Дисертації з теми "Acid-Selection"

Lactic acid bacteria were isolated from 10 samples of the traditionally fermented foods (5 samples of Vietnamese fermented pork roll and 5 samples of the salted field cabbage) and 5 samples of fresh cow milks collected from households in Vietnam. 22 strains of lactic acid bacteria were isolated for inhibition to Lactobacillus plantarum JCM 1149. Of these, only 2 strains including DC1.8 and NC1.2 have rod shape, the others have coccus shape. 7 strains showing higher antibacterial activity were selected for checking spectrum of antibacteria with indicator bacteria consistting of Bacillus subtilis ATCC 6633, Enterococcus faecium JCM 5804 and Staphylococcus aureus TLU. By which, 3 strains including NC3.5 (from Vietnamese fermented pork roll), DC1.8 (from salted field cabbage) and MC3.19 (from fresh cow milk) were selected because of their higher antibacterial ability. However, the antibacterial activity of the lactic acid bacteria can be based on their disposable compounds and some other antibacterial compounds produced during their growth (such as lactic acid, H2O2, bacteriocins, etc.). For seeking lactic acid bacteria with capability of producing bacteriocins, antibacterial compounds with protein nature, 3 above strains were checked sensitiveness to proteases (including protease K, papain, α – chymotrypsin and trypsin). Because bacteriocins are proteinaceous antibacterial compounds, so their antibacterial activity will be reduced if proteases are added. The result showed DC1.8 and MC3.19 were capable of producing bacteriocin during culture process. They were identified as Lactobacillus acidophilus and Lactococcus lactis and classified, respectively, based on analysis chemical characterisitcs by standard API 50 CHL kit and phylogeny relationship by 16s rRNA sequences.
Các chủng vi khuẩn lactic được phân lập từ 10 mẫu thực phẩm lên men truyền thống (5 mẫu nem chua, 5 mẫu dưa cải bẹ muối) và 5 mẫu sữa bò tươi được thu thập từ các hộ gia đình ở Việt Nam. 22 chủng vi khuẩn lactic đã được phân lập với tiêu chí có khả năng kháng lại vi khuẩn kiểm định Lactobacillus plantarum JCM 1149. Trong số đó, 2 chủng DC1.8 và NC1.2 có tế bào hình que, các chủng còn lại có tế bào hình cầu. 7 chủng thể hiện hoạt tính kháng khuẩn cao được lựa chọn để xác định phổ kháng khuẩn rộng hơn với ba loài vi khuẩn kiểm định Bacillus subtilis ATCC 6633, Enterococcus faecium JCM 5804 và Staphylococcus aureus TLU. Từ đó lựa chọn được 3 chủng có hoạt tính kháng khuẩn cao hơn hẳn. Các chủng này gồm NC3.5 phân lập từ nem chua, DC1.8 phân lập từ dưa cải bẹ muối và MC3.19 phân lập từ sữa bò tươi. Tuy nhiên, hoạt tính kháng khuẩn của vi khuẩn lactic bao gồm những hợp chất nội tại có trong nó và cả những hợp chất được sinh ra trong quá trình phát triển của nó (như axit lactic, H2O2, bacteriocin, …). Với định hướng tìm chủng vi khuẩn lactic có khả năng sinh bacteriocin, chất kháng khuẩn có bản chất protein, 3 chủng trên được kiểm tra độ nhạy cảm với các protease (gồm protease K, papain, α – chymotrypsin và trypsin). Do bacteriocin là chất kháng khuẩn có bản chất protein nên hoạt tính kháng khuẩn của chúng sẽ bị giảm nếu protease được bổ xung vào. Kết quả lựa chọn được chủng DC1.8 và MC3.19 có khả năng sinh bacteriocin. Hai chủng này được phân loại đến loài nhờ vào phân tích đặc điểm sinh hóa bằng kit API 50 CHL và mối quan hệ di truyền thông qua trình tự gen 16s rRNA. Kết quả phân loại đã xác định chủng DC1.8 thuộc loài Lactobacillus acidophilus và chủng MC3.19 thuộc loài Lactococcus lactis.

En aquesta tesi doctoral, es va realitzar la selecció de bacteris acètics (BA) per a dur a terme una fermentació selectiva de D-glucosa en àcid D-glucònic, sense consum de fructosa. Es seleccionaren tres soques de BA, dues pertanyents al gènere Gluconobacter i una altre del gènere Acetobacter, sent les soques de Gluconobacter les millors preparades per a aquesta oxidació. Es va mesurar l'activitat de les deshidrogenases de membrana que participen en l'oxidació completa de la D-glucosa. A més, es van seqüenciar els gens que codifiquen per aquests enzims i es van utilitzar per construir arbres filogenètics, obtenint com a resultat que els gèneres Acetobacter i Gluconobacter queden separats en diferents grups. També es van estudiar els requeriments nutricionals de les BA, centrant-se principalment en la utilització de nitrogen. El creixement d'aquests bacteris va ser millor en els medis amb una combinació completa d'aminoàcids i d'amoni. Les soques seleccionades es van seqüenciar per millorar els protocols de seguiment d'aquests bacteris durant el procés
En esta tesis doctoral, se realizó la selección de bacterias acéticas (BA) para llevar a cabo una fermentación selectiva de D-glucosa en ácido D-glucónico, sin consumo de fructosa. Se seleccionaron tres cepas de BA, dos de ellas pertenecientes al género Gluconobacter y otra del género Acetobacter, siendo las cepas de Gluconobacter las mejor preparadas para esta oxidación. Se midió la actividad de las deshidrogenasas de membrana que participan en la oxidación completa de la D-glucosa. Además, se secuenciaron los genes que codifican para estas enzimas y se utilizaron para construir árboles filogenéticos, obteniendo como resultado que los géneros Acetobacter y Gluconobacter quedan separados en diferentes grupos. También se estudiaron los requerimientos nutricionales de las BA, centrándose principalmente en la utilización de nitrógeno. El crecimiento de estas bacterias fue mejor en los medios con una combinación completa de aminoácidos y de amonio. Las cepas seleccionadas se secuenciaron para mejorar los protocolos de seguimiento de estas bacterias durante el proceso
In this PhD thesis, the selection of Acetic acid bacteria (AAB) was carried out to perform a selective fermentation of D-glucose into D-gluconic acid, without consuming fructose. Three AAB strains were selected, two of them belonging to the Gluconobacter genus and another to Acetobacter, being the Gluconobacter strains better prepared for this oxidation. The activities of the membrane-bound dehydrogenases involved in the complete oxidation of D-glucose were measured. Furthermore, the genes encoding for these enzymes were sequenced and used to construct phylogenetic trees, obtaining as a result that Acetobacter and Gluconobacter genera grouped into different clusters. The nutritional requirements, mainly focused on nitrogen, for AAB were also studied. The growth of these bacteria was better in media with a complete combination of amino acids and ammonium. The selected strains were sequenced to improve the monitoring protocols for these bacteria in the process.

Lipases are widely researched enzyme group because of their ability to catalyze a wide range of synthesis reactions. The aim of this doctoral thesis was to study and optimize fatty acid ester synthesis using lipases. The influence of enzyme choice, its preparation (immobilization), choice of substrates and reaction conditions on fat hydrolysis, biodiesel and phenethyloctanoate (flavour ester) synthesis reaction course and yield was studied. Enterobacter aerogenes lipase, which was developed in Lithuania, was immobilized and its properties were determined for the first time. Commercial lipases were used for hydrolysis and synthesis reactions, the influence of reaction parameters was determined using response surface methodology, and so the yield was optimized. Silica gel was found to be a promising reaction additive for acyl group migration catalysis in biodiesel synthesis reaction. Different lipase specificities towards different acylglycerol classes and regioisomers varied greatly; on the basis of this knowledge two-step biodiesel synthesis catalysis was proposed.
Lipazių atranka ir taikymas riebalų rūgščių esterių sintezei Lipazės yra intensyviai tiriama fermentų grupė dėl jų gebos katalizuoti platų spektrą sintezės reakcijų. Disertacinio darbo tikslas buvo ištirti ir optimizuoti riebalų rūgščių esterių sintezės, panaudojant lipazes, būdus. Tirta fermento preparato paruošimo (imobilizavimo), fermento bei substratų pasirinkimo bei reakcijos sąlygų ir priedų įtaka riebalų hidrolizės, biodyzelino, fenetiloktanoato (kvapiojo esterio) sintezės reakcijų eigai ir išeigai. Pirmą kartą ištirtos Lietuvoje paruoštos Enterobacter aerogenes lipazės savybės, ji imobilizuota ant gamtinės kilmės nešiklių. Komercinių lipazių katalizuojamų reakcijų sąlygų įtakai nustatyti ir reakcijos išeigai optimizuoti naudota atsako paviršiaus metodologija. Tiriant reakcijos priedų įtaką acilgrupės migracijai ir biodyzelino sintezės reakcijai, nustatyta, kad perspektyvus priedas yra silikagelis. Lipazių specifiškumas ženkliai skiriasi skirtingoms acilglicerolių klasėms ir regioizomerams, šių žinių pagrindu buvo pasiūlyta dviejų etapų biodyzelino sintezės katalizė.

Genetic selection to enhance muscularity in beef cattle is desirable to increase retail beef yield and the profitability of the beef industry. However it is unknown how selection for greater muscling will impact on intermediary and muscle energy metabolism which may influence certain attributes of meat quality. In order to assess these impacts of selection for greater muscling in cattle, the physiological mechanisms that underpin the increase in retail beef yield must be identified. This thesis examined the impact of selection for greater muscling on: retail beef yield; muscle glycogen; whole body insulin responsiveness; adrenaline responsiveness of muscle, adipose and liver tissue; and proportion of glycolytic and oxidative myofibres and enzyme activities. This study used 11 high (High), 10 low (Low) and 3 high muscled steers with a myostatin mutation (HighHet) from an Angus herd which had been visually selected for divergence in muscling over 15 years. The results of the yield test performed at bone-out showed that the HighHet and High muscled steers were the highest yielding with the lowest proportion of fat, while the Low muscling animals were the lowest yielding with the highest proportion of fat. Muscle glycogen and lactate concentration were analysed from four muscle biopsies, taken between 18 and 24 months of age, from the m. semimembranosus (SM), m. semitendinosus (ST) and m. longissimus thoracis et lumborum (LTL) of each animal. The muscle glycogen concentrations which were 6.1% higher in the High steers compared to the Low animals while the HighHet did not differ from either group. The effect of selection for muscling on whole body insulin responsiveness was measured using the hyperinsulineamic-euglyceamic clamp technique. Insulin was constantly infused at 2 levels, glucose was concurrently infused to maintain euglyceamia, and the steady-state glucose infusion rate (SSGIR) indicated insulin responsiveness. At the low insulin infusion rate of 0.6 mU/kg/min, the SSGIR was 73% higher for the High muscling genotype animals when compared to the Low. At the high insulin infusion rate of 6.0 mU/kg/min, these differences were proportionately less with the High and the HighHet genotypes having only 27% and 34% higher SSGIR than the Low muscled genotype. The High muscled cattle also had 30% higher plasma IGF-1 concentrations compared to the Low muscled cattle. The increased whole body insulin responsiveness in combination with higher IGF-1 concentrations in the High muscled steers is likely to initiate a greater level of protein synthesis, which may partially explain the increased muscle accretion in these animals. Increased insulin responsiveness in the High steers would also increase glycogenesis in the muscle, aligning with the glycogen results. The effect of selection for muscling on adrenaline responsiveness was measured using 7 adrenaline challenges ranging between 0.2 to 3.0 μg/kg liveweight. Plasma was analysed for NEFA, lactate, glucose and growth hormone concentration and area under curve (AUC) over time was calculated to reflect the tissue responses to adrenaline. The High steers had 30% lower lactate AUC than the Low steers at challenges greater than 2 μg/kg live weight, indicating lower muscle responsiveness at the highest adrenaline doses causing less glycogenolysis. This result also aligns with these animals having more muscle glycogen, thus more muscular animals may reduce the incidence of dark, firm, dry meat that is caused by low levels of glycogen at slaughter. At all levels of adrenaline challenge the High steers had at least 30% greater NEFA AUC, indicating that their adipose tissue was more responsive to adrenaline, resulting in greater lipolysis. In agreement with this response, the High steers had a higher plasma growth hormone concentration, which is likely to have contributed to the increased lipolysis evident in these animals in response to adrenaline. This difference in lipolysis may in part explain the reduced fatness of muscular cattle. There was no effect of selection for muscling on liver responsiveness to adrenaline. Contrary to our initial hypotheses, the High steers had less glycolytic type IIX myofibres in the LTL and larger average cross-sectional area of myofibres in the SM and ST than their Low muscled counterparts. This suggests that myofibre hypertrophy may be a possible mechanism leading to greater muscle mass of these High muscled animals. This also indicates that breeding for more muscular cattle can actually maintain the oxidative capacity of the muscle, a finding supported by the enzymatic results showing that the High muscled steers had lower activity of lactate dehydrogenase and higher activity of citrate synthase and isocitrate dehydrogenase. The High muscled cattle also had a higher concentration of iron in the LTL, and selection for increased muscling had no impact on pH decline or retail colour stability, factors which both affect meat quality. The aim of the second experiment was to determine if phenotypic measurements taken at the time of grading for Meat Standards Australia (MSA) could explain variance in ultimate pH (pHu) of carcasses and the probability of a carcass complying with MSA standards for pHu (≤5.7). Analyses of 204,072 carcass records collated by MSA at a Western Australian processor confirmed that more muscular cattle have a higher compliance rate for pHu. An increase in eye muscle area from 40 to 80 cm2, increased pHu compliance by approximately 14%. Therefore animals with greater muscularity had a lower incidence of dark, firm, dry beef supporting the results that High muscled cattle have increased insulin responsiveness, and reduced adrenaline responsiveness, leading to increased glycogen storage at slaughter. Thus, breeding more muscular cattle with eye muscle area greater than 70 cm2 may help alleviate the problem of dark, firm, dry beef. As rib fat depth increased from 0 to 20mm, pHu compliance increased by around 10%. Heavier cattle also had higher compliance than lighter cattle, and younger cattle also had higher compliance rates. This highlights the importance of good nutrition and high muscle glycogen storage prior to slaughter to maximise compliance rates. The final study examined 81 commercially managed High and Low muscled steers and showed that the effects of muscularity on muscle glycogen were variable as pasture quality and availability changed however there were no negative effects of selection for greater muscling on muscle glycogen, glycogenolysis pre-slaughter, or on the incidence of dark, firm and dry carcasses. Animal temperament assessed using crush score and flight speed measurements did however affect muscle glycogen with the more flighty animals having lower muscle glycogen concentrations.

El contingut i la composició en àcids grassos del greix intramuscular (GIM) afecten la qualitat de la carn de porc. En particular, augmentar el contingut d’àcid oleic (C18:1) milloraria la seva qualitat pel que fa a atributs organolèptics i tecnològics i propietats nutricionals. Aquesta tesi doctoral forma part d’una línia de recerca del Grup de Millora Genètica del Porcí de la Universitat de Lleida, amb l’objectiu final de trobar estratègies per millorar genèticament la qualitat de la carn de porc a través de GIM i C18:1. Es divideix en tres parts. La Part 1 discuteix les implicacions d’aplicar un enfocament estadístic específic per a dades composicionals per analitzar aquests caràcters. Es va mostrar que, com que la variabilitat de la composició del greix de la carn de porc és baixa, les tècniques estadístiques estàndards sobre percentatges bruts són suficientment robustes per la majoria d’anàlisis, incloent les que es realitzen a continuació. En la Part 2, en una línia Duroc, es va estimar que GIM i C18:1 tenen una heretabilitat alta similar (0.51−0.56, per a GIM, i 0.44−0.50, per a C18:1) i una correlació favorable entre ells (0.47). A més, existeixen escenaris de selecció en què aquests caràcters i el creixement magre es poden millorar simultàniament. Es va demostrar experimentalment que (1) GIM i C18:1 responen a la selecció basada en valors de millora a partir de dades fenotípiques de parents, i (2) l’espessor de greix dorsal es pot modificar independentment de GIM i C18:1. No obstant, la selecció per GIM i C18:1 basada en dades preses en un múscul té respostes correlacionades desiguals en altres músculs i teixits adiposos. En la Part 3 es van analitzar les variacions de la seqüència del gen estearoil-CoA desaturasa (SCD), que codifica l’enzim limitant en la biosíntesi de C18:1. Es va mostrar que hi ha una variant funcional en el gen SCD amb un efecte additiu de +0.75% en C18:1 i +1.00% en contingut total d’àcids grassos monoinsaturats, però sense efecte en GIM o engreixament de la canal. Aquesta associació es va confirmar en un estudi d’associació genòmica que també va revelar variacions de nucleòtids en el locus del gen del receptor de la leptina (LEPR) que afecten el nivell d’engreixament i, en conseqüència, la composició del greix. L’ús de marcadors en aquests dos loci va millorar substancialment la precisió en les prediccions de GIM i C18:1. Es conclou que és possible seleccionar amb èxit per GIM i C18:1 en carn de porc i es discuteixen diversos escenaris sobre com implementar aquesta selecció a la pràctica.
El contenido y la composición en ácidos grasos de la grasa intramuscular (GIM) afectan la calidad de la carne de cerdo. En particular, aumentar el contenido de ácido oleico (C18:1) mejoraría su calidad en cuanto a atributos organolépticos y tecnológicos y propiedades nutricionales. Esta tesis doctoral forma parte de una línea de investigación del Grupo de Mejora Genética del Porcino de la Universitat de Lleida, con el objetivo final de encontrar estrategias para mejorar genéticamente la calidad de la carne de cerdo a través de GIM y C18:1. Se divide en tres partes. La Parte 1 discute las implicaciones de aplicar un enfoque estadístico específico para datos composicionales para analizar estos caracteres. Se mostró que, como la variabilidad de la composición de la grasa de la carne de cerdo es baja, las técnicas estadísticas estándares sobre porcentajes brutos son suficientemente robustas para la mayoría de análisis, incluyendo los que se realizan a continuación. En la Parte 2, en una línea Duroc, se estimó que GIM y C18:1 tienen una heredabilidad alta similar (0.51−0.56, para GIM, y 0.44−0.50, para C18:1) y una correlación favorable entre ellos (0.47). Además, existen escenarios de selección en que estos caracteres y el crecimiento magro se pueden mejorar simultáneamente. Se demostró experimentalmente que (1) GIM y C18:1 responden a la selección basada en valores de mejora a partir de datos fenotípicos de parientes, y (2) el espesor de grasa dorsal se puede modificar independientemente de GIM y C18:1. No obstante, la selección por GIM y C18:1 basada en datos tomados en un músculo tiene respuestas correlacionadas desiguales en otros músculos y tejidos adiposos. En la Parte 3 se analizaron las variaciones de la secuencia del gen estearoil-CoA desaturasa (SCD), que codifica el enzima limitante en la biosíntesis de C18:1. Se mostró que hay una variante funcional en el gen SCD con un efecto aditivo de +0.75% en C18:1 y +1.00% en contenido total de ácidos grasos monoinsaturados, pero sin efecto en GIM o engrasamiento de la canal. Esta asociación se confirmó en un estudio de asociación genómica que también reveló variaciones de nucleótidos en el locus del gen del receptor de la leptina (LEPR) que afectan el nivel de engrasamiento y, en consecuencia, la composición de la grasa. El uso marcadores en estos dos loci mejoró substancialmente la precisión en las predicciones de GIM y C18:1. Se concluye que es posible seleccionar con éxito por GIM y C18:1 en carne de cerdo y se discuten varios escenarios sobre como implementar esta selección en la práctica.
Intramuscular fat (IMF) content and fatty acid composition affect the quality of pork. In particular, increasing oleic acid (C18:1) content would improve pork quality in terms of organoleptic and technological attributes and also of nutritional properties. This thesis dissertation is part of a line of research conducted by the Pig Breeding and Genetics Group of the University of Lleida, with the aim of finding strategies to genetically improve pork quality by increasing IMF and C18:1. It is divided into three parts. Part 1 discusses the implications of applying a specific statistical approach for compositional data to analyze these traits. It is shown that, because of the low variability of fatty acid composition in pork, the standard statistical techniques on raw percentages are robust enough for most genetic analyses, including those performed next. In Part 2, the genetic parameters associated to IMF and C18:1 were estimated in a purebred Duroc line. Both traits have a similar high heritability (0.51−0.56, for IMF, and 0.44−0.50, for C18:1) and a favorable genetic correlation between them (0.47). Furthermore, there exist selection scenarios where these traits and lean growth can be improved simultaneously. It was proved experimentally that (1) IMF and C18:1 respond effectively to selection on estimated breeding values based on phenotypic data of relatives, and (2) backfat thickness can be modified independently of IMF and C18:1. However, selection for IMF and C18:1 based on records from one muscle has unequal correlated responses on other muscles and fat tissues. In Part 3, the sequence variation of the stearoyl- CoA desaturase (SCD) gene, the gene producing the rate-limiting enzyme in the biosynthesis of C18:1, was analyzed. It was shown that there is a functional variant in the promoter of the SCD gene with an average additive effect of +0.75% and +1.00% on C18:1 and total monounsaturated fatty acids, respectively, but no effect on IMF or carcass fatness. This was confirmed in a genome-wide association study which also revealed nucleotide variations in the leptin receptor (LEPR) gene locus affecting overall fatness and, as a result, fat composition. The use markers at both loci substantially enhanced the accuracy of prediction of IMF and C18:1. It is concluded that it is possible to successfully select for increased IMF and C18:1 in pork. In light of the results obtained several scenarios are discussed on how to implement such selection in practice.

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The development of ―energycane‖ varieties of sugarcane for ethanol production is underway, targeting the use of both sugar juice (first generation ethanol) and bagasse (second generation ethanol). Nevertheless, identification of the preferred varieties represents the biggest challenge to the development of energycane due to large number of samples produced during breeding. In the present study, dilute acid pretreatment, enzymatic hydrolysis and fermentation processes were used to evaluate the processability of bagasse (fibrous residue generated after juice sugar extraction) from different varieties of sugarcane to select preferred varieties with the properties of improving combined ethanol yield (ethanol from juice and bagasse) per hectare. The impact of variety selection on combined ethanol yield (ethanol from juice and bagasse) per hectare was also assessed. In the first part of this study, 115 varieties of sugarcane originated from classical breeding and precision breeding (genetic engineering) were screened based on agronomic data and experimental data from biochemical processes (dilute acid pretreatment and enzymatic hydrolysis) applied to the bagasse fraction of each variety. The results showed wide variations in the chemical composition of bagasse between the varieties. Structural carbohydrates and lignin content ranged from 66.6 to 77.6% dry matter (DM) and 14.4 to 23.1% DM, respectively. The majority of precision breeding varieties showed higher arabinoxylan, lower lignin and lower ash content than most of classical breeding varieties. Combined sugar yield from the bagasse after pretreatment and enzymatic hydrolysis also varied significantly among the varieties. Up to 27.9 g/100g (dry bagasse) difference in combined sugar yield was observed. Combined sugar yield was inversely correlated with lignin as well as ash content, but it correlated positively with structural carbohydrates content. Total potential ethanol yields per hectare, calculated based on cane yield, soluble and non-soluble sugar content also differed significantly among the varieties (8,602−18,244 L/ha). Potential ethanol from bagasse contributed approximately one third of the total potential ethanol yield. Interestingly, some of the varieties had combined properties of high potential ethanol yield per hectare and improved bagasse convertibility. Thus, six varieties (3 from each breeding technology) were selected as preferred varieties for further investigation. To enhance sugar yield from bagasse, optimisation of pretreatment was conducted on the selected varieties. Industrial bagasse was included for comparison purposes. The pretreatment optimisation was based on maximising combined sugar yield from the combined pretreatment-hydrolysis process. A central composite design (CCD) was applied to investigate the effects of temperature, acid concentration and residence time on the responses and was later used to determine the maximum combined sugar yield. Pretreatment optimisation was conducted at gram scale (22.9 ml reactor) and at bench scale (1000 ml reactor). Significant differences in sugar yields (xylose, glucose, and combined sugar) between the varieties were observed. The combined sugar yields from the best performing varieties and industrial bagasse at optimal pretreatment-hydrolysis conditions differed by up to 34.1% and 33% at gram and bench scale, respectively. A high ratio of carbohydrates to lignin and low ash contents increased the release of sugar from the substrates. At mild pretreatment conditions, the differences in bioconversion efficiency between varieties were greater than at severe conditions. This observation suggests that under less severe conditions the conversion efficiency was largely determined by the properties of the biomass. Furthermore, it was demonstrated that the pretreatment conditions with temperature ranged from 184 to 200 °C and varying residence time to provide a severity factor between 3.51 and 3.96 was observed to be the area in common where 95% of maximum combined sugar yield could be obtained. Simultaneous Saccharification and Fermentation (SSF) was performed on the unwashed pressed-slurry from bagasse pretreatment at conditions for maximum combined sugar yield at bench scale. Batch and fed-batch SSF feeding strategy at different solid loadings and enzyme dosages were used aiming to reach an ethanol concentration of at least 40 g/L. The results revealed significant improvement in overall ethanol yield after SSF for the selected varieties (84.5–85.6%) compared to industrial bagasse (74.8%). The maximum ethanol concentration from the best performing varieties was 48.6−51.3 g/l and for poor performing varieties was 37.1−38.3 g/l. Ethanol concentration in the fermentation broth was inversely correlated with lignin content and the ratio of xylose to arabinose, but it showed positive correlation with glucose yield from pretreatment-enzymatic hydrolysis. The overall assessment of the varieties showed greater improvement in combined ethanol yields per hectare (71.1–90.7%) for the best performing varieties with respect to industrial sugarcane. The performance in terms of ethanol yields of selected varieties from a number harvest years was evaluated. The results showed considerable variations in ethanol yields across harvests. The results showed that the best variety in terms combined ethanol yield was not maintained across harvests. The differences in ethanol yields were greater among the varieties than across the harvests. Prolonged severe drought significantly affected the ethnol yields of all varieties represented by lower and intermediate lignin content for cane yield compared to that which had highest lignin content. However, carbohydrates content in the bagasse and sugar yield/recovery between the harvest years did not change for the most of the varieties. In summary, the present study provides evidence of the impact of cultivar selection and pretreatment optimisation in increasing conversion efficiency of bagasse. The results demonstrate that varieties with lower lignin and ash content, as well as highly substituted xylan resulted in higher sugar and ethanol yields. These results suggest that lower process requirements can be achieved without adversely affecting juice ethanol and cane yield per hectare. Nonetheless, an attempt to reduce lignin content in the bagasse, to reduce processing requirements for ethanol production, can also target the improvement of crop tolerance toward severe drought conditions.
AFRIKAANSE OPSOMMING: Die ontwikkeling van ―energie-riet‖ rasse vir etanol produksie is goed op dreef, waar beide die sap (eerste generasie etanol) en die bagasse (tweede generasie etanol) geteiken word. Die groot aantal monsters wat tydens teling geproduseer word, bied egter die grootste uitdaging vir die identifisering van nuwe rasse ten einde energie-riet te ontwikkel. In die huidige studie is verdunde suurvoorbehandeling, ensiematiese hidrolise en fermentasie-prosesse gebruik om die verwerkbaarheid van bagasse (veselagtige residu gegenereer na sap suiker ekstraksie) van verskillende suikerrietrasse te evalueer om nuwe variëteite te selekteer wat eienskappe van verbeterde gekombineerde etanolopbrengs (etanol van sap en bagasse) per hektaar toon. Die impak van variëteit-seleksie op gekombineerde etanol opbrengs (etanol van sap en bagasse) per hektaar is ook beoordeel. In die eerste deel van hierdie studie het uit ‗n siftingsproses van 115 suikerriet rasse bestaan wat deur klassieke en presisie (geneties gemodifiseerde) teling gegenereer is. Die sifting was op agronomiese data gebaseer, asook op data van verdunde suur voorafbehandeling en ensimatiese hidrolise eksperimente wat op die bagasse fraksie van elke ras uitgevoer is. Die resultate het op groot variasie in die chemiese samestelling van die bagasse van verskillende rasse gedui. Die strukturele koolhidrate het tussen 66.6 en 77.6% droë massa (DM) gewissel, terwyl die lignien inhoud ‗n variasie van 14.4 en 23.1% DM getoon het. Verder het meeste van die presisie-teling variëteite ‗n hoër arabinoxilaan, maar ‗n laer lignien en as-inhoud as meeste van die klassieke teling rasse gehad. Die gekombineerde suikeropbrengs (GSO) van die bagasse na voorafbehandeling en ensimatiese hidrolise het ook beduidend tussen rasse gewissel, waar ‗n verskil van tot 27.9 g/100g (droë bagasse) waargeneem is. Daar was ‗n omgekeerde korrelasie tussen die gekombineerde suikeropbrengs en die lignien en as-inhoud gewees, maar die opbrengs het ‗n sterk positiewe korrelasie met die strukturele koolhidrate getoon. Die totale potensiële etanol opbrengs per hektaar wat vanaf die suikerriet se oplosbare en nie-oplosbare suikerinhoud bereken is, het ook beduidend tussen rasse verskil (8,602−18,244 L/ha), waar die potensiële etanol opbrengs van die bagasse gedeelte ongeveer een derde van die totale potensiële etanol opbrengs beslaan het. Interessante bevindinge het op sommige rasse met gekombineerde eienskappe van hoë potensiële opbrengs per hektaar asook ‗n hoë omskakelingsvermoë gedui. Derhalwe is ses variëteite (drie van elke telingstegnologie) as voorkeurvariëteite vir verdere studie gekies. Om die etanol opbrengs vanaf die bagasse te verbeter was voorafbehandeling van die voorkeurvariëteite geoptimeer, en waar industriële bagasse vir vergelykingsdoeleindes ingesluit was. Vir die optimering was dit ten doel gestel om die gekombineerde suikeropbrengs van die gekombineerde voorafbehandeling-hidrolise proses te maksimeer. ‗n Sentrale saamgestelde ontwerp (SSO) is gebruik om die effek van temperatuur, suurkonsentrasie en residensietyd op die responsveranderlikes vas te stel wat uiteindelik gebruik is om die maksimum gekombineerde suikeropbrengs te bepaal. Die optimering van die voorafbehandeling is op gram-skaal in ‗n 22.9 ml reaktor, asook op bank-skaal in ‗n 1000 ml reaktor uitgevoer. Beduidende verskille in die suikeropbrengs (xilose, glukose en gekombineerde suiker) is tussen die voorkeurrasse waargeneem. Tussen die rasse wat die beste gevaar het, asook die industriële bagasse, het die gekombineerde suikeropbrengs by optimale voorafbehandeling-hidrolise toestande onderskeidelik met tot 34.1% en 33% op gram-skaal en bank-skaal gevarieer. ‗n Hoë verhouding van koolhidrate tot lignien, asook ‗n lae as-inhoud het tot ‗n toename in die vrystelling van suiker uit die substraat gelei. By matige voorafbehandelingstoestande was die verskille in omskakelingseffektiwiteit tussen rasse groter as onder hewige toestande, wat daarop gedui het dat omskakelingseffektiwiteit grotendeels deur die eienskappe van die biomassa bepaal is. Verder is daar ook gedemonstreer dat die voorbehandelingsomstandighede met temperatuur tussen 184 en 200ºC en verandering van die residensietyd om 'n hewigheidsfaktor van tussen 3.51 en 3.96 te verskaf, 'n gemeenskaplike area gelewer het waar 95% van maksimum gekombineer suiker opbrengs (GSO) verkry kon word. Gelyktydige versuikering en fermentasie (GVF) is na voorafbehandeling op ongewaste, gepersde bagasse substraat by toestande vir die maksimum gekombineerde suikeropbrengs op bank-skaal uitgevoer. Bondel en voerbondel SSF voerstrategie by verskillende vaste ladings en ensiemdoserings is gebruik om 'n etanol konsentrasie van ten minste 40 g/L te bereik. Ná GVF was die algehele etanol opbrengs vir die voorkeurvariëteite (84.5–85.6%) beduidend beter relatief tot die industriële bagasse (74.8%). Die maksimum etanol opbrengs na SSF van die rasse met die beste prestasie was 48.6-51.3 g/L en 37.1-38.3 g/L vir rasse wat swak presteer het. Die etanol konsentrasie in die fermentasiesop was omgekeerd met lignien en die verhouding van xilose tot arabinose gekorreleer, maar was duidelik positief met die glukose opbrengs vanaf voorafbehandeling-hidrolise gekorreleer. ‗n Algemene assessering het op ‗n duidelike verbetering van die voorkeurvariëteite in terme van gekombineerde etanol opbrengs per hektaar gedui (71.1–90.7%), relatief tot die industriële suikerriet. Die prestasie in terme van etanol opbrengs van geselekteerde variëteite is oor 'n reeks oesjare ge-evalueer. Die resultate het aansienlike variasies in etanol opbrengs oor oesjare getoon. Die resultate het gewys dat die beste variëteite in terme van gekombineerde etanol opbrengs nie volhou is oor oeste nie. Die verskille in etanol opbrengste tussen variëteite was groter as die verskille oor oesjare. Verlengde ernstige droogte het die etanol opbrengs van alle variëteite met laer en intermediere lignien inhoud vir rietopbrengs aansienlik beinvloed, in vergelyking met dié wat die hoogste lignien inhoud gehad het. Die koolhidraatinhoud in die bagasse en suiker opbrengs/lewering tussen die oesjare het vir die meeste variëteite egter nie gewissel nie. Ter opsomming, die huidige studie verskaf bewyse van die impak van kultivarseleksie en voorbehandelings optimisering op die verhoging van die omskakelings-doeltreffendheid van bagasse. Die resultate wys dat variëteite met laer lignien- en asinhoud, en hoogs-gesubstitueerde xilaan hoër suiker- en etanol opbrengs gelewer het. Hierdie resultate stel voor dat verminderde voorbehandelingsvereistes bereik kan word sonder om die sap etanol en rietopbrengs per hektar te benadeel. Nieteenstaande, 'n poging om die lignien inhoud van die bagasse te verminder om die verwerkingsvereistes vir etanolproduksie te verminder, kan ook die verbetering van gewas-toleransie tov ernstige droogte-toestande teiken.

Thesis advisor: Abhishek Chatterjee
Abstract Unnatural amino acid (UAA) incorporation is a powerful tool used by biochemists to discover the nature of protein structure and function. The evolution of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs enables site-specific incorporation of UAAs proteins inside of living cells. The goal of this study was to further expand the repertoire of genetically encoded unnatural amino acids in E. coli as well as eukaryotes. We first attempted to engineer an aaRS, previously evolved for p-borono-phenylalanine (pBoF), to specifically charge 3-acetyl-p-borono-phenylalanine (AcpBoF). A randomized library of the pBoF-specific synthetases was generated and it was subjected to established selection schemes in a bacterial host. This report also describes the development of a yeast-based selection system to alter the substrate specificity of bacterial leucyl-tRNA synthetase, for genetic code expansion in eukaryotes
Thesis (MS) — Boston College, 2017
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

Thesis (MSc)--Stellenbosch University, 2011.
ENGLISH ABSTRACT: The equine gastro-intestinal tract (GIT) is a relatively unexplored niche concerning the presence of natural microbiota. Studies have shown that disruption of the microbial population naturally present in the GIT leads to the onset of several forms of gastro-intestinal disorders. To maintain a balanced microbiota, probiotic bacteria need to be administered at specific levels. Beneficial microorganisms assist with digestion of the feed, absorption of nutrients from the GIT, strengthens the immune system and improves the animal‟s growth. Various combinations of lactic acid bacteria (LAB) have been administered to horses, but have failed to benefit the host in any of the latter criteria. The screening for alternative strains with probiotic properties is thus necessary. Two strains (Lactobacillus equigenerosi Le1 and Lactobacillus reuteri Lr1) were originally isolated from horse faeces. Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, both bacteriocin-producing strains, were isolated from sorghum beer and soy beans, respectively. All four strains survived growth at acidic conditions (pH 3) and the presence of 0.5%, 1.0% and 1.5% (w/v) bile salts. L. reuteri Lr1 was the most resistant to these conditions. All strains adhered to buccal (cheek) epithelium cells sampled from horses. L. equigenerosi Le1 and E. mundtii ST4SA, however, invaded the cells, but without visible signs of disrupting the cells. None of the strains contained genes encoding adhesion to collagen (Ace), resistance to vancomycin A, B and C, or, production of aggregation substance (AS), cytolysin (Cyl) and, non-cytolysin (β hemolysin III), suggesting that they are non-virulent. Of all strains, L. equigenerosi Le1 competed the best with Clostridium sp. C6 for adherence to epithelial cells. L. equigenerosi Le1 and L. reuteri Lr1, showed the highest level of co-aggregation with Clostridium sp. C6. When the four strains were administered to horses over a period of 10 days, L. reuteri Lr1 was retained the longest (8 days) in the GIT. The numbers of viable cells of Clostridium spp. and Salmonella spp. remained constant during administration of the four strains. Blood analyses showed no negative effects from administering the strains. Total white blood cell counts remained unchanged. However, a small but tentative increase in neutrophil and eosinophil cell numbers has been recorded, suggesting that the LAB may have elicited a mild, transient, intolerance reaction. The glucose, lactate and urea levels decreased during administration with the four LAB strains.
AFRIKAANSE OPSOMMING: Die spysverteringstelsel (SVS) van die perd is 'n relatief onbekende nis wat die voorkoms van natuurlike mikrobiota betref. Studies het getoon dat versteuring van die natuurlike mikrobiese populasie in die SVS aanleiding kan gee tot die ontwikkeling van menige vorms van gastro-intestinale ongesteldhede. Om 'n gebalanseerde mikrobiota te verseker, moet probiotiese bakterieë teen 'n spesifieke vlak toegedien word. Voordelige mikroorganismes bevorder vertering en absorpsie van nutriënte vanaf die SVS, versterk die immuunsisteem en bevorder die groei van die dier. Verskeie kombinasies van melksuurbakterieë is reeds aan perde toegedien, maar sonder ooglopende voordele vir die dier. Die soeke na alternatiewe stamme met probiotiese eienskappe is dus noodsaaklik. Twee melksuurbakterieë (Lactobacillus equigenerosi Le1 en Lactobacillus reuteri Lr1) is oorspronklik uit perdemis geïsoleer. Lactobacillus plantarum 423 en Enterococcus mundtii ST4SA, beide bakteriosienproduserende stamme, is afsonderlik van sorghumbier en sojabone geïsoleer. Al vier spesies groei by lae pH (pH 3) en in die teenwoordigheid van 0.5%, 1.0% en 1.5% (m/v) galsoute. L. reuteri Lr1 is die mees bestand onder hierdie toestande. Al vier stamme het aan wang epiteelselle van perde geheg. L. equigenerosi Le1 en E. mundtii ST4SA het egter die epiteelselle binnegedring, maar sonder opsigtelike vernietiging van die selle. Nie een van die stamme besit gene wat kodeer vir aanhegting aan kollageen (Ace), bestandheid teen vankomisien A, B en C, of produksie van, sel-aggregasie (AS), sitolisien (Cyl) en nie-sitolisien (β-hemolisien III), wat daarop dui dat hulle nie-virulent is. Van al die stamme het L. equigenerosi Le1 die beste met Clostridium sp. C6 vir aanhegting aan epiteelselle gekompeteer. L. equigenerosi Le1 en L. reuteri Lr1, het die beste vlak van ko-aggregasie met Clostridium sp. C6 getoon. Met die toediening van 'n kombinasie van die vier stamme aan die perde oor 'n periode van 10 dae, het L. reuteri Lr1 die langste retensie (8 dae) in die SVS getoon. Die aantal lewende selle van Clostridium spp. en Salmonella spp. het konstant gebly tydens toediening van die vier stamme. Toediening van die vier stamme het geen negatiewe effek getoon met resultate verkry van bloed analises nie. Die totale witbloed seltellings het onveranderd gebly. 'n Klein, maar tentatiewe, toename in neutrofiel- en eosinofiel selgetalle is waargeneem, wat daarop dui dat die melksuurbakterieë 'n geringe allergiese reaksie teweeggebring het. Die glukose, laktaat en ureum vlakke het gedaal tydens die toediening van die vier melksuurbakterie stamme.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The quality of wine is influenced and determined by various factors, one of which includes the process of malolactic fermentation (MLF). MLF plays an integral role in the flavour and sensory profile of most red wines as well as some white wines like Chardonnay. This process is conducted by lactic acid bacteria (LAB), specifically of the genera Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Of these, Oenococcus oeni is best adapted to survive in the harsh wine environment. MLF is defined as the conversion of L-malic acid to L-lactic acid and carbon dioxide. The conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid results in a decrease in acidity and an increase in pH, to give a softer mouthfeel and more favourable flavour profile. A further reason for conducting MLF in wine includes the improvement of microbial stability due to the removal of malic acid as a possible substrate for microorganisms. Recently, research focus has shifted to the ability of MLF and LAB to alter the aroma profile of wine via the production and/or modification of certain aroma compounds. In order for wine LAB to conduct MLF, they need to be able to survive the harsh and challenging wine environment. Conditions in South African wines are particularly challenging due to the long, hot ripening seasons resulting in high sugar concentrations which give high ethanol concentrations. Some LAB also struggle to adapt to an environment with high pH and low malic acid concentrations. These factors, combined with the use of sulphur dioxide, cause LAB to struggle in conducting and completing successful MLF. Many of the commercial starter cultures that are currently available contain LAB that have not been isolated from South African wine and are therefore not optimal for use under these challenging wine conditions. Oenococcus oeni is also the single LAB culture present in all commercially available starter cultures. The overriding goal of this study was to create a MLF starter culture containing a mixture of LAB cultures, namely O. oeni and Lactobacillus plantarum, which can successfully convert malic acid to lactic acid, ensure microbial stability, but also make a positive contribution to the wine aroma profile. Lactobacillus plantarum has previously been considered for possible use in a commercial starter culture. The LAB isolates used in this study were selected from the Institute for Wine Biotechnology culture collection as well as isolated from spontaneous MLF. The first objective was to characterise these LAB strains for important traits and for possible use as a MLF starter culture. A total of 23 strains were identified as O. oeni and 19 strains as Lb. plantarum. The identified strains were screened in a synthetic wine medium for their ability to convert malic acid to lactic acid. Based on the LAB strain performance in the synthetic wine medium, seven strains of both O. oeni and Lb. plantarum were selected. These 14 strains were screened for the presence of genes encoding for enzymes responsible for biogenic amine production and were found to contain none of the genes associated with the formation of histamine, tyramine or putrescine. The LAB strains were genetically screened for enzymes associated with aroma modification by LAB during MLF. The enzymes of interest that were screened for included β-glucosidase, esterase, protease and phenolic acid decarboxylase (PAD). The Lb. plantarum strains were found to possess more diverse enzymatic profiles related to aroma than O. oeni. The biggest differences were observed for the presence of β-glucosidase and PAD. The second objective was to perform small-scale fermentations with the individual LAB isolates. The individual isolates were evaluated in Pinotage and based on these results; three strains of each O. oeni and Lb. plantarum were selected for evaluation in mixed culture fermentations. The mixed cultures were evaluated in Pinotage, Shiraz and Cabernet Sauvignon in the 2008 vintage. As a third objective, the wines were also analytically and sensorially evaluated to investigate the changes in the aroma profile that could be attributed to the presence of the mixed LAB isolates. Based on the fermentation data as well as data pertaining to the aroma modification, three mixed cultures were selected for evaluation in the 2009 vintage in Pinotage, Cabernet Sauvignon and Chardonnay. The mixed cultures were able to successfully complete MLF in fermentation periods comparable to that of a commercial culture used as control. The different LAB cultures had distinct and diverse effects on the wine aroma profile. The O. oeni strain played a larger role in the ester concentration present after MLF, while the Lb. plantarum strain had a larger effect on the higher alcohol and volatile fatty acid concentration upon completion of MLF. The results generated by this novel study clearly indicate the potential of a mixed LAB starter culture for conducting MLF. The mixed cultures successfully completed MLF and made a positive contribution to the wine aroma profile.
AFRIKAANSE OPSOMMING: Die kwaliteit van wyn word beïnvloed en bepaal deur verskeie faktore en wynbereidings prosesse, wat die proses van appelmelksuurgisting (AMG) insluit. AMG speel ’n integrale rol in die sensoriese profiel van meeste rooiwyne, sowel as sommige witwyne soos Chardonnay. AMG word gedefinieër as die omskakeling van L-appelsuur na L-melksuur en koolstofdioksied. Hierdie omskakeling kan toegeskryf word aan die teenwoordigheid van melksuurbakterieë (MSB), spesifiek spesies van die genera Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Vanuit hierdie wyn MSB, is Oenococcus oeni die spesies wat die beste aanpas en oorleef onder stresvolle wyn kondisies. Die omskakeling van appelsuur, ’n dikarboksielsuur, na melksuur, ’n monokarboksielsuur, lei tot ‘n vermindering in suurheid en ’n verhoging in pH. Hierdie vermindering in suurheid gee ’n sagter en meer geronde mondgevoel aan die wyn en dra by tot ‘n meer aangename geurprofiel. ’n Verdere rede vir AMG in wyn is om mikrobiese stabiliteit te verseker deurdat appelsuur verwyder word as ’n moontlike koolstof substraat vir mikroörganismes. Onlangs het navorsing begin fokus op AMG en die vermoë van MSB om die aroma profiel van wyn te beïnvloed deur die produksie/modifisering van sekere aroma komponente. Vir MSB om AMG te kan deurvoer, moet hulle kan oorleef in die stresvolle wynomgewing. Wyntoestande in Suid-Afrika is veral uitdagend vir die oorlewing van mikroörganismes as gevolg van lang, warm somers wat lei tot ’n matriks met ’n hoë suikerkonsentrasie en wyn met ’n hoë etanolkonsentrasie. ‘n Omgewing met ‘n hoë pH en lae appelsuur konsentrasie, kan ook bydrae tot stresvolle kondisies vir MSB. Hierdie parameters, tesame met die gebruik van swaweldioksied, maak dit moeilik vir MSB om AMG te inisieer en te voltooi. Sommige van die kommersiële aanvangskulture wat tans beskikbaar is, bevat nie MSB wat onder Suid-Afrikaanse wyntoestande geïsoleer is nie en daarom is dit nie altyd optimaal vir gebruik nie. Oenococcus oeni is ook die enkele MSB kultuur wat in alle kommersiële kulture gebruik word. Die hoofdoelwit van hierdie studie was om ’n potensiële kommersiële aanvangskultuur te ontwikkel wat ‘n mengsel van MSB bevat. Hierdie aanvangskultuur moet AMG suksesvol kan voltooi, mikrobiologiese stabiliteit bevorder en steeds die wynaroma positief kan beïnvloed. Bakterierasse van O. oeni en Lb. plantarum is geselekteer vir gebruik in hierdie studie. Lactobacillus plantarum het reeds in vorige studies potensiaal getoon as ‘n moontlike aanvangskultuur. Die MSB isolate vir hierdie studie is geselekteer uit die Instituut vir Wynbiotegnologie se kultuurversameling en geïsoleer uit spontane AMG fermentasies. Die eerste doelwit was om hierdie MSB isolate te karakteriseer vir belangrike eienskappe en die moontlike gebruik as ’n kommersiële AMG aanvangskultuur. ‘n Totaal van 23 O. oeni en 19 Lb. plantarum isolate is geïdentifiseer. Hierdie isolate is in ’n sintetiese wynmedium geëvalueer vir hul vermoë om appelsuur na melksuur om te skakel. Op grond van hul reaksie in die sintetiese wynmedium, is sewe isolate van elk van die O. oeni en Lb. plantarum geselekteer. Hierdie 14 isolate is ondersoek vir die teenwoordigheid van die gene wat kodeer vir biogeenamien produksie en daar is gevind dat geen van die isolate enige van die biogeenamien gene wat ondersoek is, naamlik histamien, tiramien en putresien besit nie. Die MSB isolate is geneties ondersoek vir die teenwoordigheid van dié gene wat kodeer vir ensieme wat die aromaprofiel tydens AMG beïnvloed. Dié ensieme sluit β-glukosidase, esterase, protease, fenoliese suurdekarboksilase en sitraatliase in. Daar is gevind dat die Lb. plantarum isolate meer diverse ensiemprofiele as O. oeni besit. Die grootste verskille in die ensiemprofiele kan toegeskryf word aan die teenwoordigheid van β-glukosidase en fenoliese suurdekarboksilase. Die tweede doelwit was om kleinskaalse AMG fermentasies met die individuele MSB isolate uit te voer. Die individuele isolate is in Pinotage geëvalueer. Volgens hierdie resultate is drie isolate van elk van die O. oeni en Lb. plantarum geselekteer om in gemengde kulture getoets te word. Die gemengde kulture is in Pinotage, Shiraz en Cabernet Sauvignon in 2008 geëvalueer. As ’n derde doelwit is hierdie wyne ook analities en sensories geëvalueer om die veranderinge in die aromaprofiele as gevolg van die teenwoordigheid van die MSB te ondersoek. Op grond van die fermentasiedata, sowel as die data oor die aromaveranderinge, is drie gemengde kulture geselekteer vir evaluering in Pinotage, Cabernet Sauvignon en Chardonnay in 2009. Die gemengde kulture kon AMG suksesvol voltooi met fermentasietempo’s wat vergelykbaar was met dié van ‘n kommersiële AMG kultuur wat as kontrole gebruik is. Die verskillende MSB kulture het spesifieke en uiteenlopende uitwerkings op die wynaroma gehad. Die O. oeni isolaat in die gemengde kultuur blyk ‘n belangriker rol te speel in die esterkonsentrasie na AMG, terwyl die Lb. plantarum isolaat ’n groter effek het op die hoër alkohol en vlugtige vetsuurinhoud na AMG. Die resultate wat deur hierdie unieke studie gegenereer is, gee ’n aanduiding van die potensiaal van ’n gemengde MSB aanvangskultuur vir AMG. Die gemengde kulture kon AMG suksesvol voltooi en ‘n positiewe bydrae tot die aromaprofiel van die wyn lewer.

Thesis advisor: Jia Niu
Nucleic acid aptamers are promising alternatives to antibodies for a wide array of diagnostic and therapeutic applications. However, state-of-the-art aptamers suffer from poor pharmacokinetics and diversity, limiting their affinity and specificity for many therapeutically relevant targets. The emerging field of glycoscience provides opportunities to improve the utility of aptamers over antibodies. Combining synthetic chemistry with modern molecular biology and polymer science, the synthesis of Xeno Nucleic Acid monomers and chemoenzymatic polymerization via engineered polymerase enzymes allows the production of nucleic acid drugs with superior resistance to endogenous nucleases. The modular structure of nucleic acids provides for the design of sequence defined polymers capable of post-synthetically appending complex synthetic glycans, extending the catalytic geometry of aptamers. Our SELEX inspired FACS based particle display approach allows for high-throughput screening. Additionally, we expect this method has the capability of screening aptamers in human serum. Our synthetic approach utilizes a Sonogashira cross-coupling reaction to install a flexible alkyne to the major groove of 2′-deoxy-2′-fluoro-arabinose uracil base. By incorporated recent advances in nucleic acid synthesis, one-pot nucleobase activation and sugar glycosylation is achieved and bis-oxybenzyl phosphoamidite synthesis can afford gram scale HPLC-free purification of the triphosphates. The FANA C8-alkyl-uridine triphosphate will be incorporated by an engineered Tgo DNA polymerase to allow systematic introduction of alkynyl conjugation handles into a DNA-templated FANA polymer. Subsequent conjugation with azido-modified glycans via the Huisgen coppercatalyzed alkyne-azide cycloaddition (CuAAC) click reaction will generate sequence controlled nucleic acid-carbohydrate hybrid molecules amendable for directed evolution
Thesis (MS) — Boston College, 2019
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

La découverte de petites molécules capables de moduler les systèmes biologiques présente un intérêt majeur dans l'étude des mécanismes cellulaires et la mise au point de nouvelles méthodes thérapeutiques. Bien que les techniques de criblage haut débit soient régulièrement utilisées, il y a un vrai besoin de réduire le coût et le temps associés à la découverte de ligands, afin de valider la fonction de nombreuses cibles potentielles dans notre protéome ou ceux d' organismes pathogènes. A cette fm, l'émergence de technologies basées sur l'encodage de chimiothèques par des acides nucléique offre une alternative répondant à ces critères. Nous avons développé un système permettant une synthèse rapide de bibliothèques de structures variées,conjuguées à des codes PNA (acide peptidique nucléique) uniques, ainsi qu'une technologie de criblage basée sur la sélection par affinité, qui permet une étude rapide de l'interaction avec une protéine-cible et par conséquent l'identification de nouveaux ligands. Plusieurs chimiothèques ont déjà été synthétisées et criblées, et compte tenu de la stabilité chimique remarquable du PNA, nous avons également développé une nouvelle gamme de réactions compatibles avec la synthèse PNA-encodée, la voie étant maintenant ouverte pour la génération de chimiothèques plus complexes et pour l'étude de cibles biologiques très variées
The discovery of srnall molecules capable of modulating biological systems is of major interest for the understanding of cellular mechanisms as weil as for the drug discovery process. In spite of established high throughput techniques routinely used, there is a clear need to reduce the time and cost •associated to ligand discovery, in order to validate the function of numerous potential targets in our proteome or the one of pathogens. In this perspective, the emergence of technologies based on nucleic acid encoding of chemical libraries presents an alternative that fulfills these criteria. We have developed a system enabling the rapid synthesis of libraries containing various structures, conjugated to unique PNA (peptide nucleic acid) tags, a weil as a screening technique based on affinity selection that allows for the rapid study of the interaction witb a target protein and the consequent identification of new ligands. Several libraries have already been synthesized and screened, and based on the remarkable chemical stability of PNA, we have also developed a new palette of reactions compatible with PNA-encoded synthesis, the path now being open for the generation of more complex libraries, and the study of various biological targets

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Existem diferentes métodos e pseudo-fenótipos utilizados em predições genômicas, sendo necessário determinar o ideal para a característica de interesse. Os ácidos graxos da carne que contribuem de forma prejudicial para a saúde humana têm recebido considerável atenção nos últimos anos. O objetivo desse estudo foi avaliar a acurácia de predição genômica de diferentes métodos (SNP-BLUP, BayesC, BayesCπ e Bayesian Lasso) e pseudo-fenótipos (fenótipo ajustado para os efeitos fixos, valor genético estimado via pedigree e valor genético genômico obtido pelo método single step GBLUP) em dados simulados e reais de perfil de ácidos graxos no músculo Longissimus thoracis da raça Nelore terminados em confinamento. A proposta de inclusão do GEBV obtido pelo método passo único genômico BLUP (ssGBLUP) como pseudofenótipo nesta pesquisa é inédita em predição genômica, para responder um dos problemas frequentes ao utilizar a matriz advinda da informação genealógica, pela maioria dos produtores de gado de corte utilizarem o sistema de acasalamento que utiliza reprodutores multiplos. Foram utilizados cerca de 963 bovinos machos inteiros da raça Nelore terminados em confinamento e genotipados com um painel de 777.962 SNPs do Ilumina BovineSNP BeadChip. A informação genômica pode servir para melhorar o perfil de ácidos graxos em animais do grupo Zebu, por ter-se observado valores genômicos preditos com com acurácia de baixa a moderada magnitude. Nenhum dos métodos foi melhor para todos os ácidos graxos em termos de acurácia, no entanto, o método SNP-BLUP permitiu realizar avaliação menos viesada. O método ssGBLUP apresentou-se como ferramenta alternativa para obter GBVs como pseudo-fenôtipo mais acurado em situações de pedigree incompleto, pela alta proporção de de reprodutores múltiplos, sendo mais apropriado que o EBV e o fenótipo ajustado aos efeitos fixos para predizer o valor genômico dos animais.
There are different methods and pseudo-phenotypes used in genomic predictions, being necessary to determine the ideal for each trait of interest. Beef fatty acids that contribute to human health have received considerable attention in the last years. Thus, the objective of this study was to evaluate genomic predition accuracy of different methodologies (SNP-BLUP, BayesCπ, BayesC and Bayesian Lasso) pseudo-phenotypes (adjusted phenotype, estimated breeding value and genomic estimated breeding value by ssGBLUP) in simulated and real data of fatty acid profile in the Longissimus thoracis muscle of Nelore cattle finished in feedlot. The inclusion of the GEBV obtained by single step genomic BLUP (ssGBLUP) method as pseudo-phenotype in this research is innovator in genomic prediction, to answer one of the frequent problems when using the matrix derived from pedigree, by the mating system that uses multiple sires for many cattle breeders. A total of 963 Nelore bulls with phenotype for fatty acid profiles, were genotyped using the Illumina Bovine HD Bead Chip with 777,962 SNPs. Genomic information can assist in improving fatty acid profile in Zebu animals, since the use of genomic information yielded genomic values for fatty acid profile with accuracies ranging from low to moderate. None of the methods excelled in terms of accuracy, however the SNP-BLUP method allows obtaining less biased genomic evaluations. The ssGBLUP model is an appropriate alternative to obtaining more reliable and less biased GEBVs as pseudo-phenotypes in situations of missing pedigree, due to high proportion of multiple sires, being more appropriate than the EBVs or adjusted phenotypes for fixed effect to predict direct genomic values.

Thesis (Ph.D. in Biochemistry) -- University of Colorado Denver, 2007.
Typescript. Includes bibliographical references (leaves 137-161). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Sunflower (Helianthus annuus L.) high oleic acid content lines differ from conventional sunflower by an increase in oleic acid (C18:1) content of more than 60%. The current sunflower cultivars under production in South Africa are standard sunflower with high levels of linoleic acid (C18:2). The aim of this study was to improve the quality of oil produced by local sunflower germplasm with respect to oleic acid through employing a marker-assisted breeding technique to facilitate and speed up the recovery of the high oleic acid allele into the background of the recurrent parent genome. Eleven sunflower breeding genotypes with high and low oleic acid traits were obtained from the Agricultural Research Council-Grain Crops Institute (ARC-GCI) in Potchefstroom. The breeding genotypes were phenotypically characterised based on their oleic and linoleic acid levels using gas chromatography. Results demonstrated that the average mean of oleic and linoleic acid contents in high oleic acid genotypes were 72% and 17% respectively, while the average mean of oleic acid and linoleic acid contents in wild type lines were 33.5 % and 54 % respectively. These results indicated a perfect negative correlation between the amount of oleic and linoleic acids possessed in high and low oleic acid genotypes (R2 = -99.16%). Sequence characterised amplified region (SCAR) markers were tested to ascertain if any of the ten available dominant FAD2-1 markers was segregating with the high oleic acid allele. Four dominant SCAR markers (FAD2-1F4/R1; FAD2-1F4/R2; FAD2-1F13/R1; FAD2-1F14/R2) were strongly associated with the high oleic acid trait (P< 0.001). With regard to the inheritance of the high oleic acid trait, 143 plants of the F2 segregating population derived from a cross between the high oleic acid parent (AP901-95-3-4-1) and low oleic acid parent (H55-9-2-1-1) were genotyped with the four SCAR markers to determine the genetic state concerning the high oleic acid gene (Ol). Results from a Chi square analysis of the observed frequencies of each dominant FAD2-1 marker locus in 143 F2 individuals indicated that the deviation from the expected ratio of 3:1 (high to low oleic acid) was not statistically significant (P< 0.95) from the observed segregation ratio. These results were consistent with the previous finding that an incomplete dominant gene governs sunflower high oleic acid. A multiplex assay of 78 Simple sequence repeat (SSR) markers was optimised and evaluated on 143 plants of the F2 population to determine suitable SSR markers that can be used in a marker-assisted background selection. Only 14 markers were suitable for marker-assisted background selection based on their high polymorphic information content, allele frequency and maximum allele numbers. In conclusion, this study demonstrated the potential of using SSR and SCAR marker systems as a breeding tool to characterise and speed up the selection process in marker-assisted backcross breeding.
Thesis (MSc (Botany))--North-West University, Potchefstroom Campus, 2013.

A eficiência econômica da bovinocultura leiteira está relacionada à utilização de animais que apresentem, concomitantemente, bom desempenho quanto à produção, reprodução, saúde e longevidade. Nisto, o índice de seleção configura-se como ferramenta importante ao aumento da lucratividade nesse sistema, visto que permite a seleção de reprodutores para várias características simultaneamente, considerando a relação entre elas bem como a relevância econômica das mesmas. Com a recente disponibilidade de dados genômicos tornou-se ainda possível expandir a abrangência e acurácia dos índices de seleção por meio do aumento do número e qualidade das informações consideradas. Nesse contexto, dois estudos foram desenvolvidos. No primeiro, o objetivo foi estimar parâmetros genéticos e valores genéticos (VG) para características relacionadas à produção e qualidade do leite incluindo-se a informação genômica na avaliação genética. Foram utilizadas medidas de idade ao primeiro parto (IPP), produção de leite (PROD), teor de gordura (GOR), proteína (PROT), lactose, caseína, escore de células somáticas (ECS) e perfil de ácidos graxos de 4.218 vacas bem como os genótipos de 755 vacas para 57.368 polimorfismos de nucleotídeo único (SNP). Os componentes de variância e VG foram obtidos por meio de um modelo misto animal, incluindo-se os efeitos de grupos de contemporâneas, ordem de lactação, dias em lactação e os efeitos aditivo genético, ambiente permanente e residual. Duas abordagens foram desenvolvidas: uma tradicional, na qual a matriz de relacionamentos é baseada no pedigree; e uma genômica, na qual esta matriz é construída combinando-se a informação de pedigree e dos SNP. As herdabilidades variaram de 0,07 a 0,39. As correlações genéticas entre PROD e os componentes do leite variaram entre -0,45 e -0,13 enquanto correlações altas e positivas foram estimadas entre GOR e os ácidos graxos. O uso da abordagem genômica não alterou as estimativas de parâmetros genéticos; contudo, houve aumento entre 1,5% e 6,8% na acurácia dos VG, à exceção de IPP, para a qual houve uma redução de 1,9%. No segundo estudo, o objetivo foi incorporar a informação genômica no desenvolvimento de índices econômicos de seleção. Neste, os VG para PROD, GOR, PROT, teor de ácidos graxos insaturados (INSAT), ECS e vida produtiva foram combinados em índices de seleção ponderados por valores econômicos estimados sob três cenários de pagamento: exclusivamente por volume de leite (PAG1); por volume e por componentes do leite (PAG2); por volume e componentes do leite incluindo INSAT (PAG3). Esses VG foram preditos a partir de fenótipos de 4.293 vacas e genótipos de 755 animais em um modelo multi-característica sob as abordagens tradicional e genômica. O uso da informação genômica influenciou os componentes de variância, VG e a resposta à seleção. Entretanto, as correlações de ranking entre as abordagens foram altas nos três cenários, com valores entre 0,91 e 0,99. Diferenças foram principalmente observadas entre PAG1 e os demais cenários, com correlações entre 0,67 e 0,88. A importância relativa das características e o perfil dos melhores animais foram sensíveis ao cenário de remuneração considerado. Assim, verificou-se como essencial a consideração dos valores econômicos das características na avaliação genética e decisões de seleção.
The economic efficiency in dairy cattle is related to the use of animals with good performance in production, reproduction, health and longevity. This way, the selection index can be an important tool to increase profitability in this system, since it allows sire selection for multiple traits simultaneously, considering the relationship between them and their economic relevance for the activity. Also, the recent availability of genomic data has permitted to expand the coverage and accuracy of selection indexes by increasing the number and quality of the information considered. In this context, two studies were developed. In the first, the aim was to estimate genetic parameters and breeding values (BV) for milk production and quality traits, including the genomic information in genetic evaluation. Measures of age at first calving (AFC), milk yield (MY), somatic cells score (SCS) and percentages of fat (FP), protein (PP), lactose, casein, and fatty acids in milk of 4,218 cows as well as the genotypes of 755 of these cows for 57,368 single nucleotide polymorphisms (SNPs) were used. The variance components and BV were estimated from a mixed animal model which included the effects of contemporary groups, lactation order, days in lactation, and the additive genetic, permanent environmental and residual effects. Two approaches were developed: a traditional approach, in which the relationship matrix is based on pedigree information; and a genomic approach, in which the matrix is constructed by combining the pedigree and SNP information. The heritabilities ranged from 0.07 to 0.39. Genetic correlations between MY and milk components were between -0.45 and -0.13 whereas high and positive correlations were estimated between FP and fatty acids. The use of the genomic approach did not change genetic parameter estimates; however, there was an increase between 1.5% and 6.8% in BV accuracy; except for AFC, for which a reduction of 1.9% was observed. In the second study, the aim was to incorporate genomic information in the development of economic indexes for sire selection. In this, the BV for MY, FP, PP, total unsaturated fatty acids content (UFA), SCS and herd life were combined in selection indexes weighted by economic values estimated under three payment scenarios: exclusively by milk volume (PAY1); by milk volume and milk components (PAY2); and by milk volume and milk components including UFA (PAY3). These BV were predicted by using phenotypes of 4,293 cows and genotypes of 755 animals in a multi-trait model under traditional and genomic approaches. The use of genomic information influenced the estimates of variance component, BV and response to selection. However, the rank correlations between the approaches were high in all scenarios, with values between 0.91 and 0.99. Differences were mainly observed among PAY1 and the other scenarios, with correlations between 0.67 and 0.88. The relative importance of the traits and the profile of the best animals were sensitive to the scenario considered. Thus, it is essential to consider the economic values of the traits in genetic evaluation and selection decisions.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Objetivou-se identificar regiões no genôma de bovinos da raça Nelore que apresentam variações no número de cópias (CNV) e, associar estes CNV com o perfil de ácidos graxos da carne. Além disso, objetivou-se realizar associação genica ampla utilizando os método de single step (GWASss) a fim de detectar regiões genômicas associadas aos ácidos graxos dos grupos saturados, mono e poliinsaturados, assim como os omegas 3, 6 e sua relação. O estudo de caracterização e distribuição dos CNVs ao longo do genoma de bovinos Nelore, foi realizado através do software PennCNV utizando dados genotípicos de 3.794 aniamais, resultando em 399.361 CNVs identificados. Após controle de qualidade, 2.902 foram mantidos nas analises, resultando em 195.873 CNVs, com tamanho medio de 54,744 pb, maximo de 8.7 Mb e minimo 3 kb. As regiões de CNV foram geradas pela sobreposição dos CNVs através do software CNVRuler. Os cromossomos que mostraram maior incidencia de CNVR foram BTA19 (24,26%), BTA23 (18,68%) e BTA25 (18,05%). Ja os que mostraram menor incidencia foram BTA29 (1,63%), BTA13 (9,72%) and BTA8 (9,72%). As 9.805 regiões da CNV estimadas no presente estudo cobrem aproximadamente 13.05% do genoma bovino e sobrepõem-se a 5.495 genes conhecidos que envolvem processos biológicos que poderiam estar envolvidos na adaptação ambiental da subespécie a áreas tropicais. O estudo de GWASss identificou 115 janelas que explicaram mais de 1% da variação genética aditiva para os 22 ácidos graxos estudados. A identificação destas regiões e seus genes genes, tais qual ELOVL5, ESRRG, PCYT1A e os genes do grupo ABC (ABCA5, ABCA6 e ABCA10) são genes que estão relacionados direto e inderamente ao metabolismo lipídico. O GWAS entres os fenótipos de AG e os CNVs resultaram em um total de 186 CNVR siginificantivos para os grupos dos ácidos graxos saturados (43), monosaturados (42), poliinsaturados (66) e omegas (35), nas quais foram identificados 278 genes com funçao descrita. Estes resultados apontaram genes associados a AG de varias saturações, podendo ser destacados os genes SAMD8 e BSCL2, os quais estão relacionados ao metabolism lipidico; e o gene RAPGEF6, relacionado ao metabolism energetico. Assim os inúmeras regiões genômicas encontradas neste estudo, bem como os genes identificados nas mesmas, devem contribuir para a formação de uma base genética do perfil de ácidos graxos da carne de bovino Nelore (Bos indicus), podendo contribuir para uma melhor seleção das características associadas à melhora da saúde humana. saúde. O conhecimento desses CNVs deverá melhorar a compreensão dos mecanismos genéticos e fisiológicos que contribuem para as características produtivas, bem como na seleção de animais mais produtivos e eficientes, contribuindo para o melhoramento genético das características produtivas.
The objective of this study was to identify genomic regions of Nellore cattle that present variations in the number of copies (CNV) and to associate these CNV with the fatty acid profile of the meat. In addition, the objective of this study was to carry out a genome-wid association using the single step method (GWASss) to detect genomic regions associated with saturated, mono and polyunsaturated fatty acids, as well as omega 3, 6 and their relationship. The study of the characterization and distribution of CNVs along the Nellore genome was performed by PennCNV software using genotypic data of 3,794 animals, resulting in 399,361 CNVs identified. After quality control, 2,902 were maintained in the analyzes, resulting in 195,873 CNVs, with an average size of 54,744 pb, maximum of 8.7 Mb and minimum 3 kb. The CNV regions were generated by the overlap of the CNVs by CNVRuler software. The chromosomes that showed the highest incidence of CNVR were BTA19 (24.26%), BTA23 (18.68%) and BTA25 (18.05%). Those with the lowest incidence were BTA29 (1.63%), BTA13 (9.72%) and BTA8 (9.72%). The 9,805 CNV regions estimated in the present study covered approximately 13.05% of the bovine genome and overlaped 5,495 genes known to envolve in biological processes that could be involved in the environmental adaptation of the subspecies to tropical areas. The GWASss study showed 115 windows that explained more than 1% of the additive genetic variation for the 22 fatty acids studied. The identification of these regions and their genes, such as ELOVL5, ESRRG, PCYT1A and the ABC group genes (ABCA5, ABCA6 and ABCA10) are genes directly and indirectly related to lipid metabolism. The GWAS between AG phenotypes and CNVs resulted in a total of 186 CNVRs that were significant for the saturated (43), monosaturated (42), polyunsaturated (66) and omegas (35) fatty acids groups, in which 278 genes with described function. These results pointed genes associated to AG of various saturations, and the genes SAMD8 and BSCL2, which are related to lipid metabolism; and the RAPGEF6 gene, related to energetic metabolism. Thus, the numerous genomic regions found in this study, as well as the genes identified in them, should contribute to the formation of a genetic basis of the Nellore beef (Bos indicus) fatty acid profile, contributing to a better selection of the traits associated with improvement of human health. The knowledge of these CNVs could improve the understanding of the genetic and physiological mechanisms that contribute to the productive traits, as well as the selection of more productive and efficient animals.
FAPESP: 2013/11853-4

Oenococcus oeni joue un rôle essentiel dans l’élaboration du vin. Adaptée aux environnements acides et riches en alcool, elle est la bactérie lactique naturellement sélectionnée pour mener la fermentation malolactique (FML). Elle est ainsi la principale espèce recherchée et utilisée industriellement comme levain malolactique. Toutefois, il existe une grande diversité phénotypique au sein des souches d’O. oeni et notamment une variabilité des propriétés technologiques que sont la résistance à la lyophilisation, la résistance à l’inoculation dans le vin et la capacité à réaliser rapidement la FML. De nombreux gènes impliqués dans l’adaptation au vin ont déjà été identifiés mais, ne se sont pas toujours révélés efficaces pour la sélection de souches œnologiques. Dans ce contexte, cette étude a consisté à identifier des gènes spécifiques des souches d’intérêt technologique à travers l’analyse des plasmides et génomes. Face aux difficultés rencontrées pour purifier les grands plasmides, seul le plasmide pOENI-1 a été étudié. Ce travail a révélé différentes formes plasmidiques regroupées en une famille nommée « pOENI-1 ». Plusieurs gènes accessoires ont été identifiés et deux d’entre eux ont été détectés chez les souches associées aux fermentations malolactiques spontanées. La comparaison des génomes de souches aux propriétés technologiques diverses a également révélé des séquences génétiques qui leur sont spécifiques. L’ensemble de ces travaux a permis d’identifier plusieurs gènes dont la distribution statistique parmi les souches d’O. oeni a été analysée par la construction de courbes ROC. Ces courbes permettent d’évaluer la qualité des gènes en tant que marqueurs génétiques des souches d’intérêt technologique. Il est donc maintenant possible d’orienter la sélection des nouveaux levains malolactiques par l’utilisation des données génétiques et des outils statistiques décrits dans cette étude
Oenococcus oeni plays an essential role in the production of wine. Adapted to acidic and alcohol rich environments, it is the lactic acid bacterium species that is naturally selected to conduct malolactic fermentation (MLF). It is also the main species that is selected and used industrially as malolactic starter. However, there is a huge phenotypic diversity among strains of O. oeni, which includes a variability of technological properties such as resistance to freeze-drying, resistance to inoculation into the wine and the ability to quickly achieve the MLF. Many genes involved in adaptation to wine have been identified but have not always proven effective in selecting wine strains. In this context, this study aimed to identify genes that are specific strains of technological interest through the analysis of genomes and plasmids. Due to difficulties encountered to purify large plasmids, only the plasmid pOENI-1 was studied. This work has revealed several different but related plasmids that were grouped into a family named "pOENI-1". Several accessory genes have been identified and two of them were detected in O. oeni strains associated with spontaneous MLF. Comparing the genomes of strains showing various technological properties also revealed genetic sequences that are specific of those strains. Altogether, these works have revealed several genes whose statistical distribution among O. oeni strains was analyzed by constructing ROC curves. These curves are used to assess the quality of genes as genetic markers of strains of technological interest. It is now possible to guide the selection of new malolactic starters by the use of genetic data and statistical tools described in this study

The stainless steel industry is an industry which manufactures one of the most used ma-terials in the world. Even though the vast size of the industry many might not reflect werethe steel comes from when buying their IKEAcutlery etc. But in fact this industry is moreintricate and complex than one might think.During the manufacturing process, the steelgoes through several processes. One of thoseprocesses is done by treating the steel withstrong acid. This chemical process is calledpickling. The pickling process is what createsthe characteristic smooth and shiny surface ofa product made in stainless steel.This thesis is about the development of a testunit that is able to effectively test the ASRA(Acid Sludge Removal Apparatus) filter cloths.The ASRA is a filtration system developed by Scanacon in Stockholm that filtrates and puri-fies acid that is used during the pickling pro-cess of a steel manufacture process. Today, it is complicated, time consuming and dangerous totest and evaluate different filter cloths in orderto find the cloth that provides the best result,since the tests has to be conducted on the realsystems. The aim of this thesis was therefore tosolve these problems. Scanacon wanted to findan alternative solution, that would not includeinteraction with the real filtration system. Theyalso wanted a portable and safe solution. To solve the problem a iterative design pro-cess called RDCD which stands for research, design, create & delivery was developed. Theprocesses was heavily influenced by the CDIO process (Conceive, Design, Implement & Ope-rate). After the research phase was conducted it was decided that a small filter analysis productwould be the best way to solve the problem. The product was then developed one com-ponent at a time. During the project several prototypes and test was created and conductedin order to validate the design.The result is a small filtration unit speciallydesigned to simulate the ASRA system. Theproduct is able to rapidly test various filtercloths in an easy way, without putting the user in harm’s way. After the tests has been perfor-med can the user can identify which cloth that worked the best. The product is small enoughto be possible to be carried in a hard case bag,which in turn affords portability.
Stålindustrin är en industri som tillverkar ettav de mest använda materialen i världen. Trotsindustrins enorma storlek är det kanske intemånga som reflekterar varifrån stålet kommerifrån när man köper exempelvis IKEA bestick.Men faktum är att den här industrin är mycketmer invecklad och komplex än vad man kan tro. Under tillverkningsprocessen går stålet ige-nom flera processer. En av de här processerna genomförs genom att behandla stålet med starksyra. Den kemiska processen kallas för betningoch är det som ger en produkt tillverkad i stålden karaktäristiska lena och glänsande ytan. Den här examensrapporten handlar om ut-vecklandet av en test enhet som effektivt kan testa filter dukarna till ASRA (Acid SludgeRemoval Apparatus) systemet. ASRA systemetär ett filtreringssystem utvecklat av Scanaconi Stockholm som filtrerar och renar syran som används under betningsprocessen vid stål-tillverkning. Idag är det komplicerat, tidskrä-vande och farligt att testa och utvärdera olika filterdukar för att ta reda på vilken duk somger bäst resultat, eftersom att testerna måstegenomföras på de riktiga systemen. Målet fördet här examensarbetet var därför att lösa dehär problemen. Scanacon ville ha en alternativlösning som inte kräver interaktion med deriktiga systemen. De ville också ha en portabeloch säker lösning. För att lösa problemet utvecklades en itera-tiv designprocess kallad RDCD som står för research, design, create & delivery. Processen var tungt influerat av CDIO (Conceive, De-sign, Implement & Operate) processen. Efter att researchfasen var genomförd bestämdesdet att en liten filteranalysatorprodukt var detbästa sättet att lösa problemet på. Produktenutvecklades sedan en komponent i taget. Underprojektets gång har flera prototyper byggts och flera tester genomförts för att validera designen. Resultatet är en liten filtreringsenhet som är specialdesignad för att simulera ASRA syste-met. Produkten kan snabbt testa oliak filterdu-kar på ett enkelt sätt, utan utsätta användaren för fara. Efter att testerna genomförts kananvändaren identifiera vilken duk som funkatbäst. Produkten är tillräckligt liten för att fåplats i en hårdplast väska, vilket ger produktenden efterfrågade portabiliteten.

Peach [Prunus persica L. (Batsch.)] and apricot (Prunus armeniaca L.) are two Prunus species developing delicious fruits and they are mostly grown in temperate areas of the world. Both species have been cultivated since ancient times, being greatly appreciated for their unique taste and benefits to human health. Peaches and apricots can be consumed either fresh or processed through canning or drying, depending on the preference of a specific region and the use-purpose. So far, many efforts have been done in enhancing disease and pest resistance, in increasing the environmental adaptability to sub-tropical areas, in extending the harvest season or in improving fruits appearance. However, consumers frequently complain about the lack of taste in peach and apricot fruits sold on markets, encouraging the ongoing breeding projects to include organoleptic properties among their objectives. Among all the fruit quality-related traits, acidity plays a pivotal role affecting both consumers’ and market acceptance. To match with market trends, an extensive characterization of peach and apricot fruits attributes under the highlighted necessity of renewing the varieties cultivated worldwide is required. In this thesis, a peach and apricot collection of 201 and 164 accessions, respectively, was screened for many important fruit organoleptic attributes, with a specific focus on acidity and organic acids content. Fruits acidity was titrated and ten organic acids (cis-aconitate, citrate, fumarate, galacturonate, malate, oxalate, quinate, shikimate, succinate and tartrate) separation was accomplished by HPLC technique coupled to UHPLC-HRMS validation. Analyses were performed on peach pulp and on apricot pulp and peel. The final aim was in-depth dissecting the peach and apricot panel for these organoleptic parameters to exploit the existing variability within their germplasm. Malate and citrate were the most abundant organic acids in both species, with pattern more genotype than year dependent. Results suggested that seasonality effects on fruits acidity and almost all the considered-organic acids were very low. Among the other organic acids, quinate and succinate reached large concentrations in peach and apricot, respectively, while tartrate was interestingly present more in apricot peel than pulp. The availability of a reference genome in peach has allowed to further characterize peach fruit quality traits. Over the past, peach fruits acidity has been partially elucidated at genetics level, identifying one major locus and making it a breeding target. Although the great advances, the selection accuracy and the long-time required for releasing new varieties on markets still hamper peach breeding progress. To in-depth unravel acidity trait and to speed up the selection of newly developed individuals in peach, a total of 1,190 accessions were genotyped for performing genomics analyses. Two different studies were carried out in this thesis: genomic selection (GS) and genome wide association studies (GWAS). Results confirmed that GS seems feasible in peach not only for acidity but also for organic acids content, in particular for patterns of malate and citrate. GWAS confirmed the presence of one major locus acting as dominant in peach fruits but revealed other significant associations on chromosomes 1, 7 and 8. In summary, this thesis includes the first efforts in an in-depth and at multilevel dissection of acidity in peach and apricot, applying different approaches on a large panel of individuals. This thesis attempts to provide a complete overview with results that may be useful for future researches, studies and successful breeding programmes.

I batteri lattici che possiedono proprietà salutistiche, oltre a poter essere introdotti come colture starter o colture aggiunte durante la produzione di prodotti caseari, potrebbero essere utilizzati per facilitare la biosintesi in situ di molecole bioattive durante il processo di fermentazione, aumentando l’interesse nei confronti dei prodotti caseari come alimenti multifunzionali. Ultimamente, i formaggi a latte crudo sono grande oggetto di ricerca in quanto nicchie di elevata biodiversità; in seguito alla caratterizazione genotipica e tecnologica del microbiota autoctono è possibile selezionare ceppi che potrebbero essere utilizzati per migliorare il profilo sensoriale del formaggio e dare al prodotto un valore aggiunto di tipo salutistico. Il formaggio Tradizionale di Montagna (TM) viene prodotto da latte crudo di vacca e il processo di fermentazione avviene spontaneamente in piccole fattorie chiamate “Malghe”, collocate nelle aree alpine della regione Trentino. Per la prima volta, la popolazione microbica del formaggio TM è stata caratterizzata con l’obiettivo di selezionare cocchi e batteri lattici appartenenti al gruppo dei non-starter (NSLAB) che, rispettivamente, potessero essere utili per lo sviluppo di nuove colture starter o colture aggiunte. Sono state effettuate le conte microbiologiche di campioni (n=120) di latte, cagliata e formaggio a diversi tempi di stagionatura (24 ore, 1 mese e 7 mesi) su terreni di coltura selettivi. I cocchi mesofili e termofili sono risultati dominanti nelle 24 ore successive alla produzione, mentre i lattobacilli mesofili hanno mostrato la concentrazione microbica più elevata a fine stagionatura. Seicentoquaranta colonie sono state isolate da campioni di cagliata e formaggio a 24 ore e altre 95 colonie sono state isolate da campioni di formaggio a 7 mesi di stagionatura. Tutti gli isolati sono stati caratterizzati genotipicamente utilizzando la tecnica RAPD- PCR (Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction) servendosi di due primers, PCR specie-specifica e sequenziamento parziale del gene 16S rRNA. I cocchi, appartenenti a 16 specie diverse, sono stati raggruppati in 231 biotipi, mentre i NSLAB sono stati raggruppati in 70 biotipi e attribuiti a 13 specie diverse. Lactococcus lactis, Streptococcus thermophilus e Enterococcus faecalis erano le specie dominanti nei campioni di cagliata e formaggio a 24 ore; Pediococcus pentosaceus e Lactobacillus paracasei erano le specie principali a fine stagionatura. Sono state testate le caratteristiche fenotipiche, tecnologiche e salutistiche di tutti i ceppi; in particolare, lattococchi, streptococchi ed enterococchi sono stati analizzati per la loro attività acidificante e proteolitica, capacità di crescere a temperature non ottimali, produzione di acetoino, produzione di note olfattive, capacità di inibire la crescita dei coliformi autoctoni del latte e il tasso di autolisi. Il 40% degli enterococchi ha mostrato la capacità di inibire in vitro la crescita dei coliformi del latte, ma sono stati esclusi come possibili ceppi starter perchè presentavano fattori di rischio. Tra i lattococchi e gli streptococchi, 4 Lc. lactis subsp. lactis e 2 Sc. thermophilus, caratterizzati da una rapida attività acidificante e note sensoriali gradevoli, sono stati sottoposti ad un test di resistenza alla liofilizzazione. I ceppi Lc. lactis subsp. lactis 68 e Sc. thermophilus 93 hanno mostrato le proprietà migliori e, quindi, potrebbero essere adatti per la produzione di formaggi. I ceppi NSLAB sono stati testati per le loro proprietà di crescita, metabolismo dei carboidrati, attività acidificante, proteolitica e lipolitica, produzione di acetoino, attività amino peptidasica (AP) e produzione di amine biogene. Per quanto riguarda le proprietà salutistiche, l’attività di idrolisi dei sali biliari (BSH) è stata testata qualitativamente, la produzione di acidi linoleici coniugati è stata misurata spettrofotometricamente e la produzione di acido γ- aminobutirrico (GABA) è stata quantificata tramite UHPLC. Gli isolati appartenenti alla specie Lb. paracasei hanno dimostrato di essersi ben adattati all’ambiente di Malga e hanno mostrato la migliore attività AP e produzione di acetoino. Alcuni ceppi hanno mostrato proprietà salutistiche molto interessanti e hanno prodotto sostanze bioattive. In particolare, Lb. rhamnosus BT68, Lb. paracasei BT18, BT25, BT31 e Pc. pentosaceus BT3, BT13, BT51 hanno prodotto tra 70 e 130 mg/mL di CLA totali in vitro. Lb. brevis BT66 ha convertito l’acido L-glutamico in elevate concentrazioni di GABA (129 ± 8.6 mg/L) e ha mostrato attività BSH. Questi primi risultati hanno dimostrato che il formaggio TM è una riserva di elavata diversità microbica e che i batteri lattici autoctoni potrebbero essere utilizzati non solo per la produzione di prodotti caseari ma anche per le loro proprietà salutistiche. Lc. lactis subsp. lactis 68 e Sc. thermophilus 93, considerati come i ceppi autoctoni migliori, sono stati testati come colture starter per la produzione di 9 forme di formaggio TM direttamente in Malga. Tre formaggi controllo (CTRL) sono stati prodotti secondo la tradizione e senza l’aggiunta di starter o colture aggiunte, mentre tre formaggi con ceppi starter autoctoni (STR) e tre formaggi con un ceppo starter commerciale (CMS) sono stati prodotti inoculando rispettivamente nel latte di caldaia i due ceppi selezionati e un ceppo commerciale appartenente alla specie Sc. thermophilus. Dopo 24 ore, 1 mese e 7 mesi di stagionatura, è stato analizzato il contenuto microbico di tutti i formaggi sperimentali. I cocchi mesofili e i lattobacilli sono risultati dominanti nei campioni di formaggio dopo 24 ore e 1 mese di stagionatura, mentre i cocchi erano predominanti nel formaggio a fine stagionatura. Il DNA genomico totale è stato estratto e un frammento della regione V1-V3 è stato amplificato e sottoposto a pirosequenziamento-454. I lattococchi e gli streptococchi erano la specie maggiormente presenti nei formaggi CTRL e STR, e il ceppo Lc. lactis ssp. lactis 68 ha influenzato la crescita dei batteri lattici autoctoni del latte crudo appartenenti alla specie Lc. lactis ssp. cremoris durante le prime fasi di fermentazione. Inoltre, il ceppo commerciale Sc. thermophilus sembra aver predominato sui batteri della specie Lc. lactis supsp. lactis e cremoris naturalmente presenti nel latte crudo e aver portato ad una riduzione di abbondanza di Lactobacillus sp. e Enterococcus sp. La sopravvivenza del microbiota del formaggio TM alla digestione è stata testata in vitro simulando le condizioni gasto-intestinali (GI) umane. A fine stagionatura, i 9 formaggi sperimentali sono stati sottoposti ad un sistema modello che simula il processo digestivo nella bocca, nello stomaco e nell’intestino tenue e comprende delle fasi sequenziali di incubazione con i succhi gastrici e duodenali umani. Le conte microbiche sono state effettuate prima e dopo la simulazione: la conta batterica totale e i cocchi termofili sono diminuiti significativamente dopo la digestione. Trentasei lattobacilli sono stati isolati dai 9 campioni di formaggio digeriti: tra loro, 1 Lb. paracasei, 1 Lb. parabuchneri e 1 Lb. fermentum sono stati testati per la loro sopravvivenza post-transito GI. I ceppi Lc. lactis subsp. lactis 68 e Lb. parabuchneri D34 sono stati utilizzati per fermentare aliquote di latte intero che, successivamente, sono state digerite in vitro. In seguito alla simulazione della digestione, è stata registrata una riduzione maggiore (circa 1 ciclo logaritmico) della carica di Lb. parabuchneri D34 cresciuto in coltura pura piuttosto che in latte fermentato, suggerendo che Lb. parabuchneri D34 possiede la capacità intrinseca di sopravvivere alla digestione, ma il contenuto di grasso e la struttura tipici del formaggio potrebbero proteggere i batteri lattici durante il transito GI. Inoltre, il nostro interesse nei confronti dei ceppi produttori di GABA ci ha spinti a testare la capacità del ceppo Lb. brevis BT66 di produrre GABA in situ durante la produzione di formaggio, attraverso la decarbossilazione del glutammato. Venti micro-caseificazioni sperimentali sono state effettuate utilizzando un ceppo starter commerciale (107 UFC/mL) e Lb. brevis BT66 come coltura aggiunta. Lb. brevis BT66 è stato testato in quadruplicato in quattro concentrazioni diverse (102, 103, 104, 105 UFC/mL). Con l’obiettivo di seguire l’evoluzione microbica, campioni di latte, cagliata e formaggio a 20 giorni di stagionatura sono stati processati per la conta batterica su terreni selettivi. I campioni controllo e quelli contenenti i ceppi selezionati hanno mostrato un andamento simile, suggerendo che sia i ceppi autoctoni del latte che quelli aggiunti sono cresciuti durante la stagionatura. Comunque, la carica dei lattobacilli mesofili registrata nei campioni STR e CMS era più alta dei campioni CTRL. La concentrazione di GABA e acido glutammico è stata quantificata nei campioni di formaggio a 20 giorni di stagionatura tramite UHPLC-HQOMS. I profili aminoacidici hanno mostrato che mentre la carica del ceppo Lb. brevis BT66 nel latte aumentava, la concentrazione di acido glutammico (da 324 ± 37 a 202 ± 32 mg/kg) e GABA (da 154 ± 31 a 91 ± 20 mg/kg) diminuivano significativamente nel formaggio. Questi risultati ci hanno suggerito che i ceppi sperimentali hanno convertito l’acido glutammico in GABA, ma che il GABA potrebbe essere stato successivamente convertito in succinato dall’enzima GABA- transaminasi. È stato dimostrato che l’aminoacido GABA influisce sulla funzione del cervello attraverso l’asse intestino:cervello, ha effetti positivi sul diabete e l’obesità, regola il sistema immunitario, il processo infiammatorio e il metabolismo energetico nei mammiferi, stimola l’ipotensione, ha effetti diuretici e tranquillanti. A causa della sua abilità di produrre elevate concentrazioni di GABA e la sua attività BSH in vitro, il ceppo Lb. brevis BT66 è stato selezionato per essere testato in vivo in topi con obesità indotta e diabete mellito di tipo 2. Contemporaneamente, è stato testato anche il ceppo Lb. brevis DPC6108 (isolato dal tratto GI umano), che presentava le stesse proprietà. Sono stati generati i relativi mutanti rifampicina resistenti (rif), e il loro profilo genotipico, ottenuto tramite RAPD-PCR e PFGE (Pulsed-Field Gel Electrophoresis), era identico al ceppo nativo. La percentuale di conversione del glutammato monosodico in GABA è stata calcolata in seguito ad analisi aminoacidica di ultima generazione: Lb. brevis BT66rif ha prodotto 840.5 ± 266 μg/mL di GABA con un tasso di bioconversione del 73%, mentre Lb. brevis DPC6108rif ha prodotto 1,218.0 ± 393.2 μg/mL con una bioconversione dell’87%. L’attività BSH è stata positiva in seguito ad analisi quantitativa e qualitativa, e nei ceppi mutanti e in quelli nativi sono stati osservati risultati simili tra loro. I ceppi rifampicina-resistenti sono stati liofilizzati e testati per la loro stabilità a temperatura ambiente, +4 e -20 °C. Sia il metodo spettrofotometrico che le conte su piastra hanno rivelato che i ceppi liofilizzati sono sopravvissuti a temperatura ambiente per 24 ore, dopo essere stati risospesi in acqua sterile. La stabilità dei ceppi a +4 e -20 °C è stata analizzata contando le cellule vitali su terreno di coltura selettivo per 10 settimane e nessuna riduzione significativa è stata registrata nelle prime 4 settimane successive alla liofilizzazione. Entrambi i ceppi farmabiotici Lb. brevis BT66rif e DPC6108rif hanno dimostrato di essere resistenti alla liofilizzazione, sono sopravvissuti a transito attraverso il tratto GI di topo (come dimostrato da uno studio pilota) e la loro efficacia terapeutica è attualmente sotto analisi in uno studio in vivo per il trattamento dell’obesità metabolica e del diabete mellito di tipo 2.
The use of health-promoting lactic acid bacteria (LAB) strains as starter or adjunct cultures for dairy productions could facilitate the in situ bio-synthesis of bioactive molecules during the fermentation process, increasing the interest towards dairy products as multifunctional foods. Currently, there is much research about genotypic and technological characterization of raw milk cheeses microbiota, which is rich in biodiversity and could be exploited for improving the sensory attributes and add healthy benefits to the cheese. Traditional Mountain (TM) cheese is made from raw cow’s milk and spontaneously fermented in small farms called “Malga” located in the alpine areas of Trentino region. For the first time, the microbial population of TM-cheese has been characterized in order to select cocci and non-starter LAB suitable for developing new starter or adjunct cultures, respectively. Samples (n = 120) of milk, curd and cheese at different ripening times (24 hours, 1 month and 7 months) were enumerated in selective culture media. Mesophilic and thermophilic cocci dominated during the first 24 hours following production, and mesophilic lactobacilli were dominant at the end of ripening. Six hundred and forty colonies were isolated from curd and cheese 24 hours following production, and 95 more colonies were isolated from cheese after 7 months of ripening. All isolates were genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. Cocci clustered in 231 biotypes belonging to 16 different species, and non-starter LAB (NSLAB) clustered in 70 biotypes belonging to 13 different species. Lactococcus lactis, Streptococcus thermophilus and Enterococcus faecalis were dominant in curd and 24h-cheese; Pediococcus pentosaceus and Lactobacillus paracasei were the main species at the end of ripening. The phenotypic, technological and health-promoting activities of all strains were investigated. In particular, lactococci, streptococci and enterococci were tested for their acidification and proteolytic activity, ability to growth at not optimal temperatures, acetoin production, development of olfactory flavour notes, autolysis rate and ability to inhibit the growth of coliforms. Forty percent of enterococci showed the ability to inhibit raw milk resident coliforms in vitro, but they were excluded as possible starters, owing to the presence of associated risk factors. Among lactococci and streptococci, 4 Lc. lactis subsp. lactis and 2 Sc. thermophilus were fast acidifiers, produced pleasant flavours, and were subjected to the freeze-drying stability test. Lc. lactis subsp. lactis 68 and Sc. thermophilus 93 showed the best properties and might be appropriate for cheese production. NSLAB strains were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase activity (AP) and biogenic amines production. Concerning the health-promoting properties, the bile salts hydrolysis (BSH) activity was tested qualitatively, the conjugated linoleic acid production was measured spectrophotometrically, and the γ-aminobutyric acid (GABA) production was quantified by UHPLC (Ultra High Performance Liquid Cromatography). Lb. paracasei isolates resulted to be well adapted to the Malga environment and showed the highest AP activity and acetoin production. Some strains harbored very interesting health- promoting properties and produced bioactive substances. In particular, Lb. rhamnosus BT68, Lb. paracasei BT18, BT25, BT31, Pc. pentosaceus BT3, BT13, BT51 produced between 70 and 130 mg/mL of total CLA in vitro. Lb. brevis BT66 converted L-glutamate to a high concentration of GABA (129 ± 8.6 mg/L) and showed BSH activity. These first results revealed that TM-cheese is a reservoir of a high microbial diversity, and the resident LAB could be exploited not only for the applicability in dairy production but also for their health- promoting properties. Lc. lactis subsp. lactis 68 and Sc. thermophilus 93, which showed to be the best performing strains, were tested as starter and adjunct cultures, for the production of 9 experimental TM-cheese wheels in a Malga-farm, respectively. Three control (CTRL) cheeses were produced according to the tradition and any starter or adjunct culture was not added; three starter (STR) and three commercial starter (CMS) cheeses were produced inoculating the vat milk with both selected strains and a commercial Sc. thermophilus strain, respectively. After 24 hours, 1 month and 7 months of ripening the microbial content of all experimental cheeses was investigated. Mesophylic cocci and lactobacilli dominated in cheese samples after 24 hours and 1 month of ripening, while cocci dominated in full- ripened cheese. The total genomic DNA was extracted, and a fragment of the V1-V3 region was amplified and pyrosequenced. Lactococci and streptococci were the most abundant species in CTRL and STR cheese, and Lc. lactis ssp. lactis 68 affected the proliferation of the (raw milk) indigenous Lc. lactis ssp. cremoris during the early fermentation. Moreover, the commercial Sc. thermophilus showed to be dominant towards Lc. lactis supsp. lactis and cremoris naturally present in raw milk and to be responsible in decreasing the abundance of Lactobacillus sp. and Enterococcus sp. The survival of TM-cheese microbiota in vitro was investigated under simulated human gastro-intestinal (GI) conditions. The 9 full ripened experimental TM-cheeses were subjected to a model system that simulates digestive processes in the mouth, stomach and small intestine, comprising sequential incubation in human gastric and duodenal juices. Bacterial counts were performed before and after the simulation: total bacterial count and thermophilic cocci significantly decreased after the simulated digestion. Thirty-six lactobacilli were isolated from cheese after digestion: among them 1 Lb. paracasei, 1 Lb. parabuchneri and 1 Lb. fermentum were tested for their survival after GI transit. Lc. lactis subsp. lactis 68 and Lb. parabuchneri D34 strains were used to ferment whole milk and digested. The load of Lb. parabuchneri D34 decreased about one logarithmic cycle more when grown as pure culture than fermented milk after simulated digestion, suggesting that Lb. parabuchneri D34 had in itself the ability to survive to digestion, but the fat content and the cheese structure might protect LAB during the GI transit. Furthermore, our interest towards the GABA producing strains lead us to test the ability of Lb. brevis BT66 to produce GABA in situ during cheese production, through the decarboxylation of glutamate. Twenty experimental micro-cheeses were produced using a commercial starter strain (107 CFU/mL) and Lb. brevis BT66 as adjunct culture. Four different concentrations (102, 103, 104, 105 CFU/mL) of Lb. brevis BT66 were tested in quadruplicate. In order to follow the microbial evolution, samples of milk, curd and cheese after 20 days of ripening were enumerated in selective media. The control and experimental samples showed a similar trend, suggesting that both milk-resident and starter strains grew during ripening. However, the load of mesophilic lactobacilli in all experimental curd samples was higher than the control ones. The concentration of GABA and glutamic acid in cheese samples after 20 days of ripening was quantified by UHPLC-HQOMS. The amino acidic profiles showed that while the concentration of Lb. brevis BT66 in milk increased, the amount of both glutamic acid (from 324 ± 37 to 202 ± 32 mg/kg) and GABA (from 154 ± 31 to 91 ± 20 mg/kg) significantly decreased in cheese. These results suggested that the experimental strain converted glutamic acid to GABA, but that GABA may have subsequently been converted to succinate by GABA transaminases. The non-protein amino acid GABA has been reported to impact on brain function through the gut:brain axis system, to harbor an anti-obesity and antidiabetogenic effect, to regulate the immune system, the inflammation process and the energy metabolism in mammals including induction of hypotension, diuretic and tranquilizer effects, stimulation of immune cells. Owing to its ability to produce high concentrations of GABA and its BSH activity in vitro, Lb. brevis BT66 was selected to be tested in vivo in mice suffering obesity-associated type-2-diabetes. Another Lb. brevis (strain DPC6108), isolated from the human GI tract and harboring the same properties, was simultaneously investigated. The corresponding rifampicin resistant mutants (rif) were generated; their genotypic profile was obtained by RAPD-PCR and PFGE (Pulsed-Field Gel Electrophoresis) and was identical to the native strain. The conversion rates of monosodium glutamate to GABA were investigated by next- generation amino acid analysis: Lb. brevis BT66rif produced 840.5 ± 266 μg/mL of GABA with about 73% of bioconversion and Lb. brevis DPC6108rif produced 1,218.0 ± 393.2 μg/mL with about 87% of bioconversion. The BSH activity was positive to both qualitative and quantitative assays and the results were similar in both native and mutant strains. The rifampicin resistant strains were freeze-dried and tested for their stability at room temperature, +4 and -20 °C. Both spectrophotometer and plate count methods revealed that freeze-dried strains survived at room temperature during 24 hours after suspending in sterile water. The stability of freeze-dried strains at +4 and -20 °C was investigated enumerating the viable cells in selective medium during 10 weeks and any significant load reduction was not detected in the first 4 weeks following freeze-drying. Both pharmabiotic-producing Lb. brevis BT66rif and DPC6108rif were resistant to freeze-drying, survived transit through mouse GI tract (as proven by a pilot study), and their therapeutic efficiency is being assessed in vivo to treat metabolic obesity and type-2-diabetes.

Species prescriptions are developed for revegetating abandoned acidic coal overburden seepage sites in the Collie region of Western Australia. The research involved selecting appropriate plant species and determining successful methods of enhancing revegetation. Candidate species were screened for tolerance to acidic overburden materials, local climate conditions and metal toxicity. Methods tested included improving spoil conditions and trialing an alternative method for seeding.Twelve species of native plants were tested for tolerance in two acid overburden materials in pot and field trials. Eucalyptus robusta is the most tolerant, Eucalyptus camaldulensis and Eucalyptus cladocalyx are highly tolerant, Eucalyptus rudis and Melaleuca hamulosa demonstrate potential, provided adequate soil moisture is available.An important growth restriction factor in acid soils is the presence of free aluminium ions. A glasshouse trial performed on seven species for tolerance to aluminium toxicity revealed E. robusta as most tolerant and E. camaldulensis and Kunzea ericifolia a highly tolerant. E. rudis and M. hamulosa are moderately tolerant, but E. cladocalyx and Eucalyptus diversicolor are very sensitive to aluminium.Various methods were trialed to increase growth of seedlings transplanted on to acidic overburden sites. Both commercial cow manure and slow-release fertiliser tablets increase growth, whereas commercial potting mix and lime do not. Inoculation of plants with the ectomycorrhiza fungus Pisolithus tinctorius increases the amount of infection in roots but does not enhance plant growth.Supplementary fertilisation is necessary to maintain growth (nitrogen) and restore chlorophyll production (phosphorus) in fast growing eucalypt seedlings planted into typical acidic spoils. Poor levels of nutrient availability in such acidic sites appear to be the primary factor in retarding growth. In the absence of supplementation, foliage reddening is observed in several species.An alternative method of seeding dumps is fascining. Prepared dump surfaces may be covered with capsule-laden branchwood of myrtaceous species. Material of the locally available Kunzea ericifolia is effective in producing many seedlings. Subsequent seedling growth is enhanced with fertiliser and lime addition.

Groat oil content and composition are important quality traits in oats (Avena sativa L). These traits are controlled by many genes with additive effects. The chromosomal regions containing these genes, known as quantitative trait loci (QTL), can be discovered through their close association with markers. This study investigated total oil content and fatty acid components in an oat breeding population derived from a cross between high oil ('Dal') and low oil ('Exeter') parents. A genetic map consisting of 475 DArT (Diversity Array Technology) markers spanning 1271.8 cM across 40 linkage groups was constructed. QTL analysis for groat oil content and composition was conducted using grain samples grown at Aberdeen, ID in 1997. QTL analysis for multiple agronomic traits was also conducted using data collected from hill plots and field plots in Ottawa, ON in 2010. QTLs for oil content, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) were identified. Two of the QTLs associated with oil content were also associated with all of the fatty acids examined in this study, and most oil-related QTL showed similar patterns of effect on the fatty acid profile. These results suggest the presence of pleiotropic effects on oil-related traits through influences at specific nodes of the oil synthesis pathway. In addition, 12 QTL-associated markers (likely representing nine unique regions) were associated with plant height, heading date, lodging, and protein content. The results of this study will provide information for molecular breeding as well as insight into the genetic mechanisms controlling oil biosynthesis in oat.

Species prescriptions are developed for revegetating abandoned acidic coal overburden seepage sites in the Collie region of Western Australia. The research involved selecting appropriate plant species and determining successful methods of enhancing revegetation. Candidate species were screened for tolerance to acidic overburden materials, local climate conditions and metal toxicity. Methods tested included improving spoil conditions and trialing an alternative method for seeding.Twelve species of native plants were tested for tolerance in two acid overburden materials in pot and field trials. Eucalyptus robusta is the most tolerant, Eucalyptus camaldulensis and Eucalyptus cladocalyx are highly tolerant, Eucalyptus rudis and Melaleuca hamulosa demonstrate potential, provided adequate soil moisture is available.An important growth restriction factor in acid soils is the presence of free aluminium ions. A glasshouse trial performed on seven species for tolerance to aluminium toxicity revealed E. robusta as most tolerant and E. camaldulensis and Kunzea ericifolia a highly tolerant. E. rudis and M. hamulosa are moderately tolerant, but E. cladocalyx and Eucalyptus diversicolor are very sensitive to aluminium.Various methods were trialed to increase growth of seedlings transplanted on to acidic overburden sites. Both commercial cow manure and slow-release fertiliser tablets increase growth, whereas commercial potting mix and lime do not. Inoculation of plants with the ectomycorrhiza fungus Pisolithus tinctorius increases the amount of infection in roots but does not enhance plant growth.Supplementary fertilisation is necessary to maintain growth (nitrogen) and restore chlorophyll production (phosphorus) in fast growing eucalypt seedlings planted into typical acidic spoils. Poor levels of nutrient availability in such acidic sites appear to be the primary factor in ++
retarding growth. In the absence of supplementation, foliage reddening is observed in several species.An alternative method of seeding dumps is fascining. Prepared dump surfaces may be covered with capsule-laden branchwood of myrtaceous species. Material of the locally available Kunzea ericifolia is effective in producing many seedlings. Subsequent seedling growth is enhanced with fertiliser and lime addition.

Дипломний проект містить 108 сторінок, 10 ілюстрацій, 3 таблиці, 2 креслиники та 109 бібліографічних найменувань за переліком посилань. Метою даної роботи є проектування виробництва бактеріальної закваски для виготовлення кисломолочних продуктів дієтичного харчування,зокрема йогурту. Робота присвячена збільшенню обсягів виробництва вітчизняних заквасок, що дасть можливість збільшення асортименту та здешевлення молочнокислих продуктів. Обгрунтовано і запропоновано в якості продуцента бактеріальної закваски використовувати штами гомоферментативних термофільних молочнокислих бактерій Lactobacillus delbrueckii subsp. Вulgaricus та Streptococcus salivarius subsp. Thermophilus, отримані у результаті селекції з використанням природнього добору , мають пробітичні властивості і є високотехнологічними для виготовлення кисломолочної пробдукції. Обрано ефективне, економічно вигідне та надійне обладнання для виробничого культивування закваски. Розраховано та обрано апарат для виробничого культивування. Наведено технологічний, конструктивний та гідравлічний розрахунки обраного ферментеру. В роботі обґрунтовані та подані технологічна та апаратурна схеми виробництва.
The diploma project contains 108pages, 10 illustrations, 3 tables, 2 drawings and 109 bibliographic titles according to the list of references. The purpose of this work is to design the production of bacterial leaven for the manufacture of fermented milk products for dietary nutrition. The work is devoted to increasing the production of domestic leavens, which will increase the range and reduce the cost of lactic acid products. It is substantiated and proposed to use strains of homofermentative thermophilic lactic acid bacteria Lactobacillus delbrueckii subsp as a producer of bacterial yeast. Vulgaricus and Streptococcus salivarius subsp. Thermophilus, obtained by selection using natural selection, have probiotic properties and are high-tech for the manufacture of fermented milk production. Efficient, cost-effective and reliable equipment for production cultivation of sourdough has been selected. The device for industrial cultivation is calculated and selected. Technological, constructive and hydraulic calculations of the selected fermenter are given. The technological and hardware schemes of production are substantiated and presented in the work

L'adénocarcinome canalaire pancréatique (PDAC) est une maladie mortelle caractérisée par un micro-environnement tumoral (TME) extrêmement acide (˂pHe 6,5) qui joue un rôle important dans son début et sa progression. Dans ce contexte, les canaux ioniques perméables au Ca2+ représentent de bons candidats cibles en raison de leur capacité à intégrer des signaux provenant de la TME. Les canaux Ca2+ sont en effet des capteurs de pH capables d'intégrer les signaux de la TME pour activer les voies intracellulaires en aval liées à la progression du PDAC. Bien que les rôles de l'acidose tumorale et de la signalisation du Ca2+ dans la progression du cancer soient bien établis, l'hypothèse d'une TME acide utilisant la signalisation du Ca2+ comme voie préférentielle pour soutenir la progression tumorale n'a pas encore été suffisamment explorée.Mon travail de doctorat visait à étudier les changements phénotypiques et génétiques des cellules PDAC lors d'un stress acide au cours des différentes étapes de sélection et à évaluer comment l'acidose tumorale module les signaux Ca2+ et les phénotypes dans les lignées cellulaires PDAC, avec un accent particulier sur les oscillations Ca2+ et Store-Operated Ca2+ entry (SOCE). À cette fin, les cellules PANC-1 et Mia PaCa-2 ont été soumises à une pression acide à court et à long terme et à une récupération à pHe 7,4. Ce dernier traitement visait à imiter les bords du PDAC et l'évasion consécutif des cellules cancéreuses de la tumeur. L'impact de l'acidose a été évalué sur la morphologie cellulaire, la prolifération, l'adhésion, la migration, l'invasion, l'activité des invadopodes et la transition épithélio-mésenchymateuse (TEM) par des tests fonctionnels in vitro et le séquençage de l'ARN, ainsi que sur les signaux Ca2+ intracellulaires avec Fura-2. Nos résultats indiquent qu'un court traitement acide limite la croissance, l'adhésion, l'invasion et la viabilité des cellules PDAC. Au fur et à mesure que le traitement acide progresse, il sélectionne les cellules cancéreuses ayant des capacités de migration et d'invasion accrues induites par l'EMT, ce qui augmente encore leur potentiel métastatique lorsqu'elles sont réexposées à un pHe 7,4. L'analyse RNA-seq des cellules PANC-1 exposées à une acidose de courte durée et récupérées à pHe 7,4 a révélé un recâblage transcriptomique distinct. Il est intéressant de noter que les cellules PANC-1 sont caractérisées par des oscillations Ca2+ plus lentes pendant une exposition à l'acide à court terme par rapport aux cellules de contrôle et par une tendance à la régulation négative d'ORAI1 au niveau de l'ARNm, tandis que l'acidose à long terme et le rétablissement à un pH neutre déterminent le rétablissement d'oscillations Ca2+ rapides et la régulation positive d'ORAI1. Dans tous nos modèles cellulaires, les oscillations du Ca2+ sont dépendantes de SOCE, car le blocage d'ORAI1 par Synta66 et siORAI1 entraîne une altération de l'initiation et du maintien des oscillations du Ca2+. Ces données sont en corrélation avec le SOCE dans les cellules PANC-1, qui est diminuée pendant le traitement acide à court terme, et augmentée dans les cellules sélectionnées pour l'acide avec et sans récupération à pHe 7,4. Enfin, l'entrée de Ca2+ médiée par ORAI1 pourrait être impliquée dans l'activation des cascades de signalisation qui conduisent à l'augmentation de la migration et de l'invasion de tous les modèles cellulaires exposés à un pHe acide, car le traitement par Synta66 et siORAI1 n'ont pas affecté l'invasion et la migration des cellules de contrôle.En conclusion, nos résultats montrent que la sélection induite par l'acide contribue à l'acquisition d'un phénotype plus agressif dans les cellules de PDAC, caractérisé par une augmentation du SOCE, nécessaire à la génération d'oscillations rapides de Ca2+ qui peuvent activer des voies de signalisation Ca2+-dépendantes impliquées dans la progression du PDAC
Pancreatic ductal adenocarcinoma (PDAC) is the most common cancer affecting the pancreas, characterized by an unsatisfactory 5-year survival rate of around 10%, and to date, there are no effective therapeutic options for PDAC. This is in part due to a highly desmoplastic and immunosuppressive microenvironment that contributes to therapeutic failure. Moreover, the PDAC tumor microenvironment is featured by high acidosis (˂ pHe 6.5), a result of the metabolic reprogramming ("Warburg effect"), and hypoxic conditions, which offers important cues for its aggressiveness by selecting cancer cell phenotypes with competitive benefits for PDAC progression. In this context, Ca2+-permeable ion channels are known to regulate several hallmarks of cancer, including in PDAC. Therefore, they represent good target candidates due to their ability to integrate signals from the TME. Ca2+ channels are indeed pH and hypoxia sensors able to transduce TME signals to activate intracellular downstream pathways linked to PDAC progression. Although the roles of tumor acidosis and Ca2+ signaling in cancer progression are well established, the hypothesis of acidic TME employing Ca2+ signaling as a preferential route for sustaining tumor progression has not yet been sufficiently explored.My Ph.D. work aimed to study the phenotypic and genetic changes of PDAC cells upon acidic stress along the different stages of selection and to evaluate how tumor acidosis modulates Ca2+ signals and phenotypes in the PDAC cell lines, with a particular focus on Ca2+ oscillations and Store-Operated Ca2+ entry (SOCE). To this end, PANC-1 and Mia PaCa-2 cells were subjected to short- and long-term acidic pressure and recovery to pHe 7.4. The latter treatment was to mimic PDAC edges and consequent cancer cell escape from the tumor. The impact of acidosis was assessed for cell morphology, proliferation, adhesion, migration, invasion, invadopodia activity, and epithelial-mesenchymal transition (EMT) via functional in vitro assays and RNA sequencing, and for intracellular Ca2+ signals using Fura-2. Our results indicate that short acidic treatment limits the growth, adhesion, invasion, and viability of PDAC cells. As the acid treatment progresses, it selects cancer cells with enhanced migration and invasion abilities induced by EMT, thereby further enhancing their metastatic potential when re-exposed to pHe 7.4. RNA-seq analysis of PANC-1 cells exposed to short-term acidosis and pHe-selected recovered to pHe 7.4 revealed distinct transcriptome rewiring. We noted an enrichment of genes relevant to proliferation, migration, EMT, and invasion in acid-selected cells. Interestingly, PANC-1 cells are characterized by slower Ca2+ oscillations during short-term acid exposure compared to control cells and a tendency of ORAI1 downregulation at mRNA levels, while long-term acidosis and recovery to neutral pHe determine the recovery of fast Ca2+ oscillations and upregulation of ORAI1. In all our cell models, Ca2+ oscillations are SOCE-dependent, as ORAI1 blockade with Synta66 and siORAI1 results in impaired Ca2+ oscillations' initiation and maintenance. These data correlate with SOCE in PANC-1 cells, which is decreased during the short-term acid treatment, and increased in acid-selected cells with and without recovery to pHe 7.4. Finally, ORAI1-mediated Ca2+ entry might be involved in the activation of signaling cascades that lead to the increased migration and invasion of all the cell models exposed to acidic pHe, as Synta66 treatment and siORAI1 didn't affect control cells' invasion and migration.In conclusion, our findings show that acid-induced selection contributes to the acquisition of a more aggressive phenotype in PDAC cells, characterized by upregulation of SOCE, required for the generation of fast Ca2+ oscillations which may trigger Ca2+-dependent signaling pathways involved in PDAC progression

The rescue of selected adult plants and the renewing of explants constitute the greatest challenge to enable the clonal propagation of mate (Ilex paraguariensis St. Hil.). The objectives of this study were to identify and separate clones of the clonal garden by RAPD markers and to evaluate the effect of different doses of indol butyric acid (IBA) on rooting of cuttings and minicuttings of different clones of mate. Three experiments were carried out. The first, young leaves of ten clones had the DNA extracted for testing RAPD markers. The second, new shoots of stump of four clones were used to set up one bud cuttings with one half leaf. The third experiment, rooted cuttings of 20 year-old-trees were used to set up a clonal minigarden to collect minicuttings of one bud and one half leaf. All cuttings and minicuttings were treated with different doses of IBA. The RAPD technique is successful for clone separation and identification, being the primers OPP-03, OPP-06, OPP-15 and OPP-16 the most polymorphic. Single bud cuttings of new sprouts from stumps of some mate clones are capable of rooting, without the need of indole butyric acid. The clones FO10 and FO52 have different capability for rooting. Minicuttings of the clone FO10 root without indol butyric acid.
O resgate de plantas adultas selecionadas e o rejuvenescimento dos propágulos se constituem no maior desafio para viabilizar a clonagem massal da erva-mate (Ilex paraguariensis St. Hil.). Os objetivos deste trabalho foram identificar e separar clones que compõem o jardim clonal por meio de marcadores RAPD e avaliar o efeito de diferentes doses de ácido indol butírico (AIB) no enraizamento de estacas e miniestacas de diferentes clones de erva-mate. Para tanto foram realizados três experimentos. No primeiro, folhas jovens de dez clones tiveram o DNA extraído para testar marcadores RAPD. No segundo, brotações de cepas de quatro clones foram utilizadas para preparar estacas de gema única, com folha cortada pela metade. No terceiro experimento, estacas enraizadas de árvores com 20 anos de idade foram utilizadas para a formação de um minijardim clonal e produção de miniestacas com gema única e uma folha cortada pela metade. Para o enraizamento, tanto as estacas quanto as miniestacas foram tratadas com diferentes doses de AIB. A técnica RAPD é eficaz para a separação de clones de erva-mate, sendo que os iniciadores OPP-03, OPP-06, OPP-15 e OPP-16 foram os mais polimórficos. Estacas de gema única de brotações oriundas da decepa de alguns clones de erva-mate são competentes para o enraizamento, o que dispensa o uso de ácido indol butírico. Os clones FO10 e FO52 diferem quanto a competência ao enraizamento das miniestacas. O enraizamento de miniestacas do clone FO10 ocorre sem a aplicação de ácido indol butírico.

Aquesta tesi doctoral forma part d'una línia de recerca sobre la millora genètica de la qualitat de la carn en porcí. Un dels recents objectius inclòs en algunes línies paternes seleccionades per mercats de qualitat és aconseguir un nivell òptim de greix intramuscular i de composició en àcids grassos sense penalitzar el rendiment magre. Aquesta tesi està formada per quatre estudis realitzats en una línia pura de porcs Duroc destinats a la producció de càrnics d'alta qualitat. Els estudis es van dissenyar amb l'objectiu d'entendre millor la variabilitat genètica subjactent al contingut de greix i a la composició en àcids grassos i d’identificar marcadors potencials a la selecció. El primer estudi va examinar els paràmetres genètics de la ruta de l'àcid linoleic (C18:2) a l'àcid araquidònic (C20:4). En particular, es va demostrar que s’espera que la selecció pel valor absolut de C18:2 proporcioni una resposta a la selecció similar a la selecció pel greix intramuscular a greix dorsal restringit. Aquests resultats van conduir a investigar el gen de la desaturasa-2 dels àcids grassos (FADS2), com a gen candidat per a la ruta metabòlica de C18:2. Així, el segon estudi va avaluar els efectes d'una variant (el polimorfisme rs321384923 es va utilitzar com a marcador) al promotor del gen FADS2. Els resultats van demostrar que aquest polimorfisme afecta el perfil d'àcids grassos n-6 millorant l'eficiència de desaturació de C18:2 a C20:4. Addicionalment, es va avaluar l'associació dels gens de les perilipines (PLIN) i del gen de la proteína d’unió al guanilat 1 (GBP1) amb caràcters de creixement i de qualitat de la carn. Així, el tercer estudi va examinar els efectes de dos polimorfismes als gens PLIN1 i PLIN2, relacionats amb l'emmagatzematge i la mobilització de lípids. Els resultats indiquen que el polimorfisme rs333231747 a PLIN2 està associat al creixement primerenc i al pes magre. L'últim estudi va demostrar que GBP1 té dos senyals de poliadenilació actius i que el seu ús depèn del genotip rs80800372. Els porcs portadors de l'al·lel G, associat a una menor viremia després de la infecció pel virus reproductiu i respiratori porcí, van tenir transcripcions més llargues i una menor expressió gènica. En condicions no epidèmiques, l'al·lel G va augmentar el contingut de greix intramuscular, però va disminuir el pes magre. El contingut de C18:2 i els marcadors genètics investigats es poden utilitzar per dissenyar estratègies de selecció adequades per millorar la qualitat de la carn i el creixement magre.
Esta tesis doctoral es parte de una línea de investigación sobre la mejora genética de la calidad de la carne en porcino. Uno de los recientes objetivos incluido en algunas líneas paternas seleccionadas para mercados de calidad es lograr un nivel óptimo de grasa intramuscular y de composición en ácidos grasos sin penalizar el crecimiento magro. Esta tesis comprende cuatro estudios realizados en una línea pura de cerdos Duroc destinados a la producción de cárnicos de alta calidad. Los experimentos se diseñaron con el objetivo de comprender mejor la variabilidad genética subyacente en el contenido de grasa y la composición en ácidos grasos e identificar marcadores potenciales a la selección. El primer estudio examinó los parámetros genéticos de la ruta del ácido linoleico (C18:2) al ácido araquidónico (C20:4). En particular, se demostró que se espera que la selección del valor absoluto de C18:2 proporcione una respuesta similar a la selección por grasa intramuscular a grasa dorsal restringida. Estos hallazgos llevaron a investigar el gen de la desaturasa-2 de los ácidos grasos (FADS2), como gen candidato para la ruta del C18:2. Por lo tanto, el segundo estudio evaluó los efectos de una variante (el polimorfismo rs321384923 se usó como marcador) en el promotor del gen FADS2. Los resultados mostraron que este polimorfismo afecta el perfil de ácidos grasos n-6 al aumentar la eficiencia de desaturación de C18:2 a C20:4. Además, se evaluó la asociación de los genes de las perilipinas (PLIN) y el gen de la proteína de unión a guanilato 1 (GBP1) con caracteres de crecimiento y de calidad de la carne. De este modo, el tercer estudio examinó los efectos de dos polimorfismos en PLIN1 y PLIN2, relacionados con la deposición y la movilización de lípidos. Los resultados indicaron que el polimorfismo rs333231747 en PLIN2 se asocia con el crecimiento temprano y con el peso magro. El último estudio mostró que GBP1 tiene dos señales de poliadenilación activas y que su uso depende del genotipo rs80800372. Los cerdos portadores del alelo G, asociado con una menor viremia después de la infección por el virus reproductivo y respiratorio porcino, tenían transcritos más largos y una menor expresión génica. En condiciones no epidémicas, el alelo G aumentó el contenido de grasa intramuscular pero disminuyó el peso magro. El contenido de C18:2 y los marcadores genéticos investigados se pueden usar para diseñar estrategias de selección adecuadas para mejorar la calidad de la carne y el crecimiento magro.
This PhD dissertation is part of a research line on the genetic improvement of pork quality. One of the latest goals to be included in some sire lines selected for premium markets is to achieve an optimum level of intramuscular fat and fatty acid composition without penalizing lean growth performance. This thesis is comprised of four studies conducted on a purebred Duroc line used for producing high-quality meat products. The experiments were designed with the aim to better understand the genetic variability underlying fat content and fatty acid compostion and to identify potential markers for breeding. The first study examined the genetic parameters of the linoleic acid (C18:2) to arachidonic acid (C20:4) pathway. In particular, it was showed that selection for the absolute value of C18:2 is expected to deliver a similar response outcome as selection for intramuscular fat at restrained backfat thickness. These findings led to investigate the fatty acid desaturase-2 (FADS2) gene, as a candidate gene for C18:2 metabolism route. Thus, the second study evaluated the effects of a variant (rs321384923 was used as a tag single nucleotide polymorphism) in the promoter of the FADS2 gene. Results showed that this polymorphism affects the n-6 fatty acid profile by enhancing the desaturation efficiency of C18:2 to C20:4. Additionally, the association of perilipin (PLIN) genes and guanylate-binding protein-1 (GBP1) gene with growth and meat quality traits was assessed. Thus, the third study examined the effects of two polymorphisms in PLIN1 and PLIN2, which have been related to lipid storage and mobilization. Results indicated that the rs333231747 polymorphism on PLIN2 is associated to early growth and lean weight. The last study showed that GBP1 has two active polyadenylation signals and that their usage depends on the rs80800372 genotype. The pigs carrying the G allele, which has been associated with lower viraemia after porcine reproductive and respiratory virus infection, had longer transcripts and lower gene expression. In non-epidemic conditions, the G allele increased intramuscular fat content but decreased lean weight. Linoleic acid content and the investigated genetic markers can be used to design appropriate selection strategies to enhance meat quality and lean growth.

The methylotrophic yeast Pichia pastoris has become an efficient expression system for heterologous protein production. Different methods have been studied to enhance cell growth as well as the production of products of interest. Two of the major strategies for improving the product or biomass yields are optimizing bioprocess controls and cultivation conditions. In this work, the characteristics of this yeast system and of its different promoters are discussed, and the effect of operational strategies on cell growth and recombinant protein expression is also studied. The effect of different feeding strategies were studied and optimized for pGAP (glyceraldehyde-3-phosphate dehydrogenase)-regulated phytase production in P. pastoris. Alternative carbon sources were screened and the feasibility of using citric acid as a carbon source for recombinant protein production was also investigated. The effects of parameters such as the carbon source concentration and culture pH were studied using shake-flasks, and the effect of different feeding profiles on bioreactor performance was also investigated. Three feeding strategies, Stepwise feeding, Exponential feeding and DO-stat feeding were tested and DO-stat was found to be more efficient and led to a high phytase activity. A modified DO-stat method was investigated to overcome the oxygen limited condition in the standard DO-stat method. For the carbon source, citric acid showed promise in improving phytase expression. Further experiments in bioreactors performed with the presence of certain amount of citric acid showed that less glycerol could be used to achieve the same level of phytase activity.

Drosophila suzukii Matsumura è un parassita alieno invasivo dei frutti di bosco. Si è diffuso nelle Americhe e in Europa dalla fine degli anni 2000 con effetti economici negativi sui frutti di bosco. Questo parassita ha ricevuto un'attenzione speciale perché è una delle specie invasive di maggior successo del genere Drosophila, che utilizza diverse risorse alimentari e mostra un adattamento ecologico alle condizioni climatiche variabili. Il monitoraggio, come parte della gestione dei parassiti, è la chiave per controllare questo parassita. Quindi, è importante utilizzare le migliori esche e trappole disponibili per ottenere un monitoraggio affidabile. Al momento, gli attuali prodotti di fermentazione e le esche sintetiche non sono adeguatamente selettivi ed efficaci per il monitoraggio. Inoltre, non sono stati ancora sviluppati strumenti di monitoraggio efficienti. È necessario migliorare l'attrattiva delle esche disponibili in commercio che sono attualmente utilizzate per monitorare questo parassita e sviluppare un nuovo sistema di cattura per una gestione efficiente dei parassiti. Il presente lavoro mira a sviluppare il prototipo di un'esca altamente attraente per lo sviluppo di un nuovo sistema di cattura destinato al monitoraggio dei parassiti. Pertanto, studi olfattivi e di intrappolamento che includevano una serie di saggi biologici comportamentali sono stati condotti in condizioni di laboratorio e semi-campo. L'olfattometro a due scelte è stato utilizzato per valutare la risposta comportamentale delle mosche femmine verso frutti ospiti coltivati e non raccolti e ceppi di batteri lattici associati alla superficie dei frutti e al microbiota intestinale di D. suzukii. Successivamente, sono stati utilizzati saggi in gabbia per valutare la risposta comportamentale delle mosche verso composti volatili sintetici, associati ai frutti ospiti e alla fermentazione microbica, che provocano una risposta comportamentale nelle mosche adulte. Infine, una miscela sintetica è stata valutata per l'attrazione delle mosche e utilizzata nella progettazione del prototipo di un'esca altamente attraente per lo sviluppo di un nuovo sistema di cattura destinato al monitoraggio dei parassiti. I nostri risultati hanno mostrato che le mosche erano significativamente più attratte dal raccolto e dai frutti ospiti non raccolti più del controllo nel saggio biologico olfattometrico. I frutti di mora erano i frutti più attraenti. Inoltre, le mosche hanno mostrato una risposta positiva ai volatili emessi dai ceppi di batteri lattici inoculati nell'esca alimentare Droskidrink. I ceppi più attraenti, Lactobacillus kunkeei 84 e Oenococcus oeni LS, hanno mostrato un'attrazione significativa per le femmine quando combinati e inoculati in esche alimentari. Nei saggi in gabbia, l'attrattiva di un'esca commerciale, Dros'Attract, è stata migliorata utilizzando una miscela di sostanze volatili di origine vegetale (geraniolo) e volatili della fermentazione microbica (dimetil solfuro). Pertanto, è stato sviluppato un prototipo di un'esca più attraente che comprende l'esca commerciale ed entrambi i composti. Alla fine, è stato sviluppato un nuovo sistema di cattura che comprende un'esca Dros'Attract combinata con composti volatili e Droso-Trap specializzato. I dati ottenuti forniscono conoscenze sull'importanza di combinare i volatili della frutta ospite con i volatili dei microbi per aumentare l'attrattiva delle esche attraenti esistenti. Inoltre, aumenta la nostra comprensione delle risposte olfattive di D. suzukii ai composti volatili sintetici come fonti di attrattivi che possono aiutare nello sviluppo e nell'adozione di strumenti basati sul comportamento per il monitoraggio dei parassiti e strategie di gestione ecocompatibili.
Drosophila suzukii Matsumura is an alien invasive pest of soft fruits. It has spread across the Americas and Europe since the late 2000s with adverse economic effects on berries. This pest received special attention because it is one of the most successful invasive species of the genus Drosophila, utilizing different food resources and showing ecological adaptation to variable climatic conditions. Monitoring, as a component of pest management, is the key to control this pest. Hence, it is important to use the best lure and trap available to obtain reliable monitoring. At present, current fermentation products and synthetic lures are not adequately selective and effective for monitoring. Moreover, no efficient monitoring tools have been developed yet. There is a need to improve the attractiveness of commercially available lures that are currently used to monitor this pest and developing a new trapping system for efficient pest management. The present work aims to develop the prototype of a highly attractive lure for developing a new trapping system intended for pest monitoring. Therefore, olfactory and trapping studies which included a series of behavioral bioassays were conducted under laboratory and semi-field conditions. Two- choice olfactometer was used to evaluate the behavioral response of female flies towards crop and non-crop host fruits and lactic acid bacteria strains associated with fruits surface and D. suzukii gut microbiota. Subsequently, cage assays were used to evaluate the behavioral response of flies towards synthetic volatile compounds, associated with host fruits and microbial fermentation, that elicit a behavioral response in adult flies. Lastly, a synthetic blend was evaluated for fly’s attraction and used in the design of the prototype of a highly attractive lure for developing a new trapping system intended for pest monitoring. Our results showed that flies were significantly more attracted to crop and non-crop host fruits more than control in olfactometer bioassay. Blackberry fruits were the most attractive fruits. Moreover, flies exhibited a positive response to volatiles emitted by lactic acid bacteria strains inoculated into Droskidrink food bait. The most attractive strains, Lactobacillus kunkeei 84 and Oenococcus oeni LS, showed a significant attraction to females when combined and inoculated into food bait. In cage assays, the attractiveness of a commercial lure, Dros’Attract, was improved using a blend of plant-based volatiles (geraniol) and microbial fermentation volatiles (dimethyl sulfide). Therefore, a prototype of a more attractive lure was developed comprising the commercial lure and both compounds. Ultimately, a new trapping system was developed which is comprised of Dros’Attract lure combined with volatile compounds and specialized Droso-Trap. The obtained data provide knowledge on the importance of combining host fruit volatiles with microbes’ volatiles to increase the attractiveness of existing attractive lures. Also, it increases our understanding of D. suzukii olfactory responses to synthetic volatile compounds as sources of attractants which may help in the development and adoption of behaviourally based tools for pest monitoring and eco-friendly management strategies.

Thesis (MSc Food Sc)--Stellenbosch University, 2000.
ENGLISH ABSTRACT: Maas is a traditional fermented milk drink of the indigenous people of Southern Africa and can thus be used to uplift the nutritional status of the South African population, especially for the lower income groups. Furthermore, the problem of lactose intolerance among the Black population can also be addressed by the consumption of Maas. The objective of this study was to screen mesophylic lactic acid bacterial strains (25 in total) from the University of Stellenbosch Food Science Culture Collection for suitable metabolite production and then to produce traditional Maas with a starter culture combination that produces a distinctive acid and traditional flavour. The representative 25 single lactic acid starter strains were identified as Lactococcus lactis subsp. leetis biovar diacetylactis (12 strains), L. leetis subsp. leetis (four strains) and L. leetis subsp. cremoris (nine strains). These strains were inoculated into pasteurised full cream milk and activated for 8 h at 22°C. Pasteurised full cream milk was then inoculated with each of the activated starter strains, incubated at 22°C for 16 h and assessed for acid production abilities (pH = 4.6) under controlled time-temperature conditions. The results of this study showed that nine of the single strains, L. lactis subsp. leetis biovar diacetylactis (S1, S2, S3 and S5), L. teetis subsp. lactis (S13, S15 and S16) and two L. leetis subsp. cremoris strains (S17 and S22), produced sufficient acid, rendering them suitable for the use as starters in the production of traditional Maas. A pH range of 4.3 - 5.1 was reached by the nine single strains after 16 h at 22°C. Two-strain starter combinations were then formed by combining the most suitable single L. leetis subsp. leetis biovar diacetylactis, L. lactis subsp. lactis and L. lactis subsp. cremoris strains, respectively. From the data, it was concluded that acceptable Maas could be produced with four two-strain combinations (S3S 17, S3S22, S5S17 and S5S22). This selection was again based on suitable acid and metabolite production, as well as on sensory evaluation of the final product. These four two-strain combinations produced sufficient acid to reach a pH in the 4.6 - 4.8 range, and showed a high metabolite concentration for the most suitable compounds and formed a thick, smooth and creamy body texture after 16 h at 22°C. Three-strain combinations formed between the two-strain starter combinations and L. leetis subsp. teetis strains (813, 815 and 816), were also evaluated. With these combinations a lack of a pronounced Maas flavour was found. Thus, it was decided to add aroma producing strains of the species Leuconostoc mesenteroides subsp. dextranicum (strain L1) and L. mesenteroides subsp. citrovorum (strain L2) to the three-strain combinations. Four culture combinations (A, B, C and D) were then formed by combining the selected Leuconostoc strains (L1 and L2) with the most suitable Lactococcus strains (83,817,813 and 822). These combinations produced sufficient acid to reach the pH 4.5 - 4.6 range after 14 h at 22°C. Acetaldehyde was the major flavour metabolite formed in the Maas made with these four combinations, with concentrations ranging between 26.6 - 89.3 mg.l ̄ ¹, while other flavour metabolites (ethanol, acetone, diacetyl and 2-butanone) were present at lower concentrations. It was found that three of the four culture combinations (A, C and D) were characterised by a superior, but delicate flavour and a typical characteristic Maas body texture. Fruit flavoured Maas was subsequently prepared with the three most suitable culture combinations (A, C and D) using 11 flavours and a sensory evaluation performed. The statistically evaluated data showed that the appearance, smoothness, flavour intensity, sweetness and overall acceptability were influenced by the type of fruit flavour and the culture combination. Fruit flavour 4 (banana) was the most preferred flavour. The sensory panellists also indicated that the culture combination C gave the best overall acceptability over a three week study period. Data on the shelf-life study of natural unflavoured Maas, prepared with the three culture combinations (A, C and D), showed that the Maas still had an acceptable appearance, taste and good microbiological quality after 15 d at refrigerated temperatures.
AFRIKAANSE OPSOMMING: Maas is 'n tradisionele gefermenteerde melkdrankie onder die inheemse bevolking van Suid-Afrika en kan gebruik word om die voedingstatus van die Suid-Afrikaanse bevolking te verhoog, veral vir die laer inkomste groepe. Bowendien, kan die probleem van laktose intoleransie onder die Swart gemeenskap ook aangespreek word deur die verbruik van Maas. Die doel van hierdie studie was om enkelstam mesofiliese melksuur bakterieë (25 in totaal) van die Universiteit van Stellenbosch Voedselwetenskap Kultuur Versameling te ondersoek vir geskikte metaboliet produksie en tradisionele Maas met 'n kenmerkende suurheid en tradisionele geur met 'n geskikte kultuur kombinasie te produseer. Die toonaangewende 25 enkelstamme is Lactococcus lactis subsp. leetis biovar diacetylactis (12 stamme), L. lactis subsp. lactis (vier stamme) en L. lactis subsp. cremoris (nege stamme). Hierdie stamme was in gepasteuriseerde volroom melk geïnokuleer en geaktiveer vir 8 h teen 22°C. 'n Inokulum van die onderskeie geaktiveerde stamme is hierna in gepasteuriseerde volroom melk geplaas, vir 16 h teen 22°C geïnkubeer en hul vermoë om suur te produseer (pH = 4.6) onder beheerde tyd-temperatuur kondisies is bepaal. Die resultaat van die studie het aangedui dat nege enkelstamme, naamlik L. leetis subsp. lactis biovar diacetylactis (S1, S2, S3 en S5), L. lactis subsp. leetis (S13, S15 en S16) en twee L. leetis subsp. cremoris (S 17 en S22), geskikte suurheidsvlakke vir die produksie van Maas bereik het. 'n pH vlak van 4.3 - 5.1 is na 16 h teen 22°C deur hierdie nege enkelstamme bereik. Twee-stam kombinasies is onderskeidelik gevorm tussen die geskikte enkel L. lactis subsp lactis biovar diacetylactis, L. lactis subsp. lactis en L. lactis subsp. cremoris stamme. Die gevolgtrekking gemaak uit die data, is dat aanvaarbare Maas voorberei kan word met vier van die twee-stam kombinasies (S3S17, S3S22, S5S17 en S5S22) op grond van suurvorming, metaboliet produksie en sensoriese evaluasie. Hierdie vier kombinasies het genoegsame suur geproduseer om 'n pH vlak van 4.6 - 4.8 bereik, hoë metaboliet konsentrasies geproduseer en 'n dik, gladde en romerige tekstuur aangeneem na 16 h teen 22°C. Drie-stam kombinasies is gevorm tussen die onderskeie twee-stam kombinasies en L. lactis subsp. lactis stamme (813,815 en 816) en ook geëvalueer. Die tekort aan 'n skerp Maas geur in die drie-stam kombinasies het daartoe gelei dat Leuconostoc mesenteroides subsp. dextranicum (stam L1) en L. mesenteroides subsp. citrovorum (stam L2) bygevoeg is. Vier kultuur kombinasies (A, B, C en D) is gevorm deur die geselekteerde Leuconostoc stamme (L1 en L2) te kombineer met die mees gepaste Lactococcus stamme (83, 817, 813 en 822). Hierdie kombinasies het genoegsame suur geproduseer wat 'n pH vlak van 4.5 - 4.6 na 14 h teen 22°C bereik het. In die Maas wat met bovermelde kombinasies gemaak is, was die asetaldehied die mees geproduseerde geur metaboliet teen konsentrasies van 26.6 - 89.3 mg.l ̄ ¹. Ander geur metaboliete (etanol, asetoon, diasetiel, 2-butanoon) is in laer konsentrasies geproduseer. Daar is gevind dat drie uit die vier kultuur kombinasies (A, C en D) 'n superieur, delikate geur wat 'n tipies karakteristiek van die Maas gehad het. Vrugte gegeurde Maas geproduseer met die drie kultuur kombinasies (A, C en D) deur 11 geursels te gebruik, is sensories geëvalueer. Die statistiese geëvalueerde data het getoon dat die voorkoms, gladheid, geur intensiteit, soetheid en die algehele aanvaarbaarheid beïnvloed is deur die tipe vrugte geursels en die kultuur kombinasies. Die vrugte geursel 4 (piesang) het voorkeur geniet. Die sensoriese paneellede het ook aangedui dat kultuur kombinasie C die algehele mees aanvaarbare Maas geproduseer het oor die studie periode van drie weke. Data van die rakleeftyd van die natuurlike ongegeurde Maas wat geproduseer is met die drie kultuur kombinasies (A, C en D) het aangedui dat die Maas na 15 d by yskas temperatuur steeds 'n aanvaarbare voorkoms, smaak en goeie mikrobiologiese kwaliteit gehad het.

The use of health-promoting lactic acid bacteria (LAB) strains as starter or adjunct cultures for dairy productions could facilitate the in situ bio-synthesis of bioactive molecules during the fermentation process, increasing the interest towards dairy products as multifunctional foods. Currently, there is much research about genotypic and technological characterization of raw milk cheeses microbiota, which is rich in biodiversity and could be exploited for improving the sensory attributes and add healthy benefits to the cheese. Traditional Mountain (TM) cheese is made from raw cow’s milk and spontaneously fermented in small farms called “Malga” located in the alpine areas of Trentino region. For the first time, the microbial population of TM-cheese has been characterized in order to select cocci and non-starter LAB suitable for developing new starter or adjunct cultures, respectively. Samples (n = 120) of milk, curd and cheese at different ripening times (24 hours, 1 month and 7 months) were enumerated in selective culture media. Mesophilic and thermophilic cocci dominated during the first 24 hours following production, and mesophilic lactobacilli were dominant at the end of ripening. Six hundred and forty colonies were isolated from curd and cheese 24 hours following production, and 95 more colonies were isolated from cheese after 7 months of ripening. All isolates were genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. Cocci clustered in 231 biotypes belonging to 16 different species, and non-starter LAB (NSLAB) clustered in 70 biotypes belonging to 13 different species. Lactococcus lactis, Streptococcus thermophilus and Enterococcus faecalis were dominant in curd and 24h-cheese; Pediococcus pentosaceus and Lactobacillus paracasei were the main species at the end of ripening. The phenotypic, technological and health-promoting activities of all strains were investigated. In particular, lactococci, streptococci and enterococci were tested for their acidification and proteolytic activity, ability to growth at not optimal temperatures, acetoin production, development of olfactory flavour notes, autolysis rate and ability to inhibit the growth of coliforms. Forty percent of enterococci showed the ability to inhibit raw milk resident coliforms in vitro, but they were excluded as possible starters, owing to the presence of associated risk factors. Among lactococci and streptococci, 4 Lc. lactis subsp. lactis and 2 Sc. thermophilus were fast acidifiers, produced pleasant flavours, and were subjected to the freezedrying stability test. Lc. lactis subsp. lactis 68 and Sc. thermophilus 93 showed the best properties and might be appropriate for cheese production. NSLAB strains were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase activity (AP) and biogenic amines production. Concerning the health-promoting properties, the bile salts hydrolysis (BSH) activity was tested qualitatively, the conjugated linoleic acid (CLA) production was measured spectrophotometrically, and the γ-aminobutyric acid (GABA) production was quantified by UHPLC (Ultra High Performance Liquid Cromatography). Lb. paracasei isolates resulted to be well adapted to the Malga environment and showed the highest AP activity and acetoin production. Some strains harbored very interesting health-promoting properties and produced bioactive substances. In particular, Lb. rhamnosus BT68, Lb. paracasei BT18, BT25, BT31, Pc. pentosaceus BT3, BT13, BT51 produced between 70 and 130 mg/mL of total CLA in vitro. Lb. brevis BT66 converted L-glutamate to a high concentration of GABA (129 ± 8.6 mg/L) and showed BSH activity. These first results revealed that TM-cheese is a reservoir of a high microbial diversity, and the resident LAB could be exploited not only for the applicability in dairy production but also for their health-promoting properties. Lc. lactis subsp. lactis 68 and Sc. thermophilus 93, which showed to be the best performing strains, were tested as starter and adjunct cultures, for the production of 9 experimental TM-cheese wheels in a Malga-farm, respectively. Three control (CTRL) cheeses were produced according to the tradition and any starter or adjunct culture was not added; three starter (STR) and three commercial starter (CMS) cheeses were produced inoculating the vat milk with both selected strains and a commercial Sc. thermophilus strain, respectively. After 24 hours, 1 month and 7 months of ripening the microbial content of all experimental cheeses was investigated. Mesophilic cocci and lactobacilli dominated in cheese samples after 24 hours and 1 month of ripening, while cocci dominated in full-ripened cheese. The total genomic DNA was extracted, and a fragment of the V1-V3 region was amplified and pyrosequenced. Lactococci and streptococci were the most abundant species in CTRL and STR cheese, and Lc. lactis subsp. lactis 68 affected the proliferation of the (raw milk) indigenous Lc. lactis subsp. cremoris during the early fermentation. Moreover, the commercial Sc. thermophilus showed to be dominant towards Lc. lactis subsp. lactis and cremoris naturally present in raw milk and to be responsible in decreasing the abundance of Lactobacillus subp. and Enterococcus sp. The survival of TM-cheese microbiota in vitro was investigated under simulated human gastro-intestinal (GI) conditions. The 9 full ripened experimental TM-cheeses were subjected to a model system that simulates digestive processes in the mouth, stomach and small intestine, comprising sequential incubation in human gastric and duodenal juices. Bacterial counts were performed before and after the simulation: total bacterial count and thermophilic cocci significantly decreased after the simulated digestion. Thirty-six lactobacilli were isolated from cheese after digestion: among them 1 Lb. paracasei, 1 Lb. parabuchneri and 1 Lb. fermentum were tested for their survival after GI transit. Lc. lactis subsp. lactis 68 and Lb. parabuchneri D34 strains were used to ferment whole milk and digested. The load of Lb. parabuchneri D34 decreased by about one log more when grown as pure culture than fermented milk after simulated digestion, suggesting that Lb. parabuchneri D34 had in itself the ability to survive digestion, but the fat content and the cheese structure might protect LAB during the GI transit. Furthermore, our interest towards the GABA producing strains lead us to test the ability of Lb. brevis BT66 to produce GABA in situ during cheese production, through the decarboxylation of glutamate. Twenty experimental micro-cheeses were produced using a commercial starter strain (107 CFU/mL) and Lb. brevis BT66 as adjunct culture. Four different concentrations (102 , 103, 104, 105 CFU/mL) of Lb. brevis BT66 were tested in quadruplicate. In order to follow the microbial evolution, samples of milk, curd and cheese after 20 days of ripening were enumerated in selective media. The control and experimental samples showed a similar trend, suggesting that both milk-resident and starter strains grew during ripening. However, the load of mesophilic lactobacilli in all experimental curd samples was higher than the control ones. The concentration of GABA and glutamic acid in cheese samples after 20 days of ripening was quantified by UHPLC-HQOMS. The amino acidic profiles showed that while the concentration of Lb. brevis BT66 in milk increased, the amount of both glutamic acid (from 324 ± 37 to 202 ± 32 mg/kg) and GABA (from 154 ± 31 to 91 ± 20 mg/kg) significantly decreased in cheese. These results suggested that the experimental strain converted glutamic acid to GABA, but that GABA may have subsequently been converted to succinate by GABA transaminases. The non-protein amino acid GABA has been reported to impact on brain function through the gut:brain axis system, to harbor an anti-obesity and antidiabetogenic effect, to regulate the immune system, the inflammation process and the energy metabolism in mammals including induction of hypotension, diuretic and tranquilizer effects, stimulation of immune cells. Owing to its ability to produce high concentrations of GABA and its BSH activity in vitro, Lb. brevis BT66 was selected to be tested in vivo in mice suffering obesity-associated type-2-diabetes. Another Lb. brevis (strain DPC6108), isolated from the human GI tract and harboring the same properties, was simultaneously investigated. The corresponding rifampicin resistant mutants (rif) were generated; their genotypic profile was obtained by RAPD-PCR and PFGE (Pulsed-Field Gel Electrophoresis) and was identical to the native strain. The conversion rates of monosodium glutamate to GABA were investigated by nextgeneration amino acid analysis: Lb. brevis BT66rif produced 840.5 ± 266 µg/mL of GABA with about 73% of bioconversion and Lb. brevis DPC6108rif produced 1,218.0 ± 393.2 µg/mL with about 87% of bioconversion. The BSH activity was positive to both qualitative and quantitative assays and the results were similar in both native and mutant strains. The rifampicin resistant strains were freeze-dried and tested for their stability at room temperature, +4 and -20 °C. Both spectrophotometer and plate count methods revealed that freeze-dried strains survived at room temperature during 24 hours after suspending in sterile water. The stability of freezedried strains at +4 and -20 °C was investigated enumerating the viable cells in selective medium during 10 weeks and any significant load reduction was not detected in the first 4 weeks following freeze-drying. Both pharmabiotic-producing Lb. brevis BT66rif and DPC6108rif were resistant to freeze-drying, survived transit through mouse GI tract (as proven by a pilot study), and their therapeutic efficiency is being assessed in vivo to treat metabolic obesity and type-2-diabetes

The emerging evidence of preferential accumulation and long residence time of proteinaceous compounds in soil are counter to the traditional view that their structure is readily broken down through microbial activity. The shift in thinking of their residence time is, however, heavily influenced by physical and chemical protections in soil, representing an important change for understanding global biogeochemical carbon and nitrogen cycling. We investigated the accumulation patterns of proteinogenic amino acids for a long term (thousands of years) related to their sources and sinks. We found clear patterns of change in the amino acids in a 4000 year-chronosequence adjacent to Lake Michigan, USA (Michigan chronosequence) and they were tightly related to the shifts in their biological sources, namely aboveground vegetative community (r2=0.66, p<0.0001) and belowground microbial community (r2=0.71, p<0.0001). Results also showed great variations of approximately 49% between seasons (summer and winter). Moreover, seasonal dynamic patterns (22% variations) of the amino acids in soil mineral associated fraction were rather counter to the conceptual view that it represents a slow soil organic pool with long residence times. The amino acids enriched in the mineral associated fraction, (e.g., positively charged, aromatic, and sulfur containing amino acids), tended to preferentially accumulate in whole soil pool during the 4000 years of ecosystem development. Their interaction with soil minerals, therefore, may play a critical role in the long-term sink and selective accumulation of proteinaceous compounds with some degree of the displacement. This was further confirmed by another chronosequence system near Haast River, New Zealand, which is geologically separated and climatically- and ecologically- different from the Michigan chronosequence. Common trends between two chronosequences suggested that either polar interactions or redox reactions may be relatively more important in the mineral interaction of amino acids than non-polar interactions. The consistency of results at two disparate locations in the southern and northern hemispheres is strong evidence that the processes of pedogenesis and ecosystem development are parsimonious and predictable. Our research demonstrated fundamental understanding of behavior of proteinaceous compounds at the molecular species level, and further provided their partitioning mechanisms associated with soil components.
Ph. D.

Cette etude a contribue a creer une collection de streptocoques lactiques: st. Lactis subsp. Cremoris, st. Lactis subsp. Lactis, st. Lactis subsp. Diacetylactis (bacteries mesophiles) et st. Thermophilus. Onze souches de st. Cremoris appartenant a la collection existante ont ete selectionnees parmi 67 bacteries mesophiles apres des tests de lysotypie (sur 59 phages) et de lysogenie (par action de la mitomycine). Les memes tests ont ete realises sur 30 st. Thermophilus et 19 serums phagiques. Mais ces souches semblent posseder des systemes de restriction-modification puissants et sont donc moins sensibles aux phages. Le peu de phages recoltes n'ont pas permis d'etablir des groupes de lysotypie significatifs et les souches testees ne sont pas lysogenes. Afin d'ameliorer la collection, 554 bacteries mesophiles ont ete isolees; identifiees et leur pouvoir acidifiant determine

碩士
國立中興大學
食品科學系
90
Probiotics are traditionally defined as viable microorganisms that have positive health effects, and lactic acid bacteria are the most common used probiotics. The essential characteristics for lactic acid bacteria as probiotics are: (1) they should be isolated from human origin, (2) they are able to adhere to the human intestinal cells, (3) they are acid and bile stable, (4) they should be safe as used for food and clinical purpose, (5) they should be clinically validated and documented for health effects. The strains of lactic acid bacteria (LAB) used in this study were isolated from infant stool samples. When they were evaluated for their probiotic properties, it was found that four LAB strains (MRS12, TM39, RS15, RS17) were able to adhere to the Int407, Caco-2, Hela and TSGH cell lines. Three of these four LAB strains (TM39, RS15, RS17) were found to be tolerant to pH 2.0 and resistant to 0.3﹪bile salts. Pulse-field gel electrophoresis (PFGE) patterns for these three strains, ie, TM39, RS15, RS17, showed that these three strains isolated from infant stool samples belong to the same strain. These strains were identified as Enterococcus spp. with API 20 STREP system. On the part of the effect on Helicobacter pylori (H.p), it was found that the culture supernatant of some LAB strains isolated from variable origins express obviously inhibitory effect to the growth of H. pylori. For animal study, the BALB/c mice were used. Four-week-old mice were infected with H. pylori and then, they were oral-fed with different lactobacillus strains. Afterward, they were sacrificed for examination. Their stomachs were removed, and one half of the stomachs were used to count the bacteria number and to assay the urease activity. The other half were cut to several sections and stained. The results showed that strain TM39 do exert antagonistic activity against H. pylori in vitro and in vivo. On the other hand, the primary safety and the possibility for application to dairy products were tested. Results show that to this strains, ie, TM39, might be acceptable as food safety was considered. It also has the potential and reasonable shelf-life be used in fermented milk processing.

碩士
國立澎湖科技大學
食品科學系碩士班
107
127 strains of lactic acid bacteria with phytase activity were screened from 614 strains of lactic acid bacteria isolated from the algae in Penghu keeping in our laboratory. Among them, 7 strains of the highest activity lactic acid bacteria were identified as 3 strains of Lactobacillus plantarum (5-1, 1.2U/mL; 20-4-1, 1.7U/mL; 62-3, 1.72U/mL) , 1 strain of Lactobacillus brevis (12-5, 1.88 U/mL), 1 strain of Lactobacillus rhamnosus (128-1, 1.24 U/mL) and 2 strains of Lactobacillus fermentum (62-6, 0.91 U/mL; 366-1, 1.37 U/mL). After fermenting with phytase producing LAB, phytic acid in brown rice pulp, shelled wheat pulp, and whole soy milk medium can be degraded effectively. The pH was significantly reduced to 5 after 12 hours of fermentation. The number of bacteria in the fermentation for 24 hours reached a maximum. The phytic acid degradation gradually slowed down after 48 hours of fermentation. Comparing the number of viable bacteria and the amount of phytic acid degradation， Lactobacillus plantarum 20-4-1 is the best strain which can be used in grain fermentation, the phytic acid residue after fermentation for 72 hours were 13.76 mg/g (36%) of whole soybeans, 2.56 mg/g of brown rice (28%), and 5.63 mg / g (50.0%) of the shelled wheat.

碩士
國立高雄第一科技大學
環境與安全衛生工程系碩士班
106
In Taiwan, microbrewery and ethanol-to-acid business commonly use forgein microbial species for fermentation. The climate effects result in the control of fermentation process using the forgein species rather strigent. Moreover, the limited use of the native species in fermentation constrains their potential uses in fermentation business. Thus, in this study native species for fermentation were to be inoculated and preserved, and there efficiency in fermentation checked. The objectives include: (1) to select the most highly efficient ethanol-producing and ethanol-oxidizing microbial species, (2) to study the important factors in fermenting sugar and ethanol, and (3) to test the feasible method in preserving the selected microbial species. During this study, grape liquor was used to produce ethanol and to inoculate the ethanol-producing species; and a commercial red wine and Taiwan beer was used for ethanol to acid conversion and to inoculate the ethanol-oxidizing microbial species. Gas chromatograph with a flame ionization detector (GC-FID) was used to monitor the ethanol and acid content, along with pH and microbial changes. The results demonstrated that at pH=6, Temperature= 25 oC, sugar of 20 grams, initial microbial counts at 8.5×104 CFU/mL, total volume of 300 mL and no mixing conditions, the most highest ethanol production concluded. Three fruits, pineapple, banana and grape, were fermented with the obtained microbial species, and the grape run had the highest ethanol production of 2.7% on day 12. The ethanol/sugar ratio was 0.45, which was lower than the literature value of 0.85-1.37. Also after 12 days the beer-incubated ethanol oxidizing microbes produced 2-3% acid at pH=6 and temperature= 25 oC. The acid/ethanol (beer) was 1.53, which was higher than literature value of 0.63-0.69. Finally, the results of using wet or dried flour for the preservation of the obtained microbial species revealed that the initial microbial counts of wet and dried flours were 5.17×106 and 5.95×105 CFU/g, respectively, which were dropped to 1.69×106 and 1.49×105 CFU/g after 14 days, respectively. About 40 and 10% ethanol production observed if the microbes restored from wet and dried flour, respectively. Similarly, the ethanol-oxidizing bugs in the wet and dried flour were 2.17×106及1.34×106 CFU/g, and were dropped to 3.67×105及9.06×104 CFU/g after 14 days. The lost of microbes in the preserving flour deserves more attention when performing the microbial preservative operation. Key words: ethanol-producing species, sugar to ethanol, ethanol-oxidizing species, ethanol to acid, preservation of microbial species

碩士
東海大學
畜產學系
92
This study aimed at selecting homofermentative lactic acid strains for lactic acid production from whey fermentation. According to this study results, Lb. rhamnosus THSH-1 performed the better results than other strains in lactic acid productivity and lactose utilization（P＜0.05）. Optimisation of temperature for lactic acid production by Lb. rhamnosus THSH-1 was 42℃. pH adjustment and agitation culture provided beneficial effects on lactic acid production during the fermentation process（P＜0.05）. Inoculum of 10﹪Lb. rhamnosus THSH-1 was the superior to 1﹪inoculum on production of lactic acid（P＜0.05）. The addition of nutrients presented a positive effect on the lactic acid production. Among the different nitrogen sources supplemented to whey, yeast extract performed the best lactic acid productivity（P＜0.05）. At 3﹪w/v yeast extract supplementation, the lactic acid productivity was the best（P＜0.05）. The addition of pantothenic acid in whey permeate supplemented with soymilk presented a significant beneficial affect with lactic acid production（P＜0.05）.

碩士
國立嘉義大學
生化科技學系研究所
106
Inoculation of probiotics is amongst the ways to maintain and increase nutrition and bioactivity of fruits and vegetables. This study was aimed to develop the fruit and vegetable ferments with higher bioactivity for promoting product market competition. Acid-fast lactic acid bacteria were selected from the aging broths, which contain various fruit and vegetable ferments in Taiwan Enzyme Village Co., Ltd., by plating on MRS agars containing NaN3 and cycloheximide following anaerobic incubation, Gram stain test and inoculation in pineapple or papaya ferments supplemented without or with yuan zhi at 30°C incubation. After 16S rDNA sequencing, physiological and biochemical analyses, one of the best performed isolates was identified and renamed CWP217. In acid tolerance test, CWP217 showed the best growth in pH 4.2 MRS broth at 37°C for 36 and 144 hr incubation (OD600 1.46 and 1.72) than LAB a (La; OD600 1.04 and 1.35), which was the same species with CWP217 and purchased from FIRDI, and Lactobacillus rhamnosus GG (LGG; OD600 0.73 and 1.15). CWP217 was further inoculated in 5 ferments supplemented with yuan zhi at 30°C. After 14 days incubation, CWP217 showed the best growth in papaya fermentation. We further inoculated CWP217, La and LGG in bean sprouts ferments without or with yuan zhi to result B and BZ referments, respectively. Only the CWP217 inoculation showed the fermented effects on continuous growth (1.6 × 108 and 2.5 × 108 CFU/mL in 14-day referments) and continuous decrease of reducing sugars (56% and 52% after 14 days fermentation). The CWP217-inoculated B referment also showed significant 51.6% increased total flavonoid content after 6 days fermentation. Finally, in the anti-inflammatory assay on NO inhibition in LPS-induced RAW264.7 macrophages, CWP217-inoculated referments B and BZ showed 100% and 30% increases as compared with referments without any inoculation and 56% and 22% increases as compared with La-inoculated referments. This study provides the acid-resistant lactic acid bacterium CWP217 with better acid-resistant ability than the same species. The increased live bacteria and anti-inflammatory activity after CWP217 inoculation in pH 3-4 of vegetable and fruit ferments may be applied for the company to develop health benefit beverages in future.