Browsing by master's degree program "Magisterprogrammet i mikrobiologi och mikrobiell bioteknik"

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.

Catcher-protein and Tag-peptide originate from split CnaB domains of Gram-positive bacteria surface proteins, which are stabilized by spontaneous intramolecular isopeptide bonds formed between lysine and asparagine residues. However, there is a limited number of non-cross-reacting Catcher and Tag pairs available where the reaction occurs close to the diffusion limit, and which can be used in multiple fragment ligation to construct recombinant fusion proteins. Therefore, a new Catcher/Tag system – LplCatcher/LplTag – was developed in our group from CnaB domain of Lactobacillus plantarum. However, the ligation efficiency of this pair needs to be improved to expand the application possibilities. Therefore, there is a need for efficient library screening method, which allows to detect improved protein-peptide pairs where the covalent interaction takes place rapidly. In this study a new high-throughput in-vivo screening system was developed for visualizing the ligation of Catcher/Tag fusion proteins using splitFAST fluorogenic reporter system for detecting the phenotype, and Fluorescence-activated cell sorting (FACS) for separating the variants at single cell level based on fluorescence intensity. splitFAST is a system engineered by splitting a fluorescent protein named Fluorescence-Activating and absorption-Shifting Tag (FAST) into CFAST and NFAST. The system can be utilized in visualizing the protein interactions because once NFAST and CFAST associate, in the presence of a fluorogen, they form the active and highly fluorescent FAST protein. Herein, Catcher-protein was fused with CFAST and Tag-peptide with NFAST, which allowed detecting the Catcher-Tag ligation ratio based on fluorescence with splitFAST system. Next, a screening system was developed for detecting Catcher variants with improved ligation efficiency. The developed high-throughput screening system showed high potential since visualizing the protein ligation was possible, and hence the system could help in expanding the Catcher/Tag toolbox by allowing large mutant library analyzes.

The porcine gut microbiome is a complex mixture of diverse microbes. During some diseases, the microbial balance of the gut can be disturbed, and harmful bacteria might multiply to concentrations that are harmful to health. To restore balance, the increase of beneficial bacteria that have probiotic potential plays a big role in avoiding the use of antibiotics. In order to develop a probiotic product containing these beneficial bacteria, it is necessary to concretely isolate them from a fecal sample. In this pilot study, the aim was to find an optimal selective growth medium that would allow to grow bacterial species with probiotic properties serving as a potential product in pigs and which would reduce the growth of redundant bacteria. A total of 12 different media were tested with four different sole carbon sources in M9 minimal salts, of which four being supplemented with volatile fatty acids were further tested. The results suggest that cellobiose or xylose could be the best alternatives of the investigated carbon sources for the species of interest. In addition, it was found that certain volatile fatty acids can inhibit the growth of Escherichia coli and several species of the genus Bacteroides.

Dietamoeba fragilis is a very common human intestinal parasite in the world and also in Finland. D. fragilis causes dientamoebiasis in humans, and symptoms of infection may vary from mild bowel symptoms to prolonged diarrhoea and weight loss. Some of the infected patients are completely asymptomatic. Despite its prevalence, insufficient data exist on the life cycle and host organisms of D. fragilis. Interestingly, D. fragilis has been found in dogs in a few studies. Thus, more research is needed on the role of household pets as part of the parasite’s life cycle and choice of host organism. Comprehensive study on the role of dogs in the life cycle of D. fragilis is warranted. The purpose of this thesis was to design a study on the role of dogs in the D. fragilis parasite life cycle “The presence of Dientamoeba fragilis parasites and multiresistant ESBL-bacteria in dog owners and their pets”, to present research methods and to apply research permit from the ethics committee. The aim of the study is to analyze the prevalence of D. fragilis in dogs in Finland and the role of the dog in the spread of D. fragilis parasite within humans. In addition to D. fragilis, the study combines collection of samples for ESBL bacteria analyses, but this thesis focuses only on the part of study D. fragilis. Two different real-time PCR methods are tested in this project: an in-house method and an Amplidiag® Stool Parasites method, out of which Amplidiag® Stool Parasites method is selected to be used in the study. In the thesis documents for the ethics committee's referral are written and ethical permit to conduct the study from the HUS ethics committee is applied. A favourable opinion will allow a comprehensive study of dogs and humans to be carried out. Ethics permit is need to recruit participants for research on the basis of a laboratory-confirmed D. fragilis -discovery. The committee confirms that the study is ethically acceptable. During the thesis project, the committee gave the study a conditional favourable decision. The implementation of the study will start in the spring of 2023, when the final assent is received.

Antimicrobial resistance (AMR) is a crucial problem in the clinical field worldwide. The need for novel antimicrobials to tackle AMR is imminent. Image-derived data can be used to understand the mechanism of action (MoA) of newly discovered compounds and classify them within known classes of antibiotics. This study aimed to develop a simple image-based measurement method for screening new antimicrobials and simultaneously defining the MoA. In the process, Escherichia coli ATCC 25292 was selected as a model strain from the Gram-negative bacterial group. This strain was treated with antibiotics belonging to different classes. Treated bacteria were imaged with Cytation 5 Cell Imaging Reader and analyzed using ImageJ2 software. The developed workflow was validated by testing the pipeline to be applied for some ESKAPE strains categorized as antibiotic-resistant bacteria. The results revealed that this pipeline enables us to observe the bacterial single-cell phenotypic changes in response to antimicrobials, such as the elongation caused by ampicillin and ciprofloxacin treatments, which are cell wall synthesis and DNA replication inhibitors, respectively. The treated bacterial cells were significantly longer than untreated cells from the sample without antibiotic. Thus, the image-based-high throughput assay can support the drug discovery by identifying the preliminary MoA of new antimicrobials against AMR bacteria. Promising data obtained on E. coli and some pathogenic bacteria allow for pursuing similar approach with other AMR Gram-negative bacterial species.

One of the greatest challenges of our time is securing the global protein supply for the growing population in a sustainable manner. Fermentation with lactic acid bacteria has a long history of successful employment for the production of fermented foods and beverages. During this study, the ability of diverse lactic acid bacteria for fermentation and sensory improvement of leguminous and cereal protein concentrates was investigated.The main aim of this study was to overcome the sensory limitations of these plant protein ingredients by finding suitable candidates for the design of new starter cultures for their fermentation. A collection of 82 lactic acid bacteria was screened for fermentation of leguminous and cereal protein concentrates with different nutrient supplementations. Most strains required additional nutrients to adequately acidify the leguminous protein concentrate during a 24-h fermentation, while the cereal-based substrate appeared to be a more complete growth substrate. Descriptive sensory analysis also revealed differences in the aroma perceived by a panel depending on the matrix, supplementation and fermenting strain employed. Three of the strains that produced the most desirable aromas and acidified sufficiently the test matrixes were further studied. All three strains preferentially fermented glucose to lactic acid rather than any other sugar. The concentration of hexanal, one of the volatile compounds involved in grassy and beany off-flavor formation, reduced during fermentations in favor of 1-hexanol, a compound with a significantly higher odor threshold. However, only two of the cultures were able to prevent the growth of contaminating bacteria during fermentation. The results of this study can provide guidance for selecting potential starter cultures and fermentation substrate composition to improve the aroma of plant protein ingredients. Two of the selected strains especially have shown potential to be used as starter cultures for the leguminous protein concentrate. Further studies are required to optimize the performance of the selected strains in the test matrixes and to quantitatively characterize their effect on the substrates’ volatile profile, taste and antinutritional factor content

The popularity of fermented beverages is on the rise due to signature flavours, associated health and nutritional benefits and a 100% natural label. Research in this sector is currently focused on industrial-scale production of traditional homemade fermented beverages such as Kombucha, Kefir and Kvass. To expand consumer choice beyond these traditional beverages and to provide more nutritional and flavor diversity, it is essential to develop novel products by using new microbial communities and new substrates. The industrial scale-up of fermented beverages produced using microbial communities is challenging as the flavour complexity and functionality of the beverage depends on the complex fermentation processes and interactions between the microbiota species. Fermentation systems that can separate the metabolic stages into separate fermentation steps would be needed to simplify and make the complex fermentation more efficient, scalable, and reliable. The aim of the thesis was to develop and compare different fermentation strategies to control the complex fermentation of previously isolated microbes to produce a bio-flavoured, low-alcohol, malt beverage with a signature fruity flavour and aroma. During the study, green-malt microbial species: Lactiplantibacillus plantarum, Saccharomyces cerevisiae and Saprochaete suaveolens were identified as significant contributors to the development of aroma and flavour compounds in the malt fermentates. Using the optimal cell concentration of the selected species, three different fermentation strategies: simultaneous inoculation, sequential inoculation and sequential fermentation were adopted to design five different fermentation systems. Cocktail blends of individual fermentates were also created and tested for flavour and aroma. All potential production methods were compared in contrast for parameters such as ease of operation, time-efficiency, flavour and aroma, and future scalability. The results showed that complex fermentation of the novel and low alcohol malt beverage could be controlled by selecting organoleptically significant microorganisms from the complex community, controlling the time and order of inoculation and using a stagewise or modular fermentation system. Sequential fermentation produced the desired low alcohol level and flavorful, fruity malt beverages. However, this system required centrifugation at each step and thus resulted in limited ease of operation. Sequential inoculation was an optimal and efficient method of controlling the fermentation since it required a single vessel, and the metabolic stages were separated by inoculating microorganisms sequentially with a 24 h time interval between each inoculation. Creating cocktail blends from individual fermentates also produced bioflavoured, fruity, aromatic, low alcohol malt beverages. This method was time-efficient with maximum ease of operation. The resulting beverages from these different fermentation systems were novel and had fruity flavours and aroma from the metabolites synthesized by organisms S. suaveolens, L. plantarum and S. cerevisiae. Thus, bio-flavoured, low-alcohol, malt beverages with signature fruity flavour and aroma were created at VTT. For the first time S. suaveolens was used in combination with L. plantarum and S. cerevisiae for beverage production using novel three-microbe fermentation systems to control complex fermentation.

Continuous exposure to ultraviolet radiation can cause detrimental health effects in humans, including erythema, hyperpigmentation, photo-aging, and skin cancer. Sunscreens provide important protection of skin against the harmful effects of ultraviolet radiation. However, chemical and physical sunblock filters can be harmful for both human beings and the environment. There is a need for natural alternatives to commercial filters currently used in sunscreens. Mycosporine-like amino acids (MAAs) are natural microbial sunscreens produced mainly by marine organisms to protect themselves from ultraviolet radiation. There has been over three decades of interest in these natural compounds for applications in the cosmetic industry. However, low production levels in nature have hindered large-scale processes for industrial applications. Here, we demonstrates the successful expression of a MAA biosynthetic pathway from cyanobacteria in a metabolically engineered strain of Escherichia coli. Heterologous expression of the artificial codon-optimized MAA biosynthetic pathway resulted in high-level production of porphyra-334 and shinorine. These two MAAs have exceptional high molar extinction coefficients and are found in a number of commercial sunscreen formulations that rely on algal extracts. The expression of a gene encoding an ABC-transporter, which is associated with cryptic MAA biosynthetic gene clusters in cyanobacteria, in Escherichia coli resulted in the effective transport of both porphyra-334 and shinorine outside the cell. Together, these two advances improve the possibility of biotechnological production of these microbial sunscreens in industrial microbial hosts.

Enterovirukset ovat tärkeitä patogeenejä, jotka kuuluvat Picornaviridae-heimoon. Enterovirus-sukuun kuuluu lukuisia tärkeitä ihmisen patogeenejä, kuten poliovirus, enterovirus 71 ja coxsackievirukset. Yhdisteet, jotka sitoutuvat viruksen rakenteeseen ja estävät viruspartikkelin laajenemisen ovat tutkituimpia enterovirusinfektiota vastaan tarkoitettuja antiviraaleja Testasin kahden yhdisteen, CL213:n ja epigallokatekiinigallaatin (EGCG) inhibitorista aktiivisuutta coxsackievirus A9 (CVA9) -infektiota vastaan. Solukulttuureihin perustuvilla menetelmillä määritettiin molempien yhdisteiden inhibitorisen aktiivisuus CVA9-infektiota vastaan. Lisäksi aktiivisemman CL213:n vaikutus CVA9:n lämpöstabiilisuuteen määritettiin. Jotta varmistettaisiin, sitoutuuko yhdiste kapsidiin, viruksen rakenne kompleksissa yhdisteen CL213 kanssa selvitettiin cryo-elektronimikroskopian avulla. Tulokset osoittivat, että CL213 on voimakas CVA9-infektioinhibiittori in vitro ja se lisää viruksen lämpöstabiilisuutta. CVA9:n rakenne kompleksissa CL213:n kanssa selvitettiin 2.5 Ångströmin tarkkuuteen kryoelektronimikroskopialla. Vaikka lämpöstabiilisuustestauksen tulokset viittaavatkin siihen, että yhdiste sitoutuu viruksen rakenteeseen ja viruksen rakenteen ikosaedrisellä rekonstruktiolla (icosahedral reconstruction) oli korkea resoluutio, yhdisteeseen sopivaa tiheyttä (engl. density) ei löydetty viruksen rekonstruktiosta. Tämän vuoksi yhdisteen tarkka toimintamekanismi jää epäselväksi. Yllättävä tulos tutkimuksissa oli se, että EGCG, jonka on aiemmin osoitettu inhiboivan CVA9-infektiota, ei ollut lainkaan aktiivinen näissä antiviraalisen aktiivisuuden määrityksissä. Todennäköisesti ilmiö johtuu siitä, että EGCG:n toiminta perustuu puhdistetun viruksen saostamiseen ja aggregointiin liuoksesta, mutta yhdisteen aktiivisuus katoaa, kun virus on solujen kasvatusliuoksessa. Koska kumpikaan yhdiste ei tulosten perusteella sitoutunut viruksen rakenteeseen, CVA9:n ja enterovirus 71:n VP1 proteiinin hydrofobisten taskujen (engl. VP1-hydrophobic pocket) rakenteita verrattiin, jotta voitaisiin selvittää ovatko yhdisteet, joilla on nanomolaariset IC50 arvot, hyviä lähtökohtia yhdisteiden jatkokehitykselle. Vaikka molempien virusten rakenteista löytyy kyseinen hydrofobinen tasku, suurin osa yhdisteiden sitoutumiseen vaadittavista sivuketjuista eivät ole konservoituneita. Tehokkaiden antiviraalisten yhdisteiden kehitys enterovirusinfektioita vastaan on siis edelleen merkittävä haaste.

Semliki Forest virus (SFV) is a well-studied model virus of medically important mosquito-borne alphaviruses, like chikungunya virus and Sindbis virus. SFV replicates within membrane invaginations called spherules at the host plasma membrane, facilitated by the virus-encoded nsP1 protein. The objectives of this MSc thesis were to identify candidate host proteins interacting with nsP1 based on previous proteomics work and develop a screening workflow to identify stable nsP1 interactors. The overarching aim is to improve our understanding of the roles of host proteins in spherule formation and replication complex functions. The screening workflow involved knockdown assays to assess the antiviral and proviral effects of potential host interactors, followed by cell viability assays for toxicity assessment. Selected promising hits were further investigated for protein interaction with nsP1, which required cloning for mammalian expression and tagging with Myc epitope. Knockdowns resulted in several statistically significantly proviral and antiviral host factors, and all the knockdowns were non-toxic to the cells. Among the significant proviral hits, four promising candidate host proteins were cloned and expressed. Pull-down assays did not reveal stable interactions, suggesting transient or indirect interactions between these host proteins and nsP1. The lack of co-localisation with SFV replication complex supported this finding. This work sheds light on possible transient or indirect interactors of nsP1. The screening workflow effectively identified protein-protein interactions and can be applied to screen additional proteins. Future studies should employ methods suitable for studying transient interactors to gain further insights. This would enhance our understanding of key host proteins in SFV spherule formation and replication, potentially leading to novel antiviral therapies targeting alphavirus replication.