Faculty of Agriculture and Forestry
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(Helsingin yliopisto, 2021)Cyanobacteria produce a variety of toxins and a diversity of other specialized metabolites. Specialized metabolites are compounds produced by an organism to interact with the environment and provide protection against competitors, predators, or abiotic factors. The biosynthetic pathways for generating specialized metabolites are typically encoded in compact gene clusters that encode multiple biosynthetic enzymes. The structure and biosynthesis of all major cyanobacterial toxins have been resolved, but new compounds with variable functions are continuously discovered and their biosynthetic origins elucidated. Cyanobacterial specialized metabolites are widely held to have great potential in the pharmaceutical industry given the increasing need for new drugs that target infectious disease and cancer. A better understanding of the chemical structure of the compounds facilitates discovery of their biological targets and their ecological role. The aim of this study was to discover new potential drug leads from cyanobacteria, focusing on antifungal compounds, and describe their structure, activity, and biosynthetic origins. Laxaphycins are unusual specialized metabolites that consist of two distinct macrocyclic lipopeptides with either 11 or 12 amino acids. They are known to have synergistic antiproliferative and antifungal activities but unknown biosynthetic origins. Here, new chemical variants of laxaphycin family specialized metabolites were discovered from Nostoc sp. UHCC 0702 and Scytonema hofmannii PCC 7110. The laxaphycin biosynthetic gene cluster was discovered, organized as a branching pathway, with initiating enzymes participating in the biosynthesis of both different lipopeptide groups. The biosynthetic gene cluster was described in both Scytonema hofmannii PCC 7110 and Nostoc sp. UHCC 0702. New laxaphycin variants heinamides were discovered with unforeseen structural moieties and I present predictions for their origins. I confirmed that heinamides also displayed synergistic antifungal activity. The connection of the 11- and 12- amino acid residue compounds is also evident at the genetic level with the common biosynthetic enzymes of the synergistic compounds. Aeruginosins are common cyanobacterial tetrapeptides with inhibitory activity against serine proteases. Trypsin isoforms have recently been studied as a target in cancer treatment. Here pseudospumigins, new aeruginosin variants, were discovered from Nostoc sp. CENA 543. Pseudospumigins are produced through a PKS/NRPS pathway similar to known aeruginosin biosynthetic gene clusters. Pseudospumigin A acts as weak trypsin inhibitor, with time dependent IC50 value of 4.5 ȝM. Nodularin-R, a cyanotoxin, was also found from the same strain. The nodularin concentration was much higher than what has been seen in a Nostoc strain before, comparable to nodularin concentrations in the most common nodularin producers Nodularia spumigena. This study describes new cyanobacterial specialized metabolites and biosynthetic enzymes for their biosynthesis, broadening the knowledge in areas of novel structural elements, biosynthetic pathways and biological activity. The structural and activity information can help in function prediction and rational design of drug candidates or guide the screening for specific targets. The genetic information can be used in mining genomes for discovering new compounds and predicting products for cryptic biosynthetic gene clusters. Probable applications lie also in the emerging fields of combinatory biosynthesis and synthetic biology to produce engineered compounds in biological systems.
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(Helsingin yliopisto, 2021)Cyanobacteria are one of the largest and most diverse groups of prokaryotes, and the only prokaryotes capable of oxygenic photosynthesis. They inhabit a wide variety of aquatic and terrestrial light-exposed environments, where they are key players in carbon and nitrogen cycles. These organisms show vast morphological and physiological diversity, and can achieve high densities under optimal conditions due to several adaptive strategies. Such mass-accumulation events are also known as cyanobacterial blooms. One of the major concerns involving cyanobacterial blooms is the production of potent toxins that can not only negatively affect the growth of phyto/zooplankton, but are also toxic to domestic and wild animals. Aside from toxins, cyanobacteria are known for producing a wide range of natural products, i.e., compounds with unique structure and biological activities, which can have applications in medicine and biotechnology, such as antibiotics, anticancer agents, polymers, and surfactants. The enzymes involved in the biosynthesis of cyanobacterial natural products are typically encoded in contiguous stretches of genomic DNA, known as biosynthetic gene clusters. These natural product biosynthetic gene clusters have been widely explored using genome mining approaches. However, other omics tools can considerably assist the study of biosynthetic pathways and improve predictions of the chemical structures of the compounds they encode. Despite the increasing number of new genomes and described natural products, cyanobacteria remain underexplored relative to other bacteria phyla. Thus, the aim of this thesis is to expand the knowledge on genetic diversity and the natural product biosynthetic potential of cyanobacteria using multi-omic approaches. Specifically, the distribution of natural product biosynthetic gene clusters from 184 publicly available genomes from the entire phylum were first mapped, and environmental adaptations and natural products from two toxic bloom-forming genera were explored in depth. In addition, diverse cyanobacteria from Brazilian biomes were screened for antimicrobial and anticancer activity. Overall, this thesis showed that combined omic approaches are capable of providing critical insights into the genetic and biosynthetic diversity of cyanobacteria. The results advance our understanding of how natural product biosynthetic gene clusters move among cyanobacterial genomes, and how environmental factors are related to toxin production, and also identified cyanobacteria that are the source of novel natural products. This knowledge is important in preventing damage caused by toxic bloom-forming cyanobacteria, and in the discovery of novel natural products with economic interest.
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Cow's milk consumption and processing and the development of type 1 diabetes and asthma in children (Helsingin yliopisto, 2021)Cow’s milk has been associated with the development of both type 1 diabetes and asthma in children, although in different ways. Consumption of cow’s milk has been associated with an increased risk of the development of type 1 diabetes. On the other hand, several cross-sectional studies show that consumption of raw, unprocessed milk is associated with a decreased risk of asthma or atopy when compared to no consumption of raw milk or to consumption of sterilized milk. Little is known about differently processed milk products. The aim of this thesis was to study, in a birth cohort setting, whether the consumption of differently processed milk products is associated with the risk of islet autoimmunity or asthma in children. To study that, a novel processing-based classification of milk products was created in the national food composition database as part of this thesis. This classification was also used to evaluate children’s consumption of differently processed milk products, as such information was lacking. This thesis is based on the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Nutrition Study, comprising children genetically predisposed to type 1 diabetes born in Tampere or Oulu University hospitals from 1996 to 2004 (N=6081). The nutritional information of the participants was collected by food records at the age of 3 and 6 months, and 1, 2, 3, 4, 5, and 6 years. The consumption of milk products was calculated from the food records using the national food composition database. Each milk product item in the national food composition database was classified according to the adopted heat-treatment (low-pasteurized or less heated/high-pasteurized at <100°C/high-pasteurized at 100°C or sterilized) and homogenization (yes/no/fat-free). The classification was done principally based on literature. The study participants were followed for type 1 diabetes-related autoantibodies regularly until the age of 15 years. The primary outcome in the diabetes-related study was islet autoimmunity by the age of 6 years, defined as repeated positivity for islet cell autoantibodies and at least one biochemical antibody tested: insulin autoantibodies, antibodies to the 65 kD isoform of glutamic acid decarboxylase, and the tyrosine phosphatase-related islet antigen-2 molecule. The secondary outcome was islet autoimmunity by the age of 15 years. Of the 6081 children within the DIPP Nutrition Study, 4075 children were still in the DIPP follow-up at the age of 5 years, and of them 3781 participated in the DIPP Allergy and Asthma Study. The information on asthma was collected at the age of 5 years by using an ISAAC-based questionnaire. Outcomes in the asthma study were asthma, atopic asthma, and nonatopic asthma by the age of 5 years. Asthma was defined as doctor-diagnosed asthma plus wheeze or use of asthma medication during the preceding 12 months. Atopic asthma was defined as IgE-positive asthma, and nonatopic asthma as IgE-negative asthma. Joint models for longitudinal and time-to-event data were used as the statistical approach to study the association between consumption of differently processed milk products and islet autoimmunity and asthma. Almost all study participants [e.g., at the age of 3 years, 3401 of 3460 (98.3%)] consumed cow’s milk. During the first year of life, high-pasteurized at 100°C or sterilized milk products were consumed in higher amounts, while at ages 1–6 years low-pasteurized milk products were predominant. Homogenized milk products were consumed in higher amounts than nonhomogenized ones. Consumption of fat-free milk products increased by age. By the age of 6 years, islet autoimmunity developed in 246 (4.4%) of the 5545 children included in the analyses. High consumption of cow’s milk products was associated with an increased risk of islet autoimmunity [energy-adjusted hazard ratio (95% confidence intervals) 1.06 (1.02, 1.11)]. In addition, consumption of nonfermented [1.06 (1.01, 1.10)], fermented [1.35 (1.10, 1.67)], and homogenized [1.07 (1.02, 1.12)] milk products were associated with an increased risk. No processing type stood out as a particular risk factor. Asthma developed in 184 (6.0%) of the 3053 children included in the analyses, by the age of 5 years. Asthma in 101 (54.9%) children was atopic, and in 75 (40.8%) children nonatopic. The asthma type of 8 (4.3%) children could not be categorized. Consumption of high-pasteurized at 100°C or sterilized milk products [adjusted hazard ratio (95% confidence intervals) 1.06 (1.01, 1.10)] including milk-based infant formulas [1.15 (1.07, 1.23)] was associated with an increased risk of asthma. Consumption of cow’s milk products [1.09 (1.03, 1.15)], nonfermented milk products [1.08 (1.02, 1.14)], infant formulas [1.23 (1.13, 1,34)], and high-pasteurized at 100°C or sterilized milk products [1.08 (1.02, 1.15)] were associated with an increased risk of nonatopic asthma. In parallel to several previous studies, the results of this thesis suggest that high consumption of cow’s milk and cow’s milk products is associated with the development of the two immunological diseases in children. Processing of milk did not play a significant role in the association to islet autoimmunity. The consumption of high-pasteurized at 100°C or sterilized milk products was associated with an increased risk of asthma. No such association was seen for low-pasteurized milk products. This implies that processing may change the immunological properties of milk and thus play a role in the development of immunological diseases. As milk and milk products are commonly used in substantial amounts by Finnish children, future studies are needed to confirm the results and to enlighten the mechanisms behind the associations.
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(Helsingin yliopisto, 2021)This dissertation studies the stand-level economics of continuous cover and rotation forestry. The main method of this dissertation is economic-ecological optimization, where statistical-empirical size-structured ecological models are coupled with economic optimization models including fully flexible optimization between continuous cover and rotation forestry. The dissertation consists of a summary section and three original research articles. The first article compares the favourability of continuous cover forestry between pure Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) stands and studies the effects of ecological models on economically optimal solutions. The second article studies the economics of mixed-species stands with up to four tree species. The third article studies the economics of arctic forestry in the homeland region of the Sámi people using a model that simultaneously includes timber production, carbon storage, and negative externalities of forestry on reindeer husbandry. According to the results, the differences in continuous cover forestry favourability between tree species are largely dependent on both species-specific differences in natural regeneration and natural regeneration differences between ecological models. Under realistic economic parameters, our model specification yields the result that continuous cover forestry is optimal for Norway spruce in both single- and mixed-species stands. In contrast, Scots pine favours rotation forestry in both single- and mixed-species stands. Physical overyielding of a species mixture does not reveal the economic preferability of that species mixture. In addition, we demonstrate that economically optimal continuous cover forestry avoids “high grading”, i.e. selective harvesting that leads to a completely different and economically inferior outcome. Including the negative externalities of forestry on reindeer husbandry into the economic model favours continuous cover forestry in arctic Scots pine stands. A carbon price between €14–€20/tCO2 is enough to imply that saving old-growth forests as carbon storages and reindeer pastures becomes optimal.
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(Helsingin yliopisto, 2021)Below-ground carbon (C) allocation studies in boreal forests are scarce and have high levels of uncertainty in ecological and modelling studies. The uncertainty of fine root turnover and the heterogeneity of fine root distribution are the main barriers to quantifying the below-ground C allocation. Unravelling the below-ground C litter inputs of boreal forests, including fine roots and ectomycorrhizal (EcM) mycelia, could provide fundamental information for quantifying biogeochemical cycles. This thesis evaluated the below- and above-ground litter C inputs along a site type gradient of Scots pine (Pinus sylvestris) sites in southern Finland, and a distinct silver birch (Betula pendula) site in northern Finland. Furthermore, the Scots pine pioneer/fibrous root growth phenology was observed and compared with the modelled growth of the above-ground organs (predicted by the dynamic CASSIA model) in southern Finland in 2018, when there was an unusual summer drought. Fine root turnover was observed by minirhizotrons (MR) and the root growth phenology was observed by flat-bed scanners, both of which direct methods are known to provide reliable results in root research. Based on the daily root growth monitoring experiments, we found that the timing of intensive root growth lagged behind the growth of above-ground organs (shoots, secondary xylem, buds, and needles). Interestingly, we found a clear root growth increase while the needle growth decreased, which may have been caused by a shift of non-structural carbohydrates (NSC) from above-ground to below-ground. The low temperature and summer droughts may have constrained the fibrous root growth, but not influenced the pioneer root growth, which indicates that pioneer roots could be more tolerant to severe climate variations. Increasing nutrient availability could clearly increase the above-ground C allocation but not the below-ground allocation. Our study sites CT, VT, MT were named after Cajander’s Finnish site type theory in the order of increasing nutrient availability. Our study found that the nutrient-poor site CT tends to have significantly higher fine root longevity and biomass than the relatively nutrient-rich sites VT and MT. Fine roots could allocate more biomass below the ground and survive longer in nutrient-poor conditions. The distal tips of tree roots reflect the forest foraging ability, as shown by the fact that EcM root tips per basal area and fine root biomass per basal area both increased gradually from nutrient-rich to nutrient-poor sites and from low to high latitudes. Overall, we found that below-ground litter accounts for 21-58% of total litter inputs in boreal forests. This finding indicates that the C allocation pattern could be a specific effect of species and latitudes. The Scots pine in the southern sites allocated up to one third of total litter inputs below the ground but the northern silver birch allocated over half of total litter inputs below the ground. In conclusion, we suggest that the growth phenology and litter inputs of below- and above-ground organs should always be observed and quantified together. Understory species contributed significantly to litter C inputs which should not be neglected in boreal forests. Moreover, future studies should be focused on the shifting of below- and above-ground C allocation response to extreme climate and also on the need to include EcM mycelia and root exudates in the accounting of below-ground litter pools.
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(Helsingin yliopisto, 2021)Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. In this doctoral dissertation, probiotic strains Saccharomyces boulardii CNCM I-745 and Lacticaseibacillus rhamnosus GG (LGG) were developed as next-generation probiotics. The constructed S. boulardii strains, i.e., SAC4 based on the wild-type S. boulardii CNCM I-745 and SAC12 based on the URA3 auxotrophic derivative, secreted bacteriocin leucocin C, which showed inhibitory activity against the foodborne pathogen Listeria monocytogenes. Interestingly, the leucocin C secretion ability of S. boulardii SAC12 was stronger than that of SAC4. S. boulardii SAC4 killed L. monocytogenes effectively when cells of yeast and Listeria were incubated together without selection pressure, demonstrating the potential of cell mediated inhibition instead of using concentrated supernatant. Beer fermented with SAC12 was evaluated to be efficient in Listeria decontamination of chicken breast strips, with the maximum reduction of 2.2 log units from (1.8±0.3) × 10^5 CFU/g. LGG is one of the most studied probiotic strains, and it has been commercially used as a probiotic supplement in dairy products. The challenge of using LGG in dairy products is that it cannot metabolize the lactose and casein of milk, thus causing its poor growth in milk. We aimed to abolish this deficiency of LGG by bacterial conjugation, a non-GMO method. The dairy strain Lactococcus lactis NCDO 712 was used as donor, as it carries the plasmid pLP712 with the gene encoding the protease for casein degradation as well as the gene for lactose catabolism. In this study, a successful conjugation was done between L. lactis NCDO 712 and LGG. The plasmid pLP712 was conjugated into LGG, verified by plasmid-specific PCR and plasmid DNA isolation. The transconjugant L. rhamnosus LAB49 showed a clear ability of lactose utilization on indicator plate, in which lactose was the only carbon source. LAB49 was incubated in MRS, and all tested colonies (n= 80) lost their lactose-fermenting ability after 100 generations. The proteolytic activity of LAB49 was analyzed by SDS-PAGE and it showed that β-casein was fully digested in 4 h by LAB49 and NCDO 712 but not at all by LGG. The growth curve indicated that LAB49 grew well in milk, reaching stationary phase in 11 to 12 h after inoculation. These results collectively suggested that, L. rhamnosus LAB49, an upgraded food-grade and non-GMO derivative of LGG had been generated.
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(Helsingin yliopisto, 2021)Basidiomycota fungi, especially brown rot fungi, cause degradation in the wood of coniferous trees in the Northern hemisphere. The fungal species assembly in deadwood has an impact on the cycling of carbon and other nutrients in forest ecosystems. In this doctoral thesis, I first studied the species-species interactions between a common brown rot fungus of our boreal forests, Fomitopsis pinicola, and five white rot fungi Phlebia radiata, Junghuhnia luteoalba, Trichaptum abietinum, Porodaedalea laricis and Phellinus ferrugineofuscus, all belonging to the fungal phylum Basidiomycota, and able to grow on Norway spruce (Picea abies) deadwood. In mycelial interactions, the fungal species consortium influenced the production of wood-decaying enzyme activities as well as activities of enzymes important for hyphal interactions and re-cycling of cellular components. The effect of the white rot fungi P. radiata and T. abietinum on mycelial growth of F. pinicola and its brown rot decay mechanism was observed in three months on spruce wood shavings. F. pinicola demonstrated aggressive growth and rapid wood decay, which were subjective to suppression by the presence of white rot fungi, especially P. radiata. The wood decay type and efficiency during fungal interactions was observed with detection of wood decay enzyme activities, and iron reduction capacity, which is indicative for brown rot Fenton chemistry. With observations on fungal responses, their decay type and efficiency in wood decomposition, interaction studies allowed analyses of volatile organic compounds (VOCs) from fungal single-species and combination cultures on spruce wood. Among the set of identified VOCs, mainly terpenes and terpenoids released from wood by fungal action, a few signature compounds for brown rot decay and white rot decay type could be emphasized. Late-stage interaction and decay of wood was observed by 1-year long incubation of the brown rot and white rot fungi together on thin spruce wood veneer slices. Detailed analysis of the mycelial interaction zones on the spruce veneer slices showed responses at enzyme production level and at gene expression level analysed by RNA-extraction and real-time qRT-PCR. The brown rot fungus F. pinicola had a positive effect on the expression of transcripts of specific oxidoreductase-encoding genes (mnp2, lip2, lpmo1) of P. radiata. Similar responses were not observable with the hydrolytic enzyme-encoding genes (eg1, xyn1, bgl1). The white rot fungi P. radiata and T. abietinum both negatively influenced the expression of the ferric reductase encoding gene (fr1) of F. pinicola. These results, together with specific responses on enzyme activities and iron reduction ability, and the amounts of dissolved metabolites highlight that the white rot fungi start to dominate and convert the decay type from brown rot towards white rot in wood.
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(Helsingin yliopisto, 2021)The topic of the dissertation is the life of women in a rural agricultural Northern Tanzanian context. Tanzania is a rapidly growing country in East Africa and the majority of the population lives in rural areas and relies on agriculture. Despite growth in the economy and agricultural production the rural population struggles with poverty. This implies that a better understanding of the life of rural women who do most of the farm work in Tanzania is significant since an improvement in their lives has an impact on the general improvement in poverty reduction in the studied context. The purpose of this grounded theory study was to examine the lives of Northern Tanzanian rural women, and to generate a substantive theory which explains their main concerns in their lives and how they resolve them. The study involving rural women farmers and men participants (the husbands and rural actors) (N= 62, 9) in Tanzania in the spring, 02/2011 and 04/2016, was carried out through workshops, open discussions, interviews, letters, and observations. The collected data was analysed, using classic grounded theory methodology. The main concern of the rural women when living within external expectations was self-justifying related to hope of self-dignity. As a result, an emergence of a substantive theory of self-justifying shows a basic social psychological strategy experienced by rural women through sinking, awakening, toughening, and conceding. Sinking links to questions regarding the meaning of oneself. Due to this, hopelessness, caused by realising that one’s existence is only for the benefit of others, poses emptiness. During sinking, believing in oneself, a resource for continuation towards self-justification, is reduced by leaving a sense of insecurity. Additionally, the pursuit of self-dignity has lost its meaning. Awakening of hope of continuing shifts emptiness to uncertainty. Awakening is manifested by uncertainty of how to continue. During awakening, believing in one’s own efforts, a resource for continuation towards self-justification, is diminished until willingness to overcome conditions brings meaning to the future. Additionally, hope of self-dignity begins to emerge as progress advances. Toughening relates to the willingness to overcome conditions, believing in one’s own efforts and oneself, taking responsibility, internal controlling, and isolating oneself. Accordingly, the sense of being an independent responsibility bearer is created. As the progress advances a state of being pure enables believing in oneself, a source of safety, and mental freedom when living within expectations, whilst the hope of self-dignity is intensified. In conceding, there is no need to justify oneself. Diminished need to justify oneself can be caused by prolonged despair, fear, or other factors which diminish believing in oneself and the continuation towards the fulfilment of needs. By maintaining limits, generated from the expectations of how to treat others, self-justification is achieved. Self-justifying within limits, by prioritising the needs of others, is related to the level of safety. As the optimal outcome is reached, eagerness to take on more responsibilities intensifies, whilst unused physical and mental resources bring about a feeling of frustration. This is due to a limited access to self-development through material and immaterial sources because of currently living within expectations related to participation rights. The substantive theory of the coping strategy of self-justifying gives a view of how the women in this study, who live in the rural area of Northern Tanzania, resolve the main concern in their lives. Through knowledge gained, improvement of the state of an individual whilst living within expectations can be reconsidered. Keywords: Self-justifying, expectations, limits, prioritising the needs of others, grounded theory, qualitative data
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(Helsingin yliopisto, 2021)Antibiotics were once miracle cures, but because of the spread of antibiotic resistance in bacteria, their effectiveness is threatened. Although antibiotics have only been produced industrially for 70 years, antibiotic-resistant bacteria are a threat to human health. The effects of antibiotic use pass on over generations, and resistance kills an estimated 214,000 infants a year. Antibiotic-resistant bacteria have also become widespread in the environment. In my dissertation, I used a microbial ecology perspective to study how the antibiotic resistance crisis manifests itself in humans (with a focus on mothers and infants) and in the environment. My main lines of research focused on selection pressures that shape bacterial communities and the effects of the spread of resistant bacteria. I studied the amounts of antibiotic resistance genes in different environments, utilizing methods based mainly on metagenomics. Mothers pass on antibiotic-resistant bacteria to their children. However, in my study, the resistance load of infants’ intestines was most affected by infant formula use. Infants who received formula had a significantly higher proportion of bacteria carrying resistance genes than exclusively breastfed infants. Surprisingly, formula use increased the intestinal resistance load more than the antibiotic regimens given to babies, which could not be shown to have an effect in my dissertation. Antibiotic selection pressure did not explain the number of resistance genes in the environmental samples I studied either. The results suggested that fecal contamination is almost always behind the resistance load observed in the environment. It was therefore interesting that the treated wastewater discharged from European wastewater treatment plants into the environment corresponded to the types of resistance of bacterial strains isolated from infected patients. The result suggests that inadequate wastewater treatment is part of the resistance problem in Europe as well, and not just in developing countries, and potentially increases the spread of antibiotic-resistant bacteria to humans. My work shows that the most effective ways to reduce resistance may not be intuitive. Bacterial spread may play a larger role than previously thought. Efficient waste treatment and exclusive breastfeeding may reduce the number of resistant bacteria in society, the environment, and young children more effectively than reducing the use of antibiotics.
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(Helsingin yliopisto, 2021)Inflorescences are flower bearing structures that display remarkable diversity in plants. Their architecture, referring to the number and arrangement of flowers, is considered as a key attribute to the reproductive success of plants. Asteraceae is one of the largest plant families, and the evolutionary success of this family has been largely attributed to their showy inflorescence structure, the flower head (or capitulum). A flower head combines up to a thousand individual florets and numerous leaf-like bracts onto a single receptacle, and the overall structure superficially mimics a giant solitary flower. Geometrically, the individual florets are arranged in left and right turning spirals following the consecutive numbers of the Fibonacci sequence. Such a pattern has fascinated interdisciplinary researchers over centuries. Elaborating Gerbera hybrida as a study system, this thesis aims at elucidating the molecular mechanisms underlying three key aspects in the development of a flower head: the phyllotactic patterning, the inflorescence patterning, and the patterning of floral organs. This thesis first combined data from diverse microscopic methods with computational modeling and illustrated how the phyllotactic pattern is established during the growth of Gerbera flower heads. The patterning process was governed by the expansion and contraction of the organogenetic zone where new primordia arise. Earliest bract initia were found to pattern on a ‘naked’ head meristem, and to guide the emergence of Fibonacci spiral numbers. A critical character for the patterning process is the lateral displacement of auxin maxima pointing towards the older neighbor. Results from this thesis provided the first experimental basis for understanding how phyllotactic patterns are transited on a growing meristem. This thesis then demonstrated how flower meristem identity genes GhLFY and GhUFO are co-opted to regulate flower head development. While GhUFO acts as the master regulator of flower identity, GhLFY has evolved two novel functions to regulate the determinacy of inflorescence meristem and the early ray floret initiation. The results provided novel insights to explain how the flower head structures are evolved and diversified. This thesis lastly dissected functions of the SEPALLATA-like GRCDs in regulation of Gerbera flower and inflorescence development. In this study, the GRCDs were shown to have evolved specialized functions in regulating floral organ identities, among which, GRCD4 and GRCD5 are two indispensable regulators for petal development. Moreover, GRCD2 and GRCD7 show redundant functions at the inflorescence level maintaining their determinacy. The results provide an example on how gene duplications could lead to specialized and redundant functions in regulation of a highly elaborated inflorescence structure.
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(Helsingin yliopisto, 2021)Implementation of genomic evaluation is challenging for populations with limited data. The main objective of this thesis was to identify an approach to implement single-step genomic best linear unbiased prediction (ssGBLUP) in a small-scale dairy cattle population. In particular, the aims were to predict GEBVs by ssGBLUP using local genotypes, enhance the reliability of prediction by incorporating data from an external breeding population, and implement the metafounder approach. The first objective of this study was to develop state-of-the-art BLUP Animal Model for Holstein (HOL) and Russian Black & White cattle of Leningrad region (LR, Russia). The traits of focus were milk, fat, and protein yield. The data used to develop the first (FLM) and multiple (MLM) lactation models included 320,633 repeated 305d records from 49 herds. The heritability estimates for milk, fat, and protein yield were 0.24, 0.20, and 0.20 for FLM and 0.18, 0.19, and 0.18 for MLM. For cows born between 2000 and 2016, MLM estimated an annual average genetic gain of 56 kg, 1.84 kg, and 1.62 kg for milk, fat, and protein yield, respectively. The second objective was to implement ssGBLUP for LR using a set of local genotyped animals. Genomic data were available from 1080 cows and 427 bulls. MLM was improved by adding a herd by sire interaction random effect. Milk yield cross-validation analysis showed a validation reliability (R^2) of 0.21 and 0.38 for bulls and cows, respectively. The R^2 values for fat yield for bulls and cows were 0.17 and 0.41. The third objective was to enhance the LR ssGBLUP prediction by using external DFS (Denmark, Finland, and Sweden) HOL genomic and pedigree information. The inclusion of DFS genotypes did not change the milk yield R^2 for bulls but slightly decreased it for cows (0.38 to 0.36). For fat yield, R^2 increased from 0.17 to 0.18 for bulls and decreased from 0.41 to 0.34 for cows. In analysis of milk yield, the highest R^2 was realized in the ssGBLUP model simultaneously using genomic and phenotypic data from both LR and DFS: 0.30 for bulls and 0.42 for cows. In fat yield, no improvement in R^2 was observed (0.18) for bulls and an unexpected decrease was observed for cows (0.21). The fourth objective was to implement the metafounder (MF) approach in ssGBLUP. The data were a subset of Finnish Red dairy cattle, including 112,479 cows with first lactation 305d milk records. Genomic data were obtained from 3,571 bulls and 16,186 cows. MF covariance matrix (Γ) was created using base population allele frequencies estimated using a one-generation pedigree for each animal. After the estimation, markers were filtered with a minor allele frequency criterion of 0.05. Diagonal elements of the genomic relationship matrix had a lower correlation with the regular pedigree relationship matrix (A; 0.66) than with the one using Γ (A^Γ; 0.76). Validation reliability of milk GEBVs in bulls increased by 0.04 (from 0.27 to 0.31) when using the MF approach. In cows the gain was 0.01 (from 0.36 to 0.37).
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(Helsingin yliopisto, 2021)This dissertation examines current collaborative practices in wooden multistorey construction (WMC) projects through a business ecosystem approach to detect effective and ineffective practices when working with novel wooden materials and gaining knowledge of them. Furthermore, it deepens our understanding of citizen perceptions and homeowner experiences towards WMC, to ensure that professionals are able to construct buildings that are not only profitable, but also appreciated by end-users and functional in daily use. Qualitative means are used to provide an in-depth view of the matters. The findings reveal that while construction projects are essentially collaborative efforts, the novelty of WMC materials and methods requires increased communication and learning between the business ecosystem participants. The keystone of a business ecosystem has a fundamental role in enabling deeper, long-term commitment between participants through procurement practices and meetings. These allow participants to work towards a common goal and to accumulate knowledge of wooden construction also between projects. Feedback processes should be improved between the participants, but homeowner experiences should also be utilized more efficiently. The results imply that while the business ecosystem approach provides a usable lens to study collaboration in WMC projects, it should not be seen as a static system concentrating on the design and construction phases, but one that evolves throughout a building’s life cycle, incorporating end-users as the ecosystem keystones when moving to the use phase of the building. Furthermore, the results support previous literature in that end-users appreciate soft aspects of wood material, such as aesthetics and ambiance, while durability and maintenance needs create concerns. However, the qualitative approach used in this study reveals that some aspects are multifaceted, carrying both positive and negative meanings for the end-users. Homeowner experiences indicate the importance of the everyday usability of home materials. Furthermore, the ‘liveliness’ of the wooden material seemed to surprise some of the homeowners, indicating that they are more familiar with other urban construction materials. Communication with end-users should therefore be improved to decrease concerns, but also to inform about the material’s practical benefits such as pleasant soundscapes.
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(Helsingin yliopisto, 2021)The agriculture sector is facing considerable challenges in ensuring food security under projected changes in climate and pressures to reduce its environmental impacts, among others. With changes in growing season and local growing conditions already being observed, adaptation is a key factor in aiming towards climate-smart, sustainable agriculture. Process-based crop models offer a tool for understanding complex interactions associated with crop, environment and management actions, and quantifying their impacts on various outputs. In the face of uncertainties associated with impact estimates, risk assessment has become an essential part of adaptation planning. This study explored the use of a “scenario-neutral” approach for informing risk assessments in the context of crop production. Its main motivation was to examine novel insights offered by the approach for characterising uncertainties associated with modelled impacts compared to conventional scenario-based approaches, where impact estimates are tied to a given scenario. The approach utilises impact response surfaces (IRSs) to depict simulated period-mean sensitivities of cereal yields to systematic changes in baseline (1981–2010) temperature and precipitation. The analysis focused on sites in Finland, Germany and Spain, across a transect of contrasting environmental zones that hence facilitated an examination of the effect of site-specific growing conditions on the impacts of projected changes on cereal yields. The research encompassed a multi-model IRS study involving 26 crop models for wheat as well as an IRS study employing a single model for barley. In addition to analysing median responses of the model ensemble across the transect, approaches were developed for classifying and interpreting individual model responses. By combining IRSs with projections of climate interpreted probabilistically, likelihood of crop yield shortfall was estimated and its evolution throughout the 21st century visualised. This was estimated with a single crop model WOFOST for spring barley in Finland. Effects of adaptation on yield were considered through adapted sowing and cultivar choice. Evolution of future atmospheric carbon dioxide concentration [CO2] defined by representative concentration pathways also used for climate projections (RCP4.5 and RCP8.5) was also considered when estimating likelihoods. With the multi-model ensemble study of wheat yield sensitivities [CO2] was fixed at 360 ppm. Simulated cereal yields were found to decline with warming and drying and increase with higher precipitation. The yield response in Finland was dominated by temperature. Precipitation change dominated the response of spring wheat in Spain, while the response was more mixed in Germany. The multi-model ensemble median response offered a consensus view of impact sensitivities, with individual model behaviour occasionally departing markedly from the average. IRS patterns across the multi-model ensemble showed greater similarity in the pattern of modelled yield responses for Germany in comparison to Finland and Spain. Similarity in patterns was also associated with models of related genealogy. With respect to the effectiveness of tested adaptation options, results suggest that combining cultivars with short pre- and long post-anthesis phases with earlier sowing, offers most promise for obtaining the largest yield gains and smallest likelihoods of yield shortfall under future scenarios. Higher levels of [CO2] generally compensate for yield losses with warming, with the effect emphasised with the biggest increases in temperature. IRSs offer a valid alternative to conventional scenario-based approaches with many advantages for presenting and analysing results. IRSs can assist in model testing, comparison of results across models, studies and sectors and examination of various statistical characteristics of the response, greatly facilitated by the possibility to visually depict impact sensitivities in consistent ways. Use of multi-model ensembles with respect to both climate projections and crop impacts increases the robustness of results and provides information on the uncertainties around the yield estimates. The approach for estimating and visualising impact likelihoods provides improved understanding and transparency of concepts behind the likelihood estimates.
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(Helsingin yliopisto, 2021)The study focused on sensory evaluation professionals and the aim was to gain an understanding of the competencies and practices required in their work in Finnish food industry. In addition, sensory professionals’ assessments of how digitalization can change the evaluation practices were explored. The specific research questions were: 1) What kind of competencies did sensory evaluation professionals report to need in their work? 2) What kinds of knowledge-creating practices did the professionals report? and 3) How did the professionals perceive the influences of using digital technologies on certain aspects of sensory evaluation practices and sensory consumer testing? Altogether 114 sensory professionals from Finnish food companies and universities participated in the study. The first sub-study (I) examined the daily practices of sensory professionals and highlighted the perspective of competencies and contexts. The second sub-study (II) examined the routines and knowledge-creating practices at work, as well as the perceptions of the effects of digitalization on the work. The third sub-study (III) examined how did the sensory professionals perceive the possibilities of utilizing facial expression analysis in sensory evaluation work. According to the results, assessors learn extensively from each other in collaborative interactional and knowledge creation contexts. Learning and development takes place in various practical sensory evaluation assessments, test situations, quality control, and by learning from consumer feedback. Individual and collaborative knowledge-creating practices can be demonstrated in industrial sensory evaluation work. Sensory professionals described and identified practices relevant for creating knowledge. According to sensory professionals, product-specific competencies develop only through several years of long-term training, in which collaboration with other colleagues and product knowledge are key elements. According to the survey and interview results, professionals perceived that their work would change as a result of digitalization and the introduction of new methods. The research results showed that product-oriented assessment methods are accompanied by the tasks related to the management of various digital materials collected from consumer tests. New jobs require sensory professionals to have related digital competencies and understanding of related ethical responsibilities.
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(Helsingin yliopisto, 2021)Cassava (Manihot esculenta Crantz) is a staple food for millions of people in the tropics, providing high energy and nutritional value. Problems associated with cassava in the arid tropics are low yield, high cyanide concentration, and low mineral and vitamin A availability in leaves and roots. These factors hinder the utilization of cassava greatly and are highly influenced by the plant water status and potassium (K) availability in the growth environment. The productivity of cassava in such environments could be improved using irrigation strategies not based on full crop water requirements, such as deficit irrigation combined with K nutrition to ensure sustainable quantity and quality production. Additionally, employing systems that can allow non-destructive estimation of plant performance, such as the use of plant imaging techniques, could provide early information on plant performance, which would facilitate phenotyping, as well as assist in the mitigation of abiotic challenges in cassava production. This study investigated how irrigation water, K fertigation and their interactions affect the growth responses of biofortified cassava during the early growth phase. It was also investigated whether K fertigation could improve the nutritional and reduce the anti-nutritional qualities of biofortified cassava growing under typical conditions of water deficit. The ability to use red-green-blue (RGB) and multispectral sensors to detect the effects of water deficit and low K in cassava, and whether the crop quality changes due to low moisture and low K could be observed from the images, were also examined. Pot experiments were conducted at early developmental stages of cassava under controlled greenhouse conditions at the University of Helsinki. Young biofortified cassava plants were established using cuttings. The plants were subjected to three irrigation treatments (30%, 60%, 100% pot capacity) that were split into five K (0.01 mM, 1 mM, 4 mM, 16 mM, 32 mM) application rates beginning 30 days after planting (DAP) and ended at 90 DAP, when plants were harvested. Irrigation treatments and K application rates significantly affected leaf water potential, leaf osmotic potential, net photosynthesis, stomatal conductance, leaf temperature, leaf chlorophyll, water usage, plant leaf area, plant height, tuber number and whole-plant biomass. The interaction was also statistically significant for these properties. Irrigation treatments and K application rates also affected leaf turgor, but the interaction was not statistically significant. Irrigation at 30% together with 16 mM K lowered the leaf water potential by 69%, leaf osmotic potential by 41%, net photosynthesis by 35%, stomatal conductance by 41%, water usage by 50%, leaf area by 17%, and whole-plant dry mass by 41%, compared with fully irrigated plants. Lowering the K application rate below 16 mM reduced the values further. Most importantly, the growth was decreased least when irrigation was decreased to 60% together with 16 mM K, compared with optimal applications. The combined effect of irrigation treatments and K applications was statistically significant for starch, energy, minerals, total carotenoids, and cyanide, but the interaction was not statistically significant for dietary fibre and crude protein in the leaves and roots. In the leaves and roots, water deficit and lower K applications inhibited starch, carotenoids, crude protein, and fibre synthesis. Water deficit interrupted the uptake of minerals by the cassava roots from the soil and ultimately to the leaves. Improved uptake of calcium, phosphorous, magnesium, zinc, iron, sulphur, and chloride to the leaves was recorded with increased K application regardless of water deficit. K application also mitigated the effect of water deficit at 30% irrigation. Crude protein, carotenoids, and minerals were revealed to be more abundant in the leaves than in the roots, while the inverse was the case for starch content and iron. Cyanide was diminished in the leaves and roots of the cassava with increases in both irrigation and K application rates. Full irrigation (100%) with 16 mM K application produced the highest nutritional quality and the least cyanide concentration. Also, 16 mM K significantly improved the nutritional qualities and diminished the cyanide concentration irrespective of the irrigation treatment. High estimation accuracy (R2 = 0.90) for biomass, chlorophyll, and net photosynthesis were recorded. The estimated leaf area associated strongly (R = 0.98) with the measured leaf area. The estimated biomass also associated strongly with the measured biomass. Starch, energy, total carotenoids, and cyanide were estimated reasonably (R2 > 0.80) and showed strong correlations with the most of spectral indices. The estimation accuracy for all mineral elements was low, and weak relationships existed between the mineral elements and the spectral indices. The regression models identified normalized difference vegetation index, green area index, and simple ratio index as the best estimators of growth and key nutritional traits in cassava. Moreover, irrigation at 30% together with 0.01 mM K reduced all the index values but increased crop senescence index. Regardless of water deficit, increasing K application rate to 16 mM resulted in high values for all the spectral indices but low senescence index. In conclusion, adjusting K fertigation rates in combination with deficit irrigation may improve the growth and dietary quality of young cassava, and reduce cyanide concentration. Findings from this work can be utilized as a foundation to develop agronomic management practices involving K application for cassava growing in water-limited environments. The study also shows that RGB and multispectral sensors can be used to provide indirect measurements of growth and key nutritional traits in cassava, thus representing a tool that breeders may use to facilitate evaluation of cultivars, especially in the developing areas. Information from imaging data may also be used to facilitate corrective measures to avert stress, such as the decision to irrigate or apply fertilizers. Nonetheless, laboratory analysis of plant samples should support sensor estimates, especially under field conditions, when all possible factors affecting plant growth are difficult to forecast. Future studies could place emphasis on field conditions with multiple cassava cultivars and employ different imaging techniques.
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(Helsingin yliopisto, 2021)This research focuses on stand dynamics and ecological recovery in miombo woodlands, Morogoro, Tanzania. The study uses the Kitulangalo Permanent Sample Plots (PSPs) to analyse tree species’ site-specific growth, regeneration dynamics, and stand development using empirical and modeling approaches. The high number of tree species in miombo necessitated the formulation of three species groups involving 1) trees that grow relatively rapidly to be dominants in top canopy layers 2) trees that stay mainly in the lower and middle canopy levels and 3) trees that grow slowly but persistently and may eventually rise to dominant and codominant canopy positions applied in studies I and III. Study III also applies three harvesting alternatives, which align with the recommended harvesting practices for these woodlands. Diameter increment varied with the change in basal area growth across species groups, reaching a maximum of 3.2 cm (group 1) during 2008-2016. Density-dependent mortality and ingrowth also varied with species group as higher mortality rates dominated the lower and middle canopy layers due to asymmetrical competitions. Fencing the plots prompted thick grass cover. The drop in the total number of regeneration stems and the simultaneous increase in the number of main stems in fenced areas and dense plots indicated a self-thinning process induced by competition. This is linked to multi-stem regeneration undergoing a morphological transformation into single-stem saplings (main stems) and eventually becoming small trees. Harvesting intensity, density-dependent mortality, and ingrowth regulated stand basal area and therefore stand growth and development during the simulation. Stand structural development was dominated by species groups 1 and 2, indicating sustainability in species composition and structures. Stand development was affected by the addition of new stems of each species in each simulation year. Miombo stands have demonstrated the potential to attain a steady-state condition over the medium-term under-regulated stand conditions and silvicultural treatments. The developed models, treatments, and harvesting alternatives may be limited in application to Kitulangalo and similar lowland miombo woodlands in Tanzania. Future studies concerning stand conditions, silvicultural treatments, and harvesting alternatives are vital for a better understanding of stand dynamics in miombo woodlands in Tanzania. Keywords: Forest disturbance, tree growth and stand dynamics, regeneration dynamics, silvicultural treatment, harvesting alternative, miombo woodlands
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(Helsingin yliopisto, 2021)The aim of this study was to detect usability of genomic prediction for different breeding dilemmas. In order to achieve this aim, breeding data sets from oat and barley were used in the study. The studied lines were genotyped with genome-wide markers. Meanwhile, phenotypes were collected from multiple years and locations of historical breeding data. Together, the data of the line genotypic and phenotypic information formed the training population used in the analysis. The separate studies concerned genomic prediction, genome-wide association study (GWAS) and analysis on genotype by environment (GE) interaction. The studies had in common that they present ‘difficult’ topics within the breeding process. The original publication I concentrated on improving grain yield prediction for oat and barley. Grain yield presents one of the most important traits in breeding, but has low predictability due to low heritability. The prediction of genomic estimated breeding values (GEBVs) was improved by using multi-trait prediction. For this purpose, grain yield was predicted simultaneously with correlated traits. In addition, benefit of trait-assisted prediction was examined. In conclusion for oat and barley, prediction of grain yield was improved by 4% and 9% with multi-trait prediction, and by 9-14% and 11-28% with trait-assisted prediction compared to prediction of grain yield alone, respectively. The original publication II focused on Fusarium head blight (FHB) resistance in oat. FHB resistance is a troublesome trait to breed, since the disease cannot be reliably scored visually, but extensive laboratory analysis is needed to obtain resistance phenotypes. In addition, FHB resistance consists of multiple components. In the study, the correlations between FHB resistance related traits were high. Much lower correlations were seen between FHB resistance related and agronomic traits. No significant associations between FHB related traits and genetic markers were discovered with reasonable correction of population structure and genetic relationship between the studied oat lines. For this reason, using genome-wide marker information to promote resistance breeding should be done solely with genomic selection (GS), where all the marker effects are used to enrich resistance alleles within the breeding population. The original publication III explored the extent of GE interaction within breeding data sets of oat and barley. At first, the genetic correlations between trial locations within year were calculated and used to compute mean across the years. The correlations suggested that data set of oat was not as sufficient as the data set for barley to explore the quantity of GE interaction. The second step of the analysis contained genomic prediction with six different models. The prediction models contained effects due to lines, genetic, environmental covariates, GE interaction and genotype by environmental covariates interaction. The prediction accuracy was increased for both crops when GE interaction was added into the prediction model. The results from the analysis imply that GE interaction exists within the breeding data sets, and should be taken into account upon prediction. All of the conducted studies proved the usability of genomic prediction in solving principal questions in the breeding process. The studies improved prediction of central traits simultaneously enabling the prediction in the early breeding generations, and showed the significance of GE interaction, and most of all, showed that historical breeding data can be used to predict the important traits. These studies present tools for practical breeding in order to meet the demand to accelerate crop improvement.
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(Helsingin yliopisto, 2021)Water scarcity is globally a key reason for crop yield losses. Difficulties in efficient utilization of the total available precipitation cause yield limitations and even total crop failure at rainfed dryland farms. This study assessed two potentially available measures to improve the water balance of dryland agriculture; water recovery through passive dew collection and soil mulching with plant residue. Dew collection field experiments were conducted to evaluate the effectiveness of various plastic materials in dew collection in dryland conditions. The planar dew collectors were of the standard type: a 1 m2 surface tilted at 30◦ in respect to the horizontal. Dew yields were measured daily over a one-year period. The condensing surface temperatures and the meteorological conditions were monitored continuously, to calculate potential dew output. A laboratory method was prepared and tested for evaluating the attributes affecting dew condensation and droplet flow. The condensing surfaces were cooled below dewpoint by utilizing Peltier elements in controlled conditions. Dew yields measured in laboratory conditions were compared with calculated dew outputs, and with dew yields measured in field conditions. Dew occurred throughout the year, even through the dry seasons, and may be considered a small but reliable source of water. Annual dew yields were < 8% of the annual precipitation (322 mm). No significant differences were found between the tested materials. Clear skies, calm winds (0.5–2.5 m s-1), and conditions with dew point close to air temperature (Tdew ≈ Tair) favor dew condensation. Placement in the field affects the airflow characteristics at the condensing surfaces and ultimately the collected dew quantities, thus attention should be paid to the placement of the dew collectors in the field. Based on our results, a more comprehensive laboratory evaluation regime, with specific design and measurements of the airflow characteristics is needed to draw valid conclusions on the differences between the plastic foils. Mulching with plant residue was studied by measuring soil volumetric water content in vertical profiles in bare and plant residue-covered soil during a 100-day period. A one-dimensional model based on Richard’s equation was used to predict the effect of mulch over a two-year period. Compared with bare soil, mulching prolonged the time when continuously measured soil moisture content exceeded the water stress limit of maize. The predicted water-conserving effect increased with mulch thickness. However, plant residue mulch degrades naturally and the availability and competitive uses of plant residues may limit the thickness of the mulch layer. A mulch layer with a thickness of > 1 cm brought clear improvements to the soil moisture conditions and resilience against dry spells compared with bare soil. The presented models were capable of estimating the cumulative dew condensation and soil moisture behavior satisfactorily over time. For further development of the dew condensation model, more specific airflow measurements are needed for calculating the mass transfer for more accurate predictions of nightly dew quantities. This study touched upon the combined effect of irrigation with dew water and mulching on by using modeling to predict the effects on soil moisture. The results indicated that mulching improves the usability of irrigational water recovered from dew by reducing evaporation. The effect increases with layer thickness. Future research steps could include the quantification of the presented water-conserving measures on actual crop yields, especially the combined effect.
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Dry fractionation and functionalisation of cereal side streams for their improved food applicability (Helsingin yliopisto, 2021)The agro-food industry generates annually substantial amounts of side streams, resulting in the loss of high-quality protein and dietary fibre, whereas their incorporation into the food chain would positively contribute to resource sufficiency and healthier diets. However, plant-based ingredients, especially proteins, typically deliver limited performance in certain food applications, such as beverages and spoonable products, when compared with their animal-based counterparts. Therefore, fractionation and functionalisation techniques are investigated and applied to improve the applicability of the plant-origin ingredients in a wider range of food matrices where they can offer alternatives to animal-based ingredients. Dry fractionation provides a sustainable and gentle processing technology, which allows the production of multicomponent hybrid-ingredients, enriched in protein but also containing considerable amounts of dietary fibre or starch, depending on the raw material. The aim of the current work was to investigate the use of dry fractionation, more specifically, dry milling and air classification, for increasing the protein content of cereal side streams, namely, wheat, rice and rye brans, and the barley endosperm fraction. In addition, the objective was to understand the factors affecting the technological functionality and applicability of the protein-enriched ingredients in the relevant food matrices. To facilitate a more efficient fractionation, pre-treatments, including defatting with supercritical carbon dioxide (SC-CO2) for rice bran, moisture removal for wheat and rye brans and mixing with a flow aid for the barley endosperm fraction, were elucidated. The technological functionality of the protein-enriched fractions was examined, and bioprocessing and physical processing approaches for improving the ingredient applicability in high-moisture food systems were investigated with rice and barley fractions. This study revealed that the fat removal, drying and use of flowability aids were effective in enhancing dry fractionation by improving the processability, particle size reduction and dispersability of rice bran, wheat and rye brans, and the barley endosperm fraction, respectively. Pin disc milling and air classification of a SC-CO2-extracted rice bran increased the protein content from 18.5 to 25.7% with 38.0% protein separation efficiency (PSE). Alternatively, a two-step air classification of the defatted rice bran allowed to reach a slightly higher protein content (27.4%) with lower PSE (20.2%) compared with the one-step air classification approach. Air classification of the dried and pin disc-milled wheat and rye brans increased the protein content from 16.4 and 14.7%, respectively, to 30.9 and 30.7%, with PSE of 18.0 and 26.9%. Additionally, soluble-to-insoluble dietary fibre ratios were increased and phytic acid was considerably enriched in bran fractionations. The maximum protein content reached by air classification from the barley endosperm fraction, initially containing 80.0% starch and 8.3% protein, was 28.3% with 21.7% PSE, while reaching a lower protein enrichment level of 22.3% allowed obtaining PSE of 59.4%. The protein-enriched fractions, especially those from rice and wheat, exhibited higher protein solubility than the raw material brans, presumably due to the enrichment of albumin and globulin proteins from the aleurone during air classification, which was also indicated by an altered protein profile and the co-enrichment of phytic acid. When the ultra-fine milling of wheat and rye brans was explored as an alternative to fractionation, the formation of damaged starch and lowered protein solubility were observed. The protein-enriched brans and the ultra-finely milled brans both showed improved dispersion stabilities, whereas pasting viscosities, and water and oil binding capacities were lower for the hybrid ingredients compared with the pin disc-milled raw materials. The protein-enriched fraction from barley, on the other hand, exhibited low protein solubility and limited techno-functional properties. The applicability of the protein-enriched fractions in high-moisture food model systems was tested after ingredient modifications via enzyme treatment, ultrasonication and pH shifting. Phytase treatment of the protein-enriched rice bran fraction improved the behaviour of the ingredient in heat-induced gelation, especially under alkaline conditions. For the protein-enriched barley fraction, ultrasound treatment with or without pH shifting reduced particle size; improved colloidal stability at pH 3, 7 and 9; and increased protein solubility, especially at pH 9. To conclude, dry fractionation of cereal side streams allowed protein enrichment with a concurrent increase in the soluble-to-insoluble dietary fibre ratios of the brans and considerable reduction in the starch content of the barley endosperm fraction. Additionally, this thesis demonstrated for the first time that cereal side stream-derived, protein-enriched hybrid ingredients exhibit improved technological functionalities that can be further enhanced via enzymatic or physical processes that affect, for example, their gelation and dispersion stability. The bioprocessed protein-enriched rice bran fraction could find potential use as a raw material in spoonable food products delivering a good amount of protein and dietary fibre and allowing the use of the nutritional claim that the food is a ‘source of fibre’. The ultrasound-treated barley protein ingredients, on the other hand, should be further studied in the manufacturing of plant-based milk substitutes. In general, these improved ingredient properties suggest the possibility of developing novel side stream-based food ingredients with increased nutritional and technological qualities that simultaneously contribute positively to raw material resource sufficiency.
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(Helsingin yliopisto, 2020)The research documented in publications I-IV involved studies in dry, late-pregnant Ayrshire dairy cows on grass silage (GS) based diets (I-IV). The principal aim was to investigate the effect of prepartal plasma non-esterified fatty acids (NEFA) level (I) and the effect of prepartal dietary energy intake (II-IV) on the development of insulin resistance (IR) during late pregnancy (I-IV) and changes in insulin resistance in early lactation (II-IV). Detailed, extensive physiological studies were conducted to understand the mechanisms underlying the development of maternal insulin resistance and to investigate the impact of changes in dietary energy level and subsequent changes in plasma NEFA concentration prepartum. The insulin resistance was assessed by interpretation of data from intravenous glucose tolerance test (IVGTT) with minimal model (MM) approach (I-III) and by insulin challenge (IC; I, II) data. Besides insulin resistance, also the impact of prepartal energy intake on metabolic adaptations, tissue deposition and mobilisation as well as dry matter intake (DMI) and lactational performance were investigated in publications III and IV. In publication I, the key objective was to evaluate the effects of increment of plasma NEFA concentration, typically observed during the last weeks of pregnancy and in early weeks of lactation in dairy cows, on glucose tolerance and responsiveness or sensitivity to insulin as assessed by IVGTT and IC. The greater NEFA levels were achieved by abomasal infusion of tallow (TAL) or camelina oil (CAM). Compared with water infusion (CON), infusion of lipids increased basal plasma NEFA concentrations by around 50%, to an equal level than what was found in dairy cows 2 to 1 weeks prepartum on GS-based diets (II-IV). Elevation of plasma NEFA concentration impaired glucose clearance and decreased insulin secretion during metabolic challenges. These data suggest that elevated plasma NEFA concentrations impaired whole-body insulin responsiveness and sensitivity in dry cows in late pregnancy. As assessed by MM indices, both the disposition and the insulin sensitivity indices were greater after CAM than TAL infusion during IVGTT. Compared with TAL infusion, CAM had an insulin-sensitizing effect which was most likely caused by alterations in plasma profiles of major long-chain fatty acid (FA) groups. A 50% increment in the percentage of polyunsaturated FA (C18:2 and C18:3) and a similar decrease in the percentage of monounsaturated FA (C16:1 and C18:1) was found in plasma FA profiles after CAM infusion when compared with TAL. In publication II, the dietary effects on insulin resistance were assessed not only by the level of energy intake but also by comparing tissue responses to glucose and insulin in late pregnancy vs. early lactation. Compared with controlled energy intake (CEI), the effect of prepartal overfeeding and gradual restriction of energy (HEI) had a minor effect on whole-body insulin resistance during the transition period. An attenuated prepartal NEFA response to endogenous insulin was found in HEI cows suggesting a more refractory adipose tissue to insulin than in CEI. After parturition, this effect was reversed. Across the dietary treatments, both basal and stimulated insulin concentration decreased after parturition as a result of a lower response to a similar secretory stimulus than before parturition and due to increased clearance of insulin postpartum. Compared with prepartal IVGTT, glucose disposal was enhanced postpartum across the dietary treatments. A hyperbolic relationship denoted as the disposition index (DI) was observed during the IVGTT. Compared with prepartal glucose and insulin dynamics across the diets, the MM indices point to increased insulin resistance shortly before than shortly after parturition. However, low insulin concentration is the major factor regulating the use of glucose by peripheral tissues in early lactation. The lack of dietary effect on whole-body insulin resistance in publication II was most likely due to minor dietary effect on tissue accretion between treatment groups, although the lower prepartal plasma NEFA concentration in HEI than in CEI cows suggests enhanced lipid deposition in adipose tissue before parturition, facilitated by higher plasma insulin (IV). No dietary effect on plasma hormone and metabolite concentrations or total DMI was found after parturition. High energy intake during the dry period tended to decrease milk yield after calving (IV). In publication III easily applicable diets suitable for loose housing systems were compared. An ad libitum allowance of GS (HEI) induced a more pronounced BW and BCS change prepartum when compared with a GS-diet diluted with wheat straw (CEI). HEI cows demonstrated a compensatory insulin response to glucose in prepartal IVGTT which preserved glucose tolerance of peripheral tissues. The HEI diet reduced and delayed NEFA suppression suggesting decreased insulin sensitivity and responsiveness in adipose tissue prepartum. The high NDF-content in CEI diet probably decreased ruminal propionic acid production as reflected by lower prepartal glucose and insulin CEI cows. Prepartal energy level did not affect metabolic flexibility of transition dairy cows as assessed by the absence of dietary effect on mobilisation of body reserves, plasma metabolites and hormones, and DMI after calving, whereas milk yield was greater from week 5 onward in HEI than in CEI. The moderate negative effects of gradual restriction of prepartal energy and dilution of energy by mixing GS with wheat straw on early lactation production response demonstrated that these feeding practices were not optimal for transition dairy cows. A moderate or ad libitum overfeeding affected peripheral insulin resistance in the level of prepartal lipid metabolism, while ad libitum overfeeding of GS induced changes in prepartal glucose metabolism as well. Both the difference in energy intake and the composition of the diet contributed to the observed effects on glucose and NEFA dynamics orchestrated via changes in insulin concentration in the transition period. Keywords: dairy cow, transition period, grass silage, energy intake, peripheral insulin resistance, adipose tissue lipolysis, plasma hormone and metabolite, milk yield, lipid infusion, plasma NEFA, minimal model
