Browsing by Subject "hiiva"
Now showing items 1-6 of 6
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(2016)Saccharomyces cerevisiae is a popular organism in the production of biofuels, chemicals and pharmaceuticals. This is thanks to a good understanding of its metabolism, GRAS status and the ease of modification. Traditionally its genetic modification has been based on the use of selectable markers. Modifying multi gene pathways has required a sequential process consisting of multiple single gene disruptions together with marker recycling. Additionally, many industrial S. cerevisiae strains are polyploid and lack the same tools for their modification as laboratory strains. In this study we sought to develop CRISPR/Cas9 based genetic engineering method for the modification of industrial S. cerevisiae strains. The CRISPR/Cas9 system is based on the adaptive immunity system of bacteria. It makes use of the Cas9 endonuclease which produces double stranded DNA brake to any location determined by a gRNA molecule. This causes the activation of DNA repair mechanisms which can be utilized to for the genomic integration of a template DNA. This makes transformation events much more likely and thus enables producing multiple modifications at once and removes the need for the of use selectable markers. In our approach Cas9 and gRNA were transformed into the cell in a plasmid together with a separate template DNA molecule. We used this method to remove lyp1, ura3 and can1 genes from diploid and polyploid industrial S. cerevisiae strains multiple genes at a time. Simultaneously we evaluated the effect of the NHEJ repair mechanism on CRISPR/Cas9 by repeating the tests with a deletion strain missing the ku70 gene required by NHEJ. Finally the method was used for the metabolic engineering by integrating the five gene violacein metabolic pathway into two loci in a single transformation event. This study demonstrated the CRISPR/Cas9 method is well suited for the modification of industrial S. cerevisiae strains and is capable of modifying up to three loci at a time in a polyploid yeast strain.
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(2019)The percentage of silage in total dry matter consumption of cows belonging to milk recording in Finland was 51 % in 2018. Harmful microbes can be found in feed. Of these microbes’ clostridia cause problems in cheese production in dairies and yeasts affect the aerobic deterioration of silage and produce ethanol, which causes great dry matter losses. Feed microbe concentrations have traditionally been determined by cultivating, but PCR methods bring novel rapidity and sensitivity to silage microbe research. The aim of this study was to test and develop a PCR based method to detect harmful microbes in silage samples. An additional aim was to specifically identify and quantify four clostridia species and one yeast species from samples of a silage trial. First, a literature review and a database search on harmful microbes in silage were conducted. Based on the results, seven primer pairs were selected, of which three were general primer pairs and four species-specific primer pairs. The usefulness of these primers was tested on silage, cheese and yeast samples in laboratory conditions with gel electrophoresis and qPCR. Additionally, was a white lupin-spring wheat mixture ensiled without additive, with formic acid (2, 4 or 6 l/t fresh matter) and natrium nitrite (900 g/t fresh matter) -hexamine (hexamine 0, 300 or 600 g/t fresh matter) in three laboratory silos/treatment. A chemical quality analysis and quantification of harmful bacteria with qPCR were conducted on pre-ensiling forage and silage samples for Clostridium tyrobutyricum, C. butyricum, C. sporogenes, C. perfringens and Saccharomyces cerevisiae. Microbes were found in abundance. From the chosen primer pairs, two general primer pairs would have been suited for quantification on agarose gel and S. cerevisiae primers proved to be suitable for qPCR. Compared to the amount of microbe DNA, surprisingly few clostridia were found in the samples. Clostridia/spores were found only in five silos ensiled with formic acid. Butyric acid was found in more silos. Possible explanations for the small amounts of clostridia found, might be the effect of formic acid on microbe populations, minor soil contamination while mowing and collecting the forage, PCR-inhibitors in lupin, the nitrate concentration in the feed, and activity of other microbes. Yeast concentrations in the samples were high. Ethanol concentrations were small compared to yeast concentrations. There might have been too little oxygen available for the yeast. PCR-methods can be used to specify and support chemical analyses from silage. Conclusions on microbe concentrations in silage cannot be solely based on chemical analyses. Furthermore, a too specific microbe quantification might raise contradictions and problems in the interpretation of results. Primers that are genus’s specific or recognize even broader taxon might be more functional than species-specific primers.
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(2015)The aim of this thesis was to find a suitable natural antifungal substituent to improve shelf life of quark based products. Antifungal properties were analysed with four different protective cultures: HOLDBAC™ YM-B Plus, HOLDBAC™ YM-C Plus, HOLDBAC™ YM-XPM and FreshQ® 1. Also two different plant based compounds Melinat® and Verdad® F95 were examined. Different dosages and adding methods were used to study the feasibility of these compounds to quark based products. Physical and microbiological factors affecting the shelf life of fresh cheeses, manufacturing processes of cream cheese and quark based products and antifungal properties of both lactic acid and propionic bacteria as well as plant based compounds were reviewed in the literature section. The mixture of milk and quark in elimination tests and cream cheese and quark based spread in following tests were used as samples. The samples were contaminated with two different yeasts, Rhodotorula mucilaginosa and Candida utilis and a mold Aspergillus brasiliensis. The concentration of yeasts and mold were analysed after 0, 7 and 14 days storage at 9 °C. The properties of antifungal compounds were analysed also with the help of chemical and sensory analysis as well as shelf life tests. Protective cultures with recommended dosage were able to inhibit the growth of yeasts in the mixture of milk and quark. A tenfold increase in dosage did not have effect on yeast concentration. HOLDBAC™ YM-B Plus and FreshQ® 1 -cultures added at the same time with fermentation when manufacturing cream cheese and spread samples were not as efficient as potassium sorbate. However, the results from spread samples prepared with post-addition were equal to the results of potassium sorbate. Heat treatment was shown to have a significant impact on the performance of protective cultures. The heat tolerance of protective cultures is needed to investigate in the future so that the adding methods and feasibility especially to heat-treated products would be evaluated more closely.
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(2020)Tiivistelmä – Referat – Abstract Ikääntymiseen liittyvien biologisten ilmiöiden, kuten rappeutumisen ja sairauksien tutkiminen on ensiarvoisen tärkeää, koska näistä ilmiöistä on haittaa niin yksilölle kuin koko yhteiskunnalle. Erityisen kiinnostavaa on, jos sairaudet ja rappeutuminen pystytäisiin parantamaan tai niiden haitallisia vaikutuksia voitaisiin vähentää. Ikääntymiseen voidaan yhdistää monia vakavia sairauksia kuten hermostorappeumasairaudet, syöpä ja diabetes. Hermostorappeumasairauksille on tunnusomaista proteiiniaggregaattien muodostuminen. SBC003-yhdiste on luonnon yrtistä eristetty molekyyli, joka on potentiaalinen lääke hermostorappeumasairauksiin. Aikaisemmin tehdyt tutkimukset hermostorappeuma sairausmalleilla osoittavat yhdisteellä olevan voimakas konsentraatiosta riippuvainen hermosoluja suojaava vaikutus. Malliorganismit ovat tärkeitä, koska niillä pystytään testaamaan potentiaalisten molekyylien vaikutusta vahingoittamatta kuitenkaan ihmistä. Hiivasta on tullut mielenkiintoinen malliorganismi ikääntymisen ja ikääntymiseen liittyvien sairauksien tutkimuksessa. Monet biologiset prosessit ja aineenvaihduntareitit ovat konservoituneet hiivasta aina ihmiseen asti. Lisäksi geneettiset muutokset on helppo jäljittää. Ikääntymisen tutkimisessa leivinhiiva (Saccharomyces cerevisiae) on osoittautunut erinomaiseksi malliorganismiksi. Hiivan kronologinen ikääntyminen on osoittautunut myös hyväksi menetelmäksi tutkittaessa solujen post-mitoottista ikääntymistä. Hiivasoluissa pystytään havaitsemaan nopeasti, edullisesti ja helposti proteiiniaggregaattien aiheuttamat haitalliset vaikutukset. Käytettäessä hiivaa malliorganismina tutkimuksessa pystytään luomaan suora linkki geenien ja kemiallisten yhdisteiden välille. Hiivalla tehdyt solujen sairausmekanismien tutkimukset ovat vertailukelpoisia ihmisten sairauksien kanssa. Nämä seikat tekevät hiivasta kiinnostavan malliorganismin myös hermostorappeumasairauksien lääkeainetutkimuksessa. Työn tavoitteena oli selvittää, voidaanko hiivasairausmalleja käyttää SBC003-yhdisteen vaikutuksien arvioimiseen hermostorappeumasairauksissa, ja onko tutkittavalla yhdisteellä pelastava vaikutus haitallisia aggregaatteja muodostavia proteiineja vastaan hiivasairausmalleissa. Lisäksi tarkoitus oli tutkia, onko SBC003-yhdisteellä vaikutusta ikääntymisestä aiheutuvaan elinvoimaisuuden heikkenemiseen hiivasoluissa. Tutkimuksissa käytettiin seuraavia menetelmiä ja koejärjestelyjä 1. spottitestejä ja 2. elinkiertotestejä ravistelukasvatuksina, joiden elinvoimaisuutta mitattiin näytteillä, joita puolestaan analysoitiin edelleen Bioscreen-kasvatuksia apuna käyttäen. Spottitestien ja elinkiertotestien tuloksien mukaan SBC003-yhdisteellä saattaa olla pelastava vaikutus haitallisia FUS-aggregaatteja vastaan hiivan ihmisen sairausmallin soluissa. Tutkimustulosten mukaan SBC003-yhdisteellä on solun elinvoimaisuutta parantava vaikutus soluihin, jotka rappeutuvat ikääntymisen seurauksena. Saatujen tulosten varmentamiseksi pitäisi elinkierotestit ja spottitestit toistaa ja lisäksi tehdä sekä geneettisiä että epigeneettisiä tutkimuksia.
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(2019)Yeasts are significant plant pathogens, yet plant-yeast interactions are poorly understood. Mammals have immune receptors for yeast-specific MAMPs (microbe-associated molecular patterns), suggesting that plants might have similar receptors as well. Detection of MAMPs by PRRs (pattern recognition receptors) triggers the plant immune system, leading to the first phase of PTI (pattern-triggered immunity). The yeast cell wall is layered, so only some of the potential MAMP molecules, like mannans, are easily available for the plant receptors. In order to utilize yeasts and PRR-based yeast resistance in agriculture and forestry, it is crucial to identify these receptors and gain a better understanding of their functions in plant-yeast interactions. PRRs can be divided into two groups, RLPs (receptor-like proteins) and RLKs (receptor-like kinases). Some of the L-type lectin receptor kinases have already been shown to participate in the interactions between plants and fungi or fungal like pathogens, making them promising candidates for yeast receptors. G-type lectins remain less studied but have promising genes as well. In this thesis, we use forward and reverse genetics methods to show that lectin receptor kinases might include putative yeast pattern recognition receptors. We also establish the first protocol and genetic screen for the identification of plant PRRs participating in the recognition of yeast cell wall MAMPs. Only genes present in both silver birch (Betula pendula) and arabidopsis (Arabidopsis thaliana) were examined to enable following studies with an Arabidopsis model system of the interactions between silver birch and the dimorphic birch pathogen Taphrina betulina. This information can later be used in plant production with other plants and their yeasts as well.
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(2020)Beer is the world´s most popular alcohol drink, with a long history. It is commonly made with baker’s yeast Saccharomyces cerevisiae. Yeast used for fermentation has an effect on the sensory properties of the product. Saccharomyces boulardii is a probiotic yeast. The objective of the thesis was to explore the use of probiotic yeast S. boulardii in wheat beer and develop it further with the help of sensory evaluations. S. boulardii is used to prevent travelers' diarrhea and as a help to many intestinal disorders. The use of probiotic yeast could make a new interesting product with functional properties. In the experimental part of the study beer was produced with two yeasts, S. cerevisieae and S. boulardii. The beer was matured in two different temperatures (20°C and 37°C). The four samples were compared to commercial reference product. One sample (C37) did not meet quality standards so it was left from sensory analysis. The sensory analysis was performed with 9 trained panelists by using generic descriptive analysis. Concentration for alcohol was measured with High Performance Liquid Chromatography (HPLC). Also pH value and the colour shade was measured. The samples were plated on agar plates to calculate colonies forming units (CFU) to test the yeast surviving in 1, 2 and 3 months time. Eight attributes were rated using visual analogue scales, anchored verbally at the end points. Sensory evaluation showed that the beer differed statistically significantly from each other in three attributes: appearance of foam, the mouthfeel of carbon dioxide and stale, musty smell. B37 was categorized in the same group in Tukey’s test with commercial product although the alc. vol.% was lower. The samples that were fermented in the room temperature had a musty smell and they had darker colour. pH values had only minor differences and the sourness did not have statistically significant differences in sensory analysis. Yeasty taste did not vary significantly between samples. B37 maintained colonies forming units better in the first month and B20 in the second and third measurement. B37 was the most similar to the commercial product, so further studies are encouraged to assess right fermentation conditions. The survival of the probiotic yeast was good, so the product has potential to the beer market.
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