Inhibitory effects of wood extracts on the spoilage flora of marinated poultry meat

Polyphenolic compounds occurring naturally in knotwood of plants are known to have antimicrobial effects. The knots (i.e. the branch bases inside tree stems) and outer branches in pine trees contain a remarkably high concentration of phenolic stilbenes, while lignans are the major phenolic constituents of spruce knots. Large amount of these phenolic compounds can be extracted from wood knots at pulp and paper mills where their presence is undesirable. In Finland, marinating of broiler meat is done not only to increase or add value to the meat, but also to enhance the safety and shelf-life. These products are usually packed under a modified atmosphere for further protection against spoilage microorganisms. However, studies have revealed that addition of marinades to poultry products do not have an inhibitory effect on either some psychrotrophic anaerobic bacteria, such as Brochothrix thermosphacta or lactic acid bacteria associated with spoilage. Also, the activity of pathogenic Campylobacter jejuni is not affected by marinating. The objective of this study was to investigate the inhibitory and lethal activities of extracts from spruce (Picea spp.) and pine (Pinus spp.) knotwood and outer branches that are dissolved in ethanol against the spoilage microorganisms in modified atmosphere packaged marinated broiler products. Modified atmosphere packaged broiler products were separately inoculated with ‘normal’ marinades, marinades with 70% ethanol, marinades with a mixture of spruce and pine extracts dissolved in 70% ethanol or mixture of spruce and pine extracts in powder form. The bacterial colony forming units per gram obtained from each of the samples were analysed on de Man Rogosa and Sharpe agar at days 1, 6, 12 and 15. The results showed that there were significant differences in bacterial colony forming units per gram (P <0.05) between packages with ‘normal’ marinades and packages with extracts added to their marinades on the 12th and 15th day. It can be concluded that the addition of extracts from spruce and pine knotwood to marinades significantly retarded growth of spoilage microorganisms during the 15 day test period. However further research is warranted to characterise and establish the safety and suitability of the compound(s) in spruce and pine knotwood extracts that are responsible for inhibitory or lethal activity against the microbes that may be present in marinated poultry meat.

Leuconostoc spoilage of refrigerated, packaged foods

Leuconostoc spp. are lactic acid bacteria (LAB) implicated in food spoilage, especially on refrigerated, modified atmosphere packaged (MAP) meats. The overall aim of this thesis was to learn more about Leuconostoc spp. as food spoilage organisms with a focus on commercial products where LAB spoilage is considered a problem and the main factor limiting shelf-life. Therefore, we aimed to identify Leuconostoc spp. involved in food spoilage, as well as to characterise the spoilage reactions they caused and their contamination sources during poultry meat processing. In addition, we examined the distribution of strains of Leuconostoc gasicomitatum in different food commodities. Finally, we analysed the genome content of L. gasicomitatum LMG 18811 with a special focus on metabolic pathways related to food spoilage. The findings show that Leuconostoc gelidum and L.gasicomitatum were responsible for the discoloration and off-odours developed in beef steaks. Together with Leuconostoc mesenteroides, these Leuconostoc spp., also cause spoilage of vegetable sausages. In contrast, we showed that Leuconostoc spp. are not important for the shelf-life or quality of non-marinated broiler products although, in marinated broiler fillet products, Leuconostoc spp., L.gasicomitatum in particular, are considered spoilage organisms. Furthermore, the findings of the contamination survey we carried out in a poultry processing plant indicated that spoilage Leuconostoc spp. are derived from the processing environment rather than from the broilers, and that air movement distributes psychrotrophic spoilage LAB, including leuconostocs, and has an important role in meat contamination during poultry processing. Pulsed-field gel electrophoresis (PFGE) based genotyping of L. gasicomitatum strains demonstrated that certain genotypes are common in various meat products. In contrast, genotypes associated with meat were not recovered in vegetable-based sources. This suggests that these two food categories either become contaminated with, or favour the growth of different genotypes. Furthermore, the results indicated that the meat processing environment contributes to L. gasicomitatum contamination as certain genotypes were repeatedly identified from products of the same processing plant. Finally, the sequenced and annotated genome of L.gasicomitatum LMG 18811 allowed us to identify the metabolic pathways and reactions resulting in food spoilage.

Integrated climatic impacts of forestry and fibre-based packaging

The purpose of this study was to examine the integrated climatic impacts of forestry and the use fibre-based packaging materials. The responsible use of forest resources plays an integral role in mitigating climate change. Forests offer three generic mitigation strategies; conservation, sequestration and substitution. By conserving carbon reservoirs, increasing the carbon sequestration in the forest or substituting fossil fuel intensive materials and energy, it is possible to lower the amount of carbon in the atmosphere through the use of forest resources. The Finnish forest industry consumed some 78 million m3 of wood in 2009, while total of 2.4 million tons of different packaging materials were consumed that same year in Finland. Nearly half of the domestically consumed packaging materials were wood-based. Globally the world packaging material market is valued worth annually some €400 billion, of which the fibre-based packaging materials account for 40 %. The methodology and the theoretical framework of this study are based on a stand-level, steady-state analysis of forestry and wood yields. The forest stand data used for this study were obtained from Metla, and consisted of 14 forest stands located in Southern and Central Finland. The forest growth and wood yields were first optimized with the help of Stand Management Assistant software, and then simulated in Motti for forest carbon pools. The basic idea was to examine the climatic impacts of fibre-based packaging material production and consumption through different forest management and end-use scenarios. Economically optimal forest management practices were chosen as the baseline (1) for the study. In the alternative scenarios, the amount of fibre-based packaging material on the market decreased from the baseline. The reduced pulpwood demand (RPD) scenario (2) follows economically optimal management practices under reduced pulpwood price conditions, while the sawlog scenario (3) also changed the product mix from packaging to sawnwood products. The energy scenario (4) examines the impacts of pulpwood demand shift from packaging to energy use. The final scenario follows the silvicultural guidelines developed by the Forestry Development Centre Tapio (5). The baseline forest and forest product carbon pools and the avoided emissions from wood use were compared to those under alternative forest management regimes and end-use scenarios. The comparison of the climatic impacts between scenarios gave an insight into the sustainability of fibre-based packaging materials, and the impacts of decreased material supply and substitution. The results show that the use of wood for fibre-based packaging purposes is favorable, when considering climate change mitigation aspects of forestry and wood use. Fibre-based packaging materials efficiently displace fossil carbon emissions by substituting more energy intensive materials, and they delay biogenic carbon re-emissions to the atmosphere for several months up to years. The RPD and the sawlog scenarios both fared well in the scenario comparison. These scenarios produced relatively more sawnwood, which can displace high amounts of emissions and has high carbon storing potential due to the long lifecycle. The results indicate the possibility that win-win scenarios exist by shifting production from pulpwood to sawlogs; on some of the stands in the RPD and sawlog scenarios, both carbon pools and avoided emissions increased from the baseline simultaneously. On the opposite, the shift from packaging material to energy use caused the carbon pools and the avoided emissions to diminish from the baseline. Hence the use of virgin fibres for energy purposes, rather than forest industry feedstock biomass, should be critically judged if optional to each other. Managing the stands according to the silvicultural guidelines developed by the Forestry Development Centre Tapio provided the least climatic benefits, showing considerably lower carbon pools and avoided emissions. This seems interesting and worth noting, as the guidelines are the current basis for the forest management practices in Finland.

The ozone transfer between atmosphere and vegetation. A study on Scots pine in the field

Ozone (O3) is a reactive gas present in the troposphere in the range of parts per billion (ppb), i.e. molecules of O3 in 109 molecules of air. Its strong oxidative capacity makes it a key element in tropospheric chemistry and a threat to the integrity of materials, including living organisms. Knowledge and control of O3 levels are an issue in relation to indoor air quality, building material endurance, respiratory human disorders, and plant performance. Ozone is also a greenhouse gas and its abundance is relevant to global warming. The interaction of the lower troposphere with vegetated landscapes results in O3 being removed from the atmosphere by reactions that lead to the oxidation of plant-related components. Details on the rate and pattern of removal on different landscapes as well as the ultimate mechanisms by which this occurs are not fully resolved. This thesis analysed the controlling processes of the transfer of ozone at the air-plant interface. Improvement in the knowledge of these processes benefits the prediction of both atmospheric removal of O3 and its impact on vegetation. This study was based on the measurement and analysis of multi-year field measurements of O3 flux to Scots pine (Pinus sylvestris L.) foliage with a shoot-scale gas-exchange enclosure system. In addition, the analyses made use of simultaneous CO2 and H2O exchange, canopy-scale O3, CO2 and H2O exchange, foliage surface wetness, and environmental variables. All data was gathered at the SMEAR measuring station (southern Finland). Enclosure gas-exchange techniques such as those commonly used for the measure of CO2 and water vapour can be applied to the measure of ozone gas-exchange in the field. Through analysis of the system dynamics the occurring disturbances and noise can be identified. In the system used in this study, the possible artefacts arising from the ozone reactivity towards the system materials in combination with low background concentrations need to be taken into account. The main artefact was the loss of ozone towards the chamber walls, which was found to be very variable. The level of wall-loss was obtained from simultaneous and continuous measurements, and was included in the formulation of the mass balance of O3 concentration inside the chamber. The analysis of the field measurements in this study show that the flux of ozone to the Scots pine foliage is generated in about equal proportions by stomatal and non-stomatal controlled processes. Deposition towards foliage and forest is sustained also during night and winter when stomatal gas-exchange is low or absent. The non-stomatal portion of the flux was analysed further. The pattern of flux in time was found to be an overlap of the patterns of biological activity and presence of wetness in the environment. This was seen to occur both at the shoot and canopy scale. The presence of wetness enhanced the flux not only in the presence of liquid droplets but also during existence of a moisture film on the plant surfaces. The existence of these films and their relation to the ozone sinks was determined by simultaneous measurements of leaf surface wetness and ozone flux. The results seem to suggest ozone would be reacting at the foliage surface and the reaction rate would be mediated by the presence of surface wetness. Alternative mechanisms were discussed, including nocturnal stomatal aperture and emission of reactive volatile compounds. The prediction of the total flux could thus be based on a combination of a model of stomatal behaviour and a model of water absorption on the foliage surfaces. The concepts behind the division of stomatal and non-stomatal sinks were reconsidered. This study showed that it is theoretically possible that a sink located before or near the stomatal aperture prevents or diminishes the diffusion of ozone towards the intercellular air space of the mesophyll. This obstacle to stomatal diffusion happens only under certain conditions, which include a very low presence of reaction sites in the mesophyll, an extremely strong sink located on the outer surfaces or stomatal pore. The relevance, or existence, of this process in natural conditions would need to be assessed further. Potentially strong reactions were considered, including dissolved sulphate, volatile organic compounds, and apoplastic ascorbic acid. Information on the location and the relative abundance of these compounds would be valuable. The highest total flux towards the foliage and forest happens when both the plant activity and ambient moisture are high. The highest uptake into the interior of the foliage happens at large stomatal apertures, provided that scavenging reactions located near the stomatal pore are weak or non-existent. The discussion covers the methodological developments of this study, the relevance of the different controlling factors of ozone flux, the partition amongst its component, and the possible mechanisms of non-stomatal uptake.

DNA Packaging and Host Cell Lysis: Late Events in Bacteriophage PRD1 Infection

The object of this study is a tailless internal membrane-containing bacteriophage PRD1. It has a dsDNA genome with covalently bound terminal proteins required for replication. The uniqueness of the structure makes this phage a desirable object of research. PRD1 has been studied for some 30 years during which time a lot of information has accumulated on its structure and life-cycle. The two least characterised steps of the PRD1 life-cycle, the genome packaging and virus release are investigated here. PRD1 shares the main principles of virion assembly (DNA packaging in particular) and host cell lysis with other dsDNA bacteriophages. However, this phage has some fascinating individual peculiarities, such as DNA packaging into a membrane vesicle inside the capsid, absence of apparent portal protein, holin inhibitor and procapsid expansion. In the course of this study we have identified the components of the DNA packaging vertex of the capsid, and determined the function of protein P6 in packaging. We managed to purify the procapsids for an in vitro packaging system, optimise the reaction and significantly increase its efficiency. We developed a new method to determine DNA translocation and were able to quantify the efficiency and the rate of packaging. A model for PRD1 DNA packaging was also proposed. Another part of this study covers the lysis of the host cell. As other dsDNA bacteriophages PRD1 has been proposed to utilise a two-component lysis system. The existence of this lysis system in PRD1 has been proven by experiments using recombinant proteins and the multi-step nature of the lysis process has been established.

The genome packaging machinery of dsDNA bacteriophage PRD1

Viral genomes are encapsidated within protective protein shells. This encapsidation can be achieved either by a co-condensation reaction of the nucleic acid and coat proteins, or by first forming empty viral particles which are subsequently packaged with nucleic acid, the latter mechanism being typical for many dsDNA bacteriophages. Bacteriophage PRD1 is an icosahedral, non-tailed dsDNA virus that has an internal lipid membrane, the hallmark of the Tectiviridae family. Although PRD1 has been known to assemble empty particles into which the genome is subsequently packaged, the mechanism for this has been unknown, and there has been no evidence for a separate packaging vertex, similar to the portal structures used for packaging in the tailed bacteriophages and herpesviruses. In this study, a unique DNA packaging vertex was identified for PRD1, containing the packaging ATPase P9, packaging factor P6 and two small membrane proteins, P20 and P22, extending the packaging vertex to the internal membrane. Lack of small membrane protein P20 was shown to totally abolish packaging, making it an essential part of the PRD1 packaging mechanism. The minor capsid proteins P6 was shown to be an important packaging factor, its absence leading to greatly reduced packaging efficiency. An in vitro DNA packaging mechanism consisting of recombinant packaging ATPase P9, empty procapsids and mutant PRD1 DNA with a LacZ-insert was developed for the analysis of PRD1 packaging, the first such system ever for a virus containing an internal membrane. A new tectiviral sequence, a linear plasmid called pBClin15, was identified in Bacillus cereus, providing material for sequence analysis of the tectiviruses. Analysis of PRD1 P9 and other putative tectiviral ATPase sequences revealed several conserved sequence motifs, among them a new tectiviral packaging ATPase motif. Mutagenesis studies on PRD1 P9 were used to confirm the significance of the motifs. P9-type putative ATPase sequences carrying a similar sequence motif were identified in several other membrane containing dsDNA viruses of bacterial, archaeal and eukaryotic hosts, suggesting that these viruses may have similar packaging mechanisms. Interestingly, almost the same set of viruses that were found to have similar putative packaging ATPases had earlier been found to share similar coat protein folds and capsid structures, and a common origin for these viruses had been suggested. The finding in this study of similar packaging proteins further supports the idea that these viruses are descendants of a common ancestor.

Mechanism of RNA Translocation by a Viral Packaging Motor

Molecular motors are proteins that convert chemical energy into mechanical work. The viral packaging ATPase P4 is a hexameric molecular motor that translocates RNA into preformed viral capsids. P4 belongs to the ubiquitous class of hexameric helicases. Although its structure is known, the mechanism of RNA translocation remains elusive. Here we present a detailed kinetic study of nucleotide binding, hydrolysis, and product release by P4. We propose a stochastic-sequential cooperative model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive. Further, we use hydrogen/deuterium exchange detected by high resolution mass spectrometry to visualize P4 conformational dynamics during the catalytic cycle. Concerted changes of exchange kinetics reveal a cooperative unit that dynamically links ATP binding sites and the central RNA binding channel. The cooperative unit is compatible with the structure-based model in which translocation is effected by conformational changes of a limited protein region. Deuterium labeling also discloses the transition state associated with RNA loading which proceeds via opening of the hexameric ring. Hydrogen/deuterium exchange is further used to delineate the interactions of the P4 hexamer with the viral procapsid. P4 associates with the procapsid via its C-terminal face. The interactions stabilize subunit interfaces within the hexamer. The conformation of the virus-bound hexamer is more stable than the hexamer in solution, which is prone to spontaneous ring openings. We propose that the stabilization within the viral capsid increases the packaging processivity and confers selectivity during RNA loading. Finally, we use single molecule techniques to characterize P4 translocation along RNA. While the P4 hexamer encloses RNA topologically within the central channel, it diffuses randomly along the RNA. In the presence of ATP, unidirectional net movement is discernible in addition to the stochastic motion. The diffusion is hindered by activation energy barriers that depend on the nucleotide binding state. The results suggest that P4 employs an electrostatic clutch instead of cycling through stable, discrete, RNA binding states during translocation. Conformational changes coupled to ATP hydrolysis modify the electrostatic potential inside the central channel, which in turn biases RNA motion in one direction. Implications of the P4 model for other hexameric molecular motors are discussed.

Ionosphere-atmosphere interaction during solar proton events

Among the most striking natural phenomena affecting ozone are solar proton events (SPE), during which high-energy protons precipitate into the middle atmosphere in the polar regions. Ionisation caused by the protons results in changes in the lower ionosphere, and in production of neutral odd nitrogen and odd hydrogen species which then destroy ozone in well-known catalytic chemical reaction chains. Large SPEs are able to decrease the ozone concentration of upper stratosphere and mesosphere, but are not expected to significantly affect the ozone layer at 15--30~km altitude. In this work we have used the Sodankylä Ion and Neutral Chemistry Model (SIC) in studies of the short-term effects caused by SPEs. The model results were found to be in a good agreement with ionospheric observations from incoherent scatter radars, riometers, and VLF radio receivers as well as with measurements from the GOMOS/Envisat satellite instrument. For the first time, GOMOS was able to observe the SPE effects on odd nitrogen and ozone in the winter polar region. Ozone observations from GOMOS were validated against those from MIPAS/Envisat instrument, and a good agreement was found throughout the middle atmosphere. For the case of the SPE of October/November 2003, long-term ozone depletion was observed in the upper stratosphere. The depletion was further enhanced by the descent of odd nitrogen from the mesosphere inside the polar vortex, until the recovery occurred in late December. During the event, substantial diurnal variation of ozone depletion was seen in the mesosphere, caused mainly by the the strong diurnal cycle of the odd hydrogen species. In the lower ionosphere, SPEs increase the electron density which is very low in normal conditions. Therefore, SPEs make radar observations easier. In the case of the SPE of October, 1989, we studied the sunset transition of negative charge from electrons to ions, a long-standing problem. The observed phenomenon, which is controlled by the amount of solar radiation, was successfully explained by considering twilight changes in both the rate of photodetachment of negative ions and concentrations of minor neutral species. Changes in the magnetic field of the Earth control the extent of SPE-affected area. For the SPE of November 2001, the results indicated that for low and middle levels of geomagnetic disturbance the estimated cosmic radio noise absorption levels based on a magnetic field model are in a good agreement with ionospheric observations. For high levels of disturbance, the model overestimates the stretching of the geomagnetic field and the geographical extent of SPE-affected area. This work shows the importance of ionosphere-atmosphere interaction for SPE studies. By using both ionospheric and atmospheric observations, we have been able to cover for the most part the whole chain of SPE-triggered processes, from proton-induced ionisation to depletion of ozone.

Numerical approaches to new particle formation and growth in the atmosphere

Aerosols impact the planet and our daily lives through various effects, perhaps most notably those related to their climatic and health-related consequences. While there are several primary particle sources, secondary new particle formation from precursor vapors is also known to be a frequent, global phenomenon. Nevertheless, the formation mechanism of new particles, as well as the vapors participating in the process, remain a mystery. This thesis consists of studies on new particle formation specifically from the point of view of numerical modeling. A dependence of formation rate of 3 nm particles on the sulphuric acid concentration to the power of 1-2 has been observed. This suggests nucleation mechanism to be of first or second order with respect to the sulphuric acid concentration, in other words the mechanisms based on activation or kinetic collision of clusters. However, model studies have had difficulties in replicating the small exponents observed in nature. The work done in this thesis indicates that the exponents may be lowered by the participation of a co-condensing (and potentially nucleating) low-volatility organic vapor, or by increasing the assumed size of the critical clusters. On the other hand, the presented new and more accurate method for determining the exponent indicates high diurnal variability. Additionally, these studies included several semi-empirical nucleation rate parameterizations as well as a detailed investigation of the analysis used to determine the apparent particle formation rate. Due to their high proportion of the earth's surface area, oceans could potentially prove to be climatically significant sources of secondary particles. In the lack of marine observation data, new particle formation events in a coastal region were parameterized and studied. Since the formation mechanism is believed to be similar, the new parameterization was applied in a marine scenario. The work showed that marine CCN production is feasible in the presence of additional vapors contributing to particle growth. Finally, a new method to estimate concentrations of condensing organics was developed. The algorithm utilizes a Markov chain Monte Carlo method to determine the required combination of vapor concentrations by comparing a measured particle size distribution with one from an aerosol dynamics process model. The evaluation indicated excellent agreement against model data, and initial results with field data appear sound as well.