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 / Doctor of Agricultural Science / Kyoto University / DFAM

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Die Verbraucherakzeptanz von fermentierten Milchprodukten wie Joghurt oder Frischkäse ist von ihrer Textur abhängig. Zur Einstellung der produktspezifischen Textur ohne Zusatz von spezifischen Milchproteinen zur Basismilch oder milchfremden Hydrokolloiden werden in der Milchindustrie spezielle Starterkulturen eingesetzt, die während der Fermentation Exopolysaccharide (EPS) bilden. Diese EPS werden in der Regel als freie EPS ins Serum abgegeben oder verbleiben als kapsuläre EPS an der Bakterienzelle. Freie EPS lassen sich weiter anhand ihres Effektes im Produkt in fadenziehend und nicht-fadenziehend unterteilen. Aufgrund der hohen Biodiversität der EPS sowie des komplexen Zusammenhanges zwischen EPS-Eigenschaften, Substratzusammensetzung und Verfahrensbedingungen existieren jedoch widersprüchliche Ergebnisse zu ihrer Funktionalität im Produkt. Ziel dieser Arbeit war es, die wichtigsten Einflussfaktoren durch Einsatz verschiedener EPS von Lactococcus lactis und Streptococcus thermophilus in definierten Modellsystemen für stichfeste, gerührte und konzentrierte fermentierte Milchprodukte zu identifizieren und die Textureigenschaften mit den strukturellen und makromolekularen Eigenschaften der EPS in Verbindung zu bringen. Neunundzwanzig Einzelstämme von verschiedenen Starterkulturherstellern wurden auf Basis von der Fadenlänge als Indikator für den Fadenzug und der EPS-Menge in sechs Cluster eingeteilt. Aus jedem Cluster wurden Vertreter zur Herstellung von Modellsystemen genutzt, die Textur und Synärese der Produkte instrumentell erfasst und statistisch bewertet, um Einflussfaktoren auf die Eigenschaften der Modellprodukte zu identifizieren. Stichfester Modelljoghurt mit fadenziehenden EPS wies eine höhere Steifigkeit auf als jener mit nicht-fadenziehenden EPS, welche nicht mit der EPS-Menge oder der Fermentationszeit korrelierte. Unabhängig von der Art der freien EPS war beim Vorhandensein von kapsulären EPS die Gelsteifigkeit zusätzlich erhöht. Für gerührten Modelljoghurt ergab eine Komponentenanalyse zur EPS-Menge, zur Säuerungskinetik und zu Produkteigenschaften, dass fadenziehende EPS texturrelevante Eigenschaften wie Partikelgröße und Scherviskosität signifikant beeinflussen. Dies wurde auf die höhere intrinsische Viskosität für fadenziehende EPS im Vergleich zu nicht-fadenziehenden EPS zurückgeführt. Experimente mit einem Dehnrheometer ergaben, dass die Dehnviskosität stärker als die Scherviskosität mit der Fadenlänge zunahm. Ein Grund dafür sind intensivere Wechselwirkungen von fadenziehenden EPS mit Proteinen oder untereinander, was anhand höherer Relaxationszeiten in Dehnung bei höherer Fadenlänge geschlussfolgert wurde. Fadenziehende EPS erhöhten zudem die Fließgrenze von Modellfrischkäse, was hauptsächlich auf EPS-Wechselwirkungen und nicht auf die EPS-Konzentration oder Partikelgröße zurückgeführt wurde. Eine niedrigere Synärese und höhere Steifigkeit wurde entweder beim Vorhandensein von kapsulären EPS oder freien EPS beobachtet, die in Experimenten mit dynamischer Wasserdampfsorption eine höhere Feuchtebeladung aufwiesen. Fadenziehende EPS und kapsuläre EPS besaßen auch im Doppelrahmfrischkäse nach moderater Bruchhomogenisierung (0,05 MPa oder 15 MPa) eine hohe Funktionalität, was mit der höheren intrinsischen Viskosität dieser EPS korrelierte. Die Bruchhomogenisierung bei einem Druck von 30 MPa führte jedoch zu einer niedrigeren Funktionalität aufgrund der scherinduzierten Reduzierung der molekularen Masse der EPS und Umlagerungen in der Mikrostruktur der Käse. Die Ergebnisse zeigen, dass die Technofunktionalität nicht von der Konzentration der in situ produzierten EPS abhängt. Schlüsselfaktoren sind insbesondere die EPS-Lokalisierung (frei oder kapsuläre), intensivere Wechselwirkungen der fadenziehenden EPS mit Proteinen oder untereinander und ein hohes Wasserbindungsvermögen der EPS. Jedoch sollte der Energieeintrag bei der postfermentativen Verarbeitung so niedrig wie möglich sein, um die Funktionalität der fadenziehenden EPS und kapsulären EPS weitestgehend zu erhalten. Dieses Wissen ist für die Auswahl von EPS-bildenden Starterkulturen für den industriellen Maßstab entscheidend, um die gewünschten Produkteigenschaften zu erhalten.

Within the last decade, pork consumption has steadily increased and continues to be the most consumed meat globally. However, pathogenic bacterial strains resistant to antibiotics have also been increasingly found in pig farms, animals, and the environment. Bacterial food poisoning cases due to Salmonella spp. and Escherichia coli (E. coli) O157:H7 appear to be linked with a variety of pork products. The meat industry has recognized that research is needed to combat the multi-drug resistance in foodborne pathogens with alternative methods of control. This study evaluated the effects of both E. coli- and Salmonella-specific lytic bacteriophages and lactic acid (LA) on E. coli O157:H7, Salmonella Enteritidis, Salmonella Montevideo, and Salmonella Heidelberg growth in raw pork loins ready for marination. The efficacy of the treatments was determined after 1 h of application and marination. Lytic bacteriophage 5% significantly (PSalmonella spp. population by 2.30 log CFU/cm2 when compared with the initial surface attachment. Moreover, the combined treatment of LA 2.5% + phage 5% significantly (PSalmonella population by 2.35 log CFU/cm2 after 1 hour of attachment. In the post-tenderization surface samples, the combination of both phage and LA showed (PP>0.05) when analyzing the translocation of Salmonella spp. on pork loins. Similar treatment efficacy results were observed in the application of E. coli O157:H7 on pork. Following antimicrobial treatments, both control and treated loin samples were enumerated after 1 h at 4°C. Both the lytic bacteriophage 5% and the combination of lytic bacteriophage 5% with lactic acid 2.5% had a significant reduction of E. coli O157:H7 on surface attachment after 1 h of treatment application. Lytic bacteriophage 5% and Lactic acid 2.5% significantly (P < 0.05) reduced the surface bacterial population by 1.89 log CFU/cm2. Lytic bacteriophage 5% alone significantly (P < 0.05) reduced the surface bacterial population by 1.90 log CFU/cm2 when compared with the initial surface attachment groups. Moreover, in the post-tenderization surface samples, lytic bacteriophage 5% and the combination of lytic bacteriophage 5% with lactic acid 2.5% were the only treatments that had a significant reduction (P < 0.05) when compared with the control group. Interestingly, lactic acid 2.5% was the only treatment that had a significant reduction (P < 0.05) of 0.76 log CFU/cm2 when analyzing the translocation of pathogens on pork chops.

The relationship between polymer chain architecture, crystallization behavior, and morphology formation was investigated. The structures formed are highly dependent on chain configuration and crystallization kinetics. Poly(lactic acid) (PLA) and Poly(ethylene-co-vinyl acetate) (EVA) random copolymers were studied. Sample characterization was performed using a variety of techniques, including spectroscopy, scattering, and calorimetry. In PLA, structural differences between α’ and α crystalline phases were analyzed using cryogenic infrared and Raman spectroscopy. Compared to the  crystal, the ’ crystal has slightly looser packing and weaker intermolecular interactions involving carbonyl and methyl functional groups. Simulations in conjunction with Raman scattering analyzed the conformational distortion of the α’ phase. The conformation of an α’ chain was determined to have tg’t-103 conformation with tg’t-31 units randomly distributed along the chain. Departure of the O-C α dihedral angle was also confirmed. The structural disorder leads to different thermal properties for α’ and α crystalline forms, which was quantified by measuring the enthalpic change at melting for both crystals ( = 57 ± 3 J/g and =9 6 ± 3 J/g). The transformation from α’ to α and the mechanism of order formation in PLA were also elucidated. The relationship between chain configuration of EVA random copolymers and crystallization behavior was established. For three different EVA samples, the distribution of methylene sequences was calculated and compared to a distribution of crystallite sizes formed. This comparison revealed that only a small fraction of the total methylene segments present actually crystallized. Cocrystallization with highly mobile oligomers was explored to enhance the crystallization of EVA copolymers. When blended, EVA28 (28 weight percentage) cocrystallizes with C36H74 n-alkane resulting in faster crystallization kinetics and a higher degree of crystallinity. The observed increase in degree of crystallinity was directly related to the chain configuration. Compositional mapping using Raman spectroscopy provided evidence for oligomer nucleation. The cocrystallization kinetics and morphology of EVA and n-alkane blends was found to depend on the chain length of oligomer. In both systems studied, crystallization kinetics determines the morphologies formed, which are undoubtedly related to the details of molecular architecture.

Capsaicin, prostaglandin E2 (PGE2) and polydopamine (PDA) were used to target macrophage and myofibroblast activity to reduce biomaterial-induced fibrosis. The lifetime and efficacy of implantable biomedical devices are determined by the foreign body response. Immediately after implantation, proteins nonspecifically adsorb onto the material and initiate inflammation. Macrophages recruited to the site can differentiate into M1 and M2 phenotypes and upregulate inflammation and fibrosis which interferes with the intended function. M1 macrophages secrete pro-inflammatory mediators that induce chronic inflammation and promote myofibroblast differentiation while M2 macrophages are wound healing cells that suppress inflammation and regulate fibroblast activity. The fibrotic tissue is developed by myofibroblasts which produce collagen in an unregulated fashion. Collagen thickening and biomaterial encapsulation decreases efficacy and sensitive of biomedical devices. We investigated the in vitro and in vivo effects of capsaicin, PGE2 and polydopamine surface modification on macrophages and myofibroblasts. Capsaicin and PGE2 reduced poly(lactic-co-glycolic) acid (PLGA)-induced fibrosis by promoting M2 macrophage phenotype to secrete anti-inflammatory IL-10 and suppressing myofibroblast marker α-smooth muscle actin (α-SMA). Capsaicin decreased collagen by 40% and upregulated IL-10 secretion by 35% while PGE2 reduced collagen by 55% after 14 days of implantation and 40% less collagen after 42 days. PDA was used to bind an anti-fibrotic compound to the surface of a poly(dimethyl siloxane) (PDMS-PDA) to reduce fibrosis. However, PDMS-PDA controls gave an unexpected result by reducing fibrosis to the same extent as anti-fibrotic compound bound PDMS- v PDA. PDA modification reduced cellularity by 50% and significantly decreased collagen thickness by 30%. Overall, our results showed that biomaterial-induced fibrosis can be reduced by promoting M2 macrophage activity and inhibiting myofibroblast differentiation. This research demonstrates three compounds that have potential to reduce fibrosis and extend the lifetime and efficacy of implantable biomedical devices. / Thesis / Master of Applied Science (MASc) / Capsaicin, prostaglandin E2 (PGE2) and polydopamine were used to reduce scar tissue development around implanted polymers. Biomedical devices implanted in the body can undergo severe scar tissue formation, or fibrosis, and fail. Fibrosis is described by the accumulation of collagen and encapsulation of an implanted polymer. Macrophages regulate fibrosis by secreting pro-fibrotic compounds and myofibroblasts produce unregulated amounts of collagen. In this thesis, capsaicin, PGE2 and polydopamine were incorporated into implants to target macrophage and myofibroblast activity and reduce fibrosis in mice. Capsaicin and PGE2, released from a degradable polymer, altered macrophages to secrete anti-fibrotic compounds and decreased collagen by 40% and 55%, respectively. Polydopamine surface modified implants gave an unexpected result and suppressed overall cell activity to reduce fibrosis by 30%. The research conducted shows the potential of these compounds to reduce fibrosis and extend the lifetime of implantable devices.

Mitochondrial diseases are caused by gene mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) and they are among one of the most common forms of inherited disorders. It is estimated that 1 out of every 5000 individuals will develop a mitochondrial disease in their lifetime. Due to the crucial and widespread functionality of mitochondria in human cells, prolonged diseases of the mitochondria affect cells of the brain, heart, liver, muscles and kidneys and can lead to multi-organ failure in some patients. Inherited or acquired mitochondrial diseases can present at any stage of life, affecting both children and adults. Since its discovery, the mitochondrial genome has been analyzed and sequenced with increasing ease and this process has helped recognize various mitochondrial disorders as the root of genetic diseases. This paper will explore the unique properties of the mitochondrion and its genome, examine the relationship between mtDNA and some common myopathies such as Leigh syndrome (LS) or maternally inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in order to explore commonalities and differences in their inheritance patterns and their effect on mitochondrial function. Although studies have shown that these conditions generally affect the process of oxidative phosphorylation in mitochondria, because of the wide variety of presentations of this disease, further research is needed to understand the different etiologies, as well as to explore novel therapies to treat them.

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Yes / The shape memory effect of highly oriented long‐chain‐branched poly(lactic acid) (LCB‐PLA) prepared through solid‐phase die drawing technology was studied by comparison with PLA. When the recovery temperature increased from 60°C to 120°C, for PLA, only one‐step recovery at about 80°C can be observed and the recovery ratio was below 21.5%, while, for LCB‐PLA, multiple recovery behavior with high recovery ratio of 78.8% can be achieved. For oriented PLA, the recovery curve of the final sample showed the same trend with that of sample suffering just free drawing; while for oriented LCB‐PLA, the recovery curve of the final sample showed the same trend with that of sample suffering just die drawing. After shape recovery, the mechanical properties of LCB‐PLA showed a linear downward trend with the recovery temperature. Together with amorphous phase, the oriented mesomorphic phase, which formed during solid die drawing, can act as switching domains. And thus, upon heating, the chain segment of amorphous phase relaxed at first and triggered the first macroscopical shape recovery, leading to the decrease of long period (Lac) and the thickness of the amorphous layer (La). Then, with further increasing temperature, the oriented mesomorphic phase gradually relaxed resulting subsequently multi‐shape recovery, and the Lac and the La further decreased. Therefore, by regulating the recovery temperature of oriented LCB‐PLA, the shape recovery ratio and mechanical strength can be controlled effectively, and thus the self‐reinforced and self‐fastening effect can be achieved simultaneously for PLA as bone fixation material.