Browsing by Subject "yeast"

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.

Whole grain oats have a high nutritional value and a favorable taste, making oats a valid option to enhance the nutritional properties of food products. Due to the absence of gluten, baking with oat flour can be challenging but lactic acid bacteria fermentation can provide the needed functional activities and modify the sensory properties. The aim of this study was to find lactic acid bacteria (LAB) and yeast starter cultures suitable for oat flour fermentation and define their main metabolic profile. The sourdoughs were prepared with whole grain oat flour and water. Some of these also contained sprouted grain oat and fructose or sucrose to facilitate fermentation. Strains from species Lactiplantibacillus plantarum, Levilactobacillus brevis, and Saccharomyces cerevisiae were used as starters. Microbial enumeration was carried out and acidification was studied by measuring pH and total titratable acidity; organic acid content was analysed with high performance liquid chromatography. The results indicate that there are benefits to using LAB and yeast in consortium to produce oat sourdough, in that they acidify the dough and produce organic acids. The addition of sprouted oat was beneficial, allowing higher acidification and higher organic acid production. Fructose successfully allowed to increase the content of acetic acid. Sensory and baking tests are needed to draw final conclusions on the flavor of the bread.

Sorghum (Sorghum bicolor) and amaranth (Amaranthus spp.) are gluten-free cultivated crops native to Africa, and thanks to their drought-tolerating capabilities and adaptive nature, they provide an energy source in areas where cultivation of wheat is not possible. There is a need to exploit these indigenous crops and develop food products with better technological and nutritional properties to battle malnutrition on the continent. Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) have shown promising properties in improving the technological quality of gluten-free foods. The aim of this project was to identify naturally occurring LAB and yeasts from red sorghum grain, amaranth grain and amaranth leaf flour and from spontaneously fermented sourdoughs made from these flours. Possible EPS-producing LAB were of special interest. The sourdough pH and total titratable acidity were recorded daily, and microbial densities were calculated on selective media. Isolates were selected for sequencing based on morphology and biochemical properties. Twenty seven bacterial isolates, twelve of which produced EPS, were identified by partial 16S rRNA and pheS gene sequencing, and seven yeast isolates were identified by sequencing the variable D1/D2 region of the large subunit rDNA. The identified LAB belonged to five genera: Enterococcus, Lactiplantibacillus, Leuconostoc, Pediococcus and Weissella. The yeasts belonged to the genera Meyerozyma, Pichia and Rhodotorula. In general, many studies focusing on sourdough microbial communities usually concentrate on LAB, with little information on the yeasts currently being available. To the best of the author’s knowledge, this is the first report on sourdough made from amaranth leaves and its native microbial content, with 9/12 isolates producing EPS. This provides an excellent starting point for further study of the technological side of creating a gluten-free baked product using starter bacteria native to the flour itself.

Brewer's yeasts metabolize sugars and produce ethanol and CO2. This study aimed to investigate the relation between the assimilation of sugars in all-malt wort and isotopic signature of carbon and oxygen in the evolved CO2 from brewery fermentations. The isotopic composition of CO2 was measured by a tunable diode laser absorption spectrometer. The isotopic data obtained with automatic sampling, on-line, and in real-time. Wort samples were collected with 3 h intervals to quantify the residual sugars by high-performance liquid chromatography. Patterns of changes in δ13C and δ18O values were unique to experiments with each yeast type. The common overall ascending trend in δ13C and δ18O values in all experiments can be described by kinetic fractionation of isotopes, which explains that in a bioreaction the lighter isotopes participate in the reaction more readily than the heavier ones. Therefore, the early emergence of light isotopologues of CO2 may be a consequence of the fermentation of light isotopologues of sugars. A sudden decrease and then increase in delta values were observed in all experiments before the residual concentrations of glucose and fructose reach their lowest levels. This can be an indicator of the complete assimilation of monosaccharides by yeast. In the fermentations that yeast was able to consume maltose, δ13C and δ18O values raised considerably in a short period. Concurrently, maltose approached its eventual residual concentrations indicating an endpoint for its utilization by yeast. Our results confirm the hypothesis of a connection between the assimilation of sugars and the isotopic signature of evolved CO2 during brewery fermentations. The findings support the potential of off-gas isotopic analysis to monitor sugar assimilation in brewery fermentations.

Selvitin työssäni yhdeksän vapaankaupan pötsilääkkeen sisällön koostumusta ja vaikutusta pötsissä. Vertasin valmisteiden ohjeannosten mukaisia määriä ainesosakohtaisesti niiden tarpeellisuuteen, kirjallisuudessa suositeltuihin hoitoannoksiin ja lehmän päivittäiseen saantiin rehuista. Ruokintatasoksi valittiin yksinkertaisuuden vuoksi 20 kg KA/pv, joista 12 kg KA tulee säilörehusta, 8 kg KA ohrasta ja 2 kg KA rypsirouheesta. Voimakas väkirehuruokinta tai väkirehun osuuden liian nopea lisääminen ruokinnassa altistavat pötsin happamoitumiselle. Luontaisen puskuroinnin ollessa riittämätöntä, pötsin happamuutta voidaan alentaa puskuroivilla tai alkaloivilla aineilla. Yhdeksästä valmisteesta viisi sisältää happamuutta alentavia aineita. Näistä viidestä valmisteesta kahdessa (ReCovin pötsin pH pasta, Correct pH Kombi) aineiden määrä on riittävä. Natriumbikarbonaatti (ruokasooda) on puskuri ja magnesiumoksidi on alkaloiva aine, molemmat ovat tehokkaita happamuuden alentajia. Myös hiivat alentavat pötsinesteen happamuutta. Ne vähentävät maitohapon kertymistä ja lisäävät kuitua sulattavien bakteerien määrää pötsissä. Kolmessa valmisteessa yhdeksästä on riittävästi hiivaa (Super Vetrumin -jauhe, Rumelan, Correct Pötsi-Potku). Propyleeniglykoli on tehokkaana märehtijän verensokeria kohottavana aineena tarpeellinen herumiskaudella olevan syömättömän lehmän tukihoidossa. Propyleeniglykolia on kahdessa valmisteessa (Rumex pH-pasta, Correct Pötsi-Potku). Hivenaineista seleenin on todettu lisäävän alkueläinten määrää pötsissä, ja mikrobit käyttävät sitä proteiinisynteesissään. Kobolttia tarvitaan B12-vitamiinin synteesiin. B12-vitamiinia tarvitaan märehtijän energia-aineenvaihdunnalle välttämättömässä glukoneogeneesissä. Kobolttia on riittävästi viidessä valmisteessa (ReCovin Pötsin pH-pasta, Super Vetrumin -jauhe, Rumelan, Rumex, Rumevit), seleeniä vain yhdessä (ReCovin Pötsin pH-pasta). Mikrobitoiminnan häiriössä B-vitamiinien synteesi pötsissä saattaa vähentyä. Varsinkin B1-vitamiinin eli tiamiinin puute tiaminaasin tuotannon takia happamassa pötsissä ja sen yhteys kerebrokortikaali nekroosiin on hyvin tunnettu. B3-vitamiinin eli niasiinin on todettu tehostavan pötsimikrobien proteiinisynteesiä. B-vitamiineja on lisätty riittävästi neljään valmisteeseen (Biorumin, Super Vetrumin -jauhe, Rumex, Rumevit). Tutkielmassani pohdin myös millainen olisi hyvä pötsilääke. Pötsilääkkeisiin valitsin 8 edellä mainittua hyödyllistä ainetta. Happaman pötsin hoitoon suosittelen natriumbikarbonaattia ja/tai magnesiumoksidia ja hiivaa, niiden happamuutta alentavan vaikutuksen takia. Tiamiinia (B1-vitamiini), koska siitä on happamassa pötsissä todennäköisesti puutetta, ja propyleeniglykolia tukihoidoksi energiavajeeseen. Yksinkertaisen pötsihäiriön hoitoon suosittelen hiivaa, kobolttia, seleenimetioniinia (orgaaninen seleeni) ja niasiinia (B3-vitamiini) niiden pötsimikrobistoa elvyttävän vaikutuksen takia, ja propyleeniglykolia energiavajeeseen. Yhtä tärkeänä, kuin pötsihäiriöiden lääkitsemistä, pidän niiden ennaltaehkäisyä, jossa tärkeimpiä asioita ovat nopeiden ruokinnanmuutosten välttäminen ja rehujen hyvä laatu. Lisäksi on hyvä muistaa kuivan heinän edulliset vaikutukset märehtimistä, syljen erittymistä ja pötsin liikkeitä ylläpitävänä rehuna. Riittävä syljen erittyminen on tärkeä pötsinesteen happamuutta alentava tekijä.

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.

The use of sourdough has numerous benefits, including improvement of the sensory attributes of baked bread in terms of flavour, texture, volume, enhanced nutritional value and extended shelf life of bread. To achieve the desired sourdough performance and bread with optimal quality and improved flavour, it is essential to understand how the starter cultures behave in specific conditions. In a previous part of this research, the metabolic traits of lactic acid bacteria starters and yeast from the food company S.P.C. (S. Korea) were studied. This thesis aimed to explore the pro-technological properties of the selected starter associations of bacteria/yeast, i.e., Lactiplantibacillus plantarum + Fructilactibacillus sanfransciscensis + Saccharomyces cerevisiae (PSY) and Latilactobacillus curvatus + Levilactobacillus brevis + Saccharomyces cerevisiae (CBY). Consequently, analysis of acidification, proteolysis analysis (including free amino acids), and volatile compound profile were done. PSY and CBY grew at the expected cell density. pH of the sourdoughs fermented by PSY decreased along the same line and slower than that of CBY over the course of 24 h. PSY sourdough had the highest TTA value (11.12 ± 0.03 ml) and organic acid production (148.6 ± 2.4 mmol/kg and 25.1 ± 1.5 mmol/kg) than CBY sourdough TTA value (9.01 ± 0.11 ml) and organic acid production (110.6 ± 1.6 mmol/kg and 20.2 ± 0.9 mmol/kg). This shows PSY as having a relatively high capacity for producing acids during sourdough fermentation among the two associations. After assessing their proteolysis capabilities, PSY sourdough had a presumptively higher peptide content while CBY produced the highest free amino acid content (i.e., Orn having a potential repercussion on bread flavour). Several volatile compounds belonging to different chemical classes, such as acids, aldehydes, ketones, alcohols, esters, and other compounds, were produced by PSY and CBY. In PCA, the control sourdough had a distinctive volatile profile from PSY and CBY. Both PSY and CBY show much correlation with about 4% variation. Ethanol, acetic acid, benzene ethanol, 2(3H)-furanone, dihydro-5-pentyl showed their strongest influence on both sourdoughs as they are found in high amount. Finally, during sourdough fermentation, the associations performed in a desired way, and they showed differences in acidity and content of free amino acids that might have a strong influence on bread flavour. Less differences were observed in the volatile profile compounds of the two associations. Proper sensory analysis and consumer test (by the company) will be the most revealing of the differences observed in this experimental study.