Spectroscopic Imaging for the Detection and Identification of Bacterial Contaminations

There exist many types of harmful bacteria that can contaminate foods and cause serious illness, which often have their own best courses of treatment. This requires the classification of different types of bacteria. Traditional methods of bacterial identification, while able to provide accurate classification, are often very time-consuming processes. In the case of a potentially fatal bacterial infection, time is often of the essence. FTIR spectroscopy is a faster, more practical alternative that can discriminate different strains of bacteria, based on their spectral signatures, with high confidence. Bacterial contaminations on food exist as small, localized colonies that must be found and identified. As FTIR spectroscopy is a fast technique for one sample location, it is advantageous to move towards spectroscopic imaging to do FTIR spectroscopy at many locations in parallel. The amount of information obtained at one time from this technique is large, thus it can be helpful to visualize this information by means of color images. A multivariate image analysis will produce such color images depicting the different chemical properties of a material as different colors. This enables the visual discrimination of a bacterial colony from its substrate, as they will have different chemical properties. Colonies can be found in a larger sample area by merging data cubes as presented here. Once a bacterial colony is found in an extended area, it must be identified. A method of bacterial identification has been developed here that can identify bacterial colonies based on chemical information obtained from spectroscopic imaging. While spectroscopic imaging is useful for acquiring chemical information in two spatial dimensions, it inherently loses height information, which is important for investigating chemical reactions proceeding in three dimensions. This information is of key importance in the growth monitoring of bacterial colonies. Thus, novel illumination optics have been coupled with a spectroscopic imaging setup to probe the surface topography of a 3D sample while being able to acquire spectroscopic information simultaneously

Study for poly[(R)-3-hydroxybutyrate] production by bacterial strains and its modification for drug delivery systems

Nowadays biopolymers are a good alternative to petrochemical polymers. One of these biopolymers is Poly[(R)-3- Hydroxybutyrate] (PHB) which is produced from renewable resources by various microorganisms and, after its use it can be composted. It has different applications as bulk material in packing films, containers or paper coatings. Due to its excellent biocompatibility and biodegradability, PHB is a potential material for applications in drug delivery. It is therefore important to continue investigating the different strategies to ensure the competitiveness between these biopolymers and petroleumbased polymers. The first part of the thesis is mainly focused on finding a new PHB-producing bacterium capable of developing and producing a significant concentration of biopolymer using one of the conventional medium for PHB industrial production. Therefore, four water and mud samples were taken from four Bolivian salty lakes and the microorganisms were isolated in order to detect the best PHB-producing bacterial strain. A bacterium identified as a new strain of the genus Bacillus was selected for polymer production studies. This microorganism was tested under fed-batch fermentations. The results showed a high specific growth rate, and a percentage of 70% PHB in cell dry mass , with limitation in nitrogen source. The strain not only grew properly in the industrial condition proposed, but it also produced and accumulated a larger content of PHB than ever reached before for its genus. The strain was deposited in the Spanish Type Culture Collection (CECT), as Bacillus megaterium uyuni S29. The characterization of the resultant biopolymers concluded that the strain synthesized PHB homopolymer with two main molecular masses around 600 and 125 kDa with polydispersity index of 1.2 and 1.5 respectively. Moreover, the thermal analyses of the biomaterial showed different properties compared to the ones of common PHB, enlarging the application possibilities to this biopolyester. After obtaining the polymer from the bacteria, the second part was focused on its transformation. A sustainable degradation process set out for an easy industrial scale application was proposed in the second part of this thesis. A copolymer from PHA family, poly[3-hydroxybutyrate-co-4-hydroxybutyrate] , was used to carry out an enzymatic degradation of the material. Two commercially available lipases were used to decrease the polymer molecular mass to oligomers between 5 and 1 kDa. It was the first time that these triglyceride lipases were used for this type of application and due to the positive results one of them was used for degradation of PHB. The results confirmed the enzymatic reaction of the used lipase with PHB and showed a controlled decrease of the molecular mass from 300 kDa to 4 kDa. In the third part of the study, the application of the PHB as a drug delivery system was analyzed. Doxycycline is a well known broad-spectrum antibiotic but there are some concerns over its possible side effects. In order to avoid this problem and reach the infection with an effective drug concentration, protection and controlled delivery of doxycycline are desirable. Thus, different drug-entrapment and emulsification methods were studied in order to obtain optimal doxycycline -loaded PHB micro- and nano-particles. The results showed that the combination of ultrasounds with high speed stirring in the preparation of double emulsion is highly effective in obtaining doxycycline -loaded PHB particles with high drug loading and entrapment efficiency, and with great method efficiency. Furthermore, the qualitative antibacterial activity found in different formulations show that these particles are good candidates for drug delivery systems. These best methods were used with the PHAs produced throughout the thesis in order to confirm their suitability for different PHAs and to study the possibility of improving the results of drug entrapment and method efficiency.Actualmente, los biopolímeros son una buena alternativa a los polímeros derivados del petróleo. Uno de estos biopolímeros es el poli[(R)-3-hidroxibutirato] (PHB) producido a partir de recursos renovables por diversos microorganismos. Tiene diferentes aplicaciones como material a granel para films de embalaje, recipientes o revestimientos de papel. Debido a su excelente biocompatibilidad y biodegradabilidad, el PHB es un material con gran potencial para ser aplicado como vehículo para administración de fármacos. La primera parte de la tesis se centra en la búsqueda de una nueva bacteria productora de PHB capaz de desarrollar y producir una concentración significativa de biopolímero usando un medio convencional de forma industrial. Se partió de cuatro muestras de agua y barro tomadas de cuatro lagos salados de Bolivia. Los microorganismos se aislaron con el fin de detectar las mejores cepas bacterianas productoras de PHB. Para los estudios de producción de polímeros, se seleccionó una nueva cepa del género Bacillus, identificada como Bacillus megaterium uyuni S29. Se utilizó este microorganismo en diferentes fermentaciones fed-batch. Los resultados mostraron una alta tasa de crecimiento específico y se llegó a un porcentaje final de PHB de 70% en masa celular, llegando a limitar la fuente de nitrógeno. La cepa no sólo creció adecuadamente en las condiciones industriales propuestas, sino que también produjo y acumuló un gran contenido de PHB que no se había logrado antes con este género. La caracterización de los biopolímeros resultantes concluyó que Bacillus megaterium uyuni S29 sintetiza PHB homopolímero con dos masas moleculares principales de alrededor de 600 y 125 kDa con índices de polidispersidad de 1,2 y 1,5 respectivamente. Además, los análisis térmicos mostraron propiedades diferentes a las del PHB común, ampliando las posibilidades de aplicación de este biopoliéster. Después de obtener el polímero, la segunda parte de la tesis se centró en el estudio de su transformación. Por lo tanto, se estudió una propuesta de proceso sostenible de degradación de PHAs pensada para una posterior aplicación a escala industrial. Se utilizó un copolímero de la familia de los PHAs, el poli(3-hidroxibutirato-co-4- hidroxibutirato), para llevar a cabo su degradación enzimática. Se emplearon dos lipasas comerciales para disminuir la masa molecular del polímero y obtener oligómeros con masa molecular entre 5 y 1 kDa. Fue la primera vez que dichas lipasas de triglicéridos han sido utilizadas con este copolímero. Por consiguiente, se empleó una de ellas para la degradación de PHB. Se confirmó que la reacción enzimática de esta lipasa con el PHB produjo una disminución controlada de la masa molecular de 300 kDa a 4 kDa. En la tercera parte de la tesis se analizó la aplicación del PHB como sistema de liberación de fármaco. Doxiciclina es un conocido antibiótico de amplio espectro. Con el fin de evitar efectos secundarios y llegar a la infección con una concentración de fármaco eficaz, se requiere una protección y administración controlada de doxiciclina . Así que se estudiaron diferentes métodos de atrapamiento molecular y de preparación de emulsificaciones con el fin de obtener micro- y nano-partículas de PHB cargadas con doxiciclina . Los resultados mostraron que la combinación de ultrasonidos con agitación a alta velocidad en la preparación de emulsión doble es muy eficaz para la obtención de estas partículas. Además, los resultados de actividad antibacteriana llevados a cabo con estas partículas mostraron que son buenas candidatas para la liberación doxiciclina . Los mejores métodos encontrados para la formación de partículas de PHB se utilizaron con los PHAs producidos a lo largo de la tesis para confirmar su idoneidad con otros PHAs

HISTOLOGICAL STUDIES OF BREWERY SPENT GRAINS IN DIETARY PROTEIN FORMULATION IN DONRYU RATS

The increasing production of large tonnage of products in brewing industries continually generates lots of solid waste which includes spent grains, surplus yeast, malt sprout and cullet. The disposal of spent grains is often a problem and poses major health and environmental challenges, thereby making it imminently necessary to explore alternatives for its management. This paper focuses on investigating the effects of Brewery Spent Grain formulated diet on haematological, biochemical, histological and growth performance of Donryu rats. The rats were allocated into six dietary treatment groups and fed on a short-term study with diet containing graded levels of spent grains from 0, 3, 6, 9, 12 and 100% weight/weight. The outcome demonstrated that formulated diet had a positive effect on the growth performance of the rats up to levels of 6% inclusions, while the haematological and biochemical evaluation revealed that threshold limit should not exceed 9% of the grain. However, the histological study on the liver indicated a limit of 3% inclusion in feed without serious adverse effect. Thus invariably showing that blend between ranges 1-3% is appropriate for the utilization of the waste in human food without adverse effect on the liver organ. The economic advantage accruing from this waste conversion process not only solves problem of waste disposal but also handle issues of malnutrition in feeding ration

Inkorporace biosyntetizovaných nanočástic ušlechtilých kovů do vlákenných nosičů a možnosti jejich využití

Import 23/07/2015This diploma thesis is focused on polymer fibers with incorporated silver and gold biosynthesized nanoparticles prepared by different spinning methods – centrifugal spinning and electrostatic spinning (NanospiderTM technique). Theoretical part offers a brief introduction to a polymer world and introduces certain polymers often used in the spinning applications and their characteristics. Description of fiber spinning methods is included. Experimental part comprises all data about bio-metallic nanoparticles preparation and characterization by Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD), Atomic Emission Spectroscopy (AES-ICP), Infrared Spectroscopy (FTIR) and size distribution methods. Fibrous samples obtained by spinning methods are characterized by Scanning Electron Microscopy (SEM), TEM and FTIR. The efficiency of used spinning methods is compared and the quality of fibrous samples with incorporated nanoparticles is discussed. Additionally, antimicrobial properties of biosynthesized silver and gold nanoparticles are confirmed by antimicrobial evaluation.Diplomová práce je zaměřena na přípravu a vlastnosti biosyntetizovaných stříbrných a zlatých nanočástic zabudovaných v polymerních vláknech pomocí vybraných zvlákňovacích metod, a to odstředivým a elektrostatickým zvlákněním (technika NanospiderTM). Teoretická část se zabývá vlastnostmi vybraných polymerů, které jsou v praxi hojně využívány při přípravě vláken. Dále je uveden přehled různých v současnosti využívaných zvlákňovacích metod a jednotlivé metody jsou stručně charakterizovány. Experimentální část podává informace o přípravě a charakterizaci biosyntetizovaných nanočástic stříbra a zlata a zvlákněných vzorků. K analýzám byly využity následující metody: TEM, SEM, XRD, ICP-AES, FTIR a hodnocení velikostní distribuce nanočástic. Využitelnost zvlákňovacích metod a výsledných vzorků se zabudovanými nanočásticemi je rovněž diskutována. Navíc byly testovány antimikrobiální účinky připravených nanočástic pomocí stanovení minimální inhibiční koncentrace a diskového difuzního testu.9360 - Centrum nanotechnologiívýborn

Solid-state fermentation of cassava roots using cellulolytic-type alkaliphilic Bacillus spp. cultures to modify the cell walls and assist starch release

To improve cassava starch extraction by wet milling, solid-state fermentation of ground roots using cellulolytic-type alkaliphilic Bacilli spp., Bacillus akibai, B. cellulosilyticus and B. hemicellulosilyticus was investigated. Enzyme assay and scanning electron microscopy indicated that Bacillus spp. production of extracellular cellulase and polygalacturonase caused the formation of micropores through the root parenchyma cell walls and exposed the embedded cellulosic network. Gas chromatography data of the cell wall constituent sugars remaining after fermentation and Fourier transform infrared data indicated that the Bacillus treatments reduced the levels of pectin and, hemicellulose and to lesser extent cellulose. Wide-angle X-ray scattering data indicated that the Bacillus spp. cell wall degrading enzymes had partially hydrolysed the amorphous fractions of the cell wall polysaccharides. All the Bacillus spp. treatments improved starch extraction by 17–23% compared to fermentation with endogenous microflora. B. cellulosilyticus was most effective in disintegration of large root particles and as result, released marginally the most starch, probably due to it having the highest cellulase activity. Solid-state fermentation using cellulolytic-type Bacillus spp. is, therefore, promising to technology to improve the efficiency of cassava wet milling cell wall disintegration and consequent starch yield without use of commercial cell wall degrading enzymes or polluting chemicals.The International Centre for Development Oriented Research in Agriculture and a University of Pretoria Institutional Research Theme bursary.http://link.springer.com/journal/120102021-02-27hj2021Consumer ScienceFood Scienc

Characterization of chitin and chitosan derived from Hermetia illucens, a further step in a circular economy process

Due to their properties and applications, the growing demand for chitin and chitosan has stimulated the market to find more sustainable alternatives to the current commercial source (crustaceans). Bioconverter insects, such as Hermetia illucens, are the appropriate candidates, as chitin is a side stream of insect farms for feed applications. This is the first report on production and characterization of chitin and chitosan from different biomasses derived from H. illucens, valorizing the overproduced larvae in feed applications, the pupal exuviae and the dead adults. Pupal exuviae are the best biomass, both for chitin and chitosan yields and for their abundance and easy supply from insect farms. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscope analysis revealed the similarity of insect-derived polymers to commercial ones in terms of purity and structural morphology, and therefore their suitability for industrial and biomedical applications. Its fibrillary nature makes H. illucens chitin suitable for producing fibrous manufacts after conversion to chitin nanofibrils, particularly adults-derived chitin, because of its high crystallinity. A great versatility emerged from the evaluation of the physicochemical properties of chitosan obtained from H. illucens, which presented a lower viscosity-average molecular weight and a high deacetylation degree, fostering its putative antimicrobial properties

Production of Polyhydroxyalkanoates by Pseudomonas mendocina using vegetable oils and their characterisation

Synthesis of Polyhydroxyalkanoates (PHAs) by Pseudomonas mendocina, using different vegetable oils such as, coconut oil, groundnut oil, corn oil and olive oil, as the sole carbon source was investigated for the first time. The PHA yield obtained was compared with that obtained during the production of PHAs using sodium octanoate as the sole carbon source. The fermentation profiles at shaken flask and bioreactor levels revealed that vegetable oils supported the growth of Pseudomonas mendocina and PHA accumulation in this organism. Moreover, when vegetable oil (coconut oil) was used as the sole carbon source, fermentation profiles showed better growth and polymer production as compared to conditions when sodium octanoate was used as the carbon source. In addition, comparison of PHA accumulation at shaken flask and fermenter level confirmed the higher PHA yield at shaken flask level production. The highest cell mass found using sodium octanoate was 1.8 g/L, whereas cell mass as high as 5.1 g/L was observed when coconut oil was used as the feedstock at flask level production. Moreover, the maximum PHA yield of 60.5% dry cell weight (dcw) was achieved at shaken flask level using coconut oil as compared to the PHA yield of 35.1% dcw obtained using sodium octanoate as the sole carbon source. Characterisations of the chemical, physical, mechanical, surface and biocompatibility properties of the polymers produced have been carried out by performing different analyses as described in the second chapter of this study. Chemical analysis using GC and FTIR investigations showed medium chain length (MCL) PHA production in all conditions. GC-MS analysis revealed a unique terpolymer production, containing 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid when coconut oil, groundnut oil, olive oil, and corn oil were used as the carbon source. Whereas production of the homopolymer containing 3-hydroxyoctanoic acid was observed when sodium octanoate was used as the carbon source. MCL-PHAs produced in this study using sodium octanoate, coconut oil, and olive oil exhibited melting transitions, indicating that each of the PHA was crystalline or semi-crystalline polymer. In contrast, the thermal properties of PHAs produced from groundnut and corn oils showed no melting transition, indicating that they were completely amorphous or semi-crystalline, which was also confirmed by the X-Ray Diffraction (XRD) results obtained in this study. Mechanical analysis of the polymers produced showed higher stiffness of the polymer produced from coconut oil than the polymer from sodium octanoate. Surface characterisation of the polymers using Scanning Electron Microscopy (SEM) revealed a rough surface topography and surface contact angle measurement revealed their hydrophobic nature. Moreover, to investigate the potential applicability of the produced polymers as the scaffold materials for dental pulp regeneration, multipotent human Mesenchymal stem cells (hMSCs) were cultured onto the polymer films. Results indicated that these polymers are not cytotoxic towards the hMSCs and could support their attachment and proliferation. Highest cell growth was observed on the polymer samples produced from corn oil, followed by the polymer produced using coconut oil. In conclusion, this work established, for the first time, that vegetable oils are a good economical source of carbon for production of MCL-PHA copolymers effectively by Pseudomonas mendocina. Moreover, biocompatibility studies suggest that the produced polymers may have potential for dental tissue engineering application

Étude des mécanismes d’inactivation des microorganismes suite à un traitement à l’ozone

Une partie du travail a mené a un dépôt de brevet.Notre thèse s’inscrit dans le cadre de la compréhension des mécanismes d’inactivation de microorganismes, principalement des spores bactériennes, suite à un traitement à l’ozone. Les objectifs généraux, au départ, étaient de mettre en place une stratégie expérimentale dans le but de déterminer les espèces et les phénomènes impliqués dans les mécanismes d’inactivation des spores en combinant diverses techniques pluridisciplinaires. Rappelons que les récentes avancées en stérilisation par plasma sont principalement dues aux études menées sur les mécanismes d’inactivation des microorganismes. Dans ce contexte, en nous appuyant sur une synthèse des études publiées dans la littérature scientifique, nous avons élaboré une étude expérimentale en deux étapes : l’étude de la stérilisation à l’ozone en milieu sec puis en milieu humide. Au cours de ces études, nous avons principalement étudié la cinétique d’inactivation des microorganismes, les dommages occasionnés à ces derniers par de tels traitements et nous avons identifié les espèces responsables de l’inactivation pour, enfin, proposer un mécanisme d’inactivation en spécifiant le rôle et l’importance des différents phénomènes en jeu. L’originalité de notre approche s’appuie sur la corrélation faite entre ces deux types de traitement (ozone sec et humidifié) ainsi que la diversité et la complémentarité des moyens de caractérisation utilisés. Dans un second volet, nous avons mis en évidence pour la première fois la possibilité de conférer à une surface de Pétri de polystyrène (PS) une importante activité biocide suite à un traitement à l’ozone. Nous avons mené une étude de caractérisation des propriétés de ces surfaces démontrant, principalement, leur grande efficacité biocide sur une large gamme de microorganismes, l’importance de l’interaction microorganismes/surface traitée, l’existence de la rémanence biocide, les modifications physico-chimiques induites en surface… L’étude des modifications physico-chimiques et microbiologiques nous a permis d’approcher les mécanismes d’inactivation en identifiant, de manière non exhaustive, les phénomènes impliqués. Enfin, nous avons examiné l’applicabilité de cette méthode à divers types de polymères montrant que le degré d’activité biocide induite dépend de la nature du substrat.The thesis deals with the inactivation mechanisms of microorganisms (mainly endospores) after an ozone treatment. The general objective of this work was to provide an experimental strategy to identify the phenomena and chemical species involved in microorganism inactivation mechanism using multidisciplinary techniques. Recall that the recent advances in plasma sterilization are mainly due to comprehensive studies dealing with the inactivation mechanisms of microorganisms. In this context, based on a review of published studies, we proposed a two-step experimental study of ozone sterilization in both dry and humid media. In the course of this work, we have examined the inactivation kinetics of some microorganisms, their damage after treatments; we have also identified the chemical species responsible for inactivation, and proposed an inactivation mechanism (for each type of ozone treatment) by describing the importance and role of the different implicated phenomena. The originality of our approach rests on the correlation achieved between the dry and humid ozone treatment, and by the diversity and complementarity of the characterization techniques used. In a second part, we show for the first time the possibility to confer biocide activity to surfaces of polystyrene (PS) Petri dishes after their exposure to ozone. A characterization study of these treated surfaces mainly shows a high inactivation efficacy on various microorganisms, the importance of the microorganisms/treated-surface interaction, the biocide persistence of the treated surfaces and physico-chemical modifications. The study on physico-chemical and microbiological changes gives us elements for identifying the phenomena involved in the inactivation mechanisms. Finally, we have shown the possibility to confer biocide properties to polymeric surfaces in general with the same experimental process observing that the biocide efficacy depends on the nature of the polymer

Antioxidative potential of the Oyster mushroom (Pleurotus ostreatus) cultivated on grape pomace and straw supstrate

Oxidative stress caused by an imbalanced metabolism and an excess of reactive oxygen species (ROS) lead to a range of health disorders. Synthetic antioxidants can improve defence mechanisms, but their potential adverse toxic effects give priority to natural antioxidants. Pleurotus ostreatus (Jacq. ex Fr.) P. Kumer (1871) (oyster mushroom) is one of the most commonly cultivated mushrooms in the world due to its adaptibility to various substrates and having a specific mild taste of oyster in culinary processing. Because of the presence of various active ingredients it owns antidiabetic, antibacterial, anticholestrolic, antiarthritic, antioxidant, anticancer and antiviral activities. The aim of this study was to monitor the influence of grape pomace supstrate on the antioxidative potential of oyster mushroom, as well as changes of antioxidant properties from the day of harvesting during the storage period of 7 and 14 days. The substrate used for cultivation of Pleurotus ostreatus was consisted of grape pomace and straw (20:80%) The fruiting bodies were air-dried at 55°C, powdered and then prepared as crude hot water extracts in mild conditions, at 75-85°C for 1.5 h. Concentration range of 0.625-40 mg/mL of each extract was analyzed. The radical absorbance ability of the extract was tested in vitro, using DPPH free radical and ABTS+ radical scavenging capability and expressed as EC50 (mg/mL) values. Crude water extracts showed the following results: EC50 values of the DPPH scavenging ability on the 0th, 7th and 14th day were 30.25, 29.24 and 19.9 mg/mL, respectively. EC50 values of the ABTS+ scavenging activity of the extracts on the 0th, 7th and 14th day showed the difference, with following values: 8.1, 8.5 and 2.6 mg/mL, respectively. All investigated extracts showed good antioxidant abilities. Both methods confirmed decrease of the EC50 values and increasing of the antioxidative potential from harvesting day and during the storage period of 7 and 14 days