Rozprawy doktorskie na temat „Lactic acid – Metabolism”

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19766号
農博第2162号
新制||農||1040(附属図書館)
学位論文||H28||N4982(農学部図書室)
32802
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 菅原 達也, 教授 澤山 茂樹, 教授 佐藤 健司
学位規則第4条第1項該当

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19046号
農博第2124号
新制||農||1032(附属図書館)
学位論文||H27||N4928(農学部図書室)
31997
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 小川 順, 教授 加納 健司, 教授 植田 充美
学位規則第4条第1項該当

The purpose of this investigation was to compare the ventilatory threshold (T(vent)) with the lactate threshold (T(lact)) during 60 minutes of steady-state exercise at the calculated thresholds. Eight trained, male cyclists (mean age=23.3 yrs, ht=176.4 cm, wt=70.7 kg, VO₂max=61.02 ml/kg‧minˉ¹) performed a 23 W/min progressive intensity cycling test for determination of T(lact) and T(vent). T(vent) was determined by the non-linear increase in excess CO₂ (ExCO₂) while T(lact) was calculated by the 'individual anaerobic threshold' (IAT) method. Subsequently, subjects performed up to 60 minutes steady-state exercise at the threshold workloads. Results at T(vent) and T(lact) indicate significant differences (p<0.01; T(lact)>T(vent)) between VO₂, ExCO₂, HR, [BLa] and workload as calculated by Hotelling's T²-test. During the steady state exercise at each specified workload, VO₂, [BLa], heart rate and ExCO₂ were measured at 15 minute intervals. All subjects completed the steady-state exercise at T(vent) (VSS) while only 2 subjects completed the steady-state exercise at T(lact) (LSS) (avg time=48.4 min). Comparison of metabolic variables using MANOVA and multiple comparisons revealed significant differences between VSS and LSS for HR and VO₂ at all time intervals, for [BLa] at 30 and 45 minute intervals and for ExCO₂ at the 30 minute interval. Furthermore, examination of [BLa] over time using trend analysis revealed a stabilization during VSS ([formula omitted]=3.05 mmol‧Lˉ¹) whereas [BLa] continuously increased over time during LSS. Findings indicate that T(lact) (IAT method) overestimates the ability to perform prolonged work over 45 min. while T(vent) (ExCO) allows for steady-state exercise greater than 60 minutes.
Education, Faculty of
Curriculum and Pedagogy (EDCP), Department of
Graduate

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第9608号
農博第1236号
新制||農||841(附属図書館)
学位論文||H14||N3640(農学部図書室)
UT51-2002-G366
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 池田 篤治, 教授 清水 昌, 教授 加藤 暢夫
学位規則第4条第1項該当

During sustained exercise, working muscles must be supplied with adequate kinds and amounts of exogenous fuels, and the delivery rates of oxygen and oxidizable substrates should be matched. The study of metabolite fluxes and their regulation is therefore critical to the understanding of exercise metabolism. Lactate has received renewed attention from physiologists and biochemists with the realization that it is not only an end product of glycolysis, but also an important fuel for aerobic work. As an oxidizable fuel, this substrate may provide some performance advantage over other fuels such as glucose and free fatty acids. The goals of this thesis were: 1) to determine whether endurance-adapted animals can support higher plasma lactate turnover rates than sedentary animals; and 2) to investigate the major factors involved in the regulation of plasma metabolite turnover at the whole-organism level - using lactate as a model. Lactate turnover rates were measured by bolus injection of [U-¹⁴C]lactate in skipjack tuna, Katsuwonus pelamis, and in thoroughbred racehorses, Equus caballus. In tuna, turnover rates ranged from 112 to 431 umol min⁻¹ kg⁻¹ and they were positively correlated with lactate concentration (slope = 15.1, r = 0.92). This teleost is able to support higher plasma lactate turnover rates than expected for a mammalian lower temperature, and lactate is probably an important oxidizable fuel in this species. For comparative purposes, resting turnover rates of lactate and glucose were plotted versus body mass on a log-log scale for a wide range of mammalian species. These plots were linear, and they showed the same slope as the classic body mass vs metabolic rate relationship. Thoroughbred horses are likely to have an aerobic scope of 40-fold or more. One of their main physiological adaptations to exercise is the ability to increase hematocrit by more than one and a half-fold in response to exercise. In the present study, this adjustment allowed them to reach an A-V difference in 0₂ content of more than 23 vol% during maximal exercise, a much higher value than other mammals. Their lactate turnover rate and cardiac output were measured at rest and two levels of submaximal exercise (45 and 55 V0₂ max) to investigate the relationship between cardiovascular adjustments on plasma lactate turnover rate. Cardiac output ranged from 106 to 571 ml min⁻¹ kg⁻¹, and mean lactate turnover rate from 9.3 at rest, to 75.9 umol min⁻¹ kg⁻¹ at 55% V0₂ max. In contrast with the situation found in tuna, the lactate turnover rates of thoroughbreds were not elevated compared with other mammals, showing that the metabolic adaptations of these outstanding athletes do not include the ability to sustain higher lactate fluxes than sedentary animals. In horses, the contribution of plasma lactate oxidation to V0₂ is minimal, and this substrate is not an important oxidative fuel; lipid oxidation may represent their major pathway for aerobic energy production during exercise. The ability to oxidize plasma lactate at high rates is therefore not necessarily required for the "elite" performance of endurance exercise. This study also shows that both, plasma lactate concentration and cardiac output are positively correlated with turnover rate. The correlation between cardiac output and lactate turnover rate is independent of the relationship between plasma lactate concentration and turnover rate. Plasma metabolite concentration and cardiac output can be regulators of plasma metabolite turnover rate. It is proposed that these two variables are, respectively, the fine and coarse controls for flux rate adjustments during exercise.
Science, Faculty of
Zoology, Department of
Graduate

Two experiments were done to study the effects of L-carnitine supplementation (CNsup) during exercise. EXP 1, examined the effect of CNsup on lipid oxidation and muscle glycogen utilization during submaximal EX. Triglycerides were elevated by a fat feeding (90g fat), 3 h later subjects cycled for 60 min at 70% VO2max (CON). Muscle biopsies were obtained preEX, after 30 and 60 min of EX. Blood samples were taken preEX and every 15 min of EX. Subjects randomly completed two additional trials following 7 and 14 days of CNsup (6 g/day). During one of the trials, subjects received 2000 units of heparin 15 min prior to EX to elevate FFA (CNhep). There were no differences in V02, RER, HR, g of CHO and fat oxidized among the three trials. Serum total acid soluble (TASC) and free carnitine (FC) increased with CNsup (CON, 71.3 ± 2.9; CN, 92.8 ± 5.4; CNhep, 109.8 ± 3.5 mol·g'). Muscle carnitine concentration at rest was unaffected by CNsup. During EX, TASC did not change, FC decreased (p<0.05) and SCAC increased (p<0.05). With CNsup the decrease in FC was less (~50%) (p<0.05) and the increase in SCAC was greater (~200-300%) (p<0.05) compared to CON (free 65%; SCAC 150%). Pre and postEX muscle glycogens were not different. EXP 2, examined the effects of CNsup on blood lactate accumulation during maximal EX. Subjects cycled for 4 min at ~100% VO2max (CON). Exercise was repeated following 6 and 13 days of CNsup (6 g/day). Serum TASC and FC were elevated due to CNsup. Blood Lactate was measured prior to and 0, 3, 5, and 7 min postEX. CNsup resulted in less (p<0.05) lactate accumulation compared to CON. There were no differences between DAY-6 and DAY-13.
Human Performance Laboratory

Rhizopus oryzae is a filamentous fungus, which can produce high amounts of L(+)-lactic acid and produces ethanol as the main by-product. In an effort to understand the pyruvate branch point of this organism, fermentations under different inoculum and glucose concentrations were carried out. At low inoculum size (1x103 spores ml-1), high amount of lactate (78 g l-1) was produced, whereas high ethanol concentration (37 g l-1) was obtained at high inoculum sizes (1x106 spores ml-1). Decreasing working volume increased lactate production significantly at high inoculum sizes (1x105 and 1x106 spores ml-1), but did not influenced the physiology at low inoculum sizes (1x103 and 1x104 spores ml-1). In shake flask cultures, at low initial glucose concentrations biomass yield was high and lactate and ethanol yields were low. Higher lactate and ethanol and lower biomass yields were obtained by increasing the initial glucose concentrations. In alginate immobilized, semi-continuous cultures with cell retention, glucose level in the medium was kept at low values. Like in shake flask cultures, as the glucose concentration decreased lactate and ethanol yields decreased and biomass yields increased. Increasing the glucose concentration by a pulse of glucose caused increases in branch point enzyme activities, as well as in concentrations of the metabolites. In fed batch cultures higher biomass yield (0.25 g DCW g glucose-1) could be obtained. Lactate dehydrogenase was influenced by the inoculum size and glucose concentration more than pyruvate decarboxylase and alcohol dehydrogenase. It showed higher activity at lactate producing fermentations. Unlike lactate dehydrogenase, pyruvate decarboxylase and alcohol dehydrogenase showed high activity even at low glucose concentrations.

1. Brooks, G. Cell-cell and intracellular lactate shuttles. Journal of Physiology. 2009. Vol. 587 (23). P. 5591-5600. 2. Cohen, R.D. Disorders of lactic acid metabolism. Clin. Endocrinol. Metab. 1976. Vol.5(3). P. 613–625.
Lactate, or lactic acid (2-hydroxypropanoic acid), is a normal product of metabolism and is formed from pyruvic acid under anaerobic conditions in skeletal muscle tissue (25%), brain (20%), skin (25%), erythrocytes (20 %), leukocytes, mucous membrane of the small intestine and the renal medulla. Preferably lactate is metabolized in the liver (60%), kidneys (30%) and only a small amount is utilized in other tissues (10%). Measuring blood lactate levels is especially important in cases of severe sepsis and septic shock. In this context, lactate is commonly used to assess the severity of the disease, analyze the response to treatment and further prognosis. Hypoperfusion, which is most often associated with blood loss, is observed in patients with trauma. Elevated lactate levels may help to identify patients in whom normal vital signs are initially masked by tissue hypoperfusion. In patients with cardiogenic shock who require extracorporeal membrane oxygenation, lactate is a useful parameter for predicting mortality. Lactate levels increase during strenuous exercise, mainly due to anaerobic glucose metabolism. A common feature of primary and metastatic cancer is an increase in the rate of glycolysis, which leads to increased glucose uptake and lactate formation even under normal oxygen levels. Лактат або молочна кислота (2-гідроксипропанова кислота), є нормальним продуктом обміну речовин і утворюється з піровиноградної кислоти при анаеробних умовах у тканині скелетних м'язів (25%), мозку (20%), шкіри (25%), еритроцити (20%), шкіри (25%), еритроцити (20%), 20%), лейкоцити, слизової оболонки тонкого кишечника та ниркової медулі. Переважно лактат метаболізується в печінці (60%), нирках (30%), і лише невелика кількість використовуються в інших тканинах (10%). Вимірювання рівнів лактату крові особливо важлива у випадках важкого сепсису та септичного шоку. У цьому контексті, лактат зазвичай використовується для оцінки тяжкості захворювання, аналізують реакцію на лікування та подальший прогноз. Гіпоперфузія, яка найчастіше пов'язана з крововтратою, спостерігається у пацієнтів з травмою. Підвищений рівень лактатів може допомогти виявити пацієнтів, у яких звичайні життєві ознаки спочатку маскують гіпоперфузією тканин. У пацієнтів з кардіогенним шоком, які потребують екстракорпоральної мембранної оксигенації, лактат є корисним параметром для прогнозування смертності. Lactate рівень збільшується під час напружених вправ, головним чином завдяки анаеробному метаболізму глюкози.

This study examined the effects of 14 days of L-carnitine supplementation on blood and muscle lactate concentrations, and carnitine fractions, during high intensity sprint cycling exercise. Eight subjects performed three experimental trials - control I (CON I, 0 days), control II (CON II, 14 days), and L-carnitine (LCN, 28 days). Each trial consisted of a 4 min ride at 90% VO2max, followed by a rest period of 20 min, and then 5 x 1 min rides at 115% VO2max (2 min restbetween each). Following CON II, all subjects began dietary supplementation of L-carnitine for a period of 14 days (4 g/day). L-carnitine supplementation had no significant effect on either muscle carnitine or lactate concentrations following the 4 min 90% ride. Plasma total acid soluable and free carnitine concentrations were significantly higher at all time points following supplementation. Differences observed in blood hydrogen ion and lactate concentrations between CON I and CON II appear to be the result of an order effect. The data from the present investigation indicate that L-carnitine supplementation has no significant effect on blood or muscle lactate accumulation following high intensity sprint cycling exercise.
School of Physical Education

L'adage « l’union fait la force » s'applique aux aliments fermentés : l'association de plusieurs souches de bactéries lactiques (BL) a des conséquences bénéfiques sur leurs propriétés organoleptiques et sanitaires et sur leurs bienfaits pour la santé. Ces fonctionnalités peuvent dépendre des interactions entre les BL, comme démontré dans le yaourt. Créer de nouvelles associations fonctionnelles de BL est un défi, en particulier dans le contexte de la transition alimentaire où de nouvelles sources de protéines sont à utiliser. L’objectif de la thèse était de comprendre comment favoriser les interactions positives entre BL, sur la base de leur métabolisme azoté, en vue de nouvelles applications alimentaires. Les interactions ont été étudiées en associant une souche protéolytique (prot+) à une non-protéolytique (prot-), d’abord dans un milieu modèle contenant des protéines de lait et de lupin comme seule source d’azote, dans lequel la croissance des BL prot- dépendait des peptides et acides aminés libérés par la prot+. Des interactions positives fortes, faibles ou nulles ont été obtenues. Nos résultats ont montré que la libération d’acides aminés à chaine ramifiée, libres ou au sein de peptides était au cœur des interactions observées. L’impact de ces interactions a été ensuite étudié dans des yaourts alternatifs à base de lait et de lupin, sur leurs propriétés physiques et sensorielles. Les interactions positives observées dans ces mixtes ont conduit à diversifier les fonctionnalités comme la production de composés d’arôme et d’acides organiques et l’amélioration de la texture. Ces travaux apportent de nouveaux éléments de compréhension des interactions entre BL, pouvant servir à concevoir de futures aliments
The adage “in unity is strength” also applies to fermented foods: the association of lactic acid bacteria (LAB) strains brings benefits to their organoleptic properties, sanitary quality and potential health benefits. These functionalities can depend on positive interactions, as observed between LAB in yogurt. Finding ways to create new functional associations of LAB strains is challenging, especially in the context of the food transition where new resources, especially in proteins, are increasingly used. The objective of this work was to understand how to promote positive interactions between LAB, on the basis of their nitrogen metabolism, to conceive new food applications. The interactions were studied in cocultures that associated a proteolytic (prot+) and a non- proteolytic (prot-) strain. These prot+/prot- pairs were first grown in a model medium containing milk and lupinproteins as sole nitrogen nutriments, in which the growth of prot- strain thus depended on the peptides and amino acids released by the prot+ strain. Strong, weak, or no positive interactions were observed between prot+ strains and prot- strains. Our results showed that the release of branched-chain amino acids in their free form or within peptides was at the heart of the positive interactions observed. The impact of these interactions was then studied in alternative yogurts made from milk and lupin, characterized for their physical and sensory properties. The positive interactions between LAB lead to diversified functionalities of alternative yogurts, such as flavor compound and organic acid formation and texture improvement. This work brings new knowledge on the interactions between LAB, which is useful in designing future fermented foods

The metabolic pathways required for adenosine triphosphate (ATP) production within the cell are well understood, however recent publications suggest that metabolic pathways are closely linked to immune cell activation and inflammatory diseases. There has been little examination of the metabolic pathways that modulate mast cell activation and the feedback regulator lactic acid. Here we examine metabolic pathways and regulation within mast cells in the context of lipopolysaccharide (LPS) and interleukin (IL-33) activation, for which there has been little to no reported studies. First, we examine the effects of lactic acid, previously considered only a by-product of glycolysis and now understood to act as a negative feedback regulator of inflammation in the context of LPS activation and sepsis. Lactic acid is elevated in septic patients and associated with mortality, potentially due to suppressive effects on LPS signaling and contribution to late phase immunosuppression. By attenuating glycolysis and reducing ATP availability for signaling and cytokine transcription, lactic acid impairs the function of immune cells to fight the initial or subsequent infections. We support this with in vitro and in vivo data. Additionally, our lab has published that lactic acid can suppress IL-33 activation, potentially by metabolic modulation as with LPS activation; however there has been no study of the metabolic requirements for IL-33 activation. We report here that glycolysis is required for ATP and reactive oxygen species (ROS) production to augment signaling and cytokine production downstream of the IL-33 receptor. Together, these studies examine the contribution of metabolism to mast cell activation and may provide potential targets for treatments of diseases that involve LPS- or IL-33-dependent mast cell activation.

[Truncated abstract] Antimicrobial growth promoters are added to feed to prevent lactic acidosis in ruminant animals by selectively inhibiting rumen bacteria that produce lactic acid. However, recently imposed or impending bans on the use of antimicrobial growth promoters in animal production have lead to a critical need to find practical alternatives that are safe for the animal and consumer and that obtain similar production benefits. I investigated bioactive plants of Australia for their potential to prevent lactic acidosis in ruminants. The unifying hypothesis tested was that plants would be identified that selectively inhibit lactic acid-producing bacteria and consequently protect against lactic acidosis. This hypothesis was tested in a three phase process: phase 1, plant selection and collection; phase 2, a three stage protocol for screening plants and essential oils; phase 3, in vivo experiments and chemical fractionation of the most promising plant. I developed an in vitro bioassay that simulated acidosis by adding glucose to rumen fluid in Bellco tubes and incubating for 5 h (Chapter 4). The pH and gas production were used as indicators of acidosis and fermentation activity. I used this bioassay to screen ninety-five plants (dried and ground material from 79 species) and ten essential oils and included a negative control (oaten chaff) and a positive control (virginiamycin). One plant, Eremophila glabra, produced a similar pH (5.63) to the positive control (5.43) although it inhibited gas production to a moderate extent (P < 0.05). ... Seven serrulatane diterpenes were identified to be the major secondary metabolites in E. glabra. The metabolites were screened using a broth dilution and microtitre spectrophotometry method and were selective against S. bovis at between 320 and 1077 [mu]g/ mL. The serrulatanes from E. glabra were probably responsible for the activity against acidosis that I observed in vitro, because they selectively inhibited lactateproducing bacteria. It is also possible that a synergy between serrulatanes and possibly other metabolites are responsible for the activity observed in vitro. The results from my experiments support the role that bioactive plants may have to replace the antibiotics that are added to livestock feed. Australian plants were identified containing compounds that were active against the bacterial processes responsible for ruminant acidosis. To my knowledge this is the first work undertaken to identify bioactive plants of Australia for their potential to prevent acidosis. I developed in vitro screening bioassays that targeted key indicators of acidosis. These bioassays enabled me to identify 5 plants from the 104 screened that could potentially control acidosis. One of these plants in particular, E. glabra, showed a level of activity in vitro that was comparable to antibiotic protection against acidosis. The exciting in vitro results were not demonstrated in vivo but only one dose level of E. glabra was used, which was based on the in vitro work. In contrast to the in vitro system the rumen is a continuous flow system with greater complexity and it is possible that the concentration of E. glabra that I used in vivo was not optimum. This places importance on future dose response experiments to confirm the efficacy of E. glabra in vivo.

Stato dell’arte: I microrganismi probiotici negli alimenti potrebbero causare cambiamenti significativi nel sapore e nella reologia, a causa del loro metabolismo attivo. È importante, quindi, controllare il metabolismo dei microrganismi probiotici negli alimenti, senza influenzare negativamente la vitalità e le caratteristiche funzionali. Un modo per superare questo problema è l'utilizzo di colture starter attenuate attraverso l’utilizzo di metodi fisici o chimici. Alcuni autori hanno studiato l'omogeneizzazione come metodo per attenuare/modulare il metabolismo delle colture starter nei prodotti caseari (Lanciotti et al., 2004, 2006, 2007); in questo progetto di tesi ho utilizzato una nuova tecnologia emergente, gli ultrasuoni. (i) È stato effettuato uno screening delle combinazioni possibili di ultrasuoni (US) (potenza/durata) su diversi microrganismi probiotici e sono state studiate le caratteristiche probiotiche e tecnologiche dopo l'esposizione agli US. (ii) Sono stati studiati gli effetti degli US sul rilascio di componenti intracellulari. (iii) È stata valutata l'interazione dei ceppi attenuati con il microbiota intestinale utilizzando la fermentazione in vitro (batch culture fermentation). Problematiche: (i) Non si conoscono gli effetti dell’attenuazione mediante US sulle caratteristiche tecnologiche/probiotiche dei microrganismi; (ii) non è chiara la risposta delle cellule microbiche al trattamento con gli US e il danno che ne deriva, in quanto in letteratura sono disponibili pochi dati; (iii) non sono disponibili dati sull'interazione di ceppi attenuati con il microbiota intestinale. Obiettivi: (i) Scelta della migliore combinazione di ultrasuoni (potenza/durata), in grado di evitare la post-acidificazione senza compromettere la vitalità dei ceppi, studio dei cambiamenti delle caratteristiche probiotiche e tecnologiche che i microrganismi potrebbero subire a seguito dell’attenuazione; (ii) valutazione del rilascio di componenti intracellulari (acidi nucleici e proteine) dopo l'applicazione degli ultrasuoni; e (iii) studio dell’effetto di ceppi attenuati sul microbiota intestinale. Pianificazione della ricerca: Nella prima parte sono stati usati tre diversi generi di microrganismi probiotici: Lactobacillus, Bifidobacterium, Propionibacterium. I ceppi sono stati trattati con ultrasuoni e sono state studiate le caratteristiche tecnologiche e probiotiche. Nella seconda parte, sono stati studiati gli effetti degli US sul rilascio di componenti intracellulari. I ceppi sono stati studiati a seguito del trattamento fisico per valutare il rilascio di costituenti intracellulari (acidi nucleici, proteine) ed eventuali lesioni della membrana cellulare. Nell'ultima parte, è stata studiata l'interazione di ceppi attenuati con il microbiota intestinale. Questo studio è stato condotto presso l'Università di Roehampton (Regno Unito). Materiali e metodi: (i) Caratteristiche tecnologiche: Prove di acidificazione in mezzo di laboratorio, crescita a diverse temperature, pH e contenuto di sale; caratteristiche probiotiche: antibiotico resistenza, sopravvivenza a pH 2,5 e in presenza di sali biliari (0,3%), idrofobicità e formazione di biofilm. (ii) La valutazione del danno sulle cellule microbiche è stato determinato attraverso letture spettrofotometriche a 260 e 280 nm per quantificare il rilascio di DNA e proteine. (iii) L'interazione dei ceppi attenuati con il microbiota intestinale è stata valutata utilizzando la fermentazione in vitro (batch culture fermentation). Risultati: (i) Le migliori combinazioni in grado di evitare la post-acidificazione sono state le seguenti: potenza, 60%; tempo, 6 minuti; impulsi, 2 s per Lactobacillus e Bifidobacterium, 40%, 8 min P. jensenii; 60%, 4 min P. freudenreichii subsp. freudenreichii. Gli US non hanno influenzato la vitalità a 45 °C e a pH 9, ma hanno determinato una diminuzione della crescita microbica a pH 4 (lattobacilli e bifidobatteri). Tuttavia, gli US non hanno influenzato il GI dei propionibatteri. L'effetto dell'attenuazione potrebbe essere migliorato con la refrigerazione. Il trattamento con gli US non ha influenzato la maggior parte dei tratti tecnologici, ma ha causato un aumento della suscettibilità ad alcuni antibiotici. Per quanto riguarda i tratti probiotici, gli US hanno causato un aumento di idrofobicità per L. reuteri e P. freudenreichii spp. freudenreichii. Questi risultati sono stati confermati con l'adesione alle cellule Caco-2 per L. reuteri. L. reuteri attenuato ha registrato un significativo aumento di idrofobicità (dal 3 al 25%) e una maggiore adesione alle cellule Caco-2. Inoltre, gli US hanno migliorato la stabilità dei biofilm nel tempo e questo risultato ha confermato i dati ottenuti con l'idrofobicità. (ii) Il rilascio di acidi nucleici e proteine, evidenzia che la membrana cellulare potrebbe essere un altro target del trattamento fisico. (iii) Per quanto riguarda gli effetti degli US sul microbiota intestinale: i ceppi attenuati non hanno influenzato il microbiota intestinale, ma in alcuni casi sono stati evidenziati effetti positivi. Importanza e impatto della ricerca di dottorato: Il metabolismo attivo dei microrganismi probiotici potrebbe costituire un problema quando questi ultimi vengono aggiunti agli alimenti. Infatti alcuni ceppi di batteri lattici continuano a produrre acido lattico e causano post-acidificazione (diminuzione del pH durante la conservazione). Pertanto, è importante controllare il loro metabolismo. Un modo possibile per controllare il metabolismo dei probiotici negli alimenti è l'attenuazione attraverso metodi fisici o chimici. Una delle tecnologie emergenti sono gli ultrasuoni (US). Questo approccio è stato utilizzato per evitare la post-acidificazione in una bevanda commerciale di riso (Bevilacqua et al., 2016). La presente tesi di dottorato ha contribuito a valutare gli effetti dell'attenuazione mediante ultrasuoni, su alcune caratteristiche tecnologiche e probiotiche, testando tre diversi generi di ceppi probiotici. Inoltre, questo progetto di dottorato ha studiato i cambiamenti in termini di caratteristiche tecnologiche e probiotiche che i microrganismi probiotici potrebbero subire a seguito dell'attenuazione; il rilascio di proteine, acidi nucleici. Infine, la novità di questa tesi di dottorato è stata lo studio degli effetti di ceppi attenuati sul microbiota intestinale. Proposte future: una prospettiva futura potrebbe essere focalizzata sull'uso degli US per migliorare o modulare l'adesione dei ceppi probiotici, considerando l'aumento di idrofobicità e la maggiore adesione alle cellule Caco-2. È importante studiare gli effetti di altri ceppi attenuati sul microbiota intestinale modulando le variabili del trattamento.
Scientific background: Probiotics in foods could lead to significant changes in food flavor and rheology, due to their active metabolism. A possible way to overcome this problem is the attenuation of probiotics through a physical or chemical method. Some authors studied homogenization as a way to attenuate/modulate the metabolism of starter cultures in dairy products (Lanciotti et al., 2004, 2006, 2007); in this project I have used a new emerging technology, the ultrasounds. (i) A screening of the ultrasound (US) (power/duration) on different probiotic microorganisms was perfomed, and were studied the probiotic and technological characteristics after US-exposure. (ii) The effects of US on the release of intracellular components, was investigate. (iii) The interaction of attenuated strains with gut microbiota was evaluated, using in vitro batch culture fermentation. Open questions: (i) Few data are available on effects of US on probiotics and technological characteristics of probiotic strains; (ii) few data are available on the effect of attenuation with ultrasound on the sub-lethal injury; (iii) no data are available on the interaction of attenuated strains with gut microbiota. Aims: (i) Choice of the best combination of ultrasound to avoid post-acidification without affecting the viability of the strains, and study of the probiotic and technological characteristics to evaluate if attenuation could change them; (ii) study the release of intracellular components (nucleic acids and proteins) after the application of ultrasound; and (iii) evaluation the effects of attenuated strains on gut microbiota. Planning of the research: In the first part three different genera of probiotics were used: Lactobacillus, Bifidobacterium, Propionibacterium. The strains were treated with ultrasound and studied for technological and probiotic characteristics. In the second part, the effects of US on the release of intracellular components, was investigated. The strains were studied after physical treatment to assess the release of intra-cellular constituents (nucleic acids, proteins) and injury of the membrane. In the last part, the interaction of attenuated strains with gut microbiota, was studied. This study was carried out at University of Roehampton (UK). Materials and Methods: (i) Technological traits: acidification in lab medium, growth at different temperatures, pHs and salt content; probiotic traits: antibiotic-resistance, survival at pH 2.5 and in the presence of 0.3% bile salt, hydrophobicity, and biofilm formation. (ii) Injury characterization was evaluated by leakage of UV-absorbing substances. (iii) The interaction of attenuated strains with gut microbiota was evaluated, using in vitro batch culture fermentation. Results: (i) The best combinations to avoid post-acidification were the following: power, 60%; time, 6 min; pulse, 2 s for Lactobacillus and Bifidobacterium, 40%, 8 min P. jensenii; 60%, 4 min P. freudenreichii subsp. freudenreichii. US did not affect viability at 45 °C or at pH 9, but it determined a decrease of microbial growth to pH 4 (lactobacilli and bifidobacteria). However, the US did not affect the GI of propionibacteria. The effect of attenuation could be enhanced by the storage under refrigeration. US-treatment did not affect most of the technological traits, but generally caused an increase of susceptibility to some antibiotics. Concerning probiotic traits, US caused an increase of hydrophobicity for L. reuteri and P. freudenreichii spp. freudenreichii, after US-exposure. These results were confirmed with adhesion to Caco-2 cells for L. reuteri. US-attenuated L. reuteri experienced a significant increase of hydrophobicity (from 3 to 25%) and a higher adhesion to Caco-2 cells. Moreover, US improved the stability of the biofilm over the time, and this result confirmed the data obtained with hydrophobicity. (ii) The release of nucleic acids and proteins was found, highlighting that cell membrane could be another target physical treatments. (iii) Concerning the effects of US on gut microbiota, the ultrasound didn’t affect the gut microbiota, but in some cases, it could have a positive effect. Significance and Impact of PhD research: A main drawback of probiotics in foods can relate to their active metabolism, some strains of lactic acid bacteria continue to produce lactic acid and cause post-acidification (the decrease of pH within the storage). Therefore, it is important to control their metabolism. A possible way to control the metabolism of probiotic in foods is the attenuation through physical or chemical methods. One of the emerging technologies is ultrasound (US). This approach was used to avoid post-acidification in a commercial rice drink (Bevilacqua et al., 2016). The present PhD thesis contributed to evaluate the effects of attenuation with ultrasound, on some technological and probiotic strains, testing three different genera of probiotic strains. Moreover, this PhD project has investigate the changes that may affect probiotic strains after attenuation; the release of proteins, nucleic acids. Finally, the novelty of this PhD thesis was the study of the effects of attenuated strains on gut microbiota. Future trends: A future perspective could be a focus on the use of US to improve or modulate the adhesion of probiotic strains, considering the increase of hydrophobicity and the higher adhesion to Caco-2-cells. It is important to investigate the effects of other attenuated strains on gut microbiota by modulating the variables of the treatment. Key words: Hydrophobicity, acidification, growth, attenuation, gut-microbiota, proteins, nucleic acid, ultrasound, attenuated microorganisms, probiotics, lactic acid bacteria.

Lactobacillus reuteri BR11 possesses an abundant cystine uptake (Cyu) ABC-transporter that was previously found to be involved in a novel mechanism of oxidative defence mediated by cystine. The current study aimed to elucidate this mechanism with a focus on the role of the co-transcribed cystathionine ã-lyase (Cgl). Growth studies of wild-type L. reuteri BR11 and mutants inactivated in cgl and the cystine-binding protein encoding gene cyuC showed that in contrast to the Cyu transporter, whose inactivation led to growth arrest in aerated cultures, Cgl is not crucial for oxidative defence. However, the role of Cgl in oxidative defence became apparent in the presence of severe oxidative damage and cysteine deprivation. Cysteine was found to be protective against oxidative stress, and the action of Cgl in both cysteine biosynthesis and degradation poses a seemingly futile pathway that deprives the intracellular cysteine pool. To further characterise the relationship between Cgl activity and cysteine and their roles in oxidative defence, enzymatic assays were performed on purified Cgl, and intracellular concentrations of cysteine, cystathionine and methionine were determined. Cgl was highly active towards cystine and cystathionine and less active towards cysteine in vitro, suggesting the main function of Cgl to be cysteine biosynthesis. Cysteine was found at high concentrations in the cell, but the levels were not significantly affected by inactivation of cgl or growth under aerobic conditions. It was concluded that both anabolic and catabolic activities of Cgl towards cysteine contribute to oxidative defence, the former by maintaining an intracellular reservoir of thiol analogous to glutathione, and the latter by producing H2S which is readily secreted, thus creating a reducing extracellular environment. The significance of the Cyu transporter to the physiology of L. reuteri BR11 prompted a phylogenetic study to determine its presence in bacteria. Orthologs of the Cyu transporter that are closest matches to the Cyu transporter are only limited to several species of Lactobacillus and Leuconostoc. Outside the Lactobacillales order, the closest matching orthologs belong to Proteobacteria, and there are more orthologs in Proteobacteria than non-Lactobacillales Firmicutes, suggesting that the Cyu transporter locus was present in the ancestor of the Proteobacteria and Firmicutes, and over evolutionary time has been lost or diverged in many Firmicutes. The clustering of the Cyu transporter locus with a gene encoding a Cgl family protein is even rarer. It was only found in L. reuteri, Lactobacillus vaginalis, Weissella paramesenteroides, the Lactobacillus casei group, and several Campylobacter sp. An accompanying phylogenetic study of L. reuteri BR11 using multi-locus sequence analysis showed that L. reuteri BR11 had diverged from more than 100 strains of L. reuteri isolated from various hosts and geographical locations. However, comparison with other Lactobacillus species supported the current classification of BR11 as L. reuteri. The most closely related species to L. reuteri is L. vaginalis or Lactobacillus antri, depending on the housekeeping gene used for analysis. The close evolutionary relationship of L. vaginalis to L. reuteri and the high degree of sequence identity between the cgl-cyuABC loci in both species suggest that the Cyu system is highly likely to perform similar functions in L. vaginalis. In search of other genes that function in oxidative defence, a number of mutants which were inactivated in genes that confer increased resistance to oxidative stress in other bacteria were constructed. The genes targeted were ahpC (peroxidase component of the alkyl hydroperoxide reductase system), tpx (thiol peroxidase), osmC (osmotically induced protein C), mntH (Mn2+/Fe2+ transporter), gshA (ã-glutamylcysteine synthetase) and msrA (methionine sulfoxide reductase). The ahpC and mntH mutants had slightly lower minimum inhibitory concentrations of organic peroxides, suggesting these genes might be involved in resistance to organic peroxides in L. reuteri. However, none of the mutants exhibited growth defects in aerated cultures, in stark contrast to the cyuC mutant. This may be due to compensatory functions of other genes, a hypothesis which cannot be tested until a robust protocol for constructing markerless multiple gene deletion mutants in L. reuteri is developed. These results highlight the importance of the Cyu transporter in oxidative defence and provide a foundation for extending the research of this system in other bacteria.

INTRODUÇÃO: Apesar do amplo uso da medida da saturação central de oxigênio como meta terapêutica em pacientes de terapia intensiva, diferenças absolutas em relação à saturação venosa mista existem. As causas desses gradientes, bem como o comportamento das mesmas ao longo do tempo nas doenças graves não foram completamente esclarecidas. Considerando que a maioria das intervenções atualmente empregadas para reverter desequilíbrios de oxigenação tecidual presentes nos pacientes graves é direcionada, direta ou indiretamente, ao coração; a situação particular de elevada taxa de extração de oxigênio basal do miocárdio e a ausência de ferramentas de monitorização do impacto miocárdico dessas intervenções, o presente estudo, diante da possibilidade teórica da participação do efluente do seio coronário nessas diferenças centrais para pulmonares, não só para a saturação de oxigênio (SO2), analisou o comportamento da SO2, pressão parcial de dióxido de carbono (PCO2), lactato e glicose, em diferentes modelos de hipóxia e compartimentos vasculares, com ênfase na avaliação do metabolismo miocárdico e seu impacto nos gradientes centrais para pulmonares. MÉTODOS: 37 porcos, machos, com peso em torno de 35 Kg, sedados e ventilados mecanicamente, foram estudados após indução de quatro diferentes tipos de injúria hipóxica (hipóxia anêmica, estagnante, hipóxica e sepse), sendo 8 animais por grupo e mais 5 controles. Além de variáveis hemodinâmicas e de oxigenação, SO2, PCO2, lactato e glicose foram medidos, em diferentes momentos, em 9 compartimentos vasculares distintos, incluindo o seio coronário (artéria femoral, veia cava inferior e superior, átrio direito, ventrículo direito, artéria pulmonar, veia suprahepática direita e veia porta). PRINCIPAIS RESULTADOS: As concentrações de O2, lactato e glicose no efluente do seio coronário apresentaram padrões distintos entre os grupos: troca de substrato energético de lactato por glicose nos grupos hipóxia hipóxica e anêmica, aumento no consumo de ambos os substratos na sepse e ausência de tendência clara no grupo da hipóxia estagnante. Os gradientes de PCO2 entre seio coronário e artéria femoral mantiveram-se estáveis com tendência de alargamento tardio em todos os modelos. Na análise dos demais gradientes regionais, o seio coronário apresentou a menor SO2 do organismo, as menores concentrações de lactato, os maiores níveis de PCO2, e esses padrões variaram ao longo do tempo. Mesma tendência evolutiva foi percebida entre os gradientes centrais para pulmonares de O2, lactato, CO2 e glicose e a medida desses mesmos parâmetros no seio coronário. CONCLUSÕES: As concentrações de O2, lactato e glicose no efluente do seio coronário estão relacionadas ao tipo de injúria e não apenas à disponibilidade de substrato energético. Padrões de gravidade, comuns às fases tardias de todos os grupos, puderam ser identificados: qualquer redução da SO2 coronariana; incremento do metabolismo de glicose; produção de lactato pelo miocárdio e surgimento de igualdade ou inferioridade dos níveis da PCO2 coronariana em relação aos valores dos demais compartimentos vasculares do organismo (independentemente da trajetória). A tendência dos gradientes de PCO2 transmiocárdicos seguiu a do débito cardíaco e, certamente, deve refletir fluxo coronariano. A análise dos gradientes regionais se mostrou capaz de permitir a avaliação de contextos orgânicos regionais específicos, como na avaliação do metabolismo hepático, na qual foi possível demonstrar que na hipóxia, a produção de glicose hepática é mantida até o óbito, diferentemente do padrão descrito para a sepse. Por fim, com a análise dos dados do grupo sepse, foi possível demonstrar que: a) assim como os gradientes centrais para pulmonares de SO2 e lactato já foram descritos, gradientes de glicose e PCO2 também existem; b) o seio coronário participa, significativamente, na formação desses gradientes de lactato, CO2 e glicose
INTRODUCTION: Despite of the widespread use of the central venous oxygen saturation measurement as a therapeutic goal in critically ill patients, absolute differences between this measurement and the mixed venous oxygen exist. Causes of these differences, as well the behavior of these gradients in critical illness, are not completely understood. Considering current therapeutic interventions aimed to reverse tissue oxygenation impairment are mediated by increases in cardiac output; the particular scenario in which the heart is not physiologically able to further increase oxygen extraction and the absence of tools to monitoring the myocardium impact of those interventions, the present study, facing the theoretical possibility of the coronary sinus effluent participation in those central to mixed venous differences, has analyzed the oxygen saturation (SO2), carbon dioxide partial pressure (PCO2), lactate and glucose concentrations behaviors over time, in different models of tissue hypoxia and in different vascular sites. Emphasis on the myocardial energetic metabolism and its impact over central to mixed venous gradients was placed. METHODS: 37 pigs, males, weighting about 35 Kg, sedated and mechanically ventilated, were studied after induction of four different hypoxic injury models (sepsis, and anemic, stagnant, hypoxic hypoxia), eight for group and five controls. In addition to hemodynamic and oxygen variables, SO2, PCO2, lactate and glucose were measured in different phases, in 9 distinct vascular sites, including coronary sinus (femoral artery, inferior and superior vena cava, right atria, right ventricle, pulmonary artery, right suprahepatic vein and portal vein). MAIN RESULTS: Concentrations of O2, lactate and glucose in the coronary sinus effluent presented distinctive patterns among groups: shift from lactate to glucose consumption in hypoxic hypoxia and anemic hypoxia groups, increase in both glucose and lactate consumption in sepsis and absence of clear trend in stagnant hypoxia group. PCO2 gradients from systemic artery to coronary sinus presented late enlargement trend in all groups. In the regional gradients analysis comparisons, coronary sinus presented the lowest SO2, the lowest lactate concentrations, the highest PCO2 levels, and these patterns changed over time. Similar evolution trends were observed between central to mixed venous O2, PCO2, lactate and glucose gradients and the same parameters measured in coronary sinus. CONCLUSIONS: Different concentrations of O2, PCO2, lactate and glucose in coronary sinus are related to the type of hypoxic injury and not only to energetic substrate availability. Severity-related patterns, common to all groups in late phases, were identified: any reduction of coronary SO2, shift to glucose consumption, net lactate myocardial production and equality or inferiority of PCO2 levels related to other vascular compartments (independently of trend). Trends in transmyocardial PCO2 gradients followed cardiac output ones and, certainly, should mirror coronary blood flow. Regional gradients analysis showed suitable to explore specific regional metabolic settings, as in the described example of liver metabolism, in which production of glucose were maintained in all phases by this organ in hypoxic hypoxia groups, differently from the impaired production described in literature for sepsis. At last, data from sepsis group have showed: a) as to the previously known central to mixed venous SO2 and lactate gradients, PCO2 and glucose gradients also exist; b) coronary sinus has participated significantly in formation of central to mixed venous lactate, PCO2 and glucose gradients

The submitted master thesis deals with assessment of the possibility of using of cheese whey for biotechnological production. Study content composition of lyophilized whey, optimization of acid hydrolysis and preparation of cultivating mediums with different content and treatment of cheese whey. Between the aims of this thesis belong also screening of microbial producers, interesting for biotechnology and looks at their growth and production of selected metabolites of cultivation mediums containing cheese whey. All substrates and produced metabolites where examined with UHPLC-PDA-RI and GC-FID: Amount of produced microbial lipids was determined by gravimetric analysis. Examined microorganisms belonged to yeast genus Saccharomyces, Metschnikowia and bacterial genus Lactobacillus and its focused on production of ethanol, microbial lipids and lactic acid. The highest yields with using yeasts were obtained using production medium containing hydrolysed lactose in cheese whey. The highest production, in case of bacteria, was obtained using non-hydrolysed, untreated cheese whey production medium.

Lactococci are widely used in the cheese industry as a starter culture. Starter cultures face carbohydrate starvation due to the absence of a fermentable carbohydrate in the cheese curd after pressing. Starvation leads to a decreased ability to synthesize ATP, generate a proton motive force, and accumulate nutrients necessary to maintain viability. The aim of this work was to investigate the culturability of lactococci grown with and without lactose in a chemically defined medium, and to define the metabolic changes that occur during carbohydrate starvation. Lactose metabolism provided energy for logarithmic phase growth and greater cell density in L. lactis ssp. lactis ML3 and L. lactis ssp. cremoris S2. However, the rate of lactose metabolism was strain dependent in that L. lactis ssp. lactis 11454 did not metabolize lactose as rapidly as did ML3 and S2. In the absence of lactose the cells became nonculturable on agar. In addition to becoming nonculturable, the aminopeptidase and lipase/ esterase activity became nonmeasurable after 21 d, and cellular metabolism was altered because of carbohydrate starvation. Nevertheless, the cells remained viable for up to 42 d in spent media as measured by fluorescent viability stains and intracellular ATP content. Fluorescent viability staining demonstrated that the cells maintained an intact cell membrane to contain their DNA, as well as to contain enzymes and ATP necessary to maintain viability and metabolic activity. With the addition of arginine to the basal medium, the survival time, cell number, and ATP concentration increased. Amino acids, including arginine, provided energy after carbohydrate exhaustion. At the onset of lactose exhaustion, the extracellular concentrations of arginine, glycine/valine, glutamate, and glutamine decreased in the media when energy was present for their transport. There was a significant increase in serine and methionine concentrations in the spent media over the same time period. These data indicated lactococci remained viable and metabolically active, but were nonculturable in response to carbohydrate starvation. Additionally, amino acids are in a dynamic state during carbohydrate starvation, and utilization of amino acids, such as arginine and serine, could facilitate lactococcal cells in maintaining viability in harsh environments such as ripening cheese.

Genetic modification of plastids in the model plant tobacco (Nicotiana tabacum) has demonstrated that numerous foreign gene products can accumulate to high levels in this setting. Plastid biotechnology is maturing to encompass the improvement of food and feed species and the production of biopharmaceutical proteins for oral delivery necessitating development of stable transplastomic edible plants. In the interest of establishing an edible platform we have investigated the use of native and foreign regulatory elements in relation to foreign gene expression in plastids. Multiple sequence alignments of intergenic regions for 20 species of angiosperm showed that despite 95% identity in the coding region, identity in the psbA upstream region is 59% across all taxa examined, other gene coding regions displayed sequence identity of 80-97%, whereas the non-coding regions were 45-79% suggesting that our physical data can be extrapolated beyond the model presented. We found that by exchanging psbA untranslated regions (UTRs) between N. tabacum and lettuce (Lactuca sativa), the expression of the CTB-proinsulin (CTB-Pins) monocistronic transcript declined by 84% and foreign protein accumulation was reduced by as much as 97% in mature leaves. Polyribosome association assays suggest that ribosome-free transgenic transcripts are stabilized where the native UTR is employed. RNA EMSA revealed that binding proteins interacted with psbA 5' UTRs in a species specific manner and the half life of the L. sativa 5'UTR-CTB-Pins mRNA was reduced by 3.7 fold in N. tabacum stromal extracts. Our data indicate that the use of species-specific regulatory elements could lead to establishment of reproducible plastid transformation in desirable target species such as L. sativa. Using transplastomic L. sativa for oral delivery of bioencapsulated CTB-Pins we delayed the onset of diabetes in NOD mice when retinyl acetate supplement was provided compared to untouched mice. In this 30 week study we monitored blood glucose levels and evaluated the in vitro suppressive capacity of regulatory T cells isolated from diabetic mice. Whether delay or prevention was achieved appeared to be a function of antigen dose as high dose resulted in a nine week delay of onset while low dose reduced the incidence of diabetes by 36%. In addition we have evaluated metabolic engineering in the N. tabacum model where we generated cis-genic lines expressing nucleus-encoded methionine pathway enzymes in plastids. Transplastomic expression of Cystathionine gamma-Synthase led to a three-fold increase in enzyme activity and a doubling of methionine content in leaves without a deleterious phenotype. In exploring molecular mechanisms supporting gene expression in plastids and applying transplastomic technology to real human problems this work seeks address the potential of plastid biotechnology for improvement of commodity crops and production of biopharmaceuticals.
Ph.D.
Department of Biomolecular Science
Other
Biomedical Sciences PhD

Viren haben als Ursache lebensmittelassoziierter Infektionen eine große Bedeutung. Sie können vor allem über rohe oder unzureichend erhitzte Lebensmittel übertragen werden. In diesem Zusammenhang werden grüner Salat, Erdbeeren, Himbeeren, Frühlingszwiebeln, Muscheln, halbgetrocknete Tomaten, fäkal verunreinigtes Trinkwasser, Backwaren und Rohwürste als häufige Infektionsquellen genannt. Vor allem kurzgereifte Rohwürste gehören aus mikrobiologischer Sicht zu Risikoprodukten. Um eine gleichbleibende Qualität der Produkte zu gewährleisten, ist die Verwendung von Starterkulturen unerlässlich. Als sogenannte Schutzkulturen sollen sie gleichzeitig die Vermehrung unerwünschter bakterieller Pathogene unterbinden. Bisher ist allerdings nicht bekannt, inwieweit diese zur Virusinaktivierung in kurzgereiften Rohwürsten führen bzw. beitragen. Aus diesem Grund war es das Ziel dieser Arbeit, den Einfluss von rohwurstrelevanten Starter- und Schutzkulturen sowie deren Metabolite (Bacteriocine, Milchsäure) auf die Tenazität und Inaktivierungskinetik von Viren zu prüfen. Die Untersuchungen erfolgten mit dem murinen Norovirus (MNV) S 99 sowie dem humanen Influenzavirus H1N1 (A/WSN/33). Antivirale Effekte wurden zum einen anhand von in-vitro-Studien, zum anderen anhand von experimentell mit Viren kontaminierten kurzgereiften Rohwürsten (Mettwurst/Teewurst) geprüft. Die Bacteriocine Sakacin A und Nisin zeigten in phosphatgepufferter Salzlösung (PBS) keine viruzide Wirkung gegenüber MNV S 99 und H1N1 (pH 6,2; 24 °C; Exposition: 3 Tage). Weiterhin wurden anhand von in-vitro-Untersuchungen 29 verschiedene zellfreie Kulturüberstände [Milchsäurebakterien, Staphylococcus spp. (S.), Kocuria (K.) varians] hinsichtlich ihrer antiviralen Wirkung geprüft. Dabei konnte eine signifikante Titerreduktion von MNV S 99 bei Exposition mit dem Kulturüberstand eines Lactobacillus (Lb.) curvatus-Isolates festgestellt werden (p < 0,05). In mit dieser Kultur fermentiertem Tee- und Mettwurstbrät zeigte sich jedoch kein Effekt. Die Virustenazität von H1N1 und MNV S 99 konnte mit D,L-Milchsäure unter rohwurstrelevanten Bedingungen (pH 5,0 bis 6,2) sowohl in-vitro als auch im frischen Mettwurstbrät beeinflusst werden. In-vitro erzielte Titerreduktionen lagen bei 2,5 (H1N1) bzw. 3,25 log-Stufen (MNV S 99) nach drei Tagen (24 °C) Lagerung. Im Gegensatz dazu war MNV S 99 im Vergleich zu H1N1 im Mettwurstbrät stabiler. H1N1 konnte unterhalb von pH 5,5 bereits direkt nach dem Einmischen der Influenzaviren in das Wurstbrät nicht mehr nachgewiesen werden. MNV S 99 wurde hingegen erst nach einem Tag Lagerung (22 °C) maximal um 0,7 log-Stufen reduziert (pH 5,2). Die verwendeten Starter- und Schutzkulturen (Lb. sakei, Lb. curvatus, Lb. paracasei, Lb. plantarum, S. carnosus, S. xylosus, K. varians) zeigten im Mett- und Teewurstbrät im Vergleich zur Kontrolle (ohne Starterkultur) keinen zusätzlichen viruziden Effekt auf MNV S 99. Zunehmende Virustiterreduktionen konnten mit pH-Wert-Erniedrigung beobachtet werden. Nach der Reifung (1 Tag, 22 °C, pH 4,9) von Mettwurst mit Starterkulturen wurde das Virus um maximal 1,65 log-Stufen reduziert. In mit Einzel- beziehungsweise Mehrstamm-Mischkulturen fermentierter Teewurst (7 Tage, 22 °C, pH 4,9) betrug die Titerreduktion maximal 1,10 log-Stufen. Das Influenzavirus H1N1 konnte im Rohwurstbrät mit Starterkulturen auch nach Verwendung hoher Ausgangstiter bereits zu Beginn der Untersuchungen nicht mehr nachgewiesen werden. Aus den erzielten Daten kann geschlussfolgert werden, dass die Bacteriocine Sakacin A und Nisin nicht als antivirale Zusatzstoffe in Lebensmitteln (z. B. Rohwürste) geeignet sind. Das antivirale Potential von zellfreien Kulturüberständen war Bakterienstamm-spezifisch und nur in-vitro ersichtlich. Daher muss die Nutzung des Lb. curvatus 1-Stammes nicht anderen rohwurstrelevanten Starterkulturen vorgezogen werden. Die Verwendung von Milchsäure als Zusatzstoff im Rohwurstbrät eignet sich nur zum Ausschluss einer viralen Exposition im Zusammenhang mit H1N1. Frische Mettwurst muss allerdings hierzu adäquat gesäuert (pH < 5,5) werden. Neben dem antiviralen Effekt durch gebildete Säure, konnte keine weitere spezies-spezifische antivirale Wirkung verwendeter Starter- und Schutzkulturen auf MNV S 99 festgestellt werden. Die Säureleistung einzelner Kulturen ist demzufolge für eine Virusinaktivierung entscheidend. Das antivirale Potential verwendeter Starter- und Schutzkulturen in Rohwürsten ist im Zusammenhang mit MNV S 99 als gering einzuschätzen. Unter der Annahme, dass murine und humane Noroviren eine ähnliche Tenazität in kurzgereiften Rohwürsten aufweisen, sollten diese Produkte im Zusammenhang mit Noroviren als Risikoprodukte eingestuft werden.

Das Polysaccharid-Glykogen ist aus vielen Bakterien wie auch Eukaryoten bekannt. Es enthält ausschließlich über α1-4 Bindungen verknüpfte Glucoseeinheiten, die über α1-6-Bindungen verzweigt sind. Generell ist es als ein Speichermolekül anzusehen, das sowohl Kohlenstoff- als auch Energiequelle unter Stressbedingungen darstellt. Es ermöglicht die Aufrechterhaltung der Integrität der Zelle und die Erhaltung der notwendigen Stoffwechselvorgänge und führt zum Schutz einiger Zellbestandteile. In Bakterien ist die Regulation der Glykogen-Biosynthese durch die Kontrolle der Expression der Gene glg möglich. Der erste Schritt der Synthese ist die Bildung von ADP-Glucose aus Glucose-1-Phosphat durch ADP-Glucose-Pyrophosphorylase (ADP-Glc-PPase; ATP: α-d-glucose-1-Phosphat adenylyltransferase, EC 2.7.7.27), kodiert vom Gen glgC, gefolgt von den Reaktionen der Glykogen-Synthase (EC 2.4.1.21) und des Verzweigungsenzyms (EC 2.4.1.18), von dem die Genen glgA und glgB Genen kodiert sind. Der entscheidende Regulierungsschritt der Glykogen-Biosynthese in prokaryotischen Organismen, ist die durch ADP-Glc-PPase katalysierte Reaktion, die zur Bildung von ADP-Glukose und Pyrophosphat aus ATP und D-Glucose-1-Phosphat führt. Eine vergleichende Analyse des Glykogen-Biosynthese-Genclusters in Gram-negativen und Gram-positiven Bakterien zeigte, dass einige Gram-positive Spezies aus der Gattung Bacillus und Clostridium und den Milchsäurebakterien zwei Gene (glgC und glgD) besitzen. Sie kodieren für Proteine, die der ADP-Glc PPase ähnlich sind. Es wurde dokumentiert, dass GlgC und GlgD die Untereinheiten eines α2β2-Typ heterotetrameren Struktur bilden. Allerdings ist die Rolle von glgD bei Gram-positiven Bakterien noch unklar. Nur eine regulatorische Rolle des glgD in Bacillus stearothermophilus, ohne erkennbare enzymatische Aktivität des Protein-Produkts von GlgD ist bisher bekannt. In Bacillus subtilis und Streptomyces coelicolor hängt die Glykogen-Synthese mit der Sporulation und der Versorgung mit notwendigen Ressourcen zur Differenzierung zusammen. Die enzymatischen Aktivitäten der Glg Proteine, vor allem die Funktion von GlgD, welches Ähnlichkeiten in der Aminosäurensequenz mit GlgC zeigt, sind nicht charakterisiert. Demzufolge war der Schwerpunkt dieser Arbeit auf die Untersuchung der Funktion des glgC-homologen glgD Gens in einigen Michsäurebakterien (lactic acid bacteria, LAB) gerichtet. LAB sind eine Gruppe von fakultativ anaeroben, nicht pathogenen, nicht sporenbildenden grampositiven Bakterien mit wichtigen Funktionen für die menschliche Gesundheit und die Lebensmittelindustrie. Die vorliegende Arbeit ist auf die detaillierte funktionelle Analyse der beiden Gene glgC und glgD konzentriert, welche in den glgCDAP-B bzw. glgBCDAP Operons von Lactococcus lactis subsp. cremoris MG1363 und Lactobacillus plantarum WCFS1 liegen. Insbesondere war die Untersuchung der Funktion des glgC-homologen glgD Gens in LAB von Interesse für das Verständnis der Funktion des Speicher-Polysaccharids in dieser Organismengruppe. Mehr Informationen über die Funktion von glgD könnten dazu beitragen, den Mechanismus der Synthese und / oder Regulierung von Glykogen in dieser Bakteriengruppe zu verstehen und möglicherweise intrazelluläre Polysaccharidbildung (IPS) mit probiotischen Eigenschaften bestimmter Arten von LAB zu verknüpfen. Versuche zur funktionellen Charakterisierung dieser Gene schlossen derer Expression in verschiedenen E. coli Stämmen ein, dies gelang für alle Zielproteine in Form von unlöslichen Inclusion-bodies. Es wurden verschiedene Versuche unternommen, um die Bildung von Inclusion-bodies zu verhindern und eine größere Menge an löslichem Protein zu erhalten. Verschiedene Ansätze, wie Expression bei niedrigen Temperaturen, Wachstum unter Stress und Co-Expression mit verschiedenen Chaperonen sowie die Rückfaltung des Proteins aus Inclusion-bodies, waren nicht erfolgreich. Es war jedoch möglich, mittels einer Fusionsexpressionsuntersuchung der Gene glgC und glgD von Lb. plantarum WCFS1zu zeigen, dass es unter speziellen Wachstumsbedingungen eine Zunahme der Löslichkeit der Proteinfraktion um bis zu 50% im Vergleich zu den Standard-Zustand gab. Experimentell wurde nachgewiesen, dass die Proteine GlgC und GlgD stark miteinander interagieren. Beide Proteine scheinen Untereinheiten zu sein, die das voll aktive Enzym bilden. Das führt zum Modell bei dem α-und β-Untereinheiten eine heterotetrameren Struktur bilden, wie es schon zuvor im Gram-positiven Bakterium Bacillus stearothermophilus beschrieben worden ist. In dieser Studie konnte erstmals gezeigt werden, dass die GlgC und GlgD Proteine von Milchsäurebakterien miteinander interagieren. Außerdem wurde in dieser Studie auch eine niedrige, ATP-abhängige enzymatische Aktivität der GlgD Protein in Abwesenheit von GlgC beobachtet. Die Fähigkeit des GlgD Proteins ADP-Glc zu produzieren deutet auf eine mögliche auch katalytische und regulatorische Funktion des Proteins hin. Die ADP-Glc-PPase Aktivität wird offenbar in bestimmten LAB durch einen Protein-Komplex gebildet, der durch die Gene glgC und glgD kodiert wird. Diese Gene sind in folgenden LAB-Stämmen konserviert: Lactococcus lactis subsp. cremoris MG1363 und Lactobacillus plantarum WCFS1. Darüber hinaus weisen erfolglose Versuche zur von glgD getrennten Expression von glgC auf die Wichtigkeit von GlgD für die stabile und lösliche Expression von GlgC hin, der genaue Grund dafür ist unbekannt. Weitere Untersuchungen sind notwendig, um die vielen regulatorischen Aspekte des Glykogen Metabolismus in LAB sowohl auf Transkriptions- wie auch auf Translationsebene zu verstehen. Es könnte auch möglich sein, dass diese beiden Gene essentiell für das Wachstum dieser Arten sind, da sie trotz verschiedener Versuche mit verschiedenen Vektoren nicht deletiert werden konnten. Eine weitere wichtige Beobachtung dieser Studie wies darauf hin, dass UTP als Substrat für das gereinigte GlgD ist, ein Anzeichen für eine alternative Reaktion zur Produktion von UDP-Glucose. Dies könnte ein Hinweis für einen alternativen Weg der Glykogen-Biosynthese sein. Die Beobachtung, dass die glgC- und glgD-Gene offenbar essentiell sind und dass es möglicherweise einen alternativen Weg für die Glykogen-Biosynthese gibt, deutet darauf hin, dass Glykogen eine wichtige Rolle für das Überleben dieser LAB-Stämme spielt.

碩士
實踐大學
食品營養與保健生技學系碩士班
105
With increasing age, skeletal muscle mass is lost, and this aging atrophy is accompanied by a reduction in muscle strength. Probiotics have known a significant health benefit to the host. Therefore, this study was to investigate the effect of Lactic acid bacteria (LAB) on skeletal muscle energy metabolism following the long-term supplementation LAB for 12 weeks in senescence-accelerated mouse P8 (SAMP8). Sixteen-week-old; male and female; SAMP8 mice were randomly delivered into 4 groups (male = 5, female = 9), including comparison group (Non-Aging) and oral administration with water (Aging), LAB at 108 CFU/day (LAB 1X), and LPF at 109 CFU/day (LAB 10X) for 12 weeks. We evaluated body weight, food intake, body composition, open field test and muscle strength test. After sacrifice, muscle, liver and blood were collected for analysis. The result showed that LAB supplementation did not change the body weight, food intake and nutrition status, but it appeared to preserve muscle mass, motility and muscle strength function. LAB supplementation increased mitochondrial biogenesis genes expression and improve mitochondrial oxygen consumption rate. Moreover, decreased inflammatory factor in serum and protein carbonyl in muscle samples, but it was unable to significantly increased glycogen levels. In conclusion, we showed that LAB maintained the expression of mitochondrial-related transcription factors and oxidative stress in SAMP8 mice. Our results indicate that LAB is a promising candidate for relief from sarcopenia.

Dissertação de mestrado em Bioinformatics
The dairy food industry is constantly changing as novel biotechnological techniques improve the manufacturing process of dairy products. Widely used over the years in the yogurt and cheese manufacturing, Streptococcus thermophilus is now considered as an extremely valuable lactic acid bacterium for the annual market of the dairy industry. A specific, but of easy-access knowledge regarding the thermophilic bacteria metabolism would be a plus for the continuous growth of such industry. In this work, we present the Genome-Scale Metabolic (GSM) model for the LMD- 9 strain of S. thermophilus together with the detailed description of the species metabolic capabilities at the cellular level. The reconstruction of the genome-scale metabolic model, was performed using Metabolic Models Reconstruction Using Genome-Scale Information (merlin) together with COBRApy tool and OptFlux platform. S. thermophilus LMD-9 genome was functionally annotated and the encoded metabolic information was afterwards used to assemble a draft network. After extensive manual curation, the metabolic network was converted to a comprehensive metabolic model. The assembled GSM model was then validated against experimental data. The metabolism of this important stater for the dairy industry has been accessed in detail through the reconstruction. The organism possesses a simple machinery for central carbon metabolism and shows a narrow spectrum of carbohydrate utilization. The genome-scale metabolic model additionally suggests the existence of several pyruvate dissipating pathways which end in the synthesis of various compounds of interest. In silico simulations demonstrated the production of lactate and residual amounts of formate, acetolactate and acetaldehyde. Regarding the amino acid metabolism, the organism possesses complete pathways for the biosynthesis of all amino acids, except for lysine, methionine and cysteine. Furthermore, the GSM model can be used to simulate other relevant features of the S. thermophilus metabolism, such as the aroma compounds and Exopolysaccharides (EPS) synthesis, oxygen tolerance, absence of complete citrate cycle and pentose phosphate pathway, urea metabolism or amino acid catabolism.
A indústria dos lacticínios encontra-se em constante mudança devido ao aparecimento de novas técnicas biotecnológicas que permitem o melhoramento da produção dos laticínios. Amplamente utilizado ao longo dos anos na produção de iogurte e queijo, Streptococcus thermophilus é agora considerado extremamente valioso para o mercado anual desta indústria. Portanto, conhecimento especifico, mas facilmente acessível e compreensivo sobre o metabolismo da bactéria seria uma vantagem para o crescimento continuo desta industria. Nesta tese, apresentamos o modelo metabólico à escala genómica para a estirpe LMD-9 de S. thermophilus, juntamente com um estudo aprofundado das suas capacidades metabólicas. Para obter a reconstrução do modelo metabólico à escala genómica, foi usada principalmente a ferramenta merlin com o apoio da ferramenta COBRApy e a plataforma OptFlux. O genoma de S. thermophilus LMD-9 foi anotado e as informações metabólicas codificadas foram usadas para construir uma rede rascunho. Após curação manual, a rede metabólica foi convertida num modelo metabólico à escala genómica. Posteriormente, o modelo de S. thermophilus foi validado contra dados experimentais. O metabolismo desta bactéria acido láctica foi estudado em detalhe através da reconstrução. O organism dispõe de um metabolismo de carbono muito simples e um espectro de utlização de hidratos de carbono bastante reduzido. Além disso, o modelo desenvolvido sugere a existência de várias vias metabólicas que se iniciam no piruvato e terminam na síntese de vários compostos de interesse, embora as simulações in silico tenham demonstrado apenas a produção de lactato e quantidades residuais de formato, acetolactato e acetaldeído. No que diz respeito ao metabolismo dos aminoácidos, o organismo possui as vias completas para a biossíntese de todos aminoácidos, à exceção da lisina, metionina e cisteína. O modelo pode ser usado para simular outras características relevantes de S. thermophilus, tais como a síntese de EPS e compostos aromáticos, tolerância ao oxigénio, ausência de um ciclo completo do ácido cítrico ou da via das pentoses fosfato, metabolismo da ureia ou catabolismo de aminoácidos.

Proteolytic activity is very important characteristic of Lactic Acid Bacteria (LAB) They produce therapeutic benefits and also increase physiological activity of cultured dairy products by liberating a number of biologically active peptides. The main aim of this project was to determine the release of bioactive peptides from milk proteins by selected Lactobacillus species.

Sexually transmitted infections (STIs) such as gonorrhea, herpes simplex virus 2 (HSV 2), hepatitis C, human papilloma viruses (HPV) and human immunodeficiency virus type 1 (HIV) are a global health concern affecting millions of lives. Although extensive efforts have been geared towards prevention and treatment of STIs, little progress has been achieved. Recently, efforts to develop microbicides have been focused on the commensal bacterial species that comprise the vaginal microbiome and their role in immunity and disease pathogenesis. The lower FRT which includes the cervix and vagina has endogenous bacterial species that are supported by the mucosal epithelium. Lactobaccilli are the dominant endogenous bacterial species in the vagina of most women; lactobacilli convert glycogen to lactic acid (LA) which maintains a low pH environment in the vagina and serves as a deterrent to infectious organisms. The purpose of this research project was to determine the effects of LA on vaginal integrity and inflammation in a vaginal epithelial cell (VEC) tissue model, and on the ability of the broadly neutralizing anti-HIV antibody, VRCO1, to inactivate HIV in vitro. Effects of LA exposure on the viability and integrity of vaginal epithelium were determined by histology, MTT assay and measurement of transepithelial electric resistance (TEER). In addition, an enzyme-linked immunosorbent assay (ELISA) was used to measure concentrations of cytokines secreted by the VEC epithelial cells in response to different doses of LA. Using TLR agonists to simulate infection in the VEC model, we tested the hypothesis that LA has anti-inflammatory properties that modulate immune responses of the vaginal epithelium. We assessed the effect of LA on the neutralization activity of the anti-HIV-1 monoclonal antibody VRCO1 in the TZM-bl HIV neutralization assay. Tissue morphology and integrity were not affected by exposure to LA. Low concentrations of IL-1β and RANTES were expressed by VEC tissues in response to L-LA treatment. VEC tissues expressed significantly elevated concentrations of IL-1RA (p<0.0001), an anti-inflammatory cytokine, in response to LA regardless of incubation time and LA doses. In addition, treatment of VEC tissues with Poly I: C in the presence of LA dampened upregulated expression of IL-1β, TNF-alpha and IL-6 in response to the TLR 3 agonist. LA also elicited significantly higher IL-1RA concentrations when apically added to the TLR agonist-treated VEC tissues. These data suggest that LA elicits an anti-inflammatory response in the vaginal epithelium. LA acidic conditions as low as pH 3.5 did not affect the ability of VRC01, to prevent HIV infection. We found that LA, at concentrations present in vaginal secretions of normal women, inhibited the inflammatory response to a TLR agonist, possibly due to upregulated Il-1RA synthesis. In addition, the functionality of VRCO1 in an acidic milieu was not diminished, providing evidence that antibodies can function in the low pH vaginal environment. This report highlights the potential use of LA in the vagina as a microbicide due to its virucidal activity and anti-inflammatory properties. It also indicates that monoclonal antibody-based vaginal microbicides will not be neutralized by LA. There is still a need to elucidate the exact mechanisms by which LA confers immuno-modulatory properties within the female reproductive tract.

Type II diabetes mellitus (T2DM) is a chronic disease that has a worldwide prevalence which is expected to rise dramatically over the course of the next thirty years. The disease has reached pandemic stages of development in many cultures, most notably in developing countries, followed somewhat closely by developed countries with access to an overabundance of refined carbohydrates and fat (refined oils). T2DM is a condition that can be prevented or managed, but not cured; therefore a method of stymieing the development of this disease is paramount to halting its progressively increasing morbidity. In this study, bartlett pear and kiowa blackberry were investigated in relation to their ability to modify and improve both glucose metabolism and hypertension management with in vitro assay models. Effectiveness and bioactive functionality was evaluated by various in vitro assays to study the properties of: 100% bartlett pear juice, 100% kiowa blackberry juice and a ratio of 70:30 pear: blackberry juice found to have increased phenolic properties due to synergy in previous studies. These assays aimed at determining: alpha-amylase and alpha-glucosidase inhibition, angiotensin converting enzyme inhibition, total soluble phenolic content and antioxidant capabilities. These juices were also fermented with Lactobacillus helveticus and Bifidobacterium longum, common yogurt culture strains, to investigate if fermentation would improve the bioactive functionality of pear: blackberry synergies. A secondary goal of the experiment was to investigate if these fruit juices could prevent the growth of Helicobacter pylori, which is a common bacterium found in the stomach which can lead to cancer.

Kluyveromyces lactis è un lievito non convenzionale ampiamente utilizzato sia nelle applicazioni industriali che nella ricerca di base. Nel nostro laboratorio stiamo studiando il gene KlMGA2, che codifica per un modulatore della risposta all’ipossia. In lavori precedenti, abbiamo mostrato come la delezione del gene KlMGA2 in K. lactis generi un ceppo vitale, ma con difetti di crescita cellulare, alterazione in quantità e composizione degli acidi grassi, respirazione difettosa e morfologia mitocondriale alterata. Con questo lavoro mostriamo come l'assenza del gene KlMGA2 causi, oltre ai fenotipi sopra citati, una maggiore resistenza allo stress ossidativo ed una longevità molto estesa, accompagnati da un aumento dell’espressione genica delle catalasi e delle superossido dismutasi. Questo potrebbe suggerire un coinvolgimento di KlMga2 come mediatore diretto non solo della risposta ipossica, ma anche nella risposta allo stress ossidativo, ipotizzando una correlazione tra ipossia, regolazione del glucosio, biosintesi degli acidi grassi e metabolismo dei ROS. In seconda analisi, con questo lavoro si è cercato di verificare una possibile risposta di K. lactis allo stress luminoso. Negli organismi unicellulari come il lievito, che non presentano tessuti specializzati per contrastare i cambiamenti dell’ambiente esterno, l’esistenza di meccanismi cellulari adattativi alle condizioni di stress risulta essere di fondamentale importanza. Precedenti studi, hanno evidenziato che in Saccharomyces cerevisiae l’impulso luminoso rappresenta una fonte di stress, in quanto provoca l’aumento intracellulare di perossido di idrogeno (H2O2). Visti i nostri studi riguardanti KlMga2 come mediatore di risposta allo stress ossidativo, abbiamo indagato sul suo possibile coinvolgimento nella trasduzione del segnale luminoso. Pertanto, tale studio permetterebbe di esaminare dei meccanismi foto-dipendenti anche in organismi apparentemente privi di domini proteici sensibili alla luce.
The yeast Kluyveromyces lactis has been widely used in both industrial applications and basic research. We previously demonstrated that deletion of the KlMGA2, coding for a hypoxic mediator in K. lactis, generated a viable strain, although suffering of several deficiencies. We also showed that glucose signaling and glucose catabolism were involved in KlMga2 regulation. In this work, we showed that, in addition to these defects, the deletion of KlMGA2 also caused increased resistance to oxidative stress and extremely extended lifespan. These phenotypes are associated with increased expression levels of catalase and superoxide dismutase genes. We propose that KlMga2 might act as a direct mediator not only of hypoxic response, but also of oxidative stress response/adaptation, thus revealing connections between hypoxia, glucose signaling, fatty acid biosynthesis and ROS metabolism. Secondly, in this work we wanted to investigate the possible light response in this yeast. In unicellular organisms like yeasts, that cannot utilize specialized tissue for protection against environmental challenges, the presence of cellular mechanisms to respond and adapt to stress conditions is fundamental. Saccharomyces cerevisiae has been reported to respond to light by increasing hydrogen peroxide (H2O2) levels. Therefore, it could be interesting to study the possible role of oxidative stress mediator KlMga2, already studied in our laboratory, in the light response of yeast.