Diet and the development of atherosclerosis: a whole-diet approach from childhood to adulthood

Cardiovascular diseases (CVDs) are the leading cause of mortality in the world. Studies of the impact of single nutrients on the risk for CVD have often provided inconclusive results, and recent research in nutritional epidemiology with a more holistic whole-diet approach has proven fruitful. Moreover, dietary habits in childhood and adolescence may play a role in later health and disease, either independently or by tracking into adulthood. The main aims of this study were to find childhood and adulthood determinants of adulthood diet, to identify dietary patterns present among the study population and to study the associations between long-term food choices and cardiovascular health in young Finnish adults. The study is a part of the multidisciplinary Cardiovascular Risk in Young Finns study, which is an ongoing, prospective cohort study with a 21-year follow-up. At baseline in 1980, the subjects were children and adolescents aged 3 to 18 years (n included in this study = 1768), and young adults aged 24 to 39 years at the latest follow-up study in 2001 (n = 1037). Food consumption and nutrient intakes were assessed with repeated 48-hour dietary recalls. Other determinations have included comprehensive risk factor assessments using blood tests, physical measurements and questionnaires. In the latest follow-up, ultrasound examinations were performed to study early atherosclerotic vascular changes. The average intakes showed substantial changes since 1980. Intakes of fat and saturated fat had decreased, whereas the consumption of fruits and vegetables had increased. Intake of fat and consumption of vegetables in childhood and physical activity in adulthood were important health behavioural determinants of adult diet. Additionally, a principal component analysis was conducted to identify major dietary patterns at each study point. A similar set of two major patterns was recognised throughout the study. The traditional dietary pattern positively correlated with the consumption of traditional Finnish foods, such as rye, potatoes, milk, butter, sausages and coffee, and negatively correlated with fruit, berries and dairy products other than milk. This type of diet was independently associated with several risk factors of CVD, such as total and low-density lipoprotein cholesterol, apolipoprotein B and C-reactive protein concentrations among both genders, as well as with systolic blood pressure and insulin levels among women. The traditional pattern was also independently associated with intima media thickness (IMT), a subclinical predictor of CVD, in men but not in women. The health-conscious pattern, predominant among female subjects, non-smokers and urbanites, was characterised by more health-conscious food choices such as vegetables, legumes and nuts, tea, rye, fish, cheese and other dairy products, as well as by the consumption of alcoholic beverages. This pattern was inversely, but less strongly, associated with cardiovascular risk factors. Tracking of the dietary pattern scores was observed, particularly among subjects who were adolescents at baseline. Moreover, a long-term high intake of protein concurrent with a low intake of fat was positively associated with IMT. These findings suggest that food behaviour and food choices are to some extent established as early as in childhood or adolescence and may significantly track into adulthood. Long-term adherence to traditional food choices seems to increase the risk for developing CVD, especially among men. Those with intentional or unintentional low fat diets, but with high intake of protein may also be at increased risk for CVD. The findings offer practical, food-based information on the relationship between diet and CVD and encourage further use of the whole-diet approach in epidemiological research. The results support earlier findings that long-term food choices play a role in the development of CVD. The apparent influence of childhood habits is important to bear in mind when planning educational strategies for the primary prevention of CVD. Further studies on food choices over the entire lifespan are needed.

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

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.

Defining Adequate Vitamin D Intake : Cross-sectional and Intervention Studies

D-vitamiini ylläpitää normaalia luun kasvua ja uudistumista koko elämän ajan. Suomessa, kuten monissa muissakin länsimaissa, väestön D-vitamiinitilanne on riittämätön – talvisin osalla jopa puutteellinen. Tässä väitöskirjassa on tutkittu, lisääkö D-vitamiini luumassan kertymistä kasvuiässä, ja ylläpitäkö D-vitamiini luuston tasapainoista aineenvaihduntaa aikuisiällä. Nämä vaikutukset saattavat ehkäisi osteoporoosin kehittymistä eri ikäkausina. Väitöskirjatyössä tutkittiin erisuuruisten D-vitamiinilisäysten vaikutuksia kolmessa eri ikäryhmässä, jotka olivat 11-12 -vuotiaat tytöt (N=228), 21-49 -vuotiaat miehet (N=54) ja 65-85 -vuotiaat naiset (N=52). Tutkittavat satunnaistettiin ryhmiin, jotka nauttivat joko lumevalmistetta tai 5-20 µg D3-vitamiinia vitamiinilisänä. Tutkimukset olivat kaksoissokkoutettuja. Tutkimuksen aikana tutkittavilta otettiin paastoveri- ja virtsanäytteitä. Lisäksi he täyttivät tutkimuslomakkeen taustatietojen kartoittamiseksi sekä frekvenssikyselylomakkeen kalsiumin ja D-vitamiinin saannin selvittämiseksi. Tyttöjen luunmineraalitiheys (BMD) mitattiin DXA–laitteella ja miesten volumetrinen luuntiheys pQCT-menetelmällä. Näytteistä määritettiin mm. seerumin 25-hydroksi-D-vitamiinin (=S-25-OHD), lisäkilpirauhashormonin (=S-PTH) ja luun aineenvaihduntaa kuvaavien merkkiaineiden pitoisuuksia. Murrosikäisten tyttöjen poikkileikkaustutkimuksessa S-25-OHD- ja luun muodostusmerkkiaineen pitoisuudet vaihtelivat kuukausien välillä; suurimmat pitoisuudet mitattiin syyskuussa ja pienimmät maaliskuussa, mikä kuvastaa vuodenaikaisvaihtelua. Vastaava vaihtelu havaittiin lannerangan ja reisiluun BMD:ssä. D-vitamiinilisäyksellä oli myönteinen vaikutus tyttöjen luumassan lisääntymiseen. Suurin D-vitamiinilisä (10 µg/vrk) lisäsi luumassaa 17.2% enemmän reisiluussa ja 12.5% enemmän lannerangassa verrattuna lumevalmistetta nauttivien tyttöjen vastaaviin tuloksiin, mutta tulos riippui hoitomyöntyvyydestä. D-vitamiinin vaikutus luustoon välittyi vähentyneen luun hajotuksen kautta. Tutkimustuloksiin perustuen riittävä D-vitamiinin saanti murrosikäisille tytöille on 15 µg/vrk. D-vitamiinilisän vaikutus 65-85 -vuotiaiden naisten S-25-OHD-pitoisuuteen vakioitui kuudessa viikossa annoksen ollessa 5-20 µg/vrk. Näillä D-vitamiiniannoksilla ei saavutettu tavoiteltavaa S-25-OHD-pitoisuutta, joka on 80 nmol/l. Arvioimme, että 60 nmol/l -pitoisuuden, jota esiintyy kesäisin tämän ikäryhmän suomalaisilla, tämän ikäryhmän naiset saavuttaisivat 24 µg:n päivittäisellä D-vitamiinin saannilla. Terveillä miehillä havaittiin vuodenaikaisvaihtelu S-25-OHD- ja S-PTH-pitoisuudessa sekä luun hajotusta kuvaavassa merkkiainepitoisuudessa. Toisaalta vaihtelua ei havaittu radiuksen volumetrisessä luuntiheydessä eikä luun muodostusmerkkiaineen pitoisuudessa. Vuodenaikaisvaihtelu estettiin 17 µg:n päivittäisellä D-vitamiinin saannilla, mutta tämän ei havaittu vaikuttavan radiuksen luuntiheyteen kuusi kuukautta kestävän tutkimuksen aikana. Yhteenvetona todetaan, että D-vitamiinin saanti on edelleenkin riittämätöntä tutkimusten kohderyhmillä. Tämä näkyy S-25-OHD- ja PTH-pitoisuuden sekä luunaineenvaihduntaa kuvaavien merkkiaineiden vuodenaikaisvaihteluna, mikä on haitallista luuston hyvinvoinnille. D-vitamiinin saantia tulisi lisätä, jotta vähintäänkin riittävä D-vitamiinitilanne (S-25-OHD>50 nmol/l) tai mahdollisesti jopa tavoiteltava D-vitaminitilanne (S-25-OHD≥80 nmol/l) saavutettaisiin. Jotta D-vitamiinin saannin lisääminen olisi kaikissa ikäryhmissä mahdollista, on suunniteltava nykyistä enemmän D-vitamiinilla täydennettyjä elintarvikkeita.

Effects of dietary phosphorus and calcium-to-phosphorus ratio on calcium and bone metabolism in healthy 20- to 43-year-old Finnish women

Dietary habits have changed during the past decades towards an increasing consumption of processed foods, which has notably increased not only total dietary phosphorus (P) intake, but also intake of P from phosphate additives. While the intake of calcium (Ca) in many Western countries remains below recommended levels (800 mg/d), the usual daily P intake in a typical Western diet exceeds by 2- to 3-fold the dietary guidelines (600 mg/d). The effects of high P intake in healthy humans have been investigated seldom. In this thesis healthy 20- to 43-year-old women were studied. In the first controlled study (n = 14), we examined the effects of P doses, and in a cross-sectional study (n = 147) the associations of habitual P intakes with Ca and bone metabolism. In this same cross-sectional study, we also investigated whether differences exist between dietary P originating from natural P sources and phosphate additives. The second controlled study (n = 12) investigated whether by increasing the Ca intake, the effects of a high P intake could be reduced. The associations of habitual dietary calcium-to-phosphorus ratios (Ca:P ratio) with Ca and bone metabolism were determined in a cross-sectional study design (n = 147). In the controlled study, the oral intake of P doses (495, 745, 1245 and 1995 mg/d) with a low Ca intake (250 mg/d) increased serum parathyroid hormone (S-PTH) concentration in a dose-dependent manner. In addition, the highest P dose decreased serum ionized calcium (S-iCa) concentration and bone formation and increased bone resorption. In the second controlled study with a dietary P intake of 1850 mg/d, by increasing the Ca intake from 480 mg/d to 1080 mg/d and then to 1680 mg/d, the S-PTH concentration decreased, the S-iCa concentration increased and bone resorption decreased dose-dependently. However, not even the highest Ca intake could counteract the effect of high dietary P on bone formation, as indicated by unchanged bone formation activity. In the cross-sectional studies, a higher habitual dietary P intake (>1650 mg/d) was associated with lower S-iCa and higher S-PTH concentrations. The consumption of phosphate additive-containing foods was associated with a higher S-PTH concentration. Moreover, habitual low dietary Ca:P ratios (≤0.50, molar ratio) were associated with higher S-PTH concentrations and 24-h urinary Ca excretions, suggesting that low dietary Ca:P ratios may interfere with homeostasis of Ca metabolism and increase bone resorption. In summary, excessive dietary P intake in healthy Finnish women seems to be detrimental to Ca and bone metabolism, especially when dietary Ca intake is low. The results indicate that by increasing dietary Ca intake to the recommended level, the negative effects of high P intake could be diminished, but not totally prevented. These findings imply that phosphate additives may be more harmful than natural P. Thus, reduction of an excessively high dietary P intake is also beneficial for healthy individuals.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Autoxidation of conjugated linoleic acid methyl ester in the presence of α-tocopherol: the hydroperoxide pathway

The autoxidation of conjugated linoleic acid (CLA) is poorly understood in spite of increasing interest in the beneficial biological properties of CLA and growing consumption of CLA-rich foods. In this thesis, the autoxidation reactions of the two major CLA isomers, 9-cis,11-trans-octadecadienoic acid and 10-trans,12-cis-octadecadienoic acid, are investigated. The results contribute to an understanding of the early stages of the autoxidation of CLA methyl ester, and provide for the first time a means of producing and separating intact CLA methyl ester hydroperoxides as well as basic knowledge on lipid hydroperoxides and their hydroxy derivatives. Conjugated diene allylic monohydroperoxides were discovered as primary autoxidation products formed during autoxidation of CLA methyl esters in the presence and absence of α-tocopherol. This established that one of the autoxidation pathways of CLA methyl ester is the hydroperoxide pathway. Hydroperoxides were produced from the two major CLA methyl esters by taking advantage of the effect of α-tocopherol to promote hydroperoxide formation. The hydroperoxides were analysed and separated first as methyl hydroxyoctadecadienoates and then as intact hydroperoxides by HPLC. The isolated products were characterized by UV, GC-MS, and NMR techniques. In the presence of a high amount of α-tocopherol, the autoxidation of CLA methyl ester yields six kinetically-controlled conjugated diene monohydroperoxides and is diastereoselective in favour of one particular geometric isomer as a pair of enantiomers. The primary autoxidation products produced from the two major CLA isomers include new positional isomers of conjugated diene monohydroperoxides, the 8-, 10-, 12-, and 14-hydroperoxyoctadecadienoates. Furthermore, two of these new positional isomers have an unusual structure for a cis,trans lipid hydroperoxide where the allylic methine carbon is adjacent to the cis instead of the usual trans double bond. The 1H and 13C NMR spectra of nine isomeric methyl hydroxyoctadecadienoates and of ten isomeric methyl hydroperoxyoctadecadienoates including the unusual cis,trans hydroperoxides, i.e. Me 8-OOH-9c,11t and Me 14-OOH-10t,12c, were fully assigned with the aid of 2D NMR spectroscopy. The assigned NMR data enabled determination of the effects of the hydroxyl and hydroperoxyl groups on the carbon chemical shifts of CLA isomers, identification of diagnostic signals, and determination of chemical shift differences of the olefinic resonances that may help with the assignment of structure to as yet unknown lipid hydroperoxides either as hydroxy derivatives or as intact hydroperoxides. A mechanism for the hydroperoxide pathway of CLA autoxidation in the presence of a high amount of α-tocopherol was proposed based on the characterized primary products, their relative distribution, and theoretical calculations. This is an important step forward in CLA research, where exact mechanisms for the autoxidation of CLA have not been presented before. Knowledge of these hydroperoxide formation steps is of crucial importance for understanding the subsequent steps and the different pathways of the autoxidation of CLA. Moreover, a deeper understanding of the autoxidation mechanisms is required for ensuring the safety of CLA-rich foods. Knowledge of CLA oxidation and how it differs from the oxidation of nonconjugated polyunsaturated fatty acids may also be the key to understanding the biological mechanisms of CLA activity.

Development of Sample Pretreatment and Liquid Chromatographic Techniques for Antioxidative Compounds

In this study, novel methodologies for the determination of antioxidative compounds in herbs and beverages were developed. Antioxidants are compounds that can reduce, delay or inhibit oxidative events. They are a part of the human defense system and are obtained through the diet. Antioxidants are naturally present in several types of foods, e.g. in fruits, beverages, vegetables and herbs. Antioxidants can also be added to foods during manufacturing to suppress lipid oxidation and formation of free radicals under conditions of cooking or storage and to reduce the concentration of free radicals in vivo after food ingestion. There is growing interest in natural antioxidants, and effective compounds have already been identified from antioxidant classes such as carotenoids, essential oils, flavonoids and phenolic acids. The wide variety of sample matrices and analytes presents quite a challenge for the development of analytical techniques. Growing demands have been placed on sample pretreatment. In this study, three novel extraction techniques, namely supercritical fluid extraction (SFE), pressurised hot water extraction (PHWE) and dynamic sonication-assisted extraction (DSAE) were studied. SFE was used for the extraction of lycopene from tomato skins and PHWE was used in the extraction of phenolic compounds from sage. DSAE was applied to the extraction of phenolic acids from Lamiaceae herbs. In the development of extraction methodologies, the main parameters of the extraction were studied and the recoveries were compared to those achieved by conventional extraction techniques. In addition, the stability of lycopene was also followed under different storage conditions. For the separation of the antioxidative compounds in the extracts, liquid chromatographic methods (LC) were utilised. Two novel LC techniques, namely ultra performance liquid chromatography (UPLC) and comprehensive two-dimensional liquid chromatography (LCxLC) were studied and compared with conventional high performance liquid chromatography (HPLC) for the separation of antioxidants in beverages and Lamiaceae herbs. In LCxLC, the selection of LC mode, column dimensions and flow rates were studied and optimised to obtain efficient separation of the target compounds. In addition, the separation powers of HPLC, UPLC, HPLCxHPLC and HPLCxUPLC were compared. To exploit the benefits of an integrated system, in which sample preparation and final separation are performed in a closed unit, dynamic sonication-assisted extraction was coupled on-line to a liquid chromatograph via a solid-phase trap. The increased sensitivity was utilised in the extraction of phenolic acids from Lamiaceae herbs. The results were compared to those of achieved by the LCxLC system.

Sukupolvien ongelma : Tutkielmia sukupolven käsitteestä, sukupolvitietoisuudesta ja suurista ikäluokista

The aims of the thesis are (1) to present a systematic evaluation of generation and its relevance as a sociological concept, (2) to reflect on how generational consciousness, i.e. generation as an object of collective identification that has social significance, can emerge and take shape, (3) to analyze empirically the generational experiences and consciousness of one specific generation, namely Finnish baby boomers (b. 1945 1950). The thesis contributes to the discussion on the social (as distinct from its genealogical) meaning of the concept of generation, launched by Karl Mannheim s classic Das Problem der Generationen (1928), in which the central idea is that a certain group of people is bonded together by a shared experience and that this bonding can result in a distinct self-consciousness. The thesis is comprised of six original articles and an extensive summarizing chapter. In the empirical articles, the baby boomers are studied on the basis of nationally representative survey data (N = 2628) and narrative life-story interviews (N = 38). In the article that discusses the connection of generations and social movements, the analysis is based on the member survey of Attac Finland (N = 1096). Three main themes were clarified in the thesis. (1) In the social sense the concept of generation is a modern, problematic, and ultimately a political concept. It served the interests of the intellectuals who developed the concept in the early 20th century and provided them, as an alternative to the concept of social class, a new way of think about social change and progress. The concept of generation is always coupled with the concept of Zeitgeist or some other controversial way of defining what is essential, i.e. what creates generations, in a given culture. Thus generation is, as a product of definition and classification struggles, a contested concept. The concept also clearly implies elitist connotations; the idea of some kind of vanguard (the elite) that represents an entire generation by proclaiming itself as its spokesman automatically creates a counterpart, namely the others in the peer group who are thought to be represented (the masses). (2) Generational consciousness cannot emerge as a result of any kind of automatic process or endogenously; it must be made. There has to be somebody who represents the generation in order for that generation to exist in people s minds and as an object of identification; generational experiences and their meanings must be articulated. Hence, social generations are, in a fundamental manner, discursively constructed. The articulations of generational experiences (speeches, writings, manifests, labels etc.) can be called as the discursive dimension of social generations, and through this notion, how public discourse shapes people s generational consciousness can be seen. Another important element in the process is collective memory, as generational consciousness often takes form only retrospectively. (3) Finnish baby boomers are not a united or homogeneous generation but are divided into many smaller sections with specific generational experiences and consciousnesses. The content of the generational consciousness of the baby boomers is heavily politically charged. A salient dividing line inside the age group is formed by individual attitudes towards so-called 1960s radicalism. Identification with the 1960s generation functions today as a positive self-definition of a certain small leftist elite group, and the values and characteristics usually connected with the idea of the 1960s generation do not represent the whole age group. On the contrary, among some of the members of the baby boomers, the generational identification is still directed by the experience of how traditional values were disgraced in the 1960s. As objects of identification, the neutral term baby boomers and the charged 1960s generation are totally different things, and therefore they should not be used as synonyms. Although the significance of the group of the 1960s generation is often overestimated, they are however special with respect to generational consciousness because they have presented themselves as the voice of the entire generation. Their generational interpretations have spread through the media with the help of certain iconic images of the generation insomuch that 1960s radicalism has become an indirect generational experience for other parts of the baby boom cohort as well.

Survival in the Rainforest : Change and Resilience among the Punan Vuhang of Eastern Sarawak, Malaysia

This dissertation concerns the Punan Vuhang, former hunter-gatherers who are now part-time farmers living in an area of remote rainforest in the Malaysian state of Sarawak. It covers two themes: first, examining their methods of securing a livelihood in the rainforest, and second looking at their adaptation to a settled life and agriculture, and their response to rapid and large-scale commercial logging. This study engages the long-running debates among anthropologists and ecologists on whether recent hunting-gathering societies were able to survive in the tropical rainforest without dependence on farming societies for food resources. In the search for evidence, the study poses three questions: What food resources were available to rainforest hunter-gatherers? How did they hunt and gather these foods? How did they cope with periodic food shortages? In fashioning a life in the rainforest, the Punan Vuhang survived resource scarcity by developing adaptive strategies through intensive use of their knowledge of the forest and its resources. They also adopted social practices such as sharing and reciprocity, and resource tenure to sustain themselves without recourse to external sources of food. In the 1960s, the Punan Vuhang settled down in response to external influences arising in part from the Indonesian-Malaysian Confrontation. This, in turn, initiated a series of processes with political, economic and religious implications. However, elements of the traditional economy have remained resilient as the people continue to hunt, fish and gather, and are able to farm on an individual basis, unlike neighboring shifting cultivators who need to cooperate with each other. At the beginning of the 21st century, the Punan Vuhang face a new challenge arising from the issue of rights in the context of the state and national law and large-scale commercial logging in their forest habitat. The future seems bleak as they face the social problems of alcoholism, declining leadership, and dependence on cash income and commodities from the market.

Consumption in Rural Bangladesh : Households, Lifestyles, and Identities

Bangladesh, often better known to the outside world as a country of natural calamities, is one of the most densely populated countries in the world. Despite rapid urbanization, more than 75% of the people still live in rural areas. The density of the rural population is also one of the highest in the world. Being a poor and low-income country, its main challenge is to eradicate poverty through increasing equitable income. Since its independence in 1971, Bangladesh has experienced many ups and downs, but over the past three decades, its gross domestic product (GDP) has grown at an impressive rate. Consequently, the country s economy is developing and the country has outperformed many low-income countries in terms of several social indicators. Bangladesh has achieved the Millennium Development Goal (MDG) of eliminating gender disparity in primary and secondary school enrollment. A sharp decline in child and infant mortality rates, increased per capita income, and improved food security have placed Bangladesh on the track to achieving in the near future the status of a middle-income country. All these developments have influenced the consumption pattern of the country. This study explores the consumption scenario of rural Bangladesh, its changing consumption patterns, the relationship between technology and consumption in rural Bangladesh, cultural consumption in rural Bangladesh, and the myriad reasons why consumers nevertheless feel compelled to consume chemically treated foods. Data were collected in two phases in the summers of 2006 and 2008. In 2006, the empirical data were collected from the following three sources: interviews with consumers, producers/sellers, and doctors and pharmacists; observations of sellers/producers; and reviews of articles published in the national English and Bengali (the national language of Bangladesh) daily newspapers. A total of 110 consumers, 25 sellers/producers, 7 doctors, and 7 pharmacists were interviewed and observed. In 2008, data were collected through semi-structured in-depth qualitative interviews, ethnography, and unstructured conversations substantiated by secondary sources and photographs; the total number of persons interviewed was 22. -- Data were also collected on the consumption of food, clothing, housing, education, medical facilities, marriage and dowry, the division of labor, household decision making, different festivals such as Eid (for Muslims), the Bengali New Year, and Durga puja (for Hindus), and leisure. Qualitative methods were applied to the data analysis and were supported by secondary quantitative data. The findings of this study suggest that the consumption patterns of rural Bangladeshis are changing over time along with economic and social development, and that technology has rendered aspects of daily life more convenient. This study identified the perceptions and experiences of rural people regarding technologies in use and explored how culture is associated with consumption. This study identified the reasons behind the use of hazardous chemicals (e.g. calcium carbide, sodium cyclamate, cyanide and formalin, etc.) in foods as well as the extent to which food producers/sellers used such chemicals. In addition, this study assessed consumer perceptions of and attitudes toward these contaminated food items and explored how adulterated foods and food stuffs affect consumer health. This study also showed that consumers were aware that various foods and food stuffs contained hazardous chemicals, and that these adulterated foods and food stuffs were harmful to their health.

Applications of Treatment Effects Models and Semiparametric Estimation

This thesis is composed of an introductory chapter and four applications each of them constituting an own chapter. The common element underlying each of the chapters is the econometric methodology. The applications rely mostly on the leading econometric techniques related to estimation of causal effects. The first chapter introduces the econometric techniques that are employed in the remaining chapters. Chapter 2 studies the effects of shocking news on student performance. It exploits the fact that the school shooting in Kauhajoki in 2008 coincided with the matriculation examination period of that fall. It shows that the performance of men declined due to the news of the school shooting. For women the similar pattern remains unobserved. Chapter 3 studies the effects of minimum wage on employment by employing the original Card and Krueger (1994; CK) and Neumark and Wascher (2000; NW) data together with the changes-in-changes (CIC) estimator. As the main result it shows that the employment effect of an increase in the minimum wage is positive for small fast-food restaurants and negative for big fast-food restaurants. Therefore, it shows that the controversial positive employment effect reported by CK is overturned for big fast-food restaurants and that the NW data are shown, in contrast to their original results, to provide support for the positive employment effect. Chapter 4 employs the state-specific U.S. data (collected by Cohen and Einav [2003; CE]) on traffic fatalities to re-evaluate the effects of seat belt laws on the traffic fatalities by using the CIC estimator. It confirms the CE results that on the average an implementation of a mandatory seat belt law results in an increase in the seat belt usage rate and a decrease in the total fatality rate. In contrast to CE, it also finds evidence on compensating-behavior theory, which is observed especially in the states by the border of the U.S. Chapter 5 studies the life cycle consumption in Finland, with the special interest laid on the baby boomers and the older households. It shows that the baby boomers smooth their consumption over the life cycle more than other generations. It also shows that the old households smoothed their life cycle consumption more as a result of the recession in the 1990s, compared to young households.

Bacteria colonizing paper machines

Bacteria growing in paper machines can cause several problems. Biofilms detaching from paper machine surfaces may lead to holes and spots in the end product or even break the paper web leading to expensive delays in production. Heat stable endospores will remain viable through the drying section of paper machine, increasing the microbial contamination of paper and board. Of the bacterial species regularly found in the end products, Bacillus cereus is the only one classified as a pathogen. Certain B. cereus strains produce cereulide, the toxin that causes vomiting disease in food poisonings connected to B. cereus. The first aim of this thesis was to identify harmful bacterial species colonizing paper machines and to assess the role of bacteria in the formation of end product defects. We developed quantitative PCR methods for detecting Meiothermus spp. and Pseudoxanthomonas taiwanensis. Using these methods I showed that Meiothermus spp. and Psx. taiwanensis are major biofoulers in paper machines. I was the first to be able to show the connection between end product defects and biofilms in the wet-end of paper machines. I isolated 48 strains of primary-biofilm forming bacteria from paper machines. Based on one of them, strain K4.1T, I described a novel bacterial genus Deinobacterium with Deinobacterium chartae as the type species. I measured the transfer of Bacillus cereus spores from packaging paper into food. To do this, we constructed a green fluorescent protein (GFP) labelled derivative of Bacillus thuringiensis and prepared paper containing spores of this strain. Chocolate and rice were the recipient foods when transfer of the labelled spores from the packaging paper to food was examined. I showed that only minority of the Bacillus cereus spores transferred into food from packaging paper and that this amount is very low compared to the amount of B. cereus naturally occurring in foods. Thus the microbiological risk caused by packaging papers is very low. Until now, the biological function of cereulide for the producer cell has remained unknown. I showed that B. cereus can use cereulide to take up K+ from environment where K+ is scarce: cereulide binds K+ ions outside the cell with high affinity and transports these ions across cell membrane into the cytoplasm. Externally added cereulide increased the growth rate of cereulide producing strains in medium where potassium was growth limiting. In addition, cereulide producing strains outcompeted cereulide non-producing B. cereus in potassium deficient environment, but not when the potassium concentration was high. I also showed that cereulide enhances biofilm formation of B. cereus.

Ruokauskomukset Mosambikin maaseudun raskaana olevilla ja imettävillä naisilla

Ihmisille ympäri maailmaa on tyypillistä valita tiettyjä ruokia suuremmasta valikoimasta syömäkelpoista ruokaa ja luoda mieltymyksiä sekä kieltoja joitakin ruokia kohtaan. Näitä ilmiöitä kutsutaan ruokauskomuksiksi ja ne ovat tärkeä osa kulttuuria. Niiden taustalla on usein terveydelliset perustelut tai sosiaalinen arvostus yhteisössä. Afrikassa on todettu olevan paljon erilaisia ruokauskomuksia, jotka kohdistuvat usein raskaana oleviin ja imettäviin naisiin sekä lapsiin, ryhmään, joka on fysiologisesti erityisen herkässä elämänvaiheessa. Ravitsemustilan tai ruokaturvan ollessa heikko, mahdollisilla ruokauskomuksilla voi olla merkittävät seuraukset erityisesti tämän väestönosan terveydentilaan. Aliravitsemus ja virheravitsemus ovat edelleen suuria ongelmia Afrikassa ja muissa kehittyvissä maissa. Tämän työn tavoitteena oli tutkia laadullisin menetelmin, onko erityisesti raskaana olevilla ja imettävillä naisilla Keski-Mosambikin maaseudulla, Zambézian maakunnassa, ruokauskomuksia, mitä ne mahdollisesti ovat, sekä arvioida voiko ruokauskomuksilla olla ravitsemuksellista merkitystä. Menetelminä oli 5 ryhmähaastattelua kolmessa kylässä ja 10 yksilöhaastattelua kahdessa kylässä. Tutkittavat olivat 12–78-vuotiaita naisia ja heitä oli yhteensä 27. Tulkkaus, tutkimusaiheen sensitiivisyys ja haastattelijan kokemattomuus aiheuttivat haasteita haastatteluiden toteutukseen. Tämän vuoksi menetelmää muokattiin ja se vaihdettiin ryhmähaastattelusta yksilöhaastatteluun tutkimuksen aikana. Ruokauskomukset vaihtelivat sekä kylien välillä että kylien sisällä, joskin niistä löytyi samantyyppisiä pääpiirteitä. Työssä todettiin, että raskaana oleviin ja imettäviin naisiin kohdistui tutkimusalueella ruokauskomuksia. Raskauden aikana tuli välttää pääasiassa proteiinipitoisia ruokia, kuten lihaa ja kalaa ja puolestaan suosia kasvisten, hedelmien ja viljatuotteiden käyttöä. Linnun munien syöntiä sekä suositeltiin että kehotettiin välttämään raskauden aikana. Raskauden aikana ilmeni lisäksi tietoinen tapa vähentää kassavapuuron tai ruuan syömistä kokonaisuudessaan. Imetyksen aikana ei vältetty juuri mitään ruokia, vaan suositeltiin kookosta ja kasviksia. Suurin osa perusteluista liittyi äidin ja lapsen terveyteen. Perustelut voitiin jakaa seuraaviin ryhmiin: lisää äidinmaidon tuotantoa; aiheuttaa vatsakipua äidillä; pitää yllä kuntoa ja estää vatsan kasvua; äidistä tulee vahva, terve ja hän saa vitamiineja; lapsen ulkonäkö muuttuu; lapsesta tulee terve ja vahva; lapsen käyttäytyminen muuttuu; aiheuttaa keskenmenon; aiheuttaa vaikean tai helpon synnytyksen. Ruokauskomukset ja terveysviranomaisilta tullut valistus oli osittain sekoittunut keskenään. Lähes kaikki naiset kertoivat noudattavansa näitä ruokauskomuksia. Muutamat naiset kertoivat suositeltavien ruokien kohdalla, että ruokien saatavuus tekee noudattamisen joskus vaikeaksi. Proteiinipitoisten ruokien välttäminen voi lisätä proteiinialiravitsemuksen riskiä ja syömisen vähentäminen saattaa lisätä riskiä saada riittämättömästi energiaa raskauden aikana. Suositeltavat ruuat todennäköisesti edistävät terveyttä. Tutkielman perusteella voitiin todeta, että vieraassa kulttuurissa tutkiminen vaatii tutkimusmenetelmältä paljon. Tärkeää on menetelmän joustavuus, jotta sitä voi muuttaa tutkimuskentällä toimivampaan ja luotettavampaan muotoon. Johtopäätöksenä todettiin, että suositeltavin menetelmä tutkittaessa ruokauskomuksia tällä tutkimusalueella olisi ollut yksilöhaastattelut, jossa olisi ollut mahdollisimman vähän osallistujia ja osallistujat olisivat olleet samaa sukupuolta ja saman ikäisiä. Terveyden ja ravitsemuksen kannalta on tärkeää, että ruokauskomuksista ollaan tietoisia ja väestön ruokauskomuksia tutkitaan alueittain, koska ne voivat vaihdella merkittävästi jopa kylien tai ihmisryhmien sisällä.

Non-enzymatic Degradation of (1→3)(1→4)-β-D-glucan in Aqueous Processing of Oats

Cereal water-soluble β-glucan [(1→3)(1→4)-β-D-glucan] has well-evidenced health benefits and it contributes to the texture properties of foods. These functions are characteristically dependent on the excellent viscosity forming ability of this cell wall polysaccharide. The viscosity is affected by the molar mass, solubility and conformation of β-glucan molecule, which are further known to be altered during food processing. This study focused on demonstrating the degradation of β-glucan in water solutions following the addition of ascorbic acid, during heat treatments or high pressure homogenisation. Furthermore, the motivation of this study was in the non-enzymatic degradation mechanisms, particularly in oxidative cleavage via hydroxyl radicals. The addition of ascorbic acid at food-related concentrations (2-50 mM), autoclaving (120°C) treatments, and high pressure homogenisation (300-1000 bar) considerably cleaved the β-glucan chains, determined as a steep decrease in the viscosity of β-glucan solutions and decrease in the molar mass of β-glucan. The cleavage was more intense in a solution of native β-glucan with co-extracted compounds than in a solution of highly purified β-glucan. Despite the clear and immediate process-related degradation, β-glucan was less sensitive to these treatments compared to other water-soluble polysaccharides previously reported in the literature. In particular, the highly purified β-glucan was relatively resistant to the autoclaving treatments without the addition of ferrous ions. The formation of highly oxidative free radicals was detected at the elevated temperatures, and the formation was considerably accelerated by added ferrous ions. Also ascorbic acid pronounced the formation of these oxidative radicals, and oxygen was simultaneously consumed by ascorbic acid addition and by heating the β-glucan solutions. These results demonstrated the occurrence of oxidative reactions, most likely the metal catalysed Fenton-like reactions, in the β-glucan solutions during these processes. Furthermore, oxidized functional groups (carbonyls) were formed along the β-glucan chain by the treatments, including high pressure homogenisation, evidencing the oxidation of β-glucan by these treatments. The degradative forces acting on the particles in the high pressure homogenisation are generally considered to be the mechanical shear, but as shown here, carbohydrates are also easily degraded during the process, and oxidation may have a role in the modification of polysaccharides by this technique. In the present study, oat β-glucan was demonstrated to be susceptible to degradation during aqueous processing by non-enzymatic degradation mechanisms. Oxidation was for the first time shown to be a highly relevant degradation mechanism of β-glucan in food processing.