synthesis. Furthermore the physiology of the produced polysaccharides showed two possible phenotypes (liberated and capsular form) each one playing his role for the bacterial survival at his natural environment (wine) as well as at industrial level (production of malolactic starters).

Abstract The scope of this study includes aspects of phage evolution and antagonistic/mutualistic coevolution between a phage and its host. As a basic study it may provide tools for developing phage resistant starters and offer regulatory elements and factors for biotechnological applications. The LL-H anti-receptor was characterized by isolation of spontaneous LL-H host range mutants and subsequent sequencing of candidate genes. All LL-H host range mutants carried a single point mutation at the 3' end of a minor tail protein encoding gene g71. The genomic location of g71 is congruent with the other verified anti-receptor genes found in the λ supergroup. The C-terminus of Gp71 determines the adsorption specificity of phage LL-H similarly for the number of phages infecting Gram-positive and Gram-negative bacteria. A Gp71 homolog of phage JCL1032 showed 62% identity to LL-H Gp71 within the last 300 amino acids at the C-terminus. Lactobacillus delbrueckii phage receptors were investigated by the purification of different cell surface structures. Certain Lb. delbrueckii phages from homology groups a and c including LL-H, LL-H host range mutants and JCL1032, were specifically inactivated by the LTAs. In structural analyses LTAs showed differences in the degree of α-glucosyl and ᴅ-alanyl substitution. α-glucose is necessary for LL-H adsorption. A high level of ᴅ-alanine esters in LTA backbones inhibited Lb. delbrueckii phage inactivation in general. Lysogenization of strain ATCC 15808 with the temperate phage JCL1032 revealed a rarely described coexistence of phage adsorption resistance and phage immunity, which could not be explained by lysogenic conversion. In this case the role of spontaneously induced JCL1032 may be significant. The LL-H early gene region was localized between the dysfunctional lysogeny module and the terminase encoding genes. The function of five ORFs could be connected to phage DNA replication and/or homologous recombination. Transcription of LL-H genes could be divided into two, possibly three, phases in which large gene clusters were sequentially transcribed. The intensity of the late transcripts exceeded the intensity of the early transcripts by several times. Two candidate genes for transcription regulators were found. One of the two candidates is the first ORF in the LL-H early gene region.

No presente trabalho procurou-se avaliar os tipos de metabolismo existentes em espécies e linhagens de Latobacillus isoladas de ambiente de fermentação alcoólica industrial. Para tal, 17, linhagens foram crescidas por 24 horas a 32 ºC, em meio de glicose e frutose (1% de cada açúcar) suplementado com extrato de levedura, minerais e tampão. O crescimento bacteriano foi estimado por plaqueamento e abosrbância a 600 nm, enquanto que no meio de crescimento foram estimados, mediante cromatografia de alta eficiência, os teores de ácido lático, ácido acético e etanol. Os isômeros óticos D(-)- e L(+)-lactato foram dosados enzimaticamente mediante espectrofotometria a 340nm, empregando-se desidrogenases estereoespcíficas. Os resultados mostraram que entre as 17 linhagens de Latobacillus avaliadas foram observados os três tipos de metabolismo : homofermentativo obrigatório (8 linhagens), heterofermentativo facultativo (1 linhagem) e heterofermentativo obrigatório (8 linhagens). Observou-se também uma relação estequiométrica dos produtos formados (lactato, acetato e etanol), condizente com os passos metabólicos característicos de cada grupo. Os resultados também permitem deduzir que quanto à formação dos isômeros óticos, houve predominância das linhagens tipo DL (produção simultânea dos dois isômeros), sobre as linhagens D e L. No entanto foram encontradas linhagens produzindo 100% de um dado isômero (D e L), descortinando a possibilidade de que linhagens isoladas de processo industrial de produção de etanol, possam atender demandas biotecnológicas que requeiram proporções definidas dos isômeros. Foi possível deduzir que a utilização dos teores de ácido lático na avaliação da contaminação bacteriana deve ser considerada com ressalvas, especialmente no caso do emprego do ácido L(+)-lático, e notadamente no caso de comunidades bacterianas complexas. / The aim of the present work was to evaluate the metabolism type of 17 Lactobacilli strains isolated from industrial ethanol fermentation plants. The strains were grown, at 32°C for 24 hours, on a mixture of equal amounts of glucose and fructose as the carbon source, and supplemented with yeast extract, mineral nutrients and buffer. Bacterial growth was estimated either by absorbance at 600nm and by plating. The main end products of bacterial metabolism (lactate, acetate and ethanol) were measured by high performance liquid chromatography, while the stereoisomers, D(-)- and L(+)-lactate were assayed by an enzymatic methodology using stereospecific lactatedehydrogenases. According to the results, all the three types of metabolism were found among the bacteria investigated: obligately homofermentative (8 strains), facultative heterofermentative (1 strain) and obligately heterofermentative (8 strains). Additionally, it was observed a stoichiometric relationship between the major end products (lactate, acetate and ethanol) in agreement with the metabolic pathway proposed for the different bacterial types (homo- and heterofermentative strains). The results have showed a predominance of strains of DL type, regarding the stereoisomers production. However, it was found strains producing a single type of the isomeric form. These findings suggest the possibility to explore the Lactobacilli biodiversity in fuel ethanol fermentation plants for lactate production of chemically pure optical isomers. It was also observed that the use of lactate content for a chemical evaluation of bacterial contamination may be considered with some restrictions, mainly in the case of L(+)-lactate, and particularly in the case of complex bacterial communities.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22664号 / 農博第2419号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5295(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 佐藤 健司, 教授 澤山 茂樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

This thesis deals with the study of yogurt preparation and changes during its storage. The theoretical part contains information about parameters affecting both the preparation and properties of the final product. The experimental part describes methods and procedures used for determination of critical parameters such as lactic acid, lactose, glucose and galactose concentrations (HPLC-RI), amounts of streptococci and lactobacilli. The data was graphically evaluated and commented on. The work also included sensory analysis in which the prepared yogurt was compared with commercially available products. The results of this work can serve both consumers and smaller yogurt producers.

No description available.

The diploma thesis deals with the growth of biomass and production of selected metabolit–lactic acid by thermophilic bacteria Bacillus coagulans. The resulting selected metabolite was determined by HPLC method. Cultivations of this genus were performed on synthetic media, where the influence of carbohydrate used as carbon source was tested. Lactose was more suitable fot growth of biomass and glucose for production of lactic acid. On natural whey media the influence of different conditions were tested. The highest yields of biomass and production of lactic acid were observed on enriched whey medium. The last part deals with comparing the production of biomass and metabolites, depending on the volume of media. There were compared selected bioengineering characteristics of all cultivations.

No description available.