Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

<p>U doktorskoj disertaciji evaluirani su rezultati kvantifikovanih koncentracionih nivoa lipofilnih perzistentnih i emergentnih organskih polutanata u uzorcima sedimenta kolektovanih sa deset reprezentativnih lokaliteta u srednjem Podunavlju. Na osnovu sprovedenih laboratorijskih i terenskih istraživanja definisan je prostorni trend jedinjenja na ispitivanom području. Primenom multivarijantnih tehnika eksperimentalni rezultati su uspešno modelovani statističkim metodama koje su izdiferencirale izvore kontaminacije za ukupan set ispitivanih polutanata. Prikazana je procena uticaja kontaminiranog sedimenta na akvatične organizme i humanu populaciju prema stepenu kancerogenosti jedinjenja. Predstavljeni su različiti scenariji izloženosti ingestijom i dermalnim kontaktom, u zavisnosti od vremena ekspozicije i izložene površine potencijalnih receptora, različitog uzrasta i pola. Po prvi put u istraživanom području, implementirana je ex-situ ravnotežna metodologija pasivnog uzorkovanja primenom sorpcionog medijuma od silikonske gume testirane pri različitim masenim odnosima polimer-uzorak sedimenta. Primenjenom metodom uspešno su dobijene slobodno rastvorene koncentracije lipofilnh kontaminanata u pornoj vodi sedimenta, kao prediktora za određivanje biodostupnosti jedinjenja.</p> / <p>The doctoral dissertation evaluated the results of quantified concentration levels of lipophilic persistent and emergent organic pollutants in sediment samples collected from ten representative localities in the central Danube region. Based on the conducted laboratory and field research, the spatial distribution of compounds in the studied area was defined. Using multivariate techniques, the experimental results were successfully modeled by statistical methods that differentiated the sources of contamination for the total set of tested pollutants. A risk assessment of contaminated sediment on aquatic organisms and the human population according to carcinogenicity of the tested compounds is presented in thesis. Different exposure scenarios of ingestion and dermal contact, depending on the time exposure and exposed surface skin of potential receptors, of different ages and sex, are presented. For the first time in the study area, an ex-situ equilibrium passive sampling methodology was implemented using silicone rubber sorption medium tested at different polymer-sample sediment mass ratios. Using the method, the freely dissolved concentrations of lipophilic contaminants in pore water were successfully obtained as predictors for determining the bioavailability of the compounds.</p>

Filled hydrogel particles consisting of emulsified oil droplets encapsulated within a hydrogel matrix were fabricated based on the phase separation of proteins and polysaccharides through aggregative and segregative mechanisms. A 3% (wt/wt) pectin and 3% (wt/wt) caseinate mixture at pH 7 separated into an upper pectin-rich phase and a lower casein-rich phase. Casein-coated lipid droplets added to this mixture partitioned into the lower casein-rich phase. When shear was applied, an oil-in-water-in-water (O/W1/W2) emulsion consisting of oil droplets (O) contained within a casein-rich dispersed phase (W1) suspended in a pectin-rich continuous phase (W2) was formed. Acidification from pH 7 to 5 promoted adsorption of pectin onto casein-rich W1 droplets, forming filled hydrogel particles. Particles were then cross-linked using transglutaminase. Particles were assessed for stability to changes in pH, increasing levels of salts (sodium chloride and calcium chloride), and susceptibility to lipid oxidation. Both cross-linked and not cross-linked particles were stable at low pH (pH 2-5). At high pH, cross-linked particles maintained their integrity while not cross-linked particles disintegrated. Particles were stable to sodium chloride (0-500 mM). Calcium chloride levels above 4 mM resulted in system gelation. The rate of lipid oxidation for 1% (vol/vol) fish oil encapsulated within filled hydrogel particles was compared to that of oil-in-water emulsions stabilized by either Tween 20 or casein. Emulsions stabilized by Tween 20 oxidized faster than either filled hydrogel particles or casein stabilized emulsions, while filled hydrogel particles and casein stabilized emulsions showed similar oxidation rates. Using an in-vitro digestion model, the digestion of lipid encapsulated within filled hydrogel particles was compared to that of a casein stabilized oil-in-water emulsion. Results showed similar rates of digestion for both hydrogel and emulsion samples. Attempts to fabricate particles using free oil (rather than emulsified oil) were unsuccessful and resulted in the formation of large non-encapsulated oil droplets (d ~10 μm). By controlling particle concentrations of biopolymer, water, and oil, it was possible to fabricate particles that were highly resistant to gravitational separation which was attributed to the equivalent density of the continuous and particle phases. Results highlight the potential applications and versatility of this delivery system.

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13413号 / 論農博第2896号 / 新制||農||1085(附属図書館) / 学位論文||R3||N5323(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 松村 康生, 教授 白岩 立彦, 教授 丸山 伸之 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

This thesis explored how asphaltene and naphthenic amphiphile species influence the formation (morphology and size) and stability of heavy crude oil (bitumen) emulsions. It was experimentally shown that asphaltenes produce water-in-oil emulsions. Naphthenic amphiphiles on the other hand flip the emulsion morphology to oil-in-water. It was further demonstrated that the size and stability of these emulsions is influenced by physicochemical effects such as the pH, solvent-bitumen-water ratios, solvent aromaticity, and temperature. In view of these findings, the hydrophilic-lipophilic deviation (HLD) and net-average curvature (NAC) models were looked at as potential means for predicting the formation and stability of emulsions. Owing to the complexity of bitumen emulsions, however, the HLD and NAC models were instead tested against well-defined sodium dihexyl sulfosuccinate-toluene-water emulsions. The morphologies of these emulsions were predicted as a function of the formulation salinity whereas corresponding droplet sizes were predicted as a function of the continuous phase density and interfacial tension (γow). Emulsion stability trends were in turn predicted using a collision-coalescence-separation assumption. From this assumption, emulsion stability was expressed as a function of the emulsion droplet collision frequency and activation energy. The key parameters of the highly scrutinized activation energy term included the γow, interfacial rigidity, and critical film thickness. In applying the same modeling approach to the stability of other emulsions already published in the literature, it was found that the rigidity of adsorbed multilayer/liquid crystal films cannot yet be fully accounted for. This shortcoming was the reason for which only minimum stability times were reported for bitumen emulsions.

This thesis explored how asphaltene and naphthenic amphiphile species influence the formation (morphology and size) and stability of heavy crude oil (bitumen) emulsions. It was experimentally shown that asphaltenes produce water-in-oil emulsions. Naphthenic amphiphiles on the other hand flip the emulsion morphology to oil-in-water. It was further demonstrated that the size and stability of these emulsions is influenced by physicochemical effects such as the pH, solvent-bitumen-water ratios, solvent aromaticity, and temperature. In view of these findings, the hydrophilic-lipophilic deviation (HLD) and net-average curvature (NAC) models were looked at as potential means for predicting the formation and stability of emulsions. Owing to the complexity of bitumen emulsions, however, the HLD and NAC models were instead tested against well-defined sodium dihexyl sulfosuccinate-toluene-water emulsions. The morphologies of these emulsions were predicted as a function of the formulation salinity whereas corresponding droplet sizes were predicted as a function of the continuous phase density and interfacial tension (γow). Emulsion stability trends were in turn predicted using a collision-coalescence-separation assumption. From this assumption, emulsion stability was expressed as a function of the emulsion droplet collision frequency and activation energy. The key parameters of the highly scrutinized activation energy term included the γow, interfacial rigidity, and critical film thickness. In applying the same modeling approach to the stability of other emulsions already published in the literature, it was found that the rigidity of adsorbed multilayer/liquid crystal films cannot yet be fully accounted for. This shortcoming was the reason for which only minimum stability times were reported for bitumen emulsions.

Naturally occurring food antimicrobials such as plant essential oils are receiving tremendous interest as intervention systems to enhance microbiological safety and quality. Poor water solubility of essential oils makes it difficult to incorporate them in foods, impacting visual appearance, antimicrobial effectiveness, and possibly organoleptic properties. Engineered nanoscale delivery systems can principally solve these challenges, but those based on low-cost food ingredients and inexpensive and scalable processes are currently scarce. This dissertation presents a simple and scalable two-step technology to prepare nano-delivery systems. The first encapsulation step, based on emulsion-evaporation, involves preparing emulsions composed of an oil phase with thymol or eugenol, major compounds in extracts from thyme and clove respectively, in hexane and an aqueous phase with conjugates of whey protein isolate and maltodextrin, followed by evaporation of hexane by spray drying. The second step is to hydrate spray dried capsules to enable the formation of nanoscale particles. The encapsulation performance and dispersion characteristics were affected by amounts and types of conjugates (ratio of protein: maltodextrin and maltodextrin chain length), volume fraction and composition of the oil phase. The optimal conditions corresponded to 55.8 % encapsulation efficiency and 12.6 % loading for thymol and 47.9 % encapsulation efficiency and 7.9 % loading for eugenol. Dispersions prepared from the identified capsules contained particles smaller than 100 nm and were transparent at pH 3.0-7.0 and 0-50 mM before and after heating at 80°C for 15 min. Nano-dispersions and free oil were tested for antimicrobial activity against Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella typhimurium. Nano-dispersed and free antimicrobials had similar effectiveness at various pH and temperatures in tryptic soy broth and apple cider, while in 2 % reduced fat milk, nano-dispersed antimicrobials were consistently more effective than unencapsulated ones. Therefore, the commercially viable nanoscale technology presented in this study enables the delivery of lipophilic antimicrobials for enhanced microbial safety and quality, without compromising visual appearance of foods, especially clear beverages.

Abstract The SOM in boreal forests contains non-living heterogeneous components resulting from microbial and chemical transformations of organic debris from plant litter. The major components in the plant biomass all decompose at different rates and therefore, contribute variably to the stable storages of soil C. The aims of the current thesis were 1) to explore how climate, soil fertility and initial litter quality affect the decomposition rate of litter, 2) to study how the different carbon fractions found in the plant litter relate to the quality and quantity of SOM in forest soils, 3) to determine whether the recalcitrant fraction of litter is derived from lignin and other polyphenols or from lipophilic compounds and carbohydrates, and 4) to determine whether the litter originating from different plant growth forms affects SOM formation in a similar way. The study was conducted in six north boreal and six south boreal study sites, half of which were mesic and half were sub-xeric. The overall initial litter quality and decomposition rate of carbon fractions did not differ between the two fertility levels and climate regimes. Litter with high initial water-soluble extractives (WSE) and nitrogen (N) decomposed at a faster rate than litter with lower initial WSE and N concentration irrespective of the soil fertility or climate conditions. Although decomposition rate varies among litter types, decomposition rate cannot explain differences in SOM quality or quantity between the northern and southern boreal forests. The organic matter accumulation and relative proportion of acid-insoluble residue (AIR) in SOM was higher in south boreal sites both in sub-xeric and mesic sites. Detailed characterization of the AIR fraction using pyrolysis-GC demonstrated that in the litter layer the concentration of AIR contains lignin and other insoluble polyphenols, but in the F and H layers, lignin-derived and chemically modified polyphenolics and decomposition products of resin acids. Although the concentration of the AIR fraction varies among litter types, its composition varied very little among the litter types, and between a sub-xeric and a mesic site. The results of this study suggest that the differences in plant community structure and quantitative differences in the litter input by various growth forms has more impact on OM accumulation than decomposition conditions in coniferous soils. / Tiivistelmä Metsämaan orgaaninen aines koostuu eriasteisesti hajonneesta karikkeesta sekä pitkälle maatuneesta, hajottajien muokkaamasta humuksesta. Tutkimuksessa selvitettiin 1) kuinka ilmasto, maaperän ravinteisuus ja karikkeen kemialliset ominaisuudet vaikuttavat karikkeen hajoamisnopeuteen, 2) kuinka kasvien sisältämät erilaiset hiilifraktiot vaikuttavat maaperän orgaanisen aineen laatuun ja määrään, 3) millaiset hiiliyhdisteet muodostavat vaikeimmin hajoavan hiilifraktion karikkeessa ja pidemmälle maatuneessa orgaanisessa aineessa ja 4) onko erilaisten kasvien vaikutus orgaanisen aineksen muodostumiseen samanlainen kuusikoissa ja männiköissä pohjois- ja eteläboreaalisella havumetsävyöhykkeellä. Tutkimuksessa havaittiin, että karikkeen maatumisnopeudessa ei ollut eroja pohjois- ja eteläboreaalisella kasvuvyöhykkeellä eikä kuusikoiden ja männiköiden väillä. Karike, joka sisälsi runsaasti vesiliukoisia yhdisteitä ja typpeä maatui nopeammin kuin vähän vesiliukoisia yhdisteitä tai typpeä sisältävä karike. Karikkeen maatumisnopeus ei kuitenkaan kyennyt selittämään eroja pidemmälle maatuneen orgaanisen aineksen määrässä ja laadussa pohjois- ja eteläboreaalisella kasvuvyöhykkeellä. Orgaanisen aineksen määrä oli suurempi eteläboreaalisella kasvuvyöhykkeellä verrattuna pohjoisboreaaliseen. Lisäksi vaikeasti hajoavia hiiliyhdisteitä (AIR-fraktio) kertyi eteläboreaaliseen metsämaahan enemmän kuin pohjoiseen. Karikekerroksessa ligniini ja polyfenolit muodostivat suuren osan AIR-fraktiosta. Maatumisen edetessä AIR-fraktion muodostavat lähinnä ligniinin hajoamisesta peräisin olevat ja muut modifioituneet polyfenolit sekä pihkahappojen hajoamistuotteet. Vaikka AIR-fraktion konsentraatio vaihteli kariketyypeittäin, sen kemiallinen koostumus oli hyvin samanlainen karikelajista riippumatta. Tutkimuksen perusteella vallitseva kasvillisuus ja sen tuottaman karikkeen määrä ja laatu vaikuttavat enemmän metsämaahan kertyvän orgaanisen aineen määrään kuin maatumisolosuhteet.

The Caco-2 cell assay is widely used for in vitro permeability measurements. However, a draw back with the assay that this study will focus on improving, is compound adsorption to the plastic material. Lipophilic compounds such as Cyclosporin A and Peptide J, that will be used in this study, tend to bind to the plastic material in the assay. This can result in poor recovery and misleading permeability predictions. Bovine serum albumin (BSA) is an alternative used today to prevent this but is not always successful.    The aim of this study is therefore to improve the Caco-2 permeability assay by adding phospholipid covered silica beads (PLB) to the basolateral chamber. The role of the PLB is to bind the compound of interest and decrease the amount of compound bound to the plastic material and thus better predict the permeability of the compound of interest.   The PLB was produced using phosphatidylcholine and silica beads. Caco-2 cells were seeded and maintained for 21-29 days ahead of the experiment. PLB concentration of 20, 60 and 100 mg/ml were prepared. Samples were analyzed with HPLC-MSMS. The results showed that with increasing PLB concentration we had a significantly decrease in non-specific plastic binding resulting in reliable permeability predictions, concluding that the hypothesis was correct.

This diploma thesis deals with testing, optimazing and comparing methods for the identification of bacteria producing polyhydroxyalkanoates. Work included cultivation and microscopy methods, wherein the bacterial cells were stained with lipophilic dyes Nile red and Sudan black. Further, we also used flow cytometry and spectroscopic methods - Raman spectroscopy and infrared spectroscopy with Fourier transformation, and molecular biological methods, which analyzed the presence of a gene encoding PHA synthase (phaC) by polymerase chain reaction (PCR). PCR assay consist of two reactions, the firt on eis based on amplification of phaC gene along with 16S rRNDA gene, which is common for all the bacteria (multiplex PCR). The second reaction is focused on specific amplification of PHA synthase catalyzing biosynthesis of mcl-PHA. In order to overcome false positive results typical for methods analyzing genotype and also to avoid false negative results occuring in fenotype analyzing methods, the best strategy is to combine both aproaches. According to our results, analysis of presence of phaC gene by PCR can be combined with methods capable of determining presence of PHA in bacterial cells. For this purpose, Raman microspectroscopy seems to be very promising tool, since it is able to detect low content of PHA in cells and PHA can not be confused with other lipid metabolites. The results provide an overview of test methods, their advantages and disadvantages and also to compare different criteria according to which it is possible to choose the method of identification in depending on the adjustable requirements.