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(2018)Vitamin B12 is a water-soluble molecule with a complex structure. In the active form to humans B12 has to have 5,6-dimethylbenzimidazole as a lower ligand. B12 is synthesised only by certain bacteria and natural sources in the human diet are restricted mainly to foods of animal origin. The exact structure of B12 and vitamin activity, the supply from different diets and absorption in the body were discussed in the literature part of this thesis. Also B12 determination methods and B12 levels in foods of animal origin were discussed. Usually vitamin B12 contents in foods have been obtained with a microbiological method (MBA). Currently it has been of concern that MBA may overestimate results because test organism in the MBA method reacts also to compounds similar to B12. The aim of the experimental part of this thesis was to investigate content of vitamin B12 in foods of animal origin. Further, two determination methods, MBA and ultra-high performance liquid chromatography (UHPLC), were compared with each other. The aim was also to examine the effect of different extraction methods on the yield of vitamin B12. The samples chosen to this study were beef, pork meat, chicken meat, beef and pork liver, rainbow trout, Baltic herring, egg, skim milk, yogurt and Edam and Emmental cheese. Also two insects, cricket and mealworm, were chosen to this study. The extraction tests showed that it was not possible to use one extraction method to all samples. Extraction with pepsin improved the yield of vitamin B12 in rainbow trout, egg yolk, beef and milk whereas pancreatin improved the yield in cheeses and Baltic herring. As expected B12 content was the highest in livers of beef and pork. Also beef, Baltic herring and cheeses had high concentrations of B12. Chicken meat contained the lowest concentration of B12. Comparison between the MBA and the UHPLC method proved that with MBA the vitamin B12 concentrations were much higher than with UHPLC. B12 concentrations with UHPLC were 7–64% lower in meat and fish samples. Milk products and egg yolk had 20–67% lower B12 concentrations with UHPLC and insects had 71–81% lower concentrations. MBA method is sensitive and has low reagent costs but in the future UHPLC method should be chosen for B12 analysis because it can separate the active B12 form from the inactive forms.
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(2020)The usual dietary sources of vitamin B12 are animal-based foods such as meat, milk, fish and shellfish. Vitamin B12 is an essential water-soluble vitamin for humans, which have many necessary tasks in human body. People who lack animal foods like vegetarians and vegans might be at risk of vitamin B12 deficiency. Microorganisms are the only original sources of vitamin B12 in nature. Especially Propionibacterium freudenreichii synthesizes active form of vitamin B12 for humans. P. freudenreichii is GRAS -graded bacteria and it is safe to use in food matrices. Recently, it has been used for natural fortification of vitamin B12 in plant-based products by fermentation. However, the bioaccessibility of vitamin B12 of those products in human body is not very well known. In the beginning of the study, stability of different B12 vitamers were studied in different light and pH conditions. Vitamin B12 forms are very light-sensitive, especially its physiological forms methyl- and adenosylcobalamin. The aim of this research was to study bioaccessibility of vitamin B12 from P. freudenreichii cells and selected food products using a static in vitro assay. After the in vitro model, vitamin B12 content was analysed with UHPLC system. In addition of bacterial cell samples, bioaccessibility was studied also in some food samples, like bread, pasta and spray-dried powder, fortified with P. freudenreichii cells. In vitamin B12 stability studies, red light seemed to improve the stability of B12 forms. In yellow light methyl- and adenosylcobalamin degraded after 15 minutes. In red light they were detectable after hours of exposure. Methylcobalamin seems to be the most sensitive form of vitamin B12 vitamers. The study revealed that the bioaccessibility of B12 was very small (1,5 %) in P. freudenreichii cells but over 50% in whole bacterial broth. Heat treatment for the samples improved the bioaccessibility to some extent. In B12 fortified food samples, the bioaccessibility of B12 was very good (>70 %). The number of heat treatments and food structure could be one reason why bioaccessibilities in food samples are better than in cell samples. According to this research, in situ fortified food products could be a promising source of vitamin B12 in future.
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(2023)Tämän tutkielman tavoitteena oli tuottaa Propionibacterium freduenreichii -bakteerin ja Rhizopus -homesienen yhteisfermentaatiolla B12-vitamiinia härkäpavusta valmistettuun tempeen. Samalla tutkittiin, mitä aminohappoja ja orgaanisia happoja tempeissä vapautui fermentaation aikana ja vaikuttivatko ne B12-vitamiinin tuottoon. B12-vitamiinia sisältävien kasviperäisten elintarvikkeiden tuottamisen tutkiminen on tärkeää, sillä se helpottaisi vegaanista ruokavaliota noudattavien mahdollisuuksia saada B12-vitamiinia ravinnostaan. Hypoteesina oli, että B12-vitamiinia saataisiin härkäpaputempeihin, mutta oletettavasti sen pitoisuuteen valmiissa tempeissä vaikuttaisi käytetty propionibakteerikanta. Ensin kasvatettiin fermentointiin käytetyt propionibakteerikannat ja valmistettiin tempenäytteet härkäpavusta. Valmiista tempeistä määritettiin pH, kosteuspitoisuus, B12-vitamiini, vapaat aminohapot, orgaaniset hapot ja ammoniakki sekä propionibakteerien ja enterobakteerien määrät. Neljästä propionibakteerikannasta kaksi tuottivat tempeen huomattavia määriä B12-vitamiinia (1 µg/g kuivapainoa kohti). Fermentaation aikana vapaiden aminohappojen pitoisuus lisääntyi kaikilla kannoilla valmistetuissa tempeissä selvästi. Eri aminohappoja vapautui kuitenkin eri kannoilla hyvin eri määriä. Maitohappo kului näytteistä fermentoinnin aikana. Pelkällä Rhizopus -homesienellä valmistetussa näytteessä maitohapon määrä taas lisääntyi. Tämä oli tiedettävästi ensimmäinen kerta, kun tutkittiin härkäpaputempen valmistusta Rhizopus -homesienen ja propionibakteerin yhteisfermentaatiolla B12-vitamiinin tuottamiseksi. Tutkimus antoi lupaavia tuloksia B12-vitamiinipitoisen tempen valmistuksesta, sillä härkäpaputempejen vitamiinipitoisuudet olivat huomattavasti suurempia kuin mitä aikaisemmissa tutkimuksissa soija- ja lupiinitempeillä homesienen ja propionibakteerin yhteisfermentaatiossa saadut vitamiinipitoisuudet.
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(2016)Bakteereita ja eukaryootteja esiintyy erittäin runsaasti jätevedessä, jossa ne voivat muodostaa fyysisiä vuorovaikutuksia toistensa kanssa. Fyysisiä vuorovaikutuksia bakteerien ja eukaryoottien välille voi muodostua symbioosien tai saalis-saalistaja suhteen välityksellä. Bakteerit ja eukaryootit osallistuvat myös jäteveden puhdistusprosesseihin, joten näillä molemmilla on iso merkitys puhdistusprosessissa. EpicPCR-menetelmän avulla voidaan tutkia isoja mikrobipopulaatioita sekvensoimalla genomin alueita suuresta määrästä yksittäisiä soluja. Aiemmin menetelmää on käytetty selvittämään sulfaatin pelkistämiseen osallistuvien bakteerien fylogeneettista diversiteettiä järven sedimenteissä. Tämän työn tarkoituksena oli tutkia bakteerien ja eukaryoottien fyysisiä vuorovaikutuksia jätevedessä käyttäen apuna epicPCR-menetelmää. Tutkimuksessa yhdistettiin tiiviissä fyysisessä yhteydessä olleen bakteerin 16S rRNA- ja eukaryootin 18S rRNA -geeni. Samalla selvitettiin miten epicPCR-menetelmä soveltuu käytettäväksi kahden fylogeneettisen 16S rRNA- ja 18S rRNA -geenin yhdistämiseen ja miten jätevesi soveltuu näytteeksi. Tulosten perusteella pohdittiin millaisia voisivat olla bakteerien ja eukaryoottien fyysiset vuorovaikutukset ja mitä ne merkitsevät kummallekin osapuolelle. Fyysisiä yhteisvaikutuksia tutkittiin käyttämällä yksinkertaiseen teknologiaan perustuvaa epicPCR-menetelmää ja sekvensointi suoritettiin Illumina MiSeq -menetelmällä. Sekvenssien käsittely ja analyysi tehtiin käyttäen Unixin Bash -komentorivikieltä ja R-ohjelmointikieltä CSC:n Taito-laskentaklusterilla käyttäen apuna Qiime-työkalupakettia. Tulosten perusteella bakteerien ja eukaryoottien välillä havaittiin fyysisiä yhteyksiä, jotka jakaantuivat kahdella eri tavalla. Jätevedestä löytyi eukaryoottiryhmiä, jotka muodostivat yhteyden useamman bakteeriryhmän kanssa ja eukaryoottiryhmiä, jotka muodostivat yhteyden vain tietyn bakteeriryhmän kanssa. Saalistukseen pystyvät eukaryoottiryhmät muodostivat yhteyksiä useamman bakteeriryhmän kanssa kuten myös määrällisesti enemmän kuin fotosynteettiset eukaryoottiryhmät.
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(2013)Fungi play a crucial role in the ecosystem by recycling nutrients and forming mycorrhizal roots with plants. Many of the decomposer and mycorrhizal fungi are Bacidiomycetes. In the sexual reproduction stage, Bacidiomycetes produce fruiting bodies which enable them to produce and disseminate spores allowing fungi to spread to new growing sites. Fruiting bodies have been discovered to contain bacteria which may have a role in differentiation and maintenance of the fruiting body. They might also protect fruiting bodies against animals and diseases, and influence the nutritional value of the fruiting body. There is little knowledge about the amount of bacteria in the fruiting bodies. All previous research has been carried out entirely by cultivation-based methods and it shows that different fungal species contain very different amounts of bacteria. Some fruiting bodies have been shown not to contain easily cultivatable bacteria. The occurrence of archaea in fruiting bodies has not been previously studied and investigation into their function in fungi has only recently begun. In the present work significant amounts of bacterial and archaeal 16S rRNA -gene copies were discovered in the fruiting bodies of three ectomycorrhizal and three decomposer fungi species. This is the first time fruiting bodies have been shown to contain archaea. The occurrence of bacteria and archaea and the abundance of their 16S rRNA -genes in the fruiting bodies were determined using PCR ja quantitative PCR methods. Suillus bovinus and Boletus pinophilus fruiting bodies contained significantly more archaeal than bacterial gene copies. Cantharellus cibarius and Lycoperdon perlatum contained more bacterial than archaeal 16S rRNA -gene copies. In two decomposer fungi fruiting bodies, Agaricus arvensis and Piptoporus betulinus, the abundance of bacterial and archaeal gene copy numbers were equal. Suillus bovinus fruiting bodies had the largest copy number of archaeal 16S rRNA -genes from all species investigated. According to the results obtained in this work, the occurrence of bacteria and archaea might be common in fruiting bodies. The presence of bacteria and archaea in significant amounts in fruiting bodies may indicate their necessity for the development and sustainability of the fruiting body and hence to the whole life cycle of fungi.
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(2020)Milk foam is an essential part of coffee drinks such as cappuccinos. The foaming properties of milk can change during its shelf life. Milk used in coffee usually contains at least 2 % fat which can deteriorate its foaming properties. UHT milk can lose its sensory and foaming properties during its shelf life. In this thesis the change of properties of UHT-treated coffee milk called Barista milk were investigated. Barista milk is a commercial lactose free, homogenized milk meant to use with coffee. It has 2,0 % of fats and 3,5 % of proteins. The preservation of UHT barista milk was studied when milk was 4, 5, 6 and 7,5 months old. The foaming properties of barista milk were evaluated with automated foaming system. The volume of foam and its stability were measured as volume in a measuring glass. The structure of foams was also photographed and analyzed from photos. Nowadays plant-based drinks are important options for milk. Selected oat drinks for coffees were studied with same foaming methods as milk samples. The oat drink samples were kept in room temperature, fridge (+6 °C) and + 30 °C storage conditions. The tests were made when samples were 4, 8 and 10 months old. Barista milk as evaluated also with sensory evaluations. The sensory properties of barista milk were measured by tasting the milks referred to a fresh reference milk. Sweetness, bitterness, intensity of cooked flavor and fullness were evaluated from milks and cappuccino drinks made from the studied milks. In addition, a professional barista foamed the milks to makes cappuccinos and the drinks were evaluated. The preservation of milk did not affect its foaming properties or foam stability significantly. The foaming temperature affected the volume of foam more than the age of the milk. There was a slight change in the bubble structure of milk. The bubble structure was more uneven with older milk than fresh milk. Oat drinks foamed more, and the foams were more stable in all samples compared to milks of same age. The effect of foaming temperature was less meaningful with oat drinks than with milks. The sensory properties of milk did not change significantly during the study. The biggest change was the decline of cooked flavor in samples tasted as milk. The same decline was not detected when the milks were tasted as cappuccino drinks. An increase of bitterness was detected in the cappuccinos made by a barista when the milk was 6 months old. According to these results the shelf life of barista milk can be lengthened from 4 months to 5 months.
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(2019)The literature review deals with coeliac disease and wheat, rye and barley prolamins. The characteristics and analysis methods of the prolamins were discussed. Furthermore, the ELISA types, antibodies and reference material in ELISA were introduced in literature review. The aim of this master thesis study was to determine the feasibility of barley C-hordein as reference material in the quantification of wheat gluten in a sandwich ELISA method based on R5 antibody. RP-HPLC was used to determine the compositions of wheat prolamins from 27 wheat cultivars. SDS-PAGE was used for wheat prolamin subgroups identification. The R5 antibody reactivity of prolamins and the same prolamin group from different cultivars were tested. By comparing the R5 reactivity of total gluten of the 27 wheat cultivars, 10% barley C-hordein was used to calibrate the gluten content in spiking test of oat flour and oat biscuits. Omega 1,2-gliadin (Km 13) and γ-gliadin (Km 21) showed rather strong R5 reactivity while ω 5-gliadin (Km 203) and LMW glutenin subunits (Km 523) showed almost no reactivity against R5 antibody. However, the subgroup R5 reactivity differences between cultivars were not significant. The reactivity of total gluten from 27 cultivar varied from Km 14 to 192, with a logarithmic average Km 53. Thus, 10% C-hordein (Km 49) had similar reactivity of the average of all cultivars. In flour spiking test, the recoveries calibrated with PWG gliadin were 57-187%, comparing to 30-115% with 10% C-hordein calibration. In biscuit spiking test, the recoveries calibrated with PWG gliadin and 10% C-hordein were 84-145% and 44-76%.
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(2023)The construction sector is a substantial contributor to waste and global carbon emissions due to the extraction and consumption of natural resources. Scholars underscore the critical importance for the construction sector to embrace the principles of the circular economy (CE), with a central focus on the efficient and careful use of natural resources, promoting material reuse and avoiding waste. Previous research conducted on the CE transition within the construction sector has shown a gap in addressing its material perspective. In Finland, the use of wood in construction is actively promoted by the government due to its potential to reduce industry’s carbon emissions. However, most used wood materials and waste from construction end up in energy recovery processes releasing absorbed carbon. This study explores the reasons for the limited wood recirculation within the shortest and most preferable loops in construction sector in Finland. The qualitative research data is based on 12 semi-structured qualitative interviews with professionals working either directly or closely to the construction sector. Employing the method of thematic analysis, this study endeavors to discover barriers, drivers and opportunities associated with recirculation of wood in the construction industry of Finland. While the findings of this study were found to be aligned with previous research that has identified barriers and drivers to CE implementation both within the construction industry and more broadly, multiple new wood-specific challenges were identified. The research findings demonstrate the necessity of adopting a holistic and systematic approach to increasing wood recirculation in construction and underscore the significant role of mediating actors, such as management of material hubs, in facilitating its implementation. This study suggests that supported by mediating actors, the development of the business ecosystem for circulated wood, enhancement of construction industry’s experience beyond internal projects is needed. Harmonization of the industry’s practices within the interplay of CE and bioeconomy thinking would promote wood recirculation in construction.
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(2020)This study examines Finnish consumer and beef producer preferences for eight process-related beef credence attributes. A direct ranking preference method was used in surveys conducted during spring of 2019. An analysis of rank means and shares of first rankings revealed differences and similarities between respondent rankings. Ordered logistic regression was also used to analyse how rankings differ within respondent groups. The results suggest that antibiotic-free beef production was ranked as most important by consumers and second by beef producers, suggesting that such production methods may have significant market potential in the future. The results also suggest that environmentally friendly production was ranked relatively high by Finnish consumers, and higher compared to previous similar studies from other countries. In contrast to earlier studies, this study innovates with also exploring farmer preferences for credence attributes, so as to provide novel insights by contrasting consumer and producer valuations from the same country (Finland) for such attributes.
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(2019)Elintarvikkeiden komponenttien välillä olevat vuorovaikutukset voivat vaikuttaa elintarvikkeiden valmistusprosesseihin, säilyvyyteen ja terveysvaikutuksiin. Tärkkelys ja beetaglukaani molemmat sitovat vettä, ja niiden vaikutus gluteenittoman leivän rakenteen muodostumisessa on olennaista. Gluteenittoman leivän suurin haaste on rakenteen säilyminen paiston jälkeen, mikä aiheuttaa hävikkiä. Vuorovaikutuksien tuntemisen ansiosta prosessit voitaisiin optimoida niin, että gluteenittoman leivän rakenne säilyisi pidempään hyvänä. Kokeellisen työn tavoitteena oli selvittää reologisilla menetelmillä, onko tärkkelyksen ja beetaglukaanin geeliytymisessä eroja, kun näiden muodostamien seosten kuumennusaikaa ja lämpötilaa vaihdeltiin.Vuorovaikutuksia tutkittiin mittaamalla seosten viskositeettia Rapid Visco Analyzerilla (RVA), ja geelien reologiaa mitattiin taajuuspyyhkäisymittauksella. Beetaglukaania sisältävän tärkkelysliuoksen RVA:lla saadun liisteröitymiskäyrän huippuviskositeetti ja loppuviskositeetti olivat korkeampia kuin ilman beetaglukaania valmistetun tärkkelysliuoksen. Beetaglukaania ja tärkkelystä sisältävillä geeleillä oli myös vahvempi rakenne kuin pelkästä tärkkelyksestä valmistetulla geelillä. Beetaglukaanin ja tärkkelyksen liuottaminen yhdessä ei kuitenkaan johtanut vahvemman geelin muodostumiseen, kun liuosta kuumennettiin levyllä noin kahden tunnin ajan. Tutkimuksessa haluttiin tutkia tarkemmin sitä, miten liuotusaika ja -lämpötila vaikuttavat geelirakenteeseen. Kahden tunnin ajan 85 ˚C lämpötilassa liuotetun beetaglukaanin ja gelatinoidun tärkkelyksen sekoituksesta valmistetun geelin ominaisuuksia vertailtiin 15 minuuttia korkeintaan 37 ˚C:ssa liuotetun beetaglukaanin ja liisteröidyn tärkkelyksen sekoituksesta valmistettuun geeliin, ja havaittiin ettei beetaglukaanin parempi liukeneminen johtanut vahvemman geelin muodostumiseen. Tutkimuksen tulokset viittasivat siihen, että laimean beetaglukaaniliuoksen geeliytymisen taustalla on osittainen liukeneminen, mikä on todistettu jo aiemmissa tutkimuksissa. Tutkimuksen tuloksien soveltamisesta gluteenittoman leivän valmistuksessa voitaisiin saada hyötyä, kun rakenne säilyisi hyvänä pidempään prosessien optimoinnin seurauksena.
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(2014)Syanobakteerit tuottavat bioaktiivisia sekundääri- eli erikoismetabolian tuotteita. Syanobakteerien bioaktiivisia yhdisteitä on rakenteiltaan lukuisia erilaisia ja ne voidaan jakaa pääryhmiin peptidit, polyketidit, alkaloidit ja lipopolysakkaridit. Yhdisteistä on löydetty muun muassa antibakteerisia, antifungaalisia ja antiviraalisia ominaisuuksia. Lisäksi näihin bioaktiivisiin aineisiin kuuluu terveysriskin aiheuttavia toksisia yhdisteitä. Planktiset syanobakteerit ovat saaneet runsaasti huomiota niiden muodostamien massaesiintymien vuoksi, mutta myös benttiset eli sedimentissä tai erilaisilla pinnoilla elävät syanobakteerit voivat aiheuttaa terveysriskin. Benttisillä syanobakteereilla on myös potentiaalia uusien bioaktiivisten yhdisteiden löytymisessä biotekniikan tai lääketeollisuuden tarkoituksiin. Tutkimuksessa seulottiin Itämeren ja järven benttisten syanobakteerien tuottamia bioaktiivisia yhdisteitä. Syanobakteerikannat tunnistettiin 16S rRNA geenin avulla ja niiden bioaktiivisuutta tarkasteltiin maljadiffuusiomenetelmän avulla, PCR:n avulla etsittiin tunnettuja biosynteesigeenejä. Bioaktiivisia yhdisteitä pyrittiin tunnistamaan LC/MS-menetelmällä. Tunnistetut syanobakteerit muodostivat monipuolisen benttisille syanobakteereille tyypillisen lajikirjon, jossa bioaktiivisia ominaisuuksia havaittiin 24 % tutkituista kannoista. Bioaktiivisia ominaisuuksia löydettiin niin meri- kuin järviympäristöistä eristetyiltä kannoilta. Useita bioaktiivisuuksia esiintyi muun muassa Nostoc- ja Anabaena-syanobakteereilla. Benttiset syanobakteerit tuottivat antifungaalisia yhdisteitä, kuten hassallidiinia ja skytofysiiniä sekä sytotoksista anabaenolysiiniä. Lisäksi löydettiin useita syanobaktiinien biosynteesigeenejä. Benttisiltä syanobakteereilta ei havaittu mikrokystiinin tai anatoksiini-a:n biosynteesigeenejä. Antimikrobisia ominaisuuksia löydettiin kahdesta syanobakteerikannasta, joiden tuottamia bioaktiivisia yhdisteitä ei vielä tunnistettu. Benttiset syanobakteerit tuottavat siis useita tunnettuja bioaktiivisia yhdisteitä ja ovat myös potentiaalinen uusien bioaktiivisten yhdisteiden lähde.
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(2014)The aim of this study was to examine how different enrichment objects affect the behaviour of finishing pigs towards enrichment objects and other pigs and also the occurrence of tail- and ear-biting within the pens. Another aim of this study was to examine the stress level of finishing pigs by two different methods. This experiment was carried out in two piggeries; in Huittinen and Pöytyä. The pens were divided into different treatments depending on which enrichment object was in the pen. There was a straw rack in every pen and in Huittinen there was also a simple metal chain in every pen. In the control pens of both piggeries there were no other objects besides these. The enrichment objects in Huittinen were wooden pieces of young birch in different lengths, polythene pipe, simple metal chain and branching metal chain. In Pöytyä the enrichment objects were wooden pieces of young birch and polythene pipe. Data collected in both piggeries were tail and ear lesions and tear staining under the eyes. Only in Huittinen the pigs were videotaped to observe behaviours during contact with each enrichment object and oral manipulation of other pigs. Only in Pöytyä the pigs were measured the latency to approach an unfamiliar human to assess fear of humans. Statistical analysis was accomplished by SPSS-software. In Huittinen the prevalence of tail- and ear-biting and also tear staining was lower in pens with wooden enrichment objects. The use of enrichment objects was the most active in pens with wood and with polythene pipe. There were no differences between the enrichment objects on the oral manipulation of other pigs. In Pöytyä the prevalence of ear-biting was also lower in pens with wooden enrichment objects. However, in tail-biting there was only a tendency for an improvement. Also in tear staining there was only a tendency for an improvement on the left eye in pens with polythene pipe compared to the control pens and there were no differences on the right eye between the enrichment objects. Pigs in pens with wooden enrichment objects were less fearful than those with other enrichment objects and approached the unfamiliar human faster. According to this study wood had the greatest impact as enrichment object for finishing pigs. Polythene pipe as enrichment object was used as frequently as wooden enrichment objects but its efficacy in lowering the prevalence of tail- and ear-biting and also stress level was less. Metal chain was used by the pigs least and there was no difference in the activity of use when a simple metal chain was compared to a branching metal chain. There was less tail- and ear-biting and also lower stress level in pens with wooden enrichment objects compared to other pens.
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(2015)Lactobacillus rhamnosus GG is a lactic acid bacterium that is widely used as probiotic products in the dairy industry. To gain insights into the genome stability of the L. rhamnosus GG in the human gastrointestinal tract and the possible adaption mechanism under different stresses, we first examined the genotype and phenotype of the L. rhamnosus GG grown over 1000 generations under various stresses, including bile salts, osmotic stress or shearing forces. Immunoblotting analysis of L. rhamnosus grown over 1000 generations showed that the production of mucus-binding pili by L. rhamnosus GG was the most impaired when exposed to bile salts. Complementary PCR screening of 13 highly variable chromosomal regions in GG confirmed that the pilus gene cluster had been lost when exposed to bile stress over time. In vitro bile-induced genomic changes observed in GG possibly reflects the genome plasticity and stability of GG in the human intestinal tract. Still, we showed that these changes only occurred after more than 100 generations, a period of time relatively long compared to the observed time of persistence and transit of GG in the intestinal tract. Although damages and stresses may be caused by bile salts, L. rhamnosus GG still has the ability to tolerate bile salts. The resistance mechanism is still unclear but, based on previous studies, we identified one ABC transporter encoded by the gene operon called tauABC that may be involved in bile resistance. In an effort to demonstrate its function, a tauB-null mutant derivative was generated and phenotypically characterized in terms of metabolic, signaling and functional properties. The data revealed that the tauB-null mutant significantly grow slower than L. rhamnosus GG wild-type strain in the presence of ox bile extracts. Additional screenings using various bile conjugates specifically revealed that two compounds of bile salts, i.e. taurodeoxycholic and taurochenodeoxycholic acid, may be processed by the TauABC transporter, contributing at least partially to the tolerance of GG to bile salts. Overall, we showed that bile salts constitute an important stress factor for GG that causes genomic alterations, although it has bile tolerance mechanisms to bile, such as the newly-characterized tauABC operon.
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(2015)Clostridium difficile infection (CDI) is a microbiota-related disease. Typically, antibiotic-induced perturbation of gut microbiota precedes the infection, while a healthy gut microbiota provides protection, i.e. colonization resistance, against it. Furthermore, in the case of recurrent CDI that is not resolved by antibiotics, restoring the gut microbiota with a fecal microbiota transplantation (FMT) is among the few treatment options that really work. Although FMT is effective in the treatment of CDI, the factors behind treatment success remain unclear. Both, the key species and the functions that are necessary to restore the healthy microbiota and eradicate C. difficile, are a matter of speculation. This study was based on the hypothesis that the adherence of some commensal bacteria to the gut epithelial cells could play a role in eradicating C. difficile by competing for epithelial binding with it. Furthermore, the isolation of those bacteria from the donor feces would enable more detailed mechanistic studies and development of a bacterial product for the treatment of CDI in the future. As a pre-selection step, bacterial adhesion to Caco-2 cells was utilized to isolate and cultivate epithelium-adherent bacteria from the donor feces. Microbiota composition of fecal sample, and the adhered and cultured sub-populations thereof, was determined by partial 16S rDNA amplicon sequencing using MiSeq method. The pre-selection approach was successful, since the obtained populations were different, both after the adhesion and cultivation, as compared to the original fecal sample. In addition, most obtained pure isolates adhered well to enterocytes. The ability of fecal bacteria to compete with C. difficile for binding to gut epithelial cells in vitro was also studied. Isolated bacteria from Caco-2-adhered populations were applied in competition and exclusion assays with C. difficile as purified or multi-species cultures, and reduction in C. difficile binding was observed due to the certain bacteria or bacterial populations. These assays still need developing and the results must be confirmed with more repetitions. However, the results are promising and a useful ground for future work in developing bacteriotherapeutic formulations for the treatment of CDI.
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(2015)Compared to many other countries, Salmonella prevalence in finnish poultry, bovines and pigs are at a low level. This low level has been obtained because of the national salmonella control programme. However, Salmonella still occur in feeds and at broiler farms in Finland and EU. Bioactive glass (Bioglass) could be a potential feed additive for preventing Salmonella in poultry since many new feed additives have got EU ac-ceptance. Bioactive glasses have been found bactericidic when used in dentistry and artificial tissue technology as implant material. The aim of this thesis was to study if and how bioglass effects Salmonella enterica bacteria in chicken feed and gut contents. Further, the purpose was to determine if bioglass can reduce the amount of Salmonella in the sample matrixes using the spread plate method. Six strains of Salmonella enter-ica from Hambi culture collection (HAMBI 1111, 1112, 1306, 1513, 2317 ja 2331) was added to the matrixes. Commercial XLD medium was used to determine the amount of Sal-monella in each time point of the test. 0 - 10 % of bioglass was tested for bactericidic effects. The ability of bioglass to inhibit the growth of Salmonella was attempted to demonstrate with transmission electron microscopy and using gram staining and light microscopy. The results indicate that the bactericidic effects of bioglass work in the sample matrix-es nearly as well as in liquids. The added amount of Salmonella was significantly re-duced with the higher concentrations of bioglass. Lower levels of bioglass did not show bactericidic effects or the inhibition of the growth of Salmonella was minor. The bacte-ricidic effect is thought to be based on the rise of pH and the adherence to bacterial cell wall. TEM imagines illustrated the attachment of the bioglass particles to degrad-ing cells of Salmonella Typhimurium (HAMBI 1112). Bioglass could be a potential feed additive for the prevention of Salmonella in broilers.
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(2016)Sustainable development and fluctuating petroleum prices encourage manufacturing packaging materials in a more natural and cost-effective way. Green economy utilizes renewable raw materials from the nature which can be used in larger scale applications such as packaging industry. Barrier properties can be enhanced by manufacturing multilayer structures with a coating made of organic materials such as cellulose nanofibrils (CNF). With CNF it is possible to provide barrier and mechanical improvement to packaging material at low added cost. Together with hydrophobic films (e.g., HDPE, LDPE) also the low water vapor permeability and heat sealing properties can be enhanced. The aim of this thesis was to utilize CNF as a barrier layer for bio-based flexible food packaging pouches. A study consisted of examining 1-layer bio-HDPE film, 2-layer bio-HDPE/CNF film and 3-layer HDPE/CNF/LDPE film and testing their permeability (for oxygen, water vapor and aroma), sealability, tensile strength, influence of irradiation and aroma compounds (clove), and suitability for modified atmosphere packaging, MAP (with hazelnuts). The 2- and 3-layer multilayer films provided very good oxygen barrier under dry conditions, although aroma exposure increased the oxygen permeability rates. Pouches made of the 2-layer film were the most feasible for MAP, even though the 3-layer film would also have been an alternative if the film quality had been optimal. The oxidation of fatty acids in hazelnuts could be slowed down by using multilayer structure including CNF barrier under modified atmospheric conditions. This study helps to verify the feasibility of the bio-based multilayer films as a new food packaging material, and demonstrates the use of CNF as a high-barrier layer in a laminate. Bio-HDPE/CNF/bio-LDPE multilayer has potential for utilizing it in flexible food packaging pouches if the manufacture process is standardized leading into homogeneous film quality.
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(2022)In order to achieve carbon neutrality and slow down climate change, it is necessary not only to curb greenhouse gas emissions but also to remove carbon from the atmosphere. In agricultural sector, adding biochars to soils has proven to be one of the most effective methods to sequester carbon. Using biochars in urban planting soils could be simple and effective way to create carbon sinks also in urban environment. Wood based biochars are already available in the market and their viability as soil amendment is supported by an extensive body of research. Despite large evidence from agricultural and greenhouse sectors, research focused on biochars in planting soils for urban trees is scarce. To assess and demonstrate viability of biochars as component of urban planting soils, an experiment was established in 2019-2020 in Hyväntoivonpuisto-park in Helsinki. The experiment consists of four tree species and nine different planting soils, seven of which include biochars. Three of the planting soils were structural soils installed below a sealed surface. Tree growth in planting soils was followed and biomass accumulation was estimated with allometric equations. Planting soil nutrient composition was analysed at the time of soil application and nine months later after first growing season. Pyrogenic carbon fractions were analysed by BPCA analysis. Planting soil physical and hydrological properties were analysed by water retention curves with pF range of 0 – 4.2. After two growing seasons, biochars had increased tree growth in two planting soils compared to the control while in other planting soils with biochar, the growth was similar to control. In structural soils biochars had improved growth, but there were no statistically significant pairwise differences between treatments. Biochars increased the macroporosity of planting soils, indicating they could improve aeration and water conductivity in planting soils. Effects to total porosity and water retention capacity were mixed, but highest total porosity and water retention capacity was observed in planting soil with the highest amount of biochar. Planting soil nutrient composition varied a lot due to different raw materials, limiting the possibilities of making mechanistic analysis of effects of biochars. These results indicate that biochars are viable and safe constituent for planting soils which may increase tree growth by improving soil physical properties and improve carbon sinks in urban infrastructure.
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(2017)This research was conducted to examine the effect of biochar on drought tolerance and growth of a grain crop. Biochar is a multipurpose charcoal that heals the soil and promotes crop yield. The experiment was part of University of Helsinki biochar research during the summer and autumn of 2011. The effect of biochar was studied in a greenhouse experiment to determine how well it can improve sorghum (Sorghum bicolor Moench) yield. The experimental factors were amount of water given at low, intermediate and full watering levels, and the absence or presence of Acacia-biochar at level of 10 t/ha in the pots of soil used. The number of leaves per plant, leaf surface area, number of panicles, number of grains per panicle, plant and panicle height, root biomass, and whole plant (above ground) biomass was obtained. The plants were grown in a high greenhouse temperature to mimic the Sudanese extreme weather temperature according to the Khartoum Sudan weather forecast at the time of the experiment. The plants got same amount of water from cultivation until the emergence of 5 leaves. Early drought was proposed to determine its effect as the plant move from vegetative to reproductive stage and the effect of biochar on the soil water holding capacity (WHC). The drought stress started at 5 leaf stage of the plant life cycle. The data were analyzed using 2-way analysis of variance (Anova) statistical tools in SPSS (version 20.0, SPSS Inc., Chicago, IL, USA) software. Drought stress affected panicle development, grain yield, plant height, leaf surface area and overall biomass yield. Grain yield and whole plant biomass weight was higher in non-stressed plants. The results indicated no relief to drought stress by biochar addition. In this experiment, the addition of low biochar might be responsible for it inefficiency.
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(2016)Plant biomass consists largely of polymeric compounds of which diverse polysaccharides are the main components. Ferulic acid is a ubiquitous phenolic phytochemical in plant cell wall and forms the linkage between plant cell wall polymers. Therefore it is a major aspect in the recalcitrance of cell wall against microbial attack. Ferulic acid esterases (FAEs) are hydrolytic enzymes which participate in plant biomass degradation by removing ferulic acid from the polysaccharides in order to weaken the integrity of the cell wall. By using phylogenetic gene prediction strategy, three putative FAE gene models have been detected from the genome of the ascomycete fungus Aspergillus niger. The codon optimized putative FAE encoding genes have been synthetized for heterologous production in Pichia pastoris GS115. In this work, these three FAEs of A. niger, i.e. FAE796, FAE807 and FAE809, were produced in P. pastoris and their biochemical properties were characterized. The properties included substrate profiling, thermostability, pH optimum and solvent tolerance of the recombinant FAEs. The three A. niger FAEs were successfully produced in P. pastoris resulting as approximately 57 kDa molecular mass proteins. Substrate profiling was performed by using a set of 11 synthetic FAE model substrates and substrates for tannase and lipase activity. FAE796 and FAE809 preferred methoxy substrates, and thus were likely to belong to type A class of FAEs. FAE807 had activity towards a wider range of substrates including methyl sinapate, methyl cinnamate, chlorogenic acid and para-nitrophenyl ferulate, suggesting it to belong to type C class of FAEs. In addition, FAE807 had tannase activity which is a novel property described among the FAEs studied so far. The optimal temperature for FAE796, FAE807 and FAE809 were +37 °C, +55 °C and +55 °C, respectively. FAE809 was the most thermostable enzyme, and retained half of its activity up to +60 °C for 60 min. The studied FAEs were most active at pH 4.0-5.0. FAE809 was relatively stable towards the studied solvents retaining 70%-91% of its activity after solvent treatment.
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(2016)Actin is known as abundant cytoplasmic protein, which functions as a component of the cytoskeleton and in cell motility together with motor protein myosins. However, actin is also present in the nucleus, where it has been shown to take part in the control of gene expression, both independently and as part of chromatin remodeling complexes. An important aspect in the study of nuclear actin is to identify other nuclear proteins interacting with actin and to confirm these interactions in biochemical experiments. Through these interactions actin can be linked to various nuclear processes. The aim of this master’s thesis study was to express and purify five nuclear proteins that have been suggested to bind actin and to study the binding in detail both with actin filaments and monomers. The proteins of interest include four actin-related proteins (ARPs) Arp4, Arp5, Arp6 and Arp8 as well as RNA polymerase II subunit Rpb8. Out of the proteins selected for this study, the expression and purification of Arp4 and Rpb8 was successful. Neither one did bind monomeric actin with high affinity, but interestingly Rpb8 did bind actin filaments. On the other hand, Rpb8 did not have any effect on actin polymerization. These results provide new insights into nuclear actin function. It has been suggested earlier that Arp4 would form a heterocomplex with actin, but the results of this study do not support this. The binding between Rpb8 and actin in RNA polymerase II complex has not been extensively studied before, so the results provide new information about the function of actin in the polymerase complex.
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