Effect of water vapour stabilization of raw materials on the quality of the inhalation product

Generation of raw materials for dry powder inhalers by different size reduction methods can be expected to influence physical and chemical properties of the powders. This can cause differences in particle size, size distribution, shape, crystalline properties, surface texture and energy. These physical properties of powders influence the behaviour of particles before and after inhalation. Materials with an amorphous surface have different surface energy compared to materials with crystalline surface. This can affect the adhesion and cohesion of particles. Changes in the surface nature of the drug particles results in a change in product performance. By stabilization of the raw materials the amorphous surfaces are converted into crystalline surfaces. The primary aim of the study was to investigate the influence of the surface properties of the inhalation particles on the quality of the product. The quality of the inhalation product is evaluated by measuring the fine particle dose (FPD). FDP is the total dose of particles with aerodynamic diameters smaller than 5,0 μm. The secondary aim of this study was to achieve the target level of the FPD and the stability of the FPD. This study was also used to evaluate the importance of the stabilization of the inhalation powders. The study included manufacturing and analysing drug substance 200 μg/dose inhalation powder batches using non-stabilized or stabilized raw materials. The inhaler formulation consisted of micronized drug substance, lactose <100μm and micronized lactose <10μm. The inhaler device was Easyhaler®. Stabilization of the raw materials was done in different relative humidity, temperature and time. Surface properties of the raw materials were studied by dynamic vapour sorption, scanning electron microscopy and three-point nitrogen adsorption technique. Particle size was studied by laser diffraction particle size analyzer. Aerodynamic particle size distribution from inhalers was measured by new generation impactor. Stabilization of all three raw materials was successful. A clear difference between nonstabilized and stabilized raw materials was achieved for drug substance and lactose <10μm. However for lactose <100μm the difference wasn’t as clear as wanted. The surface of the non-stabilized drug substance was more irregular and the particles had more roughness on the surface compared to the stabilized drug substances particles surface. The surface of the stabilized drug particles was more regular and smoother than non-stabilized. Even though a good difference between stabilized and non-stabilized raw materials was achieved, a clear evidence of the effect of the surface properties of the inhalation particles on the quality of the product was not observed. Stabilization of the raw materials didn’t lead to a higher FPD. Possible explanations for the unexpected result might be too rough conditions in the stabilization of the drug substance or smaller than wanted difference in the degree of stabilization of the main component of the product <100μm. Despite positive effects on the quality of the product were not seen there appears to be some evidence that stabilized drug substance results in smaller particle size of dry powder inhalers.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Identification of the fungal catabolic D-galacturonate pathway

Pectin is a natural polymer consisting mainly of D-galacturonic acid monomers. Microorganisms living on decaying plant material can use D-galacturonic acid for growth. Although bacterial pathways for D-galacturonate catabolism had been described previously, no eukaryotic pathway for D-galacturonate catabolism was known at the beginning of this work. The aim of this work was to identify such a pathway. In this thesis the pathway for D-galacturonate catabolism was identified in the filamentous fungus Trichoderma reesei. The pathway consisted of four enzymes: NADPH-dependent D-galacturonate reductase (GAR1), L-galactonate dehydratase (LGD1), L-threo-3-deoxy-hexulosonate aldolase (LGA1) and NADPH-dependent glyceraldehyde reductase (GLD1). In this pathway D-galacturonate was converted to pyruvate and glycerol via L-galactonate, L-threo-3-deoxy-hexulosonate and L-glyceraldehyde. The enzyme activities of GAR1, LGD1 and LGA1 were present in crude mycelial extract only when T. reesei was grown on D-galacturonate. The activity of GLD1 was equally present on all the tested carbon sources. The corresponding genes were identified either by purifying and sequencing the enzyme or by expressing genes with homology to other similar enzymes in a heterologous host and testing the activities. The new genes that were identified were expressed in Saccharomyces cerevisiae and resulted in active enzymes. The GAR1, LGA1 and GLD1 were also produced in S. cerevisiae as active enzymes with a polyhistidine-tag, and purified and characterised. GAR1 and LGA1 catalysed reversible reactions, whereas only the forward reactions were observed for LGD1 and GLD1. When gar1, lgd1 or lga1 was deleted in T. reesei the deletion strain was unable to grow with D-galacturonate as the only carbon source, demonstrating that all the corresponding enzymes were essential for D-galacturonate catabolism and that no alternative D-galacturonate pathway exists in T. reesei. A challenge for biotechnology is to convert cheap raw materials to useful and more valuable products. Filamentous fungi are especially useful for the conversion of pectin, since they are efficient producers of pectinases. Identification of the fungal D-galacturonate pathway is of fundamental importance for the utilisation of pectin and its conversion to useful products.

Turvemaan männyn (Pinus sylvestris L.) ja kuusen (Picea abies [L.] Karst.) kanto- ja juuripuun ominaisuudet ja asetoniliukoisten uuteaineiden määrä

Tutkimus on osa Metsäklusteri Oy:n Future Biorefinery –tutkimusohjelmaa, jossa kartoitetaan mahdollisuuksia hyödyntää metsäteollisuuden raaka-aineita aiempaa tarkemmin ja uusissa tuotteissa. Tutkimuksen tavoitteena on selvittää männyn (Pinus sylvestris L.) ja kuusen (Picea abies [L.] Karst.) juurten ja kantopuun rakenne ja ominaisuudet. Tutkimuksessa selvitetään löytyykö männyn ja kuusen juurista reaktiopuuta ja määritetään asetoniliukoisten uuteaineiden osuus kantoja juuripuussa. Tutkimusaineistona oli viisi eri-ikäistä mäntyä ja viisi eri-ikäistä kuusta. Juuri- ja kantoaineisto kerättiin Metsäntutkimuslaitoksen toimesta Parkanon seudulta (62.017°N, 23.017°E) hakkuun jälkeen. Maanalaisista juurista otettiin näytteet kolmelta eri etäisyydeltä juurenniskaan nähden. Kummankaan lajin juurista ei löytynyt varsinaista reaktiopuuta, mutta joissakin näytteissä havaittiin lievää reaktiopuuta. Lievää reaktiopuuta löytyi enemmän männyn kuin kuusen juurista, eikä sitä löytynyt lainkaan kaikkein ohuimmista, noin 2 cm paksuisista juurenosista. Männyn kannoissa uuteaineprosentti oli korkeampi kuin kuusen. Männyn juurissa uuteaineprosentti kasvoi edettäessä kohti juuren kärkeä. Kuusella uuteaineprosentti laski aluksi, mutta lähellä juuren kärkeä taas kasvoi. Kuoren uuteainepitoisuus oli molemmilla puulajeilla korkeampi kuin puuaineen. Tutkimusaineisto oli suppea, eikä tutkimuksessa pyritty tilastolliseen yleistettävyyteen. Laajemmasta aineistosta tehdylle tutkimukselle on tarvetta, sillä turvekankailta saatavan puun tarjonta on Suomessa kasvussa, mutta juurten uuteaine- ja reaktiopuututkimuksia on tehty vain kivennäismailta kerätyistä aineistoista.

Studies on Stone Age Housepits in Fennoscandia (4000 - 2000 cal BC) : Changes in ground plan, site location and degree of sedentism

Housepits have a remarkably short research history as compared to Fennoscandian archaeological research on the Stone Age in general. The current understanding of the numbers and the distribution of Stone Age housepits in the Nordic countries has, for the most part, been shaped by archaeological studies carried out over the last twenty to thirty years. The main subjects of this research are Neolithic housepits, which are archaeological remains of semi-subterranean pithouses. This dissertation consists of five peer-reviewed articles and a synthesis paper. The articles deal with the development of housepits as seen in the data gathered from Finland (the Lake Saimaa area and south-eastern Finland) and Russia (the Karelian Isthmus). This synthesis expands the discussion of the changes observed in the Papers to include Fennoscandian housepit research as a whole. Certain changes in the size, shape, environmental location, and clustering of housepits extended into various cultures and ecological zones in northern Fennoscandia. Previously, the evolution of housepits has been interpreted to have been caused by the adaptation of Neolithic societies to prevailing environmental circumstances or to re-organization following contacts with the agrarian Corded Ware/Battle Axe Cultures spreading to North. This dissertation argues for two waves of change in the pithouse building tradition. Both waves brought with them certain changes in the pithouses themselves and in the practices of locating the dwellings in the environment/landscape. The changes in housepits do not go hand in hand with other changes in material culture, nor are the changes restricted to certain ecological environments. Based on current information, it appears that the changes relate primarily to the spread of new concepts of housing and possibly to new technology, as opposed to representing merely a local response to environmental factors. This development commenced already before the birth of the Corded Ware/Battle Axe Cultures. Therefore, the changes are argued to have resulted from the spreading of new ideas through the same networks that actively distributed commodities, exotic goods, and raw materials over vast areas between the southern Baltic Sea, the north-west Russian forest zone, and Fennoscandia.

Rapsista tuotetun biodieselin ja vehnästä tuotetun bioetanolin energiavirrat ja ympäristövaikutukset Euroopan unionissa

The aim of this thesis was to study the crops currently used for biofuel production from the following aspects: 1. what should be the average yield/ ha to reach an energy balance at least 0 or positive 2. what are the shares of the primary and secondary energy flows in agriculture, transport, processing and usage, and 3. overall effects of biofuel crop cultivation, transport, processing and usage. This thesis concentrated on oilseed rape biodiesel and wheat bioethanol in the European Union, comparing them with competing biofuels, such as corn and sugarcane-based ethanol, and the second generation biofuels. The study was executed by comparing Life Cycle Assessment-studies from the EU-region and by analyzing them thoroughly from the differences viewpoint. The variables were the following: energy ratio, hectare yield (l/ha), impact on greenhouse gas emissions (particularly CO2), energy consumption in crop growing and processing one hectare of a particular crop to biofuel, distribution of energy in processing and effects of the secondary energy flows, like e.g. wheat straw. Processing was found to be the most energy consuming part in the production of biofuels. So if the raw materials will remain the same, the development will happen in processing. First generation biodiesel requires esterification, which consumes approximately one third of the process energy. Around 75% of the energy consumed in manufacturing the first generation wheat-based ethanol is spent in steam and electricity generation. No breakthroughs are in sight in the agricultural sector to achieve significantly higher energy ratios. It was found out that even in ideal conditions the energy ratio of first generation wheat-based ethanol will remain slightly under 2. For oilseed rape-based biodiesel the energy ratios are better, and energy consumption per hectare is lower compared to wheat-based ethanol. But both of these are lower compared to e.g. sugarcane-based ethanol. Also the hectare yield of wheat-based ethanol is significantly lower. Biofuels are in a key position when considering the future of the world’s transport sector. Uncertainties concerning biofuels are, however, several, like the schedule of large scale introduction to consumer markets, technologies used, raw materials and their availability and - maybe the biggest - the real production capacity in relation to the fuel consumption. First generation biofuels have not been the expected answer to environmental problems. Comparisons made show that sugarcane-based ethanol is the most prominent first generation biofuel at the moment, both from energy and environment point of view. Also palmoil-based biodiesel looks promising, although it involves environmental concerns as well. From this point of view the biofuels in this study - wheat-based ethanol and oilseed rape-based biodiesel - are not very competitive options. On the other hand, crops currently used for fuel production in different countries are selected based on several factors, not only based on thier relative general superiority. It is challenging to make long-term forecasts for the biofuel sector, but it can be said that satisfying the world's current and near future traffic fuel consumption with biofuels can only be regarded impossible. This does not mean that biofuels shoud be rejected and their positive aspects ignored, but maybe this reality helps us to put them in perspective. To achieve true environmental benefits through the usage of biofuels there must first be a significant drop both in traffic volumes and overall fuel consumption. Second generation biofuels are coming, but serious questions about their availability and production capacities remain open. Therefore nothing can be taken for granted in this issue, expect the need for development.